Isolation of small extracellular vesicles from biofluids using immunoaffinity nanodiamonds

Isolation of small extracellular vesicles from biofluids using immunoaffinity nanodiamonds | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Short Report Isolation of small extracellular vesicles from biofluids using immunoaffinity nanodiamonds Su-Mei Lin, Long-Jyun Su, Liang-Yu Chen, Zhi-Xuan Zhang, Che-Yao Chang, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6654502/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Small extracellular vesicles (sEVs) are nanoscale vesicles derived from cells, known for their ability to transport a diverse array of biomolecules from their parent cells. These vesicles play a pivotal role in intercellular communication and are involved in various physiological and pathological processes. sEVs offer several advantages, including excellent biocompatibility, low immunogenicity, and widespread biodistribution. With growing insights into their biological functions, numerous strategies have emerged for leveraging sEVs in disease diagnosis and therapy. Despite this potential, standard methods for isolating sEVs from biofluids remain incompatible with routine research and clinical workflows, limiting their broader biomedical application. In this study, we present a simple and efficient immunoaffinity-based platform using nanodiamonds to isolate sEVs from a range of biological fluids. The separation efficiency was assessed using fluorescently labeled sEVs, achieving recovery rates greater than 90%. Our nanodiamond-based approach, named ExoFortius, provides a scalable, accessible, and cost-effective solution for high-yield sEV isolation. This method enables rapid, consistent, and high-purity enrichment of sEVs directly from complex biofluids. By introducing ExoFortius, this study offers a novel technological advancement aimed at accelerating sEV-related research and expanding their utility in translational medicine for both diagnostic and therapeutic applications. Biomaterials Biomedical applications Disease diagnosis Nanodiamonds Immunoaffinity Small extracellular vesicles Figures Figure 1 Figure 2 Figure 3 1 Introduction Small extracellular vesicles (sEVs), ranging from 30–150 nm in diameter, are generated via the endosomal pathway and secreted upon fusion of multivesicular bodies with the plasma membrane [ 1 ]. Morphologically cup-shaped, sEVs mediate intercellular communication by transferring bioactive cargo, including lipids, proteins, and nucleic acids [ 2 ]. They function as critical vectors for intercellular, inter-tissue, and interorgan signaling, influencing a broad spectrum of physiological and pathological processes [ 3 – 5 ]. sEVs are secreted by a wide array of cell types [ 6 – 10 ], and are selectively enriched with functional molecules reflective of their cell of origin [ 11 – 13 ]. These vesicles are abundant in body fluids, including blood, saliva, breast milk, and urine, facilitating systemic dissemination [ 14 ]. sEVs can engage target cells through ligand-receptor interactions [ 15 ], membrane fusion, or uptake via endocytosis/ phagocytosis, thereby delivering their molecular contents and modulating recipient cell signaling networks [ 16 – 18 ]. Through these mechanisms, sEVs actively participate in immunomodulation, antigen presentation, and tumorigenesis [ 2 ]. sEV biogenesis and cargo loading are tightly regulated by their parental cells to facilitate the targeted molecular information transfer, enabling specific functional outcomes in recipient cells [ 19 ]. Consequently, the molecular composition of sEVs reflects the physiological or pathological status of their cells of origin, providing a basis for their use in diagnostics, including cardiovascular diseases [ 20 , 21 ], central nervous system diseases [ 22 , 23 ], hepatic conditions [ 24 , 25 ], infectious diseases [ 26 – 28 ], and autoimmune disorders [ 29 – 31 ]. They also play a key role in non-invasive diagnostic approaches such as liquid biopsy [ 32 , 33 ]. In oncology, sEVs have garnered significant attention as tumor-specific biomarkers, offering utility in cancer detection, therapeutic monitoring, and prognosis assessment [ 34 – 37 ]. They are now considered among the most promising biomarkers for liquid biopsy applications [ 38 – 40 ]. Beyond diagnosis, sEVs are being explored across multiple biomedical domains, including targeted drug delivery [ 41 ], cell-free vaccine platforms [ 42 ], and regenerative medicine [ 43 ]. Despite substantial progress in modern medicine, significant challenges persist in the diagnosis and treatment of numerous complex diseases. Early-stage detection, before the onset of overt clinical symptoms, is widely recognized as critical for effective disease prevention, timely intervention, and improved prognostic outcomes [ 44 ]. Nonetheless, conventional early diagnostic methods face considerable limitations, including invasiveness, suboptimal sensitivity, high cost, and limited patient compliance [ 33 , 45 , 46 ]. In the therapeutic context, the management of refractory diseases, such as malignancies, neuropsychiatric conditions, autoimmune disorders, and infectious diseases, remains constrained. Despite the emergence of novel pharmacological agents, therapeutic efficacy is frequently compromised by factors such as the impermeability of the blood-brain barrier (BBB), the development of drug resistance, and inadequate target specificity [ 47 ]. The molecular content carried by sEVs offers a rich source of biomarkers that can aid in detecting the presence of disease and in identifying disease subtypes and their unique molecular profiles [ 48 ]. This capability supports the development of personalized treatment strategies, ultimately enhancing patient outcomes and minimizing unnecessary therapies [ 49 – 51 ]. sEVs have emerged as promising vectors for drug delivery, targeted therapy, and immunomodulation [ 52 ], owing to their intrinsic biocompatibility, low immunogenicity, and the structural stability provided by their lipid bilayer, which safeguards encapsulated genetic material from enzymatic degradation. Their nanoscale dimensions and membrane characteristics further enable efficient translocation across the BBB [ 53 ], positioning them as innovative tools for the diagnosis and treatment of neurological and psychiatric disorders [ 54 , 55 ]. Therefore, the specific and efficient isolation of sEVs from a mixture of cellular debris and interfering substances is essential. Traditional isolation methods, primarily based on differential centrifugation, can achieve reasonable purity, but they face limitations such as long processing times, costly instrumentation, and poor batch-to-batch reproducibility, which restrict their broader application in both research and clinical settings [ 56 ]. While rapid and precise isolation of high-purity sEVs is preferred, it remains a technical challenge. Additionally, although several innovative sEV-based detection techniques have emerged in recent years, substantial work is