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Research in the field of extracellular vesicles (EVs) is challenged by the small size of the nano-sized particles. Apart from the use of transmission and scanning electron microscopy, established technical platforms to visualize, quantify, and characterize nano-sized EVs were lacking. Recently, methodologies to characterize nano-sized EVs have been...
Citations
... Analysis may be performed by traditional Western blotting for specific EV proteins, i.e., tetraspanins CD9, CD63, CD81, tumor necrosis factor-α (TNF-α) obtained from various fluids and tissues [81]. Other methods for evaluation of morphology and size include nanoparticle tracking analysis (NTA), dynamic light scattering and electron microscopy [82,83]. Although transmission and scanning electron microscopy are wellestablished techniques to visualize, quantify and characterize nano-sized EVs, dynamic light scattering is the only one that allows intact EV characterization [82]. ...
... Other methods for evaluation of morphology and size include nanoparticle tracking analysis (NTA), dynamic light scattering and electron microscopy [82,83]. Although transmission and scanning electron microscopy are wellestablished techniques to visualize, quantify and characterize nano-sized EVs, dynamic light scattering is the only one that allows intact EV characterization [82]. The latter provides data on EV biophysical properties which includes polydispersity, aggregation, solubility, viscosity and stability. ...
Physiologically, extracellular vesicles (EVs) have been implicated as crucial mediators of immune response, cell homeostasis, angiogenesis, cell differentiation and growth, and tissue repair. In heart failure (HF) they may act as regulators of cardiac remodeling, microvascular inflammation, micro environmental changes, tissue fibrosis, atherosclerosis, neovascularization of plaques, endothelial dysfunction, thrombosis, and reciprocal heart-remote organ interaction. The chapter summaries the nomenclature, isolation, detection of EVs, their biologic role and function physiologically as well as in the pathogenesis of HF. Current challenges to the utilization of EVs as diagnostic and predictive biomarkers in HF are also discussed.
... Liver-secreted factors were collected after 16-h incubation and EVs were isolated and purified using a differential centrifugation technique. Nano-particle tracking analysis (NTA) showed that most of the EVs were of the expected size (approximately 130 nm; Fig. 1f) 24 ; cryogenic transmission electron microscopy (cryoTEM) showed that the EVs exhibited the characteristic EV morphology, consisting of a lipid bilayer and spherical structure (Fig. 1g). The presence of validated EV proteins, including TSG101, CD63, CD9, HSP70 and CD81 were confirmed using immunoblot analysis (Fig. 1h). ...
Small extracellular vesicles (EVs) are signalling messengers that regulate inter-tissue communication through delivery of their molecular cargo. Here, we show that liver-derived EVs are acute regulators of whole-body glycaemic control in mice. Liver EV secretion into the circulation is increased in response to hyperglycaemia, resulting in increased glucose effectiveness and insulin secretion through direct inter-organ EV signalling to skeletal muscle and the pancreas, respectively. This acute blood glucose lowering effect occurs in healthy and obese mice with non-alcoholic fatty liver disease, despite marked remodelling of the liver-derived EV proteome in obese mice. The EV-mediated blood glucose lowering effects were recapitulated by administration of liver EVs derived from humans with or without progressive non-alcoholic fatty liver disease, suggesting broad functional conservation of liver EV signalling and potential therapeutic utility. Taken together, this work reveals a mechanism whereby liver EVs act on peripheral tissues via endocrine signalling to restore euglycaemia in the postprandial state.
... However, because the intensity of scattered light is proportional to the sixth power of the particle size, larger particles scatter light more strongly. 90,97,98 As a result, for samples with a wide size distribution, the average particle size result tends to be biased toward larger particles. 90,97,98 The working principle of NTA is to illuminate a solution of suspended particles with a concentrated laser beam passing through a glass prism. ...
... 90,97,98 As a result, for samples with a wide size distribution, the average particle size result tends to be biased toward larger particles. 90,97,98 The working principle of NTA is to illuminate a solution of suspended particles with a concentrated laser beam passing through a glass prism. 88,91,92,97 The intensity of scattered light from each particle is detected, and the Brownian motion of the nanoparticles in the solution is observed and imaged. ...
... 90,97,98 The working principle of NTA is to illuminate a solution of suspended particles with a concentrated laser beam passing through a glass prism. 88,91,92,97 The intensity of scattered light from each particle is detected, and the Brownian motion of the nanoparticles in the solution is observed and imaged. 91,92,97 By tracking and analyzing the Brownian motion of the particles and using the Stokes-Einstein equation, the particle size of the nanoparticles is calculated. ...
