SYNTHESIS AND ENGINEERING OF POLYMERIC LATEX PARTICLES FOR
MEDICAL APPLICATIONS
By
SANGYUP KIM
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
2005
Copyright 2005
by
Sangyup Kim
To all who made this work possible
ACKNOWLEDGMENTS
I owe what I am today and this moment to many people without whom I could not
have been here First of all I would like to express my sincere and deepest appreciation
to my parents wife Mikyung daughters Heehyung and Erin whose love patience and
support helped me pursue my dream I gratefully acknowledge my advisor Dr H El-
Shall who gave me an opportunity to become part of an exciting and beneficial research
project and exert myself in the research work His guidance knowledge and stimulating
scientific and critical thinking have contributed to my success to a great extent and
inspired me to dedicate myself to research work I especially appreciate Dr R Partch as a
co-advisor who has helped me conduct experiments with valuable discussion for my
research from my first year I am greatly thankful to Dr C McDonald whose idea and
valuable experimental advice and discussion in the area of polymer chemistry and
membrane technology helped cultivate my interest in this area and were vital to
performing this research Without his contribution this work could not have been
achieved I also thank my doctoral committee members Dr R Singh Dr A Zaman and
Dr B Koopman for appreciated comments
Financial support and equipment use for research from the Particle Engineering
Research Center at the University of Florida were vital in continuing my study for more
than 4 years My sincere thanks are due to my former advisor Dr Kiryoung Ha in
Keimyung University who helped me open my eyes and broaden my thought and
introduced me into a limitless polymer field I would like to also thank to past and current
iv
members in Dr El-Shallrsquos research group for their assistance advice and support I
sincerely appreciate Stephen for the assistance of thesis writing
v
TABLE OF CONTENTS page
ACKNOWLEDGMENTS iv
LIST OF TABLES viii
LIST OF FIGURES x
1 INTRODUCTION 1
2 BACKGROUND AND LITERATURE SURVEY5
21 The Significance of End Stage Renal Disease (ESRD)5 22 Hemodialysis (HD) Treatment 6 23 Advances in Membrane Technology 8 24 Sorbent Technology16 25 Limitation of Current Hemodialysis Treatment 19 26 Latex Particles 20
261 The Components for Emulsion Polymerization 21 262 Particle Nucleation 21 263 Types of Processes for Emulsion Polymerization 23 264 Chemistry of Emulsion Polymerization 24 265 Seed Emulsion Polymerization25 266 Polystyrene Latex Particles 26 267 Various Applications of Latex Particles28
27 Proteins 30 271 Interaction Forces between Proteins33 272 β -Microglobulin (β M)2 2 35 273 Serum Albumin 36
28 Protein Adsorption38 281 Interaction between Protein Molecule and Latex Particle38
29 Hypothesis for Toxin Removal39 291 Toxin Removal by Size Sieving Based on Monodispersed Pore Size 40 292 Toxin Removal by Selective Adsorption on Engineered Latex Particles 41
3 EXPERIMENTAL AND CHARACTERIZATION METHODOLOGY 44
31 Materials 44 32 Latex Particle Preparation46
321 Preparation of Seed Latex Particles47
vi
322 Preparation of Seeded Latex Particles 48 323 Preparation of Core Shell Latex Particles 48 324 Purification of Synthesized Latex Particles48
33 Characterization49 331 Degree of Conversion49 332 Fourier Transform Infrared (FTIR) Spectroscopy49 333 Quasielastic Light Scattering (QELS) 50 334 Field Emission-Scanning Electron Microscopy (FE-SEM) 50 335 Zeta Potential Measurement 50 336 Protein Adsorption51 337 Blood Biocompatibility by Hemolysis Test 53
4 RESULTS AND DISCUSSION55
41 Polymerization of Latex Particles55 411 Polystyrene Seed Latex Particles55 412 The Growth of Polystyrene (PS) Seed Latex Particles61 413 Core Shell Latex Particles 71
42 Characterization of Latex Particles75 421 Fourier Transform Infrared Spectroscopy (FTIR)75 422 Zeta Potential Measurements 75
43 Protein Adsorption Study 85 431 Adsorption Isotherm87 432 Adsorbed Layer Thickness 103 433 Gibbs Free Energy of Protein Adsorption 104 434 Kinetics of Adsorption 107
44 Blood Compatibility 112
5 SUMMARY OF RESULTS CONCLUSION AND RECOMMENDATION FOR FUTURE WORK115
51 Summary of Results115 52 Conclusions121 53 Recommendation for Future Work121
LIST OF REFERENCES122
BIOGRAPHICAL SKETCH 141
vii
LIST OF TABLES
Table page 4-1 The polymerization recipe of polystyrene (PS) seed particles 57
4-2 Continuous addition emulsion polymerization recipe for growing polystyrene (PS) latex particles less than 500nm in size 63
4-3 Continuous addition emulsion polymerization recipe for growing polystyrene (PS) latex particles larger than 500nm in size64
4-4 The preparation recipe of PS core with various shell latex particles72
4-5 The isoelectric point (IEP) of common proteins 83
4-6 The equilibrium concentration values of BSA adsorption on polystyrene (PS) latex particles calculated the Langmuir-Freundlich isotherm model 93
4-7 The equilibrium concentration values (q ) of BSA adsorption on PSPMMA particles calculated the Langmuir-Freundlich isotherm model
m 10096
4-8 The equilibrium concentration values of BSA adsorption on PSPMMA PAA particles calculated the Langmuir-Freundlich isotherm model
90 1098
4-9 The equilibrium concentration values of BSA adsorption on PSPMMA PAA particles calculated the Langmuir-Freundlich isotherm model
75 25100
4-10 The equilibrium concentration values of β M adsorption calculated the Langmuir-Freundlich isotherm model
2102
4-11 Absorbed BSA layer thickness (Aring) of BSA in phosphate buffer (PB) at 37 Co 104
4-12 Absorbed BSA layer thickness (Aring) of BSA in phosphate buffered saline (PBS) at 37 Co 104
4-13 The values of Gibbs free energy change of BSA adsorption in phosphate buffer (PB) at 37 Co 105
4-14 The values of Gibbs free energy change of BSA adsorption in phosphate buffered saline (PBS) at 37 Co 105
viii
4-15 The values of Gibbs free energy change of β M adsorption in phosphate buffere (PB) at 37 C and pH 74
2o 106
4-16 Fitting parameters of second-order kinetic model 111
ix
LIST OF FIGURES
Figure page 1-2 Stereo drawing of the αndashcarbon backbone of β M2 2
2-1 An image of the location and cross section of a human kidney 5
2-2 A schematic draw of the hemodialysis route7
2-3 Cross-sectional SEM image view of the Polyflux S (polyamide + polyacrylethersulfone + polyvinypyrollidone) dialysis membranes 11
2-4 The chemical structure of polysulfone (PSf) 12
2-5 The chemical structure of polyamide (PA)13
2-6 Chemical structure of polyacrylonitrile (PAN) 14
2-7 Emulsion polymerization system24
2-8 L-α-amino acid 31
2-9 Ribbon diagram of human β M taken from the crystal structure of the protein bound to the heavy chain of the MHC class I complex (PDB 1DUZ)
236
2-10 Secondary structure of human serum albumin (HSA) with sub-domains 37
2-11 The relationship between pore size and particle size in body-centered cubic and closed packed arrays41
2-12 Schematic representation of the protein adsorption on the core shell latex particles at pH 7442
3-1 Chemical structure of main chemicals45
3-2 Experimental setup for semi-continuous emulsion polymerization 46
3-3 The particle preparation scheme in various types and size ranges of latex particles 47
3-4 Schematic of the procedure for a protein adsorption test 52
3-5 Separation of RBC from whole blood by centrifuge process53
x
3-6 Schematic of the procedure for hemolysis test 54
4-1 Schematic representation of semi-continuous seed latex particles preparation and growth56
4-2 Scanning Electron Micrograph (SEM) of polystyrene seed latex particles (A) PS (B) PS (C) PS (D) PSS259 S233 S207 S181 59
4-3 SEM of PS latex particles less than 500nm in size (A) 258nm (B) 320nm (C) 370nm (D) 410nm 65
4-4 SEM of PS latex particles larger than 500nm in size (A) 525nm (B) 585nm (C) 640nm (D) 790nm 68
4-5 Dependence of the particle size on the surfactant to monomer ratio70
4-6 Schematic of core shell latex particle structures 71
4-7 Scanning Electron Micrograph of latex particles (A) PS (B) PSPMMA (C) PSPMMA PAA (D) PSPMMA PAA
100
90 10 75 25 73
4-8 FTIR spectra of polymerized latex particles (A) bare polystyrene (PS) (B) PSPMMA (C) PSPMMA PAA (D) PSPMMA PAA100 90 10 75 25 76
4-9 Schematic representation of ion distribution near a positively charged surface 78
4-10 Schematic representation of zeta potential measurement79
4-11 Zeta potential of PS latex particles at 25 Co 80
4-12 Zeta potential of PSPMMA latex particles at 25 C100o 81
4-13 Zeta potential of PSPMMA PAA latex particles at 25 C90 10o 82
4-14 Zeta potential of PSPMMA PAA latex particles at 25 C75 25o 82
4-15 The decay of surface potential with distance from surface in various electrolyte concentrations (1) low (2) intermediate (3) high 84
4-16 High-affinity adsorption isotherm of typical flexible polymer on solid surface 86
4-17 Overall schematic representation of the protein adsorption on the synthesized latex particles86
4-18 The dependence of UV absorbance on BSA concentration (A) 10mgml (B) 06mgml (C) 02mgml87
4-19 Standard curve of net absorbance vs BSA sample concentration 88
xi
4-20 Fitted models for adsorption isotherm of bovine serum albumin (BSA) on polystyrene latex particles at 37 C in phosphate buffer mediao 89
4-21 Adsorption isotherm of BSA on polystyrene (PS) latex particles in phosphate buffer (PB) at 37 Co 90
4-22 Adsorption isotherm of BSA adsorption 37 C in phosphate buffered saline (PBS) on PS latex particles
o
91
4-23 Conformation of bovine serum albumin (BSA) on latex particles92
4-24 Adsorption isotherm of BSA 37 C in PB on PSPMMA core shell latex particles
o100
94
4-25 Adsorption isotherm of BSA at 37 C in PBS on PSPMMA core shell latex particles
o100
95
4-26 Adsorption isotherm of BSA at 37 C in PB on PSPMMA PAA core shell latex particles
o90 10
97
4-27 Adsorption isotherm of BSA at 37 C in PBS on PSPMMA PAA core shell latex particles
o90 10
98
4-28 Adsorption isotherm of BSA 37 C in PB on PSPMMA PAA latex particleso75 25 99
4-29 Adsorption isotherm of BSA 37 C in PBS on PSPMMA PAA core shell latex particles
o75 25
99
4-30 Adsorption isotherm of β M onto PS and PSPMMA latex particles in PB at 37 C and pH 74
2 100o 101
4-31 Adsorption isotherm of β M onto PS and PSPMMA PAA latex particles in PB at 37 C and pH 74
2 90 10o 101
4-32 Adsorption isotherm of β M onto PS and PSPMMA PAA latex particles in PB at 37 C and pH 74
2 75 25o 102
4-33 Gibbs free energy of adsorption of proteins on latex particles in PB at 37 C and pH 74
o
107
4-34 The kinetics of protein adsorption in PB on PS latex particles at 37 C and pH 74
o
109
4-35 The kinetics of protein adsorption in PB on PSPMMA latex particles at 37 C and pH 74
100o
109
4-36 The kinetics of protein adsorption in PB on PSPMMA PAA latex particles at 37 C and pH 74
90 10o 110
xii
4-37 The kinetics of protein adsorption in PB on PSPMMA PAA latex particles at 37 C and pH 74
75 25o 110
4-38 Image of red blood cells 113
4-39 Hemolysis caused by latex particles n=5114
xiii
Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy
SYNTHESIS AND ENGINEERING OF POLYMERIC LATEX PARTICLES FOR MEDICAL APPLICATIONS
By
Sangyup Kim
December 2005
Chair Hassan El-Shall Major Department Materials Science and Engineering
Latex particles with well-defined colloidal and surface characteristics have received
increasing attention due to their useful applications in many areas especially as solid
phase supports in numerous biological applications such as immunoassay DNA
diagnostic cell separation and drug delivery carrier Hemodialysis membrane using
these particles would be another potential application for the advanced separation
treatment for patients with end stage renal disease (ESRD) It is desirable to remove
middle molecular weight proteins with minimal removal of other proteins such as
albumin Thus it is necessary to understand the fundamental interactions between the
particles and blood proteins to maximize the performance of these membranes This
improvement will have significant economic and health impact
The objective of this study is to synthesize polymeric latex particles of specific
functionality to achieve the desired selective separation of target proteins from the human
blood Semi-continuous seed emulsion polymerization was used to prepare monodisperse
xiv
polystyrene seed particles ranging from 126plusmn75 to 216plusmn53 nm in size which are then
enlarged by about 800nm Surfactant amount played a key role in controlling the latex
particle size Negatively charged latex particles with a different hydrophobicity were
prepared by introduction of a sodium persulfate initiator and hydrophilic acrylic acid
monomer The prepared polymeric particles include bare polystyrene (PS) particles less
hydrophobic PS core and PMMA shell particles and more hydrophilic PS core and
PMMA-co-PAA shell latex particles with a 370nm mean diameter SEM light scattering
and zeta potential measurements were used to characterize particle size and surface
properties Adsorption isotherms of two proteins bovine serum albumin (BSA) and β2-
microglobulin (β2M) on latex particles were obtained as a function of pH and ionic
strength using the bicinchoninic acid (BCA) assay method The Langmuir-Freundlich
adsorption model was used to determine the adsorption amount of protein at equilibrium
The thickness of adsorbed BSA layer on latex particles was obtained in order to
investigate the adsorption orientation such as end-on or side-on mode Adsorption
kinetics experiments for both proteins and all latex particles were also performed The
adsorption kinetic constant determined from the Langmuir-Freundlich adsorption
isotherm model was used to calculate Gibbs free energy of adsorption to compare the
competitive adsorption of BSA and β2M Hemolysis tests were performed to investigate
the blood compatibility of synthesized latex particles PSPMMA90PAA10 and
PSPMMA75PAA25 core shell latex particles had desirable material properties with not
only a large amount and high rate of selective β2M adsorption over BSA but also high
blood compatibility showing less than 3 hemolysis
xv
CHAPTER 1 INTRODUCTION
End stage renal disease (ESRD) is a chronic condition in which kidney function is
impaired to the extent that the patientrsquos survival requires removal of toxins from the
blood by dialysis therapy or kidney transplantation The National Kidney Foundation
estimates that over 20 million Americans had chronic kidney disease in 2002 [NKF] The
number of people with ESRD is rapidly increasing in the United States with
approximately 96 295 incidents and 406 081 prevalent patients including 292 215 on
dialysis and 113 866 with a functioning graft in 2001 It is projected that there will be
more than 22 million ESRD patients by 2030 [USRDS 2003] The expenditure for the
ESRD treatment program had reached $228 billion 64 of the Medicare budget in
2001 Due in part to the limited availability of kidneys for transplantation hemodialysis
is the primary clinical treatment for the patients with ESRD
The central element of a hemodialysis instrument is the semipermeable membrane
that allows for selective transport of low molecular weight biological metabolites less
than 5000 Da such as urea and creatinine as well as excess water and electrolytes [Baker
2004] from the blood One limitation of current dialysis technologies is the inability to
efficiently remove middle molecular weight toxins such as β2-Microglobulin (β2M) and
interleukin 6 (IL-6)
β2M is a causative protein of dialysis-related amyloidosis (DRA) a disease arising
in patients with chronic kidney failure as a serious complication of long-term
hemodialysis treatment [Gejyo et al 1985] β2M deposition in tissue is the primary cause
1
2
of destructive arthritis and carpal tunnel syndrome [Vincent et al 1992 Drueke 2000]
The β2M structure is shown in Figure 1-2
Figure 1-2 Stereo drawing of the αndashcarbon backbone of β2M [Becker et al 1985]
Although attempts have been made to increase the efficiency of middle molecular
weight toxin removal by changes in the membrane pore size and the use of innovative
materials to adsorb these toxins [Samtleben et al 1996 Ronco et al 2001] removal
efficiency is not as high as those achieved by a normal healthy kidney Traditional
membranes have a number of processing and performance limitations [Westhuyzen et al
1992 Leypoldt et al 1998] such as a restricted choice of surface chemistries and limited
control of porosity The development of novel engineering membrane technology is
needed to remove middle molecule toxins
Polymeric latex particles have received increasing attention in medical application
areas especially as solid phase supports in biological applications [Piskin et al 1994]
Examples of these applications include immunoassay [Chen et al 2003 Radomske-
Galant et al 2003] DNA diagnostic [Elaїssari et al 1998] cell separation drug delivery
carrier [Luck et al 1998 Kurisawa et al 1995 Yang et al 2000] etc This is because of
3
the well-defined colloidal and surface characteristics of the particles By using a seed
emulsion polymerization method it is possible to synthesize monodisperse latex particles
with various particle size ranges and surface chemistry Functionalized core-shell latex
particles can be introduced by multi-step emulsion polymerization Core particles are
synthesized in the first stage of the polymerization and the functional monomer is added
in the second stage This is done without any emulsifier addition to prevent the
production of new homopolymer particles [Keusch et al 1973] The core-shell particles
are useful in a broad range of applications because of their improved physical and
chemical properties over their single-component counterparts [Lu et al 1996 Nelliappan
et al 1997] Through the development of a hemodialysis membrane using monodisperse
latex particles improvements in advanced separation treatment for patients with end
stage renal disease (ESRD) can be realized This requires the maximum removal of
middle molecular weight proteins with minimal removal of other beneficial proteins such
as albumin Thus an understanding of the fundamental interactions between the particles
and biopolymers is vital to maximize the performance of this membrane technology
The field of material science and biotechnology is based on fundamental chemistry
has developed over the past three decades into todayrsquos powerful discipline that enables
the development of advanced technical devices for pharmaceutical and biomedical
applications This novel and highly interdisciplinary field is closely associated with both
the physical and chemical properties of organic and inorganic particles [Niemeyer 2001]
Hemodialysis membrane using these particles would lead to improvements in the
advanced separation treatment for patients with end stage renal disease (ESRD) The
interdisciplinary nature of this approach enables a more complete understanding of the
4
phenomena of protein adsorption and the material properties necessary for selective
separation This innovative approach to membrane fabrication has the potential of making
inexpensive highly efficient membranes for both industrial and specialized separation
processes
The goal of this study is to prepare polymeric latex particles with tailored
properties to maximize separation of toxin molecules and to investigate the fundamental
interactions between the particles and molecules in the biological system in order to
optimize the performance of a membrane material for these applications
Polymeric latex particles were synthesized with specific functionality in an attempt
to achieve selective separation Seeded emulsion polymerization was used to synthesize
functionalized monodisperse latex particles in various sizes Negatively charged
hydrophobic polystyrene latex particles were synthesized by the same method Core shell
latex particles PSPMMA100 PSPMMA90PAA10 PSPMMA75PAA25 were also
synthesized to differentiate the degree of hydrophobicity of particles Scanning Electron
Micrograph (SEM) and light scattering measurements were used to characterize particle
size and shape and zeta potential measurements were conducted to measure the electrical
surface property of synthesized particles Adsorption isotherms of target proteins bovine
serum albumin (BSA) and β2M on latex particles were obtained as a function of pH
ionic strength and protein concentrations using the BCA assay method Adsorption
kinetics for both proteins on the latex particles were also measured Finally hemolysis
tests were run to determine the biocompatibility of polymer latex particles with human
blood This research will be described in more detail in chapters 3 and 4
CHAPTER 2 BACKGROUND AND LITERATURE SURVEY
21 The Significance of End Stage Renal Disease (ESRD)
The kidneys are responsible for removing excess fluid minerals and wastes from
the blood regulating electrolyte balance and blood pressure and the stimulation of red
blood cell production They also produce hormones such as erythropoietin (EPO) and
calcitriol that keep bones strong and blood healthy [Casadevall and Rossert 2005 Kurbel
et al 2003] EPO acts on the bone marrow to increase the production of red blood cell in
case of bleeding or moving to high altitudes Calcitriol mainly acts on both the cells of
the intestine to promote the absorption of calcium from food and also bone to mobilize
calcium from the bone to the blood When kidneys fail harmful waste builds up blood
pressure rises and body retains excess fluid The body also does not make enough red
blood cells When this happens treatment is needed to replace the function of failed
kidneys
Figure 2-1 An image of the location and cross section of a human kidney
5
6
The National Kidney Foundation estimates that over 20 million Americans had
chronic kidney disease in 2002 [NKF] Chronic renal disease is a gradual and progressive
loss of the function unlike acute renal disease where sudden reversible failure of kidney
function occurs Chronic renal failure usually takes place over a number of years as the
internal structures of the kidney are slowly damaged In the early stages there can be no
symptoms In fact progression may be so gradual that symptoms do not occur until
kidney function is less than one-tenth that of a normal health kidney If left untreated
chronic kidney disease may ultimately lead to kidney failure known as End Stage Renal
Disease (ESRD) ESRD is a rapidly growing heath-care problem in the United States In
2001 approximately 96295 incidents and 406081 prevalent patients were diagnosed
with ESRD including 292215 patients on dialysis and 113866 patients with a
functioning graft [USRDS 2003] The projected number of patients with ESRD is
expected to exceed 22 million patients by 2030 with much of this growth being driven by
the increasing prevalence of major contributing factors such as diabetes and high blood
pressure [USRDS 2003] A great extent of ESRD program cost and Medicare budget
have been spent in 2001 Due in part to a limited availability of kidneys for
transplantation hemodialysis (HD) is the primary method of treatment for ESRD and is
currently used for approximately 61 of US ESRD patients
22 Hemodialysis (HD) Treatment
HD removes toxins from the body by extracorporeal circulation of the blood
through a semipermeable membrane referred to as a dialyzer The toxins are removed
primarily by diffusion across the membrane to a dialysate solution which is circulated on
the opposite side of the membrane The cleaned blood is then returned to the blood
stream A surgically constructed vascular access connects the extracorporeal circuit to the
7
patientrsquos system Treatments are almost always performed three times per week in
specially equipped dialysis facilities for 3-4 hours per each treatment Figure 2-2 shows
the scheme of the hemodialysis route The critical element of a HD instrument is the
semipermeable membrane which allows for selective transport of low molecular weight
biological metabolites from blood
igure 2-2 A schematic draw of the hemodialysis route
ESRD patients who undergo dialysis therapy often experience several other
problems associated with the treatment such as anemia fatigue bone problems joint
problems itching sleep disorders and restless legs Anemia is common in patient with
kidney disease because the kidneys produce the hormone erythropoietin (EPO) which
stimulates the bone marrow to produce red blood cells Diseased kidneys often do not
produce enough EPO to stimulate the bone marrow to make a sufficient amount of red
blood cells This leads to bone disease referred to as renal osteodystrophy and causes
bones to become thin and weak or malformed and can affect both children and adults
Older patients and women who have gone through menopause are at greater risk for this
disease Uremic toxins which cannot be remove from the blood by the current dialyzer
membranes can lead to itching This problem can also be related to high levels of
F
8
parathyroid hormone (PTH) Dialysis patients can also experience day-night revers
is they have insomnia at night and sleep during the day This can be related to possible
nerve damage in the body and a chemical imbalance in the blood due to the excess toxin
Oxidative stress is a problem for patients on maintenance dialysis This problem is due to
an imbalance between pro- and antioxidant factors [Roselaar et al 1995 Cristol et al
1994] Oxidative stress affects oxidation of low-density lipoproteins which are the main
factor for atherogenesis [Huysmans et al 1998] and is also involved in the development
of malignancies and diabetes mellitus [Rice-Evans et al 1993] In order to reduce
antioxidant defense dialysis is needed to contribute to help stimulate free radical
production or eliminate antioxidants Dialysis-related amyloidosis (DRA) is also a
common and serious problem for people who have been on dialysis for more than 5
years DRA develops when proteins in the blood deposit on joints and tendons causi
pain stiffness and fluid in the joint as is the case with arthritis Normally the healthy
kidneys can filter out these proteins but dialysis filters are not as effective
23 Advances in Membrane Technology
al that
s
ng
Dialysis for blood of ESRD
Hemo filtration
s
ysis using a cellulose based membrane occurred
1913 John Abel [1990] from the Johns Hopkins Medical School described a method
purification is widely used in the treatment
dialysis (HD) techniques use a semi-permeable membrane to replace the
role of the kidney The membranes used in HD can be broadly classified into those based
on cellulose and those manufactured from synthetic copolymers These membranes come
in various shapes such as sheets tubular structures or hollow fiber arrangements The
hollow fiber type is the most popular and is incorporated into over 800 different device
in world wide [Ronco et al 2001]
The first attempt at blood dial
9
where sing
e
om
ysis was performed until the mid 1960 The basic
molec
rial in
een
benzyl)
was used for the membrane with
hepar
by the blood of a living animal may be submitted to dialysis outside the body u
a membrane based on cellulose and returned to the natural circulation without exposur
to air infection by microorganisms or any alteration that would necessarily be prejudicial
to life This same technique is still used to today however the device used has been
modified over the years as better membranes were developed and the anti-coagulant
heparin has become available
Cellulose membranes have been widely used for the treatment of renal failure fr
1928 when the first human dial
ular structure of cellulose is made of a long chain containing hydroxyl (OH)
groups The realization that such groups imparted undesirable qualities on the mate
respect to blood contact behavior was discovered in the early 1970s and since has b
the focus of development These modified cellulose membranes used the partial
substitution of benzyl groups to replace the proton of the hydroxyl groups in an attempt
to reduce their negative effect The result is a molecular mosaic of hydrophobic (
and hydrophilic (hydroxyl and cellulose) regions
Kolff [Van Noordwijk 2001] studied the rotating drum artificial kidney for patients
with acute renal failure in 1943 Cellophane tubing
in as the anticoagulant For the next 17 years hemodialysis therapy was performed
by this method but only for the patients with acute reversible renal failure Vascular
access required repeated surgical insertions of cannulas (slender tubes) into an artery and
vein and limited the number of treatments for a patient could receive in order to
minimize the amount of vascular damage
10
Initially the need for dialysis in patients with acute renal failure was determ
mainly by the development of signs and sym
ined
ptoms of uremia After dialysis some time
might
et al
ent improvement In 1960 the development of a shunt [Quinton et al
1960]
fone (PSf) polyamide (PA) and polyacrylonitrile (PAN) by phase inversion or
precip
lidone
e
elapse before uremic manifestations returned to warrant a sequential treatment of
dialysis Many patients with acute renal failure secondary to accidental or surgical
trauma were hypercatabolic but the interdialytic interval might be prolonged because of
anorexia or use of a low-protein diet However Teschan and his coworkers [Obrien
1959] showed that patient well-being and survival were improved by what they termed
prophylactic daily hemodiaysis or administration of the treatment before the patient
again became sick with uremia Their report in 1959 was the first description of daily
hemodialysis
Development of membrane accessories such as a shunt has also been an area of
focus for treatm
a flexible polytetrafluoroethylene (PTFE or Teflonreg) tubing made many more
hemodialysis treatments possible for chronic kidney failure patients PTFE has a non-
stick surface and is relative biocompatibility leading to minimized blood clotting in the
shunt
Synthetic membranes are prepared from engineered thermoplastics such as
polysul
itation of a blended mixture resulting in the formation of asymmetric and
anisotropic structures Figure 2-3 shows a fiber type of the Polyflux S membrane
consisting of poluamide (PA) polyacrylethersulfone (PAES) and polyvinylpyrro
(PVP) with the integral three-layer structure The skin layer on the inside fiber typ
membrane contacts blood and has a very high surface porosity and a narrow pore size
11
distribution This layer constitutes the discriminating barrier deciding the permeabili
and solute retention properties of the membrane The skin layer is supported by thick
sponge-type structure larger pores providing mechanical strength and very low
hydrodynamic resistance
ty
Figure 2-3 Cross-sectional SEM image view of the Polyflux S (polyamide +
polyacrylethersulfone + polyvinypyrollidone) dialysis membranes [Deppisch et al 1998]
n
[Malchesky 2004] because of its thermal stability mechanical strength and chemical
inertn ciated
ls
m
PSf is a widely used membrane material for the hemodialysis applicatio
ess According to a report from the National Surveillance of Dialysis-Asso
Disease (NSDAD) in the US over 70 of hemodialysis membranes were PSf based
[Bowry 2002] This is most likely because PSf has many advantages over other materia
This synthetic polymer is one of few materials that can withstand sterilization by stea
ethylene oxide and γ-radiation PSf membrane can be prepared by conventional
immersion precipitation methods into many different shapes including porous hollow
12
fiber or flat sheet hemodialysis membranes The material also has a high permeab
low molecular weight proteins and solute and high endotoxin retention The chemical
structure of PSf is shown in Figure 2-4
ility to
Figure 2-4 The chemical structure of polysulfone (PSf)
There is one major disadvantage to PSf The hydrophobic nature of the PSf causes
ment alternative pathway
leadin
s not
3
et al
in
O acrylate
serious complications through the activation of the comple
g to the adsorption of serum proteins onto the membranes [Singh et al 2003]
Anticoagulants are added during dialysis therapy to avoid blood clotting but this doe
completely eliminate the problem In order to overcome this disadvantage of the PSf
membrane various studies have been performed to change the materialrsquos surface
properties These investigations include hydrophilic polymer coating [Brink et al 199
Kim et al 1988 Higuchi et al 2003] layer grafting onto PSf membrane [Wavhal
2002 Song et al 2000 Pieracci et al 2002 Mok et al1994] and chemical reaction of
hydrophilic components onto the membrane surface [Higuchi et al 1990 1991 1993
Blanco et al 2001 Nabe et al 1997 Guiver et al 1993] Hydrophilic monomers 2-
hydroxy-ethylmethacrylate (HEMA) acrylic acid (AA) and methacrylic acid (MMA)
have also been grafted onto PSf membrane to increase flux and Bovine Serum Album
(BSA) retention [Ulbricht et al 1996] Hancock et al [2000] synthesized
polysulfonepoly(ethylene oxide) (PEO) block copolymers to improve the resistance to
platelet adhesion Kim et al [2003] also studied blending a sulfonated PE
diblock copolymer into PSf in order to reduce platelet adhesion and enhance
13
biocompatibility PEO is a commonly used biomaterial due to its excellent resistance
protein adsorption and inherent biocompatibility [Harris 1992] Kim et al [20
a self-transformable copolymer to enhance the hydrophilicity of an asymmetric PSf
membrane with an ultra-thin skin layer The polymer had an entrapped diblock
copolymer containing a hydrophilic block of poly (ethylene glycol) (PEG)-SO
to
05] studied
ate
lic graft
l)
e of thier
aromatic
orand
3 acryl
and a hydrophobic block of octadecylacrylate (OA) Molecular dynamic (MD)
simulations were performed as a function of copolymer density to optimize interfacial
structure information McMurry [2004] developed a strategy using an amphiphi
copolymer added to PSf membranes by introducing polysulfone-g-poly (ethylene glyco
When compared to unmodified PSf these graft copolymer and resulting blend
membranes are found to hold promise for biomedical device applications
Polyamide (PA) membranes have also been used for hemodialysis becaus
mechanical strength in both wet and dry conditions Polyamide consists of
aliphatic monomers with amide bonding (-CONH-) also known as a peptide bond
The basic amide bond in polyamide is shown in Figure 2-5 R1 and R2 can be either
aromatic or aliphatic linkage group
C N
H
O
~ R R1 2~C N
H
O
~ R R1 2~
Figure 2-5 The chemical structure of polyamide (PA)
Panichi and co-workers [1998] evaluated the biocompatibility of the PA membrane
ompatible technique due to the
use of a synthetic membrane with a sterile re-infusion fluid and the convective removal of
and concluded that PA hemofiltration was a highly bioc
14
the ac
e
e
is
n
is a semi-crystalline polymer and the mechanical properties
strong
2
CN
tivated anaphylatoxins and β2-Microglobulin (β2M) The PA based membrane
Polyfluxreg (manufactured by Gambro GmbH Germany) blended with polyamide
polyarylethersulfone and polyvinylpyrrolidone (PVP) was able to clean small molecules
such as urea dreatinine and phosphate as well as decrease β2M amount by 502
[Hoenich et al 2002] Due to the non-selectivity of the membrane removal of thes
unwanted materials also led to the undesirable loss of beneficial proteins during therapy
Meier et al [2000] evaluated different immune parameters using a modified cellulos
low-flux hemophan and synthetic high-flux PA membrane during a 1 year period in
chronic hemodialysis patients They found that the 1-year immunological evaluation of
hemodiaysis membrane biocompatibility was associated with changes in the pattern of
chronic T-cell actiovation
Polyacrylonitrile (PAN) is another commonly used membrane material because it
inherently hydrophilic and has been commercialized for ultrafiltration and microfiltratio
[Scharnagl et al 2001] PAN
ly depend on the crystalline structures The chemical structure of PAN is shown in
figure 2-6
CH CH
n2
CN
CH CH n
Chemical structure of polyacrylonitrile (PAN)
The addition of additives such as polyvinylpyrrolidone (PVP) as a pore forming
agent gives PAN membranes more flexible processing parameters and increased
e has been optimized through
copolymerization with many other vinyl monomers including glycidyl methacrylate
Figure 2-6
performance [Jung et al 2005] PAN membrane performanc
15
[Godj
acid
for
for
a non-
solub this
in
oid underneath the inner
surface of membrane decreased by increasing the amount of solvent DMSA in the
evargova et al 1999 Hicke et al 2002] N-vinylimidazole [Godjevargova et al
2000] hydroxyl ethyl methacrylate [Ray et al 1999 Bhat et al 2000] methacrylic
[Ray et al 1999] vinyl pyrrolidone [Ray et al 1999] acrylic acid [Trotta et al 2002]
acrylamide [Musale et al 1997] and vinylchloride [Broadhead et al 1998] These
monomers provide a reactive group for enzyme immobilization improved mechanical
strength solvent-resistance pervaporation permeation flux anti-fouling and bio-
compatibility Because of this PAN-based copolymer membranes have great potential
the treatment of hemodialysis in an artificial kidney This material can also be used
other applications like the treatment of wastewater the production of ultra-pure water
biocatalysis together with separation and methanol separation by pervaporation
Lin and his coworker [2004] studied the modification of PAN based dialyzer
membranes to increase the hemocompatibility by the immobilization of chitosan and
heparin conjugates on the surface of the PAN membrane When a foreign material is
exposed to blood plasma proteins are adsorbed clotting factors are activated and
le fibrin network or thrombus is formatted [Goosen et al 1980] The result of
research was that the biocompatible chitosan polymer and a blood anticoagulant hepar
prevented blood clotting They showed prolonged coagulation time reduced platelet
adsorption thrombus formation and protein adsorption
Nie et al [2004] studied PAN-based ultrafiltration hollow-fiber membranes
(UHFMs) In order to improve the membrane performance acrylonitrile (AN) was
copolymerized with other functional monomers such as maleic anhydride and α-allyl
glucoside They found that the number and size of macrov
16
intern erum
and
d a
rface The second step is the
transf after
yjak et
embrane
1978 Malchesky et al 1978] has been further developed to increase the efficiency of
al coagulant The water flux of the UHFMs also decreased while the bovine s
albumin rejection increased minutely Godjevargova et al [1992] modified the PAN
based membrane with hydroxylamine and diethylaminoethylmethacrylate to improve
membrane dialysis properties Formed functional groups like primary amine oxime
tertiary amine groups provided the membrane with more hydrophilic properties an
substantial increase in the permeability of the membranes
The wide use of filtration in practice is limited by membrane fouling Solute
molecules deposit on and in the membrane in the process of filtration causing dramatic
reduction in flux through the membrane Fouling occurs mostly in the filtration of
proteins Three kinetic steps are involved in the fouling of UF membranes according to
Nisson [1990] The first step is the transfer of solute to the su
er of solute into the membrane until it either finally adsorbs or passes through
a set of adsorption-desorption events The third step includes surface binding
accompanied by structural rearrangement in the adsorbed state [Ko et al 1993] Br
al [1998] studied the surface modification of a commercially available PAN membrane
to develop superior filtration properties with less fouling by proteins The PAN
membrane was immersed in excess NaOH solution to convert some of the surface nitrile
groups into carboxylic groups by the hydrolysis process This modified PAN m
was not so severely fouled in the Bovine Serum Albumin (BSA) filtration test The pore
size however decreased during the hydrolysis process leading to a significant reduction
in flux and made the membrane less productive in the ultrafiltration (UF) mode
24 Sorbent Technology
Over the last three decades sorbent technology [Castino et al 1976 Korshak et al
17
dialysis or replace it for the treatment of ESRD Sorbents remove solutes from solution
through specific or nonspecific adsorption depending on both the nature of the solute and
the sorbent Specific adsorption contains tailored ligands or antibodies with high
selectivity for target molecules een used in autoimmune
disord
and
e
al 1996] various resins [Ronco et al 2001]
album
e
as
Specific adsorbents have b
ers such as idiopathic thrombocytopenic purpura [Snyder et al 1992] and for the
removal of lipids in familial hyper cholesterolemia [Bosch et al 1999] Nonspecific
adsorbents such as charcoal and resins attract target molecules through various forces
including hydrophobic interactions ionic (or electrostatic) attraction hydrogen bonding
and van der Waals interactions
New dialysate with sorbents has become an accepted modification of dialysis
sorbent hemoperfusion is gaining ground as a valuable addition to dialysis especially as
new sorbents are developed [Winchester et al 2001] Hemoperfusion is defined as th
removal of toxins or metabolites from circulation by the passing of blood within a
suitable extracorpoteal circuit over semipermeable microcapsules containing adsorbents
such as activated charcoal [Samtleben et
in-conjugated agarose etc Novel adsorptive carbons with larger pore diameters
have been synthesized for potential clinical use [Mikhalovsky 1989] Newly recognized
uremic toxins [Dhondt et al 2000 Haag-Weber et al 2000] have resulted in several
investigations on alternatives to standard or high-flux hemodialysis to remove thes
molecules These methods include hemodiafilteration with [de Francisco et al 2000] or
without [Ward et al 2000 Takenaka et al 2001] dialysate regeneration using sorbents
well as hemoperfusion using such adsorbents as charcoal and resins
18
Kolarz et al [1989 1995] studied the hyper-crosslinked sorbent prepared from
styrene and divinylbenzene (DVB) for a hemoperfusion application They found that the
pore structure of a swelling sorbent was changed by additional crosslinking with α αrsquo-
dichloro-p-xylene in the presence of a tin chloride catalyst and in a di
chloroethane
soluti and
en
decyl groups that attract β2M through a hydrophobic interaction The
adsor
of
5]
d
penet
on They also realized that the hemocompatibility was useful for nemoperfusion
could be imparted to the sorbents by introducing sulfonyl groups at a concentration of
about 02mmolg
A special polymeric adsorbing material (BM-010 from Kaneka Japan) has be
investigated by another group [Furuyoshi et al 1991] for the selective removal of β2M
from the blood of dialysis partients The adsorbent consists of porous cellulose beads
modified with hexa
ption capacity of this material is 1mg of β2M per 1ml of adsorbent Using a
hemoperfusion cartridge containing 350ml of these cellulose beads in sequence with a
high-flux hemodialyzer several small clinical trials were performed During 4-5 hours
treatment about 210mg of β2M were removed thus reducing the concentration in the
blood by 60-70 of the initial level [Nakazawa et al 1993 Gejyo et al 1993 199
RenalTech developed a hemoperfusion device BetaSorbreg containing the hydrate
cross-linked polystyrene (PS) divinylbenzene (DVB) resin sorbents with a pore structure
designed to remove molecules between 4 and 30 kDa [Winchester et al 2002] In this
case solute molecules are separated according to their size based on their ability to
rate the porous network of the beaded sorbents The resin beads were prepared with
a blood compatible coating and confirmed to be biocompatible in vivo in animals
[Cowgill et al 2001]
19
25 Limitation of Current Hemodialysis Treatment
Hemodialysis is a widely used life-sustaining treatment for patients with ESRD
However it does not replace all of the complex functions of a normal healthy kidne
a result patients on dia
y As
lysis still suffer from a range of problems including infection
accelerated cardio ion anemia
chron
e
se middle
alysis
sessio
s
f
vascular disease high blood pressure chronic malnutrit
ic joint and back pain and a considerably shortened life span One significant
limitation of the current dialysis technology is the inability to efficiently remove larger
toxic molecules This is mainly because of the broad pore size distribution reducing th
selective removal of toxins and unsatisfied biocompatibility causing lots of
complications such as inflammation blood clotting calcification infection etc
Dialysis purifies the patients blood by efficiently removing small molecules like
salts urea and excess water However as toxic molecules increase in size their removal
rate by hemodialysis substantially declines Typically only 10 - 40 of the
molecular weight toxins (300-15000 Da) are removed from the blood during a di
n [Vanholder et al 1995] These toxins then reach an abnormally high level and
begin to damage the body One such toxin β2M causes destructive arthritis and carpal
tunnel syndrome by joining together like the links of chain to form a few very large
molecules and deposit damaging the surrounding tissues [Lonnemann et al 2002] This i
also a main cause of mortality for long-term dialysis patients Other middle molecule
toxins appear to inhibit the immune system and may play a significant role in the high
susceptibility to infections in dialysis patients Still others are believed to impair the
functioning of several other body systems such as the hematopoietic and other endocrine
systems This may contribute to accelerated cardiovascular disease the leading cause o
20
death among dialysis patients as well as clinical malnutrition which affects up to 50 of
this patient population
Over the last decade polymeric dialysis membranes have been developed to
increase the capacity for removing middle molecular weight toxins by changing the pore
size of dialyzer membranes and using new materials that adsorb these toxins for
impro se
styrene made during World War II in the United
States The Dow chemical compan anufacturer of polystyrene
includ
d of
on
rug
ntrol of
ved removal characteristics However removal efficiency is not as high as tho
achieved by a normal healthy kidney
26 Latex Particles
The first synthetic polymer synthesized using emulsion polymerization was a
rubber composed of 13-butadiene and
y has been a major m
ing latex which they used in paint formulations The theory of emulsion
polymerization in which a surfactant is used was established by Harkins [1948] and by
Smith and Ewart [1948] By 1956 the technology was complete including the metho
building larger diameter particles from smaller ones The product by this emulsi
polymerization is referred to as latex a colloidal dispersion of polymer particles in water
medium [Odian 1991] Latexes are currently undergoing extensive research and
development for a broad range of areas including adhesives inks paints coatings d
delivery systems medical assay kits gloves paper coatings floor polish films carpet
backing and foam mattresses to cosmetics The relatively well known and easy co
the emulsion process is one of main advantages for these applications Therefore
polymeric latex particle prepared by emulsion polymerization can be a candidate for the
medical applications because of the easy control of the particle size and morphology as
well as flexible surface chemistry to be required
21
261 The Components for Emulsion Polymerization
The main components for emulsion polymerization process are the monomer a
dispersing medium a surfactant and an initiator Available monomers are styrene
sing medium is usually water
which
ticles
n
ation To
monomer droplets but in micelles because the initiators are
insolu le
and
olution
butadiene methylmethacylate acryl acid etc The disper
will maintain a low solution viscosity provide good heat transfer and allow
transfer of the monomers from the monomer droplets into micelles and growing par
surrounded by surfactants respectively The surfactant (or emulsifier) has both
hydrophilic and hydrophobic segments Its main functions is to provide the nucleatio
sites for particles and aid in the colloidal stability of the growing particles Initiators are
water-soluble inorganic salts which dissociate into radicals to initiate polymeriz
control the molecular weight a chain transfer agent such as mercaptan may be present
262 Particle Nucleation
Free radicals are produced by dissociation of initiators at the rate on the order of
1013 radicals per milliliter per second in the water phase The location of the
polymerization is not in the
ble in the organic monomer droplets Such initiators are referred to as oil-insolub
initiators This is one of the big differences between emulsion polymerization
suspension polymerization where initiators are oil-soluble and the reaction occurs in the
monomer droplets Because the monomer droplets have a much smaller total surface area
they do not compete effectively with micelles to capture the radicals produced in s
It is in the micelles that the oil soluble monomer and water soluble initiator meet and is
favored as the reaction site because of the high monomer concentration compared to that
in the monomer droplets As polymerization proceeds the micelles grow by the addition
of monomer from the aqueous solution whose concentration is refilled by dissolution of
22
monomer from the monomer droplets There are three types of particles in the emulsion
system monomer droplets inactive micelles in which polymerization is not occurring
and active micelles in which polymerization is occurring referred to as growing polymer
particles
The mechanism of particle nucleation occurs by two simultaneous processes
micellar nucleation and homogeneous nucleation Micellar nucleation is the entry of
radicals ei
ther primary or oligomeric radicals formed by solution polymerization from
the aq ed
the
r
s
n
is coagulation with other particles rather than
polymerization of monomer The driving force for coagulation of precursor particles
ueous phase into the micelles In homogeneous nucleation solution-polymeriz
oligomeric radicals are becoming insoluble and precipitating onto themselves or onto
oligomers whose propagation has ended [Fitch et al 1969] The relative levels of micella
and homogeneous nucleation are variable with the water solubility of the monomer and
the surfactant concentration Homogeneous nucleation is favored for monomers with
higher water solubility and low surfactant concentration and micellar nucleation is
favored for monomers with low water solubility and high surfactant concentration It has
also been shown that homogeneous nucleation occurs in systems where the surfactant
concentration is below CMC [Roe 1968] A highly water insoluble monomer such a
styrene [Hansen et al 1979 Ugelstad et al 1979] has probably created by micellar
nucleation while a water soluble monomer such as vinyl acetate [Zollars 1979] has bee
formed by homogeneous nucleation
A third latex formation reaction mechanism has been proposed referred to as
coagulative nucleation In this reaction the major growth process for the first-formed
polymer particles (precursor particles)
23
sever les
yer
es
r a
l
the beginning of the polymerization As soon as the initiator is
gins with the formation and
growt
and
ved
added at one time during the later stages of the polymerization prior to complete
al nanometers in size is their relative instability compared to larger sized partic
The small size of a precursor particle with its high curvature of the electrical double la
permits the low surface charge density and high colloidal instability Once the particl
become large enough in size maintaining the high colloidal stability there is no longe
driving force for coagulation and further growth of particles takes place only by the
polymerization process
263 Types of Processes for Emulsion Polymerization
There are three types of production processes used in emulsion polymerization
batch semi-continuous (or semi-batch) and continuous In the batch type process al
components are added at
added and the temperature is increased polymerization be
h of latex particles at the same time There is no further process control possible
once the polymerization is started In the semi-continuous emulsion polymerization
process one or more components can be added continuously Various profiles of particle
nucleation and growth can be generated from different orders of component addition
during polymerization There are advantages to this process such as control of the
polymerization rate the particle number colloidal stability copolymer composition
particle morphology In the continuous process the emulsion polymerization components
are fed continuously into the reaction vessel while the product is simultaneously remo
at the same rate High production rate steady heat removal and uniform quality of
latexes are advantages of the continuous polymerization processes
Other available methods include the intermittent addition and shot addition of one
or more of the components In the shot addition process the additional components are
24
conversion of the main monomer This method has been used successfully to develo
water-soluble functional monomers such as sodium styrene sulfonat
p
e [Kim et al 1989]
264
lsion
Chemistry of Emulsion Polymerization
Emulsion polymerization is one type of free radical polymerization and can be
divided into three distinct stages initiation propagation and termination The emu
polymerization system is shown in Figure 2-7
Aqueous phaseAqueous phase
Micelle withmonomer
I Rbull
Emulsifier
Polymer particle swollenWith monomer
Monomerdroplet
I Rbull
Micelle withmonomer
I Rbull
Emulsifier
Polymer particle swollenWith monomer
MonomerdropletMonomerdroplet
I Rbull
Figure 2-7 Emulsion polymerization system [Radicals (Rmiddot) are created from initiators
(I) Monomer is transferred from larger monomer droplet into micelles by emulsifier Initiated polymer particle by radicals is keep growing until monomers are all consumed The reaction is performed in aqueous media]
In the initiation stage free radicals are created from an initiator by heat or an
ultraviolet radiation source The initiator with either peroxides groups (-O-O-) such as
odium persulfate or azo groups (-N=N-) such as azobisisobutyronitrile is commonly
used for em t
with the m er
chain ere
s
ulsion polymerization The primary free radicals created from initiator reac
onomer for initiation of polymerization In the propagation stage the polym
grows by monomer addition to the active center a free radical reactive site Th
are two possible modes of propagations head-to-head addition and head-to-tail addition
25
The head-to-tail mode is the predominant configuration of the polymer chain a result o
steric hindrance of the substitute bulky group In the termination stage polymer chain
growth is terminated by either coupling of two growing chains forming one polymer
molecule or transferring a hydrogen atom (dispropotionation) from one growing chain to
another forming two polymer molecules one having a saturated end group and the other
with an unsaturated end-group
265 Seed Emulsion Polymerization
Polymer latex particles have received increasing interest because of the versatili
of the many applications heterophase polymerization processes like emulsion dispersion
micro-emulsion seeded emulsion precipitation etc Especially to prepare well-defined
microspheres having monodispe
f
ty
rse and various particle sizes as well as surface group
ed particles prepared by emulsion polymerization
and e
s
hological design In
seede to control
n
functionalilties it is necessary to use se
nlarge them to a desired size in the further stages of reactions
Polymeric particles are required to have a uniform particle size in many
applications such as chromatography where they are used as a packing material
Morphological control of latex particle is also important for many practical application
[Schmidt 1972] Seed emulsion polymerization (or two-stage emulsion polymerization) is
a useful method to achieve both monodisperse particle size and morp
d emulsion polymerization [Gilbert 1995] preformed lsquoseedrsquo latex is used
the number of particles present in the final latex The advantage of seeded emulsio
polymerization is that the poorly reproducible process of particle nucleation can be
bypassed so that the number concentration of particles is constant and known Various
mechanisms have been proposed for the growth of latex particles [Ugelstad et al 1980
Okubo et al 1992] using this polymerization technique The initial seed particle
26
preparation step is well known and relatively easy to perform because at the relative
small particle sizes (02 to 05 micron) the particle growth process can be readily
controlled by the use of an emulsifier Enough emulsifier is used to prevent coagulation
but not enough to cause the formation of new particles As the particles are grown to
larger sizes in successive seeding steps it becomes increasingly difficult to mainta
stable uncoagulated emulsion without forming new particles and thereby destroying
monodisperisty of the latex Recently there have been several reports concerning t
reaction kinetics of seed emulsion polymerization and the development of latex
morphologies over the course of the reaction [Chern et al 1990 Delacal et al 1990 L
et al 1995 Lee 2000 2002]
266 Polystyrene Latex Particles
A number of papers have described the synthesis of the polystyrene latex particl
bearing various functional surface groups such as carboxyl [Lee et al 2000 Tun
2002 Reb et al 2000] hydroxyl [Tamai et al 1989] marcapto [Nilson 1989] epoxy
[Shimizu et al 2000 Luo et al
ly
in a
he
ee
es
cel et al
2004] acetal [Izquierdo et al 2004 Santos et al 1997]
ometyl [Izquierdo et al 2004 Park et al 2001
Sarob
h the
d
thymine [Dahman et al 2003] chlor
e et al 1998] amine [Counsin et al 1994 Ganachaud et al 1997 Ganachaud et al
1995 Miraballes-Martinez et al 2000 2001 Anna et al 2005] ester [Nagai et al 1999]
etc In order to produce these functionalized particles different methods for particle
preparation must be used The polymer emulsion with core-shell morphology of latex
particles is one of them This is a multistep emulsion polymerization process in whic
polystyrene ldquocorerdquo particle is synthesized in the first stage and the functional monomer is
added in the second stage of the polymerization without any emulsifier postfeeding to
prevent the production of new homopolymer particles thus forming the functionalize
27
polymer ldquoshellrdquo on the ldquocorerdquo particle [Keusch et al 1973] There are requirements to
limit secondary nucleation and encourage core-shell formation in seeded emulsion
polymerization including the addition of smaller seed particles at high solid content to
increase particle surface area low surfactant concentration to prevent formation of
micelles and the semi-continuous addition of monomer to create a starved-feed conditio
and keep the monomer concentration low There are some advantages [Hergeth et al
1989] of dispersions with polymeric core-shell particles First it is possible to modi
interfacial properties of polymer particles in the aqueous phase by the addition of only
very small amounts of a modifying agent during the last period of the reaction Thus
these core-shell particles are useful in a broad range of applications since they always
exhibit improved physical and chemical properties over their single-component
counterparts [Lu et al 1996 Nelliappan et al 1997] In this way the improvement of
surface properties of such dispersions is straightforward and inexpensive The other is
that polymers with a core-shell structure are perfect model systems for investigating th
material properties of polymer blends and composites because of their regular
distribution of one polymer inside a matrix polymer and because of the simple sp
geometry of the system
Their properties usually depend on the structures of latex particles Chen and his
coworkers [Chen et al 1991 1992 1993] reported the morphological development of
core shell latex particles of polystyrenepoly(methyl methacrylate) during
polymerization Before the research by Chen and his coworker Min et al [Min et al
1983] reported the morph
n
fy the
e
herical
ological development of core shell latex of polystyrene
(PS)polybuthyl acrylateat (PBA) by seeded emulsion polymerization as a function of the
28
additi
of
ity of graft
ing reagents Thus protein adsorption on polymeric solid surfaces has
st are microspheres defined as ldquofine
polym can be
n
r was
containing antigen urine serum etc The latex will become agglutinated and visibly
on method of PS They found that the percentage of grafting PS to the PBA was
greatest for the batch reaction and the PBA-PS core-shell particles with a high degree
grafting remained spherical upon aging test because of the emulsifying abil
copolymer
267 Various Applications of Latex Particles
Latex particles are applicable to a wide range of areas such as biomedical
applications [Piskin et al 1994] especially as the solid phase such as in immunoassays
[Chern et al 2003 Radomske-Galant et al 2003] DNA diagnostic drug delivery carriers
[Luck et al 1998 Kurisawa et al 1995 Yang et al 2000] blood cell separations and
column pack
become a center of attention Of particular intere
er particles having diameters in the range of 01 to several micronsrdquo which
used as functional tools by themselves or by coupling with biocompounds Singer and
Plotz [1956] firstly studied microsphere or latex agglutination test (LATrsquos) by using
monodisperse polystyrene (PS) and polyvinyltoluene polymer particles as the support o
which the biomolecules were going to adsorb The biomolecule adsorption howeve
limited by possible desorption of the adsorbed species or loss of specific activity of the
complex formed Since this work was published the latex particle applications for
immunoassay have been rapidly and widely studied and developed
Latex agglutination test or latex immunoassays start with tiny spherical latex
particles with a uniform diameter and similar surface properties The particles are coated
with antibodies (sensitized) through the hydrophobic interaction of portions of the protein
with the PS surface of the particles If sensitized particles are mixed with a sample
29
agglomerate Latex tests are inexpensive as compared with the other techniques [Ba
1988]
ngs
sed
tion Rheumatoid factor (RF) in different age subpopulations has also been
evalu
studied a
latex
Unipath [Percival 1996] has manufactured a range of immunoassay materials ba
on a chromatographic principle and deeply colored latex particles for use in the home and
clinical environments The latex particles which are already sensitized with a
monoclonal antibody can detect any antigens that bound to the surface of the latex
particles Some detectable pollutants include estrogen mimics which induce
abnormalities in the reproductive system of male fishes and lead to a total or partial male
feminiza
ated according to a patientrsquos clinical status by using a rapid slide latex agglutination
test for qualitative and semiquantitative measurement in human serum along with latex
immunoassay method [Onen et al 1998] Magalhaes and his coworkers [2004]
diagnostic method of contamination of male fishes by estrogen mimics using the
production of vitellogenin (VTG) as a biomarker This was based on a reverse
agglutination test developed with monoclonal antibodies specific to this biomarker
Premstaller and his coworkers [Premstaller et al 2000 2001] have prepared a porous
poly(styrene-divinylbenzene) (PS-DVB) polymer monolith to use for highly efficient
chromatographic separation of biomelecules such as proteins and nucleic acids They
used a porogen a mixture of tetrahydrofuran and decanol to fabricate a micropellicular
PS-DVB backbone Legido-Quigley and his colleagues [2004] have developed the
monolith column to obtain further chromatographic functionality to the column by
introducing chloromethylstyrene in place of styrene into the polymer mixture
30
Core shell type monodisperse polymer colloids have been synthesized by Sarobe
and Forcada [1998] with chloromethyl functionality in order to improve the biomolecu
adsorption through a two-step emulsion polymerization process They investigated the
functonalized particles by optimizing the experimental parameters of the functional
monomer including reaction temperature the amount and type of redox initiator sys
used the type of addition of the initiator system and the use of washing They c
le
tem
oncluded
that th
sis
serve as separation
chann
of
usually
along the polypeptide chain which contain positive or negative charges [Norde 1998]
e relation between the amount of iron sulfate and the persulfatebisulfite system
added should be controlled to obtain monodisperse particles and prevent the premature
coagulation of the polymer particles during the polymerization
A semi-continuous emulsion polymerization technique for latex particle synthe
was performed by McDonald and other researchers [Ramakrishnan et al 2004 Steve et
al 1999] They introduced a variety of particles sizes compositions morphologies and
surface modifications to fabricate latex composite membranes (LCMs) Arraying and
stabilizing latex particles on the surface of a microporous substrate form narrowly
distributed interstitial pores formed between the particles which
els They investigated the membrane performance using gas fluxes water
permeability and the retention characterization of dextran molecules From these tests
they concluded that the narrow discriminating layer made of the latex particles leads to a
highly efficient composite membrane
27 Proteins
Proteins are natural polyamides comprised of about 20 different α-amino acids
varying hydrophobicity [Norde 1998] Proteins are more or less amphiphilic and
highly surface active because of the number of amino acid residues in the side groups
31
Proteins are polymers of L-α-amino acids [Solomon and Fryhle 2000] The α refers to a
carbon with a primary amine a carboxylic acid a hydrogen and a variable side-chain
group designated as R Carbon atoms rent groups are asymmetric and can
exhib l
s
e
on
with four diffe
it two different spatial arrangements (L and D configurations) due to the tetrahedra
nature of the bonds The L refers to one of these two possible configurations Amino
acids of the D-configuration are not found in natural proteins and do not participate in
biological reactions Figure 2-8 shows the chiral carbon in 3-D as the L isomer Protein
consist of twenty different amino acids differentiated by their side-chain groups [Norde
1998] The side-chain groups have different chemical properties such as polarity charg
and size and influence the chemical properties of proteins as well as determine the
overall structure of the protein For instance the polar amino acids tend to be on the
outside of the protein when they interact with water and the nonpolar amino acids are
the inside forming a hydrophobic core
Figure 2-8 L-α-amino acid
The covalent linkage between two amino acids is known as a peptide bond A
peptide bond is formed when the amino group of one amino acid reacts with the carboxyl
group of another amino acid to form an amide bond through the elimination of water
This arrangement gives the protein chain a polarity such that one end will have a free
amino group called the N-terminus and the other end will have a free carboxyl group
called the C-terminus [Solomon and Fryhle 2000] Peptide bonds tend to be planar and
32
give the polypeptide backbone rigidity Rotation can still occur around both of the α-
carbon bonds resulting in a polypeptide backbone with different potential conformations
relative to the positions of the R groups Although many conformations are theoretically
possible interactions between the R-groups will limit the number of potential
nd to form a single functional conformation In other words
the conformation or shape of the protein is
α-helix
can interact with other
secondary structures within the sam
conformations and proteins te
due to the interactions of the chain side
groups with one another and with the polypeptide backbone The interactions can be
between amino acids that are close together as in a poly-peptide between groups that are
further apart as in amino acids or even on between groups on different polypeptides all
together These different types of interactions are often discussed in terms of primary
secondary tertiary and quaternary protein structure
The primary amino acid sequence and positions of disulfide bonds strongly
influence the overall structure of protein [Norde 1986] For example certain side-chains
will promote hydrogen-bonding between neighboring amino acids of the polypeptide
backbone resulting in secondary structures such as β-sheets or α-helices In the
conformation the peptide backbone takes on a spiral staircase shape that is stabilized by
H-bonds between carbonyl and amide groups of every fourth amino acid residue This
restricts the rotation of the bonds in the peptide backbone resulting in a rigid structure
Certain amino acids promote the formation of either α-helices or β-sheets due to the
nature of the side-chain groups Some side chain groups may prevent the formation of
secondary structures and result in a more flexible polypeptide backbone which is often
called the random coil conformation These secondary structures
e polypeptide to form motifs or domains (ie a
33
tertiary structure) A motif is a common combination of secondary structures and a
domain is a portion of a protein that folds independently Many proteins are composed o
multiple subunits and therefore exhibit quaternary structures
271 Interaction Forces between Proteins
Proteins in aqueous solution acquire compact ordered conformations In such a
compact conformation the movement along the polypeptide chain is severely restrict
implying a low conformational entropy The compact structure is possible only if
interactions within the protein molecule and interactions between the protein molecule
and its environment are sufficiently favorable to compensate for the low conformational
entropy [Malmsten 1998] Protein adsorption study is often focused on structural
rearrangements in the protein molecules because of its significance to the biological
functioning of the molecules and the important role such rearrangements play in the
mechanism of the adsorption process Knowledge of the majo
f
ed
r interaction forces that act
structures helps to understand the behavior
of pro
arge
a
ded
between protein chains and control the protein
teins at interface These forces include Coulomb interaction hydrogen bonds
hydrophobic interaction and van der Waals interactions
Coulombic interaction Most of the amino acid residues carrying electric ch
are located at the aqueous boundary of the protein molecule An the isoelectric point
(IEP) of the protein where the positive and negative charges are more or less evenly
distributed over the protein molecule intramolecular electrostatic attraction makes
compact structure favorable to proteins Deviation to either more positive or more
negative charge however leads to intramolecular repulsion and encourages an expan
structure Tanford [1967] calculated the electrostatic Gibbs energy for both a compact
impenetrable spherical molecule (protein) and a loose solvent-permeated spherical
34
molecule (protein) over which the charge is spread out From the results he found th
the repulsion force was reduced at higher ionic strength d
at
ue to the screening action of ion
ble
e
the
d the
tween
dehyd
e
ent
oles
tein
Hydrogen bond Most hydrogen bonds in proteins form between amide and
carbonyl groups of the polypeptide backbone [Malmsten 1998] The number of availa
hydrogen bonds involving peptide units is therefore far greater than that involving sid
chains Because α-helices and β-sheets are aligned more or less parallel to each other
interchain hydrogen bonds enforce each other Kresheck and Klotz [1969] examine
role of peptide-peptide hydrogen bonds and concluded that hydrogen bonds between
peptide units do not stabilize a compact structure of protein However because the
peptide chain is shielded from water due to other interactions hydrogen bonding be
peptide groups do stabilize α-helical and β-sheet structures
Hydrophobic interaction Hydrophobic interaction refers to the spontaneous
ration and subsequent aggregation of non-polar components in an aqueous
environment In aqueous solutions of proteins the various non-polar amino acid residues
will be found in the interior of the molecule thus shielded from water The
intermolecular hydrophobic interaction for the stability of a compact protein structure
was first recognized by Kauzmann [1959] If all the hydrophobic residues are buried in
the interior and all the hydrophilic residues are at the outermost border of the molecul
intramolecular hydrophobic interaction would cause a compact protein structure
However geometrical and other types of interactions generally cause a fraction of the
hydrophobic residues to be exposed to the aqueous environm
Van der Waals interaction The mutual interaction between ionic groups dip
and induced dipoles in a protein molecule cannot be established as long as the pro
35
structure is not known in great detail Moreover the surrounding aqueous medium also
contains dipoles and ions that compete for participation in the interactions w
the protein molecule Dispersion interactions favor a compact structure However
because the Hamaker constant for proteins is only a little larger than that of water the
resulting effect is relatively small [Nir 1977]
272 β
ith groups of
rgiles et al 1996
Floeg
) is degraded [Floege 2001] Renal
β2M from the serum resulting in an increase in β2M
conce well
the
)
a
al
2-Microglobulin (β2M)
The protein β2M is of particular interest because it is involved in the human
disorder dialysis-related amyloidosis (DRA) [Geiyo et al 1985 A
e 2001] DRA is a complication in end stage renal failure patients who have been on
dialysis for more than 5 years [Bardin et al 1986 Drueke 2000] DRA develops when
proteins in the blood deposit on joints and tendons causing pain stiffness and fluid in
the joints as is the case with arthritis In vivo β2M is present as the non-polymorphic
light chain of the class I major histocompatibility complex (MHC-I) As part of its
normal catabolic cycle β2M dissociates from the MHC-I complex and is transported in
the serum to the kidney where the majority (95
failure disrupts the clearance of
ntration by up to 60-fold [Floege 2001] By a mechanism that is currently not
understood β2M then self-associates into amyloid fibrils and typically accumulates in
musculoskeletal system [Homma et al 1989] Analysis of ex vivo material has shown
that the majority of amyloid fibrils in patients with Dialysis Related Amiloidosis (DRA
is present as of full-length wild-type β2M although significant amounts (~20-30) of
truncated or modified forms of the protein are also present [Floege 2001 Bellotti et al
1998] Figure 2-9 shows the ribbon diagram of human β2M Native β2M consists of
single chain of 100 amino acid residues and has a seven stranded β-sandwich fold typic
36
of the immunoglobulin superfamily [Saper et al 1991 Trinh et al 2002] β2M was fi
isolated from human urine and characterized by Berggard et al [1980] in1968 The
normal serum concentration of β
rst
re
f
e
2M is 10 to 25 mgL It is a small globular protein with
a molecular weight of 118 kDa a Strokes radius of 16Aring and a negative charge under
physiological conditions (isoelectric point IEP = 57) β2M contains two β-sheets that a
held together by a single disulphide bridge between the cysteines in positions 25 and 81
[Berggard et al 1980 Parker et al 1982 Cunningham et al 1973] β2M cannot be
removed completely by current dialysis techniques but through a better understanding o
the structure and interaction forces that lead to this structure it will be possible to mor
efficiently remove this problematic protein
Figure 2-9 Ribbon diagram of human β M taken from the crystal structure of the protein
bound to the heavy chain of the MHC class I complex (PDB 1DUZ) [Khal 2000]
273 Serum Albumin
Serum albumin is the most abundant protein found in plasma and is typically
present in the blood at a concentration of 35~
2an et
50gL According to extensive studies about
its physiological and pharmacological properties albumin has a high affinity to a very
wide range of materials such as electrolytes (Cu+2 Zn+2) fatty acids amino acids
metabolites and many drug compounds [Fehske et al 1981 Kraghhansen 1981 Putnam
37
1984 Peters 1985] The most important physiological role of the protein is therefore to
bring such solutes in the bloodstream to their target organs as well as to maintain the pH
and osmotic pressure of the plasma Bovine serum albumin (BSA) is an ellipsoidal
protein with the dimensions of 140 X 40 X 40Aring [Peter 1985] The primary structure is a
single helical polypeptide of 66 kDa (IEP = 47) with 585 residues containing 17 pairs of
as a soft an
rfaces [Kondo et al 1991 Norde et al 1992 Soderquist et al
1980 ikely
is
disulfide bridges and one free cysteine [Dugaiczyk et al 1982] BSA has been classified
d flexible protein because it has a great tendency to change its conformation
on adsorption to solid su
Carter and Ho 1994] and consists of three homologous domains (I-III) most l
derived through gene multiplication [Brown 1976] Each domain is composed of A and B
sub-domains [He et al 1992] The secondary structure of human serum albumin (HSA)
shown in Figure 2-10
Figure 2-10 Secondary structure of human serum albumin (HSA) with sub-domains
[Zunszain et al 2003]
HSA has the same structure domains with the serum albumin from other species
such as BSA [Brown 1976] Although all three domains of the albumin molecule have
similar three-dimensional structures their assembly is highly asymmetric [Sugio et al
38
1999] Domains I and II are almost perpendicular to each other to form a T-shaped
assembly in which the tail of subdomain IIA is attached to the interface region between
sub-domains IA and IB by hydrophobic interactions and hydrogen bonds In contrast
domain III protrudes from sub-domain IIB at a 45ordm angle to form the Y-shaped assembly
for domains II and III Domain III interacts only with sub-domain IIB These features
make the albumin molecule heart-shaped
28 Protein Adsorption
Protein adsorption studies date back to the 1930rsquos At the beginning these studies
mainly focused on the determination of the molecular weight electrophoretic and
ral
rearrangem
adsorption and biocompatibility of the sorbent materials were investigated [Norde 1986]
There are interaction forces at the interfaces between protein molecules and latex
particles These forces are mainly divided into the following groups hydrophobic
interaction ionic interaction hydrogen bonding and van der Waals interaction [Andrade
1985]
Hydrophobic interaction It is known that hydrophobic interaction has a major
role in protein adsorption phenomena The adsorption of proteins on the low charged
latex particles occurs by this interaction force Generally monomers such as styrene offer
hydrophobic surfaces th
by this interaction force is maximum at the isoelectric point (IEP) of the protein and the
strength [Suzawa et al 1980 1982 Shirahama et al 1989 Kondo et al 1992] By the
chromatographic applications Later the adsorption mechanism especially the structu
ents was studied Recently the studies of the relation between protein
281 Interaction between Protein Molecule and Latex Particle
at protein molecules adsorb to The amount of adsorbed protein
pH at maximum adsorption shifts to a more acidic region with an increase in ionic
39
reports [Suzawa et al 1980 1982 Lee et al 1988] protein adsorption was greater
hydrophobic surface than on a hydrophilic one implying that hydrophobic interaction is
on a
s
ydrophilic compound such as acrylic acid (AA) or methacrylic acid (MAA)
Ionic
ely
t
ces
n force is operative over small
distan e
ble
n
nt dialysis membrane therapy for end stage renal disease (ESRD)
one of most dominant forces in protein adsorption
Ionic interaction Negatively charged latex particles have ionic functional
groups on their surfaces such as salts of sulfonic and carboxylic acid Sulfate group
originate from an initiator such as sodium persulfate and carboxylic groups originate
from a h
bonds are formed between the negative charges of these latex particles and positive
surface charges of protein molecules The conventional low-charged latex particles rar
form these ionic bonds
Hydrogen bonding Hydrogen bond is a strong secondary interatomic bond tha
exists between a bound hydrogen atom (its unscreened proton) and the electrons of
adjacent atoms [Callister 1999] Protein can be adsorbed on hydrophilic polar surfa
through hydrogen bonding Hydrogen bonds are frequently formed between hydroxyl-
carbonyl or amide-hydroxyl Hydroxyl-hydroxyl or amide-hydroxyl bonds are also
formed in protein adsorption
Van der Waals interaction This interactio
ces only when water has been excluded and the two non-polar groups come clos
to each other Lewinrsquos calculation showed that the van der Waals interaction is negligi
compared with the forces involved in the entropy increases ie hydrophobic interactio
[Lewin 1974]
29 Hypothesis for Toxin Removal
As mentioned earlier insufficient removal of middle molecular range toxins is a
major drawback of curre
40
patien
is
y
ese
haracteristics over the last decade However removal
effici
ing a novel membrane design composed of an assembly of engineered
polymeric latex particles s ions Important factors
will i
e
ith a
is established it is
expec
e
ts This may cause destructive arthritis and carpal tunnel syndrome inhibit the
immune system and accelerate cardiovascular disease leading to death among dialys
patients In order to overcome these complications artificial dialysis membranes have
been developed to increase the capacity for removing middle molecular weight toxins b
changing the pore size of dialysis membranes and using new materials that adsorb th
toxins for improved removal c
ency is not as high as those achieved by a normal healthy kidney
With the knowledge obtained from all above literatures the following hypothesis
has been established for the development of a membrane for the successful removal of
the target protein β2-Microglobulin (β2M) without the removal of serum albumin This
will be done us
ynthesized to predetermined specificat
nclude pore size and surface chemistry
291 Toxin Removal by Size Sieving Based on Monodispersed Pore Size
The packing of monodispersed spherical particles can lead to the formation of
porous layers suitable for use as filters and membranes [Hsieh et al 1991] The pore siz
is defined as the largest spherical particles that can pass through the interstitial spaces
It is well known that monodispersed spherical particles can be obtained from the
seed emulsion polymerization method Many commercially available latex particles are
synthesized by this technique and are inexpensive When the defect free membrane w
monodisperse pore size distribution based on a particular particle array
ted to outperform the traditional hemodialysis membrane limitation which is a
broad range of pore size distribution When spherical particles pack in regular crystallin
arrays a number of packing geometries such as hexagonal closest packing cubic closest
41
packing and body-centered cubic packing are possible The pore size of the array
depends on these packing geometries as well as particle size Theoretical pore size can be
calculated from the simple geometry of the arr
ay Figure 2-11 shows the relationship
between particle size and pore size
0001
001
01
001 01 1 10
Por
e
1
10
microm
) S
ize
(
Particle Diameter (microm)
Body-Centered Cubic ArrayClosest Packed Array
0001
Siz
e (
Particle Diameter (microm)
001
01
001 01 1 10
Por
e
1
10
microm
)
Body-Centered Cubic ArrayClosest Packed ArrayBody-Centered Cubic ArrayClosest Packed Array
and closed packed arrays [Steve et al 1999]
McDonald and his coworkers [Ramakrishnan et al 2004 Steve et al 1999] have
described the fabrication of latex composite membranes and their performance in
previous papers It is from this prior research and knowledge gained from conversations
with Dr McDonald that this current research has been initiated [McDonald 2003] The
focus of this thesis is the extension of McDonaldrsquos research in the area of composite
membranes to medical application such as dialysis membranes
292 Toxin Removal by Selective Adsorption on Engineered Latex Particles
The flexibility in latex surface properties is a significant advantage to this
materialrsquos ability to separate proteins since the selectivity is strongly dependent on the
charge interactions of both the protein and the latex particles under the conditions of
Figure 2-11 The relationship between pore size and particle size in body-centered cubic
42
separation [Menon et al 1999 Chun et al 2002] First all sorbents were designed to
have negative surface properties because most plasma proteins in blood are negative and
should not be removed with charge interaction between proteins and sorbents at
physiological condition (pH74) It was reported that a negatively charged surface was
more blood compatible than a positive one [Srinivasan et al 1971] The toxin protein β2-
Microglobulin (β2M) can be selectively adsorbed on the surface of latex particles by the
design of a suitable hydrophobic portioned surface where only β2M protein can be
anchored with hydrophobic interactions Albumin adsorption on the latex particle is not
side-on m
adsorption of β M protein on the engineered latex particles
allowed because charge repulsion is more dominant than hydrophobic interaction with
ode The figure 2-12 shows the schematic representation of the selective
2
PMMAPAAShell
COO-
OSO3-
OSO3-
PSCore
COO-
-
-
-
-
-
-
Albumin-
Chargerepulsion
--
-
- -
- -
- --
Hydrophobic
β2-Microglobulin
2
Interaction
β -Microglobulin
PMMAPAAShell
COO-
-
-
-
-
-
-
--
-
-
-
-
-
Albumin-
Chargerepulsion
OSO3-
OSO3-
PSCore
COO-
--
-
- -
--
-
- -
- -
- --
- -
- --
Hydrophobic
β2-Microglobulin
2
particles at pH 74
Interaction
β -Microglobulin
Figure 2-12 Schematic representation of the protein adsorption on the core shell latex
43
Hydrophilichydrophobic microdomain structures were proven to be more blood
compatible [Mori et al 1982 Higuchi et al 1993 Deppisch et al 1998] The
optimization of suitable hydrophobic to hydrophilic ratio is also important for the
biocompatibility The monomers such as styrene (St) methyl methacrylate (MMA) and
acrylic acid (AA) are widely used hydrophobic and hydrophilic monomer models in
emulsion polymerization process
In summary the background and fundamental literature survey about the history
material properties and limitation of hemodialysis membrane latex particle preparation
surface chemistry and manufacturing process target proteins and protein adsorption and
finally the hypothesis for a toxin removal with high separation efficiency have been
suggested This research focuses on such hypothesis and the materials and
characterization methodology for achievement of suggested hypotheses are described in
the next chapter
CHAPTER 3 EXPERIMENTAL AND CHARACTERIZATION METHODOLOGY
As mentioned earlier the goal of this study is to prepare polymeric latex particles
with tailored properties to maximize separation of toxin molecules and to investigate
fundamental interactions between the applied particles and molecules in the
system to optimize the membrane performance for hemodialysis applications Polymeric
latex particles wit
the
biological
h monodisperse size distribution to obtain uniform pore size and
various size ranges to utilize membrane construction are necessary Surface engineering
with various combination of hydrophobichydrophilic domain on the surface of latex
particles is expected to affect the removal of target protein by selective adsorption and to
improve the biocompatibility of membrane Therefore the materials and characterization
methodology are addressed in this chapter
31 Materials
Styrene (St) monomer used for a seed and a core particle was purchased from
Fisher Scientific and used without any other purification process Acrylic acid (AA) and
methyl methacrylate (MMA) monomers introduced for shell formation were purchased
from Fisher Scientific and used without any other purification process Sodium persulfate
(SPS) and sodium bicarbonate (SBC) were obtained from Fishers Scientific and used as
received Divinylbenzene (DVB) crossliking agent was purchased from Aldrich The
anionic surfactant Aerosol regMA80-I [sodium di(13-dimethylbutyl) sulfosuccinate] was
kindly donated by Cytec 78-80 Aerosol regMA80-I is mixed with isopropanol and
water Its critical micelle concentration (CMC) is 245 mM (119) Ion exchange resin
44
45
AGreg 501-x8 Resin (20-50 mesh) was purchased from Bio-Rad Laboratories Inc It is a
mixed-bed resin with H+ and OH- as the ionic forms and is 8 crosslinked Bovine serum
albumin (BSA) (heat shock treated) was purchas Fisher Scientific The isoelectric
point of t lin
(β2M)
as
gma
ed from
he BSA is 47-49 and the molecular weight is 66300Da β2-Microglobu
was purchase from ICN Biomedicals Inc and was separated from patients with
chronic renal disease and lyophilized from ammonium bicarbonate The molecular
weight by SDS-PAGE was approximately 12000Da BSA and β2M proteins were used
received without further purification process In order to investigate the protein
adsorption evaluation bicinchoninic acid protein assay kits were purchased from Si
(Cat BCA-1) and Pierce Biotechnology (Cat 23225) The chemical structures for the
main chemicals are shown in Figure 3-1
CH2 CH
Styrene (Mw = 10415)
CH2 CHCH2 CH
Styrene (Mw = 10415)
CH2 CH
Acrylic acid (Mw = 7206)
C
OH
O
CH2 CH
Acrylic acid (Mw = 7206)
C
OH
O
CH CH2
C
OCH3
O
CH
CH CH3
2
C
OCH3
O
CH3
Methyl methacrylate (Mw = 10012)Methyl methacrylate (Mw = 10012)
CH2 CH
Divinylbenzene (Mw = 1302)
CH CH2
CH2 CH
Divinylbenzene (Mw = 1302)
CH CH2CH CH
+(Na) -(O3S)
CH -C-O-CH-CH -CH-CH3
O
2 2 3
O CH CH3
CH3 CH3
CH-C-O-CH-CH2-CH-CH3
Anionic surfactant (MA80-I) (Mw = 3885)
+(Na) -(O3S)
CH -C-O-CH-CH -CH-CH3
O
2 2 3
O CH CH3
CH3 CH3
CH-C-O-CH-CH2-CH-CH3
Anionic surfactant (MA80-I) (Mw = 3885)2
+(Na)-(O) S OO
O O
- +
Initiator sodium persulfate (Mw = 23811)
O SO
(O) (Na)+ -
O O
- +(Na) (O) S OO
O SO
(O) (Na)+ -
O O
- +
Initiator sodium persulfate (Mw = 23811) Figure 3-1 Chemical structure of main chemicals
(Na) (O) S OO
O SO
(O) (Na)
46
32 Latex Particle Preparation
The experimental set up and the particle preparation schemes for various types and
size ranges of latex particles are shown in Figure 3-2 and 3-3 In the experimental setup a
mechanical glass stirrer is connected from a motor and Teflon stirrer bar is located at
end of glass stirrer The agitation rate is precisely adjusted by controller motor Paar glas
reactor with 1L volume is partially in silicon oil bath on hot plate The reactor has fo
openings for a stirrer a thermometer a nitrogen gas inlet and a reactant feeding lnlet
The reactants for latex polymerization are fed by precisely controlled by meterin
The particle preparation is described in each latex preparation section
the
s
ur
g pump
Figure 3-2 Experimental setup for semi-continuous emulsion polymerization
1 Motor 2 Mechanical stirrer bar 3 Thermometer 4 Inlet of N2 5 Inlet for initiator and monomer 6 Glass reactor (l L) 7 Oil bath 8 Hot plate 9 Pump for initiator 10 Pump for monomer 11 Tubes for reactant feed
47
Styrene monomer
Bare PS
PSPMMA100
PSPMMA90PAA10
PSPMMA P75 AA25
Gr
win
ticle
size
PS seedparticles
og
par
Styrene monomer
Bare PS
PSPMMA100
PSPMMA90PAA10
PSPMMA P75 AA25
Gr
win
ticle
size
PS seedparticles
og
par
Figure 3-3 The particle preparation scheme in various types and size ranges of latex particles
321 Preparation of Seed Latex Particles
Polystyrene seed particles were synthesized through a typical semi-continuous
emulsion polymerization Polystyrene latex particles were prepared in a four necked Paar
glass vessel equipped with a mechanical glass stirrer thermometer glass funnel for
nitrogen gas purge and a tube for monomer feeding The reactor was placed in a silicon
oil bath on a hot plate for homogenous and stable tempe he vessel was
firstly charged with de-ionized water the emulsifier so itiator
sodium bicarbonate (SBC) styrene (St) monomer divinylbenzene (DVB) monomer
under nitrogen gas atmosphere and then heated to 72plusmn2 as
llowed to continue for another hour Second reactants St and DVB monomers SPS
initiator and SBC were fed separately using fine fluid metering pumps (Model RHSY
Fluid metering INC NY) under a nitrogen gas atmosphere for 2h After complete feeding
rature control T
dium persulfate (SPS) in
oC for 1h The reaction w
a
48
of all reactants the reaction was continued for another hour at elevated temperature 80plusmn1
oC and then cooled to room temperature by removal of the oil bath
322 Preparation of Seeded Latex Particles
The particle growth was achieved by seeded continuous emulsion polymerization
The vessel was charged with the polystyrene seeds and de-ionized water to make desired
solid content and heated up to 72plusmn2 oC for 1h under nitrogen gas atmosphere Then St
and DVB monomers and solution of SPS and SBC in de-ionized water were continuously
added using metering pump with a precisely controlled feed rate After feeding all
reactants the reaction was continued for three hours keeping temperature of 80plusmn2 oC and
then the reaction vessel was cooled down to room temperature
Monodisperse spherical polystyrene seeds with a 280nm mean diameter were used
e-polymerized polystyrene seeded emulsion
was charged in a 500m
323 Preparation of Core Shell Latex Particles
for core shell structured latex particle The pr
l glass flask under a nitrogen gas atmosphere De-ionized water
was added to achieve the desired solid content The emulsion was heated to 80plusmn2oC for
10 hour Monomers initiator and SBC to form the shell were added by fluid metering
pumps (LAB PUMP JRRHSY model Fluid metering Inc USA) with fine controlled
feed rate Flow rate can be adjusted by control ring graduated in 450 division from 0 to
100 flow which is in the range of 10mlmin respectively The reaction was sustained
for additional 30 hours at 90plusmn2oC then cooled down to room temperature
324 Purification of Synthesized Latex Particles
Synthesized latex particles were purified with an ion exchange resin 200g (10
ww polystyrene solid content) of emulsion were mixed with 4g of ion exchange resin
and stirred for 40 minute to remove unreacted monomers initiators and other impurities
49
Then the emulsion was diluted with DI water to a specific degree of solid content as
needed
33 Characterization
331
solid
termined
oscopy
d
hours
Dried latex (10mg) was mixed with
250 m on
Degree of Conversion
The degree of monomer to polymer conversion was determined gravimetrically
following the procedure described by Lee et al [1995] before the latex cleaning process
The synthesized emulsion raw materials were weighed and dried in a conventional oven
at 120oC for 30min to evaporate any unreacted monomers and water The remaining
was weighed and the degree of conversion was determined This process was repeated
three times and a mean value of the degree of conversion was de
332 Fourier Transform Infrared (FTIR) Spectr
A molecule can absorb only select frequencies (energy) of infrared radiation which
match the natural vibration frequencies The energy absorbed serves to increase the
amplitude of the vibrational motions of the bonds in the molecule [Pavin et al 1996]
Only those bonds which have a dipole moment that changes as a function of time can
absorb the infrared radiation The range of wavelengths for infrared radiation is between
25 microm (4000 cm-1) and 25 microm (400cm-1)
The synthesis of the latex particles such as polystyrene (PS) homopolymer and
PSPMMA PSPMMA90PAA10 PSPMMA75PAA25 core-shell copolymers were verifie
with FTIR Purified latex particles were dried in vacuum oven at 40oC for 24
before analysis in the FTIR (Nicolet Magma USA)
g of KBr which had also been dried in vacuum oven at 120oC for 3h Transmissi
spectra using the drift mode were plotted with 128 scans and 4cm-1 resolution
50
333 Quasielastic Light Scattering (QELS)
Latex particle size was measured by the Brookhaven ZetaPlus particle size
analyzer The solid concentration as 0001 (wv) This
ation spectroscopy (PCS) of quasielastically scattered light
For th s
ed using a FE-SEM (JEOL
JSM-
ith
ent
01
us zeta-
potential analyzer It measures the electrophoretic mobility velocity of charged colloidal
of latex particles used w
instrument uses photon correl
is technique time dependent interference patterns of light scattered from particle
in the sample cell are analyzed The interference pattern changes due to Brownian
motion giving rise to fluctuations in scattering intensity The frequency of these
fluctuations depends on the particlersquos diffusion constant that is inversely proportional to
the particle diameter
334 Field Emission-Scanning Electron Microscopy (FE-SEM)
Particle size and surface morphology were characteriz
6335F Japan) A diluted suspension of latex particles in water was dropped onto a
silicon wafer at room temperature and allowed to dry The sample was then coated w
the thinnest layer of carbon needed to obtain the required conductivity for this instrum
Secondary electron image mode was used with a 15KV of accelerating voltage The
magnification range used was between 10000X and 70000X
335 Zeta Potential Measurement
Synthesized and purified latex particles were diluted with de-ionized water to 0
wt and adjusted six different pH values 205 345 481 565 643 and 747 These
particle suspensions were then transferred to standard cuvettes for zeta potential
measurement At least two runs of ten measurements were taken for each sample and
averaged
The zeta potential measurement was carried out using Brookhaven ZetaPl
51
particles in solution and calculate zeta-potential by using the Smoluchowski equation
The f quency
gnitude
umin (BSA) as a standard model protein and β2-Microglobulin
uffer (PB) and
phosp
5
h the selected protein
e 005 01 03 05 and 07 mgml and those for
β2M
d at
teins in the supernatant after the centrifugation process using the
bicinc t
requency of laser light passing through the sample and is compared to the fre
of a reference beam The shift in the frequency called a Doppler shift and the ma
of the shift correspond to the polarity and the magnitude of the electrophoretic mobility
respectively The zeta potential is calculated from the solution conditions and the
measured mobility
336 Protein Adsorption
Protein adsorption experiments of the synthesized latex particles were performed
with bovine serum alb
(β2M) as a target protein in two types of buffer solution phosphate b
hate buffered saline (PBS) To make 5 mM of PB solution 0345g of sodium
phosphate monobasic (NaH2PO4H2O) was dissolve in 500ml of de-ionized water To
make PBS solution 0345g of sodium phosphate monobasic (NaH2PO4H2O) and 4178g
(143mM) of sodium chloride (NaCl) were dissolved in 500ml of de-ionized water The
synthesized latex particles were diluted with each buffer to have a solids content of 0
(ww) adjusted to pH values of 32 48 and 74 and mixed wit
BSA concentrations were chosen to b
were 0015 0030 0045 and 0060mgml The mixture was gently rotated using
the shaker Labquakereg (BarnsteadThermolyne Model 4002110 USA) with 8 RPM in
the incubator at 37oC for 12 hours before the latex-protein mixture was centrifuge
13000 rpm for 15min The amount of protein adsorbed was determined by quantifying
the free pro
honinic acid (BCA) assay method [Lowry et al 1951 Smith et al 1985 Baptista e
al 2003 Wiehelman et al 1988 Brown et al 1989] BCA assay kits consist of Reagent
52
A containing bicinchoninic acid sodium carbonate sodium tartrate and sodium
bicarbonate in 01N NaOH with pH=1125 and Reagent B containing 4 (wv) copper
(II) sulfate pentahydrate The BCA working reagent was prepared by mixing 50 parts of
Reagent A with 1 part of Reagent B 100microl of protein supernatant was then mixed with
2ml of BCA working reagent in a UV cuvette Incubation was allowed to continue
at room temperature
further
until color developed about 2h The absorbance at 562nm was
pectrophotometer (Perkin-Elmer Lambda 800 USA)
Unkn
Separate the p rticles in centrifuge mix supernatant with BCA reagents
measured using a UV-VIS s
own concentration of sample protein was determined by comparison to a standard
of known protein concentrations The adsorbed amount per unit surface area was
determined by the mass balance of the protein after adsorption process The simple
schematic of the procedure for a protein adsorption test is shown in Figure 3-4
Incubate mixture with agitation for 12hrs at 37oC
aand develop the color by incubation for 2hrs at room temperature
Suspend latex particles in aqueous buffer media
Add the dissolved target protein
Measure color intensity using UVVis spectroscopy at 562nm
Suspend latex particles in aqueous buffer media
Separate the p rticles in centrifuge mix supernatant with BCA reagents
Add the dissolved target protein
Incubate mixture with agitation for 12hrs at 37oC
aand develop the color by incubation for 2hrs at room temperature
Measure color intensity using UVVis spectroscopy at 562nm
Figure 3-4 Schematic of the procedure for a protein adsorption test
53
337 Blood Biocompatibility by Hemolysis Test
Hemolysis is the destruction of red blood cells which leads to the release of
hemoglobin from within the red blood cells into the blood plasma Hemolysis testing is
used to evaluate blood compatibility of the latex particles and the damaged to the red
blood cells can be determined by monitoring amount of hemoglobin released The red
blood cells (RBCs) were separated by centrifuging blood at 1500 rpm for 15 minutes
Figure 3-5 shows the phase separated blood with plasma white blood cells and red
cells after centrifugation process Separated RBSs were then washed with isotonic
phosphate buffer solution (PBS) at pH 74 to remove debris and serum protein Th
process was repeated 3 times
blood
is
Figure 3-5 Separation of RBC from whole blood by centrifuge process
Prepared latex particles were re-dispersed in PBS by sonification to obtain
homogeneously dispersed latex particles 100microl of the mixture of red blood cell (3 parts)
and PBS (11parts) was added to 1ml of 05 (ww) particle suspension PBS was used as
a negative control resulting 0 hemolysis and DI water used as a positive control to
produce 100 hemoglobin released by completely destroyed RBCs The mixture was
incubated in water bath with gentle shaking for 30 minutes at 37oC and then centrifuged
at 1500 rpm for 15 minutes 100microl of the supernatant was mixed with 2ml of the mixture
) (EtOHHCl = 2005 ww) to prevent the
White blood cells
Plasma
Red blood cells
of ethanol (99) and hydrochloric acid (37
54
precipitation of hemoglobin In order to remove remaining particles the mixture was
centri fuged again with 13000 RPM at room temperature The supernatant was then
transferred into the UV cuvette The amount of hemoglobin release was determined by
monitoring the UV absorbance at a wavelength of 397nm The schematic of the
procedure for hemolysis test is shown in Figure 3-6
Separate red blood cells (RBCs) from blood
Mix RBCs with phosphate buffer solution (PBS)
Wash and re-suspend RBC with PBS repeat three times
Add la l of RBC and incu in at 37oC
tex particle suspension into 100mbate sample with agitation for 30m
Separate particle ith EtOHHCl solutions and mix the supernatant w
Evaluate free hemoglobin in the solution using UVVis spectroscopy at 397nm
Separate red blood cells (RBCs) from bloodSeparate red blood cells (RBCs) from blood
Mix RBCs with phosphate buffer solution (PBS)Mix RBCs with phosphate buffer solution (PBS)
Wash and re-suspend RBC with PBS repeat three timesWash and re-suspend RBC with PBS repeat three times
Add latex particle suspension into 100ml of RBC and incubate sample with agitation for 30min at 37oC
Add latex particle suspension into 100ml of RBC and incubate sample with agitation for 30min at 37oC
Separate particles and mix the ith EtOHHCl solution supernatant wSeparate particles and mix the ith EtOHHCl solution supernatant w
Evaluate free hemoglobin in the solution using UVVis spectroscopy at 397nmEvaluate free hemoglobin in the solution using UVVis spectroscopy at 397nm
Figure 3-6 Schematic of the procedure for hemolysis test
In summary a description of the experimental materials and characterization
methodology has been explained In the next chapter the results and discussion of the
data obtained from these experiments is addressed
CHAPTER 4 RESULTS AND DISCUSSION
41 Polymerization of Latex Particles
411 Polystyrene Seed Latex Particles
Polymer particles have many different applications including spacer particles
lubricants packing materials for chromatography standard particles and diagnostic
drugs All applications strongly require these particles to have a uniform particle size
Uniform particle size is the first requirement for a latex composite membrane formed by
particle arrays with the interstitial spaces serving as pores for size discriminations [Jons
et al 1999 Ramakrishnan et al 2004] Although suspension polymerization has been
mainly used as one of conventional polymerization methods to prepare uniform particles
the uniformity of the recovered particles is insufficient for membrane use There is
another method known as seed emulsion polymerization where a vinyl monomer is
absorbed into fine monodiserse seed particles and polymerization causes the monomer to
crease the sizes of the seed particles uniformly In order to obtain monodisperse larger
particles by this m
particles and polymerization of the monomer is repeated
In this study cross-linked polystyrene seed latex particles were firstly synthesized
in the presence of styrene monomer divinylbenzene crosslink agent sodium persulfate
initiator sodium bicarbonate buffer and the Aerosol regMA80-I [sodium di(13-
dimethylbutyl) sulfosuccinate] surfactant using a batch type semi-continuous emulsion
polymerization process in which one or more components can be added continuously
in
ethod the procedure of absorption of monomer into fine polymer
55
56
Styrene (St) monomer and divinylbenzene (DVB) crosslink agent as well as initiator and
buffer are continuously added to enlarge the seed latex particles Various profiles of
particle nucleation and growth can be gene different orders of component
addition during polymerizati f this process such as easy
control of the polymeriza particle number
and particle morphology The main goal in
this process is to avoid secondary particle
rated from
on There are several advantages o
tion rate monodisperse particle size and
colloidal stability copolymer composition
formation leading to a monodisperse particle
size distribution Figure 4-1 shows the schematic representation of seed latex particle
preparation and growth
Monomer droplet
Seed particles Amount of surfactantsis variable
Enlarged final Add monomers
PS
PS
PSs4
particles Number of particlesare constant
Seed code
PSs1
s2
s3
Monomer droplet
Seed particles Amount of surfactantsis variable
Enlarged final Add monomers
PS
PS
PSs4
tion The
particles Number of particlesare constant
Seed code
PSs1
s2
s3
Figure 4-1 Schematic representation of semi-continuous seed latex particles preparation and growth
After the first synthesis of a small size of crosslinked polystyrene (PS) seed
particles stabilized by surfactants styrene DVB and initiator are continuously fed into
the system to enlarge the seed latex particles maintaining a narrow size distribu
recipes and reaction conditions for synthesizing these seed particles are summarized in
57
Table 4-1 The original recipe for latex particle preparation from McDonald was
modified for this emulsion system [McDonald 2003]
Table 4-1 The polymerization recipe of polystyrene (PS) seed particles Latex label PSS259 PSS233 PSS207 PSS181
Temperature (oC) 75 plusmn 5 75 plusmn 5 75 plusmn 5 75 plusmn 5
Divinylbenzene (g) 052 052 052 052
MA 80 (g) 259 233 207 181
NaHCO
Watera (g) 1315 1315 1315 1315
Styrene (g) 9948 9948 9948 9948
112 112 112 112
Na2S2O8 (g) 190 190 190 190
Waterb (g) 200 200 200 200
Reaction time (min) 140 140 140 140
Solids content 396 396 396 396
Mean particle diameter (nm) 126plusmn75 171plusmn39 182plusmn41 216plusmn53
Conversion () 965 979 957 971
3 (g)
Four different seed latex particles were synthesized based on surfactant amount in
order to determine the optimum condition for stable and monodisperse latex particles
The emulsion polymerization of latex particles has to be carried out in a narrow range of
surfactant causes flocculation or phase separation leading to broad particle
size distribution and an unstable emulsion sy
surfactant concentration where particles are stable [Nestor et al 2005] A wide range of
concentrations
stem Therefore the range of surfactant
amount was varied from 181g to 259g in increments of 026g in each latex label Other
reactants amounts were kept constant Watera is the initial charge amount and waterb
represents the amount of aqueous solution containing dissolved initiator and buffer
58
which are fed continuously after the seed particle creation The extent of monome
polymer conversion was obtained by gravimetric calcu
r to
lation and was more than 95 at
cripts such as
S207 and S181 i the surf mount added for the seed latex
nversion indi perce experi obtain
content divided by the theoretically estimated solid content and was calculated by the
all seed particles labeled PSS259 PSS233 PSS207 and PSS181 Labeled subs
S259 S233 ndicate actant a
preparation The co cates the ntage of mentally ed solid
following formula
100 colids () times=entsoliatedntentoalExperimentConversion
n the experimenta content is the weight of olid rema after
mulsion at 120oC f in and the estimated solid content is th ht
lated from the recipe
rti as di each t load d
mple of ea pension iluted by (ww)
deionized water and the mean particle diameter was determined using the Brookhaven
ZetaPlus particle size analyzer From these results the seed latex particle size increased
as the amount of surfactant decreased The amount of emulsifier also affects the
polymerization process by changing the number of micelles and their size It is known
that large amount of emulsifier produces larger numbers of smaller sized particles [Odian
1991] and was corroborated in the current work
These synthesized seed latex particles should be of narrow size distribution in order
to obtain monodisperse larger particles The SEM image showing the particle
morphology and size of a sample of polystyrene seed particles can be seen in the Figure
4-2
contdEstim (4-1)
In this equatio l solid the s ining
evaporation of e or 30m e weig
of monomers calcu
As expected the seed pa cle size w fferent for surfactan ing in see
emulsion system A sa ch sus was d 0001 with
59
Figure 4-2 Scanning Electron Micrograph (SEM) of polystyrene seed latex particles (A)
PSS259 (B) PSS233 (C) PSS207 (D) PSS181 (subscripts indicate the amount of surfactant added)
(A)
(B)
60
(C)
(D)
Figure 4-2 Continued
61
s seen in the image the seed particles are very smooth and spherical The
uniformity in the particle size distribution of these PS seed latex particles can also be
seen in the SEM image From this data we concluded that seed particles synthesized in
the presence of a surfactant with the chosen amounts are suitable for use to prepare larger
particles with high size uniformity and smooth spherical morphology
412 The Growth of Polystyrene (PS) Seed Latex Particles
Seeded emulsion polymerization has been conducted for several decades and
various mechanisms have been proposed Gracio et al [1970] suggested that the growing
polymer particles consist of an expanding polymer-rich core surrounded by a monomer-
rich shell with the outer shell providing a major locus of polymerization Seeded
mulsion polymerization is commonly used for preparing latex particles less than 1microm in
size [Cha et al 1995 Zou et al 1990 1992 Gandhi et al 1990 Park et al 1990] Such
latexes can be obtained from the growth of pre-prepared seed particles The seeds
introduced in this process serve as nucleation sites for particle growth By first swelling
the seed latex particles with additional monomer to the desired size polymerization can
then be initiated Several size ranges of monodisperse polystyrene (PS) latex particles
were prepared by this multistep seeded emulsion polymerization method For the
fabrication of particle-based membranes monodisperse latex particles with a variety of
size ranges are required
In this study seed particles from 171nm to 470nm in mean diameter and with a
highly uniform size distribution were prepared and used Watera is the initial charge
mount and waterb represents the amount aqueous solution containing dissolved initiator
and buffer that is continuously fed into system Feed time is precisely controlled by
metering pump The solid contents were maintained between 199 and 300 After
A
e
a
62
polym d as
es
ere 923 8402
n of these latex particles
are sh
ss
erization under these conditions the conversions of the latex particles labele
PS258 PS320 PS370 and PS410 were 952 957 946 and 964 respectively The recip
used in seeded emulsion polymerization to form the particles less than 500nm in mean
diameter are shown at the Table 4-2 The conversions of the latex particles larger than
500nm in mean diameter and labeled as PS525 PS585 PS640 and PS790 w
723 and 969 respectively The recipes used in the preparatio
own at the Table 4-3
A simple calculation can be used to predict the ultimate size that a latex particle can
reach after ending the polymerization Letrsquos suppose that seed density (ρs) and grown
particle density (ρgp) are the same The following equation is obtained from the ma
balance (m = ρV) where m and V are mass and volume of a particle respectively
gp
gp
s
s
Vm
Vm
In this equation m
= (4-2)
be
s and mgp are the weight of seed and total weight of seed and
added monomers respectively and Vs and Vgp are the volume of seed and grown
particle respectively
If we know the mass of monomer to be added the ultimate particle size can
calculated from the equation 4-3
3
2s D m ⎠⎝+ χ
where D
1s D m
⎟⎟⎞
⎜⎜⎛
= (4-3)
the
in
s and Dgp are the diameters for seed and grown particle respectively and χ is
weight of the monomer added Crosslinked PS latex particles with a size range from
258plusmn112 ~ 410plusmn117 in diameter polymerized by the recipes in Table 4-2 are shown
the SEM images in Figure 4-3
63
Table 4-2 Continuous addition emulsion polymerization recipe for growing polystyrene
Latex label PS
(PS) latex particles less than 500nm in size
258 PS320 PS370 PS410
Initial charge
Seed latex (g) 1000171 1000216 1000216 1000320
Watera (g) 1470 2500 1984 2600
06 07 06 06
Fee
Initiator stream
2 2 8
Feed time (min) 0-167 0-140 0-100 0-92
Solid content 284 300 300 200
Mean particle diameter (nm) 258plusmn112 320plusmn154 182plusmn71 410plusmn117
Conv
Monomer continuous
Styrene (g) 800 1200 800 600
Divinylbenzene (g)
d time (min) 0-163 0-110 0-75 0-82
Na S O (g) 07 065 05 05
NaHCO3 (g) 07 065 05 05
Waterb (g) 750 1200 200 370
ersion () 952 957 946 964 The solid content of seed latex for PS258 PS320 PS 370 and PS410 are 392 399 399 anrespectively
d 300
64
Table 4-3 Continuous addition emulsion polymerization recipe for growing polystyrene (PS) latex particles larger than 500nm in size
Latex label PS525 PS585 PS640 PS790
Initial charge
Seed latex (g) 1000390 1000470 1000470 1000320
Watera (g) 1000 520 1200 360
Monomer continuous
Styrene (g) 290 200 400 173
Divinylbenzene (g) 02 014
03 009
Feed time (min) 0-80 0-40 10-76 5-65
Initiator stream
Na2S2O8 (g) 02 014 03 012
NaHCO3 (g) 02 014 03 009
Waterb (g) 200 120 400 200
Feed time (min) 0-85 0-46 0-77 0-75
Solids content 199 200 200 201
Mean particle diameter (nm) 525plusmn151 585plusmn186 640plusmn178 790plusmn693
Conversion () 923 8402 723 969 The solid content of seed latex for PS525 PS585 PS 640 and PS790 are 200 182 193 and 173 respectively
65
Figure 4-3 SEM of PS latex particles less than 500nm in size (A) 258nm (B) 320nm (C)
370nm (D) 410nm
(A)
(B)
66
(C)
(D)
Figure 4-3 Continued
67
article size was well matched to that estimated by the geometrical calculation
From this it can be concluded that the added monomer was consumed for polymer
particle growth with a high degree of conversion From the SEM characterization newly
nucleated particles were not seen as indicated by the narrow particle size distribution
The particles less than 500nm in mean diameter were very spherical in shape with a
highly uniform particle size distribution As the particles were grown larger than 500nm
mean diameter however they became of non-spherical shape with an uneven surface
This irregularity of particle surface can be attributed to the non-homogeneous monomer
swelling into the shell of a growing polymer which can be controlled by the factors such
as temperature agitation speed initiator feeding rate and surfactant amount required to
stabilize the colloidal system The SEM image of latex particles larger than 500nm mean
iameter is shown in Figure 4-4 The particle size distribution is broader than that of the
s particles These particles can however still be used for membrane construction
as a support layer rather than skin layer The support layer does not necessarily have to be
as highly monodisperse as the skin layer which require pores of high uniformity for the
selective removal of toxins
As mentioned earlier the amount of surfactant as well as monomer in an emulsion
system is the primary determinant of the particle diameter [Odian 1991] Anionic
surfactants are generally recommended at the level of 02-30 wt based on the amount
of water [Odian 1991] The critical micelle concentration (CMC) of Aerosolreg MA 80-I
anionic surfactant is 119 The surfactant loading in this emulsion system ranged from
the 121 for SS181 to 171 for SS259
P
d
maller
68
Figure 4-4 SEM of PS latex particles larger than 500nm in size (A) 525nm (B) 585nm
(C) 640nm (D) 790nm
(A)
(B)
69
(C)
(D)
Figure 4-4 Continued
70
The surfactant to monomer ratio refers to the value of the surfactant amount
divided by monomer amount after polymerization ends The surfactant amount is the
weight of the surfactant contained in emulsion and the monomer amount is the total
weight of the monomer added for polymerization to form the ultimate polymer particles
in emulsion As the surfactant to monomer ratio decreases mean particle diameter
increases as described by Odian [1991] Generally lower surfactant concentration forms
fewer micelles resulting in larger particle size [Evans et al 1999] However there is a
plateau in particle mean diameter between a surfactant to monomer ratio of 0005 and
0013 This is due to low polymerization conversion of latex particles from monomer
leading to a smaller particle size than expected This is not a factor of surfactant amount
The dependence of the particle size on the amount of surfactant is shown in Figure 4-5
0
200
400
600
800
1000
0000 0005 0010 0015 0020 0025 0030
Surfactant to monomer ratio (ww)
Part
icle
mea
n di
amet
er (n
m)
Figure 4-5 Dependence of the particle size on the surfactant to monomer ratio
71
413
e
f bare
microdomain structures is important for biocompatibility [Mori et al 1982 Higuchi et al
1993 Deppisch et al 1998] Figure 4-6 shows the schematic of core shell latex particle
structure The recipe for core shell structures is shown in Table 4-4
Core Shell Latex Particles
The latex particles shown in Figure 4-3 (A) and labeled as S258 in Table 4-2 wer
used as seeds to prepare core-shell latex particles because of their high particle
uniformity and smooth surface morphology Methyl methacrylate (MMA) and acrylic
acid (AA) monomers were introduced to increase surface hydrophilicity over that o
polystyrene particles AA is a more hydrophilic monomer than MMA because of the
carboxyl acid group in AA is more favorable for hydrogen boding with water The
surface carboxyl groups on PAA may have many promising applications in biomedical
and biochemical fields [Kang et al 2005] MMA monomer is less hydrophobic than
styrene PMMA is known to more biocompatible than PS but still PMMA is hydrophobic
The optimization of suitable hydrophobic to hydrophilic ratios and control of
Polystyreneseed particle
Core shelllatex particles
PSPMMA100
PSPMMA90PAA10
PSPMMA75PAA25
Polystyreneseed particle
Core shelllatex particles
PSPMMA100
PSPMMA90PAA10
Figure 4-6 Schematic of core shell latex particle structures
PSPMMA75PAA25
72
Table 4-4 The preparation recipe of PS core with various shell latex particles Latex 25 label PSPMMA100 PSPMMA90PAA10 PSPMMA75PAA
Initial charge
PS seed latex (g) 500 500 500a
Monomer continuous
Methly methacylate (g) 320 288 240
Acrylic acid (g) 00 32 80
Initiator stream
Na
258 258 258
Water (g) 1570 1570 1570
Feed time (min) 0-120 0-50 0-60
b 200
Feed time (min) 0-122 0-60 0-65
952
2S2O8 (g) 02 02 02
NaHCO3 (g) 02 02 02
Water (g) 200 200
Solids content 183 183 183
Mean particle diameter (nm) 370plusmn186 370plusmn178 370plusmn191
Conversion () 955 924
Three types of core shell structures were prepared PMMA75PAA25 shell is a
copolymer consisting of the PMMA to PAA ratio of 75 to 25 by weight
PMMA90PAA10 shell is a copolymer with the PMMA to PAA ratio of 90 to 10 by
weight PMMA100 is the PMMA homopolymer shell on PS core The particle size of
these core-shell particles as well as PS latex particle was prepared to be about 370nm in
mean diameter and are characterized to determine their zeta potential protein adsorption
and biocompatibility The image of SEM of PS and core shell particles is shown at Figure
4-7 The synthesized latex particles had a high uniformity in size and a smooth spherical
surface morphology
73
Figure 4-7 Scanning
Electron Micrograph of latex particles (A) PS (B) PSPMMA100 (C) PSPMMA90PAA10 (D) PSPMMA75PAA25
(A)
(B)
74
(C)
(D)
Figure 4-7 Continued
75
42 Characterization of Latex Particles
421 Fourier Transform Infrared Spectroscopy (FTIR)
FTIR spectroscopy has proven to be a powerful tool to characterize polymeric
materials A molecule can absorb only select frequencies (energy) of infrared radiation
which match the natural vibration frequencies The energy absorbed serves to increase
the amplitude of the vibrational motions of the bonds in the molecule [Pavin et al 1996]
Only those bonds which have a dipole moment that changes as a function of time can
absorb infrared radiation The FTIR spectra of polymerized latex particles made during
this work are shown in Figure 4-8 There are number of characteristic peaks for PS latex
particles [Bhutto et al 2003 Li at al 2005] Aromatic C-H stretching vibration is shown
at 3002 cm-1 and 3103 cm-1 and aliphatic C-H asymmetrical and symmetrical stretching
shown at 2900 cm-1and 2850 cm-1 respectively There is an additional carbonyl (C=O)
adsorption at 1730cm-1 wavenumber for all core shell latex particles containing some
amount of either or both PMMA and PAA The broad OH group peak at 3400 cm-1
appear for particles containing some amount of PAA and has intensity dependent on the
amount of PAA in the shell For example the peak intensity of OH groups for
PSPMMA75PAA25 is greater than that of PSPMMA90PAA10 since there is a higher AA
monomer content in PSPMMA75PAA25 than in PSPMMA90PAA10 This OH peak is not
seen for PS and PS core PMMA shell particles because of the absence of OH groups on
PS and PMMA polymer chains
422 Zeta Potential Measurements
The electrical surface property was characterized by zeta potential measurements of
tex particles Zeta potential is an important and useful indicator of surface charge which
can be used to predict and control the stability of colloidal systems The greater the zeta
is
la
76
1520002500300035004000
Wavenumbers (cm-1)
li
Re
ativ
e
Tra
nsm
ttanc
e
Figure 4-8 FTIR spectra of polymerized latex particles (A) ba
PSPMMA100 (C) PSPMMA90PAA10 (D) PSPMM
(B)
(C) P
(D) P
C
C
OCH3
O
CH3
XCH2
CH2 n
CH
CH2 C
C
OCH3
O
XCH2 CH
OC
CH3
Y
OH
(A) Bare PS
500100000
re polystyrene (PS) (B) A75PAA25
PSPMMA100
SPMMA90PAA10
SPMMA75PAA25
77
potential the more likely the suspension is to be stable because the charged particles repel
one another and thus overcome the natural tendency to aggregate The measurement of
zeta potential is often the key ontrol of the interaction of proteins
with solid surfaces such as latex particles
Zeta potential (ζ) is the elec at exists at the shear plane of a particle
which is some small distance from the surface [Myers 1999] The zeta potential of the
particles can be determined by measuring the mobility of the particles in an applied
electric field termed electr its response to an alternating electric
field Colloidal particles dispersed in lu ically charged due to their ionic
characteristics and dipolar attributes The development of a net charge at the particle
surface affects the distribution of ions in the neighboring ng in
an increased concentration of counter ions to the surface Each particle dispersed in a
ns called fixed layer or Stern layer
Outside th compositions of ions of opposite polarities
form ble layer is form
particle-liquid interface This double layer may be considered to consist of two parts an
inner region which inclu nd relatively strongly to the surface and an outer or
diffuse region in which the ion distribution is determined by a balance of electrostatic
forces and random thermal motion The potential in this region therefore decays with the
distance from the surface until at a certain distance it becomes zero Figure 4-9 shows a
schematic representation of a typical ion distribution near a positively charged surface
The Stern surface (also referred to as the outer Helmholtz plane) is drawn through the
to understanding and c
trical potential th
ophoretic mobility or
a so tion are electr
interfacial region resulti
solution is surrounded by oppositely charged io
e Stern layer there are varying
ed in the region of the ing a cloud-like area Thus an electrical dou
des ions bou
center of those ions which are effectively adsorbed to the surface [Hunter 1981] The
78
extent of ion adsorption is determined by electrical and other long-range interactions
between the individual ions and surface of particles The ions outside of the Stern layer
form the diffuse double layer also referred to as the Gouy-Chapman layer [Burns and
Zydney 2000]
Shear plane
Stern layer
ψζ
0
ψs
ψShear plane
Stern layer
ψζ
0
ψs
ψ
t
controlled electric field is applied via electrodes immersed in a sample suspension and
Figure 4-9 Schematic representation of ion distribution near a positively charged surface
Zeta potential is a function of the surface charge of a particle any adsorbed layer a
the interface and the nature and composition of the surrounding medium in which the
particle is suspended The principle of determining zeta potential is very simple A
79
this causes the charged particles to move towards the electrode of opposite polarity
Viscous forces acting upon the moving particle tend to oppose this motion and the
equilibrium is rapidly established between the effects of the electrostatic attraction and
the viscosity dra
g The particle therefore reaches a constant terminal velocity
Figure 4-10 Schematic representation of zeta potential measurement(source
httpnitioncomenproductszeecom_shtm)
Because protein adsorption mechanisms are very complex and hard to explain in
biological systems protein adsorption is generally studied by using more ideal systems
consisting of one or more well characterized proteins a well-characterized adsorbent and
a well-defined aqueous solution Even so small changes in the experimental conditions
such as pH ionic strength and temperature generate totally different results Therefore
two media systems phosphate buffer (PB) and phosphate buffered saline (PBS) were
chosen to be used for the zeta potential measurements and protein adsorption study PB is
a simple aqueous system of 5 mM monobasic sodium phosphate (NaH2PO4H2O) to
n a constant pH PBS is used to keep the water concentration inside and outside of
the cell balanced If the water concentration is unbalanced the cell risks bursting or
shriveling up because of a phenomenon called osmosis PBS is a solution with the 5 mM
of monobasic sodium phosphate (NaH2PO4H2O) and 143 mM of sodium chloride (NaCl)
in water
maintai
80
The results of zeta potential measurement at room temperature (25plusmn1oC) as
function of pH and ionic strength (different media PB and PBS) for synthesized lat
particles are seen in Figure 4-11 Zeta potential values of polystyrene (PS) latex particle
at the Figure 4-9 are negative between -291 mV and -599 mV in PB and betwe
ex
s
en -203
mV and -278 mV in PBS at all pH ranges These negative zeta potential values of PS
latex particles are due to the sulfate groups [Vandenhu et al 1970] originated from
persulfate initiators attached via an oxygen bridge (-C-O-SO3-) at the end of polymer
chain where polymerization was initiated
-100
-80
-60
-40
-20
0
pH
Zta
pte
ntl
ζ
20
40
1 2 3 4 5 6 7 8 9
eo
ia (m
V)
PB (5mM)
PBS (5mM +143mM)
en -
Figure 4-11 Zeta potential of PS latex particles at 25oC
The zeta potential measurements of PSPMMA100 core shell latex particles as seen
in Figure 4-12 were also negative with a range of -28 and -505 mV in PB and betwe
143 mV and -186 mV in PBS at the pH 21-78 These negative values are also due to
sulfate groups on the particle surface
81
-100
-80
-60
-40
-20
0
20
40
1 2 3 4 5 6 7 8 9
Zea
pote
nti
l ζ
(mV
pH
ta
)
PB (5mM)
PBS (5mM + 143mM)
Figure 4-12 Zeta potential of PSPMMA100 latex particles at 25oC
The zeta potential for PSPMMA90PAA10 and PSPMMA75PAA25 was also
negative as seen in Figure 4-13 and 4-14 The zeta potential value for the
PSPMMA90PAA10 were between -367 mV and -678 mV in PB medium and between -
147 mV and -193 mV in PBS both at 25oC In case of the PSPMMA75PAA25 core shell
particles zeta potential values were between -291 mV and -520 mV in PB and between
-115 mV and -210 mV in PBS media Sulfate groups in PSPMMA75PAA25 and
PSPMMA90PAA10 core shell particles contribute to the negative zeta potential values
participate in forming a
negat
cid is expressed by the term pKa which is the pH
at which an acid is 50 dissociated However the contribution of the carboxylic acid
Carboxylic groups from acrylic acid monomer also would
ive surface charge of the latex particles at a pH greater than 70 because the pKa
value for the carboxylic acid is between 40 and 60 pH range [Ottewill et al 1967] This
indicates that carboxylic acid dissociation begins at a pH between pH 46-60 and
increases with pH The strength of an a
82
group to negative zeta potential values of PSPMMA-PAA core shell particles was not
detectable because there was no significant difference in zeta potential values for
synthesized latex particles at a pH greater than 70
-60
-40
-20
0
20
40
1 2 3 4 5 6 7 8 9
Zeta
pot
entia
l ζ
(mV
)
PB (5mM)PBS (5mM + 143mM)
-80
-100
pH
Figure 4-13 Zeta potential of PSPMMA90PAA10 latex particles at 25oC
-100
-80
-60
-40
-20
0
20
40
1 2 3 4 5 6 7 8 9
pH
Ze p
ont
i ζ
V)
tate
al (m
PB (5mM)PBS (5mM + 143mM)
Figure 4-14 Zeta potential of PSPMMA75PAA25 latex particles at 25oC
83
The zeta potential plots and values were similar for all latex particles This may be
due to the similar initiator density on the latex surface The initiator concentrations
monomers for shell polymerization were the same fo
to
r all core shell latex particles (062
ww from Table 4-4) A similar concentration (055 ww) of initiator to monomer was
added to prepare bare PS latex particles used in zeta potential measurements
Since the zeta potential values of all synthesized latex particles were negative
between pH=20 and pH=78 the isoelecric point (IEP) of these latex particles would be
less than pH=20 Sulfates have a pKa in the range of 10 to 20 [James 1985] indicating
that the sulfate group the conjugate base of a strong acid is protonated (-C-OSO3H) at
less than pH 20 This means that the synthesized latex particles are all negative at the
physiological pH Most serum proteins have a negative surface charge in blood
therefore when the negative latex particles are applied to blood stream fatal
tein is
avoided by charge repulsion in the blood Table 4-5 shows the common blood proteins
and their isoelectric points (IEP)
Table 4-5 The isoelectric point (IEP) of common proteins Serum proteins Isoelectric point (IEP)
complication by coagulation between applied latex particles and serum pro s
Albumin 48 α1-Globulin 20 α2-Microglobulin 54 γ1-Globulin 58 Heptoglobin 41 Hemoglobin 72 β1-lipoprotein 54 Immunoglobulin G (Ig G) 73 Fibrinogen 58 (source httpwwwfleshandbonescomreadingroompdf1085pdf
httpwwwmedalorgvisitorwww5CActive5Cch135Cch13015Cch130105aspx )
84
The absolute zeta potential values of the latex particles in phosphate buffer (PB)
was higher than that in phosphate buffered saline (PBS) This is because high
concentration of sodium and chloride electrolytes in PBS compressed the electrical
double layer causing rapid decay of surface potential Figure 4-15 shows that the
thickness of the electrical double layer is inversely proportional to the concentration of
electrolyte in the system
Surf
ace
Pote
ntia
l ψ
1κ3
10
1e
1κ 1κ
(1)(3) (2)
2 1 Z
Surf
ace
Pote
ntia
l ψ
1κ3
10
1e
1κ 1κ
(1)(3) (2)
Figure 4-15 The decay of surface potential with distance from surface in various
electrolyte concentrations (1) low (2) intermediate (3) high [Myers 1999]
2 1 Z
The electrical potential in the solution surrounding the particle surface falls off
the Debye-Huckel
yers 1999]
exponentially with distance (Z) from the surface according to
approximation [M
z) (- exp 0 κψψ = (4-4)
e reciprocal of the thickness of the electrical double layer referred to as the
The potential fallen off by a factor e The theoretical equation for the
ickness 1κ is
where κ is th
Debye length of 1
double layer th
21
22i
0
ekT 1
⎟⎟⎠
⎜⎜⎝
=sum ii zcεε
κ
⎞⎛ (4-5)
85
where
tration of
y was performed to measure the protein adsorption on the
synthesized latex particles Many physical and chemical processes occur at the boundary
between two phases and adsorption is one of the fundamental surface phenomena [Oscik
1982] This phenomenon is driven by the change in the concentration of the components
at the interface with respect to their concentrations in the bulk phase There are two
adsorption processes physical adsorption occurring when non-balanced physical forces
appear at the boundary of the phases and chemical adsorption or chemisorption
t the
interface [J
n leads
ational entropy hence adsorption takes place only if the
loss in conformational entropy is compensated
ter adsorption Cp The initial part of the
isotherm merges with the Γ-axis because at low polymer concentration the protein has a
high affinity for the surface and all of the polymer is adsorbed until the solid surface is
saturated
ε0 is the permittivity of a vacuum of free space ε is the permittivity of a medium e
is the relative permittivity or dielectric constant of the medium c is the concen
an ion (i) z is the valency of an ion (i) k is Boltzmannrsquos constant T is absolute
temperature (K)
43 Protein Adsorption Study
The adsorption stud
occurring when atoms and molecules from adjacent phases form chemical bonds a
aroniec et al 1988]
Flexible proteins in solution possess high conformational entropy as a result of the
various states each of the many segments in the protein chain can have Adsorptio
to a reduction of this conform
by sufficient attraction between polymer
segment and solid surface [Martin 1998] Isotherm shape of high affinity adsorption
between polymer and solid is shown in Figure 4-16 where the adsorbed mass Γ is plotted
against the polymer concentration in solution af
86
Figur
de
arboxyl
e 4-16 High-affinity adsorption isotherm of typical flexible polymer on solid
surface
In order to investigate the adsorption of blood proteins latex particle properties
such as electrical surface charge hydrophobicity and environmental conditions like pH
ionic strength and temperature are considered as experimental factors [Arai and Nor
1990] Figure 4-17 shows the overall schematic representation of the protein adsorption
on the synthesized latex particles with the functional groups such as sulfate and c
groups
CP
Γ
PMMAPAAShell
PS or PMMAShell OSO3
-
OSO3-
OSO3-
OSO3-
PS
-O3SO
PSCore
HOOC
-O3SO
O3SO
3
HOOC
HOOC
-
-O SO
Protein
NH3+
+
+H3N
+
COO-
Hydrophobic interaction
NH3
COO-
H3N
COO-
-OOC
Ionic interaction
Hydrophobic interaction
Hydrogen bonding
Ionic interaction
PMMAPAAShellOSO3
-
OSO3-
OSO3-
OSO3-
PS or PMMAShell -O3SOOSO3
-
PSCore
HOOC
-O3SO
O3SO
3
HOOC
HOOC
-
-O SO
-O3SO
OSO3-
OSO3-
OSO3-
PS PSCore
HOOC
-O3SO
O3SO
3
HOOC
HOOC
-
-O SO
Protein
NH3+
+
+H3N
+
COO-NH3
COO-
H3N
COO-
-OOC
NH3+
+
+H3N
+
COO-
Hydrophobic interaction
NH3
COO-
H3N
COO-
-OOC
Ionic interaction
Hydrophobic interaction
Hydrogen bonding
Figure 4-17 Overall schematic representation of the protein adsorption on the
Ionic interaction
synthesized latex particles
The surface functional groups as well as suitable surface hydrophobicity
hydrophilicity of latex particles are closely related to protein adsorption property As
indicated at Figure 4-17 various interaction forces such as hydrophobic interaction ionic
87
interaction hydrogen interaction and van der Waals interaction [Andrade 1985] exist
between protein molecule and the solid surface of latex particle
431 Adsorption Isotherm
The protein adsorption isotherm is generally defined as the relationship between the
amount of protein adsorbed by a solid (latex particle) at a constant temperature and as a
ine
the amount of protein adsorbed onto latex particles To evaluate the equilibrium
conce s
x
function of the concentration of protein Adsorption isotherms were plotted to determ
ntration of target proteins a calibration curve is firstly needed Figure 4-18 show
the UV absorbance as a factor of bovine serum albumin (BSA) concentrations The UV
absorbance of the known protein concentration was measured at 562nm which is the λma
for a protein treated by a bicinchoninic acid (BCA) assay
0
02
04
06
08
500 520 540 560 580 600
Wavelength (nm)
Abs
orba
nce
Figur gml (B)
06mgml (C) 02mgml
(A)
(C)
(B)
Max562nm
e 4-18 The dependence of UV absorbance on BSA concentration (A) 10m
Figure 4-19 shows the standard calibration curve used in adsorption isotherm
experiment of protein onto latex particles The net absorbance of each known protein
88
concentration was plotted and then a linear equation passing through a concentration of
zero protein concentration was obtained This equation is applied
to determine the
orbed on the synthesized latex particles The amount of free
protei
eported
trations (005 01 03 05
and 07mgml) were used to generate this curve as well as the adsorption isotherm
unknown protein amount ads
n was also determined using UV light at the 280nm wavelength however a
distinguishable UV intensity was too low to determine a precise value of protein
adsorption
The bicinchninic acid (BCA) assay technique [Smith et al 1985] has been r
for high efficiency of protein evaluation and was used for determination of protein
adsorption [Tangpasuthadol et al 2003 Williams et al 2003] This technique has many
advantages such as high sensitivity easy of use color complex stability and less
susceptibility to any detergents Five different BSA concen
y = 00R2 =
004741x 09939
00
01
02
03
04
0 200 400 600 800
BSA Standard (microgml)
Net
Abs
orba
nce
at 5
62nm
Figure 4-19 Standard curve of net absorbance vs BSA sample concentration
89
All isotherms were fitted to the Langmuir-Freundlich isotherms using the nonlinea
regression method Two adsorption isotherm models the Langmuir type isotherm a
Langmuir-Freundlich combination type isotherm were compared As seen the Figure 4-
20 The Langmuir isotherm model deviated further from the experimental data Th
all isotherms were fitted to the Langmuir-Freundlich isotherms [Yoon et al 1996
al 1998] usi
r
nd the
erefore
Lee et
ng the nonlinear regression method with following equation
n
n
eq
eqm
kC
kC1
1
1+
where q is the amount of adsorbed BSA per unit surface area q
qq = (4-6)
stant
were between 045 and 055 and hence n was fixed to its average 05 Actual qm values
were evaluated after fixing the n value
m is the adsorbed amount
in equilibrium Ceq is the equilibrium concentration of BSA k is the adsorption con
and n is the exponential factor for a heterogeneous system n values are empirical and
usually from zero to no more than unity [Jaekel 2002] The values of n in our system
00
10
20
30
0 02 04 06 08
C (mgml)eq
q (m
gm
2 )
pH 32Langmuir-Freundlich modelLangmuir model
Figure 4-20 Fitted models for adsorption isotherm of bovine serum albumin (BSA) on
polystyrene latex particles at 37oC in phosphate buffer media
90
The BSA protein adsorption on the synthesized latex particles was performed at
three pH levels 32 48 74 in both phosphate buffer (PB) and phosphate buffered saline
(PBS) at physiological temperature 37oC These pH levels correspond to an acidic pH
lower than the isoelectric point (IEP) of BSA the pH at the IEP and an alkaline pH
higher than the IEP of BSA respectively The IEP corresponds to the pH at which the
amino acid becomes a zwitterion Here its overall electrostatic charge is neutral and the
charge expressed by the basic amino group (-NH2 -NH3+) of the amino acid is equal to
the charge expressed by the acidic carboxyl group (-COOH -COO ) The IEP of BSA is
-
pH 48 and indicates that the side chain of the amino acids of BSA contains a greater
amount of acidic functional groups than that of basic functional groups At pH 32 and
74 BSA has a positive and negative charge respectively
Figure 4-21 and 4-22 show adsorption isotherms of BSA onto polystyrene (PS)
latex particles
00
10
20
30
40
0 02 04 06 08
Ceq (mgml)
q (m
gm
2 )
pH=32pH=48pH=74Model (pH 32)Model (pH 48)Model (pH 74)
Figure 4-21 Adsorption isotherm of BSA on polystyrene (PS) latex particles in
phosphate buffer (PB) at 37oC
91
00
10
30
40
0 02 04 06 08
Ceq (mgml)
q (m
gm
2
20
)
pH=32pH=48pH=74Model (pH 32)Model (pH 48)Model (pH 74)
(PBS) on PS latex particles
At the IEP of BSA the adsorbed amount (q
Figure 4-22 Adsorption isotherm of BSA adsorption 37oC in phosphate buffered saline
onto PS at equilibrium was
maxim
um at the IEP of BSA This is because the BSA molecules form the most
compact structures at the IEP eliminating the repulsion force between proteins A higher
number of BSA molecules can adsorb on a given surface area by hydrophobic interaction
when in this compact structure This observation agrees well with reported data by other
researchers who also indicated that the maximum adsorption from aqueous protein
solution was observed at the IEP [Mularen 1954 Watkins et al 1977 Oh at el 2000]
There was no significant difference of adsorbed BSA amount in PB and in PBS media at
pH 48 The conformational alteration of BSA was not affected significantly by the ionic
strength of the two solutions There was evidence of the effect of the electrostatic
interaction to some extent even at the IEP since the amount of BSA adsorbed in PBS is
slightly hig
m) of BSA
um indicating that hydrophobic interaction between latex particle and BSA
protein is maxim
her than in PB at this pH
92
The adsorbed amount of protein is shown to decrease at a pH 32 and 74 The
decrease in adsorption efficiency when deviating from IEP of BSA is a result of the
increase in conformational size of the protein molecules and the lateral electrostatic
repulsions between adjacent adsorbed BSA molecules Figure 4-23 shows a schematic of
possible conformational changes at each pH where hydrophobic interaction always exists
between non-polar portion of a protein and hydrophobic portion of solid surface
- - --- ---Latex surface
Zeta potential
BSA(+)
(-)
48 7432pH
(0)
+++ + ++
--+
++-
- - --- ---Latex surface
Zeta poten pH(0)
+++ + ++
--
tial
BSA(+)
(-)
48 7432
+
++-
ming
4
s
al blood Table 4-6 shows the calculated results of
Figure 4-23 Conformation of bovine serum albumin (BSA) on latex particles (Assuthat charge repulsion reduces the effect of the hydrophobic interaction at pH 74)
In addition the adsorbed amount of BSA at pH 32 was larger than that at pH 74
and still less than that at 48 This is because there is the electrostatic interaction between
the positive BSA molecules and negative PS latex particles at pH 32 and electrostatic
repulsion is dominant between negative BSA and negative PS latex particles at pH 7
The amount adsorbed increased with increasing ionic strength in pH 32 and 74 This i
because the high concentration of electrolytes reduces the electrostatic repulsion between
BSA and PS latex particle It should be noted that the BSA concentration used in these
experiments was lower than that found in normal human blood This is because the
equilibrium BSA adsorption onto the latex particles is actually reached at a much lower
concentration than that found in norm
93
adsor rm
ein
leading to a compact conformation of BSA There are two main forces affecting
adsorption amount at an acidic pH of 32 They include the ionic attraction between the
negative latex particle and the positive protein and lateral repulsion between positive
proteins The smallest amount of BSA adsorption was observed at a pH of 74 At this
point the charge repulsion between negatively charged particles and protein molecules
exists the h
of cha
bed BSA amount at equilibrium from the Langmuir-Freundich adsorption isothe
equation
In summary the hydrophobic interaction between PS latex particle and BSA
protein exists and is the dominant mechanism for the adsorption process Conformational
changes of BSA at each pH also affected the adsorption amount Adsorption was highest
at pH 48 because at this pH there is the least amount of ionic repulsion in the prot
and the lateral repulsion between proteins dominant adsorption even though there always
ydrophobic interaction between the solid latex particle and protein The effect
rge repulsion was reduced by the high concentration of electrolytes in PBS medium
Table 4-6 The equilibrium concentration values of BSA adsorption on polystyrene (PS) latex particles calculated the Langmuir-Freundlich isotherm model
Media pH qm (mgm2) 32 174 48 299 Phosphate buffer 74 136 32 260 48 283 Phosphate buffered saline 74 207
The adsorption isotherms of BSA on PSPMMA core shell particles are shown in
Figure 4-24 in phosphate buffer (PB) and in Figure 4-25 in phosphate buffered saline
(PBS) The adsorbed BSA amount was calculated using the Langmuir-Freundlich
100
94
adsorption isotherm equation and the amount listed in Table 4-7 The trend seen in the
adsorption results was very similar to the case of PS latex The overall adsorption process
was d rticle
rs
hat
0
ominated by hydrophobic interactions between the PSPMMA100 core shell pa
and BSA proteins At pH 48 the adsorbed amount (qm) of BSA on PSPMMA100 core
shell particles was 221 mgm2 in PB and 237 mgm2 in PBS and are the maximum
values recorded among the three pH levels 32 48 and 74 Suzawa and his co-worke
[1982] have obtained similar results This can be also explained by the compact
conformation of BSA molecules at the IEP of BSA which lead to a high protein
population on the solid surface At a pH of 32 and 74 BSA adsorption was less than t
at pH 48 because of the protein conformation change There was a small increase in
adsorbed BSA at pH 32 because of the charge attraction between negative PSPMMA10
core shell particles and positive BSA At pH 74 however the adsorption of BSA was
further reduced by charge repulsion between the negative PSPMMA100 core shell
particles and the negative BSA proteins
000 02 04 06 08
Ceq (mgml)
10
20q (m
gm
302 )
40
50pH=32pH=48pH=74Mode 8)l (pH 4Mode 2)l (pH 3Mode 4)
Figure 4-24 Adsorption isotherm of BSA 37oC in PB on PSPMMA core shell latex
l (pH 7
100
particles
95
The adsorbed amount of BSA increased more rapidly in PBS with increasing ioni
strength at pH 74 This shows that the low concentration of electrolytes promotes
electrostatic repulsion between negative BSA and negative PSPMMA
c
on
x
l 1980
100 core shell
particle As the ion strength of PBS is increased however the high concentration of
electrolyte leads to less electrostatic repulsion promoting more rapid protein adsorpti
By comparing the amount of adsorbed protein between bare PS latex particles and
PSPMMA100 core shell particles the amount of protein adsorption on the PS late
particles is greater than that on the PSPMMA100 core-shell particles [Suzawa et a
1982 Lee et al 1988]
00
10
20
30
40
m2 )
0 02 04 06 08
Ceq (mgml)
q (m
g
pH=32pH=48pH=74Model (pH 2) 3Model (pH 48)Model (pH 74)
Figure 4-25 Adsorption isotherm of BSA at 37oC in PBS on PSPMMA100 core shell
latex particles
96
Table particles calculated the Langmuir-Freundlich isotherm model
4-7 The equilibrium concentration values (qm) of BSA adsorption on PSPMMA100
Media pH qm (mgm ) 2
32 140
48 221 Phosphate buffer
74 130
32 169
48 237 Phosphate buffered saline
74 162
The BSA adsorption mechanism and trend on PSPMMA core shell particles was
very similar to that of the PS latex particle Unlike the case of PS and PSPMMA100 latex
particles the amount of BSA adsorbed onto PSPMMA90PAA10 and PSPMMA75PAA25
core shell particles was higher at pH 32 than pH 48 and 74 Figure 4-26 through 4-29
shows the adsorption isotherms of BSA on PSPMMA90PAA10 and PSPMMA75PAA25
core shell particles The amount of BSA adsorbed increased at pH 32 and pH 48 as the
hydrophilicity was developed by the addition of PAA On the contrary the adsorbed
amount of BSA was dramatically decreased at pH 74 The adsorption amount of BSA on
PSPMMA90PAA10 and PSPMMA75PAA25 core shell particles at equilibrium is listed in
Table 4-8 and 4-9 respectively
Carboxylic acid as well as sulfate groups are distributed on the surface of
PSPMMA90PAA10 and PSPMMA75PAA25 core shell particles and seem to largely affect
the BSA adsorption The isotherms showed very strong pH dependence to the BSA
adsorption onto these core shell latex particles The carboxylic groups (COOH) may
especially enhance the BSA adsorption prominently at pH 32 by hydrogen bonding with
protein molecules The pKa value for the PAA is 40-60 and a good indication that the
97
carboxylic acid groups are protonated at pH 32[Gebhardt et al 1983] The main
adsorption interaction force at pH 32 was generated by hydrogen bonding as well as
charge attraction between latex particle protein even th the conformational
repulsion between proteins results in a reduction of the protein
adsorption at this pH The amount of BSA adsorbed also increased as the PAA amount in
eased The a tion of BSA w inimal however less
than 07 mgm2 at pH 74 Here the carboxylic groups are ionized which increases the
charge repulsion by ionized carboxylic groups (COO-) resulting in PSPMMA-PAA latex
particle
and ough
change and charge
the core shell particles incr dsorp as m
s with much lower protein adsorption
00
20
40
q (m
g2 )
60
m
pH=32pH=48pH=74Model (pH 32)Model (pH 48)
0 02 04 06 08
Ceq (mgml)
Model (pH 74)
o shell
Figure 4-26 Adsorption isotherm of BSA at 37 C in PB on PSPMMA90PAA10 corelatex particles
98
00
40
60
80
0 02 04
C (mgm
mm
2 )
20
06 08
eq l)
q (
g
pH=32pH=48pH=74Model (pH 32)Model (pH 48)Model (pH 74)
Figure 4-27 Adsorption isotherm of BSA at 37oC in PBS on PSPMMA90PAA10 core
shell latex particles
Table 4-8 The equilibrium concentration values of BSA adsorption on PSPMMA90PAA10 particles calculated the Langmuir-Freundlich isotherm model
Media pH qm (mgm2)
32 361
48 283 Phosphate buffer
74 043
32 519
48 226 Phosphate buffered saline
74 043
99
00
20
40
60
80
100
0 02 04 06 08Ceq (mgml)
q (m
gm
2 )
pH=32pH=48pH=74Model (pH 32)Model (pH 48)Model (pH 74)
oFigure 4-28 Adsorption isotherm of BSA 37 C in PB on PSPMMA75PAA25 latex particles
00
100
20
40
60
80
0 02 04 06 08Ceq (mgml)
q (
m2 )
pH=32pH=48pH=74
mg
Model (pH 32)Model (pH 48)Model (pH 74)
Figure 4-29 Adsorption isotherm of BSA 37oC in PBS on PSPMMA75PAA25 core shell
latex particles
100
Table 4-9 The equilibrium concentration values of BSA adsorption on PSPMMA75PAA25 particles calculated the Langmuir-Freundlich isotherm model
Media pH qm (mgm2)
32 1083
48 662 Phosphate buffer (PB)
74 075
32 1077
48 681 Phosphate buffered saline (PBS)
74 057
The adsorption of β2-microglobulin (β2M) was determined under one set of
onditions in phosphate buffer at 37oC and pH 74 because of the limited amount of β2M
protein The adsorption isotherms of the β2M protein on the synthesized latex particles
are shown in Figure 4-30 4-31 and 4-32 The β2M protein concentrations for adsorption
isotherm experiment were chosen to be 0015 0030 0045 and 0060mgml since the
β2M concentration for the patients with renal failure can be elevated to similar
concentrations about 005mgml or more up to 50 times the normal level [Nissenson et
al 1995] There were steep initial slopes of the adsorption isotherm curves of β2M on
latex particles in all isotherms indicating a high affinity adsorption type It seems that the
hydrophobic interaction is enough for the adsorption process of β2M proteins to occur on
the latex particles even though charge repulsion exists between protein and latex particle
at pH 74 The complete plateau regions were not seen for all isotherms indicating that
β2M proteins were not saturated at this level and there are still available sites for β2M
c
adsorption on latex particles The initial slope of the β2M isotherms for PS was steeper
101
than that of any of the other latex particle PSPMMA100 PSPMMA90PAA10 and
PSPMMA 75PAA25
00
02
0 0002 0004 0006 0008
04
06
10
Ceq (mgml)
q (m
2 )
08
gm
PSPSPMMA100Model (PS)Model (PSPMMA100)
Figure 4-30 Adsorption isotherm of β2M onto PS and PSPMMA100 latex particles in PB
at 37 C and pH 74 o
00
02
04
06
08
10
0 0002 0004 0006 0008C (mgml)
q (m
m2 )
eq
g
PS
PSPMMA90PAA10
Model (PS)
Model (PSPMMA90PAA10)
Figure 4-31 Adsorption isotherm of β2M onto PS and PSPMMA90PAA10 latex particles
in PB at 37oC and pH 74
102
00
02
04
06
08
10
0 0002 0004 0006 0008Ceq (mgml)
q (m
gm
2 )
PS
PSPMMA75PAA25
Model (PS)Model (PSPMMA75PAA25)
Figure 4-32 Adsorption isotherm of β2M onto PS and PSPMMA75PAA25 latex particles
in PB at 37oC and pH 74
The equilibrium concentration values (qm) of β2M adsorption on latex particles are
calculated and listed in Table 4-10 The difference between the maximum amounts of
β2M adsorption on all the latex particles was not significantly different and their values
were between 069 and 08mgm2
Table 4-10 The equilibrium concentration values of β2M adsorption calculated the Langmuir-Freundlich isotherm model
Latex particles qm (mgm2)
PS 080
PSPMMA100 069
PSPMMA90PAA10 072
PSPMMA75PAA25 072
103
432 Adsorbed Layer Thickness
There are several possible explanations for large amount of BSA adsorbed at acidic
pH levels including the end-on adsorption pattern of BSA the flexibility of BSA
molecules the tilting of protein molecules due to the asymmetry of the charge
distribution or multilayer formation [Peula et al 1993] The adsorbed BSA thickness (δ)
can be calculated by the following equation [Chiu et al 1978] This equation was also
cited again by other researchers [Shirahama et al 1985]
BSA
mqρπ
δbullbull
=33
(4-7)
where π
33 is the packing factor qm is the plateau value of the adsorbed am
ρBSA is the hich corresponds to the reciprocal of its known
partial specific volume The layer thickness (δ) of BSA proteins on synthesized latex
partic
he IEP [Shirahama et al 1985] Compared to the
hydrodynamic dimensions of BSA 140 times 40 times 40 Aring3 [Fair et al 1980 Peter 1985] these
nd
ode It is unnecessary ultilayering because thickness do
exceed 140 Aring The layer thickness (δ) of BSA proteins on synthesized latex particles
e red saline (PBS) was ran 28 ~ 83 Aring at 37oC and pH 48 These
lso listed in Table 4-1
ount and
density of BSA molecule w
les in phosphate buffer (PB) ranged from 27 - 81 Aring at 37oC and pH 48 These
calculated values are listed in Table 4-11 Only δ values at pH 48 the isoelectric point
(IEP) of BSA are important because intra- and intermolecular electrostatic repulsion of
protein molecules is minimized at t
values of layer thickness indicate that the BSA molecules adsorb by side-on (40Aring) a
end-on (140Aring) m to consider m
not
in phosphat buffe ged of
calculated values are a 2
104
Table 4-11 Absorbed BSA layer thickness (Aring) of BSA in phosphate buffer (PB) at 37oC pH PS PSPMMA100 PSPMMA90PAA10 PSPMMA75PAA25
32 21 17 44 133
48 37 27 35 81
74 17 16 5 9
Table 4-12 Absorbed BSA layer thickness (Aring) of BSA in phosphate buffered saline (PBS) at 37oC
pH PS PSPMMA100 PSPMMA90PAA10 PSPMMA75PAA25
32 32 21 64 132
48 35 29
74 26 20 5 7
28 83
1n
1n
neqm
So k is closely related to the initial slope of the isotherm showing the affinity
between BSA and latex particles [Yoon et al 1996] A key point in the characterization
433 Gibbs Free Energy of Protein Adsorption
In the Langmuir isotherm model k represents the ratio of adsorption to the
desorption rate constants This definition is not applicable to the Langmuir-Freundlich
isotherm model but its meaning is similar [Yoon et al 1996] In this sense k is closely
related to the affinity between protein molecules and latex particles When the
equilibrium concentration Ceq reaches zero the value of Ceq will be very small (1gtgt
kCeq ) and then the equation 4-6 becomes
(4-8)
of adsorption processes on solid-liquid interfaces is to determine the change in Gibbsrsquos
free energy (∆Gdeg) one of the corresponding parameters for the thermodynamic driving
forces during adsorption The traditional method to evaluate adsorption thermodynamic
kCqq 1=
105
constants is based on measurement of adsorption isotherms under static conditions
[Malm ten 1998] The Langmuir-Freundlich ads lf
easily found from Then this enables calculation of the free-
energy change ∆G during adsorption expressed as
(4-9)
where R is the general gas constant (R = 8314Jmiddotmol K ) and T is the absolute
tem ture Gibb e energy A P
listed in Table 4-13 and 4-14 respectively As the ∆Gdegads ore negativ e
pro adsorption on a solid substra ill be more favorable
e of BSA adsorption in phosphate o
pH PS PSPMMA100 PSPMMA90PAA10 PSPMMA75PAA25
s orption equilibrium constant k can be
a slope of the isotherm
degads
lfads kRTG lnminus=∆ o
-1 -1
pera s fre change of BS adsorption values in B and PBS are
values are m e th
tein te w
Table 4-13 The values of Gibbs free energy changbuffer (PB) at 37 C
32 -2411 -2726 -2846 -2805
48 -2352 -2383 -2198 -1972 ∆Go
80
ads (kJmol)
74 -2452 -888 -324 -2
Table 4-14 The values of Gibbs free energy change of BSA adsorption in phosphate
pH PS PSPMMAbuffered saline (PBS) at 37oC
100 PSPMMA90PAA10 PSPMMA75PAA25
32 -2315 -2620 -2312 -2683
48 -2149 -2299 -2090 -2138 ∆Go
(kJmads ol)
74 -2430 -1992 -258 -263
Table 4-15 shows the calculated values of Gibbs free energy change of β M
adsorption on synthesized latex particles PSPMMA PAA core shell latex particle has
2
75 25
106
largest negative Gibbs free energy of β2
2
M adsorption indicating largest affinity betw
β
een
2M adsorption in phosphate buffere (PB) at 37 C and pH74
PS PSPMMA100
M and latex particle
Table 4-15 The values of Gibbs free energy change of βo
PSPMMA90PAA10 PSPMMA75PAA25
∆Go03 ads
(kJmol) -3626 -3466 -3659 -39
The Gibbs free energy of β2M was much more negative in phosphate buffer (PB)
than that of BSA in physiological condition (37 C and pH 74) o from the Table 4-13 and
the Figure 4-31 This indicates that β2M molecules more readily adsorb on latex particles
BSA This can be explained by the rate of diffusion and accessibility of β2M molecules to
the latex particle ier and faster for β2M m an BSA because of
size (molec ight) erical shape molecules It o be
due to the higher hydrophobic force and less electrostatic repulsion that contribute to the
adsorption of β2M molecules not as prevalent in the case of BSA because the IEP of β2M
(57) is clo H 74) than that of BSA (48) causing β2M to
be less electro cally lled p ro
can be seen that the ∆ β2M adsorption onto the synth article
increasing wi hy icity o x particles s the ∆Goads of the BSA
molecules is decreasing or constant This phenomenon supports the theory that the
select lizing
than BSA and that the affinity of β2M for the latex particle is much larger than that of
surface It is eas olecules th
the smaller ular we and sph of β2M may als
ser to physiological condition (p
stati repe from the latex articles than BSA F m Figure 4-33 it
Goads of esized latex p s is
th the drophil f the late wherea
ive adsorption of β2M molecules over BSA molecules can be enhanced by uti
more hydrophilic PSPMMA90PAA10 and PSPMMA75PAA25 core shell latex particles
107
-60
-50
1 2 3 4
-40∆Gad
s
-30
10
0
o (k
Jm
o-
-20l)
β2-Microglobulin (β2M)Bovine serum albumin (BSA)
Figure 4-33 Gibbs free energy of adsorption of proteins on latex particles in PB at 37
and pH 74
434 Kinetics of Adsorption
to
oC
Protein adsorption is a complex process known to have a large biological impact
and is currently not well understood quantitatively because of extreme sensitivity to pH
the concentration of other electrolytes and molecules present temperature and many
other factors that can change in the physiological system Accurate knowledge of the
adsorption kinetics under a given set of conditions is a prerequisite for elucidating the
mechanisms of many fundamental biological processes to be predicted at the molecular
level [Regner 1987] This adsorption kinetics approach is used in protein adsorption
studies because of the uncertainty related to the time needed for the interfacial proteins
reach the equilibrium It is generally accepted that the process of protein adsorption is
comprised of the following steps a) transport toward the interface b) attachment at the
interface c) eventual structural rearrangements in the adsorbed state d) detachment from
PS PSPMMA100 PSPMMA90PAA10PSPMMA75PAA25
108
the interface and transport away from the interface Each of these steps can affect the
overall rate of the adsorption process
The quantitative analysis of the protein adsorption kinetics requires that the protein
amount adsorbed is known as a function of time The kinetics tests were carried out in
phosphate buffer (PB) at 37oC and pH 74 a condition similar to physiological condition
The protein concentrations chosen were 07mgml for BSA and 006mgml for β2M
which are the maximal concentrations used in an adsorption isotherm test A time-based
evaluation of the adsorption was made by measuring the concentration of protein in
solution at different incubation times The results are shown below from Figure 4-34 to 4-
37 The adsorption kinetics of proteins onto th cond-e latex particles is expressed as a se
order model [Oumlzacar 2003] shown by the following equation
2)( tet qqk
dtdq
minus= (4-10)
Integrating above equation and applying the boundary conditions yields
ee
t tq
+=
1 (4-11)
where q
qkq
t
2
2
2
the previous adsorption isotherm results representing good reproducibility of adsorption
t and qe stand for the amount (mgm ) of protein adsorbed at time t and at
equilibrium respectively k is the equilibrium rate constant of second-order adsorption
(m mghr) and t is the incubation time (hr)
The fitting parameters calculated by using the equation 4-11 are listed in table 4-16
From this data it is seen that the amount of BSA adsorbed at equilibrium was higher than
that of β2M on PS and PSPMMA100 latex particles but less than that of β2M on
PSPMMA90PAA10 and PSPMMA75PAA25 latex particles These results are similar to
109
00
05
15
20
0 2 4 6 8 10 12 14
Time (hr)
rot
n ad
sorb
d (
10
25
Pei
em
gm
2
BSA2nd order model (BSA))β2M2nd order model (β2M)
Figure 4-34 The kinetics of protein adsorption in PB on PS latex particles at 37oC and
pH 74
00
05
10
15
20
25
30
0 2 4 6 8 10 12 14
Time (hr)
Prte
inds
oo
arb
ed (
mg
m2 )
BSA2nd order model (BSA)β2M2nd order model (β2M)
Figure 4-35 The kinetics of protein adsorption in PB on PSPMMA100 latex partic
37les at
oC and pH 74
110
00
03
06
09
12
15
0 2 4 6 8 10 12 14
Time (hr)
Prot
ein
adso
rbed
(m
gm
2 ) BSA2nd order model (BSA)β2M2nd order model (β2M)
Figure 4-36 The kinetics of protein adsorption in PB on PSPMMA90PAA10 latex
particles o at 37 C and pH 74
00
03
06
09
12
15
0 2 4 6 8 10 12 14
Time (hr)
BS
A ad
sorb
ed (
mg
m2 )
BSA2nd order model (BSA)β2M2nd order model (β2M)
Figure 4-37 The kinetics of protein adsorption in PB on PSPMMA75PAA25 latex
particles at 37oC and pH 74
111
test The variation of β2M equilibrium adsorption amount was nearly negligible for all
latex particles The plateau region is reached earlier for β2M than BSA on all latex
particles and the K values (equilibrium rate constant of second order adsorption) for the
β2M adsorption process onto latex particles are much larger than for BSA adsorption
These values indicate that the β2M molecules have a higher adsorption rate onto the latex
particles than the BSA molecules
Table 4-16 Fitting parameters of second-order kinetic model BSA β2M
Latex particles qe (mgm2) K (hr-1) qe (mgm2) K ( -1) hr
PS 144 67 074 7549
PSPMMA100 175 353 076 4438
PSPMMA90PAA10 046 97 075 5695
PSPMMA75PAA25 060 179 075 5168 K = (kqe) hr-1
There are several explanations for the phenomena observed in these experiments
First the smaller sizes of β2M molecules allow easier diffusion and a close approach to
the latex substrate Second a reduced number of hydrophobic domains on the latex
particle surface replaced by sulfate groups (-OSO3-) and ionized carboxylic groups
(COO-) are not favorable for larger BSA molecules Third the conformational change by
intra- and inter-molecular repulsion would be faster in β2M due to smaller molecular
weight Finally the ionic repulsion between β2M and the latex particles is less than that
condition (pH 74) than IEP (48) of BSA
between BSA and latex particles because IEP (57) of β2M is closer to the tested
112
44 Blood Compatibility
Blood compatibility can be defined as the property of a material or device tha
permits it to function in contact with blood without inducing any adverse reactions
Successful functioning of blood contacting biomedical materials or devices in contact
with blood strongly depends on the extent to which they damage blood during operation
High shear stresses turbulence rela
t
tively long contact times and cellular impact with
artificial surfaces are most common events experienced by blood in contact with artificial
1978 Winslow and et al
1993] Therefore before any materia e placed into the human b
biocompatibility testing must be the first performed in the view of safety The hemolysis
s a method for pr ary screening of biomaterials to determine
h blood a er circulating body fluid The tests estimat
oglobin released from th blood cell d e induced
foreign material
There are several types of blood cells in our body but the ones that make blood red
are erythrocytes or red blood cells (RBCs) Red blood cells (erythrocytes) are the most
numerous type in the blood These cells are average about 48-54 million per cubic
millimeter (mm3 same as a microliter (microl)) of blood but these values can vary over a
larger range depending on such factors as health and altitude RBCs are red because they
contain hemoglobin which is a protein that contains iron When blood circulates through
lungs oxygen binds to the hemoglobin and is then delivered to all the parts of body
RBCs are made in the bone marrow They start out as a nucleus like other cells but
when they fill up with hemoglobin the nucleus becomes smaller until it disappears
Without a nucleus RBCs are fragile and live only about 120 days In a normal healthy
devises and can cause hemolysis or thrombosis [Bernhard et al
ls can b ody
test serves a elimin the
biocompatibility wit nd oth e the
concentration of hem e red amag by a
113
body about 2 million red blood ne marrow produces new
ones j ing
dy
le
cells die per second But bo
ust as fast RBCs are about 75 micrometer across and are responsible for carry
oxygen salts and other organic substances to the cells and tissues throughout our bo
In humans and most mammals the cells are concave (indented in the middle) and flexib
enough to squeeze through even the tiniest of blood vessels the capillaries
Figure 4-38 Image of red blood cells
(Sourcehttpwwwpbrchawaiiedubemfmicroangelarbc1htm)
ch
100 90 10 75 25 f
RBCs are among the first cells that come into contact with a foreign material
injected in the blood Therefore a hemolysis assay gives information about the
biocompatibility in the case of an in vivo application The concentrations of prepared
latex particles used for the tests in this research are 05 25 and 50 (ww) and the
incubation time under moderate shaking was 30 minutes at 37oC Test results are shown
in Figure 4-39 The amount of blood cells that ruptured when in contact with the PS latex
particles was largely increased from about 30 to 91 as the solid content of PS
increased from 05 to 50 However hemolysis by the core shell latex particles su
as PSPMMA PSPMMA PAA and PSPMMA PAA was less than 35 O
114
particular interest was that hemolysis caused by PSPMMA PAA particles at 50
solid concentration was less than 02 hemolysis the lowest value among the latex
particles The low hemolysis value indicates that the PSPMMA PAA core shell latex
particles are the most blood compatible at all solid contents Other core shell particles
such as PSPMMA and PSPMMA PAA also had excellent blood comp
90 10
90 10
100 75 25 atibility with
low RBS breakage
01 2 3
20
40
60
80
100
Hem
olys
is (
)
052550
Figure 4-39 He
PS PSPMMA100 PSPMMA PAA90 10
molysis caused by latex particles n=5
4PSPMMA75PAA25
CHAPTER 5 SUMMARY OF RESULTS CONCLUSIONS AND RECOMMENDATION FOR
FUTURE WORK
51 Summary of Results
The goal of this study was to synthesize monodisperse polymeric latex particles
with tailored properties and to investigate the fundamental interactions between the
synthesized latex particles and target proteins An understanding of the fundamental
mechanism of selective adsorption is strongly required in order to maximize the
separation performance of membranes made of these materials so that it may applicable
to hemodialysis therapy for end stage renal disease (ESRD) patients For the achievement
of above research goal several investigations have been performed Analysis of the
obtained data leads to several conclusions as discussed below
Monodisperse polystyrene seed latex particles were synthesized with the size
crosslinking agent to make the latex particles more hydrodynamically stable The
conversion of monomer to polymer was over 95 and seed latex particles are spherical in
shape and have smooth surface From these small seeds bigger latex particles as large as
800nm were synthesized using a semi-continuous seeded emulsion polymerization
process The particles with a mean diameter less than 500nm are very spherical in shape
and highly uniform in particle size distribution However as the particles grow larger
than 500nm in mean diameter they became non-spherical with an uneven surface This
irregularity of particle surface can be attributed to the non-homogeneous monomer
ranges between 126plusmn75 and 216plusmn53 nm Divinylbenzene (DVB) was used as a
115
116
swelling into the shell of a growing polymer which can be controlled by the factors such
as temperature agitation speed initiator feeding rate and surfactant amount required to
stabilize the colloidal system It was determ rfactant to monomer ratio
dec ic
acid (AA) monomers were intro rticle surfaces more
hydro
to
ting of a
orm
ration is seen between
3002
0 cm-
0cm-1
-1
03
ined that as the su
reased the mean particle diameter increased Methyl methacrylate (MMA) and acryl
duced to make the latex pa
philic than bare polystyrene particles The prepared core shell latex particles were
made of PMMA100 PMMA90PAA10 and PMMA75PAA25 PMMA100 is the PMMA
homopolymer shell on a PS core PMMA90PAA10 shell is a copolymer with a PMMA
PAA ratio of 90 to 10 by weight PMMA75PAA25 shell is a copolymer consis
PMMA to PAA ratio of 75 to 25 by weight The particle size of these core-shell
particles were prepared to be about 370nm in mean diameter
The successful synthesis of latex particles was confirmed using Fourier Transf
Infrared spectroscopy (FTIR) The C-H aromatic stretching vib
cm-1 and 3103 cm-1 for PS latex particles The C-H asymmetrical stretching and
symmetrical stretching vibration peaks of CH2 for all latex particles is seen at 290
1and 2850 cm-1 respectively There is a carbonyl (C=O) characteristic band at 173
wavenumber for PSPMMA core-shell latex particles The broad OH group peak at
3400 cm
100
can be seen in the spectroscopy of the particle with PMMAPAA shell
(PSPMMA PAA and PSPMMA PAA ) The peak intensity of OH group of
PSPMMA PAA was larger than that of PSPMMA PAA relatively due to more AA
monomer content in PSPMMA PAA than in PSPMMA PAA
In the zeta potential measurements polystyrene (PS) latex particles had negative
values between -291 mV and -599 mV in the phosphate buffer (PB) and between -2
90 10 75 25
75 25 90 10
75 25 90 10
117
mV and -278 mV in the phosphate buffered saline (PBS) at 25oC and pH 21-78 These
negative zeta potential values for PS latex particles are due to ionization of the sulfate
groups (-OSO3-) originated from the initiator Zeta potential values of the PSPMMA
core shell latex particles were also negative between -28 mV and -505 mV in PB and
between -143 mV and -186 mV in PBS at pH 21-78 Negative values are also
generated by dissociation of sulfate groups on a particle surface The zeta potential value
of PSPMMA PAA and PSPMMA PAA were of negative values between -36
and -678 mV in PB medium and -147 mV and -193 mV in PBS medium at 25
100
s
7 mV
MMA75PAA25 and
PSPM
otein
g the
l change of BSA at
each w
90 10 75 25
oC For
PSPMMA PAA core shell particles zeta potential values were between -291 mV and
-520 mV in PB media and between -115 mV and -210 mV in PBS media Sulfate
groups contributed to the negative zeta potential values in PSP
75 25
MA90PAA10 core shell particles Carboxylic groups from polyacrylic acid also
contribute to the negative surface charge for the latex particles at greater than pH 70
however the degree of their contribution was not detectable from the zeta potential
measurements The absolute zeta potential values of the synthesized latex particles in PB
were higher than that in PBS This is because of the high concentration of sodium
chloride electrolyte in PBS that compress electrical double layer thickness
The bicinchoninic acid (BCA) assay technique was used to determine the pr
adsorption and all isotherms were fitted to the Langmuir-Freundlich isotherms usin
nonlinear regression method The hydrophobic interaction between PS latex particle and
BSA protein was dominant for the adsorption process Conformationa
pH also affected the adsorption amount which was highest at pH 48 because of lo
ionic repulsion making compact conformation of BSA The next larger amount of BSA
118
adsorbed onto PS was at pH 32 There were two main forces affecting the amount of
adsorption at acidic pH values One is ionic attraction between negative latex particle a
positive protein The other is lateral repulsion between positive proteins The smallest
amount of BSA adsorption was at pH 74 where charge repulsion between negative
particles and proteins is present at this pH Lateral repulsion between proteins wa
dominant for protein adsorption even though there always exists the hydrophobic
interaction between solid latex particle and protein The charge repulsion was reduced by
the high concentration of electrolytes in the PBS medium The BSA adsorption process
on PSPMMA core shell particles was very similar to the case of PS latex particle where
hydrophobic interaction between latex particle and protein was dominant However t
adsorbed BSA amount was less than that of PS in PBS The adsorbed amount of BS
PSPMMA
nd
s
he
A on
an
ed
n The main
adsor
90PAA10 and PSPMMA75PAA25 core shell particles was higher at pH 32 th
pH 48 and 74 The adsorbed BSA amount became higher at pH 32 and pH 48 as the
hydrophilicity was increased by the higher PAA amount On the contrary the adsorb
amount of BSA was dramatically decreased at pH 74 Carboxylic acid groups as well as
the sulfate groups are distributed on the surface of PSPMMA90PAA10 and
PSPMMA75PAA25 core shell particles and largely affect the BSA adsorptio
ption interaction forces at pH 32 was generated by hydrogen bonding as well as
charge attraction between latex particle and protein even when the conformational change
and charge repulsion between proteins reduce the adsorption of protein at this pH The
adsorbed BSA amount was also increased as the amount of PAA increases However the
adsorption of BSA was minimal (less than 07 mgm2) at pH 74 where the carboxylic
groups are ionized leading to more enhancement of charge repulsion by ionization of the
119
carboxylic groups (COO-) Because of this the PSPMMA-PAA latex particles have a
much lower protein adsorption
There were steep initial slopes for adsorption isotherms of β
in
s
there
M
r
e
es
o
2M on latex particles
all isotherms indicating a high affinity type adsorption The complete plateau region
were not seen for all isotherms indicating that β2M proteins were not saturated and
are still available sites for β2M adsorption on latex particles The initial slope of β2
isotherms for PS was steeper than for the other latex particles PSPMMA100
PSPMMA90PAA10 and PSPMMA75PAA25
The layer thickness (δ) of BSA proteins on synthesized latex particles in phosphate
buffer (PB) was in the range of 27 - 81 Aring at 37 C and pH 48 Only the δ values at pH
48 the isoelectric point (IEP) of BSA are important because intra- and inter-molecular
electrostatic repulsion of the protein molecules is minimized at the IEP Compared to the
hydrodynamic dimensions of BSA 140 times 40 times 40 Aring these values of the adsorbed laye
thickness indicates that the BSA molecules adsorb by the side-on (40Aring) and end-on mod
(140Aring) Consideration of the multi-layering is unnecessary because the adsorbed
thickness of BSA do not exceed 140 Aring
The Gibbs free energy of β
o
3
2M was more negative in phosphate buffer (PB) than
that of BSA in physiological condition (37 C and pH 74) indicating that βo2M molecul
are more favorably adsorbed on the latex particles than BSA and that the affinity of β2M
was much larger than BSA This may be explained by the rate of diffusion and
accessibility of β2M molecules being easier and faster than that for BSA because of
smaller size (or weight) and spherical shape of β2M molecules It may also be possible t
explain that more hydrophobic forces (less electrostatic repulsion) contributes to
120
adsorption of β2M than to that of BSA because the IEP of β2M (57) is closer to
physiological condition (pH 74) than that of BSA (pH 48) The selective adsorption of
β2M m
in β2M
larger than in BSA adsorption indicating
that fa
an that between BSA and latex particles because
IEP (
ch as
Other
olecules over BSA molecules can be enhanced by developing more hydrophilic
PSPMMA90PAA25 and PSPMMA75PAA25 core shell latex particles
The adsorption kinetics of proteins on latex particles was expressed as a second-
order model The plateau region for β2M was reached earlier than BSA on all latex
particles and the K values (equilibrium rate constant of 2nd order adsorption)
adsorption process on latex particles were much
ster adsorption rate of β2M molecules than that of the BSA molecules There are
several explanations for these phenomena First the smaller size of β2M molecules can
more easily diffuse and approach to the latex substrate Second enhanced hydrophilic
domain on the latex particle surface by sulfate groups (SO4-) and ionized carboxylic
groups (COO-) is not favorable for larger BSA molecules but is for smaller β2M to be
anchored Third the conformational change by intra- and inter-molecular repulsion
would be faster in β2M due to lower molecular weight Finally the ionic repulsion
between β2M and latex particles is less th
57) of β2M is closer to the tested condition (pH 74) than IEP (48) of BSA
In the biocompatibility test the number of red blood cells (RBCs) ruptured by PS
latex particles ranged from 30 to 91 as the solid content of PS increased from 05 to
50 respectively However hemolysis of the other core shell latex particles su
PSPMMA100 PSPMMA90PAA10 and PSPMMA75PAA25 were less than 35 The
PSPMMA90PAA10 particles at all applied solid concentrations had less than 02
hemolysis and was the most blood compatible among the prepared latex particles
121
core shell particles such as PSPMMA100 and PSPMMA75PAA25 also had excelle
compatibility with low RBC breakage
52 Conclusions
The major conclusions from this study include
nt blood
bull microglobulin (β2M) over that of the bovine serum albumin (BSA) at pH 74
was achieved by controlling the PMMA and PAA ratio to optimize
e particles posses the most desirable requirements for use in a human dialysis
ns
icles
olecule
bull
Using engineered core shell latex particles selective adsorption of β2
physiological condition was demonstrated
bull The rate of adsorption of the β2M was also much higher than that of BSA This
hydrophobichydrophilic microdomain size
bull The core shell particles also exhibited excellent biocompatibility with blood Thes
membrane
53 Recommendation for Future Work
With the results and conclusions obtained through this study several suggestio
may be given for future work as follows
bull Competitive adsorption tests of whole blood proteins on synthesized latex partunder physiological condition
bull Preparation of latex particles with optimum hydrophobic hydrophilic microdomains to maximize selective adsorption and removal of toxin molecules
bull Membrane fabrication by suitable pore size design to discriminate a toxin mby size sieving as a function of particle layers
Evaluation of membrane performance such as flow rate in water and in simulated blood fluid (SBF)
bull Application for hemodialysis as well as other technologies such as a water remediation
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al of Medicine 21 888-892
s
ilure 25 419-430
o
of protein using bicinchoninic acid Analytical Biochemistry 150 76-85
Smith on polymerization Journal of Chemical Physics 16 592-599
histocompatibility antigen HLA-A2 at 26Ǻ resolution Journal of Molecular Biology 219 277-319
chloromethyl functionality Colloids and Surfaces a-Physicochemical and Engineering Aspects 135 293-297
(1998) Functionalized monodisperse particles with chloromethyl groups for thcovalent coupling of proteins Macromolecules 31 4282-4287
and microfiltration Desalination 139 191-198
3 673 133
Hatakeyama M Ohba R Hatori H Yoshikawa T Suzuki F Oomori A Tanaka H Kawaguchi H Watan
18 877-881
acrylic acid copolymer latex Colloid and Polymer Science 263 141-146
hama H Suzuki K and Suzawa T (1989) Bovine hemoglobin adsorption opolymer lattices Journal of Colloid and Interface Science 129 4
Singer J M and Plotz C M (1956) Latex Fixation Test 1 application to the serologicdiagnosis of rheumatoid arthritis American Journ
Singh N P Bansal R Thakur A Kohli R Bansal R C and Agarwal S K (2003)Effect of membrane composition on cytokine production and clinical symptomduring hemodialysis a crossover study Renal Fa
