Extracellular vesicles (EVs) have emerged as important mediators of intercellular communication in a diverse range of biological processes. For future therapeutic applications and for EV biology research in general, understanding the in vivo fate of EVs is of utmost importance. Here we studied biodistribution of EVs in mice after systemic delivery. EVs were isolated from 3 different mouse cell sources, including dendritic cells (DCs) derived from bone marrow, and labelled with a near‐infrared lipophilic dye. Xenotransplantation of EVs was further carried out for cross‐species comparison. The reliability of the labelling technique was confirmed by sucrose gradient fractionation, organ perfusion and further supported by immunohistochemical staining using CD63‐EGFP probed vesicles. While vesicles accumulated mainly in liver, spleen, gastrointestinal tract and lungs, differences related to EV cell origin were detected. EVs accumulated in the tumour tissue of tumour‐bearing mice and, after introduction of the rabies virus glycoprotein‐targeting moiety, they were found more readily in acetylcholine‐receptor‐rich organs. In addition, the route of administration and the dose of injected EVs influenced the biodistribution pattern. This is the first extensive biodistribution investigation of EVs comparing the impact of several different variables, the results of which have implications for the design and feasibility of therapeutic studies using EVs.

Alpha-synuclein aggregation plays a central role in Parkinson's disease pathology. Direct transmission of alpha-synuclein from pathologically affected to healthy unaffected neurons may be important in the anatomical spread of the disease through the nervous system. We have demonstrated that exosomes released from alpha-synuclein over-expressing SH-SY5Y cells contained alpha-synuclein and these exosomes were capable of efficiently transferring alpha-synuclein protein to normal SH-SY5Y cells. Moreover, the incubation of cells with ammonium chloride or bafilomycin A1 to produce the lysosomal dysfunction recently reported in Parkinson's disease led to an increase in the release of alpha-synuclein in exosomes and a concomitant increase in alpha-synuclein transmission to recipient cells. This study clearly demonstrates the importance of exosomes in both the release of alpha synuclein and its transmission between cells and suggests that factors associated with PD pathology accelerate this process. These mechanisms may play an important role in PD pathology and provide a suitable target for therapeutic intervention.

Extracellular vesicles (EVs) are natural nanoparticles that mediate intercellular transfer of RNA and proteins and are of great medical interest; serving as novel biomarkers and potential therapeutic agents. However, there is little consensus on the most appropriate method to isolate high-yield and high-purity EVs from various biological fluids. Here, we describe a systematic comparison between two protocols for EV purification: ultrafiltration with subsequent liquid chromatography (UF-LC) and differential ultracentrifugation (UC). A significantly higher EV yield resulted from UF-LC as compared to UC, without affecting vesicle protein composition. Importantly, we provide novel evidence that, in contrast to UC-purified EVs, the biophysical properties of UF-LC-purified EVs are preserved, leading to a different in vivo biodistribution, with less accumulation in lungs. Finally, we show that UF-LC is scalable and adaptable for EV isolation from complex media types such as stem cell media, which is of huge significance for future clinical applications involving EVs. Recent evidence suggests extracellular vesicles (EVs) as another route of cellular communication. These EVs may be utilized for future therapeutics. In this article, the authors compared ultrafiltration with size-exclusion liquid chromatography (UF-LC) and ultra-centrifugation (UC) for EV recovery.

Exosomes are circulating nanovesicular carriers of macromolecules, increasingly used for diagnostics and therapeutics. The ability to load and target patient-derived exosomes without altering exosomal surfaces is key to unlocking their therapeutic potential. We demonstrate that a peptide (CP05) identified by phage display enables targeting, cargo loading, and capture of exosomes from diverse origins, including patient-derived exosomes, through binding to CD63—an exosomal surface protein. Systemic administration of exosomes loaded with CP05-modified, dystrophin splice–correcting phosphorodiamidate morpholino oligomer (EXO_PMO) increased dystrophin protein 18-fold in quadriceps of dystrophin-deficient mdx mice compared to CP05-PMO. Loading CP05-muscle–targeting peptide on EXO_PMO further increased dystrophin expression in muscle with functional improvement without any detectable toxicity. Our study demonstrates that an exosomal anchor peptide enables direct, effective functionalization and capture of exosomes, thus providing a tool for exosome engineering, probing gene function in vivo, and targeted therapeutic drug delivery.