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Photoactivable surface of natural poly(3-hydroxybutyrateco-3-hydroxyvalerate) for anti-adhesion applications Romain Poupart, Adnan Haider, Julien Babinot, Inn-Kyu Kang, Jean-Pierre Malval, Jacques Lalevée, Samir Abbad Andalloussi, Valérie Langlois, and Davy-Louis Versace ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.5b00002 • Publication Date (Web): 24 May 2015 Downloaded from http://pubs.acs.org on June 1, 2015

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ACS Biomaterials Science & Engineering

Photoactivable surface of natural poly(3-hydroxybutyrate-co-3-hydroxyvalerate) for anti-adhesion applications

R. Poupart a, A. Haider b, J. Babinot a, I.-K. Kang b, J.-P. Malval c, J. Lalevée c, S. Abbad Andalloussi d, V. Langlois a and D. L. Versace *a

[a]

Institut de Chimie et des Matériaux Paris-Est, Equipe Systèmes Polymères Complexes,

UMR 7182, CNRS-Université Paris-Est Créteil Val de Marne 2-8 rue Henri Dunant – 94320 Thiais, France. [b]

Department of Polymer Science and Engineering, Kyungpook National University, Daegu 702-701, South Korea

[c]

Institut de Science des Matériaux de Mulhouse, IS2M-LRC 7228, 15 rue Starcky - 68057 Mulhouse, France.

[d]

Unité Bioemco Equipe IBIOS, UMR 7618 CNRS - Université Paris-Est Créteil Val-deMarne, 61, Avenue Général de Gaulle, 94010 Créteil cedex.

Corresponding author: [email protected]

ACS Paragon Plus Environment


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ABSTRACT. A green photoinduced method for the modification of a biodegradable and biocompatible polymer, Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBHV) has been successfully carried out using two types of monomers with potential antibacterial effects, i.e. 2-[(methacryloyloxy)-ethyl] trimethylammonium chloride (META) and an ampicillin-derived monomer. The photografting process is conducted through a photoinduced free-radical process employing a thiocarbamate-based photoinitiator in an aqueous medium. Under appropriate conditions, radicals are generated from the PHBHV surface, thus initiating the UV-mediated photopolymerization of methacrylate or methylacrylamide-derived monomers from the surface of PHBHV films. The photochemical mechanism of the thiocarbamate photolysis is entirely described by the Electron Spin Resonance / Spin-Trapping technique and Laser Flash Photolysis, and the modified-PHBHV films are extensively characterized by fluorescence experiments, water contact angle and XPS measurements. Finally, a primary investigation is conducted to support the antibacterial property of the new functionalized films against Escherichia coli and Staphylococcus aureus, and their cytocompatibility with NIH3T3 fibroblastic cells is evaluated.

KEYWORDS.

Photochemistry,

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate),

photografting, antibacterial properties.


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INTRODUCTION Infections by pathogenic microorganisms are of great concern in many fields, particularly in medical devices, hospital surfaces/furniture, and surgery equipments. Statistics reveal that the average incidence of secondary infections affects 8% of all hospitalized patients (10% in Great Britain, 6.7% in Italy and 8.7% in Finland). In the United States, 1.7 million cases of nosocomial infections per year are recensed, thus causing over 90,000 deaths a year. It is estimated that the annual cost of treatment of these infections ranges from 4.5 to 11 billion US dollars1. Despite important recent progress in the development of nanobiotechnology, nanofabrication and polymerization techniques, the willingness of scientists to design and elaborate novel antibacterial surfaces remains a high research priority2. In order to limit and prevent the bacterial colonization of materials surface, intensive efforts have been focused on the modification of the surface architecture, on the improvement of the existing antibacterial surfaces or on the fabrication of new generation surfaces. Two different strategies could be proposed, i.e the passive (antifouling) and active (antimicrobial) strategies3. Passive strategies rely on inhibition of bacteria attachment, typically through physical prevention of nonspecific cell attachment by a grafted polymer coating, whereas with active strategies the antibacterial compound actively promotes bacterial killing by interfering with biological pathways leading to toxic effects. Among the passive systems, perfluorinated molecules4 or polymers based on ethylene glycol5 are the most widely used synthetic materials used to reduce nonspecific protein adsorption. However, the oxidation of PEG units into reactive aldehyde moieties in the presence of transition metal ions and oxygen found in biologically relevant solutions6 has forced the scientific community to design new strategies for creating novel active antibacterial surfaces. The first active approach has focused on developing coatings that produce reactive radical



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species which have no specific target within bacteria upon light activation7. The second approach has involved biocide leaching, namely use of antibiotics8, cytotoxic organic9, inorganic compounds10, metal nanoparticles11 or flavonoids12 which diffuse over time from a polymer material, thus inducing death of adhered bacteria. Unfortunately, antimicrobial surface treatments erode quickly which has serious adverse effects on the durability of the modified materials. Another serious issue with the use of leaching-materials is that the released compounds increase drug resistance throughout the microbial realm and materials need to be regenerated to maintain their activities. Covalently attaching antimicrobial systems to the surface presents an efficient alternative in the production of stable surface-active bactericidal materials. Therefore a final approach which induces the biochemical death of bacteria has been proposed. A wide assortment of antibacterial polymers13 exists and most rely on cationic functionalities that are theorized to penetrate cell membrane that disrupts the membrane integrity and provokes cell lysis; polymers containing phosphor- or sulfoderivatives14 and quaternary amine functionalities15 are common. Polymers presenting phenol derivatives16 and zwitterionic polymers17 have also been synthesized. Other examples of synthetic polymers based on traditional antimicrobials including fluoroquinolones18, various antifungals19, vancomycin20 or ampicillin21 could be cited. To the best of our knowledge, no reports have hitherto been published on the photografting of the common antimicrobial previously cited on biocompatible and biodegradable

surfaces such as poly(3-

hydroxyalkanoate)s (PHAs) to develop antibacterial materials with cytocompatible properties. In this study, ampicillin has been chosen because it belongs to the penicillin group of betalactam antibiotics, and remains potent against gram-positive and gram-negative bacteria. Ampicillin acts as a competitive inhibitor of the enzyme transpeptidase, which is needed by bacteria to make their cell walls. Therefore, an ampicillin-derived monomer has been


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designed, covalently grafted on a PHAs surface and its final antibacterial property has been compared with a quaternary ammonium polymer. Poly(3-hydroxyalkanoate)s (PHAs) constitute an enlarged family of biodegradable and biocompatible aliphatic polyesters22 produced by many bacterial microorganisms when subjected to stress conditions. They can be considered as promising biopolymers and have attracted much interest for a variety of medical applications23, which include controlled drug release, fracture repair, bone and cartilage remolding, and tissue engineering in general. To enhance their properties, the direct surface modification of PHBHV films appears as a real challenge. Many physical or chemical modifications have been developed in order to tune PHAs film surface properties23a, b, 24. However, in order to preserve the integrity of the film, mild grafting conditions are required. To this end, photoinduced grafting represents a promising way to introduce functional groups on PHBHV surface. Indeed, this technique is widely known to be a useful “green method” for the functionalization of polymeric materials due to its significant advantages, such as low cost of operation, innovative technology and mild conditions. The photopolymerization process is a substrate-independent method allowing for the covalent deposition of a broad range of polymers25. These technical aspects make photopolymerization a particularly useful method for surface modification strategies. Few studies have described so far the potentialities offered by the photoinduced graft polymerization method for the film surface modification of PHBHV. Its feasibility was essentially demonstrated through “grafting-from” polymerization with the use of benzophenone26, a photosensitive system based on aryl azides27, hydrogen peroxide28, triarylsulfonium salts24c or butan-2-one23b, 24b. The novelty of the results presented in this paper lies in the development of a mild and simple method, that uses an aqueous photoinitiating strategy with the use of a photoiniferter, to efficiently functionalize and tailor the surface properties of natural poly(3-hydroxybutyrate-



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co-3-hydroxyvalerate) films with two kind of polymers having antibacterial properties, i.e a quaternary ammonium and an ampicillin-derived monomers. The lack of existing research on this subject prompted us to examine more closely, both the effect of a thiocarbamate photoinitiating system on the modification of the surface of PHBHV films and the resulting antibacterial and cytocompatibility properties of these biomaterials. The first part of this study demonstrates the efficiency of the covalent grafting of the thiocarbamate-derived initiator by X-ray photoelectron spectroscopy (XPS), the mechanism of polymerization by Electron Spin Resonance Spin-Trapping and Laser Flash Photolysis, and the synthesis of an ampicillin-derived monomer (characterized by Fourier transform infrared spectroscopy (ATR-FTIR) and 1H NMR). In the second part, an effort is done to demonstrate the efficiency of the photografting of 2-(methacryloyloxy)-ethyl trimethylammonium (META) and the ampicillin methacrylamide monomer on PHBHV film surface using XPS, ATR-FTIR, and water contact angle measurements. To validate the study, the antibacterial property of the modified PHBHV films against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus), and their cytocompatibility with NIH-3T3 fibroblastic cells are finally evaluated.