still required to effectively translate these findings into clinical practice. To advance translational research and innovation in the sEV field, nanoparticles have been employed as cutting-edge technologies, evolving into highly effective tools. Nanodiamond (ND) particles, known for their chemical inertness, outstanding biocompatibility, and minimal cytotoxicity [ 57 ], have been widely utilized in biological and nanomedical applications. This is largely due to their high surface-area-to-volume ratio, adaptable surface chemistry, and capacity to emit near-infrared fluorescence from color centers [ 58 ]. A recent study by Lin et al. demonstrated that the excellent biocompatibility and multifunctional properties of NDs and fluorescent NDs (FNDs) make them promising candidates as both active and traceable vaccine adjuvants and antitumor agents. As such, NDs/FNDs offer valuable potential to the sEV field by serving as versatile tools for research and bioengineering. In clinical translational research, there remains a critical demand for sEV isolation platforms that are cost-effective to fabricate, operationally straightforward, require no specialized technical expertise, entail minimal pre-analytical processing, and provide robust reproducibility and high-resolution separation performance in terms of both purity and recovery yield. To address this unmet need, we have developed ExoFortius—a rapid, high-precision, immunoaffinity-based ND platform engineered for the selective isolation of sEVs directly from a variety of body fluids, including blood, urine, saliva, breast milk, semen, amniotic fluid, cerebrospinal fluid, bronchoalveolar fluid, as well as fluid from diseased lesions including ascites and pleural fluid, and can be subjected to multicomponent analysis. This system leverages antibodies directed against the tetraspanins CD9, CD63, and CD81, which are consistently enriched in sEV membranes [ 59 , 60 ], and are widely accepted as canonical sEV surface markers due to their broad expression across diverse cell types. ExoFortius exhibits superior recovery efficiency and particle purity, particularly under conditions of limited input biofluid volumes, thereby enabling its application in low-yield clinical specimens. Given these attributes, ExoFortius establishes a robust methodological framework for sEV-based transcriptomic profiling in a variety of body fluids and facilitates mechanistic investigations into sEV-mediated intercellular communication and disease pathophysiology. Collectively, our findings position ExoFortius as a scalable, technically accessible, and economically viable immunoaffinity-based isolation platform capable of delivering rapid, reproducible, and high-fidelity sEV enrichment directly from a variety of body fluids. 2 Materials and Methods 2.1 Chemical and materials Anti-CD9, anti-CD63, and anti-CD81 antibodies were purchased from IReal Biotechnology (Hsinchu City, Taiwan). Monocrystalline synthetic diamond powders with a nominal size of 100 nm were obtained from Element Six (Didcot, UK). 2.2 NDs and antibody-NDs Monocrystalline diamond powders were initially oxidized in air at 490°C for 2 hours to eliminate surface graphitic carbon. This was followed by microwave-assisted cleaning in a concentrated H₂SO₄–HNO₃ (3:1 v/v) solution at 100°C for 3 hours to remove metallic impurities and concurrently introduce –COOH functional groups on the surface [ 61 ]. The resulting surface-oxidized and carboxylated nanodiamonds (NDs) were then noncovalently conjugated with the antibody by simply mixing the antibody solution (5 µl, at concentrations of 1 or 5 mg/ml) with ND suspension (30 µl, at concentrations of 2 or 10 mg/ml) for 1 hour at room temperature. 2.3 FNDs and antibody-FNDs Fluorescent nanodiamonds (FNDs) were produced by subjecting monocrystalline diamond powders to 10-MeV electron irradiation to induce radiation damage, followed by vacuum annealing at 800°C to form nitrogen-vacancy centers within the diamond lattice, as previously reported [ 62 ]. Subsequently, the FNDs underwent surface oxidation, carboxylation, and noncovalent conjugation with the antibody using the same procedures described earlier for NDs. 2.4 Particle characterization The hydrodynamic sizes and ζ-potentials of both bare and antibody-conjugated NDs were determined using a particle size and ζ-potential analyzer (DelsaNano C; Beckman Coulter, CA, USA). The morphology of the NDs was examined by transmission electron microscopy (H-7650, Hitachi; Tokyo, Japan) at an accelerating voltage of 75 kV, with samples prepared on copper grids. 2.5 Protein adsorption measurement The absorption spectrum of an antibody solution (800 µg/ml) in the 200–400 nm range was recorded using a UV-Vis spectrophotometer (U-3310, Hitachi; Tokyo, Japan). The protein solution was then combined with an ND suspension (1 mg/ml) at a 1:1 volume ratio and incubated at room temperature for 30 minutes with gentle shaking. Following incubation, the antibody-ND mixture was centrifuged at 20,000 × g for 15 minutes to pellet the NDs. The concentration of unbound antibody in the supernatant was determined by measuring its absorbance at 280 nm. 2.6 Generation and characterization of human placenta pcMSCs Human placentas used in this study were collected from women undergoing cesarean sections at Taipei Medical University Hospital. The collection and use of placental tissues followed protocols approved by the Institutional Review Board of Taipei Medical University. All donors provided written informed consent, and all procedures adhered to relevant ethical guidelines and regulations. Placenta-derived mesenchymal stem cells (pcMSCs) were isolated from the choriodecidual membrane using a serum-free culture method, as previously described [ 63 ]. Briefly, choriodecidual tissues were digested overnight at 4°C in SMEM containing 1 mg/mL DNase I, 0.5 mg/mL collagenase B, and 0.5 mg/mL protease. The digested mixture was passed through a 100-µm nylon mesh to remove debris. After centrifugation, the cell pellet was resuspended in MCDB201 medium supplemented with 1% penicillin/streptomycin, 10 ng/mL epidermal growth factor, and 1% insulin-transferrin-selenium. Cells were then seeded onto culture dishes coated with human collagen type IV. Adherent cells were cultured with medium changes every 3 to 4 days to eliminate nonadherent cells. The resulting cells, identified as pcMSCs, were maintained in serum-free medium and exhibited fibroblast-like morphology upon attachment. These cells expressed surface markers CD44, CD73, CD90, and CD29, while lacking expression of CD45, CD34, CD14, and HLA-DR [ 64 ]. 