Ribbon-type and cluster-type lipoplexes constituted by a chiral lysine based cationic gemini lipid and plasmid DNA

Abstract

Lipoplexes constituted by plasmid DNA pEGFP-C3 (pDNA) or linear double-stranded calf thymus DNA (ctDNA) and mixed cationic liposomes consisting of several percentages of the cationic lysine derived lipid C₆(LL)₂ and the zwitterionic lipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) have been analyzed by both experimental and theoretical approaches. Experimental studies, consisting of electrophoretic mobility/zeta potential, small angle X-ray scattering (SAXS), cryogenic transmission electron microscopy (cryo-TEM), negatively stained transmission electron microscopy (NS-TEM), and GelRed f1uorescence intercalation assays, have been carried out at several liposome and lipoplex compositions, defined in terms of cationic lipid molar fraction and either the mass or charge ratios of the lipoplex. The electrochemical study confirms that, in the presence of the mixed lipids and in contrast with what has usually been found for linear DNA, the plasmid DNA is compacted with a large number of its Na⁺ counterions, thus yielding a much lower effective negative charge (q⁻_pDNA) than that for ctDNA (q⁻_ctDNA), as reported recently by us (J. Am. Chem. Soc., 2011) for other lipoplexes. This finding is revealed as crucial for an optimum and efficient lipoplex preparation, since a lower effective negative charge implies a lower quantity of cationic lipid and, accordingly, a potential lower cytotoxicity. TEM experiments reveal a complex scenario of multilamellar nanostructures, from ribbon-type (typically present for chiral lipids) to cluster-type structures (usually found in cationic lipid/DOPE systems), the composition of the mixed liposome playing an important role in the final morphology of the lipoplex. SAXS diffractograms confirm the existence of these two types of multilamellar structures through a deconvolution process of the first peak of diffractograms into two overlapping bands. On the other hand, a theoretical complexation model is employed to determine the net charge of the lipoplexes studied in this work. The model allows analysis and comparison of the electrochemical behaviour of lipoplexes containing linear DNA vs. those constituted by a supercoiled DNA, confirming the experimental findings.