Efficient Cellular Knockdown Mediated by siRNA Nanovectors of Gemini Cationic Lipids Having Delocalizable Headgroups and Oligo-Oxyethylene Spacers.

ABSTRACT

The use of small interfering RNAs (siRNAs) to silence specific genes is one of the most promising approaches in gene therapy, but it requires efficient nanovectors for successful cellular delivery. Recently, we reported liposomal gene carriers derived from a gemini cationic lipid (GCL) of the 1,2-bis(hexadecyl dimethyl imidazolium) oligo-oxyethylene series ((C16Im)2(C2H4O)nC2H4 with n = 1, 2, or 3) and 1,2-dioleyol phosphatidylethanolamine as highly efficient cytofectins for pDNA. On the basis of the satisfactory outcomes of the previous study, the present work focuses on the utility of coliposomes of these gemini lipids with the biocompatible neutral lipid mono oleoyl glycerol (MOG) as highly potent vectors for siRNA cellular transport in the presence of serum. The (C16Im)2(C2H4O)nC2H4/MOG-siRNA lipoplexes were characterized through (i) a physicochemical study (zeta potential, cryo-transmission electron microscopy, small-angle X-ray scattering, and fluorescence anisotropy) to establish the relationship between size, structure, fluidity, and the interaction between siRNA and the GCL/MOG gene vectors and (ii) a biological analysis (flow cytometry, fluorescence microscopy, and cell viability) to report the anti-GFP siRNA transfections in HEK 293T, HeLa, and H1299 cancer cell lines. The in vitro biological analysis confirms the cellular uptake and indicates that a short spacer, a very low molar fraction of GCL in the mixed lipid, and a moderate effective charge ratio of the lipoplex yielded maximum silencing efficacy. At these experimental conditions, the siRNA used in this work is compacted by the GCL/MOG nanovectors by forming two cubic structures (Ia3d and Pm3n) that are correlated with excellent silencing activity. These liposomal nanocarriers possess high silencing activity with a negligible cytotoxicity, which strongly supports their practical use for in vivo knockdown studies.