The biological barriers of the body, such as the blood–brain, placental, intestinal, skin, and air‐blood, protect against invading viruses and bacteria while providing necessary physical support. However, these barriers also hinder the delivery of drugs to target tissues, reducing their therapeutic efficacy. Extracellular vesicles (EVs), nanostructures with a diameter ranging from 30 nm to 10 μm secreted by cells, offer a potential solution to this challenge. These natural vesicles can effectively pass through various biological barriers, facilitating intercellular communication. As a result, artificially engineered EVs that mimic or are superior to the natural ones have emerged as a promising drug delivery vehicle, capable of delivering drugs to almost any body part to treat various diseases. This review first provides an overview of the formation and cross‐species uptake of natural EVs from different organisms, including animals, plants, and bacteria. Later, it explores the current clinical applications, perspectives, and challenges associated with using engineered EVs as a drug delivery platform. Finally, it aims to inspire further research to help bioengineered EVs effectively cross biological barriers to treat diseases.
... An oscillatory behavior of the g 2 curves is typically observed in a heterodyne detection scheme of dynamic light scattering (DLS) 46 and XPCS, 25,47−49 where an external static reference must be applied. The phase shift in the scattering due to the motion of the sample with respect to the fixed reference yields the oscillation. ...
Unraveling the mechanism of water’s glass transition and the interconnection between amorphous ices and liquid water plays an important role in our overall understanding of water. X-ray photon correlation spectroscopy (XPCS) experiments were conducted to study the dynamics and the complex interplay between the hypothesized glass transition in high-density amorphous ice (HDA) and the subsequent transition to low-density amorphous ice (LDA). Our XPCS experiments demonstrate that a heterodyne signal appears in the correlation function. Such a signal is known to originate from the interplay of a static component and a dynamic component. Quantitative analysis was performed on this heterodyne signal to extract the intrinsic dynamics of amorphous ice during the HDA–LDA transition. An angular dependence indicates non-isotropic, heterogeneous dynamics in the sample. Using the Stokes–Einstein relation to extract diffusion coefficients, the data are consistent with the scenario of static LDA islands floating within a diffusive matrix of high-density liquid water.
... 43,44 This equipment directly provides the video image of EVs in motion and simultaneously measures their size and concentrations using the Stokes-Einstein equation. [45][46][47] In this study, we report an optimized, single-step immunocapture assay combined with mini-column purification for microliter scale quantification of HCC-derived exosomes in their native state using NanoSight NS300 in fluorescence mode (F-NTA). We comprehensively validated the ability of the F-NTA method to quantify exosomes by using synthetic fluorescence beads, HCC cell line-derived EVs, and tumor-derived EVs from patients with confirmed HCC. ...
Introduction
Extracellular vesicles could serve as a non-invasive biomarker for early cancer detection. However, limited methods to quantitate cancer-derived vesicles in the native state remain a significant barrier to clinical translation.
Aim
This research aims to develop a rapid, one-step immunoaffinity approach to quantify HCC exosomes directly from a small serum volume.
Methods
HCC-derived exosomes in the serum were captured using fluorescent phycoerythrin (PE)-conjugated antibodies targeted to GPC3 and alpha-fetoprotein (AFP). Total and HCC-specific exosomes were then quantified in culture supernatant or patient-derived serums using fluorescence nanoparticle tracking analysis (F-NTA). The performance of HCC exosome quantification in the serum was compared with the tumor size determined by MRI.
Results
Initially we tested the detection limits of the F-NTA using synthetic fluorescent and non-fluorescent beads. The assay showed an acceptable sensitivity with a detection range of 10⁴–10⁸ particles/mL. Additionally, the combination of immunocapture followed by size-exclusion column purification allows the isolation of smaller-size EVs and quantification by F-NTA. Our assay demonstrated that HCC cell culture releases a significantly higher quantity of GPC3 or GPC3+AFP positive EVs (100–200 particles/cell) compared to non-HCC culture (10–40 particles/cell) (p<0.01 and p<0.05 respectively). The F-NTA enables absolute counting of HCC-specific exosomes in the clinical samples with preserved biological immunoreactivity. The performance of F-NTA was clinically validated in serum from patients ± cirrhosis and with confirmed HCC. F-NTA quantification data show selective enrichment of AFP and GPC3 positive EVs in HCC serum compared to malignancy-free cirrhosis (AUC values for GPC3, AFP, and GPC3/AFP were found 0.79, 0.71, and 0.72 respectively). The MRI-confirmed patient cohort indicated that there was a positive correlation between total tumor size and GPC3-positive exosome concentration (r:0.78 and p<0.001).
