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Cholesterol-based cationic lipids have been widely used because of biocompatibility and serum resistance. However, the reason for the effectiveness of cholesterol-based cationic lipids remains unclear. We compared the transfection route of CHOL-E, a cholesterol-based cationic lipid having an amine head and an ether linker, with that of DOTAP. The luciferase assay with chemical inhibitors and microscopic observation of pathway markers revealed that clathrin mediated endocytosis is the main pathway for CHOL-E and DOTAP. However, CHOL-E showed resistance to cholesterol depletion by methyl-β-cyclodextrin. Furthermore, CHOL-E recovered the transfection efficiency of DOTAP from cholesterol depletion. These results suggested that superior transfection of CHOL-E might be partly derived from effects on the cell membrane.

Five cholesterol-based cationic lipids were newly synthesized based on cholest-5-en-3β-oxyethane-N,N,N-trimethylammonium bromide (Chol-ETA) structure where the cholesterol backbone is linked to cationic head via various lengths of ether-linked carbon spacer. The transfection efficiency of these compounds was increased in order of three (Chol-PRO)

The new cholesterol-based cationic lipids B, C, and D with an ether linked spacer were synthesized by using aminopropyl chain extension with acrylonitrile. The cholesterol-based cationic lipid A with carbamoyl linkage were also synthesized in order to compare the difference in transfection efficiency of the two linkage types. To this end, GFP expression of these cationic lipids was confirmed respectively.