]. Cosmet. Sci.) 59, 139-150 (March/April 2008) Enhanced depigmenting effects of N-glycosylation inhibitors delivered by pH-sensitive liposomes into HM3KO melanoma cells JU YOUNG PARK, HYUNJUNG CHOI, JAE SUNG HWANG, JUNOH KIM, and IH-SEOP CHANG, Skin Research Institute) Amore Pacific Corp. R&D Center, 314-1, Bora-dong, Giheung-gu, Yongin-si, Gyeonggi-do, 446-729, Korea. Accepted for publication June 13, 2007. Presented in part at the 23rd Congress of the International Federation of Societies of Cosmetic Chemists (IFSCC), Orlando, Florida, October 2004, and in Proceedings of the IFSCC as "Enhanced Pigment Lightening Effects of N-Glycosylation Inhibitors by the CHEMS Incorporated Nano-Carrier." Synopsis Delivery activity of pH-sensitive 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE):cholesteryl hemi- succinate (CHEMS) liposomes was assessed as an in vitro intracellular carrier system to increase the bio- availability of depigmentation actives. N-glycosylation inhibitors have a glycosylation-inhibiting effect, which is useful for the skin depigmentation that operates by interfering with the maturation of tyrosinase. However, an N-glycosylation inhibitor does not easily pass through skin or even cellular membranes due to its water-soluble property. Therefore, it should be transported to target cells by an efficient delivery carrier to reduce the glycosylated tyrosinase. Glycosylation-inhibiting and depigmentation effects of N- butyldeoxynojirimycine (NB-DNJ) and 1-deoxynojirimycine (DNJ)-loaded liposomes were evaluated using Western blotting and measurement of synthesized melanin. Interestingly, it was found that the pH-sensitive liposomes increased the glycosylation-inhibiting and thus, pigment-lightening effects of N-glycosylation inhibitors in vitro. In addition, cargo materials loaded in pH-sensitive liposomes were found to be much more efficiently delivered into the cytoplasm, as observed in fluorescent-activated cell sorting (FACS) and confocal laser-scanning microscopic (CLSM) analysis. These results indicate that pH-sensitive DOPE: CHEMS liposomes have a strong potential as a carrier system to promote delivery efficiency and to enhance the biological effects of water-soluble actives for applications in cosmetics, personal care products, and pharmaceutics. INTRODUCTION Classic liposomes reaching into a cytoplasmic target site are generally first recognized, taken up by endocytosis, and eventually delivered to lysosomes. Most of these liposomes Address all correspondence to Ju Young Park. 139

140 JOURNAL OF COSMETIC SCIENCE and their cargo materials may be degraded by various hydrolases and peptidases in the lysosomes. The pH-sensitive liposomes have been designed to circumvent this lysosomal degradation by releasing their cargo contents prior to reaching the lysosomes or partly into the cytosol, where they can then diffuse to target sites (1,2). An amphiphilic stabilizer such as CHEMS incorporated in phosphatidylethanolamine (PE)-based lipo- somes is protonated, and their conformation is changed by acidic environments when they are delivered into endosomes (3,4). N-glycosylation inhibitors such as DNJ and NB-DNJ have an cx-glucosidase-inhibiting effect, which is a useful property for enhanc- ing pigment lightening on mammalian skin by interfering with the maturation of tyrosinase (5,6). However, it is difficult for these inhibitors to translocate through skin and even cellular membranes due to their hydrophilicity (7 ,8). Therefore, DNJ should be delivered across the skin and into the cytoplasmic active site by an efficient delivery carrier to facilitate biological activity for pigment-lightening effects with a minimum concentration in vitro and in vivo. In this study, we attempt to evaluate the N- glycosylation-inhibiting (GI) effects of the pH-sensitive liposomes containing N- glycosylation inhibitors on human melanoma cells, HM3KO, to see the possibility of cosmetic application as a delivery carrier of depigmentation active molecules. Further- more, the in vitro delivery efficiency of pH-sensitive liposomes was examined to confirm the location of the intracellular-delivered pH-sensitive liposomes. EXPERIMENTAL PREP ARA TI ON OF LIPOSOMES Liposomes were prepared according to lipid hydration methods. Molar ratios of lipid components of CHEMS were fixed at 3:2. Compositions of the prepared liposomes are listed in Table I. Briefly, a mixture of lipids in chloroform/methanol (95:5) was dried using a rotary evaporator under reduced pressure. Dried lipids were hydrated with PBS containing N-glycosylation inhibitors to be loaded. Hydrated lipid films were sonicated using a bath-type sonicator. The size distribution of the resulting liposomes was mea- sured by dynamic light scattering (DLS) with a vertically polarized He-Ne laser (Zeta- sizer 3000HS, Malvern, UK). Turbidity was observed by the absorbence of a liposomal Number Table I Formulations of Prepared Liposomes Lipid composition aDOPE:6CHEMS CPC:CHEMS DOPE:dfluorescein-DHPE:CHEMS PC:fluorescein-DHPE:CHEMS DOPE:fluorescein-DHPE:PEG-5 rapeseed sterol DOPE:cholesterol a 1, 2-Dioleoyl-sn-glycero-3-phosphoethanolamine. 6 Cholesteryl hemisuccinate. c L-a-Phosphatidylcholine. d N-(fl uorescein-5-thiocarbamoyl)-1,2-dihexadecanoy1-sn-glycero-3-phosphoethanolamine.