| A major problem in the clinical treatment of cancer with PTX is multidrug resistance (MDR) in tumor cells. MDR is a frequent phenomenon whereby cancer cells become resistant to the cytotoxic effects of various structurally and mechanistically unrelated chemotherapeutic agents. Once the MDR occurred, the intracellular drug accumulation reduced and the sensitivity of tumor cells to drugs was significantly decreased. Therefore, development of MDR is a major obstacle to the success of cancer chemotherapy. Overexpression of P-gp encoded by the MDRl is the most commonly encountered in successful cancer therapy with PTX. An attempt to overcome P-gp-based MDR in cancer chemotherapy is to co-administer a P-gp inhibitor along with the anticancer drug.Paclitaxel (PTX), one of the most successful anticancer drugs, is the first of a new class of microtubule stabilizing agents, and has been demonstrated significant antitumor activity in clinical trials against a broad range of solid tumors. Verapamil (VRP), a first generation P-gp inhibitor, has been reported to be able to reverse completely the resistance caused by P-gp in vitro. Therefore, to overcome the MDR of tumor cells, decrease the side effects and improve the effectiveness of PTX and VRP, it is a potential approach to co-encapsulating the two agents by using a targeted delivery system.O-carboxymethylated chitosan (OCMC) was firstly hydrophobically modified with various deoxycholic acid (DOCA) to obtain a novel kind of polymer amphiphiles, and then covalently bounded with folic acid (FA) to develop a new cancer-targeted potential drug delivery system. The characterizations of the polymers were analyzed by FTIR,1HNMR and XRD. The physicochemical properties of the self-aggregates in aqueous media were investigated by1HNMR, zetasizer, zeta potential, fluorescence spectroscopy, and transmission electron microscopy (TEM). The mean diameter of the self-aggregates in PBS solution (pH7.4) was decreased with the degree of substitution (DS) of DOCA increasing. The mean diameters of self-aggregates of DOMCs were in the range of231-383nm and DOMC-FAs were slightly smaller in the range of179-212nm, with a narrow and unimodal size distribution. The TEM images of self-aggregates showed a spherical shape. The self-aggregates covered with negatively charged OCMC shells, exhibiting zeta potentials near-20mV in PBS solution (pH7.4). The critical aggregation concentrations of DOMC conjugates were in the range of0.027-0.065mg/mL, and the cac of DOMC-FA conjugates were slightly lower than that of DOMC7. The mean diameters and the cac values depended on the DS and changed with the pH value.The self-assemble method appears to be particularly suitable for the incorporation of PTX and VRP into DOMC or DOMC-FA micelles. Both PTX and VRP were encapsulated efficiently in micelles. The average loading of PTX in DOMC and DOMC-FA micelles was27.95±0.33and33.61±0.96(64.66±1.06and84.43±3.66encapsulation efficiency), respectively, while the average loading of VRP in DOMC-FA micelles was33.42±1.28(82.28±1.59encapsulation efficiency). The drug incorporation of the optimized DOMC-FA/(PTX+VRP) micelles was improved significantly, with an entrapment efficiency of95.28±1.31for PTX and72.15±1.76for VRP, respectively. The data of our preliminary cell experiment demonstrated that5μM of VRP could achieve a higher reversal efficacy in the resistance of MCF-7/ADR to20nm of PTX. So, the molar ratio of PTX to VRP entrapped into micelles was approximately1/250, which might achieve a desirable efficacy in reversing drug resistance. The size and size distribution of drug-loaded micelles were characterized by Zetasizer and exhibited a mean particle diameter of about258-356nm for the different formulations. All the drug-loaded micelles had relatively high negative zeta potentials of around-20mV. The negative zeta potential indicated that the micelle surface was negatively charged. In our study, the drug-load micelles were stable with good fluidity when sealed and stored under conditions such as refrigeration or at room temperature for three months. The appearance and drug-loading efficiencies hardly changed.In vitro drug release study, pH7.4and pH6.2phosphate buffer solution were selected to simulate the environment of blood and internal environment of tumor cells, respectively.In vitro cumulative release profiles of PTX and VRP from different formulations displayed that the release profiles of PTX and VRP in the different pH environment were similar. In contrast with the drugs release from stock solution, there was a pronounced time prolongation of drugs release from micelles. For example, only about80wt.%PTX was released in192h and about75wt.