Physicochemical properties of cationic nanoemulsions and liposomes obtained by microfluidization complexed with a single plasmid or along with an oligonucleotide: Implications for CRISPR/Cas technology.

In this study, we investigated the effects of the association of a single plasmid or its co-complexation along with an oligonucleotide on the physicochemical properties of cationic nanoemulsions and liposomes intended for gene editing. Formulations composed of DOPE, DOTAP, DSPE-PEG (liposomes), MCT (nanoemulsions), and water were obtained by microfluidization. DSPE-PEG was found to play a crucial role on the size and polydispersity index of nanocarriers. Nucleic acids were complexated by adsorption at different charge ratios. No significant differences were noticed in the physicochemical properties of nanocarriers (i.e. droplet size, polydispersity index, or zeta potential) when a single plasmid or both plasmid and oligonucleotide were adsorbed to the formulations. Transmission electron microscopy photomicrographs suggested round nanostructures with the nucleic acids and DSPE-PEG enfolding the surface. Complexes at +4/-1 charge ratio protected nucleic acids against DNase I degradation. The oligonucleotide seemed to be released from the liposomal complexes, while nanoemulsions only released the plasmid after 24 and 48 h of incubation in DMEM supplemented or not. In vitro experiments demonstrated that complexes were highly tolerable to human fibroblasts, Hep-G2, and HEK-293 cells, demonstrating also an uptake ability of about 30%, 30%, and 90%, respectively, no matter what the formulation or the combination of nucleic acids used. Transfection efficiency of the formulations was around 25% in human fibroblasts, 32% in HEK-293, and 15% in Hep-G2 cells. The overall results demonstrated the behavior of liposomes and nanoemulsions complexed with a plasmid or a mixture of a plasmid and an oligonucleotide, and demonstrated that the association with one or two nucleic acids sequences of different length does not seem to interfere in the physicochemical characteristics of complexes or in the uptake capacity by three different types of cells.