Journal of Extracellular Biology

Extracellular vesicles (EVs) are nanoscale, cell-secreted mediators of intercellular communication with growing promise as therapeutic agents. Manufacturing practices, including EV isolation and storage approaches, are critical determinants of product consistency, purity, and potency. In this study, neural stem cell (NSC)-derived EVs were isolated from conditioned NSC culture media via oscillator-based isolation (OSC), ultracentrifugation (1 or 2 hours), and ultrafiltration, and were stored lyophilized or cryopreserved. Nanoparticle yield, size distribution, and subpopulation composition were evaluated by nano-flow cytometry, quantifying total nanoparticles, membrane-bound EVs and CD63+ EVs. Purification was calculated via particle-to-protein ratios, morphology was evaluated by transmission electron microscopy, and potency was assessed using a microglia morphology assay. Particle yield was comparable across isolation methods, though protein clearance varied, with OSC demonstrating purification relative to conditioned media. Lyophilized samples retained structural integrity, size, and population profiles comparable to cryopreserved samples. Lyophilized and cryopreserved EVs exhibited dose-dependent immunomodulatory activity in our microglia morphology assay, with significant effects observed at 200,000 EVs per cell. These findings highlight the importance of isolation method in EV product quality and support lyophilization as a viable storage strategy which overcomes the logistical limitations of cryopreservation, thereby advancing the development of a robust pipeline for therapeutic EV manufacture.

This protocol provides a standardized workflow for the isolation of extracellular vesicles (EVs) from mouse retinal tissue, and includes an assessment of EV size and concentration, marker expression, and EV visualization in accordance with the International Society for Extracellular Vesicles Minimal Information for Studies of Extracellular Vesicles (MISEV) guidelines. Most retinal EV studies rely on cell culture, which may not fully capture in vivo biology. Our approach more accurately reflects physiological and pathological EV states in vivo by enabling the extraction of EVs from intact retinal tissue. This method addresses a key gap in the field by providing a reproducible and rigorous protocol for studying retinal EVs in a biologically relevant context.

Cancer-associated fibroblasts (CAFs) secrete small extracellular vesicles (sEVs) that mediate stromal remodelling, tumour progression, and pre-metastatic niche formation. A foundational assumption in EV research is that MISEV-compliant preparations from the same conditioned medium are biologically equivalent. Here, we directly challenged this assumption through a side-by-side comparison of ultracentrifugation (UC), size exclusion chromatography (SEC), and the EXODUS nanofiltration platform using breast CAF-conditioned media, characterised in accordance with MISEV2023 guidelines using nanoparticle tracking analysis, cryogenic transmission electron microscopy (Cryo-TEM), and quantitative proteomics. EXODUS and SEC recovered approximately 7-fold more particles per mL than UC. While Cryo-TEM confirmed intact vesicle morphology across all methods, UC preparations exhibited substantial non-vesicular background, with gene ontology analysis revealing significant enrichment of ribosomal, mitochondrial, and ER-derived proteins absent from EXODUS and SEC. Matrisome profiling further uncovered method-dependent divergence in the composition of core versus matrisome-associated proteins, highlighting differences extending beyond standard purity metrics. These findings demonstrate that MISEV2023 compliance is necessary but insufficient for methodological equivalence. The isolation method should be treated as a biological variable and selected according to the EV subpopulation or cargo class under investigation.

Extracellular vesicles (EVs) carry microRNAs (miRNAs) that mediate intercellular communication and have strong potential as disease biomarkers, yet the roles of miRNA isoforms (isomiRs) in EVs remain poorly understood. Here, we analyzed 96 human EV and corresponding source samples from nine public datasets. We found that EV samples consistently contained substantially higher proportions of isomiR reads than their corresponding source samples, indicating widespread isomiR enrichment in EVs. Although individual isomiRs showed limited reproducibility across biological replicates and limited sharing between EVs and their corresponding source samples, the parent miRNAs that generated these isomiRs remained highly reproducible across replicates and strongly shared between EV-source pairs. Despite extensive isomiR diversification, EV-source pairs retained highly correlated miRNA expression profiles. Using integrated miRNA- and isomiR-related features, we further developed a random forest model that successfully associated EV samples with their corresponding source samples, with improved performance when isomiR information was included. Together, our results demonstrate that EVs are enriched for biologically meaningful isomiRs while preserving source-associated miRNA landscapes, highlighting the importance of incorporating isomiRs into future EV studies.

