搜索结果：Journal of extracellular vesicles

Epilepsy is a neurological disorder characterised by recurrent spontaneous seizures. Approximately 30% of patients are unable to achieve adequate seizure control with available medications, highlighting the need to better understand disease mechanisms and develop improved treatments. Mesial temporal lobe epilepsy (MTLE) is a common subtype of treatment-resistant epilepsy. Patients with MTLE often have experienced a prior neurological insult, such as a traumatic brain injury, or status epilepticus (SE), that is believed to contribute to epileptogenic changes that lead to MTLE. However, the molecular mechanisms underlying epileptogenesis remain incompletely understood. Extracellular vesicles (EVs) are small membrane-bound particles released by cells that are important for intercellular communication. Recent studies identified altered microRNA (miRNA) content in brain-derived EVs (BDEVs) in models of MTLE, but whether BDEV function is altered has not been explored. Using the pilocarpine mouse model of MTLE, we examined the effects of SE on the function and miRNA cargo of hippocampal BDEVs during the epileptogenic period. BDEVs were isolated from hippocampi of control and pilocarpine-treated mice 24 h after SE. To assess functional changes, we compared gene expression in N2a neuronal-like cells and BV2 microglial-like cells exposed to control BDEVs (CON-BDEVs) or BDEVs collected after SE (SE-BDEVs). SE-BDEVs induced distinct transcriptional changes in N2a cells including alterations in the expression of genes related to TGF-β signalling, amino acid regulation of mTORC1 and neurotransmitter signalling. In BV2 cells, SE-BDEVs increased the expression of genes associated with inflammatory cytokine release. Analysis of BDEV miRNA content revealed multiple differentially expressed miRNAs between SE- and CON-BDEVs, with predicted targets overlapping with genes uniquely altered by SE-BDEVs. Notably, SE-induced miRNA changes were still observed 10 days post-SE, indicating sustained modulation of BDEV cargo. These findings identify SE-induced alterations in both the function and miRNA composition of hippocampal BDEVs, suggesting that BDEVs may contribute to epileptogenesis in MTLE.

Efficient and specific delivery of mRNA to target tissues is critical for maximising therapeutic benefits while minimising off-target effects and systemic toxicity. Systemic administration of mRNA using lipid nanoparticles (LNPs) or extracellular vesicles (EVs) typically leads to predominant accumulation in the liver. We hypothesised that cardiac-specific EVs could promote enhanced relative cardiac enrichment of delivered mRNA compared with non-cardiac EVs or LNPs. In mice, intravenous administration of cardiac progenitor cell-derived EVs (CPC-EVs) achieved the greatest relative cardiac selectivity of modified mRNA encoding vascular endothelial growth factor A (VEGF-A) to the heart, with reduced liver accumulation relative to non-cardiac EVs and LNPs. Cytokine profiling across seven organs revealed that LNP delivery triggered a widespread pro-inflammatory response, whereas CPC-EVs elicited only a localised and limited cytokine activation, suggesting a more favourable safety profile. Furthermore, direct intramyocardial injection of CPC-EVs not only led to efficient mRNA uptake by cardiac tissue and robust VEGF-A protein expression, but also minimal transcriptomic perturbation in the cardiac tissue, as confirmed by RNA-seq. In contrast, LNPs and non-cardiac EVs induced widespread perturbation in the transcriptome of cardiac tissue. Functionally, VEGF-A mRNA delivery via CPC-EVs markedly increased CD31 and α-SMA expression and vessel formation in ex vivo aortic ring assays, confirming enhanced angiogenic potential. Together, these findings support CPC-EVs as a promising platform for achieving enhanced cardiac delivery of mRNA, with reduced liver accumulation, limited off-target transcriptomic perturbation, a more selective cytokine response, and enhanced angiogenic activity in ex vivo assays.

