Tungsten-doped Prussian blue nano composite material, preparation method and application

Abstract

The invention belongs to the technical field of preparation of nano composite materials, and particularly relates to a tungsten-doped Prussian blue nano composite material, a preparation method and application. The tungsten-doped Prussian blue nano composite material prepared by a one-pot hydrothermal method by taking polyvinylpyrrolidone as a reducing agent and a stabilizer and tungsten hexachloride, citric acid and potassium ferricyanide as reactants has excellent ROS (reactive oxygen species) removal capability and can protect intestinal epithelial cells and treat IBD (infectious bursal disease), and hydrogel is further taken as a carrier to prepare a colon-targeted delivery material. The W-PB is protected to be accurately delivered to the colon, the hydrogel is disintegrated to release the W-PB under the subsequent alkaline environment, and then W ions are further released, so that responsive release of the colon is achieved, and systemic toxicity of the W ions is avoided.

Description

Technical Field

[0001] The invention relates to the technical field of nanocomposite material preparation, and in particular to a tungsten-doped Prussian blue nanocomposite material, a preparation method and application thereof. Background Art

[0002] Inflammatory bowel disease (IBD) is a nonspecific chronic inflammatory disease that affects certain parts of the gastrointestinal tract and often causes extraintestinal complications. Currently, there is still a lack of effective treatment.

[0003] IBD is closely related to the dysbiosis of intestinal microbiota, which is characterized by an increase in facultative anaerobic bacteria of the Enterobacteriaceae family and a decrease in beneficial bacteria. Traditional medical treatment of IBD is based on broad-spectrum antibiotics to prevent the expansion of pathogenic bacteria, however, the widespread use of antibiotics can also destroy probiotics and increase the risk of antibiotic resistance, such as the inflammation caused by Escherichia coli K-12.

[0004] IBD and the rise of ROS (reactive oxygen species) are also closely related. ROS upregulation can be observed in the colitis tissue of patients with mouse colitis models, ultimately leading to intestinal mucosal damage. First, proinflammatory cytokines can activate immune cells and amplify inflammation and ROS generation when IBD occurs. At the same time, excessive production of ROS further activates the inflammatory / immune response through the nuclear factor κB (NF-κB) signaling pathway, leading to increased expression and secretion of proinflammatory cytokines. Not only that, high levels of ROS exposure can also cause oxidative damage to mitochondria, promote the release of mt DNA, thereby activating the STING pathway and inducing intestinal epithelial cell (IEC) apoptosis. Therefore, clearing ROS in the intestine is necessary to prevent the progression of IBD. However, several non-enzymatic and enzymatic antioxidants are currently ineffective, and many of them cause adverse immune responses. Some trials have shown that the therapeutic effects of natural antioxidants such as vitamin E, vitamin C, and coenzyme Q are not satisfactory. Furthermore, the ROS scavenger N-acetylcysteine ​​enhances the generation of helper T-17 cells in vivo in a Misshapen / Nik-related kinase-dependent manner and increases the risk of promoting autoimmune inflammatory diseases.

[0005] Rationally designed nanozymes can restore redox balance and effectively regulate intestinal flora, especially Prussian blue nanozymes (BP), which belong to TMSN and have attracted great interest in the treatment of ROS-mediated diseases due to their superior multi-antioxidant enzyme-like activities, excellent biocompatibility, easy preparation and modification, and good biocompatibility. However, Prussian blue nanozymes have no effect on nitrate reductase activity, and thus have no obvious antibacterial effect in some cases (such as against Escherichia coli K-12). Summary of the invention

[0006] The purpose of the present invention is to provide a tungsten-doped Prussian blue nanocomposite material, a preparation method and an application thereof, so as to solve the problems existing in the above-mentioned prior art, accurately edit the intestinal flora and remove ROS, protect intestinal epithelial cells, and treat IBD.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] One of the technical solutions of the present invention is to provide a method for preparing a tungsten-doped Prussian blue nanocomposite material, the steps comprising:

[0009] The tungsten-doped Prussian blue nanocomposite material (W-PB) was prepared by a one-pot hydrothermal method using polyvinylpyrrolidone (PVP) as a reducing agent and a stabilizer, and tungsten hexachloride (WCl6), citric acid and potassium ferrocyanide (K3Fe(CN)6) as reactants.

[0010] Furthermore, the mass ratio of polyvinyl pyrrolidone, tungsten hexachloride, citric acid and potassium ferrocyanide is 4:0.84:1.32:0.66.

[0011] Furthermore, the step of preparing the tungsten-doped Prussian blue nanocomposite material by the one-pot hydrothermal method comprises:

[0012] Potassium ferrocyanide and polyvinyl pyrrolidone are dissolved in water to prepare solution A;

[0013] Dissolve tungsten hexachloride and citric acid in water to prepare solution B;

[0014] The solution A and the solution B are mixed, reacted at 80° C. for 24 hours, and the reaction product is separated by centrifugation. The tungsten-doped Prussian blue nanocomposite material is obtained after washing and drying.

[0015] Preferably, the centrifugal speed is 15000 rpm and the time is 10 min.

[0016] Preferably, the washing is ultrasonic washing and resuspension, which is repeated at least once.

[0017] Preferably, the drying is vacuum freeze drying.

[0018] The second technical solution of the present invention is to provide a tungsten-doped Prussian blue nanocomposite material prepared by the above preparation method.

[0019] The third technical solution of the present invention is to provide an application of the above-mentioned tungsten-doped Prussian blue nanocomposite material in the preparation of drugs for treating inflammatory bowel disease.