2.7 Conditioned medium collection, sEV isolation, and characterization Conditioned medium was harvested from pcMSCs cultured in serum-free medium, and sEVs were isolated and purified using ultracentrifugation. Specifically, conditioned medium from pcMSCs cultured for 4 days was collected and first centrifuged at 300 × g for 10 minutes to remove dead cells. The supernatant was then sequentially centrifuged at 2,000 × g for 10 minutes to eliminate apoptotic bodies, and at 10,000 × g for 30 minutes to remove microvesicles. sEVs were subsequently isolated by ultracentrifugation at 100,000 × g for 90 minutes. To reduce contamination from soluble proteins, the sEV pellet was washed once with PBS and then resuspended in PBS after the supernatant was removed. Nanoparticle tracking analysis (NTA, NanoSight NS300) and a Litesizer DLS 500 (Anton Paar, Austria) were used to assess particle size, zeta potential, and concentration. To characterize surface markers associated with sEVs, including CD9, CD63, and CD81, NanoFCM (NanoFCM, China), a specialized flow cytometer for nanoscale particles, was employed. 2.8 Confocal fluorescence microscopy sEVs were fluorescently labeled using Alexa Fluor 488-conjugated Wheat Germ Agglutinin (WGA) (Cat. No W11261, Thermo Fisher Scientific, Rockford, IL, USA). Following incubation with ExoFortius, the antibody-conjugated FNDs, optical imaging of the sEVs was performed using a laser-scanning confocal fluorescence microscope (SP-8, Leica Microsystems; Wetzlar, Germany). The system was equipped with a supercontinuum white-light laser, providing excitation at 488 nm for the Alexa Fluor 488 dye and at 561 nm for the nitrogen-vacancy centers. Fluorescence emission was collected through a 63× oil-immersion objective (numerical aperture = 1.4) and detected in the 650–800 nm wavelength range. 3 Results and discussion 3.1 Material characterization The sizes and ζ-potentials of nanodiamonds (NDs) were analyzed before and after their conjugation with rabbit IgG. Transmission electron microscopy (TEM) images of acid-washed, bare NDs revealed particles with irregular shapes and a wide size distribution (Fig. 1 B). Dynamic light scattering (DLS) measurements in distilled deionized water (DDW) showed that these NDs had an average hydrodynamic diameter of approximately 100 nm and a polydispersity index of 0.24. Upon mixing with rabbit monoclonal antibodies in DDW, the mean particle size increased by about 20 nm (Fig. 1 A), indicating successful protein adsorption. The ζ-potential of the bare ND was measured at -45 mV, which increased to -23 mV following rabbit IgG conjugation. Rabbit monoclonal antibodies have become valuable tools in biomedical research and are increasingly adopted for diagnostic and therapeutic purposes, benefiting from the strengths of both rabbit polyclonal and mouse monoclonal antibodies [ 65 ]. Rabbit IgG has a molecular weight of approximately 150 kDa and consists of two heavy chains (~ 50 kDa each) and two light chains (~ 25 kDa each), forming a tetrameric structure. To estimate the number of rabbit IgG molecules noncovalently bound to acid-washed NDs, changes in absorbance at 280 nm were measured before and after mixing the antibody with the nanoparticles. Based on these measurements, the maximum loading capacity was estimated at 0.1 g of protein per gram of NDs. Assuming the NDs are spherical and approximately 100 nm in diameter (with an estimated mass of ~ 1.8 fg per particle), each ND can accommodate over 700 rabbit IgG molecules on its surface. 3.2 Characterization of sEVs sEVs were isolated from human placenta choriodecidual membrane-derived mesenchymal stromal cells (pcMSC) culture supernatants and subsequently characterized. Nanoparticle tracking analysis (NTA) was employed to assess the size distribution and concentration of the sEVs, revealing an average size range of 70 to 150 nm (Fig. 2 A), consistent with previous findings [ 66 ]. Flow cytometry was used to analyze surface CD markers, showing notable expression of the tetraspanins CD9 (32.6%), CD63 (10.4%), and CD81 (23.7%) (Fig. 2 B). These findings support the suitability of the isolated sEVs for evaluating the labeling efficiency of anti-sEV antibody-conjugated NDs. 3.3 Functionalization of anti-sEV antibody-conjugated nanodiamonds sEVs were employed as a model system to evaluate the capture and precipitation efficiency of NDs conjugated with anti-sEV antibodies. To facilitate visualization of the colocalization between sEVs and antibody-functionalized NDs, we substituted NDs with FNDs, leveraging their near-infrared fluorescence properties [ 58 ]. Given that sEVs express tetraspanin family proteins such as CD9, CD63, and CD81, we conjugated rabbit monoclonal antibodies against these markers to NDs to enable high-yield, high-throughput sEV isolation. Following quantification of the total sEV population, anti-sEV antibody-conjugated NDs were incubated with the sEV solution. Confocal microscopy of Alexa Fluor 488-labeled sEVs demonstrated efficient colocalization with NDs conjugated to anti-CD9, anti-CD63, and anti-CD81 antibodies, respectively (Fig. 2 C). To assess labeling efficiency, we combined all three types of antibody-conjugated NDs and incubated them with sEVs. Imaging confirmed nearly complete colocalization between sEVs and the mixed antibody-ND complexes (Fig. 2 D), indicating highly efficient capture. To further validate the isolation efficiency, equal volumes of sEVs and antibody-conjugated NDs were mixed and subjected to low-speed centrifugation (20,000 × g, 15 minutes). The supernatant retained less than 7% of the original sEV population, yielding a recovery rate exceeding 90%. These antibody-functionalized NDs, termed ExoFortius, are capable of selectively capturing and purifying specific sEV subpopulations based on their surface markers. The ExoFortius platform is modular, allowing for the attachment of pre-validated capture antibodies targeting sEV markers of interest. 4 Conclusion ExoFortius kits are compatible with a wide range of biological fluids—including serum, plasma, tumor ascites, and cell culture media—even at low concentrations and large volumes. The system offers multiple advantages: it enables rapid isolation (within 30 minutes) via a simple low-speed spin protocol, supports universal application across diverse biofluids, and provides isolated sEVs that are suitable for a range of downstream applications, including miRNA profiling, next-generation sequencing, and in vivo studies of pharmacokinetics (PK), pharmacodynamics (PD), and biodistribution (Bio-D). Importantly, the captured sEVs remain intact and bioactive, making them suitable for functional analyses and bioengineering applications. Declarations Acknowledgments Funding : This work was supported by grant 2023SKHAND011 from Shin Kong Wu Ho-Su Memorial Hospital to S.M. Lin. We would like to express our sincere gratitude to Dr. Thai-Yen Ling for his support with pcMSCs. Author contributions : M.S. Lin: Conceptualization, data curation, validation, investigation. L.J. Su: Conceptualization, data curation, validation, and investigation. L.Y. Chen: data curation, validation, and investigation. Z.X. Zhang: data curation, validation, and investigation. C.Y. Chang: Methodology, validation, and investigation. H.H. Lin: Formal analysis, validation, investigation, writing, and editing. Data availability : All data supporting the findings of this study are available within the paper. Competing interests : The authors declare no potential conflicts of interest. Ethics and consent to participate declarations : Ethics approval and consent to participate. Consent to publish : Not applicable. Clinical trial number : Not applicable. 