Preparation method of mucin thermosensitive hydrogel

Abstract

The application discloses a preparation method of mucin temperature-sensitive hydrogel and belongs to the technical field of hydrogel preparation. The method comprises the following steps: uniformly mixing pluronic F127 benzaldehyde (PF127-CHO), glycidol chitosan (Gly-CS) and a mucin solution, and adding hollow prussian blue nanoscale enzyme to obtain the mucin temperature-sensitive hydrogel HPB MPC-Gel. The application prepares a mucin hydrogel preparation loaded with hollow prussian blue nanoscale enzyme. The in-vivo and in-vitro evaluation results of the HPB MPC-Gel show that the hydrogel can improve the symptoms of IBD to a certain extent, and the hydrogel is expected to become a means for treating IBD.

Description

Technical Field

[0001] This invention relates to the field of hydrogel preparation technology, and in particular to a method for preparing mucin-based thermosensitive hydrogels. Background Technology

[0002] Inflammatory bowel disease (IBD) is a chronic, nonspecific inflammatory bowel disease that causes symptoms such as abdominal pain, diarrhea, and rectal bleeding, severely impacting patients' daily lives.

[0003] Currently, IBD cannot be completely cured. Treatment mainly focuses on relieving symptoms. Traditional drugs include immunosuppressants and antibiotics, but these have drawbacks such as low solubility and poor stability. Furthermore, these drugs may exacerbate the intestinal immune system disorders and intestinal flora imbalance caused by IBD, resulting in significant side effects.

[0004] During inflammation, on the one hand, elevated levels of reactive oxygen species (ROS) exacerbate IBD, making antioxidant enzyme therapy based on ROS level regulation an effective alternative. On the other hand, damage to the intestinal mucosa leads to loss of mucosal barrier function, resulting in IBD; therefore, ulcer repair is also a good treatment approach. Developing a formulation with mucosal protective effects and the ability to scavenge ROS is of great significance for the treatment of IBD. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing mucin-based thermosensitive hydrogels to solve the problems existing in the prior art.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] One of the technical solutions of this invention is a method for preparing a mucin-based thermosensitive hydrogel, comprising the following steps:

[0008] Prönnik F127 dibenzoaldehyde, glycidyl chitosan and mucin solution are mixed and then hollow Prussian blue nanozyme is added to form a mixed solution to obtain the mucin thermosensitive hydrogel HPB MPC-Gel.

[0009] The second technical solution of the present invention is a mucin thermosensitive hydrogel prepared by the preparation method.

[0010] The third technical solution of the present invention is the application of the mucin thermosensitive hydrogel in the preparation of a drug for treating inflammatory bowel disease.

[0011] The fourth technical solution of the present invention is a drug for treating inflammatory bowel disease, comprising the mucin thermosensitive hydrogel.

[0012] Based on the above technical solution, the present invention has the following technical effects:

[0013] (1) HPB was successfully prepared by a hydrothermal method. It was characterized using UV-Vis spectroscopy, Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, X-ray diffraction, and transmission electron microscopy. The particle size of HPB was measured to be between 180-210 nm using a particle size analyzer, and the potential was 7 mV. Transmission electron microscopy revealed that HPB consisted of regular cubes of uniform size.

[0014] (2) The formulation of HPB MPC-Gel was determined to be 15% PF127-CHO, 2.5% Gly-CS, and 3% Mucin using a single-factor method. It successfully gelled at 37℃. HPB MPC-Gel was characterized by its appearance, phase transition properties, internal structure, rheological properties, and adhesion. The results showed that the hydrogel appeared as a homogeneous blue solution, in a solution state at 25℃ and a gel state at 37℃, exhibiting good phase transition properties. The internal structure of the hydrogel was a three-dimensional network with a honeycomb appearance, which facilitated drug encapsulation and delayed drug release. Rheological analysis showed that at 25℃, G” > G’ was in a liquid state, while at 37℃, G’ > G” was in a solid state. Furthermore, swelling and in vitro degradation experiments showed that the hydrogel swelled without rapid dissolution and did not completely degrade within 24 hours, meeting the experimental expectations. Drug release results showed that only 81.14% of the drug was released within 24 hours, indicating incomplete release and suggesting that HPB MPC-Gel has a sustained-release effect. Injectability and adhesion experiments also demonstrated that HPB MPC-Gel has good injectability and adhesion, making it suitable for rectal administration. An IBD model was established to evaluate the treatment effects of each group. Compared with the model group, the HPB MPC-Gel group showed lower DAI scores, reduced body weight, inhibited colonic shortening, significantly decreased intestinal and spleen weight indices, reduced IL-1β, IL-6, TNF-α, and MPO levels, alleviated DSS-induced colitis-induced colonic pathological damage, increased mucus layer integrity, upregulated the expression of tight junction proteins Occludin and ZO-1, resisted DSS-induced intestinal mucosal barrier damage, improved the integrity of the intestinal mucosal mechanical barrier, and repaired the damaged intestinal mucosal barrier.

[0015] This invention prepares a mucin hydrogel formulation carrying hollow Prussian blue nanozymes. In vivo and in vitro evaluation results of HPB MPC-Gel show that the hydrogel has a certain effect on improving IBD symptoms and is expected to become a means of treating IBD. Attached Figure Description

[0016] Figure 1The image shows the appearance of Prussian blue nanoparticles. In this image, A represents a PB solution, and B represents an HPB solution.

[0017] Figure 2 Particle size (A) and potential (B) diagrams for PB and HPB.

[0018] Figure 3 The UV-vis spectra of PB and HPB are shown.

[0019] Figure 4 The FTIR spectra of PB and HPB are shown.

[0020] Figure 5 XPS spectra of PB and HPB.

[0021] Figure 6 The XRD spectra of PB and HPB are shown.

[0022] Figure 7 The synthesis process of MPC-Gel(A) and HPB MPC-Gel(B).

[0023] Figure 8 Scanning electron microscope images (Bar = 10 μm) of different magnifications of (A) MPC-Gel, (B) MPC-Gel, (C) HPB MPC-Gel and (D) HPB MPC-Gel.

[0024] Figure 9 FTIR plots for MPC-Gel and HPB MPC-Gel.

[0025] Figure 10 Figures showing the swelling (A) and in vitro degradation (B) of MPC-Gel and HPB MPC-Gel.

[0026] Figure 11 Rheological properties of MPC-Gel and HPB MPC-Gel.

[0027] Figure 12 Injectability diagrams for MPC-Gel (A) and HPB MPC-Gel (B).

