Synthèse de nanoparticules d'or fonctionnalisées par l'acide dihydrolipoïque (caractérisation, étude de stabilité et impact sur l'homéostasie redox cellulaire)

L'objectif de notre travail consiste en la conception de nanoparticules d'or (AuNP) monodisperses, et stables ; puis en l'évaluation de l'effet des propriétés de surface de AuNP sur leur internalisation et leur toxicité dans une lignée cellulaire (macrophages alvéolaires de rat-NR8383). Nous avons synthétisé des NP d'or soit stabilisées par les ions citrate (Au-citrate) soit fonctionnalisées par l'acide dihydrolipoïque (DHLA) (NP Au@DHLA). Nous avons montré que la densité de couverture est un paramètre crucial pour améliorer la stabilité colloïdale des AuNP dans des conditions physiologiques. Nous avons caractérisé complètement les AuNP d'un point de vue physico-chimique, en particulier en développant une méthode de dosage spectrophotocolorimétrique de l'or. Nous avons aussi étudié les effets biologiques des AuNP. Tout d'abord, en absence de cellules, nos résultats révèlent que la fonctionnalisation de surface de AuNP affecte la réactivité avec des biomolécules tels que le glutathion réduit (GSH), le S-nitrosoglutathion, et l'albumine de sérum bovin (les Au-citrate interagissant avec toutes les molécules testées), l'internalisation par les cellules (les Au-citrate sont deux fois plus internalisées) et l'état redox cellulaire (les Au-citrate diminuent de 20% le niveau intracellulaire de GSH) sans relation apparente avec la formation de ROS. En outre, les AuNP ne semblent pas induire l'expression des mRNA associées à la réponse inflammatoire (tnf), au stress oxydant (ncf1) et à l'apoptose (nfkb2), paramètres essentiels pour préserver l'intégrité de la cellule et de l'organisme, et envisager l'utilisation de ces NP comme plateforme pharmaceutiqueThe aim of our work was to produce highly monodisperse and stable gold nanoparticles (AuNP) with potential for biomedical applications, and to evaluate the effect of AuNP surface properties on their uptake and cytotoxicity in a cultured cell line (rat-alveolar macrophages-NR8383). We synthesized AuNP either stabilized with citrate (Au-citratte) or capped with a dithiol, i.e. dihydrolipoic acid (Au@DHLA NP). The present study shows that the surface packing density is a crucial parameter to enhance the colloidal stability of AuNP in physiological conditions. We fully characterized the considered AuNP from a physico-chemical point of view. Indeed, we optimized a spectrocolorimetric and a HPLC method to evaluate the properties of AuNP. We studied the biological effects of AuNP. Under cell-free conditions, our results reveal that AuNP coating affects their reactivity with biomolecules, i.e. reduced glutathione (GSH), S-nitrosoglutathione and bovine serum albumin (Au-citrate interact with all molecules), the cellular uptake (Au-citrate are two times more effective) and redox status (Au-citrate decrease the intracellular GSH level by ca 20%) with no apparent relationship with ROS formation. Furthermore, both AuNP appear not to induce mRNA expression related to inflammatory response (tnfa), oxidative stress (ncf1) and apoptosis (nfkb2), parameters of main importance to preserve cellular integrity and body safety, and to assure pharmaceutical platform functionMETZ-SCD (574632105) / SudocNANCY1-Bib. numérique (543959902) / SudocNANCY2-Bibliotheque electronique (543959901) / SudocNANCY-INPL-Bib. électronique (545479901) / SudocSudocFranceF

