Abstract Background Mechanisms governing non-vesicular extracellular particle (NVEP) biogenesis and compositional diversity remain incompletely characterized. Method This study integrates multiparametric profiling to define NVEPs. NVEPs were isolated by differential ultracentrifugation and characterized using complementary biochemical, lipidomic, and imaging approaches, including protein-to-lipid ratio analysis, lipid profiling, and advanced microscopy. Result Comprehensive characterization demonstrated that NVEPs exhibit significantly elevated protein-to-lipid ratios compared to extracellular vesicles (EVs) subtypes. Lipidomic profiling revealed pronounced enrichment in cholesteryl esters and triacylglycerols, with distinct carbon chain length signatures differentiating NVEPs from small EVs. Super-resolution microscopy confirmed marker heterogeneity across populations, with NVEPs showing specific enrichment of Arf6 and CD63. Conclusion These findings provide a robust compositional framework to distinguish non-vesicular extracellular particles from canonical EVs and underscore their emerging significance as mediators of intercellular communication and potential vehicles for targeted delivery.

ABSTRACT The concept of the “biomolecular corona” (BC) was first introduced to describe the spontaneous absorption of molecules, mainly proteins, onto the surface of synthetic nanoparticles upon exposure to biological fluids. More recently, the same term has been used to describe biomolecules that dynamically interact with the surface of extracellular vesicles (EVs) during their biogenesis, secretion, or upon exposure to intracellular or extracellular milieus, including biological fluids. The EV biomolecular corona is a key determinant of EV identity, shaping their cellular uptake, biodistribution, and functional outcomes in both physiological and pathological contexts. EVs, in contrast to synthetic nanoparticles, introduce distinct challenges and perspectives due to their biological origin, innate heterogeneity, and complex combination of physicochemical and biological properties. To address this evolving topic, the International Society for Extracellular Vesicles (ISEV) organized a dedicated workshop in Brescia, Italy, on 27th to 28th February, 2025. The event gathered selected participants from academia and industry, with expertise spanning synthetic nanomaterials, EVs, biophysics, and translational nanomedicine. The program included keynote lectures, oral presentations, posters, and a series of interactive roundtables structured around fundamentals of the biomolecular corona, EV corona biogenesis and composition, analytical and separation tools and clinical and diagnostic potential applications. This report summarizes the key scientific insights, methodological challenges, conceptual debates, and future perspectives that emerged from the workshop.

IntroductionSpirulina maxima (Sm), a blue-green microalgae, is well known for its rich nutritional composition, antioxidant, and anti-inflammatory properties. In this study, we found that small extracellular vesicles (sEVs) isolated from Sm exhibit antifibrotic activity.MethodsSm derived sEVs (Sm_sEV) were purified from the Sm culture medium using tangential flow filtration (TFF), followed by size-exclusion chromatography (SEC). Characterization of the sEVs was performed using nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), immunogold TEM, surface-enhanced IR spectroscopy (SEIRS), and flow cytometry (FC). Internalization of DiI-labeled sEVs by human primary peritoneal mesothelial cells (P-MCs) and peritoneal fibroblasts (p-FBs) was examined using fluorescence microscopy. The effect of sEVs on mesenchymal transition of P-MCs and activation of P-FBs was investigated by immunofluorescent staining, MTT cell-proliferation, and Sirius Red collagen accumulation assays, respectively. The antifibrotic relevance of Sm_sEV was further investigated in a chlorhexidine digluconate (CG)-induced mouse model of peritoneal fibrosis.ResultsThe isolated Sm_sEV exhibited a spherical morphology, with a size range of 150.0 ± 67.6 nm, and the protein-to-lipid ratio (P/L_spectr) was 2.27 ± 0.07. The sEVs cargo contained Parkinson’s disease protein 7 (PARK7), and heat shock protein 70 (HSP70). DiI-labeled sEVs were successfully internalized by both P-MCs and P-FBs and inhibited TGF-β-induced mesenchymal transition in P-MCs and the collagen production and PDGF-B-induced proliferation in P-FBs. In vivo, intraperitoneal administration of Sm_sEVs reduced CG-induced submesothelial thickening, fibronectin, and collagen type I alpha 1 immunopositivity, and increased cytokeratin 18, immunopositivity in the mesothelial layer.DiscussionThese findings highlight the antifibrotic effect of Sm_sEV and support their further investigation in the context of fibrosis.

