pH-Responsive Polymer-Stabilized ZIF-8 Nanocomposite for

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Functional Nanostructured Materials (including low-D carbon)

pH-Responsive Polymer-Stabilized ZIF-8 Nanocomposite for Fluorescence and Magnetic Resonance Dual-Modal ImagingGuided Chemo/Photodynamic Combinational Cancer Therapy Ya-Ting Qin, Hui Peng, Xi-Wen He, Wen-You Li, and YuKui Zhang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b12641 • Publication Date (Web): 27 Aug 2019 Downloaded from pubs.acs.org on August 27, 2019

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ACS Applied Materials & Interfaces

pH-Responsive Polymer-Stabilized ZIF-8 Nanocomposite for Fluorescence and Magnetic Resonance Dual-Modal Imaging-Guided Chemo/Photodynamic Combinational Cancer Therapy Ya-Ting Qin†, Hui Peng†, Xi-Wen He†, Wen-You Li*,†,§, and Yu-Kui Zhang†,‖

†College

of Chemistry, Research Center for Analytical Sciences, State Key Laboratory of

Medicinal Chemical Biology, Tianjin Key Laboratory of Biosensing and Molecular Recognition, Nankai University, Tianjin 300071, China §Collaborative

Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin

300071, China ‖National

Chromatographic Research and Analysis Center, Dalian Institute of Chemical Physics,

Chinese Academy of Sciences, Dalian 116023, China

ABSTRACT: Multifunctional diagnosis and treatment integration platform is crucial in cancer treatments. Here, we show that by integrating Gd doped silicon nanoparticles (Si-Gd NPs), chlorine e6 (Ce6), doxorubicin (DOX), zeolitic imidazolate framework-8 (ZIF-8), poly(2(diethylamino)ethyl

methacrylate)

polymers

(HOOC-PDMAEMA-SH)

and

folic

acid-

polyethylene glycol-maleimide (MaL-PEG-FA) into one single nanoplatform by self-assembly method, novel multifunctional MOFs (named FZIF-8/DOX-PD-FA) are synthesized with great biocompatibility and tumor targeting, as well as pH responsive and no drug leakage for drug delivery. In the design, Si-Gd NPs and Ce6 embedded in the nanocomposites are used for magnetic resonance and fluorescence dual-model imaging, respectively. DOX loaded by the

ACS Paragon Plus Environment

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FZIF-8/DOX-PD-FA porous structure is used for chemotherapy, while Ce6 is excited by near infrared radiation (NIR) for photodynamic therapy. In addition, the pH-responsive ability of HOOC-PDMAEMA-SH to effectively prevent drug leakage is demonstrated by drug release studies in vitro. From the results of confocal microscopy imaging in vitro and fluorescence/magnetic resonance imaging (FL/MRI) in vivo, the FZIF-8/DOX-PD-FA showed a targeting effect on MCF-7 cancer cells. More importantly, the results of treatment experiments on tumor-bearing mice showed that the tumor volume of the FZIF-8/DOX-PD-FA+NIR group is decreased the most compared to the original volume. Owing to the unique dual-modal imaging capability and excellent chemo-photodynamic combinational cancer therapy effect, the present hybrid nanocarrier provides a new research platform for a new generation of theranostic nanoparticles. Keywords: ZIF-8, Si-Gd nanoparticles, chemo/photodynamic, imaging, drug delivery

INTRODUCTION Developing multifunctional diagnostic and therapeutic integration system for cancer is crucial due to the improved accuracy of drug delivery and effectiveness of treatment, as well as minimized side effects.1-3 Nowadays, nano-biomaterials for drug delivery system have been demonstrated as promising tools for individualized diagnosis and treatment.4-6 Remarkably, advantageous properties of metal organic framework materials (MOFs) include diverse structures and flexibility, tunable pore sizes, flexibility modification and controllable synthesis.7,8 Based on these basic physicochemical properties, MOFs can offer multiple versatilities for the accommodation of active ingredients.9,10 For example, the porous structure of the MOFs can be used to load one or more drugs for simultaneous or sequential release. Their powerful


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encapsulation performance can coat a variety of inorganic nanoparticles for comprehensive diagnosis and treatment. Luminous MOFs can monitor specific targets in real time through bioimaging.11,12 Their flexibility of modification enhances the targeting and biocompatibility of nanoparticles in clinical applications, so that MOFs have become a new platform for comprehensive advanced diagnosis and therapy. Among the MOFs, nano-scaled zeolitic imidazolate framework-8 (ZIF-8) nanoparticles have been widely utilized as versatile nanocarriers due to their biocompatibility and biodegradability under acidic conditions.13,14 However, sole ZIF-8 nanoparticles do not have the unique properties of inorganic nanoparticles (fluorescence, magnetic resonance, photoacoustic, Raman), which limits their further application in biomedical fields.15,16 It is worth noting that nanocomposites based on ZIF-8 and inorganic nanoparticle synthesis have been widely used in the imaging and treatment of cancer.17-21 With the increasing demand for multifunctional imaging and therapy, existing ZIF-8 composite nanomaterials have not met the requirements. To the best of our knowledge, there is no research on ZIF-8 complex nanocarriers based on Gd doped silicon nanoparticles (Si-Gd NPs). Inspired by the excellent fluorescence and magnetic resonance properties of Si-Gd NPs, it is extremely important to apply Si-Gd NPs to the synthesis of composite ZIF-8 nanoparticles for imaging research. In addition, Si-Gd NPs have unique characteristics including high quantum yield, good biocompatibility, low cost, good photostability and non-toxicity.22-24 Chemotherapy is the most primitive and common method of treatment for all cancers. Doxorubicin (DOX) is a common broad-spectrum anticancer drug that acts on DNA.25,26 However, the drug has great side effects on the human body such as spillage of blood vessels, which can cause tissue ulceration and necrosis.27,28 Therefore, designing a nanocarrier that not


