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Extracellular vesicles are lipid-bilayer-enclosed nanoparticles present in the majority of biological fluids that mediate intercellular communication. EVs are able to transfer their contents (including nucleic acids, proteins, lipids, and small molecules) to recipient cells, and thus hold great promise as drug delivery vehicles. However, their therapeutic application is limited by lack of efficient cargo loading strategies, a need to improve EV tissue-targeting capabilities and a requirement to improve escape from the endolysosomal system. These challenges can be effectively addressed by modifying EVs with peptides which confer specific advantageous properties, thus enhancing their therapeutic potential. Here we provide an overview of the applications of peptide technology with respect to EV therapeutics. We focus on the utility of EV-modifying peptides for the purposes of promoting cargo loading, tissue-targeting and endosomal escape, leading to enhanced delivery of the EV cargo to desired cells/tissues and subcellular target locations. Both endogenous and exogenous methods for modifying EVs with peptides are considered., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Extracellular vesicles (EVs) are biological nanoparticles with important roles in intercellular communication, and potential as drug delivery vehicles. Here we demonstrate a role for the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in EV assembly and secretion. We observe high levels of GAPDH binding to the outer surface of EVs via a phosphatidylserine binding motif (G58), which promotes extensive EV clustering. Further studies in a Drosophila EV biogenesis model reveal that GAPDH is required for the normal generation of intraluminal vesicles in endosomal compartments, and promotes vesicle clustering. Fusion of the GAPDH-derived G58 peptide to dsRNA-binding motifs enables highly efficient loading of small interfering RNA (siRNA) onto the EV surface. Such vesicles efficiently deliver siRNA to multiple anatomical regions of the brain in a Huntington's disease mouse model after systemic injection, resulting in silencing of the huntingtin gene in different regions of the brain., (© 2021. The Author(s).)

Cell plasticity is a crucial hallmark leading to cancer metastasis. Upregulation of Rho/ROCK pathway drives actomyosin contractility, protrusive forces, and contributes to the occurrence of highly invasive amoeboid cells in tumors. Cancer stem cells are similarly associated with metastasis, but how these populations arise in tumors is not fully understood. Here, we show that the novel oncogene RASSF1C drives mesenchymal-to-amoeboid transition and stem cell attributes in breast cancer cells. Mechanistically, RASSF1C activates Rho/ROCK via SRC-mediated RhoGDI inhibition, resulting in generation of actomyosin contractility. Moreover, we demonstrate that RASSF1C-induced amoeboid cells display increased expression of cancer stem-like markers such as CD133, ALDH1, and Nanog, and are accompanied by higher invasive potential in vitro and in vivo. Further, RASSF1C-induced amoeboid cells employ extracellular vesicles to transfer the invasive phenotype to target cells and tissue. Importantly, the underlying RASSF1C-driven biological processes concur to explain clinical data: namely, methylation of the RASSF1C promoter correlates with better survival in early-stage breast cancer patients. Therefore, we propose the use of RASSF1 gene promoter methylation status as a biomarker for patient stratification., (©2021 The Authors. Published under the terms of the CC BY 4.0 license.)

