Preparation And Biological Activity Study Of Targeted Delivery For Platinum Drugs

Cancer, also known as malignant tumor, is an important disease of serious threat to human life and health. With cisplatin, carboplatin and oxaliplatin as the representative, the platinum antitumor drugs have been the first choice for malignant tumor chemotherapy, playing an important role in the treatment of cancer. Although the research and development of platinum antitumor drugs has made great progress, it still causes serious side effects for its lack of selectivity to tumor cells. Monoclonal antibodies can specifically recognize tumor cells, however their cell killing ability is relatively low, limiting the anti-tumor effect. Antibodies are mainly combined with non-specific chemical drugs in clinical treatment. In recent years, the targeted therapy of tumor has become a hot research field in tumor treatment. By connecting the small molecule platinum drugs with targeting antibody or antibody fragment via a chemical bond, antibody drug conjugate can be prepared to identify tumor cells, effectively kill target cells and reduce the side effect of drugs, so as to achieve high efficiency and low toxicity.In this study, two kinds of platinum prodrugs were designed and prepared, using cisplatin and oxaliplatin as model dugs. One kind is platinum (Ⅳ) prodrugs, with amino, ethylenediamine and 1R,2R diaminocyclohexane as the ligand, chloride ions or oxalate ion as the leaving group, and carboxylate groups axially symmetric introduced, which can be used for further modification. The other kind is platinum (Ⅱ) prodrugs, with amino and 1R, 2R diaminocyclohexane as the ligand, glutamic acid ion as the leaving group to introduce an amino group. It is connected to lysosomal enzyme cleavable peptide, valine-citrulline, and the carboxylate group is introduced for coupling with the targeting carrier. The structures of these compounds were characterized by IR, NMR and MS, and CCK-8 assay was used to determine the cytotoxicity of platinum prodrugs against three breast cancer cells in vitro, MCF-7, MDA-MB-231 and SK-BR-3 cells. Compared with cisplatin and oxaliplatin, the cytotoxicity of platinum prodrugs decreased significantly, showing almost non-inhibition to SK-BR-3 cells.In this paper, aiming at the HER2 antigen highly expressed on the surface of tumor cells, commercial trastuzumab was chosen as the targeting carrier to connect with platinum prodrugs mentioned above, to prepare antibody drug conjugates. High performance liquid chromatography, inductively coupled plasma atomic emission spectrometry, and BCA reagent kit were used to identify the antibody drug conjugates and calculate the ratios of drug/ antibody. In this paper, the ratios of the drug/antibody to the conjugate are in the range of 4～7, which is the ideal ratio value range of the antibody to the drug. The affinity of conjugates to Her2 antigen and target SK-BR-3 cells was measured by enzyme-linked immunosorbent assay (ELISA), and the targeting binding to Her2 negative MDA-MB-231 cells and Her2 positive SK-BR-3 cells was observed under fluorescence microscopy. After chemical cou pling with platinum compounds, the affinity of trastuzumab for both HER2 antigen and target cells decreased, but still basically maintain most of its affinity. The conjugates can specifically bind to the surface of SK-BR-3 cells with Her2 antigen located on the cell membrane, and not to Her2 negative MDA-MB-231 cells, that is the same with trastuzumab. CCK-8 kit was used to determine the in vitro cytotoxicity of conjugates against three kinds of breast cancer cells, SK-BR-3, MDA-MB-231 and MCF-7. Conjugates show significantly increased cytotoxicity against SK-BR-3 cells, almost the same as cisplatin, and better than oxaliplatin. However, for breast cancer cells MCF-7 and MDA-MB-231 with low Her2 expression, the inhibition of conjugates is still lower than 30%, even at the highest tested concertation. The cell cycle was detected by flow cytometry, showing trastuzumab-platinum (Ⅳ) conjugates mainly arrest SK-BR-3 cells at G2/M phase, while trastuzumab-platinum (Ⅱ) conjugates mainly arrest at S phase.As the experiments and analysis shows, trastuzumab-platinum drug conjugates can distinguish target cells from non-target cells, specifically bind to the surface of HER2 overexpressed breast cancer, exhibit good toxicity, and reduce damage to non-target cells. It provide a new strategy for targeted delivery design of antitumor platinum drugs.