[0028] Figure 13 The adhesion ability diagrams for HPB and HPB MPC-Gel are shown.

[0029] Figure 14 This is the standard curve for HPB.

[0030] Figure 15 The image shows the drug release curve for HPB MPC-Gel.

[0031] Figure 16 Different concentrations of HPB MPC-Gel extracts were used to detect cytotoxicity.

[0032] Figure 17 Colonic fluorescence images of mice at different time points after rectal administration of HPB@IR780 and HPB@IR780 MPC-Gel IBD.

[0033] Figure 18 The dominant bloody stool phenomenon was observed in mice in each group.

[0034] Figure 19 The DAI score (A) and body weight change rate (B) of mice in each group were recorded (n=3, ***P<0.001).

[0035] Figure 20 Photographs of the colon of mice in each group.

[0036] Figure 21 The colon length (A), intestinal weight index (B), and spleen weight index (C) of mice in each group were shown (n=3, *P<0.05).

[0037] Figure 22 The activity of IL-1β(A), IL-6(B) and TNF-α(C) in the colon tissue of mice in each group was measured (n=3, ***P<0.001).

[0038] Figure 23 MPO activity in the colon tissue of mice in each group was measured (n=3, ***P<0.001).

[0039] Figure 24 The histopathological features of the colon tissue of mice in each group are shown (scale bar is 100 μm).

[0040] Figure 25 The histopathological scores of the colon tissue of mice in each group are shown.

[0041] Figure 26 Results of Alcian Blue-glycogen staining (scale bar is 100 μm).

[0042] Figure 27 Immunohistochemistry for Occludin and ZO-1 (scale bar is 100 μm).

[0043] Figure 28 H&E staining of important organs in mice after different treatments (scale bar is 200 μm).

[0044] Figure 29 The levels of LDH(A), AST(B), BUN(C), ALT(D), UREA(E), and Cre(F) in the serum of mice in each group were measured (n=3).

[0045] Figure 30Photographs of hemolysis rate and red blood cell status in mice of each group, from left to right: Water, PBS, HPB, MPC-Gel and HPB MPC-Gel (n=3). Detailed Implementation

[0046] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field, and the reagents or raw materials used are all purchased from commercial channels or are publicly available unless otherwise specified.

[0047] This invention provides a method for preparing a mucin-based thermosensitive hydrogel, comprising the following steps:

[0048] Prönnik F127 dibenzoaldehyde, glycidyl chitosan and mucin solution are mixed and then hollow Prussian blue nanozyme is added to form a mixed solution to obtain the mucin thermosensitive hydrogel HPB MPC-Gel.

[0049] In some specific embodiments, the mass concentration of Pluronic F127 dibenzaldehyde in the mixed solution is 15%;

[0050] The mass concentration of glycidyl chitosan is 2.5%;

[0051] The mass concentration of mucin is 3%;

[0052] The concentration of hollow Prussian blue nanozyme was 1 mg / mL.

[0053] In some specific implementations, the hollow Prussian blue nanozyme is prepared by dissolving Prussian blue nanozyme and polyvinylpyrrolidone in hydrochloric acid solution, stirring at room temperature, transferring to a high-pressure reactor, and continuing the reaction in an oil bath. After the reaction is completed, the hollow Prussian blue nanozyme is obtained by centrifugation, washing, and freeze-drying.

[0054] In some specific implementations, the mass ratio of the Prussian blue nanozyme to polyvinylpyrrolidone is 1:5;

[0055] The stirring speed at room temperature is 50-200 rpm, and the time is 3.5 hours;

[0056] The conditions for continuing the reaction in the oil bath are: constant temperature reaction at 140℃ for 4 hours;

[0057] The centrifugation conditions were: 11000 rpm / min, 10 min.

[0058] In some specific implementation schemes, the Prussian blue nanozyme is prepared by dissolving potassium ferricyanide and polyvinylpyrrolidone in hydrochloric acid solution, stirring magnetically, and carrying out a high temperature and high pressure reaction. After the reaction is completed, the nanozyme is obtained by centrifugation, washing, and freeze-drying.

[0059] In some specific implementations, the mass ratio of potassium ferricyanide to polyvinylpyrrolidone is 22:500;

[0060] The magnetic stirring speed is 50-200 rpm, and the time is 0.5 h;

[0061] The conditions for the high-temperature and high-pressure reaction are: constant temperature reaction at 80℃ for 20 hours;

[0062] The centrifugation conditions were: 11000 rpm / min, 15 min.

[0063] This invention also provides a mucin thermosensitive hydrogel prepared by the aforementioned preparation method.

[0064] This invention also provides the application of the mucin thermosensitive hydrogel in the preparation of medicaments for treating inflammatory bowel disease.

[0065] This invention also provides a medicament for treating inflammatory bowel disease, comprising the mucin thermosensitive hydrogel.

[0066] Preparation method of HPB@IR780: Take 10 mg of IR780 and 100 mg of HPB, put them into dimethyl sulfoxide, stir magnetically at room temperature in the dark for 24 h, then centrifuge (11000 rpm / min, 10 min), and wash the precipitate three times with ultrapure water to obtain IR780-labeled HPB (HPB@IR780).

[0067] Preparation method of HPB@IR780 MPC-Gel: Take 6 mL of 25% PF127-CHO, add 1.7 mL of 15% Gly-CS and 2 mL of 10% Mucin solution, add an appropriate amount of 10 mg HPB@IR780, add double-distilled water to 10 mL, mix well, and obtain IR780-labeled Prussian blue nanozyme mucin thermosensitive hydrogel, denoted as HPB@IR780 MPC-Gel.

[0068] Example 1

[0069] 1.1 Synthesis of Prussian Blue Nanozyme (PB)

[0070] 660 mg of potassium ferricyanide and 15 g of polyvinylpyrrolidone (PVP-K30) were dissolved in 200 mL of hydrochloric acid solution (0.01 M). After stirring magnetically at 200 rpm for 0.5 h, the solution was transferred to a hydrothermal reactor and reacted at 80 °C for 20 h. After centrifugation (11000 rpm / min, 15 min), the resulting precipitate was washed three times with ultrapure water and then freeze-dried to obtain Prussian blue nanozyme (PB).

[0071] 1.2 Synthesis of Hollow Prussian Blue Nanozymes (HPB)

[0072] 200 mg PB and 1 g PVP were dissolved in 200 mL of hydrochloric acid solution (1 M), stirred at 200 rpm at room temperature for 3.5 h, transferred to a high-pressure reactor, reacted at 140 °C for 4 h, and then centrifuged (11000 rpm / min, 10 min). The resulting precipitate was washed three times with ultrapure water and freeze-dried to obtain hollow mesoporous Prussian blue nanozyme (HPB).

