Search Results (1,133)

Background/Objectives: Chronic kidney disease of unknown etiology (CKDu) disproportionately affects young male agricultural workers who are otherwise healthy. There is a scarcity of biomarkers for early detection of this type of kidney disease. We hypothesized that small extracellular vesicles (sEVs) released into urine may provide novel biomarkers. Methods: We obtained two urine samples at the start and the end of a workday in the fields from a limited set of workers with and without kidney impairment. Isolated sEVs were characterized for size, surface marker expression, and purity and, subsequently, their lipid composition was determined by mass spectrometry. Results: The number of particles per ml of urine normalized to osmolality and the size variance were larger in workers with possible CKDu than in control workers. Surface markers CD9, CD63, and CD81 are characteristic of sEVs and a second set of surface markers suggested the kidney as the origin. Differential expression of CD25 and CD45 suggested early inflammation in CKDu workers. Of the twenty-one lipids differentially expressed, several were bioactive, suggesting that they may have essential functions. Remarkably, fourteen of the lipids showed intermediate expression values in sEVs from healthy individuals with acute creatinine increases after a day of work. Conclusions: We identified twenty-one possible lipid biomarkers in sEVs isolated from urine that may be able to distinguish agricultural workers with early onset of CKDu. Differentially expressed surface proteins in these sEVs suggested early-stage inflammation. This pilot study was limited in the number of workers evaluated, but the approach should be further evaluated in a larger population. Full article

Extracellular vesicles (EVs), nanoscale membrane-enclosed particles, are natural carriers of proteins and nucleic acids. Microalgae are widely used as a source of bioactive substances in the food and cosmetic industries and definitely have a potential to be used as the producers of EVs for biomedical applications. In this study, the extracellular vesicles isolated from the culture medium of two unicellular microalgae, Chlamydomonas reinhardtii (Chlamy-EVs) and Parachlorella kessleri (Chlore-EVs), were characterized by atomic force microscopy (AFM), cryo-electronic microscopy (cryo-EM), and nanoparticle tracking analysis (NTA). The biocompatibility with human cells in vitro (HEK-293T, DF-2 and A172) and biodistribution in mouse organs and tissues in vivo were tested for both microalgal EVs. An exogenous therapeutic protein, human heat shock protein 70 (HSP70), was successfully loaded to Chlamy- and Chlore-EVs, and its efficient delivery to human glioma and colon carcinoma cell lines has been confirmed. Additionally, in order to search for potential therapeutic biomolecules within the EVs, their proteomes have been characterized. A total of 105 proteins were identified for Chlamy-EVs and 33 for Chlore-EVs. The presence of superoxide dismutase and catalase in the Chlamy-EV constituents allows for considering them as antioxidant agents. The effective delivery of exogenous cargo to human cells and the possibility of the particle yield optimization by varying the microalgae growth conditions make them favorable producers of EVs for biotechnology and biomedical application. Full article

Background: Melanoma metastasis, driven by tumor microenvironment (TME)-mediated crosstalk facilitated by extracellular vesicles (EVs), remains a major therapeutic challenge. A critical barrier to clinical translation is the overlap in protein cargo between tumor-derived and healthy cell EVs. Objective: To address this, we developed Scaffold-free Functional Deconvolution (SFD), a novel computational approach that leverages a comprehensive healthy cell EV protein database to deconvolute non-oncogenic background signals. Methods: Beginning with 1915 proteins (identified by MS/MS analysis on an Orbitrap Fusion Lumos Mass Spectrometer using the IonStar workflow) from melanoma EVs isolated using REIUS, SFD applies four sequential filters: exclusion of normal melanocyte EV proteins, prioritization of metastasis-linked entries (HCMDB), refinement via melanocyte-specific databases, and validation against TCGA survival data. Results: This workflow identified 21 high-confidence targets implicated in metabolic-associated acidification, immune modulation, and oncogenesis, and were analyzed for reduced disease-free and overall survival. SFD’s versatility was further demonstrated by surfaceome profiling, confirming enrichment of H7-B3 (CD276), ICAM1, and MIC-1 (GDF-15) in metastatic melanoma EV via Western blot and flow cytometry. Meta-analysis using Vesiclepedia and STRING categorized these targets into metabolic, immune, and oncogenic drivers, revealing a dense interaction network. Conclusions: Our results highlight SFD as a powerful tool for identifying clinically relevant biomarkers and therapeutic targets within melanoma EVs, with potential applications in drug development and personalized medicine. Full article