Extracellular vesicles (EVs) can be functionalized to display specific protein receptors on their surface. However, surface-display technology typically labels only a small fraction of the EV population. Here, we show that the joint display of two different therapeutically relevant protein receptors on EVs can be optimized by systematically screening EV-loading protein moieties. We used cytokine-binding domains derived from tumour necrosis factor receptor 1 (TNFR1) and interleukin-6 signal transducer (IL-6ST), which can act as decoy receptors for the pro-inflammatory cytokines tumour necrosis factor alpha (TNF-α) and IL-6, respectively. We found that the genetic engineering of EV-producing cells to express oligomerized exosomal sorting domains and the N-terminal fragment of syntenin (a cytosolic adaptor of the single transmembrane domain protein syndecan) increased the display efficiency and inhibitory activity of TNFR1 and IL-6ST and facilitated their joint display on EVs. In mouse models of systemic inflammation, neuroinflammation and intestinal inflammation, EVs displaying the cytokine decoys ameliorated the disease phenotypes with higher efficacy as compared with clinically approved biopharmaceutical agents targeting the TNF-α and IL-6 pathways., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Extracellular-vesicle-based cell-to-cell communication is conserved across all kingdoms of life. There is compelling evidence that extracellular vesicles are involved in major (patho)physiological processes, including cellular homoeostasis, infection propagation, cancer development and cardiovascular diseases. Various studies suggest that extracellular vesicles have several advantages over conventional synthetic carriers, opening new frontiers for modern drug delivery. Despite extensive research, clinical translation of extracellular-vesicle-based therapies remains challenging. Here, we discuss the uniqueness of extracellular vesicles along with critical design and development steps required to utilize their full potential as drug carriers, including loading methods, in-depth characterization and large-scale manufacturing. We compare the prospects of extracellular vesicles with those of the well established liposomes and provide guidelines to direct the process of developing vesicle-based drug delivery systems., (© 2021. Springer Nature Limited.)

The extracellular environment consists of a plethora of molecules, including extracellular miRNA that can be secreted in association with extracellular vesicles (EVs) or soluble protein complexes (non-EVs). Yet, interest in therapeutic short RNA carriers lies mainly in EVs, the vehicles conveying the great majority of the biological activity. Here, by overexpressing miRNA and shRNA sequences in parent cells and using size exclusion liquid chromatography (SEC) to separate the secretome into EV and non-EV fractions, we saw that >98% of overexpressed miRNA was secreted within the non-EV fraction. Furthermore, small RNA sequencing studies of native miRNA transcripts revealed that although the abundance of miRNAs in EVs, non-EVs and parent cells correlated well (R 2 = 0.69-0.87), quantitatively an outstanding 96.2-99.9% of total miRNA was secreted in the non-EV fraction. Nevertheless, though EVs contained only a fraction of secreted miRNAs, these molecules were stable at 37 °C in a serum-containing environment, indicating that if sufficient miRNA loading is achieved, EVs can remain delivery-competent for a prolonged period of time. This study suggests that the passive endogenous EV loading strategy might be a relatively wasteful way of loading miRNA to EVs, and active miRNA loading approaches are needed for developing advanced EV miRNA therapies in the future.

The cytokine interleukin 6 (IL6) is a key mediator of inflammation that contributes to skeletal muscle pathophysiology. IL6 activates target cells by two main mechanisms, the classical and trans-signalling pathways. While classical signalling is associated with the anti-inflammatory activities of the cytokine, the IL6 trans-signalling pathway mediates chronic inflammation and is therefore a target for therapeutic intervention. Extracellular vesicles (EVs) are natural, lipid-bound nanoparticles, with potential as targeted delivery vehicles for therapeutic macromolecules. Here, we engineered EVs to express IL6 signal transducer (IL6ST) decoy receptors to selectively inhibit the IL6 trans-signalling pathway. The potency of the IL6ST decoy receptor EVs was optimized by inclusion of a GCN4 dimerization domain and a peptide sequence derived from syntenin-1 which targets the decoy receptor to EVs. The resulting engineered EVs were able to efficiently inhibit activation of the IL6 trans-signalling pathway in reporter cells, while having no effect on the IL6 classical signalling. IL6ST decoy receptor EVs, were also capable of blocking the IL6 trans-signalling pathway in C2C12 myoblasts and myotubes, thereby inhibiting the phosphorylation of STAT3 and partially reversing the anti-differentiation effects observed when treating cells with IL6/IL6R complexes. Treatment of a Duchenne muscular dystrophy mouse model with IL6ST decoy receptor EVs resulted in a reduction in STAT3 phosphorylation in the quadriceps and gastrocnemius muscles of these mice, thereby demonstrating in vivo activity of the decoy receptor EVs as a potential therapy. Taken together, this study reveals the IL6 trans-signalling pathway as a promising therapeutic target in DMD, and demonstrates the therapeutic potential of IL6ST decoy receptor EVs., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Extracellular vesicles (EVs) form an endogenous transport system for intercellular transfer of biological cargo, including RNA, that plays a pivotal role in physiological and pathological processes. Unfortunately, whereas biological effects of EV-mediated RNA transfer are abundantly studied, regulatory pathways and mechanisms remain poorly defined due to a lack of suitable readout systems. Here, we describe a highly-sensitive CRISPR-Cas9-based reporter system that allows direct functional study of EV-mediated transfer of small non-coding RNA molecules at single-cell resolution. Using this CRISPR operated stoplight system for functional intercellular RNA exchange (CROSS-FIRE) we uncover various genes involved in EV subtype biogenesis that play a regulatory role in RNA transfer. Moreover we identify multiple genes involved in endocytosis and intracellular membrane trafficking that strongly regulate EV-mediated functional RNA delivery. Altogether, this approach allows the elucidation of regulatory mechanisms in EV-mediated RNA transfer at the level of EV biogenesis, endocytosis, intracellular trafficking, and RNA delivery.

