Sarah El Habnouni

Dr Vincent Darcos

Dr Xavier Garric

Jean-Philippe Lavigne

Prof. J Benjamin Nottelet
email: benjamin.nottelet@univ-montp1.fr

Jean Coudane

Dr S El Habnouni

Coudane

Dr J.-P Lavigne

Introduction

One of today's most-challenging fi elds is that of biomaterials, where chemical and medical scientists are working together to improve human health. Because of their various compositions, structures and excellent properties, a very-large number of polymers have been evaluated as potential biomaterials for a wide range of biomedical applications. [ 1 ] Special emphasis is placed on biodegradable polymers that are employed as drug carriers, wound dressings, medical devices and scaffolds in tissue engineering; [ 2 ] because of their excellent biocompatibility and adjustable degradability, [ 3 ] these include polyesters such as polylactide (PLA), polyglycolide (PGA) and poly( ε -caprolactone) (PCL), which are now in widespread use. As an example, PLA was approved by the US Food and Drug Administration (FDA) in the 1970s, and has since been used in the form of sutures, clips, plates and screws, ultrasound contrast agents, nerve guides and even drug-delivery devices. [ 4 ] In addition to these "classical" uses, PLA is also attractive and under investigation for innovative applications, such as devices bearing magnetic-resonance-imaging contrast agents. [ 5 ] Unfortunately, PLA has a few drawbacks, such as its crystallinity, lack of reactive side-chain groups and hydrophobicity. Generally, in order to modulate their physico-chemical and/or biological properties, polyesters are functionalized, and here two main strategies are employed. The fi rst involves the synthesis and (co)polymerization of functionalized lactones, [ 6 ] and the second, the chemical modifi cation of preformed polyesters in solution. [ 7 ] Both yield novel functional polyester chains, lending new properties. However, for implantable materials, it is not always required or even benefi cial to change all of a material's properties by modifying its polymer chains because: i) biochemical reactions occur at the surface of the material or at the interface with body fl uids; and ii) as PLA devices are FDA-approved, it is important to ensure that their intrinsic properties, such as their biocompatibility and degradation rate, are unchanged, while at the same time broadening their application possibilities. This seemingly contradictory requirement to both modifying and maintaining their properties explains why such interest focuses on a controlled modifi cation of degradable polyester surfaces.

Mild Methodology for the Versatile Chemical Modifi cation of Polylactide Surfaces: Original Combination of Anionic and Click Chemistry for Biomedical Applications

Biodegradable aliphatic polyesters such as polylactide (PLA) are widely used in medical applications. When employed as an implantable material, the control of the surface properties of PLA is of great interest because biochemical reactions occur on the surface or at interfaces. Thus, chemical modifi cations of the surface of degradable polyesters can be used to tailor the surface properties while preserving the bulk characteristics. This paper proposes a simple and versatile method of preventing polymer degradation and immobilizing simple molecules, macromolecules, and biomolecules on PLA surfaces. The method is based on a one-pot, two-step procedure: anionic activation under selected conditions followed by propargylation to form a "clickable" PLA surface. This surface is extensively characterized by SEC and fl uorescence techniques using a fl uorescent probe, confi rming both the functionalization and the absence of PLA degradation. An example of the surface immobilization of a bioactive compound is then described: a well-defi ned α α -azido-functionalized poly(quaternary ammonium) synthesized via ATRP is covalently bound to the propargylated PLA surface using "click" chemistry. This covalent grafting is confi rmed by SEC and XPS analyses. The increase in surface hydrophilicity is demonstrated by water contact-angle measurements. Finally, a primary investigation is conducted to determine the antibacterial activity of modifi ed PLA surfaces against E. coli and S. aureus . DOI: 10.1002/adfm.201100412 degradation; and ii) yield an activated stable surface that can be derivatized using a simple procedure for the immobilization of small molecules, macromolecules and biomolecules, without alterating the structure of these active compounds. These requirements are met by click chemistry, especially the Cu(I)-catalyzed Huisgen 1,3-dipolar cycloaddition of alkynes and azides (CuAAC), which has demonstrated its superiority as a binding tool in material surface science. Generally speaking, click chemistry is a versatile method, combining mild experimental conditions, tolerance of functional groups, and high yields. [ 28 ] With regard to the modifi cation of surfaces, CuAAC can effi ciently couple chemical species to surfaces, thus allowing for the formation of complex molecular architectures on a solid material. [ 29 ] In this paper, we report on the original and straightforward synthesis of a "clickable" PLA surface. An anionic activation, in the presence of lithium di-isopropyl amide (LDA) under selected conditions followed by treatment with propargyl bromide yields an activated and stable clickable PLA surface. Such surfaces were carefully characterized and used to graft a hydrophilic polymer using CuAAC. As proof of the versatility, a quaternized poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) bearing a fl uorescent probe (QFPD-MAEMA), synthesized by an ATRP process was also grafted to the PLA surface, yielding a PLA antibacterial surface of interest for implanted biomaterials. Preliminary results concerning antiadherence properties against Escherichia coli ( E. coli ) and Staphylococcus aureus ( S. aureus ) are presented and discussed.

