Durable polymeric N-halamine functionalized stainless steel surface for improved antibacterial and anti-biofilm activity

A stainless steel surface was functionalized with a novel hydantoin based copolymer and evaluated for antibacterial activity, anti-biofilm activity, and biocompatibility.


Introduction
Stainless steel (SSL) is widely used in surgical instruments, in various hospital equipments, in orthopedic implants and in water purification and food processing units due to its mechanical properties and resistant to corrosion [1][2][3][4]. However, several reports suggest that SSL surface has the ability to harbor bacterial biofilm of different genera such as Listeria, Staphylococcus, Escherichia,Bacillus and Pseudomonas that may lead to cross-contamination and fouling of the surface [5,6]. Therefore, controlling the adhesion and growth of bacteria on the SSL surface has great potential to prevent cross contamination and development of stable biofilms. In order to render the SSL surfaces antibacterial, various surface modification strategies such as deposition of biocidal metals like silver, copper, zinc and nickel, chemical vapor deposition, electroless nickel, layer-by-layer assembly of charged polyelectrolytes, sol-gel matrix of TiO 2 etc. have been employed [7][8][9][10][11][12][13]. Surface modification of SSL with natural or synthetic polymers such as antimicrobial peptides and inorganic-organic hybrid coatings of polysilsesquioxane and quaternized poly(2-(dimethyamino)ethyl methacrylate) have been reported to inhibit bacterial colonization [12,14]. Generally, the above mentioned approaches exhibit migration of antimicrobial agents from surfaces, and longer contact times are needed to inactivate the bacteria, which limit their practical application. In this context, functionalization of various surfaces using N-halamine containing polymers has emerged as a potential strategy to prepare antimicrobial surfaces due to their rapid action, broad spectrum activity, cost effectiveness and rechargeability [15][16][17][18]. The antimicrobial mechanism involves the transfer of oxidative halogen from N-halamine to the microbial receptors. Nhalamine based polymer has been mainly used in application such as in water disinfection, in antimicrobial paints and in antimicrobial treatment of textiles etc. [15][16][17][18][19][20][21][22][23][24][25][26][27][28]. There are few reports which describe the application of N-halamine in the development of antimicrobial surface for SSL [29][30][31][32]. The methods usually require prolonged exposure time to completely deactivate the microbes [29][30][31]. To the best of our knowledge, there is only one report on the surface modification of SSL using n-halamine based copolymers where a 6 log reduction (total kill) of the organisms has been reported within 15 min of contact time [32]. Generally, hydantoin (cyclic N-halamine) monomers are synthesized by attaching a polymerizable moiety to 5,5'-disubstituted Scheme 1 Hydantoin monomers with lost imide position for Cl binding (a-c), monomers containing tertiary hydrogen that can prompt the release of HCl after chlorination (d and e), a stable monomer with 2 Cl binding sites (f) and stable monomer with 3 Cl binding sites (g).
hydantoin, thereby restricting the halogen capture only at amide position [17][18][19][20][21][33][34][35][36] (Scheme 1a-c). In our previous reports, we have shown that hydantoin monomers with more than one chloramide function could be synthesized and copolymerized without sacrificing the halogen binding site in the hydantoin ring [37][38][39]. Moreover, the presence of α-H (Scheme 1d and e) close to N-Cl bond in the hydantoin moiety can lead to the elimination of HCl up on chlorination that adversely affect the bactericidal property and durability of the polymer and can cause corrosion to the surfaces [38][39][40][41]. To this end, a new hydantoin monomer (Z)-N-((4-(2,5dioxoimidazolidinn 4ylidene)methyl)phenyl)methacrylamide (DMPM) (Scheme 2, step 1) with improved chlorine binding site was synthesized and copolymerized with 3-(methacryloyloxy)propyl trimethoxysilane (MPTS) using 2,2'azobisisobutyronitrile (AIBN) as the initiator. Pretreated SSL coupons with piranha solution was immobilized with acid hydrolyzed poly(DMPM-co-MPTS) via metal-oxane bond formation. The modified SSL surface generated bactericidal chloramide function on treatment with aqueous bleach. The SSL surface challenged with Gram-positive S. aureus and Gramnegative E. coli bacteria, exhibited total kill (6 log reduction) within 10 and 12 min respectively. Moreover, the modified surface displayed potent anti-biofilm activity, long term rechargeability and durability and was nontoxic to fibroblast cell line.

