Evaluatinng The Mechanical Properties And Sorting Of Extracellular Vesicles By Atomic Force Microscopy

Extracellular vesicles(EV)are the general names of various subtypes of bilayer membrane vesicles secreted by cells into body fluids.Since carrying genetic molecules such as proteins,lipids,DNA,and RNA from parental cells,EV can not only transfer information between cells,but also serve as potential biomarkers in liquid biopsies and important carriers for targeted drug delivery.EV such as exosome,microvesicle and apoptotic body are still difficult to distinguish due to slight differences in physical properties and composition,which undoubtedly hinders the true recognition of specific EV populations and is not conducive to the application research of EV.Atomic force microscopy(AFM)is widely used in life sciences and molecular biology because of its unique advantages such as nano-scale spatial resolution,pico-level force sensitivity,sample-free labeling,and operation in multiple environments.AFM can not only characterize the three-dimensional morphology and mechanical properties of biological s amples,but also investigate the interaction between biological molecules at the single molecule level.Therefore,the mechanical properties and typing of extracellular vesicles were studied by using nanoindentation and single molecular force spectroscopy of AFM in this paper.The details were as follows:1.Exploring the mechanical properties of different extracellular vesicles by atomic force microscopyThe Young's modulus of exosomes derived from different cells was measured using the nanoindentation technique of AFM.The results were as follows:(1)The exosome of tumor cells were less rigid and more deformable than that of normal cells;(2)The exosome of metastatic tumor cells were softer and more deformable than that of non-metastatic tumor cells;(3)The exosome of Hep G2/ADM-resistant liver cancer cells were less rigid than that of Hep G2 liver cancer cells.These results might be due to differences in the molecular composition of lipids and proteins on the surface of exosome from different cells.Then,the mechanical properties of different EV subtypes(exosome,microvesicle and apoptotic body)were studied.As the size increased,the Young's modulus of exosome,microvesicle and apoptotic body decreased in turn.At the same time,the influence of p H on the mechanical properties of EV was also discussed.It was found that p H 6.5 reduced the Young's modulus of EV from normal and tumor cells compared to p H 7.4,while p H 8.0 only reduced the Young's modulus of EV from tumor cells.In addition,it was found that some metal ions can also affect the mechanical properties of EV:2.5 m M Ca²⁺and Ni²⁺can reduce the Young's modulus of EV from normal and tumor cells;2.5 m M Mg²⁺can also reduce the Young's modulus of EV from normal cells,but it increased the Young's modulus of EV from tumor cells.This work is expected to provide important information for further analysis of the mechanical properties of extracellular vesicles.2.Distinguishment of three different subtypes of extracellular vesicle by atomic force microscopyA new strategy for distinguishing different subtypes of extracellular vesicles(exosome,microvesicle,and apoptotic body)has been developed using single-molecule force spectroscopy(SMFS).Annexin A1 antib ody,annexin V and aptamer were selected as recognition probes,the interactions between the functionalized tip of the recognition probes and different extracellular vesicles(exosome,microvesicle and apoptotic body)w ere determined by single molecule force spectroscopy.Although there was no significant difference in the interaction force values between the recognition probes and different extracellular vesicles(exosome,microvesicle and apoptotic body),the binding probability was significantly different.Interesting,statistics and analysis of the binding probability of three recognition probes with different EV subtypes revealed that this method can not only distinguish exosome,microvesicle and apoptotic body of 7721 liver cancer cells,but also exosome,microvesicle and apoptotic body of L02 normal liver cells and 7721cancer cells simultaneously.Similarly,the EV of MCF10A normal breast epithelial cells and MCF-7 breast cancer cells can be also distinguished.This work utilizes multiple recognition probes to more accurately define exosomes,microvesicles,and apoptotic bodies from different cell lines,which is expected to provide important clues for in-depth analysis of the specific biological composition of different subtypes of EV.