Extracellular Vesicles (EVs) have emerged as a significant tool in therapeutic applications, exhibiting low immunogenicity and the ability to traverse biological barriers. EV-based treatments show great potential in various diseases, including oncology, neurodegeneration, and cardiovascular conditions. In cancer research, EVs play a role in tumor growth, spread, and resistance to chemotherapy, with modified EVs showing promise in improving drug delivery to challenging cancers like glioblastoma. For neurodegeneration, EVs aid in protecting neurons and restoring motor function. Likewise, in cardiovascular diseases, EVs participate in tissue repair and heart protection. The effectiveness of EV-based vaccines, exemplified by a clinically approved meningococcal serogroup B MenB-4C vaccine, further underscores the therapeutic value of this approach. This review examines the progress in EV-based therapeutics and their future potential.

Platelets are abundant in blood, where they maintain the integrity of the vasculature. Megakaryocytes, the cells responsible for platelet genesis, produce membrane protrusions from which as many as 5000 anucleate platelets can be released into the bloodstream. Platelets lack genomic DNA but contain different molecules, such as RNA, as well as organelles transmitted from the parent megakaryocyte. There is no consensus in the scientific community on whether platelets are cells or not: for example, they are sometimes called cells, small cells, anucleated cells, cell fragments, or megakaryocyte fragments. Extracellular vesicles are particles delimited by a lipid bilayer that are released from cells but cannot replicate on their own. Like platelets, extracellular vesicles lack a nucleus and carry components from their donor cell. Herein, we will explore various viewpoints suggesting that platelets may be cells, albeit not conventional cells, or may be a previously unrecognized type of extracellular vesicle. Beyond a mere debate over terminology, this perspective seeks to help properly define and classify platelets, aiming for better integration into the concept of either cells or extracellular vesicles. This will foster a clearer understanding and drive advances in platelet research.

Extracellular vesicles (EVs) are lipid membrane-enclosed particles released by all cells and can be isolated from various sources, even from solid tissues. This study focuses on isolating and characterizing EVs from mouse lymph nodes (LNs). Male C57BL/6 mice were injected with complete Freund’s adjuvant, with or without ovalbumin. Inguinal and popliteal LNs were incised 9 days after immunization, and EV isolation was carried out using a combination of differential centrifugation and size-exclusion chromatography. The characteristic morphology of small and large EVs was confirmed by transmission electron microscopy. Particle size distribution and concentration were determined by nanoparticle tracking analysis, while protein and lipid contents were measured by bicinchoninic acid assay, and sulfo-phospho-vanillin assays, respectively, to calculate the protein-to-lipid ratio. Immune and EV markers were analyzed by using flow cytometry and Western blot assay, revealing significant changes between immunized mice compared to controls. This study establishes a novel protocol for isolating and characterizing EVs from LNs and highlights the impact of immunization on EV properties, offering insights into their roles in immune processes.

Recent studies showed an unexpected complexity of extracellular vesicle (EV) biogenesis pathways. We previously found evidence that human colorectal cancer cells in vivo release large multivesicular body-like structures en bloc. Here, we tested whether this large EV type is unique to colorectal cancer cells. We found that all cell types we studied (including different cell lines and cells in their original tissue environment) released multivesicular large EVs (MV-lEVs). We also demonstrated that upon spontaneous rupture of the limiting membrane of the MV-lEVs, their intraluminal vesicles (ILVs) escaped to the extracellular environment by a ‘torn bag mechanism’. We proved that the MV-lEVs were released by ectocytosis of amphisomes (hence, we termed them amphiectosomes). Both ILVs of amphiectosomes and small EVs separated from conditioned media were either exclusively CD63 or LC3B positive. According to our model, upon fusion of multivesicular bodies with autophagosomes, fragments of the autophagosomal inner membrane curl up to form LC3B positive ILVs of amphisomes, while CD63 positive small EVs are of multivesicular body origin. Our data suggest a novel common release mechanism for small EVs, distinct from the exocytosis of multivesicular bodies or amphisomes, as well as the small ectosome release pathway.

