Research On Tumor-targeted Imaging And Application Of A Mitochondrial Fluorescence Probe In Autophagy Based On AIE Mechanism

Section I Dual-Modal MRI Contrast Agent with Aggregation-Induced Emission Characteristic for Tumor-targeted Imaging and the Toxicity TestingObject:Relying on the EPR effect, to study the effect on tumor-targeted imaging and the toxicity in cellular and whole-animal level of the dual signal nuclear magnetism contrast agent (TPE-2Gd) loaded with AIE fluorescent molecular.Methods:①Give TPE-2Gd or commercial contrast agents Gd-DTPA to mice of two different groups through caudal vein injection after building subcutaneous tumor model and then detect the tumor by using the nuclear magnetic resonance (NMR) equipment to compare the effects of subcutaneous tumor imaging between TPE-2Gd group and Gd-DTPA group.②In cellular level, observe the cell morphological changes after being dealt with TPE-2Gd by HE staining and observe the effects on cell growth and proliferation of TPE-2Gd’s through CCK8kit. Using reactive oxygen species(ROS) fluorescent probe to detect effects on intracellular ROS of TPE-2Gd by flow cytometry and fluorescent microscope. Observe the effects of TPE-2Gd on the cell apoptosis and necrosis by PI/Annexin.③In whole-animal level, take the arterial blood of SD rats to do the hemolytic test to observe the TPE-2Gd’s toxic effects on blood cells. In SD rat model, giving TPE-2Gd or saline through intravenous injection to observe the behavior and weight changes of two group rats at different time points. Drawing blood to detect the two group rats’ urinalysis performed indicators, such as blood cells, hemoglobin, inflammatory cells and so on, and inspect the indicators like intrahepatic enzymology and serum creatinine urea nitrogen to judge rats’liver and kidney function changes. At different time points, taking visceral organs to do HE staining to observe whether there are inflammatory changes or not after executing the rats through cervical dislocation method.Results:①We successfully built subcutaneous tumor model through subcutaneous i njection of H22cells from the mice. The MRI scan results showed that the tumor site’s signals of the TPE-2Gd group and Gd-DTPA group both were enhanced comp ared with that before the drug injection which is known as plain scan period. The enhanced effect of TPE-2Gd group was more obvious than that of Gd-DTPA group, and the tumor site’s signals of the TPE-2Gd group had a lower attenuation speed which led a longer detention time.②n cellular level, the cell HE staining results p rompted that TPE-2Gd had no effects on the morphological changes of HeLa cells and3T3cells. The results of CCK8experiment showed TPE-2Gd had a good bioco mpatibility and little toxicity in cell growth and proliferation. Intracellular ROS dete ction showed TPE-2Gd didn’t affect the production and increase of ROS. The resea rch about apoptosis and necrosis showed TPE-2Gd didn’t induce the occurring of ap optosis and the increase of necrosis.③In whole-animal level, the hemolytic test sh owed TPE-2Gd didn’t induce red cell rupture, nor affect the hemocyte morphology and function. Through caudal vein injection, the TPE-2Gd group and saline group h ad no statistical differences in the mental state, the growth rate of body weight in SD rats’growth process, nor did the urinalysis performed indicators. The hepatic an d renal function of TPE-2Gd group showed no differences compared with that of sa line group, and inflammatory changes were found in neither of the two groups’HE staining of various tissues and organs of different timing acquisition.Conclusions:This research successfully built mouse subcutaneous tumor model, and through the nuclear magnetic instrument we detected the targeted imaging effects of TPE-2Gd which worked as a new type dual signal nuclear magnetism contrast agents in subcutaneous tumor. TPE-2Gd has two Gd atoms and has a better signal enhanced effects in subcutaneous tumor compared with Gd-DTPA which only has one Gd atom, and the nanometer form of TPE-2Gd relying on the EPR effect is more easily to stay in tumor tissue, which increases the targeting of tumor imaging. As a living application of biological material, the material’s biocompatibility, cytotoxicity and the effects on organism’s growth and reproduction are important testing projects. This experiment demonstrated that TPE-2Gd had no effects on the cells’morphological, growth and proliferation nor the production of ROS, nor induced the the occurrence of hemolysis and cell apoptosis in cellular level. The animal experiment showed TPE-2Gd didn’t affect the SD rats’mental state, growth and development nor the hepatic and renal function, nor induce the occurrence of inflammatory in vivo. In conclusion, since the new type dual signal nuclear magnetism contrast agent combained with AIE fluorescent molecular has little cell toxicity, good biocompatibility, and enhances the signal strength of imaging, as well as extend the time of imaging, it is expected to become an excellent new generation of tumor targeting imaging material. Section II Synthesis, Characterization and Application of a Mitochondrial Fluorescence Probe in Autophagy Based on AIE MechanismObjective:To develop and characterize a mitochondrial fluorescence probe, TPE-Py-NCS. Then evaluate the physicochemical