Liver-Targeted Fetal MSC-Derived Extracellular Vesicles: A Therapeutic Approach in a Mouse Model of Acute Hepatic Failure

Liver-Targeted Fetal MSC-Derived Extracellular Vesicles: A Therapeutic Approach in a Mouse Model of Acute Hepatic Failure | 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 Method Article Liver-Targeted Fetal MSC-Derived Extracellular Vesicles: A Therapeutic Approach in a Mouse Model of Acute Hepatic Failure Alexandra Stamatopoulou, Maria Lefkaditi, Konstantinos Paschidis, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7302374/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 Background Acute hepatic failure (AHF) is a rapid deterioration of hepatocellular function, which includes coagulopathy and hepatic encephalopathy and it is related with high mortality rate. To date, liver replacement is the sole successful procedure for AHF with limitations related to donor organ shortage and lifelong immunosuppressive therapy. Mesenchymal stromal/stem cell “secretome”, including extracellular vesicles (EVs), offers considerable advantages over intact cell-based applications, such as lack of immunogenicity, standard manufacturing, and storage. However, targeted delivery of EVs to the liver still remains challenging for clinical translation. Methods We developed a novel protocol to generate liver-targeted EVs by coating EVs from umbilical cord MSCs (UC-MSCs) with albumin. EVs were isolated using ultracentrifugation and subsequently surface-modified with albumin to enhance hepatic targeting. AHF was induced in mice using carbon tetrachloride (CCl₄). The therapeutic efficacy of the albumin-coated EVs was evaluated via serum biomarker measurements and live imaging. Results The albumin-coated EVs (ALB-EVs) demonstrated higher liver bioaccumulation and reduced biodistribution to the other organs compared to naive EVs. CCL 4− induced AHF mice treated with liver specific EVs exhibited significantly decreased levels of aminotransferases and improved liver morphology and histological features. The entire protocol, from EV isolation to treatment, can be completed within one week using standard laboratory techniques. Conclusions This method provides an efficient, reproducible strategy for liver-targeted EV-based therapy in acute hepatic failure. Albumin-coated EVs represent a promising and scalable platform for developing noncellular therapeutics in regenerative medicine. Acute hepatic failure Extracellular vesicles Mesenchymal stromal cells Liver targeting Albumin coating Regenerative medicine Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction MSCs are promising candidates for therapeutic applications due to their intrinsic repertoire of anti-inflammatory and immunomodulatory factors, collectively termed the “secretome” when released extracellularly [ 1 – 8 ]. However, the direct administration of MSCs is often related to low efficacy, mainly due to immunological rejection and limited engraftment [ 9 , 10 ]. The therapeutic potential of MSC-based therapies can be enhanced through innovative strategies that prolong and optimize the delivery of their beneficial “secretome [ 11 – 13 ]. The latter offers considerable advantages over intact cell-based therapies, including reduced immunogenicity, simplified manufacturing and storage via nontoxic cryopreservation and more straightforward handling for safety and dosing, similar to conventional pharmaceutical agents [ 14 , 15 ]. MSC-derived mediators are secreted abundantly as soluble molecules and are also encapsulated within EVs of micro- and nanosized dimensions [ 16 , 17 ]. EVs are derived from the late endosomal pathway and are characterized by specific membrane markers, such as tetraspanins [ 16 ]. Importantly, MSC-derived EVs carry a functionally competent molecular cargo [ 17 ] that remains protected from degradation until it reaches neighbouring target cells, thereby effectively delivering immunomodulatory and anti-inflammatory molecules [ 18 , 19 ]. Numerous methodologies for EV isolation have been described extensively in recent studies [ 20 ]. The liver is a primary site for in vivo EV uptake; although, other organs, such as the lungs, kidneys and spleen, also demonstrate considerable accumulation of EVs [ 21 – 23 ]. Consequently, the role of the liver in EV uptake has been the focus of multiple studies, employing various methodological techniques. Recent studies have revealed that engineered EVs featuring albumin-binding domains (ABDs) on their surface exhibit significantly improved cellular uptake in the liver [ 24 ], potentially enhancing the therapeutic efficacy although prolonged circulation time of EVs by preventing premature clearance from the bloodstream [ 18 , 25 ]. Additionally, vitamin A-coupled EVs (V-EVs) have been shown to selectively target activated hepatic stellate cells, effectively reversing liver fibrosis at lower doses compared with naive EVs [ 26 , 27 ]. Similarly, the systematic release of hydrogel-encapsulated MSC-EVs[ 28 ] has been shown to promote liver regeneration in chronic liver failure models [ 29 ]. The overarching scientific objective of this method is to improve the targeted delivery of therapeutic EVs into the liver in the context of AHF. Our previously published data in Gut 2012 [ 30 ], EbioMedicine 2019 [ 31 ], Hepatology 2022 [ 14 ], and iScience 2023 [ 32 ] underscore the importance of the MSC “secretome” and EVs in AHF [ 33 ]. Therefore, EVs can be deployed as anti-inflammatory therapeutic agents for AHF. However, the nonspecific distribution of MSC-EVs following intravenous (i.v.) injection limits their efficacy [ 34 ]. To address this limitation and enhance liver-specific therapeutic effects, we propose the development of activated liver-targeting albumin-coated EVs through a straightforward, efficient and clinically available surface engineering strategy. Here, we present an integrated ultracentrifugation-based protocol to achieve high yield, pure EVs, followed by albumin surface modification to enable liver targeting [ 24 ]. Materials and Methods Cells Our laboratory routinely utilizes human amniotic fluid (AF) or umbilical cord (UC) MSCs as standard sources for the isolation and characterization of EVs [32]. A typical preparation involved three T175 flasks of 90% confluency, each containing approximately 18x10*6 cells at the time of harvest. The typical protein yield of EVs is approximately 100 µg. This technique can also be applied to different types of MSCs, including bone marrow (BM)-MSCs and their differentiated progenitor cells. However, the yield and purity of EV fractions may vary depending on the cell type, culture conditions, media composition and cell density. Additional variables, such as cell quantity and duration of serum starvation, also affect yield. Preparation of EV-depleted medium A-MEM supplemented with 20% fetal bovine serum (FBS), 1% penicillin/streptomycin, and 1% L-glutamine was filtered through 0.22 µm filter. To eliminate EVs from FBS the medium was then centrifuged at 100,000 × g for 16 h (overnight) at 4°C using T-865 fixed angle rotor. The supernatant was carefully collected without disturbing the pellet, re-filtered (0.22 µm), and stored at 4°C until use. Isolation of EVs from cell-conditioned medium We present an optimized protocol for the isolation of EVs derived from MSCs, employing a combination of centrifugation, concentration, and filtration (Fig. 1a). Specifically, 3 x 175 T flasks of UC-MSC cells were cultured until 90% confluency with A-MEM supplemented with EV-depleted FBS at a final concentration 0.5% and 2.5 µg of BSA for 48 h. After 48 h, CM