%VRP was released in144h in pH7.4. The drug-loaded DOMC-FA micelles were complex system, so that DOMC degraded very slowly and released the drug in a sustained manner. This result showed that the micelle carrier can not only solubilize the poorly soluble drugs, but also sustain controlled drug release. Where as the drugs cumulative release rates showed slight pH dependence, for instance, the PTX cumulative release rates increased from79%at pH7.4to95%at pH6.2. This observation indicated that the micelles could release more drugs in tumor tissue. The molar ratio of the PTX and VRP releasing from DOMC-FA/(PTX+VRP) micelles was still around1/250, which might not affect the reversal efficacy. These results indicated that the PTX and VRP loaded DOMC-FA micelles are useful controlled delivery system for cancer treatment.The MTT assay showed that the blank micelles were far less toxic than the Cremophor EL vehicle. There was an increased level of uptake of folate-conjugated micelles compared with plain micelles in folate receptor overexpressed human breast cancer cells, MCF-7cells, and the uptake mainly on account of the effective process of folate receptor-mediated endocytosis. The MTT assay, morphological changes and apoptosis test implied that the folate-conjugated micelles enhanced the cell-killing effect by folate-mediated active internalization, and the cytotoxicity of the FA-micellar PTX (DOMC-FA/PTX) to cancer cells was much higher than micelles without folate (DOMC/PTX) or the commercially available injectable preparation of PTX (Taxol). The results of this research demonstrated the folate-conjugated micelles could be beneficial in treatment of solid tumors by targeting delivery of micellar PTX into the tumor cells and further reducing side effects and toxicities of the drugs.Folate-functionalized micelles co-encapsulating PTX and VRP demonstrated significant inhibition of growth of drug resistant tumor cells at a PTX dose that was ineffective in the absence of VRP. We are currently investigating the floate-functionalized dual agent micelles (DOMC-FA/(PTX+VRP) formulation), with the objective of sustaining the cellular levels of both the anticancer drug and the P-gp inhibitor, thus, leading to sustained inhibition of P-gp mediated drug efflux and sustained improved PTX induced therapeutic effect. MTT assay, morphological changes, cell cycle analyz and Annexin V-FITC/PI staining suggested that dual agent micelles was a very promising approach to overcome tumor drug resistance and the micellar PTX treatment did not change the mechanism of PTX-induced apoptosis. The synergistic effect of folate receptor-mediated internalization and VRP-mediated overcoming MDR could be beneficial in treatment of MDR solid tumors by targeting delivery of micellar PTX into tumor cells, reducing side effects and toxicities to normal tissues or organs and fewer drug-drug interactions.The tissue distribution DOMC-FA/PTX micelles and Taxol injection was investigated after i.v. administrations into mouse. Compared with Taxol injection, DOMC-FA/PTX micelles distinctly changed the distribution of PTX in vivo and increased the concentration of drug in target organs greatly. The results of targeting-evaluation experiments showed DOMC-FA/PTX micelles had a marked different in recompared with Taxol injection, i.e.8.29for spleen,3.82for lung,1.78for liver,1.31for blood,0.62for heart and0.35for kidney. DOMC-FA/PTX micelles show a high liver and spleen targeting efficiency in vivo and can keep high drug levels in both tissues for relatively long time. The biodistribution of PTX in the lung and the lung targeting effect of DOMC-FA/PTX micelles is higher than Taxol injection, DOMC-FA/PTX have a better lung retentive ability. The levels of DOMC-FA/PTX micelles in the heart and kidney tissues are significantly reduced which might decrease the side effects.The in vivo pharmacokinetics of DOMC-FA/PTX micelles and Taxol injection were studied with "practical pharmacokinetic program version97". Based on the analysis of the models and parameters, it was concluded that the in vivo pharmacokinetics of those preparations in blood could be described by two-compartment model with i.v. injection. The pharmacokinetic equations of DOMC-FA/PTX micelles and Taxol injection were respectively C=30.49e-2.634t+5.641e-0.616t, C=26.422e-1157t+8.213e-0.302t. The AUC value of DOMC-FA/PTX micelles (53.217mg/L-h) were significantly higher than that (23.288mg/L-h) of Taxol injection, MRT of micelles (1.913h) higher than those (0.892h) of Taxol injection, drug clearance (CL) of micelles (0.158L/h/kg) less than those (0.345L/h/kg) of Taxol injection. The results indicated that DOMC-FA/PTX micelles could significantly lengthen the retention time of drugs in vivo and had a well-sustained release efficacy. |
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