Enhanced Therapeutic potential of AEBP1 silencing via engineered Apoptotic Mesenchymal Stem Cell- derived Nanovesicles in Atrial Fibrillation

Enhanced Therapeutic potential of AEBP1 silencing via engineered Apoptotic Mesenchymal Stem Cell- derived Nanovesicles in Atrial Fibrillation | 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 Research Article Enhanced Therapeutic potential of AEBP1 silencing via engineered Apoptotic Mesenchymal Stem Cell- derived Nanovesicles in Atrial Fibrillation Gyeongseo Yoo, Ji-Young Kang, Malgeum Park, Jaewoong Lee, Dasom Mun, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7061113/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 Chronic inflammation and fibrosis contribute markedly to the progression and recurrence of Atrial Fibrillation (AF), which is the most common form of arrhythmia. Despite ongoing therapeutic advancements, effective treatments to modulate inflammatory and fibrotic processes in AF remain limited. To overcome these limitations, we developed a novel nanotherapeutic system using apoptotic mesenchymal stem cell-derived nanovesicles (ANV) as biocompatible and immunomodulatory delivery platforms for siRNA targeting the AEBP1 gene, leading to concurrent attenuation of fibrosis. Results ANV were constructed via an extrusion method and loaded with adipocyte enhancer binding protein 1 (AEBP1)-targeting small interfering RNA (siRNA) (siAEBP1) through electroporation to form ANV-siAEBP1. ANV-siAEBP1 were then incubated with antibody-conjugated iron oxide magnetic nanoparticles (MNP), forming ANVP-siAEBP1 complex. For targeted delivery to the AF myocardium, an anti-myosin light chain 3 (MLC3) antibody was incorporated to facilitate localized accumulation in MLC3-enriched atrial cardiomyocytes. Once localized, the nanovesicles fused with cardiomyocyte membranes, allowing for the intracellular release of siAEBP1, which in turn silenced AEBP1 expression, thereby downregulating pro-fibrotic signaling and mitigating atrial fibrosis. Simultaneously, the intrinsic anti-inflammatory effects of ANV in stressed cardiomyocytes, prevented excessive inflammatory responses. Conclusions This dual mechanism of action, involving siRNA-mediated gene silencing and ANV-induced immunomodulation, results in a synergistic therapeutic effect. Thus, ANVP-siAEBP1 significantly attenuated both inflammation and fibrosis in AF myocardium with enhanced targeting efficiency, offering a promising strategy for next-generation precision therapeutics in AF. Atrial fibrillation Engineered apoptotic nanovesicles Inflammation Fibrosis AEBP1 silencing Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 INTRODUCTION Atrial fibrillation (AF) is one of the most common form of arrhythmia, with a wide spectrum of symptoms, and its incidence is increasing globally.(1) Experimental and clinical data indicates that inflammation can get induced in patients with AF, which in turn, can contribute to the progression of AF, leading to clinical phenomenon described as ‘AF begets AF’ . This can ultimately lead to severe atrial fibrosis.(2, 3) However, existing anti-arrhythmic drugs and emerging therapeutic strategies (biological approaches), exhibit limitations in target specificity, duration of action, and overall efficacy in mitigating the progression of AF.(4–6) Therefore, the development of an advanced therapeutic system capable of precise targeting and effective treatment of AF is imperative. Mesenchymal stem cell (MSC) therapy is widely used owing to its ability to rapidly respond to cellular injury and migrate to the site of damage, promoting tissue repair and regeneration.(7) MSC-derived nanovesicles are emerging as promising carriers capable of delivering therapeutic agents to specific cells and tissues. Studies have shown that these nanovesicles can enhance cardiac repair and help overcome the challenge of low cell survival in myocardial infarction,(8, 9) and myocardial ischemia/reperfusion.(10) Healthy MSC-derived nanovesicles are widely studied but the therapeutic potential of the apoptotic MSC-derived nanovesicles in AF remains unclear. Apoptotic MSC-derived nanovesicles (ANV) have been shown to express proteins associated with tissue repair and exhibit reduced levels of pro-inflammatory proteins, suggesting a potential role in modulating inflammation.(11) This minimizes the risk of inflammation and promotes controlled healing, making ANV effective for targeted therapy.(12–14) Therefore, the immunosuppressive properties of MSCs can be enhanced through apoptosis, which facilitates the retention of various cytoplasmic factors. To enhance the therapeutic efficacy, precise targeting of the desired site is essential. To date, the majority of drugs utilized for tissue injury repair have been delivered through either systemic or local administration.(15) Magnetic nanoparticles are widely used for precise targeting and molecular delivery, and their modification for enhanced specificity has already been studied.(16, 17) anti-CD63 antibody was conjugated to the magnetic nanoparticle to capture nanovesicles and anti-MLC3 antibody was conjugated to the target AF heart. MLC3 , a gene expressed in cardiomyocytes, is a potential contributor to mid-left ventricular chamber-type hypertrophic cardiomyopathy (HCM). Recent studies have identified MLC3 variants in patients with HCM, particularly in those carrying mutations in sarcomeric protein genes associated with heart failure and AF.(18, 19) Beyond its genetic association with cardiomyopathy, MLC3 has emerged as a selective marker of injured cardiomyocytes. Consequently, it has been used as a molecular target to enhance delivery to injured cardiomyocytes in various injury models, including myocardial infarct,(16) ischemic cardiomyopathy.(17, 20) However, the use of anti-MLC3 antibody strategies to specifically target AF remains underexplored. AEBP1 , an isoform of aortic carboxypeptidase-like protein,(21) is an extracellular protein involved in the remodeling of the extracellular matrix and promotes macrophage inflammatory responsiveness.(22, 23) Recent experimental evidence has shown that overexpression of AEBP1 may function in cardiac fibroblast activation, which can trigger the release of pro-fibrotic factors.(24, 25) Consistent with these findings, we observed a marked increase in AEBP1 expression in patients with AF. Thus, silencing AEBP1 gene expression may serve as a key therapeutic approach to inhibit pathways implicated in fibrosis,(22) thereby preventing cardiac fibrosis and ultimately enhancing cardiac function. Small interfering RNA (siRNA) is a promising therapeutic agent that offers a novel approach to selectively downregulate the expression of specific proteins.(26–29) Considering these characteristics, siRNA therapeutics have great potential in the treatment of cardiovascular disease.(30, 31) Despite the therapeutic potential of siRNA-based treatments, several challenges remain, particularly in the context of efficient intracellular delivery. Therefore, there is a pressing need to develop more efficient and biocompatible delivery strategies that can overcome current limitations and enable the clinical translation of siRNA therapeutics. In this study, we developed a novel nanoparticle-based therapeutic platform by encapsulating siAEBP1 within ANV and conjugating them with engineered magnetic nanoparticles to generate ANVP-siAEBP1 (Scheme. 1). ANVP can transport therapeutic agents while simultaneously conferring intrinsic therapeutic effects via their bioactive components. The conjugation of magnetic components enhances the targeting efficiency to the atrial myocardium via external magnetic guidance, while also enabling preferential accumulation through anti-MLC3 antibody to MLC3-enriched cardiomyocytes, which were found to exhibit elevated expression levels in the AF myocardium in our study. Once delivered, siAEBP1 suppressed fibrosis-associated signaling pathways by downregulating AEBP1, thereby effectively preventing fibrotic remodeling in the atrial tissue. Simultaneously, ANV themselves contribute to the anti-inflammatory effects in stressed cardiomyocytes. Through this integrated approach, ANVP-siAEBP1 offers a synergistic therapeutic effect that is anticipated to surpass the efficacy of conventional drug treatments for AF. MATERIALS AND METHODS Cell culture and Staurosporine (STS) treatment, Ang Ⅱ treatment hMSC culture and Staurosporine (STS) treatment. Human Mesenchymal Stem Cells Derived from bone marrow (hMSC) (ATCC, Manassas, VA, USA) were cultured in Dulbecco's Modified Eagle's Medium, Low Glucose (DMEM; LM001-11, Welgene, Gyeongsan, Korea) supplemented with 10% fetal bovine serum (FBS; USFBS-500, Young In Frontier, Seoul, Korea) and 1% penicillin-streptomycin (10378016, Gibco, Grand Island, NY, USA) at 37°C in a humidified atmosphere with 5% CO 2 . To induce apoptosis, hMSCs were treated with 0.25 µM Staurosporine (STS) once the cells reached 80–90% confluency (569397, Sigma) for 6h. iPSC-aCM culture and Angiotensin Ⅱ (Ang Ⅱ) treatment. The human induced pluripotent stem cell (iPSC) line CMC-hiPSC-011 was provided by the Korea National Stem Cell Bank, originally provided by Catholic University. Cells were cultured in the TeSR™-E8™ medium (STEMCELL Technologies) on vitronectin (Gibco)-coated plates at 37°C in 5% CO 2 and subcultured every 4–6 d with 10 µM Y27632 (Sigma). Through modulation of wingless-related integration site signaling, iPSCs were differentiated into iPSC-derived atrial cardiomyocytes (iPSC-aCMs), following previously described protocols with minor modifications.(32) Briefly, iPSCs began to differentiate at 80–90% confluence in Materigel (354277, Corning, Arizona, USA)-coated plates with cardio differentiation medium consists of RPMI 1640 (11875119, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with B-27 minus insulin (A1895601, Thermo Fisher Scientific) and stepwise treatment with 4 µM CHIR99021 (4423, Tocris Bioscience, Bristol, UK) for 72 h and then 2 µM Wnt-C59 (5148, Tocris Bioscience) for 48 h. On day 5 to 7 of differentiation, 1µM of RA (R2625, Sigma-Aldrich; Merck KGaA) was added for 72h. The medium was replaced with cardio culture medium composed for RPMI 1640 supplemented with B-27 (17504001, Thermo Fisher Scientific) on day 9. To mimic AF in vitro , iPSC-aCMs were treated with 0.5 µM Ang II (A9525; Sigma-Aldrich; Merck KGaA) for 24 h. Cell viability assay. Cell viability was assessed using Quanti-Max™ WST-8 Cell Viability Assay Kit (QM2500, Biomax Inc, korea) according to the manufacturer’s instructions. hMSCs were seeded in a 6-well plate and treated with different concentration of STS at multiple time point. Subsequently, cells were treated with Quanti-Max ™ for 4h in 37°C CO 2 incubator. For absorbance measurement the medium were transferred to 96 well plate and measured at a wavelength of 450 nm using a microplate reader (VersaMax). Apoptosis measurement using Fluorescence Activated Cell Sorting (FACS) assay. Apoptosis was analyzed using FITC-Annexin V Apoptosis Detection kit Ⅱ (556570, BD Biosciences, San Jose, CA, USA) according to the manufacturer’s instructions. Cells were harvested using 0.25%, 1x trypsin- ethylenediaminetetraacetic acid (LS015-10, Welgene) and washed twice with cold PBS. The pellet was resuspended in the 1x binding buffer, FITC Annexin V and PI were added sequentially. The cells were incubated for 15 min in the dark at RT and binding buffer was additionally supplemented. Right after incubation, cells were analyzed within 1 hr using a BD® LSR II SORP (BD Biosciences), and the percentage of apoptotic cells was determined using the BD FACS DIVA software. DBCO-IONP conjugation. Conjugation of DBCO-sulfo-NHS with IONPs was conducted by mixing 0.5 mg/mL amine (-NH₂)-functionalized iron oxide (IONPs; Sigma-Aldrich, 5 mg/mL, molecular weight 231.53 g/mol) solution with the 2.16 µmol DBCO-sulfo-NHS (Peptron Co., Daejeon, South Korea) DBCO solution at a 1:1 molar ratio and incubated on a rotating mixer at room temperature (RT) for 4 hours. To remove any unconjugated DBCO-sulfo-NHS, the mixture was then centrifuged at 10,000 × g for 10 minutes. 3 µM DBCO-sulfo-NHS (Peptron Co., Daejeon, South Korea) was mixed with 0.5 mg/mL amine (-NH₂)-functionalized iron oxide (IONPs; Sigma-Aldrich, 5 mg/mL) and the samples were incubated at room temperature (RT) on a rotating mixer for over 4 hours. To remove any unconjugated DBCO-sulfo-NHS the mixture was centrifuged at 10,000 x g for 10 minutes. Enzymatic modification of antibodies. Anti-CD63 (E-12) antibody (sc-365604, Santa Cruz, 0.5 mg mL − 1) and anti-Myosin light chain 3 (ab233220, Abcam, 0.2 mg mL − 1) were selectively modified at defined sites on the Fc region with an azide using the Site Click™ Antibody Azido Modification Kit (Thermofisher). In short, 100 µg of Anti-CD63 antibody, and 100 µg of anti-Myosin light chain 3 antibody were loaded into an antibody concentrator and centrifuged at 5000 x g for 6 minutes. After discarding the flow-through, 450 µL of antibody preparation buffer was added to the sample and centrifuged at 5000 x g for 8 minutes. To remove galactose residues, galactosidase was added to the antibody solution and incubated overnight at 37°C. Additionally, the azide group was enzymatically conjugated to the carbohydrate modified antibody. To initiate the reaction, Tris buffer, buffer additive, and GalT (Y289L) enzyme were added to a tube containing UDP-GalNAz, along with the carbohydrate-modified antibody, and incubated overnight at 30°C. For removing excess UDP-GalNAz, the solution was washed with 1 mL of Tris buffer and centrifuged at 1200 x g for 10 minutes. The final azide-modified antibody (∼40 µL, 2.6 mg mL − 1) was stored at 4°C before further experiments. DBCO-IONP-Antibody conjugation. To enable conjugation via click chemistry between DBCO and azide (-N₃) moieties as established in previous studies, 0.5 mg/mL DBCO-IONPs were incubated with 17.5 µg of azide-functionalized antibodies (1 mg/mL) overnight at 4°C with continuous rotation. Unconjugated antibodies were subsequently removed by centrifugation at 10,000 × g for 10 minutes, and the conjugates were stored at 4°C until further use. Preparation of Apoptotic cell derived nanovesicles conjugated with DBCO-IONP-antibody (ANVP). Once the human bone marrow hMSCs (ATCC, Manassas, VA, USA) reached ~ 90% confluence in 150 mm 2 culture dishes, the cells were harvested from the dishes by cell scraper and centrifuged at 270 x g for 5 min. The cells were subjected to sonication at 100 V with 20% power for two rounds of six cycles each, where each cycle consisted of a 4-second pulse followed by a 2-second pause. Between and after sonication, the cells were incubated on ice to minimize thermal effects. After sonication was completed, samples were centrifuged at 4°C, 800 x g, 10 min to remove cell debris. To obtain the MSC-derived nanovesicles (NVs) and Apoptotic MSC-derived nanovesicles (ANVs), sonicated hMSCs were