Thèse de doctorat en Pharmacie
Soutenue en 2006
à Paris 11 , en partenariat avec Université de Paris-Sud. Faculté de pharmacie (Châtenay-Malabry, Hauts-de-Seine) (autre partenaire) .
Cette thèse a démontré la capacité des nanocapsules à cœur aqueux à délivrer des oligonucléotides antisens et des siRNAs in vitro et in vivo. Dans un première partie de ce travail, la technique de fractionnement cellulaire a été utilisée pour étudier le trafic intracellulaire des nanocapsules chargées d'oligonucléotides (ODNs) doublement marqués. Les résultats ont montrés que ces nanocapsules sont capables non seulement d'augmenter considérablement la pénétration cellulaire des oligonucléotides, mais aussi de délivrer leur contenu dans les compartiments subcellulaires ciblés (cytoplasme, noyau). Ces résultats ont été confirmés par la microscopie confocale. De plus, les nanocapsules à cœur aqueux chargés d'ODNs anti-EWS-Fli1 se sont révélées efficace pour induire une réversion phénotypique des cellules NIH/3T3 EWS-Fli1 exprimant l'oncogène EWS-Fli1 en phénotype NIH/3T3. Cet effet est du à une inhibition spécifique et significative de l'expression de l'oncogène EWS-Fli1 détectée par la technique RT-PCR. Enfin, l'application récente de ces nanocapsules aux siRNAs, révèle pour la première fois, une inhibition significative d'une tumeur apparentée au sarcome d'Ewing, et ce en utilisant des doses très faibles en siRNAs non modifiés chimiquement. L'inhibition spécifique de l'oncogène ciblé a pu être, à nouveau, mise en évidence par la méthode RT-PCR. Des études complémentaires de microscopie confocale ont démontré la localisation cytoplasmique des siRNAs.
In vitro and in vivo fate of nucleic acids when vectorized by nanocapsules with an aqueous core
The dream of modern drug research is to discover a biologically active molecule or a class of biologically active molecules, totally specific, able to act efficiently only on the function responsible of the disease. If this dream became a reality, treatment of fatal illness (AIDS, cancer, etc. . . ) would be possible without the severe side effect and toxicities that are the main limitations of these treatments. Many drugs work by interfering with critical proteins, which have been identified as responsible for dysfunction of cells or tissues. However, conventional therapeutics agents now on the market, which tend to act on proteins in the body also often bind to non-target proteins, or exert an effect through unknown interactions. Fortunately, progress in genetics and genomics has enabled definitive studies on some of the fundamental molecular mechanisms that regulate the expression of genes, as well as the dysfunction of these mechanisms. From this mass of knowledge, the idea has emerged of drugs that may turn off genes by targeting the RNA that codes for the protein instead of the protein product. This strategy displays several advantages: (1) an active oligonucleotides (ODNs) or siRNA can be identified in a shirt time. (2) The cellular location of the proteins is not important. First results do not show any antigenicity of these nucleic acids. However, despite exciting potential for selectively modulating the expression of an individual gene, nucleic acids are still far from becoming drugs. Nucleic acids drugs are anionic macromolecules and cannot transit biological cell membranes. Additionally, they are rapidly degraded by nucleases. To overcome these limitations, various chemical modifications of nucleic acids (phosphorothioate ODNs) were synthesized. These modifications possess disadvantages, like a decreased mRNA hybridisation, higher cytotoxicity and increased unspecific effects. Therefore, a strong need for the development of unmodified acid nucleic drug delivery systems exists. The therapeutic performance of nucleic acid based drug significantly depends on the capability of carrier systems because of their fragility, impermeability to the cellular membrane and undesirable biodistribution. These delivery systems might be no viral, biocompatible and biodegradable. Carrier systems (liposomes, nanoparticles) protect these nucleic acids based drug in the harsh environment of the extracellular fluids, while inside the cells they should release incorporated drugs at a reasonable rate to remain intracellular concentration sufficient to form a complex with a target molecules such as mRNA. Actually, many academic and industrial laboratories are working on drug delivery based on nanotechnology (liposomes, nanoparticles). Our interest is based on development (unfurl) of polymeric nanocapsules with an aqueous core for nucleic acids delivery. Polyisobutylcyanoacrylate (PIBCA) nanocapsules specially designed for delivery of fragile and anionic molecules (ODNs). These nanocapsules containing in their aqueous core an active ODN induced 60% of inhibition of Ewing's sarcoma tumour after 8 I. T injections in nude mice, but the mechanism by which acts these nanocapsules remain unknown. As described in this report, our gaol was firstly focused on subcellular studies of the fate of ODNs when vectorized by nanocapsules using cellular fractionation and confocal microscopy methods (publication N°2), results showed that nanocapsules permitted enhancement of cellular penetration of oligonucleotides with a specific delivery in the targeted compartments (cytoplasm and nucleus). After fundamental mechanism investigation of delivery of oligonucleotides by nanocapsules following endocytosis way, we tested then their specific effect at cellular level in sarcoma Ewing model. Nanocapsules loaded ODNs have been proven to be highly effective tools for the specific inhibition of sarcoma Ewing's oncogene with a perfect correlation both in vitro and in vivo results in the same model (publication N°3). Recently, the discovery of RNA interference (siRNA), acting into a natural mechanism based on endogenous regulatory system that employs to silence gene expression has generated enthusiasm for cancer treatment, this new therapeutics approach more effective at low doses than other types of nucleic acid-based therapy such like oligonucleotide. But despite advantages of siRNA, obstacles to effective siRNA-based drugs remain without drug delivery systems. Finally, treatment of grafted tumor Ewing's sarcoma in nude mice using unmodified siRNA vectorized by nanocapsules leads to 80% of specific inhibition of tumor growthing after 5 I. T injection using a very low dose of siRNA (publication N°4). Nanocapsules thus appears to be an interesting system for administration of nucleic acid therapy. Further developments are now in progress in order to design such nanocapsules with specific ligands capable to recognize targets after intravenous administration.