Extracellular vesicles and circulating nucleic acids最新文献

{"title":"Optimized enrichment of circulating extracellular vesicles from whole blood samples using PROSPR.","authors":"Jose Antonio Sánchez Milán, María Mulet, Maria Font-Alberich, Magdalena Torres, Yormaris Castillo, Silvia Pico, Oriol Yuguero, Gerard Piñol-Ripoll, Filip Bellon, Montserrat Gea-Sánchez, Siu Kwan Sze, Raj N Kalaria, Xavier Gallart-Palau, Aida Serra","doi":"10.20517/evcna.2026.18","DOIUrl":"10.20517/evcna.2026.18","url":null,"abstract":"<p><p><b>Aim:</b> Whole blood constitutes a robust and readily available resource for the identification and characterization of systemically circulating substances. Despite the relevant number of blood specimens archived in biobanks globally, their utilization in extracellular vesicle (EV) research remains considerably underexploited. In this study, we present an optimized PRotein Organic Solvent PRecipitation (PROSPR) workflow, specifically designed for the rapid and standardized enrichment of EVs directly from untreated blood samples. <b>Methods:</b> PROSPR workflow was applied for EV enrichment directly from untreated blood. Comprehensive biophysical characterization was performed using dedicated flow cytometry, tunable resistive pulse sensing, and transmission electron microscopy. In parallel, discovery-driven lipidomics and high-depth proteomics were applied to assess the suitability of PROSPR-derived EVs for advanced systems biology and multi-omics integration. <b>Results:</b> PROSPR consistently yielded highly purified EV preparations that preserved canonical vesicular morphology, structural integrity, and stable ζ-potentials, indicative of intact membrane characteristics. Multi-omics analyses revealed distinct and coordinated lipid-protein signatures uniquely enriched in EV populations derived from whole blood. These signatures predominantly reflected platelet- and mitochondria-associated cargo that was not detectable in corresponding plasma-derived EV fractions, highlighting key molecular features missed by plasma-based EV profiling approaches. <b>Conclusion:</b> Consequently, the optimized PROSPR workflow presented here offers a robust and accessible methodological strategy to fully utilize dormant, archived blood resources within global and national biobanks, thereby enabling comprehensive mechanistic insights and facilitating the discovery of novel diagnostic and prognostic circulating biomarkers.</p>","PeriodicalId":520322,"journal":{"name":"Extracellular vesicles and circulating nucleic acids","volume":"7 3","pages":"1084-1107"},"PeriodicalIF":8.5,"publicationDate":"2026-07-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13673457/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149218016","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Glycosylated extracellular vesicles drive a metabolic interplay between gastric cancer cells and adipocytes.","authors":"Cátia C Ramos, Oihane E Albóniga, Adriana R Rodrigues, Clara Garcia-Vallicrosa, Joana Peixoto, Rui Fernandes, Bruno Cavadas, Alexandra M Gouveia, Celso A Reis, Juan M Falcon-Pérez, Daniela Freitas","doi":"10.20517/evcna.2026.16","DOIUrl":"10.20517/evcna.2026.16","url":null,"abstract":"<p><p><b>Aim:</b> Tumor cells and extracellular vesicles (EVs) dynamically interact with adipocytes, contributing to tumor progression and systemic metabolic dysfunctions. While the role of glycosylation in this process remains unclear, glycans facilitate communication between tumors and their surrounding environment. Here, we aimed to elucidate the pivotal role of sialyl-Tn (STn), a tumor-associated glycan, in driving a bidirectional metabolic reprogramming between gastric cancer cells and adipocytes. <b>Methods:</b> EVs were isolated from gastric cancer cells with distinct glycosylation profiles by ultracentrifugation and characterized by transmission electron microscopy, nanoparticle tracking analysis, and western blotting. Flow cytometry was used to quantify EV uptake by adipocytes. EV-mediated adipocyte browning and lipolysis were measured by real-time quantitative polymerase chain reaction, glycerol and fatty acid release, perilipin immunofluorescence, and western blotting analysis of lipid synthesis and hydrolysis enzymes. The reciprocal effects of beige adipocytes on tumor cells were evaluated using fatty acid uptake