Human adipose stem cell-derived extracellular nanovesicles for treatment of chronic liver fibrosis

Journal Pre-proof Human adipose stem cell-derived extracellular nanovesicles for treatment of chronic liver fibrosis

Hwa Seung Han, Hansang Lee, DongGil You, Van Quy Nguyen, Dae-Geun Song, Byeong Hoon Oh, Sol Shin, Ji Suk Choi, Jae Dong Kim, Cheol-Ho Pan, Dong-Gyu Jo, Yong Woo Cho, Ki Young Choi, Jae Hyung Park PII:

S0168-3659(20)30061-4

DOI:

https://doi.org/10.1016/j.jconrel.2020.01.042

Reference:

COREL 10138

To appear in:

Journal of Controlled Release

Received date:

31 July 2019

Revised date:

19 January 2020

Accepted date:

21 January 2020

Please cite this article as: H.S. Han, H. Lee, D. You, et al., Human adipose stem cellderived extracellular nanovesicles for treatment of chronic liver fibrosis, Journal of Controlled Release (2020), https://doi.org/10.1016/j.jconrel.2020.01.042

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

© 2020 Published by Elsevier.

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Human adipose stem cell-derived extracellular nanovesicles for treatment of chronic liver fibrosis Hwa Seung Hana,1 , Hansang Lee b,1 , Dong Gil Youb, Van Quy Nguyenb, Dae-Geun Songa, Byeong Hoon Ohb, Sol Shinc, Ji Suk Choid,e, Jae Dong Kime, Cheol-Ho Pana, Dong-Gyu Jof, Yong Woo Chod,e*** , Ki Young Choia,** and Jae Hyung Parkb,c,g* a

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Natural Product Informatics Research Center, Korea Institute of Science and Technology (KIST), Gangneung 25451, Republic of Korea b School of Chemical Engineering, Sungkyunkwan University, Suwon 16419, Republic of Korea c Department of Health Sciences and Technology, SAIHST, Sungkyunkwan University, Seoul 06351, Republic of Korea d Department of Chemical Engineering, Hanyang University, Ansan 15588, Republic of Korea. e Research Institute, Exostemtech Inc., Ansan 15588, Republic of Korea. f School of Pharmacy, Sungkyunkwan University, Suwon 16419, Republic of Korea. g Biomedical Institute for Convergence at SKKU (BICS), Sungkyunkwan University, S uwon 16419, Republic of Korea

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*** Corresponding author. Prof. Yong Woo Cho Department of Chemical Engineering Hanyang University, Ansan 15588, Republic of Korea Tel: +82-31-400-5279; fax: +82-31-400-3942; e-mail: [email protected] ** Corresponding author. Ki Young Choi, Ph.D. Natural Product Informatics Research Center Korea Institute of Science and Technology (KIST), Gangneung 25451, Republic of Korea Tel: +82-33-650-3510; fax: +82-2-2179-8616; e-mail: [email protected] * Corresponding author. Prof. Jae Hyung Park School of Chemical Engineering Sungkyunkwan University, Suwon 16419, Republic of Korea Tel: +82-31-290-7288; fax: +82-31-299-6857; e-mail: [email protected]

Abstract

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Liver fibrosis is an excessive wound healing process that occurs in response to liver damage depending on underlying aetiologies. Currently, there are no effective therapies and FDAapproved therapeutics for the treatment of liver fibrosis except liver tra nsplantation. Multipotent adipose-derived stem cells (ADSCs) have received significant attention as regenerative medicine for liver fibrosis owing to their advantages over stem cells with other origins. However, intrinsic limitations of stem cell therapies, such as cellular rejection and

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tumor formation, have impeded clinical applications of the ADSC-based liver therapeutics. To overcome these problems, the extracellular nanovesicles (ENVs) responsible for the

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therapeutic effect of ADSCs (A-ENVs) have shown considerable promise as cell- free

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therapeutics for liver diseases. However, A-ENVs have not been used for the treatment of intractable chronic liver diseases including liver fibrosis and cirrhosis. Therefore, in this study,

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we investigated the in vitro and in vivo antifibrotic efficacy of A-ENVs in thioacetamide-

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induced liver fibrosis models. A-ENVs significantly downregulated the expression of fibrogenic markers, such as matrix metalloproteinase-2, collagen-1, and alpha-smooth muscle

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actin. The systemic administration of A- ENVs led to high accumulation in fibrotic liver tissue

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and the restoration of liver functionality in liver fibrosis models through a marked reduction in α-SMA and collagen deposition. These results demonstrate the significant potential o f AENVs for use as extracellular nanovesicles-based therapeutics in the treatment of liver fibrosis and possibly other intractable chronic liver diseases.

