Extracellular Vesicles Isolation Research Articles

Differential extracellular vesicle concentration and their biomarker expression of integrin αv/β5, EpCAM, and glypican-1 in pancreatic cancer models

Tumor-derived extracellular vesicles (EVs) show great potential as biomarkers for several diseases, including pancreatic cancer, due to their roles in cancer development and progression. However, the challenge of utilizing EVs as biomarkers lies in their inherent heterogeneity in terms of size and concentration, making accurate quantification difficult, which is highly dependent on the isolation and quantification methods used. In our study, we compared three EV isolation techniques and two EV quantification methods. We observed variations in EV concentration, with approximately 1.5-fold differences depending on the quantification method used. Interestingly, all EV isolation techniques consistently yielded similar EV quantities, overall size distribution, and modal sizes. In contrast, we found a notable increase in total EV amounts in samples from pancreatic cancer cell lines, mouse models, and patient plasma, compared to non-cancerous conditions. Moreover, individual tumor-derived EVs exhibited at least a 3-fold increase in several EV biomarkers. Our data, obtained from EVs isolated using various techniques and quantified through different methods, as well as originating from various pancreatic cancer models, suggests that EV profiling holds promise for the identification of unique and cancer-specific biomarkers in pancreatic cancer.

Comparing the Proteomic Profiles of Extracellular Vesicles Isolated using Different Methods from Long-term Stored Plasma Samples

BackgroundThe lack of standardized protocols for isolating extracellular vesicles (EVs), especially from biobank-stored blood plasma, translates to limitations for the study of new biomarkers. This study examines whether a combination of current isolation methods could enhance the specificity and purity of isolated EVs for diagnosis and personalized medicine purposes.ResultsEVs were isolated from healthy human plasma stored for one year by ultracentrifugation (UC), size exclusion chromatography (SEC), or SEC and UC combined (SEC + UC). The EV isolates were then characterized by transmission electron microscopy imaging, nanoparticle tracking analysis (NTA) and western blotting. Proteomic procedures were used to analyze protein contents. The presence of EV markers in all isolates was confirmed by western blotting yet this analysis revealed higher albumin expression in EVs-UC, suggesting plasma protein contamination. Proteomic analysis identified 542 proteins, SEC + UC yielding the most complex proteome at 364 proteins. Through gene ontology enrichment, we observed differences in the cellular components of EVs and plasma in that SEC + UC isolates featured higher proportions of EV proteins than those derived from the other two methods. Analysis of proteins unique to each isolation method served to identify 181 unique proteins for the combined approach, including those normally appearing in low concentrations in plasma. This indicates that with this combined method, it is possible to detect less abundant plasma proteins by proteomics in the resultant isolates.ConclusionsOur findings reveal that the SEC + UC approach yields highly pure and diverse EVs suitable for comprehensive proteomic analysis with applications for the detection of new biomarkers in biobank-stored plasma samples.

Standardized Reporting of Research on Exosomes to Ensure Rigor and Reproducibility.

Significance: The study of extracellular vesicles (EVs), especially exosomes, has unlocked new avenues in understanding cellular communication and potential therapeutic applications. Recent Advances: Advancements in EV research have shown significant contributions from the International Society for Extracellular Vesicles (ISEV), in establishing methodological standards. The evolution of the Minimal Information for Studies of Extracellular Vesicles (MISEV) guidelines from 2014 to 2023 reflects enhanced research rigor and reproducibility. The launch of EV-TRACK platform promotes uniformity and reproducibility by providing a centralized repository for data sharing and standardization practices. Furthermore, databases like EVpedia and ExoCarta have facilitated data sharing and collaboration within the scientific community. Concurrently, exosome-based therapies have emerged as a forefront area within regenerative medicine and targeted drug delivery, showcasing the potential of exosomes in promoting tissue regeneration. Critical Issues: Despite advancements, the field grapples with challenges such as vesicular heterogeneity, EV isolation complexity, and standardization. These issues impact research reproducibility and clinical applications. The inconsistency in exosomal preparations in clinical trials poses significant challenges to therapeutic efficacy and safety. Future Directions: The review outlines critical areas for future research, including the need for technological innovation in EV isolation and characterization, the establishment of standardized protocols, and a deeper understanding of exosome biology. The review also highlights the need to reassess guidelines, develop new EV isolation and characterization technologies, and establish standardized protocols to overcome current limitations. Emphasis is placed on interdisciplinary research and collaboration to address the complexities of EV biology, improve clinical trial design, and ultimately realize exosome's therapeutic and diagnostic potential. Continued evaluation and rigorous scientific validation are essential for successful exosome integration.