Smith P K Krohn R I Hermanson G T Mallia A K Gartner F H ProvenzanM D Fujimoto E K Goeke N M Olson B J and Klenk D C (1985) Measurement
W V and Ewart R H (1948) Kinetics of emulsi
138
Snyder H W Cochran S K Balint J P Bertram J H Mittelman A Guthrie T Hand Jones F R (1992) Experience with protein α-immunoadsorption in treatmentresistan
t adult immune thrombocytopenic purpura Blood 79 2237-2245
Solomon T W Fryhle C B (2000) Organic chemistry 7th Ed John Wiley amp Sons
Song Y Q Sheng J Wei M and Yuan X B (2000) Surface modification of polysulfone membranes by low-temperature plasma-graft poly(ethylene glycol)
Sriniv characterizations of polymer with thier antithrombogenic characteristics Marcel Dekker New York
Suzaw ovine serum albumin on synthetic polymer lattices Journal of Colloid and Interface Science 78 266-268
Suzawa T Shirahama H and Fujimoto T (1982) Adsorption of bovine serum albumin onto homo-polymer and co-polymer lattices Journal of Colloid and
Taken itness of
9 10-14
412
Tanfo New York
Tangp ion of chitosan films- effects of hydrophobicity on protein adsorption Carbohydrate
Trinh S E (2002) Crystal structure of monomeric human beta 2 microglobulin reveals clues to its
Trotta F Drioli E Baggiani C and Lacopo D (2002) Molecular imprinted polymeric membrane for naringin recognition Journal of Membrane Science 201
Soderquist M E and Walton A G (1980) Structural changes in proteins adsorbed on polymer surfaces Journal of Colloid and Interface Science 75 386-397
New York
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a T and Murakami T (1980) Adsorption of b
Interface Science 86 144-150
aka T Itaya Y Tsuchiya Y Kobayashi K and Suzuki H (2001) Fbiocompatible high-flux hemodiafiltration for dialysis-related amyloidosis BloodPurification 1
Tamai H Hasegawa M and Suzawa T (1989) Surface characterization of hydrophilic functional polymer latex particles Journal of Applied Polymer Science 38 403-
rd C (1967) Physical chemistry of macromolecules John Wiley amp Sons
asuthadol V Pongchaisirikul N and Hoven V P (2003) Surface modificat
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C H Smith D P Kalverda A P Phillips S E V and Radford
amyloidogenic properties Proceedings of the National Academy of Sciences of the United States of America 99 9771-9776
77-84
139
Tuncel A Tuncel M Ergun B Alagoz C and Bahar T (2002) Carboxyl carrying large uniform latex particles Colloids and Surfaces a-Physicochemical and Engineering Aspects 197 79-94
Ugelstad J Kaggerud K H Fitch R M (1980) Polymer colloid II Plenum New York
USRDS (2003) United State Renal Date System (USRDS) 2003 annual report
Van Noordwijk J (2001) Dialyzing for life the development of the artificial kidney
Vandpolymerisation of styrene from characterisation of polymer end groups Chemical
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1992)
f the American Society of Nephrology 11 2344-2350
38
anes by low temperature plasma induced graft polymerization Journal of Membrane Science 209 255-269
Westhssing with renalin on serum beta2 microglobulin and
complement activation in hemodialysis patients American Journal of Nephrology
Wiec
formation Analytical Biochemistry 175 231-237
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Ward R A Schmidt S Hullin J Hillebrand G F and Samtleben W (2000) A comparison of on-line hemodiafiltration and high-flux hemodialysis A prospectiveclinical study Journal o
Watkins R W and Robertson C R (1977) Total internal reflection technique for examination of protein sdsorption Journal of Biomedical Materials Research 11915-9
Wavhal D S and Fisher E R (2002) Hydrophilic modification of polyethersulfone membr
uyzen J Foreman K Battistutta D Saltissi D and Fleming S J (1992) Effect of dialyzer reproce
12 29-36
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ms K M Arthur S J Burrell G Kelly F Phillips D W and Marshall T (2003)
140
Winchester J F Ronco C Brady J A Cowgill L D Salsberg J Yousha E Choquette M Albright R Clemmer J Davankov V Tsyurupa M PavlL Pavlov M Cohen G Horl W
ova Gotch F and Levin N W (2002) The next
step from high-flux dialysis application of sorbent technology Blood Purification
Winchester J P Ronco J A Brendolan A Davankov V Tsyurupa M Pavlova L 2001)
oglobin
Yang Q and Yang C Z (2000) Formation of an
Yoon J Y Park H Y Kim J H and Kim W S (1996) Adsorption of BSA on highly carboxylated microspheres- quantitative effects of surface functional groups
Zollars R L (1979) Kinetics of the emulsion polymerization of vinyl acetate Journal of
Zou oss-
Zou D Ma S Guan R Park M Sun L Aklonis J J and Salovey R (1992)
20 81-86
Clemme J Polaschegg H D Muller T E La Greca G Levin N W (Rationale for combined hemoperfusionhemodialysis in uremia Contribution of Nephrology 133 174-179
Winslow R M Vandergriff K D Motterlini R (1993) Mechanism of hemtoxicity Thrombosis and Haemostasis 70 36-41
S C Ge H X Hu Y Jiang Xpositively charged poly(butyl cyanoacrylate) nanoparticles stabilized with chitosColloid and Polymer Science 278 285-292
and interaction forces Journal of Colloid and Interface Science 177 613-620
Applied Polymer Science 24 1353-1370
D Derlich V Gandhi K Park M Sun L Kriz D Lee Y D Kim G Aklonis J J and Salovey R (1990) Model filled polymers 1 synthesis of crlinked monodisperse polystyrene beads Journal of Polymer Science Part A Polymer Chemistry 28 1909-1921
Model filled polymers 5 synthesis of cross-linked monodisperse polymethacrylate beads Journal of Polymer Science Part A Polymer Chemistry 30 137-144
Korea on August 7 1968 He obtained a Bachelor of Science degree in Chemistry from
Keim d graduate school and earned a
resear n Korea Institute of Science and
Coati e
ia
Busin
BIOGRAPHICAL SKETCH
Sangyup Kim was born and raised in the small town of Sung-Joo Kyungbook
yung University (Daegu Korea) in 1993 He entere
Master of Science degree in Chemical engineering in 1995 He worked as a student
cher for 2 years at Textile Polymer Division i
Technology (KIST) in Seoul Korea Next he worked over a year at the Automobile
ng Division in DongJoo-PPG in Korea In August 2001 he began studying for th
degree of Doctor of Philosophy in materials science and engineering at the University of
Florida in Gainesville After getting his PhD he plans to work at the Digital Med
ess Division of Samsung Electronics in Korea starting in January 2006
141
Copyright 2005
by
Sangyup Kim
To all who made this work possible
ACKNOWLEDGMENTS
I owe what I am today and this moment to many people without whom I could not
have been here First of all I would like to express my sincere and deepest appreciation
to my parents wife Mikyung daughters Heehyung and Erin whose love patience and
support helped me pursue my dream I gratefully acknowledge my advisor Dr H El-
Shall who gave me an opportunity to become part of an exciting and beneficial research
project and exert myself in the research work His guidance knowledge and stimulating
scientific and critical thinking have contributed to my success to a great extent and
inspired me to dedicate myself to research work I especially appreciate Dr R Partch as a
co-advisor who has helped me conduct experiments with valuable discussion for my
research from my first year I am greatly thankful to Dr C McDonald whose idea and
valuable experimental advice and discussion in the area of polymer chemistry and
membrane technology helped cultivate my interest in this area and were vital to
performing this research Without his contribution this work could not have been
achieved I also thank my doctoral committee members Dr R Singh Dr A Zaman and
Dr B Koopman for appreciated comments
Financial support and equipment use for research from the Particle Engineering
Research Center at the University of Florida were vital in continuing my study for more
than 4 years My sincere thanks are due to my former advisor Dr Kiryoung Ha in
Keimyung University who helped me open my eyes and broaden my thought and
introduced me into a limitless polymer field I would like to also thank to past and current
iv
members in Dr El-Shallrsquos research group for their assistance advice and support I
sincerely appreciate Stephen for the assistance of thesis writing
v
TABLE OF CONTENTS page
ACKNOWLEDGMENTS iv
LIST OF TABLES viii
LIST OF FIGURES x
1 INTRODUCTION 1
2 BACKGROUND AND LITERATURE SURVEY5
21 The Significance of End Stage Renal Disease (ESRD)5 22 Hemodialysis (HD) Treatment 6 23 Advances in Membrane Technology 8 24 Sorbent Technology16 25 Limitation of Current Hemodialysis Treatment 19 26 Latex Particles 20
261 The Components for Emulsion Polymerization 21 262 Particle Nucleation 21 263 Types of Processes for Emulsion Polymerization 23 264 Chemistry of Emulsion Polymerization 24 265 Seed Emulsion Polymerization25 266 Polystyrene Latex Particles 26 267 Various Applications of Latex Particles28
27 Proteins 30 271 Interaction Forces between Proteins33 272 β -Microglobulin (β M)2 2 35 273 Serum Albumin 36
28 Protein Adsorption38 281 Interaction between Protein Molecule and Latex Particle38
29 Hypothesis for Toxin Removal39 291 Toxin Removal by Size Sieving Based on Monodispersed Pore Size 40 292 Toxin Removal by Selective Adsorption on Engineered Latex Particles 41
3 EXPERIMENTAL AND CHARACTERIZATION METHODOLOGY 44
31 Materials 44 32 Latex Particle Preparation46
321 Preparation of Seed Latex Particles47
vi
322 Preparation of Seeded Latex Particles 48 323 Preparation of Core Shell Latex Particles 48 324 Purification of Synthesized Latex Particles48
33 Characterization49 331 Degree of Conversion49 332 Fourier Transform Infrared (FTIR) Spectroscopy49 333 Quasielastic Light Scattering (QELS) 50 334 Field Emission-Scanning Electron Microscopy (FE-SEM) 50 335 Zeta Potential Measurement 50 336 Protein Adsorption51 337 Blood Biocompatibility by Hemolysis Test 53
4 RESULTS AND DISCUSSION55
41 Polymerization of Latex Particles55 411 Polystyrene Seed Latex Particles55 412 The Growth of Polystyrene (PS) Seed Latex Particles61 413 Core Shell Latex Particles 71
42 Characterization of Latex Particles75 421 Fourier Transform Infrared Spectroscopy (FTIR)75 422 Zeta Potential Measurements 75
43 Protein Adsorption Study 85 431 Adsorption Isotherm87 432 Adsorbed Layer Thickness 103 433 Gibbs Free Energy of Protein Adsorption 104 434 Kinetics of Adsorption 107
44 Blood Compatibility 112
5 SUMMARY OF RESULTS CONCLUSION AND RECOMMENDATION FOR FUTURE WORK115
51 Summary of Results115 52 Conclusions121 53 Recommendation for Future Work121
LIST OF REFERENCES122
BIOGRAPHICAL SKETCH 141
vii
LIST OF TABLES
Table page 4-1 The polymerization recipe of polystyrene (PS) seed particles 57
4-2 Continuous addition emulsion polymerization recipe for growing polystyrene (PS) latex particles less than 500nm in size 63
4-3 Continuous addition emulsion polymerization recipe for growing polystyrene (PS) latex particles larger than 500nm in size64
4-4 The preparation recipe of PS core with various shell latex particles72
4-5 The isoelectric point (IEP) of common proteins 83
4-6 The equilibrium concentration values of BSA adsorption on polystyrene (PS) latex particles calculated the Langmuir-Freundlich isotherm model 93
4-7 The equilibrium concentration values (q ) of BSA adsorption on PSPMMA particles calculated the Langmuir-Freundlich isotherm model
m 10096
4-8 The equilibrium concentration values of BSA adsorption on PSPMMA PAA particles calculated the Langmuir-Freundlich isotherm model
90 1098
4-9 The equilibrium concentration values of BSA adsorption on PSPMMA PAA particles calculated the Langmuir-Freundlich isotherm model
75 25100
4-10 The equilibrium concentration values of β M adsorption calculated the Langmuir-Freundlich isotherm model
2102
4-11 Absorbed BSA layer thickness (Aring) of BSA in phosphate buffer (PB) at 37 Co 104
4-12 Absorbed BSA layer thickness (Aring) of BSA in phosphate buffered saline (PBS) at 37 Co 104
4-13 The values of Gibbs free energy change of BSA adsorption in phosphate buffer (PB) at 37 Co 105
4-14 The values of Gibbs free energy change of BSA adsorption in phosphate buffered saline (PBS) at 37 Co 105
viii
4-15 The values of Gibbs free energy change of β M adsorption in phosphate buffere (PB) at 37 C and pH 74
2o 106
4-16 Fitting parameters of second-order kinetic model 111
ix
LIST OF FIGURES
Figure page 1-2 Stereo drawing of the αndashcarbon backbone of β M2 2
2-1 An image of the location and cross section of a human kidney 5
2-2 A schematic draw of the hemodialysis route7
2-3 Cross-sectional SEM image view of the Polyflux S (polyamide + polyacrylethersulfone + polyvinypyrollidone) dialysis membranes 11
2-4 The chemical structure of polysulfone (PSf) 12
2-5 The chemical structure of polyamide (PA)13
2-6 Chemical structure of polyacrylonitrile (PAN) 14
2-7 Emulsion polymerization system24
2-8 L-α-amino acid 31
2-9 Ribbon diagram of human β M taken from the crystal structure of the protein bound to the heavy chain of the MHC class I complex (PDB 1DUZ)
236
2-10 Secondary structure of human serum albumin (HSA) with sub-domains 37
2-11 The relationship between pore size and particle size in body-centered cubic and closed packed arrays41
2-12 Schematic representation of the protein adsorption on the core shell latex particles at pH 7442
3-1 Chemical structure of main chemicals45
3-2 Experimental setup for semi-continuous emulsion polymerization 46
3-3 The particle preparation scheme in various types and size ranges of latex particles 47
3-4 Schematic of the procedure for a protein adsorption test 52
3-5 Separation of RBC from whole blood by centrifuge process53
x
3-6 Schematic of the procedure for hemolysis test 54
4-1 Schematic representation of semi-continuous seed latex particles preparation and growth56
4-2 Scanning Electron Micrograph (SEM) of polystyrene seed latex particles (A) PS (B) PS (C) PS (D) PSS259 S233 S207 S181 59
4-3 SEM of PS latex particles less than 500nm in size (A) 258nm (B) 320nm (C) 370nm (D) 410nm 65
4-4 SEM of PS latex particles larger than 500nm in size (A) 525nm (B) 585nm (C) 640nm (D) 790nm 68
4-5 Dependence of the particle size on the surfactant to monomer ratio70
4-6 Schematic of core shell latex particle structures 71
4-7 Scanning Electron Micrograph of latex particles (A) PS (B) PSPMMA (C) PSPMMA PAA (D) PSPMMA PAA
100
90 10 75 25 73
4-8 FTIR spectra of polymerized latex particles (A) bare polystyrene (PS) (B) PSPMMA (C) PSPMMA PAA (D) PSPMMA PAA100 90 10 75 25 76
4-9 Schematic representation of ion distribution near a positively charged surface 78
4-10 Schematic representation of zeta potential measurement79
4-11 Zeta potential of PS latex particles at 25 Co 80
4-12 Zeta potential of PSPMMA latex particles at 25 C100o 81
4-13 Zeta potential of PSPMMA PAA latex particles at 25 C90 10o 82
4-14 Zeta potential of PSPMMA PAA latex particles at 25 C75 25o 82
4-15 The decay of surface potential with distance from surface in various electrolyte concentrations (1) low (2) intermediate (3) high 84
4-16 High-affinity adsorption isotherm of typical flexible polymer on solid surface 86
4-17 Overall schematic representation of the protein adsorption on the synthesized latex particles86
4-18 The dependence of UV absorbance on BSA concentration (A) 10mgml (B) 06mgml (C) 02mgml87
4-19 Standard curve of net absorbance vs BSA sample concentration 88
xi
4-20 Fitted models for adsorption isotherm of bovine serum albumin (BSA) on polystyrene latex particles at 37 C in phosphate buffer mediao 89
4-21 Adsorption isotherm of BSA on polystyrene (PS) latex particles in phosphate buffer (PB) at 37 Co 90
4-22 Adsorption isotherm of BSA adsorption 37 C in phosphate buffered saline (PBS) on PS latex particles
o
91
4-23 Conformation of bovine serum albumin (BSA) on latex particles92
4-24 Adsorption isotherm of BSA 37 C in PB on PSPMMA core shell latex particles
o100
94
4-25 Adsorption isotherm of BSA at 37 C in PBS on PSPMMA core shell latex particles
o100
95
4-26 Adsorption isotherm of BSA at 37 C in PB on PSPMMA PAA core shell latex particles
o90 10
97
4-27 Adsorption isotherm of BSA at 37 C in PBS on PSPMMA PAA core shell latex particles
o90 10
98
4-28 Adsorption isotherm of BSA 37 C in PB on PSPMMA PAA latex particleso75 25 99
4-29 Adsorption isotherm of BSA 37 C in PBS on PSPMMA PAA core shell latex particles
o75 25
99
4-30 Adsorption isotherm of β M onto PS and PSPMMA latex particles in PB at 37 C and pH 74
2 100o 101
4-31 Adsorption isotherm of β M onto PS and PSPMMA PAA latex particles in PB at 37 C and pH 74
2 90 10o 101
4-32 Adsorption isotherm of β M onto PS and PSPMMA PAA latex particles in PB at 37 C and pH 74
2 75 25o 102
4-33 Gibbs free energy of adsorption of proteins on latex particles in PB at 37 C and pH 74
o
107
4-34 The kinetics of protein adsorption in PB on PS latex particles at 37 C and pH 74
o
109
4-35 The kinetics of protein adsorption in PB on PSPMMA latex particles at 37 C and pH 74
100o
109
4-36 The kinetics of protein adsorption in PB on PSPMMA PAA latex particles at 37 C and pH 74
90 10o 110
xii
4-37 The kinetics of protein adsorption in PB on PSPMMA PAA latex particles at 37 C and pH 74
75 25o 110
4-38 Image of red blood cells 113
4-39 Hemolysis caused by latex particles n=5114
xiii
Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy
SYNTHESIS AND ENGINEERING OF POLYMERIC LATEX PARTICLES FOR MEDICAL APPLICATIONS
By
Sangyup Kim
December 2005
Chair Hassan El-Shall Major Department Materials Science and Engineering
Latex particles with well-defined colloidal and surface characteristics have received
increasing attention due to their useful applications in many areas especially as solid
phase supports in numerous biological applications such as immunoassay DNA
diagnostic cell separation and drug delivery carrier Hemodialysis membrane using
these particles would be another potential application for the advanced separation
treatment for patients with end stage renal disease (ESRD) It is desirable to remove
middle molecular weight proteins with minimal removal of other proteins such as
albumin Thus it is necessary to understand the fundamental interactions between the
particles and blood proteins to maximize the performance of these membranes This
improvement will have significant economic and health impact
The objective of this study is to synthesize polymeric latex particles of specific
functionality to achieve the desired selective separation of target proteins from the human
blood Semi-continuous seed emulsion polymerization was used to prepare monodisperse
xiv
polystyrene seed particles ranging from 126plusmn75 to 216plusmn53 nm in size which are then
enlarged by about 800nm Surfactant amount played a key role in controlling the latex
particle size Negatively charged latex particles with a different hydrophobicity were
prepared by introduction of a sodium persulfate initiator and hydrophilic acrylic acid
monomer The prepared polymeric particles include bare polystyrene (PS) particles less
hydrophobic PS core and PMMA shell particles and more hydrophilic PS core and
PMMA-co-PAA shell latex particles with a 370nm mean diameter SEM light scattering
and zeta potential measurements were used to characterize particle size and surface
properties Adsorption isotherms of two proteins bovine serum albumin (BSA) and β2-
microglobulin (β2M) on latex particles were obtained as a function of pH and ionic
strength using the bicinchoninic acid (BCA) assay method The Langmuir-Freundlich
adsorption model was used to determine the adsorption amount of protein at equilibrium
The thickness of adsorbed BSA layer on latex particles was obtained in order to
investigate the adsorption orientation such as end-on or side-on mode Adsorption
kinetics experiments for both proteins and all latex particles were also performed The
adsorption kinetic constant determined from the Langmuir-Freundlich adsorption
isotherm model was used to calculate Gibbs free energy of adsorption to compare the
competitive adsorption of BSA and β2M Hemolysis tests were performed to investigate
the blood compatibility of synthesized latex particles PSPMMA90PAA10 and
PSPMMA75PAA25 core shell latex particles had desirable material properties with not
only a large amount and high rate of selective β2M adsorption over BSA but also high
blood compatibility showing less than 3 hemolysis
xv
CHAPTER 1 INTRODUCTION
End stage renal disease (ESRD) is a chronic condition in which kidney function is
impaired to the extent that the patientrsquos survival requires removal of toxins from the
blood by dialysis therapy or kidney transplantation The National Kidney Foundation
estimates that over 20 million Americans had chronic kidney disease in 2002 [NKF] The
number of people with ESRD is rapidly increasing in the United States with
approximately 96 295 incidents and 406 081 prevalent patients including 292 215 on
dialysis and 113 866 with a functioning graft in 2001 It is projected that there will be
more than 22 million ESRD patients by 2030 [USRDS 2003] The expenditure for the
ESRD treatment program had reached $228 billion 64 of the Medicare budget in
2001 Due in part to the limited availability of kidneys for transplantation hemodialysis
is the primary clinical treatment for the patients with ESRD
The central element of a hemodialysis instrument is the semipermeable membrane
that allows for selective transport of low molecular weight biological metabolites less
than 5000 Da such as urea and creatinine as well as excess water and electrolytes [Baker
2004] from the blood One limitation of current dialysis technologies is the inability to
efficiently remove middle molecular weight toxins such as β2-Microglobulin (β2M) and
interleukin 6 (IL-6)
β2M is a causative protein of dialysis-related amyloidosis (DRA) a disease arising
in patients with chronic kidney failure as a serious complication of long-term
hemodialysis treatment [Gejyo et al 1985] β2M deposition in tissue is the primary cause
1
2
of destructive arthritis and carpal tunnel syndrome [Vincent et al 1992 Drueke 2000]
The β2M structure is shown in Figure 1-2
Figure 1-2 Stereo drawing of the αndashcarbon backbone of β2M [Becker et al 1985]
Although attempts have been made to increase the efficiency of middle molecular
weight toxin removal by changes in the membrane pore size and the use of innovative
materials to adsorb these toxins [Samtleben et al 1996 Ronco et al 2001] removal
efficiency is not as high as those achieved by a normal healthy kidney Traditional
membranes have a number of processing and performance limitations [Westhuyzen et al
1992 Leypoldt et al 1998] such as a restricted choice of surface chemistries and limited
control of porosity The development of novel engineering membrane technology is
needed to remove middle molecule toxins
Polymeric latex particles have received increasing attention in medical application
areas especially as solid phase supports in biological applications [Piskin et al 1994]
Examples of these applications include immunoassay [Chen et al 2003 Radomske-
Galant et al 2003] DNA diagnostic [Elaїssari et al 1998] cell separation drug delivery
carrier [Luck et al 1998 Kurisawa et al 1995 Yang et al 2000] etc This is because of
3
the well-defined colloidal and surface characteristics of the particles By using a seed
emulsion polymerization method it is possible to synthesize monodisperse latex particles
with various particle size ranges and surface chemistry Functionalized core-shell latex
particles can be introduced by multi-step emulsion polymerization Core particles are
synthesized in the first stage of the polymerization and the functional monomer is added
in the second stage This is done without any emulsifier addition to prevent the
production of new homopolymer particles [Keusch et al 1973] The core-shell particles
are useful in a broad range of applications because of their improved physical and
chemical properties over their single-component counterparts [Lu et al 1996 Nelliappan
et al 1997] Through the development of a hemodialysis membrane using monodisperse
latex particles improvements in advanced separation treatment for patients with end
stage renal disease (ESRD) can be realized This requires the maximum removal of
middle molecular weight proteins with minimal removal of other beneficial proteins such
as albumin Thus an understanding of the fundamental interactions between the particles
and biopolymers is vital to maximize the performance of this membrane technology
The field of material science and biotechnology is based on fundamental chemistry
has developed over the past three decades into todayrsquos powerful discipline that enables
the development of advanced technical devices for pharmaceutical and biomedical
applications This novel and highly interdisciplinary field is closely associated with both
the physical and chemical properties of organic and inorganic particles [Niemeyer 2001]
Hemodialysis membrane using these particles would lead to improvements in the
advanced separation treatment for patients with end stage renal disease (ESRD) The
interdisciplinary nature of this approach enables a more complete understanding of the
4
phenomena of protein adsorption and the material properties necessary for selective
separation This innovative approach to membrane fabrication has the potential of making
inexpensive highly efficient membranes for both industrial and specialized separation
processes
The goal of this study is to prepare polymeric latex particles with tailored
properties to maximize separation of toxin molecules and to investigate the fundamental
interactions between the particles and molecules in the biological system in order to
optimize the performance of a membrane material for these applications
Polymeric latex particles were synthesized with specific functionality in an attempt
to achieve selective separation Seeded emulsion polymerization was used to synthesize
functionalized monodisperse latex particles in various sizes Negatively charged
hydrophobic polystyrene latex particles were synthesized by the same method Core shell
latex particles PSPMMA100 PSPMMA90PAA10 PSPMMA75PAA25 were also
synthesized to differentiate the degree of hydrophobicity of particles Scanning Electron
Micrograph (SEM) and light scattering measurements were used to characterize particle
size and shape and zeta potential measurements were conducted to measure the electrical
surface property of synthesized particles Adsorption isotherms of target proteins bovine
serum albumin (BSA) and β2M on latex particles were obtained as a function of pH
ionic strength and protein concentrations using the BCA assay method Adsorption
kinetics for both proteins on the latex particles were also measured Finally hemolysis
tests were run to determine the biocompatibility of polymer latex particles with human
blood This research will be described in more detail in chapters 3 and 4
CHAPTER 2 BACKGROUND AND LITERATURE SURVEY
21 The Significance of End Stage Renal Disease (ESRD)
The kidneys are responsible for removing excess fluid minerals and wastes from
the blood regulating electrolyte balance and blood pressure and the stimulation of red
blood cell production They also produce hormones such as erythropoietin (EPO) and
calcitriol that keep bones strong and blood healthy [Casadevall and Rossert 2005 Kurbel
et al 2003] EPO acts on the bone marrow to increase the production of red blood cell in
case of bleeding or moving to high altitudes Calcitriol mainly acts on both the cells of
the intestine to promote the absorption of calcium from food and also bone to mobilize
calcium from the bone to the blood When kidneys fail harmful waste builds up blood
pressure rises and body retains excess fluid The body also does not make enough red
blood cells When this happens treatment is needed to replace the function of failed
kidneys
Figure 2-1 An image of the location and cross section of a human kidney
5
6
The National Kidney Foundation estimates that over 20 million Americans had
chronic kidney disease in 2002 [NKF] Chronic renal disease is a gradual and progressive
loss of the function unlike acute renal disease where sudden reversible failure of kidney
function occurs Chronic renal failure usually takes place over a number of years as the
internal structures of the kidney are slowly damaged In the early stages there can be no
symptoms In fact progression may be so gradual that symptoms do not occur until
kidney function is less than one-tenth that of a normal health kidney If left untreated
chronic kidney disease may ultimately lead to kidney failure known as End Stage Renal
Disease (ESRD) ESRD is a rapidly growing heath-care problem in the United States In
2001 approximately 96295 incidents and 406081 prevalent patients were diagnosed
with ESRD including 292215 patients on dialysis and 113866 patients with a
functioning graft [USRDS 2003] The projected number of patients with ESRD is
expected to exceed 22 million patients by 2030 with much of this growth being driven by
the increasing prevalence of major contributing factors such as diabetes and high blood
pressure [USRDS 2003] A great extent of ESRD program cost and Medicare budget
have been spent in 2001 Due in part to a limited availability of kidneys for
transplantation hemodialysis (HD) is the primary method of treatment for ESRD and is
currently used for approximately 61 of US ESRD patients
22 Hemodialysis (HD) Treatment
HD removes toxins from the body by extracorporeal circulation of the blood
through a semipermeable membrane referred to as a dialyzer The toxins are removed
primarily by diffusion across the membrane to a dialysate solution which is circulated on
the opposite side of the membrane The cleaned blood is then returned to the blood
stream A surgically constructed vascular access connects the extracorporeal circuit to the
7
patientrsquos system Treatments are almost always performed three times per week in
specially equipped dialysis facilities for 3-4 hours per each treatment Figure 2-2 shows
the scheme of the hemodialysis route The critical element of a HD instrument is the
semipermeable membrane which allows for selective transport of low molecular weight
biological metabolites from blood
igure 2-2 A schematic draw of the hemodialysis route
ESRD patients who undergo dialysis therapy often experience several other
problems associated with the treatment such as anemia fatigue bone problems joint
problems itching sleep disorders and restless legs Anemia is common in patient with
kidney disease because the kidneys produce the hormone erythropoietin (EPO) which
stimulates the bone marrow to produce red blood cells Diseased kidneys often do not
produce enough EPO to stimulate the bone marrow to make a sufficient amount of red
blood cells This leads to bone disease referred to as renal osteodystrophy and causes
bones to become thin and weak or malformed and can affect both children and adults
Older patients and women who have gone through menopause are at greater risk for this
disease Uremic toxins which cannot be remove from the blood by the current dialyzer
membranes can lead to itching This problem can also be related to high levels of
F
8
parathyroid hormone (PTH) Dialysis patients can also experience day-night revers
is they have insomnia at night and sleep during the day This can be related to possible
nerve damage in the body and a chemical imbalance in the blood due to the excess toxin
Oxidative stress is a problem for patients on maintenance dialysis This problem is due to
an imbalance between pro- and antioxidant factors [Roselaar et al 1995 Cristol et al
1994] Oxidative stress affects oxidation of low-density lipoproteins which are the main
factor for atherogenesis [Huysmans et al 1998] and is also involved in the development
of malignancies and diabetes mellitus [Rice-Evans et al 1993] In order to reduce
antioxidant defense dialysis is needed to contribute to help stimulate free radical
production or eliminate antioxidants Dialysis-related amyloidosis (DRA) is also a
common and serious problem for people who have been on dialysis for more than 5
years DRA develops when proteins in the blood deposit on joints and tendons causi
pain stiffness and fluid in the joint as is the case with arthritis Normally the healthy
kidneys can filter out these proteins but dialysis filters are not as effective
23 Advances in Membrane Technology
al that
s
ng
Dialysis for blood of ESRD
Hemo filtration
s
ysis using a cellulose based membrane occurred
1913 John Abel [1990] from the Johns Hopkins Medical School described a method
purification is widely used in the treatment
dialysis (HD) techniques use a semi-permeable membrane to replace the
role of the kidney The membranes used in HD can be broadly classified into those based
on cellulose and those manufactured from synthetic copolymers These membranes come
in various shapes such as sheets tubular structures or hollow fiber arrangements The
hollow fiber type is the most popular and is incorporated into over 800 different device
in world wide [Ronco et al 2001]
The first attempt at blood dial
9
where sing
e
om
ysis was performed until the mid 1960 The basic
molec
rial in
een
benzyl)
was used for the membrane with
hepar
by the blood of a living animal may be submitted to dialysis outside the body u
a membrane based on cellulose and returned to the natural circulation without exposur
to air infection by microorganisms or any alteration that would necessarily be prejudicial
to life This same technique is still used to today however the device used has been
modified over the years as better membranes were developed and the anti-coagulant
heparin has become available
Cellulose membranes have been widely used for the treatment of renal failure fr
1928 when the first human dial
ular structure of cellulose is made of a long chain containing hydroxyl (OH)
groups The realization that such groups imparted undesirable qualities on the mate
respect to blood contact behavior was discovered in the early 1970s and since has b
the focus of development These modified cellulose membranes used the partial
substitution of benzyl groups to replace the proton of the hydroxyl groups in an attempt
to reduce their negative effect The result is a molecular mosaic of hydrophobic (
and hydrophilic (hydroxyl and cellulose) regions
Kolff [Van Noordwijk 2001] studied the rotating drum artificial kidney for patients
with acute renal failure in 1943 Cellophane tubing
in as the anticoagulant For the next 17 years hemodialysis therapy was performed
by this method but only for the patients with acute reversible renal failure Vascular
access required repeated surgical insertions of cannulas (slender tubes) into an artery and
vein and limited the number of treatments for a patient could receive in order to
minimize the amount of vascular damage
10
Initially the need for dialysis in patients with acute renal failure was determ
mainly by the development of signs and sym
ined
ptoms of uremia After dialysis some time
might
et al
ent improvement In 1960 the development of a shunt [Quinton et al
1960]
fone (PSf) polyamide (PA) and polyacrylonitrile (PAN) by phase inversion or
precip
lidone
e
elapse before uremic manifestations returned to warrant a sequential treatment of
dialysis Many patients with acute renal failure secondary to accidental or surgical
trauma were hypercatabolic but the interdialytic interval might be prolonged because of
anorexia or use of a low-protein diet However Teschan and his coworkers [Obrien
1959] showed that patient well-being and survival were improved by what they termed
prophylactic daily hemodiaysis or administration of the treatment before the patient
again became sick with uremia Their report in 1959 was the first description of daily
hemodialysis
Development of membrane accessories such as a shunt has also been an area of
focus for treatm
a flexible polytetrafluoroethylene (PTFE or Teflonreg) tubing made many more
hemodialysis treatments possible for chronic kidney failure patients PTFE has a non-
stick surface and is relative biocompatibility leading to minimized blood clotting in the
shunt
Synthetic membranes are prepared from engineered thermoplastics such as
polysul
itation of a blended mixture resulting in the formation of asymmetric and
anisotropic structures Figure 2-3 shows a fiber type of the Polyflux S membrane
consisting of poluamide (PA) polyacrylethersulfone (PAES) and polyvinylpyrro
(PVP) with the integral three-layer structure The skin layer on the inside fiber typ
membrane contacts blood and has a very high surface porosity and a narrow pore size
11
distribution This layer constitutes the discriminating barrier deciding the permeabili
and solute retention properties of the membrane The skin layer is supported by thick
sponge-type structure larger pores providing mechanical strength and very low
hydrodynamic resistance
ty
Figure 2-3 Cross-sectional SEM image view of the Polyflux S (polyamide +
polyacrylethersulfone + polyvinypyrollidone) dialysis membranes [Deppisch et al 1998]
n
[Malchesky 2004] because of its thermal stability mechanical strength and chemical
inertn ciated
ls
m
PSf is a widely used membrane material for the hemodialysis applicatio
ess According to a report from the National Surveillance of Dialysis-Asso
Disease (NSDAD) in the US over 70 of hemodialysis membranes were PSf based
[Bowry 2002] This is most likely because PSf has many advantages over other materia
This synthetic polymer is one of few materials that can withstand sterilization by stea
ethylene oxide and γ-radiation PSf membrane can be prepared by conventional
immersion precipitation methods into many different shapes including porous hollow
12
fiber or flat sheet hemodialysis membranes The material also has a high permeab
low molecular weight proteins and solute and high endotoxin retention The chemical
structure of PSf is shown in Figure 2-4
ility to
Figure 2-4 The chemical structure of polysulfone (PSf)
There is one major disadvantage to PSf The hydrophobic nature of the PSf causes
ment alternative pathway
leadin
s not
3
et al
in
O acrylate
serious complications through the activation of the comple
g to the adsorption of serum proteins onto the membranes [Singh et al 2003]
Anticoagulants are added during dialysis therapy to avoid blood clotting but this doe
completely eliminate the problem In order to overcome this disadvantage of the PSf
membrane various studies have been performed to change the materialrsquos surface
properties These investigations include hydrophilic polymer coating [Brink et al 199
Kim et al 1988 Higuchi et al 2003] layer grafting onto PSf membrane [Wavhal
2002 Song et al 2000 Pieracci et al 2002 Mok et al1994] and chemical reaction of
hydrophilic components onto the membrane surface [Higuchi et al 1990 1991 1993
Blanco et al 2001 Nabe et al 1997 Guiver et al 1993] Hydrophilic monomers 2-
hydroxy-ethylmethacrylate (HEMA) acrylic acid (AA) and methacrylic acid (MMA)
have also been grafted onto PSf membrane to increase flux and Bovine Serum Album
(BSA) retention [Ulbricht et al 1996] Hancock et al [2000] synthesized
polysulfonepoly(ethylene oxide) (PEO) block copolymers to improve the resistance to
platelet adhesion Kim et al [2003] also studied blending a sulfonated PE
diblock copolymer into PSf in order to reduce platelet adhesion and enhance
13
biocompatibility PEO is a commonly used biomaterial due to its excellent resistance
protein adsorption and inherent biocompatibility [Harris 1992] Kim et al [20
a self-transformable copolymer to enhance the hydrophilicity of an asymmetric PSf
membrane with an ultra-thin skin layer The polymer had an entrapped diblock
copolymer containing a hydrophilic block of poly (ethylene glycol) (PEG)-SO
to
05] studied
ate
lic graft
l)
e of thier
aromatic
orand
3 acryl
and a hydrophobic block of octadecylacrylate (OA) Molecular dynamic (MD)
simulations were performed as a function of copolymer density to optimize interfacial
structure information McMurry [2004] developed a strategy using an amphiphi
copolymer added to PSf membranes by introducing polysulfone-g-poly (ethylene glyco
When compared to unmodified PSf these graft copolymer and resulting blend
membranes are found to hold promise for biomedical device applications
Polyamide (PA) membranes have also been used for hemodialysis becaus
mechanical strength in both wet and dry conditions Polyamide consists of
aliphatic monomers with amide bonding (-CONH-) also known as a peptide bond
The basic amide bond in polyamide is shown in Figure 2-5 R1 and R2 can be either
aromatic or aliphatic linkage group
C N
H
O
~ R R1 2~C N
H
O
~ R R1 2~
Figure 2-5 The chemical structure of polyamide (PA)
Panichi and co-workers [1998] evaluated the biocompatibility of the PA membrane
ompatible technique due to the
use of a synthetic membrane with a sterile re-infusion fluid and the convective removal of
and concluded that PA hemofiltration was a highly bioc
14
the ac
e
e
is
n
is a semi-crystalline polymer and the mechanical properties
strong
2
CN
tivated anaphylatoxins and β2-Microglobulin (β2M) The PA based membrane
Polyfluxreg (manufactured by Gambro GmbH Germany) blended with polyamide
polyarylethersulfone and polyvinylpyrrolidone (PVP) was able to clean small molecules
such as urea dreatinine and phosphate as well as decrease β2M amount by 502
[Hoenich et al 2002] Due to the non-selectivity of the membrane removal of thes
unwanted materials also led to the undesirable loss of beneficial proteins during therapy
Meier et al [2000] evaluated different immune parameters using a modified cellulos
low-flux hemophan and synthetic high-flux PA membrane during a 1 year period in
chronic hemodialysis patients They found that the 1-year immunological evaluation of
hemodiaysis membrane biocompatibility was associated with changes in the pattern of
chronic T-cell actiovation
Polyacrylonitrile (PAN) is another commonly used membrane material because it
inherently hydrophilic and has been commercialized for ultrafiltration and microfiltratio
[Scharnagl et al 2001] PAN
ly depend on the crystalline structures The chemical structure of PAN is shown in
figure 2-6
CH CH
n2
CN
CH CH n
Chemical structure of polyacrylonitrile (PAN)
The addition of additives such as polyvinylpyrrolidone (PVP) as a pore forming
agent gives PAN membranes more flexible processing parameters and increased
e has been optimized through
copolymerization with many other vinyl monomers including glycidyl methacrylate
Figure 2-6
performance [Jung et al 2005] PAN membrane performanc
15
[Godj
acid
for
for
a non-
solub this
in
oid underneath the inner
surface of membrane decreased by increasing the amount of solvent DMSA in the
evargova et al 1999 Hicke et al 2002] N-vinylimidazole [Godjevargova et al
2000] hydroxyl ethyl methacrylate [Ray et al 1999 Bhat et al 2000] methacrylic
[Ray et al 1999] vinyl pyrrolidone [Ray et al 1999] acrylic acid [Trotta et al 2002]
acrylamide [Musale et al 1997] and vinylchloride [Broadhead et al 1998] These
monomers provide a reactive group for enzyme immobilization improved mechanical
strength solvent-resistance pervaporation permeation flux anti-fouling and bio-
compatibility Because of this PAN-based copolymer membranes have great potential
the treatment of hemodialysis in an artificial kidney This material can also be used
other applications like the treatment of wastewater the production of ultra-pure water
biocatalysis together with separation and methanol separation by pervaporation
Lin and his coworker [2004] studied the modification of PAN based dialyzer
membranes to increase the hemocompatibility by the immobilization of chitosan and
heparin conjugates on the surface of the PAN membrane When a foreign material is
exposed to blood plasma proteins are adsorbed clotting factors are activated and
le fibrin network or thrombus is formatted [Goosen et al 1980] The result of
research was that the biocompatible chitosan polymer and a blood anticoagulant hepar
prevented blood clotting They showed prolonged coagulation time reduced platelet
adsorption thrombus formation and protein adsorption
Nie et al [2004] studied PAN-based ultrafiltration hollow-fiber membranes
(UHFMs) In order to improve the membrane performance acrylonitrile (AN) was
copolymerized with other functional monomers such as maleic anhydride and α-allyl
glucoside They found that the number and size of macrov
16
intern erum
and
d a
rface The second step is the
transf after
yjak et
embrane
1978 Malchesky et al 1978] has been further developed to increase the efficiency of
al coagulant The water flux of the UHFMs also decreased while the bovine s
albumin rejection increased minutely Godjevargova et al [1992] modified the PAN
based membrane with hydroxylamine and diethylaminoethylmethacrylate to improve
membrane dialysis properties Formed functional groups like primary amine oxime
tertiary amine groups provided the membrane with more hydrophilic properties an
substantial increase in the permeability of the membranes
The wide use of filtration in practice is limited by membrane fouling Solute
molecules deposit on and in the membrane in the process of filtration causing dramatic
reduction in flux through the membrane Fouling occurs mostly in the filtration of
proteins Three kinetic steps are involved in the fouling of UF membranes according to
Nisson [1990] The first step is the transfer of solute to the su
er of solute into the membrane until it either finally adsorbs or passes through
a set of adsorption-desorption events The third step includes surface binding
accompanied by structural rearrangement in the adsorbed state [Ko et al 1993] Br
al [1998] studied the surface modification of a commercially available PAN membrane
to develop superior filtration properties with less fouling by proteins The PAN
membrane was immersed in excess NaOH solution to convert some of the surface nitrile
groups into carboxylic groups by the hydrolysis process This modified PAN m
was not so severely fouled in the Bovine Serum Albumin (BSA) filtration test The pore
size however decreased during the hydrolysis process leading to a significant reduction
in flux and made the membrane less productive in the ultrafiltration (UF) mode
24 Sorbent Technology
Over the last three decades sorbent technology [Castino et al 1976 Korshak et al
17
dialysis or replace it for the treatment of ESRD Sorbents remove solutes from solution
through specific or nonspecific adsorption depending on both the nature of the solute and
the sorbent Specific adsorption contains tailored ligands or antibodies with high
selectivity for target molecules een used in autoimmune
disord
and
e
al 1996] various resins [Ronco et al 2001]
album
e
as
Specific adsorbents have b
ers such as idiopathic thrombocytopenic purpura [Snyder et al 1992] and for the
removal of lipids in familial hyper cholesterolemia [Bosch et al 1999] Nonspecific
adsorbents such as charcoal and resins attract target molecules through various forces
including hydrophobic interactions ionic (or electrostatic) attraction hydrogen bonding
and van der Waals interactions
New dialysate with sorbents has become an accepted modification of dialysis
sorbent hemoperfusion is gaining ground as a valuable addition to dialysis especially as
new sorbents are developed [Winchester et al 2001] Hemoperfusion is defined as th
removal of toxins or metabolites from circulation by the passing of blood within a
suitable extracorpoteal circuit over semipermeable microcapsules containing adsorbents
such as activated charcoal [Samtleben et
in-conjugated agarose etc Novel adsorptive carbons with larger pore diameters
have been synthesized for potential clinical use [Mikhalovsky 1989] Newly recognized
uremic toxins [Dhondt et al 2000 Haag-Weber et al 2000] have resulted in several
investigations on alternatives to standard or high-flux hemodialysis to remove thes
molecules These methods include hemodiafilteration with [de Francisco et al 2000] or
without [Ward et al 2000 Takenaka et al 2001] dialysate regeneration using sorbents
well as hemoperfusion using such adsorbents as charcoal and resins
18
Kolarz et al [1989 1995] studied the hyper-crosslinked sorbent prepared from
styrene and divinylbenzene (DVB) for a hemoperfusion application They found that the
pore structure of a swelling sorbent was changed by additional crosslinking with α αrsquo-
dichloro-p-xylene in the presence of a tin chloride catalyst and in a di
chloroethane
soluti and
en
decyl groups that attract β2M through a hydrophobic interaction The
adsor
of
5]
d
penet
on They also realized that the hemocompatibility was useful for nemoperfusion
could be imparted to the sorbents by introducing sulfonyl groups at a concentration of
about 02mmolg
A special polymeric adsorbing material (BM-010 from Kaneka Japan) has be
investigated by another group [Furuyoshi et al 1991] for the selective removal of β2M
from the blood of dialysis partients The adsorbent consists of porous cellulose beads
modified with hexa
ption capacity of this material is 1mg of β2M per 1ml of adsorbent Using a
hemoperfusion cartridge containing 350ml of these cellulose beads in sequence with a
high-flux hemodialyzer several small clinical trials were performed During 4-5 hours
treatment about 210mg of β2M were removed thus reducing the concentration in the
blood by 60-70 of the initial level [Nakazawa et al 1993 Gejyo et al 1993 199
RenalTech developed a hemoperfusion device BetaSorbreg containing the hydrate
cross-linked polystyrene (PS) divinylbenzene (DVB) resin sorbents with a pore structure
designed to remove molecules between 4 and 30 kDa [Winchester et al 2002] In this
case solute molecules are separated according to their size based on their ability to
rate the porous network of the beaded sorbents The resin beads were prepared with
a blood compatible coating and confirmed to be biocompatible in vivo in animals
[Cowgill et al 2001]
19
25 Limitation of Current Hemodialysis Treatment
Hemodialysis is a widely used life-sustaining treatment for patients with ESRD
However it does not replace all of the complex functions of a normal healthy kidne
a result patients on dia
y As
lysis still suffer from a range of problems including infection
accelerated cardio ion anemia
chron
e
se middle
alysis
sessio
s
f
vascular disease high blood pressure chronic malnutrit
ic joint and back pain and a considerably shortened life span One significant
limitation of the current dialysis technology is the inability to efficiently remove larger
toxic molecules This is mainly because of the broad pore size distribution reducing th
selective removal of toxins and unsatisfied biocompatibility causing lots of
complications such as inflammation blood clotting calcification infection etc
Dialysis purifies the patients blood by efficiently removing small molecules like
salts urea and excess water However as toxic molecules increase in size their removal
rate by hemodialysis substantially declines Typically only 10 - 40 of the
molecular weight toxins (300-15000 Da) are removed from the blood during a di
n [Vanholder et al 1995] These toxins then reach an abnormally high level and
begin to damage the body One such toxin β2M causes destructive arthritis and carpal
tunnel syndrome by joining together like the links of chain to form a few very large
molecules and deposit damaging the surrounding tissues [Lonnemann et al 2002] This i
also a main cause of mortality for long-term dialysis patients Other middle molecule
toxins appear to inhibit the immune system and may play a significant role in the high
susceptibility to infections in dialysis patients Still others are believed to impair the
functioning of several other body systems such as the hematopoietic and other endocrine
systems This may contribute to accelerated cardiovascular disease the leading cause o
20
death among dialysis patients as well as clinical malnutrition which affects up to 50 of
this patient population
Over the last decade polymeric dialysis membranes have been developed to
increase the capacity for removing middle molecular weight toxins by changing the pore
size of dialyzer membranes and using new materials that adsorb these toxins for
impro se
styrene made during World War II in the United
States The Dow chemical compan anufacturer of polystyrene
includ
d of
on
rug
ntrol of
ved removal characteristics However removal efficiency is not as high as tho
achieved by a normal healthy kidney
26 Latex Particles
The first synthetic polymer synthesized using emulsion polymerization was a
rubber composed of 13-butadiene and
y has been a major m
ing latex which they used in paint formulations The theory of emulsion
polymerization in which a surfactant is used was established by Harkins [1948] and by
Smith and Ewart [1948] By 1956 the technology was complete including the metho
building larger diameter particles from smaller ones The product by this emulsi
polymerization is referred to as latex a colloidal dispersion of polymer particles in water
medium [Odian 1991] Latexes are currently undergoing extensive research and
development for a broad range of areas including adhesives inks paints coatings d
delivery systems medical assay kits gloves paper coatings floor polish films carpet
backing and foam mattresses to cosmetics The relatively well known and easy co
the emulsion process is one of main advantages for these applications Therefore
polymeric latex particle prepared by emulsion polymerization can be a candidate for the
medical applications because of the easy control of the particle size and morphology as
well as flexible surface chemistry to be required
21
261 The Components for Emulsion Polymerization
The main components for emulsion polymerization process are the monomer a
dispersing medium a surfactant and an initiator Available monomers are styrene
sing medium is usually water
which
ticles
n
ation To
monomer droplets but in micelles because the initiators are
insolu le
and
olution
butadiene methylmethacylate acryl acid etc The disper
will maintain a low solution viscosity provide good heat transfer and allow
transfer of the monomers from the monomer droplets into micelles and growing par
surrounded by surfactants respectively The surfactant (or emulsifier) has both
hydrophilic and hydrophobic segments Its main functions is to provide the nucleatio
sites for particles and aid in the colloidal stability of the growing particles Initiators are
water-soluble inorganic salts which dissociate into radicals to initiate polymeriz
control the molecular weight a chain transfer agent such as mercaptan may be present
262 Particle Nucleation
Free radicals are produced by dissociation of initiators at the rate on the order of
1013 radicals per milliliter per second in the water phase The location of the
polymerization is not in the
ble in the organic monomer droplets Such initiators are referred to as oil-insolub
initiators This is one of the big differences between emulsion polymerization
suspension polymerization where initiators are oil-soluble and the reaction occurs in the
monomer droplets Because the monomer droplets have a much smaller total surface area
they do not compete effectively with micelles to capture the radicals produced in s
It is in the micelles that the oil soluble monomer and water soluble initiator meet and is
favored as the reaction site because of the high monomer concentration compared to that
in the monomer droplets As polymerization proceeds the micelles grow by the addition
of monomer from the aqueous solution whose concentration is refilled by dissolution of
22
monomer from the monomer droplets There are three types of particles in the emulsion
system monomer droplets inactive micelles in which polymerization is not occurring
and active micelles in which polymerization is occurring referred to as growing polymer
particles
The mechanism of particle nucleation occurs by two simultaneous processes
micellar nucleation and homogeneous nucleation Micellar nucleation is the entry of
radicals ei
ther primary or oligomeric radicals formed by solution polymerization from
the aq ed
the
r
s
n
is coagulation with other particles rather than
polymerization of monomer The driving force for coagulation of precursor particles
ueous phase into the micelles In homogeneous nucleation solution-polymeriz
oligomeric radicals are becoming insoluble and precipitating onto themselves or onto
oligomers whose propagation has ended [Fitch et al 1969] The relative levels of micella
and homogeneous nucleation are variable with the water solubility of the monomer and
the surfactant concentration Homogeneous nucleation is favored for monomers with
higher water solubility and low surfactant concentration and micellar nucleation is
favored for monomers with low water solubility and high surfactant concentration It has
also been shown that homogeneous nucleation occurs in systems where the surfactant
concentration is below CMC [Roe 1968] A highly water insoluble monomer such a
styrene [Hansen et al 1979 Ugelstad et al 1979] has probably created by micellar
nucleation while a water soluble monomer such as vinyl acetate [Zollars 1979] has bee
formed by homogeneous nucleation
A third latex formation reaction mechanism has been proposed referred to as
coagulative nucleation In this reaction the major growth process for the first-formed
polymer particles (precursor particles)
23
sever les
yer
es
r a
l
the beginning of the polymerization As soon as the initiator is
gins with the formation and
growt
and
ved
added at one time during the later stages of the polymerization prior to complete
al nanometers in size is their relative instability compared to larger sized partic
The small size of a precursor particle with its high curvature of the electrical double la
permits the low surface charge density and high colloidal instability Once the particl
become large enough in size maintaining the high colloidal stability there is no longe
driving force for coagulation and further growth of particles takes place only by the
polymerization process
263 Types of Processes for Emulsion Polymerization
There are three types of production processes used in emulsion polymerization
batch semi-continuous (or semi-batch) and continuous In the batch type process al
components are added at
added and the temperature is increased polymerization be
h of latex particles at the same time There is no further process control possible
once the polymerization is started In the semi-continuous emulsion polymerization
process one or more components can be added continuously Various profiles of particle
nucleation and growth can be generated from different orders of component addition
during polymerization There are advantages to this process such as control of the
polymerization rate the particle number colloidal stability copolymer composition
particle morphology In the continuous process the emulsion polymerization components
are fed continuously into the reaction vessel while the product is simultaneously remo
at the same rate High production rate steady heat removal and uniform quality of
latexes are advantages of the continuous polymerization processes
Other available methods include the intermittent addition and shot addition of one
or more of the components In the shot addition process the additional components are
24
conversion of the main monomer This method has been used successfully to develo
water-soluble functional monomers such as sodium styrene sulfonat
p
e [Kim et al 1989]
264
lsion
Chemistry of Emulsion Polymerization
Emulsion polymerization is one type of free radical polymerization and can be
divided into three distinct stages initiation propagation and termination The emu
polymerization system is shown in Figure 2-7
Aqueous phaseAqueous phase
Micelle withmonomer
I Rbull
Emulsifier
Polymer particle swollenWith monomer
Monomerdroplet
I Rbull
Micelle withmonomer
I Rbull
Emulsifier
Polymer particle swollenWith monomer
MonomerdropletMonomerdroplet
I Rbull
Figure 2-7 Emulsion polymerization system [Radicals (Rmiddot) are created from initiators
(I) Monomer is transferred from larger monomer droplet into micelles by emulsifier Initiated polymer particle by radicals is keep growing until monomers are all consumed The reaction is performed in aqueous media]
In the initiation stage free radicals are created from an initiator by heat or an
ultraviolet radiation source The initiator with either peroxides groups (-O-O-) such as
odium persulfate or azo groups (-N=N-) such as azobisisobutyronitrile is commonly
used for em t
with the m er
chain ere
s
ulsion polymerization The primary free radicals created from initiator reac
onomer for initiation of polymerization In the propagation stage the polym
grows by monomer addition to the active center a free radical reactive site Th
are two possible modes of propagations head-to-head addition and head-to-tail addition
25
The head-to-tail mode is the predominant configuration of the polymer chain a result o
steric hindrance of the substitute bulky group In the termination stage polymer chain
growth is terminated by either coupling of two growing chains forming one polymer
molecule or transferring a hydrogen atom (dispropotionation) from one growing chain to
another forming two polymer molecules one having a saturated end group and the other
with an unsaturated end-group
265 Seed Emulsion Polymerization
Polymer latex particles have received increasing interest because of the versatili
of the many applications heterophase polymerization processes like emulsion dispersion
micro-emulsion seeded emulsion precipitation etc Especially to prepare well-defined
microspheres having monodispe
f
ty
rse and various particle sizes as well as surface group
ed particles prepared by emulsion polymerization
and e
s
hological design In
seede to control
n
functionalilties it is necessary to use se
nlarge them to a desired size in the further stages of reactions
Polymeric particles are required to have a uniform particle size in many
applications such as chromatography where they are used as a packing material
Morphological control of latex particle is also important for many practical application
[Schmidt 1972] Seed emulsion polymerization (or two-stage emulsion polymerization) is
a useful method to achieve both monodisperse particle size and morp
d emulsion polymerization [Gilbert 1995] preformed lsquoseedrsquo latex is used
the number of particles present in the final latex The advantage of seeded emulsio
polymerization is that the poorly reproducible process of particle nucleation can be
bypassed so that the number concentration of particles is constant and known Various
mechanisms have been proposed for the growth of latex particles [Ugelstad et al 1980
Okubo et al 1992] using this polymerization technique The initial seed particle
26
preparation step is well known and relatively easy to perform because at the relative
small particle sizes (02 to 05 micron) the particle growth process can be readily
controlled by the use of an emulsifier Enough emulsifier is used to prevent coagulation
but not enough to cause the formation of new particles As the particles are grown to
larger sizes in successive seeding steps it becomes increasingly difficult to mainta
stable uncoagulated emulsion without forming new particles and thereby destroying
monodisperisty of the latex Recently there have been several reports concerning t
reaction kinetics of seed emulsion polymerization and the development of latex
morphologies over the course of the reaction [Chern et al 1990 Delacal et al 1990 L
et al 1995 Lee 2000 2002]
266 Polystyrene Latex Particles
A number of papers have described the synthesis of the polystyrene latex particl
bearing various functional surface groups such as carboxyl [Lee et al 2000 Tun
2002 Reb et al 2000] hydroxyl [Tamai et al 1989] marcapto [Nilson 1989] epoxy
[Shimizu et al 2000 Luo et al
ly
in a
he
ee
es
cel et al
2004] acetal [Izquierdo et al 2004 Santos et al 1997]
ometyl [Izquierdo et al 2004 Park et al 2001
Sarob
h the
d
thymine [Dahman et al 2003] chlor
e et al 1998] amine [Counsin et al 1994 Ganachaud et al 1997 Ganachaud et al
1995 Miraballes-Martinez et al 2000 2001 Anna et al 2005] ester [Nagai et al 1999]
etc In order to produce these functionalized particles different methods for particle
preparation must be used The polymer emulsion with core-shell morphology of latex
particles is one of them This is a multistep emulsion polymerization process in whic
polystyrene ldquocorerdquo particle is synthesized in the first stage and the functional monomer is
added in the second stage of the polymerization without any emulsifier postfeeding to
prevent the production of new homopolymer particles thus forming the functionalize
27
polymer ldquoshellrdquo on the ldquocorerdquo particle [Keusch et al 1973] There are requirements to
limit secondary nucleation and encourage core-shell formation in seeded emulsion
polymerization including the addition of smaller seed particles at high solid content to
increase particle surface area low surfactant concentration to prevent formation of
micelles and the semi-continuous addition of monomer to create a starved-feed conditio
and keep the monomer concentration low There are some advantages [Hergeth et al
1989] of dispersions with polymeric core-shell particles First it is possible to modi
interfacial properties of polymer particles in the aqueous phase by the addition of only
very small amounts of a modifying agent during the last period of the reaction Thus
these core-shell particles are useful in a broad range of applications since they always
exhibit improved physical and chemical properties over their single-component
counterparts [Lu et al 1996 Nelliappan et al 1997] In this way the improvement of
surface properties of such dispersions is straightforward and inexpensive The other is
that polymers with a core-shell structure are perfect model systems for investigating th
material properties of polymer blends and composites because of their regular
distribution of one polymer inside a matrix polymer and because of the simple sp
geometry of the system
Their properties usually depend on the structures of latex particles Chen and his
coworkers [Chen et al 1991 1992 1993] reported the morphological development of
core shell latex particles of polystyrenepoly(methyl methacrylate) during
polymerization Before the research by Chen and his coworker Min et al [Min et al
1983] reported the morph
n
fy the
e
herical
ological development of core shell latex of polystyrene
(PS)polybuthyl acrylateat (PBA) by seeded emulsion polymerization as a function of the
28
additi
of
ity of graft
ing reagents Thus protein adsorption on polymeric solid surfaces has
st are microspheres defined as ldquofine
polym can be
n
r was
containing antigen urine serum etc The latex will become agglutinated and visibly
on method of PS They found that the percentage of grafting PS to the PBA was
greatest for the batch reaction and the PBA-PS core-shell particles with a high degree
grafting remained spherical upon aging test because of the emulsifying abil
copolymer
267 Various Applications of Latex Particles
Latex particles are applicable to a wide range of areas such as biomedical
applications [Piskin et al 1994] especially as the solid phase such as in immunoassays
[Chern et al 2003 Radomske-Galant et al 2003] DNA diagnostic drug delivery carriers
[Luck et al 1998 Kurisawa et al 1995 Yang et al 2000] blood cell separations and
column pack
become a center of attention Of particular intere
er particles having diameters in the range of 01 to several micronsrdquo which
used as functional tools by themselves or by coupling with biocompounds Singer and
Plotz [1956] firstly studied microsphere or latex agglutination test (LATrsquos) by using
monodisperse polystyrene (PS) and polyvinyltoluene polymer particles as the support o
which the biomolecules were going to adsorb The biomolecule adsorption howeve
limited by possible desorption of the adsorbed species or loss of specific activity of the
complex formed Since this work was published the latex particle applications for
immunoassay have been rapidly and widely studied and developed
Latex agglutination test or latex immunoassays start with tiny spherical latex
particles with a uniform diameter and similar surface properties The particles are coated
with antibodies (sensitized) through the hydrophobic interaction of portions of the protein
with the PS surface of the particles If sensitized particles are mixed with a sample
29
agglomerate Latex tests are inexpensive as compared with the other techniques [Ba
1988]
ngs
sed
tion Rheumatoid factor (RF) in different age subpopulations has also been
evalu
studied a
latex
Unipath [Percival 1996] has manufactured a range of immunoassay materials ba
on a chromatographic principle and deeply colored latex particles for use in the home and
clinical environments The latex particles which are already sensitized with a
monoclonal antibody can detect any antigens that bound to the surface of the latex
particles Some detectable pollutants include estrogen mimics which induce
abnormalities in the reproductive system of male fishes and lead to a total or partial male
feminiza
ated according to a patientrsquos clinical status by using a rapid slide latex agglutination
test for qualitative and semiquantitative measurement in human serum along with latex
immunoassay method [Onen et al 1998] Magalhaes and his coworkers [2004]
diagnostic method of contamination of male fishes by estrogen mimics using the
production of vitellogenin (VTG) as a biomarker This was based on a reverse
agglutination test developed with monoclonal antibodies specific to this biomarker
Premstaller and his coworkers [Premstaller et al 2000 2001] have prepared a porous
poly(styrene-divinylbenzene) (PS-DVB) polymer monolith to use for highly efficient
chromatographic separation of biomelecules such as proteins and nucleic acids They
used a porogen a mixture of tetrahydrofuran and decanol to fabricate a micropellicular
PS-DVB backbone Legido-Quigley and his colleagues [2004] have developed the
monolith column to obtain further chromatographic functionality to the column by
introducing chloromethylstyrene in place of styrene into the polymer mixture
30
Core shell type monodisperse polymer colloids have been synthesized by Sarobe
and Forcada [1998] with chloromethyl functionality in order to improve the biomolecu
adsorption through a two-step emulsion polymerization process They investigated the
functonalized particles by optimizing the experimental parameters of the functional
monomer including reaction temperature the amount and type of redox initiator sys
used the type of addition of the initiator system and the use of washing They c
le
tem
oncluded
that th
sis
serve as separation
chann
of
usually
along the polypeptide chain which contain positive or negative charges [Norde 1998]
e relation between the amount of iron sulfate and the persulfatebisulfite system
added should be controlled to obtain monodisperse particles and prevent the premature
coagulation of the polymer particles during the polymerization
A semi-continuous emulsion polymerization technique for latex particle synthe
was performed by McDonald and other researchers [Ramakrishnan et al 2004 Steve et
al 1999] They introduced a variety of particles sizes compositions morphologies and
surface modifications to fabricate latex composite membranes (LCMs) Arraying and
stabilizing latex particles on the surface of a microporous substrate form narrowly
distributed interstitial pores formed between the particles which
els They investigated the membrane performance using gas fluxes water
permeability and the retention characterization of dextran molecules From these tests
they concluded that the narrow discriminating layer made of the latex particles leads to a
highly efficient composite membrane
27 Proteins
Proteins are natural polyamides comprised of about 20 different α-amino acids
varying hydrophobicity [Norde 1998] Proteins are more or less amphiphilic and
highly surface active because of the number of amino acid residues in the side groups
31
Proteins are polymers of L-α-amino acids [Solomon and Fryhle 2000] The α refers to a
carbon with a primary amine a carboxylic acid a hydrogen and a variable side-chain
group designated as R Carbon atoms rent groups are asymmetric and can
exhib l
s
e
on
with four diffe
it two different spatial arrangements (L and D configurations) due to the tetrahedra
nature of the bonds The L refers to one of these two possible configurations Amino
acids of the D-configuration are not found in natural proteins and do not participate in
biological reactions Figure 2-8 shows the chiral carbon in 3-D as the L isomer Protein
consist of twenty different amino acids differentiated by their side-chain groups [Norde
1998] The side-chain groups have different chemical properties such as polarity charg
and size and influence the chemical properties of proteins as well as determine the
overall structure of the protein For instance the polar amino acids tend to be on the
outside of the protein when they interact with water and the nonpolar amino acids are
the inside forming a hydrophobic core
Figure 2-8 L-α-amino acid
The covalent linkage between two amino acids is known as a peptide bond A
peptide bond is formed when the amino group of one amino acid reacts with the carboxyl
group of another amino acid to form an amide bond through the elimination of water
This arrangement gives the protein chain a polarity such that one end will have a free
amino group called the N-terminus and the other end will have a free carboxyl group
called the C-terminus [Solomon and Fryhle 2000] Peptide bonds tend to be planar and
32
give the polypeptide backbone rigidity Rotation can still occur around both of the α-
carbon bonds resulting in a polypeptide backbone with different potential conformations
relative to the positions of the R groups Although many conformations are theoretically
possible interactions between the R-groups will limit the number of potential
nd to form a single functional conformation In other words
the conformation or shape of the protein is
α-helix
can interact with other
secondary structures within the sam
conformations and proteins te
due to the interactions of the chain side
groups with one another and with the polypeptide backbone The interactions can be
between amino acids that are close together as in a poly-peptide between groups that are
further apart as in amino acids or even on between groups on different polypeptides all
together These different types of interactions are often discussed in terms of primary
secondary tertiary and quaternary protein structure
The primary amino acid sequence and positions of disulfide bonds strongly
influence the overall structure of protein [Norde 1986] For example certain side-chains
will promote hydrogen-bonding between neighboring amino acids of the polypeptide
backbone resulting in secondary structures such as β-sheets or α-helices In the
conformation the peptide backbone takes on a spiral staircase shape that is stabilized by
H-bonds between carbonyl and amide groups of every fourth amino acid residue This
restricts the rotation of the bonds in the peptide backbone resulting in a rigid structure
Certain amino acids promote the formation of either α-helices or β-sheets due to the
nature of the side-chain groups Some side chain groups may prevent the formation of
secondary structures and result in a more flexible polypeptide backbone which is often
called the random coil conformation These secondary structures
e polypeptide to form motifs or domains (ie a
33
tertiary structure) A motif is a common combination of secondary structures and a
domain is a portion of a protein that folds independently Many proteins are composed o
multiple subunits and therefore exhibit quaternary structures
271 Interaction Forces between Proteins
Proteins in aqueous solution acquire compact ordered conformations In such a
compact conformation the movement along the polypeptide chain is severely restrict
implying a low conformational entropy The compact structure is possible only if
interactions within the protein molecule and interactions between the protein molecule
and its environment are sufficiently favorable to compensate for the low conformational
entropy [Malmsten 1998] Protein adsorption study is often focused on structural
rearrangements in the protein molecules because of its significance to the biological
functioning of the molecules and the important role such rearrangements play in the
mechanism of the adsorption process Knowledge of the majo
f
ed
r interaction forces that act
structures helps to understand the behavior
of pro
arge
a
ded
between protein chains and control the protein
teins at interface These forces include Coulomb interaction hydrogen bonds
hydrophobic interaction and van der Waals interactions
Coulombic interaction Most of the amino acid residues carrying electric ch
are located at the aqueous boundary of the protein molecule An the isoelectric point
(IEP) of the protein where the positive and negative charges are more or less evenly
distributed over the protein molecule intramolecular electrostatic attraction makes
compact structure favorable to proteins Deviation to either more positive or more
negative charge however leads to intramolecular repulsion and encourages an expan
structure Tanford [1967] calculated the electrostatic Gibbs energy for both a compact
impenetrable spherical molecule (protein) and a loose solvent-permeated spherical
34
molecule (protein) over which the charge is spread out From the results he found th
the repulsion force was reduced at higher ionic strength d
at
ue to the screening action of ion
ble
e
the
d the
tween
dehyd
e
ent
oles
tein
Hydrogen bond Most hydrogen bonds in proteins form between amide and
carbonyl groups of the polypeptide backbone [Malmsten 1998] The number of availa
hydrogen bonds involving peptide units is therefore far greater than that involving sid
chains Because α-helices and β-sheets are aligned more or less parallel to each other
interchain hydrogen bonds enforce each other Kresheck and Klotz [1969] examine
role of peptide-peptide hydrogen bonds and concluded that hydrogen bonds between
peptide units do not stabilize a compact structure of protein However because the
peptide chain is shielded from water due to other interactions hydrogen bonding be
peptide groups do stabilize α-helical and β-sheet structures
Hydrophobic interaction Hydrophobic interaction refers to the spontaneous
ration and subsequent aggregation of non-polar components in an aqueous
environment In aqueous solutions of proteins the various non-polar amino acid residues
will be found in the interior of the molecule thus shielded from water The
intermolecular hydrophobic interaction for the stability of a compact protein structure
was first recognized by Kauzmann [1959] If all the hydrophobic residues are buried in
the interior and all the hydrophilic residues are at the outermost border of the molecul
intramolecular hydrophobic interaction would cause a compact protein structure
However geometrical and other types of interactions generally cause a fraction of the
hydrophobic residues to be exposed to the aqueous environm
Van der Waals interaction The mutual interaction between ionic groups dip
and induced dipoles in a protein molecule cannot be established as long as the pro
35
structure is not known in great detail Moreover the surrounding aqueous medium also
contains dipoles and ions that compete for participation in the interactions w
the protein molecule Dispersion interactions favor a compact structure However
because the Hamaker constant for proteins is only a little larger than that of water the
resulting effect is relatively small [Nir 1977]
272 β
ith groups of
rgiles et al 1996
Floeg
) is degraded [Floege 2001] Renal
β2M from the serum resulting in an increase in β2M
conce well
the
)
a
al
2-Microglobulin (β2M)
The protein β2M is of particular interest because it is involved in the human
disorder dialysis-related amyloidosis (DRA) [Geiyo et al 1985 A
e 2001] DRA is a complication in end stage renal failure patients who have been on
dialysis for more than 5 years [Bardin et al 1986 Drueke 2000] DRA develops when
proteins in the blood deposit on joints and tendons causing pain stiffness and fluid in
the joints as is the case with arthritis In vivo β2M is present as the non-polymorphic
light chain of the class I major histocompatibility complex (MHC-I) As part of its
normal catabolic cycle β2M dissociates from the MHC-I complex and is transported in
the serum to the kidney where the majority (95
failure disrupts the clearance of
ntration by up to 60-fold [Floege 2001] By a mechanism that is currently not
understood β2M then self-associates into amyloid fibrils and typically accumulates in
musculoskeletal system [Homma et al 1989] Analysis of ex vivo material has shown
that the majority of amyloid fibrils in patients with Dialysis Related Amiloidosis (DRA
is present as of full-length wild-type β2M although significant amounts (~20-30) of
truncated or modified forms of the protein are also present [Floege 2001 Bellotti et al
1998] Figure 2-9 shows the ribbon diagram of human β2M Native β2M consists of
single chain of 100 amino acid residues and has a seven stranded β-sandwich fold typic
36
of the immunoglobulin superfamily [Saper et al 1991 Trinh et al 2002] β2M was fi
isolated from human urine and characterized by Berggard et al [1980] in1968 The
normal serum concentration of β
rst
re
f
e
2M is 10 to 25 mgL It is a small globular protein with
a molecular weight of 118 kDa a Strokes radius of 16Aring and a negative charge under
physiological conditions (isoelectric point IEP = 57) β2M contains two β-sheets that a
held together by a single disulphide bridge between the cysteines in positions 25 and 81
[Berggard et al 1980 Parker et al 1982 Cunningham et al 1973] β2M cannot be
removed completely by current dialysis techniques but through a better understanding o
the structure and interaction forces that lead to this structure it will be possible to mor
efficiently remove this problematic protein
Figure 2-9 Ribbon diagram of human β M taken from the crystal structure of the protein
bound to the heavy chain of the MHC class I complex (PDB 1DUZ) [Khal 2000]
273 Serum Albumin
Serum albumin is the most abundant protein found in plasma and is typically
present in the blood at a concentration of 35~
2an et
50gL According to extensive studies about
its physiological and pharmacological properties albumin has a high affinity to a very
wide range of materials such as electrolytes (Cu+2 Zn+2) fatty acids amino acids
metabolites and many drug compounds [Fehske et al 1981 Kraghhansen 1981 Putnam
37
1984 Peters 1985] The most important physiological role of the protein is therefore to
bring such solutes in the bloodstream to their target organs as well as to maintain the pH
and osmotic pressure of the plasma Bovine serum albumin (BSA) is an ellipsoidal
protein with the dimensions of 140 X 40 X 40Aring [Peter 1985] The primary structure is a
single helical polypeptide of 66 kDa (IEP = 47) with 585 residues containing 17 pairs of
as a soft an
rfaces [Kondo et al 1991 Norde et al 1992 Soderquist et al
1980 ikely
is
disulfide bridges and one free cysteine [Dugaiczyk et al 1982] BSA has been classified
d flexible protein because it has a great tendency to change its conformation
on adsorption to solid su
Carter and Ho 1994] and consists of three homologous domains (I-III) most l
derived through gene multiplication [Brown 1976] Each domain is composed of A and B
sub-domains [He et al 1992] The secondary structure of human serum albumin (HSA)
shown in Figure 2-10
Figure 2-10 Secondary structure of human serum albumin (HSA) with sub-domains
[Zunszain et al 2003]
HSA has the same structure domains with the serum albumin from other species
such as BSA [Brown 1976] Although all three domains of the albumin molecule have
similar three-dimensional structures their assembly is highly asymmetric [Sugio et al
38
1999] Domains I and II are almost perpendicular to each other to form a T-shaped
assembly in which the tail of subdomain IIA is attached to the interface region between
sub-domains IA and IB by hydrophobic interactions and hydrogen bonds In contrast
domain III protrudes from sub-domain IIB at a 45ordm angle to form the Y-shaped assembly
for domains II and III Domain III interacts only with sub-domain IIB These features
make the albumin molecule heart-shaped
28 Protein Adsorption
Protein adsorption studies date back to the 1930rsquos At the beginning these studies
mainly focused on the determination of the molecular weight electrophoretic and
ral
rearrangem
adsorption and biocompatibility of the sorbent materials were investigated [Norde 1986]
There are interaction forces at the interfaces between protein molecules and latex
particles These forces are mainly divided into the following groups hydrophobic
interaction ionic interaction hydrogen bonding and van der Waals interaction [Andrade
1985]
Hydrophobic interaction It is known that hydrophobic interaction has a major
role in protein adsorption phenomena The adsorption of proteins on the low charged
latex particles occurs by this interaction force Generally monomers such as styrene offer
hydrophobic surfaces th
by this interaction force is maximum at the isoelectric point (IEP) of the protein and the
strength [Suzawa et al 1980 1982 Shirahama et al 1989 Kondo et al 1992] By the
chromatographic applications Later the adsorption mechanism especially the structu
ents was studied Recently the studies of the relation between protein
281 Interaction between Protein Molecule and Latex Particle
at protein molecules adsorb to The amount of adsorbed protein
pH at maximum adsorption shifts to a more acidic region with an increase in ionic
39
reports [Suzawa et al 1980 1982 Lee et al 1988] protein adsorption was greater
hydrophobic surface than on a hydrophilic one implying that hydrophobic interaction is
on a
s
ydrophilic compound such as acrylic acid (AA) or methacrylic acid (MAA)
Ionic
ely
t
ces
n force is operative over small
distan e
ble
n
nt dialysis membrane therapy for end stage renal disease (ESRD)
one of most dominant forces in protein adsorption
Ionic interaction Negatively charged latex particles have ionic functional
groups on their surfaces such as salts of sulfonic and carboxylic acid Sulfate group
originate from an initiator such as sodium persulfate and carboxylic groups originate
from a h
bonds are formed between the negative charges of these latex particles and positive
surface charges of protein molecules The conventional low-charged latex particles rar
form these ionic bonds
Hydrogen bonding Hydrogen bond is a strong secondary interatomic bond tha
exists between a bound hydrogen atom (its unscreened proton) and the electrons of
adjacent atoms [Callister 1999] Protein can be adsorbed on hydrophilic polar surfa
through hydrogen bonding Hydrogen bonds are frequently formed between hydroxyl-
carbonyl or amide-hydroxyl Hydroxyl-hydroxyl or amide-hydroxyl bonds are also
formed in protein adsorption
Van der Waals interaction This interactio
ces only when water has been excluded and the two non-polar groups come clos
to each other Lewinrsquos calculation showed that the van der Waals interaction is negligi
compared with the forces involved in the entropy increases ie hydrophobic interactio
[Lewin 1974]
29 Hypothesis for Toxin Removal
As mentioned earlier insufficient removal of middle molecular range toxins is a
major drawback of curre
40
patien
is
y
ese
haracteristics over the last decade However removal
effici
ing a novel membrane design composed of an assembly of engineered
polymeric latex particles s ions Important factors
will i
e
ith a
is established it is
expec
e
ts This may cause destructive arthritis and carpal tunnel syndrome inhibit the
immune system and accelerate cardiovascular disease leading to death among dialys
patients In order to overcome these complications artificial dialysis membranes have
been developed to increase the capacity for removing middle molecular weight toxins b
changing the pore size of dialysis membranes and using new materials that adsorb th
toxins for improved removal c
ency is not as high as those achieved by a normal healthy kidney
With the knowledge obtained from all above literatures the following hypothesis
has been established for the development of a membrane for the successful removal of
the target protein β2-Microglobulin (β2M) without the removal of serum albumin This
will be done us
ynthesized to predetermined specificat
nclude pore size and surface chemistry
291 Toxin Removal by Size Sieving Based on Monodispersed Pore Size
The packing of monodispersed spherical particles can lead to the formation of
porous layers suitable for use as filters and membranes [Hsieh et al 1991] The pore siz
is defined as the largest spherical particles that can pass through the interstitial spaces
It is well known that monodispersed spherical particles can be obtained from the
seed emulsion polymerization method Many commercially available latex particles are
synthesized by this technique and are inexpensive When the defect free membrane w
monodisperse pore size distribution based on a particular particle array
ted to outperform the traditional hemodialysis membrane limitation which is a
broad range of pore size distribution When spherical particles pack in regular crystallin
arrays a number of packing geometries such as hexagonal closest packing cubic closest
41
packing and body-centered cubic packing are possible The pore size of the array
depends on these packing geometries as well as particle size Theoretical pore size can be
calculated from the simple geometry of the arr
ay Figure 2-11 shows the relationship
between particle size and pore size
0001
001
01
001 01 1 10
Por
e
1
10
microm
) S
ize
(
Particle Diameter (microm)
Body-Centered Cubic ArrayClosest Packed Array
0001
Siz
e (
Particle Diameter (microm)
001
01
001 01 1 10
Por
e
1
10
microm
)
Body-Centered Cubic ArrayClosest Packed ArrayBody-Centered Cubic ArrayClosest Packed Array
and closed packed arrays [Steve et al 1999]
McDonald and his coworkers [Ramakrishnan et al 2004 Steve et al 1999] have
described the fabrication of latex composite membranes and their performance in
previous papers It is from this prior research and knowledge gained from conversations
with Dr McDonald that this current research has been initiated [McDonald 2003] The
focus of this thesis is the extension of McDonaldrsquos research in the area of composite
membranes to medical application such as dialysis membranes
292 Toxin Removal by Selective Adsorption on Engineered Latex Particles
The flexibility in latex surface properties is a significant advantage to this
materialrsquos ability to separate proteins since the selectivity is strongly dependent on the
charge interactions of both the protein and the latex particles under the conditions of
Figure 2-11 The relationship between pore size and particle size in body-centered cubic
42
separation [Menon et al 1999 Chun et al 2002] First all sorbents were designed to
have negative surface properties because most plasma proteins in blood are negative and
should not be removed with charge interaction between proteins and sorbents at
physiological condition (pH74) It was reported that a negatively charged surface was
more blood compatible than a positive one [Srinivasan et al 1971] The toxin protein β2-
Microglobulin (β2M) can be selectively adsorbed on the surface of latex particles by the
design of a suitable hydrophobic portioned surface where only β2M protein can be
anchored with hydrophobic interactions Albumin adsorption on the latex particle is not
side-on m
adsorption of β M protein on the engineered latex particles
allowed because charge repulsion is more dominant than hydrophobic interaction with
ode The figure 2-12 shows the schematic representation of the selective
2
PMMAPAAShell
COO-
OSO3-
OSO3-
PSCore
COO-
-
-
-
-
-
-
Albumin-
Chargerepulsion
--
-
- -
- -
- --
Hydrophobic
β2-Microglobulin
2
Interaction
β -Microglobulin
PMMAPAAShell
COO-
-
-
-
-
-
-
--
-
-
-
-
-
Albumin-
Chargerepulsion
OSO3-
OSO3-
PSCore
COO-
--
-
- -
--
-
- -
- -
- --
- -
- --
Hydrophobic
β2-Microglobulin
2
particles at pH 74
Interaction
β -Microglobulin
Figure 2-12 Schematic representation of the protein adsorption on the core shell latex
43
Hydrophilichydrophobic microdomain structures were proven to be more blood
compatible [Mori et al 1982 Higuchi et al 1993 Deppisch et al 1998] The
optimization of suitable hydrophobic to hydrophilic ratio is also important for the
biocompatibility The monomers such as styrene (St) methyl methacrylate (MMA) and
acrylic acid (AA) are widely used hydrophobic and hydrophilic monomer models in
emulsion polymerization process
In summary the background and fundamental literature survey about the history
material properties and limitation of hemodialysis membrane latex particle preparation
surface chemistry and manufacturing process target proteins and protein adsorption and
finally the hypothesis for a toxin removal with high separation efficiency have been
suggested This research focuses on such hypothesis and the materials and
characterization methodology for achievement of suggested hypotheses are described in
the next chapter
CHAPTER 3 EXPERIMENTAL AND CHARACTERIZATION METHODOLOGY
As mentioned earlier the goal of this study is to prepare polymeric latex particles
with tailored properties to maximize separation of toxin molecules and to investigate
fundamental interactions between the applied particles and molecules in the
system to optimize the membrane performance for hemodialysis applications Polymeric
latex particles wit
the
biological
h monodisperse size distribution to obtain uniform pore size and
various size ranges to utilize membrane construction are necessary Surface engineering
with various combination of hydrophobichydrophilic domain on the surface of latex
particles is expected to affect the removal of target protein by selective adsorption and to
improve the biocompatibility of membrane Therefore the materials and characterization
methodology are addressed in this chapter
31 Materials
Styrene (St) monomer used for a seed and a core particle was purchased from
Fisher Scientific and used without any other purification process Acrylic acid (AA) and
methyl methacrylate (MMA) monomers introduced for shell formation were purchased
from Fisher Scientific and used without any other purification process Sodium persulfate
(SPS) and sodium bicarbonate (SBC) were obtained from Fishers Scientific and used as
received Divinylbenzene (DVB) crossliking agent was purchased from Aldrich The
anionic surfactant Aerosol regMA80-I [sodium di(13-dimethylbutyl) sulfosuccinate] was
kindly donated by Cytec 78-80 Aerosol regMA80-I is mixed with isopropanol and
water Its critical micelle concentration (CMC) is 245 mM (119) Ion exchange resin
44
45
AGreg 501-x8 Resin (20-50 mesh) was purchased from Bio-Rad Laboratories Inc It is a
mixed-bed resin with H+ and OH- as the ionic forms and is 8 crosslinked Bovine serum
albumin (BSA) (heat shock treated) was purchas Fisher Scientific The isoelectric
point of t lin
(β2M)
as
gma
ed from
he BSA is 47-49 and the molecular weight is 66300Da β2-Microglobu
was purchase from ICN Biomedicals Inc and was separated from patients with
chronic renal disease and lyophilized from ammonium bicarbonate The molecular
weight by SDS-PAGE was approximately 12000Da BSA and β2M proteins were used
received without further purification process In order to investigate the protein
adsorption evaluation bicinchoninic acid protein assay kits were purchased from Si
(Cat BCA-1) and Pierce Biotechnology (Cat 23225) The chemical structures for the
main chemicals are shown in Figure 3-1
CH2 CH
Styrene (Mw = 10415)
CH2 CHCH2 CH
Styrene (Mw = 10415)
CH2 CH
Acrylic acid (Mw = 7206)
C
OH
O
CH2 CH
Acrylic acid (Mw = 7206)
C
OH
O
CH CH2
C
OCH3
O
CH
CH CH3
2
C
OCH3
O
CH3
Methyl methacrylate (Mw = 10012)Methyl methacrylate (Mw = 10012)
CH2 CH
Divinylbenzene (Mw = 1302)
CH CH2
CH2 CH
Divinylbenzene (Mw = 1302)
CH CH2CH CH
+(Na) -(O3S)
CH -C-O-CH-CH -CH-CH3
O
2 2 3
O CH CH3
CH3 CH3
CH-C-O-CH-CH2-CH-CH3
Anionic surfactant (MA80-I) (Mw = 3885)
+(Na) -(O3S)
CH -C-O-CH-CH -CH-CH3
O
2 2 3
O CH CH3
CH3 CH3
CH-C-O-CH-CH2-CH-CH3
Anionic surfactant (MA80-I) (Mw = 3885)2
+(Na)-(O) S OO
O O
- +
Initiator sodium persulfate (Mw = 23811)
O SO
(O) (Na)+ -
O O
- +(Na) (O) S OO
O SO
(O) (Na)+ -
O O
- +
Initiator sodium persulfate (Mw = 23811) Figure 3-1 Chemical structure of main chemicals
(Na) (O) S OO
O SO
(O) (Na)
46
32 Latex Particle Preparation
The experimental set up and the particle preparation schemes for various types and
size ranges of latex particles are shown in Figure 3-2 and 3-3 In the experimental setup a
mechanical glass stirrer is connected from a motor and Teflon stirrer bar is located at
end of glass stirrer The agitation rate is precisely adjusted by controller motor Paar glas
reactor with 1L volume is partially in silicon oil bath on hot plate The reactor has fo
openings for a stirrer a thermometer a nitrogen gas inlet and a reactant feeding lnlet
The reactants for latex polymerization are fed by precisely controlled by meterin
The particle preparation is described in each latex preparation section
the
s
ur
g pump
Figure 3-2 Experimental setup for semi-continuous emulsion polymerization
1 Motor 2 Mechanical stirrer bar 3 Thermometer 4 Inlet of N2 5 Inlet for initiator and monomer 6 Glass reactor (l L) 7 Oil bath 8 Hot plate 9 Pump for initiator 10 Pump for monomer 11 Tubes for reactant feed
47
Styrene monomer
Bare PS
PSPMMA100
PSPMMA90PAA10
PSPMMA P75 AA25
Gr
win
ticle
size
PS seedparticles
og
par
Styrene monomer
Bare PS
PSPMMA100
PSPMMA90PAA10
PSPMMA P75 AA25
Gr
win
ticle
size
PS seedparticles
og
par
Figure 3-3 The particle preparation scheme in various types and size ranges of latex particles
321 Preparation of Seed Latex Particles
Polystyrene seed particles were synthesized through a typical semi-continuous
emulsion polymerization Polystyrene latex particles were prepared in a four necked Paar
glass vessel equipped with a mechanical glass stirrer thermometer glass funnel for
nitrogen gas purge and a tube for monomer feeding The reactor was placed in a silicon
oil bath on a hot plate for homogenous and stable tempe he vessel was
firstly charged with de-ionized water the emulsifier so itiator
sodium bicarbonate (SBC) styrene (St) monomer divinylbenzene (DVB) monomer
under nitrogen gas atmosphere and then heated to 72plusmn2 as
llowed to continue for another hour Second reactants St and DVB monomers SPS
initiator and SBC were fed separately using fine fluid metering pumps (Model RHSY
Fluid metering INC NY) under a nitrogen gas atmosphere for 2h After complete feeding
rature control T
dium persulfate (SPS) in
oC for 1h The reaction w
a
48
of all reactants the reaction was continued for another hour at elevated temperature 80plusmn1
oC and then cooled to room temperature by removal of the oil bath
322 Preparation of Seeded Latex Particles
The particle growth was achieved by seeded continuous emulsion polymerization
The vessel was charged with the polystyrene seeds and de-ionized water to make desired
solid content and heated up to 72plusmn2 oC for 1h under nitrogen gas atmosphere Then St
and DVB monomers and solution of SPS and SBC in de-ionized water were continuously
added using metering pump with a precisely controlled feed rate After feeding all
reactants the reaction was continued for three hours keeping temperature of 80plusmn2 oC and
then the reaction vessel was cooled down to room temperature
Monodisperse spherical polystyrene seeds with a 280nm mean diameter were used
e-polymerized polystyrene seeded emulsion
was charged in a 500m
323 Preparation of Core Shell Latex Particles
for core shell structured latex particle The pr
l glass flask under a nitrogen gas atmosphere De-ionized water
was added to achieve the desired solid content The emulsion was heated to 80plusmn2oC for
10 hour Monomers initiator and SBC to form the shell were added by fluid metering
pumps (LAB PUMP JRRHSY model Fluid metering Inc USA) with fine controlled
feed rate Flow rate can be adjusted by control ring graduated in 450 division from 0 to
100 flow which is in the range of 10mlmin respectively The reaction was sustained
for additional 30 hours at 90plusmn2oC then cooled down to room temperature
324 Purification of Synthesized Latex Particles
Synthesized latex particles were purified with an ion exchange resin 200g (10
ww polystyrene solid content) of emulsion were mixed with 4g of ion exchange resin
and stirred for 40 minute to remove unreacted monomers initiators and other impurities
49
Then the emulsion was diluted with DI water to a specific degree of solid content as
needed
33 Characterization
331
solid
termined
oscopy
d
hours
Dried latex (10mg) was mixed with
250 m on
Degree of Conversion
The degree of monomer to polymer conversion was determined gravimetrically
following the procedure described by Lee et al [1995] before the latex cleaning process
The synthesized emulsion raw materials were weighed and dried in a conventional oven
at 120oC for 30min to evaporate any unreacted monomers and water The remaining
was weighed and the degree of conversion was determined This process was repeated
three times and a mean value of the degree of conversion was de
332 Fourier Transform Infrared (FTIR) Spectr
A molecule can absorb only select frequencies (energy) of infrared radiation which
match the natural vibration frequencies The energy absorbed serves to increase the
amplitude of the vibrational motions of the bonds in the molecule [Pavin et al 1996]
Only those bonds which have a dipole moment that changes as a function of time can
absorb the infrared radiation The range of wavelengths for infrared radiation is between
25 microm (4000 cm-1) and 25 microm (400cm-1)
The synthesis of the latex particles such as polystyrene (PS) homopolymer and
PSPMMA PSPMMA90PAA10 PSPMMA75PAA25 core-shell copolymers were verifie
with FTIR Purified latex particles were dried in vacuum oven at 40oC for 24
before analysis in the FTIR (Nicolet Magma USA)
g of KBr which had also been dried in vacuum oven at 120oC for 3h Transmissi
spectra using the drift mode were plotted with 128 scans and 4cm-1 resolution
50
333 Quasielastic Light Scattering (QELS)
Latex particle size was measured by the Brookhaven ZetaPlus particle size
analyzer The solid concentration as 0001 (wv) This
ation spectroscopy (PCS) of quasielastically scattered light
For th s
ed using a FE-SEM (JEOL
JSM-
ith
ent
01
us zeta-
potential analyzer It measures the electrophoretic mobility velocity of charged colloidal
of latex particles used w
instrument uses photon correl
is technique time dependent interference patterns of light scattered from particle
in the sample cell are analyzed The interference pattern changes due to Brownian
motion giving rise to fluctuations in scattering intensity The frequency of these
fluctuations depends on the particlersquos diffusion constant that is inversely proportional to
the particle diameter
334 Field Emission-Scanning Electron Microscopy (FE-SEM)
Particle size and surface morphology were characteriz
6335F Japan) A diluted suspension of latex particles in water was dropped onto a
silicon wafer at room temperature and allowed to dry The sample was then coated w
the thinnest layer of carbon needed to obtain the required conductivity for this instrum
Secondary electron image mode was used with a 15KV of accelerating voltage The
magnification range used was between 10000X and 70000X
335 Zeta Potential Measurement
Synthesized and purified latex particles were diluted with de-ionized water to 0
wt and adjusted six different pH values 205 345 481 565 643 and 747 These
particle suspensions were then transferred to standard cuvettes for zeta potential
measurement At least two runs of ten measurements were taken for each sample and
averaged
The zeta potential measurement was carried out using Brookhaven ZetaPl
51
particles in solution and calculate zeta-potential by using the Smoluchowski equation
The f quency
gnitude
umin (BSA) as a standard model protein and β2-Microglobulin
uffer (PB) and
phosp
5
h the selected protein
e 005 01 03 05 and 07 mgml and those for
β2M
d at
teins in the supernatant after the centrifugation process using the
bicinc t
requency of laser light passing through the sample and is compared to the fre
of a reference beam The shift in the frequency called a Doppler shift and the ma
of the shift correspond to the polarity and the magnitude of the electrophoretic mobility
respectively The zeta potential is calculated from the solution conditions and the
measured mobility
336 Protein Adsorption
Protein adsorption experiments of the synthesized latex particles were performed
with bovine serum alb
(β2M) as a target protein in two types of buffer solution phosphate b
hate buffered saline (PBS) To make 5 mM of PB solution 0345g of sodium
phosphate monobasic (NaH2PO4H2O) was dissolve in 500ml of de-ionized water To
make PBS solution 0345g of sodium phosphate monobasic (NaH2PO4H2O) and 4178g
(143mM) of sodium chloride (NaCl) were dissolved in 500ml of de-ionized water The
synthesized latex particles were diluted with each buffer to have a solids content of 0
(ww) adjusted to pH values of 32 48 and 74 and mixed wit
BSA concentrations were chosen to b
were 0015 0030 0045 and 0060mgml The mixture was gently rotated using
the shaker Labquakereg (BarnsteadThermolyne Model 4002110 USA) with 8 RPM in
the incubator at 37oC for 12 hours before the latex-protein mixture was centrifuge
13000 rpm for 15min The amount of protein adsorbed was determined by quantifying
the free pro
honinic acid (BCA) assay method [Lowry et al 1951 Smith et al 1985 Baptista e
al 2003 Wiehelman et al 1988 Brown et al 1989] BCA assay kits consist of Reagent
52
A containing bicinchoninic acid sodium carbonate sodium tartrate and sodium
bicarbonate in 01N NaOH with pH=1125 and Reagent B containing 4 (wv) copper
(II) sulfate pentahydrate The BCA working reagent was prepared by mixing 50 parts of
Reagent A with 1 part of Reagent B 100microl of protein supernatant was then mixed with
2ml of BCA working reagent in a UV cuvette Incubation was allowed to continue
at room temperature
further
until color developed about 2h The absorbance at 562nm was
pectrophotometer (Perkin-Elmer Lambda 800 USA)
Unkn
Separate the p rticles in centrifuge mix supernatant with BCA reagents
measured using a UV-VIS s
own concentration of sample protein was determined by comparison to a standard
of known protein concentrations The adsorbed amount per unit surface area was
determined by the mass balance of the protein after adsorption process The simple
schematic of the procedure for a protein adsorption test is shown in Figure 3-4
Incubate mixture with agitation for 12hrs at 37oC
aand develop the color by incubation for 2hrs at room temperature
Suspend latex particles in aqueous buffer media
Add the dissolved target protein
Measure color intensity using UVVis spectroscopy at 562nm
Suspend latex particles in aqueous buffer media
Separate the p rticles in centrifuge mix supernatant with BCA reagents
Add the dissolved target protein
Incubate mixture with agitation for 12hrs at 37oC
aand develop the color by incubation for 2hrs at room temperature
Measure color intensity using UVVis spectroscopy at 562nm
Figure 3-4 Schematic of the procedure for a protein adsorption test
53
337 Blood Biocompatibility by Hemolysis Test
Hemolysis is the destruction of red blood cells which leads to the release of
hemoglobin from within the red blood cells into the blood plasma Hemolysis testing is
used to evaluate blood compatibility of the latex particles and the damaged to the red
blood cells can be determined by monitoring amount of hemoglobin released The red
blood cells (RBCs) were separated by centrifuging blood at 1500 rpm for 15 minutes
Figure 3-5 shows the phase separated blood with plasma white blood cells and red
cells after centrifugation process Separated RBSs were then washed with isotonic
phosphate buffer solution (PBS) at pH 74 to remove debris and serum protein Th
process was repeated 3 times
blood
is
Figure 3-5 Separation of RBC from whole blood by centrifuge process
Prepared latex particles were re-dispersed in PBS by sonification to obtain
homogeneously dispersed latex particles 100microl of the mixture of red blood cell (3 parts)
and PBS (11parts) was added to 1ml of 05 (ww) particle suspension PBS was used as
a negative control resulting 0 hemolysis and DI water used as a positive control to
produce 100 hemoglobin released by completely destroyed RBCs The mixture was
incubated in water bath with gentle shaking for 30 minutes at 37oC and then centrifuged
at 1500 rpm for 15 minutes 100microl of the supernatant was mixed with 2ml of the mixture
) (EtOHHCl = 2005 ww) to prevent the
White blood cells
Plasma
Red blood cells
of ethanol (99) and hydrochloric acid (37
54
precipitation of hemoglobin In order to remove remaining particles the mixture was
centri fuged again with 13000 RPM at room temperature The supernatant was then
transferred into the UV cuvette The amount of hemoglobin release was determined by
monitoring the UV absorbance at a wavelength of 397nm The schematic of the
procedure for hemolysis test is shown in Figure 3-6
Separate red blood cells (RBCs) from blood
Mix RBCs with phosphate buffer solution (PBS)
Wash and re-suspend RBC with PBS repeat three times
Add la l of RBC and incu in at 37oC
tex particle suspension into 100mbate sample with agitation for 30m
Separate particle ith EtOHHCl solutions and mix the supernatant w
Evaluate free hemoglobin in the solution using UVVis spectroscopy at 397nm
Separate red blood cells (RBCs) from bloodSeparate red blood cells (RBCs) from blood
Mix RBCs with phosphate buffer solution (PBS)Mix RBCs with phosphate buffer solution (PBS)
Wash and re-suspend RBC with PBS repeat three timesWash and re-suspend RBC with PBS repeat three times
Add latex particle suspension into 100ml of RBC and incubate sample with agitation for 30min at 37oC
Add latex particle suspension into 100ml of RBC and incubate sample with agitation for 30min at 37oC
Separate particles and mix the ith EtOHHCl solution supernatant wSeparate particles and mix the ith EtOHHCl solution supernatant w
Evaluate free hemoglobin in the solution using UVVis spectroscopy at 397nmEvaluate free hemoglobin in the solution using UVVis spectroscopy at 397nm
Figure 3-6 Schematic of the procedure for hemolysis test
In summary a description of the experimental materials and characterization
methodology has been explained In the next chapter the results and discussion of the
data obtained from these experiments is addressed
CHAPTER 4 RESULTS AND DISCUSSION
41 Polymerization of Latex Particles
411 Polystyrene Seed Latex Particles
Polymer particles have many different applications including spacer particles
lubricants packing materials for chromatography standard particles and diagnostic
drugs All applications strongly require these particles to have a uniform particle size
Uniform particle size is the first requirement for a latex composite membrane formed by
particle arrays with the interstitial spaces serving as pores for size discriminations [Jons
et al 1999 Ramakrishnan et al 2004] Although suspension polymerization has been
mainly used as one of conventional polymerization methods to prepare uniform particles
the uniformity of the recovered particles is insufficient for membrane use There is
another method known as seed emulsion polymerization where a vinyl monomer is
absorbed into fine monodiserse seed particles and polymerization causes the monomer to
crease the sizes of the seed particles uniformly In order to obtain monodisperse larger
particles by this m
particles and polymerization of the monomer is repeated
In this study cross-linked polystyrene seed latex particles were firstly synthesized
in the presence of styrene monomer divinylbenzene crosslink agent sodium persulfate
initiator sodium bicarbonate buffer and the Aerosol regMA80-I [sodium di(13-
dimethylbutyl) sulfosuccinate] surfactant using a batch type semi-continuous emulsion
polymerization process in which one or more components can be added continuously
in
ethod the procedure of absorption of monomer into fine polymer
55
56
Styrene (St) monomer and divinylbenzene (DVB) crosslink agent as well as initiator and
buffer are continuously added to enlarge the seed latex particles Various profiles of
particle nucleation and growth can be gene different orders of component
addition during polymerizati f this process such as easy
control of the polymeriza particle number
and particle morphology The main goal in
this process is to avoid secondary particle
rated from
on There are several advantages o
tion rate monodisperse particle size and
colloidal stability copolymer composition
formation leading to a monodisperse particle
size distribution Figure 4-1 shows the schematic representation of seed latex particle
preparation and growth
Monomer droplet
Seed particles Amount of surfactantsis variable
Enlarged final Add monomers
PS
PS
PSs4
particles Number of particlesare constant
Seed code
PSs1
s2
s3
Monomer droplet
Seed particles Amount of surfactantsis variable
Enlarged final Add monomers
PS
PS
PSs4
tion The
particles Number of particlesare constant
Seed code
PSs1
s2
s3
Figure 4-1 Schematic representation of semi-continuous seed latex particles preparation and growth
After the first synthesis of a small size of crosslinked polystyrene (PS) seed
particles stabilized by surfactants styrene DVB and initiator are continuously fed into
the system to enlarge the seed latex particles maintaining a narrow size distribu
recipes and reaction conditions for synthesizing these seed particles are summarized in
57
Table 4-1 The original recipe for latex particle preparation from McDonald was
modified for this emulsion system [McDonald 2003]
Table 4-1 The polymerization recipe of polystyrene (PS) seed particles Latex label PSS259 PSS233 PSS207 PSS181
Temperature (oC) 75 plusmn 5 75 plusmn 5 75 plusmn 5 75 plusmn 5
Divinylbenzene (g) 052 052 052 052
MA 80 (g) 259 233 207 181
NaHCO
Watera (g) 1315 1315 1315 1315
Styrene (g) 9948 9948 9948 9948
112 112 112 112
Na2S2O8 (g) 190 190 190 190
Waterb (g) 200 200 200 200
Reaction time (min) 140 140 140 140
Solids content 396 396 396 396
Mean particle diameter (nm) 126plusmn75 171plusmn39 182plusmn41 216plusmn53
Conversion () 965 979 957 971
3 (g)
Four different seed latex particles were synthesized based on surfactant amount in
order to determine the optimum condition for stable and monodisperse latex particles
The emulsion polymerization of latex particles has to be carried out in a narrow range of
surfactant causes flocculation or phase separation leading to broad particle
size distribution and an unstable emulsion sy
surfactant concentration where particles are stable [Nestor et al 2005] A wide range of
concentrations
stem Therefore the range of surfactant
amount was varied from 181g to 259g in increments of 026g in each latex label Other
reactants amounts were kept constant Watera is the initial charge amount and waterb
represents the amount of aqueous solution containing dissolved initiator and buffer
58
which are fed continuously after the seed particle creation The extent of monome
polymer conversion was obtained by gravimetric calcu
r to
lation and was more than 95 at
cripts such as
S207 and S181 i the surf mount added for the seed latex
nversion indi perce experi obtain
content divided by the theoretically estimated solid content and was calculated by the
all seed particles labeled PSS259 PSS233 PSS207 and PSS181 Labeled subs
S259 S233 ndicate actant a
preparation The co cates the ntage of mentally ed solid
following formula
100 colids () times=entsoliatedntentoalExperimentConversion
n the experimenta content is the weight of olid rema after
mulsion at 120oC f in and the estimated solid content is th ht
lated from the recipe
rti as di each t load d
mple of ea pension iluted by (ww)
deionized water and the mean particle diameter was determined using the Brookhaven
ZetaPlus particle size analyzer From these results the seed latex particle size increased
as the amount of surfactant decreased The amount of emulsifier also affects the
polymerization process by changing the number of micelles and their size It is known
that large amount of emulsifier produces larger numbers of smaller sized particles [Odian
1991] and was corroborated in the current work
These synthesized seed latex particles should be of narrow size distribution in order
to obtain monodisperse larger particles The SEM image showing the particle
morphology and size of a sample of polystyrene seed particles can be seen in the Figure
4-2
contdEstim (4-1)
In this equatio l solid the s ining
evaporation of e or 30m e weig
of monomers calcu
As expected the seed pa cle size w fferent for surfactan ing in see
emulsion system A sa ch sus was d 0001 with
59
Figure 4-2 Scanning Electron Micrograph (SEM) of polystyrene seed latex particles (A)
PSS259 (B) PSS233 (C) PSS207 (D) PSS181 (subscripts indicate the amount of surfactant added)
(A)
(B)
60
(C)
(D)
Figure 4-2 Continued
61
s seen in the image the seed particles are very smooth and spherical The
uniformity in the particle size distribution of these PS seed latex particles can also be
seen in the SEM image From this data we concluded that seed particles synthesized in
the presence of a surfactant with the chosen amounts are suitable for use to prepare larger
particles with high size uniformity and smooth spherical morphology
412 The Growth of Polystyrene (PS) Seed Latex Particles
Seeded emulsion polymerization has been conducted for several decades and
various mechanisms have been proposed Gracio et al [1970] suggested that the growing
polymer particles consist of an expanding polymer-rich core surrounded by a monomer-
rich shell with the outer shell providing a major locus of polymerization Seeded
mulsion polymerization is commonly used for preparing latex particles less than 1microm in
size [Cha et al 1995 Zou et al 1990 1992 Gandhi et al 1990 Park et al 1990] Such
latexes can be obtained from the growth of pre-prepared seed particles The seeds
introduced in this process serve as nucleation sites for particle growth By first swelling
the seed latex particles with additional monomer to the desired size polymerization can
then be initiated Several size ranges of monodisperse polystyrene (PS) latex particles
were prepared by this multistep seeded emulsion polymerization method For the
fabrication of particle-based membranes monodisperse latex particles with a variety of
size ranges are required
In this study seed particles from 171nm to 470nm in mean diameter and with a
highly uniform size distribution were prepared and used Watera is the initial charge
mount and waterb represents the amount aqueous solution containing dissolved initiator
and buffer that is continuously fed into system Feed time is precisely controlled by
metering pump The solid contents were maintained between 199 and 300 After
A
e
a
62
polym d as
es
ere 923 8402
n of these latex particles
are sh
ss
erization under these conditions the conversions of the latex particles labele
PS258 PS320 PS370 and PS410 were 952 957 946 and 964 respectively The recip
used in seeded emulsion polymerization to form the particles less than 500nm in mean
diameter are shown at the Table 4-2 The conversions of the latex particles larger than
500nm in mean diameter and labeled as PS525 PS585 PS640 and PS790 w
723 and 969 respectively The recipes used in the preparatio
own at the Table 4-3
A simple calculation can be used to predict the ultimate size that a latex particle can
reach after ending the polymerization Letrsquos suppose that seed density (ρs) and grown
particle density (ρgp) are the same The following equation is obtained from the ma
balance (m = ρV) where m and V are mass and volume of a particle respectively
gp
gp
s
s
Vm
Vm
In this equation m
= (4-2)
be
s and mgp are the weight of seed and total weight of seed and
added monomers respectively and Vs and Vgp are the volume of seed and grown
particle respectively
If we know the mass of monomer to be added the ultimate particle size can
calculated from the equation 4-3
3
2s D m ⎠⎝+ χ
where D
1s D m
⎟⎟⎞
⎜⎜⎛
= (4-3)
the
in
s and Dgp are the diameters for seed and grown particle respectively and χ is
weight of the monomer added Crosslinked PS latex particles with a size range from
258plusmn112 ~ 410plusmn117 in diameter polymerized by the recipes in Table 4-2 are shown
the SEM images in Figure 4-3
63
Table 4-2 Continuous addition emulsion polymerization recipe for growing polystyrene
Latex label PS
(PS) latex particles less than 500nm in size
258 PS320 PS370 PS410
Initial charge
Seed latex (g) 1000171 1000216 1000216 1000320
Watera (g) 1470 2500 1984 2600
06 07 06 06
Fee
Initiator stream
2 2 8
Feed time (min) 0-167 0-140 0-100 0-92
Solid content 284 300 300 200
Mean particle diameter (nm) 258plusmn112 320plusmn154 182plusmn71 410plusmn117
Conv
Monomer continuous
Styrene (g) 800 1200 800 600
Divinylbenzene (g)
d time (min) 0-163 0-110 0-75 0-82
Na S O (g) 07 065 05 05
NaHCO3 (g) 07 065 05 05
Waterb (g) 750 1200 200 370
ersion () 952 957 946 964 The solid content of seed latex for PS258 PS320 PS 370 and PS410 are 392 399 399 anrespectively
d 300
64
Table 4-3 Continuous addition emulsion polymerization recipe for growing polystyrene (PS) latex particles larger than 500nm in size
Latex label PS525 PS585 PS640 PS790
Initial charge
Seed latex (g) 1000390 1000470 1000470 1000320
Watera (g) 1000 520 1200 360
Monomer continuous
Styrene (g) 290 200 400 173
Divinylbenzene (g) 02 014
03 009
Feed time (min) 0-80 0-40 10-76 5-65
Initiator stream
Na2S2O8 (g) 02 014 03 012
NaHCO3 (g) 02 014 03 009
Waterb (g) 200 120 400 200
Feed time (min) 0-85 0-46 0-77 0-75
Solids content 199 200 200 201
Mean particle diameter (nm) 525plusmn151 585plusmn186 640plusmn178 790plusmn693
Conversion () 923 8402 723 969 The solid content of seed latex for PS525 PS585 PS 640 and PS790 are 200 182 193 and 173 respectively
65
Figure 4-3 SEM of PS latex particles less than 500nm in size (A) 258nm (B) 320nm (C)
370nm (D) 410nm
(A)
(B)
66
(C)
(D)
Figure 4-3 Continued
67
article size was well matched to that estimated by the geometrical calculation
From this it can be concluded that the added monomer was consumed for polymer
particle growth with a high degree of conversion From the SEM characterization newly
nucleated particles were not seen as indicated by the narrow particle size distribution
The particles less than 500nm in mean diameter were very spherical in shape with a
highly uniform particle size distribution As the particles were grown larger than 500nm
mean diameter however they became of non-spherical shape with an uneven surface
This irregularity of particle surface can be attributed to the non-homogeneous monomer
swelling into the shell of a growing polymer which can be controlled by the factors such
as temperature agitation speed initiator feeding rate and surfactant amount required to
stabilize the colloidal system The SEM image of latex particles larger than 500nm mean
iameter is shown in Figure 4-4 The particle size distribution is broader than that of the
s particles These particles can however still be used for membrane construction
as a support layer rather than skin layer The support layer does not necessarily have to be
as highly monodisperse as the skin layer which require pores of high uniformity for the
selective removal of toxins
As mentioned earlier the amount of surfactant as well as monomer in an emulsion
system is the primary determinant of the particle diameter [Odian 1991] Anionic
surfactants are generally recommended at the level of 02-30 wt based on the amount
of water [Odian 1991] The critical micelle concentration (CMC) of Aerosolreg MA 80-I
anionic surfactant is 119 The surfactant loading in this emulsion system ranged from
the 121 for SS181 to 171 for SS259
P
d
maller
68
Figure 4-4 SEM of PS latex particles larger than 500nm in size (A) 525nm (B) 585nm
(C) 640nm (D) 790nm
(A)
(B)
69
(C)
(D)
Figure 4-4 Continued
70
The surfactant to monomer ratio refers to the value of the surfactant amount
divided by monomer amount after polymerization ends The surfactant amount is the
weight of the surfactant contained in emulsion and the monomer amount is the total
weight of the monomer added for polymerization to form the ultimate polymer particles
in emulsion As the surfactant to monomer ratio decreases mean particle diameter
increases as described by Odian [1991] Generally lower surfactant concentration forms
fewer micelles resulting in larger particle size [Evans et al 1999] However there is a
plateau in particle mean diameter between a surfactant to monomer ratio of 0005 and
0013 This is due to low polymerization conversion of latex particles from monomer
leading to a smaller particle size than expected This is not a factor of surfactant amount
The dependence of the particle size on the amount of surfactant is shown in Figure 4-5
0
200
400
600
800
1000
0000 0005 0010 0015 0020 0025 0030
Surfactant to monomer ratio (ww)
Part
icle
mea
n di
amet
er (n
m)
Figure 4-5 Dependence of the particle size on the surfactant to monomer ratio
71
413
e
f bare
microdomain structures is important for biocompatibility [Mori et al 1982 Higuchi et al
1993 Deppisch et al 1998] Figure 4-6 shows the schematic of core shell latex particle
structure The recipe for core shell structures is shown in Table 4-4
Core Shell Latex Particles
The latex particles shown in Figure 4-3 (A) and labeled as S258 in Table 4-2 wer
used as seeds to prepare core-shell latex particles because of their high particle
uniformity and smooth surface morphology Methyl methacrylate (MMA) and acrylic
acid (AA) monomers were introduced to increase surface hydrophilicity over that o
polystyrene particles AA is a more hydrophilic monomer than MMA because of the
carboxyl acid group in AA is more favorable for hydrogen boding with water The
surface carboxyl groups on PAA may have many promising applications in biomedical
and biochemical fields [Kang et al 2005] MMA monomer is less hydrophobic than
styrene PMMA is known to more biocompatible than PS but still PMMA is hydrophobic
The optimization of suitable hydrophobic to hydrophilic ratios and control of
Polystyreneseed particle
Core shelllatex particles
PSPMMA100
PSPMMA90PAA10
PSPMMA75PAA25
Polystyreneseed particle
Core shelllatex particles
PSPMMA100
PSPMMA90PAA10
Figure 4-6 Schematic of core shell latex particle structures
PSPMMA75PAA25
72
Table 4-4 The preparation recipe of PS core with various shell latex particles Latex 25 label PSPMMA100 PSPMMA90PAA10 PSPMMA75PAA
Initial charge
PS seed latex (g) 500 500 500a
Monomer continuous
Methly methacylate (g) 320 288 240
Acrylic acid (g) 00 32 80
Initiator stream
Na
258 258 258
Water (g) 1570 1570 1570
Feed time (min) 0-120 0-50 0-60
b 200
Feed time (min) 0-122 0-60 0-65
952
2S2O8 (g) 02 02 02
NaHCO3 (g) 02 02 02
Water (g) 200 200
Solids content 183 183 183
Mean particle diameter (nm) 370plusmn186 370plusmn178 370plusmn191
Conversion () 955 924
Three types of core shell structures were prepared PMMA75PAA25 shell is a
copolymer consisting of the PMMA to PAA ratio of 75 to 25 by weight
PMMA90PAA10 shell is a copolymer with the PMMA to PAA ratio of 90 to 10 by
weight PMMA100 is the PMMA homopolymer shell on PS core The particle size of
these core-shell particles as well as PS latex particle was prepared to be about 370nm in
mean diameter and are characterized to determine their zeta potential protein adsorption
and biocompatibility The image of SEM of PS and core shell particles is shown at Figure
4-7 The synthesized latex particles had a high uniformity in size and a smooth spherical
surface morphology
73
Figure 4-7 Scanning
Electron Micrograph of latex particles (A) PS (B) PSPMMA100 (C) PSPMMA90PAA10 (D) PSPMMA75PAA25
(A)
(B)
74
(C)
(D)
Figure 4-7 Continued
75
42 Characterization of Latex Particles
421 Fourier Transform Infrared Spectroscopy (FTIR)
FTIR spectroscopy has proven to be a powerful tool to characterize polymeric
materials A molecule can absorb only select frequencies (energy) of infrared radiation
which match the natural vibration frequencies The energy absorbed serves to increase
the amplitude of the vibrational motions of the bonds in the molecule [Pavin et al 1996]
Only those bonds which have a dipole moment that changes as a function of time can
absorb infrared radiation The FTIR spectra of polymerized latex particles made during
this work are shown in Figure 4-8 There are number of characteristic peaks for PS latex
particles [Bhutto et al 2003 Li at al 2005] Aromatic C-H stretching vibration is shown
at 3002 cm-1 and 3103 cm-1 and aliphatic C-H asymmetrical and symmetrical stretching
shown at 2900 cm-1and 2850 cm-1 respectively There is an additional carbonyl (C=O)
adsorption at 1730cm-1 wavenumber for all core shell latex particles containing some
amount of either or both PMMA and PAA The broad OH group peak at 3400 cm-1
appear for particles containing some amount of PAA and has intensity dependent on the
amount of PAA in the shell For example the peak intensity of OH groups for
PSPMMA75PAA25 is greater than that of PSPMMA90PAA10 since there is a higher AA
monomer content in PSPMMA75PAA25 than in PSPMMA90PAA10 This OH peak is not
seen for PS and PS core PMMA shell particles because of the absence of OH groups on
PS and PMMA polymer chains
422 Zeta Potential Measurements
The electrical surface property was characterized by zeta potential measurements of
tex particles Zeta potential is an important and useful indicator of surface charge which
can be used to predict and control the stability of colloidal systems The greater the zeta
is
la
76
1520002500300035004000
Wavenumbers (cm-1)
li
Re
ativ
e
Tra
nsm
ttanc
e
Figure 4-8 FTIR spectra of polymerized latex particles (A) ba
PSPMMA100 (C) PSPMMA90PAA10 (D) PSPMM
(B)
(C) P
(D) P
C
C
OCH3
O
CH3
XCH2
CH2 n
CH
CH2 C
C
OCH3
O
XCH2 CH
OC
CH3
Y
OH
(A) Bare PS
500100000
re polystyrene (PS) (B) A75PAA25
PSPMMA100
SPMMA90PAA10
SPMMA75PAA25
77
potential the more likely the suspension is to be stable because the charged particles repel
one another and thus overcome the natural tendency to aggregate The measurement of
zeta potential is often the key ontrol of the interaction of proteins
with solid surfaces such as latex particles
Zeta potential (ζ) is the elec at exists at the shear plane of a particle
which is some small distance from the surface [Myers 1999] The zeta potential of the
particles can be determined by measuring the mobility of the particles in an applied
electric field termed electr its response to an alternating electric
field Colloidal particles dispersed in lu ically charged due to their ionic
characteristics and dipolar attributes The development of a net charge at the particle
surface affects the distribution of ions in the neighboring ng in
an increased concentration of counter ions to the surface Each particle dispersed in a
ns called fixed layer or Stern layer
Outside th compositions of ions of opposite polarities
form ble layer is form
particle-liquid interface This double layer may be considered to consist of two parts an
inner region which inclu nd relatively strongly to the surface and an outer or
diffuse region in which the ion distribution is determined by a balance of electrostatic
forces and random thermal motion The potential in this region therefore decays with the
distance from the surface until at a certain distance it becomes zero Figure 4-9 shows a
schematic representation of a typical ion distribution near a positively charged surface
The Stern surface (also referred to as the outer Helmholtz plane) is drawn through the
to understanding and c
trical potential th
ophoretic mobility or
a so tion are electr
interfacial region resulti
solution is surrounded by oppositely charged io
e Stern layer there are varying
ed in the region of the ing a cloud-like area Thus an electrical dou
des ions bou
center of those ions which are effectively adsorbed to the surface [Hunter 1981] The
78
extent of ion adsorption is determined by electrical and other long-range interactions
between the individual ions and surface of particles The ions outside of the Stern layer
form the diffuse double layer also referred to as the Gouy-Chapman layer [Burns and
Zydney 2000]
Shear plane
Stern layer
ψζ
0
ψs
ψShear plane
Stern layer
ψζ
0
ψs
ψ
t
controlled electric field is applied via electrodes immersed in a sample suspension and
Figure 4-9 Schematic representation of ion distribution near a positively charged surface
Zeta potential is a function of the surface charge of a particle any adsorbed layer a
the interface and the nature and composition of the surrounding medium in which the
particle is suspended The principle of determining zeta potential is very simple A
79
this causes the charged particles to move towards the electrode of opposite polarity
Viscous forces acting upon the moving particle tend to oppose this motion and the
equilibrium is rapidly established between the effects of the electrostatic attraction and
the viscosity dra
g The particle therefore reaches a constant terminal velocity
Figure 4-10 Schematic representation of zeta potential measurement(source
httpnitioncomenproductszeecom_shtm)
Because protein adsorption mechanisms are very complex and hard to explain in
biological systems protein adsorption is generally studied by using more ideal systems
consisting of one or more well characterized proteins a well-characterized adsorbent and
a well-defined aqueous solution Even so small changes in the experimental conditions
such as pH ionic strength and temperature generate totally different results Therefore
two media systems phosphate buffer (PB) and phosphate buffered saline (PBS) were
chosen to be used for the zeta potential measurements and protein adsorption study PB is
a simple aqueous system of 5 mM monobasic sodium phosphate (NaH2PO4H2O) to
n a constant pH PBS is used to keep the water concentration inside and outside of
the cell balanced If the water concentration is unbalanced the cell risks bursting or
shriveling up because of a phenomenon called osmosis PBS is a solution with the 5 mM
of monobasic sodium phosphate (NaH2PO4H2O) and 143 mM of sodium chloride (NaCl)
in water
maintai
80
The results of zeta potential measurement at room temperature (25plusmn1oC) as
function of pH and ionic strength (different media PB and PBS) for synthesized lat
particles are seen in Figure 4-11 Zeta potential values of polystyrene (PS) latex particle
at the Figure 4-9 are negative between -291 mV and -599 mV in PB and betwe
ex
s
en -203
mV and -278 mV in PBS at all pH ranges These negative zeta potential values of PS
latex particles are due to the sulfate groups [Vandenhu et al 1970] originated from
persulfate initiators attached via an oxygen bridge (-C-O-SO3-) at the end of polymer
chain where polymerization was initiated
-100
-80
-60
-40
-20
0
pH
Zta
pte
ntl
ζ
20
40
1 2 3 4 5 6 7 8 9
eo
ia (m
V)
PB (5mM)
PBS (5mM +143mM)
en -
Figure 4-11 Zeta potential of PS latex particles at 25oC
The zeta potential measurements of PSPMMA100 core shell latex particles as seen
in Figure 4-12 were also negative with a range of -28 and -505 mV in PB and betwe
143 mV and -186 mV in PBS at the pH 21-78 These negative values are also due to
sulfate groups on the particle surface
81
-100
-80
-60
-40
-20
0
20
40
1 2 3 4 5 6 7 8 9
Zea
pote
nti
l ζ
(mV
pH
ta
)
PB (5mM)
PBS (5mM + 143mM)
Figure 4-12 Zeta potential of PSPMMA100 latex particles at 25oC
The zeta potential for PSPMMA90PAA10 and PSPMMA75PAA25 was also
negative as seen in Figure 4-13 and 4-14 The zeta potential value for the
PSPMMA90PAA10 were between -367 mV and -678 mV in PB medium and between -
147 mV and -193 mV in PBS both at 25oC In case of the PSPMMA75PAA25 core shell
particles zeta potential values were between -291 mV and -520 mV in PB and between
-115 mV and -210 mV in PBS media Sulfate groups in PSPMMA75PAA25 and
PSPMMA90PAA10 core shell particles contribute to the negative zeta potential values
participate in forming a
negat
cid is expressed by the term pKa which is the pH
at which an acid is 50 dissociated However the contribution of the carboxylic acid
Carboxylic groups from acrylic acid monomer also would
ive surface charge of the latex particles at a pH greater than 70 because the pKa
value for the carboxylic acid is between 40 and 60 pH range [Ottewill et al 1967] This
indicates that carboxylic acid dissociation begins at a pH between pH 46-60 and
increases with pH The strength of an a
82
group to negative zeta potential values of PSPMMA-PAA core shell particles was not
detectable because there was no significant difference in zeta potential values for
synthesized latex particles at a pH greater than 70
-60
-40
-20
0
20
40
1 2 3 4 5 6 7 8 9
Zeta
pot
entia
l ζ
(mV
)
PB (5mM)PBS (5mM + 143mM)
-80
-100
pH
Figure 4-13 Zeta potential of PSPMMA90PAA10 latex particles at 25oC
-100
-80
-60
-40
-20
0
20
40
1 2 3 4 5 6 7 8 9
pH
Ze p
ont
i ζ
V)
tate
al (m
PB (5mM)PBS (5mM + 143mM)
Figure 4-14 Zeta potential of PSPMMA75PAA25 latex particles at 25oC
83
The zeta potential plots and values were similar for all latex particles This may be
due to the similar initiator density on the latex surface The initiator concentrations
monomers for shell polymerization were the same fo
to
r all core shell latex particles (062
ww from Table 4-4) A similar concentration (055 ww) of initiator to monomer was
added to prepare bare PS latex particles used in zeta potential measurements
Since the zeta potential values of all synthesized latex particles were negative
between pH=20 and pH=78 the isoelecric point (IEP) of these latex particles would be
less than pH=20 Sulfates have a pKa in the range of 10 to 20 [James 1985] indicating
that the sulfate group the conjugate base of a strong acid is protonated (-C-OSO3H) at
less than pH 20 This means that the synthesized latex particles are all negative at the
physiological pH Most serum proteins have a negative surface charge in blood
therefore when the negative latex particles are applied to blood stream fatal
tein is
avoided by charge repulsion in the blood Table 4-5 shows the common blood proteins
and their isoelectric points (IEP)
Table 4-5 The isoelectric point (IEP) of common proteins Serum proteins Isoelectric point (IEP)
complication by coagulation between applied latex particles and serum pro s
Albumin 48 α1-Globulin 20 α2-Microglobulin 54 γ1-Globulin 58 Heptoglobin 41 Hemoglobin 72 β1-lipoprotein 54 Immunoglobulin G (Ig G) 73 Fibrinogen 58 (source httpwwwfleshandbonescomreadingroompdf1085pdf
httpwwwmedalorgvisitorwww5CActive5Cch135Cch13015Cch130105aspx )
84
The absolute zeta potential values of the latex particles in phosphate buffer (PB)
was higher than that in phosphate buffered saline (PBS) This is because high
concentration of sodium and chloride electrolytes in PBS compressed the electrical
double layer causing rapid decay of surface potential Figure 4-15 shows that the
thickness of the electrical double layer is inversely proportional to the concentration of
electrolyte in the system
Surf
ace
Pote
ntia
l ψ
1κ3
10
1e
1κ 1κ
(1)(3) (2)
2 1 Z
Surf
ace
Pote
ntia
l ψ
1κ3
10
1e
1κ 1κ
(1)(3) (2)
Figure 4-15 The decay of surface potential with distance from surface in various
electrolyte concentrations (1) low (2) intermediate (3) high [Myers 1999]
2 1 Z
The electrical potential in the solution surrounding the particle surface falls off
the Debye-Huckel
yers 1999]
exponentially with distance (Z) from the surface according to
approximation [M
z) (- exp 0 κψψ = (4-4)
e reciprocal of the thickness of the electrical double layer referred to as the
The potential fallen off by a factor e The theoretical equation for the
ickness 1κ is
where κ is th
Debye length of 1
double layer th
21
22i
0
ekT 1
⎟⎟⎠
⎜⎜⎝
=sum ii zcεε
κ
⎞⎛ (4-5)
85
where
tration of
y was performed to measure the protein adsorption on the
synthesized latex particles Many physical and chemical processes occur at the boundary
between two phases and adsorption is one of the fundamental surface phenomena [Oscik
1982] This phenomenon is driven by the change in the concentration of the components
at the interface with respect to their concentrations in the bulk phase There are two
adsorption processes physical adsorption occurring when non-balanced physical forces
appear at the boundary of the phases and chemical adsorption or chemisorption
t the
interface [J
n leads
ational entropy hence adsorption takes place only if the
loss in conformational entropy is compensated
ter adsorption Cp The initial part of the
isotherm merges with the Γ-axis because at low polymer concentration the protein has a
high affinity for the surface and all of the polymer is adsorbed until the solid surface is
saturated
ε0 is the permittivity of a vacuum of free space ε is the permittivity of a medium e
is the relative permittivity or dielectric constant of the medium c is the concen
an ion (i) z is the valency of an ion (i) k is Boltzmannrsquos constant T is absolute
temperature (K)
43 Protein Adsorption Study
The adsorption stud
occurring when atoms and molecules from adjacent phases form chemical bonds a
aroniec et al 1988]
Flexible proteins in solution possess high conformational entropy as a result of the
various states each of the many segments in the protein chain can have Adsorptio
to a reduction of this conform
by sufficient attraction between polymer
segment and solid surface [Martin 1998] Isotherm shape of high affinity adsorption
between polymer and solid is shown in Figure 4-16 where the adsorbed mass Γ is plotted
against the polymer concentration in solution af
86
Figur
de
arboxyl
e 4-16 High-affinity adsorption isotherm of typical flexible polymer on solid
surface
In order to investigate the adsorption of blood proteins latex particle properties
such as electrical surface charge hydrophobicity and environmental conditions like pH
ionic strength and temperature are considered as experimental factors [Arai and Nor
1990] Figure 4-17 shows the overall schematic representation of the protein adsorption
on the synthesized latex particles with the functional groups such as sulfate and c
groups
CP
Γ
PMMAPAAShell
PS or PMMAShell OSO3
-
OSO3-
OSO3-
OSO3-
PS
-O3SO
PSCore
HOOC
-O3SO
O3SO
3
HOOC
HOOC
-
-O SO
Protein
NH3+
+
+H3N
+
COO-
Hydrophobic interaction
NH3
COO-
H3N
COO-
-OOC
Ionic interaction
Hydrophobic interaction
Hydrogen bonding
Ionic interaction
PMMAPAAShellOSO3
-
OSO3-
OSO3-
OSO3-
PS or PMMAShell -O3SOOSO3
-
PSCore
HOOC
-O3SO
O3SO
3
HOOC
HOOC
-
-O SO
-O3SO
OSO3-
OSO3-
OSO3-
PS PSCore
HOOC
-O3SO
O3SO
3
HOOC
HOOC
-
-O SO
Protein
NH3+
+
+H3N
+
COO-NH3
COO-
H3N
COO-
-OOC
NH3+
+
+H3N
+
COO-
Hydrophobic interaction
NH3
COO-
H3N
COO-
-OOC
Ionic interaction
Hydrophobic interaction
Hydrogen bonding
Figure 4-17 Overall schematic representation of the protein adsorption on the
Ionic interaction
synthesized latex particles
The surface functional groups as well as suitable surface hydrophobicity
hydrophilicity of latex particles are closely related to protein adsorption property As
indicated at Figure 4-17 various interaction forces such as hydrophobic interaction ionic
87
interaction hydrogen interaction and van der Waals interaction [Andrade 1985] exist
between protein molecule and the solid surface of latex particle
431 Adsorption Isotherm
The protein adsorption isotherm is generally defined as the relationship between the
amount of protein adsorbed by a solid (latex particle) at a constant temperature and as a
ine
the amount of protein adsorbed onto latex particles To evaluate the equilibrium
conce s
x
function of the concentration of protein Adsorption isotherms were plotted to determ
ntration of target proteins a calibration curve is firstly needed Figure 4-18 show
the UV absorbance as a factor of bovine serum albumin (BSA) concentrations The UV
absorbance of the known protein concentration was measured at 562nm which is the λma
for a protein treated by a bicinchoninic acid (BCA) assay
0
02
04
06
08
500 520 540 560 580 600
Wavelength (nm)
Abs
orba
nce
Figur gml (B)
06mgml (C) 02mgml
(A)
(C)
(B)
Max562nm
e 4-18 The dependence of UV absorbance on BSA concentration (A) 10m
Figure 4-19 shows the standard calibration curve used in adsorption isotherm
experiment of protein onto latex particles The net absorbance of each known protein
88
concentration was plotted and then a linear equation passing through a concentration of
zero protein concentration was obtained This equation is applied
to determine the
orbed on the synthesized latex particles The amount of free
protei
eported
trations (005 01 03 05
and 07mgml) were used to generate this curve as well as the adsorption isotherm
unknown protein amount ads
n was also determined using UV light at the 280nm wavelength however a
distinguishable UV intensity was too low to determine a precise value of protein
adsorption
The bicinchninic acid (BCA) assay technique [Smith et al 1985] has been r
for high efficiency of protein evaluation and was used for determination of protein
adsorption [Tangpasuthadol et al 2003 Williams et al 2003] This technique has many
advantages such as high sensitivity easy of use color complex stability and less
susceptibility to any detergents Five different BSA concen
y = 00R2 =
004741x 09939
00
01
02
03
04
0 200 400 600 800
BSA Standard (microgml)
Net
Abs
orba
nce
at 5
62nm
Figure 4-19 Standard curve of net absorbance vs BSA sample concentration
89
All isotherms were fitted to the Langmuir-Freundlich isotherms using the nonlinea
regression method Two adsorption isotherm models the Langmuir type isotherm a
Langmuir-Freundlich combination type isotherm were compared As seen the Figure 4-
20 The Langmuir isotherm model deviated further from the experimental data Th
all isotherms were fitted to the Langmuir-Freundlich isotherms [Yoon et al 1996
al 1998] usi
r
nd the
erefore
Lee et
ng the nonlinear regression method with following equation
n
n
eq
eqm
kC
kC1
1
1+
where q is the amount of adsorbed BSA per unit surface area q
qq = (4-6)
stant
were between 045 and 055 and hence n was fixed to its average 05 Actual qm values
were evaluated after fixing the n value
m is the adsorbed amount
in equilibrium Ceq is the equilibrium concentration of BSA k is the adsorption con
and n is the exponential factor for a heterogeneous system n values are empirical and
usually from zero to no more than unity [Jaekel 2002] The values of n in our system
00
10
20
30
0 02 04 06 08
C (mgml)eq
q (m
gm
2 )
pH 32Langmuir-Freundlich modelLangmuir model
Figure 4-20 Fitted models for adsorption isotherm of bovine serum albumin (BSA) on
polystyrene latex particles at 37oC in phosphate buffer media
90
The BSA protein adsorption on the synthesized latex particles was performed at
three pH levels 32 48 74 in both phosphate buffer (PB) and phosphate buffered saline
(PBS) at physiological temperature 37oC These pH levels correspond to an acidic pH
lower than the isoelectric point (IEP) of BSA the pH at the IEP and an alkaline pH
higher than the IEP of BSA respectively The IEP corresponds to the pH at which the
amino acid becomes a zwitterion Here its overall electrostatic charge is neutral and the
charge expressed by the basic amino group (-NH2 -NH3+) of the amino acid is equal to
the charge expressed by the acidic carboxyl group (-COOH -COO ) The IEP of BSA is
-
pH 48 and indicates that the side chain of the amino acids of BSA contains a greater
amount of acidic functional groups than that of basic functional groups At pH 32 and
74 BSA has a positive and negative charge respectively
Figure 4-21 and 4-22 show adsorption isotherms of BSA onto polystyrene (PS)
latex particles
00
10
20
30
40
0 02 04 06 08
Ceq (mgml)
q (m
gm
2 )
pH=32pH=48pH=74Model (pH 32)Model (pH 48)Model (pH 74)
Figure 4-21 Adsorption isotherm of BSA on polystyrene (PS) latex particles in
phosphate buffer (PB) at 37oC
91
00
10
30
40
0 02 04 06 08
Ceq (mgml)
q (m
gm
2
20
)
pH=32pH=48pH=74Model (pH 32)Model (pH 48)Model (pH 74)
(PBS) on PS latex particles
At the IEP of BSA the adsorbed amount (q
Figure 4-22 Adsorption isotherm of BSA adsorption 37oC in phosphate buffered saline
onto PS at equilibrium was
maxim
um at the IEP of BSA This is because the BSA molecules form the most
compact structures at the IEP eliminating the repulsion force between proteins A higher
number of BSA molecules can adsorb on a given surface area by hydrophobic interaction
when in this compact structure This observation agrees well with reported data by other
researchers who also indicated that the maximum adsorption from aqueous protein
solution was observed at the IEP [Mularen 1954 Watkins et al 1977 Oh at el 2000]
There was no significant difference of adsorbed BSA amount in PB and in PBS media at
pH 48 The conformational alteration of BSA was not affected significantly by the ionic
strength of the two solutions There was evidence of the effect of the electrostatic
interaction to some extent even at the IEP since the amount of BSA adsorbed in PBS is
slightly hig
m) of BSA
um indicating that hydrophobic interaction between latex particle and BSA
protein is maxim
her than in PB at this pH
92
The adsorbed amount of protein is shown to decrease at a pH 32 and 74 The
decrease in adsorption efficiency when deviating from IEP of BSA is a result of the
increase in conformational size of the protein molecules and the lateral electrostatic
repulsions between adjacent adsorbed BSA molecules Figure 4-23 shows a schematic of
possible conformational changes at each pH where hydrophobic interaction always exists
between non-polar portion of a protein and hydrophobic portion of solid surface
- - --- ---Latex surface
Zeta potential
BSA(+)
(-)
48 7432pH
(0)
+++ + ++
--+
++-
- - --- ---Latex surface
Zeta poten pH(0)
+++ + ++
--
tial
BSA(+)
(-)
48 7432
+
++-
ming
4
s
al blood Table 4-6 shows the calculated results of
Figure 4-23 Conformation of bovine serum albumin (BSA) on latex particles (Assuthat charge repulsion reduces the effect of the hydrophobic interaction at pH 74)
In addition the adsorbed amount of BSA at pH 32 was larger than that at pH 74
and still less than that at 48 This is because there is the electrostatic interaction between
the positive BSA molecules and negative PS latex particles at pH 32 and electrostatic
repulsion is dominant between negative BSA and negative PS latex particles at pH 7
The amount adsorbed increased with increasing ionic strength in pH 32 and 74 This i
because the high concentration of electrolytes reduces the electrostatic repulsion between
BSA and PS latex particle It should be noted that the BSA concentration used in these
experiments was lower than that found in normal human blood This is because the
equilibrium BSA adsorption onto the latex particles is actually reached at a much lower
concentration than that found in norm
93
adsor rm
ein
leading to a compact conformation of BSA There are two main forces affecting
adsorption amount at an acidic pH of 32 They include the ionic attraction between the
negative latex particle and the positive protein and lateral repulsion between positive
proteins The smallest amount of BSA adsorption was observed at a pH of 74 At this
point the charge repulsion between negatively charged particles and protein molecules
exists the h
of cha
bed BSA amount at equilibrium from the Langmuir-Freundich adsorption isothe
equation
In summary the hydrophobic interaction between PS latex particle and BSA
protein exists and is the dominant mechanism for the adsorption process Conformational
changes of BSA at each pH also affected the adsorption amount Adsorption was highest
at pH 48 because at this pH there is the least amount of ionic repulsion in the prot
and the lateral repulsion between proteins dominant adsorption even though there always
ydrophobic interaction between the solid latex particle and protein The effect
rge repulsion was reduced by the high concentration of electrolytes in PBS medium
Table 4-6 The equilibrium concentration values of BSA adsorption on polystyrene (PS) latex particles calculated the Langmuir-Freundlich isotherm model
Media pH qm (mgm2) 32 174 48 299 Phosphate buffer 74 136 32 260 48 283 Phosphate buffered saline 74 207
The adsorption isotherms of BSA on PSPMMA core shell particles are shown in
Figure 4-24 in phosphate buffer (PB) and in Figure 4-25 in phosphate buffered saline
(PBS) The adsorbed BSA amount was calculated using the Langmuir-Freundlich
100
94
adsorption isotherm equation and the amount listed in Table 4-7 The trend seen in the
adsorption results was very similar to the case of PS latex The overall adsorption process
was d rticle
rs
hat
0
ominated by hydrophobic interactions between the PSPMMA100 core shell pa
and BSA proteins At pH 48 the adsorbed amount (qm) of BSA on PSPMMA100 core
shell particles was 221 mgm2 in PB and 237 mgm2 in PBS and are the maximum
values recorded among the three pH levels 32 48 and 74 Suzawa and his co-worke
[1982] have obtained similar results This can be also explained by the compact
conformation of BSA molecules at the IEP of BSA which lead to a high protein
population on the solid surface At a pH of 32 and 74 BSA adsorption was less than t
at pH 48 because of the protein conformation change There was a small increase in
adsorbed BSA at pH 32 because of the charge attraction between negative PSPMMA10
core shell particles and positive BSA At pH 74 however the adsorption of BSA was
further reduced by charge repulsion between the negative PSPMMA100 core shell
particles and the negative BSA proteins
000 02 04 06 08
Ceq (mgml)
10
20q (m
gm
302 )
40
50pH=32pH=48pH=74Mode 8)l (pH 4Mode 2)l (pH 3Mode 4)
Figure 4-24 Adsorption isotherm of BSA 37oC in PB on PSPMMA core shell latex
l (pH 7
100
particles
95
The adsorbed amount of BSA increased more rapidly in PBS with increasing ioni
strength at pH 74 This shows that the low concentration of electrolytes promotes
electrostatic repulsion between negative BSA and negative PSPMMA
c
on
x
l 1980
100 core shell
particle As the ion strength of PBS is increased however the high concentration of
electrolyte leads to less electrostatic repulsion promoting more rapid protein adsorpti
By comparing the amount of adsorbed protein between bare PS latex particles and
PSPMMA100 core shell particles the amount of protein adsorption on the PS late
particles is greater than that on the PSPMMA100 core-shell particles [Suzawa et a
1982 Lee et al 1988]
00
10
20
30
40
m2 )
0 02 04 06 08
Ceq (mgml)
q (m
g
pH=32pH=48pH=74Model (pH 2) 3Model (pH 48)Model (pH 74)
Figure 4-25 Adsorption isotherm of BSA at 37oC in PBS on PSPMMA100 core shell
latex particles
96
Table particles calculated the Langmuir-Freundlich isotherm model
4-7 The equilibrium concentration values (qm) of BSA adsorption on PSPMMA100
Media pH qm (mgm ) 2
32 140
48 221 Phosphate buffer
74 130
32 169
48 237 Phosphate buffered saline
74 162
The BSA adsorption mechanism and trend on PSPMMA core shell particles was
very similar to that of the PS latex particle Unlike the case of PS and PSPMMA100 latex
particles the amount of BSA adsorbed onto PSPMMA90PAA10 and PSPMMA75PAA25
core shell particles was higher at pH 32 than pH 48 and 74 Figure 4-26 through 4-29
shows the adsorption isotherms of BSA on PSPMMA90PAA10 and PSPMMA75PAA25
core shell particles The amount of BSA adsorbed increased at pH 32 and pH 48 as the
hydrophilicity was developed by the addition of PAA On the contrary the adsorbed
amount of BSA was dramatically decreased at pH 74 The adsorption amount of BSA on
PSPMMA90PAA10 and PSPMMA75PAA25 core shell particles at equilibrium is listed in
Table 4-8 and 4-9 respectively
Carboxylic acid as well as sulfate groups are distributed on the surface of
PSPMMA90PAA10 and PSPMMA75PAA25 core shell particles and seem to largely affect
the BSA adsorption The isotherms showed very strong pH dependence to the BSA
adsorption onto these core shell latex particles The carboxylic groups (COOH) may
especially enhance the BSA adsorption prominently at pH 32 by hydrogen bonding with
protein molecules The pKa value for the PAA is 40-60 and a good indication that the
97
carboxylic acid groups are protonated at pH 32[Gebhardt et al 1983] The main
adsorption interaction force at pH 32 was generated by hydrogen bonding as well as
charge attraction between latex particle protein even th the conformational
repulsion between proteins results in a reduction of the protein
adsorption at this pH The amount of BSA adsorbed also increased as the PAA amount in
eased The a tion of BSA w inimal however less
than 07 mgm2 at pH 74 Here the carboxylic groups are ionized which increases the
charge repulsion by ionized carboxylic groups (COO-) resulting in PSPMMA-PAA latex
particle
and ough
change and charge
the core shell particles incr dsorp as m
s with much lower protein adsorption
00
20
40
q (m
g2 )
60
m
pH=32pH=48pH=74Model (pH 32)Model (pH 48)
0 02 04 06 08
Ceq (mgml)
Model (pH 74)
o shell
Figure 4-26 Adsorption isotherm of BSA at 37 C in PB on PSPMMA90PAA10 corelatex particles
98
00
40
60
80
0 02 04
C (mgm
mm
2 )
20
06 08
eq l)
q (
g
pH=32pH=48pH=74Model (pH 32)Model (pH 48)Model (pH 74)
Figure 4-27 Adsorption isotherm of BSA at 37oC in PBS on PSPMMA90PAA10 core
shell latex particles
Table 4-8 The equilibrium concentration values of BSA adsorption on PSPMMA90PAA10 particles calculated the Langmuir-Freundlich isotherm model
Media pH qm (mgm2)
32 361
48 283 Phosphate buffer
74 043
32 519
48 226 Phosphate buffered saline
74 043
99
00
20
40
60
80
100
0 02 04 06 08Ceq (mgml)
q (m
gm
2 )
pH=32pH=48pH=74Model (pH 32)Model (pH 48)Model (pH 74)
oFigure 4-28 Adsorption isotherm of BSA 37 C in PB on PSPMMA75PAA25 latex particles
00
100
20
40
60
80
0 02 04 06 08Ceq (mgml)
q (
m2 )
pH=32pH=48pH=74
mg
Model (pH 32)Model (pH 48)Model (pH 74)
Figure 4-29 Adsorption isotherm of BSA 37oC in PBS on PSPMMA75PAA25 core shell
latex particles
100
Table 4-9 The equilibrium concentration values of BSA adsorption on PSPMMA75PAA25 particles calculated the Langmuir-Freundlich isotherm model
Media pH qm (mgm2)
32 1083
48 662 Phosphate buffer (PB)
74 075
32 1077
48 681 Phosphate buffered saline (PBS)
74 057
The adsorption of β2-microglobulin (β2M) was determined under one set of
onditions in phosphate buffer at 37oC and pH 74 because of the limited amount of β2M
protein The adsorption isotherms of the β2M protein on the synthesized latex particles
are shown in Figure 4-30 4-31 and 4-32 The β2M protein concentrations for adsorption
isotherm experiment were chosen to be 0015 0030 0045 and 0060mgml since the
β2M concentration for the patients with renal failure can be elevated to similar
concentrations about 005mgml or more up to 50 times the normal level [Nissenson et
al 1995] There were steep initial slopes of the adsorption isotherm curves of β2M on
latex particles in all isotherms indicating a high affinity adsorption type It seems that the
hydrophobic interaction is enough for the adsorption process of β2M proteins to occur on
the latex particles even though charge repulsion exists between protein and latex particle
at pH 74 The complete plateau regions were not seen for all isotherms indicating that
β2M proteins were not saturated at this level and there are still available sites for β2M
c
adsorption on latex particles The initial slope of the β2M isotherms for PS was steeper
101
than that of any of the other latex particle PSPMMA100 PSPMMA90PAA10 and
PSPMMA 75PAA25
00
02
0 0002 0004 0006 0008
04
06
10
Ceq (mgml)
q (m
2 )
08
gm
PSPSPMMA100Model (PS)Model (PSPMMA100)
Figure 4-30 Adsorption isotherm of β2M onto PS and PSPMMA100 latex particles in PB
at 37 C and pH 74 o
00
02
04
06
08
10
0 0002 0004 0006 0008C (mgml)
q (m
m2 )
eq
g
PS
PSPMMA90PAA10
Model (PS)
Model (PSPMMA90PAA10)
Figure 4-31 Adsorption isotherm of β2M onto PS and PSPMMA90PAA10 latex particles
in PB at 37oC and pH 74
102
00
02
04
06
08
10
0 0002 0004 0006 0008Ceq (mgml)
q (m
gm
2 )
PS
PSPMMA75PAA25
Model (PS)Model (PSPMMA75PAA25)
Figure 4-32 Adsorption isotherm of β2M onto PS and PSPMMA75PAA25 latex particles
in PB at 37oC and pH 74
The equilibrium concentration values (qm) of β2M adsorption on latex particles are
calculated and listed in Table 4-10 The difference between the maximum amounts of
β2M adsorption on all the latex particles was not significantly different and their values
were between 069 and 08mgm2
Table 4-10 The equilibrium concentration values of β2M adsorption calculated the Langmuir-Freundlich isotherm model
Latex particles qm (mgm2)
PS 080
PSPMMA100 069
PSPMMA90PAA10 072
PSPMMA75PAA25 072
103
432 Adsorbed Layer Thickness
There are several possible explanations for large amount of BSA adsorbed at acidic
pH levels including the end-on adsorption pattern of BSA the flexibility of BSA
molecules the tilting of protein molecules due to the asymmetry of the charge
distribution or multilayer formation [Peula et al 1993] The adsorbed BSA thickness (δ)
can be calculated by the following equation [Chiu et al 1978] This equation was also
cited again by other researchers [Shirahama et al 1985]
BSA
mqρπ
δbullbull
=33
(4-7)
where π
33 is the packing factor qm is the plateau value of the adsorbed am
ρBSA is the hich corresponds to the reciprocal of its known
partial specific volume The layer thickness (δ) of BSA proteins on synthesized latex
partic
he IEP [Shirahama et al 1985] Compared to the
hydrodynamic dimensions of BSA 140 times 40 times 40 Aring3 [Fair et al 1980 Peter 1985] these
nd
ode It is unnecessary ultilayering because thickness do
exceed 140 Aring The layer thickness (δ) of BSA proteins on synthesized latex particles
e red saline (PBS) was ran 28 ~ 83 Aring at 37oC and pH 48 These
lso listed in Table 4-1
ount and
density of BSA molecule w
les in phosphate buffer (PB) ranged from 27 - 81 Aring at 37oC and pH 48 These
calculated values are listed in Table 4-11 Only δ values at pH 48 the isoelectric point
(IEP) of BSA are important because intra- and intermolecular electrostatic repulsion of
protein molecules is minimized at t
values of layer thickness indicate that the BSA molecules adsorb by side-on (40Aring) a
end-on (140Aring) m to consider m
not
in phosphat buffe ged of
calculated values are a 2
104
Table 4-11 Absorbed BSA layer thickness (Aring) of BSA in phosphate buffer (PB) at 37oC pH PS PSPMMA100 PSPMMA90PAA10 PSPMMA75PAA25
32 21 17 44 133
48 37 27 35 81
74 17 16 5 9
Table 4-12 Absorbed BSA layer thickness (Aring) of BSA in phosphate buffered saline (PBS) at 37oC
pH PS PSPMMA100 PSPMMA90PAA10 PSPMMA75PAA25
32 32 21 64 132
48 35 29
74 26 20 5 7
28 83
1n
1n
neqm
So k is closely related to the initial slope of the isotherm showing the affinity
between BSA and latex particles [Yoon et al 1996] A key point in the characterization
433 Gibbs Free Energy of Protein Adsorption
In the Langmuir isotherm model k represents the ratio of adsorption to the
desorption rate constants This definition is not applicable to the Langmuir-Freundlich
isotherm model but its meaning is similar [Yoon et al 1996] In this sense k is closely
related to the affinity between protein molecules and latex particles When the
equilibrium concentration Ceq reaches zero the value of Ceq will be very small (1gtgt
kCeq ) and then the equation 4-6 becomes
(4-8)
of adsorption processes on solid-liquid interfaces is to determine the change in Gibbsrsquos
free energy (∆Gdeg) one of the corresponding parameters for the thermodynamic driving
forces during adsorption The traditional method to evaluate adsorption thermodynamic
kCqq 1=
105
constants is based on measurement of adsorption isotherms under static conditions
[Malm ten 1998] The Langmuir-Freundlich ads lf
easily found from Then this enables calculation of the free-
energy change ∆G during adsorption expressed as
(4-9)
where R is the general gas constant (R = 8314Jmiddotmol K ) and T is the absolute
tem ture Gibb e energy A P
listed in Table 4-13 and 4-14 respectively As the ∆Gdegads ore negativ e
pro adsorption on a solid substra ill be more favorable
e of BSA adsorption in phosphate o
pH PS PSPMMA100 PSPMMA90PAA10 PSPMMA75PAA25
s orption equilibrium constant k can be
a slope of the isotherm
degads
lfads kRTG lnminus=∆ o
-1 -1
pera s fre change of BS adsorption values in B and PBS are
values are m e th
tein te w
Table 4-13 The values of Gibbs free energy changbuffer (PB) at 37 C
32 -2411 -2726 -2846 -2805
48 -2352 -2383 -2198 -1972 ∆Go
80
ads (kJmol)
74 -2452 -888 -324 -2
Table 4-14 The values of Gibbs free energy change of BSA adsorption in phosphate
pH PS PSPMMAbuffered saline (PBS) at 37oC
100 PSPMMA90PAA10 PSPMMA75PAA25
32 -2315 -2620 -2312 -2683
48 -2149 -2299 -2090 -2138 ∆Go
(kJmads ol)
74 -2430 -1992 -258 -263
Table 4-15 shows the calculated values of Gibbs free energy change of β M
adsorption on synthesized latex particles PSPMMA PAA core shell latex particle has
2
75 25
106
largest negative Gibbs free energy of β2
2
M adsorption indicating largest affinity betw
β
een
2M adsorption in phosphate buffere (PB) at 37 C and pH74
PS PSPMMA100
M and latex particle
Table 4-15 The values of Gibbs free energy change of βo
PSPMMA90PAA10 PSPMMA75PAA25
∆Go03 ads
(kJmol) -3626 -3466 -3659 -39
The Gibbs free energy of β2M was much more negative in phosphate buffer (PB)
than that of BSA in physiological condition (37 C and pH 74) o from the Table 4-13 and
the Figure 4-31 This indicates that β2M molecules more readily adsorb on latex particles
BSA This can be explained by the rate of diffusion and accessibility of β2M molecules to
the latex particle ier and faster for β2M m an BSA because of
size (molec ight) erical shape molecules It o be
due to the higher hydrophobic force and less electrostatic repulsion that contribute to the
adsorption of β2M molecules not as prevalent in the case of BSA because the IEP of β2M
(57) is clo H 74) than that of BSA (48) causing β2M to
be less electro cally lled p ro
can be seen that the ∆ β2M adsorption onto the synth article
increasing wi hy icity o x particles s the ∆Goads of the BSA
molecules is decreasing or constant This phenomenon supports the theory that the
select lizing
than BSA and that the affinity of β2M for the latex particle is much larger than that of
surface It is eas olecules th
the smaller ular we and sph of β2M may als
ser to physiological condition (p
stati repe from the latex articles than BSA F m Figure 4-33 it
Goads of esized latex p s is
th the drophil f the late wherea
ive adsorption of β2M molecules over BSA molecules can be enhanced by uti
more hydrophilic PSPMMA90PAA10 and PSPMMA75PAA25 core shell latex particles
107
-60
-50
1 2 3 4
-40∆Gad
s
-30
10
0
o (k
Jm
o-
-20l)
β2-Microglobulin (β2M)Bovine serum albumin (BSA)
Figure 4-33 Gibbs free energy of adsorption of proteins on latex particles in PB at 37
and pH 74
434 Kinetics of Adsorption
to
oC
Protein adsorption is a complex process known to have a large biological impact
and is currently not well understood quantitatively because of extreme sensitivity to pH
the concentration of other electrolytes and molecules present temperature and many
other factors that can change in the physiological system Accurate knowledge of the
adsorption kinetics under a given set of conditions is a prerequisite for elucidating the
mechanisms of many fundamental biological processes to be predicted at the molecular
level [Regner 1987] This adsorption kinetics approach is used in protein adsorption
studies because of the uncertainty related to the time needed for the interfacial proteins
reach the equilibrium It is generally accepted that the process of protein adsorption is
comprised of the following steps a) transport toward the interface b) attachment at the
interface c) eventual structural rearrangements in the adsorbed state d) detachment from
PS PSPMMA100 PSPMMA90PAA10PSPMMA75PAA25
108
the interface and transport away from the interface Each of these steps can affect the
overall rate of the adsorption process
The quantitative analysis of the protein adsorption kinetics requires that the protein
amount adsorbed is known as a function of time The kinetics tests were carried out in
phosphate buffer (PB) at 37oC and pH 74 a condition similar to physiological condition
The protein concentrations chosen were 07mgml for BSA and 006mgml for β2M
which are the maximal concentrations used in an adsorption isotherm test A time-based
evaluation of the adsorption was made by measuring the concentration of protein in
solution at different incubation times The results are shown below from Figure 4-34 to 4-
37 The adsorption kinetics of proteins onto th cond-e latex particles is expressed as a se
order model [Oumlzacar 2003] shown by the following equation
2)( tet qqk
dtdq
minus= (4-10)
Integrating above equation and applying the boundary conditions yields
ee
t tq
+=
1 (4-11)
where q
qkq
t
2
2
2
the previous adsorption isotherm results representing good reproducibility of adsorption
t and qe stand for the amount (mgm ) of protein adsorbed at time t and at
equilibrium respectively k is the equilibrium rate constant of second-order adsorption
(m mghr) and t is the incubation time (hr)
The fitting parameters calculated by using the equation 4-11 are listed in table 4-16
From this data it is seen that the amount of BSA adsorbed at equilibrium was higher than
that of β2M on PS and PSPMMA100 latex particles but less than that of β2M on
PSPMMA90PAA10 and PSPMMA75PAA25 latex particles These results are similar to
109
00
05
15
20
0 2 4 6 8 10 12 14
Time (hr)
rot
n ad
sorb
d (
10
25
Pei
em
gm
2
BSA2nd order model (BSA))β2M2nd order model (β2M)
Figure 4-34 The kinetics of protein adsorption in PB on PS latex particles at 37oC and
pH 74
00
05
10
15
20
25
30
0 2 4 6 8 10 12 14
Time (hr)
Prte
inds
oo
arb
ed (
mg
m2 )
BSA2nd order model (BSA)β2M2nd order model (β2M)
Figure 4-35 The kinetics of protein adsorption in PB on PSPMMA100 latex partic
37les at
oC and pH 74
110
00
03
06
09
12
15
0 2 4 6 8 10 12 14
Time (hr)
Prot
ein
adso
rbed
(m
gm
2 ) BSA2nd order model (BSA)β2M2nd order model (β2M)
Figure 4-36 The kinetics of protein adsorption in PB on PSPMMA90PAA10 latex
particles o at 37 C and pH 74
00
03
06
09
12
15
0 2 4 6 8 10 12 14
Time (hr)
BS
A ad
sorb
ed (
mg
m2 )
BSA2nd order model (BSA)β2M2nd order model (β2M)
Figure 4-37 The kinetics of protein adsorption in PB on PSPMMA75PAA25 latex
particles at 37oC and pH 74
111
test The variation of β2M equilibrium adsorption amount was nearly negligible for all
latex particles The plateau region is reached earlier for β2M than BSA on all latex
particles and the K values (equilibrium rate constant of second order adsorption) for the
β2M adsorption process onto latex particles are much larger than for BSA adsorption
These values indicate that the β2M molecules have a higher adsorption rate onto the latex
particles than the BSA molecules
Table 4-16 Fitting parameters of second-order kinetic model BSA β2M
Latex particles qe (mgm2) K (hr-1) qe (mgm2) K ( -1) hr
PS 144 67 074 7549
PSPMMA100 175 353 076 4438
PSPMMA90PAA10 046 97 075 5695
PSPMMA75PAA25 060 179 075 5168 K = (kqe) hr-1
There are several explanations for the phenomena observed in these experiments
First the smaller sizes of β2M molecules allow easier diffusion and a close approach to
the latex substrate Second a reduced number of hydrophobic domains on the latex
particle surface replaced by sulfate groups (-OSO3-) and ionized carboxylic groups
(COO-) are not favorable for larger BSA molecules Third the conformational change by
intra- and inter-molecular repulsion would be faster in β2M due to smaller molecular
weight Finally the ionic repulsion between β2M and the latex particles is less than that
condition (pH 74) than IEP (48) of BSA
between BSA and latex particles because IEP (57) of β2M is closer to the tested
112
44 Blood Compatibility
Blood compatibility can be defined as the property of a material or device tha
permits it to function in contact with blood without inducing any adverse reactions
Successful functioning of blood contacting biomedical materials or devices in contact
with blood strongly depends on the extent to which they damage blood during operation
High shear stresses turbulence rela
t
tively long contact times and cellular impact with
artificial surfaces are most common events experienced by blood in contact with artificial
1978 Winslow and et al
1993] Therefore before any materia e placed into the human b
biocompatibility testing must be the first performed in the view of safety The hemolysis
s a method for pr ary screening of biomaterials to determine
h blood a er circulating body fluid The tests estimat
oglobin released from th blood cell d e induced
foreign material
There are several types of blood cells in our body but the ones that make blood red
are erythrocytes or red blood cells (RBCs) Red blood cells (erythrocytes) are the most
numerous type in the blood These cells are average about 48-54 million per cubic
millimeter (mm3 same as a microliter (microl)) of blood but these values can vary over a
larger range depending on such factors as health and altitude RBCs are red because they
contain hemoglobin which is a protein that contains iron When blood circulates through
lungs oxygen binds to the hemoglobin and is then delivered to all the parts of body
RBCs are made in the bone marrow They start out as a nucleus like other cells but
when they fill up with hemoglobin the nucleus becomes smaller until it disappears
Without a nucleus RBCs are fragile and live only about 120 days In a normal healthy
devises and can cause hemolysis or thrombosis [Bernhard et al
ls can b ody
test serves a elimin the
biocompatibility wit nd oth e the
concentration of hem e red amag by a
113
body about 2 million red blood ne marrow produces new
ones j ing
dy
le
cells die per second But bo
ust as fast RBCs are about 75 micrometer across and are responsible for carry
oxygen salts and other organic substances to the cells and tissues throughout our bo
In humans and most mammals the cells are concave (indented in the middle) and flexib
enough to squeeze through even the tiniest of blood vessels the capillaries
Figure 4-38 Image of red blood cells
(Sourcehttpwwwpbrchawaiiedubemfmicroangelarbc1htm)
ch
100 90 10 75 25 f
RBCs are among the first cells that come into contact with a foreign material
injected in the blood Therefore a hemolysis assay gives information about the
biocompatibility in the case of an in vivo application The concentrations of prepared
latex particles used for the tests in this research are 05 25 and 50 (ww) and the
incubation time under moderate shaking was 30 minutes at 37oC Test results are shown
in Figure 4-39 The amount of blood cells that ruptured when in contact with the PS latex
particles was largely increased from about 30 to 91 as the solid content of PS
increased from 05 to 50 However hemolysis by the core shell latex particles su
as PSPMMA PSPMMA PAA and PSPMMA PAA was less than 35 O
114
particular interest was that hemolysis caused by PSPMMA PAA particles at 50
solid concentration was less than 02 hemolysis the lowest value among the latex
particles The low hemolysis value indicates that the PSPMMA PAA core shell latex
particles are the most blood compatible at all solid contents Other core shell particles
such as PSPMMA and PSPMMA PAA also had excellent blood comp
90 10
90 10
100 75 25 atibility with
low RBS breakage
01 2 3
20
40
60
80
100
Hem
olys
is (
)
052550
Figure 4-39 He
PS PSPMMA100 PSPMMA PAA90 10
molysis caused by latex particles n=5
4PSPMMA75PAA25
CHAPTER 5 SUMMARY OF RESULTS CONCLUSIONS AND RECOMMENDATION FOR
FUTURE WORK
51 Summary of Results
The goal of this study was to synthesize monodisperse polymeric latex particles
with tailored properties and to investigate the fundamental interactions between the
synthesized latex particles and target proteins An understanding of the fundamental
mechanism of selective adsorption is strongly required in order to maximize the
separation performance of membranes made of these materials so that it may applicable
to hemodialysis therapy for end stage renal disease (ESRD) patients For the achievement
of above research goal several investigations have been performed Analysis of the
obtained data leads to several conclusions as discussed below
Monodisperse polystyrene seed latex particles were synthesized with the size
crosslinking agent to make the latex particles more hydrodynamically stable The
conversion of monomer to polymer was over 95 and seed latex particles are spherical in
shape and have smooth surface From these small seeds bigger latex particles as large as
800nm were synthesized using a semi-continuous seeded emulsion polymerization
process The particles with a mean diameter less than 500nm are very spherical in shape
and highly uniform in particle size distribution However as the particles grow larger
than 500nm in mean diameter they became non-spherical with an uneven surface This
irregularity of particle surface can be attributed to the non-homogeneous monomer
ranges between 126plusmn75 and 216plusmn53 nm Divinylbenzene (DVB) was used as a
115
116
swelling into the shell of a growing polymer which can be controlled by the factors such
as temperature agitation speed initiator feeding rate and surfactant amount required to
stabilize the colloidal system It was determ rfactant to monomer ratio
dec ic
acid (AA) monomers were intro rticle surfaces more
hydro
to
ting of a
orm
ration is seen between
3002
0 cm-
0cm-1
-1
03
ined that as the su
reased the mean particle diameter increased Methyl methacrylate (MMA) and acryl
duced to make the latex pa
philic than bare polystyrene particles The prepared core shell latex particles were
made of PMMA100 PMMA90PAA10 and PMMA75PAA25 PMMA100 is the PMMA
homopolymer shell on a PS core PMMA90PAA10 shell is a copolymer with a PMMA
PAA ratio of 90 to 10 by weight PMMA75PAA25 shell is a copolymer consis
PMMA to PAA ratio of 75 to 25 by weight The particle size of these core-shell
particles were prepared to be about 370nm in mean diameter
The successful synthesis of latex particles was confirmed using Fourier Transf
Infrared spectroscopy (FTIR) The C-H aromatic stretching vib
cm-1 and 3103 cm-1 for PS latex particles The C-H asymmetrical stretching and
symmetrical stretching vibration peaks of CH2 for all latex particles is seen at 290
1and 2850 cm-1 respectively There is a carbonyl (C=O) characteristic band at 173
wavenumber for PSPMMA core-shell latex particles The broad OH group peak at
3400 cm
100
can be seen in the spectroscopy of the particle with PMMAPAA shell
(PSPMMA PAA and PSPMMA PAA ) The peak intensity of OH group of
PSPMMA PAA was larger than that of PSPMMA PAA relatively due to more AA
monomer content in PSPMMA PAA than in PSPMMA PAA
In the zeta potential measurements polystyrene (PS) latex particles had negative
values between -291 mV and -599 mV in the phosphate buffer (PB) and between -2
90 10 75 25
75 25 90 10
75 25 90 10
117
mV and -278 mV in the phosphate buffered saline (PBS) at 25oC and pH 21-78 These
negative zeta potential values for PS latex particles are due to ionization of the sulfate
groups (-OSO3-) originated from the initiator Zeta potential values of the PSPMMA
core shell latex particles were also negative between -28 mV and -505 mV in PB and
between -143 mV and -186 mV in PBS at pH 21-78 Negative values are also
generated by dissociation of sulfate groups on a particle surface The zeta potential value
of PSPMMA PAA and PSPMMA PAA were of negative values between -36
and -678 mV in PB medium and -147 mV and -193 mV in PBS medium at 25
100
s
7 mV
MMA75PAA25 and
PSPM
otein
g the
l change of BSA at
each w
90 10 75 25
oC For
PSPMMA PAA core shell particles zeta potential values were between -291 mV and
-520 mV in PB media and between -115 mV and -210 mV in PBS media Sulfate
groups contributed to the negative zeta potential values in PSP
75 25
MA90PAA10 core shell particles Carboxylic groups from polyacrylic acid also
contribute to the negative surface charge for the latex particles at greater than pH 70
however the degree of their contribution was not detectable from the zeta potential
measurements The absolute zeta potential values of the synthesized latex particles in PB
were higher than that in PBS This is because of the high concentration of sodium
chloride electrolyte in PBS that compress electrical double layer thickness
The bicinchoninic acid (BCA) assay technique was used to determine the pr
adsorption and all isotherms were fitted to the Langmuir-Freundlich isotherms usin
nonlinear regression method The hydrophobic interaction between PS latex particle and
BSA protein was dominant for the adsorption process Conformationa
pH also affected the adsorption amount which was highest at pH 48 because of lo
ionic repulsion making compact conformation of BSA The next larger amount of BSA
118
adsorbed onto PS was at pH 32 There were two main forces affecting the amount of
adsorption at acidic pH values One is ionic attraction between negative latex particle a
positive protein The other is lateral repulsion between positive proteins The smallest
amount of BSA adsorption was at pH 74 where charge repulsion between negative
particles and proteins is present at this pH Lateral repulsion between proteins wa
dominant for protein adsorption even though there always exists the hydrophobic
interaction between solid latex particle and protein The charge repulsion was reduced by
the high concentration of electrolytes in the PBS medium The BSA adsorption process
on PSPMMA core shell particles was very similar to the case of PS latex particle where
hydrophobic interaction between latex particle and protein was dominant However t
adsorbed BSA amount was less than that of PS in PBS The adsorbed amount of BS
PSPMMA
nd
s
he
A on
an
ed
n The main
adsor
90PAA10 and PSPMMA75PAA25 core shell particles was higher at pH 32 th
pH 48 and 74 The adsorbed BSA amount became higher at pH 32 and pH 48 as the
hydrophilicity was increased by the higher PAA amount On the contrary the adsorb
amount of BSA was dramatically decreased at pH 74 Carboxylic acid groups as well as
the sulfate groups are distributed on the surface of PSPMMA90PAA10 and
PSPMMA75PAA25 core shell particles and largely affect the BSA adsorptio
ption interaction forces at pH 32 was generated by hydrogen bonding as well as
charge attraction between latex particle and protein even when the conformational change
and charge repulsion between proteins reduce the adsorption of protein at this pH The
adsorbed BSA amount was also increased as the amount of PAA increases However the
adsorption of BSA was minimal (less than 07 mgm2) at pH 74 where the carboxylic
groups are ionized leading to more enhancement of charge repulsion by ionization of the
119
carboxylic groups (COO-) Because of this the PSPMMA-PAA latex particles have a
much lower protein adsorption
