Thesis presented December​ 01, 2014


Abstract:
RNA interference is a post-transcriptional inhibition mechanism, inducing the specific regulation of gene expression. This endogenous mechanism can be triggered by the transfection of exogenous interfering RNA fragments, including siRNA. This technique allows specific targeting of all the genes making up the genome, whose temporary extinction allows for the study of their function. This technique can also now be used to discover new therapeutic targets and new biomarkers. This strong research potential in vitro is also found in vivo, whereby RNA interference can be used directly as a therapeutic agent for pathological conditions such as cancer, infections or systemic diseases. However, the cytoplasmic accessibility of exogenous siRNA is required to trigger the mechanism of regulation. Currently, despite numerous transfection methods developed in the literature, this step in siRNA delivery remains an important limitation for any envisaged application.
Considering this limitation, this thesis aimed to develop a new cationic lipid-based carrier, cLNP, dedicated to cell transfection. Using a design of experiment, this formulation of cLNP was adapted from a neutral formulation encapsulating lipophilic molecules previously used for applications in fluorescence imaging and/or drug delivery. The physicochemical characterization of the cLNP particles showed strong colloidal stability, both in aqueous buffers and in cell culture media supplemented with serum. In addition, these nanovectors have proven extremely efficient in establishing and maintaining of electrostatic bonds with siRNA, thereby yielding complexes with high stability over time. Functional inhibition efficiencies of these nanoparticles have been successfully tested on three different cell lines (PC3, HeLa andU2OS). The overall results confirmed the high potential of this new nano-vector, in terms of functional inhibition and absence of cytotoxicity, and ranks among the better commercial transfection reagents tested. These features are complemented by multimodality abilities, including the encapsulation of drugs or lipophilic fluorophores. Finally, preliminary tests on difficult-to-transfect cells (primary cells, non-adherent cells, neurons) or three- dimensional cellular structures opened new promising perspectives.

Keywords:
Lipid nanocarrier, siRNA, RNAi, in vitro transfection, safety, high-throughput​ screening

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