Tungsten-doped prussian blue nanocomposite, preparation method and application thereof

Abstract

The application belongs to the technical field of nanocomposite preparation, and particularly relates to a tungsten-doped Prussian blue nanocomposite, a preparation method and application. The tungsten-doped Prussian blue nanocomposite prepared by a one-pot hydrothermal method with polyvinylpyrrolidone as a reducing agent and a stabilizer, tungsten hexachloride, citric acid and potassium ferricyanide as reactants has excellent ROS scavenging capacity, can protect intestinal epithelial cells and treat IBD, and then a colon-targeted delivery material is prepared by taking a hydrogel as a carrier, so that W-PB is accurately delivered to the colon site, the subsequent alkaline environment causes the hydrogel to disintegrate and release W-PB, and W ions are further released, so that the colon is released in a responsive manner, and the systemic toxicity of tungsten ions is avoided.

Description

Technical Field

[0001] This invention relates to the field of nanocomposite material preparation technology, and in particular to a tungsten-doped Prussian blue nanocomposite material, its preparation method, and its application. Background Technology

[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 treatments.

[0003] IBD is closely related to the dysbiosis of the gut microbiota, characterized by an increase in facultative anaerobic bacteria (Enterobacteriaceae) and a decrease in beneficial bacteria. Traditional medical treatment for IBD primarily relies on broad-spectrum antibiotics to prevent the spread of pathogenic bacteria. However, the widespread use of antibiotics can also destroy probiotics and increase the risk of antibiotic resistance, such as in cases of inflammation caused by Escherichia coli K-12.

[0004] The rise in IBD and ROS (reactive oxygen species) is also closely related. ROS upregulation was observed in colitis tissues of patients in a mouse model of colitis, ultimately leading to intestinal mucosal damage. First, pro-inflammatory cytokines can activate immune cells and amplify inflammation and ROS production during IBD. Simultaneously, excessive ROS production further activates the inflammatory / immune response through the nuclear factor-κB (NF-κB) signaling pathway, leading to increased expression and secretion of pro-inflammatory cytokines. Furthermore, high levels of ROS exposure also cause oxidative damage to mitochondria, promoting mtDNA release, thereby activating the STING pathway and inducing apoptosis in intestinal epithelial cells (IECs). Therefore, clearing ROS from the gut is essential to halting IBD progression. However, several non-enzymatic and enzymatic antioxidants are currently ineffective, many of which induce adverse immune responses. Some studies have shown that the therapeutic effects of natural antioxidants such as vitamin E, vitamin C, and coenzyme Q are unsatisfactory. In addition, the ROS scavenger N-acetylcysteine ​​enhances the production of helper T cells 17 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 gut microbiota, especially Prussian blue nanozymes (BP). Prussian blue nanozymes, a type of TMSN, have attracted significant interest in ROS-mediated disease treatment due to their superior multi-antioxidant enzyme-like activities, excellent biocompatibility, ease of preparation and modification, and good biocompatibility. However, Prussian blue nanozymes do not affect nitrate reductase activity, and therefore do not have a significant antibacterial effect in certain situations (such as against Escherichia coli K-12). Summary of the Invention

[0006] The purpose of this invention is to provide a tungsten-doped Prussian blue nanocomposite material, its preparation method, and its application, in order to solve the problems existing in the prior art, precisely edit the intestinal flora and remove ROS, protect intestinal epithelial cells, and treat IBD.

[0007] To achieve the above objectives, the present invention provides the following solution:

[0008] One of the technical solutions of this invention is to provide a method for preparing tungsten-doped Prussian blue nanocomposite materials, the steps of which include:

[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 stabilizer, and tungsten hexachloride (WCl6), citric acid and potassium ferricyanide (K3Fe(CN)6) as reactants.

[0010] Furthermore, the mass ratio of polyvinylpyrrolidone, tungsten hexachloride, citric acid and potassium ferricyanide 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 includes:

[0012] Potassium ferricyanide and polyvinylpyrrolidone were dissolved in water to prepare solution A;

[0013] Solution B is prepared by dissolving tungsten hexachloride and citric acid in water;

[0014] Solution A and solution B are mixed and reacted at 80°C for 24 hours. The reaction products are separated by centrifugation, washed, and dried to obtain the tungsten-doped Prussian blue nanocomposite material.

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

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

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

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

[0019] The third technical solution of the present invention provides 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 provides a colon-targeted delivery material, with the above-mentioned tungsten-doped Prussian blue nanocomposite material as the active ingredient.

