**5. Conclusions**

In this study, we examined the effect of cationic lipids in FA-PEG-modified cationic liposomes on gene-silencing effects in tumor cells by siRNA lipoplexes. FA-PEG-modification of cationic liposomes increased in vitro gene-silencing activity regardless of cationic lipid type in cationic liposomes compared with PEG-modification; however, the cationic lipid type in FA-PEG-modified cationic liposomes affected the optimal amount of PEG2000-DSPE or FA-PEG2000-DSPE in the liposomal formulation. Furthermore, the in vivo anti-tumor effects by PEG-modified and FA-PEG-modified siRNA lipoplexes were not correlated with their in vitro effect, indicating that the optimized formulations of FA-PEG-modified cationic liposomes by in vitro transfection were not necessarily correlated with ones by in vivo transfection. Therefore, the in vivo optimization of FA-PEG-lipids in the liposomal formulation might be important for successful in vivo FR-mediated delivery of siRNAs by FA-PEG modified cationic liposomes.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/1999-4923/11/4/181/s1, Figure S1. Effect of charge ratio (+:−) of siRNA lipoplexes on gene suppression in KB-Luc cells after transfection with siRNA lipoplexes.

**Author Contributions:** Conceptualization: Y.H.; Formal analysis: Y.H., S.S., and K.O.; Investigation: S.S., and K.O.; Project administration: Y.H.; Supervision: Y.H.; Validation: Y.H.; Writing—original draft: Y.H.; Writing—review & editing: H.O.

**Funding:** This research received no external funding.

**Acknowledgments:** We thank Yui Asami and Haruka Honma (the Department of Drug Delivery Research, Hoshi University) for their assistance with the experimental work. We thank Hiroaki Ohno (the Graduate School of Pharmaceutical Sciences, Kyoto University) for supplying OH-C-Chol.

**Conflicts of Interest:** The authors declare no conflict of interest.
