Conferring materials with antibacterial properties 1

CONFERRING MATERIALS WITH ANTIBACTERIAL PROPERTIES SHI ZHILONG (B.E., M.S. BUCT) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF CHEMICAL & BIOMOLECULAR ENGINEERING NATIONAL UNIVERSITY OF SINGAPORE 2006 ACKNOWLEDGEMENTS First of all, I would like to express my cordial gratitude to my supervisors, Prof. Neoh Koon Gee and Prof. Kang En-Tang, for their guidance, advice, support and encouragement throughout the long period of this research work. I have learnt invaluable knowledge from them on how to research work and how to enjoy in doing research. Their enthusiasm, sincerity and dedication to scientific research have greatly impressed me and will benefit me in my future career. I would like to thank all my colleagues in the laboratory and the technicians of the Department of Chemical and Biomolecular Engineering for their kind help and assistance. In particular, thanks are due to Dr. Cen Lian, Dr. Li Yali, Mr. Hu Feixiong for their helpful advice. It is my great pleasure to work with all of them. The financial support provided by the National University of Singapore is greatly appreciated. Finally, I would like to express my deepest gratitude and indebtedness to my parents for their constant concern and support. Special thanks to my wife Zhang Nan for her love, patience and encouragement. i TABLE OF CONTENTS ACKNOWLEDGEMENT ii TABLE OF CONTENTS ii SUMMARY v NOMENCLATURE vii LIST OF FIGURES viii LIST OF TABLES xiii Chapter INTRODUCTION Chapter LITERATURE SURVEY 2.1 Biofilm 2.2 Preventative Strategies of Device-related Infections 11 2.2.1 Development of antiadhesive surfaces 14 2.2.2 Incorporation of antibacterial agents 16 2.2.3 Functionalizing surfaces with antibacterial agents 17 23 2.3 Surface Functionalization 2.3.1 Surface graft copolymerization 23 2.3.2 Self-assembly polyelectrolyte multilayers 26 OF POLYMERIC ANTIBACTERIAL 28 3.1 Antibacterial Activity of Polymeric Substrate with Surface Grafted Viologen Moieties 29 Chapter SURFACE FUNCTIONALIZATION SUBSTRATE TO ACHIEVE PROPERTIES 3.1.1 Introduction 29 3.1.2 Experimental 32 3.1.3 Results and Discussion 38 3.1.4 Conclusions 50 3.2 Surface Grafted Viologen for Precipitation of Silver Nanoparticles and Their Combined Bactericidal Activities 51 3.2.1 Introduction 51 3.2.2 Experimental 53 ii 3.2.3 Results and Discussion 57 3.2.4 Conclusions 69 Chapter ANTIBACTERIAL AND ADSORPTION CHARACTERISTICS OF ACTIVATED CARBON FUNCTIONALIZED WITH QUATERNARY AMMONIUM MOIETIES 70 4.1 Introduction 71 4.2 Experimental 73 4.3 Results and Discussion 78 4.4 Conclusions 90 Chapter IN VITRO ANTIBACTERIAL AND CYTOTOXICITY ASSAY OF MULTILAYERED POLYELECTROLYTE FUNCTIONALIZED STAINLESS STEEL 91 5.1 Introduction 92 5.2 Experimental 94 5.3 Results and Discussion 100 5.4 Conclusions 114 Chapter NOVEL STRATEGIES FOR CONFERRING ANTIBACTERIAL PROPERTIES TO BONE CEMENT 115 6.1 Antibacterial and Mechanical Properties of Bone Cement Impregnated with Chitosan Nanoparticles 116 6.1.1 Introduction 116 6.1.2 Experimental 120 6.1.3 Results and Discussion 125 6.1.4 Conclusions 141 6.2 Antibacterial and Mechanical Properties of Bone Cement Containing a Monomer or Polymer with Norfloxacin Moieties 142 6.2.1 Introduction 142 6.2.2 Experimental 144 6.2.3 Results and Discussion 147 6.2.4 Conclusions 159 Chapter CONCLUSIONS 160 iii Chapter RECOMMENDATIONS FOR FUTURE STUDY 165 REFERENCES 169 LIST OF PUBLICATIONS 190 iv SUMMARY Biofilms are complex communities of surface attached microorganisms, comprising either single or multiple species. Biofilms are found ubiquitously on virtually all natural, medical, and industrial settings where bacteria exist, and they can cause device-related infections, bacterial drug resistance and microbial-induced corrosion. In this thesis, different approaches of surface and bulk functionalization to confer materials with antibacterial properties to combat biofilm were developed depending on the materials of interest. At the same time, other important properties of the materials such as mechanical property and cytotoxicity were investigated after the functionalization process. Surface modification techniques were developed for the functionalization of poly(ethylene terephthalate) (PET) film with viologen moieties, N, N'-disubstituted-4, 4'-bipyridinium, using graft copolymerization. The antibacterial property of the viologen graft copolymerized PET film was assayed against Escherichia coli (E. coli, a Gram-negative bacterium). Further, silver nanoparticles can be deposited