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The terms microparticles (MPs) and microvesicles (MVs) refer to large extracellular vesicles (EVs) generated from a broad spectrum of cells upon its activation or death by apoptosis. The unique surface antigens of MPs/MVs allow for the identification of their cellular origin as well as its functional characterization. Two basic aspects of MP/MV functions in physiology and pathological conditions are widely considered. Firstly, it has become evident that large EVs have strong procoagulant properties. Secondly, experimental and clinical studies have shown that MPs/MVs play a crucial role in the pathophysiology of inflammation-associated disorders. A cardinal feature of these disorders is an enhanced generation of platelets-, endothelial-, and leukocyte-derived EVs. Nevertheless, anti-inflammatory effects of miscellaneous EV types have also been described, which provided important new insights into the large EV-inflammation axis. Advances in understanding the biology of MPs/MVs have led to the preparation of this review article aimed at discussing the association between large EVs and inflammation, depending on their cellular origin.

The enormous library of pharmaceutical compounds presents endless research avenues. However, several factors limit the therapeutic potential of these drugs, such as drug resistance, stability, off-target toxicity, and inadequate delivery to the site of action. Extracellular vesicles (EVs) are lipid bilayer-delimited particles and are naturally released from cells. Growing evidence shows that EVs have great potential to serve as effective drug carriers. Since EVs can not only transfer biological information, but also effectively deliver hydrophobic drugs into cells, the application of EVs as a novel drug delivery system has attracted considerable scientific interest. Recently, EVs loaded with siRNA, miRNA, mRNA, CRISPR/Cas9, proteins, or therapeutic drugs show improved delivery efficiency and drug effect. In this review, we summarize the methods used for the cargo loading into EVs, including siRNA, miRNA, mRNA, CRISPR/Cas9, proteins, and therapeutic drugs. Furthermore, we also include the recent advance in engineered EVs for drug delivery. Finally, both advantages and challenges of EVs as a new drug delivery system are discussed. Here, we encourage researchers to further develop convenient and reliable loading methods for the potential clinical applications of EVs as drug carriers in the future.

Exosomes are small extracellular vesicles with diameters of 30-150 nm. In both physiological and pathological conditions, nearly all types of cells can release exosomes, which play important roles in cell communication and epigenetic regulation by transporting crucial protein and genetic materials such as miRNA, mRNA, and DNA. Consequently, exosome-based disease diagnosis and therapeutic methods have been intensively investigated. However, as in any natural science field, the in-depth investigation of exosomes relies heavily on technological advances. Historically, the two main technical hindrances that have restricted the basic and applied researches of exosomes include, first, how to simplify the extraction and improve the yield of exosomes and, second, how to effectively distinguish exosomes from other extracellular vesicles, especially functional microvesicles. Over the past few decades, although a standardized exosome isolation method has still not become available, a number of techniques have been established through exploration of the biochemical and physicochemical features of exosomes. In this work, by comprehensively analyzing the progresses in exosome separation strategies, we provide a panoramic view of current exosome isolation techniques, providing perspectives toward the development of novel approaches for high-efficient exosome isolation from various types of biological matrices. In addition, from the perspective of exosome-based diagnosis and therapeutics, we emphasize the issue of quantitative exosome and microvesicle separation.

Abstract During the last decade, there has been great interest in elucidating the biological role of extracellular vesicles (EVs), particularly, their hormone-like role in cell-to-cell communication. The field of endocrinology is uniquely placed to provide insight into the functions of EVs, which are secreted from all cells into biological fluids and carry endocrine signals to engage in paracellular and distal interactions. EVs are a heterogeneous population of membrane-bound vesicles of varying size, content, and bioactivity. EVs are specifically packaged with signaling molecules, including lipids, proteins, and nucleic acids, and are released via exocytosis into biofluid compartments. EVs regulate the activity of both proximal and distal target cells, including translational activity, metabolism, growth, and development. As such, EVs signaling represents an integral pathway mediating intercellular communication. Moreover, as the content of EVs is cell-type specific, it is a “fingerprint” of the releasing cell and its metabolic status. Recently, changes in the profile of EV and bioactivity have been described in several endocrine-related conditions including diabetes, obesity, cardiovascular diseases, and cancer. The goal of this statement is to highlight relevant aspects of EV research and their potential role in the field of endocrinology.

