Extracellular vesicles (EVs) are increasingly recognised for their roles in intercellular communication and therapeutic delivery. Yet, current labelling methods often introduce artefacts or disrupt vesicle integrity, limiting confidence in functional studies.
In this work, we explore an endogenous labelling approach using fluorogenic conjugated oligoelectrolytes (COEs), a class of water-soluble bilayer-spanning fluorescent probes. Cell lines were cultured in media supplemented with COEs, allowing the fluorophores to intercalate into the plasma membrane. During vesicle formation, these lipophilic probes were passively incorporated into EV membranes, eliminating any post-isolation staining and reducing the risks of altering EV properties introduced by exogenous labelling.
Using endogenously-labelled EVs derived from human cell lines, we demonstrate uniform and sustained fluorescence across multiple detection platforms, including flow cytometry and confocal imaging. These COE-labelled EVs retained key tetraspanin surface markers and showed efficient uptake by recipient cells, confirming compatibility with downstream functional assays.
Crucially, dye-only controls showed no aggregation or background interference, and ultrafiltration confirmed clean recovery of labelled vesicles. The method is compatible with EV release studies and requires minimal processing, supporting a simplified and biologically faithful workflow.
Overall, COE-based endogenous labeling introduces a robust strategy that preserves vesicle integrity while enhancing analytical clarity. By reducing artefacts and improving reproducibility, this method supports more accurate tracking and functional interpretation of EVs. With further validation studies, this approach may extend to other lipid-enclosed nanoscale entities, such as virus-like particles.