DELIVERY ACTIVITY OF pH-SENSITIVE LIPOSOMES 149 with previous research findings, indicating that pH-sensitive EYPC liposomes modified with SucPG increased the delivery of calcein into the cytoplasm (14). Therefore, it can be demonstrated that after cellular uptake through an endocytic path- way, the DOPE:CHEMS pH-sensitive liposomes transfer their contents into the cyto- plasm. These results indicate that the improved efficacy of the N-glycosylation inhibi- tors loaded in pH-sensitive DOPE:CHEMS liposomes was induced because of the in- creased translocation efficiency into the cytoplasm. CONCLUSIONS We prepared pH-sensitive liposomes consisting of DOPE and CHEMS with diameters in the range of 150-300 nm to deliver water-soluble N-glycosylation inhibitors into HM3KO melanoma cells. The glycosylation-inhibiting (GI) and pigment-lightening effects of NB-DNJ or DNJ were significantly increased in vitro by the pH-sensitive liposomes. It was also found that the delivery efficiency of pH-sensitive DOPE:CHEMS liposomes was higher than that of control pH-insensitive liposomes determined by evaluation using FACS and CLSM study. These results demonstrate that the increased effects of N-glycosylation inhibitors in vitro can be attributed to facilitated delivery by the pH-sensitive DOPE:CHEMS liposomes into the target cytoplasmic site. Conse- quently, these results indicate that CHEMS-incorporated pH-sensitive liposomes may have a high delivery efficiency and thus can be used as an intracellular delivery system to transport biological actives to a target site in cytoplasm. DOPE:CHEMS liposomes show promise as a nano-sized and pH-stimulant delivery carrier to enhance the bio- availability of water-soluble compounds. This pH-sensitive liposome carrier may be directly applied as a powerful delivery system for therapeutic formulations to treat skin disorders and for applications in cosmetics, personal care products, and pharmaceutics. ACKNOWLEDGMENTS We acknowledge Dr. Y. Funasaka (School of Medicine, Kobe University, Kobe, Japan) for providing HM3KO cells and Dr. Vincent J. Hearing (NIH, Bethesda, MD) for supplying PEP7, the anti-human tyrosinase antibody. We thank So Hee Kwon (NICEM, Seoul, Korea) for the CLSM analysis. REFERENCES (1) S. Simoes, V. Slepushkin, N. Diizgiines, and M. C. Pedroso de Lima, On the mechanisms of inter- nalization and intracellular delivery mediated by pH-sensitive liposomes, BBA, 1515, 23-37 (2001). (2) F. V. Bambeke, A. Kerkhois, A. Schanck, C. Remache, E. Sonveaux, P. M. Tulkens, and M.-P. Mingeot-Leclercq, Biophysical studies and intracellular destabilization of pH-sensitive liposomes, Lipids, 37, 213-223 (2000). (3) M.-Z. Lai, W. J. Vail, and F. C. Szoka, Acid- and calcium-induced structural changes in phosphati- dylethanolamine membranes stabilized by cholesteryl hemisuccinate, Biochemistry, 24, 1654-1661 (1985). (4) C. J. Chu, J. Dijkstra, M. Z. Lai, K. Hong, and F. C. Szoka Jr., Efficiency of cytoplasmic delivery by pH-sensitive liposomes to cells in culture, Pharm. Res., 7, 824-834 (1990).

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