Author: Nanofcm     Date: November 25, 2021

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 40-150 nm.On the other hand, outer membrane vesicles (OMVs) secreted by bacteria not only affect a variety of biological processes, but have also shown important applications in vaccine and anti-cancer drug development.

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 distinct markers make it extremely challenging for the single-particle analysis of EVs.

Transmission electron microscopy (TEM) is the gold standard for EV measurement, which can provide both the size and morphology information. 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 sizes are 70-90 nm and 70-100 nm, respectively. Unfortunately, none of these methods on its own has 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 40 to 150 nm.

1. Quantitative and Multiparameter Analysis of Single EVs

NanoFCM provides a versatile and powerful platform — Flow NanoAnalyzer for the multiparameter analysis of individual extracellular vesicles (40-150 nm in diameter), the fluorescence of single phycoerythrin molecules can be well detected against the background. The Flow NanoAnalyzer platform enables quantitative and multiparameter analysis 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 which have the similar refractive index with EVs, the fluorescence sensitivity of a single R-PE molecule has also been verified. The size (diameter) distribution and particle concentration of EVs could be acquired directly from the software, expression of CD9, CD63, CD81 tetraspanins could be evaluated on the single EV level, both intrinsic and extrinsic fluorescence could be detected individually. Colocalization of nucleic acids, surface markers and lipids can be achieved by simply dual-labeling. Monoclonal antibodies, aptamers and Annexin V are employed to label the surface markers and intraluminal molecules.

2. A Powerful Tool for the Early Diagnosis of Cancer

Tumor extracellular vesicles (EVs) have been shown to be carriers of abundant biomarkers 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 cancer, 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.Fluorescent-tagged monoclonal antibody against CD147 was used to label the EVs isolated from cell culture and plasma, and both the colorectal cell lines and cancer patients showed distinctly higer results than the normal cell lines/healthy donors, which agreed well with Western Blot method, indicating EVs with specific biomarkers can act as diagnostic agents for diseases.

Summary: Flow NanoAnalyzer allows simultaneous 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, and alternative probes like aptamers or nanobodies can also be used, thus making Flow NanoAnalyzer an excellent tool for the early diagnosis of diseases.

3.  Identification of EV-based Drug Delivery Platform

Extracellular vesicles (EVs) are cell-secreted nanoparticles that are playing important roles in intercellular communication. This makes them excellent candidates for targeted drug delivery vehicles. The cargo can be loaded either on the exterior surface or in the intraluminal space of EVs. Several modification strategies have been used for EV-based drug delivery, among them genetic engineering and fusion with liposomes are the most frequently used. For each drug delivery platform, physicochemical features such as the size distribution, particle concentration, purity, and biological functions are the key factors to be monitored. Meanwhile, drug loading (drug molecules per EVs and drug concentration per EVs) is also essential information that needs to be evaluated. NanoFCM offers an excellent platform for the comprehensive analysis of EV-based drug delivery system.

3.1  EngExTM Platform for Surface and Luminal Loading

ExoIL-12TM is a novel engineered exosome developed by Codiak Biosciences, which is a clinical-stage company focused on pioneering the development of exosome-based therapeutics as a new class of medicines. exoIL-12TM displays IL-12 on the surface of exosomes (engExTM Platform), and it is the first engineered exosome therapeutic candidate investigated in clinical trials. The proprietary engExTM Platform is based on the discovery of two scaffold proteins. LC-MS/MS analysis was performed on highly purified EVs and five EV proteins were identified. Flow cytometry and ELISA were used to measure cellular and exosome-associated protein expression, however, these methods fail to determine whether overexpressed scaffold proteins were uniformly distributed among EVs or enriched in subsets. By analyzing EVs at single particle level, the data suggest that overexpression of the candidate scaffolds results in abundant, uniform distribution across EVs.

3.1  Fusion with Liposomes

Liposomes have long been used to encapsulate drugs, the synthesize and encapsulation processes are simple and mature, it is easy to get high encapsulation ratio. While CD47 on exosomes has proven to release “Don’t eat me” signal, and avoid to be removed by the immune system. The combination of liposomes and exosomes will be an ideal drug delivery platform. Here, thermosensitive liposomes encapsulated with drugs were fused with genetically engineered exosomes, the resulting exosome-liposome hybrid NPs display CD47 on the surface and bear thermosensitive agents inside. NanoFCM allows the analysis of liposomes and exosomes at single particle level, and the fusion efficiency is also determined.

