Metabolic dysfunction-associated steatotic liver disease (MASLD) is highly prevalent in obesity, but scalable tools for biological risk stratification remain limited. In this cross-sectional, single-centre discovery study (MULTISITE), we profiled circulating extracellular vesicle (cEV) surface proteins by EV Array in platelet-poor plasma from individuals with obesity and MASLD (n = 36), individuals with obesity without MASLD (n = 24), and lean controls (n = 27). Liver fat was quantified by magnetic resonance imaging proton density fat fraction (MRI-PDFF). We assessed group differences, associations with continuous PDFF, and exploratory discriminatory performance using single markers, pairwise ratios, and sparse logistic models. We also performed an exploratory PDFF-extremes enrichment analysis within obesity by comparing the top versus bottom 30% of the PDFF distribution (n = 18 vs. n = 18), excluding the middle 40%. Across contrasts, single markers showed modest separation, whereas pairwise ratios provided stronger exploratory signals. In the discovery panel, uncorrected best-ratio AUCs were 0.80-0.84 and sparse-model AUCs were 0.79-0.84 under internal, non-nested cross-validation across MASLD-focused contrasts. In the focused panel, CD36 and TREM2 were the strongest single-marker signals for MASLD versus lean controls, whereas LRP-1-anchored ratios captured obesity- and liver fat-associated patterns. In the PDFF-extremes analysis, internally cross-validated AUCs reached 0.87-0.89 in the discovery panel and 0.86 for LRP-1/FATP5 in the focused panel; these estimates should be interpreted as exploratory, uncorrected discovery-stage observations rather than validated classifier performance. Exploratory benchmarking showed that FLI, but not FIB-4, discriminated MASLD-related contrasts strongly, however, these analyses provide contextual benchmarking only and do not establish incremental clinical value of cEV markers beyond routine clinical predictors. These findings nominate biologically coherent cEV surface signatures for future validation in MASLD, anchored by lipid handling/scavenger pathways and complemented by stress, immune, and coagulation biology. Trial Registration: ClinicalTrials.gov: NCT05699863.
Extracellular vesicles (EVs) hold significant promise as biomarkers, but their clinical application is constrained by variability in pre-analytical handling and isolation. EV isolation methods directly shape which plasma-derived EV-containing preparations are captured, yet systematic method comparisons across multiple analytical dimensions are limited. We comprehensively evaluated eleven EV isolation methods in pooled platelet-poor plasma (5 donors; 6 technical replicates/method). We further evaluated selected methods using five individual donors. EVs were quantified by NanoFCM, profiled for tetraspanins (CD9, CD63, CD81) via MSD assays, and further characterized by LC-MS/MS proteomics. We show that different EV isolation methods for plasma produce different EV containing preparation. EV isolation methods broadened proteome coverage in plasma but showed divergent performance. While all methods captured EVs in the 50-150 nm range, centrifugation and ultracentrifugation identified the broadest proteomes (up to 1093 proteins) driven by higher plasma protein carryover. Conversely, ExoEasy and qEV 70 isolated larger EVs and achieved stronger depletion of abundant plasma proteins but showed lower proteome coverage. A total of 117 proteins were detected across all isolation methods. Pre-clearing of samples removed contaminants but at the cost of protein identifications. We demonstrate that method selection must align with the specific analytical goal: centrifugation for comprehensive proteome profiling, affinity/size-exclusion methods for contaminant-sensitive assays, and precipitation for high-throughput applications. This systematic characterization provides an evidence-based framework and look-up resource for matching isolation strategies to downstream applications and research questions.
