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Because of its speed, sensitivity, and ability to scrutinize individual species, mass spectrometry (MS) has become an essential tool in analytical strategies aimed at studying the degradation behavior of polyesters. MS analyses can be performed prior to the degradation event for structural characterization of initial substrates or after it has occurred to measure the decreasing size of products as a function of time. Here, we show that MS can also be usefully employed during the degradation process by online monitoring the chain solvolysis induced by reactive desorption electrospray ionization (DESI). Cleavage of ester bonds in random copolymers of lactic acid (LA) and glycolic acid (GA) was achieved by electrospraying methanol-containing NaOH onto the substrates. Experimental conditions were optimized to generate methanolysis products of high abundance so that mass spectra can be conveniently processed using Kendrick-based approaches. The same reactive-DESI performance was demonstrated for two sample preparations, solvent casting for soluble samples or pressed pellets for highly crystalline substrates, permitting to compare polymers with LA/GA ratios ranging from 100/0 to 5/95. Analysis of sample fractions collected by size exclusion chromatography showed that methanolysis occurs independently of the original chain size, so data recorded for poly(LA-co-GA) (PLAGA) copolymers with the average molecular weight ranging from 10 to 180 kDa could be safely compared. The average mass of methanolysis products was observed to decrease linearly (R2 = 0.9900) as the GA content increases in PLAGA substrates, consistent with the susceptibility of ester bonds toward solvolysis being higher in GA than in LA. Because DESI only explores the surface of solids, these data do not reflect bulk degradability of the copolymers but, instead, their relative degradability at the molecular level. Based on a "reactive-DESI degradability scale" such as that established here for PLAGA, the proposed method offers interesting perspectives to qualify intrinsic degradability of different polyesters and evaluate their erosion susceptibility or to determine the degradability of those polymers known to degrade via erosion only.

Spinal cord injury is a main health issue, leading to multiple functional deficits with major consequences such as motor and sensitive impairment below the lesion. To date, all repair strategies remain ineffective. In line with the experiments showing that implanted hydrogels, immunologically inert biomaterials, from natural or synthetic origins, are promising tools and in order to reduce functional deficits, to increase locomotor recovery, and to reduce spasticity, we injected into the lesion area, 1 week after a severe T10 spinal cord contusion, a thermoresponsive physically cross-linked poly(N-isopropylacrylamide)-poly(ethylene glycol) copolymer hydrogel. The effect of postinjury intensive rehabilitation training was also studied. A group of male Sprague–Dawley rats receiving the hydrogel was enrolled in an 8 week program of physical activity (15 min/day, 5 days/week) in order to verify if the combination of a treadmill step-training and hydrogel could lead to better outcomes. The data obtained were compared to those obtained in animals with a spinal lesion alone receiving a saline injection with or without performing the same program of physical activity. Furthermore, in order to verify the biocompatibility of our designed biomaterial, an inflammatory reaction (interleukin-1β, interleukin-6, and tumor necrosis factor-α) was examined 15 days post-hydrogel injection. Functional recovery (postural and locomotor activities and sensorimotor coordination) was assessed from the day of injection, once a week, for 9 weeks. Finally, 9 weeks postinjection, the spinal reflexivity (rate-dependent depression of the H-reflex) was measured. The results indicate that the hydrogel did not induce an additional inflammation. Furthermore, we observed the same significant locomotor improvements in hydrogel-injected animals as in trained saline-injected animals. However, the combination of hydrogel with exercise did not show higher recovery compared to that evaluated by the two strategies independently. Finally, the H-reflex depression recovery was found to be induced by the hydrogel and, albeit to a lesser degree, exercise. However, no recovery was observed when the two strategies were combined. Our results highlight the effectiveness of our copolymer and its high therapeutic potential to preserve/repair the spinal cord after lesion.

Micelles from amphiphilic polylactide-block-poly(N-acryloxysuccinimide-co-N-vinylpyrrolidone) (PLA-b-P(NAS-co-NVP)) block copolymers of 105 nm in size were characterized and evaluated in a vaccine context. The micelles were non-toxic in vitro (both in dendritic cells and HeLa cells). In vitro fluorescence experiments combined with in vivo fluorescence tomography imaging, through micelle loading with the DiR near infrared probe, suggested an efficient uptake of the micelles by the immune cells. The antigenic protein p24 of the HIV-1 was successfully coupled on the micelles using the reactive N-succinimidyl ester groups on the micelle corona, as shown by SDS-PAGE analyses. The antigenicity of the coupled antigen was preserved and even improved, as assessed by the immuno-enzymatic (ELISA) test. Then, the performances of the micelles in immunization were investigated and compared to different p24-coated PLA nanoparticles, as well as Alum and MF59 gold standards, following a standardized HIV-1 immunization protocol in mice. The humoral response intensity (IgG titers) was substantially similar between the PLA micelles and all other adjuvants over an extended time range (one year). More interestingly, this immune response induced by PLA micelles was qualitatively higher than the gold standards and PLA nanoparticles analogs, expressed through an increasing avidity index over time (>60% at day 365). Taken together, these results demonstrate the potential of such small-sized micellar systems for vaccine delivery.

