Convenient Access to Biocompatible Block Copolymers from SG1

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Biomacromolecules 2009, 10, 1436–1445

Convenient Access to Biocompatible Block Copolymers from SG1-Based Aliphatic Polyester Macro-Alkoxyamines Benoıˆt Cle´ment,† Thomas Trimaille,*,† Olivier Alluin,‡,§ Didier Gigmes,† Kamel Mabrouk,† Franc¸ois Fe´ron,‡ Patrick Decherchi,§ Tanguy Marqueste,§ and Denis Bertin† Laboratoire Chimie Provence (LCP), UMR 6264, Universite´s d’Aix-Marseille I, II et III-CNRS, Equipe Chimie Radicalaire Organique et Polyme`res de Spe´cialite´, Case 542, Av. Escadrille Normandie-Niemen, 13397 Marseille Cedex 20, France, Neurobiologie des Interactions Cellulaires et Neurophysiopathologie (NICN), UMR 6184, Faculte´ de Me´decine, Universite´s d’Aix-Marseille I, II, et III, Bd Pierre Dramard, 13916 Marseille Cedex 20, France, and Institut des Sciences du Mouvement, Etienne-Jules Marey, UMR CNRS 6233, Universite´ d’Aix-Marseille II, Equipe Plasticite´ des Syste`mes Nerveux et Musculaire, Parc Scientifique et Technologique de Luminy CC910, 163, Avenue de Luminy, 13288 Marseille Cedex 09, France Received January 12, 2009; Revised Manuscript Received March 20, 2009

SG1-based poly(D,L-lactide) (PLA) or poly(ε-caprolactone) (PCL) macro-alkoxyamines were synthesized and further used as macroinitiators for nitroxide-mediated polymerization (NMP) of 2-hydroxyethyl (meth)acrylate (HE(M)A) to obtain the corresponding PLA- or PCL-PHE(M)A block copolymers. First, a PLA-SG1 macroalkoxyamine was prepared by 1,2-intermolecular radical addition (IRA) of the MAMA-SG1 (BlocBuilder) alkoxyamine onto acrylate end-capped PLA previously prepared by ring-opening polymerization. The NMP of HEA monomer from the PLA-SG1 macro-alkoxyamine appeared to be well controlled in the presence of free SG1 nitroxide, contrary to that of HEMA. In the latter case, adjustable molecular weights could be obtained by varying the HEMA to macro-alkoxyamine ratio. The versatility of our approach was then further applied to the preparation of PHEMA-b-PCL-b-PHEMA copolymers from a R,ω-di-SG1 functionalized PCL macro-alkoxyamine previously obtained from a PCL diacrylate by IRA. Preliminary studies of neuroblast cultures on these PCLbased copolymer films showed acceptable cyto-compatibility, demonstrating their potential for nerve repair applications.

Introduction Biocompatible block copolymers based on (at least) one aliphatic polyester block, such as polylactide (PLA) or poly(εcaprolactone) (PCL) have received high attention over the past decade for biomedical applications, such as drug delivery and tissue engineering. The best example is probably the PLA-bPEG copolymers which have been extensively studied as micellar particles for drug encapsulation and release1 and more recently as scaffolds in tissue engineering.2 Other interesting block copolymer architectures for biorelated applications are those consisting in one aliphatic polyester block and one vinyl polymer block, as already reported for poly(methyl methacrylate),3 poly(N-isopropropyl acrylamide),4 poly(acrylic acid),5 or even poly(N-vinyl pyrrolidone).6 These block copolymers can be now easily obtained through the combination of ring-opening polymerization (ROP) and controlled radical polymerization (CRP), such as atom transfer radical polymerization (ATRP), reversible addition-fragmentation chain transfer (RAFT), and nitroxide mediated polymerization (NMP). Up to now, ROP/ ATRP3,5 and ROP/RAFT4 combined polymerizations were particularly used to achieve these copolymer materials, and the ROP/NMP approach has been poorly explored. However, the NMP technique, particularly based on the N-(2-methylpropyl)* To whom correspondence should be addressed. E-mail: [email protected]. † Laboratoire Chimie Provence (LCP). ‡ Neurobiologie des Interactions Cellulaires et Neurophysiopathologie (NICN). § Institut des Sciences du Mouvement.

