Exploring The Application Of Mitochondria-targeted Organic Small Molecules In Photodynamic Therapy And Super-resolution Imaging

Mitochondria are one of the most important functional organelles in cells and are the site of intracellular material oxidation and energy production.Through the oxidative respiratory chain,mitochondria provide more than 80 % of the energy for cellular life activities,and a variety of enzymes in the oxidative respiratory chain are also directly involved in other life activities.Also,mitochondrial proteins and naked cyclic mitochondrial DNA are inextricably linked to cellular oxidative stress,mediating multiple modes of cell death and the generation of many major diseases.In the past decades,many researchers have gained a deeper understanding of mitochondrial components and functions with the help of electrophoresis,electron microscopy and other methods or instruments.In recent years,the booming development of confocal fluorescence microscopy has provided the possibility to observe fine structures by fluorescence means and spawned the field of fluorescence biomicroscopy.With the cross-fertilization of chemistry and biology,more and more small molecule probes,complexes and nanoparticles are being used in the field of imaging and therapy.All three types of fluorescent probes have the characteristics of being more flexible and less toxic to use,and serve different purposes depending on the synthetic design ideas.In this thesis,we have designed and synthesized two fluorescent probes in the D-A configuration of organic small molecules with large Stokes shifts and fluorescence intensities,and both probes can target mitochondria and explore different applications for different aspects of mitochondria.1.We designed and synthesized three organic small molecule structures with rigid planar theme,and modified the ether oxygen chain,acetate and hydroxyl group in the R group,respectively.To increase their water solubility and biocompatibility.The rigid planes have a large π-π conjugation system,which can effectively limit their molecular torsion and reduce energy loss,and the structure is also a D-π-A structure with both electron donor-acceptor,which can effectively accelerate the intramolecular charge transfer and enhance the fluorescence quantum yield,and the higher quantum yield is beneficial to induce the production of reactive oxygen species(ROS)under light stimulation for photodynamic therapy.After in vitro experimental assays,we found that one of the probes,U-Ts O,has excellent photocatalytic ROS conversion efficiency and has great potential for application in photodynamic therapy,and confirmed its good photodynamic therapeutic effect in cells as well through subsequent cell and tissue experiments.However,since the excitation light of U-Ts O is at 349 nm,which is in the UV region and itself has a large damage to cells,we further explored its non-linear optical effects based on single-photon photodynamic therapy and found that it has excellent two-photon and three-photon absorption at 700 nm and 940 nm,respectively,which well meet the requirements of fluorescence imaging,and in After further experiments on cells and tumor spheres,we found that the structure of the rigid plane of the large conjugate system designed by us has not been reported in photodynamic therapy compared with the common metal complexes,porphyrin rings and BODIPY derivatives,and is a new type of structure for photodynamic therapy,and we hope to have greater applications in this field in the future.2.We designed and synthesized two organic small molecules of D-A configuration with high quantum yields.Due to the introduction of benzene ring and thiophene respectively,which change the excitation light wavelength,in comparison,the excitation light wavelength of BDP containing benzene ring is blueshifted by 55 nm compared with BDTP containing thiophene,so that the Stokes shift of BDP reaches 105 nm,which also can completely exclude the effect of excitation light on emission,in In vitro tests,BDP showed a strong response to mitochondrial DNA.Further we used BDP for cellular experiments,in which we can precisely target mitochondrial DNA with the help of super-resolution confocal microscopy,and we can dynamically observe the changes of mitochondrial DNA in the cell.At low concentrations,BDP is almost nontoxic to the cell and can be used as a tool for observing mitochondrial DNA,and we can observe the changes of mitochondrial DNA when foreign stimuli are applied through the stimulation of foreign substances,and BDP At high concentrations,it can induce apoptosis,so we observed the process of mitochondrial DNA from dispersion to aggregation and adhesion to cristae during apoptosis in cells with the help of BDP super-resolution dynamics,and identified BDP as caspase family-mediated apoptotic pathway with the help of electrophoresis and fluorescence imaging.Fluorescent probes that allow dynamic observation of mitochondrial DNA have also been rarely seen in literature research.In this thesis,from the design idea of selecting suitable push-pull electron groups to enhance intramolecular charge transfer,we finally designed and synthesized two fluorescent probes of D-A conformation of organic small molecules with large Stokes shift and fluorescence intensity,both probes can target mitochondria and explore different applications for different aspects of mitochondria.