Injectable hydrogels have received high attention over the last decades in drug delivery and tissue engineering as they allow minimal invasive procedures. Among them, thermoresponsive-polymer-based hydrogels are considered of high interest owing to their simple, mild, and reproducible preparation conditions, relying only on self-reorganization upon heating. Recent advances in controlled/living polymerization techniques and chemical conjugation now offer tremendous opportunities for achieving precise engineering and control over such hydrogel properties. In this perspective, we aim at highlighting their recent inputs in the design of thermoresponsive architectures and how they can address key prerequisites of gelation behavior, degradation, biofunctionalization, and controlled drug delivery.

mRNA has recently emerged as a potent therapeutics and requires safe and effective delivery carriers, particularly prone to address its issues of poor stability and escape from endosomes. In this context, we designed poly(D,L-lactide) (PLA)-based micelles with N-succinimidyl (NS) ester decorated hydrophilic hairy corona to trap/couple a cationic fusogenic peptide and further complex mRNA. Two strategies were investigated, namely (i) sequential immobilization of peptide and mRNA onto the micelles (layer-by-layer, LbL) or (ii) direct immobilization of peptide-mRNA pre-complex (PC) on the micelles. After characterization by means of size, surface charge, peptide/mRNA coupling/complexation and mRNA serum stability, carrier cytotoxicity and transfection capacity were evaluated with dendritic cells (DCs) using both GFP and luciferase mRNAs. Whatever the approach used, the micellar assemblies afforded full protection of mRNA in serum while the peptide-mRNA complex yielded complete mRNA degradation. In addition, the micellar assemblies allowed to significantly reduce the toxicity observed with the peptide-mRNA complex. They successfully transfected hard-to transfect DCs, with a superior efficiency for the LbL made ones (whatever mRNAs studied) showing the impact of the elaboration process on the carrier properties. These results show the relevance and potential of this new PLA/peptide based micelle platform to improve mRNA stability and delivery, while offering the possibility of further multifunctionality through PLA core encapsulation.

Subunit vaccines using recombinant antigens appear as the privileged vaccination technology for safety reasons but still require the development of carriers/adjuvants ensuring optimal immunogenicity and efficacy. Micelles from self-assembled amphiphilic copolymers have recently emerged as highly relevant and promising candidates owing to their ease of preparation, low size (entering in lymphatic capillaries for reaching lymph nodes), size/surface tunability and chemical versatility enabling introduction of stimuli (e.g. pH) responsive features and biofunctionalization with dedicated molecules. In particular, research efforts have increasingly focused on dendritic cells (DCs) targeting and activation by co-delivering (with antigen) ligands of pattern recognition receptors (PRRs, e.g. toll-like receptors). Such strategy has appeared as one of the most effective for eliciting CD 8+ T-cell response, which is crucial in the eradication of tumors and numerous infectious diseases. In this short review, we highlight the recent advances in such micelle-based carriers in subunit vaccination and how their precise engineering can be a strong asset for guiding and controlling immune responses.

International audience; Injectable hydrogels are promising platforms for tissue engineering and local drug delivery as they allow minimal invasiveness. We have here developed an injectable and biodegradable hydrogel based on an amphiphilic PNIPAAm-b-PLA-b-PEG-b-PLA-b-PNIPAAm pentablock copolymer synthesized by ring-opening polymerization/nitroxide-mediated polymerization (ROP/NMP) combination. The hydrogel formation at around 30 °C was demonstrated to be mediated by intermicellar bridging through the PEG central block. Such a result was particularly highlighted by the inability of a PEG-b-PLA-b-PNIPAAm triblock analog of the same composition to gelify. The hydrogels degraded through hydrolysis of the PLA esters until complete mass loss due to the diffusion of the recovered PEG and PNIPAAm/micelle based residues in the solution. Interestingly, hydrophobic molecules such as riluzole (neuroprotective drug) or cyanine 5.5 (imaging probe) could be easily loaded in the hydrogels’ micelle cores by mixing them with the copolymer solution at room temperature. Drug release was correlated to polymer mass loss. The hydrogel was shown to be cytocompatible (neuronal cells, in vitro) and injectable through a small-gauge needle (in vivo in rats). Thus, this hydrogel platform displays highly attractive features for use in brain/soft tissue engineering as well as in drug delivery.

International audience; In line with experiments showing that implanted hydrogels are promising tools, we designed and injected, after a C2 spinal cord hemisection, a thermoresponsive and thermoreversible physically cross-linked poly(N-iso-propylacrylamide)-poly(ethylene glycol) copolymer in order to reduce functional deficits and provide a favorable environment to axotomized axons. Nasal olfactory ecto-mesenchymal stem cells were cultured on the hydrogel in order to verify its bio-compatibility. Then, inflammatory reaction (Interleukin-1β and 6, Tumor Necrosis Factor-α) was examined 15 days post-hydrogel injection. Functional recovery (postural and locomotor activities, muscle strength and tactile sensitivity) was assessed once a week, during 12 weeks. Finally, at 12 weeks post-injection, spinal reflexivity and ventilatory adjustments were measured, and the presence of glial cells and regenerated axons were determined in the injured area. Our results indicate that cells survived and proliferated on the hydrogel which, itself, did not induce an enhanced inflammation. Furthermore, we observed significant motor and sensitive improvements in hydrogel-injected animals. Hydrogel also induced H-reflex recovery close to control animals but no improved ventilatory adjustment to electrically-evoked isometric contractions. Finally, regrowing axons were visualized within the hydrogel with no glial cells colonization. Our results emphasize the effectiveness of our copolymer and its high therapeutic potential to repair the spinal cord after injury.

International audience; In spinal cord repair, the challenge consists in combining various therapies that account for multiple deleterious effects in order to induce an efficient recovery. In that context, biomaterial implantation seems to be highly relevant. Indeed, biomaterials not only serve as a growth support to promote sectioned axonal fibers, but are also used for cell transplantation and drug delivery. In this review, we discuss and put into perspective the recent results obtained in the field of spinal cord repair by synthetic hydrogel implantation. The versatility of those biomaterials is presented through the latest chemical strategies developed to enhance their therapeutic effects. (C) 2015 Academie des sciences. Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license.