from all flasks (~ 50 ml) was collected. The first centrifugation was performed at 500 × g for 5 min to remove dead cells and then at 2,000 × g for 10 min at 4°C to remove cellular debris. The supernatant was collected and centrifuged at 10,000 × g for 30 min at 4°C to exclude larger EVs. Finally, the medium was then centrifuged at 100,000 × g for 2 h at 4°C, and the resulting EV pellet was resuspended in PBS (1x) and subjected to a second ultracentrifugation at 100,000 × g for 2 h at 4°C. The EV pellet was ultimately reconstituted in PBS (1x) and stored at -80°C for subsequent use. Characterization of isolated EVs Following isolation, EVs were characterized in accordance with the guidelines established in MISEV2018 [20] to ensure the quality of EV fractions before downstream analyses. Various protocols are available to assess key characteristics, such as size, morphology, and ultrastructure, typically via electron microscopy. Additionally, the presence of EV marker proteins can be confirmed by standard immunoblotting or ELISA [20]. Nanoparticle tracking analysis (NTA) was performed to quantify particle number and size distribution of EVs, relying on light scattering. the use of detergents further improves the discrimination between EVs and non-EV molecules. As EV pellets may contain both EVs and non-EVs, caution must be taken when normalizing particle numbers for downstream analyses or functional assays [32]. Normalization based on protein quantification is the preferred method. PKH26 labeling of EVs Freshly isolated EVs by ultracentrifugation were labelled with PKH26 dye. In detail, EVs were resuspended at a concentration of 10 µg in 20 µL of 1× phosphate-buffered saline (PBS). In the EV sample was added 80 µL of Diluent C and the mixture was thoroughly resuspended by gentle pipetting to ensure uniform dispersion. In a separate tube, a PKH26 dye solution was freshly prepared by diluting 0.8 µL of PKH26 dye (final concentration 8 µM) in 100 µL of Diluent C. Equal volumes of the EV suspension and the dye solution were combined and incubated on ice for 1 to 5 minutes, with gentle mixing throughout to ensure uniform EV labeling. To terminate the staining reaction, 100 µL of 10% EV-depleted FBS in A-MEM was added. To minimize nonspecific binding, 1% bovine serum albumin (BSA) can be used as an alternative blocking agent. Importantly, the use of serum-free media or buffered salt solutions should be avoided during this step, as they can promote dye aggregation. The PKH26-labeled EVs were finally centrifuged at 100,000 × g for 2 h at 4°C using a T-865 fixed-angle rotor (Sorvall/WX 100+, MA, USA). After ultracentrifugation, the supernatant was carefully removed and kept as a negative control, and the labeled EV pellet was resuspended in 150 µL of 1× PBS for downstream applications. . Animals All the animal experiments were performed in BRFAA facilities in compliance with the European Legal Framework (European Convention 123/Council of Europe and Directive 86/609/EEC), as well as the current guidelines of International Organizations (AAALAC Int., FELASA). The facility is registered as ‘‘breeding’’ and ‘‘experimental’’ facility (Reg. Numbers: EL BIO 01 and EL 25 BIO 03, respectively) according to the Greek Presidential Decree 56/2013, which harmonizes National Legislation with the European Directive 2010/63 on the protection of animals used for scientific purposes. The present protocol was approved by the Department of Agriculture and Veterinary Service of the Prefecture of Athens (Permit Number: 350028/20-04-22 and title: Cellular therapies in liver diseases Approval No.350028/20-04-22) and according to the recommendations of the FELASA and the NIH. The number of animals used per group was determined through power analysis (G*Power 3.1.9, effective size 0.4). Liver injury was assessed through histological examination of liver sections and through monitoring aspartate aminotransferase (AST) and alanine transaminase (ALT) serum levels at 48 hours posttreatment. Establishment of an AHF Mouse Model, EV Administration, and Tissue Collection Since 2012, our laboratory has developed a CCl₄-injured mouse model for AHF [30–32]. Female Rag1-/- mice aged 6–8 weeks and weighing 22‒26 g were intraperitoneally administered (i.p.) 100 µL of CCl₄ dissolved in sunflower oil (90 µL of sunflower oil per 20 g of mouse body weight, containing 10 µL of CCL₄). For negative controls, phosphate-buffered saline (1x) (PBS) in sunflower oil was used in place of CCl₄ (90 µL sunflower oil per 20 g of mouse body weight containing 10 µL PBS). On day 2, CCL₄-treated mice (n = 4) received an an intravenous (i.v). injection of 20 µg of PKH-26-labeled Albumin-EVs dissolved in 200 µL of PBS. Conversely, CCL₄-treated mice (n = 4), which served as controls, were i.v. injected with 20 µg of PKH-26-labeled naive EVs or unlabeled naive EVs in 200 µL of PBS. Intravenous injections were conducted via the tail vein. Two hours post-injection, mice were placed in a light-tight chamber, and photon emission from the labeling dye was detected using a cooled charge-coupled device (CCD) camera apparatus [Xenogen IVIS Lumina II System (Advanced Molecular Vision, Inc., UK)]. On day 3, the animals were euthanized, and serum were collected for transaminase level analysis. Liver tissue samples were also collected in 2 ml centrifuge tubes and snap-frozen in liquid nitrogen for further analyses. Statistical analysis Τhe experimental data were presented as mean ± standard error of the mean (SEM) from three independent experiments. A one-way analysis of variance (ANOVA) was applied to determine statistically significant differences between groups. A p-value < 0.05 was considered statistically significant. The graphs presented were plotted using GraphPad Prism version 8.0.1 (GraphPad Software, San Diego, CA, USA). Results Isolation and characterization of EVs EVs were isolated from UC-MSCs through differential centrifugation, as previously reported (Fig. 1 ). Transmission electron microscopy (TEM) confirmed their size, ranging from 100 to 150 nm, (Fig. 3 a). In addition, Nanoparticle tracking analysis (NTA) revealed an average size (121.4 +/- 4.0 nm, 120.6 +/- 2.4 nm), as well as the average number of nanoparticles/mL PBS (2.75x10 10 +/- 4.74x10 9 particles/ml, 4.44x10 8 +/- 1.07x10 7 particles/ml) in UC-MSC-EV and ALB-EV samples, respectively (Fig. 3 b). Western blot analysis confirmed the presence of characteristic positive markers, including Alix, CD81, and CD9 in both EV populations (Fig. 3 c). In vivo biodistribution of ALB-PKH26-labeled EVs To assess biodistribution and homing capabilities, the isolated EVs were labeled with PKH26, a lipophilic fluorescent dye, and administered intravenously in a CCL 4 -induced AHF mouse model. Two hours post-injection ex vivo live imaging (Xenogen IVIS Lumina II System) demonstrated higher liver accumulation of modified ALB-EVs compared to naïve EVs (Fig. 4 a). Notably, ALB-EVs exhibited significantly reduced accumulation in other organs, as presented by lower fluorescent signal in the lungs and kidneys. These results suggest that albumin surface modification improves the liver-specific homing capacity of EVs (Fig. 4 b). Functional improvement of liver injury after ALB-EVs Administration of CCl 4 induced extensive inflammation, oxidative stress and hepatotoxicity. Liver function was assessed by measuring serum transaminase levels (AST and ALT). The results demonstrated that CCl₄ administration in in vivo AHF-mouse model increased AST/ALT levels to 2,339.1 ± 213.17 U/mL and 2,335.1 ± 135.14 U/mL respectively, compared with