Background: Immune-oncology has revolutionized cancer treatment, but some patients fail to benefit due to primary resistance and tumour-immune evasion. Extracellular vesicles (EVs) are secreted by both tumour and immune cells and mediate communication between cancer cells and the immune system. Our study used proteomic profiling of circulating EVs collected from NSCLC patients treated with immune checkpoint inhibitors (ICI) to identify predictive biomarkers of response as well as immune evasion mechanisms related to treatment resistance. Methods: EVs were isolated from plasma collected prior to ICI treatment using peptide-affinity purification and high-throughput proteomics was performed using Proximal Extension Assay. Differentially expressed EV proteins between durable (DR) and non-durable responders (NDR) were identified and evaluated using Cox proportional hazards regression, survival analysis, sex-stratified analysis, as well as pathway and network analysis. Results: Proteomics analysis identified 116 differentially expressed EV proteins between DR and NDR. NDR was characterized by enrichment of inflammatory, angiogenic, and immune-suppressive EV proteins, such as IL1RL1, TFRC, IL6ST, galectins, TNF superfamily death receptors, chemokines, and PCSK9. Pathway analysis revealed enrichment of angiogenesis, chemotaxis, ECM remodeling, and neutrophil degranulation associated with poor progression-free survival (PFS). In contrast, DR to ICI treatment was associated with EV proteins related to T- and B-cell activation and adaptive immunity. Sex-related differences in abundance and association with PFS was observed for certain EV proteins, including IL1RL1 and TFRC. A six protein EV model (IL1RL1, TFRC, ERI1, CCN5, IGFBPL1, and TNFRSF13C) demonstrated good prognostic performance for identifying NDR (AUC = 0.907) and stratified patients into three discrete risk groups. Conclusions: High-plex EV proteomics revealed biologically coherent tumour-immune signaling programs that are associated with ICI treatment resistance. Profiling circulating EVs may improve our understanding of EV-mediated immune evasion mechanisms and identify protein signatures that reflect the tumour immune microenvironment and predict response to immune checkpoint blockade.

Extracellular vesicles (EVs) derived from activated dendritic cells (DCs) are promising cell-free mediators capable of shaping CD8+ T-cell responses. However, their early molecular and functional effects on CD8+ T cells remain incompletely characterized, and whether engineering activated DC-derived EVs with immunomodulatory cargo can fine-tune these responses remains largely unexplored. Here, we investigated whether curcumin loading into EVs derived from CpG-activated and peptide-pulsed DC2.4 cells (EV-ACT) modulates early activation of primary CD8+ T cells. EVs were isolated by ultrafiltration coupled with size-exclusion chromatography (UF-SEC) and characterized physicochemically and molecularly. Exploratory proteomic profiling identified an activation-associated EV protein signature enriched in antigen-processing and immune-related pathways. Curcumin loading achieved an encapsulation efficiency of 16.4% while preserving EV properties, and spectral confocal fluorescence microscopy revealed heterogeneous fluorescence emission patterns consistent with distinct EV-associated curcumin microenvironments. Following rapid cellular association, EV-ACT promoted early CD8+ T-cell activation, inducing an effector-like phenotype characterized by increased CD69 expression, TNF- and Granzyme B production, and reduced Bcl-2 levels without compromising cell viability. Unlike free curcumin, EV-mediated curcumin delivery selectively reinforced these immunostimulatory responses by significantly increasing CD69 expression and STAT3 phosphorylation, sustaining early activation-associated functional and molecular reprogramming of primary CD8+ T cells.

