Topical delivery of antifungal Brazilian red propolis benzophenones-rich extract by means of cationic lipid nanoemulsions optimized by means of Box-Behnken Design

Abstract

Recent studies have demonstrated the antifungal activity of a benzophenones-rich extract (BZP) from Brazilian red propolis against non-albicans Candida strains. This study aimed to optimize the incorporation of BZP into nanoemulsions intended for the treatment of mucocutaneous fungal infections. Formulations were optimized by means of a Box-Behnken Design, which allowed evaluating simultaneously the influence of the phospholipid egg-lecithin, the cationic lipid DOTAP and BZP concentrations on the physicochemical properties of nanoemulsions, as well as on the BZP association efficiency. By using the Mini Tab® software, the optimal formulation selected, based on the smallest droplet size and highest zeta potential and association efficiency, exhibited a mean average size of 140.56 ± 5.22 nm, zeta potential of +60.72 ± 3.07 and association efficiency of 99.55 ± 1.09%. Franz-type diffusion cells were used to evaluate BZP distribution through porcine skin and mucosa. BZP were found in both mucosa and skin layers (mainly in the dermis). A higher amount of BZP (up to 3-fold) was detected in impaired skin and mucosa demonstrating the effect of the integrity of the tissue on BZP distribution, as suggested by confocal fluorescence microscopy images. BZP were detected in the receptor fluid only when esophageal mucosa was impaired. The antifungal activity of the formulations was investigated against non-albicans Candida species – C. krusei, C. glabrata, C. tropicalis and C. parapsilosis. MIC values varied from 0.654 to 2.617 μg/mL, with cell damage higher than 78% as verified by MTT assay. Such a result suggests that the optimized formulation has promising potential to be used topically for the treatment of mucocutaneous fungal infections.