IntroductionLight exposure of embryos during assisted reproduction affects embryo quality and implantation capacity in a wavelength dependent manner. We investigated the molecular mechanism of these light-induced changes through the comparative analysis of gene expression and regulatory miRNA profile of murine embryos cultured in dark environment and those exposed to white- or red filtered light. miRNA sequencing was used to assess the role of embryo-derived extracellular vesicles in the endometrium-embryo dialogue.MethodsIn vitro cultured mouse embryos at 3.5 days post coitum (dpc) were exposed to white or red filtered light. After 24 hours mRNA and miRNA content of the embryos as well as the miRNA content of embryo-derived extracellular vesicles were isolated and RNA-sequencing was performed. Differential expression analysis and functional enrichment analysis were used for evaluating the transcriptome results.ResultsLight exposure caused transcriptomic changes in the embryos. White light upregulated apoptotic pathways, while red filtered light gave rise to the activation of regeneration pathways, including DNA repair mechanisms. Embryo-derived extracellular vesicles enclosed wavelength dependently unique miRNA cargos the target genes of which play a role in embryo implantation.DiscussionWhite light upregulates apoptotic pathways, at both the transcriptome and regulatory miRNAs levels. Red filtration partially counterbalances these negative effects by shifting the cellular processes towards regeneration, including DNA repair mechanisms. Extracellular vesicles of light exposed embryos play a role in blastocyst-decidua communication through the horizontal transfer of regulatory miRNAs. Our data prove that light exposure during in vitro fertilization modifies cell function that might affect the outcome of implantation.

Recent studies have confirmed that a biomolecular corona forms around extracellular vesicles (EVs) in biofluids. However, there is limited data on how this adsorbed corona affects the accessibility of EV surface molecules. Here, we investigated various potential corona-stripping conditions for their ability to affect the immune detection of EVs. First, we artificially formed an EV corona around nascent HEK293T-PalmGFP cell-derived large EVs (lEVs) by incubating them with Cy5-labelled human plasma proteins. The co-localisation rate of plasma proteins and lEVs decreased significantly upon high-salt washing with NaCl, LiCl and KCl solutions, suggesting a considerable removal of the corona components. Additional evidence for corona modification was a significantly increased fluorescent annexin V binding to plasma lEVs and annexin V affinity capture of both THP1- and blood plasma-derived lEVs upon high-salt washing. A similar effect of high ionic strength was observed when THP1 lEVs were separated from a serum-containing medium, which allowed for corona formation, but not when EVs were produced under serum-free conditions. Using a MACSPlex kit and high-salt washing for small EVs from plasma and THP1 conditioned medium, we also demonstrated significantly improved immunodetection of 15 and 9 out of 37 surface markers, respectively. In this Technical Note, we present evidence that modifying the protein corona around EVs can significantly affect the immune detection of specific EV markers.

Extracellular vesicles (EVs) are implicated in inter-organ communication, which becomes particularly relevant during aging and exercise. DNA methylation-based aging clocks reflect lifestyle and environmental factors, while regular exercise is known to induce adaptive responses, including epigenetic adaptations. Twenty individuals with High-fitness (aged 57.7 ± 9.8 years) and twenty Medium–Low-fitness (aged 57.5 ± 9.7 years) subjects provided blood samples. EVs were isolated from the samples using a size exclusion chromatography (SEC)-based method, and their protein content was analyzed by mass spectrometry (MS). Acceleration of the biological age estimator DNAmFitAge (AgeAccelFit) was associated with the protein cargo of EVs, whereas PhenoAge and GrimAge acceleration did not show a significant relationship. This finding suggests that the epigenetic aging-modulating role of exercise may involve inter-organ communication via EVs. Set Enrichment Analysis was performed to identify enriched Gene Ontology (GO) terms for sets of proteins that were either correlated with AgeAccelFit or detected exclusively in individuals with high levels of aerobic fitness. The protein cargo of EVs further suggests that inter-organ communication influences inflammation, the immune system, cellular repair, adhesion, metabolism and coagulation. Our findings help to understand the preventive role of exercise, which could be mediated in part by EVs.