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only can be loaded with DOX but also prevents it from leaking during drug delivery, and targeted to reach the tumor site to achieve controlled release is imminent.29 After targeted delivery to the tumor site, chlorine e6 (Ce6) as a photosensitizer is promoted by near-infrared laser irradiation until the excited state reacts with oxygen in the vicinity of the cell to produce singlet oxygen (ROS) to kill the cancer cells.30-32 At present, there are more and more reports on the coalescing of the phototherapy and chemotherapy.33,34 Compared with single treatment, comprehensive treatment affords multiple advantages in terms of improving cancer therapeutic efficiency. In addition, imaging technology-guided nanoscale treatment platforms can increase the personalization and efficiency of treatment.35-37 In order to improve the accuracy of diagnosis and treatment, multimodal imaging technology has been the focus of attention. Combining fluorescence imaging (FL) and magnetic resonance imaging (MRI) can solve the defects of limited penetration of fluorescence imaging and low sensitivity of magnetic resonance imaging, thus improving imaging accuracy. The ability to prevent drug leakage is one of the most important properties of nanomedicine carriers, which can effectively reduce the side effects of drugs during delivery and can improve the therapeutic effect. Many previously reported literatures have found that ZIF-8 without any surface modification and encapsulation has poor stability in water and will release a certain amount of drug when exposed to physiological conditions.38 Poly(2-(diethylamino)ethyl methacrylate) polymers (HOOC-PDMAEMA-SH) has a pKa of 7.3, and the polymer swells under acidic pH conditions, so it can be used to achieve drug release in a tumor micro-acid environment.39,40 Folic acid (FA) is a common target molecule that is modified on the surface of nanoparticles. Folate receptors (FRs) are overexpressed on many cancer cell surfaces such as


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MCF-7 breast cancer cells, HeLa cells, SK-OV-3 ovarian cancer cells and so on.41,42 Folic acid specifically recognizes and binds to the overexpressed folate receptor on the surface of cancer cells, which promotes the phagocytosis of the nanoparticles by the cells. Polyethylene glycol (PEG) is a water-soluble polymer that can improve the biocompatibility of nanomaterials to prolong the circulation time in the body when it was modified on the surface of nanoparticles.4345 Therefore,

PEG-FA is a suitable stabilizer for drug nanocarriers.

In this work, novel multifunctional MOFs (named FZIF-8/DOX-PD-FA) are rationally designed by integrating Si-Gd nanoparticles, Ce6 photosensitizer, DOX, ZIF-8, HOOCPDMAEMA-SH and PEG-FA into one single nanoplatform. FZIF-8/DOX-PD-FA achieve pHsensitive DOX release with precise control, due to the targeted PEG-FA and the pH-responsive polymer HOOC-PDMAEMA-SH that swells under tumor micro-acid environment, which are modified on the surface of the nanoparticles. It was observed by confocal microscopy imaging that MCF-7 cells (with highly expressed FRs) were able to phagocytose more FZIF-8/DOX-PDFA than A549 cells (with lowly expressed FRs). Cytotoxicity test results showed that the FZIF8/DOX-PD-FA+NIR group causes the most MCF-7 cancer cell death compared to other groups. FZIF-8/DOX-PD-FA exhibited excellent drug delivery capacity and effectively avoided side effect caused by drug leakage under physiological environmental conditions. As expected, the FZIF-8/DOX-PD-FA nanocomposites were successfully accumulated at the tumor sites and exhibit both fluorescence and magnetic resonance bimodal imaging. In addition, the fluorescence and

magnetic

resonance

dual-modal

imaging

guided

pH-responsive

drug

release

chemical/photodynamic combination therapy was validated by in vivo tumor-bearing mice experiments. More importantly, the versatile nanoparticles showed enhanced therapeutic effect.


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The synthesis of this comprehensive nanoparticle provides a new research direction and inspiration for the construction of a comprehensive platform for diagnosis and treatment.

EXPERIMENTAL SECTION Chemicals and Materials. Trisodium citrate, diethylenetriamine pentaacetic acid (DTPA), polyvinyl pyrrolidone K30 (PVP-K30) and 3-aminopropyltriethoxysilane (APTES) were purchased from J&K scientific ltd (Beijing, China). Chlorine e6 (Ce6), 4-cyanopentanoic acid dithiobenzoate (CTP), 4,4-azobis(4-cyanovaleric acid) (ACVA), sodium borohydride (NaBH4) and GdCl3.H2O were bought from Aladdin Biochemical Technology Co. Ltd (Shanghai, China). Fetal bovine serum (FBS), RPMI-1640 medium and the Annexin FITC/PI kit were purchased from Beijing solarbio science & technology Co., Ltd (Beijing, China). 2-Methylimidazole (Hmim) and Zn(NO3)2.6H2O were purchased from Tianjin Suihua Chemical Reagent Co., Ltd (Tianjin,

China).

2-(2-Methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-

tetrazole monosodium salt (CCK-8) kit was purchased from Shanghai Qianyuan Biomedical Technology Co., Ltd (Shanghai, China). Folic acid-polyethylene glycol-maleimide (MaL-PEGFA) (MW=2000 Da) (MW=2000 Da) was purchased from Shanghai Shengsheng Biological Technology Co., Ltd (Shanghai, China). Preparation of Polyvinyl Pyrrolidone (PVP) Modified Gadolinium-Doped Silicon Nanoparticles (Si-Gd@PVP NPs). To achieve Si-Gd NPs, we employed one-pot hydrothermal approach.46 Briefly: citrate (0.4 g), DTPA (0.3 g), and GdCl3.H2O (0.2 g) were dissolved in 8 mL of deionized water (DI.H2O), and then 2 mL of APTES was added to the above solution. After stiring vigorously for 20 min, the mixture solution was placed in a autoclave and then heated for 3 h at 200 ℃. The resulting Si-Gd NPs was purified by dialysis against DI.H2O. The purified Si-


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Gd NPs was collected. PVP-K30 solution (3 mL, 25 mg/mL in DI.H2O) was added to the above Si-Gd NPs, and the mixture was allowed to react with magnetic strring for 24 h at room temperature.47,48 The Si-Gd@PVP NPs was concentrated by centrifugation with ultrafiltration centrifuge tubes (MWCO = 5000 Da). The concentrated Si-Gd@PVP NPs were collected. Preparation

of

Poly(2-(diethylamino)ethyl

ethacrylate)

(HOOC-PDMAEMA-SH).

