Extracellular vesicles (EVs) are important mediators of intercellular communication and play key roles in the regulation of pathophysiological processes. In diabetic kidney disease (DKD), it has been reported that macrophages recruited in the mesangial region may play pathogenic roles through inducing local inflammation in glomeruli. We focused on EV-mediated crosstalk between mesangial cells (MC) and macrophages as a novel therapeutic target for DKD. EVs released from MC induced inflammation in macrophages and the effect was enhanced under high-glucose conditions. For discovering novel therapeutic agents which can inhibit such EV-mediated mechanisms, drug repositioning is considered as an effective tool. We established a unique screening strategy and screened agents to aim at maximizing their specificity and potency to inhibit EV mechanisms, along with minimizing their toxicity. We succeeded in identifying alvespimycin, an HSP90 inhibitor. Treatment of diabetic rats with alvespimycin significantly suppressed mesangial expansion, inflammatory gene activation including macrophage markers, and proteinuria. The inhibitory effect on EV uptake was specific to alvespimycin compared with other known HSP90 inhibitors. MC-derived EVs are crucial for inflammation by intercellular crosstalk between MC and macrophages in DKD, and alvespimycin effectively ameliorated the progression of DKD by suppressing EV-mediated actions, suggesting that EV-targeted agents can be a novel therapeutic strategy.

Intercellular communication is an essential hallmark of multicellular organisms for their development and adult tissue homeostasis. Over the past two decades, attention has been focused on communication mechanisms based on various membrane structures, as illustrated by the burst of scientific literature in the field of extracellular vesicles (EVs). These lipid bilayer-bound nano- or microparticles, as vehicle-like devices, act as regulators in various biological and physiological processes. When EVs are internalized by recipient cells, their membrane and cytoplasmic cargoes can interfere with cellular activities, affecting pathways that regulate cell proliferation, differentiation, and migration. In cancer, EVs can transfer oncogenic factors, stimulate neo-angiogenesis and immunosuppression, reprogram stromal cells, and confer drug resistance traits, thereby remodeling the surrounding microenvironment. Although the mechanisms underlying EV biogenesis and uptake are now better understood, little is known about the spatiotemporal mechanism(s) of their actions after internalization. In this respect, we have shown that a fraction of endocytosed EVs reaches the nuclear compartment via the VOR (VAP-A-ORP3-Rab7) complex-mediated docking of late endosomes to the outer nuclear membrane in the nucleoplasmic reticulum, positioning and facilitating the transfer of EV cargoes into the nucleoplasm via nuclear pores. Here, we highlight the EV heterogeneity, the cellular pathways governing EV release and uptake by donor and recipient cells, respectively, and focus on a novel intracellular pathway leading to the nuclear transfer of EV cargoes. We will discuss how to intercept it, which could open up new avenues for clinical applications in which EVs and other small extracellular particles (e.g., retroviruses) are implicated.

Extracellular vesicles can play an important role in the processes occurring after stem cell transplantation, preventing cell apoptosis, stimulating immunological processes, and promoting the synthesis of extracellular matrix. Human follicular fluid (FF) can be a source of a subpopulation of cells with mesenchymal stem cells (MSCs) properties. Moreover these subpopulations of FF cells can differentiate into osteoblasts. In presented studies flow cytometry of ovarian FF cells confirmed positive expression of MSCs markers such as: CD44, CD90, CD105, CD73 and negative expression of a hematopoietic marker: CD45. The CD90+, CD105+, CD45- cell subpopulation has been obtained during magnetic separation using appropriate antibodies conjugated with microbeads. The extracellular vesicles (EVs) secreted by the cells during osteodifferentiation process differed from those secreted by cells culture in the basal medium. Based on the previous and current electron microscopy research, changes in size, number, and shape would support the notion that released EVs could be crucial to the ovarian FF cell subpopulation differentiation process. Osteogenic differentiation has been confirmed via Alizarin red staining. Therefore, follicular fluid (FF) can be a new source of a cell subpopulation with MSC properties, with the cells capable of differentiating into the osteogenic lineage. EVs could play a key role as mediators in tissue regeneration, especially bone tissue regeneration.

Sepsis-induced cardiomyopathy (SICM) is a complex and fatal manifestation of sepsis, characterised by myocardial dysfunction that exacerbates the clinical prognosis in septic patients. While the pathophysiology of SICM remains incompletely understood, emerging evidence highlights the multifaceted functions of exosomes, small membrane-bound extracellular vesicles, in mediating the inflammatory responses and cardiac dysfunction involved in this condition. During sepsis, exosomes are secreted by various cells, such as cardiomyocytes, endothelial cells and macrophages, which serve as critical messengers, transferring proteins, lipids and RNA molecules that influence recipient cells, thus affecting cellular functions and disease progression. This review summarises the pathophysiology of SICM and the basics of exosomes and focuses on exosome-mediated mechanisms in SICM, including their role in inflammation, oxidative stress, mitochondrial dysfunction and myocardial injury, offering novel insights into the exosome-based therapeutic strategies in SICM.

Hermansky-Pudlak syndrome type 1 (HPS-1) is a rare, autosomal recessive disorder caused by defects in the biogenesis of lysosome-related organelles complex-3 (BLOC-3). Impaired kidney function is among its clinical manifestations. To investigate HPS-1 renal involvement, we employed 1D-gel-LC-MS/MS and compared the protein composition of urinary extracellular vesicles (uEVs) from HPS-1 patients to normal control individuals. We identified 1029 proteins, 149 of which were altered in HPS-1 uEVs. Ingenuity Pathway Analysis revealed disruptions in mitochondrial function and the LXR/RXR pathway that regulates lipid metabolism, which is supported by our novel Hps1 knockout mouse. Serum concentration of the LXR/RXR pathway protein ApoA1 in our patient cohort was positively correlated with kidney function (with the estimated glomerular filtration rate or eGFR). uEVs can be used to study epithelial cell protein trafficking in HPS-1 and may provide outcome measures for HPS-1 therapeutic interventions.

