Extracellular Vesicle Analysis: Recent Technological Advances and Emerging Opportunities.

A novel immunoassay for ApoB-100, the main protein component of lipoproteins, enables the development of methods to enrich extracellular vesicles from human plasma while depleting both lipoproteins and free proteins.

Extracellular Vesicles: How a Circulating Biomarker Can Double As a Regulator of Blood Pressure.

BackgroundInfection and graft-versus-host disease (GvHD) are the major causes for mortality and morbidity of allogeneic hematopoietic stem cell transplantation (allo-HSCT). Plasma-derived extracellular vesicles (EVs) contain disease-related proteins, DNAs and RNAs, and have recently been suggested as potential biomarker candidates for transplantation complications. However, EV isolation from small plasma volumes in clinical biomarker studies using conventional methods is challenging. We therefore investigated if EVs isolated by novel automated acoustic trapping could be developed as potential biomarkers for allo-HSCT complications by performing a clinical proof-of-principle study.ResultsPlasma samples were collected from twenty consecutive patients with high-risk/relapsed hematologic malignancies undergoing allo-HSCT before transplantation and post-transplant up to 12 weeks. EVs were isolated from small plasma sample volumes (150 μl) by an automated, acoustofluidic-based particle trapping device, which utilizes a local λ/2 ultrasonic standing wave in a borosilicate glass capillary to capture plasma EVs among pre-seeded polystyrene microbeads through sound scatter interactions. We found that EVs could be reliably isolated from all plasma samples (n = 173) and that EV numbers increased more than 2-fold in the majority of patients after transplantation. Also, sufficient quantities of RNA for downstream microRNA (miRNA) analysis were obtained from all samples and EV miRNA profiles were found to differ from whole plasma profiles. As a proof of principle, expression of platelet-specific miR-142-3p in EVs was shown to correlate with platelet count kinetics after transplantation as expected. Importantly, we identified plasma EV miRNAs that were consistently positively correlated with infection and GvHD, respectively, as well as miRNAs that were consistently negatively correlated with these complications.ConclusionsThis study demonstrates that acoustic enrichment of EVs in a clinical biomarker study setting is feasible and that downstream analysis of acoustically-enriched EVs presents a promising tool for biomarker development in allo-HSCT. Certainly, these findings warrant further exploration in larger studies, which will have significant implications not only for biomarker studies in transplantation but also for the broad field of EV-based biomarker discovery.

Extracellular vesicles (EVs) derived from tumor cells have the potential to provide a much-needed source of non-invasive molecular biomarkers for liquid biopsies. However, current methods for EV isolation have limited specificity towards tumor-derived EVs that limit their clinical use. Here, we present an approach called immunomagnetic sequential ultrafiltration (iSUF) that consists of sequential stages of purification and enrichment of EVs in approximately 2 h. In iSUF, EVs present in different volumes of biofluids (0.5–100 mL) can be significantly enriched (up to 1000 times), with up to 99% removal of contaminating proteins (e.g., albumin). The EV recovery rate by iSUF for cell culture media (CCM), serum, and urine corresponded to 98.0% ± 3.6%, 96.0% ± 2.0% and 94.0% ± 1.9%, respectively (p > 0.05). The final step of iSUF enables the separation of tumor-specific EVs by incorporating immunomagnetic beads to target EV subpopulations. Serum from a cohort of clinical samples from metastatic breast cancer (BC) patients and healthy donors were processed by the iSUF platform and the isolated EVs from patients showed significantly higher expression levels of BC biomarkers (i.e., HER2, CD24, and miR21).