There were steep initial slopes for adsorption isotherms of β
in
s
there
M
r
e
es
o
2M on latex particles
all isotherms indicating a high affinity type adsorption The complete plateau region
were not seen for all isotherms indicating that β2M proteins were not saturated and
are still available sites for β2M adsorption on latex particles The initial slope of β2
isotherms for PS was steeper than for the other latex particles PSPMMA100
PSPMMA90PAA10 and PSPMMA75PAA25
The layer thickness (δ) of BSA proteins on synthesized latex particles in phosphate
buffer (PB) was in the range of 27 - 81 Aring at 37 C and pH 48 Only the δ values at pH
48 the isoelectric point (IEP) of BSA are important because intra- and inter-molecular
electrostatic repulsion of the protein molecules is minimized at the IEP Compared to the
hydrodynamic dimensions of BSA 140 times 40 times 40 Aring these values of the adsorbed laye
thickness indicates that the BSA molecules adsorb by the side-on (40Aring) and end-on mod
(140Aring) Consideration of the multi-layering is unnecessary because the adsorbed
thickness of BSA do not exceed 140 Aring
The Gibbs free energy of β
o
3
2M was more negative in phosphate buffer (PB) than
that of BSA in physiological condition (37 C and pH 74) indicating that βo2M molecul
are more favorably adsorbed on the latex particles than BSA and that the affinity of β2M
was much larger than BSA This may be explained by the rate of diffusion and
accessibility of β2M molecules being easier and faster than that for BSA because of
smaller size (or weight) and spherical shape of β2M molecules It may also be possible t
explain that more hydrophobic forces (less electrostatic repulsion) contributes to
120
adsorption of β2M than to that of BSA because the IEP of β2M (57) is closer to
physiological condition (pH 74) than that of BSA (pH 48) The selective adsorption of
β2M m
in β2M
larger than in BSA adsorption indicating
that fa
an that between BSA and latex particles because
IEP (
ch as
Other
olecules over BSA molecules can be enhanced by developing more hydrophilic
PSPMMA90PAA25 and PSPMMA75PAA25 core shell latex particles
The adsorption kinetics of proteins on latex particles was expressed as a second-
order model The plateau region for β2M was reached earlier than BSA on all latex
particles and the K values (equilibrium rate constant of 2nd order adsorption)
adsorption process on latex particles were much
ster adsorption rate of β2M molecules than that of the BSA molecules There are
several explanations for these phenomena First the smaller size of β2M molecules can
more easily diffuse and approach to the latex substrate Second enhanced hydrophilic
domain on the latex particle surface by sulfate groups (SO4-) and ionized carboxylic
groups (COO-) is not favorable for larger BSA molecules but is for smaller β2M to be
anchored Third the conformational change by intra- and inter-molecular repulsion
would be faster in β2M due to lower molecular weight Finally the ionic repulsion
between β2M and latex particles is less th
57) of β2M is closer to the tested condition (pH 74) than IEP (48) of BSA
In the biocompatibility test the number of red blood cells (RBCs) ruptured by PS
latex particles ranged from 30 to 91 as the solid content of PS increased from 05 to
50 respectively However hemolysis of the other core shell latex particles su
PSPMMA100 PSPMMA90PAA10 and PSPMMA75PAA25 were less than 35 The
PSPMMA90PAA10 particles at all applied solid concentrations had less than 02
hemolysis and was the most blood compatible among the prepared latex particles
121
core shell particles such as PSPMMA100 and PSPMMA75PAA25 also had excelle
compatibility with low RBC breakage
52 Conclusions
The major conclusions from this study include
nt blood
bull microglobulin (β2M) over that of the bovine serum albumin (BSA) at pH 74
was achieved by controlling the PMMA and PAA ratio to optimize
e particles posses the most desirable requirements for use in a human dialysis
ns
icles
olecule
bull
Using engineered core shell latex particles selective adsorption of β2
physiological condition was demonstrated
bull The rate of adsorption of the β2M was also much higher than that of BSA This
hydrophobichydrophilic microdomain size
bull The core shell particles also exhibited excellent biocompatibility with blood Thes
membrane
53 Recommendation for Future Work
With the results and conclusions obtained through this study several suggestio
may be given for future work as follows
bull Competitive adsorption tests of whole blood proteins on synthesized latex partunder physiological condition
bull Preparation of latex particles with optimum hydrophobic hydrophilic microdomains to maximize selective adsorption and removal of toxin molecules
bull Membrane fabrication by suitable pore size design to discriminate a toxin mby size sieving as a function of particle layers
Evaluation of membrane performance such as flow rate in water and in simulated blood fluid (SBF)
bull Application for hemodialysis as well as other technologies such as a water remediation
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Korea on August 7 1968 He obtained a Bachelor of Science degree in Chemistry from
Keim d graduate school and earned a
resear n Korea Institute of Science and
Coati e
ia
Busin
BIOGRAPHICAL SKETCH
Sangyup Kim was born and raised in the small town of Sung-Joo Kyungbook
yung University (Daegu Korea) in 1993 He entere
Master of Science degree in Chemical engineering in 1995 He worked as a student
cher for 2 years at Textile Polymer Division i
Technology (KIST) in Seoul Korea Next he worked over a year at the Automobile
ng Division in DongJoo-PPG in Korea In August 2001 he began studying for th
degree of Doctor of Philosophy in materials science and engineering at the University of
Florida in Gainesville After getting his PhD he plans to work at the Digital Med
ess Division of Samsung Electronics in Korea starting in January 2006
141
To all who made this work possible
ACKNOWLEDGMENTS
I owe what I am today and this moment to many people without whom I could not
have been here First of all I would like to express my sincere and deepest appreciation
to my parents wife Mikyung daughters Heehyung and Erin whose love patience and
support helped me pursue my dream I gratefully acknowledge my advisor Dr H El-
Shall who gave me an opportunity to become part of an exciting and beneficial research
project and exert myself in the research work His guidance knowledge and stimulating
scientific and critical thinking have contributed to my success to a great extent and
inspired me to dedicate myself to research work I especially appreciate Dr R Partch as a
co-advisor who has helped me conduct experiments with valuable discussion for my
research from my first year I am greatly thankful to Dr C McDonald whose idea and
valuable experimental advice and discussion in the area of polymer chemistry and
membrane technology helped cultivate my interest in this area and were vital to
performing this research Without his contribution this work could not have been
achieved I also thank my doctoral committee members Dr R Singh Dr A Zaman and
Dr B Koopman for appreciated comments
Financial support and equipment use for research from the Particle Engineering
Research Center at the University of Florida were vital in continuing my study for more
than 4 years My sincere thanks are due to my former advisor Dr Kiryoung Ha in
Keimyung University who helped me open my eyes and broaden my thought and
introduced me into a limitless polymer field I would like to also thank to past and current
iv
members in Dr El-Shallrsquos research group for their assistance advice and support I
sincerely appreciate Stephen for the assistance of thesis writing
v
TABLE OF CONTENTS page
ACKNOWLEDGMENTS iv
LIST OF TABLES viii
LIST OF FIGURES x
1 INTRODUCTION 1
2 BACKGROUND AND LITERATURE SURVEY5
21 The Significance of End Stage Renal Disease (ESRD)5 22 Hemodialysis (HD) Treatment 6 23 Advances in Membrane Technology 8 24 Sorbent Technology16 25 Limitation of Current Hemodialysis Treatment 19 26 Latex Particles 20
261 The Components for Emulsion Polymerization 21 262 Particle Nucleation 21 263 Types of Processes for Emulsion Polymerization 23 264 Chemistry of Emulsion Polymerization 24 265 Seed Emulsion Polymerization25 266 Polystyrene Latex Particles 26 267 Various Applications of Latex Particles28
27 Proteins 30 271 Interaction Forces between Proteins33 272 β -Microglobulin (β M)2 2 35 273 Serum Albumin 36
28 Protein Adsorption38 281 Interaction between Protein Molecule and Latex Particle38
29 Hypothesis for Toxin Removal39 291 Toxin Removal by Size Sieving Based on Monodispersed Pore Size 40 292 Toxin Removal by Selective Adsorption on Engineered Latex Particles 41
3 EXPERIMENTAL AND CHARACTERIZATION METHODOLOGY 44
31 Materials 44 32 Latex Particle Preparation46
321 Preparation of Seed Latex Particles47
vi
322 Preparation of Seeded Latex Particles 48 323 Preparation of Core Shell Latex Particles 48 324 Purification of Synthesized Latex Particles48
33 Characterization49 331 Degree of Conversion49 332 Fourier Transform Infrared (FTIR) Spectroscopy49 333 Quasielastic Light Scattering (QELS) 50 334 Field Emission-Scanning Electron Microscopy (FE-SEM) 50 335 Zeta Potential Measurement 50 336 Protein Adsorption51 337 Blood Biocompatibility by Hemolysis Test 53
4 RESULTS AND DISCUSSION55
41 Polymerization of Latex Particles55 411 Polystyrene Seed Latex Particles55 412 The Growth of Polystyrene (PS) Seed Latex Particles61 413 Core Shell Latex Particles 71
42 Characterization of Latex Particles75 421 Fourier Transform Infrared Spectroscopy (FTIR)75 422 Zeta Potential Measurements 75
43 Protein Adsorption Study 85 431 Adsorption Isotherm87 432 Adsorbed Layer Thickness 103 433 Gibbs Free Energy of Protein Adsorption 104 434 Kinetics of Adsorption 107
44 Blood Compatibility 112
5 SUMMARY OF RESULTS CONCLUSION AND RECOMMENDATION FOR FUTURE WORK115
51 Summary of Results115 52 Conclusions121 53 Recommendation for Future Work121
LIST OF REFERENCES122
BIOGRAPHICAL SKETCH 141
vii
LIST OF TABLES
Table page 4-1 The polymerization recipe of polystyrene (PS) seed particles 57
4-2 Continuous addition emulsion polymerization recipe for growing polystyrene (PS) latex particles less than 500nm in size 63
4-3 Continuous addition emulsion polymerization recipe for growing polystyrene (PS) latex particles larger than 500nm in size64
4-4 The preparation recipe of PS core with various shell latex particles72
4-5 The isoelectric point (IEP) of common proteins 83
4-6 The equilibrium concentration values of BSA adsorption on polystyrene (PS) latex particles calculated the Langmuir-Freundlich isotherm model 93
4-7 The equilibrium concentration values (q ) of BSA adsorption on PSPMMA particles calculated the Langmuir-Freundlich isotherm model
m 10096
4-8 The equilibrium concentration values of BSA adsorption on PSPMMA PAA particles calculated the Langmuir-Freundlich isotherm model
90 1098
4-9 The equilibrium concentration values of BSA adsorption on PSPMMA PAA particles calculated the Langmuir-Freundlich isotherm model
75 25100
4-10 The equilibrium concentration values of β M adsorption calculated the Langmuir-Freundlich isotherm model
2102
4-11 Absorbed BSA layer thickness (Aring) of BSA in phosphate buffer (PB) at 37 Co 104
4-12 Absorbed BSA layer thickness (Aring) of BSA in phosphate buffered saline (PBS) at 37 Co 104
4-13 The values of Gibbs free energy change of BSA adsorption in phosphate buffer (PB) at 37 Co 105
4-14 The values of Gibbs free energy change of BSA adsorption in phosphate buffered saline (PBS) at 37 Co 105
viii
4-15 The values of Gibbs free energy change of β M adsorption in phosphate buffere (PB) at 37 C and pH 74
2o 106
4-16 Fitting parameters of second-order kinetic model 111
ix
LIST OF FIGURES
Figure page 1-2 Stereo drawing of the αndashcarbon backbone of β M2 2
2-1 An image of the location and cross section of a human kidney 5
2-2 A schematic draw of the hemodialysis route7
2-3 Cross-sectional SEM image view of the Polyflux S (polyamide + polyacrylethersulfone + polyvinypyrollidone) dialysis membranes 11
2-4 The chemical structure of polysulfone (PSf) 12
2-5 The chemical structure of polyamide (PA)13
2-6 Chemical structure of polyacrylonitrile (PAN) 14
2-7 Emulsion polymerization system24
2-8 L-α-amino acid 31
2-9 Ribbon diagram of human β M taken from the crystal structure of the protein bound to the heavy chain of the MHC class I complex (PDB 1DUZ)
236
2-10 Secondary structure of human serum albumin (HSA) with sub-domains 37
2-11 The relationship between pore size and particle size in body-centered cubic and closed packed arrays41
2-12 Schematic representation of the protein adsorption on the core shell latex particles at pH 7442
3-1 Chemical structure of main chemicals45
3-2 Experimental setup for semi-continuous emulsion polymerization 46
3-3 The particle preparation scheme in various types and size ranges of latex particles 47
3-4 Schematic of the procedure for a protein adsorption test 52
3-5 Separation of RBC from whole blood by centrifuge process53
x
3-6 Schematic of the procedure for hemolysis test 54
4-1 Schematic representation of semi-continuous seed latex particles preparation and growth56
4-2 Scanning Electron Micrograph (SEM) of polystyrene seed latex particles (A) PS (B) PS (C) PS (D) PSS259 S233 S207 S181 59
4-3 SEM of PS latex particles less than 500nm in size (A) 258nm (B) 320nm (C) 370nm (D) 410nm 65
4-4 SEM of PS latex particles larger than 500nm in size (A) 525nm (B) 585nm (C) 640nm (D) 790nm 68
4-5 Dependence of the particle size on the surfactant to monomer ratio70
4-6 Schematic of core shell latex particle structures 71
4-7 Scanning Electron Micrograph of latex particles (A) PS (B) PSPMMA (C) PSPMMA PAA (D) PSPMMA PAA
100
90 10 75 25 73
4-8 FTIR spectra of polymerized latex particles (A) bare polystyrene (PS) (B) PSPMMA (C) PSPMMA PAA (D) PSPMMA PAA100 90 10 75 25 76
4-9 Schematic representation of ion distribution near a positively charged surface 78
4-10 Schematic representation of zeta potential measurement79
4-11 Zeta potential of PS latex particles at 25 Co 80
4-12 Zeta potential of PSPMMA latex particles at 25 C100o 81
4-13 Zeta potential of PSPMMA PAA latex particles at 25 C90 10o 82
4-14 Zeta potential of PSPMMA PAA latex particles at 25 C75 25o 82
4-15 The decay of surface potential with distance from surface in various electrolyte concentrations (1) low (2) intermediate (3) high 84
4-16 High-affinity adsorption isotherm of typical flexible polymer on solid surface 86
4-17 Overall schematic representation of the protein adsorption on the synthesized latex particles86
4-18 The dependence of UV absorbance on BSA concentration (A) 10mgml (B) 06mgml (C) 02mgml87
4-19 Standard curve of net absorbance vs BSA sample concentration 88
xi
4-20 Fitted models for adsorption isotherm of bovine serum albumin (BSA) on polystyrene latex particles at 37 C in phosphate buffer mediao 89
4-21 Adsorption isotherm of BSA on polystyrene (PS) latex particles in phosphate buffer (PB) at 37 Co 90
4-22 Adsorption isotherm of BSA adsorption 37 C in phosphate buffered saline (PBS) on PS latex particles
o
91
4-23 Conformation of bovine serum albumin (BSA) on latex particles92
4-24 Adsorption isotherm of BSA 37 C in PB on PSPMMA core shell latex particles
o100
94
4-25 Adsorption isotherm of BSA at 37 C in PBS on PSPMMA core shell latex particles
o100
95
4-26 Adsorption isotherm of BSA at 37 C in PB on PSPMMA PAA core shell latex particles
o90 10
97
4-27 Adsorption isotherm of BSA at 37 C in PBS on PSPMMA PAA core shell latex particles
o90 10
98
4-28 Adsorption isotherm of BSA 37 C in PB on PSPMMA PAA latex particleso75 25 99
4-29 Adsorption isotherm of BSA 37 C in PBS on PSPMMA PAA core shell latex particles
o75 25
99
4-30 Adsorption isotherm of β M onto PS and PSPMMA latex particles in PB at 37 C and pH 74
2 100o 101
4-31 Adsorption isotherm of β M onto PS and PSPMMA PAA latex particles in PB at 37 C and pH 74
2 90 10o 101
4-32 Adsorption isotherm of β M onto PS and PSPMMA PAA latex particles in PB at 37 C and pH 74
2 75 25o 102
4-33 Gibbs free energy of adsorption of proteins on latex particles in PB at 37 C and pH 74
o
107
4-34 The kinetics of protein adsorption in PB on PS latex particles at 37 C and pH 74
o
109
4-35 The kinetics of protein adsorption in PB on PSPMMA latex particles at 37 C and pH 74
100o
109
4-36 The kinetics of protein adsorption in PB on PSPMMA PAA latex particles at 37 C and pH 74
90 10o 110
xii
4-37 The kinetics of protein adsorption in PB on PSPMMA PAA latex particles at 37 C and pH 74
75 25o 110
4-38 Image of red blood cells 113
4-39 Hemolysis caused by latex particles n=5114
xiii
Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy
SYNTHESIS AND ENGINEERING OF POLYMERIC LATEX PARTICLES FOR MEDICAL APPLICATIONS
By
Sangyup Kim
December 2005
Chair Hassan El-Shall Major Department Materials Science and Engineering
Latex particles with well-defined colloidal and surface characteristics have received
increasing attention due to their useful applications in many areas especially as solid
phase supports in numerous biological applications such as immunoassay DNA
diagnostic cell separation and drug delivery carrier Hemodialysis membrane using
these particles would be another potential application for the advanced separation
treatment for patients with end stage renal disease (ESRD) It is desirable to remove
middle molecular weight proteins with minimal removal of other proteins such as
albumin Thus it is necessary to understand the fundamental interactions between the
particles and blood proteins to maximize the performance of these membranes This
improvement will have significant economic and health impact
The objective of this study is to synthesize polymeric latex particles of specific
functionality to achieve the desired selective separation of target proteins from the human
blood Semi-continuous seed emulsion polymerization was used to prepare monodisperse
xiv
polystyrene seed particles ranging from 126plusmn75 to 216plusmn53 nm in size which are then
enlarged by about 800nm Surfactant amount played a key role in controlling the latex
particle size Negatively charged latex particles with a different hydrophobicity were
prepared by introduction of a sodium persulfate initiator and hydrophilic acrylic acid
monomer The prepared polymeric particles include bare polystyrene (PS) particles less
hydrophobic PS core and PMMA shell particles and more hydrophilic PS core and
PMMA-co-PAA shell latex particles with a 370nm mean diameter SEM light scattering
and zeta potential measurements were used to characterize particle size and surface
properties Adsorption isotherms of two proteins bovine serum albumin (BSA) and β2-
microglobulin (β2M) on latex particles were obtained as a function of pH and ionic
strength using the bicinchoninic acid (BCA) assay method The Langmuir-Freundlich
adsorption model was used to determine the adsorption amount of protein at equilibrium
The thickness of adsorbed BSA layer on latex particles was obtained in order to
investigate the adsorption orientation such as end-on or side-on mode Adsorption
kinetics experiments for both proteins and all latex particles were also performed The
adsorption kinetic constant determined from the Langmuir-Freundlich adsorption
isotherm model was used to calculate Gibbs free energy of adsorption to compare the
competitive adsorption of BSA and β2M Hemolysis tests were performed to investigate
the blood compatibility of synthesized latex particles PSPMMA90PAA10 and
PSPMMA75PAA25 core shell latex particles had desirable material properties with not
only a large amount and high rate of selective β2M adsorption over BSA but also high
blood compatibility showing less than 3 hemolysis
xv
CHAPTER 1 INTRODUCTION
End stage renal disease (ESRD) is a chronic condition in which kidney function is
impaired to the extent that the patientrsquos survival requires removal of toxins from the
blood by dialysis therapy or kidney transplantation The National Kidney Foundation
estimates that over 20 million Americans had chronic kidney disease in 2002 [NKF] The
number of people with ESRD is rapidly increasing in the United States with
approximately 96 295 incidents and 406 081 prevalent patients including 292 215 on
dialysis and 113 866 with a functioning graft in 2001 It is projected that there will be
more than 22 million ESRD patients by 2030 [USRDS 2003] The expenditure for the
ESRD treatment program had reached $228 billion 64 of the Medicare budget in
2001 Due in part to the limited availability of kidneys for transplantation hemodialysis
is the primary clinical treatment for the patients with ESRD
The central element of a hemodialysis instrument is the semipermeable membrane
that allows for selective transport of low molecular weight biological metabolites less
than 5000 Da such as urea and creatinine as well as excess water and electrolytes [Baker
2004] from the blood One limitation of current dialysis technologies is the inability to
efficiently remove middle molecular weight toxins such as β2-Microglobulin (β2M) and
interleukin 6 (IL-6)
β2M is a causative protein of dialysis-related amyloidosis (DRA) a disease arising
in patients with chronic kidney failure as a serious complication of long-term
hemodialysis treatment [Gejyo et al 1985] β2M deposition in tissue is the primary cause
1
2
of destructive arthritis and carpal tunnel syndrome [Vincent et al 1992 Drueke 2000]
The β2M structure is shown in Figure 1-2
Figure 1-2 Stereo drawing of the αndashcarbon backbone of β2M [Becker et al 1985]
Although attempts have been made to increase the efficiency of middle molecular
weight toxin removal by changes in the membrane pore size and the use of innovative
materials to adsorb these toxins [Samtleben et al 1996 Ronco et al 2001] removal
efficiency is not as high as those achieved by a normal healthy kidney Traditional
membranes have a number of processing and performance limitations [Westhuyzen et al
1992 Leypoldt et al 1998] such as a restricted choice of surface chemistries and limited
control of porosity The development of novel engineering membrane technology is
needed to remove middle molecule toxins
Polymeric latex particles have received increasing attention in medical application
areas especially as solid phase supports in biological applications [Piskin et al 1994]
Examples of these applications include immunoassay [Chen et al 2003 Radomske-
Galant et al 2003] DNA diagnostic [Elaїssari et al 1998] cell separation drug delivery
carrier [Luck et al 1998 Kurisawa et al 1995 Yang et al 2000] etc This is because of
3
the well-defined colloidal and surface characteristics of the particles By using a seed
emulsion polymerization method it is possible to synthesize monodisperse latex particles
with various particle size ranges and surface chemistry Functionalized core-shell latex
particles can be introduced by multi-step emulsion polymerization Core particles are
synthesized in the first stage of the polymerization and the functional monomer is added
in the second stage This is done without any emulsifier addition to prevent the
production of new homopolymer particles [Keusch et al 1973] The core-shell particles
are useful in a broad range of applications because of their improved physical and
chemical properties over their single-component counterparts [Lu et al 1996 Nelliappan
et al 1997] Through the development of a hemodialysis membrane using monodisperse
latex particles improvements in advanced separation treatment for patients with end
stage renal disease (ESRD) can be realized This requires the maximum removal of
middle molecular weight proteins with minimal removal of other beneficial proteins such
as albumin Thus an understanding of the fundamental interactions between the particles
and biopolymers is vital to maximize the performance of this membrane technology
The field of material science and biotechnology is based on fundamental chemistry
has developed over the past three decades into todayrsquos powerful discipline that enables
the development of advanced technical devices for pharmaceutical and biomedical
applications This novel and highly interdisciplinary field is closely associated with both
the physical and chemical properties of organic and inorganic particles [Niemeyer 2001]
Hemodialysis membrane using these particles would lead to improvements in the
advanced separation treatment for patients with end stage renal disease (ESRD) The
interdisciplinary nature of this approach enables a more complete understanding of the
4
phenomena of protein adsorption and the material properties necessary for selective
separation This innovative approach to membrane fabrication has the potential of making
inexpensive highly efficient membranes for both industrial and specialized separation
processes
The goal of this study is to prepare polymeric latex particles with tailored
properties to maximize separation of toxin molecules and to investigate the fundamental
interactions between the particles and molecules in the biological system in order to
optimize the performance of a membrane material for these applications
Polymeric latex particles were synthesized with specific functionality in an attempt
to achieve selective separation Seeded emulsion polymerization was used to synthesize
functionalized monodisperse latex particles in various sizes Negatively charged
hydrophobic polystyrene latex particles were synthesized by the same method Core shell
latex particles PSPMMA100 PSPMMA90PAA10 PSPMMA75PAA25 were also
synthesized to differentiate the degree of hydrophobicity of particles Scanning Electron
Micrograph (SEM) and light scattering measurements were used to characterize particle
size and shape and zeta potential measurements were conducted to measure the electrical
surface property of synthesized particles Adsorption isotherms of target proteins bovine
serum albumin (BSA) and β2M on latex particles were obtained as a function of pH
ionic strength and protein concentrations using the BCA assay method Adsorption
kinetics for both proteins on the latex particles were also measured Finally hemolysis
tests were run to determine the biocompatibility of polymer latex particles with human
blood This research will be described in more detail in chapters 3 and 4
CHAPTER 2 BACKGROUND AND LITERATURE SURVEY
21 The Significance of End Stage Renal Disease (ESRD)
The kidneys are responsible for removing excess fluid minerals and wastes from
the blood regulating electrolyte balance and blood pressure and the stimulation of red
blood cell production They also produce hormones such as erythropoietin (EPO) and
calcitriol that keep bones strong and blood healthy [Casadevall and Rossert 2005 Kurbel
et al 2003] EPO acts on the bone marrow to increase the production of red blood cell in
case of bleeding or moving to high altitudes Calcitriol mainly acts on both the cells of
the intestine to promote the absorption of calcium from food and also bone to mobilize
calcium from the bone to the blood When kidneys fail harmful waste builds up blood
pressure rises and body retains excess fluid The body also does not make enough red
blood cells When this happens treatment is needed to replace the function of failed
kidneys
Figure 2-1 An image of the location and cross section of a human kidney
5
6
The National Kidney Foundation estimates that over 20 million Americans had
chronic kidney disease in 2002 [NKF] Chronic renal disease is a gradual and progressive
loss of the function unlike acute renal disease where sudden reversible failure of kidney
function occurs Chronic renal failure usually takes place over a number of years as the
internal structures of the kidney are slowly damaged In the early stages there can be no
symptoms In fact progression may be so gradual that symptoms do not occur until
kidney function is less than one-tenth that of a normal health kidney If left untreated
chronic kidney disease may ultimately lead to kidney failure known as End Stage Renal
Disease (ESRD) ESRD is a rapidly growing heath-care problem in the United States In
2001 approximately 96295 incidents and 406081 prevalent patients were diagnosed
with ESRD including 292215 patients on dialysis and 113866 patients with a
functioning graft [USRDS 2003] The projected number of patients with ESRD is
expected to exceed 22 million patients by 2030 with much of this growth being driven by
the increasing prevalence of major contributing factors such as diabetes and high blood
pressure [USRDS 2003] A great extent of ESRD program cost and Medicare budget
have been spent in 2001 Due in part to a limited availability of kidneys for
transplantation hemodialysis (HD) is the primary method of treatment for ESRD and is
currently used for approximately 61 of US ESRD patients
22 Hemodialysis (HD) Treatment
HD removes toxins from the body by extracorporeal circulation of the blood
through a semipermeable membrane referred to as a dialyzer The toxins are removed
primarily by diffusion across the membrane to a dialysate solution which is circulated on
the opposite side of the membrane The cleaned blood is then returned to the blood
stream A surgically constructed vascular access connects the extracorporeal circuit to the
7
patientrsquos system Treatments are almost always performed three times per week in
specially equipped dialysis facilities for 3-4 hours per each treatment Figure 2-2 shows
the scheme of the hemodialysis route The critical element of a HD instrument is the
semipermeable membrane which allows for selective transport of low molecular weight
biological metabolites from blood
igure 2-2 A schematic draw of the hemodialysis route
ESRD patients who undergo dialysis therapy often experience several other
problems associated with the treatment such as anemia fatigue bone problems joint
problems itching sleep disorders and restless legs Anemia is common in patient with
kidney disease because the kidneys produce the hormone erythropoietin (EPO) which
stimulates the bone marrow to produce red blood cells Diseased kidneys often do not
produce enough EPO to stimulate the bone marrow to make a sufficient amount of red
blood cells This leads to bone disease referred to as renal osteodystrophy and causes
bones to become thin and weak or malformed and can affect both children and adults
Older patients and women who have gone through menopause are at greater risk for this
disease Uremic toxins which cannot be remove from the blood by the current dialyzer
membranes can lead to itching This problem can also be related to high levels of
F
8
parathyroid hormone (PTH) Dialysis patients can also experience day-night revers
is they have insomnia at night and sleep during the day This can be related to possible
nerve damage in the body and a chemical imbalance in the blood due to the excess toxin
Oxidative stress is a problem for patients on maintenance dialysis This problem is due to
an imbalance between pro- and antioxidant factors [Roselaar et al 1995 Cristol et al
1994] Oxidative stress affects oxidation of low-density lipoproteins which are the main
factor for atherogenesis [Huysmans et al 1998] and is also involved in the development
of malignancies and diabetes mellitus [Rice-Evans et al 1993] In order to reduce
antioxidant defense dialysis is needed to contribute to help stimulate free radical
production or eliminate antioxidants Dialysis-related amyloidosis (DRA) is also a
common and serious problem for people who have been on dialysis for more than 5
years DRA develops when proteins in the blood deposit on joints and tendons causi
pain stiffness and fluid in the joint as is the case with arthritis Normally the healthy
kidneys can filter out these proteins but dialysis filters are not as effective
23 Advances in Membrane Technology
al that
s
ng
Dialysis for blood of ESRD
Hemo filtration
s
ysis using a cellulose based membrane occurred
1913 John Abel [1990] from the Johns Hopkins Medical School described a method
purification is widely used in the treatment
dialysis (HD) techniques use a semi-permeable membrane to replace the
role of the kidney The membranes used in HD can be broadly classified into those based
on cellulose and those manufactured from synthetic copolymers These membranes come
in various shapes such as sheets tubular structures or hollow fiber arrangements The
hollow fiber type is the most popular and is incorporated into over 800 different device
in world wide [Ronco et al 2001]
The first attempt at blood dial
9
where sing
e
om
ysis was performed until the mid 1960 The basic
molec
rial in
een
benzyl)
was used for the membrane with
hepar
by the blood of a living animal may be submitted to dialysis outside the body u
a membrane based on cellulose and returned to the natural circulation without exposur
to air infection by microorganisms or any alteration that would necessarily be prejudicial
to life This same technique is still used to today however the device used has been
modified over the years as better membranes were developed and the anti-coagulant
heparin has become available
Cellulose membranes have been widely used for the treatment of renal failure fr
1928 when the first human dial
ular structure of cellulose is made of a long chain containing hydroxyl (OH)
groups The realization that such groups imparted undesirable qualities on the mate
respect to blood contact behavior was discovered in the early 1970s and since has b
the focus of development These modified cellulose membranes used the partial
substitution of benzyl groups to replace the proton of the hydroxyl groups in an attempt
to reduce their negative effect The result is a molecular mosaic of hydrophobic (
and hydrophilic (hydroxyl and cellulose) regions
Kolff [Van Noordwijk 2001] studied the rotating drum artificial kidney for patients
with acute renal failure in 1943 Cellophane tubing
in as the anticoagulant For the next 17 years hemodialysis therapy was performed
by this method but only for the patients with acute reversible renal failure Vascular
access required repeated surgical insertions of cannulas (slender tubes) into an artery and
vein and limited the number of treatments for a patient could receive in order to
minimize the amount of vascular damage
10
Initially the need for dialysis in patients with acute renal failure was determ
mainly by the development of signs and sym
ined
ptoms of uremia After dialysis some time
might
et al
ent improvement In 1960 the development of a shunt [Quinton et al
1960]
fone (PSf) polyamide (PA) and polyacrylonitrile (PAN) by phase inversion or
precip
lidone
e
elapse before uremic manifestations returned to warrant a sequential treatment of
dialysis Many patients with acute renal failure secondary to accidental or surgical
trauma were hypercatabolic but the interdialytic interval might be prolonged because of
anorexia or use of a low-protein diet However Teschan and his coworkers [Obrien
1959] showed that patient well-being and survival were improved by what they termed
prophylactic daily hemodiaysis or administration of the treatment before the patient
again became sick with uremia Their report in 1959 was the first description of daily
hemodialysis
Development of membrane accessories such as a shunt has also been an area of
focus for treatm
a flexible polytetrafluoroethylene (PTFE or Teflonreg) tubing made many more
hemodialysis treatments possible for chronic kidney failure patients PTFE has a non-
stick surface and is relative biocompatibility leading to minimized blood clotting in the
shunt
Synthetic membranes are prepared from engineered thermoplastics such as
polysul
itation of a blended mixture resulting in the formation of asymmetric and
anisotropic structures Figure 2-3 shows a fiber type of the Polyflux S membrane
consisting of poluamide (PA) polyacrylethersulfone (PAES) and polyvinylpyrro
(PVP) with the integral three-layer structure The skin layer on the inside fiber typ
membrane contacts blood and has a very high surface porosity and a narrow pore size
11
distribution This layer constitutes the discriminating barrier deciding the permeabili
and solute retention properties of the membrane The skin layer is supported by thick
sponge-type structure larger pores providing mechanical strength and very low
hydrodynamic resistance
ty
Figure 2-3 Cross-sectional SEM image view of the Polyflux S (polyamide +
polyacrylethersulfone + polyvinypyrollidone) dialysis membranes [Deppisch et al 1998]
n
[Malchesky 2004] because of its thermal stability mechanical strength and chemical
inertn ciated
ls
m
PSf is a widely used membrane material for the hemodialysis applicatio
ess According to a report from the National Surveillance of Dialysis-Asso
Disease (NSDAD) in the US over 70 of hemodialysis membranes were PSf based
[Bowry 2002] This is most likely because PSf has many advantages over other materia
This synthetic polymer is one of few materials that can withstand sterilization by stea
ethylene oxide and γ-radiation PSf membrane can be prepared by conventional
immersion precipitation methods into many different shapes including porous hollow
12
fiber or flat sheet hemodialysis membranes The material also has a high permeab
low molecular weight proteins and solute and high endotoxin retention The chemical
structure of PSf is shown in Figure 2-4
ility to
Figure 2-4 The chemical structure of polysulfone (PSf)
There is one major disadvantage to PSf The hydrophobic nature of the PSf causes
ment alternative pathway
leadin
s not
3
et al
in
O acrylate
serious complications through the activation of the comple
g to the adsorption of serum proteins onto the membranes [Singh et al 2003]
Anticoagulants are added during dialysis therapy to avoid blood clotting but this doe
completely eliminate the problem In order to overcome this disadvantage of the PSf
membrane various studies have been performed to change the materialrsquos surface
properties These investigations include hydrophilic polymer coating [Brink et al 199
Kim et al 1988 Higuchi et al 2003] layer grafting onto PSf membrane [Wavhal
2002 Song et al 2000 Pieracci et al 2002 Mok et al1994] and chemical reaction of
hydrophilic components onto the membrane surface [Higuchi et al 1990 1991 1993
Blanco et al 2001 Nabe et al 1997 Guiver et al 1993] Hydrophilic monomers 2-
hydroxy-ethylmethacrylate (HEMA) acrylic acid (AA) and methacrylic acid (MMA)
have also been grafted onto PSf membrane to increase flux and Bovine Serum Album
(BSA) retention [Ulbricht et al 1996] Hancock et al [2000] synthesized
polysulfonepoly(ethylene oxide) (PEO) block copolymers to improve the resistance to
platelet adhesion Kim et al [2003] also studied blending a sulfonated PE
diblock copolymer into PSf in order to reduce platelet adhesion and enhance
13
biocompatibility PEO is a commonly used biomaterial due to its excellent resistance
protein adsorption and inherent biocompatibility [Harris 1992] Kim et al [20
a self-transformable copolymer to enhance the hydrophilicity of an asymmetric PSf
membrane with an ultra-thin skin layer The polymer had an entrapped diblock
copolymer containing a hydrophilic block of poly (ethylene glycol) (PEG)-SO
to
05] studied
ate
lic graft
l)
e of thier
aromatic
orand
3 acryl
and a hydrophobic block of octadecylacrylate (OA) Molecular dynamic (MD)
simulations were performed as a function of copolymer density to optimize interfacial
structure information McMurry [2004] developed a strategy using an amphiphi
copolymer added to PSf membranes by introducing polysulfone-g-poly (ethylene glyco
When compared to unmodified PSf these graft copolymer and resulting blend
membranes are found to hold promise for biomedical device applications
Polyamide (PA) membranes have also been used for hemodialysis becaus
mechanical strength in both wet and dry conditions Polyamide consists of
aliphatic monomers with amide bonding (-CONH-) also known as a peptide bond
The basic amide bond in polyamide is shown in Figure 2-5 R1 and R2 can be either
aromatic or aliphatic linkage group
C N
H
O
~ R R1 2~C N
H
O
~ R R1 2~
Figure 2-5 The chemical structure of polyamide (PA)
Panichi and co-workers [1998] evaluated the biocompatibility of the PA membrane
ompatible technique due to the
use of a synthetic membrane with a sterile re-infusion fluid and the convective removal of
and concluded that PA hemofiltration was a highly bioc
14
the ac
e
e
is
n
is a semi-crystalline polymer and the mechanical properties
strong
2
CN
tivated anaphylatoxins and β2-Microglobulin (β2M) The PA based membrane
Polyfluxreg (manufactured by Gambro GmbH Germany) blended with polyamide
polyarylethersulfone and polyvinylpyrrolidone (PVP) was able to clean small molecules
such as urea dreatinine and phosphate as well as decrease β2M amount by 502
[Hoenich et al 2002] Due to the non-selectivity of the membrane removal of thes
unwanted materials also led to the undesirable loss of beneficial proteins during therapy
Meier et al [2000] evaluated different immune parameters using a modified cellulos
low-flux hemophan and synthetic high-flux PA membrane during a 1 year period in
chronic hemodialysis patients They found that the 1-year immunological evaluation of
hemodiaysis membrane biocompatibility was associated with changes in the pattern of
chronic T-cell actiovation
Polyacrylonitrile (PAN) is another commonly used membrane material because it
inherently hydrophilic and has been commercialized for ultrafiltration and microfiltratio
[Scharnagl et al 2001] PAN
ly depend on the crystalline structures The chemical structure of PAN is shown in
figure 2-6
CH CH
n2
CN
CH CH n
Chemical structure of polyacrylonitrile (PAN)
The addition of additives such as polyvinylpyrrolidone (PVP) as a pore forming
agent gives PAN membranes more flexible processing parameters and increased
e has been optimized through
copolymerization with many other vinyl monomers including glycidyl methacrylate
Figure 2-6
performance [Jung et al 2005] PAN membrane performanc
15
[Godj
acid
for
for
a non-
solub this
in
oid underneath the inner
surface of membrane decreased by increasing the amount of solvent DMSA in the
evargova et al 1999 Hicke et al 2002] N-vinylimidazole [Godjevargova et al
2000] hydroxyl ethyl methacrylate [Ray et al 1999 Bhat et al 2000] methacrylic
[Ray et al 1999] vinyl pyrrolidone [Ray et al 1999] acrylic acid [Trotta et al 2002]
acrylamide [Musale et al 1997] and vinylchloride [Broadhead et al 1998] These
monomers provide a reactive group for enzyme immobilization improved mechanical
strength solvent-resistance pervaporation permeation flux anti-fouling and bio-
compatibility Because of this PAN-based copolymer membranes have great potential
the treatment of hemodialysis in an artificial kidney This material can also be used
other applications like the treatment of wastewater the production of ultra-pure water
biocatalysis together with separation and methanol separation by pervaporation
Lin and his coworker [2004] studied the modification of PAN based dialyzer
membranes to increase the hemocompatibility by the immobilization of chitosan and
heparin conjugates on the surface of the PAN membrane When a foreign material is
exposed to blood plasma proteins are adsorbed clotting factors are activated and
le fibrin network or thrombus is formatted [Goosen et al 1980] The result of
research was that the biocompatible chitosan polymer and a blood anticoagulant hepar
prevented blood clotting They showed prolonged coagulation time reduced platelet
adsorption thrombus formation and protein adsorption
Nie et al [2004] studied PAN-based ultrafiltration hollow-fiber membranes
(UHFMs) In order to improve the membrane performance acrylonitrile (AN) was
copolymerized with other functional monomers such as maleic anhydride and α-allyl
glucoside They found that the number and size of macrov
16
intern erum
and
d a
rface The second step is the
transf after
yjak et
embrane
1978 Malchesky et al 1978] has been further developed to increase the efficiency of
al coagulant The water flux of the UHFMs also decreased while the bovine s
albumin rejection increased minutely Godjevargova et al [1992] modified the PAN
based membrane with hydroxylamine and diethylaminoethylmethacrylate to improve
membrane dialysis properties Formed functional groups like primary amine oxime
tertiary amine groups provided the membrane with more hydrophilic properties an
substantial increase in the permeability of the membranes
The wide use of filtration in practice is limited by membrane fouling Solute
molecules deposit on and in the membrane in the process of filtration causing dramatic
reduction in flux through the membrane Fouling occurs mostly in the filtration of
proteins Three kinetic steps are involved in the fouling of UF membranes according to
Nisson [1990] The first step is the transfer of solute to the su
er of solute into the membrane until it either finally adsorbs or passes through
a set of adsorption-desorption events The third step includes surface binding
accompanied by structural rearrangement in the adsorbed state [Ko et al 1993] Br
al [1998] studied the surface modification of a commercially available PAN membrane
to develop superior filtration properties with less fouling by proteins The PAN
membrane was immersed in excess NaOH solution to convert some of the surface nitrile
groups into carboxylic groups by the hydrolysis process This modified PAN m
was not so severely fouled in the Bovine Serum Albumin (BSA) filtration test The pore
size however decreased during the hydrolysis process leading to a significant reduction
in flux and made the membrane less productive in the ultrafiltration (UF) mode
24 Sorbent Technology
Over the last three decades sorbent technology [Castino et al 1976 Korshak et al
17
dialysis or replace it for the treatment of ESRD Sorbents remove solutes from solution
through specific or nonspecific adsorption depending on both the nature of the solute and
the sorbent Specific adsorption contains tailored ligands or antibodies with high
selectivity for target molecules een used in autoimmune
disord
and
e
al 1996] various resins [Ronco et al 2001]
album
e
as
Specific adsorbents have b
ers such as idiopathic thrombocytopenic purpura [Snyder et al 1992] and for the
removal of lipids in familial hyper cholesterolemia [Bosch et al 1999] Nonspecific
adsorbents such as charcoal and resins attract target molecules through various forces
including hydrophobic interactions ionic (or electrostatic) attraction hydrogen bonding
and van der Waals interactions
New dialysate with sorbents has become an accepted modification of dialysis
sorbent hemoperfusion is gaining ground as a valuable addition to dialysis especially as
new sorbents are developed [Winchester et al 2001] Hemoperfusion is defined as th
removal of toxins or metabolites from circulation by the passing of blood within a
suitable extracorpoteal circuit over semipermeable microcapsules containing adsorbents
such as activated charcoal [Samtleben et
in-conjugated agarose etc Novel adsorptive carbons with larger pore diameters
have been synthesized for potential clinical use [Mikhalovsky 1989] Newly recognized
uremic toxins [Dhondt et al 2000 Haag-Weber et al 2000] have resulted in several
investigations on alternatives to standard or high-flux hemodialysis to remove thes
molecules These methods include hemodiafilteration with [de Francisco et al 2000] or
without [Ward et al 2000 Takenaka et al 2001] dialysate regeneration using sorbents
well as hemoperfusion using such adsorbents as charcoal and resins
18
Kolarz et al [1989 1995] studied the hyper-crosslinked sorbent prepared from
styrene and divinylbenzene (DVB) for a hemoperfusion application They found that the
pore structure of a swelling sorbent was changed by additional crosslinking with α αrsquo-
dichloro-p-xylene in the presence of a tin chloride catalyst and in a di
chloroethane
soluti and
en
decyl groups that attract β2M through a hydrophobic interaction The
adsor
of
5]
d
penet
on They also realized that the hemocompatibility was useful for nemoperfusion
could be imparted to the sorbents by introducing sulfonyl groups at a concentration of
about 02mmolg
A special polymeric adsorbing material (BM-010 from Kaneka Japan) has be
investigated by another group [Furuyoshi et al 1991] for the selective removal of β2M
from the blood of dialysis partients The adsorbent consists of porous cellulose beads
modified with hexa
ption capacity of this material is 1mg of β2M per 1ml of adsorbent Using a
hemoperfusion cartridge containing 350ml of these cellulose beads in sequence with a
high-flux hemodialyzer several small clinical trials were performed During 4-5 hours
treatment about 210mg of β2M were removed thus reducing the concentration in the
blood by 60-70 of the initial level [Nakazawa et al 1993 Gejyo et al 1993 199
RenalTech developed a hemoperfusion device BetaSorbreg containing the hydrate
cross-linked polystyrene (PS) divinylbenzene (DVB) resin sorbents with a pore structure
designed to remove molecules between 4 and 30 kDa [Winchester et al 2002] In this
case solute molecules are separated according to their size based on their ability to
rate the porous network of the beaded sorbents The resin beads were prepared with
a blood compatible coating and confirmed to be biocompatible in vivo in animals
[Cowgill et al 2001]
19
25 Limitation of Current Hemodialysis Treatment
Hemodialysis is a widely used life-sustaining treatment for patients with ESRD
However it does not replace all of the complex functions of a normal healthy kidne
a result patients on dia
y As
lysis still suffer from a range of problems including infection
accelerated cardio ion anemia
chron
e
se middle
alysis
sessio
s
f
vascular disease high blood pressure chronic malnutrit
ic joint and back pain and a considerably shortened life span One significant
limitation of the current dialysis technology is the inability to efficiently remove larger
toxic molecules This is mainly because of the broad pore size distribution reducing th
selective removal of toxins and unsatisfied biocompatibility causing lots of
complications such as inflammation blood clotting calcification infection etc
Dialysis purifies the patients blood by efficiently removing small molecules like
salts urea and excess water However as toxic molecules increase in size their removal
rate by hemodialysis substantially declines Typically only 10 - 40 of the
molecular weight toxins (300-15000 Da) are removed from the blood during a di
n [Vanholder et al 1995] These toxins then reach an abnormally high level and
begin to damage the body One such toxin β2M causes destructive arthritis and carpal
tunnel syndrome by joining together like the links of chain to form a few very large
molecules and deposit damaging the surrounding tissues [Lonnemann et al 2002] This i
also a main cause of mortality for long-term dialysis patients Other middle molecule
toxins appear to inhibit the immune system and may play a significant role in the high
susceptibility to infections in dialysis patients Still others are believed to impair the
functioning of several other body systems such as the hematopoietic and other endocrine
systems This may contribute to accelerated cardiovascular disease the leading cause o
20
death among dialysis patients as well as clinical malnutrition which affects up to 50 of
this patient population
Over the last decade polymeric dialysis membranes have been developed to
increase the capacity for removing middle molecular weight toxins by changing the pore
size of dialyzer membranes and using new materials that adsorb these toxins for
impro se
styrene made during World War II in the United
States The Dow chemical compan anufacturer of polystyrene
includ
d of
on
rug
ntrol of
ved removal characteristics However removal efficiency is not as high as tho
achieved by a normal healthy kidney
26 Latex Particles
The first synthetic polymer synthesized using emulsion polymerization was a
rubber composed of 13-butadiene and
y has been a major m
ing latex which they used in paint formulations The theory of emulsion
polymerization in which a surfactant is used was established by Harkins [1948] and by
Smith and Ewart [1948] By 1956 the technology was complete including the metho
building larger diameter particles from smaller ones The product by this emulsi
polymerization is referred to as latex a colloidal dispersion of polymer particles in water
medium [Odian 1991] Latexes are currently undergoing extensive research and
development for a broad range of areas including adhesives inks paints coatings d
delivery systems medical assay kits gloves paper coatings floor polish films carpet
backing and foam mattresses to cosmetics The relatively well known and easy co
the emulsion process is one of main advantages for these applications Therefore
polymeric latex particle prepared by emulsion polymerization can be a candidate for the
medical applications because of the easy control of the particle size and morphology as
well as flexible surface chemistry to be required
21
261 The Components for Emulsion Polymerization
The main components for emulsion polymerization process are the monomer a
dispersing medium a surfactant and an initiator Available monomers are styrene
sing medium is usually water
which
ticles
n
ation To
monomer droplets but in micelles because the initiators are
insolu le
and
olution
butadiene methylmethacylate acryl acid etc The disper
will maintain a low solution viscosity provide good heat transfer and allow
transfer of the monomers from the monomer droplets into micelles and growing par
surrounded by surfactants respectively The surfactant (or emulsifier) has both
hydrophilic and hydrophobic segments Its main functions is to provide the nucleatio
sites for particles and aid in the colloidal stability of the growing particles Initiators are
water-soluble inorganic salts which dissociate into radicals to initiate polymeriz
control the molecular weight a chain transfer agent such as mercaptan may be present
262 Particle Nucleation
Free radicals are produced by dissociation of initiators at the rate on the order of
1013 radicals per milliliter per second in the water phase The location of the
polymerization is not in the
ble in the organic monomer droplets Such initiators are referred to as oil-insolub
initiators This is one of the big differences between emulsion polymerization
suspension polymerization where initiators are oil-soluble and the reaction occurs in the
monomer droplets Because the monomer droplets have a much smaller total surface area
they do not compete effectively with micelles to capture the radicals produced in s
It is in the micelles that the oil soluble monomer and water soluble initiator meet and is
favored as the reaction site because of the high monomer concentration compared to that
in the monomer droplets As polymerization proceeds the micelles grow by the addition
of monomer from the aqueous solution whose concentration is refilled by dissolution of
22
monomer from the monomer droplets There are three types of particles in the emulsion
system monomer droplets inactive micelles in which polymerization is not occurring
and active micelles in which polymerization is occurring referred to as growing polymer
particles
The mechanism of particle nucleation occurs by two simultaneous processes
micellar nucleation and homogeneous nucleation Micellar nucleation is the entry of
radicals ei
ther primary or oligomeric radicals formed by solution polymerization from
the aq ed
the
r
s
n
is coagulation with other particles rather than
polymerization of monomer The driving force for coagulation of precursor particles
ueous phase into the micelles In homogeneous nucleation solution-polymeriz
oligomeric radicals are becoming insoluble and precipitating onto themselves or onto
oligomers whose propagation has ended [Fitch et al 1969] The relative levels of micella
and homogeneous nucleation are variable with the water solubility of the monomer and
the surfactant concentration Homogeneous nucleation is favored for monomers with
higher water solubility and low surfactant concentration and micellar nucleation is
favored for monomers with low water solubility and high surfactant concentration It has
also been shown that homogeneous nucleation occurs in systems where the surfactant
concentration is below CMC [Roe 1968] A highly water insoluble monomer such a
styrene [Hansen et al 1979 Ugelstad et al 1979] has probably created by micellar
nucleation while a water soluble monomer such as vinyl acetate [Zollars 1979] has bee
formed by homogeneous nucleation
A third latex formation reaction mechanism has been proposed referred to as
coagulative nucleation In this reaction the major growth process for the first-formed
polymer particles (precursor particles)
23
sever les
yer
es
r a
l
the beginning of the polymerization As soon as the initiator is
gins with the formation and
growt
and
ved
added at one time during the later stages of the polymerization prior to complete
al nanometers in size is their relative instability compared to larger sized partic
The small size of a precursor particle with its high curvature of the electrical double la
permits the low surface charge density and high colloidal instability Once the particl
become large enough in size maintaining the high colloidal stability there is no longe
driving force for coagulation and further growth of particles takes place only by the
polymerization process
263 Types of Processes for Emulsion Polymerization
There are three types of production processes used in emulsion polymerization
batch semi-continuous (or semi-batch) and continuous In the batch type process al
components are added at
added and the temperature is increased polymerization be
h of latex particles at the same time There is no further process control possible
once the polymerization is started In the semi-continuous emulsion polymerization
process one or more components can be added continuously Various profiles of particle
nucleation and growth can be generated from different orders of component addition
during polymerization There are advantages to this process such as control of the
polymerization rate the particle number colloidal stability copolymer composition
particle morphology In the continuous process the emulsion polymerization components
are fed continuously into the reaction vessel while the product is simultaneously remo
at the same rate High production rate steady heat removal and uniform quality of
latexes are advantages of the continuous polymerization processes
Other available methods include the intermittent addition and shot addition of one
or more of the components In the shot addition process the additional components are
24
conversion of the main monomer This method has been used successfully to develo
water-soluble functional monomers such as sodium styrene sulfonat
p
e [Kim et al 1989]
264
lsion
Chemistry of Emulsion Polymerization
Emulsion polymerization is one type of free radical polymerization and can be
divided into three distinct stages initiation propagation and termination The emu
polymerization system is shown in Figure 2-7
Aqueous phaseAqueous phase
Micelle withmonomer
I Rbull
Emulsifier
Polymer particle swollenWith monomer
Monomerdroplet
I Rbull
Micelle withmonomer
I Rbull
Emulsifier
Polymer particle swollenWith monomer
MonomerdropletMonomerdroplet
I Rbull
Figure 2-7 Emulsion polymerization system [Radicals (Rmiddot) are created from initiators
(I) Monomer is transferred from larger monomer droplet into micelles by emulsifier Initiated polymer particle by radicals is keep growing until monomers are all consumed The reaction is performed in aqueous media]
In the initiation stage free radicals are created from an initiator by heat or an
ultraviolet radiation source The initiator with either peroxides groups (-O-O-) such as
odium persulfate or azo groups (-N=N-) such as azobisisobutyronitrile is commonly
used for em t
with the m er
chain ere
s
ulsion polymerization The primary free radicals created from initiator reac
onomer for initiation of polymerization In the propagation stage the polym
grows by monomer addition to the active center a free radical reactive site Th
are two possible modes of propagations head-to-head addition and head-to-tail addition
25
The head-to-tail mode is the predominant configuration of the polymer chain a result o
steric hindrance of the substitute bulky group In the termination stage polymer chain
growth is terminated by either coupling of two growing chains forming one polymer
molecule or transferring a hydrogen atom (dispropotionation) from one growing chain to
another forming two polymer molecules one having a saturated end group and the other
with an unsaturated end-group
265 Seed Emulsion Polymerization
Polymer latex particles have received increasing interest because of the versatili
of the many applications heterophase polymerization processes like emulsion dispersion
micro-emulsion seeded emulsion precipitation etc Especially to prepare well-defined
microspheres having monodispe
f
ty
rse and various particle sizes as well as surface group
ed particles prepared by emulsion polymerization
and e
s
hological design In
seede to control
n
functionalilties it is necessary to use se
nlarge them to a desired size in the further stages of reactions
Polymeric particles are required to have a uniform particle size in many
applications such as chromatography where they are used as a packing material
Morphological control of latex particle is also important for many practical application
[Schmidt 1972] Seed emulsion polymerization (or two-stage emulsion polymerization) is
a useful method to achieve both monodisperse particle size and morp
d emulsion polymerization [Gilbert 1995] preformed lsquoseedrsquo latex is used
the number of particles present in the final latex The advantage of seeded emulsio
polymerization is that the poorly reproducible process of particle nucleation can be
bypassed so that the number concentration of particles is constant and known Various
mechanisms have been proposed for the growth of latex particles [Ugelstad et al 1980
Okubo et al 1992] using this polymerization technique The initial seed particle
26
preparation step is well known and relatively easy to perform because at the relative
small particle sizes (02 to 05 micron) the particle growth process can be readily
controlled by the use of an emulsifier Enough emulsifier is used to prevent coagulation
but not enough to cause the formation of new particles As the particles are grown to
larger sizes in successive seeding steps it becomes increasingly difficult to mainta
stable uncoagulated emulsion without forming new particles and thereby destroying
monodisperisty of the latex Recently there have been several reports concerning t
reaction kinetics of seed emulsion polymerization and the development of latex
morphologies over the course of the reaction [Chern et al 1990 Delacal et al 1990 L
et al 1995 Lee 2000 2002]
266 Polystyrene Latex Particles
A number of papers have described the synthesis of the polystyrene latex particl
bearing various functional surface groups such as carboxyl [Lee et al 2000 Tun
2002 Reb et al 2000] hydroxyl [Tamai et al 1989] marcapto [Nilson 1989] epoxy
[Shimizu et al 2000 Luo et al
ly
in a
he
ee
es
cel et al
2004] acetal [Izquierdo et al 2004 Santos et al 1997]
ometyl [Izquierdo et al 2004 Park et al 2001
Sarob
h the
d
thymine [Dahman et al 2003] chlor
e et al 1998] amine [Counsin et al 1994 Ganachaud et al 1997 Ganachaud et al
1995 Miraballes-Martinez et al 2000 2001 Anna et al 2005] ester [Nagai et al 1999]
etc In order to produce these functionalized particles different methods for particle
preparation must be used The polymer emulsion with core-shell morphology of latex
particles is one of them This is a multistep emulsion polymerization process in whic
polystyrene ldquocorerdquo particle is synthesized in the first stage and the functional monomer is
added in the second stage of the polymerization without any emulsifier postfeeding to
prevent the production of new homopolymer particles thus forming the functionalize
27
polymer ldquoshellrdquo on the ldquocorerdquo particle [Keusch et al 1973] There are requirements to
limit secondary nucleation and encourage core-shell formation in seeded emulsion
polymerization including the addition of smaller seed particles at high solid content to
increase particle surface area low surfactant concentration to prevent formation of
micelles and the semi-continuous addition of monomer to create a starved-feed conditio
and keep the monomer concentration low There are some advantages [Hergeth et al
1989] of dispersions with polymeric core-shell particles First it is possible to modi
interfacial properties of polymer particles in the aqueous phase by the addition of only
very small amounts of a modifying agent during the last period of the reaction Thus
these core-shell particles are useful in a broad range of applications since they always
exhibit improved physical and chemical properties over their single-component
counterparts [Lu et al 1996 Nelliappan et al 1997] In this way the improvement of
surface properties of such dispersions is straightforward and inexpensive The other is
that polymers with a core-shell structure are perfect model systems for investigating th
material properties of polymer blends and composites because of their regular
distribution of one polymer inside a matrix polymer and because of the simple sp
geometry of the system
Their properties usually depend on the structures of latex particles Chen and his
coworkers [Chen et al 1991 1992 1993] reported the morphological development of
core shell latex particles of polystyrenepoly(methyl methacrylate) during
polymerization Before the research by Chen and his coworker Min et al [Min et al
1983] reported the morph
n
fy the
e
herical
ological development of core shell latex of polystyrene
(PS)polybuthyl acrylateat (PBA) by seeded emulsion polymerization as a function of the
28
additi
of
ity of graft
ing reagents Thus protein adsorption on polymeric solid surfaces has
st are microspheres defined as ldquofine
polym can be
n
r was
containing antigen urine serum etc The latex will become agglutinated and visibly
on method of PS They found that the percentage of grafting PS to the PBA was
greatest for the batch reaction and the PBA-PS core-shell particles with a high degree
grafting remained spherical upon aging test because of the emulsifying abil
copolymer
267 Various Applications of Latex Particles
Latex particles are applicable to a wide range of areas such as biomedical
applications [Piskin et al 1994] especially as the solid phase such as in immunoassays
[Chern et al 2003 Radomske-Galant et al 2003] DNA diagnostic drug delivery carriers
[Luck et al 1998 Kurisawa et al 1995 Yang et al 2000] blood cell separations and
column pack
become a center of attention Of particular intere
er particles having diameters in the range of 01 to several micronsrdquo which
used as functional tools by themselves or by coupling with biocompounds Singer and
Plotz [1956] firstly studied microsphere or latex agglutination test (LATrsquos) by using
monodisperse polystyrene (PS) and polyvinyltoluene polymer particles as the support o
which the biomolecules were going to adsorb The biomolecule adsorption howeve
limited by possible desorption of the adsorbed species or loss of specific activity of the
complex formed Since this work was published the latex particle applications for
immunoassay have been rapidly and widely studied and developed
Latex agglutination test or latex immunoassays start with tiny spherical latex
particles with a uniform diameter and similar surface properties The particles are coated
with antibodies (sensitized) through the hydrophobic interaction of portions of the protein
with the PS surface of the particles If sensitized particles are mixed with a sample
29
agglomerate Latex tests are inexpensive as compared with the other techniques [Ba
1988]
ngs
sed
tion Rheumatoid factor (RF) in different age subpopulations has also been
evalu
studied a
latex
Unipath [Percival 1996] has manufactured a range of immunoassay materials ba
on a chromatographic principle and deeply colored latex particles for use in the home and
clinical environments The latex particles which are already sensitized with a
monoclonal antibody can detect any antigens that bound to the surface of the latex
particles Some detectable pollutants include estrogen mimics which induce
abnormalities in the reproductive system of male fishes and lead to a total or partial male
feminiza
ated according to a patientrsquos clinical status by using a rapid slide latex agglutination
test for qualitative and semiquantitative measurement in human serum along with latex
immunoassay method [Onen et al 1998] Magalhaes and his coworkers [2004]
diagnostic method of contamination of male fishes by estrogen mimics using the
production of vitellogenin (VTG) as a biomarker This was based on a reverse
agglutination test developed with monoclonal antibodies specific to this biomarker
Premstaller and his coworkers [Premstaller et al 2000 2001] have prepared a porous
poly(styrene-divinylbenzene) (PS-DVB) polymer monolith to use for highly efficient
chromatographic separation of biomelecules such as proteins and nucleic acids They
used a porogen a mixture of tetrahydrofuran and decanol to fabricate a micropellicular
PS-DVB backbone Legido-Quigley and his colleagues [2004] have developed the
monolith column to obtain further chromatographic functionality to the column by
introducing chloromethylstyrene in place of styrene into the polymer mixture
30
Core shell type monodisperse polymer colloids have been synthesized by Sarobe
and Forcada [1998] with chloromethyl functionality in order to improve the biomolecu
adsorption through a two-step emulsion polymerization process They investigated the
functonalized particles by optimizing the experimental parameters of the functional
monomer including reaction temperature the amount and type of redox initiator sys
used the type of addition of the initiator system and the use of washing They c
le
tem
oncluded
that th
sis
serve as separation
chann
of
usually
along the polypeptide chain which contain positive or negative charges [Norde 1998]
e relation between the amount of iron sulfate and the persulfatebisulfite system
added should be controlled to obtain monodisperse particles and prevent the premature
coagulation of the polymer particles during the polymerization
A semi-continuous emulsion polymerization technique for latex particle synthe
was performed by McDonald and other researchers [Ramakrishnan et al 2004 Steve et
al 1999] They introduced a variety of particles sizes compositions morphologies and
surface modifications to fabricate latex composite membranes (LCMs) Arraying and
stabilizing latex particles on the surface of a microporous substrate form narrowly
distributed interstitial pores formed between the particles which
els They investigated the membrane performance using gas fluxes water
permeability and the retention characterization of dextran molecules From these tests
they concluded that the narrow discriminating layer made of the latex particles leads to a
highly efficient composite membrane
27 Proteins
Proteins are natural polyamides comprised of about 20 different α-amino acids
varying hydrophobicity [Norde 1998] Proteins are more or less amphiphilic and
highly surface active because of the number of amino acid residues in the side groups
31
Proteins are polymers of L-α-amino acids [Solomon and Fryhle 2000] The α refers to a
carbon with a primary amine a carboxylic acid a hydrogen and a variable side-chain
group designated as R Carbon atoms rent groups are asymmetric and can
exhib l
s
e
on
with four diffe
it two different spatial arrangements (L and D configurations) due to the tetrahedra
nature of the bonds The L refers to one of these two possible configurations Amino
acids of the D-configuration are not found in natural proteins and do not participate in
biological reactions Figure 2-8 shows the chiral carbon in 3-D as the L isomer Protein
consist of twenty different amino acids differentiated by their side-chain groups [Norde
1998] The side-chain groups have different chemical properties such as polarity charg
and size and influence the chemical properties of proteins as well as determine the
overall structure of the protein For instance the polar amino acids tend to be on the
outside of the protein when they interact with water and the nonpolar amino acids are
the inside forming a hydrophobic core
Figure 2-8 L-α-amino acid
The covalent linkage between two amino acids is known as a peptide bond A
peptide bond is formed when the amino group of one amino acid reacts with the carboxyl
group of another amino acid to form an amide bond through the elimination of water
This arrangement gives the protein chain a polarity such that one end will have a free
amino group called the N-terminus and the other end will have a free carboxyl group
called the C-terminus [Solomon and Fryhle 2000] Peptide bonds tend to be planar and
32
give the polypeptide backbone rigidity Rotation can still occur around both of the α-
carbon bonds resulting in a polypeptide backbone with different potential conformations
relative to the positions of the R groups Although many conformations are theoretically
possible interactions between the R-groups will limit the number of potential
nd to form a single functional conformation In other words
the conformation or shape of the protein is
α-helix
can interact with other
secondary structures within the sam
conformations and proteins te
due to the interactions of the chain side
groups with one another and with the polypeptide backbone The interactions can be
between amino acids that are close together as in a poly-peptide between groups that are
further apart as in amino acids or even on between groups on different polypeptides all
together These different types of interactions are often discussed in terms of primary
secondary tertiary and quaternary protein structure
The primary amino acid sequence and positions of disulfide bonds strongly
influence the overall structure of protein [Norde 1986] For example certain side-chains
will promote hydrogen-bonding between neighboring amino acids of the polypeptide
backbone resulting in secondary structures such as β-sheets or α-helices In the
conformation the peptide backbone takes on a spiral staircase shape that is stabilized by
H-bonds between carbonyl and amide groups of every fourth amino acid residue This
restricts the rotation of the bonds in the peptide backbone resulting in a rigid structure
Certain amino acids promote the formation of either α-helices or β-sheets due to the
nature of the side-chain groups Some side chain groups may prevent the formation of
secondary structures and result in a more flexible polypeptide backbone which is often
called the random coil conformation These secondary structures
e polypeptide to form motifs or domains (ie a
33
tertiary structure) A motif is a common combination of secondary structures and a
domain is a portion of a protein that folds independently Many proteins are composed o
multiple subunits and therefore exhibit quaternary structures
271 Interaction Forces between Proteins
Proteins in aqueous solution acquire compact ordered conformations In such a
compact conformation the movement along the polypeptide chain is severely restrict
implying a low conformational entropy The compact structure is possible only if
interactions within the protein molecule and interactions between the protein molecule
and its environment are sufficiently favorable to compensate for the low conformational
entropy [Malmsten 1998] Protein adsorption study is often focused on structural
rearrangements in the protein molecules because of its significance to the biological
functioning of the molecules and the important role such rearrangements play in the
mechanism of the adsorption process Knowledge of the majo
f
ed
r interaction forces that act
structures helps to understand the behavior
of pro
arge
a
ded
between protein chains and control the protein
teins at interface These forces include Coulomb interaction hydrogen bonds
hydrophobic interaction and van der Waals interactions
Coulombic interaction Most of the amino acid residues carrying electric ch
are located at the aqueous boundary of the protein molecule An the isoelectric point
(IEP) of the protein where the positive and negative charges are more or less evenly
distributed over the protein molecule intramolecular electrostatic attraction makes
compact structure favorable to proteins Deviation to either more positive or more
negative charge however leads to intramolecular repulsion and encourages an expan
structure Tanford [1967] calculated the electrostatic Gibbs energy for both a compact
impenetrable spherical molecule (protein) and a loose solvent-permeated spherical
34
molecule (protein) over which the charge is spread out From the results he found th
the repulsion force was reduced at higher ionic strength d
at
ue to the screening action of ion
ble
e
the
d the
tween
dehyd
e
ent
oles
tein
Hydrogen bond Most hydrogen bonds in proteins form between amide and
carbonyl groups of the polypeptide backbone [Malmsten 1998] The number of availa
hydrogen bonds involving peptide units is therefore far greater than that involving sid
chains Because α-helices and β-sheets are aligned more or less parallel to each other
interchain hydrogen bonds enforce each other Kresheck and Klotz [1969] examine
role of peptide-peptide hydrogen bonds and concluded that hydrogen bonds between
peptide units do not stabilize a compact structure of protein However because the
peptide chain is shielded from water due to other interactions hydrogen bonding be
peptide groups do stabilize α-helical and β-sheet structures
Hydrophobic interaction Hydrophobic interaction refers to the spontaneous
ration and subsequent aggregation of non-polar components in an aqueous
environment In aqueous solutions of proteins the various non-polar amino acid residues
will be found in the interior of the molecule thus shielded from water The
intermolecular hydrophobic interaction for the stability of a compact protein structure
was first recognized by Kauzmann [1959] If all the hydrophobic residues are buried in
the interior and all the hydrophilic residues are at the outermost border of the molecul
intramolecular hydrophobic interaction would cause a compact protein structure
However geometrical and other types of interactions generally cause a fraction of the
hydrophobic residues to be exposed to the aqueous environm
Van der Waals interaction The mutual interaction between ionic groups dip
and induced dipoles in a protein molecule cannot be established as long as the pro
35
structure is not known in great detail Moreover the surrounding aqueous medium also
contains dipoles and ions that compete for participation in the interactions w
the protein molecule Dispersion interactions favor a compact structure However
because the Hamaker constant for proteins is only a little larger than that of water the
resulting effect is relatively small [Nir 1977]
272 β
ith groups of
rgiles et al 1996
Floeg
) is degraded [Floege 2001] Renal
β2M from the serum resulting in an increase in β2M
conce well
the
)
a
al
2-Microglobulin (β2M)
The protein β2M is of particular interest because it is involved in the human
disorder dialysis-related amyloidosis (DRA) [Geiyo et al 1985 A
e 2001] DRA is a complication in end stage renal failure patients who have been on
dialysis for more than 5 years [Bardin et al 1986 Drueke 2000] DRA develops when
proteins in the blood deposit on joints and tendons causing pain stiffness and fluid in
the joints as is the case with arthritis In vivo β2M is present as the non-polymorphic
light chain of the class I major histocompatibility complex (MHC-I) As part of its
normal catabolic cycle β2M dissociates from the MHC-I complex and is transported in
the serum to the kidney where the majority (95
failure disrupts the clearance of
ntration by up to 60-fold [Floege 2001] By a mechanism that is currently not
understood β2M then self-associates into amyloid fibrils and typically accumulates in
musculoskeletal system [Homma et al 1989] Analysis of ex vivo material has shown
that the majority of amyloid fibrils in patients with Dialysis Related Amiloidosis (DRA
is present as of full-length wild-type β2M although significant amounts (~20-30) of
truncated or modified forms of the protein are also present [Floege 2001 Bellotti et al
1998] Figure 2-9 shows the ribbon diagram of human β2M Native β2M consists of
single chain of 100 amino acid residues and has a seven stranded β-sandwich fold typic
36
of the immunoglobulin superfamily [Saper et al 1991 Trinh et al 2002] β2M was fi
isolated from human urine and characterized by Berggard et al [1980] in1968 The
normal serum concentration of β
rst
re
f
e
2M is 10 to 25 mgL It is a small globular protein with
a molecular weight of 118 kDa a Strokes radius of 16Aring and a negative charge under
physiological conditions (isoelectric point IEP = 57) β2M contains two β-sheets that a
held together by a single disulphide bridge between the cysteines in positions 25 and 81
[Berggard et al 1980 Parker et al 1982 Cunningham et al 1973] β2M cannot be
removed completely by current dialysis techniques but through a better understanding o
the structure and interaction forces that lead to this structure it will be possible to mor
efficiently remove this problematic protein
Figure 2-9 Ribbon diagram of human β M taken from the crystal structure of the protein
bound to the heavy chain of the MHC class I complex (PDB 1DUZ) [Khal 2000]
273 Serum Albumin
Serum albumin is the most abundant protein found in plasma and is typically
present in the blood at a concentration of 35~
2an et
50gL According to extensive studies about
its physiological and pharmacological properties albumin has a high affinity to a very
wide range of materials such as electrolytes (Cu+2 Zn+2) fatty acids amino acids
metabolites and many drug compounds [Fehske et al 1981 Kraghhansen 1981 Putnam
37
1984 Peters 1985] The most important physiological role of the protein is therefore to
bring such solutes in the bloodstream to their target organs as well as to maintain the pH
and osmotic pressure of the plasma Bovine serum albumin (BSA) is an ellipsoidal
protein with the dimensions of 140 X 40 X 40Aring [Peter 1985] The primary structure is a
single helical polypeptide of 66 kDa (IEP = 47) with 585 residues containing 17 pairs of
as a soft an
rfaces [Kondo et al 1991 Norde et al 1992 Soderquist et al
1980 ikely
is
disulfide bridges and one free cysteine [Dugaiczyk et al 1982] BSA has been classified
d flexible protein because it has a great tendency to change its conformation
on adsorption to solid su
Carter and Ho 1994] and consists of three homologous domains (I-III) most l
derived through gene multiplication [Brown 1976] Each domain is composed of A and B
sub-domains [He et al 1992] The secondary structure of human serum albumin (HSA)
shown in Figure 2-10
Figure 2-10 Secondary structure of human serum albumin (HSA) with sub-domains
[Zunszain et al 2003]
HSA has the same structure domains with the serum albumin from other species
such as BSA [Brown 1976] Although all three domains of the albumin molecule have
similar three-dimensional structures their assembly is highly asymmetric [Sugio et al
38
1999] Domains I and II are almost perpendicular to each other to form a T-shaped
assembly in which the tail of subdomain IIA is attached to the interface region between
sub-domains IA and IB by hydrophobic interactions and hydrogen bonds In contrast
domain III protrudes from sub-domain IIB at a 45ordm angle to form the Y-shaped assembly
for domains II and III Domain III interacts only with sub-domain IIB These features
make the albumin molecule heart-shaped
28 Protein Adsorption
Protein adsorption studies date back to the 1930rsquos At the beginning these studies
mainly focused on the determination of the molecular weight electrophoretic and
ral
rearrangem
adsorption and biocompatibility of the sorbent materials were investigated [Norde 1986]
There are interaction forces at the interfaces between protein molecules and latex
particles These forces are mainly divided into the following groups hydrophobic
interaction ionic interaction hydrogen bonding and van der Waals interaction [Andrade
1985]
Hydrophobic interaction It is known that hydrophobic interaction has a major
role in protein adsorption phenomena The adsorption of proteins on the low charged
latex particles occurs by this interaction force Generally monomers such as styrene offer
hydrophobic surfaces th
by this interaction force is maximum at the isoelectric point (IEP) of the protein and the
strength [Suzawa et al 1980 1982 Shirahama et al 1989 Kondo et al 1992] By the
chromatographic applications Later the adsorption mechanism especially the structu
ents was studied Recently the studies of the relation between protein
281 Interaction between Protein Molecule and Latex Particle
at protein molecules adsorb to The amount of adsorbed protein
pH at maximum adsorption shifts to a more acidic region with an increase in ionic
39
reports [Suzawa et al 1980 1982 Lee et al 1988] protein adsorption was greater
hydrophobic surface than on a hydrophilic one implying that hydrophobic interaction is
on a
s
ydrophilic compound such as acrylic acid (AA) or methacrylic acid (MAA)
Ionic
ely
t
ces
n force is operative over small
distan e
ble
n
nt dialysis membrane therapy for end stage renal disease (ESRD)
one of most dominant forces in protein adsorption
Ionic interaction Negatively charged latex particles have ionic functional
groups on their surfaces such as salts of sulfonic and carboxylic acid Sulfate group
originate from an initiator such as sodium persulfate and carboxylic groups originate
from a h
bonds are formed between the negative charges of these latex particles and positive
surface charges of protein molecules The conventional low-charged latex particles rar
form these ionic bonds
Hydrogen bonding Hydrogen bond is a strong secondary interatomic bond tha
exists between a bound hydrogen atom (its unscreened proton) and the electrons of
adjacent atoms [Callister 1999] Protein can be adsorbed on hydrophilic polar surfa
through hydrogen bonding Hydrogen bonds are frequently formed between hydroxyl-
carbonyl or amide-hydroxyl Hydroxyl-hydroxyl or amide-hydroxyl bonds are also
formed in protein adsorption
Van der Waals interaction This interactio
ces only when water has been excluded and the two non-polar groups come clos
to each other Lewinrsquos calculation showed that the van der Waals interaction is negligi
compared with the forces involved in the entropy increases ie hydrophobic interactio
[Lewin 1974]
29 Hypothesis for Toxin Removal
As mentioned earlier insufficient removal of middle molecular range toxins is a
major drawback of curre
40
patien
is
y
ese
haracteristics over the last decade However removal
effici
ing a novel membrane design composed of an assembly of engineered
polymeric latex particles s ions Important factors
will i
e
ith a
is established it is
expec
e
ts This may cause destructive arthritis and carpal tunnel syndrome inhibit the
immune system and accelerate cardiovascular disease leading to death among dialys
patients In order to overcome these complications artificial dialysis membranes have
been developed to increase the capacity for removing middle molecular weight toxins b
changing the pore size of dialysis membranes and using new materials that adsorb th
toxins for improved removal c
ency is not as high as those achieved by a normal healthy kidney
With the knowledge obtained from all above literatures the following hypothesis
has been established for the development of a membrane for the successful removal of
the target protein β2-Microglobulin (β2M) without the removal of serum albumin This
will be done us
ynthesized to predetermined specificat
nclude pore size and surface chemistry
291 Toxin Removal by Size Sieving Based on Monodispersed Pore Size
The packing of monodispersed spherical particles can lead to the formation of
porous layers suitable for use as filters and membranes [Hsieh et al 1991] The pore siz
is defined as the largest spherical particles that can pass through the interstitial spaces
It is well known that monodispersed spherical particles can be obtained from the
seed emulsion polymerization method Many commercially available latex particles are
synthesized by this technique and are inexpensive When the defect free membrane w
monodisperse pore size distribution based on a particular particle array
ted to outperform the traditional hemodialysis membrane limitation which is a
broad range of pore size distribution When spherical particles pack in regular crystallin
arrays a number of packing geometries such as hexagonal closest packing cubic closest
41
packing and body-centered cubic packing are possible The pore size of the array
depends on these packing geometries as well as particle size Theoretical pore size can be
calculated from the simple geometry of the arr
ay Figure 2-11 shows the relationship
between particle size and pore size
0001
001
01
001 01 1 10
Por
e
1
10
microm
) S
ize
(
Particle Diameter (microm)
Body-Centered Cubic ArrayClosest Packed Array
0001
Siz
e (
Particle Diameter (microm)
001
01
001 01 1 10
Por
e
1
10
microm
)
Body-Centered Cubic ArrayClosest Packed ArrayBody-Centered Cubic ArrayClosest Packed Array
and closed packed arrays [Steve et al 1999]
McDonald and his coworkers [Ramakrishnan et al 2004 Steve et al 1999] have
described the fabrication of latex composite membranes and their performance in
previous papers It is from this prior research and knowledge gained from conversations
with Dr McDonald that this current research has been initiated [McDonald 2003] The
focus of this thesis is the extension of McDonaldrsquos research in the area of composite
membranes to medical application such as dialysis membranes
292 Toxin Removal by Selective Adsorption on Engineered Latex Particles
The flexibility in latex surface properties is a significant advantage to this
materialrsquos ability to separate proteins since the selectivity is strongly dependent on the
charge interactions of both the protein and the latex particles under the conditions of
Figure 2-11 The relationship between pore size and particle size in body-centered cubic
42
separation [Menon et al 1999 Chun et al 2002] First all sorbents were designed to
have negative surface properties because most plasma proteins in blood are negative and
should not be removed with charge interaction between proteins and sorbents at
physiological condition (pH74) It was reported that a negatively charged surface was
more blood compatible than a positive one [Srinivasan et al 1971] The toxin protein β2-
Microglobulin (β2M) can be selectively adsorbed on the surface of latex particles by the
design of a suitable hydrophobic portioned surface where only β2M protein can be
anchored with hydrophobic interactions Albumin adsorption on the latex particle is not
side-on m
adsorption of β M protein on the engineered latex particles
allowed because charge repulsion is more dominant than hydrophobic interaction with
ode The figure 2-12 shows the schematic representation of the selective
2
PMMAPAAShell
COO-
OSO3-
OSO3-
PSCore
COO-
-
-
-
-
-
-
Albumin-
Chargerepulsion
--
-
- -
- -
- --
Hydrophobic
β2-Microglobulin
2
Interaction
β -Microglobulin
PMMAPAAShell
COO-
-
-
-
-
-
-
--
-
-
-
-
-
Albumin-
Chargerepulsion
OSO3-
OSO3-
PSCore
COO-
--
-
- -
--
-
- -
- -
- --
- -
- --
Hydrophobic
β2-Microglobulin
2
particles at pH 74
Interaction
β -Microglobulin
Figure 2-12 Schematic representation of the protein adsorption on the core shell latex
43
Hydrophilichydrophobic microdomain structures were proven to be more blood
compatible [Mori et al 1982 Higuchi et al 1993 Deppisch et al 1998] The
optimization of suitable hydrophobic to hydrophilic ratio is also important for the
biocompatibility The monomers such as styrene (St) methyl methacrylate (MMA) and
acrylic acid (AA) are widely used hydrophobic and hydrophilic monomer models in
emulsion polymerization process
In summary the background and fundamental literature survey about the history
material properties and limitation of hemodialysis membrane latex particle preparation
surface chemistry and manufacturing process target proteins and protein adsorption and
finally the hypothesis for a toxin removal with high separation efficiency have been
suggested This research focuses on such hypothesis and the materials and
characterization methodology for achievement of suggested hypotheses are described in
the next chapter
CHAPTER 3 EXPERIMENTAL AND CHARACTERIZATION METHODOLOGY
As mentioned earlier the goal of this study is to prepare polymeric latex particles
with tailored properties to maximize separation of toxin molecules and to investigate
fundamental interactions between the applied particles and molecules in the
system to optimize the membrane performance for hemodialysis applications Polymeric
latex particles wit
the
biological
h monodisperse size distribution to obtain uniform pore size and
various size ranges to utilize membrane construction are necessary Surface engineering
with various combination of hydrophobichydrophilic domain on the surface of latex
particles is expected to affect the removal of target protein by selective adsorption and to
improve the biocompatibility of membrane Therefore the materials and characterization
methodology are addressed in this chapter
31 Materials
Styrene (St) monomer used for a seed and a core particle was purchased from
Fisher Scientific and used without any other purification process Acrylic acid (AA) and
methyl methacrylate (MMA) monomers introduced for shell formation were purchased
from Fisher Scientific and used without any other purification process Sodium persulfate
(SPS) and sodium bicarbonate (SBC) were obtained from Fishers Scientific and used as
received Divinylbenzene (DVB) crossliking agent was purchased from Aldrich The
anionic surfactant Aerosol regMA80-I [sodium di(13-dimethylbutyl) sulfosuccinate] was
kindly donated by Cytec 78-80 Aerosol regMA80-I is mixed with isopropanol and
water Its critical micelle concentration (CMC) is 245 mM (119) Ion exchange resin
44
45
AGreg 501-x8 Resin (20-50 mesh) was purchased from Bio-Rad Laboratories Inc It is a
mixed-bed resin with H+ and OH- as the ionic forms and is 8 crosslinked Bovine serum
albumin (BSA) (heat shock treated) was purchas Fisher Scientific The isoelectric
point of t lin
(β2M)
as
gma
ed from
he BSA is 47-49 and the molecular weight is 66300Da β2-Microglobu
was purchase from ICN Biomedicals Inc and was separated from patients with
chronic renal disease and lyophilized from ammonium bicarbonate The molecular
weight by SDS-PAGE was approximately 12000Da BSA and β2M proteins were used
received without further purification process In order to investigate the protein
adsorption evaluation bicinchoninic acid protein assay kits were purchased from Si
(Cat BCA-1) and Pierce Biotechnology (Cat 23225) The chemical structures for the
main chemicals are shown in Figure 3-1
CH2 CH
Styrene (Mw = 10415)
CH2 CHCH2 CH
Styrene (Mw = 10415)
CH2 CH
Acrylic acid (Mw = 7206)
C
OH
O
CH2 CH
Acrylic acid (Mw = 7206)
C
OH
O
CH CH2
C
OCH3
O
CH
CH CH3
2
C
OCH3
O
CH3
Methyl methacrylate (Mw = 10012)Methyl methacrylate (Mw = 10012)
CH2 CH
Divinylbenzene (Mw = 1302)
CH CH2
CH2 CH
Divinylbenzene (Mw = 1302)
CH CH2CH CH
+(Na) -(O3S)
CH -C-O-CH-CH -CH-CH3
O
2 2 3
O CH CH3
CH3 CH3
CH-C-O-CH-CH2-CH-CH3
Anionic surfactant (MA80-I) (Mw = 3885)
+(Na) -(O3S)
CH -C-O-CH-CH -CH-CH3
O
2 2 3
O CH CH3
CH3 CH3
CH-C-O-CH-CH2-CH-CH3
Anionic surfactant (MA80-I) (Mw = 3885)2
+(Na)-(O) S OO
O O
- +
Initiator sodium persulfate (Mw = 23811)
O SO
(O) (Na)+ -
O O
- +(Na) (O) S OO
O SO
(O) (Na)+ -
O O
- +
Initiator sodium persulfate (Mw = 23811) Figure 3-1 Chemical structure of main chemicals
(Na) (O) S OO
O SO
(O) (Na)
46
32 Latex Particle Preparation
The experimental set up and the particle preparation schemes for various types and
size ranges of latex particles are shown in Figure 3-2 and 3-3 In the experimental setup a
mechanical glass stirrer is connected from a motor and Teflon stirrer bar is located at
end of glass stirrer The agitation rate is precisely adjusted by controller motor Paar glas
reactor with 1L volume is partially in silicon oil bath on hot plate The reactor has fo
openings for a stirrer a thermometer a nitrogen gas inlet and a reactant feeding lnlet
The reactants for latex polymerization are fed by precisely controlled by meterin
The particle preparation is described in each latex preparation section
the
s
ur
g pump
Figure 3-2 Experimental setup for semi-continuous emulsion polymerization
1 Motor 2 Mechanical stirrer bar 3 Thermometer 4 Inlet of N2 5 Inlet for initiator and monomer 6 Glass reactor (l L) 7 Oil bath 8 Hot plate 9 Pump for initiator 10 Pump for monomer 11 Tubes for reactant feed
47
Styrene monomer
Bare PS
PSPMMA100
PSPMMA90PAA10
PSPMMA P75 AA25
Gr
win
ticle
size
PS seedparticles
og
par
Styrene monomer
Bare PS
PSPMMA100
PSPMMA90PAA10
PSPMMA P75 AA25
Gr
win
ticle
size
PS seedparticles
og
par
Figure 3-3 The particle preparation scheme in various types and size ranges of latex particles
321 Preparation of Seed Latex Particles
Polystyrene seed particles were synthesized through a typical semi-continuous
emulsion polymerization Polystyrene latex particles were prepared in a four necked Paar
glass vessel equipped with a mechanical glass stirrer thermometer glass funnel for
nitrogen gas purge and a tube for monomer feeding The reactor was placed in a silicon
oil bath on a hot plate for homogenous and stable tempe he vessel was
firstly charged with de-ionized water the emulsifier so itiator
sodium bicarbonate (SBC) styrene (St) monomer divinylbenzene (DVB) monomer
under nitrogen gas atmosphere and then heated to 72plusmn2 as
llowed to continue for another hour Second reactants St and DVB monomers SPS
initiator and SBC were fed separately using fine fluid metering pumps (Model RHSY
Fluid metering INC NY) under a nitrogen gas atmosphere for 2h After complete feeding
rature control T
dium persulfate (SPS) in
oC for 1h The reaction w
a
48
of all reactants the reaction was continued for another hour at elevated temperature 80plusmn1
oC and then cooled to room temperature by removal of the oil bath
322 Preparation of Seeded Latex Particles
The particle growth was achieved by seeded continuous emulsion polymerization
The vessel was charged with the polystyrene seeds and de-ionized water to make desired
solid content and heated up to 72plusmn2 oC for 1h under nitrogen gas atmosphere Then St
and DVB monomers and solution of SPS and SBC in de-ionized water were continuously
added using metering pump with a precisely controlled feed rate After feeding all
reactants the reaction was continued for three hours keeping temperature of 80plusmn2 oC and
then the reaction vessel was cooled down to room temperature
Monodisperse spherical polystyrene seeds with a 280nm mean diameter were used
e-polymerized polystyrene seeded emulsion
was charged in a 500m
323 Preparation of Core Shell Latex Particles
for core shell structured latex particle The pr
l glass flask under a nitrogen gas atmosphere De-ionized water
was added to achieve the desired solid content The emulsion was heated to 80plusmn2oC for
10 hour Monomers initiator and SBC to form the shell were added by fluid metering
pumps (LAB PUMP JRRHSY model Fluid metering Inc USA) with fine controlled
feed rate Flow rate can be adjusted by control ring graduated in 450 division from 0 to
100 flow which is in the range of 10mlmin respectively The reaction was sustained
for additional 30 hours at 90plusmn2oC then cooled down to room temperature
324 Purification of Synthesized Latex Particles
Synthesized latex particles were purified with an ion exchange resin 200g (10
ww polystyrene solid content) of emulsion were mixed with 4g of ion exchange resin
and stirred for 40 minute to remove unreacted monomers initiators and other impurities
49
Then the emulsion was diluted with DI water to a specific degree of solid content as
needed
33 Characterization
331
solid
termined
oscopy
d
hours
Dried latex (10mg) was mixed with
250 m on
Degree of Conversion
The degree of monomer to polymer conversion was determined gravimetrically
following the procedure described by Lee et al [1995] before the latex cleaning process
The synthesized emulsion raw materials were weighed and dried in a conventional oven
at 120oC for 30min to evaporate any unreacted monomers and water The remaining
was weighed and the degree of conversion was determined This process was repeated
three times and a mean value of the degree of conversion was de
332 Fourier Transform Infrared (FTIR) Spectr
A molecule can absorb only select frequencies (energy) of infrared radiation which
match the natural vibration frequencies The energy absorbed serves to increase the
amplitude of the vibrational motions of the bonds in the molecule [Pavin et al 1996]
Only those bonds which have a dipole moment that changes as a function of time can
absorb the infrared radiation The range of wavelengths for infrared radiation is between
25 microm (4000 cm-1) and 25 microm (400cm-1)
The synthesis of the latex particles such as polystyrene (PS) homopolymer and
PSPMMA PSPMMA90PAA10 PSPMMA75PAA25 core-shell copolymers were verifie
with FTIR Purified latex particles were dried in vacuum oven at 40oC for 24
before analysis in the FTIR (Nicolet Magma USA)
g of KBr which had also been dried in vacuum oven at 120oC for 3h Transmissi
spectra using the drift mode were plotted with 128 scans and 4cm-1 resolution
50
333 Quasielastic Light Scattering (QELS)
Latex particle size was measured by the Brookhaven ZetaPlus particle size
analyzer The solid concentration as 0001 (wv) This
ation spectroscopy (PCS) of quasielastically scattered light
For th s
ed using a FE-SEM (JEOL
JSM-
ith
ent
01
us zeta-
potential analyzer It measures the electrophoretic mobility velocity of charged colloidal
of latex particles used w
instrument uses photon correl
is technique time dependent interference patterns of light scattered from particle
in the sample cell are analyzed The interference pattern changes due to Brownian
motion giving rise to fluctuations in scattering intensity The frequency of these
fluctuations depends on the particlersquos diffusion constant that is inversely proportional to
the particle diameter
334 Field Emission-Scanning Electron Microscopy (FE-SEM)
Particle size and surface morphology were characteriz
6335F Japan) A diluted suspension of latex particles in water was dropped onto a
silicon wafer at room temperature and allowed to dry The sample was then coated w
the thinnest layer of carbon needed to obtain the required conductivity for this instrum
Secondary electron image mode was used with a 15KV of accelerating voltage The
magnification range used was between 10000X and 70000X
335 Zeta Potential Measurement
Synthesized and purified latex particles were diluted with de-ionized water to 0
wt and adjusted six different pH values 205 345 481 565 643 and 747 These
particle suspensions were then transferred to standard cuvettes for zeta potential
measurement At least two runs of ten measurements were taken for each sample and
averaged
The zeta potential measurement was carried out using Brookhaven ZetaPl
51
particles in solution and calculate zeta-potential by using the Smoluchowski equation
The f quency
gnitude
umin (BSA) as a standard model protein and β2-Microglobulin
uffer (PB) and
phosp
5
h the selected protein
e 005 01 03 05 and 07 mgml and those for
β2M
d at
teins in the supernatant after the centrifugation process using the
bicinc t
requency of laser light passing through the sample and is compared to the fre
of a reference beam The shift in the frequency called a Doppler shift and the ma
of the shift correspond to the polarity and the magnitude of the electrophoretic mobility
respectively The zeta potential is calculated from the solution conditions and the
measured mobility
336 Protein Adsorption
Protein adsorption experiments of the synthesized latex particles were performed
with bovine serum alb
(β2M) as a target protein in two types of buffer solution phosphate b
hate buffered saline (PBS) To make 5 mM of PB solution 0345g of sodium
phosphate monobasic (NaH2PO4H2O) was dissolve in 500ml of de-ionized water To
make PBS solution 0345g of sodium phosphate monobasic (NaH2PO4H2O) and 4178g
(143mM) of sodium chloride (NaCl) were dissolved in 500ml of de-ionized water The
synthesized latex particles were diluted with each buffer to have a solids content of 0
(ww) adjusted to pH values of 32 48 and 74 and mixed wit
BSA concentrations were chosen to b
were 0015 0030 0045 and 0060mgml The mixture was gently rotated using
the shaker Labquakereg (BarnsteadThermolyne Model 4002110 USA) with 8 RPM in
the incubator at 37oC for 12 hours before the latex-protein mixture was centrifuge
13000 rpm for 15min The amount of protein adsorbed was determined by quantifying
the free pro
honinic acid (BCA) assay method [Lowry et al 1951 Smith et al 1985 Baptista e
al 2003 Wiehelman et al 1988 Brown et al 1989] BCA assay kits consist of Reagent
52
A containing bicinchoninic acid sodium carbonate sodium tartrate and sodium
bicarbonate in 01N NaOH with pH=1125 and Reagent B containing 4 (wv) copper
(II) sulfate pentahydrate The BCA working reagent was prepared by mixing 50 parts of
Reagent A with 1 part of Reagent B 100microl of protein supernatant was then mixed with
2ml of BCA working reagent in a UV cuvette Incubation was allowed to continue
at room temperature
further
until color developed about 2h The absorbance at 562nm was
pectrophotometer (Perkin-Elmer Lambda 800 USA)
Unkn
Separate the p rticles in centrifuge mix supernatant with BCA reagents
measured using a UV-VIS s
own concentration of sample protein was determined by comparison to a standard
of known protein concentrations The adsorbed amount per unit surface area was
determined by the mass balance of the protein after adsorption process The simple
schematic of the procedure for a protein adsorption test is shown in Figure 3-4
Incubate mixture with agitation for 12hrs at 37oC
aand develop the color by incubation for 2hrs at room temperature
Suspend latex particles in aqueous buffer media
Add the dissolved target protein
Measure color intensity using UVVis spectroscopy at 562nm
Suspend latex particles in aqueous buffer media
Separate the p rticles in centrifuge mix supernatant with BCA reagents
Add the dissolved target protein
Incubate mixture with agitation for 12hrs at 37oC
aand develop the color by incubation for 2hrs at room temperature
Measure color intensity using UVVis spectroscopy at 562nm
Figure 3-4 Schematic of the procedure for a protein adsorption test
53
337 Blood Biocompatibility by Hemolysis Test
Hemolysis is the destruction of red blood cells which leads to the release of
hemoglobin from within the red blood cells into the blood plasma Hemolysis testing is
used to evaluate blood compatibility of the latex particles and the damaged to the red
blood cells can be determined by monitoring amount of hemoglobin released The red
blood cells (RBCs) were separated by centrifuging blood at 1500 rpm for 15 minutes
Figure 3-5 shows the phase separated blood with plasma white blood cells and red
cells after centrifugation process Separated RBSs were then washed with isotonic
phosphate buffer solution (PBS) at pH 74 to remove debris and serum protein Th
process was repeated 3 times
blood
is
Figure 3-5 Separation of RBC from whole blood by centrifuge process
Prepared latex particles were re-dispersed in PBS by sonification to obtain
homogeneously dispersed latex particles 100microl of the mixture of red blood cell (3 parts)
and PBS (11parts) was added to 1ml of 05 (ww) particle suspension PBS was used as
a negative control resulting 0 hemolysis and DI water used as a positive control to
produce 100 hemoglobin released by completely destroyed RBCs The mixture was
incubated in water bath with gentle shaking for 30 minutes at 37oC and then centrifuged
at 1500 rpm for 15 minutes 100microl of the supernatant was mixed with 2ml of the mixture
) (EtOHHCl = 2005 ww) to prevent the
White blood cells
Plasma
Red blood cells
of ethanol (99) and hydrochloric acid (37
54
precipitation of hemoglobin In order to remove remaining particles the mixture was
centri fuged again with 13000 RPM at room temperature The supernatant was then
transferred into the UV cuvette The amount of hemoglobin release was determined by
monitoring the UV absorbance at a wavelength of 397nm The schematic of the
procedure for hemolysis test is shown in Figure 3-6
Separate red blood cells (RBCs) from blood
Mix RBCs with phosphate buffer solution (PBS)
Wash and re-suspend RBC with PBS repeat three times
Add la l of RBC and incu in at 37oC
tex particle suspension into 100mbate sample with agitation for 30m
Separate particle ith EtOHHCl solutions and mix the supernatant w
Evaluate free hemoglobin in the solution using UVVis spectroscopy at 397nm
Separate red blood cells (RBCs) from bloodSeparate red blood cells (RBCs) from blood
Mix RBCs with phosphate buffer solution (PBS)Mix RBCs with phosphate buffer solution (PBS)
Wash and re-suspend RBC with PBS repeat three timesWash and re-suspend RBC with PBS repeat three times
Add latex particle suspension into 100ml of RBC and incubate sample with agitation for 30min at 37oC
Add latex particle suspension into 100ml of RBC and incubate sample with agitation for 30min at 37oC
Separate particles and mix the ith EtOHHCl solution supernatant wSeparate particles and mix the ith EtOHHCl solution supernatant w
Evaluate free hemoglobin in the solution using UVVis spectroscopy at 397nmEvaluate free hemoglobin in the solution using UVVis spectroscopy at 397nm
Figure 3-6 Schematic of the procedure for hemolysis test
In summary a description of the experimental materials and characterization
methodology has been explained In the next chapter the results and discussion of the
data obtained from these experiments is addressed
CHAPTER 4 RESULTS AND DISCUSSION
41 Polymerization of Latex Particles
411 Polystyrene Seed Latex Particles
Polymer particles have many different applications including spacer particles
lubricants packing materials for chromatography standard particles and diagnostic
drugs All applications strongly require these particles to have a uniform particle size
Uniform particle size is the first requirement for a latex composite membrane formed by
particle arrays with the interstitial spaces serving as pores for size discriminations [Jons
et al 1999 Ramakrishnan et al 2004] Although suspension polymerization has been
mainly used as one of conventional polymerization methods to prepare uniform particles
the uniformity of the recovered particles is insufficient for membrane use There is
another method known as seed emulsion polymerization where a vinyl monomer is
absorbed into fine monodiserse seed particles and polymerization causes the monomer to
crease the sizes of the seed particles uniformly In order to obtain monodisperse larger
particles by this m
particles and polymerization of the monomer is repeated
In this study cross-linked polystyrene seed latex particles were firstly synthesized
in the presence of styrene monomer divinylbenzene crosslink agent sodium persulfate
initiator sodium bicarbonate buffer and the Aerosol regMA80-I [sodium di(13-
dimethylbutyl) sulfosuccinate] surfactant using a batch type semi-continuous emulsion
polymerization process in which one or more components can be added continuously
in
ethod the procedure of absorption of monomer into fine polymer
55
56
Styrene (St) monomer and divinylbenzene (DVB) crosslink agent as well as initiator and
buffer are continuously added to enlarge the seed latex particles Various profiles of
particle nucleation and growth can be gene different orders of component
addition during polymerizati f this process such as easy
control of the polymeriza particle number
and particle morphology The main goal in
this process is to avoid secondary particle
rated from
on There are several advantages o
tion rate monodisperse particle size and
colloidal stability copolymer composition
formation leading to a monodisperse particle
size distribution Figure 4-1 shows the schematic representation of seed latex particle
preparation and growth
Monomer droplet
Seed particles Amount of surfactantsis variable
Enlarged final Add monomers
PS
PS
PSs4
particles Number of particlesare constant
Seed code
PSs1
s2
s3
Monomer droplet
Seed particles Amount of surfactantsis variable
Enlarged final Add monomers
PS
PS
PSs4
tion The
particles Number of particlesare constant
Seed code
PSs1
s2
s3
Figure 4-1 Schematic representation of semi-continuous seed latex particles preparation and growth
After the first synthesis of a small size of crosslinked polystyrene (PS) seed
particles stabilized by surfactants styrene DVB and initiator are continuously fed into
the system to enlarge the seed latex particles maintaining a narrow size distribu
recipes and reaction conditions for synthesizing these seed particles are summarized in
57
Table 4-1 The original recipe for latex particle preparation from McDonald was
modified for this emulsion system [McDonald 2003]
Table 4-1 The polymerization recipe of polystyrene (PS) seed particles Latex label PSS259 PSS233 PSS207 PSS181
Temperature (oC) 75 plusmn 5 75 plusmn 5 75 plusmn 5 75 plusmn 5
Divinylbenzene (g) 052 052 052 052
MA 80 (g) 259 233 207 181
NaHCO
Watera (g) 1315 1315 1315 1315
Styrene (g) 9948 9948 9948 9948
112 112 112 112
Na2S2O8 (g) 190 190 190 190
Waterb (g) 200 200 200 200
Reaction time (min) 140 140 140 140
Solids content 396 396 396 396
Mean particle diameter (nm) 126plusmn75 171plusmn39 182plusmn41 216plusmn53
Conversion () 965 979 957 971
3 (g)
Four different seed latex particles were synthesized based on surfactant amount in
order to determine the optimum condition for stable and monodisperse latex particles
The emulsion polymerization of latex particles has to be carried out in a narrow range of
surfactant causes flocculation or phase separation leading to broad particle
size distribution and an unstable emulsion sy
surfactant concentration where particles are stable [Nestor et al 2005] A wide range of
concentrations
stem Therefore the range of surfactant
amount was varied from 181g to 259g in increments of 026g in each latex label Other
reactants amounts were kept constant Watera is the initial charge amount and waterb
represents the amount of aqueous solution containing dissolved initiator and buffer
58
which are fed continuously after the seed particle creation The extent of monome
polymer conversion was obtained by gravimetric calcu
r to
lation and was more than 95 at
cripts such as
S207 and S181 i the surf mount added for the seed latex
nversion indi perce experi obtain
content divided by the theoretically estimated solid content and was calculated by the
all seed particles labeled PSS259 PSS233 PSS207 and PSS181 Labeled subs
S259 S233 ndicate actant a
preparation The co cates the ntage of mentally ed solid
following formula
100 colids () times=entsoliatedntentoalExperimentConversion
n the experimenta content is the weight of olid rema after
mulsion at 120oC f in and the estimated solid content is th ht
lated from the recipe
rti as di each t load d
mple of ea pension iluted by (ww)
deionized water and the mean particle diameter was determined using the Brookhaven
ZetaPlus particle size analyzer From these results the seed latex particle size increased
as the amount of surfactant decreased The amount of emulsifier also affects the
polymerization process by changing the number of micelles and their size It is known
that large amount of emulsifier produces larger numbers of smaller sized particles [Odian
1991] and was corroborated in the current work
These synthesized seed latex particles should be of narrow size distribution in order
to obtain monodisperse larger particles The SEM image showing the particle
morphology and size of a sample of polystyrene seed particles can be seen in the Figure
4-2
contdEstim (4-1)
In this equatio l solid the s ining
evaporation of e or 30m e weig
of monomers calcu
As expected the seed pa cle size w fferent for surfactan ing in see
emulsion system A sa ch sus was d 0001 with
59
Figure 4-2 Scanning Electron Micrograph (SEM) of polystyrene seed latex particles (A)
PSS259 (B) PSS233 (C) PSS207 (D) PSS181 (subscripts indicate the amount of surfactant added)
(A)
(B)
60
(C)
(D)
Figure 4-2 Continued
61
s seen in the image the seed particles are very smooth and spherical The
uniformity in the particle size distribution of these PS seed latex particles can also be
seen in the SEM image From this data we concluded that seed particles synthesized in
the presence of a surfactant with the chosen amounts are suitable for use to prepare larger
particles with high size uniformity and smooth spherical morphology
412 The Growth of Polystyrene (PS) Seed Latex Particles
Seeded emulsion polymerization has been conducted for several decades and
various mechanisms have been proposed Gracio et al [1970] suggested that the growing
polymer particles consist of an expanding polymer-rich core surrounded by a monomer-
rich shell with the outer shell providing a major locus of polymerization Seeded
mulsion polymerization is commonly used for preparing latex particles less than 1microm in
size [Cha et al 1995 Zou et al 1990 1992 Gandhi et al 1990 Park et al 1990] Such
latexes can be obtained from the growth of pre-prepared seed particles The seeds
introduced in this process serve as nucleation sites for particle growth By first swelling
the seed latex particles with additional monomer to the desired size polymerization can
then be initiated Several size ranges of monodisperse polystyrene (PS) latex particles
were prepared by this multistep seeded emulsion polymerization method For the
fabrication of particle-based membranes monodisperse latex particles with a variety of
size ranges are required
In this study seed particles from 171nm to 470nm in mean diameter and with a
highly uniform size distribution were prepared and used Watera is the initial charge
mount and waterb represents the amount aqueous solution containing dissolved initiator
and buffer that is continuously fed into system Feed time is precisely controlled by
metering pump The solid contents were maintained between 199 and 300 After
A
e
a
62
polym d as
es
ere 923 8402
n of these latex particles
are sh
ss
erization under these conditions the conversions of the latex particles labele
PS258 PS320 PS370 and PS410 were 952 957 946 and 964 respectively The recip
used in seeded emulsion polymerization to form the particles less than 500nm in mean
diameter are shown at the Table 4-2 The conversions of the latex particles larger than
500nm in mean diameter and labeled as PS525 PS585 PS640 and PS790 w
723 and 969 respectively The recipes used in the preparatio
own at the Table 4-3
A simple calculation can be used to predict the ultimate size that a latex particle can
reach after ending the polymerization Letrsquos suppose that seed density (ρs) and grown
particle density (ρgp) are the same The following equation is obtained from the ma
balance (m = ρV) where m and V are mass and volume of a particle respectively
gp
gp
s
s
Vm
Vm
In this equation m
= (4-2)
be
s and mgp are the weight of seed and total weight of seed and
added monomers respectively and Vs and Vgp are the volume of seed and grown
particle respectively
If we know the mass of monomer to be added the ultimate particle size can
calculated from the equation 4-3
3
2s D m ⎠⎝+ χ
where D
1s D m
⎟⎟⎞
⎜⎜⎛
= (4-3)
the
in
s and Dgp are the diameters for seed and grown particle respectively and χ is
weight of the monomer added Crosslinked PS latex particles with a size range from
258plusmn112 ~ 410plusmn117 in diameter polymerized by the recipes in Table 4-2 are shown
the SEM images in Figure 4-3
63
Table 4-2 Continuous addition emulsion polymerization recipe for growing polystyrene
Latex label PS
(PS) latex particles less than 500nm in size
258 PS320 PS370 PS410
Initial charge
Seed latex (g) 1000171 1000216 1000216 1000320
Watera (g) 1470 2500 1984 2600
06 07 06 06
Fee
Initiator stream
2 2 8
Feed time (min) 0-167 0-140 0-100 0-92
Solid content 284 300 300 200
Mean particle diameter (nm) 258plusmn112 320plusmn154 182plusmn71 410plusmn117
Conv
Monomer continuous
Styrene (g) 800 1200 800 600
Divinylbenzene (g)
d time (min) 0-163 0-110 0-75 0-82
Na S O (g) 07 065 05 05
NaHCO3 (g) 07 065 05 05
Waterb (g) 750 1200 200 370
ersion () 952 957 946 964 The solid content of seed latex for PS258 PS320 PS 370 and PS410 are 392 399 399 anrespectively
d 300
64
Table 4-3 Continuous addition emulsion polymerization recipe for growing polystyrene (PS) latex particles larger than 500nm in size
Latex label PS525 PS585 PS640 PS790
Initial charge
Seed latex (g) 1000390 1000470 1000470 1000320
Watera (g) 1000 520 1200 360
Monomer continuous
Styrene (g) 290 200 400 173
Divinylbenzene (g) 02 014
03 009
Feed time (min) 0-80 0-40 10-76 5-65
Initiator stream
Na2S2O8 (g) 02 014 03 012
NaHCO3 (g) 02 014 03 009
Waterb (g) 200 120 400 200
Feed time (min) 0-85 0-46 0-77 0-75
Solids content 199 200 200 201
Mean particle diameter (nm) 525plusmn151 585plusmn186 640plusmn178 790plusmn693
Conversion () 923 8402 723 969 The solid content of seed latex for PS525 PS585 PS 640 and PS790 are 200 182 193 and 173 respectively
65
Figure 4-3 SEM of PS latex particles less than 500nm in size (A) 258nm (B) 320nm (C)
370nm (D) 410nm
(A)
(B)
66
(C)
(D)
Figure 4-3 Continued
67
article size was well matched to that estimated by the geometrical calculation
From this it can be concluded that the added monomer was consumed for polymer
particle growth with a high degree of conversion From the SEM characterization newly
nucleated particles were not seen as indicated by the narrow particle size distribution
The particles less than 500nm in mean diameter were very spherical in shape with a
highly uniform particle size distribution As the particles were grown larger than 500nm
mean diameter however they became of non-spherical shape with an uneven surface
This irregularity of particle surface can be attributed to the non-homogeneous monomer
swelling into the shell of a growing polymer which can be controlled by the factors such
as temperature agitation speed initiator feeding rate and surfactant amount required to
stabilize the colloidal system The SEM image of latex particles larger than 500nm mean
iameter is shown in Figure 4-4 The particle size distribution is broader than that of the
s particles These particles can however still be used for membrane construction
as a support layer rather than skin layer The support layer does not necessarily have to be
as highly monodisperse as the skin layer which require pores of high uniformity for the
selective removal of toxins
As mentioned earlier the amount of surfactant as well as monomer in an emulsion
system is the primary determinant of the particle diameter [Odian 1991] Anionic
surfactants are generally recommended at the level of 02-30 wt based on the amount
of water [Odian 1991] The critical micelle concentration (CMC) of Aerosolreg MA 80-I
anionic surfactant is 119 The surfactant loading in this emulsion system ranged from
the 121 for SS181 to 171 for SS259
P
d
maller
68
Figure 4-4 SEM of PS latex particles larger than 500nm in size (A) 525nm (B) 585nm
(C) 640nm (D) 790nm
(A)
(B)
69
(C)
(D)
Figure 4-4 Continued
70
The surfactant to monomer ratio refers to the value of the surfactant amount
divided by monomer amount after polymerization ends The surfactant amount is the
weight of the surfactant contained in emulsion and the monomer amount is the total
weight of the monomer added for polymerization to form the ultimate polymer particles
in emulsion As the surfactant to monomer ratio decreases mean particle diameter
increases as described by Odian [1991] Generally lower surfactant concentration forms
fewer micelles resulting in larger particle size [Evans et al 1999] However there is a
plateau in particle mean diameter between a surfactant to monomer ratio of 0005 and
0013 This is due to low polymerization conversion of latex particles from monomer
leading to a smaller particle size than expected This is not a factor of surfactant amount
The dependence of the particle size on the amount of surfactant is shown in Figure 4-5
0
200
400
600
800
1000
0000 0005 0010 0015 0020 0025 0030
Surfactant to monomer ratio (ww)
Part
icle
mea
n di
amet
er (n
m)
Figure 4-5 Dependence of the particle size on the surfactant to monomer ratio
71
413
e
f bare
microdomain structures is important for biocompatibility [Mori et al 1982 Higuchi et al
1993 Deppisch et al 1998] Figure 4-6 shows the schematic of core shell latex particle
structure The recipe for core shell structures is shown in Table 4-4
Core Shell Latex Particles
The latex particles shown in Figure 4-3 (A) and labeled as S258 in Table 4-2 wer
used as seeds to prepare core-shell latex particles because of their high particle
uniformity and smooth surface morphology Methyl methacrylate (MMA) and acrylic
acid (AA) monomers were introduced to increase surface hydrophilicity over that o
polystyrene particles AA is a more hydrophilic monomer than MMA because of the
carboxyl acid group in AA is more favorable for hydrogen boding with water The
surface carboxyl groups on PAA may have many promising applications in biomedical
and biochemical fields [Kang et al 2005] MMA monomer is less hydrophobic than
styrene PMMA is known to more biocompatible than PS but still PMMA is hydrophobic
The optimization of suitable hydrophobic to hydrophilic ratios and control of
Polystyreneseed particle
Core shelllatex particles
PSPMMA100
PSPMMA90PAA10
PSPMMA75PAA25
Polystyreneseed particle
Core shelllatex particles
PSPMMA100
PSPMMA90PAA10
Figure 4-6 Schematic of core shell latex particle structures
PSPMMA75PAA25
72
Table 4-4 The preparation recipe of PS core with various shell latex particles Latex 25 label PSPMMA100 PSPMMA90PAA10 PSPMMA75PAA
Initial charge
PS seed latex (g) 500 500 500a
Monomer continuous
Methly methacylate (g) 320 288 240
Acrylic acid (g) 00 32 80
Initiator stream
Na
258 258 258
Water (g) 1570 1570 1570
Feed time (min) 0-120 0-50 0-60
b 200
Feed time (min) 0-122 0-60 0-65
952
2S2O8 (g) 02 02 02
NaHCO3 (g) 02 02 02
Water (g) 200 200
Solids content 183 183 183
Mean particle diameter (nm) 370plusmn186 370plusmn178 370plusmn191
Conversion () 955 924
Three types of core shell structures were prepared PMMA75PAA25 shell is a
copolymer consisting of the PMMA to PAA ratio of 75 to 25 by weight
PMMA90PAA10 shell is a copolymer with the PMMA to PAA ratio of 90 to 10 by
weight PMMA100 is the PMMA homopolymer shell on PS core The particle size of
these core-shell particles as well as PS latex particle was prepared to be about 370nm in
mean diameter and are characterized to determine their zeta potential protein adsorption
and biocompatibility The image of SEM of PS and core shell particles is shown at Figure
4-7 The synthesized latex particles had a high uniformity in size and a smooth spherical
surface morphology
73
Figure 4-7 Scanning
Electron Micrograph of latex particles (A) PS (B) PSPMMA100 (C) PSPMMA90PAA10 (D) PSPMMA75PAA25
(A)
(B)
74
(C)
(D)
Figure 4-7 Continued
75
42 Characterization of Latex Particles
421 Fourier Transform Infrared Spectroscopy (FTIR)
FTIR spectroscopy has proven to be a powerful tool to characterize polymeric
materials A molecule can absorb only select frequencies (energy) of infrared radiation
which match the natural vibration frequencies The energy absorbed serves to increase
the amplitude of the vibrational motions of the bonds in the molecule [Pavin et al 1996]
Only those bonds which have a dipole moment that changes as a function of time can
absorb infrared radiation The FTIR spectra of polymerized latex particles made during
this work are shown in Figure 4-8 There are number of characteristic peaks for PS latex
particles [Bhutto et al 2003 Li at al 2005] Aromatic C-H stretching vibration is shown
at 3002 cm-1 and 3103 cm-1 and aliphatic C-H asymmetrical and symmetrical stretching
shown at 2900 cm-1and 2850 cm-1 respectively There is an additional carbonyl (C=O)
adsorption at 1730cm-1 wavenumber for all core shell latex particles containing some
amount of either or both PMMA and PAA The broad OH group peak at 3400 cm-1
appear for particles containing some amount of PAA and has intensity dependent on the
amount of PAA in the shell For example the peak intensity of OH groups for
PSPMMA75PAA25 is greater than that of PSPMMA90PAA10 since there is a higher AA
monomer content in PSPMMA75PAA25 than in PSPMMA90PAA10 This OH peak is not
seen for PS and PS core PMMA shell particles because of the absence of OH groups on
PS and PMMA polymer chains
422 Zeta Potential Measurements
The electrical surface property was characterized by zeta potential measurements of
tex particles Zeta potential is an important and useful indicator of surface charge which
can be used to predict and control the stability of colloidal systems The greater the zeta
is
la
76
1520002500300035004000
Wavenumbers (cm-1)
li
Re
ativ
e
Tra
nsm
ttanc
e
Figure 4-8 FTIR spectra of polymerized latex particles (A) ba
PSPMMA100 (C) PSPMMA90PAA10 (D) PSPMM
(B)
(C) P
(D) P
C
C
OCH3
O
CH3
XCH2
CH2 n
CH
CH2 C
C
OCH3
O
XCH2 CH
OC
CH3
Y
OH
(A) Bare PS
500100000
re polystyrene (PS) (B) A75PAA25
PSPMMA100
SPMMA90PAA10
SPMMA75PAA25
77
potential the more likely the suspension is to be stable because the charged particles repel
one another and thus overcome the natural tendency to aggregate The measurement of
zeta potential is often the key ontrol of the interaction of proteins
with solid surfaces such as latex particles
Zeta potential (ζ) is the elec at exists at the shear plane of a particle
which is some small distance from the surface [Myers 1999] The zeta potential of the
particles can be determined by measuring the mobility of the particles in an applied
electric field termed electr its response to an alternating electric
field Colloidal particles dispersed in lu ically charged due to their ionic
characteristics and dipolar attributes The development of a net charge at the particle
surface affects the distribution of ions in the neighboring ng in
an increased concentration of counter ions to the surface Each particle dispersed in a
ns called fixed layer or Stern layer
Outside th compositions of ions of opposite polarities
form ble layer is form
particle-liquid interface This double layer may be considered to consist of two parts an
inner region which inclu nd relatively strongly to the surface and an outer or
diffuse region in which the ion distribution is determined by a balance of electrostatic
forces and random thermal motion The potential in this region therefore decays with the
distance from the surface until at a certain distance it becomes zero Figure 4-9 shows a
schematic representation of a typical ion distribution near a positively charged surface
The Stern surface (also referred to as the outer Helmholtz plane) is drawn through the
to understanding and c
trical potential th
ophoretic mobility or
a so tion are electr
interfacial region resulti
solution is surrounded by oppositely charged io
e Stern layer there are varying
ed in the region of the ing a cloud-like area Thus an electrical dou
des ions bou
center of those ions which are effectively adsorbed to the surface [Hunter 1981] The
78
extent of ion adsorption is determined by electrical and other long-range interactions
between the individual ions and surface of particles The ions outside of the Stern layer
form the diffuse double layer also referred to as the Gouy-Chapman layer [Burns and
Zydney 2000]
Shear plane
Stern layer
ψζ
0
ψs
ψShear plane
Stern layer
ψζ
0
ψs
ψ
t
controlled electric field is applied via electrodes immersed in a sample suspension and
Figure 4-9 Schematic representation of ion distribution near a positively charged surface
Zeta potential is a function of the surface charge of a particle any adsorbed layer a
the interface and the nature and composition of the surrounding medium in which the
particle is suspended The principle of determining zeta potential is very simple A
79
this causes the charged particles to move towards the electrode of opposite polarity
Viscous forces acting upon the moving particle tend to oppose this motion and the
equilibrium is rapidly established between the effects of the electrostatic attraction and
the viscosity dra
g The particle therefore reaches a constant terminal velocity
Figure 4-10 Schematic representation of zeta potential measurement(source
httpnitioncomenproductszeecom_shtm)
Because protein adsorption mechanisms are very complex and hard to explain in
biological systems protein adsorption is generally studied by using more ideal systems
consisting of one or more well characterized proteins a well-characterized adsorbent and
a well-defined aqueous solution Even so small changes in the experimental conditions
such as pH ionic strength and temperature generate totally different results Therefore
two media systems phosphate buffer (PB) and phosphate buffered saline (PBS) were
chosen to be used for the zeta potential measurements and protein adsorption study PB is
a simple aqueous system of 5 mM monobasic sodium phosphate (NaH2PO4H2O) to
n a constant pH PBS is used to keep the water concentration inside and outside of
the cell balanced If the water concentration is unbalanced the cell risks bursting or
shriveling up because of a phenomenon called osmosis PBS is a solution with the 5 mM
of monobasic sodium phosphate (NaH2PO4H2O) and 143 mM of sodium chloride (NaCl)
in water
maintai
80
The results of zeta potential measurement at room temperature (25plusmn1oC) as
function of pH and ionic strength (different media PB and PBS) for synthesized lat
particles are seen in Figure 4-11 Zeta potential values of polystyrene (PS) latex particle
at the Figure 4-9 are negative between -291 mV and -599 mV in PB and betwe
ex
s
en -203
mV and -278 mV in PBS at all pH ranges These negative zeta potential values of PS
latex particles are due to the sulfate groups [Vandenhu et al 1970] originated from
persulfate initiators attached via an oxygen bridge (-C-O-SO3-) at the end of polymer
chain where polymerization was initiated
-100
-80
-60
-40
-20
0
pH
Zta
pte
ntl
ζ
20
40
1 2 3 4 5 6 7 8 9
eo
ia (m
V)
PB (5mM)
PBS (5mM +143mM)
en -
Figure 4-11 Zeta potential of PS latex particles at 25oC
The zeta potential measurements of PSPMMA100 core shell latex particles as seen
in Figure 4-12 were also negative with a range of -28 and -505 mV in PB and betwe
143 mV and -186 mV in PBS at the pH 21-78 These negative values are also due to
sulfate groups on the particle surface
81
-100
-80
-60
-40
-20
0
20
40
1 2 3 4 5 6 7 8 9
Zea
pote
nti
l ζ
(mV
pH
ta
)
PB (5mM)
PBS (5mM + 143mM)
Figure 4-12 Zeta potential of PSPMMA100 latex particles at 25oC
The zeta potential for PSPMMA90PAA10 and PSPMMA75PAA25 was also
negative as seen in Figure 4-13 and 4-14 The zeta potential value for the
PSPMMA90PAA10 were between -367 mV and -678 mV in PB medium and between -
147 mV and -193 mV in PBS both at 25oC In case of the PSPMMA75PAA25 core shell
particles zeta potential values were between -291 mV and -520 mV in PB and between
-115 mV and -210 mV in PBS media Sulfate groups in PSPMMA75PAA25 and
PSPMMA90PAA10 core shell particles contribute to the negative zeta potential values
participate in forming a
negat
cid is expressed by the term pKa which is the pH
at which an acid is 50 dissociated However the contribution of the carboxylic acid
Carboxylic groups from acrylic acid monomer also would
ive surface charge of the latex particles at a pH greater than 70 because the pKa
value for the carboxylic acid is between 40 and 60 pH range [Ottewill et al 1967] This
indicates that carboxylic acid dissociation begins at a pH between pH 46-60 and
increases with pH The strength of an a
82
group to negative zeta potential values of PSPMMA-PAA core shell particles was not
detectable because there was no significant difference in zeta potential values for
synthesized latex particles at a pH greater than 70
-60
-40
-20
0
20
40
1 2 3 4 5 6 7 8 9
Zeta
pot
entia
l ζ
(mV
)
PB (5mM)PBS (5mM + 143mM)
-80
-100
pH
Figure 4-13 Zeta potential of PSPMMA90PAA10 latex particles at 25oC
-100
-80
-60
-40
-20
0
20
40
1 2 3 4 5 6 7 8 9
pH
Ze p
ont
i ζ
V)
tate
al (m
PB (5mM)PBS (5mM + 143mM)
Figure 4-14 Zeta potential of PSPMMA75PAA25 latex particles at 25oC
83
The zeta potential plots and values were similar for all latex particles This may be
due to the similar initiator density on the latex surface The initiator concentrations
monomers for shell polymerization were the same fo
to
r all core shell latex particles (062
ww from Table 4-4) A similar concentration (055 ww) of initiator to monomer was
added to prepare bare PS latex particles used in zeta potential measurements
Since the zeta potential values of all synthesized latex particles were negative
between pH=20 and pH=78 the isoelecric point (IEP) of these latex particles would be
less than pH=20 Sulfates have a pKa in the range of 10 to 20 [James 1985] indicating
that the sulfate group the conjugate base of a strong acid is protonated (-C-OSO3H) at
less than pH 20 This means that the synthesized latex particles are all negative at the
physiological pH Most serum proteins have a negative surface charge in blood
therefore when the negative latex particles are applied to blood stream fatal
tein is
avoided by charge repulsion in the blood Table 4-5 shows the common blood proteins
and their isoelectric points (IEP)
Table 4-5 The isoelectric point (IEP) of common proteins Serum proteins Isoelectric point (IEP)
complication by coagulation between applied latex particles and serum pro s
Albumin 48 α1-Globulin 20 α2-Microglobulin 54 γ1-Globulin 58 Heptoglobin 41 Hemoglobin 72 β1-lipoprotein 54 Immunoglobulin G (Ig G) 73 Fibrinogen 58 (source httpwwwfleshandbonescomreadingroompdf1085pdf
httpwwwmedalorgvisitorwww5CActive5Cch135Cch13015Cch130105aspx )
84
The absolute zeta potential values of the latex particles in phosphate buffer (PB)
was higher than that in phosphate buffered saline (PBS) This is because high
concentration of sodium and chloride electrolytes in PBS compressed the electrical
double layer causing rapid decay of surface potential Figure 4-15 shows that the
thickness of the electrical double layer is inversely proportional to the concentration of
electrolyte in the system
Surf
ace
Pote
ntia
l ψ
1κ3
10
1e
1κ 1κ
(1)(3) (2)
2 1 Z
Surf
ace
Pote
ntia
l ψ
1κ3
10
1e
1κ 1κ
(1)(3) (2)
Figure 4-15 The decay of surface potential with distance from surface in various
electrolyte concentrations (1) low (2) intermediate (3) high [Myers 1999]
2 1 Z
The electrical potential in the solution surrounding the particle surface falls off
the Debye-Huckel
yers 1999]
exponentially with distance (Z) from the surface according to
approximation [M
z) (- exp 0 κψψ = (4-4)
e reciprocal of the thickness of the electrical double layer referred to as the
The potential fallen off by a factor e The theoretical equation for the
ickness 1κ is
where κ is th
Debye length of 1
double layer th
21
22i
0
ekT 1
⎟⎟⎠
⎜⎜⎝
=sum ii zcεε
κ
⎞⎛ (4-5)
85
where
tration of
y was performed to measure the protein adsorption on the
synthesized latex particles Many physical and chemical processes occur at the boundary
between two phases and adsorption is one of the fundamental surface phenomena [Oscik
1982] This phenomenon is driven by the change in the concentration of the components
at the interface with respect to their concentrations in the bulk phase There are two
adsorption processes physical adsorption occurring when non-balanced physical forces
appear at the boundary of the phases and chemical adsorption or chemisorption
t the
interface [J
n leads
ational entropy hence adsorption takes place only if the
loss in conformational entropy is compensated
ter adsorption Cp The initial part of the
isotherm merges with the Γ-axis because at low polymer concentration the protein has a
high affinity for the surface and all of the polymer is adsorbed until the solid surface is
saturated
ε0 is the permittivity of a vacuum of free space ε is the permittivity of a medium e
is the relative permittivity or dielectric constant of the medium c is the concen
an ion (i) z is the valency of an ion (i) k is Boltzmannrsquos constant T is absolute
temperature (K)
43 Protein Adsorption Study
The adsorption stud
occurring when atoms and molecules from adjacent phases form chemical bonds a
aroniec et al 1988]
Flexible proteins in solution possess high conformational entropy as a result of the
various states each of the many segments in the protein chain can have Adsorptio
to a reduction of this conform
by sufficient attraction between polymer
segment and solid surface [Martin 1998] Isotherm shape of high affinity adsorption
between polymer and solid is shown in Figure 4-16 where the adsorbed mass Γ is plotted
against the polymer concentration in solution af
86
Figur
de
arboxyl
e 4-16 High-affinity adsorption isotherm of typical flexible polymer on solid
surface
In order to investigate the adsorption of blood proteins latex particle properties
such as electrical surface charge hydrophobicity and environmental conditions like pH
ionic strength and temperature are considered as experimental factors [Arai and Nor
1990] Figure 4-17 shows the overall schematic representation of the protein adsorption
on the synthesized latex particles with the functional groups such as sulfate and c
groups
CP
Γ
PMMAPAAShell
PS or PMMAShell OSO3
-
OSO3-
OSO3-
OSO3-
PS
-O3SO
PSCore
HOOC
-O3SO
O3SO
3
HOOC
HOOC
-
-O SO
Protein
NH3+
+
+H3N
+
COO-
Hydrophobic interaction
NH3
COO-
H3N
COO-
-OOC
Ionic interaction
Hydrophobic interaction
Hydrogen bonding
Ionic interaction
PMMAPAAShellOSO3
-
OSO3-
OSO3-
OSO3-
PS or PMMAShell -O3SOOSO3
-
PSCore
HOOC
-O3SO
O3SO
3
HOOC
HOOC
-
-O SO
-O3SO
OSO3-
OSO3-
OSO3-
PS PSCore
HOOC
-O3SO
O3SO
3
HOOC
HOOC
-
-O SO
Protein
NH3+
+
+H3N
+
COO-NH3
COO-
H3N
COO-
-OOC
NH3+
+
+H3N
+
COO-
Hydrophobic interaction
NH3
COO-
H3N
COO-
-OOC
Ionic interaction
Hydrophobic interaction
Hydrogen bonding
Figure 4-17 Overall schematic representation of the protein adsorption on the
Ionic interaction
synthesized latex particles
The surface functional groups as well as suitable surface hydrophobicity
hydrophilicity of latex particles are closely related to protein adsorption property As
indicated at Figure 4-17 various interaction forces such as hydrophobic interaction ionic
87
interaction hydrogen interaction and van der Waals interaction [Andrade 1985] exist
between protein molecule and the solid surface of latex particle
431 Adsorption Isotherm
The protein adsorption isotherm is generally defined as the relationship between the
amount of protein adsorbed by a solid (latex particle) at a constant temperature and as a
ine
the amount of protein adsorbed onto latex particles To evaluate the equilibrium
conce s
x
function of the concentration of protein Adsorption isotherms were plotted to determ
ntration of target proteins a calibration curve is firstly needed Figure 4-18 show
the UV absorbance as a factor of bovine serum albumin (BSA) concentrations The UV
absorbance of the known protein concentration was measured at 562nm which is the λma
for a protein treated by a bicinchoninic acid (BCA) assay
0
02
04
06
08
500 520 540 560 580 600
Wavelength (nm)
Abs
orba
nce
Figur gml (B)
06mgml (C) 02mgml
(A)
(C)
(B)
Max562nm
e 4-18 The dependence of UV absorbance on BSA concentration (A) 10m
Figure 4-19 shows the standard calibration curve used in adsorption isotherm
experiment of protein onto latex particles The net absorbance of each known protein
88
concentration was plotted and then a linear equation passing through a concentration of
zero protein concentration was obtained This equation is applied
to determine the
orbed on the synthesized latex particles The amount of free
protei
eported
trations (005 01 03 05
and 07mgml) were used to generate this curve as well as the adsorption isotherm
unknown protein amount ads
n was also determined using UV light at the 280nm wavelength however a
distinguishable UV intensity was too low to determine a precise value of protein
adsorption
The bicinchninic acid (BCA) assay technique [Smith et al 1985] has been r
for high efficiency of protein evaluation and was used for determination of protein
adsorption [Tangpasuthadol et al 2003 Williams et al 2003] This technique has many
advantages such as high sensitivity easy of use color complex stability and less
susceptibility to any detergents Five different BSA concen
y = 00R2 =
004741x 09939
00
01
02
03
04
0 200 400 600 800
BSA Standard (microgml)
Net
Abs
orba
nce
at 5
62nm
Figure 4-19 Standard curve of net absorbance vs BSA sample concentration
89
All isotherms were fitted to the Langmuir-Freundlich isotherms using the nonlinea
regression method Two adsorption isotherm models the Langmuir type isotherm a
Langmuir-Freundlich combination type isotherm were compared As seen the Figure 4-
20 The Langmuir isotherm model deviated further from the experimental data Th
all isotherms were fitted to the Langmuir-Freundlich isotherms [Yoon et al 1996
al 1998] usi
r
nd the
erefore
Lee et
ng the nonlinear regression method with following equation
n
n
eq
eqm
kC
kC1
1
1+
where q is the amount of adsorbed BSA per unit surface area q
qq = (4-6)
stant
were between 045 and 055 and hence n was fixed to its average 05 Actual qm values
were evaluated after fixing the n value
m is the adsorbed amount
in equilibrium Ceq is the equilibrium concentration of BSA k is the adsorption con
and n is the exponential factor for a heterogeneous system n values are empirical and
usually from zero to no more than unity [Jaekel 2002] The values of n in our system
00
10
20
30
0 02 04 06 08
C (mgml)eq
q (m
gm
2 )
pH 32Langmuir-Freundlich modelLangmuir model
Figure 4-20 Fitted models for adsorption isotherm of bovine serum albumin (BSA) on
polystyrene latex particles at 37oC in phosphate buffer media
90
The BSA protein adsorption on the synthesized latex particles was performed at
three pH levels 32 48 74 in both phosphate buffer (PB) and phosphate buffered saline
(PBS) at physiological temperature 37oC These pH levels correspond to an acidic pH
lower than the isoelectric point (IEP) of BSA the pH at the IEP and an alkaline pH
higher than the IEP of BSA respectively The IEP corresponds to the pH at which the
amino acid becomes a zwitterion Here its overall electrostatic charge is neutral and the
charge expressed by the basic amino group (-NH2 -NH3+) of the amino acid is equal to
the charge expressed by the acidic carboxyl group (-COOH -COO ) The IEP of BSA is
-
pH 48 and indicates that the side chain of the amino acids of BSA contains a greater
amount of acidic functional groups than that of basic functional groups At pH 32 and
74 BSA has a positive and negative charge respectively
Figure 4-21 and 4-22 show adsorption isotherms of BSA onto polystyrene (PS)
latex particles
00
10
20
30
40
0 02 04 06 08
Ceq (mgml)
q (m
gm
2 )
pH=32pH=48pH=74Model (pH 32)Model (pH 48)Model (pH 74)
Figure 4-21 Adsorption isotherm of BSA on polystyrene (PS) latex particles in
phosphate buffer (PB) at 37oC
91
00
10
30
40
0 02 04 06 08
Ceq (mgml)
q (m
gm
2
20
)
pH=32pH=48pH=74Model (pH 32)Model (pH 48)Model (pH 74)
(PBS) on PS latex particles
At the IEP of BSA the adsorbed amount (q
Figure 4-22 Adsorption isotherm of BSA adsorption 37oC in phosphate buffered saline
onto PS at equilibrium was
maxim
um at the IEP of BSA This is because the BSA molecules form the most
compact structures at the IEP eliminating the repulsion force between proteins A higher
number of BSA molecules can adsorb on a given surface area by hydrophobic interaction
when in this compact structure This observation agrees well with reported data by other
researchers who also indicated that the maximum adsorption from aqueous protein
solution was observed at the IEP [Mularen 1954 Watkins et al 1977 Oh at el 2000]
There was no significant difference of adsorbed BSA amount in PB and in PBS media at
pH 48 The conformational alteration of BSA was not affected significantly by the ionic
strength of the two solutions There was evidence of the effect of the electrostatic
interaction to some extent even at the IEP since the amount of BSA adsorbed in PBS is
slightly hig
m) of BSA
um indicating that hydrophobic interaction between latex particle and BSA
protein is maxim
her than in PB at this pH
92
The adsorbed amount of protein is shown to decrease at a pH 32 and 74 The
decrease in adsorption efficiency when deviating from IEP of BSA is a result of the
increase in conformational size of the protein molecules and the lateral electrostatic
repulsions between adjacent adsorbed BSA molecules Figure 4-23 shows a schematic of
possible conformational changes at each pH where hydrophobic interaction always exists
between non-polar portion of a protein and hydrophobic portion of solid surface
- - --- ---Latex surface
Zeta potential
BSA(+)
(-)
48 7432pH
(0)
+++ + ++
--+
++-
- - --- ---Latex surface
Zeta poten pH(0)
+++ + ++
--
tial
BSA(+)
(-)
48 7432
+
++-
ming
4
s
al blood Table 4-6 shows the calculated results of
Figure 4-23 Conformation of bovine serum albumin (BSA) on latex particles (Assuthat charge repulsion reduces the effect of the hydrophobic interaction at pH 74)
In addition the adsorbed amount of BSA at pH 32 was larger than that at pH 74
and still less than that at 48 This is because there is the electrostatic interaction between
the positive BSA molecules and negative PS latex particles at pH 32 and electrostatic
repulsion is dominant between negative BSA and negative PS latex particles at pH 7
The amount adsorbed increased with increasing ionic strength in pH 32 and 74 This i
because the high concentration of electrolytes reduces the electrostatic repulsion between
BSA and PS latex particle It should be noted that the BSA concentration used in these
experiments was lower than that found in normal human blood This is because the
equilibrium BSA adsorption onto the latex particles is actually reached at a much lower
concentration than that found in norm
93
adsor rm
ein
leading to a compact conformation of BSA There are two main forces affecting
adsorption amount at an acidic pH of 32 They include the ionic attraction between the
negative latex particle and the positive protein and lateral repulsion between positive
proteins The smallest amount of BSA adsorption was observed at a pH of 74 At this
point the charge repulsion between negatively charged particles and protein molecules
exists the h
of cha
bed BSA amount at equilibrium from the Langmuir-Freundich adsorption isothe
equation
In summary the hydrophobic interaction between PS latex particle and BSA
protein exists and is the dominant mechanism for the adsorption process Conformational
changes of BSA at each pH also affected the adsorption amount Adsorption was highest
at pH 48 because at this pH there is the least amount of ionic repulsion in the prot
and the lateral repulsion between proteins dominant adsorption even though there always
ydrophobic interaction between the solid latex particle and protein The effect
rge repulsion was reduced by the high concentration of electrolytes in PBS medium
Table 4-6 The equilibrium concentration values of BSA adsorption on polystyrene (PS) latex particles calculated the Langmuir-Freundlich isotherm model
Media pH qm (mgm2) 32 174 48 299 Phosphate buffer 74 136 32 260 48 283 Phosphate buffered saline 74 207
The adsorption isotherms of BSA on PSPMMA core shell particles are shown in
Figure 4-24 in phosphate buffer (PB) and in Figure 4-25 in phosphate buffered saline
(PBS) The adsorbed BSA amount was calculated using the Langmuir-Freundlich
100
94
adsorption isotherm equation and the amount listed in Table 4-7 The trend seen in the
adsorption results was very similar to the case of PS latex The overall adsorption process
was d rticle
rs
hat
0
ominated by hydrophobic interactions between the PSPMMA100 core shell pa
and BSA proteins At pH 48 the adsorbed amount (qm) of BSA on PSPMMA100 core
shell particles was 221 mgm2 in PB and 237 mgm2 in PBS and are the maximum
values recorded among the three pH levels 32 48 and 74 Suzawa and his co-worke
[1982] have obtained similar results This can be also explained by the compact
conformation of BSA molecules at the IEP of BSA which lead to a high protein
population on the solid surface At a pH of 32 and 74 BSA adsorption was less than t
at pH 48 because of the protein conformation change There was a small increase in
adsorbed BSA at pH 32 because of the charge attraction between negative PSPMMA10
core shell particles and positive BSA At pH 74 however the adsorption of BSA was
further reduced by charge repulsion between the negative PSPMMA100 core shell
particles and the negative BSA proteins
000 02 04 06 08
Ceq (mgml)
10
20q (m
gm
302 )
40
50pH=32pH=48pH=74Mode 8)l (pH 4Mode 2)l (pH 3Mode 4)
Figure 4-24 Adsorption isotherm of BSA 37oC in PB on PSPMMA core shell latex
l (pH 7
100
particles
95
The adsorbed amount of BSA increased more rapidly in PBS with increasing ioni
strength at pH 74 This shows that the low concentration of electrolytes promotes
electrostatic repulsion between negative BSA and negative PSPMMA
c
on
x
l 1980
100 core shell
particle As the ion strength of PBS is increased however the high concentration of
electrolyte leads to less electrostatic repulsion promoting more rapid protein adsorpti
By comparing the amount of adsorbed protein between bare PS latex particles and
PSPMMA100 core shell particles the amount of protein adsorption on the PS late
particles is greater than that on the PSPMMA100 core-shell particles [Suzawa et a
1982 Lee et al 1988]
00
10
20
30
40
m2 )
0 02 04 06 08
Ceq (mgml)
q (m
g
pH=32pH=48pH=74Model (pH 2) 3Model (pH 48)Model (pH 74)
Figure 4-25 Adsorption isotherm of BSA at 37oC in PBS on PSPMMA100 core shell
latex particles
96
Table particles calculated the Langmuir-Freundlich isotherm model
4-7 The equilibrium concentration values (qm) of BSA adsorption on PSPMMA100
Media pH qm (mgm ) 2
32 140
48 221 Phosphate buffer
74 130
32 169
48 237 Phosphate buffered saline
74 162
The BSA adsorption mechanism and trend on PSPMMA core shell particles was
very similar to that of the PS latex particle Unlike the case of PS and PSPMMA100 latex
particles the amount of BSA adsorbed onto PSPMMA90PAA10 and PSPMMA75PAA25
core shell particles was higher at pH 32 than pH 48 and 74 Figure 4-26 through 4-29
shows the adsorption isotherms of BSA on PSPMMA90PAA10 and PSPMMA75PAA25
core shell particles The amount of BSA adsorbed increased at pH 32 and pH 48 as the
hydrophilicity was developed by the addition of PAA On the contrary the adsorbed
amount of BSA was dramatically decreased at pH 74 The adsorption amount of BSA on
PSPMMA90PAA10 and PSPMMA75PAA25 core shell particles at equilibrium is listed in
Table 4-8 and 4-9 respectively
Carboxylic acid as well as sulfate groups are distributed on the surface of
PSPMMA90PAA10 and PSPMMA75PAA25 core shell particles and seem to largely affect
the BSA adsorption The isotherms showed very strong pH dependence to the BSA
adsorption onto these core shell latex particles The carboxylic groups (COOH) may
especially enhance the BSA adsorption prominently at pH 32 by hydrogen bonding with
protein molecules The pKa value for the PAA is 40-60 and a good indication that the
97
carboxylic acid groups are protonated at pH 32[Gebhardt et al 1983] The main
adsorption interaction force at pH 32 was generated by hydrogen bonding as well as
charge attraction between latex particle protein even th the conformational
repulsion between proteins results in a reduction of the protein
adsorption at this pH The amount of BSA adsorbed also increased as the PAA amount in
eased The a tion of BSA w inimal however less
than 07 mgm2 at pH 74 Here the carboxylic groups are ionized which increases the
charge repulsion by ionized carboxylic groups (COO-) resulting in PSPMMA-PAA latex
particle
and ough
change and charge
the core shell particles incr dsorp as m
s with much lower protein adsorption
00
20
40
q (m
g2 )
60
m
pH=32pH=48pH=74Model (pH 32)Model (pH 48)
0 02 04 06 08
Ceq (mgml)
Model (pH 74)
o shell
Figure 4-26 Adsorption isotherm of BSA at 37 C in PB on PSPMMA90PAA10 corelatex particles
98
00
40
60
80
0 02 04
C (mgm
mm
2 )
20
06 08
eq l)
q (
g
pH=32pH=48pH=74Model (pH 32)Model (pH 48)Model (pH 74)
Figure 4-27 Adsorption isotherm of BSA at 37oC in PBS on PSPMMA90PAA10 core
shell latex particles
Table 4-8 The equilibrium concentration values of BSA adsorption on PSPMMA90PAA10 particles calculated the Langmuir-Freundlich isotherm model
Media pH qm (mgm2)
32 361
48 283 Phosphate buffer
74 043
32 519
48 226 Phosphate buffered saline
74 043
99
00
20
40
60
80
100
0 02 04 06 08Ceq (mgml)
q (m
gm
2 )
pH=32pH=48pH=74Model (pH 32)Model (pH 48)Model (pH 74)
oFigure 4-28 Adsorption isotherm of BSA 37 C in PB on PSPMMA75PAA25 latex particles
00
100
20
40
60
80
0 02 04 06 08Ceq (mgml)
q (
m2 )
pH=32pH=48pH=74
mg
Model (pH 32)Model (pH 48)Model (pH 74)
Figure 4-29 Adsorption isotherm of BSA 37oC in PBS on PSPMMA75PAA25 core shell
latex particles
100
Table 4-9 The equilibrium concentration values of BSA adsorption on PSPMMA75PAA25 particles calculated the Langmuir-Freundlich isotherm model
Media pH qm (mgm2)
32 1083
48 662 Phosphate buffer (PB)
74 075
32 1077
48 681 Phosphate buffered saline (PBS)
74 057
The adsorption of β2-microglobulin (β2M) was determined under one set of
onditions in phosphate buffer at 37oC and pH 74 because of the limited amount of β2M
protein The adsorption isotherms of the β2M protein on the synthesized latex particles
are shown in Figure 4-30 4-31 and 4-32 The β2M protein concentrations for adsorption
isotherm experiment were chosen to be 0015 0030 0045 and 0060mgml since the
β2M concentration for the patients with renal failure can be elevated to similar
concentrations about 005mgml or more up to 50 times the normal level [Nissenson et
al 1995] There were steep initial slopes of the adsorption isotherm curves of β2M on
latex particles in all isotherms indicating a high affinity adsorption type It seems that the
hydrophobic interaction is enough for the adsorption process of β2M proteins to occur on
the latex particles even though charge repulsion exists between protein and latex particle
at pH 74 The complete plateau regions were not seen for all isotherms indicating that
β2M proteins were not saturated at this level and there are still available sites for β2M
c
adsorption on latex particles The initial slope of the β2M isotherms for PS was steeper
101
than that of any of the other latex particle PSPMMA100 PSPMMA90PAA10 and
PSPMMA 75PAA25
00
02
0 0002 0004 0006 0008
04
06
10
Ceq (mgml)
q (m
2 )
08
gm
PSPSPMMA100Model (PS)Model (PSPMMA100)
Figure 4-30 Adsorption isotherm of β2M onto PS and PSPMMA100 latex particles in PB
at 37 C and pH 74 o
00
02
04
06
08
10
0 0002 0004 0006 0008C (mgml)
q (m
m2 )
eq
g
PS
PSPMMA90PAA10
Model (PS)
Model (PSPMMA90PAA10)
Figure 4-31 Adsorption isotherm of β2M onto PS and PSPMMA90PAA10 latex particles
in PB at 37oC and pH 74
102
00
02
04
06
08
10
0 0002 0004 0006 0008Ceq (mgml)
q (m
gm
2 )
PS
PSPMMA75PAA25
Model (PS)Model (PSPMMA75PAA25)
Figure 4-32 Adsorption isotherm of β2M onto PS and PSPMMA75PAA25 latex particles
in PB at 37oC and pH 74
The equilibrium concentration values (qm) of β2M adsorption on latex particles are
calculated and listed in Table 4-10 The difference between the maximum amounts of
β2M adsorption on all the latex particles was not significantly different and their values
were between 069 and 08mgm2
Table 4-10 The equilibrium concentration values of β2M adsorption calculated the Langmuir-Freundlich isotherm model
Latex particles qm (mgm2)
PS 080
PSPMMA100 069
PSPMMA90PAA10 072
PSPMMA75PAA25 072
103
432 Adsorbed Layer Thickness
There are several possible explanations for large amount of BSA adsorbed at acidic
pH levels including the end-on adsorption pattern of BSA the flexibility of BSA
molecules the tilting of protein molecules due to the asymmetry of the charge
distribution or multilayer formation [Peula et al 1993] The adsorbed BSA thickness (δ)
can be calculated by the following equation [Chiu et al 1978] This equation was also
cited again by other researchers [Shirahama et al 1985]
BSA
mqρπ
δbullbull
=33
(4-7)
where π
33 is the packing factor qm is the plateau value of the adsorbed am
ρBSA is the hich corresponds to the reciprocal of its known
partial specific volume The layer thickness (δ) of BSA proteins on synthesized latex
partic
he IEP [Shirahama et al 1985] Compared to the
hydrodynamic dimensions of BSA 140 times 40 times 40 Aring3 [Fair et al 1980 Peter 1985] these
nd
ode It is unnecessary ultilayering because thickness do
exceed 140 Aring The layer thickness (δ) of BSA proteins on synthesized latex particles
e red saline (PBS) was ran 28 ~ 83 Aring at 37oC and pH 48 These
lso listed in Table 4-1
ount and
density of BSA molecule w
les in phosphate buffer (PB) ranged from 27 - 81 Aring at 37oC and pH 48 These
calculated values are listed in Table 4-11 Only δ values at pH 48 the isoelectric point
(IEP) of BSA are important because intra- and intermolecular electrostatic repulsion of
protein molecules is minimized at t
values of layer thickness indicate that the BSA molecules adsorb by side-on (40Aring) a
end-on (140Aring) m to consider m
not
in phosphat buffe ged of
calculated values are a 2
104
Table 4-11 Absorbed BSA layer thickness (Aring) of BSA in phosphate buffer (PB) at 37oC pH PS PSPMMA100 PSPMMA90PAA10 PSPMMA75PAA25
32 21 17 44 133
48 37 27 35 81
74 17 16 5 9
Table 4-12 Absorbed BSA layer thickness (Aring) of BSA in phosphate buffered saline (PBS) at 37oC
pH PS PSPMMA100 PSPMMA90PAA10 PSPMMA75PAA25
32 32 21 64 132
48 35 29
74 26 20 5 7
28 83
1n
1n
neqm
So k is closely related to the initial slope of the isotherm showing the affinity
between BSA and latex particles [Yoon et al 1996] A key point in the characterization
433 Gibbs Free Energy of Protein Adsorption
In the Langmuir isotherm model k represents the ratio of adsorption to the
desorption rate constants This definition is not applicable to the Langmuir-Freundlich
isotherm model but its meaning is similar [Yoon et al 1996] In this sense k is closely
related to the affinity between protein molecules and latex particles When the
equilibrium concentration Ceq reaches zero the value of Ceq will be very small (1gtgt
kCeq ) and then the equation 4-6 becomes
(4-8)
of adsorption processes on solid-liquid interfaces is to determine the change in Gibbsrsquos
free energy (∆Gdeg) one of the corresponding parameters for the thermodynamic driving
forces during adsorption The traditional method to evaluate adsorption thermodynamic
kCqq 1=
105
constants is based on measurement of adsorption isotherms under static conditions
[Malm ten 1998] The Langmuir-Freundlich ads lf
easily found from Then this enables calculation of the free-
energy change ∆G during adsorption expressed as
(4-9)
where R is the general gas constant (R = 8314Jmiddotmol K ) and T is the absolute
tem ture Gibb e energy A P
listed in Table 4-13 and 4-14 respectively As the ∆Gdegads ore negativ e
pro adsorption on a solid substra ill be more favorable
e of BSA adsorption in phosphate o
pH PS PSPMMA100 PSPMMA90PAA10 PSPMMA75PAA25
s orption equilibrium constant k can be
a slope of the isotherm
degads
lfads kRTG lnminus=∆ o
-1 -1
pera s fre change of BS adsorption values in B and PBS are
values are m e th
tein te w
Table 4-13 The values of Gibbs free energy changbuffer (PB) at 37 C
32 -2411 -2726 -2846 -2805
48 -2352 -2383 -2198 -1972 ∆Go
80
ads (kJmol)
74 -2452 -888 -324 -2
Table 4-14 The values of Gibbs free energy change of BSA adsorption in phosphate
pH PS PSPMMAbuffered saline (PBS) at 37oC
100 PSPMMA90PAA10 PSPMMA75PAA25
32 -2315 -2620 -2312 -2683
48 -2149 -2299 -2090 -2138 ∆Go
(kJmads ol)
74 -2430 -1992 -258 -263
Table 4-15 shows the calculated values of Gibbs free energy change of β M
adsorption on synthesized latex particles PSPMMA PAA core shell latex particle has
2
75 25
106
largest negative Gibbs free energy of β2
2
M adsorption indicating largest affinity betw
β
een
2M adsorption in phosphate buffere (PB) at 37 C and pH74
PS PSPMMA100
M and latex particle
Table 4-15 The values of Gibbs free energy change of βo
PSPMMA90PAA10 PSPMMA75PAA25
∆Go03 ads
(kJmol) -3626 -3466 -3659 -39
The Gibbs free energy of β2M was much more negative in phosphate buffer (PB)
than that of BSA in physiological condition (37 C and pH 74) o from the Table 4-13 and
the Figure 4-31 This indicates that β2M molecules more readily adsorb on latex particles
BSA This can be explained by the rate of diffusion and accessibility of β2M molecules to
the latex particle ier and faster for β2M m an BSA because of
size (molec ight) erical shape molecules It o be
due to the higher hydrophobic force and less electrostatic repulsion that contribute to the
adsorption of β2M molecules not as prevalent in the case of BSA because the IEP of β2M
(57) is clo H 74) than that of BSA (48) causing β2M to
be less electro cally lled p ro
can be seen that the ∆ β2M adsorption onto the synth article
increasing wi hy icity o x particles s the ∆Goads of the BSA
molecules is decreasing or constant This phenomenon supports the theory that the
select lizing
than BSA and that the affinity of β2M for the latex particle is much larger than that of
surface It is eas olecules th
the smaller ular we and sph of β2M may als
ser to physiological condition (p
stati repe from the latex articles than BSA F m Figure 4-33 it
Goads of esized latex p s is
th the drophil f the late wherea
ive adsorption of β2M molecules over BSA molecules can be enhanced by uti
more hydrophilic PSPMMA90PAA10 and PSPMMA75PAA25 core shell latex particles
107
-60
-50
1 2 3 4
-40∆Gad
s
-30
10
0
o (k
Jm
o-
-20l)
β2-Microglobulin (β2M)Bovine serum albumin (BSA)
Figure 4-33 Gibbs free energy of adsorption of proteins on latex particles in PB at 37
and pH 74
434 Kinetics of Adsorption
to
oC
Protein adsorption is a complex process known to have a large biological impact
and is currently not well understood quantitatively because of extreme sensitivity to pH
the concentration of other electrolytes and molecules present temperature and many
other factors that can change in the physiological system Accurate knowledge of the
adsorption kinetics under a given set of conditions is a prerequisite for elucidating the
mechanisms of many fundamental biological processes to be predicted at the molecular
level [Regner 1987] This adsorption kinetics approach is used in protein adsorption
studies because of the uncertainty related to the time needed for the interfacial proteins
reach the equilibrium It is generally accepted that the process of protein adsorption is
comprised of the following steps a) transport toward the interface b) attachment at the
interface c) eventual structural rearrangements in the adsorbed state d) detachment from
PS PSPMMA100 PSPMMA90PAA10PSPMMA75PAA25
108
the interface and transport away from the interface Each of these steps can affect the
overall rate of the adsorption process
The quantitative analysis of the protein adsorption kinetics requires that the protein
amount adsorbed is known as a function of time The kinetics tests were carried out in
phosphate buffer (PB) at 37oC and pH 74 a condition similar to physiological condition
The protein concentrations chosen were 07mgml for BSA and 006mgml for β2M
which are the maximal concentrations used in an adsorption isotherm test A time-based
evaluation of the adsorption was made by measuring the concentration of protein in
solution at different incubation times The results are shown below from Figure 4-34 to 4-
37 The adsorption kinetics of proteins onto th cond-e latex particles is expressed as a se
order model [Oumlzacar 2003] shown by the following equation
2)( tet qqk
dtdq
minus= (4-10)
Integrating above equation and applying the boundary conditions yields
ee
t tq
+=
1 (4-11)
where q
qkq
t
2
2
2
the previous adsorption isotherm results representing good reproducibility of adsorption
t and qe stand for the amount (mgm ) of protein adsorbed at time t and at
equilibrium respectively k is the equilibrium rate constant of second-order adsorption
(m mghr) and t is the incubation time (hr)
The fitting parameters calculated by using the equation 4-11 are listed in table 4-16
From this data it is seen that the amount of BSA adsorbed at equilibrium was higher than
that of β2M on PS and PSPMMA100 latex particles but less than that of β2M on
PSPMMA90PAA10 and PSPMMA75PAA25 latex particles These results are similar to
109
00
05
15
20
0 2 4 6 8 10 12 14
Time (hr)
rot
n ad
sorb
d (
10
25
Pei
em
gm
2
BSA2nd order model (BSA))β2M2nd order model (β2M)
Figure 4-34 The kinetics of protein adsorption in PB on PS latex particles at 37oC and
pH 74
00
05
10
15
20
25
30
0 2 4 6 8 10 12 14
Time (hr)
Prte
inds
oo
arb
ed (
mg
m2 )
BSA2nd order model (BSA)β2M2nd order model (β2M)
Figure 4-35 The kinetics of protein adsorption in PB on PSPMMA100 latex partic
37les at
oC and pH 74
110
00
03
06
09
12
15
0 2 4 6 8 10 12 14
Time (hr)
Prot
ein
adso
rbed
(m
gm
2 ) BSA2nd order model (BSA)β2M2nd order model (β2M)
Figure 4-36 The kinetics of protein adsorption in PB on PSPMMA90PAA10 latex
particles o at 37 C and pH 74
00
03
06
09
12
15
0 2 4 6 8 10 12 14
Time (hr)
BS
A ad
sorb
ed (
mg
m2 )
BSA2nd order model (BSA)β2M2nd order model (β2M)
Figure 4-37 The kinetics of protein adsorption in PB on PSPMMA75PAA25 latex
particles at 37oC and pH 74
111
test The variation of β2M equilibrium adsorption amount was nearly negligible for all
latex particles The plateau region is reached earlier for β2M than BSA on all latex
particles and the K values (equilibrium rate constant of second order adsorption) for the
β2M adsorption process onto latex particles are much larger than for BSA adsorption
These values indicate that the β2M molecules have a higher adsorption rate onto the latex
particles than the BSA molecules
Table 4-16 Fitting parameters of second-order kinetic model BSA β2M
Latex particles qe (mgm2) K (hr-1) qe (mgm2) K ( -1) hr
PS 144 67 074 7549
PSPMMA100 175 353 076 4438
PSPMMA90PAA10 046 97 075 5695
PSPMMA75PAA25 060 179 075 5168 K = (kqe) hr-1
There are several explanations for the phenomena observed in these experiments
First the smaller sizes of β2M molecules allow easier diffusion and a close approach to
the latex substrate Second a reduced number of hydrophobic domains on the latex
particle surface replaced by sulfate groups (-OSO3-) and ionized carboxylic groups
(COO-) are not favorable for larger BSA molecules Third the conformational change by
intra- and inter-molecular repulsion would be faster in β2M due to smaller molecular
weight Finally the ionic repulsion between β2M and the latex particles is less than that
condition (pH 74) than IEP (48) of BSA
between BSA and latex particles because IEP (57) of β2M is closer to the tested
112
44 Blood Compatibility
Blood compatibility can be defined as the property of a material or device tha
permits it to function in contact with blood without inducing any adverse reactions
Successful functioning of blood contacting biomedical materials or devices in contact
with blood strongly depends on the extent to which they damage blood during operation
High shear stresses turbulence rela
t
tively long contact times and cellular impact with
artificial surfaces are most common events experienced by blood in contact with artificial
1978 Winslow and et al
1993] Therefore before any materia e placed into the human b
biocompatibility testing must be the first performed in the view of safety The hemolysis
s a method for pr ary screening of biomaterials to determine
h blood a er circulating body fluid The tests estimat
oglobin released from th blood cell d e induced
foreign material
There are several types of blood cells in our body but the ones that make blood red
are erythrocytes or red blood cells (RBCs) Red blood cells (erythrocytes) are the most
numerous type in the blood These cells are average about 48-54 million per cubic
millimeter (mm3 same as a microliter (microl)) of blood but these values can vary over a
larger range depending on such factors as health and altitude RBCs are red because they
contain hemoglobin which is a protein that contains iron When blood circulates through
lungs oxygen binds to the hemoglobin and is then delivered to all the parts of body
RBCs are made in the bone marrow They start out as a nucleus like other cells but
when they fill up with hemoglobin the nucleus becomes smaller until it disappears
Without a nucleus RBCs are fragile and live only about 120 days In a normal healthy
devises and can cause hemolysis or thrombosis [Bernhard et al
ls can b ody
test serves a elimin the
biocompatibility wit nd oth e the
concentration of hem e red amag by a
113
body about 2 million red blood ne marrow produces new
ones j ing
dy
le
cells die per second But bo
ust as fast RBCs are about 75 micrometer across and are responsible for carry
oxygen salts and other organic substances to the cells and tissues throughout our bo
In humans and most mammals the cells are concave (indented in the middle) and flexib
enough to squeeze through even the tiniest of blood vessels the capillaries
Figure 4-38 Image of red blood cells
(Sourcehttpwwwpbrchawaiiedubemfmicroangelarbc1htm)
ch
100 90 10 75 25 f
RBCs are among the first cells that come into contact with a foreign material
injected in the blood Therefore a hemolysis assay gives information about the
biocompatibility in the case of an in vivo application The concentrations of prepared
latex particles used for the tests in this research are 05 25 and 50 (ww) and the
incubation time under moderate shaking was 30 minutes at 37oC Test results are shown
in Figure 4-39 The amount of blood cells that ruptured when in contact with the PS latex
particles was largely increased from about 30 to 91 as the solid content of PS
increased from 05 to 50 However hemolysis by the core shell latex particles su
as PSPMMA PSPMMA PAA and PSPMMA PAA was less than 35 O
114
particular interest was that hemolysis caused by PSPMMA PAA particles at 50
solid concentration was less than 02 hemolysis the lowest value among the latex
particles The low hemolysis value indicates that the PSPMMA PAA core shell latex
particles are the most blood compatible at all solid contents Other core shell particles
such as PSPMMA and PSPMMA PAA also had excellent blood comp
90 10
90 10
100 75 25 atibility with
low RBS breakage
01 2 3
20
40
60
80
100
Hem
olys
is (
)
052550
Figure 4-39 He
PS PSPMMA100 PSPMMA PAA90 10
molysis caused by latex particles n=5
4PSPMMA75PAA25
CHAPTER 5 SUMMARY OF RESULTS CONCLUSIONS AND RECOMMENDATION FOR
FUTURE WORK
51 Summary of Results
The goal of this study was to synthesize monodisperse polymeric latex particles
with tailored properties and to investigate the fundamental interactions between the
synthesized latex particles and target proteins An understanding of the fundamental
mechanism of selective adsorption is strongly required in order to maximize the
separation performance of membranes made of these materials so that it may applicable
to hemodialysis therapy for end stage renal disease (ESRD) patients For the achievement
of above research goal several investigations have been performed Analysis of the
obtained data leads to several conclusions as discussed below
Monodisperse polystyrene seed latex particles were synthesized with the size
crosslinking agent to make the latex particles more hydrodynamically stable The
conversion of monomer to polymer was over 95 and seed latex particles are spherical in
shape and have smooth surface From these small seeds bigger latex particles as large as
800nm were synthesized using a semi-continuous seeded emulsion polymerization
process The particles with a mean diameter less than 500nm are very spherical in shape
and highly uniform in particle size distribution However as the particles grow larger
than 500nm in mean diameter they became non-spherical with an uneven surface This
irregularity of particle surface can be attributed to the non-homogeneous monomer
ranges between 126plusmn75 and 216plusmn53 nm Divinylbenzene (DVB) was used as a
115
116
swelling into the shell of a growing polymer which can be controlled by the factors such
as temperature agitation speed initiator feeding rate and surfactant amount required to
stabilize the colloidal system It was determ rfactant to monomer ratio
dec ic
acid (AA) monomers were intro rticle surfaces more
hydro
to
ting of a
orm
ration is seen between
3002
0 cm-
0cm-1
-1
03
ined that as the su
reased the mean particle diameter increased Methyl methacrylate (MMA) and acryl
duced to make the latex pa
philic than bare polystyrene particles The prepared core shell latex particles were
made of PMMA100 PMMA90PAA10 and PMMA75PAA25 PMMA100 is the PMMA
homopolymer shell on a PS core PMMA90PAA10 shell is a copolymer with a PMMA
PAA ratio of 90 to 10 by weight PMMA75PAA25 shell is a copolymer consis
PMMA to PAA ratio of 75 to 25 by weight The particle size of these core-shell
particles were prepared to be about 370nm in mean diameter
The successful synthesis of latex particles was confirmed using Fourier Transf
Infrared spectroscopy (FTIR) The C-H aromatic stretching vib
cm-1 and 3103 cm-1 for PS latex particles The C-H asymmetrical stretching and
symmetrical stretching vibration peaks of CH2 for all latex particles is seen at 290
1and 2850 cm-1 respectively There is a carbonyl (C=O) characteristic band at 173
wavenumber for PSPMMA core-shell latex particles The broad OH group peak at
3400 cm
100
can be seen in the spectroscopy of the particle with PMMAPAA shell
(PSPMMA PAA and PSPMMA PAA ) The peak intensity of OH group of
PSPMMA PAA was larger than that of PSPMMA PAA relatively due to more AA
monomer content in PSPMMA PAA than in PSPMMA PAA
In the zeta potential measurements polystyrene (PS) latex particles had negative
values between -291 mV and -599 mV in the phosphate buffer (PB) and between -2
90 10 75 25
75 25 90 10
75 25 90 10
117
mV and -278 mV in the phosphate buffered saline (PBS) at 25oC and pH 21-78 These
negative zeta potential values for PS latex particles are due to ionization of the sulfate
groups (-OSO3-) originated from the initiator Zeta potential values of the PSPMMA
core shell latex particles were also negative between -28 mV and -505 mV in PB and
between -143 mV and -186 mV in PBS at pH 21-78 Negative values are also
generated by dissociation of sulfate groups on a particle surface The zeta potential value
of PSPMMA PAA and PSPMMA PAA were of negative values between -36
and -678 mV in PB medium and -147 mV and -193 mV in PBS medium at 25
100
s
7 mV
MMA75PAA25 and
PSPM
otein
g the
l change of BSA at
each w
90 10 75 25
oC For
PSPMMA PAA core shell particles zeta potential values were between -291 mV and
-520 mV in PB media and between -115 mV and -210 mV in PBS media Sulfate
groups contributed to the negative zeta potential values in PSP
75 25
MA90PAA10 core shell particles Carboxylic groups from polyacrylic acid also
contribute to the negative surface charge for the latex particles at greater than pH 70
however the degree of their contribution was not detectable from the zeta potential
measurements The absolute zeta potential values of the synthesized latex particles in PB
were higher than that in PBS This is because of the high concentration of sodium
chloride electrolyte in PBS that compress electrical double layer thickness
The bicinchoninic acid (BCA) assay technique was used to determine the pr
adsorption and all isotherms were fitted to the Langmuir-Freundlich isotherms usin
nonlinear regression method The hydrophobic interaction between PS latex particle and
BSA protein was dominant for the adsorption process Conformationa
pH also affected the adsorption amount which was highest at pH 48 because of lo
ionic repulsion making compact conformation of BSA The next larger amount of BSA
118
adsorbed onto PS was at pH 32 There were two main forces affecting the amount of
adsorption at acidic pH values One is ionic attraction between negative latex particle a
positive protein The other is lateral repulsion between positive proteins The smallest
amount of BSA adsorption was at pH 74 where charge repulsion between negative
particles and proteins is present at this pH Lateral repulsion between proteins wa
dominant for protein adsorption even though there always exists the hydrophobic
interaction between solid latex particle and protein The charge repulsion was reduced by
the high concentration of electrolytes in the PBS medium The BSA adsorption process
on PSPMMA core shell particles was very similar to the case of PS latex particle where
hydrophobic interaction between latex particle and protein was dominant However t
adsorbed BSA amount was less than that of PS in PBS The adsorbed amount of BS
PSPMMA
nd
s
he
A on
an
ed
n The main
adsor
90PAA10 and PSPMMA75PAA25 core shell particles was higher at pH 32 th
pH 48 and 74 The adsorbed BSA amount became higher at pH 32 and pH 48 as the
hydrophilicity was increased by the higher PAA amount On the contrary the adsorb
amount of BSA was dramatically decreased at pH 74 Carboxylic acid groups as well as
the sulfate groups are distributed on the surface of PSPMMA90PAA10 and
PSPMMA75PAA25 core shell particles and largely affect the BSA adsorptio
ption interaction forces at pH 32 was generated by hydrogen bonding as well as
charge attraction between latex particle and protein even when the conformational change
and charge repulsion between proteins reduce the adsorption of protein at this pH The
adsorbed BSA amount was also increased as the amount of PAA increases However the
adsorption of BSA was minimal (less than 07 mgm2) at pH 74 where the carboxylic
groups are ionized leading to more enhancement of charge repulsion by ionization of the
119
carboxylic groups (COO-) Because of this the PSPMMA-PAA latex particles have a
much lower protein adsorption
There were steep initial slopes for adsorption isotherms of β
in
s
there
M
r
e
es
o
2M on latex particles
all isotherms indicating a high affinity type adsorption The complete plateau region
were not seen for all isotherms indicating that β2M proteins were not saturated and
are still available sites for β2M adsorption on latex particles The initial slope of β2
isotherms for PS was steeper than for the other latex particles PSPMMA100
PSPMMA90PAA10 and PSPMMA75PAA25