Multiple studies have described extracellular microRNAs (ex-miRNAs) as being remarkably stable despite the hostile extracellular environment, when stored at 4ºC or lower. Here we show that many ex-miRNAs are rapidly degraded when incubated at 37ºC in the presence of serum (thereby simulating physiologically relevant conditions). Stability varied widely between miRNAs, with half-lives ranging from ~1.5 hours to more than 13 hours. Notably, ex-miRNA half-lives calculated in two different biofluids (murine serum and C2C12 mouse myotube conditioned medium) were highly similar, suggesting that intrinsic sequence properties are a determining factor in miRNA stability. By contrast, ex-miRNAs associated with extracellular vesicles (isolated by size exclusion chromatography) were highly stable. The release of ex-miRNAs from C2C12 myotubes was measured over time, and mathematical modelling revealed miRNA-specific release kinetics. While some ex-miRNAs reached the steady state in cell culture medium within 24 hours, the extracellular level of miR-16 did not reach equilibrium, even after 3 days in culture. These findings are indicative of miRNA-specific release and degradation kinetics with implications for the utility of ex-miRNAs as biomarkers, and for the potential of ex-miRNAs to transfer gene regulatory information between cells.

Extracellular vesicles (EVs) are increasingly being recognized as mediators of intercellular signaling via the delivery of effector molecules. Interestingly, certain types of EVs are also capable of inducing therapeutic responses. For these reasons, the therapeutic potential of EVs is a topic of intense research, both in the context of drug delivery and regenerative medicine. However, to fully utilize EVs for therapeutic purposes, an improved understanding of the mechanisms by which they function would be highly advantageous. Here, the current state of knowledge regarding the cellular uptake and trafficking of EVs is reviewed, along with a consideration of how these pathways potentially influence the functions of therapeutic EVs. Furthermore, the natural cell-targeting abilities, biodistribution profiles, and pharmacokinetics of exogenously administered EVs, along with the components responsible for these features are discussed. An overview of the potential clinical applications and preclinical examples of their successful use is also provided. Finally, examples of EV modifications that have successfully been employed to improve their therapeutic characteristics receive a particular focus. We suggest that, in addition to investigation of EV cell targeting and routes of uptake, future research into the routes of intracellular trafficking in recipient cells is required to optimally utilize EVs for therapeutic purposes.