DMAEMA (3.68 g), ACVA (24 mg), and CTP (94 mg) were dissolved in ultra dry 1,4-dioxane. The resultant solution was stirred and purged with Ar gas for 1 h before reaction. And then, the mixture was allowed to polymerize for 24 h at 70 ℃.39,40 After being cooled to room temperature, the reaction solution was transferred to a dialysis bag for dialysis against methanol and hexane to remove impurities. PDMAEMA was dried with rotary evaporator. Next, the pH responsive polymer (HOOC-PDMAEMA-SH) was obtained by reducing PDMAEMA. Preparation of the Fluorescence ZIF-8 (FZIF-8). FZIF-8 was prepared as follows.17-20 Stock solution of Ce6 (1 mg/mL) was prepared in DI.H2O. First, the concentrated Si-Gd@PVP NPs solution was added to the Zn(NO3)2.6H2O solution (30 mL, 14.665 mg/mL) with magnetic strring, and 2-methylimidazole (30 mL, 32.4 mg/mL) solution was added to the above mixture. Then, the stock solution of Ce6 was added. This reaction mixture was stirred for 1 h. The precipitate was collected by centrifugal and washed three times with methanol and DI.H2O. FZIF-8 with different fluorescence properties and particle sizes were prepared by varying the amount of Ce6 and Si-Gd@PVP NPs. ZIF-8 was prepared using the same formulation as above except that Si-Gd@PVP NPs and Ce6 were not added. Preparation of the DOX Loaded FZIF-8 (FZIF-8/DOX). The solution of DOX (10 mL, 1 mg/mL in DI.H2O) was added to the FZIF-8 solution (20 mg/mL, 2.5 mL). This mixture solution


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was stirred for 12 h. FZIF-8/DOX was collected by centrifugal and washed with DI.H2O. The purified FZIF-8@DOX was dried by freeze-drying. Preparation of the HOOC-PDMAEMA-SH Modified FZIF-8/DOX (FZIF-8/DOX-PD). The as-synthesized 20 mg FZIF-8/DOX was dispersed in 30 mL of DI.H2O containing 10 mg of HOOC-PDMAEMA-SH, which was first sonicated for 20 min and then reacted for 48 h under room temperature.49,50 Afterward, the HOOC-PDMAEMA-SH modified FZIF-8/DOX could be obtained by the centrifugation (13000 rpm, 8 min). To remove the unreacted HOOCPDMAEMA-SH, the FZIF-8/DOX-PD was washed with DI.H2O for three times. Preparation of the PEG-FA Modified FZIF-8/DOX-PD (FZIF-8/DOX-PD-FA). Briefly, the as-synthesized 20 mg FZIF-8/DOX-PD was dispersed in 30 mL of phosphate buffer (pH=8.5) containing 10 mg of MaL-PEG-FA, which was first sonicated for 20 min and then stirred for 8 h under room temperature. Afterward, the PEG-FA modified FZIF-8/DOX-PD-FA could be obtained by the centrifugation and washed with deionized water. Determination of Reactive Oxygen Species (ROS) in Vitro. The singlet oxygen sensor green (SOSG) was used as ROS probe.51,52 The SOSG solution (10 μL, 200 μmol/L) was added to the solution of ZIF-8 and FZIF-8/DOX-PD-FA (2 mL, 0.5 mg/mL). The mixture solution was irradiated with 630 nm laser for different times. Then, the fluorescence intensity were recorded on a fluorescence spectrophotometer. The ROS quantum yields were calculated. In Vitro pH-Sensitive Release of Drugs from FZIF-8/DOX-PD-FA. DOX release test was completed by dialysis under different pH conditions.7,8 Briefly, three groups of FZIF-8/DOXPD-FA (80 mg) and three groups of FZIF-8/DOX (80 mg) were dispersed in 4 mL of PBS buffer (pH = 7.4 ) respectively. In addition, three groups of 80 mg FZIF-8/DOX-PD-FA were dispersed


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in 4 mL of PBS buffer (pH = 6.0). After the above samples were transferred to the dialysis bags (MWCO = 10000 Da), they were separately immersed in 40 mL of the corresponding different PBS buffer under magnetic stirring at 37 ℃. The supernatent (4 mL) of each group was taken at different time for testing, and corresponding fresh PBS was added. The DOX release rate were calculated by the absorption peak at 490 nm of the UV-vis spectrophotometry. Cytotoxicity Experiment. The MCF-7 cells and A549 cells were obtained from the Second Affiliated Hospital of Tianjin Medical University. The above two kinds of cell lines were cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS) under a humidified environment of 37 °C, 5% CO2 and 95% air.53,54 Cytotoxicities of FZIF-8, FZIF-8/DOX, free DOX, and FZIF-8/DOX-PD-FA were conducted on MCF-7 and A549 cancer cells using the CCK-8 kits. For experimental purposes, cells were seeded at 1.5×104 per well into 96-well cell culture plates and incubated overnight. After the medium was removed, fresh medium containing different concentrations of the above nanoparticles was added. After incubation for 4 h, cells were replaced with new medium and one group of cells incubated with FZIF-8/DOX-PD-FA was exposed to 630 nm laser irradiation for 5 min at the power of 300 mW. The cells were incubated for another 24 h. Then, 10 μL of CCK-8 was added to the culture plate for further 1 hour, and the absorbance was measured at 450 nm by a microplate reader. In order to conduct a flow cytometry test for apoptosis comparison test, cells were seeded at 1.5×105 per well into 6-well cell culture plates and incubated overnight. The remaining operations were as described above. Then, the cells were stained with the Annexin FITC/PI kit and tested by flow cytometry. In Vitro Imaging. MCF-7 cells and A549 cells were seeded into cell culture dishes at 1.2×105 cells per dish and incubated overnight.53,54 After incubation with different concentrations of the nanoparticles (FZIF-8, FZIF-8/DOX-PD-FA, FZIF-8/DOX) for a period of time at 37 °C, the