[0021] Furthermore, the colon-targeted delivery material uses a hydrogel as a carrier for the active ingredient; the hydrogel is physically cross-linked with sodium alginate and chitosan.

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

[0023] Sodium alginate was dissolved in water to obtain the first mixed solution;

[0024] A second mixed solution was obtained by dispersing tungsten-doped Prussian blue nanocomposite material in water;

[0025] Acetic acid and chitosan were added 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, which 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 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] This invention enhances the effect of Prussian blue nanomaterials on nitrate reductase activity by doping them with tungsten, thereby improving their inhibitory effect on pathogens causing inflammatory bowel disease.

[0032] The tungsten-doped Prussian blue nanocomposite material prepared by this invention can be 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] This invention utilizes a hydrogel as a carrier to load a tungsten-doped Prussian blue nanocomposite material to prepare a colon-targeted delivery material. This material protects W-PB and ensures its precise delivery to the colon. The subsequent alkaline environment causes the hydrogel to disintegrate and release W-PB, which in turn releases W ions, achieving a responsive release to the colon and avoiding the systemic toxicity of tungsten ions. Attached Figure Description

[0034] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0035] Figure 1 Figure 1 shows the morphology and phase characterization results of W-PB obtained in Example 1. In the figure, 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 to g are XRD and SC-XRD, respectively, h to i are the UV and IR spectra of W-PB and PB, respectively, j to l are the XPS spectrum, Fe 2p electronic level and W 4f electronic level of W-PB, respectively, and m to o are the XPS spectrum, Fe 2p electronic level and W 4f electronic level of W-PB after ROS removal, respectively.

[0036] Figure 2 A schematic diagram of the fabrication process for colon-targeted delivery materials;

[0037] Figure 3 The in vitro catalytic performance of W-PB is shown in Figure 1. A represents the amount of hydrogen peroxide consumed by the titanium sulfate colorimetric indicator, B represents the ESR spectrum of W-PB, C represents the ESR spectrum of W-PB, D represents the UV spectrum of DPPH radical scavenging, E represents the ability of W-PB and PB to decompose H2O2 to produce O2 in PBS, F represents the scavenging rate of W-PB and PB for ·O2 radicals, G represents the scavenging rate of W-PB and PB for ·OH radicals, and H represents the scavenging rate of W-PB and PB for DPPH radicals.

[0038] Figure 4 To illustrate the protective effect and antibacterial efficacy of W-PB on NCM460 cells, the following images are presented: a) Inverted fluorescence microscopy image of NCM460 cells stained with Calcein-AM / PI (2mM H2O2); b) Protection of NCM460 cells from H2O2-induced oxidative stress by W-PB and PB; c-d) Growth of E. coli K-12 under anaerobic conditions with different electron acceptors; e) Measurement of nitrate reductase activity of E. coli K-12 in sodium nitrate-supplemented medium; f-g) Growth of E. coli DH-5α under anaerobic conditions with different electron acceptors; h) Measurement of nitrate reductase activity of E. coli DH-5α in sodium nitrate-supplemented medium; and i) Preparation of W-PB gel.

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

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

[0041] Figure 7 ROS levels in macrophages;

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

[0043] Figure 9 Transmission electron microscopy (TEM) images of W-PB gel;

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

[0045] Figure 11 The images show scanning electron micrographs of W-PB gels under different pH conditions. SGF represents the morphology of the gel in simulated gastric fluid (pH 1.2), SIF represents the morphology of the gel in simulated intestinal fluid (pH 6.8), and SCF represents the morphology of the gel in simulated colonic fluid (pH 7.8).

[0046] Figure 12 To simulate the release of tungsten ions from W-PB gel in the gastrointestinal tract;

[0047] Figure 13The therapeutic effect of W-PB gel on DSS-induced IBD model mice is shown in the following figures: a is the experimental flowchart of W-PB gel treatment for DSS-induced IBD mice; b-c are images of colon tissue and their length quantification after 13 days; d is the daily weight change of mice in different groups; e-f are images of spleen tissue and their weight of mice in different groups on day 13; and g is H&E staining and AB / PAS staining.

[0048] Figure 14 To demonstrate the effects of W-PB gel on restoring barrier function and inhibiting intestinal inflammation, the following data is presented: a) On day 13, colon cells were collected and immunofluorescence analysis was performed to detect the expression patterns of ZO-1 and occludin; b) In the upper image: immunofluorescence analysis of the M1 macrophage marker iNOS in colon tissue (iNOS (red) and DAPI (blue); in the lower image: immunofluorescence analysis of the M2 macrophage marker CD206 in colon tissue (CD206 (red) and DAPI (blue)); c) Analysis of myeloperoxidase (MPO) and pro-inflammatory factors TNF-α, IL-6, IL-1β, and IL-10 in mouse colon tissue using ELISA.

