Extracellular Vesicles

The origin and phenotyping of extracellular vesicles

Nano-sized extracellular vesicles (EVs) released by various cell types play important roles in a plethora of (patho) physiological processes and are increasingly recognized as biomarkers for diseases. Moreover, engineered EV and EV-inspired liposomes hold great potential as drug delivery vehicles. EVs are heterogeneous in composition and size, ranging from approximately 30 to 1000 nm, with the vast majority <200 nm in size. As determined by their biogenesis, the three main classes of EVs are exosomes, microvesicles, and apoptotic bodies. In contrast to microvesicles, which are generated by budding from the plasma membrane, exosomes are derived from the endolysosomal pathway, and fall in the size range of 30-150 nm.

EVs released from different cell types (and even from a single cell type) are shown to be highly heterogeneous in size and nanostructure, a specific vesicle subtype could be solely responsible for a particular function. The function of EVs are typically evaluated on RNA level (qPCR) and protein level (ELISA, IF, Western blots). However, these ensemble-averaged techniques could not discriminate the distinct subset from other abundant EVs. Therefore, a sensitive, specific, and rapid methodology is urgently needed to measure the abundance of these specific markers on EVs at the single-particle level. However, the tiny size, heterogeneity, low refractive index as well as lacking of distinct markers make it extremely challenging for the single-particle analysis of EVs.

The gold standard for nanoparticle measurement has been electron microscopy (EM), which can determine both the size and morphology of the particles. However, the sample preparation steps and imaging techniques require dehydration, chemical fixation and/or staining of the biological specimens, which may alter the morphology of the samples. While for biological particles like EVs, cryo-electron microscopy (cryo-EM) is used to preserve the natural morphology of particles. But its routine application is prohibited due to the extremely high price and limited statistical power.

Nanoparticle tracking analysis (NTA) and tunable resistive pulse sensing (TRPS) are frequently used techniques to determine the size of nanoparticles that have been applied to analyze EVs. The minimum detectable vesicle size are 70-90 nm and 70-100 nm, respectively. Unfortunately, none of these methods on its own have the ability to reveal the biochemical properties of EVs. Flow Cytometry (FCM) is a well-established technique for high-throughput, multi-parameter, and quantitative analysis of individual cells and microscopic particles in aqueous suspension. Although FCM has been applied to analyze surface proteins of individual EVs using fluorescence threshold triggering, the minimum detectable vesicle sizes are 150-190 nm for dedicated FCM and 270-600 nm for conventional FCM, which is far from satisfaction. Flow NanoAnalyzer opens a new avenue for single EVs detection, especially for the detection of exosomes —- the subset of EVs with size ranging from 30 to 150 nm. Most of the data are unpublished, please keep an eye on the website: www.nanofcm.com.

1、Quantitative and Multiparameter Analysis of Single EVs

NanoFCM provides a versatile and powerful platform — Flow NanoAnalyzer for the multiparameteranalysis of individual extracellular vesicles (30-150 nm in diameter), the fluorescence of single phycoerythrinmolecules can be well detected against the background. The Flow NanoAnalyzer platform enables quantitative and multiparameteranalysis of single EVs down to 40 nm, which is distinctively sensitive, yet high-throughput, and shows great potential in liquid biopsy applications.

Summary: We have demonstrated the sensitivity of Flow NanoAnalyzer by detecting single silica nanoparticles, the fluorescence sensitivity of single R-PE molecule has also been verified. The size and concentration of EVs could be acquired directly from the software, both intrinsic and extrinsic fluorescence could be detected individually.

2、A Powerful Tool for the Early Diagnosis of Cancer

Tumor extracellular vesicles (EVs) have been shown to be carriers of abundant tumor material which can be used as evidence of disease, while surface proteins of EVs are of great interest in cancer diagnosis. Here we demonstrate a nanoFCM based approach for the early diagnosis of colorectal caner, which will provide an advanced technique and method for the EVs research as well as their clinical applications. Flow NanoAnalyzer will be a powerful tool to evaluate the diagnostic and therapeutic potential of EVs.

Summary: Flow NanoAnalyzer allows simultaneously acquisition of the side scatter and fluorescence signals of single EVs, it is the only flow cytometry device that has the capacity to cover the entire size range of EVs; After immunofluorescent labeling, the non-invasive early diagnosis of colorectal cancer was demonstrated based on Flow NanoAnalyzer. This strategy is feasible in other kinds of diseases, thus making Flow NanoAnalyzer an excellent tool for the early diagnosis of diseases.

Reference: [1] S Zhu, et al. ACS Nano, 2014, 8, 10998-11006. [2] Y. Tian, et al. ACS Nano, 2018, 12, 671-680.