International audience; In the development of subunit vaccines with purified or recombinant antigens for cancer and infectious diseases, the design of improved and safe adjuvants able to efficiently target the antigen presenting cells, such as dendritic cells, represents a crucial challenge. Nanoparticle-based antigen delivery systems have been identified as an innovative strategy to improve the efficacy of subunit vaccines. Among them, self-assembled micellar nanoparticles from amphiphilic (macro)molecules have recently emerged as promising candidates. In this short review, we report on the recent research findings highlighting the versatility and potential of such systems in vaccine delivery.

Background: Over the last decades, synthetic polymer-based electrospun nano/microfibers have emerged as potent materials in crucial biomedical applications such as tissue engineering, drug delivery and diagnostics. This is mainly attributed to versatility and reproducibility of the electrospinning (ES) process, as well as the high surface- to-volume ratio of the generated nanostructures. Appropriate functionalization with dedicated biomolecules (i.e. cell adhesive peptides, therapeutic molecules, bio-probes) is a critical requirement for the performances of such materials in their related application. Methods: We report on the different chemical methodologies for preparing biofunctionalized synthetic polymer fibers, on the basis of two main approaches: biomolecule introduction after ES process (post-ES) and before ES (pre-ES). We then focused on the latest implications of such materials in areas of tissue engineering, drug delivery and diagnostics. Results: This review describes the numerous immobilization strategies (either covalent or non-covalent) developed for designing biofunctionalized fibers, as well as their impact on their properties in dedicated application. The inputs of advanced conjugation tools (“clickable” chemistries, PEG linkers) for biofunctionalization are also highlighted. In the light of the literature, it appears that increasing research efforts are now devoted to multifunctional character and fiber combination with other materials (hydrogels, inorganic particles, microfluidic devices) for improved and tunable performances. Conclusion: Owing to flexibility and robustness of ES process as well as advances in conjugation and polymer/material engineering, high degree of control over biofunctionalization can now be achieved, to fit as best as possible the requirements of the targeted application. The performances reached up to now augur well for the future of such class of materials.

WOS:000427243200010; International audience; A catalyst/initiator-free radical addition reaction performed under mild conditions (water, 30 degrees C) with high yields is reported for the first time. This reaction implies simple pH-mediated alkoxyamine dissociation followed by addition onto olefinic substrates. The versatility and relevance of this selective reaction for macromolecular conjugation and engineering are shown through the syntheses of block copolymers, as well as hydrogels containing insitu-loaded proteins, which could retain biological activity. This contrasts with standard thermal radical conditions that lead to complete protein inactivation.

We report here a straightforward nanoprecipitation-based process to prepare functionalized polylactide (PLA) nanoparticles (NPs). It relies on an organic phase containing a PLA-based amphiphilic copolymer bearing N-succinimidyl esters that can spontaneously react with a peptide/protein located in the aqueous phase. The relevance of this strategy is supported by an improved antigenicity of immobilized HIV-1 Gag p24 protein.

International audience; We have here developed a straightforward one-step route to surface modified polystyrene (PS) based microfibers for protein/enzyme immobilization. Our approach consists of wet electrospinning of a poly(styrene-alt-maleic anhydride) (PSMA) polymer in an aqueous solution collector which contains the (macro) molecules to be coupled, here PEG diamine (PEGDA) or hexamethylene diamine (hexDA). The amino groups on the fiber surface were then exploited for immobilization of the horseradish peroxidase (HRP) enzyme. The immobilized HRP amounts were higher on the PEG-and hexyl-modified fibers than on the non-modified PSMA ones. The HRP catalytic activity was evaluated with 2,2'-azino-bis(3-ethyl-benzothiazoline- 6-sulfonic acid) (ABTS) as a substrate. While the retained activity of the enzyme immobilized on unmodified PSMA microfibers was only 2.4% of free enzyme, that of the enzyme immobilized on the PEGylated fibers increased to 34%. As a comparison, HRP fixed on hexyl-functionalized fibers exhibited a retention activity of 19.7%, showing the impact of a PEG spacer on HRP activity. HRP immobilization on PEG and hexyl-coated fibers had also a beneficial impact on enzyme storage stability. This study highlights the impact of surface properties on the activity of the immobilized enzyme and provides a convenient route to simultaneous elaboration/modification of fibers, for suitable protein fixation.

Glycopolymer-corona-based micelles are obtained in a one-pot procedure, through reaction of D-mannosamine or D-glucosamine with the N-succinimidyl (NS) esters of a poly(D,L-lactide)-block-poly(N-acryloxysuccinimide-co-N-vinylpyrrolidone) (PLA-b-P(NAS-co-NVP)) amphiphilic copolymer (presenting quasi-alternating NAS/NVP units) in dimethyl sulfoxide (DMSO), followed by nanoprecipitation and dialysis against water. The glycopolymer micelles exhibit a higher CMC and size than those obtained from unmodified copolymer, due to increased hydrophilicity of the external block as a result of sugar derivatization, and the availability of the sugars at the micelle surface is evidenced through interactions with Concanavalin A (Con A) lectin, as attested by turbidimetric measurements and enzyme-linked lectin assay (ELLA). Interestingly, the glycopolymer micelles can be further used for hydrophobic molecule encapsulation and release, as shown with imiquimod, while keeping their interactions with con A intact. It is concluded that the PLA-based amphiphilic/reactive copolymer represents a versatile platform for glycopolymer-based micelle constructs for drug/vaccine delivery.

International audience; An antigen probe (HIV-1 p24) immobilized onto N-succinimidyl ester based micelles was used as a solid phase coating in ELISA test, and induced a significant improvement in antibody detection sensitivity as compared to the standard free antigen coating. The relevance of this straightforward approach to improve the bioassay sensitivity was confirmed by using biotin as a generic probe.