[0020] The fourth technical solution of the present invention is to provide a colon-targeted delivery material, which uses the above-mentioned tungsten-doped Prussian blue nanocomposite material as an active ingredient.

[0021] Furthermore, the colon targeted delivery material uses hydrogel as a carrier of the active ingredient; the hydrogel is formed by physically cross-linking sodium alginate and chitosan.

[0022] A fifth technical solution of the present invention is to provide a method for preparing the above-mentioned colon targeted delivery material, comprising the following steps:

[0023] Dissolving sodium alginate in water to obtain a first mixed solution;

[0024] dispersing the tungsten-doped Prussian blue nanocomposite material in water to obtain a second mixed solution;

[0025] adding acetic acid and chitosan to the second mixed solution to obtain a third mixed solution;

[0026] The first mixed solution and the third mixed solution are mixed to obtain a reaction system, and the reaction system is stirred overnight to obtain the colon targeted delivery material.

[0027] Furthermore, the mass concentration of sodium alginate in the first mixed solution is 20 g / L.

[0028] Furthermore, the mass concentration of the tungsten-doped Prussian blue nanocomposite material in the second mixed solution is 1 g / L.

[0029] Furthermore, the dosage ratio of sodium alginate, chitosan, acetic acid and tungsten-doped Prussian blue nanocomposite material in the reaction system is 40 mg:54 mg:4 μL:3 mg.

[0030] The present invention discloses the following technical effects:

[0031] The present invention dopes tungsten into the Prussian blue nanomaterial to enhance its influence on the activity of nitrate reductase, thereby enhancing its inhibitory effect on inflammatory bowel disease pathogenic bacteria.

[0032] The tungsten-doped Prussian blue nanocomposite material prepared by the present invention is used for targeted delivery of tungsten ions, precise editing of intestinal flora and removal of ROS, protection of intestinal epithelial cells, and treatment of IBD.

[0033] The present invention uses hydrogel as a carrier to load the colon-targeted delivery material prepared by tungsten-doped Prussian blue nanocomposite material, thereby protecting W-PB and accurately delivering it to the colon. The subsequent alkaline environment causes the hydrogel to disintegrate and release W-PB, which further releases W ions, achieving responsive release in the colon and avoiding the systemic toxicity of tungsten ions. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0035] Figure 1 The morphology and phase characterization results of W-PB prepared in Example 1 are shown in FIG. a is a schematic diagram of W-PB synthesis, b is a TEM image, c is an EDX spectrum, d is a particle size distribution, e is the Zeta potential of W-PB in PBS, f to g are XRD and SC-XRD, h to i are the ultraviolet spectra and infrared spectra of W-PB and PB, j to l are the XPS spectrum, Fe 2p electronic energy level and W 4f electronic energy level of W-PB, and m to o are the XPS spectrum, Fe 2p electronic energy level and W 4f electronic energy level after scavenging ROS of W-PB, respectively;

[0036] Figure 2 Schematic diagram of the process flow for preparing colon targeted delivery materials;

[0037] Figure 3 is the in vitro catalytic performance of W-PB, wherein A is the consumption of hydrogen peroxide indicated by titanium sulfate color development, B is the ESR spectrum of W-PB, C is the ESR spectrum of W-PB, D is the UV spectrum of DPPH free radical scavenging, E is the ability of W-PB and PB to decompose H2O2 to produce O2 in PBS, F is the scavenging rate of W-PB and PB for ·O2 free radicals, G is the scavenging rate of W-PB and PB for ·OH free radicals, and H is the scavenging rate of W-PB and PB for DPPH free radicals;

[0038] Figure 4 The protective effect of W-PB on NCM460 cells and its antibacterial efficacy, wherein a is an inverted fluorescence microscope image of NCM460 cells stained with Calcein-AM / PI (2mM H2O2), b is the protection of NCM460 cells by W-PB and PB from oxidative stress caused by H2O2, c-d are the growth of E.coli K-12 in the presence of different electron acceptors under anaerobic conditions, e is the determination of nitrate reductase activity of E.coli K-12 in a medium supplemented with sodium nitrate, f-g are the growth of E.coli DH-5α in the presence of different electron acceptors under anaerobic conditions, h is the determination of nitrate reductase activity of E.coli DH-5α in a medium supplemented with sodium nitrate, and i is the preparation of W-PB gel;

[0039] Figure 5The antioxidant and anti-inflammatory effects of W-PB at the cellular level, where a is a schematic diagram of the anti-inflammatory and antioxidant properties of W-PB in vitro, b is the cell viability after co-culture of RAW264.7 cells with different concentrations of W-PB and PB for 24 hours, c is the inverted fluorescence microscope image analysis of RAW264.7 cells stained with DCFH-DA, d is the quantitative analysis of ROS by ImageJ software, e is the ROS level in RAW264.7 cells detected by flow cytometry, f~g are the effects of W-PB and PB on macrophage phenotype detected by flow cytometry, h~j are the statistical analysis of STING and p-STING expressions in RAW264.7 cells by Western Blot and ImageJ software, k~m are the levels of TNF-α, IL-6 and IL-1β in RAW264.7 cells after different treatments;

[0040] Figure 6 Cell survival rate after co-culture of NCM460 cells with different concentrations of W-PB and PB for 24 hours;

[0041] Figure 7 is the ROS level in macrophages;

[0042] Figure 8 iNOS in macrophages + and CD206 + level, where a is iNOS + , b is CD206 + ;

[0043] Fig. 9 is the transmission electron microscopy (TEM) image of W-PB gel;

[0044] Fig.10 Rheological characterization of W-PB gel, where a and b are strain sweeps, and c is the time modulus curve;

