A room-temperature ⁸⁹Zr⁴⁺ radiolabelling strategy for small extracellular vesicles with enhanced plasma stability for PET Imaging

Both for diagnostic purposes and regenerative medicine, it is essential to develop advanced imaging platforms capable of tracking the biodistribution of small extracellular vesicles (sEVs), as current methods are limited by inadequate resolution and sensitivity. In this study, we introduce a novel labeling strategy utilizing the radioisotope zirconium-89 (89Zr), which boasts a half-life of 78.4 h and is cost-effective to produce. To achieve this, we designed a new chelator tailored for 89Zr4+ that offers enhanced stability compared to the conventional deferoxamine (DFO). This chelator forms a robust complex with 89Zr4+ at room temperature, suitable for sEV labeling for PET imaging applications. The radiolabeling process involved a two-step procedure: first, conjugation of the chelator to the sEVs, and second, radiolabeling with 89Zr4+. The resulting sEV-L1-Zr demonstrated a radiochemical yield of approximately 60% and maintained around 80% stability in plasma over seven days. Importantly, our modifications did not alter the morphology, surface protein composition, internal RNA content, or bioactivity of the sEVs. We successfully visualized sEVs at very low doses in the mouse heart following intravenous injection of sEV-L1-Zr. Additionally, ex vivo experiments using a Langendorff rat heart perfusion model confirmed targeted accumulation of the vesicles in cardiomyocytes as compared to other cells in the heart compartment. This approach provides a promising platform for sensitive and stable in vivo tracking of sEVs, advancing their application in both diagnostic imaging and regenerative therapies.