Extracellular vesicles (EVs) have emerged as a promising alternative to cell-based therapies due to their benign safety profiles and favourable pharmacokinetic properties. Tumour EVs, in particular, have shown promise to function as a therapeutic cancer vaccine for various cancer types. However, the efficient and targeted delivery of EVs to dendritic cells, the prominent type of antigen-presenting cells in the body, remains a challenge. Here we report the metabolic tagging of tumour EVs with azido groups for subsequent conjugation of anti-DEC205 via efficient click chemistry. We showed that azido-sugars can metabolically label cancer cells with azido groups and that azido-labelled cells can secrete azido-tagged EVs. Anti-DEC205, upon facile modification with dibenzocyclooctyne, was successfully conjugated onto azido-tagged EVs. Anti-DEC205-conjugated EVs were preferentially internalized by dendritic cells over other types of immune cells, and improved the processing and presentation of EV-encased antigens by dendritic cells. We further demonstrated the ability of anti-DEC205-conjugated EVs to induce enhanced cytotoxic T lymphocyte responses and antitumor efficacy in comparison with control EVs. Our study provides a facile and universal approach to chemically tagging EVs and conjugating targeting moieties to EVs, for targeted modulation of cells of interest.

Pancreatic ductal adenocarcinoma (PDAC) is increasingly recognized as a systemic malignancy, characterized by profound alterations in tumor-host interactions. Small extracellular vesicles (sEVs) in peripheral blood may reflect these alterations and represent a promising minimally invasive source of biomarker information. In this proof-of-principle study, plasma-derived sEVs from patients with PDAC, healthy controls, and a comparative cohort with neuroendocrine lung cancer (NLC) were isolated by differential ultracentrifugation and characterized by western blotting and nanoparticle tracking analysis. Surface marker profiling was performed using the MACSPlex EV Kit IO, followed by univariate, multivariate, and machine-learning-based analyses. PDAC samples exhibited a distinct sEV immunophenotype with coordinated enrichment of angiogenesis-related markers (including CD105 and CD146), immune-regulatory markers (including CD25 and CD40), the coagulation-related marker CD142 and the invasion-associated marker MCSP. Principal component analysis, hierarchical clustering, and Random Forest classification showed exploratory separation of PDAC patients from healthy controls and NLC, supporting the presence of disease-specific vesicle surface marker patterns. In a very small subset of paired samples, descriptive longitudinal analyses illustrated measurable intra-individual changes during chemotherapy. Plasma sEV immunophenotyping is a technically feasible approach for capturing systemic disease-associated alterations in PDAC and provides a foundation for future biomarker-oriented validation studies.

Extracellular vesicles (EVs) released by bacteria are potent mediators of host-microbe interactions. They modulate immune responses, deliver functional molecules and influence disease progression. However, whether bacterial EVs can access the brain and functionally affect host cells remains unclear. In this study, we engineered Escherichia coli-derived EVs by electroporating Cre recombinase mRNA (Ec EVCre) and assessed their transport and functional delivery following intranasal administration. Using mT/mG reporter mice, we observed EV uptake in the olfactory epithelium and recombination-driven GFP expression in a subset of neurons in the olfactory bulb, providing proof-of-concept for the functional delivery of bacterial EV-associated mRNA into the brain. Single-cell RNA sequencing and imaging analyses of the olfactory regions revealed neuronal and immune cell subsets as key EV targets. Microfluidic biochip chamber assays with cultured sensory neurons demonstrated that EVs undergo retrograde axonal transport from neurite terminals to the soma via signalling endosomes. Pharmacological inhibition significantly impaired EV uptake, supporting the involvement of endocytic pathways. In addition to neuronal entry, we discovered that phagocytic cells, including neutrophils and macrophages, can engulf EVCre in the nasal mucosa and migrate into the brain, providing an alternative immune-mediated route for vesicle delivery. Together, these findings indicate that bacterial EVs exploit both neuronal and phagocytic pathways to deliver functional RNA cargo into the brain, providing novel insights into microbial access to the central nervous system and its implications for neuroimmune interactions.

