Investigation Of Construction And Imaging Of Fluorescent Probes Based On Three Proteases

Health problems are the focus of humans'long-term attention,and the regulation of proteases is essential in normal physiological activities and in many diseases.For example,many proteases are closely related to tumor cell growth,angiogenesis,invasion,and metastasis in the development of cancer.Identification of dynamic changes in proteases allows us to carry out early detection of cancer,intraoperative navigation,and assessment of the efficacy of anti-cancer therapies.Therefore,the analysis of proteases plays an important role in the diagnosis and treatment of cancer.At present,various kinds of chemical fluorescent probes that target proteases are flourishing.However,there are still some shortcomings such as short fluorescence wavelength,weak specificity,and the fact that small organic molecular fluorophores are easily metabolized and lose their signal.Here,we selected three cancer-related proteases as research objects,conducted corresponding probe design and synthesis for the above three problems,and conducted research on its imaging applications.The main contents of this thesis work include the following three aspects:?1?A small molecule near-infrared fluorescent probe targeting fibroblast activation protein?FAP?was designed and synthesized.The probe was successfully used for fluorescence detection of FAP-expressing cancer cells with a detection limit of 1500cells/mL.The cells were incubated with the probe for 5?M for 30 min with good confocal imaging.Successful application of fluorescence imaging in mouse breast cancer tumor models,and can distinguish between tumor and normal tissue,with tumor-specific imaging capabilities.?2?The design idea of the Dipeptidyl peptidase ??DPP ??probe was abandoned and a class of non-enzymatic probes was designed and synthesized using inhibitor affinity.The affinity of the probe to the cell DPP ? was studied by computational simulations,cell imaging,and Western blotting.The probe has a strong specificity and has been proved by molecular biology methods such as siRNA interference to eliminate the interference of proteases such as FAP,DPP ?,and DPP ?.The probe fluorescence has a good environmental stability.Experiments such as immunohistochemistry indicate that the probe can monitor abnormal expression of endogenous DPP? in cells.?3?The use of NaYbF₄:Tm upconversion nanoparticle coupled with indomethacin to synthesize a new type of composite probe based on inorganic nano-fluorophores can overcome the inherent disadvantages of some small-molecule fluorophores.The probe near-infrared 980 nm excitation up-conversion emits near-infrared fluorescence at 700-800 nm.With good water solubility and dispersibility,confocal imaging results indicate that linking inhibitors may increase the residence time of the material in the cells and the ability to target organelle Golgi apparatus.