Multiparametric Orthogonal Characterization of Extracellular Vesicles by Liquid Chromatography Combined with In-Line Light Scattering and Fluorescence Detection

Abstract

Extracellular vesicles (EVs) are membrane-enclosed biological nanoparticles with potential as diagnostic markers and carriers for therapeutics. Characterization of EVs poses severe challenges due to their complex structure and composition, requiring the combination of orthogonal analytical techniques. Here, we demonstrate how liquid chromatography combined with multi-angle light scattering (MALS) and fluorescence detection in one single apparatus can provide multiparametric characterization of EV samples, including concentration of particles, average diameter of the particles, protein amount to particle number ratio, presence of EV surface markers and lipids, EV shape, and sample purity. The method requires a small amount of sample of approximately 10⁷ EVs, limited handling of the sample and data analysis time in the order of minutes; it is fully automatable and can be applied to both crude and purified samples.

Figure 1

Schematic representation of the analytical system. Samples were injected into the chromatographic column (either size exclusion or ion exchange) to separate different species. The eluate was analyzed first by an in-line multi-angle light scattering (MALS) detector and then by a multiple wavelength fluorescence detector (FLD).

Figure 2

(A–C) Characterization of EVs derived from HEK293F cells: (A) Cryo-TEM image of purified EVs. (B) Size distribution of the EVs measured by NTA. (C) Presence of common EV surface markers CD81, CD63, and CD9 measured by bead-based flow cytometry.

Figure 3

MALS characterization of EVs. (A) Scattered light intensity (Rayleigh ratio) measured at different elution volumes at selected angles (14, 90, and 142°). (B) Scattered light intensity (Rayleigh ratio) measured at different angles θ (black dots) at 0.95 mL elution volume. The continuous line represents the fit according to eqs 1 and 2 and provides the radius of gyration r. (C) Radius of gyration (green dots) and concentration of particles measured according to eq 3 (black line) at different elution volumes. (D) Comparison between the total number of particles measured by integrating the SEC-MALS data shown in panel (C) and by NTA (see Experimental Procedure). (E) Number of particles measured by the SEC-MALS system upon progressive decrease of the injection volume. (F) Comparison between different EV sizing techniques based on light scattering. Diameter distribution of EV samples measured by SEC-MALS and NTA.

Figure 4

Characterization of EV total protein amount using intrinsic fluorescence of tryptophan residues. (A) Intrinsic fluorescence signal of an EV sample injected into the SEC column (same sample as in Figure 2A). (B) Correlation between the number of vesicles measured by MALS and the intrinsic fluorescence signal at decreasing EV sample injection volume. (C) Calibration curve of the intrinsic fluorescence signal of BSA vs injected BSA amount.

Figure 5

Characterization of vesicle markers by fluorescence detection. (A) Illustration of the strategy for the fluorescent labeling of EVs. (B–D) Chromatograms of anti-CD81 antibody (B), DiI (C), and CalceinAM (D) in the absence (red) and presence (black) of EVs. In panel (D), the data are plotted until 1.8 mL for clarity. The inset shows the full chromatogram. The eluted fluorescently labeled EV fraction is highlighted in green.

Figure 6

Characterization of vesicles directly from conditioned media by using size exclusion chromatography coupled to light scattering and fluorescence detectors. The eluted EV fraction is highlighted in green. (A) Number of particles and their radius. (B) Native fluorescence signal and (C) anti-human CD81 signal.

Figure 7

Using anion exchange chromatography (AIEX) to characterize the impurities in a SEC-purified EV sample. (A) Measured scattered light intensity (Rayleigh ratio) after AIEX at different elution volumes at selected detectors (38, 90, and 130° angles). (B) Measured EV radius of gyration (green dots) and concentrations (black line) at different elution volumes corresponding to the area highlighted in purple in panel (A). (C) Intrinsic fluorescence chromatograph after separation by AIEX. The area highlighted in purple corresponds to the elution of EVs. (D) Comparison of ng protein per particle after SEC and after additional purification by AIEX.