Placenta-derived extracellular vesicles (EVs), particularly exosomes, serve as key mediators that influence metabolic programming in offspring under adverse early nutritional conditions, such as maternal obesity or gestational diabetes. They respond to maternal nutritional disturbances-such as obesity or gestational diabetes-by altering the composition of the miRNAs and proteins they carry. Evidence from in vivo and in vitro studies suggests that these modified EVs influence offspring metabolic programming through multiple putative pathways: regulating fetal pancreatic β-cell development and function, modulating lipogenesis via PPARγ signaling, affecting placental angiogenesis, and promoting inflammation and epigenetic alterations. By transmitting maternal environmental signals to the fetus, placental EVs are hypothesized to contribute to long-term metabolic phenotypes and disease susceptibility. This review critically examines the current evidence positioning placental EVs as key messengers in maternal-fetal communication, evaluates the strength of evidence supporting their role in shaping offspring metabolic health, identifies major knowledge gaps (e.g., limited direct evidence in human offspring, lack of standardized isolation methods), and suggests their potential as early intervention biomarkers or therapeutic targets for preventing metabolic disorders in offspring. We also highlight the need for prospective cohort studies and mechanistic validation in appropriate animal models to establish causality.

Early cancer detection using minimally invasive biomarkers remains a significant challenge, particularly in early-stage disease, where circulating tumor DNA is often below the limit of detection. Extracellular vesicles (EVs), which are actively secreted by viable cancer cells and carry tumor-associated proteins, represent a promising alternative target for liquid biopsy. In this study, we developed EV-finder®, a conceptual framework for the direct detection of EV-associated proteins in serum using proximity extension assay (PEA) technology. Unlike conventional EV-based analytical methods that require prior EV isolation or enrichment, the EV-finder approach enables direct profiling of EV-associated proteins from small serum volumes without an EV isolation step, thereby simplifying the analytical workflow while preserving EV-derived molecular information. Using serum samples from patients with five cancer types (n = 193) and independent healthy controls (n = 138), we established a two-step supervised machine learning framework for cancer detection and tissue-of-origin prediction. The screening model demonstrated promising discriminative performance, with an AUC of 0.985, sensitivity of 0.929, and specificity of 0.957. Notably, no false positives were observed in an external Japanese control cohort, whereas 4 of 29 Korean control samples were classified as cancer-positive. Analysis of EV-associated protein profiles identified both pan-cancer and cancer-type-specific signatures, supporting their value for multi-cancer detection. Collectively, these findings demonstrate the potential feasibility of direct detection of EV-associated proteins from serum using PEA technology and highlight its potential as a scalable and minimally invasive strategy for multi-cancer screening.

Head and neck squamous cell carcinoma (HNSCC) is the seventh most frequently diagnosed cancer worldwide, contributing to approximately 400,000 deaths annually. Recently, the involvement of human papillomavirus (HPV) in the etiopathogenesis of this malignancy, particularly in oropharyngeal squamous cell carcinoma (OPSCC), has been emphasized. In parallel, the potential role of extracellular vesicles (EVs) in the initiation and progression of HNSCCassociated with HPV infection has emerged. EVs are small, membrane-bound vesicles secreted by all cell types, that transport biologically significant cargo, including proteins and nucleic acids. EVs produced by tumor cells interact with non-malignant cells and reprogram the tumor microenvironment, inducing immune suppression, promoting angiogenesis, and facilitating tumor metastasis. Tumor-derived EVs carry DNA and may be responsible for the horizontal transfer of viral structural and functional components, including the E6 and E7 oncoproteins to other cells. The interplay between the mechanisms associated with the HPV replication cycle, EV biogenesis and release, and carcinogenesis represents a novel and still poorly understood area in the investigation of HNSCC development and progression. This review synthesizes recent findings on the distinct roles tumor-derived EVs appear to play in both HPV-dependent and HPV-independent HNSCC.

Extracellular vesicles (EVs) are promising delivery vehicles capable of transporting therapeutic agents across biological barriers. However, native EVs primarily accumulate in liver, spleen and lungs, limiting targeted delivery to disease sites. To enhance their targeting efficiency for the plasma cell cancer multiple myeloma (MM), localized in the bone marrow (BM), we engineered HEK293-derived EVs to display a nanobody (Nb) against the MM cell surface marker CS1. We confirmed enrichment of the Nb construct on engineered EVs and binding of α-CS1 EVs to CS1. In vitro, we found enhanced α-CS1 EV uptake by MM cell cultures. In vivo, we first compared the biodistribution of HEK293-derived native EVs in healthy and MM-bearing mice. Although native EVs reached the BM in both groups, MM-bearing mice showed increased liver and lung accumulation together with reduced BM delivery. α-mCS1 EV delivery to the BM of MM-bearing mice was only slightly increased compared to native EVs, while off-target accumulation also increased. At the cellular level, no changes in EV delivery to MM cells were detected. In conclusion, while CS1 targeting enhances in vitro EV uptake by MM cells, in vivo biodistribution remains suboptimal. Further optimization is needed to improve EV-based drug delivery for MM.

