Lubricin and nano-BaSO4:: novel methods to prevent surface ......2013/11/25 · Acknowledgements...
Transcript of Lubricin and nano-BaSO4:: novel methods to prevent surface ......2013/11/25 · Acknowledgements...
Lubricin and Nano-BaSO4: Novel Methods to Prevent Surface Biofouling
GEAII Dissertation Page 1 of 117
A Dissertation Presented
by
George Ejiofor Aninwene II
to
The Graduate Engineering Program in Bioengineering
in partial fulfillment of the requirements for the degree
of
Doctor of Philosophy
in the field of
Bioengineering
November 25th, 2013
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Abstract Biofouling is a serious issue that threatens the lasting beneficial effects of many surgeries, shortens the lifespan of many implanted medical devices, and is a persistent problem for hospitals, surgeons, and patients all over the world. This set of studies is aimed to address the issue of biofouling by proposing novel surface preparation methods using lubricin and/or nano-BaSO4 as non toxic agents to prevent biofouling by inhibiting initial cellular adhesion to surfaces. Preventing initial unwanted cellular attachment and accumulation will dramatically improve outcomes and reduce instances of life threatening infections and bio-adhesions.
Lubricin is a an anti-adhesive glycoprotein that is found in the synovial fluid, which acts as a natural barrier within the body, lubricating surfaces and preventing undesirable cellular adhesion on cartilage. BaSO4 is a common additive used to make medical plastics radio opaque. Nano-formulations would retain similar radiopaque properties while imbuing the medical plastic with nano surface features which would change surface interactions with biological agents.
This research employed bacterial studies, mammalian cell studies, and mathematical modeling to better understand how these treatments will combat surface biofouling. This research proved that Lubricin was able to suppress both Staphylococcus aureus and Staphylococcus epidermidis adhesion and proliferation. The attachment and growth of Staphylococcus aureus on tissue culture polystyrene over the course of 24 hours was reduced by approximately 13.9% compared to a PBS soaked control. Under stationary conditions, lag time for Staphylococcus aureus and Staphylococcus epidermidis were increased by 27% and 36%, respectively. Additionally, surface plasmon resonance imaging (SPRi) studies determined that under flow conditions lubricin coated surfaces show 90% less Staphylococcus aureus biofouling than uncoated surfaces. These studies indicated that lubricin was able to prevent fibroblast adhesions to polystyrene surfaces, without adversely affecting the fibroblast viability. These studies indicated the once lubricin attached to surfaces the attachment of other surface proteins were blocked and thus both bacterial cells and fibroblast cells were unable to fully adhere to coated surfaces. This research demonstrated that the incorporation of nano-barium sulfate into pellethane hindered the proliferation of Staphylococcus aureus and Pseudomonas aeruginosa compared to currently used pellethane.
The results of this study will provide the medical field with novel alternative methods to reduce bio-adhesions related complications.
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Acknowledgements Special thanks those gave their time and effort to help me get to this point.
Thank you to my Committee: Dr. Thomas Jay Webster
Dr. Greg Jay Dr. Anand Asthagiri
To my friends and colleagues who aided in my experimental design, data analysis,
manuscript preparation, and overall forward progress: Erik Taylor, Vishnu Ravi, Zifan Yang, Justin Seil, Kimberly Waller, Sam McNeal, Ling Zang, Megan Creighton, Douglas Hall, Dan Hickey, Stanley Chung, David Stout, Evan Smith,
Jara Crear, Amy May, Ryan Hartigan, Ms. Barbara Kahn
To my mentors: Lamont Toliver
"Begin with the end in mind" (1963-2012)
Dr. Medeva Ghee Dr. Jabbar Bennett Dr. Valerie Wilson
Mrs. Earnestine Baker To my funding sources: Hermann Foundation
NIGMS award number R25GM083270 & R25GM083270-S1 NSF GK-12 fellowship award number 0638688
And to my Loving Family George and Doreen Aninwene
Amanda Aninwene Ana Veras
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Table of Contents
Table of Contents ............................................................................................................................ 5
0 Introduction ........................................................................................................................... 10
0.1 Biofouling ....................................................................................................................... 10
0.2 Lubricin ........................................................................................................................... 14
0.3 Nanomaterials ................................................................................................................ 16
0.4 Hypothesis ...................................................................................................................... 18
0.5 Introduction References................................................................................................. 18
1 Chapter 1: Lubricin- a Novel Means to Decrease Bacterial Proliferation on Implanted
Devices .......................................................................................................................................... 22
1.1 Keywords ........................................................................................................................ 23
1.2 Introduction.................................................................................................................... 23
1.3 Experimental details ....................................................................................................... 24
1.3.1 Substrates ............................................................................................................... 24
1.3.2 Protein Preparation ................................................................................................ 25
1.3.3 Bacteria Surface Adhesion and Proliferation Study ................................................ 25
1.3.4 Modeling ................................................................................................................. 27
1.3.5 Contact Angles ........................................................................................................ 29
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1.3.6 Statistical Analysis: .................................................................................................. 29
1.4 Results ............................................................................................................................ 30
1.4.1 Bacteria Surface Adhesion and Proliferation Study ................................................ 30
1.5 Discussion ....................................................................................................................... 41
1.5.1 Lubricin .................................................................................................................... 41
1.5.2 Mucin ...................................................................................................................... 41
1.5.3 Vitronectin .............................................................................................................. 43
1.6 Bacteria .......................................................................................................................... 44
1.6.1 Surface Interactions ................................................................................................ 44
1.7 Conclusions..................................................................................................................... 45
1.8 Acknowledgements ........................................................................................................ 46
1.9 References ...................................................................................................................... 46
2 Chapter 2: Lubricin Reduction of Biofilm Formation Under Flow as Detected by SPRi ........ 48
2.1 SPRi Background ............................................................................................................. 48
2.2 Biofilm Formation ........................................................................................................... 48
2.3 SPRi ................................................................................................................................. 49
2.4 Hypothesis ...................................................................................................................... 49
2.5 Materials and Methods .................................................................................................. 50
2.5.1 Protein Preparation ................................................................................................ 51
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2.5.2 Statistical Analysis ................................................................................................... 51
2.6 SPRi Results .................................................................................................................... 53
2.7 Discussion ....................................................................................................................... 54
2.8 References ...................................................................................................................... 54
3 Chapter 3: Lubricin Protein Coating to Reduce the Adhesion of Fibroblasts on the Surfaces
of Medical Devices ........................................................................................................................ 56
3.1 Lubricin Protein Coating to Reduce the Adhesion of Fibroblasts on the Surfaces of
Medical Devices: Abstract ......................................................................................................... 56
3.2 Background ..................................................................................................................... 56
3.3 Hypothesis ...................................................................................................................... 57
3.4 Materials and Methods .................................................................................................. 58
3.4.1 Protein Preparation ................................................................................................ 58
3.4.2 Cell Adhesion and proliferation .............................................................................. 58
3.4.3 MTS Assay ............................................................................................................... 59
3.5 Results ............................................................................................................................ 59
3.5.1 Cell Adhesion and proliferation .............................................................................. 59
3.5.2 MTS ......................................................................................................................... 63
3.6 Discussion ....................................................................................................................... 64
3.7 References ...................................................................................................................... 64
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4 Chapter 4: Nano-materials and Nano-structures: Breakthroughs in Medical Imaging ........ 66
4.1 Abstract .......................................................................................................................... 66
4.2 The need for nanomaterials in medical imaging............................................................ 66
4.3 C60 Buckeyballs .............................................................................................................. 68
4.4 Iron oxide nanoparticles................................................................................................. 72
4.5 Carbon nanotubes .......................................................................................................... 75
4.6 Quantum dots ................................................................................................................ 78
4.7 Gold Nanoparticles ......................................................................................................... 82
4.8 Nano BaSO4 .................................................................................................................... 83
4.9 Future Directions ............................................................................................................ 83
4.10 Conclusions..................................................................................................................... 84
4.11 References ...................................................................................................................... 84
5 Chapter 5: Nano-BaSO4: a Novel Antimicrobial Additive to Pellethane ............................... 89
5.1 Introduction: .................................................................................................................. 89
5.2 Material and methods: ................................................................................................... 91
5.2.1 Sample preparation ................................................................................................ 91
5.2.2 Contact angle test ................................................................................................... 91
5.2.3 Radiopacity Trials .................................................................................................... 91
5.2.4 AFM Surface Analysis .............................................................................................. 92
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5.2.5 Bacteria culture ....................................................................................................... 92
5.2.6 Bacteria growth trials .............................................................................................. 93
5.3 Results: ........................................................................................................................... 93
5.3.1 Contact angle test ................................................................................................... 93
5.3.2 Radiopacity Trials .................................................................................................... 94
5.3.3 AFM Surface analysis .............................................................................................. 95
5.3.4 Bacteria growth trials .............................................................................................. 98
5.4 Discussion: .................................................................................................................... 101
6 Conclusions: ......................................................................................................................... 102
6.1 Acknowledgements: ..................................................................................................... 102
6.2 References: ................................................................................................................... 103
7 Contributions to the Field .................................................................................................... 105
8 Appendix .............................................................................................................................. 107
8.1 Complete Reference List .............................................................................................. 107
8.2 Curriculum vitae ........................................................................................................... 117
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0 Introduction
Biofouling of implanted devices is not only a major threat to the prolonged function of
implanted devices, but it is the primary reason for their failure. This set of studies explored the
use of lubricin as a surface coating to deal with the issue of biofouling. Lubricin is an anti-
adhesive protein found in the synovial fluid, which acts as its primary lubricant. This set of
studies also investigated the use of nano-BaSO4 compared to standard currently used BaSO4
composites. BaSO4 is a commonly used radiopaque additive to medical tubing. The following
studies investigated lubricin as a surface coating and nano-BaSO4 as a radiopaque additive to
thermoplastics. These studies showed that lubricin surface coatings are effective at preventing
bacterial biofilm production of both S. aureus and S. epidermidis, as well as unwanted fibroblast
adhesion. These studies also showed that using nano-BaSO4, as opposed to conventional BaSO4,
in extruded thermoplastics yield radiopaque polymers that are less prone to adhesion and
proliferation of S. aureus and P. aeruginosa.
0.1 Biofouling
Infection after the implantation of a medical device is a continual problem in the
medical field. The success of medical devices (such as heart valves, endotracheal tubes,
pacemakers, orthopedic implants, intraocular lenses, central venous catheters, and orthopedic
joint prosthetics) is threatened by the attachment and proliferation of numerous bacteria on
their surfaces after implantation (1, 2). Post-surgical infections are costly, result in additional
harm to the patient, and can result in the need for revision surgery. Biofilms on indwelling
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medical devices and implants account for 60% of hospital-associated infections in over 1 million
patients that cost approximately 1 billion dollars per year in the United States alone (3).
Biofouling can be defined as the deposition, migration, adhesion, and/or proliferation of
unwanted biological material on the surface of an implanted device. Biofouling can lead to a
reduction or loss of device function as well as infection, inflammation, and/or damage to the
surrounding tissue. Post-surgical bacterial infection is a major form of biofouling. For example,
endophthalmitis is a serious intraocular infection that can result as a complication of intraocular
surgery (4). Endophthalmitis can cost approximately $12,580 USD (United States dollars) in
total ophthalmic medical care claims per patient treated and could lead to severe permanent
visual impairment or to a complete loss of the infected eye (4-6).
Additionally, infections from life sustaining implants can result in drastic complications.
For example, although central intravenous catheters (CVCs) are devices that are critical for
transferring fluids, delivery of medications, and monitoring of overall patient health, they are
also transvenous conduits for infection (7). The use of these devices can result in bloodstream
infections (BSI) in about 4 to 5 cases out of every 1000 CVC devices inserted (8, 9). The
estimated annual cost of caring for patients with CVC-associated BSI ranges from $296 million
USD to $2.3 billion USD (10). Post-surgical infection is costly, and potentially life threatening to
patients. One study showed that these catheter related blood stream infections can extend the
length of hospitalization by 7 to 21 days, and can increase associated costs by approximately
$37,000 (USD in 2002) (11). In the United States alone as many as 28,000 patients die each year
due to catheter-related bloodstream infections (12, 13).
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S. aureus and S. epidermidis are two major opportunistic pathogenic bacteria that
colonize a large portion of the human population (14). They are also two of the major culprits of
biofouling. Multiple strains of antibiotic resistant S. epidermidis and S. aureus have been linked
to a growing number of hospital acquired and post-operative infections (1, 14, 15). The
majority of S. epidermidis is fairly widespread throughout the cutaneous ecosystem, while S.
aureus is carried primarily on mucosal surfaces (14).
S. aureus is regarded as one of the leading causes of aggressive, persistent post-surgical
infections due to acquired resistance to antibiotics and its ability to form drug resistant biofilms
(16). Lowy et al. states that “Humans are a natural reservoir of S. aureus”, and goes on to say
that 30% to 50% of adults are colonized, with 10% to 20% persistently colonized (17). S. aureus
infections cause a range of acute and pyogenic infections, including abscesses, bacteremia,
central nervous system infections, endocarditis, osteomyelitis, pneumonia, urinary tract
infections, chronic lung infections associated with cystic fibrosis, and several syndromes caused
by exotoxins and enterotoxins, including food poisoning, scalded skin and toxic shock
syndromes (14). The overuse of methicillin and other semi-synthetic penicillins in the late 1960s
led to the emergence of methicillin-resistant S. aureus (MRSA). In 1997, approximately 60% of
S. aureus isolated from patients were resistant to methicillin (14). Additionally, new antibiotic
resistant bacteria strains continue to emerge such as vancomycin-intermediate MRSA (VISA)
and vancomycin resistant MRSA (VRSA), both of which emerged due to the overuse of
vancomycin to treat MRSA (18).
Although it is not as aggressive of a pathogen as S. aureus, S. epidermidis is a major
nosocomial pathogen that is primarily associated with infections of implanted medical devices GEAII Dissertation Page 12 of 117
(14, 19) . The overuse of methicillin and other semi-synthetic penicillins in the late 1960s also
led to the emergence of methicillin-resistant S. epidermidis (MRSE) (14). Once again, new
antibiotic resistant strains of this bacterium continue to emerge. One effective treatment
against multi-drug resistant staphylococci, including MRSE, was the glycopeptide antibiotic
vancomycin; however, resistance has developed to this treatment as well (14). S. epidermidis
will also grow robust biofilms, which may protect the bacterial colonies from antibiotic
treatments.
It has been reported that it is easier for the body to clear bacteria that are suspended in
solution, otherwise known as planktonic bacteria. Once bacteria adhere to a substrate they can
form complex polysaccharide aggregates that protect them from harsh environmental
conditions and can result in antibiotic resistance (7, 20). When bacteria attach to a substrate,
their functions change and host defenses are less able to combat resulting bacteria colonization
and biofilm formation (21). Corterton et al. defines bacterial biofilms as a collection of matrix-
enclosed bacterial populations, adherent to each other and/or surfaces or interfaces (22).
These biofilms have an inherent resistance to antimicrobial agents (21). The main challenge is
to prevent bacteria adhesion and proliferation early before biofilm production takes place,
because once the bacterial biofilm matrix forms, the bacterial infection is profoundly more
resistant to the host defenses as well as antibiotic treatment (23). Once bacteria develop a
biofilm it is very difficult for antibiotics to penetrate the polysaccharide slime layer and
effectively kill the bacteria (24). One study by Nichols et al. showed that Pseudomonas
aeruginosa (a common biofilm forming bacteria) was 15 times more susceptible to antibiotic
treatment when dispersed from a biofilm than when in a solid intact biofilm (25). A study by
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Kluytmans et al. also reported that disruptions of hydrophobic surface interactions may prevent
initial bacterial binding and inhibit bacterial colonization (26). Thus, it follows that if the
bacteria are not able to colonize the surface of a medical device and form a biofilm, it will be
much easier for the immune system to clear these bacteria before they cause an infection.
0.2 Lubricin
Lubricin (LUB) is an amphiphilic glycoprotein that is found in the synovial fluid. The
molecular weight of LUB is approximately 240 kDa and the molecular structure is similar to an
extended flexible rod with a length of approximately 200 nm and a width of approximately 1-2
nm (27, 28) (Figure 1). The half-life of this molecule is approximately 6 days when bound to
articular cartilage (29). In humans, the concentration of LUB ranges from 52-350 μg/mL in
normal joints examined post-mortem, and 276-762 μg/mL in the synovial fluid obtained from
patients undergoing arthrocentesis procedures (30). Arthrocentesis is a procedure where a
syringe is used to collect synovial fluid from the knee joint capsule to be analyzed to determine
joint health of the patient (31).
The abundance of negatively charged and highly hydrated sugars in the center mucin-
like domain of LUB contribute to its boundary lubrication properties as a result of strong
repulsion through steric and hydration forces (32). On hydrophobic surfaces, such as cartilage,
the globular tail ends will orient themselves towards the substrate and adsorb to it, while on
hydrophilic surfaces, the mucin-like domain will orient towards, and adsorb to the substrate, as
illustrated in Figure 1 (see page 24) (32, 33).
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The sugars in the central mucin-like domain of LUB are O-linked glycosylations and have
been studied by Jay et al. (34). Sugars in the central mucin-like domain have been characterized
as disaccharide β(1-3)Gal-GalNAc and trisaccharide β(1-3)Gal-GalNAc-NeuAc moieties (34). The
densely packed negatively charged sugars, along with the sialic acid residues and sulfate
groups, are responsible for the negative charge as well as the hydrophilic nature of the central
domain of LUB (35).
