Ibrahim H. A. Badr1, A. Salim1, A. M. Salem 1, M. Gouda2 ...€¦ · 1 Enhancing the...
Transcript of Ibrahim H. A. Badr1, A. Salim1, A. M. Salem 1, M. Gouda2 ...€¦ · 1 Enhancing the...
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Enhancing the biocompatibility of Sodium Membrane Electrodes by Using Chitosan-
CuO and Chitosan-CuO-Heparin Composite Membranes
Ibrahim H. A. Badr1, A. Salim
1, A. M. Salem
1, M. Gouda
2, Hossam E. M. Sayour
3.
1 Chemistry Department, Faculty of Science, Ain-Shams University, Cairo, Egypt
2 Pretreatment and Finishing department, Textile Research Division, National Research
Center, Cairo, Egypt
3 Chemistry Department, Animal Health Research Institute, Cairo, Egypt
Abstract
We investigate the effect of introducing chitosan CuO-nanoparticle and heparin
loading chitosan CuO nanoparticle composite membranes to PVC based sodium selective
membrane electrode to enhance its biocompatibility and reduce its highly thrombogenicity.
The potentiometric responses of three sets of electrodes were studied: (1) conventional PVC
membrane electrode, (2) conventional PVC membrane electrode sandwiched with chitosan-
CuO membrane (CH-CuO-PVC), and (3) conventional PVC membrane electrode sandwiched
with chitosan-CuO-heparin membrane (CH-CuO-H-PVC). Potentiometric response
characteristics of the CH-CuO-PVC and CH-CuO-H-PVC membrane electrodes (e.g.,
detection limit, linear range, response slope, and selectivity coefficients) were found to be
comparable to that of the conventional PVC-based sodium electrodes. Biocompatibility was
assessed for all membranes (PVC and CH-CuO-PVC and CH-CuO-H-PVC) through platelet
adhesion protocol. SEM and platelet counts have shown that CH-CuO and CH-CuO-H have
improved biocompatibility compared to the classical PVC membranes.
Keywords: Membrane electrodes; Heparin; Biocompatibility;
Chitosan; CuO nanoparticles. * Corresponding author: Ibrahim H. A. Badr
Chemistry Department, Faculty of Science, Ain Shams University, Cairo, Egypt
E-mail: [email protected] Tel: +201153344232
1- Introduction
The technology of potentiometric sensors based on liquid or polymer membrane materials
is well established technology that spearheaded the integration of sensing devices into the
clinical laboratory for the automated testing of physiological samples for key electrolytes such
as potassium, sodium, calcium, and chloride, polyions, as well as for measuring the pH
[Young, (1997)]. This success came from the need of simple, direct, low cost, sensitive,
reliable, selective and rapid process that introduced by potentiometric sensors [Bakker, et
al.(1999); Bühlmann, et al.( (1998); Johnson & Bachas, ( 2003)]
Implantable electrochemical sensors have a great interest and development in biomedical
and clinical application especially for real- time monitoring of important physiological species
in blood, especially when such compounds are subject to a rapid change inside the body.
Measurements of electrolytes like Ca2+
, Na+, Cl−,
HCO3
−, and K+, blood gases, metabolits and
pH play a critical role in healthcare for diagnosis, monitoring, treatment or control of diseases.
Continuous monitoring of electrolyte or blood gases or pH are badly need in emergency cases
, intensive care , surgical procedures and for critically ill hospitalized patient (e.g., liver,
kidney or coronary pulmonary or in coma). Because this represents the physiological case of
body on time to help physicians to make right decisions regarding a patient's diagnosis and
treatment. Implantable sensors offer real time measurements which is critical in such cases.
Introducing any foreign materials such as chemical sensors in the blood stream have the
potential to activate platelets and also the potential to lead to thrombus formation that can
occlude the blood vessel. Chemical sensors even in the absence of full thrombus formation,
by activating platelets often lead to cellular adhesion on the surface sensors. These adherent
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cells consume oxygen and glucose and produce CO2 as they are metabolically active.
Consequently, can cause inaccurate results for critical care analysis. The presence of platelet
adhesion and cellular activation and adhesion on sensors surface cause differences in the local
levels of PO2, PCO2, glucose, pH, etc., and on implanted sensor surface. As the platelet
adhesion and cellular activation and adhesion is dynamic process [Frost and Meyerhoff
(2015)]. This changing of the outer layer is one of the biggest practical concerns with using
intravascular sensors.
Many efforts have been made to develop materials that will resist or eliminate protein
adsorption on blood contacting polymers and utilized to prepare implantable sensors [Chen,
et al (2008); Chen, et al. (2010); Kim , et al. ( 2005); Li , et al. (2008 ); Zhang , et al.