Results and Discussion

Our approach to functionalizing the surface of a 3D PLA 94 object is based on a three-step strategy combining: 1) synthesis of a propargyl-functionalized surface via anionic activation; 2) synthesis of an azido-functionalized bioactive compound; and 3) Huisgen's 1,3-dipolar cycloaddition (click chemistry) of the bioactive compound to the propargylated surface. This strategy is expected to be versatile and offers a wide range of functionalizations. In this work, as the fi rst example of a bioactive compound, we synthesized a quaternized and fl uorescent α -azido-terminated PDMAEMA ( α -N 3 QFPDMAEMA) via atomtransfer radical polymerization (ATRP) of 2-(dimethylamino) ethyl methacrylate (DMAEMA) with a fl uorescent probe, anthracenylmethyl methacrylate (AMM), followed by quaternization of the amine group.

Synthesis of the Clickable Surface

The fi rst goal was to make the surface of the PLA 94 clickable. Several years ago, we presented a method that can be used to chemically modify aliphatic polyesters. This modifi cation route is based on activation of a polyester chain by proton extraction from the methylene groups in the a-position of the carbonyl group, using lithium N , N -di-isopropylamide (LDA) in tetrahydrofuran (THF). The activated polymer chains bearing carbanion sites are then combined with different electrophiles to generate novel copolymers. Despite the fact that the polymer chains underwent limited degradation during the fi rst step Such a surface modifi cation is the aim of current biomedical research as this would improve the biocompatibility and biointegration, avoid non-specifi c protein adsorption, control the adsorption of proteins and/or cells on engineered surfaces, or confer antibacterial properties to medical devices. [ 8 ] Surface-modifi cation methods may be classifi ed as nonpermanent (non-covalent attachment of functional groups) or permanent (covalent attachment). [ 9 ] Temporary surfacemodifi cation methods include coating, [ 10 ] entrapment [ 11 ] and migratory additives. [ 12 ] Although these methods have been used to modify PLA surfaces, they suffer from the drawbacks inherent to their non-permanent nature: the release of bioactive compounds from such modifi ed surfaces and the subsequent loss of activity potentially makes them unsuitable for most biomedical and consumer applications. For example, in the case of an antibacterial material, the release of leachable biocides that include antibiotics, phenols, iodine, quaternary ammonium or heavy metals such as silver, tin, and mercury in their composition could have serious adverse effects on the durability, useful lifetime, effi ciency and toxicity of the treated biomaterial. [ 13 ] As a consequence, it is important for the bioactive compounds to be covalently immobilized on the surface.

Up until now, permanent surface modifi cation has mainly been obtained by plasma treatment, photografting or wet chemistry, [ 14 ] but these techniques have a number of weaknesses. For instance, in plasma treatment, despite the fact that PLA wettability and cell affi nity can be improved, [ 15 ] the possibility of surface rearrangement reduces the effi ciency of the process, [ 16 ] and can lead to a non-permanent modifi cation. [ 17 ] Furthermore, plasma treatment can also affect PLA degradation. [ 18 ] Photografting has been extensively used to tailor the surface properties of PLA thanks to the advantages it offers: low cost, mild reaction conditions, selectivity of UV light and permanent alteration of surface chemistry. [ 19 ] Also, the penetration of immobilized molecules into the bulk, which may occur when organic solvents are used in the liquid-phase photografting protocol, [ 20 ] can be avoided by using vapour-phase photografting. [ 21 ] However, this technique suffers from its complexity, with new parameters and a new optimization required for each surface functionalization. The third strategy used to covalently modify the PLA surface is wet chemistry. At present, the wet-chemistry modifi cation of PLA is restricted to degrading conditions. An example is surface hydrolysis, by acid/base degradation, which is used to generate carboxylic acids (-COOH) on PLA, [ 22 ] which may then be further activated with phosphorous pentachloride (PCl 5 ), [ 23 ] thionyl chloride (SOCl 2 ), [ 24 ] or water-soluble carbodiimides [ 25 ] before subsequent conjugation with amines or alcohols. [ 26 ] Aminolysis is another means used to introduce reactive amino groups onto PLA surfaces. [ 27 ] In brief, despite the fact that these permanent surfacemodifi cation techniques are able to modify PLA surfaces, they lack versatility, since new chemistry has to be developed each time a new functionality/property is sought. Finally, given that PLA is easily degraded, modifi cations that match the complex requirements imposed by biomedical applications and tissue engineering are quite a challenge. There is therefore a need to develop a simple protocol to functionalize the surface of PLA and enable the immobilization of a wide range of bioactive compounds. Ideally, this method should: i) prevent polymer of the reaction, various functionalized PCLs of rather-high molecular weights were easily obtained in solution ( M n ≥ 20 000 g mol -1 ). [ 30 ] Preliminary studies on PLA have shown that anionic activation of the surface of a PLA plate can occur under the same conditions, but is accompanied by the degradation of the surface polymer chains. [ 31 ] In this paper, we report on a general method that is mild enough to prevent polymer degradation. This strategy is shown in Scheme 1 . The surface of a PLA plate was activated by treatment with a non-nucleophilic strong base, LDA, at low temperature in a mixture of chosen solvents.