healthy mice (99.5 ± 11.99 U/mL and 100.16 ± 10.93 U/mL, respectively), confirming liver injury and damage. Twenty-four hours after the CCL 4 injection, mice were treated with either UC-MSC-EVs or ALB-EVs. Both treatments resulted in decreased AST levels (977.167 ± 406.33 U/mL and 858 ± 280.93 U/mL respectively) and ALT levels (973.8 ± 402.98 U/mL and 955 ± 324.05 U/mL, respectively), compared with those in CCl 4 group (Fig. 5a). Additionally, ALB-EVs were more effective in lowering AST and ALT levels, indicating enhanced liver targeting and therapeutic efficacy. Statistical analysis performed by one-way ANOVA, revealed significant differences between the control and treated groups (∗∗p < 0.01, ∗∗∗∗p < 0.0001, ANOVA test). Macroscopic examination of liver morphology, demonstrated improved hepatic appearance in treated animals compared to the severely damaged livers of the untreated CCl₄ group (Fig. 5b). Histological evaluation with a haematoxylin and eosin (H&E) staining revealed severe liver damage, including necrosis and inflammatory infiltration in the CCl₄ group, which were significantly improved following UC-MSC-EVs and ALB-EVs (Fig. 5c) treatment. Further molecular analysis of inflammation-related pathways, particularly the expression of pro-inflammatory cytokines, such as TNF-α and IL-6, in liver tissues could provide valuable insight into the therapeutic potential of albumin-coated EVs in liver-related diseases requiring efficient tissue targeting and minimal immunogenicity. Discussion In this study, we established and validated an efficient approach for liver-targeted delivery of EVs using surface decoration with albumin. Serum albumin, which is the most abundant plasma protein, exhibits favorable pharmacokinetics, liver tropism, and a long circulatory half-life, and thus it could be an ideal candidate for EV surface modification aimed at liver targeting [ 24 ]. The CCl₄-induced AHF mouse model closely recapitulates features of human hepatic damage, including centrilobular necrosis, oxidative stress, lipid peroxidation, and marked elevations of serum transaminases (ALT and AST) [ 35 – 37 ]. Within twenty-four hours of CCl₄ administration, our experimental animals exhibited classical signs of acute hepatocellular injury, providing a relevant system for evaluating targeted EV biodistribution and therapeutic potential. Ex vivo live imaging, using the Xenogen IVIS Lumina II system, twenty-four hours after CCL 4 injection and two hours after treatment, demonstrated high efficacy in hepatic delivery of PKH26-labeled (ALB-EVs) compared to naive EVs. Additionally, this observation aligns with prior studies showing that albumin coating has demonstrated multiple advantages, such as improved interaction with lipid membranes, reduced cytotoxicity, enhanced cellular uptake via albumin-binding receptors (e.g., gp60, gp30, gp18, and SPARC), and extended circulation time in the bloodstream [ 24 , 38 , 39 ]. Compared to other methods requiring genetic modification or chemical conjugation [ 24 , 26 ], our albumin-coating strategy is technically straightforward, minimally disruptive and non-cytotoxic. The analysis of aminotransferases levels confirmed that administration of ALB-EVs was not associated with systemic toxicity. Instead, serum ALT and AST levels were modestly reduced compared to control, suggesting a hepatoprotective effect. While promising, this approach has limitations. In the process of isolating EVs from human MSCs, the possibility of co-isolating non-vesicular extracellular nanoparticles, including protein, nucleic acid complexes, cannot be entirely excluded [ 40 ]. However, differential ultracentrifugation ensures that the resulting pellet is enriched in EVs, thereby attributing the therapeutic outcomes observed after administration to the animal models, primarily to the EVs themselves. Our methodology presents the use of albumin-coated EVs as a promising therapeutic platform that targets specifically the liver, although a portion of intravenously administered EVs may localize to other organs, such as the lungs and spleen, and could potentially diminish their therapeutic efficacy. Therefore, it is needed to optimize the dosage across various experimental models. Lastly, regarding the application of that method to clinical practice, the isolation of EVs will require rigorous characterization and Good Manufacturing Practice (GMP) compliance. Nonetheless, the ability to selectively enrich EV localization to the liver expands the utility of EVs as delivery vehicles for RNA, proteins, or drugs in hepatic disease. By coupling a widely available protein with a non-invasive coating strategy, our method offers a valuable platform for liver-targeted therapies in the context of acute liver injury and potentially in chronic liver conditions such as fibrosis or cirrhosis. Conclusion This optimized protocol enables effective isolation, labeling, and systemic delivery of EVs in a CCl₄-induced AHF mouse model. By ensuring consistent EV preparation and precise in vivo administration, the method provides a reliable framework for studying liver-targeted EV biodistribution and their therapeutic potential in hepatic injury and regeneration. Abbreviations AHF Acute hepatic failure EVs Extracellular vesicles UC-MSCs Umbilical cord mesenchymal stromal/stem cells CCl₄ Carbon tetrachloride ALB-EVs Albumin-coated EVs Declarations Supplementary Information The online version contains supplementary material available Author contributions statements A.S. conceived the study; designed the experimental methodology; performed the experiments; analysed, interpreted and visualized the data; wrote the manuscript; and provided final approval of the manuscript. M.L. performed the experiments; analysed, interpreted and visualized the data; and approved the final manuscript. K.P. and N.K. performed and supervised the in vivo experiments and were responsible for animal licences and housekeeping; final approval of the manuscript. E.M. isolated MSCs from UC patients, analysed the data and approved the final manuscript. A.E. data analysis; financial support; edited manuscript; final approval of the manuscript. M.G.R. conceived the study; analysed and interpreted the data; provided financial support; designed the experimental methodology; supervised the research; wrote the manuscript; and provided final approval of the manuscript. ORCID for corresponding author: M.R. 0001-5790-6581 Acknowledgements We would like to thank Mr. Nikos Tsakiropoulos (Experimental Surgery, School Medicine, NKUA) for performing the biochemical analyses of mouse blood serum. We are also grateful toProfessor Sophia Havaki, Laboratory of Histology and Embryology, Medical School, NKUA, for her assistance in exosome TEM imaging and Professor Anastasios G. Kriebardis, Laboratory of Reliability and Quality Control in Laboratory Hematology (HemQcR), Department of Biomedical Sciences, Section of Medical Laboratories, School of Health & Caring Sciences, UniWA, for assistance with NTA analysis. In addition, we would like to thank Professor Vassilis G. Gorgoulis, (Laboratory of Histology and Embryology, Medical School, NKUA), and Professor Antonios Chatzigeorgiou (Laboratory of Physiology, Medical School, NKUA), for their assistance in histological processing of the samples. Funding This work was supported by the Hellenic Foundation for Research & Innovation Grant during the 1st Call for H.F.R.I. Research Projects to Support Faculty Members & Researchers and Procure High-Value Research Equipment, no 70/3/16468, 2021-2024) to MR and the action “Flagship Research Projects in challenging interdisciplinary sectors with practical applications to Greek Industry”, implemented through the National Recovery and Resilience Plan Greece 2.0 and funded by the European Union - NextGenerationEU (project code: TAEDR-0541976) to AGE. Competing interests A.G. Eliopoulos is cofounder and CSO of GENOSOPHY S.A., a spin-off company at the National and Kapodistrian University of Athens. 