Materials
Medical grade 316 SSL sheet of 0.50 mm thickness obtained from Jainex Steel and Metal, Mumbai, India was cut into 2.0 cm x 2.0 cm coupons. Diisopropylethylamine (DIPEA), 4-nitrobenzaldehyde, and ammonium acetate were obtained from Spectrochem Ltd, Mumbai, India. 3-(methacryloyloxy)propyltrimethoxysilane) (MPTS), methacryloyl chloride, palladium on activated charcoal and hydantoin were obtained from Alfa Aesar, Mumbai, India. 4-Dimethylamino pyridine (DMAP), AIBN, N,N-dimethyl formamide (DMF), barium oxide, sodium hypochlorite, silica gel (100-200 mesh) and hexane were obtained from Sigma Aldrich or HiMedia Labs, Mumbai, India. All other reagents and chemicals were of analytical grade. Milli Q water was employed throughout. MPTS was washed with aqueous sodium hydroxide followed by water to remove the inhibitors, dried over anhydrous sodium sulfate and distilled under reduced pressure before use. Dry DMF was prepared by drying the solvent overnight over barium oxide followed by vacuum distillation.

Characterization
Nuclear magnetic resonance (NMR) spectra were recorded on AVANCE III 500 MHz spectrophotometer (Bruker, Germany) with DMSO-d6 as the solvent. Fourier transform infrared (FTIR) spectra were taken using a Perkin Elmer Spectrum1 FT-IR instrument using KBr pellets. Thermo gravimetric analysis (TGA) and differential scanning calorimetry (DSC) were carried out using a Perkin Elmer (TGA7, Q500 Hi-Res TGA) and Perkin Elmer (DSC7, Q200 MDSC) instruments respectively, at a heating rate of 10 °C min-1 under a nitrogen atmosphere. X-ray photoelectron spectroscopy (XPS) was done on Ulvac-Phi Quantes (Japan) instrument with Mg Kα radiation. The elemental composition of the polymers was determined using a CHNS/O Perkin Elmer 2400 series analyser. ELISA microplate reader, Spectrum Max 5 (Molecular Devices, USA) was employed to evaluate cell viability. Gel permeation chromatography (GPC) was used to determine the weight average molar mass of the copolymer in a Shimadzu (Japan) instrument. THF was used as the mobile phase at a flow rate of 1 mL min -1 with R401 refractive index detector and a 7725 Rheodyne injector. Polystyrenes of known MW (Jordi Labs, USA) were used as the standards. 3-D surface topography of the unmodified (Control) and modified surface was visualized using AFM (Park System, XE-100 AFM) in tapping mode. Images are shown for an area of 10 μm × 10 μm.

Synthesis of hydantoin monomer
To synthesize hydantoin monomer, initially 4nitrobenzaldehyde (5 g, 33.1 mmol), ammonium acetate (5.1 g, 66.2 mmol) and hydantoin (5.1 g, 51 mmol) were dissolved in 80 ml glacial acetic acid. The reaction mixture was refluxed for 20 h and the resulting precipitate was filtered, washed with water followed by ethanol and dried under vacuum to obtain (Z)-5-(4nitrobenzylidene)imidazoline-2,4-dione (NBID). Further, NBID (3 g, 12.86 mmol) was dissolved in mixture of anhydrous THF (30 ml) and DMF (50 ml), followed by addition of 300 mg of 10% palladium on activated charcoal (Pd/C) as a catalyst. The reaction mixture was stirred for 20 h under hydrogen atmosphere to obtain (Z)-5-(4aminobenzylidene)imidazoline-2,4-dione (ABID). Finally, ABID (2 g, 7.37 mmol) was dissolved in anhydrous DMF under nitrogen atmosphere, DIPEA (1.92 ml, 11.06 mmol) and catalytic amount of DMAP were added followed by drop-wise addition of methacryloyl chloride (0.87 ml, 8.84 mmol) at 0 °C. The reaction mixture was magnetically stirred for 4 h at room temperature to complete the reaction. The mixture was diluted with ethyl acetate and washed with aqueous NaHCO 3 followed by water, dried over anhydrous Na 2 SO 4 and distilled under vacuum to remove the solvent. The obtained residue was purified through column chromatography on silica gel to yield DMPM