suspended in phosphate buffered saline (PBS), and serially extruded 8 times through 5-µm, 1-µm, 0.4-µm and 0.2-µm pore sizes polycarbonate membrane filters using a mini extruder (Avanti polar Liquids, Birmingham, USA). For density-gradient ultracentrifugation, the 2 mL ultracentrifuge tube was layered sequentially with 50% iodixanol (optiprep, Axis-shild, Oslo, Norway) at the bottom, followed by 10% iodixanol, and the sample was loaded on top and centrifuged at 100,000g for 2h at 4°C using microcentrifuge (Himac CS120GXL; Hitachi, Tokyo, Japan). The resulting pellet was carefully resuspended in cold phosphate-buffered saline (PBS) and washed twice with cold PBS using an Amicon Ultra-4 Centrifuge Filter (UFC8010; Millipore Corporation, Bedford, MA, USA) at 3,500 x g for 40 min at 4°C. The vesicle solution was stored at 4°C when not used according to the manufacturer’s instructions. Extracted ANV were combined with DBCO-IONP-antibody (hereafter referred to as MNP) and they were incubated at a 1:1 ratio via anti-CD63 interactions overnight at 4°C with rotation, resulting in the formation of ANVP. To remove unconjugated ANV and MNP, the mixture was performed at 10,000 x g for 10 minutes and stored at 4°C until needed. Characterization of ANVP. The morphology of the obtained ANVP was observed through transmission electron microscopy (TEM) (JEM-1011., JOEL Ltd., Tokyo, Japan). The zeta potential of the ANVP was evaluated with ELS-1000ZS (Otsuka Electronics, Osaka, Japan). The mean size and particle concentration of ANVP were analyzed via the nanoparticle tracking assay (NTA) using a NanoSight LM10 instrument (Malvern, Worcestershire, UK) according to the manufacturer's instructions and it was examined using NTA v.2.3 software (Malvern Panalytical, Ltd., Malvern, UK). The intermediates and final products were analyzed using Fourier transform infrared spectrometer (INVENIO., BRUKER., Texas, USA). Western blot analysis. Total protein was lysed using the EzRIPA Lysis Kit (WSE-7420, ATTO CORPORATION, Korea) with protease and phosphatase inhibitors. The protein concentration was determined using Pierce™ 660 nm Protein Assay Reagent (22660, Thermo Fisher Scientific). Proteins were separated by 10% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto PVDF membranes (IPVH00010; EMD Millipore, Bedford, MA, USA). The membranes were blocked with 5% BSA in TBS-Tween 20 (TBS-T) for 40–50 min at RT and incubated overnight at 4°C with the following primary antibodies: CD81 (1:1000, sc-166029, Santa Cruz Biotechnology), CD63 (1:1000, sc-365604, Santa Cruz Biotechnology), Lamp (1:1000, sc-18822, Santa Cruz Biotechnology), AEBP1 (1:1000, sc-271374, Santa Cruz Biotechnology) and GAPDH (1:1000, sc-25778, Santa Cruz Biotechnology). GAPDH was used as a loading control. The membranes were washed five times with TBS-T, and incubated with horseradish peroxidase-conjugated secondary antibodies (sc-2357, 1:5000; Santa Cruz Biotechnology) for 1 h. Immunoblots were visualized using an enhanced chemiluminescence detection kit (1705061; Bio-Rad Laboratories Inc., Hercules, CA, USA) and scanned using a model LAS4000 Mini Luminescent Image Analyzer (FUJIFILM, Tokyo, Japan). Band density of the experiment was quantified using ImageJ software. Study population. All procedures involving human participants were approved by the Institutional Review Board of Severance Hospital, Yonsei University (IRB No. 4-2019-0620), and written informed consent was obtained from each subject. Human tissue was obtained from patients without AF ( n = 5) and patients with AF ( n = 5) at the Yonsei University Health System (Seoul, Korea) from August 2019 to July 2021, and the clinical profiles of patients are listed in Table S1 . The two groups did not differ significantly in terms of age or body mass index (BMI). RNA isolation, cDNA synthesis and Quantitative RT-PCR (qRT-PCR). Total RNA was isolated from iPSC-aCMs, patient heart tissue and mice heart tissue using the miRNeasy® Mini Kit (217004, Qiagen, Hilden, Germany) according to the manufacturer’s protocol. For cDNA synthesis, cDNA synthesis kit (600036, Agilent Technologies, Santa Clara, CA, USA) and High-Capacity cDNA Reverse Transcription kit (4368814, Applied Biosystems, Darmstadt, Germany) was used. qRT-PCR was carried out using the PowerUp™ SYBR™ Green Master Mix (A25742, Applied Biosystems) on QuantStudio™ 3 Real-Time PCR System (A28567, Thermo fisher, USA). PCR primers were synthesized by Cosmogenetech (Daejeon, Korea) and all primer sequences are listed in Table S2, S3. The relative expression of mRNA was quantified using the ΔΔCt method, and calculated using 2 −ΔΔCq method and normalized against GAPDH.(33) siRNA transfection . iPSC-aCMs were seeded in 6-well plate and when it reached approximately 70–80% confluency, the cells were transfected with Lipofectamine™ RNAiMAX (13778150, Thermo Fisher Scientific) according to the manufacturer’s instructions. The negative control and AEBP1 siRNA were synthesized by Bioneer (Daejeon, Korea). Cells were incubated for two days in Cardio Culture Medium after transfection and harvested for subsequent analyses. Loading of siRNA into ANVP. ANV were mixed with siAEBP1 (bioneer, korea) or Negative Control siRNA at the ratio of 1:1 (wt/wt) and the mixture were electroporated using a Neon™ Transfection System (Thermo Fisher Scientific) at 1000 V, 10 ms, 2 pulses, to form ANV-siAEBP1 or ANV-NC. After siRNA loading, the transfected ANV-siAEBP1 and ANV-NC were incubated with 100 µg/ml RNase A (Thermo Fisher Scientific) for 30 min at 37°C to degrade unloaded siRNA. Thereafter, the mixture was washed twice with cold PBS using an Amicon Ultra-4 Centrifuge Filter (UFC8010; Millipore Corporation, Bedford, MA, USA) at 3,500 x g for 40 min at 4°C. Fluorescence labeling of ANVP and Immunocytochemistry. Isolated ANV and ANVP were labeled with the PKH67 green Fluorescent Cell Linker Kit (MINI67, Sigma-Aldrich; Merck KGaA) according to the manufacturer’s instructions. iPSC-aCMs were incubated with PKH labeled PBS or ANV or ANVP for 24 hours and subsequently fixed with cold 4% paraformaldehyde. MLC3 antibody (ab233220, 1:200, Abcam) was treated to cells and incubated at 4°C overnight. Next, cells were incubated with a secondary antibody (mouse anti-rabbit IgG-CFL; sc-516251, 1:5000; Santa Cruz Biotechnology) for 1h. After five times of washing with PBS, 4′,6-diamidino-2-phenylindole (DAPI) was incubated for 7 min to label the nuclei. To visualize ANVP conjugation to the cell membrane, magnetic nanoparticles (MNP) were labeled with Cy5.5 and incubated with ANV to generate Cy5.5-ANVP. Treated cells were fixed as described above, and nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Confocal images were acquired using a Zeiss LSM 710 confocal microscope (Carl Zeiss, Jena, Germany). Immunocytochemistry. iPSC-aCMs were fixed with cold 4% paraformaldehyde, permeabilized and blocked with DPBS containing 0.3% Triton X-100 and 5% bovine serum albumin. The slides were incubated overnight at 4°C with the following antibody; fluorescence-conjugated α-actin (sc-17829, 1:200; Santa Cruz Biotechnology). After five times of washing with PBS, 4′,6-diamidino-2-phenylindole (DAPI) was incubated for 7 min to label the nuclei. Anti-α-actin treated cells were washed and treated with 4′,6-diamidino-2-phenylindole (DAPI) for 7 min. The stained samples were observed using a Zeiss LSM 710 confocal microscope (Carl Zeiss, Jena, Germany). Animal experiments. All animal experiments were under ethical approval of Institutional Animal Care and Use Committee of Yonsei University College of Medicine (approval no. 2024 − 0222) and adhered to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (Publication No. 85 − 23, revised 1996). C57BL/6 male mice were purchased from Orient Bio Inc. (Seongnam, Korea), allowed unrestricted access to food and water and maintained at standard conditions (temperature, 20 ± 0.5 ◦C; humidity, 60 ± 5%; light/dark cycle, 12 h). The mice went through anesthetization with intraperitoneal injections of tiletamine-zolazepam (Zoletil® 50, 30 mg/kg) and xylazine (Rompun®, 10 mg/kg) mixed at a 1:1 ratio and implanted with Ang II-containing Alzet® 1002 micro-osmotic pumps (2 mg/kg/day; Durect Corp., Cupertino, CA, USA). The control mice were implanted with Alzet® 1002 micro-osmotic pumps containing PBS. The mice were divided into 4 groups. PBS-pump implanted group was injected with PBS, and Ang Ⅱ-pump implanted mouse group were each injected with PBS, ANVP-NC, or ANVP-siAEBP1 (at a 1:1 ratio of NC and siAEBP1 to ANVP, 300 µg each per mouse). The magnet (Ø 5mm, IV-MAG3) was applied for 30 minutes after the injection of the samples to evaluate the effect of the targeted delivery. Two days after final injection, the mice were sacrificed and the blood, heart, liver, spleen, kidney were collected. Serum isolated from blood underwent hemogram analysis and the level of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and albumin (ALB) evaluated using Fuji Dri-Chem 4000i (Fujifilm, Tokyo, Japan) according to the manufacturer’s instructions. The collected organs were used for RNA extraction, cDNA synthesis, qRT-PCR, ELISA, and immunohistochemical staining. Immunohistochemical staining. For immunohistochemical observation, the tissues were dissected, fixed in 4% paraformaldehyde, followed by paraffin embedding and sectioning into 4-µm-thick slices. To analyze the expression of MLC3 in AF mice cardiomyocytes, anti-MLC3 antibody (ab233220, Abcam) was used for primary antibody and mouse anti-rabbit (sc-516250, Santa Cruz Biotechnology) antibody was used for fluorescent secondary antibody and the nuclei was stained using DAPI (62248, Thermo Fisher Scientific). Histological analyses were performed using H&E staining to evaluate tissue pathology, iNOS (ab15323, Abcam) staining with DAB (3,3'-diaminobenzidine) to assess inflammatory responses and Masson's Trichrome (MT) for the identification of fibrotic areas. The nuclei were labeled with hematoxylin. Anti-AEBP1 (sc-271374, Santa Cruz Biotechnology) was incubated with Alexa Fluro® 488 Conjugation Kit (ab236553, Abcam) and stained to evaluate the fluorescent of AEBP1 expression. After, AEBP1 was co-stained with cardiac troponin I antibody (ab47003, Abcam) and subsequently incubated with secondary antibody (mouse anti-rabbit IgG-CFL 647, sc-516251, Santa Cruz). The tissues were observed under an inverted microscope (Olympus, Japan) and Zeiss LSM 710 confocal microscope (Carl Zeiss, Jena, Germany). To analyze fibrotic area and iNOS expression, quantification using the Image J 1.50i software (National Institutes of Health, Bethesda, MD, USA) was conducted and the quantification of MLC3 was measured using confocal microscope (Zeiss LSM 710, Carl Zeiss). In vivo biodistribution. To visualize the biodistribution, ANV and ANVP were labeled with Cy5.5. At 4h after tail vein administration (300ug of ANV and ANVP per mice), animals were euthanized and the major organs (heart, lung, liver, spleen, and kidney) were imaged using IVIS ® spectrum in vivo imaging system (PerkinElmer, Waltham, MA, USA). Echocardiography analysis. Cardiac function was evaluated by performing echocardiology using the Vevo 2100 system (VisualSonics, Toronto, Ontario, Canada). Left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) were assessed by calculating the ejection fraction (EF) and fractional shortening (FS) according to the following formulas: LVEF (%) LVEF (%) = [(left ventricular end-diastolic volume (LVEDV) ‒ left ventricular end-systolic volume (LVESV))/LVEDV] × 100; LVFS (%) = [(left ventricular end-diastolic diameter (LVEDD) ‒ left ventricular end-systolic diameter (LVESD))/LVEDD] × 100. Statistical analysis. Statistical analysis between two groups which meets the assumption of homoscedasticity were evaluated using Student's t-test and more than two groups exhibiting homoscedasticity were analyzed using the one-way analysis of variance with Tukey's post hoc test. Data that did not meet the assumption of homoscedasticity were analyzed using the Holm–Sidak method. GraphPad software 8.0 was used for statistical analysis and p values < 0.05 were considered statistically significant. Results Synthesis and characterization of magnetic engineered Apoptotic MSC-derived nanovesicles. ANV were initially generated to minimize the inflammatory risk, promote tissue healing and simultaneously function as bioactive vesicles (Fig. 1 A). To induce apoptosis in MSCs, cells were treated with varying concentrations of Staurosporine (STS) at multiple time points. The morphology of the cells began to shift after STS treatment (Figure S1 A). Time- and concentration- dependent viability tests were conducted. Viability began to decrease from 0.25 µM, and for the 6h treatment, concentrations higher than 0.25 µM did not show a significant difference in cytotoxicity (Figure S1 B). The level of apoptosis in MSCs at 6h post 0.25µM of STS treatment was assessed by FITC-Annexin/PI double staining and flow cytometry. After 6h treatment, the total percentage of early apoptotic increased to 13.4%. and late apoptosis increased to 4.2% (Figure S1 C). Based on these results, we selected a treatment with 0.25 µM STS for 6 hours to generate apoptotic MSCs for subsequent experiments. Following STS treatment, apoptotic MSCs were introduced by serial extrusion through membrane filters with diminishing pore sizes, thereby mechanically disrupting the plasma membrane of cells (34) and generated ANV. ANV were functionalized with MNP conjugated to anti-CD63 antibodies for vesicle capture and anti-MLC3 antibodies to enhance the specificity and binding affinity toward AF myocardial tissue.(35, 36) (Fig. 1 A). ANVP characterization was conducted to confirm successful MNP conjugation and the preservation of native nanovesicle properties. The Fourier Transform Infrared Spectroscopy (FT-IR) data revealed the expected characteristic peaks of functional groups from each synthetic step, including modification of the IONPs (Fe 3 O 4 ) (~ 500–600 cm -1 ), IONP-DBCO (~ 1000–1300 cm -1 ), IONP-DBCO modifications with anti-CD63 (~ 1650 cm -1 ) and anti-MLC3 (~ 1540 cm -1 ) antibodies, as well as the final conjugation with ANV, which exhibited peak shift around ~ 1200–1400 cm -1 (C-O-C bond) (Fig. 1 B). Transmission electron microscopy (TEM) images revealed that NV and ANV exhibited spherical shapes of similar sizes, consistent with previous reports.(36) The morphology of ANVP was observed to have multiple MNP conjugated to their surface (Fig. 1 C). The average zeta potential of NV was − 18.65 mV, ANV was − 23.94 mV, and ANVP was − 28.26 mv which is similar to exosomes and nanovesicles in our previous study (Fig. 1 D).(36) We used nanoparticle tracking analysis (NTA) to assess the size distribution of ANVP. As shown in Fig. 1 E, NV and ANV exhibited the most frequent particle size distributions (modes) of 111.6 nm and 116.8 nm. After conjugation with MNP, ANVP showed 166.8 nm particle size. These experimental results demonstrated that MNP were successfully conjugated to ANV, leading to the formation of ANVP. Moreover, 1 month of storage in -20°C and − 80°C had no significant effect on the average particle size of ANVP consistent with previous reports (Figure S2).