In Vitro And In Vivo Evaluation Of Multi-functional Pluronic P123/F127 Mixed Micelles To Overcome MDR Tumor

Multidrug resistance (MDR) of tumor is a major cause for the failure of cancer chemotherapy in clinical trials. To date, various anticancer drugs which are most frequently associated with MDR including taxanes, vinca alkaloids, anthracyclines and so on. Novel drug delivery systems could be used as one novel formulation approach to overcome MDR in cancer. The main objective of this study was to develop a paclitaxel (PTX)-loaded Pluronic polymeric micellar delivery system in order to overcome MDR in cancer and improve the PTX solubility, which would provide the theoretical basis for the future studies on overcoming MDR in cancer.The four chapters of this dissertation are as follows:(1) Formulation and preparation of a novel polymeric mixed micelle composed of Pluronic P123 and F127 loaded with PTX; (2) Enhanced antitumor efficacy in vitro and in vivo by PTX-loaded Pluronic P123/F127 mixed micelles (PF-PTX) in both subcutaneous MDR tumor mouse model and pulmonary metastatic mouse model; (3) Mechanisms on reversal of MDR by Pluronic P123/F127 copolymers; (4) In vitro and in vivo evaluation of folic acid conjugated PTX-loaded Pluronic P123/F127 mixed micelles (FPF-PTX) to overcome tumor MDR.At first, Doehlert matrix design was utilized to investigate the effect of four variables, namely P123 mass fraction, amount of water, feeding of PTX and hydration temperature on the responses including drug-loading coefficient (DL%), encapsulation ratio (ER%) and the percentage of PTX precipitated from the drug-loaded mixed micelles after 48h at 37℃(PTX precipitated%) for improvement of drug solubilization efficiency and micelle stability combined with desirability function. PF-PTX was prepared by thin-film hydration method. The optimized formulation showed a particle size of about 25 nm with ER%>90%, and a sustained release behavior compared to Taxol. Micelle formation was confirmed by 1HNMR and FT-IR spectroscopy. The mixed micelles had a low CMC of 0.0059% in water. In addition, micelle stability studies implied that introduction of Pluronic F127 (33 wt%) into P123 micelle system significantly increased the stability of PF-PTX.Secondly, modulation of MDR by Pluronic mixed micelles was evaluated in lung resistance protein (LRP)-overexpressing human lung adenocarcinoma A-549 cell line. Influence of PF-PTX on in vitro cytotoxicity was determined by MTT assay, while effects of Pluronic P123/F127 copolymers on R-123 and PTX accumulation were investigated. Cellular apoptosis was detected by cell nuclei staining and Annexin V-FITC apoptosis detection kit. Cell cycle arrest was also confirmed by flow cytometry. It was demonstrated that PF-PTX had superior anti-proliferation activity against A-549 cells compared with Taxol as measured by IC50. The enhanced anticancer efficacy of PF-PTX was associated with enhanced drug cellular uptake, PTX-induced apoptosis and cell arrest in the G2/M phase. Intracellular ATP depletion and decreased mitochondrial potential caused by Pluronic copolymers were found to be related to modulation of MDR. The biodistribution study indicated that PTX AUC0→12h of PF-PTX was higher in plasma, tumor, kidney and lung, lower in liver and spleen compared to Taxol. There were significantly different in these tissues between PF-PTX and Taxol (P<0.05). Therefore, the PEO hydrophilic shell of PF-PTX efficiently decreased the adsorption of plasma protein to micelles, and greatly reduced the elimination by MPS and accumulation in the liver and spleen. Therefore, the PF-PTX was expected to be a potential drug delivery system of PTX for overcoming MDR in lung cancer. PF-PTX treatment effectively prolonged survival and suppressed the tumor growth in both mouse models and reduces metastases in the pulmonary model; it also did not display any side effects.Thirdly, four different P-gp substrates including R-123, R-6g, DOX and PTX differing in hydrophobicity was used as molecular probes in this study. Effects of Pluronic P123/F127 copolymers on drug intracellular accumulation were evaluated in P-gp overexpressing human breast cancer cell line NCI/ADR-RES. These data suggested that treatment of NCI/ADR-RES cells with Pluronic P123/F127 copolymers reversed drug resistance to either hydrophilic or to hydrophobic P-gp substrates. Moreover, above the CMC, inhibition of P-gp was not affected but rather factors such as micellar trapping could contribute to decreased drug accumulation. In addition, it was shown that Pluronic P123/F127 copolymers could decrease P-gp efflux by intracellular ATP depletion. Furthermore, most of the Pluronic P123/F127 was localized to mitochondrial, as evidenced by colocalization of red fluorescence with that from MitoTracker Green. It also appeared that mitochondrial membrane potential levels all decreased in a concentration dependent manner after treatment with Pluronic P123/F127 copolymers. It seems that there might be some additional mechanisms, at least in part, through a mitochondrial apoptotic triggered pathway, for modulation of MDR in NCI/ADR-RES cells besides ATP depletion. In vitro experiments with endocytic inhibitors suggested that clathrin-mediated endocytosis and caveolae-mediated endocytosis are involved in the internalization of Pluronic P123/F127 mixed micelles.Fourthly, folic acid modified polymeric mixed micelles composed of Pluronic P123 and F127 loaded with PTX (FPF-PTX) were prepared and characterized. In order to exploit the possibility of combination of active targeting function by receptor-mediated endocytosis and modulation of MDR by Pluronic P123/F127, a number of cellular experimental works such as in vitro cytotoxicity, cell apoptosis, cellular uptake and cell cycle analysis were developed in KB and KBv cells. In vitro experiments with endocytic inhibitors suggested that clathrin-mediated endocytosis and caveolae-mediated endocytosis are involved in the internalization of folic acid modified Pluronic P123/F127 mixed micelles. The uptake of folic acid mixed micells into KBv cells were concentration and time dependent, which could be inhibited by low temperature and free folic acid, indicating that the endocytosis process was energy-driven and receptor specific. The pharmacokinetic parameters for PTX in plasma were estimated by compartmental method. In the case of PF-PTX and FPF-PTX, the AUC0→∞were 2.96 times and 2.67 times higher than that of Taxol. In addition, MRT for the formulations of PF-PTX and FPF-PTX were 4.38-fold and 2.98-fold higher than that of Taxol, respectively. CL for PF-PTX and FPF-PTX were significantly lower than that of Taxol implying a longer retention of the drug in blood circulation. These results suggested that the modification of folic acid did not evidently influence the in vivo long-circulating property of the mixed micelles. FPF-PTX treatment effectively suppressed the tumor growth in tumor-xenografted mouse model. We suggested that the combined mechanisms of folate-mediated active targeting and Pluronic-mediated overcoming MDR be beneficial in treatment of MDR solid tumors.