[0045] Fig.11 Scanning electron micrographs of W-PB gel under different pH conditions, where SGF is the morphology of the gel in simulated gastric fluid (pH 1.2), SIF is the morphology of the gel in simulated intestinal fluid (pH 6.8), and SCF is the morphology of the gel in simulated colon fluid (pH 7.8);

[0046] Fig.12 The release of tungsten ions from W-PB gel in the simulated gastrointestinal tract;

[0047] Fig.13The therapeutic effect of W-PB gel on DSS-induced IBD model mice, wherein a is the experimental flow chart of W-PB gel treatment of DSS-induced IBD mice, b-c are images of colon tissues and quantification of their lengths after 13 days, d is the daily changes in body weight of mice in different groups, e-f are images of spleen tissues and their weights of mice in different groups on the 13th day, and g is H&E staining and AB / PAS staining;

[0048] Fig.14 W-PB gel restored the barrier function and inhibited intestinal inflammation, where a was the 13th day, the colon was collected and subjected to immunofluorescence analysis to detect the expression patterns of ZO-1 and occludin; b, upper figure: immunofluorescence analysis of M1 macrophage marker iNOS in colon tissue, iNOS (red) and DAPI (blue), lower figure: immunofluorescence analysis of M2 macrophage marker CD206 in colon tissue, CD206 (red) and DAPI (blue); c was the analysis of myeloperoxidase (MPO) and proinflammatory factors TNF-α, IL-6, IL-1β and IL-10 in mouse colon tissue by ELISA;

[0049] Fig.15 It is the quantitative analysis of ZO-1 and Occludin, where a is ZO-1 and b is Occludin;

[0050] Fig.16 Quantitative analysis of iNOS and CD206, where a represents iNOS and b represents CD206. DETAILED DESCRIPTION

[0051] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0052] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0053] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0054] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0055] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0056] The approval number for animal ethics certification is: IRM2-IACUC-2409-092.

[0057] The raw materials and reagents used in the specific implementation mode of the present invention are all commercially available products, among which mouse mononuclear macrophage leukemia cells (RAW264.7) are from the cell bank of the Chinese Academy of Sciences; human normal colon epithelial cells (NCM460) are from Fenghui Bio; Escherichia coli DH5-α is from Beina Bio; Escherichia coli K-12 is from Beina Bio; the sources of other raw materials and reagents are shown in Table 1.

[0058] Table 1

[0059]

[0060]

[0061] Example 1

[0062] The preparation steps of tungsten-doped Prussian blue nanocomposite are as follows:

[0063] S1, dissolving 0.66 g of potassium ferrocyanide powder and 4 g of PVP in 100 mL of deionized water, stirring for 30 minutes, to prepare solution A;

[0064] S2, 0.84 g of WCl6 and 1.32 g of citric acid were dissolved in 100 mL of deionized water, and stirred for 30 minutes to prepare solution B;

[0065] S3. Add solution B to solution A, react at 80°C for 24 hours, then centrifuge at 15,000 rpm for 10 minutes to separate the solid product, ultrasonically wash the solid product with water and resuspend it, repeat three times, and then use a vacuum freeze dryer to treat the solid product to obtain a tungsten-doped Prussian blue nanocomposite material, recorded as W-PB.

[0066] Example 2

[0067] The preparation steps of colon targeted delivery material are as follows:

[0068] S1. Take 0.4 g of sodium alginate (SA), add it to 20 mL of water, and stir at 45° C. for 3 hours to prepare a sodium alginate solution;

[0069] S2, dissolve 30 mg of W-PB in 30 mL of water, add 40 μL of acetic acid and 0.45 g of chitosan (CS) in sequence, and stir magnetically until completely dissolved. Then add the sodium alginate solution prepared in S1, stir overnight, and obtain the colon targeted delivery material, which is recorded as W-PB gel.

[0070] Figure 2 Schematic diagram of the process flow for preparing colon targeted delivery materials.

[0071] Comparative Example 1

[0072] The preparation method of Prussian blue nanomaterial gel (PB gel) is as follows:

[0073] Compared with Example 2, the only difference is that W-PB is replaced with an equal amount of PB.

[0074] Comparative Example 2

[0075] The preparation method of a single hydrogel comprises the following steps:

[0076] Compared with Example 2, the difference is that W-PB is not added.

[0077] Test Example 1

[0078] The tungsten-doped Prussian blue nanocomposite material prepared in Example 1 was characterized in morphology and phase. The morphology and particle size of the prepared W-PB were observed using a transmission electron microscope (TEM); the lattice on the surface of the W-PB was observed using a high resolution transmission electron microscope (HRTEM); the wide-angle diffraction test of the W-PB freeze-dried powder was performed using X-ray diffraction (XRD); the valence band spectrum of the W-PB powder was determined using X-ray photoelectron spectroscopy (XPS); and the W-PB / barium sulfate pressed tablet was scanned using an ultraviolet-visible-near-infrared spectrophotometer to obtain a diffuse reflectance spectrum.

