A functional corona around extracellular vesicles

Nanoparticles can acquire a plasma protein corona defining their biological identity. Corona functions were previously considered for cell-derived extracellular vesicles (EVs). Researchers at Paracelsus Medical University have demonstrated that nano-sized EVs from therapy-grade human placental-expanded (PLX) stromal cells are surrounded by an imageable and functional protein corona when enriched with permissive technology. Scalable EV separation from cell-secreted soluble factors via tangential flow-filtration (TFF) and subtractive tandem mass-tag (TMT) proteomics revealed significant enrichment of predominantly immunomodulatory and proangiogenic proteins. Western blot, calcein-based flow cytometry, super-resolution and electron microscopy verified EV identity. PLX-EVs partly protected corona proteins from protease digestion. EVs significantly ameliorated human skin regeneration and angiogenesis in vivo, induced differential signalling in immune cells, and dose-dependently inhibited T cell proliferation in vitro. Corona removal by size-exclusion or ultracentrifugation abrogated angiogenesis. Re-establishing an artificial corona by cloaking EVs with fluorescent albumin as a model protein or defined proangiogenic factors was depicted by super-resolution microscopy, electron microscopy and zeta-potential shift, and served as a proof-of-concept. Understanding EV corona formation will improve rational EV-inspired nano-therapy design.

EV protein corona location

(a) Negative contrast TEM images of PLX EVs purified with tangential flow filtration (TFF2), depleted protein corona after TFF followed by ultracentrifugation (TUCF) and with protein corona re-established by defined factors VEGF+ IGF+ EGF in albumin solution (VIE/A). Scale bar 250 nm, small insert showing overview image with scale bar 500 nm. (b) Corona thickness based on negative contrast halo measurements. We analyzed n = 36 EV^TFF2 and n = 56 EV^TUCF+VIE/A; Mann–Whitney unpaired sample test, ****p > 0.0001. (c) New EV corona model and current/old view on EV structure (modified from (Kalluri & Lebleu, 2020) and www.exosome-rna.com/evpedia)