(37) Western blot analysis revealed that CD81, CD63, and Lamp2 are enriched in NV, ANV and ANVP showing ANVP possess the same features as typical nanovesicles (Fig. 1 F). These results confirm that ANVP was successfully modified and exhibited the characteristic features of nanovesicles, making them suitable delivery vehicles. Targeting ability of ANVP toward AF cardiomyocyte in vitro . To evaluate the potential of MLC3 as a target in AF, we assessed its expression in patient-derived AF tissue samples and in vitro models. MLC3 mRNA expression analysis of tissue samples collected from patients in the control ( n = 5) and AF groups ( n = 5) revealed a statistically significant difference between the two groups ( p < 0.01) (Fig. 2 A). The clinical profiles of the control and AF groups are listed in Table S1 , and no significant differences in age or body mass index were observed between the two groups. To examined whether MLC3 are upregulated in atrial cardiomyocytes, we treated Angiotensin Ⅱ (Ang Ⅱ) to human induced pluripotent stem cell-derived atrial cardiomyocytes (iPSC-aCMs) based on the previous studies.(38, 39) Briefly, 0.5 µM or 1 µM of Ang Ⅱ were treated to iPSC-aCMs for 24 hours, and cell area based on α-actin staining was assessed through immunocytochemistry staining. An increase in cell area was observed following drug treatment, which appears to be a consequence of AF progression in cardiomyocytes (Figure S3A, B).(40) Therefore, Ang Ⅱ concentration of 0.5 µM was chosen for subsequent experiments. MLC3 mRNA expression was augmented in Ang Ⅱ-treated iPSC-aCMs ( p < 0.0001) (Fig. 2 B) and exhibited a notable increase in relative fluorescence intensity ( p < 0.001) compared to the control group (Fig. 2 C, D). Our experimental findings demonstrate that MLC3 is significantly overexpressed in the Ang II-treated iPSC-aCM model, which mimics AF pathology. This observation was corroborated by overexpression detected in the cardiac tissues of patients with AF. Our results suggest that elevated MLC3 levels in AF tissues and iPSC-aCMs may provide a new avenue for selective targeting of atrial cardiomyocytes affected by AF. To verify whether the anti-MLC3 antibody facilitated the binding of ANVP to the cellular membrane, we conjugated Cy5.5 to the MNP, followed by conjugation with ANV, and administered it to the cells under experimental conditions. Fluorescent signals were detected on the cell surface, indicating successful membrane localization of the labeled ANVP (Figure S4). To further evaluate whether the improved binding affinity of ANVP compared to ANV enhanced the intracellular uptake of ANVP, we stained MLC3 and incubated it with PKH67 labeled ANV. Ang Ⅱ-treated iPSC-aCMs were treated with fluorescence-labeled ANV or ANVP each for 24h. The number of ANVP conjugated to iPSC-aCM membranes through MLC3 was higher than those conjugated to ANV (Fig. 2 E). This raises the possibility that MLC3-based targeting strategies can improve the precision of therapeutic delivery by directing delivery to the regions of the heart associated with AF pathology, thereby facilitating more efficient intercellular delivery. In vivo targeting of AF cardiomyocytes by ANVP . Given the strong in vitro targeting performance of ANVPs, we next evaluated their therapeutic potential in vivo using a murine model of AF. The in vivo data detailed below further support the role of MLC3-based targeting in enhancing cardiac delivery specificity and therapeutic efficacy. To verify whether Ang Ⅱ-treated AF mice showed the upregulation of MLC3, both mRNA expression levels and immunohistochemical (IHC) analyses were performed. qRT-PCR demonstrated a significant elevation in MLC3 mRNA levels in AF model mice compared to control mice ( p < 0.0001) (Fig. 3 A). Correspondingly, increased fluorescence signals were observed in heart tissues via IHC staining, confirming elevated MLC3 expression in mice with AF ( p < 0.0001) (Fig. 3 B, C). These findings suggest that targeting MLC3 using ANVP can enhance the delivery specificity and retention within the myocardium of AF model mice. For ex vivo fluorescence imaging, control mice received phosphate-buffered saline (PBS), whereas AF mice were administered either Cy5.5-labeled ANV or ANVP. Four hours post-injection, the major organs were harvested, and the fluorescent signal intensity was quantified. Notably, mice treated with ANVP exhibited the highest fluorescence retention in the myocardium under magnetic navigation, suggesting synergistic enhancement through both MLC3 targeting and external magnetic guidance ( p < 0.05) (Fig. 3 D, E). Notably, ANV injection led to a substantial reduction in accumulation within other organs, such as the kidneys and lungs, when compared to both the PBS and ANV injection groups (Figure S5A, B). Both in vitro and in vivo data confirmed significant upregulation of MLC3 under AF conditions. By targeting upregulated MLC3, we enhanced the cellular uptake of ANVP, thereby developing a delivery system with improved specificity and efficiency for cardiomyocytes targeting. Rationale for targeting AEBP1 as a therapeutic target in AF. AEBP1 is known to be upregulated in response to various stress conditions in the heart and has been implicated in promoting inflammation and fibrosis.(23, 41) Analysis of previously published single-nucleus profiling data from patients with hypertrophic cardiomyopathy as well as failing hearts revealed elevated expression of AEBP1 in myofibroblasts accompanied by increased expression of fibrosis-related genes.(25) Although a previous study has shown that AEBP1 knockdown can attenuate these pathological processes in ischemia-reperfusion injury,(42) and its regulation, particularly under AF-like conditions, remains less well defined. Given the established association between AEBP1 and stress-related signaling pathways,(43, 44) we hypothesized that AEBP1 silencing would attenuate the excessive activation of inflammatory and fibrotic pathways under AF-induced conditions. To test this hypothesis, we first examined whether AEBP1 expression was elevated under AF-induced stress and investigated the potential therapeutic effects of AEBP1 knockdown. To investigate whether AEBP1 exhibited increased expression in AF, we analyzed the mRNA expression profiles and protein levels using patient-derived tissue samples (n = 5 per group). AEBP1 showed elevated mRNA expression ( p < 0.001) (Fig. 4 A) and enhanced protein levels of AEBP1 ( p < 0.05) (Fig. 4 B, C) in AF tissues. iPSC-aCMs were treated with Ang Ⅱ, which serves as a relevant in vitro model for AF-related stress. These cells demonstrated a significant upregulation of AEBP1 mRNA expression ( p < 0.001) (Fig. 4 D), consistent with our observations in the patient samples. Subsequently, we evaluated whether AEBP1 expression was upregulated in the mouse model as expected, Ang Ⅱ-treated mice showed increased AEBP1 mRNA expression ( p < 0.0001) (Fig. 4 E). In vitro silencing of AEBP1 via ANVP-siAEBP1. To enhance the delivery of siAEBP1 to cardiomyocytes in an AF model, we encapsulated siAEBP1 in ANV we previously made by electroporation and conjugated it with MNP to generate ANVP-siAEBP1 (Fig. 4 F). The gene silencing efficacy of siAEBP1 was preserved regardless of its encapsulation within ANVP, indicating that the nanovesicle was successfully associated with the cellular membrane and delivered siRNA to effectively suppress AEBP1 gene expression (Fig. 4 G-I). This study demonstrates that ANVP is capable of efficiently transporting siAEBP1 into the intracellular environment, enabling effective gene silencing within target cells. Restoration of cardiac function via AEBP1 silencing in vivo . As previously reported, in AF, the progression of inflammatory responses, such as cytokine release and fibrosis in the cardiac tissue, is increased. To determine whether ANVP-siAEBP1 treatment attenuated the observed pathologies, we conducted an in vivo study to evaluate its therapeutic efficacy. We first generated AF mimic mice by subcutaneously implanting osmotic pumps loaded with Ang Ⅱ. Control mice were implanted with a PBS- containing pump. Seven days post-implantation, the mice were intravenously injected with PBS, ANV, ANVP-NC, or ANVP-siAEBP1 via the tail vein in Fig. 5 A. This time point was selected because AEBP1 is considered crucial for the initial fibrosis response (23) and for effective siRNA delivery. For magnetic navigation, the magnet was externally positioned over the cardiac region, 30 min after each injection. Cardiomyocytes play a crucial role in modulating the inflammatory response after cardiac injury by producing a range of pro-inflammatory mediators, including cytokines and inflammatory enzymes. Therefore, cardiomyocyte-derived inflammatory signaling may offer a safer and more effective strategy for post-injury cardiac modulation.(45, 46) Although ANV are known to exert anti-inflammatory effects, their specific effects on AF- associated inflammation remain unclear. In this study, we present evidence from mouse models of AF demonstrating that ANV treatment exerts anti-inflammatory effects. Furthermore, encapsulation of siAEBP1 enhanced these effects synergistically, leading to the suppression of both the inflammatory response and cardiac fibrosis, without observable host toxicity (Figure S8). To validate the establishment of the AF model, echocardiographic assessment was performed one week after pump implantation, confirming a decline in cardiac function in the AF model group (Figure S6). To assess the knockdown efficiency of AEBP1 by ANVP-siAEBP1, we conducted co-immunostaining for AEBP1 and cardiac troponin I (cTnI) in ANVP-targeted cardiac cells. Immunofluorescence analysis revealed a marked reduction in AEBP1 expression in the ANVP-siAEBP1 treated group compared that in the PBS- ( p < 0.001) and ANVP-NC- treated groups ( p < 0.001) (Fig. 5 B, C). These results indicate that AEBP1 was efficiently silenced by ANVP-siAEBP1 in cardiomyocytes. Consistently, mRNA expression analysis confirmed a substantial reduction in AEBP1 expression following targeted knockdown ( p < 0.0001) (Fig. 5 D). Echocardiography showed that ANVP-siAEBP1 injection has improved the ejection fraction (EF%) ( p < 0.05) and fractional shortening (FS%) ( p < 0.05) compared to the ANVP injection, and no statistically significant difference was observed compared to the control group (Fig. 5 E, F). Taken together, these results suggest that AEBP1 was effectively silenced by ANVP-siAEBP1 and that cardiac function was restored suggesting its therapeutic potential. Attenuation of inflammation and fibrosis by ANVP-siAEBP1 in vivo . Following the successful knockdown of overexpressed AEBP1 and partial restoration of cardiac function by ANVP-siAEBP1, we investigated its efficacy in reducing inflammation and fibrosis. Histological analysis using DAB staining was performed to assess the expression levels of inducible Nitric Oxide Synthase (iNOS), a well-established marker of inflammation.(47) While treatment with ANVP alone led to a modest reduction in inflammation ( p < 0.05); ANVP-siAEBP1 treatment markedly suppressed inflammation to levels comparable to those observed in the AF group ( p < 0.0001) (Fig. 6 A, B). This anti-inflammatory effect was confirmed at the mRNA level. Quantitative analysis of inflammation-related cytokines and markers in myocardial tissue revealed decreased expression of IL-1β, TNF-α, and iNOS ( p < 0.0001) following ANVP-siAEBP1 administration (Fig. 6 C). Subsequently, the extent of fibrosis was assessed. Histological staining demonstrated that ANVP-siAEBP1 treatment effectively decreased the volume fraction of total fibrosis ( p < 0.001) in the myocardium compared with that in the AF group (Fig. 6 D, E). Furthermore, the expression levels of multiple fibrosis-associated downstream factors were also significantly downregulated, including collagen Ⅰ, COL3a1, and α-SMA ( p < 0.01) indicating a broad attenuation of fibrotic signaling pathways (Fig. 6 F). No significant histological alterations were observed in major organs, including the heart, kidneys, liver, lung, and spleen following intravenous injection of each ANVP samples (Figure S7). These findings suggest that the administration of ANVP-siAEBP1 not only suppresses excessive inflammatory responses, but also inhibits the progression of fibrosis in the myocardium. DISCUSSION In this study, we developed bioengineered nanovesicles, termed ANVP, by conjugating ANV with MNP (Fig. 1 ). Although ANV have previously demonstrated therapeutic efficacy in models of cardiac injury,(48) their utility in AF, particularly in AF-induced inflammation, has not been thoroughly investigated. While IONP conjugated with anti-MLC3 antibodies have shown promising results in MI,(16) and ischemic models,(17) their application in AF has not been explored, likely because MLC3 has traditionally been identified as a ventricular marker.(49) Interestingly, our data suggested that MLC3 expression was upregulated in AF-induced cardiomyocytes, indicating a potential paradigm shift in the use of MLC3 as a viable molecular target for AF. Our in vitro data suggested that ANVP preferentially accumulated in regions with elevated MLC3 expression under AF conditions, indicating MLC3-mediated targeting (Fig. 2 ). The in vivo results validate the potential of this platform. AF-induced mouse models show elevated MLC3 expression, confirming its relevance as a target in AF pathology. Upon intravenous administration, the ANVP navigated effectively to the AF-affected myocardium under external magnetic guidance (Fig. 3 ). Given its role in AF-induced inflammation and fibrosis, AEBP1 is considered a promising therapeutic target, and these data were validated through our experiments on patient tissues, iPSC-aCMs, and an AF-induced mouse model. In addition, ANVP delivery of siAEBP1 significantly silenced AEBP1 expression in Ang Ⅱ-treated iPSC-aCMs (Fig. 4 ). The targeted fusion of ANVP facilitated the specific silencing of AEBP1 expression in vivo while partially recovering cardiac function through AEBP1 attenuation (Fig. 5 ). Concurrent with ANVP treatment and AEBP1 silencing, the secretion of pro-inflammatory cytokines was reduced, leading to an overall attenuation of the inflammatory response. Simultaneously, siAEBP1 treatment resulted in a marked reduction in fibrosis (Fig. 6 ). This strategy overcomes the major limitations associated with traditional siRNA therapy, including low transfection efficiency, limited cellular uptake, and systemic toxicity.