The recurrence of clinically advanced cancers is an evolutionary consequence of standard-of-care chemotherapies generally administered at maximum tolerated doses to kill as many cancer cells as possible. The inevitable appearance of resistance raises the possibility of shifting treatment goals from complete tumor eradication to long-term disease control. The latter approach is employed by adaptive therapy, which aims to inhibit the evolutionary dynamics governing the spread of resistant tumor phenotypes. Adaptive therapy changes focus from the cancer cells that are responsive to therapy to those that are resistant and ultimately govern outcome. This therapeutic approach retains a pool of sensitive cancer cells to compete with the therapy-resistant ones through dynamic dose modulation and/or timing. Thus, fluctuations of treatment-sensitive cells are used to control the resistant population and prolong tumor control with existing therapy agents. Here, we explore non-genetic mechanisms of resistance, including the protective role of the tumor stroma, the epithelial-to-mesenchymal transition, the overexpression of drug efflux pumps, and the extracellular vesicle-mediated transfer of them. These mechanisms can increase the size of the resistant population at the expense of the sensitive one, reducing the ability of adaptive therapy to force tumor evolution into controllable cycles.

Inflammation and immune evasion promote tumorigenesis and progression. Elevated systemic inflammation response index (SIRI) is associated with poor progression-free survival (PFS) and overall survival (OS) in non-small cell lung cancer (NSCLC) patients. Low Human Leukocyte Antigen-DR (HLA-DR) expression on monocytes is also associated with poor prognosis in NSCLC. We aimed to investigate the relationship between these two indicators and develop a predictive model based on them. SIRI was calculated and monocyte HLA-DR expression was measured by flow cytometry in 58 advanced (stage IIIB-IV) NSCLC patients. The log-rank test and multivariate Cox proportional hazard regression model were used for analysis. We confirmed that both high SIRI and low monocyte HLA-DR expression were associated with poor PFS and OS, respectively. We found a significant inverse correlation between SIRI and monocyte HLA-DR expression. In the multivariable Cox regression model, both SIRI and monocyte HLA-DR expression were identified as independent prognostic markers for PFS and OS. We also developed a nomogram for predicting PFS and OS. In conclusion, we demonstrated that the systemic inflammation response of advanced NSCLC patients, estimated by SIRI, was associated with reduced HLA-DR expression on circulating monocytes, which may influence their antigen-presenting function. Consequently, the integration of these two biomarkers into one prognostic model improves short term survival prediction in advanced NSCLC. To our knowledge, this is the first integration of SIRI and HLA-DR into a combined prognostic nomogram.

IntroductionAging is accompanied by immunoscenescence and chronic low-grade inflammation (inflammaging), contributing to age-related diseases. Physical exercise is a potent modulator of immune function and systemic inflammation, yet the effects of acute exercise intensity on immune activation, cytokine dynamics, and extracellular vesicle release in older adults remain incompletely characterized, particularly in a sex-specific context. This study investigated how a single session of acute continuous moderate versus intense exercise modulates immune cell subsets, cytokine levels, and EV profiles in healthy older individuals, with emphasis on sex-based differences.MethodsThirty-three older adults completed either a moderate (n=14, 54-79 years; 60% VO2max, 30 minutes) or an intense cycling bout (n=19, 61-85 years; incremental cardiopulmonary exercise test (CPET) to exhaustion). Peripheral blood was collected at baseline, 30 minutes, and 24 hours post-exercise. Immune cells were analyzed by flow cytometry. EVs were characterized by flow cytometry and nanoparticle tracking analysis, and cytokines were quantified by multiplex assays.ResulsModerate exercise enhanced classical monocyte activation (↑CD86, ↓CX3CR1) without altering cell counts, and selectively elevated IL-6 in females. Intense exercise induced stronger innate immune activation, increasing classical and nonclassical monocytes, CD56bright/CD16low NK cells, and sustained TNFα levels. EVs positive for tetraspanins (CD9, CD63, and CD81) were elevated 24h after intense CPET. Exploratory sex-disaggregated analyses revealed distinct profiles: females had increased CD4+ EVs, while males showed elevated HLA-ABC+ EVs.DiscussionAcute exercise modulates immune responses in an intensity- and sex-dependent manner in older adults. Extracellular vesicle release was assessed only in the high-intensity intervention, where significant changes were observed. These findings support personalized exercise regimens to enhance immune resilience and promote healthy aging.

