Facile Deposition of Manganese Dioxide to Albumin-Bound Paclitaxel

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Facile Deposition of Manganese Dioxide to Albumin Bound Paclitaxel Nanoparticles for Modulation of Hypoxic Tumor Microenvironment to Improve Chemoradiation Therapy Lingtong Meng, Yali Cheng, Shaoju Gan, Zhicheng Zhang, Xiaoning Tong, Lei Xu, Xing Jiang, Yishen Zhu, Jinhui Wu, Ahu Yuan, and Yiqiao Hu Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b00808 • Publication Date (Web): 20 Dec 2017 Downloaded from http://pubs.acs.org on December 22, 2017

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Molecular Pharmaceutics

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Facile Deposition of Manganese Dioxide to Albumin Bound Paclitaxel

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Nanoparticles for Modulation of Hypoxic Tumor Microenvironment to

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Improve Chemoradiation Therapy

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Lingtong Meng1, #, Yali Cheng1, #, Shaoju Gan1, Zhicheng Zhang1, Xiaoning Tong1, Lei Xu1,4, Xing

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Jiang1, Yishen Zhu4, Jinhui Wu1,2,3 , Ahu Yuan1,2,3,*, Yiqiao Hu1,2,3,*

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Affiliations:

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State Key Laboratory of Pharmaceutical Biotechnology, Medical School of Nanjing University,

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#

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*Author for correspondence:

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Ahu Yuan, Ph.D. Research Associate

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E-mail: [email protected]

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E-mail: [email protected]

Nanjing 210093, China; 2

Jiangsu Key Laboratory for Nano Technology, Nanjing University, Nanjing 210093, China;

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Institute of Drug R&D, Medical School of Nanjing University, Nanjing 210093, China;

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College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing 211800, China. These authors contributed equally.

Address: 22 Hankou Road, Nanjing 210093, China Phone: +86-13913801615; Fax: +86-25-83596143

Yiqiao Hu, Ph.D. Professor Address: 22 Hankou Road, Nanjing 210093, China Phone: +86-13601402829; Fax: +86-25-83596143

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ACS Paragon Plus Environment

Molecular Pharmaceutics 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Abstract

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Tumor microenvironment with hypoxia and excess hydrogen peroxide (H2O2), tremendously limits the effect of chemoradiation therapy of colorectal cancer. For the first time, we developed a facile method to deposit manganese dioxide (MnO2) on the surface of albumin bound paclitaxel nanoparticles (ANPs-PTX) to obtain MnO2 functioned ANPs-PTX (MANPs-PTX). In the tumor microenvironment, MANPs-PTX could consume excess hydrogen peroxide (H2O2) to produce abundant oxygen for tumor oxygenation and improve chemoradiation therapy. Meanwhile, the released Mn2+ from MANPs-PTX had excellent T1 magnetic resonance imaging (MRI) performances for tumor detection. Notably, the obtained MANPs-PTX would be a promising theranostic agent and have potential clinical application prospects. Keywords tumor microenvironment，manganese dioxide, albumin bound paclitaxel nanoparticles, chemoradiation therapy, magnetic resonance imaging, oxygenation


