Multi-resolution imaging using bioluminescence resonance energy transfer identifies distinct biodistribution profiles of extracellular vesicles and exomeres with redirected tropism

Extracellular particles (EP) including extracellular vesicles (EVs) and exomeres have been shown to 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. We therefore created a bioluminescence resonance energy transfer (BRET)-based reporter, PalmGRET, to enable pan-EP labelling 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, we show that EPs released by lung metastatic hepatocellular carcinoma (HCC) exhibit lung tropism with varying distributions to other major organs in immunocompetent mice. We further demonstrate that gene knockdown of lung-tropic membrane proteins, solute carrier organic anion transporter family member 2A1 (Slco2a1), alanine aminopeptidase (Cd13) and chloride intracellular channel (Clic1) decreases HCC-EP distribution to the lungs and yields distinct biodistribution profiles. We anticipate that EP-specific imaging, quantitative assays and detailed in vivo characterization to be a starting point for more accurate and comprehensive in vivo models of EP biology and therapeutic design.