Biol. Pharm. Bull. 30(6) 1117—1122 (2007)

ing exogenous nucleotides into mammalian cells is critical for both basic sciences and clinical applications, such as gene therapy. Among various methods for gene transfection, lipofection using cationic liposomes is considered to be a promising way to deliver a foreign gene to target cells. Although many kinds of cationic liposomes have been developed for lipofection, further studies are still required to achieve a transfection efficiency comparable with that of viral vectors. The results of a recent structural study indicated that a multilamellar or inverted hexagonal structure with alternating lipid bilayer and DNA monolayer might be favorable. Therefore, the structure of the lipid bilayers of cationic liposomes are probably conserved upon complexation with DNA. The width of the bilayer is estimated to be 36 Å. Recently, we have developed several cationic amphiphiles for nucleotide delivery. These are lithocholic acid-based molecules (see Chart 1) which bear cis-decalin structures, while the frequently used cholestane-based molecules (such as DC-Chol) have trans-decalin structures. The additional hydrophobic region at the 3-hydroxyl group of lithocholic acid derivatives may enforce hydrophobic interaction in the assembly, resulting in anchoring of the amphiphiles to the bilayer. We found that the hydrophobic appendant at the 3-position and the orientation and extension of the hydrophobic regions around the ether linkage both significantly influence the gene transfection. However, the mechanism of the dependence of the transfection efficiency on molecular structure was not elucidated. In liposome-mediated transfection, a liposome–DNA complex is taken into target cells by endocytosis. The internalized exogenous DNA is released by disruptive interaction between liposomal membrane and endosomal membrane. The released DNA in the cytoplasm is translocated into the nucleus, while DNA remaining in the endosomes is degraded in lysosomes. Therefore, the efficiency of cellular uptake of the liposome–DNA complex and the efficiency of release of DNA from both the endosomes and the liposomes are postulated to be major factors that determine the transfection efficiency, which can be estimated by means of luciferase assay. In the present study, we prepared cationic liposomes from lithocholic acid–polyamine conjugates (Chart 1), and investigated two chemico-physical characteristics that may affect the efficiency of the processes mentioned above. Firstly, we examined the encapsulation of DNA and compaction of the liposome–DNA complex, since the liposome–DNA interaction is thought to facilitate the uptake of liposome–DNA complexes by target cells and also to protect the plasmid DNA from enzymatic degradation. Secondly, we examined the ability of cationic liposomes to release DNA upon membrane fusion with anionic liposomes, which mimic the endosomal membrane. This release allows DNA to translocate to the nucleus, where it becomes accessible to the transcription apparatus. We also examined the relationship between these properties. Transfection efficiency was positively correlated with the ability of liposomes to release DNA, which is apparently dependent upon the molecular shape of the cationic amphiphiles.