Enhanced quantification and cell tracking of dual fluorescent labeled extracellular vesicles.

Extracellular Vesicles (EVs) are nanosized particles with significant role in disease pathogenesis and as therapeutic potential. However, the lack of reliable and efficient methods for the characterization, quantification and tracking of EVs, combined with the limitations of detection techniques in differentiating specific EVs subtypes with beneficial properties, makes these process complex and time-consuming. To address this challenge, EVs were engineered using a tricistronic plasmid that encodes fluorescent proteins fused to tetraspanins (eGFP-CD63 and mCherry-CD9), with both fluorophores localized within the luminal space. Double fluorescently labelled small EVs (sEVs) were then produced in a stably transfected HEK293SF-3F6 cell line. The fluorescently labelled sEVs were characterized using a variety of techniques. Protein expression analysis showed that the fused proteins were efficiently produced and incorporated in sEVs, as evidenced by clear fluorescence signal detected. Comparisons of the size distribution and concentration of modified sEVs with controls indicated that sEVs engineering did not affect their biogenesis and morphology. Fluorescently labelled sEVs were then quantified by flow cytometry, allowing to distinguish sEVs from other EVs subtypes or sample particles. The values were then compared to fluorometry measurements, obtaining a linear correlation what enabled a novel sEVs quantification method. The functionality of engineered sEVs was assessed by monitoring their uptake and trafficking in recipient cells, obtaining an efficient internalisation by target cells. Overall, these results demonstrate that the implementation of dual fluorescent methodology is feasible for sEVs characterization, quantification, for in vitro study of EVs interaction with cells, and intercellular communication, as well as a valuable tool in the in vitro development of targeted therapeutic EVs delivery systems.