International audience; Scorpion envenoming represents a public health issue in subtropical regions of the world. Treatment and prevention need to promote antitoxin immunity. Preserving antigenic presentation while removing toxin effect remains a major challenge in toxin vaccine development. Among particulate adjuvant, particles prepared with poly (D,L-lactide) polymer are the most extensively investigated due to their excellent biocompatibility and biodegradability. The aim of this study is to develop surfactant-free PLA nanoparticles that safely deliver venom toxic fraction to enhance specific immune response. PLA nanoparticles are coated with AahG50 (AahG50/PLA) and BotG50 (BotG50/PLA): a toxic fraction purified from Androctonus australis hector and Buthus occitanus tunetanus venoms, respectively. Residual toxicities are evaluated following injections of PLA-containing high doses of AahG50 (or BotG50). Immunization trials are performed with the detoxified fraction administered alone without adjuvant. A comparative study of the effect of Freund is also included. The neutralizing capacity of sera is determined in naive mice. Six months later, immunized mice are challenged subcutaneously with increased doses of AahG50. Subcutaneous lethal dose 50 (LD50) of AahG50 and BotG50 is of 575 μg/kg and 1300 μg/kg respectively. By comparison, BotG50/PLA is totally innocuous while 50% of tested mice survive 2875 μg AahG50/kg. Alhydrogel and Freund are not able to detoxify such a high dose. Cross-antigenicity between particulate and soluble fraction is also, ensured. AahG50/PLA and BotG50/PLA induce high antibody levels in mice serum. The neutralizing capacity per mL of anti-venom was 258 μg/mL and 186 μg/mL calculated for anti-AahG50/PLA and anti-BotG50/PLA sera, respectively. Animals immunized with AahG50/PLA are protected against AahG50 injected dose of 3162 μg/kg as opposed all non-immunized mice died at this dose. We find that the detoxification approach based PLA nanoparticles, benefit the immunogenicity and protective efficacy of venom immunogen.

International audience; To develop bioactive scaffolds of targeted properties for tissue repair or biomedical applications, hybrid microfiber-nanoparticle (MF-NP) matrices capable of controlled nanoparticle (NP) delivery were prepared through two novel approaches. In a first strategy, the suppleness of the jet-spraying method to produce polymer microfibers (MF) was used to deposit poly(D,L-lactide) (PLA) NP on poly(lactic-co-glycolic acid) (PLGA) MF by direct co-projection. The second approach relied on the post-incubation of PLA NP aqueous dispersion with MF preliminarily prepared by jet-spraying. NP coverage density onto MF and NP release was assessed by scanning electron microscopy and fluorescence measurements using coumarin-6 loaded NP. The first process was shown to allow high coverage density of NP onto MF (300 mu g/mg MF) and strong association, with no NP release observed over time. In the second approach, direct incubation of PLA NP with PLA MF led to lower NP coverage density (40 mu g/mg MF) with very fast release of NP from MF. The pre-coating of MF with poly-L-lysine (PLL) or the one of NP with lysozyme as a model protein drug afforded a higher coverage density and stronger association, coupled with a more sustained release of NP from MF over time. These results show the possibility to control the immobilization density and release of NP through appropriate preparation process and surface modification, and are of prime interest for the development of complex scaffolds with orchestrated bioactivity. (C) 2015. Elsevier B.V. All rights reserved.

International audience; The properties of poly(D,L-lactide)-block-poly(2-hydroxyethyl acrylate) (PLA-b-PHEA) block copolymers by means of in vitro / in vivo (rat) degradation are investigated and compared to those of PLA homopolymer. Over 12 weeks, we observe mass loss and molecular weight decrease. In vitro and in vivo findings are very similar for each polymer tested. When a short PHEA block is used (PLA-b-PHEA 15 000-3 000 g.mol(-1), 85/15 wt%), the degradation process is found to be very similar to that of homo-PLA, and to be typical of a bulk erosion mechanism, with no mass loss observed until week 7 and continuous decrease of molar mass within this timeframe. For a longer PHEA block length within the block copolymer (PLA-b-PHEA 15 000-7 500 g.mol(-1), 65/35 wt%), the degradation mechanism is modified, with a significant mass loss observed at early times and only a slight decrease in molar mass. The latter finding is related to the pronounced hydrophilicity and softness of the material induced by the PHEA block, which allow easy diffusion and rapid leakage of the degradation residues from the material towards the aqueous medium. Schwann cells are found to better adhere on spin-coated films of PLA-b-PHEA (85/15 wt%) than on PLA ones. These results show the potential of such hydrophilized PLA-based copolymers for use in peripheral nerve repair.

Poly(N-acryloxysuccinimide) (PNAS) and poly(N-acryloxysuccinimide-co-N-vinylpyrrolidone) (P(NAS-co-NVP)) of adjustable molecular weights and narrow polydispersities were prepared by nitroxide-mediated polymerization (NMP) in N,N-dimethylformamide in the presence of free SG1 (N-tert-butyl-N-1-diethylphosphono-)2,2-dimethylpropyl) nitroxide), with MAMA-SG 1 (N-(2-methylpropyl)-N-(1-diethylphosphono-2,2-dimethylpropyl)-O-(2-carboxylprop-2-yl)hydroxylamine) alkoxyamine as initiator. The reactivity ratios of NAS and NVP were determined to be r(NAS) = 0.12 and r(NVP) = 0, indicating a strong alternating tendency for the P(NAS-co-NVP) copolymer. NAS/NVP copolymerization was then performed from a SG1-functionalized poly(D,L-lactice) (PLA-SG1) macro-alkoxyamine as initiator, leading to the corresponding PLA-b-P(NAS-co-NVP) block copolymer, with similar NAS and NVP reactivity ratios as mentioned above. The copolymer was used as a surface modifier for the PLA diafiltration and nanoprecipitation processes to achieve nanoparticles in the range of 450 and 150 nm, respectively. The presence of the functional/hydrophilic P(NAS-co-NVP) block, and particularly the N-succinimidyl (NS) ester moieties at the particle surface, was evidenced by ethanolamine derivatization and zeta potential measurements.