EXPERIMENTAL SECTION Materials. Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBHV) with 12% of 3hydroxyvalerate (HV) and a molar mass of 90,000 g/mol was purchased from GoodFellow. PHBHV was first purified by dissolution in chloroform for 2 h (10 % w / v) and precipitated in

ethanol

for

removing

citric

ester

used

as

plasticizer.

2-(methacryloyloxy)-

ethyltrimethylammonium (80%, META), N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (>98%, EDC), (N,N-diethylthiocarbamoylthio)-acetic acid; toluidine blue; acetic acid (>99,7%), methacrylic acid (MAA, 99%), sodium phosphate buffer (pH=8) and


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methacrylic acid N-hydroxysuccinimide ester (98%, NHS META) were purchased from Sigma-Aldrich. Ampicillin, ethylenediamine (>99%) and N-hydroxysuccinimide (98%, NHS) was provided by Alfa Aesar. Methylene chloride, propan-2-ol and ethanol were supplied by Carlo Erba. All of the aqueous solutions were prepared using ultrapure water. The structures of the respective monomers and the photoinitiating system are shown in Table 1.

Table 1. Structure of the monomers/polymer and the photoinitiating system used in this study. Compounds

Function

Poly(3-hydroxybutyrate-co-3hydroxyvalerate)

Polymer

Structure O H

O

O O

n

HB

[2(Methacryloyloxy)ethyl]trimethylammonium chloride(META). Ampicillin

Methacrylate monomer

p

OH

HV

Cl O

N

O

-

(N,N-diethylthiocarbamoylthio)-acetic acid

Photoinitiating system

S N S O HO

Preparation of PHBHV films.To get rid of the plasticizer (citric acid), PHBHV granules (20 g) were dissolved in 200 mL of methylene chloride, and stirred at 50 °C until the solution became completely homogenous. The solution was then precipitated in ethanol solution (1.5 L). The pure PHBHV was obtained after filtration and dried under vacuum at 40°C during one night. Pure PHBHV powder was placed between two teflon films and baked at 170°C for 5



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min under pressure of 2 bars. Films with a thickness of 30 µm were obtained and cut into pieces of 1.5 cm x 1.5 cm. Aminolysis of the PHBHV films. Two PHBHV films were introduced into a round-bottom flask containing 4 mL of ethylenediamine and 59 mL of propan-2-ol during 1 hour at 37°C. Films are washed 24 hours in water under agitation and dried one night under vacuum at room temperature. The morphology of the PHBHV surface does not change. These results are confirmed by the review of Gao et al29 concerning the aminolysis-based surface modification of polyesters for biomedical applications. Synthesis of the Ampicillin monomer. The synthesis is a two-step reaction. In a first step, the ampicillin is dissolved in a pH=9 water solution (pH is adjusted with a 1M NaOH solution) and then lyophilized in order to collect the ionised form of the ampicillin (COONa+). In a second step, 750 mg of ionized ampicillin and 430 mg of NHS-META are dissolved in 20 mL of water then pH is adjusted to 9. The reaction took one night at room temperature and the solution was then lyophilized in order to prevent Ampicillin degradation. The product was analysed with 1H NMR (Brüker Ultra Shield 400, the proton Larmor frequency is at 400 MHz). Ampicillin monomer (C20H22O5N3S): 1H NMR (D2O, 400 MHz): δ- 1.41 (s, 3H), 1.47 (s, 3H), 1.93 (s, 3H), 4.16 (s, 1H), 5.47 (s, 2H), 5.50 (s, 2H), 5.72 (s, 1H), 7.44 (m, 5H). To test the stability of the monomer, 30 mg of the ampicillin-derived monomer was dissolved in 1 mL of D2O and irradiated with the UV lamp during 360s. The irradiated monomer was analyzed by 1H NMR, and showed no difference with the not irradiated, thus proving its stability toward light irradiation. Photografting procedures. Aminolysed filmswere placed in an acetonitrile solution (4 mL) containing 9.1 mg of EDC, 5.7 mg of NHS and 5.0 mg of (N,N-diethylthiocarbamoylthio)acetic acid during 60 h. The solution was protected from the light with an aluminium film.


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Films were intensively washed during 4 h with acetonitrile and dried one night under vacuum at room temperature. The PBHHV films were soaked respectively into two water solutions of META (0.1 M) and ampicillin-derived monomer (0.1 M). During the irradiation, a polypropylene film, which is transparent to UV light, was laid on the top of the PHBHV film to avoid oxygen diffusion inside the solution. Both sides of the PHBHV films were irradiated at room temperature at a distance of 11 cm of a flexible guide coupled with a Lightningcure LC8 (L8251) from Hamamatsu equipped with a mercury-xenon lamp (200W). The maximum UV light intensity at the sample position was measured by radiometry (International Light Technologies ILT 393) to be 180 mW.cm-2 in the 250−450 nm. The irradiation time was 240 s for the META solution and 360 s for the ampicillin methacrylamide solution. Then the films were washed during 4 h in water then dried one night under vacuum at room temperature. Quantitation of surface COOH Density. To demonstrate the feasibility of the grafting method, experiments were first done with methacrylic acid (MAA). The density of carboxylate groups on the MAA-grafted PHBHV films was determined using the toluidine blue method. Toluidine blue is a dye which possesses positively charge amine groups and can absorb to the negatively charged carboxylate groups of grafted-MAA, but not to the native PHBHV film surface. Samples were dipped in toluidine blue (0.1 wt %) in sodium phosphate buffer (20 mM, pH=8) for 5 min, rinsed with water and dried in a nitrogen stream. Toluidine blue is then desorbed from the PHBHV-g-PMAA surface by dipping the film in 25 mL of 10 wt % of acetic acid for 10 min. The maximal optical absorption of the dye (at 630 nm) released from the PHBHV-g-PMAA surface was measured using a spectrophotometer (Varian, Cary 50 Bio). The density of COO- groups on the PHBHV-g-PMAA surface (expressed as mol/cm2) was determined on the hypothesis of a 1:1 ratio between COO- and bound toluidine blue.



