Prussian Blue Nanozyme with Multienzyme Activity Reduces Colitis in

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Biological and Medical Applications of Materials and Interfaces

Prussian Blue Nanozyme with Multi-enzyme Activity Reduces Colitis in Mice Jiulong Zhao, Xiaojun Cai, Wei Gao, Linlin Zhang, Duowu Zou, Yuanyi Zheng, Zhaoshen Li, and Hangrong Chen ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b10345 • Publication Date (Web): 20 Jul 2018 Downloaded from http://pubs.acs.org on July 22, 2018

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ACS Applied Materials & Interfaces

Prussian Blue Nanozyme with Multi-enzyme Activity Reduces Colitis in Mice

Jiulong Zhao1, Xiaojun Cai2,3*, Wei Gao3, Linlin Zhang2, Duowu Zou1, Yuanyi Zheng3, Zhaoshen Li1*, Hangrong Chen2*

1

Department of Gastroenterology, Changhai Hospital, Second Military Medical University,

200433, Shanghai, P. R. China E-mail: [email protected] 2

State Key Laboratory of High Performance Ceramics and Superfine Microstructure,

Shanghai Institute of Ceramics, Chinese Academy of Sciences 200050, Shanghai, P. R. China E-mail: [email protected] 3

Shanghai Institute of Ultrasound in Medicine, Shanghai Jiao Tong University Affiliated

Sixth people's Hospital, 200050, Shanghai, P. R. China E-mail: [email protected]

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Abstract: The over production of reactive oxygen species (ROS) is central to the progression of Inflammatory bowel disease (IBD), which may be the potential therapeutic target. Prussian blue nanoparticles (PB) with good biosafety, can act as an artificial nanozyme, effectively scavenging ROS. To date, PB-based nanomaterials have not been developed and utilized for treatment of IBD. In this study, polyvinylpyrrolidone (PVP)-modified Prussian blue nanoparticles (PPBs) are constructed with good physiological stability and biosafety by a simple and efficient method. The prepared PPBs with capabilities of scavenging ROS and inhibiting proinflammatory cytokine, significantly reduce colitis in mice without distinct side effects via intravenous administration. This report provides a demonstration of the protective effect of PB-based nanomedicine against IBD in living animals, offering hope and a potential alternative treatment option for patients suffering from IBD. Keywords: Prussian blue, reactive oxygen species, inflammatory bowel disease, colitis, nanozyme


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Table of Content



1. INTRODUCTION Inflammation is a defensive immune response against foreign pathogens, which is mediated by the innate and adaptive immune systems in vertebrates.1-2 Inflammatory bowel disease (IBD), including ulcerative colitis (UC) and Crohn's disease, is an autoimmune disease, in which the immune system attacks the cells of the digestive system, and involves chronic inflammation of the gastrointestinal tract.3-6 The production of reactive oxygen species (ROS) including hydrogen peroxide (H2O2), hydroxyl radicals (•OH), and superoxide anions (•OOH), is central to the progression of many inflammatory diseases.7-9 ROS act as both signaling molecules and the mediator of inflammation. They are classically defined as partially reduced metabolites of oxygen, which act as strong oxidants, inducing damage to proteins, lipids, and DNA.10-12 Nanoparticulate drug delivery is a promising strategy for treatment of IBD, which can increase the targeting efficacy and reduce side effects.13-14 Bilirubin nanoparticles with a size of approximately 110 nm, were reported to act as efficient hydrogen peroxide scavengers, accumulating at the site of inflammation and reducing colitis in mice via intravenous administration.15 In addition, Vong et al. developed a nitroxide radical-containing nanoparticle with antioxidative nitroxide radicals for treatment of mice with dextran sulfate sodium (DSS)-induced colitis.16 To scavenge ROS at the site of inflammation is a promising strategy for treatment of IBD.10, 17 Prussian blue nanoparticles (PBs) have been widely applied as a cancer theranostic agent1823

and biosensor 24, due to its excellent magnetic, photothermal conversion, and multi-enzyme

mimetic capabilities. In our previous researches, PB-based nanomedicines were designed to achieve ultrasound/magnetic resonance imaging/photoacoustic imaging and photothermalchemo therapy for tumor successfully.19-21 Importanly, we found that PBs could effectively scavenge ROS, including •OH, H2O2, and •OOH, through peroxidase (POD), catalase (CAT), and superoxide dismutase (SOD) activities,19 which is consistent with the other reported researches.25 Thus, PB may possess great potential in the treatment of inflammatory diseases 4 ACS Paragon Plus Environment

