| Vincristine (VCR) is a widely used anti-cancer drug. The application of VCR in cancer chemotherapy has mostly been limited by dose-dependent toxicity and development of drug resistance resulting from repeated administrations. Quinine (QN) is one of the traditional antimalarial drugs, and it is found to be a good candidate for reversing multi drug resistance (MDR). Therefore, in order to improve the therapeutic effect of VCR for drug-resistance tumor and reduce the side effect, we investigated the mechanisms of QN reversing multi drug resistance and prepared the VCR and QN codelivery liposome (VQL). The specific research work is divided into the following several parts:In this study, we first clarified the action mechanism of QN as a potential sensitizer for VCR on three kinds of MDR cancer cells through the several pathways including induced intracellular depletion of ATP, increased ATPase activity and decreased P-gp expression in cells. The results show that QN can reduce the content of ATP in three resistant cells and QN has high affinity with P-gp, compared with other resistance reversal agents. Additional, QN can reduce the expression of P-gp. Therefore, QN is a good candidate for reversing MDR.According to the physical and chemical properties of VCR and QN, we used thin-film hydration method to prepare the liposomes by. Then we separated the liposome and free drugs by Sephadex G50 gel column chromatography. We used PBS (pH 7.4) to elute liposomes and free drugs and then used methanol solution to destroy the structure of liposomes. HPLC was developed for VCR determination and UV was developed for QN determination in vitro and it was validated by characterization of linearity. Both the drugs in eluted liposomes and in the initial liposomes were respectively determined by HPLC and UV methods and then the encapsulation efficiency (EE) was calculated. Furthermore, effect of Drug-loading content on the EE% and the size of liposome were investigated in order to optimize the VCR and QN liposomes (VQL) with the high encapsulation efficiency.After preparing, we investigated the characterization of the liposome, including the appearance, size and electron microscope scanning, and then examined the in vitro release behavior of three proportions of liposomes. We found that when the pH of release media declined from 7.4 to 5.5, an obviously faster release occurred for both drugs.Then, we also investigated the antitumor effect of the liposomes in vitro and in vivo. First, we detected the cytotoxicity of the liposome and free drugs on A549/T, HCT-8/V and MCF-7/A cells by MTT experiment, the results showed that VQL has a strong reversal resistance effect on three resistance cells, the IC50 value decreased to 0.095±0.008μg/mL、1.36±0.25μg/mL and 2.95±1.17μg/mL. The cellular uptake of VCR in A549/T, HCT-8/V and MCF-7/ADR cells was determined by HPLC. The uptake of VCR in co-delivery groups increased over time while the free drug teams decreased.And we constructed the multi-cellular tumor spheroids (MTS) as a new model to study the cytotoxicity of liposome and conducted animal experiments to verify the in vivo efficacy of the VQL. And the in vivo anti-tumor evaluation was performed on MDR tumor bearing mice, which was further displayed the prominent capability of VQL to reverse VCR resistance with the highest tumor growth inhibitionAll the findings suggest that VQL could be a promising nano-scaled therapeutic agent to overcome the MDR, and may have important clinical implications against cancer treatment. |
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