IWL LIPOSOMES AND SKIN BARRIER IMPROVEMENT 237 scan of 85% to quantify and eliminate the apolar lipids, a second development, again to a distance of 10 cm, was performed with chloroform/n-hexane/methanol/acetone (55:5:3:7, by vol) to separate the ceramides. Following a partial scan of 85% to quantify and elimi- nate the ceramides, a third development, again to a distance of 10 cm, was performed with chloroform/methanol/formic acid (57:12:0.3, by vol) to separate and quantify, after a total scan of 100%, the glycosilceramides and sterol sulfate. After each elution, the rods were heated for 5 min at 60°C to dry the remaining solvent. LIPOSOME FORMATION AND EVALUATION IWL liposomes were prepared with the three extracts by evaporating the organic solvent to dryness under a stream of dry nitrogen to form a thin fi lm on the fl ask. The fi lm was hydrated with 0.9% NaCl solution to give a fi nal lipid concentration of 2% for each ex- tract. Liposomes were formed by sonication of the suspension in a sonicator, a Labsonic 1510 (B. Braun, Melsungen, Germany), at 100W for about 15 min, with the temperature maintained at 65°C. Evaluation of vesicle size distribution and the polydispersity index of liposomes was car- ried out at 25°C by dynamic light scattering, employing an Autosizer Ilc photon correla- tion spectrometer (Malvern Instruments Limited, Malvern, UK). Samples were diluted from 20 to 0.1 mg/ml. Quartz cuvettes were fi lled with the samples, and all the experi- ments were thermostatically controlled (25°C). The samples were measured at a scatter- ing angle of 90°. Data thus obtained were analyzed using a version of the program CONTIN provided by Malvern Instruments. SUBJECTS Nine healthy Caucasian volunteers (all females), phototype (Fitzpatrick skin type) III–IV with a mean age of 29.9 ± 5.0 years (range 24–39 years), participated in all studies. All the subjects were advised to avoid the use of topical drugs or moisturizers on the volar forearm for one week prior to the experiments. To obtain reliable measurements, the volunteers were acclimatized for 15 min in a conditioned room (20°C, 60% RH) before the experiments. NON-INVASIVE BIOPHYSICAL MEASUREMENTS The effect of topically applied liposomes on skin properties was evaluated by non-invasive biophysical techniques. TEWL is a sensitive index of skin barrier integrity. This parameter evaluates the water loss in g/m2h, measured using the TewameterTM210 (Courage & Khazaka, Cologne, Germany). Moreover, skin hydration was determined using a Corneometer CM 85 (Courage & Khazaka), which measures skin capacitance in arbitrary units (AU). Both parameters were recorded in accordance with established guidelines (18–21). EFFICACY OF LIPOSOMES ON INTACT HUMAN SKIN A long-term study was performed to test the effect of the liposome solutions when ap- plied repeatedly to intact skin. Baseline measurements of TEWL and skin capacitance

JOURNAL OF COSMETIC SCIENCE 238 were taken before 10 μl of the solutions was topically applied onto fi ve marked zones of the volar forearm: three zones for topical treatment (the three IWL liposome samples), one zone for the placebo solution (0.9% NaCl solution), and one untreated zone (control). The placebo and liposome solutions were randomly applied (10 μl) onto marked areas of 4 cm2 using an Exmire microsyringe (ITO Corp., Fuji, Japan). After 24 h (day 1), both biophysical parameters, TEWL and skin capacitance, were evaluated and then 10 μl of the solutions was applied again. The procedure was repeated once daily for two more days and the parameters were measured (days 2, 3, and 4). PROTECTION OF INTACT HUMAN SKIN AGAINST DETERGENT ACTION A test was performed to evaluate the protective effect of IWL liposomes applied to intact skin followed by sodium lauryl sulfate (SLS) exposure. The baseline measurements of TEWL and skin capacitance were taken, in this study, in the fi ve treated areas of the volar forearm of the volunteers submitted to the effi cacy study described above. The fi ve zones were then exposed to a 2% SLS aqueous solution for two hours (see SLS exposure), and the resultant irritation reaction was assessed 2.5 hours after SLS exposure by measuring TEWL and skin capacitance (22). SLS EXPOSURE SLS was chosen as a surfactant to provoke a chemical disruption of healthy skin since it is fre- quently present in detergent-based products of daily use such as household or body cleanser products. Ten microliters of an aqueous solution of 2% SLS was pipetted onto a layer of fi lter paper placed in each of several aluminium chambers (d=12 mm, large Finn chambers, Epitest Oy, Finland). The chambers were fi xed to the skin for two hours with adhesive tape. Upon removal of the patch, the skin was gently rinsed with water and allowed to dry. DATA TREATMENT The mean values were calculated and Dixon’s test was used to detect outliers, which were excluded from the data. The parameters were normalized, with each value divided by the baseline value. Parameter changes in the fi gures were simultaneously evaluated versus control and placebo values. ANOVA variance analysis was used to determine signifi cant differences between values obtained from different treatments (signifi cance level accepted: p 0.05) using the Statgraphics program. RESULTS AND DISCUSSION LIPID EXTRACTION AND ANALYSIS Raw Spanish Merino wool was extracted at the pilot plant scale in order to obtain a lipid extract rich in ceramides. Merino wool from Spain was used because its internal lipid composition resembles that found in the skin stratum corneum (23). The extraction was