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Imaging extracellular vesicles: current and emerging methods
摘要： Extracellular vesicles (EVs) are lipid bilayer-enclosed nanoparticles released by cells. They range from 30 nm to several micrometers in diameter, and ferry biological cargos such as proteins, lipids, RNAs and DNAs for local and distant intercellular communications. EVs have since been found to play a role in development, as well as in diseases including cancers. To elucidate the roles of EVs, researchers have established different methods to visualize and study their spatiotemporal properties. However, since EV are nanometer-sized, imaging them demands a full understanding of each labeling strategy to ensure accurate monitoring. This review covers current and emerging strategies for EV imaging for prospective studies.
关键词： Bioluminescence,MRI,Microvesicles,SPECT,Biodistribution,Imaging,Extracellular vesicles,Fluorescence,Dyes,exosomes
更新于2025-09-23 15:23:52
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Quality of extracellular vesicle images by transmission electron microscopy is operator and protocol dependent
摘要： Transmission electron microscopy (TEM) has nanometre resolution and can be used to distinguish single extracellular vesicles (EVs) from non-EV particles. TEM images of EVs are a result of operator image selection. To which extent operator image selection reflects the overall sample quality, and to which extent the images are comparable and reproducible, is unclear. In a first attempt to improve the comparability and reproducibility of TEM to visualise EVs, we compared operator image selection to images taken at predefined locations from the same grids, using four EV TEM preparation protocols, a single EV-containing sample and a single TEM instrument. Operator image selection leads to high-quality images that are more similar between the protocols. In contrast, images taken at predefined locations reveal differences between the protocols, for example in number of EVs per image and background quality. From the evaluated protocols, for only one protocol the operator image selection is comparable to the TEM images taken at predefined locations. Taken together, operator image selection can be used to demonstrate the presence of EVs in a sample, but seem less suitable to demonstrate the quality of a sample. Because images taken at predefined locations reflect the overall quality of the EV-containing sample rather than the presence of EVs alone, this is a first step to improve the comparability and reproducibility of TEM for monitoring the quality of EV-containing samples.
关键词： urine,negative staining,transmission electron microscopy (TEM),Exosomes,extracellular vesicles (EVs),microvesicles
更新于2025-09-23 15:23:52
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Extracellular vesicles based self-grown gold nanopopcorn for combinatorial chemo-photothermal therapy
摘要： Here, we generated a popcorn-like gold nanostructure exploiting extracellular vesicles (EVs). EVs can first serve as the vehicle for chemotherapeutic drug doxorubicin (DOX). Taking advantages of EVs, gold nanoparticles can be then self-grown surrounding the EVs, assembling into popcorn-like nanostructure. The formulated nanopopcorn, consisting of self-grown gold nanoparticles and EVs encapsulated with DOX, retained the photothermal transduction from gold nanoparticle assemblies and cytotoxicity of DOX. Under external near infrared irradiation, gold nanopopcorn can produce hyperthermia to induce tumor ablation and trigger drug release, achieving combinatorial chemo-photothermal therapy. The nanoplatform demonstrated improved cellular internalization, controlled drug release, enhanced antitumor efficacy with tumor inhibitory rate up to 98.6% and reduced side effects. Our design of popcorn-like nanostructure will contribute a novel modality for facile and green synthesis of complex metal nanostructures exploiting natural properties of EVs for combinational therapy.
关键词： photothermal therapy,self-growth,gold nanopopcorn,chemotherapy,extracellular vesicles
更新于2025-09-23 15:22:29
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High-fidelity probing of the structure and heterogeneity of extracellular vesicles by resonance-enhanced atomic force microscopy infrared spectroscopy
摘要： Extracellular vesicles (EVs) are highly specialized nanoscale assemblies that deliver complex biological cargos to mediate intercellular communication. EVs are heterogeneous, and characterization of this heterogeneity is paramount to understanding EV biogenesis and activity, as well as to associating them with biological responses and pathologies. Traditional approaches to studying EV composition generally lack the resolution and/or sensitivity to characterize individual EVs, and therefore the assessment of EV heterogeneity has remained challenging. We have recently developed an atomic force microscope IR spectroscopy (AFM-IR) approach to probe the structural composition of single EVs with nanoscale resolution. Here, we provide a step-by-step procedure for our approach and show its power to reveal heterogeneity across individual EVs, within the same population of EVs and between different EV populations. Our approach is label free and able to detect lipids, proteins and nucleic acids within individual EVs. After isolation of EVs from cell culture medium, the protocol involves incubation of the EV sample on a suitable substrate, setup of the AFM-IR instrument and collection of nano-IR spectra and nano-IR images. Data acquisition and analyses can be completed within 24 h, and require only a basic knowledge of spectroscopy and chemistry. We anticipate that new understanding of EV composition and structure through AFM-IR will contribute to our biological understanding of EV biology and could find application in disease diagnosis and the development of EV therapies.
