Ragelle, Héloïse
[UCL]
The mechanism of RNA silencing has first been evidenced in plants. Then, studies on the nematode Caenorhabditis elegans identified a similar phenomenon, called RNA interference (RNAi). RNAi was described as a naturally occurring mechanism that induced sequence-specific post-transcriptional gene silencing and that was mediated by long double stranded RNAs. Few years later, the administration of exogenous small interfering RNA (siRNA) of 21-22 nucleotides in mammalian cells successfully induced the silencing of endogenous and heterologous genes. This discovery has created new perspectives for the development of gene medicines, especially for the treatment of diseases that are characterized by an overexpression of specific proteins. The RNAi technology promised the rapid development of highly specific therapeutics that could theoretically silence any gene, thus overcoming the issue of undruggable targets. However, after the initial enthusiasm, it clearly appeared that the delivery of small RNAs molecules required precisely engineered nanosystems. Although recent progresses have been made in the understanding of the siRNA delivery modalities, as well as in the design of siRNA delivery systems, the translational development of RNAi-based drugs remains challenging and researchers are still looking for powerful delivery approaches. This work investigated the biopolymer chitosan as the basis material for the elaboration of a siRNA delivery system for tumor targeting. Chitosan exhibits attractive properties, such as its biocompatibility and biodegradability, its cationic nature and its chemical versatility that allows the grafting of targeting ligand. These characteristics make chitosan an ideal basis for nanoparticles (NPs) construction. The research was conducted in three steps. First, the formulation parameters for the design of optimal siRNA NPs using chitosan were established. The principle requirements for ideal NPs were suitable physico-chemical characteristics, especially size and polydispersity, high gene silencing activity without cytotoxicity and stability in plasma. This first part provided the basis for the further design of ligand-grafted NPs. The second part focused on the in vitro evaluation of the targeted nanoparticles and, more precisely, on the investigation of their intracellular delivery mechanisms using a new method of stem-loop RT-qPCR. This phase of the project provided a proof of principle of the cancer-cell targeting ability of our delivery system. Finally, different ligand structures, grafting strategies (clip versus chain-end coupling) and linker properties (hydrophilic versus lipophilic) were investigated to determine whether these parameters influence NP efficacy. Moreover, with the prospect of future in vivo testing, the NPs were evaluated in vitro for their ability to silence the therapeutic targets implicated in cancer metabolism.
Bibliographic reference |
Ragelle, Héloïse. Targeted chitosan-based delivery systems of siRNA. Prom. : Vandermeulen, Gaëlle ; Preat, Véronique |
Permanent URL |
http://hdl.handle.net/2078.1/135379 |