Isolation and Characterization of Extracellular Vesicles of Chick Embryo Blood.

Exosomes from plants or animals are a cheap, available, and promising option in medicine, which can be used for the detection or treatment of various diseases. This study aims to evaluate the antitoxic and antioxidant properties of Extracellular vesicle (EVs) extracted from chicken embryo blood using a fibroblast cell line (NIH/3T3). EVs from chick embryos were extracted in this experimental investigation using the sedimentation method and examined using dynamic light scattering (DLS) and field emission electron microscopy (FE-SEM). The protein concentration and overall antioxidant capacity of the EVs were determined using bicinchoninic acid (BCA) and antioxidant capacity (FRAP). EVs were added to NIH/3T3 cells at varying concentrations (1, 2, and 10 mg/ml), and the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay test was used to measure cell survival. The size of the isolated EVs was confirmed to be less than 100 nm by electron microscopy and DLS. The quantity of protein in these EVs was 3200 µg/ml, and their total antioxidant capacities were 3130.17, 1914.122, and 976.9 μMol/L. The MTT test findings demonstrated that NIH/3T3 cells survived treatment with EVs (P ≤ 0.001) compared to the control group. Antioxidant-rich and protein-rich exosomes in chicken embryos may be valuable in managing oxidative stress.

EPEN-20. LEVERAGING ABERRANT EXTRACELLULAR VESICLE SIGNALING FOR TARGETED DRUG REPURPOSING IN PEDIATRIC EPENDYMOMA

Abstract Increasing evidence highlights the diverse functions of extracellular vesicles (EVs) as intercellular messengers in all facets of cancer progression, pointing towards a potential new avenue in cancer treatment. Limited understanding of tumor-specific deregulations and lack of suitable drug compounds have hampered clinical translation of targeted interference with EV signaling. The most aggressive types of intracranial ependymoma (EPN) in children and adults are resistant to chemotherapy. Despite enormous diagnostic advancements in classification and stratification, EPN lack recurrent effective molecular targets precluding access of affected patients to precision-based oncology therapeutics. Here, we characterized the proteome of ninety-five EPN and five matching healthy control brain tissue samples. Employing a mulit-omic data integration approach, we identified EVs as an excessively augmented pathway on the functional level. Isolation and molecular characterization of EPN EVs combined with single cell sequencing analysis, and ultra-high content imaging unraveled perivascular mesenchymal cell populations as predominant EV originators. Targeting EV pathways through chemical inhibition of sphingomyelinases (SMPD1, SMPD2, SMPD3) including re-purposed FDA approved compounds revealed growth inhibition, reduced invasion capacity in vitro and significant survival probability in vivo. Transcriptomic analysis and scanning electron microscopy delineated transition of mesenchymal-to neuroepithelial subpopulations in cells and patient-derived xenograft (PDX) models of EPN upon reduction of EV release. This study showcases the inhibition of EV signaling as a promising therapeutic strategy against mesenchymal tumor persister cells, which are the suspected origins of repeated EPN relapses. Our study provides a framework to leverage cell state-specific vulnerabilities and drug repurposing to attack the most aggressive cell populations in cancers with unfavorable prognosis.

