Research On Magnetic Beads Modified On The Surface Using For Extration Of Exosomes

With the deepening of research,the great potential of exosomes in the fields of liquid biopsy and targeted therapy has attracted more and more attention.However,due to the size of exosomes ranging from 20 to 200 nm,and the extracellular vesicles secreted by cells include not only exosomes,but also microvesicles with larger size,separation of exosomes is very difficult.At present commonly used methods include overspeed centrifugal separation,chemical precipitation and immuno separation.However,overspeed centrifugal need expensive ultracentrifuge and the efficiency is low.Chemical precipitation will introduce protein impurities.immuno separation can secrete body outside the membrane structure,impact secrete biological activity of the body,so on a secrete body outside are widely used in the separation and extraction has certain limitation.Studies have shown that the membrane structure surface of extracellular vesicles,including exosomes,contains phosphatidylserine,which can bind to Tim4 protein in sufficient Ca2+ conditions and separate when Ca2+ is depleted.Since the combination of the two is reversible and will not affect the exosome activity,it can be applied to the separation and enrichment of highly active exosomes.In this study,we tried to study the method of extracting exosomes with high purity and activity from human peripheral blood by using magnetic nano-spheres modified on the basis of previous studies.In this study,Tim4 protein-specific grafting was performed on the surface of modified nanometer magnetic beads,combined with extracellular vesicles containing exosomes in the condition of rich in Ca Cl2,and then Ca2+ chelating agent was used to remove Ca2+ and isolate vesicles.Meanwhile,mi RNA in extracellular vesicles was analyzed and compared with RNA in plasma background.Through a series of characterization,this study confirmed that the method of Tim4 magnetic bead affinity can obtain abundant extracellular vesicles with high activity including exosomes.This method has low cost and simple operation,and is more convenient and widely used than the traditional extraction method,which provides new technical support for the exosome research.The activity and content of extracellular vesicles obtained are pretty high,which provide practical significance for liquid biopsy and clinical treatment.Moreover,the combination of the magnetic beads and vesicles is reversible and may be used for repeatedly extraction of the same kind of sample.Moreover,we studied the contents of several types of mi RNA extracted from extracellular vesicles and confirmed that the obtained extracellular vesicles contained a considerable number of mi RNAs that could be usedfor gene analysis.Future studies can calculate the proportion of exosomes based on the obtained mi RNA content through deconvolution algorithm,providing further support for clinical diagnosis and treatment.