Studies On Docetaxel-loaded Micelles For Overcoming Multidrug Resistance And Tumor-targeting

Docetaxel (DTX) is an important anticancer taxane which has been used with success in patients with various human malignancies. However, there are some shortcomings have considerably impeded its clinical use. The obstacles can be sued up in the following aspects. One cause is the low bio-availability due to the low solubility in water. Another is the emergence of multidrug resistance (MDR), which leads to low drug concentration in tumor cells. A third cause is the lack of target to tumor, thus evoking serious side effects. Therefore, the new formulation is urgent to improve the solubility of DTX as well as to avoid the MDR and minimize undesirable side-effects due to non-target. In present study, we have used the synthesized copolymer PEO-PPO-PCL to construct polymer micelles for loading the anticancer drug DTX. The work is as follows:Part I:It is known that DTX is a substrate of P-gp. The P-gp function is the basis of MDR in tumor cells. DTX can be pumped out of the cell by P-gp and fail to accumulate to cytotoxic levels. TPGS is widely used as P-gp inhibitor in overcoming MDR. Herein, PEO-PPO-PCL was used in combination with TPGS to form mixed micelles to overcome MDR. DTX-loaded micelles were prepared by thin film hydration method. The prepared spherical mixed micelles were found to possess nanoscale size (25 nm-135 nm). The PEO-PPO-PCL/TPGS mixed micelles had low critical micelle concentration (CMC) (～10-6 g/mL) and low hemolysis rate (< 5%), which has proved they were safe for use in vivo. Moreover, they had obvious sustained release behavior in vitro and longer circulation time than free DTX in vivo. The cellular uptake and MTT assay in MCF-7 and A549 cells exhibited that DTX-loaded micelles could overcome MDR, get higher cellular uptake and higher antitumor efficacy than free DTX. The IC50 values demonstrated that the three series DTX-loaded were 69,82 and 100 folds effective than free DTX after 72 h treatment with MCF-7 cells, respectively.Part Ⅱ:The most extensively studied mechanism of MDR is the overexpression of P-glycoprotein (P-gp) which can purge a lot of drugs from cells. In addition, autophagy function is another important resistance mechanism. The combination of two or more drug is a promising strategy to suppress the multidrug resistance (MDR) through different action mechanisms. Here, we co-encapsulate anticancer drug docetaxel (DTX) and autophagy inhibitor chloroquine (CQ) in PEO-PPO-PCL/TPGS micelles. In vitro cytotoxicity with different DTX/CQ mass ratio was studied and the sample with a DTX/CQ ratio of 0.8/0.2 was found to have the strongest synergism effect. The micelles were characterized in terms of morphology, particle size, drug loading content and hemolysis rate. In vitro release was performed in phosphate buffered solution (PBS) at pH 7.4. The in vitro antitumor activity of (DTX/CQ)-loaded PEO-PPO-PCL/TPGS micelles against MCF-7 and MCF-7/Adr cells were evaluated by the MTT method. The cellular uptake was also studied in MCF-7/Adr cells. The dual-drug micelles possessed nanosize and sustained release profile in vitro. Dual-drug loaded micelles have low hemolysis rate (< 5%), indicating that they are safe for use in vivo. Studies on cellular uptake demonstrate that the micelles can effectively accumulate in cancer cells. Furthermore, the co-delivery micelles have obviously higher therapeutic effects against MCF-7 and MCF-7/Adr cells than either free drug or individually DTX-loaded micelles. The IC50 values of (DTX/CQ) loaded PEO68-PPO34-PCL18/TPGS and PEO68-PPO34-PCL36/TPGS micelles are 134.16 and 194.74 fold smaller than that of free DTX after 48 h treatment with MCF-7/Adr cells, respectively.Part III:Flolate was introduced to the PEO-PPO-PCL micelles and we have constructed DTX-loaded FA-PEG2k/PEO-PPO-PCL micelles. The morphologies showed that the micelles were spherical with a narrow size distribution. The mean particle sizes were 136 nm. The micelles possessed sustained release profile in vitro and low hemolysis rate (< 5%). Results of the MTT assay performed against A549 cells and MCF-7 cells indicated that the inhibition capacity was significantly enhanced with DTX-loaded FA-PEG2k/PEO-PPO-PCL in comparison to free DTX. And compared with A549 cells (FR-negative), DTX-loaded FA-PEG2k/PEO-PPO-PCL micelles exhibited a higher inhibitory effect on MCF-7cells (FR-positive). Results of the cellular uptake process evidenced an increasing fluorescence intensity of the cells treated with FA-PEG2k/PEO-PPO-PCL ccompared with free coumarin-6 against A549 cells and MCF-7 cells. For MCF-7 cells (FR-positive), the intake of coumarin-6 loaded FA-PEG2k/PEO-PPO-PCL micelles was much stronger than the non-flolate micelles. However, for A549 cells (FR-negative), the uptake was not much different between flolate micelles and non-flolate micelles.