Cerebrospinal fluid (CSF) extracellular vesicles (EVs) show promise as a source of neurological disease biomarkers, although their precise origin is poorly understood. Current extraction techniques produce disappointing yield and purity. This study describes the application of ultrafiltration LC (UFLC) to CSF-EVs, compared with ultracentrifugation (UC), and explores CSF-EV origin. EVs are extracted from human CSF by UC and UFLC and characterized using nanoparticle tracking analysis, electron microscopy, and immunoblotting. EV and CSF proteomes are analyzed by LC-MS/MS. UFLC-isolated particles have size, morphology, and marker expression characteristic of EVs. UFLC provides greater EV yield (UFLC 7.90 × 10 8  ± SD 1.31 × 10 8 EVs mL -1 CSF, UC 1.06 × 10 8  ± 0.57 × 10 8 p < 0.001). UFLC enhances purity, proteomic depth (UFLC 622 ± 49, UC 298 ± 50, p = 0.001), and consistency of quantification (CV 17% vs 23%). EVs contain more intracellular proteins (Odds ratio [OR] 2.63 p < 0.001) and fewer plasma proteins than CSF (OR 0.60, p < 0.001). CSF and EV-enriched proteomes show overrepresentation of brain-specific proteins (EV OR 3.18, p < 0.001; CSF OR 3.37, p < 0.001). Overrepresentation of cerebral white matter (OR 1.99, p = 0.015) and choroid plexus proteins (OR 1.87, p<0.001) is observed in EVs. UFLC improves yield and purity of CSF-EVs. The EV-enriched proteome better reflects the intracellular and white matter proteome than whole CSF., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Extracellular vesicles (EVs) mediate cell-to-cell communication by delivering or displaying macromolecules to their recipient cells. While certain broad-spectrum EV effects reflect their protein cargo composition, others have been attributed to individual EV-loaded molecules such as specific miRNAs. In this work, we have investigated the contents of vesicular cargo using small RNA sequencing of cells and EVs from HEK293T, RD4, C2C12, Neuro2a and C17.2. The majority of RNA content in EVs (49-96%) corresponded to rRNA-, coding- and tRNA fragments, corroborating with our proteomic analysis of HEK293T and C2C12 EVs which showed an enrichment of ribosome and translation-related proteins. On the other hand, the overall proportion of vesicular small RNA was relatively low and variable (2-39%) and mostly comprised of miRNAs and sequences mapping to piRNA loci. Importantly, this is one of the few studies, which systematically links vesicular RNA and protein cargo of vesicles. Our data is particularly useful for future work in unravelling the biological mechanisms underlying vesicular RNA and protein sorting and serves as an important guide in developing EVs as carriers for RNA therapeutics.

Cells release membrane enclosed nano-sized vesicles termed extracellular vesicles (EVs) that function as mediators of intercellular communication by transferring biological information between cells. Tumor-derived EVs have emerged as important mediators in cancer development and progression, mainly through transfer of their bioactive content which can include oncoproteins, oncogenes, chemokine receptors, as well as soluble factors, transcripts of proteins and miRNAs involved in angiogenesis or inflammation. This transfer has been shown to influence the metastatic behavior of primary tumors. Moreover, tumor-derived EVs have been shown to influence distant cellular niches, establishing favorable microenvironments that support growth of disseminated cancer cells upon their arrival at these pre-metastatic niches. It is generally accepted that cells release a number of major EV populations with distinct biophysical properties and biological functions. Exosomes, microvesicles, and apoptotic bodies are EV populations most widely studied and characterized. They are discriminated based primarily on their intracellular origin. However, increasing evidence suggests that even within these EV populations various subpopulations may exist. This heterogeneity introduces an extra level of complexity in the study of EV biology and function. For example, EV subpopulations could have unique roles in the intricate biological processes underlying cancer biology. Here, we discuss current knowledge regarding the role of subpopulations of EVs in cancer development and progression and highlight the relevance of EV heterogeneity. The position of tetraspanins and integrins therein will be highlighted. Since addressing EV heterogeneity has become essential for the EV field, current and novel techniques for isolating EV subpopulations will also be discussed. Further dissection of EV heterogeneity will advance our understanding of the critical roles of EVs in health and disease.

Extracellular vesicles (EVs) play a pivotal role in cell-to-cell communication and have been shown to take part in several physiological and pathological processes. EVs have traditionally been purified by ultracentrifugation (UC), however UC has limitations, including resulting in, operator-dependant yields, EV aggregation and altered EV morphology, and moreover is time consuming. Here we show that commercially available bind-elute size exclusion chromatography (BE-SEC) columns purify EVs with high yield (recovery ~ 80%) in a time-efficient manner compared to current methodologies. This technique is reproducible and scalable, and surface marker analysis by bead-based flow cytometry revealed highly similar expression signatures compared with UC-purified samples. Furthermore, uptake of eGFP labelled EVs in recipient cells was comparable between BE-SEC and UC samples. Hence, the BE-SEC based EV purification method represents an important methodological advance likely to facilitate robust and reproducible studies of EV biology and therapeutic application.