Keywords: Liver fibrosis, Adipose-derived stem cells, Extracellular nanovesicles 1. Introduction Liver fibrosis is one of the major causes of morbidity and mortality worldwide. It is a

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chronic liver disease that arises from an excessive wound healing response caused by liver injury depending on underlying aetiologies—viral hepatitis, alcoholic hepatitis, nonalcoholic steatohepatitis, autoimmune liver diseases, and metabolic disorders [1-5]. During hepatic fibrogenesis, liver tissue undergoes fundamental remodeling characterized by hepatocyte necrosis, inflammation, oxidative stress, and fibrillary extracellular matrix (ECM) deposition, which may culminate in cirrhosis and liver cancer [6, 7]. Over the past three decades, a

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number of researchers have studied the mechanisms underlying the critical events during fibrogenesis to find effective therapeutics for liver fibrosis, which inhibit the activation of

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hepatic stellate cells (HSCs) to prevent ECM deposition or induce the degradation of ECM to

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achieve antifibrotic effects [8, 9]. However, until now, there are no effective therapies or FDA-approved antifibrogenic agents, making liver transplantation the only curative treatment

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for decompensated cirrhotic liver disease [10].

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Multipotent mesenchymal stem cell (MSC)-based regenerative medicine has gained considerable attention as an effective treatment for various refractory diseases owing to the

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ability of MSCs to repair damaged tissues and restore functionality by differentiation into

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multiple cells [11-13]. Based on these characteristics, various stem cells derived from human bone marrow (BMSCs), umbilical cord (UCSCs), and adipose tissue (ADSCs) have been used for the treatment of liver disease [14-18]. Interestingly, previous studies revealed that MSC transplantation could protect hepatocytes by reducing ROS damage and downregulate myofibroblast activity, leading to an antifibrotic effect in fibrotic tissue [19-21]. Nevertheless, these cell-based transplantations have numerous limitations including the reduction in the capacity of engrafted cells caused by immune- mediated rejection and iatrogenic tumor formation [22]. In particular, the clinical use of BMSCs and UCSCs for future cell-based therapies might not satisfy patients’ requirements because of the highly invasive aspiration procedure, the decline in the proliferation and differentiation of cells by senescence, and

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difficulties in mass production and extremely high costs [23, 24]. To overcome these problems, a novel cell- free therapy based on stem cell-derived extracellular membrane vesicles may provide a new therapeutic strategy owing to the ability of the vesicles to circumvent the risks of using viable replicating cells without compromising the adva ntages of therapeutic stem cells [25-27]. Extracellular nanovesicles (ENVs), which are nano-sized membrane vesicles with a

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diameter of 30–200 nm endogenously released by cells, have recently received considerable attention as alternative therapeutics because it is believed that MSCs achieve a therapeutic

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effect in vivo primarily through the ENVs responsible for the paracrine effects of MSCs [28-

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32]. These unique characteristics of ENVs have led researchers to investigate their potential for the treatment of liver failure [32-36]. It has been reported that the ENVs derived from

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BMSCs and UCSCs demonstrate an improved therapeutic effect in treating acute and chronic

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liver injuries when compared to MSCs [37, 38]. However, these approaches have some intrinsic limitations, which have also been found in the BMSC- or UCSC-based therapeutic

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approaches, hampering their clinical applications; large amounts of ENVs required for

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clinical applications cannot be easily harvested from these types of MSCs, which also leads to significant increases in costs [39]. To circumvent the obstacles, we hypothesized that ADSC-derived ENVs (A-ENVs) can be exploited as an alternative and optimal therapeutic ENV for treatment of liver fibrosis owing to the following intrinsic advantages of ADSCs over other types of MSCs. (1) ADSCs have shown excellent therapeutic potential for the treatment of liver fibrosis animal models. (2) A larger number of stem cells can be collected from adipose tissues than from other tissues (e.g. 40 times more than bone marrow). (3) ADSCs can also be harvested from adipose tissues more easily than other types of MSCs harvested from other tissues; the aspiration procedure for ADSCs is relatively less invasive than that for other types of MSCs.

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Moreover, (4) low risk of complications for patients and (5) their superior self- renewing ability are also seminal merits of ADSCs. Despite the fascinating features of ADSCs, however, clinical applications of ADSCs are still hampered by intrinsic drawbacks of t he MSC transplantation approach including cellular rejection and iatrogenic tumorigenesis. Therefore, A-ENV—an avatar of ADSC—can be considered as a safer and more effective therapeutic approach for liver diseases. The abundance of ADSCs are expected to facilitate

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large-scale ENV production and lower the cost for ENV-based therapeutics. The easy and relatively noninvasive harvesting procedure can provide patients with a safe therapeutic

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option [40-45]. Although engineered A- ENVs have been used as a miRNA delivery vehicle

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for treatment of liver fibrosis, or as ENV-therapeutics for acute liver failure in a few preliminary studies [46, 47], the use of A-ENVs as natural therapeutics for the treatment of

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chronic liver diseases like liver fibrosis or liver cirrhosis has not been reported to the best of

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our knowledge.

