In Vivo Tracking of Extracellular Vesicles in Mice Using Fusion Protein Comprising Lactadherin and Gaussia Luciferase.

Extracellular vesicles (EVs) are cell-derived vesicles comprising a lipid bilayer and are found in body fluids, such as blood, sweat, and urine. As EVs, especially exosomes, function as endogenous intercellular delivery tools, their roles in various biological events have been extensively investigated. In addition, they are expected to become safe and effective drug delivery systems (DDS) because of their intrinsic nature. In the development of EV-based DDS, as well as in the investigation of the biological functions of EVs, it is important to analyze the in vivo behavior of EVs by tracking them. Therefore, we have developed a sensitive EV-labeling method to track EVs in vivo by designing a fusion protein comprising lactadherin (LA) (alias milk fat globule-EGF factor 8), a protein that binds to EV membranes through interaction with phosphatidylserine, and Gaussia luciferase (gLuc), a chemiluminescent protein. gLuc-LA-labeled EVs are easily obtained by transfecting EV-producing cells with a gLuc-LA-encoding plasmid vector. Here, we describe methods to label EVs with the fusion protein and to track the labeled EVs in vivo.