| Primary hepatoma is one of the most common malignant tumors worldwide and the third leading cause of cancer-related mortality. Liver resection is the preferred treatment of hepatoma and can effectively improve the life quality and prolong the survival. However, for the reasons of hedden start and rapid development, most of the hepatoma patients were diagnosed in their middle or late stage, and lose the opportunity of surgical treatment. Act as another potential means to cure hepatoma, liver transplantation is difficult to benefit the majority of patients due to liver source scarcity and high cost. Although chemotherapy plays an important role in the treatment of tumor, its effects in the treatment of hepatoma are not ideal. For the first reason, the drawbacks of traditional chemotherapy agents, such as the poor specificit of tissue distribution and the short biologic half-life in the circulation, make it hard to maintain effective drug concentration in the tumor site. For another, most of the hepatomas are not sensitive to the cytotoxic agents. Morever, the intolerable side effects are also an obstacle for the application of traditional chemotherapy agents. The development of nanoscale drug delivery system in the clinical field provides a new mothed to overcome the defects of traditional chemotherapy agents. Especially, polymeric nanocarriers, which have perfect biocompatibility and high ability of modification, can targetedly deliver and intelligently release its cargos, so the pharmacokinetic characteristics and circulating half-life of traditional chemotherapy agents may be dramatically improved. Because of the controllable biodegradability and precise secondary conformations, the amphiphilic poly(amino acid)-based copolymers may be an ideal choice for the construction of polymeric nanocarrier. Nanoscale micelle loaded with epirubicin(EPI) was prepared based on the amphiphilic methoxy poly(ethylene glycol)-block-poly(L-glutamic acid)(m PEG-b-PGA) copolymer. And then, various experimental methods in vivo and in vitro were employed to evaluate the advantages of this nanomedicine in the treatment of hepatoma. Specific experiments are as follows.Section 1. Preparation, characterization, and intracellular drug delivery of p H-sensitive m PEG-b-PGA/EPI.(I) The amphiphilic diblock m PEG-b-PGA copolymer was synthesized through the ring-opening polymerization(ROP) of L-benzyl-L-glutamate-N-carboxyanhydride(BLG NCA) initiated by the amino-terminated m PEG and then the deprotection of benzyl group. The copolymer was characterized by proton nuclear magnetic resonance(1H NMR) spectra and Fourier transform infrared chromatography(FT-IR), the results demonstrated the successful synthesis of m PEG-b-PGA. Based on the electrostatic interaction between m PEG-b-PGA and EPI, the drug-loaded polymeric micelle was prepared by dialysis method. The results of dynamic laser scattering(DLS) and transmission electron microscope show that the micelle was spherical with the diameter around 80- 90 nm, which was well consistent with the enhanced permeability and resistance(EPR) effect of solid tumor. The drug-loading content(DLC) and drug-loading efficiency(DLE) of m PEG-b-PGA/EPI were 14.68 and 72.5 wt.%, respectively, which indicated that the amphiphilic poly(amino acid)-based micelle could load the hydrophobic traditional chemotherapy agents efficiently. The later release experiment in vitro confirmed the nicely delayed and p H-sensitive drug release property of m PEG-b-PGA/EPI.(II) The confocal laser scanning microscopy(CLSM) and flow cytometry were employed to detect the Hep G2 uptake offree EPIand m PEG-b-PGA/EPI. The results showed that the drug-loaded micelles could effectively enter the hepatoma cells, and the intracellular drug accumulation was improved with the treatment of m PEG-b-PGA/EPI with the extension of time. The cytotoxicities of parent copolymer and m PEG-b-PGA/EPI were detected by MTT assay. m PEG-b-PGA exhibited nice biocompatibility with the maximum concentration of 100 μg/m L for 72 h, the cells could keep their viability more than 90%. m PEG-b-PGA/EPI showed a typical timeand does-dependent inhibition of hepatoma cells.Section 2. Toxicological analysis and tissue distribution