Conclusion
We developed an immunocapture assay that can be used for simultaneous isolation and quantification of HCC-derived exosomes from a small serum volume with high accuracy.
... 96 This method involves light scattering measurements and tracking the Brownian motion of irradiated single particles using an optical microscope equipped with a high-definition camera. 97 Cryopreservation (4 • C for the short term and −80 • C for the long term) is the most commonly used method for storing isolated exosomes. 98 However, it is important to avoid repeated freeze-thaw cycles to maintain their integrity. ...
Exosomes, membrane‐enclosed vesicles, are secreted by all types of cells. Exosomes can transport various molecules, including proteins, lipids, functional mRNAs, and microRNAs, and can be circulated to various recipient cells, leading to the production of local paracrine or distal systemic effects. Numerous studies have proved that exosomes can pass through the blood—brain barrier, thus, enabling the transfer of peripheral substances into the central nervous system (CNS). Consequently, exosomes may be a vital factor in the exchange of information between the periphery and CNS. This review will discuss the structure, biogenesis, and functional characterization of exosomes and summarize the role of peripheral exosomes deriving from tissues like the lung, gut, skeletal muscle, and various stem cell types in communicating with the CNS and influencing the brain's function. Then, we further discuss the potential therapeutic effects of exosomes in brain diseases and the clinical opportunities and challenges. Gaining a clearer insight into the communication between the CNS and the external areas of the body will help us to ascertain the role of the peripheral elements in the maintenance of brain health and illness and will facilitate the design of minimally invasive techniques for diagnosing and treating brain diseases.
... When bacteria are not present, these nutrients will be unused and thus visible at NTA, as shown. Therefore, it is not possible to completely distinguish the different particle populations from each other (Giebel & Helmbrecht, 2017;Guerreiro et al., 2018). Le et al. (2023) detected dark structures on TEM pictures of EV-like particles (EVLPs) from BHI, but after using eukaryotic EV markers they concluded that the EVLPs in BHI probably are large protein aggregates and not true EVs. ...
Periodontitis is an inflammatory condition caused by bacteria and represents a serious health problem worldwide as the inflammation damages the supporting tissues of the teeth and may predispose to systemic diseases. Porphyromonas gingivalis is considered a keystone periodontal pathogen that releases bacterial extracellular vesicles (bEVs) containing virulence factors, such as gingipains, that may contribute to the pathogenesis of periodontitis. This study aimed to isolate and characterize bEVs from three strains of P. gingivalis, investigate putative bEV uptake into human oral fibroblasts, and determine the gingipain activity of the bEVs. bEVs from three bacterial strains, ATCC 33277, A7A1-28, and W83, were isolated through ultrafiltration and size-exclusion chromatography. Vesicle size distribution was measured by nano-tracking analysis (NTA). Transmission electron microscopy was used for bEV visualization. Flow cytometry was used to detect bEVs and gingipain activity was measured with an enzyme assay using a substrate specific for arg-gingipain. The uptake of bEVs into oral fibroblasts was visualized using confocal microscopy. NTA showed bEV concentrations from 10 8 to 10 11 particles/mL and bEV diameters from 42 to 356 nm. TEM pictures demonstrated vesicle-like structures. bEV-gingipains were detected both by flow cytometry and enzyme assay. Fibroblasts incubated with bEVs labeled with fluorescent dye displayed intracellular localization consistent with bEV internalization. In conclusion, bEVs from P. gingivalis were successfully isolated and characterized, and their uptake into human oral fibroblasts was documented. The bEVs displayed active gingipains demonstrating their origin from P. gingivalis and the potential role of bEVs in periodontitis. K E Y W O R D S bacterial vesicle, Porphyromonas gingivalis, virulence factors MicrobiologyOpen. 2023;12:e1388. www.MicrobiologyOpen.com |
... In addition, the molecular composition of MVs primarily consists of cytoplasmic and plasma membrane-associated proteins since MVs are formed by the outward budding of the membrane, and they may vary greatly depending on the cell type (24). In addition, MVs were first described as subcellular material originating from platelets and were demonstrated to play a role in blood coagulation (25,26). More recently, they have been reported to transfer cargo to target cells, thus playing an essential role in cell communication (27). ...