Synthesis of copolymer and surface modification of SSL
A 25 ml pressure tube was charged with different concentrations of DMPM, MPTS and 0.1 % AIBN in dry THF (10 ml) and purged with N 2 for 30 min. The polymerization was allowed to proceed at 60 °C in an oil bath for 24 h. The resulting polymer was precipitated in methanol and dried under vacuum to obtain hydantoin based polymer, poly(DMPM-co-MPTS). The SSL coupons were treated with acetone, ethanol and water for 10 min in each solvent. After drying, the coupons were treated with piranha solution (H 2 O 2 /H 2 SO 4 in 7:3 volumetric ratio) for 30 min and then dried in an air oven [42]. The coupons were then immersed in 20 ml acetone/methanol (1:1) containing 1.5 g of poly(DMPM-co-MPTS) for 1h to obtain the uniform adsorption of polymer on surface [43]. The adsorbed polymer was hydrolyzed with 0.1 M HCl to convert Si-OCH3 group to Si-OH. Finally, the coupons were washed with ethanol and water and dried in N 2 atmosphere and cured at 90 °C for 1.5 h to promote the metal-oxane bond formation between SSL surface and coated polymer (Scheme 2, step 2).

Chlorination and determination of oxidative chlorine content
500 mg of the finely powdered hydantoin containing copolymer, poly(DMPM-co-MPTS) was treated with 9-12% sodium hypochlorite solution at pH 7 (adjusted by 6 N HCl) for 1.5 h. After chlorination, the polymer was washed thoroughly with water and dried at 45 °C for 1 h to remove free chlorine [37]. The oxidative chlorine content of the polymer was determined by standard iodometric/thiosulfate titration method [39]. Blank titration was performed under similar condition as control. The percentage chlorine content was calculated using following equation, Where V Cl and Vo are the volumes (mL) of the sodium thiosulfate solution consumed in the titration of the copolymer solution and the blank, respectively, and Wg is the weight of the polymer. Each test was repeated three times and the average was taken.

Antibacterial efficacy testing
Bactericidal activity of SSL surface modified with hydantoin based polymer was evaluated against representative Gram-positive S. aureus (ATCC 29213) and Gram-negative E. coli (BL21) bacteria. Initially, the target bacterial strains were allowed to grow in nutrient broth overnight at 37 °C in a rotatory shaker at 180 rpm. The bacterial cultures were harvested and washed with phosphate buffered saline (PBS, pH 7.4). A "sandwich method" was adopted to evaluate the antibacterial efficacy of the modified SSL surface. A Scheme 2 Synthesis of hydantion based polymer and functionalization on SSL surface. surface modified SSL coupons was exposed to 30 µL of bacterial suspension containing 6-6.5 log 10 CFU mL -1 , and a second surface modified SSL coupon was placed on the top of first with a sterile beaker on top to ensure the good contact of bacteria with the surface. After predetermined contact time the coupons were immersed in 5 mL 0.03 wt% sterilized aqueous sodium thiosulfate solution to quench the active chlorine and thus terminate the disinfection activity [39]. The quenched samples were vortexed, serially diluted and 100 µL of each dilution was placed on nutrient agar plate. The plates were incubated at 37 °C for 24 h to grow the colony. Unchlorinated surface was taken as negative control. The percentage reduction was calculated using the following formula, Where X is the population of CFU on the control plate and Y is the population of CFU on the test plate. The log reduction was then calculated as, The plate counting experiments were performed in triplicate using fresh bacteria inocula and statistical analysis was performed using Microsoft Excel 2013.