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1. Introduction

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Colorectal cancer is the third most common form of cancer worldwide and about half of the patients die of the disease in five years1. Current treatments of late colorectal cancer are not always successful in terms of single therapy including chemotherapy and radiotherapy, which lead to distant metastasis, resistance and local recurrence2-3. Therefore, radiotherapy combined with chemotherapy has attracted great interests from the basic research and clinical practice4-5. Paclitaxel is one of the most promising candidates combined with radiotherapy among the abundant chemotherapeutic agents, which is contributed to its remarkable effect of cell cycle arrest at G2/M phase6-8. Meanwhile, albumin-bound paclitaxel nanoparticles (ANPs-PTX, FDA approved in 2005), are developed to maintain the therapeutic benefits of paclitaxel but avoid polyoxyethylated castor oil related toxicity of the paclitaxel injection (Taxol™). In a certain sense, ANPs-PTX is a superior substitute of traditional paclitaxel chemotherapy agent which is used for chemoradiation therapy9-10. However, the characteristic abnormalities of tumor microenvironment with hypoxia and excess reactive oxygen species (ROS) like H2O2 (~100µM), tremendously limit the chemoradiation therapeutic efficacy of colorectal cancer11-12. Compared with normal cells, elevated ROS level has widely been detected which is mainly due to the rapid metabolism in the mitochondria of multiple cancer cells13. In addition, some studies have also suggested that radiation or chemotherapy could produce abundant ROS, including H2O214-15. Notably, excess H2O2 in the tumor regions can stabilize hypoxia-inducible factor-1α (HIF-1α) by inhibiting prolyl hydroxylase (PHD) which leads to various genes expression responsible for proliferation and survival of cancer cells16-17. Hence, decreasing tumor H2O2 level could potentially benefit the effect of cancer therapy. Meanwhile, lack of oxygen also inhibit PHD activity leading to HIF-1α subunit stabilization and recognition with HIF-1β in the nucleus for further genetic transcription18. Additionally, hypoxic tumor cells become resistant to radiation therapy, where oxygen molecules could immobilize radiation-induced DNA damage and then prevent damaged DNA repair19. Meanwhile, increased tumor oxygenation could also arrest more tumor cells at G2/M phase which are the most radiosensitive of all cell cycle phases20. Also, increasing tumor oxygenation would observably improve cancer treatment. Therefore, attractive benefits during tumor chemoradiation therapy would be achieved if excess H2O2 could be converted into oxygen. Recently, some strategies have been exploited for this conversion, including delivering catalase, manganese dioxide (MnO2) and prussian blue nanoparticles21-23. It is noteworthy that MnO2 has high reactivity and specificity toward acidic pH and H2O2 for simultaneous and sustained production of oxygen to overcome tumor hypoxia24-26. Meanwhile, the produced Mn2+ from MnO2 is a kind of strong T1 magnetic resonance (MR) contrast agent for tumor detection27. The multiple benefits of MnO2 are closely considered to be combined with ANPs-PTX to improve chemoradiation therapy of colorectal cancer. In our research, we aimed at developing a facile method to prepare MnO2



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functioned ANPs-PTX. Inspired by the process of deposition of MnO2 onto graphene oxide and albumin25, 28-29, we firstly deposited manganese dioxide on the surface of the albumin bound paclitaxel nanoparticles (ANPs-PTX) via a facile oxidation preparation to obtain MnO2 functioned ANPs-PTX (MANPs-PTX). Then we detailedly detected the characterization of the obtained MANPs-PTX including particle size, zeta potential, morphology and oxygen generation. After intravenous injection, MANPs-PTX could accumulate within the tumor regions via EPR effect and release Mn2+, oxygen and paclitaxel in response to tumor microenvironment including excess H2O2 and acidic pH. Abundant oxygen generation could overcome the hypoxia of tumor, thus effectively enhance the therapeutic efficacy of chemoradiation therapy. Meanwhile, the released Mn2+ had excellent T1-MRI performances for tumor imaging. Additionally, MANPs-PTX exhibited no obvious toxicity to normal tissues. Notably, the obtained MANPs-PTX via our facile preparation would be a promising theranostic agent for colorectal cancer treatment (Figure 1).

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Figure 1. (a) Scheme illustration of facile preparation of manganese dioxide deposited albumin bound paclitaxel nanoparticles (MANPs-PTX). (b) The mechanism of MANPs-PTX for enhanced chemoradiation therapy and tumor MR imaging.

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2. Materials and methods

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2.1 Materials and Reagents

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Paclitaxel was obtained from Hongdoushan Biomedical (Jiangsu, China). KMnO4 was purchased from Sinopharm Chemical Reagent Co. Human serum albumin (HSA), IR-775, Coumarin 6 and cell uptake inhibitors were bought from Sigma-Aldrich (St. Louis, MO). Cell-Counting Kit-8 (CCK-8) was obtained from Dojindo Laboratories (Kumamoto, Japan). TUNEL assay kit, HIF-1α antibody and Ki67 antibody were purchased from Thermo Fisher Scientific (Shanghai, China).

