Micelles complexes de polyions (optimisation d'un vecteur pour la thérapie cellulaire Ex Vivo par les cellules dentritiques)

MONTPELLIER-BU Pharmacie (341722105) / SudocSudocFranceF

Mise au point et développement de microparticules biodégradables vecteurs de protéines (application au GDNF (Glial Cell Line Derived Neurotrophic Factor))

Ce travail repose sur l'élaboration de vecteurs microparticulaires biodégradables de protéines et leur caractérisation in vitro. Implantables en intra-cérébral, ces systèmes permettent la libération d'un principe actif in situ de façon prolongée et contrôlée. Ils offrent ainsi une alternative thérapeutique intéressante, notamment pour les pathologies neurodégénératives, grâce à la vectorisation de facteurs neurotrophiques. La première partie de notre travail expérimental concerne le développement d'un outil permettant l'étude de la libération in vitro de vecteurs de protéines. Dans une seconde partie, des microsphères de GDNF (Glial Cell Line Derived Neurotrophic Factor), candidat potentiel au traitement de la maladie de Parkinson, ont été réalisées par une technique de double émulsion extraction-évaporation de solvant. Ces microsphères de PLGA ont permis la libération de GDNF biologiquement actif sur une période prolongée de 56 jours. Enfin, dans une dernière partie, nous avons développé une technique de préparation de microsphères caractérisée par l'emploi d'excipients utilisés en médecine humaine. Cette étude a été menée afin de s'affranchir des solvants chlorés toxiques, couramment employés dans les procédés classiques de microencapsulation. Par ailleurs, ces particules ont montré leur aptitude à encapsuler une protéine.This work was based on the development of biodegradable microparticles of proteins and on their characterization in vitro. The studied protein was a neurotrophic factor, Glial Cell Line Derived Neurotrophic Factor (GDNF), a potential candidate to the Parkinson disease treatment. The first part of our experimental work was the development of a novel in vitro system. It was required to assess the stability and the release kinetics of the protein microspheres. In second part, GDNF-loaded microspheres were prepared by a double emulsion technique. They allowed the release over 56 days of biologically active GDNF at clinically relevant doses. In third part, we developed biodegradable PLGA microparticles prepared by an original solvent diffusion process, without the use of unacceptable organic solvent, and aimed to encapsulate proteins.ANGERS-BU Médecine-Pharmacie (490072105) / SudocSudocFranceF

Equilibrium protein adsorption on nanometric vegetable-oil hybrid film/water interface using neutron reflectometry

International audienceNanofilms of thickness of about two nanometers have been formed at the air-water interface using functionalized castor oil (ICO) with cross-linkable silylated groups. These hybrid films represent excellent candidates for replacing conventional polymeric materials in biomedical applications, but they need to be optimized in terms of biocompatibility which is highly related to protein adsorption. Neutron reflectivity has been used to study the adsorption of two model proteins, bovine serum albumin and lysozyme, at the silylated oil (ICO)-water interface in the absence and presence of salt at physiologic ionic strength and pH and at different protein concentrations. These measurements are compared to adsorption at the air-water interface. While salt enhances adsorption by a similar degree at the air-water and the oil-water interface, the impact of the oil film is significant, with adsorption at the oil-water interface three-to four-fold higher compared to the air-water interface. Under these conditions, the concentration profiles of the adsorbed layers for both proteins indicate multilayer adsorption: The thickness of the outer layer (oil-side) is close to the dimension of the minor axis of the protein molecule, ~ 30 Å, suggesting a side-way orientation with the long axis parallel to the interface. The inner layer extends to 55-60 Å. Interestingly, in all cases, the composition of oil film remains intact without significant protein penetration into the film. The optimal adsorption on these nanofilms, 1.7-2.0 mg•m-2 , is comparable to the results obtained recently on thick solid cross-linked films using quartz crystal microbalance and atomic force microscopy, showing in particular that adsorption at these ICO film interfaces under standard physiological conditions is non-specific. These results furnish useful information towards the elaboration of vegetable oil-based nanofilms, in direct nanoscale applications or as precursor films in the fabrication of thicker macroscopic films for biomedical applications.

Microfluidic Systems for Droplet Generation in Aqueous Continuous Phases: A Focus Review

International audienceMicrofluidics is one of the most fascinating fields that researchers have been trying to apply in a large number of scientific disciplines over the past two decades. Among them, the discipline of food and pharmaceutical formulation encountered several obstacles when combining microfluidics with aqueous media. Indeed, the physical properties of liquids at micrometric volumes being particular, the droplet generation within microfluidic devices is a big challenge to be met. This focus review is intended to be an initiation for those who would like to generate microdroplets in microfluidic systems involving aqueous continuous phases. It provides a state-of-the-art look at such systems while focusing on the microfluidic devices used, their applications to form a wide variety of emulsions and particles, and the key role held by the interface between the device channels and the emulsion. This review also leads to reflections on new materials that can be used in microfluidic systems with aqueous continuous phases

pH-sensitive double-hydrophilic block copolymer micelles for biological applications.