Biofilm growth inhibition assay
Anti-biofilm activity of SSL surface modified with hydantoin based copolymer was determined using metabolic activity based colorimetric assay [44]. Initially, the SSL coupon was placed in 24 well plate and 200 µL of bacterial culture containing S. aureus or E. coli supplemented with 0.4% of glucose was spread on the coupon surface. The Plate was then incubated under dark and humid condition for 24 h at 37 °C to allow the growth of biofilm. Further, the coupon was removed from the well and gently washed with sterile PBS to remove non adhered planktonic cells. Washed coupon was transferred into fresh 24 well plate with the addition of 1 mL of nutrient broth containing 50 µM of resazurin sodium salt to quantify the metabolic activity of the biofilm formed on the surface. The fluorescence intensity of the solution was measured at room temperature using an excitation filter at 530 nm and an emission filter at 580 nm in a fluorescence reader.
SEM based imaging of E.coli and S. aureus were also employed to determine the potential of SSL surfaces for inhibition of biofilm growth [45]. 200 µL of targeted bacterial suspension supplemented with 0.4 % of glucose was spread on SSL coupon modified with chlorinated polymer. Further, the coupon was incubated under dark at 37 °C for 24 h to grow the biofilm. The coupon was removed from the well plate, gently washed with water, fixed with 2.5% glutaraldehyde followed by drying in a sterile environment, sputtered with gold imaged using SEM. The same procedure was applied for pristine or SSL surface modified with unchlorinated polymer that served as control.

Durability and regenerability testing
Recharge and discharge cycles were performed to evaluate the durability and regenerability of SSL coupons modified with chlorinated hydantoin polymer as reported before [46]. Immersion of chlorinated coupons to quench the active chlorine in 0.03% of aqueous sodium thiosulfate solution is termed as "discharge". Further, the coupons were washed and treated with 9-12 % of sodium hypochlorite solution to load the polymer with active chlorine which is termed as "recharge". The chlorine content and bactericidal activity were determined after 5, 15, 25 and 45 cycles to determine the durability and regenerability of the modified SSL surface.

In vitro cytotoxicity assay
The cytotoxicity assays were performed using the MTT based colorimetric method [47]. The NIH/3T3 cells were initially propagated in 25 ml tissue culture flask containing DMEM supplemented 10% FBS. Subsequently, the cells were seeded in 48well plates (1.0 × 10 4 cells / well) and incubated in 5% CO 2 at 37 °C for 24 h. The medium was replaced with fresh 10% FBS-containing DMEM, followed by the addition of polymer coated with chlorinated or unchlorinated SSL surface. After 48 h, MTT solution (5 mg mL −1 ) was added and the plates were incubated for 4 h at 37 °C. The supernatant was gently removed and the formazan crystals were solubilized in DMSO and the optical density was read at 570 nm in a mutiplate reader.
For fluorescence-based imaging, NIH/3T3 cells were seeded in 48-well plate and incubated at 37 °C for 24 h. Further, the medium was replaced with fresh 10% FBS-containing DMEM, followed by the addition of polymer coated with chlorinated or unchlorinated SSL surface. After 48 h, the modified SSL coupon was removed, the media was aspirated and cells were washed 4 times with PBS to remove dead cells. To determine cell viability and nucleus integrity, FDA and DAPI staining methods were employed [48]. Stained cells were excited at 488 nm (FDA) and 360 nm (DAPI) to visualize and image under a fluorescence microscope.

Statistical Analysis
Statistical analyses were performed using Prism (v5.0, GraphPad Software, USA). The data were assessed with ordinary one-way ANOVA followed by a Tukey posthoc test. A value of p < 0.005 was considered as significant.

Synthesis hydantoin monomer
Initially, (Z)-5-(4-nitrobenzylidene)imidazoline-2,4-dione (NBID) was synthesized and Characterized by NMR ( 1 H and 13 C) and FTIR. Tethering of the hydantoin moiety to 4-nitrobenzaldehyde was confirmed by amide and imide proton signals at 11.41 ppm and 10.89 ppm for hydantoin ring in 1 H NMR spectrum ( Figure S1). In addition, 13 C NMR spectrum showed the presence of amide and imide carbonyl carbon signals at 165.70 ppm and 156.23 ppm respectively ( Figure S2). In the FTIR spectrum, the peaks at 3225 cm -1 and 1748 cm -1 were attributed to NH and C=O respectively to confirm the synthesis of NBID ( Figure S3a). Further, the appearance of two characteristic bands for primary amine around 3230 cm -1 confirmed the reduction of nitro group to amine suggesting the formation of ABID ( Figure S3b). The introduction of the singlet peak at 4.89 ppm for amine group in the 1 H NMR also confirms the synthesis of the compound ( Figure  S4). Finally, the polymerizable moiety was tethered by amide bond formation with the methacryloyl chloride to give rise to hydantoin monomer. In the 1 H NMR, the appearance of signals at 6.33 ppm, 5.79 ppm and 1.94 ppm for olefinic protons and -CH 3 respectively suggested the attachment of polymerizable moiety (Figure 1). In addition, the arrival of peaks at 167.30 ppm, 140.71 ppm, 114.50 ppm and 19.12 ppm for C=O, =C-, =CH 2 and CH 3 respectively in the 13 C NMR reflected the formation of hydantoin monomer ( Figure  S6). Further the presence of a peak at 1640 cm -1 for -C=C stretching in FTIR spectrum suggested the formation hydantoin monomer (Figure 2 a).