MP18-13 IDENTIFICATION OF TUMOR-SPECIFIC MARKERS ON EXTRACELLULAR VESICLES IN PATIENTS WITH RENAL CELL CARCINOMA

Extracellular vesicles (EVs) are bilayer lipid membrane-enclosed vesicles shed by cells into physiological fluids. EVs serve as important mediators of cell-cell communication, which can transfer proteins, messenger RNA, microRNAs, metabolites and other biological molecules. There is a significant interest in EVs as potential novel tools for liquid biopsy-based disease diagnostics and therapy delivery. In this work, we developed and evaluated new techniques for EV enrichment from human blood plasma using size-exclusion chromatography (SEC) and multimodal chromatography. In brief: 1. we evaluated different column bed volumes for the SEC-based EV enrichment, and the column packed with 4 mL of the SEC stationary phase Sepharose CL-2B showed the most efficient EV separation; 2. The maximum protein binding capacity of multimodal chromatography resins CaptoCore 400 and CaptoCore 700 in both suspension mode and the column format were determined. 3. We combined the size-exclusion chromatography and multimode chromatography approaches to further increase the purity of EV enrichment from blood plasma and decrease the levels of free plasma proteins in EV isolates. The developed biphasic 'sandwich' columns packed with two layers of SEC and multimode CaptoCore stationary phases placed on top of each other and sequential depletion of plasma proteins by the CaptoCore beads were evaluated for EV enrichment from blood plasma. Higher purity of EV isolation by the 'sandwich' column approach was confirmed by EV count/size distribution and free protein concentration measurements. However, the "sandwich" columns packed with a layer of CaptoCore 700 demonstrated a better plasma protein binding capacity but lower recovery rate of vesicles, while "sandwich" columns packed with a layer of CaptoCore 700 showed a higher EV recovery rate, but a little lower plasma protein binding capacity. The EV isolates prepared using the developed enrichment techniques were evaluated by means of transmission electron microscopy (TEM), nano-flow cytometry, proteomics profiling using nanoscale liquid chromatography coupled to tandem mass spectrometry, and glycomic profiling of released N-glycans using capillary electrophoresis coupled to tandem mass spectrometry. The microscopy- and molecular profiling-based techniques confirmed that the developed techniques could effectively enrich EVs from complex physiological fluids at high purity.

<p dir="ltr">Extracellular vesicles (EVs) are membrane-enclosed nanoparticles secreted by all cell types. They carry a variety of bioactive molecules, including lipids, proteins, and nucleic acids, which can act as signaling messengers to other cells. The two main types of EVs under investigation are microvesicles and exosomes. Microvesicles are formed by outward budding of the plasma membrane, while exosomes originate from inward budding within multivesicular bodies and are released upon fusion with the plasma membrane. EVs are found in various biological fluids such as blood, saliva, and urine, as well as in cell culture media. Their roles in intercellular communication have made them a focus of research in fundamental biology, biomarker discovery, and therapeutic development, including engineering EVs for clinical applications.</p><p dir="ltr">Studying EVs often requires their isolation, which presents several challenges. The physical and molecular similarities between EV subtypes make them difficult to separate, and common isolation protocols often co-isolate contaminants such as protein aggregates and lipoproteins. In Study I, we compared five fundamentally different EV isolation techniques, each based on distinct EV properties such as density, size, affinity, and hydrophobicity. These methods were applied to two biofluids, cell culture supernatant and plasma, at varying volumes to assess whether the optimal isolation method depends on the biofluid type. Plasma, for instance, contains high levels of lipoproteins that overlap with EVs in size and density, complicating isolation. EV isolates were characterized using transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), Bioanalyzer, flow cytometry, and proteomic profiling. All methods successfully enriched for EVs, but the composition and quality of the isolates varied depending on the biofluid, sample volume, and isolation technique. This study emphasized the importance of method selection, as each technique isolates different EV subpopulations with different contaminants, which can affect study outcomes and comparability. It also highlighted that some components initially considered as contaminants may be biologically relevant.</p><p dir="ltr">In Studies II and III, we explored the therapeutic potential of dendritic cell (DC)- derived EVs, particularly their use in cancer immunotherapy. Using EVs loaded with the model antigen ovalbumin (OVA), we investigated strategies to enhance their immunogenicity. In Study II, we focused on the immune checkpoint molecule PD-L1, which was present on our EVs. PD-L1 present on Tumor-derived EVs is known to suppress T cell activation, but its role on therapeutic DC-EVs was unclear. We generated PD-L1-deficient (PD-L1-/-) EVs and compared their immunogenicity to wild-type EVs in multiple mouse tumor models. While most models showed a trend toward stronger immune activation with PD-L1-/- EVs, a significant improvement was observed only in a prophylactic tumor model. These findings suggest that removing PD-L1 may enhance the efficacy of EV-based immunotherapies.</p><p dir="ltr">In Study III, we tested whether targeting EVs to B cells could further improve their immunogenicity. A soluble fusion protein (C1C2-D123) was developed to bind phosphatidylserine on EVs and CD21 on B cells. This protein successfully directed EVs to B cells in vitro and in vivo without altering their biodistribution. In an in vivo immunization model, EVs decorated with the fusion protein induced a higher frequency of antigen-specific CD8+ T cells compared to non-targeted EVs, supporting B cell targeting as a strategy to enhance immune responses.</p><p dir="ltr">In conclusion, this thesis highlights the importance of EV isolation strategy and cargo engineering in optimizing EV-based therapies. It demonstrates that both the method of isolation and the molecular composition of EVs significantly influence their biological and therapeutic potential.</p><h3>List of scientific papers</h3><p dir="ltr">I. Rosanne E. Veerman, <b>Loes Teeuwen</b>, Paulo Czarnewski, Gözde Güclüler Akpinar, AnnSofi Sandberg, Xiaofang Cao, Maria Pernemalm, Lukas M. Orre, Susanne Gabrielsson, Maria Eldh. Molecular evaluation of five different isolation methods for extracellular vesicles reveals different clinical applicability and subcellular origin. J Extracellular Vesicles. Volume 10, Epub e12128, (2021). <a href="https://doi.org/10.1002/jev2.12128" rel="noreferrer" target="_blank">https://doi.org/10.1002/jev2.12128</a></p><p dir="ltr">II. <b>Teeuwen*, L.</b>, Steiner*, L., Reinhardt, C., Offens, A., Kuipers, J.E., Martínez- Martínez, D., Mazouin, J., Chambers, B.J., Güçluler Akpinar, G., Gabrielsson, S. Removal of PD-L1 on extracellular vesicles for cancer vaccination modulates anti-tumor responses in a murine immunotherapy model. [Manuscript]</p><p dir="ltr">III. Annemarijn Offens, <b>Loes Teeuwen</b>, Gozde Gucluler Akpinar, Loïc Steiner, Sander Kooijmans, Doste Mamand, Hannah Weissinger, Alexander Kall, Maria Eldh, Oscar P.B. Wiklander, Samir El-Andaloussi, Mikael C.I. Karlsson, Pieter Vader, Susanne Gabrielsson. A fusion protein that targets antigen-loaded extracellular vesicles to B cells enhances antigen-specific T cell expansion. J Controlled Release, Volume 382, 113665 (2025). <a href="https://doi.org/10.1016/j.jconrel.2025.113665" rel="noreferrer" target="_blank">https://doi.org/10.1016/j.jconrel.2025.113665</a></p><p dir="ltr">* Shared first authorship.</p>