[0079] Figure 1 The morphology and phase characterization results of W-PB prepared in Example 1, wherein a is a schematic diagram of W-PB synthesis, b is a TEM image, c is an EDX spectrum, d is the particle size distribution, e is the Zeta potential of W-PB in PBS, f~g are XRD and SC-XRD, h~i are the UV spectra and infrared spectra of W-PB and PB, j~l are the XPS spectrum, Fe 2p electronic energy level and W 4f electronic energy level of W-PB, m~o are the XPS spectrum, Fe 2p electronic energy level and W 4f electronic energy level after ROS removal of W-PB. It can be seen from the figure that the hydrochloric acid produced by the hydrolysis of tungsten chloride creates an acid environment, making PVP reducible, and the iron ions in potassium ferrocyanide are reduced, and Fe 3+ / Fe 2+ and CN - Coordination to form W-PB nanoparticles ( Figure 1 As shown in a). As shown in b and c, W-PB is cubic and well dispersed, and C, N, Fe and W are evenly distributed. The W element content was 7.28% ± 0.65 by ICP-OES quantitative determination, thus formally successfully preparing W-PB. Due to the stabilizing effect of PVP, W-PB has relatively excellent monodispersity in aqueous solution, with an average particle size of 197.84nm ( Figure 1 According to the Zeta potential measurement, W-PB has a highly negative charge (-21.05 mV) ( Figure 1(e). The XRD pattern in g shows that the synthesized PB NPs have obvious diffraction peaks at 17.49°, 24.84°, 35.42° and 39.76°, corresponding to the (200), (220), (400) and (420) crystal planes of the standard structure (PDF#97-007-7340). No other characteristic peaks assigned to W or tungsten oxide were observed in f. In addition, a single crystal XRD diffraction experiment was performed, showing the structure of the Debye diffraction ring ( Figure 1 The W-PB concentration was determined by linear fitting using the relationship between the absorbance at the 710 nm absorption peak and the W-PB concentration. Figure 1 The chemical bond characteristics were analyzed by infrared spectra of W-PB and PB, which showed that the stretching characteristic C=N was located at 2090 cm -1 ( Figure 1 XPS shows that Fe, W, C, N and O elements exist simultaneously in W-PB ( Figure 1 The high-resolution spectrum of Fe 2p shows four peaks at 708.6 eV (Fe 2p 1 / 2 ) and 721.3 eV (Fe 2p 3 / 2 ) belongs to Fe 2+ , at 709.7 eV (Fe 2p 3 / 2 ) and 723.1eV(Fe2p 1 / 2 ) belongs to Fe 3+ ( Figure 1 (k in Fig. 2+ and Fe 3+ The ratio of about 2.38 is also very beneficial to the ROS removal mediated by W-PB. The high-resolution spectrum of W4f shows four peaks. The peaks at 38.1eV and 36.1eV belong to W 6+ 4f 7 / 2 , at 36.5 eV (W 4f 5 / 2 ) and 34.1eV(W 4f 7 / 2 ) belongs to W 4+ ( Figure 1 W 4+ With W 6+ The ratio is about 0.15. 6+ / W 4+ and Fe 3+ / Fe 2+ The REDOX coupling of W-PB may provide abundant redox reaction sites for reactive oxygen species and improve their ability to scavenge ROS. 2+ with Fe 3+and W 4+ With W 6+ The ratio of has changed, indicating that transition metals can transfer electrons through changes in valence state and produce catalysis ( Figure 1 (shown as m~o in the figure).

[0080] Test Example 2

[0081] In vitro catalytic performance study of W-PB: catalytic H2O2, ·O2, ·OH and DPPH free radical scavenging ability test, the results are as follows Figure 3 shown.

[0082] The inhibition percentage was detected using a superoxide dismutase (SOD) activity detection kit, a hydroxyl radical scavenging ability detection kit, and a DPPH free radical scavenging ability detection kit according to the instructions. The inhibition percentage was calculated according to formula 1-1.

[0083]

[0084] For the removal of hydrogen peroxide: prepare 9 mL of 3% hydrogen peroxide PBS solution, add W-PB and PB respectively, so that the final concentration of W-PB and PB is 100 μg / mL, and use a dissolved oxygen meter to detect the oxygen concentration in the solution.

[0085] Detection of remaining hydrogen peroxide: Using a hydrogen peroxide (H2O2) content detection kit, samples were taken from the above solution at different time points (0, 10 min, 20 min, 30 min, 40 min, 50 min) for detection.

[0086] ESR hydroxyl radical: Prepare a 5mg / mL FeSO4 solution, take 200μL of the solution, add 20μL DMPO and 160μL deionized water, then add 20μL 30% hydrogen peroxide, mix well and react for 5 minutes, take samples for testing, and use them as control group data. For other sample test groups, replace 160μL deionized water with 160μL sample solution.

[0087] ESR superoxide radicals: Use PBS buffer as solvent, prepare 10mM xanthine solution and 1U / mL xanthine oxidase solution, take 100μL xanthine solution and 100μL xanthine oxidase solution, add 20μL DMPO and 180μL buffer, incubate for 10min and take samples for testing. For other sample test groups, replace 180μL buffer with 180μL sample solution.