Extracellular vesicles (EVs) are cell-derived, membranous nanoparticles that mediate intercellular communication by transferring biomolecules, including proteins and RNA, between cells. As a result of their suggested natural capability to functionally deliver RNA, EVs may be harnessed as therapeutic RNA carriers. One major limitation for their translation to therapeutic use is the lack of an efficient, robust, and scalable method to load EVs with RNA molecules of interest. Here, we evaluated and optimized methods to load EVs with cholesterol-conjugated small interfering RNAs (cc-siRNAs) by systematic evaluation of the influence of key parameters, including incubation time, volume, temperature, and EV:cc-siRNA ratio. EV loading under conditions that resulted in the highest siRNA retention percentage, incubating 15 molecules of cc-siRNA per EV at 37°C for 1 hr in 100 μL, facilitated concentration-dependent silencing of human antigen R (HuR), a therapeutic target in cancer, in EV-treated cells. These results may accelerate the development of EV-based therapeutics., (Copyright © 2017 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

RNA interference (RNAi) has tremendous potential for specific silencing of disease-causing genes. Its clinical usage however critically depends on the development of carrier systems that can transport the RNAi-mediating small interfering RNA (siRNA) molecules to the cytosol of target cells. Recent reports have suggested that extracellular vesicles (EVs) form a natural transport system through which biomolecules, including RNA, is exchanged between cells. Therefore, EVs are increasingly being considered as potential therapeutic siRNA delivery systems.In this chapter we describe a method for preparing siRNA-loaded EVs, including a robust, scalable method to isolate them from cell culture supernatants.

To develop effective disease-modifying therapies for neurodegenerative diseases, reliable markers of diagnosis, disease activity and progression are a research priority. The fact that neurodegenerative pathology is primarily associated with distinct subsets of cells in discrete areas of the CNS makes the identification of relevant biomarker molecules a challenge. The trafficking of macromolecules from the CNS to the cerebrospinal fluid and blood, mediated by extracellular vesicles (EVs), presents a promising source of CNS-specific biomarkers. EVs are released by almost all cell types and carry a cargo of protein and nucleic acid that varies according to the cell of origin. EV output changes with cell status and reflects intracellular events, so surface marker expression can be used to identify the cell type from which EVs originate. EVs could, therefore, provide an enriched pool of information about core neuropathogenic, cell-specific processes. This Review examines the current knowledge of the biology and function of EVs, discusses the evidence for their involvement in the pathogenesis of neurodegenerative diseases, and considers their potential as biomarkers of disease.

Background: Currently, even the most sophisticated methods of assisted reproductive technology (ART) allow us to achieve live births in only approximately 30% of patients, indicating that our understanding of the fine mechanisms underlying reproduction is far from ideal. One of the main challenges associated with studies of gamete structure and function is that these cells are remarkably resistant towards the uptake of exogenous substances, including 'molecular research tools' such as drugs, biomolecules and intracellular markers. This phenomenon can affect not only the performance of reproductive biology research techniques, but also the outcomes of the in vitro handling of gametes, which forms the cornerstone of ART. Improvement of intra-gamete delivery in a non-aggressive fashion is vital for the investigation of gamete physiology, and the advancement of infertility treatment. In this review, we outline the current state of nanomaterial-mediated delivery into gametes and embryos in vitro, and discuss the potential of a novel exciting drug delivery technology, based upon the use of targeted 'natural' nanoparticles known as extracellular vesicles (EVs), for reproductive science and ART, given the promising emerging data from other fields., Methods: A comprehensive electronic search of PubMed and Web of Science databases was performed using the following keywords: 'nanoparticles', 'nanomaterials', 'cell-penetrating peptides', 'sperm', 'oocyte', 'egg', 'embryo', 'exosomes', 'microvesicles', 'extracellular vesicles', 'delivery', 'reproduction', to identify the relevant research and review articles, published in English up to January 2015. The reference lists of identified publication were then scanned to extract additional relevant publications., Results: Biocompatible engineered nanomaterials with high loading capacity, stability and selective affinity represent a potential versatile tool for the minimally invasive internalization of molecular cargo into gametes and embryos. However, it is becoming increasingly clear that the translation of these experimental tools into clinical applications is likely to be limited by their non-biodegradable nature. To allow the subsequent use of these methodologies for clinical ART, studies should utilize biodegradable delivery platforms, which mimic natural mechanisms of molecular cargo trafficking as closely as possible. Currently, EVs represent the most physiological intracellular delivery tools for reproductive science and medicine. These natural mediators of cell communication combine the benefits of engineered nanomaterials, such as the potential for in vitro production, targeting and loading, with the essential feature of biodegradability., Conclusion: We anticipate that future investigations into the possibility of applying EVs for the intentional intracellular delivery of molecular compounds into gametes and embryos will open new horizons for reproductive science and clinical ART, ultimately leading to improvements in patient care., (© The Author 2015. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

