Copolymer-epirubicin Conjugate Micelles Hepatic Arterial Infusion For Targeting Therapy Of Rat Transplanted Hepatocarcinoma

Primary hepatocellular carcinoma is one of the most malignant diseases in our country, It is characterized by hidden start, high malignancy degree and high recurrence rete, early diagnosis is very difficult, and now surgical reseetion rate is low, so chemotherapy is still the commonly used treatment in clinic. Traditional drugs have less specificity to the tumor tissues. While, interval impact chemotherapy can kill tumor cell, which can also have unpleasant side-effects as well as induce drug resistance. So the long-term effect of chemotherapy is not satisfied. Therefore, a targeted and controlled-release drug delivery system, generated with the aim to improve the pharmaceutical effects and decrease the side effect, having been an international hot research topic on the chemotherapy of hepatocarcinoma now.Epirubicin is a newmember of Anthracyclines drugs, which gradually takes the place of adriamycin and becomes the first-line agents in the treatment of diverse malignant tumors such as hepatocarcinoma, breast cancer, gastric cancer and pancreatic carcinoma. In this study, we used EPI as prodrug, and copolymer-epirubicin conjugate micelles were made through ultrafiltration with PEG-b-P(LA-co-DHP/NPC) which possess the tumor-targeting effect as the carrier. We investigated the controlled release character and the proliferation suppress effect on walker-256 cells of EPI-NPs in vitro, and further studied the mechanism on walker-256 cells apoptosis induced by EPI-NPs. We set a rat transplanted hepatocarcinoma model, and investigate d the distribution and effect of EPI-NPs in vivo by delivery the drug through hepatic arterial infusion, and we also studied the mechanism on the targeting and controlled release of EPI-NPs in rat, which may provides a theory basis for the clinical use of EPI-NPs.Purpose: To prepare the EPI-NPs with targeting and controlled release quality, study the controlled release and anti-tumor effect of EPI-NPs in vitro and in vivo, further discuss the mechanisms on the above characters of nano-carrier system, with the hope to provide a new way for the targeting therapy of hepatocarcinoma.Method:1. EPI-NPs were made through ultrafiltration with PEG-b-P (LA-co-DHP/NPC) as the carrier. The physic-chemical properties of EPI-NPs, such as the shape, the size and the distribution of particle size, were detected in vitro. The drug-releasing characteristics was also studied in vitro, the releasing curve was draw according to the result.2. Walker-256 cells were cultured in vitro, EPI was used as control drug. Confocal Laser Scanning Microscope was used to detect the EPI-NPs uptake by Walker-256 cells; MTT test and Flow cytometry were used to study the effect of EPI-NPs on the proliferation, apoptosis and cell cycle of Walker-256 cells. RT-PCR and Western Blot were used to detect the effect of EPI-NPs on the expression of apoptisis releated gene, p53 gene and bcl-2 gene, at the mRNA and protein levels. And the mechanisms on the controlled-release and anti-tumor effect of EPI-NPs were preliminarily discussed.3.①The transplanted hepatocarcinoma rat models were made by implantation of tumor particles or direct injection of cancerous ascites, we compared these two method and analysis the blood supply character of the transplanted tumor, and lay a foundation for the drug test in vivo.②The drug was delivered to the rat model through hepatic arterial infusion. Based on the autofluorescence character of EPI, we used tissue fluorescence analysis and homogenate fluorescence analysis to investigate the distribute character of EPI-NPs in the hepatocarcinoma rat model. The results were compared with that of small-molecule EPI.③Pharmacodynamic Study:analysis the effect of EPI-NPs hepatic arterial infusion on the survival rate of the rat model, and analysis the suppression effect of EPI-NPs on the transplanted hepatic tumor by volume anti-tumor ratio and quality anti-tumor ratio, and further discuss the anti-tumor effect of EPI-NPs in vivo.Results:1. Preparation of EPI-NPs:①The EPI-NPs obtained by ultrafiltration method present a uniformed spherical shape, the CMC value was 8.27mg/L the average particle size measured by DLS was 86nm, the drug-loading rate was 14.7%.