HaloTag display enables quantitative single-particle characterization and functionalization of engineered extracellular vesicles

Extracellular vesicles (EVs) play key roles in diverse biological processes, transport biomolecules between cells, and have been engineered for therapeutic applications. A useful EV bioengineering strategy is to express engineered proteins on the EV surface to confer targeting, bioactivity, and other properties. Measuring how incorporation varies across a population of EVs is important for characterizing such materials and understanding their function, yet it remains challenging to quantitatively characterize the absolute number of engineered proteins incorporated at single-EV resolution. To address these needs, Northwestern University researchers developed a HaloTag-based characterization platform in which dyes or other synthetic species can be covalently and stoichiometrically attached to engineered proteins on the EV surface. To evaluate this system, the researchers employed several orthogonal quantification methods, including flow cytometry and fluorescence microscopy, and found that HaloTag-mediated quantification is generally robust across EV analysis methods. They compared HaloTag-labeling to antibody-labeling of EVs using single vesicle flow cytometry, enabling us to quantify the substantial degree to which antibody labeling can underestimate the absolute number of proteins present on an EV. Finally, the researchers demonstrate use of HaloTag to compare between protein designs for EV bioengineering. Overall, the HaloTag system is a useful EV characterization tool which complements and expands existing methods.

Validation of a HaloTag display system for functionalizing the EV surface


A) Cartoon illustrating how HaloTag-expressing HEK293FT cells produce HaloTag EVs which are isolated via differential centrifugation. The HaloTag protein can be used to display a variety of moieties on the surface of EVs via conjugation with different HaloTag ligands. B) HaloTag EVs display classical EV morphology. Transmission electron micrographs of HaloTag EV subpopulations show classic cup-shaped morphology. Top: Exosomes. Bottom: MVs. Scale bar: 100 nm. C,D) Western blots yield expected patterns of common EV markers in purified vesicles vs producer cells. EVs contain expected markers, calnexin is only present in cell lysate, and the FLAG tag fused to N-terminus of engineered HaloTag yields an expected band of 44.8 kDa and is present in engineered cell lysate and EVs populations. E) Representative histogram of nanoparticle tracking analysis of EVs derived from HEK293FT cells with or without HaloTag expression, normalized to the mode in each population. F) HaloTag expressing cells (bottom) but not unmodified cells (top) increase in fluorescence after exposure to AF 488 HaloTag ligand. G) HaloTag EVs adsorbed on polystyrene beads react with and conjugate AF 488 ligand, but unmodified EVs and the mock condition yield no such signal. Cell experiments were performed in biological triplicate. EV experiments were performed in technical triplicate. Error bars indicate standard error of the mean. Multicomparison statistical analysis was performed using a one-way ANOVA test, followed by Tukey’s multiple comparison test to evaluate specific comparisons (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). 

Mitrut RE, Stranford DM, Chan M, Bailey MD, Luo M, Meade TJ, Wang M, Leonard JN. (2023) HaloTag display enables quantitative single-particle characterization and functionalization of engineered extracellular vesicles. bioRXiv [online preprint]. [abstract]

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