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2.2 Preparation of ANPs-PTX、MnO2-HSA and MANPs-PTX


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ANPs-PTX was prepared according to our previous report30. MANPs-PTX was obtained via facile one-step oxidation preparation method. 6mL of KMnO4 solution (2.43mg/mL) was dropped into 14mL of ANPs-PTX (1.14mg/mL of PTX) suspension at 37 ºC for 30min under vigorous stirring to form MANPs-PTX. Then, MANPs-PTX was dialyzed with distilled deionized water for three times and condensed by an ultrafiltration device (Millipore 8400, ultrafiltration membrane MW:10KD). MANPs-PTX/coumarin 6 (PTX: coumarin 6=10:1) and MANPs-PTX/IR775 (PTX: IR775=10:1) were formed with the similar method. MnO2-HSA was prepared by dropwise adding 6mL of KMnO4 solution (1.05mg/mL) into 14mL of HSA suspension (3.57mg/mL) under vigorous stirring at 37 ºC. Then, MnO2-HSA was dialyzed with distilled deionized water for three times and condensed in the same way for further use.

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2.3 Characterization of MANPs-PTX

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In order to determine the concentration of PTX in ANPs-PTX and MANPs-PTX, both nanoparticles were extracted by adding acetonitrile to the suspension with a volume ration of 9:1, then shaken vigorously. The supernatant solution was collected by centrifugation at 5000rpm for 10min and the paclitaxel concentration was detected by High Performance Liquid Chromatography (HPLC, LC20-AT, SHIMADZU, Japan). Particle size distribution of nanoparticles and zeta potentials were measured by Zetasizer Nano (Malvern Instruments, Malvern, UK). The morphology and lattice information of MANPs-PTX were characterized using Transmission Electron Microscopy (TEM, JEM-200CX, JEOL, Japan). UV-Vis spectra of nanoparticles were detected by a UV-vis Spectrometer (UV 2450, SHIMADZU). The concentrations of MnO2 were also measured by UV-vis spectrometer using an established standard absorbance curve. In order to avoid the influence of protein, 400nm wavelength was selected.

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2.4 Oxygen generation and hydrogen peroxide consumption in vitro

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In order to determine the ability of oxygen generation from MANPs-PTX, a Clark Oxygen Probe (OX25, Unisense, Denmark) was applied where constant monitoring production of O2 was achieved. Oxygen probe was immersed in the test solution of MANPs-PTX (PTX:MnO2=70µg/mL:35µg/mL) or equivalent ANPs-PTX for several minutes in order to balance the system. Then 100µM of H2O2 was injected into test suspension. After a period of 10 minutes monitoring, another injection of H2O2 was conducted for four cycles. In addition, the contrary relationship between H2O2 consumption and oxygen generation was further confirmed in another test system. Briefly, MANPs-PTX (10µM of MnO2) was injected into 100µM of H2O2 and the production of oxygen was recorded. The process was repeated nine times and accumulated oxygen was calculated. In addition, concentrations of H2O2 were determined by Hydrogen Peroxide Assay Kit (Nanjing Jiancheng Bioengineering Institute) according to manufacturer's instructions.

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2.5 Cytotoxicity and intracellular uptake