A hydroxyl-functionalized alkoxyamine derived from the SG1 nitroxide was used as a dual initiator for ring-opening polymerization (ROP) of e-caprolactone (CL) and nitroxide-mediated polymerization ...

International audience; SG1-based poly(d,l-lactide) (PLA) or poly(epsilon-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 macro-alkoxyamine 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 alpha,omega-di-SG1 functionalized PCL macro-alkoxyamine previously obtained from a PCL diacrylate by IRA. Preliminary studies of neuroblast cultures on these PCL-based copolymer films showed acceptable cyto-compatibility, demonstrating their potential for nerve repair applications.

Novel “second generation” alkoxyamines, derived from N -(2-methylpropyl)- N -(1-diethylphosphono-2,2-dimethylpropyl)- N -oxyl (so-called SG1) as initiators for nitroxide-mediated polymerization (NMP) were synthesized by intermolecular radical 1,2-addition (IRA) of a high dissociation rate constant alkoxyamine (BlocBuilder ® , also called MAMA-SG1) onto various activated olefins, such as n -butyl acrylate, acrylic acid, dimethylacrylamide, 2-hydroxyethylacrylate and styrene. The potential of this radical addition was further applied to the synthesis of multifunctional alkoxyamines as precursors for complex macromolecular architectures, namely 3- and 4-arm star polymers. For this, tri- and tetra-acrylates were synthesized by reaction of acryloyl chloride with the 1,1,1-tris(hydroxymethyl)ethane and pentaerythritol, respectively, in the presence of triethylamine. The addition of MAMA-SG1 onto these olefins led to the tri- and tetra-functional SG1-based alkoxyamines which were further used to prepare polystyrene stars of controlled molecular weights and polydispersity values not exceeding 2. The individual arms were recovered by hydrolysis of the ester groups of the star core originating from the alkoxyamine initiator under basic conditions. The decreasing molecular weight determined by GPC during hydrolysis demonstrated the star architecture of the polymers.

New monoalkyl-substituted lactides were synthesized by reaction of α-hydroxy acids with 2-bromopropionyl bromide, and polymerized with various catalysts in the presence of benzyl alcohol by ring-opening polymerization (ROP). The classic tin(II) 2-ethylhexanoate (Sn(Oct)2) catalyst was leading to polymers with narrow distribution and predictable molecular weights, in polymerizations in bulk or toluene at 100 °C. The polymerization rate was corresponding to the steric hindrance of the alkyl substituents, such as butyl, hexyl, benzyl, isopropyl, and dimethyl groups. A yield of 83% was obtained with the hexyl-substituted lactide after 1 h of polymerization. Excellent conversions (97%) could be achieved by using the alternative catalyst 4-(dimethylamino)pyridine (DMAP). This latter organic catalyst was most efficient in polymerizing the more steric-hindered lactides with good molecular weight and polydispersity control, in comparison to the tin(II) 2-ethylhexanoate and tin(II) trifluoromethane sulfonate [Sn(OTf)2] catalysts. The efficiency of the DMAP catalyst and the variability of the monomer synthesis route for new alkyl-substituted lactides allow to prepare and to envision a wide range of new functionalized polylactides for the elaboration of tailored materials. © 2004 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 42: 4379–4391, 2004

Triglyceride-based emulsions were first prepared by a solvent displacement procedure which was modified to achieve their functionalization by surface deposition of various amphiphilic comb-like copolymers. These emulsions have been characterized as regards to hydrodynamic particle size and surface charges using dynamic light scattering and electrophoretic mobility measurements. The adsorption isotherms of a polydT15 oligonucleotide and a model plasmid showed that the process was dependent on the nature of the interfaces, the affinity for the nucleic acid increasing with more cationic charges, together with improved accessibility. The binding process was found to proceed according to two regimes: one at low nucleic acid coverage, independent of the initial plasmid concentration, and the second one at high coverage, which was nucleic-acid-concentration dependent. This behavior was considered to occur because of the development of repulsive interactions upon increasing the amount of immobilized nucleic acid. The complexation of plasmid complexed at the interface was finally investigated using the ethidium bromide displacement technique. The level of compaction of plasmid complexed onto the functionalized emulsions was lower than that obtained with the parent free polymer.

International audience; Activation of immune cells through pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs) or NOD-like receptors (NLRs), has been identified as a key issue in the development of new efficient vaccine adjuvants. We report here on the elaboration and immunostimulatory potential of polylactide (PLA)-based micelles core-loaded with imiquimod TLR7 ligand and able to be further surface-functionalized with antigenic protein (HIV-1 Gag p24) for antigen delivery purpose. Micelles prepared from poly(D,L-lactide)-b-poly(N-acryloxysuccinimide-co-N-vinylpyrrolidone) amphiphilic copolymer were incubated in the presence of imiquimod, leading to 1.2 wt% loading, and further conjugated to p24 antigen through reaction of p24 lysines and N-terminal amine with the N-succinimidyl pendant groups of the micelle corona. The impact of imiquimod encapsulation in the micelles on its immunostimulatory properties was investigated in vitro, by monitoring: (i) the NF-kappa B and mitogen-activated protein kinases (MAPK) pathways through experiments with RAW-Blue (TM) cells, a mouse macrophage cell line encoding an NF-kappa B/AP-1-inducible reporter construct; (ii) human dendritic cells (DCs) maturation markers by flow cytometry. RAW-Blue (TM) cells based experiments showed that imiquimod encapsulated in the micelles was much more efficient to activate the NF-kappa B and MAPK pathways than free imiquimod. Furthermore, encapsulated imiquimod was found to induce much higher maturation of DCs than the free analog. Finally, these immunostimulatory properties of the loaded imiquimod were shown to be conserved when the p24 antigen was coupled at the micelle surface. Taken together, these data regarding improved immunostimulatory efficiency suggest the strong potential of our micelle-based nano-system for vaccine delivery.

