Hydrolyzable p(DMAPEMA) polymers for gene delivery.

The efficiency of cationic polymers as transfectants is thought to be closely related to their DNA association/dissociation properties. An incomplete polymer-DNA dissociation could explain the relatively low gene expression obtained with p(DMAEMA) polymers. Our approach was to synthesize a p(DMAEMA) analogue, p(DMAPEMA), bearing an hydrolyzable cationic group incorporated into the pendant chain with a view to improving transfection. The complexation of DNA with both polymers was studied by agarose gel electrophoresis, size and zeta potential measurements, as well as the dissociation of the polyplexes, after treatment by an anionic polymer, sodium hydroxide or heat. The transfection efficiencies of the polyplexes were evaluated with 293T and BHK21 cells in comparison with Exgen 500. P(DMAPEMA) polymers were able to complex DNA and to release it in a free intact form after an alkaline treatment or storage at 37 degrees C. Poly(aspartic acid) was unable to dissociate p(DMAPEMA) based polyplexes, in contrast to p(DMAEMA) ones. No transfection was obtained with p(DMAPEMA) with both cell lines. A slow hydrolysis under physiological conditions resulting in the absence of DNA unpacking or endosomal entrapment could explain these results. Better transfection results were obtained with polyplexes which were able to be dissociated by electrostatic interactions rather than ones which required the hydrolysis mechanism to release free DNA into cells. Scheme of hydrolyzable p(DMAPEMA) polymer.

Rapid Bacterial Identification, Resistance, Virulence and Type Profiling using Selected Reaction Monitoring Mass Spectrometry.

Mass spectrometry (MS) in Selected Reaction Monitoring (SRM) mode is proposed for in-depth characterisation of microorganisms in a multiplexed analysis. Within 60-80 minutes, the SRM method performs microbial identification (I), antibiotic-resistance detection (R), virulence assessment (V) and it provides epidemiological typing information (T). This SRM application is illustrated by the analysis of the human pathogen Staphylococcus aureus, demonstrating its promise for rapid characterisation of bacteria from positive blood cultures of sepsis patients.

New hydrolyzable pH-responsive cationic polymers for gene delivery: a preliminary study.

Here we want to report the synthesis and the characterization of 2-methylacrylic acid 2-(3-imidazol-1-yl-propionyloxy)ethyl ester (IPEMA), a new methacrylate derivative monomer bearing an hydrolyzable side chain terminated by an imidazole group. The kp/kt(1/2) value for its homopolymerization in N,N-dimethylformamide at 60 degrees C was found to be 0.120 mol(-1/2) x L(1/2) x s(-1/2). The free radical copolymerization of N,N-dimethylaminoethyl methacrylate and this monomer was studied in N,N-dimethylformamide at 60 degrees C, the reactivity ratios of this couple of monomers were determined to be r(DMAEMA) = 1.13 +/- 0.09 and r(IPEMA) = 0.82 +/- 0.09 (using distinct calculation methods). Molecular weights analysis, parallely with refractive index increments measurements, were performed to characterize the obtained polymers. Potentiometric titrations showed the ability of these copolymers to act as a 'proton sponge'. Preliminary study of the copolymers hydrolysis proved that imidazole units could be slowly cleaved from the polymer backbone at 37 degrees C in neutral aqueous buffer. Agarose gel electrophoresis of plasmid DNA/polymer complexes demonstrated the DNA complexing properties of these imidazole-based copolymers.

Self-assemblies on chitosan nanohydrogels.