Cell-derived extracellular vesicles (EVs) are promising nanocarriers for therapeutic delivery platforms owing to their biocompatibility and capacity to protect and efficiently transport bioactive molecules. However, EV-based therapeutics remain constrained by inefficient cargo loading and low production yields, which limit scalable biomanufacturing. To overcome these limitations, we exploited the use of the mechanosensitive ion channel Piezo1 as a robust regulator of EV biogenesis using HEK293FT cells co-transfected with Piezo1 and the bioluminescent EV reporter PalmReNL. Activation of Piezo1 with Yoda1 (30 µM) increased PalmReNL-EV release by 3-fold, while GsMTx4 inhibited EV release by 80.7%. This effect was unaffected by removal of extracellular Ca2+ but was suppressed by intracellular Ca2+ chelation with BAPTA-AM, indicating a reliance on intracellular Ca2+ mobilisation. Small EVs (sEVs) from Piezo1-activated cells were purified by anion exchange chromatography and analysed by proteomics, identifying 48 proteins exclusively in Piezo1-induced sEVs preparations among 148 total detected, including cytoskeletal and stress-related factors, while preserving enrichment of extracellular matrix (ECM) structural components prominent in both conditions. Yoda1 treatment increased the release of both large EVs (lEVs) and sEVs, with a particularly pronounced increase in sEV production. As a proof of concept for therapeutic cargo delivery, Yoda1 stimulation increased the incorporation of exogenously expressed interleukin-10 (IL-10) into sEVs by up to 4-fold, and the bioactivity of sEV-associated IL-10 was validated using IL-10-CyCLoPs reporter cells. However, exposing Piezo1-overexpressing cells to 30 µM Yoda1 markedly delayed cell adhesion and spreading, indicating that excessive Piezo1 activation may constrain sustained production of therapeutic sEVs. Collectively, these results identify mechanotransduction as a key regulator of sEV biogenesis and underscore the need for precise temporal control, potentially achievable through ultrasound-based modulation, for the rational engineering of next-generation sEV therapeutics.
Recent studies have shown that adipose tissue (AT) secretes elevated levels of extracellular vesicles (EVs) in obesity, and these EVs play roles in metabolic diseases. The inhibition of calpains has anti-inflammatory and anti-fibrotic effects on AT in mice and reduces EV secretion in some cell types in vitro. However, its effects on human AT and adipocyte EV secretion remain unexplored. This study aimed to investigate calpeptin's effects on EV-mediated communication and adipocyte function, offering potential insights into therapeutic approaches for metabolic diseases. Human Simpson Golabi Behmel Syndrome (SGBS) preadipocytes were differentiated and treated with calpeptin. EVs were isolated by standard ultracentrifugation, and studied by nanoparticle tracking analysis, electron microscopy, and mass spectrometry. Diverse analyses, including RNA-sequencing, liquid chromatography-mass spectrometry (LC-MS), and confocal microscopy were utilized to study calpeptin's effects on SGBS cells. AT samples from bariatric surgery patients were cultured ex vivo to assess calpeptin's effects on primary AT. We demonstrated for the first time that calpeptin reduces EV secretion in human SGBS adipocytes. Proteomic analyses revealed that calpeptin alters the abundances of proteins related to EV secretory pathways. While reduced EV secretion was accompanied by anti-inflammatory effects, calpeptin also altered insulin signalling pathways and reduced adiponectin expression, suggesting negative effects on adipocyte metabolism. Indeed, LC-MS analyses of cells and EVs revealed that calpeptin altered proteins-both in cells and EVs-that are associated with stress responses. Notably, calpeptin upregulated HO-1 in vitro and in ex vivo AT cultures, indicating induced oxidative stress in adipocytes and AT. While calpeptin shows anti-inflammatory promise in human SGBS adipocytes, its adverse effects on insulin signalling, adiponectin expression, and signs of oxidative stress raise concerns about its therapeutic potential against obesity-related pathologies in humans. Our results highlight the need to understand the broader impact of calpeptin on adipocyte metabolism.
