Microfluidic Engineering for the Development of Ozonized Oil Microemulsions Aimed at Canine Leishmania Treatment: Implementation of 3D Skin Models

The skin is the primary interface between the host and Leishmania parasites, where initial interactions can lead to severe cutaneous lesions [1]. In vitro 3D skin models allow for studying these interactions and exploring new therapies like ozone therapy, recognized for its antimicrobial and regenerative properties [2, 3]. However, ensuring the stability and bioavailability of ozonederived compounds in ozonated vegetable oils remains a challenge, requiring innovative engineering approaches. This study aims to establish and evaluate the viability of canine skin explants in vitro as a platform for testing the efficacy of ozonized oil against Leishmania infection. Additionally, we explore different approaches for developing ozonized oil microemulsions. In this initial phase, a pre-existing microfluidic device was used to evaluate formulation stability. Skin explants from five dogs (n=5) were maintained in culture and assessed at T0, T3, T5, T7, and T10. Metabolic Activity was analyzed using the resazurin assay, while histological integrity was assessed through hematoxylin-eosin staining. In parallel, a microfluidic-based device was used to produce microemulsions of ozonized olive oil at 400 IP and 800 IP in an aqueous solution of surfactant (Tween 80 at 2.5%) and study formulation stability. Preliminary results demonstrated that the culture system maintained viable skin explants for at least five days, supporting its feasibility for Leishmania infection studies. Microfluidic systems showed promise in developing ozonized oil microemulsions, with further optimisation needed. Future applications may extend beyond cutaneous treatments, potentially exploring ocular and other therapeutic uses.