Synthesis of copolymer and surface modification of SSL
The copolymerization of hydantoin monomer DMPM with MPTS was carried out via free radical polymerization. The resulting copolymer was characterized by GPC, FTIR, NMR, and XPS. The weight average molecular weight (Mw) and number average molecular weight (Mn) of the polymers containing 10 mol% of hydantoin monomer were 13500 Da and 5300 Da respectively with polydispersity index of 2.54 ( Figure S7). The FTIR spectrum of hydantoin monomer (DMPM) showed the characteristic peaks of -NH, C=O, -C=C and corrugated signal for aromatic ring at 2980 cm -1 , 1750 cm -1 , 1640 cm -1 and 698 cm -1 respectively (Figure 2a). Spectrum of pristine polymer (PMPTS) showed the peak for -CH, C=O and Si-OCH 3 at 3200 cm -1 , 1750 cm -1 1200 cm -1 respectively (Figure 2b). In the copolymer poly(DMPM-co-MPTS), the disappearance of stretching vibration of -C=C suggested the introduction of hydantoin monomer in the copolymer. Other characteristic peaks of hydantoin monomer were merged in the polymer peak (Figure 2c). In the 1 H NMR spectrum, the appearance of signals at 11.28 ppm and 10.59 ppm confirmed the incorporation of hydantoin monomer in the polymer ( Figure 3). Moreover, the disappearance of olefinic proton from hydantoin monomers (DMPM) and commercial monomer (MPTS) in the NMR spectrum suggested the formation of copolymer via free radical polymerization. It should be pointed out that we have not determined the copolymer composition in these copolymers since the reactivity of these two monomers may be different. In addition, XPS analysis was performed to understand the surface chemistry of the polymer (Figure 4a). The XPS spectrum of poly(DMPM-co-MPTS) exhibited an additional peak for N1s at 400 eV along with C1s and O1s at 400 eV and 285 eV respectively suggesting the incorporation of hydantoin monomer in the copolymer. Further, the deconvolution of C1s peak revealed the peak division for C-N along with C-C, C-O and C=O again suggesting the incorporation of the hydantoin monomer in the copolymer. Signal processing of N1s revealed the peak division for amide and imide nitrogen. Peaks assigned to the photoelectrons from Si 2s and Si 2p at 154 and 101eV from the base polymer confirmed the siloxane moiety. The synthesized polymer was employed for surface modification of SSL. Surface was characterized using XPS figure 4b). SSL surface treated with the piranha solution exhibited the intense peak of O 1s (43.8 %) at 532 eV attributed to surface oxidation treatment (Figure 4bi) as compared to (35. 9%) for the C 1s peak. Further, the SSL surface modified with polymer exhibited the N 1s, Si 2s and Si 2p peaks at 400, 154 and 101 eV respectively along with C 1s and O 1s confirming the coating of polymer over surface (Figure 4bii). In this, oxygen (O 1s at 532 eV, 36.4%), carbon (C 1s at 285 eV 57.4%) nitrogen (N 1s at 400 eV, 2%) and Silicon (Si 2s and 2p at 101 and 154 eV, 4.2%) were obtained.   Figure 4biii). Further, the surface topography of control and modified stainless steel were visualized in tapping mode using atomic force microscopy ( Figure S9). Control surface exhibited the surface roughness of 4.5 nm, whereas the surface after functionalization with hydantoin based polymer showed surface roughness of 6.4 nm. The increase in roughness is possibly due to formation of small patches at few places. Most of the coated surface exhibited the uniform distribution of the polymer that may provide active bactericidal activity across the surface.