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Eliopoulos is cofounder and CSO of GENOSOPHY S.A., a spin-off company at the National and Kapodistrian University of Athens. Supplementary Files SupplementaryInformation5.8.251.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-7302374","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Method Article","associatedPublications":[],"authors":[{"id":498411708,"identity":"97e7ed82-ac64-4a13-9fee-b17f3c59baa8","order_by":0,"name":"Alexandra Stamatopoulou","email":"","orcid":"","institution":"National and Kapodistrian University of Athens (NKUA)","correspondingAuthor":false,"prefix":"","firstName":"Alexandra","middleName":"","lastName":"Stamatopoulou","suffix":""},{"id":498411709,"identity":"bf14ef52-a3fa-49f3-ada2-13eb4cc1db35","order_by":1,"name":"Maria Lefkaditi","email":"","orcid":"","institution":"National and Kapodistrian University of Athens (NKUA)","correspondingAuthor":false,"prefix":"","firstName":"Maria","middleName":"","lastName":"Lefkaditi","suffix":""},{"id":498411711,"identity":"e082a29b-9c0c-48a8-a903-b1f945873a4b","order_by":2,"name":"Konstantinos Paschidis","email":"","orcid":"","institution":"Academy of Athens (BRFAA)","correspondingAuthor":false,"prefix":"","firstName":"Konstantinos","middleName":"","lastName":"Paschidis","suffix":""},{"id":498411714,"identity":"aea00785-2acf-49f4-97b4-8f9d70dc50f9","order_by":3,"name":"Εfstathios Michalopoulos","email":"","orcid":"","institution":"Biomedical Research Foundation of the Academy of Athens (BRFAA)","correspondingAuthor":false,"prefix":"","firstName":"Εfstathios","middleName":"","lastName":"Michalopoulos","suffix":""},{"id":498411715,"identity":"1299d0ba-359e-4cec-9ac4-348b4d742dc6","order_by":4,"name":"Nikolaos Kostomitsopoulos","email":"","orcid":"","institution":"Academy of Athens (BRFAA)","correspondingAuthor":false,"prefix":"","firstName":"Nikolaos","middleName":"","lastName":"Kostomitsopoulos","suffix":""},{"id":498411716,"identity":"9ed61839-1cda-422b-899d-3cd0324eec08","order_by":5,"name":"Aristides G. Eliopoulos","email":"","orcid":"","institution":"National and Kapodistrian University of Athens (NKUA)","correspondingAuthor":false,"prefix":"","firstName":"Aristides","middleName":"G.","lastName":"Eliopoulos","suffix":""},{"id":498411717,"identity":"fda9dcd7-1d77-44c8-bcb2-e477b42f526a","order_by":6,"name":"Maria G. Roubelakis","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAv0lEQVRIiWNgGAWjYHACNgaGAhsgnUCSFoM00rUcJkGLeXuP2YMPBuej+dkT2CQ+1DDYbW8goEXmzBlzwxkGt3Nn9jxgk5xxjCF5zgECWiQkcsykeYBaNtxIYJPmYWNIliDkMLCWPwbncveDtfwjVguDwYHcDRJALbxtDHaEtfAcKzfsMUjOnXHmYbPlzD6JBMJa2Ju3PfhRYZfb35588MaHbzb2BLUgAcYGkBGJDSRogQB7knWMglEwCkbBsAcAMQw41a/ppUsAAAAASUVORK5CYII=","orcid":"","institution":"National and Kapodistrian University of Athens (NKUA)","correspondingAuthor":true,"prefix":"","firstName":"Maria","middleName":"G.","lastName":"Roubelakis","suffix":""}],"badges":[],"createdAt":"2025-08-05 15:38:25","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7302374/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7302374/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":88893737,"identity":"4abc4f59-1747-427d-832b-f747f28cc536","added_by":"auto","created_at":"2025-08-12 12:57:59","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":550984,"visible":true,"origin":"","legend":"\u003cp\u003eOverview of EV isolation steps from conditioned media. a. Schematic representation of the isolation process of both large and small EV pellets. b. Representative photographs capturing the critical steps through the isolation procedure, showing the methodology and equipment involved.\u003cem\u003e \u003c/em\u003e(Figure created with BioRender.com).\u003c/p\u003e","description":"","filename":"OnlineFigure1Roubelakis.png","url":"https://assets-eu.researchsquare.com/files/rs-7302374/v1/68159118bfc280f0b49d2fbf.png"},{"id":88891347,"identity":"5177e969-ca20-4247-8e58-53d945bccf6a","added_by":"auto","created_at":"2025-08-12 12:49:59","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":66122,"visible":true,"origin":"","legend":"\u003cp\u003eOverview of the steps for the \u003cem\u003ein vivo\u003c/em\u003e administration of PKH26-labeled EVs in the AHF mouse model. (Figure created with BioRender.com)\u003c/p\u003e","description":"","filename":"OnlineFigure2Roubelakis.png","url":"https://assets-eu.researchsquare.com/files/rs-7302374/v1/13430332a0ae4e863c644d98.png"},{"id":88891348,"identity":"1184cc2c-77a5-4a52-802f-08200aedd9fe","added_by":"auto","created_at":"2025-08-12 12:49:59","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":234739,"visible":true,"origin":"","legend":"\u003cp\u003eCharacterization of EVs. a. Evaluation of morphology via transmission electron microscopy (TEM) and b. size distribution and concentration via nanoparticle tracking analysis (NTA), c. Western blotting (WB).\u003c/p\u003e","description":"","filename":"OnlineFigure3Roubelakis.png","url":"https://assets-eu.researchsquare.com/files/rs-7302374/v1/d49cc576c43e26b001f3e1b9.png"},{"id":88891350,"identity":"4f84446d-fc5e-4122-a85c-70f1a241766b","added_by":"auto","created_at":"2025-08-12 12:49:59","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":378168,"visible":true,"origin":"","legend":"\u003cp\u003eBiodistribution of PKH26-labeled UC-MSC-EVs and ALB-EVs following i.v. administration in mice, as determined by imaging via the Xenogen IVIS Lumina II System. a. Representative fluorescent images captured 2 hours post-injection of CCL\u003csub\u003e4\u003c/sub\u003e-induced AHF mice treated with PKH26 labeled either UC-MSC- EVs (left) or ALB-EVs (right). The fluorescence signal corresponds to PKH26 labeling and the color scale represents the signal intensity in counts. b. Quantification of fluorescence intensity (total flux, photons/sec) in the liver, kidneys, and lungs. Data are presented as means ± SEMs (\u003cem\u003en\u003c/em\u003e = 4 mice per group).\u003c/p\u003e","description":"","filename":"OnlineFigure4Roubelakis.png","url":"https://assets-eu.researchsquare.com/files/rs-7302374/v1/d02612aabe094b8e2e3d4ffc.png"},{"id":88895997,"identity":"6d592558-6d15-42a7-9b14-3a3e6ff63936","added_by":"auto","created_at":"2025-08-12 13:06:00","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":345080,"visible":true,"origin":"","legend":"\u003cp\u003eEvaluation of liver function and morphology following treatment.\u003cbr\u003e\na. Levels of serological AST and ALT transaminases after CCl\u003csub\u003e4\u003c/sub\u003e\u0026nbsp;injection and administration of UC-MSC-EVs and ALB-EVs (∗∗p \u0026lt; 0.01, ∗∗∗∗p \u0026lt; 0.0001, ANOVA test). b. Representative images of livers from each group, demonstrating improved liver morphology and reduced hepatic damage in treated animals (n=4/group). c. Representative H\u0026amp;E stained images in liver tissue from CCl₄-induced and treated CCl\u003csub\u003e4\u003c/sub\u003e-induced AHF mice with UC-MSC-EV and ALB-EV. White arrows indicate areas of necrosis and inflammation. Original magnification: 20×. Data are presented as the means ± SEMs. P values were calculated relative to the CCl₄-induced group.