Systematic optimization and comparative evaluation of exosome fluorescence labeling workflows based on cholesterol anchoring and copper-free click chemistry

Three exosome fluorescence-labeling workflows based on azide-functionalized cholesterol anchoring and copper-free click chemistry were systematically optimized and comparatively evaluated. A549 cell-derived exosomes were used as the primary model. By varying the sequence of azide introduction and fluorescent click reaction, one-step, two-step, and three-step protocols were compared. Under the tested conditions, the two-step protocol provided the most favorable overall balance among exosome recovery, fluorescence intensity normalized to exosome particle concentration, dye consumption, reaction volume, and operational simplicity. After labeling, the exosomes retained their typical morphology, particle size distribution, while the representative exosome-associated proteins CD63, CD81, and TSG101 remained detectable. The fluorescence intensity of the labeled exosomes showed a strong linear correlation with particle concentration, supporting its potential for rapid quantitative analysis. Cellular uptake experiments confirmed the feasibility of the labeled exosomes for in vitro imaging, and fluorescence remained detectable during the 24-h in vivo observation period. Overall, this study identifies an optimized two-step workflow for exosome fluorescence labeling and provides a practical approach for exosome visualization and tracking under the evaluated experimental conditions. Graphical abstract