of m PEG-b-PGA/EPI. We speculate that m PEG-b-PGA/EPI also has improved security in vivo and can improve the pharmacokinetic characteristic of free EPI. So tissue compatibility hemolysis test, maximum tolerated dose(MTD) test, and drug tissue distribution assay were carried out. Intravenous injection will be the manner of m PEG-b-PGA/EPI administration eventually, so good blood compatibility is an important basis for its further application. In the hemolysis test of our study, a large dose of m PEG-b-PGA(5 mg/m L) did not induce apparent hemolytic reaction, while m PEG can improve the risk of hemolysis of high does free EPI. Furthermore, the maximum tolerated dose(MTD) experiment confirmed that m PEG-b-PGA/EPI had the ability to reduce the toxicity of EPI, so high dosage of drug could be administrated safely. What’s more, the results of tissue distribution test with subcutaneous hepatoma mouse model showed that m PEG-b-PGA/EPI could significantly improve the drug accumulation in tumor tissue compared with free EPI, which might prompt that it would have a better anti-hepatoma efficacy.Section 3. Anti-hepatoma efficacy and safety of m PEG-b-PGA/EPI in vivo. The improvement of antitumor efficacy and reduction of the side effects from traditional chemotherapy agents are the ultimate goals of nanocarriers. In our study, mice hepatoma H22 cells were injected subcutaneously in BALB/c mice to construct hepatoma model, and the antitumor efficacies of free EPI and m PEG-b-PGA/EPI were assessed. The mouse models were treated with free EPI and m PEG-b-PGA/EPI with a EPI equivalent dose of 8.0 mg/kg, or m PEG-b-PGA with the corresponding material dose in m PEG-b-PGA/EPI group though intravenous injection,and the normal saline was used as a negative control. The treatment was repeated every 4 days for total 16 days, the tumor volume and body weight were measured every day. At the end of treatment, the mice were sacrificed. The tumor tissues and important organs(i.e., heart, liver, spleen, lung, and kidney) were separated and weighed. Hematoxylin and eosin(H&E) staining, TUNEL assay, and immunofluorescence detection were employed to evaluate organ injuries, tumor tissue necrosis, and apoptosis caused by different treatments.In the aspect of anti-hepatoma treatment, the two EPI formulations both inhibited the growth of tumor, while the m PEG-b-PGA/EPI treatment showed a significantly better efficacy than free EPI for the EPR effect of solid tumors, and slow and p H-sensitive drug release. The tumor inhibitory rate and tumor index of m PEG-b-PGA/EPI group are both better than that of free EPI group. The rusults of H&E staining and TUNEL assays further clarified the advantage of m PEG-b-PGA/EPI. The necrosis areas of tumors and the average fluorescence intensity of apoptotic cells showed that m PEG-b-PGA/EPI could significantly enhance the tumor necrosis and apoptosis induced by EPI. At the same time, the expression of Caspase-3 and Ki-67 in tumor tissues of various treatment groups were detected by immunofluorescence detection, and their average fluorescence intensity in free EPI and m PEG-b-PGA/EPI groups were 2.60±0.49 and 0.67±0.19, and 1.75±0.41 and 0.40±0.13, repectively. The difference was statistically significant(P< 0.05). These results reflect the enhancement effect of m PEG-b-PGA/EPI to EPI on another side. In the aspect of safety, compared with the control group, the repeated injection of m PEG-b-PGA materials did not induce significant changes in body weight, organ index and pathology detection of model mice. It proved again that the m PEG-b-PGA had nice biocompatibility and can be applied in nanocarrier preparation. Although the two drug formulations both caused body weight reduction and organ damage more or less, the extents in m PEG-b-PGA/EPI group were lower. What’s more, m PEG-b-PGA/EPI significantly improved the situation of liver and spleen index of free EPI group.In conclusion, on the basis of good biocompatibility of m PEG-b-PGA copolymer, EPI-loaded micelle, i.e., m PEG-b-PGA/EPI, could significantly enhance the therapeutic effect and reduce side effects of EPI, and hold potential for the hepotama treatment. |
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