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targeting ROS. However, until now, PB-based nanomedicine has not been explored and applied in the treatment of IBD. Here, we provide a demonstration of

PB nanozyme for treatment of IBD. The

monodispersive polyvinylpyrrolidone (PVP)-modified PB nanoparticles (PPBs) was constructed via a one pot strategy to address the insolubility of PBs (Figure 1a). The prepared PPBs show excellent stability in various environments because of the stabilization of PVP. The PPBs could act as an artificial nanoenzyme, effectively scavenging ROS including •OH, H2O2, and •OOH via POD, CAT and SOD activity (Figure 1a). PPBs alleviate the progression of mouse models of DSS-induced colitis without distinct side effect (Figure 1b) via scavenging ROS and inhibiting pro-inflammatory cytokine. Our study provides a proof of concept of PB nanozyme as a safe and potentially effective nanomedicine for colonic IBD.

Figure 1. (a) PPBs were generated through a hydrothermal method, by direct dissociation of a single-source precursor K3[Fe(CN)6] in the presence of PVP as a capping and reducing agent. The PPBs could act as an artificial nanoenzyme, effectively scavenging ROS including •OH, H2O2, and •OOH via peroxide, catalase and SOD activity. (b) Prussian blue (PB) can be oxidized into Berlin Green (BG), and even Prussian yellow (PY) via scavenging ROS. In addition, PY can be reduced into BG, and even PB by H2O2. These characteristics endow PPBs with good ROS scavenging capability. The PPBs were remarkably efficient nanozyme to reverse the effects of DSS-induced colitis in mice, which provides a potential alternative strategy for treatment of IBD.



2. EXPERIMENTAL SECTION

2.1 Materials. Polyvinylpyrrolidone (PVP, K30), hydrochloric acid (HCl, 36.0% ~ 38.0%), and potassium ferricyanide (K3[Fe(CN)6]) were purchased from Adamas-beta®.

2.2 Synthesis of PPBs. Typically, under magnetic stirring, 8.0 g PVP and 696 mg K3[Fe(CN)6] were added into HCl solution (1 M, 40 mL) for 60 min.Then, the mixed solution was placed in an electric oven (100 °C, 24 h). Finally, Prussian blue nanoparticles modified with PVP (PPBs) were obtained after centrifugation.

2.3 Measurement of peroxidase-like activity. The peroxidase (POD)-like activity assays of PPBs were carried out at room temperature using 3,5,3’,5’-tetramethylbenzidine (TMB) as substrates in the presence of H2O2 in 0.2 M HAc-NaAc buffer solution. The absorbance of the color reaction (at 650 nm for TMB) was recorded at a certain reaction time via a Microplate Reader (Model 680, BIO-RAD, USA) to express the POD-like activity. Typically, reaction systems containing 6 µg/mL PPBs solution, 30% H2O2, 10 mg/mL TMB/ABTS and buffer solutions with the volume ratio of 1:3:1:20 were used to show the chromogenic reactions implying POD-like activity.

2.4 Measurement of the effect of PPBs on H2O2. 1.2 mL 30% H2O2 solution (1.2 M) was added to PBS buffer solution (pH 7.4) and then 0.2 mL 120 µg/mL PPBs solution was added to the above mixture, and the generated oxygen is measured by using a specific oxygen electrode on Multi-Parameter Analyzer (DZS-708, Cany, China).

2.5 Measurement of the effect of PPBs on hydroxyl radicals. Different concentrations of PPBs were added to 0.1 mg/mL TiO2 (P25, Degussa Huls Corporation, Germany) and 50 mM 5-tert-butoxycarbonyl 5-methyl-1-pyrroline-N-oxide (BMPO), placed in quartz capillary tubes. After 5 min exposure to UV light at 340 nm, Bruker EMX spectrometer is used to obtain the ESR spetra.

2.6 Measurement of the effect of PPBs on superoxide anions. Xanthine/xanthine oxidase system was used to generate superoxide anions, and BMPO was used as a superoxide anions 6 ACS Paragon Plus Environment

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trapping agent. Before ESR tests, various concentrations of PPBs were added into the Xanthine/xanthine oxidase system.

2.7 Animals. The animal experiments were performed in compliance with guidelines set by the Animal Care Committee of Changhai hospital for IBD Research. Balb/c mice (clean class, 8 weeks old) were provided by Shanghai Laboratory Animal Center (SLAC, CAS).