Elemental analysis
Carbon, hydrogen and nitrogen contents of the polymer were quantified using CHNOS analyzer in CHN mode. Iodometric titration method was employed to quantify the oxidative chlorine content in the chlorinated polymer. Expected enhancement of active chlorine and nitrogen content was observed upon increasing the feed ratio of hydantoin monomer in the copolymerization reaction (Table S1).

Thermal analysis
Thermal stability of the pristine polymer and the copolymer were analyzed using thermogravimetry (TGA) and differential scanning calorimetry (DSC). The TGA exhibited similar degradation pattern for both the polymers [ Figure S7a), The thermograms of both the polymers showed 10% degradation around 340 °C , whereas more than 50 % degradation was observed around 500 °C and only 30 % of the polymer was left at 900 °C. The observed weight loss in the polymers can be attributed to the condensation and combustion of the organic groups. From these results, it was concluded that there was no additional thermal instability observed due to hydantoin monomer. The DSC of the pristine polymer and its corresponding copolymer are shown in (Figure S7b). The glass transition temperature (Tg) of the copolymer modified with the hydantoin monomer was found to be 115 °C. The Tg of the copolymer was similar to the pristine polymer suggesting that the introduction of the hydantoin monomer did not create thermal instability in the copolymer.

Antibacterial assay
The antibacterial activity of the modified SSL surface was evaluated through plate counting method. This study was performed with fresh inoculums (varying between 6-6.5 log 10 CFU mL -1 for both the strains) for each experiment in triplicates. The pristine SSL was treated as the control and compared to modified  and chlorinated surface for both S. aureus and E. coli. Surface modified with chlorinated hydantoin polymer exhibited time dependent decrease in bacterial population attributed to its exceptional antibacterial activity against both the strains (Figure 5a and b). Surface modified with the copolymer containing 10 mol% of hydantoin monomer exhibited improved bactericidal activity than the surface modified with 5 mol% of hydantoin monomer. Copolymer with 10 mol% of hydantoin monomer showed 2.1 log reduction and 4.6 log reduction in 5 min and 8 min respectively. While a total kill was observed in 10 min of exposure to S. aureus, in the case of E. coli, total kill was observed in 12 min of exposure due to the presence of characteristic extracellular polysaccharide layer. However, copolymer with 5 mol% of hydantoin monomer required 15 min to exhibit the total kill against both the strains. The antibacterial study of hydantoin polymer suggested that an increase in Cl binding sites or chlorine content in polymer improved the biocidal property. Further, the optical micrograph images were taken for the pristine SSL (control) and surface modified with the copolymer containing 10 mol% of hydantoin monomer. The control surfaces exhibited the dense growth of bacterial cell for both the strains. However, the surfaces modified with 10 % hydantoin monomer exhibited time dependent decrease in the colony formation on the plates (Fig 5 c and d).
In order to examine whether a higher hydantoin monomer content in the copolymer would give rise to better bactericidal properties in the copolymer, we raised the hydantoin monomer content to 15 mol%. The oxidative chlorine content of copolymer containing 15 mol% hydantoin monomer was found to be 1.68 % compared to 1.50 % of the 10 mol% containing hydantoin monomer. We performed the antibacterial assessment with copolymer containing of 15 mol% hydantoin monomer and could not observe significant difference as compared to the polymer containing 10 mol% of hydantoin monomer ( Figure S10). Moreover, on increasing the monomer content to 15 mol% in the feed, the copolymer was found to lose its physical strength and was not suitable for multiple antibacterial tests in the durability and rechargeability assay.

Anti-biofilm assay
Biofilm growth on pristine or modified SSL surface was quantified by measuring the metabolic activity using resazurin based colorimetric assay (Figure 6a). SSL surface modified with unchlorinated polymer containing 10 mol% of hydantoin monomer exhibited similar metabolic activity to the control (pristine SSL surface). The surface modified with chlorinated hydantoin polymer showed significant reduction in the metabolic activity and hence biofilm growth. Moreover, the biofilm growth inhibition increased with increase in the hydantoin monomer ratio in the copolymer.
Surface modified with the chlorinated copolymer containing 5 or 10 mol% of hydantoin monomer exhibited 12 % (S. aureus) and 15% (E. coli) or 5 % (S. aureus) and 7 % (E. coli) of biofilm growth respectively. The surface modified with chlorinated hydantoin polymer showed significant reduction in the metabolic activity and hence biofilm growth. Moreover, the biofilm growth inhibition increased with increase in the hydantoin monomer ratio in the copolymer. Surface modified with the chlorinated copolymer containing 5 or 10 mol% of hydantoin monomer exhibited 12 % (S. aureus) and 15% (E. coli) or 5 % (S. aureus) and 7 % (E. coli) of biofilm growth respectively. SEM based image analysis undertaken to complement the metabolic activity measurements also suggested that surface modified with chlorinated polymer exhibited comprehensive damage in the biofilm growth (Figure 6b and c). The surface modified with unchlorinated polymer displayed compact protrusions and surface corrugations typically associated with mature biofilm architecture. Whereas, the surface modified with chlorinated hydantoin polymer showed the disaggregated biofilm growth.

