Extracellular vesicles (EVs) play a key role in many physiological and pathophysiological processes and hold great potential for therapeutic and diagnostic use. Despite significant advances within the last decade, the key issue of EV storage stability remains unresolved and under investigated. Here, we aimed to identify storage conditions stabilizing EVs and comprehensively compared the impact of various storage buffer formulations at different temperatures on EVs derived from different cellular sources for up to 2 years. EV features including concentration, diameter, surface protein profile and nucleic acid contents were assessed by complementary methods, and engineered EVs containing fluorophores or functionalized surface proteins were utilized to compare cellular uptake and ligand binding. We show that storing EVs in PBS over time leads to drastically reduced recovery particularly for pure EV samples at all temperatures tested, starting already within days. We further report that using PBS as diluent was found to result in severely reduced EV recovery rates already within minutes. Several of the tested new buffer conditions largely prevented the observed effects, the lead candidate being PBS supplemented with human albumin and trehalose (PBS‐HAT). We report that PBS‐HAT buffer facilitates clearly improved short‐term and long‐term EV preservation for samples stored at ‐80°C, stability throughout several freeze‐thaw cycles, and drastically improved EV recovery when using a diluent for EV samples for downstream applications.

Extracellular vesicles (EVs) function as natural delivery vectors and mediators of biological signals across tissues. Here, by leveraging these functionalities, we show that EVs decorated with an antibody-binding moiety specific for the fragment crystallizable (Fc) domain can be used as a modular delivery system for targeted cancer therapy. The Fc-EVs can be decorated with different types of immunoglobulin G antibody and thus be targeted to virtually any tissue of interest. Following optimization of the engineered EVs by screening Fc-binding and EV-sorting moieties, we show the targeting of EVs to cancer cells displaying the human epidermal receptor 2 or the programmed-death ligand 1, as well as lower tumour burden and extended survival of mice with subcutaneous melanoma tumours when systemically injected with EVs displaying an antibody for the programmed-death ligand 1 and loaded with the chemotherapeutic doxorubicin. EVs with Fc-binding domains may be adapted to display other Fc-fused proteins, bispecific antibodies and antibody–drug conjugates.

Intracellular delivery of protein and RNA therapeutics represents a major challenge. Here, we develop highly potent engineered extracellular vesicles (EVs) by incorporating bio-inspired attributes required for effective delivery. These comprise an engineered mini-intein protein with self-cleavage activity for active cargo loading and release, and fusogenic VSV-G protein for endosomal escape. Combining these components allows high efficiency recombination and genome editing in vitro following EV-mediated delivery of Cre recombinase and Cas9/sgRNA RNP cargoes, respectively. In vivo, infusion of a single dose Cre loaded EVs into the lateral ventricle in brain of Cre-LoxP R26-LSL-tdTomato reporter mice results in greater than 40% and 30% recombined cells in hippocampus and cortex respectively. In addition, we demonstrate therapeutic potential of this platform by showing inhibition of LPS-induced systemic inflammation via delivery of a super-repressor of NF-ĸB activity. Our data establish these engineered EVs as a platform for effective delivery of multimodal therapeutic cargoes, including for efficient genome editing.

Extracellular vesicles (EVs) are gaining ground as next-generation drug delivery modalities. Genetic fusion of the protein of interest to a scaffold protein with high EV-sorting ability represents a robust cargo loading strategy. To address the paucity of such scaffold proteins, we leverage a simple and reliable assay that can distinguish intravesicular cargo proteins from surface- as well as non-vesicular proteins and compare the EV-sorting potential of 244 candidate proteins. We identify 24 proteins with conserved EV-sorting abilities across five types of producer cells. TSPAN2 and TSPAN3 emerge as lead candidates and outperform the well-studied CD63 scaffold. Importantly, these engineered EVs show promise as delivery vehicles in cell cultures and mice as demonstrated by efficient transfer of luminal cargo proteins as well as surface display of different functional entities. The discovery of these scaffolds provides a platform for EV-based engineering.

Extracellular vesicles (EVs) are promising carriers for the delivery of a variety of chemical and biological drugs. However, their efficacy is limited by the lack of cellular specificity. Available methods to improve the tissue specificity of EVs predominantly rely on surface display of proteins and peptides, largely overlooking the dense glycocalyx that constitutes the outermost layer of EVs. In the present study, we report a reconfigurable glycoengineering strategy that can endogenously display glycans of interest on EV surface. Briefly, EV producer cells are genetically engineered to co‐express a glycosylation domain (GD) inserted into the large extracellular loop of CD63 (a well‐studied EV scaffold protein) and fucosyltransferase VII (FUT7) or IX (FUT9), so that the engineered EVs display the glycan of interest. Through this strategy, we showcase surface display of two types of glycan ligands, sialyl Lewis X (sLeX) and Lewis X, on EVs and achieve high specificity towards activated endothelial cells and dendritic cells, respectively. Moreover, the endothelial cell‐targeting properties of sLeX‐EVs were combined with the intrinsic therapeutic effects of mesenchymal stem cells (MSCs), leading to enhanced attenuation of endothelial damage. In summary, this study presents a reconfigurable glycoengineering strategy to produce EVs with strong cellular specificity and highlights the glycocalyx as an exploitable trait for engineering EVs.

Extracellular vesicles (EVs) are nanosized cell‐derived vesicles produced by all cells, which provide a route of intercellular communication by transmitting biological cargo. While EVs offer promise as therapeutic agents, the molecular mechanisms of EV biogenesis are not yet fully elucidated, in part due to the concurrence of numerous interwoven pathways which give rise to heterogenous EV populations in vitro. The equilibrium between the EV‐producing pathways is heavily influenced by factors in the extracellular environment, in such a way that can be taken advantage of to boost production of engineered EVs. In this study, a quantifiable EV‐engineering approach is used to investigate how different cell media conditions alter EV production. The presence of serum, exogenous EVs, and other signaling factors in cell media alters EV production at the physical, molecular, and transcriptional levels. Further, it is demonstrated that the ceramide‐dependent EV biogenesis route is the major pathway to production of engineered EVs during optimized EV‐production. These findings suggest a novel understanding to the mechanisms underlying EV production in cell culture which can be applied to develop advanced EV production methods.