N-(1-diethylphosphono-2,2-dimethylpropyl)-N-oxyl (so-called SG1) nitroxide, is considered as environmentally attractive compared to ATRP and RAFT7 and can be advantageous regarding applications in biomaterials.8 Additionally, the SG1 nitroxide is known to control a large range of vinyl monomers.9 In previous papers, we described the potential of the SG1-based alkoxyamine MAMA-SG1,10 commercially referred as BlocBuilder (from Arkema company) as a versatile precursor for complex macromolecular architectures. We particularly reported the synthesis of an OH-functionalized alkoxyamine from simple reaction of the N-hydrosuccinimide (NHS) ester activated MAMA-SG1 with ethanolamine, which was further used as a dual initiator to obtain PS-b-PLA block copolymers by combining the NMP and ROP techniques.11 This NMP/ROP dual initiator approach had been initiated by Hawker et al.12 using a TEMPO-based alkoxyamine obtained in a multiple step synthesis. In the present paper, we exploited the ability of the MAMA-SG1 to be involved in 1,2-intermolecular radical addition (IRA) reactions13 for combining ROP and NMP for block copolymer preparation. Our strategy relies on the IRA of the MAMA-SG1 on acrylate end-capped aliphatic polyesters (PLA, PCL) to obtain the corresponding SG1-based polyester macro-alkoxyamines as initiators for NMP technique. The advantage of this approach over the dual initiator strategy relies on the possibility to prepare relatively easily more complex architectures, such as ABA triblock copolymers (starting from a R,ω-diacrylated polyester), and to use commercially available aliphatic polyesters that just need to present hydroxyl end groups to generate the acrylate moiety (by simple reaction with acryloyl

10.1021/bm900003f CCC: $40.75  2009 American Chemical Society Published on Web 04/27/2009

Biocompatible Block Copolymers

chloride). The potential of our approach is illustrated by the synthesis of PLA- and PCL-PHE(M)A based block copolymers. Several works focused on the synthesis PHEMA-g-PLA or PHEMA-g-PCL grafted copolymers14 but, to the best of our knowledge, the block copolymer analogs were not reported up to now, except very recently for R,ω-brominated oligo-CL used as initiators for HEMA ATRP.15 PHE(M)A is widely investigated as scaffold in the field of tissue engineering, and particularly nerve repair,16 due to its hydrophilic and hydrogellike properties. Aliphatic polyesters are also choice candidates in this field due to their biodegradability.17 The strategy described here combines the properties of both polymers in a single homogeneous material for the latter application.

Experimental Section Materials. MAMA-SG1 (BlocBuilder) was kindly provided by Arkema (France). 2-Hydroxyethyl acrylate (HEA, 96%), 2-hydroxyethyl methacrylate (HEMA, 97%), R,ω-dihydroxyl terminated polycaprolactone (10000 g · mol-1, PDI ) 1.4), tin(II) 2-ethylhexanoate (Sn(Oct)2), acetic anhydride, and pyridine were purchased from Sigma-Aldrich and used as received. D,L-Lactide was purchased, packaged under vacuum, from Purac Biochem (The Netherlands), and then stored under argon. Toluene was distilled over sodium/benzophenone and other solvents were used as received. Synthesis of Acrylate End-Capped Aliphatic Polyesters. Acrylate End-Capped PLA. ROP of D,L-lactide was run in toluene at 100 °C with HEA as initiator and Sn(Oct)2 as catalyst. Typically, D,L-lactide (10 g, 69.4 mmol), HEA (125 mg, 1.07 mmol, targeted Mn∼10000 g · mol-1), and Sn(Oct)2 (250 mg, 0.55 mmol) were placed in a Schlenk, submitted to vacuum/argon cycles. Dry toluene (35 mL, [D,L-LA] ) 2 mol · L-1) was then added through the septum under an argon atmosphere in the Schlenk, which was placed in an oil bath preheated at 100 °C. The mixture was stirred for 2 h and then quenched with nondistilled THF. The polymer was precipitated in cold methanol and dried at 30 °C under vacuum. Diacrylate End-Capped PCL. R,ω-Dihydroxyl terminated polycaprolactone (5 g, 0.5 mmol) was allowed to react with acryloyl chloride (1 mL, 12.3 mmol) in dichloromethane (30 mL) at room temperature. After 16 h reaction, the polymer was precipitated in cold methanol and dried under vacuum. Incomplete functionalization was observed by 1H NMR. The product was then allowed to react again with acryloyl chloride (12 equiv/OH functions) at room temperature for 3 days to ensure complete functionalization before being precipitated in cold methanol and dried under vacuum. Synthesis of PLA and PCL-SG1 Macro-Alkoxyamines. A solution of PLA acrylate (4 g, 0.4 mmol) and MAMA-SG1 (1.52 g, 4 mmol) in THF (5 mL) was introduced in a Schlenk tube equipped with a rotaflo, deoxygenated by argon bubbling and heated at 100 °C for 1 h under stirring. The polymer was then precipitated twice in cold methanol. PCL-di-SG1 was synthesized following the same procedure as reported above for PLA-SG1. NMP from PLA and PCL-SG1 Macro-Alkoxyamines. NMP of Styrene. Styrene (8 g, 76.9 mmol) and PLA-SG1 alkoxyamine (2 g, 0.2 mmol) for a Mn (PS) ) 40000 g · mol-1 were introduced in a 50 mL two-neck round-bottom flask fitted with a septum and condenser and degassed for 20 min by argon bubbling. The mixture was then heated to 120 °C under argon with vigorous stirring. After polymerization, the mixture was cooled down and precipitated in cold methanol. NMP of HEA. HEA (8.9 g, 76.9 mmol), PLA-SG1 alkoxyamine (2 g, ∼0.2 mmol), and SG1 nitroxide (7.1 mg, 0.024 mmol) for a Mn (PHEA) ) 40000 g · mol-1 were introduced in a 50 mL two-neck round-bottom flask fitted with a septum and condenser and degassed for 20 min by argon