PLA degradation was avoided by a careful choice of the anionic activation conditions. Various solvent mixtures, reaction times and reaction temperatures were tested. The best results were obtained when the PLA plates were functionalized using LDA in a mixture of THF and diethyl ether (1/2 v/v) for half an hour at -50 ° C to -30 ° C, followed by the addition of propargyl bromide in two-fold excess versus LDA (Scheme 1a ). The electrophile was added over a period of one hour at a temperature rising from -30 ° C to room temperature (RT). The plate was then washed with water and diethyl ether to remove any non-covalently grafted species.

The challenge here was to maintain the physico-chemical properties of the core of the plate while modifying the surface. However, since functionalization occurred only at the surface, conventional characterization techniques such as IR or Raman spectroscopy (which are both penetrating techniques), were unable to detect the propargyl group. The presence of propargyl groups on the surface and their reactivity were therefore investigated by an indirect detection method using a fl uorescent probe (Scheme 1b ) and 9-azidomethylanthracene was prepared for this purpose in accordance with a described procedure. [ 32 ] This compound was used to detect the presence of propargyl groups on the surface of the PLA and to check their chemical availability for CuAAC. The click reaction of the probe was performed under aqueous click conditions. [ 33 ] The extensively washed plate ( 2 ) was analyzed using size-exclusion chromatography (SEC) with refractometric and fl uorescence double detection. The SEC traces obtained from the surface to the core showed that only the external layers were fl uorescent ( Figure 1 ). Therefore, the core was unaffected and the bulk properties were conserved. In addition, SEC using refractometric detection of molecular weight and polydispersity. The second route, called the "grafting-onto" technique, involves the reaction between a functional group of a premade polymer and an active site on the surface. The advantage is that control can be exerted over the molecular weight and polydispersity of the grafted polymer. However, this technique is limited by the low grafting density due to poor accessibility to the reactive surface groups caused by steric hindrance. Thanks to its very-high effi ciency, CuAAC is often considered as the tool of choice to overcome this limitation. As an initial demonstration of our methodology, we synthesized a poly(quaternary ammonium) with antibacterial activity and grafted it by CuAAC onto a clickable PLA surface. [ 34 ] PDMAEMA derivatives were therefore synthesized in accordance with well-known chemistry. [ 35 ] A fl uorescent-azide-containing PDMAEMA ( α -N 3 FPDMAEMA) ( 5) precursor was prepared following a similar procedure to that reported by Agut et al. [ 36 ] Well-defi ned α -N 3 FPDMAEMA was synthesized by ATRP at 60 ° C in toluene in the presence of CuBr/ N , N , N ′ , N ″ , N ″ -pentamethyldiethylenetriamine (PMDETA) as a catalytic system, using a specifi c azide-functionalized initiator, (3-azidopropyl)bromoisobutyrate ( 3 ), and a fl uorescent probe ( 4 ) ( Scheme 2 ).

SEC showed a monomodal trace with polydispersity below 1.2. The molecular weights ( M n ) determined by SEC were relative to polystyrene standards, so more-accurate values were provided by 1 H-NMR spectroscopy. Accordingly, M n,NMR of 5 was determined to be 6500 g mol -1 , in good agreement with showed that the molecular weight and polydispersity of the surface were unchanged compared to that of the core. The external layers and the core had the same molecular weight ( M n = 100 000 g mol -1 ) and polydispersity (PDI = 1.9).

Our specifi c conditions may therefore be considered to yield a stable intermediate, which is the clickable surface of the PLA 94 ( 1 ), with no degradation and without altering the 3D shape of the plate. To the best of our knowledge, this is the fi rst example of a modifi ed PLA surface obtained in a simple and mild onestep procedure, allowing the specifi c coupling of azido-modifi ed bioactive molecules by click chemistry.

Synthesis of a Fluorescent, Quaternized and Clickable

PDMAEMA ( α α -N 3 QFPDMAEMA)
The grafting of 9-azidomethylanthracene as a fl uorescent probe to the clickable PLA surface demonstrated that functionalization by small molecules is possible. Surface modifi cations with macromolecules are far more challenging. Typically, two routes are used to bind macromolecules to the surface of a polymeric device. The fi rst is the "grafting-from" procedure, involving the polymerization of monomers from surfaceanchored initiators. This method ensures the synthesis of polymer chains covalently attached to the surface and can produce a high grafting density. The major drawback of this method is the lack of control over the grafted polymer in terms the theoretical value of 7000 g mol -1 . In addition to the molecular weight, the SEC chromatograms, using a photodiode-array (PDA) detector, confi rmed the incorporation of the fl uorescent methacrylate dye during the ATRP process. The presence of azide groups was evidenced by the characteristic azide absorbance peak at ≈ 2100 cm -1 in the FTIR spectrum ( S1-S4 , Supporting Information). Finally, quaternized α -N 3 FPDMAEMA ( α -N 3 QFPDMAEMA) was obtained by a conventional technique, using methyl iodide or heptyl iodide as an alkylating agent, in THF for 18 h at room temperature.

