Abstract
In recent decades, the recurrence and mortality rates of fungal infections have increased, likely due to antifungal resistance and insufficient attention from healthcare authorities. This trend highlights the urgent need for new antifungal treatments, with essential oils (EOs) emerging as promising alternatives. This study focuses on the characterization, nanoencapsulation, and evaluation of the EO of Lippia origanoides Kunth - pepper-rosemary - on toxicity and antifungal activity against filamentous fungi and yeasts, with a particular emphasis on the influence of surfactants. The EO was characterized by GC-MS and encapsulated in Nanostructured Lipid Carriers (NLCs) using either a nonionic surfactant or a combination with a cationic surfactant. NLCs were further characterized by the determination of the retention of the marker compound by high-performance liquid chromatography (HPLC), of morphology by transmission electronic microscopy (TEM), and their stability was assessed under thermal stress over 28 days. Minimum inhibitory concentrations (MIC) were determined against four yeast fungi - Candida albicans (ATCC 64548), Candida auris (CDC B11903), Candida parapsilosis (ATCC 22019), and Malassezia furfur (ATCC 14521) - and three filamentous fungi - Aspergillus flavus (ATCC 204304), Fusarium keratoplasticum (ATCC 36031), and Trichophyton rubrum (ATCC 28188). The NLC's acute toxicity was evaluated in Galleria mellonella larvae. The results demonstrated the stability, safety, and potent antifungal efficacy of EO-loaded NLCs. The charge of the NLCs played a critical role in their antifungal performance for most fungal species. The differential responses observed suggest that CTAB enhances antifungal activity by imparting a positive charge to the nanoparticles, creating an additive effect with thymol. CTAB's ability to reverse the fungal cell surface charge from negative to positive was significant. However, C. auris, A. flavus, and F. keratoplasticum showed no sensitivity to CTAB, indicating that surface charge was not a factor for these fungi. The EO and its NLC formulations exhibited significant in vitro antifungal activity, suggesting their potential as alternative therapies for fungal infections.