[0049] Figure 15 Quantitative analysis of ZO-1 and Occludin, where a represents ZO-1 and b represents Occludin;

[0050] Figure 16 Quantitative analysis of iNOS and CD206, where a represents iNOS and b represents CD206. Detailed Implementation

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

[0052] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0053] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0054] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

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

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

[0057] In the specific embodiments of this invention, all raw materials and reagents used are commercially available products. Among them, mouse mononuclear macrophage leukemia cells (RAW264.7) are from the Cell Bank of the Chinese Academy of Sciences; human normal colonic epithelial cells (NCM460) are from Fenghui Biotechnology; Escherichia coli DH5-α is from Beina Biotechnology; Escherichia coli K-12 is from Beina Biotechnology; and 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 materials are as follows:

[0063] S1. Dissolve 0.66g of potassium ferricyanide powder and 4g of PVP in 100mL of deionized water and stir for 30 minutes to prepare solution A.

[0064] S2. Dissolve 0.84g of WCl6 and 1.32g of citric acid in 100mL of deionized water and stir for 30 minutes to prepare solution B.

[0065] S3. Add solution B to solution A and react at 80℃ for 24 h. Then centrifuge at 15000 rpm for 10 min to separate the solid product. Wash the solid product with ultrasonic water and resuspend it. Repeat this process three times. Then process the solid product with a vacuum freeze dryer to obtain tungsten-doped Prussian blue nanocomposite material, denoted as W-PB.

[0066] Example 2

[0067] The preparation steps for colon-targeted delivery materials are as follows:

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

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

[0070] Figure 2 A 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] The only difference from Example 2 is that W-PB is replaced with an equal amount of PB.

[0074] Comparative Example 2

[0075] The preparation method of a single hydrogel is as follows:

[0076] The difference compared to Example 2 is that W-PB was not added.

[0077] Experimental Example 1

[0078] The morphology and phase composition of the tungsten-doped Prussian blue nanocomposite material prepared in Example 1 were characterized. The morphology and particle size of the prepared W-PB were observed using a transmission electron microscope (TEM). The lattice of the W-PB surface was observed using a high-resolution transmission electron microscope (HRTEM). Wide-angle diffraction of the W-PB lyophilized 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). The diffuse reflectance spectrum of the W-PB / barium sulfate pellet was obtained by scanning with a UV-Vis-NIR spectrophotometer.

[0079] Figure 1 Figure 1 shows the morphology and phase characterization results of W-PB obtained in Example 1. In the figure, 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, respectively, h-i are the UV and IR spectra of W-PB and PB, respectively, j-l are the XPS spectrum, Fe 2p electronic level, and W 4f electronic level of W-PB, respectively, and m-o are the XPS spectrum, Fe 2p electronic level, and W 4f electronic level after ROS removal of W-PB, respectively. As can be seen from the figure, the acidic environment created by the hydrolysis of tungsten chloride produces hydrochloric acid, which makes PVP reducible. The iron ions in potassium ferricyanide are reduced, and Fe... 3+ / Fe 2+ and CN - Coordination to form W-PB nanoparticles ( Figure 1 As shown in a). b and c show that W-PB is cubic, well dispersed, and has a uniform distribution of C, N, Fe, and W. Quantitative determination by ICP-OES showed that the W element content was 7.28% ± 0.65%, confirming the successful preparation of W-PB. Due to the stabilizing effect of PVP, W-PB exhibits relatively excellent monodispersity in aqueous solution, with an average particle size of 197.84 nm. Figure 1 (As shown in d). According to Zeta potential measurements, W-PB has a highly negative charge (-21.05 mV) ( Figure 1As shown in e). XRD patterns in g show that the synthesized PB NPs have distinct 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 were observed assigned to W or tungsten oxide in f. In addition, single-crystal XRD diffraction experiments were performed, revealing the structure of the Debye diffraction rings ( Figure 1 As shown in g), this indicates that W ions are doped into the lattice of PB without significantly affecting its phase structure. Using the relationship between the absorbance value at the 710 nm absorption peak and the W-PB concentration, a standard curve was obtained through linear fitting to accurately determine the mass concentration of W-PB. Figure 1 (As shown in h). Chemical bond characteristics were analyzed using the infrared spectra of W-PB and PB, revealing a stretching feature where C=N is located at 2090 cm⁻¹. -1 ( Figure 1 As shown in Figure i). XPS shows that Fe, W, C, N and O elements are present simultaneously in W-PB ( Figure 1 (As shown in j). 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 The peak of ) belongs to Fe 2+ At 709.7 eV (Fe 2p 3 / 2 ) and 723.1 eV (Fe2p 1 / 2 The peak of ) belongs to Fe 3+ ( Figure 1 (As shown in k). Fe 2+ and Fe 3+ The ratio of approximately 2.38 is also highly favorable for W-PB-mediated ROS scavenging. High-resolution spectra of W4f show four peaks, with the peaks at 38.1 eV and 36.1 eV belonging to W... 6+ 4f 7 / 2 At 36.5 eV (W 4f) 5 / 2 ) and 34.1eV (W 4f 7 / 2 The peak of ) belongs to W 4+ ( Figure 1 (As shown in Figure 1). W 4+ With W 6+ The ratio is approximately 0.15. W 6+ / W 4+ and Fe 3+ / Fe 2+ The REDOX coupling may provide abundant redox reaction sites for reactive oxygen species, enhancing their ability to scavenge ROS. After co-incubation of W-PB with hydrogen peroxide, Fe was found... 2+ with Fe 3+and W 4+ With W 6+ The change in the ratio indicates that transition metals can transfer electrons through changes in valence state, thus exhibiting catalytic activity. Figure 1 (as shown in m~o).