This work compared the potential effects of bone marrow mesenchymal stem cells (BM-MSCs) with BM-MSCs-derived exosomes against impaired autophagy in streptozotocin (STZ)-induced diabetic rats.

Three days after STZ injection, a single dose of (3 ​× ​10^6) BM- MSCs or BM-MSCs-derived exosomes (80 μg/rat) was administered to evaluate their effects against nondiabetic and diabetic control rats. We assessed pancreatic structure via light and electron microscopy and evaluated its staining for insulin and the autophagy marker P62 immunohistochemically. Moreover, autophagy marker LC3 gene expression was examined by PCR.

Both BM-MSCs and BM-MSCs derived exosomes showed histological restoration of pancreatic tissues. Both treatments markedly increased the amount of insulin and significantly decreased the autophagy markers P62 and LC3.

Our findings suggest that both BM-MSCs and BM-MSCs-derived exosomes provides a potential alternative to modulate diabetes mellitus.

Exosomes have emerged as promising tools for targeted drug delivery in biomedical applications and medicine. This review delves into the scientific advancements, challenges, and future prospects specifically associated with these technologies. In this work, we trace the research milestones that led to the discovery and characterization of exosomes and extracellular vesicles, and discuss strategies for optimizing the synthetic yield and the loading of these particles with various therapeutics. In addition, we report the current major issues affecting the field and hampering the clinical translation of these technologies. Highlighting the pivotal role of imaging techniques, we explore how they drive exosome therapy and development by offering insights into biodistribution and cellular trafficking dynamics. Methodologies for vesicle isolation, characterization, loading, and delivery mechanisms are thoroughly examined, alongside strategies aimed at enhancing their therapeutic efficacy. Special emphasis was dedicated to their therapeutic properties, particularly to their ability to deliver biologics into the cytoplasm. Furthermore, we delve into the intricate balance between surface modifications and targeting properties including also transgenic methods aimed at their functionalization and visualization within biological systems. This review underscores the transformative potential of these carriers in targeted drug delivery and identifies crucial areas for further research and clinical translation.

Extracellular vesicles (EVs) are nanoscale, membrane-enclosed structures released by cells into the extracellular milieu. These vesicles encapsulate a diverse array of molecular constituents, including nucleic acids, proteins, and lipids, which provide insights into the physiological or pathological conditions of their parent cells. Despite their potential, the study of EV-derived DNA (EV-DNA) has gathered relatively limited attention. This review aims to present a thorough examination of the emerging knowledge surrounding the utility of EV-DNA as a non-invasive biomarker across a spectrum of diseases. The review delves into various mechanisms underlying DNA packaging within EVs and the prevalent methodologies employed for extraction of EV-DNA. The relevance of EV-DNA is assessed across numerous health conditions, notably cancer, cardiovascular diseases, neurodegenerative disorders, infectious diseases, and pregnancy-related complications. The use of EV-DNA for cancer mutation detection has demonstrated remarkable sensitivity and specificity, thereby enhancing both diagnostic accuracy and therapeutic monitoring. In the context of cardiovascular diseases, EV-DNA serves as a predictive marker for events such as myocardial infarctions and shows a correlation with the severity of the disease. With respect to neurodegenerative conditions, including Parkinson's and Alzheimer's, EV-DNA contributes to the understanding of disease mechanisms and progression. Additionally, it plays an essential role in modulating immune tolerance and facilitating communication between maternal and fetal systems. Although there is a pressing need for standardized protocols for EV isolation and DNA analysis to facilitate clinical implementation, the prospect of EV-DNA as a non-invasive biomarker for diagnostic and prognostic purposes across diverse pathological conditions is considerable.

Small extracellular vesicles (EVs) are a diverse group of lipid-based particles that are ≤200 nm in diameter and contain an aqueous core. EVs have been shown to mediate intercellular communications between a wide array of immune cells; the downstream effects are diverse and have potential implications for the development of novel immunotherapeutic treatments. Despite a high volume of studies addressing the role EVs play in the immune system, our understanding of the crosstalk between T cells and cancer cells remains limited. Here, we discuss how EVs derived from cancer cells modulate T cell functions and conversely, how T cell derived EVs are crucial in modulating adaptive immune functions. In the context of cancer, tumor derived EVs (TD-EVs) halt T cell-mediated immunity by interfering with effector functions and enhancing regulatory T cell (Treg) functions. In contrast, EVs derived from effector T cells can serve to stimulate anticancer immunity, curbing metastasis and tumor growth. These findings highlight important aspects of how EVs can both mediate the therapeutic effects of T cells as well as impair T cell-mediated immunity. This calls for a deeper understanding of EV-mediated effects in order to advance them as next-generation therapeutics and nanocarriers.

Cardiovascular disease (CVD) is a leading cause of mortality, affecting ∼18 million individuals each year. Obesity and type 2 diabetes mellitus in particular, both chronic metabolic disorders, are risk factors for CVD. The salutary effects of physical activity in preventing and ameliorating CVD have long been acknowledged, as it improves glucose and lipid homeostasis, alongside attenuating oxidative damage, increasing mitochondrial function, and ultimately improving cardiac function. Exercise serves as a catalyst for the secretion of extracellular vesicles (EVs), facilitating inter-tissue communication, by which tissues can deliver important signals from one tissue to another. In recent years, an increasing number of studies have focused on the cargo encapsulated within exercise-derived EVs, as well as the orchestration of inter-tissue crosstalk aimed at modulating metabolism and tissue function in CVDs. The precise mechanisms underpinning the cardioprotective properties of exercise-derived EVs, however, remains only partially elucidated. This review explores novel EV based therapeutic options in CVD and, in particular, EVs derived from models of exercise to alter metabolism and enhance cardiovascular outcomes.