Intercellular communication was long thought to be regulated exclusively through direct contact between cells or via release of soluble molecules that transmit the signal by binding to a suitable receptor on the target cell, and/or via uptake into that cell. With the discovery of small secreted vesicular structures that contain complex cargo, both in their lumen and the lipid membrane that surrounds them, a new frontier of signal transduction was discovered. These “extracellular vesicles” (EV) were initially thought to be garbage bags through which the cell ejected its waste. Whilst this is a major function of one type of EV, i.e., apoptotic bodies, many EVs have intricate functions in intercellular communication and compound exchange; although their physiological roles are still ill-defined. Additionally, it is now becoming increasingly clear that EVs mediate disease progression and therefore studying EVs has ignited significant interests among researchers from various fields of life sciences. Consequently, the research effort into the pathogenic roles of EVs is significantly higher even though their protective roles are not well established. The “Focus on extracellular vesicles” series of reviews highlights the current state of the art regarding various topics in EV research, whilst this review serves as an introductory overview of EVs, their biogenesis and molecular composition.

Exosomes have been proposed as vehicles for microRNA (miRNA) -based intercellular communication and a source of miRNA biomarkers in bodily fluids. Although exosome preparations contain miRNAs, a quantitative analysis of their abundance and stoichiometry is lacking. In the course of studying cancer-associated extracellular miRNAs in patient blood samples, we found that exosome fractions contained a small minority of the miRNA content of plasma. This low yield prompted us to perform a more quantitative assessment of the relationship between miRNAs and exosomes using a stoichiometric approach. We quantified both the number of exosomes and the number of miRNA molecules in replicate samples that were isolated from five diverse sources (i.e., plasma, seminal fluid, dendritic cells, mast cells, and ovarian cancer cells). Regardless of the source, on average, there was far less than one molecule of a given miRNA per exosome, even for the most abundant miRNAs in exosome preparations (mean ± SD across six exosome sources: 0.00825 ± 0.02 miRNA molecules/exosome). Thus, if miRNAs were distributed homogenously across the exosome population, on average, over 100 exosomes would need to be examined to observe one copy of a given abundant miRNA. This stoichiometry of miRNAs and exosomes suggests that most individual exosomes in standard preparations do not carry biologically significant numbers of miRNAs and are, therefore, individually unlikely to be functional as vehicles for miRNA-based communication. We propose revised models to reconcile the exosome-mediated, miRNA-based intercellular communication hypothesis with the observed stoichiometry of miRNAs associated with exosomes.

Increasing evidence indicates that the ability of cancer cells to convey biological information to recipient cells within the tumor microenvironment (TME) is crucial for tumor progression. Microvesicles (MVs) are heterogenous vesicles formed by budding of the cellular membrane, which are secreted in larger amounts by cancer cells than normal cells. Recently, several reports have also disclosed that MVs function as important mediators of intercellular communication between cancerous and stromal cells within the TME, orchestrating complex pathophysiological processes. Chemokines are a family of small inflammatory cytokines that are able to induce chemotaxis in responsive cells. MVs which selective incorporate chemokines as their molecular cargos may play important regulatory roles in oncogenic processes including tumor proliferation, apoptosis, angiogenesis, metastasis, chemoresistance and immunomodulation, et al. Therefore, it is important to explore the association of MVs and chemokines in TME, identify the potential prognostic marker of tumor, and develop more effective treatment strategies. Here we review the relevant literature regarding the role of MVs and chemokines in TME.

