Construction And Imaging Tracking Of Mitochondrial/lipid Droplet Fluorescent Probes With Ultra-high Photostability

As the basic structure of organisms,cells have highly differentiated organelles,including lysosomes,mitochondria,endoplasmic reticulum,Golgi bodies,nuclei and lipid droplets.Each organelle has a complex and ordered structure and plays a definite and indispensable role in various cellular processes.Mitochondria are active organelles with various forms,which provide energy for cell life activities,participate in cell proliferation and metabolism,and play an important role in cell life.Studies have found that the occurrence of human degenerative diseases is often accompanied by changes in mitochondrial morphology,size and even location.Therefore,visualization of mitochondria and tracking their dynamic changes have important physiological and pathological significance.Fluorescence microscopic imaging techniques with advantages of simple operation,in situ operation and excellent spatial and temporal resolution have been widely used in mitochondrial visualization.The classical cationic probe relies on electrostatic action to target mitochondria rapidly,but it is easy to lose its targeting due to the influence of mitochondrial membrane potential;the reactive-based probe can improve the leakage of the probe when the mitochondrial membrane potential is decreased by chemical reaction with proteins on the mitochondria,but it has high cytotoxicity;long-alkyl-chain mitochondrial probes can stably target mitochondria by hydrophobic interaction and have low cytotoxicity.However,their poor photostability still cannot meet the demand for long-term tracking of mitochondrial dynamic changes.Lipid droplets are organelles that store neutral lipids in cells and are involved in many biological processes,playing a crucial role in energy storage and production,construction of cell membranes,and intracellular signaling.In response to different cellular metabolic states,lipid droplets exhibit highly dynamic cyclic behaviors,such as growth,selective degradation,and regeneration.Lipid droplets are also regarded as indicators for various diseases(such as fatty liver disease and obesity),so monitoring the dynamic behavior of intracellular lipid droplets is of great significance for early disease diagnosis.Although various fluorescent probes targeting lipid droplets have been developed,it is still a great challenge to track the dynamic changes of lipid droplets under the microscope due to their poor photostability.In this paper,based on the current research status of mitochondria targeting and lipid droplet targeting fluorescent probes,fluorophores with excellent performance were precisely selected to build mitochondrial and lipid droplet fluorescent probes with ultra-photostability.Furthermore,the dynamic changes of mitochondria and lipid droplets in living cells were tracked by the probes with excellent photostability.Firstly,this work has successfully designed and synthesized three fluorescent probes(DQPI-8,DQPI-10 and DQPI-12)with mitochondrial targeting by selecting fluorophore purposefully and tuning the side chain of styryl-pyridinium precisely.DQPI-12 can accurately and firmly target mitochondria in living cells independent of mitochondrial membrane potential changes.Compared with the commercial mitochondrial probes,DQPI-12 has better photostability and can be used to observe the mitochondrial filamentous structure under STED microscope.Benefit from its ultra-photostability and leakage-free ability,DQPI-12 can realize real-time tracking of mitochondrial dynamic change in HeLa cells and neurons,and can monitor the occurrence of mitophagy in-situ for a long time.Secondly,a novel lipid droplet targeted fluorescent probe N-CN-BD was synthesized by coupling 4-(diethylamino)-2-hydroxybenzaldehyde with 4-cyanopyridine.The molecule showed different absorption and fluorescence emission properties in PBS solution with different pH values.It can not only target lipid droplets in living cells,but also stain lipid droplets in fixed cells.N-CN-BD has excellent photostability and biocompatibility,and can track the dynamic changes of lipid droplets in living cells in real time and in situ.Thirdly,pyrene was selected as the fluorophore and vinylpyridine cationic salt as the targeting group of mitochondria.Regulate the length of hydrophobic alkyl chain of pyridine salt,two dyes(BI-BD-12 and BI-BD-18)were designed and synthesized.Co-localization experiments with commercial mitochondria showed that BI-BD-12 could stain mitochondria in living cells,and could still target mitochondria when mitochondrial membrane potential decreased or disappeared.BI-BD-12 has low cytotoxicity and is suitable for imaging living cells.In addition,BI-BD-12 has excellent photostability and is a potential tool for tracking mitochondrial dynamic changes.