[0073] The appearance of the prepared Prussian blue nanoparticles is as follows: Figure 1 As shown, A is PB solution and B is HPB solution.

[0074] Characterization of 2 nanoparticles

[0075] The particle size and potential of PB and HPB were determined using a NanoZS90 laser particle size analyzer. The morphology of PB and HPB was observed and photographed using a JEM-2000EX transmission electron microscope. The ultraviolet-visible spectra of PB and HPB solutions were determined using a UV-vis spectrophotometer, with a scanning range of 400–1000 nm. The Fourier transform infrared spectra of PB and HPB were determined using a Fourier transform infrared spectrophotometer (FTIR). Potassium bromide was ground and dried at 120 °C for 12 h using the potassium bromide pelleting method. The sample was mixed with potassium bromide powder, ground, and then pelleted. The wavenumber range was 4000–500 cm⁻¹. -1 The resolution is 4cm. -1 The obtained PB and HPB were characterized by X-ray diffraction (XRD). Appropriate amounts of lyophilized PB and HPB were ground into a powder without any grainy texture using a mortar and pestle. The sample was then added to a sample well, flattened, and analyzed using the instrument.

[0076] The crystal structure was characterized using X-ray photoelectron spectrometer (XPS). Appropriate amounts of lyophilized PB and HPB were ground into a powder with no grainy texture using a mortar and pestle. The sample was added to the sample cell, flattened, and then analyzed using XPS. The crystal structure was measured using X-ray photoelectron spectrometer (XPS).

[0077] As shown in Table 1 and Figure 2 As shown, the nanoparticles prepared in this experiment exhibit a narrow single-size distribution, with diameters ranging from 180 to 210 nm and a PDI < 0.3. The low PDI value indicates uniform particle size and good dispersibility. The average particle size of PB is 186.13 ± 0.23 nm, while that of HPB is 208.87 ± 0.60 nm. The larger particle size of HPB compared to PB is due to its hollow mesoporous structure, which creates cavities and pores within the particles, thus increasing the overall particle size. Nanoparticles carry surface charges, leading to electrostatic interactions that affect their stability and bioactivity. The absolute value of the Zeta potential reflects the magnitude of the electrostatic repulsion between nanoparticles; a higher value indicates a stronger electrostatic repulsion and better physical stability. In this experiment, both PB and HPB exhibit electronegativity. The increased Zeta potential value of HPB indicates improved physical stability.

[0078] Table 1. Particle size, PDI, and Zeta potential of nanoparticles

[0079]

[0080] The TEM images show that PB and HPB are cubic in shape and uniformly arranged. The average particle size of HPB is larger than that of PB, which is consistent with the trend of the particle size results.

[0081] The UV-vis absorption spectra of PB and HPB solutions are as follows: Figure 3 As shown in the results, PB and HPB have a relatively broad absorption peak in the range of 600 nm to 1000 nm. The maximum absorption peaks of PB and HPB appear at 710 nm and 730 nm, respectively, which fully reflects the absorption characteristics of Fe. 2+ and Fe 3+ The existence of.

[0082] FTIR results as follows Figure 4 As shown, PB and HPB at 2060cm -1 The presence of absorption peaks is caused by the stretching vibrations of the Fe-CN-Fe bonds.

[0083] The chemical composition and elemental valence states of PB and HPB were analyzed using XPS. Figure 5The XPS full spectrum shows that both PB and HPB contain characteristic peaks of C1s, N1s, Ols, and Fe2p.

[0084] The crystal structures of PB and HPB were analyzed using XRD, such as... Figure 6 As shown, diffraction peaks at 2θ angles of 17.43°, 24.74°, 35.25°, and 39.58° are revealed; these can be attributed to different diffraction planes of 200, 220, 400, and 420, and PB and HPB are consistent.

[0085] Example 2

[0086] Preparation, characterization and efficacy of Prussian blue nanozyme mucin thermosensitive hydrogel

[0087] 1. Preparation of mucin-based thermosensitive hydrogel (MPC-Gel)

[0088] Different volumes of Pluronic F127 dibenzoaldehyde (PF127-CHO), glycidyl chitosan (Gly-CS), and mucin solutions were mixed and heated in a water bath at 37°C to obtain Mucin / PF127-CHO / Gly-CS Hydrogel (MPC-Gel).

[0089] To determine the gel-forming concentration required for the formation of a stable hydrogel, the selection of concentrations of various substances in the hydrogel preparation system was analyzed, and the gel-forming effect of the gel system was compared through a vial inversion experiment.

[0090] (1) Selection of Gly-CS concentration (unless otherwise specified, all concentrations mentioned in this invention are mass concentrations)

[0091] To determine the concentration of Gly-CS, different concentrations of glycidyl chitosan (Gly-CS) were prepared following the general procedures for hydrogel preparation, and gelation was observed through a vial inversion experiment. As shown in Table 2, the concentration of PF127-CHO was 15%, the concentration of mucin was 3%, and the concentrations of Gly-CS were set to 0%, 0.5%, 1%, 1.5%, 2%, and 2.5%, respectively. The vials were placed in water at room temperature and 37°C, and the vial inversion experiment was conducted to observe the gelation process.

[0092] Table 2. Gel formation at different concentrations of Gly-CS

[0093]

[0094] With the concentration of PF127-CHO fixed at 15% and the concentration of Mucin constant at 2%, the gelation behavior of MPC-Gel at 25℃ and 37℃ was observed using different concentrations of Gly-CS. The results showed that when the concentration of Gly-CS was below 2.5%, it remained liquid at both 25℃ and 37℃. When the concentration of Gly-CS was 2.5%, it was a flowing liquid at 25℃ but transformed into a gel at 37℃. Therefore, 2.5% was chosen as the optimal concentration for Gly-CS.

[0095] (2) Selection of PF127-CHO concentration

[0096] To determine the concentration of PF127-CHO, different concentrations of PF127-CHO were prepared according to the general procedures for hydrogel preparation, and the gelation was observed through a vial inversion experiment. As shown in Table 3, the concentration of Gly-CS was 2.5%, the concentration of mucin was 3%, and the concentrations of PF127-CHO were set to 0%, 5%, 7.5%, 10%, 12.5%, and 15%, respectively. The vials were placed in water at room temperature and 37°C, and the vial inversion experiment was conducted to observe the gelation.