(50, 51) Moreover, the magnetic navigation system and MLC3 targeting strategy provide improved delivery efficiency to the AF heart, which is a critical advancement over passive targeting approaches. This approach not only introduces MLC3 as a novel therapeutic target in AF, but also exerts inflammation reduction and synergistic anti-fibrotic effects through gene silencing, offering significant translational potential for future cardiac therapies. Abbreviations AF Atrial Fibrillation ANV Apoptotic mesenchymal stem cell-derived nanovesicle AEBP1 Adipocyte enhancer binding protein 1 siRNA small interfering RNA ANVP Apoptotic mesenchymal stem cell-derived nanovesicle conjugated with iron oxide magnetic nanoparticle MLC3 myosin light chain 3 MSC mesenchymal stem cell HCM Hypertrophic cardiomyopathy ACLP aortic carboxypeptidase-like protein STS staurosporine FT-IR Fourier Transform Infrared Spectroscopy IONP iron oxide magnetic nanoparticle DBCO Dibenzocyclooctyne TEM Transmission electron microscopy NTA nanoparticle tracking analysis iPSC-aCM human induced pluripotent stem cell-derived atrial cardiomyocyte Ang Ⅱ angiotensin Ⅱ Cy5.5 Sulfo-Cyanine5.5 IHC immunohistochemical qRT-PCR quantitative reverse transcription polymerase chain reaction PBS Phosphate Buffered Saline NC negative control cTnI cardiac troponin I DAB 3,3'-Diaminobenzidine iNOS Nitric Oxide Synthase IL-1β Interleukin-1 beta TNF-α Tumor Necrosis Factor alpha COL3a1 collagen type III alpha 1 chain α-SMA alpha-smooth muscle actin Declarations Ethics approval and consent to participate All procedures involving human participants were approved by the Institutional Review Board of Severance Hospital, Yonsei University (IRB No. 4-2019-0620), and written informed consent was obtained from each subject. All animal experiments were under ethical approval of Institutional Animal Care and Use Committee of Yonsei University College of Medicine (approval no. 2024-0222) and adhered to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (Publication No. 85-23, revised 1996). Consent for publication All participants provided consent for the publication of this manuscript. Availability of data and materials The dataset(s) supporting the conclusions of this article is(are) included within the article (and its additional file(s)). Competing interests The authors declare no competing interests. Funding Korean government (the Ministry of Science and ICT, the Ministry of Health & Welfare, KFRM21B0604L1-01 and 24A0202L1), National Research Foundation of Korea grants funded by the Korean government (MSIT) (2021R1C1C2094541 and 2023R1A2C3003320). Korean Cardiac Research Foundation (202101-02). Department of Cardiology, Graduate School of Medical Science, Brain Korea 21 Project, Yonsei University College of Medicine. Authors’ contributions Gyeongseo Yoo: Conceptualization; Investigation; Validation; Writing—original draft. Ji-Young Kang: Investigation. Malgeum Park: Investigation. Jaewoong Lee: Validation. Dasom Mun: Conceptualization; Supervision. Nuri Yun: Conceptualization; Supervision. Boyoung Joung: Conceptualization; Supervision Acknowledgements This study was supported by the National Research Foundation of Korea grants funded by the Korean government (MSIT) (2021R1C1C2094541 and 2023R1A2C3003320); the Korean Fund for Regenerative Medicine grant funded by the Korean government (the Ministry of Science and ICT, the Ministry of Health & Welfare, KFRM21B0604L1-01 and 24A0202L1); the Korean Cardiac Research Foundation (202101-02); and the Department of Cardiology, Graduate School of Medical Science, Brain Korea 21 Project, Yonsei University College of medicine. 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Adv Funct Mater. 2023;33(23). Kane C, Terracciano CMN. Concise Review: Criteria for Chamber-Specific Categorization of Human Cardiac Myocytes Derived from Pluripotent Stem Cells. Stem Cells. 2017;35(8):1881-97. Reischl D, Zimmer A. Drug delivery of siRNA therapeutics: potentials and limits of nanosystems. Nanomedicine. 2009;5(1):8-20. Xue HY, Liu S, Wong HL. Nanotoxicity: a key obstacle to clinical translation of siRNA-based nanomedicine. Nanomedicine (Lond). 2014;9(2):295-312. Scheme 1 Scheme 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Additionalfile1.pdf scheme1.png Scheme 1. Schematic illustration of AF treatment via targeted delivery of ANVP-siAEBP1. Apoptotic MSCs, pre-treated with STS, were processed to produce ANV. These ANV were subsequently electroporated with siAEBP1 and conjugated with MNP, forming ANVP-siAEBP1. Upon intravenous administration, ANVP-siAEBP1 actively targeted cardiomyocytes through anti-MLC3 antibodies and magnetic guidance. This targeted delivery system effectively suppressed inflammation and fibrosis, thereby preventing cardiac dysfunction and promoting the resolution of AF. 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-7061113","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":499269560,"identity":"1357ca39-1bcb-4d2f-bd33-905bf605dc2e","order_by":0,"name":"Gyeongseo Yoo","email":"","orcid":"","institution":"Yonsei University College of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Gyeongseo","middleName":"","lastName":"Yoo","suffix":""},{"id":499269561,"identity":"5f5b84e4-8273-443d-a981-ae3d921a7cdb","order_by":1,"name":"Ji-Young Kang","email":"","orcid":"","institution":"Yonsei University College of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Ji-Young","middleName":"","lastName":"Kang","suffix":""},{"id":499269562,"identity":"e1fe0d9a-72a9-4c48-9f8c-17c35b003868","order_by":2,"name":"Malgeum Park","email":"","orcid":"","institution":"Yonsei University College of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Malgeum","middleName":"","lastName":"Park","suffix":""},{"id":499269563,"identity":"139fefae-eb4f-4b2a-9d10-ad409980a5c6","order_by":3,"name":"Jaewoong Lee","email":"","orcid":"","institution":"Yonsei University College of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Jaewoong","middleName":"","lastName":"Lee","suffix":""},{"id":499269564,"identity":"0b92f7c5-465a-48f8-bb1a-65b819af823c","order_by":4,"name":"Dasom Mun","email":"","orcid":"","institution":"Yonsei University College of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Dasom","middleName":"","lastName":"Mun","suffix":""},{"id":499269569,"identity":"6aa44cea-ef74-40b7-90ef-891d92f2c9a6","order_by":5,"name":"Nuri Yun","email":"","orcid":"","institution":"GNTPharma Science and Technology Center for Health","correspondingAuthor":false,"prefix":"","firstName":"Nuri","middleName":"","lastName":"Yun","suffix":""},{"id":499269571,"identity":"aa956206-d48c-473c-b64c-e8c36ec8459a","order_by":6,"name":"Boyoung Joung","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyElEQVRIiWNgGAWjYBAC/vk9hg8+GNjIMTDwEKlF4sYZY8MZFWnGxGsxiEhLE+Y5czixgXgtEoePMfO2paVvuJF7+ANDjR0RWuQb2x7ObbPJ3XAjL02C4VgyMbYcbDd425YG1JJjxsDAdoAYLYltErxth9MNbuQYf2D4R4yWiMQ2SaD3E4BaDCQY24jQInHjYDMokA1nnnljJpHYR4Rf+Oc3NoKiUp7vONBhH74REWJwoAByUgIJGhgY5BtIUj4KRsEoGAUjCQAA15RCELsTm80AAAAASUVORK5CYII=","orcid":"","institution":"Yonsei University College of Medicine","correspondingAuthor":true,"prefix":"","firstName":"Boyoung","middleName":"","lastName":"Joung","suffix":""}],"badges":[],"createdAt":"2025-07-07 04:23:05","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7061113/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7061113/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":89369054,"identity":"bc8581d7-6758-4af3-bac6-0871e0fae715","added_by":"auto","created_at":"2025-08-19 09:49:22","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":583994,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePreparation and characterization of ANVP.\u003c/strong\u003e A) Generation of ANV by serial extrusion of apoptotic MSCs, followed by conjugation with MNP to form ANVP. B) Infrared spectral analysis confirming the distinct chemical composition at each stage of ANVP development. C) Representative TEM images of NV, IONP, ANV, and ANVP. Scale bar = 200 nm D) Zeta potential measurements of NV, IONP, ANV, and ANVP. E) Size distribution profiles of NV, IONP, ANV, and ANVP. F) Western blot analyses showing various NV-associated and cellular marker expressions.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7061113/v1/5ea8d84eb4bb5110f4ffbab9.png"},{"id":89370175,"identity":"5872dd08-e911-4fb0-9308-64dd82f50976","added_by":"auto","created_at":"2025-08-19 09:57:23","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":920920,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEvaluating MLC3 expression changes in AF and enhanced targeting efficiency via MLC3 recognition.\u003c/strong\u003e A) qRT-PCR analysis of MLC3 levels in heart tissue samples from patients without AF (n = 5) and with AF (n = 5). B) qRT-PCR analysis of MLC3 mRNA expression in control iPSC-aCMs and Ang II-treated iPSC-aCMs. C, D) Representative immunofluorescence images and quantification showing MLC3 upregulation in Ang II-treated iPSC-aCMs. Nuclei were stained with DAPI (blue); green fluorescence indicates MLC3 expression. Scale bar = 20 µm. E) Representative immunofluorescence images showing enhanced cellular uptake of ANVP via MLC3 targeting in iPSC-aCMs. ANV were labeled with PKH67 (green), and nuclei were stained with DAPI (blue). Scale bar = 20 µm. **P \u0026lt; 0.01, ***P \u0026lt; 0.001, ****P \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7061113/v1/974d7bbd335ca755f1daabe0.png"},{"id":89370172,"identity":"9e94da45-fe41-438c-a901-0739bce80dfe","added_by":"auto","created_at":"2025-08-19 09:57:22","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":411361,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTargeting AF-induced heart via anti-MLC3 antibody and magnetic guidance.\u003c/strong\u003e A) qRT-PCR analysis of MLC3 mRNA levels in mouse heart tissue, normalized to GAPDH. B, C) Representative immunofluorescence images and corresponding quantification showing MLC3 expression (red) in mouse heart tissue. Nuclei were counterstained with DAPI (blue). Scale bar = 20 μm. D, E) Representative IVIS images and quantification of heart fluorescence intensity at 4 h post-intravenous injection of PBS (control), ANV, or ANVP in AF-induced mice (\u003cem\u003en\u003c/em\u003e = 3 per group). *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, ****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7061113/v1/23097c0c5f6e9c224b629ba4.png"},{"id":89371586,"identity":"b5d6e484-8856-4f72-bfe0-9a7aa04aa3c2","added_by":"auto","created_at":"2025-08-19 10:13:22","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":311276,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAEBP1 identified as a therapeutic target in AF.\u003c/strong\u003e A) qRT-PCR analysis of AEBP1 expression in heart tissues from patients without AF (n = 5) and with AF (n = 5). B, C) Representative western blots and corresponding quantification of AEBP1 protein levels in patients without AF (n = 4) and with AF (n = 4). D) Quantification of AEBP1 mRNA levels in iPSC-aCMs. E) Quantification of AEBP1 mRNA levels in mouse heart tissues. F) Stepwise construction of ANVP-siAEBP1 by electroporating siAEBP1 into ANV, followed by MNP conjugation. G) Assessment of siAEBP1 knockdown efficiency after encapsulation within ANVP. H, I) Representative immunofluorescence images and corresponding quantification showing AEBP1 expression (green) following ANVP-siAEBP1 treatment in iPSC-aCMs. Nuclei were counterstained with Hoechst 33342 (blue). Scale bar = 20μm. Fluorescence intensities were normalized using control values to account for background and variability. *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001, ****P \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7061113/v1/36bd3f0f87cbba9b9fe169c9.png"},{"id":89369063,"identity":"354b9238-999e-4260-8591-8c6c136c2fb1","added_by":"auto","created_at":"2025-08-19 09:49:23","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":845635,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eANVP-siAEBP1 attenuates inflammatory responses in an \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003emodel.\u003c/strong\u003e A) Schematic overview of the \u003cem\u003ein vivo\u003c/em\u003e experimental design and workflow. B, C) Representative confocal microscopy images in cardiac tissue. Cardiomyocytes were stained with cTnI (red), and AEBP1 (green), while nuclei were counter-stained with DAPI (blue). Scale bar = 20 μm. Fluorescence intensities were normalized using control values to account for background and variability. D) Knockdown efficiency of AEBP1 by ANVP-siAEBP1 evaluated using qRT-PCR. Data normalized by GAPDH. E, F) Representative M-mode echocardiographic images and corresponding quantification of ejection fraction and fractional shortening in the indicated groups (\u003cem\u003en\u003c/em\u003e = 3 per group). *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, ***\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.001, ****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7061113/v1/81694e552ebc7b9526156a8b.png"},{"id":89370485,"identity":"2aa521f1-d8ca-40cd-a3ee-ca3147b22338","added_by":"auto","created_at":"2025-08-19 10:05:23","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1276373,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eANVP-siAEBP1 attenuates inflammatory and fibrotic responses in an \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e model.\u003c/strong\u003e A, B) Representative 3,3-diaminobenzidine (DAB)-stained immunohistochemical images and quantification of iNOS protein expression in mice heart tissue. Scale bar = 100 μm. C) mRNA expression levels of inflammatory markers IL-1β, TNF-α, and iNOS assessed by qRT-PCR. D, E) Masson's trichrome staining and quantification of fibrotic area in AF-injured mice. Scale bar = 100 μm. F) qRT-PCR analysis of fibrosis-related markers including Collagen I, COL3a1, and α-SMA in the indicated groups. All data normalized to GAPDH. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, ****\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-7061113/v1/98e1f341d456068d8b5627b6.png"},{"id":91414565,"identity":"5eec43d5-86ef-4086-a110-fb7f0efa4be4","added_by":"auto","created_at":"2025-09-16 09:09:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5382680,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7061113/v1/69e3858d-536d-4758-870a-952da195be4f.pdf"},{"id":89369061,"identity":"714388e3-8f4d-4813-b861-3e341c7e807a","added_by":"auto","created_at":"2025-08-19 09:49:23","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1061701,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfile1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7061113/v1/402cca77b1050a19de1488fd.pdf"},{"id":89369056,"identity":"223d67e5-9320-49d7-bfbf-3acf78c5ada6","added_by":"auto","created_at":"2025-08-19 09:49:22","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":363194,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScheme 1. Schematic illustration of AF treatment via targeted delivery of ANVP-siAEBP1.