<p dir="ltr">Extracellular vesicles (EVs) are membrane-enclosed nanoparticles secreted by all cell types. They carry a variety of bioactive molecules, including lipids, proteins, and nucleic acids, which can act as signaling messengers to other cells. The two main types of EVs under investigation are microvesicles and exosomes. Microvesicles are formed by outward budding of the plasma membrane, while exosomes originate from inward budding within multivesicular bodies and are released upon fusion with the plasma membrane. EVs are found in various biological fluids such as blood, saliva, and urine, as well as in cell culture media. Their roles in intercellular communication have made them a focus of research in fundamental biology, biomarker discovery, and therapeutic development, including engineering EVs for clinical applications.</p><p dir="ltr">Studying EVs often requires their isolation, which presents several challenges. The physical and molecular similarities between EV subtypes make them difficult to separate, and common isolation protocols often co-isolate contaminants such as protein aggregates and lipoproteins. In Study I, we compared five fundamentally different EV isolation techniques, each based on distinct EV properties such as density, size, affinity, and hydrophobicity. These methods were applied to two biofluids, cell culture supernatant and plasma, at varying volumes to assess whether the optimal isolation method depends on the biofluid type. Plasma, for instance, contains high levels of lipoproteins that overlap with EVs in size and density, complicating isolation. EV isolates were characterized using transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), Bioanalyzer, flow cytometry, and proteomic profiling. All methods successfully enriched for EVs, but the composition and quality of the isolates varied depending on the biofluid, sample volume, and isolation technique. This study emphasized the importance of method selection, as each technique isolates different EV subpopulations with different contaminants, which can affect study outcomes and comparability. It also highlighted that some components initially considered as contaminants may be biologically relevant.</p><p dir="ltr">In Studies II and III, we explored the therapeutic potential of dendritic cell (DC)- derived EVs, particularly their use in cancer immunotherapy. Using EVs loaded with the model antigen ovalbumin (OVA), we investigated strategies to enhance their immunogenicity. In Study II, we focused on the immune checkpoint molecule PD-L1, which was present on our EVs. PD-L1 present on Tumor-derived EVs is known to suppress T cell activation, but its role on therapeutic DC-EVs was unclear. We generated PD-L1-deficient (PD-L1-/-) EVs and compared their immunogenicity to wild-type EVs in multiple mouse tumor models. While most models showed a trend toward stronger immune activation with PD-L1-/- EVs, a significant improvement was observed only in a prophylactic tumor model. These findings suggest that removing PD-L1 may enhance the efficacy of EV-based immunotherapies.</p><p dir="ltr">In Study III, we tested whether targeting EVs to B cells could further improve their immunogenicity. A soluble fusion protein (C1C2-D123) was developed to bind phosphatidylserine on EVs and CD21 on B cells. This protein successfully directed EVs to B cells in vitro and in vivo without altering their biodistribution. In an in vivo immunization model, EVs decorated with the fusion protein induced a higher frequency of antigen-specific CD8+ T cells compared to non-targeted EVs, supporting B cell targeting as a strategy to enhance immune responses.</p><p dir="ltr">In conclusion, this thesis highlights the importance of EV isolation strategy and cargo engineering in optimizing EV-based therapies. It demonstrates that both the method of isolation and the molecular composition of EVs significantly influence their biological and therapeutic potential.</p><h3>List of scientific papers</h3><p dir="ltr">I. Rosanne E. Veerman, <b>Loes Teeuwen</b>, Paulo Czarnewski, Gözde Güclüler Akpinar, AnnSofi Sandberg, Xiaofang Cao, Maria Pernemalm, Lukas M. Orre, Susanne Gabrielsson, Maria Eldh. Molecular evaluation of five different isolation methods for extracellular vesicles reveals different clinical applicability and subcellular origin. J Extracellular Vesicles. Volume 10, Epub e12128, (2021). <a href="https://doi.org/10.1002/jev2.12128" rel="noreferrer" target="_blank">https://doi.org/10.1002/jev2.12128</a></p><p dir="ltr">II. <b>Teeuwen*, L.</b>, Steiner*, L., Reinhardt, C., Offens, A., Kuipers, J.E., Martínez- Martínez, D., Mazouin, J., Chambers, B.J., Güçluler Akpinar, G., Gabrielsson, S. Removal of PD-L1 on extracellular vesicles for cancer vaccination modulates anti-tumor responses in a murine immunotherapy model. [Manuscript]</p><p dir="ltr">III. Annemarijn Offens, <b>Loes Teeuwen</b>, Gozde Gucluler Akpinar, Loïc Steiner, Sander Kooijmans, Doste Mamand, Hannah Weissinger, Alexander Kall, Maria Eldh, Oscar P.B. Wiklander, Samir El-Andaloussi, Mikael C.I. Karlsson, Pieter Vader, Susanne Gabrielsson. A fusion protein that targets antigen-loaded extracellular vesicles to B cells enhances antigen-specific T cell expansion. J Controlled Release, Volume 382, 113665 (2025). <a href="https://doi.org/10.1016/j.jconrel.2025.113665" rel="noreferrer" target="_blank">https://doi.org/10.1016/j.jconrel.2025.113665</a></p><p dir="ltr">* Shared first authorship.</p>