Click Reaction of α α -N 3 QFPDMAEMA on Propargylated PLA 94 Surface

The next critical step was the "click-chemistry" reaction of α -N 3 QFPDMAEMA on the propargyl-functionalized PLA surface. Although already described for a number of other surface types, we sought mild conditions for the heterogeneous click reaction of a macromolecule onto the surface of the propargylated PLA. CuAAC was performed under aqueous conditions, following the same procedure as that described for the labelling of the surface 2 . The reaction was carried out for 48 h at 40 ° C ( Scheme 3 ).

The plate was then extensively washed with water and ethanol to remove all physically adsorbed molecules. SEC analysis of the surface, using refractometric and fl uorescence double detection, showed this approach to be effective. No degradation was observed by refractometric detection and the peak corresponding to the PLA exhibited fl uorescence properties due to the covalent binding of the fl uorescent PDMAEMA on the plate ( S5 , Supporting Information). The same experiment, carried out without the propargylic functionalization step, was performed as a blank. Specifi cally, after reaction of the PLA plate with LDA, all of the carbanionic sites were quenched with water and immersed in the "click" reaction medium with α -N 3 QFPDMAEMA under the same conditions as described above. This control reaction was carried out to ensure that no fl uorescence was detected at the PLA retention time, and that washings were able to remove all of the α -N 3 QFPDMAEMA that was potentially adsorbed on the surface. SEC analysis of the surface with fl uorescence detection showed that no peaks were present ( S6 , Supporting Information). It may therefore be stated that the fl uorescence signal observed for the surface of 7 (see Scheme 3) was solely due to the covalent binding of the QFPDMAEMA to the surface.

X-Ray-Photoelectron-Spectroscopy (XPS) Analysis

XPS was used to characterize the surfaces further. Given its high sensitivity and non-destructive nature, XPS was chosen to analyse the surface composition after the grafting of the QFP-DMAEMA. Table 1 gives the XPS binding energies, and the full-widths-at-half-maximum (fwhms) for the C 1s, O 1s, and N It may be calculated from the XPS survey scans of the neat PLA that the C/O ratio was 1.7 (i.e., close to the theoretical value of 1.5, which is based on the chemical structure of PLA). The small difference is due to the socalled adventitious carbon induced by the adsorption of impurity hydrocarbons, and is used for binding-energy calibration by setting its energy to 284.8 eV, to correct for sample charging. [ 37 ] XPS survey scans of the PLAgraft -QFPDMAEMA showed one additional element: N 1s at 398 eV. Since PLA does not contain nitrogen, it was used as an elemental marker for the QFPDMAEMA immobilization. Therefore, the presence of nitrogen confi rmed successful QFPDMAEMA binding. It is important to mention here that the use of copper and its partial removal from materials is often a limitation of "click" chemistry, when targeting biomedical applications. Our experiments showed that extensive washing removed all copper, as none was detected in the 933 eV region by the XPS analyses of the PLA surfaces.

QFPDMAEMA immobilization on the surface was further investigated by high-resolution XPS in the C 1s, O 1s and N 1s regions ( Figure 3 ). The high-resolution scan of the neat PLA was deconvoluted into three C 1s peak components of approximately the same composition, corresponding to the three types b) Theoretical atomic composition of the grafted chain based on the chemical structure.

C-H/C-C C-N C-N + C-O O-C = O N-C N + -C O-C O = C BE (
the triazole chain-ends and the DMAEMA units. The C 1s core-level spectrum shows fi ve peak components at BE ≈ 284.76, 285.66, 286.02, 286.82 and 288.88 eV, which were attributed to C-H/C-C, C-N, C-N + , C-O and O = C-O, respectively, in accordance with the literature. [ 42 ] The two additional peaks corresponding to the binding energies of C-N and C-N + confi rmed the QFPDMAEMA grafting onto the surface of the PLA. Finally, the highresolution scan of O 1s showed two peaks at BE ≈ 532.08 and 533.52 eV. These were assigned to O-C and O = C-O. These results confi rmed that the PLA surface had been chemically modifi ed by the covalent immobilization of QFPDAEMA, without any degradation of this fragile material. Two surface properties were then investigated, namely wettability and antiadherence against bacteria.

Static Contact-Angle Measurements

The surface wettability was measured using a static angle technique ( S7 , Supporting Information). As expected, the PLA plate was hydrophobic, as demonstrated by an 84 ° contact angle. After immobilization of the FPDMAEMA quaternized with methyl iodide (C1 QFPDMAEMA M n,NMR = 6500 g mol -1 ), the contact angle decreased to 55 ° because of the presence of hydrophilic quaternary ammonium (QA).

