The Preparation And Anti-tumor Effect Evaluation Of Doxorubicin And Choroquine Co-loaded Liposomes

Doxorubicin (DOX) as a widely used chemotherapeutics always causes multidrug resistance (MDR) in human cancer cells, such as human breast cancer. MDR is a major obstacle to successful clinical cancer chemotherapy. The over-expression of P-glycoprotein (P-gp) and the multidrug-resistance-associated protein (MRP) have been confirmed to confer broad drug resistance in tumor cells. To deal with this problem, we developed a novel formulation that DOX and chloroquine (CQ) were simultaneously loaded into liposomes by a pH gradient method where CQ plays the role of a chemical sensitizer.Firstly, HPLC determination was developed both for DOX and CQ in vitro and it was validated by characterization of linearity, precision as well as accuracy. PBS (pH7.4) was used to elute liposomes and then methanol solution was applied to destroy the structure of liposomes. Both the drugs in eluted liposomes and in the initial liposomes were respectively determined by HPLC and then the encapsulation efficiency (EE) was calculated.Furthermore, various manufacture factors were investigated in order to optimize the DOX and CQ liposomes (DCL) with the high encapsulation efficiencies greater than90%. These factors included effect of Chol/SPC on the EE, effect of Drug-loading content on the EE%and the size of liposome, effect of internal buffer on the EE, effect of internal pH on the EE, effect of molar concentration of the internal buffer on the EE, effect of exterior phase on the EE, effect of incubation temperature and time on the EE and effect of the ratio (DOX:CQ:SPC) on the EE.Thirdly, the selected formulations with different ratios were investigated in PBS with different pH (5.5,6.5and7.4) for their drug release behaviors. Besides, the release behavior of four formulations, DCL, free drug, DOXL and CQL were also tested for compare ration. We also evaluated different formulations of free drug and liposomes by dose-dependent MTT experimentation on MCF-7, MCF-7/ADR, HL60and HL60/ADR cell lines to obtain various IC50values and the CI. It showed that the DCL could efficiently reversal the P-gp and MRP1mediated MDR. The IC50of DCL to P-gp-overexpressing MCF-7/ADR cells and MRP1-overexpressing HL60/ADR cells were5.7and19.5times less than that of free DOX, respectively.Finally, flow cytometry was used to determine the quantities drugs entrapped by the cells and confocal laser-scanning microscopy was used to evaluate DOX cellular uptake and localization. In addition, from the results of the western blot, the expression of P-gp and MRP1of MCF-7/ADR and HL60/ADR cells was significantly inhibited after treatment with DCL. Therefore, we proposed that liposomes simultaneously loaded with the anticancer drug DOX and the chemosensitizer CQ might be a suitable strategy for cancer therapy by the reversal of P-gp-and MRP1-mediated multidrug resistance.