| Extracellular vesicles(EVs,30-1,000 nm)are nano-scaled and lipid-bilayer-enelosed vesicles,which are secreted by all cells in the process of their life and widely exist in cell culture supernatant and body fluids such as blood,urine and latex.EVs are implicated in trafficking of molecules(e.g.,proteins,lipids,nucleic acids and metabolites)from their parental cells to recipient cell and have an effect on physiologic function.EVs have been mainly classified as exosomes(30-150 nm)and microvesicles(100-1000 nm)with regard to their biogenesis pathways,being secreted from late endosomal compartments named multivesicular bodies or budded from the cells5 plasma membrane,respectively.EVs are involved in the physiological and pathological processes such as antigen presentation,angiogenesis,tumor growth and metastasis,showing great application prospects in the diagnosis and treatment of diseases.However,EVs released from different cell types(and even from a single cell type)are highly heterogeneous in size and in their contents.In addition,a specific vesicle subtype could be solely responsible for a particular function.Important limitations-including practical difficulties in assaying low concentrations of extracellular vesicles in circulation,identifying their tissue of origin,and specifying which molecular cargo is most relevant-have restrained enthusiasm tor research into the role of extracellular vesicles in vivo.Therefore,a sensitive,specific,and rapid nlethodology is urgently needed to measure the multiple parameters of EVs at the single-particle level.Nevertheless,the small size and extremely low contents of proteins,nucleic acids and other molecules make the analysis of individual EVs to be a major technical challenge.Although electron microscopy can intuitively characterize the physical properties of EVs,such as morphology and particle size,they still have shortcomings such as cumbersome sample preparation and slow analysis speed.In addition,immuno-electron microscopy(IEM)with gold-labeled antibody has been used to confirm and quantify specific protein expression on individual EVs,yet its routine application is also prohibited due to the tedious procedures and limited statistical power.Nanoparticle tracking analysis and tunable resistive pulse sensing are often used in the rapid determination of EV particle size and concentration,but it is difficult to characterize the biochemical properties of EVs.The heterogeneity of EVs and the biochemical information of the subpopulation could be easily masked by other abundant EVs using ensemble averaged techniques,such as Western blotting,enzyme-linked immunesorbent assay(ELISA)and PCR.Flow cytometry(FCM)is a well-established technique for high-throughput,multi0 parameter,and quantitative analysis of individual cells and microscopic particles in aqueous suspension.Physical information regarding cell size,shape,as well as morphology of particles can be provided via light scattering measurements,and biochemical properties such as nucleic acid content,and antigenic determinants of biological cells can be gathered through fluorescent labeling.Nevertheless,it has been extremely difricult for conventional flow cytometry to detect EVs smaller than 300 nm-500 nm in diameter based on light scattering.Employing strategies for singlemolecule fluorescence detection in a sheathed flow,we have recently developed nano-flow cytometry(nFCM)that enables light scattering detection of single silica nanoparticles,viruses,and gold nanaparticles as small as 24 nm,27 nm,and 7 nm in diameter,respectively.The light scattering detection is 4-6 order of magnitudes more sensitive than traditional FCMs.In this dissertation,we applied nFCM to build a rapid approach for quantitative multiparameter analysis of single EVs down to 40 nm with an analysis rate up to 10,000 particles per minute.nFCM allows researchers to analyze and characterize EVs in the same way as traditional flow cytometry analysis of cells.This dissertation consists of the following sections:In the first chapter,we make an overview of the application of the main functions and important applications of EVs and the recent advanced characterization technology in EV analysis.The second chapter introduces the construction of unlabeled analysis method for extracellular vesicles by using scattering light.By using Triton X-100 to lyse the phospholipid membranes of EVs while no effect on proteins and other particles,the purity of EV samples is determined by using nFCM to enumerate the particle events before and after the treatment.To examine the performance of nFCM in the absolute size distribution analysis of EVs,a series of monodisperse silica nanoparticles(SiNPs)are synthesized and used as the size reference standards.The deviation of SSC intensity induced by the slight refractive index mismatch between SiNPs(1.461)and EVs(1.400)is also corrected based on Mie theory.The analysis rate of nFCM is up to 10,000 particles per minute,and it only takes a few minutes to obtain highly statistically representative EVs particle size distribution characteristics,with a resolution and accuracy comparable to that of cryo-TEM,but more statistically.The third chapter demonstrates the application of nFCM in the multi-parameter analysis of individual EVs.Combined with fluorescence labeling strategy,simultaneous insights into the physical and biochemical properties of EVs is elucidated.Phospholipid bilayer membrane is the main component and common structural characteristic of EVs,the labeling efficiency of various lipophilic probes on EVs is determined by using lipid membrane labeling strategy.By labeling DNA/RNA with nucleic acid dyes,the distribution of nucleic acid contents in single EVs was determined.By clicking chemistry and metabolic incorporation,the analysis of sialic acids expressed on EVs was performed.In combination with immunofluorescence labeling,the ratio of the EV subpopulation expressing one or two specific surface proteins and the copy number and distribution of specific proteins on a single EVs surface are determined.The fourth chapter is the construction of multi-parameter detection method for EVs in clinical blood samples.Since the diameter of the sample flow is much smaller than the diameter of the laser spot in the sample detection process of nFCM,the detection efficiency of particles passing through the detection area is close to 100%.The concentration of total EVs and the subpopulation expressing specific proteins from platelet free plasma(PFP)was measured via single particle enumeration.With only 50?L PFP,diagnosis and treatment monitoring of colorectal cancer is also demonstrated by CD 147-positive EV concentration.nFCM will provide an advanced technical platform for basic research and clinical application of EVs.In the fifth chapter,the work of this thesis is summarized and the future prospect of nFCM is discussed. |
PDF Full Text Request