[0080] Experimental Example 2

[0081] In vitro catalytic performance study of W-PB: Testing of its ability to scavenge H2O2, ·O2, ·OH, and DPPH free radicals. Results are as follows: Figure 3 As shown.

[0082] The inhibition percentage was measured using a superoxide dismutase (SOD) activity assay kit, a hydroxyl radical scavenging assay kit, and a DPPH radical scavenging assay kit, respectively, according to the manufacturer's instructions. The inhibition percentage was calculated using Equation 1-1.

[0083]

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

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

[0086] ESR hydroxyl radical: Prepare a 5 mg / mL FeSO4 solution. Take 200 μL of this solution, add 20 μL of LMPO and 160 μL of deionized water, then add 20 μL of 30% hydrogen peroxide. Mix well and react for 5 min. Take a sample for testing as the control group data. For other sample test groups, simply replace the 160 μL of deionized water with 160 μL of sample solution.

[0087] ESR superoxide radical: Using PBS buffer as a solvent, prepare a 10mM xanthine solution and a 1U / mL xanthine oxidase solution. Take 100μL of xanthine solution and 100μL of xanthine oxidase solution, then add 20μL DMPO and 180μL buffer. After incubating for 10 minutes, take samples for testing. For other sample test groups, simply replace the 180μL buffer with 180μL of sample solution.

[0088] Figure 3The in vitro catalytic performance of W-PB is shown in the figure. A represents the amount of hydrogen peroxide consumed by titanium sulfate colorimetric analysis; B and C represent the ESR spectra of W-PB; D represents the UV spectrum of DPPH radical scavenging; E represents the ability of W-PB and PB to decompose H₂O₂ to produce O₂ in PBS; F represents the scavenging rates of W-PB and PB for ·O₂ radicals; G represents the scavenging rates of W-PB and PB for ·OH radicals; and H represents the scavenging rates of W-PB and PB for DPPH radicals. The figure shows that in A, using titanium sulfate colorimetric analysis and UV quantification to detect hydrogen peroxide consumption, W-PB's ability to consume hydrogen peroxide is approximately twice that of PB. This is because tungsten atoms have empty d orbitals, which easily form low-barrier transition states, exposing more catalytic sites and thus enhancing its catalytic activity. During this process, a large number of bubbles were observed to be generated in the solution, with oxygen as the product. To confirm 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 both W-PB and PB concentrations were 10 μg / mL, the oxygen production rate of W-PB was significantly higher than that of PB. At 19 min, the oxygen production concentration of W-PB was 28.1 mg / L, while that of PB was 10.52 mg / L, demonstrating the strong oxygen production capacity of W-doped Prussian blue. In B, hydroxyl radicals were scavenged in the presence of DMPO as a radical scavenger; in C, superoxide anion radicals were eliminated in the presence of DMPO as a radical scavenger. Further analysis using ESR was conducted to determine 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 min and 10 min. The results are shown below. Figure 3 As shown in B and C, 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, its catalytic activity was quantified using a hydroxyl radical scavenging ability assay kit and a superoxide dismutase (SOD) activity assay kit, respectively. The results are shown in Figure 1. Figure 3 As shown in F and G, at a concentration of 80 μg / mL for both W-PB and PB, the average inhibition percentages of both for hydroxyl radicals were 36.16% and 44.65%, respectively, and for superoxide anions, they were 40.00% and 47.57%, respectively. W-doped Prussian blue did not significantly inhibit the scavenging activity against hydroxyl radicals and superoxide anions compared to undoped Prussian blue. Reactive nitrogen can also damage body tissues and amplify inflammatory responses. Furthermore, the scavenging effect of W-PB on reactive nitrogen DPPH radicals was also characterized, as shown in the results... Figure 3As shown in H, W-PB has an average inhibition percentage of 39.80% on DPPH radicals, while PB has an average inhibition percentage of 13.92%. The data indicate that doping with W can significantly improve the scavenging ability of Prussian blue for DPPH radicals.