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Attenuated Total Reflection-FTIR (ATR-FTIR) Spectroscopy: The FTIR spectra of PHBHV and PHBHV-modified films were recorded with a Bruker Tensor 27 spectrometer, equipped with an attenuated internal reflection accessory using a diamond crystal. Infrared spectra were collected at a resolution of 4 cm-1 with an accumulation of 32 scans. For the Fourier transformation of the interferogram, a Blackman-Harris-3-term apodization function was selected as well as a zerofilling factor of 2 and a standard Mertz procedure for phase correction. Electron Spin Resonance / Spin-trapping (ESR-ST) experiments were carried out using an X-Band spectrometer (Bruker EMX-plus Biospin). The radicals were generated under argon at room temperature using a polychromatic light irradiation (Xe-Hg lamp; Hamamatsu, L8252, 150 W) and trapped by phenyl-N-tertbutylnitrone (PBN). The ESR spectra simulations were done with the PEST WINSIM program. All of the samples were prepared in a 6 mm quartz cylindrical tube and dissolved in tert-butylbenzene as an inert solvent. Laser Flash Photolysis (LFP) analysis. The nanosecond laser flash photolysis set up working at 266 nm is based on a nanosecond Nd:TAG laser (Powerlite 9010, Continuum) operating at 10 Hz with a 5 mJ / pulse energy. The analyzing system (LP900, Edinburgh Instruments) used a 450-W pulsed xenon arc lamp, a Czerny-Turner monochromator, a fast photomultiplier and a transient digitizer (TDS 340, Tektronix). The sample was contained in a 1-cm cell. Measurements were done at room temperature. UV-visible spectroscopy. UV-Vis spectra were recorded on a Varian spectrophotometer (Cary 50 bio) in the range 250 to 800 nm. All the solutions were degassed with argon during 5 min before use. Fluorescence microscopy. Inverted microscope IX73 from Olympus equipped with a 75W Xe Lamp housing. The excitation and emission light is filtered with a fluorescence mirror


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unit (U-FUN from Olympus) associating a band pass filter centered at 365 nm (BP360-370), dichroic mirror (DM410) and long pass filter (BA420IF). Static contact angle measurements. The water contact angles were measured using standard methods. In all the experiments described in this study, static contact angles were measured using a goniometer from Krüss (Easy Drop Krüss). The first step of the measurement was to deposit a water drop of defined volume on the PHBHV film surface. To apply reproducible and uniform volume drops of deionized water, calibrated micropipettes were used. The volume of the water drop was in the range of 15-20 µL. Drop shape was automatically recorded with a high speed framing camera, images were then processed by a computer and stored. The uncertainty in the measurements depends on the light-dark contrasts of the drop picture and an error of 3 - 4° could be assumed. Measurements have been done 12 times for each film. Anti-adherence property. Initial adhesion assays were performed using two strains of bacteria, namely E. coli ATCC25922 and S. aureus ATCC6538 on the PHBHV modified films.

Prior to in vitro antibacterial tests, the bacterial strains were grown aerobically

overnight in Luria–Bertani broth at 37 °C under stirring. Overnight cultures of E. coli and S. aureus grown in Luria–Bertani (LB) broth were diluted to an optical density (OD 600 nm) of 0.05 in sterile LB broth. At this point, the reference and modified films (1.5cm x 1.5cm) were immersed in the culture; the corresponding vials were placed on a slantwise rotating wheel to avoid sedimentation of bacteria, incubated for 1 h at 37 °C and shaken at 150 rpm to allow initial adhesion to occur (INFORS AG-CH 4103, Bottmingen-Basel, Switzerland). Following initial adhesion (1 h), the samples were rinsed seven times with sterile saline solution (NaCl, 0.9% w/v) to remove any non-adherent cells. Colonized native and treated PHBHV films were then transferred to 2 mL sterile saline (solution A) and vortexed vigorously for 30 s. The samples were then transferred to 2 mL



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sterile saline (solution B) and sonicated in a Branson 2200 sonicator for 3 min. Samples were transferred once more to 2 mL sterile saline (solution C) and vortexed vigorously for 30 s. Suspensions A, B and C were pooled, serially diluted and plated on PCA medium for viable counting. The cells removed during these three phases represent the loosely attached biofilm population. A 100 µL volume of the detached viable bacteria solution was introduced onto the surface of a Plat Count agar plate. The process was repeated through a succession of 24 predried plates. Finally, the total bacterial adhesion was determined by a counting of the CFUs, after overnight statically incubation of the agar plates at 37 °C. Each experiment was done four times. Levels of adhesion were given as numbers of cells per square centimeter. Statistical analysis. All values corresponding to the anti-adherence property of E. coli and S. aureus are expressed as mean ± standard deviation. Statistical analysis was performed using Student’s t-test for the calculation of significance level of the data. Differences were considered statistically significant at P < 0.05. Ten samples per group were evaluated. Cell adhesion. In order to examine the interactions of the native and modified PHBHV films with NIH-3T3 fibroblasts, cell adhesion experiment was performed according to the method previously reported in

30

. Briefly, samples were fitted in a 4 well culture dish and

subsequently immersed in a Dulbecco’s Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS) (Gibco, Japan) and 1% penicillin G-streptomycin (Gibco, Japan). To the samples, 1 mL of the NIH-3T3 fibroblastic cells solution (3 × 104 cells.cm-2) were seeded on the sample and incubated in a humidified atmosphere (5% CO2 and at 37°C) for 1 and 3 days to determine the cell adhesion on the PHBHV films. After incubation, the supernatant was removed, washed twice with phosphate buffer saline (PBS) (Gibco, USA), and fixed with an aqueous 2.5% glutardialdehyde solution for 20 min. The sample sheet was then dehydrated, dried in a critical point drier. The surface morphology of the samples was then observed with FE-SEM (400 Hitachi; Tokyo, Japan).


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Cell proliferation with BrdU assay protocol. Proliferation of NIH-3T3 fibroblastic cells seeded PHBHVs films was determined using a colorimetric immune assay, based on the measurement of BrdU, which was incorporated during DNA synthesis. BrdU enzyme-linked immunosorbent assay (ELISA; Roche Molecular Biochemicals) was performed according to the manufacturer's instructions. Briefly, after cell culture for 48 h, BrdU-labeling solution was added to each well. The solution was allowed to incorporate into the cells in a CO2 incubator at 37°C for 20 h. Subsequently, the supernatant in each well was removed by pipetting and washed twice with PBS. The cells were treated with 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA) (Gibco, Tokyo, Japan) and harvested by centrifugation of the cell solution at 1,000 rpm for 15 min. The harvested cells were mixed with FixDenat solution to fix the cells and denature the DNA and then incubated for 30 min. Subsequently, diluted anti-BrdU peroxidase (dilution ratio of 1:100) was added to the cells and incubated at 20 °C for 120 min. After removing the unbound antibody conjugate, 100 µL substrate was added and allowed to stand for 20 min. The reaction was completed by adding 25 µL H2SO4 solution (1 M). The solution was then transferred to a 96-well plate and measured within 5 min at 490 nm with a reference wavelength of 690 nm, using an ELISA plate reader (EL 9800). The blank reading corresponded to 100 µL of culture medium with or without BrdU. Cell viability. A standard live/dead assay was used to evaluate cell viability after culturing NIH-3T3 fibroblast cells on the native PHBHV and modified PHBHV films for 3 days based on the previously reported method31.

Briefly, the fibroblast cells were harvested using

trypsin/ehtylenediamine tetraacetic acid (TE/EDTA) and then centrifuged before suspending them in PBS with a cell density of 1×105 - 1×106 cells/mL. Subsequently, 200 µL of a cell suspension was mixed with a 100 µL assay solution [10 µL calcein-AM solution (1 mM in dimethyl sulfoxide (DMSO)), and 5 µL propidium iodide (1.5 mM in H2O) was mixed with 5



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mL PBS] and incubated for 15 min at 37 °C. The cells were examined by fluorescence microscopy with using a band-pass filter (Nikon Eclipse E600-POL, Japan).