(2008)]. Poly(ethylene glycol) (PEG)-based materials have shown the ability to decrease
protein adsorption [ Zhao , et al. (2010); Kim , et al. (2005)]. Zwitterionic surfaces have also
demonstrated a reduction in protein adsorption [Kim, et al. (2005); Li, ,et al. (2008); Zhang,
et al. (2008)]. Also studies to reduce protein adsorption create polymer brushes with tethered
pCBMA on surfaces [Zhang, Chen, Jiang (2006)]. Oligo (ethylene glycol) polymer brushes
tuned to thicknesses of 5–50 nm [Ma, Hyun, et al. (2004)], and tethered pSBMA on the
surface [Zhang, et al.( (2006)]. Those represent promising success at reducing protein
adsorption in bench top experiments. But, it still remains to be seen whether these approaches
can significantly improve in vivo performance of intravascular sensors. Approach of nitric
oxide (NO) release coatings that cause inhibition of platelet activation and adhesion has also
been explored [Frost, et. Al. (2002)]. Also combination of nitric oxide (NO) release coatings
with immobilized heparin improved blood compatibility by two means [Zhou, Meyerhoff
(2005); Wu et al.(2007)]. NO release coatings has also been applied recently to prepare
intravascular glucose sensors that yielded improved analytical performance and reduced
surface thrombosis when placed in the veins of rabbits [YanQ, et al. (2011)], also
immobilized heparin (to prevent bulk thrombosis/clot formation), and immobilized CD47 has
been proven successful [Finley, Rauova (2012); Finley, et al. (2013)].
Implanted devices suffer from biofilm formation and biofouling. Bacterial
colonization in the form of biofilms on surfaces causes persistent infections and is an issue of
considerable concern to healthcare providers [Mohankandhasamy and Jintae (2016)].
Biofouling is the accumulation of unwanted proteins and other analytes or organisms (cells
and bacteria) on surface of host materials [Banerjee, et al.(2011)]. The performance of
biomedical implants and devices can be changed by the adsorption of contaminating matter
and lead to patient infection, shortened durability, and increased healthcare cost by
replacement of devices. As a result there is an urgent clinical need to develop long-lasting
biomedical materials or devices with antibacterial and antibiofilm surfaces
[Mohankandhasamy and Jintae (2016)].
Different methodologies have been used to can enhance device biocompatibility and
functionality and material properties and surfaces can be modified to reduce microbial
contamination and prevent biofilm infections by different used include : antifouling coatings
[Banerjee, et al (2011)], antiadhesive surface modifications [Neoh and Kang (2011)],
addition of antimicrobials to the surfaces of medical devices [Shintani (2004); Kwok, et al.(
(1999); Sodhi, et al.( (2001)], Coating devices with polymer products [ Li, et al.( (2013)]
coating, lamination, adsorption, or immobilization of biomolecules [Khoo, Hamilton,
Grinstaff, , et al (2009), Lee, Wang, Yang,, et al. (2013); Arciola, , et al. (2012)] surface
engineering with chemical moieties [ Antoci ,et al. (2008); Gottenbos, et al. (2002); Hume,
Baveja, Muir et al.(2004); Pavlukhina, et al.(2012)]. Many approaches of Nanotechnology
provided a promising advancement to prevent drug-resistant biofilm infections of medical
devices and biomaterials. Copper, silver, gold, titanium, and zinc are known to have
antibacterial and antibiofilm properties that offer alternatives to antibiotics without increasing
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the risk of resistance development. It has been established that metal-based nanoparticles have
much better antimicrobial activities than their micro-sized counterparts [Colon, Ward,
Webster (2006); Jones, et al.( (2008)].
CuO NPs exhibit effective antimicrobial activity against various bacteria, CuO
nanoparticles have effective antimicrobial action against a wide range of pathogens and also
drug resistant bacteria [ Alexandru et al. (2016), Kelly et al.( (2003); Raffi , et al. (2010).].
Chitosan is linear, semi-crystalline poly(aminosaccharide) cationic polymer. It consists of
two repeating units, N-acetyl-d-glucosamine and d-glucosamine linked by (1-4)-β-glycosidic
linkage with pKa of 6.5. Chitosan is obtained by alkaline deacetylation of chitin [Azuma et
al.( (2015)]. Chitin is the second important naturally occurring polysaccharide .It is found in
exoskeleton of arthropods mostly in crustaceans (shrimps ,lobster and crabs), insects, radulae
of molluscs and in beaks and internal shells of cephalopods (e.g. octopus and squid), cell wall
of fungi. Chitosan gained enormous importance in many filed specially in pharmaceutical and
biomedical applications (e.g. drug delivery, tissue engineering, gene delivery) chitosan is also
used in treating major burns, preparation of artificial skin and bones, surgical sutures, contact
lenses, blood dialysis membranes and artificial blood vessels etc. Chitosan has unique
properties due to its biological properties include biocompatibility, biodegradability, and non-
toxicity, mucoadhesive and antimicrobial activity. Besides this, it is fungistatic, bacteriostatic,
spermicidal, anticholestermic and anticarcinogenic. antitumor, blood anticoagulant,
antigastritis, hypochlesterolaemic and antithrombogenic agents [Pillai et al.( (2009), Saikia
et al. (2015); Croisier & Jérôme (2013)]. Chitosan is considered as promising polymer in
formulation of drug delivery system due to its unique chemical, physical and biological
properties like non- toxicity and ease of modification. The cationic nature of chitosan due to
primary amino groups impart a lot of properties such as controlled drug release, transfection,
mucoadhesion, and permeation enhancement [Felt , et al.( (1998); Park, et al.( (2010)].