Vision impairment caused by age-related macular degeneration (AMD) is a global health priority. Retinal pathology is driven by ageing and exacerbated by genetic and environmental risk factors. Peripheral blood circulation is increasingly recognised as a contributor to disease initiation and progression, and a source of biomarkers to support earlier diagnosis and personalised treatment strategies. Here, we performed an analysis of plasma-derived extracellular vesicle (EV)-enriched samples from 30 AMD and 30 controls. Proteomics identified significantly altered proteins in AMD, the majority of which were downregulated with a strong interaction network involving complement proteins and endopeptidase inhibitors. Importantly, many of the altered proteins are known AMD biomarker candidates and act in pathways affecting AMD: oxidative stress response, immune function and proteolysis dysregulation. Lipidomics revealed an increase in total sphingomyelin to ceramide ratio in AMD, indicating lipid metabolism defects. Raman spectroscopy complemented these findings by demonstrating protein and lipid oxidative modifications, alongside compositional abnormalities in AMD samples. Thus, AMD plasma EV-enriched samples carry a systemic signature of complement and coagulation dysregulation, impaired redox homeostasis, and altered sphingolipid metabolism, reflecting established mechanisms of AMD retinal pathology. These AMD-associated biochemical profiles form a promising source for developing new diagnostics and mechanistic insights for precision medicine.
Pharmacological strategies to modulate extracellular vesicle (EV) release in vivo are gaining traction, yet their broader effects on local immune microenvironments remain unclear. In this study, we assessed how repeated intraperitoneal (i.p.) dosing of the neutral sphingomyelinase inhibitor GW4869 reshapes the peritoneal cavity. GW4869 administration triggered a strong local inflammatory reaction, was accompanied by a selective depletion of resident peritoneal macrophages and was associated with higher EV counts 24 h after the final injection. Targeted macrophage depletion in the absence of GW4869 produced a comparable increase in EV levels, implicating shifts in cellular composition and inflammatory state as key drivers of EV accumulation. By applying single-particle flow cytometry, we differentiated small EV subsets from ApoB+ lipoproteins and uncovered substantial heterogeneity within CD9+ particles, ruling out lipoprotein co-detection as the main explanation for the elevated EV signal. Overall, our results highlight that GW4869 treatment can reshape the local immune landscape and that such context-dependent changes must be considered when using this compound to infer EV biogenesis in vivo.
ABSTRACT Metabolic dysfunction‐associated steatotic liver disease (MASLD) is the worldwide leading cause of liver‐related mortality with a prevalence of 75% among individuals with obesity and lacking screenings tools. Extracellular vesicles (EVs)‐based microRNAs (miRNAs) have emerged as promising biomarkers with important roles in the pathogenesis of MASLD. This study aimed to characterize EV‐based miRNA profiles in individuals with obesity and MASLD before and during weight loss intervention. Small RNA sequencing was used to profile EV‐miRNAs from plasma across three groups: individuals with obesity and MASLD (n = 35), individuals with obesity without hepatic steatosis (n = 24) and lean controls without hepatic steatosis (n = 26). Further, the MASLD group underwent a personalized weight‐loss intervention. The liver and MASLD‐related miR‐122‐5p and three other miRNAs were differentially expressed in the MASLD group compared with the obesity control group at baseline. Moreover, miR‐122‐5p correlated with liver fat and liver enzymes (ALT, AST and GGT) at baseline, revealed solid predictability for MASLD in a combined panel (AUC = 0.8), and lastly demonstrated the highest accuracy in identifying individuals with high liver fat among all individuals with obesity. Finally, several MASLD‐ and hepatocellular carcinoma‐related miRNAs decreased significantly following weight loss and liver fat reduction in the MASLD group.
