Journal of Extracellular Biology

Extracellular vesicles (EVs) and non-vesicular extracellular particles (NVEPs) have been recently discovered as part of the diverse components of the secretome. EVs and NVEPs have been shown to differ not only in size and morphology but also in their cargo and biological functions. NVEPs are a newly discovered group of multimolecular assemblies with potential roles in physiological and pathological states. However, very little is known about this new class of particles. Here, for the first time, we present a method for simultaneously isolating and characterizing EVs and NVEPs from primary mouse tissues.

Bladder cancer exhibits molecular heterogeneity that complicates early diagnosis and prognosis, and drives confounding clinical outcomes. Non-muscle invasive and muscle-invasive subtypes, especially for intermediate to high grade, carry a 25 - 50% progression-free survival rate, underscoring the need for high precision prognostic strategy. Urinary extracellular vesicles (uEVs) are promising carriers of tumor-derived RNAs and proteins. However, significant challenges in studying uEVs arise from the diverse cellular origin of uEVs associated with the dynamic composition of urine, which presents roadblocks for developing the clinical utility of uEVs. We introduced an AI-driven EV liquid biopsy pipeline that integrates (1) standardized EV isolation via NanoPom magnetic beads, (2) transcriptomic profiling for molecular subtyping, and (3) prognostic scoring algorithm. In a discovery cohort of 16 bladder cancer patients including both MIBC and NMIBC, we compared NanoPom isolated uEVs with ExoEasy and Fujifilm MagCapture isolated uEVs, for identifying bladder cancer subtype-specific gene signatures, and externally validated them using UCSC Xena. The result outperformed currently reported bladder cancer diagnostic biomarkers from assays including Galeas, CxBladder, and Xpert. In a validation cohort of matched 7 patient plasma samples, we confirmed with plasma derived EVs for correlating with urinary EV biomarkers from NGS sequencing. The prognostic score stratified patients into low-, intermediate-, and high-grade risk groups based on Xenas bladder cancer survival outcomes. Our AI-driven uEV liquid biopsy pipeline proves the concept for high precision bladder cancer subtyping and prognosis, which could potentially facilitate treatment decision and lead to advanced profiling of bladder tumor biology using uEV liquid biopsy.

Extracellular vesicles (EVs) are subcellular particles surrounded by a lipid bilayer membrane and incorporating various additional biomolecules derived from their donor cell. In many disease contexts circulating EVs have received increasing scientific attention due to their potential diagnostic and prognostic value. Additionally, EVs have been ascribed multiple biological functions, ranging from cellular waste disposal to sophisticated, intercellular communication. Consequently, EVs involved in pathological processes may represent therapeutic targets, whereas EV-based therapeutics are being developed for targeted delivery of molecular cargoes in vivo. Detailed knowledge of the molecular content of natural EVs derived from diverse cellular origins is crucial to identify biomarkers, dissect EV functions, and optimize EV engineering for therapeutic purposes. Although the lipid composition of biological membranes has a significant impact on their biophysical and -chemical properties and may affect signaling and interactions at the molecular and cellular level, relatively little is known about the lipid composition of EV membranes. Here, we applied high resolution mass spectrometry to deeply and quantitatively characterize the lipidome of EVs isolated from a panel of malignant and non-malignant cell lines, providing a comprehensive data resource for biomarker research and EV engineering efforts. Furthermore, subset comparisons indicate striking differences between lipid profiles of EVs isolated from cells of different tissue origin, suggesting distinct membrane characteristics that could affect EV biodistribution and function in vivo.