We recently demonstrated that intra-pancreatic (iPan) delivery of conditioned media (CM) generated by human bone marrow-derived multipotent stromal cells (hBM-MSC) can act as a biotherapeutic to reduce hyperglycemia in STZ-treated mice by stimulating endogenous islet regeneration. We have established analogous stromal cell lines derived from human islet cultures that grow adherent to plastic and possess multipotent mesodermal differentiation in vitro. Furthermore, CM generated by these human pancreas-derived MSC (hPanc-MSC) contained proangiogenic stimuli within extracellular vesicles (EVs) that accelerated blood vessel regeneration after injection into ischemic hindlimbs. Herein, we investigated whether iPan-injection of CM generated by hPanc-MSC could stimulate islet regeneration in STZ-treated mice. We hypothesized that islet regenerative stimuli would be harboured within EVs secreted by hPanc-MSC. iPan-injection of unfractioned CM (6µg) generated by hBM-MSC and hPanc-MSC equally reduced hyperglycemia in STZ-treated mice, compared to mice injected with media control (*p<0.05). To determine whether islet regenerative stimuli was harboured within EVs, ultrafiltration was used to separate CM into EV enriched (EV+) or depleted (EV-) subfractions. Enrichment of EVs was validated by nanoscale flow cytometry and atomic force microscopy, and the EV+ and EV- hPANC-MSC subfractions were compared for protein content by label-free mass spectrometry. Expression of typical EV-associated proteins (CD9, CD81, CD63) were exclusive to the EV+ fraction and putative islet regenerative factors were detected in both the EV- and EV+ subfractions C. Mice iPan-injected with either EV+ or EV- CM sub-fractions showed significantly reduced hyperglycemia compared to unconditioned media controls (*p<0.05). These initial studies provide a foundation to further explore the use the hPanc-MSC secretome or related small-molecule pharmaceuticals to enhance islet-regeneration in situ. Disclosure T. Cooper: None. J. Ma: None. G.I. Bell: None. G. Lajoie: None. D.A. Hess: None.

