Solvent Effects on Skin Penetration and Spatial Distribution of the Hydrophilic Nitroxide Spin Probe PCA Investigated by EPR.

Oxidative stress occurs in extrinsic skin aging processes and diseases when the enhanced production of free radicals exceeds the homeostatic antioxidant capacity of the skin. The spin probe, 3-(carboxy)-2,2,5,5-tetramethylpyrrolidin-1-oxyl (PCA), is frequently used to study the cutaneous radical production by electron paramagnetic resonance (EPR) spectroscopy. This approach requires delivering PCA into the skin, yet solvent effects on the skin penetration and spatial distribution of PCA have not been thoroughly investigated. Three solvents of ethanol, phosphate-buffered saline (PBS) and ethanol-PBS (1:1) were studied. For both human and porcine skin ex vivo, the amount of PCA in the stratum corneum (SC) was the lowest when using ethanol and very similar for PBS and ethanol-PBS. The highest amount of PCA in the viable skin layers was detected for ethanol-PBS, yet it only took up less than 5% of the total amount. The majority of PCA was localized in the SC, among which PCA with high mobility was predominantly distributed in the hydrophilic microenvironment of corneocytes and PCA with lower mobility was mainly in the less hydrophilic microenvironment of intercellular skin lipids. A higher ethanol concentration in the solvent could improve the distribution of PCA in the hydrophilic microenvironments of the SC. The results suggest that ethanol-PBS (1:1) is best-suited for delivering most PCA deep into the skin. This work enhances the understanding of solvent effects on the skin penetration and distribution of PCA and supports the utilization of PCA in studying cutaneous radical production.