Serum-free synthetic media are frequently used as an alternative to extracellular vesicle-depleted serum containing media (EV-DEP) for EV production and collection. Here, we fully characterised the EVs released by MDA-MB-231 or HEK-293T cells cultured in 10% EV-DEP DMEM or a commonly used serum-free synthetic medium, Opti-MEM. Cells cultured in Opti-MEM released a significantly higher yield of CD9- and CD63-positive EVs as compared to cells grown in EV-DEP DMEM. In MDA-MB-231 cells the increased EV release was likely driven by nutrient deprivation (decreased phospho-S6) and increased cell stress (increased phospho-AKT). MDA-MB-231 cells grown in EV-DEP DMEM relied upon actin cytoskeleton dynamics for up to 50% of EVs released, while cells in Opti-MEM did not use ROCK kinases for EV biogenesis. Mass spectrometry analysis showed that the EV proteomes from both conditions were largely unchanged other than EV-DEP DMEM EVs contained more histones and bovine proteins. Addition of 2.5 - 10% serum or 2.5 mg/mL albumin to Opti-MEM medium partially rescued some cell stress phenotypes and returned EV release rates to that of cells in EV-DEP DMEM. The choice of media is an important consideration when designing EV studies and their downstream applications. While Opti-MEM increased cell stress, it achieved a high yield of EVs that were depleted of histones and contaminating bovine proteins - useful for therapeutic applications. Alternatively, EV-DEP DMEM produced a decreased yield of EVs in lower stress conditions, however, the EVs were histone and bovine protein rich which may have important implications for use for use in therapeutic or immunological experiments.

Extracellular vesicles (EV) surface proteins have important extracellular functions and determine cellular tropism; however, characterizing the EV surfaceome remains challenging with available methods. EV-mediated intercellular communication takes place primarily through interactions at the recipient cell membrane, underscoring the importance of methodological advances to map this interplay. Here, we leverage the proximity labeling enzyme APEX2 (Apurinic/Apyrimidinic Endodeoxyribonuclease 2) for high-fidelity analysis of the EV surfaceome and cellular tropism. Surface display of APEX2 on EVs is achieved through its genetic fusion with EV-sorting domains, such as CD63 and TSPAN2. Upon adding the substrates biotin-phenol and hydrogen peroxide, vesicle surface APEX2 enables biotinylation of EV integral and corona proteins as well as target cells in vitro. Further data mining of the EV surfaceome reveals potential scaffolds for the bioengineering of EVs. Altogether, we introduce a robust tool for EV surfaceome and target cell mapping and uncover novel EV-sorting domains for bioengineering.

BackgroundExtracellular vesicle (EV)-based liquid biopsy remains challenging due to the limited specificity and low abundance of EV cargoes. This study aimed to identify cancer-specific molecules from tissue-derived EVs and evaluate their potential as circulating biomarkers for non-small cell lung cancer (NSCLC). MethodsTissue-derived EVs were isolated by density gradient flotation and subjected to proteomic profiling to identify differentially expressed proteins. Public transcriptomic data, functional enrichment, and variable importance scores were integrated to select candidate biomarkers. Immunogold labeling transmission electron microscopy (TEM) was used to visualize protein expression on the EV membrane using, and an ultrasensitive modified digital EV screening technique (DEST) was employed to quantify protein signals in circulating EVs. The diagnostic performance was evaluated by the area under the curves (AUC). ResultsTissue-derived EVs were isolated from cancer tissues and paired non-cancer lung tissues from patients with NSCLC. Proteomic profiling identified 1,568 tissue-derived EV proteins, of which 904 were differentially expressed. Integrated transcriptomic and proteomic analyses yielded eight cancer-specific, membrane-localized candidate biomarkers, with GLUT1 showing the highest variable importance score. Immunogold labeling TEM confirmed the localization of GLUT1 on the EV membrane. Modified DEST analysis of 47 patients revealed significantly elevated levels of CD63+ EV-derived GLUT1 in the cancer group than in the non-cancer group (P < 0.01). Diagnostic performance reached an AUC of 0.817 in the whole cohort and 0.898 in the stage-I cohort, exceeding that of conventional serum biomarkers. A three-marker panel (CD63+ EV-derived GLUT1, CYFRA 21-1, and CEA) further improved discrimination, with AUCs of 0.853 in the whole cohort and 0.944 in the stage-I cohort. ConclusionCirculating EV-derived GLUT1 was preliminarily identified as a novel biomarker for NSCLC, and its combination with conventional serum biomarkers enhanced diagnostic performance, particularly in early-stage cases.