assays, metabolomics, RNA sequencing, Seahorse metabolic flux, organelle number, proliferation, and migration assays. <b>Results:</b> STn-positive gastric cancer cells and EVs induced white-into-beige transdifferentiation. These cells exhibited an enhanced capacity to release glycerol and fatty acids, which were predominantly taken up by gastric cancer cells displaying STn. As a feedback mechanism, gastric cancer cells shift their metabolism towards fatty acid oxidation, concomitant with increased mitochondrial and endosomal biogenesis, and enhanced motility. <b>Conclusion:</b> Our findings reveal a key role for the tumor-associated glycan STn in metabolic reprogramming of adipocytes by tumor cells and EVs, opening new avenues for targeted therapies in cancer patients experiencing metabolic dysfunctions, such as cancer cachexia.</p>","PeriodicalId":520322,"journal":{"name":"Extracellular vesicles and circulating nucleic acids","volume":"7 3","pages":"1056-1083"},"PeriodicalIF":8.5,"publicationDate":"2026-07-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13673474/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149218055","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Bacterial membrane vesicles as effective immunotherapeutic agents in melanoma, colon, and breast cancer.","authors":"Jan Lötvall, Negar Ordouzadeh, Kyong-Su Park","doi":"10.20517/evcna.2026.07","DOIUrl":"10.20517/evcna.2026.07","url":null,"abstract":"<p><p><b>Aim:</b> Bacterial outer membrane vesicles are highly immunogenic due to their native bacterial antigens, however，their toxic components trigger systemic inflammation. We previously developed non-toxic synthetic bacterial vesicles (SyBV), and confirmed their anti-tumor effects when co-administered with tumor antigens. Building on this, the present study aimed to extensively assess the immunotherapeutic potential of local SyBV administration, leveraging endogenous tumor antigens across multiple cancer models. <b>Methods:</b> SyBV were generated from <i>Escherichia coli</i> membranes via lysozyme-induced cell lysis and ionic stress. Multiple intratumoral administrations of SyBV at different doses were evaluated in melanoma-bearing mice. Therapeutic efficacy was further assessed according to initial tumor size and across multiple tumor models, including melanoma, colon, breast, kidney, and lung cancers. Systemic anti-tumor effects were also examined by evaluating tumor growth at distant, non-injected tumor sites following local SyBV immunization. <b>Results:</b> High-dose SyBV treatment effectively suppressed tumor growth in melanoma-bearing mice. Anti-tumor efficacy varied depending on initial tumor size, with the strongest effects observed when treatment was initiated at smaller tumor volumes. Local SyBV immunization also induced systemic anti-tumor responses, reducing tumor growth at distant tumor sites. Furthermore, SyBV demonstrated significant immunotherapeutic efficacy in colon and breast cancer models, but not in kidney or lung cancer models, highlighting their therapeutic potential in selected cancer types. <b>Conclusion:</b> Our findings show that engineered bacterial vesicles serve as effective standalone cancer immunotherapeutics, allowing simpler drug formulations and potentially improving their clinical translation.</p>","PeriodicalId":520322,"journal":{"name":"Extracellular vesicles and circulating nucleic acids","volume":"7 3","pages":"1043-1055"},"PeriodicalIF":8.5,"publicationDate":"2026-07-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13678171/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149225705","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Immunoregulatory extracellular vesicles in multiple myeloma patients' peripheral blood.","authors":"Irma Airoldi, Debora Soncini, Danilo Marimpietri, Fabio Malavasi, Alberto L Horenstein, Matteo Garibotto, Michele Cea, Fabio Morandi","doi":"10.20517/evcna.2025.180","DOIUrl":"10.20517/evcna.2025.180","url":null,"abstract":"<p><p><b>Aim:</b> Extracellular vesicles (EVs) mediate cell-to-cell communication and co-contribute in cancer progression and modulation of immune responses. The aim of this work was to characterize the phenotype and function of EVs isolated from the peripheral blood (PB) of multiple myeloma (MM) patients, with particular attention to adenosinergic