RESULTS AND DISCUSSION The first step of our methodology concerns the bottom-up growth of different polymers tethered chains from the modified PHBHV film surface, according to a photoinduced freeradical method. The different stages of the free-radical reaction are presented hereafter. 1. Photolysis of the thiocarbamate derivative under UV irradiation (N, N-diethylthiocarbamoylthio)-acetic acid was used because of its capability to anchor with the carboxylic acid group to the pendant –NH2 group on the PHBHV-modified surface, and because any investigation related to its reactivity under light activation on PHBHV surface have not been described yet. The ability of the thiocarbamate derivative to generate efficient radicals in the “grafting-from” process was described using Laser Flash Photolysis and ESR / Spin Trapping. Figures 1A and 1B display the transition absorption spectrum and the ESR-ST signals of the thiocarbamate derivative respectively, upon Xe-Hg lamp UV irradiation (200400 nm) under argon saturated solution. Figures 1A and 1B provide spectra which consist of a mixture of two radical species derived both from carbon and sulfur-centered radicals. Laser flash photolysis (266 nm) of the thiocarbamate derivative results in a maximum absorption band at 575 nm (Figure 1A) which is assigned to the dithiocarbamyl radical25b, 32 (•SCSN(C2H5)2). To confirm the later process upon light irradiation, ESR-ST experiments were performed: the carbon-centered radical (•CH2COOH) generated by the homolytic C–S single bond cleavage is well observed (Figure 1B): the hyperfine coupling constants of the carbon radical adduct (aN = 14.6 G, aH = 4.4 G) are in agreement with the literature data33. It should be pointed out that the experimental data (Figure 1B-1) are in excellent agreement with the simulated results


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(Figure 1B-2). The global mechanism of the thiocarbamate derivative photolysis is thus described in Scheme 1.

Scheme 1. Photolysis of the thiocarbamate derivative upon light activation S N

+

N

S O

H

S

hν

S

H C O HO

HO

Figure 1. (A) Transition absorption spectrum of the thiyl radical (•SCSN(C2H5)2) and (B) ESR spin-trapping spectrum (1) and simulated spectrum (2) obtained by irradiation of thiocarbamate derivative in tert-butylbenzene (Xe–Hg lamp, PBN 0.05 M). Inset: lifetime of the thiyl radical



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2. Evidence of the photografting process on the surface of the PHBHV films In most of the studies done in UV photografting, benzophenone and its derivatives have been used as photoinitiating systems28,

34

. However, such compounds are only solubilized into

organic solvents. Only a few studies described this process in water medium. The photografting investigation in this study will be performed in accordance with a “green chemistry” procedure with the use of a photoactivable PHBHV surface. The photoactivable PHBHV surface was developed according to a « two-step » procedure, i.e. the aminolysis of the native PHBHV surface following by the covalent grafting of the thiocarbamate derivative via an amidification reaction. In order to investigate the chemical changes of the aminolyzed and the thiocarbamate-derived PHBHV surfaces, XPS measurements have been performed using the Mg Kα X-ray source operated in a low power mode (24 W) to avoid degradation and chemical modification induced by irradiation. Data acquisitions have mainly been focused on the S2s and N1s core level lines in this case. The elemental composition as well as the element chemical bonding can be deduced from peaks shapes as binding energy of the atomic orbital is strongly influenced by local potential of the emitting atom. The XPS high resolution spectra of N1s and S2s regions of the aminolyzed PHBHV film and the thiocarbamate-derived PHBHV film are more specifically described in Figure 2. For nonmodified PHBHV film (not shown), the C1s spectrum demonstrates the appearance of three main components, aliphatic carbon (C-H/C-C) at 285.1 eV, C-O bond at 286.7 eV and carbon from carbonyl group (O-C=O) at 289.2 eV in good agreement with the literature23a, 24b. In the case of PHBHV films modified with ethylenediamine (Figure 2A), a new peak attributed to the binding energy of the N1s core level of the amine group (C-N) is located at 399 eV. Concerning the thiocarbamate derivated PHBHV film, two new peaks appear at 399 eV


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(Figure 2B) and 232 eV (Figure 2C) respectively for the N1s and S2s core level of the thiocarbamate group. These results evidence the covalent grafting of the thiocarbamate initiator at the surface of the PHBHV film.

Figure 2. XPS spectra of (A) aminolyzed PHBHV film (N1s core level), B) thiocarbamate derived PHBHV film (N1s core level) and (C) thiocarbamate derived PHBHV film (S2s core level). Irradiation time = 300 s. Light intensity = 180 mW.cm-2. Hg-Xe lamp.

To demonstrate the efficiency of the photoactivable PHBHV surface (with thiocarbamate function) for covalently photografting different type of monomers, methacrylic acid was used as a reference and was grafted upon light activation. The photografting of MAA was revealed by the fluorescence image (Figure 3A). The fluorescence spectrum of the PHBHV-g-PMAA surface according to the toluidine blue method was demonstrated in Figure 3B. The calibration curve of the toluidine blue allows us to determine the density of COO- function on the PHBHV-g-PMAA surface (Figure 3C). According to this method, the density of COO− grafted on the PHBHV-g-PMAA was evaluated at 13x10-9 mol.cm-2, thus confirming the



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efficiency of the photoinduced grafting. It is also interesting to point out that the fluorescence of the films only occurs when PMAA is grafted. The general mechanism of the “graftingfrom” process from the modified PHBHV film is described on Scheme 2. Under light activation, the photolysis of the photoiniferter yields a pair of radicals: the reactive one (the carbon-centered radical, Scheme 2A) can initiate monomers polymerization to produce a polymer end radical; the less reactive one (the dithiocarbamyl radical, Scheme 2A) cannot initiate the polymerization of monomers, but prefers to terminate the growing polymer chains, forming dithiocarbamyl end-capped chain (Scheme 2B).

Figure 3. A) Fluorescence image of the PHBHV-g-PMAA film modified with toluidine blue, B-1) Fluorescence background spectrum of the PHBHV film introduced in the toluidine blue solution and intensively washed with water, B-2) fluorescence spectrum of the corresponding modified PHBHV-g-PMAA film with toluidine blue and C) toluidine blue calibration curve.


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Scheme 2. A) Homolytic bond cleavage of the thiocarbamate derivative on the PHBHV film upon light activation and B) “grafting-from” process from the modified PHBHV surface.

3. Synthesis of modified PHBHV films with antibacterial property 3.1. Synthesis of an ampicillin-derived monomer The synthetic route of the ampicillin-derived monomer is given in Scheme 3. The success of the reaction was demonstrated by 1H NMR (Figure 4) that showed the disappearance of the proton in the α-position of the primary amine of the ampicillin at δ = 4.67 ppm (f in Figure 4A) along with the appearance of two ethylenic signals (δ = 5.72 ppm and δ = 5.47 ppm, l, l’ in Figure 4B) and methylene proton in the α-position of the methacrylamide function (δ = 5.50 ppm, f in Figure 4B). The presence of a single signal at δ = 2.62 ppm is attributed to the free N-hydroxysuccinimide as a reaction sub product. The later is not photosensible and cannot afford the photopolymerization alone but it can be removed after the photografting process by the multiple rinsing of the modified-PHBHV surface with water and acetonitrile. It is also interesting to underline that the synthesized ampicillin methacrylamide monomer is stable under irradiation and do not homopolymerize during the time of irradiation (360 s).



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Scheme 3. Synthesis of the ampicillin-based monomer

Figure 4. 1H NMR in D2O (400 MHz) of A) the ampicillin and B) the ampicillin-derived monomer after the amidification reaction.