International audienceIn the recent years, double-hydrophilic block copolymer (DHBC) micelles have appeared as potential vectors for pharmaceutical applications due to their simple preparation method in aqueous solvent. The present study aims at underscoring the strategy for the choice of the partners in the formulation of DHBC micelles presenting a good stability in physiological conditions (pH 7.4, 0.15mol/L NaCl) and a pH-sensitivity allowing their disassembly at pH 5. Using light scattering and laser-doppler electrophoresis, micelles of polymethacrylic acid-b-polyethylene oxide complexing either poly-L-lysine (PLL) or an oligochitosan were characterised. Whatever the polyamine counter-polyion considered, the micelles were perfectly formed for an amine/methacrylic acid molar charge ratio of one. They were characterised by a hydrodynamic diameter of 28nm for PLL and 60nm for oligochitosan and by a neutral zeta potential. The stability study as a function of the pH and of the ionic strength revealed different behaviours. Oligochitosan micelles were stable until pH 7 and unstable at 0.15mol/L NaCl. On the contrary, PLL micelles were stable in physiological conditions and disassembled at pH 5. As a conclusion, the choice of the partners to formulate double-hydrophilic block copolymer based-micelles is strategic in order to obtain well-adapted vectors applied to the pharmaceutical field

How to achieve sustained and complete protein release from PLGA-based microparticles?

International audienceOne of the most challenging tasks in the delivery of therapeutic proteins from PLGA-based microparticles is the sustained and complete release of the protein in its native form. The mechanisms responsible for incomplete protein release from these devices are numerous and complex; the beneficial effect of different formulations has often been evaluated in vitro. Strategies employed for overcoming protein destabilization during the release step are reviewed in this paper. Proteins have been protected in the deleterious environment by adding stabilizers to the formulation, or by modifying the protein or the polymer. Alternatively, some strategies have aimed at avoiding the formation of the destabilizing environment. As experimental conditions may influence the results from in vitro release studies, we initially report precautions to avoid adverse effects

Effect of GDNF-releasing biodegradable microspheres on the function and the survival of intrastriatal fetal ventral mesencephalic cell grafts.

International audienceThe transplantation of fetal ventral mesencephalic (FVM) cell suspensions into the brain striatal system is an alternative approach for the treatment of Parkinson's disease (PD). However, one objection to this procedure is the relatively poor survival of implanted cells. Attempts have been made to improve the survival of grafted dopaminergic neurons using glial cell line-derived neurotrophic factor (GDNF). Nevertheless, the clinical application of GDNF is limited, due to the difficulties in administering a protein to the brain tissue and due to the ubiquity of its receptor, thus leading to neurological side effects. A strategy to deliver GDNF in the brain based on the intracerebral implantation of biodegradable poly(D,L-lactic acid-co-glycolic acid) sustained release microspheres has been developed. Such microparticles can be easily implanted by sterotaxy in precise and functional areas of the brain without causing damage to the surrounding tissue. Moreover, the release profile of the GDNF-loaded microspheres showed a sustained release over 56 days of biologically active GDNF at clinically relevant doses. The present study shows that the implantation of GDNF-loaded microspheres at a distance to the site of FVM cells in the 6-hydroxydopamine-lesioned rat model of PD improves dopaminergic graft survival and function. Furthermore, the unloaded and the GDNF-loaded microspheres, when they are mixed with FVM cells, may provide a mechanical support and a 3D environment inducing differentiation and increased function of dopaminergic neurons. Taken together, these results show that GDNF microspheres represent an efficient delivery system for cell transplantation studies

Liposil, a promising composite material for drug storage and release

International audiencePreliminary tests in the field of drug storage and release of composite materials known as liposils were described. These silica-based particles were obtained via liposome templating. The non-porous amorphous silica cladding of liposils protected the liposomes which retained the fundamental properties of their phospholipid bilayer. In an improved synthesis, two formulations were used, one with and the other without cholesterol in the phospholipid bilayer. Stability tests were done using carboxyfluorescein as a model hydrophilic drug loaded in the liposomes aqueous phase before the templating process. The stability of the loaded liposils was analyzed at two different pH (1.2 and 7.4) in a flow cell, according to the USP 28 norm. At pH 1.2, the silica shell was stable and prevented their rapid degradation. Interestingly, at pH 7.4 the analysis of the release kinetics revealed that the hydrolysis of the silica shell initially released intact liposomes. Characterizations of liposils were done at various steps of these processes. The stability observed for liposils make them good starting material for drug storage and release schemes. For instance, functionalization of their external surface should improve their capture by cells whereby drug release could then be induced by external stimuli, such as ultrasounds or microwaves