BackgroundExtracellular vesicles (EVs) are nano and macro-sized, lipid-bound particles, involved in cellular communication. Interestingly, cancer-derived EVs show a heterologous and cross-species tumour tropism which makes them a potential tool for efficient delivery of therapeutic small interfering RNA (siRNA) to the tumour cells. MethodsEVs derived from glioblastoma cells (U373P and U373vIII) were loaded with EGFRvIII siRNA to develop a targeted therapeutic strategy against glioblastoma. EV biodistribution was evaluated using fluorescent indocyanine green (ICG) staining followed by ex vivo imaging. Different loading strategies, including passive loading, sonication, saponin-mediated membrane permeabilization, electroporation, and transfection were assessed for their efficiency in loading siRNA into EVs. The efficiency of each method was evaluated by nano flowcytometry, in vitro uptake assay followed by immunoblot (western blot) analysis. Eventually, the most effective formulation was tested for the systemic siRNA administration and selective tumour delivery in vivo, followed by evaluation of tumour size and EGFRvIII expression. ResultsHere, we showed that siRNA transfection into EVs was the most effective loading strategy, as confirmed by nano-flow cytometry, uptake assays, and western blot analysis, achieving over 90% knockdown efficiency in vitro for EVs carrying EGFRvIII siRNA. In vivo, EGFRvIII siRNA-loaded EVs homed to the tumour site and downregulated EGFRvIII expression compared with the PBS-siRNA control group; however, no significant tumour shrinkage was observed. ConclusionEGFRvIII-targeting, glioblastoma cell-derived EVs can be used as siRNA delivery carriers for targeted gene therapy in glioblastoma. However, further optimization of siRNA delivery and treatment duration is required.

Extracellular Vesicles (EVs), membrane vesicles released by all cells, are emerging mediators of cell-cell communication. By carrying biomolecules from tissues to biofluids, EVs have attracted attention as non-invasive sources of clinical biomarkers in liquid biopsies. Although frequently employed for content characterization of EVs, the study of bulk preparations lacks information on sub-populations and the intrinsic heterogeneity of vesicles. Importantly, these strategies also difficult the characterization of EVs from small quantities of samples. We here present a Flow Cytometry strategy that enables detailed population analysis of EVs, at the same time decreasing sample volume requirements and accelerating the overall processing time. We show its unique application for quality control of isolates of EVs by comparing the proportion of vesicular and non-vesicular particles in samples prepared by different protocols. In addition, we demonstrate its suitability for the study of populations of EVs from samples characterized by challenging small volumes. To illustrate that, we perform longitudinal non-lethal analysis of EVs in mouse plasma and in single-animal collections of murine vitreous humor. By allowing for the analysis of EVs from minimal amounts of sample, our Flow Cytometry strategy has an unexplored potential in the study of EVs in clinical samples with intrinsically limited volumes. When compared to conventional methods, it also multiplies by several times the number of different analytes that can be studied from a single collection of biofluid.

Nanoparticles can acquire a protein corona defining their biological identity. Corona functions were not yet considered for cell-derived extracellular vesicles (EVs). Here we demonstrate that nanosized EVs from therapy-grade human placental-expanded (PLX) stromal cells are surrounded by an imageable and functional protein corona when enriched with permissive technology. Scalable EV separation from cell-secreted soluble factors via tangential flow-filtration and subtractive tandem mass-tag proteomics revealed significant enrichment of predominantly immunomodulatory and proangiogenic proteins. Western blot, calcein-based flow cytometry, super-resolution and electron microscopy verified EV identity. PLX-EVs protected corona proteins from protease digestion. EVs significantly ameliorated human skin regeneration and angiogenesis in vivo, induced differential signaling in immune cells, and dose-dependently inhibited T cell proliferation in vitro. Corona removal by size-exclusion or ultracentrifugation abrogated angiogenesis. Re-establishing an artificial corona by cloaking EVs with defined proangiogenic proteins served as a proof-of-concept. Understanding EV corona formation will improve rational EV-inspired nanotherapy design.

The majority of studies on extracellular vesicles (EVs) focused on samples isolated from liquid matrices, such as cell culture media and blood, due to their accessibility. However, recent research highlights the emerging roles of EVs derived from solid tissues, including the brain, muscles and tumors. Investigating EVs from the extracellular matrix of solid tissues offers insights into their microenvironment and potential biological influences on surrounding cells. This study presents a universal method for comparing EV-enriched samples from solid (human skeletal muscle biopsy) and liquid (human plasma) matrices, addressing technical challenges and minimizing biases in separation techniques. By employing optimized protocols and advanced analytical techniques, the study reveals differences in biomolecular composition, nanomechanical properties, particle yield, size distribution, and colloidal stability between human skeletal muscle and plasma EVs. Understanding these distinctions may contribute to the development of novel diagnostic assays for muscular pathologies and shed light on the roles of EVs in diverse tissue environments.