on the modified film through the photoinduced reduction capability of the viologen moieties in silver salt solution. The size and distribution of the silver nanoparticles with varying reaction time were investigated. The combined antibacterial effect of viologen and silver nanoparticles was assessed. The stability of the modified film was carried out after aging in a weathering chamber. Two types of quaternary ammonium group were also successfully covalently coupled on the activated carbon (AC) using wet chemistry method. Both types of v functionalized ACs show highly effective antibacterial activities against E. coli and Staphylococcus aureus (S. aureus, a Gram-positive bacterium). Furthermore, the functionalized ACs can be used in repeated antibacterial applications with little loss in efficacy. Using phenol as a model compound, the adsorption capacity of the different ACs was also investigated. A simple and versatile method was then developed to confer stainless steel (SS) with antibacterial property via the alternate deposition of quaternized polyethylenimine (PEI) or quaternized PEI-silver complex and poly(acrylic acid) (PAA). The antibacterial activity was assessed using E. coli and S. aureus. The inhibition of bacterial growth on the surface of functionalized SS was investigated using fluorescence microscopy after staining with a combination dye. The cytotoxicity of the functionalized SS towards mammalian cells was evaluated by the MTT assay. Two new strategies were developed to confer antibacterial properties to bone cement using bulk modification. The first strategy employs chitosan nanoparticles which may be further derivatized with quaternary ammonium groups, while the second uses norfloxacin moieties, as one of the components in the commercial poly(methyl methacrylate) bone cement. Antibacterial assay using S. aureus and Staphylococcus epidermidis (S. epidermidis) was carried out. The results showed promising bactericidal activities. The studies also addressed the issue of mechanical property and cytotoxicity of the bone cement after functionalization. vi NOMENCLATURE AC Activated carbon ALBC Antibiotic-load bone cement BE Binding energy BV Benzyl viologen DMEM Dulbecco's modified eagle’s medium DMF Dimethylformamide EDX Energy dispersive X-ray spectroscopy E. coli Escherichia coli FE-SEM Field emission scanning electron microscopy FTIR Fourier transform infrared GMA Glycidyl methacrylate HVV N-hexyl-N′-(4-vinylbenzyl)-4,4′-bipyridinium bromide chloride ICP Induced coupled plasma MTT 3-[4,5-dimethyl-thiazol-2-yl]-2,5-diphenyltetrazolium bromide NOR Norfloxacin PAA Poly(acrylic acid) PEI Polyethylenimine PET Poly(ethylene terephthalate) PMMA Poly(methyl methacrylate) PVP Poly(4-vinyl pyridine) PBS Phosphate buffer solution QAS 3-(trimethoxysilyl)- propyldimethyloctadecylammonium chloride S. aureus Staphylococcus aureus S. epidermidis Staphylococcus epidermidis SEM Scanning electron microscopy SS Stainless steel UV Ultraviolet VBC 4-vinyl benzyl chloride VBV N,N′-bis(4-vinylbenzyl)-4,4′-bipyridinium dichloride XPS X-ray photoelectron spectroscopy vii LIST OF FIGURES Figure 3.1 Scheme of synthesis of HVV. Figure 3.2 Schematic illustration of the surface functionalization of PET films with HVV. Figure 3.3 FTIR spectrum of the as-synthesized HVV powder. Figure 3.4 XPS: (a) N 1s, (b) Cl 2p, and (c) Br 3d core-level spectra of assynthesized HVV powder. Figure 3.5 XPS wide scan, N 1s and C 1s core-level spectra (a, b, c) of pristine PET film, wide scan, N 1s and C 1s core-level spectra (d, e, f) of PET film after HVV graft-copolymerization using 40 wt% HVV monomer concentration. Figure 3.6 UV-visible absorption spectra of PET film graft copolymerized with HVV (using 40 wt% HVV monomer concentration): base spectrum, after 10 irradiation (0 spectrum) and bleaching in air (10 spectrum). Figure 3.7 Optical micrographs of (a) pristine PET and (b) HVV graftcopolymerized PET surface after exposure to waterborne E. coli and subsequent incubation in solid growth agar for 24h，respectively. Figure 3.8 Scanning electron micrographs of (a) pristine PET film and (b) PET film after HVV graft-copolymerization after exposure to waterborne E. coli and subsequently incubated with solid growth agar for 24h, respectively. Figure 3.9 Effect of HVV monomer concentration on (a) surface concentration of N+ , and (b) antibacterial activity of the HVV graft-copolymerized PET. For the antibacterial test, the film was in contact with the E. coli in PBS solution for 18h. Figure 3.10 