\u003c/strong\u003e Apoptotic MSCs, pre-treated with STS, were processed to produce ANV. These ANV were subsequently electroporated with siAEBP1 and conjugated with MNP, forming ANVP-siAEBP1. Upon intravenous administration, ANVP-siAEBP1 actively targeted cardiomyocytes through anti-MLC3 antibodies and magnetic guidance. This targeted delivery system effectively suppressed inflammation and fibrosis, thereby preventing cardiac dysfunction and promoting the resolution of AF.\u003c/p\u003e","description":"","filename":"scheme1.png","url":"https://assets-eu.researchsquare.com/files/rs-7061113/v1/32a1a5e50cbddc980b849736.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Enhanced Therapeutic potential of AEBP1 silencing via engineered Apoptotic Mesenchymal Stem Cell- derived Nanovesicles in Atrial Fibrillation","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eAtrial fibrillation (AF) is one of the most common form of arrhythmia, with a wide spectrum of symptoms, and its incidence is increasing globally.(1) Experimental and clinical data indicates that inflammation can get induced in patients with AF, which in turn, can contribute to the progression of AF, leading to clinical phenomenon described as \u003cem\u003e\u0026lsquo;AF begets AF\u0026rsquo;\u003c/em\u003e. This can ultimately lead to severe atrial fibrosis.(2, 3) However, existing anti-arrhythmic drugs and emerging therapeutic strategies (biological approaches), exhibit limitations in target specificity, duration of action, and overall efficacy in mitigating the progression of AF.(4\u0026ndash;6) Therefore, the development of an advanced therapeutic system capable of precise targeting and effective treatment of AF is imperative.\u003c/p\u003e\u003cp\u003eMesenchymal stem cell (MSC) therapy is widely used owing to its ability to rapidly respond to cellular injury and migrate to the site of damage, promoting tissue repair and regeneration.(7) MSC-derived nanovesicles are emerging as promising carriers capable of delivering therapeutic agents to specific cells and tissues. Studies have shown that these nanovesicles can enhance cardiac repair and help overcome the challenge of low cell survival in myocardial infarction,(8, 9) and myocardial ischemia/reperfusion.(10) Healthy MSC-derived nanovesicles are widely studied but the therapeutic potential of the apoptotic MSC-derived nanovesicles in AF remains unclear. Apoptotic MSC-derived nanovesicles (ANV) have been shown to express proteins associated with tissue repair and exhibit reduced levels of pro-inflammatory proteins, suggesting a potential role in modulating inflammation.(11) This minimizes the risk of inflammation and promotes controlled healing, making ANV effective for targeted therapy.(12\u0026ndash;14) Therefore, the immunosuppressive properties of MSCs can be enhanced through apoptosis, which facilitates the retention of various cytoplasmic factors.\u003c/p\u003e\u003cp\u003eTo enhance the therapeutic efficacy, precise targeting of the desired site is essential. To date, the majority of drugs utilized for tissue injury repair have been delivered through either systemic or local administration.(15) Magnetic nanoparticles are widely used for precise targeting and molecular delivery, and their modification for enhanced specificity has already been studied.(16, 17) anti-CD63 antibody was conjugated to the magnetic nanoparticle to capture nanovesicles and anti-MLC3 antibody was conjugated to the target AF heart.\u003c/p\u003e\u003cp\u003e\u003cem\u003eMLC3\u003c/em\u003e, a gene expressed in cardiomyocytes, is a potential contributor to mid-left ventricular chamber-type hypertrophic cardiomyopathy (HCM). Recent studies have identified MLC3 variants in patients with HCM, particularly in those carrying mutations in sarcomeric protein genes associated with heart failure and AF.(18, 19) Beyond its genetic association with cardiomyopathy, MLC3 has emerged as a selective marker of injured cardiomyocytes. Consequently, it has been used as a molecular target to enhance delivery to injured cardiomyocytes in various injury models, including myocardial infarct,(16) ischemic cardiomyopathy.(17, 20) However, the use of anti-MLC3 antibody strategies to specifically target AF remains underexplored.\u003c/p\u003e\u003cp\u003e\u003cem\u003eAEBP1\u003c/em\u003e, an isoform of aortic carboxypeptidase-like protein,(21) is an extracellular protein involved in the remodeling of the extracellular matrix and promotes macrophage inflammatory responsiveness.(22, 23) Recent experimental evidence has shown that overexpression of AEBP1 may function in cardiac fibroblast activation, which can trigger the release of pro-fibrotic factors.(24, 25) Consistent with these findings, we observed a marked increase in AEBP1 expression in patients with AF. Thus, silencing AEBP1 gene expression may serve as a key therapeutic approach to inhibit pathways implicated in fibrosis,(22) thereby preventing cardiac fibrosis and ultimately enhancing cardiac function.\u003c/p\u003e\u003cp\u003eSmall interfering RNA (siRNA) is a promising therapeutic agent that offers a novel approach to selectively downregulate the expression of specific proteins.(26\u0026ndash;29) Considering these characteristics, siRNA therapeutics have great potential in the treatment of cardiovascular disease.(30, 31) Despite the therapeutic potential of siRNA-based treatments, several challenges remain, particularly in the context of efficient intracellular delivery. Therefore, there is a pressing need to develop more efficient and biocompatible delivery strategies that can overcome current limitations and enable the clinical translation of siRNA therapeutics.\u003c/p\u003e\u003cp\u003eIn this study, we developed a novel nanoparticle-based therapeutic platform by encapsulating siAEBP1 within ANV and conjugating them with engineered magnetic nanoparticles to generate ANVP-siAEBP1 (Scheme. 1). ANVP can transport therapeutic agents while simultaneously conferring intrinsic therapeutic effects via their bioactive components. The conjugation of magnetic components enhances the targeting efficiency to the atrial myocardium via external magnetic guidance, while also enabling preferential accumulation through anti-MLC3 antibody to MLC3-enriched cardiomyocytes, which were found to exhibit elevated expression levels in the AF myocardium in our study. Once delivered, siAEBP1 suppressed fibrosis-associated signaling pathways by downregulating AEBP1, thereby effectively preventing fibrotic remodeling in the atrial tissue. Simultaneously, ANV themselves contribute to the anti-inflammatory effects in stressed cardiomyocytes. Through this integrated approach, ANVP-siAEBP1 offers a synergistic therapeutic effect that is anticipated to surpass the efficacy of conventional drug treatments for AF.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cp\u003e\u003cb\u003eCell culture and Staurosporine (STS) treatment, Ang Ⅱ treatment\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003ehMSC culture and Staurosporine (STS) treatment.\u003c/b\u003e Human Mesenchymal Stem Cells Derived from bone marrow (hMSC) (ATCC, Manassas, VA, USA) were cultured in Dulbecco's Modified Eagle's Medium, Low Glucose (DMEM; LM001-11, Welgene, Gyeongsan, Korea) supplemented with 10% fetal bovine serum (FBS; USFBS-500, Young In Frontier, Seoul, Korea) and 1% penicillin-streptomycin (10378016, Gibco, Grand Island, NY, USA) at 37\u0026deg;C in a humidified atmosphere with 5% CO\u003csub\u003e2\u003c/sub\u003e. To induce apoptosis, hMSCs were treated with 0.25 \u0026micro;M Staurosporine (STS) once the cells reached 80\u0026ndash;90% confluency (569397, Sigma) for 6h.\u003c/p\u003e\u003cp\u003e\u003cb\u003eiPSC-aCM culture and Angiotensin Ⅱ (Ang Ⅱ) treatment.\u003c/b\u003e The human induced pluripotent stem cell (iPSC) line CMC-hiPSC-011 was provided by the Korea National Stem Cell Bank, originally provided by Catholic University. Cells were cultured in the TeSR\u0026trade;-E8\u0026trade; medium (STEMCELL Technologies) on vitronectin (Gibco)-coated plates at 37\u0026deg;C in 5% CO\u003csub\u003e2\u003c/sub\u003e and subcultured every 4\u0026ndash;6 d with 10 \u0026micro;M Y27632 (Sigma). Through modulation of wingless-related integration site signaling, iPSCs were differentiated into iPSC-derived atrial cardiomyocytes (iPSC-aCMs), following previously described protocols with minor modifications.(32) Briefly, iPSCs began to differentiate at 80\u0026ndash;90% confluence in Materigel (354277, Corning, Arizona, USA)-coated plates with cardio differentiation medium consists of RPMI 1640 (11875119, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with B-27 minus insulin (A1895601, Thermo Fisher Scientific) and stepwise treatment with 4 \u0026micro;M CHIR99021 (4423, Tocris Bioscience, Bristol, UK) for 72 h and then 2 \u0026micro;M Wnt-C59 (5148, Tocris Bioscience) for 48 h. On day 5 to 7 of differentiation, 1\u0026micro;M of RA (R2625, Sigma-Aldrich; Merck KGaA) was added for 72h. The medium was replaced with cardio culture medium composed for RPMI 1640 supplemented with B-27 (17504001, Thermo Fisher Scientific) on day 9.\u003c/p\u003e\u003cp\u003eTo mimic AF \u003cem\u003ein vitro\u003c/em\u003e, iPSC-aCMs were treated with 0.5 \u0026micro;M Ang II (A9525; Sigma-Aldrich; Merck KGaA) for 24 h.\u003c/p\u003e\u003cp\u003e\u003cb\u003eCell viability assay.\u003c/b\u003e Cell viability was assessed using Quanti-Max\u0026trade; WST-8 Cell Viability Assay Kit (QM2500, Biomax Inc, korea) according to the manufacturer\u0026rsquo;s instructions. hMSCs were seeded in a 6-well plate and treated with different concentration of STS at multiple time point. Subsequently, cells were treated with Quanti-Max\u003csup\u003e\u0026trade;\u003c/sup\u003e for 4h in 37\u0026deg;C CO\u003csub\u003e2\u003c/sub\u003e incubator. For absorbance measurement the medium were transferred to 96 well plate and measured at a wavelength of 450 nm using a microplate reader (VersaMax).\u003c/p\u003e\u003cp\u003e\u003cb\u003eApoptosis measurement using Fluorescence Activated Cell Sorting (FACS) assay.\u003c/b\u003e Apoptosis was analyzed using FITC-Annexin V Apoptosis Detection kit Ⅱ (556570, BD Biosciences, San Jose, CA, USA) according to the manufacturer\u0026rsquo;s instructions. Cells were harvested using 0.25%, 1x trypsin- ethylenediaminetetraacetic acid (LS015-10, Welgene) and washed twice with cold PBS. The pellet was resuspended in the 1x binding buffer, FITC Annexin V and PI were added sequentially. The cells were incubated for 15 min in the dark at RT and binding buffer was additionally supplemented. Right after incubation, cells were analyzed within 1 hr using a BD\u0026reg; LSR II SORP (BD Biosciences), and the percentage of apoptotic cells was determined using the BD FACS DIVA software.\u003c/p\u003e\u003cp\u003e\u003cb\u003eDBCO-IONP conjugation.\u003c/b\u003e Conjugation of DBCO-sulfo-NHS with IONPs was conducted by mixing 0.5 mg/mL amine (-NH₂)-functionalized iron oxide (IONPs; Sigma-Aldrich, 5 mg/mL, molecular weight 231.53 g/mol) solution with the 2.16 \u0026micro;mol DBCO-sulfo-NHS (Peptron Co., Daejeon, South Korea) DBCO solution at a 1:1 molar ratio and incubated on a rotating mixer at room temperature (RT) for 4 hours. To remove any unconjugated DBCO-sulfo-NHS, the mixture was then centrifuged at 10,000 \u0026times; g for 10 minutes. 3 \u0026micro;M DBCO-sulfo-NHS (Peptron Co., Daejeon, South Korea) was mixed with 0.5 mg/mL amine (-NH₂)-functionalized iron oxide (IONPs; Sigma-Aldrich, 5 mg/mL) and the samples were incubated at room temperature (RT) on a rotating mixer for over 4 hours. To remove any unconjugated DBCO-sulfo-NHS the mixture was centrifuged at 10,000 x g for 10 minutes.\u003c/p\u003e\u003cp\u003e\u003cb\u003eEnzymatic modification of antibodies.\u003c/b\u003e Anti-CD63 (E-12) antibody (sc-365604, Santa Cruz, 0.5 mg mL\u0026thinsp;\u0026minus;\u0026thinsp;1) and anti-Myosin light chain 3 (ab233220, Abcam, 0.2 mg mL\u0026thinsp;\u0026minus;\u0026thinsp;1) were selectively modified at defined sites on the Fc region with an azide using the Site Click\u0026trade; Antibody Azido Modification Kit (Thermofisher). In short, 100 \u0026micro;g of Anti-CD63 antibody, and 100 \u0026micro;g of anti-Myosin light chain 3 antibody were loaded into an antibody concentrator and centrifuged at 5000 x g for 6 minutes. After discarding the flow-through, 450 \u0026micro;L of antibody preparation buffer was added to the sample and centrifuged at 5000 x g for 8 minutes. To remove galactose residues, galactosidase was added to the antibody solution and incubated overnight at 37\u0026deg;C. Additionally, the azide group was enzymatically conjugated to the carbohydrate modified antibody. To initiate the reaction, Tris buffer, buffer additive, and GalT (Y289L) enzyme were added to a tube containing UDP-GalNAz, along with the carbohydrate-modified antibody, and incubated overnight at 30\u0026deg;C. For removing excess UDP-GalNAz, the solution was washed with 1 mL of Tris buffer and centrifuged at 1200 x g for 10 minutes. The final azide-modified antibody (\u0026sim;40 \u0026micro;L, 2.6 mg mL\u0026thinsp;\u0026minus;\u0026thinsp;1) was stored at 4\u0026deg;C before further experiments.\u003c/p\u003e\u003cp\u003e\u003cb\u003eDBCO-IONP-Antibody conjugation.\u003c/b\u003e To enable conjugation via click chemistry between DBCO and azide (-N₃) moieties as established in previous studies, 0.5 mg/mL DBCO-IONPs were incubated with 17.5 \u0026micro;g of azide-functionalized antibodies (1 mg/mL) overnight at 4\u0026deg;C with continuous rotation. Unconjugated antibodies were subsequently removed by centrifugation at 10,000 \u0026times; g for 10 minutes, and the conjugates were stored at 4\u0026deg;C until further use.\u003c/p\u003e\u003cp\u003e\u003cb\u003ePreparation of Apoptotic cell derived nanovesicles conjugated with DBCO-IONP-antibody (ANVP).\u003c/b\u003e Once the human bone marrow hMSCs (ATCC, Manassas, VA, USA) reached\u0026thinsp;~\u0026thinsp;90% confluence in 150 mm\u003csup\u003e2\u003c/sup\u003e culture dishes, the cells were harvested from the dishes by cell scraper and centrifuged at 270 x g for 5 min. The cells were subjected to sonication at 100 V with 20% power for two rounds of six cycles each, where each cycle consisted of a 4-second pulse followed by a 2-second pause. Between and after sonication, the cells were incubated on ice to minimize thermal effects. After sonication was completed, samples were centrifuged at 4\u0026deg;C, 800 x g, 10 min to remove cell debris.\u003c/p\u003e\u003cp\u003eTo obtain the MSC-derived nanovesicles (NVs) and Apoptotic MSC-derived nanovesicles (ANVs), sonicated hMSCs were suspended in phosphate buffered saline (PBS), and serially extruded 8 times through 5-\u0026micro;m, 1-\u0026micro;m, 0.4-\u0026micro;m and 0.2-\u0026micro;m pore sizes polycarbonate membrane filters using a mini extruder (Avanti polar Liquids, Birmingham, USA).