The discovery that extracellular RNAs (exRNA) can act as endocrine signalling molecules established a novel paradigm in intercellular communication. ExRNAs can be transported, both locally and systemically in virtually all body fluids. In association with an array of carrier vehicles of varying complexity, exRNA can alter target cell phenotype. This highlights the important role secreted exRNAs have in regulating human health and disease. The NIH Common Fund exRNA Communication program was established in 2012 to accelerate and catalyze progress in the exRNA biology field. The program addressed both exRNA and exRNA carriers, and served to generate foundational knowledge for the field from basic exRNA biology to future potential clinical applications as biomarkers and therapeutics. To address scientific challenges, the exRNA Communication program developed novel tools and technologies to isolate exRNA carriers and analyze their cargo. Here, we discuss the outcomes of the NIH Common Fund exRNA Communication program, as well as the evolution of exRNA as a scientific field through the analysis of scientific publications and NIH funding. ExRNA and associated carriers have potential clinical use as biomarkers, diagnostics, and therapeutics. Recent translational applications include exRNA-related technologies repurposed as novel diagnostics in response to the COVID-19 pandemic, the clinical use of extracellular vesicle-based biomarker assays, and exRNA carriers as drug delivery platforms. This comprehensive landscape analysis illustrates how discoveries and innovations in exRNA biology are being translated both into the commercial market and the clinic. Analysis of program outcomes and NIH funding trends demonstrate the impact of this NIH Common Fund program.

Mitochondrial dysfunction has been identified as a trigger for cellular autophagy dysfunction and programmed cell death. Emerging studies have revealed that, in pathological contexts, intercellular transfer of mitochondria takes place, facilitating the restoration of mitochondrial function, energy metabolism, and immune homeostasis. Extracellular vesicles, membranous structures released by cells, exhibit reduced immunogenicity and enhanced stability during the transfer of mitochondria. Thus, this review provides a concise overview of mitochondrial dysfunction related diseases and the mechanism of mitochondrial dysfunction in diseases progression, and the composition and functions of the extracellular vesicles, along with elucidating the principal mechanisms underlying intercellular mitochondrial transfer. In this article, we will focus on the advancements in both animal models and clinical trials concerning the therapeutic efficacy of extracellular vesicle-mediated mitochondrial transplantation across various systemic diseases in neurodegenerative diseases and cardiovascular diseases. Additionally, the review delves into the multifaceted roles of extracellular vesicle-transplanted mitochondria, encompassing anti-inflammatory actions, promotion of tissue repair, enhancement of cellular function, and modulation of metabolic and immune homeostasis within diverse pathological contexts, aiming to provide novel perspectives for extracellular vesicle transplantation of mitochondria in the treatment of various diseases.

Mutual interaction between doxorubicin (DOX) and cardiomyocytes is crucial for cardiotoxicity progression. Cardiomyocyte injury is an important pathological feature of DOX-induced cardiomyopathy, and its molecular pathogenesis is multifaceted. In addition to the direct toxic effects of DOX on cardiomyocytes, DOX-induced exosomes in the extracellular microenvironment also regulate the pathophysiological states of cardiomyocytes. However, the mechanisms by which DOX regulates exosome secretion and subsequent pathogenesis remain incompletely understood. Here, we found that DOX significantly increased exosome secretion from cardiomyocytes, and inhibiting this release could alleviate cardiomyocyte injury. DOX promoted exosome secretion by reducing cardiomyocyte silencing information regulator 1 (SIRT1) expression, exacerbating cardiotoxicity. DOX impaired lysosomal acidification in cardiomyocytes, reducing the degradation of intracellular multivesicular bodies (MVBs), resulting in an increase in MVB volume before fusing with the plasma membrane to release their contents. Mechanistically, SIRT1 loss inhibited lysosomal acidification by reducing the expression of the ATP6V1A subunit of the lysosomal vacuolar-type H+ ATPase (V-ATPase) proton pump. Overexpressing SIRT1 increased ATP6V1A expression, improved lysosomal acidification, inhibited exosome secretion, and thereby alleviated DOX-induced cardiotoxicity. Interestingly, DOX also induced mitochondrial-derived vesicle formation in cardiomyocytes, which may further increase the abundance of MVBs and promote exosome release. Collectively, this study identified SIRT1-mediated impairment of lysosomal acidification as a key mechanism underlying the increased exosome secretion from cardiomyocytes induced by DOX, providing new insights into DOX-induced cardiotoxicity pathogenesis.

Extracellular vesicles (EVs), particularly microvesicles (MVs), have gained significant attention for their role as mediators of intercellular communication in both physiological and pathological contexts, including diabetes mellitus (DM) and its complications. This review provides a comprehensive analysis of the emerging roles of MVs in the pathogenesis of diabetes and associated complications such as nephropathy, retinopathy, cardiomyopathy, and neuropathy. MVs, through their cargo of proteins, lipids, mRNAs, and miRNAs, regulate critical processes like inflammation, oxidative stress, immune responses, and tissue remodeling, all of which contribute to the progression of diabetes and its complications. We examine the molecular mechanisms underlying MVs' involvement in these pathological processes and discuss their potential as biomarkers and therapeutic tools, particularly for drug delivery. Despite promising evidence, challenges remain in isolating and characterizing MVs, understanding their molecular mechanisms, and validating them for clinical use. Advanced techniques such as single-cell RNA sequencing and proteomics are required to gain deeper insights. Improved isolation and purification methods are essential for translating MVs into clinical applications, with potential to develop novel diagnostic and therapeutic strategies to improve patient outcomes in diabetes.

