Studies On Syntheses, Crystal Structures,And Inhibition Of Tumor Angiogenesis Of Copper-based Complexes

Tumorigenesis, invasion, deterioration, and metastasis of tumor cells are closely related to angiogenesis. Targeting inhibition of tumor angiogenesis has been regarded as an important strategy in oncotherapy, which has quickly developed from the chemotherapy into a gene era using RNAinterference technique. The copper-based complexes were regarded as the succedaneum of cisplatin drugs. The researches showed that the copper-based complexes acted as gene carrier, and had a bigger development on the biomedical applications. The research of copper-based complexes on inhibiting tumor angiogenesis was very little. The antitumor activities of copper-based complexes were investigated from varied views by targeting to tumor angiogenesis, vascular endothelial growth factor(VEGF), and tumor cells, and vascular endothelial cells, which had the very novelty and vital significance.Based on such a research background, in this thesis, a dipeptide copper-based complex and three different series copper-based complexes with Schiff base ligands were designed and synthesized, and their crystal structures were obtained. The properties and mechanisms of anti-angiogenesis and anti-tumor and cellular uptake were investigated from varied views by targeting to tumor angiogenesis, tumor cells, and vascular endothelial growth factor(VEGF). Importantly, a copper-based complex si RNA vehicle’s system was built, and loaded the angiogenesis-targeted VEGF-si RNA to release into the cytoplasm of target cells. The nanoscale copper-based complex si RNA system was investigated the efficacy and mechanism in inhibiting tumor angiogenesis. Eventually, the new nanoscale copper-based complex si RNA carrier was obtained, and enhanced anti-angiogenesis and anti-cancer efficacy by virtue of the combination of chemotherapeutic e?cacy and si RNA gene silencing efficacy.This thesis mainly includes the following five chapters and conclusion:In chapter I, the important research targets on anti-angiogenesis and anti-tumor, the mechanisms of inhibiting angiogenesis and delaying tumor growth,the research development of copper-based complexes anti-tumor activities and anti-angiogenesis drugs, were mainly summarized. The application and limitation of MOFs carriers in loading drugs and si RNA in the field of anti-cancer were introduced. Based on such the research background, we put forward the purpose and meaning for the topic selection,the contents of research, and the discussed questions.In chapter Ⅱ, we synthesized two dinuclear copper-based complexes bridged by 4,4’-bipyridine(Cu-1 and Cu-2).Their single crystals were abtained, and their crystal structures were characterized, and their cytotoxicities were tested by the MTT method. We used the flow cytometry analysis method to investigate the properties of inducing apoptosis, and used the HUVECs migration and tube formation assays, and the chicken chorioallantoic membrane(CAM) assays to investigate the inhibition effects on VEGF mediating migration and angiogenesis. The cell absorption and absorption mechanism of Cu-2 were studied by the combination with flow cytometry analysis and living cell imaging of laser confocal microscope. The effects on the important signal molecules Akt/p-Akt and Erk1/2 /p-Erk1/2 were investigated by Western Blot assays, and the flow cytometry analysis and living cell imaging of laser confocal microscope were used to study their effects on intracellular reactive oxygen species(ROS). Our experimental results showed that Cu-2 was more effective than Cu-1 in terms of in suppressing angiogenesis and tumor growth. Furthermore, cancer cells and HUVECs can undergo energydependent and non-endocytotic Cu-2 uptake into the cell nucleus. Cu-2 can inhibit the expression of p-Akt and p-Erk1/2 protein molecules and reduce the levels of ROS to inhibit angiogenesis and induce apoptosis. Cu-2 is a potential tumor angiogenesis inhibitor and anti-tumor drug.In chapter Ⅲ, a copper-based complex(PSBCu)with mixed-ligands was synthesized, and its single crystals were obtained, and the crystal structure was thoroughly characterized. Using the same research methods as in chapter Ⅱ, we investigated the cellular uptakes of PSBCu, the properties of anti-angiogenesis and anti-cancer, and the mechanism of anti-angiogenesis and anti-cancer. Moreover, we investigated the interactions beween PSBCu and the human serum albumin( HSA) by fluorescence and CD