Recently, metal oxides have been investigated in drug delivery applications because they have
the ability to improve drug loading, Control the drug release rate and targeting to specific
location in the body. For example ZnO nanoparticles stabilized with cationic chitosan coating
have been applied for preparation of doxorubicin loaded ZnO quantam dots [AbdElhady
(2012)]. Copper oxide (CuO) has attracted attention because of its antimicrobial and biocide
properties and they can be used in many biomedical applications [Kelly, Coronado, Zhao,
Schatz (2003); Raffi, et al. (2010)]. Therefore, CuO nanoparticles could be used to enhance
loading heparin into chitosan film and it also could be used to improve release of heparin
from the chitosan film.
In this study, we investigated the utility of chitosan-CuO nanoparticle membrane to
improve the blood compatibility of ion selective electrodes based on highly thrombogenic
polymers (e.g., PVC). Chitosan-CuO nanoparticle membranes used to the sensing PVC film
to prevent the contact between the highly thrombogenic PVC and the sample solution, and
therefore enhance the biocompatibility of PVC-based membrane electrodes. Furthermore,
improvement of chitosan biocompatibility could be achieved by adding heparin
(anticoagulant) to chitosan CuO nano particles.
2- Experimental
2.1. Reagents and Materials
Sodium ionophore-X, o-nitrophenyl octy ether (o-NPOE), potassium
tetrakis(chlorophenyl) borate KTClPB , bis (2-ethylhexyl) sebacate (DOS), Tridodecyl-
methylammonium chloride (TDMAC) and porcine intestinal mucosal heparin were obtained
from Fluka (Ronkonkoma, NY), and poly(vinyl chloride) was from Polysciences
(Warrington, PA). Tetrahydrofuran (THF) was purchased from Fisher (Fair Lawn, NJ) and
tris-(hydroxymethyl) amino methane (Tris) was obtained from sigma (St. Louis, MO).
Anticoagulant solution CPDA-1 was obtained as a gift from the central laboratory of the
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armed forces (Cairo, Egypt). Each 100 ml of CPDA-1 contains: 3.1900 g dextrose, 2.6300 g
sodium citrate dihydrate, 0.2990 g of anhydrous citric acid, 0.2220 g of monobasic sodium
phosphate monohydrate, and 0.0275 g of adenine. Blood samples were collected from jugular
vein of healthy sheep (Ministry of agricultural, Cairo, Egypt). All standard solutions and
buffers were prepared with de-ionized distilled water.
2.2. Preparation Heparin-selective electrode:
Polymer membrane were prepared [Meyerhoff, Yang, (1992); Meyerhoff, Yang,
(1992)] by dissolving 4mg (crossponding 2%) of TDMAC, 32.5% (65mg) DOS, 65.5 % (131
mg) PVC in 2.00 ml of tetrahydrofuran (THF). This cocktail was poured into 25-mm internal
diameter glass ring placed onto a glass plate, and the membrane was formed after evaporation
of THF overnight. Working electrode was designed by cutting out disks from the membrane
using a cork borer (7mm diameter) and then the small disks are glued to a PVC tubing using
THF. Working electrodes were soaked in 15 mM NaCl overnight prior to use. 15 mM NaCl
also use as the internal filling solution. A double-junction Ag/AgCl electrode (Orion Model
90-02-00) with an Orion (90-02-02) internal filling solution was used as the reference
electrode. The outer compartment of the reference electrode was filled with saturated
potassium nitrate. All measurements were carried out in 0.12 M NaCl. Membrane potentials
were monitored at room temperature using data acquisition system, eight-channel electrode-
computer interface (Nico2000 Ltd., London, UK) controlled by Nico-2000 software. All
potentiometric calibrations shown are the average of three trials.
2.3. Study of released Heparin using Heparin-selective electrode:
Heparin-selective electrode was used to detect amount of heparin released,
Approximately 0.1 g from every of chitosan-CuO- heparin membrane of different heparin
loadings (0.28 wt %, 1.41 wt % and 2.78 wt % heparin conc) was soaked in 20 ml of 0.12 M
NaCl (saline buffer). Membranes were left in this solution for specific time. Heparin sensor
was utilized to detect the amount of released heparin at time intervals 0, 3, 6, 12, 36, 48, 72
and 96 hours. Three electrodes were used to detect the amount of released heparin for each
measurement.
2.4 Preparation of conventional sodium selective poly (vinyl chloride) (PVC) membranes:
Sodium selective membrane was prepared by dissolving 2.0 mg of sodium ionophore-X
(corresponding to 0.70 wt%), 0.50 mg of the lipophilic salt potassium tetrakis (chlorophenyl)
borate KTClPB (corresponding to 0.20 wt%), 100.0 mg of poly vinyl chloride (corresponding
to 33.0 wt%) and 200.0 mg of plasticizer nitrophenyl octy ether (NPOE) (corresponding to
66.1 wt%) in 2.00 ml of tetrhydrofuran (THF). The cocktail was poured into 25-mm internal
diameter glass ring placed onto a glass plate, and the membrane was formed after evaporation
of THF overnight.