Survival ratio of E. coli cells in PBS at 37 oC as a function of time in contact with the different substrates: (a) pristine PET, (b) HVV graft copolymerized PET (40 wt% HVV monomer used in graft copolymerization) and (c) HVV graft copolymerization PET after 10min UV irradiation and then left to bleach in air. The cell number was determined by spread plate method. Figure 3.11 XPS N 1s (a) and C 1s (b) core-level spectra of PET film after HVVN graft-copolymerization using 40 wt% HVVN monomer concentration. viii Figure 3.12 Amount of silver deposited on the surface of the HVVN-PET film after reaction with a 200 mg/l AgNO3 solution under UV irradiation for various time periods. Figure 3.13 XPS Ag 3d core-level spectrum of the HVVN-PET film after reaction with a 200 mg/l AgNO3 solution for 15 min. Figure 3.14 UV-visible absorption spectra of the HVV-PET films before and after reaction with a 200 mg/l AgNO3 solution under UV irradiation for different periods of time. Figure 3.15 Scanning electron micrographs of the HVV-PET films before and after reaction with a 200 mg/l AgNO3 solution for (a) 15 min, and (b) 30 under UV irradiation. Figure 3.16 Viable E. coli cell number as a function of time in contact with the different substrates: Pristine PET (█), VBV-PET (◆), HVVN-PET (●), HVVN-PET after reaction in AgNO3 for 30 (▼) and HVVN-PET after reaction in AgNO3 for 15 (▲). The cell number was determined by the surface-spread method. Figure 3.17 Comparison of antibacterial effect of the silver coated HVVN-PET film before and after aging in a weathering chamber for 48 h. The silver coated HVV-PET film was obtained after 15 photoinduced reaction in a 200 mg/l AgNO3 solution. Figure 4.1 Schematic representation of the two routes for the functionalization of AC with quaternary ammonium groups. Figure 4.2 FTIR spectra of (a) AC and (b) AC-COOH. Figure 4.3 XPS wide scan of AC (a), Q-AC (b) and P-AC (c); N 1s core-level spectra of AC (d), Q-AC (e) and P-AC (f). Figure 4.4 FE-SEM micrographs of AC (a), Q-AC (b) and P-AC (c). Figure 4.5 Viable E. coli cell number (a) and viable S. aureus cell number (b) as a function of time in contact with the different substrates: Control (■), AC (●), Q-AC (Œ), and P-AC (▲). The cell number was determined by the surface spread method. Figure 4.6 Repeated antibacterial assay using 100 mg of P-AC in contact with 30 ml E. coli suspension (107 cells/ml). Figure 4.7 Adsorption isotherms of phenol on AC (●), Q-AC (Œ) and P-AC (▲). ix Chapter subjected to thorough washing with water to remove viologen which was not graft copolymerized on the substrate. This substrate will be referred to as the HVVN-PET substrate in the subsequent discussion. Silver deposition on HVVN-PET The HVVN-PET film was then inserted into a Pyrex® tube which contained 20 ml silver nitrate solution at a concentration of 200 mg/l. The solution was degassed for 20 with argon, and then sealed under an argon atmosphere. The substrate in the silver nitrate solution was subsequently exposed to UV irradiation on each surface in the rotary photochemical reactor at 25-28 oC for various periods of time. After irradiation, the substrates were washed with doubly distilled water and dried under reduced pressure. Tests and characterization The surface compositions were measured using XPS as described in Section 3.1.2. FE-SEM imaging and composition determination of the surface were carried out on a JEOL JSM 6700F scanning electron microscope with energy dispersive X-ray (EDX) accessory. The amount of N+ immobilized on the surface of the PET film was determined by the modified dye interaction method as described in Section 3.1.2. The amount of silver immobilized on the surface of the HVVN functionalized PET film was determined from the difference in the concentration of silver ions before and after the photoinduced reaction of the silver nitrate solution with the HVVN-PET film. The silver concentration in the solution was measured using a inductively coupled plasma 54 Chapter (ICP) emission spectrometer (Perkin Elmer Optima 3000DV) at a wavelength of 328.068 nm. The detection limit of this instrument is specified to be 0.9 ppb. Determination of antibacterial effect of HVVN functionalized PET films. E. coli was cultivated as described in Section 3.1.2. The E. coli-containing broth was centrifuged at 2700 rpm for 10 min, and after the removal of the supernatant, the cells were washed twice with sterile PBS and resuspended in PBS at a concentration of 107 cells/ml. One piece of PET film (either