[0097] Table 3. Gel formation of PF127-CHO at different concentrations

[0098]

[0099] With the concentration of Mucin fixed at 2% and the concentration of Gly-CS at 2.5%, the gelation behavior of HPB MPC-Gel at 25℃ and 37℃ was observed using different concentrations of PF127-CHO. The results showed that when the concentration of PF127-CHO was below 10%, it remained liquid at both 25℃ and 37℃. When the concentration of PF127-CHO was 10%, it was a flowing liquid at 25℃ and gelled at 37℃. The gelation time gradually shortened with increasing concentration. To ensure successful conversion to a gel in vivo, 15% was chosen as the concentration of PF127-CHO for subsequent experiments.

[0100] (3) Selection of mucin concentration

[0101] To determine the concentration of mucin, different concentrations of mucin were prepared following the general procedures for hydrogel preparation. The gelation effect and stability were observed through a vial inversion experiment. As shown in Table 4, the concentrations of Gly-CS were 2.5% and PF127-CHO were 15%. Mucin concentrations of 0%, 1%, 2%, 3%, 4%, and 5% were set, and vials were placed in water at room temperature and 37°C for the vial inversion experiment to observe their gelation behavior.

[0102] Table 4. Mucin at different concentrations

[0103]

[0104] With the concentration of Gly-CS fixed at 2.5% and the concentration of PF127-CHO constant at 15%, the gelation behavior of MPC-Gel at 25℃ and 37℃ was observed using different concentrations of Mucin. The results showed that when the concentration of Mucin was above 4%, it remained liquid at both 25℃ and 37℃. When the concentration of Mucin was below 4%, it was a flowing liquid at 25℃ but gelled at 37℃. The gelation time and gelation temperature gradually increased with increasing concentration. To ensure successful gelation in vivo, 3% Mucin was chosen as the concentration for subsequent experiments.

[0105] 2. Preparation of Prussian blue nanozyme mucin thermosensitive hydrogel

[0106] Take 6 mL of 25% PF127-CHO, add 1.7 mL of 15% Gly-CS and 2 mL of 10% Mucin solution, add 10 mg of HPB, add double-distilled water to 10 mL, mix well, and you will get a Prussian blue nanozyme mucin thermosensitive hydrogel, denoted as HPBMucin / PF127-CHO / Gly-CS Hydrogel (HPB MPC-Gel). In the Prussian blue nanozyme mucin thermosensitive hydrogel mixture, the mass concentration of Prussian blue nanozyme F127 dibenzaldehyde is 15%; the mass concentration of glycidyl chitosan is 2.5%; the mass concentration of mucin is 3%; and the concentration of hollow Prussian blue nanozyme is 1 mg / mL.

[0107] The synthesis process of hydrogels is as follows Figure 7 As shown, after MPC-Gel and HPB MPC-Gel were synthesized into pregels, they were converted into homogeneous gels after being bathed in a water bath at 37°C for 5 minutes.

[0108] Characterization of 3HPB MPC-Gel

[0109] 3.1 Microscopic morphology detection: Take appropriate amounts of freeze-dried HPB MPC-Gel and MPC-Gel samples respectively, and observe the morphology of the gels by scanning electron microscopy (SEM).

[0110] The cross-sectional morphology of MPC-Gel and HPB MPC-Gel was observed using SEM. The results are as follows: Figure 8 As shown, both MPC-Gel and HPB MPC-Gel exhibit a porous three-dimensional structure with a uniform and tightly interconnected network. The added HPB is distributed in the honeycomb-like MPC-Gel. This network structure gives the hydrogel a large specific surface area, which is more conducive to drug diffusion.

[0111] 3.2 FT-IR Detection: Appropriate amounts of Gly-CS, PF127-CHO, Mucin, HPB MPC-Gel, and MPC-Gel powders were placed in a vacuum drying oven at 60℃ for 48 hours. The dried sample was then ground into a fine powder, and the infrared spectrum was recorded using the KBr pellet method. The detection range was 4000-500 cm⁻¹. -1 .

[0112] FTIR values ​​for HPB, Mucin, PF127-CHO, Gly-CS, MPC-Gel, and HPB MPC-Gel are as follows: Figure 9 As shown: 1717 cm⁻¹ in the PF127-CHO spectrum -1 and 2737cm -1 The value at 3290 cm⁻¹ represents the stretching vibration of the C=O and CH bonds on the aldehyde group. (Mucin spectrum, 3290 cm⁻¹) -1 The peak represents the amino-symmetric stretching vibration of the amide bond, at 1632 cm⁻¹. -1 The region represents the stretching vibration band of the carbonyl group (amide absorption band I). (3700-3000 cm⁻¹ in the Gly-CS spectrum) -1 The peak at 1644 cm⁻¹ represents the stretching vibrations of the OH group on the hydroxyl group and the NH group on the amino group. (HPB spectrum, 1644 cm⁻¹) -1 The peak at 1700 cm⁻¹ corresponds to the stretching vibration of the carbonyl group (C=O). The MPC-Gel spectrum at 1700 cm⁻¹... -1 2700cm -1 3000-3700cm -1 The presence of absorption peaks similar to those of PF127-CHO, Mucin, and Gly-CS in the vicinity indicates successful synthesis of MPC-Gel. The HPB MPC-Gel spectrum shows a peak similar to HPB around 1600 nm, indicating successful synthesis of HPB MPC-Gel.

[0113] 3.3 Swelling and in vitro degradation behavior:

[0114] The swelling properties of hydrogels and nanocomposite hydrogels were evaluated using a gravimetric method. HPB MPC-Gel and MPC-Gel were prepared. After recording the dry weight (W0) of the samples, the samples were placed in 20 mL centrifuge tubes containing 10 mL of PBS solution (pH = 7.4) and incubated with gentle shaking (37 °C, 55 rpm) to reach swelling equilibrium. The samples were weighed at 0.5 h, 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, and 7 h (W0). s The swelling percentage (SP) is calculated using the following formula:

[0115]

[0116] In the formula: Ws is the mass of the sample after swelling, and W0 is the mass of the sample before swelling.

[0117] The in vitro degradation behavior of hydrogels and nanocomposite hydrogels was evaluated using a gravimetric method. Two mL of HPB MPC-Gel and MPC-Gel were taken and stabilized in a 37°C water bath for 30 min. The sample weight was recorded as W0. An equal volume of PBS was then added to the surface of the hydrogel. The gel was then placed in a constant-temperature shaking environment (37°C, 55 rpm). The remaining mass (Wt) was recorded at 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, 48 h, and 72 h. The weight remaining (WR) was calculated using the following formula:

[0118]

[0119] In the formula: Wt is the mass of the sample after degradation, and W0 is the initial mass of the sample.