2.5 Preparation of sodium selective membrane electrodes based on PVC, chitosan-CuO and
modified with (CH-CuO-PVC), and Chitosan CuO-heparin (CH-CuO-H)
A schematic representation of the three sets of membrane electrodes used in this study is
shown in (Fig.1). Small disks of about 7.00 mm diameter were cut from PVC, chitosan-CuO
(CH-CuO) and chitosan CuO-heparin (CH-CuO-H) master membranes. PVC membrane
electrode was prepared by gluing the PVC membrane to a PVC tubing in using THF to give
the control PVC membrane electrodes. CH-CuO-PVC and CH-CuO-H-PVC were prepared
by physically attaching CH-CuO, CH-CuO-H film, respectively to the tip of the PVC
membrane using Teflon tape, so that the CH-CuO or the CH-CuO-H would face the sample
solution. The internal filling solution for all electrodes was 1.0 mM NaCl prepared in the
measuring buffer solution. A double-junction Ag/AgCl electrode (Orion Model 90-02-00)
with an Orion (90-02-02) internal filling solution was used as the reference electrode. All
potentiometric measurements were performed in 0.05 M Tris-HCl, pH 7.4. Membrane
potentials were monitored at room temperature using data acquisition system, eight-channel
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electrode-computer interface (Nico2000 Ltd., London, UK) controlled by Nico-2000
software. Combination glass electrode [Orion model (8-02] was used for all pH
measurements. The detection limit, linear range, and selectivity coefficients were determined
according to the reported methods [Bakker, et al., (2000); Guilbault, et al., (1976)], and the
data presented in this paper are average of three ion–selective electrodes measurement.
Figure 1: Schematic diagram for the three sets of sodium selective membrane electrodes used in this
study: (a) conventional PVC, (b) CH-CuO-PVC, and (c) CH-CuO-H-PVC.
2.6 Biocompatibility studies
The collected blood was obtained from the jugular vein of a healthy sheep. Each pint of
blood was drawn into a blood collection bag (JMS, Singapore, PTE Ltd.) containing 70.00 ml
of CPDA-1 anti-coagulant solution. Within 30 min after collection, blood was centrifuged at
240 × g for 15 min at 4.0ᵒC. Platelet was determined by Exigo vet hematology analyzer with
a concentration of approximately 9.5×104 cells /µL. The PRP was preincubated at 37◦C for
20 min. PVC, CH-CuO, CH-CuO-H membranes were conditioned in phosphate-buffered
saline (PBS was formulated with 10 mM phosphate, 138 mM sodium chloride, and 2.7 mM
potassium chloride, pH 7.4) for three days and were immersed in PRP for 2 h at 37◦C. The
membranes were then rinsed in PBS and fixed in a PBS solution containing 2% (w/v)
glutaraldehyde for 2 h at room temperature.The membranes were dehydrated by immersion in
serial dilutions of ethanol (30, 50, 70, 90, and 95%, v/v), two immersions in absolute ethanol,
and two immersions in hexamethyldisilazane. After solvent evaporation within desiccator, the
membranes were coated with gold and examined with QUANTA FEG 250 scanning electron
microscope (SEM).
3- Results and discussion
In this study we introduce CH-CuO-H and CH-CuO to PVC electrode to enhance the
biocompatibility and reduce the thrombogenicity of PVC by sustained release of anticoagulant
heparin fom CH-CuO-H and reduce of biofilm formation and biofouling by CuO
nanoparticles (see below).
Many efforts have been focused on the development of materials that will be sufficiently
biocompatible with blood for the several days of use in critically ill hospital patients.
Utilization of more biocompatible polymers in the fabrication of sensing film [Cha and
Meyerhoff, (1989);Cosofret, et al., (1994); Cha, et al., (1995); Liu, et al., (1993); Linder,
et al. (1995); Cosofret, et al. (1996); Yun, et al. (1997); Berrocal, et al. (2001); Bratov, et
al., (1995); Ambrose and Meyerhoff (1996); Heng and Hall, (1996); Heng and Hall,
(2000), Shin, et al. (1996); Högg, et al. (1996); Tsujimura, et al., (1996); Poplawski, et al.