ectoenzymes. <b>Methods:</b> EVs were isolated by centrifugation of PB plasma samples from newly diagnosed (ND) MM patients and healthy donors (HD). EVs were characterized by nanoparticle tracking analysis (NTA). Flow cytometry was used to assess the expression of CD138 (a tumor-associated antigen), nicotinamide phosphoribosyltransferase/pre-B cell colony enhancing factor (NAMPT/PBEF1), and adenosinergic ectoenzymes CD38, CD39, CD203a, and CD73. Functional studies were performed by assessing T-cell proliferation <i>in vitro</i> using carboxyfluorescein succinimidyl ester (CFSE) dilution and flow cytometry, with or without EVs to the culture. <b>Results:</b> The PB EVs size was comparable between MM patients and HDs, whereas EV concentration was significantly higher in MM patients. EVs from MM patients expressed higher levels of CD138, PBEF, CD38, and CD73 than those from HDs. In addition, CD138⁺ EVs from MM patients expressed higher levels of PBEF, CD38, CD39, and CD73 than CD138^- counterparts. Finally, MM-derived EVs inhibited T-cell proliferation <i>in vitro</i>. This inhibition correlated with the expression of CD138 and CD203a. <b>Conclusion:</b> This study provides the first phenotypic and functional characterization of EVs obtained from the PB of MM patients. The presence of functional adenosinergic molecules may represent a novel immune-escape mechanism operating at distant sites. Nonetheless, further studies are required to define specific contributions of adenosinergic ectoenzymes and other immune-regulatory mechanisms.</p>","PeriodicalId":520322,"journal":{"name":"Extracellular vesicles and circulating nucleic acids","volume":"7 3","pages":"1029-1042"},"PeriodicalIF":8.5,"publicationDate":"2026-07-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13673463/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149218034","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Terpenoid-enriched <i>Curcuma wenyujin</i> nanovesicles for suppressing inflammation and restoring lipid homeostasis in MASH.","authors":"Lin Liu, Jiale Niu, Yulong Sun, Zhuoyan He, Ziming Jiao, Guoen Li, Ganglin Wang, Fangjun Luo, Wei Li","doi":"10.20517/evcna.2025.181","DOIUrl":"10.20517/evcna.2025.181","url":null,"abstract":"<p><p><b>Aim:</b> This study investigates the potential of <i>Curcuma wenyujin</i>-derived nanovesicles (CW-DNVs) to ameliorate metabolic dysfunction-associated steatohepatitis (MASH) and explores their underlying mechanism, focusing on hepatic macrophage accumulation and the regulation of lipid metabolism. <b>Methods:</b> CW-DNVs were isolated via ultracentrifugation and sucrose gradient purification, and their physicochemical properties, cellular uptake, and <i>in vivo</i> biodistribution were characterized. Anti-inflammatory and lipid-lowering effects were evaluated in liver macrophages, hepatocytes, and a high-fat diet (HFD)-induced MASH mouse model. Lipidomic, small-molecule, and small RNA (sRNA) cargoes were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and RNA sequencing. <b>Results:</b> CW-DNVs were spherical (~211 nm diameter), had a zeta potential of -27.4 mV, and were enriched in lipids, proteins, sRNAs, and terpenoids like well-known bioactive curcumenol and germacrone. Following intraperitoneal injection, they preferentially accumulated in Kupffer cells and were cleared within 7 days. In HFD-fed mice, CW-DNVs reduced body weight gain, hepatic steatosis, serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), triglycerides (TG), total cholesterol (TC), and hepatic interleukin (IL)-6 levels. Mechanistically, they upregulated peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) and microsomal triglyceride transfer protein (MTTP), and downregulated fatty acid synthase (FASN), promoting lipid oxidation and export. <i>In vitro</i>, CW-DNVs suppressed lipopolysaccharide-activated IL-6, IL-1β, and tumor necrosis factor-alpha (TNF-α) in macrophages and reduced oleic acid-induced lipid accumulation in hepatocytes. sRNA sequencing identified predominantly rRNA-derived fragments (not canonical miRNAs); however, anti-inflammatory activity was primarily attributed to terpenoid components. <b>Conclusion:</b> CW-DNVs exert dual functionality in modulating macrophage inflammation and lipid metabolism. Loaded with bioactive terpenoids, they represent an effective natural nanoplatform for MASH therapy.