Enzyme-Assisted Covalent Labeling Method Of Extracellular Vesicles And Its Application In Cellular Imaging

In the past several years,extracellular vesicles(EVs)have become important mediators of intercellular communication,participating in the transmission of biological signals from prokaryotes to higher eukaryotes and regulating a variety of biological processes.In addition,the pathophysiology of EVs has also been found in many diseases,including cancer,infectious diseases,and neurodegenerative diseases.It has highlighted the status of EVs as potential new targets for therapeutic intervention.But due to the lack of appropriate tracking technology,little is known about the interaction between EVs and recipient cells.Based on this,we constructed a new model of covalent labeling of EVs by using a chemical enzyme-assisted method.By this way,we also can achieve the visualization of cellular uptake process of EVs.The specific research contents are as follows:1.Construction of a new method for chemical enzyme-assisted extracellular vesicle labeling.We reported a new chemical enzyme labeling technique that first used phospholipase D(PLD)to catalyze phosphatidylcholine and alkynes in situ exchange of choline on natural vesicle membranes.Subsequent alkynyl-azide click chemistry enabled fluorescent dye molecules to be labeled on the vesicles,and images of the labeled vesicles can be obtained by confocal fluorescence microscopy.The subsequent morphological and biological function characterizations showed that enzyme-assisted fluorescent labeling method of EVs was efficient and biocompatible.What's more,this process didn't affect the physical morphology and biological activity of EVs.In addition,we found that vesicles labeled with this method had better retention effects than traditional membrane-inserted dye-labeled vesicles through stability experiments,and didn't dissociate for a relatively long time even in a biological environment.Therefore,we think the method has broad application prospects in the fields of cell imaging and biological analysis.2.Imaging study of enzyme-assisted fluorescently labeled EVs during cell internalization.We obtained Cy5-EVs by using the above-mentioned chemical enzyme labeling strategy,and applied it to monitor the cell internalization process of cancer cell-derived EVs.The uptake process of immune cells such as RAW264.7 cells for vesicles was monitored shortly and muchly.We also explored the real-time process of vesicles entering the cell and the uptake effect with time gradient.Subsequently we treated the cells with a variety of highly specific uptake pathway inhibitors,further revealing the possible related mechanisms of the above internalization process.This powerful biocompatible labeling strategy provides an indispensable tool of EVs from chemical biology research to drug delivery for clinical research.