Prolonged release microparticles able to reduce the initial burst effect

Les formes multiparticulaires injectables présentent l inconvénient d une libération initiale prématurée dont les conséquences sont une toxicité systémique si les concentrations sanguines du principe actif deviennent importantes ainsi qu une modification de la libération. Pour résoudre ce problème, des microparticules composites ont été mises au point : il s agit de microparticules encapsulant des nanoparticules. Le concept a d abord été démontré in vitro en encapsulant des nanoparticules de poly(epsilon-caprolactone) dans un polymère non biodégradable en choisissant comme modèles une molécule de faible masse moléculaire (ibuprofène) et un peptide (acétate de triptoréline). L originalité du travail réside dans le choix des polymères et des solvants retenus pour la fabrication des microparticules. Le solvant utilisé pour fabriquer les microparticules doit être un non-solvant du polymère des nanoparticules. L acétate d éthyle répondait à ces conditions puisqu il ne dissout pas la poly(epsilon-caprolactone) mais que c est un excellent solvant de l éthylcellulose ou du polymère polycationique utilisé dans la première partie du travail. Sur la base d études de libération in vitro, il a ainsi été démontré que les microparticules composites permettaient effectivement de fortement réduire cette libération précoce tout en continuant d assurer une libération prolongée. Dans un deuxième temps, la réduction de la libération initiale a été confirmée par une étude in vivo chez le rat avec 2 principes actifs modèles : ibuprofène et insuline. Toutefois, le polymère de la matrice des microparticules a été remplacé par un copolymère biodégradable constitué d acides lactique et glycolique. Il a été démontré que le nouveau concept de microparticules composites permettait de proposer une forme originale limitant la libération initiale des principes actifs suite à leur administration sous-cutanée ou intramusculaire tout en assurant une libération prolongéeMultiparticular injectable dosage forms present a burst effect known to lead to i) a systemic toxicoligal critical issue if blood concentrations of the drug are too high and ii) a change in the release profile due to a lower loading charge in microparticles. In order to solve this problem, composite microparticles have been developed: they consist in nanoparticles encapsulated in microparticles. Such a concept has been demonstrated in vitro by encapsulating poly(epsiloncaprolactone) nanoparticles in a non-biodegradable polymeric matrix with two model drugs: a small molecular weight drug (ibuprofene) and a peptide (triptorelin acetate). The novelty of the research work lies on the adequate choice of polymers and solvents used for microparticles manufacturing. Indeed, the solvent used to manufacture microparticles has to be a non-solvent of the nanoparticles polymer. Ethyl acetate was a good candidate since it does not dissolve poly(epsilon-caprolactone) nanoparticles but is an excellent solvent for ethylcellulose and the polycationic polymer used in the first part of the work. Based on in vitro release studies, it was demonstrated that composite microparticles allowed the initial release to be strongly reduced together with a prolonged release. In a second part, the burst release reduction has been confirmed in vivo in rats with 2 drug models: ibuprofen and insulin. However, the microparticles polymer matrix was replaced by a biodegradable copolymer made of lactic and glycolic acids. It has been demonstrated that the novel composite microparticles were an innovative dosage form able to control the initial burst release often associated to microparticles after sub-cutaneous or intramuscular administration while still maintaining the prolonged release of the encapsulated drugs. Such a result can be associated with the more difficult diffusion of the drug through the two consecutive polymeric barriers of nanoparticles and microparticles.NANCY1-Bib. numérique (543959902) / SudocSudocFranceF

Systèmes injectables biodégradables pour la libération prolongée d'ivermectine

Des systèmes injectables de formation in situ ont été utilisés dans les dernières années pour l'obtention de formulations de préparation facile et permettant la libération prolongée de principes actifs. Ces systèmes utilisant des solvants biocompatibles et des polymères biodégradables sont des liquides (solutions ou émulsions) qui une fois injectés dans l'organisme donnent lieu à des implants (ISI) ou à des microparticules (ISM) solides. La formation de ces systèmes est induite par la précipitation du polymère à partir des solutions polymériques qu'ils contiennent lors du contact avec les fluides corporaux aqueux. Dans ce travail, des ISI et des ISM, réalisés à partir des polymères de l'acide lactique et/ou glycolique (PLA et PLGA) et des différents solvants biocompatibles, pour la libération prolongée d?ivermectine (IVM), un principe actif antiparasitaire faiblement biodisponible par la voie orale, ont été développés. Les profils de libération du principe actif in vitro et in vivo à partir de ces systèmes, ont été comparés avec ceux obtenus à partir de microparticules réalisées par la méthode classique dite d'émulsion simple - évaporation de solvant ; il s'agit d'une technique aux multiples étapes, à coût élevé et dont l'utilisation de solvants toxiques la font difficilement industrialisable. La libération du principe actif à partir des microparticules obtenues par émulsion simple/évaporation du solvant a été influencée par la forte interaction du principe actif avec les polymères mais aussi par la porosité. Dans le cas des systèmes in situ, la vitesse de libération d'IVM a été conditionnée par la solubilité dans l'eau du solvant biocompatible sélectionné et par les interactions solvant/polymère. Pour les ISM, des paramètres tels que la nature de la phase externe, aqueuse (ISM-O/W) ou huileuse (ISM-O/O), la solubilité dans l'eau du solvant de la phase interne, l'affinité entre les phases et l'affinité de l'IVM pour chacune des phases, ont déterminé la vitesse de libération du principe actif. La bonne stabilité ainsi que les profils de libération plus prolongés et présentant une faible libération initiale du principe actif in vivo et in vitro, ont montré que les ISI et les ISM réalisés à partir de solvants biocompatibles de faible solubilité dans l'eau tels que la triacetine sont les plus indiqués pour l'encapsulation d'IVM par rapport à ceux plus solubles dans l'eau comme la N-methyl-2-pyrrolidone et la 2-pyrrolidone. Ces systèmes représentent donc une alternative intéressante par rapport aux formulations conventionnelles d'IVMIn situ forming injectable systems have been used in the past years to obtain sustained drug release formulations which are easy to prepare. These systems using biocompatible solvents and biodegradable polymers are liquids (solutions or emulsions) that upon injection on the body lead to solid implants (ISI) or microparticles (ISM). These systems are formed in contact with water body fluids by polymer precipitation from the polymeric solution. In this work, ISI and ISM made from lactide and/or glycolide polymers (PLA and PLGA) and different biocompatible solvents were performed to obtain sustained release of ivermectin (IVM), an antiparasitic drug with a low oral bioavailability. In vitro and in vivo drug release profiles from these systems were compared with those from microparticles obtained by the classical simple emulsion/solvent evaporation method, which is difficult to propose in industry because of its multiple steps, high cost and the solvent toxicity. Drug release from simple emulsion/solvent evaporation microparticles was affected by the strong polymer/drug interactions and porosity. Concerning to in situ forming systems, the rate of IVM release was dependent on solvent water solubility and solvent/polymer interactions. The nature of the external phase, water (ISM-O/W) or oil (ISM-O/O), the water solubility of the solvent in the internal phase, phase affinity and IVM/phase affinity determined drug release from ISM. The good stability, the in vitro and in vivo sustained release and the low burst effect of IVM, indicated that ISI and ISM formulated from low hydrosoluble biocompatible solvents such as triacetin are more appropriated to IVM formulation instead of those based on more hydrophilic solvent (N-methyl-2-pyrrolidone and 2-pyrrolidone). These systems are an interesting alternative to conventional IVM formulationsNANCY1-Bib. numérique (543959902) / SudocSudocFranceF

