Paclitaxel Loaded Micellar Delivery System Based On Pluronic Block Copolymer For Overcoming Multidrug Resistance In Cancer

Multidrug resistance of cancer cells is a major cause for the failure of anti-cancerchemotherapy in the treatment of cancer patients. So far, various anticancer drugswhich are most frequently associated with multidrug resistance (MDR) includetaxanes, vinca alkaloids, anthracyclines, epipodophyllotoxins, antimetabolites,topotecan, dactinomycin and mitomycin C. Establishment of various strategies andapproaches to inhibit or circumvent MDR and hence to enhance the efficacy ofanticancer drugs is one of the significant missions in the area of tumor therapy. Drugdelivery system (DDS) is a novel approach to overcoming MDR in cancer nowadays.The main aims of this research were to investigate a novel mieellar delivery system toovercome MDR in cancer, based on Pluronic and paxlitaxel, which will provide thebasis for future studies of overcoming drug resistance and ultimately improvingchemotherapy and the outcome of cancer patients.The contents of this thesis were including (1) preparation and characterization ofpolymeric mieellar delivery systems of paelitaxel; (2) evaluation of reversing MDReffect of the polymeric micelles with the resistant cancer cell lines MCF-7/ADR orSKOV-3/PTX; (3) investigation of the mechanism of overcoming MDR with theresistant cancer cell lines SKOV-3/PTX; (4) study of pharmaeokineties,biodistribution and in vivo anticaneer efficacy of the micellar delivery system ofpaclitaxel.PTX-loaded polymeric micelles were prepared with Pluronic P105 by thin-filmhydration methods. Based on the results of single factor experiments, with theevaluation index of entrapment efficiency, drug loading coefficient and drugconcentration of the micelle solution, micelle formulation was optimized employingthe central composite design-response surface methodology. The final optimizedformulation was 6mg of PTX, 300mg cartier, 5 mL of water phase and 70℃temperature of hydration. Studies of the physieo-chemical property of micellesshowed that the mieelle size was about ca. 24 nm with drug loading coefficient of ca.1ï¼…and PTX concentration of ca. 650μg/ml. In order to improve the ability ofsolubilizing PTX, Pluronic P105 was modified with two strategies. One approach wasto prepare a mixed P105/L101 micelle with P105:L101=8:1(v/v) based on the resultof interaction of binary Pluronic P105 and L101 in aqueous solution. Another approach was to synthesis PCL-P105-PCL and then to prepare the micelles with threedifferent PCL-P 105-PCL block copolymers(P 105/PCL5, P 105/PCL20, P 105/PCL50).The mixed P105/L101 micelle showed high solubilization capacity for PTX with drugloading coefficient of ca. 2ï¼…, PTX concentration of ca. 1000μg/ml and micelle size ofca. 185nm. The P105/PCL50 micelle showed higher solubilization capacity for PTXwith drug loading coefficient of ca. 5ï¼…, PTX concentration of ca. 1000μg/ml andmicelle size of ca. 150nm. The in vitro release experiment showed that three micellarPTXs (P105/PTX, P105/L101/PTX P105/PCL50/PTX) sustained the release of PTXfrom micelles. The release profile of PTX from Taxol was similar with the PTX stocksolution. The cumulative release amount of PTX from Taxol in 6 h was around 95.2ï¼….Pluronic P105 micelles released only 45.4ï¼…PTX in 6 h (P＜0.05) and 79.6ï¼…PTX in24 h, which was much slower than Taxol. The release of PTX from P105/PCL50/PTXand P105/L101/PTX were faster than P105/PTX, but they were much slower thanTaxol(P＜0.05). The release rates of four PTX formulations in an ascending order wereP105/PTX, P105/PCL50/PTX, P105/L101/PTX, Taxol.The cytotoxicity of polymeric micellar PTX was assessed against human breastcancer cell line (MCF-7, MCF-7/ADR) or human ovarian