Durability and regenerability
The durability and regenerability were determined by estimating the oxidative chlorine content and the antibacterial activity of the surface modified with copolymer containing 10 mol% hydantoin monomer. The modified surface was stable for 45 dischargerecharge cycles, and was able to retain 70 % chlorine content. The 30 % loss of chlorine binding ability (structural integrity) could be due to repetitive exposure of the polymer to the harsh bleaching condition (Table 1). Surfaces remain active for 3 cycles of antibacterial testing after each charge. After 3 cycles, surfaces start losing its activity and after 5 cycles, it does not show any activity.

In Vitro Cytotoxicity Assay
Materials with applicability in clinical setting, water purification systems and food processing and storage units need to go through the evaluation for biocompatibility. To this end, modified surface was tested for cytotoxicity on NIH/3T3 fibroblast cells using MTT based assay. In comparison with the control (tissue culture plate), SSL surface modified with the unchlorinated or chlorinated copolymer containing 10 mol% of hydantoin monomer exhibited negligible cytotoxicity against the fibroblast cell line (Figure 7a). However, when the surface was modified with the chlorinated copolymer containing 15 mol% hydantoin monomer it exhibited about 20 % cytotoxicity possibly due to higher chlorine content. In support of MTT based cytotoxicity assay, fluorescence microscopic analysis of cells treated with modified surface was also carried out. Cells treated with the unchlorinated or chlorinated copolymer containing 10 mol% of hydantoin monomer showed the large number of FDA and DAPI stained cells as control, indicating the comparable viable cells in treated and control. Whereas, cells treated with the surface modified with the chlorinated copolymer containing 15 mol% of hydantoin monomer showed lesser number of FDA or DAPI stained cell (viable cells), indicating the slight cytotoxic nature of the copolymer. MTT assay and fluorescence analysis indicated the nontoxic nature of the surface modified with the copolymer containing 10 mol % of hydantoin monomer suggesting potential application in biomedical and food processing industries.

Conclusion
In this study, a new hydantoin monomer containing three Cl binding sites was synthesized and copolymerized with the commercially available siloxane based monomer, MPTS. The Figure 7. Cytotoxicity assay of SSL surface modified with unchlorinated and chlorinated polymer. a) MTT based cytotoxicity assay of unchlorinated polymer containing 10 mol% (i) 15 mol% (ii) and chlorinated polymer containing 10 mol% (iii) 15 mol% (iv) of hydantoin monomer; b) Fluorescence based cytotoxicity assay of unchlorinated polymer containing 10 mol% (i) and 15 mole % (ii), chlorinated polymer containing 10 mol% (iii) and 15 mol% of hydantoin monomer (iv). synthesized copolymer was covalently functionalized onto SSL surface via metal oxane bond to provide antibacterial and antibiofilm coating. Surface functionalized with copolymer containing 10 mol% of hydantoin monomer exhibited total kill in 10 min against S. aureus and 12 min against E.coli. Fluorescence based metabolic activity along with SEM imaging revealed the excellent anti-biofilm activity of the SSL surface modified with the hydantoin based copolymer. Lack of tertiary hydrogen in the hydantoin ring aborted the possible release of HCl and improved the rechargeability and durability of coating even up to 45 dischargerecharge cycles. The coating was found to be biocompatible when tested against fibroblast cell line. The excellent antibacterial and anti-biofilm activity, long-term rechargeability and biocompatibility of modified SSL surface makes it a good candidate material for use in food processing and other biomedical and health-care applications where prevention of bacterial adhesion and biofilm formation are highly desirable.