Desenvolvimento e avaliação biológica in vitro de nanopartículas poliméricas ph-sensíveis contendo metotrexato

Abstract

The traditional cancer treatments act in a non-specific way, and cause side effects through loss of normal cell functions. Therefore, the development of new treatments with greater selectivity for the tumoral tissue is very important for the improvement of the anticancer therapy. In the last years, many studies have shown that an effective approach to enhancing the pharmacological action of drugs is conjugating the active molecule with a nanostructured delivery system. These news drug delivery systems have the capacity to improve the therapeutic index of several drugs, increasing the efficiency, decreasing the toxicity and achieving therapeutic indexes for an extended period. Therefore, the main objective of this research was to develop polymeric nanoparticles ( Ps) with the polyme poli(ε-caprolactone) and incorporating the surfactant 77 KL as a pH-responsive adjuvant , in order to entrap the antitumor drug methotrexate (MTX). After an exhaustive formulation study, the NPs was optimized with the surfactant poloxamer 407 as a stabilizer, which has the capacity to improve the entrapment efficiency (EE) of nanoparticulate systems, as well as to decrease the resistance of tumor cells. The NPs showed mean hydrodynamic diameter, polydispersity index and zeta potential of 178.5 nm, 0.135, -1.98 mV, respectively. A high-performance liquid chromatography method was developed and validated to measure the MTX in the NPs. The stability and photostability assessments, the EE and the in vitro release studies of MTX were determined using the validated analytical method. The NPs were freeze-dried with trehalose at 10% (w/v) as a cryoprotectant to improve the NPs stability, showing EE of 20.52%. In vitro release studies were performed in media with pH 7.4 and 5.4, exhibiting slightly accelerated release patterns under acidic conditions. The pH-dependent membrane-lytic activity of NPs was assessed using the hemolysis assay, with the erythrocytes as a model of the endosomal membrane. The inclusion of 77KL on the formulation gives it a pH-sensitive membrane-lytic behavior in the acidic environment found in the intracellular compartments. Moreover, the in vitro antitumor activity of the NPs was evaluated in physiological pH and in the pH characteristic of the tumor tissue (pHe ~ 6.6), using tumor cell lines (MCF-7 and HepG2) and different cell viability assays (MTT and NRU). Our results proved that the nanoencapsulated MTX was more active against the tumor cell lines than the free drug, as determined by both viability assays. Therefore, the overall results indicated that the NPs have pH-sensitive properties and a greater in vitro antitumor activity than the free drug, suggesting that these nanodevices could be potentially useful as a carrier system in cancer therapy.