Preparação, caracterização físico-química e avaliação in vitro da atividade anti-leishmania de nanoparticulas de PLA encapsuladas com o derivado 2 amino tiofênico SB83

Abstract

Leishmaniasis is a neglected disease that affects millions of people globally and is a serious public health problem, especially in tropical and subtropical regions. This study aimed to develop and characterize polymeric nanospheres of poly(lactic acid) (PLA) encapsulated with the 2-amino-thiophene derivative SB83, aiming at improving the efficacy and/or safety of the treatment of cutaneous leishmaniasis. The nanospheres were prepared using the nanoprecipitation method, followed by the validation of a quantification method by High Performance Liquid Chromatography (HPLC) according to the ICH (Q2) and ANVISA guidelines. The nanospheres were characterized by size, polydispersity index (PDI), zeta potential, morphology, encapsulation efficiency (EE) and drug release kinetics. In vitro assays were performed to determine cytotoxicity (CC50) in macrophages, efficacy against promastigotes (IC50) and axenic amastigotes (EC50) in Leishmania amazonensis strains. The results indicated that the nanospheres with SB83 in suspension (NPSB83), lyophilized (NPSB83FD) and lyophilized with trehalose (NPSB83TFD) presented average sizes of 205.7 ± 4.75 nm, 273.2 ± 0.11 nm and 207.6 ± 0.08 nm, respectively, with PDI of 0.274 ± 0.04, 0.425 ± 0.03 and 0.355 ± 0.03, and zeta potentials (mV) of -20.4 ± 0.65, -19.6 ± 0.12 and -18.1 ± 0.19, respectively. The EE was 64.92% ± 0.12 and 88.43% ± 0.8 for the direct and indirect methods, respectively. Transmission electron microscopy analyses confirmed the spherical morphology and uniform distribution of the particles, with no evidence of aggregation. The in vitro release kinetics revealed a slow release of SB83 from the nanospheres, reaching 72.82% for NPSB83, 69.47% for NPSB83FD and 90.48% for NPSB83TFD over 96 hours. The development of the analytical method by HPLC for SB83 quantification was linear (R² of 0.9999) and demonstrated selectivity with the addition of white nanosphere excipient. The repeatability and intermediate precision demonstrated precision, presenting RSD% lower than 2%. The method was accurate and demonstrated recovery rates between 101.75% and 103.75%, with RSD% lower than 2%. The detection and quantification limits were 0.12 µg/mL and 0.36 µg/mL, respectively, indicating sensitivity in the quantification and detection of SB83. However, the method was not robust to variations in flow, temperature and mobile phase ratio. In vitro cytotoxicity tests in RAW 264.7 macrophages revealed a significant reduction (p < 0.0001) in the cytotoxic effect of SB83 encapsulated in nanospheres (CC50 between 182.25 ± 8.27 and 194.63 ± 9.42) compared to free SB83 (CC50 = 105.51 ± 6.11) and miltefosine (CC50 = 119.74 ± 8.53). Antileishmanial activity assays demonstrated that both free SB83 and SB83 encapsulated in nanospheres were more efficient in inhibiting the growth of promastigote forms of Leishmania amazonensis than miltefosine; however, SB83 presented the lowest IC50 values (5.72 ± 0.46 µM). Against axenic amastigote forms, no statistically significant difference was observed between the activities of free SB83, nanoencapsulated SB83 and miltefosine, however all nanospheres presented selectivity indices 50% higher, promoting a significant gain in therapeutic safety. Therefore, PLA nanospheres encapsulated with SB83 are a promising strategy for the treatment of cutaneous leishmaniasis, offering sustained drug release, colloidal stability and gains in therapeutic safety.