When Functionalization of PLA Surfaces Meets ThiolâYne

Article pubs.acs.org/Biomac

When Functionalization of PLA Surfaces Meets Thiol−Yne Photochemistry: Case Study with Antibacterial Polyaspartamide Derivatives Carla Sardo,‡ Benjamin Nottelet,*,† Daniela Triolo,‡ Gaetano Giammona,‡,§ Xavier Garric,† Jean-Philippe Lavigne,∥ Gennara Cavallaro,*,‡ and Jean Coudane† †

Institut des Biomolécules Max Mousseron (CNRS UMR 5247), Département des Biopolymères Artificiels, UFR Pharmacie Université Montpellier I, Université Montpellier 2-15, Avenue Charles Flahaut, 34093 Montpellier, France ‡ Dipartimento di Scienze e Tecnologie Biologiche, Chimiche e Farmaceutiche, Sez. CTF, University of Palermo, via Archirafi 32, 90123 Palermo, Italy § IBF-CNR, via Ugo La Malfa, 153, 90143 Palermo, Italy ∥ Bacterial virulence and infectious disease, INSERM U1047, University of Montpellier 1 - UFR Médecine Site de Nîmes, 186 Chemin du Carreau de Lanes, CS 83021, 30908 NIMES Cedex 2, France S Supporting Information *

ABSTRACT: In this work we wish to report on the covalent functionalization of polylactide (PLA) surfaces by photoradical thiol−yne to yield antibacterial surfaces. At first, hydrophilic and hydrophobic thiol fluorescent probes are synthesized and used to study and optimize the conditions of ligation on alkynePLA surfaces. In a second part, a new antibacterial polyaspartamide copolymer is covalently grafted. The covalent surface modification and the density of surface functionalization are evaluated by SEC and XPS analyses. No degradation of PLA chains is observed, whereas covalent grafting is confirmed by the presence of S2p and N1s signals. Antiadherence and antibiofilm activities are assessed against four bacterial strains, including Gram-negative and Gram-positive bacteria. A strong activity is observed with adherence reduction factors superior to 99.98% and biofilm formation decreased by 80%. Finally, in vitro cytocompatibility tests of the antibacterial surfaces are performed with L929 murine fibroblasts and show cell viability without promoting proliferation.

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infections. The first approach is the noncovalent immobilization of antibacterial agents. It includes drug releasing coatings6,7 and mixing techniques8−10 but intrinsically leads to loss of activity after drug elution and risk of promoting the emergence of multidrug resistant bacteria. The second approach is the covalent immobilization of antibacterial agents. It includes plasma treatment,11,12 photografting,13 and chemical modification by aminolysis or hydrolysis.14−17 However, these techniques run into two main problems that are (i) the nature of the degradable aliphatic polyesters that cannot be modified by these techniques without considering their relative fragility, and (ii) the fact that they are generally not designed to provide a stable, shelf-ready, and general platform for specific postfunctionalization. To address this problem, we recently reported on a mild chemical modification technique allowing for the propargylation of PLA surfaces.18 Beside its nondegrading nature, this methodology gave access to a large

INTRODUCTION

As a direct result of the life expectancy increase, we have witnessed in recent times an exponential use of implants and devices for the restoration and maintenance of human anatomy and functions after trauma, surgery, or general wear.1 However, several drawbacks associated with the use of implants are generally reported, including the risks of limited healing, inflammation, and the development of biomaterial-associated septic failures.2,3 To limit these risks, the modulation of the surface properties of prosthetic materials appears therefore as a convenient and efficient strategy.4 In this context, various strategies have been proposed to modify the surface properties of aliphatic polyesters. This family of degradable polymers is indeed widely used in clinics and includes the well-known poly(lactide) (PLA), poly(glycolide) (PGA), poly(ε-caprolactone) (PCL), and their copolymers. However, although classically used for temporary implants, these polymers, like others, are prone to infection with, for example, a 4% infection rate for PLA screws and pins for orthopedic and trauma surgery.5 Two main approaches have thus been retained to make them less susceptible or even resistant to bacterial © 2014 American Chemical Society

Received: September 15, 2014 Revised: October 15, 2014 Published: October 16, 2014 4351

dx.doi.org/10.1021/bm5013772 | Biomacromolecules 2014, 15, 4351−4362

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Scheme 1. Fluorescent Probes and PHEA Copolymer Used for the Thiol−Yne Surface Photochemical Functionalization of PLA

modification of PLA are only related to photoinitiation of surface polymerizations.32,33 For these reasons, the main objective of this work is therefore to propose new avenue for the photochemical thiol−yne postpolymerization surface modification of PLA and to apply this approach to functionalize PLA surfaces with a cationic polyhydroxyethylaspartamide derivative to confer antibacterial activity to the surface. To this aim, in a first part of the project, model fluorescent probes have been used to optimize the conditions of covalent functionalization under various conditions, including aqueous and organic medium. In a second part, the versatility of this strategy has been exemplified with the surface functionalization of PLA with a novel antibacterial polymer based on α,β-poly(N-2-hydroxyethyl)-D,L-aspartamide (PHEA). PHEA is a water-soluble and biocompatible polymer whose derivatives were successfully used for various biomedical applications.34−36 In the present work PHEA copolymers bearing (i) disulfide moieties for grafting on propargylated PLA surfaces, and (ii) free ammonium and primary amino groups for antibacterial activity, have been synthesized and grafted on PLA surfaces. Finally, antibacterial activity and biocompatibility of the antibacterial surfaces are discussed.

variety of possible postfunctionalization, thanks to the use of the highly efficient and orthogonal Cu(I)-catalyzed Huisgen 1,3-dipolar cycloaddition of alkynes and azides (CuAAC) to covalently conjugate bioactive compounds to the PLA propargylated surface. This strategy led us to produce potent and yet biocompatible antibacterial PLA biomaterials with both bactericidal and antibiofilm activities.19 In the continuity of this work, and in a constant quest to provide versatile and easy routes toward bioactive polyester surfaces, we were interested in extrapolating this approach to the reaction of photochemical thiol−yne addition. This reaction presents the advantage of being a metal free reaction and to be a more appropriate chemistry in the frame of biomedical applications. Thiol−ene and thiol−yne reactions, although basically known for over 100 years, have recently been the cornerstone of tremendous research work due to the recognition for their “‘click’” characteristics and the ability of thiols to react via either radical or catalyzed processes under very mild conditions and, depending on the conditions, within short reaction times.20,21 In parallel, while thiol−ene/thiol−yne surface functionalization has been largely applied to nondegradable polymer materials (silicone, polypropylene etc.),22−24 to the best of our knowledge no study was reported on the photochemical thiol−ene/thiol−yne functionalization of aliphatic polyesters surfaces. Indeed, efforts dedicated to thiol− ene/thiol−yne post-polymerization modification of polyesters are to date focused on PLA or PCL chains prefunctionalized by mean of copolymerization of alkene or alkyne functional lactones,25,26 or by use of alkene or alkyne functional ring opening polymerization initiators.27,28 In particular, our group recently reported on the synthesis of PLA and PCL bearing alkyne groups on the main chain obtained by copolymerization of propargyl caprolactone or propargyl glycolide.29−31 On the other hand, studies dedicated to the photochemical surface

