Development of a simple labeling method using fluorescent protein fusion proteins targeting the membrane lipids of...

Extracellular vesicles (EV) are lipid-based nanoparticles naturally released by cells, exhibiting considerable heterogeneity in size, surface charge, and biomolecular composition. Recently, increasing attention has been directed toward the characterization of distinct EV-subpopulations, particularly based on unique surface antigen expression profiles. Therefore, a method for analyzing EV-subpopulations using versatile equipment would be highly valuable. In this study, we developed a labeling method for analyzing specific populations in small EVs (sEVs) distinguished by their levels phosphatidylserine (PS) exposure. For visualization, sEVs were labeled with two fusion proteins (enhanced green fluorescent protein [EGFP] linked to lactadherin [LA] and mCherry-Vn96) comprising a PS-binding protein or sEV-tropic peptide (Vn96) combined with fluorescent proteins. Using ultracentrifugation, bulk sEVs were collected, and a fraction of PS^(-) sEVs (PS⁽⁺⁾ sEV-depleted fraction) were isolated by depleting PS⁽⁺⁾ sEVs from bulk sEVs. In bulk sEVs, the colocalization of EGFP-LA and mCherry-Vn96-derived signals was detected. In contrast, the PS⁽⁺⁾ sEV-depleted fraction exhibited reduced EGFP-LA fluorescence signal, with only mCherry-Vn96 fluorescence remaining detectable. In conclusion, our labeling technique facilitates the identification and analysis of sEV-subpopulations using fluorescence microscopy in small sample volumes. This platform can also be adapted for broader applications by incorporating additional protein markers.