[0089] Experimental Example 3

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

[0091] The cytotoxicity and antibacterial efficacy of W-PB were evaluated, and the results are as follows: Figure 4 As shown.

[0092] Cytotoxicity evaluation methods:

[0093] The cytotoxic effects of W-PB and PB on NCM460 and RAW264.7 cells were evaluated using the CCK8 assay. Cells in logarithmic growth phase were collected and seeded into 96-well plates at a density of 1000 cells / well (100 μL per well) and incubated for 24 h. The culture medium was then replaced with drug-containing medium (100 μL per well) at different concentrations and formulations, and incubation continued for another 24 h. After treatment with W-PB or PB at concentrations of 0, 6.25, 12.25, 25, 50, 75, 100, and 200 μg / mL, the culture supernatant was discarded, and the cells were gently washed twice with PBS. 100 μL of medium containing 10% CCK8 was added to each well, and the cells were incubated in the dark for 0.5 h to 4 h. The absorbance was recorded at 450 nm using a microplate reader. The optimal incubation time for CCK8 was observed when the absorbance value of the negative control group was near 1.0. Calculate cell viability according to Equation 2-1

[0094]

[0095] Antibacterial test:

[0096] a. Nitrate reductase activity test

[0097] To induce nitrate reductase expression, overnight cultures of *E. coli* K-12 or *E. coli* DH5α were diluted 100:40 in fresh NB or LB broth containing 1 mM sodium nitrate. Then, W-PB (0.1 mg / L) was added. -1 Alternatively, other materials (PB) can be added to the broth medium. The culture is incubated aerobically at 37°C for 3 hours. Active proteins are extracted from *E. coli* K-12 or *E. coli* DH5-α by sonication. The relative nitrate reductase activity is determined using an NR assay kit.

[0098] b. Anaerobic growth experiment

[0099] To induce nitrate reductase expression, overnight cultures of *E. coli* K-12 or *E. coli* DH5α were diluted 100:40 in fresh NB or LB broth containing 1 mM sodium nitrate. Then, W-PB (0.1 mg / L) was added. -1 Alternatively, other materials (PB) can be added to the broth medium. The culture is then anaerobically incubated at 37°C for 12 hours, followed by colony counting.

[0100] Figure 4 To illustrate the protective effect and antibacterial efficacy of W-PB on NCM460 cells, the figures are as follows: a) inverted fluorescence microscopy image of NCM460 cells stained with Calcein-AM / PI (2mM H2O2); b) protection of NCM460 cells from H2O2-induced oxidative stress by W-PB and PB; c-d) growth of E. coli K-12 under anaerobic conditions with different electron acceptors; e) nitrate reductase activity of E. coli K-12 in sodium nitrate-supplemented medium; f-g) growth of E. coli DH-5α under anaerobic conditions with different electron acceptors; h) nitrate reductase activity of E. coli DH-5α in sodium nitrate-supplemented medium; and i) preparation of the W-PB gel. The figures show that reactive oxygen species (ROS) are overexpressed and released by immune cells accumulated in diseased tissues, leading to damage to colonic epithelial cells. To visually demonstrate the antioxidant properties of W-PB, a 2mM H2O2-induced oxidative damage experiment was further conducted on NCM460 cells, and the damage was characterized using a live-dead cell staining method. Figure 4 (a) In the H2O2 group, there were a large number of dead cells marked with red signals. Interestingly, the intensity of the red signal was significantly reduced after treatment with W-PB or PB, confirming that W-PB and PB can effectively scavenge ROS, thereby protecting cells from oxidative damage. CCK8 assay results showed that cell viability decreased to 70.9% after H2O2 treatment, while pretreatment with W-PB and PB increased cell viability to 79.9% and 77.5%, respectively. Figure 4(b) All these results indicate that W-PB has excellent protective effects 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 *Enterobacteria*. To investigate whether W-PB can inhibit bacterial nitrate reductase activity, the effects of PB and W-PB on wild-type *E. coli* strains (DH-5α and K-12) were compared. The results showed that tungsten-doped PB can significantly inhibit bacterial nitrate reductase activity, thereby inhibiting the growth of *Enterobacteria*. Figure 4 (c-h). Considering the complex gastrointestinal environment, including highly acidic gastric juice, bile salts, digestive enzymes, and diet, biocompatible and biodegradable chitosan and sodium alginate were selected, and a gel was formed through electrostatic cross-linking ( Figure 4 (i)