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The ATR-FTIR spectrum of the ampicillin and the ampicillin methacrylamide monomer are displayed in Figure 5. In the IR spectrum of ampicillin, a strong band observed at 3033 cm-1 is attributed to the C-H stretching vibrations of the phenyl ring. The vibration bond at 2733 cm-1 is due to the symmetric stretching of the methyl groups. In the case of cyclic molecule an increase of the carbonyl frequency, as the ring size decreases below six atoms, is observed. Hence, a band visualized at 1766 cm-1 is assigned to the carbonyl vibrations of the four member ring. A second absorption band is displayed at 1692 cm-1 which corresponds to the carbonyl absorption of the COO- group. An intense absorption band is observed at 1578 cm-1 which is attributed to the bending vibration of N-H bond and an additional peak at 1645 cm-1 is assigned to the ring C=C stretching/C=O stretching vibration. A broad absorption frequency extending from 2300 cm-1 to 3300 cm-1 is also displayed in Figure 5A. The factor which is responsible from the diffuse shape and the relatively low frequency is attributed to the hydrogen bonding. Finally, an N-H symmetric stretching band appears at 3220 cm-1. For the methacrylic acid N-hydroxysuccinimide ester (not shown here), the shoulder peaks at 1734 and 1778 cm-1 are assigned to the carbonyl stretching of succinimidyl ester. The carbon double bond of this compound is located at 1636 cm-1. After the amidification reaction between the primary amine –NH2 of the ampicillin and the methacrylic acid N-hydroxysuccinimide ester, the intensity of absorption band between 2300 and 3300 cm-1 strongly decreases, thus revealing the diminution of the hydrogen bonding in the new synthesized ampicillin monomer. Two new peaks at 1636 cm-1 and 1609 cm-1 corresponding respectively to the carbon double bond (C=C) and the carbonyl absorption peak of the new peptide bond (C(=O)-NH-R) of the ampicillin methylacrylamide monomer appear. Moreover, the carbonyl stretching vibration of the succinimidyl ester (from the methacrylic acid N-hydroxysuccinimide ester) entirely disappears.



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Figure 5. ATR-FTIR spectra of ampicillin (A and A’) and ampicillin methacrylamide monomer (B and B’).

3.2. Synthesis of multiple modified PHBHV surfaces with antibacterial property The ATR-FTIR of the native PHBHV film shows typical absorption bands such as –C-H aliphatic and asymmetric stretching band likewise a –C=O stretching band from an ester group at, respectively, 2870−3010 cm−1 and at 1720 cm-1. In the aminolyzed film, a broad band between 2900-3400 cm-1 and attributed to the appearance of hydrogen bondings indicates that the amidification reaction occurred. When grafting the photoiniferter (N,Ndiethylthiocarbamoylthio)-acetic acid onto the modified-PHBHV surface, the broad band between 2900 and 3400 cm-1 no longer appears. When the ammonium methacrylate (META) or ampicillin methacrylamide monomers are grafted on PHBHV films, the characterized methacrylate (or methacrylamide) band at 1636 cm-1 disappears. This demonstrates the consumption of the methacrylate (or methacrylamide) double bonds by the “grafting-from” process and, therefore, the efficiency of the photografting method. The overall IR spectra are displayed in supporting information (Figure S1).


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To confirm the grafting of META and ampicillin-derived monomer on the PHBHV films, both the water contact angle measurements and XPS analysis on the modified PHBHV surface have been done. The water contact angles of the different PHBHV surfaces are compared with the native PHBHV film. Table 2 summarizes the water contact angle for the native and the modified PHBHV surfaces. For the native PHBHV film, the water contact angle was evaluated at 93°. When the aminolysis reaction occurred, the contact angle drops at approximately 80°, indicating that the film is more hydrophilic due to the –NH2 function at the surface. When the photoiniferter is grafted, the contact angle remains stable at 80°, whereas it falls down at 70° with the grafting of a water solution of META at 0.1M. The hydrophilic properties are assumed to be due to the ammonium groups. Concerning the ampicillin grafted PHBHV films, the water contact angle drastically falls to 20°: it could be explained by the fact that ampicillin is a very hydrophilic component, thus indicating that the grafting occurred.

Table 2. Evolution of the water contact angles of the native and the modified PHBHV films.

Modified PHBHV films PHBHV

93° ± 1.5°

Aminolyzed PHBHV

80° ± 2.3°

Thiocarbamate grafted on PHBHV surface

80° ± 1.8°

PHBHV-g-PMETA PHBHV-g-P(Ampicillin) a

Water contact angle a

70° ± 3° 20° ± 3.8°

Average on 12 samples

In order to investigate the chemical changes of the modified PHBHV surfaces, XPS measurements have been performed. Data acquisitions have mainly been focused on the C1s, S2p3/2, N1s and O1s core level lines. XPS survey-scan spectra for the unmodified PHBHV



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film and the PHBHV-modified films with META and ampicillin are displayed in Figure 6. XPS survey-scans of the aminolyzed and the thiocarbamate modified PHBHV films are displayed in supporting information (Figure S2). The XPS high resolution spectra of N1s, S2p3/2 and C1s regions of the PHBHV, PHBHV-g-PMETA and PHBHV-g-P(Ampicillin) films are more specifically described in Figure 7. XPS assignment for the C1s, N1s, S2p3/2, and O1s detailled spectra are summarized in Table 3. For the native PHBHV film, the C1s spectrum demonstrates the appearance of three main components, aliphatic carbon (C-H/C-C) at 285.1 eV, C-O bond at 286.7 eV and carbon from carbonyl group (O-C=O) at 289.2 eV in good agreement with the literature24b (Figures 6A and 7E). In the case of PHBHV films modified with META (Figure 6B), the C1s core-level spectrum show four peak components with binding energies at 285.1, 286.1, 286.7 and 289.2 eV, which are respectively attributed to C-H/C-C, C-N+, C-O and O-C=O. For the same sample, the binding energy of the N1s core level of the ammonium group15a, 24b (C-N+) is located at 403.1 eV (Figures 6B and 7A) whereas this of the (C-N) bond (present in the thiocarbamate group) is visualized at 399 eV (Figures 6B and 7A). Concerning the PHBHV-g-P(Ampicillin) films (Figure 6C and Figures 7B-D), the C1s core level spectrum show four peaks components with binding energies at 284, 285.1, 287 and 288.3 eV, which were attributed to C=C, C-H/C-C, S-C=S and N-C=O, respectively. The appearance of both the carbon double bonds peak (C=C, benzylic cycle) at 284 eV and the carbon peak (N-C=O) from the amide group of ampicillin confirmed the grafting of ampicillin on the PHBHV surface. Therefore, the O1s signal from the amide group of the ampicillin appears at 531.2 eV. The S2p3/2 detailed spectrum (Figure 7C) can be deconvoluted into two main components, the C-S bond35 at 163.7 eV and the C=S double bond36 at 162.4


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eV, thus demonstrating the presence of ampicillin and the thiocarbamate group on the surface of the modified-PHBHV films.

Figure 6. XPS survey-scan spectra of (A) PHBHV film, (B) PHBHV-g-PMETA film, and (C) PHBHV-g-P(Ampicillin) film. Irradiation time = 300 s. Light intensity = 180 mW.cm-2.HgXe lamp.



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Figure 7. XPS high resolution spectra of the (A) N1s region of the PHBHV-g-PMETA film, B) N1s region of the PHBHV-g-P(Ampicillin) film, C) S2p3/2 region of the PHBHV-gP(Ampicillin) film, D) C1s region of the PHBHV-g-P(Ampicillin) film and E) C1s region of the PHBHV film.


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Table 3. XPS assignment for the native PHBHV film, PHBHV-g-PMETA film, PHBHV-gP(ampicillin) films.

Nature of the films

C1s

O1s

N1s

S2p3/2

PHBHV

PHBHV-g-PMETA

PHBHV-g-P(ampicillin)

C=C

-

-

284

C-C / C-H

285.1

285.1

285.1

C-O

286.7

286.7

-

S-C=S

-

-

287

O-C=O

289.2

289.2

-

N-C=O

-

-

288.3

C-N+

-

286.1

-

O*-C=O

532.1

532.1

-

O-C=O*

533.4

533.6

533

N-C=O*

-

-

531.2

C-N

-

399

399.3

C-N+

-

403.1

-

C-S

-

163.7

163.7

C=S

-

162.4

162.4

3.3. Anti-adhesion property The anti-adhesion property of polymers grafted with ammonium group proved to be of great interest for the prevention of adhesion and proliferation of bacteria on material surfaces. The efficiency of the modified PHBHV films to inhibit the bacterial adhesion (Figure 8) were investigated against Gram-Negative bacteria (E. coli) and Gram-Positive bacteria (S. aureus) in comparison with the native PHBHV films.