The therapeutic potential of mesenchymal stromal cells (MSCs) is evident by the number of new and ongoing trials targeting an impressive variety of conditions. In bone and cartilage repair, MSCs are expected to replace the damaged tissue, while in other therapies they modulate a therapeutic response by the secretion of bioactive molecules. MSCs possess a phenotypic plasticity and harbor an arsenal of bioactive molecules that can be released upon sensing signals in the local milieu either directly or packaged in extracellular vesicles (EVs). The reported paracrine effects comprise many of the important functions of MSCs, including supporting hematopoietic stem cells in the bone marrow, promoting angiogenesis, and modulating the immune system. The major drawback in MSC therapy is the incomplete understanding of cell fate following systemic administration as well as the mechanisms by which these cells correct disease. In this review we discuss what is known about MSC engraftment, hemocompatibility, and immunomodulation, as well as the potential of bringing the MSC-EV field toward a clinical translation.

Extracellular vesicles (EVs) are cell derived membranous nanoparticles. EVs are important mediators of cell–cell communication via the transfer of bioactive content and as such they are being investigated for disease diagnostics as biomarkers and for potential therapeutic cargo delivery to recipient cells. However, existing methods for isolating EVs from biological samples suffer from challenges related to co‐isolation of unwanted materials such as proteins, nucleic acids, and lipoproteins. In the pursuit of improved EV isolation techniques, we introduce multimodal flowthrough chromatography (MFC) as a scalable alternative to size exclusion chromatography (SEC). The use of MFC offers significant advantages for purifying EVs, resulting in enhanced yields and increased purity with respect to protein and nucleic acid co‐isolates from conditioned 3D cell culture media. Compared to SEC, significantly higher EV yields with similar purity and preserved functionality were also obtained with MFC in 2D cell cultures. Additionally, MFC yielded EVs from serum with comparable purity to SEC and similar apolipoprotein B content. Overall, MFC presents an advancement in EV purification yielding EVs with high recovery, purity, and functionality, and offers an accessible improvement to researchers currently employing SEC. [ABSTRACT FROM AUTHOR]

Extracellular vesicles (EVs) are nano-sized particles constitutively released from cells into all biological fluids. Interestingly, these vesicles contain genetic cargoes including proteins, RNA and bioactive lipids that can be functionally delivered and affect recipient cells. As a result, there is growing interest in studying EVs in pathological conditions, including central nervous system (CNS)-related diseases, as EVs may be used for diagnostic purposes or as therapeutic agents. However, one major bottleneck is the need for better EV purification strategies when considering complex biological sources such as serum/protein-rich media or plasma. In this study, we have performed a systematic comparison study between the current gold-standard method: ultracentrifugation, to an alternative: size-exclusion chromatography (LC), using induced pluripotent stem cell (iPSC) derived complex media as a model system. We demonstrate that LC allows for derivation of purer EVs from iPSCs, which was previously impossible with the original UC method. Importantly, our study further highlights the various drawbacks when using the conventional UC approach that lead to misinterpretation of EV data. Lastly, we describe novel data on our iPSC-EVs; how they could relate to stem cell biology and discuss their potential use as EV therapeutics for CNS diseases. [ABSTRACT FROM AUTHOR]