pristine or functionalized) of cm × cm in size was immersed in 30 ml of this suspension in an conical flask and shaken at 200 rpm at 37oC. The viable cell counts of the E. coli were measured by the surface spread-plate method. At the predetermined time, ml of bacteria culture was taken from the flask and decimal serial dilutions with PBS were repeated with each initial sample. A drop of 0.1 ml of the diluted sample was then spread onto triplicate solid growth agar plates. After incubation of the plates at 37oC for 24 h, the number of viable cells (colonies) was counted manually and the results after multiplication with the dilution factor were expressed as mean colony forming units (CFU) per ml. The stability of the functionalized films was tested in a Ci3000 Xenon Weather-Ometer (Atlas Electric Device Co., Chicago, USA). During the test, the weathering chamber was maintained at a relative humidity of 70%, and a dry bulb temperature and black panel temperature of 40 oC and 70 oC, respectively. The simulated solar irradiation was directed at the film’s surface with an intensity of 0.56 W/m2 at 340 nm. A water spray was activated for in each 30 cycle. After 48 h, the film was removed from the chamber and dried under reduced pressure before being subjected to antibacterial 55 Chapter assay. The bactericidal efficiency of the film after weathering was then compared to that before the weathering test. 56 Chapter 3.2.3 Results and Discussion HVVN PET films As shown in Section 3.1, the success of the UV-induced surface graft copolymerization of HVVN on the PET films can be ascertained by comparing the XPS spectra of the film before and after the grafting process. The presence of the surface grafted HVVN polymer after the UV-induced graft copolymerization step can be deduced from the presence of the N 1s signal in the XPS wide scan spectra. The corresponding N 1s core-level spectrum (Figure 3.11(a)) shows well resolved peaks due to the nitrogen radical (N•) at 399.6 eV formed during X-ray excitation in the analysis chamber (Sampanthar et al., 2000), positively charged nitrogen (N+) at 401.8 eV and NO3 at - 406.2 eV (Moulder et al., 1992). The almost one to one peak area ratio of the nitrogen from the viologen chain backbone (N• and N+) and the NO3- counterion confirms that charge neutrality is maintained. The C 1s core-level spectrum (Figure 3.11(b)) after HVVN grafting shows a peak at 285.5 eV attributable to the C-N species of the HVVN in addition to the C-C, C-H peak at 284.6 eV, and the C-O and O-C=O peak components of the pristine PET film are masked. This implies that the surface HVVN copolymer is thicker than the probing depth of the XPS technique (~10nm). No Cl and Br signals can be detected in HVVN-PET film, which shows that the exchange of the halide anions by NO3- anions was complete. The quantitative amount of N+ present on the film surfaces was obtained from the titration method using fluorescein (Na salt) as described in Section 3.1.1. The amount of surface N+ increases from to 25 nmol/cm2 as the concentration of HVVN monomer used in the grafting process increases from 10 to 40 wt%. A further increase in monomer concentration beyond this value results in only a small increase in [N+]. 57 Chapter Hence, for the present work, a 40 wt% concentration of HVVN was used in the grafting process. Figure 3.11 XPS N 1s (a) and C 1s (b) core-level spectra of PET film after HVVN graft-copolymerization using 40 wt% HVVN monomer concentration. 58 Chapter Photoinduced reduction of silver on HVVN-PET films The amount of silver deposited on the surface of HVVN-PET films was calculated from the difference in the silver ion concentration of silver nitrate solution before and after reaction. Figure 3.12 shows the silver uptake by the HVVN-PET film as a function of the UV irradiation time. After a reaction time of 30 min, the uptake of silver has almost reached a plateau of 0.08 μmol/cm2. This amount of silver deposited on the HVVN-PET film represents 4% of the original silver ions from solution. Ag Uptake (μmol/cm2) 0.12 0.08 0.04 0.00 20 40 60 Time (min) Figure 3.12 Amount of silver deposited on the surface of the HVVN-PET film after reaction with a 200 mg/l AgNO3 solution under UV irradiation for various time periods. 