Designing dedicated rapamycin nanoparticles for articular vectorization: A holistic pharmacological approach

International audienceIntroduction: Rapamycin, an immunosuppressant, is a mTOR inhibitor leading to autophagy restoration. It is a candidate (10 μM) for experimental intra-articular (IA) treatment of osteoarthritis (OA), autophagy being defective in OA. Material and methods: We performed in vitro cytotoxicity on human chondrocytes, synoviocytes, and mesenchymal stem cells (MSCs) after 24 h exposure in hypoxia and normoxia with various rapamycin concentrations (10 nM to 100 μM). We then evaluated autophagic (beclin1 and Atg7) and catabolic (MMP13) genes with 10 μM rapamycin exposure for 24 h. We also developed rapamycin-loaded nanoparticles (NPs) for IA injection to assess NPs' cytotoxicity vs free rapamycin. We finally studied in vivo pharmacokinetic parameters of free (IV & IA) or NPs-loaded rapamycin (IA) (10 μM) injections in blood and synovial tissue in rat knees: AUC, T1/2, and MRT. Results: In vitro, we observed the concentrationdependent toxicity of free rapamycin for chondrocytes, synoviocytes, and MSCs. The IC50 was: 68.7, 79.7, and 79.7 μM in normoxia and 79.1, 29.9 and 38.5 μM in hypoxia, respectively. No difference was observed for autophagic genes in the three cellular types, but the IL1β-induced MMP13 level decreased. Rapamycin NPs showed similar dose-dependent toxicity on articular cells. In vivo, we determined (10 μM) an AUC higher for IV free rapamycin than for IA injection (AUCIA free and AUCIA NP), with respectively 4248, 28, and 74 μg/ min/L in blood. We observed a difference between AUCIA free and AUCIA NP (3735 and 10513 μg/min/L) concerning synovium. T1/2 and MRT were higher after NPs than free rapamycin injections: 57.8-5.0 h, vs. 80.6-5.5 h, respectively. Histology revealed no knee injury. Discussion/Conclusion: In vitro, rapamycin (10 μM) did not induce articular cytotoxicity and reduced IL1β's phlogistic effects. Rapamycin NPs did not induce cytotoxicity on articular cells at 10 μM. In vivo, IA administration of 10 μM rapamycin NPs increased its local residency vs. free rapamycin with lower systemic diffusion

Development of enoxaparin sodium polymeric microparticles for colon-specific delivery

