JOURNAL OF COSMETIC SCIENCE 236 of forming liposomes with a stable bilayer structure (8,9). This vesicular structure, which mimics the organized lipid structures of the stratum corneum, offers a suitable strategy for achieving an accurate vehiculization of a particular compound and for incorporating additional lipid content that may reinforce the barrier function of the skin (10–12). Fur- thermore, earlier studies (13,14) have demonstrated the ability of these IWL liposomes to improve skin barrier properties when applied onto intact and compromised skin. Slightly better results have been obtained with these liposomes, containing a mixture of natural ceramides, when compared with stratum corneum liposomes (modeling stratum corneum lipids using synthetic lipid mixtures), which had only one type of ceramide present in the formulation. Accordingly, IWL could be regarded as a new natural extract that is benefi - cial to topical application and suitable for incorporation into pharmaceutical or cosmetic formulations in the treatment and care of skin (15). Therefore, IWL have been extracted at the pilot plant scale both by organic solvent extrac- tion (OSE) using methanol or acetone (16) and by supercritical fl uid extraction (SFE) with CO2, using 10% methanol or ethanol as polarity modifi ers (17). In the present work, lipo- somes containing IWL extracts with different lipid compositions were formed. Vesicular diameter, polydispersity index, and stability were determined. The effi cacy of these IWL liposomes when applied topically onto intact skin on a long-term basis was studied. To this end, in vivo changes in transepidermal water loss (TEWL) were measured as an index of bar- rier repair, whereas the water-holding capacity was measured as changes in skin capacitance. Finally, the protection of intact skin against detergent action was evaluated after topical application of the IWL liposome samples by measuring the aforementioned parameters. MATERIALS AND METHODS EXTRACTION PROCEDURES Raw Spanish Merino wool samples supplied by SAIPEL (Terrassa, Spain) were used for lipid extraction. Prior to the extraction, raw wool was industrially cleaned following a procedure previously described (16). IWL were extracted at the pilot plant scale by OSE using metha- nol. The extraction procedure consisted of a pump-forced refl ow system. Five kilograms of wool were extracted for four hours at 56°C (16). IWL were also obtained at the pilot plant scale by SFE using a CSFF (Iberfl uid Instruments/ICP-CSIC, Spain). Fifty grams of wool were extracted with CO2 at 60°C and 160 atm using 10% methanol or ethanol as polarity modifi ers of CO2 (17). IWL extracts obtained at the pilot plant scale were concentrated and stored in chloroform/methanol (2:1, v/v) at −20°C until their analysis. LIPID ANALYSIS The quantitative analysis of the samples was performed by thin-layer chromatography coupled to an automated fl ame ionization detector (TLC-FID), an Iatroscan MK-5 ana- lyzer (Iatron, Tokyo, Japan). Samples were applied on silica gel S-III Chromarods using an SES (Nieder-Olm, Germany) 3202/15-01 sample spotter. The determination of the composition was made using an optimized TLC-FID protocol to analyze lipid content (16). The rods were developed initially to a distance of 10 cm with n-hexane/diethyl ether/formic acid (53:17:0.3, by vol) to separate apolar and polar lipids. After a partial

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