Injectable hydrogels are attractive for tissue engineering due to their minimally invasive administration and their ability to provide localized and controlled drug delivery. In this study, we investigated an amphiphilic PNIPAAm-b-PLA-b-PEG-b-PLA-b-PNIPAAm pentablock copolymer hydrogel, previously developed as a thermoresponsive system forming via intermicellar bridging at physiological temperature. Its capacity to load and release neuroprotective drugs with distinct physicochemical properties was evaluated. Hydrophobic (riluzole) and hydrophilic (minocycline, apelin-13) model drugs were incorporated, and in vitro release profiles were monitored under physiological conditions. Drug release behavior depended on drug hydrophobicity: hydrophilic compounds exhibited rapid, diffusion-driven release with a pronounced burst effect, whereas the hydrophobic drug showed a slower and more sustained release, correlated with hydrogel degradation (mass loss) at low drug loadings. The hydrogel showed no detectable cytotoxicity toward neuronal cells in vitro. In addition, hydrogel-mediated drug delivery modulated cellular responses depending on the compound, reducing the cytotoxic effects of minocycline and enhancing neuronal survival in the presence of apelin. Importantly, hydrogel-mediated delivery enabled modulation of drug exposure kinetics, thereby influencing the resulting cellular responses. Altogether, these results highlight the ability of this thermoresponsive hydrogel to accommodate drugs with diverse physicochemical properties and to modulate their release and associated biological effects, supporting its relevance as a platform for controlled delivery of neuroactive compounds.

IntroductionMicelle-based delivery systems provide a promising platform for vaccine adjuvants by encapsulating active compounds and enabling targeted delivery, thereby limiting systemic side effects. Here, we investigated the immune response, biodistribution, and antiviral effects of micelles loaded with the TLR7/8 agonist, 3M-052, in zebrafish (Danio rerio) larvae.MethodsFluorescently labelled micelles were injected to transgenic reporter zebrafish larvae, and localization of micelles as well as neutrophils, macrophages, or mxa-expressing, was monitored by live confocal microscopy. Host response was also assessed at the whole-body level by qRT-PCR. In addition, larvae were infected with a recombinant Sindbis Virus to monitor the localization of viral infection. ResultsMicelles demonstrated progressive clearance from the injection site and promoted local recruitment of neutrophils and macrophages, indicating immune activation. Whole-larva analysis showed no significant upregulation of key immune genes following unloaded or 3M-052-loaded micelle injection. However, 3M-052-micelles triggered a transient and localized mxa response, confirming induction of a local type I interferon response. Furthermore, zebrafish larvae exhibited significantly reduced virus replication near the site of 3M-052-micelle injection. DiscussionThese findings indicate that micelles effectively deliver 3M-052 and elicit a localized antiviral response without broad systemic immune activation.

ABSTRACT Photoresponsive polymers have emerged as a dynamic class of materials exhibiting a response to light. On the other hand, amphiphilic polymers are materials possessing both hydrophobic and hydrophilic sides in their macromolecular structure. The particularity of amphiphilic polymers is that they can simultaneously interact with hydrophobic and hydrophilic environments, leading to self‐assembly. The combination of these intriguing photosensitive and amphiphilic properties has spurred the development of amphiphilic photoresponsive polymers with adaptable features. Exposure to light triggers changes in the polymers’ properties, which can be exploited to influence the formation and stability of diverse nanoscale structures such as core–shell micelles, worm‐like micellar assemblies, vesicles, and other complex macromolecular architectures. This precise control over polymer behavior has propelled these materials to the forefront of research and innovation. This review sheds light and classifies the main photosensitive chemical groups used to design such photoresponsive polymers. Furthermore, a concise overview and a discussion about the synthesis pathways of photoresponsive polymers, with or without amphiphilic behavior, are presented, followed by a projection of potential opportunities raised by these polymers to improve controlled agrochemicals release area.

A polyethylene glycol (PEG) grafted copolymer based on a N-vinyl pyrrolidone (NVP) backbone was designed for conjugating antibodies (Abs) at PEG chain ends, leading to multivalent (“multi-antibody”) bioconjugate for use as a detection system in immunoassays. For this purpose, a poly(N-vinyl pyrrolidone-co-N-acryloxysuccinimide) (P(NVP-co-NAS)) copolymer was prepared by nitroxide mediated polymerization (NMP) and the N-succinimidyl activated esters of the NAS units were used for grafting heterobifunctional amine-PEG-maleimide by amidation. The PEG maleimide end-groups were further conjugated to Abs through thiol-maleimide addition. Enzyme-linked immunosorbent assay (ELISA) performed with the obtained bioconjugate in the detection phase showed significant improvement in antigen detection as compared to standard protocol with free Ab. These results point out the relevance of this straightforward approach to improve antibody accessibility and reduce background noise (thanks to anti-fouling PEG) for enhanced sensitivity in the field of immunoassays.