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medium was carefully removed, and the cells were washed 3 times with PBS (0.1 M, pH 7.4), and then the cells were fixed with 4% paraformaldehyde for 0.5 hours. Next, a laser confocal microscope was used to observe the state in which these nanoparticles are phagocytosed by the cells and the growth state of the cells. In Vivo Imaging Detection. The FZIF-8/DOX-PD-FA nanoparticles were injected into the MCF-7 tumor bearing nude mice via intraperitoneal (50 mg/kg) when the tumor volume reached 100-150 mm3. The near-infrared (NIR) imaging was conducted at 0 min, 10 min, 1 h, 2 h, 14 h and 24 h after injection. The nuclear magnetic resonance imaging was conducted at 14 h after injection.7,8 In Vivo Chemo/Photodynamic Combinational Cancer Therapy. To assess the anti-tumor effect of FZIF-8/DOX-PD-FA, we established a MCF-7 tumor model. 100 μL (12106) of MCF7 cells were implanted subcutaneously in nude mice (8 weeks old), and the tumors were grown to 140-160 mm3. These tumor-bearing mice were used to study the antitumor efficacy of FZIF8/DOX-PD-FA, FZIF-8/DOX, DOX, FZIF-8/DOX-PD-FA+NIR. Briefly, MCF-7 tumor-bearing mice were divided into 5 groups, each group containing 5 mice. Two groups were intraperitonealy injected with 200 μL of FZIF-8/DOX-PD-FA (50 mg/kg), and the third group (PBS) as a control experiment. FZIF-8/DOX (200 μL, 50 mg/kg) was intraperitonealy injected into the fourth group and the last group was intraperitonealy injected with DOX. The above preparation was injected every other day, and one group of intraperitonealy injected wih FZIF8/DOX-PD-FA (50 mg/kg) was irradiated with 630 nm laser for 10 min. In order to assess the treatment effect, the tumor sizes and weights of these mice were monitored during the process.7,8 To test the in vivo toxicity of these nanoparticles, the internal organs were stained with hematoxylin and eosin (H&E) for histological analysis. All procedures involving animals were


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approved by the Institutional Animal Care Committee of Nankai University and conformed to the National Institutes of Health guidelines.22-24

RESULTS AND DISCUSSION Preparation and Characterization of FZIF-8/DOX-PD-FA. The complete synthesis process of FZIF-8/DOX-PD-FA and the application of fluorescence and magnetic resonance dual-model imaging-guided chemo/photodynamic combinational cancer therapy are illustrated in Scheme 1 and 2. Firstly, Si-Gd NPs with good optical properties and magnetic resonance capabilities were synthesized by one-pot hydrothermal approach.46 Secondly, the PVP-K30 was modified on the surface of Si-Gd NPs to successfully prepare Si-Gd@PVP nanoparticles.47,48 Next, Zn2+, Hmin, Ce6 and Si-Gd@PVP NPs self-assembled by one-pot method to form multifunctional MOFs (FZIF-8).49,50 After FZIF-8 loaded with DOX (FZIF-8/DOX), in order to achieve controlled drug release, HOOC-PDMAEMA-SH (Scheme 2A) was wrapped on the FZIF-8/DOX surface (FZIF8/DOX-PD) through the coordination bonds formed by the Zn2+ and the -COOH (Scheme 2B). In addition, the precise tumor targeting capability and biocompatibility of the nanocomposites were achieved via MaL-PEG-FA targeting ligands that was further modified on the FZIF-8/DOX-PD surface (FZIF-8/DOX-PD-FA). This is because the thiol group on the FZIF-8/DOX-PD surface can react with the MaL at the end of the MaL-PEG-FA to form a covalent bond under alkaline conditions

(Scheme

2C).

Finally,

intraperitoneal

injection

of

FZIF-8/DOX-PD-FA

nanocomposites into tumor-bearing mice were conducted for diagnosis and treatment. The morphology and fluorescence characteristics of the Si-Gd NPs were characterized by TEM (Figure 1A,B) and fluorescence spectroscopy (Figure 1C and S1A,B), respectively. Comparable to Si-Gd NPs (black line in Figure S1C), the Si-Gd@PVP NPs exhibit the infrared


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characteristic absorption peak of PVP -N-C=O (1290 cm-1), -CH2 (1420 cm-1), -C=O (1671 cm-1), confirming the successful modification of PVP (red line in Figure S1C). From the photoluminescence spectra (Figure S1D), the optimal excitation wavelength and emission wavelength of Si-Gd@PVP NPs are red-shifted compared to Si-Gd NPs. Next, we synthesized FZIF-8 under different reaction conditions. As shown in Figures S2 to S5, the concentration of Si-Gd@PVP NPs and Ce6 do have a great influence on the morphology and fluorescence properties of FZIF-8 nanoparticles. Considering the need for imaging and treatment, we chose a reaction system with 1 mL of Si-Gd@PVP NPs and 2 mL (1 mg/mL) of Ce6. In order to prevent Scheme 1. (A) The synthesis route of FZIF-8/DOX-PD-FA. (B) Schematic illustration of fluorescence and magnetic resonance dual-modal imaging-guided chemo/photodynamic combinational cancer therapy.


12

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Scheme 2. (A) The synthesis route of PDMAEMA-SH. (B) The process that PDMAEMA-SH is wrapped on the surface of FZIF-8/DOX. (C) The process that PEG-FA is modified on the surface of FZIF-8/DOX-PD.


