Quantum Dot Labeling and Visualization of Extracellular Vesicles

Abstract

Extracellular vesicles (EVs) are important mediators of intercellular communication. Their role in disease processes, uncovered mostly over the last two decades, makes them potential biomarkers, leading to a need to fundamentally understand EV biology. Direct visualization of EVs can provide insights into EV behavior, but current labeling techniques are often restricted by false-positive signals and rapid photobleaching. Hence, we developed a method of labeling EVs through conjugation with quantum dots (QDs)-high photoluminescent nanosized semi-conductors-using click chemistry. We showed that QD-EV conjugation could be tailored by altering QD to EV ratio or by using a catalyst. This conjugation chemistry was stable in a biological environment and upon storage for up to a week. Using size-exclusion chromatography, QD-EV conjugates could be separated from unconjugated QDs, enabling EV-specific signal detection. We demonstrate that these QD-EV conjugates can be live- and fixed-imaged in high resolution on cells and in tissue sheets, and the conjugates have better photostability compared with the commonly used EV dye DiI. We labeled two distinct EV populations: human semen EVs (sEVs) from fresh semen samples donated by healthy volunteers and brain EVs (bEVs) from excised rat brain tissues. We visualized QD-sEVs in epithelial sheets isolated from human vaginal mucosa and time-lapse imaged QD-bEV interactions with microglial BV-2 cells. The development of the QD-EV conjugate will benefit the study of EV localization, movement, and function and accelerate their potential use as biomarkers, therapeutic agents, or drug-delivery vehicles.

Keywords: click chemistry; extracellular vesicles; labeling; live imaging; quantum dots.

Figure 1.

Schematic of QD-EV conjugation chemistry. PEG-NH₂-modified QDs are first conjugated with Sulfo-S-4FB (QD-4FB). EVs, which contain a surface rich in primary amines, are modified with Sulfo-S-HyNic (EV-HyNic). Under mild conditions at room temperature, the QD-4FB and EV-HyNic are reacted together to form a stable bis-aryl bond.

Figure 2.

Characterization of purified sEVs and QD-sEV conjugates. (A) Representative data of sEV particle quantity (black) and protein concentration (red) distribution in elution f1-f25 of qEV column purification. f7-f11 contain ~97% of EVs with only 11% of protein, while f14-f23 contain 86% of protein. (B) Absorbance intensity at 354 nm of Sulfo-S-4FB (green), sEV-HyNic (orange), and sEV-HyNic + Sulfo-S-4FB (gray) during the first 30 min after chemical addition and mixing. sEV-HyNic modification was confirmed by increased absorbance at 354 nm (n = 3). (C) Absorbance intensity at 354 nm of Sulfo-S-4FB (green), Sulfo-S-HyNic (orange), and Sulfo-S-4FB + Sulfo-S-HyNic (gray) during the first 30 min after chemical mixing (n = 3). (D) Hydrodynamic sizes (left) and zeta potential (right) of QD-PEG-NH₂ (black) and QD-4FB (green) in 1×PBS (n = 3). (E) Representative data of QD-sEV conjugates (blue) and QD-4FB alone (green) fluorescence intensity at 585 nm in elution f7–30 of qEV column purification of QD-sEV conjugates. (F) Size distribution of sEV-HyNic and QD-sEV (n = 3) - black lines represent SEM for each size reported. (G) Zeta potential of sEV-HyNic and QD-sEV conjugates in 1×PBS (n = 3). (H) Representative fluorescence spectra of QD-PEG-NH₂ and QD-sEVs.

Figure 3.

TEM imaging of QDs, sEVs, and QD-sEVs. (A) QDs, sEVs, and QD-sEVs conjugates were imaged using TEM. QDs alone (left panel), sEVs alone (middle panel), and QD-sEV conjugates (right panel) were presented. QDs on the surface of sEVs are pointed out by white arrows. A magnified TEM image further indicates colocalization of QDs and sEVs at the single EV level (right insert). Scale bar: 100 nm in left panel, 200 nm in middle and right pane, and 100 nm in right insert. (B) QDs were conjugated to sEVs using click chemistry in varying ratios of QD to sEV and imaged using TEM. QD:sEV ratios of 40:1 (left), 70:1 (middle), 100:1 (right) were evaluated. Altering QD:sEV ratio changes the uniformity of QD distribution on the sEV surface. White arrows point to QDs on the surface of sEVs. Scale bars: 100 nm in left panel, 200 nm in middle and right panel.

Figure 4.

Characterization of QD-bEV conjugates. (A) Representative data of bEV particle quantity (black) and protein concentration (red) in f5-f15 of qEV column purification. f7-f10 contained both bEVs and proteins, while f12-f15 only contain proteins. (B) Representative data of particle/protein ratio of bEVs in f6-f15. f7-f10 contain the majority of pure bEVs and were collected for further conjugation experiments. (C) Absorbance intensity at 354 nm of Sulfo-S-4FB, bEV-HyNic, and Sulfo-S-4FB + bEV-HyNic during the first 30 min after chemical mixing (n = 3). bEV-HyNic modification was confirmed by increased absorbance at 354 nm. (D) Size distribution of bEV-HyNic and QD-bEV (n = 3) - black lines represent SEM for each size reported. (E) Zeta potential of bEV-HyNic and QD-bEV conjugates in 1×PBS (n = 3).

Figure 5.

Photostability and imaging of QD-bEVs on BV-2 cells. (A) The fluorescence intensity of QD-bEVs (n = 6) and DiI-bEVs (n = 3) during 10 min laser exposure under the same laser power. (B) Representative images of QD-bEVs on single BV-2 cell (top) and DiI-bEVs on single BV-2 cell (bottom) at 0, 5, and 10 min of laser exposure. Images were converted to grayscale for better visualization. Scale bars: 10 μm. (C) Representative 60× maximum intensity projection images of QD-bEVs (red) distribution and cellular interaction on Lectin stained BV-2 cells (green). Scale bars: 50 μm. (D) Higher magnification of an area in (C). QD-bEVs localized on the BV-2 cell membrane and throughout the individual cell. Scale bars: 10 μm. (E) Representative 60× maximum intensity projection images of pQDs (red) negative control distribution and cellular interaction on Lectin stained BV-2 cells (green). No obvious QD signals were found on BV-2 cells using the same laser power and imaging settings as (C-D), indicating unconjugated QDs can be successfully removed through SEC purification. Scale bars: 50 μm.