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Amin, S.; Massoumi, H.; Tewari, D.; Roy, A.; Chaudhuri, M.; Jazayerli, C.; Krishan, A.; Singh, M.; Soleimani, M.; Karaca, E.E.; et al. Cell Type-Specific Extracellular Vesicles and Their Impact on Health and Disease. Int. J. Mol. Sci. 2024 , 25 , 2730. Al-Madhagi, H. The Landscape of Exosomes Biogenesis to Clinical Applications. Int. J. Nanomedicine 2024 , Volume 19 , 3657–3675. Van Den Boorn, J.G.; Schlee, M.; Coch, C.; Hartmann, G. SiRNA delivery with exosome nanoparticles. Nat. Biotechnol. 2011 , 29 . Liu, C.G.; Song, J.; Zhang, Y.Q.; Wang, P.C. MicroRNA-193b is a regulator of amyloid precursor protein in the blood and cerebrospinal fluid derived exosomal microRNA-193b is a biomarker of Alzheimer’s disease. Mol. Med. Rep. 2014 , 10 . Théry, C.; Amigorena, S.; Raposo, G.; Clayton, A. Isolation and Characterization of Exosomes from Cell Culture Supernatants and Biological Fluids. Curr. Protoc. Cell Biol. 2006 , 30 . Ho, D. Nanodiamonds : applications in biology and nanoscale medicine ; Springer, 2010; ISBN 9781441905314. Chang, H.-C.; Hsiao, W.W.-W.; Su, M.-C. Fluorescent Nanodiamonds ; John Wiley & Sons, Ltd: Chichester, UK, 2018; Kowal, J.; Arras, G.; Colombo, M.; Jouve, M.; Morath, J.P.; Primdal-Bengtson, B.; Dingli, F.; Loew, D.; Tkach, M.; Théry, C. Proteomic comparison defines novel markers to characterize heterogeneous populations of extracellular vesicle subtypes. Proc. Natl. Acad. Sci. 2016 , 113 . Andreu, Z.; Yáñez-Mà 3 , M. Tetraspanins in Extracellular Vesicle Formation and Function. Front. Immunol. 2014 , 5 . Huang, L.-C.L.; Chang, H.-C. Adsorption and Immobilization of Cytochrome c on Nanodiamonds. Langmuir 2004 . Lu, H.C.; Peng, Y.C.; Chou, S.L.; Lo, J.I.; Cheng, B.M.; Chang, H.C. Far-UV-Excited Luminescence of Nitrogen-Vacancy Centers: Evidence for Diamonds in Space. Angew. Chemie - Int. Ed. 2017 , 56 . Lu, Y.; Chen, T.; Lin, H.; Chen, Y.; Lin, Y.; Le, D.; Huang, Y.; Wang, A.H. ‐J.; Lee, C.; Ling, T. Small Extracellular Vesicles Engineered Using Click Chemistry to Express Chimeric Antigen Receptors Show Enhanced Efficacy in Acute Liver Failure. J. Extracell. Vesicles 2025 , 14 . Su, L.-J.; Wu, M.-S.; Hui, Y.Y.; Chang, B.-M.; Pan, L.; Hsu, P.-C.; Chen, Y.-T.; Ho, H.-N.; Huang, Y.-H.; Ling, T.-Y.; et al. Fluorescent nanodiamonds enable quantitative tracking of human mesenchymal stem cells in miniature pigs. Sci. Rep. 2017 , 7 , 45607. Weber, J.; Peng, H.; Rader, C. From rabbit antibody repertoires to rabbit monoclonal antibodies. Exp. Mol. Med. 2017, 49 . Veerman, R.E.; Teeuwen, L.; Czarnewski, P.; Güclüler Akpinar, G.; Sandberg, A.S.; Cao, X.; Pernemalm, M.; Orre, L.M.; Gabrielsson, S.; Eldh, M. Molecular evaluation of five different isolation methods for extracellular vesicles reveals different clinical applicability and subcellular origin. J. Extracell. Vesicles 2021 , 10 . 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6654502","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Short Report","associatedPublications":[],"authors":[{"id":471473265,"identity":"e369bc0b-b18b-4d8e-98e4-cfe689379029","order_by":0,"name":"Su-Mei Lin","email":"","orcid":"","institution":"Shin Kong Wu Ho-Su Memorial Hospital","correspondingAuthor":false,"prefix":"","firstName":"Su-Mei","middleName":"","lastName":"Lin","suffix":""},{"id":471473266,"identity":"677ab006-70b2-4487-9018-ba8abcd329c3","order_by":1,"name":"Long-Jyun Su","email":"","orcid":"","institution":"MediDiamond Inc","correspondingAuthor":false,"prefix":"","firstName":"Long-Jyun","middleName":"","lastName":"Su","suffix":""},{"id":471473267,"identity":"a24ba3e5-0d92-47db-b395-d687dccffa1a","order_by":2,"name":"Liang-Yu Chen","email":"","orcid":"","institution":"LuminX Biotech Co., Ltd","correspondingAuthor":false,"prefix":"","firstName":"Liang-Yu","middleName":"","lastName":"Chen","suffix":""},{"id":471473268,"identity":"12af137f-0e27-45db-9c24-e767ed38e1fd","order_by":3,"name":"Zhi-Xuan Zhang","email":"","orcid":"","institution":"LuminX Biotech Co., Ltd","correspondingAuthor":false,"prefix":"","firstName":"Zhi-Xuan","middleName":"","lastName":"Zhang","suffix":""},{"id":471473271,"identity":"4e2e2dd3-3701-408a-9eaf-57aa8cd00743","order_by":4,"name":"Che-Yao Chang","email":"","orcid":"","institution":"National Kaohsiung Normal University","correspondingAuthor":false,"prefix":"","firstName":"Che-Yao","middleName":"","lastName":"Chang","suffix":""},{"id":471473274,"identity":"0eff8d7d-e217-4a6c-beff-b68ae25d035a","order_by":5,"name":"Hsin-Hung Lin","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2UlEQVRIiWNgGAWjYBACgxs5DAcYKoCswwwGB8BCh4nScoZULQyMbUDWAQYDiNABwloYDxfO2ybHd5x544GfOTZ5fMd5D35gqLGJxqXF/v4bhsMzt902ljzMVnCwd1taseRhvmQJhmNpuQ14HHaYd9vtxA2HeQwO8G47DGKYMTA2HCagZc7tepCWg3+J19JwO8EAqOUw8bbwHLttOBPol8Oy29ISZx7mMZZIwOuX/MefeWpuy/OdP7z549ttNol9588YfvhQY4NTCw6QQJryUTAKRsEoGAVoAACpaWnN1Wkr8QAAAABJRU5ErkJggg==","orcid":"","institution":"MediDiamond Inc","correspondingAuthor":true,"prefix":"","firstName":"Hsin-Hung","middleName":"","lastName":"Lin","suffix":""}],"badges":[],"createdAt":"2025-05-13 10:38:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6654502/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6654502/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":84712512,"identity":"2f6d5c32-6384-4382-9a8e-a9dc2a5fd2f5","added_by":"auto","created_at":"2025-06-16 13:39:55","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":211680,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCharacterization of nanodiamonds and antibody-conjugated nanodiamonds. (A) \u003c/strong\u003eHydrodynamic size of bare NDs and antibody-conjugated NDs in distilled deionized water. Values given in the parentheses of annotations are ζ-potentials. mAb, the monoclonal antibody. \u003cstrong\u003e(B) \u003c/strong\u003eTransmission electron microscopy image of bare NDs. Scale bar = 100 nm.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6654502/v1/589e076a0101fa4bb7a8ae8a.png"},{"id":84711749,"identity":"ecf0ce97-ee0d-4868-a5e0-fba06f6999c8","added_by":"auto","created_at":"2025-06-16 13:31:54","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":195562,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExoFortius captured sEVs with high efficiency. (A\u003c/strong\u003e) Nanoparticle tracking analysis (NTA): Quantification and size of sEVs isolated from conditioned medium of pcMSCs, ranging from 70 to 150 nm. \u003cstrong\u003e(B\u003c/strong\u003e) Flow cytometry was used to analyze EV surface markers, including CD9, CD63, and CD81. The gating strategy for each marker is shown, with the respective fluorescence intensities on the y-axis and side scatter on the x-axis. \u003cstrong\u003e(C) \u003c/strong\u003eConfocal images of colocalizations of anti-CD9, anti-CD63, and anti-CD81-conjugated NDs interacting with sEVs, respectively. sEVs were stained with Alexa Fluor 488 WGA dye. Scale bar = 10 μm.