The layer thickness (δ) of BSA proteins on synthesized latex particles in phosphate
buffer (PB) was in the range of 27 - 81 Aring at 37 C and pH 48 Only the δ values at pH
48 the isoelectric point (IEP) of BSA are important because intra- and inter-molecular
electrostatic repulsion of the protein molecules is minimized at the IEP Compared to the
hydrodynamic dimensions of BSA 140 times 40 times 40 Aring these values of the adsorbed laye
thickness indicates that the BSA molecules adsorb by the side-on (40Aring) and end-on mod
(140Aring) Consideration of the multi-layering is unnecessary because the adsorbed
thickness of BSA do not exceed 140 Aring
The Gibbs free energy of β
o
3
2M was more negative in phosphate buffer (PB) than
that of BSA in physiological condition (37 C and pH 74) indicating that βo2M molecul
are more favorably adsorbed on the latex particles than BSA and that the affinity of β2M
was much larger than BSA This may be explained by the rate of diffusion and
accessibility of β2M molecules being easier and faster than that for BSA because of
smaller size (or weight) and spherical shape of β2M molecules It may also be possible t
explain that more hydrophobic forces (less electrostatic repulsion) contributes to
120
adsorption of β2M than to that of BSA because the IEP of β2M (57) is closer to
physiological condition (pH 74) than that of BSA (pH 48) The selective adsorption of
β2M m
in β2M
larger than in BSA adsorption indicating
that fa
an that between BSA and latex particles because
IEP (
ch as
Other
olecules over BSA molecules can be enhanced by developing more hydrophilic
PSPMMA90PAA25 and PSPMMA75PAA25 core shell latex particles
The adsorption kinetics of proteins on latex particles was expressed as a second-
order model The plateau region for β2M was reached earlier than BSA on all latex
particles and the K values (equilibrium rate constant of 2nd order adsorption)
adsorption process on latex particles were much
ster adsorption rate of β2M molecules than that of the BSA molecules There are
several explanations for these phenomena First the smaller size of β2M molecules can
more easily diffuse and approach to the latex substrate Second enhanced hydrophilic
domain on the latex particle surface by sulfate groups (SO4-) and ionized carboxylic
groups (COO-) is not favorable for larger BSA molecules but is for smaller β2M to be
anchored Third the conformational change by intra- and inter-molecular repulsion
would be faster in β2M due to lower molecular weight Finally the ionic repulsion
between β2M and latex particles is less th
57) of β2M is closer to the tested condition (pH 74) than IEP (48) of BSA
In the biocompatibility test the number of red blood cells (RBCs) ruptured by PS
latex particles ranged from 30 to 91 as the solid content of PS increased from 05 to
50 respectively However hemolysis of the other core shell latex particles su
PSPMMA100 PSPMMA90PAA10 and PSPMMA75PAA25 were less than 35 The
PSPMMA90PAA10 particles at all applied solid concentrations had less than 02
hemolysis and was the most blood compatible among the prepared latex particles
121
core shell particles such as PSPMMA100 and PSPMMA75PAA25 also had excelle
compatibility with low RBC breakage
52 Conclusions
The major conclusions from this study include
nt blood
bull microglobulin (β2M) over that of the bovine serum albumin (BSA) at pH 74
was achieved by controlling the PMMA and PAA ratio to optimize
e particles posses the most desirable requirements for use in a human dialysis
ns
icles
olecule
bull
Using engineered core shell latex particles selective adsorption of β2
physiological condition was demonstrated
bull The rate of adsorption of the β2M was also much higher than that of BSA This
hydrophobichydrophilic microdomain size
bull The core shell particles also exhibited excellent biocompatibility with blood Thes
membrane
53 Recommendation for Future Work
With the results and conclusions obtained through this study several suggestio
may be given for future work as follows
bull Competitive adsorption tests of whole blood proteins on synthesized latex partunder physiological condition
bull Preparation of latex particles with optimum hydrophobic hydrophilic microdomains to maximize selective adsorption and removal of toxin molecules
bull Membrane fabrication by suitable pore size design to discriminate a toxin mby size sieving as a function of particle layers
Evaluation of membrane performance such as flow rate in water and in simulated blood fluid (SBF)
bull Application for hemodialysis as well as other technologies such as a water remediation
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Korea on August 7 1968 He obtained a Bachelor of Science degree in Chemistry from
Keim d graduate school and earned a
resear n Korea Institute of Science and
Coati e
ia
Busin
BIOGRAPHICAL SKETCH
Sangyup Kim was born and raised in the small town of Sung-Joo Kyungbook
yung University (Daegu Korea) in 1993 He entere
Master of Science degree in Chemical engineering in 1995 He worked as a student
cher for 2 years at Textile Polymer Division i
Technology (KIST) in Seoul Korea Next he worked over a year at the Automobile
ng Division in DongJoo-PPG in Korea In August 2001 he began studying for th
degree of Doctor of Philosophy in materials science and engineering at the University of
Florida in Gainesville After getting his PhD he plans to work at the Digital Med
ess Division of Samsung Electronics in Korea starting in January 2006
141
ACKNOWLEDGMENTS
I owe what I am today and this moment to many people without whom I could not
have been here First of all I would like to express my sincere and deepest appreciation
to my parents wife Mikyung daughters Heehyung and Erin whose love patience and
support helped me pursue my dream I gratefully acknowledge my advisor Dr H El-
Shall who gave me an opportunity to become part of an exciting and beneficial research
project and exert myself in the research work His guidance knowledge and stimulating
scientific and critical thinking have contributed to my success to a great extent and
inspired me to dedicate myself to research work I especially appreciate Dr R Partch as a
co-advisor who has helped me conduct experiments with valuable discussion for my
research from my first year I am greatly thankful to Dr C McDonald whose idea and
valuable experimental advice and discussion in the area of polymer chemistry and
membrane technology helped cultivate my interest in this area and were vital to
performing this research Without his contribution this work could not have been
achieved I also thank my doctoral committee members Dr R Singh Dr A Zaman and
Dr B Koopman for appreciated comments
Financial support and equipment use for research from the Particle Engineering
Research Center at the University of Florida were vital in continuing my study for more
than 4 years My sincere thanks are due to my former advisor Dr Kiryoung Ha in
Keimyung University who helped me open my eyes and broaden my thought and
introduced me into a limitless polymer field I would like to also thank to past and current
iv
members in Dr El-Shallrsquos research group for their assistance advice and support I
sincerely appreciate Stephen for the assistance of thesis writing
v
TABLE OF CONTENTS page
ACKNOWLEDGMENTS iv
LIST OF TABLES viii
LIST OF FIGURES x
1 INTRODUCTION 1
2 BACKGROUND AND LITERATURE SURVEY5
21 The Significance of End Stage Renal Disease (ESRD)5 22 Hemodialysis (HD) Treatment 6 23 Advances in Membrane Technology 8 24 Sorbent Technology16 25 Limitation of Current Hemodialysis Treatment 19 26 Latex Particles 20
261 The Components for Emulsion Polymerization 21 262 Particle Nucleation 21 263 Types of Processes for Emulsion Polymerization 23 264 Chemistry of Emulsion Polymerization 24 265 Seed Emulsion Polymerization25 266 Polystyrene Latex Particles 26 267 Various Applications of Latex Particles28
27 Proteins 30 271 Interaction Forces between Proteins33 272 β -Microglobulin (β M)2 2 35 273 Serum Albumin 36
28 Protein Adsorption38 281 Interaction between Protein Molecule and Latex Particle38
29 Hypothesis for Toxin Removal39 291 Toxin Removal by Size Sieving Based on Monodispersed Pore Size 40 292 Toxin Removal by Selective Adsorption on Engineered Latex Particles 41
3 EXPERIMENTAL AND CHARACTERIZATION METHODOLOGY 44
31 Materials 44 32 Latex Particle Preparation46
321 Preparation of Seed Latex Particles47
vi
322 Preparation of Seeded Latex Particles 48 323 Preparation of Core Shell Latex Particles 48 324 Purification of Synthesized Latex Particles48
33 Characterization49 331 Degree of Conversion49 332 Fourier Transform Infrared (FTIR) Spectroscopy49 333 Quasielastic Light Scattering (QELS) 50 334 Field Emission-Scanning Electron Microscopy (FE-SEM) 50 335 Zeta Potential Measurement 50 336 Protein Adsorption51 337 Blood Biocompatibility by Hemolysis Test 53
4 RESULTS AND DISCUSSION55
41 Polymerization of Latex Particles55 411 Polystyrene Seed Latex Particles55 412 The Growth of Polystyrene (PS) Seed Latex Particles61 413 Core Shell Latex Particles 71
42 Characterization of Latex Particles75 421 Fourier Transform Infrared Spectroscopy (FTIR)75 422 Zeta Potential Measurements 75
43 Protein Adsorption Study 85 431 Adsorption Isotherm87 432 Adsorbed Layer Thickness 103 433 Gibbs Free Energy of Protein Adsorption 104 434 Kinetics of Adsorption 107
44 Blood Compatibility 112
5 SUMMARY OF RESULTS CONCLUSION AND RECOMMENDATION FOR FUTURE WORK115
51 Summary of Results115 52 Conclusions121 53 Recommendation for Future Work121
LIST OF REFERENCES122
BIOGRAPHICAL SKETCH 141
vii
LIST OF TABLES
Table page 4-1 The polymerization recipe of polystyrene (PS) seed particles 57
4-2 Continuous addition emulsion polymerization recipe for growing polystyrene (PS) latex particles less than 500nm in size 63
4-3 Continuous addition emulsion polymerization recipe for growing polystyrene (PS) latex particles larger than 500nm in size64
4-4 The preparation recipe of PS core with various shell latex particles72
4-5 The isoelectric point (IEP) of common proteins 83
4-6 The equilibrium concentration values of BSA adsorption on polystyrene (PS) latex particles calculated the Langmuir-Freundlich isotherm model 93
4-7 The equilibrium concentration values (q ) of BSA adsorption on PSPMMA particles calculated the Langmuir-Freundlich isotherm model
m 10096
4-8 The equilibrium concentration values of BSA adsorption on PSPMMA PAA particles calculated the Langmuir-Freundlich isotherm model
90 1098
4-9 The equilibrium concentration values of BSA adsorption on PSPMMA PAA particles calculated the Langmuir-Freundlich isotherm model
75 25100
4-10 The equilibrium concentration values of β M adsorption calculated the Langmuir-Freundlich isotherm model
2102
4-11 Absorbed BSA layer thickness (Aring) of BSA in phosphate buffer (PB) at 37 Co 104
4-12 Absorbed BSA layer thickness (Aring) of BSA in phosphate buffered saline (PBS) at 37 Co 104
4-13 The values of Gibbs free energy change of BSA adsorption in phosphate buffer (PB) at 37 Co 105
4-14 The values of Gibbs free energy change of BSA adsorption in phosphate buffered saline (PBS) at 37 Co 105
viii
4-15 The values of Gibbs free energy change of β M adsorption in phosphate buffere (PB) at 37 C and pH 74
2o 106
4-16 Fitting parameters of second-order kinetic model 111
ix
LIST OF FIGURES
Figure page 1-2 Stereo drawing of the αndashcarbon backbone of β M2 2
2-1 An image of the location and cross section of a human kidney 5
2-2 A schematic draw of the hemodialysis route7
2-3 Cross-sectional SEM image view of the Polyflux S (polyamide + polyacrylethersulfone + polyvinypyrollidone) dialysis membranes 11
2-4 The chemical structure of polysulfone (PSf) 12
2-5 The chemical structure of polyamide (PA)13
2-6 Chemical structure of polyacrylonitrile (PAN) 14
2-7 Emulsion polymerization system24
2-8 L-α-amino acid 31
2-9 Ribbon diagram of human β M taken from the crystal structure of the protein bound to the heavy chain of the MHC class I complex (PDB 1DUZ)
236
2-10 Secondary structure of human serum albumin (HSA) with sub-domains 37
2-11 The relationship between pore size and particle size in body-centered cubic and closed packed arrays41
2-12 Schematic representation of the protein adsorption on the core shell latex particles at pH 7442
3-1 Chemical structure of main chemicals45
3-2 Experimental setup for semi-continuous emulsion polymerization 46
3-3 The particle preparation scheme in various types and size ranges of latex particles 47
3-4 Schematic of the procedure for a protein adsorption test 52
3-5 Separation of RBC from whole blood by centrifuge process53
x
3-6 Schematic of the procedure for hemolysis test 54
4-1 Schematic representation of semi-continuous seed latex particles preparation and growth56
4-2 Scanning Electron Micrograph (SEM) of polystyrene seed latex particles (A) PS (B) PS (C) PS (D) PSS259 S233 S207 S181 59
4-3 SEM of PS latex particles less than 500nm in size (A) 258nm (B) 320nm (C) 370nm (D) 410nm 65
4-4 SEM of PS latex particles larger than 500nm in size (A) 525nm (B) 585nm (C) 640nm (D) 790nm 68
4-5 Dependence of the particle size on the surfactant to monomer ratio70
4-6 Schematic of core shell latex particle structures 71
4-7 Scanning Electron Micrograph of latex particles (A) PS (B) PSPMMA (C) PSPMMA PAA (D) PSPMMA PAA
100
90 10 75 25 73
4-8 FTIR spectra of polymerized latex particles (A) bare polystyrene (PS) (B) PSPMMA (C) PSPMMA PAA (D) PSPMMA PAA100 90 10 75 25 76
4-9 Schematic representation of ion distribution near a positively charged surface 78
4-10 Schematic representation of zeta potential measurement79
4-11 Zeta potential of PS latex particles at 25 Co 80
4-12 Zeta potential of PSPMMA latex particles at 25 C100o 81
4-13 Zeta potential of PSPMMA PAA latex particles at 25 C90 10o 82
4-14 Zeta potential of PSPMMA PAA latex particles at 25 C75 25o 82
4-15 The decay of surface potential with distance from surface in various electrolyte concentrations (1) low (2) intermediate (3) high 84
4-16 High-affinity adsorption isotherm of typical flexible polymer on solid surface 86
4-17 Overall schematic representation of the protein adsorption on the synthesized latex particles86
4-18 The dependence of UV absorbance on BSA concentration (A) 10mgml (B) 06mgml (C) 02mgml87
4-19 Standard curve of net absorbance vs BSA sample concentration 88
xi
4-20 Fitted models for adsorption isotherm of bovine serum albumin (BSA) on polystyrene latex particles at 37 C in phosphate buffer mediao 89
4-21 Adsorption isotherm of BSA on polystyrene (PS) latex particles in phosphate buffer (PB) at 37 Co 90
4-22 Adsorption isotherm of BSA adsorption 37 C in phosphate buffered saline (PBS) on PS latex particles
o
91
4-23 Conformation of bovine serum albumin (BSA) on latex particles92
4-24 Adsorption isotherm of BSA 37 C in PB on PSPMMA core shell latex particles
o100
94
4-25 Adsorption isotherm of BSA at 37 C in PBS on PSPMMA core shell latex particles
o100
95
4-26 Adsorption isotherm of BSA at 37 C in PB on PSPMMA PAA core shell latex particles
o90 10
97
4-27 Adsorption isotherm of BSA at 37 C in PBS on PSPMMA PAA core shell latex particles
o90 10
98
4-28 Adsorption isotherm of BSA 37 C in PB on PSPMMA PAA latex particleso75 25 99
4-29 Adsorption isotherm of BSA 37 C in PBS on PSPMMA PAA core shell latex particles
o75 25
99
4-30 Adsorption isotherm of β M onto PS and PSPMMA latex particles in PB at 37 C and pH 74
2 100o 101
4-31 Adsorption isotherm of β M onto PS and PSPMMA PAA latex particles in PB at 37 C and pH 74
2 90 10o 101
4-32 Adsorption isotherm of β M onto PS and PSPMMA PAA latex particles in PB at 37 C and pH 74
2 75 25o 102
4-33 Gibbs free energy of adsorption of proteins on latex particles in PB at 37 C and pH 74
o
107
4-34 The kinetics of protein adsorption in PB on PS latex particles at 37 C and pH 74
o
109
4-35 The kinetics of protein adsorption in PB on PSPMMA latex particles at 37 C and pH 74
100o
109
4-36 The kinetics of protein adsorption in PB on PSPMMA PAA latex particles at 37 C and pH 74
90 10o 110
xii
4-37 The kinetics of protein adsorption in PB on PSPMMA PAA latex particles at 37 C and pH 74
75 25o 110
4-38 Image of red blood cells 113
4-39 Hemolysis caused by latex particles n=5114
xiii
Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy
SYNTHESIS AND ENGINEERING OF POLYMERIC LATEX PARTICLES FOR MEDICAL APPLICATIONS
By
Sangyup Kim
December 2005
Chair Hassan El-Shall Major Department Materials Science and Engineering
Latex particles with well-defined colloidal and surface characteristics have received
increasing attention due to their useful applications in many areas especially as solid
phase supports in numerous biological applications such as immunoassay DNA
diagnostic cell separation and drug delivery carrier Hemodialysis membrane using
these particles would be another potential application for the advanced separation
treatment for patients with end stage renal disease (ESRD) It is desirable to remove
middle molecular weight proteins with minimal removal of other proteins such as
albumin Thus it is necessary to understand the fundamental interactions between the
particles and blood proteins to maximize the performance of these membranes This
improvement will have significant economic and health impact
The objective of this study is to synthesize polymeric latex particles of specific
functionality to achieve the desired selective separation of target proteins from the human
blood Semi-continuous seed emulsion polymerization was used to prepare monodisperse
xiv
polystyrene seed particles ranging from 126plusmn75 to 216plusmn53 nm in size which are then
enlarged by about 800nm Surfactant amount played a key role in controlling the latex
particle size Negatively charged latex particles with a different hydrophobicity were
prepared by introduction of a sodium persulfate initiator and hydrophilic acrylic acid
monomer The prepared polymeric particles include bare polystyrene (PS) particles less
hydrophobic PS core and PMMA shell particles and more hydrophilic PS core and
PMMA-co-PAA shell latex particles with a 370nm mean diameter SEM light scattering
and zeta potential measurements were used to characterize particle size and surface
properties Adsorption isotherms of two proteins bovine serum albumin (BSA) and β2-
microglobulin (β2M) on latex particles were obtained as a function of pH and ionic
strength using the bicinchoninic acid (BCA) assay method The Langmuir-Freundlich
adsorption model was used to determine the adsorption amount of protein at equilibrium
The thickness of adsorbed BSA layer on latex particles was obtained in order to
investigate the adsorption orientation such as end-on or side-on mode Adsorption
kinetics experiments for both proteins and all latex particles were also performed The
adsorption kinetic constant determined from the Langmuir-Freundlich adsorption
isotherm model was used to calculate Gibbs free energy of adsorption to compare the
competitive adsorption of BSA and β2M Hemolysis tests were performed to investigate
the blood compatibility of synthesized latex particles PSPMMA90PAA10 and
PSPMMA75PAA25 core shell latex particles had desirable material properties with not
only a large amount and high rate of selective β2M adsorption over BSA but also high
blood compatibility showing less than 3 hemolysis
xv
CHAPTER 1 INTRODUCTION
End stage renal disease (ESRD) is a chronic condition in which kidney function is
impaired to the extent that the patientrsquos survival requires removal of toxins from the
blood by dialysis therapy or kidney transplantation The National Kidney Foundation
estimates that over 20 million Americans had chronic kidney disease in 2002 [NKF] The
number of people with ESRD is rapidly increasing in the United States with
approximately 96 295 incidents and 406 081 prevalent patients including 292 215 on
dialysis and 113 866 with a functioning graft in 2001 It is projected that there will be
more than 22 million ESRD patients by 2030 [USRDS 2003] The expenditure for the
ESRD treatment program had reached $228 billion 64 of the Medicare budget in
2001 Due in part to the limited availability of kidneys for transplantation hemodialysis
is the primary clinical treatment for the patients with ESRD
The central element of a hemodialysis instrument is the semipermeable membrane
that allows for selective transport of low molecular weight biological metabolites less
than 5000 Da such as urea and creatinine as well as excess water and electrolytes [Baker
2004] from the blood One limitation of current dialysis technologies is the inability to
efficiently remove middle molecular weight toxins such as β2-Microglobulin (β2M) and
interleukin 6 (IL-6)
β2M is a causative protein of dialysis-related amyloidosis (DRA) a disease arising
in patients with chronic kidney failure as a serious complication of long-term
hemodialysis treatment [Gejyo et al 1985] β2M deposition in tissue is the primary cause
1
2
of destructive arthritis and carpal tunnel syndrome [Vincent et al 1992 Drueke 2000]
The β2M structure is shown in Figure 1-2
Figure 1-2 Stereo drawing of the αndashcarbon backbone of β2M [Becker et al 1985]
Although attempts have been made to increase the efficiency of middle molecular
weight toxin removal by changes in the membrane pore size and the use of innovative
materials to adsorb these toxins [Samtleben et al 1996 Ronco et al 2001] removal
efficiency is not as high as those achieved by a normal healthy kidney Traditional
membranes have a number of processing and performance limitations [Westhuyzen et al
1992 Leypoldt et al 1998] such as a restricted choice of surface chemistries and limited
control of porosity The development of novel engineering membrane technology is
needed to remove middle molecule toxins
Polymeric latex particles have received increasing attention in medical application
areas especially as solid phase supports in biological applications [Piskin et al 1994]
Examples of these applications include immunoassay [Chen et al 2003 Radomske-
Galant et al 2003] DNA diagnostic [Elaїssari et al 1998] cell separation drug delivery
carrier [Luck et al 1998 Kurisawa et al 1995 Yang et al 2000] etc This is because of
3
the well-defined colloidal and surface characteristics of the particles By using a seed
emulsion polymerization method it is possible to synthesize monodisperse latex particles
with various particle size ranges and surface chemistry Functionalized core-shell latex
particles can be introduced by multi-step emulsion polymerization Core particles are
synthesized in the first stage of the polymerization and the functional monomer is added
in the second stage This is done without any emulsifier addition to prevent the
production of new homopolymer particles [Keusch et al 1973] The core-shell particles
are useful in a broad range of applications because of their improved physical and
chemical properties over their single-component counterparts [Lu et al 1996 Nelliappan
et al 1997] Through the development of a hemodialysis membrane using monodisperse
latex particles improvements in advanced separation treatment for patients with end
stage renal disease (ESRD) can be realized This requires the maximum removal of
middle molecular weight proteins with minimal removal of other beneficial proteins such
as albumin Thus an understanding of the fundamental interactions between the particles
and biopolymers is vital to maximize the performance of this membrane technology
The field of material science and biotechnology is based on fundamental chemistry
has developed over the past three decades into todayrsquos powerful discipline that enables
the development of advanced technical devices for pharmaceutical and biomedical
applications This novel and highly interdisciplinary field is closely associated with both
the physical and chemical properties of organic and inorganic particles [Niemeyer 2001]
Hemodialysis membrane using these particles would lead to improvements in the
advanced separation treatment for patients with end stage renal disease (ESRD) The
interdisciplinary nature of this approach enables a more complete understanding of the
4
phenomena of protein adsorption and the material properties necessary for selective
separation This innovative approach to membrane fabrication has the potential of making
inexpensive highly efficient membranes for both industrial and specialized separation
processes
The goal of this study is to prepare polymeric latex particles with tailored
properties to maximize separation of toxin molecules and to investigate the fundamental
interactions between the particles and molecules in the biological system in order to
optimize the performance of a membrane material for these applications
Polymeric latex particles were synthesized with specific functionality in an attempt
to achieve selective separation Seeded emulsion polymerization was used to synthesize
functionalized monodisperse latex particles in various sizes Negatively charged
hydrophobic polystyrene latex particles were synthesized by the same method Core shell
latex particles PSPMMA100 PSPMMA90PAA10 PSPMMA75PAA25 were also
synthesized to differentiate the degree of hydrophobicity of particles Scanning Electron
Micrograph (SEM) and light scattering measurements were used to characterize particle
size and shape and zeta potential measurements were conducted to measure the electrical
surface property of synthesized particles Adsorption isotherms of target proteins bovine
serum albumin (BSA) and β2M on latex particles were obtained as a function of pH
ionic strength and protein concentrations using the BCA assay method Adsorption
kinetics for both proteins on the latex particles were also measured Finally hemolysis
tests were run to determine the biocompatibility of polymer latex particles with human
blood This research will be described in more detail in chapters 3 and 4
CHAPTER 2 BACKGROUND AND LITERATURE SURVEY
21 The Significance of End Stage Renal Disease (ESRD)
The kidneys are responsible for removing excess fluid minerals and wastes from
the blood regulating electrolyte balance and blood pressure and the stimulation of red
blood cell production They also produce hormones such as erythropoietin (EPO) and
calcitriol that keep bones strong and blood healthy [Casadevall and Rossert 2005 Kurbel
et al 2003] EPO acts on the bone marrow to increase the production of red blood cell in
case of bleeding or moving to high altitudes Calcitriol mainly acts on both the cells of
the intestine to promote the absorption of calcium from food and also bone to mobilize
calcium from the bone to the blood When kidneys fail harmful waste builds up blood
pressure rises and body retains excess fluid The body also does not make enough red
blood cells When this happens treatment is needed to replace the function of failed
kidneys
Figure 2-1 An image of the location and cross section of a human kidney
5
6
The National Kidney Foundation estimates that over 20 million Americans had
chronic kidney disease in 2002 [NKF] Chronic renal disease is a gradual and progressive
loss of the function unlike acute renal disease where sudden reversible failure of kidney
function occurs Chronic renal failure usually takes place over a number of years as the
internal structures of the kidney are slowly damaged In the early stages there can be no
symptoms In fact progression may be so gradual that symptoms do not occur until
kidney function is less than one-tenth that of a normal health kidney If left untreated
chronic kidney disease may ultimately lead to kidney failure known as End Stage Renal
Disease (ESRD) ESRD is a rapidly growing heath-care problem in the United States In
2001 approximately 96295 incidents and 406081 prevalent patients were diagnosed
with ESRD including 292215 patients on dialysis and 113866 patients with a
functioning graft [USRDS 2003] The projected number of patients with ESRD is
expected to exceed 22 million patients by 2030 with much of this growth being driven by
the increasing prevalence of major contributing factors such as diabetes and high blood
pressure [USRDS 2003] A great extent of ESRD program cost and Medicare budget
have been spent in 2001 Due in part to a limited availability of kidneys for
transplantation hemodialysis (HD) is the primary method of treatment for ESRD and is
currently used for approximately 61 of US ESRD patients
22 Hemodialysis (HD) Treatment
HD removes toxins from the body by extracorporeal circulation of the blood
through a semipermeable membrane referred to as a dialyzer The toxins are removed
primarily by diffusion across the membrane to a dialysate solution which is circulated on
the opposite side of the membrane The cleaned blood is then returned to the blood
stream A surgically constructed vascular access connects the extracorporeal circuit to the
7
patientrsquos system Treatments are almost always performed three times per week in
specially equipped dialysis facilities for 3-4 hours per each treatment Figure 2-2 shows
the scheme of the hemodialysis route The critical element of a HD instrument is the
semipermeable membrane which allows for selective transport of low molecular weight
biological metabolites from blood
igure 2-2 A schematic draw of the hemodialysis route
ESRD patients who undergo dialysis therapy often experience several other
problems associated with the treatment such as anemia fatigue bone problems joint
problems itching sleep disorders and restless legs Anemia is common in patient with
kidney disease because the kidneys produce the hormone erythropoietin (EPO) which
stimulates the bone marrow to produce red blood cells Diseased kidneys often do not
produce enough EPO to stimulate the bone marrow to make a sufficient amount of red
blood cells This leads to bone disease referred to as renal osteodystrophy and causes
bones to become thin and weak or malformed and can affect both children and adults
Older patients and women who have gone through menopause are at greater risk for this
disease Uremic toxins which cannot be remove from the blood by the current dialyzer
membranes can lead to itching This problem can also be related to high levels of
F
8
parathyroid hormone (PTH) Dialysis patients can also experience day-night revers
is they have insomnia at night and sleep during the day This can be related to possible
nerve damage in the body and a chemical imbalance in the blood due to the excess toxin
Oxidative stress is a problem for patients on maintenance dialysis This problem is due to
an imbalance between pro- and antioxidant factors [Roselaar et al 1995 Cristol et al
1994] Oxidative stress affects oxidation of low-density lipoproteins which are the main
factor for atherogenesis [Huysmans et al 1998] and is also involved in the development
of malignancies and diabetes mellitus [Rice-Evans et al 1993] In order to reduce
antioxidant defense dialysis is needed to contribute to help stimulate free radical
production or eliminate antioxidants Dialysis-related amyloidosis (DRA) is also a
common and serious problem for people who have been on dialysis for more than 5
years DRA develops when proteins in the blood deposit on joints and tendons causi
pain stiffness and fluid in the joint as is the case with arthritis Normally the healthy
kidneys can filter out these proteins but dialysis filters are not as effective
23 Advances in Membrane Technology
al that
s
ng
Dialysis for blood of ESRD
Hemo filtration
s
ysis using a cellulose based membrane occurred
1913 John Abel [1990] from the Johns Hopkins Medical School described a method
purification is widely used in the treatment
dialysis (HD) techniques use a semi-permeable membrane to replace the
role of the kidney The membranes used in HD can be broadly classified into those based
on cellulose and those manufactured from synthetic copolymers These membranes come
in various shapes such as sheets tubular structures or hollow fiber arrangements The
hollow fiber type is the most popular and is incorporated into over 800 different device
in world wide [Ronco et al 2001]
The first attempt at blood dial
9
where sing
e
om
ysis was performed until the mid 1960 The basic
molec
rial in
een
benzyl)
was used for the membrane with
hepar
by the blood of a living animal may be submitted to dialysis outside the body u
a membrane based on cellulose and returned to the natural circulation without exposur
to air infection by microorganisms or any alteration that would necessarily be prejudicial
to life This same technique is still used to today however the device used has been
modified over the years as better membranes were developed and the anti-coagulant
heparin has become available
Cellulose membranes have been widely used for the treatment of renal failure fr
1928 when the first human dial
ular structure of cellulose is made of a long chain containing hydroxyl (OH)
groups The realization that such groups imparted undesirable qualities on the mate
respect to blood contact behavior was discovered in the early 1970s and since has b
the focus of development These modified cellulose membranes used the partial
substitution of benzyl groups to replace the proton of the hydroxyl groups in an attempt
to reduce their negative effect The result is a molecular mosaic of hydrophobic (
and hydrophilic (hydroxyl and cellulose) regions
Kolff [Van Noordwijk 2001] studied the rotating drum artificial kidney for patients
with acute renal failure in 1943 Cellophane tubing
in as the anticoagulant For the next 17 years hemodialysis therapy was performed
by this method but only for the patients with acute reversible renal failure Vascular
access required repeated surgical insertions of cannulas (slender tubes) into an artery and
vein and limited the number of treatments for a patient could receive in order to
minimize the amount of vascular damage
10
Initially the need for dialysis in patients with acute renal failure was determ
mainly by the development of signs and sym
ined
ptoms of uremia After dialysis some time
might
et al
ent improvement In 1960 the development of a shunt [Quinton et al
1960]
fone (PSf) polyamide (PA) and polyacrylonitrile (PAN) by phase inversion or
precip
lidone
e
elapse before uremic manifestations returned to warrant a sequential treatment of
dialysis Many patients with acute renal failure secondary to accidental or surgical
trauma were hypercatabolic but the interdialytic interval might be prolonged because of
anorexia or use of a low-protein diet However Teschan and his coworkers [Obrien
1959] showed that patient well-being and survival were improved by what they termed
prophylactic daily hemodiaysis or administration of the treatment before the patient
again became sick with uremia Their report in 1959 was the first description of daily
hemodialysis
Development of membrane accessories such as a shunt has also been an area of
focus for treatm
a flexible polytetrafluoroethylene (PTFE or Teflonreg) tubing made many more
hemodialysis treatments possible for chronic kidney failure patients PTFE has a non-
stick surface and is relative biocompatibility leading to minimized blood clotting in the
shunt
Synthetic membranes are prepared from engineered thermoplastics such as
polysul
itation of a blended mixture resulting in the formation of asymmetric and
anisotropic structures Figure 2-3 shows a fiber type of the Polyflux S membrane
consisting of poluamide (PA) polyacrylethersulfone (PAES) and polyvinylpyrro
(PVP) with the integral three-layer structure The skin layer on the inside fiber typ
membrane contacts blood and has a very high surface porosity and a narrow pore size
11
distribution This layer constitutes the discriminating barrier deciding the permeabili
and solute retention properties of the membrane The skin layer is supported by thick
sponge-type structure larger pores providing mechanical strength and very low
hydrodynamic resistance
ty
Figure 2-3 Cross-sectional SEM image view of the Polyflux S (polyamide +
polyacrylethersulfone + polyvinypyrollidone) dialysis membranes [Deppisch et al 1998]
n
[Malchesky 2004] because of its thermal stability mechanical strength and chemical
inertn ciated
ls
m
PSf is a widely used membrane material for the hemodialysis applicatio
ess According to a report from the National Surveillance of Dialysis-Asso
Disease (NSDAD) in the US over 70 of hemodialysis membranes were PSf based
[Bowry 2002] This is most likely because PSf has many advantages over other materia
This synthetic polymer is one of few materials that can withstand sterilization by stea
ethylene oxide and γ-radiation PSf membrane can be prepared by conventional
immersion precipitation methods into many different shapes including porous hollow
12
fiber or flat sheet hemodialysis membranes The material also has a high permeab
low molecular weight proteins and solute and high endotoxin retention The chemical
structure of PSf is shown in Figure 2-4
ility to
Figure 2-4 The chemical structure of polysulfone (PSf)
There is one major disadvantage to PSf The hydrophobic nature of the PSf causes
ment alternative pathway
leadin
s not
3
et al
in
O acrylate
serious complications through the activation of the comple
g to the adsorption of serum proteins onto the membranes [Singh et al 2003]
Anticoagulants are added during dialysis therapy to avoid blood clotting but this doe
completely eliminate the problem In order to overcome this disadvantage of the PSf
membrane various studies have been performed to change the materialrsquos surface
properties These investigations include hydrophilic polymer coating [Brink et al 199
Kim et al 1988 Higuchi et al 2003] layer grafting onto PSf membrane [Wavhal
2002 Song et al 2000 Pieracci et al 2002 Mok et al1994] and chemical reaction of
hydrophilic components onto the membrane surface [Higuchi et al 1990 1991 1993
Blanco et al 2001 Nabe et al 1997 Guiver et al 1993] Hydrophilic monomers 2-
hydroxy-ethylmethacrylate (HEMA) acrylic acid (AA) and methacrylic acid (MMA)
have also been grafted onto PSf membrane to increase flux and Bovine Serum Album
(BSA) retention [Ulbricht et al 1996] Hancock et al [2000] synthesized
polysulfonepoly(ethylene oxide) (PEO) block copolymers to improve the resistance to
platelet adhesion Kim et al [2003] also studied blending a sulfonated PE
diblock copolymer into PSf in order to reduce platelet adhesion and enhance
13
biocompatibility PEO is a commonly used biomaterial due to its excellent resistance
protein adsorption and inherent biocompatibility [Harris 1992] Kim et al [20
a self-transformable copolymer to enhance the hydrophilicity of an asymmetric PSf
membrane with an ultra-thin skin layer The polymer had an entrapped diblock
copolymer containing a hydrophilic block of poly (ethylene glycol) (PEG)-SO
to
05] studied
ate
lic graft
l)
e of thier
aromatic
orand
3 acryl
and a hydrophobic block of octadecylacrylate (OA) Molecular dynamic (MD)
simulations were performed as a function of copolymer density to optimize interfacial
structure information McMurry [2004] developed a strategy using an amphiphi
copolymer added to PSf membranes by introducing polysulfone-g-poly (ethylene glyco
When compared to unmodified PSf these graft copolymer and resulting blend
membranes are found to hold promise for biomedical device applications
Polyamide (PA) membranes have also been used for hemodialysis becaus
mechanical strength in both wet and dry conditions Polyamide consists of
aliphatic monomers with amide bonding (-CONH-) also known as a peptide bond
The basic amide bond in polyamide is shown in Figure 2-5 R1 and R2 can be either
aromatic or aliphatic linkage group
C N
H
O
~ R R1 2~C N
H
O
~ R R1 2~
Figure 2-5 The chemical structure of polyamide (PA)
Panichi and co-workers [1998] evaluated the biocompatibility of the PA membrane
ompatible technique due to the
use of a synthetic membrane with a sterile re-infusion fluid and the convective removal of
and concluded that PA hemofiltration was a highly bioc
14
the ac
e
e
is
n
is a semi-crystalline polymer and the mechanical properties
strong
2
CN
tivated anaphylatoxins and β2-Microglobulin (β2M) The PA based membrane
Polyfluxreg (manufactured by Gambro GmbH Germany) blended with polyamide
polyarylethersulfone and polyvinylpyrrolidone (PVP) was able to clean small molecules
such as urea dreatinine and phosphate as well as decrease β2M amount by 502
[Hoenich et al 2002] Due to the non-selectivity of the membrane removal of thes
unwanted materials also led to the undesirable loss of beneficial proteins during therapy
Meier et al [2000] evaluated different immune parameters using a modified cellulos
low-flux hemophan and synthetic high-flux PA membrane during a 1 year period in
chronic hemodialysis patients They found that the 1-year immunological evaluation of
hemodiaysis membrane biocompatibility was associated with changes in the pattern of
chronic T-cell actiovation
Polyacrylonitrile (PAN) is another commonly used membrane material because it
inherently hydrophilic and has been commercialized for ultrafiltration and microfiltratio
[Scharnagl et al 2001] PAN
ly depend on the crystalline structures The chemical structure of PAN is shown in
figure 2-6
CH CH
n2
CN
CH CH n
Chemical structure of polyacrylonitrile (PAN)
The addition of additives such as polyvinylpyrrolidone (PVP) as a pore forming
agent gives PAN membranes more flexible processing parameters and increased
e has been optimized through
copolymerization with many other vinyl monomers including glycidyl methacrylate
Figure 2-6
performance [Jung et al 2005] PAN membrane performanc
15
[Godj
acid
for
for
a non-
solub this
in
oid underneath the inner
surface of membrane decreased by increasing the amount of solvent DMSA in the
evargova et al 1999 Hicke et al 2002] N-vinylimidazole [Godjevargova et al
2000] hydroxyl ethyl methacrylate [Ray et al 1999 Bhat et al 2000] methacrylic
[Ray et al 1999] vinyl pyrrolidone [Ray et al 1999] acrylic acid [Trotta et al 2002]
acrylamide [Musale et al 1997] and vinylchloride [Broadhead et al 1998] These
monomers provide a reactive group for enzyme immobilization improved mechanical
strength solvent-resistance pervaporation permeation flux anti-fouling and bio-
compatibility Because of this PAN-based copolymer membranes have great potential
the treatment of hemodialysis in an artificial kidney This material can also be used
other applications like the treatment of wastewater the production of ultra-pure water
biocatalysis together with separation and methanol separation by pervaporation
Lin and his coworker [2004] studied the modification of PAN based dialyzer
membranes to increase the hemocompatibility by the immobilization of chitosan and
heparin conjugates on the surface of the PAN membrane When a foreign material is
exposed to blood plasma proteins are adsorbed clotting factors are activated and
le fibrin network or thrombus is formatted [Goosen et al 1980] The result of
research was that the biocompatible chitosan polymer and a blood anticoagulant hepar
prevented blood clotting They showed prolonged coagulation time reduced platelet
adsorption thrombus formation and protein adsorption
Nie et al [2004] studied PAN-based ultrafiltration hollow-fiber membranes
(UHFMs) In order to improve the membrane performance acrylonitrile (AN) was
copolymerized with other functional monomers such as maleic anhydride and α-allyl
glucoside They found that the number and size of macrov
16
intern erum
and
d a
rface The second step is the
transf after
yjak et
embrane
1978 Malchesky et al 1978] has been further developed to increase the efficiency of
al coagulant The water flux of the UHFMs also decreased while the bovine s
albumin rejection increased minutely Godjevargova et al [1992] modified the PAN
based membrane with hydroxylamine and diethylaminoethylmethacrylate to improve
membrane dialysis properties Formed functional groups like primary amine oxime
tertiary amine groups provided the membrane with more hydrophilic properties an
substantial increase in the permeability of the membranes
The wide use of filtration in practice is limited by membrane fouling Solute
molecules deposit on and in the membrane in the process of filtration causing dramatic
reduction in flux through the membrane Fouling occurs mostly in the filtration of
proteins Three kinetic steps are involved in the fouling of UF membranes according to
Nisson [1990] The first step is the transfer of solute to the su
er of solute into the membrane until it either finally adsorbs or passes through
a set of adsorption-desorption events The third step includes surface binding
accompanied by structural rearrangement in the adsorbed state [Ko et al 1993] Br
al [1998] studied the surface modification of a commercially available PAN membrane
to develop superior filtration properties with less fouling by proteins The PAN
membrane was immersed in excess NaOH solution to convert some of the surface nitrile
groups into carboxylic groups by the hydrolysis process This modified PAN m
was not so severely fouled in the Bovine Serum Albumin (BSA) filtration test The pore
size however decreased during the hydrolysis process leading to a significant reduction
in flux and made the membrane less productive in the ultrafiltration (UF) mode
24 Sorbent Technology
Over the last three decades sorbent technology [Castino et al 1976 Korshak et al
17
dialysis or replace it for the treatment of ESRD Sorbents remove solutes from solution
through specific or nonspecific adsorption depending on both the nature of the solute and
the sorbent Specific adsorption contains tailored ligands or antibodies with high
selectivity for target molecules een used in autoimmune
disord
and
e
al 1996] various resins [Ronco et al 2001]
album
e
as
Specific adsorbents have b
ers such as idiopathic thrombocytopenic purpura [Snyder et al 1992] and for the
removal of lipids in familial hyper cholesterolemia [Bosch et al 1999] Nonspecific
adsorbents such as charcoal and resins attract target molecules through various forces
including hydrophobic interactions ionic (or electrostatic) attraction hydrogen bonding
and van der Waals interactions
New dialysate with sorbents has become an accepted modification of dialysis
sorbent hemoperfusion is gaining ground as a valuable addition to dialysis especially as
new sorbents are developed [Winchester et al 2001] Hemoperfusion is defined as th
removal of toxins or metabolites from circulation by the passing of blood within a
suitable extracorpoteal circuit over semipermeable microcapsules containing adsorbents
such as activated charcoal [Samtleben et
in-conjugated agarose etc Novel adsorptive carbons with larger pore diameters
have been synthesized for potential clinical use [Mikhalovsky 1989] Newly recognized
uremic toxins [Dhondt et al 2000 Haag-Weber et al 2000] have resulted in several
investigations on alternatives to standard or high-flux hemodialysis to remove thes
molecules These methods include hemodiafilteration with [de Francisco et al 2000] or
without [Ward et al 2000 Takenaka et al 2001] dialysate regeneration using sorbents
well as hemoperfusion using such adsorbents as charcoal and resins
18
Kolarz et al [1989 1995] studied the hyper-crosslinked sorbent prepared from
styrene and divinylbenzene (DVB) for a hemoperfusion application They found that the
pore structure of a swelling sorbent was changed by additional crosslinking with α αrsquo-
dichloro-p-xylene in the presence of a tin chloride catalyst and in a di
chloroethane
soluti and
en
decyl groups that attract β2M through a hydrophobic interaction The
adsor
of
5]
d
penet
on They also realized that the hemocompatibility was useful for nemoperfusion
could be imparted to the sorbents by introducing sulfonyl groups at a concentration of
about 02mmolg
A special polymeric adsorbing material (BM-010 from Kaneka Japan) has be
investigated by another group [Furuyoshi et al 1991] for the selective removal of β2M
from the blood of dialysis partients The adsorbent consists of porous cellulose beads
modified with hexa
ption capacity of this material is 1mg of β2M per 1ml of adsorbent Using a
hemoperfusion cartridge containing 350ml of these cellulose beads in sequence with a
high-flux hemodialyzer several small clinical trials were performed During 4-5 hours
treatment about 210mg of β2M were removed thus reducing the concentration in the
blood by 60-70 of the initial level [Nakazawa et al 1993 Gejyo et al 1993 199
RenalTech developed a hemoperfusion device BetaSorbreg containing the hydrate
cross-linked polystyrene (PS) divinylbenzene (DVB) resin sorbents with a pore structure
designed to remove molecules between 4 and 30 kDa [Winchester et al 2002] In this
case solute molecules are separated according to their size based on their ability to
rate the porous network of the beaded sorbents The resin beads were prepared with
a blood compatible coating and confirmed to be biocompatible in vivo in animals
[Cowgill et al 2001]
19
25 Limitation of Current Hemodialysis Treatment
Hemodialysis is a widely used life-sustaining treatment for patients with ESRD
However it does not replace all of the complex functions of a normal healthy kidne
a result patients on dia
y As
lysis still suffer from a range of problems including infection
accelerated cardio ion anemia
chron
e
se middle
alysis
sessio
s
f
vascular disease high blood pressure chronic malnutrit
ic joint and back pain and a considerably shortened life span One significant
limitation of the current dialysis technology is the inability to efficiently remove larger
toxic molecules This is mainly because of the broad pore size distribution reducing th
selective removal of toxins and unsatisfied biocompatibility causing lots of
complications such as inflammation blood clotting calcification infection etc
Dialysis purifies the patients blood by efficiently removing small molecules like
salts urea and excess water However as toxic molecules increase in size their removal
rate by hemodialysis substantially declines Typically only 10 - 40 of the
molecular weight toxins (300-15000 Da) are removed from the blood during a di
n [Vanholder et al 1995] These toxins then reach an abnormally high level and
begin to damage the body One such toxin β2M causes destructive arthritis and carpal
tunnel syndrome by joining together like the links of chain to form a few very large
molecules and deposit damaging the surrounding tissues [Lonnemann et al 2002] This i
also a main cause of mortality for long-term dialysis patients Other middle molecule
toxins appear to inhibit the immune system and may play a significant role in the high
susceptibility to infections in dialysis patients Still others are believed to impair the
functioning of several other body systems such as the hematopoietic and other endocrine
systems This may contribute to accelerated cardiovascular disease the leading cause o
20
death among dialysis patients as well as clinical malnutrition which affects up to 50 of
this patient population
Over the last decade polymeric dialysis membranes have been developed to
increase the capacity for removing middle molecular weight toxins by changing the pore
size of dialyzer membranes and using new materials that adsorb these toxins for
impro se
styrene made during World War II in the United
States The Dow chemical compan anufacturer of polystyrene
includ
d of
on
rug
ntrol of
ved removal characteristics However removal efficiency is not as high as tho
achieved by a normal healthy kidney
26 Latex Particles
The first synthetic polymer synthesized using emulsion polymerization was a
rubber composed of 13-butadiene and
y has been a major m
ing latex which they used in paint formulations The theory of emulsion
polymerization in which a surfactant is used was established by Harkins [1948] and by
Smith and Ewart [1948] By 1956 the technology was complete including the metho
building larger diameter particles from smaller ones The product by this emulsi
polymerization is referred to as latex a colloidal dispersion of polymer particles in water
medium [Odian 1991] Latexes are currently undergoing extensive research and
development for a broad range of areas including adhesives inks paints coatings d
delivery systems medical assay kits gloves paper coatings floor polish films carpet
backing and foam mattresses to cosmetics The relatively well known and easy co
the emulsion process is one of main advantages for these applications Therefore
polymeric latex particle prepared by emulsion polymerization can be a candidate for the
medical applications because of the easy control of the particle size and morphology as
well as flexible surface chemistry to be required
21
261 The Components for Emulsion Polymerization
The main components for emulsion polymerization process are the monomer a
dispersing medium a surfactant and an initiator Available monomers are styrene
sing medium is usually water
which
ticles
n
ation To
monomer droplets but in micelles because the initiators are
insolu le
and
olution
butadiene methylmethacylate acryl acid etc The disper
will maintain a low solution viscosity provide good heat transfer and allow
transfer of the monomers from the monomer droplets into micelles and growing par
surrounded by surfactants respectively The surfactant (or emulsifier) has both
hydrophilic and hydrophobic segments Its main functions is to provide the nucleatio
sites for particles and aid in the colloidal stability of the growing particles Initiators are
water-soluble inorganic salts which dissociate into radicals to initiate polymeriz
control the molecular weight a chain transfer agent such as mercaptan may be present
262 Particle Nucleation
Free radicals are produced by dissociation of initiators at the rate on the order of
1013 radicals per milliliter per second in the water phase The location of the
polymerization is not in the
ble in the organic monomer droplets Such initiators are referred to as oil-insolub
initiators This is one of the big differences between emulsion polymerization
suspension polymerization where initiators are oil-soluble and the reaction occurs in the
monomer droplets Because the monomer droplets have a much smaller total surface area
they do not compete effectively with micelles to capture the radicals produced in s
It is in the micelles that the oil soluble monomer and water soluble initiator meet and is
favored as the reaction site because of the high monomer concentration compared to that
in the monomer droplets As polymerization proceeds the micelles grow by the addition
of monomer from the aqueous solution whose concentration is refilled by dissolution of
22
monomer from the monomer droplets There are three types of particles in the emulsion
system monomer droplets inactive micelles in which polymerization is not occurring
and active micelles in which polymerization is occurring referred to as growing polymer
particles
The mechanism of particle nucleation occurs by two simultaneous processes
micellar nucleation and homogeneous nucleation Micellar nucleation is the entry of
radicals ei
ther primary or oligomeric radicals formed by solution polymerization from
the aq ed
the
r
s
n
is coagulation with other particles rather than
polymerization of monomer The driving force for coagulation of precursor particles
ueous phase into the micelles In homogeneous nucleation solution-polymeriz
oligomeric radicals are becoming insoluble and precipitating onto themselves or onto
oligomers whose propagation has ended [Fitch et al 1969] The relative levels of micella
and homogeneous nucleation are variable with the water solubility of the monomer and
the surfactant concentration Homogeneous nucleation is favored for monomers with
higher water solubility and low surfactant concentration and micellar nucleation is
favored for monomers with low water solubility and high surfactant concentration It has
also been shown that homogeneous nucleation occurs in systems where the surfactant
concentration is below CMC [Roe 1968] A highly water insoluble monomer such a
styrene [Hansen et al 1979 Ugelstad et al 1979] has probably created by micellar
nucleation while a water soluble monomer such as vinyl acetate [Zollars 1979] has bee
formed by homogeneous nucleation
A third latex formation reaction mechanism has been proposed referred to as
coagulative nucleation In this reaction the major growth process for the first-formed
polymer particles (precursor particles)
23
sever les
yer
es
r a
l
the beginning of the polymerization As soon as the initiator is
gins with the formation and
growt
and
ved
added at one time during the later stages of the polymerization prior to complete
al nanometers in size is their relative instability compared to larger sized partic
The small size of a precursor particle with its high curvature of the electrical double la
permits the low surface charge density and high colloidal instability Once the particl
become large enough in size maintaining the high colloidal stability there is no longe
driving force for coagulation and further growth of particles takes place only by the
polymerization process
263 Types of Processes for Emulsion Polymerization
There are three types of production processes used in emulsion polymerization
batch semi-continuous (or semi-batch) and continuous In the batch type process al
components are added at
added and the temperature is increased polymerization be
h of latex particles at the same time There is no further process control possible
once the polymerization is started In the semi-continuous emulsion polymerization
process one or more components can be added continuously Various profiles of particle
nucleation and growth can be generated from different orders of component addition
during polymerization There are advantages to this process such as control of the
polymerization rate the particle number colloidal stability copolymer composition
particle morphology In the continuous process the emulsion polymerization components
are fed continuously into the reaction vessel while the product is simultaneously remo
at the same rate High production rate steady heat removal and uniform quality of
latexes are advantages of the continuous polymerization processes
Other available methods include the intermittent addition and shot addition of one
or more of the components In the shot addition process the additional components are
24
conversion of the main monomer This method has been used successfully to develo
water-soluble functional monomers such as sodium styrene sulfonat
p
e [Kim et al 1989]
264
lsion
Chemistry of Emulsion Polymerization
Emulsion polymerization is one type of free radical polymerization and can be
divided into three distinct stages initiation propagation and termination The emu
polymerization system is shown in Figure 2-7
Aqueous phaseAqueous phase
Micelle withmonomer
I Rbull
Emulsifier
Polymer particle swollenWith monomer
Monomerdroplet
I Rbull
Micelle withmonomer
I Rbull
Emulsifier
Polymer particle swollenWith monomer
MonomerdropletMonomerdroplet
I Rbull
Figure 2-7 Emulsion polymerization system [Radicals (Rmiddot) are created from initiators
(I) Monomer is transferred from larger monomer droplet into micelles by emulsifier Initiated polymer particle by radicals is keep growing until monomers are all consumed The reaction is performed in aqueous media]
In the initiation stage free radicals are created from an initiator by heat or an
ultraviolet radiation source The initiator with either peroxides groups (-O-O-) such as
odium persulfate or azo groups (-N=N-) such as azobisisobutyronitrile is commonly
used for em t
with the m er
chain ere
s
ulsion polymerization The primary free radicals created from initiator reac
onomer for initiation of polymerization In the propagation stage the polym
grows by monomer addition to the active center a free radical reactive site Th
are two possible modes of propagations head-to-head addition and head-to-tail addition
25
The head-to-tail mode is the predominant configuration of the polymer chain a result o
steric hindrance of the substitute bulky group In the termination stage polymer chain
growth is terminated by either coupling of two growing chains forming one polymer
molecule or transferring a hydrogen atom (dispropotionation) from one growing chain to
another forming two polymer molecules one having a saturated end group and the other
with an unsaturated end-group
265 Seed Emulsion Polymerization
Polymer latex particles have received increasing interest because of the versatili
of the many applications heterophase polymerization processes like emulsion dispersion
micro-emulsion seeded emulsion precipitation etc Especially to prepare well-defined
microspheres having monodispe
f
ty
rse and various particle sizes as well as surface group
ed particles prepared by emulsion polymerization
and e
s
hological design In
seede to control
n
functionalilties it is necessary to use se
nlarge them to a desired size in the further stages of reactions
Polymeric particles are required to have a uniform particle size in many
applications such as chromatography where they are used as a packing material
Morphological control of latex particle is also important for many practical application
[Schmidt 1972] Seed emulsion polymerization (or two-stage emulsion polymerization) is
a useful method to achieve both monodisperse particle size and morp
d emulsion polymerization [Gilbert 1995] preformed lsquoseedrsquo latex is used
the number of particles present in the final latex The advantage of seeded emulsio
polymerization is that the poorly reproducible process of particle nucleation can be
bypassed so that the number concentration of particles is constant and known Various
mechanisms have been proposed for the growth of latex particles [Ugelstad et al 1980
Okubo et al 1992] using this polymerization technique The initial seed particle
26
preparation step is well known and relatively easy to perform because at the relative
small particle sizes (02 to 05 micron) the particle growth process can be readily
controlled by the use of an emulsifier Enough emulsifier is used to prevent coagulation
but not enough to cause the formation of new particles As the particles are grown to
larger sizes in successive seeding steps it becomes increasingly difficult to mainta
stable uncoagulated emulsion without forming new particles and thereby destroying
monodisperisty of the latex Recently there have been several reports concerning t
reaction kinetics of seed emulsion polymerization and the development of latex
morphologies over the course of the reaction [Chern et al 1990 Delacal et al 1990 L
et al 1995 Lee 2000 2002]
266 Polystyrene Latex Particles
A number of papers have described the synthesis of the polystyrene latex particl
bearing various functional surface groups such as carboxyl [Lee et al 2000 Tun
2002 Reb et al 2000] hydroxyl [Tamai et al 1989] marcapto [Nilson 1989] epoxy
[Shimizu et al 2000 Luo et al
ly
in a
he
ee
es
cel et al
2004] acetal [Izquierdo et al 2004 Santos et al 1997]
ometyl [Izquierdo et al 2004 Park et al 2001
Sarob
h the
d
thymine [Dahman et al 2003] chlor
e et al 1998] amine [Counsin et al 1994 Ganachaud et al 1997 Ganachaud et al
1995 Miraballes-Martinez et al 2000 2001 Anna et al 2005] ester [Nagai et al 1999]
etc In order to produce these functionalized particles different methods for particle
preparation must be used The polymer emulsion with core-shell morphology of latex
particles is one of them This is a multistep emulsion polymerization process in whic
polystyrene ldquocorerdquo particle is synthesized in the first stage and the functional monomer is
added in the second stage of the polymerization without any emulsifier postfeeding to
prevent the production of new homopolymer particles thus forming the functionalize
27
polymer ldquoshellrdquo on the ldquocorerdquo particle [Keusch et al 1973] There are requirements to
limit secondary nucleation and encourage core-shell formation in seeded emulsion
polymerization including the addition of smaller seed particles at high solid content to
increase particle surface area low surfactant concentration to prevent formation of
micelles and the semi-continuous addition of monomer to create a starved-feed conditio
and keep the monomer concentration low There are some advantages [Hergeth et al
1989] of dispersions with polymeric core-shell particles First it is possible to modi
interfacial properties of polymer particles in the aqueous phase by the addition of only
very small amounts of a modifying agent during the last period of the reaction Thus
these core-shell particles are useful in a broad range of applications since they always
exhibit improved physical and chemical properties over their single-component
counterparts [Lu et al 1996 Nelliappan et al 1997] In this way the improvement of
surface properties of such dispersions is straightforward and inexpensive The other is
that polymers with a core-shell structure are perfect model systems for investigating th
material properties of polymer blends and composites because of their regular
distribution of one polymer inside a matrix polymer and because of the simple sp
geometry of the system
Their properties usually depend on the structures of latex particles Chen and his
coworkers [Chen et al 1991 1992 1993] reported the morphological development of
core shell latex particles of polystyrenepoly(methyl methacrylate) during
polymerization Before the research by Chen and his coworker Min et al [Min et al
1983] reported the morph
n
fy the
e
herical
ological development of core shell latex of polystyrene
(PS)polybuthyl acrylateat (PBA) by seeded emulsion polymerization as a function of the
28
additi
of
ity of graft
ing reagents Thus protein adsorption on polymeric solid surfaces has
st are microspheres defined as ldquofine
polym can be
n
r was
containing antigen urine serum etc The latex will become agglutinated and visibly
on method of PS They found that the percentage of grafting PS to the PBA was
greatest for the batch reaction and the PBA-PS core-shell particles with a high degree
grafting remained spherical upon aging test because of the emulsifying abil
copolymer
267 Various Applications of Latex Particles
Latex particles are applicable to a wide range of areas such as biomedical
applications [Piskin et al 1994] especially as the solid phase such as in immunoassays
[Chern et al 2003 Radomske-Galant et al 2003] DNA diagnostic drug delivery carriers
[Luck et al 1998 Kurisawa et al 1995 Yang et al 2000] blood cell separations and
column pack
become a center of attention Of particular intere
er particles having diameters in the range of 01 to several micronsrdquo which
used as functional tools by themselves or by coupling with biocompounds Singer and
Plotz [1956] firstly studied microsphere or latex agglutination test (LATrsquos) by using
monodisperse polystyrene (PS) and polyvinyltoluene polymer particles as the support o
which the biomolecules were going to adsorb The biomolecule adsorption howeve
limited by possible desorption of the adsorbed species or loss of specific activity of the
complex formed Since this work was published the latex particle applications for
immunoassay have been rapidly and widely studied and developed
Latex agglutination test or latex immunoassays start with tiny spherical latex
particles with a uniform diameter and similar surface properties The particles are coated
with antibodies (sensitized) through the hydrophobic interaction of portions of the protein
with the PS surface of the particles If sensitized particles are mixed with a sample
29
agglomerate Latex tests are inexpensive as compared with the other techniques [Ba
1988]
ngs
sed
tion Rheumatoid factor (RF) in different age subpopulations has also been
evalu
studied a
latex
Unipath [Percival 1996] has manufactured a range of immunoassay materials ba
on a chromatographic principle and deeply colored latex particles for use in the home and
clinical environments The latex particles which are already sensitized with a
monoclonal antibody can detect any antigens that bound to the surface of the latex
particles Some detectable pollutants include estrogen mimics which induce
abnormalities in the reproductive system of male fishes and lead to a total or partial male
feminiza
ated according to a patientrsquos clinical status by using a rapid slide latex agglutination
test for qualitative and semiquantitative measurement in human serum along with latex
immunoassay method [Onen et al 1998] Magalhaes and his coworkers [2004]
diagnostic method of contamination of male fishes by estrogen mimics using the
production of vitellogenin (VTG) as a biomarker This was based on a reverse
agglutination test developed with monoclonal antibodies specific to this biomarker
Premstaller and his coworkers [Premstaller et al 2000 2001] have prepared a porous
poly(styrene-divinylbenzene) (PS-DVB) polymer monolith to use for highly efficient
chromatographic separation of biomelecules such as proteins and nucleic acids They
used a porogen a mixture of tetrahydrofuran and decanol to fabricate a micropellicular
PS-DVB backbone Legido-Quigley and his colleagues [2004] have developed the
monolith column to obtain further chromatographic functionality to the column by
introducing chloromethylstyrene in place of styrene into the polymer mixture
30
Core shell type monodisperse polymer colloids have been synthesized by Sarobe
and Forcada [1998] with chloromethyl functionality in order to improve the biomolecu
adsorption through a two-step emulsion polymerization process They investigated the
functonalized particles by optimizing the experimental parameters of the functional
monomer including reaction temperature the amount and type of redox initiator sys
used the type of addition of the initiator system and the use of washing They c
le
tem
oncluded
that th
sis
serve as separation
chann
of
usually
along the polypeptide chain which contain positive or negative charges [Norde 1998]
e relation between the amount of iron sulfate and the persulfatebisulfite system
added should be controlled to obtain monodisperse particles and prevent the premature
coagulation of the polymer particles during the polymerization
A semi-continuous emulsion polymerization technique for latex particle synthe
was performed by McDonald and other researchers [Ramakrishnan et al 2004 Steve et
al 1999] They introduced a variety of particles sizes compositions morphologies and
surface modifications to fabricate latex composite membranes (LCMs) Arraying and
stabilizing latex particles on the surface of a microporous substrate form narrowly
distributed interstitial pores formed between the particles which
els They investigated the membrane performance using gas fluxes water
permeability and the retention characterization of dextran molecules From these tests
they concluded that the narrow discriminating layer made of the latex particles leads to a
highly efficient composite membrane
27 Proteins
Proteins are natural polyamides comprised of about 20 different α-amino acids
varying hydrophobicity [Norde 1998] Proteins are more or less amphiphilic and
highly surface active because of the number of amino acid residues in the side groups
31
Proteins are polymers of L-α-amino acids [Solomon and Fryhle 2000] The α refers to a
carbon with a primary amine a carboxylic acid a hydrogen and a variable side-chain
group designated as R Carbon atoms rent groups are asymmetric and can
exhib l
s
e
on
with four diffe
it two different spatial arrangements (L and D configurations) due to the tetrahedra
nature of the bonds The L refers to one of these two possible configurations Amino
acids of the D-configuration are not found in natural proteins and do not participate in
biological reactions Figure 2-8 shows the chiral carbon in 3-D as the L isomer Protein
consist of twenty different amino acids differentiated by their side-chain groups [Norde
1998] The side-chain groups have different chemical properties such as polarity charg
and size and influence the chemical properties of proteins as well as determine the
overall structure of the protein For instance the polar amino acids tend to be on the
outside of the protein when they interact with water and the nonpolar amino acids are
the inside forming a hydrophobic core
Figure 2-8 L-α-amino acid
The covalent linkage between two amino acids is known as a peptide bond A
peptide bond is formed when the amino group of one amino acid reacts with the carboxyl
group of another amino acid to form an amide bond through the elimination of water
This arrangement gives the protein chain a polarity such that one end will have a free
amino group called the N-terminus and the other end will have a free carboxyl group
called the C-terminus [Solomon and Fryhle 2000] Peptide bonds tend to be planar and
32
give the polypeptide backbone rigidity Rotation can still occur around both of the α-
carbon bonds resulting in a polypeptide backbone with different potential conformations
relative to the positions of the R groups Although many conformations are theoretically
possible interactions between the R-groups will limit the number of potential
nd to form a single functional conformation In other words
the conformation or shape of the protein is
α-helix
can interact with other
secondary structures within the sam
conformations and proteins te
due to the interactions of the chain side
groups with one another and with the polypeptide backbone The interactions can be
between amino acids that are close together as in a poly-peptide between groups that are
further apart as in amino acids or even on between groups on different polypeptides all
together These different types of interactions are often discussed in terms of primary
secondary tertiary and quaternary protein structure
The primary amino acid sequence and positions of disulfide bonds strongly
influence the overall structure of protein [Norde 1986] For example certain side-chains
will promote hydrogen-bonding between neighboring amino acids of the polypeptide
backbone resulting in secondary structures such as β-sheets or α-helices In the
conformation the peptide backbone takes on a spiral staircase shape that is stabilized by
H-bonds between carbonyl and amide groups of every fourth amino acid residue This
restricts the rotation of the bonds in the peptide backbone resulting in a rigid structure
Certain amino acids promote the formation of either α-helices or β-sheets due to the
nature of the side-chain groups Some side chain groups may prevent the formation of
secondary structures and result in a more flexible polypeptide backbone which is often
called the random coil conformation These secondary structures
e polypeptide to form motifs or domains (ie a
33
tertiary structure) A motif is a common combination of secondary structures and a
domain is a portion of a protein that folds independently Many proteins are composed o
multiple subunits and therefore exhibit quaternary structures
271 Interaction Forces between Proteins
Proteins in aqueous solution acquire compact ordered conformations In such a
compact conformation the movement along the polypeptide chain is severely restrict
implying a low conformational entropy The compact structure is possible only if
interactions within the protein molecule and interactions between the protein molecule
and its environment are sufficiently favorable to compensate for the low conformational
entropy [Malmsten 1998] Protein adsorption study is often focused on structural
rearrangements in the protein molecules because of its significance to the biological
functioning of the molecules and the important role such rearrangements play in the
mechanism of the adsorption process Knowledge of the majo
f
ed
r interaction forces that act
structures helps to understand the behavior
of pro
arge
a
ded
between protein chains and control the protein
teins at interface These forces include Coulomb interaction hydrogen bonds
hydrophobic interaction and van der Waals interactions
Coulombic interaction Most of the amino acid residues carrying electric ch
are located at the aqueous boundary of the protein molecule An the isoelectric point
(IEP) of the protein where the positive and negative charges are more or less evenly
distributed over the protein molecule intramolecular electrostatic attraction makes
compact structure favorable to proteins Deviation to either more positive or more
negative charge however leads to intramolecular repulsion and encourages an expan
structure Tanford [1967] calculated the electrostatic Gibbs energy for both a compact
impenetrable spherical molecule (protein) and a loose solvent-permeated spherical
34
molecule (protein) over which the charge is spread out From the results he found th
the repulsion force was reduced at higher ionic strength d
at
ue to the screening action of ion
ble
e
the
d the
tween
dehyd
e
ent
oles
tein
Hydrogen bond Most hydrogen bonds in proteins form between amide and
carbonyl groups of the polypeptide backbone [Malmsten 1998] The number of availa
hydrogen bonds involving peptide units is therefore far greater than that involving sid
chains Because α-helices and β-sheets are aligned more or less parallel to each other
interchain hydrogen bonds enforce each other Kresheck and Klotz [1969] examine
role of peptide-peptide hydrogen bonds and concluded that hydrogen bonds between
peptide units do not stabilize a compact structure of protein However because the
peptide chain is shielded from water due to other interactions hydrogen bonding be
peptide groups do stabilize α-helical and β-sheet structures
Hydrophobic interaction Hydrophobic interaction refers to the spontaneous
ration and subsequent aggregation of non-polar components in an aqueous
environment In aqueous solutions of proteins the various non-polar amino acid residues
will be found in the interior of the molecule thus shielded from water The
intermolecular hydrophobic interaction for the stability of a compact protein structure
was first recognized by Kauzmann [1959] If all the hydrophobic residues are buried in
the interior and all the hydrophilic residues are at the outermost border of the molecul
intramolecular hydrophobic interaction would cause a compact protein structure
However geometrical and other types of interactions generally cause a fraction of the
hydrophobic residues to be exposed to the aqueous environm
Van der Waals interaction The mutual interaction between ionic groups dip
and induced dipoles in a protein molecule cannot be established as long as the pro
35
structure is not known in great detail Moreover the surrounding aqueous medium also
contains dipoles and ions that compete for participation in the interactions w
the protein molecule Dispersion interactions favor a compact structure However
because the Hamaker constant for proteins is only a little larger than that of water the
resulting effect is relatively small [Nir 1977]
272 β
ith groups of
rgiles et al 1996
Floeg
) is degraded [Floege 2001] Renal
β2M from the serum resulting in an increase in β2M
conce well
the
)
a
al
2-Microglobulin (β2M)
The protein β2M is of particular interest because it is involved in the human
disorder dialysis-related amyloidosis (DRA) [Geiyo et al 1985 A
e 2001] DRA is a complication in end stage renal failure patients who have been on
dialysis for more than 5 years [Bardin et al 1986 Drueke 2000] DRA develops when
proteins in the blood deposit on joints and tendons causing pain stiffness and fluid in
the joints as is the case with arthritis In vivo β2M is present as the non-polymorphic
light chain of the class I major histocompatibility complex (MHC-I) As part of its
normal catabolic cycle β2M dissociates from the MHC-I complex and is transported in
the serum to the kidney where the majority (95
failure disrupts the clearance of
ntration by up to 60-fold [Floege 2001] By a mechanism that is currently not
understood β2M then self-associates into amyloid fibrils and typically accumulates in
musculoskeletal system [Homma et al 1989] Analysis of ex vivo material has shown
that the majority of amyloid fibrils in patients with Dialysis Related Amiloidosis (DRA
is present as of full-length wild-type β2M although significant amounts (~20-30) of
truncated or modified forms of the protein are also present [Floege 2001 Bellotti et al
1998] Figure 2-9 shows the ribbon diagram of human β2M Native β2M consists of
single chain of 100 amino acid residues and has a seven stranded β-sandwich fold typic
36
of the immunoglobulin superfamily [Saper et al 1991 Trinh et al 2002] β2M was fi
isolated from human urine and characterized by Berggard et al [1980] in1968 The
normal serum concentration of β
rst
re
f
e
2M is 10 to 25 mgL It is a small globular protein with
a molecular weight of 118 kDa a Strokes radius of 16Aring and a negative charge under
physiological conditions (isoelectric point IEP = 57) β2M contains two β-sheets that a
held together by a single disulphide bridge between the cysteines in positions 25 and 81
[Berggard et al 1980 Parker et al 1982 Cunningham et al 1973] β2M cannot be
removed completely by current dialysis techniques but through a better understanding o
the structure and interaction forces that lead to this structure it will be possible to mor
efficiently remove this problematic protein
Figure 2-9 Ribbon diagram of human β M taken from the crystal structure of the protein
bound to the heavy chain of the MHC class I complex (PDB 1DUZ) [Khal 2000]
273 Serum Albumin
Serum albumin is the most abundant protein found in plasma and is typically
present in the blood at a concentration of 35~
2an et
50gL According to extensive studies about
its physiological and pharmacological properties albumin has a high affinity to a very
wide range of materials such as electrolytes (Cu+2 Zn+2) fatty acids amino acids
metabolites and many drug compounds [Fehske et al 1981 Kraghhansen 1981 Putnam
37
1984 Peters 1985] The most important physiological role of the protein is therefore to
bring such solutes in the bloodstream to their target organs as well as to maintain the pH
and osmotic pressure of the plasma Bovine serum albumin (BSA) is an ellipsoidal
protein with the dimensions of 140 X 40 X 40Aring [Peter 1985] The primary structure is a
single helical polypeptide of 66 kDa (IEP = 47) with 585 residues containing 17 pairs of
as a soft an
rfaces [Kondo et al 1991 Norde et al 1992 Soderquist et al
1980 ikely
is
disulfide bridges and one free cysteine [Dugaiczyk et al 1982] BSA has been classified
d flexible protein because it has a great tendency to change its conformation
on adsorption to solid su
Carter and Ho 1994] and consists of three homologous domains (I-III) most l
derived through gene multiplication [Brown 1976] Each domain is composed of A and B
sub-domains [He et al 1992] The secondary structure of human serum albumin (HSA)
shown in Figure 2-10
Figure 2-10 Secondary structure of human serum albumin (HSA) with sub-domains
[Zunszain et al 2003]
HSA has the same structure domains with the serum albumin from other species
such as BSA [Brown 1976] Although all three domains of the albumin molecule have
similar three-dimensional structures their assembly is highly asymmetric [Sugio et al
38
1999] Domains I and II are almost perpendicular to each other to form a T-shaped
assembly in which the tail of subdomain IIA is attached to the interface region between
sub-domains IA and IB by hydrophobic interactions and hydrogen bonds In contrast
domain III protrudes from sub-domain IIB at a 45ordm angle to form the Y-shaped assembly
for domains II and III Domain III interacts only with sub-domain IIB These features
make the albumin molecule heart-shaped
28 Protein Adsorption
Protein adsorption studies date back to the 1930rsquos At the beginning these studies
mainly focused on the determination of the molecular weight electrophoretic and
ral
rearrangem
adsorption and biocompatibility of the sorbent materials were investigated [Norde 1986]
There are interaction forces at the interfaces between protein molecules and latex
particles These forces are mainly divided into the following groups hydrophobic
interaction ionic interaction hydrogen bonding and van der Waals interaction [Andrade
1985]
Hydrophobic interaction It is known that hydrophobic interaction has a major
role in protein adsorption phenomena The adsorption of proteins on the low charged
latex particles occurs by this interaction force Generally monomers such as styrene offer
hydrophobic surfaces th
by this interaction force is maximum at the isoelectric point (IEP) of the protein and the
strength [Suzawa et al 1980 1982 Shirahama et al 1989 Kondo et al 1992] By the
chromatographic applications Later the adsorption mechanism especially the structu
ents was studied Recently the studies of the relation between protein
281 Interaction between Protein Molecule and Latex Particle
at protein molecules adsorb to The amount of adsorbed protein
pH at maximum adsorption shifts to a more acidic region with an increase in ionic
39
reports [Suzawa et al 1980 1982 Lee et al 1988] protein adsorption was greater
hydrophobic surface than on a hydrophilic one implying that hydrophobic interaction is
on a
s
ydrophilic compound such as acrylic acid (AA) or methacrylic acid (MAA)
Ionic
ely
t
ces
n force is operative over small
distan e
ble
n
nt dialysis membrane therapy for end stage renal disease (ESRD)
one of most dominant forces in protein adsorption
Ionic interaction Negatively charged latex particles have ionic functional
groups on their surfaces such as salts of sulfonic and carboxylic acid Sulfate group
originate from an initiator such as sodium persulfate and carboxylic groups originate
from a h
bonds are formed between the negative charges of these latex particles and positive
surface charges of protein molecules The conventional low-charged latex particles rar
form these ionic bonds
Hydrogen bonding Hydrogen bond is a strong secondary interatomic bond tha
exists between a bound hydrogen atom (its unscreened proton) and the electrons of
adjacent atoms [Callister 1999] Protein can be adsorbed on hydrophilic polar surfa
through hydrogen bonding Hydrogen bonds are frequently formed between hydroxyl-
carbonyl or amide-hydroxyl Hydroxyl-hydroxyl or amide-hydroxyl bonds are also
formed in protein adsorption
Van der Waals interaction This interactio
ces only when water has been excluded and the two non-polar groups come clos
to each other Lewinrsquos calculation showed that the van der Waals interaction is negligi
compared with the forces involved in the entropy increases ie hydrophobic interactio
[Lewin 1974]
29 Hypothesis for Toxin Removal
As mentioned earlier insufficient removal of middle molecular range toxins is a
major drawback of curre
40
patien
is
y
ese
haracteristics over the last decade However removal
effici
ing a novel membrane design composed of an assembly of engineered
polymeric latex particles s ions Important factors
will i
e
ith a
is established it is
expec
e
ts This may cause destructive arthritis and carpal tunnel syndrome inhibit the
immune system and accelerate cardiovascular disease leading to death among dialys
patients In order to overcome these complications artificial dialysis membranes have
been developed to increase the capacity for removing middle molecular weight toxins b
changing the pore size of dialysis membranes and using new materials that adsorb th
toxins for improved removal c
ency is not as high as those achieved by a normal healthy kidney
With the knowledge obtained from all above literatures the following hypothesis
has been established for the development of a membrane for the successful removal of
the target protein β2-Microglobulin (β2M) without the removal of serum albumin This
will be done us
ynthesized to predetermined specificat
nclude pore size and surface chemistry
291 Toxin Removal by Size Sieving Based on Monodispersed Pore Size
The packing of monodispersed spherical particles can lead to the formation of
porous layers suitable for use as filters and membranes [Hsieh et al 1991] The pore siz
is defined as the largest spherical particles that can pass through the interstitial spaces
It is well known that monodispersed spherical particles can be obtained from the
seed emulsion polymerization method Many commercially available latex particles are
synthesized by this technique and are inexpensive When the defect free membrane w
monodisperse pore size distribution based on a particular particle array
ted to outperform the traditional hemodialysis membrane limitation which is a
broad range of pore size distribution When spherical particles pack in regular crystallin
arrays a number of packing geometries such as hexagonal closest packing cubic closest
41
packing and body-centered cubic packing are possible The pore size of the array
depends on these packing geometries as well as particle size Theoretical pore size can be
calculated from the simple geometry of the arr
ay Figure 2-11 shows the relationship
between particle size and pore size
0001
001
01
001 01 1 10
Por
e
1
10
microm
) S
ize
(
Particle Diameter (microm)
Body-Centered Cubic ArrayClosest Packed Array
0001
Siz
e (
Particle Diameter (microm)
001
01
001 01 1 10
Por
e
1
10
microm
)
Body-Centered Cubic ArrayClosest Packed ArrayBody-Centered Cubic ArrayClosest Packed Array
and closed packed arrays [Steve et al 1999]
McDonald and his coworkers [Ramakrishnan et al 2004 Steve et al 1999] have
described the fabrication of latex composite membranes and their performance in
previous papers It is from this prior research and knowledge gained from conversations
with Dr McDonald that this current research has been initiated [McDonald 2003] The
focus of this thesis is the extension of McDonaldrsquos research in the area of composite
membranes to medical application such as dialysis membranes
292 Toxin Removal by Selective Adsorption on Engineered Latex Particles
The flexibility in latex surface properties is a significant advantage to this
materialrsquos ability to separate proteins since the selectivity is strongly dependent on the
charge interactions of both the protein and the latex particles under the conditions of
Figure 2-11 The relationship between pore size and particle size in body-centered cubic
42
separation [Menon et al 1999 Chun et al 2002] First all sorbents were designed to
have negative surface properties because most plasma proteins in blood are negative and
should not be removed with charge interaction between proteins and sorbents at
physiological condition (pH74) It was reported that a negatively charged surface was
more blood compatible than a positive one [Srinivasan et al 1971] The toxin protein β2-
Microglobulin (β2M) can be selectively adsorbed on the surface of latex particles by the
design of a suitable hydrophobic portioned surface where only β2M protein can be
anchored with hydrophobic interactions Albumin adsorption on the latex particle is not
side-on m
adsorption of β M protein on the engineered latex particles
allowed because charge repulsion is more dominant than hydrophobic interaction with
ode The figure 2-12 shows the schematic representation of the selective
2
PMMAPAAShell
COO-
OSO3-
OSO3-
PSCore
COO-
-
-
-
-
-
-
Albumin-
Chargerepulsion
--
-
- -
- -
- --
Hydrophobic
β2-Microglobulin
2
Interaction
β -Microglobulin
PMMAPAAShell
COO-
-
-
-
-
-
-
--
-
-
-
-
-
Albumin-
Chargerepulsion
OSO3-
OSO3-
PSCore
COO-
--
-
- -
--
-
- -
- -
- --
- -
- --
Hydrophobic
β2-Microglobulin
2
particles at pH 74
Interaction
β -Microglobulin
Figure 2-12 Schematic representation of the protein adsorption on the core shell latex
43
Hydrophilichydrophobic microdomain structures were proven to be more blood
compatible [Mori et al 1982 Higuchi et al 1993 Deppisch et al 1998] The
optimization of suitable hydrophobic to hydrophilic ratio is also important for the
biocompatibility The monomers such as styrene (St) methyl methacrylate (MMA) and
acrylic acid (AA) are widely used hydrophobic and hydrophilic monomer models in
emulsion polymerization process
In summary the background and fundamental literature survey about the history
material properties and limitation of hemodialysis membrane latex particle preparation
surface chemistry and manufacturing process target proteins and protein adsorption and
finally the hypothesis for a toxin removal with high separation efficiency have been
suggested This research focuses on such hypothesis and the materials and
characterization methodology for achievement of suggested hypotheses are described in
the next chapter
CHAPTER 3 EXPERIMENTAL AND CHARACTERIZATION METHODOLOGY
As mentioned earlier the goal of this study is to prepare polymeric latex particles
with tailored properties to maximize separation of toxin molecules and to investigate
fundamental interactions between the applied particles and molecules in the
system to optimize the membrane performance for hemodialysis applications Polymeric
latex particles wit
the
biological
h monodisperse size distribution to obtain uniform pore size and
various size ranges to utilize membrane construction are necessary Surface engineering
with various combination of hydrophobichydrophilic domain on the surface of latex
particles is expected to affect the removal of target protein by selective adsorption and to
improve the biocompatibility of membrane Therefore the materials and characterization
methodology are addressed in this chapter
31 Materials
Styrene (St) monomer used for a seed and a core particle was purchased from
Fisher Scientific and used without any other purification process Acrylic acid (AA) and
methyl methacrylate (MMA) monomers introduced for shell formation were purchased
from Fisher Scientific and used without any other purification process Sodium persulfate
(SPS) and sodium bicarbonate (SBC) were obtained from Fishers Scientific and used as
received Divinylbenzene (DVB) crossliking agent was purchased from Aldrich The
anionic surfactant Aerosol regMA80-I [sodium di(13-dimethylbutyl) sulfosuccinate] was
kindly donated by Cytec 78-80 Aerosol regMA80-I is mixed with isopropanol and
water Its critical micelle concentration (CMC) is 245 mM (119) Ion exchange resin
44
45
AGreg 501-x8 Resin (20-50 mesh) was purchased from Bio-Rad Laboratories Inc It is a
mixed-bed resin with H+ and OH- as the ionic forms and is 8 crosslinked Bovine serum
albumin (BSA) (heat shock treated) was purchas Fisher Scientific The isoelectric
point of t lin
(β2M)
as
gma
ed from
he BSA is 47-49 and the molecular weight is 66300Da β2-Microglobu
was purchase from ICN Biomedicals Inc and was separated from patients with
chronic renal disease and lyophilized from ammonium bicarbonate The molecular
weight by SDS-PAGE was approximately 12000Da BSA and β2M proteins were used
received without further purification process In order to investigate the protein
adsorption evaluation bicinchoninic acid protein assay kits were purchased from Si
(Cat BCA-1) and Pierce Biotechnology (Cat 23225) The chemical structures for the
main chemicals are shown in Figure 3-1
CH2 CH
Styrene (Mw = 10415)
CH2 CHCH2 CH
Styrene (Mw = 10415)
CH2 CH
Acrylic acid (Mw = 7206)
C
OH
O
CH2 CH
Acrylic acid (Mw = 7206)
C
OH
O
CH CH2
C
OCH3
O
CH
CH CH3
2
C
OCH3
O
CH3
Methyl methacrylate (Mw = 10012)Methyl methacrylate (Mw = 10012)
CH2 CH
Divinylbenzene (Mw = 1302)
CH CH2
CH2 CH
Divinylbenzene (Mw = 1302)
CH CH2CH CH
+(Na) -(O3S)
CH -C-O-CH-CH -CH-CH3
O
2 2 3
O CH CH3
CH3 CH3
CH-C-O-CH-CH2-CH-CH3
Anionic surfactant (MA80-I) (Mw = 3885)
+(Na) -(O3S)
CH -C-O-CH-CH -CH-CH3
O
2 2 3
O CH CH3
CH3 CH3
CH-C-O-CH-CH2-CH-CH3
Anionic surfactant (MA80-I) (Mw = 3885)2
+(Na)-(O) S OO
O O
- +
Initiator sodium persulfate (Mw = 23811)
O SO
(O) (Na)+ -
O O
- +(Na) (O) S OO
O SO
(O) (Na)+ -
O O
- +
Initiator sodium persulfate (Mw = 23811) Figure 3-1 Chemical structure of main chemicals
(Na) (O) S OO
O SO
(O) (Na)
46
32 Latex Particle Preparation
The experimental set up and the particle preparation schemes for various types and
size ranges of latex particles are shown in Figure 3-2 and 3-3 In the experimental setup a
mechanical glass stirrer is connected from a motor and Teflon stirrer bar is located at
end of glass stirrer The agitation rate is precisely adjusted by controller motor Paar glas
reactor with 1L volume is partially in silicon oil bath on hot plate The reactor has fo
openings for a stirrer a thermometer a nitrogen gas inlet and a reactant feeding lnlet
The reactants for latex polymerization are fed by precisely controlled by meterin
The particle preparation is described in each latex preparation section
the
s
ur
g pump
Figure 3-2 Experimental setup for semi-continuous emulsion polymerization
1 Motor 2 Mechanical stirrer bar 3 Thermometer 4 Inlet of N2 5 Inlet for initiator and monomer 6 Glass reactor (l L) 7 Oil bath 8 Hot plate 9 Pump for initiator 10 Pump for monomer 11 Tubes for reactant feed
47
Styrene monomer
Bare PS
PSPMMA100
PSPMMA90PAA10
PSPMMA P75 AA25
Gr
win
ticle
size
PS seedparticles
og
par
Styrene monomer
Bare PS
PSPMMA100
PSPMMA90PAA10
PSPMMA P75 AA25
Gr
win
ticle
size
PS seedparticles
og
par
Figure 3-3 The particle preparation scheme in various types and size ranges of latex particles
321 Preparation of Seed Latex Particles
Polystyrene seed particles were synthesized through a typical semi-continuous
emulsion polymerization Polystyrene latex particles were prepared in a four necked Paar
glass vessel equipped with a mechanical glass stirrer thermometer glass funnel for
nitrogen gas purge and a tube for monomer feeding The reactor was placed in a silicon
oil bath on a hot plate for homogenous and stable tempe he vessel was
firstly charged with de-ionized water the emulsifier so itiator
sodium bicarbonate (SBC) styrene (St) monomer divinylbenzene (DVB) monomer
under nitrogen gas atmosphere and then heated to 72plusmn2 as
llowed to continue for another hour Second reactants St and DVB monomers SPS
initiator and SBC were fed separately using fine fluid metering pumps (Model RHSY
Fluid metering INC NY) under a nitrogen gas atmosphere for 2h After complete feeding
rature control T
dium persulfate (SPS) in
oC for 1h The reaction w
a
48
of all reactants the reaction was continued for another hour at elevated temperature 80plusmn1
oC and then cooled to room temperature by removal of the oil bath
322 Preparation of Seeded Latex Particles
The particle growth was achieved by seeded continuous emulsion polymerization
The vessel was charged with the polystyrene seeds and de-ionized water to make desired
solid content and heated up to 72plusmn2 oC for 1h under nitrogen gas atmosphere Then St
and DVB monomers and solution of SPS and SBC in de-ionized water were continuously
added using metering pump with a precisely controlled feed rate After feeding all
reactants the reaction was continued for three hours keeping temperature of 80plusmn2 oC and
then the reaction vessel was cooled down to room temperature
Monodisperse spherical polystyrene seeds with a 280nm mean diameter were used
e-polymerized polystyrene seeded emulsion
was charged in a 500m
323 Preparation of Core Shell Latex Particles
for core shell structured latex particle The pr
l glass flask under a nitrogen gas atmosphere De-ionized water
was added to achieve the desired solid content The emulsion was heated to 80plusmn2oC for
10 hour Monomers initiator and SBC to form the shell were added by fluid metering
pumps (LAB PUMP JRRHSY model Fluid metering Inc USA) with fine controlled
feed rate Flow rate can be adjusted by control ring graduated in 450 division from 0 to
100 flow which is in the range of 10mlmin respectively The reaction was sustained
for additional 30 hours at 90plusmn2oC then cooled down to room temperature
324 Purification of Synthesized Latex Particles
Synthesized latex particles were purified with an ion exchange resin 200g (10
ww polystyrene solid content) of emulsion were mixed with 4g of ion exchange resin
and stirred for 40 minute to remove unreacted monomers initiators and other impurities
49
Then the emulsion was diluted with DI water to a specific degree of solid content as
needed
33 Characterization
331
solid
termined
oscopy
d
hours
Dried latex (10mg) was mixed with
250 m on
Degree of Conversion
The degree of monomer to polymer conversion was determined gravimetrically
following the procedure described by Lee et al [1995] before the latex cleaning process
The synthesized emulsion raw materials were weighed and dried in a conventional oven
at 120oC for 30min to evaporate any unreacted monomers and water The remaining
was weighed and the degree of conversion was determined This process was repeated
three times and a mean value of the degree of conversion was de
332 Fourier Transform Infrared (FTIR) Spectr
A molecule can absorb only select frequencies (energy) of infrared radiation which
match the natural vibration frequencies The energy absorbed serves to increase the
amplitude of the vibrational motions of the bonds in the molecule [Pavin et al 1996]
Only those bonds which have a dipole moment that changes as a function of time can
absorb the infrared radiation The range of wavelengths for infrared radiation is between
25 microm (4000 cm-1) and 25 microm (400cm-1)
The synthesis of the latex particles such as polystyrene (PS) homopolymer and
PSPMMA PSPMMA90PAA10 PSPMMA75PAA25 core-shell copolymers were verifie
with FTIR Purified latex particles were dried in vacuum oven at 40oC for 24
before analysis in the FTIR (Nicolet Magma USA)
g of KBr which had also been dried in vacuum oven at 120oC for 3h Transmissi
spectra using the drift mode were plotted with 128 scans and 4cm-1 resolution
50
333 Quasielastic Light Scattering (QELS)
Latex particle size was measured by the Brookhaven ZetaPlus particle size
analyzer The solid concentration as 0001 (wv) This
ation spectroscopy (PCS) of quasielastically scattered light
For th s
ed using a FE-SEM (JEOL
JSM-
ith
ent
01
us zeta-
potential analyzer It measures the electrophoretic mobility velocity of charged colloidal
of latex particles used w
instrument uses photon correl
is technique time dependent interference patterns of light scattered from particle
in the sample cell are analyzed The interference pattern changes due to Brownian
motion giving rise to fluctuations in scattering intensity The frequency of these
fluctuations depends on the particlersquos diffusion constant that is inversely proportional to
the particle diameter
334 Field Emission-Scanning Electron Microscopy (FE-SEM)
Particle size and surface morphology were characteriz
6335F Japan) A diluted suspension of latex particles in water was dropped onto a
silicon wafer at room temperature and allowed to dry The sample was then coated w
the thinnest layer of carbon needed to obtain the required conductivity for this instrum
Secondary electron image mode was used with a 15KV of accelerating voltage The
magnification range used was between 10000X and 70000X
335 Zeta Potential Measurement
Synthesized and purified latex particles were diluted with de-ionized water to 0
wt and adjusted six different pH values 205 345 481 565 643 and 747 These
particle suspensions were then transferred to standard cuvettes for zeta potential
measurement At least two runs of ten measurements were taken for each sample and
averaged
The zeta potential measurement was carried out using Brookhaven ZetaPl
51
particles in solution and calculate zeta-potential by using the Smoluchowski equation
The f quency
gnitude
umin (BSA) as a standard model protein and β2-Microglobulin
uffer (PB) and
phosp
5
h the selected protein
e 005 01 03 05 and 07 mgml and those for
β2M
d at
teins in the supernatant after the centrifugation process using the
bicinc t
requency of laser light passing through the sample and is compared to the fre
of a reference beam The shift in the frequency called a Doppler shift and the ma
of the shift correspond to the polarity and the magnitude of the electrophoretic mobility
respectively The zeta potential is calculated from the solution conditions and the
measured mobility
336 Protein Adsorption
Protein adsorption experiments of the synthesized latex particles were performed
with bovine serum alb
(β2M) as a target protein in two types of buffer solution phosphate b
hate buffered saline (PBS) To make 5 mM of PB solution 0345g of sodium
phosphate monobasic (NaH2PO4H2O) was dissolve in 500ml of de-ionized water To
make PBS solution 0345g of sodium phosphate monobasic (NaH2PO4H2O) and 4178g
(143mM) of sodium chloride (NaCl) were dissolved in 500ml of de-ionized water The
synthesized latex particles were diluted with each buffer to have a solids content of 0
(ww) adjusted to pH values of 32 48 and 74 and mixed wit
BSA concentrations were chosen to b
were 0015 0030 0045 and 0060mgml The mixture was gently rotated using
the shaker Labquakereg (BarnsteadThermolyne Model 4002110 USA) with 8 RPM in
the incubator at 37oC for 12 hours before the latex-protein mixture was centrifuge
13000 rpm for 15min The amount of protein adsorbed was determined by quantifying
the free pro
honinic acid (BCA) assay method [Lowry et al 1951 Smith et al 1985 Baptista e
al 2003 Wiehelman et al 1988 Brown et al 1989] BCA assay kits consist of Reagent
52
A containing bicinchoninic acid sodium carbonate sodium tartrate and sodium
bicarbonate in 01N NaOH with pH=1125 and Reagent B containing 4 (wv) copper
(II) sulfate pentahydrate The BCA working reagent was prepared by mixing 50 parts of
Reagent A with 1 part of Reagent B 100microl of protein supernatant was then mixed with
2ml of BCA working reagent in a UV cuvette Incubation was allowed to continue
at room temperature
further
until color developed about 2h The absorbance at 562nm was
pectrophotometer (Perkin-Elmer Lambda 800 USA)
Unkn
Separate the p rticles in centrifuge mix supernatant with BCA reagents
measured using a UV-VIS s
own concentration of sample protein was determined by comparison to a standard
of known protein concentrations The adsorbed amount per unit surface area was
determined by the mass balance of the protein after adsorption process The simple
schematic of the procedure for a protein adsorption test is shown in Figure 3-4
Incubate mixture with agitation for 12hrs at 37oC
aand develop the color by incubation for 2hrs at room temperature
Suspend latex particles in aqueous buffer media
Add the dissolved target protein
Measure color intensity using UVVis spectroscopy at 562nm
Suspend latex particles in aqueous buffer media
Separate the p rticles in centrifuge mix supernatant with BCA reagents
Add the dissolved target protein
Incubate mixture with agitation for 12hrs at 37oC
aand develop the color by incubation for 2hrs at room temperature
Measure color intensity using UVVis spectroscopy at 562nm
Figure 3-4 Schematic of the procedure for a protein adsorption test
53
337 Blood Biocompatibility by Hemolysis Test
Hemolysis is the destruction of red blood cells which leads to the release of
hemoglobin from within the red blood cells into the blood plasma Hemolysis testing is
used to evaluate blood compatibility of the latex particles and the damaged to the red
blood cells can be determined by monitoring amount of hemoglobin released The red
blood cells (RBCs) were separated by centrifuging blood at 1500 rpm for 15 minutes
Figure 3-5 shows the phase separated blood with plasma white blood cells and red
cells after centrifugation process Separated RBSs were then washed with isotonic
phosphate buffer solution (PBS) at pH 74 to remove debris and serum protein Th
process was repeated 3 times
blood
is
Figure 3-5 Separation of RBC from whole blood by centrifuge process
Prepared latex particles were re-dispersed in PBS by sonification to obtain
homogeneously dispersed latex particles 100microl of the mixture of red blood cell (3 parts)
and PBS (11parts) was added to 1ml of 05 (ww) particle suspension PBS was used as
a negative control resulting 0 hemolysis and DI water used as a positive control to
produce 100 hemoglobin released by completely destroyed RBCs The mixture was
incubated in water bath with gentle shaking for 30 minutes at 37oC and then centrifuged
at 1500 rpm for 15 minutes 100microl of the supernatant was mixed with 2ml of the mixture
) (EtOHHCl = 2005 ww) to prevent the
White blood cells
Plasma
Red blood cells
of ethanol (99) and hydrochloric acid (37
54
precipitation of hemoglobin In order to remove remaining particles the mixture was
centri fuged again with 13000 RPM at room temperature The supernatant was then
transferred into the UV cuvette The amount of hemoglobin release was determined by
monitoring the UV absorbance at a wavelength of 397nm The schematic of the
procedure for hemolysis test is shown in Figure 3-6
Separate red blood cells (RBCs) from blood
Mix RBCs with phosphate buffer solution (PBS)
Wash and re-suspend RBC with PBS repeat three times
Add la l of RBC and incu in at 37oC
tex particle suspension into 100mbate sample with agitation for 30m
Separate particle ith EtOHHCl solutions and mix the supernatant w
Evaluate free hemoglobin in the solution using UVVis spectroscopy at 397nm
Separate red blood cells (RBCs) from bloodSeparate red blood cells (RBCs) from blood
Mix RBCs with phosphate buffer solution (PBS)Mix RBCs with phosphate buffer solution (PBS)
Wash and re-suspend RBC with PBS repeat three timesWash and re-suspend RBC with PBS repeat three times
Add latex particle suspension into 100ml of RBC and incubate sample with agitation for 30min at 37oC
Add latex particle suspension into 100ml of RBC and incubate sample with agitation for 30min at 37oC
Separate particles and mix the ith EtOHHCl solution supernatant wSeparate particles and mix the ith EtOHHCl solution supernatant w
Evaluate free hemoglobin in the solution using UVVis spectroscopy at 397nmEvaluate free hemoglobin in the solution using UVVis spectroscopy at 397nm
Figure 3-6 Schematic of the procedure for hemolysis test
In summary a description of the experimental materials and characterization
methodology has been explained In the next chapter the results and discussion of the
data obtained from these experiments is addressed
CHAPTER 4 RESULTS AND DISCUSSION
41 Polymerization of Latex Particles
411 Polystyrene Seed Latex Particles
Polymer particles have many different applications including spacer particles
lubricants packing materials for chromatography standard particles and diagnostic
drugs All applications strongly require these particles to have a uniform particle size
Uniform particle size is the first requirement for a latex composite membrane formed by
particle arrays with the interstitial spaces serving as pores for size discriminations [Jons
et al 1999 Ramakrishnan et al 2004] Although suspension polymerization has been
mainly used as one of conventional polymerization methods to prepare uniform particles
the uniformity of the recovered particles is insufficient for membrane use There is
another method known as seed emulsion polymerization where a vinyl monomer is
absorbed into fine monodiserse seed particles and polymerization causes the monomer to
crease the sizes of the seed particles uniformly In order to obtain monodisperse larger
particles by this m
particles and polymerization of the monomer is repeated
In this study cross-linked polystyrene seed latex particles were firstly synthesized
in the presence of styrene monomer divinylbenzene crosslink agent sodium persulfate
initiator sodium bicarbonate buffer and the Aerosol regMA80-I [sodium di(13-
dimethylbutyl) sulfosuccinate] surfactant using a batch type semi-continuous emulsion
polymerization process in which one or more components can be added continuously
in
ethod the procedure of absorption of monomer into fine polymer
55
56
Styrene (St) monomer and divinylbenzene (DVB) crosslink agent as well as initiator and
buffer are continuously added to enlarge the seed latex particles Various profiles of
particle nucleation and growth can be gene different orders of component
addition during polymerizati f this process such as easy
control of the polymeriza particle number
and particle morphology The main goal in
this process is to avoid secondary particle
rated from
on There are several advantages o
tion rate monodisperse particle size and
colloidal stability copolymer composition
formation leading to a monodisperse particle
size distribution Figure 4-1 shows the schematic representation of seed latex particle
preparation and growth
Monomer droplet
Seed particles Amount of surfactantsis variable
Enlarged final Add monomers
PS
PS
PSs4
particles Number of particlesare constant
Seed code
PSs1
s2
s3
Monomer droplet
Seed particles Amount of surfactantsis variable
Enlarged final Add monomers
PS
PS
PSs4
tion The
particles Number of particlesare constant
Seed code
PSs1
s2
s3
Figure 4-1 Schematic representation of semi-continuous seed latex particles preparation and growth
After the first synthesis of a small size of crosslinked polystyrene (PS) seed
particles stabilized by surfactants styrene DVB and initiator are continuously fed into
the system to enlarge the seed latex particles maintaining a narrow size distribu
recipes and reaction conditions for synthesizing these seed particles are summarized in
57
Table 4-1 The original recipe for latex particle preparation from McDonald was
modified for this emulsion system [McDonald 2003]
Table 4-1 The polymerization recipe of polystyrene (PS) seed particles Latex label PSS259 PSS233 PSS207 PSS181
Temperature (oC) 75 plusmn 5 75 plusmn 5 75 plusmn 5 75 plusmn 5
Divinylbenzene (g) 052 052 052 052
MA 80 (g) 259 233 207 181
NaHCO
Watera (g) 1315 1315 1315 1315
Styrene (g) 9948 9948 9948 9948
112 112 112 112
Na2S2O8 (g) 190 190 190 190
Waterb (g) 200 200 200 200
Reaction time (min) 140 140 140 140
Solids content 396 396 396 396
Mean particle diameter (nm) 126plusmn75 171plusmn39 182plusmn41 216plusmn53
Conversion () 965 979 957 971
3 (g)
Four different seed latex particles were synthesized based on surfactant amount in
order to determine the optimum condition for stable and monodisperse latex particles
The emulsion polymerization of latex particles has to be carried out in a narrow range of
surfactant causes flocculation or phase separation leading to broad particle
size distribution and an unstable emulsion sy
surfactant concentration where particles are stable [Nestor et al 2005] A wide range of
concentrations
stem Therefore the range of surfactant
amount was varied from 181g to 259g in increments of 026g in each latex label Other
reactants amounts were kept constant Watera is the initial charge amount and waterb
represents the amount of aqueous solution containing dissolved initiator and buffer
58
which are fed continuously after the seed particle creation The extent of monome
polymer conversion was obtained by gravimetric calcu
r to
lation and was more than 95 at
cripts such as
S207 and S181 i the surf mount added for the seed latex
nversion indi perce experi obtain
content divided by the theoretically estimated solid content and was calculated by the
all seed particles labeled PSS259 PSS233 PSS207 and PSS181 Labeled subs
S259 S233 ndicate actant a
preparation The co cates the ntage of mentally ed solid
following formula
100 colids () times=entsoliatedntentoalExperimentConversion
n the experimenta content is the weight of olid rema after
mulsion at 120oC f in and the estimated solid content is th ht
lated from the recipe
rti as di each t load d
mple of ea pension iluted by (ww)
deionized water and the mean particle diameter was determined using the Brookhaven
ZetaPlus particle size analyzer From these results the seed latex particle size increased
as the amount of surfactant decreased The amount of emulsifier also affects the
polymerization process by changing the number of micelles and their size It is known
that large amount of emulsifier produces larger numbers of smaller sized particles [Odian
1991] and was corroborated in the current work
These synthesized seed latex particles should be of narrow size distribution in order
to obtain monodisperse larger particles The SEM image showing the particle
morphology and size of a sample of polystyrene seed particles can be seen in the Figure
4-2
contdEstim (4-1)
In this equatio l solid the s ining
evaporation of e or 30m e weig
of monomers calcu
As expected the seed pa cle size w fferent for surfactan ing in see
emulsion system A sa ch sus was d 0001 with
59
Figure 4-2 Scanning Electron Micrograph (SEM) of polystyrene seed latex particles (A)
PSS259 (B) PSS233 (C) PSS207 (D) PSS181 (subscripts indicate the amount of surfactant added)
(A)
(B)
60
(C)
(D)
Figure 4-2 Continued
61
s seen in the image the seed particles are very smooth and spherical The
uniformity in the particle size distribution of these PS seed latex particles can also be
seen in the SEM image From this data we concluded that seed particles synthesized in
the presence of a surfactant with the chosen amounts are suitable for use to prepare larger
particles with high size uniformity and smooth spherical morphology
412 The Growth of Polystyrene (PS) Seed Latex Particles
Seeded emulsion polymerization has been conducted for several decades and
various mechanisms have been proposed Gracio et al [1970] suggested that the growing
polymer particles consist of an expanding polymer-rich core surrounded by a monomer-
rich shell with the outer shell providing a major locus of polymerization Seeded
mulsion polymerization is commonly used for preparing latex particles less than 1microm in
size [Cha et al 1995 Zou et al 1990 1992 Gandhi et al 1990 Park et al 1990] Such
latexes can be obtained from the growth of pre-prepared seed particles The seeds
introduced in this process serve as nucleation sites for particle growth By first swelling
the seed latex particles with additional monomer to the desired size polymerization can
then be initiated Several size ranges of monodisperse polystyrene (PS) latex particles
were prepared by this multistep seeded emulsion polymerization method For the
fabrication of particle-based membranes monodisperse latex particles with a variety of
size ranges are required
In this study seed particles from 171nm to 470nm in mean diameter and with a
highly uniform size distribution were prepared and used Watera is the initial charge
mount and waterb represents the amount aqueous solution containing dissolved initiator
and buffer that is continuously fed into system Feed time is precisely controlled by
metering pump The solid contents were maintained between 199 and 300 After
A
e
a
62
polym d as
es
ere 923 8402
n of these latex particles
are sh
ss
erization under these conditions the conversions of the latex particles labele
PS258 PS320 PS370 and PS410 were 952 957 946 and 964 respectively The recip
used in seeded emulsion polymerization to form the particles less than 500nm in mean
diameter are shown at the Table 4-2 The conversions of the latex particles larger than
500nm in mean diameter and labeled as PS525 PS585 PS640 and PS790 w
723 and 969 respectively The recipes used in the preparatio
own at the Table 4-3
A simple calculation can be used to predict the ultimate size that a latex particle can
reach after ending the polymerization Letrsquos suppose that seed density (ρs) and grown
particle density (ρgp) are the same The following equation is obtained from the ma
balance (m = ρV) where m and V are mass and volume of a particle respectively
gp
gp
s
s
Vm
Vm
In this equation m
= (4-2)
be
s and mgp are the weight of seed and total weight of seed and
added monomers respectively and Vs and Vgp are the volume of seed and grown
particle respectively
If we know the mass of monomer to be added the ultimate particle size can
calculated from the equation 4-3
3
2s D m ⎠⎝+ χ
where D
1s D m
⎟⎟⎞
⎜⎜⎛
= (4-3)
the
in
s and Dgp are the diameters for seed and grown particle respectively and χ is
weight of the monomer added Crosslinked PS latex particles with a size range from
258plusmn112 ~ 410plusmn117 in diameter polymerized by the recipes in Table 4-2 are shown
the SEM images in Figure 4-3
63
Table 4-2 Continuous addition emulsion polymerization recipe for growing polystyrene
Latex label PS
(PS) latex particles less than 500nm in size
258 PS320 PS370 PS410
Initial charge
Seed latex (g) 1000171 1000216 1000216 1000320
Watera (g) 1470 2500 1984 2600
06 07 06 06
Fee
Initiator stream
2 2 8
Feed time (min) 0-167 0-140 0-100 0-92
Solid content 284 300 300 200
Mean particle diameter (nm) 258plusmn112 320plusmn154 182plusmn71 410plusmn117
Conv
Monomer continuous
Styrene (g) 800 1200 800 600
Divinylbenzene (g)
d time (min) 0-163 0-110 0-75 0-82
Na S O (g) 07 065 05 05
NaHCO3 (g) 07 065 05 05
Waterb (g) 750 1200 200 370
ersion () 952 957 946 964 The solid content of seed latex for PS258 PS320 PS 370 and PS410 are 392 399 399 anrespectively
d 300
64
Table 4-3 Continuous addition emulsion polymerization recipe for growing polystyrene (PS) latex particles larger than 500nm in size
Latex label PS525 PS585 PS640 PS790
Initial charge
Seed latex (g) 1000390 1000470 1000470 1000320
Watera (g) 1000 520 1200 360
Monomer continuous
Styrene (g) 290 200 400 173
Divinylbenzene (g) 02 014
03 009
Feed time (min) 0-80 0-40 10-76 5-65
Initiator stream
Na2S2O8 (g) 02 014 03 012
NaHCO3 (g) 02 014 03 009
Waterb (g) 200 120 400 200
Feed time (min) 0-85 0-46 0-77 0-75
Solids content 199 200 200 201
Mean particle diameter (nm) 525plusmn151 585plusmn186 640plusmn178 790plusmn693
Conversion () 923 8402 723 969 The solid content of seed latex for PS525 PS585 PS 640 and PS790 are 200 182 193 and 173 respectively
65
Figure 4-3 SEM of PS latex particles less than 500nm in size (A) 258nm (B) 320nm (C)
370nm (D) 410nm
(A)
(B)
66
(C)
(D)
Figure 4-3 Continued
67
article size was well matched to that estimated by the geometrical calculation
From this it can be concluded that the added monomer was consumed for polymer
particle growth with a high degree of conversion From the SEM characterization newly
nucleated particles were not seen as indicated by the narrow particle size distribution
The particles less than 500nm in mean diameter were very spherical in shape with a
highly uniform particle size distribution As the particles were grown larger than 500nm
mean diameter however they became of non-spherical shape with an uneven surface
This irregularity of particle surface can be attributed to the non-homogeneous monomer
swelling into the shell of a growing polymer which can be controlled by the factors such
as temperature agitation speed initiator feeding rate and surfactant amount required to
stabilize the colloidal system The SEM image of latex particles larger than 500nm mean
iameter is shown in Figure 4-4 The particle size distribution is broader than that of the
s particles These particles can however still be used for membrane construction
as a support layer rather than skin layer The support layer does not necessarily have to be
as highly monodisperse as the skin layer which require pores of high uniformity for the
selective removal of toxins
As mentioned earlier the amount of surfactant as well as monomer in an emulsion
system is the primary determinant of the particle diameter [Odian 1991] Anionic
surfactants are generally recommended at the level of 02-30 wt based on the amount
of water [Odian 1991] The critical micelle concentration (CMC) of Aerosolreg MA 80-I
anionic surfactant is 119 The surfactant loading in this emulsion system ranged from
the 121 for SS181 to 171 for SS259
P
d
maller
68
Figure 4-4 SEM of PS latex particles larger than 500nm in size (A) 525nm (B) 585nm
(C) 640nm (D) 790nm
(A)
(B)
69
(C)
(D)
Figure 4-4 Continued
70
The surfactant to monomer ratio refers to the value of the surfactant amount
divided by monomer amount after polymerization ends The surfactant amount is the
weight of the surfactant contained in emulsion and the monomer amount is the total
weight of the monomer added for polymerization to form the ultimate polymer particles
in emulsion As the surfactant to monomer ratio decreases mean particle diameter
increases as described by Odian [1991] Generally lower surfactant concentration forms
fewer micelles resulting in larger particle size [Evans et al 1999] However there is a
plateau in particle mean diameter between a surfactant to monomer ratio of 0005 and
0013 This is due to low polymerization conversion of latex particles from monomer
leading to a smaller particle size than expected This is not a factor of surfactant amount
The dependence of the particle size on the amount of surfactant is shown in Figure 4-5
0
200
400
600
800
1000
0000 0005 0010 0015 0020 0025 0030
Surfactant to monomer ratio (ww)
Part
icle
mea
n di
amet
er (n
m)
Figure 4-5 Dependence of the particle size on the surfactant to monomer ratio
71
413
e
f bare
microdomain structures is important for biocompatibility [Mori et al 1982 Higuchi et al
1993 Deppisch et al 1998] Figure 4-6 shows the schematic of core shell latex particle
structure The recipe for core shell structures is shown in Table 4-4
Core Shell Latex Particles
The latex particles shown in Figure 4-3 (A) and labeled as S258 in Table 4-2 wer
used as seeds to prepare core-shell latex particles because of their high particle
uniformity and smooth surface morphology Methyl methacrylate (MMA) and acrylic
acid (AA) monomers were introduced to increase surface hydrophilicity over that o
polystyrene particles AA is a more hydrophilic monomer than MMA because of the
carboxyl acid group in AA is more favorable for hydrogen boding with water The
surface carboxyl groups on PAA may have many promising applications in biomedical
and biochemical fields [Kang et al 2005] MMA monomer is less hydrophobic than
styrene PMMA is known to more biocompatible than PS but still PMMA is hydrophobic
The optimization of suitable hydrophobic to hydrophilic ratios and control of
Polystyreneseed particle
Core shelllatex particles
PSPMMA100
PSPMMA90PAA10
PSPMMA75PAA25
Polystyreneseed particle
Core shelllatex particles
PSPMMA100
PSPMMA90PAA10
Figure 4-6 Schematic of core shell latex particle structures
PSPMMA75PAA25
72
Table 4-4 The preparation recipe of PS core with various shell latex particles Latex 25 label PSPMMA100 PSPMMA90PAA10 PSPMMA75PAA
Initial charge
PS seed latex (g) 500 500 500a
Monomer continuous
Methly methacylate (g) 320 288 240
Acrylic acid (g) 00 32 80
Initiator stream
Na
258 258 258
Water (g) 1570 1570 1570
Feed time (min) 0-120 0-50 0-60
b 200
Feed time (min) 0-122 0-60 0-65
952
2S2O8 (g) 02 02 02
NaHCO3 (g) 02 02 02
Water (g) 200 200
Solids content 183 183 183
Mean particle diameter (nm) 370plusmn186 370plusmn178 370plusmn191
Conversion () 955 924
Three types of core shell structures were prepared PMMA75PAA25 shell is a
copolymer consisting of the PMMA to PAA ratio of 75 to 25 by weight
PMMA90PAA10 shell is a copolymer with the PMMA to PAA ratio of 90 to 10 by
weight PMMA100 is the PMMA homopolymer shell on PS core The particle size of
these core-shell particles as well as PS latex particle was prepared to be about 370nm in
mean diameter and are characterized to determine their zeta potential protein adsorption
and biocompatibility The image of SEM of PS and core shell particles is shown at Figure
4-7 The synthesized latex particles had a high uniformity in size and a smooth spherical
surface morphology
73
Figure 4-7 Scanning
Electron Micrograph of latex particles (A) PS (B) PSPMMA100 (C) PSPMMA90PAA10 (D) PSPMMA75PAA25
(A)
(B)
74
(C)
(D)
Figure 4-7 Continued
75
42 Characterization of Latex Particles
421 Fourier Transform Infrared Spectroscopy (FTIR)
FTIR spectroscopy has proven to be a powerful tool to characterize polymeric
materials A molecule can absorb only select frequencies (energy) of infrared radiation
which match the natural vibration frequencies The energy absorbed serves to increase
the amplitude of the vibrational motions of the bonds in the molecule [Pavin et al 1996]
Only those bonds which have a dipole moment that changes as a function of time can
absorb infrared radiation The FTIR spectra of polymerized latex particles made during
this work are shown in Figure 4-8 There are number of characteristic peaks for PS latex
particles [Bhutto et al 2003 Li at al 2005] Aromatic C-H stretching vibration is shown
at 3002 cm-1 and 3103 cm-1 and aliphatic C-H asymmetrical and symmetrical stretching
shown at 2900 cm-1and 2850 cm-1 respectively There is an additional carbonyl (C=O)
adsorption at 1730cm-1 wavenumber for all core shell latex particles containing some
amount of either or both PMMA and PAA The broad OH group peak at 3400 cm-1
appear for particles containing some amount of PAA and has intensity dependent on the
amount of PAA in the shell For example the peak intensity of OH groups for
PSPMMA75PAA25 is greater than that of PSPMMA90PAA10 since there is a higher AA
monomer content in PSPMMA75PAA25 than in PSPMMA90PAA10 This OH peak is not
seen for PS and PS core PMMA shell particles because of the absence of OH groups on
PS and PMMA polymer chains
422 Zeta Potential Measurements
The electrical surface property was characterized by zeta potential measurements of
tex particles Zeta potential is an important and useful indicator of surface charge which
can be used to predict and control the stability of colloidal systems The greater the zeta
is
la
76
1520002500300035004000
Wavenumbers (cm-1)
li
Re
ativ
e
Tra
nsm
ttanc
e
Figure 4-8 FTIR spectra of polymerized latex particles (A) ba
PSPMMA100 (C) PSPMMA90PAA10 (D) PSPMM
(B)
(C) P
(D) P
C
C
OCH3
O
CH3
XCH2
CH2 n
CH
CH2 C
C
OCH3
O
XCH2 CH
OC
CH3
Y
OH
(A) Bare PS
500100000
re polystyrene (PS) (B) A75PAA25
PSPMMA100
SPMMA90PAA10
SPMMA75PAA25
77
potential the more likely the suspension is to be stable because the charged particles repel
one another and thus overcome the natural tendency to aggregate The measurement of
zeta potential is often the key ontrol of the interaction of proteins
with solid surfaces such as latex particles
Zeta potential (ζ) is the elec at exists at the shear plane of a particle
which is some small distance from the surface [Myers 1999] The zeta potential of the
particles can be determined by measuring the mobility of the particles in an applied
electric field termed electr its response to an alternating electric
field Colloidal particles dispersed in lu ically charged due to their ionic
characteristics and dipolar attributes The development of a net charge at the particle
surface affects the distribution of ions in the neighboring ng in
an increased concentration of counter ions to the surface Each particle dispersed in a
ns called fixed layer or Stern layer
Outside th compositions of ions of opposite polarities
form ble layer is form
particle-liquid interface This double layer may be considered to consist of two parts an
inner region which inclu nd relatively strongly to the surface and an outer or
diffuse region in which the ion distribution is determined by a balance of electrostatic
forces and random thermal motion The potential in this region therefore decays with the
distance from the surface until at a certain distance it becomes zero Figure 4-9 shows a
schematic representation of a typical ion distribution near a positively charged surface
The Stern surface (also referred to as the outer Helmholtz plane) is drawn through the
to understanding and c
trical potential th
ophoretic mobility or
a so tion are electr
interfacial region resulti
solution is surrounded by oppositely charged io
e Stern layer there are varying
ed in the region of the ing a cloud-like area Thus an electrical dou
des ions bou
center of those ions which are effectively adsorbed to the surface [Hunter 1981] The
78
extent of ion adsorption is determined by electrical and other long-range interactions
between the individual ions and surface of particles The ions outside of the Stern layer
form the diffuse double layer also referred to as the Gouy-Chapman layer [Burns and
Zydney 2000]
Shear plane
Stern layer
ψζ
0
ψs
ψShear plane
Stern layer
ψζ
0
ψs
ψ
t
controlled electric field is applied via electrodes immersed in a sample suspension and
Figure 4-9 Schematic representation of ion distribution near a positively charged surface
Zeta potential is a function of the surface charge of a particle any adsorbed layer a
the interface and the nature and composition of the surrounding medium in which the
particle is suspended The principle of determining zeta potential is very simple A
79
this causes the charged particles to move towards the electrode of opposite polarity
Viscous forces acting upon the moving particle tend to oppose this motion and the
equilibrium is rapidly established between the effects of the electrostatic attraction and
the viscosity dra
g The particle therefore reaches a constant terminal velocity
Figure 4-10 Schematic representation of zeta potential measurement(source
httpnitioncomenproductszeecom_shtm)
Because protein adsorption mechanisms are very complex and hard to explain in
biological systems protein adsorption is generally studied by using more ideal systems
consisting of one or more well characterized proteins a well-characterized adsorbent and
a well-defined aqueous solution Even so small changes in the experimental conditions
such as pH ionic strength and temperature generate totally different results Therefore
two media systems phosphate buffer (PB) and phosphate buffered saline (PBS) were
chosen to be used for the zeta potential measurements and protein adsorption study PB is
a simple aqueous system of 5 mM monobasic sodium phosphate (NaH2PO4H2O) to
n a constant pH PBS is used to keep the water concentration inside and outside of
the cell balanced If the water concentration is unbalanced the cell risks bursting or
shriveling up because of a phenomenon called osmosis PBS is a solution with the 5 mM
of monobasic sodium phosphate (NaH2PO4H2O) and 143 mM of sodium chloride (NaCl)
in water
maintai
80
The results of zeta potential measurement at room temperature (25plusmn1oC) as
function of pH and ionic strength (different media PB and PBS) for synthesized lat
particles are seen in Figure 4-11 Zeta potential values of polystyrene (PS) latex particle
at the Figure 4-9 are negative between -291 mV and -599 mV in PB and betwe
ex
s
en -203
mV and -278 mV in PBS at all pH ranges These negative zeta potential values of PS
latex particles are due to the sulfate groups [Vandenhu et al 1970] originated from
persulfate initiators attached via an oxygen bridge (-C-O-SO3-) at the end of polymer
chain where polymerization was initiated
-100
-80
-60
-40
-20
0
pH
Zta
pte
ntl
ζ
20
40
1 2 3 4 5 6 7 8 9
eo
ia (m
V)
PB (5mM)
PBS (5mM +143mM)
en -
Figure 4-11 Zeta potential of PS latex particles at 25oC
The zeta potential measurements of PSPMMA100 core shell latex particles as seen
in Figure 4-12 were also negative with a range of -28 and -505 mV in PB and betwe
143 mV and -186 mV in PBS at the pH 21-78 These negative values are also due to
sulfate groups on the particle surface
81
-100
-80
-60
-40
-20
0
20
40
1 2 3 4 5 6 7 8 9
Zea
pote
nti
l ζ
(mV
pH
ta
)
PB (5mM)
PBS (5mM + 143mM)
Figure 4-12 Zeta potential of PSPMMA100 latex particles at 25oC
The zeta potential for PSPMMA90PAA10 and PSPMMA75PAA25 was also
negative as seen in Figure 4-13 and 4-14 The zeta potential value for the
PSPMMA90PAA10 were between -367 mV and -678 mV in PB medium and between -
147 mV and -193 mV in PBS both at 25oC In case of the PSPMMA75PAA25 core shell
particles zeta potential values were between -291 mV and -520 mV in PB and between
-115 mV and -210 mV in PBS media Sulfate groups in PSPMMA75PAA25 and
PSPMMA90PAA10 core shell particles contribute to the negative zeta potential values
participate in forming a
negat
cid is expressed by the term pKa which is the pH
at which an acid is 50 dissociated However the contribution of the carboxylic acid
Carboxylic groups from acrylic acid monomer also would
ive surface charge of the latex particles at a pH greater than 70 because the pKa
value for the carboxylic acid is between 40 and 60 pH range [Ottewill et al 1967] This
indicates that carboxylic acid dissociation begins at a pH between pH 46-60 and
increases with pH The strength of an a
82
group to negative zeta potential values of PSPMMA-PAA core shell particles was not
detectable because there was no significant difference in zeta potential values for
synthesized latex particles at a pH greater than 70
-60
-40
-20
0
20
40
1 2 3 4 5 6 7 8 9
Zeta
pot
entia
l ζ
(mV
)
PB (5mM)PBS (5mM + 143mM)
-80
-100
pH
Figure 4-13 Zeta potential of PSPMMA90PAA10 latex particles at 25oC
-100
-80
-60
-40
-20
0
20
40
1 2 3 4 5 6 7 8 9
pH
Ze p
ont
i ζ
V)
tate
al (m
PB (5mM)PBS (5mM + 143mM)
Figure 4-14 Zeta potential of PSPMMA75PAA25 latex particles at 25oC
83
The zeta potential plots and values were similar for all latex particles This may be
due to the similar initiator density on the latex surface The initiator concentrations
monomers for shell polymerization were the same fo
to
r all core shell latex particles (062
ww from Table 4-4) A similar concentration (055 ww) of initiator to monomer was
added to prepare bare PS latex particles used in zeta potential measurements
Since the zeta potential values of all synthesized latex particles were negative
between pH=20 and pH=78 the isoelecric point (IEP) of these latex particles would be
less than pH=20 Sulfates have a pKa in the range of 10 to 20 [James 1985] indicating
that the sulfate group the conjugate base of a strong acid is protonated (-C-OSO3H) at
less than pH 20 This means that the synthesized latex particles are all negative at the
physiological pH Most serum proteins have a negative surface charge in blood
therefore when the negative latex particles are applied to blood stream fatal
tein is
avoided by charge repulsion in the blood Table 4-5 shows the common blood proteins
and their isoelectric points (IEP)
Table 4-5 The isoelectric point (IEP) of common proteins Serum proteins Isoelectric point (IEP)
complication by coagulation between applied latex particles and serum pro s
Albumin 48 α1-Globulin 20 α2-Microglobulin 54 γ1-Globulin 58 Heptoglobin 41 Hemoglobin 72 β1-lipoprotein 54 Immunoglobulin G (Ig G) 73 Fibrinogen 58 (source httpwwwfleshandbonescomreadingroompdf1085pdf
httpwwwmedalorgvisitorwww5CActive5Cch135Cch13015Cch130105aspx )
84
The absolute zeta potential values of the latex particles in phosphate buffer (PB)
was higher than that in phosphate buffered saline (PBS) This is because high
concentration of sodium and chloride electrolytes in PBS compressed the electrical
double layer causing rapid decay of surface potential Figure 4-15 shows that the
thickness of the electrical double layer is inversely proportional to the concentration of
electrolyte in the system
Surf
ace
Pote
ntia
l ψ
1κ3
10
1e
1κ 1κ
(1)(3) (2)
2 1 Z
Surf
ace
Pote
ntia
l ψ
1κ3
10
1e
1κ 1κ
(1)(3) (2)
Figure 4-15 The decay of surface potential with distance from surface in various
electrolyte concentrations (1) low (2) intermediate (3) high [Myers 1999]
2 1 Z
The electrical potential in the solution surrounding the particle surface falls off
the Debye-Huckel
yers 1999]
exponentially with distance (Z) from the surface according to
approximation [M
z) (- exp 0 κψψ = (4-4)
e reciprocal of the thickness of the electrical double layer referred to as the
The potential fallen off by a factor e The theoretical equation for the
ickness 1κ is
where κ is th
Debye length of 1
double layer th
21
22i
0
ekT 1
⎟⎟⎠
⎜⎜⎝
=sum ii zcεε
κ
⎞⎛ (4-5)
85
where
tration of
y was performed to measure the protein adsorption on the
synthesized latex particles Many physical and chemical processes occur at the boundary
between two phases and adsorption is one of the fundamental surface phenomena [Oscik
1982] This phenomenon is driven by the change in the concentration of the components
at the interface with respect to their concentrations in the bulk phase There are two
adsorption processes physical adsorption occurring when non-balanced physical forces
appear at the boundary of the phases and chemical adsorption or chemisorption
t the
interface [J
n leads
ational entropy hence adsorption takes place only if the
loss in conformational entropy is compensated
ter adsorption Cp The initial part of the
isotherm merges with the Γ-axis because at low polymer concentration the protein has a
high affinity for the surface and all of the polymer is adsorbed until the solid surface is
saturated
ε0 is the permittivity of a vacuum of free space ε is the permittivity of a medium e
is the relative permittivity or dielectric constant of the medium c is the concen
an ion (i) z is the valency of an ion (i) k is Boltzmannrsquos constant T is absolute
temperature (K)
43 Protein Adsorption Study
The adsorption stud
occurring when atoms and molecules from adjacent phases form chemical bonds a
aroniec et al 1988]
Flexible proteins in solution possess high conformational entropy as a result of the
various states each of the many segments in the protein chain can have Adsorptio
to a reduction of this conform
by sufficient attraction between polymer
segment and solid surface [Martin 1998] Isotherm shape of high affinity adsorption
between polymer and solid is shown in Figure 4-16 where the adsorbed mass Γ is plotted
against the polymer concentration in solution af
86
Figur
de
arboxyl
e 4-16 High-affinity adsorption isotherm of typical flexible polymer on solid
surface
In order to investigate the adsorption of blood proteins latex particle properties
such as electrical surface charge hydrophobicity and environmental conditions like pH
ionic strength and temperature are considered as experimental factors [Arai and Nor
1990] Figure 4-17 shows the overall schematic representation of the protein adsorption
on the synthesized latex particles with the functional groups such as sulfate and c
groups
CP
Γ
PMMAPAAShell
PS or PMMAShell OSO3
-
OSO3-
OSO3-
OSO3-
PS
-O3SO
PSCore
HOOC
-O3SO
O3SO
3
HOOC
HOOC
-
-O SO
Protein
NH3+
+
+H3N
+
COO-
Hydrophobic interaction
NH3
COO-
H3N
COO-
-OOC
Ionic interaction
Hydrophobic interaction
Hydrogen bonding
Ionic interaction
PMMAPAAShellOSO3
-
OSO3-
OSO3-
OSO3-
PS or PMMAShell -O3SOOSO3
-
PSCore
HOOC
-O3SO
O3SO
3
HOOC
HOOC
-
-O SO
-O3SO
OSO3-
OSO3-
OSO3-
PS PSCore
HOOC
-O3SO
O3SO
3
HOOC
HOOC
-
-O SO
Protein
NH3+
+
+H3N
+
COO-NH3
COO-
H3N
COO-
-OOC
NH3+
+
+H3N
+
COO-
Hydrophobic interaction
NH3
COO-
H3N
COO-
-OOC
Ionic interaction
Hydrophobic interaction
Hydrogen bonding
Figure 4-17 Overall schematic representation of the protein adsorption on the
Ionic interaction
synthesized latex particles
The surface functional groups as well as suitable surface hydrophobicity
hydrophilicity of latex particles are closely related to protein adsorption property As
indicated at Figure 4-17 various interaction forces such as hydrophobic interaction ionic
87
interaction hydrogen interaction and van der Waals interaction [Andrade 1985] exist
between protein molecule and the solid surface of latex particle
431 Adsorption Isotherm
The protein adsorption isotherm is generally defined as the relationship between the
amount of protein adsorbed by a solid (latex particle) at a constant temperature and as a
ine
the amount of protein adsorbed onto latex particles To evaluate the equilibrium
conce s
x
function of the concentration of protein Adsorption isotherms were plotted to determ
ntration of target proteins a calibration curve is firstly needed Figure 4-18 show
the UV absorbance as a factor of bovine serum albumin (BSA) concentrations The UV
absorbance of the known protein concentration was measured at 562nm which is the λma
for a protein treated by a bicinchoninic acid (BCA) assay
0
02
04
06
08
500 520 540 560 580 600
Wavelength (nm)
Abs
orba
nce
Figur gml (B)
06mgml (C) 02mgml
(A)
(C)
(B)
Max562nm
e 4-18 The dependence of UV absorbance on BSA concentration (A) 10m
Figure 4-19 shows the standard calibration curve used in adsorption isotherm
experiment of protein onto latex particles The net absorbance of each known protein
88
concentration was plotted and then a linear equation passing through a concentration of
zero protein concentration was obtained This equation is applied
to determine the
orbed on the synthesized latex particles The amount of free
protei
eported
trations (005 01 03 05
and 07mgml) were used to generate this curve as well as the adsorption isotherm
unknown protein amount ads
n was also determined using UV light at the 280nm wavelength however a
distinguishable UV intensity was too low to determine a precise value of protein
adsorption
The bicinchninic acid (BCA) assay technique [Smith et al 1985] has been r
for high efficiency of protein evaluation and was used for determination of protein
adsorption [Tangpasuthadol et al 2003 Williams et al 2003] This technique has many
advantages such as high sensitivity easy of use color complex stability and less
susceptibility to any detergents Five different BSA concen
y = 00R2 =
004741x 09939
00
01
02
03
04
0 200 400 600 800
BSA Standard (microgml)
Net
Abs
orba
nce
at 5
62nm
Figure 4-19 Standard curve of net absorbance vs BSA sample concentration
89
All isotherms were fitted to the Langmuir-Freundlich isotherms using the nonlinea
regression method Two adsorption isotherm models the Langmuir type isotherm a
Langmuir-Freundlich combination type isotherm were compared As seen the Figure 4-
20 The Langmuir isotherm model deviated further from the experimental data Th
all isotherms were fitted to the Langmuir-Freundlich isotherms [Yoon et al 1996
al 1998] usi
r
nd the
erefore
Lee et
ng the nonlinear regression method with following equation
n
n
eq
eqm
kC
kC1
1
1+
where q is the amount of adsorbed BSA per unit surface area q
qq = (4-6)
stant
were between 045 and 055 and hence n was fixed to its average 05 Actual qm values
were evaluated after fixing the n value
m is the adsorbed amount
in equilibrium Ceq is the equilibrium concentration of BSA k is the adsorption con
and n is the exponential factor for a heterogeneous system n values are empirical and
usually from zero to no more than unity [Jaekel 2002] The values of n in our system
00
10
20
30
0 02 04 06 08
C (mgml)eq
q (m
gm
2 )
pH 32Langmuir-Freundlich modelLangmuir model
Figure 4-20 Fitted models for adsorption isotherm of bovine serum albumin (BSA) on
polystyrene latex particles at 37oC in phosphate buffer media
90
The BSA protein adsorption on the synthesized latex particles was performed at
three pH levels 32 48 74 in both phosphate buffer (PB) and phosphate buffered saline
(PBS) at physiological temperature 37oC These pH levels correspond to an acidic pH
lower than the isoelectric point (IEP) of BSA the pH at the IEP and an alkaline pH
higher than the IEP of BSA respectively The IEP corresponds to the pH at which the
amino acid becomes a zwitterion Here its overall electrostatic charge is neutral and the
charge expressed by the basic amino group (-NH2 -NH3+) of the amino acid is equal to
the charge expressed by the acidic carboxyl group (-COOH -COO ) The IEP of BSA is
-
pH 48 and indicates that the side chain of the amino acids of BSA contains a greater
amount of acidic functional groups than that of basic functional groups At pH 32 and
74 BSA has a positive and negative charge respectively
Figure 4-21 and 4-22 show adsorption isotherms of BSA onto polystyrene (PS)
latex particles
00
10
20
30
40
0 02 04 06 08
Ceq (mgml)
q (m
gm
2 )
pH=32pH=48pH=74Model (pH 32)Model (pH 48)Model (pH 74)
Figure 4-21 Adsorption isotherm of BSA on polystyrene (PS) latex particles in
phosphate buffer (PB) at 37oC
91
00
10
30
40
0 02 04 06 08
Ceq (mgml)
q (m
gm
2
20
)
pH=32pH=48pH=74Model (pH 32)Model (pH 48)Model (pH 74)
(PBS) on PS latex particles
At the IEP of BSA the adsorbed amount (q
Figure 4-22 Adsorption isotherm of BSA adsorption 37oC in phosphate buffered saline
onto PS at equilibrium was
maxim
um at the IEP of BSA This is because the BSA molecules form the most
compact structures at the IEP eliminating the repulsion force between proteins A higher
number of BSA molecules can adsorb on a given surface area by hydrophobic interaction
when in this compact structure This observation agrees well with reported data by other
researchers who also indicated that the maximum adsorption from aqueous protein
solution was observed at the IEP [Mularen 1954 Watkins et al 1977 Oh at el 2000]
There was no significant difference of adsorbed BSA amount in PB and in PBS media at
pH 48 The conformational alteration of BSA was not affected significantly by the ionic
strength of the two solutions There was evidence of the effect of the electrostatic
interaction to some extent even at the IEP since the amount of BSA adsorbed in PBS is
slightly hig
m) of BSA
um indicating that hydrophobic interaction between latex particle and BSA
protein is maxim
her than in PB at this pH
92
The adsorbed amount of protein is shown to decrease at a pH 32 and 74 The
decrease in adsorption efficiency when deviating from IEP of BSA is a result of the
increase in conformational size of the protein molecules and the lateral electrostatic
repulsions between adjacent adsorbed BSA molecules Figure 4-23 shows a schematic of
possible conformational changes at each pH where hydrophobic interaction always exists
between non-polar portion of a protein and hydrophobic portion of solid surface
- - --- ---Latex surface
Zeta potential
BSA(+)
(-)
48 7432pH
(0)
+++ + ++
--+
++-
- - --- ---Latex surface
Zeta poten pH(0)
+++ + ++
--
tial
BSA(+)
(-)
48 7432
+
++-
ming
4
s
al blood Table 4-6 shows the calculated results of
Figure 4-23 Conformation of bovine serum albumin (BSA) on latex particles (Assuthat charge repulsion reduces the effect of the hydrophobic interaction at pH 74)
In addition the adsorbed amount of BSA at pH 32 was larger than that at pH 74
and still less than that at 48 This is because there is the electrostatic interaction between
the positive BSA molecules and negative PS latex particles at pH 32 and electrostatic
repulsion is dominant between negative BSA and negative PS latex particles at pH 7
The amount adsorbed increased with increasing ionic strength in pH 32 and 74 This i
because the high concentration of electrolytes reduces the electrostatic repulsion between
BSA and PS latex particle It should be noted that the BSA concentration used in these
experiments was lower than that found in normal human blood This is because the
equilibrium BSA adsorption onto the latex particles is actually reached at a much lower
concentration than that found in norm
93
adsor rm
ein
leading to a compact conformation of BSA There are two main forces affecting
adsorption amount at an acidic pH of 32 They include the ionic attraction between the
negative latex particle and the positive protein and lateral repulsion between positive
proteins The smallest amount of BSA adsorption was observed at a pH of 74 At this
point the charge repulsion between negatively charged particles and protein molecules
exists the h
of cha
bed BSA amount at equilibrium from the Langmuir-Freundich adsorption isothe
equation
In summary the hydrophobic interaction between PS latex particle and BSA
protein exists and is the dominant mechanism for the adsorption process Conformational
changes of BSA at each pH also affected the adsorption amount Adsorption was highest
at pH 48 because at this pH there is the least amount of ionic repulsion in the prot
and the lateral repulsion between proteins dominant adsorption even though there always
ydrophobic interaction between the solid latex particle and protein The effect
rge repulsion was reduced by the high concentration of electrolytes in PBS medium
Table 4-6 The equilibrium concentration values of BSA adsorption on polystyrene (PS) latex particles calculated the Langmuir-Freundlich isotherm model
Media pH qm (mgm2) 32 174 48 299 Phosphate buffer 74 136 32 260 48 283 Phosphate buffered saline 74 207
The adsorption isotherms of BSA on PSPMMA core shell particles are shown in
Figure 4-24 in phosphate buffer (PB) and in Figure 4-25 in phosphate buffered saline
(PBS) The adsorbed BSA amount was calculated using the Langmuir-Freundlich
100
94
adsorption isotherm equation and the amount listed in Table 4-7 The trend seen in the
adsorption results was very similar to the case of PS latex The overall adsorption process
was d rticle
rs
hat
0
ominated by hydrophobic interactions between the PSPMMA100 core shell pa
and BSA proteins At pH 48 the adsorbed amount (qm) of BSA on PSPMMA100 core
shell particles was 221 mgm2 in PB and 237 mgm2 in PBS and are the maximum
values recorded among the three pH levels 32 48 and 74 Suzawa and his co-worke
[1982] have obtained similar results This can be also explained by the compact
conformation of BSA molecules at the IEP of BSA which lead to a high protein
population on the solid surface At a pH of 32 and 74 BSA adsorption was less than t
at pH 48 because of the protein conformation change There was a small increase in
adsorbed BSA at pH 32 because of the charge attraction between negative PSPMMA10
core shell particles and positive BSA At pH 74 however the adsorption of BSA was
further reduced by charge repulsion between the negative PSPMMA100 core shell
particles and the negative BSA proteins
000 02 04 06 08
Ceq (mgml)
10
20q (m
gm
302 )
40
50pH=32pH=48pH=74Mode 8)l (pH 4Mode 2)l (pH 3Mode 4)
Figure 4-24 Adsorption isotherm of BSA 37oC in PB on PSPMMA core shell latex
l (pH 7
100
particles
95
The adsorbed amount of BSA increased more rapidly in PBS with increasing ioni
strength at pH 74 This shows that the low concentration of electrolytes promotes
electrostatic repulsion between negative BSA and negative PSPMMA
c
on
x
l 1980
100 core shell
particle As the ion strength of PBS is increased however the high concentration of
electrolyte leads to less electrostatic repulsion promoting more rapid protein adsorpti
By comparing the amount of adsorbed protein between bare PS latex particles and
PSPMMA100 core shell particles the amount of protein adsorption on the PS late
particles is greater than that on the PSPMMA100 core-shell particles [Suzawa et a
1982 Lee et al 1988]
00
10
20
30
40
m2 )
0 02 04 06 08
Ceq (mgml)
q (m
g
pH=32pH=48pH=74Model (pH 2) 3Model (pH 48)Model (pH 74)
Figure 4-25 Adsorption isotherm of BSA at 37oC in PBS on PSPMMA100 core shell
latex particles
96
Table particles calculated the Langmuir-Freundlich isotherm model
4-7 The equilibrium concentration values (qm) of BSA adsorption on PSPMMA100
Media pH qm (mgm ) 2
32 140
48 221 Phosphate buffer
74 130
32 169
48 237 Phosphate buffered saline
74 162
The BSA adsorption mechanism and trend on PSPMMA core shell particles was
very similar to that of the PS latex particle Unlike the case of PS and PSPMMA100 latex
particles the amount of BSA adsorbed onto PSPMMA90PAA10 and PSPMMA75PAA25
core shell particles was higher at pH 32 than pH 48 and 74 Figure 4-26 through 4-29
shows the adsorption isotherms of BSA on PSPMMA90PAA10 and PSPMMA75PAA25
core shell particles The amount of BSA adsorbed increased at pH 32 and pH 48 as the
hydrophilicity was developed by the addition of PAA On the contrary the adsorbed
amount of BSA was dramatically decreased at pH 74 The adsorption amount of BSA on
PSPMMA90PAA10 and PSPMMA75PAA25 core shell particles at equilibrium is listed in
Table 4-8 and 4-9 respectively
Carboxylic acid as well as sulfate groups are distributed on the surface of
PSPMMA90PAA10 and PSPMMA75PAA25 core shell particles and seem to largely affect
the BSA adsorption The isotherms showed very strong pH dependence to the BSA
adsorption onto these core shell latex particles The carboxylic groups (COOH) may
especially enhance the BSA adsorption prominently at pH 32 by hydrogen bonding with
protein molecules The pKa value for the PAA is 40-60 and a good indication that the
97
carboxylic acid groups are protonated at pH 32[Gebhardt et al 1983] The main
adsorption interaction force at pH 32 was generated by hydrogen bonding as well as
charge attraction between latex particle protein even th the conformational
repulsion between proteins results in a reduction of the protein
adsorption at this pH The amount of BSA adsorbed also increased as the PAA amount in
eased The a tion of BSA w inimal however less
than 07 mgm2 at pH 74 Here the carboxylic groups are ionized which increases the
charge repulsion by ionized carboxylic groups (COO-) resulting in PSPMMA-PAA latex
particle
and ough
change and charge
the core shell particles incr dsorp as m
s with much lower protein adsorption
00
20
40
q (m
g2 )
60
m
pH=32pH=48pH=74Model (pH 32)Model (pH 48)
0 02 04 06 08
Ceq (mgml)
Model (pH 74)
o shell
Figure 4-26 Adsorption isotherm of BSA at 37 C in PB on PSPMMA90PAA10 corelatex particles
98
00
40
60
80
0 02 04
C (mgm
mm
2 )
20
06 08
eq l)
q (
g
pH=32pH=48pH=74Model (pH 32)Model (pH 48)Model (pH 74)
Figure 4-27 Adsorption isotherm of BSA at 37oC in PBS on PSPMMA90PAA10 core
shell latex particles
Table 4-8 The equilibrium concentration values of BSA adsorption on PSPMMA90PAA10 particles calculated the Langmuir-Freundlich isotherm model
Media pH qm (mgm2)
32 361
48 283 Phosphate buffer
74 043
32 519
48 226 Phosphate buffered saline
74 043
99
00
20
40
60
80
100
0 02 04 06 08Ceq (mgml)
q (m
gm
2 )
pH=32pH=48pH=74Model (pH 32)Model (pH 48)Model (pH 74)
oFigure 4-28 Adsorption isotherm of BSA 37 C in PB on PSPMMA75PAA25 latex particles
00
100
20
40
60
80
0 02 04 06 08Ceq (mgml)
q (
m2 )
pH=32pH=48pH=74
mg
Model (pH 32)Model (pH 48)Model (pH 74)
Figure 4-29 Adsorption isotherm of BSA 37oC in PBS on PSPMMA75PAA25 core shell
latex particles
100
Table 4-9 The equilibrium concentration values of BSA adsorption on PSPMMA75PAA25 particles calculated the Langmuir-Freundlich isotherm model
Media pH qm (mgm2)
32 1083
48 662 Phosphate buffer (PB)
74 075
32 1077
48 681 Phosphate buffered saline (PBS)
74 057
The adsorption of β2-microglobulin (β2M) was determined under one set of
onditions in phosphate buffer at 37oC and pH 74 because of the limited amount of β2M
protein The adsorption isotherms of the β2M protein on the synthesized latex particles
are shown in Figure 4-30 4-31 and 4-32 The β2M protein concentrations for adsorption
isotherm experiment were chosen to be 0015 0030 0045 and 0060mgml since the
β2M concentration for the patients with renal failure can be elevated to similar
concentrations about 005mgml or more up to 50 times the normal level [Nissenson et
al 1995] There were steep initial slopes of the adsorption isotherm curves of β2M on
latex particles in all isotherms indicating a high affinity adsorption type It seems that the
hydrophobic interaction is enough for the adsorption process of β2M proteins to occur on
the latex particles even though charge repulsion exists between protein and latex particle
at pH 74 The complete plateau regions were not seen for all isotherms indicating that
β2M proteins were not saturated at this level and there are still available sites for β2M
c
adsorption on latex particles The initial slope of the β2M isotherms for PS was steeper
101
than that of any of the other latex particle PSPMMA100 PSPMMA90PAA10 and
PSPMMA 75PAA25
00
02
0 0002 0004 0006 0008
04
06
10
Ceq (mgml)
q (m
2 )
08
gm
PSPSPMMA100Model (PS)Model (PSPMMA100)
Figure 4-30 Adsorption isotherm of β2M onto PS and PSPMMA100 latex particles in PB
at 37 C and pH 74 o
00
02
04
06
08
10
0 0002 0004 0006 0008C (mgml)
q (m
m2 )
eq
g
PS
PSPMMA90PAA10
Model (PS)
Model (PSPMMA90PAA10)
Figure 4-31 Adsorption isotherm of β2M onto PS and PSPMMA90PAA10 latex particles
in PB at 37oC and pH 74
102
00
02
04
06
08
10
0 0002 0004 0006 0008Ceq (mgml)
q (m
gm
2 )
PS
PSPMMA75PAA25
Model (PS)Model (PSPMMA75PAA25)
Figure 4-32 Adsorption isotherm of β2M onto PS and PSPMMA75PAA25 latex particles
in PB at 37oC and pH 74
The equilibrium concentration values (qm) of β2M adsorption on latex particles are
calculated and listed in Table 4-10 The difference between the maximum amounts of
β2M adsorption on all the latex particles was not significantly different and their values
were between 069 and 08mgm2
Table 4-10 The equilibrium concentration values of β2M adsorption calculated the Langmuir-Freundlich isotherm model
Latex particles qm (mgm2)
PS 080
PSPMMA100 069
PSPMMA90PAA10 072
PSPMMA75PAA25 072
103
432 Adsorbed Layer Thickness
There are several possible explanations for large amount of BSA adsorbed at acidic
pH levels including the end-on adsorption pattern of BSA the flexibility of BSA
molecules the tilting of protein molecules due to the asymmetry of the charge
distribution or multilayer formation [Peula et al 1993] The adsorbed BSA thickness (δ)
can be calculated by the following equation [Chiu et al 1978] This equation was also
cited again by other researchers [Shirahama et al 1985]
BSA
mqρπ
δbullbull
=33
(4-7)
where π
33 is the packing factor qm is the plateau value of the adsorbed am
ρBSA is the hich corresponds to the reciprocal of its known
partial specific volume The layer thickness (δ) of BSA proteins on synthesized latex
partic
he IEP [Shirahama et al 1985] Compared to the
hydrodynamic dimensions of BSA 140 times 40 times 40 Aring3 [Fair et al 1980 Peter 1985] these
nd
ode It is unnecessary ultilayering because thickness do
exceed 140 Aring The layer thickness (δ) of BSA proteins on synthesized latex particles
e red saline (PBS) was ran 28 ~ 83 Aring at 37oC and pH 48 These
lso listed in Table 4-1
ount and
density of BSA molecule w
les in phosphate buffer (PB) ranged from 27 - 81 Aring at 37oC and pH 48 These
calculated values are listed in Table 4-11 Only δ values at pH 48 the isoelectric point
(IEP) of BSA are important because intra- and intermolecular electrostatic repulsion of
protein molecules is minimized at t
values of layer thickness indicate that the BSA molecules adsorb by side-on (40Aring) a
end-on (140Aring) m to consider m
not
in phosphat buffe ged of
calculated values are a 2
104
Table 4-11 Absorbed BSA layer thickness (Aring) of BSA in phosphate buffer (PB) at 37oC pH PS PSPMMA100 PSPMMA90PAA10 PSPMMA75PAA25
32 21 17 44 133
48 37 27 35 81
74 17 16 5 9
Table 4-12 Absorbed BSA layer thickness (Aring) of BSA in phosphate buffered saline (PBS) at 37oC
pH PS PSPMMA100 PSPMMA90PAA10 PSPMMA75PAA25
32 32 21 64 132
48 35 29
74 26 20 5 7
28 83
1n
1n
neqm
So k is closely related to the initial slope of the isotherm showing the affinity
between BSA and latex particles [Yoon et al 1996] A key point in the characterization
433 Gibbs Free Energy of Protein Adsorption
In the Langmuir isotherm model k represents the ratio of adsorption to the
desorption rate constants This definition is not applicable to the Langmuir-Freundlich
isotherm model but its meaning is similar [Yoon et al 1996] In this sense k is closely
related to the affinity between protein molecules and latex particles When the
equilibrium concentration Ceq reaches zero the value of Ceq will be very small (1gtgt
kCeq ) and then the equation 4-6 becomes
(4-8)
of adsorption processes on solid-liquid interfaces is to determine the change in Gibbsrsquos
free energy (∆Gdeg) one of the corresponding parameters for the thermodynamic driving
forces during adsorption The traditional method to evaluate adsorption thermodynamic
kCqq 1=
105
constants is based on measurement of adsorption isotherms under static conditions
[Malm ten 1998] The Langmuir-Freundlich ads lf
easily found from Then this enables calculation of the free-
energy change ∆G during adsorption expressed as
(4-9)
where R is the general gas constant (R = 8314Jmiddotmol K ) and T is the absolute
tem ture Gibb e energy A P
listed in Table 4-13 and 4-14 respectively As the ∆Gdegads ore negativ e
pro adsorption on a solid substra ill be more favorable
e of BSA adsorption in phosphate o
pH PS PSPMMA100 PSPMMA90PAA10 PSPMMA75PAA25
s orption equilibrium constant k can be
a slope of the isotherm
degads
lfads kRTG lnminus=∆ o
-1 -1
pera s fre change of BS adsorption values in B and PBS are
values are m e th
tein te w
Table 4-13 The values of Gibbs free energy changbuffer (PB) at 37 C
32 -2411 -2726 -2846 -2805
48 -2352 -2383 -2198 -1972 ∆Go
80
ads (kJmol)
74 -2452 -888 -324 -2
Table 4-14 The values of Gibbs free energy change of BSA adsorption in phosphate
pH PS PSPMMAbuffered saline (PBS) at 37oC
100 PSPMMA90PAA10 PSPMMA75PAA25
32 -2315 -2620 -2312 -2683
48 -2149 -2299 -2090 -2138 ∆Go
(kJmads ol)
74 -2430 -1992 -258 -263
Table 4-15 shows the calculated values of Gibbs free energy change of β M
adsorption on synthesized latex particles PSPMMA PAA core shell latex particle has
2
75 25
106
largest negative Gibbs free energy of β2
2
M adsorption indicating largest affinity betw
β
een
2M adsorption in phosphate buffere (PB) at 37 C and pH74
PS PSPMMA100
M and latex particle
Table 4-15 The values of Gibbs free energy change of βo
PSPMMA90PAA10 PSPMMA75PAA25
∆Go03 ads
(kJmol) -3626 -3466 -3659 -39
The Gibbs free energy of β2M was much more negative in phosphate buffer (PB)
than that of BSA in physiological condition (37 C and pH 74) o from the Table 4-13 and
the Figure 4-31 This indicates that β2M molecules more readily adsorb on latex particles
BSA This can be explained by the rate of diffusion and accessibility of β2M molecules to
the latex particle ier and faster for β2M m an BSA because of
size (molec ight) erical shape molecules It o be
due to the higher hydrophobic force and less electrostatic repulsion that contribute to the
adsorption of β2M molecules not as prevalent in the case of BSA because the IEP of β2M
(57) is clo H 74) than that of BSA (48) causing β2M to
be less electro cally lled p ro
can be seen that the ∆ β2M adsorption onto the synth article
increasing wi hy icity o x particles s the ∆Goads of the BSA
molecules is decreasing or constant This phenomenon supports the theory that the
select lizing
than BSA and that the affinity of β2M for the latex particle is much larger than that of
surface It is eas olecules th
the smaller ular we and sph of β2M may als
ser to physiological condition (p
stati repe from the latex articles than BSA F m Figure 4-33 it
Goads of esized latex p s is
th the drophil f the late wherea
ive adsorption of β2M molecules over BSA molecules can be enhanced by uti
more hydrophilic PSPMMA90PAA10 and PSPMMA75PAA25 core shell latex particles
107
-60
-50
1 2 3 4
-40∆Gad
s
-30
10
0
o (k
Jm
o-
-20l)
β2-Microglobulin (β2M)Bovine serum albumin (BSA)
Figure 4-33 Gibbs free energy of adsorption of proteins on latex particles in PB at 37
and pH 74
434 Kinetics of Adsorption
to
oC
Protein adsorption is a complex process known to have a large biological impact
and is currently not well understood quantitatively because of extreme sensitivity to pH
the concentration of other electrolytes and molecules present temperature and many
other factors that can change in the physiological system Accurate knowledge of the
adsorption kinetics under a given set of conditions is a prerequisite for elucidating the
mechanisms of many fundamental biological processes to be predicted at the molecular
level [Regner 1987] This adsorption kinetics approach is used in protein adsorption
studies because of the uncertainty related to the time needed for the interfacial proteins
reach the equilibrium It is generally accepted that the process of protein adsorption is
comprised of the following steps a) transport toward the interface b) attachment at the
interface c) eventual structural rearrangements in the adsorbed state d) detachment from
PS PSPMMA100 PSPMMA90PAA10PSPMMA75PAA25
108
the interface and transport away from the interface Each of these steps can affect the
overall rate of the adsorption process
The quantitative analysis of the protein adsorption kinetics requires that the protein
amount adsorbed is known as a function of time The kinetics tests were carried out in
phosphate buffer (PB) at 37oC and pH 74 a condition similar to physiological condition
The protein concentrations chosen were 07mgml for BSA and 006mgml for β2M
which are the maximal concentrations used in an adsorption isotherm test A time-based
evaluation of the adsorption was made by measuring the concentration of protein in
solution at different incubation times The results are shown below from Figure 4-34 to 4-
37 The adsorption kinetics of proteins onto th cond-e latex particles is expressed as a se
order model [Oumlzacar 2003] shown by the following equation
2)( tet qqk
dtdq
minus= (4-10)
Integrating above equation and applying the boundary conditions yields
ee
t tq
+=
1 (4-11)
where q
qkq
t
2
2
2
the previous adsorption isotherm results representing good reproducibility of adsorption
t and qe stand for the amount (mgm ) of protein adsorbed at time t and at
equilibrium respectively k is the equilibrium rate constant of second-order adsorption
(m mghr) and t is the incubation time (hr)
The fitting parameters calculated by using the equation 4-11 are listed in table 4-16
From this data it is seen that the amount of BSA adsorbed at equilibrium was higher than
that of β2M on PS and PSPMMA100 latex particles but less than that of β2M on
PSPMMA90PAA10 and PSPMMA75PAA25 latex particles These results are similar to
109
00
05
15
20
0 2 4 6 8 10 12 14
Time (hr)
rot
n ad
sorb
d (
10
25
Pei
em
gm
2
BSA2nd order model (BSA))β2M2nd order model (β2M)
Figure 4-34 The kinetics of protein adsorption in PB on PS latex particles at 37oC and
pH 74
00
05
10
15
20
25
30
0 2 4 6 8 10 12 14
Time (hr)
Prte
inds
oo
arb
ed (
mg
m2 )
BSA2nd order model (BSA)β2M2nd order model (β2M)
Figure 4-35 The kinetics of protein adsorption in PB on PSPMMA100 latex partic
37les at
oC and pH 74
110
00
03
06
09
12
15
0 2 4 6 8 10 12 14
Time (hr)
Prot
ein
adso
rbed
(m
gm
2 ) BSA2nd order model (BSA)β2M2nd order model (β2M)
Figure 4-36 The kinetics of protein adsorption in PB on PSPMMA90PAA10 latex
particles o at 37 C and pH 74
00
03
06
09
12
15
0 2 4 6 8 10 12 14
Time (hr)
BS
A ad
sorb
ed (
mg
m2 )
BSA2nd order model (BSA)β2M2nd order model (β2M)
Figure 4-37 The kinetics of protein adsorption in PB on PSPMMA75PAA25 latex
particles at 37oC and pH 74
111
test The variation of β2M equilibrium adsorption amount was nearly negligible for all
latex particles The plateau region is reached earlier for β2M than BSA on all latex
particles and the K values (equilibrium rate constant of second order adsorption) for the
β2M adsorption process onto latex particles are much larger than for BSA adsorption
These values indicate that the β2M molecules have a higher adsorption rate onto the latex
particles than the BSA molecules
Table 4-16 Fitting parameters of second-order kinetic model BSA β2M
Latex particles qe (mgm2) K (hr-1) qe (mgm2) K ( -1) hr
PS 144 67 074 7549
PSPMMA100 175 353 076 4438
PSPMMA90PAA10 046 97 075 5695
PSPMMA75PAA25 060 179 075 5168 K = (kqe) hr-1
There are several explanations for the phenomena observed in these experiments
First the smaller sizes of β2M molecules allow easier diffusion and a close approach to
the latex substrate Second a reduced number of hydrophobic domains on the latex
particle surface replaced by sulfate groups (-OSO3-) and ionized carboxylic groups
(COO-) are not favorable for larger BSA molecules Third the conformational change by
intra- and inter-molecular repulsion would be faster in β2M due to smaller molecular
weight Finally the ionic repulsion between β2M and the latex particles is less than that
condition (pH 74) than IEP (48) of BSA
between BSA and latex particles because IEP (57) of β2M is closer to the tested
112
44 Blood Compatibility
Blood compatibility can be defined as the property of a material or device tha
permits it to function in contact with blood without inducing any adverse reactions
Successful functioning of blood contacting biomedical materials or devices in contact
with blood strongly depends on the extent to which they damage blood during operation
High shear stresses turbulence rela
t
tively long contact times and cellular impact with
artificial surfaces are most common events experienced by blood in contact with artificial
1978 Winslow and et al
1993] Therefore before any materia e placed into the human b
biocompatibility testing must be the first performed in the view of safety The hemolysis
s a method for pr ary screening of biomaterials to determine
h blood a er circulating body fluid The tests estimat
oglobin released from th blood cell d e induced
foreign material
There are several types of blood cells in our body but the ones that make blood red
are erythrocytes or red blood cells (RBCs) Red blood cells (erythrocytes) are the most
numerous type in the blood These cells are average about 48-54 million per cubic
millimeter (mm3 same as a microliter (microl)) of blood but these values can vary over a
larger range depending on such factors as health and altitude RBCs are red because they
contain hemoglobin which is a protein that contains iron When blood circulates through
lungs oxygen binds to the hemoglobin and is then delivered to all the parts of body
RBCs are made in the bone marrow They start out as a nucleus like other cells but
when they fill up with hemoglobin the nucleus becomes smaller until it disappears
Without a nucleus RBCs are fragile and live only about 120 days In a normal healthy
devises and can cause hemolysis or thrombosis [Bernhard et al
ls can b ody
test serves a elimin the
biocompatibility wit nd oth e the
concentration of hem e red amag by a
113
body about 2 million red blood ne marrow produces new
ones j ing
dy
le
cells die per second But bo
ust as fast RBCs are about 75 micrometer across and are responsible for carry
oxygen salts and other organic substances to the cells and tissues throughout our bo
In humans and most mammals the cells are concave (indented in the middle) and flexib
enough to squeeze through even the tiniest of blood vessels the capillaries
Figure 4-38 Image of red blood cells
(Sourcehttpwwwpbrchawaiiedubemfmicroangelarbc1htm)
ch
100 90 10 75 25 f
RBCs are among the first cells that come into contact with a foreign material
injected in the blood Therefore a hemolysis assay gives information about the
biocompatibility in the case of an in vivo application The concentrations of prepared
latex particles used for the tests in this research are 05 25 and 50 (ww) and the
incubation time under moderate shaking was 30 minutes at 37oC Test results are shown
in Figure 4-39 The amount of blood cells that ruptured when in contact with the PS latex
particles was largely increased from about 30 to 91 as the solid content of PS
increased from 05 to 50 However hemolysis by the core shell latex particles su
as PSPMMA PSPMMA PAA and PSPMMA PAA was less than 35 O
114
particular interest was that hemolysis caused by PSPMMA PAA particles at 50
solid concentration was less than 02 hemolysis the lowest value among the latex
particles The low hemolysis value indicates that the PSPMMA PAA core shell latex
particles are the most blood compatible at all solid contents Other core shell particles
such as PSPMMA and PSPMMA PAA also had excellent blood comp
90 10
90 10
100 75 25 atibility with
low RBS breakage
01 2 3
20
40
60
80
100
Hem
olys
is (
)
052550
Figure 4-39 He
PS PSPMMA100 PSPMMA PAA90 10
molysis caused by latex particles n=5
4PSPMMA75PAA25
CHAPTER 5 SUMMARY OF RESULTS CONCLUSIONS AND RECOMMENDATION FOR
FUTURE WORK
51 Summary of Results
The goal of this study was to synthesize monodisperse polymeric latex particles
with tailored properties and to investigate the fundamental interactions between the
synthesized latex particles and target proteins An understanding of the fundamental
mechanism of selective adsorption is strongly required in order to maximize the
separation performance of membranes made of these materials so that it may applicable
to hemodialysis therapy for end stage renal disease (ESRD) patients For the achievement
of above research goal several investigations have been performed Analysis of the
obtained data leads to several conclusions as discussed below
Monodisperse polystyrene seed latex particles were synthesized with the size
crosslinking agent to make the latex particles more hydrodynamically stable The
conversion of monomer to polymer was over 95 and seed latex particles are spherical in
shape and have smooth surface From these small seeds bigger latex particles as large as
800nm were synthesized using a semi-continuous seeded emulsion polymerization
process The particles with a mean diameter less than 500nm are very spherical in shape
and highly uniform in particle size distribution However as the particles grow larger
than 500nm in mean diameter they became non-spherical with an uneven surface This
irregularity of particle surface can be attributed to the non-homogeneous monomer
ranges between 126plusmn75 and 216plusmn53 nm Divinylbenzene (DVB) was used as a
115
116
swelling into the shell of a growing polymer which can be controlled by the factors such
as temperature agitation speed initiator feeding rate and surfactant amount required to
stabilize the colloidal system It was determ rfactant to monomer ratio
dec ic
acid (AA) monomers were intro rticle surfaces more
hydro
to
ting of a
orm
ration is seen between
3002
0 cm-
0cm-1
-1
03
ined that as the su
reased the mean particle diameter increased Methyl methacrylate (MMA) and acryl
duced to make the latex pa
philic than bare polystyrene particles The prepared core shell latex particles were
made of PMMA100 PMMA90PAA10 and PMMA75PAA25 PMMA100 is the PMMA
homopolymer shell on a PS core PMMA90PAA10 shell is a copolymer with a PMMA
PAA ratio of 90 to 10 by weight PMMA75PAA25 shell is a copolymer consis
PMMA to PAA ratio of 75 to 25 by weight The particle size of these core-shell
particles were prepared to be about 370nm in mean diameter
The successful synthesis of latex particles was confirmed using Fourier Transf
Infrared spectroscopy (FTIR) The C-H aromatic stretching vib
cm-1 and 3103 cm-1 for PS latex particles The C-H asymmetrical stretching and
symmetrical stretching vibration peaks of CH2 for all latex particles is seen at 290
1and 2850 cm-1 respectively There is a carbonyl (C=O) characteristic band at 173
wavenumber for PSPMMA core-shell latex particles The broad OH group peak at
3400 cm
100
can be seen in the spectroscopy of the particle with PMMAPAA shell
(PSPMMA PAA and PSPMMA PAA ) The peak intensity of OH group of
PSPMMA PAA was larger than that of PSPMMA PAA relatively due to more AA
monomer content in PSPMMA PAA than in PSPMMA PAA
In the zeta potential measurements polystyrene (PS) latex particles had negative
values between -291 mV and -599 mV in the phosphate buffer (PB) and between -2
90 10 75 25
75 25 90 10
75 25 90 10
117
mV and -278 mV in the phosphate buffered saline (PBS) at 25oC and pH 21-78 These
negative zeta potential values for PS latex particles are due to ionization of the sulfate
groups (-OSO3-) originated from the initiator Zeta potential values of the PSPMMA
core shell latex particles were also negative between -28 mV and -505 mV in PB and
between -143 mV and -186 mV in PBS at pH 21-78 Negative values are also
generated by dissociation of sulfate groups on a particle surface The zeta potential value
of PSPMMA PAA and PSPMMA PAA were of negative values between -36
and -678 mV in PB medium and -147 mV and -193 mV in PBS medium at 25
100
s
7 mV
MMA75PAA25 and
PSPM
otein
g the
l change of BSA at
each w
90 10 75 25
oC For
PSPMMA PAA core shell particles zeta potential values were between -291 mV and
-520 mV in PB media and between -115 mV and -210 mV in PBS media Sulfate
groups contributed to the negative zeta potential values in PSP
75 25
MA90PAA10 core shell particles Carboxylic groups from polyacrylic acid also
contribute to the negative surface charge for the latex particles at greater than pH 70
however the degree of their contribution was not detectable from the zeta potential
measurements The absolute zeta potential values of the synthesized latex particles in PB
were higher than that in PBS This is because of the high concentration of sodium
chloride electrolyte in PBS that compress electrical double layer thickness
The bicinchoninic acid (BCA) assay technique was used to determine the pr
adsorption and all isotherms were fitted to the Langmuir-Freundlich isotherms usin
nonlinear regression method The hydrophobic interaction between PS latex particle and
BSA protein was dominant for the adsorption process Conformationa
pH also affected the adsorption amount which was highest at pH 48 because of lo
ionic repulsion making compact conformation of BSA The next larger amount of BSA
118
adsorbed onto PS was at pH 32 There were two main forces affecting the amount of
adsorption at acidic pH values One is ionic attraction between negative latex particle a
positive protein The other is lateral repulsion between positive proteins The smallest
amount of BSA adsorption was at pH 74 where charge repulsion between negative
particles and proteins is present at this pH Lateral repulsion between proteins wa
dominant for protein adsorption even though there always exists the hydrophobic
interaction between solid latex particle and protein The charge repulsion was reduced by
the high concentration of electrolytes in the PBS medium The BSA adsorption process
on PSPMMA core shell particles was very similar to the case of PS latex particle where
hydrophobic interaction between latex particle and protein was dominant However t
adsorbed BSA amount was less than that of PS in PBS The adsorbed amount of BS
PSPMMA
nd
s
he
A on
an
ed
n The main
adsor
90PAA10 and PSPMMA75PAA25 core shell particles was higher at pH 32 th
pH 48 and 74 The adsorbed BSA amount became higher at pH 32 and pH 48 as the
hydrophilicity was increased by the higher PAA amount On the contrary the adsorb
amount of BSA was dramatically decreased at pH 74 Carboxylic acid groups as well as
the sulfate groups are distributed on the surface of PSPMMA90PAA10 and
PSPMMA75PAA25 core shell particles and largely affect the BSA adsorptio
ption interaction forces at pH 32 was generated by hydrogen bonding as well as
charge attraction between latex particle and protein even when the conformational change
and charge repulsion between proteins reduce the adsorption of protein at this pH The
adsorbed BSA amount was also increased as the amount of PAA increases However the
adsorption of BSA was minimal (less than 07 mgm2) at pH 74 where the carboxylic
groups are ionized leading to more enhancement of charge repulsion by ionization of the
119
carboxylic groups (COO-) Because of this the PSPMMA-PAA latex particles have a
much lower protein adsorption
There were steep initial slopes for adsorption isotherms of β
in
s
there
M
r
e
es
o
2M on latex particles
all isotherms indicating a high affinity type adsorption The complete plateau region
were not seen for all isotherms indicating that β2M proteins were not saturated and
are still available sites for β2M adsorption on latex particles The initial slope of β2
isotherms for PS was steeper than for the other latex particles PSPMMA100
PSPMMA90PAA10 and PSPMMA75PAA25
The layer thickness (δ) of BSA proteins on synthesized latex particles in phosphate
buffer (PB) was in the range of 27 - 81 Aring at 37 C and pH 48 Only the δ values at pH
48 the isoelectric point (IEP) of BSA are important because intra- and inter-molecular
electrostatic repulsion of the protein molecules is minimized at the IEP Compared to the
hydrodynamic dimensions of BSA 140 times 40 times 40 Aring these values of the adsorbed laye
thickness indicates that the BSA molecules adsorb by the side-on (40Aring) and end-on mod
