DELIVERY ACTIVITY OF pH-SENSITIVE LIPOSOMES 141 suspension at 500 nm using a Cary 3E UV-VIS spectrophotometer (Varian, Victoria, Australia) to evaluate the pH-sensitivity of the prepared liposomes (15). To quantify intracellular delivery of pH-sensitive liposomes by FACS measurement, either a component of the lipid bilayer or a loading material was marked with fluores- cence. To label the lipid bilayer, DOPE, CHEMS and 1 % N-(fluorescein-5-thiocar- bamoyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (fluorescein-DHPE) (w/w) (Ex/Em = 495/519 nm Molecular Probes Inc., OR) was dissolved in chloroform/ methanol, and then dried and hydrated (L3 liposome). Fluorescein-encapsulated pH- sensitive liposomes were made by hydration of DOPE:CHEMS (3:2) film with 75 µM calcein (Ex/Em = 494/517) in PBS. 1-cx-Phosphatidylcholine (PC):CHEMS (3:2) was used as a control. After hydration, the procedure of liposome preparation was as previ- ously described. For the CLSM study, a dried film of DOPE:CHEMS (3:2) containing 1 % fluorescein- DHPE (Ex/Em= 495/519 nm) (L3 liposome) was hydrated with PBS containing 2.5 µM dextran-rhodamine B (10,000 mw (Ex/Em = 572/589 nm Molecular Probes Inc., OR). Therefore, the lipid bilayer and loading materials were labeled simultaneously to trace the cellular uptake of the pH-sensitive liposomes. PEG-5 rapeseed sterol (Cognis GmbH, Diisseldorf, Germany) was used instead of CHEMS as an amphiphilic stabilizer for a control L5 liposome. Unencapsulated fluorescence was separated from the liposomes by using MicroSpin™ G-25 columns (Amersham Biosciences Corp., NJ). CELL CULTURE HM3KO, a pigmented melanoma cell line (9), was cultured with minimum essential medium (MEM) supplemented with 1 % (v/v) antibiotics (streptomycin, 10,000 µg/ml penicillin, 10,000 IU/ml) and 10% (v/v) FBS (Gibco BRL, MD) at 37°C in a humidified atmosphere containing 5% CO2 . The cells were subcultured every five days. For the cytotoxicity assay, the suspension of the cells was poured into a 96-well flat-bottomed plate. After adhesion to the plate, cells were incubated with a 2.8-360 µM lipid concentration of liposomes in 100 µl of culture media for an additional 48 hr, and the cytotoxicity was then evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo- lium bromide (MTT) assay (10). Briefly, MTT was dissolved in phosphate buffer at 5 mg/ml and filtered for sterilization. Ten microliters of the MTT solution was added to the medium, resulting in a final concentration at 500 µg/ml, and then the cells were incubated for 4 h at 3 7°C. The purple formazan product was dissolved by 100 µ1/well of 40 mN HCl in isopropyl alcohol. Absorbence was measured at 550 nm in a microplate reader (Elx800, Bio-TEK Instr. Inc.). PIGMENT LIGHTENING Pre-cultured HM3KO cells were incubated with culture media containing 1 to 200 µM of NB-DNJ or DNJ-loaded DOPE:CHEMS liposomes (L1 ), for an additional five days. Concentration of the added liposomes was determined by the MIT assay evaluating the viability of the cells. The medium containing liposomes was renewed every two days.

142 JOURNAL OF COSMETIC SCIENCE After incubation with the liposomes for five days, the cells were washed with PBS and harvested with trypsin/EDTA. The collected cells in 0.1 M ofTris-HCl (pH 7.2) buffer containing 1 % Nonidet P-40, 0.01 % SDS, and protease inhibitors (Complete™ protease inhibitor mixture Roche, Mannheim, Germany) were lysated by probe-type sonication. Synthesized melanin was separated from cell lysates by centrifugation. The quantity of melanin was measured by the absorbance at 490 nm. The amount of melanin obtained from the non-treated HM3KO cells was assumed as 100% melanin synthesis as a control. Thus, melanin synthesis of cells treated with liposomes or free N-glycosylation inhibitors was presented as a relative % to the control. Endoglycosidase H (EndoH) and peptide-N-glycosidase F (PNGaseF) digestion and Western blotting were followed as previously described (11). Proteins of the cell lysates (10 µg of protein in 5 µl of cell lysis buffer) were hydrolyzed with EndoH according to the manufacturer's instructions (New England Biolabs, MA). Carbohydrate cleavage of the proteins by Endo H was stopped with the sample buffer at 70°C for ten minutes, and the proteins were subjected to Western blotting. The con- centration of hydrolyzed proteins was measured using a protein assay kit (Pierce Bio- technology, Inc., Rockford, IL) in comparison with bovine serum albumin as a standard. Hydrolyzed proteins were separated on a 10% polyacrylamide gel by electrophoresis. Following the transblotting of polyacrylamide gels onto nitrocellulose membranes, the membranes were incubated with PEP7, a polyclonal antibody (1: 1000 dilution) specific for humans, followed by incubation with horseradish peroxidase-conjugated anti-rabbit IgG (Amersham, Bucks., U .K. 1: 1000 dilution). The immunoreactive bands were detected by Chemiluminescence, using ECL Western blotting detection reagents (Am- ersham Biosciences, Piscataway, NJ). INTRACELLULAR DELIVERY The cellular delivery of pH-sensitive liposomes was quantified by FACS measurements. Cells were incubated with pH-sensitive liposomes containing 1 % fluorescein-DHPE (L3 ) for examination of the cellular-delivered amount of carrier components. PC:CHEMS containing 1 % fluorescein-DHPE liposomes (L4), pH-insensitive liposomes, was used as a control. Furthermore, calcein was encapsulated in DOPE:CHEMS liposomes (L1) to estimate the delivery efficiency of biologically active molecules loaded in pH-sensitive liposomes. Fluorescence intensity was compared with results from cells incubated with calcein-loaded PC:CHEMS liposomes (L 2 ). After one hour of incubation, the cells were washed with PBS three times, detached by treatment with trypsin/EDTA, and centri- fuged at 1000 rpm for five minutes. The resulting cell pellet was resuspended in PBS and analyzed by FACS Vantage (Beckton Dickinson, NJ). Intracellular delivery of the fluorescence-labeled pH-sensitive liposomes was monitored by a confocal laser-scanning microscope (CLSM). HM3KO cells grown in a Lab-Tek II chamber slide (Nunc Inc, IL) were incubated with dextran-rhodamine-B-loaded DOPE: CHEMS (L3 ) or DOPE:PEG-5 rapeseed sterol (L 5 ) liposomes containing 1 % fluorescein- DHPE for one hour. After incubation with the liposomes, the cells were washed with PBS three times and incubated with 2 µg/ml of DAPI (Ex/Em = 358/461) in PBS for two minutes to stain the nucleic acids. Then, the cells were washed with PBS. For fixation, pre-cooled methanol at -20°C was added to the cells for five minutes. After