In Vitro Antibacterial Activity

The ability of the two modifi ed surfaces to prevent bacterial adherence was tested with E. coli and S. aureus . The data are reported in Table 2 . The two surfaces modifi ed by FPDMAEMA quaternized with either a methyl (C1 QFP-DMAEMA) or a heptyl (C7 QFPDMAEMA) group reduced the adherence of E. coli and S. aureus compared with neat PLA. For the C1-QFPDMAEMA-grafted surfaces, the colony-forming-unit (CFU) ratio was 2.7 × 10 -4 for E. coli and and 3.3 × 10 -5 for S. aureus , as compared with genuine PLA. This anti-adherence activity was of carbon atoms present in PLA. The peaks at 284.77, 286.79 and 288.86 eV were assigned to C-H/C-C, C-O and O-C = O bonds, respectively. The O 1s peaks at 532.23 and 533.67 eV correspond to the binding energies of the C-O and O-C = O groups in PLA, repsectively. These results were expected and are consistent with previous reports. [ 38 ] The high-resolution scan in the N 1s region shows two peaks of approximately equal intensity. The low binding energy at 399.55 eV is characteristic of the C-N species in QFPDMAEMA, and the peak at 402.28 eV was attributed to C-N ± . [ 39 ] The presence of neutral nitrogen on the QFPDMAEMA chains indicates partial quaternization. The absence of a peak corresponding to free azido groups at BE = 405 eV [ 40 ] is additional evidence that the QFPDMAEMA was covalently attached on the surface. No peaks corresponding to the nitrogen atoms of the triazole ring (doubly bonded, 400.3 eV and singly bonded, 398.2 eV) [ 41 ] were detected as a result of the low grafting density of the PDMAEMA on the PLA surface, and of the 1/50 ratio between Binding Energy (eV) as compared to the bare PLA. This study is ongoing with investigations focusing on other bacterial strains, the formation of biofi lms, and an evaluation of the surface biocompatibility using cell cultures. Other biomedical applications for PLA will also be investigated using this strategy for the synthesis and surface immobilization of other azido-modifi ed bioactive compounds.

Experimental Section

Materials : PLA 94 was synthesized by the ring-opening copolymerization of L -lactide (88%) and DL -lactide (12%) (PURAC, Lyon, France), in bulk, using tin 2-ethylhexanoate as a catalyst. ( M n = 100 000 g mol -1 ; PDI = 1.9). The PLA 94 plate was shaped using a Carver hydraulic heated press (4120-289). The plates were heated at 130 ° C and PLA powder was pressed for 5 min before cooling under pressure (1.5 × 10 7 Pa). PLA 94 plates with a thickness of 500 μ m were obtained. DMAEMA (98%, Aldrich, St Quentin Fallavier, France) was passed through a column of basic alumina to remove the stabilizing agents. Toluene (Aldrich) and dichloromethane (DCM) (Aldrich) were dried by refl uxing over CaH 2 and were then distilled before use. Triethylamine (TEA) (Aldrich) was distilled over KOH. THF (Aldrich) was dried by refl uxing over a benzophenone/ sodium mixture until a deep-blue color was obtained and was then distilled. All of the other solvents and chemicals were purchased from Aldrich and were used as received.

NMR Spectroscopy : The 1 H-NMR and 13 C-NMR spectra were recorded using a Bruker spectrometer (AMX300) operating at 300 MHz and 75 MHz, respectively. Deuterated chloroform or deuterated dimethyl sulfoxide (DMSO) were used as solvents. The chemical shifts are expressed in ppm with respect to tetramethylsilane (TMS).

Attenuated Total Refl ection (ATR)-FTIR Spectroscopy : The FTIR spectra were recorded using a Perkin-Elmer Spectrum 100 FTIR spectrometer using the attenuated-total-refl ectance (ATR) method.

Size-Exclusion Chromatography : SEC was performed at room temperature using 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 fl uorescence detector. Calibration was established with polystyrene standards from Polymer Laboratories. THF and THF/TEA (95/5; v/v)were used as eluents for the PDMAEMA analyses, with a fl ow rate of 1 mL min -1 .

XPS : XPS was performed using an ESCALAB 250 photoelectron spectrometer with Al K α radiation (1486.6 eV) and an overall instrument resolution of 1.1 eV. All of the spectra were collected at an electron take-off angle of 90 ° to the sample surface. Surfaces of 400 μ m 2 were analyzed. The binding energies were corrected by referencing the binding energy of the C-C component of C 1s to 284.8 eV.

Contact-Angle Measurements : The water contact angles were measured using the sessile-drop method. The system was composed of a sample stage to hold the substrate, a syringe to apply a droplet of ultra-pure water, a light source on the back and a camera to capture the profi le of the drop on the PLA surfaces. Image J software (NIH, USA) was used to measure the contact angle. A water-drop volume of 50 μ L was placed on the fi lm surface and allowed to stabilize for 30 s.