International audienceBackground and aimsRecent studies have shown that low molecular weight heparins are effective in the treatment of inflammatory bowel disease. Therefore, there is considerable interest in the development of an oral colonic delivery pharmaceutical system allowing targeted release of heparin in the inflamed tissue. The objective of this study was to prepare microparticles for the oral administration and colonic release of enoxaparin and to evaluate the influence of certain formulation factors on their characteristics.MethodsMicroparticles were prepared by water/oil/water double emulsion technique followed by solvent evaporation. The influence of several formulation factors on the characteristics of microparticles were evaluated. The formulation factors were alginate concentration in the inner aqueous phase, polymer (Eudragit® FS 30D and Eudragit® RS PO) concentration in the organic phase and ratios between the two polymers. The microparticles were characterized in terms of morphology, size, entrapment efficiency and enoxaparin release.ResultsThe results showed that increasing sodium alginate percentage reduced the encapsulation efficiency of enoxaparin and accelerated enoxaparin release. Regarding the influence of the two polymers, reducing polymer concentration in the organic phase led to a smaller size of microparticles, a lower entrapment efficiency and an important retardation of enoxaparin release. The formulation prepared with Eudragit® FS 30D limited the release to a maximum of 3% in gastric simulated environment, a specific characteristic of oral systems for colonic delivery, and fulfilled our objective to delay the release.ConclusionsMicroparticles prepared with Eudragit® FS 30D represent a suitable and potential oral system for the colonic delivery of enoxaparin

Antioxidant Properties of S-Nitrosoglutathione and Nanotechnologies

International audienceCardiovascular diseases are associated with oxidative stress and a reduced bioavailability of nitric oxide (NO). To counteract both processes, the administration of S-nitrosoglutathione (GSNO) can be envisaged. GSNO is able to induce protein S-nitrosation (Pr-SNO), which is a post-translational modification of proteins, participating in the storage of NO in tissues, and protect thiol functions from oxidation. However, GSNO antioxidant power is poorly studied, which is probably linked to its low stability. This low stability can be addressed by nanotechnologies that will increase GSNO protection and provide a sustained release of the drug

Polymer nanocomposites enhance S-nitrosoglutathione intestinal absorption and promote the formation of releasable nitric oxide stores in rat aorta

International audienceAlginate/chitosan nanocomposite particles (GSNO-acNCPs), i.e. S-nitrosoglutathione (GSNO) loaded polymeric nanoparticles incorporated into an alginate and chitosan matrix, were developed to increase the effective GSNO loading capacity, a nitric oxide (NO) donor, and to sustain its release from the intestine following oral administration. Compared with free GSNO and GSNO loaded nanoparticles, GSNO-acNCPs promoted 2.7-fold GSNO permeation through a model of intestinal barrier (Caco-2 cells). After oral administration to Wistar rats, GSNO-acNCPs promoted NO storage into the aorta during at least 17 h, as highlighted by (i) a long-lasting hyporeactivity to phenylephrine (decrease in maximum vasoconstrictive effect of aortic rings) and (ii) N-acetylcysteine (a thiol which can displace NO from tissues)-induced vasodilation of aortic rings preconstricted with phenylephrine. In conclusion, GSNO-acNCPs enhance GSNO intestinal absorption and promote the formation of releasable NO stores into the rat aorta. GSNO-acNCPs are promising carriers for chronic oral application devoted to the treatment of cardiovascular diseases. In the cardiovascular system, deficiency of endogenous nitric oxide (NO) is the consequence of either insufficient synthesis (endothelium dysfunction) 1,2 or excessive NO degradation 3,4 (increased oxidative or nitrosative stresses, decreased antioxidant enzyme activity). NO depletion is one of the key factors in the initiation and progress of many diseases, such as atherosclerosis, 5 pulmonary hypertension, 6 thrombosis, 7 ischemia 8 and cardiac arrhythmia. 9 To maintain an appropriate level of NO and treat NO deficiency, several NO-related therapeutics have been developed such as nitrosamines, organic nitrates, metal–NO complexes, N-diazeniumdiolates. However, all act at very short term and lead to tolerance phenomena. S-nitrosothiols (RSNOs) present the advantage of a longer half-life, with no tolerance nor oxidative stress induction. Under physiological conditions, S-nitrosoglutathione (GSNO), a major endogenous RSNO, is one of the main storage forms of NO in tissues. 10 GSNO has been investigated for its powerful antiplatelet activity, 11,12 arterio/venous selective vasodilator effects, 13,14 antimicrobial 15 and antithrombotic effects. 16 Despite such therapeutic potencies, GSNO pharmaceutical forms are still lacking. This may be related to the fast and often unpredictable rate of decomposition of GSNO. In vitro, because of pH-, light-and temperature-dependent sensitivities , GSNO is susceptible to many degradation processe

