Enzyme Mimics Of Cu-MOF And Fe-MIL-101 And Their Effects On Cancer Cells

Artificial enzyme mimetics have become an increasingly important focus for research, because natural enzymes bear some considerable disadvantages, such as a sensitivity of catalytic activity in environmental conditions, a relatively low stability and a poor recovery. The construction of efficient enzyme mimetics is challenging due to the importance of enzyme in biology and industry.Higher incidence and mortality rates of cancer are increasing. Particularly, ovarian cancer is the second most common gynaecological malignancy worldwide and the leading cause of death among gynaecologic neoplasms. Early diagnosis, novel and more effective agents for treatment of cancer are key to improving patient survival rates at present. Increasing evidence suggests that the tumour microenvironment also plays critical roles in tumour progression and metastasis. The tumour microenvironment comprises numerous signalling molecules and pathways that influence the angiogenic response. Tumour growth and proliferation depends on the formation of new angiogenesis for the supply of oxygen and nutrients. Matrix metalloproteinases (MMP), a family of zinc-binding proteins including the gelatinases MMP-2 and MMP-9, have been shown to play a central role in angiogenesis and tumour cell invasion and metastasis due to their ability to degrade the extracellular matrix. Increasing evidence suggests that the tumour microenvironment also plays critical roles in tumour progression and metastasis. So, synergistic tumour therapeutics target both tumour microenvironment and tumour cells are new strategies.Metal-organic frameworks (MOFs) are a unique class of hybrid porous solids based on metal centers or clusters and organic linkers. Compared to traditional porous materials, they possess outstanding properties of high surface areas, high porosity, tunable pore sizes and topologies, which enable them unique advantages for applications in many areas. With the number of type and structure increasing, the application range of MOFs is extending. Until now, the proteasemimic and available toxicity information regarding MOFs have remained scarce and the contemporary use of MOFs for cancer treatment has been largely limited to serving as contrast agents for imaging techniques and carriers for drug delivery. Few reports have directly used MOFs as anticancer agents. As well as no reports has directly used MOFs as intrinsic proteasemimic.Based on the above reasons, this paper is focused on Cu-MOF and Fe-MIL-101, the study of activities of mimic enzyme and their effects on cancer cells, including cell digestion and separation, cell detection, cell toxicity, cell migration, anti-angiogenesis, inhibition of matrix metalloproteinases (MMP-2/9) and actin expression. It was reported firstly that Cu-MOF possesses an intrinsic enzyme mimicking activity and was applied into dissociate cells in cell culture. We also found that Fe-MIL-101 possesses an intrinsic peroxidase-like activity and shows highly catalytic activity even at neutral pH. We take advantage of folic acid conjugated Fe-MIL-101 without any surface modication to design and develop a simple, cheap, highly selective and sensitive colorimetric assay to detect cancer cells based on its intrinsic peroxidase-like activity. The thesis also investigated the mechanism of their effects on cancer cells. The main contents of the present research are as follows:(1) Cu-MOF was used in mimic enzyme to catalytic hydrolysis of proteins (BSA, casine). The effects of temperature, pH and reaction time on the reaction, as well as catalytic kinetics and the reaction mechanism were investigated. It was found that Cu-MOF possesses an intrinsic enzyme mimicking activity similar to t natural trypsin. The Michaelis constant (Km) of Cu-MOF is about 26,000-fold smaller than that of free trypsin, suggesting that Cu-MOF has a higher affinity for BSA than trypsin. Cu-MOF also exhibited significantly higher catalytic efficiency than homogeneous artificial metalloprotease Cu(II) complexes and could be reused for ten times without losing in its activity. Moreover, Cu-MOF was successfully used to simulate trypsinization in cell culture since it dissociated cells in culture even without EDTA. These findings will open an avenue for using metal-organic frameworks as an artificial protease.(2) Fe-MIL-101 was used in mimic enzyme to catalytic oxidation of 3,3’,5, 5’-tetramethylbenzidine (TMB). The effects of amount of catalyst and H2O2, temperature and pH on the reaction, as well as catalytic kinetics and the reaction mechanism were investigated. It was shown that Fe-MIL-101 possesses an intrinsic enzyme mimicking activity over a broad pH range similar to that found in natural horseradish peroxidase and shows highly catalytic activity even at neutral pH. The Michaelis constant (Km) of Fe-MIL-101 with H2O2 as the substrate is about 616-fold (at pH 4.0) and 20-fold (at pH 7.0) smaller than free natural horseradish peroxidase (HRP), indicating a much higher affinity for H2O2 than HRP and most of the peroxidase mimetics. Moreover, Fe-MIL-101 was successfully used to detect cancer cells by conjugating folic acid onto Fe-MIL-101 without any surface modification. The detection limit of the method for HeLa cells was estimated to be 50 cells and the reaction colour produced with 10 cells could also be observed by the naked eye. The proposed method holds considerable potential for simple, sensitive, universal, and specific cancer cell detection.(3) Evaluation of the antitumor activity of Fe-MIL-101. The cytotoxicity of Fe-MIL-101 in four cell lines (human cervical cancer cells HeLa, human lung adenocarcinoma cells A549, human ovarian cancer cells SK.OV3 and human umbilical vein endothelial cells HUVEC cells) and control normal mouse embryonic fibroblast BABL-3T3 cells was evaluated by the MTT assay. Fe-MIL-101 has selective toxicity against SKOV3 cells (IC50= 23.6μg mL-1) and HUVEC cells (IC50= 9.9μg mL-1) and shows less toxicity against normal BABL-3T3 cells (IC50= 77.8μg mL-1). AO/EB, Hoechst 33342 and JC-lstaining and flow cytometry analyses indicate that Fe-MIL-101 induced apoptosis of SKOV3 via G0/G1 cell cycle arrest and decreased the mitochondrial membrane potential. In addition, western blot results shown that Fe-MIL-101 exhibited stronger anti-migration and antiangiogenic effects in SKOV3 cells via downregulation the expression of MMP-2/9.(4) The cytotoxicity of Cu-MOF was evaluated by the MTT assay using four cell lines (human lung adenocarcinoma cells (A549), colon adenocarcinoma cells (HT-29), human mouth epidermal carcinoma cells (KB), and human ovarian cancer cells (SKOV3) and with normal mouse embryonic fibroblasts BABL-3T3 cells as control. Cu-MOF may have a potential utility to distinguish effectively between tumor cells and normal cells. AO/EB, Hoechst 33342 and JC-1 staining and flow cytometry analyses indicate that Cu-MOF induced apoptosis of SKOV3 via G2/M cell cycle arrest and decreased the mitochondrial membrane potential. In addition, western blot results shown that Cu-MOF exhibited stronger anti-migration effects in SKOV3 cells via downregulation the expression of MMP-9, active-MMP-2 and actin.