property, AIE character, photo-stability and biocompatibility and explore the application in cell autophagyMethod:We developed an AIE molecular TPE-Py-NCS after modifying TPE-Py-N3and characterize molecular structure by using NMR and HRMS, photo-physical properties by UV spectrophotometer and fluorescence spectrophotometer and the diameters and dispersibility of of TPE-Py-NCS nanoparticals. Then we recorded PL spectra of DMSO/H2O mixtures of TPE-Py-NCS with different water fractions (/w) to identify the AIE character and detect the biocompatibility by MTT assay. Based on the similar compatible principle, we washed the cells using acetone after staining to verify the combination mode between TPE-Py-NCS and mitochondria. We supposed that if the fluorescence faded after washing by acetone, the combination mode between TPE-Py-NCS and mitochondria should be electrostatic interaction; if the fluorescence was still strong after washing by acetone, it should be chemical bonding that makes TPE-Py-NCS band to the mitochondria tightly. In addition, the membrane potential of mitochondria decreased after the cells being treated with CCCP, then we observed the staining result after changing the microenvironment of mitochondria. To quantitatively investigate the photostability of TPE-Py-NCS, cells were continuously scanned by confocal microscope. After inducing HeLa cells autophagy by rapamycin, we stained the cells using AIE probe TPE-Py-NCS and commercial dye MitoTracker(?) Red CMXRos, then continuous scanning image was collected using CLSM to study the fusion process of mitochondria and lysosome and make a comparison of AIE probe TPE-Py-NCS and commercial dye MitoTracker(?) Red CMXRos in the application of autophagy research.Result:In this work, a new fluorescent probe, TPE-Py-NCS, were successfully synthesized. The result was proved by the result of NMR and HRMS. Particle size analysis indicate the average particle size of TPE-Py-NCS is44nm, the particle size is uniform and monodispersed in the0.1%DMSO in water. The absorption and emission maxima of TPE-Py-NCS in DMSO solution are located at397nm and638nm, respectively. The PL spectra of DMSO/H2O mixtures of TPE-Py-NCS with different water fractions (fa) show the TPE-Py-NCS is AIE-active. The emission in pure DMSO solution is weak and it is progressive enhanced when up to99%of water is added to the DMSO solution. The MTT experiment suggest that the TPE-Py-NCS does not affect the proliferation and have a good biocompatibility and hypotoxicity. The intracellular imaging show the TPE-Py-NCS can pass through cell membrane, enter the cell and specifically stain mitochondria. The selectivity and the sensitivity is almost perfect. To further demonstrate that the TPE-Py-NCS is targeted to mitochondria, MitoTracker(?) Red CMXRos, a commercially available mitochondria imaging agent, was used to co-stain the HeLa cells for comparison, the Pearsons correlation coefficient between the two fluorescent images.was0.97. The probe can stain live cell and fixed cell. The probe still can high specifically stain mitochondria, despite the membrane potential of mitochondria was decreased by CCCP. The fluorescence in mitochondria can been observed in HeLa cell which was stained TPE-Py-NCS and washed by acetone. The result indicate the TPE-Py-NCS can covalently bond the mitochondria through the reaction between-NCS group and amino in protein of mitochondria. This covalent connection with mitochondria can resist the environment change. Continuous scanning by confocal microscope was used to quantitatively investigate the photostability of TPE-Py-NCS. During10minutes scanning, the signal loss of TPE-Py-NCS is a little, and no significant difference was observed between the first and the last scan. The photo-stability of TPE-Py-NCS is good. For observed the process of mitophagy, TPE-Py-NCS and LysoTracker Red was utilized to label the mitochondria and lysosome, separately. At the72minutes after the cell was induced autophagy, autolysosomes was appeared. After about8min, the autolysosomes was disappeared. The CLSM indicate the performance of TPE-Py-NCS is better than MitoTracker(?) Red CMXRos in the study of autophagy.Conclusion:The TPE-Py-NCS fluorescent probe was obtained by TPE-Py-N3’s reaction with CS2. It is positively charged and can specifically bind to negatively charged organelle membrane-mitochondria membrane. After the probe is combined with mitochondria by electrostatic attraction, NCS group will reaction with the mitochondrial protein functional-group--NH2and bond tightly to the mitochondria, which can withstand a variety of changes in the microenvironment surrounding the mitochondria, such as depolarization and regional acidification occuring in the process of autophagy. TPE-Py-NCS has a characteristic of AIE. Namely, weak fluorescence is displayed in a good solvent, strong fluorescence in a poor solvent such as PBS, cell culture medium. The fluorescent probes for cell staining is biocompatible and can exhibit good light stability. In a continuous observation of dynamic processes of autophagy, good anti-photobleaching is a key feature fluorescent probes must have. Good resistance to photobleaching effect was showed in the process of autophagy observation by using TPE-Py-NCS, which providing assistance for the researches of autophagy process.