Obtenção e avaliação in vitro de nanopartículas de Poli(ε- Caprolactona) revestidas com quitosana para carreamento de Anfotericina B

Abstract

In recent decades, invasive fungal infections (IFIs) have increased significantly in immunocompromised patients in intensive care units, thus being one of the major causes of morbidity and mortality associated with this type of disease, where the species of Candida represents the fourth most common cause of IFI in humans. Among the antifungal agents approved for the treatment of IFI, the most widely used is amphotericin B (AmB), which is characterized by being broad spectrum antifungal drug. However, its clinical use has been limited due to its side effects being the most adverse the nephrotoxicity. Furthermore, AmB has a low solubility, low permeability in the membranes and poor stability in the gastric environment, which makes it impossible to be administered by the oral route. In this study, the aim was to develop nanoparticles of poly (ε-caprolactone) coated with chitosan (CS) for AmB carrier with the goal reducing toxicity to human cells and improving the oral bioavailability. Nanoparticles (Nps) were obtained by the nanoprecipitation method, and parameters such as type and concentration of surfactant, organic phase proportion: aqueous phase, chitosan concentration and amount of drug were optimized. The Nps were characterized in terms of size, polydispersity index, zeta potential, morphology, in vitro release (physiological pH and gastrointestinal fluids), aggregation status, hemolysis, cytotoxicity over Vero cell line and antifungal activity. The Nps-AnBCS obtained were nanometric sized, with a mean diameter of 318 ± 35 nm, low polidispersion of 0.24 ± 0.02, zeta potential of + 36.2 ± 1.8 mV and high entrapment efficiency (% EE) of 69% ± 0.02. The study about the state of aggregation of the encapsulated AmB revealed that it is in a state of low molecular aggregation. The kinetic profile of the AmB released from the Nps-AnBCS was of second order and governed by diffusion. The release in the gastrointestinal fluids showed that the Nps-AnBCS presented good stability in the simulated gastric and intestinal fluid obtaining percentages of AmB release of 10.08% (pH = 1.2) and 29.15% (pH= 6, 8) at the time of 6 h. The results of the in vitro hemolysis study revealed that AmB encapsulated significantly reduced the hemolytic toxicity of AmB (p <0.05) compared with free drug. The cytotoxicity of Nps-AnBCS over the Vero cell line showed a significant reduction in renal toxicity of AmB. In addition, the AmB associated with the Nps presented antifungal activity against the Candida parapsilosis strain, being therefore an excellent proposal for the oral delivery of AmB.