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EXPERIMENTAL SECTION

Materials. PLA94 was synthesized by bulk ring-opening copolymerization of L-lactide (88%) and D,L-lactide (12%) (PURAC, Lyon, France), using tin 2-ethylhexanoate as catalyst (M̅ n = 200 000 g/mol; Đ = 1.9). PLA94 plates were shaped using a hydraulic heated press (Carver press 4120-289). The plates were heated at 130 °C and PLA powder, was pressed for 5 min before cooling under pressure (1.5 × 107 Pa). PLA94 plates 500 μm thick were obtained. (3-Carboxypropyl)trimethylammonium chloride (CPTA), N-(3-(dimethylamino)propyl)-N′-ethylcarbodiimide hydrochloride (EDC.HCl), 1-hydroxybenzotriazole hydrate (HOBT), (±)-α-lipoic acid (LA), N-hydroxysuccinimide (NHS), ethylenediamine (EDA), bis(44352


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C(OH)−CH, −CHC(OH)−CH and NH−CS−NH−CH2); 3.9 (2H, −NH−CH2−CH2−SS−); 3.0 (2H, −CH2−SS−) (Supporting Information Figure S1b). Synthesis of PHEA Copolymers. Synthesis of PHEA-EDA-CPTA. PHEA-EDA-CPTA derivative was synthesized starting from α,βpoly(N-2-hydroxyethyl)-D,L-aspartamide (PHEA) as described by Licciardi at al. (Scheme 1).38 The starting PHEA (Mw = 45 000 Da, Đ = 1.8) was prepared and purified according to the previously reported procedure.39 Spectroscopic data, FTIR, and 1H NMR (data not shown) were in agreement with previous results. First, ethylenediamine was bounded to PHEA hydroxyl groups, by the formation of an urethane bond, using bis(4-nitrophenyl)carbonate (BNPC) as condensing agent. To perform this first step, a solution of PHEA (250 mg, 1.58 mmol of repeating units) in 3 mL of anhydrous DMF was added dropwise to a solution of BNPC (480 mg, 1.58 mmol) in 4 mL of the same solvent under constant stirring and the mixture was kept at 40 °C for 4 h. After, the activated PHEA solution was added under stirring to 530 μL of ethylenediamine (7.90 mmol) and the mixture kept at room temperature for 4 h. The reaction mixture was then precipitated into acetone and the precipitate isolated by centrifugation and washed several times with the same solvent. The product was further purified by exhaustive dialysis against water, using a Spectrapor dialysis membrane with molecular weight cutoff of 12 000−14 000 Da and finally freeze-dried. The obtained PHEA-EDA was further derivatized with CPTA, by carbodiimide-mediated coupling, leading to the formation of an amide bond between carboxylic group of CPTA and primary amine groups of PHEA-EDA side chains. Briefly, PHEA-EDA (100 mg, corresponding to 0.26 mmol of pendant −NH2) was dissolved in 5 mL of 1:1 H2O/DMSO mixture followed by the addition of CPTA (24.16 mg, 0.13 mmol, R = 0.5 with R = mmol CPTA/mmol of NH2 groups) and HOBT (26.95 mg, 0.19 mmol). After adjusting pH at 6.8 by NaOH 0.1 N, EDC·HCl (38.34 mg, 0.19 mmol) was added and pH was maintained constant using HCl 0.1 N for at least 2 h. The reaction mixture was left at room temperature overnight and then purified by exhaustive dialysis using a Spectrapor dialysis membrane with molecular weight cutoff of 12 000− 14 000 Da. Finally, freeze-drying of the solution led to solid pure product (Supporting Information Figure S2). Obtained PHEA-EDACPTA was characterized by 1H NMR analysis (300 MHz, D2O): δ = 1.99 (m, 2 H, −CO−CH2−CH2−CH2−N+(CH3)3); 2.27 (m, 2 H, −CO−CH2−CH2−CH2−N+(CH3)3); 2.71 (m, 2 H, −CO−CH− CH2−CO−NH−); 3.06 (s, 9 H, −C−N+(CH3)3); 3.15−3.25 (m, 4 H, −NH−CH2−CH2−OH, −CO−CH2−CH2−CH2−N+(CH3)3), 3.36 (m, 2 H, −NH−CH2−CH2−NH−CO−); 3.57 (m, 2 H, −NH− CH2−CH2−OH); 4.02 (m, 2 H, −NH−CH2−CH2−O(CO)NH− CH2−CH2−NH−); 4.72 (m, 1 H, −NH−CH(CO)CH2−). Synthesis of PHEA-EDA-CPTA-LA. Coupling of lipoic acid was performed by a two-step reaction. Lipoic acid (24.74 mg, 0.12 mmol), dissolved in 375 μL of dried DMSO, was first activated by formation of a hydroxy succinimidyl ester in the presence of NHS (16.57 mg, 0.144 mmol) and EDC (34.50 mg, 0.18 mmol), at 40 °C for 4 h in a thermostatically controlled water bath under constant stirring. After this activation time, a PHEA-EDA-CPTA solution (100 mg, corresponding to 0.12 mmol of amine groups, in 2.5 mL of dried DMSO) was added dropwise into the activated lipoic acid solution and the reaction mixture was kept at 40 °C. After 24 h, the mixture was precipitated into an excess of diethyl ether/acetone 1:1. The suspension was collected by centrifugation (4 °C, 9800 rpm, 10 min), and the solid washed several times with the same mixture of solvents. Residual organic solvents were removed under vacuum, and the solid was dissolved in bidistilled water and purified exhaustively by dialysis using a Spectrapor dialysis membrane with molecular weight cutoff of 12 000−14 000 Da. The product was finally freeze-dried (Supporting Information Figure S2). PHEA-EDA-CPTA-LA was characterized by 1H NMR analysis (300 MHz, D2O): δ = 1.33 (m, 2 H, −NH−CO−(CH2)2−CH2−CH2−cCH−SS−CH2−CH2−); 1.55 (m, 4 H, −NH−CO−CH2−CH2−CH2−CH2−cCH−SS−CH2− CH2−); 1.90 (m, 1 H, −NH−CO−(CH2)4−cCH−SS−CH2− CH2−); 2.01 (m, 2 H, −CO−CH2−CH2−CH2−N+(CH3)3); 2.17 (m 2 H, −NH−CO−CH2−(CH2)3−cCH−SS−CH2−CH2−); 2.29