\u003c/p\u003e\u003cp\u003eFor density-gradient ultracentrifugation, the 2 mL ultracentrifuge tube was layered sequentially with 50% iodixanol (optiprep, Axis-shild, Oslo, Norway) at the bottom, followed by 10% iodixanol, and the sample was loaded on top and centrifuged at 100,000g for 2h at 4\u0026deg;C using microcentrifuge (Himac CS120GXL; Hitachi, Tokyo, Japan). The resulting pellet was carefully resuspended in cold phosphate-buffered saline (PBS) and washed twice with cold PBS using an Amicon Ultra-4 Centrifuge Filter (UFC8010; Millipore Corporation, Bedford, MA, USA) at 3,500 x g for 40 min at 4\u0026deg;C. The vesicle solution was stored at 4\u0026deg;C when not used according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e\u003cp\u003eExtracted ANV were combined with DBCO-IONP-antibody (hereafter referred to as MNP) and they were incubated at a 1:1 ratio via anti-CD63 interactions overnight at 4\u0026deg;C with rotation, resulting in the formation of ANVP. To remove unconjugated ANV and MNP, the mixture was performed at 10,000 x g for 10 minutes and stored at 4\u0026deg;C until needed.\u003c/p\u003e\u003cp\u003e\u003cb\u003eCharacterization of ANVP.\u003c/b\u003e The morphology of the obtained ANVP was observed through transmission electron microscopy (TEM) (JEM-1011., JOEL Ltd., Tokyo, Japan). The zeta potential of the ANVP was evaluated with ELS-1000ZS (Otsuka Electronics, Osaka, Japan). The mean size and particle concentration of ANVP were analyzed via the nanoparticle tracking assay (NTA) using a NanoSight LM10 instrument (Malvern, Worcestershire, UK) according to the manufacturer's instructions and it was examined using NTA v.2.3 software (Malvern Panalytical, Ltd., Malvern, UK). The intermediates and final products were analyzed using Fourier transform infrared spectrometer (INVENIO., BRUKER., Texas, USA).\u003c/p\u003e\u003cp\u003e\u003cb\u003eWestern blot analysis.\u003c/b\u003e Total protein was lysed using the EzRIPA Lysis Kit (WSE-7420, ATTO CORPORATION, Korea) with protease and phosphatase inhibitors. The protein concentration was determined using Pierce\u0026trade; 660 nm Protein Assay Reagent (22660, Thermo Fisher Scientific). Proteins were separated by 10% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto PVDF membranes (IPVH00010; EMD Millipore, Bedford, MA, USA). The membranes were blocked with 5% BSA in TBS-Tween 20 (TBS-T) for 40\u0026ndash;50 min at RT and incubated overnight at 4\u0026deg;C with the following primary antibodies: CD81 (1:1000, sc-166029, Santa Cruz Biotechnology), CD63 (1:1000, sc-365604, Santa Cruz Biotechnology), Lamp (1:1000, sc-18822, Santa Cruz Biotechnology), AEBP1 (1:1000, sc-271374, Santa Cruz Biotechnology) and GAPDH (1:1000, sc-25778, Santa Cruz Biotechnology). GAPDH was used as a loading control. The membranes were washed five times with TBS-T, and incubated with horseradish peroxidase-conjugated secondary antibodies (sc-2357, 1:5000; Santa Cruz Biotechnology) for 1 h. Immunoblots were visualized using an enhanced chemiluminescence detection kit (1705061; Bio-Rad Laboratories Inc., Hercules, CA, USA) and scanned using a model LAS4000 Mini Luminescent Image Analyzer (FUJIFILM, Tokyo, Japan). Band density of the experiment was quantified using ImageJ software.\u003c/p\u003e\u003cp\u003e\u003cb\u003eStudy population.\u003c/b\u003e All procedures involving human participants were approved by the Institutional Review Board of Severance Hospital, Yonsei University (IRB No. 4-2019-0620), and written informed consent was obtained from each subject. Human tissue was obtained from patients without AF (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5) and patients with AF (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5) at the Yonsei University Health System (Seoul, Korea) from August 2019 to July 2021, and the clinical profiles of patients are listed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. The two groups did not differ significantly in terms of age or body mass index (BMI).\u003c/p\u003e\u003cp\u003e\u003cb\u003eRNA isolation, cDNA synthesis and Quantitative RT-PCR (qRT-PCR).\u003c/b\u003e Total RNA was isolated from iPSC-aCMs, patient heart tissue and mice heart tissue using the miRNeasy\u0026reg; Mini Kit (217004, Qiagen, Hilden, Germany) according to the manufacturer\u0026rsquo;s protocol. For cDNA synthesis, cDNA synthesis kit (600036, Agilent Technologies, Santa Clara, CA, USA) and High-Capacity cDNA Reverse Transcription kit (4368814, Applied Biosystems, Darmstadt, Germany) was used. qRT-PCR was carried out using the PowerUp\u0026trade; SYBR\u0026trade; Green Master Mix (A25742, Applied Biosystems) on QuantStudio\u0026trade; 3 Real-Time PCR System (A28567, Thermo fisher, USA). PCR primers were synthesized by Cosmogenetech (Daejeon, Korea) and all primer sequences are listed in Table S2, S3. The relative expression of mRNA was quantified using the ΔΔCt method, and calculated using 2\u003csup\u003e\u0026minus;ΔΔCq\u003c/sup\u003e method and normalized against GAPDH.(33)\u003c/p\u003e\u003cp\u003e\u003cb\u003esiRNA transfection\u003c/b\u003e. iPSC-aCMs were seeded in 6-well plate and when it reached approximately 70\u0026ndash;80% confluency, the cells were transfected with Lipofectamine\u0026trade; RNAiMAX (13778150, Thermo Fisher Scientific) according to the manufacturer\u0026rsquo;s instructions. The negative control and AEBP1 siRNA were synthesized by Bioneer (Daejeon, Korea). Cells were incubated for two days in Cardio Culture Medium after transfection and harvested for subsequent analyses.\u003c/p\u003e\u003cp\u003e\u003cb\u003eLoading of siRNA into ANVP.\u003c/b\u003e ANV were mixed with siAEBP1 (bioneer, korea) or Negative Control siRNA at the ratio of 1:1 (wt/wt) and the mixture were electroporated using a Neon\u0026trade; Transfection System (Thermo Fisher Scientific) at 1000 V, 10 ms, 2 pulses, to form ANV-siAEBP1 or ANV-NC. After siRNA loading, the transfected ANV-siAEBP1 and ANV-NC were incubated with 100 \u0026micro;g/ml RNase A (Thermo Fisher Scientific) for 30 min at 37\u0026deg;C to degrade unloaded siRNA. Thereafter, the mixture was washed twice with cold PBS using an Amicon Ultra-4 Centrifuge Filter (UFC8010; Millipore Corporation, Bedford, MA, USA) at 3,500 x g for 40 min at 4\u0026deg;C.\u003c/p\u003e\u003cp\u003e\u003cb\u003eFluorescence labeling of ANVP and Immunocytochemistry.\u003c/b\u003e Isolated ANV and ANVP were labeled with the PKH67 green Fluorescent Cell Linker Kit (MINI67, Sigma-Aldrich; Merck KGaA) according to the manufacturer\u0026rsquo;s instructions. iPSC-aCMs were incubated with PKH labeled PBS or ANV or ANVP for 24 hours and subsequently fixed with cold 4% paraformaldehyde. MLC3 antibody (ab233220, 1:200, Abcam) was treated to cells and incubated at 4\u0026deg;C overnight. Next, cells were incubated with a secondary antibody (mouse anti-rabbit IgG-CFL; sc-516251, 1:5000; Santa Cruz Biotechnology) for 1h. After five times of washing with PBS, 4\u0026prime;,6-diamidino-2-phenylindole (DAPI) was incubated for 7 min to label the nuclei.\u003c/p\u003e\u003cp\u003eTo visualize ANVP conjugation to the cell membrane, magnetic nanoparticles (MNP) were labeled with Cy5.5 and incubated with ANV to generate Cy5.5-ANVP. Treated cells were fixed as described above, and nuclei were counterstained with 4\u0026prime;,6-diamidino-2-phenylindole (DAPI). Confocal images were acquired using a Zeiss LSM 710 confocal microscope (Carl Zeiss, Jena, Germany).\u003c/p\u003e\u003cp\u003e\u003cb\u003eImmunocytochemistry.\u003c/b\u003e iPSC-aCMs were fixed with cold 4% paraformaldehyde, permeabilized and blocked with DPBS containing 0.3% Triton X-100 and 5% bovine serum albumin. The slides were incubated overnight at 4\u0026deg;C with the following antibody; fluorescence-conjugated α-actin (sc-17829, 1:200; Santa Cruz Biotechnology). After five times of washing with PBS, 4\u0026prime;,6-diamidino-2-phenylindole (DAPI) was incubated for 7 min to label the nuclei. Anti-α-actin treated cells were washed and treated with 4\u0026prime;,6-diamidino-2-phenylindole (DAPI) for 7 min. The stained samples were observed using a Zeiss LSM 710 confocal microscope (Carl Zeiss, Jena, Germany).\u003c/p\u003e\u003cp\u003e\u003cb\u003eAnimal experiments.\u003c/b\u003e All animal experiments were under ethical approval of Institutional Animal Care and Use Committee of Yonsei University College of Medicine (approval no. 2024\u0026thinsp;\u0026minus;\u0026thinsp;0222) and adhered to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (Publication No. 85\u0026thinsp;\u0026minus;\u0026thinsp;23, revised 1996). C57BL/6 male mice were purchased from Orient Bio Inc. (Seongnam, Korea), allowed unrestricted access to food and water and maintained at standard conditions (temperature, 20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 ◦C; humidity, 60\u0026thinsp;\u0026plusmn;\u0026thinsp;5%; light/dark cycle, 12 h). The mice went through anesthetization with intraperitoneal injections of tiletamine-zolazepam (Zoletil\u0026reg; 50, 30 mg/kg) and xylazine (Rompun\u0026reg;, 10 mg/kg) mixed at a 1:1 ratio and implanted with Ang II-containing Alzet\u0026reg; 1002 micro-osmotic pumps (2 mg/kg/day; Durect Corp., Cupertino, CA, USA). The control mice were implanted with Alzet\u0026reg; 1002 micro-osmotic pumps containing PBS.\u003c/p\u003e\u003cp\u003eThe mice were divided into 4 groups. PBS-pump implanted group was injected with PBS, and Ang Ⅱ-pump implanted mouse group were each injected with PBS, ANVP-NC, or ANVP-siAEBP1 (at a 1:1 ratio of NC and siAEBP1 to ANVP, 300 \u0026micro;g each per mouse). The magnet (\u0026Oslash; 5mm, IV-MAG3) was applied for 30 minutes after the injection of the samples to evaluate the effect of the targeted delivery.\u003c/p\u003e\u003cp\u003eTwo days after final injection, the mice were sacrificed and the blood, heart, liver, spleen, kidney were collected. Serum isolated from blood underwent hemogram analysis and the level of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and albumin (ALB) evaluated using Fuji Dri-Chem 4000i (Fujifilm, Tokyo, Japan) according to the manufacturer\u0026rsquo;s instructions. The collected organs were used for RNA extraction, cDNA synthesis, qRT-PCR, ELISA, and immunohistochemical staining.\u003c/p\u003e\u003cp\u003e\u003cb\u003eImmunohistochemical staining.\u003c/b\u003e For immunohistochemical observation, the tissues were dissected, fixed in 4% paraformaldehyde, followed by paraffin embedding and sectioning into 4-\u0026micro;m-thick slices. To analyze the expression of MLC3 in AF mice cardiomyocytes, anti-MLC3 antibody (ab233220, Abcam) was used for primary antibody and mouse anti-rabbit (sc-516250, Santa Cruz Biotechnology) antibody was used for fluorescent secondary antibody and the nuclei was stained using DAPI (62248, Thermo Fisher Scientific). Histological analyses were performed using H\u0026amp;E staining to evaluate tissue pathology, iNOS (ab15323, Abcam) staining with DAB (3,3'-diaminobenzidine) to assess inflammatory responses and Masson's Trichrome (MT) for the identification of fibrotic areas. The nuclei were labeled with hematoxylin. Anti-AEBP1 (sc-271374, Santa Cruz Biotechnology) was incubated with Alexa Fluro\u0026reg; 488 Conjugation Kit (ab236553, Abcam) and stained to evaluate the fluorescent of AEBP1 expression. After, AEBP1 was co-stained with cardiac troponin I antibody (ab47003, Abcam) and subsequently incubated with secondary antibody (mouse anti-rabbit IgG-CFL 647, sc-516251, Santa Cruz).\u003c/p\u003e\u003cp\u003eThe tissues were observed under an inverted microscope (Olympus, Japan) and Zeiss LSM 710 confocal microscope (Carl Zeiss, Jena, Germany). To analyze fibrotic area and iNOS expression, quantification using the Image J 1.50i software (National Institutes of Health, Bethesda, MD, USA) was conducted and the quantification of MLC3 was measured using confocal microscope (Zeiss LSM 710, Carl Zeiss).\u003c/p\u003e\u003cp\u003e\u003cb\u003eIn vivo\u003c/b\u003e \u003cb\u003ebiodistribution.\u003c/b\u003e To visualize the biodistribution, ANV and ANVP were labeled with Cy5.5. At 4h after tail vein administration (300ug of ANV and ANVP per mice), animals were euthanized and the major organs (heart, lung, liver, spleen, and kidney) were imaged using IVIS \u0026reg; spectrum \u003cem\u003ein vivo\u003c/em\u003e imaging system (PerkinElmer, Waltham, MA, USA).\u003c/p\u003e\u003cp\u003e\u003cb\u003eEchocardiography analysis.\u003c/b\u003e Cardiac function was evaluated by performing echocardiology using the Vevo 2100 system (VisualSonics, Toronto, Ontario, Canada). Left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) were assessed by calculating the ejection fraction (EF) and fractional shortening (FS) according to the following formulas: LVEF (%) LVEF (%) = [(left ventricular end-diastolic volume (LVEDV) ‒ left ventricular end-systolic volume (LVESV))/LVEDV] \u0026times; 100; LVFS (%) = [(left ventricular end-diastolic diameter (LVEDD) ‒ left ventricular end-systolic diameter (LVESD))/LVEDD] \u0026times; 100.\u003c/p\u003e\u003cp\u003e\u003cb\u003eStatistical analysis.\u003c/b\u003e Statistical analysis between two groups which meets the assumption of homoscedasticity were evaluated using Student's t-test and more than two groups exhibiting homoscedasticity were analyzed using the one-way analysis of variance with Tukey's post hoc test. Data that did not meet the assumption of homoscedasticity were analyzed using the Holm\u0026ndash;Sidak method. GraphPad software 8.0 was used for statistical analysis and p values\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered statistically significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eSynthesis and characterization of magnetic engineered Apoptotic MSC-derived nanovesicles.\u003c/b\u003e ANV were initially generated to minimize the inflammatory risk, promote tissue healing and simultaneously function as bioactive vesicles (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). To induce apoptosis in MSCs, cells were treated with varying concentrations of Staurosporine (STS) at multiple time points. The morphology of the cells began to shift after STS treatment (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA). Time- and concentration- dependent viability tests were conducted. Viability began to decrease from 0.25 \u0026micro;M, and for the 6h treatment, concentrations higher than 0.25 \u0026micro;M did not show a significant difference in cytotoxicity (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB). The level of apoptosis in MSCs at 6h post 0.25\u0026micro;M of STS treatment was assessed by FITC-Annexin/PI double staining and flow cytometry. After 6h treatment, the total percentage of early apoptotic increased to 13.4%. and late apoptosis increased to 4.2% (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eC). Based on these results, we selected a treatment with 0.25 \u0026micro;M STS for 6 hours to generate apoptotic MSCs for subsequent experiments. Following STS treatment, apoptotic MSCs were introduced by serial extrusion through membrane filters with diminishing pore sizes, thereby mechanically disrupting the plasma membrane of cells (34) and generated ANV.