ABSTRACT Takayasu arteritis (TAK) is a large‐vessel vasculitis that can lead to aneurysmal dilation, yet reliable circulating biomarkers for vascular remodelling remain lacking. Extracellular vesicles (EVs) carry miRNAs implicated in vascular biology, but their relevance to TAK has not been fully explored. Circulating EVs were isolated from serum of 54 patients with TAK and healthy controls (HC), characterized, and profiled for miRNA content. Effects on endothelial cells (ECs) and monocytes were examined in vitro, and the diagnostic performance of EV‐associated miR‐223‐3p for aortic dilation was assessed by ROC analysis. Circulating EVs from patients with TAK exhibited a distinct miRNA profile compared with HC. HC‐EVs reduced the transcript abundance of ICAM‐1 and VCAM‐1 in ECs, an effect attenuated in TAK‐EVs and accompanied by enhanced monocyte adhesion. EV‐derived miR‐223‐3p was upregulated in TAK but selectively downregulated in patients with aortic dilation. EV‐associated miR‐223‐3p discriminated aortic dilation with an AUC of 0.748, exceeding that of CRP and ESR. Flow cytometric profiling revealed a reduction in platelet‐derived EVs in patients with aortic dilation. Reduced EV‐associated miR‐223‐3p is associated with aortic dilation in TAK and may serve as a candidate biomarker as an adjunct to imaging‐based assessment, warranting prospective validation in larger cohorts.
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.
ABSTRACT Biofilm‐associated staphylococcal infections remain exceptionally difficult to treat due to the presence of resilient staphylococcal biofilms and the limited effectiveness of currently available antibiotics. To combat persistent infections, providers are often forced to use prolonged courses of combination antimicrobial therapies that have significant toxicities with limited effectiveness, leading to increased hospital stays, substantial health care costs and a rise in patient morbidity and mortality. Staphylococcus aureus is a dominant cause of recalcitrant biofilms, for which novel non‐antibiotic therapeutics are critically needed. Lysins, a class of protein‐based antimicrobials, rapidly kill staphylococci, exhibit potent anti‐biofilm activity, have a low propensity of resistance development and synergy with antibiotics. To address this unmet need, we evaluate an extracellular vesicle (EV)‐based delivery platform for the engineered anti‐staphylococcal lysin LYSG101, designed to improve stability and localization at sites of infection. Initial proof‐of‐concept work is provided here, demonstrating that human serum‐derived EVs can be loaded with LYSG101 to exert a potent in vitro antimicrobial effect against both planktonic and biofilm forms of S. aureus . EV‐mediated delivery achieved activity equivalent to free lysin, with additional translational advantages including stability and the potential for sustained intra‐articular retention. This work supports further development of the EV‐mediated lysin delivery as a broadly applicable antimicrobial platform, with potential future applications in biofilm‐associated infections, including prosthetic joint infection (PJI).
ABSTRACT Head and neck cancer (HNC) is among the top ten malignancies worldwide and is associated with high morbidity and mortality. Oral potentially malignant disorders (OPMDs) predispose to certain HNCs. Recent studies suggest emerging roles of extracellular vesicles (EVs) in malignant transformation and provide potential in the management of HNC. This systematic review aimed to evaluate the role of EVs in HNC and OPMDs. The systematic review followed PRISMA‐2020 guidelines and was registered with PROSPERO. A comprehensive literature search was conducted in five electronic databases (Medline, Scopus, Embase, Cochrane and Web of Science), yielding 3705 initial records. Following de‐duplication and independent screening, 237 eligible articles were included. Data extraction was performed by two independent reviewers. Risk of bias was assessed using the Joanna Briggs Institute critical appraisal tools. Thematic analysis with narrative data synthesis was conducted and the results were organised under the following subthemes: EV isolation and characterisation methods, EV role in cancer and OPMDs progression, immune modulation, biomarker potential, and therapeutic applications. Studies included in the review were published between “1985–2025” and showed an accelerated growth in EV research after 2020. Most of the studies relied on traditional isolation and characterisation techniques including ultracentrifugation, transmission electron microscopy and flow cytometry. Most evidence was reported from preclinical studies and demonstrated EVs’ promise as biomarkers for early detection, prognostication, and therapeutic targets. However, the findings also underscored the need for standardised protocols. Current evidence on EVs’ role as drivers of epithelial mesenchymal transition, invasion, immune escape, metabolic reprogramming, and angiogenesis in the pathogenesis of HNC are summarised together with the role of EVs as biomarkers in HNC and potential applications in HNC therapy and therapy resistance. Mechanistic insights into the biological behaviour of EVs in HNC are provided and evidence‐based recommendations for future research are proposed in this review.