nitrophenyl)carbonate (4-BNPC), tin 2-ethylhexanoate, 3-mercaptopropionic acid, 1-pyrenebutanol, p-toluenesulfonic acid, fluorescein isothiocyanate (FITC), cystamine dihydrochloride (≥98%), N,Ndiisopropylethylamine (DIPEA), tris(2-carboxyethyl)phosphine hydrochloride solution (TCEP, 0.5 M), α,α-dimethoxy-α-phenylacetophenone (Irgacure651), 2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone (Irgacure2959), and all solvents were purchased from Sigma-Aldrich. PrestoBlueTM, Dulbecco’s modified Eagle’s medium (DMEM/F-12), phosphate buffered saline (PBS), sterile Dulbecco’s phosphate buffered saline (DBPS), fetal bovin serum (FBS), penicillin, streptomycin, and glutamine were purchased from Invitrogen (Cergy Pontoise, France). BD Falcon tissue culture polystyrene (TCPS) 24well plates were purchased from Becton Dickinson (Le Pont de Claix, France). Characterization of Polymers. NMR. 1H NMR and 13C NMR spectra were recorded on a Bruker spectrometer (AMX300) operating at 300 and 75 MHz, respectively. Deuterated chloroform or deuterated dimethyl sulfoxide (DMSO) were used as solvents. Chemical shifts were expressed in ppm with respect to tetramethylsilane (TMS). ATR-FTIR Spectroscopy. Infrared spectra were recorded on a PerkinElmer Spectrum 100 Fourier transform infrared (FTIR) spectrometer using the attenuated total reflectance (ATR) method. Size Exclusion Chromatography (SEC). The molecular weights and dispersities (Đ) of PLA and functionalized PLA chains were determined at room temperature on a Waters system equipped with a guard column, a 600 mm PLgel 5 mm Mixed C column (Polymer Laboratories), a Waters 410 refractometric detector, and a Waters 470 scanning fluorescence detector. Calibration was established with poly(styrene) standards from Polymer Laboratories. THF or THF/ TEA (95/5; v/v) were used as eluent at a flow rate of 1 mL/min. The weight-average molecular weights (Mw’s) and dispersity (Đ) of PHEA and PHEA-EDA were determined by aqueous SEC. The protocol involved a PolySep-GFC-P3000 gel column from Phenomenex connected to a Waters 2410 refractive-index detector. Phosphate buffer solution 0.05 M at pH 4 was used as the eluent at 35 °C with a flow rate of 0.6 mL/min and poly(ethylene oxide) standards (in the range 6−271 kg/mol) were used for calibration. For the determination of Mw’s and Đ of PHEA-EDA-CPTA and PHEA-EDA-CPTA-LA, organic SEC was performed. In this case, a Phenogel GPC column (5 μm, 104 Å pores size) from Phenomenex, connected to Waters 2410 refractive-index detector, was used. DMSO was employed as the eluent at 50 °C with a flow rate of 0.6 mL/min, and poly(ethylene oxide) standards (in the range 6−271 kg/mol) were used for calibration. All samples and standard solutions were prepared at a concentration of 2.5 mg/mL in the mobile phase and filtered using 0.45 μm nylon syringe filters Synthesis of Fluorescent Probes. Synthesis of Pyrene Thiol Derivative (Py-SH). (4-Pyren-2-yl)butyl 3-mercaptopropionate, referred as the pyrene thiol derivative (Py-SH, Scheme 1), was synthesized according to a procedure already described elsewhere with benzene being replaced by toluene as the solvent.37 Details are provided in the Supporting Information. Synthesis of Fluorescein Thiol Derivative ((FITC-S)2). A dimer of fluorescein containing a disulfide bond ((FITC-S)2, Scheme 1) was synthesized by direct reaction of FITC and cystamine. In a typical reaction, cystamine (10.12 mg, 44.9 μmol) and DIPEA (23 μL, 135 μmol) were solubilized in 7 mL of anhydrous ethanol. Meanwhile, FITC (35 mg, 89.9 μmol) was solubilized in 4 mL of anhydrous acetone before dropwise addition to the cystamine solution under stirring and argon flow. The resulting mixture was stirred in the dark at room temperature for 2 h. The crude medium was purified by flash chromatography, and the solvents were distilled off under vacuum. The solid was dissolved in water, and the solution was subjected to an extensive dialysis against water using Spectra Pore Tubing with a molecular weight cutoff of 500 Da. Finally, the solution was freezedried to yield the fluoresceine dimer (yield 60%) that was stored in the dark. (FITC-S)2 was characterized by 1H NMR analysis (300 MHz, d6DMSO): δ = 8.7 (1H, −NH−CS−NH−CH2); 8.4 (1H, CH− C(NH)C); 7.9 (1H, CCH−CHC(NH)); 7.2 (1H, CCH− CHC(NH)); 6.7 (2H, CHCH−C(C)); 6.6 (5H, −CH 4353


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(m, 2 H, −CO−CH2−CH2−CH2−N+(CH3)3); 2.36 (m, 2 H, −NH− CO−(CH2)4−cCH−SS−CH2−CH2−); 2.73 (m, 4 H, −CO−CH− CH2−CO−NH−); 3.06 (s, 9 H, −C−N+(CH3)3); 3.17−3.24 (m, 4 H, −NH−CH2−CH2−OH, −CO−CH2−CH2−CH2−N+(CH3)3); 3.37 (m, 2 H, −NH−CH2−CH2−NH−CO−); 3.57 (m, 2 H, −NH− CH2−CH2−OH); 4.02 (m, 2 H, −NH−CH2−CH2−O(CO)NH− CH2−CH2−NH−); 4.63 (m, 1 H, −NH−CH(CO)CH2−). FITC-Labeling of PHEA-EDA-CPTA-LA. FITC labeled polymer (FITC-PHEA-EDA-CPTA-LA) was obtained under conditions adapted from the Sigma-Aldrich protocol for proteins labeling. Details are provided in the Supporting Information. Propargylation of PLA Plates. Clickable PLA surfaces were prepared in accordance with a recently described procedure.18 Typically, a PLA94 plate (m = 125 mg, thickness = 500 μm, area = 1.76 cm2) was immersed in a stirred solution of 2.0 M lithium diisopropyl amide (LDA) (4 mL, 8 mmol) in a mixture of anhydrous tetrahydrofuran (60 mL)/diethyl ether (120 mL) at −50 °C in an argon inert atmosphere. After 30 min, propargyl bromide (1.4 mL, 12 mmol) was added at a temperature of −30 °C. The mixture was stirred for 1 h with a ramp temperature from −30 °C to room temperature. The PLA plate was quenched and washed with water, diethyl ether, and methanol. Residual solvents were removed under vacuum. Thiol−Yne Photochemical Surface Functionalization. All thiol−yne reactions were photoinitiated by UV irradiation (∼44 mW/cm2, λmax = 365 nm, 15 min irradiation (ca. 40 J/cm2) on each plate side) under ambient laboratory conditions (room temperature and normal atmosphere). Reaction mixtures were not degassed prior to use. The influence of irradiation time, thiol group concentration, and presence and nature of photoinitiator has been studied. Details of the various thiol−yne reactions are given in the following paragraphs. UV irradiations were carried out with a DYMAX 2000-EC Series UV curing flood lamps equipped with a light shield, and UV intensity measurements were measured with a DYMAX ACCU-CAL 50 radiometer. Py-SH Fluorescent Probe. A 200 mM stock solution of Py-SH in acetone was prepared and used for all experiments. A 1.3 mM stock solution of α,α-dimethoxy-α-phenylacetophenone (DMAP, Irgacure651) in c-hexane has been prepared and used when required. In a typical procedure, 375 μL of Py-SH stock solution was added to 1.125 mL of Irgacure651 stock solution to obtain a final concentration of 50 mM of fluorescent probe in an acetone/c-hexane (1:3) mixture in the presence of 2 mol % of photoinitiator with respect to thiol groups. A 500 μL aliquot was placed in a quartz reaction vessel containing a propargylated PLA plate and irradiated with UV light for 15 min on each side. After thiol−yne reaction, the plate was washed extensively with diethyl ether and acetone/c-hexane (1:3) mixture before drying in vacuo. The same protocol was used for photoiniator-free reactions by replacing the photoinitiator solution by pure c-hexane. All conditions are provided in the Supporting Information (Table S2). (FITC-S) 2 Fluorescent Probe. A 5 mM stock solution of Irgacure2959 in deionized water was prepared and used when required. Two reactions media have been tested according to the following typical procedures. For DMSO/water medium (1:4), in a typical experiment 10.5 mg of (FITC-S)2 was dissolved in 100 μL of DMSO ((FITC-S)23, Table S3), then 300 μL of a TCEP 0.5 M solution was added, and the mixture was stirred in dark for 2.5 h at room temperature. Just before irradiation, 100 μL of Irgacure2959 stock solution was added. For ethanol/water medium (1:1), in a typical experiment, 10.5 mg of (FITC-S)2 was dissolved in a solvent mixture of HCl 0.1 N (250 μL) and ethanol (500 μL) ((FITC-S)24, Table S3). After solubilization, 250 μL of a TCEP 0.5 M solution was added and the mixture was stirred in the dark for 2.5 h at room temperature. Just before irradiation, 100 μL of Irgacure2959 stock solution was added. In all cases, a 500 μL aliquot of the chosen medium was placed in a quartz reaction vessel containing a propargylated PLA plate and irradiated with UV light for 15 min on each side. After thiol−yne reaction, the samples were washed extensively with diethyl ether, ethanol, ethanol/water (1:1), and water.