[0088] Figure 3The in vitro catalytic performance of W-PB, where A is the consumption of hydrogen peroxide indicated by titanium sulfate color development, B is the ESR spectrum of W-PB, C is the ESR spectrum of W-PB, D is the UV spectrum of DPPH free radical scavenging, E is the ability of W-PB and PB to decompose H2O2 to produce O2 in PBS, F is the scavenging rate of W-PB and PB for ·O2 free radicals, G is the scavenging rate of W-PB and PB for ·OH free radicals, and H is the scavenging rate of W-PB and PB for DPPH free radicals. It can be found from the figure that titanium sulfate color development and UV quantification are used to detect the consumption of hydrogen peroxide in A. The results show that the ability of W-PB to consume hydrogen peroxide is about twice that of PB. This is because the tungsten atom itself has an empty d orbital, which is easy to form a transition state with a low energy barrier, exposing more catalytic sites, and thus improving its catalytic activity. In this process, it was found that a large number of bubbles were produced in the solution, and the product was oxygen. To verify that the product was oxygen, W-PB was dispersed in PBS, H2O2 was added and the oxygen content of the solution was recorded using a dissolved oxygen analyzer. Figure 3 As shown in E, when the concentrations of W-PB and PB were both 10 μg / mL, the oxygen production rate of W-PB was significantly higher than that of PB. At 19 minutes, the oxygen production concentration of W-PB was 28.1 mg / L, while that of PB was 10.52 mg / L. Prussian blue doped with W element showed strong oxygen production capacity. In B, in the presence of DMPO as a free radical scavenger, hydroxyl radicals were scavenged; in C, in the presence of DMPO as a free radical scavenger, superoxide anion radicals were eliminated. ESR was further used to analyze whether W-PB could also catalyze the decomposition of other types of ROS. DMPO was used to capture ·O2 and ·OH, and their intensities were measured at 5 minutes and 10 minutes. The results are shown in the figure. Figure 3 As shown in B and C in Figure 5, at 5 min, the signal intensity of the W-PB group was significantly lower than that of the blank group, indicating that W-PB has the ability to catalyze the decomposition of ·O2 and ·OH. Next, the hydroxyl radical scavenging ability detection kit and the superoxide dismutase (SOD) activity detection kit were used to quantify its catalytic activity. The results are shown in Figure 3 As shown in F and G, when the concentration of W-PB and PB was 80 μg / mL, the average inhibition percentages of the two on hydroxyl radicals were 36.16 and 44.65, and the inhibition percentages on superoxide anions were 40.00 and 47.57. The Prussian blue doped with W did not significantly inhibit the scavenging activity of hydroxyl radicals and superoxide anions compared to the undoped Prussian blue. Reactive nitrogen can also damage body tissues and amplify inflammatory reactions. The scavenging of active nitrogen DPPH free radicals by W-PB was also characterized. The results are shown in Figure 3As shown in H, the average inhibition percentage of W-PB on DPPH free radicals is 39.80, while that of PB is 13.92. The data show that doping with W element can greatly improve the scavenging ability of Prussian blue on DPPH free radicals.

[0089] Test Example 3

[0090] Based on the above excellent in vitro ROS scavenging effect, in order to further evaluate the anti-inflammatory ability of W-PB at the cellular level, the CCK8 method was used to detect the biocompatibility of the two nanomaterials, W-PB and PB, with normal cells.

[0091] The cytotoxicity and antibacterial effects of W-PB were evaluated. Figure 4 shown.

[0092] Cytotoxicity evaluation method:

[0093] The CCK8 method was used to evaluate the killing effect of W-PB and PB on NCM460 cells and RAW264.7. Cells in the logarithmic growth phase were collected and seeded into 96-well plates (100 μL per well) at a density of 1000 cells / well and incubated for 24 hours. The culture medium was replaced with drug-containing culture medium (100 μL per well) containing different concentrations and different preparations, and the incubation continued for 24 hours. Among them, the concentrations of W-PB or PB were 0, 6.25, 12.25, 25, 50, 75, 100 and 200 μg / mL. After the drug treatment, the culture supernatant was discarded and gently washed twice with PBS. 100 μL of culture medium containing 10% CCK8 was added to each well and incubated in the incubator in the dark for 0.5h to 4h. The absorbance value was recorded at a wavelength of 450nm using an enzyme reader. The incubation time of CCK8 was optimal when the absorbance value of the negative control group was around 1.0. Calculate cell survival rate according to formula 2-1

[0094]

[0095] Antibacterial experiment:

[0096] a. Nitrate reductase activity test

[0097] To induce nitrate reductase expression, an overnight culture of E. coli K-12 or E. coli DH5-α was diluted 100:40 in fresh NB or LB broth containing 1 mM sodium nitrate. Then W-PB (0.1 mg L -1 ) or other materials (PB) were added to the broth medium. The culture was incubated aerobically at 37°C for 3 hours, and the active protein was extracted from E. coli K-12 or E. coli DH5-α by ultrasonic disruption, and the relative nitrate reductase activity was determined using the NR detection kit and the relative nitrate reductase activity was determined using the NR detection kit.

[0098] b. Anaerobic growth test

[0099] To induce the expression of nitrate reductase, an overnight culture of E. coli K-12 or E. coli DH5-α was diluted 100:40 in fresh NB or LB broth containing 1 mM sodium nitrate. Then W-PB (0.1 mg L -1 ) or other materials (PB) were added to the broth medium. The culture was incubated anaerobically at 37°C for 12 h and the colonies were counted.

