Cationic Liposomes Carrying siRNA: Impact of Lipid Composition on Physicochemical Properties, Cytotoxicity and Endosomal Escape

Abstract

In recent year, cationic liposomes have gained a lot of attention for siRNA delivery. Despite this, intracellular barriers as endosomal escape and cytosolic delivery of siRNA still represent a challeng, as well as the cytotoxicity due to cationic lipids. To address these issues, we developed four liposomal formulations, composed of two different cationic lipids (DOTAP and DC-Cholesterol) and different ratio of co-lipids (cholesterol and DOPE). The objective is to dissect these impacts on siRNA efficacy and cytotoxicity. Liposomes were complexed to siRNA at six different N/P molar ratios, physico-chemical properties were characterized, and consequently, N/P 2.5, 5 and 10 were selected for in vitro experiments. We have shown that cytotoxicity is influenced by the N/P ratio, the concentration of cationic lipid, as well as the nature of the cationic lipid. For instance, cell viability decreased by 70% with liposomes composed of DOTAP/Cholesterol/DOPE 1/0.75/0.5 at a N/P ratio 10, whereas the same formulation at a N/P ratio of 2.5 was safe. Interestingly, we have observed differences in terms of mRNA knock-down efficiency, whereas the transfection rate was quite similar for each formulation. Liposomes containing 50% of DOPE induced a mRNA silencing of around 80%. This study allowed us to highlight crucial parameters in order to develop lipoplexes which are safe, and which induce an efficient intracytoplasmic release of siRNA.

Systematic Screening of Commonly Used Commercial Transfection Reagents towards Efficient Transfection of Single-Stranded Oligonucleotides

“…Because oligonucleotides are able to continuously shuttle between the nucleus and the cytoplasm through passive diffusion and active transport [ 9 ], once SSOs are internalised, escaping from endosomes becomes a rate limiting step. To this day, although the detailed mechanism of endosome escape is still unclear [ 3 , 13 ], it is conceivable to assume that for the same cell type, the amount of SSOs functionally react with their cytoplasm/nucleus targets should closely correlate with the amount of internalised SSO molecules. Therefore, conjugating SSO with fluorescent dyes and measuring the relative fluorescence intensity provides a simple way to monitor the efficacy of transfection reagents studied.…”

Lipid Nanoparticle-Delivered Chemically Modified mRNA Restores Chloride Secretion in Cystic Fibrosis

“…[32][33][34][35][36][37][38][39] LNPs contain an ionizable lipid that facilitates cmRNA packaging through complexation and aids in triggering endosomal escape through membrane destabilization. 33,[40][41][42][43][44][45][46][47] Formulations also consist of a phospholipid and cholesterol to maintain structural integrity and an outer lipid that is decorated with polyethylene glycol (PEG). 40,41,48,49 This coating cloaks the LNP from the host immune response, imparts serum stability, and extends in vivo circulation time.…”

An artificial cationic oligosaccharide combined with phosphorothioate linkages strongly improves siRNA stability

“…In general, higher N/P ratios of these polycation complexes enable more effective stabilisation of the nucleotides caused by stronger interaction between the carriers and the nucleotides 54 . However, cell viability is gradually decreased with the increase of N/P ratios 55 – 57 , due to nonspecific binding of the polycations to other biomolecules. In addition, polycation complexes with excessively high N/P ratios show low siRNA efficiency for gene-silencing by interrupting siRNA release from the complexes 58 .…”