In the present study, we revealed that transfection efficiency of the pDNA/γ-CyD/AF-liposomes, not N-liposomes, was inhibited by the addition of AF in HepG2 cells ( Figure 6 ). Meanwhile, in NIH3T3 cells, transfection efficiency of the pDNA/AF-liposomes was not suppressed by the addition of AF. These results strongly suggest that pDNA/γ-CyD/AFliposomes can be entered HepG2 cells through ASGP-Rmediated endocytosis, consistent with Aramaki and his colleague's report [41] , and the enhancing effect of the γ-CyD may by associated with the ASGP-R-mediated endocytosis. However, γ-CyD also enhanced gene transfer activity of pDNA/AF-liposomes even in A549 cells and NIH3T3 cells, ASGP-R negative cells (Figure 7) . These results suggest that the enhancing effect of γ-CyD on transfection efficiency of pDNA/AF-liposomes is in an ASGP-R-independent manner. The charge ratio of AF-liposomes/pDNA was 1.6. γ-CyDs were added to AF-liposomes suspension before freeze-drying. The concentrations of γ-CyDs were 1 μM/μM lipids. Cells were incubated with Alexa-pDNA/γ-CyDs/RH-AF-liposomes for 3 h in FCS-free medium. After washing twice, the cells were observed using a confocal laser scanning microscopy.