P32-05 Characterization and Biosafety Profile of a Betulin Hydrogel for Potential Skin Applications – An In vitro-In Ovo Study

The skin is the largest organ of the human body, composed of epidermis, dermis, and hypodermis, with each of them being involved in different functions. Prolonged exposure to risk factors or unsafe products can lead to serious diseases ( i.e. , cancer). Gels have a wide range of uses, including in the medical-pharmaceutical, cosmetic, or food fields, and are differentiated by the nature of the liquid phase. Hydrogels are hydrophilic, three-dimensional networks, capable of absorbing large amounts of water or biological fluids, and have been intensively researched in recent decades as potential drug delivery systems. Moreover, betulin (BET) is a pentacyclic triterpene with anti-melanoma, anti-inflammatory, anti-aging, and antioxidant properties, with applications in dermatological pathologies and skincare. Therefore, the objective of the current study was the development of a BET hydrogel formulation with potential cutaneous applications and its biosafety evaluation in terms of cytotoxicity in HaCaT and JB6 Cl 41-5a skin healthy cell lines, and irritant potential in 3D EpiDerm microtissues and on the chorioallantoic membrane (CAM) of embryonated chicken eggs. Following the meticulous characterization of the blank and BET hydrogels, their cytotoxicity at concentrations of 100-500 µg/ml was performed by investigating the HaCaT and JB6 Cl 41-5a cells’ viability (MTT method), morphology (bright-field microscopy), and nuclear aspect (Hoescht 33342 staining). The potential irritant effect was evaluated in vitro , by applying the Skin Irritation Test in EpiDerm microtissues and in ovo, by performing the Hen's Egg Test on the CAM. The results indicated that the hydrogels did not produce cytotoxicity after 24 hours of treatment, as the decrease in cell viability did not exceed 75%. The cell morphology and nuclear appearance did not change significantly after treatment, and the apoptotic index remained similar to control for treated cells. In the 3D experimental model, the viability of the microtissue treated with blank and BET hydrogels was higher than 70%. In addition, these formulations presented a low irritation score in ovo, being categorized as non-irritants. These results suggested that the blank and the BET hydrogels present increased biosafety, are biocompatible, and did not induce signs of cytotoxicity or signs of irritation. Based on these analyses, future directions should focus on the therapeutic efficacy of these formulations.