[0100] Figure 4 Figure 1 is the protective effect of W-PB on NCM460 cells and its antibacterial efficacy, where a is an inverted fluorescence microscope image of NCM460 cells stained with Calcein-AM / PI (2mM H2O2), b is the protection of NCM460 cells from oxidative stress caused by H2O2 by W-PB and PB, c-d are the growth of E.coli K-12 in the presence of different electron acceptors under anaerobic conditions, e is the determination of nitrate reductase activity of E.coli K-12 in a medium supplemented with sodium nitrate, f-g are the growth of E.coli DH-5α in the presence of different electron acceptors under anaerobic conditions, h is the determination of nitrate reductase activity of E.coli DH-5α in a medium supplemented with sodium nitrate, and i is the preparation of W-PB gel. As can be seen from the figure, reactive oxygen species (ROS) are overexpressed and released by immune cells accumulated in diseased tissues, which will lead to damage to colonic epithelial cells. In order to intuitively demonstrate the antioxidant properties of W-PB, a 2 mM H2O2-induced NCM460 cell oxidative damage experiment was further conducted and characterized by live-dead cell staining ( Figure 4 a). In the H2O2 group, there were a large number of dead cells marked by red signals. Interestingly, after treatment with W-PB or PB, the intensity of the red signal was significantly reduced, confirming that W-PB and PB can effectively remove ROS and protect cells from oxidative damage. The results of the CCK8 experiment showed that the cell survival rate decreased to 70.9% after H2O2 treatment, while the cell survival rates increased to 79.9% and 77.5% after pre-treatment with W-PB and PB, respectively ( Figure 4b). All these results indicate that W-PB has an excellent protective effect on NCM460 cells. Abnormal proliferation of harmful bacteria (such as E. coli) can occupy ecological niches and prevent the colonization of beneficial bacteria, but this proliferation is inhibited when tungstate is added for treatment because tungsten can replace molybdenum in molybdenum pterin cofactor, inactivating the latter in Enterobacteriaceae. In order to explore whether W-PB can inhibit the nitrate reductase activity of bacteria, the effects of PB and W-PB on wild-type E. coli strains (DH-5α and K-12) were compared. The experimental results showed that PB doped with tungsten can significantly inhibit the activity of bacterial nitrate reductase, thereby inhibiting the growth of Enterobacteriaceae ( Figure 4 Considering the complex gastrointestinal environment, including the highly acidic gastric juice, bile salts, digestive enzymes and diet, biocompatible and degradable chitosan and sodium alginate were selected to form a gel through electrostatic cross-linking ( Figure 4 i) in the text box.

[0101] Figure 5 The antioxidant and anti-inflammatory effects of W-PB at the cellular level, where a is a schematic diagram of the anti-inflammatory and antioxidant properties of W-PB in vitro, b is the cell viability after co-culture of RAW264.7 cells with different concentrations of W-PB and PB for 24 hours, c is the inverted fluorescence microscope image analysis of RAW264.7 cells stained with DCFH-DA, d is the quantitative analysis of ROS by ImageJ software, e is the ROS level in RAW264.7 cells detected by flow cytometry, f~g are the effects of W-PB and PB on macrophage phenotype detected by flow cytometry, h~j are the statistical analysis of STING and p-STING expressions in RAW264.7 cells by Western Blot and ImageJ software, k~m are the levels of TNF-α, IL-6 and IL-1β in RAW264.7 cells after different treatments; Figure 6 Cell survival rate after co-culture of NCM460 cells with different concentrations of W-PB and PB for 24 hours; Figure 7 is the ROS level in macrophages; where a is iNOS + , b is CD206 + .Depend on Figures 5 to 8 It is known that in the IBD model, high levels of reactive oxygen and nitrogen species (RONS) can damage the mitochondria of macrophages, leading to DNA leakage and subsequent activation of the STING pathway ( Figure 5a). Given the excellent ROS scavenging ability of W-PB nanozymes, the antioxidant and anti-inflammatory properties of W-PB at the cellular level were further investigated. First, to evaluate the cytotoxicity of W-PB and PB nanozymes, CCK8 experiments were performed by co-culturing W-PB and PB nanozymes with various cell types for 24 hours. The results showed that when the concentration of both materials was 100 μg / mL, the cell viability exceeded 90%, indicating that W-PB nanozymes have excellent cytocompatibility ( Figure 5 b and Figure 6 ). Therefore, W-PB and PB concentrations of 100 μg / mL were selected for further experiments. In order to intuitively demonstrate the antioxidant properties of W-PB, 2 mM H2O2 was further used to induce oxidative damage in Raw264.7 cells and characterized by cell viability staining experiments. The intracellular ROS level was further tested using the ROS-sensitive probe 2,7-dichlorofluorescein diacetate (DCFH-DA) fluorescent dye. The results showed that the intracellular ROS level increased significantly after H2O2 treatment. In contrast, W-PB pretreatment significantly reduced the green fluorescence caused by ROS, which was better than PB, confirming its powerful ROS scavenging ability ( Figure 5 In addition, flow cytometry was used to detect intracellular ROS levels, and the results were consistent with those obtained previously ( Figure 5 e and Figure 7 ). Excessive ROS generation can affect the differentiation of macrophages, prompting them to switch to the M1 phenotype and secrete proinflammatory cytokines, further exacerbating local inflammation and tissue damage. To understand the effect of W-PB on macrophage phenotype, macrophages were co-cultured with LPS, W-PB or PB for 24 hours. Then, the polarization state of macrophages was evaluated by flow cytometry ( Figure 5 f-g). Compared with the LPS group, both PB and W-PB treatments resulted in a decrease in the expression level of iNOS, a marker of M1 macrophages (approximately 8.8% and 16.7%, respectively), while the expression of CD206, a marker of M2 macrophages, increased by 5.4% and 15.74%, respectively ( Figure 8 To further confirm the downregulation of the cGAS-STING pathway, the expression and activation of STING were determined by Western Blot ( Figure 5 h). The blotting images showed that the expression of STING and p-STING was significantly upregulated in RAW264.7 cells after H2O2 treatment. The upregulation of p-STING indicated the activation of the cGAS-STING pathway. Both PB and W-PB treatments inhibited the overexpression of STING and p-STING, especially W-PB almost reversed the upregulation of STING and p-STING and restored them to the normal group level ( Figure 5These results indicate that W-PB effectively inhibited inflammation mediated by the cGAS-STING pathway. Finally, ELISA kits were used to measure the levels of cytokines in the supernatant of LPS and cell co-culture. As expected, W-PB significantly reduced the levels of TNF-α, IL-6, and IL-1β, and the effect was better than that of PB ( Figure 5 These findings indicate that W-PB exhibits excellent antioxidant and cytoprotective properties and is able to break the ROS-inflammation vicious cycle, suggesting its potential application in the in vivo treatment of IBD.