spectroscopy, and investigated the abilities of inducing VEGF DNA to form the G-quadruplex structures and stabilize these G-quadruplex structures with CD spectroscopy. The results showed that PSBCu undergone a good cell uptake in HUVECs and cancer cells, down-regulated the expression of protein molecules p-Akt and p-Erk1/2, and increased the levels of ROS, and more effectively inhibited proliferation of cancer cells and HUVECs, and promoted induced-apoptosis, and inhibited angiogenesis to enhance the anti-cancer effect. In addition, PSBCu can induce VEGF DNA to form the G-quadruplex structures and stabilize these G-quadruplex structures, maybe can inhibit VEGF expression at the transcriptional level, more effectively inhibit angiogenesis. PSBCu is also a potential multi-function tumor angiogenesis inhibitor and anti-tumor drug.In chapter Ⅳ, a couple of chiral and water-soluble tetranuclear copper(II)-based complexes(L-Cu and D-Cu) were synthesized, and the single crystals with hexagonal-plate morphologies were obtained, and the absolute structures of crystals were thoroughly characterized. Remarkably, the nanocrystals were prepared only by the simple way which dissolved the crystals into the defined amount solvent. Importantly, we built a si RNA vehicle’s system using the copper-based complex nanocrystals. The efficiency of si RNA loading and release were investigated by the agarose gel electrophoresis, high resolution transmission electron microscopy(HRTEM), cellular uptake and transfection assays. The research results showed that the L-Cu and D-Cu successfully loaded si RNA into the cytoplasm of target cells. We used the tube formations assay, chicken chorioallantoic membrane assay, and aortic ring microtubule formation assay to analysize the inhibiting effect on VEGF mediating angiogenesis, and analyzed the mechanisms of anti-angiogenesis and anti-cancer from the VEGF/VEGFR2 signaling pathway,the level of reactive oxygen species, and the mitochondrial membrane potential views. All the results indicated that these nanoscale copper(II)-based complexes loaded VEGF-si RNA, and the efficient VEGF-si RNA loading was closely related to the chiral selectivity with especial preference to L-Cu; and the nanoscale MOFs protected VEGF-si RNA from nuclease degradation, and enhanced its cellular uptake, and promoted it to escape from endosomes to silence VEGF genes and activated Erk/p-Erk1/2, Akt/p-Akt, and FAK/p-FAK signallings to enhance anti-angiogenesis and anti-cancer efficacy. This work also showed that nanoscale copper-based complexes hold great promise in the combination of chemotherapy and gene therapy for enhancing anti-cancer efficacy, for not only the excellent anti-angiogenesis and anti-cancerr drug candidates, but also the good non-virus gene carriers.In chapter Ⅴ, a couple of chiral and novel tetranuclear copper(II)-based complexes(L-TNBr Cu and D-TNBr Cu) were synthesized, and their single crystals with block morphologies were obtained,and the absolute structures of crystals were thoroughly characterized. Flow cytometry analysis method was used to inspect the induced-apoptosis, the level of the ROS, and the influence of mitochondrial transmembrane potential. Three important signalling pathways which are related to angiogenesis in VEGF/VEGFR2 signal transduction pathway were investigated by Western Blot assays. The results showed that the chiral L-TNBr Cu and D-TNBr Cu exhibited the wide cytotoxicities against cancer cells without significant difference in term of chiral choice. They performed the anti-cancer function by simultaneously targeting to the cancer cells and vascular endothelial cells to regulate the levels of intracellular ROS, reduce the mitochondrial membrane potentials, and down-regulate the expressions of p-AKT, p-Erk1/2 and p-FAK, resulting in the inhibition of proliferation, metastasis, and angiogenesis, and the promotion of induced-apoptosis. They are also the potential anti-cancer drugs.In the conclusion part, the similarities and differences in the four kinds of copper-based complexes in term of anti-angiogenesis and anti-cancer and their functional mechanism were summarized,and the close relations and differences were illuminated on the bioactivities of copper-based complexes with the different ligand, coordination number, and geometric configuration. The potential prospects of copper-based complexes with Schiff base ligand were expected as a new type of the multi-functional anti-angiogenesis and anti-cancer drugs and gene carriers.