Obtenção, caracterização e avaliação da atividade antifúngica de nanopartículas de quitosana contendo o peptídio TistH identificado na peçonha do escorpião Tityus stigmurus

Abstract

The Tityus stigmurus scorpion venom has been studied in recent years and revealed many toxins (peptides) with high potential for biotechnological applications. The transcriptomics approach from the venom glands of T. stigmurus, realized by our scientific group, identified several bioactive peptides with pharmacological potential. Among the molecules identified, the activity of peptide like-hypotensins class (TistH, Tityus stigmurus hypotensin), reported in the literature, evidenced the TistH with bradykinin-potentiating peptide, in addition exert antifungal effect. The maximum efficacy of this class of compounds can be achieved by immobilizing it in specific and suitable biomaterials or suitable carriers. This study objective to obtained and characterized physiochemically TistH-loading cross-linked chitosan nanoparticles for incorporation and adsorption, evaluate the biocompatibility as well as improve of antifungal effect. The nanossystems containing the TistH by incorporation (CN-TistH-Inc) and absorption (CN-TistH-Ads) methods at concentration of 0.5 and 1.0% were obtained by ionic gelation technique. The nanosystems at 0.5 and 1.0% were analyzed by dynamic light scattering showed positive zeta potential, with size particles less than 160 nm and polydispersity index less than 0.3. The encapsulation efficiency were greater than 96.5% and the success was confirmed by polyacrylamide gel electrophoresis and Fourier transform infrared spectroscopy. The CNTistH-Inc and CN-TistH-Ads at 0.5 and 1.0% were analyzed by atomic force microscopy and scanning electronic microscopy evidenced nanometric particles, spherical shape with smooth surface and homogeneous aspects. The stability was evaluated by particle size and polydispersity index can possible observed stable colloidal dispersions for 4 months. According in vitro release profile was observed for all formulations prolonged and sustained release for 24 hours, with kinetic parabolic diffusion mechanism. The biocompatibility was evaluated in erythrocytes, normal cells originated from murine macrophage (RAW 264.7) and kidney of African green monkeys (Vero E6) suggested biocompatibility that both nanosystems, in concentration used for antifungal assay, not demonstrated be hemolytic and cytotoxic, respectively. The antifungal activity was evaluated through of determination of the Minimum inhibitory concentration (MIC), minimum fungicidal concentration (MFC) and antibiofilm activity used yeast of genus Candida. The CN-TistH-Inc and CN-TistH-Ads showed a minimal inhibitory concentration of 89.2 µg/mL against Candida albicans, 11.1 µg/mL for C. parapsilosis and C. tropicalis, confirmed by minimum fungicidal concentrations assay. Moreover, the TistH-loaded cross-linked chitosan nanoparticles significantly reduced the biofilm formation of clinical yeast sepsis of C. tropicalis and C. krusei, as well as clinical yeasts of vulvovaginal candidiasis of C. albicans. Thus, this study demonstrated that chitosan nanoparticles are promising for carrying and delivering the peptides TistH from Tityus stigmurus, with improve antifungal activity against genus Candida spp. may eventually be applied in the future as a possible antifungal agent.