Nanohydrogels of pure chitosan, containing neither potentially toxic solvent nor chemical cross-linker, were obtained by an ammonia-induced physical gelation of a reverse emulsion of a chitosan solution in a triglyceride mixture as an organic phase. The resulting colloids were obtained with a controlled size distribution and displayed a positive surface charge. Assemblies with various macromolecules were investigated as a first step toward new nano-carriers for bioactive molecules. Chondroitin sulfate formed polyelectrolyte complexes with the positively charged surface of the nanogels, leading to negative chitosan-based colloidal hydrogels with preservation of the original average size of the dispersion. The mode of assembly of HIV-1 p24 protein with these colloids relied on multiple interactions between the protein and the hydrogels, irrespective of their surface charges. Anyhow, the amounts of loaded protein remained limited, suggesting a surface association. The assembly of an immunoglobulin (IgG) was markedly different from p24. No association was detected with the positive colloidal hydrogels whereas a very high loading capacity could be obtained with the negative ones. So, this work reports that fully biodegradable submicrometric physical hydrogels could be obtained from naturally occurring polymers. These gels could cargo a variety of biomolecules making them versatile carriers with many potential applications in Life Sciences.

Synthesis and structural characterization of chitosan nanogels.

Colloidal physical gels of pure chitosan were obtained via an ammonia-induced gelation in a reverse phase emulsion. The water weight fraction and the chitosan concentration in the water phase were optimized so as to yield nanogels with controlled particle size and size distribution. The spherical morphology of the nanogels was established by transmission electron microscopy with negative staining. Wide-angle X-ray scattering experiments showed that these gels were partially crystalline. The electrophoretic mobilities of the particles remained positive up to pH 7, above which the particles aggregated due to the charge neutralization. From the investigation on the colloidal stability of these nanogels in various conditions (pH, salt concentration, temperature), an electrosteric stabilization process of the particles was pointed out, related to the conformation of mobile chitosan chains at the gel-liquid interface. Therefore, the structure of the nanogels was deduced as being core-shell type, a gelified core of neutralized chitosan chains surrounded by partially protonated chains.

A novel synthesis of chitosan nanoparticles in reverse emulsion.

Physical hydrogels of chitosan in the colloidal domain were obtained in the absence of both cross-linker and toxic organic solvent. The approach was based on a reverse emulsion of a chitosan solution in a Miglyol/Span 80 mixture, generally regarded as safe. Temperature and surfactant concentration were optimized, and the impact of the degree of acetylation (DA) and the molar mass of chitosan was investigated. When chitosan had a DA above 30%, only macroscopic gels were obtained, because of the predominance of attractive Van der Waals forces. The lower the molar mass of chitosan, the better the control over particle size and size distribution, probably as a result of either a reduction in the viscosity of the internal aqueous phase or an increase in the disentanglement of the polymer chain during the process. After extraction and redispersion of the colloid in an ammonium acetate buffer, the composition of the particles was around 80% of pure chitosan corresponding to a recovery of 60% of the original input. These new and safe colloids offer wide perspectives of development in further applications.

New insights into self-organization of a model lipid mixture and quantification of its adsorption on spherical polymer particles.

The adsorption of lipids onto spherical polymer colloids led to original assemblies presenting structural characteristics adjustable with the lipid formulation. The model system selected for this work involved sulfate-charged poly(styrene) submicrometer particles and zwitterionic/cationic lipid mixtures composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dipalmitoyl-3-trimethylammonium-propane (DPTAP). According to the theoretical packing parameter calculations and whatever the DPPC/DPTAP ratio, the two lipids self-assembled in aqueous media to spontaneously form vesicles. A phase transition investigation of these DPPC/DPTAP vesicles using differential scanning calorimetry revealed particular thermotropic behaviors, especially for the equimolar formulation where very strong interactions occurred between DPPC and DPTAP. Furthermore, the coating of the lipids around particles was monitored versus DPPC/DPTAP ratio by means of numerous appropriate techniques. First, a thermogravimetric analysis, providing decomposition profiles of lipid/polymer particle assemblies with temperature, was atypically carried out for such nanostructures. Then, 1H NMR spectroscopy enabled the exact DPPC/DPTAP molar ratios adsorbed on particles to be determined by differentiating both lipids. Subsequently, it also pointed out the major role of electrostatic interactions as driving forces in the assembly elaboration process. In addition to these findings, quantitative information has been collected and correlated with chemical lipid assays and permitted the statement of a lipid bilayer coverage for the assemblies prepared in water, in agreement with quasi-elastic light scattering data.