Genetic versus chemical labeling: A quantitative comparison of tracking strategies for extracellular vesicles

Extracellular vesicles (EVs) play critical roles in intercellular communication and represent promising drug delivery vectors. A major challenge in the field is the lack of labeling strategies that enable quantitative, stable, and non-perturbing tracking of individual EVs.

We systematically compared genetic and chemical labeling strategies for single-vesicle tracking. A HaloTag-based genetic approach was evaluated against two chemical methods-cholesterol-modified antisense oligonucleotides (ASO) and the lipophilic dye DiD. Labeling efficiency, stability, aggregation effects, and cellular uptake were assessed using nanoflow cytometry (NanoFCM), medium angle light scatter (MALS), and cell fluorescence microscopy.

The HaloTag system achieved 98% labeling efficiency without inducing aggregation and exhibited exceptional stability. In contrast, both chemical labels showed severe limitations: DiD dye tends to aggregate significantly, forming nanoparticles, which affects both the recovery efficiency and labeling efficiency of EVs, but this issue is improved by our genetic engineering modifications to EVs. Although the novel Chol-ASO strategy achieves a high initial labeling efficiency of up to 99% without inducing aggregation, the ASO-EV complex dissociates rapidly within hours under cell culture conditions. Consequently, the observed fluorescent signal partially originates from cellular uptake of free ASO rather than EV-mediated delivery, which severely confounds the interpretation of genuine EV uptake.

Genetic labeling using HaloTag provides a superior approach for quantitative and stable single-vesicle tracking, making it ideal for mechanistic studies of EV function. Chemical labels, though convenient for clinical EV samples, exhibit limitations. Further improvement of chemical labeling approaches is required to meet the demands of reliable EV tracking.