ABSTRACT The intestine plays critical roles in nutrient homeostasis and systemic health. The small intestine is the major organ that packages dietary lipids into chylomicrons and secretes them into the mesenteric lymph. Extracellular vesicles (EVs) as a mechanism for cell‐to‐cell communication often exhibit organ specific characteristics. EV secretion from the intestine into the mesenteric lymph—a unique biofluid—remains unexplored. To characterize gut‐derived EVs, Sprague‐Dawley rats were surgically implanted with cannula in the mesenteric lymph duct (for lymph collection) and the duodenum (for lipid infusion). Lymph fluid was collected for assessment of EV secretion. EVs in lymph fluids collected before and after lipid infusion were characterized by transmission electron microscopy, nanoparticle tracking analysis, and further analyzed by flow cytometry with antibodies against CD63, CD81, CD9 and apolipoprotein B (ApoB). Lipid infusion increased lymph triglyceride output peaking at 2 h. Lymph fluids contained EVs with diameters in the range of 20 to 300 nm and the signals of specific EV markers CD63, CD81 and CD9 were significantly elevated following lipid infusion. Depletion of chylomicrons from lymph had differential effects on the percentage and intensity of CD63, CD81 and CD9. In addition, these EVs exhibited distinct patterns in ApoB+ particle population, with the median fluorescence intensity of CD63 and CD81 being significantly higher than that of CD9. To conclude, EVs secreted into the mesenteric lymph could rapidly respond to lipid supply. EVs may bind to and co‐secrete with chylomicrons. Finally, gut‐derived EVs during active lipid absorption may include multiple subtypes with different affinities to chylomicrons. Collectively, these findings highlight the intestine as an EV secretion organ and the mesenteric lymph as an important biofluid harboring EVs, which opens a new venue for future investigation of biological functions of gut‐derived EVs.
ABSTRACT Ischaemic brain stroke is among the leading causes of death and disability worldwide. However, the current treatments have a limited time window and regeneration potential. Clinically relevant human platelet‐derived extracellular vesicles (EVs) offer potential neuroprotective treatment for stroke. Here, the neuronal uptake of carboxyfluorescein succinimidyl ester (CFSE)‐labelled EVs was confirmed by confocal imaging and three‐dimensional (3D) image analysis with Imaris software. The results showed that human induced pluripotent stem cell (hiPSC)‐derived neurons can internalize EVs. We also show the colocalization of EVs with cellular organelles: early endosomes and lysosomes. We used an in vitro human model of stroke to study the effects of hypoxia on neurons. After hypoxia, the activity of the neurons, including spiking and bursting, decreased. However, the activity was restored after 72 h of reperfusion. EVs did not affect neuronal activity acutely, but during long‐term follow‐up, neurons showed increased activity. Together, our findings provide insights into the effects of platelet‐derived EVs on neuronal uptake, morphology and functionality and changes after hypoxic insult in an in vitro human model.
By preserving molecular information inherited from the source cell, extracellular vesicles (EVs) can serve as biomarkers for tissue health or disease, paving the way for liquid biopsy applications. The enrichment of tissue-specific EVs (TS-EVs) from human biofluids can be challenging due to technical and methodological limitations. Here, we use single and sequential immunoaffinity capture workflows to enrich antigen-specific EVs circulating in human blood. We demonstrate the specificity, efficiency, and consistency of our approach for enriching blood plasma EV subpopulations from the nervous system, alveolar cells and hepatocytes. The enriched subpopulations are characterized by canonical EV features and markers, as well as co-localization of tissue-specific and general markers on the surface. We provide a validated workflow to derive multiple EV subpopulations from circulation, leveraging their promise as informative biomarkers of tissue status and enabling liquid biopsy and biomarker discovery.
