The extent of EGFP-expressing cells in the pDNA/AFliposomes system without CyDs was found to be 8%, while that with γ-CyD and HP-γ-CyD were 19% and 10%, respectively ( Figure 5(a) ). Additionally, the enhancing effect of γ-CyD was observed in the pGL3-control vector encoding firefly luciferase and having a SV40 promoter ( Figure 5(b) ). These results suggest that the enhancing effect of γ-CyD on gene transfer activity of AF-liposomes is a geneand promoter-independent manner. To confirm whether pDNA/γ-CyD/AF-liposomes have ASGP-R-mediated gene transfer activity, we performed transfection experiments in HepG2 cells in the presence and absence of AF, as an ASGP-R competitive inhibitor. Here, we confirmed that ASGP-R are expressed in HepG2 cells by the RT-PCR method (data not shown), which is consistent with previous findings [33] [34] [35] . As shown in Figure 6 , gene transfer activity of AF-liposomes was markedly inhibited by the addition of AF, but not BSA, a control protein. These results suggest that AF-liposomes had the ASGP-R-mediated gene transfer activity. Interestingly, the similar enhancing effects of γ-CyD on Renilla luciferase protein expression after transfection of pDNA/AF-liposomes were, however, observed in A549 cells and NIH3T3 cells, ASGP-R negative cells (Figure 7) . These results suggest that γ-CyD enhances the transfection efficiency of pDNA/AFliposomes in ASGP-R-independent manner.