Cell-derived extracellular vesicles (EVs), which carry diverse biomolecules such as nucleic acids, proteins, metabolites, and lipids reflecting their cell of origin, are released under both physiological and pathological conditions. EVs have been demonstrated to mediate cell-to-cell communication and serve as biomarkers. EV-associated DNA (EV-DNA) comprises genomic and mitochondrial DNA (i.e., gDNA and mtDNA) fragments. Some studies have revealed that EV-DNA can represent the full nuclear genome and mitochondrial genome of parental cells. Furthermore, DNA fragments loaded into EVs are stable and can be transferred to recipient cells to regulate their biological functions. In this review, we summarized and discussed EV-DNA research advances with an emphasis on EV-DNA detection at the population-EV and single-EV levels, gene transfer-associated biological functions, and clinical applications as biomarkers for disease liquid biopsy. We hope that this review will provide potential directions or guidance for future EV-DNA investigations.

The skin is an essential organ that protects the body from external aggressions; therefore, damage from various wounds can significantly impair its function, and effective methods for regenerating and restoring its barrier function are crucial. This study aimed to mass-produce wound-healing exosomes using a fragment of the fibroblast growth factor 2 (FGF2)-derived peptide (FP2) to enhance cell proliferation and exosome production. Our experiments demonstrated increased cell proliferation when Wharton's jelly mesenchymal stem cells (WJ MSCs) were coated with FP2. Exosomes from FP2-coated WJ MSCs were analyzed using nanoparticle-tracking analysis, transmission electron microscopy, and Western blotting. Subsequently, fibroblasts were treated with these exosomes, and their viability and migration effects were compared. Anti-inflammatory effects were also evaluated by inducing pro-inflammatory factors in RAW264.7 cells. The treatment of fibroblasts with FP2-coated WJ MSC-derived exosomes (FP2-exo) increased the expression of FGF2, confirming their wound-healing effect in vivo. Overall, the results of this study highlight the significant impact of FP2 on the proliferation of WJ MSCs and the anti-inflammatory and wound-healing effects of exosomes, suggesting potential applications beyond wound healing.

Gene overexpression by transient transfection of in vitro cultured model cell lines with plasmid DNA is a commonly used method for studying molecular aspects of human biology and pathobiology. However, there is accumulating evidence suggesting that human cells may actively secrete fragments of DNA and the implications of this phenomenon for in vitro cultured cells transiently transfected with foreign nucleic acids has been overlooked. Therefore, in the current study we investigated whether a cell-to-cell transmission of acquired plasmid DNA takes place in a commonly used human cell line model. We transiently transfected HEK293 cells with EGFP encoding plasmids to serve as donor cells and either co-cultured these with stably mCherry expressing recipient cells in different set-ups or transferred their culture medium to the recipient cells. We found that recipient cells produced EGFP after being co-cultured with donor cells but not when they were exposed to their culture medium. The employment of different co-culture set-ups excluded that the observed effect stemmed from technical artefacts and provided evidence that an intercellular plasmid transfer takes place requiring physical proximity between living cells. This phenomenon could represent a significant biological artefact for certain studies such as those addressing protein transmissions in prion diseases.

Extracellular vesicles (EVs) have emerged as key intercellular communication and pathogenesis mediators. Parasitic organisms' helminths, cause widespread infections with significant health impacts worldwide. Recent research has shed light on the role of EVs in the lifecycle, immune evasion, and disease progression of these parasitic organisms. These tiny membrane-bound organelles including microvesicles and exosomes, facilitate the transfer of proteins, lipids, mRNAs, and microRNAs between cells. EVs have been isolated from various bodily fluids, offering a potential diagnostic and therapeutic avenue for combating infectious agents. According to recent research, EVs from helminths hold great promise in the diagnosis of parasitic infections due to their specificity, early detection capabilities, accessibility, and the potential for staging and monitoring infections, promote intercellular communication, and are a viable therapeutic tool for the treatment of infectious agents. Exploring host-parasite interactions has identified promising new targets for diagnostic, therapy, and vaccine development against helminths. This literature review delves into EVS's origin, nature, biogenesis, and composition in these parasitic organisms. It also highlights the proteins and miRNAs involved in EV release, providing a comprehensive summary of the latest findings on the significance of EVs in the biology of helminths, promising targets for therapeutic and diagnostic biomarkers.

This historical review of extracellular vesicles in the setting of exposure to ionizing radiation (IR) traces our understanding of how vesicles were initially examined and reported in the literature in the late 1970s (for secreted exosomes) and early 1980s (for plasma membrane-derived, exfoliated vesicles) to where we are now and where we may be headed in the next decade. An emphasis is placed on biophysical properties of extracellular vesicles, energy consumption and the role of vesiculation as an essential component of membrane turnover. The impact of intercellular signal trafficking by vesicle surface and intra-vesicular lipids, proteins, nucleic acids and metabolites is reviewed in the context of biomarkers for estimating individual radiation dose after exposure to radiation, pathogenesis of disease and development of cell-free therapeutics. Since vesicles express both growth stimulatory and inhibitory molecules, a hypothesis is proposed to consider superposition in a shared space and entanglement of molecules by energy sources that are external to human cells. Implications of this approach for travel in deep space are briefly discussed in the context of clinical disorders that have been observed after space travel.

The post-transcriptional control of gene expression is a complex and evolving field in adipocyte biology, with the premise that the delivery of microRNA (miRNA) species to the obese adipose tissue may facilitate weight loss. Cells shed extracellular vesicles (EVs) that may deliver miRNAs as intercellular messengers. However, we know little about the miRNA profile of EVs secreted by adipocytes during postnatal development. Here, we defined the miRNA cargo of EVs secreted by mouse adipocytes in two distinct phases of development: on postnatal day 6, when adipocytes are lipolytic and thermogenic, and on postnatal day 56, when adipocytes have active lipogenesis. EVs were collected from cell culture supernatants, and their miRNA profile was defined by small RNA sequencing. The most abundant miRNA of mouse adipocyte-derived EVs was mmu-miR-148a-3p. Adipocyte EVs on postnatal day 6 were hallmarked with mmu-miR-98-5p, and some miRNAs were specific to this developmental stage, such as mmu-miR-466i-5p and 12 novel miRNAs. Adipocytes on postnatal day 56 secreted mmu-miR-365-3p, and 16 miRNAs were specific to this developmental stage. The miRNA cargo of adipocyte EVs targeted gene networks of cell proliferation, insulin signaling, interferon response, thermogenesis, and lipogenesis. We provided here a database of miRNAs secreted by developing mouse adipocytes, which may be a tool for further studies on the regulation of gene networks that control mouse adipocyte development.

