A functional corona around extracellular vesicles enhances angiogenesis, skin regeneration and immunomodulation

Abstract

Nanoparticles can acquire a plasma protein corona defining their biological identity. Corona functions were previously considered for cell-derived extracellular vesicles (EVs). Here we demonstrate 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.

Keywords: EV corona; EV function; angiogenesis; extracelular vesicle; placenta derived stromal cells; tangential flow filtration.

FIGURE 1

Experimental workflow. ALB, albumin; EVs, extracellular vesicles; MISEV, minimal information for studies of extracellular vesicles, updated 2018; SEC, size‐exclusion chromatography; sol. F., soluble factors; TEM, transmission electron microscopy; TFF, tangential flow filtration; TRPS, tunable resistive pulse sensing; TSEC, TFF x 2, followed by SEC; TUCF, TFF x 2 followed by ultracentrifugation (UCF); VIE/A, VEGF + IGF + EGF in albumin for corona reconstitution; EV characterization following MISEV2018 recommendations was performed as indicated in the results section and the respective figures. EV track ID: EV210462

FIGURE 2

EV identity and purity. (a) Cryo‐TEM showing representative images of PLX‐EVs after TFF2 (EV^TFF2). Scale bars 100 nm. (b) Super‐resolution microscopy topology display of tetraspanins CD81 (red), CD63 (green) and CD9 (blue), via single‐molecule fluorescence of individual EV^TFF2 (left) and the overall distribution of double and triple marker positive EVs as indicated in text inserts, based on 99,209 EV areas analysed. Scale bars 100 nm. (c) Western blots comparing placental‐expanded (PLX) cells (from lots P15, P25, P27) and corresponding EV preparations after one or two TFF cycles (TFF1, TFF2). Results for tetraspanins CD9, CD63, CD81, EV‐specific flotillin, cell markers calnexin and GRP94, as well as culture medium supplement‐derived apolipoprotein A1 (ApoA1) and human serum albumin (HSA). (d) Densitometry of blots shown in (c) normalised to total protein/lane. Significant differences (*p < 0.0332, **p < 0.0021 and ***p < 0.0002) were identified based on two‐tailed t‐test with 95% confidence level. The complete western blot membranes including also control endothelial cells are shown in a separate Western blot supplement

FIGURE 3

Quantitative proteomics of PLX secretome fractions. (a) Heatmap comparing the proteome of soluble factor factors (sol. F.) and EV^TFF2 with human platelet lysate‐supplemented defibrinised unconditioned cell culture medium (α‐MEM). (b) Volcano plot comparing protein expression signal significance to levels of enrichment in sol. F. vs. EV^TFF2. Functional categories according to corresponding GO terms as listed in Table S4 are depicted by color‐coded dots as indicated; additional highly over‐represented proteins marked as open circles (see also Figure S6). (c) Pathway enrichment analysis for significantly differentially detected proteins (n = 814, adjusted p < 0.05). Enriched proteins (n = 118) and corresponding WIKI pathways highlighted in the same colour. Proteins most significantly (absolute log2‐fold‐change > 1) enriched in soluble factors (bottom left; n = 10) and proteins enriched in the EV^TFF2 fractions (top; n = 47) shown. Proteins found in several pathways are shown with one bar with different corresponding colour codes. The remaining differentially detected proteins are highlighted as grey barcode on the bottom (category ‘others’; n = 696). Abbreviations: Net., network; path., pathway; sign., signalling

FIGURE 4

PLX‐EVs capture proangiogenic factors. (a) Angiogenic potential of different PLX secretome fractions as analysed by endothelial network formation in a matrigel assay. Total length of the endothelial networks in the presence of PLX‐EVs at the indicated EV per endothelial cell ratio is shown (e.g., Log10 [3] = 1000:1). Volumes of soluble factors (sol. F.) added to the assay were calculated accordingly corresponding to EV number as described in the methods section. Results pooled from three independent donors (***p < 0.0002, ****p < 0.0001). (b) Separation of EV^TFF2 from their adjacent proteins by size exclusion chromatography (EV^SEC) revealed significant loss of proangiogenic function