Extracellular vesicles (EVs) have emerged as highly promising natural nanomedicines and nanocarriers, holding transformative potential for the treatment of various diseases. However, the lack of rapid and comprehensive characterization techniques for EV preparation analysis, coupled with the absence of efficient quality control methods, significantly hinders process optimization and large-scale production. To address these challenges, we developed a label-free clustering analysis (LFCA) platform that integrates nano-flow cytometry for particle size distribution analysis with a clustering algorithm to deconvolute EV subpopulations and distinguish them from impurities. This platform enables the rapid quantification of EV component distribution and composition within 5 min using minimal sample input. Leveraging the high-throughput capabilities of LFCA, we established a cascaded workflow incorporating a microcarrier-based 3D culture system, a custom tangential flow filtration device, and multimodal size exclusion chromatography for EV preparation from adipose mesenchymal stem cells. This approach achieves a 4-fold increase in EV yield compared to ultracentrifugation while maintaining comparable purity and preserving EV integrity. Critically, the resulting EVs exhibited enhanced functional potency in pro-angiogenic and anti-inflammatory assays, confirming the clinical relevance of our optimized production system. These advancements provide a scalable solution for EV production, paving the way for clinical applications.

Small extracellular vesicles (sEV) are increasingly reported as biomarkers for the early diagnosis of pancreatic cancer (PC), but the current techniques for isolation and detection of sEV rely on expensive instruments and tedious protocols. In this work, a facile and rapid sEV isolation and detection method (LAPT-sEViso) was developed, which is based on the specific aggregation of GPC-1-positive PC-derived sEV and an aptamer-functionalized DNA long chain produced by rolling circle amplification (RCA-APT). The LAPT-sEViso can efficiently isolate sEV from cell culture medium and serum, showing 45 times higher yield (5.5 × 106 particles mL-1), 1.1 times higher purity (1.66 × 1010 particles mg-1) and 4.9 times higher recovery (80.9%) comparing to the traditional ultracentrifugation method, with only $1000 common instruments and $2.88 reagents/materials in 1 h. Moreover, after simple filtration and on-membrane ELISA, sEV concentration can be instrument-free detected with a limit of detection of 5.62 × 103 particles μL-1 (linear range from 5.0 × 103 to 5.0 × 107 μL-1). The LAPT-sEViso provides an efficient and practical approach for the rapid isolation and detection of sEV, providing a novel approach for the sEV-based liquid biopsy.

Ischemic stroke is a major cause of death and disability, in which neuroinflammation exacerbates injury. Mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs) offer therapeutic potential but face translational hurdles in scalable production, rapid systemic clearance, and inefficient targeted delivery. We engineered an implantable alginate-microsphere system encapsulating EV-secreting MSCs and displaying the RGD-4C peptide (ACDCRGDCFC) on its surface. This platform functions as a bioreactor that sustains the release of functionalized EVs with enhanced targeting to the ischemic brain. Proteomics analyses compared EVs derived from 3D-microsphere cultures and conventional 2D cultures. Efficacy was evaluated in a mouse stroke model with intraperitoneal microsphere implantation, assessing biodistribution, neuroinflammation, microglial polarization, and recovery. The system sustained the release of targeted EVs, demonstrating proteomic enrichment of anti-inflammatory cargo. In vivo, the platform enhanced EV accumulation in the ischemic brain, reduced neuroinflammation, shifted microglia toward a reparative phenotype, and significantly improved neuronal survival and functional recovery. This integrated platform represents a promising preclinical strategy for treating ischemic stroke and has potential applications in other neuroinflammatory diseases. This system circumvents the need for EV extraction and storage while eliminating the peak-and-trough kinetics of bolus injections, and suggests potential for future translation pending further validation.

