Study On Drug-Pluronic Conjugation Mixed Micelles For Overcoming Multidrug Resistance In Cancer

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. However, the drug-loading content and curative effect to MDR tumor are still unsatisfactory.In present work, a methotrexate (MTX) conjugated polymeric mixed micelles for MDR cancer therapy was developed to improve the drug loading content and antitumor efficacy in MDR tumor of MTX. The dual drugs loaded mixed micelles could be prepared through chemically conjugated one hydrophilic drug and physically entrapped the other hydrophobic drug. And in this way, a much higher drug-loading content and synergistic effect will be achieved. Some clinical studies have shown that incorporation of doxorubicin (DOX) and paclitaxel (PTX) increases tumor regression rates relative to the individual drugs and has been used as first-line treatment for metastatic breast cancer. Therefore, dual drug loaded mixed micelles containing chemically conjugated DOX and physically entrapped PTX.In order to increase the drug delivery efficiency to the integrin proteins predominantly expressed tumor angiogenesis and MDR tumor, c(RGDyK) ligand, a cyclic RGD peptide with high affinity and specificity, was conjugated to PEO chain of F127and was used as the platform for actively targeted drug delivery.In the first chapter, MTX was conjugated with Pluronic P105(P105-MTX). The Pluronic F127and P105-MTX polymeric mixed micelles (F127/P105-MTX) were fabricated by thin-film hydration technique, the drug loading of MTX in F127/P105-MTX was found to be3.3-fold higher than that of physically entrapped MTX mixed micelles (PF-MTX). The optimized formulation of PF-MTX showed the particle size around22nm, while F127/P105-MTX was100nm. The physical status of MTX encapsulated in polymeric matrix was investigated by DSC analysis. F127/P105-MTX had a sustained and pH-dependent release profile for MTX. In vitro cytotoxicity, cell apoptosis and cell cycle arrest studies also demonstrated that F127/P105-MTX had better antitumor efficacy in KBv MDR cells compared to that of PF-MTX. In vitro experiments with endocytic inhibitors suggested that clathrin-mediated endocytosis and caveolae-mediated endocytosis are involved in the internalization of F127/P105-MTX.In the second chapter, the mechanism of F127/P105-MTX has been studied with KBv MDR cell line. The uptake of F127/P105-MTX into KBv cells could be inhibited by free folic acid, indicating that the endocytosis process was receptor specific. In vitro,3D KBv spheroids were developed to evaluate the solid tumor penetration ability, and F127/P105-MTX exhibited the strongest activity in tumor penetration and reduction of KBv tumor spheroid volume and in the apoptosis of the spheroids. The pharmacokinetic parameters for MTX in plasma were estimated by compartmental method. In the case of F127/P105-MTX, the AUC0â†'∞were2.43times higher than that of PF-MTX. In addition, MRT for the formulations of F127/P105-MTX was1.34-fold higher than that of PF-MTX. CL for F127/P105-MTX was significantly lower than that of PF-MTX implying a longer retention of the drug in blood circulation. In vivo multispectral fluorescent imaging indicated that F127/P105-MTX had high specificity and efficiency in tumor targeting in KBv subcutaneous xenograft nude mice model. Furthermore, the in vivo antitumoral activity of F127/P105-MTX against KBv xenograft tumor was assessed. F127/P105-MTX reflected stronger tumor growth inhibition than PF-MTX without any side effects. All of these results demonstrated that chemical conjugation of drug in mixed micelles for overcoming multidrug resistance in cancer is quite feasible.In the third chapter, dual drug loaded mixed micelles (PF-DP) was developed with chemical conjugation of DOX (P105-DOX) and physically entrapped PTX, due to their excellent anti-tumor efficiency in clinical against various solid tumors, particularly breast cancer. The ratio of Pluronic, PTX and DOX in the mixed micellces was300mg:4mg:6mg, when achieving the highest encapsulation efficiency, drug loading content and cytotoxicity. The releases of DOX and PTX from PF-DP at neutral pH were slow and sustained; however, the drug releases were much faster at acidic environment. PF-DP obviously enhanced DOX and PTX accumulation in both KBv and Human Breast Cancer (MCF-7/ADR) MDR cells. The uptake of PF-DP into MCF-7/ADR cells was energy-driven clathrin-mediated endocytosis and caveolae-mediated endocytosis. The results of intracellular distribution by confocal laser microscopy showed that the slightly acid condition in lysosomes triggered DOX release from PF-DP, and enable the following entry into nucleus. Furthermore, the dual drug loaded mixed micelles suppresses sensitive and MDR tumor cells growth more efficiently than the delivery of either DOX or PTX at the same concentrations, indicating a synergistic effect.In the fourth chapter, c(RGDyK) modified dual drug loaded mixed micelles composed of Pluronic P105and F127loaded with DOX and PTX (c(RGDyK)) was prepared and characterized. The c(RGDyK) ligand, a cyclic RGD peptide that can bind to the integrin proteins detected in the activate endothelial cells and some types of cancer cells with high affinity and specificity, was conjugated to the NHS-activated PEO terminus of F127. The c(RGDyK)-functionalized dual drug loaded mixed micelles was fabricated by the thin film hydration technique and was used as the platform for actively targeted DOX and PTX delivery to integrin αvβ3-rich angiogenesis endothelial cells and MDR cancer cells. Human umbilical vein endothelial cell (HUVEC) was included into the cellular level experiments as the endothelial cell model of tumor neovasculature. c(RGDyK) modified mixed micelles obviously enhanced integrin-receptor rich HUVEC uptake, and these uptake could be inhibited by free c(RGDyK), indicating that the internalization of c(RGDyK) modified mixed micelles by HUVEC was mediated by integrin proteins transporter. A HUVEC monolayer and MCF-7/ADR3D spheroids co-culture model was developed to evaluate the dual actively drug delivery function. The results revealed that c(RGDyK) modified mixed micelles exhibited the strongest ability in tumor spheroid penetration after crossing the HUVEC monolayer. Without cytotoxicity to HUVECs, c(RGDyK) modified mixed micelles can inhibit of tubular formation, their vital activities to induce the angiogenesis. Moreover, cytotoxicity to KBv cells was enhanced by c(RGDyK)-FP-DP, due to enhanced cell uptake through integrin receptor. In vivo NIR fluorescent imaging indicated that c(RGDyK) modified mixed micelles has much stronger tumor targeting effects than PF-DP.The biodistribution and pharmacodynamic evaluations on KBv tumor bearing nude mice were carried out in the final chapter. It was demonstrated that DOX or PTX AUC0â†'48h of mixed micelles was higher in plasma and tumor, lower in heart, liver and spleen compared to free drugs. There were significantly different in these tissues between mixed micelles and free drug (P<0.05). Compared with PF-DP group, more drugs accumulated in tumor tissues in c(RGDyK)-FP-DP group. c(RGDyK)-FP-DP treatment effectively suppressed the tumor growth and prolonged survival in KBv MDR tumor-xenografted mice model with low cardiotoxicity, suggesting that the combined mechanisms of c(RGDyK)-mediated active targeting, induced tumor neovasculature endothelial cell apoptosis and Pluronic-mediated overcoming MDR be beneficial in treatment of MDR solid tumors.