[0120] like Figure 10 As shown in Figure A, both MPC-Gel and HPB MPC-Gel exhibited water absorption in PBS solution at pH 7.4, rapidly swelling within 3 hours. The equilibrium swelling rates of MPC-Gel and HPB MPC-Gel reached 337.18% and 398.87%, respectively, reaching swelling equilibrium after 6 hours, demonstrating good water absorption capacity. The swelling rate of HPB MPC-Gel was lower than that of MPC-Gel. Therefore, the degradation performance of MPC-Gel and HPB MPC-Gel was evaluated. Figure 10 As shown in Figure B, when MPC-Gel and HPB MPC-Gel were in PBS solution, their weight retention rates at 48 h were 53.33% and 39.42%, respectively, indicating rapid degradation. At 72 h, their weight retention rates were 32.85% and 29.57%, respectively, showing a slower degradation rate. This ensured that the gel maintained sufficient colloidal mass in the first few hours, allowing for the full release of HPB while allowing other intestinal contents to pass through smoothly until a new gel was injected.

[0121] 3.4 Rheological Performance Testing: To investigate the mechanical properties of the hydrogels, a rotational rheometer was used for testing. HPB MPC-Gel and MPC-Gel were prepared and placed on the sample stage after the nanocomposite hydrogels were formed. The test rotor was a 50mm parallel plate, and the measurement gap was set to 1mm. The angular frequency variation range was set to 0.1-200 rad / s at 25℃ and 37℃, respectively, to obtain the corresponding curves of storage modulus (G') and loss modulus (G'').

[0122] The MPC-Gel and HPB MPC-Gel were scanned and tested using a rheometer at 25°C and 37°C. Figure 11As shown, at 25℃, the values ​​of G' and G” are both very small, with G' being lower than G”, indicating a sol state. At 37℃, G” > G', with G' value approximately 71480 Pa and G” value approximately 10930 Pa, a difference of nearly 60000 Pa. This indicates that the hydrogel transforms into a high-strength and stable gel state, and this temperature also meets the requirements for intraperitoneal injection.

[0123] 3.5 Injectability test: The freshly prepared hydrogel was drawn into a 1mL syringe and injected into a 5mL sample bottle containing 37℃ ultrapure water. The results were observed and photographed.

[0124] The injectability of MPC-Gel and HPB MPC-Gel was investigated by loading them into 1 mL syringes and observing their state in water at 37°C after injection. Figure 12 As shown, both MPC-Gel and HPB MPC-Gel are filamentous and have good flowability in water at 37°C, indicating that they can be used for rectal administration.

[0125] 3.6 Adhesion evaluation: The composite hydrogel HPB MPC-Gel was placed directly on the colonic segment between the cecum and rectum of SD rats for 10 min. Then, 30 mL of PBS solution (pH=7.4) was injected within 30 s to continuously subject the nanocomposite hydrogel to the impact of water flow in order to determine whether HPB MPC-Gel would migrate.

[0126] The strong adhesion of hydrogels can improve the therapeutic effect of drugs and increase drug retention. To further explore the adhesion of HPB MPC-Gel to biological tissues, rat colon tissue was used as a carrier in the study. The results are as follows: Figure 13 The diagram illustrates the adhesion of HPB and HPB MPC-Gel in the intestine. After continuously injecting 30 mL of PBS solution over 30 seconds to impact the gel, HPB was easily washed away, while HPB-hydrogel remained stationary. This indicates that despite continuous impact, HPB MPC-Gel can remain in the intestine for a certain period, increasing drug retention time.

[0127] 4. Evaluation of in vitro drug release performance

[0128] 4.1 Establishment of analytical method for HPB content determination

[0129] The content of HPB hydrogel was determined by ultraviolet-visible spectrophotometry. HPB standard lines were established by diluting HPB with PBS to different concentration gradients (15.625 μg / mL, 31.25 μg / mL, 65.25 μg / mL, 125 μg / mL, 250 μg / mL, 500 μg / mL, and 1 mg / mL), and measuring the absorbance of the solutions at 720 nm. A standard curve was plotted with absorbance on the ordinate and drug concentration on the abscissa.

[0130] Choosing 720 nm as the measurement wavelength, a linear relationship was established between HPB absorbance (A) and concentration (C), and a standard curve was plotted. Figure 14 The linear regression equation was obtained as A = 0.0032C - 0.0041, R0. 2 =0.9997. The absorbance of HPB showed a good linear relationship with concentration, with a linear range of 15.625 μg / mL to 1 mg / mL.

[0131] 4.2 In vitro release behavior study: 2 mL of HPB MPC-Gel was completely gelled at 37°C, and 20 mL of PBS buffer was added. The mixture was placed in a constant-temperature shaking environment at 37°C and 55 rpm. 2 mL of the supernatant was collected at 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, 48 h, and 72 h for content determination, and an equal volume of PBS buffer was added to maintain a constant volume of the release system. The experiment was repeated three times in parallel. The release solution was injected and analyzed according to the conditions described in "2.4.1". The cumulative percentage of drug release was calculated using the following formula:

[0132]

[0133] In the formula: Cn is the concentration of HPB in the released liquid at the nth sampling, V0 is the volume of the released medium, and V is the volume of the released liquid taken each time.

[0134] SEM images and in vitro degradation behavior of HPB-hydrogel indicate that it possesses a suitable pore size and also exhibits certain in vitro degradation capabilities. This experiment further investigated the drug release process of HPB MPC-Gel in PBS solution at pH 7.4. Figure 15 As shown, HPB release in HPB MPC-Gel increased over time. Within 12 hours, HPB exhibited a burst release behavior, with a cumulative drug release percentage reaching 81.14%. After 24 hours, the release gradually slowed down, and at 72 hours, the drug release percentage was 94.27%. The drug release was continuous throughout the process, indicating that hydrogel is an ideal drug controlled-release carrier.