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PHBHV film and thiocarbamate derivated PHBHV films did not exhibit antibacterial property which neither significantly inhibited nor affected the Escherichia coli and Staphyloccoccus aureus adhesion. However, there is a weak decrease of the number of CFUs on modified PHBHV film with amine groups in comparison with the native PHBHV film against Escherichia coli. When amine groups come into contact with bacteria, they can penetrate the cell membrane, disrupt its integrity and provokes cell lysis. The later results have already demonstrated in some investigations37. In contrast, the presence of ammonium groups onto the surface led to a drastic reduction by 97 % and 92 % of the adhesion of E. coli and S. aureus, respectively. Such results are in accordance with literature data. Indeed, surfaces containing quaternary ammonium groups have been shown to damage both Gram-positive and Gram-negative cells by the disruption of their cellular membranes2a. The positively charged nitrogen in the ammonium group interacts with the negatively charged head groups of acidic phospholipids in the bacterial cellular membrane, causing general perturbations in the lipid bilayers38. This causes the cells to release potassium ions, which in turn causes the cell to lose its ability to undergo osmoregulation and other physiological functions38-39, therefore leading to the cell death. With the PHBHV-g-P(Ampicillin) films, the reduction of the E. coli and S. aureus adhesion reaches 90 % and 60 % respectively. Ampicillin acts as a competitive inhibitor of the enzyme transpeptidase, which is needed by bacteria to make their cell walls. The inhibition of transpeptidation leads to the formation of a weakened peptidoglycan. As autolysins continue to act, further damage is done to the bacteria cell wall which will result in cell lysis and its death40. Even if the primary –NH2 group in ampicillin helps penetrating the cell walls of the Gram negative bacteria, it is interesting to point out that the grafted ampicillin does not completely lose its antibacterial property.


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These results clearly indicated that the grafting of ammonium group or ampicillin moieties on the surface of PHBHV films is a powerful method for substantially enhancing the antibacterial property (this is particularly true with E. coli) and can prevent from the adhesion of bacteria on material surfaces.

Figure 8. Comparison of anti-adherence property of native PHBHV, PHBHV-g-PMETA (0.1 M), PHBHV-g-P(Ampicillin) (0.1 M), PHBHV-g-NH2 and PHBHV-g-thiocarbamate films against E. coli and S. aureus. Data is shown as mean ± standard deviation (n=6). Results indicate significant difference obtained by t-test (P < 0.05).

4. Cytocompatibility. The biocompatibility of antibacterial quaternary ammonium compounds (QACs) has been recently reviewed39a and some of them (for example poly(4-vinylpyridine), PVP) are known to be toxic to mammalian cells41. Therefore, further work is required to assess accurately and compare the biocompatibility of different cell lines and the antibacterial activity of such polymers42. In our case, PHBHV modified films could be used as a film coating and be



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eligible for applications where biocompatibility and antibacterial properties are needed such as dental or orthopedic devices. This lack of biocompatibility investigations prompted us to evaluate in this study the cytotoxicity of the modified-PHBHV films with NIH-3T3 fibroblastic cells. Figure 9 shows the NIH-3T3 fibroblasts adhesion on the surface of the pristine PHBHV and modifiedPHBHV films after 1 and 3 days respectively. From the start, fibroblastic cells adhered to the surface of the pristine and the modified PHBHV films. It was clearly observed from Figures 9A, 9B and 9C that the NIH-3T3 fibroblastic cells were well adhered to the pristine and modified PHBHV films after 1 day, whatever the nature of the PHBHV surface used. The numbers of cells cultured for 1 day significantly increased after 3 days of culture (Figures 9D, 9E and 9F) and are independent of the type of films. After 3 days, Figures 9D, 9E and 9F illustrate the presence of flattened and spread cells on the surface of PHBHV-derived films. In the process of cell development, the inter-cellular communication is essential in order to exchange signals concerning differentiation or other cell informations. The generation of these communication channels was already noticeable for PHBHV-modified films. The production of slender cytoplasmic projections forming focal adhesions on the surface of the films was observed for all the films. These results indicated that the cell compatibility of PHBHV-derived films was not influenced by the grafting of META or ampicillin monomer.


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Figure 9. SEM images of the fibroblast cells (NIH 3T3) adhesion after 1 and 3 days on the native PHBV films (A, D) and on modified PHBHV films : PHBHV-g-PMETA (0.1 M) (B, E), and PHBHV-g-P(Ampicillin) (0.1 M) (C, F).

Many calorimetric methods have been implemented for the estimation of exact cell number. Among them, calorimetric immune assay based on the measurement of BrdU, which was incorporated during DNA synthesis, is widely employed43. The data obtained from the BrdU assay showed that the NIH-3T3 fibroblastic cells proliferated on all the PHBHV films (Figure 10). The cell proliferation on the surface of the modified PHBHV films (PHBHV-g-PMETA and PHBHV-g-P(Ampicillin)) was not significantly different from that on the PHBHV control and was within the standard deviation (Figure 10).



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Figure 10. Proliferation of NIH-3T3 cells cultured on the native and the surface-modified PHBHV films for 3 days as determined by a BrdU assay. Data expressed as mean ± standard deviation (n=6) for the specific absorbance.

For further assessment of the biocompatible nature of the pristine and surface modified PHBHV films, cell viability experiment using calcein-AM and propidium iodide (live/dead) assay was performed. The fluorescence color of the cells cultured on the native PHBHV films was totally green, thus indicating a good viability of the cells (Figure 11A). Only a few dead cells showed red fluorescence by the staining of propidium iodide when cultured on the PHBHV-modified films with META and ampicillin (Figures 11B and 11C, see the white arrows). The data obtained from the cell viability experiment suggests that the pristine and the surface modified PHBHV films provide a native environment to the NIH-3T3 fibroblastic cells.


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Figure 11. Live/dead assays: Fluorescence microscopy images of NIH-3T3 cells stained with calcein-AM (green) and propidium iodide after 3 days culture on A) PHBV, B) PHBHV-gPMETA (0.1 M) and C) PHBHV-g-P(Ampicillin) (0.1 M) films. (White arrays show the dead NIH-3T3 fibroblastic cells).

CONCLUSIONS PHBHV-derived polymer films with antibacterial property were successfully engineered according to an environmentally sustainable “green chemistry” approach. Our study has demonstrated an efficient photografting method for the covalent surface modification of photoactivable PHBHV films with two types of monomers, under light activation and in aqueous medium. Under light activation, the thiocarbamate-derived initiator which has been grafted onto the PHBHV surface can afford the generation of carbon-centered radicals which



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are able to initiate the free-radical polymerization of methacrylate or methacrylamide monomers according to a “grafting from” process. Such PHBHV-derived materials led to a tremendous reduction of 97 % and 90 % of the adherence of Escherichia coli respectively with META and with an ampicillin-derived monomer, without affecting their cytocompatibility. Concerning the S. aureus adhesion, its reduction was evaluated at 92 % with META but only 60 % on the PHBHV-g-P(Ampicillin) films. This new route of grafting could be further used to broaden the application portfolio of PHAs. The possible uses of a permanent, nonbiofouling surface such as described in this investigation would include for example treatment of food packaging or coating for biomedical devices.

AUTHOR INFORMATION Corresponding Author *D. L. Versace, E-mail: [email protected], Tel: +33 1 49 78 12 28, Fax: +33 1 49 78 12 01 ACKNOWLEDGMENTS The authors would like to thank the CNRS institute and UPEC for financial supports.

SUPPORTING INFORMATION AVAILABLE Figure S1 displays the ATR-FTIR spectra of the native and the different modified PHBHV films. Figure S2 shows the XPS survey-scan spectra of PHBHV-g-NH2 and PHBHV-gthiocarbamate films. This information is available free of charge via the Internet at http://pubs.acs.org/.


