Fluorogenic labelling for tracking extracellular vesicle nanocarriers in the brain

BackgroundReliable tracking of extracellular vesicles (EVs), key biological nanocarriers in nanomedicine, remains a major technical challenge due to the limitations of conventional lipophilic dyes, including aggregation, micelle formation, and nonspecific background signals that compromise biodistribution analyses. MethodsHere, we present a fluorogenic labeling strategy based on Aco-600, a water-soluble probe exhibiting a "light-on" activation in hydrophobic environments. Medium/large EVs (m/lEVs) derived from murine BV2 microglial cells were labeled and intranasally administered to adult C57BL/6 mice. EV biodistribution and brain uptake were quantitatively assessed by ex vivo fluorescence imaging on brain cryosections at multiple time points (5-1440 min), focusing on the cortex and hippocampus. ResultsAco-600 labeling enabled high signal-to-noise detection with minimal background and no evidence of dye aggregation artifacts. Quantitative analysis revealed a consistent spatiotemporal distribution profile across brain regions, with peak signal intensity at 60 minutes post-administration, followed by progressive clearance. This approach provided reproducible and sensitive tracking of EV biodistribution following a clinically relevant intranasal delivery route. ConclusionsOur findings establish fluorogenic labeling as a robust and artifact-minimizing strategy for in vivo EV tracking. This method enhances the accuracy of biodistribution studies and supports the development of EV-based nanomedicine platforms, particularly for central nervous system delivery applications.