(1997), Badr, et.al (2014), Badr, et.al (2015)]. Coating the PVC membrane surface with a
biocompatible polymer [Zhang, et al., (1996); Berrocal, et al., (2000), Badr, et al., (2014),
Badr, et al., (2015)], or immobilization of anticoagulants to the membrane surface (to prevent
thrombosis/clot formation) [Badr, et al., (2005)]. Implantable devices suffer from biofilm
formation and biofouling. In vivo, nonspecific protein adsorption facilitates bacterial
attachment to surfaces and leads to colonization, subsequent biofilm formation, and infectious
disease. Protein fouling followed by microbial attachment with biofilm development is a
dormant factor of the failure of biomedical devices and implants. Also, microbial attachment
reduces the sensitivities and efficacies of devices [Zhang and Chiao (2015)]. Different
methodologies have been used to can enhance device biocompatibility and functionality and
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material properties and surfaces can be modified to reduce microbial contamination and
prevent biofilm infections by different used include : antifouling coatings [Banerjee, et al.(
(2011)], antiadhesive surface modifications [Neoh and Kang (2011)], addition of
antimicrobials to the surfaces of medical devices [Shintani (2004), Kwok, et al.( (1999);
Sodhi, et al.( (2001)], Coating devices with polymer products [Molin, Yang, and Ndoni
(2013)] coating, lamination, adsorption, or immobilization of biomolecules [Khoo et al.(
(2009); Lee, et al.( (2013); Arciola, et al. (2012)] surface engineering with chemical moieties
[Antoci, et al.(2008); Gottenbos et al.( (2002); Hume, et al (2004); Pavlukhina,et
al.(2012)]. Many approaches of Nanotechnology provided a promising advancement to
prevent drug-resistant biofilm infections of medical devices and biomaterials. Copper, silver,
gold, titanium, and zinc are known to have antibacterial and antibiofilm properties that offer
alternatives to antibiotics without increasing the risk of resistance development. It has been
established that metal-based nanoparticles have much better antimicrobial activities than their
micro-sized counterparts [Colon, Ward, and Webster (2006); Jones, Ray, Ranjit, and
Manna (2008)]. Exhibit quite different bacterial adhesions, protein adsorptions, and tissue
integration characteristics [Puckett, Taylor & Raimondo (2010); Salwiczek, Qu, Gardiner
et al. (2014), Singh, Vyas, Patil et al., (2011)].
CuO NPs exhibit effective antimicrobial activity against various bacteria, CuO
nanoparticles have effective antimicrobial action against a wide range of pathogens and also
drug resistant bacteria [ Grumezescu et. al, (2016); Kelly, Coronado, Zhao, Schatz (2003),
Raffi, Mehrwan, et al. (2010)]. Therefore, introduction of CuO into Chitosan film could
reduce formation of biofilm and befouling on the surface on medical devices.
In this study the biocompatibility of membrane electrodes based on PVC was improved by
using CH-CuO and CH-CuO-H films. Heparin and CuO are expected to reduce biofilm
formation and biofouling. Thin film of CH-CuO or CH-CuO-H was sandwiched to PVC.
Furthermore, we investigated the effect of introducing CH-CuO or CH-CuO-H layer on the
potentiometric response characteristics of PVC-based sodium-selective membrane electrodes
formulated with sodium ionophore-X.
Heparin-selective electrode was used to detect amount of released heparin. A measurement of
heparin clotting time is not suitable for direct and rapid measurements. Heparin-selective
electrode enables direct, rapid and selective detection of free heparin with lower detection
limits of 0.1-1 unit/ml. (Fig 2) shows the time trace for three successive calibration of heparin
selective electrode as measured in 0.12 M NaCl. After each calibration the heparin sensor was
washed in 15 mM NaCl for 45 min. As can be seen the same heparin sensors showed good
reproducibility of each calibration. Approximately 0.1 g from each of chitosan-CuO- heparin
membrane of different heparin loadings (0.28%, 1.41% and 2.78% heparin conc) soaked in 20
ml of 0.12 M NaCl for specific time. Heparin sensor was then utilized to detect the amount of
released heparin at time intervals 0, 36, 12, 36, 48, 72 and 96 hours.
Figure 2: Dynamic response time of heparin selective membrane electrodes toward increasing heparin concentration,
as measured in 0.12 M NaCl.
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Figure 3 Potentiometric responses of heparin selective membrane electrodes as function time for CH-CuO-H A) 0.28
wt % B) 1.41 wt % C) 2.78 wt % heparin.
Figure 3 shows, change in mV as function of time due to the heparin released from
CH-CuO-H in 20 mL of buffer 0.12 M NaCl for three different heparin loading. As shown in
this figure the potentiometric response of released heparin from the three different heparin
loading of CH-CuO-H membrane (0.28 wt %, 1.41 wt % and 2.78 wt % heparin) showed a
decrease in signal was observed with time and all membranes continue to release heparin up
to 72 hours and then reached a plateau.The amounts of released heparin were calculated after
72 hours for each membrane film as summarized in Table 1. This table Summarized
calculations of cumulative amount or heparin release form different CH-CuO-H membranes
(0.28%, 1.41% and 2.78% heparin conc) up to 72 hours. The releases after 72 hours were
respectively 64.68, 71.11 and 79.21 µg/mL. As shown in (Fig 3) the release heparin from the
three membranes was more rapid at initial stage of experiments. The highest rate of release
was at the first 10 hours after that the rate of released were decreased till reach to plateau. 72
hours. Metal oxides NPs have the ability to improve drug loading and control the drug release
rate [Xiaoli , Yanan Luo et al. (2016), AbdElhady , (2012)]. Respectively, presence of CuO
stabilizes heparin in membrane phase. This is because CuO NPs have great affinity to
carboxyl and amine groups [Ruparelia et al. (2008)]. Each disaccharide repeating unit of
heparin contains a carboxyl group and therefore, it can be attached to heparin carboxyl
function group, which in turns enhances the attachment of heparin to chitosan.