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bubbling. The mixture was then heated to 120 °C under argon with vigorous stirring. After polymerization, the mixture was cooled down, dissolved in minimum DMSO, and precipitated in diethyl ether. NMP of HEMA from PLA-SG1 (and PCL-SG1) Macro-Alkoxyamine. Typically, HEMA (0.65 g, 5 mmol), PLA-SG1 alkoxyamine (1 g, 0.1 mmol), and tert-butyl benzene (13.5 mL) were introduced in a 50 mL two-neck round-bottom flask fitted with a septum and condenser and degassed for 20 min by argon bubbling. The mixture was then heated to 100 °C under argon with vigorous stirring. After polymerization, the mixture was cooled down leading to the precipitation of the polymer, sticking to the walls of the flask. tert-Butyl benzene was removed from the flask and the polymer was washed with diethyl ether, then dissolved in minimum DMSO, and precipitated in diethyl ether. NMP of HEMA from the PCL dimacro-alkoxyamine was performed in the same manner. PHE(M)A Block Acetylation for GPC and NP-GPEC Analysis. A 0.3-0.4 mL PHEMA-PLA/PCL sample (or 0.1 mL for PHEA-PLA sample) was withdrawn from the flask at a given time of the polymerization, and 0.2 mL of pyridine and 0.2 mL of acetic anhydride were added. The mixture was stirred overnight for reaction, followed by precipitation in diethyl ether (except for PHEA-PLA) and drying under vacuum before GPC analysis. Acetylation was also performed on the purified copolymers for NP-GPEC and some GPC analysis, following the same procedure. Analytical Techniques. 13C, 31P, and 1H NMR analyses were performed on a Bruker Advance 300 spectrometer in CDCl3 or DMSOd6. Polymer molecular weights and polydispersities were determined by gel permeation chromatography (GPC) at 30 °C on a system comprising of a Waters 515 HPLC pump equipped with three “Styragel” columns used in series [HR 3 (4.6 × 300 mm, separation between 500 and 30000 g · mol-1), HR 4 (4.6 × 300 mm, separation between 5000 and 600000 g · mol-1), and HR 5 (4.6 × 300 mm, separation between 2000 and 4 × 106 g · mol-1)] and two detectors: UV/visible (Waters 2487) and RI (Waters 2414). THF was the mobile phase with a flow rate of 1 mL · min-1. Calibration was based on polystyrene standards. Normal phase gradient polymer elution chromatography (NP-GPEC) was carried out with a system consisting in an Waters Alliance 2695 module, a column oven, and an evaporative light scattering detector PL-ELS 2100 (Polymer Laboratories). A Nucleosil column, 100 Å pore size, 7 µm particle size (Macherey Nagel), was used at 30 °C. Dilute polymer solutions were made in THF (2.5 mg/mL). At a constant flow rate of 0.8 mL · min-1, a linear binary gradient in heptane/THF (nonsolvent/solvent for the polymers used in the analysis, respectively) starting from 100:0 at t ) 0 min to 0:100 at t ) 20 min was used, followed by a reverse gradient from 0:100 to 100:0 (heptane/THF) in 10 min for the subsequent analysis. Preparation of Copolymer Films. PCL homopolymer and the block copolymers of PCL and PHEMA were spin coated on glass coverslips (previously washed with 0.1 M HCl) from 2 wt % THF solutions at 1000 trs/min. The films were then dried at 40 °C under vacuum and were sterilized by autoclaving (120 °C, 20 min). Neuroblast Cultures. Dorsal root ganglion (DRG) cultures were prepared following the procedure described by Rougon and coworkers.18 In brief, spinal cords were removed from E18 C57BL/6 mice and the attached DRGs were removed and dissociated by trypsin treatment before plating on polymer-coated coverslips. Dissociated DRG were plated in DMEM culture medium supplemented with fetal bovine serum (Invitrogen). Five days after plating, neuroblasts were observed using an inverted phase light microscope (Zeiss).

