High efficiency preparation of monodisperse plasma membrane derived extracellular vesicles for therapeutic applications

Extracellular vesicles (EVs) are highly interesting for the design of next-generation therapeutics. However, their preparation methods face challenges in standardization, yield, and reproducibility. University of Basel researchers have developed a highly efficient and reproducible EV preparation method for monodisperse nano plasma membrane vesicles (nPMVs), which yields 10 to 100 times more particles per cell and hour than conventional EV preparation methods. nPMVs are produced by homogenizing giant plasma membrane vesicles following cell membrane blebbing and apoptotic body secretion induced by chemical stressors. nPMVs showed no significant differences compared to native EVs from the same cell line in cryo-TEM analysis, in vitro cellular interactions, and in vivo biodistribution studies in zebrafish larvae. Proteomics and lipidomics, on the other hand, suggested substantial differences consistent with the divergent origin of these two EV types and indicated that nPMVs primarily derive from apoptotic extracellular vesicles. nPMVs may provide an attractive source for developing EV-based pharmaceutical therapeutics.

Schematic representation of the generation of nano plasma membrane vesicles (nPMVs), their characterization, and in vitro/in vivo studies for comparison to native extracellular vesicles (EVs)

Donor cells are treated with chemical stressors (i.e., paraformaldehyde (PFA), dithiothreitol (DTT)) to induce apoptosis, which results in membrane blebbing and the production of ca. 1–3 µm sized apoptotic bodies (ApoBDs), namely giant plasma membrane vesicles (GPMVs). GPMVs are isolated and processed into unilamellar nPMVs through extrusion and purification to obtain a defined, monodisperse, and reproducible size distribution. nPMVs possibly differ in cargo and protein composition. The production rate and yield of the nPMV and native EV preparation method were evaluated. nPMVs were thereafter directly compared to native EVs using physico-chemical methods, 2D- and 3D-cryo-TEM, proteomics, lipidomics, in vitro recipient cell interactions using several human cell lines (i.e., Huh7 cells and THP-1 M0 macrophages), and biodistribution in transgenic (Tg) zebrafish larvae (ZFL) (i.e., Tg(kdrl:EGFP), Tg(mpeg:Kaede) (short for Tg(mpeg1:Gal4:UAS:Kaede)) as an in vivo vertebrate model.