LUB’s amino acid sequence has significant similarities to the adhesive protein
vitronectin (36). Both proteins contain somatomedin B (SMB) and hemopexin-like (PEX)
domains and there is approximately a 60% sequence similarity between both proteins (36). In
vitronectin, SMB and PEX sequences are known to regulate the complement and coagulation
systems, mediate extracellular matrix attachment, and promote cell attachment and
proliferation (36). The PEX and SMB domains are found in the globular tails of LUB as seen in
Figure 1 (36-38).
Previous studies have shown that in addition to providing boundary layer lubrication,
LUB plays a role in reducing or preventing undesirable cellular attachment. Rhee et al. showed
that LUB was able to reduce synovial cell overgrowth within joints and Noyori et al. indicated
that LUB may be responsible for the inability of fibroblasts to adhere to healthy cartilage (36,
39). However, previous studies from other research groups have not investigated the
antibacterial or bacteriostatic properties of LUB.
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0.3 Nanomaterials
This study also investigated preventing biofouling through the use of nano-materials. A
nano-material has features or dimensions less than 100 nm (40, 41). This change in feature size
can create materials drastically different from their micron counterparts. That is, once a
material (such as a particle, grain, or tube) reaches the nanoscale, they have the potential to
possess vastly different mechanical, chemical, optical, electrical, thermal, catalytic, magnetic,
and biological properties (40-42). Nanometer-sized particles have structural and functional
properties that are not available in either singular molecules or bulk materials (43). The
differences between nanomaterials and their macroscopic counterparts are the result of their
increased relative surface area and quantum effects (42). For example, a 30 nm particle may
have 5% of its atoms on the surface, while a 10 nm particle of the same composition may have
20% of its atoms on its surface and a 3 nm particle of the same composition may have up to
50% of its atoms on the surface (42). As a result of this additional available surface area, a given
mass of a particular nano material may be much more reactive than the same mass of the same
material without nano features (42). The large surface area to volume ratio of nanomaterials
allow these materials to encapsulate, act as, or be conjugated to diagnostic agents (whether
they are optical, radioisotopic, or magnetic) or therapeutic agents to the surface (43, 44).
Additionally, quantum effects begin to dominate the properties of a substance as the size is
reduced into the nanoscale (42). These quantum effects (size dependent shifts in the physical
properties of a material) can be responsible for shifts in magnetic, electrical, optical, and/or
biological behavior (42). Additional studies have shown that the use of nano structures on
surfaces can lead to surfaces that are antimicrobial or resistant to bacterial proliferation (7).
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Currently, silver is the primary antimicrobial active ingredient being developed into
many products; however, there remains a need for more cost effective solutions. Specifically,
some studies cite the emergence of silver resistant bacteria and provide indications of
mammalian cell toxicity (45). A study by Silver et al. investigated genetic mechanisms in
Enterobacteriaceae which confer resistance silver treatment (46). One Enterobacteriaceae
investigated was Salmonella typhimurium. An Ag(I)-resistant Salmonella strain was linked to the
death of several patients and required the shutdown of the burn ward of Massachusetts
General Hospital (46, 47). Salmonella typhimurium was also found to be resistant to silver
nitrate, mercuric chloride, ampicillin, chloramphenicol, tetracycline, streptomycin, and
sulphonamides (47). Silver's study also went on to criticize the near ubiquitous use of silver in
consumer products from burn dressings and antiseptic ointments to baby pacifiers and drinking
water additives (46). This overuse of silver will result in selective pressures, which will promote
the emergence of more silver resistant bacterial strains. In contrast, there has been an
emergence of studies which demonstrated slowed or stopped growth of bacteria on materials
with nanostructured surface features which alter surface energetics or nanoscale surface
roughness to repel bacteria, without the use of antibiotics (7). Barium sulfate (BaSO4) is a
common agent used to make medical tubing radiopaque; however, in addition to this, BaSO4
polymeric formulations have been shown to exhibit antimicrobial activity (Foster Corporation,
email communication, 2011). One of the goals of this study was to investigate if nano-BaSO4
pellethane composites are able to effectively act as antimicrobial surfaces (surfaces that
prevent initial bacterial adhesion and proliferation) while still remaining radio opaque.
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0.4 Hypothesis
These studies investigated modifying implant surfaces to reduce the occurrence of post-
operative infection based complications. These sets of studies hypothesized that the
glycoprotein lubricin could be used as a surface coating to reduce biofouling. Additionally, these
studies hypothesized that nano-BaSO4 could act as a thermally stable bacteriostatic additive to
thermoplastics to be used in medical tubing.
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37. D. P. Chang, N. I. Abu-Lail, F. Guilak, G. D. Jay, S. Zauscher, Conformational mechanics, adsorption, and normal force interactions of lubricin and hyaluronic acid on model surfaces. Langmuir 24, 1183 (Feb, 2008).
38. B. Zappone, G. W. Greene, E. Oroudjev, G. D. Jay, J. N. Israelachvili, Molecular Aspects of Boundary Lubrication by Human Lubricin: Effect of Disulfide Bonds and Enzymatic Digestion†. Langmuir 24, 1495 (2008/02/01, 2007).
39. K. Noyori, H. E. Jasin, Inhibition of human fibroblast adhesion by cartilage surface proteoglycans. Arthritis & Rheumatism 37, 1656 (1994).
40. D. M. Goncalves, R. de Liz, D. Girard, Activation of Neutrophils by Nanoparticles. The Scientific World Journal 11, 1877 (2011).
41. M. Vijayakumar, K. Priya, F. T. Nancy, A. Noorlidah, A. B. A. Ahmed, Biosynthesis, characterisation and anti-bacterial effect of plant-mediated silver nanoparticles using Artemisia nilagirica. Industrial Crops and Products 41, 235 (2013).
42. S. Lanone, J. Boczkowski, Biomedical applications and potential health risks of nanomaterials: Molecular mechanisms. Curr. Mol. Med. 6, 651 (September, 2006).
43. X. M. Qian et al., In vivo tumor targeting and spectroscopic detection with surface-enhanced Raman nanoparticle tags. Nat. Biotechnol. 26, 83 (Jan, 2008).
44. A. Wang, F. Gu, O. Farokhzad, in Safety of Nanoparticles, T. J. Webster, Ed. (Springer New York, 2009), pp. 209-235.
45. I. Chopra, The increasing use of silver-based products as antimicrobial agents: a useful development or a cause for concern? Journal of Antimicrobial Chemotherapy 59, 587 (April 1, 2007, 2007).
46. S. Silver, Bacterial silver resistance: molecular biology and uses and misuses of silver compounds. FEMS Microbiology Reviews 27, 341 (2003).
47. G. Larkin Mchugh, R. Moellering, C. Hopkins, M. Swartz, Salmonella typhimurium Resistant to Silver nitrate, Chloramphenicol, and Ampicillin: A New Threat in Burn Units ? The Lancet 305, 235 (1975).
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1 Chapter 1: Lubricin- a Novel Means to Decrease Bacterial
Proliferation on Implanted Devices
Staphylococcus aureus (S. aureus) and Staphylococcus epidermidis (S. epidermidis)
infections after the implantation of a prosthetic device can lead to major complications.
Reducing bacterial adhesion to the surface of implanted materials will help to reduce the
incidence of infection. Lubricin is an amphiphilic glycoprotein that is found in the synovial fluid.
This study investigated lubricin’s ability to prevent bacterial attachment and proliferation on
polymer surfaces. The findings from this study indicated that lubricin was able to reduce the
attachment and growth of Staphylococcus aureus on tissue culture polystyrene over the course
of 24 hours by approximately 13.9% compared to the PBS soaked control. Lubricin also
increased the lag time (the period of time between the introduction of bacteria to a new
environment and their exponential growth) of S. aureus by approximately 27% compared to the
PBS soaked control. This study also indicated that vitronectin, a protein homologous to lubricin,
was able to reduce bacterial growth and adhesion on tissue culture polystyrene by
approximately 11% compared to the PBS soaked control. Vitronectin was also able to increase
the lag time by approximately 43% compared to the PBS soaked control. Mucin was studied
because there are similarities between it and the center domain of lubricin. Results showed
that the reduction of bacterial proliferation on mucin-coated surfaces was not as drastic as that
seen with lubricin. In summary, this study provided the first evidence that lubricin reduces the
initial growth of bacteria on a surface and suppresses initial biofilm formation. This can be very
beneficial for medical implants and lead to improved patient outcomes.
GEAII Dissertation Page 22 of 117
1.1 Keywords
Lubricin, Bio-fouling, Infection, Implant, Protein Coating, Bacterial Adhesion, Staphylococcus
aureus, Staphylococcus epidermidis, Kinetic Bacterial Growth modeling
1.2 Introduction
Lubricin (LUB) is a glycoprotein found in the synovial fluid that plays a major role in its
lubricating and anti-cell-adhesive properties (33). LUB has a mucin-like center domain and
globular tail domains (Figure 1) (48). The purpose of this in vitro study was to determine: LUB’s
ability to prevent bacterial adhesion and proliferation under tissue culture conditions on a
model tissue culture surface (polystyrene), its ability to serve as an effective non-immune
opsonification agent for resisting bacteria colonization, and to present evidence that LUB is a
viable means to reduce bacterial infection of medical implants. Due to structure similarity, in
this study, mucin and vitronectin were also used to investigate the possible bacteriostatic
nature of the center and the globular tail domains of lubricin, respectively.
GEAII Dissertation Page 23 of 117
Figure 1: Schematic representations of lubricin structure (32, 33, 49). A.) Mucin-like domain is found in center and vitronectin like domains (analogues to SMB (stomatomedin B) and PEX (haemopexin)) are found at the ends (32, 33). Lubricin is similar to vitronectin but it contains a negatively charged mucin domain. Lubricin may exist as a monomer or form dimers through disulfide bonds of the cysteine groups found in the globular tail regions. B.) Lubricin covers surfaces with end-grafted brushes (38). Hydrophobic domains orient themselves towards hydrophobic surfaces and lubricin may form a loop structure with its mucin domain (38). LUB coating imparts a negatively charged hydrophilic nature on to the coated surface.
1.3 Experimental details
1.3.1 Substrates
Standard tissue culture polystyrene was used as the polymer substrate. Sterile tissue
culture polystyrene plates were obtained from Becton, Dickinson and Company. The LUB used
GEAII Dissertation Page 24 of 117
in these trials was extracted under sterile conditions from bovine synovial fluid obtained from
Pel-Freez (Arkansas, USA) (50). The sterile extraction procedure was described fully by Jay et al.,
but in short it involves extraction of LUB from bovine synovial fluid through a hyaluronate
digestion, followed by anion exchange chromatography, and affinity chromatography to purify
and concentrate the protein (50).
1.3.2 Protein Preparation
50 μg of bovine vitronectin (VTN) (Sigma-Aldrich) were dissolved in 1 mL of sterile PBS.
LUB and bovine submaxillary mucin (BSM) (Sigma-Aldrich) were used at a concentration of 200
μg/mL. Prior to use, both BSM and VTN solutions were filtered through low protein binding
sterile filters with a pore size of 0.2 μm (Corning). 50 μL of each protein solution were dried on
the well surface overnight.
1.3.3 Bacteria Surface Adhesion and Proliferation Study
1.3.3.1 S. aureus
96 well polystyrene tissue culture plates were treated with LUB, BSM, or VTN. BSM was
used in this trial due to the similarities between mucin and the center domain of LUB. The main
differences between mucin and LUB is that mucin lacks the globular hydrophobic end domains
found in LUB and mucin does not display LUB's boundary lubricating properties (51). Vitronectin
was used since it is a homologous protein to LUB. Although LUB and VTN have a 60% sequence
similarity, VTN lacks the center mucin-like domain found in LUB (36). LUB and BSM were used in
the crystal violet end point trials, while LUB, BSM, and VTN were used in the 24-hour optical
density trials.
GEAII Dissertation Page 25 of 117
Staphylococcus aureus (S. aureus) obtained from the American Type Culture Collection
(25923) was cultured in tryptic soy broth (TSB) (Sigma Aldrich, St. Louis, MO, USA) for 18 hours
to reach stationary phase, then diluted to a density of 1 × 107 bacteria/mL (as estimated by the
McFarland scale which corresponded to an optical density of 0.52 at 562 nm and then further
diluting at a ratio of 1:90) (1). Bacterial solutions were seeded into the wells and were allowed
to incubate for 15 minutes in a stationary incubator maintained at 37°C. After 15 minutes the
bacterial solution was removed, the plates were rinsed three times with sterile PBS, and the
wells were filled with 200 μL of fresh TSB. For crystal violet trials plates were incubated for 24
hours before crystal violet was used to determine the quantity of bacterial biofilm formed. For
this, plates were rinsed once with PBS, then 175 μL of a crystal violet solution (Sigma) was
added to each well and allowed to sit for 15 minutes to stain the biofilm. Solutions were then
removed and plates were again rinsed 3 times with PBS and plates were allowed to dry. Once
dry, 200 μL of EtOH were added and after 15 minutes optical density readings were read at 562
nm with a spectrophotometer (Spectramax 340PC). For the 24 hour optical density trials after
the initial bacterial seeding and rinsing steps, optical density measurements were taken every 4
minutes for 24 hours while maintaining the temperature at 37°C. Comparisons were done
between the PBS coated samples and the protein coated samples and the percent difference
was calculated for every data point over the course of the experiment. A Student's t-test was
done at each time point in the experiment to compare the protein soaked samples to the PBS
soaked samples. P-values of less than 0.05 indicated that any observed differences could be
considered significant.
GEAII Dissertation Page 26 of 117
1.3.3.2 S. epidermidis
96 well polystyrene tissue culture plates were treated in a manner similar to the S.
aureus trials. However, the initial seeding volume of the bacterial solution was 50 μL of S.
epidermidis (American Type Culture Collection (35984)) diluted in the same manner mentioned
above. After 15 minutes, the bacterial solution was removed, the plates were rinsed three
times with sterile PBS, and the wells were filled with 200 μL of fresh TSB. Crystal violet end
point trials and 24hr optical density trials were done in the same manner as seen the above.
Comparisons were done between the PBS coated samples and the protein coated samples and
the percent difference was calculated for every data point over the course of the experiment. A
student's t-test was done at each time point in the experiment to compare the protein soaked
samples to the PBS soaked samples.
1.3.4 Modeling
The ability of LUB to inhibit bacterial proliferation was quantified in this study by
determining the effect that LUB and protein sub-regions of LUB (mucin and vitronectin) have on
the growth curve of each bacterium. This curve is generally separated into several phases. The
first is the lag phase during which the bacteria are adapting to the conditions of their
environment and not dividing. The lag time (λ) is the length of this phase. Then, there is an
exponential phase during which the bacteria are dividing at a constant rate, leading to
exponential growth. If the natural logarithm of the number of organisms in this phase is plotted
against time, a straight line will result. The slope of this line is the maximum specific growth
rate (μm). Depletion of essential nutrients and/or formation of toxins and inhibitory products
causes the bacteria to enter the stationary phase during which the growth and death rate are
GEAII Dissertation Page 27 of 117
equal (52). The ratio of number of organisms in this equilibrium state to initial number of
organisms is the upper asymptote (A).
The three values (λ, μm, and A) were determined here by fitting the bacterial growth data
to two sigmoid growth functions: the logistic function and the Gompertz function. These are
both mathematical models of a time series where the growth at the beginning and end of the
period of time is the smallest. The logistic function is symmetrical about its inflection point,
while the Gompertz function approaches its upper asymptote more slowly than its lower
asymptote.
The growth data (optical density vs. time) obtained from these bacterial trials were fit to a
modified forms of the logistic model (Equation 1) and the Gompertz model (Equation 2) using
MATLAB R2013a.
ln �𝑂𝐷𝑡𝑂𝐷0
� = 𝐴
�1+ 𝑒[4𝜇𝑚𝐴 (λ−𝑡)+2]� (1)
ln �𝑂𝐷𝑡𝑂𝐷0
� = 𝐴𝑒−𝑒µ𝑚+𝑒𝐴 �𝜆 −𝑡� + 1
(2)
The model equations used were modified from their generic forms to contain coefficients
for the three bacterial growth parameters lag time (λ), maximum specific growth rate (µm), and
the asymptote of the stationary phase (A) by deriving formulas to represent the mathematical
parameters in terms of the bacterial growth parameters (53). 𝑂𝐷𝑡 is the optical density at time
t and 𝑂𝐷0 is the optical density at the start of the experiment. The goodness-of-fit was
determined using R-square and the sum of squares for error (SSE). The models with the best fit
GEAII Dissertation Page 28 of 117
were used to determine the value of the three parameters for each combination of bacteria
and protein.
1.3.5 Contact Angles
Contact angle measurements were made on a Krüss Easy Drop contact angle instrument
(Hamburg, Germany) connected to an image analysis program (Drop Shape Analysis (Version
1.8)). For surface energy experiments, the tissue culture polystyrene samples were dried
overnight under vacuum after coating. The Krüss Easy Drop apparatus was used to measure the
contact angles that resulted when a 10 μL drop of either H2O, glycerol, or ethylene glycol was
dispensed onto the surface of the sample (54). The Krüss Trackman software was used to
record the contact angle after the drop was placed on the surface within 30 seconds of the
droplet being dispensed. All readings were taken at ambient room temperature.