Synthesis of the Clickable Surface of PLA 94 ( 1 ) : Typically, a PLA 94 plate ( m = 125 mg, thickness = 500 μ m, surface = 1.76 cm 2 ) was immersed in a stirred solution of 2.0 M lithium di-isopropyl amide (1.5 mL, 3 mmol) in an anhydrous THF (60 mL)/diethyl ether (120 mL) mixture at -50 ° C under an inert argon atmosphere. This activation step was carried out for 30 min before the addition of propargyl bromide (700 μ L, 6 mmol) at -30 ° C. The mixture was stirred for 1 h at -30 ° C and then raised to room temperature. The PLA plate was quenched and washed with water, diethyl ether and methanol. Any residual solvent was removed under vacuum.

Synthesis of 9-Azidomethylanthracene : 9 -Azidomethylanthracene was synthesized from commercially available 9-chloromethylanthracene according to a previous report. [ 32 ] (99% yield): 1 H NMR (300 MHz, CDCl 3 , improved with the C7-QFPDMAEMA-grafted surfaces, which gave ratios of 2.7 × 10 -7 and 6.7 × 10 -7 , respectively.

Another experiment using a genetically modifi ed strain of E. coli expressing the gfp gene ( E.coli gfp + ) was performed to visualize the bacterial adherence on the modifi ed surfaces and on neat PLA by fl uorescence microscopy. The microscopy images are shown in Figure 4 . Fewer adherent bacteria were present on the C1-QFPDMAEMA-modifi ed surface than on the PLA unmodifi ed surface, and this phenomenon was even more marked with C7 QFPDMAEMA, where no fl uorescence was detected, showing that no bacteria were retained on this surface.

These preliminary results for the antibacterial activity of the modifi ed PLA surfaces are encouraging, and biological investigations, such as biofi lm assays and cell cultures, will follow. Furthermore, this antibacterial activity demonstrates that the strategy proposed herein enables PLA surfaces to be functionalized via the covalent grafting of (macro)molecules while preserving the bioactivity of these (macro)molecules and the bulk properties of the material.

Conclusions

A novel method has been developed to chemically modify the surface of biodegradable polyesters. Using carefully selected anionic reaction conditions, we have demonstrated that wet chemistry can be used to obtain propargyl-functionalized surfaces without altering the core. It is expected that this bioorthogonal functionalization enables the covalent immobilization of simple molecules, macromolecules and biomolecules by a click methodology, while preserving the other functionalities and/or activity. As an initial example, we have reported the grafting of an α -N 3 -functionalized poly(quaternary ammonium) using click chemistry. We then used SEC and XPS analyses to show that: i) only the external layer was modifi ed, without any degradation of the PLA core; ii) poly(quaternary ammonium) immobilization without adsorption was achieved by click chemistry in heterogeneous medium; and iii) no copper, used as a catalyst, was detected, which is of importance to ensure biocompatibility. This surface modifi cation fi rstly improved the hydrophilicity of the modifi ed PLA, with its water contact angle decreasing by approximately 29 ° . The biological activity of this modifi ed surface was also determined in terms of its potential antibacterial properties against E. coli and S. aureus . A marked antiadherent effect was shown, with up to 10 7 fewer bacteria on the treated surfaces Quaternization of α -Azido-FPDMAEMA ( 5 ) : α -Azido FPDMAEMA (500 mg) was dissolved in THF (100 mL) in a round-bottom fl ask. The mixture was stirred and an excess (2 eq.) of quaternizing agent (heptyl iodide or methyl iodide) was added. The solution was stirred at room temperature overnight. The crude copolymer was then purifi ed by dialysis against deionized water using a dialysis tube (cut-off = 3 500 Daltons). The fi nal aqueous solution was freeze-dried to yield the quaternized αazido FPDMAEMA.

Synthesis of PLA-graft-QFPDMAEMA Surface ( 6 ) : A clickable plate of PLA ( 1 ) was immersed in a solution of αazido QFPDMAEMA ( 5 ) (100 mg, 0.6 mmol) in a water (1 mL)/ethanol (3 mL) mixture. A Cu I catalyst was then generated in situ using copper sulfate (300 μ L of a 1 M aqueous solution) and sodium ascorbate (600 μ L of a 1 M solution). The reaction was performed at 40 ° C for 2 days. The PLA plate was extensively washed by dialysis against water until no fl uorescence was detected in the dialysis medium. The plate was then rinsed with ethanol and methanol, and was fi nally dried under vacuum prior to analysis. The surface was analyzed by water-contact-angle measurements and XPS. SEC analyses were performed using refractometric and fl uorescence double detection after dissolution of the surface in THF (30 s in 1 mL, 5 mg).

