Finally, we investigated the role of free γ-CyD on transfection efficiency of pDNA/γ-CyD/AF-liposomes in HepG2 cells. The physical mixture of pDNA/AF-liposomes and γ-CyD in culture medium had no enhancing effect on transfection efficiency of pDNA/AF-liposomes (data not shown). Therefore, encapsulation of γ-CyD into AF-liposomes may be pivotal for enhancing gene transfer activity. To reveal the detailed mechanism for the enhancing effect of γ-CyD associated in AF-liposomes on transfection efficiency of pDNA/AF-liposomes, further elaborate study should be necessary.