Table 1. Summary of calculations of released heparin from different CH-CuO-H film
Film weight,
g
wt % Heparin in Film
Released heparin after 72 hr
Unit/mL
Released heparin after 72 µg/mL
%Released heparin after
72
0.11 0.28 % 0.58 64.68 21.10 %
0.10 1.41 % 0.63 70.11 4.81 %
0.10 2.78 % 0.71 79.21 2.73 %
We have studied the potentiometric response characteristics of the three sodium selective
membrane electrodes prepared in this study in order to probe the effect of adding CH-CuO
and CH-CuO-H layer on the potentiometric response behaviour of PVC based sodium
selective membrane electrodes. Sensing PVC membranes in three types of electrodes (PVC,
CH-CuO-PVC and CH-CuO-H -PVC) were prepared using 0.7 wt % sodium ionophore X,
33.0 wt% polyvinyl chloride, 0.2 wt% KTClPB and 66.1 wt% NPOE. The potentiometric
responses of the three membrane electrodes (PVC, CH-CuO-PVC and CH-CuO-H-PVC)
towards different cations are shown in (Figure 4-6). The response characteristics (e.g.,
selectivity, response time, linear range...etc.) of the these sets of sodium selective membrane
electrodes (PVC, CH-CuO-PVC and CH-CuO-H-PVC) are summarized in Table 2. Sodium
selective membrane electrode based on PVC (control membrane) showed sodium detection
limit of 8.0×10-6
M and had a linear response range from 1.5×10-5
to 0.135 M as shown in
(Figure 4). Also as shown (Figure 5) and (Figure 6) the detection limits of CH-CuO-PVC
and CH-CuO-H–PVC membranes are slightly higher than PVC based sodium selective
membrane electrodes. Table 2 showed that the detection limits (CH-CuO-H–PVC >CH-CuO-
PVC > PVC) shifted to higher sodium ion concentrations. Also, the lower limit of the linear
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range (CH-CuO-H–PVC >CH-CuO-PVC > PVC) was shifted to higher concentrations.
Although there is a shift to higher concentration in case of CH-CuO-H–PVC and CH-CuO–
PVC, it is still much lower than sodium concentrations in physiological fluid which is 135-
145 mM. In case of CH-CuO and CH-CuO-H the diffusion layer thickness is larger than in
the classical PVC membrane electrode, due to the smaller stirring effect on the thin film of
aqueous buffer solution located between CH-CuO or CH-CuO-H and PVC. Due to increasing
in thickness in the diffusion layer, it is expected that sodium concentration at PVC sample
solution interface will increase. Therefore, the lower detection limit is shifted to high
concentrations of sodium. Similarly, shifting in the detection limits to high sodium
concentrations was notices in in case of heparin-modified CTA compared to unmodified CTA
based membrane electrodes formulated with sodium ionophore-X [Badr, et al., (2005)], as
well as in heparin-modified (HCH-PVC) and unmodified (CH-PVC) membrane electrode
[Badr, et al., (2014)]. The response slopes of CH-CuO membrane electrodes towards sodium
was 55.2.mV/decade and 56.4 for CH-CuO-H which are near-Nernstian. These observed
slopes of CH-CuO and CH-CuO -H are close to the control PVC membrane, (59mV/decade)
as shown in Table 2.
Table (2): Summary of response characteristics of sodium membrane electrode
NA: not applicable, amaximal selectivity limit(<) was used for ions that exhibited minimal response
towards interfernet ions [Bakker, et al., (2000)], bRequired selectivity coefficient for blood [Oesch, et
al., (1986)], ctherabutic level of lithium ion.
The response time is an important factor for any ion selective electrode. The effect of
introducing CH-CuO-H and CH-CuO on the response time of conventional PVC membrane
was studied to control PVC. As shown in Figure 7 PVC shows equilibrium response in a
short time with t90 value of 0.5 min for 21 mM sodium concentration. While it took more time
to reach the equilibrium for CH-CuO and CH-CuO-H with t90 value of 2.5 min for both
membrane electrodes. The slight increase in the response time of PVC- CH-CuO and PVC-
CH-CuO-H compared to control PVC is consistent with the higher detection limit observed
for those membrane electrodes. This is because more time is required for sodium ions to
penetrate CH-CuO and CH-CuO-H and reach PVC sensing film. It is worth mentioning that
modified membrane electrodes exhibited good reversibility (see data in Figure 8).
Figure 4. Potentiometric responses of sodium selective PVC membrane electrode toward different ions
measured in 50 mM Tris-HCl, pH 7.4. The cations tested are: (♦) sodium, (■) potassiom, (+)lithium,
(×)ammonium, (*)magnesium, and (-) calcium.
log K ± SD a Slope,
mV/de
cade
Detectio
n limit,
μM
Linear
range, M Na+ K
+ Li
+ NH4
+ Ca
2+ Mg
2+
PVC 0 -2.0±0.1 <-
2.8±0.2
<-3.6±
0.1
-2.4±
0.2
<-3.8±
0.1 59 8.5
1.5×10-5
to
0.135
CH-CuO
PVC 0
-
1.87±0.2
<-
2.8±0.2
<-
3.4±0.4
-2.2±
0.2
<-3.3±
0.3 55.2 22
5×10-5
to
0.135
CH-CuO–H
PVC 0
-
1.99±0.2
<-
2.6±0.3
<-
3.2±0.1
-2.2±
0.2
<-3.1±
0.4 56.4 39.8
7.9×10-5
to
0.135
Required
selectivity
coefficient b
0 < -0.6 < 0.1
<2.1c
<1.6 <-1.3 <-1.2 NA NA NA
9
Figure 5 Potentiometric responses of sodium selective CH-CuO-PVC membrane electrode toward
different ions measured in 50 mM Tris-HCl, pH 7.4.
