Skin penetration of active folate salts and their effects on wound healing _in vitro_: new approaches for dermal folate application

Abstract

# Objectives The skin has a high demand for folates because of its constant renewal and growth. Folates play an important role in the metabolism of healthy skin and in the regeneration of irritated or injured skin. The high proliferation rate supports wound healing in chronic and degenerative skin diseases. In contrast to systemic folate administration, there is little known on skin penetration of folates after topical application. Therefore, skin penetration with L-formyltetrahydrofolate di-arginine salt (L-FTHF-di-arginine), L-formyltetrahydrofolate calcium gluconate mixture (L-FTHF-Ca) and L-methyltetrahydrofolate dicholine salt (L-MTHF-dicholine) was studied. # Methods We investigated the epidermal penetration of 2.5% aqueous solution of L-FTHF di-arginine, L-FTHF Ca and L-MTHF dicholine using *in vitro* reconstructed epidermal skin models. These multi-layered epidermis models closely emulate the barrier of human skin. Aqueous solutions of - L-FTHF di-arginine, L-FTHF Ca and L-MTHF dicholine were applied topically to the keratinized layer. The folate concentration below the multi-layer was analyzed after 24 hours by high-performance liquid chromatography (HPLC)-analysis to detect penetration. In order to assess an effect on wound healing *in vitro*, the selected folates were tested in a keratinocyte scratch assay. # Results Analysis of the examined folates in the cell culture medium located opposite of the application site showed their penetration through the reconstructed epidermal model. In epidermal models to which L-FTHF di-arginine was added, L-MTHF dicholine was detected on the opposite site. L-FTHF salts were partially converted to L-MTHF. Improved wound healing was shown in keratinocyte scratch assay when the selected folates were added to the medium. # Conclusion The *in vitro* experiments on an epidermal skin model have revealed the following new findings: a) the different reduced folates investigated (L-FTHF di-arginine, L-FTHF Ca and L-MTHF dicholine) penetrate well into and through the skin, in contrast to folic acid, which is commonly used; b) they neither disturb the viability of skin cells nor their barrier function, but even improve it; c) the proliferation and migration of epidermal cells, which is important for wound healing, is promoted; d) epidermal cells are able to convert L-FTHF locally into L-MTHF, i.e., to the dominant biologically active form in the circulation. This creates important prerequisites for a future topical application of folates for the treatment of certain skin diseases and the prophylaxis of skin aging processes.