IWL LIPOSOMES AND SKIN BARRIER IMPROVEMENT 239 performed by OSE with pure methanol and by SFE with CO2, using 10% methanol or ethanol as polarity modifi ers, at the conditions described in the experimental procedure section (16,17). These solvents were chosen as extractors to avoid the use of chlorinated solvents, which were previously used at laboratory scale (23). Quantitative analysis of the three different lipid extracts was carried out by TLC-FID fol- lowing the methodology previously reported (16). The different lipid families were quan- tifi ed as sterol esters (ST-ES), free fatty acids (FFA), sterols (ST), ceramides and glycosilceramides (CER), and sterol sulfate (ST-SUL). The amount of these components analyzed, expressed in percentages of total lipid analyzed (% o.l.a.), is shown in Figure 1. Comparison of the three extracts obtained at the pilot plant scale showed little variation in the percentage of the three main lipid families (free fatty acids, sterols, and ceramides). However, it should be noted that the percentage of sterol sulfate for the extract obtained by OSE with pure methanol (about 10%) is much higher than the sterol sulfate content in the two other extracts obtained by SFE (about 1%). LIPOSOME CHARACTERISTICS AND STABILITY IWL extracted with chloroform/methanol azeotrope by Soxhlet extraction at laboratory scale, whose composition resembles that in the stratum corneum, have been demonstrated to be capable of forming liposomes with a stable bilayer structure (8,9). In the present work, liposomes were formed using the different IWL extracts obtained at the pilot plant scale by evaporating the organic solvent to dryness, hydrating with 0.9% NaCl solution, and by sonication as detailed in the Experimental section. Liposomes were easily formed with the IWL extract obtained by OSE with pure methanol. However, some lipids re- mained as aggregates in the fl ask when liposomes were formed with the IWL extracts obtained by SFE using 10% methanol or ethanol as modifi ers. These aggregates, analyzed by TLC-FID, were richer in free fatty acids and ceramides, whereas sterol and sterol sul- fate were practically absent. Figure 2 shows the lipid composition of liposomes really Figure 1. Amount of sterol esters (ST-ES), free fatty acids (FFA), sterols (ST), ceramides and glycosilcer- amides (CER), and sterol sulphate (ST-SUL) quantifi ed in the three different IWL extracts, expressed as percentages of the total lipid weight analyzed (% o.l.a).

JOURNAL OF COSMETIC SCIENCE 240 formed with the three wool lipid extracts, expressed in percentages of total lipid analyzed (% o.l.a.) The increase in percentage of sterol in the two extracts obtained by SFE should be pointed out. As a consequence, percentages of free fatty acids and ceramides in these SFE extracts decreased. Therefore, the composition of liposomes prepared with IWL extracted by OSE became richer in polar lipids such as ceramides, glycosilceramides, and sterol sulfate. The ceramides alone are known to be insuffi cient for bilayer formation, perhaps because they are not ionized at physiological pH. However, free fatty acids and cholesteryl sulfate, which are also present in stratum corneum lipids, are ionized at physiological pH and their presence together with ceramides is necessary for bilayer formation (24). Therefore, the fact that the formation of liposomes with lipids extracted by SFE is more complicated than that of liposomes with lipids extracted by OSE may be due to the lower amount of sterol sulfate contained in lipids extracted by SFE (Figure 1). Besides, the composition of lipids extracted by OSE using pure methanol is very similar to that obtained with labo- ratory-scale Soxhlet extraction using chloroform/methanol azeotrope, which has been demonstrated to form stable liposomes (8,9). The vesicle size distribution and polydispersity index of liposomes were determined by dynamic light scattering in order to characterize these liposomes and to study their sta- bility for 14 days. The results are indicated in Table I. Figure 2. Amount of sterol esters (ST-ES), free fatty acids (FFA), sterols (ST), ceramides and glycosilcer- amides (CER), and sterol sulphate (ST-SUL) quantifi ed in the three different IWL liposome samples, ex- pressed as percentages of the total lipid weight analyzed (% o.l.a.). Table I Characteristics and Stability of Liposomes: Vesicular Diameter (d) and Polydispersity Index (P.I) Day 0 Day 7 Day 14 Extract d (nm) P.I. d (nm) P.I. d (nm) P.I. OSE MeOH 173.2 0.284 186.4 0.231 189.7 0.256 SFE MeOH 233.8 0.573 266.1 0.556 297.8 0.711 SFE EtOH 298.7 0.620 610.6 0.728 636.8 0.756