Redirected tropisms of extracellular vesicles and exomeres yield distinct biodistribution profiles

Bionanoparticles including extracellular vesicles and exomeres (collectively termed EVs), 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. Here we report the development of a multi-resolution EV reporter, and reveal distinct biodistribution profiles following redirection of EV tropisms. We created a bioluminescence resonance energy transfer (BRET)-based reporter, PalmGRET, to enable pan-bionanoparticle labelling ranging from exomeres ( 200 nm) EVs. PalmGRET emits robust, sustained signals and allows the visualization, tracking and quantification of bionanoparticles from whole-animal to nanoscopic resolutions under different imaging modalities, including bioluminescence, BRET and fluorescence. Using PalmGRET, we show that EVs released by lung metastatic hepatocellular carcinoma (HCC) exhibit lung tropism with varying distributions to other major organs in immunocompetent mice. EV proteomics identified HCC-EV lung tropic protein candidates associated with cancer progression, in which SLCO2A1 and CLIC1 expression on non-tropic EVs conferred lung-tropism, while CD13 gave spleen tropism. Our results further demonstrate that redirected lung tropism decreases EV distribution to the liver, whereas the spleen tropism significantly reduces over time delivery to most major organs including the liver, kidneys, heart and brain. We anticipate our assays to be a starting point for more accurate and comprehensive in vivo models of EV biology and design. Furthermore, detailed in vivo EV characterization is critical for EV-based therapeutic developments, and a well-defined and quantitative assay for EV biodistribution profiles will be relevant for such development.