This study investigates T cell subsets in pericardial fluid samples obtained from heart transplantation donors, heart transplantation recipients, and coronary artery bypass graft patients. Using flow cytometry, we characterized regulatory T cells (Tregs), tissue-resident memory T cells (Trm), and exhausted T cells based on specific markers. Our results showed significant alterations in the CD4+ and CD8+ T cell subsets, migration (CXCR3, CCR5), and exhaustion markers (PD-1, TIM3) across the groups. Notably, Tregs and Trm cells were enriched in recipients, while markers of T cell exhaustion showed a complex regulation. These findings provide novel insights into the local immune regulation in cardiac disease and transplantation.

This study sought to compare the behavior of Treg subsets displaying different coexpression patterns of Neuropilin-1 (Nrp1) and Helios, under the influence of gut stress unrelated to hematopoietic stem cell transplantation, pretransplantation conditioning, and posttransplant gastrointestinal acute graft versus host disease (GI-aGvHD). Host CD4+/CD25hi/Foxp3+ Treg cells, identified by flow cytometry, were isolated from various tissues of mice affected by these stressors. Expression of CD25, CTLA-4, CD39, OX40, integrin-β7, LAG3, TGFβ/LAP, granzyme-A, -B, and interleukin-10 was compared in four Treg subsets displaying Helios or Nrp1 only, both or none. Fluorescence-activated cell sorter-sorted Treg subsets, displaying markers affected in a conditioning- and GI-aGVHD-restricted manner, were further investigated by transcriptome profiling and T-cell suppression assays. We found that conditioning by irradiation greatly diminished the relative frequency of Helios+/Nrp1+ Treg, shifting the balance toward Helios-/Nrp1- Treg in the host. Upregulation of integrin-β7 and OX40 occurred in GI-aGvHD-dependent manner in Helios+/Nrp1+ cells but not in Helios-/Nrp1- Treg. Sorted Treg subsets, confirmed to overexpress Nrp1, Helios, OX40, or integrin-β7, displayed superior immunosuppressive activity and enrichment in activation-related messenger RNA transcripts. Our data suggest that conditioning-induced shrinkage of the Nrp1+/Helios+ Treg subset may contribute to the development of GI-GvHD by impairing gut homing and decreasing the efficiency of Treg-mediated immunosuppression.

Objectives: We aimed to assess the relationship among circulating extracellular vesicles (EVs), hypoxia-related proteins, and the conventional risk factors of life-threatening coronary artery disease (CAD) to find more precise novel biomarkers. Methods: Patients were categorized based on coronary CT angiography. Patients with a Segment Involvement Score > 5 were identified as CAD patients. Individuals with a Segment Involvement Score < 5 were considered control subjects. The characterization of EVs and analysis of the plasma concentration of growth differentiation factor-15 were performed using multicolor or bead-based flow cytometry. The plasma protein levels of glycogen phosphorylase, muscle form, clusterin, and carboxypeptidase N subunit 1 were determined using an enzyme-linked immunosorbent assay. Multiple logistic regression was used to determine the association of the biomarkers with the CAD outcome after accounting for established risk factors. The analysis was built in three steps: first, we included the basic clinical and laboratory variables (Model 1), then we integrated the plasma protein values (Model 2), and finally, we complemented it with the circulating EV pattern (Model 3). To assess the discrimination value of the models, an area under (AUC) the receiver operating curve was calculated and compared across the three models. Results: The area under the curve (AUC) values were 0.68, 0.77, and 0.84 in Models 1, 2, and 3, respectively. The variables with the greatest impact on the AUC values were hemoglobin (0.2 (0.16-0.26)) in Model 1, carboxypeptidase N subunit 1 (0.12 (0.09-0.14)) in Model 2, and circulating CD41+/CD61+ EVs (0.31 (0.15-0.5)) in Model 3. A correlation analysis showed a significant impact of circulating CD41+/CD61+ platelet-derived EVs (p = 0.03, r = -0.4176) in Model 3. Conclusions: Based on our results, the circulating EV profile can be used as a supportive biomarker, along with the conventional laboratory markers of CAD, and it enables a more sensitive, non-invasive diagnostic analysis of CAD.