Antiadherence Activity: i) Two strains of bacteria were used, E. coli NECS19923, genetically modifi ed to express the gfp gene ( E. Coli gfp + ) and S. aureus NSA4201. Before in vitro antibacterial tests, the bacterial strains were grown aerobically overnight on Müller-Hinton medium at 37 ° C under stirring.

ii) Evaluation of the bacterial adherence on the modifi ed plates was performed following a procedure described previously. [ 42 ] The PLA plates were immersed in a bacterial solution (DO 600 = 0.05) for 1 h; then, nonadherent bacteria on the sample surface were removed by washing several times with sterilized water before incubation of the PLA objects in a neutral medium. After 24 h of incubation at 37 ° C, the plates were submitted to a vortex and ultrasound in physiological saline buffer to remove any adherent bacteria from the surface. Detached viable bacteria were counted by series dilutions, whereas adherent viable bacteria were quantifi ed by pressing the samples about fi fteen times directly onto the surface of a Müller-Hinton-type agar plate. Finally, the total bacterial adherence was calculated by adding the count of the CFUs, after overnight incubation of the agar plates at 37 ° C, to all cultivated bacteria.

iii) For the visualization of the bacteria on the surfaces, the samples were immersed in a diluted culture solution of E.coli gfp + (DO 600 = 0.05). After 1 h, the PLA objects were washed with sterilized water and observed using a fl uorescence microscope.

Figure 1

 1 Scheme 1 . a) Synthesis of a clickable surface of PLA 94 : i) LDA/ET 2 O/THF, -50 ° C to -30 ° C, 30 min; ii) propargyl bromide, -30 ° C to room temperature (RT), 1 h. b) Fluorescent labelling of the surface by CuAAC: iii) CuSO 4 /sodium ascorbate H 2 O/EtOH, 40 ° C, 48 h.

  Scheme 2 . a) Synthesis of α -azido initiator: i) NaN 3 , tetrabutylammonium hydrogen sulfate (TBAHS)/water, 80 ° C, 24 h then RT, 12 h; ii) TEA/DCM 0 ° C, 1 h then RT, 17 h. b) Synthesis of fl uorescent comonomer: iii) TEA, DCM, 0 ° C, 1 h then RT, 17 h. c) Copolymerization with DMAEMA by ATRP and quaternization: iv) CuBr, PMDETA, toluene, 60 ° C, 20 min; v) CH 3 I or C 7 H 15 I, THF, RT, 17 h. The table insert provides an overview of the proportions of 3 / 4 /DMAEMA, M n determined by 1 H-NMR spectroscopy and the PDI determined by SEC.

Scheme 3 .

 3 Scheme 3 . a) Synthesis of a clickable surface of PLA 94 : i) LDA/ET 2 O/THF, -50 ° C to -30 ° C, 30 min; ii) propargyl bromide, -30 ° C to RT, 1 h. b) Covalent immobilization of α -N 3 QFPDMAEMA by CuAAC: iii) CuSO 4 /sodium ascorbate H 2 O/EtOH, 40 ° C, 48 h.

Figure 2

 2 -b shows XPS survey-scan spectra for neat PLA and PLAgraft -QFPDMAEMA, respectively. Figure 2 a shows two peaks located at binding energies of 283 and 531 eV, corresponding to C 1s and O 1s, respectively.

Figure 2 .

 2 Figure 2 . a-b) XPS survey scan spectra of PLA (a) and PLAgraft -QFPDMAEMA (b) surfaces.

Figure 3 .

 3 Figure 3 . a-c) XPS high-resolution spectra of the C 1s (a), O 1s (b), and N 1s (c) regions of the PLA (left) and PLAgraft -QFPDMAEMA (right) surfaces.

Figure 4 .

 4 Figure 4 . a-c) Visualization of adherence of E. coli gfp + on: neat PLA (a) and PLAgraft -QFPDMAEMA plate with -CH 3 as an alkylating agent (b) and -C 7 H 15 as alkylating agent (c).

Table 1 .

 1 Comparison of assignments (eV) of XPS peaks, fwhm for PLA, and PLAgraft -QFPDMAEMA surface and composit N 3 QFPDMAEMA.

	PLA 94 a)

Table 2 .

 2 Comparison of antiadherence activity against E. coli and S. aureus of PLA, PLAgraft -QFPDMAEMA plate with -CH 3 as alkylating agent and PLAgraft -QFPDMAEMA plate with -C 7 H 15 as alkylating agent.

	CFU E. coli	CFU S. aureus

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Acknowledgements

The authors thank the French Ministry of Education and Research for Sarah El Habnouni's fellowship, and Sylvie Hunger for her help with the NMR spectroscopy.

Supporting Information

Supporting Information is available from the Wiley Online Library or from the author.

Received: February 22, 2011 Revised: April 22, 2011 Published online: July 6, 2011 δ ): 8.49 (s, 1H, H 10 anthracene), 8.29-8.26 (m, 2H, H 1 H 8 anthracene), 8.05-8.02 (m, 2H, H 4 H 5 anthracene), 7.60-7.24 (m, 4H, H 2 H 3 H 6 H 7 anthracene), 5.32 (s, 2H, C H 2 ); FTIR (ATR): ν = 2093 cm -1 (N 3 ).

