A Novel Method For Quantification Of Extracellular Vesicles Based On Lanthanide Labeling

Extracellular Vesicles（EVs）have been reported to be involved in the regulation of various physiological and pathological processes and are closely related to the occurrence and development of various diseases in recent years,and researches on EVs have made much progress in developing new methods for enrichment of EVs,omics analysis,disease diagnosis and treatment,etc.In particular,the analysis of EVs proteins has received widespread attention.To deeply understand the biological functions and significance of EVs,it is still necessary to continue to explore the interactions between EVs and cells.In recent years,EVs have received widespread attention from researchers as an important means of intercellular communication.Under physiological or pathological conditions,donor cells release EVs that containing bioactive molecules and then the EVs can be internalized into corresponding recipient cells and affect their biological functions,which is an important way of intercellular communication.To further study the interactions between EVs and cells,it is necessary to quantify the uptake of EVs at the cellular level.However,there are limited quantitative methods for EVs especially for measuring their intracellular uptake,there is still lacking of sensitive methods to accurately quantify the internalized level of EVs by cells.Small Extracellular Vesicles（s EVs）are EVs with a particle size range of 30-200nm and are the most studied subgroup of EVs in recent years.In view of the deficiencies of current quantitative methods of EVs,we established a novel method based on lanthanide element labeling and inductively coupled plasma mass spectrometry（ICP-MS）analysis for absolute quantification of EVs.Utilizing the chelation reaction between lanthanide metal europium（Eu）and the phosphate groups on the EV membrane,Eu labeling of s EVs can be realized through a simple one-step reaction.Then,absolute quantification of s EVs and their cellular uptake can be achieved using the highly sensitive response of Eu in ICP-MS analysis.Characterization of s EVs with and without Eu labeling shows that this labeling method does not affect the classic physical and chemical properties and the inherent biological activities of EVs.Subsequently,ICP-MS and Nanoparticle Flow Cytometry（Nano FCM）were used to detect the Eu content and particle number of Eu-s EVs,respectively.A quantitative standard curve of the Eu content in Eu-s EVs versus the number of s EVs particles was obtained with a quantitative linear range spans three orders of magnitudes.After calculation,we found that the average mass of Eu loaded on the surface of each s EV is 0.09 fg,which is equivalent to 3961 Eu³⁺atoms per s EV particle.Furthermore,we utilized this method to quantitatively study the uptake of s EVs by He La cells.It was found that He La cells uptake s EVs in a time-dependent manner.Compared with the average number of s EV particles taken up per cell at 37°C,which was 4020±171,the cellular uptake of s EVs under low temperature conditions（4°C）was significantly inhibited（reduced by 61%）.In addition,through quantitative analysis of s EVs uptake by He La cells under various treatment of endocytosis inhibitors,it was proved that He La cells internalized s EVs mainly through macropinocytosis rather than the clathrin-dependent endocytosis pathway.We also used two glycosidases（PNGF and neuraminidase）to deglycosylate s EVs and found that the deglycosylation treatment on the s EV surface made them more easily internalized by He La cells.Treatment with PNGF and neuraminidase resulted in an increase of the cellular uptake efficiency of s EVs by He La cells by 47%and 76%,respectively.In summary,we have established a new method for the labeling and quantification of s EVs,which can be used for absolute quantitative analysis of the internalization of s EVs at cellular levels.This method is simple to operate with high sensitivity and solves the problem of the lack of high-precision quantitative methods in the current study of the cellular uptake of EVs.This new method is expected to provide a new technique and ideas for the in-depth research on the interaction between EVs and recipient cells in cargo delivery,as well as the evaluations of dosage and drug efficacy when using EVs as drugs or drug delivery vehicles.As an emerging target of liquid biopsy,EVs have received widespread attention in recent years.And proteomics has now become an important strategy to study the dynamic changes in protein abundance of EVs in body fluids for biomarker discovery and therefore benefiting prediction of the occurrence and development of diseases.Plasma is known as the most commonly used body fluid for clinical testing.Containing a large amount of physiological and pathological information,plasma is recognized as an important source of disease biomarkers.However,due to the interference of large number of high-abundance protein that naturally present in plasma,it is difficult to achieve deep coverage of blood proteomics.To circumvent this difficulty and to construct an efficient and highly selective strategy for plasma biomarker screening,proteomics analysis of plasma EVs received most attention in recent years.We developed a fast EVs enrichment strategy based on tandem utilization of size exclusion chromatography（SEC）and ultracentrifugation.This strategy achieves efficient enrichment of plasma s EVs with high purity by combination of two EV enrichment methods to increase the identification scale of plasma EVs proteome.This strategy was applied to proteomic study of EVs in the plasma of patients with post-stroke cognitive normal（PSCN）and post-stroke cognitive impairment（PSCI）as well as healthy people.Through subsequent mass spectrometry identification and corresponding proteomic quantitative analysis,we discovered potential EV protein markers associated with the occurrence and development of ischemic stroke-related diseases,providing valuable information for studying its pathogenesis and treatment-related targets.