1.3.6 Statistical Analysis:
All of the above trials were performed with a minimum of three replicates each. The
standard error of the means was used to describe variance about the mean. Statistical analysis
of numerical data in this study was completed using an unpaired t-test assuming unequal
variances. Statistical significance was considered at p<0.05. For the bacteria surface adhesion
and proliferation study, a 95% confidence interval was determined at each time point and was
used as an additional method to ensure statistical differences between experimental and
control trials.
GEAII Dissertation Page 29 of 117
1.4 Results
1.4.1 Bacteria Surface Adhesion and Proliferation Study
1.4.1.1 S. aureus
The polystyrene bacteria trials showed that treatment with 200 μg/mL LUB resulted in a
reduced S. aureus optical density when compared with the control (Figure 2B). Over the course
of the experiment, the protein-coated samples showed a significant decrease in growth when
compared to the PBS coated samples at approximately 3.9 hours (near the start of the
exponential growth phase) as indicated by the growth curve. This trend of a decrease at the
start of the exponential phase was seen with samples coated with LUB, VTN, and BSM.
Mathematical modeling of the data showed that all three proteins increased the length of the
lag phase (Table 1, Figure 3A).
Between the period of 3.9 and 5.1 hours there was a significant depression in the LUB
growth curve versus the PBS growth curve (p<0.05) (see Figure 2B). A divergence of growth for
LUB treated surfaces compared to PBS treated surfaces persisted (p<0.05) (see Figure 2B). At
the end of 24 hours, there was a 13.87% difference between the growth curves of LUB coated
samples and the PBS coated samples. This indicated that the LUB suppressed attachment and
proliferation of S. aureus during the exponential phase of bacteria growth, and reduced the
overall amount of bacterial proliferation once the bacteria reached the stationary phase. The
mathematical model showed that there was a 27% (51.6 minute) increase in lag time (Figure
3A), a 13% reduction in the ratio of equilibrium number of bacteria in the stationary phase to
GEAII Dissertation Page 30 of 117
initial number of bacteria (Figure 3C), and a decrease in the maximum specific growth rate
(Figure 3B).
In the VTN S. aureus trial, the first period of significant reduction of the bacterial growth
curve was between 3.9 and 5.6 hours (p<0.05) (see Figure 2D). After 10.9 hours, the VTN
growth curve continued to show a significant difference from the PBS soaked samples. Between
13 and 14.4 hours, the results indicated some significance (p<0.058); however, after 14.4 hours
a significant depression in the growth curve compared to the control PBS soaked samples was
seen until the end of the trial (p<0.05). At the end of 24 hours there was a 10.96% difference
between the growth curves of VTN soaked sample and the PBS soaked samples. Again, this
indicated that the protein disrupted the start of the exponential growth phase as well as the
stationary phase. The mathematical model showed an increase in lag time of 43% (82.2
minutes) (Figure 3A), a 13% reduction in the ratio of equilibrium number of bacteria in the
stationary phase to the initial number of bacteria (Figure 3C) and an increase in the maximum
specific growth rate (Figure 3B).
In the case of BSM, there was a significant reduction in bacterial proliferation between
4.13-4.9 hours (p<0.05) (see Figure 2C). While some disruption of the exponential growth
phase was observed, there was no prolonged effect of BSM on the S. aureus load in the
stationary phase. However, the crystal violet trials showed that while the LUB resulted in a
minor reduction in biofilm production, the BSM coated samples showed a 58% reduction in
adherent biofilms when compared to the PBS coated samples (see Figure 2A). The difference in
the crystal violet results indicated that although BSM may not reduce the initial adhesion and
proliferation as well as LUB, biofilms grown on BSM coated surfaces appeared to be drastically GEAII Dissertation Page 31 of 117
less adherent than those grown on LUB coated or PBS coated surfaces. The mathematical
model showed a 29% (55.4 minute) increase in lag time (Figure 3A), a significant decrease in
maximum specific growth rate (Figure 3C), and no significant change in asymptote between
BSM and the PBS soaked control (Figure 3B).
Figure 2: A.) Crystal violet results after the 24hr S. aureus surface adhesion and proliferation trial. BSM treated samples showed a significant reduction in biofilm formation. Approximately 58% less biofilm was measured on BSM coated samples vs. PBS treated samples. B.) S. aureus surface adhesion and proliferation with LUB (200μg/mL) over 24 hours determined by optical density readings. LUB treatment significantly suppressed bacterial growth over the course of 24 hrs by 13.9%. Data = mean +/- SEM; N = 3 (@24hr p<0.01) C.) S. aureus surface adhesion and proliferation with VTN (50 μg/ml) over 24 hours determined by optical density readings. VTN treatment significantly suppressed bacterial growth over the course of 24 hrs by 11%. Data = mean +/- SEM; N = 3 (@24hr p<0.03) D.) S. aureus surface adhesion and proliferation with BSM (200μg/mL) over 24 hours determined by optical density readings. Some significant reduction in proliferation was seen between the 4.2 and 4.9hr time points. Data = mean +/- SEM; N = 3;
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[*indicates areas of significant difference between protein treated trials and PBS treated trials] as determined by p<0.05]; [♦ indicates areas where using a confidence interval of 95% a significance reduction was seen between protein treated trials and PBS treated trials].
Figure 3: A.) Lag times for S. aureus with each protein derived from the mathematical model. B.) Maximum specific growth rate for S. aureus with each protein derived from the
GEAII Dissertation Page 33 of 117
mathematical model. C.) Optical density ratio (asymptote value/initial value) for S. aureus with each protein derived from the mathematical model. (Error bars represent 95% confidence intervals.)
Table 1: A summary of the effect of mucin, vitronectin and lubricin on the growth curve parameters of S. aureus.
Mucin Vitronectin Lubricin
Lag Time (λ) ↑ ↑ ↑
Maximum specific
growth rate (μm)
↓ ↑ ↓
Asymptote of growth ↑ ↓ ~
1.4.1.2 S. epidermidis
The polystyrene bacterial trials showed that treatment with 200 μg/mL LUB resulted in a
retardation of the S. epidermidis growth curve when compared with the control (Figure 4B).
When compared to PBS coated samples, p-values of less than 0.05 indicated that any observed
differences could be considered significant. Over the course of the experiment, the LUB and
BSM coated samples showed a significant decrease at approximately 6.2 hours, near the start
of the exponential growth phase, as indicated by the growth curve. Mathematical modeling
showed that treatment with LUB and BSM resulted in an increase in lag time, whereas
treatment with VTN resulted in a small decrease in lag time (Table 2, Figure 5A).
GEAII Dissertation Page 34 of 117
As seen in Figure 4B, between the period of 6.2 and 12.2 hours there was a significant
depression in the LUB growth curve versus the PBS growth curve (p<0.05). This indicated that
the LUB initially suppressed attachment and retarded the initial proliferation of S. epidermidis.
The crystal violet trials showed that while the LUB resulted in a minor reduction in biofilm
production, the BSM coated samples showed a 15.5% reduction in adherent biofilms when
compared to the PBS coated samples (Figure 4A). The mathematical model showed a 36%
(105.7 minute) increase in lag time (Figure 5A), no significant difference in the asymptote
(Figure 5B), and a slight increase in maximum specific growth rate (Figure 5C).
VTN did not appear to significantly decrease S. epidermidis proliferation (Figure 4D).
From the mathematical model, no significant difference in the lag time was observed between
the VTN coated samples and the PBS coated samples. However, there was a 3% decrease in
asymptote (Figure 5B), and a significant decrease in maximum specific growth rate between the
VTN coated samples and the PBS coated samples (Figure 5C).
In the case of BSM, there was a significant reduction in bacterial proliferation between
6.3 and 9.6 hours (p<0.05) (Figure 4C). While some disruption of the exponential growth phase
was observed there was no prolonged bacteriostatic effect of BSM, indicating a significant S.
epidermidis reduction once the stationary phase was reached. The mathematical model showed
a 30% increase in lag time (Figure 5A), no significant reduction in asymptote (Figure 5B), and no
significant reduction in maximum specific growth rate (Figure 5C).
GEAII Dissertation Page 35 of 117
Figure 4: A.) Crystal violet results after 24hr S. epidermidis surface adhesion and proliferation trial. BSM treated samples showed significant reduction in biofilm formation. Approximately 15.5% less biofilm was measured on BSM coated samples vs. PBS treated samples. B.) S. epidermidis Surface Adhesion and Proliferation with LUB (200μg/mL) over 24 hours determined by optical density readings. LUB treatment significantly suppressed bacterial from 6.2 to 12.3 hour time points (p<0.05) Data = mean +/- SEM; N = 4 C.) S. epidermidis Adhesion and Proliferation with BSM (200μg/mL) over 24 hours determined by optical density readings. Some significant reduction in proliferation seen between 6.3 and 9.6 hour time points Data = mean +/- SEM; N = 4 D.) S. epidermidis Surface Adhesion and Proliferation with VTN (50 μg/ml) over 24 hours determined by optical density readings. VTN did not appear to significantly decrease bacterial proliferation. Although t-test indicated a significance decrease starting at 21.7hrs this was not confirmed by the confidence interval calculations. Data = mean +/- SEM; N = 4 [*indicates areas of significant difference between protein treated trials and PBS treated trials] as determined by p<0.05] [♦ indicates areas where using a confidence interval of 95% a significance reduction was seen between protein treated trials and PBS treated trials]
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GEAII Dissertation Page 36 of 117
Figure 5: A.) Lag times for S. epidermidis with each protein derived from the mathematical model. B.) Optical density ratio (asymptote value/initial value) for S. epidermidis with each protein derived from the mathematical model. C.) Maximum specific growth rate for S. epidermidis with each protein derived from the mathematical model. (Error bars represent 95% confidence interval.) GEAII Dissertation Page 37 of 117
Table 2: Summary of the effects of mucin, vitronectin and lubricin on the growth curve parameters of S. epidermidis.
Mucin Vitronectin Lubricin
Lag Time (λ) ↑ ↓ ↑
Maximum specific
growth rate (μm)
↑ ↓ ↑
Asymptote of growth ↑ ↓ ~
1.4.1.2.1 Modeling
The modified Gompertz and Logistic models both fit the bacterial growth data very
closely (Figure 6, Figure 7). The modified Gompertz model had a slightly better fit for 8 out of 10
data sets based on the R-square and Sum of squared errors (SSE). Therefore, we used the
Gompertz model to compare the growth parameters between different protein treatments of
each bacterium. A summary of results derived from the models can be found in Table 1 and
Table 2.
GEAII Dissertation Page 38 of 117
Figure 6: A) S. aureus + PBS growth data (blue) fitted to modified Logistic and Gompertz growth curves (red). B) S. aureus + 200 µg/mL BSM growth data (blue) fitted to modified Logistic and Gompertz growth curves (red). C) S. aureus + 50 µg/mL VTN growth data (blue) fitted to modified Logistic and Gompertz growth curves (red). D) S. aureus + 200 µg/mL LUB growth data (blue) fitted to modified Logistic and Gompertz growth curves (red). (95% confidence interval for the models is shown by the dashed red lines.)
GEAII Dissertation Page 39 of 117
Figure 7: A) S. epidermidis + PBS growth data (blue) fitted to modified Logistic and Gompertz growth curves (red). B) S. epidermidis + BSM growth data (blue) fitted to modified Logistic and Gompertz growth curves (red). C) S. epidermidis + VTN growth data (blue) fitted to modified Logistic and Gompertz growth curves (red). D) S. epidermidis + LUB growth data (blue) fitted to modified Logistic and Gompertz growth curves (red). 95% confidence interval for the models is shown by the dashed red lines.
GEAII Dissertation Page 40 of 117
1.4.1.2.2 Surface Energy
In our trials, all samples that were soaked in PBS showed rapid and complete wetting
and the initial contact angle could not be measured with our equipment. Since LUB was
dissolved in PBS, all the tissue culture polystyrene samples soaked in LUB solutions showed
complete wetting. Table 3 shows the contact angle measurements for uncoated tissue culture
treated polystyrene.
Table 3: Contact angle results for uncoated tissue culture polystyrene. Coated polystyrene samples showed complete wetting and thus are not displayed.
Blank/Untreated PS Contact Angle STD Error
Water 55.0 1.37 Ethylene Glycol 32.4 0.20
Glycerol 58.8 0.38
1.5 Discussion
1.5.1 Lubricin
This set of studies showed, for the first time, that lubricin significantly reduced the initial
attachment and proliferation of bacteria. The most striking change was the significant increase
in the length of the lag phase (36% for S. epidermidis and 27% for S. aureus) indicating that
lubricin made it more difficult for the bacteria to adapt to the surface and begin to grow.
1.5.2 Mucin
As previously mentioned, LUB has a central mucin-like domain. Mucins are large
glycoproteins that are the major components in the mucus that cover the luminal surfaces of
epithelial organs (55). Additionally, mucins are found in mucus which forms a physical barrier
between plasma membranes and the extracellular environment (55). Their structure can be
GEAII Dissertation Page 41 of 117
described as a threadlike peptide backbone, with densely packed carbohydrate side chains (55).
Mucins have unique properties as surfactants. They adsorb to hydrophobic surfaces via surface-
protein interactions while clinging to water molecules through their hydrophilic oligosaccharide
clusters (Figure 1) (55).
Secretory mucins typically have a very high molecular mass and contain hundreds of O-
linked saccharide side chains, constituting between 50%-90% of their molecular weight (35, 56).
The oligosaccharide side chains are about 5–15 monomers long and are O-linked to the serine
and threonine residues of the protein core with a sufficiently high density that steric
interactions force them to stretch away from the central protein core in a “bottlebrush”
configuration (35). These oligosaccharide side chains are most commonly composed of N-acetyl
glucosamine, N-acetyl galactosamine, galactose, fucose, and sialic acid, and other carbohydrate
residues have also been reported (35). Sialic acid residues are negatively charged and work in
concert with sulfate groups to give mucins a net negative charge (35). Due to their hydrophilic
(glycosylated) and hydrophobic (unglycosylated) domains (i.e., an amphiphilic, block-copolymer
structure), mucins are able to adhere strongly to a wide range of surfaces by hydrogen bonding,
hydrophobic interactions, and by electrostatic interactions (35). Mucins can vary in size and
length due to differing numbers of tandem repeats in the central domain. Even with the same
type of mucin, the number of tandem repeats can differ from individual to individual (57). The
hydroxyl groups of the serines and threonines in the tandem repeats, as well as of those in the
flanking domains, are the sites of O-glycosylation.
Work by Shi et al. indicated that mucin surface coatings could decrease bacterial
adhesion of Staphylococcus aureus and Staphylococcus epidermidis by reducing the GEAII Dissertation Page 42 of 117
hydrophobic binding forces and by providing a barrier which prevents the bacteria from making
close contact with the surfaces (55). That study showed that when a 1 mg/mL bovine
submaxillary mucin solution was used to coat poly (methyl methacrylate) and polystyrene over
a 90% reduction in bacterial adhesion was seen when compared to the uncoated control (55).
The literature indicates their negatively charged glycosylated regions may be responsible for
this effect, and noted a relationship between higher mucin concentration on the surface, lower
hydrophobicity, and lower bacterial adhesion (55). However, this study did not investigate the
effects this protein had on bacterial adhesion on the surface past 3 hours, nor did it examine
the effect this protein coating had on the growth curve of the bacteria. This study showed 24
hour kinetic data of bacterial growth on mucin-coated surfaces.
1.5.3 Vitronectin
As stated previously there is approximately 60% sequence similarity between LUB and
vitronectin (36). Vitronectin is a glycoprotein found in the blood and extracellular matrix (58).
Vitronectin is a known ‘serum spreading factor' and inhibitor of the membrane attack complex
of the complement response (58). The molecular weight of human vitronectin is 75 kDa. It
contains three glycosylation sites and its carbohydrate moiety contributes to approximately
30% of its molecular mass (58). Vitronectin is also a multifunctional protein, but it is highly
homologous to LUB. The main difference between vitronectin and LUB is that vitronectin lacks a
mucin-like central domain.
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1.6 Bacteria
1.6.1 Surface Interactions
Hydrophobic adsorption has been shown to be an important mechanism in the physical
interactions of Staphylococcus aureus (S. aureus) (55). Previous studies have shown that
bacteria bear negative charges on their surfaces (55) and that S. aureus have a high relative
surface charge and high hydrophobicity (55). Strong interactions take place between the
membrane lipids of bacteria and hydrophobic surfaces (55). L. Shi et al. state that the degree of
hydrophobicity will determine how well bacteria will adhere to a surface and how extensively
they will proliferate (55). Thus, it follows that reducing the hydrophobicity of a surface could
result in surfaces that are less prone to bacterial adhesion.
In order to adhere to a surface, bacteria need to establish firm interactions to prevent
their rapid elimination by physicochemical mechanisms (26). Bacterial adherence may be
nonspecifically mediated by physicochemical forces, such as hydrophobic interactions (26).
Disruptions of these hydrophobic interactions may prevent bacterial binding and lead to a
prevention of bacterial colonization. If the bacteria are not able to colonize a surface and form a
biofilm, it will be much easier for the immune system to clear these bacteria before they cause
an infection. This study shows that LUB inhibits bacterial colonization of surfaces by blocking
bacteria surface interaction. This study proposes that the negative charge of LUB's central
mucin domain repels the bacteria and that the LUB protein coating of the blocked other surface
proteins from interacting with the substrate, further preventing the cells from adhering. Future
studies will be needed would be needed to confirm this mechanism of repulsion.