Cirrhosis is characterized by progressive immune dysregulation, endothelial dysfunction, and haemostatic imbalance. Circulating extracellular vesicles (EVs) have emerged as potential biomarkers reflecting these pathophysiological processes. We aimed to determine whether EVs mirror disease severity and predict liver-related outcomes in cirrhosis. In this prospective single-centre study, patients with compensated, stable decompensated, or acutely decompensated cirrhosis were enrolled. EVs were isolated from platelet-poor plasma and quantified by flow cytometry to characterize platelet-, endothelial-, immune-, and CK18+ EVs, EVs expressing markers associated with endothelial anticoagulant pathways and tissue remodelling. Primary endpoints were first hepatic decompensation in compensated cirrhosis and a composite of further decompensation, acute-on-chronic liver failure, or liver-related mortality in acutely decompensated cirrhosis. Associations were analysed using Fine-Grey competing-risk models. We included 228 patients, including 75 compensated, 44 stable decompensated, and 109 acutely decompensated. Median follow-up was 418 days. EV profiling showed progressive increases in total, platelet-derived, endothelial-, immune-derived, and tissue remodelling-associated EVs across Child-Pugh stages, suggestive of increasing thrombo-inflammatory and endothelial perturbation. First hepatic decompensation occurred in 7 patients with compensated cirrhosis and was associated with higher MELD and Child-Pugh scores, alcohol-related aetiology, and lower platelet count. In univariate competing-risk analyses, higher levels of several EV subpopulations were associated with first decompensation, but these associations disappeared after adjustment for MELD. Among patients with decompensated cirrhosis, 65 developed further decompensation, ACLF, or liver-related death; higher CRP levels were associated with these events, whereas no EV subpopulation was associated with the composite outcome. Circulating EVs reflect cirrhosis severity and are suggestive of progressive thrombo-inflammatory, endothelial, and tissue-remodelling changes. However, EVs were not independently associated with clinical outcomes. Patients with liver cirrhosis demonstrate progressive alterations in inflammation, endothelial function, and haemostasis with progression of liver disease. In this study, circulating extracellular vesicles reflected the severity of cirrhosis, suggestive of progressive thrombo‐inflammatory, endothelial, and tissue‐remodelling processes. However, extracellular vesicles were not independently associated with clinical outcomes.

Poxvirus-based vectors provide a versatile cancer immunotherapy platform, enabling the expression of immunostimulatory molecules and cancer-specific antigens. While infections with pathogenic viruses are well known to modulate extracellular vesicle (EV) biogenesis and function, the extent to which therapeutic poxviral vectors influence EV secretion by immune cells and thereby affect therapeutic efficacy remains underexplored. In this study, we showed that poxviruses, including the clinically relevant Modified Vaccinia Ankara (MVA), stimulate the secretion of small EVs (sEVs) containing viral proteins and immune-related signatures from peripheral blood mononuclear cells (PBMCs). Using an engineered MVA vector, we demonstrated the transfer of virus-encoded therapeutic payloads to sEVs, including the model ovalbumin (OVA)-derived peptide SIINFEKL presented by the class I major histocompatibility complex (MHC I) and the immune activators interleukin-12 (IL-12) and CD40 ligand (CD40L). Depending on the isolation method, these sEVs stimulated SIINFEKL-specific CD8+ T cells with varying efficiencies in vitro. Remarkably, intravenous injection of these sEVs, but also of the soluble secretome from the same cells, into E.G7-OVA lymphoma-bearing mice reduced tumor growth to an extent comparable to the virus itself. Taken together, our findings indicate that EVs released from immune cells infected with engineered therapeutic poxviruses exert potent antitumor activity. These vesicles represent actionable mediators whose secretion and functionalization can be harnessed to improve viral vector-based immunotherapies, as well as being considered as therapeutic vectors in their own.