2.8 The in vivo biosafety of PPBs in mouse model. The healthy mice were divided into three groups randomly. The mice in the one group were used as the control group. PPBs (40 mg/kg) were injected via intravenous administration into healthy mice of the other two groups. After PPBs injection of different time (7 days and 30 days), the mice were respectively sacrificed. The blood of mouse was collected for serum biochemistry tests and blood routine index tests. The main organs were taken out for the histological analysis. 2.9 The effect of PPBs on DSS-induced colitis mouse model. Mice were randomly divided into three groups including normal control group, DSS-induced colitis group, DSS-induced colitis treated with PPBs group. Mice with colitis were induced by DSS (3 wt%, MW ： 36000-50000; MP Biomedicals, USA) added in the drinking water for 7 days. The PPBs (10 mg/Kg) were intravenously injected on day 1, 3, 5, 7. On day 9, all mice were sacrificed under isoflurane anesthesia. During 9 days, visible stool consistency, change in body weight, and fecal bleeding were evaluated daily. The disease activity index (DAI) including the stool consistency index (0–3), weight loss index (0–4), and fecal bleeding index (0–3) were measured (Table S1). On day 9, mice were sacrificed under isoflurane anesthesia, and the entire colon was excised. The length of colon was tested and washed with saline solution mildly. Part of the colon was prepared for histologic analyses. MPO activity was measured by using a Myeloperoxidase assay kit (Nanjing Jiancheng Bioengineering Institute, China ). ROS levels were performed using ROS/RNS Assay Kit (Green Fluorescence; cellbiolab, San Diego, CA).



The concentrations of proinflammatory cytokines including IL-6, IFN-γ, IL-1β, and TNF-α were measured using Mouse ELISA Kit (Anogen-Yesbiotech, Canada).

2.9 Statistical analysis. All data were presented as mean ± SEM unless otherwise indicated. For a single comparison, the significance of differences between means was measured by the Student’s t-test. All analyses were carried out using SPSS 19.0 software (p < 0.05). 3. RESULTS AND DISSCUSSION 3.1 Characteristic of PPBs PPBs were prepared through a hydrothermal method, by direct dissociation of a singlesource precursor K3[Fe(CN)6] in the presence of PVP. On the one hand, PVP acts as a reducing agent to react with K3[Fe(CN)6] for achieving PPBs. On the other hand, PVP serves as a capping agent to address the insolubility of PPBs. Transmission electron microscopy (TEM) images (Figure 2a) demonstrate the formation of uniform PPBs with cube morphology. The prepared PPBs have an average size of 60 nm and good monodispersity (the inset of Figure 2a). The distinct crystal lattice interspersing the whole nanoparticle can be observed in the high-resolution TEM image (Figure 2b). High resolution TEM images (Figure 2b) and electron diffraction of selected areas (Figure 2c) display the lattice fringe spacing of 0.454, 0.333, and 0.253 nm, corresponding to the (220), (200), and (400) lattice planes, respectively. The prepared PPBs have a crystalline structure with strong typical diffraction spots (Figure 2c). Element mapping confirms the composition of PPBs with C, N, Fe, and K element (Figure 2d). In addition, the X-ray powder diffraction pattern of PPBs reveals the PB crystal structure (JCPDS no. 73-0687, Figure 2e). Fourier transform infrared spectroscopy shows the absorbance peaks at 2090 cm-1 and 1670 cm-1 (Figure 2f), which indicates -CN- stretching in the Fe-CN-Fe bond of PPBs, and C=O stretching in the PVP amide unit, respectively. The UV-vis-NIR absorbance spectrum of PPBs discovers an obvious characteristic PB peak at approximately 706 nm (Figure 2g), which can be attributed to the intermetallic charge-transfer band from Fe(II) to Fe(III) in PB. A plausible mechanism for the 8 ACS Paragon Plus Environment

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formation of the PPBs may be as follows: first, [Fe(CN)6]3- ions are changed into [Fe(CN)6]4ions with the present of a reductant under acidic condition. In the meantime, [Fe(CN)6]3- ions release Fe3+ ions through dissociation ionization. The reduced [Fe(CN)6]4- ions react with the released Fe3+ ions to form small PB crystals with a PVP shell. Afterwards, the prepared PPBs could be formed via the self-assembling of small PB crystals (Figure 1a).