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CT26 cells (murine colon cells) were cultured in 1640 medium supplemented with 10% FBS and 1% penicillin/streptomycin at 37ºC with 5% CO2. To detect the cytotoxicity, CT26 cells were seeded into 96-well plates at a density of 5×103 cells per well. After attachment, the cells were covered with paraffin for 12 hours. The medium was separated from the normoxic atmosphere by the overlying liquid paraffin film, which provided a hypoxic environment to tumor cells. Then, CT26 cells were incubated for 12h with vehicle, MnO2-HSA (0.22µg/mL of MnO2), ANPs-PTX (0.43µg/mL of PTX), MANPs-PTX (PTX:MnO2=0.43µg/mL:0.22µg/mL), respectively. Different solutions were added beneath the liquid paraffin film. The radiation efficiency was set as 3Gy/min and radiation lasted for 100s. After radiation (0 or 5Gy), different treatments were removed and CT26 cells were cultured continuously for another 72 hours. A mixed solution consisted of CCK-8 (10µL) and fresh culture medium (90µL) was added to each well and incubated for an additional 2 hours. Finally, the absorbance was measured at 450 nm using a microplate reader. Cells treated with vehicle (HSA) were set as control. To detect the intracellular uptake, CT26 cells were seeded with a density of 1×105 per well in 6-well plates. The cells were treated with MANPs-PTX/Coumarin 6 for 0, 1, 2, 4, 8h, respectively. After the drug removed, CT26 cells were trypsinized and analyzed by flow cytometry (Ex=488nm, FL1 channel, BD FACS Calibur). To investigate the endocytosis pathway, CT26 cells were pretreated with different inhibitors (dynasore 30µM, chlorpromazine 12.5µM and methyl-β-cyclodextrin 1.3 mg/mL) 0.5h prior to the incubation of MANPs-PTX/Coumarin 6 for 4h at 37 ºC. Meanwhile, CT26 cells were incubated with MANPs-PTX/Coumarin 6 at 4 ºC to determine whether the cellular uptake of MANPs-PTX was energy dependent. Coumarin 6 fluorescence in endocytosis assay was analyzed by FLOWJO.

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2.6 Intracellular trafficking of MANPs-PTX

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CT26 cells were seeded with a density of 7×104 per well in 24-well plates covered by glass disk. After attachment, the cells were incubated with MANPs-PTX/Coumarin 6 for 4 hours. The lysosomal marker Lysotracker Red and DAPI were used to label lysosome and nucleus of CT26 cells. The cells were washed twice with PBS. Images were obtained and analyzed by Olympus FV3000 laser scanning confocal microscope.

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2.7 Animals and CT26 transplanted tumor model

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Male Balb/c mice were purchased from Yangzhou University Medical Center (Yangzhou, China) and received care in accordance with Institution Animal Care and Use Committee (IACUC) of Nanjing University. CT26 tumors were firstly developed by subcutaneously implanting 5×106 CT26 cells suspension in the lower flanks of mice. Fourteen days later, tumor mass was isolated and cut into small pieces of about 2 mm3 which were subcutaneously implanted in the right axilla of mice to establish CT26 colon cancer model.

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2.8 Imaging of MANPs-PTX in vivo

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To determine the distribution of MANPs-PTX in CT26 colon cancer model, NIR


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dye IR775 was encapsulated in MANPs-PTX to obtain MANPs-PTX/IR775. IVIS Lumina imaging system (Ex=745nm, Em=820nm, Xenogen Corporation-Caliper, Alameda, CA, USA) was used to detect the IR775 fluorescent signal in vivo. Tumor bearing mice were treated with MANPs-PTX/IR775 via intravenous injection. Anesthetic mice were placed on an animal plate at 37 ºC and images were captured from 15 minutes to 96 hours post injection. For tissue distribution assay, the mice were sacrificed at 8h post-injection and the major organs were isolated and determined similar with the whole animal imaging. Data was analyzed using IVIS Living Imaging Software.

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2.9 MR imaging properties measurement

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To investigate the effect of MANPs-PTX on T1-weighted MR signals in vitro and in vivo, T1 relaxivity characterization and T1-weighted imaging were carried out on MR scanner (Biospec 7T/20 USR, Germany). Tumor bearing mice (350-400mm3) were treated with MANPs-PTX (PTX:MnO2=30mg/kg:15 mg/kg) via intravenous injection. Anesthetic mice were fixed in an animal groove and images were obtained from 0h to 48h. For in vitro detection, MANPs-PTX (PTX:MnO2=70µg/mL:35µg/mL) was pretreated with different concentrations of H2O2 (0,50,100,150, 200, 300µM, pH=6.8) for 30min. The T1 phantom images and T1 relaxation time were recorded. Phantom images were analyzed using Nikon NIS-Elements and ImageJ Software.