\u003c/p\u003e\u003cp\u003eANV were functionalized with MNP conjugated to anti-CD63 antibodies for vesicle capture and anti-MLC3 antibodies to enhance the specificity and binding affinity toward AF myocardial tissue.(35, 36) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). ANVP characterization was conducted to confirm successful MNP conjugation and the preservation of native nanovesicle properties. The Fourier Transform Infrared Spectroscopy (FT-IR) data revealed the expected characteristic peaks of functional groups from each synthetic step, including modification of the IONPs (Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e) (~\u0026thinsp;500\u0026ndash;600 cm\u003csup\u003e-1\u003c/sup\u003e), IONP-DBCO (~\u0026thinsp;1000\u0026ndash;1300 cm\u003csup\u003e-1\u003c/sup\u003e), IONP-DBCO modifications with anti-CD63 (~\u0026thinsp;1650 cm\u003csup\u003e-1\u003c/sup\u003e) and anti-MLC3 (~\u0026thinsp;1540 cm\u003csup\u003e-1\u003c/sup\u003e) antibodies, as well as the final conjugation with ANV, which exhibited peak shift around ~\u0026thinsp;1200\u0026ndash;1400 cm\u003csup\u003e-1\u003c/sup\u003e(C-O-C bond) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Transmission electron microscopy (TEM) images revealed that NV and ANV exhibited spherical shapes of similar sizes, consistent with previous reports.(36) The morphology of ANVP was observed to have multiple MNP conjugated to their surface (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). The average zeta potential of NV was \u0026minus;\u0026thinsp;18.65 mV, ANV was \u0026minus;\u0026thinsp;23.94 mV, and ANVP was \u0026minus;\u0026thinsp;28.26 mv which is similar to exosomes and nanovesicles in our previous study (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD).(36) We used nanoparticle tracking analysis (NTA) to assess the size distribution of ANVP. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE, NV and ANV exhibited the most frequent particle size distributions (modes) of 111.6 nm and 116.8 nm. After conjugation with MNP, ANVP showed 166.8 nm particle size. These experimental results demonstrated that MNP were successfully conjugated to ANV, leading to the formation of ANVP. Moreover, 1 month of storage in -20\u0026deg;C and \u0026minus;\u0026thinsp;80\u0026deg;C had no significant effect on the average particle size of ANVP consistent with previous reports (Figure S2).(37) Western blot analysis revealed that CD81, CD63, and Lamp2 are enriched in NV, ANV and ANVP showing ANVP possess the same features as typical nanovesicles (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). These results confirm that ANVP was successfully modified and exhibited the characteristic features of nanovesicles, making them suitable delivery vehicles.\u003c/p\u003e\u003cp\u003e\u003cb\u003eTargeting ability of ANVP toward AF cardiomyocyte\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e. To evaluate the potential of MLC3 as a target in AF, we assessed its expression in patient-derived AF tissue samples and \u003cem\u003ein vitro\u003c/em\u003e models. MLC3 mRNA expression analysis of tissue samples collected from patients in the control (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5) and AF groups (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5) revealed a statistically significant difference between the two groups (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). The clinical profiles of the control and AF groups are listed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, and no significant differences in age or body mass index were observed between the two groups. To examined whether MLC3 are upregulated in atrial cardiomyocytes, we treated Angiotensin Ⅱ (Ang Ⅱ) to human induced pluripotent stem cell-derived atrial cardiomyocytes (iPSC-aCMs) based on the previous studies.(38, 39) Briefly, 0.5 \u0026micro;M or 1 \u0026micro;M of Ang Ⅱ were treated to iPSC-aCMs for 24 hours, and cell area based on α-actin staining was assessed through immunocytochemistry staining. An increase in cell area was observed following drug treatment, which appears to be a consequence of AF progression in cardiomyocytes (Figure S3A, B).(40) Therefore, Ang Ⅱ concentration of 0.5 \u0026micro;M was chosen for subsequent experiments.\u003c/p\u003e\u003cp\u003eMLC3 mRNA expression was augmented in Ang Ⅱ-treated iPSC-aCMs (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB) and exhibited a notable increase in relative fluorescence intensity (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) compared to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, D). Our experimental findings demonstrate that MLC3 is significantly overexpressed in the Ang II-treated iPSC-aCM model, which mimics AF pathology. This observation was corroborated by overexpression detected in the cardiac tissues of patients with AF. Our results suggest that elevated MLC3 levels in AF tissues and iPSC-aCMs may provide a new avenue for selective targeting of atrial cardiomyocytes affected by AF.\u003c/p\u003e\u003cp\u003eTo verify whether the anti-MLC3 antibody facilitated the binding of ANVP to the cellular membrane, we conjugated Cy5.5 to the MNP, followed by conjugation with ANV, and administered it to the cells under experimental conditions. Fluorescent signals were detected on the cell surface, indicating successful membrane localization of the labeled ANVP (Figure S4). To further evaluate whether the improved binding affinity of ANVP compared to ANV enhanced the intracellular uptake of ANVP, we stained MLC3 and incubated it with PKH67 labeled ANV. Ang Ⅱ-treated iPSC-aCMs were treated with fluorescence-labeled ANV or ANVP each for 24h. The number of ANVP conjugated to iPSC-aCM membranes through MLC3 was higher than those conjugated to ANV (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). This raises the possibility that MLC3-based targeting strategies can improve the precision of therapeutic delivery by directing delivery to the regions of the heart associated with AF pathology, thereby facilitating more efficient intercellular delivery.\u003c/p\u003e\u003cp\u003e\u003cb\u003eIn vivo targeting of AF cardiomyocytes by ANVP\u003c/b\u003e. Given the strong \u003cem\u003ein vitro\u003c/em\u003e targeting performance of ANVPs, we next evaluated their therapeutic potential \u003cem\u003ein vivo\u003c/em\u003e using a murine model of AF. The \u003cem\u003ein vivo\u003c/em\u003e data detailed below further support the role of MLC3-based targeting in enhancing cardiac delivery specificity and therapeutic efficacy. To verify whether Ang Ⅱ-treated AF mice showed the upregulation of MLC3, both mRNA expression levels and immunohistochemical (IHC) analyses were performed. qRT-PCR demonstrated a significant elevation in MLC3 mRNA levels in AF model mice compared to control mice (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Correspondingly, increased fluorescence signals were observed in heart tissues via IHC staining, confirming elevated MLC3 expression in mice with AF (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, C). These findings suggest that targeting MLC3 using ANVP can enhance the delivery specificity and retention within the myocardium of AF model mice.\u003c/p\u003e\u003cp\u003eFor \u003cem\u003eex vivo\u003c/em\u003e fluorescence imaging, control mice received phosphate-buffered saline (PBS), whereas AF mice were administered either Cy5.5-labeled ANV or ANVP. Four hours post-injection, the major organs were harvested, and the fluorescent signal intensity was quantified. Notably, mice treated with ANVP exhibited the highest fluorescence retention in the myocardium under magnetic navigation, suggesting synergistic enhancement through both MLC3 targeting and external magnetic guidance (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD, E). Notably, ANV injection led to a substantial reduction in accumulation within other organs, such as the kidneys and lungs, when compared to both the PBS and ANV injection groups (Figure S5A, B). Both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e data confirmed significant upregulation of MLC3 under AF conditions. By targeting upregulated MLC3, we enhanced the cellular uptake of ANVP, thereby developing a delivery system with improved specificity and efficiency for cardiomyocytes targeting.\u003c/p\u003e\u003cp\u003e\u003cb\u003eRationale for targeting AEBP1 as a therapeutic target in AF.\u003c/b\u003e AEBP1 is known to be upregulated in response to various stress conditions in the heart and has been implicated in promoting inflammation and fibrosis.(23, 41) Analysis of previously published single-nucleus profiling data from patients with hypertrophic cardiomyopathy as well as failing hearts revealed elevated expression of AEBP1 in myofibroblasts accompanied by increased expression of fibrosis-related genes.(25) Although a previous study has shown that AEBP1 knockdown can attenuate these pathological processes in ischemia-reperfusion injury,(42) and its regulation, particularly under AF-like conditions, remains less well defined. Given the established association between AEBP1 and stress-related signaling pathways,(43, 44) we hypothesized that AEBP1 silencing would attenuate the excessive activation of inflammatory and fibrotic pathways under AF-induced conditions. To test this hypothesis, we first examined whether AEBP1 expression was elevated under AF-induced stress and investigated the potential therapeutic effects of AEBP1 knockdown.\u003c/p\u003e\u003cp\u003eTo investigate whether AEBP1 exhibited increased expression in AF, we analyzed the mRNA expression profiles and protein levels using patient-derived tissue samples (n\u0026thinsp;=\u0026thinsp;5 per group). AEBP1 showed elevated mRNA expression (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA) and enhanced protein levels of AEBP1 (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, C) in AF tissues. iPSC-aCMs were treated with Ang Ⅱ, which serves as a relevant \u003cem\u003ein vitro\u003c/em\u003e model for AF-related stress. These cells demonstrated a significant upregulation of AEBP1 mRNA expression (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD), consistent with our observations in the patient samples. Subsequently, we evaluated whether AEBP1 expression was upregulated in the mouse model as expected, Ang Ⅱ-treated mice showed increased AEBP1 mRNA expression (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE).\u003c/p\u003e\u003cp\u003e\u003cb\u003eIn vitro\u003c/b\u003e \u003cb\u003esilencing of AEBP1 via ANVP-siAEBP1.\u003c/b\u003e To enhance the delivery of siAEBP1 to cardiomyocytes in an AF model, we encapsulated siAEBP1 in ANV we previously made by electroporation and conjugated it with MNP to generate ANVP-siAEBP1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). The gene silencing efficacy of siAEBP1 was preserved regardless of its encapsulation within ANVP, indicating that the nanovesicle was successfully associated with the cellular membrane and delivered siRNA to effectively suppress AEBP1 gene expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG-I). This study demonstrates that ANVP is capable of efficiently transporting siAEBP1 into the intracellular environment, enabling effective gene silencing within target cells.\u003c/p\u003e\u003cp\u003e\u003cb\u003eRestoration of cardiac function via AEBP1 silencing\u003c/b\u003e \u003cb\u003ein vivo\u003c/b\u003e. As previously reported, in AF, the progression of inflammatory responses, such as cytokine release and fibrosis in the cardiac tissue, is increased. To determine whether ANVP-siAEBP1 treatment attenuated the observed pathologies, we conducted an \u003cem\u003ein vivo\u003c/em\u003e study to evaluate its therapeutic efficacy. We first generated AF mimic mice by subcutaneously implanting osmotic pumps loaded with Ang Ⅱ. Control mice were implanted with a PBS- containing pump. Seven days post-implantation, the mice were intravenously injected with PBS, ANV, ANVP-NC, or ANVP-siAEBP1 via the tail vein in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA. This time point was selected because AEBP1 is considered crucial for the initial fibrosis response (23) and for effective siRNA delivery. For magnetic navigation, the magnet was externally positioned over the cardiac region, 30 min after each injection. Cardiomyocytes play a crucial role in modulating the inflammatory response after cardiac injury by producing a range of pro-inflammatory mediators, including cytokines and inflammatory enzymes. Therefore, cardiomyocyte-derived inflammatory signaling may offer a safer and more effective strategy for post-injury cardiac modulation.(45, 46) Although ANV are known to exert anti-inflammatory effects, their specific effects on AF- associated inflammation remain unclear. In this study, we present evidence from mouse models of AF demonstrating that ANV treatment exerts anti-inflammatory effects. Furthermore, encapsulation of siAEBP1 enhanced these effects synergistically, leading to the suppression of both the inflammatory response and cardiac fibrosis, without observable host toxicity (Figure S8). To validate the establishment of the AF model, echocardiographic assessment was performed one week after pump implantation, confirming a decline in cardiac function in the AF model group (Figure S6).\u003c/p\u003e\u003cp\u003eTo assess the knockdown efficiency of AEBP1 by ANVP-siAEBP1, we conducted co-immunostaining for AEBP1 and cardiac troponin I (cTnI) in ANVP-targeted cardiac cells. Immunofluorescence analysis revealed a marked reduction in AEBP1 expression in the ANVP-siAEBP1 treated group compared that in the PBS- (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and ANVP-NC- treated groups (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, C). These results indicate that AEBP1 was efficiently silenced by ANVP-siAEBP1 in cardiomyocytes. Consistently, mRNA expression analysis confirmed a substantial reduction in AEBP1 expression following targeted knockdown (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). Echocardiography showed that ANVP-siAEBP1 injection has improved the ejection fraction (EF%) (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and fractional shortening (FS%) (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) compared to the ANVP injection, and no statistically significant difference was observed compared to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE, F). Taken together, these results suggest that AEBP1 was effectively silenced by ANVP-siAEBP1 and that cardiac function was restored suggesting its therapeutic potential.