Ca~(2+)-Mediated Anionic Lipid-Plasmid DNA Lipoplexes. Electrochemical, Structural, and Biochemical Studies

摘要

Several experimental methods, such as zeta potential, gel electrophoresis, small-angle X-ray scattering, gene transfection, fluorescence microscopy, flow cytometry, and cell viability/cytotoxicity assays, have been used to analyze the potential of anionic lipids (AL) as effective nontoxic and nonviral DNA vectors, assisted by divalent cations. The lipoplexes studied are those comprised of the green fluorescent protein-encoding plasmid DNA pEGFP-C3, an anionic lipid as 1,2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DOPG) or 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), and a zwitterionic lipid, the 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE, not charged at physiological pH). The studies have been carried on at different liposome and lipoplex compositions and in the presence of a variety of [Ca~(2+)]. Electrochemical experiments reveal that DOPG/DOPE and DOPS/DOPE anionic liposomes may compact more effectively pDNA at low molar fractions (with an excess of DOPE) and at AL/pDNA ratios ≈20. Calcium concentrations around 15-20 mM are needed to yield lipoplexes neutral or slightly positive. From a structural standpoint, DOPG/DOPE-Ca~(2+)-pDNA lipoplexes are self-assembled into a HII c phase (inverted cylindrical micelles in hexagonal ordering with plasmid supercoils inside the cylinders), while DOPS/DOPE-Ca~(2+)-pDNA lipoplexes show two phases in coexistence: one classical H_(II)~c phase which contains pDNA supercoils and one Lα phase without pDNA among the lamellae, i.e., a lamellar stack of lipidic bilayers held together by Ca~(2+) bridges. Transfection and cell viability studies were done with HEK293T and HeLa cells in the presence of serum. Lipoplexes herein studied show moderate-to-low transfection levels combined with moderate-to-high cell viability, comparable to those yield by Lipofectamine2000*, which is a cationic lipid (CL) standard formulation, but none of them improve the output of typical CL gen vectors, mostly if they are gemini or dendritic. This fact would be indicating that, nowadays, lipofection via anionic lipids and divalent cations as mediators still needs to enhance transfection levels in order to be considered as a real and plausible alternative to lipofection through improved CLs-based lipoplexes.