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

Yuki Kobayashi,Yuki Takahashi,Hiroto Otera,Yuriko Higuchi,Y. Takakura

Published 2026 in Journal of Pharmaceutical and Biomedical Analysis

ABSTRACT

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.

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