cancer cell line (SKOV-3,SKOV-3/PTX) by a standard MTT assay. The results demonstrated that Pluronicmicellar PTX (P105/PTX or P105/L101/PTX) were able to reverse resistance to PTXin MCF-7/ADR and SKOV-3/PTX tumor cells compared with free PTX solution. Inthe case of human ovarian cancer cell, the IC₅₀ of PTX solution(1ï¼…DMSO), Taxol,P105/PTX and P105/L101/PTX against MCF-7/ADR were ca 425.7±69.8, 88.4±6.5,30.1±4.4, 19.3±5.4ng/ml, respectively, comparing with the IC₅₀ value ranging from10.8 to 12.4ng/ml against MCF-7. The resistance reversion indexes (RRI) of Taxol,P105/PTX and P105/L101/PTX against MCF-7/ADR were ca. 4.82, 14.10, 22.10,respectively. In the ease of human ovarian cancer cell line, the IC₅₀ of PTXsolution(1ï¼…DMSO), Taxol, P105/PTX and P105/L101/PTX against SKOV-3/PTXwere ca 11.1±2.9, 5.11±1.78, 1.14±0.07, 0.47±0.11μg/ml, respectively, comparingwith the IC₅₀ value ranging from 0.11-0.17μg/ml against SKOV-3. The resistancereversion index (RRI) of Taxol, P105/PTX and P105/L101/PTX againstSKOV-3/PTX was ca. 2.15, 9.65, 23.40, respectively. Three P 105-PCL micellar PTXs(i.e. P105/PCL5/PTX, P105/PCL20/PTX or P105/PCL50 /PTX) were also able toreverse resistance to PTX in SKOV-3/PTX tumor cells compared with free PTXsolution. IC₅₀ of P105-PCL50/PTX, P105-PCL20/PTX and P105-PCL5/PTX against SKOV-3/PTX were ca 0.20±0.05, 0.41±0.08, 0.67±0.24μg/ml, respectively,comparing with the IC₅₀ ranging from 0.06-0.17μg/ml against SKOV-3. Theresistance reversion indexes (RRI) of three micelles against SKOV-3/PTX were ca.55.00, 26.83, 16.42, respectively.Based on the experimental results mentioned above, two folate-mediatedPluronic/PTX micelles (FOL-P105/PTX, FOL-PI05/L101/PTX) were prepared inorder to investigate whether folate-mediated micellar PTXs increase theircytotoxicities in tumor cells. The results demonstrated that FOL-micellar PTX had asignificant increase in cellular uptake compared with a plain micellar PTX during 2.5h incubation with MCF-7/ADR cells (P＜0.05). 1 mM free folic acid significantlyreduced the PTX uptake in MCF-7/ADR cells incubated with FOL-micellar PTX for90 min (P＜0.05), but had no significant effect on PTX uptake in the case of plainmicellar PTX or free PTX. Two FOL-micellar PTXs significantly enhanced thecytotoxicity of PTX against MCF-7 and MCF-7/ADR cells compared with plainmicellar PTX or free PTX solution. IC₅₀ of FOL-micellar PTXs against MCF-7 andMCF-7/ADR cells were 6.4±0.6, 9.4±2.6 ng/ml (FOL-P105/PTX) and 5.8±2.6,7.5±1.5 ng/ml(FOL-P105/L101/PTX), respectively.With PTX-resistant SKOV-3 tumor cells as an in vitro model of MDR tumor cells,a combination of experiments examining effects of five carriers (P 105, P 105/L 101,P105/PCL5, P105/PCL20, P105/PCL50 ) on R123 accumulation, membranemicroviscosity, Pgp ATPase activity, intracellular ATP and mitochondrialtransmembrane potential in SKOV-3/PTX tumor cells was used to uncover themechanism of action of the micellar formulation based on Pluronic P105. The resultsshowed the five carriers over the different experimental concentration range enhancedthe R123 accumulation in SKOV-3/PTX tumor cells, which was believed to correlatewith inhibition of Pgp in SKOV3/PTX cells, because R123, a substrate of Pgp, wascommonly used for evaluation of the Pgp mediated drug efflux in MDR cancer cells.Five carriers not only caused a dramatic decrease in the intracellular ATP levels and aloss in mitochondrial transmembrane potential, but also induced drastic decrease inthe membrane microviscosity and inhibitory effect on Pgp ATPase activity over thedifferent experimental concentration range. These results indicated that themechanism of reversal of the MDR cancer cells by these polymers involved ATPdepletion, the decrease in membrane microviscosity, and the