Exosomes are a subgroup of extracellular vesicles that function as transmitters, allowing cells to communicate more effectively with each other. However, exosomes may have both beneficial and harmful impacts on central nervous system disorders. Hence, the fundamental molecular mechanisms of the origin of illness and its progression are currently being investigated. The involvement of exosomes in the origin and propagation of neurodegenerative illness has been demonstrated recently. Exosomes provide a representation of the intracellular environment since they include various essential bioactive chemicals. The latest studies have demonstrated that exosomes transport several proteins. Additionally, these physiological vesicles are important in the regeneration of nervous tissue and the healing of neuronal lesions. They also offer a microenvironment to stimulate the conformational variation of concerning proteins for aggregation, resulting in neurodegenerative diseases. The biosynthesis, composition, and significance of exosomes as extracellular biomarkers in neurodegenerative disorders are discussed in this article, with a particular emphasis on their neuroprotective effects.

This review focusses on the significance of fluorescent, phosphorescent labelling and tracking of extracellular vesicles (EVs) for unravelling their biology, pathophysiology, and potential diagnostic and therapeutic uses. Various labeling strategies, such as lipid membrane, surface protein, luminal, nucleic acid, radionuclide, quantum dot labels, and metal complex-based stains, are evaluated for visualizing and characterizing EVs. Direct labelling with fluorescent lipophilic dyes is simple but generally lacks specificity, while surface protein labelling offers selectivity but may affect EV-cell interactions. Luminal and nucleic acid labelling strategies have their own advantages and challenges. Each labelling approach has strengths and weaknesses, which require a suitable probe and technique based on research goals, but new tetranuclear polypyridylruthenium(II) complexes as phosphorescent probes have strong phosphorescence, selective staining, and stability. Future research should prioritize the design of novel fluorescent probes and labelling platforms that can significantly enhance the efficiency, accuracy, and specificity of EV labeling, while preserving their composition and functionality. It is crucial to reduce false positive signals and explore the potential of multimodal imaging techniques to gain comprehensive insights into EVs.

Exosomes are small extracellular vesicles that play a crucial role in intercellular communication and offer significant potential for a wide range of biomedical applications. However, conventional methods for exosome isolation have limitations in terms of purity, scalability, and preservation of exosome structural integrity. To address these challenges, an exosome isolation platform using chitosan oligosaccharide lactate conjugated 1-pyrenecarboxylic acid (COL-Py) based self-assembled magnetic nanoclusters (CMNCs), is presented. CMNCs are characterized to optimize their size, stability, and interaction dynamics with exosomes. The efficiency of CMNCs in isolating exosomes is systematically evaluated using various analytical methods to demonstrate their ability to capture exosomes based on amphiphilic lipid bilayers. CMNC-based exosome isolation consistently yields exosomes with structural integrity and purity similar to those obtained using traditional methods. The reusability of CMNCs over multiple exosome isolation cycles underscores their scalability and offers an efficient solution for biomedical applications. These results are supported by western blot analysis, which demonstrated the superiority of CMNC-based isolation in terms of purity compared to conventional methods. By providing a scalable and efficient exosome isolation process that preserves both structural integrity and purity, CMNCs can constitute a new platform that can contribute to the field of exosome studies.

Cystatin B (CSTB) is a small protease inhibitor protein being involved in cell proliferation and neuronal differentiation. Loss-of-function mutations in CSTB gene cause progressive myoclonic epilepsy 1 (EPM1). We previously demonstrated that CSTB is locally synthesized in synaptic nerve terminals from rat brain and secreted into the media, indicating its role in synaptic plasticity. In this work, we have further investigated the involvement of CSTB in synaptic plasticity, using synaptosomes from human cerebral organoids (hCOs) as well as from rodents' brain. Our data demonstrate that CSTB is released from synaptosomes in two ways: (i) as a soluble protein and (ii) in extracellular vesicles-mediated pathway. Synaptosomes isolated from hCOs are enriched in pre-synaptic proteins and contain CSTB at all developmental stages analyzed. CSTB presence in the synaptic territories was also confirmed by immunostaining on human neurons in vitro. To investigate if the depletion of CSTB affects synaptic plasticity, we characterized the synaptosomes from EPM1 hCOs. We found that the levels of presynaptic proteins and of an initiation factor linked to local protein synthesis were both reduced in EPM1 hCOs and that the extracellular vesicles trafficking pathway was impaired. Moreover, EPM1 neurons displayed anomalous morphology with longer and more branched neurites bearing higher number of intersections and nodes, suggesting connectivity alterations. In conclusion, our data strengthen the idea that CSTB plays a critical role in the synapse physiology and reveal that pathologically low levels of CSTB may affect synaptic plasticity, leading to synaptopathy and altered neuronal morphology.

