Membrane-binding peptides for extracellular vesicles on-chip analysis

Abstract

Small extracellular vesicles (sEVs) present fairly distinctive lipid membrane features in the extracellular environment. These include high curvature, lipid-packing defects and a relative abundance in lipids such as phosphatidylserine and ceramide. sEV membrane could be then considered as a "universal" marker, alternative or complementary to traditional, characteristic, surface-associated proteins. Here, we introduce the use of membrane-sensing peptides as new, highly efficient ligands to directly integrate sEV capturing and analysis on a microarray platform. Samples were analysed by label-free, single-particle counting and sizing, and by fluorescence co-localisation immune staining with fluorescent anti-CD9/anti-CD63/anti-CD81 antibodies. Peptides performed as selective yet general sEV baits and showed a binding capacity higher than anti-tetraspanins antibodies. Insights into surface chemistry for optimal peptide performances are also discussed, as capturing efficiency is strictly bound to probes surface orientation effects. We anticipate that this new class of ligands, also due to the versatility and limited costs of synthetic peptides, may greatly enrich the molecular toolbox for EV analysis.

Keywords: Extracellular vesicles; membrane binding; membrane curvature; microarrays; peptides.

Figure 1.

Common mechanisms involved in membrane recognition by curvature-sensing peptides. (a) Purely electrostatic interactions are typical of cationic peptides; (b) specific binding to lipids particularly abundant in small vesicles (e.g. phosphatidylserine) can drive the interaction; (c) amphipathic peptides usually approach highly curved membranes through electrostatics, and subsequently insert into lipid-packing defects. Binding can be stabilised by peptide folding within the membrane, facilitated by the presence of hydrophobic groups.

Scheme 1.

Membrane-sensing peptides are used to capture EV on sensing surfaces. Peptidic probes are immobilised on chips through chemoselective click-type reaction between azido groups provided by MCP-6 surface coating and propargyl-glycine-terminated peptides. Peptidic probes are synthesised in a linear form (BP) and in two multivalent presentation: branched (BPb) and tandem (BPt). As a negative control (BPn), a peptide where arginine residues were mutated to (oppositely charged) glutamic acid residues was synthesised.

Figure 2.

Characterisation of UC-isolated EVs from HEK cell-line culture performed by TEM, NTA and WB. (a) TEM imaging of the bulk UC-isolated HEK EVs after negative staining by phosphotungstic acid. (b) Results of the analysis by NTA providing a mean particle size of 180 ± 1 nm and a concentration of 1.2 × 10¹² particles/mL. (c) The presence of transmembrane protein CD63 and CD9 and luminal proteins ALIX and TSG101 was assessed by Western blotting. The UC preparation resulted positive to all the four proteins. (d) HEK UC particle density per mm² detected on BP and BPn peptide spots in a blank sample (filtered PBS) and in 1 × 10⁶–1 × 10⁹ particles/mL concentrations range. A clear dose-response effect is visible. Signal on BPn peptide is negligible. (e) Representative images of BP and BPn peptide spots incubated with 1 × 10⁷–1 × 10⁹ particles/mL: blue dots indicate detected particles. (f) HEK UC particle density per mm² detected on antibody microarray (anti CD81/CD63/CD9). Only 1 × 10⁹ particles/mL concentration provides on CD antibodies spots a signal distinguishable from that on the negative control antibody. (g) Observed size distribution of captured particles reported as the number of counts detected in each 5 nm bin. Representative peptide spot images are reported in the supplementary information (Figure S1).

Scheme 2.

Scheme of the assay for label-free and fluorescence detection of EV captured on microarray chips. A silicon chip is arrayed with spots of capturing peptides and incubated with the EV sample. SP-IRIS platform images the chip and provides a label-free counting and sizing of the captured EV. The same chip can then be further incubated with fluorescent antibodies for immune staining of EV-associated proteins and three-colour fluorescence based co-localisation of EV surface markers.

Figure 3.

NTA analysis of (a) sEV-enriched sample obtained by centrifugation at 100.000 g for 2 h (mean particle diameter 143 ± 1 nm) and (b) MVs-enriched sample obtained at 10.000 g for 1 h (mean particle diameter 236 ± 5 nm). (c) Western blotting analysis for sEVs (lane 1) and MVs-enriched sample (lane 2). CD63, CD9 and CD81 markers are confirmed for both samples. Similarly, luminal proteins Alix and TSG101 are detected for both sEVs and MVs samples. (d) Observed size distribution on peptide chips for captured vesicles from the sEVs and (e) MVs-enriched sample obtained at 10.000 g. The size is reported as the number of counts detected in each 5 nm bin. (f) Size distribution on tetraspanins antibody chip for the sEVS sample and (g) MVs sample.

Figure 4.

(a) EV density after incubation with HEK UC sample at the concentration of 1 × 10⁹ particles/mL label free detected on BP peptides; (b) correspondent EV density detected by fluorescence on BP peptides; (c) representative BP spot and fluorescence immune staining. Images were acquired on the three different fluorescence channels: green particles are vesicles captured by BP and positive for CD81; blue particles are vesicles captured by BP and positive for CD63 whereas red particles are vesicles captured by BP and positive for CD9.

Figure 5.

(a) TEM imaging of EVs obtained from human serum by UC followed by combined polymer precipitation and SEC showed results comparable with analogous analysis reported elsewhere [36]; (b) NTA analysis provided a mean particle size of 203 ± 3 nm and a concentration of 8.2 × 10¹¹ particles/mL; (c) the presence of transmembrane protein CD63, CD81 and CD9 and luminal proteins ALIX and TSG101 was assessed by Western blotting of serum (lane 1) and after combined isolation of EVs. Contamination by lipoproteins is assessed by WB of Apo AI in serum (lane 1) and in the purified EVs (lane 2). (d) Analysis of EV isolated by ultracentrifugation, polymer precipitation and SEC from human serum incubated on peptide microarrays at 1 × 10⁹ particles/mL concentration. Density of particles captured by BP peptides (left panel) is confirmed by fluorescence staining using CD81/CD/63/CD9 fluorescent antibodies (right panel). (e) Analysis performed on unpurified human serum diluted 1:8. Density of particles captured by BP peptides is detected label free (left panel) and by fluorescence staining using CD81/CD/63/CD9 fluorescent antibodies (right panel). Observed size distribution of captured EV is reported in the supplementary information (Figure S3).

Figure 6.

(a) EV density on BP peptides immobilised either chemoselectively on MCP-6 or randomly on MCP-2. EV capturing capacity is abolished when peptides are not oriented on the microarray surface. (b) Comparison of representative images of BP spots either chemoselectively or randomly immobilised. Spot size is smaller on MCP-6, edges well defined and particle counting after incubation higher than on the random bound peptide.

Figure 7.

(a) NTA analysis of the SEC-isolated EVs. Mean particle size: 208 ± 3 nm. (b) WB analysis of SEC-isolated EVs from human serum. The presence of the transmembrane proteins CD9 and CD63 and the luminal proteins ALIX and TSG101 is checked and positivity confirmed for CD63 and TSG101; (c) TEM imaging after negative staining showed results in line with analogous analysis reported elsewhere [36,39]; (d) NTA analysis of SEC-isolated EVs after 6 h incubation at 37°C in presence of trypsin mean particle size: 247 ± 3 nm; (e) TEM imaging after trypsin treatment. (f) Particle density of EV from human serum incubated at the concentration of 1 × 10⁹ particles/mL. EV capturing by BP peptides is not affected by trypsin treatment. (g) EV binding on CD81/CD63/CD9 antibody chip is abolished by surface protein digestion using trypsin.