Figure 2. (a) TEM image of PPBs, the inset: size distribution of PPBs measured from TEM image. (b) Highresolution TEM image of PPBs. (c) An electron diffraction image of PPBs. (d) Element mapping of PPBs. Blue: K, pink: Fe, green: N, yellow: C. (e) The XRD pattern of PPBs. (f) The FTIR spectrum of PPBs. (g) The UV-visNIR absorbance curve of PPBs.

3.2 The effect of PPBs on ROS A hydroxyl radical-generating TiO2/UV system was used to investigate the ROS scavenging ability of PPBs. The TiO2/UV system exhibits the characteristic signal peak of BMPO/•OH without PPBs (Figure 3a). As the concentration of PPBs increases from 0 to 10 µg/mL, the signal intensity of BMPO/•OH displays a steep decline. When the PPB 9 ACS Paragon Plus Environment


concentration further increases to 50 µg/mL, the signal intensity of BMPO/•OH remains only 1/16 that of the TiO2/UV system alone, indicating the excellent scavenging capability of PPBs against •OH (Figure 3a). The redox potential of Prussian blue (PB)/Berlin Green (BG), BG/Prussian yellow (PY), and •OH/H2O are about 0.9, 1.4 V, and 2.9 V, respectively. Therefore, the effect of PPBs on •OH can be represented in Reaction 3.

(Ⅲ) [(Ⅱ)( ) ] (PB) (Ⅲ) [(Ⅲ)( ) ] [(Ⅱ)( ) ] (Ⅲ) [(Ⅲ)( ) ]

(BG)

(PY)

→ +

(1)

→ + ( − )

(2)

+ + ∙→ +

(3)

The addition of H2O2 into the PPBs solution generated a number of observable bubbles (the inset of Figure 3a), which were verified to be O2.26 PPBs therefore can act as a catalase, catalyzing the decomposition of H2O2 to produce oxygen. The CAT-like activity of PPBs can be shown in the following reaction:

+ → +

(4)

+ → +

(5)

+ + → + +

(6)

+ + → + +

(7)

Furthermore, we assayed the commonly used natural POD substrate 3,5,3,5tetramethylbenzidine (TMB) to investigate the POD activity of PPBs. The absorbance of TMB at 650 nm was recorded with a Microplate Reader. The absorbance increases after the addition of PPBs, indicative of POD-like activity (Figure 3b, Reaction 8). !"

+ + #$ (%&'& (') +


(8)

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We used the xanthine/xanthine oxidase system to explore the effect of PPBs on superoxide radicals (•OOH). With the concentration of PPBs increases, the BMPO/•OOH signal intensity decreases dramatically (Figure 3c), demonstrating that PPBs can act as a SOD-like nanoenzyme to scavenge •OOH. *+,/!.

• #////$ +

(9)

Taken together, the results suggest that the prepared PPBs can act as artificial nanoenzymes to efficiently change the detrimental ROS into H2O and O2, avoiding lipid peroxidation, protein oxidation and DNA damage (Figure 3d). The hydrodynamic diameters and UV-visNIR spectra of PPBs showed no significant change, indicating the good stabilities of PPBs after reaction with ROS (Figure S1). Therefore, PPBs possess great potential in targeting ROS for treatment of IBD.

Figure 3. (a) The effect of PPBs on ·OH generated by a TiO2/UV system. The signal intensities of the second line in the BMPO/·OH spectrum are disclosed. Insets: images of PPBs solution and PPBs solution with H2O2 for 5 min. (b) The POD-like activity of PPBs. (c) The effect of PPBs on ·OOH produced by the xanthine/xanthine oxidase system. The signal intensities of BMPO/·OH are shown. (d) The effect of PPBs on ROS and the potential application in nanomodulating inflammation of PPBs.



3.3 The physiological stability of PPBs In clinical practice, intravenous administration is used to treat those patients suffering from moderate-to-severe IBD. The prepared PPBs must have good physiological stability. We investigated the colloid stability of PPBs in various environments by dynamic light scattering (DLS) and UV-vis-NIR spectroscopy. The diameters and absorbance value at 706 nm of PPBs in the saline solution have no significant changes for even 7 days at 4, 25 and 60 °C, respectively (Figure 4a,b). The good stability of the prepared PPBs under different temperatures indicated that they could be easily stored without any special environment. Then,

Figure 4. (a) Hydrodynamic diameter and (b) absorbance at 706 nm of PPBs in normal saline solution at different time point and various temperature. (c) Hydrodynamic diameter and (d) absorbance at 706 nm of PPBs in different solutions at various time point. (e) Hydrodynamic diameter and (f) absorbance at 706 nm of PPBs in solution with different pH values at various time point. TEM image of PPBs (g) before and (h) after incubated in normal saline solution of 60 °C for 7 days.


