Injected extracellular vesicles and other nanoparticles hitchhike on erythrocytes and platelets from circulation towards organ clearance

The application of extracellular vesicles (EVs) for targeted delivery of drugs, proteins, and RNA to specific cell types or organs is a promising approach in molecular therapy. However, to achieve efficient and targeted delivery, it is crucial to improve our understanding of the fate of injected EVs in terms of tissue and cellular biodistribution. Studies by us and others in mouse models have demonstrated that EVs are rapidly cleared from the blood circulation following distribution to different organs, with large fractions ending up in liver, spleen and lungs within minutes after injection. In this study, we investigated the key steps and kinetics of this clearance process and particularly focused on understanding how intra-venously injected EVs would interact with circulating blood cells shortly after injection. Following the injection of fluorescently tagged EVs into mice, we utilized high sensitivity imaging flow cytometry to study the binding or association of EVs to different blood cell types, including platelets (PLTs) and red blood cells (RBCs), both in vitro and in vivo. Notably, PLTs and RBCs are regularly excluded in EV biodistribution and targeting studies even though they account for more than 99 % of all blood cells. We demonstrate that a significant proportion of injected EVs binds to PLTs and RBCs within minutes, and we further show that EVs ‘hitchhike’ on both PLTs and RBCs to the liver and spleen, where they are collectively cleared by macrophages. Detailed assessment of this process in a newly established ex vivo PLT/RBC binding assay revealed that this process is independent of EV purity, EV source and species, and highlights that it also applies to other nanoparticles such as lipid nanoparticles (LNPs) and liposomes. Interestingly, EVs engineered to bind albumin show clearly reduced PLT/RBC association. In summary, we report a highly relevant yet largely overlooked mechanism that significantly contributes to clearance of EVs and impacts their biodistribution and pharmacokinetics.