Cu-labeling of small extracellular vesicle surfaces via a cross-bridged macrocyclic chelator for pharmacokinetic study by positron emission tomography imaging.

We developed a method of labeling the surfaces of small extracellular vesicles (sEVs) with Cu using a cross-bridged, macrocyclic chelator (CB-TE1A1P) and applied to pharmacokinetics study with positron emission tomography (PET). After incubation in 20% plasma for 10 min, approximately a half of the Cu was desorbed from Cu-labeled sEVs purified by phosphate-buffered saline wash, suggesting partly weak interaction without coordinating to CB-TE1A1P. After subsequent purification with albumin, Cu desorption was greatly reduced, resulting in a radiochemical stability of 95.7%. Notably, labeling did not alter the physicochemical and biological properties of sEVs. After intravenous injection, Cu-labeled sEVs rapidly disappeared from the systemic blood circulation and accumulated mainly in the liver and spleen of macrophage-competent mice. In macrophage-depleted mice, Cu-labeled sEVs remained in the blood circulation for a longer period and gradually accumulated in the liver and spleen, suggesting mechanisms of hepatic and splenic accumulation other than macrophage-dependent phagocytosis. The comparison of tissue uptake clearance between macrophage-competent and macrophage-depleted mice suggests that macrophages contributed to 67% and 76% of sEV uptake in the liver and spleen, respectively. The application of this method in pharmacokinetics PET studies can be useful in preclinical and clinical research and the development of sEV treatment modalities.