\u003cstrong\u003e (D) \u003c/strong\u003eA\u003cstrong\u003e \u003c/strong\u003econfocal image of colocalizations of mixed anti-CD9, anti-CD63, and anti-CD81-conjugated NDs interacting with sEVs. sEVs were stained with Alexa Fluor 488 WGA dye. Scale bar = 10 μm.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6654502/v1/2b0324ee8ea2f73b9f74d02e.png"},{"id":84711765,"identity":"e9be9c6d-4ec7-4b25-9b03-3c1bba95d722","added_by":"auto","created_at":"2025-06-16 13:31:57","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":161507,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic representation of the ExoFortius applications. \u003c/strong\u003eExoFortius is a straightforward, one-step platform developed for the efficient isolation and precipitation of total sEVs from biofluids using antibody-conjugated nanodiamonds (NDs). The ExoFortius kits are compatible with a broad spectrum of biological fluids, including serum, plasma, tumor ascites, and cell culture media, even when working with dilute samples or large volumes. This system offers several key advantages: it enables rapid sEV isolation in under 30 minutes using a simple low-speed centrifugation step, supports broad applicability across various biofluids, and yields sEVs suitable for diverse downstream analyses such as miRNA profiling, next-generation sequencing, and in vivo pharmacokinetic (PK), pharmacodynamic (PD), and biodistribution (Bio-D) studies. Crucially, the isolated sEVs retain their structural integrity and biological activity, making them ideal for functional studies and engineering applications.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6654502/v1/90a2cf977619fc5c733144e9.png"},{"id":91193847,"identity":"ada7579f-19f9-4069-a678-911738d89339","added_by":"auto","created_at":"2025-09-12 14:46:56","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1408082,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6654502/v1/2c42d45b-bcbc-4785-b08b-47c871870838.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Isolation of small extracellular vesicles from biofluids using immunoaffinity nanodiamonds","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eSmall extracellular vesicles (sEVs), ranging from 30\u0026ndash;150 nm in diameter, are generated via the endosomal pathway and secreted upon fusion of multivesicular bodies with the plasma membrane [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Morphologically cup-shaped, sEVs mediate intercellular communication by transferring bioactive cargo, including lipids, proteins, and nucleic acids [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. They function as critical vectors for intercellular, inter-tissue, and interorgan signaling, influencing a broad spectrum of physiological and pathological processes [\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. sEVs are secreted by a wide array of cell types [\u003cspan additionalcitationids=\"CR7 CR8 CR9\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], and are selectively enriched with functional molecules reflective of their cell of origin [\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. These vesicles are abundant in body fluids, including blood, saliva, breast milk, and urine, facilitating systemic dissemination [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. sEVs can engage target cells through ligand-receptor interactions [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], membrane fusion, or uptake via endocytosis/ phagocytosis, thereby delivering their molecular contents and modulating recipient cell signaling networks [\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Through these mechanisms, sEVs actively participate in immunomodulation, antigen presentation, and tumorigenesis [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003esEV biogenesis and cargo loading are tightly regulated by their parental cells to facilitate the targeted molecular information transfer, enabling specific functional outcomes in recipient cells [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Consequently, the molecular composition of sEVs reflects the physiological or pathological status of their cells of origin, providing a basis for their use in diagnostics, including cardiovascular diseases [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], central nervous system diseases [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], hepatic conditions [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], infectious diseases [\u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], and autoimmune disorders [\u003cspan additionalcitationids=\"CR30\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. They also play a key role in non-invasive diagnostic approaches such as liquid biopsy [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. In oncology, sEVs have garnered significant attention as tumor-specific biomarkers, offering utility in cancer detection, therapeutic monitoring, and prognosis assessment [\u003cspan additionalcitationids=\"CR35 CR36\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. They are now considered among the most promising biomarkers for liquid biopsy applications [\u003cspan additionalcitationids=\"CR39\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Beyond diagnosis, sEVs are being explored across multiple biomedical domains, including targeted drug delivery [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e], cell-free vaccine platforms [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e], and regenerative medicine [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDespite substantial progress in modern medicine, significant challenges persist in the diagnosis and treatment of numerous complex diseases. Early-stage detection, before the onset of overt clinical symptoms, is widely recognized as critical for effective disease prevention, timely intervention, and improved prognostic outcomes [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Nonetheless, conventional early diagnostic methods face considerable limitations, including invasiveness, suboptimal sensitivity, high cost, and limited patient compliance [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. In the therapeutic context, the management of refractory diseases, such as malignancies, neuropsychiatric conditions, autoimmune disorders, and infectious diseases, remains constrained. Despite the emergence of novel pharmacological agents, therapeutic efficacy is frequently compromised by factors such as the impermeability of the blood-brain barrier (BBB), the development of drug resistance, and inadequate target specificity [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe molecular content carried by sEVs offers a rich source of biomarkers that can aid in detecting the presence of disease and in identifying disease subtypes and their unique molecular profiles [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. This capability supports the development of personalized treatment strategies, ultimately enhancing patient outcomes and minimizing unnecessary therapies [\u003cspan additionalcitationids=\"CR50\" citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. sEVs have emerged as promising vectors for drug delivery, targeted therapy, and immunomodulation [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e], owing to their intrinsic biocompatibility, low immunogenicity, and the structural stability provided by their lipid bilayer, which safeguards encapsulated genetic material from enzymatic degradation. Their nanoscale dimensions and membrane characteristics further enable efficient translocation across the BBB [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e], positioning them as innovative tools for the diagnosis and treatment of neurological and psychiatric disorders [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. Therefore, the specific and efficient isolation of sEVs from a mixture of cellular debris and interfering substances is essential. Traditional isolation methods, primarily based on differential centrifugation, can achieve reasonable purity, but they face limitations such as long processing times, costly instrumentation, and poor batch-to-batch reproducibility, which restrict their broader application in both research and clinical settings [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. While rapid and precise isolation of high-purity sEVs is preferred, it remains a technical challenge. Additionally, although several innovative sEV-based detection techniques have emerged in recent years, substantial work is still required to effectively translate these findings into clinical practice.\u003c/p\u003e \u003cp\u003eTo advance translational research and innovation in the sEV field, nanoparticles have been employed as cutting-edge technologies, evolving into highly effective tools. Nanodiamond (ND) particles, known for their chemical inertness, outstanding biocompatibility, and minimal cytotoxicity [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e], have been widely utilized in biological and nanomedical applications. This is largely due to their high surface-area-to-volume ratio, adaptable surface chemistry, and capacity to emit near-infrared fluorescence from color centers [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. A recent study by Lin et al. demonstrated that the excellent biocompatibility and multifunctional properties of NDs and fluorescent NDs (FNDs) make them promising candidates as both active and traceable vaccine adjuvants and antitumor agents. As such, NDs/FNDs offer valuable potential to the sEV field by serving as versatile tools for research and bioengineering.\u003c/p\u003e \u003cp\u003eIn clinical translational research, there remains a critical demand for sEV isolation platforms that are cost-effective to fabricate, operationally straightforward, require no specialized technical expertise, entail minimal pre-analytical processing, and provide robust reproducibility and high-resolution separation performance in terms of both purity and recovery yield. To address this unmet need, we have developed ExoFortius\u0026mdash;a rapid, high-precision, immunoaffinity-based ND platform engineered for the selective isolation of sEVs directly from a variety of body fluids, including blood, urine, saliva, breast milk, semen, amniotic fluid, cerebrospinal fluid, bronchoalveolar fluid, as well as fluid from diseased lesions including ascites and pleural fluid, and can be subjected to multicomponent analysis. This system leverages antibodies directed against the tetraspanins CD9, CD63, and CD81, which are consistently enriched in sEV membranes [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e], and are widely accepted as canonical sEV surface markers due to their broad expression across diverse cell types.\u003c/p\u003e \u003cp\u003eExoFortius exhibits superior recovery efficiency and particle purity, particularly under conditions of limited input biofluid volumes, thereby enabling its application in low-yield clinical specimens. Given these attributes, ExoFortius establishes a robust methodological framework for sEV-based transcriptomic profiling in a variety of body fluids and facilitates mechanistic investigations into sEV-mediated intercellular communication and disease pathophysiology. Collectively, our findings position ExoFortius as a scalable, technically accessible, and economically viable immunoaffinity-based isolation platform capable of delivering rapid, reproducible, and high-fidelity sEV enrichment directly from a variety of body fluids.\u003c/p\u003e"},{"header":"2 Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Chemical and materials\u003c/h2\u003e \u003cp\u003eAnti-CD9, anti-CD63, and anti-CD81 antibodies were purchased from IReal Biotechnology (Hsinchu City, Taiwan). Monocrystalline synthetic diamond powders with a nominal size of 100 nm were obtained from Element Six (Didcot, UK).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 NDs and antibody-NDs\u003c/h2\u003e \u003cp\u003eMonocrystalline diamond powders were initially oxidized in air at 490\u0026deg;C for 2 hours to eliminate surface graphitic carbon. This was followed by microwave-assisted cleaning in a concentrated H₂SO₄\u0026ndash;HNO₃ (3:1 v/v) solution at 100\u0026deg;C for 3 hours to remove metallic impurities and concurrently introduce \u0026ndash;COOH functional groups on the surface [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. The resulting surface-oxidized and carboxylated nanodiamonds (NDs) were then noncovalently conjugated with the antibody by simply mixing the antibody solution (5 \u0026micro;l, at concentrations of 1 or 5 mg/ml) with ND suspension (30 \u0026micro;l, at concentrations of 2 or 10 mg/ml) for 1 hour at room temperature.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 FNDs and antibody-FNDs\u003c/h2\u003e \u003cp\u003eFluorescent nanodiamonds (FNDs) were produced by subjecting monocrystalline diamond powders to 10-MeV electron irradiation to induce radiation damage, followed by vacuum annealing at 800\u0026deg;C to form nitrogen-vacancy centers within the diamond lattice, as previously reported [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. Subsequently, the FNDs underwent surface oxidation, carboxylation, and noncovalent conjugation with the antibody using the same procedures described earlier for NDs.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Particle characterization\u003c/h2\u003e \u003cp\u003eThe hydrodynamic sizes and ζ-potentials of both bare and antibody-conjugated NDs were determined using a particle size and ζ-potential analyzer (DelsaNano C; Beckman Coulter, CA, USA). The morphology of the NDs was examined by transmission electron microscopy (H-7650, Hitachi; Tokyo, Japan) at an accelerating voltage of 75 kV, with samples prepared on copper grids.