The same protocol was used for photoiniator free reactions by replacing the photoinitiator solution by pure water. The same protocol was used for acidic free reactions by replacing HCl solution by deionized water. All conditions are provided in the Supporting Information in Table S3 where the notation “(HCl X N)” refers to the use of an HCl solution of the corresponding normality to prepare the reaction medium. PHEA-EDA-CPTA-LA and FITC-PHEA-EDA-CPTA-LA. The same protocol as the one described for the water-soluble (FITC-S)2 probe was used for the functionalization of PLA surfaces with PHEA-EDACPTA-LA and FITC-PHEA-EDA-CPTA-LA. All conditions are provided in the Supporting Information (Table S3). Evaluation of the Extent of Functionalization. Fluorimetric Evaluation. For each set of conditions, the extent of functionalization was assessed as follows: after irradiation and extensive washings, 5 mg samples were cut in the PLA plates. Samples were dissolved in THF, filtered, and analyzed by SEC. The ratio between the areas of the peaks corresponding to the polymer obtained from the fluorescence detector and from the refractive index detector, referred as Fluo/RI in Tables S2 and S3, was then calculated and used to evaluate the grafting conditions. The wavelength used were λem = 380 nm, λex = 340 nm for Py-SH and λem = 447 nm, λex = 437 nm for the FITC derivatives. X-ray Photoelectron Spectrometry (XPS). XPS data were obtained on an ESCALAB 250 photoelectron spectrometer with Al Kα radiation (1486.6 eV) and overall instrument resolution of 1.1 eV. All spectra were collected at an electron takeoff angle of 90° to the sample surface. An area of 400 μm2 of each surface was analyzed. The binding energies were corrected by referencing binding energy of the C−C component of C 1s to 284.8 eV. Bacterial Strains. Four clinical bacterial strains in the early stationary phase were used: Escherichia coli NECS19923, Staphylococcus aureus NSA4201, Staphylococcus epidermidis NSE175861, and Pseudomonas aeruginosa NPA01. NECS19923 strain was genetically modified to express green fluorescent protein (GFP) using a pBBRderived nonmobilizable plasmid carrying a GFP expression cassette. Antiadherence Activity. Before conducting any in vitro antibacterial tests, the bacterial strains were first grown aerobically overnight on Muller Hinton medium at 37 °C with stirring. Bacterial adherence to modified plates was assessed in accordance with a previously described procedure. PLA plates were immersed in a bacterial solution (OD600 = 0.05) for 1 h, then nonadherent bacteria on the sample surface were removed by repeated washings with sterilized water. Plates were then incubated in neutral medium for 24 h at 37 °C under static conditions, rinsed in sterile saline to remove any nonbiofilm population cells that may have deposited on the samples, and then transferred to 2 mL saline (suspension A) and vortexed vigorously for 30 s. Samples were then transferred to 2 mL sterile saline (suspension B) and sonicated for 3 min. Samples were transferred once more to 2 mL of sterile saline (suspension C) and vortexed vigorously for 30 s. Suspensions A, B, and C were pooled, serially diluted, and plated onto Luria agar for viable counting. The cells removed during these three phases represent the loosely attached biofilm population. Following the final vortex step, the samples were blotted onto the surface of a predried Mueller Hinton agar plate for 1 min. The process was repeated through a succession of 15 predried plates and colony counts were performed, following an overnight incubation at 37 °C. Finally, total bacterial adherence was calculated by adding the colony forming unit (CFU) count after overnight incubation of the agar plates at 37 °C, to all cultivated bacteria. Biofilm Formation. The susceptibility of the modified PLA plates to biofilm formation was evaluated by creating a liquid−air interface between the plate and a bacterial suspension (OD600 = 0.05, i.e., ≈1 × 105 CFU/mL). To do this, the different plates were placed vertically in a 12-well Greiner plate containing 2 mL of Muller−Hinton growth medium, and 20 μL of cell suspension in PBS was added. After incubating under static conditions for 72 h at 37 °C in a 100% humidity atmosphere, unattached bacteria were removed from the plates by repeated washings with sterilized water. A biofilm formed under these conditions on the bare plates, at the liquid−air interface. Biofilm-forming bacteria were recovered by immersion in DMSO, and 4354


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250 μL of this DMSO-containing bacterial solution was analyzed in a Mithras LB940 luminometer (Berthold). Biofilm formation was expressed in relative luminescence units (RLU). Alternatively, biofilm formation was evaluated by examination under a fluorescence microscope. In this case, after rinsing with PBS, the plates were placed between a slide and cover glass with moviol and examined under a Leica fluorescence microscope (×20 and ×100). All these tests were performed in triplicate. Cytocompatibility. Murine fibroblasts cells (designated L929) were used to assess the in vitro cytocompatibility of the materials as recommended by the International and European Standards (ISO 10993-5:2009). Cells were cultured in modified Eagle’s medium (MEM) containing 10% horse serum, penicillin (100 μg/mL), streptomycin (100 μg/mL), and Glutamax (1%). The PLA plates were disinfected in ethanol for 30 min before being immersed in a solution of sterile PBS containing penicillin and streptomycin (1 mg/ mL) and incubated for 48 h at 37 °C. The plates were then rinsed three times with sterile PBS before being soaked for 12 h in sterile PBS. These sterile PLA plates were stamped to fit the wells of 24-well cell culture plates. In vitro cytocompatibility was assessed by monitoring the proliferation of L929 fibroblasts on the surface of the plates. To do this, the plates were placed in polystyrene 24-well tissue culture plates (TCPS) and seeded with 1 × 104 L929 cells. Cell viability after 1, 4, and 12 days was evaluated using the PrestoBlue assay that reflects the number of living cells present on a surface at a given time point. Culture medium was removed at scheduled time points and replaced by 1 mL of fresh medium containing 10% PrestoBlue. Fluorescence at 530 nm (ex.) and 615 nm (em.) was measured on a Victor X3 photometer (PerkinElmer). All data points and standard deviations correspond to measurements in triplicate. Data Analysis. All biological data are expressed as means ± SD and correspond to measurements in triplicate. For cytocompatibility tests, one-way ANOVA was applied to determine the statistical significance for multiple comparisons (a level of p < 0.05 was considered statistically significant).

