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 and exomeres play a significant role in diseases and therapeutic applications. However, the spatiotemporal dynamics of EPs in vivo remains to be elucidated with a suitable method. In this study, we developed a bioluminescence resonance energy transfer (BRET)-based reporter, PalmGRET, to visualize, track and quantify EPs both in vitro and in vivo. To explore the tropism effect of membrane proteins on EPs from lung metastatic hepatocellular carcimoma (HCC), we established PalmGRET in a mouse hepatocellular carcinoma cell line, HCA1, termed HCA1-PalmGRET. EP harvested from HCA1-PalmGRET cells were processed with gel electrophoresis, followed by in-gel digestion and LC-MS/MS analysis. The identified protein list was compared with published lung tropism databases and four membrane proteins were selected for subsequent gene knockdown experiments: solute carrier organic anion transporter family member 2A1 (Slco2a1), alanine aminopeptidase (Anpep/Cd13), chloride intracellular channel 1 (Clic1), and sodium-hydrogen antiporter 3 regulator 1 (Nherf1). Using PalmGRET, we revealed that knockdown of Slco2a1, Cd13 and Clic1 significantly reduced lung tropism of HCC-EPs with varying redirected delivery to other organs.