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Figure 1. (A) TEM image of Si-Gd NPs. (B) Particle size distribution of Si-Gd NPs. (C) Optical properties of the Si-Gd NPs aqueous solution. Photo-luminescence excitation spectrum (EX, red line), and PL spectrum (EM, black line). TEM image of ZIF-8 (D), FZIF-8/DOX (E), FZIF8/DOX-PD-FA (F). (G) Dark-field STEM image and EDS elemental mapping of one FZIF8/DOX-PD-FA.


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the drug from being leaked during the delivery process, a pH-responsive polymer (HOOCPDMAEMA-SH) was synthesized. First of all, the polymer of PDMAEMA can be easily confirmed via 1H NMR (Figure S6). Then, the NMR spectra (Figure S7) and UV–vis characteristic (Figure S8) change of PDMAEMA before and after reduction by NaBH4 prove the successful preparation of HOOC-PDMAEMA-SH. Gel permeation chromatography (GPC) results show that the calculated DPn was 52, and the Mw/Mn value was 1.22. TEM (Figure 1) shows that the particle sizes are 45 nm of ZIF-8 (Figure 1D), 65 nm of FZIF8/DOX (Figure 1E) and 70 nm of FZIF-8/DOX-PD-FA (Figure 1F), respectively. It is clear that the particle size of FZIF-8/DOX-PD-FA is larger than that of FZIF-8/DOX due to the coating of the pH-responsive polymer HOOC-PDMAEMA-SH and PEG-FA. The composition of the FZIF8/DOX-PD-FA structure was evident from the dark-field scanning transmission electron microscopy (DF-STEM) and EDS elemental mapping data (Figure 1G and S9). As shown in Figure 2A, the emission wavelengths of the FZIF-8/DOX-PD-FA are concentrated at 470 nm (Si-Gd NPs) and 675 nm (Ce6), respectively22,31. The appearance of fluorescent signals further proves that Si-Gd NPs and Ce6 are doped in the FZIF-8/DOX-PD-FA. The difference between the infrared characteristic peaks of FZIF-8 and ZIF-8 can further proves that Si-Gd NPs and Ce6 are successfully embedded (Figure S10A). Comparable to FZIF-8/DOX (red line in Figure 2B), FZIF-8/DOX-PD exhibits the infrared characteristic absorption peak of ester carbonyl group (C=O 1734 cm-1), confirming the successful modification of HOOC-PDMAEMA-SH. Comparable to FZIF-8/DOX-PD (blue line in Figure 2B), FZIF-8/DOX-PD-FA exhibits the infrared characteristic absorption peak of amide group (-N=C=O 1571 cm-1), confirming the successful modification of MaL-PEG-FA. In addition (Figure 2B), HOOC-PDMAEMA-SH exhibits the infrared characteristic absorption peak of ester carbonyl group (-C=O 1734 cm-1),


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Figure 2. (A) The emission spectrum of FZIF-8/DOX-PD-FA (ex = 408 nm). (B) FT-IR spectra of the HOOC-PDMAEMA-SH polymer (black line), FZIF-8/DOX (red line), FZIF8/DOX-PD (blue line) and FZIF-8/DOX-PD-FA (pink line). (C) XRD analyses for ZIF-8 (green line), FZIF-8 (pink line), FZIF-8/DOX-PD (blue line), FZIF-8/DOX-PD-FA (red line), and SiGd NPs (black line) which enlarged view is in the upper right corner of Figure C. (D) TGA analyses for FZIF-8/DOX (black line), FZIF-8/DOX-PD (red line) and FZIF-8/DOX-PD-FA (blue line). (E) Pore size distribution of FZIF-8. (F) Analysis of adsorption and degassing of nitrogen by FZIF-8.


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also confirming the successful preparation39. We further demonstrated the successful modification of HOOC-PDMAEMA-SH and MaL-PEG-FA by the characterization of XRD (Figure 2C), TGA (Figure 2D) and zeta potential (Figure S10B). N2 adsorption-desorption isotherm measurements of FZIF-8 at 77 K shows type-I isotherms (Figure 2E,F), and characteristic with Brunauer–Emmett–Teller-specific surface areas (SBET) of 845.8 m2/g. These results further indicate that as-synthesized FZIF-8 possesses significantly higher specific surface areas and larger pore volume. Owing to mesoporous structure, FZIF-8 can be supposed as ideal carriers to encapsulate anticancer drug. Herein, DOX, a broad-spectrum anti-tumor drug is chosen as an anticancer drug model with a high loading capacity of 258.8 mg/g. FZIF-8 increases the load of DOX and is not quickly removed from the body. In Vitro ROS Test and pH-Sensitive Release of DOX. Ce6 is a promising photosensitizer or PDT agent that mediates the photo-induced conversion of oxygen to ROS with therapeutic effects. The ROS generation of FZIF-8/DOX-PD-FA induced by the embedded Ce6 upon laser irradiation was detected using singlet oxygen sensor green (SOSG) as the indicator, whose ﬂuorescence at 525 nm.51,52 It can be found from Figure 3A that the fluorescence intensity of SOSG in the FZIF-8/DOX-PD-FA solution is increased by 8.3 times after irradiated by 630 nm laser for 10 minutes. However, there is no significant change in the fluorescence intensity of SOSG in the ZIF-8 solution, suggesting the ROS could be effectively produced by FZIF-8/DOXPD-FA and the nanoparticle can be used for photodynamic therapy. As a drug-delivery system, pH-responsive release is pivotal for the biological application.7,8 Therefore, cumulative release profiles were investigated in a series of buffers of pH 7.4 and 6.0. As shown in Figure 3B, FZIF-8/DOX-PD-FA has almost no release of DOX at pH 7.4 compared


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Figure 3. (A) 1O2 generation of FZIF-8/DOX-PD-FA and ZIF-8 under 300 mW/cm2 of NIR light irradiation. SOSG was used as the ROS probe. (B) Cumulative release of DOX from the FZIF8/DOX-PD-FA at different pHs (pH=7.4, pH=6.0), and cumulative release of DOX from the FZIF-8/DOX at pH=7.4. All statistical data are represented as mean ± standard deviation (SD) (n = 3). Cytotoxicity assay of MCF-7 cancer cells (C) and A549 cancer cells (D) after incubation with different concentrations of nanoparticles with or without laser irradiation by standard CCK8 assays. All statistical data are represented as mean ± standard deviation (SD) (n = 8). (E) FACS analysis of apoptosis and necrosis of MCF-7 cancer cells by staining with Annexin V-FITC and PI after various treatments (Nanoparticles concentration: 80 μg/mL).