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2.10 Oxygen generation and hydrogen peroxide (H2O2) consumption in vivo

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Tumor bearing mice were intratumorally injected with MANPs-PTX (PTX:MnO2= 3mg/mL:1.5mg/mL) or equivalent ANPs-PTX when tumor volume reached about 350mm3. In order to investigate oxygen generation of MANPs-PTX, the oxygen concentrations within tumor tissues were carried out using Clark Oxygen Probe (OX25, Unisense) for consistent monitoring. The mice were anesthetized with intraperitoneal injection of amytal sodium (60mg/kg) and fixed on the bench board. Clark oxygen probe controlled with a fine-control elevator, was inserted into tumor tissue carefully. The oxygen probe was equilibrated for several minutes before i.t. injection of 50µL MANPs-PTX or ANPs-PTX. Then the oxygen concentration was automatically recorded, respectively. For the detection of H2O2 consumption, mice were sacrificed at 30min post intratumorally injection of 50µL MANPs-PTX or ANPs-PTX, respectively. Tumor tissues were isolated and weighted. Tissue lysate was added and homogenized using an IKA-Ultra Turrax T25 Homogenizer. The suspensions were centrifuged at 12000g for 5min. Supernatants were tested with Hydrogen Peroxide Assay Kit according to the manufacture’s guidance and all the procedures were performed at 4 ºC.

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2.11 Enhanced chemoradiotherapy by MANPs-PTX in vivo

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Treatments were started when tumor volume of Balb/c mice reached approximately 70-100mm3. The mice were divided into eight groups, which were treated with HSA (vehicle), MnO2-HSA, ANPs-PTX, MANPs-PTX with or without



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radiation (0 or 5Gy), respectively. All the samples were intravenously administered via tail vein and X-ray radiation therapy was performed 8 hours post i.v. injection. Treatments were conducted on day 0 and day 7, respectively. The tumor size and body weight were recorded every other day. Tumor volume was calculated according to the following formula: width2 ×length/2. The blood for serum biochemistry was collected before sacrifice. Serum biochemistry data was measured using Chemray 430 (Rayto, China). Meanwhile, Hematoxylin and Eosin (HE) staining of major organs and tumors from sacrificed mice were stained according to the manufacturer's instructions on day 15.

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2.12 Immunofluorescence and immunochemistry

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To determine tumor hypoxia, rabbit monoclonal HIF-1α antibody was used for staining HIF-1α. The tumor tissues for HIF-1α staining were obtained from mice sacrificed at 12h after i.v. injection of vehicle, MnO2-HSA, ANPs-PTX and MANPs-PTX, respectively. HIF-1α antibody (Dylight 488) was diluted 1:200 with 3%BSA blocking buffer before usage and DAPI (1µg/mL) was used to label cell nucleus. To estimate the enhanced effect of chemoradiotherapy, TUNEL assay kit and rabbit monoclonal Ki67 antibody were used for the staining of apoptosis and proliferative cells, respectively. The mice were treated with different samples via intravenous injection 12h prior to radiotherapy (5Gy). Tumor sections were prepared from mice sacrificed at 24h after radiation treatments. For Ki67 immunochemistry, primary rabbit Ki67 antibody was diluted 1:200 with 3%BSA and the second goat anti-rabbit antibody was diluted 1:5000 with blocking buffer which was conjugated with Horseradish Peroxidase (HRP). The DAB (3，3’-diaminobenzidine) reaction time was set as the time of the tumor sections treated with vehicle+RT appearing abundant tan precipitation which was controlled within 8 minutes. The manufacturer's instructions were followed for all procedures for TUNEL assay. Images were captured using Nikon Eclipse Ti (Japan) and analyzed with Nikon NIS-Elements.

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2.13 Statistical analysis

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Statistical significance was performed by two-tail Student’s t-test for two groups and one-way analysis of variance for multiple groups. A value of p

Facile Deposition of Manganese Dioxide to Albumin-Bound Paclitaxel

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