An anion exchange membrane sensor detects EGFR and its activity state in plasma CD63 extracellular vesicles from patients with glioblastoma

We present a quantitative sandwich immunoassay for CD63 Extracellular Vesicles (EVs) and a constituent surface cargo, EGFR and its activity state, that provides a sensitive, selective, fluorophore-free and rapid alternative to current EV-based diagnostic methods. Our sensing design utilizes a charge-gating strategy, with a hydrophilic anion exchange membrane functionalized with capture antibodies and a charged silica nanoparticle reporter functionalized with detection antibodies. With sensitivity and robustness enhancement by the ion-depletion action of the membrane, this hydrophilic design with charged reporters minimizes interference from dispersed proteins, thus enabling direct plasma analysis without the need for EV isolation or sensor blocking. With a LOD of 30 EVs/μL and a high relative sensitivity of 0.01% for targeted proteomic subfractions, our assay enables accurate quantification of the EV marker, CD63, with colocalized EGFR by an operator/sample insensitive universal normalized calibration. We analysed untreated clinical samples of Glioblastoma to demonstrate this new platform. Notably, we target both total and “active” EGFR on EVs; with a monoclonal antibody mAb806 that recognizes a normally hidden epitope on overexpressed or mutant variant III EGFR. Analysis of samples yielded an area-under-the-curve (AUC) value of 0.99 and a low p-value of 0.000033, surpassing the performance of existing assays and markers.

Isolation and quantification of L1CAM-positive extracellular vesicles on a chip as a potential biomarker for Parkinson's Disease.

Extracellular vesicles (EVs) carry disease-specific molecular profiles, demonstrating massive potential in biomarker discovery. In this study, we developed an integrated biochip platform, termed EVID-biochip (EVs identification and detection biochip), which integrates in situ electrochemical protein detection with on-chip antifouling-immunomagnetic beads modified with CD81 antibodies and zwitterion molecules, enabling efficient isolation and detection of neuronal EVs. The capability of the EVID-biochip to isolate common EVs and detect neuronal EVs associated with Parkinson's disease in human serum is successfully demonstrated, using the transmembrane protein L1-cell adhesion molecule (L1CAM) as a target biomarker. The EVID-biochip exhibited high efficiency and specificity for the detection of L1CAM with a sensitivity of 1pg/mL. Based on the validation of 76 human serum samples, for the first time, this study discovered that the level of L1CAM/neuronal EV particles in serum could serve as a reliable indicator to distinguish Parkinson's disease from control groups with AUC=0.973. EVID-biochip represents a reliable and rapid liquid biopsy platform for the analysis of complex biofluids offering EVs isolation and detection in a single chip, requiring a small sample volume (300µL) and an assay time of 1.5h. This approach has the potential to advance the diagnosis and biomarker discovery of various neurological disorders and other diseases.

Single-extracellular vesicle (EV) analyses validate the use of L1 Cell Adhesion Molecule (L1CAM) as a reliable biomarker of neuron-derived EVs.

Isolation of neuron-derived extracellular vesicles (NDEVs) with L1 Cell Adhesion Molecule (L1CAM)-specific antibodies has been widely used to identify blood biomarkers of CNS disorders. However, full methodological validation requires demonstration of L1CAM in individual NDEVs and lower levels or absence of L1CAM in individual EVs from other cells. Here, we used multiple single-EV techniques to establish the neuronal origin and determine the abundance of L1CAM-positive EVs in human blood. L1CAM epitopes of the ectodomain are shown to be co-expressed on single-EVs with the neuronal proteins β-III-tubulin, GAP43, and VAMP2, the levels of which increase in parallel with the enrichment of L1CAM-positive EVs. Levels of L1CAM-positive EVs carrying the neuronal proteins VAMP2 and β-III-tubulin range from 30% to 63%, in contrast to 0.8%-3.9% of L1CAM-negative EVs. Plasma fluid-phase L1CAM does not bind to single-EVs. Our findings support the use of L1CAM as a target for isolating plasma NDEVs and leveraging their cargo to identify biomarkers reflecting neuronal function.

Simultaneous isolation of intact brain cells and cell-specific extracellular vesicles from cryopreserved Alzheimer’s disease cortex

