A NIR-II quantum dot-assisted dual-color imaging strategy enables simultaneous tracking of two subtypes of extracellular vesicles in vivo

Extracellular vesicles (EVs) are naturally occurring nano-sized membrane-bound vesicles with remarkable heterogeneity. To effectively elucidate their various biological behaviors, it is imperative to label EVs with fluorophores. Traditional EV labeling within the visible spectrum leads to autofluorescence interference and emission wavelength limitations, hindering the study of diverse EVs and necessitating separate analyses in different subjects which affects research accuracy. In this study, we leveraged an innovative near-infrared-II (NIR-II) QDassisted dual-color imaging technique to simultaneously monitor the distribution patterns of EVs in single animals, discerning differences based on size, surface proteins, and inter-vesicular interactions. We revealed that EVs smaller than 200 nm (small EVs, sEVs) predominantly localize to the spleen and lymph nodes as opposed to their larger counterparts (large EVs, lEVs, > 200 nm). This preferential positioning in peripheral immune organs for sEVs appears to be intricately linked to their surface composition, exemplified by the presence of biomolecules like programmed death-ligand 1 (PD-L1). Results also revealed that tumor cell-derived sEVs and immunocyte-derived sEVs significantly affect each other's biodistribution in vivo. In summary, present study provides a refined method for the accurate evaluation of the in vivo biological behaviors of heterogeneous EVs. This advancement is expected to deepen our understanding of EV diversity and their complex roles within biological systems.