Plant-derived extracellular vesicles (PDEVs) are nanoscale, phospholipid bilayer-enclosed vesicles secreted by living cells through cytokinesis under physiological and pathological conditions. Owing to their high biocompatibility and stability, PDEVs have attracted considerable interest in regenerative medicine applications. They can exhibit the capacity to enhance cellular proliferation, migration, and multi-lineage differentiation through immunomodulation, anti-inflammation effects, antioxidative protection, and tissue regeneration mechanisms. Given their abundant availability, favorable safety profile, and low immunogenicity risks, PDEVs have been successfully employed in therapeutic interventions for skeletal muscle disorders, cardiovascular diseases, neurodegenerative conditions, and tissue regeneration applications. This review mainly provides a comprehensive overview of PDEVs, systematically examining their biological properties, standardized isolation and characterization methodologies, preservation techniques, and current applications in regenerative medicine. Furthermore, we critically discuss future research directions and clinical translation potential, aiming to facilitate the advancement of PDEV-based therapeutic strategies. Full article

Neuroinflammation, driven by activated microglia, contributes to the progression of neurodegenerative diseases. Extracellular vesicles mediate intercellular communication and influence immune responses. Chrysin, a natural flavone found in fruits and propolis, has demonstrated anti-inflammatory effects. This study explored the immunomodulatory potential of chrysin-loaded EVs (EVs-Chry) derived from BV2 microglial cells. BV2 cells were treated with chrysin for 24 h to assess cytotoxicity and proliferation. EVs were isolated from treated and untreated cells, characterized by nanoparticle tracking analysis, and applied to naïve BV2 cells prior to LPS stimulation. Effects on cell morphology, migration, cytokine expression (IL-1β, IL-6), inflammasome activity (caspase-1), and apoptosis-related protein Bcl-xL were investigated. Our results show that EVs-Chry significantly reduced LPS-induced cell proliferation, restored resting microglial morphology, and reduced migratory capacity. Furthermore, co-treatment with EVs-Chry and LPS reduced pro-inflammatory cytokines such as IL-1β, IL-6, and caspase-1 expression while enhancing anti-apoptotic Bcl-xL levels, indicating a shift toward an anti-inflammatory, neuroprotective micro-glial phenotype. Together, our results demonstrated that EVs-Chry have neuroprotective effects on LPS-induced microglial activation and modulate microglial responses to inflammatory stimuli, attenuating pro-inflammatory signaling and promoting cellular homeostasis. These findings support the therapeutic potential of EVs-Chry in the context of neuroinflammatory and neurodegenerative disorders. Full article

Reparative tertiary dentinogenesis requires the recruitment and odontogenic differentiation of dental pulp stem cells (DPSCs). Extracellular vesicles (EVs) as bioactive molecules have gained attention in regenerative medicine for their ability to mediate tissue repair through intercellular communication, influencing cell recruitment, proliferation, and differentiation. This study aimed to evaluate the effects of EVs on DPSC homing and odontogenic differentiation for dentin regeneration. DPSC-derived EVs were cultured in either growth (EV-G) or odontogenic differentiation (EV-O) conditions and isolated using a modified precipitation method. EVs were characterized by nanoparticle tracking analysis, scanning electron microscopy, antibody array, and cellular uptake assay. Treatment with 5 × 10⁸ EVs/mL significantly enhanced DPSC chemotaxis and proliferation compared with a no-treatment control and a lower dosage of EV (5 × 10⁷ EVs/mL). Gene expression and biochemical analyses revealed that EV-O up-regulated odontogenic markers including collagen type 1A1 (COL1A1), runt-related transcription factor 2 (RUNX2), and alkaline phosphatase (ALP). EV-O enhanced dentin regeneration by approximately 55% over vehicle controls in a rabbit partial dentinotomy/pulpotomy model. We identified key microRNAs (miR-21-5p, miR-221-3p, and miR-708-3p) in EV-O involved in cell homing and odontogenesis. In conclusion, our EV-based cell homing and odontogenic differentiation strategy has significant therapeutic potential for dentin regeneration. Full article

