Lipossomas ph-sensíveis contendo complexo deciclodextrina catiônica/DNA plasmidial: otimização do método de preparo, caracterização físico-química eestudo de transfecção

Abstract

Among non-viral vectors, pH-sensitive liposomes are an attractive alternative in gene therapy, because they undergo a phase transition, leading to destabilization of the bilayer and release of encapsulated material at acid pH, as it occurs within the endosoma. In this paper, we report the development of pH-sensitive liposomes composed of DOPE:CHEMS (molar ratio 6:4, respectively) containing pAct plasmid -able to express the gene of activin - which reduces prostate cancer cell growth. These liposomes, called LpAct, were produced by reverse phase method, and pAct plasmid was previously complexed with -monodeoxy-6-monoamine--cyclodextrin (Am--CD+) at a 1/1 charge ratio (+/-). The 24 full factorial design was used to evaluate the influence of rotation speed, vacuum intensity, total lipid concentrationand DNA concentration on encapsulation efficiency of pAct in liposomes. According to the results, the intensity of the vacuum and the pAct concentration were the main factors affecting the encapsulation of pAct in liposomes. This study allowed to establish the optimal conditions to the production of LpAct. These conditions are: rotation speed of 120 rpm, vaccum intensity of 300 mbar, lipid concentration of 10mM and pAct concentration of 30 g/mL. The percentage of pAct encapsulated was approximately 85% (25 g/mL) using this protocol. Energy dispersive X-ray diffraction (EDXD) was used to investigate the supramolecular organization of DOPE molecules in the presence of Am--CD+/pAct-, culture medium components and proteins of fetalbovine serum, at pH 7.4 and 5.0. At physiological pH, the study revealed that the Am- -CD+/pAct- complex interacts with the lipid membrane resulting in the presence of lamellar and hexagonal phases. However, at pH 5.0, only the hexagonal phase was observed. At pH 5.0 and 7.0, the presence of the culture medium components and proteins led to the formation of less ordered structures, however, the pH-sensitivity ofthe system was maintained. The MTT method was used to evaluate the cytotoxicity of LpAct on prostate cancer cell line LNCaP. The transfection capacity of LpAct in these cells was investigated by expression of green fluorescent protein (EGFP). This study included the evaluation of different time intervals of transfection (4, 6, 24 and 48 hours) and the influence of the presence of fetal bovine serum. According to the results, LpAct were able to transfect LNCaP cells, but with lower efficiency than thatobserved with cationic liposomes used as control (Lipofectamine 2000).