Study On Polymeric Micelle Drug Delivery Systems For Reversing Multidrug Resistance In Cancer

Multidrug resistance of cancer cells to anti-tumor drugs remains a major impediment to successful chemotherapy. Looking for various strategies and tactics to reverse or overcome multidrug resistance and hence to enhance the efficacy of anticancer drugs is one of the significant mission in the area of tumor therapy. In this thesis the polymeric micelle drug delivery system with the effect of reversal of multidrug resistance was studied and the reversing mechanism was explained to explore a new routine of reversing multidrug resistance by carriers in terms of formulation.The contents of this thesis were including establishment of resistant cancer cell lines, screening of the polymer carrier with potential of reversing multidrug resistance of cancer cells, preparation of the polymeric micelle drug delivery systems of paclitaxel and 9-nitrocamptothecin; investigation of reversing multidrug resistance effect and mechanism of the polymeric micelle drug delivery systems using the resistant cancer cell lines, study of pharmacokinetics and biodistribution of polymeric micelle drug delivery system.Caco-2 cell line with the high level expression of P-glycoprotein and resistant human ovarian cancer cell line, Skov-3/PTX, were established as the resistant cancer cell models. Skov-3/PTX resistant cell line was established by stepwise exposure of ovarian cancer cell line, Skov-3 to paclitaxel for 11 months with stable growth and proliferation in 50 ng/mL PTX. The biological characters of Caco-2 and Skov-3/PTX cell lines were detected. After 14 days cultivation, the Caco-2 cells expressed high activity of P-glycoprotein and showed an obvious resistance to RH-123 and PTX. CsA as P-glycoprotein substrate could partially reverse this resistance. The resistance index (RI) of Skov-3/PTX cells was up to 45.90. They showed an obvious cross-resistance to gefitinib, 9-nitrocamptothecin and adriamycin. PT-PCR studies showed that the levels of MDR1, MRP and GST-p mRNA expression in the Skov-3/PTX cells were higher than the sensitive cells'. The results of uptake of RH-123 and PTX further proved that Skov-3/PTX cells had significant resistance to RH-123 and PTX, and P-glycoprotein substrate, CsA can partially reverse the resistance and increase the uptake of drug in resistant cells.The study of physico-chemical property showed that PTX was a poorly water-soluble drug. Its solubility in water was only about 0.48 μg/mL which did notvary significantly with pH (4-8). It was easy to dissolve in n-octanol and had a good liposolubility. So PTX is suitable as a model drug for amphipathic polymeric micelles. Then the effects of Pluronic series including L61, P123, P105 and F127 on cell uptake were studied with two types of resistant cells. The screening of carriers provided the experiment foundations for the preparation of micelle drug delivery system with property of reversal of multidrug resistance. The results showed that PTX accumulations in resistant cells were enhanced by Pluronic P123, L61 and P105 when the concentration of carriers was above critical micelle concentration (CMC), and the effects of P123 and L61 were more obvious than P105. However, F127 had no effect on uptake of PTX in resistant cells. So Pluronic P123 and L61 were chosed as alternative carriers for preparation studies of polymeric micelles.The hydrophilic block of Pluronic L61 is too short to dissolve in water at room temperature. So it is hard to formulate into micelle preparations. So the PTX-loaded polymeric micelles were prepared with Pluronic P123 by thin-film hydration methods. Based on the results of single factor experiments, with the evaluation index of entrapment efficiency, drug loading coefficient and drug concentration of the micelle solution, micelle formulation was optimized employing the central composite design-response surface methodology. The final optimized formulation was 2mg of PTX and 5 mL of water phase with 100mg carrier. To enhance the storage stability, the micelles were freeze dried. The initial investigation of freeze drying artwork was carried out and 5% trehalose was used as supporting agent. Studies of the physico-chemical property of micelles showed that the micelles were round shaped with a diameter of about 25.2nm and a relative uniform size. In vitro release of PTX from the micelles was sustained and the polymeric micelles were structurally stable in human plasma.Pluronic P123 micelles could enhance the uptake of PTX and RH-123 in Caco-2 and Skov-3/PTX cell lines and this effect was concentration dependent of carriers. The micelles could also increase the sensitivity of resistant cells to anti-tumor drugs, and the reversing resistance indexes to Caco-2 and Skov-3/PTX cell lines for PTX were 5.04 and 13.77, respectively. Therefore, Pluronic P123 micelles possessed the ability of reversing resistance in cancer cells. In the study of the reversing mechanism, the influence of micelles to cell membrane fluidity, expression of MDR1, MRP, GST mRNA and ATP level in cells were investigated. It showed that once Pluronic P123 formed micelles, it could increase the cell membrane fluidity, and this effect wasconcentration dependant. It also could inhibit the expression of MDR1, MRP, and GST mRNA and deplete the ATP level in resistant cells.After iv administration of Taxol injection and Pluronic P123 micelles to rats and mice, the pharmacokinetics and biodistribution were assessed, respectively, to investigate the in vivo drug release behavior from Pluronic P123 micelles. The result indicated that the polymeric micelles with Pluronic P123 could alter the parameters of pharmacokinetics and biodistribution of paclitaxel. The blood circulation time of paclitaxel in rats and mice was significantly prolonged, and the accumulation of paclitaxel in plasma, kidney, ovary & uterus and lung was increased and the drug accumulation in liver was decreased. The t_1/2β of micelles and Taxol injection in rats were 5.85±1.52 h and 2.50±0.63 h (P<0.05) ; AUC of plasma in rats were 2916.8±873.6 μg·L^-1·h and 1007.9±192.6 μg·L^-1·h; AUC of plasma in mice were 3.35 μg·h·g^-1 and 1.82 μg·h·g^-1,respectively.9-nitrocamptothecin (9-NC) was a poorly water-soluble drug. Its solubility in water was only about 5 μg/mL. This value was not effectively affected by change of pH value (pH1～7). The apparent partition coefficients of 9-NC in n-octanol/water was increased with the increase of pH at first. The partition coefficient reached highest level at pH5.0 with the LogP value of 2.08, and then it decreased as the pH value increased continually. So the liposolubility was relatively higher at pH5.0. The central composite design-response surface methodology with two factors and five levels was employed to optimize the preparation of 9-NC-Pluronic P123 micelles. And the optimized formulation consisted of 1 mg 9-NC and 15 mL water phase with 100 mg carrier. The physico-chemical studies of 9-NC-loaded micelles showed that the micelles had the mean particle size about 23.2nm with narrow size distribution and a spherical shape. 9-NC was sustained release from the formulation in vitro. In addition, the micelle drug delivery system remained stable in human plasma.The drug uptake and cytotoxicity tests of 9-NC micelles in Skov-3/PTX cell lines were carried out. It demonstrated that Pluronic P123 could reverse the cross-resistance of Skov-3/PTX cell lines to 9-NC, increase the drug uptake in resistant cells, enhance the cytotoxicity of 9-NC to resistant cells with the reversing resistant index of 3.57. The pharmacokinetics study in rats showed that the micelle formation could significantly prolong the plasma circulation time of 9-NC, the plasma drug concentration of 9-NC micelles was much higher than the 9-NC solution at the same dose (P<0.05). The AUC_0～t and AUC_0～8 of test group were 3.6 and 3.7 folds higherthan that of control group, respectively. The study of biodistribution in mice indicated that the micelles could significantly increase the drug accumulation in lung, ovary & uterus, kidney and pancreas compared to 9-NC solution. In addition, the accumulation in plasma and spleen were also increased in different levels. But the drug accumulation was lower than the 9-NC solution in liver with no significant change in heart.