A moon jellyfish (Aurelia aurita) is a representative of the phylum Cnidaria, commonly found in the northern seas of the globe. The regenerative abilities of cnidarians have recently been associated with extracellular vesicles (EVs) secreted by these organisms. In this study, a method for the isolation of EVs from the oral arms of A. aurita is presented. The methodology includes differential centrifugation, size-exclusion chromatography, and ultrafiltration. The isolates were characterized with tunable resistive pulse sensing, cryogenic transmission electron microscopy, capillary electrophoresis (CE), and electrophoretic light scattering (ELS). Small (<150 nm in diameter) EVs were abundant in the isolates. The EVs were found to carry nucleic acids, indicating their role in signaling. Additionally, the difference in zeta potential values measured with ELS and CE indicates high glycation in the vesicles analyzed. Although the method developed was effective in isolating EVs from small sample volumes (0.5 mL), the EV yield was insufficient for omics analysis. Thus, the scaling up of the isolation process is required for comprehensive biochemical analysis and biological activity assessment in A. aurita-derived EVs. Full article

Extracellular vesicles (EVs) have emerged as promising acellular tools for modulating immune responses for tissue engineering applications. This study explores the potential of human fibroblast-derived EVs delivered within a three-dimensional (3D) injectable scaffold composed of polycaprolactone (PCL) nanofibers and collagen (PNCOL) to reprogram macrophage behavior and support scaffold integrity under inflammatory conditions. EVs were successfully isolated from human fibroblasts using ultracentrifugation and characterized for purity, size distribution and surface markers (CD63 and CD9). Macrophage-laden PNCOL scaffolds were prepared under three conditions: macrophage-only (MP), fibroblast co-encapsulated (F-MP), and EV-encapsulated (EV-MP) groups. Structural integrity was assessed via scanning electron microscopy and Masson’s trichrome staining, while immunomodulatory effects were evaluated through metabolic assays, gene expression profiling, and immunohistochemistry for macrophage polarization markers (CD80, CD206). When co-encapsulated with pro-inflammatory (M1) macrophages in PNCOL scaffolds, fibroblast-derived EVs preserved scaffold structure and significantly enhanced macrophage metabolic activity compared to the control (MP) and other experimental group (F-MP). The gene expression and immunohistochemistry data demonstrated substantial upregulation of anti-inflammatory markers (TGF-β, CD163, and CCL18) and surface protein CD206, indicating a phenotypic shift toward M2-like macrophages for EV-encapsulated scaffolds relative to the other groups. The findings of this study demonstrate that fibroblast-derived EVs integrated into injectable PCL–collagen scaffolds offer a viable, cell-free approach to modulate inflammation, preserve scaffold structure, and support regenerative healing. This strategy holds significant promise for advancing immuno-instructive platforms in regenerative medicine, particularly in settings where conventional cell therapies face limitations in survival, cost, or safety. Full article

Asthma, the most common chronic respiratory condition in children, involves airway inflammation, hyper-responsiveness, and frequent exacerbation that worsen the airflow and inflammation. Exosomes, extracellular vesicles carrying microRNAs (miRNAs), play a key role in cell communication alongside other types of communication and are promising markers of asthma severity. This study compares exosomal miRNA and long non-coding RNA (lncRNA) profiles in boys with asthma, focusing on differences between those with normal lung functions and those with severe airflow obstruction. This study enrolled 20 boys aged 9–18 years with asthma, split into two groups based on their lung function. Ten had normal lung function (NLF; FEV1/FVC > 0.84, FEF75% > 69% predicted), while ten had severe airflow obstruction (SAO; FEV1/FVC < 0.70, FEF75 < 50% predicted). Saliva and blood samples were collected. Exosomes were isolated, quantified, and analyzed via small RNA sequencing to identify differentially expressed (DE) miRNA and lncRNA profiles. Bioinformatic tools were then used to explore potential miRNA biomarkers linked to asthma severity. SAO subjects were more likely to exhibit allergen sensitization, higher IgE levels, and more eosinophils. We identified 27 DE miRNAs in plasma and 40 DE miRNAs in saliva. Additionally, five key miRNAs were identified in both saliva and plasma which underline important pathways such as neurotrophins, T-cell receptor, and B-cell receptor signaling. We further outlined key features and functions of miRNAs and long non-coding RNAS (lncRNAs) and their interactions in children with asthma. This study identified DE miRNAs and lncRNAs in children with SAO when compared to those with NLF. Exosomal miRNAs show strong potential as non-invasive biomarkers for personalized asthma diagnosis, treatment, and monitoring. These RNA markers may also aid in tracking disease progression and response to therapy, thereby supporting the need for future studies aimed at applications in precision medicine. Full article