[0101] Figure 5 The images show the antioxidant and anti-inflammatory effects of W-PB at the cellular level. Specifically, a) is a schematic diagram of the in vitro anti-inflammatory and antioxidant properties of W-PB; b) shows the cell viability of RAW264.7 cells after co-culturing with different concentrations of W-PB and PB for 24 hours; c) shows the inverted fluorescence microscope images of RAW264.7 cells stained with DCFH-DA; d) shows the quantitative analysis of ROS using ImageJ software; e) shows the ROS level in RAW264.7 cells detected by flow cytometry; f–g show the effects of W-PB and PB on macrophage phenotype detected by flow cytometry; h–j show the statistical analysis of STING and p-STING expression in RAW264.7 cells using Western blotting and ImageJ software; and k–m show the levels of TNF-α, IL-6, and IL-1β in RAW264.7 cells after different treatments. Figure 6 Cell viability after co-culturing NCM460 cells with different concentrations of W-PB and PB for 24 hours; Figure 7 The ROS level in macrophages; where a is iNOS. + b is CD206 + .Depend on Figures 5-8 It is known that in the IBD model, high levels of reactive oxygen species (RONS) can damage macrophage mitochondria, leading to DNA leakage and subsequent activation of the STING pathway. Figure 5(a) 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, a CCK8 assay was 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 cell compatibility (a). Figure 5 b and Figure 6 Therefore, W-PB and PB concentrations of 100 μg / mL were selected for further experiments. To visually demonstrate the antioxidant properties of W-PB, oxidative damage in Raw264.7 cells was induced using 2 mM H2O2, and the cells were characterized by cell viability staining. Intracellular ROS levels were further tested using the ROS-sensitive probe 2,7-dichlorofluorescein diacetate (DCFH-DA) fluorescent dye. The results showed that intracellular ROS levels significantly increased after H2O2 treatment. In contrast, W-PB pretreatment significantly reduced ROS-induced green fluorescence, showing better results than PB, confirming its strong ROS scavenging ability. Figure 5 (c-d in the original text). Furthermore, intracellular ROS levels were detected using flow cytometry, and the results were consistent with previously obtained results. Figure 5 e and Figure 7 Excessive ROS production can affect macrophage differentiation, prompting them to transform into the M1 phenotype, secrete pro-inflammatory cytokines, and further exacerbate 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. Subsequently, the polarization status of macrophages was assessed by flow cytometry. Figure 5 (f-g). Compared with the LPS group, both PB and W-PB treatments led to a decrease in the expression level of the M1 macrophage marker iNOS (approximately 8.8% and 16.7%, respectively), while the expression of the M2 macrophage marker CD206 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 blotting. Figure 5 (h in the image). Blot images showed that the expression of STING and p-STING in RAW264.7 cells was significantly upregulated after H2O2 treatment. The upregulation of p-STING indicates activation of the cGAS-STING pathway. Both PB and W-PB treatments inhibited the overexpression of STING and p-STING, especially W-PB, which almost reversed the upregulation of STING and p-STING, restoring them to the levels of the normal group (h in the image). Figure 5These results indicate that W-PB effectively inhibits cGAS-STING pathway-mediated inflammation. Finally, cytokine levels in LPS and cell co-culture supernatant were measured using an ELISA kit. As expected, W-PB significantly reduced the levels of TNF-α, IL-6, and IL-1β, with better results than PB (i~j). Figure 5 These findings indicate that W-PB exhibits superior antioxidant and cytoprotective properties, capable of breaking the vicious cycle of ROS-inflammation, suggesting its potential application in in vivo treatment of IBD.