②In vitro release process study of EPI-NPs revealed that the conjugated drug was sensitive to acid, the release rate was elevated along with the decrease of pH value, the drug was almost totally released within 9d under the environment of pH5.0, as to the the environment of pH7.4, the drug release rate was only 10% on 20d.2. The antitumor test in vitro:(1)By confocal microscope observation, we detected that the EPI-NPs concentration in Walker-256 cells was higher than that of EPI; (2)The MTT test indicate that the proliferation suppression effect of EPI-NPs on the Walker-256 cells was higher than that of free EPI, the inhibition ratio of EPI-NPs was increased along with the prolonging of drug action and the increase of drug concentration. (3)Flow cytometry assay suggested that both EPI and EPI-NPs can induce apoptosis of Walker-256 cells, and the cell cycle was restricted to the G2/M phase. The apoptosis induction effect of EPI-NPs was strong than EPI. (4)RT-PCR and Western Blot analysis revealed that the expression of p53 gene was elevated in both mRNA and protein level after EPI-NPs action,while the expression of bcl-2 gene decreased.3. Drug distribution test and pharmacodynamic test in vivo.(1)The establish of rat transplanted hepatocarcinoma model:The transplanted hepatocarcinoma rat models made by implantation of tumor particles or direct injection of cancerous ascites were all successed with the rate of 100%, with no difference on the survival time of the tumor-bearing rat. Compared with implantation of tumor particles, the direct injection of cancerous ascites was more simple, and took less time for tumor formation(8d). So, the direct injection method was better for the in vivo test which had strong demand for animal models.(2)In vivo drug distribution test:①Drug intrahepatic distribution:the fluorescence intensity of tumor tissue in EPI-NPs group was more intense than that of normal tissue at different time points. As to the EPI group, the fluorescence distributed equably on tumor and normal tissue.②Besides heart and brain, the drug fluorescence on each tissue of rat model in EPI-NPs group was more intensive than that of EPI group. The drug fluorescence on tissues of rats in EPI-NPs group took an gradient changes as follows:tumor >liver>spleen, lung, kidney>heart> brain, while the fluorescence distributed equably on each tissue in EPI group.③After hepatic arterial infusion, besides brain tissue, each tissue had a fluorescence peak at 15min and 2h respectively.(3)Pharmacodynamic test:The survival rate, the volume anti-tumor ratio and the quality anti-tumor ratio in EPI-NPs group were higher than that of EPI group.Conclusion:①The micelle modified by PEG has good biocompatibility and hydrophilicity, as well as passive targeting and pH targeting, which ensured the good controlled-release essence and intracellur targeting function.②EPI-NPs had lower cytotoxicity and good intracellur targeting activity, its antitumor effect may achieved by apoptosis induction through upregulation of p53 gene expression and downregulation of Bcl-2 gene expression on mRNA and protein levels, and the apoptosis may partly caused by restriction of cell cycle to the G2/M phase.③Redistribution of EPI in liver and the whole body was achieved by EPI-NPs hepatic arterial infusion, the distribution and metabolism regularity of EPI were changed, which helped to increase the bioavailability of EPI at the targets and relieve the cardiotoxicity at the same time.④The "double peak" phenomenon of drug fluorescence intensity within liver may prolonged the action time of the drug and increase the propotion of drug distribution in liver and tumor, which may be helpful for the treatment of tumor.⑤EPI-NPs had favourable suppression effect on the rat transplanted hepatocarcinoma.Above all, we used the passive targeting and pH targeting characters of the EPI-NPs. According to the blood supply specialty of walker-256 hepatic transplanted carcinoma, we used hepatic arterial infusion as the drug delivery pattern. We realized the targeting and controlled release of EPI-NPs in the animal model, and the bioavailability of EPI was also increased, thus my study may provide a new drug targeting therapy method for hepatocarcinoma and other malignant tumors.