Concentrations of extracellular vesicles (EVs) and other particles are measured in plasma for biomarker exploration. A commonly used method, flow cytometry, requires plasma dilution to ensure single-particle detection. Since plasma EVs are outnumbered by variable concentrations of lipoproteins, dilution differs between the plasma samples. Dilution can result in misidentification of fluorescent background signals as labeled EVs. This phenomenon, called the dilution paradox, leads to overestimation of plasma EV concentrations, and likely impacts conclusions from earlier performed biomarker studies. This study reevaluated earlier conclusions from our clinical biomarker studies Antiplatelet Therapy Effect on Extracellular Vesicles (AFFECT EV) and Circulating Nanotraces to Identify the Cause of Stroke (CINTICS), by taking the dilution paradox into account. We developed a model that quantifies the fluorescent background and estimates whether a flow cytometry measurement is dominated by fluorescent background, that is, if the measurement is unreliable. This model was applied to the original datasets of the AFFECT EV and CINTICS studies to identify and exclude unreliable measurements. We investigated whether exclusion of unreliable data affects the original conclusions. Our model estimated that 47% (1156/2457) of the evaluated measurements are unreliable, and conclusions from both biomarker studies required adjustment. Our model improves reliability and reproducibility of EV concentration measurements using flow cytometry. We recommend to reanalyze earlier EV flow cytometry studies using our model and to use a fixed dilution factor in future EV flow cytometry studies to enable reliable EV concentration measurements.

Extracellular vesicles (EVs) contain RNA, protein, lipids, and other molecules from their parental cell type, which led to the hypothesis that urine-derived stem cell EVs (USC-EVs) could have potent regenerative effects in the setting of acute kidney injury (AKI). We tested this hypothesis in two different in vitro AKI models and an in vivo AKI model. To test USC-EVs ability to recover human kidney cells from nephrotoxicity, HK-2 human proximal tubular epithelial cell line and human kidney organoids derived from induced pluripotent stem cells were treated with 5 &#x3bc;M cisplatin for 48 h followed by 10 &#x3bc;g USC-EVs for 48 h. We also tested USC-EVs in the unilateral nephrectomy followed by ischemia reperfusion model (UNIRI) AKI model to explore systemic effects in animals. USC-EV treatment of human kidney cells resulted in increased cellular proliferation (P &#x2264; 0.0001), decreased cytotoxicity (P < 0.0001), and lowered oxidative stress (P &#x2264; 0.01) as compared with cisplatin alone groups. In mouse models, we found improved pathology as well as reduced kidney injury marker KIM-1 expression in the USC-EV treated group as compared with the UNIRI alone group. Analysis of USC-EV miRNA-targeting pathways was associated with epithelial cell proliferation, migration, and positive regulation of MAPK signaling pathway. As predicted by this analysis, we found increased phosphorylation of the MAPK downstream target ERK1/2 in USC-EV-treated HK-2 cells. This study suggests that much of the reported benefit of USCs is due to their EVs and supports further development of USC-EVs as a potential next-generation therapeutic for AKI.NEW & NOTEWORTHY Urine-derived stem cell extracellular vesicles (USC-EVs) increased proliferation while lowering apoptosis and lipid peroxidation in human kidney cells and organoids treated with toxic chemotherapy. In the acute kidney injury (AKI) model mice, we found lowered inflammation, tubular dilation, casts, and kidney injury marker neutrophil gelatinase-associated lipocalin (NGAL) with USC-EV treatment. The USC-EVs carry microRNA cargo that bioinformatic tools predict will affect multiple pathways, including the MAPK cascade. Altogether, our results suggest that USC-EVs may improve recovery from AKI.