\u003c/p\u003e","description":"","filename":"OnlineFigure5RoubelakisnewFinal.png","url":"https://assets-eu.researchsquare.com/files/rs-7302374/v1/604525d67a81558e9f74675e.png"},{"id":89334075,"identity":"40729848-d245-40ff-8e5f-73f6113b3a2f","added_by":"auto","created_at":"2025-08-19 01:16:29","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2647607,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7302374/v1/8d1f18e4-6db6-459b-a734-b2a713ebfbd7.pdf"},{"id":88893736,"identity":"0b21c5d1-b1e5-404c-b60a-5594266808e7","added_by":"auto","created_at":"2025-08-12 12:57:59","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":115971,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformation5.8.251.docx","url":"https://assets-eu.researchsquare.com/files/rs-7302374/v1/ec229ce2b3c3d0bac4cb8da1.docx"}],"financialInterests":"Competing interest reported. A.G. Eliopoulos is cofounder and CSO of GENOSOPHY S.A., a spin-off company at the National and Kapodistrian University of Athens.","formattedTitle":"Liver-Targeted Fetal MSC-Derived Extracellular Vesicles: A Therapeutic Approach in a Mouse Model of Acute Hepatic Failure","fulltext":[{"header":"Introduction","content":"\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eMSCs are promising candidates for therapeutic applications due to their intrinsic repertoire of anti-inflammatory and immunomodulatory factors, collectively termed the \u0026ldquo;secretome\u0026rdquo; when released extracellularly [\u003cspan additionalcitationids=\"CR2 CR3 CR4 CR5 CR6 CR7\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. However, the direct administration of MSCs is often related to low efficacy, mainly due to immunological rejection and limited engraftment [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. The therapeutic potential of MSC-based therapies can be enhanced through innovative strategies that prolong and optimize the delivery of their beneficial \u0026ldquo;secretome [\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. The latter offers considerable advantages over intact cell-based therapies, including reduced immunogenicity, simplified manufacturing and storage via nontoxic cryopreservation and more straightforward handling for safety and dosing, similar to conventional pharmaceutical agents [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eMSC-derived mediators are secreted abundantly as soluble molecules and are also encapsulated within EVs of micro- and nanosized dimensions [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. EVs are derived from the late endosomal pathway and are characterized by specific membrane markers, such as tetraspanins [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Importantly, MSC-derived EVs carry a functionally competent molecular cargo [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] that remains protected from degradation until it reaches neighbouring target cells, thereby effectively delivering immunomodulatory and anti-inflammatory molecules [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eNumerous methodologies for EV isolation have been described extensively in recent studies [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The liver is a primary site for \u003cem\u003ein vivo\u003c/em\u003e EV uptake; although, other organs, such as the lungs, kidneys and spleen, also demonstrate considerable accumulation of EVs [\u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Consequently, the role of the liver in EV uptake has been the focus of multiple studies, employing various methodological techniques. Recent studies have revealed that engineered EVs featuring albumin-binding domains (ABDs) on their surface exhibit significantly improved cellular uptake in the liver [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], potentially enhancing the therapeutic efficacy although prolonged circulation time of EVs by preventing premature clearance from the bloodstream [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Additionally, vitamin A-coupled EVs (V-EVs) have been shown to selectively target activated hepatic stellate cells, effectively reversing liver fibrosis at lower doses compared with naive EVs [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Similarly, the systematic release of hydrogel-encapsulated MSC-EVs[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] has been shown to promote liver regeneration in chronic liver failure models [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe overarching scientific objective of this method is to improve the targeted delivery of therapeutic EVs into the liver in the context of AHF. Our previously published data in Gut 2012 [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], EbioMedicine 2019 [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], Hepatology 2022 [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], and iScience 2023 [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e] underscore the importance of the MSC \u0026ldquo;secretome\u0026rdquo; and EVs in AHF [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Therefore, EVs can be deployed as anti-inflammatory therapeutic agents for AHF. However, the nonspecific distribution of MSC-EVs following intravenous (i.v.) injection limits their efficacy [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. To address this limitation and enhance liver-specific therapeutic effects, we propose the development of activated liver-targeting albumin-coated EVs through a straightforward, efficient and clinically available surface engineering strategy. Here, we present an integrated ultracentrifugation-based protocol to achieve high yield, pure EVs, followed by albumin surface modification to enable liver targeting [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cb\u003eCells\u003c/b\u003e\u003c/p\u003e\n\u003cp\u003eOur laboratory routinely utilizes human amniotic fluid (AF) or umbilical cord (UC) MSCs as standard sources for the isolation and characterization of EVs [32]. A typical preparation involved three T175 flasks of 90% confluency, each containing approximately 18x10*6 cells at the time of harvest. The typical protein yield of EVs is approximately 100 µg. This technique can also be applied to different types of MSCs, including bone marrow (BM)-MSCs and their differentiated progenitor cells. However, the yield and purity of EV fractions may vary depending on the cell type, culture conditions, media composition and cell density. Additional variables, such as cell quantity and duration of serum starvation, also affect yield.\u003c/p\u003e\n\u003cp\u003e\u003cb\u003ePreparation of EV-depleted medium\u003c/b\u003e\u003c/p\u003e\n\u003cp\u003eA-MEM supplemented with 20% fetal bovine serum (FBS), 1% penicillin/streptomycin, and 1% L-glutamine was filtered through 0.22 µm filter. To eliminate EVs from FBS the medium was then centrifuged at 100,000 × g for 16 h (overnight) at 4°C using T-865 fixed angle rotor. The supernatant was carefully collected without disturbing the pellet, re-filtered (0.22 µm), and stored at 4°C until use.\u003c/p\u003e\n\u003cp\u003e\u003cb\u003eIsolation of EVs from cell-conditioned medium\u003c/b\u003e\u003c/p\u003e\n\u003cp\u003eWe present an optimized protocol for the isolation of EVs derived from MSCs, employing a combination of centrifugation, concentration, and filtration (Fig.