Extracellular vesicles (EVs) are promising nanocarriers for therapeutic delivery; however, the factors governing EV uptake by recipient cells remain incompletely understood. In this study, we investigated whether EV internalization is primarily influenced by donor-cell origin or recipient-cell phenotype. Fluorescently labeled EVs derived from HEK293T, or SKBR-3 cells were incubated with a range of human epithelial, immune, and murine cancer cell lines at different doses and time points. HEK293T-derived EVs showed highly variable uptake across recipient cells, with hepatocellular carcinoma cell lines Huh7 and HepG2 exhibiting the highest internalization, while parental HEK293T cells showed the lowest. THP-1 immune cells also demonstrated strong uptake, whereas Jurkat cells showed moderate uptake. In murine melanoma models, Yummer cells internalized more EVs than B16F10 cells. Importantly, similar uptake trends were observed using SKBR-3-derived EVs, where Huh7 and HepG2 again displayed the highest uptake despite originating from a different donor cell source. EV internalization increased with dose and incubation time until saturation at higher concentrations. Together, these results demonstrate that EV uptake is predominantly determined by recipient-cell characteristics rather than EV source. These findings provide important mechanistic insight for the development of EV-based therapeutics and suggest that optimizing recipient-cell targeting is essential for efficient vesicle-mediated delivery. Graphical abstractEV uptake is determined by cell membrane properties rather than by the source of the EVs. The image was created by Biorender. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=122 SRC="FIGDIR/small/726167v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@f5c1cborg.highwire.dtl.DTLVardef@860962org.highwire.dtl.DTLVardef@1d20239org.highwire.dtl.DTLVardef@9003af_HPS_FORMAT_FIGEXP M_FIG C_FIG

Ovarian cancer (OC) remains a leading cause of gynecologic cancer mortality due to late-stage diagnosis and limited early detection strategies. Ascites fluid, a pathological hallmark of OC, is a rich source of tumor-derived extracellular vesicles (EVs) that reflect the tumor microenvironment and hold promise for biomarker discovery. However, isolating EVs from minimal ascites volumes (<100 {micro}L) poses technical challenges using conventional methods like ultracentrifugation or size-exclusion chromatography (SEC). This study explores the application of strong anion exchange (SAX) magnetic beads (Mag-Net) for efficient EV isolation from as little as 2 {micro}L of ascites fluid from both murine models and a human patient with mucinous borderline tumor. We demonstrate that SAX achieves robust EV capture at 10{micro}l of input volume, enabling comprehensive proteomic profiling and single-EV surface-enhanced Raman spectroscopy (SERS) with a >2-fold increase in proteomic depth compared to raw ascites. Notably, this study was able to identify 1000 proteins not previously annotated in Vesiclepedia for OC-derived EVs, alongside distinct SERS signatures, highlighting the potential for multiomic analysis. Comparative analysis with UC revealed enhanced proteomic depth obtained with SAX beads, albeit we also observed differential detection of canonical markers (e.g., CD9, CD81) between input volumes of ascites fluid. These findings establish SAX as a scalable, low-input platform for EV-based biomarker discovery, paving the way for improved early detection and molecular insights into OC progression.

Extracellular vesicles (EVs) are mediators of intercellular communication, yet their impact on Chinese Hamster Ovary (CHO) cell physiology and bioprocess performance remains poorly understood. Here, we investigated whether small EVs (sEVs) and large EVs (LgEVs) that accumulate during fed-batch and perfusion cultures modulate CHO cell growth, metabolism, apoptosis, and monoclonal antibody (mAb) production. EVs isolated from early- and late-stage cultures were added to fresh CHO cultures grown with or without glutamine supplementation. Only LgEVs had a significant impact. Late-stage LgEVs markedly altered CHO-cell behavior, reducing cell proliferation, increasing apoptosis under glutamine-limited conditions, and substantially enhancing mAb productivity in a dose-dependent manner. Glutamine supplementation largely alleviated the growth-inhibitory and pro-apoptotic effects of LgEVs while preserving their positive impact on productivity, suggesting that glutamine decouples EV-mediated stress from productivity enhancement. Metabolic analyses revealed increased glucose consumption, a glutamine-dependent shift between glycine and alanine overflow metabolism, and remodeling of amino-acid utilization. Metabolic flux analysis further demonstrated enhanced glycolytic overflow and increased reliance on amino acid-supported anaplerosis. Conversely, selective removal of LgEVs from perfusion medium significantly improved cell expansion without reducing antibody production, supporting an inhibitory role for late-stage LgEVs. These LgEVs were enriched in let-7 family miRNAs and miR-21, consistent with RNAseq analyses demonstrating stress-associated enrichment of these miRNAs in CHO EVs and with functional studies showing that let-7a and miR-21reduce CHO-cell growth. Together, these observations suggest that selective miRNA loading contributes to the growth, metabolic, and productivity phenotypes elicited by late-stage LgEVs. Our findings identify LgEVs as endogenous regulators of CHO-cell physiology and potential targets for optimizing high-density fed-batch and perfusion biomanufacturing processes. HighlightsO_LIEndogenous late-stage Large Extracellular Vesicles (LgEVs) reduce CHO cell growth but boost specific mAb productivity. C_LIO_LIGlutamine supplementation rescues LgEV-mediated growth inhibition and apoptosis. C_LIO_LIMetabolic Flux Analysis (MFA) based on the dynamic behavior of amino acid and other metabolite and substrate concentrations reveals the pyruvate node as a metabolic bottleneck and the associated lactate overflow metabolism as resulting from LgEV exposure. C_LIO_LIStress-associated let-7 and miR-21 microRNAs are highly enriched on a per-EV basis in late-stage LgEVs. C_LIO_LISelective removal of LgEVs improves perfusion cell growth without impacting antibody titer. C_LI