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to FZIF-8/DOX. However, FZIF-8/DOX-PD-FA can release up to 57% of DOX at pH 6.0 (tumor microenvironment). The above datas show that the modification of HOOC-PDMAEMASH does play a role in preventing drug leakage during drug delivery. Moreover, it also shows that the FZIF-8/DOX-PD-FA can be applied to drug delivery. In Vitro Toxicity and in Vitro Chemo/photodynamic Combinational Antitumor Effect. To assess the effect of FZIF-8/DOX-PD-FA on chemo/photodynamic therapy in vitro, we selected MCF-7 (with highly expressed FRs) and A549 (with lowly expressed FRs) cancer cell lines.53,54 Cytotoxicity experiments were evaluated by CCK-8 immunoassay. As observed in Figure 3C and 3D, the survival rates of MCF-7 and A549 cells are higher than 90% after incubation for 24 h with different concentrations of the as-prepared FZIF-8 (black). This result indicates that FZIF-8 is non-toxic and has good biocompatibility and can be used for in vivo research. Figure 3C and 3D also shows the viability of MCF-7 and A549 cancer cells after incubation with different concentrations of FZIF-8/DOX-PD-FA and FZIF-8/DOX with or without laser irradiation (300 mW/cm2). The survival rates of all formulation groups gradually decrease along with the increase in FZIF-8/DOX-PD-FA and FZIF-8/DOX concentration, indicating a concentration-dependent therapeutic effect. Notably, the apoptotic rate of MCF-7 cells is higher than that of A549 cells. This result demonstrates that FA on the surface of FZIF8/DOX-PD-FA can promote the phagocytosis of the nanoparticles by cells overexpressing FRs. As shown in Figure 3C, the FZIF-8/DOX-PD-FA group shows a much higher cytotoxicity effect on MCF-7 cells than free DOX group and FZIF-8/DOX, which demonstrates that the FZIF-8/DOX-PD-FA effectively avoids drug leakage and has the ability to target. For the laser irradiated groups, the cell viability decreases significantly as the concentration of FZIF-8/DOXPD-FA increased. At the concentration of 80 μg/mL, the cell viability of FZIF-8/DOX-PD-FA


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groups with NIR laser irradiation decreased to 20.1%. In comparison, for the FZIF-8/DOX-PDFA group with NIR laser irradiation, the cell viability is lower than the FZIF-8/DOX-PD-FA group without NIR laser irradiation. This is because the near-infrared laser irradiation of FZIF8/DOX-PD-FA can stimulate the photosensitizer Ce6 to produce ROS which can also have a killing effect on cells. This strongly reveals that the synergy of chemotherapy and photothermal therapy shows obvious superiority. To further demonstrate the chemo/photodynamic combinational antitumor effect, MCF-7 cancer cells were co-cultured with FZIF-8/DOX-PD-FA, FZIF-8/DOX and FZIF-8 (80 μg/mL) for 4 h, respectively. Then, one FZIF-8/DOX-PD-FA group was irradiated with NIR laser for 10 minutes, and then all groups of cells were cultured for 24 hours and analyzed by flow cytometry (FACS) with Annexin V-FITC/PI fluorescence staining to further verify the level of apoptosis and necrosis. From the results shown in Figure 3E, the FZIF-8/DOX-PD-FA group with NIR laser irradiation shows significant apoptosis and necrosis than other groups of cells, which is mainly due to the synergistic effect of chemical drug and photodynamic therapy.33,55 This result is consistent with the above results of CCK-8 assay. Taken together, FZIF-8/DOX-PD-FA can be used as a potential therapeutic agent based on a combination of chemotherapy and photodynamic therapy. In Vitro Multicolor Fluorescence Imaging Application. To evaluate the efficacy of FZIF-8PD-FA on the targeted fluorescence imaging, the effects of concentration of FZIF-8-PD-FA were first investigated. After incubation with the FZIF-8-PD-FA for 4 h (Figure 4 and 5), the cancer cells became brightly illuminated with dual-color when they were excited with 408 nm and 561 nm. From the confocal microscopy images shown in Figure 4, the higher the concentration of FZIF-8-PD-FA incubated with MCF-7 cancer cells, the stronger the fluorescence intensity. In


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contrast (Figure 5), MCF-7 cancer cells incubated with FZIF-8 and A549 cancer cells incubated with FZIF-8-PD-FA show weaker fluorescence intensity than MCF-7 cells incubated with FZIF8-PD-FA. This is attributed to the specific binding of the FA on the FZIF-8-PD-FA surface to folate receptor on the surface of MCF-7 cells, which promotes the phagocytosis of FZIF-8-PDFA. To further illustrate the targeting of FZIF-8-PD-FA to MCF-7 cancer cells, we performed analysis using flow cytometry (FACS). From the results shown in Figure S11, when FZIF-8-PDFA is co-cultured with MCF-7 cells, more cells show fluorescent signals, indicating that more FZIF-8-PD-FA nanoparticles are phagocytized. This result agrees with previous studies on the apoptosis of cells.

Figure 4. Confocal microscopy images (excitation wavelengths were 408 nm and 561 nm) of MCF-7 cancer cells after incubation with different concentrations of FZIF-8-PD-FA (20 μg/mL and 40 μg/mL) for 4 h.


