PERCUTANEOUS ABSORPTION OF OMC 387 In this study we incorporated OMC, as a UV absorber sunscreen, into liposomes. The aim of our work was to establish a comparison between the skin permeation of one o/w lotion vehicle versus the liposomal formulations and also to determine the effect of liposome size reduction on the skin percutaneous absorption. Meanwhile, the SPF of the formulations was determined by an in vivo method in human volunteers, to evaluate the actual efficacy of OMC formulations as a potentially more efficient sunscreen. MATERIALS AND METHODS REAGENTS AND CHEMICALS OMC, cholesterol, and vitamin E were purchased from Merck (Darmstadt, Germany). Lanolin, white petrolatum, stearic acid, propylparaben, methylparaben, disodium EDTA, propylene glycol, and triethanolamine were purchased from Sigma (USA). Soya phosphatidylcholine (Soya PC) was obtained from Avanti Polar Lipids (Alabaster, Ala­ bama, USA). Phosphate-buffered saline (PBS) ingredients were supplied from Sigma (USA). All solvents used in this study were high-performance liquid chromatography (HPLC) grade. All chemicals were of the purest grade available. PREPARATION OF OIL/WATER EMULSION CONTAINING OMC OMC (insoluble in water, 290.40 g/mol, 7 .5%), lanolin (5%), white petrolatum (2.5%), stearic acid (4%), and propylparaben (0.05%) were mixed and heated at 77°-82°C until all the ingredients were melted and dissolved (oil phase). Methylparaben (0.1 %), diso­ dium EDTA (0.05%), and propylene glycol (5%) were dissolved in PBS (pH= 7.2, 75.8%) using indirect heat (77°-82°C) (aqueous phase). The aqueous phase was added to the oil phase at 77°-82°C, and the mixture was stirred until it cooled down to room temperature (27). PREPARATION OF LIPOSOMES CONTAINING OMC Multilamellar liposomes containing OMC were prepared by the fusion method (28). Briefly, the lipid components consisted of Soya PC (15%), cholesterol (2%), vitamin E (0.3%), and propylparaben (0.05%). They were melted in propylene glycol at 77°-82°C (lipid melt). When the lipid melt cooled down to 50°C, OMC (7 .5%) was added and mixed completely. PBS (up to 100%) and methylparaben (0.1 %) were heated separately at 5 5 ° C and added to the previously heated (50°C) lipid melt and vigorously stirred until it cooled down to room temperature. PREPARATION OF SUV LIPOSOMES CONTAINING OMC SUV liposomes were prepared by probe sonication (Soniprep 150 Ultrasonic, England) of ML Vs containing OMC. The probe sonication was performed at 4°C (on ice), at 20 cycles, with 15 seconds of sonication separated by intervals of 15 seconds (29).

388 JOURNAL OF COSMETIC SCIENCE MORPHOLOGY AND SIZE ANALYSIS OF LIPOSOMES An optical microscope (Olympus, Germany) was used for studying the morphological features of ML Vs containing OMC. The mean diameter and particle size distribution of liposomes were determined by a particle size analyzer (PSA) (Klotz, Germany). The mean size of the SUVs was determined using transmission electron microscopy (TEM) by the negative staining method. Liposomes (10 µl) were mixed with 10 µl of 1 % sodium phosphotungstate solution (pH 7.0) for 10-15 seconds. They were then pipetted onto Formvar-coated copper grids. After the excess stain was removed by filter paper, the samples were viewed and photographed with a LEO 912 AB transmission electron microscope at an accelerating voltage of 80 KV (29). DETERMINATION OF OMC BY HPLC A Hitachi 1-7100 liquid chromatograph equipped with a 20-µl loop injector and a Hitachi 1-7420 UV-VIS detector (Hitachi, Tokyo, Japan) was used to carry out OMC determination. The column was a LiChrospher 100 RP-18 (12.5 cm x 4 mm, 5-µm particle size) (Merck, Darmstadt, Germany). OMC was determined at 313 nm. The mobile phase was H 2 O/HAc/EtOH, 29.5:0.5:70 (v/v/v), filtered through a 0.45-µm membrane filter (Durape, Millipore, USA), with a flow rate of 0.5 ml/min (30). The calibration curve was prepared with methanol solutions of OMC at concentrations ranging from 2 to 18 µg/ml (n = 6). The standard curves were linear (r2 = 0.999). The intra- and interday variation for OMC was performed and there were no significant differences among the day-to-day analyses. The validation results were established for three injections per concentration, using six different concentrations. The unknown concentrations were determined by using the standard curve as a reference (15 ). DETERMINATION OF ENCAPSULATION EFFICIENCY OF OMC IN LIPOSOMES In order to determine the encapsulation efficiency, liposomes were separated from un­ encapsulated OMC using ultracentrifugation (Beckman Optima 190K, USA) at 100,000.0 x g for 1 hour at 4°C and subsequently washed three times with PBS. The supernatant and precipitate were analyzed by HPLC to determine the encapsulation percentage. The entrapment efficiency of liposomes was calculated by the following equation: [(T-C)/11 x 100, where T is the total amount of drug that is detected both in the supernatant and sediment, and C is the amount of drug detected only in the supernatant (29,31-33). Since the encapsulation efficiency of OMC in ML V and SUV liposomes was high (around 90%) we used the liposomes containing OMC for the human studies without separation of unencapsulated OMC. SPF DETERMINATION OF THE HOMOSALATE REFERENCE, COLIPA STANDARD, O/W EMULSION, AND LIPOSOMES CONTAINING OMC BY IN VIVO METHOD In this study, ten subjects, both female and male, whose skin types were classified as types I, II or III, were tested for each sample. Informed consent was obtained from each subject. The backs of these subjects were used for UV exposure. The radiation was