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1.7 Conclusions
Trials with LUB on polystyrene show that at a coating with a working concentration of
200 μg/mL, LUB is able to reduce S. aureus adhesion and growth by approximately 13.9% over
24 hours and that with a coating concentration of 50 μg/mL VTN was able to reduce bacterial
adhesion and proliferation by approximately 11% over 24 hours. Trials with S. epidermidis
showed that LUB and BSM were able to significantly retard the growth process and delay the
start of the exponential growth phases as well. BSM trials indicated that the mucin domain of
LUB may provide a mechanism of LUB’s bacteriostatic nature. Additionally, the crystal violet
trials indicate the mucin domain may play a major role in the reduction of adhesive bacterial
biofilm.
Mathematical modeling showed that lubricin significantly increased the length of the lag
phase of Staphylococcus aureus by 27% and Staphylococcus epidermidis by 36% (Table 1, Table
2). The lag phase is the period in which the bacteria are adapting to their environment and not
dividing. In this phase they are preparing for exponential growth. The results suggest that LUB
makes it more difficult for bacteria to adapt to their environment, and as a result it takes longer
for the bacteria to enter the phase of exponential growth. We hypothesize that this is
significant because a longer lag phase will allow more time for the immune system to recognize
and clear bacteria either before they start to divide or in the early stages of the exponential
growth phase. It will also allow more time for certain antimicrobials to work. Since our study
used far greater concentrations of bacteria than are normally found in a physiological
environment, we hypothesize that these effects would much greater in vivo.
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In summary, because of LUB's ability to reduce bacterial attachment and proliferation
using only a low concentration of protein and a very simple drying technique for surface
coating, LUB shows promise as an anti-biofouling agent and bacteriostatic coating. Thus, LUB
should be further studied as a means to prevent bacterial infections without risking the
development of additional drug resistant bacteria strains.
1.8 Acknowledgements
Special thanks to Evan Smith, Ling Zang, Kimberly Waller, Jara Crear, Justin Seil, and Megan
Creighton for aid in experimental design and data analysis. Additionally, the authors would like
to thank the Hermann Foundation, NIGMS award number R25GM083270 and R25GM083270-
S1, and the NSF GK-12 fellowship award number 0638688 for funding.
1.9 References
1. S. D. Puckett, E. Taylor, T. Raimondo, T. J. Webster, The relationship between the nanostructure of titanium surfaces and bacterial attachment. Biomaterials 31, 706 (2010).
26. J. Kluytmans, A. van Belkum, H. Verbrugh, Nasal carriage of Staphylococcus aureus: epidemiology, underlying mechanisms, and associated risks. Clin. Microbiol. Rev. 10, 505 (July 1, 1997, 1997).
32. D. P. Chang, N. I. Abu-Lail, F. Guilak, G. D. Jay, S. Zauscher, Conformational Mechanics, Adsorption, and Normal Force Interactions of Lubricin and Hyaluronic Acid on Model Surfaces†Langmuir 24, 1183 (01/09, 2008).
33. B. Zappone, M. Ruths, G. W. Greene, G. D. Jay, J. N. Israelachvili, Adsorption, Lubrication, and Wear of Lubricin on Model Surfaces: Polymer Brush-Like Behavior of a Glycoprotein. Biophysical Journal 92, 1693 (2007).
35. J. M. Coles, D. P. Chang, S. Zauscher, Molecular mechanisms of aqueous boundary lubrication by mucinous glycoproteins. Current Opinion in Colloid & Interface Science 15, 406 (2010).
36. D. K. Rhee et al., The secreted glycoprotein lubricin protects cartilage surfaces and inhibits synovial cell overgrowth. The Journal of Clinical Investigation 115, 622 (03/01, 2005).
GEAII Dissertation Page 46 of 117
38. B. Zappone, G. W. Greene, E. Oroudjev, G. D. Jay, J. N. Israelachvili, Molecular Aspects of Boundary Lubrication by Human Lubricin: Effect of Disulfide Bonds and Enzymatic Digestion†. Langmuir 24, 1495 (2008/02/01, 2007).
48. G. D. Jay, J. R. Torres, M. L. Warman, M. C. Laderer, K. S. Breuer, The role of lubricin in the mechanical behavior of synovial fluid. Proc. Natl. Acad. Sci. U. S. A. 104, 6194 (April, 2007).
49. A. R. C. Jones et al., Binding and localization of recombinant lubricin to articular cartilage surfaces. Journal of Orthopaedic Research 25, 283 (2007).
50. G. D. Jay, K. Haberstroh, C.-J. Cha, Comparison of the boundary-lubricating ability of bovine synovial fluid, lubricin, and Healon. Journal of Biomedical Materials Research 40, 414 (1998).
51. G. D. Jay, B.-S. Hong, Characterization of a bovine synovial fluid lubricating factor. II. Comparison with purified ocular and salivary mucin. Connective Tissue Research 28, 89 (1992).
52. M. L. Shuler, F. Kargi, Bioprocess Engineering: Basic Concepts (Second Edition). (Prentice Hall PTR, Upper Saddle River, NJ, 2002).
53. M. H. Zwietering, I. Jongenburger, F. M. Rombouts, K. Vantriet, Modeling of the Bacterial-Growth Curve. Applied and Environmental Microbiology 56, 1875 (Jun, 1990).
54. S. D. Puckett, P. P. Lee, D. M. Ciombor, R. K. Aaron, T. J. Webster, Nanotextured titanium surfaces for enhancing skin growth on transcutaneous osseointegrated devices. Acta Biomaterialia 6, 2352 (2009).
55. L. Shi, R. Ardehali, K. D. Caldwell, P. Valint, Mucin coating on polymeric material surfaces to suppress bacterial adhesion. Colloids and Surfaces B: Biointerfaces 17, 229 (2000).
56. W. Jiang, J. Woitach, R. Keil, V. Bhavanandan, Bovine submaxillary mucin contains multiple domains and tandemly repeated non-identical sequences. Biochemical journal 331, 193 (1998).
57. W. P. Jiang, D. Gupta, D. Gallagher, S. Davis, V. P. Bhavanandan, The central domain of bovine submaxillary mucin consists of over 50 tandem repeats of 329 amino acids - Chromosomal localization of the BSM1 gene and relations to ovine and porcine counterparts. Eur. J. Biochem. 267, 2208 (Apr, 2000).
58. I. Schvartz, D. Seger, S. Shaltiel, Vitronectin. The International Journal of Biochemistry & Cell Biology 31, 539 (1999).
GEAII Dissertation Page 47 of 117
2 Chapter 2: Lubricin Reduction of Biofilm Formation Under
Flow as Detected by SPRi
2.1 SPRi Background
Surface plasmon resonance imaging (SPRi) is a powerful tool that allows real time
measurements of biological adhesion events on the surface of gold-coated glass prisms.
Historically this tool has been used to examine protein binding and kinetic data; however, this
study investigated using this technology to understand biofilm formation underflow.
2.2 Biofilm Formation
Once bacteria adhere to a substrate they can form a biofilm, a complex polysaccharide
aggregate that protect them from harsh environmental conditions and can result in antibiotic
resistance (Figure 8) (20, 23)).
Figure 8: Bacterial biofilm formation (adapted from Otto et. al (20))
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A biofilm is a collection of matrix-enclosed bacterial populations, adherent to each other
and/or surfaces or interfaces (22). It is very difficult for antibiotics to penetrate the
polysaccharide slime layers of the biofilm and thus many antibiotics are unable to treat the
resulting persistent infection (24). The best method to prevent biofilm formation is to prevent
the initial attachment of bacteria to the surface
2.3 SPRi
Surface plasmon resonance imaging (SPRi) provides label-free quantitative and
qualitative information about events occurring on and near the surface (~200nm) over a
relatively large area (~1cm2) (59, 60). SPRi instruments operate by shining light through a prism
onto a gold surface and detecting the intensity of the reflected light that exits the prism using a
charge coupled device (CCD) camera (60). Light of a certain wavelength is projected on the
metal surface through the glass prism, the light is then converted to surface plasmon polaritons
(SPPs). If a SPP is not created the light is reflected back and is detected using a charge coupled
device (CCD). If the refractive index changes at the interface at the surface of the metal coating
(due to any attachment to the surface), then SPPs will not be generated at those locations, the
light will be reflected back to the CCD surface and bright spots will appear on the image (60).
The work by the Goluch lab group at Northeastern University has focused on using this
technology to get real time images and quantitative data about biofilm growth on a surface.
2.4 Hypothesis
The hypothesis of this study is that coating the surface of the gold-coated prism used in
SPRi with lubricin will prevent biofilm formation under flow conditions.
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2.5 Materials and Methods
Surface plasmon resonance imaging (SPRi), performed in a HORIBA Scientific SPRi-Lab+
device (HORIBA Scientific, USA), provided label-free a quantitative and qualitative information
about events occurring on and near the surface (~200nm) over a relatively large area (~1cm2)
(59, 60). SPRi instruments operate by light transmission through a high refractive index glass
prism onto a gold surface, followed by subsequent detection events that manifest themselves
as changes in the intensity of reflected light that exits the prism, which is measured using a
charge coupled device (CCD) camera (60). Light of a certain wavelength is projected onto the
metal surface through the glass prism. At the surface interface the light is converted into
surface plasmon polaritons (SPPs). Attachment events on the surface of the metal result in
changes in the refractive index, resulting in the loss of SPP generation at the specific
attachment site. Thus, if there is an attachment at the surface of the metal, the light will be
reflected back and detected by a CCD (charge coupled device) camera and bright spots will
appear on the image (60).
A glass prism was purchased with a 50nm layer of bare gold on top (SPRi-Biochip,
HORIBA Scientific, USA). Polydimethylsiloxane (PDMS) (Sylgard 184 PDMS kit, Dow Chemical,
USA) was used to create a 300 µL growth chamber over the surface of the prism. Prior to trials,
the prism and flow chamber were cleaned and sterilized using 70% EtOH and rinsed with
deionized water. Once the flow chamber and prism were cleaned and aligned, approximately
5µL of each protein solution were streaked onto the growth chamber and allowed to dry for 3
hours at room temperature under sterile conditions. For these trials, both LUB and BSM were
used at a concentration of 200µg/mL. Once dry, the growth chamber was seeded with 300 µL of
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S. aureus using 6mL of Difco Luria Bertani Broth (LB) Growth Media (Miller) inoculated with S.
aureus incubated for 18 hours at 37°C and then diluted 1:100 in LB, yielding approximately
5*106 bacteria/ml. Thereafter, the growth chamber was inserted into the SPRI device and a
flow rate of LB growth media was maintained at 10 µl/minute (Figure 9). The trial was run for
one day, and the Horiba SPRi apparatus was set to take measurements and images every 3
minutes. The changes in reflectivity of the surface indicated the adherence of bacteria and
growth of a biofilm on the surface(59, 60). The numerical values were paired with the visual
images obtained during the trials to measure the relative quantity of biofilm formed on the gold
surface of the prism in the coated and uncoated regions.
2.5.1 Protein Preparation
Lubricin (LUB) and bovine submaxillary mucin (BSM) were used at a concentration of
200 μg/mL. Prior to use, BSM and VTN solutions were filtered through low protein binding
sterile filters with a pore size of 0.2 μm (Corning).
2.5.2 Statistical Analysis
Error in these trials was calculated through the use of standard error of means.
Statistical analysis of numerical data in this study was completed using an unpaired t-test
assuming unequal variances. Statistical significance was considered at p<0.05.
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Figure 9: A. SPRi schematic: growth chamber was treated with protein coatings, then seeded with bacteria. Fresh sterile LB growth media was pumped through the growth chamber. Light was projected through glass onto the gold-coated surface. A charge coupled device (CCD) camera was used to detect shifts in the intensity of light that exited the prism. B. Bacterial Growth Chamber: Lubricin and bovine submaxillary mucin were streaked in the bacteria growth chamber prior to bacterial seeding.
Bare Gold
Lubricin
Mucin
Bacteria Growth Chamber
A.
B.
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2.6 SPRi Results
Lubricin coated areas of the gold surface were almost completely devoid of biofilm production,
while mucin-coated areas initially resisted biofilm production (Figure 10). At the end of the
trials, lubricin showed over a 90% clearance when compared to bare gold, while mucin only
resulted in approximately 7% reduction in biofilm adherence and proliferation (Figure 11).
Figure 10: SPRi images taken at time 0, 6, 12, 18, and 24 hr time points: Lubricin coatings resulted in near complete blockage of biofilm adherence on the surface.
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Figure 11: SPRi change in reflectivity at trial endpoint: Lubricin showed over 90% reduction of bacterial biofilm when compared to bare gold. Mucin only resulted in approximately 7% reduction. (data=mean +SEM; N=3; *indicates a significant difference between lubricin coated areas and both bare gold and mucin as determined by p<0.05)
2.7 Discussion
These trials showed simply drying lubricin onto a surface may be enough to stop over
90% biofilm production under flow conditions. Although mucin showed some initial blockage of
biofilm formation, the effects could not be considered significant after 24 hours. This study
shows that lubricin may be able to act as a quick and effective method to coat medical implants
and used in flow systems such as hemodialysis devices to prevent the accumulation of biofilm
and reduce the chances of related blood born infections.
2.8 References
20. M. Otto, Staphylococcus epidermidis - the 'accidental' pathogen. Nat. Rev. Microbiol. 7, 555 (Aug, 2009).
22. J. W. Costerton, Z. Lewandowski, D. E. Caldwell, D. R. Korber, H. M. Lappinscott, Microbial Biofilms. Annu. Rev. Microbiol. 49, 711 (1995).
0
2
4
6
8
10
12
14
16
18
Lubricin Bare Gold Mucin
Chan
ge in
Ref
lect
ivity
*
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23. L. Hall-Stoodley, J. W. Costerton, P. Stoodley, Bacterial biofilms: From the natural environment to infectious diseases. Nat. Rev. Microbiol. 2, 95 (Feb, 2004).
24. R. M. Donlan, Role of Biofilms in Antimicrobial Resistance. ASAIO Journal 46, S47 (2000).
59. P. N. Abadian, N. Tandogan, J. J. Jamieson, E. D. Goluch, Using Surface Plasmon Resonance Imaging (SPRi) to Study Bacterial Biofilms. 8, 021804 (2014).
60. E. Goluch, P. Abadian, G. E. Aninwene II, T. J. Webster. (USA, 2013).
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3 Chapter 3: Lubricin Protein Coating to Reduce the Adhesion
of Fibroblasts on the Surfaces of Medical Devices
3.1 Lubricin Protein Coating to Reduce the Adhesion of Fibroblasts on the
Surfaces of Medical Devices: Abstract
Excessive postsurgical cellular or tissue adhesions on the surface of medical implants or
surgical site can lead to major post surgical complications (61-64). This study showed that
lubricin, an anti-adhesive protein found in the synovial fluid, resulted in surfaces that were non-
adherent to fibroblasts. Similar results were found for bovine submaxillary mucin (BSM), a
protein similar to the central sub-region of lubricin. However, lubricin showed much greater
efficacy than BSM.
3.2 Background
Cellular adhesion can result in major post operative complications. For example, one
major complication following cataract surgery is posterior capsular opacification of the
implanted intraocular lenses (IOLs) due to the adhesion, migration, collagen deposition, and
lens fiber generation of lens epithelial cells that remain in the surgery site after the natural lens
is removed (64). The result is that additional procedures must be performed to remove the
obstructions from the viewing frame of the lens. According to a study by Awasthi et al. the only
effective procedure to treat this posterior capsular opacification is using a Nd:YAG laser
capsulotomy, to clear the visual axis (64). This procedure represents a significant cost burden to
national healthcare systems and can result in retinal detachment, iris hemorrhage, corneal
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edema, extreme dislocation or damage of the IOL, and exacerbation of localized
endophthalmitis (infection of the inner eye) (64).
In addition to excess cellular adhesion, tissue adhesions are a major cause of failure of
surgical therapies (65). These tissue adhesions can result in a wide range of clinical issues for
the patient, from abdominal discomfort to complete intestinal blockage (65). These adhesions
also make revision surgery more time consuming, and increase the risk to the patient. One
study estimated that the economic burden of hospitalization for lower abdominal-pelvic
adhesiolysis in the united states (including hospital cost and surgeon's fees) was well over
$1179 million (65).
Thus, there is a need to control unwanted cellular adhesion and extracellular matrix
formation on numerous medical devices in a safe yet effective way that will not disrupt the
function of the medical device or damage surrounding healthy tissue. LUB forms natural
lubricating barrier layers within the body (36). Additionally, a study by Rhee et al. showed that
LUB was able to prevent the adhesion of synoviocytes on tissue culture plates.
3.3 Hypothesis
The hypothesis of this study is that LUB could be used as a simple medical device coating
to prevent fibroblast adhesion and growth, without adversely affecting the overall health of the
unattached cells which could lead to significant toxicity.
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3.4 Materials and Methods
3.4.1 Protein Preparation
The LUB used in these trials was extracted under sterile conditions from bovine synovial
fluid obtained from Pel-Freez (Arkansas, USA) (50). The sterile extraction procedure was
described fully by Jay et al., but in short it involves extraction of LUB from bovine synovial fluid
through a hyaluronate digestion, followed by anion exchange chromatography, and affinity
chromatography to purify and concentrate the protein (50). LUB and bovine submaxillary mucin
(BSM) (Sigma-Aldrich) were used at a concentration of 200 μg/mL. Prior to use, BSM solutions
were filtered through low protein binding sterile filters with a pore size of 0.2 μm (Corning).