Rationale: Extracellular vesicles (EVs) are thought to mediate intercellular communication during development and disease. Yet, biological insight to intercellular EV transfer remains elusive, also in the heart, and is technically challenging to demonstrate. Here, we aimed to investigate biological transfer of cardiomyocyte-derived EVs in the neonatal heart. Methods: We exploited CD9 as a marker of EVs, and generated two lines of cardiomyocyte specific EV reporter mice: Tnnt2-Cre; double-floxed inverted CD9/EGFP and αMHC-MerCreMer; double-floxed inverted CD9/EGFP. The two mouse lines were utilized to determine whether developing cardiomyocytes transfer EVs to other cardiac cells (non-myocytes and cardiomyocytes) in vitro and in vivo and investigate the intercellular transport pathway of cardiomyocyte-derived EVs. Results: Genetic tagging of cardiomyocytes was confirmed in both reporter mouse lines and proof of concept in the postnatal heart showed that, a fraction of EGFP+/MYH1- non-myocytes exist firmly demonstrating in vivo cardiomyocyte-derived EV transfer. However, two sets of direct and indirect EGFP +/- cardiac cell co-cultures showed that cardiomyocyte-derived EGFP+ EV transfer requires cell-cell contact and that uptake of EGFP+ EVs from the medium is limited. The same was observed when co-cultiring with mouse macrophages. Further mechanistic insight showed that cardiomyocyte EV transfer occurs through type I tunneling nanotubes. Conclusion: While the current notion assumes that EVs are transferred through secretion to the surroundings, our data show that cardiomyocyte-derived EV transfer in the developing heart occurs through nanotubes between neighboring cells. Whether these data are fundamental and relate to adult hearts and other organs remains to be determined, but they imply that the normal developmental process of EV transfer goes through cell-cell contact rather than through the extracellular compartment.

Selective staining of extracellular vesicles (EVs) is a major challenge for diagnostic and therapeutic applications. Herein, the EV labeling properties of a new class of tetranuclear polypyridylruthenium(II) complexes, Rubb7-TNL and Rubb7-TL, as phosphorescent stains are described. These new stains have many advantages over standard stains to detect and characterize EVs, including: high specificity for EV staining versus cell staining; high phosphorescence yields; photostability; and a lack of leaching from EVs until incorporation with target cells. As an example of their utility, large EVs released from control (basal) or lipopolysaccharide (LPS)-stimulated THP-1 monocytic leukemia cells were studied as a model of immune system EVs released during bacterial infection. Key findings from EV staining combined with flow cytometry were as follows: (i) LPS-stimulated THP-1 cells generated significantly larger and more numerous large EVs, as compared with those from unstimulated cells; (ii) EVs retained native EV physical properties after staining; and (iii) the new stains selectively differentiated intact large EVs from artificial liposomes, which are models of cell membrane fragments or other lipid-containing debris, as well as distinguished two distinct subpopulations of monocytic EVs within the same experiment, as a result of biochemical differences between unstimulated and LPS-stimulated monocytes. Comparatively, the staining patterns of A549 epithelial lung carcinoma-derived EVs closely resembled those of THP-1 cell line-derived EVs, which highlighted similarities in their selective staining despite their distinct cellular origins. This is consistent with the hypothesis that these new phosphorescent stains target RNA within the EVs.

Exosomes (EXOs) are a subgroup of extracellular vesicles (EVs) that contain numerous biologically active molecules. They exhibit an essential mode of cell communication, primarily between distinct cell populations, for the maintenance of tissue homeostasis and coordination of adaptive responses to various stresses. These intercellular communications are vital for the complex, multicellular cardiovascular system. In the last ten years, their potential role as effective tissue-to-tissue communicators has received increasing attention in cardiovascular physiology and pathology. There is growing evidence that repair of the heart and regeneration can be promoted by EXOs derived from cardiomyocytes or stem/progenitor cells. However, the underlying mechanisms remain unclear. EVs derived from different stem/progenitor cell populations have been used as cell-free therapies in different preclinical models involving cardiovascular diseases and have shown promising results. In this review, we have summarized the recent developments in EXOs research, the impact of EXOs derived from different cells on the cardiovascular system, their potential therapeutic roles as well as new diagnostic biomarkers, and the possible clinical translational outcomes.

Extracellular vesicles in human milk are critical in supporting newborn growth and development. Bioavailability of dietary extracellular vesicles may depend on the composition of membrane lipids. Single-nucleotide polymorphisms (SNPs) in the fatty acid desaturase gene cluster impact the content of long-chain polyunsaturated fatty acids in human milk phospholipids. This study investigated the relation between variation in FADS1 and FADS2 with the content of polyunsaturated fatty acids in extracellular vesicles from human milk.

Milk was obtained from a cohort of mothers (N = 70) at 2-4 weeks of lactation. SNPs in the FADS gene locus were determined using pyrosequencing for rs174546 in FADS1 and rs174575 in FADS2. Quantitative lipidomic analysis of polyunsaturated fatty acids in human milk and extracellular vesicles from human milk was completed by gas chromatography-mass spectrometry.

The rs174546 and rs174575 genotypes were independent predictors of the arachidonic acid content in extracellular vesicles. The rs174546 genotype also predicted eicosapentaenoic acid and docosahexaenoic acid in extracellular vesicles. The reduced content of long-chain polyunsaturated fatty acids in extracellular vesicles in human milk may be due to lower fatty acid desaturase activity in mothers who are carriers of the A allele in rs174546 or the G allele in rs174575.

The polyunsaturated fatty acid composition of milk extracellular vesicles is predicted by the FADS genotype. These findings yield novel insights regarding extracellular vesicle content and composition that can inform the design of future research to explore how lipid metabolites impact the bioavailability of human milk extracellular vesicles.

Degenerative orthopaedic diseases pose a notable worldwide public health issue attributable to the global aging population. Conventional medical approaches, encompassing physical therapy, pharmaceutical interventions, and surgical methods, face obstacles in halting or reversing the degenerative process. In recent times, exosome-based therapy has gained widespread acceptance and popularity as an effective treatment for degenerative orthopaedic diseases. This therapeutic approach holds the potential for "cell-free" tissue regeneration. Exosomes, membranous vesicles resulting from the fusion of intracellular multivesicles with the cell membrane, are released into the extracellular matrix. Addressing challenges such as the rapid elimination of natural exosomes in vivo and the limitation of drug concentration can be effectively achieved through various strategies, including engineering modification, gene overexpression modification, and biomaterial binding. This review provides a concise overview of the source, classification, and preparation methods of exosomes, followed by an in-depth analysis of their functions and potential applications. Furthermore, the review explores various strategies for utilizing exosomes in the treatment of degenerative orthopaedic diseases, encompassing engineering modification, gene overexpression, and biomaterial binding. The primary objective is to provide a fresh viewpoint on the utilization of exosomes in addressing bone degenerative conditions and to support the practical application of exosomes in the theranosis of degenerative orthopaedic diseases.