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Figure 5. Confocal microscopy images (excitation wavelengths were 408 nm and 561 nm) of MCF-7 cancer cells after incubation with FZIF-8-PD-FA (40 μg/mL) and FZIF-8 (40 μg/mL) for 4 h, and A549 cancer cells after incubation with FZIF-8-PD-FA (40 μg/mL). In addition, we explored the ability of FZIF-8/DOX-PD-FA to deliver DOX to MCF-7 cells via fluorescent signals. After incubation with the FZIF-8/DOX-PD-FA for 2 h and 4 h (Figure 6A), the cells became brightly illuminated with multicolor when they were excited with 408 nm, 486 nm and 561 nm, respectively. At the same time, it can be observed that DOX is still in the cytoplasm during co-culture for 2 hours, but DOX almost completely enters the nucleus after 4 hours. As shown in Figure S12, the higher the concentration of FZIF-8/DOX-PD-FA incubated with MCF-7 cancer cells, the stronger the fluorescence. Futhermore, MCF-7 cells incubated with FZIF-8/DOX-PD-FA exhibits the strongest fluorescence intensity compared to FZIF-8/DOXincubated MCF-7 cells and FZIF-8/DOX-PD-FA-incubated A549 cells (Figure 6B).


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Figure 6. (A) Confocal microscopy images of MCF-7 cancer cells after incubation with FZIF8/DOX-PD-FA (40 μg/mL) for 4 h and 2 h. (B) Confocal images of MCF-7 cancer cells after incubation with FZIF-8/DOX-PD-FA (40 μg/mL), FZIF-8/DOX (40 μg/mL) for 4 h, and A549 cancer cells after incubation with FZIF-8/DOX-PD-FA (40 μg/mL) for 4 h. (Excitation wavelengths were 408 nm, 486 nm and 561 nm.)


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Figure 7. Confocal microscopy images (excitation wavelengths were 408 nm, 486 nm and 561 nm) of MCF-7 cancer cells after co-cultured with FZIF-8/DOX-PD-FA (80 μg/mL) for 4 h and irradiated by 630 nm NIR laser light at 300 mW/cm2 (0 min, 5 min and 10 min).

More importantly, the effect of chemo/photodynamic combinational therapy was observed by laser confocal imaging. First, FZIF-8/DOX-PD-FA (80 μg/mL) was co-cultured with MCF-7 cells for 4 hours. Furthermore, the cells were exposed to NIR laser for 0, 5 and 10 minutes, respectively. After 24 hours of culture, laser confocal observation of cell growth status was performed. As shown in Figure 7, with the prolongation of NIR laser irradiation time, there are more and more apoptotic cells. When the irradiation time reached 10 minutes, the cells are almost all apoptosis. The results observed in this confocal image are consistent with previous cytotoxicity assays, which further demonstrates the effectiveness of the chemo/photodynamic combinational antitumor.


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Figure 8. (A) In vivo NIR florescence imaging of MCF-7 tumor bearing mouse at 0, 10 min, 1 h, 2 h, 14 h and 24 h after intraperitoneal injection of FZIF-8/DOX-PD-FA or FZIF-8/DOX. (B) T1-Weighted MR images of FZIF-8/DOX-PD-FA with different concentrations of Gd: 4, 2.5, 1.54, 0.75, 0.4 and 0 mM. (C) The r1 relaxivity curve of FZIF-8/DOX-PD-FA. (D) In vivo MR images of mouse before and after intraperitoneal injection of FZIF-8/DOX-PD-FA (Red circles are the tumor sites). In Vivo Studies in Tumor Fluorescence and MRI Dual-Model Imaging. To study the targeted behaviors of FZIF-8/DOX-PD-FA in vivo, the mice bearing tumors derived from MCF7 cells were administered with FZIF-8/DOX-PD-FA and FZIF-8/DOX intraperitoneally, which were then monitored using a NIR fluorescence imaging system. The in vivo NIR fluorescence images of mice at 0 min, 10 min, 1 h, 2 h, 14 h, and 24 h following the injection of FZIF-8/DOX-


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PD-FA or FZIF-8/DOX are shown in Figure 8. As shown in Figure 8A, the strongest remarkable fluorescence signal of FZIF-8/DOX-PD-FA is observed at the tumor site 14 h postinjection, which reflects target selectivity and effective enrichment. Subsequently, the fluorescence signal of the tumor decrease progressively during the following 10 h, which indicates that FZIF8/DOX-PD-FA is gradually metabolized. As a control experiment, FZIF-8/DOX was intraperitoneally injected into tumor-bearing mice, and it can be seen from Figure 8A that no FZIF-8/DOX-PD-FA nanoparticles are enriched at the tumor site at the same time. In addition, T1-weighted MRI studies were performed on tumor-bearing mice to affirm the high penetration and enhanced accumulation of FZIF-8/DOX-PD-FA inside the solid tumor. Gadolinium ions (Gd3+) is one of the most widely used magnetic resonance agents today. Since FZIF-8/DOX-PD-FA is doped with Si-Gd NPs, magnetic resonance imaging can be performed. T1-Weighted MR images of FZIF-8/DOX-PD-FA with different concentrations of Gd (4, 2.5, 1.54, 0.75, 0.4 and 0 mM) are shown in Figure 8B. T1-weighted MR images of the FZIF8/DOX-PD-FA reveal a concentration-dependent brightening effect. The r1 relaxivity curve of FZIF-8/DOX-PD-FA is shown in Figure 8C. At the same time, the FZIF-8/DOX-PD-FA solution was injected via intraperitoneal administration. As shown in Figure 8D, a strong MRI signal is observed at the tumor site, indicating the ability of magnetic resonance contrast strengthening effect of FZIF-8/DOX-PD-FA nanocomposites. However, no signal is observed in the control group. In summary, the FZIF-8/DOX-PD-FA can achieve targeted fluorescence and magnetic resonance dual-modal imaging in tumor-bearing mice. More importantly, the targeted accumulation of the nanoparticles in the tumor and great imaging property enable the utilization of FZIF-8/DOX-PD-FA to locate the tumor, thereby providing a precise imaging-guided nanoplatform for precision medicine.