A subset of extracellular vehicles (EVs) with particle diameters >200 nm, large vesicles (lEVs) contain mitochondria that increase recipient cell bioenergetics. To date, sequential centrifugation (SC) is the most reported protocol to separate lEVs from the smaller EVs (<200 nm)/exosomes. We have previously demonstrated that lEVs derived from brain endothelial cells (BECs) using the standard SC method transferred their innate mitochondria to recipient BECs, increased recipient BEC bioenergetics, reduced brain infarct volume, and improved behavioral outcomes in a mouse model of transient ischemic stroke. Despite their promising therapeutic activity, SC-isolated lEVs are likely a mixture of mitochondria-containing lEVs and non-mitochondria-containing lEVs. We hypothesized that subsequent purification of SC-isolated lEVs using density-gradient centrifugation (DGC) may yield a purer sample of mitochondria-containing lEVs. We established a DGC protocol to purify lEVs. In this pilot study, lEVs isolated using SC and DGC protocols were compared to determine their physicochemical characteristics and their effects on recipient BEC bioenergetics. SC-lEVs and DGC-lEVs both significantly restored ATP levels in OGD-injured BECs with no difference between groups. However, a Seahorse mitochondrial function assay revealed distinct functional effects: SC-lEVs did not significantly alter respiration, whereas DGC-lEVs induced a dose-dependent increase in oxygen consumption rate, indicating enhanced oxidative phosphorylation. These findings demonstrate that DGC purification yields a more mitochondria-enriched and functionally potent lEV preparation with an enhanced capacity to restore oxidative phosphorylation in ischemic BECs. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=132 SRC="FIGDIR/small/732469v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@12d5e90org.highwire.dtl.DTLVardef@19b44a5org.highwire.dtl.DTLVardef@b7ad75org.highwire.dtl.DTLVardef@dd1a3d_HPS_FORMAT_FIGEXP M_FIG C_FIG

While engineered nanomaterials offer unprecedented precision in targeting tumor cells, their efficacy is often limited by rapid clearance from the bloodstream via the mononuclear phagocyte system (MPS). To overcome this limitation, a promising strategy known as MPS-cytoblockade has been developed. This approach involves administering antibodies against host erythrocytes. The resulting saturation of the MPS with erythrocyte clearance creates a critical window, allowing subsequently administered nanoparticles to evade immune surveillance and circulate for a significantly extended period. However, MPS-cytoblockade induces a transient reduction in hematocrit, which can lead to adverse effects. Here, we demonstrate that approaches to restore hematocrit, specifically through the administration of donor erythrocyte suspension or the hormone erythropoietin, effectively prevent this drop while maintaining the efficacy of the MPS-cytoblockade. Notably, these interventions do not compromise the prolonged circulation time of the nanoparticles or alter their biodistribution, preserving high accumulation in tumors. Our findings establish a viable strategy to mitigate a key side effect of MPS-cytoblockade, thereby enhancing its therapeutic potential and safety profile.