evidenced by 1H NMR analysis with the appearance of a peak at 8.7 ppm characteristic of the thiourea bond, and by the shift from 3.0 to 3.85 ppm of the peak corresponding to the methylene of the cystamine close to the nitrogen (Figure S1b). Propargylated PLA Surfaces. Propargyl-functionalized PLA surfaces have been prepared according to the procedure recently reported by our group (Scheme 2). PLA plates were Scheme 2. Schematic Strategy for the Versatile Thiol−Yne Photochemical Modification of PLA Surface

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RESULTS AND DISCUSSION Synthesis of Fluorescent Probes. The aim of this work was to study the versatility of the thiol−yne photochemical surface modification of PLA and to evaluate this approach to functionalize PLA surfaces with cationic antibacterial PHEA derivative. For this purpose, two fluorescent probes able to react under thiol−yne conditions were first synthesized. As a first probe, a hydrophobic thiol pyrene derivative was chosen (Py-SH, Scheme 1) to (i) confirm the propargylation of the PLA plates and (ii) study thiol−yne photochemical reaction under organic conditions that ensure the conservation of the PLA shape. This probe was synthesized by esterification reaction between 1-pyrenebutanol and 3-mercaptopropionic acid.37 The quantitative esterification was characterized by 1H NMR analysis with shift of a peak at 3.7 ppm corresponding to the methylene in α-position of the hydroxyl group in 1pyrenebutanol to 4.2 ppm corresponding to the methylene in the formed ester bond (Figure S1a). As a second probe, a hydrophilic and water-soluble dimer of fluoresceine having a disulfide bond was chosen ((FITC-S)2 (Scheme 1) to establish thiol−yne reaction conditions that may be extrapolated to other water-soluble compounds including the antibacterial PHEAEDA-CPTA-LA copolymers. The probe was synthesized by reaction between FITC and cystamine, quantitatively yielding the corresponding thiourea. This strategy is simple and often used in peptide synthesis as the presence of disulfide allows an easier workup compared to free thiol containing molecules. The corresponding derivative with free thiol groups can easily be recovered by in situ reduction as it will be shown in the following. The full conversion of isocyanate to thiourea was

modified via anionic activation using optimized mild conditions that guarantee the three-dimensional shape of the modified object while avoiding hydrolytic degradation of the polyester as previously demonstrated.18 Briefly, PLA plates were functionalized through a first activation step using LDA in a solvent/ nonsolvent mixture of tetrahydrofuran/diethyl ether (1/2 v/v), followed by the addition of propargyl bromide and its grafting on the activated PLA surface. After extensive washings of the surface to remove the potentially nonreacted and adsorbed species, the extent of propargylation was assessed. Because the chemical modification takes place only at the very surface of the plates, classical penetrating characterization techniques (FTIR or Raman spectroscopies) are unable to detect the surface propargyl groups. Effective propragylation and availability of the propargyl groups located on the PLA surface were therefore indirectly evidenced thanks to the ligation of the Py-SH fluorescent probe (see subsection titled Photochemical Thiol−Yne Surface Functionalization of PLA under Organic Conditions). 4355


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Figure 1. 1H NMR (D2O, 300 MHz) spectrum of PHEA-EDA-CPTA-LA copolymer.

Synthesis of PHEA Copolymers. PHEA is a highly watersoluble synthetic polymer having important properties such as hydrophilicity and biocompatibility. Moreover, PHEA and its derivatives are bioeliminable and do not induce immunogenic response.40 The large number of hydroxyl groups makes PHEA a versatile platform for the derivatization with molecules of various molecular weights. For example, it was recently demonstrated that grafting polymethacrylate side chains on a PHEA backbone gives a polymeric system for oral delivery of proteins.36 Coupling short oligoamine, such as spermine or diethylene triamine, leads to simple and biocompatible nucleic acid based drug delivery systems.34 In this work a PHEA derivative, namely, PHEA-EDA-CPTALA copolymer bearing lipoic acid groups (Scheme 1), has been synthesized in high yields (86%) to give a polycation enable to be grafted on the clickable propargylated PLA94 surfaces. The rationale for this approach was to use the biocompatibility and biodegradability of PHEA-based copolymers and the biocidal properties of polycations to design an implantable antibacterial PLA94 surface, which would prevent the initial attachment of bacteria, therefore preventing the subsequent formation of a biofilm. The activation reaction of hydroxyl groups of PHEA with 4BNPC, followed by the reaction with ethylenediamine (EDA) in order to functionalize at least 50% of hydroxyl groups of PHEA with EDA, was performed by using a equimolar ratio between 4-BNPC and repeating units of PHEA using a molar excess of EDA in DMF solution. Under these conditions, PHEA-EDA copolymer was obtained with a molar derivatization degree (DDmol %) of 50%, calculated by analysis of the 1H NMR spectrum, comparing the integral of the peak at 4.0 ppm related to EDA-functionalized hydroxyethyl groups of PHEA with the integral of the signal at 2.7 ppm, related to methylene groups of PHEA backbone. PHEA-EDA was easily reacted with 3-(carboxypropyl)-trimethylammonium chloride (CPTA), in very mild conditions by a one-step carbodiimide-mediated coupling in the presence of HOBT. The conditions used to functionalize at least 50% of amine pendent groups of EDA

(corresponding to about 25% of repeating units of PHEA) involved molar ratios CPTA/amine groups = 0.5 and HOBT/ amine groups = 0.7. A DDmol % in CPTA of 25% was calculated in PHEA-EDA-CPTA by comparing the integral of the peak at 3.2 ppm related to trimethylammonium groups of CPTA with the integral of the peak at 3.0 ppm assignable to methylene groups of polymer backbone. (Supporting Information Figure S2) To introduce side chain groups suitable for thiol−yne photoclick coupling on propargylated PLA94 surface, lipoic acid was finally linked to PHEA-EDA-CPTA copolymer. Lipoic acid contains a dithiolane ring that is prone to react with alkyne groups after proper reduction, while ensuring in the disulfide form the stability of the polymer before use. This molecule is also well-known for its metal-chelating capacity, its ability to scavenge reactive oxygen species (ROS) to regenerate endogenous antioxidants and to repair oxidative damage.41 The reaction was conducted in mild and simple conditions performing a carbodiimide-mediated conjugation between the carboxylic group of LA and free primary amino groups of EDA in PHEA-EDA-CPTA. The reaction was performed in two steps to improve efficiency. The first step consisted in the activation of LA carboxyl groups in DMSO with EDC in the presence of N-hydroxysuccinimide (NHS), that allow to obtain a considerably more stable amine-reactive intermediate with respect to the O-acylisourea intermediate (Supporting Information Figure S2). The experimental conditions (molar ratio LA/free amine groups = 1) gave a DDmol % in LA equal to ca. 13% as calculated (Figure 1) by comparing the 1H NMR signal at 1.33 ppm assigned to LA (−NH−CO−(CH2)2−CH2− CH2−cCH−SS−CH2−CH2−) with the signal at 4.6 ppm of PHEA backbone (−NH−CH(CO)CH2−). The average molecular weights of PHEA and its derivatives, PHEA-EDA, PHEA-EDA-CPTA, and PHEA-EDA-CPTA-LA, determined by size exclusion chromatography (SEC), ranged from 30 to 80 kg/mol (Supporting Information Table S1). SEC traces also confirmed that the CPTA and LA groups were linked to the 4356