3.3  EVs for Loading Oncolytic Adenoviruses

Extracellular vesicles with low immune response are able to target tumor cells through bioengineering, which in turn becomes one of the most ideal carriers for drug treatment of tumor diseases. Oncolytic adenoviruses (OA) have been explored in both preclinical and clinical therapies. While the poor targeting delivery is a major obstacle that limits OA application. Bioengineered cell membrane nanovesicles (BCMNs) that are genetically engineered with targeting ligands specific for cancer cells was developed, and OA was encapsulated in the BCMNs afterwards, the resulting OA@BCMNs demonstrated enhanced targeting delivery, viral oncolysis, and survival benefits in multiple xenograft models. Nano-flow cytometry was used to evaluate the encapsulation efficiency of OA@BCMNs, CFSE and SYTO 62 probes were applied to label the BCMNs and nucleic acids of OA, respectively. The positive ratio of 60% demonstrate the successful preparation of the OA-encapsulated BCMNs nanostructure.

3.4  Milk-Derived EVs for Drug Delivery

Foods are expected to be the ideal sources for massive production of EVs. Among them, bovine milk-derived EVs (mEVs) have emerged as a novel class of drug-delivery system, as mEVs and their cargos can be absorbed upon oral adiministration in humans. In this study, a method called “Sonication and Extrusion-assisted Active Loading” (SEAL) was developed to actively load drugs (Doxorubicin) to mEVs, which showed ~10-fold enhancement of drug encapsulation efficiency compared with passive loading. Nano-flow cytometry was employed to reveal the heterogeneous encapsulation behaviour of Dox-mEVs on the single particle basis, and the recovery rate could be provided as well. The as-developed cargo-loading approach and nano-flow cytometry-based characterization method will provide an instructive insight in the development of EV-based delivery systems.

4.  Comprehensive Evaluation of Isolation Methods for Extracellular Vesicles

Extracellular vesicles (EVs) are increasingly developed as both diagnostic and therapeutic agents for a variety of diseases models. For translating them effectively into the clinics, isolation of EVs from complex biological fluids with high purity is essential. Generally differential ultracentrifugation is the standard method for EV isolation, density gradient centrifugation (DGC) is able to get highly purifed EVs. Commercial available strategies such as size exclusion chromatography (SEC), polymer-based precipitation technologies, ultrafiltration, and immunoaffinity-based methods are used for the isolation of low to mediate volumes of EVs, including body fluids. While Tangential flow filtration (TFF), ion exchange chromatography (IEX) have been applied for the scalable production and enrichment of EVs. Upon evaluating the methods for the EV preparation/isolation, important parameters are the particle concentration, recovery efficiency/yield and purification factors as well as the quality, which is the biological activity of obtained products. Triton X-100 treatment, particles per protein amount, membrane-based staining, and biomarker profiling are the most frequently used strategies for purity assessment of EVs. Summary: Compared with ensemble-averaged analysis strategies, NanoFCM exhibits distinct advantages for the comprehensive comparison of EV isolation methods. NanoFCM provides quantitative analysis of EV size distribution, concentration, purity and phenotype without prejudice, thus serving as a new benchmark for the quality and efficiency assessment of EV isolation methods and will be a great asset to the further development of EV isolation protocols.

5. Outer Membrane Vesicles/Bacterial Membrane Vesicles

Outer membrane vesicles (OMVs) are nano-scale lipid vesicles secreted by bacteria during the growth process. OMVs carry nucleic acids and proteins, especially virulence factors and immune regulatory factor for the inter and intra-species communication. OMVs not only affect a variety of biological processes, but have also shown important applications in vaccine and anti-cancer drug development. However, the purification and characterization of OMVs face serious challenges due to their high individual variability, small size and sparse contents. Obtaining high purity samples of OMVs and assessing their purity quantitatively are also urgent challenges for OMVs research. Summary: The OMVs secreted by Gram negative bacterium (E. coli O157:H7) and Gram positive strain (S. aureus) were isolated and analyzed. This study established a quantitative characterization method for the purity, particle concentration and size distribution of OMVs at the single particle level based on Flow NanoAnalyzer, and the separation and purification of OMVs by combining differential centrifugation and density gradient centrifugation.

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