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer that lacks effective targeted therapies and is frequently associated with chemotherapy resistance and immunosuppression. Inducible nitric oxide synthase (iNOS) is overexpressed in breast cancer and has been strongly correlated with poor clinical outcomes, owing to its role in promoting tumor progression, invasiveness, and resistance to therapy. Although highly selective iNOS inhibitors such as N-(3-(Aminomethyl)benzyl)acetamidine (1400W) exhibit considerable therapeutic promise, their clinical translation has been hindered by unfavorable pharmacokinetic properties. To overcome these limitations, we developed a pH-responsive nanoscale formulation based on Schiff base conjugation between 1400W and oxidized PEGylated alginate (OPA), in combination with ionic interactions. The resulting nanoparticles (NPs) exhibited efficient release of 1400W under acidic conditions and effectively suppressed nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. While the NPs alone did not induce significant cytotoxicity, they synergistically enhanced the anticancer efficacy of paclitaxel (PTX) in MDA-MB-231 TNBC cells, significantly inhibiting cell viability and migration. In addition, the NP-PTX combination markedly reduced endothelial tube formation in HUVECs, compared to PTX alone indicating potentiation of the anti-angiogenic activity of PTX. In conclusion, the pH-responsive NPs enables effective modulation of NO signaling and enhances the therapeutic activity of PTX in TNBC cells. These findings support the potential of iNOS-targeted nanomedicine as an adjuvant strategy for TNBC treatment and warrant further investigation using in vivo models to evaluate pharmacokinetics, tumor accumulation, and antitumor efficacy of the NPs.

Extrahepatic delivery of lipid nanoparticles (LNPs) to non-phagocytic cells is a major challenge, with the leading strategy involving surface functionalization with target-specific monoclonal antibody (mAb) ligands. We investigate the stability of mAb-conjugated LNPs using two anchoring systems: the commonly used DSPE-PEG2kDa-maleimide and a block copolymer, PCL5kDa-b-PEG2kDa -maleimide, with the hypothesis that conjugation to a 150,000 Da antibody could overwhelm the relatively small ~600 Da aliphatic anchor on the PEG-lipid in vivo. Shedding of the mAB would compromise targeting. Conjugation integrity following IV injection was assessed by tagging LNPs and mAbs with metal ion tracers that could be quantified by ICP-MS. Results show that DSPE-PEG-mAb rapidly (within 1h) dissociates from LNPs in blood, leading to accelerated LNP clearance. In contrast, mAbs conjugated using PCL-b-PEG remained stably associated with the LNP over the 24h circulation and clearance of the construct. Results are connected to a thermodynamic model that reproduces experimental findings for PEG-anchor(-mAb) shedding in vitro and in vivo. This study identifies anchoring strength as a critical, unconsidered parameter for in vivo performance when conjugating mAbs to LNPs for extrahepatic delivery.

Retinal and choroidal vascular diseases are major causes of vision loss that require frequent intravitreal anti-VEGF therapy. Anti-angiogenic peptide AXT107 demonstrated efficacy in preclinical studies and was advanced to the clinical stage. To provide for sustained delivery of the peptide and avoid complications with intravitreal injection, we evaluated suprachoroidal delivery of AXT107 microparticles (MP-AXT107). The original, soluble AXT107 formulation was ineffective at inhibiting laser-induced choroidal neovascularization (CNV) in our rat model and was consequently reformulated as microparticles. MP-AXT107 demonstrated high peptide incorporation efficiency, reproducible morphology, and physical and chemical stability for at least 9 months under refrigerated storage. In the rat CNV model, suprachoroidal MP-AXT107 significantly reduced neovascular area by approximately 60% relative to vehicle controls. Safety and durability were evaluated in a 9-month GLP toxicology study in Gottingen minipigs following a single suprachoroidal injection of vehicle or MP-AXT107 (0.125-1.25 mg/eye). Transient increases in IOP and mild ocular inflammatory findings were observed immediately following administration but resolved rapidly without lasting effects. No treatment-related adverse ocular findings were observed during the remainder of the study, and the highest tested dose (1.25 mg/eye) was established as the no-observed-adverse-effect level. Bioanalysis at study completion demonstrated persistent AXT107 localization primarily within choroid/RPE and scleral tissues, with no signs of systemic exposure. Collectively, these findings demonstrate that suprachoroidal delivery of MP-AXT107 enables sustained anti-angiogenic activity with favorable ocular safety and prolonged tissue retention, supporting further clinical development as a durable therapy for retinal and choroidal vascular diseases.

