Background: Reliable 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. Methods: Here, 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 (from 5 to 1440 min), focusing on the cortex and hippocampus. Results: Aco-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. Conclusions: Our 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.
Rinaldi, A., Catalano, M.
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