Characterization of extracellular vesicles and synthetic nanoparticles with four orthogonal single-particle analysis platforms

Abstract

We compared four orthogonal technologies for sizing, counting, and phenotyping of extracellular vesicles (EVs) and synthetic particles. The platforms were: single-particle interferometric reflectance imaging sensing (SP-IRIS) with fluorescence, nanoparticle tracking analysis (NTA) with fluorescence, microfluidic resistive pulse sensing (MRPS), and nanoflow cytometry measurement (NFCM). EVs from the human T lymphocyte line H9 (high CD81, low CD63) and the promonocytic line U937 (low CD81, high CD63) were separated from culture conditioned medium (CCM) by differential ultracentrifugation (dUC) or a combination of ultrafiltration (UF) and size exclusion chromatography (SEC) and characterized by transmission electron microscopy (TEM) and Western blot (WB). Mixtures of synthetic particles (silica and polystyrene spheres) with known sizes and/or concentrations were also tested. MRPS and NFCM returned similar particle counts, while NTA detected counts approximately one order of magnitude lower for EVs, but not for synthetic particles. SP-IRIS events could not be used to estimate particle concentrations. For sizing, SP-IRIS, MRPS, and NFCM returned similar size profiles, with smaller sizes predominating (per power law distribution), but with sensitivity typically dropping off below diameters of 60 nm. NTA detected a population of particles with a mode diameter greater than 100 nm. Additionally, SP-IRIS, MRPS, and NFCM were able to identify at least three of four distinct size populations in a mixture of silica or polystyrene nanoparticles. Finally, for tetraspanin phenotyping, the SP-IRIS platform in fluorescence mode was able to detect at least two markers on the same particle, while NFCM detected either CD81 or CD63. Based on the results of this study, we can draw conclusions about existing single-particle analysis capabilities that may be useful for EV biomarker development and mechanistic studies.

Keywords: ectosomes; exosomes; extracellular vesicles; microvesicles; nanoflow cytometry; nanoparticle tracking analysis; resistive pulse sensing; single particle interferometric reflectance imaging sensing.

FIGURE 1

Methodology and EV separation. [(a) EVs were separated from H9 and U937 culture‐conditioned media by a combination of ultrafiltration and size exclusion chromatography (SEC EVs) or by differential ultracentrifugation (100K EVs). (b) Immunoblots of cell lysates from H9 and U937, EVs separated by ultracentrifugation (100K EVs) and SEC (SEC EVs), and later fractions of SEC (enriched for protein; SEC‐P). Antibodies are specified in Table 1; see also Supplementary Figure 1. (c) Electron micrograph of SEC EVs and 100K EVs from both cell lines. As indicated for each subpanel, leftmost scale bars represent 500 nm at magnification 40,000×; rightmost scale bars are 100 nm at magnification 100,000×. (d) EM of SS and PS. Leftmost scale bars are 500 nm at magnification 17,500×; rightmost scale bars are 100 nm at magnification 65,000×.]

FIGURE 2

SS and PS size distribution. [Size distributions for SS (n = 3) with standard deviation for (a) SP‐IRIS, (b) NTA, (c) MRPS, and (d) NFCM. Nominal SS diameters are indicated by vertical dotted lines: 68 nm, 91 nm, 113 nm, and 151 nm. Size distributions for PS (n = 3; with SD) for (e) SP‐IRIS, (f) NTA, (g) MRPS, and (h) NFCM. Nominal PS diameters are indicated by vertical dotted lines: 70 nm, 90 nm, 125 nm, and 150 nm. Inset in Figure 2c shows a single MRPS measurement of the size distribution; see also Supplementary Figure 3 for individual readings. 5‐nm bin sizes were used for all graphs.]

FIGURE 3

SS and PS quantification. [(a) SP‐IRIS label‐free capture for SS and PS using four capture spots (n = 3 per group; mean particle count per spot with SD). b) SS quantification (n = 3; mean particles/ml with SD). (c) PS quantification (n = 3; mean particles/ml with SD). In panels b and d, nominal PS concentration is indicated by a horizontal dotted line (1.0×10¹² particles/ml).]

FIGURE 4

H9 and U937 particle size distribution. [Diameters of particles for H9 SEC EVs and 100K EVs (n = 3 per group, with standard deviation) for (a) SP‐IRIS, (b) NTA, (c) MRPS, and (d) NFCM. Size distributions for U937 SEC EVs and 100K EVs (n = 3 per group; with SD) for (e) SP‐IRIS, (f) NTA, (g) MRPS, and (h) NFCM. Default bin sizes were retained for each method: 5 nm (SP‐IRIS), 30 nm (NTA), 1 nm (MRPS), 0.5 nm (NFCM). Please see Supplementary Figure 7 for graphs without error bars.]

FIGURE 5

H9 and U937 particle quantification. [SP‐IRIS label‐free capture for (a) H9 SEC EVs and 100K EVs and (b) U937 SEC EVs and 100K EVs using CD81, CD63, and mouse isotype control capture antibodies (measured on n = 3 SP‐IRIS chips and with n = 3 antibody spots each; mean particle count/spot with SD). H9 and U937 particle quantification (n = 3; mean particles/ml with SD) for (c) SEC EVs and (d) 100K EVs using NTA, MRPS, and NFCM.]

FIGURE 6

Particle phenotyping. [SP‐IRIS fluorescence detection using labelled anti‐CD81 and anti‐CD63 after particle capture with CD81, CD63, and mouse isotype control (n = 3 per group; mean and SD) for (a) H9 SEC EVs, (b) H9 100K EVs, (c) U937 SEC EVs, and (d) U937 100K EVs. Percent of particles detected with fluorescently‐labelled anti‐CD81 and anti‐CD63 by NTA and NFCM (n = 3 per group; mean and SD) for (e) H9 SEC EVs, (f) H9 100K EVs, (g) U937 SEC EVs, and (h) U937 100K EVs. Asterisk. An asterisk indicates that, in the authors’ view, an antibody did not perform on the instrument; it does not necessarily mean that the antibody would not perform in another context or with additional optimization.]