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Protein adsorption measurement\u003c/h2\u003e \u003cp\u003eThe absorption spectrum of an antibody solution (800 \u0026micro;g/ml) in the 200\u0026ndash;400 nm range was recorded using a UV-Vis spectrophotometer (U-3310, Hitachi; Tokyo, Japan). The protein solution was then combined with an ND suspension (1 mg/ml) at a 1:1 volume ratio and incubated at room temperature for 30 minutes with gentle shaking. Following incubation, the antibody-ND mixture was centrifuged at 20,000 \u0026times; g for 15 minutes to pellet the NDs. The concentration of unbound antibody in the supernatant was determined by measuring its absorbance at 280 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Generation and characterization of human placenta pcMSCs\u003c/h2\u003e \u003cp\u003eHuman placentas used in this study were collected from women undergoing cesarean sections at Taipei Medical University Hospital. The collection and use of placental tissues followed protocols approved by the Institutional Review Board of Taipei Medical University. All donors provided written informed consent, and all procedures adhered to relevant ethical guidelines and regulations. Placenta-derived mesenchymal stem cells (pcMSCs) were isolated from the choriodecidual membrane using a serum-free culture method, as previously described [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. Briefly, choriodecidual tissues were digested overnight at 4\u0026deg;C in SMEM containing 1 mg/mL DNase I, 0.5 mg/mL collagenase B, and 0.5 mg/mL protease. The digested mixture was passed through a 100-\u0026micro;m nylon mesh to remove debris. After centrifugation, the cell pellet was resuspended in MCDB201 medium supplemented with 1% penicillin/streptomycin, 10 ng/mL epidermal growth factor, and 1% insulin-transferrin-selenium. Cells were then seeded onto culture dishes coated with human collagen type IV. Adherent cells were cultured with medium changes every 3 to 4 days to eliminate nonadherent cells. The resulting cells, identified as pcMSCs, were maintained in serum-free medium and exhibited fibroblast-like morphology upon attachment. These cells expressed surface markers CD44, CD73, CD90, and CD29, while lacking expression of CD45, CD34, CD14, and HLA-DR [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Conditioned medium collection, sEV isolation, and characterization\u003c/h2\u003e \u003cp\u003eConditioned medium was harvested from pcMSCs cultured in serum-free medium, and sEVs were isolated and purified using ultracentrifugation. Specifically, conditioned medium from pcMSCs cultured for 4 days was collected and first centrifuged at 300 \u0026times; g for 10 minutes to remove dead cells. The supernatant was then sequentially centrifuged at 2,000 \u0026times; g for 10 minutes to eliminate apoptotic bodies, and at 10,000 \u0026times; g for 30 minutes to remove microvesicles. sEVs were subsequently isolated by ultracentrifugation at 100,000 \u0026times; g for 90 minutes. To reduce contamination from soluble proteins, the sEV pellet was washed once with PBS and then resuspended in PBS after the supernatant was removed. Nanoparticle tracking analysis (NTA, NanoSight NS300) and a Litesizer DLS 500 (Anton Paar, Austria) were used to assess particle size, zeta potential, and concentration. To characterize surface markers associated with sEVs, including CD9, CD63, and CD81, NanoFCM (NanoFCM, China), a specialized flow cytometer for nanoscale particles, was employed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Confocal fluorescence microscopy\u003c/h2\u003e \u003cp\u003esEVs were fluorescently labeled using Alexa Fluor 488-conjugated Wheat Germ Agglutinin (WGA) (Cat. No W11261, Thermo Fisher Scientific, Rockford, IL, USA). Following incubation with ExoFortius, the antibody-conjugated FNDs, optical imaging of the sEVs was performed using a laser-scanning confocal fluorescence microscope (SP-8, Leica Microsystems; Wetzlar, Germany). The system was equipped with a supercontinuum white-light laser, providing excitation at 488 nm for the Alexa Fluor 488 dye and at 561 nm for the nitrogen-vacancy centers. Fluorescence emission was collected through a 63\u0026times; oil-immersion objective (numerical aperture\u0026thinsp;=\u0026thinsp;1.4) and detected in the 650\u0026ndash;800 nm wavelength range.\u003c/p\u003e \u003c/div\u003e"},{"header":"3 Results and discussion","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Material characterization\u003c/h2\u003e \u003cp\u003eThe sizes and ζ-potentials of nanodiamonds (NDs) were analyzed before and after their conjugation with rabbit IgG. Transmission electron microscopy (TEM) images of acid-washed, bare NDs revealed particles with irregular shapes and a wide size distribution (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Dynamic light scattering (DLS) measurements in distilled deionized water (DDW) showed that these NDs had an average hydrodynamic diameter of approximately 100 nm and a polydispersity index of 0.24. Upon mixing with rabbit monoclonal antibodies in DDW, the mean particle size increased by about 20 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA), indicating successful protein adsorption. The ζ-potential of the bare ND was measured at -45 mV, which increased to -23 mV following rabbit IgG conjugation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eRabbit monoclonal antibodies have become valuable tools in biomedical research and are increasingly adopted for diagnostic and therapeutic purposes, benefiting from the strengths of both rabbit polyclonal and mouse monoclonal antibodies [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. Rabbit IgG has a molecular weight of approximately 150 kDa and consists of two heavy chains (~\u0026thinsp;50 kDa each) and two light chains (~\u0026thinsp;25 kDa each), forming a tetrameric structure. To estimate the number of rabbit IgG molecules noncovalently bound to acid-washed NDs, changes in absorbance at 280 nm were measured before and after mixing the antibody with the nanoparticles. Based on these measurements, the maximum loading capacity was estimated at 0.1 g of protein per gram of NDs. Assuming the NDs are spherical and approximately 100 nm in diameter (with an estimated mass of ~\u0026thinsp;1.8 fg per particle), each ND can accommodate over 700 rabbit IgG molecules on its surface.