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REFERENCES 1. (a) Ducel, G.; Fabry, J.; Nicolle, L., Prevention of hospital-acquired infections : a practical guide. 2nd ed.; Geneva, Switzerland : World Health Organization: 2002; (b) Pace, J. L.; Rupp, M. E.; Finch, R. G., Biofilms, Infection, and Antimicrobial Therapy. CRC Press Taylor and Francis Group: 2006. 2. (a) Tiller, J. C.; Liao, C.-J.; Lewis, K.; Klibanov, A. M., Designing surfaces that kill bacteria on contact. Proc. Natl Acad. Sci. 2001, 98 (11), 5981-5985; (b) Bazaka, K.; Jacob, M.; Crawford, R.; Ivanova, E., Efficient surface modification of biomaterial to prevent biofilm formation and the attachment of microorganisms. Appl Microbiol Biotechnol 2012, 95 (2), 299-311; (c) Arciola, C. R.; Campoccia, D.; Speziale, P.; Montanaro, L.; Costerton, J. W., Biofilm formation in Staphylococcus implant infections. A review of molecular mechanisms and implications for biofilm-resistant materials. Biomaterials 2012, 33 (26), 5967-5982. 3. (a) Lok, C.-N.; Ho, C.-M.; Chen, R.; He, Q.-Y.; Yu, W.-Y.; Sun, H.; Tam, P.-H.; Chiu, J.-F.; Che, C.-M., Silver nanoparticles: partial oxidation and antibacterial activities. J Biol Inorg Chem 2007, 12 (4), 527-534; (b) Ye, S.; Majumdar, P.; Chisholm, B.; Stafslien, S.; Chen, Z., Antifouling and Antimicrobial Mechanism of Tethered Quaternary Ammonium Salts in a Cross-linked Poly(dimethylsiloxane) Matrix Studied Using Sum Frequency Generation Vibrational Spectroscopy. Langmuir 2010, 26 (21), 16455-16462; (c) Kim, J. S.; Kuk, E.; Yu, K. N.; Kim, J.-H.; Park, S. J.; Lee, H. J.; Kim, S. H.; Park, Y. K.; Park, Y. H.; Hwang, C.-Y.; Kim, Y.-K.; Lee, Y.-S.; Jeong, D. H.; Cho, M.-H., Antimicrobial effects of silver nanoparticles. Nanomedicine: Nanotechnology, Biology and Medicine 2007, 3 (1), 95101; (d) Yu, Q.; Wu, Z.; Chen, H., Dual-function antibacterial surfaces for biomedical applications. Acta Biomaterialia 2015, 16 (0), 1-13. 4. Genzer, J.; Efimenko, K., Recent developments in superhydrophobic surfaces and their relevance to marine fouling: a review. Biofouling 2006, 22, 339-360. 5. (a) Park, K. D.; Kim, Y. S.; Han, D. K.; Kim, Y. H.; Lee, E. H. B.; Suh, H.; Choi, K. S., Bacterial adhesion on PEG modified polyurethane surfaces. Biomaterials 1998, 19 (7–9), 851-859; (b) Ostuni, E.; Chapman, R. G.; Liang, M. N.; Meluleni, G.; Pier, G.; Ingber, D. E.; Whitesides, G. M., Self-Assembled Monolayers That Resist the Adsorption of Proteins and the Adhesion of Bacterial and Mammalian Cells. Langmuir 2001, 17 (20), 6336-6343. 6. (a) Ostuni, E.; Chapman, R. G.; Holmlin, R. E.; Takayama, S.; Whitesides, G. M., A Survey of Structure−Property Relationships of Surfaces that Resist the Adsorption of Protein. Langmuir 2001, 17 (18), 5605-5620; (b) Li, L.; Chen, S.; Jiang, S., Protein interactions with oligo(ethylene glycol) (OEG) self-assembled monolayers: OEG stability, surface packing density and protein adsorption. J. Biomater. Sci., Polym. Ed. 2007, 18 (11), 1415-1427. 7. (a) Lim, K. S.; Oh, K. W.; Kim, S. H., Antimicrobial activity of organic photosensitizers embedded in electrospun nylon 6 nanofibers. Polym. Int. 2012, 61 (10), 1519-1524; (b) Perni, S.; Piccirillo, C.; Pratten, J.; Prokopovich, P.; Chrzanowski, W.; Parkin, I. P.; Wilson, M., The antimicrobial properties of light-activated polymers containing methylene blue and gold nanoparticles. Biomaterials 2009, 30 (1), 89-93; (c) Decraene, V.; Pratten, J.; Wilson, M., Cellulose Acetate Containing Toluidine Blue and Rose Bengal Is an Effective Antimicrobial Coating when Exposed to White Light. Appl. Environ. Microbiol. 2006, 72 (6), 4436-4439. 8. Ruckh, T.; Oldinski, R.; Carroll, D.; Mikhova, K.; Bryers, J.; Popat, K., Antimicrobial effects of nanofiber poly(caprolactone) tissue scaffolds releasing rifampicin. J. Mater. Sci.: Mater. Med. 2012, 23 (6), 1411-1420. 9. Chung, D.; Papadakis, S. E.; Yam, K. L., Evaluation of a polymer coating containing triclosan as the antimicrobial layer for packaging materials. Int. J. Food Sci. Technol. 2003, 38 (2), 165-169.



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25. (a) Fouassier, J.-P.; Rabek, J.-F., Radiation Curing in Polymer Science and Technology. Elsevier Applied Science: London, New-York, 1993; (b) Fouassier, J.-P.; Lalevée, J., Photoinitiators for Polymer Synthesis: Scope, Reactivity and Efficiency. Wiley VCH: 2013. 26. (a) Lao, H.-K.; Renard, E.; Fagui, A. E.; Langlois, V.; Vallée-Rehel, K.; Linossier, I., Functionalization of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) films via surface-initiated atom transfer radical polymerization: Comparison with the conventional free-radical grafting procedure. J. Appl. Polym. Sci. 2011, 120 (1), 184-194; (b) Ma, H.; Davis, R. H.; Bowman, C. N., A Novel Sequential Photoinduced Living Graft Polymerization. Macromolecules 2000, 33 (2), 331-335. 27. Rupp, B.; Ebner, C.; Rossegger, E.; Slugovc, C.; Stelzer, F.; Wiesbrock, F., UVinduced crosslinking of the biopolyester poly(3-hydroxybutyrate)-co-(3-hydroxyvalerate). Green Chem. 2010, 12 (10), 1796-1802. 28. Lao, H.-K.; Renard, E.; Langlois, V.; Vallée-Rehel, K.; Linossier, I., Surface functionalization of PHBV by HEMA grafting via UV treatment: Comparison with thermal free radical polymerization. J. Appl. Polym. Sci. 2010, 116 (1), 288-297. 29. Zhu, Y.; Mao, Z.; Gao, C., Aminolysis-based surface modification of polyesters for biomedical applications. RSC Advances 2013, 3 (8), 2509-2519. 30. Haider, A.; Gupta, K. C.; Kang, I.-K., Morphological Effects of HA on the Cell Compatibility of Electrospun HA/PLGA Composite Nanofiber Scaffolds. BioMed Research International 2014, 2014, 308306. 31. Shin, Y.-S.; Borah, J. S.; Haider, A.; Kim, S.; Huh, M.-W.; Kang, I.-K., Fabrication of pamidronic acid-immobilized TiO2/hydroxyapatite composite nanofiber mats for biomedical applications. J. Nanomaterials 2013, 2013, 152-152. 32. Otsu, T., Iniferter concept and living radical polymerization. J. Polym. Sci. Part A: Polym. Chem. 2000, 38 (12), 2121-2136. 33. Ettinger, K. V.; Forrester, A. R.; Hunter, C. H., Lyoluminescence and spin trapping. Can. J. Chem. 1982, 60 (12), 1549-1559. 34. (a) Yang, W.; Rånby, B., Photoinitiation performance of some ketones in the LDPE– acrylic acid surface photografting system. Eur. Polym. J. 1999, 35 (8), 1557-1568; (b) Ma, H.; Davis, R. H.; Bowman, C. N., A Novel Sequential Photoinduced Living Graft Polymerization. Macromolecules 1999, 33 (2), 331-335; (c) Ke, Y.; Wang, Y.; Ren, L.; Lu, L.; Wu, G.; Chen, X.; Chen, J., Photografting polymerization of polyacrylamide on PHBV films (I). J. Appl. Polym. Sci. 2007, 104 (6), 4088-4095. 35. Clark, D. T.; Thomas, H. R., Applications of ESCA to polymer chemistry. XVII. Systematic investigation of the core levels of simple homopolymers. J. Polym. Sci.: Polym. Chem. Ed. 1978, 16 (4), 791-820. 36. Briggs, D.; Seah, M. P., Practical Surface Analysis. 2nd ed.; John Wiley & Sons: New York, NY, USA, 1993; Vol. 1. 37. (a) Kabara, J. J.; Conley, A. J.; Truant, J. P., Relationship of Chemical Structure and Antimicrobial Activity of Alkyl Amides and Amines. Antimicrobial Agents and Chemotherapy 1972, 2 (6), 492-498; (b) Endo, Y.; Tani, T.; Kodama, M., Antimicrobial activity of tertiary amine covalently bonded to a polystyrene fiber. Appl. Environ. Microbiol. 1987, 53 (9), 2050-2055; (c) Eldin, M. S. M.; Soliman, E. A.; Hashem, A. I.; Tamer, T. M., Antibacterial Activity of Chitosan Chemically Modified with New Technique. Trends Biomater. Artif. Organs 2008, 22 (3), 125-137. 38. Gilbert, P.; Moore, L. E., Cationic antiseptics: diversity of action under a common epithet. J. Appl. Microbiol. 2005, 99 (4), 703-715. 39. (a) Buffet-Bataillon, S.; Tattevin, P.; Bonnaure-Mallet, M.; Jolivet-Gougeon, A., Emergence of resistance to antibacterial agents: the role of quaternary ammonium


