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

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... 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.