JOURNAL OF COSMETIC SCIENCE 10 (AVO) (trade name Parsol 1789), which is one of the most frequently used ingredients in sunscreens products. Sunscreens have also been used along with whitening agents to main- tain light skin tone. Whitening agents protect the skin against the negative effects of UVR including skin blemishes and brown spots. Arbutin (AR), a potent tyrosinase in- hibitor and an antioxidant (1), has been widely used as a skin-whitening agent. It can ef- fectively restrain the activity of tyrosinase and the formation of melanin in the skin (2,3). Although AVO and AR are widely used as sunscreen and whitening agents, respectively, their physicochemical properties limit their effectiveness, especially when used concur- rently. AVO, for example, undergoes photodegradation under sunlight illumination and therefore loses part of its protection capacity during usage (4,5). AR, on the other hand, is very hydrophilic and therefore has limited permeability across the outermost epidermis through the hydrophobic stratum corneum into the deeper layers of the skin (6). Several strategies have been reported to address some of these limitations. The addition of photostabilizing agents, complexation with cyclodextrins, and encapsulation in poly- meric or lipid microparticles were used to enhance the stability of AVO (7,8). Studies have shown that inclusion of a carrier in sunscreen formulations may enhance photopro- tection by reducing both skin penetration and photodecomposition of UV absorbers (9). Carriers such as liposomes have also been used to facilitate the penetration of hydrophilic compounds such as AR through the skin. Liposomes are spherical, bilayered structures composed mainly of phospholipids and have been widely used in drug delivery and cos- metic applications (10). Liposomal formulations emerged as attractive alternatives for the topical delivery of drugs and cosmetic ingredients not only because of their biocompati- bility, nontoxicity, and suitability for encapsulation of both hydrophilic and lipophilic compounds but also because of their ability to enhance skin penetration. The objective of this study was, therefore, to address the physical properties of AVO and AR that limits their use in cosmetic applications by coencapsulating the two ingredients into liposomal formulations. Due to their differences in lipophilicity, two liposome preparation meth- ods, that is, thin fi lm hydration and reverse-phase evaporation, were used to coencapsu- late AVO and AR. The physical properties of liposomes prepared by each method were compared, and their in vitro permeation was evaluated using the Franz diffusion cells. MATERIAL AND METHODS MATERIALS AR was purchased from Alfa Aesar (Ward Hill, MA), AVO was purchased from Merck KGaA (Darmstadt, Germany), and L-α-phosphatidylcholine (EPC) was purchased from Avanti (Alabaster, AL). Sephadex cartridges prepacked with G-50 gel were obtained from Pharmacia Biotech (Uppsala, Sweden). Cellulose acetate membranes (pore diameter 2 μm) were purchased from Millipore (Bedford, MA). SPECTRAL ANALYSIS OF AR AND AVO The absorption spectra of AR and AVO alone or in combination were carried out in 95% ethanol or methanol. Absorption spectra were measured using Hitachi U-3300

PREPARATION OF LIPOSOMES LOADED WITH AVO AND AR 11 spectrophotometer (Hitachi, Japan). Calibration curves using 95% ethanol or methanol as the solvent were subsequently used for quantifying AR and AVO in the liposomes and the compartments of the in vitro permeation assembly, respectively. PREPARATION OF LIPOSOMES AR and AVO were encapsulated in the liposomes by the thin fi lm hydration and/or re- versed phase evaporation method. The initial step for either method is similar and in- volves the formation of layered lipid fi lm. Thin fi lm hydration method. (a) Lipo-AVO: 10–20 μmol EPC was fi rst dissolved in 100– 200 μl chloroform containing 125 μg/ml AVO. After evaporating chloroform, the mix- ture formed a thin fi lm at the bottom of the test tube. The fi lm was then hydrated in 1 ml of 0.9% NaCl by vortexing the dispersion at 37°C for 3 min. (b) Lipo-AR-AVO: 10–20 μmol EPC was fi rst dissolved in 100–200 μl chloroform containing 125 μg/ml AVO. After evaporating chloroform, the mixture formed a thin fi lm at the bottom of the test tube. The fi lm was then hydrated in 1 ml of AR stock solution in 0.9% NaCl by vortexing the dispersion at 37°C for 3 min. (c) Lipo-AR: 10–20 μmol EPC was fi rst dis- solved in 100–200 μl chloroform. After evaporating chloroform, the mixture formed a thin fi lm at the bottom of the test tube. The fi lm was then hydrated in 1 ml of AR stock solution in 0.9% NaCl by vortexing the dispersion at 37°C for 3 min. Reverse-phase evaporation method. (a) Lipo-AVO: 10–20 μmol EPC was fi rst dissolved in 100– 200 μl chloroform containing 125 μg/ml AVO. After evaporating chloroform, the mixture formed a thin fi lm at the bottom of the test tube. The dry lipid fi lm was then redissolved in 1 ml of diethyl ether to which 1 ml of 0.9% NaCl solution was added. A stable emulsion was created by vortexing the mixture for 5 min at 37°C. The liposome solution was subse- quently formed by slowly removing diethyl ether using a rotary evaporator. (b) Lipo-AR- AVO: 10–20 μmol EPC was fi rst dissolved in 100–200 μl chloroform containing 125 μg/ml AVO. After evaporating chloroform, the mixture formed a thin fi lm at the bottom of the test tube. The dry lipid fi lm was then redissolved in 1 ml of diethyl ether to which 1 ml of AR stock solution in 0.9% NaCl solution was added. A stable emulsion was created by vortex- ing the mixture for 5 min at 37°C. The liposome solution was subsequently formed by slowly removing diethyl ether using a rotary evaporator. (c) Lipo-AR: 10–20 μmol EPC was fi rst dissolved in 100–200 μl chloroform. After evaporating chloroform, the mixture formed a thin fi lm at the bottom of the test tube. The dry lipid fi lm was then redissolved in 1 ml of diethyl ether to which 1 ml of AR stock solution in 0.9% NaCl solution was added. A stable emulsion was created by vortexing the mixture for 5 min at 37°C. The liposome solution was subsequently formed by slowly removing diethyl ether using a rotary evaporator. For all liposome preparations, untrapped AR and AVO were removed by size-exclusion chromatography using Sephadex G-50 column. The fi nal liposome preparations were stored at 4°C, unless otherwise specifi ed. CHARACTERIZATION OF LIPOSOMES Vesicle size and distribution was measured by dynamic light scattering using Coulter N4 Plus submicron particle size analyzer (Beckman Coulter, Irvine, CA). The amount of AR