WOS:000358766800025; International audience; The functionalization of alkoxyamines prior to nitroxide-mediated polymerization (NMP) induces important structural variations when compared to the parent molecules. This may have important consequences on the design of functionalized materials by pre-functionalization. In this context, a wide range of amide-functionalized alkoxyamines (a functionality often obtained after conjugation from COOH- and N-succinimidyl-containing alkoxyamines) based on the nitroxide SG1 (N-tert-butyl-N-(1-diethyl phosphono-2,2-dimethylpropyl) nitroxide) have been synthesized and their dissociation rate constants (k(d)) have been determined. To rationalize their reactivity, a multi-parameter procedure was applied and enabled to discriminate disubstituted amide-functionalized alkoxyamines from monosubstituted ones. Monosubstituted alkoxyamines exhibited lower k(d) than their disubstituted counterparts (E-a increase of similar to 7-10 kJ mol(-1)) because of the occurrence of intramolecular hydrogen bonding (IHB) between the alkyl and the nitroxide fragments. NMP of styrene, n-butyl acrylate and methyl methacrylate with a small amount of acrylonitrile was then successfully performed from two representative secondary SG1-based alkoxyamines employed for conjugation: namely AMA (COOH-containing) and AMA-NHS (N-succinimidyl derivative), and compared to polymerizations initiated with AMA-Gem, an AMA-based alkoxyamine pre-functionalized with the anticancer drug Gemcitabine (Gem) and subjected to IHB. Although AMA-NHS showed the best results due to its lower Ea, the strong polarity of the Gem moiety that counter-balanced the detrimental effect of IHB over its kd still allowed for a reasonable control.

International audience; Intermolecular radical 1,2-addition (IRA) of N-tert-butyl-N-(1-diethylphosphono-2,2-dimethylpropyl)aminoxyl (SG1) based alkoxyamines onto activated olefins is used as a tool for peptide ligation. This strategy relies on simple peptide pre-derivatization to obtain (i) a SG1 nitroxide functionalized resin peptide at its N-terminus (SG1-peptide alkoxyamine), (ii) a vinyl functionalized peptide (either at its C-terminus or N-terminus), and does not require any coupling agents.

International audience; The present study is designed to assess the properties of a new degradable PLA-b-PHEMA block copolymer hydrogel and its therapeutic effectiveness after implantation following a thoracic spinal cord hemisection on rats. Degradable characteristics and porous aspect of the scaffold are respectively analyzed by the evaluation of its mass loss and by electron microscopy. The biomaterial toxicity is measured through in vitro tests based on motoneuron survival and neurite growth on copolymer sub-strate. Functional measurements are assessed by the Basso, Beattie and Bresnahan (BBB) and the Dynamic Weight Bearing (DWB) tests during 8 weeks post-surgery. Histological analyses are achieved to evaluate the presence of blood vessels and axons, the density of the glial scar, the inflammatory reaction and the myelination at the lesion site and around it. The results indicate that the synthetic PLA-b-PHEMA block copolymer is a non-toxic and degradable biomaterial that provides support for regenerating axons and seems to limit scar tissue formation. Additionally, the implantation of the porous PLA-b-PHEMA scaffold enhances locomotor improvement. The observed functional recovery highlights the potential benefits of plain tissue engineering material, which can further be optimized by bioactive molecule functionalization or transplanted cell encapsulation.

Poly(methyl methacrylate)-b-poly(butyl acrylate)-b-poly(ε-capro-lactone) (PMMA-PBA-PCL) triblock copolymers of typical number-averaged block molecular weights of 45 to 100 kg/mol for PMMA, 25 kg/mol for PBA and 10 kg/mol for PCL were synthesized by sequential nitroxide-mediated polymerization (NMP) of BA and MMA from a SG1 (N-(tert-butyl)-N-(1-diethylphosphono-2,2-dimethylpropyl)) nitroxide-functionalized PCL macroalkoxyamine initiator. This alkoxy-amine was obtained by transformation of an OH-terminated PCL into acrylate-end capped PCL, followed by 1,2-intermolecular radical addition (IRA) of MAMA-SG1 (so-called BlocBuilder® MA). NMP of BA from the macroalkoxyamine proceeded in a controlled manner. The high living chain ratio of PBA-PCL allowed further initiation of NMP of MMA, leading to triblock copolymers with adjustable PMMA block molecular weights. The samples prepared by solvent casting were found to organize in a lamellar morphology for the pure triblock copolymers, and in a spherical morphology when blended with a PMMA homo-polymer of Mn = 40 kg/mol and polydispersity index of 2.1. Finally, the mechanical properties and the local deformation mechanisms of the pure triblock copolymers and their blends with PMMA were investigated by time-resolved SAXS measurements during tensile testing. The dominant microscopic deformation mode of the triblock copolymer was found to change from crazing to cavitation-induced shear yielding, due to the easy-cavitating PCL block of the copolymer.

