Distinct targeting and uptake of platelet and red blood cell-derived extracellular vesicles into immune cells.

Blood-derived extracellular vesicles (EVs) hold great therapeutic potential. As blood contains mixed EV populations, it is challenging to study EVs originating from different cells separately. Blood cell concentrates manufactured in blood banks offer an excellent non-invasive source of blood cell-specific EV populations. To study blood cell-specific EVs, we isolated EVs from platelet (TREVs) and red blood cell (EryEVs) concentrates and characterized them using nanoparticle tracking analysis, imaging flow cytometry, electron microscopy and western blot analysis and co-cultured them with peripheral blood mononuclear cells (PBMCs). Our aim was to use imaging flow cytometry to investigate EV interaction with PBMCs as well as study their effects on T-lymphocyte populations to better understand their possible biological functions. As a conclusion, TREVs interacted with PBMCs more than EryEVs. Distinctively, TREVs were uptaken into CD11c+ monocytes rapidly and into CD19+ B-lymphocytes in 24 h. EryEVs were not uptaken into CD11c+ monocytes before the 24-h time point, and they were only seen on the surface of lymphocytes. Neither TREVs nor EryEV were uptaken into CD3+ T-lymphocytes and no effect on T-cell populations was detected. We have previously seen similar differences in targeting PC-3 cancer cells. Further studies are needed to address the functional properties of blood cell concentrate-derived EVs. This study demonstrates that imaging flow cytometry can be used to study the distinctive differences in the interaction and uptake of EVs. Considering our current and previous results, EVs present a new valuable component for the future development of blood-derived therapeutics.

Development of tandem cation exchange chromatography for high purity extracellular vesicle isolation: The effect of ligand steric availability.

Purification of extracellular vesicles for research and therapeutic applications requires updated methodology to address the limitations of traditional ultracentrifugation and other size-based separation techniques. Their downfalls include induced extracellular vesicle aggregation, low yields, poor scalability and one-dimensionality of the separation process, as the size or sedimentation speed of extracellular vesicles is often the only selection criterion. Ion exchange chromatography is a promising alternative or supplementary method candidate, as it offers a different approach for extracellular vesicle separation, which is surface charge. For now, mostly anion exchange chromatography has been evaluated for extracellular vesicle purification, as it successfully relies on the strongly negative surface charge of extracellular vesicles. However, as extracellular vesicles are very complex in their structure, also cation exchange chromatography could be applicable, due to individual cationic domains on the extracellular vesicle surface. Here, we compare anion exchange chromatography to different types of cation exchange chromatography for the purification of platelet extracellular vesicle samples also containing plasma-derived impurities. We found that the choice of resin structure used for cation exchange chromatography is critical for binding platelet extracellular vesicles, as a conventional-type cation exchanger was found to only capture and elute less than 20% of extracellular vesicles. With the tentacle-type resin, it was possible to obtain comparable platelet extracellular vesicle yields (over 90%) with cation exchange chromatography compared to anion exchange chromatography, as well as superior purity, especially when it was combined to conventional cation exchange resin.