Investigação da atividade antitumoral de lipossomas pH-sensíveis de circulação prolongada contendo doxorrubicina em modelo animal experimental

Abstract

Breast cancer is the most common cancer in women worldwide, with 20% of all new cases of cancer each year, both in developing or developed countries. Anthracyclines, especially doxorubicin, play an important role in the treatment of breast cancer, however, the high toxicity, especially cardiotoxicity, and low penetration in solid tumors are the main limitations for its use. The encapsulation of doxorubicin in long-circulating liposomes helped to reduce the incidence of adverse effects, including cardiac toxicity, which led to the approval by FDA in 1995 of the first liposomal formulation for cancer treatment, Doxil®. Despite its advantages, generally long-circulating liposomes exhibit a low drug release rate and tend not to be able to merge with the endosomal membrane upon cell internalization. These factors reduce the intracytoplasmic concentration of the drug, which can compromise the therapeutic efficacy. Therefore, pH-sensitive liposomal formulations containing anticancer drugs have been developed to take advantage of the endosomal acidification, which leads to destabilization of the liposomal membranes followed by intracytoplasmic release of its internal content. In this work, it was developed a long-circulating and pH-sensitive liposomal formulation containing doxorubicin (SpHL-DXR) for evaluation of cytotoxicity against the murine breast tumor cell line 4T1 and the antitumor activity in a breast cancer animal model. SpHL-DXR were characterized by average diameter, polydispersity index (IP), zeta potential and encapsulation efficacy analyses. The average diameter was below 200 nm with IP no higher than 0.2 and zeta potential close to neutrality, allowing the application of this formulation for in vivo studies. Storage stability evaluation of blank liposomes (without doxorubicin) at 4°C confirmed the maintenance of the physico-chemical characteristics for up to six months, suggesting the possibility of production of a kit containing, separately, the blank formulation and the lyophilized doxorubicin for administration in patients. pH-sensitivity studies have shown responsiveness of liposomes to pH variations. Cytotoxicity of SpHL-DXR against 4T1 cell line was evaluated by MTT assay. IC50 obtained for SpHL-DXR treatment was equal to 1563 nM. This value corresponds to 35% reduction when compared to free DXR (IC50 equal to 2413 nM), indicating a strong improvement of cytotoxic activity upon liposomal encapsulation. For antitumor activity studies, breast tumor-bearing mice were intravenously treated with HEPES-buffered saline pH 7.4 (control group), blank liposomes, free doxorubicin, liposomal formulation similar to Doxil® or SpHL-DXR (dose equal to 4 mg of doxorrubicin.Kg-1) every two days, in a total of four administrations. The following parameters were evaluated during the treatment period: tumor volume, tumor weight, and animal’s body weight. Histological examination of tumor and organs of interest (liver, spleen, kidney, lung, bone and heart) were performed. Considering the tumor volume analyses, SpHL-DXR were able to stabilize tumor growth in contrast to free doxorubicin. For tumor histological analyses, it were observed large areas of necrosis with cell karyolysis and karyorrhexis in tumors treated with SpHL-DXR, while for the other groups less extensive areas of necrosis and larger areas of viable cells were observed. The histological analyses of organs (liver, spleen, lung, kidney, bone and heart) of animals treated with SpHL-DXR showed no alterations in cell architecture, suggesting lack of toxicity.