Extracellular vesicles (EVs) may contain a variety of molecular cargo including proteins and nucleic acids. Vault particle components have been repeatedly reported in the literature as EV cargo. Here, we demonstrated by small RNA sequencing that vault RNA (vtRNA) were highly abundant in EV pellets enriched by differential centrifugation. EVs were prepared by commonly used enrichment methods and biochemical assays used to determine whether vault particle components were bona fide EV cargo. EVs were isolated by differential centrifugation, size exclusion chromatography (SEC) and Dynabead immunocapture. RNase and proteinase treatment of EV preparations demonstrated that most vtRNA and major vault protein (MVP) were not enclosed and protected within the EV membrane. Vault-like particles were visualised in differential centrifugation pellets by cryo-transmission electron microscopy. EVs enriched by size exclusion chromatography and those isolated by immunocapture post-ultracentrifugation showed co-purification of MVP, whereas EVs isolated by direct immunocapture from conditioned medium were MVP-negative. Taken together, commonly used isolation techniques, such as differential centrifugation and SEC, can lead to contamination of EVs with vault particles. The current study highlights the importance of determining the topology of putative EV-associated components to determine if they are EV cargo or contaminants that have been co-purified.

Malignant pleural effusion (MPE) is a common clinical manifestation of advanced non-small cell lung cancer (NSCLC) and represents a valuable source of tumor-derived components, including extracellular vesicles (EVs). The aim of this study was to compare three different EV enrichment methods with potential applicability in clinical practice: a commercial Norgen kit (NOR), immunoaffinity capture (IA), and ultracentrifugation (UC). Following EV characterization, IA demonstrated the best overall performance in terms of EV yield and purity, NOR showed intermediate efficiency, while UC was the least effective method. The IA method exhibited characteristics suitable for potential clinical implementation, whereas NOR and UC may require combination with additional enrichment approaches. Advancing pleural effusion-based liquid biopsy toward clinical application will depend on the development of robust, scalable, and user-friendly EV enrichment workflows, along with harmonized guidelines for sample collection, preprocessing, and data reporting. Establishing such standards will enhance reproducibility, enable meaningful cross-study comparisons, and accelerate the integration of EV-based biomarkers into precision oncology.

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.

ObjectiveThe objective of this project was to determine if extracellular vesicles (EVs) were present in FACTORFIVEs EXOFECTA LyoBeads to validate flash freezing and subsequent lyophilization as an effective EV preservation method. SummaryEVs were isolated from resuspended EXOFECTA LyoBeads using differential ultracentrifugation and subjected to further characterization techniques such as nanoparticle tracking analysis (NTA), single particle flow cytometry, and cryo-electron microscopy (cryo-EM). First, NTA revealed particles with size distributions and concentrations consistent with the expected characteristics of EVs, indicating successful isolation from samples. Secondly, flow cytometry was performed using antibodies targeting the canonical EV tetraspanins CD9, CD63, and CD81. Detection of these markers supported the presence of enriched EVs in the samples. Finally, cryo-EM was performed to confirm the existence of vesicular bodies and their morphology. Visualization of the samples revealed membrane-enclosed particles with a visible lipid bilayer, consistent with EVs. Overall, the results support EVs were in EXOFECTA LyoBeads, indicating EV retention in the final product after FACTORFIVEs method of flash freezing, stabilizing, and lyophilization.

Follicular fluid extracellular vesicles (FF EVs) facilitate communication between oocytes and somatic cells within the ovarian follicle, playing a pivotal role in follicular development. This study highlights the molecular and functional distinctions between small (SEV) and large (LEV) FF EV subpopulations, revealing their specialized regulatory roles in granulosa cell (GC) biology and their consequential impact on ovarian function. Single-EV profiling uncovered distinct tetraspanin distributions, with LEVs containing a lower proportion of CD9/CD63/CD81-positive particles compared to SEVs. Functionally, SEVs reduced estradiol secretion by GCs, whereas LEVs enhanced progesterone production, demonstrating their differential effects on steroidogenesis. Transcriptomic analysis revealed extensive SEV-induced changes in GC gene expression, affecting pathways involved in transcription, TGF-{beta} signaling, extracellular matrix (ECM) remodeling, and cell cycle regulation. In contrast, LEVs elicited minimal transcriptional changes, primarily modulating genes associated with immune regulation and oxidative stress defense. Small RNA sequencing further revealed distinct non-coding RNA (ncRNA) profiles, with SEVs enriched in miRNAs targeting pathways critical for GC differentiation, while LEVs carried higher levels of piRNAs implicated in maintaining genomic stability. These findings advance our understanding of FF EV-mediated intercellular communication and underscore the importance of investigating EV subpopulations independently.