59 Chapter The chemical state of the silver on the HVVN-PET film was investigated using the XPS technique. Figure 3.13 shows the XPS Ag 3d core-level spectrum of the HVVN-PET film after photoinduced reaction with a 200 mg/l AgNO3 solution for 15 min. Since no Ag 3d signal was discernible on the HVVN-PET film surface before the photoinduced reaction in AgNO3, the peaks in this spectrum are attributable to the silver uptake from the AgNO3 solution. The peaks at 368.0 eV(3d5/2) and 374.0 eV (3d3/2) attributable to the Ag0 species (Beamson and Briggs, 1992; Moulder et al., 1992; Stathatos et al., 2000) show that Ag+ ions from the solution have been reduced to the metallic state upon reaction with the viologen on the HVVN-PET film surface. Our previous study has postulated that upon UV irradiation the viologen dication can be Intensity (arb. units) readily reduced to radical cation via the transfer of an electron from the counteranion. Ag 3d 5/2 365 370 Ag 3d 3/2 375 Binding Energy (eV) Figure 3.13 XPS Ag 3d core-level spectrum of the HVVN-PET film after reaction with a 200 mg/l AgNO3 solution for 15 min. 60 Chapter The viologen radical cations can then react with the metal ions in solution or adsorbed on the film surface, and concomitantly the radical cations undergo oxidation back to the dication state (Zhao et al., 2003). The N 1s core-level spectrum of the HVVN-PET film after 15 photoinduced reaction with AgNO3 is nearly the same as that before reaction (as shown in Figure 3.11(a)). For this film, the Ag/N (N from viologen chain backbone) ratio as determined by XPS is 0.18. This ratio has been calculated from the ratio of the areas of the respective peaks after correction with the sensitivity factors. It has been reported that when silver particles are reduced to nanometer dimensions, they exhibit unique optical properties in the visible spectral range due to the excitation of the collective oscillations of conducting electrons known as surface plasmons (Malynych and Chumanov, 2003). The wavelength of the absorption maximum depends on the size, shape, and dielectric environment of nanoparticles. The UV-visible absorption spectra of the HVVN-PET film before and after reaction with the silver nitrate solution under UV irradiation for various time periods are shown in Figure 3.14. No distinct absorption bands between 350 nm to 750 nm can be observed in the spectrum of the pristine PET and HVVN-PET films. After reaction of HVVN-PET with silver nitrate solution under UV irradiation, a new band appears in the 400 nm region. This absorption is the characteristic plasmon peak of silver nanoparticles (Chen et al., 1998; Michaels et al., 1999; Doremus, 2002; Lin et al., 2003). As can be seen from Figure 3.14, there is a red shift of the absorption band from 420 nm to 440 nm with increasing reaction time. At the same time, the full width at half maximum of the absorption band also increases. This result indicates that with increasing reaction time, the size of the nanoparticles increases and the size distribution also becomes wider. 61 Chapter Absorbance (arb. units) 30 15 min HVVN-PET PET 300 400 500 600 700 Wavelength (nm) Figure 3.14 UV-visible absorption spectra of the HVV-PET films before and after reaction with a 200 mg/l AgNO3 solution under UV irradiation for different periods of time. The FE-SEM images of the HVVN PET film after reaction with a 200 mg/l AgNO3 solution for 15 and 30 under UV irradiation are shown in Figure 3.15. Using the software package (Smart-view®) available with the FE-SEM, the mean diameter (Dm) of the nanoparticles was calculated from 20 particles chosen arbitrarily from each image. For a reaction time of 15 min, the Dm of the silver nanoparticles is 25 nm (Figure 3.15(a)). After a reaction time of 30 min, the silver nanoparticles have started to aggregate (Figure 3.15(b)). EDX analysis of the HVVN-PET film surface confirms the extensive presence of silver on the surface of the HVVN-PET films. 62 Chapter (a) (b) Figure 3.15 Scanning electron micrographs of the HVV-PET films before and after reaction with a 200 mg/l AgNO3 solution for (a) 15 min, and (b) 30 under UV irradiation. 63 Chapter Antibacterial effect of the functionalized PET films. The antibacterial effect of the functionalized PET films was investigated by a comparison of the number of viable cells in the suspension in contact with the different substrates. From Figure 3.16, it can be seen that with the pristine PET film, the number Log (viable cell number/ml) of viable cell in the suspension decreased by [...]