(140Aring) Consideration of the multi-layering is unnecessary because the adsorbed
thickness of BSA do not exceed 140 Aring
The Gibbs free energy of β
o
3
2M was more negative in phosphate buffer (PB) than
that of BSA in physiological condition (37 C and pH 74) indicating that βo2M molecul
are more favorably adsorbed on the latex particles than BSA and that the affinity of β2M
was much larger than BSA This may be explained by the rate of diffusion and
accessibility of β2M molecules being easier and faster than that for BSA because of
smaller size (or weight) and spherical shape of β2M molecules It may also be possible t
explain that more hydrophobic forces (less electrostatic repulsion) contributes to
120
adsorption of β2M than to that of BSA because the IEP of β2M (57) is closer to
physiological condition (pH 74) than that of BSA (pH 48) The selective adsorption of
β2M m
in β2M
larger than in BSA adsorption indicating
that fa
an that between BSA and latex particles because
IEP (
ch as
Other
olecules over BSA molecules can be enhanced by developing more hydrophilic
PSPMMA90PAA25 and PSPMMA75PAA25 core shell latex particles
The adsorption kinetics of proteins on latex particles was expressed as a second-
order model The plateau region for β2M was reached earlier than BSA on all latex
particles and the K values (equilibrium rate constant of 2nd order adsorption)
adsorption process on latex particles were much
ster adsorption rate of β2M molecules than that of the BSA molecules There are
several explanations for these phenomena First the smaller size of β2M molecules can
more easily diffuse and approach to the latex substrate Second enhanced hydrophilic
domain on the latex particle surface by sulfate groups (SO4-) and ionized carboxylic
groups (COO-) is not favorable for larger BSA molecules but is for smaller β2M to be
anchored Third the conformational change by intra- and inter-molecular repulsion
would be faster in β2M due to lower molecular weight Finally the ionic repulsion
between β2M and latex particles is less th
57) of β2M is closer to the tested condition (pH 74) than IEP (48) of BSA
In the biocompatibility test the number of red blood cells (RBCs) ruptured by PS
latex particles ranged from 30 to 91 as the solid content of PS increased from 05 to
50 respectively However hemolysis of the other core shell latex particles su
PSPMMA100 PSPMMA90PAA10 and PSPMMA75PAA25 were less than 35 The
PSPMMA90PAA10 particles at all applied solid concentrations had less than 02
hemolysis and was the most blood compatible among the prepared latex particles
121
core shell particles such as PSPMMA100 and PSPMMA75PAA25 also had excelle
compatibility with low RBC breakage
52 Conclusions
The major conclusions from this study include
nt blood
bull microglobulin (β2M) over that of the bovine serum albumin (BSA) at pH 74
was achieved by controlling the PMMA and PAA ratio to optimize
e particles posses the most desirable requirements for use in a human dialysis
ns
icles
olecule
bull
Using engineered core shell latex particles selective adsorption of β2
physiological condition was demonstrated
bull The rate of adsorption of the β2M was also much higher than that of BSA This
hydrophobichydrophilic microdomain size
bull The core shell particles also exhibited excellent biocompatibility with blood Thes
membrane
53 Recommendation for Future Work
With the results and conclusions obtained through this study several suggestio
may be given for future work as follows
bull Competitive adsorption tests of whole blood proteins on synthesized latex partunder physiological condition
bull Preparation of latex particles with optimum hydrophobic hydrophilic microdomains to maximize selective adsorption and removal of toxin molecules
bull Membrane fabrication by suitable pore size design to discriminate a toxin mby size sieving as a function of particle layers
Evaluation of membrane performance such as flow rate in water and in simulated blood fluid (SBF)
bull Application for hemodialysis as well as other technologies such as a water remediation
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Vincent C Chanard J Caudwell V Lavaud S Wong T and Revillard J P (Kinetics of I-125 beta2 microglobulin turnover in dialyzed patients Kidney International 42 1434-1443
Ward R A Schmidt S Hullin J Hillebrand G F and Samtleben W (2000) A comparison of on-line hemodiafiltration and high-flux hemodialysis A prospectiveclinical study Journal o
Watkins R W and Robertson C R (1977) Total internal reflection technique for examination of protein sdsorption Journal of Biomedical Materials Research 11915-9
Wavhal D S and Fisher E R (2002) Hydrophilic modification of polyethersulfone membr
uyzen J Foreman K Battistutta D Saltissi D and Fleming S J (1992) Effect of dialyzer reproce
12 29-36
helman K J Braun R D and Fitzpatrick J D (1988) Investigation of the bicinchoninic acid protein assay Identification of the groups responsible for color
ms K M Arthur S J Burrell G Kelly F Phillips D W and Marshall T (2003)
140
Winchester J F Ronco C Brady J A Cowgill L D Salsberg J Yousha E Choquette M Albright R Clemmer J Davankov V Tsyurupa M PavlL Pavlov M Cohen G Horl W
ova Gotch F and Levin N W (2002) The next
step from high-flux dialysis application of sorbent technology Blood Purification
Winchester J P Ronco J A Brendolan A Davankov V Tsyurupa M Pavlova L 2001)
oglobin
Yang Q and Yang C Z (2000) Formation of an
Yoon J Y Park H Y Kim J H and Kim W S (1996) Adsorption of BSA on highly carboxylated microspheres- quantitative effects of surface functional groups
Zollars R L (1979) Kinetics of the emulsion polymerization of vinyl acetate Journal of
Zou oss-
Zou D Ma S Guan R Park M Sun L Aklonis J J and Salovey R (1992)
20 81-86
Clemme J Polaschegg H D Muller T E La Greca G Levin N W (Rationale for combined hemoperfusionhemodialysis in uremia Contribution of Nephrology 133 174-179
Winslow R M Vandergriff K D Motterlini R (1993) Mechanism of hemtoxicity Thrombosis and Haemostasis 70 36-41
S C Ge H X Hu Y Jiang Xpositively charged poly(butyl cyanoacrylate) nanoparticles stabilized with chitosColloid and Polymer Science 278 285-292
and interaction forces Journal of Colloid and Interface Science 177 613-620
Applied Polymer Science 24 1353-1370
D Derlich V Gandhi K Park M Sun L Kriz D Lee Y D Kim G Aklonis J J and Salovey R (1990) Model filled polymers 1 synthesis of crlinked monodisperse polystyrene beads Journal of Polymer Science Part A Polymer Chemistry 28 1909-1921
Model filled polymers 5 synthesis of cross-linked monodisperse polymethacrylate beads Journal of Polymer Science Part A Polymer Chemistry 30 137-144
Korea on August 7 1968 He obtained a Bachelor of Science degree in Chemistry from
Keim d graduate school and earned a
resear n Korea Institute of Science and
Coati e
ia
Busin
BIOGRAPHICAL SKETCH
Sangyup Kim was born and raised in the small town of Sung-Joo Kyungbook
yung University (Daegu Korea) in 1993 He entere
Master of Science degree in Chemical engineering in 1995 He worked as a student
cher for 2 years at Textile Polymer Division i
Technology (KIST) in Seoul Korea Next he worked over a year at the Automobile
ng Division in DongJoo-PPG in Korea In August 2001 he began studying for th
degree of Doctor of Philosophy in materials science and engineering at the University of
Florida in Gainesville After getting his PhD he plans to work at the Digital Med
ess Division of Samsung Electronics in Korea starting in January 2006
141
members in Dr El-Shallrsquos research group for their assistance advice and support I
sincerely appreciate Stephen for the assistance of thesis writing
v
TABLE OF CONTENTS page
ACKNOWLEDGMENTS iv
LIST OF TABLES viii
LIST OF FIGURES x
1 INTRODUCTION 1
2 BACKGROUND AND LITERATURE SURVEY5
21 The Significance of End Stage Renal Disease (ESRD)5 22 Hemodialysis (HD) Treatment 6 23 Advances in Membrane Technology 8 24 Sorbent Technology16 25 Limitation of Current Hemodialysis Treatment 19 26 Latex Particles 20
261 The Components for Emulsion Polymerization 21 262 Particle Nucleation 21 263 Types of Processes for Emulsion Polymerization 23 264 Chemistry of Emulsion Polymerization 24 265 Seed Emulsion Polymerization25 266 Polystyrene Latex Particles 26 267 Various Applications of Latex Particles28
27 Proteins 30 271 Interaction Forces between Proteins33 272 β -Microglobulin (β M)2 2 35 273 Serum Albumin 36
28 Protein Adsorption38 281 Interaction between Protein Molecule and Latex Particle38
29 Hypothesis for Toxin Removal39 291 Toxin Removal by Size Sieving Based on Monodispersed Pore Size 40 292 Toxin Removal by Selective Adsorption on Engineered Latex Particles 41
3 EXPERIMENTAL AND CHARACTERIZATION METHODOLOGY 44
31 Materials 44 32 Latex Particle Preparation46
321 Preparation of Seed Latex Particles47
vi
322 Preparation of Seeded Latex Particles 48 323 Preparation of Core Shell Latex Particles 48 324 Purification of Synthesized Latex Particles48
33 Characterization49 331 Degree of Conversion49 332 Fourier Transform Infrared (FTIR) Spectroscopy49 333 Quasielastic Light Scattering (QELS) 50 334 Field Emission-Scanning Electron Microscopy (FE-SEM) 50 335 Zeta Potential Measurement 50 336 Protein Adsorption51 337 Blood Biocompatibility by Hemolysis Test 53
4 RESULTS AND DISCUSSION55
41 Polymerization of Latex Particles55 411 Polystyrene Seed Latex Particles55 412 The Growth of Polystyrene (PS) Seed Latex Particles61 413 Core Shell Latex Particles 71
42 Characterization of Latex Particles75 421 Fourier Transform Infrared Spectroscopy (FTIR)75 422 Zeta Potential Measurements 75
43 Protein Adsorption Study 85 431 Adsorption Isotherm87 432 Adsorbed Layer Thickness 103 433 Gibbs Free Energy of Protein Adsorption 104 434 Kinetics of Adsorption 107
44 Blood Compatibility 112
5 SUMMARY OF RESULTS CONCLUSION AND RECOMMENDATION FOR FUTURE WORK115
51 Summary of Results115 52 Conclusions121 53 Recommendation for Future Work121
LIST OF REFERENCES122
BIOGRAPHICAL SKETCH 141
vii
LIST OF TABLES
Table page 4-1 The polymerization recipe of polystyrene (PS) seed particles 57
4-2 Continuous addition emulsion polymerization recipe for growing polystyrene (PS) latex particles less than 500nm in size 63
4-3 Continuous addition emulsion polymerization recipe for growing polystyrene (PS) latex particles larger than 500nm in size64
4-4 The preparation recipe of PS core with various shell latex particles72
4-5 The isoelectric point (IEP) of common proteins 83
4-6 The equilibrium concentration values of BSA adsorption on polystyrene (PS) latex particles calculated the Langmuir-Freundlich isotherm model 93
4-7 The equilibrium concentration values (q ) of BSA adsorption on PSPMMA particles calculated the Langmuir-Freundlich isotherm model
m 10096
4-8 The equilibrium concentration values of BSA adsorption on PSPMMA PAA particles calculated the Langmuir-Freundlich isotherm model
90 1098
4-9 The equilibrium concentration values of BSA adsorption on PSPMMA PAA particles calculated the Langmuir-Freundlich isotherm model
75 25100
4-10 The equilibrium concentration values of β M adsorption calculated the Langmuir-Freundlich isotherm model
2102
4-11 Absorbed BSA layer thickness (Aring) of BSA in phosphate buffer (PB) at 37 Co 104
4-12 Absorbed BSA layer thickness (Aring) of BSA in phosphate buffered saline (PBS) at 37 Co 104
4-13 The values of Gibbs free energy change of BSA adsorption in phosphate buffer (PB) at 37 Co 105
4-14 The values of Gibbs free energy change of BSA adsorption in phosphate buffered saline (PBS) at 37 Co 105
viii
4-15 The values of Gibbs free energy change of β M adsorption in phosphate buffere (PB) at 37 C and pH 74
2o 106
4-16 Fitting parameters of second-order kinetic model 111
ix
LIST OF FIGURES
Figure page 1-2 Stereo drawing of the αndashcarbon backbone of β M2 2
2-1 An image of the location and cross section of a human kidney 5
2-2 A schematic draw of the hemodialysis route7
2-3 Cross-sectional SEM image view of the Polyflux S (polyamide + polyacrylethersulfone + polyvinypyrollidone) dialysis membranes 11
2-4 The chemical structure of polysulfone (PSf) 12
2-5 The chemical structure of polyamide (PA)13
2-6 Chemical structure of polyacrylonitrile (PAN) 14
2-7 Emulsion polymerization system24
2-8 L-α-amino acid 31
2-9 Ribbon diagram of human β M taken from the crystal structure of the protein bound to the heavy chain of the MHC class I complex (PDB 1DUZ)
236
2-10 Secondary structure of human serum albumin (HSA) with sub-domains 37
2-11 The relationship between pore size and particle size in body-centered cubic and closed packed arrays41
2-12 Schematic representation of the protein adsorption on the core shell latex particles at pH 7442
3-1 Chemical structure of main chemicals45
3-2 Experimental setup for semi-continuous emulsion polymerization 46
3-3 The particle preparation scheme in various types and size ranges of latex particles 47
3-4 Schematic of the procedure for a protein adsorption test 52
3-5 Separation of RBC from whole blood by centrifuge process53
x
3-6 Schematic of the procedure for hemolysis test 54
4-1 Schematic representation of semi-continuous seed latex particles preparation and growth56
4-2 Scanning Electron Micrograph (SEM) of polystyrene seed latex particles (A) PS (B) PS (C) PS (D) PSS259 S233 S207 S181 59
4-3 SEM of PS latex particles less than 500nm in size (A) 258nm (B) 320nm (C) 370nm (D) 410nm 65
4-4 SEM of PS latex particles larger than 500nm in size (A) 525nm (B) 585nm (C) 640nm (D) 790nm 68
4-5 Dependence of the particle size on the surfactant to monomer ratio70
4-6 Schematic of core shell latex particle structures 71
4-7 Scanning Electron Micrograph of latex particles (A) PS (B) PSPMMA (C) PSPMMA PAA (D) PSPMMA PAA
100
90 10 75 25 73
4-8 FTIR spectra of polymerized latex particles (A) bare polystyrene (PS) (B) PSPMMA (C) PSPMMA PAA (D) PSPMMA PAA100 90 10 75 25 76
4-9 Schematic representation of ion distribution near a positively charged surface 78
4-10 Schematic representation of zeta potential measurement79
4-11 Zeta potential of PS latex particles at 25 Co 80
4-12 Zeta potential of PSPMMA latex particles at 25 C100o 81
4-13 Zeta potential of PSPMMA PAA latex particles at 25 C90 10o 82
4-14 Zeta potential of PSPMMA PAA latex particles at 25 C75 25o 82
4-15 The decay of surface potential with distance from surface in various electrolyte concentrations (1) low (2) intermediate (3) high 84
4-16 High-affinity adsorption isotherm of typical flexible polymer on solid surface 86
4-17 Overall schematic representation of the protein adsorption on the synthesized latex particles86
4-18 The dependence of UV absorbance on BSA concentration (A) 10mgml (B) 06mgml (C) 02mgml87
4-19 Standard curve of net absorbance vs BSA sample concentration 88
xi
4-20 Fitted models for adsorption isotherm of bovine serum albumin (BSA) on polystyrene latex particles at 37 C in phosphate buffer mediao 89
4-21 Adsorption isotherm of BSA on polystyrene (PS) latex particles in phosphate buffer (PB) at 37 Co 90
4-22 Adsorption isotherm of BSA adsorption 37 C in phosphate buffered saline (PBS) on PS latex particles
o
91
4-23 Conformation of bovine serum albumin (BSA) on latex particles92
4-24 Adsorption isotherm of BSA 37 C in PB on PSPMMA core shell latex particles
o100
94
4-25 Adsorption isotherm of BSA at 37 C in PBS on PSPMMA core shell latex particles
o100
95
4-26 Adsorption isotherm of BSA at 37 C in PB on PSPMMA PAA core shell latex particles
o90 10
97
4-27 Adsorption isotherm of BSA at 37 C in PBS on PSPMMA PAA core shell latex particles
o90 10
98
4-28 Adsorption isotherm of BSA 37 C in PB on PSPMMA PAA latex particleso75 25 99
4-29 Adsorption isotherm of BSA 37 C in PBS on PSPMMA PAA core shell latex particles
o75 25
99
4-30 Adsorption isotherm of β M onto PS and PSPMMA latex particles in PB at 37 C and pH 74
2 100o 101
4-31 Adsorption isotherm of β M onto PS and PSPMMA PAA latex particles in PB at 37 C and pH 74
2 90 10o 101
4-32 Adsorption isotherm of β M onto PS and PSPMMA PAA latex particles in PB at 37 C and pH 74
2 75 25o 102
4-33 Gibbs free energy of adsorption of proteins on latex particles in PB at 37 C and pH 74
o
107
4-34 The kinetics of protein adsorption in PB on PS latex particles at 37 C and pH 74
o
109
4-35 The kinetics of protein adsorption in PB on PSPMMA latex particles at 37 C and pH 74
100o
109
4-36 The kinetics of protein adsorption in PB on PSPMMA PAA latex particles at 37 C and pH 74
90 10o 110
xii
4-37 The kinetics of protein adsorption in PB on PSPMMA PAA latex particles at 37 C and pH 74
75 25o 110
4-38 Image of red blood cells 113
4-39 Hemolysis caused by latex particles n=5114
xiii
Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy
SYNTHESIS AND ENGINEERING OF POLYMERIC LATEX PARTICLES FOR MEDICAL APPLICATIONS
By
Sangyup Kim
December 2005
Chair Hassan El-Shall Major Department Materials Science and Engineering
Latex particles with well-defined colloidal and surface characteristics have received
increasing attention due to their useful applications in many areas especially as solid
phase supports in numerous biological applications such as immunoassay DNA
diagnostic cell separation and drug delivery carrier Hemodialysis membrane using
these particles would be another potential application for the advanced separation
treatment for patients with end stage renal disease (ESRD) It is desirable to remove
middle molecular weight proteins with minimal removal of other proteins such as
albumin Thus it is necessary to understand the fundamental interactions between the
particles and blood proteins to maximize the performance of these membranes This
improvement will have significant economic and health impact
The objective of this study is to synthesize polymeric latex particles of specific
functionality to achieve the desired selective separation of target proteins from the human
blood Semi-continuous seed emulsion polymerization was used to prepare monodisperse
xiv
polystyrene seed particles ranging from 126plusmn75 to 216plusmn53 nm in size which are then
enlarged by about 800nm Surfactant amount played a key role in controlling the latex
particle size Negatively charged latex particles with a different hydrophobicity were
prepared by introduction of a sodium persulfate initiator and hydrophilic acrylic acid
monomer The prepared polymeric particles include bare polystyrene (PS) particles less
hydrophobic PS core and PMMA shell particles and more hydrophilic PS core and
PMMA-co-PAA shell latex particles with a 370nm mean diameter SEM light scattering
and zeta potential measurements were used to characterize particle size and surface
properties Adsorption isotherms of two proteins bovine serum albumin (BSA) and β2-
microglobulin (β2M) on latex particles were obtained as a function of pH and ionic
strength using the bicinchoninic acid (BCA) assay method The Langmuir-Freundlich
adsorption model was used to determine the adsorption amount of protein at equilibrium
The thickness of adsorbed BSA layer on latex particles was obtained in order to
investigate the adsorption orientation such as end-on or side-on mode Adsorption
kinetics experiments for both proteins and all latex particles were also performed The
adsorption kinetic constant determined from the Langmuir-Freundlich adsorption
isotherm model was used to calculate Gibbs free energy of adsorption to compare the
competitive adsorption of BSA and β2M Hemolysis tests were performed to investigate
the blood compatibility of synthesized latex particles PSPMMA90PAA10 and
PSPMMA75PAA25 core shell latex particles had desirable material properties with not
only a large amount and high rate of selective β2M adsorption over BSA but also high
blood compatibility showing less than 3 hemolysis
xv
CHAPTER 1 INTRODUCTION
End stage renal disease (ESRD) is a chronic condition in which kidney function is
impaired to the extent that the patientrsquos survival requires removal of toxins from the
blood by dialysis therapy or kidney transplantation The National Kidney Foundation
estimates that over 20 million Americans had chronic kidney disease in 2002 [NKF] The
number of people with ESRD is rapidly increasing in the United States with
approximately 96 295 incidents and 406 081 prevalent patients including 292 215 on
dialysis and 113 866 with a functioning graft in 2001 It is projected that there will be
more than 22 million ESRD patients by 2030 [USRDS 2003] The expenditure for the
ESRD treatment program had reached $228 billion 64 of the Medicare budget in
2001 Due in part to the limited availability of kidneys for transplantation hemodialysis
is the primary clinical treatment for the patients with ESRD
The central element of a hemodialysis instrument is the semipermeable membrane
that allows for selective transport of low molecular weight biological metabolites less
than 5000 Da such as urea and creatinine as well as excess water and electrolytes [Baker
2004] from the blood One limitation of current dialysis technologies is the inability to
efficiently remove middle molecular weight toxins such as β2-Microglobulin (β2M) and
interleukin 6 (IL-6)
β2M is a causative protein of dialysis-related amyloidosis (DRA) a disease arising
in patients with chronic kidney failure as a serious complication of long-term
hemodialysis treatment [Gejyo et al 1985] β2M deposition in tissue is the primary cause
1
2
of destructive arthritis and carpal tunnel syndrome [Vincent et al 1992 Drueke 2000]
The β2M structure is shown in Figure 1-2
Figure 1-2 Stereo drawing of the αndashcarbon backbone of β2M [Becker et al 1985]
Although attempts have been made to increase the efficiency of middle molecular
weight toxin removal by changes in the membrane pore size and the use of innovative
materials to adsorb these toxins [Samtleben et al 1996 Ronco et al 2001] removal
efficiency is not as high as those achieved by a normal healthy kidney Traditional
membranes have a number of processing and performance limitations [Westhuyzen et al
1992 Leypoldt et al 1998] such as a restricted choice of surface chemistries and limited
control of porosity The development of novel engineering membrane technology is
needed to remove middle molecule toxins
Polymeric latex particles have received increasing attention in medical application
areas especially as solid phase supports in biological applications [Piskin et al 1994]
Examples of these applications include immunoassay [Chen et al 2003 Radomske-
Galant et al 2003] DNA diagnostic [Elaїssari et al 1998] cell separation drug delivery
carrier [Luck et al 1998 Kurisawa et al 1995 Yang et al 2000] etc This is because of
3
the well-defined colloidal and surface characteristics of the particles By using a seed
emulsion polymerization method it is possible to synthesize monodisperse latex particles
with various particle size ranges and surface chemistry Functionalized core-shell latex
particles can be introduced by multi-step emulsion polymerization Core particles are
synthesized in the first stage of the polymerization and the functional monomer is added
in the second stage This is done without any emulsifier addition to prevent the
production of new homopolymer particles [Keusch et al 1973] The core-shell particles
are useful in a broad range of applications because of their improved physical and
chemical properties over their single-component counterparts [Lu et al 1996 Nelliappan
et al 1997] Through the development of a hemodialysis membrane using monodisperse
latex particles improvements in advanced separation treatment for patients with end
stage renal disease (ESRD) can be realized This requires the maximum removal of
middle molecular weight proteins with minimal removal of other beneficial proteins such
as albumin Thus an understanding of the fundamental interactions between the particles
and biopolymers is vital to maximize the performance of this membrane technology
The field of material science and biotechnology is based on fundamental chemistry
has developed over the past three decades into todayrsquos powerful discipline that enables
the development of advanced technical devices for pharmaceutical and biomedical
applications This novel and highly interdisciplinary field is closely associated with both
the physical and chemical properties of organic and inorganic particles [Niemeyer 2001]
Hemodialysis membrane using these particles would lead to improvements in the
advanced separation treatment for patients with end stage renal disease (ESRD) The
interdisciplinary nature of this approach enables a more complete understanding of the
4
phenomena of protein adsorption and the material properties necessary for selective
separation This innovative approach to membrane fabrication has the potential of making
inexpensive highly efficient membranes for both industrial and specialized separation
processes
The goal of this study is to prepare polymeric latex particles with tailored
properties to maximize separation of toxin molecules and to investigate the fundamental
interactions between the particles and molecules in the biological system in order to
optimize the performance of a membrane material for these applications
Polymeric latex particles were synthesized with specific functionality in an attempt
to achieve selective separation Seeded emulsion polymerization was used to synthesize
functionalized monodisperse latex particles in various sizes Negatively charged
hydrophobic polystyrene latex particles were synthesized by the same method Core shell
latex particles PSPMMA100 PSPMMA90PAA10 PSPMMA75PAA25 were also
synthesized to differentiate the degree of hydrophobicity of particles Scanning Electron
Micrograph (SEM) and light scattering measurements were used to characterize particle
size and shape and zeta potential measurements were conducted to measure the electrical
surface property of synthesized particles Adsorption isotherms of target proteins bovine
serum albumin (BSA) and β2M on latex particles were obtained as a function of pH
ionic strength and protein concentrations using the BCA assay method Adsorption
kinetics for both proteins on the latex particles were also measured Finally hemolysis
tests were run to determine the biocompatibility of polymer latex particles with human
blood This research will be described in more detail in chapters 3 and 4
CHAPTER 2 BACKGROUND AND LITERATURE SURVEY
21 The Significance of End Stage Renal Disease (ESRD)
The kidneys are responsible for removing excess fluid minerals and wastes from
the blood regulating electrolyte balance and blood pressure and the stimulation of red
blood cell production They also produce hormones such as erythropoietin (EPO) and
calcitriol that keep bones strong and blood healthy [Casadevall and Rossert 2005 Kurbel
et al 2003] EPO acts on the bone marrow to increase the production of red blood cell in
case of bleeding or moving to high altitudes Calcitriol mainly acts on both the cells of
the intestine to promote the absorption of calcium from food and also bone to mobilize
calcium from the bone to the blood When kidneys fail harmful waste builds up blood
pressure rises and body retains excess fluid The body also does not make enough red
blood cells When this happens treatment is needed to replace the function of failed
kidneys
Figure 2-1 An image of the location and cross section of a human kidney
5
6
The National Kidney Foundation estimates that over 20 million Americans had
chronic kidney disease in 2002 [NKF] Chronic renal disease is a gradual and progressive
loss of the function unlike acute renal disease where sudden reversible failure of kidney
function occurs Chronic renal failure usually takes place over a number of years as the
internal structures of the kidney are slowly damaged In the early stages there can be no
symptoms In fact progression may be so gradual that symptoms do not occur until
kidney function is less than one-tenth that of a normal health kidney If left untreated
chronic kidney disease may ultimately lead to kidney failure known as End Stage Renal
Disease (ESRD) ESRD is a rapidly growing heath-care problem in the United States In
2001 approximately 96295 incidents and 406081 prevalent patients were diagnosed
with ESRD including 292215 patients on dialysis and 113866 patients with a
functioning graft [USRDS 2003] The projected number of patients with ESRD is
expected to exceed 22 million patients by 2030 with much of this growth being driven by
the increasing prevalence of major contributing factors such as diabetes and high blood
pressure [USRDS 2003] A great extent of ESRD program cost and Medicare budget
have been spent in 2001 Due in part to a limited availability of kidneys for
transplantation hemodialysis (HD) is the primary method of treatment for ESRD and is
currently used for approximately 61 of US ESRD patients
22 Hemodialysis (HD) Treatment
HD removes toxins from the body by extracorporeal circulation of the blood
through a semipermeable membrane referred to as a dialyzer The toxins are removed
primarily by diffusion across the membrane to a dialysate solution which is circulated on
the opposite side of the membrane The cleaned blood is then returned to the blood
stream A surgically constructed vascular access connects the extracorporeal circuit to the
7
patientrsquos system Treatments are almost always performed three times per week in
specially equipped dialysis facilities for 3-4 hours per each treatment Figure 2-2 shows
the scheme of the hemodialysis route The critical element of a HD instrument is the
semipermeable membrane which allows for selective transport of low molecular weight
biological metabolites from blood
igure 2-2 A schematic draw of the hemodialysis route
ESRD patients who undergo dialysis therapy often experience several other
problems associated with the treatment such as anemia fatigue bone problems joint
problems itching sleep disorders and restless legs Anemia is common in patient with
kidney disease because the kidneys produce the hormone erythropoietin (EPO) which
stimulates the bone marrow to produce red blood cells Diseased kidneys often do not
produce enough EPO to stimulate the bone marrow to make a sufficient amount of red
blood cells This leads to bone disease referred to as renal osteodystrophy and causes
bones to become thin and weak or malformed and can affect both children and adults
Older patients and women who have gone through menopause are at greater risk for this
disease Uremic toxins which cannot be remove from the blood by the current dialyzer
membranes can lead to itching This problem can also be related to high levels of
F
8
parathyroid hormone (PTH) Dialysis patients can also experience day-night revers
is they have insomnia at night and sleep during the day This can be related to possible
nerve damage in the body and a chemical imbalance in the blood due to the excess toxin
Oxidative stress is a problem for patients on maintenance dialysis This problem is due to
an imbalance between pro- and antioxidant factors [Roselaar et al 1995 Cristol et al
1994] Oxidative stress affects oxidation of low-density lipoproteins which are the main
factor for atherogenesis [Huysmans et al 1998] and is also involved in the development
of malignancies and diabetes mellitus [Rice-Evans et al 1993] In order to reduce
antioxidant defense dialysis is needed to contribute to help stimulate free radical
production or eliminate antioxidants Dialysis-related amyloidosis (DRA) is also a
common and serious problem for people who have been on dialysis for more than 5
years DRA develops when proteins in the blood deposit on joints and tendons causi
pain stiffness and fluid in the joint as is the case with arthritis Normally the healthy
kidneys can filter out these proteins but dialysis filters are not as effective
23 Advances in Membrane Technology
al that
s
ng
Dialysis for blood of ESRD
Hemo filtration
s
ysis using a cellulose based membrane occurred
1913 John Abel [1990] from the Johns Hopkins Medical School described a method
purification is widely used in the treatment
dialysis (HD) techniques use a semi-permeable membrane to replace the
role of the kidney The membranes used in HD can be broadly classified into those based
on cellulose and those manufactured from synthetic copolymers These membranes come
in various shapes such as sheets tubular structures or hollow fiber arrangements The
hollow fiber type is the most popular and is incorporated into over 800 different device
in world wide [Ronco et al 2001]
The first attempt at blood dial
9
where sing
e
om
ysis was performed until the mid 1960 The basic
molec
rial in
een
benzyl)
was used for the membrane with
hepar
by the blood of a living animal may be submitted to dialysis outside the body u
a membrane based on cellulose and returned to the natural circulation without exposur
to air infection by microorganisms or any alteration that would necessarily be prejudicial
to life This same technique is still used to today however the device used has been
modified over the years as better membranes were developed and the anti-coagulant
heparin has become available
Cellulose membranes have been widely used for the treatment of renal failure fr
1928 when the first human dial
ular structure of cellulose is made of a long chain containing hydroxyl (OH)
groups The realization that such groups imparted undesirable qualities on the mate
respect to blood contact behavior was discovered in the early 1970s and since has b
the focus of development These modified cellulose membranes used the partial
substitution of benzyl groups to replace the proton of the hydroxyl groups in an attempt
to reduce their negative effect The result is a molecular mosaic of hydrophobic (
and hydrophilic (hydroxyl and cellulose) regions
Kolff [Van Noordwijk 2001] studied the rotating drum artificial kidney for patients
with acute renal failure in 1943 Cellophane tubing
in as the anticoagulant For the next 17 years hemodialysis therapy was performed
by this method but only for the patients with acute reversible renal failure Vascular
access required repeated surgical insertions of cannulas (slender tubes) into an artery and
vein and limited the number of treatments for a patient could receive in order to
minimize the amount of vascular damage
10
Initially the need for dialysis in patients with acute renal failure was determ
mainly by the development of signs and sym
ined
ptoms of uremia After dialysis some time
might
et al
ent improvement In 1960 the development of a shunt [Quinton et al
1960]
fone (PSf) polyamide (PA) and polyacrylonitrile (PAN) by phase inversion or
precip
lidone
e
elapse before uremic manifestations returned to warrant a sequential treatment of
dialysis Many patients with acute renal failure secondary to accidental or surgical
trauma were hypercatabolic but the interdialytic interval might be prolonged because of
anorexia or use of a low-protein diet However Teschan and his coworkers [Obrien
1959] showed that patient well-being and survival were improved by what they termed
prophylactic daily hemodiaysis or administration of the treatment before the patient
again became sick with uremia Their report in 1959 was the first description of daily
hemodialysis
Development of membrane accessories such as a shunt has also been an area of
focus for treatm
a flexible polytetrafluoroethylene (PTFE or Teflonreg) tubing made many more
hemodialysis treatments possible for chronic kidney failure patients PTFE has a non-
stick surface and is relative biocompatibility leading to minimized blood clotting in the
shunt
Synthetic membranes are prepared from engineered thermoplastics such as
polysul
itation of a blended mixture resulting in the formation of asymmetric and
anisotropic structures Figure 2-3 shows a fiber type of the Polyflux S membrane
consisting of poluamide (PA) polyacrylethersulfone (PAES) and polyvinylpyrro
(PVP) with the integral three-layer structure The skin layer on the inside fiber typ
membrane contacts blood and has a very high surface porosity and a narrow pore size
11
distribution This layer constitutes the discriminating barrier deciding the permeabili
and solute retention properties of the membrane The skin layer is supported by thick
sponge-type structure larger pores providing mechanical strength and very low
hydrodynamic resistance
ty
Figure 2-3 Cross-sectional SEM image view of the Polyflux S (polyamide +
polyacrylethersulfone + polyvinypyrollidone) dialysis membranes [Deppisch et al 1998]
n
[Malchesky 2004] because of its thermal stability mechanical strength and chemical
inertn ciated
ls
m
PSf is a widely used membrane material for the hemodialysis applicatio
ess According to a report from the National Surveillance of Dialysis-Asso
Disease (NSDAD) in the US over 70 of hemodialysis membranes were PSf based
[Bowry 2002] This is most likely because PSf has many advantages over other materia
This synthetic polymer is one of few materials that can withstand sterilization by stea
ethylene oxide and γ-radiation PSf membrane can be prepared by conventional
immersion precipitation methods into many different shapes including porous hollow
12
fiber or flat sheet hemodialysis membranes The material also has a high permeab
low molecular weight proteins and solute and high endotoxin retention The chemical
structure of PSf is shown in Figure 2-4
ility to
Figure 2-4 The chemical structure of polysulfone (PSf)
There is one major disadvantage to PSf The hydrophobic nature of the PSf causes
ment alternative pathway
leadin
s not
3
et al
in
O acrylate
serious complications through the activation of the comple
g to the adsorption of serum proteins onto the membranes [Singh et al 2003]
Anticoagulants are added during dialysis therapy to avoid blood clotting but this doe
completely eliminate the problem In order to overcome this disadvantage of the PSf
membrane various studies have been performed to change the materialrsquos surface
properties These investigations include hydrophilic polymer coating [Brink et al 199
Kim et al 1988 Higuchi et al 2003] layer grafting onto PSf membrane [Wavhal
2002 Song et al 2000 Pieracci et al 2002 Mok et al1994] and chemical reaction of
hydrophilic components onto the membrane surface [Higuchi et al 1990 1991 1993
Blanco et al 2001 Nabe et al 1997 Guiver et al 1993] Hydrophilic monomers 2-
hydroxy-ethylmethacrylate (HEMA) acrylic acid (AA) and methacrylic acid (MMA)
have also been grafted onto PSf membrane to increase flux and Bovine Serum Album
(BSA) retention [Ulbricht et al 1996] Hancock et al [2000] synthesized
polysulfonepoly(ethylene oxide) (PEO) block copolymers to improve the resistance to
platelet adhesion Kim et al [2003] also studied blending a sulfonated PE
diblock copolymer into PSf in order to reduce platelet adhesion and enhance
13
biocompatibility PEO is a commonly used biomaterial due to its excellent resistance
protein adsorption and inherent biocompatibility [Harris 1992] Kim et al [20
a self-transformable copolymer to enhance the hydrophilicity of an asymmetric PSf
membrane with an ultra-thin skin layer The polymer had an entrapped diblock
copolymer containing a hydrophilic block of poly (ethylene glycol) (PEG)-SO
to
05] studied
ate
lic graft
l)
e of thier
aromatic
orand
3 acryl
and a hydrophobic block of octadecylacrylate (OA) Molecular dynamic (MD)
simulations were performed as a function of copolymer density to optimize interfacial
structure information McMurry [2004] developed a strategy using an amphiphi
copolymer added to PSf membranes by introducing polysulfone-g-poly (ethylene glyco
When compared to unmodified PSf these graft copolymer and resulting blend
membranes are found to hold promise for biomedical device applications
Polyamide (PA) membranes have also been used for hemodialysis becaus
mechanical strength in both wet and dry conditions Polyamide consists of
aliphatic monomers with amide bonding (-CONH-) also known as a peptide bond
The basic amide bond in polyamide is shown in Figure 2-5 R1 and R2 can be either
aromatic or aliphatic linkage group
C N
H
O
~ R R1 2~C N
H
O
~ R R1 2~
Figure 2-5 The chemical structure of polyamide (PA)
Panichi and co-workers [1998] evaluated the biocompatibility of the PA membrane
ompatible technique due to the
use of a synthetic membrane with a sterile re-infusion fluid and the convective removal of
and concluded that PA hemofiltration was a highly bioc
14
the ac
e
e
is
n
is a semi-crystalline polymer and the mechanical properties
strong
2
CN
tivated anaphylatoxins and β2-Microglobulin (β2M) The PA based membrane
Polyfluxreg (manufactured by Gambro GmbH Germany) blended with polyamide
polyarylethersulfone and polyvinylpyrrolidone (PVP) was able to clean small molecules
such as urea dreatinine and phosphate as well as decrease β2M amount by 502
[Hoenich et al 2002] Due to the non-selectivity of the membrane removal of thes
unwanted materials also led to the undesirable loss of beneficial proteins during therapy
Meier et al [2000] evaluated different immune parameters using a modified cellulos
low-flux hemophan and synthetic high-flux PA membrane during a 1 year period in
chronic hemodialysis patients They found that the 1-year immunological evaluation of
hemodiaysis membrane biocompatibility was associated with changes in the pattern of
chronic T-cell actiovation
Polyacrylonitrile (PAN) is another commonly used membrane material because it
inherently hydrophilic and has been commercialized for ultrafiltration and microfiltratio
[Scharnagl et al 2001] PAN
ly depend on the crystalline structures The chemical structure of PAN is shown in
figure 2-6
CH CH
n2
CN
CH CH n
Chemical structure of polyacrylonitrile (PAN)
The addition of additives such as polyvinylpyrrolidone (PVP) as a pore forming
agent gives PAN membranes more flexible processing parameters and increased
e has been optimized through
copolymerization with many other vinyl monomers including glycidyl methacrylate
Figure 2-6
performance [Jung et al 2005] PAN membrane performanc
15
[Godj
acid
for
for
a non-
solub this
in
oid underneath the inner
surface of membrane decreased by increasing the amount of solvent DMSA in the
evargova et al 1999 Hicke et al 2002] N-vinylimidazole [Godjevargova et al
2000] hydroxyl ethyl methacrylate [Ray et al 1999 Bhat et al 2000] methacrylic
[Ray et al 1999] vinyl pyrrolidone [Ray et al 1999] acrylic acid [Trotta et al 2002]
acrylamide [Musale et al 1997] and vinylchloride [Broadhead et al 1998] These
monomers provide a reactive group for enzyme immobilization improved mechanical
strength solvent-resistance pervaporation permeation flux anti-fouling and bio-
compatibility Because of this PAN-based copolymer membranes have great potential
the treatment of hemodialysis in an artificial kidney This material can also be used
other applications like the treatment of wastewater the production of ultra-pure water
biocatalysis together with separation and methanol separation by pervaporation
Lin and his coworker [2004] studied the modification of PAN based dialyzer
membranes to increase the hemocompatibility by the immobilization of chitosan and
heparin conjugates on the surface of the PAN membrane When a foreign material is
exposed to blood plasma proteins are adsorbed clotting factors are activated and
le fibrin network or thrombus is formatted [Goosen et al 1980] The result of
research was that the biocompatible chitosan polymer and a blood anticoagulant hepar
prevented blood clotting They showed prolonged coagulation time reduced platelet
adsorption thrombus formation and protein adsorption
Nie et al [2004] studied PAN-based ultrafiltration hollow-fiber membranes
(UHFMs) In order to improve the membrane performance acrylonitrile (AN) was
copolymerized with other functional monomers such as maleic anhydride and α-allyl
glucoside They found that the number and size of macrov
16
intern erum
and
d a
rface The second step is the
transf after
yjak et
embrane
1978 Malchesky et al 1978] has been further developed to increase the efficiency of
al coagulant The water flux of the UHFMs also decreased while the bovine s
albumin rejection increased minutely Godjevargova et al [1992] modified the PAN
based membrane with hydroxylamine and diethylaminoethylmethacrylate to improve
membrane dialysis properties Formed functional groups like primary amine oxime
tertiary amine groups provided the membrane with more hydrophilic properties an
substantial increase in the permeability of the membranes
The wide use of filtration in practice is limited by membrane fouling Solute
molecules deposit on and in the membrane in the process of filtration causing dramatic
reduction in flux through the membrane Fouling occurs mostly in the filtration of
proteins Three kinetic steps are involved in the fouling of UF membranes according to
Nisson [1990] The first step is the transfer of solute to the su
er of solute into the membrane until it either finally adsorbs or passes through
a set of adsorption-desorption events The third step includes surface binding
accompanied by structural rearrangement in the adsorbed state [Ko et al 1993] Br
al [1998] studied the surface modification of a commercially available PAN membrane
to develop superior filtration properties with less fouling by proteins The PAN
membrane was immersed in excess NaOH solution to convert some of the surface nitrile
groups into carboxylic groups by the hydrolysis process This modified PAN m
was not so severely fouled in the Bovine Serum Albumin (BSA) filtration test The pore
size however decreased during the hydrolysis process leading to a significant reduction
in flux and made the membrane less productive in the ultrafiltration (UF) mode
24 Sorbent Technology
Over the last three decades sorbent technology [Castino et al 1976 Korshak et al
17
dialysis or replace it for the treatment of ESRD Sorbents remove solutes from solution
through specific or nonspecific adsorption depending on both the nature of the solute and
the sorbent Specific adsorption contains tailored ligands or antibodies with high
selectivity for target molecules een used in autoimmune
disord
and
e
al 1996] various resins [Ronco et al 2001]
album
e
as
Specific adsorbents have b
ers such as idiopathic thrombocytopenic purpura [Snyder et al 1992] and for the
removal of lipids in familial hyper cholesterolemia [Bosch et al 1999] Nonspecific
adsorbents such as charcoal and resins attract target molecules through various forces
including hydrophobic interactions ionic (or electrostatic) attraction hydrogen bonding
and van der Waals interactions
New dialysate with sorbents has become an accepted modification of dialysis
sorbent hemoperfusion is gaining ground as a valuable addition to dialysis especially as
new sorbents are developed [Winchester et al 2001] Hemoperfusion is defined as th
removal of toxins or metabolites from circulation by the passing of blood within a
suitable extracorpoteal circuit over semipermeable microcapsules containing adsorbents
such as activated charcoal [Samtleben et
in-conjugated agarose etc Novel adsorptive carbons with larger pore diameters
have been synthesized for potential clinical use [Mikhalovsky 1989] Newly recognized
uremic toxins [Dhondt et al 2000 Haag-Weber et al 2000] have resulted in several
investigations on alternatives to standard or high-flux hemodialysis to remove thes
molecules These methods include hemodiafilteration with [de Francisco et al 2000] or
without [Ward et al 2000 Takenaka et al 2001] dialysate regeneration using sorbents
well as hemoperfusion using such adsorbents as charcoal and resins
18
Kolarz et al [1989 1995] studied the hyper-crosslinked sorbent prepared from
styrene and divinylbenzene (DVB) for a hemoperfusion application They found that the
pore structure of a swelling sorbent was changed by additional crosslinking with α αrsquo-
dichloro-p-xylene in the presence of a tin chloride catalyst and in a di
chloroethane
soluti and
en
decyl groups that attract β2M through a hydrophobic interaction The
adsor
of
5]
d
penet
on They also realized that the hemocompatibility was useful for nemoperfusion
could be imparted to the sorbents by introducing sulfonyl groups at a concentration of
about 02mmolg
A special polymeric adsorbing material (BM-010 from Kaneka Japan) has be
investigated by another group [Furuyoshi et al 1991] for the selective removal of β2M
from the blood of dialysis partients The adsorbent consists of porous cellulose beads
modified with hexa
ption capacity of this material is 1mg of β2M per 1ml of adsorbent Using a
hemoperfusion cartridge containing 350ml of these cellulose beads in sequence with a
high-flux hemodialyzer several small clinical trials were performed During 4-5 hours
treatment about 210mg of β2M were removed thus reducing the concentration in the
blood by 60-70 of the initial level [Nakazawa et al 1993 Gejyo et al 1993 199
RenalTech developed a hemoperfusion device BetaSorbreg containing the hydrate
cross-linked polystyrene (PS) divinylbenzene (DVB) resin sorbents with a pore structure
designed to remove molecules between 4 and 30 kDa [Winchester et al 2002] In this
case solute molecules are separated according to their size based on their ability to
rate the porous network of the beaded sorbents The resin beads were prepared with
a blood compatible coating and confirmed to be biocompatible in vivo in animals
[Cowgill et al 2001]
19
25 Limitation of Current Hemodialysis Treatment
Hemodialysis is a widely used life-sustaining treatment for patients with ESRD
However it does not replace all of the complex functions of a normal healthy kidne
a result patients on dia
y As
lysis still suffer from a range of problems including infection
accelerated cardio ion anemia
chron
e
se middle
alysis
sessio
s
f
vascular disease high blood pressure chronic malnutrit
ic joint and back pain and a considerably shortened life span One significant
limitation of the current dialysis technology is the inability to efficiently remove larger
toxic molecules This is mainly because of the broad pore size distribution reducing th
selective removal of toxins and unsatisfied biocompatibility causing lots of
complications such as inflammation blood clotting calcification infection etc
Dialysis purifies the patients blood by efficiently removing small molecules like
salts urea and excess water However as toxic molecules increase in size their removal
rate by hemodialysis substantially declines Typically only 10 - 40 of the
molecular weight toxins (300-15000 Da) are removed from the blood during a di
n [Vanholder et al 1995] These toxins then reach an abnormally high level and
begin to damage the body One such toxin β2M causes destructive arthritis and carpal
tunnel syndrome by joining together like the links of chain to form a few very large
molecules and deposit damaging the surrounding tissues [Lonnemann et al 2002] This i
also a main cause of mortality for long-term dialysis patients Other middle molecule
toxins appear to inhibit the immune system and may play a significant role in the high
susceptibility to infections in dialysis patients Still others are believed to impair the
functioning of several other body systems such as the hematopoietic and other endocrine
systems This may contribute to accelerated cardiovascular disease the leading cause o
20
death among dialysis patients as well as clinical malnutrition which affects up to 50 of
this patient population
Over the last decade polymeric dialysis membranes have been developed to
increase the capacity for removing middle molecular weight toxins by changing the pore
size of dialyzer membranes and using new materials that adsorb these toxins for
impro se
styrene made during World War II in the United
States The Dow chemical compan anufacturer of polystyrene
includ
d of
on
rug
ntrol of
ved removal characteristics However removal efficiency is not as high as tho
achieved by a normal healthy kidney
26 Latex Particles
The first synthetic polymer synthesized using emulsion polymerization was a
rubber composed of 13-butadiene and
y has been a major m
ing latex which they used in paint formulations The theory of emulsion
polymerization in which a surfactant is used was established by Harkins [1948] and by
Smith and Ewart [1948] By 1956 the technology was complete including the metho
building larger diameter particles from smaller ones The product by this emulsi
polymerization is referred to as latex a colloidal dispersion of polymer particles in water
medium [Odian 1991] Latexes are currently undergoing extensive research and
development for a broad range of areas including adhesives inks paints coatings d
delivery systems medical assay kits gloves paper coatings floor polish films carpet
backing and foam mattresses to cosmetics The relatively well known and easy co
the emulsion process is one of main advantages for these applications Therefore
polymeric latex particle prepared by emulsion polymerization can be a candidate for the
medical applications because of the easy control of the particle size and morphology as
well as flexible surface chemistry to be required
21
261 The Components for Emulsion Polymerization
The main components for emulsion polymerization process are the monomer a
dispersing medium a surfactant and an initiator Available monomers are styrene
sing medium is usually water
which
ticles
n
ation To
monomer droplets but in micelles because the initiators are
insolu le
and
olution
butadiene methylmethacylate acryl acid etc The disper
will maintain a low solution viscosity provide good heat transfer and allow
transfer of the monomers from the monomer droplets into micelles and growing par
surrounded by surfactants respectively The surfactant (or emulsifier) has both
hydrophilic and hydrophobic segments Its main functions is to provide the nucleatio
sites for particles and aid in the colloidal stability of the growing particles Initiators are
water-soluble inorganic salts which dissociate into radicals to initiate polymeriz
control the molecular weight a chain transfer agent such as mercaptan may be present
262 Particle Nucleation
Free radicals are produced by dissociation of initiators at the rate on the order of
1013 radicals per milliliter per second in the water phase The location of the
polymerization is not in the
ble in the organic monomer droplets Such initiators are referred to as oil-insolub
initiators This is one of the big differences between emulsion polymerization
suspension polymerization where initiators are oil-soluble and the reaction occurs in the
monomer droplets Because the monomer droplets have a much smaller total surface area
they do not compete effectively with micelles to capture the radicals produced in s
It is in the micelles that the oil soluble monomer and water soluble initiator meet and is
favored as the reaction site because of the high monomer concentration compared to that
in the monomer droplets As polymerization proceeds the micelles grow by the addition
of monomer from the aqueous solution whose concentration is refilled by dissolution of
22
monomer from the monomer droplets There are three types of particles in the emulsion
system monomer droplets inactive micelles in which polymerization is not occurring
and active micelles in which polymerization is occurring referred to as growing polymer
particles
The mechanism of particle nucleation occurs by two simultaneous processes
micellar nucleation and homogeneous nucleation Micellar nucleation is the entry of
radicals ei
ther primary or oligomeric radicals formed by solution polymerization from
the aq ed
the
r
s
n
is coagulation with other particles rather than
polymerization of monomer The driving force for coagulation of precursor particles
ueous phase into the micelles In homogeneous nucleation solution-polymeriz
oligomeric radicals are becoming insoluble and precipitating onto themselves or onto
oligomers whose propagation has ended [Fitch et al 1969] The relative levels of micella
and homogeneous nucleation are variable with the water solubility of the monomer and
the surfactant concentration Homogeneous nucleation is favored for monomers with
higher water solubility and low surfactant concentration and micellar nucleation is
favored for monomers with low water solubility and high surfactant concentration It has
also been shown that homogeneous nucleation occurs in systems where the surfactant
concentration is below CMC [Roe 1968] A highly water insoluble monomer such a
styrene [Hansen et al 1979 Ugelstad et al 1979] has probably created by micellar
nucleation while a water soluble monomer such as vinyl acetate [Zollars 1979] has bee
formed by homogeneous nucleation
A third latex formation reaction mechanism has been proposed referred to as
coagulative nucleation In this reaction the major growth process for the first-formed
polymer particles (precursor particles)
23
sever les
yer
es
r a
l
the beginning of the polymerization As soon as the initiator is
gins with the formation and
growt
and
ved
added at one time during the later stages of the polymerization prior to complete
al nanometers in size is their relative instability compared to larger sized partic
The small size of a precursor particle with its high curvature of the electrical double la
permits the low surface charge density and high colloidal instability Once the particl
become large enough in size maintaining the high colloidal stability there is no longe
driving force for coagulation and further growth of particles takes place only by the
polymerization process
263 Types of Processes for Emulsion Polymerization
There are three types of production processes used in emulsion polymerization
batch semi-continuous (or semi-batch) and continuous In the batch type process al
components are added at
added and the temperature is increased polymerization be
h of latex particles at the same time There is no further process control possible
once the polymerization is started In the semi-continuous emulsion polymerization
process one or more components can be added continuously Various profiles of particle
nucleation and growth can be generated from different orders of component addition
during polymerization There are advantages to this process such as control of the
polymerization rate the particle number colloidal stability copolymer composition
particle morphology In the continuous process the emulsion polymerization components
are fed continuously into the reaction vessel while the product is simultaneously remo
at the same rate High production rate steady heat removal and uniform quality of
latexes are advantages of the continuous polymerization processes
Other available methods include the intermittent addition and shot addition of one
or more of the components In the shot addition process the additional components are
24
conversion of the main monomer This method has been used successfully to develo
water-soluble functional monomers such as sodium styrene sulfonat
p
e [Kim et al 1989]
264
lsion
Chemistry of Emulsion Polymerization
Emulsion polymerization is one type of free radical polymerization and can be
divided into three distinct stages initiation propagation and termination The emu
polymerization system is shown in Figure 2-7
Aqueous phaseAqueous phase
Micelle withmonomer
I Rbull
Emulsifier
Polymer particle swollenWith monomer
Monomerdroplet
I Rbull
Micelle withmonomer
I Rbull
Emulsifier
Polymer particle swollenWith monomer
MonomerdropletMonomerdroplet
I Rbull
Figure 2-7 Emulsion polymerization system [Radicals (Rmiddot) are created from initiators
(I) Monomer is transferred from larger monomer droplet into micelles by emulsifier Initiated polymer particle by radicals is keep growing until monomers are all consumed The reaction is performed in aqueous media]
In the initiation stage free radicals are created from an initiator by heat or an
ultraviolet radiation source The initiator with either peroxides groups (-O-O-) such as
odium persulfate or azo groups (-N=N-) such as azobisisobutyronitrile is commonly
used for em t
with the m er
chain ere
s
ulsion polymerization The primary free radicals created from initiator reac
onomer for initiation of polymerization In the propagation stage the polym
grows by monomer addition to the active center a free radical reactive site Th
are two possible modes of propagations head-to-head addition and head-to-tail addition
25
The head-to-tail mode is the predominant configuration of the polymer chain a result o
steric hindrance of the substitute bulky group In the termination stage polymer chain
growth is terminated by either coupling of two growing chains forming one polymer
molecule or transferring a hydrogen atom (dispropotionation) from one growing chain to
another forming two polymer molecules one having a saturated end group and the other
with an unsaturated end-group
265 Seed Emulsion Polymerization
Polymer latex particles have received increasing interest because of the versatili
of the many applications heterophase polymerization processes like emulsion dispersion
micro-emulsion seeded emulsion precipitation etc Especially to prepare well-defined
microspheres having monodispe
f
ty
rse and various particle sizes as well as surface group
ed particles prepared by emulsion polymerization
and e
s
hological design In
seede to control
n
functionalilties it is necessary to use se
nlarge them to a desired size in the further stages of reactions
Polymeric particles are required to have a uniform particle size in many
applications such as chromatography where they are used as a packing material
Morphological control of latex particle is also important for many practical application
[Schmidt 1972] Seed emulsion polymerization (or two-stage emulsion polymerization) is
a useful method to achieve both monodisperse particle size and morp
d emulsion polymerization [Gilbert 1995] preformed lsquoseedrsquo latex is used
the number of particles present in the final latex The advantage of seeded emulsio
polymerization is that the poorly reproducible process of particle nucleation can be
bypassed so that the number concentration of particles is constant and known Various
mechanisms have been proposed for the growth of latex particles [Ugelstad et al 1980
Okubo et al 1992] using this polymerization technique The initial seed particle
26
preparation step is well known and relatively easy to perform because at the relative
small particle sizes (02 to 05 micron) the particle growth process can be readily
controlled by the use of an emulsifier Enough emulsifier is used to prevent coagulation
but not enough to cause the formation of new particles As the particles are grown to
larger sizes in successive seeding steps it becomes increasingly difficult to mainta
stable uncoagulated emulsion without forming new particles and thereby destroying
monodisperisty of the latex Recently there have been several reports concerning t
reaction kinetics of seed emulsion polymerization and the development of latex
morphologies over the course of the reaction [Chern et al 1990 Delacal et al 1990 L
et al 1995 Lee 2000 2002]
266 Polystyrene Latex Particles
A number of papers have described the synthesis of the polystyrene latex particl
bearing various functional surface groups such as carboxyl [Lee et al 2000 Tun
2002 Reb et al 2000] hydroxyl [Tamai et al 1989] marcapto [Nilson 1989] epoxy
[Shimizu et al 2000 Luo et al
ly
in a
he
ee
es
cel et al
2004] acetal [Izquierdo et al 2004 Santos et al 1997]
ometyl [Izquierdo et al 2004 Park et al 2001
Sarob
h the
d
thymine [Dahman et al 2003] chlor
e et al 1998] amine [Counsin et al 1994 Ganachaud et al 1997 Ganachaud et al
1995 Miraballes-Martinez et al 2000 2001 Anna et al 2005] ester [Nagai et al 1999]
etc In order to produce these functionalized particles different methods for particle
preparation must be used The polymer emulsion with core-shell morphology of latex
particles is one of them This is a multistep emulsion polymerization process in whic
polystyrene ldquocorerdquo particle is synthesized in the first stage and the functional monomer is
added in the second stage of the polymerization without any emulsifier postfeeding to
prevent the production of new homopolymer particles thus forming the functionalize
27
polymer ldquoshellrdquo on the ldquocorerdquo particle [Keusch et al 1973] There are requirements to
limit secondary nucleation and encourage core-shell formation in seeded emulsion
polymerization including the addition of smaller seed particles at high solid content to
increase particle surface area low surfactant concentration to prevent formation of
micelles and the semi-continuous addition of monomer to create a starved-feed conditio
and keep the monomer concentration low There are some advantages [Hergeth et al
1989] of dispersions with polymeric core-shell particles First it is possible to modi
interfacial properties of polymer particles in the aqueous phase by the addition of only
very small amounts of a modifying agent during the last period of the reaction Thus
these core-shell particles are useful in a broad range of applications since they always
exhibit improved physical and chemical properties over their single-component
counterparts [Lu et al 1996 Nelliappan et al 1997] In this way the improvement of
surface properties of such dispersions is straightforward and inexpensive The other is
that polymers with a core-shell structure are perfect model systems for investigating th
material properties of polymer blends and composites because of their regular
distribution of one polymer inside a matrix polymer and because of the simple sp
geometry of the system
Their properties usually depend on the structures of latex particles Chen and his
coworkers [Chen et al 1991 1992 1993] reported the morphological development of
core shell latex particles of polystyrenepoly(methyl methacrylate) during
polymerization Before the research by Chen and his coworker Min et al [Min et al
1983] reported the morph
n
fy the
e
herical
ological development of core shell latex of polystyrene
(PS)polybuthyl acrylateat (PBA) by seeded emulsion polymerization as a function of the
28
additi
of
ity of graft
ing reagents Thus protein adsorption on polymeric solid surfaces has
st are microspheres defined as ldquofine
polym can be
n
r was
containing antigen urine serum etc The latex will become agglutinated and visibly
on method of PS They found that the percentage of grafting PS to the PBA was
greatest for the batch reaction and the PBA-PS core-shell particles with a high degree
grafting remained spherical upon aging test because of the emulsifying abil
copolymer
267 Various Applications of Latex Particles
Latex particles are applicable to a wide range of areas such as biomedical
applications [Piskin et al 1994] especially as the solid phase such as in immunoassays
[Chern et al 2003 Radomske-Galant et al 2003] DNA diagnostic drug delivery carriers
[Luck et al 1998 Kurisawa et al 1995 Yang et al 2000] blood cell separations and
column pack
become a center of attention Of particular intere
er particles having diameters in the range of 01 to several micronsrdquo which
used as functional tools by themselves or by coupling with biocompounds Singer and
Plotz [1956] firstly studied microsphere or latex agglutination test (LATrsquos) by using
monodisperse polystyrene (PS) and polyvinyltoluene polymer particles as the support o
which the biomolecules were going to adsorb The biomolecule adsorption howeve
limited by possible desorption of the adsorbed species or loss of specific activity of the
complex formed Since this work was published the latex particle applications for
immunoassay have been rapidly and widely studied and developed
Latex agglutination test or latex immunoassays start with tiny spherical latex
particles with a uniform diameter and similar surface properties The particles are coated
with antibodies (sensitized) through the hydrophobic interaction of portions of the protein
with the PS surface of the particles If sensitized particles are mixed with a sample
29
agglomerate Latex tests are inexpensive as compared with the other techniques [Ba
1988]
ngs
sed
tion Rheumatoid factor (RF) in different age subpopulations has also been
evalu
studied a
latex
Unipath [Percival 1996] has manufactured a range of immunoassay materials ba
on a chromatographic principle and deeply colored latex particles for use in the home and
clinical environments The latex particles which are already sensitized with a
monoclonal antibody can detect any antigens that bound to the surface of the latex
particles Some detectable pollutants include estrogen mimics which induce
abnormalities in the reproductive system of male fishes and lead to a total or partial male
feminiza
ated according to a patientrsquos clinical status by using a rapid slide latex agglutination
test for qualitative and semiquantitative measurement in human serum along with latex
immunoassay method [Onen et al 1998] Magalhaes and his coworkers [2004]
diagnostic method of contamination of male fishes by estrogen mimics using the
production of vitellogenin (VTG) as a biomarker This was based on a reverse
agglutination test developed with monoclonal antibodies specific to this biomarker
Premstaller and his coworkers [Premstaller et al 2000 2001] have prepared a porous
poly(styrene-divinylbenzene) (PS-DVB) polymer monolith to use for highly efficient
chromatographic separation of biomelecules such as proteins and nucleic acids They
used a porogen a mixture of tetrahydrofuran and decanol to fabricate a micropellicular
PS-DVB backbone Legido-Quigley and his colleagues [2004] have developed the
monolith column to obtain further chromatographic functionality to the column by
introducing chloromethylstyrene in place of styrene into the polymer mixture
30
Core shell type monodisperse polymer colloids have been synthesized by Sarobe
and Forcada [1998] with chloromethyl functionality in order to improve the biomolecu
adsorption through a two-step emulsion polymerization process They investigated the
functonalized particles by optimizing the experimental parameters of the functional
monomer including reaction temperature the amount and type of redox initiator sys
used the type of addition of the initiator system and the use of washing They c
le
tem
oncluded
that th
sis
serve as separation
chann
of
usually
along the polypeptide chain which contain positive or negative charges [Norde 1998]
e relation between the amount of iron sulfate and the persulfatebisulfite system
added should be controlled to obtain monodisperse particles and prevent the premature
coagulation of the polymer particles during the polymerization
A semi-continuous emulsion polymerization technique for latex particle synthe
was performed by McDonald and other researchers [Ramakrishnan et al 2004 Steve et
al 1999] They introduced a variety of particles sizes compositions morphologies and
surface modifications to fabricate latex composite membranes (LCMs) Arraying and
stabilizing latex particles on the surface of a microporous substrate form narrowly
distributed interstitial pores formed between the particles which
els They investigated the membrane performance using gas fluxes water
permeability and the retention characterization of dextran molecules From these tests
they concluded that the narrow discriminating layer made of the latex particles leads to a
highly efficient composite membrane
27 Proteins
Proteins are natural polyamides comprised of about 20 different α-amino acids
varying hydrophobicity [Norde 1998] Proteins are more or less amphiphilic and
highly surface active because of the number of amino acid residues in the side groups
31
Proteins are polymers of L-α-amino acids [Solomon and Fryhle 2000] The α refers to a
carbon with a primary amine a carboxylic acid a hydrogen and a variable side-chain
group designated as R Carbon atoms rent groups are asymmetric and can
exhib l
s
e
on
with four diffe
it two different spatial arrangements (L and D configurations) due to the tetrahedra
nature of the bonds The L refers to one of these two possible configurations Amino
acids of the D-configuration are not found in natural proteins and do not participate in
biological reactions Figure 2-8 shows the chiral carbon in 3-D as the L isomer Protein
consist of twenty different amino acids differentiated by their side-chain groups [Norde
1998] The side-chain groups have different chemical properties such as polarity charg
and size and influence the chemical properties of proteins as well as determine the
overall structure of the protein For instance the polar amino acids tend to be on the
outside of the protein when they interact with water and the nonpolar amino acids are
the inside forming a hydrophobic core
Figure 2-8 L-α-amino acid
The covalent linkage between two amino acids is known as a peptide bond A
peptide bond is formed when the amino group of one amino acid reacts with the carboxyl
group of another amino acid to form an amide bond through the elimination of water
This arrangement gives the protein chain a polarity such that one end will have a free
amino group called the N-terminus and the other end will have a free carboxyl group
called the C-terminus [Solomon and Fryhle 2000] Peptide bonds tend to be planar and
32
give the polypeptide backbone rigidity Rotation can still occur around both of the α-
carbon bonds resulting in a polypeptide backbone with different potential conformations
relative to the positions of the R groups Although many conformations are theoretically
possible interactions between the R-groups will limit the number of potential
nd to form a single functional conformation In other words
the conformation or shape of the protein is
α-helix
can interact with other
secondary structures within the sam
conformations and proteins te
due to the interactions of the chain side
groups with one another and with the polypeptide backbone The interactions can be
between amino acids that are close together as in a poly-peptide between groups that are
further apart as in amino acids or even on between groups on different polypeptides all
together These different types of interactions are often discussed in terms of primary
secondary tertiary and quaternary protein structure
The primary amino acid sequence and positions of disulfide bonds strongly
influence the overall structure of protein [Norde 1986] For example certain side-chains
will promote hydrogen-bonding between neighboring amino acids of the polypeptide
backbone resulting in secondary structures such as β-sheets or α-helices In the
conformation the peptide backbone takes on a spiral staircase shape that is stabilized by
H-bonds between carbonyl and amide groups of every fourth amino acid residue This
restricts the rotation of the bonds in the peptide backbone resulting in a rigid structure
Certain amino acids promote the formation of either α-helices or β-sheets due to the
nature of the side-chain groups Some side chain groups may prevent the formation of
secondary structures and result in a more flexible polypeptide backbone which is often
called the random coil conformation These secondary structures
e polypeptide to form motifs or domains (ie a
33
tertiary structure) A motif is a common combination of secondary structures and a
domain is a portion of a protein that folds independently Many proteins are composed o
multiple subunits and therefore exhibit quaternary structures
271 Interaction Forces between Proteins
Proteins in aqueous solution acquire compact ordered conformations In such a
compact conformation the movement along the polypeptide chain is severely restrict
implying a low conformational entropy The compact structure is possible only if
interactions within the protein molecule and interactions between the protein molecule
and its environment are sufficiently favorable to compensate for the low conformational
entropy [Malmsten 1998] Protein adsorption study is often focused on structural
rearrangements in the protein molecules because of its significance to the biological
functioning of the molecules and the important role such rearrangements play in the
mechanism of the adsorption process Knowledge of the majo
f
ed
r interaction forces that act
structures helps to understand the behavior
of pro
arge
a
ded
between protein chains and control the protein
teins at interface These forces include Coulomb interaction hydrogen bonds
hydrophobic interaction and van der Waals interactions
Coulombic interaction Most of the amino acid residues carrying electric ch
are located at the aqueous boundary of the protein molecule An the isoelectric point
(IEP) of the protein where the positive and negative charges are more or less evenly
distributed over the protein molecule intramolecular electrostatic attraction makes
compact structure favorable to proteins Deviation to either more positive or more
negative charge however leads to intramolecular repulsion and encourages an expan
structure Tanford [1967] calculated the electrostatic Gibbs energy for both a compact
impenetrable spherical molecule (protein) and a loose solvent-permeated spherical
34
molecule (protein) over which the charge is spread out From the results he found th
the repulsion force was reduced at higher ionic strength d
at
ue to the screening action of ion
ble
e
the
d the
tween
dehyd
e
ent
oles
tein
Hydrogen bond Most hydrogen bonds in proteins form between amide and
carbonyl groups of the polypeptide backbone [Malmsten 1998] The number of availa
hydrogen bonds involving peptide units is therefore far greater than that involving sid
chains Because α-helices and β-sheets are aligned more or less parallel to each other
interchain hydrogen bonds enforce each other Kresheck and Klotz [1969] examine
role of peptide-peptide hydrogen bonds and concluded that hydrogen bonds between
peptide units do not stabilize a compact structure of protein However because the
peptide chain is shielded from water due to other interactions hydrogen bonding be
peptide groups do stabilize α-helical and β-sheet structures
Hydrophobic interaction Hydrophobic interaction refers to the spontaneous
ration and subsequent aggregation of non-polar components in an aqueous
environment In aqueous solutions of proteins the various non-polar amino acid residues
will be found in the interior of the molecule thus shielded from water The
intermolecular hydrophobic interaction for the stability of a compact protein structure
was first recognized by Kauzmann [1959] If all the hydrophobic residues are buried in
the interior and all the hydrophilic residues are at the outermost border of the molecul
intramolecular hydrophobic interaction would cause a compact protein structure
However geometrical and other types of interactions generally cause a fraction of the
hydrophobic residues to be exposed to the aqueous environm
Van der Waals interaction The mutual interaction between ionic groups dip
and induced dipoles in a protein molecule cannot be established as long as the pro
35
structure is not known in great detail Moreover the surrounding aqueous medium also
contains dipoles and ions that compete for participation in the interactions w
the protein molecule Dispersion interactions favor a compact structure However
because the Hamaker constant for proteins is only a little larger than that of water the
resulting effect is relatively small [Nir 1977]
272 β
ith groups of
rgiles et al 1996
Floeg
) is degraded [Floege 2001] Renal
β2M from the serum resulting in an increase in β2M
conce well
the
)
a
al
2-Microglobulin (β2M)
The protein β2M is of particular interest because it is involved in the human
disorder dialysis-related amyloidosis (DRA) [Geiyo et al 1985 A
e 2001] DRA is a complication in end stage renal failure patients who have been on
dialysis for more than 5 years [Bardin et al 1986 Drueke 2000] DRA develops when
proteins in the blood deposit on joints and tendons causing pain stiffness and fluid in
the joints as is the case with arthritis In vivo β2M is present as the non-polymorphic
light chain of the class I major histocompatibility complex (MHC-I) As part of its
normal catabolic cycle β2M dissociates from the MHC-I complex and is transported in
the serum to the kidney where the majority (95
failure disrupts the clearance of
ntration by up to 60-fold [Floege 2001] By a mechanism that is currently not
understood β2M then self-associates into amyloid fibrils and typically accumulates in
musculoskeletal system [Homma et al 1989] Analysis of ex vivo material has shown
that the majority of amyloid fibrils in patients with Dialysis Related Amiloidosis (DRA
is present as of full-length wild-type β2M although significant amounts (~20-30) of
truncated or modified forms of the protein are also present [Floege 2001 Bellotti et al
1998] Figure 2-9 shows the ribbon diagram of human β2M Native β2M consists of
single chain of 100 amino acid residues and has a seven stranded β-sandwich fold typic
36
of the immunoglobulin superfamily [Saper et al 1991 Trinh et al 2002] β2M was fi
isolated from human urine and characterized by Berggard et al [1980] in1968 The
normal serum concentration of β
rst
re
f
e
2M is 10 to 25 mgL It is a small globular protein with
a molecular weight of 118 kDa a Strokes radius of 16Aring and a negative charge under
physiological conditions (isoelectric point IEP = 57) β2M contains two β-sheets that a
held together by a single disulphide bridge between the cysteines in positions 25 and 81
[Berggard et al 1980 Parker et al 1982 Cunningham et al 1973] β2M cannot be
removed completely by current dialysis techniques but through a better understanding o
the structure and interaction forces that lead to this structure it will be possible to mor
efficiently remove this problematic protein
Figure 2-9 Ribbon diagram of human β M taken from the crystal structure of the protein
bound to the heavy chain of the MHC class I complex (PDB 1DUZ) [Khal 2000]
273 Serum Albumin
Serum albumin is the most abundant protein found in plasma and is typically
present in the blood at a concentration of 35~
2an et
50gL According to extensive studies about
its physiological and pharmacological properties albumin has a high affinity to a very
wide range of materials such as electrolytes (Cu+2 Zn+2) fatty acids amino acids
metabolites and many drug compounds [Fehske et al 1981 Kraghhansen 1981 Putnam
37
1984 Peters 1985] The most important physiological role of the protein is therefore to
bring such solutes in the bloodstream to their target organs as well as to maintain the pH
and osmotic pressure of the plasma Bovine serum albumin (BSA) is an ellipsoidal
protein with the dimensions of 140 X 40 X 40Aring [Peter 1985] The primary structure is a
single helical polypeptide of 66 kDa (IEP = 47) with 585 residues containing 17 pairs of
as a soft an
rfaces [Kondo et al 1991 Norde et al 1992 Soderquist et al
1980 ikely
is
disulfide bridges and one free cysteine [Dugaiczyk et al 1982] BSA has been classified
d flexible protein because it has a great tendency to change its conformation
on adsorption to solid su
Carter and Ho 1994] and consists of three homologous domains (I-III) most l
derived through gene multiplication [Brown 1976] Each domain is composed of A and B
sub-domains [He et al 1992] The secondary structure of human serum albumin (HSA)
shown in Figure 2-10
Figure 2-10 Secondary structure of human serum albumin (HSA) with sub-domains
[Zunszain et al 2003]
HSA has the same structure domains with the serum albumin from other species
such as BSA [Brown 1976] Although all three domains of the albumin molecule have
similar three-dimensional structures their assembly is highly asymmetric [Sugio et al
38
1999] Domains I and II are almost perpendicular to each other to form a T-shaped
assembly in which the tail of subdomain IIA is attached to the interface region between
sub-domains IA and IB by hydrophobic interactions and hydrogen bonds In contrast
domain III protrudes from sub-domain IIB at a 45ordm angle to form the Y-shaped assembly
for domains II and III Domain III interacts only with sub-domain IIB These features
make the albumin molecule heart-shaped
28 Protein Adsorption
Protein adsorption studies date back to the 1930rsquos At the beginning these studies
mainly focused on the determination of the molecular weight electrophoretic and
ral
rearrangem
adsorption and biocompatibility of the sorbent materials were investigated [Norde 1986]
There are interaction forces at the interfaces between protein molecules and latex
particles These forces are mainly divided into the following groups hydrophobic
interaction ionic interaction hydrogen bonding and van der Waals interaction [Andrade
1985]
Hydrophobic interaction It is known that hydrophobic interaction has a major
role in protein adsorption phenomena The adsorption of proteins on the low charged
latex particles occurs by this interaction force Generally monomers such as styrene offer
hydrophobic surfaces th
by this interaction force is maximum at the isoelectric point (IEP) of the protein and the
strength [Suzawa et al 1980 1982 Shirahama et al 1989 Kondo et al 1992] By the
chromatographic applications Later the adsorption mechanism especially the structu
ents was studied Recently the studies of the relation between protein
281 Interaction between Protein Molecule and Latex Particle
at protein molecules adsorb to The amount of adsorbed protein
pH at maximum adsorption shifts to a more acidic region with an increase in ionic
39
reports [Suzawa et al 1980 1982 Lee et al 1988] protein adsorption was greater
hydrophobic surface than on a hydrophilic one implying that hydrophobic interaction is
on a
s
ydrophilic compound such as acrylic acid (AA) or methacrylic acid (MAA)
Ionic
ely
t
ces
n force is operative over small
distan e
ble
n
nt dialysis membrane therapy for end stage renal disease (ESRD)
one of most dominant forces in protein adsorption
Ionic interaction Negatively charged latex particles have ionic functional
groups on their surfaces such as salts of sulfonic and carboxylic acid Sulfate group
originate from an initiator such as sodium persulfate and carboxylic groups originate
from a h
bonds are formed between the negative charges of these latex particles and positive
surface charges of protein molecules The conventional low-charged latex particles rar
form these ionic bonds
Hydrogen bonding Hydrogen bond is a strong secondary interatomic bond tha
exists between a bound hydrogen atom (its unscreened proton) and the electrons of
adjacent atoms [Callister 1999] Protein can be adsorbed on hydrophilic polar surfa
through hydrogen bonding Hydrogen bonds are frequently formed between hydroxyl-
carbonyl or amide-hydroxyl Hydroxyl-hydroxyl or amide-hydroxyl bonds are also
formed in protein adsorption
Van der Waals interaction This interactio
ces only when water has been excluded and the two non-polar groups come clos
to each other Lewinrsquos calculation showed that the van der Waals interaction is negligi
compared with the forces involved in the entropy increases ie hydrophobic interactio
[Lewin 1974]
29 Hypothesis for Toxin Removal
As mentioned earlier insufficient removal of middle molecular range toxins is a
major drawback of curre
40
patien
is
y
ese
haracteristics over the last decade However removal
effici
ing a novel membrane design composed of an assembly of engineered
polymeric latex particles s ions Important factors
will i
e
ith a
is established it is
expec
e
ts This may cause destructive arthritis and carpal tunnel syndrome inhibit the
immune system and accelerate cardiovascular disease leading to death among dialys
patients In order to overcome these complications artificial dialysis membranes have
been developed to increase the capacity for removing middle molecular weight toxins b
changing the pore size of dialysis membranes and using new materials that adsorb th
toxins for improved removal c
ency is not as high as those achieved by a normal healthy kidney
With the knowledge obtained from all above literatures the following hypothesis
has been established for the development of a membrane for the successful removal of
the target protein β2-Microglobulin (β2M) without the removal of serum albumin This
will be done us
ynthesized to predetermined specificat
nclude pore size and surface chemistry
291 Toxin Removal by Size Sieving Based on Monodispersed Pore Size
The packing of monodispersed spherical particles can lead to the formation of
porous layers suitable for use as filters and membranes [Hsieh et al 1991] The pore siz
is defined as the largest spherical particles that can pass through the interstitial spaces
It is well known that monodispersed spherical particles can be obtained from the
seed emulsion polymerization method Many commercially available latex particles are
synthesized by this technique and are inexpensive When the defect free membrane w
monodisperse pore size distribution based on a particular particle array
ted to outperform the traditional hemodialysis membrane limitation which is a
broad range of pore size distribution When spherical particles pack in regular crystallin
arrays a number of packing geometries such as hexagonal closest packing cubic closest
41
packing and body-centered cubic packing are possible The pore size of the array
depends on these packing geometries as well as particle size Theoretical pore size can be
calculated from the simple geometry of the arr
ay Figure 2-11 shows the relationship
between particle size and pore size
0001
001
01
001 01 1 10
Por
e
1
10
microm
) S
ize
(
Particle Diameter (microm)
Body-Centered Cubic ArrayClosest Packed Array
0001
Siz
e (
Particle Diameter (microm)
001
01
001 01 1 10
Por
e
1
10
microm
)
Body-Centered Cubic ArrayClosest Packed ArrayBody-Centered Cubic ArrayClosest Packed Array
and closed packed arrays [Steve et al 1999]
McDonald and his coworkers [Ramakrishnan et al 2004 Steve et al 1999] have
described the fabrication of latex composite membranes and their performance in
previous papers It is from this prior research and knowledge gained from conversations
with Dr McDonald that this current research has been initiated [McDonald 2003] The
focus of this thesis is the extension of McDonaldrsquos research in the area of composite
membranes to medical application such as dialysis membranes
292 Toxin Removal by Selective Adsorption on Engineered Latex Particles
The flexibility in latex surface properties is a significant advantage to this
materialrsquos ability to separate proteins since the selectivity is strongly dependent on the
charge interactions of both the protein and the latex particles under the conditions of
Figure 2-11 The relationship between pore size and particle size in body-centered cubic
42
separation [Menon et al 1999 Chun et al 2002] First all sorbents were designed to
have negative surface properties because most plasma proteins in blood are negative and
should not be removed with charge interaction between proteins and sorbents at
physiological condition (pH74) It was reported that a negatively charged surface was
more blood compatible than a positive one [Srinivasan et al 1971] The toxin protein β2-
Microglobulin (β2M) can be selectively adsorbed on the surface of latex particles by the
design of a suitable hydrophobic portioned surface where only β2M protein can be
anchored with hydrophobic interactions Albumin adsorption on the latex particle is not
side-on m
adsorption of β M protein on the engineered latex particles
allowed because charge repulsion is more dominant than hydrophobic interaction with
ode The figure 2-12 shows the schematic representation of the selective
2
PMMAPAAShell
COO-
OSO3-
OSO3-
PSCore
COO-
-
-
-
-
-
-
Albumin-
Chargerepulsion
--
-
- -
- -
- --
Hydrophobic
β2-Microglobulin
2
Interaction
β -Microglobulin
PMMAPAAShell
COO-
-
-
-
-
-
-
--
-
-
-
-
-
Albumin-
Chargerepulsion
OSO3-
OSO3-
PSCore
COO-
--
-
- -
--
-
- -
- -
- --
- -
- --
Hydrophobic
β2-Microglobulin
2
particles at pH 74
Interaction
β -Microglobulin
Figure 2-12 Schematic representation of the protein adsorption on the core shell latex
43
Hydrophilichydrophobic microdomain structures were proven to be more blood
compatible [Mori et al 1982 Higuchi et al 1993 Deppisch et al 1998] The
optimization of suitable hydrophobic to hydrophilic ratio is also important for the
biocompatibility The monomers such as styrene (St) methyl methacrylate (MMA) and
acrylic acid (AA) are widely used hydrophobic and hydrophilic monomer models in
emulsion polymerization process
In summary the background and fundamental literature survey about the history
material properties and limitation of hemodialysis membrane latex particle preparation
surface chemistry and manufacturing process target proteins and protein adsorption and
finally the hypothesis for a toxin removal with high separation efficiency have been
suggested This research focuses on such hypothesis and the materials and
characterization methodology for achievement of suggested hypotheses are described in
the next chapter
CHAPTER 3 EXPERIMENTAL AND CHARACTERIZATION METHODOLOGY
As mentioned earlier the goal of this study is to prepare polymeric latex particles
with tailored properties to maximize separation of toxin molecules and to investigate
fundamental interactions between the applied particles and molecules in the
system to optimize the membrane performance for hemodialysis applications Polymeric
latex particles wit
the
biological
h monodisperse size distribution to obtain uniform pore size and
various size ranges to utilize membrane construction are necessary Surface engineering
with various combination of hydrophobichydrophilic domain on the surface of latex
particles is expected to affect the removal of target protein by selective adsorption and to
improve the biocompatibility of membrane Therefore the materials and characterization
methodology are addressed in this chapter
31 Materials
Styrene (St) monomer used for a seed and a core particle was purchased from
Fisher Scientific and used without any other purification process Acrylic acid (AA) and
methyl methacrylate (MMA) monomers introduced for shell formation were purchased
from Fisher Scientific and used without any other purification process Sodium persulfate
(SPS) and sodium bicarbonate (SBC) were obtained from Fishers Scientific and used as
received Divinylbenzene (DVB) crossliking agent was purchased from Aldrich The
anionic surfactant Aerosol regMA80-I [sodium di(13-dimethylbutyl) sulfosuccinate] was
kindly donated by Cytec 78-80 Aerosol regMA80-I is mixed with isopropanol and
water Its critical micelle concentration (CMC) is 245 mM (119) Ion exchange resin
44
45
AGreg 501-x8 Resin (20-50 mesh) was purchased from Bio-Rad Laboratories Inc It is a
mixed-bed resin with H+ and OH- as the ionic forms and is 8 crosslinked Bovine serum
albumin (BSA) (heat shock treated) was purchas Fisher Scientific The isoelectric
point of t lin
(β2M)
as
gma
ed from
he BSA is 47-49 and the molecular weight is 66300Da β2-Microglobu
was purchase from ICN Biomedicals Inc and was separated from patients with
chronic renal disease and lyophilized from ammonium bicarbonate The molecular
weight by SDS-PAGE was approximately 12000Da BSA and β2M proteins were used
received without further purification process In order to investigate the protein
adsorption evaluation bicinchoninic acid protein assay kits were purchased from Si
(Cat BCA-1) and Pierce Biotechnology (Cat 23225) The chemical structures for the
main chemicals are shown in Figure 3-1
CH2 CH
Styrene (Mw = 10415)
CH2 CHCH2 CH
Styrene (Mw = 10415)
CH2 CH
Acrylic acid (Mw = 7206)
C
OH
O
CH2 CH
Acrylic acid (Mw = 7206)
C
OH
O
CH CH2
C
OCH3
O
CH
CH CH3
2
C
OCH3
O
CH3
Methyl methacrylate (Mw = 10012)Methyl methacrylate (Mw = 10012)
CH2 CH
Divinylbenzene (Mw = 1302)
CH CH2
CH2 CH
Divinylbenzene (Mw = 1302)
CH CH2CH CH
+(Na) -(O3S)
CH -C-O-CH-CH -CH-CH3
O
2 2 3
O CH CH3
CH3 CH3
CH-C-O-CH-CH2-CH-CH3
Anionic surfactant (MA80-I) (Mw = 3885)
+(Na) -(O3S)
CH -C-O-CH-CH -CH-CH3
O
2 2 3
O CH CH3
CH3 CH3
CH-C-O-CH-CH2-CH-CH3
Anionic surfactant (MA80-I) (Mw = 3885)2
+(Na)-(O) S OO
O O
- +
Initiator sodium persulfate (Mw = 23811)
O SO
(O) (Na)+ -
O O
- +(Na) (O) S OO
O SO
(O) (Na)+ -
O O
- +
Initiator sodium persulfate (Mw = 23811) Figure 3-1 Chemical structure of main chemicals
(Na) (O) S OO
O SO
(O) (Na)
46
32 Latex Particle Preparation
The experimental set up and the particle preparation schemes for various types and
size ranges of latex particles are shown in Figure 3-2 and 3-3 In the experimental setup a
mechanical glass stirrer is connected from a motor and Teflon stirrer bar is located at
end of glass stirrer The agitation rate is precisely adjusted by controller motor Paar glas
reactor with 1L volume is partially in silicon oil bath on hot plate The reactor has fo
openings for a stirrer a thermometer a nitrogen gas inlet and a reactant feeding lnlet
The reactants for latex polymerization are fed by precisely controlled by meterin
The particle preparation is described in each latex preparation section
the
s
ur
g pump
Figure 3-2 Experimental setup for semi-continuous emulsion polymerization
1 Motor 2 Mechanical stirrer bar 3 Thermometer 4 Inlet of N2 5 Inlet for initiator and monomer 6 Glass reactor (l L) 7 Oil bath 8 Hot plate 9 Pump for initiator 10 Pump for monomer 11 Tubes for reactant feed
47
Styrene monomer
Bare PS
PSPMMA100
PSPMMA90PAA10
PSPMMA P75 AA25
Gr
win
ticle
size
PS seedparticles
og
par
Styrene monomer
Bare PS
PSPMMA100
PSPMMA90PAA10
PSPMMA P75 AA25
Gr
win
ticle
size
PS seedparticles
og
par
Figure 3-3 The particle preparation scheme in various types and size ranges of latex particles
321 Preparation of Seed Latex Particles
Polystyrene seed particles were synthesized through a typical semi-continuous
emulsion polymerization Polystyrene latex particles were prepared in a four necked Paar
glass vessel equipped with a mechanical glass stirrer thermometer glass funnel for
nitrogen gas purge and a tube for monomer feeding The reactor was placed in a silicon
oil bath on a hot plate for homogenous and stable tempe he vessel was
firstly charged with de-ionized water the emulsifier so itiator
sodium bicarbonate (SBC) styrene (St) monomer divinylbenzene (DVB) monomer
under nitrogen gas atmosphere and then heated to 72plusmn2 as
llowed to continue for another hour Second reactants St and DVB monomers SPS
initiator and SBC were fed separately using fine fluid metering pumps (Model RHSY
Fluid metering INC NY) under a nitrogen gas atmosphere for 2h After complete feeding
rature control T
dium persulfate (SPS) in
oC for 1h The reaction w
a
48
of all reactants the reaction was continued for another hour at elevated temperature 80plusmn1
oC and then cooled to room temperature by removal of the oil bath
322 Preparation of Seeded Latex Particles
The particle growth was achieved by seeded continuous emulsion polymerization
The vessel was charged with the polystyrene seeds and de-ionized water to make desired
solid content and heated up to 72plusmn2 oC for 1h under nitrogen gas atmosphere Then St
and DVB monomers and solution of SPS and SBC in de-ionized water were continuously
added using metering pump with a precisely controlled feed rate After feeding all
reactants the reaction was continued for three hours keeping temperature of 80plusmn2 oC and
then the reaction vessel was cooled down to room temperature
Monodisperse spherical polystyrene seeds with a 280nm mean diameter were used
e-polymerized polystyrene seeded emulsion
was charged in a 500m
323 Preparation of Core Shell Latex Particles
for core shell structured latex particle The pr
l glass flask under a nitrogen gas atmosphere De-ionized water
was added to achieve the desired solid content The emulsion was heated to 80plusmn2oC for
10 hour Monomers initiator and SBC to form the shell were added by fluid metering
pumps (LAB PUMP JRRHSY model Fluid metering Inc USA) with fine controlled
feed rate Flow rate can be adjusted by control ring graduated in 450 division from 0 to
100 flow which is in the range of 10mlmin respectively The reaction was sustained
for additional 30 hours at 90plusmn2oC then cooled down to room temperature
324 Purification of Synthesized Latex Particles
Synthesized latex particles were purified with an ion exchange resin 200g (10
ww polystyrene solid content) of emulsion were mixed with 4g of ion exchange resin
and stirred for 40 minute to remove unreacted monomers initiators and other impurities
49
Then the emulsion was diluted with DI water to a specific degree of solid content as
needed
33 Characterization
331
solid
termined
oscopy
d
hours
Dried latex (10mg) was mixed with
250 m on
Degree of Conversion
The degree of monomer to polymer conversion was determined gravimetrically
following the procedure described by Lee et al [1995] before the latex cleaning process
The synthesized emulsion raw materials were weighed and dried in a conventional oven
at 120oC for 30min to evaporate any unreacted monomers and water The remaining
was weighed and the degree of conversion was determined This process was repeated
three times and a mean value of the degree of conversion was de
332 Fourier Transform Infrared (FTIR) Spectr
A molecule can absorb only select frequencies (energy) of infrared radiation which
match the natural vibration frequencies The energy absorbed serves to increase the
amplitude of the vibrational motions of the bonds in the molecule [Pavin et al 1996]
Only those bonds which have a dipole moment that changes as a function of time can
absorb the infrared radiation The range of wavelengths for infrared radiation is between
25 microm (4000 cm-1) and 25 microm (400cm-1)
The synthesis of the latex particles such as polystyrene (PS) homopolymer and
PSPMMA PSPMMA90PAA10 PSPMMA75PAA25 core-shell copolymers were verifie
with FTIR Purified latex particles were dried in vacuum oven at 40oC for 24
before analysis in the FTIR (Nicolet Magma USA)
g of KBr which had also been dried in vacuum oven at 120oC for 3h Transmissi
spectra using the drift mode were plotted with 128 scans and 4cm-1 resolution
50
333 Quasielastic Light Scattering (QELS)
Latex particle size was measured by the Brookhaven ZetaPlus particle size
analyzer The solid concentration as 0001 (wv) This
ation spectroscopy (PCS) of quasielastically scattered light
For th s
ed using a FE-SEM (JEOL
JSM-
ith
ent
01
us zeta-
potential analyzer It measures the electrophoretic mobility velocity of charged colloidal
of latex particles used w
instrument uses photon correl
is technique time dependent interference patterns of light scattered from particle
in the sample cell are analyzed The interference pattern changes due to Brownian
motion giving rise to fluctuations in scattering intensity The frequency of these
fluctuations depends on the particlersquos diffusion constant that is inversely proportional to
the particle diameter
334 Field Emission-Scanning Electron Microscopy (FE-SEM)
Particle size and surface morphology were characteriz
6335F Japan) A diluted suspension of latex particles in water was dropped onto a
silicon wafer at room temperature and allowed to dry The sample was then coated w
the thinnest layer of carbon needed to obtain the required conductivity for this instrum
Secondary electron image mode was used with a 15KV of accelerating voltage The
magnification range used was between 10000X and 70000X
335 Zeta Potential Measurement
Synthesized and purified latex particles were diluted with de-ionized water to 0
wt and adjusted six different pH values 205 345 481 565 643 and 747 These
particle suspensions were then transferred to standard cuvettes for zeta potential
measurement At least two runs of ten measurements were taken for each sample and
averaged
The zeta potential measurement was carried out using Brookhaven ZetaPl
51
particles in solution and calculate zeta-potential by using the Smoluchowski equation
The f quency
gnitude
umin (BSA) as a standard model protein and β2-Microglobulin
uffer (PB) and
phosp
5
h the selected protein
e 005 01 03 05 and 07 mgml and those for
β2M
d at
teins in the supernatant after the centrifugation process using the
bicinc t
requency of laser light passing through the sample and is compared to the fre
of a reference beam The shift in the frequency called a Doppler shift and the ma
of the shift correspond to the polarity and the magnitude of the electrophoretic mobility
respectively The zeta potential is calculated from the solution conditions and the
measured mobility
336 Protein Adsorption
Protein adsorption experiments of the synthesized latex particles were performed
with bovine serum alb
(β2M) as a target protein in two types of buffer solution phosphate b
hate buffered saline (PBS) To make 5 mM of PB solution 0345g of sodium
phosphate monobasic (NaH2PO4H2O) was dissolve in 500ml of de-ionized water To
make PBS solution 0345g of sodium phosphate monobasic (NaH2PO4H2O) and 4178g
(143mM) of sodium chloride (NaCl) were dissolved in 500ml of de-ionized water The
synthesized latex particles were diluted with each buffer to have a solids content of 0
(ww) adjusted to pH values of 32 48 and 74 and mixed wit
BSA concentrations were chosen to b
were 0015 0030 0045 and 0060mgml The mixture was gently rotated using
the shaker Labquakereg (BarnsteadThermolyne Model 4002110 USA) with 8 RPM in
the incubator at 37oC for 12 hours before the latex-protein mixture was centrifuge
13000 rpm for 15min The amount of protein adsorbed was determined by quantifying
the free pro
honinic acid (BCA) assay method [Lowry et al 1951 Smith et al 1985 Baptista e
al 2003 Wiehelman et al 1988 Brown et al 1989] BCA assay kits consist of Reagent
52
A containing bicinchoninic acid sodium carbonate sodium tartrate and sodium
bicarbonate in 01N NaOH with pH=1125 and Reagent B containing 4 (wv) copper
(II) sulfate pentahydrate The BCA working reagent was prepared by mixing 50 parts of
Reagent A with 1 part of Reagent B 100microl of protein supernatant was then mixed with
2ml of BCA working reagent in a UV cuvette Incubation was allowed to continue
at room temperature
further
until color developed about 2h The absorbance at 562nm was
pectrophotometer (Perkin-Elmer Lambda 800 USA)
Unkn
Separate the p rticles in centrifuge mix supernatant with BCA reagents
measured using a UV-VIS s
own concentration of sample protein was determined by comparison to a standard
of known protein concentrations The adsorbed amount per unit surface area was
determined by the mass balance of the protein after adsorption process The simple
schematic of the procedure for a protein adsorption test is shown in Figure 3-4
Incubate mixture with agitation for 12hrs at 37oC
aand develop the color by incubation for 2hrs at room temperature
Suspend latex particles in aqueous buffer media
Add the dissolved target protein
Measure color intensity using UVVis spectroscopy at 562nm
Suspend latex particles in aqueous buffer media
Separate the p rticles in centrifuge mix supernatant with BCA reagents
Add the dissolved target protein
Incubate mixture with agitation for 12hrs at 37oC
aand develop the color by incubation for 2hrs at room temperature
Measure color intensity using UVVis spectroscopy at 562nm
Figure 3-4 Schematic of the procedure for a protein adsorption test
53
337 Blood Biocompatibility by Hemolysis Test
Hemolysis is the destruction of red blood cells which leads to the release of
hemoglobin from within the red blood cells into the blood plasma Hemolysis testing is
used to evaluate blood compatibility of the latex particles and the damaged to the red
blood cells can be determined by monitoring amount of hemoglobin released The red
blood cells (RBCs) were separated by centrifuging blood at 1500 rpm for 15 minutes
Figure 3-5 shows the phase separated blood with plasma white blood cells and red
cells after centrifugation process Separated RBSs were then washed with isotonic
phosphate buffer solution (PBS) at pH 74 to remove debris and serum protein Th
process was repeated 3 times
blood
is
Figure 3-5 Separation of RBC from whole blood by centrifuge process
Prepared latex particles were re-dispersed in PBS by sonification to obtain
homogeneously dispersed latex particles 100microl of the mixture of red blood cell (3 parts)
and PBS (11parts) was added to 1ml of 05 (ww) particle suspension PBS was used as
a negative control resulting 0 hemolysis and DI water used as a positive control to
produce 100 hemoglobin released by completely destroyed RBCs The mixture was
incubated in water bath with gentle shaking for 30 minutes at 37oC and then centrifuged
at 1500 rpm for 15 minutes 100microl of the supernatant was mixed with 2ml of the mixture
) (EtOHHCl = 2005 ww) to prevent the
White blood cells
Plasma
Red blood cells
of ethanol (99) and hydrochloric acid (37
54
precipitation of hemoglobin In order to remove remaining particles the mixture was
centri fuged again with 13000 RPM at room temperature The supernatant was then
transferred into the UV cuvette The amount of hemoglobin release was determined by
monitoring the UV absorbance at a wavelength of 397nm The schematic of the
procedure for hemolysis test is shown in Figure 3-6
Separate red blood cells (RBCs) from blood
Mix RBCs with phosphate buffer solution (PBS)
Wash and re-suspend RBC with PBS repeat three times
Add la l of RBC and incu in at 37oC
tex particle suspension into 100mbate sample with agitation for 30m
Separate particle ith EtOHHCl solutions and mix the supernatant w
Evaluate free hemoglobin in the solution using UVVis spectroscopy at 397nm
Separate red blood cells (RBCs) from bloodSeparate red blood cells (RBCs) from blood
Mix RBCs with phosphate buffer solution (PBS)Mix RBCs with phosphate buffer solution (PBS)
Wash and re-suspend RBC with PBS repeat three timesWash and re-suspend RBC with PBS repeat three times
Add latex particle suspension into 100ml of RBC and incubate sample with agitation for 30min at 37oC
Add latex particle suspension into 100ml of RBC and incubate sample with agitation for 30min at 37oC
Separate particles and mix the ith EtOHHCl solution supernatant wSeparate particles and mix the ith EtOHHCl solution supernatant w
Evaluate free hemoglobin in the solution using UVVis spectroscopy at 397nmEvaluate free hemoglobin in the solution using UVVis spectroscopy at 397nm
Figure 3-6 Schematic of the procedure for hemolysis test
In summary a description of the experimental materials and characterization
methodology has been explained In the next chapter the results and discussion of the
data obtained from these experiments is addressed
CHAPTER 4 RESULTS AND DISCUSSION
41 Polymerization of Latex Particles
411 Polystyrene Seed Latex Particles
Polymer particles have many different applications including spacer particles
lubricants packing materials for chromatography standard particles and diagnostic
drugs All applications strongly require these particles to have a uniform particle size
Uniform particle size is the first requirement for a latex composite membrane formed by
particle arrays with the interstitial spaces serving as pores for size discriminations [Jons
et al 1999 Ramakrishnan et al 2004] Although suspension polymerization has been
mainly used as one of conventional polymerization methods to prepare uniform particles
the uniformity of the recovered particles is insufficient for membrane use There is
another method known as seed emulsion polymerization where a vinyl monomer is
absorbed into fine monodiserse seed particles and polymerization causes the monomer to
crease the sizes of the seed particles uniformly In order to obtain monodisperse larger
particles by this m
particles and polymerization of the monomer is repeated
In this study cross-linked polystyrene seed latex particles were firstly synthesized
in the presence of styrene monomer divinylbenzene crosslink agent sodium persulfate
initiator sodium bicarbonate buffer and the Aerosol regMA80-I [sodium di(13-
dimethylbutyl) sulfosuccinate] surfactant using a batch type semi-continuous emulsion
polymerization process in which one or more components can be added continuously
in
ethod the procedure of absorption of monomer into fine polymer
55
56
Styrene (St) monomer and divinylbenzene (DVB) crosslink agent as well as initiator and
buffer are continuously added to enlarge the seed latex particles Various profiles of
particle nucleation and growth can be gene different orders of component
addition during polymerizati f this process such as easy
control of the polymeriza particle number
and particle morphology The main goal in
this process is to avoid secondary particle
rated from
on There are several advantages o
tion rate monodisperse particle size and
colloidal stability copolymer composition
formation leading to a monodisperse particle
size distribution Figure 4-1 shows the schematic representation of seed latex particle
preparation and growth
Monomer droplet
Seed particles Amount of surfactantsis variable
Enlarged final Add monomers
PS
PS
PSs4
particles Number of particlesare constant
Seed code
PSs1
s2
s3
Monomer droplet
Seed particles Amount of surfactantsis variable
Enlarged final Add monomers
PS
PS
PSs4
tion The
particles Number of particlesare constant
Seed code
PSs1
s2
s3
Figure 4-1 Schematic representation of semi-continuous seed latex particles preparation and growth
After the first synthesis of a small size of crosslinked polystyrene (PS) seed
particles stabilized by surfactants styrene DVB and initiator are continuously fed into
the system to enlarge the seed latex particles maintaining a narrow size distribu
recipes and reaction conditions for synthesizing these seed particles are summarized in
57
Table 4-1 The original recipe for latex particle preparation from McDonald was
modified for this emulsion system [McDonald 2003]
Table 4-1 The polymerization recipe of polystyrene (PS) seed particles Latex label PSS259 PSS233 PSS207 PSS181
Temperature (oC) 75 plusmn 5 75 plusmn 5 75 plusmn 5 75 plusmn 5
Divinylbenzene (g) 052 052 052 052
MA 80 (g) 259 233 207 181
NaHCO
Watera (g) 1315 1315 1315 1315
Styrene (g) 9948 9948 9948 9948
112 112 112 112
Na2S2O8 (g) 190 190 190 190
Waterb (g) 200 200 200 200
Reaction time (min) 140 140 140 140
Solids content 396 396 396 396
Mean particle diameter (nm) 126plusmn75 171plusmn39 182plusmn41 216plusmn53
Conversion () 965 979 957 971
3 (g)
Four different seed latex particles were synthesized based on surfactant amount in
order to determine the optimum condition for stable and monodisperse latex particles
The emulsion polymerization of latex particles has to be carried out in a narrow range of
surfactant causes flocculation or phase separation leading to broad particle
size distribution and an unstable emulsion sy
surfactant concentration where particles are stable [Nestor et al 2005] A wide range of
concentrations
stem Therefore the range of surfactant
amount was varied from 181g to 259g in increments of 026g in each latex label Other
reactants amounts were kept constant Watera is the initial charge amount and waterb
represents the amount of aqueous solution containing dissolved initiator and buffer
58
which are fed continuously after the seed particle creation The extent of monome
polymer conversion was obtained by gravimetric calcu
r to
lation and was more than 95 at
cripts such as
S207 and S181 i the surf mount added for the seed latex
nversion indi perce experi obtain
content divided by the theoretically estimated solid content and was calculated by the
all seed particles labeled PSS259 PSS233 PSS207 and PSS181 Labeled subs
S259 S233 ndicate actant a
preparation The co cates the ntage of mentally ed solid
following formula
100 colids () times=entsoliatedntentoalExperimentConversion
n the experimenta content is the weight of olid rema after
mulsion at 120oC f in and the estimated solid content is th ht
lated from the recipe
rti as di each t load d
mple of ea pension iluted by (ww)
deionized water and the mean particle diameter was determined using the Brookhaven
ZetaPlus particle size analyzer From these results the seed latex particle size increased
as the amount of surfactant decreased The amount of emulsifier also affects the
polymerization process by changing the number of micelles and their size It is known
that large amount of emulsifier produces larger numbers of smaller sized particles [Odian
1991] and was corroborated in the current work
These synthesized seed latex particles should be of narrow size distribution in order
to obtain monodisperse larger particles The SEM image showing the particle
morphology and size of a sample of polystyrene seed particles can be seen in the Figure
4-2
contdEstim (4-1)
In this equatio l solid the s ining
evaporation of e or 30m e weig
of monomers calcu
As expected the seed pa cle size w fferent for surfactan ing in see
emulsion system A sa ch sus was d 0001 with
59
Figure 4-2 Scanning Electron Micrograph (SEM) of polystyrene seed latex particles (A)
PSS259 (B) PSS233 (C) PSS207 (D) PSS181 (subscripts indicate the amount of surfactant added)
(A)
(B)
60
(C)
(D)
Figure 4-2 Continued
61
s seen in the image the seed particles are very smooth and spherical The
uniformity in the particle size distribution of these PS seed latex particles can also be
seen in the SEM image From this data we concluded that seed particles synthesized in
the presence of a surfactant with the chosen amounts are suitable for use to prepare larger
particles with high size uniformity and smooth spherical morphology
412 The Growth of Polystyrene (PS) Seed Latex Particles
Seeded emulsion polymerization has been conducted for several decades and
various mechanisms have been proposed Gracio et al [1970] suggested that the growing
polymer particles consist of an expanding polymer-rich core surrounded by a monomer-
rich shell with the outer shell providing a major locus of polymerization Seeded
mulsion polymerization is commonly used for preparing latex particles less than 1microm in
size [Cha et al 1995 Zou et al 1990 1992 Gandhi et al 1990 Park et al 1990] Such
latexes can be obtained from the growth of pre-prepared seed particles The seeds
introduced in this process serve as nucleation sites for particle growth By first swelling
the seed latex particles with additional monomer to the desired size polymerization can
then be initiated Several size ranges of monodisperse polystyrene (PS) latex particles
were prepared by this multistep seeded emulsion polymerization method For the
fabrication of particle-based membranes monodisperse latex particles with a variety of
size ranges are required
In this study seed particles from 171nm to 470nm in mean diameter and with a
highly uniform size distribution were prepared and used Watera is the initial charge
mount and waterb represents the amount aqueous solution containing dissolved initiator
and buffer that is continuously fed into system Feed time is precisely controlled by
metering pump The solid contents were maintained between 199 and 300 After
A
e
a
62
polym d as
es
ere 923 8402
n of these latex particles
are sh
ss
erization under these conditions the conversions of the latex particles labele
PS258 PS320 PS370 and PS410 were 952 957 946 and 964 respectively The recip
used in seeded emulsion polymerization to form the particles less than 500nm in mean
diameter are shown at the Table 4-2 The conversions of the latex particles larger than
500nm in mean diameter and labeled as PS525 PS585 PS640 and PS790 w
723 and 969 respectively The recipes used in the preparatio
own at the Table 4-3
A simple calculation can be used to predict the ultimate size that a latex particle can
reach after ending the polymerization Letrsquos suppose that seed density (ρs) and grown
particle density (ρgp) are the same The following equation is obtained from the ma
balance (m = ρV) where m and V are mass and volume of a particle respectively
gp
gp
s
s
Vm
Vm
In this equation m
= (4-2)
be
s and mgp are the weight of seed and total weight of seed and
added monomers respectively and Vs and Vgp are the volume of seed and grown
particle respectively
If we know the mass of monomer to be added the ultimate particle size can
calculated from the equation 4-3
3
2s D m ⎠⎝+ χ
where D
1s D m
⎟⎟⎞
⎜⎜⎛
= (4-3)
the
in
s and Dgp are the diameters for seed and grown particle respectively and χ is
weight of the monomer added Crosslinked PS latex particles with a size range from
258plusmn112 ~ 410plusmn117 in diameter polymerized by the recipes in Table 4-2 are shown
the SEM images in Figure 4-3
63
Table 4-2 Continuous addition emulsion polymerization recipe for growing polystyrene
Latex label PS
(PS) latex particles less than 500nm in size
258 PS320 PS370 PS410
Initial charge
Seed latex (g) 1000171 1000216 1000216 1000320
Watera (g) 1470 2500 1984 2600
06 07 06 06
Fee
Initiator stream
2 2 8
Feed time (min) 0-167 0-140 0-100 0-92
Solid content 284 300 300 200
Mean particle diameter (nm) 258plusmn112 320plusmn154 182plusmn71 410plusmn117
Conv
Monomer continuous
Styrene (g) 800 1200 800 600
Divinylbenzene (g)
d time (min) 0-163 0-110 0-75 0-82
Na S O (g) 07 065 05 05
NaHCO3 (g) 07 065 05 05
Waterb (g) 750 1200 200 370
ersion () 952 957 946 964 The solid content of seed latex for PS258 PS320 PS 370 and PS410 are 392 399 399 anrespectively
d 300
64
Table 4-3 Continuous addition emulsion polymerization recipe for growing polystyrene (PS) latex particles larger than 500nm in size
Latex label PS525 PS585 PS640 PS790
Initial charge
Seed latex (g) 1000390 1000470 1000470 1000320
Watera (g) 1000 520 1200 360
Monomer continuous
Styrene (g) 290 200 400 173
Divinylbenzene (g) 02 014
03 009
Feed time (min) 0-80 0-40 10-76 5-65
Initiator stream
Na2S2O8 (g) 02 014 03 012
NaHCO3 (g) 02 014 03 009
Waterb (g) 200 120 400 200
Feed time (min) 0-85 0-46 0-77 0-75
Solids content 199 200 200 201
Mean particle diameter (nm) 525plusmn151 585plusmn186 640plusmn178 790plusmn693
Conversion () 923 8402 723 969 The solid content of seed latex for PS525 PS585 PS 640 and PS790 are 200 182 193 and 173 respectively
65
Figure 4-3 SEM of PS latex particles less than 500nm in size (A) 258nm (B) 320nm (C)
370nm (D) 410nm
(A)
(B)
66
(C)
(D)
Figure 4-3 Continued
67
article size was well matched to that estimated by the geometrical calculation
From this it can be concluded that the added monomer was consumed for polymer
particle growth with a high degree of conversion From the SEM characterization newly
nucleated particles were not seen as indicated by the narrow particle size distribution
The particles less than 500nm in mean diameter were very spherical in shape with a
highly uniform particle size distribution As the particles were grown larger than 500nm
mean diameter however they became of non-spherical shape with an uneven surface
This irregularity of particle surface can be attributed to the non-homogeneous monomer
swelling into the shell of a growing polymer which can be controlled by the factors such
as temperature agitation speed initiator feeding rate and surfactant amount required to
stabilize the colloidal system The SEM image of latex particles larger than 500nm mean
iameter is shown in Figure 4-4 The particle size distribution is broader than that of the
s particles These particles can however still be used for membrane construction
as a support layer rather than skin layer The support layer does not necessarily have to be
as highly monodisperse as the skin layer which require pores of high uniformity for the
selective removal of toxins
As mentioned earlier the amount of surfactant as well as monomer in an emulsion
system is the primary determinant of the particle diameter [Odian 1991] Anionic
surfactants are generally recommended at the level of 02-30 wt based on the amount
of water [Odian 1991] The critical micelle concentration (CMC) of Aerosolreg MA 80-I
anionic surfactant is 119 The surfactant loading in this emulsion system ranged from
the 121 for SS181 to 171 for SS259
P
d
maller
68
Figure 4-4 SEM of PS latex particles larger than 500nm in size (A) 525nm (B) 585nm
(C) 640nm (D) 790nm
(A)
(B)
69
(C)
(D)
Figure 4-4 Continued
70
The surfactant to monomer ratio refers to the value of the surfactant amount
divided by monomer amount after polymerization ends The surfactant amount is the
weight of the surfactant contained in emulsion and the monomer amount is the total
weight of the monomer added for polymerization to form the ultimate polymer particles
in emulsion As the surfactant to monomer ratio decreases mean particle diameter
increases as described by Odian [1991] Generally lower surfactant concentration forms
fewer micelles resulting in larger particle size [Evans et al 1999] However there is a
plateau in particle mean diameter between a surfactant to monomer ratio of 0005 and
0013 This is due to low polymerization conversion of latex particles from monomer
leading to a smaller particle size than expected This is not a factor of surfactant amount
The dependence of the particle size on the amount of surfactant is shown in Figure 4-5
0
200
400
600
800
1000
0000 0005 0010 0015 0020 0025 0030
Surfactant to monomer ratio (ww)
Part
icle
mea
n di
amet
er (n
m)
Figure 4-5 Dependence of the particle size on the surfactant to monomer ratio
71
413
e
f bare
microdomain structures is important for biocompatibility [Mori et al 1982 Higuchi et al
1993 Deppisch et al 1998] Figure 4-6 shows the schematic of core shell latex particle
structure The recipe for core shell structures is shown in Table 4-4
Core Shell Latex Particles
The latex particles shown in Figure 4-3 (A) and labeled as S258 in Table 4-2 wer
used as seeds to prepare core-shell latex particles because of their high particle
uniformity and smooth surface morphology Methyl methacrylate (MMA) and acrylic
acid (AA) monomers were introduced to increase surface hydrophilicity over that o
polystyrene particles AA is a more hydrophilic monomer than MMA because of the
carboxyl acid group in AA is more favorable for hydrogen boding with water The
surface carboxyl groups on PAA may have many promising applications in biomedical
and biochemical fields [Kang et al 2005] MMA monomer is less hydrophobic than
styrene PMMA is known to more biocompatible than PS but still PMMA is hydrophobic
The optimization of suitable hydrophobic to hydrophilic ratios and control of
Polystyreneseed particle
Core shelllatex particles
PSPMMA100
PSPMMA90PAA10
PSPMMA75PAA25
Polystyreneseed particle
Core shelllatex particles
PSPMMA100
PSPMMA90PAA10
Figure 4-6 Schematic of core shell latex particle structures
PSPMMA75PAA25
72
Table 4-4 The preparation recipe of PS core with various shell latex particles Latex 25 label PSPMMA100 PSPMMA90PAA10 PSPMMA75PAA
Initial charge
PS seed latex (g) 500 500 500a
Monomer continuous
Methly methacylate (g) 320 288 240
Acrylic acid (g) 00 32 80
Initiator stream
Na
258 258 258
Water (g) 1570 1570 1570
Feed time (min) 0-120 0-50 0-60
b 200
Feed time (min) 0-122 0-60 0-65
952
2S2O8 (g) 02 02 02
NaHCO3 (g) 02 02 02
Water (g) 200 200
Solids content 183 183 183
Mean particle diameter (nm) 370plusmn186 370plusmn178 370plusmn191
Conversion () 955 924
Three types of core shell structures were prepared PMMA75PAA25 shell is a
copolymer consisting of the PMMA to PAA ratio of 75 to 25 by weight
PMMA90PAA10 shell is a copolymer with the PMMA to PAA ratio of 90 to 10 by
weight PMMA100 is the PMMA homopolymer shell on PS core The particle size of
these core-shell particles as well as PS latex particle was prepared to be about 370nm in
mean diameter and are characterized to determine their zeta potential protein adsorption
and biocompatibility The image of SEM of PS and core shell particles is shown at Figure
4-7 The synthesized latex particles had a high uniformity in size and a smooth spherical
surface morphology
73
Figure 4-7 Scanning
Electron Micrograph of latex particles (A) PS (B) PSPMMA100 (C) PSPMMA90PAA10 (D) PSPMMA75PAA25
(A)
(B)
74
(C)
(D)
Figure 4-7 Continued
75
42 Characterization of Latex Particles
421 Fourier Transform Infrared Spectroscopy (FTIR)
FTIR spectroscopy has proven to be a powerful tool to characterize polymeric
materials A molecule can absorb only select frequencies (energy) of infrared radiation
which match the natural vibration frequencies The energy absorbed serves to increase
the amplitude of the vibrational motions of the bonds in the molecule [Pavin et al 1996]
Only those bonds which have a dipole moment that changes as a function of time can
absorb infrared radiation The FTIR spectra of polymerized latex particles made during
this work are shown in Figure 4-8 There are number of characteristic peaks for PS latex
particles [Bhutto et al 2003 Li at al 2005] Aromatic C-H stretching vibration is shown
at 3002 cm-1 and 3103 cm-1 and aliphatic C-H asymmetrical and symmetrical stretching
shown at 2900 cm-1and 2850 cm-1 respectively There is an additional carbonyl (C=O)
adsorption at 1730cm-1 wavenumber for all core shell latex particles containing some
amount of either or both PMMA and PAA The broad OH group peak at 3400 cm-1
appear for particles containing some amount of PAA and has intensity dependent on the
amount of PAA in the shell For example the peak intensity of OH groups for
PSPMMA75PAA25 is greater than that of PSPMMA90PAA10 since there is a higher AA
monomer content in PSPMMA75PAA25 than in PSPMMA90PAA10 This OH peak is not
seen for PS and PS core PMMA shell particles because of the absence of OH groups on
PS and PMMA polymer chains
422 Zeta Potential Measurements
The electrical surface property was characterized by zeta potential measurements of
tex particles Zeta potential is an important and useful indicator of surface charge which
can be used to predict and control the stability of colloidal systems The greater the zeta
is
la
76
1520002500300035004000
Wavenumbers (cm-1)
li
Re
ativ
e
Tra
nsm
ttanc
e
Figure 4-8 FTIR spectra of polymerized latex particles (A) ba
PSPMMA100 (C) PSPMMA90PAA10 (D) PSPMM
(B)
(C) P
(D) P
C
C
OCH3
O
CH3
XCH2
CH2 n
CH
CH2 C
C
OCH3
O
XCH2 CH
OC
CH3
Y
OH
(A) Bare PS
500100000
re polystyrene (PS) (B) A75PAA25
PSPMMA100
SPMMA90PAA10
SPMMA75PAA25
77
potential the more likely the suspension is to be stable because the charged particles repel
one another and thus overcome the natural tendency to aggregate The measurement of
zeta potential is often the key ontrol of the interaction of proteins
with solid surfaces such as latex particles
Zeta potential (ζ) is the elec at exists at the shear plane of a particle
which is some small distance from the surface [Myers 1999] The zeta potential of the
particles can be determined by measuring the mobility of the particles in an applied
electric field termed electr its response to an alternating electric
field Colloidal particles dispersed in lu ically charged due to their ionic
characteristics and dipolar attributes The development of a net charge at the particle
surface affects the distribution of ions in the neighboring ng in
an increased concentration of counter ions to the surface Each particle dispersed in a
ns called fixed layer or Stern layer
Outside th compositions of ions of opposite polarities
form ble layer is form
particle-liquid interface This double layer may be considered to consist of two parts an
inner region which inclu nd relatively strongly to the surface and an outer or
diffuse region in which the ion distribution is determined by a balance of electrostatic
forces and random thermal motion The potential in this region therefore decays with the
distance from the surface until at a certain distance it becomes zero Figure 4-9 shows a
schematic representation of a typical ion distribution near a positively charged surface
The Stern surface (also referred to as the outer Helmholtz plane) is drawn through the
to understanding and c
trical potential th
ophoretic mobility or
a so tion are electr
interfacial region resulti
solution is surrounded by oppositely charged io
e Stern layer there are varying
ed in the region of the ing a cloud-like area Thus an electrical dou
des ions bou
center of those ions which are effectively adsorbed to the surface [Hunter 1981] The
78
extent of ion adsorption is determined by electrical and other long-range interactions
between the individual ions and surface of particles The ions outside of the Stern layer
form the diffuse double layer also referred to as the Gouy-Chapman layer [Burns and
Zydney 2000]
Shear plane
Stern layer
ψζ
0
ψs
ψShear plane
Stern layer
ψζ
0
ψs
ψ
t
controlled electric field is applied via electrodes immersed in a sample suspension and
Figure 4-9 Schematic representation of ion distribution near a positively charged surface
Zeta potential is a function of the surface charge of a particle any adsorbed layer a
the interface and the nature and composition of the surrounding medium in which the
particle is suspended The principle of determining zeta potential is very simple A
79
this causes the charged particles to move towards the electrode of opposite polarity
Viscous forces acting upon the moving particle tend to oppose this motion and the
equilibrium is rapidly established between the effects of the electrostatic attraction and
the viscosity dra
g The particle therefore reaches a constant terminal velocity
Figure 4-10 Schematic representation of zeta potential measurement(source
httpnitioncomenproductszeecom_shtm)
Because protein adsorption mechanisms are very complex and hard to explain in
biological systems protein adsorption is generally studied by using more ideal systems
consisting of one or more well characterized proteins a well-characterized adsorbent and
a well-defined aqueous solution Even so small changes in the experimental conditions
such as pH ionic strength and temperature generate totally different results Therefore
two media systems phosphate buffer (PB) and phosphate buffered saline (PBS) were
chosen to be used for the zeta potential measurements and protein adsorption study PB is
a simple aqueous system of 5 mM monobasic sodium phosphate (NaH2PO4H2O) to
n a constant pH PBS is used to keep the water concentration inside and outside of
the cell balanced If the water concentration is unbalanced the cell risks bursting or
shriveling up because of a phenomenon called osmosis PBS is a solution with the 5 mM
of monobasic sodium phosphate (NaH2PO4H2O) and 143 mM of sodium chloride (NaCl)
in water
maintai
80
The results of zeta potential measurement at room temperature (25plusmn1oC) as
function of pH and ionic strength (different media PB and PBS) for synthesized lat
particles are seen in Figure 4-11 Zeta potential values of polystyrene (PS) latex particle
at the Figure 4-9 are negative between -291 mV and -599 mV in PB and betwe
ex
s
en -203
mV and -278 mV in PBS at all pH ranges These negative zeta potential values of PS
latex particles are due to the sulfate groups [Vandenhu et al 1970] originated from
persulfate initiators attached via an oxygen bridge (-C-O-SO3-) at the end of polymer
chain where polymerization was initiated
-100
-80
-60
-40
-20
0
pH
Zta
pte
ntl
ζ
20
40
1 2 3 4 5 6 7 8 9
eo
ia (m
V)
PB (5mM)
PBS (5mM +143mM)
en -
Figure 4-11 Zeta potential of PS latex particles at 25oC
The zeta potential measurements of PSPMMA100 core shell latex particles as seen
in Figure 4-12 were also negative with a range of -28 and -505 mV in PB and betwe
143 mV and -186 mV in PBS at the pH 21-78 These negative values are also due to
sulfate groups on the particle surface
81
-100
-80
-60
-40
-20
0
20
40
1 2 3 4 5 6 7 8 9
Zea
pote
nti
l ζ
(mV
pH
ta
)
PB (5mM)
PBS (5mM + 143mM)
Figure 4-12 Zeta potential of PSPMMA100 latex particles at 25oC
The zeta potential for PSPMMA90PAA10 and PSPMMA75PAA25 was also
negative as seen in Figure 4-13 and 4-14 The zeta potential value for the
PSPMMA90PAA10 were between -367 mV and -678 mV in PB medium and between -
147 mV and -193 mV in PBS both at 25oC In case of the PSPMMA75PAA25 core shell
particles zeta potential values were between -291 mV and -520 mV in PB and between
-115 mV and -210 mV in PBS media Sulfate groups in PSPMMA75PAA25 and
PSPMMA90PAA10 core shell particles contribute to the negative zeta potential values
participate in forming a
negat
cid is expressed by the term pKa which is the pH
at which an acid is 50 dissociated However the contribution of the carboxylic acid
Carboxylic groups from acrylic acid monomer also would
ive surface charge of the latex particles at a pH greater than 70 because the pKa
value for the carboxylic acid is between 40 and 60 pH range [Ottewill et al 1967] This
indicates that carboxylic acid dissociation begins at a pH between pH 46-60 and
increases with pH The strength of an a
82
group to negative zeta potential values of PSPMMA-PAA core shell particles was not
detectable because there was no significant difference in zeta potential values for
synthesized latex particles at a pH greater than 70
-60
-40
-20
0
20
40
1 2 3 4 5 6 7 8 9
Zeta
pot
entia
l ζ
(mV
)
PB (5mM)PBS (5mM + 143mM)
-80
-100
pH
Figure 4-13 Zeta potential of PSPMMA90PAA10 latex particles at 25oC
-100
-80
-60
-40
-20
0
20
40
1 2 3 4 5 6 7 8 9
pH
Ze p
ont
i ζ
V)
tate
al (m
PB (5mM)PBS (5mM + 143mM)
Figure 4-14 Zeta potential of PSPMMA75PAA25 latex particles at 25oC
83
The zeta potential plots and values were similar for all latex particles This may be
due to the similar initiator density on the latex surface The initiator concentrations
monomers for shell polymerization were the same fo
to
r all core shell latex particles (062
ww from Table 4-4) A similar concentration (055 ww) of initiator to monomer was
added to prepare bare PS latex particles used in zeta potential measurements
Since the zeta potential values of all synthesized latex particles were negative
between pH=20 and pH=78 the isoelecric point (IEP) of these latex particles would be
less than pH=20 Sulfates have a pKa in the range of 10 to 20 [James 1985] indicating
that the sulfate group the conjugate base of a strong acid is protonated (-C-OSO3H) at
less than pH 20 This means that the synthesized latex particles are all negative at the
physiological pH Most serum proteins have a negative surface charge in blood
therefore when the negative latex particles are applied to blood stream fatal
tein is
avoided by charge repulsion in the blood Table 4-5 shows the common blood proteins
and their isoelectric points (IEP)
Table 4-5 The isoelectric point (IEP) of common proteins Serum proteins Isoelectric point (IEP)
complication by coagulation between applied latex particles and serum pro s
Albumin 48 α1-Globulin 20 α2-Microglobulin 54 γ1-Globulin 58 Heptoglobin 41 Hemoglobin 72 β1-lipoprotein 54 Immunoglobulin G (Ig G) 73 Fibrinogen 58 (source httpwwwfleshandbonescomreadingroompdf1085pdf
httpwwwmedalorgvisitorwww5CActive5Cch135Cch13015Cch130105aspx )
84
The absolute zeta potential values of the latex particles in phosphate buffer (PB)
was higher than that in phosphate buffered saline (PBS) This is because high
concentration of sodium and chloride electrolytes in PBS compressed the electrical
double layer causing rapid decay of surface potential Figure 4-15 shows that the
thickness of the electrical double layer is inversely proportional to the concentration of
electrolyte in the system
Surf
ace
Pote
ntia
l ψ
1κ3
10
1e
1κ 1κ
(1)(3) (2)
2 1 Z
Surf
ace
Pote
ntia
l ψ
1κ3
10
1e
1κ 1κ
(1)(3) (2)
Figure 4-15 The decay of surface potential with distance from surface in various
electrolyte concentrations (1) low (2) intermediate (3) high [Myers 1999]
2 1 Z
The electrical potential in the solution surrounding the particle surface falls off
the Debye-Huckel
yers 1999]
exponentially with distance (Z) from the surface according to
approximation [M
z) (- exp 0 κψψ = (4-4)
e reciprocal of the thickness of the electrical double layer referred to as the
The potential fallen off by a factor e The theoretical equation for the
ickness 1κ is
where κ is th
Debye length of 1
double layer th
21
22i
0
ekT 1
⎟⎟⎠
⎜⎜⎝
=sum ii zcεε
κ
⎞⎛ (4-5)
85
where
tration of
y was performed to measure the protein adsorption on the
synthesized latex particles Many physical and chemical processes occur at the boundary
between two phases and adsorption is one of the fundamental surface phenomena [Oscik
1982] This phenomenon is driven by the change in the concentration of the components
at the interface with respect to their concentrations in the bulk phase There are two
adsorption processes physical adsorption occurring when non-balanced physical forces
appear at the boundary of the phases and chemical adsorption or chemisorption
t the
interface [J
n leads
ational entropy hence adsorption takes place only if the
loss in conformational entropy is compensated
ter adsorption Cp The initial part of the
isotherm merges with the Γ-axis because at low polymer concentration the protein has a
high affinity for the surface and all of the polymer is adsorbed until the solid surface is
saturated
ε0 is the permittivity of a vacuum of free space ε is the permittivity of a medium e
is the relative permittivity or dielectric constant of the medium c is the concen
an ion (i) z is the valency of an ion (i) k is Boltzmannrsquos constant T is absolute
temperature (K)
43 Protein Adsorption Study
The adsorption stud
occurring when atoms and molecules from adjacent phases form chemical bonds a
aroniec et al 1988]
Flexible proteins in solution possess high conformational entropy as a result of the
various states each of the many segments in the protein chain can have Adsorptio
to a reduction of this conform
by sufficient attraction between polymer
segment and solid surface [Martin 1998] Isotherm shape of high affinity adsorption
between polymer and solid is shown in Figure 4-16 where the adsorbed mass Γ is plotted
against the polymer concentration in solution af
86
Figur
de
arboxyl
e 4-16 High-affinity adsorption isotherm of typical flexible polymer on solid
surface
In order to investigate the adsorption of blood proteins latex particle properties
such as electrical surface charge hydrophobicity and environmental conditions like pH
ionic strength and temperature are considered as experimental factors [Arai and Nor
1990] Figure 4-17 shows the overall schematic representation of the protein adsorption
on the synthesized latex particles with the functional groups such as sulfate and c
groups
CP
Γ
PMMAPAAShell
PS or PMMAShell OSO3
-
OSO3-
OSO3-
OSO3-
PS
-O3SO
PSCore
HOOC
-O3SO
O3SO
3
HOOC
HOOC
-
-O SO
Protein
NH3+
+
+H3N
+
COO-
Hydrophobic interaction
NH3
COO-
H3N
COO-
-OOC
Ionic interaction
Hydrophobic interaction
Hydrogen bonding
Ionic interaction
PMMAPAAShellOSO3
-
OSO3-
OSO3-
OSO3-
PS or PMMAShell -O3SOOSO3
-
PSCore
HOOC
-O3SO
O3SO
3
HOOC
HOOC
-
-O SO
-O3SO
OSO3-
OSO3-
OSO3-
PS PSCore
HOOC
-O3SO
O3SO
3
HOOC
HOOC
-
-O SO
Protein
NH3+
+
+H3N
+
COO-NH3
COO-
H3N
COO-
-OOC
NH3+
+
+H3N
+
COO-
Hydrophobic interaction
NH3
COO-
H3N
COO-
-OOC
Ionic interaction
Hydrophobic interaction
Hydrogen bonding
Figure 4-17 Overall schematic representation of the protein adsorption on the
Ionic interaction
synthesized latex particles
The surface functional groups as well as suitable surface hydrophobicity
hydrophilicity of latex particles are closely related to protein adsorption property As
indicated at Figure 4-17 various interaction forces such as hydrophobic interaction ionic
87
interaction hydrogen interaction and van der Waals interaction [Andrade 1985] exist
between protein molecule and the solid surface of latex particle
431 Adsorption Isotherm
The protein adsorption isotherm is generally defined as the relationship between the
amount of protein adsorbed by a solid (latex particle) at a constant temperature and as a
ine
the amount of protein adsorbed onto latex particles To evaluate the equilibrium
conce s
x
function of the concentration of protein Adsorption isotherms were plotted to determ
ntration of target proteins a calibration curve is firstly needed Figure 4-18 show
the UV absorbance as a factor of bovine serum albumin (BSA) concentrations The UV
absorbance of the known protein concentration was measured at 562nm which is the λma
for a protein treated by a bicinchoninic acid (BCA) assay
0
02
04
06
08
500 520 540 560 580 600
Wavelength (nm)
Abs
orba
nce
Figur gml (B)
06mgml (C) 02mgml
(A)
(C)
(B)
Max562nm
e 4-18 The dependence of UV absorbance on BSA concentration (A) 10m
Figure 4-19 shows the standard calibration curve used in adsorption isotherm
experiment of protein onto latex particles The net absorbance of each known protein
88
concentration was plotted and then a linear equation passing through a concentration of
zero protein concentration was obtained This equation is applied
to determine the
orbed on the synthesized latex particles The amount of free
protei
eported
trations (005 01 03 05
and 07mgml) were used to generate this curve as well as the adsorption isotherm
unknown protein amount ads
n was also determined using UV light at the 280nm wavelength however a
distinguishable UV intensity was too low to determine a precise value of protein
adsorption
The bicinchninic acid (BCA) assay technique [Smith et al 1985] has been r
for high efficiency of protein evaluation and was used for determination of protein
adsorption [Tangpasuthadol et al 2003 Williams et al 2003] This technique has many
advantages such as high sensitivity easy of use color complex stability and less
susceptibility to any detergents Five different BSA concen
y = 00R2 =
004741x 09939
00
01
02
03
04
0 200 400 600 800
BSA Standard (microgml)
Net
Abs
orba
nce
at 5
62nm
Figure 4-19 Standard curve of net absorbance vs BSA sample concentration
89
All isotherms were fitted to the Langmuir-Freundlich isotherms using the nonlinea
regression method Two adsorption isotherm models the Langmuir type isotherm a
Langmuir-Freundlich combination type isotherm were compared As seen the Figure 4-
20 The Langmuir isotherm model deviated further from the experimental data Th
all isotherms were fitted to the Langmuir-Freundlich isotherms [Yoon et al 1996
al 1998] usi
r
nd the
erefore
Lee et
ng the nonlinear regression method with following equation
n
n
eq
eqm
kC
kC1
1
1+
where q is the amount of adsorbed BSA per unit surface area q
qq = (4-6)
stant
were between 045 and 055 and hence n was fixed to its average 05 Actual qm values
were evaluated after fixing the n value
m is the adsorbed amount
in equilibrium Ceq is the equilibrium concentration of BSA k is the adsorption con
and n is the exponential factor for a heterogeneous system n values are empirical and
usually from zero to no more than unity [Jaekel 2002] The values of n in our system
00
10
20
30
0 02 04 06 08
C (mgml)eq
q (m
gm
2 )
pH 32Langmuir-Freundlich modelLangmuir model
Figure 4-20 Fitted models for adsorption isotherm of bovine serum albumin (BSA) on
polystyrene latex particles at 37oC in phosphate buffer media
90
The BSA protein adsorption on the synthesized latex particles was performed at
three pH levels 32 48 74 in both phosphate buffer (PB) and phosphate buffered saline
(PBS) at physiological temperature 37oC These pH levels correspond to an acidic pH
lower than the isoelectric point (IEP) of BSA the pH at the IEP and an alkaline pH
higher than the IEP of BSA respectively The IEP corresponds to the pH at which the
amino acid becomes a zwitterion Here its overall electrostatic charge is neutral and the
charge expressed by the basic amino group (-NH2 -NH3+) of the amino acid is equal to
the charge expressed by the acidic carboxyl group (-COOH -COO ) The IEP of BSA is
-
pH 48 and indicates that the side chain of the amino acids of BSA contains a greater
amount of acidic functional groups than that of basic functional groups At pH 32 and
74 BSA has a positive and negative charge respectively
Figure 4-21 and 4-22 show adsorption isotherms of BSA onto polystyrene (PS)
latex particles
00
10
20
30
40
0 02 04 06 08
Ceq (mgml)
q (m
gm
2 )
pH=32pH=48pH=74Model (pH 32)Model (pH 48)Model (pH 74)
Figure 4-21 Adsorption isotherm of BSA on polystyrene (PS) latex particles in
phosphate buffer (PB) at 37oC
91
00
10
30
40
0 02 04 06 08
Ceq (mgml)
q (m
gm
2
20
)
pH=32pH=48pH=74Model (pH 32)Model (pH 48)Model (pH 74)
(PBS) on PS latex particles
At the IEP of BSA the adsorbed amount (q
Figure 4-22 Adsorption isotherm of BSA adsorption 37oC in phosphate buffered saline
onto PS at equilibrium was
maxim
um at the IEP of BSA This is because the BSA molecules form the most
compact structures at the IEP eliminating the repulsion force between proteins A higher
number of BSA molecules can adsorb on a given surface area by hydrophobic interaction
when in this compact structure This observation agrees well with reported data by other
researchers who also indicated that the maximum adsorption from aqueous protein
solution was observed at the IEP [Mularen 1954 Watkins et al 1977 Oh at el 2000]
There was no significant difference of adsorbed BSA amount in PB and in PBS media at
pH 48 The conformational alteration of BSA was not affected significantly by the ionic
strength of the two solutions There was evidence of the effect of the electrostatic
interaction to some extent even at the IEP since the amount of BSA adsorbed in PBS is
slightly hig
m) of BSA
um indicating that hydrophobic interaction between latex particle and BSA
protein is maxim
her than in PB at this pH
92
The adsorbed amount of protein is shown to decrease at a pH 32 and 74 The
decrease in adsorption efficiency when deviating from IEP of BSA is a result of the
increase in conformational size of the protein molecules and the lateral electrostatic
repulsions between adjacent adsorbed BSA molecules Figure 4-23 shows a schematic of
possible conformational changes at each pH where hydrophobic interaction always exists
between non-polar portion of a protein and hydrophobic portion of solid surface
- - --- ---Latex surface
Zeta potential
BSA(+)
(-)
48 7432pH
(0)
+++ + ++
--+
++-
- - --- ---Latex surface
Zeta poten pH(0)
+++ + ++
--
tial
BSA(+)
(-)
48 7432
+
++-
ming
4
s
al blood Table 4-6 shows the calculated results of
Figure 4-23 Conformation of bovine serum albumin (BSA) on latex particles (Assuthat charge repulsion reduces the effect of the hydrophobic interaction at pH 74)
In addition the adsorbed amount of BSA at pH 32 was larger than that at pH 74
and still less than that at 48 This is because there is the electrostatic interaction between
the positive BSA molecules and negative PS latex particles at pH 32 and electrostatic
repulsion is dominant between negative BSA and negative PS latex particles at pH 7
The amount adsorbed increased with increasing ionic strength in pH 32 and 74 This i
because the high concentration of electrolytes reduces the electrostatic repulsion between
BSA and PS latex particle It should be noted that the BSA concentration used in these
experiments was lower than that found in normal human blood This is because the
equilibrium BSA adsorption onto the latex particles is actually reached at a much lower
concentration than that found in norm
93
adsor rm
ein
leading to a compact conformation of BSA There are two main forces affecting
adsorption amount at an acidic pH of 32 They include the ionic attraction between the
negative latex particle and the positive protein and lateral repulsion between positive
proteins The smallest amount of BSA adsorption was observed at a pH of 74 At this
point the charge repulsion between negatively charged particles and protein molecules
exists the h
of cha
bed BSA amount at equilibrium from the Langmuir-Freundich adsorption isothe
equation
In summary the hydrophobic interaction between PS latex particle and BSA
protein exists and is the dominant mechanism for the adsorption process Conformational
changes of BSA at each pH also affected the adsorption amount Adsorption was highest
at pH 48 because at this pH there is the least amount of ionic repulsion in the prot
and the lateral repulsion between proteins dominant adsorption even though there always
ydrophobic interaction between the solid latex particle and protein The effect
rge repulsion was reduced by the high concentration of electrolytes in PBS medium
Table 4-6 The equilibrium concentration values of BSA adsorption on polystyrene (PS) latex particles calculated the Langmuir-Freundlich isotherm model
Media pH qm (mgm2) 32 174 48 299 Phosphate buffer 74 136 32 260 48 283 Phosphate buffered saline 74 207
The adsorption isotherms of BSA on PSPMMA core shell particles are shown in
Figure 4-24 in phosphate buffer (PB) and in Figure 4-25 in phosphate buffered saline
(PBS) The adsorbed BSA amount was calculated using the Langmuir-Freundlich
100
94
adsorption isotherm equation and the amount listed in Table 4-7 The trend seen in the
adsorption results was very similar to the case of PS latex The overall adsorption process
was d rticle
rs
hat
0
ominated by hydrophobic interactions between the PSPMMA100 core shell pa
and BSA proteins At pH 48 the adsorbed amount (qm) of BSA on PSPMMA100 core
shell particles was 221 mgm2 in PB and 237 mgm2 in PBS and are the maximum
values recorded among the three pH levels 32 48 and 74 Suzawa and his co-worke
[1982] have obtained similar results This can be also explained by the compact
conformation of BSA molecules at the IEP of BSA which lead to a high protein
population on the solid surface At a pH of 32 and 74 BSA adsorption was less than t
at pH 48 because of the protein conformation change There was a small increase in
adsorbed BSA at pH 32 because of the charge attraction between negative PSPMMA10
core shell particles and positive BSA At pH 74 however the adsorption of BSA was
further reduced by charge repulsion between the negative PSPMMA100 core shell
particles and the negative BSA proteins
000 02 04 06 08
Ceq (mgml)
10
20q (m
gm
302 )
40
50pH=32pH=48pH=74Mode 8)l (pH 4Mode 2)l (pH 3Mode 4)
Figure 4-24 Adsorption isotherm of BSA 37oC in PB on PSPMMA core shell latex
l (pH 7
100
particles
95
The adsorbed amount of BSA increased more rapidly in PBS with increasing ioni
strength at pH 74 This shows that the low concentration of electrolytes promotes
electrostatic repulsion between negative BSA and negative PSPMMA
c
on
x
l 1980
100 core shell
particle As the ion strength of PBS is increased however the high concentration of
electrolyte leads to less electrostatic repulsion promoting more rapid protein adsorpti
By comparing the amount of adsorbed protein between bare PS latex particles and
PSPMMA100 core shell particles the amount of protein adsorption on the PS late
particles is greater than that on the PSPMMA100 core-shell particles [Suzawa et a
1982 Lee et al 1988]
00
10
20
30
40
m2 )
0 02 04 06 08
Ceq (mgml)
q (m
g
pH=32pH=48pH=74Model (pH 2) 3Model (pH 48)Model (pH 74)
Figure 4-25 Adsorption isotherm of BSA at 37oC in PBS on PSPMMA100 core shell
latex particles
96
Table particles calculated the Langmuir-Freundlich isotherm model
4-7 The equilibrium concentration values (qm) of BSA adsorption on PSPMMA100
Media pH qm (mgm ) 2
32 140
48 221 Phosphate buffer
74 130
32 169
48 237 Phosphate buffered saline
74 162
The BSA adsorption mechanism and trend on PSPMMA core shell particles was
very similar to that of the PS latex particle Unlike the case of PS and PSPMMA100 latex
particles the amount of BSA adsorbed onto PSPMMA90PAA10 and PSPMMA75PAA25
core shell particles was higher at pH 32 than pH 48 and 74 Figure 4-26 through 4-29
shows the adsorption isotherms of BSA on PSPMMA90PAA10 and PSPMMA75PAA25
core shell particles The amount of BSA adsorbed increased at pH 32 and pH 48 as the
hydrophilicity was developed by the addition of PAA On the contrary the adsorbed
amount of BSA was dramatically decreased at pH 74 The adsorption amount of BSA on
PSPMMA90PAA10 and PSPMMA75PAA25 core shell particles at equilibrium is listed in
Table 4-8 and 4-9 respectively
Carboxylic acid as well as sulfate groups are distributed on the surface of
PSPMMA90PAA10 and PSPMMA75PAA25 core shell particles and seem to largely affect
the BSA adsorption The isotherms showed very strong pH dependence to the BSA
adsorption onto these core shell latex particles The carboxylic groups (COOH) may
especially enhance the BSA adsorption prominently at pH 32 by hydrogen bonding with
protein molecules The pKa value for the PAA is 40-60 and a good indication that the
97
carboxylic acid groups are protonated at pH 32[Gebhardt et al 1983] The main
adsorption interaction force at pH 32 was generated by hydrogen bonding as well as
charge attraction between latex particle protein even th the conformational
repulsion between proteins results in a reduction of the protein
adsorption at this pH The amount of BSA adsorbed also increased as the PAA amount in
eased The a tion of BSA w inimal however less
than 07 mgm2 at pH 74 Here the carboxylic groups are ionized which increases the
charge repulsion by ionized carboxylic groups (COO-) resulting in PSPMMA-PAA latex
particle
and ough
change and charge
the core shell particles incr dsorp as m
s with much lower protein adsorption
00
20
40
q (m
g2 )
60
m
pH=32pH=48pH=74Model (pH 32)Model (pH 48)
0 02 04 06 08
Ceq (mgml)
Model (pH 74)
o shell
Figure 4-26 Adsorption isotherm of BSA at 37 C in PB on PSPMMA90PAA10 corelatex particles
98
00
40
60
80
0 02 04
C (mgm
mm
2 )
20
06 08
eq l)
q (
g
pH=32pH=48pH=74Model (pH 32)Model (pH 48)Model (pH 74)
Figure 4-27 Adsorption isotherm of BSA at 37oC in PBS on PSPMMA90PAA10 core
shell latex particles
Table 4-8 The equilibrium concentration values of BSA adsorption on PSPMMA90PAA10 particles calculated the Langmuir-Freundlich isotherm model
Media pH qm (mgm2)
32 361
48 283 Phosphate buffer
74 043
32 519
48 226 Phosphate buffered saline
74 043
99
00
20
40
60
80
100
0 02 04 06 08Ceq (mgml)
q (m
gm
2 )
pH=32pH=48pH=74Model (pH 32)Model (pH 48)Model (pH 74)
oFigure 4-28 Adsorption isotherm of BSA 37 C in PB on PSPMMA75PAA25 latex particles
00
100
20
40
60
80
0 02 04 06 08Ceq (mgml)
q (
m2 )
pH=32pH=48pH=74
mg
Model (pH 32)Model (pH 48)Model (pH 74)
Figure 4-29 Adsorption isotherm of BSA 37oC in PBS on PSPMMA75PAA25 core shell
latex particles
100
Table 4-9 The equilibrium concentration values of BSA adsorption on PSPMMA75PAA25 particles calculated the Langmuir-Freundlich isotherm model
Media pH qm (mgm2)
32 1083
48 662 Phosphate buffer (PB)
74 075
32 1077
48 681 Phosphate buffered saline (PBS)
74 057
The adsorption of β2-microglobulin (β2M) was determined under one set of
onditions in phosphate buffer at 37oC and pH 74 because of the limited amount of β2M
protein The adsorption isotherms of the β2M protein on the synthesized latex particles
are shown in Figure 4-30 4-31 and 4-32 The β2M protein concentrations for adsorption
isotherm experiment were chosen to be 0015 0030 0045 and 0060mgml since the
β2M concentration for the patients with renal failure can be elevated to similar
concentrations about 005mgml or more up to 50 times the normal level [Nissenson et
al 1995] There were steep initial slopes of the adsorption isotherm curves of β2M on
latex particles in all isotherms indicating a high affinity adsorption type It seems that the
hydrophobic interaction is enough for the adsorption process of β2M proteins to occur on
the latex particles even though charge repulsion exists between protein and latex particle
at pH 74 The complete plateau regions were not seen for all isotherms indicating that
β2M proteins were not saturated at this level and there are still available sites for β2M
c
adsorption on latex particles The initial slope of the β2M isotherms for PS was steeper
101
than that of any of the other latex particle PSPMMA100 PSPMMA90PAA10 and
PSPMMA 75PAA25
00
02
0 0002 0004 0006 0008
04
06
10
Ceq (mgml)
q (m
2 )
08
gm
PSPSPMMA100Model (PS)Model (PSPMMA100)
Figure 4-30 Adsorption isotherm of β2M onto PS and PSPMMA100 latex particles in PB
at 37 C and pH 74 o
00
02
04
06
08
10
0 0002 0004 0006 0008C (mgml)
q (m
m2 )
eq
g
PS
PSPMMA90PAA10
Model (PS)
Model (PSPMMA90PAA10)
Figure 4-31 Adsorption isotherm of β2M onto PS and PSPMMA90PAA10 latex particles
in PB at 37oC and pH 74
102
00
02
04
06
08
10
0 0002 0004 0006 0008Ceq (mgml)
q (m
gm
2 )
PS
PSPMMA75PAA25
Model (PS)Model (PSPMMA75PAA25)
Figure 4-32 Adsorption isotherm of β2M onto PS and PSPMMA75PAA25 latex particles
in PB at 37oC and pH 74
The equilibrium concentration values (qm) of β2M adsorption on latex particles are
calculated and listed in Table 4-10 The difference between the maximum amounts of
β2M adsorption on all the latex particles was not significantly different and their values
were between 069 and 08mgm2
Table 4-10 The equilibrium concentration values of β2M adsorption calculated the Langmuir-Freundlich isotherm model
Latex particles qm (mgm2)
PS 080
PSPMMA100 069
PSPMMA90PAA10 072
PSPMMA75PAA25 072
103
432 Adsorbed Layer Thickness
There are several possible explanations for large amount of BSA adsorbed at acidic
pH levels including the end-on adsorption pattern of BSA the flexibility of BSA
molecules the tilting of protein molecules due to the asymmetry of the charge
distribution or multilayer formation [Peula et al 1993] The adsorbed BSA thickness (δ)
can be calculated by the following equation [Chiu et al 1978] This equation was also
cited again by other researchers [Shirahama et al 1985]
BSA
mqρπ
δbullbull
=33
(4-7)
where π
33 is the packing factor qm is the plateau value of the adsorbed am
ρBSA is the hich corresponds to the reciprocal of its known
partial specific volume The layer thickness (δ) of BSA proteins on synthesized latex
partic
he IEP [Shirahama et al 1985] Compared to the
hydrodynamic dimensions of BSA 140 times 40 times 40 Aring3 [Fair et al 1980 Peter 1985] these
nd
ode It is unnecessary ultilayering because thickness do
exceed 140 Aring The layer thickness (δ) of BSA proteins on synthesized latex particles
e red saline (PBS) was ran 28 ~ 83 Aring at 37oC and pH 48 These
lso listed in Table 4-1
ount and
density of BSA molecule w
les in phosphate buffer (PB) ranged from 27 - 81 Aring at 37oC and pH 48 These
calculated values are listed in Table 4-11 Only δ values at pH 48 the isoelectric point
(IEP) of BSA are important because intra- and intermolecular electrostatic repulsion of
protein molecules is minimized at t
values of layer thickness indicate that the BSA molecules adsorb by side-on (40Aring) a
end-on (140Aring) m to consider m
not
in phosphat buffe ged of
calculated values are a 2
104
Table 4-11 Absorbed BSA layer thickness (Aring) of BSA in phosphate buffer (PB) at 37oC pH PS PSPMMA100 PSPMMA90PAA10 PSPMMA75PAA25
32 21 17 44 133
48 37 27 35 81
74 17 16 5 9
Table 4-12 Absorbed BSA layer thickness (Aring) of BSA in phosphate buffered saline (PBS) at 37oC
pH PS PSPMMA100 PSPMMA90PAA10 PSPMMA75PAA25
32 32 21 64 132
48 35 29
74 26 20 5 7
28 83
1n
1n
neqm
So k is closely related to the initial slope of the isotherm showing the affinity
between BSA and latex particles [Yoon et al 1996] A key point in the characterization
433 Gibbs Free Energy of Protein Adsorption
In the Langmuir isotherm model k represents the ratio of adsorption to the
desorption rate constants This definition is not applicable to the Langmuir-Freundlich
isotherm model but its meaning is similar [Yoon et al 1996] In this sense k is closely
related to the affinity between protein molecules and latex particles When the
equilibrium concentration Ceq reaches zero the value of Ceq will be very small (1gtgt
kCeq ) and then the equation 4-6 becomes
(4-8)
of adsorption processes on solid-liquid interfaces is to determine the change in Gibbsrsquos
free energy (∆Gdeg) one of the corresponding parameters for the thermodynamic driving
forces during adsorption The traditional method to evaluate adsorption thermodynamic
kCqq 1=
105
constants is based on measurement of adsorption isotherms under static conditions
[Malm ten 1998] The Langmuir-Freundlich ads lf
easily found from Then this enables calculation of the free-
energy change ∆G during adsorption expressed as
(4-9)
where R is the general gas constant (R = 8314Jmiddotmol K ) and T is the absolute
tem ture Gibb e energy A P
listed in Table 4-13 and 4-14 respectively As the ∆Gdegads ore negativ e
pro adsorption on a solid substra ill be more favorable
e of BSA adsorption in phosphate o
pH PS PSPMMA100 PSPMMA90PAA10 PSPMMA75PAA25
s orption equilibrium constant k can be
a slope of the isotherm
degads
lfads kRTG lnminus=∆ o
-1 -1
pera s fre change of BS adsorption values in B and PBS are
values are m e th
tein te w
Table 4-13 The values of Gibbs free energy changbuffer (PB) at 37 C
32 -2411 -2726 -2846 -2805
48 -2352 -2383 -2198 -1972 ∆Go
80
ads (kJmol)
74 -2452 -888 -324 -2
Table 4-14 The values of Gibbs free energy change of BSA adsorption in phosphate
pH PS PSPMMAbuffered saline (PBS) at 37oC
100 PSPMMA90PAA10 PSPMMA75PAA25
32 -2315 -2620 -2312 -2683
48 -2149 -2299 -2090 -2138 ∆Go
(kJmads ol)
74 -2430 -1992 -258 -263
Table 4-15 shows the calculated values of Gibbs free energy change of β M
adsorption on synthesized latex particles PSPMMA PAA core shell latex particle has
2
75 25
106
largest negative Gibbs free energy of β2
2
M adsorption indicating largest affinity betw
β
een
2M adsorption in phosphate buffere (PB) at 37 C and pH74
PS PSPMMA100
M and latex particle
Table 4-15 The values of Gibbs free energy change of βo
PSPMMA90PAA10 PSPMMA75PAA25
∆Go03 ads
(kJmol) -3626 -3466 -3659 -39
The Gibbs free energy of β2M was much more negative in phosphate buffer (PB)
than that of BSA in physiological condition (37 C and pH 74) o from the Table 4-13 and
the Figure 4-31 This indicates that β2M molecules more readily adsorb on latex particles
BSA This can be explained by the rate of diffusion and accessibility of β2M molecules to
the latex particle ier and faster for β2M m an BSA because of
size (molec ight) erical shape molecules It o be
due to the higher hydrophobic force and less electrostatic repulsion that contribute to the
adsorption of β2M molecules not as prevalent in the case of BSA because the IEP of β2M
(57) is clo H 74) than that of BSA (48) causing β2M to
be less electro cally lled p ro
can be seen that the ∆ β2M adsorption onto the synth article
increasing wi hy icity o x particles s the ∆Goads of the BSA
molecules is decreasing or constant This phenomenon supports the theory that the
select lizing
than BSA and that the affinity of β2M for the latex particle is much larger than that of
surface It is eas olecules th
the smaller ular we and sph of β2M may als
ser to physiological condition (p
stati repe from the latex articles than BSA F m Figure 4-33 it
Goads of esized latex p s is
th the drophil f the late wherea
ive adsorption of β2M molecules over BSA molecules can be enhanced by uti
more hydrophilic PSPMMA90PAA10 and PSPMMA75PAA25 core shell latex particles
107
-60
-50
1 2 3 4
-40∆Gad
s
-30
10
0
o (k
Jm
o-
-20l)
β2-Microglobulin (β2M)Bovine serum albumin (BSA)
Figure 4-33 Gibbs free energy of adsorption of proteins on latex particles in PB at 37
and pH 74
434 Kinetics of Adsorption
to
oC
Protein adsorption is a complex process known to have a large biological impact
and is currently not well understood quantitatively because of extreme sensitivity to pH
the concentration of other electrolytes and molecules present temperature and many
other factors that can change in the physiological system Accurate knowledge of the
adsorption kinetics under a given set of conditions is a prerequisite for elucidating the
mechanisms of many fundamental biological processes to be predicted at the molecular
level [Regner 1987] This adsorption kinetics approach is used in protein adsorption
studies because of the uncertainty related to the time needed for the interfacial proteins
reach the equilibrium It is generally accepted that the process of protein adsorption is
comprised of the following steps a) transport toward the interface b) attachment at the
interface c) eventual structural rearrangements in the adsorbed state d) detachment from
PS PSPMMA100 PSPMMA90PAA10PSPMMA75PAA25
108
the interface and transport away from the interface Each of these steps can affect the
overall rate of the adsorption process
The quantitative analysis of the protein adsorption kinetics requires that the protein
amount adsorbed is known as a function of time The kinetics tests were carried out in
phosphate buffer (PB) at 37oC and pH 74 a condition similar to physiological condition
The protein concentrations chosen were 07mgml for BSA and 006mgml for β2M
which are the maximal concentrations used in an adsorption isotherm test A time-based
evaluation of the adsorption was made by measuring the concentration of protein in
solution at different incubation times The results are shown below from Figure 4-34 to 4-
37 The adsorption kinetics of proteins onto th cond-e latex particles is expressed as a se
order model [Oumlzacar 2003] shown by the following equation
2)( tet qqk
dtdq
minus= (4-10)
Integrating above equation and applying the boundary conditions yields
ee
t tq
+=
1 (4-11)
where q
qkq
t
2
2
2
the previous adsorption isotherm results representing good reproducibility of adsorption
t and qe stand for the amount (mgm ) of protein adsorbed at time t and at
equilibrium respectively k is the equilibrium rate constant of second-order adsorption
(m mghr) and t is the incubation time (hr)
The fitting parameters calculated by using the equation 4-11 are listed in table 4-16
From this data it is seen that the amount of BSA adsorbed at equilibrium was higher than
that of β2M on PS and PSPMMA100 latex particles but less than that of β2M on
PSPMMA90PAA10 and PSPMMA75PAA25 latex particles These results are similar to
109
00
05
15
20
0 2 4 6 8 10 12 14
Time (hr)
rot
n ad
sorb
d (
10
25
Pei
em
gm
2
BSA2nd order model (BSA))β2M2nd order model (β2M)
Figure 4-34 The kinetics of protein adsorption in PB on PS latex particles at 37oC and
pH 74
00
05
10
15
20
25
30
0 2 4 6 8 10 12 14
Time (hr)
Prte
inds
oo
arb
ed (
mg
m2 )
BSA2nd order model (BSA)β2M2nd order model (β2M)
Figure 4-35 The kinetics of protein adsorption in PB on PSPMMA100 latex partic
37les at
oC and pH 74
110
00
03
06
09
12
15
0 2 4 6 8 10 12 14
Time (hr)
Prot
ein
adso
rbed
(m
gm
2 ) BSA2nd order model (BSA)β2M2nd order model (β2M)
Figure 4-36 The kinetics of protein adsorption in PB on PSPMMA90PAA10 latex
particles o at 37 C and pH 74
00
03
06
09
12
15
0 2 4 6 8 10 12 14
Time (hr)
BS
A ad
sorb
ed (
mg
m2 )
BSA2nd order model (BSA)β2M2nd order model (β2M)
Figure 4-37 The kinetics of protein adsorption in PB on PSPMMA75PAA25 latex
particles at 37oC and pH 74
111
test The variation of β2M equilibrium adsorption amount was nearly negligible for all
latex particles The plateau region is reached earlier for β2M than BSA on all latex
particles and the K values (equilibrium rate constant of second order adsorption) for the
β2M adsorption process onto latex particles are much larger than for BSA adsorption
These values indicate that the β2M molecules have a higher adsorption rate onto the latex
particles than the BSA molecules
Table 4-16 Fitting parameters of second-order kinetic model BSA β2M
Latex particles qe (mgm2) K (hr-1) qe (mgm2) K ( -1) hr
PS 144 67 074 7549
PSPMMA100 175 353 076 4438
PSPMMA90PAA10 046 97 075 5695
PSPMMA75PAA25 060 179 075 5168 K = (kqe) hr-1
There are several explanations for the phenomena observed in these experiments
First the smaller sizes of β2M molecules allow easier diffusion and a close approach to
the latex substrate Second a reduced number of hydrophobic domains on the latex
particle surface replaced by sulfate groups (-OSO3-) and ionized carboxylic groups
(COO-) are not favorable for larger BSA molecules Third the conformational change by
intra- and inter-molecular repulsion would be faster in β2M due to smaller molecular
weight Finally the ionic repulsion between β2M and the latex particles is less than that
condition (pH 74) than IEP (48) of BSA
between BSA and latex particles because IEP (57) of β2M is closer to the tested
112
44 Blood Compatibility
Blood compatibility can be defined as the property of a material or device tha
permits it to function in contact with blood without inducing any adverse reactions
Successful functioning of blood contacting biomedical materials or devices in contact
with blood strongly depends on the extent to which they damage blood during operation
High shear stresses turbulence rela
t
tively long contact times and cellular impact with
artificial surfaces are most common events experienced by blood in contact with artificial
1978 Winslow and et al
1993] Therefore before any materia e placed into the human b
biocompatibility testing must be the first performed in the view of safety The hemolysis
s a method for pr ary screening of biomaterials to determine
h blood a er circulating body fluid The tests estimat
oglobin released from th blood cell d e induced
foreign material
There are several types of blood cells in our body but the ones that make blood red
are erythrocytes or red blood cells (RBCs) Red blood cells (erythrocytes) are the most
numerous type in the blood These cells are average about 48-54 million per cubic
millimeter (mm3 same as a microliter (microl)) of blood but these values can vary over a
larger range depending on such factors as health and altitude RBCs are red because they
contain hemoglobin which is a protein that contains iron When blood circulates through
lungs oxygen binds to the hemoglobin and is then delivered to all the parts of body
RBCs are made in the bone marrow They start out as a nucleus like other cells but
when they fill up with hemoglobin the nucleus becomes smaller until it disappears
Without a nucleus RBCs are fragile and live only about 120 days In a normal healthy
devises and can cause hemolysis or thrombosis [Bernhard et al
ls can b ody
test serves a elimin the
biocompatibility wit nd oth e the
concentration of hem e red amag by a
113
body about 2 million red blood ne marrow produces new
ones j ing
dy
le
cells die per second But bo
ust as fast RBCs are about 75 micrometer across and are responsible for carry
oxygen salts and other organic substances to the cells and tissues throughout our bo
In humans and most mammals the cells are concave (indented in the middle) and flexib
enough to squeeze through even the tiniest of blood vessels the capillaries
Figure 4-38 Image of red blood cells
(Sourcehttpwwwpbrchawaiiedubemfmicroangelarbc1htm)
ch
100 90 10 75 25 f
RBCs are among the first cells that come into contact with a foreign material
injected in the blood Therefore a hemolysis assay gives information about the
biocompatibility in the case of an in vivo application The concentrations of prepared
latex particles used for the tests in this research are 05 25 and 50 (ww) and the
incubation time under moderate shaking was 30 minutes at 37oC Test results are shown
in Figure 4-39 The amount of blood cells that ruptured when in contact with the PS latex
particles was largely increased from about 30 to 91 as the solid content of PS
increased from 05 to 50 However hemolysis by the core shell latex particles su
as PSPMMA PSPMMA PAA and PSPMMA PAA was less than 35 O
114
particular interest was that hemolysis caused by PSPMMA PAA particles at 50
solid concentration was less than 02 hemolysis the lowest value among the latex
particles The low hemolysis value indicates that the PSPMMA PAA core shell latex
particles are the most blood compatible at all solid contents Other core shell particles
such as PSPMMA and PSPMMA PAA also had excellent blood comp
90 10
90 10
100 75 25 atibility with
low RBS breakage
01 2 3
20
40
60
80
100
Hem
olys
is (
)
052550
Figure 4-39 He
PS PSPMMA100 PSPMMA PAA90 10
molysis caused by latex particles n=5
4PSPMMA75PAA25
CHAPTER 5 SUMMARY OF RESULTS CONCLUSIONS AND RECOMMENDATION FOR
FUTURE WORK
51 Summary of Results
The goal of this study was to synthesize monodisperse polymeric latex particles
with tailored properties and to investigate the fundamental interactions between the
synthesized latex particles and target proteins An understanding of the fundamental
mechanism of selective adsorption is strongly required in order to maximize the
separation performance of membranes made of these materials so that it may applicable
to hemodialysis therapy for end stage renal disease (ESRD) patients For the achievement
of above research goal several investigations have been performed Analysis of the
obtained data leads to several conclusions as discussed below
Monodisperse polystyrene seed latex particles were synthesized with the size
crosslinking agent to make the latex particles more hydrodynamically stable The
conversion of monomer to polymer was over 95 and seed latex particles are spherical in
shape and have smooth surface From these small seeds bigger latex particles as large as
800nm were synthesized using a semi-continuous seeded emulsion polymerization
process The particles with a mean diameter less than 500nm are very spherical in shape
and highly uniform in particle size distribution However as the particles grow larger
than 500nm in mean diameter they became non-spherical with an uneven surface This
irregularity of particle surface can be attributed to the non-homogeneous monomer
ranges between 126plusmn75 and 216plusmn53 nm Divinylbenzene (DVB) was used as a
115
116
swelling into the shell of a growing polymer which can be controlled by the factors such
as temperature agitation speed initiator feeding rate and surfactant amount required to
stabilize the colloidal system It was determ rfactant to monomer ratio
dec ic
acid (AA) monomers were intro rticle surfaces more
hydro
to
ting of a
orm
ration is seen between
3002
0 cm-
0cm-1
-1
03
ined that as the su
reased the mean particle diameter increased Methyl methacrylate (MMA) and acryl
duced to make the latex pa
philic than bare polystyrene particles The prepared core shell latex particles were
made of PMMA100 PMMA90PAA10 and PMMA75PAA25 PMMA100 is the PMMA
homopolymer shell on a PS core PMMA90PAA10 shell is a copolymer with a PMMA
PAA ratio of 90 to 10 by weight PMMA75PAA25 shell is a copolymer consis
PMMA to PAA ratio of 75 to 25 by weight The particle size of these core-shell
particles were prepared to be about 370nm in mean diameter
The successful synthesis of latex particles was confirmed using Fourier Transf
Infrared spectroscopy (FTIR) The C-H aromatic stretching vib
cm-1 and 3103 cm-1 for PS latex particles The C-H asymmetrical stretching and
symmetrical stretching vibration peaks of CH2 for all latex particles is seen at 290
1and 2850 cm-1 respectively There is a carbonyl (C=O) characteristic band at 173
wavenumber for PSPMMA core-shell latex particles The broad OH group peak at
3400 cm
100
can be seen in the spectroscopy of the particle with PMMAPAA shell
(PSPMMA PAA and PSPMMA PAA ) The peak intensity of OH group of
PSPMMA PAA was larger than that of PSPMMA PAA relatively due to more AA
monomer content in PSPMMA PAA than in PSPMMA PAA
In the zeta potential measurements polystyrene (PS) latex particles had negative
values between -291 mV and -599 mV in the phosphate buffer (PB) and between -2
90 10 75 25
75 25 90 10
75 25 90 10
117
mV and -278 mV in the phosphate buffered saline (PBS) at 25oC and pH 21-78 These
negative zeta potential values for PS latex particles are due to ionization of the sulfate
groups (-OSO3-) originated from the initiator Zeta potential values of the PSPMMA
core shell latex particles were also negative between -28 mV and -505 mV in PB and
between -143 mV and -186 mV in PBS at pH 21-78 Negative values are also
generated by dissociation of sulfate groups on a particle surface The zeta potential value
of PSPMMA PAA and PSPMMA PAA were of negative values between -36
and -678 mV in PB medium and -147 mV and -193 mV in PBS medium at 25
100
s
7 mV
MMA75PAA25 and
PSPM
otein
g the
l change of BSA at
each w
90 10 75 25
oC For
PSPMMA PAA core shell particles zeta potential values were between -291 mV and
-520 mV in PB media and between -115 mV and -210 mV in PBS media Sulfate
groups contributed to the negative zeta potential values in PSP
75 25
MA90PAA10 core shell particles Carboxylic groups from polyacrylic acid also
contribute to the negative surface charge for the latex particles at greater than pH 70
however the degree of their contribution was not detectable from the zeta potential
measurements The absolute zeta potential values of the synthesized latex particles in PB
were higher than that in PBS This is because of the high concentration of sodium
chloride electrolyte in PBS that compress electrical double layer thickness
The bicinchoninic acid (BCA) assay technique was used to determine the pr
adsorption and all isotherms were fitted to the Langmuir-Freundlich isotherms usin
nonlinear regression method The hydrophobic interaction between PS latex particle and
BSA protein was dominant for the adsorption process Conformationa
pH also affected the adsorption amount which was highest at pH 48 because of lo
ionic repulsion making compact conformation of BSA The next larger amount of BSA
118
adsorbed onto PS was at pH 32 There were two main forces affecting the amount of
adsorption at acidic pH values One is ionic attraction between negative latex particle a
positive protein The other is lateral repulsion between positive proteins The smallest
amount of BSA adsorption was at pH 74 where charge repulsion between negative
particles and proteins is present at this pH Lateral repulsion between proteins wa
dominant for protein adsorption even though there always exists the hydrophobic
interaction between solid latex particle and protein The charge repulsion was reduced by
the high concentration of electrolytes in the PBS medium The BSA adsorption process
on PSPMMA core shell particles was very similar to the case of PS latex particle where
hydrophobic interaction between latex particle and protein was dominant However t
adsorbed BSA amount was less than that of PS in PBS The adsorbed amount of BS
PSPMMA
nd
s
he
A on
an
ed
n The main
adsor
90PAA10 and PSPMMA75PAA25 core shell particles was higher at pH 32 th
pH 48 and 74 The adsorbed BSA amount became higher at pH 32 and pH 48 as the
hydrophilicity was increased by the higher PAA amount On the contrary the adsorb
amount of BSA was dramatically decreased at pH 74 Carboxylic acid groups as well as
the sulfate groups are distributed on the surface of PSPMMA90PAA10 and
PSPMMA75PAA25 core shell particles and largely affect the BSA adsorptio
ption interaction forces at pH 32 was generated by hydrogen bonding as well as
charge attraction between latex particle and protein even when the conformational change
and charge repulsion between proteins reduce the adsorption of protein at this pH The
adsorbed BSA amount was also increased as the amount of PAA increases However the
adsorption of BSA was minimal (less than 07 mgm2) at pH 74 where the carboxylic
groups are ionized leading to more enhancement of charge repulsion by ionization of the
119
carboxylic groups (COO-) Because of this the PSPMMA-PAA latex particles have a
much lower protein adsorption
There were steep initial slopes for adsorption isotherms of β
in
s
there
M
r
e
es
o
2M on latex particles
all isotherms indicating a high affinity type adsorption The complete plateau region
were not seen for all isotherms indicating that β2M proteins were not saturated and
are still available sites for β2M adsorption on latex particles The initial slope of β2
isotherms for PS was steeper than for the other latex particles PSPMMA100
PSPMMA90PAA10 and PSPMMA75PAA25
The layer thickness (δ) of BSA proteins on synthesized latex particles in phosphate