BackgroundThe neuronal and gliaI populations within the brain are tightly interwoven, making isolation and study of large populations of a single cell type from brain tissue a major technical challenge. Concurrently, cell-type specific extracellular vesicles (EVs) hold enormous diagnostic and therapeutic potential in neurodegenerative disorders including Alzheimer’s disease (AD). New MethodPostmortem AD cortical samples were thawed and gently dissociated. Following filtration, myelin and red blood cell removal, cell pellets were immunolabeled with fluorescent antibodies and analyzed by flow cytometry. The cell pellet supernatant was applied to a triple sucrose cushion for brain EV isolation. ResultsNeuronal, astrocyte and microglial cell populations were identified. Cell integrity was demonstrated using calcein AM, which is retained by cells with esterase activity and an intact membrane. For some experiments cell pellets were fixed, permeabilized, and immunolabeled for cell-specific markers. Characterization of brain small EV fractions showed the expected size, depletion of EV negative markers, and enrichment in positive and cell-type specific markers. Comparison with existing methods and conclusionsWe optimized and integrated established protocols, aiming to maximize information obtained from each human autopsy brain sample. The uniqueness of our method lies in its capability to isolate cells and EVs from a single cryopreserved brain sample. Our results not only demonstrate the feasibility of isolating specific brain cell subpopulations for RNA-seq but also validate these subpopulations at the protein level. The accelerated study of EVs from human samples is crucial for a better understanding of their contribution to neuron/glial crosstalk and disease progression.

Altered Extracellular Vesicle-Derived Protein and microRNA Signatures in Bronchoalveolar Lavage Fluid from Patients with Chronic Obstructive Pulmonary Disease.

Chronic obstructive pulmonary disease (COPD) is a progressive lung disease for which there is no cure. Accumulating research results suggest a role for extracellular vesicles (EVs) in the pathogenesis of COPD. This study aimed to uncover the involvement of EVs and their molecular cargo in the progression of COPD by identification of EV-associated protein and microRNA (miRNA) profiles. We isolated EVs from the bronchial alveolar lavage fluid (BALF) of 18 patients with COPD and 11 healthy controls using size-exclusion chromatography. EV isolates were characterized using nanoparticle tracking analysis and protein content. Proteomic analysis revealed a higher abundance of 284 proteins (log2FC > 1) and a lower abundance of 3 proteins (log2FC < -1) in EVs derived from patients with COPD. Ingenuity pathway analysis showed that proteins enriched in COPD-associated EVs trigger inflammatory responses, including neutrophil degranulation. Variances in surface receptors and ligands associated with COPD EVs suggest a preferential interaction with alveolar cells. Small RNAseq analysis identified a higher abundance of ten miRNAs and a lower abundance of one miRNA in EVs from COPD versus controls (Basemean > 100, FDR < 0.05). Our data indicate that the molecular composition of EVs in the BALF of patients with COPD is altered compared to healthy control EVs. Several components in COPD EVs were identified that may perpetuate inflammation and alveolar tissue destruction.

Buoyant Metal-Organic Framework Corona-Driven Fast Isolation and Ultrasensitive Profiling of Circulating Extracellular Vesicles.

Accurately assaying tumor-derived circulating extracellular vesicles (EVs) is fundamental in noninvasive cancer diagnosis and therapeutic monitoring but limited by challenges in efficient EV isolation and profiling. Here, we report a bioinspired buoyancy-driven metal-organic framework (MOF) corona that leverages on-bubble coordination and dual-encoded surface-enhanced Raman scattering (SERS) nanotags to streamline rapid isolation and ultrasensitive profiling of plasma EVs in a single assay for cancer diagnostics. This integrated bubble-MOF-SERS EV assay (IBMsv) allows barnacle-like high-density adhesion of MOFs on a self-floating bubble surface to enable fast isolation (2 min, near 90% capture efficiency) of tumor EVs via enhanced EV-MOF binding. Also, IBMsv harnesses four-plexed SERS nanotags to profile the captured EV surface protein markers at a single-particle level. Such a sensitive assay allows multiplexed profiling of EVs across five cancer types, revealing heterogeneous EV surface expression patterns. Furthermore, the IBMsv assay enables cancer diagnosis in a pilot clinical cohort (n = 55) with accuracies >95%, improves discrimination between cancer and noncancer patients via an algorithm, and monitors the surgical treatment response from hepatocellular carcinoma patients. This assay provides a fast, sensitive, streamlined, multiplexed, and portable blood test tool to enable cancer diagnosis and response monitoring in clinical settings.

