Absolute Quantification of Dynamic Cellular Uptake of Small Extracellular Vesicles via Lanthanide Element Labeling and ICP–MS

Small extracellular vesicles (sEVs) are increasingly reported to play important roles in numerous physiological and pathological processes. Cellular uptake of sEVs is of great significance for functional regulation in recipient cells. Although various sEV quantification, labeling, and tracking metho...

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Veröffentlicht in:Analytical chemistry (Washington) 2023-08, Vol.95 (32), p.11934-11942
Hauptverfasser: Yang, Ningli, Zhao, Chuanping, Kong, Linlin, Zhang, Baoying, Han, Chunguang, Zhang, Yangjun, Qian, Xiaohong, Qin, Weijie
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container_end_page 11942
container_issue 32
container_start_page 11934
container_title Analytical chemistry (Washington)
container_volume 95
creator Yang, Ningli
Zhao, Chuanping
Kong, Linlin
Zhang, Baoying
Han, Chunguang
Zhang, Yangjun
Qian, Xiaohong
Qin, Weijie
description Small extracellular vesicles (sEVs) are increasingly reported to play important roles in numerous physiological and pathological processes. Cellular uptake of sEVs is of great significance for functional regulation in recipient cells. Although various sEV quantification, labeling, and tracking methods have been reported, it is still highly challenging to quantify the absolute amount of cellular uptake of sEVs and correlate this information with phenotypic variations in the recipient cell. Therefore, we developed a novel strategy using lanthanide element labeling and inductively coupled plasma–mass spectrometry (ICP–MS) for the absolute and sensitive quantification of sEVs. This strategy utilizes the chelation interaction between Eu3+ and the phosphate groups on the sEV membrane for specific labeling. sEVs internalized by cells can then be quantified by ICP–MS using a previously established linear relationship between the europium content and the particle numbers. High Eu labeling efficiency and stability were demonstrated by various evaluations, and no structural or functional alterations in the sEVs were discovered after Eu labeling. Application of this method revealed that 4020 ± 171 sEV particles/cell were internalized by HeLa cells at 37 °C and 61% uptake inhibition at 4 °C. Further investigation led to the quantitative differential analysis of sEV cellular uptake under the treatment of several chemical endocytosis inhibitors. A 23% strong inhibition indicated that HeLa cells uptake sEVs mainly through the macropinocytosis pathway. This facile labeling and absolute quantification strategy of sEVs with ppb-level high sensitivity is expected to become a potential tool for studying the functions of sEVs in intracellular communication and cargo transportation.
doi_str_mv 10.1021/acs.analchem.3c01421
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Cellular uptake of sEVs is of great significance for functional regulation in recipient cells. Although various sEV quantification, labeling, and tracking methods have been reported, it is still highly challenging to quantify the absolute amount of cellular uptake of sEVs and correlate this information with phenotypic variations in the recipient cell. Therefore, we developed a novel strategy using lanthanide element labeling and inductively coupled plasma–mass spectrometry (ICP–MS) for the absolute and sensitive quantification of sEVs. This strategy utilizes the chelation interaction between Eu3+ and the phosphate groups on the sEV membrane for specific labeling. sEVs internalized by cells can then be quantified by ICP–MS using a previously established linear relationship between the europium content and the particle numbers. High Eu labeling efficiency and stability were demonstrated by various evaluations, and no structural or functional alterations in the sEVs were discovered after Eu labeling. Application of this method revealed that 4020 ± 171 sEV particles/cell were internalized by HeLa cells at 37 °C and 61% uptake inhibition at 4 °C. Further investigation led to the quantitative differential analysis of sEV cellular uptake under the treatment of several chemical endocytosis inhibitors. A 23% strong inhibition indicated that HeLa cells uptake sEVs mainly through the macropinocytosis pathway. 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source American Chemical Society Journals
subjects Analytical chemistry
Chelation
Chemistry
Endocytosis
Europium
Extracellular vesicles
Inductively coupled plasma mass spectrometry
Intracellular signalling
Labeling
Mass spectrometry
Mass spectroscopy
Phenotypic variations
Stability analysis
Structure-function relationships
Vesicles
title Absolute Quantification of Dynamic Cellular Uptake of Small Extracellular Vesicles via Lanthanide Element Labeling and ICP–MS
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