HaloTag display enables quantitative single-particle characterisation and functionalisation of engineered extracellular vesicles

Extracellular vesicles (EVs) are tiny bubbles released by cells that transport biomolecules between cells, playing crucial roles in biological processes and emerging as promising tools for therapeutic applications. One of the key challenges in EV research is accurately quantifying the incorporation of engineered proteins on their surface, a critical step in enhancing their targeting and therapeutic potential.

EVs as Biological Couriers: EVs are natural carriers that shuttle proteins, nucleic acids, and other biomolecules between cells, influencing diverse biological functions from immune response regulation to cancer progression.

Engineering EVs for Precision: In bioengineering EVs for therapeutic use, scientists manipulate these vesicles to express specific proteins on their surface. This modification can confer targeting abilities, enhance bioactivity, or impart other desired properties crucial for therapeutic efficacy.

The Challenge of Quantification: Measuring the precise number of engineered proteins incorporated into EVs at the single-vesicle level has been a persistent challenge. Accurate quantification is essential for understanding how these modified EVs function and ensuring consistent quality in therapeutic applications.

Introducing the HaloTag Platform: To address this challenge, researchers at Northwestern University have developed a novel characterisation platform based on HaloTag technology. HaloTag allows for the covalent and stoichiometric attachment of dyes or synthetic species to engineered proteins on the EV surface, enabling precise quantification.

Validation of a HaloTag display system for functionalising 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 and d) Western blots yield expected patterns of common EV markers in purified vesicles versus 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, normalised 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).

Robust Quantification Methods: The HaloTag-based approach has been validated using various quantification techniques such as flow cytometry and fluorescence microscopy. These methods have demonstrated robustness across different EV analysis protocols, providing reliable insights into the distribution and quantity of engineered proteins on EVs.

Comparative Analysis: The researchers compared HaloTag labelling with traditional antibody-based labelling using single vesicle flow cytometry. This comparison highlighted significant discrepancies, showing that antibody labelling can underestimate the actual protein content on EV surfaces.

Advancing EV Bioengineering: Moreover, the HaloTag system facilitates comparative analyses between different protein designs for EV bioengineering. This capability allows researchers to optimize engineered EVs for enhanced therapeutic efficacy and specificity.

Future Directions: As EV-based therapies continue to evolve, the HaloTag characterisation platform promises to be instrumental. Its ability to accurately quantify engineered proteins on EVs not only enhances our understanding of these vesicles but also accelerates the development of targeted therapies for various diseases.

Conclusion: The HaloTag-based characterisation platform represents a significant advancement in EV research and bioengineering. By enabling precise measurement of engineered protein incorporation on EVs, this technology unlocks new possibilities for tailored therapeutics and biomedical applications.

Looking Ahead: Future research will focus on further refining HaloTag methodologies, expanding its applications across different EV types and cargo, and translating these advancements into clinical settings. This journey holds promise for transforming how we harness EVs as powerful tools in medicine and biotechnology.

Mitrut RE, Stranford DM, DiBiase BN, Chan JM, Bailey MD, Luo M, Harper CS, Meade TJ, Wang M, Leonard JN. (2024) HaloTag display enables quantitative single-particle characterisation and functionalisation of engineered extracellular vesicles. J Extracell Vesicles 13(7):e12469. [article]

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