</p>","PeriodicalId":520322,"journal":{"name":"Extracellular vesicles and circulating nucleic acids","volume":"7 2","pages":"1010-1028"},"PeriodicalIF":8.5,"publicationDate":"2026-06-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13367161/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148451280","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Mesenchymal stem cell-derived extracellular vesicles for dry eye disease: principles, progress, and challenges.","authors":"Yuting Feng, Mingqi Zhang","doi":"10.20517/evcna.2025.174","DOIUrl":"10.20517/evcna.2025.174","url":null,"abstract":"<p><p>Dry eye disease (DED) is a multifactorial ocular surface disorder characterized by disruption of tear film and/or ocular surface homeostasis that represents a significant global public health concern. This review summarizes the pathogenesis of DED, with particular emphasis on the central role of ocular surface inflammation. It further comprehensively evaluates the therapeutic rationale, preclinical advances, and translational challenges associated with mesenchymal stem cell-derived extracellular vesicle (MSC-EV) therapy for DED. MSC-EVs exhibit numerous properties, including anti-inflammatory, immunomodulatory, reparative, and regenerative effects, and offer key advantages such as a cell-free nature, low immunogenicity, minimal tumorigenic risk, high stability, and suitability for topical administration. Through advanced strategies such as cargo engineering, hybrid design, biomaterial-assisted delivery, and genetic engineering, the therapeutic performance of MSC-EVs can be further optimized, enabling targeted delivery, improved retention and bioavailability, and precise immunomodulation. In addition, three-dimensional ocular surface and <i>ex vivo</i> models help overcome the limitations of traditional animal models in replicating human ocular physiology. Among available sources, umbilical cord-derived MSC-EVs represent one of the most promising candidates for clinical translation. Despite these advances, several challenges remain, including ocular-specific anatomical and physiological barriers, limited pharmacological characterization, lack of standardized large-scale production and storage protocols, incomplete toxicological and microbiological safety evaluation, and the absence of a unified regulatory framework. Overall, MSC-EVs represent a potentially transformative therapeutic strategy for the management of refractory DED. However, as most current evidence remains preclinical, further validation in human-relevant models and clinical studies is essential before definitive conclusions can be drawn.</p>","PeriodicalId":520322,"journal":{"name":"Extracellular vesicles and circulating nucleic acids","volume":"7 2","pages":"973-1009"},"PeriodicalIF":8.5,"publicationDate":"2026-06-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13367160/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148451393","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Advances in microbial extracellular vesicles: synthetic biology platforms and medical applications.","authors":"Zi-Yue-Er Tang, Xin-Lu Yang, Yan-Wen Zhou, Hang Zou, Ying Li, Hong Wu, Dai-Xu Wei","doi":"10.20517/evcna.2025.103","DOIUrl":"10.20517/evcna.2025.103","url":null,"abstract":"<p><p>Extracellular vesicles (EVs), typically ranging from 20 to 400 nanometers in diameter, are membrane-bound structures released into the extracellular environment by bacteria via specific secretion mechanisms. Consequently, these vesicles play a crucial role in bacterial physiological regulation and communication with hosts. Compared with EVs derived from plants and animals, Microbial extracellular vesicles (MEVs) offer distinct advantages, including lower production costs, higher yields, and greater abundance. This review outlines the biogenesis and release mechanisms of MEVs, and highlights how synthetic biology tools and platforms can be leveraged to engineer these vesicles, such as enhancing their production, modifying their cargo, and tailoring their surface properties. Furthermore, this article examines the promising biomedical applications of engineered MEVs, including targeted drug delivery, immune and inflammatory modulation, the discovery of disease biomarkers and therapeutic development. However, clinical translation of MEVs faces considerable challenges, primarily due to the lack of standardized, universally applicable isolation and purification protocols. This review therefore summarises contemporary extraction methods, functional characteristics and applications of MEVs alongside examples of recent MEV modifications. Ultimately, this work aims to bridge existing knowledge gaps and facilitate the development of MEV-based therapeutic strategies.