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the DLS distribution curves (Figure 4c) and UV-vis-NIR spectra (Figure 4d) of PPBs with the same concentration in various solutions including water, normal saline, normal saline with the pH value of 5.0, and cell culture medium (Dulbecco’s modified Eagle’s medium (DMEM) supplemented with fetal bovine serum) were almost identical at different time points. We compared TEM image of PPBs before and after storing at 60 °C for 7 days. In addition, the prepared PPBs could remain stable in various pH solutions (Figure 4e,f). The morphology of PPBs remained no change, which demonstrated PPBs own excellent physiological stability (Figure 4g,h). Taken together, these results fully indicated the good colloidal stability of PPBs in physiological circumstances, demonstrating PPBs could be suitable for intravenous administration. In the other reported studies of constructing PB nanoparticles for biomedical application via intravenous administration, complex post-modification or costly polyethylene glycol was needed to address the insoluble of PB.22, 27 Herein, PPBs were constructed via a one-pot strategy without complex process and costly raw materials to address the insolubility of PB. 3.4 The therapeutic effect of PPBs on DSS-induced mice with colitis Encouraged by the excellent nanoenzyme-like performance of the PPBs, the therapeutic efficacy in the DSS-induced colitis mice model was investigated. Due to the well-established enhanced permeability and retention effect and the uptake by the infiltrated inflammatory cells, nanoparticles can accumulate at the site of inflammation from the blood through the endothelial pathway.28 Subsequently, the study of whether the PPBs could accumulate in the inflamed colon was conducted. Mice were treated with a 3% DSS solution instead of drinking water for 5 days before intravenous administration with PPBs. Few PPBs were observed in the colons of normal mice, while PPBs were located distinctly in the colons of mice with colitis, indicating that PPBs preferentially localized to the site of inflammation (Figure 5a,b). In addition, approximate 3.3 percentage of the injected PPBs can accumulate in the inflamed colon according to inductively coupled plasma optical emission spectrometer (ICP-OES). The preferential accumulation at the site of inflammation colons may be ascribed to the wellestablished enhanced permeability and retention effect and the uptake by the infiltrated inflammatory cells. We supplied the mice 13 ACS Paragon Plus Environment


Figure 5. (a) Through intravenous administration, PPBs target the inflamed colon of DSS-induced colitis mouse model from the endothelium via mechanism mediated by size. (b) Eosin-stained colon sections from mice of the control and DSS-induced colitis group. The PPBs accumulated at the site of inflammatory colons (the blue dots in the images). (c) The overall experimental procedure. Mice were provided with normal water or water containing 3% DSS for 7 days. On days 1, 3, 5, and 7, mice were treated with PPBs or normal saline (NS). (d) The changes of body weight of mice in various groups. (e) Colon lengths and (f) the corresponding images of the colons in each group. (g) Hematoxylin and Eosin (H&E) staining colon sections from each group on day 9.