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polymer chain and not present as low molecular weight contaminants. Photochemical Thiol−Yne Surface Functionalization of PLA under Organic Conditions. As mentioned above, PySH was used as a probe to confirm the propargylation of the PLA plates and to study thiol−yne photochemical reaction under organic conditions. The main difficulty was to find a solvent with a good transparency to UV and that would ensure the conservation of the plates morphology. This last point is of crucial importance if this strategy is aimed at the modification of the surface of prosthetic materials that have a defined shape (interference screws, surgical meshes, etc.). Various solvents have therefore been tested, and the mixture acetone/c-hexane (1:3) was selected. Based on the literature, a 50 mM concentration of free thiol groups was chosen. A study of the parameters was then carried out, focusing on the addition mode of the probe (drop deposition vs plate immersion), on the irradiation time, and on the amount of photoinitiator. Detailed conditions are provided in the Supporting Information (Table S2). As described in the Experimental Section, the extent of reaction was assessed thanks to the Fluo/RI ratio between the signals given by the fluorescence detector and the refractive index detector in SEC analyses. It should be noted that the relative low intensity of the fluorescence detector compared to the concentration detector is ascribed to the high amount of none-substituted bulk PLA (detected by the RI concentration detector) over the substituted surface PLA (detected by the fluorescence detector), as well as the inherent low fluorescence of the probes in the THF used for SEC.42 First, it is to note that the effective grafting of the fluorescent probe on the polymer chain was clearly demonstrated by the same retention time observed for fluorimetric and refractometric detections. For illustration purposes, the fluorescence and refractive index chromatograms of Py-SH2 are provided in Figure 2a. In addition, although easy and more economical in terms of product used, which may be of interest for expensive or scarce substituent, drop deposition was abandoned as a result of the inhomogeneity of the surface modification (data not shown). Other parameters have thus been studied under immersion conditions with propargylated PLA plates being soaked in the reaction medium containing the fluorescent probe and the photoinitiator. Different irradiation times, ranging from minutes to a few hours, can be found in the literature. In this work, each side of the propargylated PLA plate has been irradiated 5 or 15 min with no significant difference in the absence of photoinitiator (Table S2, Py-SH1 and Py-SH3), whereas in the presence of photoinitiator higher grafting ratios were obtained for longer irradiation times (Table S2, Py-SH2 and Py-SH4). The comparison of the reactions carried out in the presence (2 mol %) or in the absence of Irgacure651 also showed better results when using the photoinitiator (Py-SH1 vs Py-SH2 and Py-SH3 vs Py-SH4). No further improvement was observed with a higher (20 mol %) photoinitiator concentration (data not shown). As a consequence, for all other reactions, and especially to confirm the propargylation of all batches of PLA plates prior use in other photochemical reactions, a 15 min irradiation time on each side of the PLA plates and a 2 mol % concentration of photoinitiator have been used. Finally, in order to confirm that the fluorescence was due to the thiol−yne reaction, a control was also carried out on pristine PLA plates (Table S2, Control). No fluorescence signal was detectable as shown on the corresponding chromatogram in Figure 2a. This control also confirmed the absence of polymer chain

Figure 2. Chromatograms of modified PLA surfaces and pristine PLA surfaces (control) obtained by SEC analyses with a coupled fluorescence/refractive index detection after photochemical radical thiol−yne functionalization with (a) Py-SH probe, (b) (FITC-S)2 probe, and (c) FITC-PHEA-EDA-CPTA-LA.

degradation during the modification process as illustrated by the comparison between the control and Py-SH2 SEC chromatograms. At this point, one should note, that when considering surface functionalization an important advantage of the thiol−yne 4357


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Experimental Section for detailed procedures). All conditions are provided in the Supporting Information (Table S3), and for the sake of clarity results are illustrated in Figure 3. In

reaction over the thiol−ene reaction, is the ability for an ynebond to react with 2 equiv of thiol and form a double addition product as illustrated in Scheme 2. This double addition requires the formation of a vinylthioether (mono addition) that undergoes a second radical addition. The determination of the extent of mono- and diaddition product, which depends on a variety of factors, was not aimed in this study and the presence of mono adduct at the surface cannot be excluded. However, based on the alkyne and thiol structures used and on the previous studies of Fairbanks et al.,43 we can expect to have a predominant diaddition over monoaddition. For clarity, Scheme 2 shows therefore only the double addition product. Photochemical Thiol−Yne Surface Functionalization of PLA under Aqueous Conditions. (FITC-S)2 Fluorescent Probe. We focused on the possibility to extrapolate this methodology to aqueous media, which is of particular interest in the frame of functionalization with biomolecules that are generally water-soluble and may be denaturated in organic solvents. The same approach was used, with the Py-SH probe being replaced by the (FITC-S)2 dimer and the organo-soluble Irgacure651 by the water-soluble Irgacure2959. Beside solvents and as a result of the dimeric nature of the probe, additional parameters, that is, reduction time and concentration of reductive TCEP, were studied, whereas the irradiation time (15 min for each side of plates), the concentration of photoinitiator (2 mol %/SH), and the concentration of free thiol groups in the reaction medium (50 mM) were kept constant. Detailed conditions are provided in the Supporting Information (Table S3). Reduction time was set to 2.5 h. In practice, all compounds were mixed and stirred for 2.5 h except for the photoinitiator that was added just before irradiation. The impact of the TCEP concentration, 3 or 6 equiv with respect to the thiol groups, was not clearly demonstrated in our set of experiments with the (FITC-S)2 dimer. However, with regard to the final use of PHEA copolymers, 6 equiv was finally chosen to ensure a full reduction of the disulfide bridges that may be less accessible on the polymer backbone. On the opposite, the choice of solvent was critical. With the aim to establish conditions that may be used for a large number of hydrophilic compounds, various mixtures of water, ethanol, and dimethyl sulfoxide were tested. Slightly higher extents of functionalization were obtained when adding DMSO to the reaction medium (Table S3, (FITC-S)22 and (FITC-S)23) with SEC peaks ratios Fluo/RI around 6.5 × 10−4. However, an efficient functionalization was also observed with water/ethanol mixtures (Table S3, (FITC-S)21 and (FITC-S)24) with SEC peaks ratios Fluo/RI around 5.5 × 10−4. It should be noted that no fluorescence was detected in the control experiments run under the same conditions with pristine PLA plates (Figure 2b). Because of the limited improvement in terms of functionalization when DMSO was used as cosolvent, one should preferably use a water/ethanol mixture for the photochemical modification of surfaces, as DMSO could partially dissolve PLA chains. FITC-PHEA-EDA-CPTA-LA. A second set of experiments focused on the modification of PLA surface with a macromolecular species of interest, namely, the FITC-labeled PHEAEDA-CPTA-LA copolymer. The impact of slighlty acidic aqueous media was assessed as it should favor the solubilization of PHEA-EDA-CPTA-LA thanks to the partial protonation of the copolymeric free amino groups. Mixtures of water/ethanol (1:1) were therefore used as reaction medium and prepared with HCl solutions with normality in the range 0.1 to 1 N (see