</p>","PeriodicalId":520322,"journal":{"name":"Extracellular vesicles and circulating nucleic acids","volume":"7 2","pages":"945-972"},"PeriodicalIF":8.5,"publicationDate":"2026-06-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13367158/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148451368","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Neuropilin-1 enriched mesenchymal stem cell derived-exosomes alleviate vascular hyperpermeability in acute lung injury.","authors":"Qinyi Deng, Yifan Lu, Linyan Yuan, Shiyu Gao, Xiaoyan Hu, Van Minh Le, Xiaoyan Chen, Xin Liang, Yun Feng","doi":"10.20517/evcna.2025.169","DOIUrl":"10.20517/evcna.2025.169","url":null,"abstract":"<p><p><b>Aim:</b> Mesenchymal stem cells (MSCs) have shown therapeutic potential in acute lung injury (ALI); however, the key functional components and their roles in regulating pulmonary vascular endothelial permeability remain unclear. This study aimed to identify functional proteins within MSC-derived exosomes (MSC-Exo) and elucidate their roles in regulating pulmonary vascular endothelial permeability to enhance MSC-Exo-based therapy. <b>Methods:</b> Proteomic analysis identified neuropilin-1 (NRP1) as a candidate functional protein in MSC-Exo. Functional assays, including annexin V flow cytometry for apoptosis, colony formation assays for proliferation, and Transwell migration and wound healing assays, were performed to assess these processes in injured pulmonary microvascular endothelial cells (PMVECs). Immunofluorescence and Western blotting were used to evaluate NRP1 localization, p130Cas phosphorylation and matrix metalloproteinases 1 (MMP1)/9 expression. In ALI rat models, histopathology, Evans blue extravasation, and immunohistochemistry were used to assess lung injury and vascular permeability. <b>Results:</b> NRP1-enriched MSC-Exo reduced apoptosis, enhanced colony formation, and promoted migration in PMVECs. Mechanistically, NRP1 modulated the Bcl-2/Bax ratio, inhibited caspase 3/9 activation, and promoted p130Cas phosphorylation with increased MMP1/9 expression. NRP1 also colocalized with PDGFR-α, suggesting a potential role in PDGF-BB signaling. <i>In vivo</i>, NRP1-overexpressing exosome showed superior efficacy in reducing pulmonary edema, vascular leakage, and restoring tight junction proteins, whereas NRP1 depletion impaired these effects. <b>Conclusion:</b> NRP1 in MSC-Exo enhances endothelial repair by promoting proliferation and migration while inhibiting apoptosis. <i>In vivo</i>, NRP1-enriched MSC-Exo improve vascular barrier function and attenuate lung injury, supporting their therapeutic potential in ALI.</p>","PeriodicalId":520322,"journal":{"name":"Extracellular vesicles and circulating nucleic acids","volume":"7 2","pages":"904-926"},"PeriodicalIF":8.5,"publicationDate":"2026-06-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13367150/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148451386","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Molecular profiling and functional analyses of umbilical cord mesenchymal stem cell-derived extracellular vesicles for dry eye diseases.","authors":"Qiao-Yu Hsu, Chia-Ni Hsiung, Hsin-Hung Cheng, Wen-Yu Lien, Wen-Hsien Lin, Martin Sieber, Chi-Chih Kang","doi":"10.20517/evcna.2025.177","DOIUrl":"10.20517/evcna.2025.177","url":null,"abstract":"<p><p><b>Aim:</b> Dry eye disease (DED) is a multifactorial disorder characterized by tear film instability, ocular surface damage, and inflammation. This study investigates the molecular landscape and the therapeutic potential of umbilical cord mesenchymal stem cell-derived extracellular vesicles (UCMSC-EVs) as a standardized, cell-free treatment for DED. <b>Methods:</b> UCMSC-EVs were produced from UCMSCs in a scalable 3D bioreactor system and enriched via tangential flow filtration (TFF). The physicochemical properties of UCMSC-EVs were characterized. The molecular cargo of UCMSC-EVs