[0102] Fig. 9 is the transmission electron microscopy (TEM) image of W-PB gel; Fig.10 Rheological characterization of W-PB gel, where a and b are strain sweeps, and c is the time modulus curve; Fig.11 Scanning electron micrographs of W-PB gel under different pH conditions, where SGF is the morphology of the gel in simulated gastric fluid (pH 1.2), SIF is the morphology of the gel in simulated intestinal fluid (pH 6.8), and SCF is the morphology of the gel in simulated colon fluid (pH 7.8); Fig.12 The release of tungsten ions from W-PB gel in the simulated gastrointestinal tract. Figures 9 to 12 It can be seen that after the gel was prepared, its morphology was observed by transmission electron microscopy (TEM). It can be seen that the gel has a network structure, and W-PB nanoparticles are embedded in it, indicating that W-PB has been successfully loaded ( Fig. 9 ). Subsequently, the gel was rheologically characterized. To evaluate the viscoelastic properties of the hydrogel, a dynamic strain sweep was first performed to set the appropriate conditions for the dynamic frequency sweep. In the lower strain range (0.0001%-0.01%), the values ​​of the storage modulus (G') and loss modulus (G") remained constant, indicating that the gel was in an undisturbed state. The intersection represents the critical point between the gel and fluid states, indicating that the sample is a hydrogel ( Fig.10 However, at higher shear strains (>0.01%), the value of G' decreases, and when the shear strain is 0.06%, the values ​​of G' and G" intersect, indicating the transition from gel to sol. It is worth noting that when the stress is greater than 4Pa, the structure of the sample becomes unstable ( Fig.10 b). The strain was kept at 1% and a dynamic time scan was performed. The linear scan was completed from 0 to 10 min. G' was always greater than G", indicating that the surface hydrogel had good stability ( Fig.10c). Based on the rheological characterization results, it can be seen that W-PB gel with certain stability and good oral gavage performance was successfully prepared. The stability of W-PB gel in gastrointestinal simulation was further tested. In simulated gastric fluid (SGF) at pH 1.2, almost no tungsten ion was released within 2 hours. In simulated intestinal fluid (SIF) at pH 6.8, the cumulative release of tungsten ions was about 19% within 4 hours, while in simulated colonic fluid (SCF) at pH 7.8, the cumulative release of the drug reached about 78% within 2 hours ( Fig.12 In addition, combined with scanning electron micrographs of W-PB gels at different pH values, it was found that in simulated gastric fluid (SGF) at pH 1.2, the hydrogel collapsed to form a dense structure ( Fig.11 ). In contrast, in simulated intestinal fluid (SIF) at pH 6.8, the hydrogel swelled and the pore size increased; in simulated colonic fluid (SCF) at pH 7.8, the hydrogel swelled more. These results suggest that W-PB gel may be better for delivering W-PB to the colon.

[0103] Fig.13 The therapeutic effect of W-PB gel on DSS-induced IBD model mice, where a is the experimental flow chart of W-PB gel treatment of DSS-induced IBD mice, b~c are images of colon tissue and quantification of its length after 13 days, d is the daily change of body weight of mice in different groups, e~f are images of spleen tissue and its weight of mice in different groups on the 13th day, and g is H&E staining and AB / PAS staining. As can be seen from the figure, the experimental process is that the normal group received ultrapure water for 7 consecutive days; the DSS group received 2.5% DSS drinking water for the first 7 days; the treatment group was given the corresponding drugs by oral gavage every day for the next 7 days. Dextran sulfate sodium (DSS) is known to cause damage to the integrity of the intestinal mucosa and produce direct toxicity to intestinal epithelial cells. In order to induce IBD in mice, C57BL / 6 mice were exposed to sterile drinking water containing 2.5% DSS for 7 consecutive days ( Fig.13 a in the figure). Subsequently, the mice were randomly divided into 7 groups (n=5 in each group): healthy group (Healthy), control group (DSS+PBS), DSS+W-PB gel group (DSS+W-PB gel), DSS+W-PB group, DSS+PB gel group (DSS+PB gel), DSS+gel group (DSS+gel), and DSS+5-aminosalicylic acid (5-ASA) group (DSS+5-ASA). All mice were given the corresponding drugs by oral gavage every day for the next 7 days. The recovery of intestinal length was evaluated, as shown in Figure 5. Fig.13As shown in Figures b to c, mice in the W-PB gel group showed significant protection against DSS-induced intestinal length loss compared with the PB gel group (P < 0.001 compared with the W-PB gel group and the DSS group; P < 0.001 compared with the PB gel group). The body weight of mice in the PB gel and W-PB gel groups increased significantly compared with the mice in the 5-ASA, gel, and DSS groups ( Fig.13 In addition, the volume and weight of the spleen were examined ( Fig.13 e to f) to visually track changes in the immune system. Significantly, the spleen volume and weight of the W-PB gel-treated group were almost maintained at healthy levels, while the spleen of mice in the IBD model group was significantly enlarged, indicating a systemic immune response. The therapeutic effect of W-PB gel was further evaluated by histological analysis ( Fig.13 g). Hematoxylin and eosin (H&E) staining showed that the intestinal mucosa of IBD model mice was destroyed, manifested by rupture, separation, and immune cell infiltration, and the colonic crypts were severely damaged, including atrophy and loss of parallel pit structures. In contrast, these microstructures in the W-PB gel group remained intact, and the colonic epithelium remained intact, showing normal colonic crypt morphology with no signs of inflammatory cell infiltration. Alcian blue / periodic acid Schiff (AB / PAS) staining highlighted the presence of a large number of goblet cells and mucin in normal healthy colon tissue.