Which casein micelle removal method is suitable for studies of human milk extracellular vesicles? A systematic comparison of four different treatments for casein depletion before extracellular vesicle isolation from human milk

Aim: This study aimed to systematically compare four casein micelle removal methods on the particle and protein characteristics of the isolated human milk EVs. Methods: The defatted milk was treated with 1% sodium citrate, 20 mM ethylenediaminetetraacetic acid (EDTA), 1% acetic acid, or 1% chymosin/calcium chloride for 30 min at 4 °C to remove casein micelles. EV isolation was performed using qEV size exclusion chromatography. Milk turbidity at the optical density 350 nm and dot immunoblot with casein antibody were applied to monitor the qEV fractions. Particle analyses were performed using transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). The enrichment of human milk EV markers, i.e., tetraspanins, Alix, lactadherin, butyrophilin, and xanthine dehydrogenase, and casein depletion capabilities were evaluated by proteomics and immunoblotting. Results: Compared to the untreated condition, sodium citrate and EDTA decreased milk turbidity by disrupting casein micelles, while acetic acid and chymosin removed them by inducing precipitation/coagulation. All treatments shifted casein immunoreactivity in the qEV fractions from large micelles (the exclusion volume) to small molecular sizes (gel-infiltrated fractions). Acidification affected human milk EV morphology, while EDTA, acetic acid, and chymosin methods slightly altered EV particle numbers. Different casein micelle removal methods confer different degrees of human milk EV marker enrichment and casein depletion. The method performances could be ranked as follows: chymosin &gt; EDTA &gt; acetic acid &gt; sodium citrate. Conclusion: Our findings suggest that chymosin and EDTA should be considered as the method of choice for casein micelle removal in future studies involving human milk EV isolation and characterization.

Development of a Sampling and Storage Protocol of Extracellular Vesicles (EVs)-Establishment of the First EV Biobank for Polytraumatized Patients.

In the last few years, several studies have emphasized the existence of injury-specific EV "barcodes" that could have significant importance for the precise diagnosis of different organ injuries in polytrauma patients. To expand the research potential of the NTF (network trauma research) biobank of polytraumatized patients, the NTF research group decided to further establish a biobank for EVs. However, until now, the protocols for the isolation, characterization, and storage of EVs for biobank purposes have not been conceptualized. Plasma and serum samples from healthy volunteers (n = 10) were used. Three EV isolation methods of high relevance for the work with patients' samples (ultracentrifugation, size exclusion chromatography, and immune magnetic bead-based isolation) were compared. EVs were quantified using nanoparticle tracking analysis, EV proteins, and miRNAs. The effects of different isolation solutions; the long storage of samples (up to 3 years); and the sensibility of EVs to serial freezing-thawing cycles and different storage conditions (RT, 4/-20/-80 °C, dry ice) were evaluated. The SEC isolation method was considered the most suitable for EV biobanking. We did not find any difference in the quantity of EVs between serum and plasma-EVs. The importance of particle-free PBS as an isolation solution was confirmed. Plasma that has been frozen for a long time can also be used as a source of EVs. Serial freezing-thawing cycles were found to affect the mean size of EVs but not their amount. The storage of EV samples for 5 days on dry ice significantly reduced the EV protein concentration.

Therapeutic Potential of Bovine Milk-Derived Extracellular Vesicles.

Milk is a fundamental component of the human diet, owing to its substantial nutritional content. In addition, milk contains nanoparticles called extracellular vesicles (EVs), which have indicated their potential beneficial roles such as cell-to-cell communication, disease biomarkers, and therapeutics agents. Amidst other types of EVs, milk EVs (MEVs) have their significance due to their high abundance, easy access, and stability in harsh environmental conditions, such as low pH in the gut. There have been plenty of studies conducted to evaluate the therapeutic potential of bovine MEVs over the past few years, and attention has been given to their engineering for drug delivery and targeted therapy. However, there is a gap between the experimental findings available and clinical trials due to the many challenges related to EV isolation, cargo, and the uniformity of the material. This review aims to provide a comprehensive comparison of various techniques for the isolation of MEVs and offers a summary of the therapeutic potential of bovine MEVs described over the last decade, analyzing potential challenges and further applications. Although a number of aspects still need to be further elucidated, the available data point to the role of MEVs as a potential candidate with therapeutics potential, and the supplementation of MEVs would pave the way to understanding their in-depth effects.