Extracellular vesicles (EVs) secreted by glioblastoma (GBM) cells carry tumor-specific biomarkers such as microRNA-1246 (miR-1246), offering significant potential for noninvasive GBM detection. However, the efficient isolation of EVs from complex biofluids and ultrasensitive detection of their low-abundance miRNAs encapsulated in EVs remain formidable challenges. To address this, we designed a multifunctional nanoplatform of UiO-66-NH2@Au@MSDC (UAM), which integrates EV capture and in situ miRNA analysis. The initial construction of UiO-66-NH2@Au (UA) provided a high density of thiol-binding sites for the attachment of mercapto (SH)-spacer-DNA-cholesterol (MSDC) probes to form UiO-66-NH2@Au@MSDC (UAM). The abundant cholesterol motifs on these probes subsequently enabled highly efficient EV capture, achieving a recovery rate of 84.8 &#xb1; 2.4% under a low centrifugal force of 8000g while preserving EV structural integrity and bioactivity. For the downstream analysis of EVs, we developed an innovative EXPAR-based strategy, termed Y-EXPAR, to address the specificity challenge in detecting miR-1246. The entire detection system employs a temperature-synergistic strategy at 55 &#xb0;C, which simultaneously triggers the membrane fusion of EVs with synthetic cationic liposomes (preloaded with Y-EXPAR reagents) and activates the Y-EXPAR. The resulting fusion creates confined nanoreactors that concentrate trace amounts of miR-1246, thereby enabling ultrasensitive detection with detection limits of 1.4 fM for miR-1246 and 22 particles/&#x3bc;L for EVs. This integrated platform overcomes key bottlenecks in EV-based liquid biopsy, providing a practical, low-equipment-demand approach for the early diagnosis of GBM and laying a foundation for advancing precision oncology diagnostics.

Cell-derived extracellular vesicles (EVs) attract growing interest as biologically active, acellular platforms for therapeutic and diagnostic use in regenerative medicine, immunomodulation, and drug delivery. While EV-based products advance into clinical development worldwide, their acellular nature maps non-uniformly onto pre-existing categories. In the European Union, the 2025 European Medicines Agency/Committee for Advanced Therapies (EMA/CAT) guideline clarifies that "not substantially modified extracellular vesicles" fall outside the current advanced therapy medicinal products (ATMPs) definition, requiring case-by-case development within other medicinal-product frameworks. Conversely, the United States Food and Drug Administration (FDA) regulates exosome/EV products for disease treatment as drugs and biological products subject to premarket requirements, while other regions apply existing drug and regenerative-medicine-related instruments using jurisdiction-specific classification criteria. This heterogeneity creates challenges for regulatory positioning, quality assessment, comparability, and cross-regional clinical development. We critically examine the evolving landscape of EV-based therapeutics, focusing on regulatory, technical, safety, and ethical considerations from a global perspective. We argue EV-based products should be developed within existing medicinal product frameworks (e.g., biologics, ATMP-related instruments), provided regulatory tools are applied consistently, and operational grey zones (e.g., classification criteria, critical quality attributes (CQAs), potency strategies, and comparability) are identified for clarification. Without proposing normative frameworks, this work provides a state-of-the-art synthesis intended to support ongoing regulatory dialogue across regions. The discussion is relevant for international agencies such as the EMA, FDA, the Pharmaceuticals and Medical Devices Agency (PMDA) of Japan, the World Health Organization (WHO), and the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH), as well as for scientific societies such as the International Society for Extracellular Vesicles (ISEV) promoting methodological convergence. By aligning scientific insight with regulatory awareness, this review supports advancing EV-based therapies toward safe, reproducible, and internationally credible clinical use.

Gut microbial dysbiosis has been observed in several diseases. Although causal links and direct effects on host cells remain unclear, bacteria-derived extracellular vesicles (BEVs) from the gut microbiota may regulate the host immune response. We examined the impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection on the gut microbiome and BEVs release, and the effects of released BEVs on cytokine responses in monocyte-derived cell lines. Fecal samples from 17 patients with coronavirus disease 2019 (COVID-19) and 20 healthy individuals were collected to isolate bacterial and BEV fractions. Parental BEV-releasing bacteria were identified from vesicle-encapsulated bacterial DNA by 16S rRNA gene sequencing. Patients with COVID-19 exhibited altered gut microbiota composition and the profile of bacterial DNA-containing BEVs (dcBEVs) release compared to healthy controls. BEVs from patients, but not from healthy individuals, significantly changed cytokine levels in U937 monocyte cells. Following COVID-19 recovery, dcBEV profiles diverged into two distinct groups: those that retained the capacity to induce cytokines in monocytes and those that lost this functionality. BEVs from single bacterial cultures within families altered after COVID-19 onset affected the expression of genes in monocytes, primarily immune-response genes, notably chemokine ligands and G protein-coupled receptors. SARS-CoV-2-induced dysbiosis alters the profile of dcBEVs release, thereby modulating the host immune response and potentially contributing to COVID-19 pathogenesis.