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

Smallextracellular vesicles (sEVs) are increasinglyreported toplay important roles in numerous physiological and pathological processes.Cellular uptake of sEVs is of great significance for functional regulationin recipient cells. Although various sEV quantification, labeling,and tracking methods have been reported, it is still highly challengingto quantify the absolute amount of cellular uptake of sEVs and correlatethis information with phenotypic variations in the recipient cell.Therefore, we developed a novel strategy using lanthanide elementlabeling and inductively coupled plasma-mass spectrometry (ICP-MS)for the absolute and sensitive quantification of sEVs. This strategyutilizes the chelation interaction between Eu3+ and thephosphate groups on the sEV membrane for specific labeling. sEVs internalizedby cells can then be quantified by ICP-MS using a previouslyestablished linear relationship between the europium content and theparticle numbers. High Eu labeling efficiency and stability were demonstratedby various evaluations, and no structural or functional alterationsin the sEVs were discovered after Eu labeling. Application of thismethod revealed that 4020 & PLUSMN; 171 sEV particles/cell were internalizedby HeLa cells at 37 & DEG;C and 61% uptake inhibition at 4 & DEG;C.Further investigation led to the quantitative differential analysisof sEV cellular uptake under the treatment of several chemical endocytosisinhibitors. A 23% strong inhibition indicated that HeLa cells uptakesEVs mainly through the macropinocytosis pathway. This facile labelingand absolute quantification strategy of sEVs with ppb-level high sensitivityis expected to become a potential tool for studying the functionsof sEVs in intracellular communication and cargo transportation.

Automated summary

Extracellular vesicles (sEVs) play important roles in physiological and pathological processes, and their cellular uptake is crucial for regulating recipient cells. A novel strategy using lanthanide element labeling and ICP-MS was developed for the quantification of sEVs. This method demonstrated high labeling efficiency and stability, and revealed the internalization of sEVs by HeLa cells. The strategy can be used as a potential tool for studying sEVs in intracellular communication and cargo transportation.