Sterile phosphate buffered saline (PBS) was used in the dilution process for both LUB and BSM.
3.4.2 Cell Adhesion and proliferation
24 well polystyrene plates (Fisher) were coated with 125 μL of LUB, BSM, or PBS
(controls) and plates were rocked overnight at room temperature to ensure uniform coating.
Fibroblast cells were chosen for this study due to their ubiquitous nature in the body, relatively
high growth rate, and strong adhesive properties. Human fibroblast cells (ATTC# CCL-110) were
grown in tissue culture flasks in Eagle's minimal essential medium (EMEM) containing 10% fetal
bovine serum (FBS) and 1% penicillin/streptomycin (all supplies from Gibco). Once fibroblasts
reached confluence on the tissue culture flasks, trypsin (Sigma) was used to suspend the cells.
Then, the cells were counted using a Beckman Coulter counter. The cells were diluted to 7*103
cells per mL and 1mL of the solution was added to each of the treated wells. The plates were
then incubated overnight in 5% CO2 at 37°C. Phase contrast microscope images were taken at
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5X and 10X after 1 day. On day 2, the plates were rinsed with PBS and fresh media was added
prior to a second set of images being taken. Additionally, to determine the toxicity of the LUB
and BSM solutions, at that time, non-adherent fibroblasts were re-seeded onto uncoated tissue
culture polystyrene plates and after an additional day of culture, phase contrast images of
attached cells were once again acquired.
3.4.3 MTS Assay
To quantify fibroblast viability, an MTS kit (Sigma) was also used in the present study. For
this, 96-well tissue culture plates (Fisher) were treated with 50 µL of PBS, 0.2 mg/mL BSM, or
0.2 mg/mL LUB. Blank controls were also used. The coatings were allowed to dry overnight
similar to the procedure described in the previous section. Each well was then seeded with
80,000 cells/well and fibroblasts were cultured as described above for 1 and 2 days under
standard conditions. At the end of the prescribed time period, the wells were rinsed to remove
all of the non-adherent or loosely attached cells, then 30 µL of an MTS dye was added to each
well and the plate was incubated under standard conditions for an additional 4 hours.
Absorbance values were obtained through a spectrophotometer (Spectramax) at a wavelength
of 490 nm. Absorbance readings at different cell concentrations (2.5 × 103, 1 × 104, 4 × 104, 8 ×
104, and 1.15 × 105 cells/mL) were used to make a standard curve.
3.5 Results
3.5.1 Cell Adhesion and proliferation
After one day of incubation, fibroblast on untreated and PBS soaked tissue culture
plates showed healthy cell adhesion and spreading, while cells plated on mucin coated samples
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remained unattached and clumped up, and cells on LUB coated samples remained unattached
(Figure 12) .
Figure 12: Phase Contrast Images of Fibroblasts Grown on Tissue Culture Plates: A. Untreated, B. PBS Coated, C. Bovine Serum Mucin (BSM) Coated D. Lubricin (LUB) Coated.
On the second day the media in the wells was aspirated and the wells were rinsed once
with PBS, then fresh media was added. The trial untreated (this makes no sense) and PBS
coated wells continued to show standard cellular attachment and proliferation (Figure 13 A&B).
However, the cellular clumps from day one in the BSM wells deposit (this makes no sense) and
attach in thick "cellular mats" on the well surface (Figure 13C). In the case of LUB coated
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samples, the cells remained unattached and were almost completely removed with the
aspirated media (Figure 13D).
Figure 13: Day 2- Phase Images of Fibroblast Grown on Tissue Culture Plates- A. Untreated Well, B. PBS Coated Well, C.BSM Coated Well, D. LUB Coated Well
The aspirates from the second day trials were re-plated on fresh sterile uncoated plates.
The aspirate from the uncoated and PBS coated wells behaved in a similar manner; there was
very little adhesion and the wells were filled with mostly dead floating cells (Figure 14A&B). The
cells from the BSM coated wells showed some clumping and adhesion, but were mostly non-
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adherent and dead (Figure 14C). The wells containing aspirates from the LUB coated plates
showed a great deal of adhesion and spreading (Figure 14D).
Figure 14: Day 3- Phase Images of Fibroblast Aspirated from Day 2 of – A. Untreated, B. PBS Coated Tissue, C.BSM Coated, D. LUB Coated
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3.5.2 MTS
Figure 15: Decreased fibroblast density on bovine serum mucin (BSM) and lubricin (LUB) coated tissue culture polystyrene as determined using the MTS assay. Original seeding density: 80,000 cells per sample. * p < 0.05 comparing day 2 to day 1 on the same sample. Both LUB and BSM coated wells experienced significantly lower (p < 0.05) cell density compared to PBS after 1 and 2 days of culture. ‡ p < 0.01 compared to PBS at the same day. ◊ p < 0.01 compared to BSM at the same day. Media (BSM) and Media (LUB) refer to media containing these respective proteins.
The MTS trials confirmed the qualitative results and showed that both LUB and BSM
significantly decreased fibroblast densities when compared to the PBS coated samples (Figure
15). In fact, compared to PBS coated controls, results demonstrated that LUB and BSM coated
tissue culture polystyrene decreased fibroblast density by approximately 10% and 30% after just
1 day and approximately 30%and 30%after just 2 days of culture, respectively. BSM inhibited
fibroblast density better than LUB after 1 day, however, both proteins inhibited fibroblast density
0
10000
20000
30000
40000
50000
60000
70000
80000
Blank PBS BSM LUB Media (BSM) Media (LUB)
Fibr
obla
st D
ensi
ty (c
ells
/mL)
Well Type
Day 1
Day 2
* * *
*
‡ ‡
‡
◊
◊
‡
◊
‡
‡
‡ ‡
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the same after 2 days of culture. Interestingly, adding BSM and LUB to the cell culture media
completely inhibited fibroblast growth on the tissue culture polystyrene after 1 day and both
inhibited fibroblast density approximately 30% compared to PBS controls after 2 days of culture.
3.6 Discussion
The results of these trials showed that lubricin acted as an effective non-toxic surface
treatment to reduce fibroblast adhesion. Lubricin coated surfaces completely blocked fibroblast
adhesion and proliferation, however the non-adherent fibroblast were still able to be re-plated
and grow in a healthy normal manner. The bovine submaxillary mucin acted as a less effective
surface treatment. While the BSM prevented the initial adhesion on the first day of growth, on
the second day, cells formed clumps and began to stick and proliferate on the surface. It is
possible the BSM on the surface degraded overtime, which resulted in the fibroblast being able
to attach to the surface on day two. The aspirated fibroblast from BSM wells could be viably
grown, but not as well as the fibroblast extracted from the lubricin wells. Overall, lubricin is the
superior treatment to prevent fibroblast adhesion. These trials indicated that once LUB adhered
to a surface, the interactions of other surface proteins were blocked and thus cellular
attachment was prevented without harming the cells. These trials also indicated that lubricin
may be a viable cellular anti-adhesive option for medical device surfaces.
3.7 References
36. D. K. Rhee et al., The secreted glycoprotein lubricin protects cartilage surfaces and inhibits synovial cell overgrowth. The Journal of Clinical Investigation 115, 622 (03/01, 2005).
GEAII Dissertation Page 64 of 117
50. G. D. Jay, K. Haberstroh, C.-J. Cha, Comparison of the boundary-lubricating ability of bovine synovial fluid, lubricin, and Healon. Journal of Biomedical Materials Research 40, 414 (1998).
61. M. F. Cordeiro et al., Modulating conjunctival wound healing. Eye 14, 536 (2000).
62. A. H. DeCherney, G. S. diZerega, Clinical problem of intraperitoneal postsurgical adhesion formation following general surgery and the use of adhesion prevention barriers. Surg. Clin.-North Am. 77, 671 (Jun, 1997).
63. M. M. Binda, C. R. Molinas, P. R. Koninckx, Reactive oxygen species and adhesion formation: Clinical implications in adhesion prevention. Human Reproduction 18, 2503 (December 1, 2003, 2003).
64. G. S. W. B. J. Awasthi N, Posterior capsular opacification: A problem reduced but not yet eradicated. Arch. Ophthalmol. 127, 555 (2009).
65. A. H. DeCherney, G. S. diZerega, Clinical Problem of Intraperitoneal Postsurgical Adhesion Formation Following General Urgery and the use of Adhesion Prevention Barriers. Surg. Clin.-North Am. 77, 671 (1997).
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4 Chapter 4: Nano-materials and Nano-structures:
Breakthroughs in Medical Imaging
4.1 Abstract
The key to understanding many ailments in the human body is to be able to look inside
the body with extreme precision and resolution. As medical imaging techniques progress, the
need to perform invasive exploratory surgeries will decrease. The emerging research in
nanotechnology has shown that it is possible to create nano-particles that may be able to image
more areas in the body with better resolution, target tissues on-demand, and/or behave in a
more efficient manner than their macro-counterparts, offering clear images with a smaller
quantity of the material needed. The use of nanotechnology can bring brand new levels of
accuracy and clarity to the field of medical imaging. This review will delve into some of the
novel advances in nano-technology and how they can be potentially applied to the field of
medical imaging.
4.2 The need for nanomaterials in medical imaging
Nanomaterials offer powerful new tools that can be tuned to identify and label many
tissues or even distinct cell types within a single tissue to help in disease diagnosis and
treatment. For example, cancer remains a serious medical problem that presents many
challenges for detection and treatment. Cancers also can metastasize and form tumors in other
sites within the body. Nanoparticles can offer the opportunity to: i) detect cancer cells before
other techniques are able to ii) deliver directed therapeutics to the cancerous cell cluster
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before the formation of a major tumor, and iii) track the migration of mobile cancerous cells
before they have an opportunity to take hold and form a tumor in another part of the body (43,
66). Due to their size, these nanoparticles can be transported through cell membranes, remain
longer in the blood stream, and reach areas where micron-sized particles cannot (67). The
result is that nanoparticles can be finely tuned to enter and internally label living cells (67).
Nanoparticles may offer the opportunity to differentiate between various cell types and offer
highly tuned differential labeling of tissues by functionalizing them with specific cell-targeting
antibodies (68). This can help physicians clearly see the interactions between various tissues in
close proximity, and in the case of surgery, may help physicians determine the best locations to
make surgical implantations. Additionally, as a result of their increased surface area to volume
ratio, nanoparticles may also be used as part of polymer composites to render them more
visible to radiographs, clearer MRIs, or other non-invasive imaging techniques (69-71). Table 4
shows several nanomaterials with their possible biomedical imaging functions; each
nanomaterial is expanded on below.
GEAII Dissertation Page 67 of 117
Table 4: List of nanomaterials and potential biomedical applications (adapted from (71))
Material Applications Details
C60 Buckeyballs Imaging and tissue targeting
First nano-structure to be intentionally fabricated. Useful in imaging and can
be functionalized with various different contrasts agents. (72)
Iron Oxide Nanoparticles
Imaging and tracking by MRI
Useful as multifunctional particles. Can also be used to generate localized heat
to kill targeted tissue.
Carbon Nanotubes Bimolecular sensors
Useful in cell tracking, optical labeling, as an MRI contrast agent, and as an
electrocatalyst (e.g., glucose sensing).
Quantum Dots Imaging and tracking by
fluorescence
Exhibit broad emission spectra and can be coupled to antibodies, however
toxicity is a concern.
Gold Nanoparticles
Imaging and drug delivery
Excellent light scattering and absorbing capabilities. Can be used for
photothermal ablation of targeted tissues.
BaSO4 Nano Particles
Radiopaque polymer additive
Excellent for scattering or absorbing X-rays when embedded in other
materials. (73)
4.3 C60 Buckeyballs
Carbon nano-structured buckminsterfullerenes (fullerenes; structures of C60 or C80)
were the first nano-structure to be intentionally fabricated, resulting in the 1996 Nobel prize in
chemistry to Robert F. Curl Jr., Sir Harold W. Kroto and Richard E. Smalley "for their discovery of
GEAII Dissertation Page 68 of 117
fullerenes," and as made famous from images of the molecular structure likened to a soccer-
ball (72). Fullerenes are particularly attractive for applications in imaging because the carbon
surfaces can be functionalized with contrast agents (Iodine, 99mTc, Gd) and, thus, they have
found imaging applications in X-ray imaging, single photon emission computed tomography
(SPECT), and in MRI (with respective contrast agents from references: (74-76)).
Fullerenes (C60 or C80) can also be used to encapsulate metals, called an endohedral
metallofullerene, and the structure has been found useful for MRI imaging (77-80). The ability
to encapsulate a metal atom in the fullerene C60 is unique from other nanomaterials, this is
because the metal atom is caged within the carbon ball structure, and thus the surface is
pristine (from the perspective of surface chemistry; Figure 16) (77). Furthermore, fullerene C80
has been used to encapsulate multiple metal atoms due to a larger size than C60, through the
application of trimetallic nitride template (TNT) endohedral fullerenes (78-80). Fullerene C80
containing gadolinium and scandium ([Sc1Gd2N@C80Om(OH)n (m ≈ 12; n ≈ 26)] has been
shown to increase the MRI relativity time substantially (up to 20.7 mM-1 s-1) when compared
to standard clinically used gadolinium agents (Gd-DTPA, 3.2 mM-1 s-1), or as compared to C60
with encapsulation of Gd (4.6 mM-1 s-1) and also demonstrated better water solubility than
C60 (77-80). This enhanced relativity might be due to the larger area allowing the encapsulation
of multiple Gd or other metal atoms (78-80).
GEAII Dissertation Page 69 of 117
Figure 16: Ball-and-stick depiction of Gd@C60[C(COOH)2]10, illustrating a possible arrangement of 10 C(COOH)2 addends on a single C60 cage (light blue, C; red, O; white, H; dark blue, Gd) (adapted from (77))
The main problems stopping fullerenes from being used for medical imaging have been
water insolubility, aggregation, and localization to the respiratory system. The issues of
insolubility and aggregation have been overcome by modifying the fullerene surface using
carboxylate derivatization chemistry, or by functionalizing the surface with polyethylene glycol
(PEG), an agent commonly used to prevent particle agglomeration (77, 79, 80). Other studies
have examined creating different chemical moieties on the surfaces of fullerenes to enhance
water solubility, and found a highly soluble malonodiserinolamide group enhanced the water
solubility of C60 (C60-ser), are non-ionic, and are based on the biologically-stable serinolamide
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structures used to water solubilize commercially available X-ray contrast agents (such as
iohexol and iopamidol) which are also designed to be non-ionic and non-toxic (81). Later studies
with C60-ser determined that water-soluble fullerenes were localized in the nucleus of liver
cancer cells through the nuclear pore complex, indicating that these nanostructures may be
used as nanovectors for the delivery of diagnostic or therapeutic agents across cell membranes
(82). Furthermore, in a mouse model of liver cancer, the C60-serPF conjugate was detected in
most tissues, permeating through the altered vasculature of the tumor and the tightly
regulated blood brain barrier while evading the reticulo-endothelial system (RES) (82). Further
studies must be carried out to determine where fullerenes end up in the human body, what
quantity of fullerenes are necessary for imaging in humans, and if fullerenes that are able to
cross such biological barriers pose a threat to non-diseased tissues of the body.
Conjugation of fullerenes to biomolecules may offer a solution to the issue of tissue
specific targeting required for imaging applications. Conjugation of fullerenes to antibodies is a
promising strategy to target fullerenes, and has been achieved for fullerene
immunoconjugation to a murine anti-gp240 melanoma antibody, an antigen found on the
surface of >80% of human melanoma cell lines and biopsy specimens (83). In this study, water
soluble C60-ser were attached to the cross-linker, N-succinimidyl-3-(2-pyridyldithio) propionate
(or SPDP) (83). The fullerene attached to the antibody after addition of 2-iminothiolane, which
produced new disulfide bonds between the Fc fragment of the antibody and the SPDP cross-
linker group on C60 (83). Further studies will be required to determine if the C60
immunoconjugates localize at the targeted site of the body when used in imaging applications
(83). Fullerenes have also been attached to the surface of 30 nm viral nanoparticles (VNP) to
GEAII Dissertation Page 71 of 117
enhance water solubility using the concept of hydrophilic “chaperones” and to prevent
aggregation of fullerenes by site-specific bindings on the VNP surface (84). The site specific
attachment was enabled by using surface derivatized fullerenes and carbodiimide-N-
hydroxysuccinimide chemistry, resulting in linking of the fullerene to the lysine amino acid
groups on the surface of the virus, and allowing delivery to cancer cells (84). Future studies with
VNP and fullerenes also need to determine where they localize in the body, and if RES clearance
is still a concern.