[0104] Fig.14 W-PB gel restored the barrier function and inhibited intestinal inflammation, where a was the 13th day, the colon was collected and subjected to immunofluorescence analysis to detect the expression patterns of ZO-1 and occludin; b, upper figure: immunofluorescence analysis of M1 macrophage marker iNOS in colon tissue, iNOS (red) and DAPI (blue), lower figure: immunofluorescence analysis of M2 macrophage marker CD206 in colon tissue, CD206 (red) and DAPI (blue); c was the ELISA analysis of myeloperoxidase (MPO) and proinflammatory factors TNF-α, IL-6, IL-1β and IL-10 in mouse colon tissue. Fig.15 It is a quantitative analysis of ZO-1 and Occludin, wherein a is ZO-1 and b is Occludin. Fig.16 Quantitative analysis of iNOS and CD206, where a is iNOS and b is CD206. Figure 14 to Figure 16It can be seen that, in contrast, the colon tissue of the model group showed a significant loss of goblet cells. However, after treatment with W-PB gel, the goblet cell structure was well preserved, and mucus secretion increased significantly, exceeding that of the model group. These findings suggest that W-PB gel can provide a protective effect on intestinal tissue by promoting mucus secretion from goblet cells. Subsequently, the integrity of the intestinal barrier was assessed by immunofluorescence analysis of the expression of two tight junction proteins, ZO-1 and Occludin, which are responsible for closing the intercellular space between intestinal epithelial cells. In the colon tissue of the IBD model group, the levels of these two proteins were significantly lower than those in the healthy group. However, after treatment with W-PB gel, the expression levels of these two proteins were significantly upregulated, indicating the successful restoration of intestinal barrier function ( Fig.14 a and Fig.15 The types of colonic macrophages were also evaluated. Immunofluorescence analysis showed a shift in macrophage types in colonic tissue after W-PB treatment ( Fig.14 b and Fig.16 ). Compared with the IBD model group, the number of pro-inflammatory M1 macrophages decreased by 93%, while the number of anti-inflammatory M2 macrophages increased by approximately 70.5 times. These findings indicate that W-PB gel can effectively polarize macrophages. Since myeloperoxidase (MPO) not only reflects the level of oxidative stress but also reflects the infiltration of inflammatory cells in IBD colon tissue, the activity of MPO was also analyzed in IBD colon tissue. MPO in feces has been used as a powerful indicator of disease severity in IBD patients. W-PB gel significantly reduced MPO activity in IBD colon tissue to normal levels, with better results than PB gel. Finally, the effects of W-PB gel in reducing pro-inflammatory cytokines (including TNF-α, IL-6, and IL-1β) and upregulating the anti-inflammatory cytokine IL-10 were further confirmed by enzyme-linked immunosorbent assay (ELISA) ( Fig.14 c) in.

[0105] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for preparing a tungsten-doped Prussian blue nanocomposite material, characterized in that the steps include: The tungsten-doped Prussian blue nanocomposite material is prepared by a one-pot hydrothermal method using polyvinyl pyrrolidone as a reducing agent and a stabilizer, and tungsten hexachloride, citric acid and potassium ferrocyanide as reactants.

2. The preparation method according to claim 1, characterized in that The mass ratio of the polyvinyl pyrrolidone, tungsten hexachloride, citric acid and potassium ferrocyanide is 4:0.84:1.32:0.

66.

3. The preparation method according to claim 1, characterized in that: The step of preparing the tungsten-doped Prussian blue nanocomposite material by a one-pot hydrothermal method comprises: Potassium ferrocyanide and polyvinyl pyrrolidone are dissolved in water to prepare solution A; Dissolve tungsten hexachloride and citric acid in water to prepare solution B; The solution A and the solution B are mixed, reacted at 80° C. for 24 hours, and the reaction product is separated by centrifugation. The tungsten-doped Prussian blue nanocomposite material is obtained after washing and drying.

4. The preparation method according to claim 3, characterized in that: The centrifugal speed is 15000 rpm and the time is 10 min; the washing is ultrasonic washing and resuspension, which is repeated at least once; and the drying is vacuum freeze drying.

5. The tungsten-doped Prussian blue nanocomposite material obtained by the preparation method according to claim 1.

6. Use of the tungsten-doped Prussian blue nanocomposite material as claimed in claim 5 in preparing a drug for treating inflammatory bowel disease.

7. A colon targeted delivery material, characterized in that: The tungsten-doped Prussian blue nanocomposite material as claimed in claim 5 is used as an active ingredient.

8. The colon targeted delivery material according to claim 7, characterized in that: The colon targeted delivery material uses hydrogel as a carrier of the active ingredient; the hydrogel is formed by physical cross-linking of sodium alginate and chitosan.

9. The method for preparing the colon targeted delivery material according to claim 7, characterized in that the steps include: Dissolving sodium alginate in water to obtain a first mixed solution; dispersing the tungsten-doped Prussian blue nanocomposite material in water to obtain a second mixed solution; adding acetic acid and chitosan to the second mixed solution to obtain a third mixed solution; The first mixed solution and the third mixed solution are mixed to obtain a reaction system, and the reaction system is stirred overnight to obtain the colon targeted delivery material.

10. The preparation method according to claim 9, characterized in that: The mass concentration of sodium alginate in the first mixed solution is 20 g / L; and / or, The mass concentration of the tungsten-doped Prussian blue nanocomposite material in the second mixed solution is 1 g / L; and / or The dosage ratio of sodium alginate, chitosan, acetic acid and tungsten-doped Prussian blue nanocomposite material in the reaction system is 40 mg:54 mg:4 μL:3 mg.

Images