buffer (PB) was in the range of 27 - 81 Aring at 37 C and pH 48 Only the δ values at pH
48 the isoelectric point (IEP) of BSA are important because intra- and inter-molecular
electrostatic repulsion of the protein molecules is minimized at the IEP Compared to the
hydrodynamic dimensions of BSA 140 times 40 times 40 Aring these values of the adsorbed laye
thickness indicates that the BSA molecules adsorb by the side-on (40Aring) and end-on mod
(140Aring) Consideration of the multi-layering is unnecessary because the adsorbed
thickness of BSA do not exceed 140 Aring
The Gibbs free energy of β
o
3
2M was more negative in phosphate buffer (PB) than
that of BSA in physiological condition (37 C and pH 74) indicating that βo2M molecul
are more favorably adsorbed on the latex particles than BSA and that the affinity of β2M
was much larger than BSA This may be explained by the rate of diffusion and
accessibility of β2M molecules being easier and faster than that for BSA because of
smaller size (or weight) and spherical shape of β2M molecules It may also be possible t
explain that more hydrophobic forces (less electrostatic repulsion) contributes to
120
adsorption of β2M than to that of BSA because the IEP of β2M (57) is closer to
physiological condition (pH 74) than that of BSA (pH 48) The selective adsorption of
β2M m
in β2M
larger than in BSA adsorption indicating
that fa
an that between BSA and latex particles because
IEP (
ch as
Other
olecules over BSA molecules can be enhanced by developing more hydrophilic
PSPMMA90PAA25 and PSPMMA75PAA25 core shell latex particles
The adsorption kinetics of proteins on latex particles was expressed as a second-
order model The plateau region for β2M was reached earlier than BSA on all latex
particles and the K values (equilibrium rate constant of 2nd order adsorption)
adsorption process on latex particles were much
ster adsorption rate of β2M molecules than that of the BSA molecules There are
several explanations for these phenomena First the smaller size of β2M molecules can
more easily diffuse and approach to the latex substrate Second enhanced hydrophilic
domain on the latex particle surface by sulfate groups (SO4-) and ionized carboxylic
groups (COO-) is not favorable for larger BSA molecules but is for smaller β2M to be
anchored Third the conformational change by intra- and inter-molecular repulsion
would be faster in β2M due to lower molecular weight Finally the ionic repulsion
between β2M and latex particles is less th
57) of β2M is closer to the tested condition (pH 74) than IEP (48) of BSA
In the biocompatibility test the number of red blood cells (RBCs) ruptured by PS
latex particles ranged from 30 to 91 as the solid content of PS increased from 05 to
50 respectively However hemolysis of the other core shell latex particles su
PSPMMA100 PSPMMA90PAA10 and PSPMMA75PAA25 were less than 35 The
PSPMMA90PAA10 particles at all applied solid concentrations had less than 02
hemolysis and was the most blood compatible among the prepared latex particles
121
core shell particles such as PSPMMA100 and PSPMMA75PAA25 also had excelle
compatibility with low RBC breakage
52 Conclusions
The major conclusions from this study include
nt blood
bull microglobulin (β2M) over that of the bovine serum albumin (BSA) at pH 74
was achieved by controlling the PMMA and PAA ratio to optimize
e particles posses the most desirable requirements for use in a human dialysis
ns
icles
olecule
bull
Using engineered core shell latex particles selective adsorption of β2
physiological condition was demonstrated
bull The rate of adsorption of the β2M was also much higher than that of BSA This
hydrophobichydrophilic microdomain size
bull The core shell particles also exhibited excellent biocompatibility with blood Thes
membrane
53 Recommendation for Future Work
With the results and conclusions obtained through this study several suggestio
may be given for future work as follows
bull Competitive adsorption tests of whole blood proteins on synthesized latex partunder physiological condition
bull Preparation of latex particles with optimum hydrophobic hydrophilic microdomains to maximize selective adsorption and removal of toxin molecules
bull Membrane fabrication by suitable pore size design to discriminate a toxin mby size sieving as a function of particle layers
Evaluation of membrane performance such as flow rate in water and in simulated blood fluid (SBF)
bull Application for hemodialysis as well as other technologies such as a water remediation
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dra
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krishnan S McDonald C J Prudhomme R K and Carbeck J D (2004) Latex composite membranes structure and pro
S K Sawant S B Joshi J B and Pangarkar V G (1999) Methanol semembranes for separation of methanol-ethylene glycol mixtures by pe
P Margarit-Puri K Klapper M and Mullen K (2000) Polymerizable and
polymerization Macromolecules 33 7718-7723
CRC Baca Raton (FL)
Riceevans C A and Diplock A T (1993) Current status of antioxidant therapy FRadical Biology and Medicine 15 77-96
C P (1968a) Surface chemistry asp
C P (1968b) Surface chemistry relationships in emulsion polymerization Industrial and Engi
Ronco C Brendolan A Winchester J F Golds E Clemmer J Polaschegg H D Muller T E La Greca G and Levin N W (2001) First clinical experience wan adjunctive hemoperfusion device designed specifically to remove beta(2)
o C Ghezzi P M Hoenich N A Delfino P G (2001) Membranes and filters for hemodialysis Database 2001 Karger Basel
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20 408-413
Samtleben W Gurland H J Lysaght M J Winchester J F (1996) Plasma exchanand hemoperfusion Kluwer Academic Dordrecht
137
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or
Saper M A Bjorkman P J and Wiley D C (1991) Refined structure of the human
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Shimizu N Sugimoto K Tang J W Nishi T Sato I Hiramoto M Aizawa S
abe H and Handa H (2000) high-performance affinity beads for identifying drug receptors Nature Biotechnology
Shirahama H and Suzawa T (1985) Adsorption of bovine serum albumin onto styrene
Shira nto 83-490
al of Medicine 21 888-892
s
ilure 25 419-430
o
of protein using bicinchoninic acid Analytical Biochemistry 150 76-85
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3 673 133
Hatakeyama M Ohba R Hatori H Yoshikawa T Suzuki F Oomori A Tanaka H Kawaguchi H Watan
18 877-881
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138
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t adult immune thrombocytopenic purpura Blood 79 2237-2245
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Suzaw ovine serum albumin on synthetic polymer lattices Journal of Colloid and Interface Science 78 266-268
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412
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Tangp ion of chitosan films- effects of hydrophobicity on protein adsorption Carbohydrate
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C H Smith D P Kalverda A P Phillips S E V and Radford
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77-84
139
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1992)
f the American Society of Nephrology 11 2344-2350
38
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Wiec
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12 29-36
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140
Winchester J F Ronco C Brady J A Cowgill L D Salsberg J Yousha E Choquette M Albright R Clemmer J Davankov V Tsyurupa M PavlL Pavlov M Cohen G Horl W
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step from high-flux dialysis application of sorbent technology Blood Purification
Winchester J P Ronco J A Brendolan A Davankov V Tsyurupa M Pavlova L 2001)
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Yang Q and Yang C Z (2000) Formation of an
Yoon J Y Park H Y Kim J H and Kim W S (1996) Adsorption of BSA on highly carboxylated microspheres- quantitative effects of surface functional groups
Zollars R L (1979) Kinetics of the emulsion polymerization of vinyl acetate Journal of
Zou oss-
Zou D Ma S Guan R Park M Sun L Aklonis J J and Salovey R (1992)
20 81-86
Clemme J Polaschegg H D Muller T E La Greca G Levin N W (Rationale for combined hemoperfusionhemodialysis in uremia Contribution of Nephrology 133 174-179
Winslow R M Vandergriff K D Motterlini R (1993) Mechanism of hemtoxicity Thrombosis and Haemostasis 70 36-41
S C Ge H X Hu Y Jiang Xpositively charged poly(butyl cyanoacrylate) nanoparticles stabilized with chitosColloid and Polymer Science 278 285-292
and interaction forces Journal of Colloid and Interface Science 177 613-620
Applied Polymer Science 24 1353-1370
D Derlich V Gandhi K Park M Sun L Kriz D Lee Y D Kim G Aklonis J J and Salovey R (1990) Model filled polymers 1 synthesis of crlinked monodisperse polystyrene beads Journal of Polymer Science Part A Polymer Chemistry 28 1909-1921
Model filled polymers 5 synthesis of cross-linked monodisperse polymethacrylate beads Journal of Polymer Science Part A Polymer Chemistry 30 137-144
Korea on August 7 1968 He obtained a Bachelor of Science degree in Chemistry from
Keim d graduate school and earned a
resear n Korea Institute of Science and
Coati e
ia
Busin
BIOGRAPHICAL SKETCH
Sangyup Kim was born and raised in the small town of Sung-Joo Kyungbook
yung University (Daegu Korea) in 1993 He entere
Master of Science degree in Chemical engineering in 1995 He worked as a student
cher for 2 years at Textile Polymer Division i
Technology (KIST) in Seoul Korea Next he worked over a year at the Automobile
ng Division in DongJoo-PPG in Korea In August 2001 he began studying for th
degree of Doctor of Philosophy in materials science and engineering at the University of
Florida in Gainesville After getting his PhD he plans to work at the Digital Med
ess Division of Samsung Electronics in Korea starting in January 2006
141
TABLE OF CONTENTS page
ACKNOWLEDGMENTS iv
LIST OF TABLES viii
LIST OF FIGURES x
1 INTRODUCTION 1
2 BACKGROUND AND LITERATURE SURVEY5
21 The Significance of End Stage Renal Disease (ESRD)5 22 Hemodialysis (HD) Treatment 6 23 Advances in Membrane Technology 8 24 Sorbent Technology16 25 Limitation of Current Hemodialysis Treatment 19 26 Latex Particles 20
261 The Components for Emulsion Polymerization 21 262 Particle Nucleation 21 263 Types of Processes for Emulsion Polymerization 23 264 Chemistry of Emulsion Polymerization 24 265 Seed Emulsion Polymerization25 266 Polystyrene Latex Particles 26 267 Various Applications of Latex Particles28
27 Proteins 30 271 Interaction Forces between Proteins33 272 β -Microglobulin (β M)2 2 35 273 Serum Albumin 36
28 Protein Adsorption38 281 Interaction between Protein Molecule and Latex Particle38
29 Hypothesis for Toxin Removal39 291 Toxin Removal by Size Sieving Based on Monodispersed Pore Size 40 292 Toxin Removal by Selective Adsorption on Engineered Latex Particles 41
3 EXPERIMENTAL AND CHARACTERIZATION METHODOLOGY 44
31 Materials 44 32 Latex Particle Preparation46
321 Preparation of Seed Latex Particles47
vi
322 Preparation of Seeded Latex Particles 48 323 Preparation of Core Shell Latex Particles 48 324 Purification of Synthesized Latex Particles48
33 Characterization49 331 Degree of Conversion49 332 Fourier Transform Infrared (FTIR) Spectroscopy49 333 Quasielastic Light Scattering (QELS) 50 334 Field Emission-Scanning Electron Microscopy (FE-SEM) 50 335 Zeta Potential Measurement 50 336 Protein Adsorption51 337 Blood Biocompatibility by Hemolysis Test 53
4 RESULTS AND DISCUSSION55
41 Polymerization of Latex Particles55 411 Polystyrene Seed Latex Particles55 412 The Growth of Polystyrene (PS) Seed Latex Particles61 413 Core Shell Latex Particles 71
42 Characterization of Latex Particles75 421 Fourier Transform Infrared Spectroscopy (FTIR)75 422 Zeta Potential Measurements 75
43 Protein Adsorption Study 85 431 Adsorption Isotherm87 432 Adsorbed Layer Thickness 103 433 Gibbs Free Energy of Protein Adsorption 104 434 Kinetics of Adsorption 107
44 Blood Compatibility 112
5 SUMMARY OF RESULTS CONCLUSION AND RECOMMENDATION FOR FUTURE WORK115
51 Summary of Results115 52 Conclusions121 53 Recommendation for Future Work121
LIST OF REFERENCES122
BIOGRAPHICAL SKETCH 141
vii
LIST OF TABLES
Table page 4-1 The polymerization recipe of polystyrene (PS) seed particles 57
4-2 Continuous addition emulsion polymerization recipe for growing polystyrene (PS) latex particles less than 500nm in size 63
4-3 Continuous addition emulsion polymerization recipe for growing polystyrene (PS) latex particles larger than 500nm in size64
4-4 The preparation recipe of PS core with various shell latex particles72
4-5 The isoelectric point (IEP) of common proteins 83
4-6 The equilibrium concentration values of BSA adsorption on polystyrene (PS) latex particles calculated the Langmuir-Freundlich isotherm model 93
4-7 The equilibrium concentration values (q ) of BSA adsorption on PSPMMA particles calculated the Langmuir-Freundlich isotherm model
m 10096
4-8 The equilibrium concentration values of BSA adsorption on PSPMMA PAA particles calculated the Langmuir-Freundlich isotherm model
90 1098
4-9 The equilibrium concentration values of BSA adsorption on PSPMMA PAA particles calculated the Langmuir-Freundlich isotherm model
75 25100
4-10 The equilibrium concentration values of β M adsorption calculated the Langmuir-Freundlich isotherm model
2102
4-11 Absorbed BSA layer thickness (Aring) of BSA in phosphate buffer (PB) at 37 Co 104
4-12 Absorbed BSA layer thickness (Aring) of BSA in phosphate buffered saline (PBS) at 37 Co 104
4-13 The values of Gibbs free energy change of BSA adsorption in phosphate buffer (PB) at 37 Co 105
4-14 The values of Gibbs free energy change of BSA adsorption in phosphate buffered saline (PBS) at 37 Co 105
viii
4-15 The values of Gibbs free energy change of β M adsorption in phosphate buffere (PB) at 37 C and pH 74
2o 106
4-16 Fitting parameters of second-order kinetic model 111
ix
LIST OF FIGURES
Figure page 1-2 Stereo drawing of the αndashcarbon backbone of β M2 2
2-1 An image of the location and cross section of a human kidney 5
2-2 A schematic draw of the hemodialysis route7
2-3 Cross-sectional SEM image view of the Polyflux S (polyamide + polyacrylethersulfone + polyvinypyrollidone) dialysis membranes 11
2-4 The chemical structure of polysulfone (PSf) 12
2-5 The chemical structure of polyamide (PA)13
2-6 Chemical structure of polyacrylonitrile (PAN) 14
2-7 Emulsion polymerization system24
2-8 L-α-amino acid 31
2-9 Ribbon diagram of human β M taken from the crystal structure of the protein bound to the heavy chain of the MHC class I complex (PDB 1DUZ)
236
2-10 Secondary structure of human serum albumin (HSA) with sub-domains 37
2-11 The relationship between pore size and particle size in body-centered cubic and closed packed arrays41
2-12 Schematic representation of the protein adsorption on the core shell latex particles at pH 7442
3-1 Chemical structure of main chemicals45
3-2 Experimental setup for semi-continuous emulsion polymerization 46
3-3 The particle preparation scheme in various types and size ranges of latex particles 47
3-4 Schematic of the procedure for a protein adsorption test 52
3-5 Separation of RBC from whole blood by centrifuge process53
x
3-6 Schematic of the procedure for hemolysis test 54
4-1 Schematic representation of semi-continuous seed latex particles preparation and growth56
4-2 Scanning Electron Micrograph (SEM) of polystyrene seed latex particles (A) PS (B) PS (C) PS (D) PSS259 S233 S207 S181 59
4-3 SEM of PS latex particles less than 500nm in size (A) 258nm (B) 320nm (C) 370nm (D) 410nm 65
4-4 SEM of PS latex particles larger than 500nm in size (A) 525nm (B) 585nm (C) 640nm (D) 790nm 68
4-5 Dependence of the particle size on the surfactant to monomer ratio70
4-6 Schematic of core shell latex particle structures 71
4-7 Scanning Electron Micrograph of latex particles (A) PS (B) PSPMMA (C) PSPMMA PAA (D) PSPMMA PAA
100
90 10 75 25 73
4-8 FTIR spectra of polymerized latex particles (A) bare polystyrene (PS) (B) PSPMMA (C) PSPMMA PAA (D) PSPMMA PAA100 90 10 75 25 76
4-9 Schematic representation of ion distribution near a positively charged surface 78
4-10 Schematic representation of zeta potential measurement79
4-11 Zeta potential of PS latex particles at 25 Co 80
4-12 Zeta potential of PSPMMA latex particles at 25 C100o 81
4-13 Zeta potential of PSPMMA PAA latex particles at 25 C90 10o 82
4-14 Zeta potential of PSPMMA PAA latex particles at 25 C75 25o 82
4-15 The decay of surface potential with distance from surface in various electrolyte concentrations (1) low (2) intermediate (3) high 84
4-16 High-affinity adsorption isotherm of typical flexible polymer on solid surface 86
4-17 Overall schematic representation of the protein adsorption on the synthesized latex particles86
4-18 The dependence of UV absorbance on BSA concentration (A) 10mgml (B) 06mgml (C) 02mgml87
4-19 Standard curve of net absorbance vs BSA sample concentration 88
xi
4-20 Fitted models for adsorption isotherm of bovine serum albumin (BSA) on polystyrene latex particles at 37 C in phosphate buffer mediao 89
4-21 Adsorption isotherm of BSA on polystyrene (PS) latex particles in phosphate buffer (PB) at 37 Co 90
4-22 Adsorption isotherm of BSA adsorption 37 C in phosphate buffered saline (PBS) on PS latex particles
o
91
4-23 Conformation of bovine serum albumin (BSA) on latex particles92
4-24 Adsorption isotherm of BSA 37 C in PB on PSPMMA core shell latex particles
o100
94
4-25 Adsorption isotherm of BSA at 37 C in PBS on PSPMMA core shell latex particles
o100
95
4-26 Adsorption isotherm of BSA at 37 C in PB on PSPMMA PAA core shell latex particles
o90 10
97
4-27 Adsorption isotherm of BSA at 37 C in PBS on PSPMMA PAA core shell latex particles
o90 10
98
4-28 Adsorption isotherm of BSA 37 C in PB on PSPMMA PAA latex particleso75 25 99
4-29 Adsorption isotherm of BSA 37 C in PBS on PSPMMA PAA core shell latex particles
o75 25
99
4-30 Adsorption isotherm of β M onto PS and PSPMMA latex particles in PB at 37 C and pH 74
2 100o 101
4-31 Adsorption isotherm of β M onto PS and PSPMMA PAA latex particles in PB at 37 C and pH 74
2 90 10o 101
4-32 Adsorption isotherm of β M onto PS and PSPMMA PAA latex particles in PB at 37 C and pH 74
2 75 25o 102
4-33 Gibbs free energy of adsorption of proteins on latex particles in PB at 37 C and pH 74
o
107
4-34 The kinetics of protein adsorption in PB on PS latex particles at 37 C and pH 74
o
109
4-35 The kinetics of protein adsorption in PB on PSPMMA latex particles at 37 C and pH 74
100o
109
4-36 The kinetics of protein adsorption in PB on PSPMMA PAA latex particles at 37 C and pH 74
90 10o 110
xii
4-37 The kinetics of protein adsorption in PB on PSPMMA PAA latex particles at 37 C and pH 74
75 25o 110
4-38 Image of red blood cells 113
4-39 Hemolysis caused by latex particles n=5114
xiii
Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy
SYNTHESIS AND ENGINEERING OF POLYMERIC LATEX PARTICLES FOR MEDICAL APPLICATIONS
By
Sangyup Kim
December 2005
Chair Hassan El-Shall Major Department Materials Science and Engineering
Latex particles with well-defined colloidal and surface characteristics have received
increasing attention due to their useful applications in many areas especially as solid
phase supports in numerous biological applications such as immunoassay DNA
diagnostic cell separation and drug delivery carrier Hemodialysis membrane using
these particles would be another potential application for the advanced separation
treatment for patients with end stage renal disease (ESRD) It is desirable to remove
middle molecular weight proteins with minimal removal of other proteins such as
albumin Thus it is necessary to understand the fundamental interactions between the
particles and blood proteins to maximize the performance of these membranes This
improvement will have significant economic and health impact
The objective of this study is to synthesize polymeric latex particles of specific
functionality to achieve the desired selective separation of target proteins from the human
blood Semi-continuous seed emulsion polymerization was used to prepare monodisperse
xiv
polystyrene seed particles ranging from 126plusmn75 to 216plusmn53 nm in size which are then
enlarged by about 800nm Surfactant amount played a key role in controlling the latex
particle size Negatively charged latex particles with a different hydrophobicity were
prepared by introduction of a sodium persulfate initiator and hydrophilic acrylic acid
monomer The prepared polymeric particles include bare polystyrene (PS) particles less
hydrophobic PS core and PMMA shell particles and more hydrophilic PS core and
PMMA-co-PAA shell latex particles with a 370nm mean diameter SEM light scattering
and zeta potential measurements were used to characterize particle size and surface
properties Adsorption isotherms of two proteins bovine serum albumin (BSA) and β2-
microglobulin (β2M) on latex particles were obtained as a function of pH and ionic
strength using the bicinchoninic acid (BCA) assay method The Langmuir-Freundlich
adsorption model was used to determine the adsorption amount of protein at equilibrium
The thickness of adsorbed BSA layer on latex particles was obtained in order to
investigate the adsorption orientation such as end-on or side-on mode Adsorption
kinetics experiments for both proteins and all latex particles were also performed The
adsorption kinetic constant determined from the Langmuir-Freundlich adsorption
isotherm model was used to calculate Gibbs free energy of adsorption to compare the
competitive adsorption of BSA and β2M Hemolysis tests were performed to investigate
the blood compatibility of synthesized latex particles PSPMMA90PAA10 and
PSPMMA75PAA25 core shell latex particles had desirable material properties with not
only a large amount and high rate of selective β2M adsorption over BSA but also high
blood compatibility showing less than 3 hemolysis
xv
CHAPTER 1 INTRODUCTION
End stage renal disease (ESRD) is a chronic condition in which kidney function is
impaired to the extent that the patientrsquos survival requires removal of toxins from the
blood by dialysis therapy or kidney transplantation The National Kidney Foundation
estimates that over 20 million Americans had chronic kidney disease in 2002 [NKF] The
number of people with ESRD is rapidly increasing in the United States with
approximately 96 295 incidents and 406 081 prevalent patients including 292 215 on
dialysis and 113 866 with a functioning graft in 2001 It is projected that there will be
more than 22 million ESRD patients by 2030 [USRDS 2003] The expenditure for the
ESRD treatment program had reached $228 billion 64 of the Medicare budget in
2001 Due in part to the limited availability of kidneys for transplantation hemodialysis
is the primary clinical treatment for the patients with ESRD
The central element of a hemodialysis instrument is the semipermeable membrane
that allows for selective transport of low molecular weight biological metabolites less
than 5000 Da such as urea and creatinine as well as excess water and electrolytes [Baker
2004] from the blood One limitation of current dialysis technologies is the inability to
efficiently remove middle molecular weight toxins such as β2-Microglobulin (β2M) and
interleukin 6 (IL-6)
β2M is a causative protein of dialysis-related amyloidosis (DRA) a disease arising
in patients with chronic kidney failure as a serious complication of long-term
hemodialysis treatment [Gejyo et al 1985] β2M deposition in tissue is the primary cause
1
2
of destructive arthritis and carpal tunnel syndrome [Vincent et al 1992 Drueke 2000]
The β2M structure is shown in Figure 1-2
Figure 1-2 Stereo drawing of the αndashcarbon backbone of β2M [Becker et al 1985]
Although attempts have been made to increase the efficiency of middle molecular
weight toxin removal by changes in the membrane pore size and the use of innovative
materials to adsorb these toxins [Samtleben et al 1996 Ronco et al 2001] removal
efficiency is not as high as those achieved by a normal healthy kidney Traditional
membranes have a number of processing and performance limitations [Westhuyzen et al
1992 Leypoldt et al 1998] such as a restricted choice of surface chemistries and limited
control of porosity The development of novel engineering membrane technology is
needed to remove middle molecule toxins
Polymeric latex particles have received increasing attention in medical application
areas especially as solid phase supports in biological applications [Piskin et al 1994]
Examples of these applications include immunoassay [Chen et al 2003 Radomske-
Galant et al 2003] DNA diagnostic [Elaїssari et al 1998] cell separation drug delivery
carrier [Luck et al 1998 Kurisawa et al 1995 Yang et al 2000] etc This is because of
3
the well-defined colloidal and surface characteristics of the particles By using a seed
emulsion polymerization method it is possible to synthesize monodisperse latex particles
with various particle size ranges and surface chemistry Functionalized core-shell latex
particles can be introduced by multi-step emulsion polymerization Core particles are
synthesized in the first stage of the polymerization and the functional monomer is added
in the second stage This is done without any emulsifier addition to prevent the
production of new homopolymer particles [Keusch et al 1973] The core-shell particles
are useful in a broad range of applications because of their improved physical and
chemical properties over their single-component counterparts [Lu et al 1996 Nelliappan
et al 1997] Through the development of a hemodialysis membrane using monodisperse
latex particles improvements in advanced separation treatment for patients with end
stage renal disease (ESRD) can be realized This requires the maximum removal of
middle molecular weight proteins with minimal removal of other beneficial proteins such
as albumin Thus an understanding of the fundamental interactions between the particles
and biopolymers is vital to maximize the performance of this membrane technology
The field of material science and biotechnology is based on fundamental chemistry
has developed over the past three decades into todayrsquos powerful discipline that enables
the development of advanced technical devices for pharmaceutical and biomedical
applications This novel and highly interdisciplinary field is closely associated with both
the physical and chemical properties of organic and inorganic particles [Niemeyer 2001]
Hemodialysis membrane using these particles would lead to improvements in the
advanced separation treatment for patients with end stage renal disease (ESRD) The
interdisciplinary nature of this approach enables a more complete understanding of the
4
phenomena of protein adsorption and the material properties necessary for selective
separation This innovative approach to membrane fabrication has the potential of making
inexpensive highly efficient membranes for both industrial and specialized separation
processes
The goal of this study is to prepare polymeric latex particles with tailored
properties to maximize separation of toxin molecules and to investigate the fundamental
interactions between the particles and molecules in the biological system in order to
optimize the performance of a membrane material for these applications
Polymeric latex particles were synthesized with specific functionality in an attempt
to achieve selective separation Seeded emulsion polymerization was used to synthesize
functionalized monodisperse latex particles in various sizes Negatively charged
hydrophobic polystyrene latex particles were synthesized by the same method Core shell
latex particles PSPMMA100 PSPMMA90PAA10 PSPMMA75PAA25 were also
synthesized to differentiate the degree of hydrophobicity of particles Scanning Electron
Micrograph (SEM) and light scattering measurements were used to characterize particle
size and shape and zeta potential measurements were conducted to measure the electrical
surface property of synthesized particles Adsorption isotherms of target proteins bovine
serum albumin (BSA) and β2M on latex particles were obtained as a function of pH
ionic strength and protein concentrations using the BCA assay method Adsorption
kinetics for both proteins on the latex particles were also measured Finally hemolysis
tests were run to determine the biocompatibility of polymer latex particles with human
blood This research will be described in more detail in chapters 3 and 4
CHAPTER 2 BACKGROUND AND LITERATURE SURVEY
21 The Significance of End Stage Renal Disease (ESRD)
The kidneys are responsible for removing excess fluid minerals and wastes from
the blood regulating electrolyte balance and blood pressure and the stimulation of red
blood cell production They also produce hormones such as erythropoietin (EPO) and
calcitriol that keep bones strong and blood healthy [Casadevall and Rossert 2005 Kurbel
et al 2003] EPO acts on the bone marrow to increase the production of red blood cell in
case of bleeding or moving to high altitudes Calcitriol mainly acts on both the cells of
the intestine to promote the absorption of calcium from food and also bone to mobilize
calcium from the bone to the blood When kidneys fail harmful waste builds up blood
pressure rises and body retains excess fluid The body also does not make enough red
blood cells When this happens treatment is needed to replace the function of failed
kidneys
Figure 2-1 An image of the location and cross section of a human kidney
5
6
The National Kidney Foundation estimates that over 20 million Americans had
chronic kidney disease in 2002 [NKF] Chronic renal disease is a gradual and progressive
loss of the function unlike acute renal disease where sudden reversible failure of kidney
function occurs Chronic renal failure usually takes place over a number of years as the
internal structures of the kidney are slowly damaged In the early stages there can be no
symptoms In fact progression may be so gradual that symptoms do not occur until
kidney function is less than one-tenth that of a normal health kidney If left untreated
chronic kidney disease may ultimately lead to kidney failure known as End Stage Renal
Disease (ESRD) ESRD is a rapidly growing heath-care problem in the United States In
2001 approximately 96295 incidents and 406081 prevalent patients were diagnosed
with ESRD including 292215 patients on dialysis and 113866 patients with a
functioning graft [USRDS 2003] The projected number of patients with ESRD is
expected to exceed 22 million patients by 2030 with much of this growth being driven by
the increasing prevalence of major contributing factors such as diabetes and high blood
pressure [USRDS 2003] A great extent of ESRD program cost and Medicare budget
have been spent in 2001 Due in part to a limited availability of kidneys for
transplantation hemodialysis (HD) is the primary method of treatment for ESRD and is
currently used for approximately 61 of US ESRD patients
22 Hemodialysis (HD) Treatment
HD removes toxins from the body by extracorporeal circulation of the blood
through a semipermeable membrane referred to as a dialyzer The toxins are removed
primarily by diffusion across the membrane to a dialysate solution which is circulated on
the opposite side of the membrane The cleaned blood is then returned to the blood
stream A surgically constructed vascular access connects the extracorporeal circuit to the
7
patientrsquos system Treatments are almost always performed three times per week in
specially equipped dialysis facilities for 3-4 hours per each treatment Figure 2-2 shows
the scheme of the hemodialysis route The critical element of a HD instrument is the
semipermeable membrane which allows for selective transport of low molecular weight
biological metabolites from blood
igure 2-2 A schematic draw of the hemodialysis route
ESRD patients who undergo dialysis therapy often experience several other
problems associated with the treatment such as anemia fatigue bone problems joint
problems itching sleep disorders and restless legs Anemia is common in patient with
kidney disease because the kidneys produce the hormone erythropoietin (EPO) which
stimulates the bone marrow to produce red blood cells Diseased kidneys often do not
produce enough EPO to stimulate the bone marrow to make a sufficient amount of red
blood cells This leads to bone disease referred to as renal osteodystrophy and causes
bones to become thin and weak or malformed and can affect both children and adults
Older patients and women who have gone through menopause are at greater risk for this
disease Uremic toxins which cannot be remove from the blood by the current dialyzer
membranes can lead to itching This problem can also be related to high levels of
F
8
parathyroid hormone (PTH) Dialysis patients can also experience day-night revers
is they have insomnia at night and sleep during the day This can be related to possible
nerve damage in the body and a chemical imbalance in the blood due to the excess toxin
Oxidative stress is a problem for patients on maintenance dialysis This problem is due to
an imbalance between pro- and antioxidant factors [Roselaar et al 1995 Cristol et al
1994] Oxidative stress affects oxidation of low-density lipoproteins which are the main
factor for atherogenesis [Huysmans et al 1998] and is also involved in the development
of malignancies and diabetes mellitus [Rice-Evans et al 1993] In order to reduce
antioxidant defense dialysis is needed to contribute to help stimulate free radical
production or eliminate antioxidants Dialysis-related amyloidosis (DRA) is also a
common and serious problem for people who have been on dialysis for more than 5
years DRA develops when proteins in the blood deposit on joints and tendons causi
pain stiffness and fluid in the joint as is the case with arthritis Normally the healthy
kidneys can filter out these proteins but dialysis filters are not as effective
23 Advances in Membrane Technology
al that
s
ng
Dialysis for blood of ESRD
Hemo filtration
s
ysis using a cellulose based membrane occurred
1913 John Abel [1990] from the Johns Hopkins Medical School described a method
purification is widely used in the treatment
dialysis (HD) techniques use a semi-permeable membrane to replace the
role of the kidney The membranes used in HD can be broadly classified into those based
on cellulose and those manufactured from synthetic copolymers These membranes come
in various shapes such as sheets tubular structures or hollow fiber arrangements The
hollow fiber type is the most popular and is incorporated into over 800 different device
in world wide [Ronco et al 2001]
The first attempt at blood dial
9
where sing
e
om
ysis was performed until the mid 1960 The basic
molec
rial in
een
benzyl)
was used for the membrane with
hepar
by the blood of a living animal may be submitted to dialysis outside the body u
a membrane based on cellulose and returned to the natural circulation without exposur
to air infection by microorganisms or any alteration that would necessarily be prejudicial
to life This same technique is still used to today however the device used has been
modified over the years as better membranes were developed and the anti-coagulant
heparin has become available
Cellulose membranes have been widely used for the treatment of renal failure fr
1928 when the first human dial
ular structure of cellulose is made of a long chain containing hydroxyl (OH)
groups The realization that such groups imparted undesirable qualities on the mate
respect to blood contact behavior was discovered in the early 1970s and since has b
the focus of development These modified cellulose membranes used the partial
substitution of benzyl groups to replace the proton of the hydroxyl groups in an attempt
to reduce their negative effect The result is a molecular mosaic of hydrophobic (
and hydrophilic (hydroxyl and cellulose) regions
Kolff [Van Noordwijk 2001] studied the rotating drum artificial kidney for patients
with acute renal failure in 1943 Cellophane tubing
in as the anticoagulant For the next 17 years hemodialysis therapy was performed
by this method but only for the patients with acute reversible renal failure Vascular
access required repeated surgical insertions of cannulas (slender tubes) into an artery and
vein and limited the number of treatments for a patient could receive in order to
minimize the amount of vascular damage
10
Initially the need for dialysis in patients with acute renal failure was determ
mainly by the development of signs and sym
ined
ptoms of uremia After dialysis some time
might
et al
ent improvement In 1960 the development of a shunt [Quinton et al
1960]
fone (PSf) polyamide (PA) and polyacrylonitrile (PAN) by phase inversion or
precip
lidone
e
elapse before uremic manifestations returned to warrant a sequential treatment of
dialysis Many patients with acute renal failure secondary to accidental or surgical
trauma were hypercatabolic but the interdialytic interval might be prolonged because of
anorexia or use of a low-protein diet However Teschan and his coworkers [Obrien
1959] showed that patient well-being and survival were improved by what they termed
prophylactic daily hemodiaysis or administration of the treatment before the patient
again became sick with uremia Their report in 1959 was the first description of daily
hemodialysis
Development of membrane accessories such as a shunt has also been an area of
focus for treatm
a flexible polytetrafluoroethylene (PTFE or Teflonreg) tubing made many more
hemodialysis treatments possible for chronic kidney failure patients PTFE has a non-
stick surface and is relative biocompatibility leading to minimized blood clotting in the
shunt
Synthetic membranes are prepared from engineered thermoplastics such as
polysul
itation of a blended mixture resulting in the formation of asymmetric and
anisotropic structures Figure 2-3 shows a fiber type of the Polyflux S membrane
consisting of poluamide (PA) polyacrylethersulfone (PAES) and polyvinylpyrro
(PVP) with the integral three-layer structure The skin layer on the inside fiber typ
membrane contacts blood and has a very high surface porosity and a narrow pore size
11
distribution This layer constitutes the discriminating barrier deciding the permeabili
and solute retention properties of the membrane The skin layer is supported by thick
sponge-type structure larger pores providing mechanical strength and very low
hydrodynamic resistance
ty
Figure 2-3 Cross-sectional SEM image view of the Polyflux S (polyamide +
polyacrylethersulfone + polyvinypyrollidone) dialysis membranes [Deppisch et al 1998]
n
[Malchesky 2004] because of its thermal stability mechanical strength and chemical
inertn ciated
ls
m
PSf is a widely used membrane material for the hemodialysis applicatio
ess According to a report from the National Surveillance of Dialysis-Asso
Disease (NSDAD) in the US over 70 of hemodialysis membranes were PSf based
[Bowry 2002] This is most likely because PSf has many advantages over other materia
This synthetic polymer is one of few materials that can withstand sterilization by stea
ethylene oxide and γ-radiation PSf membrane can be prepared by conventional
immersion precipitation methods into many different shapes including porous hollow
12
fiber or flat sheet hemodialysis membranes The material also has a high permeab
low molecular weight proteins and solute and high endotoxin retention The chemical
structure of PSf is shown in Figure 2-4
ility to
Figure 2-4 The chemical structure of polysulfone (PSf)
There is one major disadvantage to PSf The hydrophobic nature of the PSf causes
ment alternative pathway
leadin
s not
3
et al
in
O acrylate
serious complications through the activation of the comple
g to the adsorption of serum proteins onto the membranes [Singh et al 2003]
Anticoagulants are added during dialysis therapy to avoid blood clotting but this doe
completely eliminate the problem In order to overcome this disadvantage of the PSf
membrane various studies have been performed to change the materialrsquos surface
properties These investigations include hydrophilic polymer coating [Brink et al 199
Kim et al 1988 Higuchi et al 2003] layer grafting onto PSf membrane [Wavhal
2002 Song et al 2000 Pieracci et al 2002 Mok et al1994] and chemical reaction of
hydrophilic components onto the membrane surface [Higuchi et al 1990 1991 1993
Blanco et al 2001 Nabe et al 1997 Guiver et al 1993] Hydrophilic monomers 2-
hydroxy-ethylmethacrylate (HEMA) acrylic acid (AA) and methacrylic acid (MMA)
have also been grafted onto PSf membrane to increase flux and Bovine Serum Album
(BSA) retention [Ulbricht et al 1996] Hancock et al [2000] synthesized
polysulfonepoly(ethylene oxide) (PEO) block copolymers to improve the resistance to
platelet adhesion Kim et al [2003] also studied blending a sulfonated PE
diblock copolymer into PSf in order to reduce platelet adhesion and enhance
13
biocompatibility PEO is a commonly used biomaterial due to its excellent resistance
protein adsorption and inherent biocompatibility [Harris 1992] Kim et al [20
a self-transformable copolymer to enhance the hydrophilicity of an asymmetric PSf
membrane with an ultra-thin skin layer The polymer had an entrapped diblock
copolymer containing a hydrophilic block of poly (ethylene glycol) (PEG)-SO
to
05] studied
ate
lic graft
l)
e of thier
aromatic
orand
3 acryl
and a hydrophobic block of octadecylacrylate (OA) Molecular dynamic (MD)
simulations were performed as a function of copolymer density to optimize interfacial
structure information McMurry [2004] developed a strategy using an amphiphi
copolymer added to PSf membranes by introducing polysulfone-g-poly (ethylene glyco
When compared to unmodified PSf these graft copolymer and resulting blend
membranes are found to hold promise for biomedical device applications
Polyamide (PA) membranes have also been used for hemodialysis becaus
mechanical strength in both wet and dry conditions Polyamide consists of
aliphatic monomers with amide bonding (-CONH-) also known as a peptide bond
The basic amide bond in polyamide is shown in Figure 2-5 R1 and R2 can be either
aromatic or aliphatic linkage group
C N
H
O
~ R R1 2~C N
H
O
~ R R1 2~
Figure 2-5 The chemical structure of polyamide (PA)
Panichi and co-workers [1998] evaluated the biocompatibility of the PA membrane
ompatible technique due to the
use of a synthetic membrane with a sterile re-infusion fluid and the convective removal of
and concluded that PA hemofiltration was a highly bioc
14
the ac
e
e
is
n
is a semi-crystalline polymer and the mechanical properties
strong
2
CN
tivated anaphylatoxins and β2-Microglobulin (β2M) The PA based membrane
Polyfluxreg (manufactured by Gambro GmbH Germany) blended with polyamide
polyarylethersulfone and polyvinylpyrrolidone (PVP) was able to clean small molecules
such as urea dreatinine and phosphate as well as decrease β2M amount by 502
[Hoenich et al 2002] Due to the non-selectivity of the membrane removal of thes
unwanted materials also led to the undesirable loss of beneficial proteins during therapy
Meier et al [2000] evaluated different immune parameters using a modified cellulos
low-flux hemophan and synthetic high-flux PA membrane during a 1 year period in
chronic hemodialysis patients They found that the 1-year immunological evaluation of
hemodiaysis membrane biocompatibility was associated with changes in the pattern of
chronic T-cell actiovation
Polyacrylonitrile (PAN) is another commonly used membrane material because it
inherently hydrophilic and has been commercialized for ultrafiltration and microfiltratio
[Scharnagl et al 2001] PAN
ly depend on the crystalline structures The chemical structure of PAN is shown in
figure 2-6
CH CH
n2
CN
CH CH n
Chemical structure of polyacrylonitrile (PAN)
The addition of additives such as polyvinylpyrrolidone (PVP) as a pore forming
agent gives PAN membranes more flexible processing parameters and increased
e has been optimized through
copolymerization with many other vinyl monomers including glycidyl methacrylate
Figure 2-6
performance [Jung et al 2005] PAN membrane performanc
15
[Godj
acid
for
for
a non-
solub this
in
oid underneath the inner
surface of membrane decreased by increasing the amount of solvent DMSA in the
evargova et al 1999 Hicke et al 2002] N-vinylimidazole [Godjevargova et al
2000] hydroxyl ethyl methacrylate [Ray et al 1999 Bhat et al 2000] methacrylic
[Ray et al 1999] vinyl pyrrolidone [Ray et al 1999] acrylic acid [Trotta et al 2002]
acrylamide [Musale et al 1997] and vinylchloride [Broadhead et al 1998] These
monomers provide a reactive group for enzyme immobilization improved mechanical
strength solvent-resistance pervaporation permeation flux anti-fouling and bio-
compatibility Because of this PAN-based copolymer membranes have great potential
the treatment of hemodialysis in an artificial kidney This material can also be used
other applications like the treatment of wastewater the production of ultra-pure water
biocatalysis together with separation and methanol separation by pervaporation
Lin and his coworker [2004] studied the modification of PAN based dialyzer
membranes to increase the hemocompatibility by the immobilization of chitosan and
heparin conjugates on the surface of the PAN membrane When a foreign material is
exposed to blood plasma proteins are adsorbed clotting factors are activated and
le fibrin network or thrombus is formatted [Goosen et al 1980] The result of
research was that the biocompatible chitosan polymer and a blood anticoagulant hepar
prevented blood clotting They showed prolonged coagulation time reduced platelet
adsorption thrombus formation and protein adsorption
Nie et al [2004] studied PAN-based ultrafiltration hollow-fiber membranes
(UHFMs) In order to improve the membrane performance acrylonitrile (AN) was
copolymerized with other functional monomers such as maleic anhydride and α-allyl
glucoside They found that the number and size of macrov
16
intern erum
and
d a
rface The second step is the
transf after
yjak et
embrane
1978 Malchesky et al 1978] has been further developed to increase the efficiency of
al coagulant The water flux of the UHFMs also decreased while the bovine s
albumin rejection increased minutely Godjevargova et al [1992] modified the PAN
based membrane with hydroxylamine and diethylaminoethylmethacrylate to improve
membrane dialysis properties Formed functional groups like primary amine oxime
tertiary amine groups provided the membrane with more hydrophilic properties an
substantial increase in the permeability of the membranes
The wide use of filtration in practice is limited by membrane fouling Solute
molecules deposit on and in the membrane in the process of filtration causing dramatic
reduction in flux through the membrane Fouling occurs mostly in the filtration of
proteins Three kinetic steps are involved in the fouling of UF membranes according to
Nisson [1990] The first step is the transfer of solute to the su
er of solute into the membrane until it either finally adsorbs or passes through
a set of adsorption-desorption events The third step includes surface binding
accompanied by structural rearrangement in the adsorbed state [Ko et al 1993] Br
al [1998] studied the surface modification of a commercially available PAN membrane
to develop superior filtration properties with less fouling by proteins The PAN
membrane was immersed in excess NaOH solution to convert some of the surface nitrile
groups into carboxylic groups by the hydrolysis process This modified PAN m
was not so severely fouled in the Bovine Serum Albumin (BSA) filtration test The pore
size however decreased during the hydrolysis process leading to a significant reduction
in flux and made the membrane less productive in the ultrafiltration (UF) mode
24 Sorbent Technology
Over the last three decades sorbent technology [Castino et al 1976 Korshak et al
17
dialysis or replace it for the treatment of ESRD Sorbents remove solutes from solution
through specific or nonspecific adsorption depending on both the nature of the solute and
the sorbent Specific adsorption contains tailored ligands or antibodies with high
selectivity for target molecules een used in autoimmune
disord
and
e
al 1996] various resins [Ronco et al 2001]
album
e
as
Specific adsorbents have b
ers such as idiopathic thrombocytopenic purpura [Snyder et al 1992] and for the
removal of lipids in familial hyper cholesterolemia [Bosch et al 1999] Nonspecific
adsorbents such as charcoal and resins attract target molecules through various forces
including hydrophobic interactions ionic (or electrostatic) attraction hydrogen bonding
and van der Waals interactions
New dialysate with sorbents has become an accepted modification of dialysis
sorbent hemoperfusion is gaining ground as a valuable addition to dialysis especially as
new sorbents are developed [Winchester et al 2001] Hemoperfusion is defined as th
removal of toxins or metabolites from circulation by the passing of blood within a
suitable extracorpoteal circuit over semipermeable microcapsules containing adsorbents
such as activated charcoal [Samtleben et
in-conjugated agarose etc Novel adsorptive carbons with larger pore diameters
have been synthesized for potential clinical use [Mikhalovsky 1989] Newly recognized
uremic toxins [Dhondt et al 2000 Haag-Weber et al 2000] have resulted in several
investigations on alternatives to standard or high-flux hemodialysis to remove thes
molecules These methods include hemodiafilteration with [de Francisco et al 2000] or
without [Ward et al 2000 Takenaka et al 2001] dialysate regeneration using sorbents
well as hemoperfusion using such adsorbents as charcoal and resins
18
Kolarz et al [1989 1995] studied the hyper-crosslinked sorbent prepared from
styrene and divinylbenzene (DVB) for a hemoperfusion application They found that the
pore structure of a swelling sorbent was changed by additional crosslinking with α αrsquo-
dichloro-p-xylene in the presence of a tin chloride catalyst and in a di
chloroethane
soluti and
en
decyl groups that attract β2M through a hydrophobic interaction The
adsor
of
5]
d
penet
on They also realized that the hemocompatibility was useful for nemoperfusion
could be imparted to the sorbents by introducing sulfonyl groups at a concentration of
about 02mmolg
A special polymeric adsorbing material (BM-010 from Kaneka Japan) has be
investigated by another group [Furuyoshi et al 1991] for the selective removal of β2M
from the blood of dialysis partients The adsorbent consists of porous cellulose beads
modified with hexa
ption capacity of this material is 1mg of β2M per 1ml of adsorbent Using a
hemoperfusion cartridge containing 350ml of these cellulose beads in sequence with a
high-flux hemodialyzer several small clinical trials were performed During 4-5 hours
treatment about 210mg of β2M were removed thus reducing the concentration in the
blood by 60-70 of the initial level [Nakazawa et al 1993 Gejyo et al 1993 199
RenalTech developed a hemoperfusion device BetaSorbreg containing the hydrate
cross-linked polystyrene (PS) divinylbenzene (DVB) resin sorbents with a pore structure
designed to remove molecules between 4 and 30 kDa [Winchester et al 2002] In this
case solute molecules are separated according to their size based on their ability to
rate the porous network of the beaded sorbents The resin beads were prepared with
a blood compatible coating and confirmed to be biocompatible in vivo in animals
[Cowgill et al 2001]
19
25 Limitation of Current Hemodialysis Treatment
Hemodialysis is a widely used life-sustaining treatment for patients with ESRD
However it does not replace all of the complex functions of a normal healthy kidne
a result patients on dia
y As
lysis still suffer from a range of problems including infection
accelerated cardio ion anemia
chron
e
se middle
alysis
sessio
s
f
vascular disease high blood pressure chronic malnutrit
ic joint and back pain and a considerably shortened life span One significant
limitation of the current dialysis technology is the inability to efficiently remove larger
toxic molecules This is mainly because of the broad pore size distribution reducing th
selective removal of toxins and unsatisfied biocompatibility causing lots of
complications such as inflammation blood clotting calcification infection etc
Dialysis purifies the patients blood by efficiently removing small molecules like
salts urea and excess water However as toxic molecules increase in size their removal
rate by hemodialysis substantially declines Typically only 10 - 40 of the
molecular weight toxins (300-15000 Da) are removed from the blood during a di
n [Vanholder et al 1995] These toxins then reach an abnormally high level and
begin to damage the body One such toxin β2M causes destructive arthritis and carpal
tunnel syndrome by joining together like the links of chain to form a few very large
molecules and deposit damaging the surrounding tissues [Lonnemann et al 2002] This i
also a main cause of mortality for long-term dialysis patients Other middle molecule
toxins appear to inhibit the immune system and may play a significant role in the high
susceptibility to infections in dialysis patients Still others are believed to impair the
functioning of several other body systems such as the hematopoietic and other endocrine
systems This may contribute to accelerated cardiovascular disease the leading cause o
20
death among dialysis patients as well as clinical malnutrition which affects up to 50 of
this patient population
Over the last decade polymeric dialysis membranes have been developed to
increase the capacity for removing middle molecular weight toxins by changing the pore
size of dialyzer membranes and using new materials that adsorb these toxins for
impro se
styrene made during World War II in the United
States The Dow chemical compan anufacturer of polystyrene
includ
d of
on
rug
ntrol of
ved removal characteristics However removal efficiency is not as high as tho
achieved by a normal healthy kidney
26 Latex Particles
The first synthetic polymer synthesized using emulsion polymerization was a
rubber composed of 13-butadiene and
y has been a major m
ing latex which they used in paint formulations The theory of emulsion
polymerization in which a surfactant is used was established by Harkins [1948] and by
Smith and Ewart [1948] By 1956 the technology was complete including the metho
building larger diameter particles from smaller ones The product by this emulsi
polymerization is referred to as latex a colloidal dispersion of polymer particles in water
medium [Odian 1991] Latexes are currently undergoing extensive research and
development for a broad range of areas including adhesives inks paints coatings d
delivery systems medical assay kits gloves paper coatings floor polish films carpet
backing and foam mattresses to cosmetics The relatively well known and easy co
the emulsion process is one of main advantages for these applications Therefore
polymeric latex particle prepared by emulsion polymerization can be a candidate for the
medical applications because of the easy control of the particle size and morphology as
well as flexible surface chemistry to be required
21
261 The Components for Emulsion Polymerization
The main components for emulsion polymerization process are the monomer a
dispersing medium a surfactant and an initiator Available monomers are styrene
sing medium is usually water
which
ticles
n
ation To
monomer droplets but in micelles because the initiators are
insolu le
and
olution
butadiene methylmethacylate acryl acid etc The disper
will maintain a low solution viscosity provide good heat transfer and allow
transfer of the monomers from the monomer droplets into micelles and growing par
surrounded by surfactants respectively The surfactant (or emulsifier) has both
hydrophilic and hydrophobic segments Its main functions is to provide the nucleatio
sites for particles and aid in the colloidal stability of the growing particles Initiators are
water-soluble inorganic salts which dissociate into radicals to initiate polymeriz
control the molecular weight a chain transfer agent such as mercaptan may be present
262 Particle Nucleation
Free radicals are produced by dissociation of initiators at the rate on the order of
1013 radicals per milliliter per second in the water phase The location of the
polymerization is not in the
ble in the organic monomer droplets Such initiators are referred to as oil-insolub
initiators This is one of the big differences between emulsion polymerization
suspension polymerization where initiators are oil-soluble and the reaction occurs in the
monomer droplets Because the monomer droplets have a much smaller total surface area
they do not compete effectively with micelles to capture the radicals produced in s
It is in the micelles that the oil soluble monomer and water soluble initiator meet and is
favored as the reaction site because of the high monomer concentration compared to that
in the monomer droplets As polymerization proceeds the micelles grow by the addition
of monomer from the aqueous solution whose concentration is refilled by dissolution of
22
monomer from the monomer droplets There are three types of particles in the emulsion
system monomer droplets inactive micelles in which polymerization is not occurring
and active micelles in which polymerization is occurring referred to as growing polymer
particles
The mechanism of particle nucleation occurs by two simultaneous processes
micellar nucleation and homogeneous nucleation Micellar nucleation is the entry of
radicals ei
ther primary or oligomeric radicals formed by solution polymerization from
the aq ed
the
r
s
n
is coagulation with other particles rather than
polymerization of monomer The driving force for coagulation of precursor particles
ueous phase into the micelles In homogeneous nucleation solution-polymeriz
oligomeric radicals are becoming insoluble and precipitating onto themselves or onto
oligomers whose propagation has ended [Fitch et al 1969] The relative levels of micella
and homogeneous nucleation are variable with the water solubility of the monomer and
the surfactant concentration Homogeneous nucleation is favored for monomers with
higher water solubility and low surfactant concentration and micellar nucleation is
favored for monomers with low water solubility and high surfactant concentration It has
also been shown that homogeneous nucleation occurs in systems where the surfactant
concentration is below CMC [Roe 1968] A highly water insoluble monomer such a
styrene [Hansen et al 1979 Ugelstad et al 1979] has probably created by micellar
nucleation while a water soluble monomer such as vinyl acetate [Zollars 1979] has bee
formed by homogeneous nucleation
A third latex formation reaction mechanism has been proposed referred to as
coagulative nucleation In this reaction the major growth process for the first-formed
polymer particles (precursor particles)
23
sever les
yer
es
r a
l
the beginning of the polymerization As soon as the initiator is
gins with the formation and
growt
and
ved
added at one time during the later stages of the polymerization prior to complete
al nanometers in size is their relative instability compared to larger sized partic
The small size of a precursor particle with its high curvature of the electrical double la
permits the low surface charge density and high colloidal instability Once the particl
become large enough in size maintaining the high colloidal stability there is no longe
driving force for coagulation and further growth of particles takes place only by the
polymerization process
263 Types of Processes for Emulsion Polymerization
There are three types of production processes used in emulsion polymerization
batch semi-continuous (or semi-batch) and continuous In the batch type process al
components are added at
added and the temperature is increased polymerization be
h of latex particles at the same time There is no further process control possible
once the polymerization is started In the semi-continuous emulsion polymerization
process one or more components can be added continuously Various profiles of particle
nucleation and growth can be generated from different orders of component addition
during polymerization There are advantages to this process such as control of the
polymerization rate the particle number colloidal stability copolymer composition
particle morphology In the continuous process the emulsion polymerization components
are fed continuously into the reaction vessel while the product is simultaneously remo
at the same rate High production rate steady heat removal and uniform quality of
latexes are advantages of the continuous polymerization processes
Other available methods include the intermittent addition and shot addition of one
or more of the components In the shot addition process the additional components are
24
conversion of the main monomer This method has been used successfully to develo
water-soluble functional monomers such as sodium styrene sulfonat
p
e [Kim et al 1989]
264
lsion
Chemistry of Emulsion Polymerization
Emulsion polymerization is one type of free radical polymerization and can be
divided into three distinct stages initiation propagation and termination The emu
polymerization system is shown in Figure 2-7
Aqueous phaseAqueous phase
Micelle withmonomer
I Rbull
Emulsifier
Polymer particle swollenWith monomer
Monomerdroplet
I Rbull
Micelle withmonomer
I Rbull
Emulsifier
Polymer particle swollenWith monomer
MonomerdropletMonomerdroplet
I Rbull
Figure 2-7 Emulsion polymerization system [Radicals (Rmiddot) are created from initiators
(I) Monomer is transferred from larger monomer droplet into micelles by emulsifier Initiated polymer particle by radicals is keep growing until monomers are all consumed The reaction is performed in aqueous media]
In the initiation stage free radicals are created from an initiator by heat or an
ultraviolet radiation source The initiator with either peroxides groups (-O-O-) such as
odium persulfate or azo groups (-N=N-) such as azobisisobutyronitrile is commonly
used for em t
with the m er
chain ere
s
ulsion polymerization The primary free radicals created from initiator reac
onomer for initiation of polymerization In the propagation stage the polym
grows by monomer addition to the active center a free radical reactive site Th
are two possible modes of propagations head-to-head addition and head-to-tail addition
25
The head-to-tail mode is the predominant configuration of the polymer chain a result o
steric hindrance of the substitute bulky group In the termination stage polymer chain
growth is terminated by either coupling of two growing chains forming one polymer
molecule or transferring a hydrogen atom (dispropotionation) from one growing chain to
another forming two polymer molecules one having a saturated end group and the other
with an unsaturated end-group
265 Seed Emulsion Polymerization
Polymer latex particles have received increasing interest because of the versatili
of the many applications heterophase polymerization processes like emulsion dispersion
micro-emulsion seeded emulsion precipitation etc Especially to prepare well-defined
microspheres having monodispe
f
ty
rse and various particle sizes as well as surface group
ed particles prepared by emulsion polymerization
and e
s
hological design In
seede to control
n
functionalilties it is necessary to use se
nlarge them to a desired size in the further stages of reactions
Polymeric particles are required to have a uniform particle size in many
applications such as chromatography where they are used as a packing material
Morphological control of latex particle is also important for many practical application
[Schmidt 1972] Seed emulsion polymerization (or two-stage emulsion polymerization) is
a useful method to achieve both monodisperse particle size and morp
d emulsion polymerization [Gilbert 1995] preformed lsquoseedrsquo latex is used
the number of particles present in the final latex The advantage of seeded emulsio
polymerization is that the poorly reproducible process of particle nucleation can be
bypassed so that the number concentration of particles is constant and known Various
mechanisms have been proposed for the growth of latex particles [Ugelstad et al 1980
Okubo et al 1992] using this polymerization technique The initial seed particle
26
preparation step is well known and relatively easy to perform because at the relative
small particle sizes (02 to 05 micron) the particle growth process can be readily
controlled by the use of an emulsifier Enough emulsifier is used to prevent coagulation
but not enough to cause the formation of new particles As the particles are grown to
larger sizes in successive seeding steps it becomes increasingly difficult to mainta
stable uncoagulated emulsion without forming new particles and thereby destroying
monodisperisty of the latex Recently there have been several reports concerning t
reaction kinetics of seed emulsion polymerization and the development of latex
morphologies over the course of the reaction [Chern et al 1990 Delacal et al 1990 L
et al 1995 Lee 2000 2002]
266 Polystyrene Latex Particles
A number of papers have described the synthesis of the polystyrene latex particl
bearing various functional surface groups such as carboxyl [Lee et al 2000 Tun
2002 Reb et al 2000] hydroxyl [Tamai et al 1989] marcapto [Nilson 1989] epoxy
[Shimizu et al 2000 Luo et al
ly
in a
he
ee
es
cel et al
2004] acetal [Izquierdo et al 2004 Santos et al 1997]
ometyl [Izquierdo et al 2004 Park et al 2001
Sarob
h the
d
thymine [Dahman et al 2003] chlor
e et al 1998] amine [Counsin et al 1994 Ganachaud et al 1997 Ganachaud et al
1995 Miraballes-Martinez et al 2000 2001 Anna et al 2005] ester [Nagai et al 1999]
etc In order to produce these functionalized particles different methods for particle
preparation must be used The polymer emulsion with core-shell morphology of latex
particles is one of them This is a multistep emulsion polymerization process in whic
polystyrene ldquocorerdquo particle is synthesized in the first stage and the functional monomer is
added in the second stage of the polymerization without any emulsifier postfeeding to
prevent the production of new homopolymer particles thus forming the functionalize
27
polymer ldquoshellrdquo on the ldquocorerdquo particle [Keusch et al 1973] There are requirements to
limit secondary nucleation and encourage core-shell formation in seeded emulsion
polymerization including the addition of smaller seed particles at high solid content to
increase particle surface area low surfactant concentration to prevent formation of
micelles and the semi-continuous addition of monomer to create a starved-feed conditio
and keep the monomer concentration low There are some advantages [Hergeth et al
1989] of dispersions with polymeric core-shell particles First it is possible to modi
interfacial properties of polymer particles in the aqueous phase by the addition of only
very small amounts of a modifying agent during the last period of the reaction Thus
these core-shell particles are useful in a broad range of applications since they always
exhibit improved physical and chemical properties over their single-component
counterparts [Lu et al 1996 Nelliappan et al 1997] In this way the improvement of
surface properties of such dispersions is straightforward and inexpensive The other is
that polymers with a core-shell structure are perfect model systems for investigating th
material properties of polymer blends and composites because of their regular
distribution of one polymer inside a matrix polymer and because of the simple sp
geometry of the system
Their properties usually depend on the structures of latex particles Chen and his
coworkers [Chen et al 1991 1992 1993] reported the morphological development of
core shell latex particles of polystyrenepoly(methyl methacrylate) during
polymerization Before the research by Chen and his coworker Min et al [Min et al
1983] reported the morph
n
fy the
e
herical
ological development of core shell latex of polystyrene
(PS)polybuthyl acrylateat (PBA) by seeded emulsion polymerization as a function of the
28
additi
of
ity of graft
ing reagents Thus protein adsorption on polymeric solid surfaces has
st are microspheres defined as ldquofine
polym can be
n
r was
containing antigen urine serum etc The latex will become agglutinated and visibly
on method of PS They found that the percentage of grafting PS to the PBA was
greatest for the batch reaction and the PBA-PS core-shell particles with a high degree
grafting remained spherical upon aging test because of the emulsifying abil
copolymer
267 Various Applications of Latex Particles
Latex particles are applicable to a wide range of areas such as biomedical
applications [Piskin et al 1994] especially as the solid phase such as in immunoassays
[Chern et al 2003 Radomske-Galant et al 2003] DNA diagnostic drug delivery carriers
[Luck et al 1998 Kurisawa et al 1995 Yang et al 2000] blood cell separations and
column pack
become a center of attention Of particular intere
er particles having diameters in the range of 01 to several micronsrdquo which
used as functional tools by themselves or by coupling with biocompounds Singer and
Plotz [1956] firstly studied microsphere or latex agglutination test (LATrsquos) by using
monodisperse polystyrene (PS) and polyvinyltoluene polymer particles as the support o
which the biomolecules were going to adsorb The biomolecule adsorption howeve
limited by possible desorption of the adsorbed species or loss of specific activity of the
complex formed Since this work was published the latex particle applications for
immunoassay have been rapidly and widely studied and developed
Latex agglutination test or latex immunoassays start with tiny spherical latex
particles with a uniform diameter and similar surface properties The particles are coated
with antibodies (sensitized) through the hydrophobic interaction of portions of the protein
with the PS surface of the particles If sensitized particles are mixed with a sample
29
agglomerate Latex tests are inexpensive as compared with the other techniques [Ba
1988]
ngs
sed
tion Rheumatoid factor (RF) in different age subpopulations has also been
evalu
studied a
latex
Unipath [Percival 1996] has manufactured a range of immunoassay materials ba
on a chromatographic principle and deeply colored latex particles for use in the home and
clinical environments The latex particles which are already sensitized with a
monoclonal antibody can detect any antigens that bound to the surface of the latex
particles Some detectable pollutants include estrogen mimics which induce
abnormalities in the reproductive system of male fishes and lead to a total or partial male
feminiza
ated according to a patientrsquos clinical status by using a rapid slide latex agglutination
test for qualitative and semiquantitative measurement in human serum along with latex
immunoassay method [Onen et al 1998] Magalhaes and his coworkers [2004]
diagnostic method of contamination of male fishes by estrogen mimics using the
production of vitellogenin (VTG) as a biomarker This was based on a reverse
agglutination test developed with monoclonal antibodies specific to this biomarker
Premstaller and his coworkers [Premstaller et al 2000 2001] have prepared a porous
poly(styrene-divinylbenzene) (PS-DVB) polymer monolith to use for highly efficient
chromatographic separation of biomelecules such as proteins and nucleic acids They
used a porogen a mixture of tetrahydrofuran and decanol to fabricate a micropellicular
PS-DVB backbone Legido-Quigley and his colleagues [2004] have developed the
monolith column to obtain further chromatographic functionality to the column by
introducing chloromethylstyrene in place of styrene into the polymer mixture
30
Core shell type monodisperse polymer colloids have been synthesized by Sarobe
and Forcada [1998] with chloromethyl functionality in order to improve the biomolecu
adsorption through a two-step emulsion polymerization process They investigated the
functonalized particles by optimizing the experimental parameters of the functional
monomer including reaction temperature the amount and type of redox initiator sys
used the type of addition of the initiator system and the use of washing They c
le
tem
oncluded
that th
sis
serve as separation
chann
of
usually
along the polypeptide chain which contain positive or negative charges [Norde 1998]
e relation between the amount of iron sulfate and the persulfatebisulfite system
added should be controlled to obtain monodisperse particles and prevent the premature
coagulation of the polymer particles during the polymerization
A semi-continuous emulsion polymerization technique for latex particle synthe
was performed by McDonald and other researchers [Ramakrishnan et al 2004 Steve et
al 1999] They introduced a variety of particles sizes compositions morphologies and
surface modifications to fabricate latex composite membranes (LCMs) Arraying and
stabilizing latex particles on the surface of a microporous substrate form narrowly
distributed interstitial pores formed between the particles which
els They investigated the membrane performance using gas fluxes water
permeability and the retention characterization of dextran molecules From these tests
they concluded that the narrow discriminating layer made of the latex particles leads to a
highly efficient composite membrane
27 Proteins
Proteins are natural polyamides comprised of about 20 different α-amino acids
varying hydrophobicity [Norde 1998] Proteins are more or less amphiphilic and
highly surface active because of the number of amino acid residues in the side groups
31
Proteins are polymers of L-α-amino acids [Solomon and Fryhle 2000] The α refers to a
carbon with a primary amine a carboxylic acid a hydrogen and a variable side-chain
group designated as R Carbon atoms rent groups are asymmetric and can
exhib l
s
e
on
with four diffe
it two different spatial arrangements (L and D configurations) due to the tetrahedra
nature of the bonds The L refers to one of these two possible configurations Amino
acids of the D-configuration are not found in natural proteins and do not participate in
biological reactions Figure 2-8 shows the chiral carbon in 3-D as the L isomer Protein
consist of twenty different amino acids differentiated by their side-chain groups [Norde
1998] The side-chain groups have different chemical properties such as polarity charg
and size and influence the chemical properties of proteins as well as determine the
overall structure of the protein For instance the polar amino acids tend to be on the
outside of the protein when they interact with water and the nonpolar amino acids are
the inside forming a hydrophobic core
Figure 2-8 L-α-amino acid
The covalent linkage between two amino acids is known as a peptide bond A
peptide bond is formed when the amino group of one amino acid reacts with the carboxyl
group of another amino acid to form an amide bond through the elimination of water
This arrangement gives the protein chain a polarity such that one end will have a free
amino group called the N-terminus and the other end will have a free carboxyl group
called the C-terminus [Solomon and Fryhle 2000] Peptide bonds tend to be planar and
32
give the polypeptide backbone rigidity Rotation can still occur around both of the α-
carbon bonds resulting in a polypeptide backbone with different potential conformations
relative to the positions of the R groups Although many conformations are theoretically
possible interactions between the R-groups will limit the number of potential
nd to form a single functional conformation In other words
the conformation or shape of the protein is
α-helix
can interact with other
secondary structures within the sam
conformations and proteins te
due to the interactions of the chain side
groups with one another and with the polypeptide backbone The interactions can be
between amino acids that are close together as in a poly-peptide between groups that are
further apart as in amino acids or even on between groups on different polypeptides all
together These different types of interactions are often discussed in terms of primary
secondary tertiary and quaternary protein structure
The primary amino acid sequence and positions of disulfide bonds strongly
influence the overall structure of protein [Norde 1986] For example certain side-chains
will promote hydrogen-bonding between neighboring amino acids of the polypeptide
backbone resulting in secondary structures such as β-sheets or α-helices In the
conformation the peptide backbone takes on a spiral staircase shape that is stabilized by
H-bonds between carbonyl and amide groups of every fourth amino acid residue This
restricts the rotation of the bonds in the peptide backbone resulting in a rigid structure
Certain amino acids promote the formation of either α-helices or β-sheets due to the
nature of the side-chain groups Some side chain groups may prevent the formation of
secondary structures and result in a more flexible polypeptide backbone which is often
called the random coil conformation These secondary structures
e polypeptide to form motifs or domains (ie a
33
tertiary structure) A motif is a common combination of secondary structures and a
domain is a portion of a protein that folds independently Many proteins are composed o
multiple subunits and therefore exhibit quaternary structures
271 Interaction Forces between Proteins
Proteins in aqueous solution acquire compact ordered conformations In such a
compact conformation the movement along the polypeptide chain is severely restrict
implying a low conformational entropy The compact structure is possible only if
interactions within the protein molecule and interactions between the protein molecule
and its environment are sufficiently favorable to compensate for the low conformational
entropy [Malmsten 1998] Protein adsorption study is often focused on structural
rearrangements in the protein molecules because of its significance to the biological
functioning of the molecules and the important role such rearrangements play in the
mechanism of the adsorption process Knowledge of the majo
f
ed
r interaction forces that act
structures helps to understand the behavior
of pro
arge
a
ded
between protein chains and control the protein
teins at interface These forces include Coulomb interaction hydrogen bonds
hydrophobic interaction and van der Waals interactions
Coulombic interaction Most of the amino acid residues carrying electric ch
are located at the aqueous boundary of the protein molecule An the isoelectric point
(IEP) of the protein where the positive and negative charges are more or less evenly
distributed over the protein molecule intramolecular electrostatic attraction makes
compact structure favorable to proteins Deviation to either more positive or more
negative charge however leads to intramolecular repulsion and encourages an expan
structure Tanford [1967] calculated the electrostatic Gibbs energy for both a compact
impenetrable spherical molecule (protein) and a loose solvent-permeated spherical
34
molecule (protein) over which the charge is spread out From the results he found th
the repulsion force was reduced at higher ionic strength d
at
ue to the screening action of ion
ble
e
the
d the
tween
dehyd
e
ent
oles
tein
Hydrogen bond Most hydrogen bonds in proteins form between amide and
carbonyl groups of the polypeptide backbone [Malmsten 1998] The number of availa
hydrogen bonds involving peptide units is therefore far greater than that involving sid
chains Because α-helices and β-sheets are aligned more or less parallel to each other
interchain hydrogen bonds enforce each other Kresheck and Klotz [1969] examine
role of peptide-peptide hydrogen bonds and concluded that hydrogen bonds between
peptide units do not stabilize a compact structure of protein However because the
peptide chain is shielded from water due to other interactions hydrogen bonding be
peptide groups do stabilize α-helical and β-sheet structures
Hydrophobic interaction Hydrophobic interaction refers to the spontaneous
ration and subsequent aggregation of non-polar components in an aqueous
environment In aqueous solutions of proteins the various non-polar amino acid residues
will be found in the interior of the molecule thus shielded from water The
intermolecular hydrophobic interaction for the stability of a compact protein structure
was first recognized by Kauzmann [1959] If all the hydrophobic residues are buried in
the interior and all the hydrophilic residues are at the outermost border of the molecul
intramolecular hydrophobic interaction would cause a compact protein structure
However geometrical and other types of interactions generally cause a fraction of the
hydrophobic residues to be exposed to the aqueous environm
Van der Waals interaction The mutual interaction between ionic groups dip
and induced dipoles in a protein molecule cannot be established as long as the pro
35
structure is not known in great detail Moreover the surrounding aqueous medium also
contains dipoles and ions that compete for participation in the interactions w
the protein molecule Dispersion interactions favor a compact structure However
because the Hamaker constant for proteins is only a little larger than that of water the
resulting effect is relatively small [Nir 1977]
272 β
ith groups of
rgiles et al 1996
Floeg
) is degraded [Floege 2001] Renal
β2M from the serum resulting in an increase in β2M
conce well
the
)
a
al
2-Microglobulin (β2M)
The protein β2M is of particular interest because it is involved in the human
disorder dialysis-related amyloidosis (DRA) [Geiyo et al 1985 A
e 2001] DRA is a complication in end stage renal failure patients who have been on
dialysis for more than 5 years [Bardin et al 1986 Drueke 2000] DRA develops when
proteins in the blood deposit on joints and tendons causing pain stiffness and fluid in
the joints as is the case with arthritis In vivo β2M is present as the non-polymorphic
light chain of the class I major histocompatibility complex (MHC-I) As part of its
normal catabolic cycle β2M dissociates from the MHC-I complex and is transported in
the serum to the kidney where the majority (95
failure disrupts the clearance of
ntration by up to 60-fold [Floege 2001] By a mechanism that is currently not
understood β2M then self-associates into amyloid fibrils and typically accumulates in
musculoskeletal system [Homma et al 1989] Analysis of ex vivo material has shown
that the majority of amyloid fibrils in patients with Dialysis Related Amiloidosis (DRA
is present as of full-length wild-type β2M although significant amounts (~20-30) of
truncated or modified forms of the protein are also present [Floege 2001 Bellotti et al
1998] Figure 2-9 shows the ribbon diagram of human β2M Native β2M consists of
single chain of 100 amino acid residues and has a seven stranded β-sandwich fold typic
36
of the immunoglobulin superfamily [Saper et al 1991 Trinh et al 2002] β2M was fi
isolated from human urine and characterized by Berggard et al [1980] in1968 The
normal serum concentration of β
rst
re
f
e
2M is 10 to 25 mgL It is a small globular protein with
a molecular weight of 118 kDa a Strokes radius of 16Aring and a negative charge under
physiological conditions (isoelectric point IEP = 57) β2M contains two β-sheets that a
held together by a single disulphide bridge between the cysteines in positions 25 and 81
[Berggard et al 1980 Parker et al 1982 Cunningham et al 1973] β2M cannot be
removed completely by current dialysis techniques but through a better understanding o
the structure and interaction forces that lead to this structure it will be possible to mor
efficiently remove this problematic protein
Figure 2-9 Ribbon diagram of human β M taken from the crystal structure of the protein
bound to the heavy chain of the MHC class I complex (PDB 1DUZ) [Khal 2000]
273 Serum Albumin
Serum albumin is the most abundant protein found in plasma and is typically
present in the blood at a concentration of 35~
2an et
50gL According to extensive studies about
its physiological and pharmacological properties albumin has a high affinity to a very
wide range of materials such as electrolytes (Cu+2 Zn+2) fatty acids amino acids
metabolites and many drug compounds [Fehske et al 1981 Kraghhansen 1981 Putnam
37
1984 Peters 1985] The most important physiological role of the protein is therefore to
bring such solutes in the bloodstream to their target organs as well as to maintain the pH
and osmotic pressure of the plasma Bovine serum albumin (BSA) is an ellipsoidal
protein with the dimensions of 140 X 40 X 40Aring [Peter 1985] The primary structure is a
single helical polypeptide of 66 kDa (IEP = 47) with 585 residues containing 17 pairs of
as a soft an
rfaces [Kondo et al 1991 Norde et al 1992 Soderquist et al
1980 ikely
is
disulfide bridges and one free cysteine [Dugaiczyk et al 1982] BSA has been classified
d flexible protein because it has a great tendency to change its conformation
on adsorption to solid su
Carter and Ho 1994] and consists of three homologous domains (I-III) most l
derived through gene multiplication [Brown 1976] Each domain is composed of A and B
sub-domains [He et al 1992] The secondary structure of human serum albumin (HSA)
shown in Figure 2-10
Figure 2-10 Secondary structure of human serum albumin (HSA) with sub-domains
[Zunszain et al 2003]
HSA has the same structure domains with the serum albumin from other species
such as BSA [Brown 1976] Although all three domains of the albumin molecule have
similar three-dimensional structures their assembly is highly asymmetric [Sugio et al
38
1999] Domains I and II are almost perpendicular to each other to form a T-shaped
assembly in which the tail of subdomain IIA is attached to the interface region between
sub-domains IA and IB by hydrophobic interactions and hydrogen bonds In contrast
domain III protrudes from sub-domain IIB at a 45ordm angle to form the Y-shaped assembly
for domains II and III Domain III interacts only with sub-domain IIB These features
make the albumin molecule heart-shaped
28 Protein Adsorption
Protein adsorption studies date back to the 1930rsquos At the beginning these studies
mainly focused on the determination of the molecular weight electrophoretic and
ral
rearrangem
adsorption and biocompatibility of the sorbent materials were investigated [Norde 1986]
There are interaction forces at the interfaces between protein molecules and latex
particles These forces are mainly divided into the following groups hydrophobic
interaction ionic interaction hydrogen bonding and van der Waals interaction [Andrade
1985]
Hydrophobic interaction It is known that hydrophobic interaction has a major
role in protein adsorption phenomena The adsorption of proteins on the low charged
latex particles occurs by this interaction force Generally monomers such as styrene offer
hydrophobic surfaces th
by this interaction force is maximum at the isoelectric point (IEP) of the protein and the
strength [Suzawa et al 1980 1982 Shirahama et al 1989 Kondo et al 1992] By the
chromatographic applications Later the adsorption mechanism especially the structu
ents was studied Recently the studies of the relation between protein
281 Interaction between Protein Molecule and Latex Particle
at protein molecules adsorb to The amount of adsorbed protein
pH at maximum adsorption shifts to a more acidic region with an increase in ionic
39
reports [Suzawa et al 1980 1982 Lee et al 1988] protein adsorption was greater
hydrophobic surface than on a hydrophilic one implying that hydrophobic interaction is
on a
s
ydrophilic compound such as acrylic acid (AA) or methacrylic acid (MAA)
Ionic
ely
t
ces
n force is operative over small
distan e
ble
n
nt dialysis membrane therapy for end stage renal disease (ESRD)
one of most dominant forces in protein adsorption
Ionic interaction Negatively charged latex particles have ionic functional
groups on their surfaces such as salts of sulfonic and carboxylic acid Sulfate group
originate from an initiator such as sodium persulfate and carboxylic groups originate
from a h
bonds are formed between the negative charges of these latex particles and positive
surface charges of protein molecules The conventional low-charged latex particles rar
form these ionic bonds
Hydrogen bonding Hydrogen bond is a strong secondary interatomic bond tha
exists between a bound hydrogen atom (its unscreened proton) and the electrons of
adjacent atoms [Callister 1999] Protein can be adsorbed on hydrophilic polar surfa
through hydrogen bonding Hydrogen bonds are frequently formed between hydroxyl-
carbonyl or amide-hydroxyl Hydroxyl-hydroxyl or amide-hydroxyl bonds are also
formed in protein adsorption
Van der Waals interaction This interactio
ces only when water has been excluded and the two non-polar groups come clos
to each other Lewinrsquos calculation showed that the van der Waals interaction is negligi
compared with the forces involved in the entropy increases ie hydrophobic interactio
[Lewin 1974]
29 Hypothesis for Toxin Removal
As mentioned earlier insufficient removal of middle molecular range toxins is a
major drawback of curre
40
patien
is
y
ese
haracteristics over the last decade However removal
effici
ing a novel membrane design composed of an assembly of engineered
polymeric latex particles s ions Important factors
will i
e
ith a
is established it is
expec
e
ts This may cause destructive arthritis and carpal tunnel syndrome inhibit the
immune system and accelerate cardiovascular disease leading to death among dialys
patients In order to overcome these complications artificial dialysis membranes have
been developed to increase the capacity for removing middle molecular weight toxins b
changing the pore size of dialysis membranes and using new materials that adsorb th
toxins for improved removal c
ency is not as high as those achieved by a normal healthy kidney
With the knowledge obtained from all above literatures the following hypothesis
has been established for the development of a membrane for the successful removal of
the target protein β2-Microglobulin (β2M) without the removal of serum albumin This
will be done us
ynthesized to predetermined specificat
nclude pore size and surface chemistry
291 Toxin Removal by Size Sieving Based on Monodispersed Pore Size
The packing of monodispersed spherical particles can lead to the formation of
porous layers suitable for use as filters and membranes [Hsieh et al 1991] The pore siz
is defined as the largest spherical particles that can pass through the interstitial spaces
It is well known that monodispersed spherical particles can be obtained from the
seed emulsion polymerization method Many commercially available latex particles are
synthesized by this technique and are inexpensive When the defect free membrane w
monodisperse pore size distribution based on a particular particle array
ted to outperform the traditional hemodialysis membrane limitation which is a
broad range of pore size distribution When spherical particles pack in regular crystallin
arrays a number of packing geometries such as hexagonal closest packing cubic closest
41
packing and body-centered cubic packing are possible The pore size of the array
depends on these packing geometries as well as particle size Theoretical pore size can be
calculated from the simple geometry of the arr
ay Figure 2-11 shows the relationship
between particle size and pore size
0001
001
01
001 01 1 10
Por
e
1
10
microm
) S
ize
(
Particle Diameter (microm)
Body-Centered Cubic ArrayClosest Packed Array
0001
Siz
e (
Particle Diameter (microm)
001
01
001 01 1 10
Por
e
1
10
microm
)
Body-Centered Cubic ArrayClosest Packed ArrayBody-Centered Cubic ArrayClosest Packed Array
and closed packed arrays [Steve et al 1999]
McDonald and his coworkers [Ramakrishnan et al 2004 Steve et al 1999] have
described the fabrication of latex composite membranes and their performance in
previous papers It is from this prior research and knowledge gained from conversations
with Dr McDonald that this current research has been initiated [McDonald 2003] The
focus of this thesis is the extension of McDonaldrsquos research in the area of composite
membranes to medical application such as dialysis membranes
292 Toxin Removal by Selective Adsorption on Engineered Latex Particles
The flexibility in latex surface properties is a significant advantage to this
materialrsquos ability to separate proteins since the selectivity is strongly dependent on the
charge interactions of both the protein and the latex particles under the conditions of
Figure 2-11 The relationship between pore size and particle size in body-centered cubic
42
separation [Menon et al 1999 Chun et al 2002] First all sorbents were designed to
have negative surface properties because most plasma proteins in blood are negative and
should not be removed with charge interaction between proteins and sorbents at
physiological condition (pH74) It was reported that a negatively charged surface was
more blood compatible than a positive one [Srinivasan et al 1971] The toxin protein β2-
Microglobulin (β2M) can be selectively adsorbed on the surface of latex particles by the
design of a suitable hydrophobic portioned surface where only β2M protein can be
anchored with hydrophobic interactions Albumin adsorption on the latex particle is not
side-on m
adsorption of β M protein on the engineered latex particles
allowed because charge repulsion is more dominant than hydrophobic interaction with
ode The figure 2-12 shows the schematic representation of the selective
2
PMMAPAAShell
COO-
OSO3-
OSO3-
PSCore
COO-
-
-
-
-
-
-
Albumin-
Chargerepulsion
--
-
- -
- -
- --
Hydrophobic
β2-Microglobulin
2
Interaction
β -Microglobulin
PMMAPAAShell
COO-
-
-
-
-
-
-
--
-
-
-
-
-
Albumin-
Chargerepulsion
OSO3-
OSO3-
PSCore
COO-
--
-
- -
--
-
- -
- -
- --
- -
- --
Hydrophobic
β2-Microglobulin
2
particles at pH 74
Interaction
β -Microglobulin
Figure 2-12 Schematic representation of the protein adsorption on the core shell latex
43
Hydrophilichydrophobic microdomain structures were proven to be more blood
compatible [Mori et al 1982 Higuchi et al 1993 Deppisch et al 1998] The
optimization of suitable hydrophobic to hydrophilic ratio is also important for the
biocompatibility The monomers such as styrene (St) methyl methacrylate (MMA) and
acrylic acid (AA) are widely used hydrophobic and hydrophilic monomer models in
emulsion polymerization process
In summary the background and fundamental literature survey about the history
material properties and limitation of hemodialysis membrane latex particle preparation
surface chemistry and manufacturing process target proteins and protein adsorption and
finally the hypothesis for a toxin removal with high separation efficiency have been
suggested This research focuses on such hypothesis and the materials and
characterization methodology for achievement of suggested hypotheses are described in
the next chapter
CHAPTER 3 EXPERIMENTAL AND CHARACTERIZATION METHODOLOGY
As mentioned earlier the goal of this study is to prepare polymeric latex particles
with tailored properties to maximize separation of toxin molecules and to investigate
fundamental interactions between the applied particles and molecules in the
system to optimize the membrane performance for hemodialysis applications Polymeric
latex particles wit
the
biological
h monodisperse size distribution to obtain uniform pore size and
various size ranges to utilize membrane construction are necessary Surface engineering
with various combination of hydrophobichydrophilic domain on the surface of latex
particles is expected to affect the removal of target protein by selective adsorption and to
improve the biocompatibility of membrane Therefore the materials and characterization
methodology are addressed in this chapter
31 Materials
Styrene (St) monomer used for a seed and a core particle was purchased from
Fisher Scientific and used without any other purification process Acrylic acid (AA) and
methyl methacrylate (MMA) monomers introduced for shell formation were purchased
from Fisher Scientific and used without any other purification process Sodium persulfate
(SPS) and sodium bicarbonate (SBC) were obtained from Fishers Scientific and used as
received Divinylbenzene (DVB) crossliking agent was purchased from Aldrich The
anionic surfactant Aerosol regMA80-I [sodium di(13-dimethylbutyl) sulfosuccinate] was
kindly donated by Cytec 78-80 Aerosol regMA80-I is mixed with isopropanol and
water Its critical micelle concentration (CMC) is 245 mM (119) Ion exchange resin
44
45
AGreg 501-x8 Resin (20-50 mesh) was purchased from Bio-Rad Laboratories Inc It is a
mixed-bed resin with H+ and OH- as the ionic forms and is 8 crosslinked Bovine serum
albumin (BSA) (heat shock treated) was purchas Fisher Scientific The isoelectric
point of t lin
(β2M)
as
gma
ed from
he BSA is 47-49 and the molecular weight is 66300Da β2-Microglobu
was purchase from ICN Biomedicals Inc and was separated from patients with
chronic renal disease and lyophilized from ammonium bicarbonate The molecular
weight by SDS-PAGE was approximately 12000Da BSA and β2M proteins were used
received without further purification process In order to investigate the protein
adsorption evaluation bicinchoninic acid protein assay kits were purchased from Si
(Cat BCA-1) and Pierce Biotechnology (Cat 23225) The chemical structures for the
main chemicals are shown in Figure 3-1
CH2 CH
Styrene (Mw = 10415)
CH2 CHCH2 CH
Styrene (Mw = 10415)
CH2 CH
Acrylic acid (Mw = 7206)
C
OH
O
CH2 CH
Acrylic acid (Mw = 7206)
C
OH
O
CH CH2
C
OCH3
O
CH
CH CH3
2
C
OCH3
O
CH3
Methyl methacrylate (Mw = 10012)Methyl methacrylate (Mw = 10012)
CH2 CH
Divinylbenzene (Mw = 1302)
CH CH2
CH2 CH
Divinylbenzene (Mw = 1302)
CH CH2CH CH
+(Na) -(O3S)
CH -C-O-CH-CH -CH-CH3
O
2 2 3
O CH CH3
CH3 CH3
CH-C-O-CH-CH2-CH-CH3
Anionic surfactant (MA80-I) (Mw = 3885)
+(Na) -(O3S)
CH -C-O-CH-CH -CH-CH3
O
2 2 3
O CH CH3
CH3 CH3
CH-C-O-CH-CH2-CH-CH3
Anionic surfactant (MA80-I) (Mw = 3885)2
+(Na)-(O) S OO
O O
- +
Initiator sodium persulfate (Mw = 23811)
O SO
(O) (Na)+ -
O O
- +(Na) (O) S OO
O SO
(O) (Na)+ -
O O
- +
Initiator sodium persulfate (Mw = 23811) Figure 3-1 Chemical structure of main chemicals
(Na) (O) S OO
O SO
(O) (Na)
46
32 Latex Particle Preparation
The experimental set up and the particle preparation schemes for various types and
size ranges of latex particles are shown in Figure 3-2 and 3-3 In the experimental setup a
mechanical glass stirrer is connected from a motor and Teflon stirrer bar is located at
end of glass stirrer The agitation rate is precisely adjusted by controller motor Paar glas
reactor with 1L volume is partially in silicon oil bath on hot plate The reactor has fo
openings for a stirrer a thermometer a nitrogen gas inlet and a reactant feeding lnlet
The reactants for latex polymerization are fed by precisely controlled by meterin
The particle preparation is described in each latex preparation section
the
s
ur
g pump
Figure 3-2 Experimental setup for semi-continuous emulsion polymerization
1 Motor 2 Mechanical stirrer bar 3 Thermometer 4 Inlet of N2 5 Inlet for initiator and monomer 6 Glass reactor (l L) 7 Oil bath 8 Hot plate 9 Pump for initiator 10 Pump for monomer 11 Tubes for reactant feed
47
Styrene monomer
Bare PS
PSPMMA100
PSPMMA90PAA10
PSPMMA P75 AA25
Gr
win
ticle
size
PS seedparticles
og
par
Styrene monomer
Bare PS
PSPMMA100
PSPMMA90PAA10
PSPMMA P75 AA25
Gr
win
ticle
size
PS seedparticles
og
par
Figure 3-3 The particle preparation scheme in various types and size ranges of latex particles
321 Preparation of Seed Latex Particles
Polystyrene seed particles were synthesized through a typical semi-continuous
emulsion polymerization Polystyrene latex particles were prepared in a four necked Paar
glass vessel equipped with a mechanical glass stirrer thermometer glass funnel for
nitrogen gas purge and a tube for monomer feeding The reactor was placed in a silicon
oil bath on a hot plate for homogenous and stable tempe he vessel was
firstly charged with de-ionized water the emulsifier so itiator
sodium bicarbonate (SBC) styrene (St) monomer divinylbenzene (DVB) monomer
under nitrogen gas atmosphere and then heated to 72plusmn2 as
llowed to continue for another hour Second reactants St and DVB monomers SPS
initiator and SBC were fed separately using fine fluid metering pumps (Model RHSY
Fluid metering INC NY) under a nitrogen gas atmosphere for 2h After complete feeding
rature control T
dium persulfate (SPS) in
oC for 1h The reaction w
a
48
of all reactants the reaction was continued for another hour at elevated temperature 80plusmn1
oC and then cooled to room temperature by removal of the oil bath
322 Preparation of Seeded Latex Particles
The particle growth was achieved by seeded continuous emulsion polymerization
The vessel was charged with the polystyrene seeds and de-ionized water to make desired
solid content and heated up to 72plusmn2 oC for 1h under nitrogen gas atmosphere Then St
and DVB monomers and solution of SPS and SBC in de-ionized water were continuously
added using metering pump with a precisely controlled feed rate After feeding all
reactants the reaction was continued for three hours keeping temperature of 80plusmn2 oC and
then the reaction vessel was cooled down to room temperature
Monodisperse spherical polystyrene seeds with a 280nm mean diameter were used
e-polymerized polystyrene seeded emulsion
was charged in a 500m
323 Preparation of Core Shell Latex Particles
for core shell structured latex particle The pr
l glass flask under a nitrogen gas atmosphere De-ionized water
was added to achieve the desired solid content The emulsion was heated to 80plusmn2oC for
10 hour Monomers initiator and SBC to form the shell were added by fluid metering
pumps (LAB PUMP JRRHSY model Fluid metering Inc USA) with fine controlled
feed rate Flow rate can be adjusted by control ring graduated in 450 division from 0 to
100 flow which is in the range of 10mlmin respectively The reaction was sustained
for additional 30 hours at 90plusmn2oC then cooled down to room temperature
324 Purification of Synthesized Latex Particles
Synthesized latex particles were purified with an ion exchange resin 200g (10
ww polystyrene solid content) of emulsion were mixed with 4g of ion exchange resin
and stirred for 40 minute to remove unreacted monomers initiators and other impurities
49
Then the emulsion was diluted with DI water to a specific degree of solid content as
needed
33 Characterization
331
solid
termined
oscopy
d
hours
Dried latex (10mg) was mixed with
250 m on
Degree of Conversion
The degree of monomer to polymer conversion was determined gravimetrically
following the procedure described by Lee et al [1995] before the latex cleaning process
The synthesized emulsion raw materials were weighed and dried in a conventional oven
at 120oC for 30min to evaporate any unreacted monomers and water The remaining
was weighed and the degree of conversion was determined This process was repeated
three times and a mean value of the degree of conversion was de
332 Fourier Transform Infrared (FTIR) Spectr
A molecule can absorb only select frequencies (energy) of infrared radiation which
match the natural vibration frequencies The energy absorbed serves to increase the
amplitude of the vibrational motions of the bonds in the molecule [Pavin et al 1996]
Only those bonds which have a dipole moment that changes as a function of time can
absorb the infrared radiation The range of wavelengths for infrared radiation is between
25 microm (4000 cm-1) and 25 microm (400cm-1)
The synthesis of the latex particles such as polystyrene (PS) homopolymer and
PSPMMA PSPMMA90PAA10 PSPMMA75PAA25 core-shell copolymers were verifie
with FTIR Purified latex particles were dried in vacuum oven at 40oC for 24
before analysis in the FTIR (Nicolet Magma USA)
g of KBr which had also been dried in vacuum oven at 120oC for 3h Transmissi
spectra using the drift mode were plotted with 128 scans and 4cm-1 resolution
50
333 Quasielastic Light Scattering (QELS)
Latex particle size was measured by the Brookhaven ZetaPlus particle size
analyzer The solid concentration as 0001 (wv) This
ation spectroscopy (PCS) of quasielastically scattered light
For th s
ed using a FE-SEM (JEOL
JSM-
ith
ent
01
us zeta-
potential analyzer It measures the electrophoretic mobility velocity of charged colloidal
of latex particles used w
instrument uses photon correl
is technique time dependent interference patterns of light scattered from particle
in the sample cell are analyzed The interference pattern changes due to Brownian
motion giving rise to fluctuations in scattering intensity The frequency of these
fluctuations depends on the particlersquos diffusion constant that is inversely proportional to
the particle diameter
334 Field Emission-Scanning Electron Microscopy (FE-SEM)
Particle size and surface morphology were characteriz
6335F Japan) A diluted suspension of latex particles in water was dropped onto a
silicon wafer at room temperature and allowed to dry The sample was then coated w
the thinnest layer of carbon needed to obtain the required conductivity for this instrum
Secondary electron image mode was used with a 15KV of accelerating voltage The
magnification range used was between 10000X and 70000X
335 Zeta Potential Measurement
Synthesized and purified latex particles were diluted with de-ionized water to 0
wt and adjusted six different pH values 205 345 481 565 643 and 747 These
particle suspensions were then transferred to standard cuvettes for zeta potential
measurement At least two runs of ten measurements were taken for each sample and
averaged
The zeta potential measurement was carried out using Brookhaven ZetaPl
51
particles in solution and calculate zeta-potential by using the Smoluchowski equation
The f quency
gnitude
umin (BSA) as a standard model protein and β2-Microglobulin
uffer (PB) and
phosp
5
h the selected protein
e 005 01 03 05 and 07 mgml and those for
β2M
d at
teins in the supernatant after the centrifugation process using the
bicinc t
requency of laser light passing through the sample and is compared to the fre
of a reference beam The shift in the frequency called a Doppler shift and the ma
of the shift correspond to the polarity and the magnitude of the electrophoretic mobility
respectively The zeta potential is calculated from the solution conditions and the
measured mobility
336 Protein Adsorption
Protein adsorption experiments of the synthesized latex particles were performed
with bovine serum alb
(β2M) as a target protein in two types of buffer solution phosphate b
hate buffered saline (PBS) To make 5 mM of PB solution 0345g of sodium
phosphate monobasic (NaH2PO4H2O) was dissolve in 500ml of de-ionized water To
make PBS solution 0345g of sodium phosphate monobasic (NaH2PO4H2O) and 4178g
(143mM) of sodium chloride (NaCl) were dissolved in 500ml of de-ionized water The
synthesized latex particles were diluted with each buffer to have a solids content of 0
(ww) adjusted to pH values of 32 48 and 74 and mixed wit
BSA concentrations were chosen to b
were 0015 0030 0045 and 0060mgml The mixture was gently rotated using
the shaker Labquakereg (BarnsteadThermolyne Model 4002110 USA) with 8 RPM in
the incubator at 37oC for 12 hours before the latex-protein mixture was centrifuge
13000 rpm for 15min The amount of protein adsorbed was determined by quantifying
the free pro
honinic acid (BCA) assay method [Lowry et al 1951 Smith et al 1985 Baptista e
al 2003 Wiehelman et al 1988 Brown et al 1989] BCA assay kits consist of Reagent
52
A containing bicinchoninic acid sodium carbonate sodium tartrate and sodium
bicarbonate in 01N NaOH with pH=1125 and Reagent B containing 4 (wv) copper
(II) sulfate pentahydrate The BCA working reagent was prepared by mixing 50 parts of
Reagent A with 1 part of Reagent B 100microl of protein supernatant was then mixed with
2ml of BCA working reagent in a UV cuvette Incubation was allowed to continue
at room temperature
further
until color developed about 2h The absorbance at 562nm was
pectrophotometer (Perkin-Elmer Lambda 800 USA)
Unkn
Separate the p rticles in centrifuge mix supernatant with BCA reagents
measured using a UV-VIS s
own concentration of sample protein was determined by comparison to a standard
of known protein concentrations The adsorbed amount per unit surface area was
determined by the mass balance of the protein after adsorption process The simple
schematic of the procedure for a protein adsorption test is shown in Figure 3-4
Incubate mixture with agitation for 12hrs at 37oC
aand develop the color by incubation for 2hrs at room temperature
Suspend latex particles in aqueous buffer media
Add the dissolved target protein
Measure color intensity using UVVis spectroscopy at 562nm
Suspend latex particles in aqueous buffer media
Separate the p rticles in centrifuge mix supernatant with BCA reagents
Add the dissolved target protein
Incubate mixture with agitation for 12hrs at 37oC
aand develop the color by incubation for 2hrs at room temperature
Measure color intensity using UVVis spectroscopy at 562nm
Figure 3-4 Schematic of the procedure for a protein adsorption test
53
337 Blood Biocompatibility by Hemolysis Test
Hemolysis is the destruction of red blood cells which leads to the release of
hemoglobin from within the red blood cells into the blood plasma Hemolysis testing is
used to evaluate blood compatibility of the latex particles and the damaged to the red
blood cells can be determined by monitoring amount of hemoglobin released The red
blood cells (RBCs) were separated by centrifuging blood at 1500 rpm for 15 minutes
Figure 3-5 shows the phase separated blood with plasma white blood cells and red
cells after centrifugation process Separated RBSs were then washed with isotonic
phosphate buffer solution (PBS) at pH 74 to remove debris and serum protein Th
process was repeated 3 times
blood
is
Figure 3-5 Separation of RBC from whole blood by centrifuge process
Prepared latex particles were re-dispersed in PBS by sonification to obtain
homogeneously dispersed latex particles 100microl of the mixture of red blood cell (3 parts)
and PBS (11parts) was added to 1ml of 05 (ww) particle suspension PBS was used as
a negative control resulting 0 hemolysis and DI water used as a positive control to
produce 100 hemoglobin released by completely destroyed RBCs The mixture was
incubated in water bath with gentle shaking for 30 minutes at 37oC and then centrifuged
at 1500 rpm for 15 minutes 100microl of the supernatant was mixed with 2ml of the mixture
) (EtOHHCl = 2005 ww) to prevent the
White blood cells
Plasma
Red blood cells
of ethanol (99) and hydrochloric acid (37
54
precipitation of hemoglobin In order to remove remaining particles the mixture was
centri fuged again with 13000 RPM at room temperature The supernatant was then
transferred into the UV cuvette The amount of hemoglobin release was determined by
monitoring the UV absorbance at a wavelength of 397nm The schematic of the
procedure for hemolysis test is shown in Figure 3-6
Separate red blood cells (RBCs) from blood
Mix RBCs with phosphate buffer solution (PBS)
Wash and re-suspend RBC with PBS repeat three times
Add la l of RBC and incu in at 37oC
tex particle suspension into 100mbate sample with agitation for 30m
Separate particle ith EtOHHCl solutions and mix the supernatant w
Evaluate free hemoglobin in the solution using UVVis spectroscopy at 397nm
Separate red blood cells (RBCs) from bloodSeparate red blood cells (RBCs) from blood
Mix RBCs with phosphate buffer solution (PBS)Mix RBCs with phosphate buffer solution (PBS)
Wash and re-suspend RBC with PBS repeat three timesWash and re-suspend RBC with PBS repeat three times
Add latex particle suspension into 100ml of RBC and incubate sample with agitation for 30min at 37oC
Add latex particle suspension into 100ml of RBC and incubate sample with agitation for 30min at 37oC
Separate particles and mix the ith EtOHHCl solution supernatant wSeparate particles and mix the ith EtOHHCl solution supernatant w
Evaluate free hemoglobin in the solution using UVVis spectroscopy at 397nmEvaluate free hemoglobin in the solution using UVVis spectroscopy at 397nm
Figure 3-6 Schematic of the procedure for hemolysis test
In summary a description of the experimental materials and characterization
methodology has been explained In the next chapter the results and discussion of the
data obtained from these experiments is addressed
CHAPTER 4 RESULTS AND DISCUSSION
41 Polymerization of Latex Particles
411 Polystyrene Seed Latex Particles
Polymer particles have many different applications including spacer particles
lubricants packing materials for chromatography standard particles and diagnostic
drugs All applications strongly require these particles to have a uniform particle size
Uniform particle size is the first requirement for a latex composite membrane formed by
particle arrays with the interstitial spaces serving as pores for size discriminations [Jons
et al 1999 Ramakrishnan et al 2004] Although suspension polymerization has been
mainly used as one of conventional polymerization methods to prepare uniform particles
the uniformity of the recovered particles is insufficient for membrane use There is
another method known as seed emulsion polymerization where a vinyl monomer is
absorbed into fine monodiserse seed particles and polymerization causes the monomer to
crease the sizes of the seed particles uniformly In order to obtain monodisperse larger
particles by this m
particles and polymerization of the monomer is repeated
In this study cross-linked polystyrene seed latex particles were firstly synthesized
in the presence of styrene monomer divinylbenzene crosslink agent sodium persulfate
initiator sodium bicarbonate buffer and the Aerosol regMA80-I [sodium di(13-
dimethylbutyl) sulfosuccinate] surfactant using a batch type semi-continuous emulsion
polymerization process in which one or more components can be added continuously
in
ethod the procedure of absorption of monomer into fine polymer
55
56
Styrene (St) monomer and divinylbenzene (DVB) crosslink agent as well as initiator and
buffer are continuously added to enlarge the seed latex particles Various profiles of
particle nucleation and growth can be gene different orders of component
addition during polymerizati f this process such as easy
control of the polymeriza particle number
and particle morphology The main goal in
this process is to avoid secondary particle
rated from
on There are several advantages o
tion rate monodisperse particle size and
colloidal stability copolymer composition
formation leading to a monodisperse particle
size distribution Figure 4-1 shows the schematic representation of seed latex particle
preparation and growth
Monomer droplet
Seed particles Amount of surfactantsis variable
Enlarged final Add monomers
PS
PS
PSs4
particles Number of particlesare constant
Seed code
PSs1
s2
s3
Monomer droplet
Seed particles Amount of surfactantsis variable
Enlarged final Add monomers
PS
PS
PSs4
tion The
particles Number of particlesare constant
Seed code
PSs1
s2
s3
Figure 4-1 Schematic representation of semi-continuous seed latex particles preparation and growth
After the first synthesis of a small size of crosslinked polystyrene (PS) seed
particles stabilized by surfactants styrene DVB and initiator are continuously fed into
the system to enlarge the seed latex particles maintaining a narrow size distribu
recipes and reaction conditions for synthesizing these seed particles are summarized in
57
Table 4-1 The original recipe for latex particle preparation from McDonald was
modified for this emulsion system [McDonald 2003]
Table 4-1 The polymerization recipe of polystyrene (PS) seed particles Latex label PSS259 PSS233 PSS207 PSS181
Temperature (oC) 75 plusmn 5 75 plusmn 5 75 plusmn 5 75 plusmn 5
Divinylbenzene (g) 052 052 052 052
MA 80 (g) 259 233 207 181
NaHCO
Watera (g) 1315 1315 1315 1315
Styrene (g) 9948 9948 9948 9948
112 112 112 112
Na2S2O8 (g) 190 190 190 190
Waterb (g) 200 200 200 200
Reaction time (min) 140 140 140 140
Solids content 396 396 396 396
Mean particle diameter (nm) 126plusmn75 171plusmn39 182plusmn41 216plusmn53
Conversion () 965 979 957 971
3 (g)
Four different seed latex particles were synthesized based on surfactant amount in
order to determine the optimum condition for stable and monodisperse latex particles
The emulsion polymerization of latex particles has to be carried out in a narrow range of
surfactant causes flocculation or phase separation leading to broad particle
size distribution and an unstable emulsion sy
surfactant concentration where particles are stable [Nestor et al 2005] A wide range of
concentrations
stem Therefore the range of surfactant
amount was varied from 181g to 259g in increments of 026g in each latex label Other
reactants amounts were kept constant Watera is the initial charge amount and waterb
represents the amount of aqueous solution containing dissolved initiator and buffer
58
which are fed continuously after the seed particle creation The extent of monome
polymer conversion was obtained by gravimetric calcu
r to
lation and was more than 95 at
cripts such as
S207 and S181 i the surf mount added for the seed latex
nversion indi perce experi obtain
content divided by the theoretically estimated solid content and was calculated by the
all seed particles labeled PSS259 PSS233 PSS207 and PSS181 Labeled subs
S259 S233 ndicate actant a
preparation The co cates the ntage of mentally ed solid
following formula
100 colids () times=entsoliatedntentoalExperimentConversion
n the experimenta content is the weight of olid rema after
mulsion at 120oC f in and the estimated solid content is th ht
lated from the recipe
rti as di each t load d
mple of ea pension iluted by (ww)
deionized water and the mean particle diameter was determined using the Brookhaven
ZetaPlus particle size analyzer From these results the seed latex particle size increased
as the amount of surfactant decreased The amount of emulsifier also affects the
polymerization process by changing the number of micelles and their size It is known
that large amount of emulsifier produces larger numbers of smaller sized particles [Odian
1991] and was corroborated in the current work
These synthesized seed latex particles should be of narrow size distribution in order
to obtain monodisperse larger particles The SEM image showing the particle
morphology and size of a sample of polystyrene seed particles can be seen in the Figure
4-2
contdEstim (4-1)
In this equatio l solid the s ining
evaporation of e or 30m e weig
of monomers calcu
As expected the seed pa cle size w fferent for surfactan ing in see
emulsion system A sa ch sus was d 0001 with
59
Figure 4-2 Scanning Electron Micrograph (SEM) of polystyrene seed latex particles (A)
PSS259 (B) PSS233 (C) PSS207 (D) PSS181 (subscripts indicate the amount of surfactant added)
(A)
(B)
60
(C)
(D)
Figure 4-2 Continued
61
s seen in the image the seed particles are very smooth and spherical The
uniformity in the particle size distribution of these PS seed latex particles can also be
seen in the SEM image From this data we concluded that seed particles synthesized in
the presence of a surfactant with the chosen amounts are suitable for use to prepare larger
particles with high size uniformity and smooth spherical morphology
412 The Growth of Polystyrene (PS) Seed Latex Particles
Seeded emulsion polymerization has been conducted for several decades and
various mechanisms have been proposed Gracio et al [1970] suggested that the growing
polymer particles consist of an expanding polymer-rich core surrounded by a monomer-
rich shell with the outer shell providing a major locus of polymerization Seeded
mulsion polymerization is commonly used for preparing latex particles less than 1microm in
size [Cha et al 1995 Zou et al 1990 1992 Gandhi et al 1990 Park et al 1990] Such
latexes can be obtained from the growth of pre-prepared seed particles The seeds
introduced in this process serve as nucleation sites for particle growth By first swelling
the seed latex particles with additional monomer to the desired size polymerization can
then be initiated Several size ranges of monodisperse polystyrene (PS) latex particles
were prepared by this multistep seeded emulsion polymerization method For the
fabrication of particle-based membranes monodisperse latex particles with a variety of
size ranges are required
In this study seed particles from 171nm to 470nm in mean diameter and with a
highly uniform size distribution were prepared and used Watera is the initial charge
mount and waterb represents the amount aqueous solution containing dissolved initiator
and buffer that is continuously fed into system Feed time is precisely controlled by
metering pump The solid contents were maintained between 199 and 300 After
A
e
a
62
polym d as
es
ere 923 8402
n of these latex particles
are sh
ss
erization under these conditions the conversions of the latex particles labele
PS258 PS320 PS370 and PS410 were 952 957 946 and 964 respectively The recip
used in seeded emulsion polymerization to form the particles less than 500nm in mean
diameter are shown at the Table 4-2 The conversions of the latex particles larger than
500nm in mean diameter and labeled as PS525 PS585 PS640 and PS790 w
723 and 969 respectively The recipes used in the preparatio
own at the Table 4-3
A simple calculation can be used to predict the ultimate size that a latex particle can
reach after ending the polymerization Letrsquos suppose that seed density (ρs) and grown
particle density (ρgp) are the same The following equation is obtained from the ma
balance (m = ρV) where m and V are mass and volume of a particle respectively
gp
gp
s
s
Vm
Vm
In this equation m
= (4-2)
be
s and mgp are the weight of seed and total weight of seed and
added monomers respectively and Vs and Vgp are the volume of seed and grown
particle respectively
If we know the mass of monomer to be added the ultimate particle size can
calculated from the equation 4-3
3
2s D m ⎠⎝+ χ
where D
1s D m
⎟⎟⎞
⎜⎜⎛
= (4-3)
the
in
s and Dgp are the diameters for seed and grown particle respectively and χ is
weight of the monomer added Crosslinked PS latex particles with a size range from
258plusmn112 ~ 410plusmn117 in diameter polymerized by the recipes in Table 4-2 are shown
the SEM images in Figure 4-3
63
Table 4-2 Continuous addition emulsion polymerization recipe for growing polystyrene
Latex label PS
(PS) latex particles less than 500nm in size
258 PS320 PS370 PS410
Initial charge
Seed latex (g) 1000171 1000216 1000216 1000320
Watera (g) 1470 2500 1984 2600
06 07 06 06
Fee
Initiator stream
2 2 8
Feed time (min) 0-167 0-140 0-100 0-92
Solid content 284 300 300 200
Mean particle diameter (nm) 258plusmn112 320plusmn154 182plusmn71 410plusmn117
Conv
Monomer continuous
Styrene (g) 800 1200 800 600
Divinylbenzene (g)
d time (min) 0-163 0-110 0-75 0-82
Na S O (g) 07 065 05 05
NaHCO3 (g) 07 065 05 05
Waterb (g) 750 1200 200 370
ersion () 952 957 946 964 The solid content of seed latex for PS258 PS320 PS 370 and PS410 are 392 399 399 anrespectively
d 300
64
Table 4-3 Continuous addition emulsion polymerization recipe for growing polystyrene (PS) latex particles larger than 500nm in size
Latex label PS525 PS585 PS640 PS790
Initial charge
Seed latex (g) 1000390 1000470 1000470 1000320
Watera (g) 1000 520 1200 360
Monomer continuous
Styrene (g) 290 200 400 173
Divinylbenzene (g) 02 014
03 009
Feed time (min) 0-80 0-40 10-76 5-65
Initiator stream
Na2S2O8 (g) 02 014 03 012
NaHCO3 (g) 02 014 03 009
Waterb (g) 200 120 400 200
Feed time (min) 0-85 0-46 0-77 0-75
Solids content 199 200 200 201
Mean particle diameter (nm) 525plusmn151 585plusmn186 640plusmn178 790plusmn693
Conversion () 923 8402 723 969 The solid content of seed latex for PS525 PS585 PS 640 and PS790 are 200 182 193 and 173 respectively
65
Figure 4-3 SEM of PS latex particles less than 500nm in size (A) 258nm (B) 320nm (C)
370nm (D) 410nm
(A)
(B)
66
(C)
(D)
Figure 4-3 Continued
67
article size was well matched to that estimated by the geometrical calculation
From this it can be concluded that the added monomer was consumed for polymer
particle growth with a high degree of conversion From the SEM characterization newly
nucleated particles were not seen as indicated by the narrow particle size distribution
The particles less than 500nm in mean diameter were very spherical in shape with a
highly uniform particle size distribution As the particles were grown larger than 500nm
mean diameter however they became of non-spherical shape with an uneven surface
This irregularity of particle surface can be attributed to the non-homogeneous monomer
swelling into the shell of a growing polymer which can be controlled by the factors such
as temperature agitation speed initiator feeding rate and surfactant amount required to
stabilize the colloidal system The SEM image of latex particles larger than 500nm mean
iameter is shown in Figure 4-4 The particle size distribution is broader than that of the
s particles These particles can however still be used for membrane construction
as a support layer rather than skin layer The support layer does not necessarily have to be
as highly monodisperse as the skin layer which require pores of high uniformity for the
selective removal of toxins
As mentioned earlier the amount of surfactant as well as monomer in an emulsion
system is the primary determinant of the particle diameter [Odian 1991] Anionic
surfactants are generally recommended at the level of 02-30 wt based on the amount
of water [Odian 1991] The critical micelle concentration (CMC) of Aerosolreg MA 80-I
anionic surfactant is 119 The surfactant loading in this emulsion system ranged from
the 121 for SS181 to 171 for SS259
P
d
maller
68
Figure 4-4 SEM of PS latex particles larger than 500nm in size (A) 525nm (B) 585nm
(C) 640nm (D) 790nm
(A)
(B)
69
(C)
(D)
Figure 4-4 Continued
70
The surfactant to monomer ratio refers to the value of the surfactant amount
divided by monomer amount after polymerization ends The surfactant amount is the
weight of the surfactant contained in emulsion and the monomer amount is the total
weight of the monomer added for polymerization to form the ultimate polymer particles
in emulsion As the surfactant to monomer ratio decreases mean particle diameter
increases as described by Odian [1991] Generally lower surfactant concentration forms
fewer micelles resulting in larger particle size [Evans et al 1999] However there is a
plateau in particle mean diameter between a surfactant to monomer ratio of 0005 and
0013 This is due to low polymerization conversion of latex particles from monomer
leading to a smaller particle size than expected This is not a factor of surfactant amount
The dependence of the particle size on the amount of surfactant is shown in Figure 4-5
0
200
400
600
800
1000
0000 0005 0010 0015 0020 0025 0030
Surfactant to monomer ratio (ww)
Part
icle
mea
n di
amet
er (n
m)
Figure 4-5 Dependence of the particle size on the surfactant to monomer ratio
71
413
e
f bare
microdomain structures is important for biocompatibility [Mori et al 1982 Higuchi et al
1993 Deppisch et al 1998] Figure 4-6 shows the schematic of core shell latex particle
structure The recipe for core shell structures is shown in Table 4-4
Core Shell Latex Particles
The latex particles shown in Figure 4-3 (A) and labeled as S258 in Table 4-2 wer
used as seeds to prepare core-shell latex particles because of their high particle
uniformity and smooth surface morphology Methyl methacrylate (MMA) and acrylic
acid (AA) monomers were introduced to increase surface hydrophilicity over that o
polystyrene particles AA is a more hydrophilic monomer than MMA because of the
carboxyl acid group in AA is more favorable for hydrogen boding with water The
surface carboxyl groups on PAA may have many promising applications in biomedical
and biochemical fields [Kang et al 2005] MMA monomer is less hydrophobic than
styrene PMMA is known to more biocompatible than PS but still PMMA is hydrophobic
The optimization of suitable hydrophobic to hydrophilic ratios and control of
Polystyreneseed particle
Core shelllatex particles
PSPMMA100
PSPMMA90PAA10
PSPMMA75PAA25
Polystyreneseed particle
Core shelllatex particles
PSPMMA100
PSPMMA90PAA10
Figure 4-6 Schematic of core shell latex particle structures
PSPMMA75PAA25
72
Table 4-4 The preparation recipe of PS core with various shell latex particles Latex 25 label PSPMMA100 PSPMMA90PAA10 PSPMMA75PAA
Initial charge
PS seed latex (g) 500 500 500a
Monomer continuous
Methly methacylate (g) 320 288 240
Acrylic acid (g) 00 32 80
Initiator stream
Na
258 258 258
Water (g) 1570 1570 1570
Feed time (min) 0-120 0-50 0-60
b 200
Feed time (min) 0-122 0-60 0-65
952
2S2O8 (g) 02 02 02
NaHCO3 (g) 02 02 02
Water (g) 200 200
Solids content 183 183 183
Mean particle diameter (nm) 370plusmn186 370plusmn178 370plusmn191
Conversion () 955 924
Three types of core shell structures were prepared PMMA75PAA25 shell is a
copolymer consisting of the PMMA to PAA ratio of 75 to 25 by weight
PMMA90PAA10 shell is a copolymer with the PMMA to PAA ratio of 90 to 10 by
weight PMMA100 is the PMMA homopolymer shell on PS core The particle size of
these core-shell particles as well as PS latex particle was prepared to be about 370nm in
mean diameter and are characterized to determine their zeta potential protein adsorption
and biocompatibility The image of SEM of PS and core shell particles is shown at Figure
4-7 The synthesized latex particles had a high uniformity in size and a smooth spherical
surface morphology
73
Figure 4-7 Scanning
Electron Micrograph of latex particles (A) PS (B) PSPMMA100 (C) PSPMMA90PAA10 (D) PSPMMA75PAA25
(A)
(B)
74
(C)
(D)
Figure 4-7 Continued
75
42 Characterization of Latex Particles
421 Fourier Transform Infrared Spectroscopy (FTIR)
FTIR spectroscopy has proven to be a powerful tool to characterize polymeric
materials A molecule can absorb only select frequencies (energy) of infrared radiation
which match the natural vibration frequencies The energy absorbed serves to increase
the amplitude of the vibrational motions of the bonds in the molecule [Pavin et al 1996]
Only those bonds which have a dipole moment that changes as a function of time can
absorb infrared radiation The FTIR spectra of polymerized latex particles made during
this work are shown in Figure 4-8 There are number of characteristic peaks for PS latex
particles [Bhutto et al 2003 Li at al 2005] Aromatic C-H stretching vibration is shown
at 3002 cm-1 and 3103 cm-1 and aliphatic C-H asymmetrical and symmetrical stretching
shown at 2900 cm-1and 2850 cm-1 respectively There is an additional carbonyl (C=O)
adsorption at 1730cm-1 wavenumber for all core shell latex particles containing some
amount of either or both PMMA and PAA The broad OH group peak at 3400 cm-1
appear for particles containing some amount of PAA and has intensity dependent on the
amount of PAA in the shell For example the peak intensity of OH groups for
PSPMMA75PAA25 is greater than that of PSPMMA90PAA10 since there is a higher AA
monomer content in PSPMMA75PAA25 than in PSPMMA90PAA10 This OH peak is not
seen for PS and PS core PMMA shell particles because of the absence of OH groups on
PS and PMMA polymer chains
422 Zeta Potential Measurements
The electrical surface property was characterized by zeta potential measurements of
tex particles Zeta potential is an important and useful indicator of surface charge which
can be used to predict and control the stability of colloidal systems The greater the zeta
is
la
76
1520002500300035004000
Wavenumbers (cm-1)
li
Re
ativ
e
Tra
nsm
ttanc
e
Figure 4-8 FTIR spectra of polymerized latex particles (A) ba
PSPMMA100 (C) PSPMMA90PAA10 (D) PSPMM
(B)
(C) P
(D) P
C
C
OCH3
O
CH3
XCH2
CH2 n
CH
CH2 C
C
OCH3
O
XCH2 CH
OC
CH3
Y
OH
(A) Bare PS
500100000
re polystyrene (PS) (B) A75PAA25
PSPMMA100
SPMMA90PAA10
SPMMA75PAA25
77
potential the more likely the suspension is to be stable because the charged particles repel
one another and thus overcome the natural tendency to aggregate The measurement of
zeta potential is often the key ontrol of the interaction of proteins
with solid surfaces such as latex particles
Zeta potential (ζ) is the elec at exists at the shear plane of a particle
which is some small distance from the surface [Myers 1999] The zeta potential of the
particles can be determined by measuring the mobility of the particles in an applied
electric field termed electr its response to an alternating electric
field Colloidal particles dispersed in lu ically charged due to their ionic
characteristics and dipolar attributes The development of a net charge at the particle
surface affects the distribution of ions in the neighboring ng in
an increased concentration of counter ions to the surface Each particle dispersed in a
ns called fixed layer or Stern layer
Outside th compositions of ions of opposite polarities
form ble layer is form
particle-liquid interface This double layer may be considered to consist of two parts an
inner region which inclu nd relatively strongly to the surface and an outer or
diffuse region in which the ion distribution is determined by a balance of electrostatic
forces and random thermal motion The potential in this region therefore decays with the
distance from the surface until at a certain distance it becomes zero Figure 4-9 shows a
schematic representation of a typical ion distribution near a positively charged surface
The Stern surface (also referred to as the outer Helmholtz plane) is drawn through the
to understanding and c
trical potential th
ophoretic mobility or
a so tion are electr
interfacial region resulti
solution is surrounded by oppositely charged io
e Stern layer there are varying
ed in the region of the ing a cloud-like area Thus an electrical dou
des ions bou
center of those ions which are effectively adsorbed to the surface [Hunter 1981] The
78
extent of ion adsorption is determined by electrical and other long-range interactions
between the individual ions and surface of particles The ions outside of the Stern layer
form the diffuse double layer also referred to as the Gouy-Chapman layer [Burns and
Zydney 2000]
Shear plane
Stern layer
ψζ
0
ψs
ψShear plane
Stern layer
ψζ
0
ψs
ψ
t
controlled electric field is applied via electrodes immersed in a sample suspension and
Figure 4-9 Schematic representation of ion distribution near a positively charged surface
Zeta potential is a function of the surface charge of a particle any adsorbed layer a
the interface and the nature and composition of the surrounding medium in which the
particle is suspended The principle of determining zeta potential is very simple A
79
this causes the charged particles to move towards the electrode of opposite polarity
Viscous forces acting upon the moving particle tend to oppose this motion and the
equilibrium is rapidly established between the effects of the electrostatic attraction and
the viscosity dra
g The particle therefore reaches a constant terminal velocity
Figure 4-10 Schematic representation of zeta potential measurement(source
httpnitioncomenproductszeecom_shtm)
Because protein adsorption mechanisms are very complex and hard to explain in
biological systems protein adsorption is generally studied by using more ideal systems
consisting of one or more well characterized proteins a well-characterized adsorbent and
a well-defined aqueous solution Even so small changes in the experimental conditions
such as pH ionic strength and temperature generate totally different results Therefore
two media systems phosphate buffer (PB) and phosphate buffered saline (PBS) were
chosen to be used for the zeta potential measurements and protein adsorption study PB is
a simple aqueous system of 5 mM monobasic sodium phosphate (NaH2PO4H2O) to
n a constant pH PBS is used to keep the water concentration inside and outside of
the cell balanced If the water concentration is unbalanced the cell risks bursting or
shriveling up because of a phenomenon called osmosis PBS is a solution with the 5 mM
of monobasic sodium phosphate (NaH2PO4H2O) and 143 mM of sodium chloride (NaCl)
in water
maintai
80
The results of zeta potential measurement at room temperature (25plusmn1oC) as
function of pH and ionic strength (different media PB and PBS) for synthesized lat
particles are seen in Figure 4-11 Zeta potential values of polystyrene (PS) latex particle
at the Figure 4-9 are negative between -291 mV and -599 mV in PB and betwe
ex
s
en -203
mV and -278 mV in PBS at all pH ranges These negative zeta potential values of PS
latex particles are due to the sulfate groups [Vandenhu et al 1970] originated from
persulfate initiators attached via an oxygen bridge (-C-O-SO3-) at the end of polymer
chain where polymerization was initiated
-100
-80
-60
-40
-20
0
pH
Zta
pte
ntl
ζ
20
40
1 2 3 4 5 6 7 8 9
eo
ia (m
V)
PB (5mM)
PBS (5mM +143mM)
en -
Figure 4-11 Zeta potential of PS latex particles at 25oC
The zeta potential measurements of PSPMMA100 core shell latex particles as seen
in Figure 4-12 were also negative with a range of -28 and -505 mV in PB and betwe
143 mV and -186 mV in PBS at the pH 21-78 These negative values are also due to
sulfate groups on the particle surface
81
-100
-80
-60
-40
-20
0
20
40
1 2 3 4 5 6 7 8 9
Zea
pote
nti
l ζ
(mV
pH
ta
)
PB (5mM)
PBS (5mM + 143mM)
Figure 4-12 Zeta potential of PSPMMA100 latex particles at 25oC
The zeta potential for PSPMMA90PAA10 and PSPMMA75PAA25 was also
negative as seen in Figure 4-13 and 4-14 The zeta potential value for the
PSPMMA90PAA10 were between -367 mV and -678 mV in PB medium and between -
147 mV and -193 mV in PBS both at 25oC In case of the PSPMMA75PAA25 core shell
particles zeta potential values were between -291 mV and -520 mV in PB and between
-115 mV and -210 mV in PBS media Sulfate groups in PSPMMA75PAA25 and
PSPMMA90PAA10 core shell particles contribute to the negative zeta potential values
participate in forming a
negat
cid is expressed by the term pKa which is the pH
at which an acid is 50 dissociated However the contribution of the carboxylic acid
Carboxylic groups from acrylic acid monomer also would
ive surface charge of the latex particles at a pH greater than 70 because the pKa
value for the carboxylic acid is between 40 and 60 pH range [Ottewill et al 1967] This
indicates that carboxylic acid dissociation begins at a pH between pH 46-60 and
increases with pH The strength of an a
82
group to negative zeta potential values of PSPMMA-PAA core shell particles was not
detectable because there was no significant difference in zeta potential values for
synthesized latex particles at a pH greater than 70
-60
-40
-20
0
20
40
1 2 3 4 5 6 7 8 9
Zeta
pot
entia
l ζ
(mV
)
PB (5mM)PBS (5mM + 143mM)
-80
-100
pH
Figure 4-13 Zeta potential of PSPMMA90PAA10 latex particles at 25oC
-100
-80
-60
-40
-20
0
20
40
1 2 3 4 5 6 7 8 9
pH
Ze p
ont
i ζ
V)
tate
al (m
PB (5mM)PBS (5mM + 143mM)
Figure 4-14 Zeta potential of PSPMMA75PAA25 latex particles at 25oC
83
The zeta potential plots and values were similar for all latex particles This may be
due to the similar initiator density on the latex surface The initiator concentrations
monomers for shell polymerization were the same fo
to
r all core shell latex particles (062
ww from Table 4-4) A similar concentration (055 ww) of initiator to monomer was
added to prepare bare PS latex particles used in zeta potential measurements
Since the zeta potential values of all synthesized latex particles were negative
between pH=20 and pH=78 the isoelecric point (IEP) of these latex particles would be
less than pH=20 Sulfates have a pKa in the range of 10 to 20 [James 1985] indicating
that the sulfate group the conjugate base of a strong acid is protonated (-C-OSO3H) at
less than pH 20 This means that the synthesized latex particles are all negative at the
physiological pH Most serum proteins have a negative surface charge in blood
therefore when the negative latex particles are applied to blood stream fatal
tein is
avoided by charge repulsion in the blood Table 4-5 shows the common blood proteins
and their isoelectric points (IEP)
Table 4-5 The isoelectric point (IEP) of common proteins Serum proteins Isoelectric point (IEP)
complication by coagulation between applied latex particles and serum pro s
Albumin 48 α1-Globulin 20 α2-Microglobulin 54 γ1-Globulin 58 Heptoglobin 41 Hemoglobin 72 β1-lipoprotein 54 Immunoglobulin G (Ig G) 73 Fibrinogen 58 (source httpwwwfleshandbonescomreadingroompdf1085pdf
httpwwwmedalorgvisitorwww5CActive5Cch135Cch13015Cch130105aspx )
84
The absolute zeta potential values of the latex particles in phosphate buffer (PB)
was higher than that in phosphate buffered saline (PBS) This is because high
concentration of sodium and chloride electrolytes in PBS compressed the electrical
double layer causing rapid decay of surface potential Figure 4-15 shows that the
thickness of the electrical double layer is inversely proportional to the concentration of
electrolyte in the system
Surf
ace
Pote
ntia
l ψ
1κ3
10
1e
1κ 1κ
(1)(3) (2)
2 1 Z
Surf
ace
Pote
ntia
l ψ
1κ3
10
1e
1κ 1κ
(1)(3) (2)
Figure 4-15 The decay of surface potential with distance from surface in various
electrolyte concentrations (1) low (2) intermediate (3) high [Myers 1999]
2 1 Z
The electrical potential in the solution surrounding the particle surface falls off
the Debye-Huckel
yers 1999]
exponentially with distance (Z) from the surface according to
approximation [M
z) (- exp 0 κψψ = (4-4)
e reciprocal of the thickness of the electrical double layer referred to as the
The potential fallen off by a factor e The theoretical equation for the
ickness 1κ is
where κ is th
Debye length of 1
double layer th
21
22i
0
ekT 1
⎟⎟⎠
⎜⎜⎝
=sum ii zcεε
κ
⎞⎛ (4-5)
85
where
tration of
y was performed to measure the protein adsorption on the
synthesized latex particles Many physical and chemical processes occur at the boundary
between two phases and adsorption is one of the fundamental surface phenomena [Oscik
1982] This phenomenon is driven by the change in the concentration of the components
at the interface with respect to their concentrations in the bulk phase There are two
adsorption processes physical adsorption occurring when non-balanced physical forces
appear at the boundary of the phases and chemical adsorption or chemisorption
t the
interface [J
n leads
ational entropy hence adsorption takes place only if the
loss in conformational entropy is compensated
ter adsorption Cp The initial part of the
isotherm merges with the Γ-axis because at low polymer concentration the protein has a
high affinity for the surface and all of the polymer is adsorbed until the solid surface is
saturated
ε0 is the permittivity of a vacuum of free space ε is the permittivity of a medium e
is the relative permittivity or dielectric constant of the medium c is the concen
an ion (i) z is the valency of an ion (i) k is Boltzmannrsquos constant T is absolute
temperature (K)
43 Protein Adsorption Study
The adsorption stud
occurring when atoms and molecules from adjacent phases form chemical bonds a
aroniec et al 1988]
Flexible proteins in solution possess high conformational entropy as a result of the
various states each of the many segments in the protein chain can have Adsorptio
to a reduction of this conform
by sufficient attraction between polymer
segment and solid surface [Martin 1998] Isotherm shape of high affinity adsorption
between polymer and solid is shown in Figure 4-16 where the adsorbed mass Γ is plotted
against the polymer concentration in solution af
86
Figur
de
arboxyl
e 4-16 High-affinity adsorption isotherm of typical flexible polymer on solid
surface
In order to investigate the adsorption of blood proteins latex particle properties
such as electrical surface charge hydrophobicity and environmental conditions like pH
ionic strength and temperature are considered as experimental factors [Arai and Nor
1990] Figure 4-17 shows the overall schematic representation of the protein adsorption
on the synthesized latex particles with the functional groups such as sulfate and c
groups
CP
Γ
PMMAPAAShell
PS or PMMAShell OSO3
-
OSO3-
OSO3-
OSO3-
PS
-O3SO
PSCore
HOOC
-O3SO
O3SO
3
HOOC
HOOC
-
-O SO
Protein
NH3+
+
+H3N
+
COO-
Hydrophobic interaction
NH3
COO-
H3N
COO-
-OOC
Ionic interaction
Hydrophobic interaction
Hydrogen bonding
Ionic interaction
PMMAPAAShellOSO3
-
OSO3-
OSO3-
OSO3-
PS or PMMAShell -O3SOOSO3
-
PSCore
HOOC
-O3SO
O3SO
3
HOOC
HOOC
-
-O SO
-O3SO
OSO3-
OSO3-
OSO3-
PS PSCore
HOOC
-O3SO
O3SO
3
HOOC
HOOC
-
-O SO
Protein
NH3+
+
+H3N
+
COO-NH3
COO-
H3N
COO-
-OOC
NH3+
+
+H3N
+
COO-
Hydrophobic interaction
NH3
COO-
H3N
COO-
-OOC
Ionic interaction
Hydrophobic interaction
Hydrogen bonding
Figure 4-17 Overall schematic representation of the protein adsorption on the
Ionic interaction
synthesized latex particles
The surface functional groups as well as suitable surface hydrophobicity
hydrophilicity of latex particles are closely related to protein adsorption property As
indicated at Figure 4-17 various interaction forces such as hydrophobic interaction ionic
87
interaction hydrogen interaction and van der Waals interaction [Andrade 1985] exist
between protein molecule and the solid surface of latex particle
431 Adsorption Isotherm
The protein adsorption isotherm is generally defined as the relationship between the
amount of protein adsorbed by a solid (latex particle) at a constant temperature and as a
ine
the amount of protein adsorbed onto latex particles To evaluate the equilibrium
conce s
x
function of the concentration of protein Adsorption isotherms were plotted to determ
ntration of target proteins a calibration curve is firstly needed Figure 4-18 show
the UV absorbance as a factor of bovine serum albumin (BSA) concentrations The UV
absorbance of the known protein concentration was measured at 562nm which is the λma
for a protein treated by a bicinchoninic acid (BCA) assay
0
02
04
06
08
500 520 540 560 580 600
Wavelength (nm)
Abs
orba
nce
Figur gml (B)
06mgml (C) 02mgml
(A)
(C)
(B)
Max562nm
e 4-18 The dependence of UV absorbance on BSA concentration (A) 10m
Figure 4-19 shows the standard calibration curve used in adsorption isotherm
experiment of protein onto latex particles The net absorbance of each known protein
88
concentration was plotted and then a linear equation passing through a concentration of
zero protein concentration was obtained This equation is applied
to determine the
orbed on the synthesized latex particles The amount of free
protei
eported
trations (005 01 03 05
and 07mgml) were used to generate this curve as well as the adsorption isotherm
unknown protein amount ads
n was also determined using UV light at the 280nm wavelength however a
distinguishable UV intensity was too low to determine a precise value of protein
adsorption
The bicinchninic acid (BCA) assay technique [Smith et al 1985] has been r
for high efficiency of protein evaluation and was used for determination of protein
adsorption [Tangpasuthadol et al 2003 Williams et al 2003] This technique has many
advantages such as high sensitivity easy of use color complex stability and less
susceptibility to any detergents Five different BSA concen
y = 00R2 =
004741x 09939
00
01
02
03
04
0 200 400 600 800
BSA Standard (microgml)
Net
Abs
orba
nce
at 5
62nm
Figure 4-19 Standard curve of net absorbance vs BSA sample concentration
89
All isotherms were fitted to the Langmuir-Freundlich isotherms using the nonlinea
regression method Two adsorption isotherm models the Langmuir type isotherm a
Langmuir-Freundlich combination type isotherm were compared As seen the Figure 4-
20 The Langmuir isotherm model deviated further from the experimental data Th
all isotherms were fitted to the Langmuir-Freundlich isotherms [Yoon et al 1996
al 1998] usi
r
nd the
erefore
Lee et
ng the nonlinear regression method with following equation
n
n
eq
eqm
kC
kC1
1
1+
where q is the amount of adsorbed BSA per unit surface area q
qq = (4-6)
stant
were between 045 and 055 and hence n was fixed to its average 05 Actual qm values
were evaluated after fixing the n value
m is the adsorbed amount
in equilibrium Ceq is the equilibrium concentration of BSA k is the adsorption con
and n is the exponential factor for a heterogeneous system n values are empirical and
usually from zero to no more than unity [Jaekel 2002] The values of n in our system
00
10
20
30
0 02 04 06 08
C (mgml)eq
q (m
gm
2 )
pH 32Langmuir-Freundlich modelLangmuir model
Figure 4-20 Fitted models for adsorption isotherm of bovine serum albumin (BSA) on
polystyrene latex particles at 37oC in phosphate buffer media
90
The BSA protein adsorption on the synthesized latex particles was performed at
three pH levels 32 48 74 in both phosphate buffer (PB) and phosphate buffered saline
(PBS) at physiological temperature 37oC These pH levels correspond to an acidic pH
lower than the isoelectric point (IEP) of BSA the pH at the IEP and an alkaline pH
higher than the IEP of BSA respectively The IEP corresponds to the pH at which the
amino acid becomes a zwitterion Here its overall electrostatic charge is neutral and the
charge expressed by the basic amino group (-NH2 -NH3+) of the amino acid is equal to
the charge expressed by the acidic carboxyl group (-COOH -COO ) The IEP of BSA is
-
pH 48 and indicates that the side chain of the amino acids of BSA contains a greater
amount of acidic functional groups than that of basic functional groups At pH 32 and
74 BSA has a positive and negative charge respectively
Figure 4-21 and 4-22 show adsorption isotherms of BSA onto polystyrene (PS)
latex particles
00
10
20
30
40
0 02 04 06 08
Ceq (mgml)
q (m
gm
2 )
pH=32pH=48pH=74Model (pH 32)Model (pH 48)Model (pH 74)
Figure 4-21 Adsorption isotherm of BSA on polystyrene (PS) latex particles in
phosphate buffer (PB) at 37oC
91
00
10
30
40
0 02 04 06 08
Ceq (mgml)
q (m
gm
2
20
)
pH=32pH=48pH=74Model (pH 32)Model (pH 48)Model (pH 74)
(PBS) on PS latex particles
At the IEP of BSA the adsorbed amount (q
Figure 4-22 Adsorption isotherm of BSA adsorption 37oC in phosphate buffered saline
onto PS at equilibrium was
maxim
um at the IEP of BSA This is because the BSA molecules form the most
compact structures at the IEP eliminating the repulsion force between proteins A higher
number of BSA molecules can adsorb on a given surface area by hydrophobic interaction
when in this compact structure This observation agrees well with reported data by other
researchers who also indicated that the maximum adsorption from aqueous protein
solution was observed at the IEP [Mularen 1954 Watkins et al 1977 Oh at el 2000]
There was no significant difference of adsorbed BSA amount in PB and in PBS media at
pH 48 The conformational alteration of BSA was not affected significantly by the ionic
strength of the two solutions There was evidence of the effect of the electrostatic
interaction to some extent even at the IEP since the amount of BSA adsorbed in PBS is
slightly hig
m) of BSA
um indicating that hydrophobic interaction between latex particle and BSA
protein is maxim
her than in PB at this pH
92
The adsorbed amount of protein is shown to decrease at a pH 32 and 74 The
decrease in adsorption efficiency when deviating from IEP of BSA is a result of the
increase in conformational size of the protein molecules and the lateral electrostatic
repulsions between adjacent adsorbed BSA molecules Figure 4-23 shows a schematic of
possible conformational changes at each pH where hydrophobic interaction always exists
between non-polar portion of a protein and hydrophobic portion of solid surface
- - --- ---Latex surface
Zeta potential
BSA(+)
(-)
48 7432pH
(0)
+++ + ++
--+
++-
- - --- ---Latex surface
Zeta poten pH(0)
+++ + ++
--
tial
BSA(+)
(-)
48 7432
+
++-
ming
4
s
al blood Table 4-6 shows the calculated results of
Figure 4-23 Conformation of bovine serum albumin (BSA) on latex particles (Assuthat charge repulsion reduces the effect of the hydrophobic interaction at pH 74)
In addition the adsorbed amount of BSA at pH 32 was larger than that at pH 74
and still less than that at 48 This is because there is the electrostatic interaction between
the positive BSA molecules and negative PS latex particles at pH 32 and electrostatic
repulsion is dominant between negative BSA and negative PS latex particles at pH 7
The amount adsorbed increased with increasing ionic strength in pH 32 and 74 This i
because the high concentration of electrolytes reduces the electrostatic repulsion between
BSA and PS latex particle It should be noted that the BSA concentration used in these
experiments was lower than that found in normal human blood This is because the
equilibrium BSA adsorption onto the latex particles is actually reached at a much lower
concentration than that found in norm
93
adsor rm
ein
leading to a compact conformation of BSA There are two main forces affecting
adsorption amount at an acidic pH of 32 They include the ionic attraction between the
negative latex particle and the positive protein and lateral repulsion between positive
proteins The smallest amount of BSA adsorption was observed at a pH of 74 At this
point the charge repulsion between negatively charged particles and protein molecules
exists the h
of cha
bed BSA amount at equilibrium from the Langmuir-Freundich adsorption isothe
equation
In summary the hydrophobic interaction between PS latex particle and BSA
protein exists and is the dominant mechanism for the adsorption process Conformational
changes of BSA at each pH also affected the adsorption amount Adsorption was highest
at pH 48 because at this pH there is the least amount of ionic repulsion in the prot
and the lateral repulsion between proteins dominant adsorption even though there always
ydrophobic interaction between the solid latex particle and protein The effect
rge repulsion was reduced by the high concentration of electrolytes in PBS medium
Table 4-6 The equilibrium concentration values of BSA adsorption on polystyrene (PS) latex particles calculated the Langmuir-Freundlich isotherm model
Media pH qm (mgm2) 32 174 48 299 Phosphate buffer 74 136 32 260 48 283 Phosphate buffered saline 74 207
The adsorption isotherms of BSA on PSPMMA core shell particles are shown in
Figure 4-24 in phosphate buffer (PB) and in Figure 4-25 in phosphate buffered saline
(PBS) The adsorbed BSA amount was calculated using the Langmuir-Freundlich
100
94
adsorption isotherm equation and the amount listed in Table 4-7 The trend seen in the
adsorption results was very similar to the case of PS latex The overall adsorption process
was d rticle
rs
hat
0
ominated by hydrophobic interactions between the PSPMMA100 core shell pa
and BSA proteins At pH 48 the adsorbed amount (qm) of BSA on PSPMMA100 core
shell particles was 221 mgm2 in PB and 237 mgm2 in PBS and are the maximum
values recorded among the three pH levels 32 48 and 74 Suzawa and his co-worke
[1982] have obtained similar results This can be also explained by the compact
conformation of BSA molecules at the IEP of BSA which lead to a high protein
population on the solid surface At a pH of 32 and 74 BSA adsorption was less than t
at pH 48 because of the protein conformation change There was a small increase in
adsorbed BSA at pH 32 because of the charge attraction between negative PSPMMA10
core shell particles and positive BSA At pH 74 however the adsorption of BSA was
further reduced by charge repulsion between the negative PSPMMA100 core shell
particles and the negative BSA proteins
000 02 04 06 08
Ceq (mgml)
10
20q (m
gm
302 )
40
50pH=32pH=48pH=74Mode 8)l (pH 4Mode 2)l (pH 3Mode 4)
Figure 4-24 Adsorption isotherm of BSA 37oC in PB on PSPMMA core shell latex
l (pH 7
100
particles
95
The adsorbed amount of BSA increased more rapidly in PBS with increasing ioni
strength at pH 74 This shows that the low concentration of electrolytes promotes
electrostatic repulsion between negative BSA and negative PSPMMA
c
on
x
l 1980
100 core shell
particle As the ion strength of PBS is increased however the high concentration of
electrolyte leads to less electrostatic repulsion promoting more rapid protein adsorpti
By comparing the amount of adsorbed protein between bare PS latex particles and
PSPMMA100 core shell particles the amount of protein adsorption on the PS late
particles is greater than that on the PSPMMA100 core-shell particles [Suzawa et a
1982 Lee et al 1988]
00
10
20
30
40
m2 )
0 02 04 06 08
Ceq (mgml)
q (m
g
pH=32pH=48pH=74Model (pH 2) 3Model (pH 48)Model (pH 74)
Figure 4-25 Adsorption isotherm of BSA at 37oC in PBS on PSPMMA100 core shell
latex particles
96
Table particles calculated the Langmuir-Freundlich isotherm model
4-7 The equilibrium concentration values (qm) of BSA adsorption on PSPMMA100
Media pH qm (mgm ) 2
32 140
48 221 Phosphate buffer
74 130
32 169
48 237 Phosphate buffered saline
74 162
The BSA adsorption mechanism and trend on PSPMMA core shell particles was
very similar to that of the PS latex particle Unlike the case of PS and PSPMMA100 latex
particles the amount of BSA adsorbed onto PSPMMA90PAA10 and PSPMMA75PAA25
core shell particles was higher at pH 32 than pH 48 and 74 Figure 4-26 through 4-29
shows the adsorption isotherms of BSA on PSPMMA90PAA10 and PSPMMA75PAA25
core shell particles The amount of BSA adsorbed increased at pH 32 and pH 48 as the
hydrophilicity was developed by the addition of PAA On the contrary the adsorbed
amount of BSA was dramatically decreased at pH 74 The adsorption amount of BSA on
PSPMMA90PAA10 and PSPMMA75PAA25 core shell particles at equilibrium is listed in
Table 4-8 and 4-9 respectively
Carboxylic acid as well as sulfate groups are distributed on the surface of
PSPMMA90PAA10 and PSPMMA75PAA25 core shell particles and seem to largely affect
the BSA adsorption The isotherms showed very strong pH dependence to the BSA
adsorption onto these core shell latex particles The carboxylic groups (COOH) may
especially enhance the BSA adsorption prominently at pH 32 by hydrogen bonding with
protein molecules The pKa value for the PAA is 40-60 and a good indication that the
97
carboxylic acid groups are protonated at pH 32[Gebhardt et al 1983] The main
adsorption interaction force at pH 32 was generated by hydrogen bonding as well as
charge attraction between latex particle protein even th the conformational
repulsion between proteins results in a reduction of the protein
adsorption at this pH The amount of BSA adsorbed also increased as the PAA amount in
eased The a tion of BSA w inimal however less
than 07 mgm2 at pH 74 Here the carboxylic groups are ionized which increases the
charge repulsion by ionized carboxylic groups (COO-) resulting in PSPMMA-PAA latex
particle
and ough
change and charge
the core shell particles incr dsorp as m
s with much lower protein adsorption
00
20
40
q (m
g2 )
60
m
pH=32pH=48pH=74Model (pH 32)Model (pH 48)
0 02 04 06 08
Ceq (mgml)
Model (pH 74)
o shell
Figure 4-26 Adsorption isotherm of BSA at 37 C in PB on PSPMMA90PAA10 corelatex particles
98
00
40
60
80
0 02 04
C (mgm
mm
2 )
20
06 08
eq l)
q (
g
pH=32pH=48pH=74Model (pH 32)Model (pH 48)Model (pH 74)
Figure 4-27 Adsorption isotherm of BSA at 37oC in PBS on PSPMMA90PAA10 core
shell latex particles
Table 4-8 The equilibrium concentration values of BSA adsorption on PSPMMA90PAA10 particles calculated the Langmuir-Freundlich isotherm model
Media pH qm (mgm2)
32 361
48 283 Phosphate buffer
74 043
32 519
48 226 Phosphate buffered saline
74 043
99
00
20
40
60
80
100
0 02 04 06 08Ceq (mgml)
q (m
gm
2 )
pH=32pH=48pH=74Model (pH 32)Model (pH 48)Model (pH 74)
oFigure 4-28 Adsorption isotherm of BSA 37 C in PB on PSPMMA75PAA25 latex particles
00
100
20
40
60
80
0 02 04 06 08Ceq (mgml)
q (
m2 )
pH=32pH=48pH=74
mg
Model (pH 32)Model (pH 48)Model (pH 74)
Figure 4-29 Adsorption isotherm of BSA 37oC in PBS on PSPMMA75PAA25 core shell
latex particles
100
Table 4-9 The equilibrium concentration values of BSA adsorption on PSPMMA75PAA25 particles calculated the Langmuir-Freundlich isotherm model
Media pH qm (mgm2)
32 1083
48 662 Phosphate buffer (PB)
74 075
32 1077
48 681 Phosphate buffered saline (PBS)
74 057
The adsorption of β2-microglobulin (β2M) was determined under one set of
onditions in phosphate buffer at 37oC and pH 74 because of the limited amount of β2M
protein The adsorption isotherms of the β2M protein on the synthesized latex particles
are shown in Figure 4-30 4-31 and 4-32 The β2M protein concentrations for adsorption
isotherm experiment were chosen to be 0015 0030 0045 and 0060mgml since the
β2M concentration for the patients with renal failure can be elevated to similar
concentrations about 005mgml or more up to 50 times the normal level [Nissenson et
al 1995] There were steep initial slopes of the adsorption isotherm curves of β2M on
latex particles in all isotherms indicating a high affinity adsorption type It seems that the
hydrophobic interaction is enough for the adsorption process of β2M proteins to occur on
the latex particles even though charge repulsion exists between protein and latex particle
at pH 74 The complete plateau regions were not seen for all isotherms indicating that
β2M proteins were not saturated at this level and there are still available sites for β2M
c
adsorption on latex particles The initial slope of the β2M isotherms for PS was steeper
101
than that of any of the other latex particle PSPMMA100 PSPMMA90PAA10 and
PSPMMA 75PAA25
00
02
0 0002 0004 0006 0008
04
06
10
Ceq (mgml)
q (m
2 )
08
gm
PSPSPMMA100Model (PS)Model (PSPMMA100)
Figure 4-30 Adsorption isotherm of β2M onto PS and PSPMMA100 latex particles in PB
at 37 C and pH 74 o
00
02
04
06
08
10
0 0002 0004 0006 0008C (mgml)
q (m
m2 )
eq
g
PS
PSPMMA90PAA10
Model (PS)
Model (PSPMMA90PAA10)
Figure 4-31 Adsorption isotherm of β2M onto PS and PSPMMA90PAA10 latex particles
in PB at 37oC and pH 74
102
00
02
04
06
08
10
0 0002 0004 0006 0008Ceq (mgml)
q (m
gm
2 )
PS
PSPMMA75PAA25
Model (PS)Model (PSPMMA75PAA25)
Figure 4-32 Adsorption isotherm of β2M onto PS and PSPMMA75PAA25 latex particles
in PB at 37oC and pH 74
The equilibrium concentration values (qm) of β2M adsorption on latex particles are
calculated and listed in Table 4-10 The difference between the maximum amounts of
β2M adsorption on all the latex particles was not significantly different and their values
were between 069 and 08mgm2
Table 4-10 The equilibrium concentration values of β2M adsorption calculated the Langmuir-Freundlich isotherm model
Latex particles qm (mgm2)
PS 080
PSPMMA100 069
PSPMMA90PAA10 072
PSPMMA75PAA25 072
103
432 Adsorbed Layer Thickness
There are several possible explanations for large amount of BSA adsorbed at acidic
pH levels including the end-on adsorption pattern of BSA the flexibility of BSA
molecules the tilting of protein molecules due to the asymmetry of the charge
distribution or multilayer formation [Peula et al 1993] The adsorbed BSA thickness (δ)
can be calculated by the following equation [Chiu et al 1978] This equation was also
cited again by other researchers [Shirahama et al 1985]
BSA
mqρπ
δbullbull
=33
(4-7)
where π
33 is the packing factor qm is the plateau value of the adsorbed am
ρBSA is the hich corresponds to the reciprocal of its known
partial specific volume The layer thickness (δ) of BSA proteins on synthesized latex
partic
he IEP [Shirahama et al 1985] Compared to the
hydrodynamic dimensions of BSA 140 times 40 times 40 Aring3 [Fair et al 1980 Peter 1985] these
nd
ode It is unnecessary ultilayering because thickness do
exceed 140 Aring The layer thickness (δ) of BSA proteins on synthesized latex particles
e red saline (PBS) was ran 28 ~ 83 Aring at 37oC and pH 48 These
lso listed in Table 4-1
ount and
density of BSA molecule w
les in phosphate buffer (PB) ranged from 27 - 81 Aring at 37oC and pH 48 These
calculated values are listed in Table 4-11 Only δ values at pH 48 the isoelectric point
(IEP) of BSA are important because intra- and intermolecular electrostatic repulsion of
protein molecules is minimized at t
values of layer thickness indicate that the BSA molecules adsorb by side-on (40Aring) a
end-on (140Aring) m to consider m
not
in phosphat buffe ged of
calculated values are a 2
104
Table 4-11 Absorbed BSA layer thickness (Aring) of BSA in phosphate buffer (PB) at 37oC pH PS PSPMMA100 PSPMMA90PAA10 PSPMMA75PAA25
32 21 17 44 133
48 37 27 35 81
74 17 16 5 9
Table 4-12 Absorbed BSA layer thickness (Aring) of BSA in phosphate buffered saline (PBS) at 37oC
pH PS PSPMMA100 PSPMMA90PAA10 PSPMMA75PAA25
32 32 21 64 132
48 35 29
74 26 20 5 7
28 83
1n
1n
neqm
So k is closely related to the initial slope of the isotherm showing the affinity
between BSA and latex particles [Yoon et al 1996] A key point in the characterization
433 Gibbs Free Energy of Protein Adsorption
In the Langmuir isotherm model k represents the ratio of adsorption to the
desorption rate constants This definition is not applicable to the Langmuir-Freundlich
isotherm model but its meaning is similar [Yoon et al 1996] In this sense k is closely
related to the affinity between protein molecules and latex particles When the
equilibrium concentration Ceq reaches zero the value of Ceq will be very small (1gtgt
kCeq ) and then the equation 4-6 becomes
(4-8)
of adsorption processes on solid-liquid interfaces is to determine the change in Gibbsrsquos
free energy (∆Gdeg) one of the corresponding parameters for the thermodynamic driving
forces during adsorption The traditional method to evaluate adsorption thermodynamic
kCqq 1=
105
constants is based on measurement of adsorption isotherms under static conditions
[Malm ten 1998] The Langmuir-Freundlich ads lf
easily found from Then this enables calculation of the free-
energy change ∆G during adsorption expressed as
(4-9)
where R is the general gas constant (R = 8314Jmiddotmol K ) and T is the absolute
tem ture Gibb e energy A P
listed in Table 4-13 and 4-14 respectively As the ∆Gdegads ore negativ e
pro adsorption on a solid substra ill be more favorable
e of BSA adsorption in phosphate o
pH PS PSPMMA100 PSPMMA90PAA10 PSPMMA75PAA25
s orption equilibrium constant k can be
a slope of the isotherm
degads
lfads kRTG lnminus=∆ o
-1 -1
pera s fre change of BS adsorption values in B and PBS are
values are m e th
tein te w
Table 4-13 The values of Gibbs free energy changbuffer (PB) at 37 C
32 -2411 -2726 -2846 -2805
48 -2352 -2383 -2198 -1972 ∆Go
80
ads (kJmol)
74 -2452 -888 -324 -2
Table 4-14 The values of Gibbs free energy change of BSA adsorption in phosphate
pH PS PSPMMAbuffered saline (PBS) at 37oC
100 PSPMMA90PAA10 PSPMMA75PAA25
32 -2315 -2620 -2312 -2683
48 -2149 -2299 -2090 -2138 ∆Go
(kJmads ol)
74 -2430 -1992 -258 -263
Table 4-15 shows the calculated values of Gibbs free energy change of β M
adsorption on synthesized latex particles PSPMMA PAA core shell latex particle has
2
75 25
106
largest negative Gibbs free energy of β2
2
M adsorption indicating largest affinity betw
β
een
2M adsorption in phosphate buffere (PB) at 37 C and pH74
PS PSPMMA100
M and latex particle
Table 4-15 The values of Gibbs free energy change of βo
PSPMMA90PAA10 PSPMMA75PAA25
∆Go03 ads
(kJmol) -3626 -3466 -3659 -39
The Gibbs free energy of β2M was much more negative in phosphate buffer (PB)
than that of BSA in physiological condition (37 C and pH 74) o from the Table 4-13 and
the Figure 4-31 This indicates that β2M molecules more readily adsorb on latex particles
BSA This can be explained by the rate of diffusion and accessibility of β2M molecules to
the latex particle ier and faster for β2M m an BSA because of
size (molec ight) erical shape molecules It o be
due to the higher hydrophobic force and less electrostatic repulsion that contribute to the
adsorption of β2M molecules not as prevalent in the case of BSA because the IEP of β2M
(57) is clo H 74) than that of BSA (48) causing β2M to
be less electro cally lled p ro
can be seen that the ∆ β2M adsorption onto the synth article
increasing wi hy icity o x particles s the ∆Goads of the BSA
molecules is decreasing or constant This phenomenon supports the theory that the
select lizing
than BSA and that the affinity of β2M for the latex particle is much larger than that of
surface It is eas olecules th
the smaller ular we and sph of β2M may als
ser to physiological condition (p
stati repe from the latex articles than BSA F m Figure 4-33 it
Goads of esized latex p s is
th the drophil f the late wherea
ive adsorption of β2M molecules over BSA molecules can be enhanced by uti
more hydrophilic PSPMMA90PAA10 and PSPMMA75PAA25 core shell latex particles
107
-60
-50
1 2 3 4
-40∆Gad
s
-30
10
0
o (k
Jm
o-
-20l)
β2-Microglobulin (β2M)Bovine serum albumin (BSA)
Figure 4-33 Gibbs free energy of adsorption of proteins on latex particles in PB at 37
and pH 74
434 Kinetics of Adsorption
to
oC
Protein adsorption is a complex process known to have a large biological impact
and is currently not well understood quantitatively because of extreme sensitivity to pH
the concentration of other electrolytes and molecules present temperature and many
other factors that can change in the physiological system Accurate knowledge of the
adsorption kinetics under a given set of conditions is a prerequisite for elucidating the
mechanisms of many fundamental biological processes to be predicted at the molecular
level [Regner 1987] This adsorption kinetics approach is used in protein adsorption
studies because of the uncertainty related to the time needed for the interfacial proteins
reach the equilibrium It is generally accepted that the process of protein adsorption is
comprised of the following steps a) transport toward the interface b) attachment at the
interface c) eventual structural rearrangements in the adsorbed state d) detachment from
PS PSPMMA100 PSPMMA90PAA10PSPMMA75PAA25
108
the interface and transport away from the interface Each of these steps can affect the
overall rate of the adsorption process
The quantitative analysis of the protein adsorption kinetics requires that the protein
amount adsorbed is known as a function of time The kinetics tests were carried out in
phosphate buffer (PB) at 37oC and pH 74 a condition similar to physiological condition
The protein concentrations chosen were 07mgml for BSA and 006mgml for β2M
which are the maximal concentrations used in an adsorption isotherm test A time-based
evaluation of the adsorption was made by measuring the concentration of protein in
solution at different incubation times The results are shown below from Figure 4-34 to 4-
37 The adsorption kinetics of proteins onto th cond-e latex particles is expressed as a se
order model [Oumlzacar 2003] shown by the following equation
2)( tet qqk
dtdq
minus= (4-10)
Integrating above equation and applying the boundary conditions yields
ee
t tq
+=
1 (4-11)
where q
qkq
t
2
2
2
the previous adsorption isotherm results representing good reproducibility of adsorption
t and qe stand for the amount (mgm ) of protein adsorbed at time t and at
equilibrium respectively k is the equilibrium rate constant of second-order adsorption
(m mghr) and t is the incubation time (hr)
The fitting parameters calculated by using the equation 4-11 are listed in table 4-16
From this data it is seen that the amount of BSA adsorbed at equilibrium was higher than
that of β2M on PS and PSPMMA100 latex particles but less than that of β2M on
PSPMMA90PAA10 and PSPMMA75PAA25 latex particles These results are similar to
109
00
05
15
20
0 2 4 6 8 10 12 14
Time (hr)
rot
n ad
sorb
d (
10
25
Pei
em
gm
2
BSA2nd order model (BSA))β2M2nd order model (β2M)
Figure 4-34 The kinetics of protein adsorption in PB on PS latex particles at 37oC and
pH 74
00
05
10
15
20
25
30
0 2 4 6 8 10 12 14
Time (hr)
Prte
inds
oo
arb
ed (
mg
m2 )
BSA2nd order model (BSA)β2M2nd order model (β2M)
Figure 4-35 The kinetics of protein adsorption in PB on PSPMMA100 latex partic
37les at
oC and pH 74
110
00
03
06
09
12
15
0 2 4 6 8 10 12 14
Time (hr)
Prot
ein
adso
rbed
(m
gm
2 ) BSA2nd order model (BSA)β2M2nd order model (β2M)
Figure 4-36 The kinetics of protein adsorption in PB on PSPMMA90PAA10 latex
particles o at 37 C and pH 74
00
03
06
09
12
15
0 2 4 6 8 10 12 14
Time (hr)
BS
A ad
sorb
ed (
mg
m2 )
BSA2nd order model (BSA)β2M2nd order model (β2M)
Figure 4-37 The kinetics of protein adsorption in PB on PSPMMA75PAA25 latex
particles at 37oC and pH 74
111
test The variation of β2M equilibrium adsorption amount was nearly negligible for all
latex particles The plateau region is reached earlier for β2M than BSA on all latex
particles and the K values (equilibrium rate constant of second order adsorption) for the
β2M adsorption process onto latex particles are much larger than for BSA adsorption
These values indicate that the β2M molecules have a higher adsorption rate onto the latex
particles than the BSA molecules
Table 4-16 Fitting parameters of second-order kinetic model BSA β2M
Latex particles qe (mgm2) K (hr-1) qe (mgm2) K ( -1) hr
PS 144 67 074 7549
PSPMMA100 175 353 076 4438
PSPMMA90PAA10 046 97 075 5695
PSPMMA75PAA25 060 179 075 5168 K = (kqe) hr-1
There are several explanations for the phenomena observed in these experiments
First the smaller sizes of β2M molecules allow easier diffusion and a close approach to
the latex substrate Second a reduced number of hydrophobic domains on the latex
particle surface replaced by sulfate groups (-OSO3-) and ionized carboxylic groups
(COO-) are not favorable for larger BSA molecules Third the conformational change by
intra- and inter-molecular repulsion would be faster in β2M due to smaller molecular
weight Finally the ionic repulsion between β2M and the latex particles is less than that
condition (pH 74) than IEP (48) of BSA
between BSA and latex particles because IEP (57) of β2M is closer to the tested
112
44 Blood Compatibility
Blood compatibility can be defined as the property of a material or device tha
permits it to function in contact with blood without inducing any adverse reactions
Successful functioning of blood contacting biomedical materials or devices in contact
with blood strongly depends on the extent to which they damage blood during operation
High shear stresses turbulence rela
t
tively long contact times and cellular impact with
artificial surfaces are most common events experienced by blood in contact with artificial
1978 Winslow and et al
1993] Therefore before any materia e placed into the human b
biocompatibility testing must be the first performed in the view of safety The hemolysis
s a method for pr ary screening of biomaterials to determine
h blood a er circulating body fluid The tests estimat
oglobin released from th blood cell d e induced
foreign material
There are several types of blood cells in our body but the ones that make blood red
are erythrocytes or red blood cells (RBCs) Red blood cells (erythrocytes) are the most
numerous type in the blood These cells are average about 48-54 million per cubic
millimeter (mm3 same as a microliter (microl)) of blood but these values can vary over a
larger range depending on such factors as health and altitude RBCs are red because they
contain hemoglobin which is a protein that contains iron When blood circulates through
lungs oxygen binds to the hemoglobin and is then delivered to all the parts of body
RBCs are made in the bone marrow They start out as a nucleus like other cells but
when they fill up with hemoglobin the nucleus becomes smaller until it disappears
Without a nucleus RBCs are fragile and live only about 120 days In a normal healthy
devises and can cause hemolysis or thrombosis [Bernhard et al
ls can b ody
test serves a elimin the
biocompatibility wit nd oth e the
concentration of hem e red amag by a
113
body about 2 million red blood ne marrow produces new
ones j ing
dy
le
cells die per second But bo
ust as fast RBCs are about 75 micrometer across and are responsible for carry
oxygen salts and other organic substances to the cells and tissues throughout our bo
In humans and most mammals the cells are concave (indented in the middle) and flexib
enough to squeeze through even the tiniest of blood vessels the capillaries
Figure 4-38 Image of red blood cells
(Sourcehttpwwwpbrchawaiiedubemfmicroangelarbc1htm)
ch
100 90 10 75 25 f
RBCs are among the first cells that come into contact with a foreign material
injected in the blood Therefore a hemolysis assay gives information about the
biocompatibility in the case of an in vivo application The concentrations of prepared
latex particles used for the tests in this research are 05 25 and 50 (ww) and the
incubation time under moderate shaking was 30 minutes at 37oC Test results are shown
in Figure 4-39 The amount of blood cells that ruptured when in contact with the PS latex
particles was largely increased from about 30 to 91 as the solid content of PS
increased from 05 to 50 However hemolysis by the core shell latex particles su
as PSPMMA PSPMMA PAA and PSPMMA PAA was less than 35 O
114
particular interest was that hemolysis caused by PSPMMA PAA particles at 50
solid concentration was less than 02 hemolysis the lowest value among the latex
particles The low hemolysis value indicates that the PSPMMA PAA core shell latex
particles are the most blood compatible at all solid contents Other core shell particles
such as PSPMMA and PSPMMA PAA also had excellent blood comp
90 10
90 10
100 75 25 atibility with
low RBS breakage
01 2 3
20
40
60
80
100
Hem
olys
is (
)
052550
Figure 4-39 He
PS PSPMMA100 PSPMMA PAA90 10
molysis caused by latex particles n=5
4PSPMMA75PAA25
CHAPTER 5 SUMMARY OF RESULTS CONCLUSIONS AND RECOMMENDATION FOR
FUTURE WORK
51 Summary of Results
The goal of this study was to synthesize monodisperse polymeric latex particles
with tailored properties and to investigate the fundamental interactions between the
synthesized latex particles and target proteins An understanding of the fundamental
mechanism of selective adsorption is strongly required in order to maximize the
separation performance of membranes made of these materials so that it may applicable
to hemodialysis therapy for end stage renal disease (ESRD) patients For the achievement
of above research goal several investigations have been performed Analysis of the
obtained data leads to several conclusions as discussed below
Monodisperse polystyrene seed latex particles were synthesized with the size
crosslinking agent to make the latex particles more hydrodynamically stable The
conversion of monomer to polymer was over 95 and seed latex particles are spherical in
shape and have smooth surface From these small seeds bigger latex particles as large as
800nm were synthesized using a semi-continuous seeded emulsion polymerization
process The particles with a mean diameter less than 500nm are very spherical in shape
and highly uniform in particle size distribution However as the particles grow larger
than 500nm in mean diameter they became non-spherical with an uneven surface This
irregularity of particle surface can be attributed to the non-homogeneous monomer
ranges between 126plusmn75 and 216plusmn53 nm Divinylbenzene (DVB) was used as a
115
116
swelling into the shell of a growing polymer which can be controlled by the factors such
as temperature agitation speed initiator feeding rate and surfactant amount required to
stabilize the colloidal system It was determ rfactant to monomer ratio
dec ic
acid (AA) monomers were intro rticle surfaces more
hydro
to
ting of a
orm
ration is seen between
3002
0 cm-
0cm-1
-1
03
ined that as the su
reased the mean particle diameter increased Methyl methacrylate (MMA) and acryl
duced to make the latex pa
philic than bare polystyrene particles The prepared core shell latex particles were
made of PMMA100 PMMA90PAA10 and PMMA75PAA25 PMMA100 is the PMMA
homopolymer shell on a PS core PMMA90PAA10 shell is a copolymer with a PMMA
PAA ratio of 90 to 10 by weight PMMA75PAA25 shell is a copolymer consis
PMMA to PAA ratio of 75 to 25 by weight The particle size of these core-shell
particles were prepared to be about 370nm in mean diameter
The successful synthesis of latex particles was confirmed using Fourier Transf
Infrared spectroscopy (FTIR) The C-H aromatic stretching vib
cm-1 and 3103 cm-1 for PS latex particles The C-H asymmetrical stretching and
symmetrical stretching vibration peaks of CH2 for all latex particles is seen at 290
1and 2850 cm-1 respectively There is a carbonyl (C=O) characteristic band at 173
wavenumber for PSPMMA core-shell latex particles The broad OH group peak at
3400 cm
100
can be seen in the spectroscopy of the particle with PMMAPAA shell
(PSPMMA PAA and PSPMMA PAA ) The peak intensity of OH group of
PSPMMA PAA was larger than that of PSPMMA PAA relatively due to more AA
monomer content in PSPMMA PAA than in PSPMMA PAA
In the zeta potential measurements polystyrene (PS) latex particles had negative
values between -291 mV and -599 mV in the phosphate buffer (PB) and between -2
90 10 75 25
75 25 90 10
75 25 90 10
117
mV and -278 mV in the phosphate buffered saline (PBS) at 25oC and pH 21-78 These
negative zeta potential values for PS latex particles are due to ionization of the sulfate
groups (-OSO3-) originated from the initiator Zeta potential values of the PSPMMA
core shell latex particles were also negative between -28 mV and -505 mV in PB and
between -143 mV and -186 mV in PBS at pH 21-78 Negative values are also
generated by dissociation of sulfate groups on a particle surface The zeta potential value
of PSPMMA PAA and PSPMMA PAA were of negative values between -36
and -678 mV in PB medium and -147 mV and -193 mV in PBS medium at 25
100
s
7 mV
MMA75PAA25 and
PSPM
otein
g the
l change of BSA at
each w
90 10 75 25
oC For
PSPMMA PAA core shell particles zeta potential values were between -291 mV and
-520 mV in PB media and between -115 mV and -210 mV in PBS media Sulfate
groups contributed to the negative zeta potential values in PSP
75 25
MA90PAA10 core shell particles Carboxylic groups from polyacrylic acid also
contribute to the negative surface charge for the latex particles at greater than pH 70
however the degree of their contribution was not detectable from the zeta potential
measurements The absolute zeta potential values of the synthesized latex particles in PB
were higher than that in PBS This is because of the high concentration of sodium
chloride electrolyte in PBS that compress electrical double layer thickness
The bicinchoninic acid (BCA) assay technique was used to determine the pr
adsorption and all isotherms were fitted to the Langmuir-Freundlich isotherms usin
nonlinear regression method The hydrophobic interaction between PS latex particle and
BSA protein was dominant for the adsorption process Conformationa
pH also affected the adsorption amount which was highest at pH 48 because of lo
ionic repulsion making compact conformation of BSA The next larger amount of BSA
118
adsorbed onto PS was at pH 32 There were two main forces affecting the amount of
adsorption at acidic pH values One is ionic attraction between negative latex particle a
positive protein The other is lateral repulsion between positive proteins The smallest
amount of BSA adsorption was at pH 74 where charge repulsion between negative
