Abstract Extracellular vesicles (EVs), through their complex cargo, can reflect the state of their cell of origin and change the functions and phenotypes of other cells. These features indicate strong biomarker and therapeutic potential and have generated broad interest, as evidenced by the steady year‐on‐year increase in the numbers of scientific publications about EVs. Important advances have been made in EV metrology and in understanding and applying EV biology. However, hurdles remain to realising the potential of EVs in domains ranging from basic biology to clinical applications due to challenges in EV nomenclature, separation from non‐vesicular extracellular particles, characterisation and functional studies. To address the challenges and opportunities in this rapidly evolving field, the International Society for Extracellular Vesicles (ISEV) updates its ‘Minimal Information for Studies of Extracellular Vesicles’, which was first published in 2014 and then in 2018 as MISEV2014 and MISEV2018, respectively. The goal of the current document, MISEV2023, is to provide researchers with an updated snapshot of available approaches and their advantages and limitations for production, separation and characterisation of EVs from multiple sources, including cell culture, body fluids and solid tissues. In addition to presenting the latest state of the art in basic principles of EV research, this document also covers advanced techniques and approaches that are currently expanding the boundaries of the field. MISEV2023 also includes new sections on EV release and uptake and a brief discussion of in vivo approaches to study EVs. Compiling feedback from ISEV expert task forces and more than 1000 researchers, this document conveys the current state of EV research to facilitate robust scientific discoveries and move the field forward even more rapidly.

Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches

Current update on nanoplatforms as therapeutic and diagnostic tools: A review for the materials used as nanotheranostics and imaging modalities.

Recently, antimicrobial peptides (AMPs), also called host defence peptides (HDPs), are attracting great interest, as they are a highly viable alternative in the search of new approaches to the resistance presented by bacteria against antibiotics in infectious diseases. However, due to their nature, they present a series of disadvantages such as low bioavailability, easy degradability by proteases, or low solubility, among others, which limits their use as antimicrobial agents. For all these reasons, the use of vehicles for the delivery of AMPs, such as polymers, nanoparticles, micelles, carbon nanotubes, dendrimers, and other types of systems, allows the use of AMPs as a real alternative to treatment with antibiotics.

Nanosystems as Vehicles for the Delivery of Antimicrobial Peptides (AMPs)

Abstract It has been demonstrated recently that extracellular vesicles (EVs) carry DNA; however, many fundamental features of DNA in EVs (EV‐DNA) remain elusive. In this study, a laboratory‐built nano‐flow cytometer (nFCM) that can detect single EVs as small as 40 nm in diameter and single DNA fragments of 200 bp upon SYTO 16 staining was used to study EV‐DNA at the single‐vesicle level. Through simultaneous side‐scatter and fluorescence (FL) detection of single particles and with the combination of enzymatic treatment, present study revealed that: (1) naked DNA or DNA associated with non‐vesicular entities is abundantly presented in EV samples prepared from cell culture medium by ultracentrifugation; (2) the quantity of EV‐DNA in individual EVs exhibits large heterogeneity and the population of DNA positive (DNA+) EVs varies from 30% to 80% depending on the cell type; (3) external EV‐DNA is mainly localized on relatively small size EVs (e.g. <100 nm for HCT‐15 cell line) and the secretion of external DNA+ EVs can be significantly reduced by exosome secretion pathway inhibition; (4) internal EV‐DNA is mainly packaged inside the lumen of relatively large EVs (e.g. 80–200 nm for HCT‐15 cell line); (5) double‐stranded DNA (dsDNA) is the predominant form of both the external and internal EV‐DNA; (6) histones (H3) are not found in EVs, and EV‐DNA is not associated with histone proteins and (7) genotoxic drug induces an enhanced release of DNA+ EVs, and the number of both external DNA+ EVs and internal DNA+ EVs as well as the DNA content in single EVs increase significantly. This study provides direct and conclusive experimental evidence for an in‐depth understanding of how DNA is associated with EVs.

Analysis of extracellular vesicle DNA at the single‐vesicle level by nano‐flow cytometry

Abstract Flow cytometry (FCM) offers a multiparametric technology capable of characterizing single extracellular vesicles (EVs). However, most flow cytometers are designed to detect cells, which are larger than EVs. Whereas cells exceed the background noise, signals originating from EVs partly overlap with the background noise, thereby making EVs more difficult to detect than cells. This technical mismatch together with complexity of EV‐containing fluids causes limitations and challenges with conducting, interpreting and reproducing EV FCM experiments. To address and overcome these challenges, researchers from the International Society for Extracellular Vesicles (ISEV), International Society for Advancement of Cytometry (ISAC), and the International Society on Thrombosis and Haemostasis (ISTH) joined forces and initiated the EV FCM working group. To improve the interpretation, reporting, and reproducibility of future EV FCM data, the EV FCM working group published an ISEV position manuscript outlining a framework of minimum information that should be reported about an FCM experiment on single EVs (MIFlowCyt‐EV). However, the framework contains limited background information. Therefore, the goal of this compendium is to provide the background information necessary to design and conduct reproducible EV FCM experiments. This compendium contains background information on EVs, the interaction between light and EVs, FCM hardware, experimental design and preanalytical procedures, sample preparation, assay controls, instrument data acquisition and calibration, EV characterization, and data reporting. Although this compendium focuses on EVs, many concepts and explanations could also be applied to FCM detection of other particles within the EV size range, such as bacteria, lipoprotein particles, milk fat globules, and viruses.