Figure 6 Potentiometric responses of sodium selective CH-CuO-H-PVC membrane electrode toward
different ions measured in 50 mM Tris-HCl, pH 7.4.
Figure 7. Dynamic response time of sodium selective membrane electrodes toward increasing sodium ion
concentrations: PVC (solid black line), CH-CuO-PVC (dashed blak-line ) and CH-CuO-H-PVC (dashed
gray line).
Figure 8 Dynamic reversibility of sodium selective electrode PVC (solid line), CH-CuO-PVC (dashed
black-line ). The concentration of sodium ions switched for m 10-4
and 10-2
several times.
The most important character of membrane electrodes is the selectivity of the
membrane so it is important to probe the effect of CH-CuO or CH-CuO-H layer on the
selectivity of PVC-based sodium selective membrane electrodes. All of membrane electrodes
selectivity coefficients (PVC, CH-CuO -PVC and CH-CuO-H -PVC) were calculated at 1.0 M
concentrations and maximal selectivity coefficients are reported for interferent ions that give
significantly lower than Nernstian slopes [Bakker, et al., (2000)]. All data of selectivity are
shown in (Table 2). and all membrane electrodes potentiometric responses towards different
ions are represented in (Figures 4-6) as recommended [Bakker, et al., (2000)] the reported
data in Table 2 showed that the selectivity coefficients of CH-CuO or CH-CuO-H are
comparable to the selectivity coefficients of controlled PVC-based sodium selective
membrane electrodes. This proves that physical attachment as a layer of CH-CuO or CH-
CuO-H to PVC sensing film did not interfere with the ion-ionophore binding chemistry.
Presence of CH-CuO or CH-CuO-H did not change the binding constants of ion-ionophore or
single ion partition coefficient. It is worth mentioning that the selectivity coefficient of the
three sets of membrane electrodes (PVC, CH-CuO-PVC or CH-CuO-H-PVC) easily meet the
selectivity requirements for sodium analysis in physiological fluids (see data in the Table 2).
Platelet adhesion to the polymeric membranes pictured using scanning electron
microscopy (SEM) can be used to compare the thrombogenicity of different surfaces. Platelets
10
play a key role in the coagulation cascade and clot formation. Therefore, the adhesion and
activation of platelets on a surface is an indicator of its thrombogenicity. Platelet adhesion is
routinely used to investigate the interactions between materials and blood in vitro [Badr et
al., 2014; Berrocal, et al., (2001); Berrocal, et al., (2000); Espadas-Torre ,et al (1995);
Espadas-Torre, et al., (1997); Frost, et al., (2003); Schenfisch, et al., (2000)]. The relative
thrombogenicity of different membranes (PVC, CH-CuO-PVC or CH-CuO-H-PVC) were
investigated using the platelet adhesion protocol. Blood was obtained from the jugular vein
of a healthy sheep at the ministry of agriculture, (Cairo, Egypt). Each pint of blood was drawn
into a blood collection bag (JMS, Singapore, PTE Ltd.) containing 70.00 mL of CPDA-1 anti-
coagulant solution. Within 30 min after collection, blood was centrifuged at 240 g for 15 min
at 4 ºC. The supernatant was platelet-rich plasma (PRP) with a concentration of
approximately 9.5 ×104 cells /µL as determined with Exigo vet hematology analyzer. The
PRP was preincubated at 37 ºC for 20 min. All membranes (PVC, chitosan, heparin-modified
chitosan) were conditioned in phosphate-buffered saline (PBS, containing 10 mM phosphate,
138 mM sodium chloride, and 2.7 mM potassium chloride, pH 7.4) for 3 days, and were
immersed in PRP for 2 h at 37 ºC. They were then rinsed in PBS and fixed in a PBS solution
containing 2 % (w/v) glutaraldehyde for 2 h at room temperature. Membranes were
dehydrated by immersion in serial dilutions of ethanol (30, 50, 70, 90, and 95% v/v), two
immersions in absolute ethanol, and two immersions in hexamethyldisilazane. After solvent
evaporation within desiccators, the membranes were coated with gold and examined using
QUANTA FEG 250 scanning electron microscope (SEM). Micrographs and results showed
below representative of various membranes materials subjected to this procedure.
In vitro blood compatibility results are shown in SEM micrographs in Figure 9 A-C.
