Folate Receptor-mediated Anti-tumor Activity Of Biodegradable Paclitaxel-polymer Conjugate Micelles On Esophageal Cancer Cell Line EC9706: In Vitro And In Vivo

It is estimated that there were482,300new esophageal cancer cases and406,800deathsin2008worldwide, and incidence rates vary from country to country by nearly16-fold, withthe highest rates found in Southern and Eastern Africa and Eastern Asia, such as Ethiopia,China, and Mongolia. Esophageal cancer is the6th most common malignancy worldwide. It ischaracterized by rapid progression and fatal prognosis in most cases. The overall five-yearsurvival rate is approximately15%, with most patients dying within the first year ofdiagnosis.The folate receptor (FR) is over-expressed in many types of human cancers. It canmediate endocytosis of folic acid (FA) or FA-carrying drugs. Based on this specific interactionbetween FA and FR, folic acid and paclitaxel (PTX) containing micelles (FA-M(PTX)) wereprepared by co-assembling an FA-polymer conjugate, poly(ethylene glycol)-b-poly(L-lactide-co-2,2-dihydroxylmethyl-propylene carbonate/FA)[PEG-b-P(LA-co-DHP/FA), FAcontent1.4wt%] and a PTX-polymer conjugate, poly(ethylene glycol)-b-poly(L-lactide-co-2-methyl-2-carboxyl-propylene carbonate/PTX)[PEG-b-P(LA-co-DCC/PTX), PTXcontent25wt%] with a mass ratio of1:9. Anti-tumor activity of FA-M(PTX) on esophagealEC9706cancers were evaluated on EC9706xenograft nude mice models by tumor size andweight measurements, survival time analysis, flow cytometry and TUNEL (terminaldeoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling) assay,immunohistochemistry (IHC) and histopathology. The results showed that FA-M(PTX)exhibits significant anti-tumor effects in vivo compared to pure PTX and simplePTX-polymer micelles at an equivalent PTX dose of20mg/kg. This enhanced efficacy ofFA-M(PTX) is ascribed to the targeting effect of FA moieties in the micelles although theFA-content in FA-(PTX) is only1.4wt%. Therefore, conjugation of both PTX and FA ontothe hydrophobic segments of the block copolymer and co-assembling of the PTX-polymerand FA-polymer conjugates are successful strategies to construct cancer cell targeting drugdelivery systems. The antitumor drug Paclitaxel (Taxol) stabilizes microtubules and reduces their dynamicity, promoting mitotic arrest and cell death, which is widely used in chemotherapy for various carcinomas. However, the clinical application of paclitaxel in cancer therapy is limited due to its low water solubility and non-selective toxicity. However, its clinical utility is hampered by poor aqueous solubility and non-selective toxicity. Delivery systems that specifically target paclitaxel to the tumor site are anticipated to (a) reduce systemic toxicity,(b) improve efficiency of paclitaxel delivery to tumors and increase its dwell time within the tumor,(c) lower the paclitaxel dosage required to achieve effective tumor growth reduction, and (d) improve paclitaxel's solubility. Additionally, increased accumulation of paclitaxel is observed in tumors due to passive targeting of the nanoparticle that occurs as a result of the enhanced permeability and retention effect (EPR). Antitumor agent-encapsulated nano-micelles from amphiphilic block copolymers have attracted considerable attention due to their mechanical strength and properties, effective protection of the drugs, low toxicity, prolonged circulation in the blood, enhanced permeation and retention in tumor, etc.. To overcome initial burst drug-release problem, polymer-drug conjugates, especially biodegradable amphiphilic block copolymerdrug conjugates are developed. In these conjugates, the drug molecules are chemically combined to the polymer ends or side chains, so that their release based on physical diffusion is eliminated. They can self-assemble into nanoscale micelles in aqueous medium and therefore, they can be administrated by intravenous injection easily. The medicated micelles are taken up by the cells via endocytosis and the drug molecules are released from the conjugates rapidly once they enter the cells because of the acidic circumstance and the acid hydrolyses in the lysosomes And these medicated micelles show high cytotoxicity to many cancer cells in vitro and anti-cancer activity in vivo.As an active targeting moiety, folic acid or folate has been intensively studied for its unique advantages over other ligands or antibodies:(1) It does not exhibit immunogenicity as many proteins do;(2) It has been found that folate receptor (FR) is up-regulated in more than90%of non-mucinous ovarian carcinomas. It is over-expressed in kidney, brain, lung, and breast carcinomas whereas it is expressed at very low level in most normal tissues. The FR also has a positive correlation with the pathological stage of the tumors. Because the interaction of folate and FR is specific, folic acid or folate-drugs can be delivered to FR-over-expressing cancer cells;(3) Its influence on pharmacokinetics is negligible owing to its simple structure and small size;(4) The cost is low because its synthesis and purification are quite simple;(5) Its carboxyl group is easy to react with OH or NH2, and the reaction products retain its intrinsic activity. Therefore, it has been conjugated to several drug molecules, liposomes or polymers. Recently, it was attached to a triblock copolymer methoxy-poly(ethylene glycol)-b-poly(L-lactide)-b-poly(L-lysine)(PEG-b-PLA-b-PLL) via the reaction of its COOH with the pendant NH2groups on PLL block, preferred endocytosis in HeLa cells and enrichment in H22tumor tissue were observed for the folate-conjugated micelles.In this research, folic acid (FA) was chosen as