[0135] 5. Cell compatibility assessment

[0136] The cytotoxicity of HPB MPC-Gel was analyzed using the CCK-8 assay. RAW 264.7 cells were cultured in vitro with HPB MPC-Gel extract to evaluate the cytotoxicity. The HPB MPC-Gel gel was irradiated with UV for 2 h to kill bacteria, and then extracted with 50 mL of DMEM complete medium at 37 °C for 24 h. After extraction, the extract was filtered through a 0.22 μm microporous membrane and stored at 4 °C. Before use, the extract was diluted with culture medium to a concentration range of 0.34-5.4 mg / mL. Different concentrations of HPB MPC-Gel extract were added to the experimental groups during medium change, while the control group received an equal volume of culture medium. After culturing for 24 or 48 hours, the culture medium and extract were removed, and 100 μL of DMEM complete medium containing 10% CCK-8 was added to each well. The cells were incubated at 37°C with 5% CO2 for 30 minutes, and the absorbance at 450 nm was measured using a microplate reader. Cell viability was calculated.

[0137] The cytotoxicity of HPB MPC-Gel against RAW 264.7 cells was detected using the CCK-8 assay. The results are as follows: Figure 16 As shown, after incubating RAW 264.7 cells with different concentrations of HPB MPC-Gel extract for 24 h and 48 h, the cell viability was above 90%, indicating that HPB MPC-Gel has good biocompatibility with cells.

[0138] In vivo pharmacodynamic evaluation of 6HPB MPC-Gel

[0139] 6.1 Establishment of the IBD mouse model: Eight-week-old male C57BL / 6J mice, weighing 20-22g, were used. The ambient temperature was 20-22℃, and the relative humidity was approximately 55%. The light and dark periods alternated for 12 hours each. The animal experiments complied with international ethical standards. A certain mass of DSS was weighed and mixed with double-distilled water to prepare a 3% DSS solution. Mice had free access to water, and the DSS solution was changed fresh daily. The modeling period was 7 days to establish the mouse enteritis model. During this period, the mouse weight, stool characteristics, and fecal blood were recorded daily. The rate of change in mouse weight was calculated. The Disease Activity Index (DAI) score of the IBD model was calculated according to Table 5, and the severity of the model lesions was assessed.

[0140]

[0141] DAI = Weight Loss Score + Stool Shape Score + Stool Bleeding Score;

[0142] Table 5 DAI Scoring Criteria

[0143]

[0144] 6.2 Retention Effect in the Colon: IR780 iodide was used as a fluorescent probe in the experiment. Mice with colitis were randomly divided into two groups: the experimental group (HPB@IR780 group) and the HPB@IR780 MPC-Gel group. HPB@IR780 and HPB@IR780 MPC-Gel (HPB@IR780: 10 mg / mL) were administered rectally. Mice were euthanized at 0, 2, 6, 10, 12, 24, 48, 72, and 96 h after rectal administration, and the colon was imaged to assess the distribution of HPB@IR780 MPC-Gel in the colon.

[0145] To determine whether HPB MPC-Gel could prolong HPB retention in the colonic lumen, HPB@IR780 and HPB@IR780 MPC-Gel were prepared, and retention after rectal perfusion was evaluated using in vivo imaging. Figure 17 As shown, HPB@IR780 and HPB@IR780 MPC-Gel exhibited strong fluorescence in the colon, with the fluorescence intensity decreasing over time. However, compared to HPB@IR780 MPC-Gel, the signal of HPB@IR780 decreased rapidly. Only a weak fluorescence signal was observed in HPB@IR780 12 hours after rectal injection, and no fluorescence signal was detected at 24 hours. In contrast, HPB@IR780 MPC-Gel showed a strong fluorescence signal 12 hours after rectal instillation. Even at 24 hours, HPB@IR780 MPC-Gel still exhibited a fluorescence signal. HPB@IR780 MPC-Gel can prolong the retention time of HPB, thus better exerting its anti-inflammatory effect.

[0146] 6.3 In vivo efficacy experiment: Twenty-five healthy SPF-grade male C57BL / 6J mice were randomly divided into 5 groups (n=5 per group): Control group, 3% DSS model group, HPB group, MPC-Gel group, and HPB MPC-Gel group. Administered rectally at a volume of 200 μL every two days for 8 days, with treatment 24 hours after the last administration. Day 0 was designated as the first day of administration. Mice in each group received HPB, MPC-Gel, and HPB MPC-Gel rectally on days 0, 2, 4, 6, and 8, respectively. The HPB dosage was 10 mg / kg. The normal control group and DSS model group received the same volume of physiological saline daily. Mouse weight, stool characteristics, and fecal blood were observed and recorded daily. On the eighth day of the experiment, mice were euthanized by cervical dislocation. The entire intestinal segment was removed from the cecum to the anus. The length of the colon was measured and photographed. After weighing the colonic segment, about 0.5 cm of tissue was cut from 1 cm above the anus and fixed in 4% paraformaldehyde for H&E staining. The remaining colonic segment was collected and quickly stored in an ultra-low temperature freezer at -80°C.

[0147] On the third day of the experiment, mice in the DSS group developed loose stools. On the fifth day, the mice in the DSS group experienced significant weight loss and bloody stools were observed. Figure 18 This indicates that a DSS-induced IBD model has been successfully established. Figure 19 As shown in Figure B, compared with the DSS group, the HPB group, MPC-Gel group, and HPB MPC-Gel group all showed a slower rate of weight loss, indicating a certain therapeutic effect. The DAI score was calculated based on the mice's weight loss, changes in stool characteristics, and fecal bleeding. The results are as follows: Figure 19 As shown in Figure A, the DSS group score increased significantly over time, reaching 11.8 on day 8. Although the DAI scores of mice in the HPB group, MPC-Gel group, and HPB MPC-Gel group also increased to some extent, the upward trend was more moderate. Among them, the HPB MPC-Gel group had the lowest score of 5.8, indicating that HPB MPC-Gel has a certain therapeutic effect on colitis.

[0148] 6.4 Determination of colon length, intestinal weight index, and spleen weight index in mice

[0149] The intestinal weight index and spleen index of mice were calculated using the following formulas:

[0150]

[0151] like Figure 20 and 21Figure A shows images of the colon appearance and colon length statistics for each group of mice. In the normal control group, the colon surface was smooth, without edema or congestion, and the feces were formed; the colon length reached 6.44 cm. In the DSS group, the colon length was significantly shortened, with no formed feces, obvious congestion, and an intestinal length of only 3.02 cm. In the HPB, MPC-Gel, and HPB MPC-Gel groups, the degree of colon shrinkage was less severe; bloody contents were visible in the intestines, the feces were relatively formed, and some cecal segments showed loose stools and slight edema, but the degree was milder than in the DSS group. The colon length in the HPB MPC-Gel group was 6.06 cm, a more significant increase compared to the Blank-hydrogel and HPB-hydrogel groups, indicating that the HPB-hydrogel group had a better therapeutic effect on colitis. Figure 21 As shown in Figures B and C, the intestinal weight index and spleen weight index of the DSS group were 34.42 and 5.12, respectively, both significantly higher than those of the normal control group. After treatment, the intestinal weight index and spleen weight index showed varying degrees of decrease, with the HPB MPC-Gel group exhibiting the lowest values ​​(25.41 and 3.25, respectively). This indicates that the HPB MPC-Gel group had the best therapeutic effect on colitis. In conclusion, HPB MPC-Gel can inhibit colonic shortening, reduce intestinal and spleen weight indices, and effectively improve DSS-induced IBD symptoms.

