A Supramolecular salicylic acid – betaine with superior epidermal benefits and better tolerance: in vitro proof of concept studies

Introduction: Recent progress in supramolecule research has led to a surge in its application in cosmetic products. However, comprehensive investigation of the potential benefits, especially differentiating biological activities and associating that with physical-chemical properties, remains to be established. In the current study, a supramolecular salicylic acid-betaine (Supra SA-B) was benchmarked to its physical mixture counterpart for its impact on epidermal homeostasis as well as insult potential when applied topically. Methods: The structure of supra-SA-B and physical mixture of SA+B (1:1 molar ratio) were characterized by a powder X-ray diffractometer before and after dissolution in aqueous alcohol solution. For skin tolerance evaluation, SA, Supra-SA-B, and SA+B were applied systematically to primary human keratinocytes or topically on the surface of SkinEthic™ RHE reconstructed human epidermal models. Cell and model viabilities were assessed using MTT assay, respectively. Inflammatory cytokines produced by models were assessed using Luminex multi-plex assay. Histological morphology was evaluated by hematoxylin-eosin (HE) staining, with aquaporin-3 (AQP-3) and Ki67 measured by immunofluorescence (IF). Finally, RNA-sequencing was conducted to characterize overall biological response based on non-targeted transcriptomic profiles. Results: Characteristic peaks remained the same for Supra-SA-B after freeze drying verified that their crystalline structures, including supramolecular, were retained in the current solution. With the addition of betaine and introduction of supramolecular format, Supra-SA-B treated groups demonstrated significantly higher cell viabilities comparing to SA or SA+B. With 18-hrs of treatment, Supra-SA-B treated group released less inflammatory cytokines. In addition, Supra-SA-B demonstrated better hydration effect with increased AQP-3 expression and more organized epidermal structure, in comparison to either SA alone or SA+B. Consistent result was observed in the RNA transcriptomic profiles of RHE models after treatment. Supra-SA-B did not induce major transcriptome drift as compared to SA or SA+B, with less evident negative impact on loricrin, and late cornified envelope (LCE) gene expression. At the same time, the boosting effect on hyaluronic acid synthase (HAS) was maintained across all the treatments vs. control. Conclusion: Our findings indicated that Supra-SA-B ameliorated skin tolerance risk for SA, enhanced hydration with less insult on barrier functions, suggesting promising potential for cosmetic application.