Mesenchymal stromal cell extracellular vesicles (MSC EVs) hold great therapeutic potential. Their immunomodulatory abilities make them suitable candidates to treat autoimmune diseases, such as rheumatoid arthritis. However, MSC EV production must be reproducibly scaled to meet the demand of research and therapeutics. To achieve this, four human umbilical cord MSC (UC-MSC) donors were pooled, a method known to generate a large cell source, that averages heterogeneous cell attributes. Upon generating MSC EV enrichments, donor pooling proved advantageous by increasing EV yield, both by increasing particle number and the presence of EV defining characteristics. Protein analysis suggests this could be due to an upregulation of protein transport mechanisms but requires further work to confirm. When applied to an inflammatory model of arthritis, pooled UC-MSC EV enrichments surpassed their parental cells and single donor UC-MSC EV enrichments, in alleviating arthritic pathophysiology, albeit the particle input was doubled since this was normalised by cell number. Therefore, we propose donor pooling as a simple and effective method to generate MSC EV enrichments, with potential to increase EV production without compromising therapeutic efficacy.
ABSTRACT Extracellular vesicles (EVs), lipid bilayer nanoparticles released by virtually all cells, serve as essential messengers for intercellular communication. Due to their involvement in several pathophysiological processes, EVs have recently gained considerable attention as potentially diagnostic and prognostic biomarkers for various illnesses. The widespread distribution of EVs across all biofluids positions them as ideal, minimally invasive biomarkers for disease progression using a liquid biopsy approach. Among biofluids, saliva is uniquely accessible and has a low soluble protein content, making its EV population a highly promising source of diagnostic biomarkers. Salivary EVs have been investigated for their promising potential for diagnosing local and systemic diseases, including cancers, autoimmune diseases and neuropsychiatric disorders. In this review, we present a synopsis of the current landscape related to salivary EVs, highlighting the unique characteristics and potential of these vesicles, the technical challenges related to their application, and their future prospects for clinical translation as powerful diagnostic tools.
Temozolomide (TMZ) is a first-line chemotherapeutic agent for the treatment of glioblastoma (GBM), while a majority of patients do not effectively respond to TMZ owing to the multiple resistance mechanisms. In this study, we found that TMZ treatment substantially increased the exosome secretion from GBM cell line. The secreted exosomes from TMZ-treated C6 cells maintained typical physicochemical properties, with only slight fluctuation in expression profile of surface markers compared to untreated cell-derived exosomes. Further investigation revealed that increased expression of p53 might be the predominant reason to promote exosome secretion, potentially through the upregulation of six-transmembrane epithelial antigen of prostate 3 (STEAP3) expression under stress condition. Moreover, we identified that pre-treatment with TMZ-induced exosomes could reduce the sensitivity of C6 cells to TMZ and considered that exosome secretion may serve as a potent TMZ resistance mechanism via enhancing anti-apoptotic ability of GBM cells towards TMZ exposure. Proteomics analysis revealed a strategic mechanism to resist TMZ-induced apoptosis by upregulating exosomal proteins associated with biogenesis, chemoresistance, and therapeutic adaption. This finding revealed a considerable TMZ resistance mechanism and provided a new inspiration for preventing exosome biogenesis to resensitise TMZ cytotoxicity towards GBM.