Adipose tissue (AT) is a complicated metabolic organ consisting of a heterogeneous population of cells that exert wide‑ranging effects on the regulation of systemic metabolism and in maintaining metabolic homeostasis. Various obesity‑related complications are associated with the development of dysfunctional AT. As an essential transmitter of intercellular information, extracellular vesicles (EVs) have recently been recognized as crucial in regulating multiple physiological functions. AT‑derived extracellular vesicles (ADEVs) have been shown to facilitate cellular communication both inside and between ATs and other peripheral organs. Here, the role of EVs released from ATs in the homeostasis of metabolic and cardiovascular diseases, cancer, and neurological disorders by delivering lipids, proteins, and nucleic acids between different cells is summarized. Furthermore, the differences in the sources of ADEVs, such as adipocytes, AT macrophages, AT‑derived stem cells, and AT‑derived mesenchymal stem cells, are also discussed. This review may provide valuable information for the potential application of ADEVs in metabolic syndrome, cardiovascular diseases, cancer, and neurological disorders.
... NTA is a straightforward technique for semi-quantitative particle characterization, which allows the analysis of exosome size distribution and concentration [30] . The method involves light scattering measurements and tracking of the Brownian motion of irradiated single particles using an optical microscope equipped with a high-definition camera [31] . ...
In the inflammatory microenvironment, there are numerous exosomes secreted by immune cells (Macrophages, neutrophils, dendritic cells), mesenchymal stem cells (MSCs) and platelets as intercellular communicators, which participate in the regulation of inflammation by modulating gene expression and releasing anti-inflammatory factors. Due to their good biocompatibility, accurate targeting, low toxicity and immunogenicity, these exosomes are able to selectively deliver therapeutic drugs to the site of inflammation through interactions between their surface-antibody or modified ligand with cell surface receptors. Therefore, the role of exosome-based biomimetic delivery strategies in inflammatory diseases has attracted increasing attention. Here we review current knowledge and techniques for exosome identification, isolation, modification and drug loading. More importantly, we highlight progress in using exosomes to treat chronic inflammatory diseases such as rheumatoid arthritis (RA), osteoarthritis (OA), atherosclerosis (AS), and inflammatory bowel disease (IBD). Finally, we also discuss their potential and challenges as anti-inflammatory drug carriers.
... However, EVs are very heterogenous and this heterogeneity cannot be dissected with conventional technologies such as molecular analyses of EV preparation homogenates or by particle quantification such as nanoparticle tracking analysis (NTA) (Dragovic et al., 2011;Koliha et al., 2016;Kowal et al., 2016;Sokolova et al., 2011). Thus, it is a current challenge in the EV field to improve our EV analysis methodology and define methods allowing complex analysis at the single EV level (Giebel & Helmbrecht, 2017). In this context, we have recently qualified imaging flow cytometry (IFCM) as a next generation EV characterization platform (Gögens et al., 2019). ...
COVID-19 is characterized by a wide spectrum of disease severity, whose indicators and underlying mechanisms need to be identified. The role of extracellular vesi-cles (EVs) in COVID-19 and their biomarker potential, however, remains largely unknown. Aiming to identify specific EV signatures of patients with mild compared to severe COVID-19, we characterized the EV composition of 20 mild and 26 severe COVID-19 patients along with 16 sex and age-matched healthy donors with a panel of eight different antibodies by imaging flow cytometry (IFCM). We correlated the obtained data with 37 clinical, prerecorded biochemical and immunological parameters. Severe patients' sera contained increased amounts of CD13 + and CD82 + EVs, which positively correlated with IL-6-producing and circulating myeloid-derived suppressor cells (MDSCs) and with the serum concentration of proinflammatory cytokines, respectively. Sera of mild COVID-19 patients contained more HLA-ABC + EVs than sera of the healthy donors and more CD24 + EVs than severe COVID-19 patients. Their increased abundance negatively correlated with disease severity and accumulation of MDSCs, being considered as key drivers of immunopathogenesis in COVID-19. Altogether, our results support the potential of serum EVs as powerful biomarkers for COVID-19 severity and pave the way for future investigations aiming to unravel the role of EVs in COVID-19 progression.