\u003c/p\u003e\u003cp\u003e\u003cb\u003eAttenuation of inflammation and fibrosis by ANVP-siAEBP1\u003c/b\u003e \u003cb\u003ein vivo\u003c/b\u003e. Following the successful knockdown of overexpressed AEBP1 and partial restoration of cardiac function by ANVP-siAEBP1, we investigated its efficacy in reducing inflammation and fibrosis. Histological analysis using DAB staining was performed to assess the expression levels of inducible Nitric Oxide Synthase (iNOS), a well-established marker of inflammation.(47) While treatment with ANVP alone led to a modest reduction in inflammation (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05); ANVP-siAEBP1 treatment markedly suppressed inflammation to levels comparable to those observed in the AF group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, B). This anti-inflammatory effect was confirmed at the mRNA level. Quantitative analysis of inflammation-related cytokines and markers in myocardial tissue revealed decreased expression of IL-1β, TNF-α, and iNOS (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) following ANVP-siAEBP1 administration (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003eSubsequently, the extent of fibrosis was assessed. Histological staining demonstrated that ANVP-siAEBP1 treatment effectively decreased the volume fraction of total fibrosis (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) in the myocardium compared with that in the AF group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD, E). Furthermore, the expression levels of multiple fibrosis-associated downstream factors were also significantly downregulated, including collagen Ⅰ, COL3a1, and α-SMA (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) indicating a broad attenuation of fibrotic signaling pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF). No significant histological alterations were observed in major organs, including the heart, kidneys, liver, lung, and spleen following intravenous injection of each ANVP samples (Figure S7). These findings suggest that the administration of ANVP-siAEBP1 not only suppresses excessive inflammatory responses, but also inhibits the progression of fibrosis in the myocardium.\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eIn this study, we developed bioengineered nanovesicles, termed ANVP, by conjugating ANV with MNP (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Although ANV have previously demonstrated therapeutic efficacy in models of cardiac injury,(48) their utility in AF, particularly in AF-induced inflammation, has not been thoroughly investigated. While IONP conjugated with anti-MLC3 antibodies have shown promising results in MI,(16) and ischemic models,(17) their application in AF has not been explored, likely because MLC3 has traditionally been identified as a ventricular marker.(49) Interestingly, our data suggested that MLC3 expression was upregulated in AF-induced cardiomyocytes, indicating a potential paradigm shift in the use of MLC3 as a viable molecular target for AF. Our \u003cem\u003ein vitro\u003c/em\u003e data suggested that ANVP preferentially accumulated in regions with elevated MLC3 expression under AF conditions, indicating MLC3-mediated targeting (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The \u003cem\u003ein vivo\u003c/em\u003e results validate the potential of this platform. AF-induced mouse models show elevated MLC3 expression, confirming its relevance as a target in AF pathology. Upon intravenous administration, the ANVP navigated effectively to the AF-affected myocardium under external magnetic guidance (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eGiven its role in AF-induced inflammation and fibrosis, AEBP1 is considered a promising therapeutic target, and these data were validated through our experiments on patient tissues, iPSC-aCMs, and an AF-induced mouse model. In addition, ANVP delivery of siAEBP1 significantly silenced AEBP1 expression in Ang Ⅱ-treated iPSC-aCMs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The targeted fusion of ANVP facilitated the specific silencing of AEBP1 expression \u003cem\u003ein vivo\u003c/em\u003e while partially recovering cardiac function through AEBP1 attenuation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Concurrent with ANVP treatment and AEBP1 silencing, the secretion of pro-inflammatory cytokines was reduced, leading to an overall attenuation of the inflammatory response. Simultaneously, siAEBP1 treatment resulted in a marked reduction in fibrosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThis strategy overcomes the major limitations associated with traditional siRNA therapy, including low transfection efficiency, limited cellular uptake, and systemic toxicity.(50, 51) Moreover, the magnetic navigation system and MLC3 targeting strategy provide improved delivery efficiency to the AF heart, which is a critical advancement over passive targeting approaches.\u003c/p\u003e\u003cp\u003eThis approach not only introduces MLC3 as a novel therapeutic target in AF, but also exerts inflammation reduction and synergistic anti-fibrotic effects through gene silencing, offering significant translational potential for future cardiac therapies.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eAF\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eAtrial Fibrillation\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eANV\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eApoptotic mesenchymal stem cell-derived nanovesicle\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eAEBP1\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eAdipocyte enhancer binding protein 1\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003esiRNA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003esmall interfering RNA\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eANVP\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eApoptotic mesenchymal stem cell-derived nanovesicle conjugated with iron oxide magnetic nanoparticle\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eMLC3\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003emyosin light chain 3\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eMSC\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003emesenchymal stem cell\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eHCM\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eHypertrophic cardiomyopathy\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eACLP\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eaortic carboxypeptidase-like protein\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eSTS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003estaurosporine\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eFT-IR\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eFourier Transform Infrared Spectroscopy\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eIONP\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eiron oxide magnetic nanoparticle\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eDBCO\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eDibenzocyclooctyne\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eTEM\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eTransmission electron microscopy\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eNTA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003enanoparticle tracking analysis\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eiPSC-aCM\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ehuman induced pluripotent stem cell-derived atrial cardiomyocyte\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eAng Ⅱ\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eangiotensin Ⅱ\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCy5.5\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eSulfo-Cyanine5.5\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eIHC\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eimmunohistochemical\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eqRT-PCR\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003equantitative reverse transcription polymerase chain reaction\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ePBS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ePhosphate Buffered Saline\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eNC\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003enegative control\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ecTnI\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ecardiac troponin I\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eDAB\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003e3,3'-Diaminobenzidine\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eiNOS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eNitric Oxide Synthase\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eIL-1β\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eInterleukin-1 beta\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eTNF-α\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eTumor Necrosis Factor alpha\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCOL3a1\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ecollagen type III alpha 1 chain\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eα-SMA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ealpha-smooth muscle actin\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll procedures involving human participants were approved by the Institutional Review Board of Severance Hospital, Yonsei University (IRB No. 4-2019-0620), and written informed consent was obtained from each subject.\u003c/p\u003e\n\u003cp\u003eAll animal experiments were under ethical approval of Institutional Animal Care and Use Committee of Yonsei University College of Medicine (approval no. 2024-0222) and adhered to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (Publication No. 85-23, revised 1996).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll participants provided consent for the publication of this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe dataset(s) supporting the conclusions of this article is(are) included within the article (and its additional file(s)).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eKorean government (the Ministry of Science and ICT, the Ministry of Health \u0026amp; Welfare, KFRM21B0604L1-01 and 24A0202L1), National Research Foundation of Korea grants funded by the Korean government (MSIT) (2021R1C1C2094541 and 2023R1A2C3003320). Korean Cardiac Research Foundation (202101-02). Department of Cardiology, Graduate School of Medical Science, Brain Korea 21 Project, Yonsei University College of Medicine.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGyeongseo Yoo: Conceptualization; Investigation; Validation; Writing\u0026mdash;original draft. Ji-Young Kang: Investigation. Malgeum Park: Investigation. Jaewoong Lee: Validation. Dasom Mun: Conceptualization; Supervision. Nuri Yun: Conceptualization; Supervision. Boyoung Joung: Conceptualization; Supervision\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the National Research Foundation of Korea grants funded by the Korean government (MSIT) (2021R1C1C2094541 and 2023R1A2C3003320); the Korean Fund for Regenerative Medicine grant funded by the Korean government (the Ministry of Science and ICT, the Ministry of Health \u0026amp; Welfare, KFRM21B0604L1-01 and 24A0202L1); the Korean Cardiac Research Foundation (202101-02); and the Department of Cardiology, Graduate School of Medical Science, Brain Korea 21 Project, Yonsei University College of medicine. The author would like to thank BioRender (www.biorender.com) for figure preparation assistance, and Editage (www.editage.co.kr) for English language editing.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eKornej J, Borschel CS, Benjamin EJ, Schnabel RB. 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Nanomedicine (Lond). 2014;9(2):295-312.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Scheme 1","content":"\u003cp\u003eScheme 1 is available in the Supplementary Files section.\u003c/p\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":"Atrial fibrillation, Engineered apoptotic nanovesicles, Inflammation, Fibrosis, AEBP1 silencing","lastPublishedDoi":"10.21203/rs.3.rs-7061113/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7061113/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eChronic inflammation and fibrosis contribute markedly to the progression and recurrence of Atrial Fibrillation (AF), which is the most common form of arrhythmia. Despite ongoing therapeutic advancements, effective treatments to modulate inflammatory and fibrotic processes in AF remain limited. To overcome these limitations, we developed a novel nanotherapeutic system using apoptotic mesenchymal stem cell-derived nanovesicles (ANV) as biocompatible and immunomodulatory delivery platforms for siRNA targeting the AEBP1 gene, leading to concurrent attenuation of fibrosis.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eANV were constructed via an extrusion method and loaded with adipocyte enhancer binding protein 1 (AEBP1)-targeting small interfering RNA (siRNA) (siAEBP1) through electroporation to form ANV-siAEBP1. ANV-siAEBP1 were then incubated with antibody-conjugated iron oxide magnetic nanoparticles (MNP), forming ANVP-siAEBP1 complex. For targeted delivery to the AF myocardium, an anti-myosin light chain 3 (MLC3) antibody was incorporated to facilitate localized accumulation in MLC3-enriched atrial cardiomyocytes. Once localized, the nanovesicles fused with cardiomyocyte membranes, allowing for the intracellular release of siAEBP1, which in turn silenced AEBP1 expression, thereby downregulating pro-fibrotic signaling and mitigating atrial fibrosis. Simultaneously, the intrinsic anti-inflammatory effects of ANV in stressed cardiomyocytes, prevented excessive inflammatory responses.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003eThis dual mechanism of action, involving siRNA-mediated gene silencing and ANV-induced immunomodulation, results in a synergistic therapeutic effect. Thus, ANVP-siAEBP1 significantly attenuated both inflammation and fibrosis in AF myocardium with enhanced targeting efficiency, offering a promising strategy for next-generation precision therapeutics in AF.\u003c/p\u003e","manuscriptTitle":"Enhanced Therapeutic potential of AEBP1 silencing via engineered Apoptotic Mesenchymal Stem Cell- derived Nanovesicles in Atrial Fibrillation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-19 09:49:18","doi":"10.21203/rs.3.rs-7061113/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":"679759b8-0034-484c-9f65-31c9d0053864","owner":[],"postedDate":"August 19th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-09-16T09:08:28+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-19 09:49:18","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7061113","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7061113","identity":"rs-7061113","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}