1.Exosomes are nanosized extracellular vesicles that carry bioactive molecules reflective of their cells of origin. Developing methods to detect functional enzymatic activity within exosomes can provide a new generation of rapid and informative diagnostic tools. Aldehyde dehydrogenase (ALDH) enzymes, particularly ALDH1A3, are overexpressed in several cancers and contribute to tumor aggressiveness and drug resistance. However, their presence and functionality in cancer-derived exosomes remain poorly characterized. Here, we developed a nano-flow cytometry-based method to detect ALDH activity directly within individual exosomes derived from breast cancer cell lines (SKBR3, MDA-MB-231, and MCF7). Exosomes were isolated by differential ultracentrifugation and validated by nanoparticle tracking analysis, cryogenic transmission electron microscopy, and bead-based immunophenotyping of canonical markers. ALDH enzymatic activity was detected using a resorufin-based fluorescent substrate capable of crossing the exosomal membrane. To ensure specificity, assays were performed in the presence or absence of a selective ALDH inhibitor, confirming that the fluorescent signal originated from ALDH activity within the vesicles. This work provides the first functional evidence of ALDH1A3 enzymatic activity in cancer-derived exosomes and establishes a proof-of-concept platform for rapid, activity-based detection of exosomal enzymes, opening new perspectives for exosome-based diagnostics in breast cancer.

Whole-tissue spatial proteomics level provides critical insights into region-specific biological regulations but remains challenging. Previously, we introduced the Micro-scaffold Assisted Spatial Proteomics (MASP) concept for whole-tissue mapping. However, this prototype required substantial development in spatial resolution, practicality, and throughput for practical application. Here we present a next-generation MASP technique (hex-MASP) featuring i) a new design of hexagonal-micro-wells fabricated with optimized Projection Micro-Stereolithography (P{micro}SL) 3D-printing, achieving high spatial resolution, sampling robustness and mechanical strength for reproducibly compartmentalizing even tough tissues; ii) enhanced throughput/effectiveness in sample preparation and LC-MS analysis with high quantitative quality. Applied to mouse brain, hex-MASP for the first time achieved in-depth, whole-tissue mapping for >6,000 proteins in mouse brains, with high spatial accuracy and excellent data quality. The substantially improved resolution revealed critical regional details across the entire brain, that were not previously captured, enabling precise depiction of protein distribution heterogeneity. This technique enabled the discovery of many unreported regionally-enriched proteins across brain structures. We further applied hex-MASP to investigate the intra-brain distribution of intracerebroventricularly-dosed antibody therapeutics and related proteins, which to our knowledge, enabled whole tissue mapping of protein drugs for the first time and revealed novel mechanistic insights into antibody distribution and localized treatment effects. Hex-MASP represent a robust, scalable platform for whole-tissue spatial proteomics.

The gut microbiome can contribute to anti-tumour immunity and cancer therapy responses, but translating live microbe-based interventions remains challenging due to safety, controllability, and delivery constraints. Bacterial extracellular vesicles (BEVs) are an attractive cell-free alternative, as they package bacterial cargo into a nanoscale format capable of host-cell engagement, immunological activation, and systemic distribution. Here, we investigated the anti-tumour potential of BEVs derived from the human gut commensal Bacteroides thetaiotaomicron (Bt). We show that delivery route is a major determinant of efficacy. Intravenous, but not intraperitoneal, administration produced robust anti-tumour activity in a B16F10 melanoma mouse model. Intravenously delivered Bt BEVs suppressed primary tumour growth in a dose-dependent manner and reduced metastatic outgrowth in the lung. Bt BEVs did not directly impair tumour-cell viability in vitro, but they activated NF-{kappa}B and Toll-like receptor signalling in innate immune reporter systems and localised to tumour tissue following systemic administration. Together, these data support a model in which Bt BEVs act via host immune modulation rather than direct tumour cytotoxicity. These findings identify naturally produced commensal-derived Bt BEVs as a potential microbial therapeutic modality and as an alternative to the use of live bacterial administration in cancer therapy.