... incorporation of antibacterial agents and functionalized surfaces with antibacterial agents 2.2 .1 Development of antiadhesive surfaces Several research groups have tried to modify devices with new surface properties that would lead to a reduction of bacterial adhesion Bridgett et al (19 92) studied the adherence of three isolates of S epidermidis to polystyrene surfaces that were modified with a copolymer... lifetime of the device 2.2.3 Functionalized surfaces with antibacterial agents Device surfaces functionalized with antibacterial agents have significant advantages They can incorporate a large variety of agents on the surface and the existing devices can be modified easily and inexpensively without changing the device bulk properties Coating with antibacterial agents have shown promising results and... reduction of biomaterials-centered infections 17 Chapter 2 McLean et al (19 93) reported that silver-copper surface films, sputter-coated onto materials, show antibacterial activity against biofilm formation Sioshansi et al (19 94) used the technique of ion implantation to deposit silver-based coatings on a silicone rubber which thereafter demonstrated antibacterial activity Recently, Woodyard et al (19 96) compared... preventing device-related infections (Bach et al., 19 93; Greenfeld et al., 19 95) Clinical studies on the performance of the catheters have shown inconsistent outcomes (Civetta et al., 19 96; Maki et al., 19 97) 19 Chapter 2 Covalent immobilization of quaternary ammonium on surfaces The methods described above focus on the technologies that provide biomaterials with antibacterial agents or antibiotics which leach... material properties The specific aims of this study were as follows: 1) To develop a technique of covalently immobilizing quaternary ammonium salt on polymeric and inorganic materials and explore the antibacterial effect of these materials 2 Chapter 1 2) To develop a simple and versatile method to functionalize stainless steel to confer it with antibacterial property and investigate the cytotoxicity of the... dental materials) , textile manufacturing, biofouling, antimicrobial filters and food packaging The choice of materials in any application is closely related to the desired performance The materials selected in this project include polymeric and inorganic materials which are used as biomaterials and in industries The different surface and bulk modifications were developed according to the material properties. .. nanoparticles or norfloxacin moieties can be used to confer antibacterial properties to plain bone cement or to further enhance the effects of antibiotic-loaded cement These modified cements offered two distinct advantages: high mechanical strength even with an additive to bone cement powder loading of 15 %, and long lasting antibacterial effect 5 Chapter 1 Chapter 7 gives the overall conclusion of the present... growing within complex communities has been shown to offer protection (Costerton et al., 19 99) The biofilms most often encountered include dental plaque and the slime on surfaces within both natural and man-made water systems, including domestic water supplies and drains It is only recently that biofilms have been implicated in many medical conditions and infections (Gristina, 19 87; Donlan, 20 01) With. .. associated with such infections exceed $3 billion annually in the U.S alone The number of device-associated infections will continue to rise as more patients receive biomedical implants From 19 96 to 20 01, the number of hip and knee joint replacements increased by 14 % (Deyo el at 2004) The majority of these implant procedures were performed on patients 65 years of age and older (Moore et al .19 91) The worldwide... mechanism as those in solution More recently, Tiller et al (20 01, 2002) described methods for treating flat surfaces such as glass, high-density polyethylene, low-density polyethylene, polypropylene, and nylon, poly(ethylene terephthalate) with poly(4-vinyl-pyridine) modified with pendant quaternary ammonium salts The antibacterial properties of these materials were assessed by spraying aqueous suspensions . Mechanical Properties of Bone Cement Impregnated with Chitosan Nanoparticles 11 6 6 .1. 1 Introduction 11 6 6 .1. 2 Experimental 12 0 6 .1. 3 Results and Discussion 12 5 6 .1. 4 Conclusions 14 1 6.2 Antibacterial. 91 5 .1 Introduction 92 5.2 Experimental 94 5.3 Results and Discussion 10 0 5.4 Conclusions 11 4 Chapter 6 NOVEL STRATEGIES FOR CONFERRING ANTIBACTERIAL PROPERTIES TO BONE CEMENT 11 5 6 .1 Antibacterial. SUBSTRATE TO ACHIEVE ANTIBACTERIAL PROPERTIES 28 3 .1 Antibacterial Activity of Polymeric Substrate with Surface Grafted Viologen Moieties 3 .1. 1 Introduction 3 .1. 2 Experimental 3 .1. 3 Results and