In this study, we aimed to evaluate whether natural A-ENVs could effectively

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attenuate liver fibrosis in a thioacetamide (TAA)-induced animal model by altering the

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microenvironment of activated HSCs in fibrotic tissue (Figure 1A). The experimental conditions for the investigation of the antifibrotic effect were optimized through in vitro comparison screening with ENVs of other origins. Based on in vitro studies, we further investigated the organ distribution and in vivo antifibrotic effect of A-ENVs in a fibrosis model using near- infrared fluorescence (NIRF) imaging, blood chemistry tests, and stained liver tissue analyses. Overall, the results indicated that natural A-ENVs could have significant potential as extracellular nanovesicles-based therapeutics for treating liver fibrosis.

2. Materials and methods 2.1 Materials

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The Radioimmunoprecipitation assay (RIPA) lysis buffer was purchased fro m Thermo Fisher Scientific lnc (MA, USA). Cy5.5 N-hydroxysuccimide(NHS) ester monoreactive CyDye was purchased from GE Healthcare (NJ, USA). Dulbecco’s modified eagle medium (DMEM), fetal bovine serum (FBS), Dulbecco's phosphate-buffered saline (DPBS), antibiotic-antimycotic (AA) solution (100X), and trypsin- EDTA were obtained from WelGENE (Gyeongsan, Korea). Anti-CD9 antibody (ab92726), Anti-CD63 antibody

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(ab59479), Anti-cytokeratin 18 antibody (ab668) and Anti-CD68 antibody (ab201340) were purchased from Abcam (Cambridge, UK). Anti- TGF-β1 (AF-100-21C) was obtained from

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PeproTech (CA, USA). Anti-Collagen 1A1 (#39952), Anti-p-SMAD2 (#3108), Anti-p-

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SMAD3 (#9520), Anti-SMAD2/3 (#5678), Anti-Snail (#3895), and Anti- GAPDH (#2118) were obtained from Cell Signaling Technology (MA, USA). Tangential flow filtration (TFF)

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with a 300-kDa MWCO ultrafiltration membrane filter capsule was purchased from Pall

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Corporation (NY, USA).

TAA, thiazolyl blue tetrazolium bromide (MTT), anti-β-actin (A2228), and anti-α-

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smooth muscle actin (α-SMA) were obtained from Sigma-Aldrich Co. (St. Louis, USA). CD9

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and CD63 ENV ELISA Complete Kit was purchased from System Bioscience (CA, USA). TRIzol reagent was obtained from Invitrogen (CA, USA). cDNA Reverse Transcription Kit and SYBR® Green PCR Master Mix were obtained from Applied Biosystems (CA, USA). Primary ADSCs were obtained from Cefobio Inc. (Seoul, Korea). For A-ENV isolation, a conditioned medium (CM) was collected by proliferating ADSCs with passages 7–9. The primers for matrix metalloproteinase-2 (MMP-2), collagen-1 (COL-1), and TGF-β1 were purchased from Macrogen (Seoul, Korea). 2.2 Isolation of A-ENVs ADSCs were seeded in a T-175 flask at a density of 1 × 106 cells with DMEM containing 10% FBS and 1% AA at 37℃ in a humidified 5% CO 2 atmosphere. After 4 d of

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culture (2 × 106 cells per flask), the cells were washed twice with DPBS and incubated with serum- free DMEM at 37℃ for 24 h. The CM was collected through centrifugation at 2,000 g for 20 min, followed by filtration using 0.22 μm filters to remove the cell debris from the medium. Subsequently, the filtered CM was concentrated via TFF with a 500 kDa MWCO ultrafiltration membrane filter capsule to remove contaminants, as previously reported [48]. Finally, purified A-ENVs were suspended in PBS and stored at -70℃ until further use. For the

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ex vivo organ imaging, A-ENVs were labelled with cyanine 5.5 N-hydroxysuccinimide

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(NHS) monoester. In brief, 10 μL of Cy5.5 NHS monoester dye (1 mg/ml) was mixed with 1

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ml of A-ENVs (1 × 1010 ) at 4℃ in the dark for 12 h. The solution was purified using

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Sephadex PD-10 column (GE Healthcare, NJ, USA) and subsequently used for in vivo

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imaging.

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2.3 Characterization of A-ENVs

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The morphology of A-ENVs was observed via transition electron microscopy (TEM)

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(JEOL-2100F, Tokyo, Japan) performed at an accelerating voltage of 200 kV. For TEM sample preparation, A-ENVs were dropped on a 200- mesh carbon- film-coated grid (Samchang Inc., Seoul, Korea), which was negatively stained with 1% uranyl acetate for 1 min and washed with deionized water. The size distribution and number of A-ENVs were measured using particle size analyzer (Nano ZS90, Malvern Instruments, Worcs, UK) and nanoparticle tracking analysis (NTA) (NanoSight LM10, Malvern Instruments Ltd., Malvern, UK). The samples were diluted 10-fold to 200- fold with PBS to reach the optimal concentration (20–30 particles/frame), and readings were performed in triplicates of 30 s at 30 frames per second. The surface zeta potential of A- ENVs was determined at 25℃ using Zetasizer Nano ZS90 (Malvern Instruments, Worcs, UK). The A-ENV proteins were

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quantitatively measured using Pierce BCA Protein Assay Kit (Thermo Fisher Scientific lnc, MA, USA) according to the manufacturer ’s manual.