ABSTRACT Small extracellular vesicles (EVs) from bovine milk and whey are emerging as biologically active carriers of microRNA (miRNA), yet their composition and variability among different dairy sources remain incompletely understood. We conducted an integrated characterisation of EV‐associated miRNAs isolated from milk and whey collected from three dairy farms. EVs were purified and validated according to MISEV2023 criteria, and small RNA sequencing was performed on milk and whey samples collected on three different days at each farm (18 samples). After quality filtering and adaptor trimming, mapped reads were normalised to counts per million (CPM). To ensure robust detection, only miRNAs present in at least 67% of samples were retained, resulting in a high‐confidence set of 329 miRNAs for downstream analyses. Milk‐derived EVs showed a higher overall miRNA read count than whey‐derived EVs (9.45 ± 5.3 million vs. 5.03 ± 2.5 million reads; p < 0.10), with Farm Z displaying the greatest overall abundance. Stability assessment using Z‐scored coefficients of variation demonstrated that miRNA reproducibility varied considerably between farms and collections, indicating that farm‐ and collection‐specific factors, rather than matrix origin alone, were major contributors to expression variability. Differential expression analysis (DESeq2) identified 32 miRNAs significantly modulated between milk and whey EVs, with most (n = 29) enriched in whey. Functional enrichment of predicted targets indicated involvement in immune and inflammation‐related pathways, including NF‐κB signalling, cytokine–receptor interaction and Ras signalling. Pairwise comparison of farms revealed only a small number of differentially expressed miRNAs in both matrices, and KEGG analyses did not identify significant pathways after multiple‐testing correction. This study presents a comparative overview of bovine milk and whey EV‐associated miRNAs, highlighting higher miRNA abundance in milk, significant farm‐dependent variability, and enrichment of immune‐related pathways that distinguish the two matrices.
ABSTRACT Extracellular vesicles (EVs) are nanosized lipid bilayer particles naturally secreted by cells, mediating intercellular communication and transporting diverse bioactive molecules. Among alternative EV sources, bovine milk‐derived EVs (BMEVs) have emerged as promising platforms for drug delivery due to their accessibility, scalability, stability and promising biocompatibility, although their long‐term safety profile, particularly for engineered or cargo‐loaded formulations, still requires further investigation. BMEVs can encapsulate small molecules, natural polyphenols and nucleic acids, enhancing bioavailability, protecting cargo from degradation and facilitating functional delivery. Recent studies demonstrate their potential for oral administration and their intrinsic therapeutic properties, including antioxidant, anti‐inflammatory, and immunomodulatory activities. Moreover, the increasing number of patents highlights their translational and commercial relevance in therapeutic, cosmetic and nutraceutical applications. Despite these promising features, challenges remain in standardizing isolation protocols, optimizing cargo loading, ensuring batch reproducibility and evaluating long‐term safety. Addressing these gaps is essential to enable clinical translation and establish BMEVs as versatile drug delivery platforms. Trial Registration: ClinicalTrials.gov identifier: NCT07402083
The concept of protein corona formation around extracellular vesicles (EVs) has given birth to new insights into how cells may recognize EVs by proteins presented at the EV surface. Here we present spatially resolved proteomics using the biotin ligase TurboID to map proteins interacting at the EV surface, without a need for physical isolation of the EV-corona complexes. TurboID promiscuously biotinylates nearby proteins within a few nm distance from the fused 'bait' protein. We genetically engineered EVs by modifying CD63 as the bait protein to which TurboID was fused facing outward to map EV corona proteins. Biotinylated proteins were then analysed by Western blotting and liquid chromatography-mass spectrometry. Western blots revealed protein patterns that are distinct depending on the localization of the fused TurboID. The mass spectrometry analysis identified many of the serum proteins commonly known to form a corona around a synthetic solid nanoparticle, also supporting those previously reported through the physical isolation approaches. Rather striking is, however, the EV corona footprint of endogenous proteins that tells us about the EV biogenesis and what the intrinsic endogenous corona might look like. This approach, which we coined EV-SPEC (Spatial Proteomics of Endogenous Corona), has thus the potential to revolutionize our understanding of EV biology by shifting the focus from the EVs themselves to the proteins that make up the corona, or how the cell 'sees' them, in analogy with the biomolecular corona extensively characterized for synthetic nanoparticles.