the targeting moiety, and it is attached to a carrier polymer via an ester linkage. Based on our previous work, paclitaxel (PTX) is conjugated to another carrier polymer. These two carrier polymers are co-assembled into composite micelles which are expected to have many properties including water solubility, prolonged circulation, biodegradability, folate-receptor targeting and rapid cell uptake. To examine the anti-tumor efficacy of these composite micelles, human esophageal EC9706cancer xenograft nude mice models were constructed and a series of evaluation was performed such as tumor growth inhibition rate, tumor cell apoptosis rate by flow cytometry and TUNEL assay, survival time analysis, immunohistochemistry (IHC) and histopathology. The results showed that FA-M(PTX) exhibits The results showed that FA-M(PTX) exhibits significant anti-tumor efficacy in vivo compared to pure PTX and simple PTX-polymer micelles at an equivalent PTX dose of20mg/kg. This enhanced efficacy of FA-M(PTX) is ascribed to the targeting effect of FA moieties in the micelles although the FA-content in FA-(PTX)isonly1.4wt%.Results and conclusions of this study:1. In vitro1.1MTT assay:The cytotoxic effects of pure PTX, M(PTX) and FA-M(PTX) on EC9706cells evaluated by MTT are depicted in Fig.1as a function of equivalent PTX concentration. It is seen that the three types of PTX drugs, i.e., pure PTX, M(PTX) and FA-M(PTX) are all inhibitory to EC9706at the three time points examined. Their cytotoxicities increase with increasing drug concentration and exposure time. The half maximal inhibitary concentration(IC50) of PTX to EC9706at24h were (13.96±0.73)|jg/ml. And the IC50ofPTX, M(PTX) and FA-M(PTX) to EC9706were (0.11±0.03),(22.75±0.84), and (21.07±0.38)|jg/ml at48h, and (0.04±0.01),(7.75±0.12), and (2.13±0.07)|jg/ml at72h, respectively. Thecell growth inhibition rates follow the following order: M(PTX) FA-M(PTX)<PTX.T hat is to say, pure PTX is the most efficacious among the three in vitro.1.2Annexin V/PI double-staining assay: As shown above, the early and late apoptosis ratesof the drug groups were significantly higher compared with the control group at24h,48and72h. The three drug groups follow the order of M(PTX) FA-M(PTX) PTX at24h andPTX <M(PTX) FA-M(PTX) at48hand72h.2. Invivo2.1Tumor growth inhibition:57nude mice were used for observation of tumor growth andinhibition. The average initial volumes of the xenograft tumors of the four groups werecomparable and there was no significant difference between the groups (data not shown).Obviously, the tumor growth inhibition displays following order: FA-M(PTX)> M(PTX)>pure PTX> Control. On the12th day after drug administration, the mice were sacrificed andtheir tumors were excised. Their sizes and weights were measured to calculate the tumorinhibition rate. The drug groups showed obvious antitumor efficacy and the FA-M(PTX) wassuperior to pure PTX and M(PTX) groups (P<0.05) but there was no significant statisticaldifference between pure PTX group and M(PTX) group.2.2Flow cytometry assay of xenograft tumors: The apoptosis rates of the esophageal cancercells were also detected using flow cytometer (FCM) after Annexin V OTC/PI double staining.The apoptosis rate of the control group was significantly lower than those of the drug groups(P<0.05), and there were also significant differences between the FA-M(PTX) group and theother two drug groups (P<0.05), but there was no statistical difference between PTX groupand M(PTX) group (P=0.122). In summary, the apoptosis rate followed the order of Control <PTX M(PTX)<FA-M(PTX) on the12th day after drug administration.2.3TUNEL assay of tumor sections: the TUNEL assay revealed significant statisticaldifferences between the drug groups and the control group (P<0.01) and between theFA-M(PTX) group and the other two drug groups (P<0.01) on the12th day after drugadministration. The grey values followed the order of Control> PTX> M(PTX)>FA-M(PTX). 2.4Immunohistochemistry (IHC) examination: The expression levels of the proteinsassociated with cell apoptosis in57excised tumors were semi-quantitatively evaluated byIHC method and the results were shown as the grey values of the IHC photos calculated usingImagePlus software. The apoptosis-related proteins examined included Bax, Bcl-2, Caspase-3and survivin. The grey values of the groups were in the order of Control> PTX M(PTX)>FA-M(PTX) for Bax and Caspase-3, whereas survivin, Bcl-2showed the order of Control <PTX M(PTX)<FA-M(PTX), indicating significant statistical difference of FA-M(PTX)with the other two drug groups. This was in agreement with the results of FCM and TUNELassays as well as the tumor size and weight measurement.2.5Histopathological examination: The xenograft tumor was histopathologically examinedby HE staining, and the slices were obtained along the margin of the excised tumors where theblood supply were theoretically better than that within the tumor.2.6Survival analysis:101nude mice divided into four groups randomly were used forsurvival analysis. The subcutaneous tumors were measured every two days. The drug groupsshowed significant tumor growth inhibition efficacy. The Kaplan-Meier survival curve wasdrawn. The median/mean survival time of the groups was compared using the log-rank test.The difference in survival time between the FA-M(PTX) and the other groups was statisticallysignificant. The tumor bearing models in drug groups showed a prolonged survival timecompared with the control group and followed the order of pure PTX <M(PTX)<FA-M(PTX). Furthermore, the FA-M(PTX) was significantly more effective than pure PTXand M(PTX), which urged us to step forward.