particles and proteins is present at this pH Lateral repulsion between proteins wa
dominant for protein adsorption even though there always exists the hydrophobic
interaction between solid latex particle and protein The charge repulsion was reduced by
the high concentration of electrolytes in the PBS medium The BSA adsorption process
on PSPMMA core shell particles was very similar to the case of PS latex particle where
hydrophobic interaction between latex particle and protein was dominant However t
adsorbed BSA amount was less than that of PS in PBS The adsorbed amount of BS
PSPMMA
nd
s
he
A on
an
ed
n The main
adsor
90PAA10 and PSPMMA75PAA25 core shell particles was higher at pH 32 th
pH 48 and 74 The adsorbed BSA amount became higher at pH 32 and pH 48 as the
hydrophilicity was increased by the higher PAA amount On the contrary the adsorb
amount of BSA was dramatically decreased at pH 74 Carboxylic acid groups as well as
the sulfate groups are distributed on the surface of PSPMMA90PAA10 and
PSPMMA75PAA25 core shell particles and largely affect the BSA adsorptio
ption interaction forces at pH 32 was generated by hydrogen bonding as well as
charge attraction between latex particle and protein even when the conformational change
and charge repulsion between proteins reduce the adsorption of protein at this pH The
adsorbed BSA amount was also increased as the amount of PAA increases However the
adsorption of BSA was minimal (less than 07 mgm2) at pH 74 where the carboxylic
groups are ionized leading to more enhancement of charge repulsion by ionization of the
119
carboxylic groups (COO-) Because of this the PSPMMA-PAA latex particles have a
much lower protein adsorption
There were steep initial slopes for adsorption isotherms of β
in
s
there
M
r
e
es
o
2M on latex particles
all isotherms indicating a high affinity type adsorption The complete plateau region
were not seen for all isotherms indicating that β2M proteins were not saturated and
are still available sites for β2M adsorption on latex particles The initial slope of β2
isotherms for PS was steeper than for the other latex particles PSPMMA100
PSPMMA90PAA10 and PSPMMA75PAA25
The layer thickness (δ) of BSA proteins on synthesized latex particles in phosphate
buffer (PB) was in the range of 27 - 81 Aring at 37 C and pH 48 Only the δ values at pH
48 the isoelectric point (IEP) of BSA are important because intra- and inter-molecular
electrostatic repulsion of the protein molecules is minimized at the IEP Compared to the
hydrodynamic dimensions of BSA 140 times 40 times 40 Aring these values of the adsorbed laye
thickness indicates that the BSA molecules adsorb by the side-on (40Aring) and end-on mod
(140Aring) Consideration of the multi-layering is unnecessary because the adsorbed
thickness of BSA do not exceed 140 Aring
The Gibbs free energy of β
o
3
2M was more negative in phosphate buffer (PB) than
that of BSA in physiological condition (37 C and pH 74) indicating that βo2M molecul
are more favorably adsorbed on the latex particles than BSA and that the affinity of β2M
was much larger than BSA This may be explained by the rate of diffusion and
accessibility of β2M molecules being easier and faster than that for BSA because of
smaller size (or weight) and spherical shape of β2M molecules It may also be possible t
explain that more hydrophobic forces (less electrostatic repulsion) contributes to
120
adsorption of β2M than to that of BSA because the IEP of β2M (57) is closer to
physiological condition (pH 74) than that of BSA (pH 48) The selective adsorption of
β2M m
in β2M
larger than in BSA adsorption indicating
that fa
an that between BSA and latex particles because
IEP (
ch as
Other
olecules over BSA molecules can be enhanced by developing more hydrophilic
PSPMMA90PAA25 and PSPMMA75PAA25 core shell latex particles
The adsorption kinetics of proteins on latex particles was expressed as a second-
order model The plateau region for β2M was reached earlier than BSA on all latex
particles and the K values (equilibrium rate constant of 2nd order adsorption)
adsorption process on latex particles were much
ster adsorption rate of β2M molecules than that of the BSA molecules There are
several explanations for these phenomena First the smaller size of β2M molecules can
more easily diffuse and approach to the latex substrate Second enhanced hydrophilic
domain on the latex particle surface by sulfate groups (SO4-) and ionized carboxylic
groups (COO-) is not favorable for larger BSA molecules but is for smaller β2M to be
anchored Third the conformational change by intra- and inter-molecular repulsion
would be faster in β2M due to lower molecular weight Finally the ionic repulsion
between β2M and latex particles is less th
57) of β2M is closer to the tested condition (pH 74) than IEP (48) of BSA
In the biocompatibility test the number of red blood cells (RBCs) ruptured by PS
latex particles ranged from 30 to 91 as the solid content of PS increased from 05 to
50 respectively However hemolysis of the other core shell latex particles su
PSPMMA100 PSPMMA90PAA10 and PSPMMA75PAA25 were less than 35 The
PSPMMA90PAA10 particles at all applied solid concentrations had less than 02
hemolysis and was the most blood compatible among the prepared latex particles
121
core shell particles such as PSPMMA100 and PSPMMA75PAA25 also had excelle
compatibility with low RBC breakage
52 Conclusions
The major conclusions from this study include
nt blood
bull microglobulin (β2M) over that of the bovine serum albumin (BSA) at pH 74
was achieved by controlling the PMMA and PAA ratio to optimize
e particles posses the most desirable requirements for use in a human dialysis
ns
icles
olecule
bull
Using engineered core shell latex particles selective adsorption of β2
physiological condition was demonstrated
bull The rate of adsorption of the β2M was also much higher than that of BSA This
hydrophobichydrophilic microdomain size
bull The core shell particles also exhibited excellent biocompatibility with blood Thes
membrane
53 Recommendation for Future Work
With the results and conclusions obtained through this study several suggestio
may be given for future work as follows
bull Competitive adsorption tests of whole blood proteins on synthesized latex partunder physiological condition
bull Preparation of latex particles with optimum hydrophobic hydrophilic microdomains to maximize selective adsorption and removal of toxin molecules
bull Membrane fabrication by suitable pore size design to discriminate a toxin mby size sieving as a function of particle layers
Evaluation of membrane performance such as flow rate in water and in simulated blood fluid (SBF)
bull Application for hemodialysis as well as other technologies such as a water remediation
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38
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140
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20 81-86
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Model filled polymers 5 synthesis of cross-linked monodisperse polymethacrylate beads Journal of Polymer Science Part A Polymer Chemistry 30 137-144
Korea on August 7 1968 He obtained a Bachelor of Science degree in Chemistry from
Keim d graduate school and earned a
resear n Korea Institute of Science and
Coati e
ia
Busin
BIOGRAPHICAL SKETCH
Sangyup Kim was born and raised in the small town of Sung-Joo Kyungbook
yung University (Daegu Korea) in 1993 He entere
Master of Science degree in Chemical engineering in 1995 He worked as a student
cher for 2 years at Textile Polymer Division i
Technology (KIST) in Seoul Korea Next he worked over a year at the Automobile
ng Division in DongJoo-PPG in Korea In August 2001 he began studying for th
degree of Doctor of Philosophy in materials science and engineering at the University of
Florida in Gainesville After getting his PhD he plans to work at the Digital Med
ess Division of Samsung Electronics in Korea starting in January 2006
141
322 Preparation of Seeded Latex Particles 48 323 Preparation of Core Shell Latex Particles 48 324 Purification of Synthesized Latex Particles48
33 Characterization49 331 Degree of Conversion49 332 Fourier Transform Infrared (FTIR) Spectroscopy49 333 Quasielastic Light Scattering (QELS) 50 334 Field Emission-Scanning Electron Microscopy (FE-SEM) 50 335 Zeta Potential Measurement 50 336 Protein Adsorption51 337 Blood Biocompatibility by Hemolysis Test 53
4 RESULTS AND DISCUSSION55
41 Polymerization of Latex Particles55 411 Polystyrene Seed Latex Particles55 412 The Growth of Polystyrene (PS) Seed Latex Particles61 413 Core Shell Latex Particles 71
42 Characterization of Latex Particles75 421 Fourier Transform Infrared Spectroscopy (FTIR)75 422 Zeta Potential Measurements 75
43 Protein Adsorption Study 85 431 Adsorption Isotherm87 432 Adsorbed Layer Thickness 103 433 Gibbs Free Energy of Protein Adsorption 104 434 Kinetics of Adsorption 107
44 Blood Compatibility 112
5 SUMMARY OF RESULTS CONCLUSION AND RECOMMENDATION FOR FUTURE WORK115
51 Summary of Results115 52 Conclusions121 53 Recommendation for Future Work121
LIST OF REFERENCES122
BIOGRAPHICAL SKETCH 141
vii
LIST OF TABLES
Table page 4-1 The polymerization recipe of polystyrene (PS) seed particles 57
4-2 Continuous addition emulsion polymerization recipe for growing polystyrene (PS) latex particles less than 500nm in size 63
4-3 Continuous addition emulsion polymerization recipe for growing polystyrene (PS) latex particles larger than 500nm in size64
4-4 The preparation recipe of PS core with various shell latex particles72
4-5 The isoelectric point (IEP) of common proteins 83
4-6 The equilibrium concentration values of BSA adsorption on polystyrene (PS) latex particles calculated the Langmuir-Freundlich isotherm model 93
4-7 The equilibrium concentration values (q ) of BSA adsorption on PSPMMA particles calculated the Langmuir-Freundlich isotherm model
m 10096
4-8 The equilibrium concentration values of BSA adsorption on PSPMMA PAA particles calculated the Langmuir-Freundlich isotherm model
90 1098
4-9 The equilibrium concentration values of BSA adsorption on PSPMMA PAA particles calculated the Langmuir-Freundlich isotherm model
75 25100
4-10 The equilibrium concentration values of β M adsorption calculated the Langmuir-Freundlich isotherm model
2102
4-11 Absorbed BSA layer thickness (Aring) of BSA in phosphate buffer (PB) at 37 Co 104
4-12 Absorbed BSA layer thickness (Aring) of BSA in phosphate buffered saline (PBS) at 37 Co 104
4-13 The values of Gibbs free energy change of BSA adsorption in phosphate buffer (PB) at 37 Co 105
4-14 The values of Gibbs free energy change of BSA adsorption in phosphate buffered saline (PBS) at 37 Co 105
viii
4-15 The values of Gibbs free energy change of β M adsorption in phosphate buffere (PB) at 37 C and pH 74
2o 106
4-16 Fitting parameters of second-order kinetic model 111
ix
LIST OF FIGURES
Figure page 1-2 Stereo drawing of the αndashcarbon backbone of β M2 2
2-1 An image of the location and cross section of a human kidney 5
2-2 A schematic draw of the hemodialysis route7
2-3 Cross-sectional SEM image view of the Polyflux S (polyamide + polyacrylethersulfone + polyvinypyrollidone) dialysis membranes 11
2-4 The chemical structure of polysulfone (PSf) 12
2-5 The chemical structure of polyamide (PA)13
2-6 Chemical structure of polyacrylonitrile (PAN) 14
2-7 Emulsion polymerization system24
2-8 L-α-amino acid 31
2-9 Ribbon diagram of human β M taken from the crystal structure of the protein bound to the heavy chain of the MHC class I complex (PDB 1DUZ)
236
2-10 Secondary structure of human serum albumin (HSA) with sub-domains 37
2-11 The relationship between pore size and particle size in body-centered cubic and closed packed arrays41
2-12 Schematic representation of the protein adsorption on the core shell latex particles at pH 7442
3-1 Chemical structure of main chemicals45
3-2 Experimental setup for semi-continuous emulsion polymerization 46
3-3 The particle preparation scheme in various types and size ranges of latex particles 47
3-4 Schematic of the procedure for a protein adsorption test 52
3-5 Separation of RBC from whole blood by centrifuge process53
x
3-6 Schematic of the procedure for hemolysis test 54
4-1 Schematic representation of semi-continuous seed latex particles preparation and growth56
4-2 Scanning Electron Micrograph (SEM) of polystyrene seed latex particles (A) PS (B) PS (C) PS (D) PSS259 S233 S207 S181 59
4-3 SEM of PS latex particles less than 500nm in size (A) 258nm (B) 320nm (C) 370nm (D) 410nm 65
4-4 SEM of PS latex particles larger than 500nm in size (A) 525nm (B) 585nm (C) 640nm (D) 790nm 68
4-5 Dependence of the particle size on the surfactant to monomer ratio70
4-6 Schematic of core shell latex particle structures 71
4-7 Scanning Electron Micrograph of latex particles (A) PS (B) PSPMMA (C) PSPMMA PAA (D) PSPMMA PAA
100
90 10 75 25 73
4-8 FTIR spectra of polymerized latex particles (A) bare polystyrene (PS) (B) PSPMMA (C) PSPMMA PAA (D) PSPMMA PAA100 90 10 75 25 76
4-9 Schematic representation of ion distribution near a positively charged surface 78
4-10 Schematic representation of zeta potential measurement79
4-11 Zeta potential of PS latex particles at 25 Co 80
4-12 Zeta potential of PSPMMA latex particles at 25 C100o 81
4-13 Zeta potential of PSPMMA PAA latex particles at 25 C90 10o 82
4-14 Zeta potential of PSPMMA PAA latex particles at 25 C75 25o 82
4-15 The decay of surface potential with distance from surface in various electrolyte concentrations (1) low (2) intermediate (3) high 84
4-16 High-affinity adsorption isotherm of typical flexible polymer on solid surface 86
4-17 Overall schematic representation of the protein adsorption on the synthesized latex particles86
4-18 The dependence of UV absorbance on BSA concentration (A) 10mgml (B) 06mgml (C) 02mgml87
4-19 Standard curve of net absorbance vs BSA sample concentration 88
xi
4-20 Fitted models for adsorption isotherm of bovine serum albumin (BSA) on polystyrene latex particles at 37 C in phosphate buffer mediao 89
4-21 Adsorption isotherm of BSA on polystyrene (PS) latex particles in phosphate buffer (PB) at 37 Co 90
4-22 Adsorption isotherm of BSA adsorption 37 C in phosphate buffered saline (PBS) on PS latex particles
o
91
4-23 Conformation of bovine serum albumin (BSA) on latex particles92
4-24 Adsorption isotherm of BSA 37 C in PB on PSPMMA core shell latex particles
o100
94
4-25 Adsorption isotherm of BSA at 37 C in PBS on PSPMMA core shell latex particles
o100
95
4-26 Adsorption isotherm of BSA at 37 C in PB on PSPMMA PAA core shell latex particles
o90 10
97
4-27 Adsorption isotherm of BSA at 37 C in PBS on PSPMMA PAA core shell latex particles
o90 10
98
4-28 Adsorption isotherm of BSA 37 C in PB on PSPMMA PAA latex particleso75 25 99
4-29 Adsorption isotherm of BSA 37 C in PBS on PSPMMA PAA core shell latex particles
o75 25
99
4-30 Adsorption isotherm of β M onto PS and PSPMMA latex particles in PB at 37 C and pH 74
2 100o 101
4-31 Adsorption isotherm of β M onto PS and PSPMMA PAA latex particles in PB at 37 C and pH 74
2 90 10o 101
4-32 Adsorption isotherm of β M onto PS and PSPMMA PAA latex particles in PB at 37 C and pH 74
2 75 25o 102
4-33 Gibbs free energy of adsorption of proteins on latex particles in PB at 37 C and pH 74
o
107
4-34 The kinetics of protein adsorption in PB on PS latex particles at 37 C and pH 74
o
109
4-35 The kinetics of protein adsorption in PB on PSPMMA latex particles at 37 C and pH 74
100o
109
4-36 The kinetics of protein adsorption in PB on PSPMMA PAA latex particles at 37 C and pH 74
90 10o 110
xii
4-37 The kinetics of protein adsorption in PB on PSPMMA PAA latex particles at 37 C and pH 74
75 25o 110
4-38 Image of red blood cells 113
4-39 Hemolysis caused by latex particles n=5114
xiii
Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy
SYNTHESIS AND ENGINEERING OF POLYMERIC LATEX PARTICLES FOR MEDICAL APPLICATIONS
By
Sangyup Kim
December 2005
Chair Hassan El-Shall Major Department Materials Science and Engineering
Latex particles with well-defined colloidal and surface characteristics have received
increasing attention due to their useful applications in many areas especially as solid
phase supports in numerous biological applications such as immunoassay DNA
diagnostic cell separation and drug delivery carrier Hemodialysis membrane using
these particles would be another potential application for the advanced separation
treatment for patients with end stage renal disease (ESRD) It is desirable to remove
middle molecular weight proteins with minimal removal of other proteins such as
albumin Thus it is necessary to understand the fundamental interactions between the
particles and blood proteins to maximize the performance of these membranes This
improvement will have significant economic and health impact
The objective of this study is to synthesize polymeric latex particles of specific
functionality to achieve the desired selective separation of target proteins from the human
blood Semi-continuous seed emulsion polymerization was used to prepare monodisperse
xiv
polystyrene seed particles ranging from 126plusmn75 to 216plusmn53 nm in size which are then
enlarged by about 800nm Surfactant amount played a key role in controlling the latex
particle size Negatively charged latex particles with a different hydrophobicity were
prepared by introduction of a sodium persulfate initiator and hydrophilic acrylic acid
monomer The prepared polymeric particles include bare polystyrene (PS) particles less
hydrophobic PS core and PMMA shell particles and more hydrophilic PS core and
PMMA-co-PAA shell latex particles with a 370nm mean diameter SEM light scattering
and zeta potential measurements were used to characterize particle size and surface
properties Adsorption isotherms of two proteins bovine serum albumin (BSA) and β2-
microglobulin (β2M) on latex particles were obtained as a function of pH and ionic
strength using the bicinchoninic acid (BCA) assay method The Langmuir-Freundlich
adsorption model was used to determine the adsorption amount of protein at equilibrium
The thickness of adsorbed BSA layer on latex particles was obtained in order to
investigate the adsorption orientation such as end-on or side-on mode Adsorption
kinetics experiments for both proteins and all latex particles were also performed The
adsorption kinetic constant determined from the Langmuir-Freundlich adsorption
isotherm model was used to calculate Gibbs free energy of adsorption to compare the
competitive adsorption of BSA and β2M Hemolysis tests were performed to investigate
the blood compatibility of synthesized latex particles PSPMMA90PAA10 and
PSPMMA75PAA25 core shell latex particles had desirable material properties with not
only a large amount and high rate of selective β2M adsorption over BSA but also high
blood compatibility showing less than 3 hemolysis
xv
CHAPTER 1 INTRODUCTION
End stage renal disease (ESRD) is a chronic condition in which kidney function is
impaired to the extent that the patientrsquos survival requires removal of toxins from the
blood by dialysis therapy or kidney transplantation The National Kidney Foundation
estimates that over 20 million Americans had chronic kidney disease in 2002 [NKF] The
number of people with ESRD is rapidly increasing in the United States with
approximately 96 295 incidents and 406 081 prevalent patients including 292 215 on
dialysis and 113 866 with a functioning graft in 2001 It is projected that there will be
more than 22 million ESRD patients by 2030 [USRDS 2003] The expenditure for the
ESRD treatment program had reached $228 billion 64 of the Medicare budget in
2001 Due in part to the limited availability of kidneys for transplantation hemodialysis
is the primary clinical treatment for the patients with ESRD
The central element of a hemodialysis instrument is the semipermeable membrane
that allows for selective transport of low molecular weight biological metabolites less
than 5000 Da such as urea and creatinine as well as excess water and electrolytes [Baker
2004] from the blood One limitation of current dialysis technologies is the inability to
efficiently remove middle molecular weight toxins such as β2-Microglobulin (β2M) and
interleukin 6 (IL-6)
β2M is a causative protein of dialysis-related amyloidosis (DRA) a disease arising
in patients with chronic kidney failure as a serious complication of long-term
hemodialysis treatment [Gejyo et al 1985] β2M deposition in tissue is the primary cause
1
2
of destructive arthritis and carpal tunnel syndrome [Vincent et al 1992 Drueke 2000]
The β2M structure is shown in Figure 1-2
Figure 1-2 Stereo drawing of the αndashcarbon backbone of β2M [Becker et al 1985]
Although attempts have been made to increase the efficiency of middle molecular
weight toxin removal by changes in the membrane pore size and the use of innovative
materials to adsorb these toxins [Samtleben et al 1996 Ronco et al 2001] removal
efficiency is not as high as those achieved by a normal healthy kidney Traditional
membranes have a number of processing and performance limitations [Westhuyzen et al
1992 Leypoldt et al 1998] such as a restricted choice of surface chemistries and limited
control of porosity The development of novel engineering membrane technology is
needed to remove middle molecule toxins
Polymeric latex particles have received increasing attention in medical application
areas especially as solid phase supports in biological applications [Piskin et al 1994]
Examples of these applications include immunoassay [Chen et al 2003 Radomske-
Galant et al 2003] DNA diagnostic [Elaїssari et al 1998] cell separation drug delivery
carrier [Luck et al 1998 Kurisawa et al 1995 Yang et al 2000] etc This is because of
3
the well-defined colloidal and surface characteristics of the particles By using a seed
emulsion polymerization method it is possible to synthesize monodisperse latex particles
with various particle size ranges and surface chemistry Functionalized core-shell latex
particles can be introduced by multi-step emulsion polymerization Core particles are
synthesized in the first stage of the polymerization and the functional monomer is added
in the second stage This is done without any emulsifier addition to prevent the
production of new homopolymer particles [Keusch et al 1973] The core-shell particles
are useful in a broad range of applications because of their improved physical and
chemical properties over their single-component counterparts [Lu et al 1996 Nelliappan
et al 1997] Through the development of a hemodialysis membrane using monodisperse
latex particles improvements in advanced separation treatment for patients with end
stage renal disease (ESRD) can be realized This requires the maximum removal of
middle molecular weight proteins with minimal removal of other beneficial proteins such
as albumin Thus an understanding of the fundamental interactions between the particles
and biopolymers is vital to maximize the performance of this membrane technology
The field of material science and biotechnology is based on fundamental chemistry
has developed over the past three decades into todayrsquos powerful discipline that enables
the development of advanced technical devices for pharmaceutical and biomedical
applications This novel and highly interdisciplinary field is closely associated with both
the physical and chemical properties of organic and inorganic particles [Niemeyer 2001]
Hemodialysis membrane using these particles would lead to improvements in the
advanced separation treatment for patients with end stage renal disease (ESRD) The
interdisciplinary nature of this approach enables a more complete understanding of the
4
phenomena of protein adsorption and the material properties necessary for selective
separation This innovative approach to membrane fabrication has the potential of making
inexpensive highly efficient membranes for both industrial and specialized separation
processes
The goal of this study is to prepare polymeric latex particles with tailored
properties to maximize separation of toxin molecules and to investigate the fundamental
interactions between the particles and molecules in the biological system in order to
optimize the performance of a membrane material for these applications
Polymeric latex particles were synthesized with specific functionality in an attempt
to achieve selective separation Seeded emulsion polymerization was used to synthesize
functionalized monodisperse latex particles in various sizes Negatively charged
hydrophobic polystyrene latex particles were synthesized by the same method Core shell
latex particles PSPMMA100 PSPMMA90PAA10 PSPMMA75PAA25 were also
synthesized to differentiate the degree of hydrophobicity of particles Scanning Electron
Micrograph (SEM) and light scattering measurements were used to characterize particle
size and shape and zeta potential measurements were conducted to measure the electrical
surface property of synthesized particles Adsorption isotherms of target proteins bovine
serum albumin (BSA) and β2M on latex particles were obtained as a function of pH
ionic strength and protein concentrations using the BCA assay method Adsorption
kinetics for both proteins on the latex particles were also measured Finally hemolysis
tests were run to determine the biocompatibility of polymer latex particles with human
blood This research will be described in more detail in chapters 3 and 4
CHAPTER 2 BACKGROUND AND LITERATURE SURVEY
21 The Significance of End Stage Renal Disease (ESRD)
The kidneys are responsible for removing excess fluid minerals and wastes from
the blood regulating electrolyte balance and blood pressure and the stimulation of red
blood cell production They also produce hormones such as erythropoietin (EPO) and
calcitriol that keep bones strong and blood healthy [Casadevall and Rossert 2005 Kurbel
et al 2003] EPO acts on the bone marrow to increase the production of red blood cell in
case of bleeding or moving to high altitudes Calcitriol mainly acts on both the cells of
the intestine to promote the absorption of calcium from food and also bone to mobilize
calcium from the bone to the blood When kidneys fail harmful waste builds up blood
pressure rises and body retains excess fluid The body also does not make enough red
blood cells When this happens treatment is needed to replace the function of failed
kidneys
Figure 2-1 An image of the location and cross section of a human kidney
5
6
The National Kidney Foundation estimates that over 20 million Americans had
chronic kidney disease in 2002 [NKF] Chronic renal disease is a gradual and progressive
loss of the function unlike acute renal disease where sudden reversible failure of kidney
function occurs Chronic renal failure usually takes place over a number of years as the
internal structures of the kidney are slowly damaged In the early stages there can be no
symptoms In fact progression may be so gradual that symptoms do not occur until
kidney function is less than one-tenth that of a normal health kidney If left untreated
chronic kidney disease may ultimately lead to kidney failure known as End Stage Renal
Disease (ESRD) ESRD is a rapidly growing heath-care problem in the United States In
2001 approximately 96295 incidents and 406081 prevalent patients were diagnosed
with ESRD including 292215 patients on dialysis and 113866 patients with a
functioning graft [USRDS 2003] The projected number of patients with ESRD is
expected to exceed 22 million patients by 2030 with much of this growth being driven by
the increasing prevalence of major contributing factors such as diabetes and high blood
pressure [USRDS 2003] A great extent of ESRD program cost and Medicare budget
have been spent in 2001 Due in part to a limited availability of kidneys for
transplantation hemodialysis (HD) is the primary method of treatment for ESRD and is
currently used for approximately 61 of US ESRD patients
22 Hemodialysis (HD) Treatment
HD removes toxins from the body by extracorporeal circulation of the blood
through a semipermeable membrane referred to as a dialyzer The toxins are removed
primarily by diffusion across the membrane to a dialysate solution which is circulated on
the opposite side of the membrane The cleaned blood is then returned to the blood
stream A surgically constructed vascular access connects the extracorporeal circuit to the
7
patientrsquos system Treatments are almost always performed three times per week in
specially equipped dialysis facilities for 3-4 hours per each treatment Figure 2-2 shows
the scheme of the hemodialysis route The critical element of a HD instrument is the
semipermeable membrane which allows for selective transport of low molecular weight
biological metabolites from blood
igure 2-2 A schematic draw of the hemodialysis route
ESRD patients who undergo dialysis therapy often experience several other
problems associated with the treatment such as anemia fatigue bone problems joint
problems itching sleep disorders and restless legs Anemia is common in patient with
kidney disease because the kidneys produce the hormone erythropoietin (EPO) which
stimulates the bone marrow to produce red blood cells Diseased kidneys often do not
produce enough EPO to stimulate the bone marrow to make a sufficient amount of red
blood cells This leads to bone disease referred to as renal osteodystrophy and causes
bones to become thin and weak or malformed and can affect both children and adults
Older patients and women who have gone through menopause are at greater risk for this
disease Uremic toxins which cannot be remove from the blood by the current dialyzer
membranes can lead to itching This problem can also be related to high levels of
F
8
parathyroid hormone (PTH) Dialysis patients can also experience day-night revers
is they have insomnia at night and sleep during the day This can be related to possible
nerve damage in the body and a chemical imbalance in the blood due to the excess toxin
Oxidative stress is a problem for patients on maintenance dialysis This problem is due to
an imbalance between pro- and antioxidant factors [Roselaar et al 1995 Cristol et al
1994] Oxidative stress affects oxidation of low-density lipoproteins which are the main
factor for atherogenesis [Huysmans et al 1998] and is also involved in the development
of malignancies and diabetes mellitus [Rice-Evans et al 1993] In order to reduce
antioxidant defense dialysis is needed to contribute to help stimulate free radical
production or eliminate antioxidants Dialysis-related amyloidosis (DRA) is also a
common and serious problem for people who have been on dialysis for more than 5
years DRA develops when proteins in the blood deposit on joints and tendons causi
pain stiffness and fluid in the joint as is the case with arthritis Normally the healthy
kidneys can filter out these proteins but dialysis filters are not as effective
23 Advances in Membrane Technology
al that
s
ng
Dialysis for blood of ESRD
Hemo filtration
s
ysis using a cellulose based membrane occurred
1913 John Abel [1990] from the Johns Hopkins Medical School described a method
purification is widely used in the treatment
dialysis (HD) techniques use a semi-permeable membrane to replace the
role of the kidney The membranes used in HD can be broadly classified into those based
on cellulose and those manufactured from synthetic copolymers These membranes come
in various shapes such as sheets tubular structures or hollow fiber arrangements The
hollow fiber type is the most popular and is incorporated into over 800 different device
in world wide [Ronco et al 2001]
The first attempt at blood dial
9
where sing
e
om
ysis was performed until the mid 1960 The basic
molec
rial in
een
benzyl)
was used for the membrane with
hepar
by the blood of a living animal may be submitted to dialysis outside the body u
a membrane based on cellulose and returned to the natural circulation without exposur
to air infection by microorganisms or any alteration that would necessarily be prejudicial
to life This same technique is still used to today however the device used has been
modified over the years as better membranes were developed and the anti-coagulant
heparin has become available
Cellulose membranes have been widely used for the treatment of renal failure fr
1928 when the first human dial
ular structure of cellulose is made of a long chain containing hydroxyl (OH)
groups The realization that such groups imparted undesirable qualities on the mate
respect to blood contact behavior was discovered in the early 1970s and since has b
the focus of development These modified cellulose membranes used the partial
substitution of benzyl groups to replace the proton of the hydroxyl groups in an attempt
to reduce their negative effect The result is a molecular mosaic of hydrophobic (
and hydrophilic (hydroxyl and cellulose) regions
Kolff [Van Noordwijk 2001] studied the rotating drum artificial kidney for patients
with acute renal failure in 1943 Cellophane tubing
in as the anticoagulant For the next 17 years hemodialysis therapy was performed
by this method but only for the patients with acute reversible renal failure Vascular
access required repeated surgical insertions of cannulas (slender tubes) into an artery and
vein and limited the number of treatments for a patient could receive in order to
minimize the amount of vascular damage
10
Initially the need for dialysis in patients with acute renal failure was determ
mainly by the development of signs and sym
ined
ptoms of uremia After dialysis some time
might
et al
ent improvement In 1960 the development of a shunt [Quinton et al
1960]
fone (PSf) polyamide (PA) and polyacrylonitrile (PAN) by phase inversion or
precip
lidone
e
elapse before uremic manifestations returned to warrant a sequential treatment of
dialysis Many patients with acute renal failure secondary to accidental or surgical
trauma were hypercatabolic but the interdialytic interval might be prolonged because of
anorexia or use of a low-protein diet However Teschan and his coworkers [Obrien
1959] showed that patient well-being and survival were improved by what they termed
prophylactic daily hemodiaysis or administration of the treatment before the patient
again became sick with uremia Their report in 1959 was the first description of daily
hemodialysis
Development of membrane accessories such as a shunt has also been an area of
focus for treatm
a flexible polytetrafluoroethylene (PTFE or Teflonreg) tubing made many more
hemodialysis treatments possible for chronic kidney failure patients PTFE has a non-
stick surface and is relative biocompatibility leading to minimized blood clotting in the
shunt
Synthetic membranes are prepared from engineered thermoplastics such as
polysul
itation of a blended mixture resulting in the formation of asymmetric and
anisotropic structures Figure 2-3 shows a fiber type of the Polyflux S membrane
consisting of poluamide (PA) polyacrylethersulfone (PAES) and polyvinylpyrro
(PVP) with the integral three-layer structure The skin layer on the inside fiber typ
membrane contacts blood and has a very high surface porosity and a narrow pore size
11
distribution This layer constitutes the discriminating barrier deciding the permeabili
and solute retention properties of the membrane The skin layer is supported by thick
sponge-type structure larger pores providing mechanical strength and very low
hydrodynamic resistance
ty
Figure 2-3 Cross-sectional SEM image view of the Polyflux S (polyamide +
polyacrylethersulfone + polyvinypyrollidone) dialysis membranes [Deppisch et al 1998]
n
[Malchesky 2004] because of its thermal stability mechanical strength and chemical
inertn ciated
ls
m
PSf is a widely used membrane material for the hemodialysis applicatio
ess According to a report from the National Surveillance of Dialysis-Asso
Disease (NSDAD) in the US over 70 of hemodialysis membranes were PSf based
[Bowry 2002] This is most likely because PSf has many advantages over other materia
This synthetic polymer is one of few materials that can withstand sterilization by stea
ethylene oxide and γ-radiation PSf membrane can be prepared by conventional
immersion precipitation methods into many different shapes including porous hollow
12
fiber or flat sheet hemodialysis membranes The material also has a high permeab
low molecular weight proteins and solute and high endotoxin retention The chemical
structure of PSf is shown in Figure 2-4
ility to
Figure 2-4 The chemical structure of polysulfone (PSf)
There is one major disadvantage to PSf The hydrophobic nature of the PSf causes
ment alternative pathway
leadin
s not
3
et al
in
O acrylate
serious complications through the activation of the comple
g to the adsorption of serum proteins onto the membranes [Singh et al 2003]
Anticoagulants are added during dialysis therapy to avoid blood clotting but this doe
completely eliminate the problem In order to overcome this disadvantage of the PSf
membrane various studies have been performed to change the materialrsquos surface
properties These investigations include hydrophilic polymer coating [Brink et al 199
Kim et al 1988 Higuchi et al 2003] layer grafting onto PSf membrane [Wavhal
2002 Song et al 2000 Pieracci et al 2002 Mok et al1994] and chemical reaction of
hydrophilic components onto the membrane surface [Higuchi et al 1990 1991 1993
Blanco et al 2001 Nabe et al 1997 Guiver et al 1993] Hydrophilic monomers 2-
hydroxy-ethylmethacrylate (HEMA) acrylic acid (AA) and methacrylic acid (MMA)
have also been grafted onto PSf membrane to increase flux and Bovine Serum Album
(BSA) retention [Ulbricht et al 1996] Hancock et al [2000] synthesized
polysulfonepoly(ethylene oxide) (PEO) block copolymers to improve the resistance to
platelet adhesion Kim et al [2003] also studied blending a sulfonated PE
diblock copolymer into PSf in order to reduce platelet adhesion and enhance
13
biocompatibility PEO is a commonly used biomaterial due to its excellent resistance
protein adsorption and inherent biocompatibility [Harris 1992] Kim et al [20
a self-transformable copolymer to enhance the hydrophilicity of an asymmetric PSf
membrane with an ultra-thin skin layer The polymer had an entrapped diblock
copolymer containing a hydrophilic block of poly (ethylene glycol) (PEG)-SO
to
05] studied
ate
lic graft
l)
e of thier
aromatic
orand
3 acryl
and a hydrophobic block of octadecylacrylate (OA) Molecular dynamic (MD)
simulations were performed as a function of copolymer density to optimize interfacial
structure information McMurry [2004] developed a strategy using an amphiphi
copolymer added to PSf membranes by introducing polysulfone-g-poly (ethylene glyco
When compared to unmodified PSf these graft copolymer and resulting blend
membranes are found to hold promise for biomedical device applications
Polyamide (PA) membranes have also been used for hemodialysis becaus
mechanical strength in both wet and dry conditions Polyamide consists of
aliphatic monomers with amide bonding (-CONH-) also known as a peptide bond
The basic amide bond in polyamide is shown in Figure 2-5 R1 and R2 can be either
aromatic or aliphatic linkage group
C N
H
O
~ R R1 2~C N
H
O
~ R R1 2~
Figure 2-5 The chemical structure of polyamide (PA)
Panichi and co-workers [1998] evaluated the biocompatibility of the PA membrane
ompatible technique due to the
use of a synthetic membrane with a sterile re-infusion fluid and the convective removal of
and concluded that PA hemofiltration was a highly bioc
14
the ac
e
e
is
n
is a semi-crystalline polymer and the mechanical properties
strong
2
CN
tivated anaphylatoxins and β2-Microglobulin (β2M) The PA based membrane
Polyfluxreg (manufactured by Gambro GmbH Germany) blended with polyamide
polyarylethersulfone and polyvinylpyrrolidone (PVP) was able to clean small molecules
such as urea dreatinine and phosphate as well as decrease β2M amount by 502
[Hoenich et al 2002] Due to the non-selectivity of the membrane removal of thes
unwanted materials also led to the undesirable loss of beneficial proteins during therapy
Meier et al [2000] evaluated different immune parameters using a modified cellulos
low-flux hemophan and synthetic high-flux PA membrane during a 1 year period in
chronic hemodialysis patients They found that the 1-year immunological evaluation of
hemodiaysis membrane biocompatibility was associated with changes in the pattern of
chronic T-cell actiovation
Polyacrylonitrile (PAN) is another commonly used membrane material because it
inherently hydrophilic and has been commercialized for ultrafiltration and microfiltratio
[Scharnagl et al 2001] PAN
ly depend on the crystalline structures The chemical structure of PAN is shown in
figure 2-6
CH CH
n2
CN
CH CH n
Chemical structure of polyacrylonitrile (PAN)
The addition of additives such as polyvinylpyrrolidone (PVP) as a pore forming
agent gives PAN membranes more flexible processing parameters and increased
e has been optimized through
copolymerization with many other vinyl monomers including glycidyl methacrylate
Figure 2-6
performance [Jung et al 2005] PAN membrane performanc
15
[Godj
acid
for
for
a non-
solub this
in
oid underneath the inner
surface of membrane decreased by increasing the amount of solvent DMSA in the
evargova et al 1999 Hicke et al 2002] N-vinylimidazole [Godjevargova et al
2000] hydroxyl ethyl methacrylate [Ray et al 1999 Bhat et al 2000] methacrylic
[Ray et al 1999] vinyl pyrrolidone [Ray et al 1999] acrylic acid [Trotta et al 2002]
acrylamide [Musale et al 1997] and vinylchloride [Broadhead et al 1998] These
monomers provide a reactive group for enzyme immobilization improved mechanical
strength solvent-resistance pervaporation permeation flux anti-fouling and bio-
compatibility Because of this PAN-based copolymer membranes have great potential
the treatment of hemodialysis in an artificial kidney This material can also be used
other applications like the treatment of wastewater the production of ultra-pure water
biocatalysis together with separation and methanol separation by pervaporation
Lin and his coworker [2004] studied the modification of PAN based dialyzer
membranes to increase the hemocompatibility by the immobilization of chitosan and
heparin conjugates on the surface of the PAN membrane When a foreign material is
exposed to blood plasma proteins are adsorbed clotting factors are activated and
le fibrin network or thrombus is formatted [Goosen et al 1980] The result of
research was that the biocompatible chitosan polymer and a blood anticoagulant hepar
prevented blood clotting They showed prolonged coagulation time reduced platelet
adsorption thrombus formation and protein adsorption
Nie et al [2004] studied PAN-based ultrafiltration hollow-fiber membranes
(UHFMs) In order to improve the membrane performance acrylonitrile (AN) was
copolymerized with other functional monomers such as maleic anhydride and α-allyl
glucoside They found that the number and size of macrov
16
intern erum
and
d a
rface The second step is the
transf after
yjak et
embrane
1978 Malchesky et al 1978] has been further developed to increase the efficiency of
al coagulant The water flux of the UHFMs also decreased while the bovine s
albumin rejection increased minutely Godjevargova et al [1992] modified the PAN
based membrane with hydroxylamine and diethylaminoethylmethacrylate to improve
membrane dialysis properties Formed functional groups like primary amine oxime
tertiary amine groups provided the membrane with more hydrophilic properties an
substantial increase in the permeability of the membranes
The wide use of filtration in practice is limited by membrane fouling Solute
molecules deposit on and in the membrane in the process of filtration causing dramatic
reduction in flux through the membrane Fouling occurs mostly in the filtration of
proteins Three kinetic steps are involved in the fouling of UF membranes according to
Nisson [1990] The first step is the transfer of solute to the su
er of solute into the membrane until it either finally adsorbs or passes through
a set of adsorption-desorption events The third step includes surface binding
accompanied by structural rearrangement in the adsorbed state [Ko et al 1993] Br
al [1998] studied the surface modification of a commercially available PAN membrane
to develop superior filtration properties with less fouling by proteins The PAN
membrane was immersed in excess NaOH solution to convert some of the surface nitrile
groups into carboxylic groups by the hydrolysis process This modified PAN m
was not so severely fouled in the Bovine Serum Albumin (BSA) filtration test The pore
size however decreased during the hydrolysis process leading to a significant reduction
in flux and made the membrane less productive in the ultrafiltration (UF) mode
24 Sorbent Technology
Over the last three decades sorbent technology [Castino et al 1976 Korshak et al
17
dialysis or replace it for the treatment of ESRD Sorbents remove solutes from solution
through specific or nonspecific adsorption depending on both the nature of the solute and
the sorbent Specific adsorption contains tailored ligands or antibodies with high
selectivity for target molecules een used in autoimmune
disord
and
e
al 1996] various resins [Ronco et al 2001]
album
e
as
Specific adsorbents have b
ers such as idiopathic thrombocytopenic purpura [Snyder et al 1992] and for the
removal of lipids in familial hyper cholesterolemia [Bosch et al 1999] Nonspecific
adsorbents such as charcoal and resins attract target molecules through various forces
including hydrophobic interactions ionic (or electrostatic) attraction hydrogen bonding
and van der Waals interactions
New dialysate with sorbents has become an accepted modification of dialysis
sorbent hemoperfusion is gaining ground as a valuable addition to dialysis especially as
new sorbents are developed [Winchester et al 2001] Hemoperfusion is defined as th
removal of toxins or metabolites from circulation by the passing of blood within a
suitable extracorpoteal circuit over semipermeable microcapsules containing adsorbents
such as activated charcoal [Samtleben et
in-conjugated agarose etc Novel adsorptive carbons with larger pore diameters
have been synthesized for potential clinical use [Mikhalovsky 1989] Newly recognized
uremic toxins [Dhondt et al 2000 Haag-Weber et al 2000] have resulted in several
investigations on alternatives to standard or high-flux hemodialysis to remove thes
molecules These methods include hemodiafilteration with [de Francisco et al 2000] or
without [Ward et al 2000 Takenaka et al 2001] dialysate regeneration using sorbents
well as hemoperfusion using such adsorbents as charcoal and resins
18
Kolarz et al [1989 1995] studied the hyper-crosslinked sorbent prepared from
styrene and divinylbenzene (DVB) for a hemoperfusion application They found that the
pore structure of a swelling sorbent was changed by additional crosslinking with α αrsquo-
dichloro-p-xylene in the presence of a tin chloride catalyst and in a di
chloroethane
soluti and
en
decyl groups that attract β2M through a hydrophobic interaction The
adsor
of
5]
d
penet
on They also realized that the hemocompatibility was useful for nemoperfusion
could be imparted to the sorbents by introducing sulfonyl groups at a concentration of
about 02mmolg
A special polymeric adsorbing material (BM-010 from Kaneka Japan) has be
investigated by another group [Furuyoshi et al 1991] for the selective removal of β2M
from the blood of dialysis partients The adsorbent consists of porous cellulose beads
modified with hexa
ption capacity of this material is 1mg of β2M per 1ml of adsorbent Using a
hemoperfusion cartridge containing 350ml of these cellulose beads in sequence with a
high-flux hemodialyzer several small clinical trials were performed During 4-5 hours
treatment about 210mg of β2M were removed thus reducing the concentration in the
blood by 60-70 of the initial level [Nakazawa et al 1993 Gejyo et al 1993 199
RenalTech developed a hemoperfusion device BetaSorbreg containing the hydrate
cross-linked polystyrene (PS) divinylbenzene (DVB) resin sorbents with a pore structure
designed to remove molecules between 4 and 30 kDa [Winchester et al 2002] In this
case solute molecules are separated according to their size based on their ability to
rate the porous network of the beaded sorbents The resin beads were prepared with
a blood compatible coating and confirmed to be biocompatible in vivo in animals
[Cowgill et al 2001]
19
25 Limitation of Current Hemodialysis Treatment
Hemodialysis is a widely used life-sustaining treatment for patients with ESRD
However it does not replace all of the complex functions of a normal healthy kidne
a result patients on dia
y As
lysis still suffer from a range of problems including infection
accelerated cardio ion anemia
chron
e
se middle
alysis
sessio
s
f
vascular disease high blood pressure chronic malnutrit
ic joint and back pain and a considerably shortened life span One significant
limitation of the current dialysis technology is the inability to efficiently remove larger
toxic molecules This is mainly because of the broad pore size distribution reducing th
selective removal of toxins and unsatisfied biocompatibility causing lots of
complications such as inflammation blood clotting calcification infection etc
Dialysis purifies the patients blood by efficiently removing small molecules like
salts urea and excess water However as toxic molecules increase in size their removal
rate by hemodialysis substantially declines Typically only 10 - 40 of the
molecular weight toxins (300-15000 Da) are removed from the blood during a di
n [Vanholder et al 1995] These toxins then reach an abnormally high level and
begin to damage the body One such toxin β2M causes destructive arthritis and carpal
tunnel syndrome by joining together like the links of chain to form a few very large
molecules and deposit damaging the surrounding tissues [Lonnemann et al 2002] This i
also a main cause of mortality for long-term dialysis patients Other middle molecule
toxins appear to inhibit the immune system and may play a significant role in the high
susceptibility to infections in dialysis patients Still others are believed to impair the
functioning of several other body systems such as the hematopoietic and other endocrine
systems This may contribute to accelerated cardiovascular disease the leading cause o
20
death among dialysis patients as well as clinical malnutrition which affects up to 50 of
this patient population
Over the last decade polymeric dialysis membranes have been developed to
increase the capacity for removing middle molecular weight toxins by changing the pore
size of dialyzer membranes and using new materials that adsorb these toxins for
impro se
styrene made during World War II in the United
States The Dow chemical compan anufacturer of polystyrene
includ
d of
on
rug
ntrol of
ved removal characteristics However removal efficiency is not as high as tho
achieved by a normal healthy kidney
26 Latex Particles
The first synthetic polymer synthesized using emulsion polymerization was a
rubber composed of 13-butadiene and
y has been a major m
ing latex which they used in paint formulations The theory of emulsion
polymerization in which a surfactant is used was established by Harkins [1948] and by
Smith and Ewart [1948] By 1956 the technology was complete including the metho
building larger diameter particles from smaller ones The product by this emulsi
polymerization is referred to as latex a colloidal dispersion of polymer particles in water
medium [Odian 1991] Latexes are currently undergoing extensive research and
development for a broad range of areas including adhesives inks paints coatings d
delivery systems medical assay kits gloves paper coatings floor polish films carpet
backing and foam mattresses to cosmetics The relatively well known and easy co
the emulsion process is one of main advantages for these applications Therefore
polymeric latex particle prepared by emulsion polymerization can be a candidate for the
medical applications because of the easy control of the particle size and morphology as
well as flexible surface chemistry to be required
21
261 The Components for Emulsion Polymerization
The main components for emulsion polymerization process are the monomer a
dispersing medium a surfactant and an initiator Available monomers are styrene
sing medium is usually water
which
ticles
n
ation To
monomer droplets but in micelles because the initiators are
insolu le
and
olution
butadiene methylmethacylate acryl acid etc The disper
will maintain a low solution viscosity provide good heat transfer and allow
transfer of the monomers from the monomer droplets into micelles and growing par
surrounded by surfactants respectively The surfactant (or emulsifier) has both
hydrophilic and hydrophobic segments Its main functions is to provide the nucleatio
sites for particles and aid in the colloidal stability of the growing particles Initiators are
water-soluble inorganic salts which dissociate into radicals to initiate polymeriz
control the molecular weight a chain transfer agent such as mercaptan may be present
262 Particle Nucleation
Free radicals are produced by dissociation of initiators at the rate on the order of
1013 radicals per milliliter per second in the water phase The location of the
polymerization is not in the
ble in the organic monomer droplets Such initiators are referred to as oil-insolub
initiators This is one of the big differences between emulsion polymerization
suspension polymerization where initiators are oil-soluble and the reaction occurs in the
monomer droplets Because the monomer droplets have a much smaller total surface area
they do not compete effectively with micelles to capture the radicals produced in s
It is in the micelles that the oil soluble monomer and water soluble initiator meet and is
favored as the reaction site because of the high monomer concentration compared to that
in the monomer droplets As polymerization proceeds the micelles grow by the addition
of monomer from the aqueous solution whose concentration is refilled by dissolution of
22
monomer from the monomer droplets There are three types of particles in the emulsion
system monomer droplets inactive micelles in which polymerization is not occurring
and active micelles in which polymerization is occurring referred to as growing polymer
particles
The mechanism of particle nucleation occurs by two simultaneous processes
micellar nucleation and homogeneous nucleation Micellar nucleation is the entry of
radicals ei
ther primary or oligomeric radicals formed by solution polymerization from
the aq ed
the
r
s
n
is coagulation with other particles rather than
polymerization of monomer The driving force for coagulation of precursor particles
ueous phase into the micelles In homogeneous nucleation solution-polymeriz
oligomeric radicals are becoming insoluble and precipitating onto themselves or onto
oligomers whose propagation has ended [Fitch et al 1969] The relative levels of micella
and homogeneous nucleation are variable with the water solubility of the monomer and
the surfactant concentration Homogeneous nucleation is favored for monomers with
higher water solubility and low surfactant concentration and micellar nucleation is
favored for monomers with low water solubility and high surfactant concentration It has
also been shown that homogeneous nucleation occurs in systems where the surfactant
concentration is below CMC [Roe 1968] A highly water insoluble monomer such a
styrene [Hansen et al 1979 Ugelstad et al 1979] has probably created by micellar
nucleation while a water soluble monomer such as vinyl acetate [Zollars 1979] has bee
formed by homogeneous nucleation
A third latex formation reaction mechanism has been proposed referred to as
coagulative nucleation In this reaction the major growth process for the first-formed
polymer particles (precursor particles)
23
sever les
yer
es
r a
l
the beginning of the polymerization As soon as the initiator is
gins with the formation and
growt
and
ved
added at one time during the later stages of the polymerization prior to complete
al nanometers in size is their relative instability compared to larger sized partic
The small size of a precursor particle with its high curvature of the electrical double la
permits the low surface charge density and high colloidal instability Once the particl
become large enough in size maintaining the high colloidal stability there is no longe
driving force for coagulation and further growth of particles takes place only by the
polymerization process
263 Types of Processes for Emulsion Polymerization
There are three types of production processes used in emulsion polymerization
batch semi-continuous (or semi-batch) and continuous In the batch type process al
components are added at
added and the temperature is increased polymerization be
h of latex particles at the same time There is no further process control possible
once the polymerization is started In the semi-continuous emulsion polymerization
process one or more components can be added continuously Various profiles of particle
nucleation and growth can be generated from different orders of component addition
during polymerization There are advantages to this process such as control of the
polymerization rate the particle number colloidal stability copolymer composition
particle morphology In the continuous process the emulsion polymerization components
are fed continuously into the reaction vessel while the product is simultaneously remo
at the same rate High production rate steady heat removal and uniform quality of
latexes are advantages of the continuous polymerization processes
Other available methods include the intermittent addition and shot addition of one
or more of the components In the shot addition process the additional components are
24
conversion of the main monomer This method has been used successfully to develo
water-soluble functional monomers such as sodium styrene sulfonat
p
e [Kim et al 1989]
264
lsion
Chemistry of Emulsion Polymerization
Emulsion polymerization is one type of free radical polymerization and can be
divided into three distinct stages initiation propagation and termination The emu
polymerization system is shown in Figure 2-7
Aqueous phaseAqueous phase
Micelle withmonomer
I Rbull
Emulsifier
Polymer particle swollenWith monomer
Monomerdroplet
I Rbull
Micelle withmonomer
I Rbull
Emulsifier
Polymer particle swollenWith monomer
MonomerdropletMonomerdroplet
I Rbull
Figure 2-7 Emulsion polymerization system [Radicals (Rmiddot) are created from initiators
(I) Monomer is transferred from larger monomer droplet into micelles by emulsifier Initiated polymer particle by radicals is keep growing until monomers are all consumed The reaction is performed in aqueous media]
In the initiation stage free radicals are created from an initiator by heat or an
ultraviolet radiation source The initiator with either peroxides groups (-O-O-) such as
odium persulfate or azo groups (-N=N-) such as azobisisobutyronitrile is commonly
used for em t
with the m er
chain ere
s
ulsion polymerization The primary free radicals created from initiator reac
onomer for initiation of polymerization In the propagation stage the polym
grows by monomer addition to the active center a free radical reactive site Th
are two possible modes of propagations head-to-head addition and head-to-tail addition
25
The head-to-tail mode is the predominant configuration of the polymer chain a result o
steric hindrance of the substitute bulky group In the termination stage polymer chain
growth is terminated by either coupling of two growing chains forming one polymer
molecule or transferring a hydrogen atom (dispropotionation) from one growing chain to
another forming two polymer molecules one having a saturated end group and the other
with an unsaturated end-group
265 Seed Emulsion Polymerization
Polymer latex particles have received increasing interest because of the versatili
of the many applications heterophase polymerization processes like emulsion dispersion
micro-emulsion seeded emulsion precipitation etc Especially to prepare well-defined
microspheres having monodispe
f
ty
rse and various particle sizes as well as surface group
ed particles prepared by emulsion polymerization
and e
s
hological design In
seede to control
n
functionalilties it is necessary to use se
nlarge them to a desired size in the further stages of reactions
Polymeric particles are required to have a uniform particle size in many
applications such as chromatography where they are used as a packing material
Morphological control of latex particle is also important for many practical application
[Schmidt 1972] Seed emulsion polymerization (or two-stage emulsion polymerization) is
a useful method to achieve both monodisperse particle size and morp
d emulsion polymerization [Gilbert 1995] preformed lsquoseedrsquo latex is used
the number of particles present in the final latex The advantage of seeded emulsio
polymerization is that the poorly reproducible process of particle nucleation can be
bypassed so that the number concentration of particles is constant and known Various
mechanisms have been proposed for the growth of latex particles [Ugelstad et al 1980
Okubo et al 1992] using this polymerization technique The initial seed particle
26
preparation step is well known and relatively easy to perform because at the relative
small particle sizes (02 to 05 micron) the particle growth process can be readily
controlled by the use of an emulsifier Enough emulsifier is used to prevent coagulation
but not enough to cause the formation of new particles As the particles are grown to
larger sizes in successive seeding steps it becomes increasingly difficult to mainta
stable uncoagulated emulsion without forming new particles and thereby destroying
monodisperisty of the latex Recently there have been several reports concerning t
reaction kinetics of seed emulsion polymerization and the development of latex
morphologies over the course of the reaction [Chern et al 1990 Delacal et al 1990 L
et al 1995 Lee 2000 2002]
266 Polystyrene Latex Particles
A number of papers have described the synthesis of the polystyrene latex particl
bearing various functional surface groups such as carboxyl [Lee et al 2000 Tun
2002 Reb et al 2000] hydroxyl [Tamai et al 1989] marcapto [Nilson 1989] epoxy
[Shimizu et al 2000 Luo et al
ly
in a
he
ee
es
cel et al
2004] acetal [Izquierdo et al 2004 Santos et al 1997]
ometyl [Izquierdo et al 2004 Park et al 2001
Sarob
h the
d
thymine [Dahman et al 2003] chlor
e et al 1998] amine [Counsin et al 1994 Ganachaud et al 1997 Ganachaud et al
1995 Miraballes-Martinez et al 2000 2001 Anna et al 2005] ester [Nagai et al 1999]
etc In order to produce these functionalized particles different methods for particle
preparation must be used The polymer emulsion with core-shell morphology of latex
particles is one of them This is a multistep emulsion polymerization process in whic
polystyrene ldquocorerdquo particle is synthesized in the first stage and the functional monomer is
added in the second stage of the polymerization without any emulsifier postfeeding to
prevent the production of new homopolymer particles thus forming the functionalize
27
polymer ldquoshellrdquo on the ldquocorerdquo particle [Keusch et al 1973] There are requirements to
limit secondary nucleation and encourage core-shell formation in seeded emulsion
polymerization including the addition of smaller seed particles at high solid content to
increase particle surface area low surfactant concentration to prevent formation of
micelles and the semi-continuous addition of monomer to create a starved-feed conditio
and keep the monomer concentration low There are some advantages [Hergeth et al
1989] of dispersions with polymeric core-shell particles First it is possible to modi
interfacial properties of polymer particles in the aqueous phase by the addition of only
very small amounts of a modifying agent during the last period of the reaction Thus
these core-shell particles are useful in a broad range of applications since they always
exhibit improved physical and chemical properties over their single-component
counterparts [Lu et al 1996 Nelliappan et al 1997] In this way the improvement of
surface properties of such dispersions is straightforward and inexpensive The other is
that polymers with a core-shell structure are perfect model systems for investigating th
material properties of polymer blends and composites because of their regular
distribution of one polymer inside a matrix polymer and because of the simple sp
geometry of the system
Their properties usually depend on the structures of latex particles Chen and his
coworkers [Chen et al 1991 1992 1993] reported the morphological development of
core shell latex particles of polystyrenepoly(methyl methacrylate) during
polymerization Before the research by Chen and his coworker Min et al [Min et al
1983] reported the morph
n
fy the
e
herical
ological development of core shell latex of polystyrene
(PS)polybuthyl acrylateat (PBA) by seeded emulsion polymerization as a function of the
28
additi
of
ity of graft
ing reagents Thus protein adsorption on polymeric solid surfaces has
st are microspheres defined as ldquofine
polym can be
n
r was
containing antigen urine serum etc The latex will become agglutinated and visibly
on method of PS They found that the percentage of grafting PS to the PBA was
greatest for the batch reaction and the PBA-PS core-shell particles with a high degree
grafting remained spherical upon aging test because of the emulsifying abil
copolymer
267 Various Applications of Latex Particles
Latex particles are applicable to a wide range of areas such as biomedical
applications [Piskin et al 1994] especially as the solid phase such as in immunoassays
[Chern et al 2003 Radomske-Galant et al 2003] DNA diagnostic drug delivery carriers
[Luck et al 1998 Kurisawa et al 1995 Yang et al 2000] blood cell separations and
column pack
become a center of attention Of particular intere
er particles having diameters in the range of 01 to several micronsrdquo which
used as functional tools by themselves or by coupling with biocompounds Singer and
Plotz [1956] firstly studied microsphere or latex agglutination test (LATrsquos) by using
monodisperse polystyrene (PS) and polyvinyltoluene polymer particles as the support o
which the biomolecules were going to adsorb The biomolecule adsorption howeve
limited by possible desorption of the adsorbed species or loss of specific activity of the
complex formed Since this work was published the latex particle applications for
immunoassay have been rapidly and widely studied and developed
Latex agglutination test or latex immunoassays start with tiny spherical latex
particles with a uniform diameter and similar surface properties The particles are coated
with antibodies (sensitized) through the hydrophobic interaction of portions of the protein
with the PS surface of the particles If sensitized particles are mixed with a sample
29
agglomerate Latex tests are inexpensive as compared with the other techniques [Ba
1988]
ngs
sed
tion Rheumatoid factor (RF) in different age subpopulations has also been
evalu
studied a
latex
Unipath [Percival 1996] has manufactured a range of immunoassay materials ba
on a chromatographic principle and deeply colored latex particles for use in the home and
clinical environments The latex particles which are already sensitized with a
monoclonal antibody can detect any antigens that bound to the surface of the latex
particles Some detectable pollutants include estrogen mimics which induce
abnormalities in the reproductive system of male fishes and lead to a total or partial male
feminiza
ated according to a patientrsquos clinical status by using a rapid slide latex agglutination
test for qualitative and semiquantitative measurement in human serum along with latex
immunoassay method [Onen et al 1998] Magalhaes and his coworkers [2004]
diagnostic method of contamination of male fishes by estrogen mimics using the
production of vitellogenin (VTG) as a biomarker This was based on a reverse
agglutination test developed with monoclonal antibodies specific to this biomarker
Premstaller and his coworkers [Premstaller et al 2000 2001] have prepared a porous
poly(styrene-divinylbenzene) (PS-DVB) polymer monolith to use for highly efficient
chromatographic separation of biomelecules such as proteins and nucleic acids They
used a porogen a mixture of tetrahydrofuran and decanol to fabricate a micropellicular
PS-DVB backbone Legido-Quigley and his colleagues [2004] have developed the
monolith column to obtain further chromatographic functionality to the column by
introducing chloromethylstyrene in place of styrene into the polymer mixture
30
Core shell type monodisperse polymer colloids have been synthesized by Sarobe
and Forcada [1998] with chloromethyl functionality in order to improve the biomolecu
adsorption through a two-step emulsion polymerization process They investigated the
functonalized particles by optimizing the experimental parameters of the functional
monomer including reaction temperature the amount and type of redox initiator sys
used the type of addition of the initiator system and the use of washing They c
le
tem
oncluded
that th
sis
serve as separation
chann
of
usually
along the polypeptide chain which contain positive or negative charges [Norde 1998]
e relation between the amount of iron sulfate and the persulfatebisulfite system
added should be controlled to obtain monodisperse particles and prevent the premature
coagulation of the polymer particles during the polymerization
A semi-continuous emulsion polymerization technique for latex particle synthe
was performed by McDonald and other researchers [Ramakrishnan et al 2004 Steve et
al 1999] They introduced a variety of particles sizes compositions morphologies and
surface modifications to fabricate latex composite membranes (LCMs) Arraying and
stabilizing latex particles on the surface of a microporous substrate form narrowly
distributed interstitial pores formed between the particles which
els They investigated the membrane performance using gas fluxes water
permeability and the retention characterization of dextran molecules From these tests
they concluded that the narrow discriminating layer made of the latex particles leads to a
highly efficient composite membrane
27 Proteins
Proteins are natural polyamides comprised of about 20 different α-amino acids
varying hydrophobicity [Norde 1998] Proteins are more or less amphiphilic and
highly surface active because of the number of amino acid residues in the side groups
31
Proteins are polymers of L-α-amino acids [Solomon and Fryhle 2000] The α refers to a
carbon with a primary amine a carboxylic acid a hydrogen and a variable side-chain
group designated as R Carbon atoms rent groups are asymmetric and can
exhib l
s
e
on
with four diffe
it two different spatial arrangements (L and D configurations) due to the tetrahedra
nature of the bonds The L refers to one of these two possible configurations Amino
acids of the D-configuration are not found in natural proteins and do not participate in
biological reactions Figure 2-8 shows the chiral carbon in 3-D as the L isomer Protein
consist of twenty different amino acids differentiated by their side-chain groups [Norde
1998] The side-chain groups have different chemical properties such as polarity charg
and size and influence the chemical properties of proteins as well as determine the
overall structure of the protein For instance the polar amino acids tend to be on the
outside of the protein when they interact with water and the nonpolar amino acids are
the inside forming a hydrophobic core
Figure 2-8 L-α-amino acid
The covalent linkage between two amino acids is known as a peptide bond A
peptide bond is formed when the amino group of one amino acid reacts with the carboxyl
group of another amino acid to form an amide bond through the elimination of water
This arrangement gives the protein chain a polarity such that one end will have a free
amino group called the N-terminus and the other end will have a free carboxyl group
called the C-terminus [Solomon and Fryhle 2000] Peptide bonds tend to be planar and
32
give the polypeptide backbone rigidity Rotation can still occur around both of the α-
carbon bonds resulting in a polypeptide backbone with different potential conformations
relative to the positions of the R groups Although many conformations are theoretically
possible interactions between the R-groups will limit the number of potential
nd to form a single functional conformation In other words
the conformation or shape of the protein is
α-helix
can interact with other
secondary structures within the sam
conformations and proteins te
due to the interactions of the chain side
groups with one another and with the polypeptide backbone The interactions can be
between amino acids that are close together as in a poly-peptide between groups that are
further apart as in amino acids or even on between groups on different polypeptides all
together These different types of interactions are often discussed in terms of primary
secondary tertiary and quaternary protein structure
The primary amino acid sequence and positions of disulfide bonds strongly
influence the overall structure of protein [Norde 1986] For example certain side-chains
will promote hydrogen-bonding between neighboring amino acids of the polypeptide
backbone resulting in secondary structures such as β-sheets or α-helices In the
conformation the peptide backbone takes on a spiral staircase shape that is stabilized by
H-bonds between carbonyl and amide groups of every fourth amino acid residue This
restricts the rotation of the bonds in the peptide backbone resulting in a rigid structure
Certain amino acids promote the formation of either α-helices or β-sheets due to the
nature of the side-chain groups Some side chain groups may prevent the formation of
secondary structures and result in a more flexible polypeptide backbone which is often
called the random coil conformation These secondary structures
e polypeptide to form motifs or domains (ie a
33
tertiary structure) A motif is a common combination of secondary structures and a
domain is a portion of a protein that folds independently Many proteins are composed o
multiple subunits and therefore exhibit quaternary structures
271 Interaction Forces between Proteins
Proteins in aqueous solution acquire compact ordered conformations In such a
compact conformation the movement along the polypeptide chain is severely restrict
implying a low conformational entropy The compact structure is possible only if
interactions within the protein molecule and interactions between the protein molecule
and its environment are sufficiently favorable to compensate for the low conformational
entropy [Malmsten 1998] Protein adsorption study is often focused on structural
rearrangements in the protein molecules because of its significance to the biological
functioning of the molecules and the important role such rearrangements play in the
mechanism of the adsorption process Knowledge of the majo
f
ed
r interaction forces that act
structures helps to understand the behavior
of pro
arge
a
ded
between protein chains and control the protein
teins at interface These forces include Coulomb interaction hydrogen bonds
hydrophobic interaction and van der Waals interactions
Coulombic interaction Most of the amino acid residues carrying electric ch
are located at the aqueous boundary of the protein molecule An the isoelectric point
(IEP) of the protein where the positive and negative charges are more or less evenly
distributed over the protein molecule intramolecular electrostatic attraction makes
compact structure favorable to proteins Deviation to either more positive or more
negative charge however leads to intramolecular repulsion and encourages an expan
structure Tanford [1967] calculated the electrostatic Gibbs energy for both a compact
impenetrable spherical molecule (protein) and a loose solvent-permeated spherical
34
molecule (protein) over which the charge is spread out From the results he found th
the repulsion force was reduced at higher ionic strength d
at
ue to the screening action of ion
ble
e
the
d the
tween
dehyd
e
ent
oles
tein
Hydrogen bond Most hydrogen bonds in proteins form between amide and
carbonyl groups of the polypeptide backbone [Malmsten 1998] The number of availa
hydrogen bonds involving peptide units is therefore far greater than that involving sid
chains Because α-helices and β-sheets are aligned more or less parallel to each other
interchain hydrogen bonds enforce each other Kresheck and Klotz [1969] examine
role of peptide-peptide hydrogen bonds and concluded that hydrogen bonds between
peptide units do not stabilize a compact structure of protein However because the
peptide chain is shielded from water due to other interactions hydrogen bonding be
peptide groups do stabilize α-helical and β-sheet structures
Hydrophobic interaction Hydrophobic interaction refers to the spontaneous
ration and subsequent aggregation of non-polar components in an aqueous
environment In aqueous solutions of proteins the various non-polar amino acid residues
will be found in the interior of the molecule thus shielded from water The
intermolecular hydrophobic interaction for the stability of a compact protein structure
was first recognized by Kauzmann [1959] If all the hydrophobic residues are buried in
the interior and all the hydrophilic residues are at the outermost border of the molecul
intramolecular hydrophobic interaction would cause a compact protein structure
However geometrical and other types of interactions generally cause a fraction of the
hydrophobic residues to be exposed to the aqueous environm
Van der Waals interaction The mutual interaction between ionic groups dip
and induced dipoles in a protein molecule cannot be established as long as the pro
35
structure is not known in great detail Moreover the surrounding aqueous medium also
contains dipoles and ions that compete for participation in the interactions w
the protein molecule Dispersion interactions favor a compact structure However
because the Hamaker constant for proteins is only a little larger than that of water the
resulting effect is relatively small [Nir 1977]
272 β
ith groups of
rgiles et al 1996
Floeg
) is degraded [Floege 2001] Renal
β2M from the serum resulting in an increase in β2M
conce well
the
)
a
al
2-Microglobulin (β2M)
The protein β2M is of particular interest because it is involved in the human
disorder dialysis-related amyloidosis (DRA) [Geiyo et al 1985 A
e 2001] DRA is a complication in end stage renal failure patients who have been on
dialysis for more than 5 years [Bardin et al 1986 Drueke 2000] DRA develops when
proteins in the blood deposit on joints and tendons causing pain stiffness and fluid in
the joints as is the case with arthritis In vivo β2M is present as the non-polymorphic
light chain of the class I major histocompatibility complex (MHC-I) As part of its
normal catabolic cycle β2M dissociates from the MHC-I complex and is transported in
the serum to the kidney where the majority (95
failure disrupts the clearance of
ntration by up to 60-fold [Floege 2001] By a mechanism that is currently not
understood β2M then self-associates into amyloid fibrils and typically accumulates in
musculoskeletal system [Homma et al 1989] Analysis of ex vivo material has shown
that the majority of amyloid fibrils in patients with Dialysis Related Amiloidosis (DRA
is present as of full-length wild-type β2M although significant amounts (~20-30) of
truncated or modified forms of the protein are also present [Floege 2001 Bellotti et al
1998] Figure 2-9 shows the ribbon diagram of human β2M Native β2M consists of
single chain of 100 amino acid residues and has a seven stranded β-sandwich fold typic
36
of the immunoglobulin superfamily [Saper et al 1991 Trinh et al 2002] β2M was fi
isolated from human urine and characterized by Berggard et al [1980] in1968 The
normal serum concentration of β
rst
re
f
e
2M is 10 to 25 mgL It is a small globular protein with
a molecular weight of 118 kDa a Strokes radius of 16Aring and a negative charge under
physiological conditions (isoelectric point IEP = 57) β2M contains two β-sheets that a
held together by a single disulphide bridge between the cysteines in positions 25 and 81
[Berggard et al 1980 Parker et al 1982 Cunningham et al 1973] β2M cannot be
removed completely by current dialysis techniques but through a better understanding o
the structure and interaction forces that lead to this structure it will be possible to mor
efficiently remove this problematic protein
Figure 2-9 Ribbon diagram of human β M taken from the crystal structure of the protein
bound to the heavy chain of the MHC class I complex (PDB 1DUZ) [Khal 2000]
273 Serum Albumin
Serum albumin is the most abundant protein found in plasma and is typically
present in the blood at a concentration of 35~
2an et
50gL According to extensive studies about
its physiological and pharmacological properties albumin has a high affinity to a very
wide range of materials such as electrolytes (Cu+2 Zn+2) fatty acids amino acids
metabolites and many drug compounds [Fehske et al 1981 Kraghhansen 1981 Putnam
37
1984 Peters 1985] The most important physiological role of the protein is therefore to
bring such solutes in the bloodstream to their target organs as well as to maintain the pH
and osmotic pressure of the plasma Bovine serum albumin (BSA) is an ellipsoidal
protein with the dimensions of 140 X 40 X 40Aring [Peter 1985] The primary structure is a
single helical polypeptide of 66 kDa (IEP = 47) with 585 residues containing 17 pairs of
as a soft an
rfaces [Kondo et al 1991 Norde et al 1992 Soderquist et al
1980 ikely
is
disulfide bridges and one free cysteine [Dugaiczyk et al 1982] BSA has been classified
d flexible protein because it has a great tendency to change its conformation
on adsorption to solid su
Carter and Ho 1994] and consists of three homologous domains (I-III) most l
derived through gene multiplication [Brown 1976] Each domain is composed of A and B
sub-domains [He et al 1992] The secondary structure of human serum albumin (HSA)
shown in Figure 2-10
Figure 2-10 Secondary structure of human serum albumin (HSA) with sub-domains
[Zunszain et al 2003]
HSA has the same structure domains with the serum albumin from other species
such as BSA [Brown 1976] Although all three domains of the albumin molecule have
similar three-dimensional structures their assembly is highly asymmetric [Sugio et al
38
1999] Domains I and II are almost perpendicular to each other to form a T-shaped
assembly in which the tail of subdomain IIA is attached to the interface region between
sub-domains IA and IB by hydrophobic interactions and hydrogen bonds In contrast
domain III protrudes from sub-domain IIB at a 45ordm angle to form the Y-shaped assembly
for domains II and III Domain III interacts only with sub-domain IIB These features
make the albumin molecule heart-shaped
28 Protein Adsorption
Protein adsorption studies date back to the 1930rsquos At the beginning these studies
mainly focused on the determination of the molecular weight electrophoretic and
ral
rearrangem
adsorption and biocompatibility of the sorbent materials were investigated [Norde 1986]
There are interaction forces at the interfaces between protein molecules and latex
particles These forces are mainly divided into the following groups hydrophobic
interaction ionic interaction hydrogen bonding and van der Waals interaction [Andrade
1985]
Hydrophobic interaction It is known that hydrophobic interaction has a major
role in protein adsorption phenomena The adsorption of proteins on the low charged
latex particles occurs by this interaction force Generally monomers such as styrene offer
hydrophobic surfaces th
by this interaction force is maximum at the isoelectric point (IEP) of the protein and the
strength [Suzawa et al 1980 1982 Shirahama et al 1989 Kondo et al 1992] By the
chromatographic applications Later the adsorption mechanism especially the structu
ents was studied Recently the studies of the relation between protein
281 Interaction between Protein Molecule and Latex Particle
at protein molecules adsorb to The amount of adsorbed protein
pH at maximum adsorption shifts to a more acidic region with an increase in ionic
39
reports [Suzawa et al 1980 1982 Lee et al 1988] protein adsorption was greater
hydrophobic surface than on a hydrophilic one implying that hydrophobic interaction is
on a
s
ydrophilic compound such as acrylic acid (AA) or methacrylic acid (MAA)
Ionic
ely
t
ces
n force is operative over small
distan e
ble
n
nt dialysis membrane therapy for end stage renal disease (ESRD)
one of most dominant forces in protein adsorption
Ionic interaction Negatively charged latex particles have ionic functional
groups on their surfaces such as salts of sulfonic and carboxylic acid Sulfate group
originate from an initiator such as sodium persulfate and carboxylic groups originate
from a h
bonds are formed between the negative charges of these latex particles and positive
surface charges of protein molecules The conventional low-charged latex particles rar
form these ionic bonds
Hydrogen bonding Hydrogen bond is a strong secondary interatomic bond tha
exists between a bound hydrogen atom (its unscreened proton) and the electrons of
adjacent atoms [Callister 1999] Protein can be adsorbed on hydrophilic polar surfa
through hydrogen bonding Hydrogen bonds are frequently formed between hydroxyl-
carbonyl or amide-hydroxyl Hydroxyl-hydroxyl or amide-hydroxyl bonds are also
formed in protein adsorption
Van der Waals interaction This interactio
ces only when water has been excluded and the two non-polar groups come clos
to each other Lewinrsquos calculation showed that the van der Waals interaction is negligi
compared with the forces involved in the entropy increases ie hydrophobic interactio
[Lewin 1974]
29 Hypothesis for Toxin Removal
As mentioned earlier insufficient removal of middle molecular range toxins is a
major drawback of curre
40
patien
is
y
ese
haracteristics over the last decade However removal
effici
ing a novel membrane design composed of an assembly of engineered
polymeric latex particles s ions Important factors
will i
e
ith a
is established it is
expec
e
ts This may cause destructive arthritis and carpal tunnel syndrome inhibit the
immune system and accelerate cardiovascular disease leading to death among dialys
patients In order to overcome these complications artificial dialysis membranes have
been developed to increase the capacity for removing middle molecular weight toxins b
changing the pore size of dialysis membranes and using new materials that adsorb th
toxins for improved removal c
ency is not as high as those achieved by a normal healthy kidney
With the knowledge obtained from all above literatures the following hypothesis
has been established for the development of a membrane for the successful removal of
the target protein β2-Microglobulin (β2M) without the removal of serum albumin This
will be done us
ynthesized to predetermined specificat
nclude pore size and surface chemistry
291 Toxin Removal by Size Sieving Based on Monodispersed Pore Size
The packing of monodispersed spherical particles can lead to the formation of
porous layers suitable for use as filters and membranes [Hsieh et al 1991] The pore siz
is defined as the largest spherical particles that can pass through the interstitial spaces
It is well known that monodispersed spherical particles can be obtained from the
seed emulsion polymerization method Many commercially available latex particles are
synthesized by this technique and are inexpensive When the defect free membrane w
monodisperse pore size distribution based on a particular particle array
ted to outperform the traditional hemodialysis membrane limitation which is a
broad range of pore size distribution When spherical particles pack in regular crystallin
arrays a number of packing geometries such as hexagonal closest packing cubic closest
41
packing and body-centered cubic packing are possible The pore size of the array
depends on these packing geometries as well as particle size Theoretical pore size can be
calculated from the simple geometry of the arr
ay Figure 2-11 shows the relationship
between particle size and pore size
0001
001
01
001 01 1 10
Por
e
1
10
microm
) S
ize
(
Particle Diameter (microm)
Body-Centered Cubic ArrayClosest Packed Array
0001
Siz
e (
Particle Diameter (microm)
001
01
001 01 1 10
Por
e
1
10
microm
)
Body-Centered Cubic ArrayClosest Packed ArrayBody-Centered Cubic ArrayClosest Packed Array
and closed packed arrays [Steve et al 1999]
McDonald and his coworkers [Ramakrishnan et al 2004 Steve et al 1999] have
described the fabrication of latex composite membranes and their performance in
previous papers It is from this prior research and knowledge gained from conversations
with Dr McDonald that this current research has been initiated [McDonald 2003] The
focus of this thesis is the extension of McDonaldrsquos research in the area of composite
membranes to medical application such as dialysis membranes
292 Toxin Removal by Selective Adsorption on Engineered Latex Particles
The flexibility in latex surface properties is a significant advantage to this
materialrsquos ability to separate proteins since the selectivity is strongly dependent on the
charge interactions of both the protein and the latex particles under the conditions of
Figure 2-11 The relationship between pore size and particle size in body-centered cubic
42
separation [Menon et al 1999 Chun et al 2002] First all sorbents were designed to
have negative surface properties because most plasma proteins in blood are negative and
should not be removed with charge interaction between proteins and sorbents at
physiological condition (pH74) It was reported that a negatively charged surface was
more blood compatible than a positive one [Srinivasan et al 1971] The toxin protein β2-
Microglobulin (β2M) can be selectively adsorbed on the surface of latex particles by the
design of a suitable hydrophobic portioned surface where only β2M protein can be
anchored with hydrophobic interactions Albumin adsorption on the latex particle is not
side-on m
adsorption of β M protein on the engineered latex particles
allowed because charge repulsion is more dominant than hydrophobic interaction with
ode The figure 2-12 shows the schematic representation of the selective
2
PMMAPAAShell
COO-
OSO3-
OSO3-
PSCore
COO-
-
-
-
-
-
-
Albumin-
Chargerepulsion
--
-
- -
- -
- --
Hydrophobic
β2-Microglobulin
2
Interaction
β -Microglobulin
PMMAPAAShell
COO-
-
-
-
-
-
-
--
-
-
-
-
-
Albumin-
Chargerepulsion
OSO3-
OSO3-
PSCore
COO-
--
-
- -
--
-
- -
- -
- --
- -
- --
Hydrophobic
β2-Microglobulin
2
particles at pH 74
Interaction
β -Microglobulin
Figure 2-12 Schematic representation of the protein adsorption on the core shell latex
43
Hydrophilichydrophobic microdomain structures were proven to be more blood
compatible [Mori et al 1982 Higuchi et al 1993 Deppisch et al 1998] The
optimization of suitable hydrophobic to hydrophilic ratio is also important for the
biocompatibility The monomers such as styrene (St) methyl methacrylate (MMA) and
acrylic acid (AA) are widely used hydrophobic and hydrophilic monomer models in
emulsion polymerization process
In summary the background and fundamental literature survey about the history
material properties and limitation of hemodialysis membrane latex particle preparation
surface chemistry and manufacturing process target proteins and protein adsorption and
finally the hypothesis for a toxin removal with high separation efficiency have been
suggested This research focuses on such hypothesis and the materials and
characterization methodology for achievement of suggested hypotheses are described in
the next chapter
CHAPTER 3 EXPERIMENTAL AND CHARACTERIZATION METHODOLOGY
As mentioned earlier the goal of this study is to prepare polymeric latex particles
with tailored properties to maximize separation of toxin molecules and to investigate
fundamental interactions between the applied particles and molecules in the
system to optimize the membrane performance for hemodialysis applications Polymeric
latex particles wit
the
biological
h monodisperse size distribution to obtain uniform pore size and
various size ranges to utilize membrane construction are necessary Surface engineering
with various combination of hydrophobichydrophilic domain on the surface of latex
particles is expected to affect the removal of target protein by selective adsorption and to
improve the biocompatibility of membrane Therefore the materials and characterization
methodology are addressed in this chapter
31 Materials
Styrene (St) monomer used for a seed and a core particle was purchased from
Fisher Scientific and used without any other purification process Acrylic acid (AA) and
methyl methacrylate (MMA) monomers introduced for shell formation were purchased
from Fisher Scientific and used without any other purification process Sodium persulfate
(SPS) and sodium bicarbonate (SBC) were obtained from Fishers Scientific and used as
received Divinylbenzene (DVB) crossliking agent was purchased from Aldrich The
anionic surfactant Aerosol regMA80-I [sodium di(13-dimethylbutyl) sulfosuccinate] was
kindly donated by Cytec 78-80 Aerosol regMA80-I is mixed with isopropanol and
water Its critical micelle concentration (CMC) is 245 mM (119) Ion exchange resin
44
45
AGreg 501-x8 Resin (20-50 mesh) was purchased from Bio-Rad Laboratories Inc It is a
mixed-bed resin with H+ and OH- as the ionic forms and is 8 crosslinked Bovine serum
albumin (BSA) (heat shock treated) was purchas Fisher Scientific The isoelectric
point of t lin
(β2M)
as
gma
ed from
he BSA is 47-49 and the molecular weight is 66300Da β2-Microglobu
was purchase from ICN Biomedicals Inc and was separated from patients with
chronic renal disease and lyophilized from ammonium bicarbonate The molecular
weight by SDS-PAGE was approximately 12000Da BSA and β2M proteins were used
received without further purification process In order to investigate the protein
adsorption evaluation bicinchoninic acid protein assay kits were purchased from Si
(Cat BCA-1) and Pierce Biotechnology (Cat 23225) The chemical structures for the
main chemicals are shown in Figure 3-1
CH2 CH
Styrene (Mw = 10415)
CH2 CHCH2 CH
Styrene (Mw = 10415)
CH2 CH
Acrylic acid (Mw = 7206)
C
OH
O
CH2 CH
Acrylic acid (Mw = 7206)
C
OH
O
CH CH2
C
OCH3
O
CH
CH CH3
2
C
OCH3
O
CH3
Methyl methacrylate (Mw = 10012)Methyl methacrylate (Mw = 10012)
CH2 CH
Divinylbenzene (Mw = 1302)
CH CH2
CH2 CH
Divinylbenzene (Mw = 1302)
CH CH2CH CH
+(Na) -(O3S)
CH -C-O-CH-CH -CH-CH3
O
2 2 3
O CH CH3
CH3 CH3
CH-C-O-CH-CH2-CH-CH3
Anionic surfactant (MA80-I) (Mw = 3885)
+(Na) -(O3S)
CH -C-O-CH-CH -CH-CH3
O
2 2 3
O CH CH3
CH3 CH3
CH-C-O-CH-CH2-CH-CH3
Anionic surfactant (MA80-I) (Mw = 3885)2
+(Na)-(O) S OO
O O
- +
Initiator sodium persulfate (Mw = 23811)
O SO
(O) (Na)+ -
O O
- +(Na) (O) S OO
O SO
(O) (Na)+ -
O O
- +
Initiator sodium persulfate (Mw = 23811) Figure 3-1 Chemical structure of main chemicals
(Na) (O) S OO
O SO
(O) (Na)
46
32 Latex Particle Preparation
The experimental set up and the particle preparation schemes for various types and
size ranges of latex particles are shown in Figure 3-2 and 3-3 In the experimental setup a
mechanical glass stirrer is connected from a motor and Teflon stirrer bar is located at
end of glass stirrer The agitation rate is precisely adjusted by controller motor Paar glas
reactor with 1L volume is partially in silicon oil bath on hot plate The reactor has fo
openings for a stirrer a thermometer a nitrogen gas inlet and a reactant feeding lnlet
The reactants for latex polymerization are fed by precisely controlled by meterin
The particle preparation is described in each latex preparation section
the
s
ur
g pump
Figure 3-2 Experimental setup for semi-continuous emulsion polymerization
1 Motor 2 Mechanical stirrer bar 3 Thermometer 4 Inlet of N2 5 Inlet for initiator and monomer 6 Glass reactor (l L) 7 Oil bath 8 Hot plate 9 Pump for initiator 10 Pump for monomer 11 Tubes for reactant feed
47
Styrene monomer
Bare PS
PSPMMA100
PSPMMA90PAA10
PSPMMA P75 AA25
Gr
win
ticle
size
PS seedparticles
og
par
Styrene monomer
Bare PS
PSPMMA100
PSPMMA90PAA10
PSPMMA P75 AA25
Gr
win
ticle
size
PS seedparticles
og
par
Figure 3-3 The particle preparation scheme in various types and size ranges of latex particles
321 Preparation of Seed Latex Particles
Polystyrene seed particles were synthesized through a typical semi-continuous
emulsion polymerization Polystyrene latex particles were prepared in a four necked Paar
glass vessel equipped with a mechanical glass stirrer thermometer glass funnel for
nitrogen gas purge and a tube for monomer feeding The reactor was placed in a silicon
oil bath on a hot plate for homogenous and stable tempe he vessel was
firstly charged with de-ionized water the emulsifier so itiator
sodium bicarbonate (SBC) styrene (St) monomer divinylbenzene (DVB) monomer
under nitrogen gas atmosphere and then heated to 72plusmn2 as
llowed to continue for another hour Second reactants St and DVB monomers SPS
initiator and SBC were fed separately using fine fluid metering pumps (Model RHSY
Fluid metering INC NY) under a nitrogen gas atmosphere for 2h After complete feeding
rature control T
dium persulfate (SPS) in
oC for 1h The reaction w
a
48
of all reactants the reaction was continued for another hour at elevated temperature 80plusmn1
oC and then cooled to room temperature by removal of the oil bath
322 Preparation of Seeded Latex Particles
The particle growth was achieved by seeded continuous emulsion polymerization
The vessel was charged with the polystyrene seeds and de-ionized water to make desired
solid content and heated up to 72plusmn2 oC for 1h under nitrogen gas atmosphere Then St
and DVB monomers and solution of SPS and SBC in de-ionized water were continuously
added using metering pump with a precisely controlled feed rate After feeding all
reactants the reaction was continued for three hours keeping temperature of 80plusmn2 oC and
then the reaction vessel was cooled down to room temperature
Monodisperse spherical polystyrene seeds with a 280nm mean diameter were used
e-polymerized polystyrene seeded emulsion
was charged in a 500m
323 Preparation of Core Shell Latex Particles
for core shell structured latex particle The pr
l glass flask under a nitrogen gas atmosphere De-ionized water
was added to achieve the desired solid content The emulsion was heated to 80plusmn2oC for
10 hour Monomers initiator and SBC to form the shell were added by fluid metering
pumps (LAB PUMP JRRHSY model Fluid metering Inc USA) with fine controlled
feed rate Flow rate can be adjusted by control ring graduated in 450 division from 0 to
100 flow which is in the range of 10mlmin respectively The reaction was sustained
for additional 30 hours at 90plusmn2oC then cooled down to room temperature
324 Purification of Synthesized Latex Particles
Synthesized latex particles were purified with an ion exchange resin 200g (10
ww polystyrene solid content) of emulsion were mixed with 4g of ion exchange resin
and stirred for 40 minute to remove unreacted monomers initiators and other impurities
49
Then the emulsion was diluted with DI water to a specific degree of solid content as
needed
33 Characterization
331
solid
termined
oscopy
d
hours
Dried latex (10mg) was mixed with
250 m on
Degree of Conversion
The degree of monomer to polymer conversion was determined gravimetrically
following the procedure described by Lee et al [1995] before the latex cleaning process
The synthesized emulsion raw materials were weighed and dried in a conventional oven
at 120oC for 30min to evaporate any unreacted monomers and water The remaining
was weighed and the degree of conversion was determined This process was repeated
three times and a mean value of the degree of conversion was de
332 Fourier Transform Infrared (FTIR) Spectr
A molecule can absorb only select frequencies (energy) of infrared radiation which
match the natural vibration frequencies The energy absorbed serves to increase the
amplitude of the vibrational motions of the bonds in the molecule [Pavin et al 1996]
Only those bonds which have a dipole moment that changes as a function of time can
absorb the infrared radiation The range of wavelengths for infrared radiation is between
25 microm (4000 cm-1) and 25 microm (400cm-1)
The synthesis of the latex particles such as polystyrene (PS) homopolymer and
PSPMMA PSPMMA90PAA10 PSPMMA75PAA25 core-shell copolymers were verifie
with FTIR Purified latex particles were dried in vacuum oven at 40oC for 24
before analysis in the FTIR (Nicolet Magma USA)
g of KBr which had also been dried in vacuum oven at 120oC for 3h Transmissi
spectra using the drift mode were plotted with 128 scans and 4cm-1 resolution
50
333 Quasielastic Light Scattering (QELS)
Latex particle size was measured by the Brookhaven ZetaPlus particle size
analyzer The solid concentration as 0001 (wv) This
ation spectroscopy (PCS) of quasielastically scattered light
For th s
ed using a FE-SEM (JEOL
JSM-
ith
ent
01
us zeta-
potential analyzer It measures the electrophoretic mobility velocity of charged colloidal
of latex particles used w
instrument uses photon correl
is technique time dependent interference patterns of light scattered from particle
in the sample cell are analyzed The interference pattern changes due to Brownian
motion giving rise to fluctuations in scattering intensity The frequency of these
fluctuations depends on the particlersquos diffusion constant that is inversely proportional to
the particle diameter
334 Field Emission-Scanning Electron Microscopy (FE-SEM)
Particle size and surface morphology were characteriz
6335F Japan) A diluted suspension of latex particles in water was dropped onto a
silicon wafer at room temperature and allowed to dry The sample was then coated w
the thinnest layer of carbon needed to obtain the required conductivity for this instrum
Secondary electron image mode was used with a 15KV of accelerating voltage The
magnification range used was between 10000X and 70000X
335 Zeta Potential Measurement
Synthesized and purified latex particles were diluted with de-ionized water to 0
wt and adjusted six different pH values 205 345 481 565 643 and 747 These
particle suspensions were then transferred to standard cuvettes for zeta potential
measurement At least two runs of ten measurements were taken for each sample and
averaged
The zeta potential measurement was carried out using Brookhaven ZetaPl
51
particles in solution and calculate zeta-potential by using the Smoluchowski equation
The f quency
gnitude
umin (BSA) as a standard model protein and β2-Microglobulin
uffer (PB) and
phosp
5
h the selected protein
e 005 01 03 05 and 07 mgml and those for
β2M
d at
teins in the supernatant after the centrifugation process using the
bicinc t
requency of laser light passing through the sample and is compared to the fre
of a reference beam The shift in the frequency called a Doppler shift and the ma
of the shift correspond to the polarity and the magnitude of the electrophoretic mobility
respectively The zeta potential is calculated from the solution conditions and the
measured mobility
336 Protein Adsorption
Protein adsorption experiments of the synthesized latex particles were performed
with bovine serum alb
(β2M) as a target protein in two types of buffer solution phosphate b
hate buffered saline (PBS) To make 5 mM of PB solution 0345g of sodium
phosphate monobasic (NaH2PO4H2O) was dissolve in 500ml of de-ionized water To
make PBS solution 0345g of sodium phosphate monobasic (NaH2PO4H2O) and 4178g
(143mM) of sodium chloride (NaCl) were dissolved in 500ml of de-ionized water The
synthesized latex particles were diluted with each buffer to have a solids content of 0
(ww) adjusted to pH values of 32 48 and 74 and mixed wit
BSA concentrations were chosen to b
were 0015 0030 0045 and 0060mgml The mixture was gently rotated using
the shaker Labquakereg (BarnsteadThermolyne Model 4002110 USA) with 8 RPM in
the incubator at 37oC for 12 hours before the latex-protein mixture was centrifuge
13000 rpm for 15min The amount of protein adsorbed was determined by quantifying
the free pro
honinic acid (BCA) assay method [Lowry et al 1951 Smith et al 1985 Baptista e
al 2003 Wiehelman et al 1988 Brown et al 1989] BCA assay kits consist of Reagent
52
A containing bicinchoninic acid sodium carbonate sodium tartrate and sodium
bicarbonate in 01N NaOH with pH=1125 and Reagent B containing 4 (wv) copper
(II) sulfate pentahydrate The BCA working reagent was prepared by mixing 50 parts of
Reagent A with 1 part of Reagent B 100microl of protein supernatant was then mixed with
2ml of BCA working reagent in a UV cuvette Incubation was allowed to continue
at room temperature
further
until color developed about 2h The absorbance at 562nm was
pectrophotometer (Perkin-Elmer Lambda 800 USA)
Unkn
Separate the p rticles in centrifuge mix supernatant with BCA reagents
measured using a UV-VIS s
own concentration of sample protein was determined by comparison to a standard
of known protein concentrations The adsorbed amount per unit surface area was
determined by the mass balance of the protein after adsorption process The simple
schematic of the procedure for a protein adsorption test is shown in Figure 3-4
Incubate mixture with agitation for 12hrs at 37oC
aand develop the color by incubation for 2hrs at room temperature
Suspend latex particles in aqueous buffer media
Add the dissolved target protein
Measure color intensity using UVVis spectroscopy at 562nm
Suspend latex particles in aqueous buffer media
Separate the p rticles in centrifuge mix supernatant with BCA reagents
Add the dissolved target protein
Incubate mixture with agitation for 12hrs at 37oC
aand develop the color by incubation for 2hrs at room temperature
Measure color intensity using UVVis spectroscopy at 562nm
Figure 3-4 Schematic of the procedure for a protein adsorption test
53
337 Blood Biocompatibility by Hemolysis Test
Hemolysis is the destruction of red blood cells which leads to the release of
hemoglobin from within the red blood cells into the blood plasma Hemolysis testing is
used to evaluate blood compatibility of the latex particles and the damaged to the red
blood cells can be determined by monitoring amount of hemoglobin released The red
blood cells (RBCs) were separated by centrifuging blood at 1500 rpm for 15 minutes
Figure 3-5 shows the phase separated blood with plasma white blood cells and red
cells after centrifugation process Separated RBSs were then washed with isotonic
phosphate buffer solution (PBS) at pH 74 to remove debris and serum protein Th
process was repeated 3 times
blood
is
Figure 3-5 Separation of RBC from whole blood by centrifuge process
Prepared latex particles were re-dispersed in PBS by sonification to obtain
homogeneously dispersed latex particles 100microl of the mixture of red blood cell (3 parts)
and PBS (11parts) was added to 1ml of 05 (ww) particle suspension PBS was used as
a negative control resulting 0 hemolysis and DI water used as a positive control to
produce 100 hemoglobin released by completely destroyed RBCs The mixture was
incubated in water bath with gentle shaking for 30 minutes at 37oC and then centrifuged
at 1500 rpm for 15 minutes 100microl of the supernatant was mixed with 2ml of the mixture
) (EtOHHCl = 2005 ww) to prevent the
White blood cells
Plasma
Red blood cells
of ethanol (99) and hydrochloric acid (37
54
precipitation of hemoglobin In order to remove remaining particles the mixture was
centri fuged again with 13000 RPM at room temperature The supernatant was then
transferred into the UV cuvette The amount of hemoglobin release was determined by
monitoring the UV absorbance at a wavelength of 397nm The schematic of the
procedure for hemolysis test is shown in Figure 3-6
Separate red blood cells (RBCs) from blood
Mix RBCs with phosphate buffer solution (PBS)
Wash and re-suspend RBC with PBS repeat three times
Add la l of RBC and incu in at 37oC
tex particle suspension into 100mbate sample with agitation for 30m
Separate particle ith EtOHHCl solutions and mix the supernatant w
Evaluate free hemoglobin in the solution using UVVis spectroscopy at 397nm
Separate red blood cells (RBCs) from bloodSeparate red blood cells (RBCs) from blood
Mix RBCs with phosphate buffer solution (PBS)Mix RBCs with phosphate buffer solution (PBS)
Wash and re-suspend RBC with PBS repeat three timesWash and re-suspend RBC with PBS repeat three times
Add latex particle suspension into 100ml of RBC and incubate sample with agitation for 30min at 37oC
Add latex particle suspension into 100ml of RBC and incubate sample with agitation for 30min at 37oC
Separate particles and mix the ith EtOHHCl solution supernatant wSeparate particles and mix the ith EtOHHCl solution supernatant w
Evaluate free hemoglobin in the solution using UVVis spectroscopy at 397nmEvaluate free hemoglobin in the solution using UVVis spectroscopy at 397nm
Figure 3-6 Schematic of the procedure for hemolysis test
In summary a description of the experimental materials and characterization
methodology has been explained In the next chapter the results and discussion of the
data obtained from these experiments is addressed
CHAPTER 4 RESULTS AND DISCUSSION
41 Polymerization of Latex Particles
411 Polystyrene Seed Latex Particles
Polymer particles have many different applications including spacer particles
lubricants packing materials for chromatography standard particles and diagnostic
drugs All applications strongly require these particles to have a uniform particle size
Uniform particle size is the first requirement for a latex composite membrane formed by
particle arrays with the interstitial spaces serving as pores for size discriminations [Jons
et al 1999 Ramakrishnan et al 2004] Although suspension polymerization has been
mainly used as one of conventional polymerization methods to prepare uniform particles
the uniformity of the recovered particles is insufficient for membrane use There is
another method known as seed emulsion polymerization where a vinyl monomer is
absorbed into fine monodiserse seed particles and polymerization causes the monomer to
crease the sizes of the seed particles uniformly In order to obtain monodisperse larger
particles by this m
particles and polymerization of the monomer is repeated
In this study cross-linked polystyrene seed latex particles were firstly synthesized
in the presence of styrene monomer divinylbenzene crosslink agent sodium persulfate
initiator sodium bicarbonate buffer and the Aerosol regMA80-I [sodium di(13-
dimethylbutyl) sulfosuccinate] surfactant using a batch type semi-continuous emulsion
polymerization process in which one or more components can be added continuously
in
ethod the procedure of absorption of monomer into fine polymer
55
56
Styrene (St) monomer and divinylbenzene (DVB) crosslink agent as well as initiator and
buffer are continuously added to enlarge the seed latex particles Various profiles of
particle nucleation and growth can be gene different orders of component
addition during polymerizati f this process such as easy
control of the polymeriza particle number
and particle morphology The main goal in
this process is to avoid secondary particle
rated from
on There are several advantages o
tion rate monodisperse particle size and
colloidal stability copolymer composition
formation leading to a monodisperse particle
size distribution Figure 4-1 shows the schematic representation of seed latex particle
preparation and growth
Monomer droplet
Seed particles Amount of surfactantsis variable
Enlarged final Add monomers
PS
PS
PSs4
particles Number of particlesare constant
Seed code
PSs1
s2
s3
Monomer droplet
Seed particles Amount of surfactantsis variable
Enlarged final Add monomers
PS
PS
PSs4
tion The
particles Number of particlesare constant
Seed code
PSs1
s2
s3
Figure 4-1 Schematic representation of semi-continuous seed latex particles preparation and growth
After the first synthesis of a small size of crosslinked polystyrene (PS) seed
particles stabilized by surfactants styrene DVB and initiator are continuously fed into
the system to enlarge the seed latex particles maintaining a narrow size distribu
recipes and reaction conditions for synthesizing these seed particles are summarized in
57
Table 4-1 The original recipe for latex particle preparation from McDonald was
modified for this emulsion system [McDonald 2003]
Table 4-1 The polymerization recipe of polystyrene (PS) seed particles Latex label PSS259 PSS233 PSS207 PSS181
Temperature (oC) 75 plusmn 5 75 plusmn 5 75 plusmn 5 75 plusmn 5
Divinylbenzene (g) 052 052 052 052
MA 80 (g) 259 233 207 181
NaHCO
Watera (g) 1315 1315 1315 1315
Styrene (g) 9948 9948 9948 9948
112 112 112 112
Na2S2O8 (g) 190 190 190 190
Waterb (g) 200 200 200 200
Reaction time (min) 140 140 140 140
Solids content 396 396 396 396
Mean particle diameter (nm) 126plusmn75 171plusmn39 182plusmn41 216plusmn53
Conversion () 965 979 957 971
3 (g)
Four different seed latex particles were synthesized based on surfactant amount in
order to determine the optimum condition for stable and monodisperse latex particles
The emulsion polymerization of latex particles has to be carried out in a narrow range of
surfactant causes flocculation or phase separation leading to broad particle
size distribution and an unstable emulsion sy
surfactant concentration where particles are stable [Nestor et al 2005] A wide range of
concentrations
stem Therefore the range of surfactant
amount was varied from 181g to 259g in increments of 026g in each latex label Other
reactants amounts were kept constant Watera is the initial charge amount and waterb
represents the amount of aqueous solution containing dissolved initiator and buffer
58
which are fed continuously after the seed particle creation The extent of monome
polymer conversion was obtained by gravimetric calcu
r to
lation and was more than 95 at
cripts such as
S207 and S181 i the surf mount added for the seed latex
nversion indi perce experi obtain
content divided by the theoretically estimated solid content and was calculated by the
all seed particles labeled PSS259 PSS233 PSS207 and PSS181 Labeled subs
S259 S233 ndicate actant a
preparation The co cates the ntage of mentally ed solid
following formula
100 colids () times=entsoliatedntentoalExperimentConversion
n the experimenta content is the weight of olid rema after
mulsion at 120oC f in and the estimated solid content is th ht
lated from the recipe
rti as di each t load d
mple of ea pension iluted by (ww)
deionized water and the mean particle diameter was determined using the Brookhaven
ZetaPlus particle size analyzer From these results the seed latex particle size increased
as the amount of surfactant decreased The amount of emulsifier also affects the
polymerization process by changing the number of micelles and their size It is known
that large amount of emulsifier produces larger numbers of smaller sized particles [Odian
1991] and was corroborated in the current work
These synthesized seed latex particles should be of narrow size distribution in order
to obtain monodisperse larger particles The SEM image showing the particle
morphology and size of a sample of polystyrene seed particles can be seen in the Figure
4-2
contdEstim (4-1)
In this equatio l solid the s ining
evaporation of e or 30m e weig
of monomers calcu
As expected the seed pa cle size w fferent for surfactan ing in see
emulsion system A sa ch sus was d 0001 with
59
Figure 4-2 Scanning Electron Micrograph (SEM) of polystyrene seed latex particles (A)
PSS259 (B) PSS233 (C) PSS207 (D) PSS181 (subscripts indicate the amount of surfactant added)
(A)
(B)
60
(C)
(D)
Figure 4-2 Continued
61
s seen in the image the seed particles are very smooth and spherical The
uniformity in the particle size distribution of these PS seed latex particles can also be
seen in the SEM image From this data we concluded that seed particles synthesized in
the presence of a surfactant with the chosen amounts are suitable for use to prepare larger
particles with high size uniformity and smooth spherical morphology
412 The Growth of Polystyrene (PS) Seed Latex Particles
Seeded emulsion polymerization has been conducted for several decades and
various mechanisms have been proposed Gracio et al [1970] suggested that the growing
polymer particles consist of an expanding polymer-rich core surrounded by a monomer-
rich shell with the outer shell providing a major locus of polymerization Seeded
mulsion polymerization is commonly used for preparing latex particles less than 1microm in
size [Cha et al 1995 Zou et al 1990 1992 Gandhi et al 1990 Park et al 1990] Such
latexes can be obtained from the growth of pre-prepared seed particles The seeds
introduced in this process serve as nucleation sites for particle growth By first swelling
the seed latex particles with additional monomer to the desired size polymerization can
then be initiated Several size ranges of monodisperse polystyrene (PS) latex particles
were prepared by this multistep seeded emulsion polymerization method For the
fabrication of particle-based membranes monodisperse latex particles with a variety of
size ranges are required
In this study seed particles from 171nm to 470nm in mean diameter and with a
highly uniform size distribution were prepared and used Watera is the initial charge
mount and waterb represents the amount aqueous solution containing dissolved initiator
and buffer that is continuously fed into system Feed time is precisely controlled by
metering pump The solid contents were maintained between 199 and 300 After
A
e
a
62
polym d as
es
ere 923 8402
n of these latex particles
are sh
ss
erization under these conditions the conversions of the latex particles labele
PS258 PS320 PS370 and PS410 were 952 957 946 and 964 respectively The recip
used in seeded emulsion polymerization to form the particles less than 500nm in mean
diameter are shown at the Table 4-2 The conversions of the latex particles larger than
500nm in mean diameter and labeled as PS525 PS585 PS640 and PS790 w
723 and 969 respectively The recipes used in the preparatio
own at the Table 4-3
A simple calculation can be used to predict the ultimate size that a latex particle can
reach after ending the polymerization Letrsquos suppose that seed density (ρs) and grown
particle density (ρgp) are the same The following equation is obtained from the ma
balance (m = ρV) where m and V are mass and volume of a particle respectively
gp
gp
s
s
Vm
Vm
In this equation m
= (4-2)
be
s and mgp are the weight of seed and total weight of seed and
added monomers respectively and Vs and Vgp are the volume of seed and grown
particle respectively
If we know the mass of monomer to be added the ultimate particle size can
calculated from the equation 4-3
3
2s D m ⎠⎝+ χ
where D
1s D m
⎟⎟⎞
⎜⎜⎛
= (4-3)
the
in
s and Dgp are the diameters for seed and grown particle respectively and χ is
weight of the monomer added Crosslinked PS latex particles with a size range from
258plusmn112 ~ 410plusmn117 in diameter polymerized by the recipes in Table 4-2 are shown
the SEM images in Figure 4-3
63
Table 4-2 Continuous addition emulsion polymerization recipe for growing polystyrene
Latex label PS
(PS) latex particles less than 500nm in size
258 PS320 PS370 PS410
Initial charge
Seed latex (g) 1000171 1000216 1000216 1000320
Watera (g) 1470 2500 1984 2600
06 07 06 06
Fee
Initiator stream
2 2 8
Feed time (min) 0-167 0-140 0-100 0-92
Solid content 284 300 300 200
Mean particle diameter (nm) 258plusmn112 320plusmn154 182plusmn71 410plusmn117
Conv
Monomer continuous
Styrene (g) 800 1200 800 600
Divinylbenzene (g)
d time (min) 0-163 0-110 0-75 0-82
Na S O (g) 07 065 05 05
NaHCO3 (g) 07 065 05 05
Waterb (g) 750 1200 200 370
ersion () 952 957 946 964 The solid content of seed latex for PS258 PS320 PS 370 and PS410 are 392 399 399 anrespectively
d 300
64
Table 4-3 Continuous addition emulsion polymerization recipe for growing polystyrene (PS) latex particles larger than 500nm in size
Latex label PS525 PS585 PS640 PS790
Initial charge
Seed latex (g) 1000390 1000470 1000470 1000320
Watera (g) 1000 520 1200 360
Monomer continuous
Styrene (g) 290 200 400 173
Divinylbenzene (g) 02 014
03 009
Feed time (min) 0-80 0-40 10-76 5-65
Initiator stream
Na2S2O8 (g) 02 014 03 012
NaHCO3 (g) 02 014 03 009
Waterb (g) 200 120 400 200
Feed time (min) 0-85 0-46 0-77 0-75
Solids content 199 200 200 201
Mean particle diameter (nm) 525plusmn151 585plusmn186 640plusmn178 790plusmn693
Conversion () 923 8402 723 969 The solid content of seed latex for PS525 PS585 PS 640 and PS790 are 200 182 193 and 173 respectively
65
Figure 4-3 SEM of PS latex particles less than 500nm in size (A) 258nm (B) 320nm (C)
370nm (D) 410nm
(A)
(B)
66
(C)
(D)
Figure 4-3 Continued
67
article size was well matched to that estimated by the geometrical calculation
From this it can be concluded that the added monomer was consumed for polymer
particle growth with a high degree of conversion From the SEM characterization newly
nucleated particles were not seen as indicated by the narrow particle size distribution
The particles less than 500nm in mean diameter were very spherical in shape with a
highly uniform particle size distribution As the particles were grown larger than 500nm
mean diameter however they became of non-spherical shape with an uneven surface
This irregularity of particle surface can be attributed to the non-homogeneous monomer
swelling into the shell of a growing polymer which can be controlled by the factors such
as temperature agitation speed initiator feeding rate and surfactant amount required to
stabilize the colloidal system The SEM image of latex particles larger than 500nm mean
iameter is shown in Figure 4-4 The particle size distribution is broader than that of the
s particles These particles can however still be used for membrane construction
as a support layer rather than skin layer The support layer does not necessarily have to be
as highly monodisperse as the skin layer which require pores of high uniformity for the
selective removal of toxins
As mentioned earlier the amount of surfactant as well as monomer in an emulsion
system is the primary determinant of the particle diameter [Odian 1991] Anionic
surfactants are generally recommended at the level of 02-30 wt based on the amount
of water [Odian 1991] The critical micelle concentration (CMC) of Aerosolreg MA 80-I
anionic surfactant is 119 The surfactant loading in this emulsion system ranged from
the 121 for SS181 to 171 for SS259
P
d
maller
68
Figure 4-4 SEM of PS latex particles larger than 500nm in size (A) 525nm (B) 585nm
(C) 640nm (D) 790nm
(A)
(B)
69
(C)
(D)
Figure 4-4 Continued
70
The surfactant to monomer ratio refers to the value of the surfactant amount
divided by monomer amount after polymerization ends The surfactant amount is the
weight of the surfactant contained in emulsion and the monomer amount is the total
weight of the monomer added for polymerization to form the ultimate polymer particles
in emulsion As the surfactant to monomer ratio decreases mean particle diameter
increases as described by Odian [1991] Generally lower surfactant concentration forms
fewer micelles resulting in larger particle size [Evans et al 1999] However there is a
plateau in particle mean diameter between a surfactant to monomer ratio of 0005 and
0013 This is due to low polymerization conversion of latex particles from monomer
leading to a smaller particle size than expected This is not a factor of surfactant amount
The dependence of the particle size on the amount of surfactant is shown in Figure 4-5
0
200
400
600
800
1000
0000 0005 0010 0015 0020 0025 0030
Surfactant to monomer ratio (ww)
Part
icle
mea
n di
amet
er (n
m)
Figure 4-5 Dependence of the particle size on the surfactant to monomer ratio
71
413
e
f bare
microdomain structures is important for biocompatibility [Mori et al 1982 Higuchi et al
1993 Deppisch et al 1998] Figure 4-6 shows the schematic of core shell latex particle
structure The recipe for core shell structures is shown in Table 4-4
Core Shell Latex Particles
The latex particles shown in Figure 4-3 (A) and labeled as S258 in Table 4-2 wer
used as seeds to prepare core-shell latex particles because of their high particle
uniformity and smooth surface morphology Methyl methacrylate (MMA) and acrylic
acid (AA) monomers were introduced to increase surface hydrophilicity over that o
polystyrene particles AA is a more hydrophilic monomer than MMA because of the
carboxyl acid group in AA is more favorable for hydrogen boding with water The
surface carboxyl groups on PAA may have many promising applications in biomedical
and biochemical fields [Kang et al 2005] MMA monomer is less hydrophobic than
styrene PMMA is known to more biocompatible than PS but still PMMA is hydrophobic
The optimization of suitable hydrophobic to hydrophilic ratios and control of
Polystyreneseed particle
Core shelllatex particles
PSPMMA100
PSPMMA90PAA10
PSPMMA75PAA25
Polystyreneseed particle
Core shelllatex particles
PSPMMA100
PSPMMA90PAA10
Figure 4-6 Schematic of core shell latex particle structures
PSPMMA75PAA25
72
Table 4-4 The preparation recipe of PS core with various shell latex particles Latex 25 label PSPMMA100 PSPMMA90PAA10 PSPMMA75PAA
Initial charge
PS seed latex (g) 500 500 500a
Monomer continuous
Methly methacylate (g) 320 288 240
Acrylic acid (g) 00 32 80
Initiator stream
Na
258 258 258
Water (g) 1570 1570 1570
Feed time (min) 0-120 0-50 0-60
b 200
Feed time (min) 0-122 0-60 0-65
952
2S2O8 (g) 02 02 02
NaHCO3 (g) 02 02 02
Water (g) 200 200
Solids content 183 183 183
Mean particle diameter (nm) 370plusmn186 370plusmn178 370plusmn191
Conversion () 955 924
Three types of core shell structures were prepared PMMA75PAA25 shell is a
copolymer consisting of the PMMA to PAA ratio of 75 to 25 by weight
PMMA90PAA10 shell is a copolymer with the PMMA to PAA ratio of 90 to 10 by
weight PMMA100 is the PMMA homopolymer shell on PS core The particle size of
these core-shell particles as well as PS latex particle was prepared to be about 370nm in
mean diameter and are characterized to determine their zeta potential protein adsorption
and biocompatibility The image of SEM of PS and core shell particles is shown at Figure
4-7 The synthesized latex particles had a high uniformity in size and a smooth spherical
surface morphology
73
Figure 4-7 Scanning
Electron Micrograph of latex particles (A) PS (B) PSPMMA100 (C) PSPMMA90PAA10 (D) PSPMMA75PAA25
(A)
(B)
74
(C)
(D)
Figure 4-7 Continued
75
42 Characterization of Latex Particles
421 Fourier Transform Infrared Spectroscopy (FTIR)
FTIR spectroscopy has proven to be a powerful tool to characterize polymeric
materials A molecule can absorb only select frequencies (energy) of infrared radiation
which match the natural vibration frequencies The energy absorbed serves to increase
the amplitude of the vibrational motions of the bonds in the molecule [Pavin et al 1996]
Only those bonds which have a dipole moment that changes as a function of time can
absorb infrared radiation The FTIR spectra of polymerized latex particles made during
this work are shown in Figure 4-8 There are number of characteristic peaks for PS latex
particles [Bhutto et al 2003 Li at al 2005] Aromatic C-H stretching vibration is shown
at 3002 cm-1 and 3103 cm-1 and aliphatic C-H asymmetrical and symmetrical stretching
shown at 2900 cm-1and 2850 cm-1 respectively There is an additional carbonyl (C=O)
adsorption at 1730cm-1 wavenumber for all core shell latex particles containing some
amount of either or both PMMA and PAA The broad OH group peak at 3400 cm-1
appear for particles containing some amount of PAA and has intensity dependent on the
amount of PAA in the shell For example the peak intensity of OH groups for
PSPMMA75PAA25 is greater than that of PSPMMA90PAA10 since there is a higher AA
monomer content in PSPMMA75PAA25 than in PSPMMA90PAA10 This OH peak is not
seen for PS and PS core PMMA shell particles because of the absence of OH groups on
PS and PMMA polymer chains
422 Zeta Potential Measurements
The electrical surface property was characterized by zeta potential measurements of
tex particles Zeta potential is an important and useful indicator of surface charge which
can be used to predict and control the stability of colloidal systems The greater the zeta
is
la
76
1520002500300035004000
Wavenumbers (cm-1)
li
Re
ativ
e
Tra
nsm
ttanc
e
Figure 4-8 FTIR spectra of polymerized latex particles (A) ba
PSPMMA100 (C) PSPMMA90PAA10 (D) PSPMM
(B)
(C) P
(D) P
C
C
OCH3
O
CH3
XCH2
CH2 n
CH
CH2 C
C
OCH3
O
XCH2 CH
OC
CH3
Y
OH
(A) Bare PS
500100000
re polystyrene (PS) (B) A75PAA25
PSPMMA100
SPMMA90PAA10
SPMMA75PAA25
77
potential the more likely the suspension is to be stable because the charged particles repel
one another and thus overcome the natural tendency to aggregate The measurement of
zeta potential is often the key ontrol of the interaction of proteins
with solid surfaces such as latex particles
Zeta potential (ζ) is the elec at exists at the shear plane of a particle
which is some small distance from the surface [Myers 1999] The zeta potential of the
particles can be determined by measuring the mobility of the particles in an applied
electric field termed electr its response to an alternating electric
field Colloidal particles dispersed in lu ically charged due to their ionic
characteristics and dipolar attributes The development of a net charge at the particle
surface affects the distribution of ions in the neighboring ng in
an increased concentration of counter ions to the surface Each particle dispersed in a
ns called fixed layer or Stern layer
Outside th compositions of ions of opposite polarities
form ble layer is form
particle-liquid interface This double layer may be considered to consist of two parts an
inner region which inclu nd relatively strongly to the surface and an outer or
diffuse region in which the ion distribution is determined by a balance of electrostatic
forces and random thermal motion The potential in this region therefore decays with the
distance from the surface until at a certain distance it becomes zero Figure 4-9 shows a
schematic representation of a typical ion distribution near a positively charged surface
The Stern surface (also referred to as the outer Helmholtz plane) is drawn through the
to understanding and c
trical potential th
ophoretic mobility or
a so tion are electr
interfacial region resulti
solution is surrounded by oppositely charged io
e Stern layer there are varying
ed in the region of the ing a cloud-like area Thus an electrical dou
des ions bou
center of those ions which are effectively adsorbed to the surface [Hunter 1981] The
78
extent of ion adsorption is determined by electrical and other long-range interactions
between the individual ions and surface of particles The ions outside of the Stern layer
form the diffuse double layer also referred to as the Gouy-Chapman layer [Burns and
Zydney 2000]
Shear plane
Stern layer
ψζ
0
ψs
ψShear plane
Stern layer
ψζ
0
ψs
ψ
t
controlled electric field is applied via electrodes immersed in a sample suspension and
Figure 4-9 Schematic representation of ion distribution near a positively charged surface
Zeta potential is a function of the surface charge of a particle any adsorbed layer a
the interface and the nature and composition of the surrounding medium in which the
particle is suspended The principle of determining zeta potential is very simple A
79
this causes the charged particles to move towards the electrode of opposite polarity
Viscous forces acting upon the moving particle tend to oppose this motion and the
equilibrium is rapidly established between the effects of the electrostatic attraction and
the viscosity dra
g The particle therefore reaches a constant terminal velocity
Figure 4-10 Schematic representation of zeta potential measurement(source
httpnitioncomenproductszeecom_shtm)
Because protein adsorption mechanisms are very complex and hard to explain in
biological systems protein adsorption is generally studied by using more ideal systems
consisting of one or more well characterized proteins a well-characterized adsorbent and
a well-defined aqueous solution Even so small changes in the experimental conditions
such as pH ionic strength and temperature generate totally different results Therefore
two media systems phosphate buffer (PB) and phosphate buffered saline (PBS) were
chosen to be used for the zeta potential measurements and protein adsorption study PB is
a simple aqueous system of 5 mM monobasic sodium phosphate (NaH2PO4H2O) to
n a constant pH PBS is used to keep the water concentration inside and outside of
the cell balanced If the water concentration is unbalanced the cell risks bursting or
shriveling up because of a phenomenon called osmosis PBS is a solution with the 5 mM
of monobasic sodium phosphate (NaH2PO4H2O) and 143 mM of sodium chloride (NaCl)
in water
maintai
80
The results of zeta potential measurement at room temperature (25plusmn1oC) as
function of pH and ionic strength (different media PB and PBS) for synthesized lat
particles are seen in Figure 4-11 Zeta potential values of polystyrene (PS) latex particle
at the Figure 4-9 are negative between -291 mV and -599 mV in PB and betwe
ex
s
en -203
mV and -278 mV in PBS at all pH ranges These negative zeta potential values of PS
latex particles are due to the sulfate groups [Vandenhu et al 1970] originated from
persulfate initiators attached via an oxygen bridge (-C-O-SO3-) at the end of polymer
chain where polymerization was initiated
-100
-80
-60
-40
-20
0
pH
Zta
pte
ntl
ζ
20
40
1 2 3 4 5 6 7 8 9
eo
ia (m
V)
PB (5mM)
PBS (5mM +143mM)
en -
Figure 4-11 Zeta potential of PS latex particles at 25oC
The zeta potential measurements of PSPMMA100 core shell latex particles as seen
in Figure 4-12 were also negative with a range of -28 and -505 mV in PB and betwe
143 mV and -186 mV in PBS at the pH 21-78 These negative values are also due to
sulfate groups on the particle surface
81
-100
-80
-60
-40
-20
0
20
40
1 2 3 4 5 6 7 8 9
Zea
pote
nti
l ζ
(mV
pH
ta
)
PB (5mM)
PBS (5mM + 143mM)
Figure 4-12 Zeta potential of PSPMMA100 latex particles at 25oC
The zeta potential for PSPMMA90PAA10 and PSPMMA75PAA25 was also
negative as seen in Figure 4-13 and 4-14 The zeta potential value for the
PSPMMA90PAA10 were between -367 mV and -678 mV in PB medium and between -
147 mV and -193 mV in PBS both at 25oC In case of the PSPMMA75PAA25 core shell
particles zeta potential values were between -291 mV and -520 mV in PB and between
-115 mV and -210 mV in PBS media Sulfate groups in PSPMMA75PAA25 and
PSPMMA90PAA10 core shell particles contribute to the negative zeta potential values
participate in forming a
negat
cid is expressed by the term pKa which is the pH
at which an acid is 50 dissociated However the contribution of the carboxylic acid
Carboxylic groups from acrylic acid monomer also would
ive surface charge of the latex particles at a pH greater than 70 because the pKa
value for the carboxylic acid is between 40 and 60 pH range [Ottewill et al 1967] This
indicates that carboxylic acid dissociation begins at a pH between pH 46-60 and
increases with pH The strength of an a
82
group to negative zeta potential values of PSPMMA-PAA core shell particles was not
detectable because there was no significant difference in zeta potential values for
synthesized latex particles at a pH greater than 70
-60
-40
-20
0
20
40
1 2 3 4 5 6 7 8 9
Zeta
pot
entia
l ζ
(mV
)
PB (5mM)PBS (5mM + 143mM)
-80
-100
pH
Figure 4-13 Zeta potential of PSPMMA90PAA10 latex particles at 25oC
-100
-80
-60
-40
-20
0
20
40
1 2 3 4 5 6 7 8 9
pH
Ze p
ont
i ζ
V)
tate
al (m
PB (5mM)PBS (5mM + 143mM)
Figure 4-14 Zeta potential of PSPMMA75PAA25 latex particles at 25oC
83
The zeta potential plots and values were similar for all latex particles This may be
due to the similar initiator density on the latex surface The initiator concentrations
monomers for shell polymerization were the same fo
to
r all core shell latex particles (062
ww from Table 4-4) A similar concentration (055 ww) of initiator to monomer was
added to prepare bare PS latex particles used in zeta potential measurements
Since the zeta potential values of all synthesized latex particles were negative
between pH=20 and pH=78 the isoelecric point (IEP) of these latex particles would be
less than pH=20 Sulfates have a pKa in the range of 10 to 20 [James 1985] indicating
that the sulfate group the conjugate base of a strong acid is protonated (-C-OSO3H) at
less than pH 20 This means that the synthesized latex particles are all negative at the
physiological pH Most serum proteins have a negative surface charge in blood
therefore when the negative latex particles are applied to blood stream fatal
tein is
avoided by charge repulsion in the blood Table 4-5 shows the common blood proteins
and their isoelectric points (IEP)
Table 4-5 The isoelectric point (IEP) of common proteins Serum proteins Isoelectric point (IEP)
complication by coagulation between applied latex particles and serum pro s
Albumin 48 α1-Globulin 20 α2-Microglobulin 54 γ1-Globulin 58 Heptoglobin 41 Hemoglobin 72 β1-lipoprotein 54 Immunoglobulin G (Ig G) 73 Fibrinogen 58 (source httpwwwfleshandbonescomreadingroompdf1085pdf
httpwwwmedalorgvisitorwww5CActive5Cch135Cch13015Cch130105aspx )
84
The absolute zeta potential values of the latex particles in phosphate buffer (PB)
was higher than that in phosphate buffered saline (PBS) This is because high
concentration of sodium and chloride electrolytes in PBS compressed the electrical
double layer causing rapid decay of surface potential Figure 4-15 shows that the
thickness of the electrical double layer is inversely proportional to the concentration of
electrolyte in the system
Surf
ace
Pote
ntia
l ψ
1κ3
10
1e
1κ 1κ
(1)(3) (2)
2 1 Z
Surf
ace
Pote
ntia
l ψ
1κ3
10
1e
1κ 1κ
(1)(3) (2)
Figure 4-15 The decay of surface potential with distance from surface in various
electrolyte concentrations (1) low (2) intermediate (3) high [Myers 1999]
2 1 Z
The electrical potential in the solution surrounding the particle surface falls off
the Debye-Huckel
yers 1999]
exponentially with distance (Z) from the surface according to
approximation [M
z) (- exp 0 κψψ = (4-4)
e reciprocal of the thickness of the electrical double layer referred to as the
The potential fallen off by a factor e The theoretical equation for the
ickness 1κ is
where κ is th
Debye length of 1
double layer th
21
22i
0
ekT 1
⎟⎟⎠
⎜⎜⎝
=sum ii zcεε
κ
⎞⎛ (4-5)
85
where
tration of
y was performed to measure the protein adsorption on the
synthesized latex particles Many physical and chemical processes occur at the boundary
between two phases and adsorption is one of the fundamental surface phenomena [Oscik
1982] This phenomenon is driven by the change in the concentration of the components
at the interface with respect to their concentrations in the bulk phase There are two
adsorption processes physical adsorption occurring when non-balanced physical forces
appear at the boundary of the phases and chemical adsorption or chemisorption
t the
interface [J
n leads
ational entropy hence adsorption takes place only if the
loss in conformational entropy is compensated
ter adsorption Cp The initial part of the
isotherm merges with the Γ-axis because at low polymer concentration the protein has a
high affinity for the surface and all of the polymer is adsorbed until the solid surface is
saturated
ε0 is the permittivity of a vacuum of free space ε is the permittivity of a medium e
is the relative permittivity or dielectric constant of the medium c is the concen
an ion (i) z is the valency of an ion (i) k is Boltzmannrsquos constant T is absolute
temperature (K)
43 Protein Adsorption Study
The adsorption stud
occurring when atoms and molecules from adjacent phases form chemical bonds a
aroniec et al 1988]
Flexible proteins in solution possess high conformational entropy as a result of the
various states each of the many segments in the protein chain can have Adsorptio
to a reduction of this conform
by sufficient attraction between polymer
segment and solid surface [Martin 1998] Isotherm shape of high affinity adsorption
between polymer and solid is shown in Figure 4-16 where the adsorbed mass Γ is plotted
against the polymer concentration in solution af
86
Figur
de
arboxyl
e 4-16 High-affinity adsorption isotherm of typical flexible polymer on solid
surface
In order to investigate the adsorption of blood proteins latex particle properties
such as electrical surface charge hydrophobicity and environmental conditions like pH
ionic strength and temperature are considered as experimental factors [Arai and Nor
1990] Figure 4-17 shows the overall schematic representation of the protein adsorption
on the synthesized latex particles with the functional groups such as sulfate and c
groups
CP
Γ
PMMAPAAShell
PS or PMMAShell OSO3
-
OSO3-
OSO3-
OSO3-
PS
-O3SO
PSCore
HOOC
-O3SO
O3SO
3
HOOC
HOOC
-
-O SO
Protein
NH3+
+
+H3N
+
COO-
Hydrophobic interaction
NH3
COO-
H3N
COO-
-OOC
Ionic interaction
Hydrophobic interaction
Hydrogen bonding
Ionic interaction
PMMAPAAShellOSO3
-
OSO3-
OSO3-
OSO3-
PS or PMMAShell -O3SOOSO3
-
PSCore
HOOC
-O3SO
O3SO
3
HOOC
HOOC
-
-O SO
-O3SO
OSO3-
OSO3-
OSO3-
PS PSCore
HOOC
-O3SO
O3SO
3
HOOC
HOOC
-
-O SO
Protein
NH3+
+
+H3N
+
COO-NH3
COO-
H3N
COO-
-OOC
NH3+
+
+H3N
+
COO-
Hydrophobic interaction
NH3
COO-
H3N
COO-
-OOC
Ionic interaction
Hydrophobic interaction
Hydrogen bonding
Figure 4-17 Overall schematic representation of the protein adsorption on the
Ionic interaction
synthesized latex particles
The surface functional groups as well as suitable surface hydrophobicity
hydrophilicity of latex particles are closely related to protein adsorption property As
indicated at Figure 4-17 various interaction forces such as hydrophobic interaction ionic
87
interaction hydrogen interaction and van der Waals interaction [Andrade 1985] exist
between protein molecule and the solid surface of latex particle
431 Adsorption Isotherm
The protein adsorption isotherm is generally defined as the relationship between the
amount of protein adsorbed by a solid (latex particle) at a constant temperature and as a
ine
the amount of protein adsorbed onto latex particles To evaluate the equilibrium
conce s
x
function of the concentration of protein Adsorption isotherms were plotted to determ
ntration of target proteins a calibration curve is firstly needed Figure 4-18 show
the UV absorbance as a factor of bovine serum albumin (BSA) concentrations The UV
absorbance of the known protein concentration was measured at 562nm which is the λma
for a protein treated by a bicinchoninic acid (BCA) assay
0
02
04
06
08
500 520 540 560 580 600
Wavelength (nm)
Abs
orba
nce
Figur gml (B)
06mgml (C) 02mgml
(A)
(C)
(B)
Max562nm
e 4-18 The dependence of UV absorbance on BSA concentration (A) 10m
Figure 4-19 shows the standard calibration curve used in adsorption isotherm
experiment of protein onto latex particles The net absorbance of each known protein
88
concentration was plotted and then a linear equation passing through a concentration of
zero protein concentration was obtained This equation is applied
to determine the
orbed on the synthesized latex particles The amount of free
protei
eported
trations (005 01 03 05
and 07mgml) were used to generate this curve as well as the adsorption isotherm
unknown protein amount ads
n was also determined using UV light at the 280nm wavelength however a
distinguishable UV intensity was too low to determine a precise value of protein
adsorption
The bicinchninic acid (BCA) assay technique [Smith et al 1985] has been r
for high efficiency of protein evaluation and was used for determination of protein
adsorption [Tangpasuthadol et al 2003 Williams et al 2003] This technique has many
advantages such as high sensitivity easy of use color complex stability and less
susceptibility to any detergents Five different BSA concen
y = 00R2 =
004741x 09939
00
01
02
03
04
0 200 400 600 800
BSA Standard (microgml)
Net
Abs
orba
nce
at 5
62nm
Figure 4-19 Standard curve of net absorbance vs BSA sample concentration
89
All isotherms were fitted to the Langmuir-Freundlich isotherms using the nonlinea
regression method Two adsorption isotherm models the Langmuir type isotherm a
Langmuir-Freundlich combination type isotherm were compared As seen the Figure 4-
20 The Langmuir isotherm model deviated further from the experimental data Th
all isotherms were fitted to the Langmuir-Freundlich isotherms [Yoon et al 1996
al 1998] usi
r
nd the
erefore
Lee et
ng the nonlinear regression method with following equation
n
n
eq
eqm
kC
kC1
1
1+
where q is the amount of adsorbed BSA per unit surface area q
qq = (4-6)
stant
were between 045 and 055 and hence n was fixed to its average 05 Actual qm values
were evaluated after fixing the n value
m is the adsorbed amount
in equilibrium Ceq is the equilibrium concentration of BSA k is the adsorption con
and n is the exponential factor for a heterogeneous system n values are empirical and
usually from zero to no more than unity [Jaekel 2002] The values of n in our system
00
10
20
30
0 02 04 06 08
C (mgml)eq
q (m
gm
2 )
pH 32Langmuir-Freundlich modelLangmuir model
Figure 4-20 Fitted models for adsorption isotherm of bovine serum albumin (BSA) on
polystyrene latex particles at 37oC in phosphate buffer media
90
The BSA protein adsorption on the synthesized latex particles was performed at
three pH levels 32 48 74 in both phosphate buffer (PB) and phosphate buffered saline
(PBS) at physiological temperature 37oC These pH levels correspond to an acidic pH
lower than the isoelectric point (IEP) of BSA the pH at the IEP and an alkaline pH
higher than the IEP of BSA respectively The IEP corresponds to the pH at which the
amino acid becomes a zwitterion Here its overall electrostatic charge is neutral and the
charge expressed by the basic amino group (-NH2 -NH3+) of the amino acid is equal to
the charge expressed by the acidic carboxyl group (-COOH -COO ) The IEP of BSA is
-
pH 48 and indicates that the side chain of the amino acids of BSA contains a greater
amount of acidic functional groups than that of basic functional groups At pH 32 and
74 BSA has a positive and negative charge respectively
Figure 4-21 and 4-22 show adsorption isotherms of BSA onto polystyrene (PS)
latex particles
00
10
20
30
40
0 02 04 06 08
Ceq (mgml)
q (m
gm
2 )
pH=32pH=48pH=74Model (pH 32)Model (pH 48)Model (pH 74)
Figure 4-21 Adsorption isotherm of BSA on polystyrene (PS) latex particles in
phosphate buffer (PB) at 37oC
91
00
10
30
40
0 02 04 06 08
Ceq (mgml)
q (m
gm
2
20
)
pH=32pH=48pH=74Model (pH 32)Model (pH 48)Model (pH 74)
(PBS) on PS latex particles
At the IEP of BSA the adsorbed amount (q
Figure 4-22 Adsorption isotherm of BSA adsorption 37oC in phosphate buffered saline
onto PS at equilibrium was
maxim
um at the IEP of BSA This is because the BSA molecules form the most
compact structures at the IEP eliminating the repulsion force between proteins A higher
number of BSA molecules can adsorb on a given surface area by hydrophobic interaction
when in this compact structure This observation agrees well with reported data by other
researchers who also indicated that the maximum adsorption from aqueous protein
solution was observed at the IEP [Mularen 1954 Watkins et al 1977 Oh at el 2000]
There was no significant difference of adsorbed BSA amount in PB and in PBS media at
pH 48 The conformational alteration of BSA was not affected significantly by the ionic
strength of the two solutions There was evidence of the effect of the electrostatic
interaction to some extent even at the IEP since the amount of BSA adsorbed in PBS is
slightly hig
m) of BSA
um indicating that hydrophobic interaction between latex particle and BSA
protein is maxim
her than in PB at this pH
92
The adsorbed amount of protein is shown to decrease at a pH 32 and 74 The
decrease in adsorption efficiency when deviating from IEP of BSA is a result of the
increase in conformational size of the protein molecules and the lateral electrostatic
repulsions between adjacent adsorbed BSA molecules Figure 4-23 shows a schematic of
possible conformational changes at each pH where hydrophobic interaction always exists
between non-polar portion of a protein and hydrophobic portion of solid surface
- - --- ---Latex surface
Zeta potential
BSA(+)
(-)
48 7432pH
(0)
+++ + ++
--+
++-
- - --- ---Latex surface
Zeta poten pH(0)
+++ + ++
--
tial
BSA(+)
(-)
48 7432
+
++-
ming
4
s
al blood Table 4-6 shows the calculated results of
Figure 4-23 Conformation of bovine serum albumin (BSA) on latex particles (Assuthat charge repulsion reduces the effect of the hydrophobic interaction at pH 74)
In addition the adsorbed amount of BSA at pH 32 was larger than that at pH 74
and still less than that at 48 This is because there is the electrostatic interaction between
the positive BSA molecules and negative PS latex particles at pH 32 and electrostatic
repulsion is dominant between negative BSA and negative PS latex particles at pH 7
The amount adsorbed increased with increasing ionic strength in pH 32 and 74 This i
because the high concentration of electrolytes reduces the electrostatic repulsion between
BSA and PS latex particle It should be noted that the BSA concentration used in these
experiments was lower than that found in normal human blood This is because the
equilibrium BSA adsorption onto the latex particles is actually reached at a much lower
concentration than that found in norm
93
adsor rm
ein
leading to a compact conformation of BSA There are two main forces affecting
adsorption amount at an acidic pH of 32 They include the ionic attraction between the
negative latex particle and the positive protein and lateral repulsion between positive
proteins The smallest amount of BSA adsorption was observed at a pH of 74 At this
point the charge repulsion between negatively charged particles and protein molecules
exists the h
of cha
bed BSA amount at equilibrium from the Langmuir-Freundich adsorption isothe
equation
In summary the hydrophobic interaction between PS latex particle and BSA
protein exists and is the dominant mechanism for the adsorption process Conformational
changes of BSA at each pH also affected the adsorption amount Adsorption was highest
at pH 48 because at this pH there is the least amount of ionic repulsion in the prot
and the lateral repulsion between proteins dominant adsorption even though there always
ydrophobic interaction between the solid latex particle and protein The effect
rge repulsion was reduced by the high concentration of electrolytes in PBS medium
Table 4-6 The equilibrium concentration values of BSA adsorption on polystyrene (PS) latex particles calculated the Langmuir-Freundlich isotherm model
Media pH qm (mgm2) 32 174 48 299 Phosphate buffer 74 136 32 260 48 283 Phosphate buffered saline 74 207
The adsorption isotherms of BSA on PSPMMA core shell particles are shown in
Figure 4-24 in phosphate buffer (PB) and in Figure 4-25 in phosphate buffered saline
(PBS) The adsorbed BSA amount was calculated using the Langmuir-Freundlich
100
94
adsorption isotherm equation and the amount listed in Table 4-7 The trend seen in the
adsorption results was very similar to the case of PS latex The overall adsorption process
was d rticle
rs
hat
0
ominated by hydrophobic interactions between the PSPMMA100 core shell pa
and BSA proteins At pH 48 the adsorbed amount (qm) of BSA on PSPMMA100 core
shell particles was 221 mgm2 in PB and 237 mgm2 in PBS and are the maximum
values recorded among the three pH levels 32 48 and 74 Suzawa and his co-worke
[1982] have obtained similar results This can be also explained by the compact
conformation of BSA molecules at the IEP of BSA which lead to a high protein
population on the solid surface At a pH of 32 and 74 BSA adsorption was less than t
at pH 48 because of the protein conformation change There was a small increase in
adsorbed BSA at pH 32 because of the charge attraction between negative PSPMMA10
core shell particles and positive BSA At pH 74 however the adsorption of BSA was
further reduced by charge repulsion between the negative PSPMMA100 core shell
particles and the negative BSA proteins
000 02 04 06 08
Ceq (mgml)
10
20q (m
gm
302 )
40
50pH=32pH=48pH=74Mode 8)l (pH 4Mode 2)l (pH 3Mode 4)
Figure 4-24 Adsorption isotherm of BSA 37oC in PB on PSPMMA core shell latex
l (pH 7
100
particles
95
The adsorbed amount of BSA increased more rapidly in PBS with increasing ioni
strength at pH 74 This shows that the low concentration of electrolytes promotes
electrostatic repulsion between negative BSA and negative PSPMMA
c
on
x
l 1980
100 core shell
particle As the ion strength of PBS is increased however the high concentration of
electrolyte leads to less electrostatic repulsion promoting more rapid protein adsorpti
By comparing the amount of adsorbed protein between bare PS latex particles and
PSPMMA100 core shell particles the amount of protein adsorption on the PS late
particles is greater than that on the PSPMMA100 core-shell particles [Suzawa et a
1982 Lee et al 1988]
00
10
20
30
40
m2 )
0 02 04 06 08
Ceq (mgml)
q (m
g
pH=32pH=48pH=74Model (pH 2) 3Model (pH 48)Model (pH 74)
Figure 4-25 Adsorption isotherm of BSA at 37oC in PBS on PSPMMA100 core shell
latex particles
96
Table particles calculated the Langmuir-Freundlich isotherm model
4-7 The equilibrium concentration values (qm) of BSA adsorption on PSPMMA100
Media pH qm (mgm ) 2
32 140
48 221 Phosphate buffer
74 130
32 169
48 237 Phosphate buffered saline
74 162
The BSA adsorption mechanism and trend on PSPMMA core shell particles was
very similar to that of the PS latex particle Unlike the case of PS and PSPMMA100 latex
particles the amount of BSA adsorbed onto PSPMMA90PAA10 and PSPMMA75PAA25
core shell particles was higher at pH 32 than pH 48 and 74 Figure 4-26 through 4-29
shows the adsorption isotherms of BSA on PSPMMA90PAA10 and PSPMMA75PAA25
core shell particles The amount of BSA adsorbed increased at pH 32 and pH 48 as the
hydrophilicity was developed by the addition of PAA On the contrary the adsorbed
amount of BSA was dramatically decreased at pH 74 The adsorption amount of BSA on
PSPMMA90PAA10 and PSPMMA75PAA25 core shell particles at equilibrium is listed in
Table 4-8 and 4-9 respectively
Carboxylic acid as well as sulfate groups are distributed on the surface of
PSPMMA90PAA10 and PSPMMA75PAA25 core shell particles and seem to largely affect
the BSA adsorption The isotherms showed very strong pH dependence to the BSA
adsorption onto these core shell latex particles The carboxylic groups (COOH) may
especially enhance the BSA adsorption prominently at pH 32 by hydrogen bonding with
protein molecules The pKa value for the PAA is 40-60 and a good indication that the
97
carboxylic acid groups are protonated at pH 32[Gebhardt et al 1983] The main
adsorption interaction force at pH 32 was generated by hydrogen bonding as well as
charge attraction between latex particle protein even th the conformational
repulsion between proteins results in a reduction of the protein
adsorption at this pH The amount of BSA adsorbed also increased as the PAA amount in
eased The a tion of BSA w inimal however less
than 07 mgm2 at pH 74 Here the carboxylic groups are ionized which increases the
charge repulsion by ionized carboxylic groups (COO-) resulting in PSPMMA-PAA latex
particle
and ough
change and charge
the core shell particles incr dsorp as m
s with much lower protein adsorption
00
20
40
q (m
g2 )
60
m
pH=32pH=48pH=74Model (pH 32)Model (pH 48)
0 02 04 06 08
Ceq (mgml)
Model (pH 74)
o shell
Figure 4-26 Adsorption isotherm of BSA at 37 C in PB on PSPMMA90PAA10 corelatex particles
98
00
40
60
80
0 02 04
C (mgm
mm
2 )
20
06 08
eq l)
q (
g
pH=32pH=48pH=74Model (pH 32)Model (pH 48)Model (pH 74)
Figure 4-27 Adsorption isotherm of BSA at 37oC in PBS on PSPMMA90PAA10 core
shell latex particles
Table 4-8 The equilibrium concentration values of BSA adsorption on PSPMMA90PAA10 particles calculated the Langmuir-Freundlich isotherm model
Media pH qm (mgm2)
32 361
48 283 Phosphate buffer
74 043
32 519
48 226 Phosphate buffered saline
74 043
99
00
20
40
60
80
100
0 02 04 06 08Ceq (mgml)
q (m
gm
2 )
pH=32pH=48pH=74Model (pH 32)Model (pH 48)Model (pH 74)
oFigure 4-28 Adsorption isotherm of BSA 37 C in PB on PSPMMA75PAA25 latex particles
00
100
20
40
60
80
0 02 04 06 08Ceq (mgml)
q (
m2 )
pH=32pH=48pH=74
mg
Model (pH 32)Model (pH 48)Model (pH 74)
Figure 4-29 Adsorption isotherm of BSA 37oC in PBS on PSPMMA75PAA25 core shell
latex particles
100
Table 4-9 The equilibrium concentration values of BSA adsorption on PSPMMA75PAA25 particles calculated the Langmuir-Freundlich isotherm model
Media pH qm (mgm2)
32 1083
48 662 Phosphate buffer (PB)
74 075
32 1077
48 681 Phosphate buffered saline (PBS)
74 057
The adsorption of β2-microglobulin (β2M) was determined under one set of
onditions in phosphate buffer at 37oC and pH 74 because of the limited amount of β2M
protein The adsorption isotherms of the β2M protein on the synthesized latex particles
are shown in Figure 4-30 4-31 and 4-32 The β2M protein concentrations for adsorption
isotherm experiment were chosen to be 0015 0030 0045 and 0060mgml since the
β2M concentration for the patients with renal failure can be elevated to similar
concentrations about 005mgml or more up to 50 times the normal level [Nissenson et
al 1995] There were steep initial slopes of the adsorption isotherm curves of β2M on
latex particles in all isotherms indicating a high affinity adsorption type It seems that the
hydrophobic interaction is enough for the adsorption process of β2M proteins to occur on
the latex particles even though charge repulsion exists between protein and latex particle
at pH 74 The complete plateau regions were not seen for all isotherms indicating that
β2M proteins were not saturated at this level and there are still available sites for β2M
c
adsorption on latex particles The initial slope of the β2M isotherms for PS was steeper
101
than that of any of the other latex particle PSPMMA100 PSPMMA90PAA10 and
PSPMMA 75PAA25
00
02
0 0002 0004 0006 0008
04
06
10
Ceq (mgml)
q (m
2 )
08
gm
PSPSPMMA100Model (PS)Model (PSPMMA100)
Figure 4-30 Adsorption isotherm of β2M onto PS and PSPMMA100 latex particles in PB
at 37 C and pH 74 o
00
02
04
06
08
10
0 0002 0004 0006 0008C (mgml)
q (m
m2 )
eq
g
PS
PSPMMA90PAA10
Model (PS)
Model (PSPMMA90PAA10)
Figure 4-31 Adsorption isotherm of β2M onto PS and PSPMMA90PAA10 latex particles
in PB at 37oC and pH 74
102
00
02
04
06
08
10
0 0002 0004 0006 0008Ceq (mgml)
q (m
gm
2 )
PS
PSPMMA75PAA25
Model (PS)Model (PSPMMA75PAA25)
Figure 4-32 Adsorption isotherm of β2M onto PS and PSPMMA75PAA25 latex particles
in PB at 37oC and pH 74
The equilibrium concentration values (qm) of β2M adsorption on latex particles are
calculated and listed in Table 4-10 The difference between the maximum amounts of
β2M adsorption on all the latex particles was not significantly different and their values
were between 069 and 08mgm2
Table 4-10 The equilibrium concentration values of β2M adsorption calculated the Langmuir-Freundlich isotherm model
Latex particles qm (mgm2)
PS 080
PSPMMA100 069
PSPMMA90PAA10 072
PSPMMA75PAA25 072
103
432 Adsorbed Layer Thickness
There are several possible explanations for large amount of BSA adsorbed at acidic
pH levels including the end-on adsorption pattern of BSA the flexibility of BSA
molecules the tilting of protein molecules due to the asymmetry of the charge
distribution or multilayer formation [Peula et al 1993] The adsorbed BSA thickness (δ)
can be calculated by the following equation [Chiu et al 1978] This equation was also
cited again by other researchers [Shirahama et al 1985]
BSA
mqρπ
δbullbull
=33
(4-7)
where π
33 is the packing factor qm is the plateau value of the adsorbed am
ρBSA is the hich corresponds to the reciprocal of its known
partial specific volume The layer thickness (δ) of BSA proteins on synthesized latex
partic
he IEP [Shirahama et al 1985] Compared to the
hydrodynamic dimensions of BSA 140 times 40 times 40 Aring3 [Fair et al 1980 Peter 1985] these
nd
ode It is unnecessary ultilayering because thickness do
exceed 140 Aring The layer thickness (δ) of BSA proteins on synthesized latex particles
e red saline (PBS) was ran 28 ~ 83 Aring at 37oC and pH 48 These
lso listed in Table 4-1
ount and
density of BSA molecule w
les in phosphate buffer (PB) ranged from 27 - 81 Aring at 37oC and pH 48 These
calculated values are listed in Table 4-11 Only δ values at pH 48 the isoelectric point
(IEP) of BSA are important because intra- and intermolecular electrostatic repulsion of
protein molecules is minimized at t
values of layer thickness indicate that the BSA molecules adsorb by side-on (40Aring) a
end-on (140Aring) m to consider m
not
in phosphat buffe ged of
calculated values are a 2
104
Table 4-11 Absorbed BSA layer thickness (Aring) of BSA in phosphate buffer (PB) at 37oC pH PS PSPMMA100 PSPMMA90PAA10 PSPMMA75PAA25
32 21 17 44 133
48 37 27 35 81
74 17 16 5 9
Table 4-12 Absorbed BSA layer thickness (Aring) of BSA in phosphate buffered saline (PBS) at 37oC
pH PS PSPMMA100 PSPMMA90PAA10 PSPMMA75PAA25
32 32 21 64 132
48 35 29
74 26 20 5 7
28 83
1n
1n
neqm
So k is closely related to the initial slope of the isotherm showing the affinity
between BSA and latex particles [Yoon et al 1996] A key point in the characterization
433 Gibbs Free Energy of Protein Adsorption
In the Langmuir isotherm model k represents the ratio of adsorption to the
desorption rate constants This definition is not applicable to the Langmuir-Freundlich
isotherm model but its meaning is similar [Yoon et al 1996] In this sense k is closely
related to the affinity between protein molecules and latex particles When the
equilibrium concentration Ceq reaches zero the value of Ceq will be very small (1gtgt
kCeq ) and then the equation 4-6 becomes
(4-8)
of adsorption processes on solid-liquid interfaces is to determine the change in Gibbsrsquos
free energy (∆Gdeg) one of the corresponding parameters for the thermodynamic driving
forces during adsorption The traditional method to evaluate adsorption thermodynamic
kCqq 1=
105
constants is based on measurement of adsorption isotherms under static conditions
[Malm ten 1998] The Langmuir-Freundlich ads lf
easily found from Then this enables calculation of the free-
energy change ∆G during adsorption expressed as
(4-9)
where R is the general gas constant (R = 8314Jmiddotmol K ) and T is the absolute
tem ture Gibb e energy A P
listed in Table 4-13 and 4-14 respectively As the ∆Gdegads ore negativ e
pro adsorption on a solid substra ill be more favorable
e of BSA adsorption in phosphate o
pH PS PSPMMA100 PSPMMA90PAA10 PSPMMA75PAA25
s orption equilibrium constant k can be
a slope of the isotherm
degads
lfads kRTG lnminus=∆ o
-1 -1
pera s fre change of BS adsorption values in B and PBS are
values are m e th
tein te w
Table 4-13 The values of Gibbs free energy changbuffer (PB) at 37 C
32 -2411 -2726 -2846 -2805
48 -2352 -2383 -2198 -1972 ∆Go
80
ads (kJmol)
74 -2452 -888 -324 -2
Table 4-14 The values of Gibbs free energy change of BSA adsorption in phosphate
pH PS PSPMMAbuffered saline (PBS) at 37oC
100 PSPMMA90PAA10 PSPMMA75PAA25
32 -2315 -2620 -2312 -2683
48 -2149 -2299 -2090 -2138 ∆Go
(kJmads ol)
74 -2430 -1992 -258 -263
Table 4-15 shows the calculated values of Gibbs free energy change of β M
adsorption on synthesized latex particles PSPMMA PAA core shell latex particle has
2
75 25
106
largest negative Gibbs free energy of β2
2
M adsorption indicating largest affinity betw
β
een
2M adsorption in phosphate buffere (PB) at 37 C and pH74
PS PSPMMA100
M and latex particle
Table 4-15 The values of Gibbs free energy change of βo
PSPMMA90PAA10 PSPMMA75PAA25
∆Go03 ads
(kJmol) -3626 -3466 -3659 -39
The Gibbs free energy of β2M was much more negative in phosphate buffer (PB)
than that of BSA in physiological condition (37 C and pH 74) o from the Table 4-13 and
the Figure 4-31 This indicates that β2M molecules more readily adsorb on latex particles
BSA This can be explained by the rate of diffusion and accessibility of β2M molecules to
the latex particle ier and faster for β2M m an BSA because of
size (molec ight) erical shape molecules It o be
due to the higher hydrophobic force and less electrostatic repulsion that contribute to the
adsorption of β2M molecules not as prevalent in the case of BSA because the IEP of β2M
(57) is clo H 74) than that of BSA (48) causing β2M to
be less electro cally lled p ro
can be seen that the ∆ β2M adsorption onto the synth article
increasing wi hy icity o x particles s the ∆Goads of the BSA
molecules is decreasing or constant This phenomenon supports the theory that the
select lizing
than BSA and that the affinity of β2M for the latex particle is much larger than that of
surface It is eas olecules th
the smaller ular we and sph of β2M may als
ser to physiological condition (p
stati repe from the latex articles than BSA F m Figure 4-33 it
Goads of esized latex p s is
th the drophil f the late wherea
ive adsorption of β2M molecules over BSA molecules can be enhanced by uti
more hydrophilic PSPMMA90PAA10 and PSPMMA75PAA25 core shell latex particles
107
-60
-50
1 2 3 4
-40∆Gad
s
-30
10
0
o (k
Jm
o-
-20l)
β2-Microglobulin (β2M)Bovine serum albumin (BSA)
Figure 4-33 Gibbs free energy of adsorption of proteins on latex particles in PB at 37
and pH 74
434 Kinetics of Adsorption
to
oC
Protein adsorption is a complex process known to have a large biological impact
and is currently not well understood quantitatively because of extreme sensitivity to pH
the concentration of other electrolytes and molecules present temperature and many
other factors that can change in the physiological system Accurate knowledge of the
adsorption kinetics under a given set of conditions is a prerequisite for elucidating the
mechanisms of many fundamental biological processes to be predicted at the molecular
level [Regner 1987] This adsorption kinetics approach is used in protein adsorption
studies because of the uncertainty related to the time needed for the interfacial proteins
reach the equilibrium It is generally accepted that the process of protein adsorption is
comprised of the following steps a) transport toward the interface b) attachment at the
interface c) eventual structural rearrangements in the adsorbed state d) detachment from
PS PSPMMA100 PSPMMA90PAA10PSPMMA75PAA25
108
the interface and transport away from the interface Each of these steps can affect the
overall rate of the adsorption process
The quantitative analysis of the protein adsorption kinetics requires that the protein
amount adsorbed is known as a function of time The kinetics tests were carried out in
phosphate buffer (PB) at 37oC and pH 74 a condition similar to physiological condition
The protein concentrations chosen were 07mgml for BSA and 006mgml for β2M
which are the maximal concentrations used in an adsorption isotherm test A time-based
evaluation of the adsorption was made by measuring the concentration of protein in
solution at different incubation times The results are shown below from Figure 4-34 to 4-
37 The adsorption kinetics of proteins onto th cond-e latex particles is expressed as a se
order model [Oumlzacar 2003] shown by the following equation
2)( tet qqk
dtdq
minus= (4-10)
Integrating above equation and applying the boundary conditions yields
ee
t tq
+=
1 (4-11)
where q
qkq
t
2
2
2
the previous adsorption isotherm results representing good reproducibility of adsorption
t and qe stand for the amount (mgm ) of protein adsorbed at time t and at
equilibrium respectively k is the equilibrium rate constant of second-order adsorption
(m mghr) and t is the incubation time (hr)
The fitting parameters calculated by using the equation 4-11 are listed in table 4-16
From this data it is seen that the amount of BSA adsorbed at equilibrium was higher than
that of β2M on PS and PSPMMA100 latex particles but less than that of β2M on
PSPMMA90PAA10 and PSPMMA75PAA25 latex particles These results are similar to
109
00
05
15
20
0 2 4 6 8 10 12 14
Time (hr)
rot
n ad
sorb
d (
10
25
Pei
em
gm
2
BSA2nd order model (BSA))β2M2nd order model (β2M)
Figure 4-34 The kinetics of protein adsorption in PB on PS latex particles at 37oC and
pH 74
00
05
10
15
20
25
30
0 2 4 6 8 10 12 14
Time (hr)
Prte
inds
oo
arb
ed (
mg
m2 )
BSA2nd order model (BSA)β2M2nd order model (β2M)
Figure 4-35 The kinetics of protein adsorption in PB on PSPMMA100 latex partic
37les at
oC and pH 74
110
00
03
06
09
12
15
0 2 4 6 8 10 12 14
Time (hr)
Prot
ein
adso
rbed
(m
gm
2 ) BSA2nd order model (BSA)β2M2nd order model (β2M)
Figure 4-36 The kinetics of protein adsorption in PB on PSPMMA90PAA10 latex
particles o at 37 C and pH 74
00
03
06
09
12
15
0 2 4 6 8 10 12 14
Time (hr)
BS
A ad
sorb
ed (
mg
m2 )
BSA2nd order model (BSA)β2M2nd order model (β2M)
Figure 4-37 The kinetics of protein adsorption in PB on PSPMMA75PAA25 latex
particles at 37oC and pH 74
111
test The variation of β2M equilibrium adsorption amount was nearly negligible for all
latex particles The plateau region is reached earlier for β2M than BSA on all latex
particles and the K values (equilibrium rate constant of second order adsorption) for the
β2M adsorption process onto latex particles are much larger than for BSA adsorption
These values indicate that the β2M molecules have a higher adsorption rate onto the latex
particles than the BSA molecules
Table 4-16 Fitting parameters of second-order kinetic model BSA β2M
Latex particles qe (mgm2) K (hr-1) qe (mgm2) K ( -1) hr
PS 144 67 074 7549
PSPMMA100 175 353 076 4438
PSPMMA90PAA10 046 97 075 5695
PSPMMA75PAA25 060 179 075 5168 K = (kqe) hr-1
There are several explanations for the phenomena observed in these experiments
First the smaller sizes of β2M molecules allow easier diffusion and a close approach to
the latex substrate Second a reduced number of hydrophobic domains on the latex
particle surface replaced by sulfate groups (-OSO3-) and ionized carboxylic groups
(COO-) are not favorable for larger BSA molecules Third the conformational change by
intra- and inter-molecular repulsion would be faster in β2M due to smaller molecular
weight Finally the ionic repulsion between β2M and the latex particles is less than that
condition (pH 74) than IEP (48) of BSA
between BSA and latex particles because IEP (57) of β2M is closer to the tested
112
44 Blood Compatibility
Blood compatibility can be defined as the property of a material or device tha
permits it to function in contact with blood without inducing any adverse reactions
Successful functioning of blood contacting biomedical materials or devices in contact
with blood strongly depends on the extent to which they damage blood during operation
High shear stresses turbulence rela
t
tively long contact times and cellular impact with
artificial surfaces are most common events experienced by blood in contact with artificial
1978 Winslow and et al
1993] Therefore before any materia e placed into the human b
biocompatibility testing must be the first performed in the view of safety The hemolysis
s a method for pr ary screening of biomaterials to determine
h blood a er circulating body fluid The tests estimat
oglobin released from th blood cell d e induced
foreign material
There are several types of blood cells in our body but the ones that make blood red
are erythrocytes or red blood cells (RBCs) Red blood cells (erythrocytes) are the most
numerous type in the blood These cells are average about 48-54 million per cubic
millimeter (mm3 same as a microliter (microl)) of blood but these values can vary over a
larger range depending on such factors as health and altitude RBCs are red because they
contain hemoglobin which is a protein that contains iron When blood circulates through
lungs oxygen binds to the hemoglobin and is then delivered to all the parts of body
RBCs are made in the bone marrow They start out as a nucleus like other cells but
when they fill up with hemoglobin the nucleus becomes smaller until it disappears
Without a nucleus RBCs are fragile and live only about 120 days In a normal healthy
devises and can cause hemolysis or thrombosis [Bernhard et al
ls can b ody
test serves a elimin the
biocompatibility wit nd oth e the
concentration of hem e red amag by a
113
body about 2 million red blood ne marrow produces new
ones j ing
dy
le
cells die per second But bo
ust as fast RBCs are about 75 micrometer across and are responsible for carry
oxygen salts and other organic substances to the cells and tissues throughout our bo
In humans and most mammals the cells are concave (indented in the middle) and flexib
enough to squeeze through even the tiniest of blood vessels the capillaries
Figure 4-38 Image of red blood cells
(Sourcehttpwwwpbrchawaiiedubemfmicroangelarbc1htm)
ch
100 90 10 75 25 f
RBCs are among the first cells that come into contact with a foreign material
injected in the blood Therefore a hemolysis assay gives information about the
biocompatibility in the case of an in vivo application The concentrations of prepared
latex particles used for the tests in this research are 05 25 and 50 (ww) and the
incubation time under moderate shaking was 30 minutes at 37oC Test results are shown
in Figure 4-39 The amount of blood cells that ruptured when in contact with the PS latex
particles was largely increased from about 30 to 91 as the solid content of PS
increased from 05 to 50 However hemolysis by the core shell latex particles su
as PSPMMA PSPMMA PAA and PSPMMA PAA was less than 35 O
114
particular interest was that hemolysis caused by PSPMMA PAA particles at 50
solid concentration was less than 02 hemolysis the lowest value among the latex
particles The low hemolysis value indicates that the PSPMMA PAA core shell latex
particles are the most blood compatible at all solid contents Other core shell particles
such as PSPMMA and PSPMMA PAA also had excellent blood comp
90 10
90 10
100 75 25 atibility with
low RBS breakage
01 2 3
20
40
60
80
100
Hem
olys
is (
)
052550
Figure 4-39 He
PS PSPMMA100 PSPMMA PAA90 10
molysis caused by latex particles n=5
4PSPMMA75PAA25
CHAPTER 5 SUMMARY OF RESULTS CONCLUSIONS AND RECOMMENDATION FOR
FUTURE WORK
51 Summary of Results
The goal of this study was to synthesize monodisperse polymeric latex particles
with tailored properties and to investigate the fundamental interactions between the
synthesized latex particles and target proteins An understanding of the fundamental
mechanism of selective adsorption is strongly required in order to maximize the
separation performance of membranes made of these materials so that it may applicable
to hemodialysis therapy for end stage renal disease (ESRD) patients For the achievement
of above research goal several investigations have been performed Analysis of the
obtained data leads to several conclusions as discussed below
Monodisperse polystyrene seed latex particles were synthesized with the size
crosslinking agent to make the latex particles more hydrodynamically stable The
conversion of monomer to polymer was over 95 and seed latex particles are spherical in
shape and have smooth surface From these small seeds bigger latex particles as large as
800nm were synthesized using a semi-continuous seeded emulsion polymerization
process The particles with a mean diameter less than 500nm are very spherical in shape
and highly uniform in particle size distribution However as the particles grow larger
than 500nm in mean diameter they became non-spherical with an uneven surface This
irregularity of particle surface can be attributed to the non-homogeneous monomer
ranges between 126plusmn75 and 216plusmn53 nm Divinylbenzene (DVB) was used as a
115
116
swelling into the shell of a growing polymer which can be controlled by the factors such
as temperature agitation speed initiator feeding rate and surfactant amount required to
stabilize the colloidal system It was determ rfactant to monomer ratio
dec ic
acid (AA) monomers were intro rticle surfaces more
hydro
to
ting of a
orm
ration is seen between
3002
0 cm-
0cm-1
-1
03
ined that as the su
reased the mean particle diameter increased Methyl methacrylate (MMA) and acryl
duced to make the latex pa
philic than bare polystyrene particles The prepared core shell latex particles were
made of PMMA100 PMMA90PAA10 and PMMA75PAA25 PMMA100 is the PMMA
homopolymer shell on a PS core PMMA90PAA10 shell is a copolymer with a PMMA
PAA ratio of 90 to 10 by weight PMMA75PAA25 shell is a copolymer consis
PMMA to PAA ratio of 75 to 25 by weight The particle size of these core-shell
particles were prepared to be about 370nm in mean diameter
The successful synthesis of latex particles was confirmed using Fourier Transf
Infrared spectroscopy (FTIR) The C-H aromatic stretching vib
cm-1 and 3103 cm-1 for PS latex particles The C-H asymmetrical stretching and
symmetrical stretching vibration peaks of CH2 for all latex particles is seen at 290
1and 2850 cm-1 respectively There is a carbonyl (C=O) characteristic band at 173
wavenumber for PSPMMA core-shell latex particles The broad OH group peak at
3400 cm
100
can be seen in the spectroscopy of the particle with PMMAPAA shell
(PSPMMA PAA and PSPMMA PAA ) The peak intensity of OH group of
PSPMMA PAA was larger than that of PSPMMA PAA relatively due to more AA
monomer content in PSPMMA PAA than in PSPMMA PAA
In the zeta potential measurements polystyrene (PS) latex particles had negative
values between -291 mV and -599 mV in the phosphate buffer (PB) and between -2
90 10 75 25
75 25 90 10
75 25 90 10
117
mV and -278 mV in the phosphate buffered saline (PBS) at 25oC and pH 21-78 These
negative zeta potential values for PS latex particles are due to ionization of the sulfate
groups (-OSO3-) originated from the initiator Zeta potential values of the PSPMMA
core shell latex particles were also negative between -28 mV and -505 mV in PB and
between -143 mV and -186 mV in PBS at pH 21-78 Negative values are also
generated by dissociation of sulfate groups on a particle surface The zeta potential value
of PSPMMA PAA and PSPMMA PAA were of negative values between -36
and -678 mV in PB medium and -147 mV and -193 mV in PBS medium at 25
100
s
7 mV
MMA75PAA25 and
PSPM
otein
g the
l change of BSA at
each w
90 10 75 25
oC For
PSPMMA PAA core shell particles zeta potential values were between -291 mV and
-520 mV in PB media and between -115 mV and -210 mV in PBS media Sulfate
groups contributed to the negative zeta potential values in PSP
75 25
MA90PAA10 core shell particles Carboxylic groups from polyacrylic acid also
contribute to the negative surface charge for the latex particles at greater than pH 70
however the degree of their contribution was not detectable from the zeta potential
measurements The absolute zeta potential values of the synthesized latex particles in PB
were higher than that in PBS This is because of the high concentration of sodium
chloride electrolyte in PBS that compress electrical double layer thickness
The bicinchoninic acid (BCA) assay technique was used to determine the pr
adsorption and all isotherms were fitted to the Langmuir-Freundlich isotherms usin
nonlinear regression method The hydrophobic interaction between PS latex particle and
BSA protein was dominant for the adsorption process Conformationa
pH also affected the adsorption amount which was highest at pH 48 because of lo
ionic repulsion making compact conformation of BSA The next larger amount of BSA
118
adsorbed onto PS was at pH 32 There were two main forces affecting the amount of
adsorption at acidic pH values One is ionic attraction between negative latex particle a
positive protein The other is lateral repulsion between positive proteins The smallest
amount of BSA adsorption was at pH 74 where charge repulsion between negative
particles and proteins is present at this pH Lateral repulsion between proteins wa
dominant for protein adsorption even though there always exists the hydrophobic
interaction between solid latex particle and protein The charge repulsion was reduced by
the high concentration of electrolytes in the PBS medium The BSA adsorption process
on PSPMMA core shell particles was very similar to the case of PS latex particle where
hydrophobic interaction between latex particle and protein was dominant However t
adsorbed BSA amount was less than that of PS in PBS The adsorbed amount of BS
PSPMMA
nd
s
he
A on
an
ed
n The main
adsor
90PAA10 and PSPMMA75PAA25 core shell particles was higher at pH 32 th
pH 48 and 74 The adsorbed BSA amount became higher at pH 32 and pH 48 as the
hydrophilicity was increased by the higher PAA amount On the contrary the adsorb
amount of BSA was dramatically decreased at pH 74 Carboxylic acid groups as well as
the sulfate groups are distributed on the surface of PSPMMA90PAA10 and
PSPMMA75PAA25 core shell particles and largely affect the BSA adsorptio
ption interaction forces at pH 32 was generated by hydrogen bonding as well as
charge attraction between latex particle and protein even when the conformational change
and charge repulsion between proteins reduce the adsorption of protein at this pH The
adsorbed BSA amount was also increased as the amount of PAA increases However the
adsorption of BSA was minimal (less than 07 mgm2) at pH 74 where the carboxylic
groups are ionized leading to more enhancement of charge repulsion by ionization of the
119
carboxylic groups (COO-) Because of this the PSPMMA-PAA latex particles have a
much lower protein adsorption
There were steep initial slopes for adsorption isotherms of β
in
s
there
M
r
e
es
o
2M on latex particles
all isotherms indicating a high affinity type adsorption The complete plateau region
were not seen for all isotherms indicating that β2M proteins were not saturated and
are still available sites for β2M adsorption on latex particles The initial slope of β2
isotherms for PS was steeper than for the other latex particles PSPMMA100
PSPMMA90PAA10 and PSPMMA75PAA25
The layer thickness (δ) of BSA proteins on synthesized latex particles in phosphate
buffer (PB) was in the range of 27 - 81 Aring at 37 C and pH 48 Only the δ values at pH
48 the isoelectric point (IEP) of BSA are important because intra- and inter-molecular
electrostatic repulsion of the protein molecules is minimized at the IEP Compared to the
hydrodynamic dimensions of BSA 140 times 40 times 40 Aring these values of the adsorbed laye
thickness indicates that the BSA molecules adsorb by the side-on (40Aring) and end-on mod
(140Aring) Consideration of the multi-layering is unnecessary because the adsorbed
thickness of BSA do not exceed 140 Aring
The Gibbs free energy of β
o
3
2M was more negative in phosphate buffer (PB) than
that of BSA in physiological condition (37 C and pH 74) indicating that βo2M molecul
are more favorably adsorbed on the latex particles than BSA and that the affinity of β2M
