Comprehensive FRET-based tracking reveals extracellular vesicle fate across diverse administration routes and...

While extracellular vesicles (EVs) are promising drug delivery platforms, their in vivo fate remains poorly characterized. Most current studies using fluorescence resonance energy transfer (FRET)-based labeling strategies focus only on the proportion of intact particles, which fails to comprehensively depict the fate of EVs. In this study, we employed a simple and practical FRET-based tracking strategy to investigate the in vivo and intracellular distribution of human umbilical mesenchymal stem cell-derived EVs (uMSC-EVs). For the first time, we categorized the structural disruption of EVs into three distinct states: intact vesicles, non-intact vesicles, and the complete destruction of vesicle components. We systematically compared the spatiotemporal distribution of uMSC-EVs following five different administration routes in mice and assessed their degradation behavior and lysosomal colocalization in multiple cell types. Our results demonstrated route-dependent distribution and integrity of uMSC-EVs: intratracheal administration led to lung enrichment with high integrity; intravenous injection caused rapid liver accumulation with gradual integrity loss; intranasal delivery resulted in nasal accumulation of partially disrupted EVs; while intramuscular and subcutaneous injections promoted prolonged retention at injection sites. Ex vivo imaging confirmed these patterns and provided organ-specific dynamics. At the cellular level, uMSC-EVs were rapidly internalized and degraded within lysosomes within 6 h in A549 cells. HUVECs showed slower uptake but sustained degradation. In immune cells, both DC2.4 and RAW264.7 cells continuously trafficked EVs to lysosomes within 4 h, with DC2.4 degrading them slowly and RAW264.7 exhibiting the most efficient degradation. In summary, we present a novel strategy to monitor uMSC-EVs fate with broad applicability. By enabling detailed investigation into the spatiotemporal distribution of various nanocarriers-including LNPs and liposomes-this approach offers critical insights for both fundamental research and the rational design of efficient delivery platforms.