https://onlinelibrary.wiley.com/doi/pdfdirect/10.1002/jev2.12299

A compendium of single extracellular vesicle flow cytometry

Active targeting has been hailed as one of the most promising strategies to further enhance the therapeutic efficacy of liposomal nanomedicines. Owing to the critical role of ligand density in mediating cellular uptake and the intrinsic heterogeneity of liposomal formulations, precise quantification of the surface ligand density on a single-particle basis is of fundamental importance. In this work, we report a method to simultaneously measure the particle size and the number of ligands on the same liposomal nanoparticles by nanoflow cytometry. Then the ligand density for each individual liposome can be determined. With an analysis rate up to 10 000 particles per minute, a statistically representative distribution of ligand density could be determined in minutes. By utilizing fluorescently labeled recombinant receptors as the detection probe against the conjugated ligands, only those available for cell targeting can be exclusively detected. The influence of ligand input, conjugation strategy, and the polyethylene glycol spacer length on the available ligand density of folate-modified liposomes was investigated. The correlation between the available ligand density and cell targeting capability was assessed in a quantitative perspective for liposomes modified with three different targeting moieties. The optimal ligand density was determined to be 0.5-2.0, 0.7, and 0.2 ligand per 100 nm2 for folate-, transferrin-, and HER2-antibody-conjugated liposomes, respectively. These optimal values agreed well with the spike density of the natural counterparts, viruses. The as-developed approach is generally applicable to a wide range of active-targeting nanocarriers.

Quantification of Available Ligand Density on the Surface of Targeted Liposomal Nanomedicines at the Single-Particle Level.

Single-particle characterization of theranostic liposomes with stimulus sensing and controlled drug release properties.

Lipid-based nanomedicines (LBNMs), including liposomes, lipid nanoparticles (LNPs) and extracellular vesicles (EVs), are recognized as one of the most clinically acceptable nano-formulations. However, the bench-to-bedside translation efficiency is far from satisfactory, mainly due to the lack of in-depth understanding of their physical and biochemical attributes at the single-particle level. In this review, we first give a brief introduction of LBNMs, highlighting some milestones and related scientific and clinical achievements in the past several decades, as well as the grand challenges in the characterization of LBNMs. Next, we present an overview of each category of LBNMs as well as the core properties that largely dictate their biological characteristics and clinical performance, such as size distribution, particle concentration, morphology, drug encapsulation and surface properties. Then, the recent applications of several analytical techniques including electron microscopy, atomic force microscopy, fluorescence microscopy, Raman microscopy, nanoparticle tracking analysis, tunable resistive pulse sensing and flow cytometry on the single-particle characterization of LBNMs are thoroughly discussed. Particularly, the comparative advantages of the newly developed nano-flow cytometry that enables quantitative analysis of both the physical and biochemical characteristics of LBNMs smaller than 40 nm with high throughput and statistical robustness are emphasized. The overall aim of this review article is to illustrate the importance, challenges and achievements associated with single-particle characterization of LBNMs. 

Characterization of lipid-based nanomedicines at the single-particle level

Although lipophilic membrane dyes (LMDs) or probes (LMPs) are widely used to label extracellular vesicles (EVs) for detection and purification, their labelling performance has not been systematically characterized. Through concurrent side scattering and fluorescence detection of single EVs as small as 40 nm in diameter by a laboratory-built nano-flow cytometer (nFCM), present study identified that (1) PKH67 and PKH26 could maximally label ∼60%-80% of EVs isolated from the conditioned cell culture medium (purity of ∼88%) and ∼40%-70% of PFP-EVs (purity of ∼73%); (2) excessive PKH26 could cause damage to the EV structure; (3) di-8-ANEPPS and high concentration of DiI could achieve efficient and uniform labelling of EVs with nearly 100% labelling efficiency for di-8-ANEPPS and 70%-100% for DiI; (4) all the four tested LMDs can aggregate and form micelles that exhibit comparable side scatter and fluorescence intensity with those of labelled EVs and thus hardly be differentiate from each other; (5) as the LMD concentration went up, the particle number of self-aggregates increased while the fluorescence intensity of aggregates remained constant; (6) PKH67 and PKH26 tend to form more aggregated micelles than di-8-ANEPPS and DiI, and the effect of LMD self-aggregation can be negligible at optimal staining conditions. (7) All the four tested LMDs can label almost all the very-low-density lipoprotein (VLDL) particles, indicating potential confounding factor in plasma-EV labelling. Besides, it was discovered that DSPE-PEG2000 -biotin can only label ∼50% of plasma-EVs. The number of LMP inserted into the membrane of single EVs was measured for the first time and it was confirmed that membrane labelling by lipophilic dyes did not interfere with the immunophenotyping of EVs. nFCM provides a unique perspective for a better understanding of EV labelling by LMD/LMP. 

Quantitative assessment of lipophilic membrane dye‐based labelling of extracellular vesicles by nano‐flow cytometry

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