[0152] 6.5 Colon Tissue Cytokine Detection: Weigh colon tissue samples and add 0.01M pre-chilled PBS at a weight-to-volume ratio of 1:9. Add three 3mm grinding beads, grind using a low-temperature homogenizer, centrifuge at 5000g for 10 min, and collect the supernatant. Subsequent experimental procedures were strictly performed according to the instructions for TNF-α, IL-6, and IL-1β.

[0153] The levels of inflammatory factors were measured, and the results were as follows: Figure 22 As shown, the IL-1β level in the blank control group was 49.15 pg / mL, but it increased to 308.85 pg / mL in the DSS model group. After different drug treatments, the levels in the HPN-NPs group, MPC-Gel group, and HPB MPC-Gel group were 128.20, 138.40, and 78.60 pg / mL, respectively, all showing varying degrees of decrease. Similarly, colitis increases the levels of two inflammatory factors, IL-6 and TNF-α, which decreased to varying degrees after drug treatment. Notably, among the three inflammatory factors, the HPB MPC-Gel group had significantly lower levels than the DSS model group and was closest to the normal control group, indicating that HPB MPC-Gel has the best ability to inhibit the expression of inflammatory factors such as IL-1β, IL-6, and TNF-α.

[0154] 6.6 Detection of myeloperoxidase (MPO) activity in colon tissue: Mouse colon samples were taken, and the MPO activity in mouse colon tissue was detected according to the instructions of the corresponding MPO kit.

[0155] The results are as follows Figure 23 As shown, the MPO values ​​of the normal control group, DSS model group, HPN-NPs group, MPC-Gel group, and HPB MPC-Gel group were 0.13, 0.52, 0.23, 0.24, and 0.15 U / g, respectively. It can be seen that the MPO values ​​decreased after drug treatment. Among them, the value of the HPB MPC-Gel group was closest to that of the control group, indicating that it had basically recovered to the normal value. This shows that HPB MPC-Gel can effectively reduce the MPO activity in the mouse colon.

[0156] 6.7 Colonic histopathological experiment: The colonic tissue was observed under a fluorescence upright microscope to see whether there were pathological changes. Referring to Table 6, the morphology of the mouse intestinal tissue was observed and histopathological scores were made. The scores consisted of the severity of inflammation, the degree of lesion, the degree of crypt destruction, and the extent of lesion.

[0157] Table 6. Pathological and Histological Scores

[0158]

[0159] To observe intestinal damage, the colon was stained with H&E and the histopathological condition of the colon tissue of mice in each group was observed. Results are as follows: Figure 24 As shown, the colonic tissue structure of normal control group mice was intact, with defects in mucosal structures, rich in goblet cells, and without edema or inflammatory cell infiltration. There were no obvious ulcers or inflammation. In the DSS model group, the integrity of the colonic mucosa was disrupted, crypts disappeared, glands decreased, and a large number of inflammatory cells diffusely infiltrated. Compared with the DSS group, the colonic tissue integrity of the HPB and MPC-Gel groups was better, with reduced inflammatory cell infiltration, indicating that the HPB and MPC-Gel groups could alleviate colitis. Comparison of pathological sections from each group revealed that the internal colonic structure of the HPB MPC-Gel group was more substantial, denser, and more intact than that of the HPB and MPC-Gel groups. Its pathological histological score ( Figure 25 The levels were significantly lower in the HPB-hydrogel group compared to the DSS group. These results indicate that HPB-hydrogel has a better therapeutic effect on colitis in mice.

[0160] 6.8 Alcian blue-glycogen staining for mucin expression.

[0161] The mucus layer is the colon's first line of defense against harmful substances and microorganisms. This mucus, produced by goblet cells, prevents microbial invasion, separates parasites from intestinal epithelial cells, protects the integrity of the intestinal barrier, and plays a crucial role in maintaining intestinal homeostasis. The colonic mucus layer is primarily composed of mucin MUC2, a glycosylated protein produced by intestinal goblet cells. Therefore, analyzing the distribution of mucin MUC2 using Alixin blue staining, detecting the number of goblet cells, and observing the number of goblet cells in the intestinal mucosa and mucin secretion can further assess the degree of intestinal barrier damage. Figure 26 The results of Alixin Blue-glycogen staining for each group showed that normal mice had abundant, neatly arranged, and tightly packed goblet cells in their colons, with the goblet cells surrounded by a mucus layer. Compared with the control group, the DSS model group mice showed almost no goblet cells in their colons, disrupted crypt structures, and significant mucus layer damage. The HPB and MPC-Gel groups showed an increase in goblet cells and a relatively intact mucus layer. The HPB MPC-Gel group showed a significant increase in the number of goblet cells, with a mucus layer similar to the control group. This indicates that HPB MPC-Gel can provide good protection for intestinal epithelial cells, increase the number of goblet cells, secrete more mucin to improve the intestinal mucus layer, and alleviate IBD.

[0162] 6.9 Immunohistochemical experiments.

[0163] The intestinal mucosal barrier comprises mechanical, chemical, immune, and biological barriers, forming a highly selective dynamic barrier that prevents the invasion of pathogenic antigens. Cell junctions are also an important part of the intestinal mechanical barrier, with tight junction proteins being key components. These proteins connect epithelial cells, regulate epithelial polarity and the bacterial transport of solutes and fluids in the intercellular space, and prevent microbial invasion via cellular bypass pathways. Abnormal expression of these proteins increases intestinal epithelial permeability, leading to the invasion of foreign antigens and causing mucosal damage. Therefore, immunohistochemical staining of the tight junction proteins Occludin and ZO-1 in the colon tissue of mice in each group was used to assess the integrity of the mucosal barrier. Results are as follows: Figure 27 As shown, Occludin and ZO-1 were almost not expressed in the DSS model group, reflecting impaired intestinal barrier function and disruption of intercellular connections, leading to increased intestinal permeability and thus promoting inflammation and aggravating mucosal damage. Compared with the DSS model group, the HPB group, MPC-Gel group, and HPB MPC-Gel group all upregulated the expression of Occludin and ZO-1, with the HPB MPC-Gel group showing significant Occludin and ZO-1 expression. This indicates that HPB-hydrogel can regulate the epithelial barrier, improve the integrity of the intestinal mechanical barrier, repair the damaged intestinal mucosal barrier, and has a certain promoting effect on the improvement of colitis.