Results and Discussion Synthesis of the PLA-SG1 Macro-Alkoxyamine. Our strategy to obtain aliphatic polyester-b-PHE(M)A block copolymers was based on the synthesis of SG1-based polyester macro-alkoxyamines as initiators for further NMP of HE(M)A (Scheme 1 for PLA). This methodology is based on the derivatization of a previously obtained acrylate end-capped PLA (1) by 1,2-intermolecular radical addition (IRA) of the MAMA-

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Additionally, the vinyl protons (5.5-6.5 ppm) totally disappeared. 31P NMR of the polymer further revealed one single peak (24.4 ppm) showing that the PLA-SG1 was quite pure and that MAMA-SG1 was efficiently removed during the purification steps (precipitation in methanol). 31P NMR analysis using diethylphosphite as internal standard (7.9 ppm) confirmed the high functionalization yield (85-90%). The molecular weight after IRA was checked by GPC and remained nearly unchanged (Mn PLA-SG1 ) 9500 g.mol-1, PDI ) 1.29). Preparation of the Block Copolymers from PLA-SG1 Macro-Alkoxyamine. In a preliminary experiment the ability of the PLA macro-alkoxyamine to initiate NMP (bulk, 120 °C) was checked using the styrene monomer as model, targeting a Mn of 40000 g · mol-1. Polymerization was controlled as shown by the linear evolution of the Mn versus conversion and the good agreement between experimental and theoretical Mn (Figure 3a). Polydispersity index values remained quite acceptable (1.3-1.6). At 48% conversion, the PLA-b-PS copolymer was precipitated in cold methanol. The GPC trace of the copolymer was clearly shifted to higher molecular weights, comparing to the PLASG1 precursor (Figure 3b), with a PS block of about 19000 g · mol-1, close to that expected (19200 g · mol-1).

Figure 1. (a) Ln([M]0/[M]) vs time and (b) Mn and PDI vs conversion curves for the ROP of D,L-lactide in toluene at 100 °C (dotted line is the theoretical curve). Molecular weights were obtained from universal calibration.

SG1 (BlocBuilder) to obtain the corresponding PLA-SG1 macroalkoxyamine (2). The latter was then further used to initiate the NMP of the monomer of interest (HEA in the scheme) to achieve the desired block copolymer (3). Acrylate end-capped PLA was first obtained by ROP of D,Llactide with 2-hydroxyethyl acrylate as initiator and Sn(Oct)2 as catalyst in toluene ([D,L-LA] ) 2 mol · L-1) at 100 °C with a catalyst to initiator molar ratio of 1:2. The kinetics was followed, revealing a linear evolution of the Ln[M]0/[M] versus time (Figure 1a). About 90% conversion was reached in 1.5 h. A linear evolution of the Mn (determined from GPC in THF by universal calibration using Mark-Houwink coefficients19) with conversion was also observed, in good agreement with those expected from the theory, and polydispersities remained quite narrow (PDI ∼ 1.3), showing the controlled character of the polymerization (Figure 1b). The typical 1H NMR spectrum of the PLA after precipitation in cold methanol is presented in Figure 2a. The molecular weights can be determined comparing the peak integral value of the vinyl protons (5.5-6.5 ppm) to that of the methine of the PLA backbone (5-5.3 ppm), and are in good agreement with that obtained from GPC. The molecular weight of further used PLA was typically close to 10000 g · mol-1 (PDI ) 1.3). In the next step, the IRA of the MAMA-SG1 was performed on the acrylate end-capped PLA to obtain the SG1 terminated PLA. The reaction was run in THF at 100 °C with an excess of MAMA-SG1 (10 equiv/double bond). The functionalization yield was high (∼90%), as shown by 1H NMR on the precipitated polymer in methanol (Figure 2b), comparing the peak integrals of the t-butyl protons of the SG1 (1-1.2 ppm) with the methine ones from the PLA backbone (5-5.3 ppm).