Historically used for the delivery of hydrophobic drugs through core encapsulation, amphiphilic copolymer micelles have also more recently appeared as potent nano-systems to deliver protein and peptide therapeutics. In addition to ease and reproducibility of preparation, micelles are chemically versatile as hydrophobic/hydrophilic segments can be tuned to afford protein immobilization through different approaches, including non-covalent interactions (e.g., electrostatic, hydrophobic) and covalent conjugation, while generally maintaining protein biological activity. Similar to many other drugs, protein/peptide delivery is increasingly focused on stimuli-responsive nano-systems able to afford triggered and controlled release in time and space, thereby improving therapeutic efficacy and limiting side effects. This short review discusses advances in the design of such micelles over the past decade, with an emphasis on stimuli-responsive properties for optimized protein/peptide delivery.

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.

Leishmaniasis is the 3rd most challenging vector-borne disease after malaria and lymphatic filariasis. Currently, no vaccine candidate is approved or marketed against leishmaniasis due to difficulties in eliciting broad immune responses when using sub-unit vaccines. The aim of this work was the design of a particulate sub-unit vaccine for vaccination against leishmaniasis. The poly (D,L-lactide) nanoparticles (PLA-NPs) were developed in order to efficiently adsorb a recombinant L. major histone H2B (L. major H2B) and to boost its immunogenicity. Firstly, a study was focused on the production of well-formed nanoparticles by the nanoprecipitation method without using a surfactant and on the antigen adsorption process under mild conditions. The set-up preparation method permitted to obtain H2B-adsorbed nanoparticles H2B/PLA (adsorption capacity of about 2.8% (w/w)) with a narrow size distribution (287 nm) and a positive zeta potential (30.9 mV). Secondly, an in vitro release assay performed at 37 °C, pH 7.4, showed a continuous release of the adsorbed H2B for almost 21 days (30%) from day 7. The immune response of H2B/PLA was investigated and compared to H2B + CpG7909 as a standard adjuvant. The humoral response intensity (IgG) was substantially similar between both formulations. Interestingly, when challenged with the standard parasite strain (GLC94) isolated from a human lesion of cutaneous leishmaniasis, mice showed a significant reduction in footpad swelling compared to unvaccinated ones, and no deaths occurred until week 17th. Taken together, these results demonstrate that PLA-NPs represent a stable, cost-effective delivery system adjuvant for use in vaccination against leishmaniasis.

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.

Micelles from self-assembled amphiphilic copolymers are highly attractive in drug delivery, due to their small size and hydrophilic stealth corona allowing prolonged lifetimes in the bloodstream and thus improved drug bioavailability. Polylactide (PLA)-based amphiphilic copolymer micelles are key candidates in this field, owing to the well-established biodegradability and biocompatibility of PLA. While PLA-b-poly(ethylene glycol) (PEG) block copolymer micelles can be seen as the "gold standard" in drug delivery research so far, the progresses in controlled radical polymerizations (Atom Transfer Radical Polymerization, Reversible Addition-Fragmentation Transfer and Nitroxide Mediated Polymerization) have offered new opportunities in the design of advanced amphiphilic copolymers for drug delivery due to their flexibility in many regards: (i) they can be easily combined with ring-opening polymerization (ROP) of lactide, with a diversity in types of architectures (e.g., block, graft, star), (ii) they allow (co)polymerization of a wide range of vinyl monomers, possibly circumventing PEG limitations, (iii) functionalization (with biomolecules or stimuli-cleavable moieties) is versatile due to end-group fidelity and copolymerization ability with reactive/functional comonomers. In this review, we report on the advances in the past decade of such amphiphilic PLA/vinyl polymer based nano-carriers, regarding key properties such as stealth character, cell targeting and stimuli-responsiveness.

PURPOSE: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.METHODS: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.RESULTS: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.CONCLUSIONS: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.

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.

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.

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-isopropylacrylamide)-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 biocompatibility. 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.

Thermoresponsive poly(N-isopropylacrylamide) (PNIPAAm)-based injectable hydrogels represent highly attractive materials in tissue engineering and drug/vaccine delivery but face the problem of long-term bioaccumulation due to non-degradability. In this context, we developed an amphiphilic poly(D,L-lactide)-b-poly(NIPAAm-co-polyethylene glycol methacrylate) (PLA-b-P(NIPAAm-co-PEGMA)) copolymer architecture, through a combination of ring-opening and nitroxide-mediated polymerizations, undergoing gelation in aqueous solution near 30 °C. Complete hydrogel mass loss was observed under physiological conditions after few days upon PLA hydrolysis. This was due to the inability of the resulting P(NIPAAm-co-PEGMA) segment, that contains sufficiently high PEG content, to gel. The copolymer was shown to be non-toxic on dendritic cells. These results thus provide a new way to engineer safe PNIPAAm-based injectable hydrogels with PNIPAAm-reduced content and a degradable feature.

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.

A catalyst/initiator-free radical addition reaction performed under mild conditions (water, 30 °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 in situ-loaded proteins, which could retain biological activity. This contrasts with standard thermal radical conditions that lead to complete protein inactivation.

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.

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.

Surface modified microfibers were prepared in a one-step process, and were prone to retain the activity and improve the stability of immobilized enzymes.

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.