Construction And Biological Application Of Mitochondrial Targeting Cell Microenvironment Fluorescent Probes

Mitochondria,known as the"cell powerhouses,"are the main sites of cellular aerobic respiration and participate in various cellular processes including differentiation,information transmission,and apoptosis.The microenvironment within cells,especially the mitochondrial microenvironment,such as temperature,polarity,acidity,viscosity,and hypoxia,plays a crucial role in the functioning of cells.For example,mitochondrial viscosity is closely related to the transport of substances within cells,the transport of chemical signals,and the interaction of biological macromolecules.The level of hypoxia affects the effectiveness of cancer treatments like radiation therapy and chemotherapy,and can even lead to drug resistance in tumors.Abnormalities in the mitochondrial microenvironment are closely related to many diseases such as heart failure,liver damage,and cancer development.Therefore,the development of methods that can detect changes in cell viscosity and hypoxia levels in real-time is crucial for understanding cell functions and clarifying the mechanisms behind various related diseases.Fluorescence imaging methods have advantages such as high detection sensitivity,good identification selectivity,non-invasive detection,and minimal damage to cells,which make them the ideal method for in-situ and real-time research into cellular and in vivo biological events.This thesis focuses on the mitochondrial viscosity and hypoxic microenvironment,and designs and synthesizes a series of near-infrared fluorescence probes for in situ real-time imaging and monitoring of viscosity and hypoxia in cells and live mice.The specific research contents are as follows:1.Based on the DHX dye,which is a dihydroxyanthraquinone derivative,various electron-donating groups were linked to the pyridinium salt electron acceptor group to design and synthesize a series of near-infrared（NIR）viscosity probes,which enhance intra-molecular charge transfer（ICT）and introduce more molecular rotors.The probe DHX-V-3 was selected for its high signal-to-noise ratio,good recognition selectivity,and good mitochondrial targeting and immobilization ability.By using the probe DHX-V-3,real-time imaging of viscosity changes within the mitochondria of He La cells under lipopolysaccharide（LPS）or nystatin stimulation was achieved.In addition,based on the excellent tissue imaging depth of NIR probes,DHX-V-3was successfully applied to visualize the viscosity changes in an inflammation model mouse in vivo.2.Using biotin as a tumor targeting group and nitrobenzyl bromide as a nitroreductase responsive group,a near-infrared fluorescent probe,DHX-Bio-NO₂,was designed and synthesized for evaluating the hypoxia level in tumor cells by detecting the nitroreductase level.The probe DHX-Bio-NO₂ showed a good linear response to nitroreductase,was not interfered by common biological active species,and exhibited low cytotoxicity.The cell imaging experiment results demonstrated that the probe DHX-Bio-NO₂ could specifically target tumor cells and had the ability to quantitatively measure the nitroreductase levels in tumor cells.By using this probe,normal cells and tumor cells could be distinguished based on the difference in hypoxia levels.