A slight shoulder toward low molecular weight was however observed on the GPC trace obtained from RI detection (explaining the polydispersity value ∼ 1.5). This can be attributed to residual PLA chains, since a quite symmetric GPC trace obtained by UV detection at 254 nm for the copolymer. Normal phase gradient polymer elution chromatography (NP-GPEC) analysis was further carried out, which consists in eluting the copolymer from an apolar solvent (heptane, 100% at t ) 0 min) to more polar one (THF, 100% at t ) 20 min), performing a linear binary solvent gradient (Figure 3c). The PLA-SG1 precursor and a PS homopolymer were injected as controls, and the PS homopolymer was eluted before the PLA since it is less polar (t ∼ 15 min for PS, t ∼ 17.8 min for PLA). As expected, the PLA-b-PS copolymer sample was eluted between the PS and PLA homopolymers (t ∼ 16 min). The presence of PLA precursor in the copolymer sample seems to be also detected (minor peak at ∼17.5 min, corresponding to the shoulder of the PLA precursor), corroborating the results from GPC. The residual PLA species could be of two types: (i) residual PLASG1 species due to an incomplete initiation of NMP of styrene or (ii) PLA chains, which would not have been initiated by the HEA during ROP (initiation by water, impurities, etc.) and consequently unable to be functionalized with the SG1 moiety through IRA. The first hypothesis of incomplete initiation of NMP from PLA-SG1 is, however, less probable than the second one. Indeed, the dissociation rate constant (kd) of PLA-SG1 was determined by electron paramagnetic resonance (EPR), following a previously described procedure,20 to be about 1.09 × 10-2 s-1 at 120 °C. This value is higher than that of the MONAMS alkoxyamine (CH3-O-C(dO)-CH(CH3)-SG1), which was proved in earlier studies21 to allow good initiation efficiency and control for the NMP of styrene at 120 °C. This strongly suggests that the kd value of PLA-SG1 is thus high enough to ensure an efficient initiation of the styrene NMP from this macroalkoxyamine at 120 °C. As a result, the residual PLA species are rather thought to be chains that are not functionalized with SG1 (i.e., presumably chains not initiated from HEA but from impurities during ROP). This is in good agreement with the previous 31P NMR assay on the PLA-SG1 which revealed ∼85-90% SG1-functionalization yield, as mentioned above. Finally, the determination of the PS block molecular weight



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Figure 2. 1H NMR spectrum of purified (a) acrylated end-capped PLA (1) and (b) PLA-SG1 (2) obtained after intermolecular radical addition of MAMA-SG1. Scheme 1. Synthesis Methodology for the Formation of the PLA-Based Block Copolymers (PLA-b-PHEA as an Example)

from 1H NMR peak integrations gave a value of 22000 g · mol-1, relatively close to that obtained by GPC. With a view to produce PLA based amphiphilic block copolymers, the NMP of the HEA from the PLA-SG1 was first investigated. SG1 nitroxide is known to control acrylate monomers,21 which exhibit relatively high propagation rate constant (kp) values, but when using a starting alkoxyamine presenting a relatively low dissociation rate constant, a certain amount of free SG1 has to be added in the polymerization medium to help the persistent radical effect to take place.21 It is typically the case for the PLA-SG1 alkoxyamine, with its kd value of about 1.09 × 10-2 s-1 at 120 °C. Moreover, the few data in the literature show an even higher kp value for HEA than for classical acrylates such as butyl,22 which is not in favor of a good polymerization control (enhanced propagation rate compared to initiation). The NMP of HEA from PLA-SG1 was then performed in bulk at 120 °C using 12 mol % free SG1 compared to the alkoxyamine targeting a block molecular weight of 40000 g · mol-1. Kinetics was of first order with respect to monomer as indicated by linear evolution of the ln[M]0/[M]