Urinary extracellular vesicles (uEVs) represent a promising source of non-invasive biomarkers; however, their clinical translation is still limited by suboptimal isolation strategies, which often suffer from poor scalability, co-isolation of abundant urinary proteins, and bias toward specific EV subpopulations. Here, we employ a membrane-sensing peptide (MSP)-based affinity approach for uEVs isolation, that exploits the highly lipid membranes curvature of EV as universal target, enabling pan-specific capture independent of surface marker expression. MSP-functionalized beads were applied to minimally processed urine samples and benchmarked against differential ultracentrifugation (dUC) and size-exclusion chromatography (SEC). Comprehensive characterization by nanoparticle tracking analysis, transmission electron microscopy, high-sensitivity flow cytometry, single-molecule array (SiMoA), and fluorescence nanoparticle tracking analysis, demonstrated that MSP-based isolation preserves vesicle integrity and maintains the native distribution of canonical tetraspanins (CD9, CD63, CD81), without evidence of subpopulation bias. Notably, MSP-based isolation significantly reduced co-isolated contaminants, such as uromodulin, resulting in improved sample purity. By combining high recovery, improved purity, and operational simplicity, MSP workflow offers practical advantages, including reduced processing time, scalability, and compatibility with standard laboratory equipment, without the need for extensive pre-processing. These properties characterize MSP-based affinity capture as a robust and versatile alternative to conventional uEVs isolation approaches, with strong potential for translational and clinical applications.

Microvascular endothelial cell-derived extracellular vesicles (EVs) mediate local intercellular communication relevant to wound healing and inflammation, yet their proteomic cargo and functional properties remain poorly characterized. Here, EVs were isolated from human microvascular endothelial cells (HMEC-1) by standard ultracentrifugation (UC Bulk) or ultracentrifugation combined with size exclusion chromatography (UC+SEC) and characterized by nanoparticle tracking analysis, immunoblotting, cryogenic electron microscopy, and label-free mass spectrometry. UC+SEC achieved a 77-fold improvement in particle-to-protein ratio with 70-93% depletion of serum and extracellular matrix contaminants while preserving canonical EV markers (ALIX, CD9). Mass spectrometry identified 673 proteins in UC+SEC versus 336 in UC Bulk, with both preparations enriched in wound healing, hemostasis, and angiogenesis pathways. Despite dramatic purity differences, both isolation methods produced functionally comparable EVs that significantly enhanced dermal fibroblast wound closure. Functional assays on primary human dermal microvascular endothelial cells (HDMECs) revealed that HMEC-1-derived EVs exert inflammation-dependent dual effects on TNF- pre-treated endothelium: upregulating VCAM-1 expression while simultaneously preserving VE-cadherin-mediated junction integrity. These effects were strictly inflammatory-dependent, with no detectable activity on healthy endothelial cells. This research uncovers a paradoxical phenotype in which microvascular endothelial EVs enhance immune cell recruitment signals while protecting barrier function exclusively under inflammatory conditions, suggesting a regulatory mechanism that may contribute to vascular homeostasis during inflammatory challenges.

Cetaceans are key sentinel species for environmental health monitoring. Although sampling from free-ranging animals is challenging, the analysis of cetacean blow offers a minimally invasive approach to assess their health status. Extracellular vesicles (EVs) are cell-derived nanostructures present in biological fluids and widely studied as disease biomarkers in humans. Despite the potential for similar uses, EVs have not been studied in cetacean blow to date . This proof-of-concept study aims to assess the feasibility of the isolation and characterization of EVs from blow samples collected from five bottlenose dolphins (Tursiops truncatus) kept under human care and from a free ranging specimen of long-finned pilot whale (Globicephala melas). EVs were purified from bottlenose dolphin blows by ultracentrifugation (UC) or size exclusion chromatography (SEC) and from the long-finned pilot whale by SEC. Particle concentration and size distribution were assessed by Nanoparticle Tracking Analysis (NTA), morphology by Air-atomic force microscopy (AFM) and protein expression by Western Blotting (WB). NTA revealed a higher mean particle concentration in bottlenose dolphin EVs isolated by UC compared to SEC, while EVs isolated from the long-finned pilot whale presented a lower particle concentration. AFM confirmed the presence of EV-like particles within the typical EV size range in bottlenose dolphins EVs obtained both by SEC or UC. All EV samples were positive for CD9 and integrin-{beta} and negative to Calnexin. SEC was more sensitive to detect OmpA, a membrane protein of Gram-negative bacteria, in EVs from both species. Our pilot study demonstrates that EVs are present in cetacean blow and can be isolated and characterized. Future investigations focused on characterizing and quantifying a wider array of EV associated molecules may further the application of blow EV analysis for cetacean health assessments.