Polymeric Nanoparticles for Increasing Oral Bioavailability of Curcumin

International audienceDespite the promising biological and antioxidant properties of curcumin, its medical applications are limited due to poor solubility in water and low bioavailability. Polymeric nanoparticles (NPs) adapted to oral delivery may overcome these drawbacks. Properties such as particle size, zeta potential, morphology and encapsulation efficiency were assessed. Then, the possibility of storing these NPs in a solid-state form obtained by freeze-drying, in vitro curcumin dissolution and cytocompatibility towards intestinal cells were evaluated. Curcumin-loaded Eudragit ® RLPO (ERL) NPs showed smaller particle diameters (245 ± 2 nm) and better redispersibility after freeze-drying than either poly(lactic-co-glycolic acid) (PLGA) or polycaprolactone (PCL) NPs. The former NPs showed lower curcumin encapsulation efficiency (62%) than either PLGA or PCL NPs (90% and 99%, respectively). Nevertheless, ERL NPs showed rapid curcumin release with 91 ± 5% released over 1 h. The three curcumin-loaded NPs proposed in this work were also compatible with intestinal cells. Overall, ERL NPs are the most promising vehicles for increasing the oral bioavailability of curcumin

Screening of process parameters for the formulation of alginate microparticles with efficient encapsulation of fragile molecules using a Plackett-Burman design.

Présentation PosterNational audiencePeptide drugs efficacy and safety are often limited by instability, short half-life, immunogenicity and their hydrophilic nature which compromises their transport across biological membranes. These limitations could be addressed by polymeric formulations produced with processes adapted to these hydrophilic and labile drugs. For example, S-nitrosoglutathione (GSNO) is a tripeptide, highly hydrophilic, and physiological form of storage of nitric oxide (NO). NO is involved in several physiological processes, particularly in the cardiovascular system. It inhibits platelet aggregation, prevents endothelial dysfunction and promotes vasodilation but NO bioavailability is decreased both by ageing and various diseases. GSNO has been shown to be safe in clinical trials and effective in various preclinical models. However, GSNO needs to be presented in an adapted galenicform for using as a prodrug for oral administration. Consequently, we have developed several polymeric particles based on alginate obtained by emulsion/gelation process that target the following specifications: efficient encapsulation of hydrophilic molecules, with a particle size control, release control. An optimization of the formulation is currently proposed. A model compound (FCF sun yellow) with similar physicochemical properties to GSNO is used during particle development, to facilitate the analytical steps. A 4-factor Plackett-Burman experimental design was carried out to optimize the formulation process using the Quality by Design development paradigm, including one critical material attribute and three process parameters. The physico-chemical characterization of particles was performed to identify the optimized process and formulation parameters

Doxorubicin-Loaded Glycyrrhetinic Acid Modified Recombinant Human Serum Albumin Nanoparticles for Targeting Liver Tumor Chemotherapy

Due to overexpression of glycyrrhetinic acid (GA) receptor in liver cancer cells, glycyrrhetinic acid modified recombinant human serum albumin (rHSA) nanoparticles for targeting liver tumor cells may result in increased therapeutic efficacy and decreased adverse effects of cancer therapy. In this study, doxorubicin (DOX) loaded and glycyrrhetinic acid modified recombinant human serum albumin nanoparticles (DOX/GA-rHSA NPs) were prepared for targeting therapy for liver cancer. GA was covalently coupled to recombinant human serum albumin nanoparticles, which could efficiently deliver DOX into liver cancer cells. The resultant GA-rHSA NPs exhibited uniform spherical shape and high stability in plasma with fixed negative charge (∼−25 mV) and a size about 170 nm. DOX was loaded into GA-rHSA NPs with a maximal encapsulation efficiency of 75.8%. Moreover, the targeted NPs (DOX/GA-rHSA NPs) showed increased cytotoxic activity in liver tumor cells compared to the nontargeted NPs (DOX/rHSA NPs, DOX loaded recombinant human serum albumin nanoparticles without GA conjugating). The targeted NPs exhibited higher cellular uptake in a GA receptor-positive liver cancer cell line than nontargeted NPs as measured by both flow cytometry and confocal laser scanning microscopy. Biodistribution experiments showed that DOX/GA-rHSA NPs exhibited a much higher level of tumor accumulation than nontargeted NPs at 1 h after injection in hepatoma-bearing Balb/c mice. Therefore, the DOX/GA-rHSA NPs could be considered as an efficient nanoplatform for targeting drug delivery system for liver cancer