was much larger than BSA This may be explained by the rate of diffusion and
accessibility of β2M molecules being easier and faster than that for BSA because of
smaller size (or weight) and spherical shape of β2M molecules It may also be possible t
explain that more hydrophobic forces (less electrostatic repulsion) contributes to
120
adsorption of β2M than to that of BSA because the IEP of β2M (57) is closer to
physiological condition (pH 74) than that of BSA (pH 48) The selective adsorption of
β2M m
in β2M
larger than in BSA adsorption indicating
that fa
an that between BSA and latex particles because
IEP (
ch as
Other
olecules over BSA molecules can be enhanced by developing more hydrophilic
PSPMMA90PAA25 and PSPMMA75PAA25 core shell latex particles
The adsorption kinetics of proteins on latex particles was expressed as a second-
order model The plateau region for β2M was reached earlier than BSA on all latex
particles and the K values (equilibrium rate constant of 2nd order adsorption)
adsorption process on latex particles were much
ster adsorption rate of β2M molecules than that of the BSA molecules There are
several explanations for these phenomena First the smaller size of β2M molecules can
more easily diffuse and approach to the latex substrate Second enhanced hydrophilic
domain on the latex particle surface by sulfate groups (SO4-) and ionized carboxylic
groups (COO-) is not favorable for larger BSA molecules but is for smaller β2M to be
anchored Third the conformational change by intra- and inter-molecular repulsion
would be faster in β2M due to lower molecular weight Finally the ionic repulsion
between β2M and latex particles is less th
57) of β2M is closer to the tested condition (pH 74) than IEP (48) of BSA
In the biocompatibility test the number of red blood cells (RBCs) ruptured by PS
latex particles ranged from 30 to 91 as the solid content of PS increased from 05 to
50 respectively However hemolysis of the other core shell latex particles su
PSPMMA100 PSPMMA90PAA10 and PSPMMA75PAA25 were less than 35 The
PSPMMA90PAA10 particles at all applied solid concentrations had less than 02
hemolysis and was the most blood compatible among the prepared latex particles
121
core shell particles such as PSPMMA100 and PSPMMA75PAA25 also had excelle
compatibility with low RBC breakage
52 Conclusions
The major conclusions from this study include
nt blood
bull microglobulin (β2M) over that of the bovine serum albumin (BSA) at pH 74
was achieved by controlling the PMMA and PAA ratio to optimize
e particles posses the most desirable requirements for use in a human dialysis
ns
icles
olecule
bull
Using engineered core shell latex particles selective adsorption of β2
physiological condition was demonstrated
bull The rate of adsorption of the β2M was also much higher than that of BSA This
hydrophobichydrophilic microdomain size
bull The core shell particles also exhibited excellent biocompatibility with blood Thes
membrane
53 Recommendation for Future Work
With the results and conclusions obtained through this study several suggestio
may be given for future work as follows
bull Competitive adsorption tests of whole blood proteins on synthesized latex partunder physiological condition
bull Preparation of latex particles with optimum hydrophobic hydrophilic microdomains to maximize selective adsorption and removal of toxin molecules
bull Membrane fabrication by suitable pore size design to discriminate a toxin mby size sieving as a function of particle layers
Evaluation of membrane performance such as flow rate in water and in simulated blood fluid (SBF)
bull Application for hemodialysis as well as other technologies such as a water remediation
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Korea on August 7 1968 He obtained a Bachelor of Science degree in Chemistry from
Keim d graduate school and earned a
resear n Korea Institute of Science and
Coati e
ia
Busin
BIOGRAPHICAL SKETCH
Sangyup Kim was born and raised in the small town of Sung-Joo Kyungbook
yung University (Daegu Korea) in 1993 He entere
Master of Science degree in Chemical engineering in 1995 He worked as a student
cher for 2 years at Textile Polymer Division i
Technology (KIST) in Seoul Korea Next he worked over a year at the Automobile
ng Division in DongJoo-PPG in Korea In August 2001 he began studying for th
degree of Doctor of Philosophy in materials science and engineering at the University of
Florida in Gainesville After getting his PhD he plans to work at the Digital Med
ess Division of Samsung Electronics in Korea starting in January 2006
141
LIST OF TABLES
Table page 4-1 The polymerization recipe of polystyrene (PS) seed particles 57
4-2 Continuous addition emulsion polymerization recipe for growing polystyrene (PS) latex particles less than 500nm in size 63
4-3 Continuous addition emulsion polymerization recipe for growing polystyrene (PS) latex particles larger than 500nm in size64
4-4 The preparation recipe of PS core with various shell latex particles72
4-5 The isoelectric point (IEP) of common proteins 83
4-6 The equilibrium concentration values of BSA adsorption on polystyrene (PS) latex particles calculated the Langmuir-Freundlich isotherm model 93
4-7 The equilibrium concentration values (q ) of BSA adsorption on PSPMMA particles calculated the Langmuir-Freundlich isotherm model
m 10096
4-8 The equilibrium concentration values of BSA adsorption on PSPMMA PAA particles calculated the Langmuir-Freundlich isotherm model
90 1098
4-9 The equilibrium concentration values of BSA adsorption on PSPMMA PAA particles calculated the Langmuir-Freundlich isotherm model
75 25100
4-10 The equilibrium concentration values of β M adsorption calculated the Langmuir-Freundlich isotherm model
2102
4-11 Absorbed BSA layer thickness (Aring) of BSA in phosphate buffer (PB) at 37 Co 104
4-12 Absorbed BSA layer thickness (Aring) of BSA in phosphate buffered saline (PBS) at 37 Co 104
4-13 The values of Gibbs free energy change of BSA adsorption in phosphate buffer (PB) at 37 Co 105
4-14 The values of Gibbs free energy change of BSA adsorption in phosphate buffered saline (PBS) at 37 Co 105
viii
4-15 The values of Gibbs free energy change of β M adsorption in phosphate buffere (PB) at 37 C and pH 74
2o 106
4-16 Fitting parameters of second-order kinetic model 111
ix
LIST OF FIGURES
Figure page 1-2 Stereo drawing of the αndashcarbon backbone of β M2 2
2-1 An image of the location and cross section of a human kidney 5
2-2 A schematic draw of the hemodialysis route7
2-3 Cross-sectional SEM image view of the Polyflux S (polyamide + polyacrylethersulfone + polyvinypyrollidone) dialysis membranes 11
2-4 The chemical structure of polysulfone (PSf) 12
2-5 The chemical structure of polyamide (PA)13
2-6 Chemical structure of polyacrylonitrile (PAN) 14
2-7 Emulsion polymerization system24
2-8 L-α-amino acid 31
2-9 Ribbon diagram of human β M taken from the crystal structure of the protein bound to the heavy chain of the MHC class I complex (PDB 1DUZ)
236
2-10 Secondary structure of human serum albumin (HSA) with sub-domains 37
2-11 The relationship between pore size and particle size in body-centered cubic and closed packed arrays41
2-12 Schematic representation of the protein adsorption on the core shell latex particles at pH 7442
3-1 Chemical structure of main chemicals45
3-2 Experimental setup for semi-continuous emulsion polymerization 46
3-3 The particle preparation scheme in various types and size ranges of latex particles 47
3-4 Schematic of the procedure for a protein adsorption test 52
3-5 Separation of RBC from whole blood by centrifuge process53
x
3-6 Schematic of the procedure for hemolysis test 54
4-1 Schematic representation of semi-continuous seed latex particles preparation and growth56
4-2 Scanning Electron Micrograph (SEM) of polystyrene seed latex particles (A) PS (B) PS (C) PS (D) PSS259 S233 S207 S181 59
4-3 SEM of PS latex particles less than 500nm in size (A) 258nm (B) 320nm (C) 370nm (D) 410nm 65
4-4 SEM of PS latex particles larger than 500nm in size (A) 525nm (B) 585nm (C) 640nm (D) 790nm 68
4-5 Dependence of the particle size on the surfactant to monomer ratio70
4-6 Schematic of core shell latex particle structures 71
4-7 Scanning Electron Micrograph of latex particles (A) PS (B) PSPMMA (C) PSPMMA PAA (D) PSPMMA PAA
100
90 10 75 25 73
4-8 FTIR spectra of polymerized latex particles (A) bare polystyrene (PS) (B) PSPMMA (C) PSPMMA PAA (D) PSPMMA PAA100 90 10 75 25 76
4-9 Schematic representation of ion distribution near a positively charged surface 78
4-10 Schematic representation of zeta potential measurement79
4-11 Zeta potential of PS latex particles at 25 Co 80
4-12 Zeta potential of PSPMMA latex particles at 25 C100o 81
4-13 Zeta potential of PSPMMA PAA latex particles at 25 C90 10o 82
4-14 Zeta potential of PSPMMA PAA latex particles at 25 C75 25o 82
4-15 The decay of surface potential with distance from surface in various electrolyte concentrations (1) low (2) intermediate (3) high 84
4-16 High-affinity adsorption isotherm of typical flexible polymer on solid surface 86
4-17 Overall schematic representation of the protein adsorption on the synthesized latex particles86
4-18 The dependence of UV absorbance on BSA concentration (A) 10mgml (B) 06mgml (C) 02mgml87
4-19 Standard curve of net absorbance vs BSA sample concentration 88
xi
4-20 Fitted models for adsorption isotherm of bovine serum albumin (BSA) on polystyrene latex particles at 37 C in phosphate buffer mediao 89
4-21 Adsorption isotherm of BSA on polystyrene (PS) latex particles in phosphate buffer (PB) at 37 Co 90
4-22 Adsorption isotherm of BSA adsorption 37 C in phosphate buffered saline (PBS) on PS latex particles
o
91
4-23 Conformation of bovine serum albumin (BSA) on latex particles92
4-24 Adsorption isotherm of BSA 37 C in PB on PSPMMA core shell latex particles
o100
94
4-25 Adsorption isotherm of BSA at 37 C in PBS on PSPMMA core shell latex particles
o100
95
4-26 Adsorption isotherm of BSA at 37 C in PB on PSPMMA PAA core shell latex particles
o90 10
97
4-27 Adsorption isotherm of BSA at 37 C in PBS on PSPMMA PAA core shell latex particles
o90 10
98
4-28 Adsorption isotherm of BSA 37 C in PB on PSPMMA PAA latex particleso75 25 99
4-29 Adsorption isotherm of BSA 37 C in PBS on PSPMMA PAA core shell latex particles
o75 25
99
4-30 Adsorption isotherm of β M onto PS and PSPMMA latex particles in PB at 37 C and pH 74
2 100o 101
4-31 Adsorption isotherm of β M onto PS and PSPMMA PAA latex particles in PB at 37 C and pH 74
2 90 10o 101
4-32 Adsorption isotherm of β M onto PS and PSPMMA PAA latex particles in PB at 37 C and pH 74
2 75 25o 102
4-33 Gibbs free energy of adsorption of proteins on latex particles in PB at 37 C and pH 74
o
107
4-34 The kinetics of protein adsorption in PB on PS latex particles at 37 C and pH 74
o
109
4-35 The kinetics of protein adsorption in PB on PSPMMA latex particles at 37 C and pH 74
100o
109
4-36 The kinetics of protein adsorption in PB on PSPMMA PAA latex particles at 37 C and pH 74
90 10o 110
xii
4-37 The kinetics of protein adsorption in PB on PSPMMA PAA latex particles at 37 C and pH 74
75 25o 110
4-38 Image of red blood cells 113
4-39 Hemolysis caused by latex particles n=5114
xiii
Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy
SYNTHESIS AND ENGINEERING OF POLYMERIC LATEX PARTICLES FOR MEDICAL APPLICATIONS
By
Sangyup Kim
December 2005
Chair Hassan El-Shall Major Department Materials Science and Engineering
Latex particles with well-defined colloidal and surface characteristics have received
increasing attention due to their useful applications in many areas especially as solid
phase supports in numerous biological applications such as immunoassay DNA
diagnostic cell separation and drug delivery carrier Hemodialysis membrane using
these particles would be another potential application for the advanced separation
treatment for patients with end stage renal disease (ESRD) It is desirable to remove
middle molecular weight proteins with minimal removal of other proteins such as
albumin Thus it is necessary to understand the fundamental interactions between the
particles and blood proteins to maximize the performance of these membranes This
improvement will have significant economic and health impact
The objective of this study is to synthesize polymeric latex particles of specific
functionality to achieve the desired selective separation of target proteins from the human
blood Semi-continuous seed emulsion polymerization was used to prepare monodisperse
xiv
polystyrene seed particles ranging from 126plusmn75 to 216plusmn53 nm in size which are then
enlarged by about 800nm Surfactant amount played a key role in controlling the latex
particle size Negatively charged latex particles with a different hydrophobicity were
prepared by introduction of a sodium persulfate initiator and hydrophilic acrylic acid
monomer The prepared polymeric particles include bare polystyrene (PS) particles less
hydrophobic PS core and PMMA shell particles and more hydrophilic PS core and
PMMA-co-PAA shell latex particles with a 370nm mean diameter SEM light scattering
and zeta potential measurements were used to characterize particle size and surface
properties Adsorption isotherms of two proteins bovine serum albumin (BSA) and β2-
microglobulin (β2M) on latex particles were obtained as a function of pH and ionic
strength using the bicinchoninic acid (BCA) assay method The Langmuir-Freundlich
adsorption model was used to determine the adsorption amount of protein at equilibrium
The thickness of adsorbed BSA layer on latex particles was obtained in order to
investigate the adsorption orientation such as end-on or side-on mode Adsorption
kinetics experiments for both proteins and all latex particles were also performed The
adsorption kinetic constant determined from the Langmuir-Freundlich adsorption
isotherm model was used to calculate Gibbs free energy of adsorption to compare the
competitive adsorption of BSA and β2M Hemolysis tests were performed to investigate
the blood compatibility of synthesized latex particles PSPMMA90PAA10 and
PSPMMA75PAA25 core shell latex particles had desirable material properties with not
only a large amount and high rate of selective β2M adsorption over BSA but also high
blood compatibility showing less than 3 hemolysis
xv
CHAPTER 1 INTRODUCTION
End stage renal disease (ESRD) is a chronic condition in which kidney function is
impaired to the extent that the patientrsquos survival requires removal of toxins from the
blood by dialysis therapy or kidney transplantation The National Kidney Foundation
estimates that over 20 million Americans had chronic kidney disease in 2002 [NKF] The
number of people with ESRD is rapidly increasing in the United States with
approximately 96 295 incidents and 406 081 prevalent patients including 292 215 on
dialysis and 113 866 with a functioning graft in 2001 It is projected that there will be
more than 22 million ESRD patients by 2030 [USRDS 2003] The expenditure for the
ESRD treatment program had reached $228 billion 64 of the Medicare budget in
2001 Due in part to the limited availability of kidneys for transplantation hemodialysis
is the primary clinical treatment for the patients with ESRD
The central element of a hemodialysis instrument is the semipermeable membrane
that allows for selective transport of low molecular weight biological metabolites less
than 5000 Da such as urea and creatinine as well as excess water and electrolytes [Baker
2004] from the blood One limitation of current dialysis technologies is the inability to
efficiently remove middle molecular weight toxins such as β2-Microglobulin (β2M) and
interleukin 6 (IL-6)
β2M is a causative protein of dialysis-related amyloidosis (DRA) a disease arising
in patients with chronic kidney failure as a serious complication of long-term
hemodialysis treatment [Gejyo et al 1985] β2M deposition in tissue is the primary cause
1
2
of destructive arthritis and carpal tunnel syndrome [Vincent et al 1992 Drueke 2000]
The β2M structure is shown in Figure 1-2
Figure 1-2 Stereo drawing of the αndashcarbon backbone of β2M [Becker et al 1985]
Although attempts have been made to increase the efficiency of middle molecular
weight toxin removal by changes in the membrane pore size and the use of innovative
materials to adsorb these toxins [Samtleben et al 1996 Ronco et al 2001] removal
efficiency is not as high as those achieved by a normal healthy kidney Traditional
membranes have a number of processing and performance limitations [Westhuyzen et al
1992 Leypoldt et al 1998] such as a restricted choice of surface chemistries and limited
control of porosity The development of novel engineering membrane technology is
needed to remove middle molecule toxins
Polymeric latex particles have received increasing attention in medical application
areas especially as solid phase supports in biological applications [Piskin et al 1994]
Examples of these applications include immunoassay [Chen et al 2003 Radomske-
Galant et al 2003] DNA diagnostic [Elaїssari et al 1998] cell separation drug delivery
carrier [Luck et al 1998 Kurisawa et al 1995 Yang et al 2000] etc This is because of
3
the well-defined colloidal and surface characteristics of the particles By using a seed
emulsion polymerization method it is possible to synthesize monodisperse latex particles
with various particle size ranges and surface chemistry Functionalized core-shell latex
particles can be introduced by multi-step emulsion polymerization Core particles are
synthesized in the first stage of the polymerization and the functional monomer is added
in the second stage This is done without any emulsifier addition to prevent the
production of new homopolymer particles [Keusch et al 1973] The core-shell particles
are useful in a broad range of applications because of their improved physical and
chemical properties over their single-component counterparts [Lu et al 1996 Nelliappan
et al 1997] Through the development of a hemodialysis membrane using monodisperse
latex particles improvements in advanced separation treatment for patients with end
stage renal disease (ESRD) can be realized This requires the maximum removal of
middle molecular weight proteins with minimal removal of other beneficial proteins such
as albumin Thus an understanding of the fundamental interactions between the particles
and biopolymers is vital to maximize the performance of this membrane technology
The field of material science and biotechnology is based on fundamental chemistry
has developed over the past three decades into todayrsquos powerful discipline that enables
the development of advanced technical devices for pharmaceutical and biomedical
applications This novel and highly interdisciplinary field is closely associated with both
the physical and chemical properties of organic and inorganic particles [Niemeyer 2001]
Hemodialysis membrane using these particles would lead to improvements in the
advanced separation treatment for patients with end stage renal disease (ESRD) The
interdisciplinary nature of this approach enables a more complete understanding of the
4
phenomena of protein adsorption and the material properties necessary for selective
separation This innovative approach to membrane fabrication has the potential of making
inexpensive highly efficient membranes for both industrial and specialized separation
processes
The goal of this study is to prepare polymeric latex particles with tailored
properties to maximize separation of toxin molecules and to investigate the fundamental
interactions between the particles and molecules in the biological system in order to
optimize the performance of a membrane material for these applications
Polymeric latex particles were synthesized with specific functionality in an attempt
to achieve selective separation Seeded emulsion polymerization was used to synthesize
functionalized monodisperse latex particles in various sizes Negatively charged
hydrophobic polystyrene latex particles were synthesized by the same method Core shell
latex particles PSPMMA100 PSPMMA90PAA10 PSPMMA75PAA25 were also
synthesized to differentiate the degree of hydrophobicity of particles Scanning Electron
Micrograph (SEM) and light scattering measurements were used to characterize particle
size and shape and zeta potential measurements were conducted to measure the electrical
surface property of synthesized particles Adsorption isotherms of target proteins bovine
serum albumin (BSA) and β2M on latex particles were obtained as a function of pH
ionic strength and protein concentrations using the BCA assay method Adsorption
kinetics for both proteins on the latex particles were also measured Finally hemolysis
tests were run to determine the biocompatibility of polymer latex particles with human
blood This research will be described in more detail in chapters 3 and 4
CHAPTER 2 BACKGROUND AND LITERATURE SURVEY
21 The Significance of End Stage Renal Disease (ESRD)
The kidneys are responsible for removing excess fluid minerals and wastes from
the blood regulating electrolyte balance and blood pressure and the stimulation of red
blood cell production They also produce hormones such as erythropoietin (EPO) and
calcitriol that keep bones strong and blood healthy [Casadevall and Rossert 2005 Kurbel
et al 2003] EPO acts on the bone marrow to increase the production of red blood cell in
case of bleeding or moving to high altitudes Calcitriol mainly acts on both the cells of
the intestine to promote the absorption of calcium from food and also bone to mobilize
calcium from the bone to the blood When kidneys fail harmful waste builds up blood
pressure rises and body retains excess fluid The body also does not make enough red
blood cells When this happens treatment is needed to replace the function of failed
kidneys
Figure 2-1 An image of the location and cross section of a human kidney
5
6
The National Kidney Foundation estimates that over 20 million Americans had
chronic kidney disease in 2002 [NKF] Chronic renal disease is a gradual and progressive
loss of the function unlike acute renal disease where sudden reversible failure of kidney
function occurs Chronic renal failure usually takes place over a number of years as the
internal structures of the kidney are slowly damaged In the early stages there can be no
symptoms In fact progression may be so gradual that symptoms do not occur until
kidney function is less than one-tenth that of a normal health kidney If left untreated
chronic kidney disease may ultimately lead to kidney failure known as End Stage Renal
Disease (ESRD) ESRD is a rapidly growing heath-care problem in the United States In
2001 approximately 96295 incidents and 406081 prevalent patients were diagnosed
with ESRD including 292215 patients on dialysis and 113866 patients with a
functioning graft [USRDS 2003] The projected number of patients with ESRD is
expected to exceed 22 million patients by 2030 with much of this growth being driven by
the increasing prevalence of major contributing factors such as diabetes and high blood
pressure [USRDS 2003] A great extent of ESRD program cost and Medicare budget
have been spent in 2001 Due in part to a limited availability of kidneys for
transplantation hemodialysis (HD) is the primary method of treatment for ESRD and is
currently used for approximately 61 of US ESRD patients
22 Hemodialysis (HD) Treatment
HD removes toxins from the body by extracorporeal circulation of the blood
through a semipermeable membrane referred to as a dialyzer The toxins are removed
primarily by diffusion across the membrane to a dialysate solution which is circulated on
the opposite side of the membrane The cleaned blood is then returned to the blood
stream A surgically constructed vascular access connects the extracorporeal circuit to the
7
patientrsquos system Treatments are almost always performed three times per week in
specially equipped dialysis facilities for 3-4 hours per each treatment Figure 2-2 shows
the scheme of the hemodialysis route The critical element of a HD instrument is the
semipermeable membrane which allows for selective transport of low molecular weight
biological metabolites from blood
igure 2-2 A schematic draw of the hemodialysis route
ESRD patients who undergo dialysis therapy often experience several other
problems associated with the treatment such as anemia fatigue bone problems joint
problems itching sleep disorders and restless legs Anemia is common in patient with
kidney disease because the kidneys produce the hormone erythropoietin (EPO) which
stimulates the bone marrow to produce red blood cells Diseased kidneys often do not
produce enough EPO to stimulate the bone marrow to make a sufficient amount of red
blood cells This leads to bone disease referred to as renal osteodystrophy and causes
bones to become thin and weak or malformed and can affect both children and adults
Older patients and women who have gone through menopause are at greater risk for this
disease Uremic toxins which cannot be remove from the blood by the current dialyzer
membranes can lead to itching This problem can also be related to high levels of
F
8
parathyroid hormone (PTH) Dialysis patients can also experience day-night revers
is they have insomnia at night and sleep during the day This can be related to possible
nerve damage in the body and a chemical imbalance in the blood due to the excess toxin
Oxidative stress is a problem for patients on maintenance dialysis This problem is due to
an imbalance between pro- and antioxidant factors [Roselaar et al 1995 Cristol et al
1994] Oxidative stress affects oxidation of low-density lipoproteins which are the main
factor for atherogenesis [Huysmans et al 1998] and is also involved in the development
of malignancies and diabetes mellitus [Rice-Evans et al 1993] In order to reduce
antioxidant defense dialysis is needed to contribute to help stimulate free radical
production or eliminate antioxidants Dialysis-related amyloidosis (DRA) is also a
common and serious problem for people who have been on dialysis for more than 5
years DRA develops when proteins in the blood deposit on joints and tendons causi
pain stiffness and fluid in the joint as is the case with arthritis Normally the healthy
kidneys can filter out these proteins but dialysis filters are not as effective
23 Advances in Membrane Technology
al that
s
ng
Dialysis for blood of ESRD
Hemo filtration
s
ysis using a cellulose based membrane occurred
1913 John Abel [1990] from the Johns Hopkins Medical School described a method
purification is widely used in the treatment
dialysis (HD) techniques use a semi-permeable membrane to replace the
role of the kidney The membranes used in HD can be broadly classified into those based
on cellulose and those manufactured from synthetic copolymers These membranes come
in various shapes such as sheets tubular structures or hollow fiber arrangements The
hollow fiber type is the most popular and is incorporated into over 800 different device
in world wide [Ronco et al 2001]
The first attempt at blood dial
9
where sing
e
om
ysis was performed until the mid 1960 The basic
molec
rial in
een
benzyl)
was used for the membrane with
hepar
by the blood of a living animal may be submitted to dialysis outside the body u
a membrane based on cellulose and returned to the natural circulation without exposur
to air infection by microorganisms or any alteration that would necessarily be prejudicial
to life This same technique is still used to today however the device used has been
modified over the years as better membranes were developed and the anti-coagulant
heparin has become available
Cellulose membranes have been widely used for the treatment of renal failure fr
1928 when the first human dial
ular structure of cellulose is made of a long chain containing hydroxyl (OH)
groups The realization that such groups imparted undesirable qualities on the mate
respect to blood contact behavior was discovered in the early 1970s and since has b
the focus of development These modified cellulose membranes used the partial
substitution of benzyl groups to replace the proton of the hydroxyl groups in an attempt
to reduce their negative effect The result is a molecular mosaic of hydrophobic (
and hydrophilic (hydroxyl and cellulose) regions
Kolff [Van Noordwijk 2001] studied the rotating drum artificial kidney for patients
with acute renal failure in 1943 Cellophane tubing
in as the anticoagulant For the next 17 years hemodialysis therapy was performed
by this method but only for the patients with acute reversible renal failure Vascular
access required repeated surgical insertions of cannulas (slender tubes) into an artery and
vein and limited the number of treatments for a patient could receive in order to
minimize the amount of vascular damage
10
Initially the need for dialysis in patients with acute renal failure was determ
mainly by the development of signs and sym
ined
ptoms of uremia After dialysis some time
might
et al
ent improvement In 1960 the development of a shunt [Quinton et al
1960]
fone (PSf) polyamide (PA) and polyacrylonitrile (PAN) by phase inversion or
precip
lidone
e
elapse before uremic manifestations returned to warrant a sequential treatment of
dialysis Many patients with acute renal failure secondary to accidental or surgical
trauma were hypercatabolic but the interdialytic interval might be prolonged because of
anorexia or use of a low-protein diet However Teschan and his coworkers [Obrien
1959] showed that patient well-being and survival were improved by what they termed
prophylactic daily hemodiaysis or administration of the treatment before the patient
again became sick with uremia Their report in 1959 was the first description of daily
hemodialysis
Development of membrane accessories such as a shunt has also been an area of
focus for treatm
a flexible polytetrafluoroethylene (PTFE or Teflonreg) tubing made many more
hemodialysis treatments possible for chronic kidney failure patients PTFE has a non-
stick surface and is relative biocompatibility leading to minimized blood clotting in the
shunt
Synthetic membranes are prepared from engineered thermoplastics such as
polysul
itation of a blended mixture resulting in the formation of asymmetric and
anisotropic structures Figure 2-3 shows a fiber type of the Polyflux S membrane
consisting of poluamide (PA) polyacrylethersulfone (PAES) and polyvinylpyrro
(PVP) with the integral three-layer structure The skin layer on the inside fiber typ
membrane contacts blood and has a very high surface porosity and a narrow pore size
11
distribution This layer constitutes the discriminating barrier deciding the permeabili
and solute retention properties of the membrane The skin layer is supported by thick
sponge-type structure larger pores providing mechanical strength and very low
hydrodynamic resistance
ty
Figure 2-3 Cross-sectional SEM image view of the Polyflux S (polyamide +
polyacrylethersulfone + polyvinypyrollidone) dialysis membranes [Deppisch et al 1998]
n
[Malchesky 2004] because of its thermal stability mechanical strength and chemical
inertn ciated
ls
m
PSf is a widely used membrane material for the hemodialysis applicatio
ess According to a report from the National Surveillance of Dialysis-Asso
Disease (NSDAD) in the US over 70 of hemodialysis membranes were PSf based
[Bowry 2002] This is most likely because PSf has many advantages over other materia
This synthetic polymer is one of few materials that can withstand sterilization by stea
ethylene oxide and γ-radiation PSf membrane can be prepared by conventional
immersion precipitation methods into many different shapes including porous hollow
12
fiber or flat sheet hemodialysis membranes The material also has a high permeab
low molecular weight proteins and solute and high endotoxin retention The chemical
structure of PSf is shown in Figure 2-4
ility to
Figure 2-4 The chemical structure of polysulfone (PSf)
There is one major disadvantage to PSf The hydrophobic nature of the PSf causes
ment alternative pathway
leadin
s not
3
et al
in
O acrylate
serious complications through the activation of the comple
g to the adsorption of serum proteins onto the membranes [Singh et al 2003]
Anticoagulants are added during dialysis therapy to avoid blood clotting but this doe
completely eliminate the problem In order to overcome this disadvantage of the PSf
membrane various studies have been performed to change the materialrsquos surface
properties These investigations include hydrophilic polymer coating [Brink et al 199
Kim et al 1988 Higuchi et al 2003] layer grafting onto PSf membrane [Wavhal
2002 Song et al 2000 Pieracci et al 2002 Mok et al1994] and chemical reaction of
hydrophilic components onto the membrane surface [Higuchi et al 1990 1991 1993
Blanco et al 2001 Nabe et al 1997 Guiver et al 1993] Hydrophilic monomers 2-
hydroxy-ethylmethacrylate (HEMA) acrylic acid (AA) and methacrylic acid (MMA)
have also been grafted onto PSf membrane to increase flux and Bovine Serum Album
(BSA) retention [Ulbricht et al 1996] Hancock et al [2000] synthesized
polysulfonepoly(ethylene oxide) (PEO) block copolymers to improve the resistance to
platelet adhesion Kim et al [2003] also studied blending a sulfonated PE
diblock copolymer into PSf in order to reduce platelet adhesion and enhance
13
biocompatibility PEO is a commonly used biomaterial due to its excellent resistance
protein adsorption and inherent biocompatibility [Harris 1992] Kim et al [20
a self-transformable copolymer to enhance the hydrophilicity of an asymmetric PSf
membrane with an ultra-thin skin layer The polymer had an entrapped diblock
copolymer containing a hydrophilic block of poly (ethylene glycol) (PEG)-SO
to
05] studied
ate
lic graft
l)
e of thier
aromatic
orand
3 acryl
and a hydrophobic block of octadecylacrylate (OA) Molecular dynamic (MD)
simulations were performed as a function of copolymer density to optimize interfacial
structure information McMurry [2004] developed a strategy using an amphiphi
copolymer added to PSf membranes by introducing polysulfone-g-poly (ethylene glyco
When compared to unmodified PSf these graft copolymer and resulting blend
membranes are found to hold promise for biomedical device applications
Polyamide (PA) membranes have also been used for hemodialysis becaus
mechanical strength in both wet and dry conditions Polyamide consists of
aliphatic monomers with amide bonding (-CONH-) also known as a peptide bond
The basic amide bond in polyamide is shown in Figure 2-5 R1 and R2 can be either
aromatic or aliphatic linkage group
C N
H
O
~ R R1 2~C N
H
O
~ R R1 2~
Figure 2-5 The chemical structure of polyamide (PA)
Panichi and co-workers [1998] evaluated the biocompatibility of the PA membrane
ompatible technique due to the
use of a synthetic membrane with a sterile re-infusion fluid and the convective removal of
and concluded that PA hemofiltration was a highly bioc
14
the ac
e
e
is
n
is a semi-crystalline polymer and the mechanical properties
strong
2
CN
tivated anaphylatoxins and β2-Microglobulin (β2M) The PA based membrane
Polyfluxreg (manufactured by Gambro GmbH Germany) blended with polyamide
polyarylethersulfone and polyvinylpyrrolidone (PVP) was able to clean small molecules
such as urea dreatinine and phosphate as well as decrease β2M amount by 502
[Hoenich et al 2002] Due to the non-selectivity of the membrane removal of thes
unwanted materials also led to the undesirable loss of beneficial proteins during therapy
Meier et al [2000] evaluated different immune parameters using a modified cellulos
low-flux hemophan and synthetic high-flux PA membrane during a 1 year period in
chronic hemodialysis patients They found that the 1-year immunological evaluation of
hemodiaysis membrane biocompatibility was associated with changes in the pattern of
chronic T-cell actiovation
Polyacrylonitrile (PAN) is another commonly used membrane material because it
inherently hydrophilic and has been commercialized for ultrafiltration and microfiltratio
[Scharnagl et al 2001] PAN
ly depend on the crystalline structures The chemical structure of PAN is shown in
figure 2-6
CH CH
n2
CN
CH CH n
Chemical structure of polyacrylonitrile (PAN)
The addition of additives such as polyvinylpyrrolidone (PVP) as a pore forming
agent gives PAN membranes more flexible processing parameters and increased
e has been optimized through
copolymerization with many other vinyl monomers including glycidyl methacrylate
Figure 2-6
performance [Jung et al 2005] PAN membrane performanc
15
[Godj
acid
for
for
a non-
solub this
in
oid underneath the inner
surface of membrane decreased by increasing the amount of solvent DMSA in the
evargova et al 1999 Hicke et al 2002] N-vinylimidazole [Godjevargova et al
2000] hydroxyl ethyl methacrylate [Ray et al 1999 Bhat et al 2000] methacrylic
[Ray et al 1999] vinyl pyrrolidone [Ray et al 1999] acrylic acid [Trotta et al 2002]
acrylamide [Musale et al 1997] and vinylchloride [Broadhead et al 1998] These
monomers provide a reactive group for enzyme immobilization improved mechanical
strength solvent-resistance pervaporation permeation flux anti-fouling and bio-
compatibility Because of this PAN-based copolymer membranes have great potential
the treatment of hemodialysis in an artificial kidney This material can also be used
other applications like the treatment of wastewater the production of ultra-pure water
biocatalysis together with separation and methanol separation by pervaporation
Lin and his coworker [2004] studied the modification of PAN based dialyzer
membranes to increase the hemocompatibility by the immobilization of chitosan and
heparin conjugates on the surface of the PAN membrane When a foreign material is
exposed to blood plasma proteins are adsorbed clotting factors are activated and
le fibrin network or thrombus is formatted [Goosen et al 1980] The result of
research was that the biocompatible chitosan polymer and a blood anticoagulant hepar
prevented blood clotting They showed prolonged coagulation time reduced platelet
adsorption thrombus formation and protein adsorption
Nie et al [2004] studied PAN-based ultrafiltration hollow-fiber membranes
(UHFMs) In order to improve the membrane performance acrylonitrile (AN) was
copolymerized with other functional monomers such as maleic anhydride and α-allyl
glucoside They found that the number and size of macrov
16
intern erum
and
d a
rface The second step is the
transf after
yjak et
embrane
1978 Malchesky et al 1978] has been further developed to increase the efficiency of
al coagulant The water flux of the UHFMs also decreased while the bovine s
albumin rejection increased minutely Godjevargova et al [1992] modified the PAN
based membrane with hydroxylamine and diethylaminoethylmethacrylate to improve
membrane dialysis properties Formed functional groups like primary amine oxime
tertiary amine groups provided the membrane with more hydrophilic properties an
substantial increase in the permeability of the membranes
The wide use of filtration in practice is limited by membrane fouling Solute
molecules deposit on and in the membrane in the process of filtration causing dramatic
reduction in flux through the membrane Fouling occurs mostly in the filtration of
proteins Three kinetic steps are involved in the fouling of UF membranes according to
Nisson [1990] The first step is the transfer of solute to the su
er of solute into the membrane until it either finally adsorbs or passes through
a set of adsorption-desorption events The third step includes surface binding
accompanied by structural rearrangement in the adsorbed state [Ko et al 1993] Br
al [1998] studied the surface modification of a commercially available PAN membrane
to develop superior filtration properties with less fouling by proteins The PAN
membrane was immersed in excess NaOH solution to convert some of the surface nitrile
groups into carboxylic groups by the hydrolysis process This modified PAN m
was not so severely fouled in the Bovine Serum Albumin (BSA) filtration test The pore
size however decreased during the hydrolysis process leading to a significant reduction
in flux and made the membrane less productive in the ultrafiltration (UF) mode
24 Sorbent Technology
Over the last three decades sorbent technology [Castino et al 1976 Korshak et al
17
dialysis or replace it for the treatment of ESRD Sorbents remove solutes from solution
through specific or nonspecific adsorption depending on both the nature of the solute and
the sorbent Specific adsorption contains tailored ligands or antibodies with high
selectivity for target molecules een used in autoimmune
disord
and
e
al 1996] various resins [Ronco et al 2001]
album
e
as
Specific adsorbents have b
ers such as idiopathic thrombocytopenic purpura [Snyder et al 1992] and for the
removal of lipids in familial hyper cholesterolemia [Bosch et al 1999] Nonspecific
adsorbents such as charcoal and resins attract target molecules through various forces
including hydrophobic interactions ionic (or electrostatic) attraction hydrogen bonding
and van der Waals interactions
New dialysate with sorbents has become an accepted modification of dialysis
sorbent hemoperfusion is gaining ground as a valuable addition to dialysis especially as
new sorbents are developed [Winchester et al 2001] Hemoperfusion is defined as th
removal of toxins or metabolites from circulation by the passing of blood within a
suitable extracorpoteal circuit over semipermeable microcapsules containing adsorbents
such as activated charcoal [Samtleben et
in-conjugated agarose etc Novel adsorptive carbons with larger pore diameters
have been synthesized for potential clinical use [Mikhalovsky 1989] Newly recognized
uremic toxins [Dhondt et al 2000 Haag-Weber et al 2000] have resulted in several
investigations on alternatives to standard or high-flux hemodialysis to remove thes
molecules These methods include hemodiafilteration with [de Francisco et al 2000] or
without [Ward et al 2000 Takenaka et al 2001] dialysate regeneration using sorbents
well as hemoperfusion using such adsorbents as charcoal and resins
18
Kolarz et al [1989 1995] studied the hyper-crosslinked sorbent prepared from
styrene and divinylbenzene (DVB) for a hemoperfusion application They found that the
pore structure of a swelling sorbent was changed by additional crosslinking with α αrsquo-
dichloro-p-xylene in the presence of a tin chloride catalyst and in a di
chloroethane
soluti and
en
decyl groups that attract β2M through a hydrophobic interaction The
adsor
of
5]
d
penet
on They also realized that the hemocompatibility was useful for nemoperfusion
could be imparted to the sorbents by introducing sulfonyl groups at a concentration of
about 02mmolg
A special polymeric adsorbing material (BM-010 from Kaneka Japan) has be
investigated by another group [Furuyoshi et al 1991] for the selective removal of β2M
from the blood of dialysis partients The adsorbent consists of porous cellulose beads
modified with hexa
ption capacity of this material is 1mg of β2M per 1ml of adsorbent Using a
hemoperfusion cartridge containing 350ml of these cellulose beads in sequence with a
high-flux hemodialyzer several small clinical trials were performed During 4-5 hours
treatment about 210mg of β2M were removed thus reducing the concentration in the
blood by 60-70 of the initial level [Nakazawa et al 1993 Gejyo et al 1993 199
RenalTech developed a hemoperfusion device BetaSorbreg containing the hydrate
cross-linked polystyrene (PS) divinylbenzene (DVB) resin sorbents with a pore structure
designed to remove molecules between 4 and 30 kDa [Winchester et al 2002] In this
case solute molecules are separated according to their size based on their ability to
rate the porous network of the beaded sorbents The resin beads were prepared with
a blood compatible coating and confirmed to be biocompatible in vivo in animals
[Cowgill et al 2001]
19
25 Limitation of Current Hemodialysis Treatment
Hemodialysis is a widely used life-sustaining treatment for patients with ESRD
However it does not replace all of the complex functions of a normal healthy kidne
a result patients on dia
y As
lysis still suffer from a range of problems including infection
accelerated cardio ion anemia
chron
e
se middle
alysis
sessio
s
f
vascular disease high blood pressure chronic malnutrit
ic joint and back pain and a considerably shortened life span One significant
limitation of the current dialysis technology is the inability to efficiently remove larger
toxic molecules This is mainly because of the broad pore size distribution reducing th
selective removal of toxins and unsatisfied biocompatibility causing lots of
complications such as inflammation blood clotting calcification infection etc
Dialysis purifies the patients blood by efficiently removing small molecules like
salts urea and excess water However as toxic molecules increase in size their removal
rate by hemodialysis substantially declines Typically only 10 - 40 of the
molecular weight toxins (300-15000 Da) are removed from the blood during a di
n [Vanholder et al 1995] These toxins then reach an abnormally high level and
begin to damage the body One such toxin β2M causes destructive arthritis and carpal
tunnel syndrome by joining together like the links of chain to form a few very large
molecules and deposit damaging the surrounding tissues [Lonnemann et al 2002] This i
also a main cause of mortality for long-term dialysis patients Other middle molecule
toxins appear to inhibit the immune system and may play a significant role in the high
susceptibility to infections in dialysis patients Still others are believed to impair the
functioning of several other body systems such as the hematopoietic and other endocrine
systems This may contribute to accelerated cardiovascular disease the leading cause o
20
death among dialysis patients as well as clinical malnutrition which affects up to 50 of
this patient population
Over the last decade polymeric dialysis membranes have been developed to
increase the capacity for removing middle molecular weight toxins by changing the pore
size of dialyzer membranes and using new materials that adsorb these toxins for
impro se
styrene made during World War II in the United
States The Dow chemical compan anufacturer of polystyrene
includ
d of
on
rug
ntrol of
ved removal characteristics However removal efficiency is not as high as tho
achieved by a normal healthy kidney
26 Latex Particles
The first synthetic polymer synthesized using emulsion polymerization was a
rubber composed of 13-butadiene and
y has been a major m
ing latex which they used in paint formulations The theory of emulsion
polymerization in which a surfactant is used was established by Harkins [1948] and by
Smith and Ewart [1948] By 1956 the technology was complete including the metho
building larger diameter particles from smaller ones The product by this emulsi
polymerization is referred to as latex a colloidal dispersion of polymer particles in water
medium [Odian 1991] Latexes are currently undergoing extensive research and
development for a broad range of areas including adhesives inks paints coatings d
delivery systems medical assay kits gloves paper coatings floor polish films carpet
backing and foam mattresses to cosmetics The relatively well known and easy co
the emulsion process is one of main advantages for these applications Therefore
polymeric latex particle prepared by emulsion polymerization can be a candidate for the
medical applications because of the easy control of the particle size and morphology as
well as flexible surface chemistry to be required
21
261 The Components for Emulsion Polymerization
The main components for emulsion polymerization process are the monomer a
dispersing medium a surfactant and an initiator Available monomers are styrene
sing medium is usually water
which
ticles
n
ation To
monomer droplets but in micelles because the initiators are
insolu le
and
olution
butadiene methylmethacylate acryl acid etc The disper
will maintain a low solution viscosity provide good heat transfer and allow
transfer of the monomers from the monomer droplets into micelles and growing par
surrounded by surfactants respectively The surfactant (or emulsifier) has both
hydrophilic and hydrophobic segments Its main functions is to provide the nucleatio
sites for particles and aid in the colloidal stability of the growing particles Initiators are
water-soluble inorganic salts which dissociate into radicals to initiate polymeriz
control the molecular weight a chain transfer agent such as mercaptan may be present
262 Particle Nucleation
Free radicals are produced by dissociation of initiators at the rate on the order of
1013 radicals per milliliter per second in the water phase The location of the
polymerization is not in the
ble in the organic monomer droplets Such initiators are referred to as oil-insolub
initiators This is one of the big differences between emulsion polymerization
suspension polymerization where initiators are oil-soluble and the reaction occurs in the
monomer droplets Because the monomer droplets have a much smaller total surface area
they do not compete effectively with micelles to capture the radicals produced in s
It is in the micelles that the oil soluble monomer and water soluble initiator meet and is
favored as the reaction site because of the high monomer concentration compared to that
in the monomer droplets As polymerization proceeds the micelles grow by the addition
of monomer from the aqueous solution whose concentration is refilled by dissolution of
22
monomer from the monomer droplets There are three types of particles in the emulsion
system monomer droplets inactive micelles in which polymerization is not occurring
and active micelles in which polymerization is occurring referred to as growing polymer
particles
The mechanism of particle nucleation occurs by two simultaneous processes
micellar nucleation and homogeneous nucleation Micellar nucleation is the entry of
radicals ei
ther primary or oligomeric radicals formed by solution polymerization from
the aq ed
the
r
s
n
is coagulation with other particles rather than
polymerization of monomer The driving force for coagulation of precursor particles
ueous phase into the micelles In homogeneous nucleation solution-polymeriz
oligomeric radicals are becoming insoluble and precipitating onto themselves or onto
oligomers whose propagation has ended [Fitch et al 1969] The relative levels of micella
and homogeneous nucleation are variable with the water solubility of the monomer and
the surfactant concentration Homogeneous nucleation is favored for monomers with
higher water solubility and low surfactant concentration and micellar nucleation is
favored for monomers with low water solubility and high surfactant concentration It has
also been shown that homogeneous nucleation occurs in systems where the surfactant
concentration is below CMC [Roe 1968] A highly water insoluble monomer such a
styrene [Hansen et al 1979 Ugelstad et al 1979] has probably created by micellar
nucleation while a water soluble monomer such as vinyl acetate [Zollars 1979] has bee
formed by homogeneous nucleation
A third latex formation reaction mechanism has been proposed referred to as
coagulative nucleation In this reaction the major growth process for the first-formed
polymer particles (precursor particles)
23
sever les
yer
es
r a
l
the beginning of the polymerization As soon as the initiator is
gins with the formation and
growt
and
ved
added at one time during the later stages of the polymerization prior to complete
al nanometers in size is their relative instability compared to larger sized partic
The small size of a precursor particle with its high curvature of the electrical double la
permits the low surface charge density and high colloidal instability Once the particl
become large enough in size maintaining the high colloidal stability there is no longe
driving force for coagulation and further growth of particles takes place only by the
polymerization process
263 Types of Processes for Emulsion Polymerization
There are three types of production processes used in emulsion polymerization
batch semi-continuous (or semi-batch) and continuous In the batch type process al
components are added at
added and the temperature is increased polymerization be
h of latex particles at the same time There is no further process control possible
once the polymerization is started In the semi-continuous emulsion polymerization
process one or more components can be added continuously Various profiles of particle
nucleation and growth can be generated from different orders of component addition
during polymerization There are advantages to this process such as control of the
polymerization rate the particle number colloidal stability copolymer composition
particle morphology In the continuous process the emulsion polymerization components
are fed continuously into the reaction vessel while the product is simultaneously remo
at the same rate High production rate steady heat removal and uniform quality of
latexes are advantages of the continuous polymerization processes
Other available methods include the intermittent addition and shot addition of one
or more of the components In the shot addition process the additional components are
24
conversion of the main monomer This method has been used successfully to develo
water-soluble functional monomers such as sodium styrene sulfonat
p
e [Kim et al 1989]
264
lsion
Chemistry of Emulsion Polymerization
Emulsion polymerization is one type of free radical polymerization and can be
divided into three distinct stages initiation propagation and termination The emu
polymerization system is shown in Figure 2-7
Aqueous phaseAqueous phase
Micelle withmonomer
I Rbull
Emulsifier
Polymer particle swollenWith monomer
Monomerdroplet
I Rbull
Micelle withmonomer
I Rbull
Emulsifier
Polymer particle swollenWith monomer
MonomerdropletMonomerdroplet
I Rbull
Figure 2-7 Emulsion polymerization system [Radicals (Rmiddot) are created from initiators
(I) Monomer is transferred from larger monomer droplet into micelles by emulsifier Initiated polymer particle by radicals is keep growing until monomers are all consumed The reaction is performed in aqueous media]
In the initiation stage free radicals are created from an initiator by heat or an
ultraviolet radiation source The initiator with either peroxides groups (-O-O-) such as
odium persulfate or azo groups (-N=N-) such as azobisisobutyronitrile is commonly
used for em t
with the m er
chain ere
s
ulsion polymerization The primary free radicals created from initiator reac
onomer for initiation of polymerization In the propagation stage the polym
grows by monomer addition to the active center a free radical reactive site Th
are two possible modes of propagations head-to-head addition and head-to-tail addition
25
The head-to-tail mode is the predominant configuration of the polymer chain a result o
steric hindrance of the substitute bulky group In the termination stage polymer chain
growth is terminated by either coupling of two growing chains forming one polymer
molecule or transferring a hydrogen atom (dispropotionation) from one growing chain to
another forming two polymer molecules one having a saturated end group and the other
with an unsaturated end-group
265 Seed Emulsion Polymerization
Polymer latex particles have received increasing interest because of the versatili
of the many applications heterophase polymerization processes like emulsion dispersion
micro-emulsion seeded emulsion precipitation etc Especially to prepare well-defined
microspheres having monodispe
f
ty
rse and various particle sizes as well as surface group
ed particles prepared by emulsion polymerization
and e
s
hological design In
seede to control
n
functionalilties it is necessary to use se
nlarge them to a desired size in the further stages of reactions
Polymeric particles are required to have a uniform particle size in many
applications such as chromatography where they are used as a packing material
Morphological control of latex particle is also important for many practical application
[Schmidt 1972] Seed emulsion polymerization (or two-stage emulsion polymerization) is
a useful method to achieve both monodisperse particle size and morp
d emulsion polymerization [Gilbert 1995] preformed lsquoseedrsquo latex is used
the number of particles present in the final latex The advantage of seeded emulsio
polymerization is that the poorly reproducible process of particle nucleation can be
bypassed so that the number concentration of particles is constant and known Various
mechanisms have been proposed for the growth of latex particles [Ugelstad et al 1980
Okubo et al 1992] using this polymerization technique The initial seed particle
26
preparation step is well known and relatively easy to perform because at the relative
small particle sizes (02 to 05 micron) the particle growth process can be readily
controlled by the use of an emulsifier Enough emulsifier is used to prevent coagulation
but not enough to cause the formation of new particles As the particles are grown to
larger sizes in successive seeding steps it becomes increasingly difficult to mainta
stable uncoagulated emulsion without forming new particles and thereby destroying
monodisperisty of the latex Recently there have been several reports concerning t
reaction kinetics of seed emulsion polymerization and the development of latex
morphologies over the course of the reaction [Chern et al 1990 Delacal et al 1990 L
et al 1995 Lee 2000 2002]
266 Polystyrene Latex Particles
A number of papers have described the synthesis of the polystyrene latex particl
bearing various functional surface groups such as carboxyl [Lee et al 2000 Tun
2002 Reb et al 2000] hydroxyl [Tamai et al 1989] marcapto [Nilson 1989] epoxy
[Shimizu et al 2000 Luo et al
ly
in a
he
ee
es
cel et al
2004] acetal [Izquierdo et al 2004 Santos et al 1997]
ometyl [Izquierdo et al 2004 Park et al 2001
Sarob
h the
d
thymine [Dahman et al 2003] chlor
e et al 1998] amine [Counsin et al 1994 Ganachaud et al 1997 Ganachaud et al
1995 Miraballes-Martinez et al 2000 2001 Anna et al 2005] ester [Nagai et al 1999]
etc In order to produce these functionalized particles different methods for particle
preparation must be used The polymer emulsion with core-shell morphology of latex
particles is one of them This is a multistep emulsion polymerization process in whic
polystyrene ldquocorerdquo particle is synthesized in the first stage and the functional monomer is
added in the second stage of the polymerization without any emulsifier postfeeding to
prevent the production of new homopolymer particles thus forming the functionalize
27
polymer ldquoshellrdquo on the ldquocorerdquo particle [Keusch et al 1973] There are requirements to
limit secondary nucleation and encourage core-shell formation in seeded emulsion
polymerization including the addition of smaller seed particles at high solid content to
increase particle surface area low surfactant concentration to prevent formation of
micelles and the semi-continuous addition of monomer to create a starved-feed conditio
and keep the monomer concentration low There are some advantages [Hergeth et al
1989] of dispersions with polymeric core-shell particles First it is possible to modi
interfacial properties of polymer particles in the aqueous phase by the addition of only
very small amounts of a modifying agent during the last period of the reaction Thus
these core-shell particles are useful in a broad range of applications since they always
exhibit improved physical and chemical properties over their single-component
counterparts [Lu et al 1996 Nelliappan et al 1997] In this way the improvement of
surface properties of such dispersions is straightforward and inexpensive The other is
that polymers with a core-shell structure are perfect model systems for investigating th
material properties of polymer blends and composites because of their regular
distribution of one polymer inside a matrix polymer and because of the simple sp
geometry of the system
Their properties usually depend on the structures of latex particles Chen and his
coworkers [Chen et al 1991 1992 1993] reported the morphological development of
core shell latex particles of polystyrenepoly(methyl methacrylate) during
polymerization Before the research by Chen and his coworker Min et al [Min et al
1983] reported the morph
n
fy the
e
herical
ological development of core shell latex of polystyrene
(PS)polybuthyl acrylateat (PBA) by seeded emulsion polymerization as a function of the
28
additi
of
ity of graft
ing reagents Thus protein adsorption on polymeric solid surfaces has
st are microspheres defined as ldquofine
polym can be
n
r was
containing antigen urine serum etc The latex will become agglutinated and visibly
on method of PS They found that the percentage of grafting PS to the PBA was
greatest for the batch reaction and the PBA-PS core-shell particles with a high degree
grafting remained spherical upon aging test because of the emulsifying abil
copolymer
267 Various Applications of Latex Particles
Latex particles are applicable to a wide range of areas such as biomedical
applications [Piskin et al 1994] especially as the solid phase such as in immunoassays
[Chern et al 2003 Radomske-Galant et al 2003] DNA diagnostic drug delivery carriers
[Luck et al 1998 Kurisawa et al 1995 Yang et al 2000] blood cell separations and
column pack
become a center of attention Of particular intere
er particles having diameters in the range of 01 to several micronsrdquo which
used as functional tools by themselves or by coupling with biocompounds Singer and
Plotz [1956] firstly studied microsphere or latex agglutination test (LATrsquos) by using
monodisperse polystyrene (PS) and polyvinyltoluene polymer particles as the support o
which the biomolecules were going to adsorb The biomolecule adsorption howeve
limited by possible desorption of the adsorbed species or loss of specific activity of the
complex formed Since this work was published the latex particle applications for
immunoassay have been rapidly and widely studied and developed
Latex agglutination test or latex immunoassays start with tiny spherical latex
particles with a uniform diameter and similar surface properties The particles are coated
with antibodies (sensitized) through the hydrophobic interaction of portions of the protein
with the PS surface of the particles If sensitized particles are mixed with a sample
29
agglomerate Latex tests are inexpensive as compared with the other techniques [Ba
1988]
ngs
sed
tion Rheumatoid factor (RF) in different age subpopulations has also been
evalu
studied a
latex
Unipath [Percival 1996] has manufactured a range of immunoassay materials ba
on a chromatographic principle and deeply colored latex particles for use in the home and
clinical environments The latex particles which are already sensitized with a
monoclonal antibody can detect any antigens that bound to the surface of the latex
particles Some detectable pollutants include estrogen mimics which induce
abnormalities in the reproductive system of male fishes and lead to a total or partial male
feminiza
ated according to a patientrsquos clinical status by using a rapid slide latex agglutination
test for qualitative and semiquantitative measurement in human serum along with latex
immunoassay method [Onen et al 1998] Magalhaes and his coworkers [2004]
diagnostic method of contamination of male fishes by estrogen mimics using the
production of vitellogenin (VTG) as a biomarker This was based on a reverse
agglutination test developed with monoclonal antibodies specific to this biomarker
Premstaller and his coworkers [Premstaller et al 2000 2001] have prepared a porous
poly(styrene-divinylbenzene) (PS-DVB) polymer monolith to use for highly efficient
chromatographic separation of biomelecules such as proteins and nucleic acids They
used a porogen a mixture of tetrahydrofuran and decanol to fabricate a micropellicular
PS-DVB backbone Legido-Quigley and his colleagues [2004] have developed the
monolith column to obtain further chromatographic functionality to the column by
introducing chloromethylstyrene in place of styrene into the polymer mixture
30
Core shell type monodisperse polymer colloids have been synthesized by Sarobe
and Forcada [1998] with chloromethyl functionality in order to improve the biomolecu
adsorption through a two-step emulsion polymerization process They investigated the
functonalized particles by optimizing the experimental parameters of the functional
monomer including reaction temperature the amount and type of redox initiator sys
used the type of addition of the initiator system and the use of washing They c
le
tem
oncluded
that th
sis
serve as separation
chann
of
usually
along the polypeptide chain which contain positive or negative charges [Norde 1998]
e relation between the amount of iron sulfate and the persulfatebisulfite system
added should be controlled to obtain monodisperse particles and prevent the premature
coagulation of the polymer particles during the polymerization
A semi-continuous emulsion polymerization technique for latex particle synthe
was performed by McDonald and other researchers [Ramakrishnan et al 2004 Steve et
al 1999] They introduced a variety of particles sizes compositions morphologies and
surface modifications to fabricate latex composite membranes (LCMs) Arraying and
stabilizing latex particles on the surface of a microporous substrate form narrowly
distributed interstitial pores formed between the particles which
els They investigated the membrane performance using gas fluxes water
permeability and the retention characterization of dextran molecules From these tests
they concluded that the narrow discriminating layer made of the latex particles leads to a
highly efficient composite membrane
27 Proteins
Proteins are natural polyamides comprised of about 20 different α-amino acids
varying hydrophobicity [Norde 1998] Proteins are more or less amphiphilic and
highly surface active because of the number of amino acid residues in the side groups
31
Proteins are polymers of L-α-amino acids [Solomon and Fryhle 2000] The α refers to a
carbon with a primary amine a carboxylic acid a hydrogen and a variable side-chain
group designated as R Carbon atoms rent groups are asymmetric and can
exhib l
s
e
on
with four diffe
it two different spatial arrangements (L and D configurations) due to the tetrahedra
nature of the bonds The L refers to one of these two possible configurations Amino
acids of the D-configuration are not found in natural proteins and do not participate in
biological reactions Figure 2-8 shows the chiral carbon in 3-D as the L isomer Protein
consist of twenty different amino acids differentiated by their side-chain groups [Norde
1998] The side-chain groups have different chemical properties such as polarity charg
and size and influence the chemical properties of proteins as well as determine the
overall structure of the protein For instance the polar amino acids tend to be on the
outside of the protein when they interact with water and the nonpolar amino acids are
the inside forming a hydrophobic core
Figure 2-8 L-α-amino acid
The covalent linkage between two amino acids is known as a peptide bond A
peptide bond is formed when the amino group of one amino acid reacts with the carboxyl
group of another amino acid to form an amide bond through the elimination of water
This arrangement gives the protein chain a polarity such that one end will have a free
amino group called the N-terminus and the other end will have a free carboxyl group
called the C-terminus [Solomon and Fryhle 2000] Peptide bonds tend to be planar and
32
give the polypeptide backbone rigidity Rotation can still occur around both of the α-
carbon bonds resulting in a polypeptide backbone with different potential conformations
relative to the positions of the R groups Although many conformations are theoretically
possible interactions between the R-groups will limit the number of potential
nd to form a single functional conformation In other words
the conformation or shape of the protein is
α-helix
can interact with other
secondary structures within the sam
conformations and proteins te
due to the interactions of the chain side
groups with one another and with the polypeptide backbone The interactions can be
between amino acids that are close together as in a poly-peptide between groups that are
further apart as in amino acids or even on between groups on different polypeptides all
together These different types of interactions are often discussed in terms of primary
secondary tertiary and quaternary protein structure
The primary amino acid sequence and positions of disulfide bonds strongly
influence the overall structure of protein [Norde 1986] For example certain side-chains
will promote hydrogen-bonding between neighboring amino acids of the polypeptide
backbone resulting in secondary structures such as β-sheets or α-helices In the
conformation the peptide backbone takes on a spiral staircase shape that is stabilized by
H-bonds between carbonyl and amide groups of every fourth amino acid residue This
restricts the rotation of the bonds in the peptide backbone resulting in a rigid structure
Certain amino acids promote the formation of either α-helices or β-sheets due to the
nature of the side-chain groups Some side chain groups may prevent the formation of
secondary structures and result in a more flexible polypeptide backbone which is often
called the random coil conformation These secondary structures
e polypeptide to form motifs or domains (ie a
33
tertiary structure) A motif is a common combination of secondary structures and a
domain is a portion of a protein that folds independently Many proteins are composed o
multiple subunits and therefore exhibit quaternary structures
271 Interaction Forces between Proteins
Proteins in aqueous solution acquire compact ordered conformations In such a
compact conformation the movement along the polypeptide chain is severely restrict
implying a low conformational entropy The compact structure is possible only if
interactions within the protein molecule and interactions between the protein molecule
and its environment are sufficiently favorable to compensate for the low conformational
entropy [Malmsten 1998] Protein adsorption study is often focused on structural
rearrangements in the protein molecules because of its significance to the biological
functioning of the molecules and the important role such rearrangements play in the
mechanism of the adsorption process Knowledge of the majo
f
ed
r interaction forces that act
structures helps to understand the behavior
of pro
arge
a
ded
between protein chains and control the protein
teins at interface These forces include Coulomb interaction hydrogen bonds
hydrophobic interaction and van der Waals interactions
Coulombic interaction Most of the amino acid residues carrying electric ch
are located at the aqueous boundary of the protein molecule An the isoelectric point
(IEP) of the protein where the positive and negative charges are more or less evenly
distributed over the protein molecule intramolecular electrostatic attraction makes
compact structure favorable to proteins Deviation to either more positive or more
negative charge however leads to intramolecular repulsion and encourages an expan
structure Tanford [1967] calculated the electrostatic Gibbs energy for both a compact
impenetrable spherical molecule (protein) and a loose solvent-permeated spherical
34
molecule (protein) over which the charge is spread out From the results he found th
the repulsion force was reduced at higher ionic strength d
at
ue to the screening action of ion
ble
e
the
d the
tween
dehyd
e
ent
oles
tein
Hydrogen bond Most hydrogen bonds in proteins form between amide and
carbonyl groups of the polypeptide backbone [Malmsten 1998] The number of availa
hydrogen bonds involving peptide units is therefore far greater than that involving sid
chains Because α-helices and β-sheets are aligned more or less parallel to each other
interchain hydrogen bonds enforce each other Kresheck and Klotz [1969] examine
role of peptide-peptide hydrogen bonds and concluded that hydrogen bonds between
peptide units do not stabilize a compact structure of protein However because the
peptide chain is shielded from water due to other interactions hydrogen bonding be
peptide groups do stabilize α-helical and β-sheet structures
Hydrophobic interaction Hydrophobic interaction refers to the spontaneous
ration and subsequent aggregation of non-polar components in an aqueous
environment In aqueous solutions of proteins the various non-polar amino acid residues
will be found in the interior of the molecule thus shielded from water The
intermolecular hydrophobic interaction for the stability of a compact protein structure
was first recognized by Kauzmann [1959] If all the hydrophobic residues are buried in
the interior and all the hydrophilic residues are at the outermost border of the molecul
intramolecular hydrophobic interaction would cause a compact protein structure
However geometrical and other types of interactions generally cause a fraction of the
hydrophobic residues to be exposed to the aqueous environm
Van der Waals interaction The mutual interaction between ionic groups dip
and induced dipoles in a protein molecule cannot be established as long as the pro
35
structure is not known in great detail Moreover the surrounding aqueous medium also
contains dipoles and ions that compete for participation in the interactions w
the protein molecule Dispersion interactions favor a compact structure However
because the Hamaker constant for proteins is only a little larger than that of water the
resulting effect is relatively small [Nir 1977]
272 β
ith groups of
rgiles et al 1996
Floeg
) is degraded [Floege 2001] Renal
β2M from the serum resulting in an increase in β2M
conce well
the
)
a
al
2-Microglobulin (β2M)
The protein β2M is of particular interest because it is involved in the human
disorder dialysis-related amyloidosis (DRA) [Geiyo et al 1985 A
e 2001] DRA is a complication in end stage renal failure patients who have been on
dialysis for more than 5 years [Bardin et al 1986 Drueke 2000] DRA develops when
proteins in the blood deposit on joints and tendons causing pain stiffness and fluid in
the joints as is the case with arthritis In vivo β2M is present as the non-polymorphic
light chain of the class I major histocompatibility complex (MHC-I) As part of its
normal catabolic cycle β2M dissociates from the MHC-I complex and is transported in
the serum to the kidney where the majority (95
failure disrupts the clearance of
ntration by up to 60-fold [Floege 2001] By a mechanism that is currently not
understood β2M then self-associates into amyloid fibrils and typically accumulates in
musculoskeletal system [Homma et al 1989] Analysis of ex vivo material has shown
that the majority of amyloid fibrils in patients with Dialysis Related Amiloidosis (DRA
is present as of full-length wild-type β2M although significant amounts (~20-30) of
truncated or modified forms of the protein are also present [Floege 2001 Bellotti et al
1998] Figure 2-9 shows the ribbon diagram of human β2M Native β2M consists of
single chain of 100 amino acid residues and has a seven stranded β-sandwich fold typic
36
of the immunoglobulin superfamily [Saper et al 1991 Trinh et al 2002] β2M was fi
isolated from human urine and characterized by Berggard et al [1980] in1968 The
normal serum concentration of β
rst
re
f
e
2M is 10 to 25 mgL It is a small globular protein with
a molecular weight of 118 kDa a Strokes radius of 16Aring and a negative charge under
physiological conditions (isoelectric point IEP = 57) β2M contains two β-sheets that a
held together by a single disulphide bridge between the cysteines in positions 25 and 81
[Berggard et al 1980 Parker et al 1982 Cunningham et al 1973] β2M cannot be
removed completely by current dialysis techniques but through a better understanding o
the structure and interaction forces that lead to this structure it will be possible to mor
efficiently remove this problematic protein
Figure 2-9 Ribbon diagram of human β M taken from the crystal structure of the protein
bound to the heavy chain of the MHC class I complex (PDB 1DUZ) [Khal 2000]
273 Serum Albumin
Serum albumin is the most abundant protein found in plasma and is typically
present in the blood at a concentration of 35~
2an et
50gL According to extensive studies about
its physiological and pharmacological properties albumin has a high affinity to a very
wide range of materials such as electrolytes (Cu+2 Zn+2) fatty acids amino acids
metabolites and many drug compounds [Fehske et al 1981 Kraghhansen 1981 Putnam
37
1984 Peters 1985] The most important physiological role of the protein is therefore to
bring such solutes in the bloodstream to their target organs as well as to maintain the pH
and osmotic pressure of the plasma Bovine serum albumin (BSA) is an ellipsoidal
protein with the dimensions of 140 X 40 X 40Aring [Peter 1985] The primary structure is a
single helical polypeptide of 66 kDa (IEP = 47) with 585 residues containing 17 pairs of
as a soft an
rfaces [Kondo et al 1991 Norde et al 1992 Soderquist et al
1980 ikely
is
disulfide bridges and one free cysteine [Dugaiczyk et al 1982] BSA has been classified
d flexible protein because it has a great tendency to change its conformation
on adsorption to solid su
Carter and Ho 1994] and consists of three homologous domains (I-III) most l
derived through gene multiplication [Brown 1976] Each domain is composed of A and B
sub-domains [He et al 1992] The secondary structure of human serum albumin (HSA)
shown in Figure 2-10
Figure 2-10 Secondary structure of human serum albumin (HSA) with sub-domains
[Zunszain et al 2003]
HSA has the same structure domains with the serum albumin from other species
such as BSA [Brown 1976] Although all three domains of the albumin molecule have
similar three-dimensional structures their assembly is highly asymmetric [Sugio et al
38
1999] Domains I and II are almost perpendicular to each other to form a T-shaped
assembly in which the tail of subdomain IIA is attached to the interface region between
sub-domains IA and IB by hydrophobic interactions and hydrogen bonds In contrast
domain III protrudes from sub-domain IIB at a 45ordm angle to form the Y-shaped assembly
for domains II and III Domain III interacts only with sub-domain IIB These features
make the albumin molecule heart-shaped
28 Protein Adsorption
Protein adsorption studies date back to the 1930rsquos At the beginning these studies
mainly focused on the determination of the molecular weight electrophoretic and
ral
rearrangem
adsorption and biocompatibility of the sorbent materials were investigated [Norde 1986]
There are interaction forces at the interfaces between protein molecules and latex
particles These forces are mainly divided into the following groups hydrophobic
interaction ionic interaction hydrogen bonding and van der Waals interaction [Andrade
1985]
Hydrophobic interaction It is known that hydrophobic interaction has a major
role in protein adsorption phenomena The adsorption of proteins on the low charged
latex particles occurs by this interaction force Generally monomers such as styrene offer
hydrophobic surfaces th
by this interaction force is maximum at the isoelectric point (IEP) of the protein and the
strength [Suzawa et al 1980 1982 Shirahama et al 1989 Kondo et al 1992] By the
chromatographic applications Later the adsorption mechanism especially the structu
ents was studied Recently the studies of the relation between protein
281 Interaction between Protein Molecule and Latex Particle
at protein molecules adsorb to The amount of adsorbed protein
pH at maximum adsorption shifts to a more acidic region with an increase in ionic
39
reports [Suzawa et al 1980 1982 Lee et al 1988] protein adsorption was greater
hydrophobic surface than on a hydrophilic one implying that hydrophobic interaction is
on a
s
ydrophilic compound such as acrylic acid (AA) or methacrylic acid (MAA)
Ionic
ely
t
ces
n force is operative over small
distan e
ble
n
nt dialysis membrane therapy for end stage renal disease (ESRD)
one of most dominant forces in protein adsorption
Ionic interaction Negatively charged latex particles have ionic functional
groups on their surfaces such as salts of sulfonic and carboxylic acid Sulfate group
originate from an initiator such as sodium persulfate and carboxylic groups originate
from a h
bonds are formed between the negative charges of these latex particles and positive
surface charges of protein molecules The conventional low-charged latex particles rar
form these ionic bonds
Hydrogen bonding Hydrogen bond is a strong secondary interatomic bond tha
exists between a bound hydrogen atom (its unscreened proton) and the electrons of
adjacent atoms [Callister 1999] Protein can be adsorbed on hydrophilic polar surfa
through hydrogen bonding Hydrogen bonds are frequently formed between hydroxyl-
carbonyl or amide-hydroxyl Hydroxyl-hydroxyl or amide-hydroxyl bonds are also
formed in protein adsorption
Van der Waals interaction This interactio
ces only when water has been excluded and the two non-polar groups come clos
to each other Lewinrsquos calculation showed that the van der Waals interaction is negligi
compared with the forces involved in the entropy increases ie hydrophobic interactio
[Lewin 1974]
29 Hypothesis for Toxin Removal
As mentioned earlier insufficient removal of middle molecular range toxins is a
major drawback of curre
40
patien
is
y
ese
haracteristics over the last decade However removal
effici
ing a novel membrane design composed of an assembly of engineered
polymeric latex particles s ions Important factors
will i
e
ith a
is established it is
expec
e
ts This may cause destructive arthritis and carpal tunnel syndrome inhibit the
immune system and accelerate cardiovascular disease leading to death among dialys
patients In order to overcome these complications artificial dialysis membranes have
been developed to increase the capacity for removing middle molecular weight toxins b
changing the pore size of dialysis membranes and using new materials that adsorb th
toxins for improved removal c
ency is not as high as those achieved by a normal healthy kidney
With the knowledge obtained from all above literatures the following hypothesis
has been established for the development of a membrane for the successful removal of
the target protein β2-Microglobulin (β2M) without the removal of serum albumin This
will be done us
ynthesized to predetermined specificat
nclude pore size and surface chemistry
291 Toxin Removal by Size Sieving Based on Monodispersed Pore Size
The packing of monodispersed spherical particles can lead to the formation of
porous layers suitable for use as filters and membranes [Hsieh et al 1991] The pore siz
is defined as the largest spherical particles that can pass through the interstitial spaces
It is well known that monodispersed spherical particles can be obtained from the
seed emulsion polymerization method Many commercially available latex particles are
synthesized by this technique and are inexpensive When the defect free membrane w
monodisperse pore size distribution based on a particular particle array
ted to outperform the traditional hemodialysis membrane limitation which is a
broad range of pore size distribution When spherical particles pack in regular crystallin
arrays a number of packing geometries such as hexagonal closest packing cubic closest
41
packing and body-centered cubic packing are possible The pore size of the array
depends on these packing geometries as well as particle size Theoretical pore size can be
calculated from the simple geometry of the arr
ay Figure 2-11 shows the relationship
between particle size and pore size
0001
001
01
001 01 1 10
Por
e
1
10
microm
) S
ize
(
Particle Diameter (microm)
Body-Centered Cubic ArrayClosest Packed Array
0001
Siz
e (
Particle Diameter (microm)
001
01
001 01 1 10
Por
e
1
10
microm
)
Body-Centered Cubic ArrayClosest Packed ArrayBody-Centered Cubic ArrayClosest Packed Array
and closed packed arrays [Steve et al 1999]
McDonald and his coworkers [Ramakrishnan et al 2004 Steve et al 1999] have
described the fabrication of latex composite membranes and their performance in
previous papers It is from this prior research and knowledge gained from conversations
with Dr McDonald that this current research has been initiated [McDonald 2003] The
focus of this thesis is the extension of McDonaldrsquos research in the area of composite
membranes to medical application such as dialysis membranes
292 Toxin Removal by Selective Adsorption on Engineered Latex Particles
The flexibility in latex surface properties is a significant advantage to this
materialrsquos ability to separate proteins since the selectivity is strongly dependent on the
charge interactions of both the protein and the latex particles under the conditions of
Figure 2-11 The relationship between pore size and particle size in body-centered cubic
42
separation [Menon et al 1999 Chun et al 2002] First all sorbents were designed to
have negative surface properties because most plasma proteins in blood are negative and
should not be removed with charge interaction between proteins and sorbents at
physiological condition (pH74) It was reported that a negatively charged surface was
more blood compatible than a positive one [Srinivasan et al 1971] The toxin protein β2-
Microglobulin (β2M) can be selectively adsorbed on the surface of latex particles by the
design of a suitable hydrophobic portioned surface where only β2M protein can be
anchored with hydrophobic interactions Albumin adsorption on the latex particle is not
side-on m
adsorption of β M protein on the engineered latex particles
allowed because charge repulsion is more dominant than hydrophobic interaction with
ode The figure 2-12 shows the schematic representation of the selective
2
PMMAPAAShell
COO-
OSO3-
OSO3-
PSCore
COO-
-
-
-
-
-
-
Albumin-
Chargerepulsion
--
-
- -
- -
- --
Hydrophobic
β2-Microglobulin
2
Interaction
β -Microglobulin
PMMAPAAShell
COO-
-
-
-
-
-
-
--
-
-
-
-
-
Albumin-
Chargerepulsion
OSO3-
OSO3-
PSCore
COO-
--
-
- -
--
-
- -
- -
- --
- -
- --
Hydrophobic
β2-Microglobulin
2
particles at pH 74
Interaction
β -Microglobulin
Figure 2-12 Schematic representation of the protein adsorption on the core shell latex
43
Hydrophilichydrophobic microdomain structures were proven to be more blood
compatible [Mori et al 1982 Higuchi et al 1993 Deppisch et al 1998] The
optimization of suitable hydrophobic to hydrophilic ratio is also important for the
biocompatibility The monomers such as styrene (St) methyl methacrylate (MMA) and
acrylic acid (AA) are widely used hydrophobic and hydrophilic monomer models in
emulsion polymerization process
In summary the background and fundamental literature survey about the history
material properties and limitation of hemodialysis membrane latex particle preparation
surface chemistry and manufacturing process target proteins and protein adsorption and
finally the hypothesis for a toxin removal with high separation efficiency have been
suggested This research focuses on such hypothesis and the materials and
characterization methodology for achievement of suggested hypotheses are described in
the next chapter
CHAPTER 3 EXPERIMENTAL AND CHARACTERIZATION METHODOLOGY
As mentioned earlier the goal of this study is to prepare polymeric latex particles
with tailored properties to maximize separation of toxin molecules and to investigate
fundamental interactions between the applied particles and molecules in the
system to optimize the membrane performance for hemodialysis applications Polymeric
latex particles wit
the
biological
h monodisperse size distribution to obtain uniform pore size and
various size ranges to utilize membrane construction are necessary Surface engineering
with various combination of hydrophobichydrophilic domain on the surface of latex
particles is expected to affect the removal of target protein by selective adsorption and to
improve the biocompatibility of membrane Therefore the materials and characterization
methodology are addressed in this chapter
31 Materials
Styrene (St) monomer used for a seed and a core particle was purchased from
Fisher Scientific and used without any other purification process Acrylic acid (AA) and
methyl methacrylate (MMA) monomers introduced for shell formation were purchased
from Fisher Scientific and used without any other purification process Sodium persulfate
(SPS) and sodium bicarbonate (SBC) were obtained from Fishers Scientific and used as
received Divinylbenzene (DVB) crossliking agent was purchased from Aldrich The
anionic surfactant Aerosol regMA80-I [sodium di(13-dimethylbutyl) sulfosuccinate] was
kindly donated by Cytec 78-80 Aerosol regMA80-I is mixed with isopropanol and
water Its critical micelle concentration (CMC) is 245 mM (119) Ion exchange resin
44
45
AGreg 501-x8 Resin (20-50 mesh) was purchased from Bio-Rad Laboratories Inc It is a
mixed-bed resin with H+ and OH- as the ionic forms and is 8 crosslinked Bovine serum
albumin (BSA) (heat shock treated) was purchas Fisher Scientific The isoelectric
point of t lin
(β2M)
as
gma
ed from
he BSA is 47-49 and the molecular weight is 66300Da β2-Microglobu
was purchase from ICN Biomedicals Inc and was separated from patients with
chronic renal disease and lyophilized from ammonium bicarbonate The molecular
weight by SDS-PAGE was approximately 12000Da BSA and β2M proteins were used
received without further purification process In order to investigate the protein
adsorption evaluation bicinchoninic acid protein assay kits were purchased from Si
(Cat BCA-1) and Pierce Biotechnology (Cat 23225) The chemical structures for the
main chemicals are shown in Figure 3-1
CH2 CH
Styrene (Mw = 10415)
CH2 CHCH2 CH
Styrene (Mw = 10415)
CH2 CH
Acrylic acid (Mw = 7206)
C
OH
O
CH2 CH
Acrylic acid (Mw = 7206)
C
OH
O
CH CH2
C
OCH3
O
CH
CH CH3
2
C
OCH3
O
CH3
Methyl methacrylate (Mw = 10012)Methyl methacrylate (Mw = 10012)
CH2 CH
Divinylbenzene (Mw = 1302)
CH CH2
CH2 CH
Divinylbenzene (Mw = 1302)
CH CH2CH CH
+(Na) -(O3S)
CH -C-O-CH-CH -CH-CH3
O
2 2 3
O CH CH3
CH3 CH3
CH-C-O-CH-CH2-CH-CH3
Anionic surfactant (MA80-I) (Mw = 3885)
+(Na) -(O3S)
CH -C-O-CH-CH -CH-CH3
O
2 2 3
O CH CH3
CH3 CH3
CH-C-O-CH-CH2-CH-CH3
Anionic surfactant (MA80-I) (Mw = 3885)2
+(Na)-(O) S OO
O O
- +
Initiator sodium persulfate (Mw = 23811)
O SO
(O) (Na)+ -
O O
- +(Na) (O) S OO
O SO
(O) (Na)+ -
O O
- +
Initiator sodium persulfate (Mw = 23811) Figure 3-1 Chemical structure of main chemicals
(Na) (O) S OO
O SO
(O) (Na)
46
32 Latex Particle Preparation
The experimental set up and the particle preparation schemes for various types and
size ranges of latex particles are shown in Figure 3-2 and 3-3 In the experimental setup a
mechanical glass stirrer is connected from a motor and Teflon stirrer bar is located at
end of glass stirrer The agitation rate is precisely adjusted by controller motor Paar glas
reactor with 1L volume is partially in silicon oil bath on hot plate The reactor has fo
openings for a stirrer a thermometer a nitrogen gas inlet and a reactant feeding lnlet
The reactants for latex polymerization are fed by precisely controlled by meterin
The particle preparation is described in each latex preparation section
the
s
ur
g pump
Figure 3-2 Experimental setup for semi-continuous emulsion polymerization
1 Motor 2 Mechanical stirrer bar 3 Thermometer 4 Inlet of N2 5 Inlet for initiator and monomer 6 Glass reactor (l L) 7 Oil bath 8 Hot plate 9 Pump for initiator 10 Pump for monomer 11 Tubes for reactant feed
47
Styrene monomer
Bare PS
PSPMMA100
PSPMMA90PAA10
PSPMMA P75 AA25
Gr
win
ticle
size
PS seedparticles
og
par
Styrene monomer
Bare PS
PSPMMA100
PSPMMA90PAA10
PSPMMA P75 AA25
Gr
win
ticle
size
PS seedparticles
og
par
Figure 3-3 The particle preparation scheme in various types and size ranges of latex particles
321 Preparation of Seed Latex Particles
Polystyrene seed particles were synthesized through a typical semi-continuous
emulsion polymerization Polystyrene latex particles were prepared in a four necked Paar
glass vessel equipped with a mechanical glass stirrer thermometer glass funnel for
nitrogen gas purge and a tube for monomer feeding The reactor was placed in a silicon
oil bath on a hot plate for homogenous and stable tempe he vessel was
firstly charged with de-ionized water the emulsifier so itiator
sodium bicarbonate (SBC) styrene (St) monomer divinylbenzene (DVB) monomer
under nitrogen gas atmosphere and then heated to 72plusmn2 as
llowed to continue for another hour Second reactants St and DVB monomers SPS
initiator and SBC were fed separately using fine fluid metering pumps (Model RHSY
Fluid metering INC NY) under a nitrogen gas atmosphere for 2h After complete feeding
rature control T
dium persulfate (SPS) in
oC for 1h The reaction w
a
48
of all reactants the reaction was continued for another hour at elevated temperature 80plusmn1
oC and then cooled to room temperature by removal of the oil bath
322 Preparation of Seeded Latex Particles
The particle growth was achieved by seeded continuous emulsion polymerization
The vessel was charged with the polystyrene seeds and de-ionized water to make desired
solid content and heated up to 72plusmn2 oC for 1h under nitrogen gas atmosphere Then St
and DVB monomers and solution of SPS and SBC in de-ionized water were continuously
added using metering pump with a precisely controlled feed rate After feeding all
reactants the reaction was continued for three hours keeping temperature of 80plusmn2 oC and
then the reaction vessel was cooled down to room temperature
Monodisperse spherical polystyrene seeds with a 280nm mean diameter were used
e-polymerized polystyrene seeded emulsion
was charged in a 500m
323 Preparation of Core Shell Latex Particles
for core shell structured latex particle The pr
l glass flask under a nitrogen gas atmosphere De-ionized water
was added to achieve the desired solid content The emulsion was heated to 80plusmn2oC for
10 hour Monomers initiator and SBC to form the shell were added by fluid metering
pumps (LAB PUMP JRRHSY model Fluid metering Inc USA) with fine controlled
feed rate Flow rate can be adjusted by control ring graduated in 450 division from 0 to
100 flow which is in the range of 10mlmin respectively The reaction was sustained
for additional 30 hours at 90plusmn2oC then cooled down to room temperature
324 Purification of Synthesized Latex Particles
Synthesized latex particles were purified with an ion exchange resin 200g (10
ww polystyrene solid content) of emulsion were mixed with 4g of ion exchange resin
and stirred for 40 minute to remove unreacted monomers initiators and other impurities
49
Then the emulsion was diluted with DI water to a specific degree of solid content as
needed
33 Characterization
331
solid
termined
oscopy
d
hours
Dried latex (10mg) was mixed with
250 m on
Degree of Conversion
The degree of monomer to polymer conversion was determined gravimetrically
following the procedure described by Lee et al [1995] before the latex cleaning process
The synthesized emulsion raw materials were weighed and dried in a conventional oven
at 120oC for 30min to evaporate any unreacted monomers and water The remaining
was weighed and the degree of conversion was determined This process was repeated
three times and a mean value of the degree of conversion was de
332 Fourier Transform Infrared (FTIR) Spectr
A molecule can absorb only select frequencies (energy) of infrared radiation which
match the natural vibration frequencies The energy absorbed serves to increase the
amplitude of the vibrational motions of the bonds in the molecule [Pavin et al 1996]
Only those bonds which have a dipole moment that changes as a function of time can
absorb the infrared radiation The range of wavelengths for infrared radiation is between
25 microm (4000 cm-1) and 25 microm (400cm-1)
The synthesis of the latex particles such as polystyrene (PS) homopolymer and
PSPMMA PSPMMA90PAA10 PSPMMA75PAA25 core-shell copolymers were verifie
with FTIR Purified latex particles were dried in vacuum oven at 40oC for 24
before analysis in the FTIR (Nicolet Magma USA)
g of KBr which had also been dried in vacuum oven at 120oC for 3h Transmissi
spectra using the drift mode were plotted with 128 scans and 4cm-1 resolution
50
333 Quasielastic Light Scattering (QELS)
Latex particle size was measured by the Brookhaven ZetaPlus particle size
analyzer The solid concentration as 0001 (wv) This
ation spectroscopy (PCS) of quasielastically scattered light
For th s
ed using a FE-SEM (JEOL
JSM-
ith
ent
01
us zeta-
potential analyzer It measures the electrophoretic mobility velocity of charged colloidal
of latex particles used w
instrument uses photon correl
is technique time dependent interference patterns of light scattered from particle
in the sample cell are analyzed The interference pattern changes due to Brownian
motion giving rise to fluctuations in scattering intensity The frequency of these
fluctuations depends on the particlersquos diffusion constant that is inversely proportional to
the particle diameter
334 Field Emission-Scanning Electron Microscopy (FE-SEM)
Particle size and surface morphology were characteriz
6335F Japan) A diluted suspension of latex particles in water was dropped onto a
silicon wafer at room temperature and allowed to dry The sample was then coated w
the thinnest layer of carbon needed to obtain the required conductivity for this instrum
Secondary electron image mode was used with a 15KV of accelerating voltage The
magnification range used was between 10000X and 70000X
335 Zeta Potential Measurement
Synthesized and purified latex particles were diluted with de-ionized water to 0
wt and adjusted six different pH values 205 345 481 565 643 and 747 These
particle suspensions were then transferred to standard cuvettes for zeta potential
measurement At least two runs of ten measurements were taken for each sample and
averaged
The zeta potential measurement was carried out using Brookhaven ZetaPl
51
particles in solution and calculate zeta-potential by using the Smoluchowski equation
The f quency
gnitude
umin (BSA) as a standard model protein and β2-Microglobulin
uffer (PB) and
phosp
5
h the selected protein
e 005 01 03 05 and 07 mgml and those for
β2M
d at
teins in the supernatant after the centrifugation process using the
bicinc t
requency of laser light passing through the sample and is compared to the fre
of a reference beam The shift in the frequency called a Doppler shift and the ma
of the shift correspond to the polarity and the magnitude of the electrophoretic mobility
respectively The zeta potential is calculated from the solution conditions and the
measured mobility
336 Protein Adsorption
Protein adsorption experiments of the synthesized latex particles were performed
with bovine serum alb
(β2M) as a target protein in two types of buffer solution phosphate b
hate buffered saline (PBS) To make 5 mM of PB solution 0345g of sodium
phosphate monobasic (NaH2PO4H2O) was dissolve in 500ml of de-ionized water To
make PBS solution 0345g of sodium phosphate monobasic (NaH2PO4H2O) and 4178g
(143mM) of sodium chloride (NaCl) were dissolved in 500ml of de-ionized water The
synthesized latex particles were diluted with each buffer to have a solids content of 0
(ww) adjusted to pH values of 32 48 and 74 and mixed wit
BSA concentrations were chosen to b
were 0015 0030 0045 and 0060mgml The mixture was gently rotated using
the shaker Labquakereg (BarnsteadThermolyne Model 4002110 USA) with 8 RPM in
the incubator at 37oC for 12 hours before the latex-protein mixture was centrifuge
13000 rpm for 15min The amount of protein adsorbed was determined by quantifying
the free pro
honinic acid (BCA) assay method [Lowry et al 1951 Smith et al 1985 Baptista e
al 2003 Wiehelman et al 1988 Brown et al 1989] BCA assay kits consist of Reagent
52
A containing bicinchoninic acid sodium carbonate sodium tartrate and sodium
bicarbonate in 01N NaOH with pH=1125 and Reagent B containing 4 (wv) copper
(II) sulfate pentahydrate The BCA working reagent was prepared by mixing 50 parts of
Reagent A with 1 part of Reagent B 100microl of protein supernatant was then mixed with
2ml of BCA working reagent in a UV cuvette Incubation was allowed to continue
at room temperature
further
until color developed about 2h The absorbance at 562nm was
pectrophotometer (Perkin-Elmer Lambda 800 USA)
Unkn
Separate the p rticles in centrifuge mix supernatant with BCA reagents
measured using a UV-VIS s
own concentration of sample protein was determined by comparison to a standard
of known protein concentrations The adsorbed amount per unit surface area was
determined by the mass balance of the protein after adsorption process The simple
schematic of the procedure for a protein adsorption test is shown in Figure 3-4
Incubate mixture with agitation for 12hrs at 37oC
aand develop the color by incubation for 2hrs at room temperature
Suspend latex particles in aqueous buffer media
Add the dissolved target protein
Measure color intensity using UVVis spectroscopy at 562nm
Suspend latex particles in aqueous buffer media
Separate the p rticles in centrifuge mix supernatant with BCA reagents
Add the dissolved target protein
Incubate mixture with agitation for 12hrs at 37oC
aand develop the color by incubation for 2hrs at room temperature
Measure color intensity using UVVis spectroscopy at 562nm
Figure 3-4 Schematic of the procedure for a protein adsorption test
53
337 Blood Biocompatibility by Hemolysis Test
Hemolysis is the destruction of red blood cells which leads to the release of
hemoglobin from within the red blood cells into the blood plasma Hemolysis testing is
used to evaluate blood compatibility of the latex particles and the damaged to the red
blood cells can be determined by monitoring amount of hemoglobin released The red
blood cells (RBCs) were separated by centrifuging blood at 1500 rpm for 15 minutes
Figure 3-5 shows the phase separated blood with plasma white blood cells and red
cells after centrifugation process Separated RBSs were then washed with isotonic
phosphate buffer solution (PBS) at pH 74 to remove debris and serum protein Th
process was repeated 3 times
blood
is
Figure 3-5 Separation of RBC from whole blood by centrifuge process
Prepared latex particles were re-dispersed in PBS by sonification to obtain
homogeneously dispersed latex particles 100microl of the mixture of red blood cell (3 parts)
and PBS (11parts) was added to 1ml of 05 (ww) particle suspension PBS was used as
a negative control resulting 0 hemolysis and DI water used as a positive control to
produce 100 hemoglobin released by completely destroyed RBCs The mixture was
incubated in water bath with gentle shaking for 30 minutes at 37oC and then centrifuged
at 1500 rpm for 15 minutes 100microl of the supernatant was mixed with 2ml of the mixture
) (EtOHHCl = 2005 ww) to prevent the
White blood cells
Plasma
Red blood cells
of ethanol (99) and hydrochloric acid (37
54
precipitation of hemoglobin In order to remove remaining particles the mixture was
centri fuged again with 13000 RPM at room temperature The supernatant was then
transferred into the UV cuvette The amount of hemoglobin release was determined by
monitoring the UV absorbance at a wavelength of 397nm The schematic of the
procedure for hemolysis test is shown in Figure 3-6
Separate red blood cells (RBCs) from blood
Mix RBCs with phosphate buffer solution (PBS)
Wash and re-suspend RBC with PBS repeat three times
Add la l of RBC and incu in at 37oC
tex particle suspension into 100mbate sample with agitation for 30m
Separate particle ith EtOHHCl solutions and mix the supernatant w
Evaluate free hemoglobin in the solution using UVVis spectroscopy at 397nm
Separate red blood cells (RBCs) from bloodSeparate red blood cells (RBCs) from blood
Mix RBCs with phosphate buffer solution (PBS)Mix RBCs with phosphate buffer solution (PBS)
Wash and re-suspend RBC with PBS repeat three timesWash and re-suspend RBC with PBS repeat three times
Add latex particle suspension into 100ml of RBC and incubate sample with agitation for 30min at 37oC
Add latex particle suspension into 100ml of RBC and incubate sample with agitation for 30min at 37oC
Separate particles and mix the ith EtOHHCl solution supernatant wSeparate particles and mix the ith EtOHHCl solution supernatant w
Evaluate free hemoglobin in the solution using UVVis spectroscopy at 397nmEvaluate free hemoglobin in the solution using UVVis spectroscopy at 397nm
Figure 3-6 Schematic of the procedure for hemolysis test
In summary a description of the experimental materials and characterization
methodology has been explained In the next chapter the results and discussion of the
data obtained from these experiments is addressed
CHAPTER 4 RESULTS AND DISCUSSION
41 Polymerization of Latex Particles
411 Polystyrene Seed Latex Particles
Polymer particles have many different applications including spacer particles
lubricants packing materials for chromatography standard particles and diagnostic
drugs All applications strongly require these particles to have a uniform particle size
Uniform particle size is the first requirement for a latex composite membrane formed by
particle arrays with the interstitial spaces serving as pores for size discriminations [Jons
et al 1999 Ramakrishnan et al 2004] Although suspension polymerization has been
mainly used as one of conventional polymerization methods to prepare uniform particles
the uniformity of the recovered particles is insufficient for membrane use There is
another method known as seed emulsion polymerization where a vinyl monomer is
absorbed into fine monodiserse seed particles and polymerization causes the monomer to
crease the sizes of the seed particles uniformly In order to obtain monodisperse larger
particles by this m
particles and polymerization of the monomer is repeated
In this study cross-linked polystyrene seed latex particles were firstly synthesized
in the presence of styrene monomer divinylbenzene crosslink agent sodium persulfate
initiator sodium bicarbonate buffer and the Aerosol regMA80-I [sodium di(13-
dimethylbutyl) sulfosuccinate] surfactant using a batch type semi-continuous emulsion
polymerization process in which one or more components can be added continuously
in
ethod the procedure of absorption of monomer into fine polymer
55
56
Styrene (St) monomer and divinylbenzene (DVB) crosslink agent as well as initiator and
buffer are continuously added to enlarge the seed latex particles Various profiles of
particle nucleation and growth can be gene different orders of component
addition during polymerizati f this process such as easy
control of the polymeriza particle number
and particle morphology The main goal in
this process is to avoid secondary particle
rated from
on There are several advantages o
tion rate monodisperse particle size and
colloidal stability copolymer composition
formation leading to a monodisperse particle
size distribution Figure 4-1 shows the schematic representation of seed latex particle
preparation and growth
Monomer droplet
Seed particles Amount of surfactantsis variable
Enlarged final Add monomers
PS
PS
PSs4
particles Number of particlesare constant
Seed code
PSs1
s2
s3
Monomer droplet
Seed particles Amount of surfactantsis variable
Enlarged final Add monomers
PS
PS
PSs4
tion The
particles Number of particlesare constant
Seed code
PSs1
s2
s3
Figure 4-1 Schematic representation of semi-continuous seed latex particles preparation and growth
After the first synthesis of a small size of crosslinked polystyrene (PS) seed
particles stabilized by surfactants styrene DVB and initiator are continuously fed into
the system to enlarge the seed latex particles maintaining a narrow size distribu
recipes and reaction conditions for synthesizing these seed particles are summarized in
57
Table 4-1 The original recipe for latex particle preparation from McDonald was
modified for this emulsion system [McDonald 2003]
Table 4-1 The polymerization recipe of polystyrene (PS) seed particles Latex label PSS259 PSS233 PSS207 PSS181
Temperature (oC) 75 plusmn 5 75 plusmn 5 75 plusmn 5 75 plusmn 5
Divinylbenzene (g) 052 052 052 052
MA 80 (g) 259 233 207 181
NaHCO
Watera (g) 1315 1315 1315 1315
Styrene (g) 9948 9948 9948 9948
112 112 112 112
Na2S2O8 (g) 190 190 190 190
Waterb (g) 200 200 200 200
Reaction time (min) 140 140 140 140
Solids content 396 396 396 396
Mean particle diameter (nm) 126plusmn75 171plusmn39 182plusmn41 216plusmn53
Conversion () 965 979 957 971
3 (g)
Four different seed latex particles were synthesized based on surfactant amount in
order to determine the optimum condition for stable and monodisperse latex particles
The emulsion polymerization of latex particles has to be carried out in a narrow range of
surfactant causes flocculation or phase separation leading to broad particle
size distribution and an unstable emulsion sy
surfactant concentration where particles are stable [Nestor et al 2005] A wide range of
concentrations
stem Therefore the range of surfactant
amount was varied from 181g to 259g in increments of 026g in each latex label Other
reactants amounts were kept constant Watera is the initial charge amount and waterb
represents the amount of aqueous solution containing dissolved initiator and buffer
58
which are fed continuously after the seed particle creation The extent of monome
polymer conversion was obtained by gravimetric calcu
r to
lation and was more than 95 at
cripts such as
S207 and S181 i the surf mount added for the seed latex
nversion indi perce experi obtain
content divided by the theoretically estimated solid content and was calculated by the
all seed particles labeled PSS259 PSS233 PSS207 and PSS181 Labeled subs
S259 S233 ndicate actant a
preparation The co cates the ntage of mentally ed solid
following formula
100 colids () times=entsoliatedntentoalExperimentConversion
n the experimenta content is the weight of olid rema after
mulsion at 120oC f in and the estimated solid content is th ht
lated from the recipe
rti as di each t load d
mple of ea pension iluted by (ww)
deionized water and the mean particle diameter was determined using the Brookhaven
ZetaPlus particle size analyzer From these results the seed latex particle size increased
as the amount of surfactant decreased The amount of emulsifier also affects the
polymerization process by changing the number of micelles and their size It is known
that large amount of emulsifier produces larger numbers of smaller sized particles [Odian
1991] and was corroborated in the current work
These synthesized seed latex particles should be of narrow size distribution in order
to obtain monodisperse larger particles The SEM image showing the particle
morphology and size of a sample of polystyrene seed particles can be seen in the Figure
4-2
contdEstim (4-1)
In this equatio l solid the s ining
evaporation of e or 30m e weig
of monomers calcu
As expected the seed pa cle size w fferent for surfactan ing in see
emulsion system A sa ch sus was d 0001 with
59
Figure 4-2 Scanning Electron Micrograph (SEM) of polystyrene seed latex particles (A)
PSS259 (B) PSS233 (C) PSS207 (D) PSS181 (subscripts indicate the amount of surfactant added)
(A)
(B)
60
(C)
(D)
Figure 4-2 Continued
61
s seen in the image the seed particles are very smooth and spherical The
uniformity in the particle size distribution of these PS seed latex particles can also be
seen in the SEM image From this data we concluded that seed particles synthesized in
the presence of a surfactant with the chosen amounts are suitable for use to prepare larger
particles with high size uniformity and smooth spherical morphology
412 The Growth of Polystyrene (PS) Seed Latex Particles
Seeded emulsion polymerization has been conducted for several decades and
various mechanisms have been proposed Gracio et al [1970] suggested that the growing
polymer particles consist of an expanding polymer-rich core surrounded by a monomer-
rich shell with the outer shell providing a major locus of polymerization Seeded
mulsion polymerization is commonly used for preparing latex particles less than 1microm in
size [Cha et al 1995 Zou et al 1990 1992 Gandhi et al 1990 Park et al 1990] Such
latexes can be obtained from the growth of pre-prepared seed particles The seeds
introduced in this process serve as nucleation sites for particle growth By first swelling
the seed latex particles with additional monomer to the desired size polymerization can
then be initiated Several size ranges of monodisperse polystyrene (PS) latex particles
were prepared by this multistep seeded emulsion polymerization method For the
fabrication of particle-based membranes monodisperse latex particles with a variety of
size ranges are required
In this study seed particles from 171nm to 470nm in mean diameter and with a
highly uniform size distribution were prepared and used Watera is the initial charge
mount and waterb represents the amount aqueous solution containing dissolved initiator
and buffer that is continuously fed into system Feed time is precisely controlled by
metering pump The solid contents were maintained between 199 and 300 After
A
e
a
62
polym d as
es
ere 923 8402
n of these latex particles
are sh
ss
erization under these conditions the conversions of the latex particles labele
PS258 PS320 PS370 and PS410 were 952 957 946 and 964 respectively The recip
used in seeded emulsion polymerization to form the particles less than 500nm in mean
diameter are shown at the Table 4-2 The conversions of the latex particles larger than
500nm in mean diameter and labeled as PS525 PS585 PS640 and PS790 w
723 and 969 respectively The recipes used in the preparatio
own at the Table 4-3
A simple calculation can be used to predict the ultimate size that a latex particle can
reach after ending the polymerization Letrsquos suppose that seed density (ρs) and grown
particle density (ρgp) are the same The following equation is obtained from the ma
balance (m = ρV) where m and V are mass and volume of a particle respectively
gp
gp
s
s
Vm
Vm
In this equation m
= (4-2)
be
s and mgp are the weight of seed and total weight of seed and
added monomers respectively and Vs and Vgp are the volume of seed and grown
particle respectively
If we know the mass of monomer to be added the ultimate particle size can
calculated from the equation 4-3
3
2s D m ⎠⎝+ χ
where D
1s D m
⎟⎟⎞
⎜⎜⎛
= (4-3)
the
in
s and Dgp are the diameters for seed and grown particle respectively and χ is
weight of the monomer added Crosslinked PS latex particles with a size range from
258plusmn112 ~ 410plusmn117 in diameter polymerized by the recipes in Table 4-2 are shown
the SEM images in Figure 4-3
63
Table 4-2 Continuous addition emulsion polymerization recipe for growing polystyrene
Latex label PS
(PS) latex particles less than 500nm in size
258 PS320 PS370 PS410
Initial charge
Seed latex (g) 1000171 1000216 1000216 1000320
Watera (g) 1470 2500 1984 2600
06 07 06 06
Fee
Initiator stream
2 2 8
Feed time (min) 0-167 0-140 0-100 0-92
Solid content 284 300 300 200
Mean particle diameter (nm) 258plusmn112 320plusmn154 182plusmn71 410plusmn117
Conv
Monomer continuous
Styrene (g) 800 1200 800 600
Divinylbenzene (g)
d time (min) 0-163 0-110 0-75 0-82
Na S O (g) 07 065 05 05
NaHCO3 (g) 07 065 05 05
Waterb (g) 750 1200 200 370
ersion () 952 957 946 964 The solid content of seed latex for PS258 PS320 PS 370 and PS410 are 392 399 399 anrespectively
d 300
64
Table 4-3 Continuous addition emulsion polymerization recipe for growing polystyrene (PS) latex particles larger than 500nm in size
Latex label PS525 PS585 PS640 PS790
Initial charge
Seed latex (g) 1000390 1000470 1000470 1000320
Watera (g) 1000 520 1200 360
Monomer continuous
Styrene (g) 290 200 400 173
Divinylbenzene (g) 02 014
03 009
Feed time (min) 0-80 0-40 10-76 5-65
Initiator stream
Na2S2O8 (g) 02 014 03 012
NaHCO3 (g) 02 014 03 009
Waterb (g) 200 120 400 200
Feed time (min) 0-85 0-46 0-77 0-75
Solids content 199 200 200 201
Mean particle diameter (nm) 525plusmn151 585plusmn186 640plusmn178 790plusmn693
Conversion () 923 8402 723 969 The solid content of seed latex for PS525 PS585 PS 640 and PS790 are 200 182 193 and 173 respectively
65
Figure 4-3 SEM of PS latex particles less than 500nm in size (A) 258nm (B) 320nm (C)
370nm (D) 410nm
(A)
(B)
66
(C)
(D)
Figure 4-3 Continued
67
article size was well matched to that estimated by the geometrical calculation
From this it can be concluded that the added monomer was consumed for polymer
particle growth with a high degree of conversion From the SEM characterization newly
nucleated particles were not seen as indicated by the narrow particle size distribution
The particles less than 500nm in mean diameter were very spherical in shape with a
highly uniform particle size distribution As the particles were grown larger than 500nm
mean diameter however they became of non-spherical shape with an uneven surface
This irregularity of particle surface can be attributed to the non-homogeneous monomer
swelling into the shell of a growing polymer which can be controlled by the factors such
as temperature agitation speed initiator feeding rate and surfactant amount required to
stabilize the colloidal system The SEM image of latex particles larger than 500nm mean
iameter is shown in Figure 4-4 The particle size distribution is broader than that of the
s particles These particles can however still be used for membrane construction
as a support layer rather than skin layer The support layer does not necessarily have to be
as highly monodisperse as the skin layer which require pores of high uniformity for the
selective removal of toxins
As mentioned earlier the amount of surfactant as well as monomer in an emulsion
system is the primary determinant of the particle diameter [Odian 1991] Anionic
surfactants are generally recommended at the level of 02-30 wt based on the amount
of water [Odian 1991] The critical micelle concentration (CMC) of Aerosolreg MA 80-I
anionic surfactant is 119 The surfactant loading in this emulsion system ranged from
the 121 for SS181 to 171 for SS259
P
d
maller
68
Figure 4-4 SEM of PS latex particles larger than 500nm in size (A) 525nm (B) 585nm
(C) 640nm (D) 790nm
(A)
(B)
69
(C)
(D)
Figure 4-4 Continued
70
The surfactant to monomer ratio refers to the value of the surfactant amount
divided by monomer amount after polymerization ends The surfactant amount is the
weight of the surfactant contained in emulsion and the monomer amount is the total
weight of the monomer added for polymerization to form the ultimate polymer particles
in emulsion As the surfactant to monomer ratio decreases mean particle diameter
increases as described by Odian [1991] Generally lower surfactant concentration forms
fewer micelles resulting in larger particle size [Evans et al 1999] However there is a
plateau in particle mean diameter between a surfactant to monomer ratio of 0005 and
0013 This is due to low polymerization conversion of latex particles from monomer
leading to a smaller particle size than expected This is not a factor of surfactant amount
The dependence of the particle size on the amount of surfactant is shown in Figure 4-5
0
200
400
600
800
1000
0000 0005 0010 0015 0020 0025 0030
Surfactant to monomer ratio (ww)
Part
icle
mea
n di
amet
er (n
m)
Figure 4-5 Dependence of the particle size on the surfactant to monomer ratio
71
413
e
f bare
microdomain structures is important for biocompatibility [Mori et al 1982 Higuchi et al
1993 Deppisch et al 1998] Figure 4-6 shows the schematic of core shell latex particle
structure The recipe for core shell structures is shown in Table 4-4
Core Shell Latex Particles
The latex particles shown in Figure 4-3 (A) and labeled as S258 in Table 4-2 wer
used as seeds to prepare core-shell latex particles because of their high particle
uniformity and smooth surface morphology Methyl methacrylate (MMA) and acrylic
acid (AA) monomers were introduced to increase surface hydrophilicity over that o
polystyrene particles AA is a more hydrophilic monomer than MMA because of the
carboxyl acid group in AA is more favorable for hydrogen boding with water The
surface carboxyl groups on PAA may have many promising applications in biomedical
and biochemical fields [Kang et al 2005] MMA monomer is less hydrophobic than
styrene PMMA is known to more biocompatible than PS but still PMMA is hydrophobic
The optimization of suitable hydrophobic to hydrophilic ratios and control of
Polystyreneseed particle
Core shelllatex particles
PSPMMA100
PSPMMA90PAA10
PSPMMA75PAA25
Polystyreneseed particle
Core shelllatex particles
PSPMMA100
PSPMMA90PAA10
Figure 4-6 Schematic of core shell latex particle structures
PSPMMA75PAA25
72
Table 4-4 The preparation recipe of PS core with various shell latex particles Latex 25 label PSPMMA100 PSPMMA90PAA10 PSPMMA75PAA
Initial charge
PS seed latex (g) 500 500 500a
Monomer continuous
Methly methacylate (g) 320 288 240
Acrylic acid (g) 00 32 80
Initiator stream
Na
258 258 258
Water (g) 1570 1570 1570
Feed time (min) 0-120 0-50 0-60
b 200
Feed time (min) 0-122 0-60 0-65
952
2S2O8 (g) 02 02 02
NaHCO3 (g) 02 02 02
Water (g) 200 200
Solids content 183 183 183
Mean particle diameter (nm) 370plusmn186 370plusmn178 370plusmn191
Conversion () 955 924
Three types of core shell structures were prepared PMMA75PAA25 shell is a
copolymer consisting of the PMMA to PAA ratio of 75 to 25 by weight
PMMA90PAA10 shell is a copolymer with the PMMA to PAA ratio of 90 to 10 by
weight PMMA100 is the PMMA homopolymer shell on PS core The particle size of
these core-shell particles as well as PS latex particle was prepared to be about 370nm in
mean diameter and are characterized to determine their zeta potential protein adsorption
and biocompatibility The image of SEM of PS and core shell particles is shown at Figure
4-7 The synthesized latex particles had a high uniformity in size and a smooth spherical
surface morphology
73
Figure 4-7 Scanning
Electron Micrograph of latex particles (A) PS (B) PSPMMA100 (C) PSPMMA90PAA10 (D) PSPMMA75PAA25
(A)
(B)
74
(C)
(D)
Figure 4-7 Continued
75
42 Characterization of Latex Particles
421 Fourier Transform Infrared Spectroscopy (FTIR)
FTIR spectroscopy has proven to be a powerful tool to characterize polymeric
materials A molecule can absorb only select frequencies (energy) of infrared radiation
which match the natural vibration frequencies The energy absorbed serves to increase
the amplitude of the vibrational motions of the bonds in the molecule [Pavin et al 1996]
Only those bonds which have a dipole moment that changes as a function of time can
absorb infrared radiation The FTIR spectra of polymerized latex particles made during
this work are shown in Figure 4-8 There are number of characteristic peaks for PS latex
particles [Bhutto et al 2003 Li at al 2005] Aromatic C-H stretching vibration is shown
at 3002 cm-1 and 3103 cm-1 and aliphatic C-H asymmetrical and symmetrical stretching
shown at 2900 cm-1and 2850 cm-1 respectively There is an additional carbonyl (C=O)
adsorption at 1730cm-1 wavenumber for all core shell latex particles containing some
amount of either or both PMMA and PAA The broad OH group peak at 3400 cm-1
appear for particles containing some amount of PAA and has intensity dependent on the
amount of PAA in the shell For example the peak intensity of OH groups for
PSPMMA75PAA25 is greater than that of PSPMMA90PAA10 since there is a higher AA
monomer content in PSPMMA75PAA25 than in PSPMMA90PAA10 This OH peak is not
seen for PS and PS core PMMA shell particles because of the absence of OH groups on
PS and PMMA polymer chains
422 Zeta Potential Measurements
The electrical surface property was characterized by zeta potential measurements of
tex particles Zeta potential is an important and useful indicator of surface charge which
can be used to predict and control the stability of colloidal systems The greater the zeta
is
la
76
1520002500300035004000
Wavenumbers (cm-1)
li
Re
ativ
e
Tra
nsm
ttanc
e
Figure 4-8 FTIR spectra of polymerized latex particles (A) ba
PSPMMA100 (C) PSPMMA90PAA10 (D) PSPMM
(B)
(C) P
(D) P
C
C
OCH3
O
CH3
XCH2
CH2 n
CH
CH2 C
C
OCH3
O
XCH2 CH
OC
CH3
Y
OH
(A) Bare PS
500100000
re polystyrene (PS) (B) A75PAA25
PSPMMA100
SPMMA90PAA10
SPMMA75PAA25
77
potential the more likely the suspension is to be stable because the charged particles repel
one another and thus overcome the natural tendency to aggregate The measurement of
zeta potential is often the key ontrol of the interaction of proteins
with solid surfaces such as latex particles
Zeta potential (ζ) is the elec at exists at the shear plane of a particle
which is some small distance from the surface [Myers 1999] The zeta potential of the
particles can be determined by measuring the mobility of the particles in an applied
electric field termed electr its response to an alternating electric
field Colloidal particles dispersed in lu ically charged due to their ionic
characteristics and dipolar attributes The development of a net charge at the particle
surface affects the distribution of ions in the neighboring ng in
an increased concentration of counter ions to the surface Each particle dispersed in a
ns called fixed layer or Stern layer
Outside th compositions of ions of opposite polarities
form ble layer is form
particle-liquid interface This double layer may be considered to consist of two parts an
inner region which inclu nd relatively strongly to the surface and an outer or
diffuse region in which the ion distribution is determined by a balance of electrostatic
forces and random thermal motion The potential in this region therefore decays with the
distance from the surface until at a certain distance it becomes zero Figure 4-9 shows a
schematic representation of a typical ion distribution near a positively charged surface
The Stern surface (also referred to as the outer Helmholtz plane) is drawn through the
to understanding and c
trical potential th
ophoretic mobility or
a so tion are electr
interfacial region resulti
solution is surrounded by oppositely charged io
e Stern layer there are varying
ed in the region of the ing a cloud-like area Thus an electrical dou
des ions bou
center of those ions which are effectively adsorbed to the surface [Hunter 1981] The
78
extent of ion adsorption is determined by electrical and other long-range interactions
between the individual ions and surface of particles The ions outside of the Stern layer
form the diffuse double layer also referred to as the Gouy-Chapman layer [Burns and
Zydney 2000]
Shear plane
Stern layer
ψζ
0
ψs
ψShear plane
Stern layer
ψζ
0
ψs
ψ
t
controlled electric field is applied via electrodes immersed in a sample suspension and
Figure 4-9 Schematic representation of ion distribution near a positively charged surface
Zeta potential is a function of the surface charge of a particle any adsorbed layer a
the interface and the nature and composition of the surrounding medium in which the
particle is suspended The principle of determining zeta potential is very simple A
79
this causes the charged particles to move towards the electrode of opposite polarity
Viscous forces acting upon the moving particle tend to oppose this motion and the
equilibrium is rapidly established between the effects of the electrostatic attraction and
the viscosity dra
g The particle therefore reaches a constant terminal velocity
Figure 4-10 Schematic representation of zeta potential measurement(source
httpnitioncomenproductszeecom_shtm)
Because protein adsorption mechanisms are very complex and hard to explain in
biological systems protein adsorption is generally studied by using more ideal systems
consisting of one or more well characterized proteins a well-characterized adsorbent and
a well-defined aqueous solution Even so small changes in the experimental conditions
such as pH ionic strength and temperature generate totally different results Therefore
two media systems phosphate buffer (PB) and phosphate buffered saline (PBS) were
chosen to be used for the zeta potential measurements and protein adsorption study PB is
a simple aqueous system of 5 mM monobasic sodium phosphate (NaH2PO4H2O) to
n a constant pH PBS is used to keep the water concentration inside and outside of
the cell balanced If the water concentration is unbalanced the cell risks bursting or
shriveling up because of a phenomenon called osmosis PBS is a solution with the 5 mM
of monobasic sodium phosphate (NaH2PO4H2O) and 143 mM of sodium chloride (NaCl)
in water
maintai
80
The results of zeta potential measurement at room temperature (25plusmn1oC) as
function of pH and ionic strength (different media PB and PBS) for synthesized lat
particles are seen in Figure 4-11 Zeta potential values of polystyrene (PS) latex particle
at the Figure 4-9 are negative between -291 mV and -599 mV in PB and betwe
ex
s
en -203
mV and -278 mV in PBS at all pH ranges These negative zeta potential values of PS
latex particles are due to the sulfate groups [Vandenhu et al 1970] originated from
persulfate initiators attached via an oxygen bridge (-C-O-SO3-) at the end of polymer
chain where polymerization was initiated
-100
-80
-60
-40
-20
0
pH
Zta
pte
ntl
ζ
20
40
1 2 3 4 5 6 7 8 9
eo
ia (m
V)
PB (5mM)
PBS (5mM +143mM)
en -
Figure 4-11 Zeta potential of PS latex particles at 25oC
The zeta potential measurements of PSPMMA100 core shell latex particles as seen
in Figure 4-12 were also negative with a range of -28 and -505 mV in PB and betwe
143 mV and -186 mV in PBS at the pH 21-78 These negative values are also due to
sulfate groups on the particle surface
81
-100
-80
-60
-40
-20
0
20
40
1 2 3 4 5 6 7 8 9
Zea
pote
nti
l ζ
(mV
pH
ta
)
PB (5mM)
PBS (5mM + 143mM)
Figure 4-12 Zeta potential of PSPMMA100 latex particles at 25oC
The zeta potential for PSPMMA90PAA10 and PSPMMA75PAA25 was also
negative as seen in Figure 4-13 and 4-14 The zeta potential value for the
PSPMMA90PAA10 were between -367 mV and -678 mV in PB medium and between -
147 mV and -193 mV in PBS both at 25oC In case of the PSPMMA75PAA25 core shell
particles zeta potential values were between -291 mV and -520 mV in PB and between
-115 mV and -210 mV in PBS media Sulfate groups in PSPMMA75PAA25 and
PSPMMA90PAA10 core shell particles contribute to the negative zeta potential values
participate in forming a
negat
cid is expressed by the term pKa which is the pH
at which an acid is 50 dissociated However the contribution of the carboxylic acid
Carboxylic groups from acrylic acid monomer also would
ive surface charge of the latex particles at a pH greater than 70 because the pKa
value for the carboxylic acid is between 40 and 60 pH range [Ottewill et al 1967] This
indicates that carboxylic acid dissociation begins at a pH between pH 46-60 and
increases with pH The strength of an a
82
group to negative zeta potential values of PSPMMA-PAA core shell particles was not
detectable because there was no significant difference in zeta potential values for
synthesized latex particles at a pH greater than 70
-60
-40
-20
0
20
40
1 2 3 4 5 6 7 8 9
Zeta
pot
entia
l ζ
(mV
)
PB (5mM)PBS (5mM + 143mM)
-80
-100
pH
Figure 4-13 Zeta potential of PSPMMA90PAA10 latex particles at 25oC
-100
-80
-60
-40
-20
0
20
40
1 2 3 4 5 6 7 8 9
pH
Ze p
ont
i ζ
V)
tate
al (m
PB (5mM)PBS (5mM + 143mM)
Figure 4-14 Zeta potential of PSPMMA75PAA25 latex particles at 25oC
83
The zeta potential plots and values were similar for all latex particles This may be
due to the similar initiator density on the latex surface The initiator concentrations
monomers for shell polymerization were the same fo
to
r all core shell latex particles (062
ww from Table 4-4) A similar concentration (055 ww) of initiator to monomer was
added to prepare bare PS latex particles used in zeta potential measurements
Since the zeta potential values of all synthesized latex particles were negative
between pH=20 and pH=78 the isoelecric point (IEP) of these latex particles would be
less than pH=20 Sulfates have a pKa in the range of 10 to 20 [James 1985] indicating
that the sulfate group the conjugate base of a strong acid is protonated (-C-OSO3H) at
less than pH 20 This means that the synthesized latex particles are all negative at the
physiological pH Most serum proteins have a negative surface charge in blood
therefore when the negative latex particles are applied to blood stream fatal
tein is
avoided by charge repulsion in the blood Table 4-5 shows the common blood proteins
and their isoelectric points (IEP)
Table 4-5 The isoelectric point (IEP) of common proteins Serum proteins Isoelectric point (IEP)
complication by coagulation between applied latex particles and serum pro s
Albumin 48 α1-Globulin 20 α2-Microglobulin 54 γ1-Globulin 58 Heptoglobin 41 Hemoglobin 72 β1-lipoprotein 54 Immunoglobulin G (Ig G) 73 Fibrinogen 58 (source httpwwwfleshandbonescomreadingroompdf1085pdf
httpwwwmedalorgvisitorwww5CActive5Cch135Cch13015Cch130105aspx )
84
The absolute zeta potential values of the latex particles in phosphate buffer (PB)
was higher than that in phosphate buffered saline (PBS) This is because high
concentration of sodium and chloride electrolytes in PBS compressed the electrical
double layer causing rapid decay of surface potential Figure 4-15 shows that the
thickness of the electrical double layer is inversely proportional to the concentration of
electrolyte in the system
Surf
ace
Pote
ntia
l ψ
1κ3
10
1e
1κ 1κ
(1)(3) (2)
2 1 Z
Surf
ace
Pote
ntia
l ψ
1κ3
10
1e
1κ 1κ
(1)(3) (2)
Figure 4-15 The decay of surface potential with distance from surface in various
electrolyte concentrations (1) low (2) intermediate (3) high [Myers 1999]
2 1 Z
The electrical potential in the solution surrounding the particle surface falls off
the Debye-Huckel
yers 1999]
exponentially with distance (Z) from the surface according to
approximation [M
z) (- exp 0 κψψ = (4-4)
e reciprocal of the thickness of the electrical double layer referred to as the
The potential fallen off by a factor e The theoretical equation for the
ickness 1κ is
where κ is th
Debye length of 1
double layer th
21
22i
0
ekT 1
⎟⎟⎠
⎜⎜⎝
=sum ii zcεε
κ
⎞⎛ (4-5)
85
where
tration of
y was performed to measure the protein adsorption on the
synthesized latex particles Many physical and chemical processes occur at the boundary
between two phases and adsorption is one of the fundamental surface phenomena [Oscik
1982] This phenomenon is driven by the change in the concentration of the components
at the interface with respect to their concentrations in the bulk phase There are two
adsorption processes physical adsorption occurring when non-balanced physical forces
appear at the boundary of the phases and chemical adsorption or chemisorption
t the
interface [J
n leads
ational entropy hence adsorption takes place only if the
loss in conformational entropy is compensated
ter adsorption Cp The initial part of the
isotherm merges with the Γ-axis because at low polymer concentration the protein has a
high affinity for the surface and all of the polymer is adsorbed until the solid surface is
saturated
ε0 is the permittivity of a vacuum of free space ε is the permittivity of a medium e
is the relative permittivity or dielectric constant of the medium c is the concen
an ion (i) z is the valency of an ion (i) k is Boltzmannrsquos constant T is absolute
temperature (K)
43 Protein Adsorption Study
The adsorption stud
occurring when atoms and molecules from adjacent phases form chemical bonds a
aroniec et al 1988]
Flexible proteins in solution possess high conformational entropy as a result of the
various states each of the many segments in the protein chain can have Adsorptio
to a reduction of this conform
by sufficient attraction between polymer
segment and solid surface [Martin 1998] Isotherm shape of high affinity adsorption
between polymer and solid is shown in Figure 4-16 where the adsorbed mass Γ is plotted
against the polymer concentration in solution af
86
Figur
de
arboxyl
e 4-16 High-affinity adsorption isotherm of typical flexible polymer on solid
surface
In order to investigate the adsorption of blood proteins latex particle properties
such as electrical surface charge hydrophobicity and environmental conditions like pH
ionic strength and temperature are considered as experimental factors [Arai and Nor
1990] Figure 4-17 shows the overall schematic representation of the protein adsorption
on the synthesized latex particles with the functional groups such as sulfate and c
groups
CP
Γ
PMMAPAAShell
PS or PMMAShell OSO3
-
OSO3-
OSO3-
OSO3-
PS
-O3SO
PSCore
HOOC
-O3SO
O3SO
3
HOOC
HOOC
-
-O SO
Protein
NH3+
+
+H3N
+
COO-
Hydrophobic interaction
NH3
COO-
H3N
COO-
-OOC
Ionic interaction
Hydrophobic interaction
Hydrogen bonding
Ionic interaction
PMMAPAAShellOSO3
-
OSO3-
OSO3-
OSO3-
PS or PMMAShell -O3SOOSO3
-
PSCore
HOOC
-O3SO
O3SO
3
HOOC
HOOC
-
-O SO
-O3SO
OSO3-
OSO3-
OSO3-
PS PSCore
HOOC
-O3SO
O3SO
3
HOOC
HOOC
-
-O SO
Protein
NH3+
+
+H3N
+
COO-NH3
COO-
H3N
COO-
-OOC
NH3+
+
+H3N
+
COO-
Hydrophobic interaction
NH3
COO-
H3N
COO-
-OOC
Ionic interaction
Hydrophobic interaction
Hydrogen bonding
Figure 4-17 Overall schematic representation of the protein adsorption on the
Ionic interaction
synthesized latex particles
The surface functional groups as well as suitable surface hydrophobicity
hydrophilicity of latex particles are closely related to protein adsorption property As
indicated at Figure 4-17 various interaction forces such as hydrophobic interaction ionic
87
interaction hydrogen interaction and van der Waals interaction [Andrade 1985] exist
between protein molecule and the solid surface of latex particle
431 Adsorption Isotherm
The protein adsorption isotherm is generally defined as the relationship between the
amount of protein adsorbed by a solid (latex particle) at a constant temperature and as a
ine
the amount of protein adsorbed onto latex particles To evaluate the equilibrium
conce s
x
function of the concentration of protein Adsorption isotherms were plotted to determ
ntration of target proteins a calibration curve is firstly needed Figure 4-18 show
the UV absorbance as a factor of bovine serum albumin (BSA) concentrations The UV
absorbance of the known protein concentration was measured at 562nm which is the λma
for a protein treated by a bicinchoninic acid (BCA) assay
0
02
04
06
08
500 520 540 560 580 600
Wavelength (nm)
Abs
orba
nce
Figur gml (B)
06mgml (C) 02mgml
(A)
(C)
(B)
Max562nm
e 4-18 The dependence of UV absorbance on BSA concentration (A) 10m
Figure 4-19 shows the standard calibration curve used in adsorption isotherm
experiment of protein onto latex particles The net absorbance of each known protein
88
concentration was plotted and then a linear equation passing through a concentration of
zero protein concentration was obtained This equation is applied
to determine the
orbed on the synthesized latex particles The amount of free
protei
eported
trations (005 01 03 05
and 07mgml) were used to generate this curve as well as the adsorption isotherm
unknown protein amount ads
n was also determined using UV light at the 280nm wavelength however a
distinguishable UV intensity was too low to determine a precise value of protein
adsorption
The bicinchninic acid (BCA) assay technique [Smith et al 1985] has been r
for high efficiency of protein evaluation and was used for determination of protein
adsorption [Tangpasuthadol et al 2003 Williams et al 2003] This technique has many
advantages such as high sensitivity easy of use color complex stability and less
susceptibility to any detergents Five different BSA concen
y = 00R2 =
004741x 09939
00
01
02
03
04
0 200 400 600 800
BSA Standard (microgml)
Net
Abs
orba
nce
at 5
62nm
Figure 4-19 Standard curve of net absorbance vs BSA sample concentration
89
All isotherms were fitted to the Langmuir-Freundlich isotherms using the nonlinea
regression method Two adsorption isotherm models the Langmuir type isotherm a
Langmuir-Freundlich combination type isotherm were compared As seen the Figure 4-
20 The Langmuir isotherm model deviated further from the experimental data Th
all isotherms were fitted to the Langmuir-Freundlich isotherms [Yoon et al 1996
al 1998] usi
r
nd the
erefore
Lee et
ng the nonlinear regression method with following equation
n
n
eq
eqm
kC
kC1
1
1+
where q is the amount of adsorbed BSA per unit surface area q
qq = (4-6)
stant
were between 045 and 055 and hence n was fixed to its average 05 Actual qm values
were evaluated after fixing the n value
m is the adsorbed amount
in equilibrium Ceq is the equilibrium concentration of BSA k is the adsorption con
and n is the exponential factor for a heterogeneous system n values are empirical and
usually from zero to no more than unity [Jaekel 2002] The values of n in our system
00
10
20
30
0 02 04 06 08
C (mgml)eq
q (m
gm
2 )
pH 32Langmuir-Freundlich modelLangmuir model
Figure 4-20 Fitted models for adsorption isotherm of bovine serum albumin (BSA) on
polystyrene latex particles at 37oC in phosphate buffer media
90
The BSA protein adsorption on the synthesized latex particles was performed at
three pH levels 32 48 74 in both phosphate buffer (PB) and phosphate buffered saline
(PBS) at physiological temperature 37oC These pH levels correspond to an acidic pH
lower than the isoelectric point (IEP) of BSA the pH at the IEP and an alkaline pH
higher than the IEP of BSA respectively The IEP corresponds to the pH at which the
amino acid becomes a zwitterion Here its overall electrostatic charge is neutral and the
charge expressed by the basic amino group (-NH2 -NH3+) of the amino acid is equal to
the charge expressed by the acidic carboxyl group (-COOH -COO ) The IEP of BSA is
-
pH 48 and indicates that the side chain of the amino acids of BSA contains a greater
amount of acidic functional groups than that of basic functional groups At pH 32 and
74 BSA has a positive and negative charge respectively
Figure 4-21 and 4-22 show adsorption isotherms of BSA onto polystyrene (PS)
latex particles
00
10
20
30
40
0 02 04 06 08
Ceq (mgml)
q (m
gm
2 )
pH=32pH=48pH=74Model (pH 32)Model (pH 48)Model (pH 74)
Figure 4-21 Adsorption isotherm of BSA on polystyrene (PS) latex particles in
phosphate buffer (PB) at 37oC
91
00
10
30
40
0 02 04 06 08
Ceq (mgml)
q (m
gm
2
20
)
pH=32pH=48pH=74Model (pH 32)Model (pH 48)Model (pH 74)
(PBS) on PS latex particles
At the IEP of BSA the adsorbed amount (q
Figure 4-22 Adsorption isotherm of BSA adsorption 37oC in phosphate buffered saline
onto PS at equilibrium was
maxim
um at the IEP of BSA This is because the BSA molecules form the most
compact structures at the IEP eliminating the repulsion force between proteins A higher
number of BSA molecules can adsorb on a given surface area by hydrophobic interaction
when in this compact structure This observation agrees well with reported data by other
researchers who also indicated that the maximum adsorption from aqueous protein
solution was observed at the IEP [Mularen 1954 Watkins et al 1977 Oh at el 2000]
There was no significant difference of adsorbed BSA amount in PB and in PBS media at
pH 48 The conformational alteration of BSA was not affected significantly by the ionic
strength of the two solutions There was evidence of the effect of the electrostatic
interaction to some extent even at the IEP since the amount of BSA adsorbed in PBS is
slightly hig
m) of BSA
um indicating that hydrophobic interaction between latex particle and BSA
protein is maxim
her than in PB at this pH
92
The adsorbed amount of protein is shown to decrease at a pH 32 and 74 The
decrease in adsorption efficiency when deviating from IEP of BSA is a result of the
increase in conformational size of the protein molecules and the lateral electrostatic
repulsions between adjacent adsorbed BSA molecules Figure 4-23 shows a schematic of
possible conformational changes at each pH where hydrophobic interaction always exists
between non-polar portion of a protein and hydrophobic portion of solid surface
- - --- ---Latex surface
Zeta potential
BSA(+)
(-)
48 7432pH
(0)
+++ + ++
--+
++-
- - --- ---Latex surface
Zeta poten pH(0)
+++ + ++
--
tial
BSA(+)
(-)
48 7432
+
++-
ming
4
s
al blood Table 4-6 shows the calculated results of
Figure 4-23 Conformation of bovine serum albumin (BSA) on latex particles (Assuthat charge repulsion reduces the effect of the hydrophobic interaction at pH 74)
In addition the adsorbed amount of BSA at pH 32 was larger than that at pH 74
and still less than that at 48 This is because there is the electrostatic interaction between
the positive BSA molecules and negative PS latex particles at pH 32 and electrostatic
repulsion is dominant between negative BSA and negative PS latex particles at pH 7
The amount adsorbed increased with increasing ionic strength in pH 32 and 74 This i
because the high concentration of electrolytes reduces the electrostatic repulsion between
BSA and PS latex particle It should be noted that the BSA concentration used in these
experiments was lower than that found in normal human blood This is because the
equilibrium BSA adsorption onto the latex particles is actually reached at a much lower
concentration than that found in norm
93
adsor rm
ein
leading to a compact conformation of BSA There are two main forces affecting
adsorption amount at an acidic pH of 32 They include the ionic attraction between the
negative latex particle and the positive protein and lateral repulsion between positive
proteins The smallest amount of BSA adsorption was observed at a pH of 74 At this
point the charge repulsion between negatively charged particles and protein molecules
exists the h
of cha
bed BSA amount at equilibrium from the Langmuir-Freundich adsorption isothe
equation
In summary the hydrophobic interaction between PS latex particle and BSA
protein exists and is the dominant mechanism for the adsorption process Conformational
changes of BSA at each pH also affected the adsorption amount Adsorption was highest
at pH 48 because at this pH there is the least amount of ionic repulsion in the prot
and the lateral repulsion between proteins dominant adsorption even though there always
ydrophobic interaction between the solid latex particle and protein The effect
rge repulsion was reduced by the high concentration of electrolytes in PBS medium
Table 4-6 The equilibrium concentration values of BSA adsorption on polystyrene (PS) latex particles calculated the Langmuir-Freundlich isotherm model
Media pH qm (mgm2) 32 174 48 299 Phosphate buffer 74 136 32 260 48 283 Phosphate buffered saline 74 207
The adsorption isotherms of BSA on PSPMMA core shell particles are shown in
Figure 4-24 in phosphate buffer (PB) and in Figure 4-25 in phosphate buffered saline
(PBS) The adsorbed BSA amount was calculated using the Langmuir-Freundlich
100
94
adsorption isotherm equation and the amount listed in Table 4-7 The trend seen in the
adsorption results was very similar to the case of PS latex The overall adsorption process
was d rticle
rs
hat
0
ominated by hydrophobic interactions between the PSPMMA100 core shell pa
and BSA proteins At pH 48 the adsorbed amount (qm) of BSA on PSPMMA100 core
shell particles was 221 mgm2 in PB and 237 mgm2 in PBS and are the maximum
values recorded among the three pH levels 32 48 and 74 Suzawa and his co-worke
[1982] have obtained similar results This can be also explained by the compact
conformation of BSA molecules at the IEP of BSA which lead to a high protein
population on the solid surface At a pH of 32 and 74 BSA adsorption was less than t
at pH 48 because of the protein conformation change There was a small increase in
adsorbed BSA at pH 32 because of the charge attraction between negative PSPMMA10
core shell particles and positive BSA At pH 74 however the adsorption of BSA was
further reduced by charge repulsion between the negative PSPMMA100 core shell
particles and the negative BSA proteins
000 02 04 06 08
Ceq (mgml)
10
20q (m
gm
302 )
40
50pH=32pH=48pH=74Mode 8)l (pH 4Mode 2)l (pH 3Mode 4)
Figure 4-24 Adsorption isotherm of BSA 37oC in PB on PSPMMA core shell latex
l (pH 7
100
particles
95
The adsorbed amount of BSA increased more rapidly in PBS with increasing ioni
strength at pH 74 This shows that the low concentration of electrolytes promotes
electrostatic repulsion between negative BSA and negative PSPMMA
c
on
x
l 1980
100 core shell
particle As the ion strength of PBS is increased however the high concentration of
electrolyte leads to less electrostatic repulsion promoting more rapid protein adsorpti
By comparing the amount of adsorbed protein between bare PS latex particles and
PSPMMA100 core shell particles the amount of protein adsorption on the PS late
particles is greater than that on the PSPMMA100 core-shell particles [Suzawa et a
1982 Lee et al 1988]
00
10
20
30
40
m2 )
0 02 04 06 08
Ceq (mgml)
q (m
g
pH=32pH=48pH=74Model (pH 2) 3Model (pH 48)Model (pH 74)
Figure 4-25 Adsorption isotherm of BSA at 37oC in PBS on PSPMMA100 core shell
latex particles
96
Table particles calculated the Langmuir-Freundlich isotherm model
4-7 The equilibrium concentration values (qm) of BSA adsorption on PSPMMA100
Media pH qm (mgm ) 2
32 140
48 221 Phosphate buffer
74 130
32 169
48 237 Phosphate buffered saline
74 162
The BSA adsorption mechanism and trend on PSPMMA core shell particles was
very similar to that of the PS latex particle Unlike the case of PS and PSPMMA100 latex
particles the amount of BSA adsorbed onto PSPMMA90PAA10 and PSPMMA75PAA25
core shell particles was higher at pH 32 than pH 48 and 74 Figure 4-26 through 4-29
shows the adsorption isotherms of BSA on PSPMMA90PAA10 and PSPMMA75PAA25
core shell particles The amount of BSA adsorbed increased at pH 32 and pH 48 as the
hydrophilicity was developed by the addition of PAA On the contrary the adsorbed
amount of BSA was dramatically decreased at pH 74 The adsorption amount of BSA on
PSPMMA90PAA10 and PSPMMA75PAA25 core shell particles at equilibrium is listed in
Table 4-8 and 4-9 respectively
Carboxylic acid as well as sulfate groups are distributed on the surface of
PSPMMA90PAA10 and PSPMMA75PAA25 core shell particles and seem to largely affect
the BSA adsorption The isotherms showed very strong pH dependence to the BSA
adsorption onto these core shell latex particles The carboxylic groups (COOH) may
especially enhance the BSA adsorption prominently at pH 32 by hydrogen bonding with
protein molecules The pKa value for the PAA is 40-60 and a good indication that the
97
carboxylic acid groups are protonated at pH 32[Gebhardt et al 1983] The main
adsorption interaction force at pH 32 was generated by hydrogen bonding as well as
charge attraction between latex particle protein even th the conformational
repulsion between proteins results in a reduction of the protein
adsorption at this pH The amount of BSA adsorbed also increased as the PAA amount in
eased The a tion of BSA w inimal however less
than 07 mgm2 at pH 74 Here the carboxylic groups are ionized which increases the
charge repulsion by ionized carboxylic groups (COO-) resulting in PSPMMA-PAA latex
particle
and ough
change and charge
the core shell particles incr dsorp as m
s with much lower protein adsorption
00
20
40
q (m
g2 )
60
m
pH=32pH=48pH=74Model (pH 32)Model (pH 48)
0 02 04 06 08
Ceq (mgml)
Model (pH 74)
o shell
Figure 4-26 Adsorption isotherm of BSA at 37 C in PB on PSPMMA90PAA10 corelatex particles
98
00
40
60
80
0 02 04
C (mgm
mm
2 )
20
06 08
eq l)
q (
g
pH=32pH=48pH=74Model (pH 32)Model (pH 48)Model (pH 74)
Figure 4-27 Adsorption isotherm of BSA at 37oC in PBS on PSPMMA90PAA10 core
shell latex particles
Table 4-8 The equilibrium concentration values of BSA adsorption on PSPMMA90PAA10 particles calculated the Langmuir-Freundlich isotherm model
Media pH qm (mgm2)
32 361
48 283 Phosphate buffer
74 043
32 519
48 226 Phosphate buffered saline
74 043
99
00
20
40
60
80
100
0 02 04 06 08Ceq (mgml)
q (m
gm
2 )
pH=32pH=48pH=74Model (pH 32)Model (pH 48)Model (pH 74)
oFigure 4-28 Adsorption isotherm of BSA 37 C in PB on PSPMMA75PAA25 latex particles
00
100
20
40
60
80
0 02 04 06 08Ceq (mgml)
q (
m2 )
pH=32pH=48pH=74
mg
Model (pH 32)Model (pH 48)Model (pH 74)
Figure 4-29 Adsorption isotherm of BSA 37oC in PBS on PSPMMA75PAA25 core shell
latex particles
100
Table 4-9 The equilibrium concentration values of BSA adsorption on PSPMMA75PAA25 particles calculated the Langmuir-Freundlich isotherm model
Media pH qm (mgm2)
32 1083
48 662 Phosphate buffer (PB)
74 075
32 1077
48 681 Phosphate buffered saline (PBS)
74 057
The adsorption of β2-microglobulin (β2M) was determined under one set of
onditions in phosphate buffer at 37oC and pH 74 because of the limited amount of β2M
protein The adsorption isotherms of the β2M protein on the synthesized latex particles
are shown in Figure 4-30 4-31 and 4-32 The β2M protein concentrations for adsorption
isotherm experiment were chosen to be 0015 0030 0045 and 0060mgml since the
β2M concentration for the patients with renal failure can be elevated to similar
concentrations about 005mgml or more up to 50 times the normal level [Nissenson et
al 1995] There were steep initial slopes of the adsorption isotherm curves of β2M on
latex particles in all isotherms indicating a high affinity adsorption type It seems that the
hydrophobic interaction is enough for the adsorption process of β2M proteins to occur on
the latex particles even though charge repulsion exists between protein and latex particle
at pH 74 The complete plateau regions were not seen for all isotherms indicating that
β2M proteins were not saturated at this level and there are still available sites for β2M
c
adsorption on latex particles The initial slope of the β2M isotherms for PS was steeper
101
than that of any of the other latex particle PSPMMA100 PSPMMA90PAA10 and
PSPMMA 75PAA25
00
02
0 0002 0004 0006 0008
04
06
10
Ceq (mgml)
q (m
2 )
08
gm
PSPSPMMA100Model (PS)Model (PSPMMA100)
Figure 4-30 Adsorption isotherm of β2M onto PS and PSPMMA100 latex particles in PB
at 37 C and pH 74 o
00
02
04
06
08
10
0 0002 0004 0006 0008C (mgml)
q (m
m2 )
eq
g
PS
PSPMMA90PAA10
Model (PS)
Model (PSPMMA90PAA10)
Figure 4-31 Adsorption isotherm of β2M onto PS and PSPMMA90PAA10 latex particles
in PB at 37oC and pH 74
102
00
02
04
06
08
10
0 0002 0004 0006 0008Ceq (mgml)
q (m
gm
2 )
PS
PSPMMA75PAA25
Model (PS)Model (PSPMMA75PAA25)
Figure 4-32 Adsorption isotherm of β2M onto PS and PSPMMA75PAA25 latex particles
in PB at 37oC and pH 74
The equilibrium concentration values (qm) of β2M adsorption on latex particles are
calculated and listed in Table 4-10 The difference between the maximum amounts of
β2M adsorption on all the latex particles was not significantly different and their values
were between 069 and 08mgm2
Table 4-10 The equilibrium concentration values of β2M adsorption calculated the Langmuir-Freundlich isotherm model
Latex particles qm (mgm2)
PS 080
PSPMMA100 069
PSPMMA90PAA10 072
PSPMMA75PAA25 072
103
432 Adsorbed Layer Thickness
There are several possible explanations for large amount of BSA adsorbed at acidic
pH levels including the end-on adsorption pattern of BSA the flexibility of BSA
molecules the tilting of protein molecules due to the asymmetry of the charge
distribution or multilayer formation [Peula et al 1993] The adsorbed BSA thickness (δ)
can be calculated by the following equation [Chiu et al 1978] This equation was also
cited again by other researchers [Shirahama et al 1985]
BSA
mqρπ
δbullbull
=33
(4-7)
where π
33 is the packing factor qm is the plateau value of the adsorbed am
ρBSA is the hich corresponds to the reciprocal of its known
partial specific volume The layer thickness (δ) of BSA proteins on synthesized latex
partic
he IEP [Shirahama et al 1985] Compared to the
hydrodynamic dimensions of BSA 140 times 40 times 40 Aring3 [Fair et al 1980 Peter 1985] these
nd
ode It is unnecessary ultilayering because thickness do
exceed 140 Aring The layer thickness (δ) of BSA proteins on synthesized latex particles
e red saline (PBS) was ran 28 ~ 83 Aring at 37oC and pH 48 These
lso listed in Table 4-1
ount and
density of BSA molecule w
les in phosphate buffer (PB) ranged from 27 - 81 Aring at 37oC and pH 48 These
calculated values are listed in Table 4-11 Only δ values at pH 48 the isoelectric point
(IEP) of BSA are important because intra- and intermolecular electrostatic repulsion of
protein molecules is minimized at t
values of layer thickness indicate that the BSA molecules adsorb by side-on (40Aring) a
end-on (140Aring) m to consider m
not
in phosphat buffe ged of
calculated values are a 2
104
Table 4-11 Absorbed BSA layer thickness (Aring) of BSA in phosphate buffer (PB) at 37oC pH PS PSPMMA100 PSPMMA90PAA10 PSPMMA75PAA25
32 21 17 44 133
48 37 27 35 81
74 17 16 5 9
Table 4-12 Absorbed BSA layer thickness (Aring) of BSA in phosphate buffered saline (PBS) at 37oC
pH PS PSPMMA100 PSPMMA90PAA10 PSPMMA75PAA25
32 32 21 64 132
48 35 29
74 26 20 5 7
28 83
1n
1n
neqm
So k is closely related to the initial slope of the isotherm showing the affinity
between BSA and latex particles [Yoon et al 1996] A key point in the characterization
433 Gibbs Free Energy of Protein Adsorption
In the Langmuir isotherm model k represents the ratio of adsorption to the
desorption rate constants This definition is not applicable to the Langmuir-Freundlich
isotherm model but its meaning is similar [Yoon et al 1996] In this sense k is closely
related to the affinity between protein molecules and latex particles When the
equilibrium concentration Ceq reaches zero the value of Ceq will be very small (1gtgt
kCeq ) and then the equation 4-6 becomes
(4-8)
of adsorption processes on solid-liquid interfaces is to determine the change in Gibbsrsquos
free energy (∆Gdeg) one of the corresponding parameters for the thermodynamic driving
forces during adsorption The traditional method to evaluate adsorption thermodynamic
kCqq 1=
105
constants is based on measurement of adsorption isotherms under static conditions
[Malm ten 1998] The Langmuir-Freundlich ads lf
easily found from Then this enables calculation of the free-
energy change ∆G during adsorption expressed as
(4-9)
where R is the general gas constant (R = 8314Jmiddotmol K ) and T is the absolute
tem ture Gibb e energy A P
listed in Table 4-13 and 4-14 respectively As the ∆Gdegads ore negativ e
pro adsorption on a solid substra ill be more favorable
e of BSA adsorption in phosphate o
pH PS PSPMMA100 PSPMMA90PAA10 PSPMMA75PAA25
s orption equilibrium constant k can be
a slope of the isotherm
degads
lfads kRTG lnminus=∆ o
-1 -1
pera s fre change of BS adsorption values in B and PBS are
values are m e th
tein te w
Table 4-13 The values of Gibbs free energy changbuffer (PB) at 37 C
32 -2411 -2726 -2846 -2805
48 -2352 -2383 -2198 -1972 ∆Go
80
ads (kJmol)
74 -2452 -888 -324 -2
Table 4-14 The values of Gibbs free energy change of BSA adsorption in phosphate
pH PS PSPMMAbuffered saline (PBS) at 37oC
100 PSPMMA90PAA10 PSPMMA75PAA25
32 -2315 -2620 -2312 -2683
48 -2149 -2299 -2090 -2138 ∆Go
(kJmads ol)
74 -2430 -1992 -258 -263
Table 4-15 shows the calculated values of Gibbs free energy change of β M
adsorption on synthesized latex particles PSPMMA PAA core shell latex particle has
2
75 25
106
largest negative Gibbs free energy of β2
2
M adsorption indicating largest affinity betw
β
een
2M adsorption in phosphate buffere (PB) at 37 C and pH74
PS PSPMMA100
M and latex particle
Table 4-15 The values of Gibbs free energy change of βo
PSPMMA90PAA10 PSPMMA75PAA25
∆Go03 ads
(kJmol) -3626 -3466 -3659 -39
The Gibbs free energy of β2M was much more negative in phosphate buffer (PB)
than that of BSA in physiological condition (37 C and pH 74) o from the Table 4-13 and
the Figure 4-31 This indicates that β2M molecules more readily adsorb on latex particles
BSA This can be explained by the rate of diffusion and accessibility of β2M molecules to
the latex particle ier and faster for β2M m an BSA because of
size (molec ight) erical shape molecules It o be
due to the higher hydrophobic force and less electrostatic repulsion that contribute to the
adsorption of β2M molecules not as prevalent in the case of BSA because the IEP of β2M
(57) is clo H 74) than that of BSA (48) causing β2M to
be less electro cally lled p ro
can be seen that the ∆ β2M adsorption onto the synth article
increasing wi hy icity o x particles s the ∆Goads of the BSA
molecules is decreasing or constant This phenomenon supports the theory that the
select lizing
than BSA and that the affinity of β2M for the latex particle is much larger than that of
surface It is eas olecules th
the smaller ular we and sph of β2M may als
ser to physiological condition (p
stati repe from the latex articles than BSA F m Figure 4-33 it
Goads of esized latex p s is
th the drophil f the late wherea
ive adsorption of β2M molecules over BSA molecules can be enhanced by uti
more hydrophilic PSPMMA90PAA10 and PSPMMA75PAA25 core shell latex particles
107
-60
-50
1 2 3 4
-40∆Gad
s
-30
10
0
o (k
Jm
o-
-20l)
β2-Microglobulin (β2M)Bovine serum albumin (BSA)
Figure 4-33 Gibbs free energy of adsorption of proteins on latex particles in PB at 37
and pH 74
434 Kinetics of Adsorption
to
oC
Protein adsorption is a complex process known to have a large biological impact
and is currently not well understood quantitatively because of extreme sensitivity to pH
the concentration of other electrolytes and molecules present temperature and many
other factors that can change in the physiological system Accurate knowledge of the
adsorption kinetics under a given set of conditions is a prerequisite for elucidating the
mechanisms of many fundamental biological processes to be predicted at the molecular
level [Regner 1987] This adsorption kinetics approach is used in protein adsorption
studies because of the uncertainty related to the time needed for the interfacial proteins
reach the equilibrium It is generally accepted that the process of protein adsorption is
comprised of the following steps a) transport toward the interface b) attachment at the
interface c) eventual structural rearrangements in the adsorbed state d) detachment from
PS PSPMMA100 PSPMMA90PAA10PSPMMA75PAA25
108
the interface and transport away from the interface Each of these steps can affect the
overall rate of the adsorption process
The quantitative analysis of the protein adsorption kinetics requires that the protein
amount adsorbed is known as a function of time The kinetics tests were carried out in
phosphate buffer (PB) at 37oC and pH 74 a condition similar to physiological condition
The protein concentrations chosen were 07mgml for BSA and 006mgml for β2M
which are the maximal concentrations used in an adsorption isotherm test A time-based
evaluation of the adsorption was made by measuring the concentration of protein in
solution at different incubation times The results are shown below from Figure 4-34 to 4-
37 The adsorption kinetics of proteins onto th cond-e latex particles is expressed as a se
order model [Oumlzacar 2003] shown by the following equation
2)( tet qqk
dtdq
minus= (4-10)
Integrating above equation and applying the boundary conditions yields
ee
t tq
+=
1 (4-11)
where q
qkq
t
2
2
2
the previous adsorption isotherm results representing good reproducibility of adsorption
t and qe stand for the amount (mgm ) of protein adsorbed at time t and at
equilibrium respectively k is the equilibrium rate constant of second-order adsorption
(m mghr) and t is the incubation time (hr)
The fitting parameters calculated by using the equation 4-11 are listed in table 4-16
From this data it is seen that the amount of BSA adsorbed at equilibrium was higher than
that of β2M on PS and PSPMMA100 latex particles but less than that of β2M on
PSPMMA90PAA10 and PSPMMA75PAA25 latex particles These results are similar to
109
00
05
15
20
0 2 4 6 8 10 12 14
Time (hr)
rot
n ad
sorb
d (
10
25
Pei
em
gm
2
BSA2nd order model (BSA))β2M2nd order model (β2M)
Figure 4-34 The kinetics of protein adsorption in PB on PS latex particles at 37oC and
pH 74
00
05
10
15
20
25
30
0 2 4 6 8 10 12 14
Time (hr)
Prte
inds
oo
arb
ed (
mg
m2 )
BSA2nd order model (BSA)β2M2nd order model (β2M)
Figure 4-35 The kinetics of protein adsorption in PB on PSPMMA100 latex partic
37les at
oC and pH 74
110
00
03
06
09
12
15
0 2 4 6 8 10 12 14
Time (hr)
Prot
ein
adso
rbed
(m
gm
2 ) BSA2nd order model (BSA)β2M2nd order model (β2M)
Figure 4-36 The kinetics of protein adsorption in PB on PSPMMA90PAA10 latex
particles o at 37 C and pH 74
00
03
06
09
12
15
0 2 4 6 8 10 12 14
Time (hr)
BS
A ad
sorb
ed (
mg
m2 )
BSA2nd order model (BSA)β2M2nd order model (β2M)
Figure 4-37 The kinetics of protein adsorption in PB on PSPMMA75PAA25 latex
particles at 37oC and pH 74
111
test The variation of β2M equilibrium adsorption amount was nearly negligible for all
latex particles The plateau region is reached earlier for β2M than BSA on all latex
particles and the K values (equilibrium rate constant of second order adsorption) for the
β2M adsorption process onto latex particles are much larger than for BSA adsorption
These values indicate that the β2M molecules have a higher adsorption rate onto the latex
particles than the BSA molecules
Table 4-16 Fitting parameters of second-order kinetic model BSA β2M
Latex particles qe (mgm2) K (hr-1) qe (mgm2) K ( -1) hr
PS 144 67 074 7549
PSPMMA100 175 353 076 4438
PSPMMA90PAA10 046 97 075 5695
PSPMMA75PAA25 060 179 075 5168 K = (kqe) hr-1
There are several explanations for the phenomena observed in these experiments
First the smaller sizes of β2M molecules allow easier diffusion and a close approach to
the latex substrate Second a reduced number of hydrophobic domains on the latex
particle surface replaced by sulfate groups (-OSO3-) and ionized carboxylic groups
(COO-) are not favorable for larger BSA molecules Third the conformational change by
intra- and inter-molecular repulsion would be faster in β2M due to smaller molecular
weight Finally the ionic repulsion between β2M and the latex particles is less than that
condition (pH 74) than IEP (48) of BSA
between BSA and latex particles because IEP (57) of β2M is closer to the tested
112
44 Blood Compatibility
Blood compatibility can be defined as the property of a material or device tha
permits it to function in contact with blood without inducing any adverse reactions
Successful functioning of blood contacting biomedical materials or devices in contact
with blood strongly depends on the extent to which they damage blood during operation
High shear stresses turbulence rela
t
tively long contact times and cellular impact with
artificial surfaces are most common events experienced by blood in contact with artificial
1978 Winslow and et al
1993] Therefore before any materia e placed into the human b
biocompatibility testing must be the first performed in the view of safety The hemolysis
s a method for pr ary screening of biomaterials to determine
h blood a er circulating body fluid The tests estimat
oglobin released from th blood cell d e induced
foreign material
There are several types of blood cells in our body but the ones that make blood red
are erythrocytes or red blood cells (RBCs) Red blood cells (erythrocytes) are the most
numerous type in the blood These cells are average about 48-54 million per cubic
millimeter (mm3 same as a microliter (microl)) of blood but these values can vary over a
larger range depending on such factors as health and altitude RBCs are red because they
contain hemoglobin which is a protein that contains iron When blood circulates through
lungs oxygen binds to the hemoglobin and is then delivered to all the parts of body
RBCs are made in the bone marrow They start out as a nucleus like other cells but
when they fill up with hemoglobin the nucleus becomes smaller until it disappears
Without a nucleus RBCs are fragile and live only about 120 days In a normal healthy
devises and can cause hemolysis or thrombosis [Bernhard et al
ls can b ody
test serves a elimin the
biocompatibility wit nd oth e the
concentration of hem e red amag by a
113
body about 2 million red blood ne marrow produces new
ones j ing
dy
le
cells die per second But bo
ust as fast RBCs are about 75 micrometer across and are responsible for carry
oxygen salts and other organic substances to the cells and tissues throughout our bo
In humans and most mammals the cells are concave (indented in the middle) and flexib
enough to squeeze through even the tiniest of blood vessels the capillaries
Figure 4-38 Image of red blood cells
(Sourcehttpwwwpbrchawaiiedubemfmicroangelarbc1htm)
ch
100 90 10 75 25 f
RBCs are among the first cells that come into contact with a foreign material
injected in the blood Therefore a hemolysis assay gives information about the
biocompatibility in the case of an in vivo application The concentrations of prepared
latex particles used for the tests in this research are 05 25 and 50 (ww) and the
incubation time under moderate shaking was 30 minutes at 37oC Test results are shown
in Figure 4-39 The amount of blood cells that ruptured when in contact with the PS latex
particles was largely increased from about 30 to 91 as the solid content of PS
increased from 05 to 50 However hemolysis by the core shell latex particles su
as PSPMMA PSPMMA PAA and PSPMMA PAA was less than 35 O
114
particular interest was that hemolysis caused by PSPMMA PAA particles at 50
solid concentration was less than 02 hemolysis the lowest value among the latex
particles The low hemolysis value indicates that the PSPMMA PAA core shell latex
particles are the most blood compatible at all solid contents Other core shell particles
such as PSPMMA and PSPMMA PAA also had excellent blood comp
90 10
90 10
100 75 25 atibility with
low RBS breakage
01 2 3
20
40
60
80
100
Hem
olys
is (
)
052550
Figure 4-39 He
PS PSPMMA100 PSPMMA PAA90 10
molysis caused by latex particles n=5
4PSPMMA75PAA25
CHAPTER 5 SUMMARY OF RESULTS CONCLUSIONS AND RECOMMENDATION FOR
FUTURE WORK
51 Summary of Results
The goal of this study was to synthesize monodisperse polymeric latex particles
with tailored properties and to investigate the fundamental interactions between the
synthesized latex particles and target proteins An understanding of the fundamental
mechanism of selective adsorption is strongly required in order to maximize the
separation performance of membranes made of these materials so that it may applicable
to hemodialysis therapy for end stage renal disease (ESRD) patients For the achievement
of above research goal several investigations have been performed Analysis of the
obtained data leads to several conclusions as discussed below
Monodisperse polystyrene seed latex particles were synthesized with the size
crosslinking agent to make the latex particles more hydrodynamically stable The
conversion of monomer to polymer was over 95 and seed latex particles are spherical in
shape and have smooth surface From these small seeds bigger latex particles as large as
800nm were synthesized using a semi-continuous seeded emulsion polymerization
process The particles with a mean diameter less than 500nm are very spherical in shape
and highly uniform in particle size distribution However as the particles grow larger
than 500nm in mean diameter they became non-spherical with an uneven surface This
irregularity of particle surface can be attributed to the non-homogeneous monomer
ranges between 126plusmn75 and 216plusmn53 nm Divinylbenzene (DVB) was used as a
115
116
swelling into the shell of a growing polymer which can be controlled by the factors such
as temperature agitation speed initiator feeding rate and surfactant amount required to
stabilize the colloidal system It was determ rfactant to monomer ratio
dec ic
acid (AA) monomers were intro rticle surfaces more
hydro
to
ting of a
orm
ration is seen between
3002
0 cm-
0cm-1
-1
03
ined that as the su
reased the mean particle diameter increased Methyl methacrylate (MMA) and acryl
duced to make the latex pa
philic than bare polystyrene particles The prepared core shell latex particles were
made of PMMA100 PMMA90PAA10 and PMMA75PAA25 PMMA100 is the PMMA
homopolymer shell on a PS core PMMA90PAA10 shell is a copolymer with a PMMA
PAA ratio of 90 to 10 by weight PMMA75PAA25 shell is a copolymer consis
PMMA to PAA ratio of 75 to 25 by weight The particle size of these core-shell
particles were prepared to be about 370nm in mean diameter
The successful synthesis of latex particles was confirmed using Fourier Transf
Infrared spectroscopy (FTIR) The C-H aromatic stretching vib
cm-1 and 3103 cm-1 for PS latex particles The C-H asymmetrical stretching and
symmetrical stretching vibration peaks of CH2 for all latex particles is seen at 290
1and 2850 cm-1 respectively There is a carbonyl (C=O) characteristic band at 173
wavenumber for PSPMMA core-shell latex particles The broad OH group peak at
3400 cm
100
can be seen in the spectroscopy of the particle with PMMAPAA shell
(PSPMMA PAA and PSPMMA PAA ) The peak intensity of OH group of
PSPMMA PAA was larger than that of PSPMMA PAA relatively due to more AA
monomer content in PSPMMA PAA than in PSPMMA PAA
In the zeta potential measurements polystyrene (PS) latex particles had negative
values between -291 mV and -599 mV in the phosphate buffer (PB) and between -2
90 10 75 25
75 25 90 10
75 25 90 10
117
mV and -278 mV in the phosphate buffered saline (PBS) at 25oC and pH 21-78 These
negative zeta potential values for PS latex particles are due to ionization of the sulfate
groups (-OSO3-) originated from the initiator Zeta potential values of the PSPMMA
core shell latex particles were also negative between -28 mV and -505 mV in PB and
between -143 mV and -186 mV in PBS at pH 21-78 Negative values are also
generated by dissociation of sulfate groups on a particle surface The zeta potential value
of PSPMMA PAA and PSPMMA PAA were of negative values between -36
and -678 mV in PB medium and -147 mV and -193 mV in PBS medium at 25
100
s
7 mV
MMA75PAA25 and
PSPM
otein
g the
l change of BSA at
each w
90 10 75 25
oC For
PSPMMA PAA core shell particles zeta potential values were between -291 mV and
-520 mV in PB media and between -115 mV and -210 mV in PBS media Sulfate
groups contributed to the negative zeta potential values in PSP
75 25
MA90PAA10 core shell particles Carboxylic groups from polyacrylic acid also
contribute to the negative surface charge for the latex particles at greater than pH 70
however the degree of their contribution was not detectable from the zeta potential
measurements The absolute zeta potential values of the synthesized latex particles in PB
were higher than that in PBS This is because of the high concentration of sodium
chloride electrolyte in PBS that compress electrical double layer thickness
The bicinchoninic acid (BCA) assay technique was used to determine the pr
adsorption and all isotherms were fitted to the Langmuir-Freundlich isotherms usin
nonlinear regression method The hydrophobic interaction between PS latex particle and
BSA protein was dominant for the adsorption process Conformationa
pH also affected the adsorption amount which was highest at pH 48 because of lo
ionic repulsion making compact conformation of BSA The next larger amount of BSA
118
adsorbed onto PS was at pH 32 There were two main forces affecting the amount of
adsorption at acidic pH values One is ionic attraction between negative latex particle a
positive protein The other is lateral repulsion between positive proteins The smallest
amount of BSA adsorption was at pH 74 where charge repulsion between negative
particles and proteins is present at this pH Lateral repulsion between proteins wa
dominant for protein adsorption even though there always exists the hydrophobic
interaction between solid latex particle and protein The charge repulsion was reduced by
the high concentration of electrolytes in the PBS medium The BSA adsorption process
on PSPMMA core shell particles was very similar to the case of PS latex particle where
hydrophobic interaction between latex particle and protein was dominant However t
adsorbed BSA amount was less than that of PS in PBS The adsorbed amount of BS
PSPMMA
nd
s
he
A on
an
ed
n The main
adsor
90PAA10 and PSPMMA75PAA25 core shell particles was higher at pH 32 th
pH 48 and 74 The adsorbed BSA amount became higher at pH 32 and pH 48 as the
hydrophilicity was increased by the higher PAA amount On the contrary the adsorb
amount of BSA was dramatically decreased at pH 74 Carboxylic acid groups as well as
the sulfate groups are distributed on the surface of PSPMMA90PAA10 and
PSPMMA75PAA25 core shell particles and largely affect the BSA adsorptio
ption interaction forces at pH 32 was generated by hydrogen bonding as well as
charge attraction between latex particle and protein even when the conformational change
and charge repulsion between proteins reduce the adsorption of protein at this pH The
adsorbed BSA amount was also increased as the amount of PAA increases However the
adsorption of BSA was minimal (less than 07 mgm2) at pH 74 where the carboxylic
groups are ionized leading to more enhancement of charge repulsion by ionization of the
119
carboxylic groups (COO-) Because of this the PSPMMA-PAA latex particles have a
much lower protein adsorption
There were steep initial slopes for adsorption isotherms of β
in
s
there
M
r
e
es
o
2M on latex particles
all isotherms indicating a high affinity type adsorption The complete plateau region
were not seen for all isotherms indicating that β2M proteins were not saturated and
are still available sites for β2M adsorption on latex particles The initial slope of β2
isotherms for PS was steeper than for the other latex particles PSPMMA100
PSPMMA90PAA10 and PSPMMA75PAA25
The layer thickness (δ) of BSA proteins on synthesized latex particles in phosphate
buffer (PB) was in the range of 27 - 81 Aring at 37 C and pH 48 Only the δ values at pH
48 the isoelectric point (IEP) of BSA are important because intra- and inter-molecular
electrostatic repulsion of the protein molecules is minimized at the IEP Compared to the
hydrodynamic dimensions of BSA 140 times 40 times 40 Aring these values of the adsorbed laye
thickness indicates that the BSA molecules adsorb by the side-on (40Aring) and end-on mod
(140Aring) Consideration of the multi-layering is unnecessary because the adsorbed
thickness of BSA do not exceed 140 Aring
The Gibbs free energy of β
o
3
2M was more negative in phosphate buffer (PB) than
that of BSA in physiological condition (37 C and pH 74) indicating that βo2M molecul
are more favorably adsorbed on the latex particles than BSA and that the affinity of β2M
was much larger than BSA This may be explained by the rate of diffusion and
accessibility of β2M molecules being easier and faster than that for BSA because of
smaller size (or weight) and spherical shape of β2M molecules It may also be possible t
explain that more hydrophobic forces (less electrostatic repulsion) contributes to
120
adsorption of β2M than to that of BSA because the IEP of β2M (57) is closer to
physiological condition (pH 74) than that of BSA (pH 48) The selective adsorption of
β2M m
in β2M
larger than in BSA adsorption indicating
that fa
an that between BSA and latex particles because
IEP (
ch as
Other
olecules over BSA molecules can be enhanced by developing more hydrophilic
PSPMMA90PAA25 and PSPMMA75PAA25 core shell latex particles
The adsorption kinetics of proteins on latex particles was expressed as a second-
order model The plateau region for β2M was reached earlier than BSA on all latex
particles and the K values (equilibrium rate constant of 2nd order adsorption)
adsorption process on latex particles were much
ster adsorption rate of β2M molecules than that of the BSA molecules There are
several explanations for these phenomena First the smaller size of β2M molecules can
more easily diffuse and approach to the latex substrate Second enhanced hydrophilic
domain on the latex particle surface by sulfate groups (SO4-) and ionized carboxylic
groups (COO-) is not favorable for larger BSA molecules but is for smaller β2M to be
anchored Third the conformational change by intra- and inter-molecular repulsion
would be faster in β2M due to lower molecular weight Finally the ionic repulsion
between β2M and latex particles is less th
57) of β2M is closer to the tested condition (pH 74) than IEP (48) of BSA
In the biocompatibility test the number of red blood cells (RBCs) ruptured by PS
latex particles ranged from 30 to 91 as the solid content of PS increased from 05 to
50 respectively However hemolysis of the other core shell latex particles su
PSPMMA100 PSPMMA90PAA10 and PSPMMA75PAA25 were less than 35 The
PSPMMA90PAA10 particles at all applied solid concentrations had less than 02
hemolysis and was the most blood compatible among the prepared latex particles
121
core shell particles such as PSPMMA100 and PSPMMA75PAA25 also had excelle
compatibility with low RBC breakage
52 Conclusions
The major conclusions from this study include
nt blood
bull microglobulin (β2M) over that of the bovine serum albumin (BSA) at pH 74
was achieved by controlling the PMMA and PAA ratio to optimize
e particles posses the most desirable requirements for use in a human dialysis
ns
icles
olecule
bull
Using engineered core shell latex particles selective adsorption of β2
physiological condition was demonstrated
bull The rate of adsorption of the β2M was also much higher than that of BSA This
hydrophobichydrophilic microdomain size
bull The core shell particles also exhibited excellent biocompatibility with blood Thes
membrane
53 Recommendation for Future Work
With the results and conclusions obtained through this study several suggestio
may be given for future work as follows
bull Competitive adsorption tests of whole blood proteins on synthesized latex partunder physiological condition
bull Preparation of latex particles with optimum hydrophobic hydrophilic microdomains to maximize selective adsorption and removal of toxin molecules
bull Membrane fabrication by suitable pore size design to discriminate a toxin mby size sieving as a function of particle layers
Evaluation of membrane performance such as flow rate in water and in simulated blood fluid (SBF)
bull Application for hemodialysis as well as other technologies such as a water remediation
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Korea on August 7 1968 He obtained a Bachelor of Science degree in Chemistry from
Keim d graduate school and earned a
resear n Korea Institute of Science and
Coati e
ia
Busin
BIOGRAPHICAL SKETCH
Sangyup Kim was born and raised in the small town of Sung-Joo Kyungbook
yung University (Daegu Korea) in 1993 He entere
Master of Science degree in Chemical engineering in 1995 He worked as a student
cher for 2 years at Textile Polymer Division i
Technology (KIST) in Seoul Korea Next he worked over a year at the Automobile
ng Division in DongJoo-PPG in Korea In August 2001 he began studying for th
degree of Doctor of Philosophy in materials science and engineering at the University of
Florida in Gainesville After getting his PhD he plans to work at the Digital Med
ess Division of Samsung Electronics in Korea starting in January 2006
141
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