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with a 3wt% solution of DSS instead of drinking water for 7 days to obtain the DSS-induced colitis mice model (Figure 5c). Normal saline and PPBs were injected intravenously into mice in the colitis and treatment groups, respectively, and the therapeutic efficacy was evaluated on day 9. The disease activity index (DAI), body weight, colon length, and histological analysis are important parameters for the assessment of therapeutic efficacy in the colitis mice model. Mice in the colitis group exhibit a significant increase in the DAI compared to the control group, while mice in the treatment group have lower DAI compared to the colitis group (Figure 5d). Mice in the colitis group display significantly shorter colons than that of the control mice and reveal the signs of bleeding, indicative of severe inflammation (Figure 5e,f). Interestingly, mice treated by intravenous injection of PPBs possess little evidences of colon shortening, bleeding, or abnormal stools (Figure 5f). In the histological analysis, mice with colitis show severe destruction of mucosal areas, massive infiltration of immune cells, and severe colonic epithelial damage (Figure 5g). Conversely, the mucosal areas of the colons display only minor damage, and the immune cells in the mucosa, muscle or submucosa reveal little infiltration (Figure 5g), demonstrating the significant therapeutic effect of PPBs on mice with DSS-induced colitis. Furthermore, we measured proinflammatory mediators in the colonic mucosa including myeloperoxidase (MPO), ROS, TNF-α, IL-6, and IL-1β. These proinflammatory mediators play important roles in UC. Compared with the control group, MPO activity highly raise in DSS-induced colitis mice model, but significantly reduced in the PPB treatment group (Figure 6a). Owning to the excellent scavenging properties of PPBs, ROS levels in the treatment group are dramatically decreased compared with the colitis group (Figure 6b), and depict no significant differences from the control levels. Levels of the proinflammatory cytokines (TNF-α, IL-6, and IL-1β) are much higher in the colitis group than those in the control group, indicative of severity of the disease (Figure 6c-e). Fortunately, with the aid of PPBs, these up-regulated cytokines are considerably reduced (Figure 6c-e). Since NF-kB is a major inducer of inflammatory cytokines including IL-6 and TNF-α, the prepared PPBs is considered to be as a potent NF-kB inhibitor, suppressing NF-kB activation path way. Taken together, these results clearly illustrate that PPBs have outstanding inflammation nanomodulation properties, and could be used in the efficient treatment of IBD. 15 ACS Paragon Plus Environment


Figure 6. (a) The myeloperoxidase (MPO) activity of colonic tissues from mice in each group. (b) ROS, (c) IL-6, (d) IL-1β, and (e) TNF-α levels in colon homogenates from the control group, DSS-induced colitis group, and DSS-induced colitis treated with PPBs group.

3.4 The potential toxicity in vivo of PPBs To investigate the potential toxicity of PPBs via intravenous administration, blood routine tests and biochemistry indexes were measured before and after various time. The total platelet, hemoglobin, white blood cell and red blood cell counts are unaffected 7 d and 30 d after PPBs injection (Figure 7a-d). Alanine transaminase (ALT) and aspartate transaminase (AST), markers of hepatocellular injury, demonstrate no significant changes (Figure 7e,f). IL-6 production levels and TNF-α in the serum remain unchange (Figure 7g,h). Furthermore, histopathology assays of the main organs including the spleen, heart, liver, kidney, and lung demonstrate no observable impairments induced by PPBs (Figure 7i,j). Taken together, the results suggest that the prepared PPBs possess a considerably good safety profile in vivo and do not induce an immune response, enabling them suitable for clinical application via intravenous administration. 16 ACS Paragon Plus Environment

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Figure 7. Hematologic analyses exposure to PPBs after 7 days and 30 days. (a) total white blood cell count (b) total hemoglobin. (c) total red blood cell count. (d) total platelet count.. (e) Alanine transaminase (ALT) and (f) aspartate transaminase (AST) levels in the livers of the three groups. (g) IL-6 and (h) TNF-α production in the serum of the three groups. (i) Histopathological images and (j) TUNEL assay of mouse tissue after intravenous administration exposure to PPBs.



4. Conclusion In summary, we have generated PPBs with good physiological stability and excellent biosafety via a simple and low-cost strategy. The prepared PPBs acted as an efficient ROS scavenger, changing harmful ROS including •OH, H2O2, and •OOH into H2O and O2 to alleviate oxidative stress. PPBs with the average size of approximate 60 nm, show preferential accumulation at the sites of inflamed colonic mucosa, due to the well-established enhanced permeability and retention effect. Thanks to the capabilities of scavenging ROS and inhibiting proinflammatory cytokine, PPBs significantly accumulated at the inflammed site and reduce colitis in mice without adverse side effect. These PPBs may enable the development of even more chemically potent and biologically compatible materials. This study provides a significant demonstration of the protective effects of PPBs against IBD in living animals, offering hope and a potential alternative treatment option for patients with IBD.

ASSOCIATED CONTENT Supporting Information The table of DAI, the stabilities of PPBs before and after ROS scavenging,

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] *E-mail: [email protected] *E-mail: [email protected] Notes The authors declare no competing financial interests. Acknowledgements This work was supported by the National Natural Science Foundation of China (Grant No.81470800, No.51772316, No.81670485, No.81370493), the National Key Research and Development Program of China (Grant No. 2017YFB0702602), and the Key Projects of International Cooperation and Exchanges of NSFC (No.81720108023). 18 ACS Paragon Plus Environment

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Prussian Blue Nanozyme with Multienzyme Activity Reduces Colitis in

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