Figure 3. Extent of surface functionalization of PLA surfaces by FITCPHEA-EDA-CPTA-LA as a function of the reaction conditions: acidic H2O/EtOH (1:1) medium, [SH] = 10 mM (dark blue bars); acidic H2O/EtOH (1:1) medium, [SH] = 25 mM (light blue bar); acidic H2O/DMSO (4:1), [SH] = 50 mM (red bars) (see Experimental Section and Table S3 for further details).

opposition to the cases of Py-SH and (FITC-S)2, the concentration of free thiol groups was initially fixed to a 10 mM concentration as a 50 mM concentration would represent a high amount of the macromolecular FITC-PHEA-EDACPTA-LA (DDLA ≈ 13 mol %). Functionalization was first carried out in increasing acidity media (Table S3, FITC-Pol1, FITC-Pol2, FITC-Pol3). It can be concluded that the more acidic the reaction medium, the higher the extent of functionalization (Figure 3). This is due to the reduction of disulfide by TCEP that proceeds readily at low pH as thiolate−disulfide interchange is prevented because only low levels of thiolate are present.44 However, for the sake of having mild conditions that could be extrapolated to a large number of biomolecules, the less acidic conditions have been retained for the rest of this work (HCl 0.1 N). Under these mild conditions, one can still improve the surface thiol−yne modification by simply increasing the amount of initial thiol groups as demonstrated by the reaction FITC-Pol4 where a 25 mM concentration was used instead of 10 mM. Even with a high 50 mM concentration of FITC-PHEA-EDA-CPTA-LA, the extent of functionalization in the presence of DMSO was lower than under the mild acidic conditions used in FITC-Pol5 (ratio Fluo/RI = 7.6 × 10−4 against 9.4 × 10−4, respectively). XPS Analyses of PHEA-EDA-CPTA-LA Functionalized PLA Surfaces. Finally, in anticipation of the antibacterial tests, PLA surfaces have been functionalized by the nonfluorescent PHEAEDA-CPTA-LA copolymer and analyzed by XPS to quantitatively evaluate the extent of functionalization. Conditions were the same as the one used for the FITC-labeled polymer (irradiation time = 2 × 15 min, [Irgacure 2959] = 2 mol %/SH, [SH] = 25 mM, TCEP/SH = 6, reduction time = 2.5 h, H2O/ EtOH (1:1) (HCl 0.1 N)). Table 1 gives the XPS binding energies, the full-widths-at-half-maximum (fwhm’s) and the atomic compositions for the C 1s, O 1s, N 1s, and S 2p species. Figure 4 shows XPS survey-scan spectra for pristine PLA, PHEA-EDA-CPTA-LA copolymer, and PLA surface grafted with PHEA-EDA-CPTA-LA, as well as the high-resolution 4358


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Table 1. Comparison of Assignments (eV) of XPS Peaks, Fwhm, and Composition for PLA, PHEA-EDA-CPTA-LA, Propargylated PLA Surfaces Functionalized with PHEA-EDA-CPTA-LA and Control Pristine PLA Plate Functionalized with PHEA-EDA-CPTA-LAa C 1s

O 1s

N 1s

S 2p

PLA

BE (eV) fwhm composition (atom %)b

285.00 2.08 63.78

532.88 2.87 36.22

PHEA-EDA-CPTA-LA

BE (eV) fwhm composition (atom %)b composition (atom %)c

285.53 2.45 61.74 54.98

531.14 1.98 22.25 22.80

399.46 1.40 14.05 19.08

163.12 1.23 1.56 1.57

PLA-g-PHEA-EDA-CPTA-LA


285.02 2.02 62.81

532.60 3.10 32.37

399.45 1.43 4.78

162.98 0.55 0.06

PLA control plate/PHEA-EDA-CPTA-LA


285.13 2.28 62.15

532.68 3.02 34.63

399.47 1.54 3.23

a Irradiation time = 2 × 15 min, concentration of photoinitiator [Irg 2959] = 2 mol %/SH, [SH] = 25 mM, TCEP/SH = 6, reduction time = 2.5 h, H2O/EtOH (1:1) (HCL 0.1 N). bExperimental results obtained from XPS analysis. cTheoretical atomic composition of the grafted chain based on the chemical structure.

scans in the S2p region for the PHEA-EDA-CPTA-LA copolymer and the PLA/PHEA-EDA-CPTA-LA grafted surface. For all samples, survey scan spectra show two major peaks located at 285 and 531 eV binding energies, corresponding to C1s and O1s, respectively. PHEA-EDA-CPTA-LA copolymer and PLA/PHEA-EDA-CPTA-LA grafted surface spectra show additional peaks at 399 and 163 eV assigned to N1s and S2p, respectively. Since PLA does not contain nitrogen and sulfur, they were used as elemental markers for the PHEA-EDACPTA-LA immobilization. Indeed, it is generally recognized that the appearance of S2p3/2 peak (162.8 eV) is a direct evidence to justify the immobilization of thiol in thiol−yne click reaction.45 A control experiment was done with nonpropargylated PLA (Table 1). No signal corresponding to sulfur could be found, which confirmed the absence of residual adsorbed copolymer after the washings steps. One should note the presence of nitrogen; however, this was assigned to a surface contamination as no corresponding sulfur atom was detected and that control experiments did not show any adsorption of FITC-PHEA-EDA-CPTA-LA under the same conditions on pristine PLA (Figure 2c). High resolution spectra were carried out in the S2p region in an attempt to evaluate the extent of grafting and reduction step efficiency. No further information could be obtained as typical S2p3/2 BEs for alkylthiol, thioether, and dialkyl disulfides are between 163 and 164 eV for all species.46 Instead, information on the extent of quaternization of the grafted PHEA-EDACPTA-LA was obtained by deconvolution of the high resolution spectra in the N1s region (Supporting Information Figure S3). Three contributions were found, with a binding energy at 399 eV characteristic of the C−N amide, at 400 eV characteristic of the C−N amine, and at 402 eV attributed to C−N+ of ammonium groups. The two peaks at 400 and 402 eV were approximately of equal intensity on the deconvoluted spectrum, which confirms the 50% functionalization of EDA amine groups by CPTA. Antiadherence Activity of the Modified PLA Surfaces. Bacterial infection and proliferation on the surface of

Figure 4. XPS survey scan spectra of pristine PLA (black line), PHEAEDA-CPTA-LA copolymer (blue line), and modified PLA/PHEAEDA-CPTA-LA surface (red line). Insets correspond to the high resolution spectra in the S2p region.