[0102] Figure 9 Transmission electron microscopy (TEM) images of W-PB gel; Figure 10 Rheological characterization of W-PB gel, where a and b are strain scans, and c is the time modulus curve; Figure 11 The images show scanning electron micrographs of W-PB gels under different pH conditions. SGF represents the morphology of the gel in simulated gastric fluid (pH 1.2), SIF represents the morphology of the gel in simulated intestinal fluid (pH 6.8), and SCF represents the morphology of the gel in simulated colonic fluid (pH 7.8). Figure 12 This describes the release of tungsten ions from W-PB gel in a simulated gastrointestinal tract. Figures 9-12 As can be seen, after the gel was prepared, its morphology was observed using transmission electron microscopy (TEM). It can be seen that the gel has a network structure with W-PB nanoparticles embedded within it, indicating that W-PB was successfully loaded (…). Figure 9 Subsequently, the gel was rheologically characterized. To evaluate the viscoelasticity of the hydrogel, dynamic strain scanning was first performed to set appropriate conditions for dynamic frequency scanning. Within a low strain range (0.0001%–0.01%), the storage modulus (G') and loss modulus (G") remained constant, indicating that the gel was in an undisturbed state. The intersection point represents the critical point between the gel and fluid states, indicating that the sample is a hydrogel. Figure 10 (a) 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 a gel-to-sol transition. Notably, when the stress is greater than 4 Pa, the sample structure becomes unstable. Figure 10 (b) The strain was maintained at 1%, and a dynamic time scan was performed. Linear scans were completed within 0–10 minutes. G' was consistently greater than G", indicating good stability of the surface hydrogel. Figure 10(c) 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. Further testing was conducted on the stability of the W-PB gel in a gastrointestinal simulation. In simulated gastric fluid (SGF) at pH 1.2, almost no tungsten ion release was observed within 2 hours. In simulated intestinal fluid (SIF) at pH 6.8, the cumulative release of tungsten ions was approximately 19% within 4 hours, while in simulated colonic fluid (SCF) at pH 7.8, the cumulative release of the drug reached approximately 78% within 2 hours. Figure 12 Furthermore, combining scanning electron micrographs of W-PB gels at different pH values, it was found that in simulated gastric juice (SGF) at pH 1.2, the hydrogel collapsed to form a dense structure. Figure 11 In contrast, the hydrogel swelled and its pore size increased in simulated intestinal fluid (SIF) at pH 6.8; and it swelled even more significantly in simulated colonic fluid (SCF) at pH 7.8. These results suggest that W-PB gels may deliver W-PB to the colon more effectively.

[0103] Figure 13 The figure shows the therapeutic effect of W-PB gel on DSS-induced IBD model mice. Figure a shows the experimental flowchart of W-PB gel treatment for DSS-induced IBD mice; figures b-c show images of colon tissue and their length quantification after 13 days; figure d shows the daily weight changes of mice in different groups; figures e-f show images of spleen tissue and their weight on day 13; and figure g shows H&E staining and AB / PAS staining. As shown in the figure, the experimental procedure was as follows: the normal group received ultrapure water for 7 consecutive days; the DSS group received 2.5% DSS in drinking water for the first 7 days; and the treatment group received the corresponding drug orally via gavage for the next 7 days. Sodium dextran sulfate (DSS) is known to cause damage to the integrity of the intestinal mucosa and has direct toxicity to intestinal epithelial cells. To induce IBD in mice, C57BL / 6 mice were exposed to sterile drinking water containing 2.5% DSS for 7 consecutive days. Figure 13 (a) Subsequently, mice were randomly assigned to 7 groups (n=5 per group): Healthy group, control group (DSS+PBS), DSS+W-PB gel group, DSS+W-PB group, DSS+PB gel group, DSS+gel group, and DSS+5-aminosalicylic acid (5-ASA) group. All mice were administered the corresponding drugs orally by gavage daily for the next 7 days. Recovery of intestinal length was assessed, as shown in Figure 1. Figure 13As shown in b-c, compared with the PB gel group, mice in the W-PB gel group showed a significant protective effect, preventing DSS-induced intestinal length loss (P<0.001 compared with the W-PB gel group and the DSS group; P<0.001 compared with the PB gel group). Compared with mice in the 5-ASA, gel, and DSS groups, mice in the PB gel and W-PB gel groups showed a significant increase in body weight. Figure 13 (d) In addition, the volume and weight of the spleen were examined. Figure 13 The e-f sequences were used to visually track changes in the immune system. Notably, the spleen volume and weight in the W-PB gel treatment group remained almost at healthy levels, while the spleens of mice in the IBD model group were significantly enlarged, indicating a systemic immune response. The therapeutic effect of W-PB gel was further evaluated through histological analysis. Figure 13 (g) Hematoxylin and eosin (H&E) staining revealed intestinal mucosal disruption in IBD model mice, characterized by rupture, separation, and immune cell infiltration, with severe damage to the colonic crypts, including atrophy and loss of parallel concave structures. In contrast, these microstructures remained intact in the W-PB gel group, with the colonic epithelium remaining intact, showing normal colonic crypt morphology without signs of inflammatory cell infiltration. Alixin blue / periodic acid Schiff (AB / PAS) staining highlighted the abundance of goblet cells and mucin in normal, healthy colonic tissue.