Simple SummaryProstate cancer is the second most commonly diagnosed cancer and the fifth leading cause of cancer-related death in men. It is a generally slow-growing cancer that—when detected in its early stages—has high chances of successful treatment. Just like all cells, cancerously degenerated cells release extracellular vesicles (EVs) to communicate with other cells. The aim of our research was to specifically isolate prostate cancer-derived EVs from urine, characterize the EV surface markers of a prostate cancer cohort, and assess the potential value of the prostate-specific membrane antigen (PSMA) as a biomarker for liquid biopsy in early cancer diagnostics. Our findings demonstrate that the automated isolation of EVs allows for an overall improvement of the precision in sample purification in comparison to manual isolation, thus optimizing the further characterization of EV surface markers as well as evaluating their use in clinical application.All cells release extracellular vesicles (EVs) to communicate with adjacent and distant cells. Consequently, circulating EVs are found in all bodily fluids, providing information applicable for liquid biopsy in early cancer diagnosis. Studies observed an overexpression of the membrane-bound prostate-specific membrane antigen (PSMA) on prostate cancer cells. To investigate whether EVs derived from communicating prostate cells allow for reliable conclusions on prostate cancer development, we isolated PSMA-positive, as well as CD9-positive, EVs from cell-free urine with the use of magnetic beads. These populations of EVs were subsequently compared to CD9-positive EVs isolated from female urine in Western blotting, indicating the successful isolation of prostate-derived and ubiquitous EVs, respectively. Furthermore, we developed a device with an adapted protocol that enables an automated immunomagnetic enrichment of EVs of large sample volumes (up to 10 mL), while simultaneously reducing the overall bead loss and hands-on time. With an in-house spotted antibody microarray, we characterized PSMA as well as other EV surface markers of a prostate cohort of 44 urine samples in a more simplified way. In conclusion, the automated and specific enrichment of EVs from urine has a high potential for future diagnostic applications.

An optimized method for enrichment of whole brain-derived extracellular vesicles reveals insight into neurodegenerative processes in a mouse model of Alzheimer’s disease