miRNAs are small, noncoding RNAs that not only regulate gene expression within cells, but might also constitute promising extracellular biomarkers for a variety of pathologies, including the progressive muscle-wasting disorder Duchenne Muscular Dystrophy (DMD). A set of muscle-enriched miRNAs, the myomiRs (miR-1, miR-133, and miR-206) are highly elevated in the serum of patients with DMD and in dystrophin-deficient animal models. Furthermore, circulating myomiRs might be used as pharmacodynamic biomarkers, given that their levels can be restored towards wild-type levels following exon skipping therapy in dystrophic mice. The relationship between muscle pathology and extracellular myomiR release is complex, and incompletely understood. Here, we discuss current progress leading towards the clinical utility of extracellular miRNAs as putative DMD biomarkers, and their possible contribution to muscle physiology. [ABSTRACT FROM AUTHOR]

Extracellular vesicles (EVs) are natural nanoparticles that mediate intercellular transfer of RNA and proteins and are of great medical interest; serving as novel biomarkers and potential therapeutic agents. However, there is little consensus on the most appropriate method to isolate high-yield and high-purity EVs from various biological fluids. Here, we describe a systematic comparison between two protocols for EV purification: ultrafiltration with subsequent liquid chromatography (UF-LC) and differential ultracentrifugation (UC). A significantly higher EV yield resulted from UF-LC as compared to UC, without affecting vesicle protein composition. Importantly, we provide novel evidence that, in contrast to UC-purified EVs, the biophysical properties of UF-LC-purified EVs are preserved, leading to a different in vivo biodistribution, with less accumulation in lungs. Finally, we show that UF-LC is scalable and adaptable for EV isolation from complex media types such as stem cell media, which is of huge significance for future clinical applications involving EVs. From the Clinical Editor Recent evidence suggests extracellular vesicles (EVs) as another route of cellular communication. These EVs may be utilized for future therapeutics. In this article, the authors compared ultrafiltration with size-exclusion liquid chromatography (UF-LC) and ultra-centrifugation (UC) for EV recovery. [ABSTRACT FROM AUTHOR]

Extracellular vesicles (EVs) play a significant role in cell–cell communication in numerous physiological processes and pathological conditions, and offer promise as novel biomarkers and therapeutic agents for genetic diseases. Many recent studies have described different molecular mechanisms that contribute to EV biogenesis and release from cells. However, little is known about how external stimuli such as cell culture conditions can affect the quantity and content of EVs. While N2a neuroblastoma cells cultured in serum-free (OptiMEM) conditions did not result in EVs with significant biophysical or size differences compared with cells cultured in serum-containing (pre-spun) conditions, the quantity of isolated EVs was greatly increased. Moreover, the expression levels of certain vesicular proteins (e.g. small GTPases, G-protein complexes, mRNA processing proteins and splicing factors), some of which were previously reported to be involved in EV biogenesis, were found to be differentially expressed in EVs under different culture conditions. These data, therefore, contribute to the understanding of how extracellular factors and intracellular molecular pathways affect the composition and release of EVs. [ABSTRACT FROM AUTHOR]

