BINDING OF SURFACTANTS 187 EXPERIMENTAL MATERIALS The materials used in these binding experiments were •4C-labeled SDS and SLI, ob- tained from Unilever Research, Colworth Laboratory. Potassium hydrogen phthalate and potassium dihydrogen phosphate were from Aldrich Chemicals Co. Sodium borate, TEA (triethanolamine), and sodium hydroxide used for buffer preparations were pur- chased from Fisher Scientific Corp. Hairless guinea pig skin was obtained from Charles River Laboratories (Wilmington, MA), and the stratum corneum was isolated from it by a trypsin separation procedure. Human stratum corneum was also obtained by trypsin separation from excised cadaver skin obtained from the International Institute for the Advancement of Science. METHODS Adsorption procedure. The procedure used to evaluate surfactant binding to guinea pig and human stratum corneum required that three 8-mm punches of SC be placed in 25.4-mm (1-in) square Teflon screens. These screens were placed in treatment solutions containing a combination of •4C-labeled (1 }xCi/ml) and nonlabeled surfactant, ranging in total concentration from 1 mM to 100 mM. Before the addition of the SC sample to the treatment solution, 50 }xl of the treatment solution was removed to provide initial radioactivity values. After the selected treatment time (for example, one hour), the screens were rinsed by moving them back and forth five times in a dish of distilled water. This was to essentially wash off the excess bulk solution that would have been associated with the corneum. Since the objective of the work was to determine the thermodynamic binding of the surfactant to the corneum, excessive rinsing was not done to avoid desorption. The screens were then blotted and dried with desiccation overnight. The weight of each individual SC sample was recorded using a Perkin-Elmer AD4 Autobalance, and the sample was placed into scintillation vials containing 0.5 ml of distilled water. Scintiverse BD (Fisher Scientific) was added to each vial, and the radioactivity of the stratum corneum samples was determined using the Beckman Scin- tillation Counter. The amount of surfactant bound to the SC was calculated over the final weight of SC. This was particularly important since in certain systems a weight loss was observed upon surfactant treatment due to the extraction of corneocytes and other surfactant-soluble components. For experiments with soaps, it was necessary to buffer the Na oleate and Na laurate solutions with 0.4 M TEA. The pH of the system was around 9.5. Stratum corneum samples for delipidized experiments were prepared by treating the Teflon screens containing the 8-mm punches with a 2:1 chloroform/methanol solution for one hour and drying with nitrogen. RESULTS SURFACTANT BINDING TO GUINEA PIG STRATUM CORNEUM The initial experiments of SDS and SLI binding were done on hairless guinea pig stratum corneum (GPSC). Figure 2 shows the binding isotherm of SDS and SLI to GPSC

188 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS Hairless Guinea Pig Stratum Corneum pH 5.5, 1 hr Treatment Time, RT CMC ß SDS 0 0 20 40 60 80 100 Surfactant Concentration (mM) Figure 2. Binding of SDS and SLI to hairless guinea pig stratum corneum. after a one-hour treatment at pH 5.5 and room temperature (RT). The shape of the SDS binding isotherm to GPSC is similar to the typical isotherm given in Figure 1. The main difference between the binding to skin and to water-soluble proteins appears to be that in the former case the binding continues to occur above the surfactant CMC. The amount of binding to GPSC (1-2 mg/g) is significantly higher than the reported values of surfactant binding to keratinous protein such as wool (0.4 mg/g ref. 20). A comparison of SDS to SLI (one-hour treatment, pH 5.5, RT) shows that less SLI binds to GPSC than SDS over this concentration range (Figure 2). These results are consistent with the known clinical mildness of alkyl isethionate-based bars (14). Extending the treatment time to six hours shows some increase in the SDS binding, especially at high concentrations (Figure 3). In contrast to this, the 24-hour binding isotherm is similar to the one-hour binding in the low concentration region and slightly lower than the one-hour binding at 100 mM SDS. The longer treatments affected the physical integrity of the GPSC. We suspect that the observed low binding at 24 hours is because of the physical extraction/removal of parts of the corneum by the surfactant. For this reason, further experiments were limited to one hour or less. Removal of lipids from the GPSC with a 2:1 chloroform/methanol solution prior to surfactant treatment results in a decrease in surfactant binding (Figure 4). These results are consistent with the reported effects of delipidization on the structure of stratum corneum (29,30). It is suggested that delipidization primarily removes the fluid lipids and that upon drying, the lipids covalently linked to the corneocytes form a tighter network that reduces the surfactant penetration to protein regions, i.e., prevents the surfactants from accessing the binding sites (29). SURFACTANT BINDING TO HUMAN STRATUM CORNEUM Binding isotherms. As with GPSC, the results given in Figure 5 show that at pH 5.5,