EVs can be categorized into three principal subtypes based on their size and biological origins: exosomes (30-150 nm), microvesicles (100-1000 nm), and apoptotic bodies (1000-5000 nm). Notably, in the context of cancer, tumor-derived EVs (TDEVs) are prolifically released into bodily fluids, exerting multifaceted roles in tumorigenesis. TDEVs transport tumor-promoting signaling cues, leading to the reprogramming of the tumor microenvironment (TME). These cues stimulate immunosuppression, establish metastatic niches, enhance resistance to antitumor therapies, induce metabolic alterations in other tumor cells, reprogram endothelial cells, and promote angiogenesis. Furthermore, EVs have emerged as a promising next-generation platform for antitumor therapeutics, offering several advantages over conventional delivery systems. Leveraging the principles of existing knowledge from artificial lipid-based delivery systems like liposomes, EV-based research can capitalize on their natural nanoscopic lipid-based nanocarrier properties. Moreover, endogenously derived EVs possess unique advantages in overcoming therapeutic delivery-related challenges for precise tumor targeting, including low immunogenicity, the ability to traverse the blood-brain barrier, high target specificity through surface receptorligand interactions, and biocompatibility. However, numerous challenges persist in scaling up EV production, developing standardized characterization protocols, minimizing batch-to-batch variations, extending shelf life, implementing novel EV bioengineering techniques for target specificity, formulating regulatory guidelines, and achieving efficient clinical translation.This special issue collection serves as a comprehensive resource, offering recent insights into the roles of EVs in tumorigenesis, advancements in EV isolation techniques, applications of EVs in therapeutic delivery and proteomics, and discussions on potential sources of therapeutic EVs.They employed agarose beads with cation chelates to precisely bind to the 6His-tagged membrane-sensing peptide, standardizing the isolation protocol for versatile EV applications.Hadizadeh et al. conducted a comprehensive exploration of exosome biogenesis and its critical role as a biomarker in metabolic disorders. They also delved into cutting-edge technologies for exosome detection and isolation, highlighting the growing recognition of small extracellular vesicles (EVs) as versatile entities that can be harnessed for precise targeting of cellular signaling pathways, thereby offering therapeutic potential for mitigating pathological conditions. Additionally, the inherent vehicle-like properties of exosomes position them as advantageous platforms for drug and gene delivery due to their low immunogenicity.In their study, Hou et al. shed light on the pivotal role of exosomes in shaping the physiological processes and microenvironment of melanoma through intricate cell-to-cell communication networks. These small vesicles hold immense promise as innovative vehicles for drug delivery in melanoma treatment, especially when coupled with advanced bioengineering strategies such as surface modification and diverse loading approaches. Their review systematically categorizes advancements in exosome-based therapies for melanoma, spanning a spectrum of drug categories including chemotherapy, immunotherapy, photothermal therapy, and radiotherapy. However, they also address the substantial challenges in translating exosome-based therapies into clinical practice, including the absence of standardized isolation methods, storage protocols, and safety concerns associated with exosome-based nanomedicines. Overcoming these hurdles promises to unlock the full therapeutic potential of exosome-based strategies for melanoma in the future.derived from pleural effusion (PE) in lung cancer patients. They evaluated three techniques: ultracentrifugation (UC), a combination of UC and size exclusion chromatography (UC-SEC), and a combination of UC and density gradient ultracentrifugation (UC-DGU). Their results demonstrated that the UC-SEC method exhibited the highest purity when isolating pEVs. Moreover, proteomic analysis revealed that UC-SEC isolated a greater number of proteins from pEVs compared to UC and UC-DGU. Notably, they identified novel protein markers (CD11C, HLA DPA1, and HLA DRB1) enriched in pEVs, offering valuable insights for studying diseases associated with pleural effusion.In their review, Zhu et al. delve into the evolving role of Plant-Derived Extracellular Vesicles (PDEVs) as a highly promising nano-delivery system in the context of tumor treatment. They highlight the substantial potential of PDEVs for transporting a diverse range of cargoes, including nucleic acids, proteins, and chemotherapeutic agents, both in laboratory settings and clinical cancer treatment scenarios. However, the progress of PDEVs as a drug delivery platform faces formidable challenges, including the lack of standardized isolation and purification methods and the time-consuming nature of current techniques, which yield limited quantities. Moreover, a deeper understanding of PDEVs' biological properties and transport mechanisms is imperative, including the identification of characteristic markers and surface proteins. They conclude that robust pre-clinical and large-scale clinical studies are essential to ensure the safety and efficacy of PDEVs in large-scale production. Efforts to enhance drug loading efficiency, establish optimal storage conditions, and extend in vivo circulation time also warrant attention.We firmly believe that the articles featured in this research topic make significant contributions to our understanding of the diagnostic and therapeutic potential of EVs. These contributions equip our readers with invaluable insights, inspire innovative ideas, and foster a resolute determination to advance in this direction. Further investigations remain imperative to refine EV-based protocols, ensuring their optimal efficiency and efficacy in therapeutic delivery.

Assessing the role of extracellular vesicles in renin-angiotensin system signalling in cardiomyocyte hypertrophy

Cholesterol affinity recognition for extracellular vesicle isolation and metabolomics analysis in liver disease.

Extracellular vesicles (EVs), crucial in facilitating the transport of diverse molecular cargoes for intercellular communication, have shown great potential in diagnostics, therapeutics, and drug delivery. The challenge of developing effective preparation methods for EVs is heightened by their intrinsic heterogeneity and complexity. Here, a novel strategy for high EV enrichment is developed by utilizing EV-affinitive-modified cellulose nanofibrils. Specifically, modified cellulose with rich carboxyl groups has outstanding dispersing properties, able to be dispersed into cellulose nanofibrils in solution. These cellulose nanofibrils are utilized as scaffolds for the immobilization of EV-affinitive antibody of CD63 by chemical conjugation. The CD63-modified nanofibrils demonstrate a superior EV capture efficiency of 86.4% compared with other reported methods. The high performance of this system is further validated by the efficient capture of EVs from biological blood plasma, allowing the detection of bioactive markers from EV-derived miRNAs and proteins. The authors envision that these modified cellulose nanofibrils of enhanced capability on EV enrichment will open new avenues in various biomedical applications.