BackgroundAtherosclerotic plaque formation is the underlying cause of heart attack and stroke. Macrophages play a key role in plaque progression. Neutrophils are rarely detected in plaques but have been shown to play an integral role in plaque development. We have previously shown that microvesicles released from stimulated neutrophils are present in plaques and enhance plaque formation. Neutrophil microvesicles (NMV) have been found to modulate macrophage activity in atherosclerosis and are known to contain microRNA that can influence macrophage behaviour. We hypothesise that NMV can interact with macrophages within atherosclerotic plaques and alter macrophage phenotype and function.MethodsTo determine whether NMV can cross the endothelium, human coronary artery endothelial cells (HCAEC) were cultured ± TNF on transwell inserts. NMV were stained with PKH lipophilic membrane dye and added to the upper compartment of the transwells at ratios of 1:10, 1:50, and 1:100 HCAEC: NMV. After 24h NMV in the basal chamber were visualised using fluorescent microscopy and quantified using Fiji. To investigate NMV internalisation by macrophages, M0 human monocyte-derived macrophages (HMDM) were treated at a ratio of 1:100 HMDM:PKH stained NMV for periods of 24, 6 and 1h. Following treatment, NMV internalisation was quantified by flow cytometry and visualised using confocal microscopy. Trypan blue was used to quench surface fluorescence and distinguish between adherent and internalised NMV. Modulation of macrophage polarisation by NMV was assessed by RT- qPCR analysis of CD68, CD86, MRC1 and CD163 expression after 24h.ResultsNMV were able to cross the endothelium in a dose-dependent manner. NMV also interacted with both stimulated and unstimulated HCAECs. NMV interaction with HMDM occurred rapidly with approximately 10% HMDM being identified as PKH+ve after 1h. Internalization of NMVs by HMDMs increased over time with approximately 64% of PKH+ve cells containing NMV after 24h. However, incubation of HMDM with NMVs did not result in any significant changes in polarisation marker expression after 24h.ConclusionsNMV can cross the endothelial monolayer and interact with macrophages. They are internalised by macrophages but are not able to induce changes in polarisation in the absence of other polarising stimuli. Further investigation is underway to elucidate the mechanisms of NMV internalisation and determine whether NMV can act synergistically with cytokines present in the plaque to influence macrophage polarisation.Conflict of InterestNone

Introduction Atherosclerosis is the major underlying cause of heart attack and stroke and, as such, understanding the underlying pathological mechanisms of the disease remains a priority. Whilst neutrophils are the most abundant circulating leukocyte, they are rarely detected within developing plaques. Neutrophil microvesicles (NMVs), large (>0.1µm) extracellular vesicles derived from the plasma membrane, were shown to increase miR-155 in endothelial cells (ECs) at atheroprone sites. This led to exacerbation of plaque formation in a mouse model of atherosclerosis. We hypothesise that NMVs contain other microRNA (miRNA) that may influence atherosclerosis initiation and progression.MethodsNMV were isolated from human peripheral blood neutrophils that were stimulated with 10 ng/mL native (n)LDL, 10 ng/mL oxidised (ox)LDL, or PBS only (unstimulated control). Small RNAs were isolated from NMV using miRNeasy mini kit (Qiagen, Germany) and quantity and quality of isolated RNA determined using NanoPhotometer® N60 spectrophotometer (Geneflow, UK) and a 2100 Bioanalyser (Agilent, UK). Small RNA sequencing analysis was performed using Illumina sequencing platform by Novogen, UK. Sequencing data was processed to obtain the clean reads that aligned to the human reference genome. Data was further processed and visualised using R software.Results757 miRNAs were detected in NMVs. Of these, 527 miRNAs were expressed in all NMV groups, 55 uniquely expressed in NMVs from n/oxLDL stimulated neutrophils and 36 only in unstimulated control NMVs. The most abundant miRNA in all samples was miR-148a-3p, a miRNA previously shown to enhance plaque formation. High levels of miR-155 were also detected in all samples.ConclusionNMVs have been found to contain a high abundance of miRNAs, with some expression dependent on the stimulus used for induction of MV release. Target and pathway analysis of these data are ongoing. miRNA previously shown to play a role in plaque formation were among the most highly abundant within NMV suggesting that NMV delivery of miRNA to atherosclerotic plaques may play an important role in exacerbating plaque formation.Conflict of InterestThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this poster.

Extracellular vesicles (EVs) are cell-derived membranous vesicles secreted by cells into the extracellular space, which play a role in cell to cell communication. EVs are categorized into 3 groups depending on their size, surface marker, and method of release from the host cell. Recently, EVs have become of interest in the study of multiple disease etiologies and are believed to be potential biomarkers for many diseases. Multiple different methods have been developed to isolate EVs from different samples such as cell culture medium, serum, blood, and urine. Once isolated, EVs can be characterized by technology such as nanotracking analysis, dynamic light scattering, and nanoscale flow cytometry. In this review, we summarize the current methods of EV isolation, provide details into the three methods of EV characterization, and provide insight into which isolation approaches are most suitable for EV isolation from bronchoalveolar lavage fluid (BALF).