High-throughput surface epitope immunoaffinity isolation of extracellular vesicles and downstream analysis.

Extracellular vesicles (EVs), including exosomes, have significant potential for diagnostic and therapeutic applications. The lack of standardized methods for efficient and high-throughput isolation and analysis of EVs, however, has limited their widespread use in clinical practice. Surface epitope immunoaffinity (SEI) isolation utilizes affinity ligands, including antibodies, aptamers, or lectins, that target specific surface proteins present on EVs. Paramagnetic bead-SEI isolation represents a fit-for-purpose solution for the reproducible, high-throughput isolation of EVs from biofluids and downstream analysis of RNA, protein, and lipid biomarkers that is compatible with clinical laboratory workflows. This study evaluates a new SEI isolation method for enriching subpopulations of EVs. EVs were isolated from human plasma using a bead-based SEI method designed for on-bead and downstream analysis of EV-associated RNA and protein biomarkers. Western blot analysis confirmed the presence of EV markers in the captured nanoparticles. Mass spectrometry analysis of the SEI lysate identified over 1500 proteins, with the top 100 including known EV-associated proteins. microRNA (miRNA) sequencing followed by RT-qPCR analysis identified EV-associated miRNA transcripts. Using SEI, EVs were isolated using automated high-throughput particle moving instruments, demonstrating equal or higher protein and miRNA yield and recovery compared to manual processing. SEI is a rapid, efficient, and high-throughput method for isolating enriched populations of EVs; effectively reducing contamination and enabling the isolation of a specific subpopulation of EVs. In this study, high-throughput EV isolation and RNA extraction have been successfully implemented. This technology holds great promise for advancing the field of EV research and facilitating their application for biomarker discovery and clinical research.

Proteomic Analysis of Umbilical Cord Mesenchymal Stem Cell-Derived Extracellular Vesicles: A Systematic Review.

Numerous challenges remain within conventional cell-based therapy despite the growing trend of stem cells used to treat various life-debilitating diseases. These limitations include batch-to-batch heterogeneity, induced alloreactivity, cell survival and integration, poor scalability, and high cost of treatment, thus hindering successful translation from lab to bedside. However, recent pioneering technology has enabled the isolation and enrichment of small extracellular vesicles (EVs), canonically known as exosomes. EVs are described as a membrane-enclosed cargo of functional biomolecules not limited to lipids, nucleic acid, and proteins. Interestingly, studies have correlated the biological role of MSC-EVs to the paracrine activity of MSCs. This key evidence has led to rigorous studies on MSC-EVs as an acellular alternative. Using EVs as a therapy was proposed as a model leading to improvements through increased safety; enhanced bioavailability due to size and permeability; reduced heterogeneity by selective and quantifiable properties; and prolonged shelf-life via long-term freezing or lyophilization. Yet, the identity and potency of EVs are still relatively unknown due to various methods of preparation and to qualify the final product. This is reflected by the absence of regulatory strategies overseeing manufacturing, quality control, clinical implementation, and product registration. In this review, the authors review the various production processes and the proteomic profile of MSC-EVs.

Aspects of conceptualization, development, and simulation of an extracellular vesicles' bulk isolation device for cosmetic and therapeutic applications

This paper discusses the concept and model to develop a prototype extracellular vesicle bulk-isolation device (EBID). It proposes an engineering design and potential ways to mitigate limitations in the currently available extracellular vesicle (EV) isolation techniques. A more convenient isolation technique is developed and presented to ensure production-scale isolation and purification of the specific size of EV (approximately 0.1 to 0.34 µm) from a large quantity of cell-culture media or biological fluid sources. A more appropriate design concept was established based on the chitosan-assisted precipitation of EV techniques and the membrane filtration technology-based prototype. After that, a novel theoretical model for bulk isolation of EVs was developed and presented using pressure-driven circulation and particulate flow in the EBID loop, where the EV production results from the biofluid flowing through the EBID membrane filter module (MFM). Governing equations are coupled with Darcy's law to investigate the biofluid flow behavior in the EBID MFM, therefore simulating the isolation process of EVs from their biofluids. The flow characteristics of the EV isolation process within the EBID MFM are theoretically simulated and discussed for improved isolation yield with a high degree of quality and purity.