4.4 Iron oxide nanoparticles
Nanoscale superparamagnetic iron oxide particles have been used as magnetic probes
for many biomedical applications (85). These nanoparticles may be composed of a magnetite
(Fe3O4) and/or a maghemite (γFe2O3) core coated in a hydrophobic coating, such as a monomer
coating, a polysaccharide coating, or a synthetic polymer coating (85). Due to their ability to
cause changes in the spin-relaxation times of neighboring water molecules, superparamagnetic
iron oxide nanoparticles are well suited as contrast agents in magnetic resonance imaging (MRI)
(42). MRI is a powerful noninvasive tool which can provide high image resolution as well as
detailed contrast of soft tissue allowing for the observation of tissue morphology and
anatomical details (86). Additionally, MRI can be used to image the entire body of a subject,
and is currently one of the primary tools in oncology imaging (86). Superparamagnetic iron
oxide nanoparticles allow for MRI techniques that can investigate down to a cellular, and even
molecular level (86). Yilmaz et al. have completed work using superparamagnetic iron oxide
particles as MRI agents for targeting macrophages to explore the visualization of myocardial
(peri-) infarct zones (87). Park et al. investigated the use of using superparamagnetic iron oxide
GEAII Dissertation Page 72 of 117
nanoparticles as a method to monitor cell migration and proliferation using noninvasive MRI
techniques (88). Additionally iron oxide nanoparticles can be used to monitor gene expression
or detect pathologies such as cancer, arthritis, atherosclerotic plaques, or brain inflammation
(42). Neuwelt et al. have completed work to show that dextran coated iron oxide nanoparticles
can be used to enhance MRI images of intracranial tumors, as well as stain these tissues to be
visualized through both light microscopy and electron microscopy (89). The results of the above
study indicated that due to their ability to breach the blood brain barrier, persist up to five
days, and be observed through various different imaging techniques, these coated iron oxide
nanoparticles are a better contrast option than the standard contrast agents such as
gadolinium (89).
A recent advance in imaging with iron oxide nanoparticles is the potential for dual
contrast imaging using ultrasound (along with MRI) and real time imaging with guided therapy.
Recent studies have found that nanoparticles targeted to specific tissues of the body with
magnets increase the contrast of that region in both ultrasound and MRI (90). For example,
several studies have shown that nanoparticles delivered to the brains of rats increased the
tissue contrast in both ultrasound and MRI, giving the prospects for guided surgery (90).
Current MRI contrast agents, such as gadolinium, did increase MRI contrast, but had no
diagnostic benefit during sonography, indicating new prospects for the use of SPIONs (or
superparamagnetic iron oxide nanoparticles) in neurosurgery (90). Moreover, studies with
SPIONs incorporated into microbubbles found that SPIONs enhanced the contrast in ultrasound
images compared to microbubbles without SPIONs in both tissue phantoms and rabbit livers,
GEAII Dissertation Page 73 of 117
while simultaneously reducing T2 and T2 weighted signals during MRI imaging (these studies
were conducted in vitro) (91).
More recent studies have combined SPIONs with micro-bubbles towards guided
therapy, such as poly(lactic-co-glycolic acid) (PLGA) micro-particles incorporating SPIONs, as
used for breast tumor imaging and ultrasound tumor ablation therapy (92). Similar studies used
SPIONs for combined ultrasound and MRI imaging to visualize tumors, while using ultrasound in
real time to guide ultrasound-triggered release (93, 94). One such study used acoustic droplets
to incorporate SPIONs, along with the anti-cancer drug doxorubicin (93). The acoustic droplets
were targeted using a combination of ultrasound mediated acoustic droplet vaporization and
magnetic-assisted targeting in tissue phantoms. Furthermore, tumor-targeting anti-VEGFR2
antibodies were conjugated to the phospholipid-based acoustic droplets for cancer cell-specific
chemotherapeutic targeting (93). Such combination methods show promise for using the most
advanced knowledge about SPIONs to comprehensively target, treat, and image tumors in real
time during tumor therapy (93, 94).
Finally, another recent advance for the combination of ultrasound and magnetic imaging
is using magneto-motive techniques during ultrasound imaging (95-98). In this method,
magnets are used to move tissues during ultrasound imaging to distinguish SPION laden tissues
from others in the body (95-98). A more recent advance uses phase-locked imaging along with
frequency tracking to create a clearer image without background movement caused by indirect
motion of areas outside of nanoparticle laden areas. These advances move towards in vivo
tissue imaging with targeted contrast agents, along with future potentials for non-invasive
magnetic manipulations of tissues inside of the body (96). GEAII Dissertation Page 74 of 117
While the iron oxide in the nanoparticles is unlikely to cause long term toxicity due to iron
concentration alone, these particles may produce reactive oxygen species (ROS) which could
negatively affect cell function (99). Studies have also shown that the toxicity of iron oxide
nanoparticles may also vary by cell type, this will have to be considered determining
appropriate delivery methods of these particles into patient tissues (99). Currently only
dextran-coated SPIONs are approved for human in vivo use by the Food and Drug
Administration (FDA) (99). However, care needs to be taken developing new coating techniques
for these particles to ensure that these coatings will not result in added cytotoxic effects.
4.5 Carbon nanotubes
Carbon nanotubes were first discovered in Japan by Sumio Iijima (100). As seen in Figure
17, carbon nanotubes can be described as rolled graphene sheets that are held together by van
der Waals interactions, and these strong interactions dictate the bundling of the carbon
nanotubes and their formation into large aggregates (70, 101). A single rolled layer of graphene
forms a single walled carbon nanotube with diameters that can range from 0.4-2nm (see Figure
2) (70). Additionally, these graphene layers can create coaxial cylinders, thus, forming multi-
walled carbon nanotubes with diameters which can vary from 2-100 nm (70). Typical carbon
nanotubes are a few microns in length and can have length to diameter aspect ratios of
approximately 1:1000 (70).
GEAII Dissertation Page 75 of 117
Figure 17: Conceptual diagram of (A) a single walled carbon nanotube ((102)) and (B) a multiwall carbon nanotube ((103)) along with typical lengths and dimensions (adapted from (101))
One of the key advantages of carbon nanotubes is that they can easily enter cells and
act as a delivery vehicles for various molecules relevant to therapy or diagnosis (104). Studies
have shown that carbon nanotubes have the ability to pass through the cell membrane of
cancer cells and localize around the nuclear membrane, thus, increasing the therapeutic effect
of any functionalized drugs and creating another possible labeling technique for cancerous cells
which can later be excised surgically (101). Also, once in the cell, the unique physical properties
of carbon nanotubes allow for efficient electromagnetic stimulation through the use of various
imaging methods, such as MRI (104). The large surface area and internal volume of carbon
nanotubes allows a variety of drugs and small molecules, such as contrast agents, to be loaded
onto the nanotube (104). Functionalizing molecules to these nanotubes make them powerful GEAII Dissertation Page 76 of 117
agents in both cancer imaging and cancer treatment using both systematic and localized
methods (104).
Ultrasound is a commonly used non-invasive method of visualizing organ systems and
tissues within the body. When investigating possible ultrasound contrast agents, several
factors need to be considered: ease of administration into the blood pool or cavity to be
examined, high stability, ease of elimination from the body, level of toxicity, and ability to
reflect ultrasonic waves (105). Functionalized carbon nanotubes have been shown to be easily
administrable through injection, highly stable in biological solutions, nontoxic, and able to be
eliminated from the body when appropriately functionalized (105). Functionalization of the
surface of carbon nanotubes is necessary to inhibit non-specific binding of biomolecules to the
hydrophobic nanotube surface, and to allow for ultrasensitive detection of biological species
(106). In their study, Delogu et al. used multiwalled carbon nanotubes that were oxidized and
subsequently functionalized by 1,3-dipolar cyclo-addition of azomethine ylides (105). Delogu et
al. found that these functionalized multiwalled nanotubes could be imaged in ultrasonography,
and displayed higher signals than graphene oxide, pristine multiwalled nanotubes, or
functionalized single walled nanotubes (105). Studies have also shown that these carbon
nanotubes can demonstrate near-infrared photoluminescence and can be used as multicolor
contrast agents in Raman imaging (105, 106). Additionally, work has been done to use single
wall carbon nanotubes conjugated with targeting peptides to enhance tissue targeting and
imaging using photoacoustic imaging techniques (105). Although the functionalized carbon
nanotubes used in the study by Delpgu et al. have been shown to be nontoxic, pristine water-
soluble carbon nanotubes have been found to be toxic under in vitro conditions (42, 105). As
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this technology develops, great care needs to be taken to ensure that these nanotubes can be
excreted or removed from the body once their period of usefulness has passed.
4.6 Quantum dots
Quantum dots are semi-conducting, light-emitting nano crystals (107). Generally
quantum dots contain a central core that is surrounded by an organic coating that protects the
core from physiological environments (see Figure 18) (108). This organic coating may have
proteins and oligonucleotides attached to the surface for targeting purposes (71). Quantum
dots are fluorescent nanoparticles of about 2-10 nm, their central cores are composed of atoms
that belong to elemental groups of II-VI (e.g., cadmium and selenium) or III-V (e.g., indium) (71).
Quantum dots are powerful imaging tools due to their size, superior signal brightness,
resistance to photo-bleaching, simultaneous excitation of multiple fluorescence colors, and
highly tunable light emission based on composition (107). Due to these features, different color
sets of quantum dots can be excited from a single light source with minimal spectral overlap
(107). Quantum dots are highly controllable; by altering the core size, core compositions, shell
compositions or surface coatings, one can vastly alter their optical properties (107).
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Figure 18: (A) Schematic illustration of the quantum dot structure (adapted from (108)) (B) Ten distinguishable emission colors of ZnS-capped CdSe QDs excited with a near-UV lamp. From left to right (blue to red), the emission maxima are located at 443, 473, 481, 500, 518, 543, 565, 587, 610, and 655 nm. (adapted from (109))
By altering core size and composition, one can create a customized quantum dot
emission profile with a specific maximum that can be tuned to almost any wavelength of the
electromagnetic spectrum, from the ultraviolet region to the near infrared region (450-850nm)
(71, 107). Alterations in the shell compositions and surface coatings can increase photo
luminescence without significantly affecting the emission range (107). Quantum dots have GEAII Dissertation Page 79 of 117
emission peaks which are substantially narrower than those of organic dyes, and thus more
quantum dot emissions are resolvable within the visible light spectrum than would be possible
with standard fluorophores (107). Therefore, the emission spectra from different quantum dots
can be easily distinguished from each other (see Figure 19) (42, 110).
Figure 19: (A) Size- and material- dependent emission spectra of several surfactant-coated semiconductor nanocrystals in a variety of sizes. The blue series represents different sizes of CdSe nanocrystals with diameters of 2.1, 2.4, 3.1, 3.6, and 4.6 nm (from right to left). The green series is InP nanocrystals with diameters of 3.0, 3.5, and 4.6 nm. The red series is InAs nanocrystals with diameters of 2.8, 3.6, 4.6, and 6.0 nm. (B) A true-color image of a series of silica-coated core (CdSe)-shell (ZnS or CdS) nanocrystal probes in aqueous buffer, all illuminated simultaneously with a handheld ultraviolet lamp. (adapted from (111))
Additionally, since quantum dots have a broader absorption spectra than organic dyes,
quantum dots can be excited over a broad range of wavelengths (71). The result is that one
could have highly tuned quantum dots target different cell types in a tissue network to allow a
GEAII Dissertation Page 80 of 117
physician to easily identify malignant tissues or cells by inspection and possibly remove them
without damaging the surrounding healthy cells. For example, Zdobnova et al. developed
methods of cell imaging where the tumor targeting antibody would either conjugate to the
cancer cell and attract the functionalized quantum dot to result in tissue labeling, or the tumor
targeting antibody could conjugate to the quantum dot and the complex would recognize and
stain cancerous cells (see Figure 20) (112).
Figure 20: Two approaches for tumor cells imaging using quantum dot-scFv antibody complexes based on barnase-barstar as discussed in Zdobnova et al. (adapted from (112)).
These quantum dots can also be conjugated with therapeutic agents and be used for both
cancer detection and treatment (107). Due to their semi-conductive nature, quantum dots can
also be used to label biological systems for detection by optical or electrical means (42).
Quantum dots have a great potential for long-term multicolor in vivo imaging (110). However
there is concern due to the potential toxicity of metals used in the core of quantum dots, such
GEAII Dissertation Page 81 of 117
as cadmium-selenium (110). Therefore, great care needs to be taken when designing these
quantum dots to make sure that any potentially toxic metals are completely sequestered from
physiological systems.
4.7 Gold Nanoparticles
Nanostructures made of gold have been employed as photoactive labels in a variety of
biosensing systems (113). Gold is a noble metal and like other noble metals gold nanoparticles
have strong size dependent optical properties and UV-visible extinction bands (113). Depending
on particle size, gold nanoparticles can appear red, blue, green, or brown (114). Because they
are noble metals, gold nanoparticles benefit from strongly enhanced plasmon resonance at
optical frequencies, as a result they are excellent for absorbing or scattering visible light (115).
Nanoscale gold has great potential as imaging and photothermal therapy agents in living
systems (114). Additionally, gold nanoparticles have applications as quenchers in fluorescence
resonance energy transfer studies (42). For example, gold nanoparticles can exhibit distance-
dependent optical properties. Because of this, they have been used to evaluate the binding of
DNA-conjugated gold nanoparticles to complimentary RNA sequences (42). Guan et al. have
developed a colorimetric method to determine melamine levels based on the aggregation of
chitosan-stabilized gold nanoparticles (116). While pure gold nanoparticles larger than roughly
5nm in diameter may have little or no toxic effects due to the inert nature of the element, the
various surface modifications which may be required to create functional nanoparticle
composites may result in potentially toxic particles (113).
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4.8 Nano BaSO4
The use of nanotechnology can also provide products that are already used in the field
of imaging to have a dual purpose. BaSO4 can absorb X-rays by either scattering or absorbing
electromagnetic waves (73). Thus, BaSO4 is a common agent incorporated into medical tubing
and implanted devices to provide radiopacity. Studies described later in this document have
been done to determine if changing the dimensions of the BaSO4 powder used in extruded
pellethane polymer emulsions into the nano regime, as opposed to micron particles, would be
able to reduce bacterial proliferation on the resulting plastic (69). Initial studies showed that
certain percentages of these nano particles in the polymer formulation resulted in materials
which were able to reduce bacterial proliferation in solution while still remaining radiopaque
(69). Additional studies using nano-BaSO4 as a possible bone cement additive showed greater
radiopacity than unmodified cements, while enhancing osteoblast (bone forming cell) growth
on the implant surface (117). Future directions of these studies should involve optimizing the
concentrations of these nano materials to enhance their antibiotic nature and retain
radiopaque qualities, without changing the tensile characteristics of the material.
4.9 Future Directions
As these technologies advance it is important to understand the possible toxicity of
these new nanomaterials. Once these materials are in the nanoscale their toxicity profiles may
be much different from their macro counterparts. Standard toxicity tests look for inflammation
or clear modification of cell function but nanomaterials may cause changes that are more
subtle (118). Pathogenic reactions from nanomaterials may include, but are not limited to,
interference in cell metabolism, alteration of cell signal transduction pathways, and/or subtle
GEAII Dissertation Page 83 of 117
changes to cellular biochemical functions which may not be readily apparent during short term
assays (118). As this field progresses, the need to investigate all aspects of toxicity before a
nanomaterial, before it is used in clinical practice, will only increase.
4.10 Conclusions
The use of nanomaterials in medical imaging will: open up new levels of early detection
of disease, allow for more accurate assessment of illness, better inform physicians so they can
design personalized treatments, allow for better monitoring of the efficacy of treatment, and
will improve understanding of tissue function even down to a cellular level (85). As this field
expands, more methods and techniques will be discovered which will not only fine tune current
methods of imagining, but will open up whole new methods to see within a patient without
opening them up. Through their special properties, nano materials are poised to become the
leading materials in medical imaging as well as medical treatment.
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105. L. G. Delogu et al., Functionalized multiwalled carbon nanotubes as ultrasound contrast agents. Proceedings of the National Academy of Sciences 109, 16612 (October 9, 2012, 2012).
106. Z. Liu, S. Tabakman, K. Welsher, H. Dai, Carbon nanotubes in biology and medicine: In vitro and in vivo detection, imaging and drug delivery. Nano Res. 2, 85 (2009/02/01, 2009).
107. P. Pericleous et al., Quantum dots hold promise for early cancer imaging and detection. International Journal of Cancer 131, 519 (2012).
108. Y. Zhang, T. H. Wang, Quantum Dot Enabled Molecular Sensing and Diagnostics. Theranostics 2, 631 (2012).
109. M. Han, X. Gao, J. Z. Su, S. Nie, Quantum-dot-tagged microbeads for multiplexed optical coding of biomolecules. Nat Biotech 19, 631 (2001).
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110. E. B. Voura, J. K. Jaiswal, H. Mattoussi, S. M. Simon, Tracking metastatic tumor cell extravasation with quantum dot nanocrystals and fluorescence emission-scanning microscopy. Nature Medicine 10, 993 (2004).
111. M. Bruchez, M. Moronne, P. Gin, S. Weiss, A. P. Alivisatos, Semiconductor Nanocrystals as Fluorescent Biological Labels. Science 281, 2013 (September 25, 1998, 1998).
112. T. A. Zdobnova, O. A. Stremovskiy, E. N. Lebedenko, S. M. Deyev, Self-Assembling Complexes of Quantum Dots and scFv Antibodies for Cancer Cell Targeting and Imaging. PLoS ONE 7, 1 (2012).
113. Y. Jin, X. Zhao, in Safety of Nanoparticles: From Manufacturing to Medical Applications, T. J. Webster, Ed. (Springer New York, 2009), pp. 19-31.
114. A. Alkilany, C. Murphy, Toxicity and cellular uptake of gold nanoparticles: what we have learned so far? J Nanopart Res 12, 2313 (2010/09/01, 2010).