Platelet-derived growth factor (PDGF)-induced changes in vascular smooth muscle cells (VSMCs) stimulate vascular remodeling, resulting in vascular diseases such as pulmonary arterial hypertension. VSMCs communicate with endothelial cells through extracellular vesicles (EVs) carrying cargos, including microRNAs. To understand the molecular mechanisms through which PDGF-stimulated pulmonary artery smooth muscle cells (PASMCs) interact with pulmonary artery endothelial cells (PAECs) under pathological conditions, we investigated the crosstalk between PASMCs and PAECs via extracellular vesicle miR-409-5p under PDGF stimulation. miR-409-5p expression was upregulated in PASMCs upon PDGF signaling, and it was released into EVs. The elevated expression of miR-409-5p was transported to PAECs and led to their impaired function, including reduced NO release, which consequentially resulted in enhanced PASMC proliferation. We propose that the positive regulatory loop of PASMC-extracellular vesicle miR-409-5p-PAEC is a potential mechanism underlying the proliferation of PASMCs under PDGF stimulation. Therefore, miR-409-5p may be a novel therapeutic target for the treatment of vascular diseases, including pulmonary arterial hypertension.

Cardiovascular disease constitutes a noteworthy public health challenge characterized by a pronounced incidence, frequency, and mortality rate, particularly impacting specific demographic groups, and imposing a substantial burden on the healthcare infrastructure. Certain risk factors, such as age, gender, and smoking, contribute to the prevalence of fatal cardiovascular disease, highlighting the need for targeted interventions. Current challenges in clinical practice involve medication complexities, the lack of a systematic decision-making approach, and prevalent drug therapy problems. Stem cell-derived extracellular vesicles stand as versatile entities with a unique molecular fingerprint, holding significant therapeutic potential across a spectrum of applications, particularly in the realm of cardio-protection. Their lipid, protein, and nucleic acid compositions, coupled with their multifaceted functions, underscore their role as promising mediators in regenerative medicine and pave the way for further exploration of their intricate contributions to cellular physiology and pathology. Here, we overview our current understanding of the possible role of stem cell-derived extracellular vesicles in the clinical management of human cardiovascular pathologies.

Peripheral nerve injury can result in significant clinical complications that have uncertain prognoses. Currently, there is a lack of effective pharmacological interventions for nerve damage, despite the existence of several small compounds, peptides, hormones, and growth factors that have been suggested as potential enhancers of neuron regeneration. Despite the objective of achieving full functional restoration by surgical intervention, the persistent challenge of inadequate functional recovery remains a significant concern in the context of peripheral nerve injuries.

To examine the impact of exosomes on the process of functional recovery following a complete radial nerve damage.

A male individual, aged 24, who is right-hand dominant and an immigrant, arrived with an injury caused by a knife assault. The cut is located on the left arm, specifically below the elbow. The neurological examination and electrodiagnostic testing reveal evidence of left radial nerve damage. The sural autograft was utilized for repair, followed by the application of 1 mL of mesenchymal stem cell-derived exosome, comprising 5 billion microvesicles. This exosome was split into four equal volumes of 0.25 mL each and delivered microsurgically to both the proximal and distal stumps using the subepineural pathway. The patient was subjected to a period of 180 d during which they had neurological examination and electrodiagnostic testing.

The duration of the patient's follow-up period was 180 d. An increasing Tinel's sign and sensory-motor recovery were detected even at the 10th wk following nerve grafting. Upon the conclusion of the 6-mo post-treatment period, an evaluation was conducted to measure the extent of improvement in motor and sensory functions of the nerve. This assessment was based on the British Medical Research Council scale and the Mackinnon-Dellon scale. The results indicated that the level of improvement in motor function was classified as M5, denoting an excellent outcome. Additionally, the level of improvement in sensory function was classified as S3+, indicating a good outcome. It is noteworthy that these assessments were conducted in the absence of physical therapy. At the 10th wk post-injury, despite the persistence of substantial axonal damage, the nerve exhibited indications of nerve re-innervation as evidenced by control electromyography (EMG). In contrast to the preceding. EMG analysis revealed a significant electrophysiological enhancement in the EMG conducted at the 6th-mo follow-up, indicating ongoing regeneration.

Enhanced comprehension of the neurobiological ramifications associated with peripheral nerve damage, as well as the experimental and therapy approaches delineated in this investigation, holds the potential to catalyze future clinical progress.

Cholangiocarcinoma (CCA) is a highly aggressive malignant tumor that originates from the biliary system. With restricted treatment options at hand, the challenging aspect of early CCA diagnosis leads to a bleak prognosis. Besides the intrinsic characteristics of tumor cells, the generation and progression of CCA are profoundly influenced by the tumor microenvironment, which engages in intricate interactions with cholangiocarcinoma cells. Of notable significance is the role of extracellular vesicles as key carriers in enabling communication between cancer cells and the tumor microenvironment. This review aims to provide a comprehensive overview of current research examining the interplay between extracellular vesicles and the tumor microenvironment in the context of CCA. Specifically, we will emphasize the significant contributions of extracellular vesicles in molding the CCA microenvironment and explore their potential applications in the diagnosis, prognosis assessment, and therapeutic strategies for this aggressive malignancy.