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compounds—a critical review. Int. J. Antimicrob. Agents 2012, 39 (5), 381-389; (b) Kenawy, E.-R.; Worley, S. D.; Broughton, R., The Chemistry and Applications of Antimicrobial Polymers: A State-of-the-Art Review. Biomacromolecules 2007, 8 (5), 1359-1384; (c) Timofeeva, L.; Kleshcheva, N., Antimicrobial polymers: mechanism of action, factors of activity, and applications. Appl Microbiol Biotechnol 2011, 89 (3), 475-492. 40. Kotra, L. P.; Mobashery, S., β-Lactam antibiotics, β-lactamases and bacterial resistance. Bull. Inst. Pasteur 1998, 96 (3), 139-150. 41. Li, G.; Shen, J.; Zhu, Y., Study of pyridinium-type functional polymers. II. Antibacterial activity of soluble pyridinium-type polymers. J. Appl. Polym. Sci. 1998, 67 (10), 1761-1768. 42. Stratton, T. R.; Rickus, J. L.; Youngblood, J. P., In Vitro Biocompatibility Studies of Antibacterial Quaternary Polymers. Biomacromolecules 2009, 10 (9), 2550-2555. 43. Haider, A.; Gupta, K.; Kang, I.-K., PLGA/nHA hybrid nanofiber scaffold as a nanocargo carrier of insulin for accelerating bone tissue regeneration. NanoScale Research Lett. 2014, 9 (1), 314-326.



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Photoactivable surface of natural poly(3-hydroxybutyrate-co-3hydroxyvalerate) for anti-adhesion applications

R. Poupart, A. Haider, J. Babinot, I.-K. Kang, J.-P. Malval, J. Lalevée, S. Abbad Andalloussi, V. Langlois and D. L. Versace*

“For Table of Contents Use Only”

Development of a new functionalized and non-cytotoxic biomaterial with anti-adherence properties according to a photochemistry process


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Photoactivable surface of natural poly(3-hydroxybutyrate-co-3-hydroxyvalerate) for anti-adhesion applications

R. Poupart, A. Haider, J. Babinot, I.-K. Kang, J.-P. Malval, J. Lalevée, S. Abbad Andalloussi, V. Langlois and D. L. Versace



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Figures

Figure 1. (A) Transition absorption spectrum of the thiyl radical (•SCSN(C2H5)2) and (B) ESR spin-trapping spectrum (1) and simulated spectrum (2) obtained by irradiation of thiocarbamate derivative in tert-butylbenzene (Xe–Hg lamp, PBN 0.05 M). Inset: lifetime of the thiyl radical


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Figure 2. XPS spectra of (A) aminolyzed PHBHV film (N1s core level), B) thiocarbamate derived PHBHV film (N1s core level) and (C) thiocarbamate derived PHBHV film (S2s core level). Irradiation time = 300 s. Light intensity = 180 mW.cm-2. Hg-Xe lamp.



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Figure 3. A) Fluorescence image of the PHBHV-g-PMAA film modified with toluidine blue, B-1) Fluorescence background spectrum of the PHBHV film introduced in the toluidine blue solution and intensively washed with water, B-2) fluorescence spectrum of the corresponding modified PHBHV-g-PMAA film with toluidine blue and C) toluidine blue calibration curve.


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Figure 4. 1H NMR in D2O of A) the ampicillin and B) the ampicillin-derived monomer after the amidification reaction.



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Figure 5. ATR-FTIR spectra of ampicillin (A and A’) and ampicillin methacrylamide monomer (B and B’).


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Figure 6. XPS survey-scan spectra of (A) PHBHV film, (B) PHBHV-g-PMETA film, and (C) PHBHV-g-P(Ampicillin) film. Irradiation time = 300 s. Light intensity = 180 mW.cm-2.Hg-Xe lamp.



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Figure 7. XPS high resolution spectra of the (A) N1s region of the PHBHV-g-PMETA film, B) N1s region of the PHBHV-g-P(Ampicillin) film, C) S2p3/2 region of the PHBHV-gP(Ampicillin) film, D) C1s region of the PHBHV-g-P(Ampicillin) film and E) C1s region of the PHBHV film.


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Figure 8. Comparison of anti-adherence property of native PHBHV, PHBHV-g-PMETA (0.1 M), PHBHV-g-P(Ampicillin) (0.1 M), PHBHV-g-NH2 and PHBHV-g-thiocarbamate films against E. coli and S. aureus. Data is shown as mean ± standard deviation (n=6). Results indicate significant difference obtained by t-test (P < 0.05).



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Figure 9. SEM images of the fibroblast cells (NIH 3T3) adhesion after 1 and 3 days on the native PHBV films (A, D) and on modified PHBHV films : PHBHV-g-PMETA (0.1 M) (B, E), and PHBHV-g-P(Ampicillin) (0.1 M) (C, F).


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Figure 10. Proliferation of NIH-3T3 cells cultured on the native and the surface-modified PHBHV films for 3 days as determined by a BrdU assay. Data expressed as mean ± standard deviation (n=6) for the specific absorbance.



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Figure 11. Live/dead assays: Fluorescence microscopy images of NIH-3T3 cells stained with calcein-AM (green) and propidium iodide after 3 days culture on A) PHBV, B) PHBHV-gPMETA (0.1 M) and C) PHBHV-g-P(Ampicillin) (0.1 M) films. (White arrays show the dead NIH-3T3 fibroblastic cells).


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Schemes

Scheme 1. Photolysis of the thiocarbamate derivative upon light activation S

H S

hν

N

+

N

S O

S

H C O HO

HO

Scheme 2. A) Homolytic bond cleavage of the thiocarbamate derivative on the PHBHV film upon light activation and B) “grafting-from” process from the modified PHBHV surface.



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Scheme 3. Synthesis of the ampicillin-based monomer


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Photoactivable Surface of Natural Poly(3 ... - ACS Publications

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