2.4 Western blotting To examine the presence of ENV marker proteins, A-ENVs with the same

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concentration as that in the micro BCA protein assay were mixed with the RIPA buffer and

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boiled for 10 min at 95℃. Next, the samples with 100 μg proteins were loaded onto SDS-

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PAGE, followed by transfer onto PVDF membranes (Bio-Rad, CA, USA) at 90 V for 1 h.

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Then, the membranes were blocked with 5% nonfat dry milk (w/v) in Tris-buffered saline with 0.1% Tween-20 (TBS-T) for 2 h at room temperature and subsequently incubated with

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primary antibodies against CD9 and CD63 under non-reducing conditions and β-actin for 12 h at 4℃. After vigorous washing in TBS-T, the blots were incubated with horseradish-

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peroxidase- labeled secondary antibodies (Sigma-Aldrich Co., St. Louis, USA) for 1 h. The

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labeled proteins were visualized using the enhanced chemiluminescence plus kit (Thermo

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Fisher Scientific lnc, MA, USA), and the development was observed using ChemiDoc-IT2 (UVP, CA, USA).

To confirm antifibrotic effect of A-ENVs using western blot analysis, cells or animal tissues were harvested for whole-cell or tissue extracts using ice-cold RIPA buffer containing protease inhibitor cocktail (#78425, Thermo Fisher Scientific) and phosphatase inhibitor cocktail (#78428, Thermo Fisher Scientific). The cells were centrifuges and the supernatants were measured for protein concentration by micro BCA assay. The lysates were separated in Tris- glycine SDS-polyacrylamide gels at concentrations ranging from 4 to 15% and transferred to PVDF membranes. After blocking the membrane with 3% bovine serum albumin (BSA, Sigma-Aldrich), the membranes were incubated with the primary antibodies

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overnight. Protein bands were detected using ChemiDoc T M XRS+ with Image LabT M software (Bio-Rad, CA, USA).

2.5 Quantitative RT-PCR To investigate the change in fibrogenic gene expression, such as α-SMA, COL-1, and MMP-2, LX-2 human hepatic stellate cells were treated with 1 ng/ml TGF-β1 alone or in

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combination with different numbers (1 × 106 –108 ) of A-ENVs per well (A-ENVs6 - A-

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ENVs8 ) in 6-well plates with a density of 1 × 10 6 cells for 48 h. The total fibrogenic RNAs from LX-2 cells were extracted using TRIzol reagent, and the concentration of RNAs was

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determined using NanoDrop2000 (Thermo Fisher Scientific Inc, DE, USA). RNAs (1 μg)

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were reverse transcribed by employing a high-capacity cDNA Reverse Transcription Kit, after which real-time PCR amplification was performed by utilizing SYBR® Green PCR

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Master Mix using a Stepone™ Real- Time PCR system (Applied Biosystems, CA, USA). The

5’-GCTTCACAGGATTCCCGTCTTAA-3’ (reverse),

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3’ (forward),

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following sequences of the primers were used: α-SMA 5’-TCGCATCAAGGCCCAAGAAA-

AGCAGGTCCTTGGAAACCTT-3’ (reverse),

MMP-2

(forward),

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5’-

5’-GAAAAGGAGTTGGACTTGGC-3’

5’-GTATTTGATGGCATCGCTCA-3’

CATTCCCTGCAAAGAACACA-3’

Collagen

(reverse),

(forward), GAPDH

5’5′-

TTGATGGCAACAATCTCCAC-3′ (forward), and 5′-CGTCCCGTAGACAAAATGGT-3′ (reverse). All data were analyzed according to the comparative Ct method and were normalized to GAPDH. 2.6 In vitro cell viability To investigate the cytotoxicity of A-ENVs, LX-2 cells were seeded at a density of 2 × 103 cells/well in 96-well plates and stabilized for 24 h at 37℃ in a humidified 5% CO 2

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atmosphere. The cells were incubated with 1 ng/ml TGF-β1 alone or in combination with AENVs6 - A-ENVs8 for 24 h or 48 h. Afterward, cell viability was evaluated using an MTT assay, and absorbance was measured at a wavelength of 570 nm using a microplate reader.

2.7 In vitro immunofluorescence imaging

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To observe the changes in α-SMA expression on cells, LX-2 cells were seeded at a

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density of 1 × 10 5 cells/well on 6-well plates and stabilized at 37℃ in a humidified 5% CO 2

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atmosphere for 24 h. The cells were treated with A-ENVs6 - A-ENVs8 in the presence of 1

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ng/ml TGF-β1. After 48 h, the cells were washed twice with DPBS and then fixed with 4% paraformaldehyde for 15 min. Subsequently, the cells were washed twice with DPBS and

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then permeabilized with PBS containing 0.5% Triton X-100 for 5 min. Then, the cells were washed twice with PBS and incubated with blocking solution (5% BSA) for 1 h. Primary

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antibodies (α-SMA, diluted 1:200 in blocking solution) were incubated with the cells

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overnight at 4℃. The cells were washed thrice with PBS and then incubated with

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fluorescence- labeled second antibodies (diluted 1:400 in blocking solution) for 1 h at room temperature. Finally, the cells were washed three times with PBS and mounted with 4,6diamidino-2-phenylindole. The fluorescence signal of α-SMA was observed using confocal laser microscopy (Zeiss LSM 700, Carl Zeiss, Oberkochen, Germany).