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Characterization of sEVs\u003c/h2\u003e \u003cp\u003esEVs were isolated from human placenta choriodecidual membrane-derived mesenchymal stromal cells (pcMSC) culture supernatants and subsequently characterized. Nanoparticle tracking analysis (NTA) was employed to assess the size distribution and concentration of the sEVs, revealing an average size range of 70 to 150 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), consistent with previous findings [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. Flow cytometry was used to analyze surface CD markers, showing notable expression of the tetraspanins CD9 (32.6%), CD63 (10.4%), and CD81 (23.7%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). These findings support the suitability of the isolated sEVs for evaluating the labeling efficiency of anti-sEV antibody-conjugated NDs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Functionalization of anti-sEV antibody-conjugated nanodiamonds\u003c/h2\u003e \u003cp\u003esEVs were employed as a model system to evaluate the capture and precipitation efficiency of NDs conjugated with anti-sEV antibodies. To facilitate visualization of the colocalization between sEVs and antibody-functionalized NDs, we substituted NDs with FNDs, leveraging their near-infrared fluorescence properties [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. Given that sEVs express tetraspanin family proteins such as CD9, CD63, and CD81, we conjugated rabbit monoclonal antibodies against these markers to NDs to enable high-yield, high-throughput sEV isolation.\u003c/p\u003e \u003cp\u003eFollowing quantification of the total sEV population, anti-sEV antibody-conjugated NDs were incubated with the sEV solution. Confocal microscopy of Alexa Fluor 488-labeled sEVs demonstrated efficient colocalization with NDs conjugated to anti-CD9, anti-CD63, and anti-CD81 antibodies, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). To assess labeling efficiency, we combined all three types of antibody-conjugated NDs and incubated them with sEVs. Imaging confirmed nearly complete colocalization between sEVs and the mixed antibody-ND complexes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD), indicating highly efficient capture.\u003c/p\u003e \u003cp\u003eTo further validate the isolation efficiency, equal volumes of sEVs and antibody-conjugated NDs were mixed and subjected to low-speed centrifugation (20,000 \u0026times; g, 15 minutes). The supernatant retained less than 7% of the original sEV population, yielding a recovery rate exceeding 90%. These antibody-functionalized NDs, termed ExoFortius, are capable of selectively capturing and purifying specific sEV subpopulations based on their surface markers. The ExoFortius platform is modular, allowing for the attachment of pre-validated capture antibodies targeting sEV markers of interest.\u003c/p\u003e \u003c/div\u003e"},{"header":"4 Conclusion","content":"\u003cp\u003eExoFortius kits are compatible with a wide range of biological fluids\u0026mdash;including serum, plasma, tumor ascites, and cell culture media\u0026mdash;even at low concentrations and large volumes. The system offers multiple advantages: it enables rapid isolation (within 30 minutes) via a simple low-speed spin protocol, supports universal application across diverse biofluids, and provides isolated sEVs that are suitable for a range of downstream applications, including miRNA profiling, next-generation sequencing, and in vivo studies of pharmacokinetics (PK), pharmacodynamics (PD), and biodistribution (Bio-D). Importantly, the captured sEVs remain intact and bioactive, making them suitable for functional analyses and bioengineering applications.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e: This work was supported by grant 2023SKHAND011 from Shin Kong Wu Ho-Su Memorial Hospital to S.M. Lin. We would like to express our sincere gratitude to Dr. Thai-Yen Ling for his support with pcMSCs.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e: M.S. Lin: Conceptualization, data curation, validation, investigation. L.J. Su: Conceptualization, data curation, validation, and investigation. L.Y. Chen: data curation, validation, and investigation. Z.X. Zhang: data curation, validation, and investigation. C.Y. Chang: Methodology, validation, and investigation. H.H. Lin: Formal analysis, validation, investigation, writing, and editing.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e: All data supporting the findings of this study are available within the paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e: The authors declare no potential conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics and consent to participate declarations\u003c/strong\u003e: Ethics approval and consent to participate.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to publish\u003c/strong\u003e: Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical trial number\u003c/strong\u003e: Not applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eKalluri, R.; LeBleu, V.S. 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Vesicles\u003c/em\u003e \u003cstrong\u003e2021\u003c/strong\u003e, \u003cem\u003e10\u003c/em\u003e.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Biomaterials, Biomedical applications, Disease diagnosis, Nanodiamonds, Immunoaffinity, Small extracellular vesicles","lastPublishedDoi":"10.21203/rs.3.rs-6654502/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6654502/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSmall extracellular vesicles (sEVs) are nanoscale vesicles derived from cells, known for their ability to transport a diverse array of biomolecules from their parent cells. These vesicles play a pivotal role in intercellular communication and are involved in various physiological and pathological processes. sEVs offer several advantages, including excellent biocompatibility, low immunogenicity, and widespread biodistribution. With growing insights into their biological functions, numerous strategies have emerged for leveraging sEVs in disease diagnosis and therapy. Despite this potential, standard methods for isolating sEVs from biofluids remain incompatible with routine research and clinical workflows, limiting their broader biomedical application. In this study, we present a simple and efficient immunoaffinity-based platform using nanodiamonds to isolate sEVs from a range of biological fluids. The separation efficiency was assessed using fluorescently labeled sEVs, achieving recovery rates greater than 90%. Our nanodiamond-based approach, named ExoFortius, provides a scalable, accessible, and cost-effective solution for high-yield sEV isolation. This method enables rapid, consistent, and high-purity enrichment of sEVs directly from complex biofluids. By introducing ExoFortius, this study offers a novel technological advancement aimed at accelerating sEV-related research and expanding their utility in translational medicine for both diagnostic and therapeutic applications.\u003c/p\u003e","manuscriptTitle":"Isolation of small extracellular vesicles from biofluids using immunoaffinity nanodiamonds","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-16 13:31:50","doi":"10.21203/rs.3.rs-6654502/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7e663573-9c4d-4f41-96ba-d3cd852409f8","owner":[],"postedDate":"June 16th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-09-12T14:38:45+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-16 13:31:50","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6654502","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6654502","identity":"rs-6654502","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}