International audience; Poly(L-lysine) (PLL) dendrigrafts (DGLs) are arborescent biosynthetic polymers of regular and controlled structures. They have specific properties such as biocompatibility and non-immunogenicity, and their surface density of NH2 functions can be easily modified and therefore appears as a powerful tool for the functionalization of hydrophobic polymers used in the context of tissue engineering. In this study, we evaluated several criteria of human skin fibroblasts when cultured with DGL of generations 2, 3 and 4, with linear PLL polymer as reference. In aqueous phase, DGLs and PLL displayed a similar cytotoxicity towards fibroblasts. Plastic culture plates grafted with DGLs were further characterized as homogeneous surfaces by atomic force microscopy and surface characterization by amino density estimation by colorimetric assay. Proliferation of fibroblasts was increased when cultured onto PLL and DGLs monolayers when compared with crude plates. Cellular adhesion was increased by 20% on DGLs in comparison to PLL. Integrin α5 subunit protein expression level was increased after 48 h of culture on DGLs, in comparison to control or PLL-coated surfaces. The presence of DGLs did not lead to overexpression or activation of matrix metalloproteinases 2 and 9. Finally, fibroblasts adhesion was increased by 40% on poly-(lactic-co-glycolic acid) matrices functionalized with DGLs when compared to PLL. Overall, these features make DGL promising candidates for the surface engineering of biomaterials in tissue engineering.

Glycopolymer-corona-based micelles are obtained in a one-pot procedure, through reaction of D-mannosamine or D-glucosamine with the N-succinimidyl (NS) esters of a poly(D,L-lactide)-block-poly(N-acryloxysuccinimide-co-N-vinylpyrrolidone) (PLA-b-P(NAS-co-NVP)) amphiphilic copolymer (presenting quasi-alternating NAS/NVP units) in dimethyl sulfoxide (DMSO), followed by nanoprecipitation and dialysis against water. The glycopolymer micelles exhibit a higher CMC and size than those obtained from unmodified copolymer, due to increased hydrophilicity of the external block as a result of sugar derivatization, and the availability of the sugars at the micelle surface is evidenced through interactions with Concanavalin A (Con A) lectin, as attested by turbidimetric measurements and enzyme-linked lectin assay (ELLA). Interestingly, the glycopolymer micelles can be further used for hydrophobic molecule encapsulation and release, as shown with imiquimod, while keeping their interactions with con A intact. It is concluded that the PLA-based amphiphilic/reactive copolymer represents a versatile platform for glycopolymer-based micelle constructs for drug/vaccine delivery.

International audience; A sample pretreatment was evaluated to enable the production of intact cationic species of synthetic polymers holding a labile end-group using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. More specifically, polymers obtained by nitroxide-mediated polymerization involving the MAMA-SG1 alkoxyamine were stirred for a few hours in trifluoroacetic acid (TFA) to induce the substitution of a tert-butyl group on the nitrogen of nitroxide end-group by a hydrogen atom. Nuclear magnetic resonance, electrospray ionization tandem mass spectrometry, and theoretical calculations were combined to scrutinize this sample pretreatment from both mechanistic and energetic points of view. The substitution reaction was found to increase the dissociation energy of the fragile C?ON bond to a sufficient extent to prevent this bond to be spontaneously cleaved during MALDI analysis. This TFA treatment is shown to be very efficient regardless of the nature of the polymer, as evidenced by reliable MALDI mass spectrometric data obtained for poly(ethylene oxide), polystyrene and poly(butylacrylate).

International audience; We report here a straightforward route to polymer?peptide conjugates via the convenient synthesis of SG1-functionalized peptides able to initiate nitroxide-mediated polymerization (NMP). The NMP initiator SG1-functionalized peptide (SG1-GGGWIKVAV) was prepared by solid-phase peptide synthesis (SPPS) using an automated synthesizer, with a last step consisting of reaction between the carboxylic acid function of the MAMA-SG1 (BlocBuilder) alkoxyamine with the peptide terminal amine in the presence of benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP) as coupling agent. The resulting SG1-functionalized resin-attached peptide was used to perform the controlled NMP of styrene, leading after trifluoroacetic acid cleavage to the corresponding polystyrene?peptide conjugate. The latter was thoroughly characterized by UV spectrometry, gel permeation chromatography (GPC), 1H NMR, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS).

This paper reports the preparation of a MAMA-SG1 (BlocBuilder™) derived alkoxyamine bearing a N -succinimidyl (NHS) ester group 1 , valuable for functional and advanced polymer synthesis. This alkoxyamine was exploited following two strategies: (i) a post-functionalization approach based on the transformation of α-NHS chain ends of polymers previously obtained by nitroxide mediated polymerization (NMP) from 1 (path A) and (ii) a pre-functionalization approach based on the functionalization of alkoxyamine 1 prior to NMP (path B). Path A was demonstrated by derivatization of α-NHS functionalized polystyrenes with ethanolamine, yielding hydroxyl-functionalized polystyrenes. Path B was illustrated by two examples: first, a OH functional alkoxyamine initiator, prepared by reaction of 1 with ethanolamine, was used for the synthesis of polystyrene- b -poly( d , l -lactide) by combining NMP and ring-opening polymerization. Secondly, a poly(propylene oxide)–SG1 macroalkoxyamine, obtained from reaction of 1 with NH 2 -functionalized poly(propylene oxide), was used as a macroinitiator for NMP of styrene to obtain a PS- b -PPO block copolymer.