versus time (Figure 4a). In Figure 4b is plotted the evolution of the copolymer molecular weight and PDI determined by GPC (in THF) as a function of conversion for the polymerization. These molecular weights correspond to the acetylated form of the PHEA (PHEA(Ac)) of the block copolymer. Indeed, PHEA is insoluble in THF, but we aimed to perform GPC analysis of the block copolymer in this solvent for comparison with the PLA-SG1 precursor. That is why the alcohol functions of the HEA were acetylated through reaction with acetic anhydride, following a previously reported procedure.23 The quantitative acetylation yield was attested by 1H NMR, by checking the total disappearance of CH2 protons next to the alcohol (3.5 ppm) and CH2 protons next to the ester of the HEA (4.0 ppm), which were both shifted to a single peak at 4.2 ppm (-CO-O-CH2-CH2-O-CO-Me). Molecular weights increased linearly with conversion and were close to the expected ones, demonstrating a good control of the polymerization. Nevertheless, PDI values tend to increase with the conversion, as observed on the GPC traces by the broadening of the molecular weight distributions observed along the conversion (Figure 4c).

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Figure 3. Nitroxide-mediated bulk polymerization of styrene at 120 °C from PLA-SG1 alkoxyamine (targeted Mn ) 40000 g · mol-1); (a) Mn (b) and PDI (O) versus conversion (theoretical Mn in dotted line); (b) GPC traces of the purified PLA-b-PS by precipitation in methanol from RI detector (continuous left trace) and from UV detector at 254 nm (dotted left trace) compared to PLA-SG1 precursor (right trace, RI detector); (c) NP-GPEC analysis of the PLA-SG1 precursor (dotted line), PS homopolymer control of Mn ) 20000 g · mol-1 (gray line), and PLA-b-PS copolymer sample (black line).

As we used nonpurified HEA for polymerization, the diacrylate impurities, known to be present in HEA commercial monomer, might be partially responsible for this PDI increase. NP-GPEC analysis was further performed on the acetylated copolymer sample (PHEA block is not soluble in THF). The PLA-SG1 precursor and an acetylated PHEA of similar molecular weight to that of the PHEA(Ac) block (Mn ∼ 14000 g · mol-1) were used as controls. Being more polar, PHEA(Ac) was eluted at a higher elution time than PLA-SG1 (t ∼ 20.6 min for PHEA(Ac) and 17.8 min for PLA), as shown in Figure 5. The presence of

Cle´ment et al.

Figure 4. Nitroxide-mediated polymerization of 2-hydroxyethyl acrylate (HEA) at 120 °C from PLA-SG1 alkoxyamine (targeted Mn ) 40000 g · mol-1); (a) ln([M]0/[M]) vs time; (b) Mn and PDI vs conversion; (c) GPC traces (THF) along the conversion; from right to left: PLASG1 precursor and PLA-b-PHEA(Ac) at 60, 120, 180, and 200 min of polymerization.

the PLA-b-PHEA copolymer was attested because it was eluted at intermediate elution time between both homopolymers (∼20 min). This elution time was closer to that of PHEA(Ac) homopolymer control than to that of PLA precursor, corroborating the copolymer composition obtained from GPC (PLA-bPHEA(Ac) ∼ 9000-b-14000 g · mol-1). Interestingly, GPEC analysis shows that the copolymer sample seems to be devoid of residual PLA precursor. Finally, the minor side peak at 20.6 min of the copolymer sample might correspond to high molecular weight species arising from partial cross-linking of the block copolymer due to the diacrylate impurities rather than



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Table 1. Characteristics of the PLA and PCL Based Block Copolymers Obtained from the PLA-SG1 and PCL-di-SG1 Macro-Alkoxyamine Precursors copolymer

Mn (g · mol-1) macro-alkox.

HEMA equiv/ macro-alkox.

Mna (g · mol-1) GPC

Mn (g · mol-1) 1H NMR

PDI

PLA-b-PHEMA PLA-b-PHEMA PHEMA-b-PCL-b-PHEMA PHEMA-b-PCL-b-PHEMA

9500 9500 10000 10000

50 100 50 100

17600 34500 16300 33400

14600 25500 14400a 28200a

1.6 2.0 1.7 2.1

a

Values obtained from acetylated block copolymer and corrected by subtracting the acetyl moiety molecular weight (42) * DPPHEMA(Ac).