Host antimicrobial peptides (AMPs) and extracellular vesicles (EVs) are known to play important roles as part of the immune system, from antimicrobial actions to immune regulation. Recent results also demonstrate that EVs could serve as carriers for AMPs. Related, it was shown that some AMPs can remove the protein corona (PC), the externally adsorbed layer of proteins, from EVs which can be exploited for subtractive proteomics strategies. The interaction of these compounds is thus interesting for multiple reasons from better insight to natural processes to direct applications in EV-based bioengineering.However, we have only limited information on the various ways these species may interact with each other. To reach a broader overview, here we selected twenty-six AMPs, including cell-penetrating peptides (CPPs), and investigated their interactions with red blood cell-derived vesicles (REVs). For this, we employed a complex lipid biophysics including linearly polarized light spectroscopy, flow cytometry, nanoparticle tracking analysis, electron microscopy and also zeta-potential measurements. This enabled the categorization of these peptides into distinct groups. At specific low concentrations, peptides such as LL-37 and lasioglossin-III were effective in PC elimination with minimal disruption of the membrane. In contrast, AMPs like KLA, bradykinin, histatin-5, and most of the tested CPPs (e.g. octa-arginine, penetratin, and buforin II), demonstrate cell-penetrating mechanisms as they could sustain large peptide concentrations with minimal membrane damage. The systematic overview presented here shows the potential mechanism of how AMPs and EVs could interact in vivo, and also how certain peptides may be employed to manipulate EVs for specific applications.

Aims The association and co-isolation of low-density lipoproteins (LDL) and extracellular vesicles (EVs) have been shown in blood plasma. Here we explore this relationship to better understand the role of EVs in atherogenesis. Methods and results Wild type (WT), PCSK9(-/-), and LDLR-/- C57BL/6 mice were used in this study. Eleven week-old male mice were fed high-fat diet (HFD) for 12 weeks or kept on normal diet until old age (22-months). Cardiac function was assessed by ultrasound, cholesterol was quantified with a colorimetric kit and circulating EVs were measured using flow cytometry. Plaques were analysed post-mortem using Oil-Red-O staining of the aortic arch. EVs were measured from platelet free blood plasma samples of normal and hypercholesterolaemic clinical patients. Based on annexin V and CD63 staining, we found a significant increase in EV levels in LDLR-/- and PCSK9(-/-) mice after HFD, but CD81 showed no significant change in either group. There was no significant change in plaque formation after HFD. PCSK9(-/-) mice show a favourable cardiac function after HFD. Blood cholesterol levels progressively increased during HFD, with LDLR-/- mice showing high levels while PCSK9(-/-) were significantly lowered compared to WT animals. In mice at old age, similar cholesterol levels were observed as in young mice. In old age, LDLR-/- mice showed significantly increased plaques. At old age, ejection fraction was decreased in all groups of mice, as were CD63(+) EVs. Similarly to mice, in patients with hypercholesterolaemia, CD63(+) EVs were significantly depleted. Conclusions This research demonstrates an inverse relationship between circulating EVs and cholesterol, making EVs a potential marker for cardiovascular disease (CVD). HFD causes reduced cardiac function, but atherosclerotic development is slowly progressing in hypercholesterolaemic models and only observed with old animals. These results also bring further evidence for the benefit of using of PCSK9 inhibitors as therapeutic agents in CVD.