2.8 Ex vivo organ distribution of A-ENVs in liver fibrosis mouse model All experiments performed with live animals complied with the relevant laws and institutional guidelines of Sungkyunkwan University, and institutional committees approved all experiments. To observe the ex vivo organ distribution of A-ENVs in the liver fibrosis

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model, male C57BL/6 mice were prepared with 200 mg/kg of TAA three times per week through intraperitoneal injection for 12 wk. The solution containing Cy5.5-A-ENVs (200 μl) was injected into the mice via the tail vein. Each group of mice was sacrificed 3 or 24 h postinjection. Then, major organs were obtained and visualized using an IVIS Lumina III In Vivo Imaging System (Caliper Life Sciences, MA, USA) with a 670- nm pulsed laser diode. All data were quantitatively calculated using the region of interest function of embedded software

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in IVIS Imaging System, and the values were presented as means with standard deviations for groups of at least three animals. For histological immunostaining, the dissected livers were

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stained with Anti-cytokeratin 18 (anti-CK18), Anti-CD68 and α-SMA according to the

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manufacturer’s IHC protocols. In brief, deparaffinized liver tissue were heated in 1 mM EDMA solution for antigen retrieval. Slides were washed three times with PBST and treated

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with 1% BSA solution for 1 h. The liver tissues were incubated with primary antibodies

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overnight. Slides were washed three times with PBST and incubated with FITC anti- mouse IgG antibody (#406001, Biolegend) for 2 h at 4℃. After washing the slides, samples were

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mounted with DAPI mounting solution and visualized using Zeiss microscope (Zeiss LSM

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700, Carl Zeiss, Oberkochen, Germany). For quantification of hydroxyproline content in liver tissue, a Hydroxyproline Assay Kit (#ab222941, Abcam) was used according to the manufacturer ’s instructions. Briefly, liver tissues were homogenized and incubated for 1 h at 120℃ in 10 N NaOH. After hydrolysis, samples were sequentially incubated with oxidation reagent mix for 20 min and developer for 5 min. Each sample was incubated with the diluted dimethyl aminobenaldehyde (DMAB) for 45 min at 65°C. The samples were measured at 560 nm absorbance using a microplate reader (Bio-Tek Instruments Inc).

2.9 Blood biochemical assay

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To investigate the liver function test in blood, TAA- induced liver fibrosis mice were prepared as described above. The blood collected from the mice was centrifuged for 10 min at 13,000 rpm at 4℃. The isolated serum was analyzed for aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatease (ALP), total bilirubin (TBIL), and total protein (TP). These parameters of the serum were evaluated by ChemOn Inc (Suwon,

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Korea).

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2.10 Liver histology

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For histological analysis, the dissected liver tissues were recovered and immediately fixed in 4% (v/v) paraformaldehyde, embedded in paraffin, and then sliced to a thickness of 6

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mm. Then, the tissue sections were stained with hematoxylin and eosin (H&E), Masson’s trichrome (MT), anti-α-SMA following manufacturer instructions. The images of stained

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liver tissues were visualized using a light microscope and image transfer software (Olympus,

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Tokyo, Japan). For the quantification of the fibrotic area, stained liver tissues were imaged

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randomly by collecting 20–30 images per mouse. The images were analyzed using integrated optical density quantification (Image-Pro Plus, Media Cybernetics, Inc., MD. USA)

2.11 Statistical analysis All values were expressed as the mean ± standard error of the mean or the mean ± standard deviation. The differences between experimental and control groups were assessed using Student’s t-test and or one-way ANOVA considered statistically significant (marked with an asterisk (*) in figures) if p < 0.05. 3. Results and discussion

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3.1 Preparation and characterization of A-ENVs Rapid, efficient, and large-scale production of ENVs is imperative for their successful clinical application. To meet these requirements, in this study, we employed an automated TFF system, which can facilitate effective ENV isolation and purification. For the preparation of A-ENVs, the CM collected from proliferating ADSCs was isolated using TFF with a 500-kDa MWCO ultrafiltration membrane filter system [48]. The physicobiochemical

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characteristics of the A-ENVs obtained from ADSCs are shown in Figure 1B, C, and D.

Figure. 1. Physicochemical characteristics of extracellular vesicles derived from adipose stem cells (A-ENVs). A) Schematic illustration for A-ENV preparation and treatment of a liver fibrosis mouse model. B) Size distribution and transmission electron microscope (TEM) images of A-ENVs. C) Surface charge and D) western blot analyses of A-ENVs for an ENV marker. Quantitated values were normalized using loading control.

