| Cancer has been a great threat to human health, and chemotherapy has been an important means of cancer treatment. Taxol(?), a preparation of paclitaxel (PTX) which has bas been widely used in clinical treatment, has a strong hematologic toxicity and high incidence of allergic reaction. Genexol(?)-PM, produced by Samyang Company in Korea has decreased the blood toxicity and the possibility of allergic reaction through the application of PEG-PLA micelle drug delivery system, and improved the safety of drug. But compared with Taxol(?), Genexol(?)-PM does not increase the relative drug accumulation in tumor, oppositely less than Taxol(?), also fails to solve the multi-drug resistance (MDR) problem which is regarded as a significant obstacle of administration of PTX to cancer patients. To increase anti-tumor efficiency of Genexol(?)-PM by overcoming MDR in tumor and achieving active target effect is a new challenge. Vitamin E-TPGS (VE-TPGS) is an amphiphilic block copolymer which has short chain and low CMC, proved to be an inhibitor of P-gp that the main cause of MDR. Tumor-specific cell receptor AnnexinAl (AnnAl) and its specific ligand IF-7 can be used to achieve active tumor targeting effect. We choose PEG-PLA (Genexol(?)-PM original vehicle) and VE-TPGS as carrier materials, preparing PTX-loaded mixed micelle, modified with the peptide IF-7 partially on its surface, to develop an AnnAl receptor-mediated and PTX-loaded PEG-PLA/VE-TPGS mixed micellar delivery system (IF-7-PV-PTX) ultimately. We hope it could enhance the treatment of MDR tumors by the double effects of overcoming MDR and active tumor targeting.In the first part, the PTX-loaded PEG-PLA/VE-TPGS (PV-PTX) mixed micelle was prepared by film hydration method. The formulation and preparing process was optimized by single factor method and a central composite design, evaluated with particle size, encapsulation ratio (ER%), drug-loading coefficient (DL%) and the percentage of PTX precipitated from the micelles after 24h at 37℃。Ultimately we obtained an optimized formulation:VE-TPGS:PEG-PLA=13:87; PTX dosage was 10mg, dissolved in acetonitrile; the film was hydrated by 10ml deionized water at 25℃. The optimized mixed micelle showed a particle size of about 17nm with 24% for DL% and 95% for ER%. The value of CMC was between the two materials, respectively. PTX was contained in the hydrophobic core of the mixed micelles in the form of amorphous molecule or solid solution. The sustained release behavior of mixed micelles in vitro was similar to PEG-PLA-PTX.In the second part, we chose A549 and KBv as MDR cell model to evaluate the modulation of MDR by mixed micelles through the cellular uptake and cytotoxicity experiments in vitro. We also investigated the endocytic pathway and the position of the micelles inside the cells. The results showed that the improved anticancer efficacy of PV-PTX micelles was associated with the enhanced cellular uptake by overcoming MDR. After internalized by A549 cell, the mixed micelle was delivered to lysosome through caveolae-mediated endocytosis and clathrin-mediated endocytosis, which consumed energy. Based on the results of cell experiments, we can conclude that PV-PTX mixed micelle is an ideal drug delivery system for overcoming MDR.In the third part, IF-7 was conjugated to the mal-activated PEG terminus of PEG-PLA, and we replaced 10%(w/w) PEG-PLA with IF-7-PEG-PLA to prepare IF-7 functionalized mixed micelle. The peptide IF-7 which was exposed on the micellar surface could help to enhance the micelle accumulation in the tumor by receptor-ligand interaction. Physiochemical characterization of IF-7-PV-PTX showed that the particle size was about 48nm, which is increased significantly compared with that of PV-PTX. However, the appearance is still round and uniform. A similar release behavior in vitro was observed compared to PV-PTX. Coumarin 6 and DiR were used as fluorescent probes to investigate the tumor targeting ability of IF-7-PV-PTX micelle in vitro and in vivo. A549, B16F10 and HUVEC cells on which the AnnA1 receptors predominantly expressed were chose as cell model. The results of cell experiments demonstrated that IF-7-PV-PTX not only maintained the characteristics of PV-PTX to overcome MDR of tumor cells, but also targeted to tumor cells and tumor vascular endothelial cells actively.3D A549 spheroids were developed to evaluate the solid tumor penetration ability in vitro, and IF-7-PV-PTX exhibited the strongest activity in tumor penetration and the apoptosis of the tumor spheroids. In vivo multispectral fluorescent imaging indicated that IF-7-PV micelle had high specificity and efficiency in active tumor targeting in both subcutaneous tumor-bearing nude mice model and melanoma pulmonary metastatic mice model. The results showed that 2h after administration, IF-7 significantly improved the distribution rate of micelles to tumor and the amount of micelles accumulated in tumor site.In the final part, we studied the pharmacokinetic parameters of IF-7-PV-PTX micelles in beagle dogs, and applied subcutaneous A549 tumor-bearing nude mice model to further assess the antitumor ability of IF-7-PV-PTX. The pharmacokinetic behaviors in vivo of PEG-PLA-PTX, PV-PTX and IF-7-PV-PTX micelles were similar. However, Cmax of IF-7-PV-PTX micelle was significantly lower than PEG-PLA-PTX micelle, and the distribution half-life t1/2(α) of IF-7-PV-PTX was significantly shorter (P<0.05) than PEG-PLA-PTX, which indicated that the distribution rate of IF-7-PV-PTX from the plasma to tissue become faster. The pharmacodynamic results showed that IF-7-PV-PTX could significantly enhance the inhibition efficacy of MDR tumor growth compared with PEG-PLA-PTX MDR. All results confirmed that IF-7-PV-PTX micelle could enhance the treatment of MDR tumors by the double effects of overcoming MDR and active tumor targeting. |
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