Mesenchymal stromal cells (MSCs) have demonstrated therapeutic efficacy across numerous clinical applications, with evidence suggesting their paracrine effects, particularly through extracellular vesicles (EVs), possibly driving functional outcomes. In this study we perform the comprehensive characterization of microRNA expression profiles in human MSC-derived EVs (MSC-EV) compared to their parental cells, cultured under clinically relevant xeno-free conditions. MSCs were isolated from the bone marrows of healthy donors and characterised according to the International Society for Cellular Therapy criteria, while MSC-EVs were isolated using differential ultracentrifugation and validated according to the International Society for Extracellular Vesicle guidelines. NanoString profiling identified 590 mature microRNAs expressed across both populations, with 42 being significantly differentially expressed between MSC-EVs and parental MSCs. Five microRNAs were distinctly highly expressed in MSCs and five in MSC-EVs, while fifteen of the top twenty most abundant microRNAs showed high expression in both populations. MicroRNA expression patterns were validated in an independent cohort. Functional pathway analysis of differentially expressed microRNAs showed enrichment of key biological processes including cell proliferation, differentiation, and immune regulation. This standardised profiling approach develops our understanding of MSC/MSC-EV microRNA cargo, using a transparent methodological approach that allows for the improved comparability of datasets for the development and advancement of MSC-EV therapeutics. Full article

Exosomes, small extracellular vesicles secreted by various cell types, have gained significant attention in cancer investigations. Isolation and characterization of exosomes derived from DOK (dysplastic oral keratinocyte), SCC (squamous cell carcinoma) and HaCaT (normal skin keratinocyte) cell lines and microRNA profiling were conducted. Magnetic sorting was applied to obtain pure exosomes. Morphology and size were characterized by transmission electron microscopy and nanoparticle tracking analysis. Validation of membrane exosomal markers (CD9, CD63) was performed via Western blotting. MiR-21, miR-31, and miR-133 levels were analyzed in exosomes and parent cells by qPCR. Biological effects of the exosomes were tested by adding them to fibroblast cultures and determining the expression of relevant carcinogenesis markers by qPCR. Exosomes appeared as cup-shaped nano-sized particles, and there was no difference regarding particle diameter and concentration between the three types of exosomes. The oncogenic miR-21 was significantly upregulated both in SCC and SCC-derived exosomes compared to DOK and HaCaT cells and their respective exosomes. However, miR-31 unexpectedly showed the highest expression in normal cells and the lowest in HaCaT exosomes. MiR-133, the tumor suppressor miRNA, was downregulated in both SCC and DOK cells compared to normal (HaCaT) cells, while the opposite situation was observed in exosomes, with HaCaT cells showing the lowest levels of miR-133. The differences in exosome content were reflected in signaling pathway activation in exosome-treated fibroblasts, with SCC exosomes exerting the most potent effect on several cancer-related pathways, notably PIK3/AKT, PTEN, and NOTCH signaling cascades. Full article

Novel therapeutic approaches for wound healing have included biomaterials from the Atlantic cod (Gadus morhua L.), with promising results in wound management. The use of extracellular vesicles (EVs), which can be isolated from cod biofluids, remains to be studied. EVs play key roles in cellular communication, and their use both as biomarkers and as therapeutic agents is widely reported in human pathologies, particularly with respect to mesenchymal stem cells. This pilot study characterized the total proteomic cargo content of EVs from cod serum and mucus and assessed the EVs’ potential for regenerative activity in wound-healing processes, using human and mouse fibroblast and keratinocyte in vitro scratch injury models. The pro-regenerative potential of both cod serum EVs and mucus EVs was identified, with differing capacities for accelerating wound closure in fibroblast and keratinocyte cells. This was further supported by varying effects of the cod serum EVs and mucus EVs on cellular vimentin and FGF-2 levels. The serum EV and mucus EV protein cargoes differed with respect to abundance of protein hits and associated enriched functional GO and KEGG pathways, but both were associated with immune, stress and wound-healing processes. Cod EVs may present as innovative therapeutic options for regenerative medicine applications, and our reported findings provide valuable insights for future in-depth studies. Full article