关键词： spectroscopy,heterogeneity,nanoscale characterization,AFM-IR,extracellular vesicles
更新于2025-09-23 15:22:29
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Augmented COlorimetric NANoplasmonic (CONAN) Method for Grading Purity and Determine Concentration of EV Microliter Volume Solutions
摘要： This protocol paper describes how to assign a purity grade and to subsequently titrate extracellular vesicle (EV) solutions of a few microliters in volume by microplate COlorimetric NANoplasmonic (CONAN) assay. The CONAN assay consists of a solution of gold nanoparticles (AuNPs) into which the EV preparation is added. The solution turns blue if the EV preparation is pure, whereas it stays red if soluble exogenous single and aggregated proteins (SAPs; often referred to as protein contaminants) are present. The color change is visible by the naked eye or can be quantified by UV-Vis spectroscopy, providing an index of purity (a unique peculiarity to date). The assay specifically targets SAPs, and not the EV-related proteins, with a detection limit <50 ng/μl (an order of magnitude higher resolution than that of the Bradford protein assay). For pure solutions, the assay also allows for determining the EV number, as the color shift is linearly dependent on the AuNP/EV molar ratio. Instead, it automatically reports if the solution bears SAP contaminants, thus avoiding counting artifacts. The CONAN assay proves to be robust and reliable and displays very interesting performances in terms of cost (inexpensive reagents, run by standard microplate readers), working volumes (1–2 μl of sample required), and time (full procedure takes <1 h). The assay is applicable to all classes of natural and artificial lipid microvesicles and nanovesicles.
关键词： extracellular vesicles,particle number,nanoplasmonics,titration,liposomes,synthetic vesicles,nanoparticles,purity
更新于2025-09-16 10:30:52
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Editorial: Plasmonic Technologies for Bioanalytical Applications
摘要： Bioanalytical technologies that provide quantitative information about bioanalytes of interest in biological systems are becoming widespread in biomedical applications, environment monitoring, and food quality and safety. Among recent developments, plasmonic-based technologies made significant strides toward real life applications. They are based on surface plasmons, which are collective oscillations of conductive electrons on noble metal film or nanoparticle surfaces induced by electromagnetic radiation at the metal-dielectric interface. Plasmonic nanostructures have the ability to control and manipulate visible light at the nanometer scale and have been used to create plasmonic filters, wave-guides, nanoscopic light sources, and other devices with unique or improved properties. In addition, plasmonics has helped further our understanding of the interaction of molecules with nanostructures and has been used in biosensing applications. Their low ohmic and optical losses, chemical activity, tunable optical properties, specific response to incident light, and the ability to enhance signals such as fluorescence and Raman scattering, have been exploited in biomedical applications. Specific plasmonic-based technologies include nanoscale optical absorption spectroscopy, surface plasmon resonance (SPR), localized surface plasmon resonance (LSPR), surface-enhanced Raman scattering spectroscopy (SERS), tip-enhanced Raman spectroscopy (TERS), surface-enhanced infrared absorption spectroscopy (SEIRAS), single molecule plasmonics (SMP), and chiral plasmonics (CP). This special issue is needed for chemists, biologists, and materials scientists who work in the area of plasmonics applications in bioanalytical sensing.
关键词： plasmonic materials,plasmonics,bioanalytical,medical diagnosis,extracellular vesicles
更新于2025-09-16 10:30:52
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ZnO nanocrystals shuttled by extracellular vesicles as effective Trojan nano-horses against cancer cells
摘要： The effective application of nanoparticles in cancer theranostics is jeopardized by their aggregation in biological media, rapid degradation and clearance. The design of biomimetic nanoconstructs with enhanced colloidal stability and non-immunogenicity is therefore essential. We propose naturally stable cell-derived extracellular vesicles to encapsulate zinc oxide (ZnO) nanocrystals as efficacious nanodrugs, to obtain highly biomimetic and stable Trojan nano-horses (TNHs). Materials & methods: Coupling efficiency, biostability, cellular cytotoxicity and internalization were tested. Results: In vitro studies showed a high internalization of TNHs into cancer cells and efficient cytotoxic activity thanks to ZnO intracellular release. Conclusion: TNHs represent an efficient biomimetic platform for future nanotheranostic applications, with biomimetic extracellular vesicle-lipid envelope, facilitated ZnO cellular uptake and potential therapeutic implications.
关键词： biomimetics,extracellular vesicles,colloidal stability,cytotoxicity,nanocrystals,zinc oxide
更新于2025-09-11 14:15:04
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Intraoperative visualization of the tumor microenvironment and quantification of extracellular vesicles by label-free nonlinear imaging
摘要： Characterization of the tumor microenvironment, including extracellular vesicles (EVs), is important for understanding cancer progression. EV studies have traditionally been performed on dissociated cells, lacking spatial information. Since the distribution of EVs in the tumor microenvironment is associated with cellular function, there is a strong need for visualizing EVs in freshly resected tissues. We intraoperatively imaged untreated human breast tissues using a custom nonlinear imaging system. Label-free optical contrasts of the tissue, correlated with histological findings, enabled point-of-procedure characterization of the tumor microenvironment. EV densities from 29 patients with breast cancer were found to increase with higher histologic grade and shorter tumor-to-margin distance and were significantly higher than those from 7 cancer-free patients undergoing breast reduction surgery. Acquisition and interpretation of these intraoperative images not only provide real-time visualization of the tumor microenvironment but also offer the potential to use EVs as a label-free biomarker for cancer diagnosis and prognosis.
关键词： extracellular vesicles,label-free nonlinear imaging,breast cancer,intraoperative imaging,tumor microenvironment
更新于2025-09-04 15:30:14