AVALIAÇÃO DO POTENCIAL ANTIFÚNGICO DE NANOPARTÍCULAS DE PRATA DE Musa x paradisiaca CONTRA Paracoccidioides spp

Abstract

Paracoccidioidomycosis (PCM) is the most prevalent systemic mycosis in Latin America, caused by fungi of the genus Paracoccidioides. Despite its clinical relevance, therapeutic options remain limited to three classes of antifungal agents, which are often associated with adverse effects. In this context, the search for new therapeutic alternatives with activity against Paracoccidioides becomes essential. Silver nanoparticles (AgNPs) stand out as promising candidates due to their recognized antimicrobial potential, including antifungal activity against different pathogens.In this study, we evaluated the antifungal activity of silver nanoparticles synthesized from Musa x paradisiaca (AgNP-MP) against isolates of Paracoccidioides spp. Initially, a literature review was carried out to assess the methodological variables employed in broth microdilution assays against this fungal genus. Among the 59 studies analyzed, variations were observed in culture medium, inoculum concentration, incubation time, and reading method. The most frequently used media were RPMI-1640, RPMI-1640 supplemented with glucose, and McVeigh & Morton. The inoculum concentration ranged from 10³ to 10⁶ cells/mL, and the incubation period from 72 hours to 15 days. As for the endpoint, visual analysis and the use of resazurin predominated. Based on this systematization, experimental assays were conducted against Paracoccidioides brasiliensis (Pb18) and P. lutzii (Pb01). The results demonstrated that an inoculum of 10⁵ cells/mL, three days of incubation, and visual reading combined with resazurin constitute adequate and reproducible parameters for MIC determination.These parameters were employed in the antifungal analysis of AgNP-MP against different Paracoccidioides species. Broth microdilution assays were performed to determine the Minimum Inhibitory Concentration (MIC) and Minimum Fungicidal Concentration (MFC), according to the Clinical and Laboratory Standards Institute (CLSI) protocol M27-A3, with modifications. The susceptibility of isolates to conventional antifungal agentes, amphotericin B (AmB), itraconazole (ITZ), and sulfamethoxazole (SMX), as well as the interaction between AgNP-MP and these drugs, was also evaluated using the checkerboard method. Biofilm inhibition assays were assessed by biomass quantification using crystal violet, metabolic activity through MTT reduction, and cell viability by colony-forming unit (CFU) counts. Additional assays with ergosterol and sorbitol were performed to investigate possible mechanisms of action of AgNP-MP. Scanning electron microscopy (SEM) and Calcofluor White staining were employed to observe morphological alterations after treatment. Moreover, in vitro cytotoxicity assays were performed on murine macrophage cells. MIC and MFC values of AgNP MP ranged from 0.41 to 6.68 µg/mL. Overall, MIC50 and MIC90 were 3.34 µg/mL for the Paracoccidioides genus, and fungicidal activity was observed for all isolates tested. Susceptibility to conventional antifungal agents ranged from 0.01 to 0.5 µg/mL for AmB, 0.0002 to 0.015 µg/mL for ITZ, and 37.5 to 2,400 µg/mL for SMX. No synergism was observed between AgNP-MP and antifungal agents, suggesting that the nanoparticle acts independently, without additive effects in the tested combinations. At a concentration of 3.34 µg/mL, cell viability was reduced by 89.8% for P. lutzii (Pb01) and 90.7% for P. brasiliensis (Pb18). Biofilm biomass was reduced by 69.6% (Pb01) and 64.5% (Pb18), while metabolic activity decreased by 93.1% and 83.3%, respectively. Assays with ergosterol and sorbitol suggested the cell wall as a possible target, while SEM analyses confirmed these findings by showing morphological alterations consistent with antifungal effects. Calcofluor White staining further corroborated the observed cell wall alterations. Although no cytotoxicity was detected, AgNP-MP, despite its antifungal potential demonstrated against different Paracoccidioides species, still requires further studies to elucidate its safetyile profile.