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In Vivo Studies on Chemo/Photodynamic Combinational Therapy. In order to prove that FZIF-8/DOX-PD-FA has a good anticancer efficacy through the combination of PDT and chemotherapy, we performed an in vivo anti-tumor experiment. MCF-7 tumor models were established by subcutaneous injection of 100 μL (1×107) MCF-7 cells in the front or hind legs of 8-week-old nude mice. When the tumor volume reached 140-160 mm3, the tumor-bearing mice were divided into five groups, i.e., PBS group, FZIF-8-PD-FA+NIR group, free DOX group, FZIF-8/DOX-PD-FA group, FZIF-8/DOX-PD-FA+NIR group. During the treatment of tumors, 200 μL of PBS, FZIF-8-PD-FA (50 mg/kg), FZIF-8/DOX-PD-FA (50 mg/kg), free DOX (equivalent to the release amount from FZIF-8/DOX-PD-FA) were intraperitoneally injected into tumor-bearing mice. After 14 h, the tumor-bearing mice in the FZIF-8-PD-FA+NIR group and the FZIF-8/DOX-PD-FA+NIR group were irradiated with 630 nm (300 mW/cm2) laser for 10 minutes for photodynamic therapy. These samples were injected every two days and NIR illumination was performed. In addition, tumor volume and mass changes were recorded every other day. And analyze the statistics between each group. As shown in Figure 9A and B, compared to the PBS group, all the treatment groups show inhibited tumor growth. The tumor volume of the PBS control group increase to 5.4 folds at day 11. After the treatment with free DOX, the tumor volume increase to 4.01 times of original size, while the tumor volume of FZIF8/DOX-PD-FA group reduce to 0.87 times. This is because FZIF-8/DOX-PD-FA has a targeting and pH responsiveness as a drug nanocarrier, and is more easily enriched at the tumor site, so that more DOX can be brought to the tumor site to more effectively inhibit the growth of tumor. Compared with the PBS group, the FZIF-8-PD-FA+NIR group exhibite an effect of inhibiting tumor growth. This is attributed to the fact that the singlet oxygen generated by the doping of Ce6 in the FZIF-8-PD-FA under the NIR laser irradiation inhibits the growth of the tumor cells.


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Excitingly, the maximum inhibition rate can be observed for the FZIF-8/DOX-PD-FA+NIR group, in which the tumor volume reduce to 0.22 times of original size. This is attributed to the synergistic treatment of chemical drugs and photodynamics. Tumor morphology picture after treatment also demonstrates an enhanced synergistic therapy of PDT and chemotherapy (Figure 9B). Therefore, FZIF-8/DOX-PD-FA+NIR nanoplatforms show excellent chemo/photodynamic combinational therapy effects for cancer in vivo under irradiation of an NIR laser, far exceeding the effects of photodynamic therapy or chemotherapy alone.

Figure 9. In vivo anti-tumor effects. All intraperitoneal injection (50 mg/kg of nanoparticles and equivalent DOX) were performed once every other day. (A, B) The tumors from different mice groups treated with PBS, DOX, FZIF-8-PD-FA+NIR, FZIF-8/DOX-PD-FA and FZIF-8/DOXPD-FA+NIR. (C) Body weight monitoring during the process. All statistical data are represented as mean ± standard deviation (SD) (n = 5).


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Figure 10. Histological analysis of major organs (hearts, livers, spleens, lungs, and kidneys) of tumor-bearing mice from different treated groups. (DOX, FZIF-8-PD-FA+NIR, FZIF-8/DOXPD-FA and FZIF-8/DOX-PD-FA+NIR.) Organ sections were stained with H&E. Moreover, we monitored the body weight of the tumor-bearing mice for all groups. As shown in Figure 9C, the weight monitoring curves show that the body weight is normal during the treatment. To further assess systemic toxicity in the animals, the H&E staining analysis of heart, liver, spleen, lung and kidney of these tumor-bearing mice were performed. Figure 10 shows that there are no obvious lesions in various tissues and organs of mice during the treatment. The above experimental results indicate that FZIF-8/DOX-PD-FA+NIR has lower side effects on organisms and excellent hemo/histocompatibility both in vitro and in vivo for tumor therapy.

CONCLUSION In summary, we have successfully prepared versatile FZIF-8/DOX-PD-FA by rapid and simple self-assembly method and further modification with pH responsive polymers and targeting ligands. Confocal microscopy images and cytotoxicity experimental results


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demonstrate the effectiveness of FZIF-8/DOX-PD-FA in imaging and treatment in vitro. Tumor targeting ability of FZIF-8/DOX-PD-FA nanocomposites has been confirmed through FL/MRI imaging. In vivo therapeutic experimental results on tumor-bearing mice are sufficient to demonstrate that FZIF-8/DOX-PD-FA nanocomposites have the ability to avoid drug leakage and combine chemotherapy with PDT to enhance the efficacy of cancer treatment efficacy. To the best of our knowledge, this is the first report on ZIF-8, which combines Si-Gd NPs with the photosensitizer Ce6 for comprehensive treatment guided by dual-modal imaging of tumors. The design of FZIF-8/DOX-PD-FA not only broadens the application of ZIF-8 nanoparticles, but also opens up new possibilities for the development of effective and safe therapeutic treatments for cancer. ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: Fluorescence spectra, FT-IR spectra, TEM and SEM images, 1H NMR spectra, Ultraviolet absorption spectra, EDS element analysis, FACS analysis, Confocal microscopy images. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] ORCID wenyouLi NOTES The authors declare no competing financial interest. ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (nos. 21775077 and 21475069) and the Tianjin Natural Science Foundation (no. 16JCZDJC37200).


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pH-Responsive Polymer-Stabilized ZIF-8 Nanocomposite for

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