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

PERCUTANEOUS ABSORPTION OF OMC 389 produced by a xenon arc lamp, and unwanted spectral regions were removed by filter systems. The application amount of the tested sunscreen samples and the standard formulations was 2 mg/cm2 . The sunscreen was spread over the area with a finger stall in order to achieve a uniform film. After application, the product was allowed to dry for 15 minutes before irradiation. Air was conditioned between 20°C and 25°C during the waiting period. Each test subsite in a series was exposed to controlled amounts of simulated sunlight using the solar simulator. The MEDs (minimal erythemal doses) of the unprotected and protected skin were recorded and then the SPFs of the samples were determined (34,3 5 ). DETERMINATION OF PERCUTANEOUS ABSORPTION OF OMC USING THE STRIPPING METHOD After filing an informed consent, six healthy Caucasian volunteers (three women, three men), aged 37 ± 8 years, participated in this study. For 15 minutes they were held in a stripping room where the temperature was maintained at 20°C and the relative humidity was 50-60%. Preparations were applied onto areas of 4 cm2 , on the flexor surface of the central area of the forearm. Each assay was repeated three times. The product was applied at a dose of 2 mg/cm2 with a glass spatula, weighed before and after the deposit, and was spread uniformly over the whole area. Non-treated skin surround­ ing the application site was protected with adhesive tape. Protected non-occluding guards were used over the skin sites to prevent accidental product removal. Subjects wore loose long-sleeved lab coats throughout the study to minimize light exposure to the product-treated skin sites, ensuring that the sites were protected but not occluded. The Transpore tapes™ adhesive tapes were applied to the treated areas by application of a consistent pressure generated by stroking the thumb ten times along the tape (36,3 7). Stripping was performed at 0.5, 3, 5, and 7 hours after treatment under controlled conditions over 10 seconds on the same volunteers. The stratum corneum was removed by 22 tape strips with transparent adhesive tape (Transpore tape™, 3M). Strips 1-2, 3-7, 8-12, 13-17, and 18-22 were pooled separately in different groups. These strips were called group 1 (strips 1-2), group 2 (strips 3-7), group 3 (strips 8-12), group 4 (strips 13-1 7), and group 5 (strips 18-22), which introduce the different layers of the stratum corneum. Ten milliliters of methanol was added to the strips, and each sample was shaken with a vortex shaker. The amount of OMC was then determined in these solutions by HPLC (6,7 ,13,36). STATISTICAL ANALYSIS A one-way ANOV A statistical test was used to assess the significance of the differences in the concentrations of OMC in different groups of strips. In the case of a significant F value, multiple-comparison Tukey-Kramer tests was used to compare the means of the different treatment groups. Results with p 0.05 were considered to be statistically significant. RESULTS CHARACTERIZATION OF THE LIPOSOMES The liposomes made by the fusion method were morphologically homogenous multi-