Mitochondria-Targeting Microenvironmental Fluorescent Probes For Visualization Of Autophagy

Mitochondria are an important organelle that maintains the normal life activities of the entire life body.They are distributed in all eukaryotic cells.They are responsible for a variety of including respiration,energy production,as a place for redox reactions and regulating the balance of Ca²⁺in the entire life body.And other important functions.Mitochondrial tissue is in a dynamic network of mutual flow and interconnection.Their structure and position are not fixed,but change with cell type,developmental stage,and other physiological conditions and environments.Mitochondria,as the only maternally inherited organelle,and generating signals to communicate with other organelles,are considered to be one of the most complex and dynamic feedback sensory systems in human cells.The autophagy process is a normal process of phagocytosis of cells.It starts when cells form a kind of autophagosome in the human body,which can devour the cytoplasmic components in the human body purposefully and non-purposely.Autophagy can also prevent various diseases by regulating cell morphology.It plays a vital role in maintaining the balance of human cell health and regulating various human diseases.The autophagy process of mitochondria is often accompanied by changes in various cell microenvironments,such as polarity,viscosity,p H,and so on.The complex environment of mitochondria is related to a variety of human diseases.Therefore,the monitoring of changes in the microenvironment during mitochondrial autophagy is of great significance for future research on mitochondrial functions and diseases.So far,the classic methods for monitoring autophagy include transmission electron microscopy(TEM),western blot,plasmid transfection and so on.However,due to technical difficulties,the use of TEM and western blot cannot be used to detect autophagy in living cells.Plasmid transfection is a biological method for visually detecting autophagy,but its operation requires complicated transfection technology and takes a long time.The reagents and drugs used for plasmid transfection are also quite expensive.In order to overcome the limitations of existing methods,there is an urgent need for efficient and low-cost tools to monitor autophagy in real time.Fluorescent probes are a tool for detecting autophagy that only responds to signal molecules,has high detection accuracy,and high imaging resolution.Therefore,it has attracted more and more public attention.The application of fluorescent probes to biological tissue imaging has promoted the development of cell biology and therapeutic imaging technology.(1)Carbazole is often used as an excellent fluorophore precursor because of its lyotropic effect,high quantum yield,and large Stokes shift.The carbazole group can be modified at multiple sites to provide different optical properties for the developed probes,which provides a good basis for the design of ideal fluorescent probes.We know that the typical intramolecular charge transfer(ICT)type fluorescent probe is a strong push-pull system,which is very sensitive to the polar environment.Therefore,we synthesized a fluorescent probe KMP of ICT system based on carbazole derivatives.As designed,the carbazole fluorophore and the tetramethoxyphenylacetylene group constitute a large D-?-A system,and the probe shows a good response to the polar environment.The probe KMP has two maximum ultraviolet absorption peaks at 360 nm and 470 nm.And tested in a variety of polar solvents,the probe KMP has a good lyotropic effect.Under the illumination of 360 nm excitation light,the probe shows an excellent response to the polar environment.On the other hand,the positively charged benzothiazole bromide gives the probe a good mitochondrial localization ability.In cell fluorescence microscopy imaging,we can clearly observe the shape of the mitochondria.At the same time,the cytotoxicity experiment proved that the probe KMP has lower cytotoxicity,less damage to cells,and can be used to study cells for a long time without affecting cell survival.By using DMSO solvent to change the polarity of mitochondria,the change process of mitochondrial polarity can be clearly detected by the probe.In addition,in the experiment of inducing cells to autophagy in the culture lacking serum,the visual study of mitochondrial autophagy was successfully realized by observing the changes in the fluorescence intensity of the cells.(2)As we all know,phenolic hydroxyl is a group sensitive to p H response.Under acidic conditions,hydroxyl is a weaker electron donor;under alkaline conditions,deprotonated O^-is a stronger electron-donating group.Therefore,based on this characteristic,we used7-hydroxycoumarin as the fluorescent matrix to synthesize a fluorescent probe CMPH that specifically responds to mitochondrial p H.The phenolic hydroxyl group is a group that specifically responds to p H.Connecting benzothiazole benzyl bromide to the probe CMPH can not only serve as a positioning group for the mitochondria,so that the probe successfully enters the mitochondria,but also is quaternized.The amino group can endow the probe with better biocompatibility.The probe showed two maximum absorption peaks in PBS buffer solutions of different p H.And under the excitation of 380 nm excitation light,the probe showed excellent response performance to p H environment.When the p H range is about 6.5to 8.2,the fluorescence intensity of the probe has an excellent proportional relationship with the change of p H,and its p K_a=7.79 corresponds exactly to the slightly alkaline p H range of mitochondria.In the research on the stability of the probe,it is found that the fluorescence intensity of the probe basically does not change during the test process,and the interference of other ions and most biological components can be excluded.Biological studies have shown that the probe CMPH has low toxicity to cells and can observe cells for a long time without damage.The different p H of mitochondria was adjusted by high-K⁺buffer solution and nigericin,which proved that the probe can detect the change of mitochondrial p H through the difference of cell fluorescence intensity.The most important thing is to realize the visual study of mitochondrial autophagy.