TEM images show that A-ENVs have a spherical shape. Their hydrodynamic diameters and particle concentration, which were examined by DLS and NTA, were found to be 94.2 ± 4.7 nm and 1.9 × 108 ± 3.8 × 107 particles/mL, respectively (Figure 1B and Table S1). As shown in Figure 1C, A-ENVs have a zeta potential of -23.7 ± 6.7 mV owing to the

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presence of a negatively charged phospholipid membrane [49, 50]. A-ENVs were characterized by western blotting to investigate the expression of ENV marker proteins. The result showed that A-ENVs were positive for the marker proteins (CD9 and CD63) on their surface membranes, confirming the intact membrane structure of A-ENVs harvested via the TFF ultrafiltration method (Figure 1D).

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3.2 In vitro antifibrotic effect and cytotoxicities of A-ENVs LX-2 human hepatic stellate cell was used for in vitro hepatic fibrosis studies. For

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HSC activation, TGF-β1 was used as a stimulator in the in vitro experiment [51, 52]. To

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investigate the in vitro antifibrotic effects of A- ENVs, LX-2 human hepatic stellate cells were

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treated with 1 ng/mL of TGF-β1 alone or in combination with A-ENVs. To visualize the expression level of α-SMA in the activated HSC, α-SMA proteins in LX-2 cells were

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immunostained with an anti-α-SMA antibody, and then the cells were examined under a

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confocal microscope (Figure 2A).

Figure. 2. In vitro antifibrotic effects of A-ENVs in LX-2 cells. A) Immunofluorescence images of LX-2 cells incubated with A-ENVs for 48 h in the presence of TGF-β1 (1 ng/ml). B) Quantification of a-SMA-positive areas. Hepatic fibrosis-associated C) α-SMA, D) COL-1, and E) MMP-2 gene expression in LX-2 cells treated with A-ENVs. LX-2 cells were treated with ENVs for 48 h in the presence of TGF-β1 (1 ng/ml). Data expressed as the mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001 versus the TGF-β1 treated control group. ###p <

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0.001 versus the control group. ‡‡p < 0.001 compared with the A-ENVs7 . (F) Western blot analysis of LX-2 cell incubated with A-ENVs for 48 h in the presence of TGF-β1 (1 ng/ml). A-ENVs6 , A-ENVs7 , and A-ENVs8 represent different numbers (1 × 106 – 108 ) of A-ENVs. Strong fluorescence signals of α-SMA were observed in the TGF-β1 group, but prominent fluorescence signals were not detected in the control group without the TGF-β1 treatment. On the other hand, when the TGF-β1 group was treated with different numbers (1 × 106 – 108 ) of A-ENVs (A- ENVs6 , A-ENVs7 , and A-ENVs8 ), the fluorescence signals of α-

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SMA significantly diminished in a dose-dependent manner, indicating excellent antifibrotic

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activities of A- ENVs. Among the three A- ENVs, A-ENVs8 exhibited the highest antifibrotic

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activity. These results were further supported by quantitative analys es (Figure 2B).

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Compared to the TGF-β1 group, A- ENVs8 led to the most significant reduction in α-SMA expression. In particular, A-ENVs8 -induced downregulation of α-SMA levels, which was

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higher than that of TGF-β1 (3.4 times) and A-ENVs6 (1.7 times), respectively.

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To test if A-ENVs suppressed other types of fibrotic markers, the antifibrotic effects

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of A-ENVs were further studied using another quantitative analysis method. In this

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experiment, the expression levels of three different types of fibrosis-associated markers (αSMA, COL-1, and MMP-2) in the activated LX-2 cells treated with A-ENVs were analyzed using RT-PCR (Figure 2C, D, and E). Upon activation of LX-2 cells with TGF-β1, the expression levels of α-SMA, COL-1, and MMP-2 increased by 2.7, 2.8, and 9.3 times compared to the control groups, respectively. Interestingly, the upregulated expression of the fibrotic markers (α-SMA, COL-1 and MMP-2) in activated LX-2 cells became downregulated following treatment with A-ENVs. For instance, A-ENVs suppressed the αSMA expression level in a number-dependent fashion; A-ENVs8 showed the strongest suppression of α-SMA, which was 1.4 and 1.2 times higher than that of A-ENVs6 and AENVs7 , respectively. The results are in good agreement with the imaging analysis results shown in Figure 2B. A-ENVs also significantly suppressed the other fibrotic markers (COL-

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1 and MMP-2) (Figure 2D and E). To further explore the mechanism of A-ENVs for the treatment of liver fibrosis, we performed western blot analyses. The treatment of TGF-β1 into LX-2 cells induced the upregulation of collagen type 1 alpha 1 (Col1A1), phosphorylated Smad2 (p-SMAD2), phosphorylated Smad3 (p-SMAD3) and Snail. However, the treatment of A-ENVs dose-dependently attenuated Col1A1 and Snail expression and decreased the phosphorylation level of SMAD2 and SMAD3, suggesting that A-ENVs effectively inhibit

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TGF-β1 signaling via SMAD2/3 in LX-2 cells. Taken together, the downregulation of TGF-

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β1 signaling via SMAD2/3 can be one of the key mechanisms by which the A-ENVs

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treatment ameliorates liver fibrosis.

