JOURNAL OF COSMETIC SCIENCE 40 MATERIALS AND METHODS MATERIALS The EPDME used in this study was synthesized as previously reported (1). Figure 1 shows the chemical formula of EPDME. Liposomes (1 mg/ml), prepared from lecithin, were a gift from Professor Kumazawa of Ibaraki University. METHODS Experimental dry skin and preparation of stripped SC samples. The Shiseido Research Center Internal Review Board approved all protocols used in this study. The ethical principles for non-clinical biomedical research involving human subjects, as stated in the Declaration of Helsinki Principles, were adhered to in every respect. The mid-volar forearms of ten volunteers (male, average age 38) were treated with 150 μ1 of 1% SDS (Wako Pure Chemical Industries, Osaka, Japan) and covered with adhesive tape for one hour. The application site was washed with distilled water, and then 10% EPDME aqueous solution or water was applied in appropriate amounts. Application of 10% EPDME aqueous solu- tion was carried out before the subjects’ going to bed and on rising the following morn- ing. On the following day, the sample-application area was washed with soap, and the physiological parameters of the skin were evaluated 20 min later at 22°C and 45% RH. The SC sheets for ex vivo measurement were prepared as reported (2,3). A surface biopsy was conducted with a quartz glass plate (8 mm × 70 mm Matsunami, Tokyo, Japan) on which a single drop of cyanoacrylate resin had been spread uniformly. Four SC specimens were successively removed in this way from the mid-volar arm treated with SDS and EPDME, SDS and distilled water, or without treatment (control). TEWL was measured using a Vapometer SWL3N (Delfi n, Finland) before and after strip- ping the SC. The water content of the SC was measured with a Corneometer CM825 (C+K Electric, Koln, Germany) and a Skicon 200 (ISBS Co, Hamamatsu, Japan) before and after stripping the SC. These instruments utilize measurements of capacitance and conductance, respectively, to determine water content. Preparation of SC sheets for ex vivo measurements. A single-chain aliphatic spin probe, 5-doxylstearic acid (5-DSA) was purchased from Aldrich-Sigma Chemical Co. Inc., and was used as received. A 5-DSA ethanol solution (26 mM) was prepared, and diluted 1000-fold with distilled water. Stripped SC samples were incubated with 100 μl of the 5-DSA ethanol-aqueous solution (26 μM) for 1 hour at 37°C. Excess spin probe was washed off with distilled water, and the SC samples were mounted in the EPR cavity (3). Figure 1. Chemical formula of polyoxyethylene/polyoxypropylene dimethyl ether (EPDME). EPDME is a random copolymer of ethylene oxide and propylene oxide, and the numbers 14 and 7 represent the average molecular ratio of the two monomers.
EPDME IMPROVES LAMELLAR LIPID STRUCTURE 41 Preparation of EPDME-loaded liposomes. Liposomes (0.5 mg/0.5 ml) were incubated with the 5-DSA ethanol-aqueous solution (26 μM fi nal concentration) in a microtube for 1 hour at 37°C then the EPDME solution was added (1% fi nal concentration) and the mixture was incubated for ten minutes at 37°C. SDS aqueous solution was added (0.02% fi nal concentration) and incubation was continued for 30 minutes at 37°C. Excess spin probe was washed off with distilled water by centrifugation, and disper- sions were transferred to 50 μl capillaries, which were used as sample cells for the EPR measurement. EPR measurements and spectral analysis. A JEOL JES-RE1X X-band (9 GHz) EPR spec- trometer was used to measure all samples. Before measurements of the SC from volun- teers, ex vivo specimens were treated with distilled water for fi ve minutes to have suffi cient hydration (3,4). Typical spectrometer settings were as follows: microwave power, 10 mW time constant, one second sweep time, eight minutes modulation, 0.2 mT sweep width, 15 mT. All measurements were performed at ambient tempera- ture. The obtained EPR spectra were analyzed by conventional calculation of the order parameter. The order parameter S provides a measure of the fl exibility of the spin label in the mem- brane. The fl exibility refl ects motility and orientation, and S = 1 for a highly ordered system and S = 0 for completely isotropic motion (5). The conventional S value is deter- mined from the hyperfi ne couplings of the EPR signals according to the following rela- tions (6): A A c 2A . , , . 1 3 3 ( ) 2 II II XX YY ZZ zz XX A A a A A A A S a a ac A A AYY In calculation from experimental spectra, the principal components of (AXX + AYY + AZZ) = (0.66, 0.55, 3.45) mT were used (7). Multiple comparison assays were carried out using Fisher’s protected least signifi cant dif- ference (Fisher’s PLSD) in the case of the order parameter S of SC from volunteers, and Dunnett’s test in the case of the order parameter S of liposomes. Multiple linear regres- sion analysis was carried out using JMP Ver.6 (SAS Institute Inc.). RESULTS Figure 2 shows EPR spectra of SC from the fi rst stripping from one volunteer. Water treatment resulted in an order parameter (S) value of 0.57, while SDS treatment decreased it to 0.53 and EPDME treatment restored it to 0.56. Figure 3 shows the average values of the order parameter of ten subjects. The parameter was decreased signifi cantly upon SDS treatment, but was signifi cantly restored when EPDME was applied. Figure 4 shows the changes in the mean values of the order parameter of SC obtained in successive strippings (i.e., increasing depth). In all four strippings, SDS treatment sig- nifi cantly decreased the order parameter S, showing that the structural order of the SC lipids was disrupted, while EPDME signifi cantly, although not completely, restored the order parameter.
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