115. P. K. Jain, K. S. Lee, I. H. El-Sayed, M. A. El-Sayed, Calculated Absorption and Scattering Properties of Gold Nanoparticles of Different Size, Shape, and Composition: Applications in Biological Imaging and Biomedicine. The Journal of Physical Chemistry B 110, 7238 (2006/04/01, 2006).
116. H. Guan, J. Yu, D. Chi, Label-free colorimetric sensing of melamine based on chitosan-stabilized gold nanoparticles probes. Food Control 32, 35 (2013).
117. R. Gillani, B. Ercan, A. Qiao, T. J. Webster, Nanofunctionalized zirconia and barium sulfate particles as bone cement additives. Int. J. Nanomed. 5, 1 (2010).
118. R. H. Hurt, M. Monthioux, A. Kane, Toxicology of carbon nanomaterials: Status, trends, and perspectives on the special issue. Carbon 44, 1028 (2006).
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5 Chapter 5: Nano-BaSO4: a Novel Antimicrobial Additive to
Pellethane
5.1 Introduction:
Each year, there are approximately 1.7 million hospital-acquired infections in the US,
90,000 of which are fatal (119). More than half of these infections can be attributed to
contamination of life-sustaining medical devices such as endotracheal tubes, bladder catheters,
and central venous catheters, as well as other medical implants (23). These infections can
prolong hospital stay, increase medical costs, and result in the death of the patient (120). In the
United States alone as many as 28,000 patients die each year due to catheter-related
bloodstream infections (12, 13). Each bloodstream infection can cost the health care system
more than US$35,000 per case, with a resulting total potential burden of US$35 billion (121).
Clearly, there is a great need to develop more efficient antimicrobial and bacteriostatic
products for the medical device community.
The main challenge is to prevent bacterial adhesion and proliferation before biofilm
production takes place. Once the bacterial biofilm matrix is formed the bacterial infection can
become profoundly more resistant to the host’s defenses as well as antibiotic treatment (23).
Corterton et al. defines a biofilm as a collection of matrix-enclosed bacterial populations
adherent to each other and/or surfaces or interfaces (22). Once bacteria develop a biofilm it is
very difficult for antibiotics to penetrate the polysaccharide slime layer and effectively kill the
bacteria (24). One study by Nichols et al. showed that Pseudomonas aeruginosa (common
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biofilm forming bacteria) was 15 times more susceptible to antibiotic treatment when
dispersed from a biofilm than when in a solid intact biofilm (25).
Currently silver is the primary antimicrobial active ingredient being developed into many
products; however, there remains a need for more cost effective solutions (Foster Corporation,
email communication, 2011). In addition, some studies cite the emergence of silver resistant
bacteria and provide indications of mammalian cell toxicity (45). In contrast, there has been an
emergence of studies which demonstrate slowed or stopped growth of bacteria on materials
with nanostructured surface features which alter surface energetics or use nanoscale surface
roughness to repel bacteria. (7) Additional studies have shown that the use of nanostructures
on surfaces can lead to surfaces that were antimicrobial or resistant to bacterial
proliferation.(7)
Barium sulfate (BaSO4) is a common agent used to make medical tubing radiopaque;
however, in addition to this, BaSO4 polymeric formulations have been shown to exhibit
antimicrobial activity (Foster Corporation, email communication, 2011). Prior studies with
nano-BaSO4 indicate that these partials are not toxic to mammalian cells (122). The goal of this
study is to investigate if nano-BaSO4 pellethane composites are able to effectively act as
antimicrobial surfaces—surfaces that prevent initial bacterial adhesion and proliferation while
remaining radio-opaque.
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5.2 Material and methods:
5.2.1 Sample preparation
Pellethane pellets were mixed with various weight percentages of BaSO4 powder (7
micron in diameter) or nano-BaSO4 (73 nm in diameter for nano-BaSO4) [sizes as reported by
Foster Corporation]. Seven different sample groups were made (0% BaSO4, 20% nano- BaSO4,
30% nano-BaSO4, 40% nano-BaSO4, 20% BaSO4, 30% BaSO4, and 40% BaSO4). These mixtures
were mixed, melted, and extruded into tapes. Tapes were then cut into disks that fit within the
12-well plate (approximately 22mm in diameter). These disks were then sterilized through
successive 5 minute soaks in 100% EtOH, 70% EtOH, and sterile DI water. DI water soaks were
done at least twice to ensure complete removal of EtOH. Samples were then exposed to UV
light for 1 hour and allowed to dry under sterile conditions. Dry samples were stored at room
temperature under sterile conditions.
5.2.2 Contact angle test
Contact angle measurements were made on a Krüss Easy Drop contact angle instrument
(Krüss, Germany) connected to an image analysis program (Drop Shape Analysis (Version 1.8)).
The Krüss Easy Drop apparatus was used to measure the contact angles that resulted when a
10μL drop of either H2O, glycerol, or ethylene glycol was placed on the surface of a sample disk
(123).
5.2.3 Radiopacity Trials
Polymer samples were labeled and x-ray using an infinity XMA HF-30AP set to Manual
technique mode with an exposure time of 0.016 seconds and [email protected] and 70KV. Images were
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taken of each sample individually and s-values (numeric values of exposure received by the
receptors in the digital system) were recorded (124). For analysis, the s-value for the 0% BaSO4
sample was used as a base and subtracted from all the other values to normalize the results.
5.2.4 AFM Surface Analysis
AFM images of polymer samples were obtained on an Agilent 5500 AFM/SPM
microscope under acoustic AC mode using Si probes operating at a resonant frequency of 154
kHz. All measurements were carried out at room temperature and acquired images had a
resolution of 512 x 512 pixels collected at a speed of 1 line/minute. Post image processing of
AFM images were done using Pico Image software provided with the instrument. The images
were subjected to standard image processing techniques that included line correction, form
removal, leveling and threshold adjusting. In all the AFM measurements, topography, phase
and amplitude images were obtained. For this study, only the topography images were
compared and presented.
5.2.5 Bacteria culture
Stock solutions of Staphylococcus aureus (S. aureus) (ATCC# 25923) and Pseudomonas
aeruginosa (Schroeter) Migula (P. aeruginosa) (ATCC# 27853) were obtained from the American
Type Culture Collection. Stock solutions were diluted and frozen. Bacteria from stock solutions
were streaked out for isolation on agar plates. Approximately 3 mL of sterile tryptic soy broth
(TSB) (Sigma Aldrich) were inoculated with one colony of desired bacteria, then incubated at
37°C while on a shaker set to 200 rpm. These solutions were incubated for 18 hours to reach
stationary phase and were then diluted to a density of 1×107 bacteria/mL (as estimated by the
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McFarland scale which corresponded to an optical density of 0.52 at 562 nm and was then
further diluted at a ratio of 1:100) (1).
5.2.6 Bacteria growth trials
All bacterial growth trials with polymer samples were done in 12 well tissue culture
plates. For trials where polymer samples were used, the sterile polymer disks were placed in
separate labeled wells of a 12 well tissue culture plate. They were then covered in 1 mL of the
bacteria solution. The plates were then sealed with parafilm and incubated at 37°C while
shaking at 200 rpm for 1.5 hours. Next, 10 μL samples of each solution were added to separate
990 μL of fresh sterile TSB in a micro-centrifuge tube, creating a 1:100 dilution. The tubes were
then vortexed and 100 μL were removed from each tube and added to 900 μL of sterile TSB in a
micro-centrifuge tube, creating a 1:1000 dilution. To quantify the colony forming units (CFUs), 5
separate 20 μL samples were plated in TSB-Agar plates, and allowed to incubate overnight. In
addition to experimental trials, a set of control growth trials were conducted where bacteria
grown under similar conditions to the experimental samples but without the polymer disk were
diluted and plated at 15 minute intervals to track the bacteria growth rate at room
temperature. The next day, the CFUs were counted and this count was used to calculate the
total number of CFUs in each solution.
5.3 Results:
5.3.1 Contact angle test
Trials showed that as the percentage of conventional BaSO4 increased there was
decrease in the H2O contact angle. Additionally the 20% BaSO4 nano was significantly more
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hydrophilic than the conventional 20% BaSO4. Other than the results at 20% there was no
significant predictable change in contact angle between the nano 20% BaSO4 and the
conventional 20% BaSO4. (Figure 21).
Figure 21: Contact Angle measurements on pellethane samples. Data = mean +/- SEM. (x-axis indicates weight percentages of BaSO4 or nano-BaSO4 in pellethane composites)
5.3.2 Radiopacity Trials
These trials indicate that the nano-BaSO4 samples were still able to make the samples
radiopaque. Figure 22 shows s-values for each one of the samples. The high s-value of the 40%
nano-BaSO4 sample may be due to nanoparticle agglomeration during the extrusion process
(See Figure 22 )
0.00
20.00
40.00
60.00
80.00
100.00
120.00
0% BaSO4
20% (nano) BaSO4
30% (nano) BaSO4
40% (nano) BaSO4
20% BaSO4
30% BaSO4
40% BaSO4
Angl
e (ϴ
)
Amount of BaSO4 in Pellethane
dH2O
Ethylene Glycol
Gycerol
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Figure 22: Sample Radiopacity s-values. 0% BaSO4 was used as a control, and the value for the control was subtracted for all the other experiential s-values. (x-axis indicates weight percentages of BaSO4 or nano-BaSO4 in pellethane composites)
5.3.3 AFM Surface analysis
AFM results, seen in Figure 23, show that the samples with the nano-BaSO4 resulted in
rougher surfaces with more nano features. However, the 40% nano-BaSO4 was extremely
challenging to image due to the tip of the imaging cantilever repeatedly getting stuck on the
sample. The RMS (root mean square height) values are a calculation of the standard deviation
of the height distribution, or surface roughness. The RMS values from this study indicated that
as the percentage of nano particles increased the surface roughness actually decreased. This
may be the result of the agglomeration of the nano particles into larger macro-particles as their
percentage in the polymer increased. Figure 24 shows a compilation of all the numerical data
collected from the AFM trials. Table 5 displays the descriptions of the height parameters as
defined by ISO 25178 (adapted from (125)). The results of Figure 23indicated that the 40%
0
10
20
30
40
50
60
70
0% BaSO4 20% (nano) BaSO4
30% (nano) BaSO4
40% (nano) BaSO4
20% BaSO4 30% BaSO4 40% BaSO4
S-Va
lue
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nano-BaSO4 was the "smoothest" sample with the smallest height parameters in every category
other than skewness (Ssk) (Figure 24).
Figure 23: AFM images of 5 µm x 5 µm areas of polymer samples. (*For 40% (nano) BaSO4 sample images of smaller areas was unattainable due to was repeated interaction between the
GEAII Dissertation Page 96 of 117
sample surface and imaging tip.) RMS= Standard deviation of the height distribution, surface roughness.
Figure 24: Compiled numerical measurements from AFM analysis of polymer samples
Table 5: AFM Height Parameters (adapted from (125))
Sq Root mean
square height Standard deviation of the height distribution, or RMS surface roughness. Computes the standard deviation for the amplitudes of the surface (RMS).
Ssk Skewness Skewness of the height distribution. Third statistical moment, qualifying the symmetry of the height distribution. A negative Ssk indicates that the surface is composed with principally one plateau and deep and fine valleys. In this case, the distribution is sloping to the top. A positive Ssk indicates a surface with lots of peaks on a plane.
Sku Kurtosis Kurtosis of the height distribution. Fourth statistical moment, qualifying the flatness of the height distribution.
Sp Maximum peak
height Height between the highest peak and the mean plane.
Sv Maximum pit
height Depth between the mean plane and the deepest valley.
0
10
20
30
40
50
60
Sq Ssk Sku Sp Sv Sz Sa
0% BaSO4
20% (nano) BaSO4
30% (nano) BaSO4
40% (nano) BaSO4
20% BaSO4
30% BaSO4
40% BaSO4
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Sz Maximum
height Height between the highest peak and the deepest valley.
Sa Arithmetical
mean height Mean surface roughness. This parameter is deprecated and shall be replaced by Sq in the future.
5.3.4 Bacteria growth trials
Bacterial trials are displayed in Figure 25 - Figure 28. The red line on Figure 27 and Figure
28 displays the growth curve of S. aureus and P. aeruginosa, respectively, at room temperature
over the approximate time required to complete the sampling (approximately 110 minutes).
The results from Figure 25 and Figure 26were used to create the red growth curves in Figure 27
and Figure 28. The data in Figure 25 and Figure 26 was used to ensure that the results seen in
Figure 27 and Figure 28 were not affected by bacterial growth during the sample dilution phase
of the experiment. Figure 26 and Figure 28 are the results for Pseudomonas aeruginosa. The
bacteria results indicated significant decrease in bacteria proliferation at certain concentrations
of nano BaSO4. In the case of S. aureus significant decreases were observed when 20% and 40%
nano BaSO4 polymers were used (Figure 27). In the case of P. aeruginosa, significant decreases
were observed when 0% BaSO4 as well as 30%, and 40% nano BaSO4 polymers were used(Figure
28). Additionally the 40% nano BaSO4 led to a significant decrease in P. aeruginosa compared to
the 40% BaSO4 polymer (Figure 28).
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Figure 25: Room temperature growth of S. aureus. Data = mean +/- SEM
Figure 26: Room temperature growth of Pseudomonas aeruginosa. Data = mean +/- SEM
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Figure 27: S. aureus (SA) colony count after 1.5 hours of contact with pellethane polymers. Data = mean +/- SEM nmin=13 N=2 [*indicates a significant decrease when the marked sample is compared to the samples of 0% BaSO4, as well as the empty well control samples containing no polymer sample, as determined by p<0.05] [**indicates a significant decrease when the marked sample is compared to the samples of 30% BaSO4 as well as the samples of 40% BaSO4 , as determined by p<0.05]
Figure 28: P. aeruginosa (PA) colony count after 1.5 hours of contact with pellethane polymers. Data = mean +/- SEM nmin=11 N=2 [*indicates a significant decrease when the
GEAII Dissertation Page 100 of 117
marked sample is compared to the empty well control samples containing no polymer sample, as well as the samples of 40% BaSO4 as determined by p<0.05]
5.4 Discussion:
The results of these trials indicated that adding nano-BaSO4 to the extrusion process of
pellethane was able to change the surface dynamics and microbial surface interactions of the
resulting polymer sample. This study indicates that there may be an optimal nano-BaSO4
concentration in these polymers, beyond which the benefits of the nanoparticle additives are
lost and the material’s integrity is jeopardized due to particle agglomeration. Prior studies
indicated that certain nanoscale roughness will decrease bacteria function due to the inability
of the stiff membrane of bacteria being unable to adjust and adhere to the nano surface
features (See Figure 29)
Figure 29: Illustration comparing bacteria surface interactions with nano-rough composites and conventional or nano-smooth composites: Due to the high degree of roughness at the nanoscale on nanomaterial composites, rigid bacteria cell membranes cannot lay flush against the material surface(adapted from (126)). This may inhibit the preliminary steps leading to bacterial adhesion. As a result, bacterial activity on a nano-composite surface is reduced. The exact nanoscale surface roughness that inhibits bacteria activity remains to be tested, but results from this study suggests that the 40% BaSO4 composite with an RMS roughness of 2 nm (when measured on 5 μm × 5 μm AFM scans) was the best at inhibiting both Pseudomonas aeruginosa and Staphylococcus aureus activity.
GEAII Dissertation Page 101 of 117
From the results it appears that the nano 20% and the nano 40% polymer blends yielded a
marked reduction in S. aureus proliferation, while the 30% and 40% blends showed a marked
decrease in P. aeruginosa proliferation. When this information is compared to the collected
AFM data, it is clear that the relative "smoothness" of the 40% nano-BaSO4 polymer blend may
have played a role in its ability to hinder bacterial proliferation. Additionally the skewness (ssk
value) may have negated the effects of the relative "smoothness of the samples" in the case of
S. aureus. For P. aeruginosa the bacterial proliferation appeared to be directly correlated to the
surface roughness (Sq value). These results indicated that decreasing surface roughness can
play a major role in decreasing bacterial proliferation; however for bacteria (such as S. aureus)
if there are many peaks on the surface, the benefit of a nano-smooth surface is lost. These
results indicate that longer antimicrobial trials should be conducted to investigate the potency
and longevity of the antimicrobial effects seen.
6 Conclusions:
These trials indicate that although the nano-BaSO4 did not change the hydrodynamic
nature of the samples there is a significant change when nano- BaSO4 is present in the polymer.
The result was a reduction in bacterial proliferation. Further trials need to be done to better
characterize the polymers as well as understand bacterial growth on samples over prolonged
periods of time.
6.1 Acknowledgements:
Special thanks to Adriana Noemí Santiago and Gozde Durmus for their help in sample
preparation and experimental procedure development. Additionally, thank you to Becky Sustak,
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Lindsay DuPont, and Advanced Radiology, RI, for use of their x-ray equipment. Thank you
Dattatri Nagesha, for your help in the AFM studies.
6.2 References:
1. S. D. Puckett, E. Taylor, T. Raimondo, T. J. Webster, The relationship between the nanostructure of titanium surfaces and bacterial attachment. Biomaterials 31, 706 (2010).
7. E. Taylor, T. J. Webster, Reducing infections through nanotechnology and nanoparticles. Int. J. Nanomed. 6, 1463 (2011).
