ConspectusExtracellular vesicles are nanoparticles produced by cells. They are composed of cellular membrane with associated membrane proteins that surrounds an aqueous core containing soluble molecules such as proteins and nucleic acids, like miRNA and mRNA. They are important in many physiological and pathological processes as they can transfer biological molecules from producer cells to acceptor cells. Preparation of the niche for cancer metastasis, stimulation of tissue regeneration and orchestration of the immune response are examples of the diverse processes in which extracellular vesicles have been implicated. As a result, these vesicles have formed a source of inspiration for many scientific fields. They could be used, for example, as liquid biopsies in diagnostics, as therapeutics in regenerative medicine, or as drug delivery vehicles for transport of medicines. In this Account, we focus on drug delivery applications.As we learn more and more about these vesicles, the complexity increases. What originally appeared to be a relatively uniform population of cellular vesicles is increasingly subdivided into different subsets. Cells make various distinct vesicle types whose physicochemical aspects and composition is influenced by parental cell type, cellular activation state, local microenvironment, biogenesis pathway, and intracellular cargo sorting routes. It has proven difficult to assess the effects of changes in production protocol on the characteristics of the cell-derived vesicle population. On top of that, each isolation method for vesicles necessarily enriches certain vesicle classes and subpopulations while depleting others. Also, each method is associated with a varying degree of vesicle purity and concomitant coisolation of nonvesicular material. What emerges is a staggering heterogeneity. This constitutes one of the main challenges of the field as small changes in production and isolation protocols may have large impact on the vesicle characteristics and on subsequent vesicle activity.We try to meet this challenge by careful experimental design and development of tools that enable robust readouts. By engineering the surface and cargo of extracellular vesicles through chemical and biological techniques, favorable characteristics can be enforced while unfavorable qualities can be overruled or masked. This is coupled to the precise evaluation of the interaction of extracellular vesicles with cells to determine the extracellular vesicle uptake routes and intracellular routing. Sensitive reporter assays enable reproducible analysis of functional delivery.This systematic evaluation and optimization of extracellular vesicles improves our insight into the critical determinants of extracellular vesicle activity and should improve translation into clinical application of engineered extracellular vesicles as a new class of drug delivery systems.

Extracellular vesicles (EVs) have potential in disease treatment since they can be loaded with therapeutic molecules and engineered for retention by specific tissues. However, questions remain on optimal dosing, administration and pharmacokinetics. Previous studies have addressed biodistribution and pharmacokinetics in rodents, but little evidence is available for larger animals. Here, we investigated the pharmacokinetics and biodistribution of Expi293F-derived EVs labelled with a highly sensitive nanoluciferase reporter (palmGRET) in a non-human primate model (Macaca nemestrina), comparing intravenous (IV) and intranasal (IN) administration over a 125-fold dose range. We report that EVs administered IV had longer circulation times in plasma than previously reported in mice and were detectable in cerebrospinal fluid after 30-60 min. EV association with peripheral blood mononuclear cells, especially B-cells, was observed as early as 1-min post-administration. EVs were detected in liver and spleen within 1 h of IV administration. However, IN delivery was minimal, suggesting that pretreatment approaches may be needed in large animals. Furthermore, EV circulation times strongly decreased after repeated IV administration, possibly due to immune responses and with clear implications for xenogeneic EV-based therapeutics. We hope that our findings from this baseline study in macaques will help to inform future research and therapeutic development of EVs.Tom Driedonks and Linglei Jiang contributed equally to this study.