As can be seen in this figure a great difference of thrombogenicity or platelet adhesiveness for
different polymer materials is present under the same conditions. Large number of platelets
deposition and clusters presented in conventional PVC-based sodium-selective membranes
(Figure 9A). That refers to high thrombogenicity and less compatibility of blood to the PVC
membrane. Whereas, CH-CuO (Figure 9B) and CH-CuO-H (Figure 9C) showed smaller
number of platelets adhesion to the surface. A few platelets were observed on chitosan CH-
CuO or CH-CuO-H. As chitosan has rich free amino group with pKa value of about 6.3
[Rinaudo, et al (1999)]. When the pH is above the pKa, chitosan is uncharged due to the
protonation of the amino groups (e.g., measurement conditions, pH 7.4). As reported, the
neutralization of this charge decreased the platelet adhesion [Sagnella & Mai-Ngam (2005)].
Also, it is well-documented in the literature that blending polymers with surfactants exhibited
improved blood compatibility [Amiji (1995); Espadas-Torre& Meyerhoff (1995)].
Therefore, blending chitosan-CuO with softener that has surfactant properties, also the
measurement of the platelet adhesion was done at pH 7.4 is expected to improve chitosan-
CuO biocompatibility. Moreover, CuO nanoparticles has a good antifouling performance
[Chapman, et al (2013)] that expected to improve the biocompatibility by reducing or
limiting protein and cell adsorption as show in Figure 9B. Platelets are central in the blood
clotting cascade; preventing platelets from reaching PVC would effectively prevent
coagulation on the surface of PVC and improve the blood compatibility of ISEs based on
PVC. Slow release of heparin from CH-CuO-H and presence of CuO nanoparticles will
improve biocompatibility by minimize platelet adhesion and inhibit biofilm formation or
biofouling. This was further confirmed by platelet counts of PVC, CH-CuO and CH-CuO–H
membranes. A cell count form SEM micrograghs of five membrane segments of the same
membrane yielded an average 13.5 (±5.3) × 103 cells/mm
2, 0.21 (±0.14) × 10
3 cells/mm
2 (n =
5), and 0.15 (±0.05) × 103 cells/mm
2 (n = 5) for PVC, CH-CuO and CH-CuO–H membranes,
respectively (see data in Figure 10). This indicates that the number of platelets adhered to
PVC is about 64-fold higher than that found in CH-CuO and about 90-fold higher than that
11
observed for CH-CuO–H. As expected, SEM micrographs of PVC film showed large platelet
aggregates indicating a high thrombogenicity of this membrane [Badr et al. (2014)].
Figure 9. Scanning electron micrographs of various membranes after 2-h contact with PRP: (A)
PVC, (B) CH-CuO, and (C) CH-CuO-H
Figure. 10 Scanning electron platelet count for different membranes (PVC, CH-CuO, and CH-
CuO-H) after contact 2 hours PRP.
4- Conclusion
CH-CuO, CH-CuO-H were used to improve the blood compatibility of PVC-based
membrane electrodes. CH-CuO, CH-CuO-H were physically attached to the sensing PVC
layer. Three sets of electrodes were prepared: (1) conventional PVC membrane electrode, (2)
conventional PVC membrane electrode sandwiched with CH-CuO membrane (CH-CuO-
PVC), and (3) conventional PVC membrane electrode sandwiched with Chitosan-CuO-
Heparin membrane (CH-CuO-H-PVC). All potentiometric response characterization (e.g.,
detection limit, linear range, response slope, and selectivity coefficients) were studied to all
membranes and were found to be comparable to control PVC. This indicates that physical
attachment of CH-CuO and CH-CuO-H to PVC sensing film did not affect the potentiometric
behavior of sodium electrode.
Acknowledgement
The authors are grateful to Ain Shams University Scientific Research Sector (Grant 08-010)
for providing financial support to undertake this work.
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بالجسيمات النانوية لأكسيذ النحاس اغشية الكيتوزان المحمل تحسين التوافق الحيوى لغشاء قطب الصوديوم باستخذام
والهيبارينالنانوية لأكسيذ النحاس المحمل بالجسيمات والكيتوزان
3 حساو سس2يحذ جد عبذ انحهى 1 سانى يحذعبذ انب 1 أيش سانى حسا 1إبشاى حس عه بذس
س جامعة عين شم- كمية العموم-1 انشكز انقي نهبحث -2
يعذ صحت انحا- 3
:انهخص
وغشاء الكيتوزانبانجساث انات لأكسذ انحاس في هذا البحث نقوم بدراسة تاثير ادخال غشاء الكيتوزان المحمل
نهصدو رنك كهساذ ر انغشاء الإتقائ فم انبن غشاءوالهيبارين إلىانات لأكسذ انحاس المحمل بالجسيمات
كهساذ عه تخثش انذو فم نتحس انتافك انح تقهم انقذسة انعانت نهبن
( 2)كهساذ ، فم انغشاء الانكتشد انتقهذ نهبن( 1): تمت دراسة استجابة القوة القياسية لثلاثة مجموعات من المجسات
انجساث انات لأكسذ انحاس ، مركب الكيتوزانكهساذ انغط بغشاء ي فم انغشاء الانكتشد انتقهذ نهبن
جساث انات لأكسذ انحاس الكيتوزاني مركب كهساذ انغط بغشاء فم انغشاء الانكتشد انتقهذ نهبن( 3)
. انباس
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.كزنك تى تقى انتافك انح نجع الأغشت ي خلال دساساث انتصاق انصفائح انذيت