Design,Synthesis And Cell Imaging Of Activatable Autophagy Fluorescence Probes

In the past ten years,fluorescent probes have developed into powerful tools for labeling and studying biological analytes.Fluorescent probes based on small organic molecules have become more and more popular due to their small molecular structure and easy design,adjustable fluorescence performance,relatively fa st response speed and excellent biocompatibility.Many fluorescent probes have been reported for detecting disease-related substances,and many probes can also detect changes in the intracellular mico-environment.Lipid droplets(LDs)are dynamic organelles that are irreplaceable in regulating cell lipid metabolism and ensuring cell energy homeostasis.Lipophagy is an essential catabolic pathway for regulating the dynamics and metabolism of LDs.However,tools for imaging lipophagy in live cells remain un explored.Here we report the development of a new NIR fluorescence probe(LD-OH)for activatable imaging of lipophagy in live cells.LD-OH is designed by conjugating a xanthene scaffold with a quinoline moiety.In vitro results show that LD-OH exhibits green emission at neutral pH,and at acidic pH its NIR fluorescence is activated.Colocalization assay reveals that LD-OH specifically targets LDs.LD-OH is successfully utilized to image the dynamic production and movements of LDs with its green emission.Fluorescence images demonstrate that LD-OH is capable of imaging of lipophagy with high contrast and NIR fluorescence.We further introduce LD-OH to study the effect of different conditions on lipophagy in live cells.Our probe is the first NIR fluorescence probe for activatable imaging lipophagy,holding a great potential for elucidating its biological roles in live cells and animal models.As the power source of cells,mitochondria play a vital role in cell metabolism.Mitophagy is the process of removing damaged mitochondria through their own lysosomes to maintain mitochondrial health.Abnormal mitophagy is related to various pathological processes.Therefore,the detection of mitophagy is crucial for understanding the main functions of mitochondria under pathological and physiological conditions.Here we have successfully synthesized three fluorescent probes for imaging mitophagy in living cells.Through the comparison of spectral properties,Mito-3 is selected as the ideal fluorescent rotor probe for imaging mitochondria in living cells.In vitro results show that Mito-3 is sensitive to pH with good specificity,biocompatibility,photostability,and real-time and cyclical response to pH.Colocalization assay reveals that the fluorescence from Mito-3 overlaps with the fluorescence from Mito Tracker Green(a mitochondrial dye),indicating that Mito-3 can target mitochondria.Fluorescence images demonstrate that Mito-3 can respond to changes in mitochondrial pH and be used for mitophagy imaging.Our probes can image mitophagy in living cells,providing a promising tool for studying the physiological functions of mitochondria in living cells and animal models.