Extracellular vesicles (EVs) have emerged as important mediators of intercellular communication in a diverse range of biological processes. For future therapeutic applications and for EV biology research in general, understanding the in vivo fate of EVs is of utmost importance. Here we studied biodistribution of EVs in mice after systemic delivery. EVs were isolated from 3 different mouse cell sources, including dendritic cells (DCs) derived from bone marrow, and labelled with a near-infrared lipophilic dye. Xenotransplantation of EVs was further carried out for cross-species comparison. The reliability of the labelling technique was confirmed by sucrose gradient fractionation, organ perfusion and further supported by immunohistochemical staining using CD63-EGFP probed vesicles. While vesicles accumulated mainly in liver, spleen, gastrointestinal tract and lungs, differences related to EV cell origin were detected. EVs accumulated in the tumour tissue of tumour-bearing mice and, after introduction of the rabies virus glycoprotein-targeting moiety, they were found more readily in acetylcholine-receptor-rich organs. In addition, the route of administration and the dose of injected EVs influenced the biodistribution pattern. This is the first extensive biodistribution investigation of EVs comparing the impact of several different variables, the results of which have implications for the design and feasibility of therapeutic studies using EVs. [ABSTRACT FROM AUTHOR]

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Remyelination is a key aspect in multiple sclerosis pathology and a special effort is being made to promote it. However, there is still no available treatment to regenerate myelin and several strategies are being scrutinized. Myelination is naturally performed by oligodendrocytes and microRNAs have been postulated as a promising tool to induce oligodendrocyte precursor cell differentiation and therefore remyelination. Herein, DSPC liposomes and PLGA nanoparticles were studied for miR-219a-5p encapsulation, release and remyelination promotion. In parallel, they were compared with biologically engineered extracellular vesicles overexpressing miR-219a-5p. Interestingly, extracellular vesicles showed the highest oligodendrocyte precursor cell differentiation levels and were more effective than liposomes and polymeric nanoparticles crossing the blood–brain barrier. Finally, extracellular vesicles were able to improve EAE animal model clinical evolution. Our results indicate that the use of extracellular vesicles as miR-219a-5p delivery system can be a feasible and promising strategy to induce remyelination in multiple sclerosis patients. [ABSTRACT FROM AUTHOR]

Extracellular vesicles (EVs), including exosomes, microvesicles, and apoptotic bodies, are released by almost all cell types, including tumour cells. Through transfer of their molecular contents, EVs are capable of altering the function of recipient cells. Increasing evidence suggests a key role for EV mediated intercellular communication in a variety of cellular processes involved in tumour development and progression, including immune suppression, angiogenesis, and metastasis. Aspects of EV biogenesis or function are therefore increasingly being considered as targets for anticancer therapy. Here, we summarise the current knowledge on the contributions of EVs to cancer pathogenesis and discuss novel therapeutic strategies to target EVs to prevent tumour growth and spread. [ABSTRACT FROM AUTHOR]

Abstract: Extracellular vesicles (EVs) are specialised endogenous carriers of proteins and nucleic acids and are involved in intercellular communication. EVs are therefore proposed as candidate drug delivery systems for the delivery of nucleic acids and other macromolecules. However, the preparation of EV-based drug delivery systems is hampered by the lack of techniques to load the vesicles with nucleic acids. In this work we have now characterised in detail the use of an electroporation method for this purpose. When EVs were electroporated with fluorescently labelled siRNA, siRNA retention was comparable with previously published results (20–25% based on fluorescence spectroscopy and fluorescence fluctuation spectroscopy), and electroporation with unlabelled siRNA resulted in significant siRNA retention in the EV pellet as measured by RT-PCR. Remarkably, when siRNA was electroporated in the absence of EVs, a similar or even greater siRNA retention was measured. Nanoparticle tracking analysis and confocal microscopy showed extensive formation of insoluble siRNA aggregates after electroporation, which could be dramatically reduced by addition of EDTA. Other strategies to reduce aggregate formation, including the use of cuvettes with conductive polymer electrodes and the use of an acidic citrate electroporation buffer, resulted in a more efficient reduction of siRNA precipitation than EDTA. However, under these conditions, siRNA retention was below 0.05% and no significant differences in siRNA retention could be measured between samples electroporated in the presence or absence of EVs. Our results show that electroporation of EVs with siRNA is accompanied by extensive siRNA aggregate formation, which may cause overestimation of the amount of siRNA actually loaded into EVs. Moreover, our data clearly illustrate that electroporation is far less efficient than previously described, and highlight the necessity for alternative methods to prepare siRNA-loaded EVs. [Copyright &y& Elsevier]