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implantable materials relies on a four-step mechanism with (i) bacterial attachment to the surface, (ii) bacterial accumulation in multiple layers, (iii) biofilm maturation that includes the appearance of sessile bacteria not sensitive to conventional antibiotics, and finally, (iv) cell detachment from the biofilm in a planktonic state that causes the dispersal of small bacterial colonies and results in a more general and pronounced infection.47 In a previous study, we demonstrated that PLA surfaces functionalized with quaternized poly(2(dimethylamino)ethyl methacrylate)s (QPDMAEMAs) have a strong antibacterial activity with reduction factors (ASTM E 2149-01) superior to 99.999%.19 QPDMAEMAs are biostable compounds that are known to be cytotoxic depending on their macromolecular parameters and use,48 but in this case only few QPDMAEMAs chains were required at the PLA surface and these chains were immobilized. However, in the present work, we were interested in assessing the antibacterial activity of the more biocompatible PHEA derivatives that have already been used in various biomedical applications. The antiadherence efficiency of the modified PLA surfaces was evaluated against four strains including two Gram-negative (E. coli, P. aeruginosa) and two Gram-positive (S. aureus, S. epidermidis) isolates, which are among the most representative bacterial strains responsible for nosocomial infections. PHEAEDA-CPTA-LA modified PLA surfaces showed similar and high antiadherence efficiency toward all strains (Figure 5). No

Figure 6. Quantification of biofilms on PLA/PHEA-EDA-CPTA-LA surface (red bars) after 72 h by crystal violet staining and measurement of the released stained bacteria at OD 600. Results are expressed relative to the biofilm on control PLA plates (blue bars) (data are expressed as means ± SD and correspond to measurements in triplicate).

of the modified PLA surfaces. In addition, this result can be compared to the 60% decrease in OD600 observed in our previous work with QPDMAEMAs modified PLA surfaces.19 A higher antibacterial activity was observed despite the use of the small methyl groups of the trimethylammonium, which is known to be less active toward bacteria than heptyl or octyl groups.49 Indeed, Mw, alkylating agent, but also charge density are critical parameters with regard to the polyquaternary ammoniums (PQAs) antibacterial activity.50−52 PQA binding to the bacterial cytoplasmic membrane and their diffusion through the cell wall are believed to be linked both to electrostatic (Mw and charge density) and hydrophobic (alkylating agent chain length) interactions. Thus, an optimal range of alkyl chain lengths and Mw’s exists for the antibacterial action of these polymers, which may differ depending on the substrate/PQA couple. Therefore, the observed higher antibiofilm activity may be ascribed to the higher molecular weight of the PHEA-EDA-CPTA-LA copolymer (Mn > 50 000 g/mol, Supporting Information Table S1) compared to the QPDMAEMAs (Mn ≤ 10 000 g/mol) and to a more favorable overall hydrophobic/hydrophilic balance of the PHEA-EDACPTA-LA copolymers. Cytocompatibility of the PHEA-EDA-CPTA-LA Modified PLA Surfaces. Given their final potential application as antibacterial implants, we were interested in evaluating the cytocompatibility of the proposed antibacterial PLA surfaces, which in addition to exerting potent antibacterial effects should also be nontoxic for the tissues surrounding the implantation site. Cytocompatibility tests of PHEA-EDA-CPTA-LA modified PLA surfaces were conducted on the L-929 fibroblasts cell line and the antibacterial surfaces were compared to pristine PLA surfaces and TCPS control plates. At 1 day, similar level of L929 fibroblasts viability was observed with no statistical differences in cell number on the antibacterial PLA surfaces and on pristine PLA (Figure 7). This result suggests that L929 correctly adhered onto the antibacterial PLA surfaces and the cell adhesion process is equivalent for all the tested surfaces. For both surfaces, cell proliferation was slower than on the TCPS control but nevertheless high enough, with a relative proliferation of about 63% for PHEA-EDA-CPTA-LA modified PLA surfaces and 70% for PLA. However, at 12 days, proliferation on PHEA-

Figure 5. Comparison of antibacterial activity of pristine PLA surface (blue bars) versus modified PLA/PHEA-EDA-CPTA-LA surface (red bars) against four bacterial strains: E. coli, P. aeruginosa, S. aureus, and S. epidermidis (data are expressed as means ± SD and correspond to measurements in triplicate).

significant difference could be found between the various bacterial strains, with a very potent antibacterial activity of the modified surfaces corresponding to reduction factors superior to 99.98% (Supporting Information Table S4). Biofilm formation was also evaluated by luminometry and fluorescence microscopy. Marked antibiofilm activity was found on all tested bacterial strains. Figure 6 shows that biofilm formation was strongly inhibited by the modification of PLA surfaces. This was evidenced by a strong decrease of the total number of bacteria present in the biofilm formed after 72 h of incubation. About 80% decrease in OD600 was obtained with the four strains on the modified PLA surfaces compared to the control PLA surfaces. Interestingly, similar high efficiencies were found against the Gram-negative and Gram-positive isolates, which confirmed the nonspecific antibacterial activity 4360


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photochemical immobilization of (FITC-S)2 and FITCPHEA-EDA-CPTA-LA). This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Fondazione Cariplo (Project 2010-0454) and MIUR (PRIN 2010-2011, 20109P1MH2_003) for funding, Valér ie Flaud from the Institut Charles Gerhardt de Montpellier (ICGM - CNRS UMR 5253), Plateforme d’Analyses et de Caractérisation) for the XPS analyses, and Sylvie Hunger for the NMR characterizations.

Figure 7. L929 proliferation on PLA/PHEA-EDA-CPTA-LA surface (green bars) compared to pristine PLA surface (blue bars) and TCPS control (gray bars) at 1, 4, and 12 days (data are expressed as means ± SD and correspond to measurements in triplicate).

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EDA-CPTA-LA modified PLA surfaces only represented 30% of the proliferation on TCPS control against 75% on pristine PLA. The number of cells after 12 days on the antibacterial surface was the same than after 4 days. This set of results tends to demonstrate that the PHEA-EDA-CPTA-LA modified PLA surfaces, although not cytotoxic, are not prone to high proliferation as a probable result of the presence of ammoniums groups on PHEA-EDA-CPTA-LA. Therefore, it appears that these antibacterial surfaces tend to guarantee cell viability without promoting proliferation. To address this specific point, one could consider decreasing the PHEA-EDA-CPTA-LA molecular weight and try to find a best compromise between the potent antibacterial activity (high molecular weight PQAs) and the absence of cytotoxic effects (low molecular weight PQAs). However, this is beyond the scope of this work and should be the matter of further studies.

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CONCLUSION To summarize, we describe a novel approach toward functional PLA surfaces. The use of thiol−yne photochemical reaction leads to an easy surface functionalization of the degradable PLA in a rapid and versatile manner that guarantees the PLA chains integrity and the PLA shape integrity. A methodological screening demonstrated the possibility to covalently bind hydrophobic and hydrophilic molecules at the PLA surface. The synthesis of clickable PHEA-EDA-CPTA-LA and its grafting on PLA to generate antibacterial surfaces further demonstrated the potential of this approach. The PLA modified by this new cationic PHEA derivative showed marked antiadherence and antibiofilm activities without cytotoxicity. The photochemical thiol−yne post-polymerization modification of PLA shows therefore great promise and will be further considered for various biomedical applications.

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REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

Synthetic procedures of pyrene thiol derivative (Py-SH) and FITC-labeled PHEA-EDA-CPTA-LA; 1H NMR of Py-SH and (FITC-S)2; synthetic scheme for the synthesis of PHEA-EDACPTA-LA; XPS high resolution spectrum of PHEA-EDACPTA-LA; Table S1 (characterizations of PHEA copolymer), Table S2 (conditions of thiol−yne photochemical immobilization of Py-SH), and Table S3 (conditions of thiol−yne 4361


Biomacromolecules

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