12. S. M. Berenholtz et al., Eliminating catheter-related bloodstream infections in the intensive care unit. Crit Care Med 32, 2014 (October, 2004).
13. T. Stockton. (Johns Hopkins Medicine Office of Corporate Communications, 2004), vol. 2012.
22. J. W. Costerton, Z. Lewandowski, D. E. Caldwell, D. R. Korber, H. M. Lappinscott, Microbial Biofilms. Annu. Rev. Microbiol. 49, 711 (1995).
23. L. Hall-Stoodley, J. W. Costerton, P. Stoodley, Bacterial biofilms: From the natural environment to infectious diseases. Nat. Rev. Microbiol. 2, 95 (Feb, 2004).
24. R. M. Donlan, Role of Biofilms in Antimicrobial Resistance. ASAIO Journal 46, S47 (2000).
25. W. W. Nichols, M. J. Evans, M. P. E. Slack, H. L. Walmsley, The penetration of antibiotics into aggregates of mucoid and non-mucoid Pseudomonas aeruginosa. J. Gen. Microbiol. 135, 1291 (May, 1989).
45. I. Chopra, The increasing use of silver-based products as antimicrobial agents: a useful development or a cause for concern? Journal of Antimicrobial Chemotherapy 59, 587 (April 1, 2007, 2007).
119. J. P. Burke, Infection Control — A Problem for Patient Safety. New England Journal of Medicine 348, 651 (2003).
120. W. H. Sheng et al., Comparative impact of hospital-acquired infections on medical costs, length of hospital stay and outcome between community hospitals and medical centres. Journal of Hospital Infection 59, 205 (2005).
121. R. Fears, J. W. M. van der Meer, V. ter Meulen, The Changing Burden of Infectious Disease in Europe. Science translational medicine 3, (Oct, 2011).
GEAII Dissertation Page 103 of 117
122. A. Kroll et al., Cytotoxicity screening of 23 engineered nanomaterials using a test matrix of ten cell lines and three different assays. Part. Fibre Toxicol. 8, (Feb, 2011).
123. S. D. Puckett, P. P. Lee, D. M. Ciombor, R. K. Aaron, T. J. Webster, Nanotextured titanium surfaces for enhancing skin growth on transcutaneous osseointegrated devices. Acta Biomaterialia 6, 2352.
124. P. Sprawls. (American Chiropractic Registry of Radiologic Technologists, 2012), vol. 2012.
125. I. O. f. Standardization. (2012), vol. ISO 25178-2:2012.
126. J. T. Seil, T. J. Webster, Antimicrobial applications of nanotechnology: methods and literature. Int. J. Nanomed. 7, 2767 (2012).
GEAII Dissertation Page 104 of 117
7 Contributions to the Field
The above studies introduced lubricin as a novel means to prevent both bacterial
adhesion and fibroblast adhesions. This thesis provides evidence that lubricin is a viable
candidate to solve two of the major post operative complications associated with cataract
surgery, cellular biofouling and infection. These findings place lubricin in a prime position to be
a leading anti-adhesive surface coating in the medical and research industry. The above work
indicates that lubricin would an ideal candidate for further research as a prophylactic agent to
prevent bacterial adhesion on implanted and transdermal medical devices such as endotracheal
tubes, central venous catheters, stents, and neural implants. The stability and relatively ease of
coating a surface make lubricin an ideal candidate to be used in clinical settings.
The nano-BaSO4 trials showed that simply changing the size of a common agent in
medical tubing can have pronounced effects bacterial surface interactions. This work indicates
that nano-BaSO4 holds potential as a non-toxic antimicrobial additive to many medical devices
which need to be radiopaque. Although further optimization may be needed to ensure the
mechanical properties of the nano-BaSO4 polymers are appropriate to their uses,it is certain
that through blending and optimization an ideal antimicrobial polymer could developed for use
in a wide array of medical devices.
This body of work opens up whole new possibilities of both the polymer composition
and the protein coating of medical devices. The use of these non-toxic additives will avoid the
development of additional drug resistant bacteria, while also avoiding damage to surrounding
tissue. These treatments each have their advantages. Nano BaSO4 appears to be a stable
GEAII Dissertation Page 105 of 117
additive during the polymer extrusion process, and although lubricin may need to be coated on
a medical device close to the time of delivery, lubricin appears to be more effective at
preventing biofouling. The next phases for this work would be to explore combining lubricin
with nano-BaSO4 polymers, to determine the optimal conditions for creating and coating
various medical devices in a clinical setting.
GEAII Dissertation Page 106 of 117
8 Appendix
8.1 Complete Reference List
1. S. D. Puckett, E. Taylor, T. Raimondo, T. J. Webster, The relationship between the nanostructure of titanium surfaces and bacterial attachment. Biomaterials 31, 706 (2010).
2. D. J. Weber, K. L. Hoffman, R. A. Thoft, A. S. Baker, Endophthalmitis Following Intraocular Lens Implantation: Report of 30 Cases and Review of the Literature. Review of Infectious Diseases 8, 12 (January 1, 1986, 1986).
3. M. Nymer, E. Cope, R. Brady, M. E. Shirtliff, J. G. Leid, in Springer Series on Biofilms, M. Shirtliff, J. G. Leid, Eds. (Springer-Verlag Berlin, Heidelberger Platz 3, D-14197 Berlin, Germany, 2009), vol. 3, pp. 239-264.
4. M. Taban et al., Acute Endophthalmitis Following Cataract Surgery: A Systematic Review of the Literature. Arch Ophthalmol 123, 613 (May 1, 2005, 2005).
5. E. Sharifi, T. C. Porco, A. Naseri, Cost-Effectiveness Analysis of Intracameral Cefuroxime Use for Prophylaxis of Endophthalmitis after Cataract Surgery. Ophthalmology 116, 1887 (2009).
6. J. K. Schmier, M. T. Halpern, D. W. Covert, E. C. Lau, A. L. Robin, Evaluation of Medicare Costs of Endophthalmitis among Patients after Cataract Surgery. Ophthalmology 114, 1094 (2007).
7. E. Taylor, T. J. Webster, Reducing infections through nanotechnology and nanoparticles. Int. J. Nanomed. 6, 1463 (2011).
8. A. r. f. t. N. System, National Nosocomial Infections Surveillance (NNIS) System Report, data summary from January 1992 through June 2004, issued October 2004. American Journal of Infection Control 32, 470 (2004).
9. N. P. O'Grady et al., Guidelines for the prevention of intravascular catheter-related infections. Centers for Disease Control and Prevention. Recommendations and reports : Morbidity and mortality weekly report. Recommendations and reports / Centers for Disease Control 51, 1 (2002 Aug, 2002).
10. L. A. Mermel, Prevention of intravascular catheter-related infections. Ann. Intern. Med. 132, 391 (Mar, 2000).
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11. M. Vineet Chopra, Sarah L Krein, Russell N Olmsted, Nasia Safdar, Sanjay Saint, in Making Health Care Safer II: An Updated Critical Analysis of the Evidence for Patient Safety Practices. (Agency for Healthcare Research and Quality (US), Rockville (MD), 2013 ).
12. S. M. Berenholtz et al., Eliminating catheter-related bloodstream infections in the intensive care unit. Crit Care Med 32, 2014 (October, 2004).
13. T. Stockton. (Johns Hopkins Medicine Office of Corporate Communications, 2004), vol. 2012.
14. S. R. Gill et al., Insights on Evolution of Virulence and Resistance from the Complete Genome Analysis of an Early Methicillin-Resistant Staphylococcus aureus Strain and a Biofilm-Producing Methicillin-Resistant Staphylococcus epidermidis Strain. J. Bacteriol. 187, 2426 (April 1, 2005, 2005).
15. R. J. Rubin et al., The economic impact of Staphylococcus aureus infection in New York City hospitals. Emerg. Infect. Dis 5, 9 (Jan-Feb, 1999).
16. P. A. Suci, Z. Varpness, E. Gillitzer, T. Douglas, M. Young, Targeting and Photodynamic Killing of a Microbial Pathogen Using Protein Cage Architectures Functionalized with a Photosensitizer. Langmuir 23, 12280 (10/19, 2007).
17. F. D. Lowy, Staphylococcus aureus Infections. New England Journal of Medicine 339, 520 (1998).
18. P. C. Appelbaum, The emergence of vancomycin-intermediate and vancomycin-resistant Staphylococcus aureus. Clinical Microbiology and Infection 12, 16 (2006).
19. J. P. O'Gara, H. Humphreys, Staphylococcus epidermidis biofilms: importance and implications. Journal of Medical Microbiology 50, 582 (July 1, 2001, 2001).
20. M. Otto, Staphylococcus epidermidis - the 'accidental' pathogen. Nat. Rev. Microbiol. 7, 555 (Aug, 2009).
21. J. W. Costerton, P. S. Stewart, E. P. Greenberg, Bacterial Biofilms: A Common Cause of Persistent Infections. Science 284, 1318 (May 21, 1999, 1999).
22. J. W. Costerton, Z. Lewandowski, D. E. Caldwell, D. R. Korber, H. M. Lappinscott, Microbial Biofilms. Annu. Rev. Microbiol. 49, 711 (1995).
23. L. Hall-Stoodley, J. W. Costerton, P. Stoodley, Bacterial biofilms: From the natural environment to infectious diseases. Nat. Rev. Microbiol. 2, 95 (Feb, 2004).
24. R. M. Donlan, Role of Biofilms in Antimicrobial Resistance. ASAIO Journal 46, S47 (2000).
GEAII Dissertation Page 108 of 117
25. W. W. Nichols, M. J. Evans, M. P. E. Slack, H. L. Walmsley, The penetration of antibiotics into aggregates of mucoid and non-mucoid Pseudomonas aeruginosa. J. Gen. Microbiol. 135, 1291 (May, 1989).
26. J. Kluytmans, A. van Belkum, H. Verbrugh, Nasal carriage of Staphylococcus aureus: epidemiology, underlying mechanisms, and associated risks. Clin. Microbiol. Rev. 10, 505 (July 1, 1997, 1997).
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8.2 Curriculum vitae
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George E. Aninwene II [email protected]
(732)485-5078 21 Dudley St, Apt #3,
Boston, MA 02119 41 Dunbar Avenue,
Piscataway, NJ 08854
Profile: Biochemical/Biomedical Engineering major with excellent problem-solving and communication skills. Excels in presentations of ideas and topics through a variety of means and media: Task-oriented, with the ability to communicate and collaborate with others to accomplish goals: Diplomatic, skilled in leadership and teaching.
Education:
University of Maryland, Baltimore County, Baltimore, MD o Biochemical Engineering; Minor in Biology – 2008
Brown University, Providence RI o Program in Innovation Management and Entrepreneurship: M.S. in
Innovation Management and Entrepreneurship Engineering – May 2010 o PhD in Biomedical Engineering – Continued at Northeastern University
Northeastern University, Boston MA o PhD in Bio-Engineering – May 2014
Honors/Awards: ● Meyerhoff Scholarship (UMBC 2003-2008) ● National Merit Scholarship (2003) ● UMBC Honors College ● National Society of Collegiate Scholars
● Brown University Graduate Student Recognition Award (2011 & 2012) ● SFB STAR Award Honorable Mention(2012)
Research Experience: Rutgers Nanotechnology Program –
Rutgers University New Brunswick, NJ June – August 2002 o Assisted a research team under the direction of Dr. Richard Haber to research the rheological
properties of Alumina mixtures. o Presented my summer research project before an audience of faculty, staff, and colleagues,
and received honored recognition. Pre-Marc U*Star Research Program –
University of Maryland, Baltimore County, Baltimore, MD January – August 2005 o Used Spectrophotometric Quantification to attempt to determine the amount of
Polysaccharide secreted by sessile Staphylococcus aureus Biofilms into their flowing nutrient medias.
o Aided in developing procedures for quantifying Staphylococcus aureus cells recovered from frozen solutions.
Leadership Alliance Summer Research Early Identification Program/REU – Brown University, Providence, RI June 5th – August 4th 2006, June 3rd – August 3rd 2007
o Anodized titanium samples, cultured osteoblast cells, coated titanium samples, performed cell adhesion assays, and analyzed the data.
o Improved existing experimental procedures and proposed various different coating methods for the samples.
o Presented my research findings in the Leadership Alliance National Symposium, at Brown University’s annual Summer Research Symposium, at Eli Lilly Headquarters in Indianapolis, Indiana as an Eli Lilly scholar (2007), and presented an award-winning oral presentation at ABRCMS (2006).
Eli Lilly and Company Summer Internship – Brown University, Providence, RI
Eli Lilly, Indianapolis, IN May – August 2008
o Optimization of a Scale-down Model for Bioproduct Production in Bioreactor.
Coursework: UMBC: Organic Chemistry I&II; Thermodynamics; Fluid-Mechanics I&II; Kinetics; Process Engineering; Separation Processes; Biochemical Engineering; Biochemical Engineering Lab; Process Engineering Economics & Design 1&2; Chemical Engineering Systems Analysis; Genetics; Biochemistry; Math 152&225&251; Differential-Equations; Physics 121&122; Psychology 100; Abnormal Psychology.
Brown: Drug and Gene Delivery; Analytical Methods in Biomaterials; Innovation/Technology Management I&II; Emerging and Breakthrough Tech; Business Engineering Fundamentals I&II; Innovation and Entrepreneurship; Globalization Immersion Experience and Entrepreneurship Laboratory; Science, Technology, and Human Health; Impacts of Nanomaterials; Toxicity of Nanoparticles.
Graduate Research: Projects: Lubricin as a Means to Prevent Bacteria Adhesion, Cellular Encroachment, & Mineral Deposition o Mentored two undergraduate students. o Currently mentoring one undergraduate student.
Nano-BaSO4 as a novel antimicrobial additive to medical polymers o Currently mentoring one undergraduate student.
Grants/Fellowships: Initiative for Maximizing Student Development (IMSD) Grant September 2008 – May 2010
GK-12 Fellowship: Graduate STEM Fellow in K-12 Education May 2010 – June 2012
Conferences: Society for Biomaterials – April 21st – April 24th, 2010 – Presented poster April 13th – April 16th, 2011 – Oral Presentation April 10th – April 13th, 2013 – Oral Presentation
Biomedical Engineering Society – October 6th – October 9th , 2010 – Presented poster October 12th – October 15th , 2011 – Presented poster October 24th – October 27th , 2012 – Oral Presentation September 25th - September 28th, 2013– Oral Presentation
Materials Research Society – November 29th – December 3rd, 2010 – Presented poster November 28th – December 2nd, 2011 – Presented poster
Northeast Bioengineering Conference – April 1st – April 3rd, 2011 – Presented poster April 5th – April 7th, 2013 – Presented poster
9th World Biomaterials Congress – June 1st – April 5th, 2012 – Oral Presentation Society for Biomaterials Fall Symposium– October 24th – October 27th , 2012 – Oral Presentation
Publications: 2013 – Nanotechnology: Special Edition on Biomaterials(Vol.11)- Studium Press LLC (To be released September 2013) Use of Nano-materials and Nano Structures to further the Medical Imaging Techniques George E. Aninwene II, Erik N. Taylor, and Thomas J. Webster 2013 – Northeast Bioengineering conference proceedings paper Nano-BaSO4: A Novel Bacteriostatic Polymer Additive George E. Aninwene II, and Thomas J. Webster 2012 – Dove Medical Press Nano- BaSO4: A Novel Antimicrobial Additive to Pellethane George E. Aninwene II, David Stout, Zifan Yan, and Thomas J. Webster 2012 – Powder Metallurgy & Mining Nanostructured and Nanoparticulate Metals: Redefining the Field of Medical Devices George E. Aninwene II and Thomas J. Webster 2012 – MRS conference proceedings paper Lubricin as a Surface Treatment to Reduce Post-operative Biofouling and Infection George E. Aninwene II, Doug Hall, Amy Mei, Gregory D. Jay, and Thomas J. Webster 2011-Book Chapter Nanomedicine: Principles and Perspectives, Volume 1 “Tissue engineering in vivo with nanotechnology.” Erik N. Taylor, David A. Stout, George E. Aninwene II, and Thomas J. Webster 2011 – Northeast Bioengineering conference proceedings paper Preventing bacterial adhesion and cellular encroachment on intraocular lenses with lubricin George E. Aninwene II, Erik Taylor, Amy Mei, Gregory D. Jay, and Thomas J. Webster 2011 – MRS conference proceedings paper Using Antibiotic Conjugated Magnetic Nanoparticles and a Magnetic Field for the Treatment of Bone Prosthetic Infections, Erik N. Taylor, George E. Aninwene II, and Thomas J. Webster 2011 – MRS conference proceedings paper Decreased Attachment of Bacteria to Lubricin Coated Intraocular Lenses George E. Aninwene II, Erik Taylor, Amy Mei, Gregory D. Jay, and Thomas J. Webster 2010 – NANOSMAT conference proceedings paper Decreased Attachment of Epithelial Cells and Bacteria to Lubricin Coated Intraocular Lenses George E. Aninwene II, Erik Taylor, Amy Mei, Gregory D. Jay, and Thomas J. Webster
Extracurricular UMBC Game Development Club, Jujitsu, ECM JeetKuneDo, WRIK Karaoke, Activities: National Society of Black Engineers Skills: Microsoft Power Point, Microsoft Word, Microsoft Excel, ImageJ, AutoCAD
2002, MatLab, Photoshop, Photo suite, Audacity
References available upon request.