Poly(hexyl-substituted lactides) (PHLA) as new hydrophobic polyesters with controlled molecular weights and narrow distributions were synthesized by ring-opening polymerization (ROP) using tin(II) 2-ethylhexanoate (Sn(Oct)(2)) and benzyl alcohol as catalyst and initiator. Glass transition temperatures (T(g)) and zero shear viscosities (eta(0)) at 25 degrees C could be modulated from T(g)= -42 degrees C to -10 degrees C and 40 to 4850 Pa s, respectively, by varying the polymer molecular weight and the number of hexyl groups along the polymer chain. Degradation studies were performed in terms of both mass and molecular weight loss in the course of time. The degradation mechanism is shown to be of the "bulk erosion" type, and comparable to standard poly(D,L-lactide) (PLA). Despite the increased steric hindrance in the poly(monohexyl-substituted lactide) (PmHLA) due to the hexyl side groups, its degradation rate at pH 7.4 and 37 degrees C was found to be slightly higher than observed for the analogue standard PLA. This could be attributed to the flexible rubbery state of the hexyl-substituted polymer (T(g) approximately -15 degrees C) at the physiological temperature, which is favoring the degradation in comparison to the rigid and glassy standard PLA (T(g) approximately 40 degrees C). In contrast, degradation studies performed at 60 degrees C, where both polymers are above their glass transition temperature, confirmed that the degradation rate is lower for the sterically more hindered PmHLA. The degradation products were analyzed by ESI-MS. Hydrolysis lead first to the corresponding oligo-ester fragments and finally to the nontoxic 2-hydroxyoctanoic acid and lactic acid. Tetracycline was tested as a model drug for release studies. This drug was found to be released faster and in higher amounts in its active form from the PHLA matrix than from standard PLA. The results presented in this work demonstrate the potential of these hydrophobic polylactide-based semisolid materials as an alternative to conventional PLA/PLGA for injectable drug delivery systems.

Novel amphiphilic methoxy-poly(ethylene glycol)-poly(hexyl-substituted lactides) block copolymers were synthesized by ring-opening polymerization (ROP) of mono and dihexyl-substituted lactide (mHLA and diHLA) in bulk at 100 degrees C in the presence of tin(II) 2-ethylhexanoate (Sn(Oct)(2)) as catalyst and methoxy-poly(ethylene glycol) (MPEG) as initiator. MPEG-PmHLA and MPEG-PdiHLA copolymers of predictable molecular weights and narrow polydispersities were obtained, as shown by (1)H NMR and GPC. DSC experiments showed that the MPEG-PHLA block-copolymer presents a bulk microstructure containing MPEG domains segregated from the PHLA domains. Micelles were successfully prepared from these block copolymers, with sizes ranging from 30 to 80 nm. The critical micellar concentration (CMC) was found to decrease with the increasing number of hexyl groups on the polyester block (MPEG-PLA > MPEG-PmHLA > MPEG-PdiHLA) for copolymers of the same composition and molecular weight. The hydrophobicity of the micelle core in dependence of the number of hexyl groups along the PLA chain was evidenced by absorbance experiments with the incorporation of the dye Nile Red. These novel amphiphilic copolymers are interesting for micellar drug delivery and especially in regard to optimized hydrophobic drug loadings, as it was shown for griseofulvin as a model drug.

Homolytic dissociation of alkoxyamines yields two radicals, a nitroxide and an alkyl radical. This reversible reaction was used to control radical polymerization of various olefins or to perform intermolecular radical addition (IRA). In a first part, we aimed at developing a cheaper alternative to SG1 radical able to control polymerization of a broad range of monomers including methacrylates. We developed several linear aliphatic and aromatic nitroxide structures. Contrary to SG1 nitroxide, theses structures were not suitable to control radical polymerization of styrene and n-butyl acrylate. As for the control of methacrylates, we studied DPAIO’s analogues, wich were suitable for the preparation of methacrylate based block copolymers.In a second part, we extended the field of IRA to peptide ligation and polymer coupling. Two peptides prefunctionnalized with an olefin and a MAMA-SG1 alkoxyamine were coupled by IRA with good yields. This method was called Alkoxyamine Peptide Ligation (APL). The development of triggered 4-VP-SG1 alkoxyamine allowed performing polymer clicking through IRA in mild conditions without any initiator/catalyst or irradiation source. The synthesis of an hydrogel allowing biological activity retention of in situ encapsulated biomolecule showed that this methodology is relevant for biomedical applications., La dissociation homolytique des alcoxyamines conduit à la formation de deux radicaux, un nitroxyde et un radical alkyle. Cette réaction réversible a été mise à profit pour contrôler les réactions de polymérisation radicalaire de monomères vinyliques ou pour réaliser des additions radicalaires intermoléculaires (IRA). Dans une première partie, notre but a été de développer une alternative plus économique au nitroxyde SG1 pour le contrôle de la polymérisation d’une large gamme de monomères y compris des méthacrylates. Nous avons développé plusieurs structures linéaires aliphatiques et aromatiques mais sans que celles-ci ne présentent les qualités du nitroxyde SG1 pour le contrôle de la polymérisation du styrène et de l’acrylate de n-butyle. Pour le contrôle de la polymérisation des méthacrylates, nous avons étudié des analogues du DPAIO qui se sont montrés aptes à la préparation de copolymères à blocs à base méthacrylate.Dans un second temps nous avons étendu l’IRA à la ligation peptidique et au couplage de polymères. Deux peptides préfonctionnalisés par une oléfine et par l’alcoxyamine MAMA-SG1™ ont été couplés par IRA avec de bons rendements. Cette méthode a été nommé Alcoxyamine Peptide Ligation (APL). Le développement de l’alcoxyamine activable 4-VP-SG1 a permis le couplage de type clic par IRA de polymères dans des conditions douces et sans catalyseur ou irradiation lumineuse. La synthèse d’un hydrogel encapsulant in situ une protéine tout en préservant son activité, a validé la compatibilité de cette méthode avec des applications biomédicales