Multiresolution imaging identifies distinct biodistribution profiles of extracellular vesicles and exomeres

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. Researchers from the National Taiwan University have created a bioluminescence resonance energy transfer (BRET)‐based reporter, PalmGRET, 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, the researchers show that EPs released by lung metastatic hepatocellular carcinoma (HCC) exhibit lung tropism with varying distributions to other major organs in immunocompetent mice. They further demonstrate 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. The researchers anticipate 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.

PalmGRET labels cellular and EV membranes

a) Representation of multimodal PalmGRET labeling the inner leaflet of the plasma membrane of cells and EVs to emit BLI and FL using BRET excitation (BRET‐FL), as well as FL from external light excitation. b) Lentiviral constructs for PalmGRET and GRET (control) for EP labeling. c) Multiresolution EP imaging. PalmGRET labels cellular membranes to enable imaging of EV donor cells and EVs from nanoscopic to macroscopic resolutions by FL and/or BLI signals. d) 3D reconstruction of Z‐stack, live‐cell confocal micrographs of 293T‐PalmGRET cells, revealing uneven, budding membrane surfaces (arrows) and protrusions (arrowheads) that may be precursors of EV release. e) Coculture of 293T‐PalmGRET and 293T‐PalmtdTomato cells showing released EPs in the extracellular space. 293T‐PalmGRET EPs (arrows); 293T‐PalmtdTomato EPs (arrowheads). Bar, 40 µm.