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In addition, we evaluated the in vitro cytotoxicity and cell proliferation of A-ENVs

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using an MTT assay (Figure S2). The control groups and TGF-β1 group did not exhibit significant cytotoxicity. As expected, A-ENVs or the other groups did not show significant

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cytotoxicity on activated LX-2 cells. Instead, proliferation of the activated LX-2 cells was

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observed as the incubation time increased, which is consistent with previously reported

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results [53, 54]. These results imply that A-ENVs might inactivate the activated HSCs through downregulation of fibrogenic signaling without inducing significant toxicity to the HSCs, indicating their potential for repairing injured liver tissue [33, 55, 56]. The A-ENV groups enabled us to further investigate their antifibrotic activities in vivo using animal models owing to their favorable physicochecmical and biological features—excellent biocompatibility, easy production, and excellent antifibrotic effects. 3.3 Ex vivo organ distribution of A-ENVs in liver fibrosis model To investigate the organ distribution of A- ENVs, we prepared liver fibrosis animal models through intraperitoneal injection of TAA into C57BL/6 thrice per week for 12 weeks (Figure 3A). The TAA- induced model is considered more similar to human liver fibrosis and

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cirrhosis compared to CCl4 -induced models because of prominent regenerative nodules and rapid development from periportal fibrosis [57]. Next, we injected Cy5.5- labeled A-ENVs into the TAA- induced liver fibrosis animal models to image their ex vivo organ distribution using an NIRF fluorescence imaging system. Ex vivo organ distribution data demonstrated that higher fluorescence signals of A-ENVs in normal and fibrosis groups were initially detected at liver tissue than other organs. Interestingly, the fluorescence signals of A-ENVs in

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the liver tissue of the fibrosis group was higher than that of the normal group 24 h postinjection (Figure 3B). As shown in Figure 3C, the A-ENV in fibrosis group exhibited 1.8

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times and 1.7 times higher fluorescence intensity compared to the normal group 3 h and 24 h

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post-injection, respectively. To investigate the fate of A- ENVs, we further examined the accumulation of A-ENVs in different types of cells such as hepatic stellate cells, hepatocyte,

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and Kupffer cells in liver tissues (Figure S3). Strong colocalization signals of A-ENVs and

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CD68 were observed implying that the enhanced phagocytic capacity of hepatic macrophages

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fibrotic liver tissue.

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associated with liver fibrosis can be responsible for the effective targeting of A-ENVs in

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Figure. 3. Ex vivo organ distribution of A-ENVs in a liver fibrosis mice model. A) Cy5.5labeled-A-ENVs (Cy5.5-A-ENVs) were administered to normal and fibrosis mouse models induced by thioacetamide (TAA) injection three times per week for 12 weeks. B) Ex vivo organ imaging of Cy5.5-A-ENVs at predetermined time intervals (Li: Liver, Lu: Lung, Sp: Spleen, Ki: Kidney, He: Heart). C) Quantification of fluorescence intensity in major organs of normal and fibrosis mouse models. D) Confocal microscopic images of A-ENVs in fibrotic liver tissue. Scale bar, 30 μm. *p < 0.001 versus 24 h post-injection group in normal model.

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3.4 Blood chemistry tests after treatment of A-ENVs in liver fibrosis model

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Blood chemistry analysis was performed to evaluate the restoration of liver function in animal models (Figure 4A). A-EXO 7 and A-EXO 8 were injected into the liver fibrosis animal models to test the antifibrotic effects of A-ENVs; ADSCs were also treated as the positive control. After A-ENV treatment, blood samples were collected, and representative blood markers related to the liver function were analyzed. The serum levels of AST, ALT, ALP, and TBIL in the TAA-treated group were 2.0 times to 5.4 times higher, and those of the TP were 1.2 times lower than those of the control group, implying that the mice model of liver fibrosis was successfully established. However, A-ENVs8 treatment not only significantly suppressed the serum levels of AST, ALT, ALP, and TBIL but also restored the serum levels of the TP compared to other groups (Figure 4B and Table S2).

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Figure. 4. Blood chemistry analyses of A-ENVs 24 h after injection into liver fibrosis mice. A) A-ENVs were administered to normal and fibrosis mouse models induced by TAA injection three times per week for 12 weeks. B) Serum levels of AST, ALT, ALP, TBIL, and TP. Data expressed as the mean ± SEM and normalized to the value in the TAA-treated fibrosis group. *p < 0.05, **p < 0.01, and ***p < 0.001 versus the PBS-treated group. ###p < 0.001 versus the control group. C) Liver weight/body weight (LW/BW) ratio in normal and fibrosis mouse models. **p < 0.01 and ***p < 0.001 versus the PBS-treated group in fibrosis mouse models. ‡p < 0.001 versus the control group. #p < 0.001 compared with ADSC.