\u0026nbsp;1a). Specifically, 3 x 175 T flasks of UC-MSC cells were cultured until 90% confluency with A-MEM supplemented with EV-depleted FBS at a final concentration 0.5% and 2.5 µg of BSA for 48 h. After 48 h, CM from all flasks (~ 50 ml) was collected. The first centrifugation was performed at 500 × \u003cem\u003eg\u003c/em\u003e for 5 min to remove dead cells and then at 2,000 × g for 10 min at 4°C to remove cellular debris. The supernatant was collected and centrifuged at 10,000 × g for 30 min at 4°C to exclude larger EVs. Finally, the medium was then centrifuged at 100,000 × g for 2 h at 4°C, and the resulting EV pellet was resuspended in PBS (1x) and subjected to a second ultracentrifugation at 100,000 × g for 2 h at 4°C. The EV pellet was ultimately reconstituted in PBS (1x) and stored at -80°C for subsequent use.\u003c/p\u003e\n\u003cp\u003e\u003cb\u003eCharacterization of isolated EVs\u003c/b\u003e\u003c/p\u003e\n\u003cp\u003eFollowing isolation, EVs were characterized in accordance with the guidelines established in MISEV2018 [20] to ensure the quality of EV fractions before downstream analyses. Various protocols are available to assess key characteristics, such as size, morphology, and ultrastructure, typically via electron microscopy. Additionally, the presence of EV marker proteins can be confirmed by standard immunoblotting or ELISA [20]. Nanoparticle tracking analysis (NTA) was performed to quantify particle number and size distribution of EVs, relying on light scattering. the use of detergents further improves the discrimination between EVs and non-EV molecules. As EV pellets may contain both EVs and non-EVs, caution must be taken when normalizing particle numbers for downstream analyses or functional assays [32]. Normalization based on protein quantification is the preferred method.\u003c/p\u003e\n\u003cdiv\u003e\n \u003cp\u003e\u003cb\u003ePKH26 labeling of EVs\u003c/b\u003e\u003c/p\u003e\n \u003cp\u003eFreshly isolated EVs by ultracentrifugation were labelled with PKH26 dye. In detail, EVs were resuspended at a concentration of 10 µg in 20 µL of 1× phosphate-buffered saline (PBS). In the EV sample was added 80 µL of Diluent C and the mixture was thoroughly resuspended by gentle pipetting to ensure uniform dispersion. In a separate tube, a PKH26 dye solution was freshly prepared by diluting 0.8 µL of PKH26 dye (final concentration 8 µM) in 100 µL of Diluent C. Equal volumes of the EV suspension and the dye solution were combined and incubated on ice for 1 to 5 minutes, with gentle mixing throughout to ensure uniform EV labeling. To terminate the staining reaction, 100 µL of 10% EV-depleted FBS in A-MEM was added. To minimize nonspecific binding, 1% bovine serum albumin (BSA) can be used as an alternative blocking agent. Importantly, the use of serum-free media or buffered salt solutions should be avoided during this step, as they can promote dye aggregation. The PKH26-labeled EVs were finally centrifuged at 100,000 × g for 2 h at 4°C using a T-865 fixed-angle rotor (Sorvall/WX 100+, MA, USA). After ultracentrifugation, the supernatant was carefully removed and kept as a negative control, and the labeled EV pellet was resuspended in 150 µL of 1× PBS for downstream applications.\u003c/p\u003e\n\u003c/div\u003e\n\u003cp\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cb\u003eAnimals\u003c/b\u003e\u003c/p\u003e\n\u003cp\u003eAll the animal experiments were performed in BRFAA facilities in compliance with the European Legal Framework (European Convention 123/Council of Europe and Directive 86/609/EEC), as well as the current guidelines of International Organizations (AAALAC Int., FELASA). The facility is registered as ‘‘breeding’’ and ‘‘experimental’’ facility (Reg. Numbers: EL BIO 01 and EL 25 BIO 03, respectively) according to the Greek Presidential Decree 56/2013, which harmonizes National Legislation with the European Directive 2010/63 on the protection of animals used for scientific purposes. The present protocol was approved by the Department of Agriculture and Veterinary Service of the Prefecture of Athens (Permit Number: 350028/20-04-22 and title: Cellular therapies in liver diseases Approval No.350028/20-04-22) and according to the recommendations of the FELASA and the NIH. The number of animals used per group was determined through power analysis (G*Power 3.1.9, effective size 0.4). Liver injury was assessed through histological examination of liver sections and through monitoring aspartate aminotransferase (AST) and alanine transaminase (ALT) serum levels at 48 hours posttreatment.\u003c/p\u003e\n\u003cp\u003e\u003cb\u003eEstablishment of an AHF Mouse Model, EV Administration, and Tissue Collection\u003c/b\u003e\u003c/p\u003e\n\u003cp\u003eSince 2012, our laboratory has developed a CCl₄-injured mouse model for AHF [30–32]. Female Rag1-/- mice aged 6–8 weeks and weighing 22‒26 g were intraperitoneally administered (i.p.) 100 µL of CCl₄ dissolved in sunflower oil (90 µL of sunflower oil per 20 g of mouse body weight, containing 10 µL of CCL₄). For negative controls, phosphate-buffered saline (1x) (PBS) in sunflower oil was used in place of CCl₄ (90 µL sunflower oil per 20 g of mouse body weight containing 10 µL PBS). On day 2, CCL₄-treated mice (n = 4) received an an intravenous (i.v). injection of 20 µg of PKH-26-labeled Albumin-EVs dissolved in 200 µL of PBS. Conversely, CCL₄-treated mice (n = 4), which served as controls, were i.v. injected with 20 µg of PKH-26-labeled naive EVs or unlabeled naive EVs in 200 µL of PBS. Intravenous injections were conducted via the tail vein. Two hours post-injection, mice were placed in a light-tight chamber, and photon emission from the labeling dye was detected using a cooled charge-coupled device (CCD) camera apparatus [Xenogen IVIS Lumina II System (Advanced Molecular Vision, Inc., UK)]. On day 3, the animals were euthanized, and serum were collected for transaminase level analysis. Liver tissue samples were also collected in 2 ml centrifuge tubes and snap-frozen in liquid nitrogen for further analyses.\u003c/p\u003e\n\u003cdiv\u003e\n \u003ch2\u003eStatistical analysis\u003c/h2\u003e\n \u003cp\u003eΤhe experimental data were presented as mean ± standard error of the mean (SEM) from three independent experiments. A one-way analysis of variance (ANOVA) was applied to determine statistically significant differences between groups. A p-value \u0026lt; 0.05 was considered statistically significant. The graphs presented were plotted using GraphPad Prism version 8.0.1 (GraphPad Software, San Diego, CA, USA).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eIsolation and characterization of EVs\u003c/b\u003e\u003c/p\u003e\u003cp\u003eEVs were isolated from UC-MSCs through differential centrifugation, as previously reported (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Transmission electron microscopy (TEM) confirmed their size, ranging from 100 to 150 nm, (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). In addition, Nanoparticle tracking analysis (NTA) revealed an average size (121.4 +/- 4.0 nm, 120.6 +/- 2.4 nm), as well as the average number of nanoparticles/mL PBS (2.75x10\u003csup\u003e10\u003c/sup\u003e +/- 4.74x10\u003csup\u003e9\u003c/sup\u003eparticles/ml, 4.44x10\u003csup\u003e8\u003c/sup\u003e +/- 1.07x10\u003csup\u003e7\u003c/sup\u003e particles/ml) in UC-MSC-EV and ALB-EV samples, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). Western blot analysis confirmed the presence of characteristic positive markers, including Alix, CD81, and CD9 in both EV populations (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eIn vivo\u003c/b\u003e \u003cb\u003ebiodistribution of ALB-PKH26-labeled EVs\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo assess biodistribution and homing capabilities, the isolated EVs were labeled with PKH26, a lipophilic fluorescent dye, and administered intravenously in a