Academic Journal
Systematic optimization and comparative evaluation of exosome fluorescence labeling workflows based on cholesterol anchoring and copper-free click chemistry.
| Τίτλος: | Systematic optimization and comparative evaluation of exosome fluorescence labeling workflows based on cholesterol anchoring and copper-free click chemistry. |
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| Συγγραφείς: | Li C; School of Pharmacy, Shanghai Jiao Tong University, Shanghai, 200240, China., Zeng Y; School of Pharmacy, Shanghai Jiao Tong University, Shanghai, 200240, China., Wei K; School of Pharmacy, Shanghai Jiao Tong University, Shanghai, 200240, China., Li X; School of Pharmacy, Shanghai Jiao Tong University, Shanghai, 200240, China. lixy@sjtu.edu.cn., Wang Y; School of Pharmacy, Shanghai Jiao Tong University, Shanghai, 200240, China. wangyan11@sjtu.edu.cn. |
| Πηγή: | Mikrochimica acta [Mikrochim Acta] 2026 Aug 21; Vol. 193 (9). Date of Electronic Publication: 2026 Aug 21. |
| Τύπος έκδοσης: | Journal Article; Comparative Study; Research Support, Non-U.S. Gov't |
| Γλώσσα: | English |
| Στοιχεία περιοδικού: | Publisher: Springer-Verlag Country of Publication: Austria NLM ID: 7808782 Publication Model: Electronic Cited Medium: Internet ISSN: 1436-5073 (Electronic) Linking ISSN: 00263672 NLM ISO Abbreviation: Mikrochim Acta Subsets: MEDLINE |
| Imprint Name(s): | Original Publication: Wien ; New York : Springer-Verlag. |
| Ιατρικοί όροι (MeSH): | Exosomes*/chemistry , Exosomes*/metabolism , Cholesterol*/chemistry , Fluorescent Dyes*/chemistry, Azides/chemistry ; Copper/chemistry ; Click Chemistry ; Humans ; A549 Cells |
| Περίληψη: | 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. (© 2026. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.) |
| Competing Interests: | Declarations. Competing interests: The authors declare no competing interests. |
| References: | RSC Adv. 2025 Apr 4;15(14):10583-10601. (PMID: 40190630) ACS Nano. 2021 Feb 23;15(2):3212-3227. (PMID: 33470092) Chem Soc Rev. 2024 Jul 1;53(13):6779-6829. (PMID: 38828885) Small. 2025 Dec;21(51):e10386. (PMID: 41208337) J Lipid Res. 2019 Jan;60(1):9-18. (PMID: 30076207) ACS Nano. 2024 Aug 13;18(32):21009-21023. (PMID: 39087239) Biomaterials. 2025 Aug;319:123208. (PMID: 40023928) Science. 2020 Feb 7;367(6478):. (PMID: 32029601) Bioconjug Chem. 2020 May 20;31(5):1562-1574. (PMID: 32369345) Phys Chem Chem Phys. 2021 Mar 21;23(11):6408-6421. (PMID: 33705506) Nucleic Acids Res. 2025 Jan 6;53(D1):D1670-D1676. (PMID: 39470721) Anal Chim Acta. 2024 May 29;1305:342527. (PMID: 38677835) Theranostics. 2021 Jan 1;11(7):3183-3195. (PMID: 33537081) Theranostics. 2026 Jan 1;16(1):545-579. (PMID: 41328354) Biosens Bioelectron. 2024 May 1;251:116104. (PMID: 38368644) Pharmaceutics. 2023 Feb 07;15(2):. (PMID: 36839873) J Extracell Vesicles. 2015 May 14;4:27066. (PMID: 25979354) Talanta. 2024 May 1;271:125700. (PMID: 38277965) J Thromb Haemost. 2023 Aug;21(8):2032-2044. (PMID: 37201724) Biochim Biophys Acta Biomembr. 2018 Jun;1860(6):1350-1361. (PMID: 29551275) Nanomedicine (Lond). 2020 Sep;15(22):2149-2170. (PMID: 32885720) Anal Chem. 2017 Dec 5;89(23):12968-12975. (PMID: 29139297) |
| Grant Information: | 81874307, 21874088 the National Natural Science Foundation of China; 20142200400, 21440730300,19DZ2202200 Shanghai Science and Technology Commission Scientific Research Project |
| Contributed Indexing: | Keywords: Cholesterol anchoring; Copper-free click chemistry; Exosomes; Fluorescent labeling; Workflow optimization |
| Substance Nomenclature: | 97C5T2UQ7J (Cholesterol) 0 (Fluorescent Dyes) 0 (Azides) 789U1901C5 (Copper) |
| Entry Date(s): | Date Created: 20260821 Date Completed: 20260821 Latest Revision: 20261008 |
| Update Code: | 20261008 |
| DOI: | 10.1007/s00604-026-08353-4 |
| PMID: | 42629546 |
| Βάση Δεδομένων: | MEDLINE |
| ISSN: | 1436-5073 |
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| DOI: | 10.1007/s00604-026-08353-4 |