VISUALIZATION OF SKIN BARRIER PERTURBATION 287 This may further induce the creation of intra-corneocyte penetration pathways once SDS "opens-up" the cross-linked keratin structure of the corneocytes. Therefore, a group of such damaged adjacent corneocytes, taken together, may exhibit a large number of intra-corneocyte penetration pathways that may result in a localized transport region (LTR). A simultaneous quantitative analysis of the red- and the green-channel TPM skin images showed that an aqueous contacting solution of 1 wt% SDS + 10 wt% glycerol does not significantly induce corneocyte damage or L TR formation. Therefore, taken together with the results reported by us recently (2,45), these dual-channel TPM skin images provide additional evidence that adding 10 wt% glycerol to a 1 wt% SDS aqueous contacting solution significantly mitigates the ability of SDS to penetrate into the SC and interact with the keratins of the corneocytes and induce corneocyte damage. The dual-channel TPM images of p-FTS exposed to an aqueous 1 wt% SCI contacting solution showed a lower extent of SRB penetration into the corneocytes and into the intercellular lipid bilayers relative to the aqueous contacting solution of 1 wt% SDS. The PBS control solution induced localization of the SRB probe within the intercellular lipid bilayers surrounding the corneocytes of the SC. In addition, aqueous contacting solutions containing 10 wt% glycerol showed the least extent oflipid bilayer perturbation, and no effect on the corneocytes of the SC, relative to the other surfactant-humectant aqueous contacting solutions considered here. This important finding is consistent with in vitro skin barrier measurements reported by us in the literature (2,45), where an aqueous contacting solution of 10 wt% glycerol was shown to reduce the porosity-to-tortuosity ratio and the average radius of the aqueous pores relative to a PBS control. Therefore, these results indicate that glycerol, at an appropriate concentration, closes aqueous pores in the SC, which should reduce the extent of skin penetration of a hydrophilic irritant found in a skin cleansing formulation, such as a surfactant micelle, thereby mitigating skin barrier perturbation induced by the surfactant micelle. Consequently, cosmetic formulators may be able to use this mechanistic knowledge to tune the skin mildness of a formulation containing surfactants and humectants. For the five aqueous contacting solutions considered here, most of the SRB probe that penetrates into the skin barrier is present in the SC, and the probe intensity decays significantly as one visualizes the layers in the epidermis below the SC. We have quantified the amount of SRB that penetrated into the SC as a function of the SC depth upon contacting p-FTS separately with the five aqueous contacting solutions (i-v). This TPM analysis revealed that SDS enhances the probe partition coefficient the most, and that the extent of skin barrier perturbation induced by the five aqueous contacting solutions considered follows the order (from the highest to the lowest): i ii iii iv v. Therefore, a cosmetic formulator can use the TPM skin visualization and quan­ tification methodology presented here to screen new cosmetic formulations containing surfactants and humectants based on their ability to perturb the aqueous pores of the SC. The development of such an in vitro visual ranking methodology, including quantifica­ tion, can potentially reduce many costly and time-consuming in vivo human and animal testing procedures, thereby significantly reducing the cost and time-to-market of new cosmetic formulations containing surfactants and humectants. ACKNOWLEDGMENTS We thank Dr. Sidney Hornby and Dr. Yohini Appa from Neutrogena Corporation for useful discussions, and for providing partial financial support for this work.

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