FIGURE 5

PLX‐EVs promote angiogenesis during skin regeneration in vivo. (a) Graphic illustration of the skin cell transplantation strategy testing EV vs. soluble factor (sol. F.) function during in vivo full thickness skin wound regeneration (Ebner‐Peking et al., 2021). Keratinocyte + fibroblast (KC + FB) cell suspension grafts, transplanted in the absence or presence of either TFF2‐isolated PLX‐EVs (+EV^TFF2) or sol. F. as described in the methods section (step I + II). Human skin biopsy sampling (III) and representative immunohistochemistry (IV; antihuman CD44, red; antihuman vimentin, green). (b‐m) Histology of day 14 post grafting. Hematoxylin and eosin (HE) and Masson Goldner trichrome (MG3C) staining confirmed the layered cell organization into epidermis and dermis. Dermal analysis showed vessel enrichment predominantly when supporting the cell grafts with EV^TFF2. Serum only and sol. F.‐driven cell transplants revealed limited murine vessel sprouting and tissue haemorrhage. Vessels were stabilised by pericytes (arrow in L). Dotted boxes in (b‐d; h‐j) indicate magnified areas in (e‐g; k‐m). (n) Quantification showing significantly increased epidermal thickness (Cox & Mann, 2008) in cell grafts in the absence of EVs^TFF2 in transplants of KC+FB ‘cells only’ or KC+FB transplanted in the presence of sol.F:, compared to control human skin (ctrl). (o) Vessel density in grafted dermis. Mean ± SD results (n, o). One Way‐ANOVA, multiple comparison of four biological and three technical replicates (*p < 0.05, ****p < 0.0001)

FIGURE 6

Immunomodulation and cell signalling by EVs: (a) Inhibition of phytohemagglutinin (PHA)‐induced T cell proliferation by PLX stromal cells, EV^TFF1 and EV^TFF2 as compared to soluble factors (sol. F.) added as indicated in limited dilution at defined ratio to mononuclear leukocytes. The percentage of inhibition was calculated relative to the maximum proliferation induced by PHA. Pooled results of three independent donors measured at day four. The first four bars show inhibition of T cell proliferation in a dose‐response to decreasing number of PLX:T cells of 1:1, 1:3, 1:9, 1:27, from left to right. (b). Assay format as used in (a) but testing the inhibition of T cell proliferation by size‐exclusion‐purified EV^TSEC and their former corona separated by size‐exclusion chromatography, compared to parental EV^TFF2. (c) Peripheral blood mononuclear cells were incubated with bodipy‐labelled EV^TFF2 at predetermined ratio of 1:5000 for one, 24 and 48 h as indicated. The bodipy signal was located to CD3⁺ T cells, CD19⁺ b cells, CD56⁺ NK cells and CD14⁺ monocytes by polychromatic flow cytometry. Pooled results from three independent donors performed in triplicate were analysed (in a‐c). Statistical analysis was done in Graph‐Pad Prizm 7.03 using one way ANOVA analysis with Sidak correction for multiple samples (** p < 0.002; *** p < 0.0002; **** p < 0.0001)

FIGURE 7

EV protein corona analysis: (a) Endothelial cell network formation comparing EVs^TFF2 with ultracentrifuged, that is, corona‐depleted EVs (EV^TUCF) and free growth factors and growth factor corona‐cloaked EVs. Representative experiment, quadruplicates. (b) Dose dependent endothelial cell network formation in the presence (+) or absence (‐) of EV^TUCF in an 8% human albumin solution containing VEGF, IGF and EGF (VIE/A) (n = 3; individual additional experiments colour coded in red, green, blue, and covering a broader dose range than in (a)). Statistics were calculated using a linear mixed model with treatment as fixed effect and dilution as random effect (a, b). (c) Fold‐change of VEGF concentration comparing EV^TFF2 to EV^TUCF (mean ± SD, n = 3, T‐test, p < 0.05). (d) Zeta potential of EV^TUCF in the absence or presence of a re‐established protein corona (+ corona) measured in quadruplicates by TRPS. Mode of distribution was used for statistical analysis (T test; **p < 0.01). (e) Zeta potential distribution vs. individual EV size (pooled dataset; n = 4)

FIGURE 8

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 as shown in detail in Figure S12. 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)