Inclusion of DOPE in lipoplex formulations has hampered the establishment of a correlation between cationic lipid structure, biological specificity, and transfection activity, simply because the presence of a helper lipid not only alters the physicochemical properties of the lipoplex but also modifies cell surface specific interactions during the process of transfection. To this end, four cationic cholesterol-based derivatives were synthesized by systematically varying the methylation of the polar headgroup, after which the physicochemical properties, in the absence of DOPE and serum, were correlated with their transfection activity and interaction with cell membranes. It was found that only the primary and secondary amine derivatives, AC-Chol and MC-Chol, respectively, are able to mediate in vitro cell transfection. These results were consistent with fusion experiments and cell internalization studies which illustrated that although cell surface binding occurs for all of the cationic lipids, only the active analogues were able to gain entry into the cytosol. Given the minute differences in the physical properties of these cationic derivatives, we speculate that the biological specificity of the active cationic derivatives either triggers endocytotic pathways leading to eventual endosomal fusion allowing cytoplasmic access to the packaged DNA or other endocytotic pathways that avoid lysosomal degradation.

In this paper, a novel series of bis [(aminoethyl)]-amine cationic lipid derivatives have been synthesized and identified to purity by NMR and Elemental analysis. B16-F0 cells were transfected with cationic lipid/pEGFP-N1 and cationic lipid/β-gal lipoplexes complexed at +/− charge ratios of 1:1, 2:1, and 4:1. Dimyristoyl derivative showed highest activity at charge ratio 2:1 and both dimyristoyl and dioleoyl derivatives showed similar β-gal activity at charge ratios 4:1. In 40 mM tris buffer pH 7.2 the dioleoyl derivative was able to fully complex with and retard pDNA at charge ratios above 2:1. None of the other lipid derivatives, dilauroyl, dimyristoyl, dipalmitoyl and distearoyl were able to fully neutralize the plasmid DNA at charge ratios similar to those used in the transfection experiment. The gel-to-liquid phase transition temperatures for dimyristoyl, dipalmitoyl and distearoyl were determined by a fluorescence anisotropy method to be 27.5˚C, 32.5˚C and 39˚C, respectively. A gel-to-liquid crystalline phase transition temperature below 37˚C, appears to be the crucial property that cationic lipids have to possess in order to mediate high levels of in vitro transfection activity in the absence of other helper lipids.