Multiresolution Imaging Using Bioluminescence Resonance Energy Transfer Identifies Distinct Biodistribution Profiles of Extracellular Vesicles and Exomeres with Redirected Tropism

Extracellular particles (EPs) including extracellular vesicles (EVs) and exomeres play significant roles in diseases and therapeutic applications. However, their spatiotemporal dynamics in vivo have remained largely unresolved in detail due to the lack of a suitable method. Therefore, a bioluminescence resonance energy transfer (BRET)‐based reporter, PalmGRET, is created to enable pan‐EP labeling ranging from exomeres (<50 nm) to small (<200 nm) and medium and large (>200 nm) EVs. PalmGRET emits robust, sustained signals and allows the visualization, tracking, and quantification of the EPs from whole animal to nanoscopic resolutions under different imaging modalities, including bioluminescence, BRET, and fluorescence. Using PalmGRET, it is shown that EPs released by lung metastatic hepatocellular carcinoma (HCC) exhibit lung tropism with varying distributions to other major organs in immunocompetent mice. It is further demonstrated that gene knockdown of lung‐tropic membrane proteins, solute carrier organic anion transporter family member 2A1, alanine aminopeptidase/Cd13, and chloride intracellular channel 1 decreases HCC‐EP distribution to the lungs and yields distinct biodistribution profiles. It is anticipated that EP‐specific imaging, quantitative assays, and detailed in vivo characterization are a starting point for more accurate and comprehensive in vivo models of EP biology and therapeutic design.