Figure 5. NP-GPEC analysis of the PLA-SG1 precursor (dotted line), PHEA(Ac) homopolymer control (gray line), and PLA-b-PHEA(Ac) copolymer sample (black line).

to PHEA homopolymer. Indeed, a reference polymerization in the absence of PLA-SG1 showed no autopolymerization of HEA. Finally,1H NMR spectrum (in DMSO-d6) of the block copolymer purified by precipitation in diethyl ether is presented in Figure 6. Peaks characteristics of both blocks are observed and the PHEA block molecular weight was determined from integration calculations to be 13500 g · mol-1, close to that obtained by GPC. NMP of HEMA was further performed from this macroalkoxyamine in tert-butyl benzene at 100 °C. The polymerization was not controlled, the Mn values remaining unchanged whatever the conversion (which reached typically 80% within 80 min.). This result was expected since the NMP of the pure methacrylates is known to be, up to now, not controlled in the presence of SG1 nitroxide.24 However, the molecular weight of the

PHEMA block could be adjusted by increasing the monomer to macro-alkoxyamine ratio (i.e., monomer concentration) as shown in Table 1 (entries 1 and 2). Polydispersity index then slightly increased but remained acceptable. As an example, the 1H NMR spectrum of the PLAb-PHEMA in DMSO-d6 after purification by precipitation in diethyl ether is presented in Figure 7a. The PHEMA molecular weight in the copolymer, calculated from peak integration, was about 5100 g · mol-1. The chromatogram of the acetylated form of this copolymer analyzed by GPC in THF is presented in Figure 7b. A shift toward higher molecular weights was observed comparing with the trace of the PLA-SG1 precursor, as a result of formation of the PLA-b-PHEMA copolymer. The slightly higher Mn values found by GPC compared to 1H NMR, whatever the HEMA/macro-alkoxyamine ratio used (Table 1), could be explained by the relative calibration used (PS standards). Synthesis of PHEMA-b-PCL-b-PHEMA Triblock Copolymer from PCL-di SG1 Macro-Alkoxyamine and Preliminary Cytocompatibility Studies. PCL-based materials have been more studied as guide scaffolds for nerve repair than the PLA derivatives and seem to be more promising in this field.25,26 Our strategy could be used to obtain PCL-b-PHEMA diblock copolymers in a straightforward manner, namely, performing ROP of the ε-caprolactone with HEA as initiator, followed by the IRA of the MAMA-SG1 for further initiation of NMP of HEMA. However, to further show the versatility of our approach, we focused on the synthesis of the PHEMA-b-PCLb-PHEMA triblock copolymer, which is expected to exhibit better mechanical properties than the diblock analog. For this, a R,ω-dihydroxyl PCL (Mn ) 10000 g · mol-1, PDI ) 1.4) was allowed to react with large excess of acryloyl chloride in

Figure 6. 1H NMR spectrum of the PLA-b-PHEA in DMSO-d6 after precipitation in diethyl ether.

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Figure 7. (a) 1H NMR (in DMSO-d6) and (b) GPC trace of the PLA-b-PHEMA block copolymer (9500-5100 g · mol-1 by 1H NMR, Table 1, entry 1); GPC was performed in THF on the acetylated PHEMA block (PLA-SG1 precursor in dotted lines).

dichloromethane, yielding a R,ω-diacrylated PCL (100% functionalization attested by 1H NMR integration, Figure 8). The IRA of the MAMA-SG1 on the obtained PCL was further

performed to obtain a PCL-di-SG1 in quantitative yields, as attested by 1H NMR (Figure 8) showing the total disappearance of the vinyl proton peaks (5.5-6.5 ppm) and appearance of the

Figure 8. 1H NMR spectrum (in CDCl3) of diacrylate end-capped PCL and PCL-di-SG1 obtained after intermolecular radical addition of MAMASG1.



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Figure 9. (a) 1H NMR (in DMSO-d6); (b) 1H NMR (in CDCl3, after acetylation); (c) GPC (THF) trace; and (d) NP-GPEC trace of the PHEMAb-PCL-b-PHEMA block copolymer (∼10000-10000-10000 g · mol-1, Table 1, entry 4); GPC and NP-GPEC were performed on the acetylated PHEMA block (PCL-di-SG1 precursor in dotted line and PHEMA(Ac) control for NP-GPEC in gray line).