In addition, the liver weight-to-body weight ratio in the A-ENVs8 -treated group was 1.3 times lower than that of the TAA group (Figure 4C). These results demonstrated that A-

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EXO 8 treatment successfully restored the liver function of the TAA-induced liver fibrosis animal models.

3.5 In vivo antifibrotic effect of A-ENVs and tissue analyses Fibrotic liver tissues of mice were visually assessed to evaluate morphological alterations to verify the effectiveness of A- ENVs in treating liver fibrosis (Figure 5A). In the

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normal group, the livers showed a smooth surface with a reddish co lor and soft texture. On the contrary, the livers from the TAA group exhibited hard, pale, and rough surfaces with a

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brownish color and nodule formation, indicating serious liver injury. Importantly, A-ENVs8

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showed clear morphological restoration along with a smooth surface and deep red color in

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macroscopic images, suggesting that A-ENVs8 distinctly alleviated TAA- induced liver fibrosis. These results were further supported by the histopathological examination after H&E,

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MT, and α-SMA staining (Figure 5B). The collagen-stained area of the A-ENVs8 group was

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6.6 times lower than that of the TAA group; the area of the A-ENVs7 group was 2.6 times

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lower than that of the TAA group. These results were consistent with those of hydroxyproline assay, showing the collagen turnover in the fibrotic liver tissue after the treatment of A-ENVs (Figure 5C and 5E). α-SMA staining results also showed a similar tendency to the collagenstaining results. A remarkable reduction was found in the α-SMA stained areas of the AENVs8 -treated group (Figure 5D). Downregulation of collagen and α-SMA expression in the fibrotic liver tissue was further confirmed by the western blot analysis ( Figure 5F). Taken together, these data substantiated that systemic administration of A-ENVs could histologically and functionally alleviate liver fibrosis in TAA-induced mice.

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Figure. 5. Antifibrotic effects of intravenously injected A-ENVs determined by liver histology. A) Representative photographs of normal and fibrotic livers from mice. Livers were collected at 24 h after injection. B) Representative photomicrographs of liver sections stained with hematoxylin and eosin (H&E), immunohistochemistry for an activated HSC marker (α-SMA), and collagen depositions (MT) treated with A-ENVs. Quantification of histological analyses of C) collagen and D) α-SMA. Percentage of fibrotic and immunoreactive areas were determined using Image-Pro Plus. #p < 0.05 and ##p < 0.001 compared with ADSC. (E) Quantification of the expression level of collagen by hydroxyproline assay in total livers. (F) Western blot analyses of fibrotic markers. Control is PBS-treated normal liver samples.

Notably, treatment using 108 A-ENVs (A-ENVs8 ) turned out to be the most effective treatment for liver fibrosis, indicated by a remarkable reduction in collagen deposition and αSMA expression. However, treatment using a larger number of A- ENVs (A-EXO 9 ) was found

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to be less effective than that using A-ENVs8 (data are not shown); this implies that the optimal administration dose of A-ENVs can be 108 . Although it was found that A-ENVs in this work may reduce collagen deposition and attenuated HSC activation by TGF-β1 via Smad2/3 signaling pathway, recent studies demonstrated that the antifibrotic acitivity is likely associated with the following biological events: (i) enhanced proliferation and reduced apoptosis of hepatocytes through the HGF/c-Met pathway in fibrotic tissue, and (ii) enhanced

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ant-inflammatory responses of natural killer T-cells in liver disease through the Wnt/β-catenin

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signaling pathway [47, 58].

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4. Conclusions

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We report a novel therapeutic role of A-ENVs in this study, which can be employed as potent antifibrotic agents for the treatment of liver fibrosis. The in vitro studies

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demonstrated that A-ENV treatment effectively suppressed the multiple fibrotic proteins

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associated with liver fibrosis. Upon systemic administration, A-ENVs preferentially

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accumulated in fibrotic liver tissue. Treatment with A-ENVs successfully inhibited the activity of activated HSCs, leading to reductions in collagen deposition and α-SMA expression levels without noticeable structural damages to the liver tissue and restored liver functionality in TAA- induced liver fibrosis mice models. Overall, this cell- free therapeutic approach based on ADSC-derived extracellular vesicles poses great potential as a safe and effective therapeutic, which can facilitate clinical applications of extracellular vesicle-based therapeutics for treating intractable chronic liver diseases.

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Declaration of Competing Interest Dong-Gyu Jo, Yong Woo Cho, and Jae Hyung Park are stockholders of Exostemtech Inc. Ji Suk Choi and Jae Dong Kim are employees of Exostemtech Inc. The other authors have no conflicts of interest.

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Acknowledgements

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This research was supported by Basic Science Research Programs through the

the Industrial Core

Technology

Development Program

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(2017R1D1A1B03034888),

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National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT

(10078392) funded by the Ministry of Trade, Industry and Energy (MOTIE, Korea), and a

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Korea Institute of Science and Technology (KIST) intramural research grant.

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Appendix A. Supplementary data

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Supplementary data to this article can be found online at …

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