Conventional methods for isolating extracellular vesicles (EVs) are often limited by long processing times, a low purity, and a reliance on specialized equipment. To overcome these challenges, we developed the DeSEI (amine-functionalized Diatomaceous earth-based Syringe platform for EV Isolation), a novel platform employing low-cost, amine-functionalized diatomaceous earth (ADe) within a simple syringe–filter system. The capture mechanism leverages the electrostatic interaction between the positively charged ADe and the negatively charged EV surface, enabling a rapid and efficient isolation. The optimized 30 min protocol yields intact EVs with morphology, size, and protein markers comparable to those from ultracentrifugation, ensuring minimal cellular contamination. Notably, DeSEI exhibited a nearly 60-fold higher recovery efficiency of EV-derived miRNA compared to ultracentrifugation. The platform further proved its versatility with a rapid one-step miRNA extraction protocol and a user-friendly cartridge format. The direct miRNA extraction capability is particularly advantageous for a streamlined biomarker analysis, while the cartridge design illustrates a clear pathway toward developing point-of-care diagnostic tools. The DeSEI offers a promising alternative to existing methods for EV-based research by providing a combination of speed, simplicity, and procedural flexibility that does not require specialized equipment. Full article

Background: Increased IL-1β levels may promote carcinogenesis and metastasis by affecting tumor biology and the tumor microenvironment (TME). In this context, extracellular vesicles (EVs) play a key role in cell-to-cell communication, thus modulating the TME and immune response. Here, we aimed to test whether tumor-derived small EVs (TEVs) isolated from sensitive and osimertinib-resistant (OR) non-small-cell lung cancer (NSCLC) cells may promote EMT via fibronectin binding to α5β1 integrin as well as suppress the immune system and if these effects may be favored by IL-1β. Methods: TEVs were isolated from control, OR, and IL-1β-stimulated NSCLC cells. Expressions of fibronectin and PD-L1 were screened in TEVs and the mRNA levels of vimentin and SMAD3 were also assessed in cancer cells after TEV co-culturing. Furthermore, to detect the effect on immune cells, we co-cultured TEVs with lung cancer patients’ peripheral blood mononuclear cells (PBMCs). Results: TEVs were positive for fibronectin and the highest protein levels were found in TEVs obtained from the OR and IL-1β-stimulated cells. TEV-mediated activation of α5β1 signaling led to the upregulation of vimentin and SMAD3 mRNA in NSCLC cells and stimulated cell migration. EVs also increased PD-1, CTLA-4, FOXP3, TNF-α, IL-12, and INF-γ mRNA in lung cancer patients’ immune cells. Conclusions: Our findings indicate that TEVs promote EMT in NSCLC cells by the activation of the fibronectin–α5β1 axis. Finally, IL-1β stimulation induces TEV release with biological properties similar to OR TEVs, thus leading to cancer invasion and immune suppression and suggesting that inflammation can promote tumor spreading. Full article

Despite notable advancements in clinical care, cardiovascular disease (CVD) remains a leading global cause of mortality. Encompassing a wide range of heart and blood vessel disorders, CVD requires targeted prevention and treatment strategies to mitigate its public health impact. In recent years, extracellular vesicles (EVs) have emerged as crucial mediators of intercellular communication, influencing key processes such as vascular remodeling, inflammation, and immune responses in CVDs. EVs, including exosomes and microvesicles, carry bioactive molecules such as miRNAs, proteins, and lipids that contribute to disease progression. They are released by various cell types, including platelets, erythrocytes, leukocytes, endothelial cells, and cardiomyocytes, each playing distinct roles in cardiovascular homeostasis and pathology. Given their presence in circulating blood and other body fluids, EVs are increasingly recognized as promising non-invasive biomarkers for CVD diagnosis and prognosis. Furthermore, EV-based therapeutic strategies, including engineered EVs for targeted drug delivery, are being explored for treating atherosclerosis, myocardial infarction, heart failure, and hypertension. However, challenges remain regarding the standardization of EV isolation and characterization techniques, which are critical for their clinical implementation. This review highlights the diverse roles of EVs in CVD pathophysiology, their potential as diagnostic and prognostic biomarkers, and emerging therapeutic applications, clearing the way for their integration into cardiovascular precision medicine. Full article