was analyzed via proteomics analysis and microRNA (miRNA) microarray profiling across multiple production lots. Functional potency of UCMSC-EVs was assessed using a human corneal epithelial cell line (HCE-S) wound healing assay and two human inflammation models. <b>Results:</b> UCMSC-EVs exhibited a consistent cargo profile enriched with proteins and miRNAs targeting DED-related pathways, with high proteomic correlation (Pearson's <i>r</i> > 0.8) across independent lots. Functional assays demonstrated that UCMSC-EVs promoted HCE-S wound healing and suppressed pro-inflammatory cytokines in a dose-dependent manner. Furthermore, a significant negative correlation was identified between PEDF (Pigment Epithelium-Derived Factor) cargo concentration and interferon gamma (IFN-γ) secretion (Pearson's <i>r</i> = -0.724, <i>P</i> = 0.005), providing a quantitative link between molecular identity and anti-inflammatory potency. <b>Conclusion:</b> UCMSC-EVs possess favorable physicochemical and molecular characteristics, and demonstrate robust regenerative and anti-inflammatory properties <i>in vitro</i>. The high manufacturing consistency and the identification of key molecular drivers, such as PEDF, support the potential of UCMSC-EV as a well-characterized and viable cell-free therapy for mitigating DED progression.</p>","PeriodicalId":520322,"journal":{"name":"Extracellular vesicles and circulating nucleic acids","volume":"7 2","pages":"927-944"},"PeriodicalIF":8.5,"publicationDate":"2026-06-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13369709/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148458309","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Comparison of extracellular vesicle isolation methods reveals method-dependent protein and miRNA profiles in saliva.","authors":"Elodie Simphor, Jérémy Boulestreau, Romane Marchal, Thi Nhu Ngoc Van, Franck Molina, Malik Kahli","doi":"10.20517/evcna.2025.176","DOIUrl":"10.20517/evcna.2025.176","url":null,"abstract":"<p><p><b>Aim:</b> Salivary extracellular vesicles (EVs) are promising non-invasive biomarkers for disease diagnosis and monitoring, reflecting both systemic and local physiological states. However, the diversity of EV isolation protocols and the possibility that each method enriches distinct EV subpopulations remain major barriers to reproducibility and data comparability. This study aimed to evaluate the impact of different EV isolation methods on the yield, purity, and molecular composition of salivary EVs from healthy volunteers, thereby clarifying how these methods affect the potential use of salivary EVs as a non-invasive biomarker source for disease diagnosis and monitoring. <b>Methods:</b> We conducted a comprehensive comparison of three EV isolation methods - ultracentrifugation (UC), polyethylene glycol (PEG)-based co-precipitation (Q), and immunoaffinity capture (M) - to evaluate their impact on EV yield, purity, and molecular composition. Salivary EVs from healthy volunteers were analyzed using proteomic and small RNA sequencing approaches. <b>Results:</b> Principal component analysis revealed clear isolation method-dependent clustering, where M-derived EVs displayed the most distinct profile. The UC and Q methods yielded broader proteomic repertoires with higher total protein content, while M-isolated EVs exhibited greater purity and enrichment of trafficking- and lysosome-associated proteins. Among the 731 microRNAs (miRNAs) analyzed, 28 were consistently altered across methods and 65 uniquely enriched in M isolates. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) confirmed the key directional trends. These 93 method-dependent miRNAs were predicted to target genes associated with synaptic structure and neurodegenerative pathways. <b>Conclusion:</b> These findings show that EV isolation methodology profoundly influences the cargo composition of salivary EVs and suggest that M can isolate specific EV populations that may better meet diagnostic requirements.</p>","PeriodicalId":520322,"journal":{"name":"Extracellular vesicles and circulating nucleic acids","volume":"7 2","pages":"878-903"},"PeriodicalIF":8.5,"publicationDate":"2026-06-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13367162/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148451334","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}