tert-butyl proton peaks of the SG1 moiety (1-1.2 ppm) with the expected integration compared to those of CH2 of the central core of PCL. NMP of HEMA was then performed from this PCL dialkoxyamine in the same conditions as previously reported for the PLA precursor. The molecular weight of the PHEMA block could be here also adjusted by the HEMA to alkoxyamine molar ratio (Table 1, entries 3 and 4). As before, the copolymers purified by precipitation in diethyl ether were characterized by 1 H NMR and GPC. The presence of the targeted copolymer was evidenced by 1H NMR analysis in DMSO-d6, with a clear

assignment of the peaks (Figure 9a). However, molecular weights could not be determined by integration from this spectrum, due to peak overlapping and also due to the poor solubility of PCL block in DMSO, which can lead to overestimated PHEMA block molecular weight. To overcome this drawback, a common solvent for both blocks had to be chosen. CDCl3 was satisfactory, provided the PHEMA was transformed into the acetylated form (PHEMA is not soluble in CHCl3). The accurate determination was then performed by integration from 1 H NMR analysis in CDCl3 on the acetylated block copolymer (Figure 9b).

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Cle´ment et al.

Figure 10. Neuroblast culture on films spin coated with (a) PCL and (b) PHEMA-b-PCL-b-PHEMA. Most neurons are attached and extend neuritis. Some cells exhibit a round shape with a diameter of 10 µm ( 5.

By GPC, a clear shift was observed comparing the acetylated block copolymer with the PCL precursor (Figure 9c). The obtained values were in a good agreement with those obtained by 1H NMR (Table 1). Similarly, as observed for PLA-bPHEMA, the PDI values tended to increase when increasing the HEMA to macro-alkoxyamine ratio (Table 1). As shown in Figure 9d, the formation of the copolymer was finally checked by NP-GPEC, with an intermediate elution time (major peak at t ) 19.8 min) between the PCL precursor (t ) 16.4 min) and the PHEMA(Ac) as control (t ) 20.7 min). The presence of side diblock PCL-PHEMA with the triblock could not be totally excluded. Finally, non-negligible amounts of residual PCL precursor were detected in the copolymer sample (t ) 16.4 min) that could be attributed to noncomplete initiation of the NMP of HEMA. The copolymer (PHEMA-b-PCL-b-PHEMA with each block of about 10000 g · mol-1, entry 4) and PCL homopolymer, as a reference, were then used to prepare films by spin-coating as supports for preliminary tests of culture of neuroblasts from dorsal root ganglia. After a few hours, the neurones adhered quite well and homogeneously on the films and developed extensions (axons, dendrites), whatever the polymer used, showing that they were in a quite favorable environment to grow (Figure 10). When zones of naked glass surface were artificially created (with a scalpel) on some copolymer film samples (before cell culture), it appeared that the cells did not tend to migrate from the copolymer regions to the glass ones, further showing their good accommodation with the polymer surface. After seven days, no significant cellular death was observed, which will have to be further confirmed by MTT tests. These preliminary results suggest acceptable cyto-compatibilty and open the door to further use of these copolymer materials as nerve guides. This validates our synthesis methodology and the use of the SG1 nitroxide-mediated polymerization for preparation of copolymer architectures for biorelated applications. A new study based on an in vitro assessment of cell proliferation and cell death as well as an in vivo evaluation of polymer biocompatibility is currently in progress.

Conclusion In this work, SG1-based aliphatic polyester macroalkoxyamines were produced by the IRA of the MAMA-SG1 on the previously obtained acrylate end-capped polyesters and successfully used as macroinitiators for the NMP of HEA and HEMA to obtain the corresponding block copolymers. Contrary to the HEA case, the NMP of HEMA was not controlled. Nevertheless, adjustable PHEMA molecular weights could be obtained, tuning the HEMA to macro-alkoxyamine ratio, whatever its nature (PLA or PCL).

This work demonstrates the versatility of our approach based on the IRA of the MAMA-SG1 regarding possibilities of combining ROP and NMP to achieve valuable novel copolymers for biomaterials applications, as shown by preliminary cytocompatibility tests. Further studies are underway to synthesize PLA or PCL-b-PHE(M)A copolymers derived block copolymers of different molecular weights and compositions to find the best compromise in terms of degradation, mechanical properties and biocompatibility for further use particularly in nerve repair. Acknowledgment. The authors are grateful to Arkema, Universite´ de Provence, and CNRS for financial support and M. Rollet (UMR 6264) for NP-GPEC experiments.

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BM900003F

Convenient Access to Biocompatible Block Copolymers from SG1

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