Synthesis of PLA 94 Fluorescently Labelled Surface ( 2 ) : A clickable plate of PLA ( 1 ) was immersed in a solution of 9-azidomethylanthracene (100 mg, 0.4 mmol) in an ethanol (3 mL)/water (1 mL) mixture. The Cu I catalyst was then generated in situ by the addition of copper sulfate (300 μ L of a 1 M aqueous solution) and sodium ascorbate (600 μ L of a 1 M aqueous solution). The reaction was performed at 40 ° C for 2 days. The PLA plate was then extensively washed with water and ethanol, rinsed with methanol and fi nally dried under vacuum prior to analysis. The plate was immersed for 30 s in three successive THF baths and each solution (S1, S2, and S3) was analyzed by SEC. Afterwards, the plate was entirely dissolved in THF in order to analyse the bulk (B).

Synthesis of 3-Azidopropanol : 3-Chloropropanol (5.1 mL, 5.77 g, 61 mmol), sodium azide (7.94 g, 122 mmol), tetrabutylammonium hydrogen sulfate (TBAHS) (40 mg), and water (5 mL) were placed in a round-bottom fl ask. The reaction mixture was stirred fi rst at 80 ° C for 24 h, and then at room temperature for 12 h. The mixture was extracted with diethyl ether (3 × 100 mL). The organic phase was dried over MgSO 4 and the solvent was removed under vacuum. 4.8 g of 3-azidopropanol were obtained after distillation (78% yield). 1 H NMR (300 MHz, CDCl 3 ,

Synthesis of (3-Azidopropyl)bromoisobutyrate (APBIB) ( α -Functionalized ATRP Initiator) ( 3 ) : 3-Azidopropanol (2 g, 19.8 mmol), triethylamine (4.1 mL, 29.7 mmol) and dry CH 2 Cl 2 (50 mL) were placed in a roundbottom fl ask. Bromoisobutyryl bromide (3.67 mL, 29.7 mmol) was then added slowly at 0 ° C, and the mixture was stirred at room temperature for 17 h. The solution was fi ltered to remove the triethylammonium bromide and the solvent was removed under vacuum. The viscous residue was dissolved in DCM and washed with a saturated sodium hydrogenocarbonate (NaHCO 3 ) solution. The product was dried over MgSO 4 and the volatiles were removed under vacuum. The crude product was purifi ed by column chromatography on silica using a heptane/ethyl acetate (9:1) mixture as the eluent. (90% yield). 1 H NMR (300 MHz, CDCl 3 , δ ): 4.24 (t, 2H, C H 2 OC = O), 3.41 (t, 2H, C H 2 N 3 ), 1.92 (m, 2H, CH 2 C H 2 CH 2 ), 1.91 (s, 6H, Br(C H 3 ) 2 CC = O).

Synthesis of Anthracenylmethyl Methacrylate ( 4 ) (AMM) (Fluorescent Probe) : A solution of 9-(methanol)anthracene (3 g, 14.4 mmol, 1 eq.), triethylamine (3.04 mL, 21.6 mmol, 1.5 eq.) and hydroquinone (0.622 g, 5.6 mmol, 0.4 eq.) in dry DCM (75 mL) was cooled at 0 ° C. Then, methacryloylchloride (2.18 mL, 21.6 mmol, 1.5 eq.) was added dropwise under an inert atmosphere. After stirring at room temperature for 17 h the crude product was washed three times with 1 M hydrochloric acid (3 × 100 mL), 1 M sodium hydrogenocarbonate (3 × 100 mL) and deionized water (100 mL). The product was dried over MgSO 4 and fi ltered, and the solvent was removed under vacuum. The product was purifi ed by column chromatography on silica gel using chloroform as the eluent. ( m = 3.2 mg, 82% yield). 1 H NMR (300 MHz, CDCl 3 , δ ): 8.49 (s, 1H, H 10 anthracene), 8. 37-8.34

Synthesis of α -Azido FPDMAEMA : Polymerization was carried out using the standard Schlenk technique under an argon atmosphere. CuBr (118 mg, 0.82 mmol, 1 eq.), the initiator ( 3 ) (206 mg, 0.82 mmol, 1 eq.) and the fl uorescent probe ( 4 ) (115 mg, 0.41 mmol, 0.5 equiv) were placed in an oven-dried Schlenk tube. The tube was fi tted with a rubber septum. Degassed toluene (7 mL) and DMAEMA (7 mL, 41.5 mmol, 50 equiv) were transferred to the tube via a degassed syringe. The solution was further degassed by three freeze-thaw-pump cycles. The mixture was stirred rapidly under argon and PMDETA was added. The resulting mixture was placed in a thermostatically controlled oil bath at 60 ° C for 20 min. The reaction was stopped by immersion in liquid nitrogen, and the catalyst was removed by passing through a column of activated basic alumina prior to molecular-weight analysis. The conversion was measured using 1 H-NMR spectroscopy.