Isolation of Extracellular Vesicles from Agri-Food Wastes: A Novel Perspective in the Valorization of Agri-Food Wastes and By-Products.

Agri-food wastes generated by industrial food processing are valorized through the extraction of biomolecules to obtain value-added products useful for various industrial applications. In the present review, we describe the valuable by-products and bioactive molecules that can be obtained from agricultural wastes and propose extracellular vesicles (EVs) as innovative nutraceutical and therapeutic compounds that could be derived from agriculture residues. To support this idea, we described the general features and roles of EVs and focused on plant-derived extracellular vesicles (PDEVs) that are considered natural carriers of bioactive molecules and are involved in intercellular communication between diverse kingdoms of life. Consistently, PDEVs exert beneficial effects (anti-inflammatory, anti-tumor, and immune-modulatory) on mammalian cells. Although this research field is currently in its infancy, in the near future, the isolation of EVs and their use as nutraceutical tools could represent a new and innovative way to valorize waste from the agri-food industry in an ecofriendly way.

The role of the size of affinity ligands in the detection and characterization of extracellular vesicles

Surface proteins on the membrane of nano-sized extracellular vesicles (EVs) not only play crucial roles in cell-to-cell communication, but also are specific binding targets for EV detection, isolation and tracking. The low abundance of protein biomarkers on EV surface, the formation of clusters and the complex EV surface network impose significant challenges to the study of EVs. Employing bulky sized affinity ligands, such as antibodies, in the detection and characterization of these vesicles often result in reduced sensitivity of detection or poor quantification of proteins on the EV surface. By virtue of their small size and high specificity, Affibody molecules emerge as a potential alternative to their monoclonal antibody counterparts as robust affinity ligands in EV research. In this study, we present a theoretical framework on the superiority of anti-HER2 Affibodies over anti-HER2 antibodies in labeling and detecting HER2-positive EVs, followed by the demonstration of the advantages of HER2 Affibodies in accessing EV surface and the detection of EVs through multiple types of approaches including fluorescence intensity, colorimetry, and fluorescence polarization. HER2 Affibodies outperformed by 10-fold over three HER2 antibody clones in accessing HER2-positive EVs derived from different human cancer cell lines. Furthermore, HRP-Affibody molecules could detect EVs from cancer cells spiked into human serum with at least a 2-fold higher sensitivity compared with that of their antibody counterparts. In addition, in fluorescence polarization assays in which no separation of free from bound ligand is required, FITC-labeled HER2 Affibodies could sensitively detect HER2-positive EVs with a clinically relevant limit of detection, whilst HER2 antibodies failed to detect EVs in the same conditions. With the demonstrated superiority in accessing and detecting surface targets over bulky-sized antibodies in EVs, Affibodies may become the next-generation of affinity ligands in the precise characterization and quantification of molecular architecture on the surface of EVs.

Recent Advances in Microfluidic-Based Extracellular Vesicle Analysis.

Extracellular vesicles (EVs) serve as vital messengers, facilitating communication between cells, and exhibit tremendous potential in the diagnosis and treatment of diseases. However, conventional EV isolation methods are labor-intensive, and they harvest EVs with low purity and compromised recovery. In addition, the drawbacks, such as the limited sensitivity and specificity of traditional EV analysis methods, hinder the application of EVs in clinical use. Therefore, it is urgent to develop effective and standardized methods for isolating and detecting EVs. Microfluidics technology is a powerful and rapidly developing technology that has been introduced as a potential solution for the above bottlenecks. It holds the advantages of high integration, short analysis time, and low consumption of samples and reagents. In this review, we summarize the traditional techniques alongside microfluidic-based methodologies for the isolation and detection of EVs. We emphasize the distinct advantages of microfluidic technology in enhancing the capture efficiency and precise targeting of extracellular vesicles (EVs). We also explore its analytical role in targeted detection. Furthermore, this review highlights the transformative impact of microfluidic technology on EV analysis, with the potential to achieve automated and high-throughput EV detection in clinical samples.