BackgroundP-glycoprotein (P-gp/ABCB1) is a key efflux transporter that maintains barrier integrity by clearing xenobiotics and toxic metabolites. At the feto-maternal interface, trophoblast-derived extracellular vesicles (CTC-EVs) naturally and transiently transfer functional P-gp to maternal decidual cells, restoring lost and or reduced P-gp function (exofection) to sustain pregnancy homeostasis. A similar loss of P-gp at the blood brain barrier (BBB) contributes to impaired amyloid-{beta} (A{beta}) clearance and neuroinflammation in Alzheimers disease. We investigated whether CTC-EV-mediated exofection could restore P-gp function in human brain endothelial cells (hBECs) and enhance A{beta} clearance under inflammatory and neurodegenerative conditions. MethodsCTC-EVs were isolated and characterized by nanoparticle tracking analysis and western blotting for P-gp and EV markers. Transcriptomic profiling of CTC-EVs identified enrichment of transporter-related genes, including solute carriers and ABC transporters, along with inflammatory mediators. Network analysis revealed coordinated modules linking EV cargo to transporter regulation, endocytosis/trafficking pathways, and inflammatory remodeling processes converging on BBB efflux activity. hBECs were exposed to LPS (500 ng/mL, 48 h) with or without CTC-EVs. P-gp expression was assessed by immunofluorescence (mean fluorescence intensity, MFI) and western blotting, while functional efflux was measured using Calcein-AM assays. A{beta} oligomer transport was evaluated using a transwell hBEC model. In vivo, 3xTg-AD mice received intravenous CTC-EVs (1x10L/day for 5 days), followed by assessment of P-gp expression, A{beta} burden, and neuroinflammatory markers. Pharmacokinetic studies in P-gp knockout mice were conducted to confirm functional transporter recovery. ResultsLPS exposure significantly reduced P-gp expression in hBECs (41.3% decrease in MFI, p=0.0084), which was restored by CTC-EVs (46.7% increase vs. LPS, p=0.0121). Exofection increased P-gp by a 2.1-fold following EV treatment as determined by western blot. Functional assays demonstrated enhanced efflux, with a 38.5% reduction in intracellular Calcein fluorescence (p<0.001). Network-informed mechanisms supported coordinated regulation of transporter and trafficking pathways. CTC-EVs improved A{beta} transport across inflamed hBEC monolayers. In vivo, EV-treated 3xTg-AD mice exhibited increased P-gp expression in the frontal cortex (38.6%) and hippocampus (42.1%), reduced A{beta} plaque burden (27.9%), and decreased inflammatory markers (IL-1{beta} and TNF-, p<0.05). In P-gp knockout mice, EVs reduced brain drug accumulation by 22.4% (p=0.032), confirming restoration of transporter function. ConclusionCTC derived EVs are natural carriers of functional transporter proteins and restore efflux capacity in compromised endothelial barriers. Integration of transcriptomic and network analyses highlights coordinated regulation of transporter, trafficking, and inflammatory pathways underlying exofection. This reproductive biology inspired strategy offers a promising therapeutic approach for enhancing A{beta} clearance and mitigating neuroinflammation in Alzheimers disease.