CCL\u003csub\u003e4\u003c/sub\u003e-induced AHF mouse model. Two hours post-injection ex vivo live imaging (Xenogen IVIS Lumina II System) demonstrated higher liver accumulation of modified ALB-EVs compared to na\u0026iuml;ve EVs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). Notably, ALB-EVs exhibited significantly reduced accumulation in other organs, as presented by lower fluorescent signal in the lungs and kidneys. These results suggest that albumin surface modification improves the liver-specific homing capacity of EVs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eFunctional improvement of liver injury after ALB-EVs\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAdministration of CCl\u003csub\u003e4\u003c/sub\u003e induced extensive inflammation, oxidative stress and hepatotoxicity. Liver function was assessed by measuring serum transaminase levels (AST and ALT). The results demonstrated that CCl₄ administration in \u003cem\u003ein vivo\u003c/em\u003e AHF-mouse model increased AST/ALT levels to 2,339.1\u0026thinsp;\u0026plusmn;\u0026thinsp;213.17 U/mL and 2,335.1\u0026thinsp;\u0026plusmn;\u0026thinsp;135.14 U/mL respectively, compared with healthy mice (99.5\u0026thinsp;\u0026plusmn;\u0026thinsp;11.99 U/mL and 100.16\u0026thinsp;\u0026plusmn;\u0026thinsp;10.93 U/mL, respectively), confirming liver injury and damage. Twenty-four hours after the CCL\u003csub\u003e4\u003c/sub\u003e injection, mice were treated with either UC-MSC-EVs or ALB-EVs. Both treatments resulted in decreased AST levels (977.167\u0026thinsp;\u0026plusmn;\u0026thinsp;406.33 U/mL and 858\u0026thinsp;\u0026plusmn;\u0026thinsp;280.93 U/mL respectively) and ALT levels (973.8\u0026thinsp;\u0026plusmn;\u0026thinsp;402.98 U/mL and 955\u0026thinsp;\u0026plusmn;\u0026thinsp;324.05 U/mL, respectively), compared with those in CCl\u003csub\u003e4\u003c/sub\u003e group (Fig.\u0026nbsp;5a). Additionally, ALB-EVs were more effective in lowering AST and ALT levels, indicating enhanced liver targeting and therapeutic efficacy. Statistical analysis performed by one-way ANOVA, revealed significant differences between the control and treated groups (\u0026lowast;\u0026lowast;p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, \u0026lowast;\u0026lowast;\u0026lowast;\u0026lowast;p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, ANOVA test). Macroscopic examination of liver morphology, demonstrated improved hepatic appearance in treated animals compared to the severely damaged livers of the untreated CCl₄ group (Fig.\u0026nbsp;5b). Histological evaluation with a haematoxylin and eosin (H\u0026amp;E) staining revealed severe liver damage, including necrosis and inflammatory infiltration in the CCl₄ group, which were significantly improved following UC-MSC-EVs and ALB-EVs (Fig.\u0026nbsp;5c) treatment.\u003c/p\u003e\u003cp\u003eFurther molecular analysis of inflammation-related pathways, particularly the expression of pro-inflammatory cytokines, such as TNF-α and IL-6, in liver tissues could provide valuable insight into the therapeutic potential of albumin-coated EVs in liver-related diseases requiring efficient tissue targeting and minimal immunogenicity.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we established and validated an efficient approach for liver-targeted delivery of EVs using surface decoration with albumin. Serum albumin, which is the most abundant plasma protein, exhibits favorable pharmacokinetics, liver tropism, and a long circulatory half-life, and thus it could be an ideal candidate for EV surface modification aimed at liver targeting [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThe CCl₄-induced AHF mouse model closely recapitulates features of human hepatic damage, including centrilobular necrosis, oxidative stress, lipid peroxidation, and marked elevations of serum transaminases (ALT and AST) [\u003cspan additionalcitationids=\"CR36\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Within twenty-four hours of CCl₄ administration, our experimental animals exhibited classical signs of acute hepatocellular injury, providing a relevant system for evaluating targeted EV biodistribution and therapeutic potential. Ex vivo live imaging, using the Xenogen IVIS Lumina II system, twenty-four hours after CCL\u003csub\u003e4\u003c/sub\u003e injection and two hours after treatment, demonstrated high efficacy in hepatic delivery of PKH26-labeled (ALB-EVs) compared to naive EVs. Additionally, this observation aligns with prior studies showing that albumin coating has demonstrated multiple advantages, such as improved interaction with lipid membranes, reduced cytotoxicity, enhanced cellular uptake via albumin-binding receptors (e.g., gp60, gp30, gp18, and SPARC), and extended circulation time in the bloodstream [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Compared to other methods requiring genetic modification or chemical conjugation [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], our albumin-coating strategy is technically straightforward, minimally disruptive and non-cytotoxic. The analysis of aminotransferases levels confirmed that administration of ALB-EVs was not associated with systemic toxicity. Instead, serum ALT and AST levels were modestly reduced compared to control, suggesting a hepatoprotective effect. While promising, this approach has limitations. In the process of isolating EVs from human MSCs, the possibility of co-isolating non-vesicular extracellular nanoparticles, including protein, nucleic acid complexes, cannot be entirely excluded [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. However, differential ultracentrifugation ensures that the resulting pellet is enriched in EVs, thereby attributing the therapeutic outcomes observed after administration to the animal models, primarily to the EVs themselves. Our methodology presents the use of albumin-coated EVs as a promising therapeutic platform that targets specifically the liver, although a portion of intravenously administered EVs may localize to other organs, such as the lungs and spleen, and could potentially diminish their therapeutic efficacy. Therefore, it is needed to optimize the dosage across various experimental models. Lastly, regarding the application of that method to clinical practice, the isolation of EVs will require rigorous characterization and Good Manufacturing Practice (GMP) compliance. Nonetheless, the ability to selectively enrich EV localization to the liver expands the utility of EVs as delivery vehicles for RNA, proteins, or drugs in hepatic disease. By coupling a widely available protein with a non-invasive coating strategy, our method offers a valuable platform for liver-targeted therapies in the context of acute liver injury and potentially in chronic liver conditions such as fibrosis or cirrhosis.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis optimized protocol enables effective isolation, labeling, and systemic delivery of EVs in a CCl₄-induced AHF mouse model. By ensuring consistent EV preparation and precise \u003cem\u003ein vivo\u003c/em\u003e administration, the method provides a reliable framework for studying liver-targeted EV biodistribution and their therapeutic potential in hepatic injury and regeneration.