[0104] Figure 14 To demonstrate the effects of W-PB gel on restoring barrier function and inhibiting intestinal inflammation, the following data are presented: a) On day 13, colon cells were collected and immunofluorescence analysis was performed to detect the expression patterns of ZO-1 and occludin; b) In the upper image: immunofluorescence analysis of the M1 macrophage marker iNOS in colon tissue (iNOS (red) and DAPI (blue); in the lower image: immunofluorescence analysis of the M2 macrophage marker CD206 in colon tissue (CD206 (red) and DAPI (blue)); c) Analysis of myeloperoxidase (MPO) and pro-inflammatory factors TNF-α, IL-6, IL-1β, and IL-10 in mouse colon tissue using ELISA. Figure 15 Quantitative analysis of ZO-1 and Occludin, where a represents ZO-1 and b represents Occludin. Figure 16 Quantitative analysis of iNOS and CD206, where a represents iNOS and b represents CD206. Figures 14-16It was observed that, in contrast, the colonic tissue of the model group showed a significant absence of goblet cells. However, after treatment with W-PB gel, the goblet cell structure was well preserved, and mucus secretion significantly increased, exceeding that of the model group. These findings suggest that W-PB gel can provide protection for 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 sealing the intercellular space between intestinal epithelial cells. In the colonic 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 successful restoration of intestinal barrier function. Figure 14 a and Figure 15 The types of colonic macrophages were also assessed. Immunofluorescence analysis showed a change in macrophage types in colonic tissue after W-PB treatment. Figure 14 b and Figure 16 Compared to 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-fold. These findings indicate that W-PB gel can effectively polarize macrophages. Since myeloperoxidase (MPO) reflects not only the level of oxidative stress but also the infiltration of inflammatory cells in IBD colonic tissue, MPO activity was also analyzed in IBD colonic tissue. Fecal MPO has been used as a powerful indicator of disease severity in IBD patients. W-PB gel significantly reduced MPO activity in IBD colonic tissue to normal levels, with better results than PB gel. Finally, enzyme-linked immunosorbent assay (ELISA) further confirmed 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. Figure 14 (c)

[0105] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined 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... include: Potassium ferricyanide and polyvinylpyrrolidone were dissolved in water to prepare solution A; Solution B is prepared by dissolving tungsten hexachloride and citric acid in water; The solutions A and B were mixed and reacted at 80°C for 24 hours. The reaction products were separated by centrifugation, and the products were washed and dried to obtain the tungsten-doped Prussian blue nanocomposite material. The mass ratio of polyvinylpyrrolidone, tungsten hexachloride, citric acid and potassium ferricyanide is 4:0.84:1.32:0.

66.

2. The preparation method according to claim 1, characterized in that, The centrifugation speed is 15000 rpm and the time is 10 min; the washing is ultrasonic water washing and resuspension, repeated at least once; the drying is vacuum freeze drying.

3. The tungsten-doped Prussian blue nanocomposite material prepared by the preparation method described in claim 1.

4. The application of the tungsten-doped Prussian blue nanocomposite material as described in claim 3 in the preparation of drugs for treating inflammatory bowel disease.

5. A colon-targeted delivery material, characterized in that, The tungsten-doped Prussian blue nanocomposite material described in claim 3 is used as the active ingredient; The colon-targeted delivery material uses a hydrogel as a carrier for the active ingredient; the hydrogel is physically cross-linked with sodium alginate and chitosan. The preparation steps of the colon-targeted delivery material include: Sodium alginate was dissolved in water to obtain the first mixed solution; A second mixed solution was obtained by dispersing tungsten-doped Prussian blue nanocomposite material in water; Acetic acid and chitosan were added 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, which is stirred overnight to obtain the colon-targeted delivery material.

6. The colon-targeted delivery material as described in claim 5, 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 in the second mixed solution is 1 g / L; and / or The 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.

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