Enhancing protocols and methods for producing therapeutic extracellular vesicles (EVs) in bioreactors is crucial to achieve scalable production while ensuring both quality and quantity. Studies have shown that mechanical stress can promote EV release, although the underlying mechanisms remain largely unclear. Here we investigated which mechanisms are responsible for the increase of EV production under shear stress. EVs were produced from adipose tissue-derived stromal cells (ASCs), also described as mesenchymal stromal cells (MSCs) that can support the regeneration of injured tissues via several paracrine factors. The cultures were treated with GsMTx4, an inhibitor which blocks the Piezo1 ion channels, or with YODA1, its agonist, to assess if mechanical or shear forces pathways are involved in enhancing the EV release. EVs were quantified and characterized after high shear (HS) stimulation compared with no shear stress stimulation (3D) and standard cultures methods (2D). These experiments showed that, after mechanical stimulation by shear stress, EV production increased in bioreactors and this effect was blocked by the inhibition of Piezo1 ion channels with GsMTx4 (88%) with no impact on cell viability. Consistently, the agonist YODA1 increased the EV production (149%). The implications of these findings are significant, especially for regenerative medicine and cellular therapies, where the efficient production of high-quality EVs is crucial. By understanding turbulence-induced shear stress and the natural mechanotransductive pathways within cells, it may be possible to optimize the production of therapeutic EVs, paving the way for more effective treatments in the future.

Current extracellular vesicle (EV) isolation methods depend on large expensive equipment like ultracentrifuges and are laborious and time consuming. There is also currently no method available for high throughput isolation to meet clinical demands. Here, we present a method that combines our previous published ExtraPEG method and magnetic beads. Western blot and nanoparticle tracking analysis (NTA) of the purified EVs revealed higher or equivalent recovery and purity with this method compared to ExtraPEG or size exclusion chromatography (SEC) methods. With this newly developed workflow and automated liquid handling instrument, we have successfully isolated up to 96 EV samples from 5 {micro}L pre-cleared serum in 45 minutes. Moreover, DNA / small RNA / protein purification and profiling steps could be seamlessly integrated into the isolation workflow. To profile EV protein markers, EVs were lysed from the binding step and covalently bound to the surface of the beads. TotalSeq or ELISA antibody can be applied with under a standard protocol. With this extended protocol, researchers can easily complete EV isolation and protein profiling experiment within 8 hours. Taken together, we provide a high throughput method for EV isolation and molecular analyses that may be used for sensitive biomarker detection from biological fluids.

Extracellular vesicles (EVs) have been lauded as next generation medicines, but very few EV-based therapeutics have progressed to clinical use. Limited clinical translation is largely due to technical barriers that hamper our ability to mass-produce EVs, i.e. to isolate, purify and characterise them effectively. Technical limitations in comprehensive characterisation of EVs leads to unpredicted biological effects of EVs. Here, using a range of optical and non-optical techniques, we showed that the differences in molecular composition of EVs isolated using two isolation methods correlated with the differences in their biological function. Our results demonstrated that the isolation method determines the composition of isolated EVs at single and sub-population levels. Besides the composition, we measured for the first time the dry mass and predicted sedimentation of EVs. These parameters were shown to correlate well with the biological and functional effects of EVs on single cell and cell cultures. We anticipate that our new multiscale characterisation approach, which goes beyond traditional experimental methodology, will support fundamental understanding of EVs as well as elucidate the functional effects of EVs in in vitro and in vivo studies. Our findings and methodology will be pivotal for developing optimal isolation methods and establishing EVs as mainstream therapeutics and diagnostics. This innovative approach is applicable to a wide range of sectors including biopharma and biotechnology as well as to regulatory agencies.

The biological functions of extracellular vesicles (EVs) depend on their cellular source. Further, different subpopulations of EVs from the same cells carry different cargo, but differences in their biological functions are less understood. We here identify a very small EV subpopulation released by HEK293F cells (miniEVs). These EVs, in contrast to the larger EVs, were found to have anti-inflammatory properties. Quantitative proteomics identified a potential anti-inflammatory molecule, Syndecan-4 (SDC4), on the surface of the miniEVs, but not larger EVs. We engineered HEK293F cells to overexpress SDC4, which results in the molecule being highly expressed in all EV subpopulations. Expression of SDC4, a proteoglycan, also increased the presence of heparan sulfate on the EV surface. Furthermore, these EVs were found to have potent anti-inflammatory effects in vitro, which heparinase treatment could slightly reduce. Furthermore, the SDC4 EVs showed anti-inflammatory effects in vivo in a model of peritonitis. We conclude that HEK293F EVs can be engineered to become anti-inflammatory, and that SDC4-expressing HEK293F-EV potentially could become an anti-inflammatory therapeutic.