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eAHF \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Acute hepatic failure\u003c/p\u003e\n\u003cp\u003eEVs \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Extracellular vesicles\u003c/p\u003e\n\u003cp\u003eUC-MSCs \u0026nbsp; \u0026nbsp;Umbilical cord mesenchymal stromal/stem cells\u003c/p\u003e\n\u003cp\u003eCCl₄ \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Carbon tetrachloride\u003c/p\u003e\n\u003cp\u003eALB-EVs \u0026nbsp; \u0026nbsp; \u0026nbsp;Albumin-coated EVs\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eSupplementary Information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe online version contains supplementary material available\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions statements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA.S. conceived the study; designed the experimental methodology; performed the experiments; analysed, interpreted and visualized the data; wrote the manuscript; and provided final approval of the manuscript. M.L. performed the experiments; analysed, interpreted and visualized the data; and approved the final manuscript. K.P. and N.K. performed and supervised the \u003cem\u003ein vivo\u003c/em\u003e experiments and were responsible for animal licences and housekeeping; final approval of the manuscript. E.M. isolated MSCs from UC patients, analysed the data and approved the final manuscript. A.E. data analysis; financial support; edited manuscript; final approval of the manuscript. M.G.R. conceived the study; analysed and interpreted the data; provided financial support; designed the experimental methodology; supervised the research; wrote the manuscript; and provided final approval of the manuscript.\u003c/p\u003e\n\u003cp\u003eORCID for corresponding author:\u0026nbsp;M.R. 0001-5790-6581\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank Mr. Nikos Tsakiropoulos (Experimental Surgery, School Medicine, NKUA) for performing the biochemical analyses of mouse blood serum.\u0026nbsp;We are also grateful toProfessor Sophia Havaki, Laboratory of Histology and Embryology, Medical School, NKUA, for her assistance in exosome TEM imaging and Professor\u0026nbsp;Anastasios G. Kriebardis, Laboratory of Reliability and Quality Control in Laboratory Hematology (HemQcR), Department of Biomedical Sciences, Section of Medical Laboratories, School of Health \u0026amp; Caring Sciences, UniWA, for assistance with NTA analysis.\u0026nbsp;In addition, we would like to thank\u0026nbsp;Professor Vassilis G. Gorgoulis, (Laboratory of Histology and Embryology, Medical School, NKUA), and Professor Antonios Chatzigeorgiou (Laboratory of Physiology, Medical School, NKUA), for their assistance in histological processing of the samples.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Hellenic Foundation for Research \u0026amp; Innovation Grant during the 1st Call for H.F.R.I. Research Projects to Support Faculty Members \u0026amp; Researchers and Procure High-Value Research Equipment, no 70/3/16468, 2021-2024) to MR and the action “Flagship Research Projects in challenging interdisciplinary sectors with practical applications to Greek Industry”, implemented through the National Recovery and Resilience Plan Greece 2.0 and funded by the European Union - NextGenerationEU (project code: TAEDR-0541976) to AGE.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA.G. Eliopoulos is cofounder and CSO of GENOSOPHY S.A., a spin-off company at the National and Kapodistrian University of Athens.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePatient consent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEthics approval is provided in the respective sections (Materials and Methods]\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eChen P, Zhou YK, Han CS, Chen LJ, Wang YM, Zhuang ZM et al. Stem Cells From Human Exfoliated Deciduous Teeth Alleviate Liver Cirrhosis via Inhibition of Gasdermin D-Executed Hepatocyte Pyroptosis. Front Immunol. 2022;13.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWang H, Wang D, Yang L, Wang Y, Jia J, Na D, et al. 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Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.sciencedirect.com/science/article/pii/S0142961213009307?via%3Dihub\u003c/span\u003e\u003cspan address=\"https://www.sciencedirect.com/science/article/pii/S0142961213009307?via%3Dihub\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWelsh JA, Goberdhan DCI, O\u0026rsquo;Driscoll L, Buzas EI, Blenkiron C, Bussolati B et al. Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches. J Extracell Vesicles. 2024;13(2).\u003c/span\u003e\u003c/li\u003e\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":"Acute hepatic failure, Extracellular vesicles, Mesenchymal stromal cells, Liver targeting, Albumin coating, Regenerative medicine","lastPublishedDoi":"10.21203/rs.3.rs-7302374/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7302374/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cb\u003eBackground\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAcute hepatic failure (AHF) is a rapid deterioration of hepatocellular function, which includes coagulopathy and hepatic encephalopathy and it is related with high mortality rate. To date, liver replacement is the sole successful procedure for AHF with limitations related to donor organ shortage and lifelong immunosuppressive therapy. Mesenchymal stromal/stem cell \u0026ldquo;secretome\u0026rdquo;, including extracellular vesicles (EVs), offers considerable advantages over intact cell-based applications, such as lack of immunogenicity, standard manufacturing, and storage. However, targeted delivery of EVs to the liver still remains challenging for clinical translation.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMethods\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWe developed a novel protocol to generate liver-targeted EVs by coating EVs from umbilical cord MSCs (UC-MSCs) with albumin. EVs were isolated using ultracentrifugation and subsequently surface-modified with albumin to enhance hepatic targeting. AHF was induced in mice using carbon tetrachloride (CCl₄). The therapeutic efficacy of the albumin-coated EVs was evaluated via serum biomarker measurements and live imaging.\u003c/p\u003e\u003cp\u003e\u003cb\u003eResults\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe albumin-coated EVs (ALB-EVs) demonstrated higher liver bioaccumulation and reduced biodistribution to the other organs compared to naive EVs. CCL\u003csub\u003e4\u0026minus;\u003c/sub\u003einduced AHF mice treated with liver specific EVs exhibited significantly decreased levels of aminotransferases and improved liver morphology and histological features. The entire protocol, from EV isolation to treatment, can be completed within one week using standard laboratory techniques.\u003c/p\u003e\u003cp\u003e\u003cb\u003eConclusions\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThis method provides an efficient, reproducible strategy for liver-targeted EV-based therapy in acute hepatic failure. Albumin-coated EVs represent a promising and scalable platform for developing noncellular therapeutics in regenerative medicine.\u003c/p\u003e","manuscriptTitle":"Liver-Targeted Fetal MSC-Derived Extracellular Vesicles: A Therapeutic Approach in a Mouse Model of Acute Hepatic Failure","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-12 12:49:55","doi":"10.21203/rs.3.rs-7302374/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":"4867f8a0-d1a3-4438-91bc-b63de905631f","owner":[],"postedDate":"August 12th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-08-19T01:08:19+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-12 12:49:55","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7302374","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7302374","identity":"rs-7302374","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}