[0164] 6.10 Hydrogel Biosafety Experiment:

[0165] The heart, liver, spleen, lungs, and kidneys of mice were fixed with 4% paraformaldehyde fixative and observed under a fluorescence upright microscope to see if any pathological changes occurred.

[0166] Plasma samples were collected from each mouse, and the levels of AST, ALT, LDH, BUN, Cre, and UREA in the mouse plasma were detected. The tests were performed according to the instructions of the corresponding kits.

[0167] Hemolysis in each group of samples was assessed using mouse erythrocytes. 1 mL of blood was collected from the mouse eyeball using a sodium citrate anticoagulant tube. The blood was centrifuged at 5000 rpm for 15 min at 4°C, and the supernatant was discarded. 0.6 mL of H2O, PBS, HPB, MPC-Gel, and HPB MPC-Gel were added to 2% (0.6 mL) of erythrocytes. The mixture was incubated at 37°C for 30 min, and the supernatant was collected after centrifugation. The absorbance was measured at 545 nm using a microplate reader, and the hemolysis rate was calculated.

[0168]

[0169] To investigate the biosafety of HPB, MPC-Gel, and HPB MPC-Gel, heart, liver, spleen, lung, and kidney tissues from mice were first sectioned and subjected to H&E staining. The results are as follows: Figure 28 As shown, the organ and tissue structures in each group remained intact, with no abnormalities such as inflammation, necrosis, congestion, or hemorrhage observed. This indicates that HPB, MPC-Gel, and HPB MPC-Gel have good tissue safety and no toxic side effects.

[0170] To more comprehensively evaluate biosafety, blood samples were collected from each group of mice at the end of the experiment, and the levels of biochemical indicators such as ALT, AST, LDH, BUN, UREA, and Cre were measured. The blood biochemical analysis results are as follows: Figure 29 As shown, the ALT results for each group were 44.37, 43.07, 41.87, 40.90, and 41.30 U / L; the AST results were 153.73, 161.07, 157.50, 156.10, and 155.13 U / L; the LDH results were 328.00, 267.33, 409.00, 348.67, and 320.33 g / mL; the BUN results were 6.64, 6.66, 6.40, 6.72, and 5.98 mM / L; and the Cre results were 21.10, 21.47, 21.00, 21.60, and 20.63 μM / L. There were no statistically significant differences among these groups. Furthermore, the hemolysis rate under physiological conditions was further investigated using a hemolysis test. Figure 30As shown, blood samples incubated with deionized water turned into a red, transparent solution, indicating that red blood cells ruptured and dissolved, resulting in hemolysis. However, blood samples incubated with HPB, MPC-Gel, and HPB-MPC-Gel did not exhibit visible hemolysis, and the relative hemolysis rate of each group was less than 1%, indicating that HPB, MPC-Gel, and HPB-hydrogel all have good blood compatibility.

[0171] In summary, this invention prepared a mucin hydrogel formulation carrying hollow Prussian blue nanozymes. In vivo and in vitro evaluation results of HPBMPC-Gel showed that the hydrogel had a certain effect on improving IBD symptoms and is expected to become a means of treating IBD.

[0172] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A method for preparing a mucin temperature-sensitive hydrogel, characterized in that, The method comprises the following steps: The Pluronic F127 benzaldehyde, glycidyl chitosan and mucin solution are mixed, the hollow Prussian blue nanoscale enzyme is added to form a mixed solution, and a mucin temperature-sensitive hydrogel HPB MPC-Gel is obtained; In the mixed solution, the mass concentration of Pluronic F127 benzaldehyde is 15%; The mass concentration of glycidyl chitosan is 2.5%; The mass concentration of mucin is 3%; The concentration of the hollow Prussian blue nanoscale enzyme is 1 mg / mL.

2. The production method according to claim 1, characterized by, The preparation method of the hollow Prussian blue nanoscale enzyme is as follows: the Prussian blue nanoscale enzyme and polyvinylpyrrolidone are dissolved in a hydrochloric acid solution, stirred at room temperature, transferred to a high-pressure reaction kettle, and continuously reacted in an oil bath kettle, and then centrifuged, washed and freeze-dried in sequence to obtain the hollow Prussian blue nanoscale enzyme.

3. The preparation method according to claim 2, characterized in that, The mass ratio of the Prussian blue nanoscale enzyme to polyvinylpyrrolidone is 1:5; The stirring speed at room temperature is 50-200 rpm, and the stirring time is 3.5 h; The continuous reaction in the oil bath kettle is carried out at 140℃ for 4 h; The centrifugation is carried out at 11 000 rpm for 10 min.

4. The production method according to claim 3, characterized by, The preparation method of the Prussian blue nanoscale enzyme is as follows: potassium ferricyanide and polyvinylpyrrolidone are dissolved in a hydrochloric acid solution, magnetically stirred, transferred into a hydrothermal reaction kettle, and then subjected to high-temperature and high-pressure reaction, and then centrifuged, washed and freeze-dried in sequence to obtain the Prussian blue nanoscale enzyme.

5. The preparation method according to claim 4, characterized in that, In the preparation method of the Prussian blue nanoscale enzyme, the mass ratio of potassium ferricyanide to polyvinylpyrrolidone is 22:500; The magnetic stirring speed is 50-200 rpm, and the stirring time is 0.5 h; The high-temperature and high-pressure reaction of the hydrothermal reaction kettle is carried out at 80℃ for 20 h; The centrifugation is carried out at 11 000 rpm for 15 min.

6. The mucin temperature-sensitive hydrogel prepared by the preparation method of any one of claims 1-5.

7. The use of the mucin temperature-sensitive hydrogel of claim 6 in the preparation of a medicament for treating inflammatory bowel disease.

8. A medicament for treating inflammatory bowel disease, characterized by comprising a compound of the formula (I) or a pharmaceutically acceptable salt thereof. The mucin temperature-sensitive hydrogel of claim 6.

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