Hyaluronic acid skin penetration evaluated by tape stripping using ELISA kit assay

Abstract

Hyaluronic Acid (HA) is an endogenous skin matrix component with moisturizing and anti-inflammatory and healing properties. Cosmetic formulations containing HA aim to enhance skin structure, hydrate skin and reduce wrinkles. Therefore, the skin diffusion of HA into stratum corneum after application of a formulation containing two different size of HA, High Molecular weight (HMW-HA) and Low Molecular Weight (LMW-HA)) was evaluated. Ex vivo human skin samples were used to validate an ELISA assay measuring HA in the stratum corneum (SC), viable epidermis and dermis, and to identify optimal washing and extraction methods. These methods were used to measure HA levels in the SC of subjects before and after daily topical application of an HA-containing formulation for 7 days. Samples of SC (5 tape strips) were taken before and 2 h after the application on D0, D1 and D7. The ELISA assay was suitable for measuring HA in the SC but not epidermal or dermal layers. The upper and lower limits of quantitation were the same for both sizes of HA (200 and 3.1 ng/ml, respectively). In both ex vivo human skin and human volunteers, the "dry method" of removing the formulation led to much higher levels of HA in the SC samples, whereas the "wet method" involving one cotton swab soaked with an aqueous solution containing 10% soap and a second cotton swab for drying, was effective in removing the formulation and more relevant to simulate washing/showering. In the clinical study, the amount of HA in SC layers 3-5 were used to represent the HA level in the SC, whereas layers 1 and 2 were considered as surface "residual film". After each application, there was a significantly higher amount of HA compared to the amount before application, which was observed using both wash methods. The residual level 24 h after the first application was at least 8 times higher than before the first application and at least 31 times higher after 7 applications. In conclusion, these investigations validated the use of the ELISA method for the measurement of HA in SC samples. The ex vivo experiments provided recommendations for the clinical study, including the preferred cleansing and optimal sampling methods. The clinical study demonstrated the diffusion, accumulation and maintenance of HA levels in the SC after repeated application of the formulation containing HMW-HA and LMW-HA.

Keywords: Clinical study; ELISA; Hyaluronic acid; Skin penetration; Stratum corneum strips; Validation.