Among multiple pathways of intercellular communication operative in multicellular organisms, the trafficking of extracellular vesicles (EVs) and particles (EP) represents a unique mode of cellular information exchange with emerging roles in health and disease, including cancer. A distinctive feature of EV/EP-mediated cell-cell communication is that it involves simultaneous short- or long-range transfer of numerous molecular constituents (cargo) from donor to recipient cells. EV/EP uptake by donor cells elicits signalling or metabolic responses, or else leads to EV-re-emission or degradation. EVs are heterogeneous membranous structures released from cells via increasingly defined mechanisms involving either formation of multivesicular endosomes (exosomes) or budding from the plasma membrane (ectosomes). EPs (exomeres, supermeres) are membraneless complex particles, smaller than EVs and of less defined biogenesis and function. EVs/EPs carry complex assemblies of proteins, lipids and nucleic acids (RNA, DNA), which they shuttle into intercellular milieu, body fluids and recipient cells, via surface contact, fusion and different forms of internalization (endocytosis, micropinocytosis). While the physiological functions of EVs/EPs communication pathways continue to be investigated, their roles in cancer are increasingly well-defined. For example, EVs are involved in the transmission of cancer-specific molecular cargo, including mutant, oncogenic, transforming, or regulatory macromolecules to indolent, or normal cells, sometimes triggering their quasi-transformation-like states, or phenotypic alterations. Conversely, a reciprocal and avid uptake of stromal EVs by cancer cells may be responsible for modulating their oncogenic repertoire, as exemplified by the angiocrine effects of endothelial EVs influencing cancer cell stemness. EV exchanges during cancer progression have also been implicated in the formation of tumour stroma, angiogenesis and non-angiogenic neovascularization processes, immunosuppression, colonization of metastatic organ sites (premetastatic niche), paraneoplastic and systemic pathologies (thrombosis, diabetes, hepatotoxicity). Thus, an EV/EP-mediated horizontal transfer of cellular content emerges as a new dimension in cancer pathogenesis with functional, diagnostic, and therapeutic implications.

The landscape of exosome research has undergone a significant paradigm shift, with a departure from early conceptions of exosomes as vehicles for cellular waste disposal towards their recognition as integral components of cellular communication with therapeutic potential. This chapter presents an exhaustive elucidation of exosome biology, detailing the processes of exosome biogenesis, release, and uptake, and their pivotal roles in signal transduction, tissue repair, regeneration, and intercellular communication. Additionally, the chapter highlights recent innovations and anticipates future directions in exosome research, emphasizing their applicability in clinical settings. Exosomes have the unique ability to navigate through tissue spaces to enter the circulatory system, positioning them as key players in tissue repair. Their contributory role in various processes of tissue repair, although in the nascent stages of investigation, stands out as a promising area of research. These vesicles function as a complex signaling network for intracellular and organ-level communication, critical in both pathological and physiological contexts. The chapter further explores the tissue-specific functionality of exosomes and underscores the advancements in methodologies for their isolation and purification, which have been instrumental in expanding the scope of exosome research. The differential cargo profiles of exosomes, dependent on their cellular origin, position them as prospective diagnostic biomarkers for tissue damage and regenerative processes. Looking ahead, the trajectory of exosome research is anticipated to bring transformative changes to biomedical fields. This includes advancing diagnostic and prognostic techniques that utilize exosomes as non-invasive biomarkers for a plethora of diseases, such as cancer, neurodegenerative, and cardiovascular conditions. Additionally, engineering exosomes through alterations of their native content or surface properties presents a novel frontier, including the synthesis of artificial or hybrid variants with enhanced functional properties. Concurrently, the ethical and regulatory frameworks surrounding exosome research, particularly in clinical translation, will require thorough deliberation. In conclusion, the diverse aspects of exosome research are coalescing to redefine the frontiers of diagnostic and therapeutic methodologies, cementing its importance as a discipline of considerable consequence in the biomedical sciences.

Exosomes, a type of extracellular vesicles, are effective tools for delivering small-molecule drugs and biological therapeutics into cells and tissues. Surface modifications with targeting ligands ensure precise delivery to specific cells, minimizing accumulation in healthy organs and reducing the side effects. This is a rapidly growing area in drug delivery research and this review aims to comprehensively discuss the recent advances in the field.

Recent studies have presented compelling evidence supporting the application of exosomes as efficient delivery vehicles that escape endosome trapping, achieving effective in vivo delivery in animal models. This review provides a systemic discussion on the exosome-based delivery technology, with topics covering exosome purification, surface modification, and targeted delivery of various cargos ranging from siRNAs, miRNAs, and proteins, to small molecule drugs.

Exosome-based gene and drug delivery has low toxicity and low immunogenicity. Surface modifications of the exosomes can effectively avoid endosome trapping and increase delivery efficiency. This exciting technology can be applied to improve the treatments for a wide variety of diseases.

The transmission of DNA through extracellular vesicles (EVs) represents a novel genetic material transfer mechanism that may impact genome evolution and tumorigenesis. We aimed to investigate the potential for vertical DNA transmission within maternal endometrial EVs to the pre-implantation embryo and describe any effect on embryo bioenergetics. We discovered that the human endometrium secretes all three general subtypes of EV - apoptotic bodies (ABs), microvesicles (MVs), and exosomes (EXOs) - into the human endometrial fluid (EF) within the uterine cavity. EVs become uniformly secreted into the EF during the menstrual cycle, with the proportion of different EV populations remaining constant; however, MVs contain significantly higher levels of mitochondrial (mt)DNA than ABs or EXOs. During the window of implantation, MVs contain an eleven-fold higher level of mtDNA when compared to cells-of-origin within the receptive endometrium, which possesses a lower mtDNA content and displays the upregulated expression of mitophagy-related genes. Furthermore, we demonstrate the internalization of EV-derived nuclear-encoded (n)DNA/mtDNA by trophoblast cells of murine embryos, which associates with a reduction in mitochondrial respiration and ATP production. These findings suggest that the maternal endometrium suffers a reduction in mtDNA content during the preconceptional period, that nDNA/mtDNA become packaged into secreted EVs that the embryo uptakes, and that the transfer of DNA to the embryo within EVs occurs alongside the modulation of bioenergetics during implantation.