interaction of thepolymer molecules with cell membranes accompanied by significant inhibition of Pgp ATPase activity, which all had a combined result of potent inhibition of Pgp inSKOV-3/PTX tumor cells. In addition to inhibition of Pgp efflux system, PluronicP105/L101/PTX formulation (PTX=10μg/mL Pluronic=0.25ï¼…) induced apoptosis inthe resistant SKOV-3/PTX cancer cells, which was believed to be another mechanismof action of the micellar formulation sensitizing MDR tumor cells. Decrease ofmitochondrial transmembrane potential was believed to be one of the earlyintracellular events of apoptosis induced by Pluronic P 105/L101 micelles.In order to understand the mechanisms of Pluronic effects in various cells better,further studies were carried out to correlate the global gene expression profiles ofSKOV-3/PTX and sensitizing responses to Pluronic formulations applying thepharmacogenomic approach. The results showed that there were significantlydifferent changes in the global gene expression profiles of SKOV-3/PTX exposure tomixed P105/L101/PTX micelle (PTX=10μg/mL Pluronic=0.25ï¼…), PTX solution(1ï¼…DMSO) or blank control (DMEM medium). There were 2293 differentlyexpressed genes with four samples. Further analysis was carried out to investigate thedifferently expressed genes involved in apoptosis, drug resistance/metabolism and cellcycle. The results demonstrated that formulation of the drug with Pluronic P 105/L101could affect the expression of those genes, of which most of genes as a growth-suppressing signal could inhibit tumor growth and enhanced apoptosis, butsimultaneously some genes as a growth-enhancing signal could enhance tumorgrowth.Pharmacokinetics and biodistribution of the three polymeric micelles (P105/PTX,P105/L101/PTX, P105/PCL50/PTX) were assessed by i.v. administration of Taxolinjection and the three polymeric micelles to rats and mice, respectively. The resultsof pharmacokinetics study in rats indicated that the three polymeric micelles couldalter the parameters of pharmacokinetics of paclitaxel. The blood circulation time ofpaclitaxel loaded in the three polymeric micelles was significantly prolongedcompared to Taxol injection. The AUCs of four PTX formulations in a descendingorder were P105/PTX＞P105/L101/PTX＞P105/PCL50/PTX＞Taxol; The CLs in adescending order were Taxol＞P105/PCL50/PTX＞P105/L101/PTX＞P105/PTX; t_/2ain a descending order were P105/PCL50/PTX＞P105/L101/PTX＞P105/PTX＞Taxol, t_1/2β in a descending order were P105/L101/PTX＞P105/PTX＞P105/PCL50/PTX＞Taxolo The in vitro release behaviors of the three polymericmicelles were basically correlated to the prolonged blood circulation time of three micellar paclitaxel in vivo. The study of biodistrabution in mice showed the drugaccumulation of the three polymeric micelles in plasma was increased and the drugaccumulation of the three in liver was decreased, which also indicated the sustainedrelease of the three micelles in vivo. The drug accumulation of P105/PTX andP105/L101/PTX in lung, spleen and kindey was significantly increased. Like theP123/PTX, the drug accumulation of P105/PCL50/PTX in lung, spleen, kindey andovary/uterus was increased.Further studies were carried out to investigate whether P105/PCL50/PTX couldinhibit the growth of s.c. human ovarian SKOV-3/PTX carcinoma xenografts inBALB/c nude mice compared with reference Taxol injection. The results indicatedthat the polymeric micellar PTX showed more potent inhibition of tumor growth thanTaxol injection with 12 mg/kg PTX equivalent dose (P＜0.05). Inhibition ratio(ï¼…) oftumor growth was 43.9ï¼…and 63.4ï¼…for Taxol injection and the polymeric micellarPTX, respectively, which demonstrated that P105/PCL50/PTX could significantlyinhibit the growth of resistant SKOV-3/PTX tumor in BALB/c nude mice.