Preparation method of mucoprotein temperature-sensitive hydrogel

Abstract

The invention discloses a preparation method of mucoprotein temperature-sensitive hydrogel, and belongs to the technical field of hydrogel preparation. Comprising the following steps: uniformly mixing pluronic F127 dibenzaldehyde (PF127-CHO), glycidyl chitosan (Gly-CS) and a mucoprotein solution, and adding hollow Prussian blue nano-enzyme, so as to obtain the mucoprotein temperature-sensitive hydrogel HPB MPC-Gel. According to the mucoprotein hydrogel preparation loaded with the hollow Prussian blue nano-enzyme, the in-vivo and in-vitro evaluation results of HPB MPC-Gel show that the hydrogel has a certain improvement effect on IBD symptoms and is expected to become a means for treating IBD.

Description

Technical Field

[0001] The present invention relates to the technical field of hydrogel preparation, and particularly to a preparation method of mucin thermosensitive hydrogel. Background Art

[0002] Inflammatory bowel diseases (IBD) is a chronic non-specific intestinal inflammation, with symptoms such as abdominal pain, diarrhea, and bloody stools during onset, seriously affecting the daily life of patients.

[0003] Currently, IBD cannot be completely cured, and the treatment methods mainly focus on relieving symptoms. Traditional drugs include immunosuppressants and antibiotics, etc., but they have defects such as low solubility and poor stability, and these drugs may exacerbate the intestinal immune system disorder and intestinal flora imbalance caused by IBD, with relatively large side effects.

[0004] During the process of inflammation, on the one hand, the level of reactive oxygen species (ROS) increases, exacerbating the occurrence of IBD. The antioxidant enzyme treatment strategy based on ROS level regulation has become an effective alternative strategy; on the other hand, the intestinal mucosa is damaged, resulting in the loss of mucosal barrier function and causing IBD. Therefore, the repair of ulcer wounds is also a good treatment method. Constructing a preparation with mucosal protection and the ability to scavenge ROS is of greater significance for the treatment of IBD. Summary of the Invention

[0005] The purpose of the present invention is to provide a preparation method of mucin thermosensitive hydrogel to solve the problems existing in the above-mentioned prior art.

[0006] To achieve the above purpose, the present invention provides the following solutions:

[0007] One of the technical solutions of the present invention, a preparation method of mucin thermosensitive hydrogel, includes the following steps:

[0008] Mix Pluronic F127 dibenzaldehyde, glycidyl chitosan, and mucin solution, and add hollow Prussian blue nanozyme to form a mixed solution, then the mucin thermosensitive hydrogel HPB MPC-Gel can be obtained.

[0009] Two of the technical solutions of the present invention, the mucin thermosensitive hydrogel prepared by the above preparation method.

[0010] Three of the technical solutions of the present invention, the application of the mucin thermosensitive hydrogel in the preparation of drugs for treating inflammatory bowel disease.

[0011] Four of the technical solutions of the present invention, a drug for treating inflammatory bowel disease, including the above-mentioned mucin thermosensitive hydrogel.

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

[0013] (1) HPB was successfully prepared by the hydrothermal method. It was characterized by ultraviolet-visible spectroscopy, Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, X-ray diffraction pattern, and transmission electron microscopy. The particle size of HPB was detected by a particle size analyzer to be between 180 - 210 nm, and the potential was 7 mV. HPB was observed to be regular cubes with uniform size by transmission electron microscopy.

[0014] (2) The formulation of HPB MPC-Gel was determined by the single-factor method as 15% PF127-CHO, 2.5% Gly-CS, 3% Mucin, and it could gel smoothly at 37°C. HPB MPC-Gel was characterized in terms of appearance morphology, phase transition characteristics, internal structure, rheological properties, and adhesiveness. The results showed that the hydrogel presented a blue homogeneous solution in appearance, was in a solution state at 25°C, and in a gel state at 37°C, having good phase transition characteristics; the interior of the hydrogel was a three-dimensional network structure with a honeycomb-like appearance, which was beneficial for drug encapsulation and delayed drug release; rheological analysis showed that at 25°C, G” > G' and it was in a liquid state, at 37°C, G' > G” and it was in a solid state. In addition, the results of the swelling experiment and in vitro degradation experiment showed that the hydrogel would swell and would not undergo rapid erosion, and it would not degrade completely within 24 h, meeting the experimental expectations. The drug release results showed that the drug release only reached 81.14% within 24 h and did not release completely, indicating that HPB MPC-Gel had a sustained-release effect. At the same time, the injectability and adhesion experiments showed that HPB MPC-Gel had good injectability and adhesiveness and could be applied to rectal administration. By establishing an IBD model, the therapeutic effects of each group were evaluated. Compared with the model group, the HPB MPC-Gel group had a lower DAI score, less weight loss, inhibited colon shortening, significantly reduced intestinal weight index and spleen weight index of mice, reduced the contents of IL-1β, IL-6, TNF-α, and MPO, alleviated the colon pathological damage caused by DSS-induced colitis, increased the integrity of the mucus layer, upregulated the expression of tight junction proteins Occludin and ZO-1, resisted the damage of DSS to the intestinal mucosal barrier, and improved the integrity of the intestinal mucosal mechanical barrier and repaired the damaged intestinal mucosal barrier.

[0015] The present invention prepared a mucin hydrogel preparation loaded with hollow Prussian blue nanozyme. The in vivo and in vitro evaluation results of HPB MPC-Gel showed that the hydrogel played a certain role in improving IBD symptoms and was expected to become a means for treating IBD. Description of the Drawings

[0016] Figure 1Appearance of Prussian blue nanoparticles. Among them, A is the PB solution and B is the HPB solution.

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

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

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

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

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

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

[0023] Figure 8 SEM images of MPC-Gel and HPB MPC-Gel at different magnifications (Bar = 10 μm).

[0024] Figure 9 FTIR diagrams of MPC-Gel and HPB MPC-Gel.

[0025] Figure 10 Swelling (A) and in vitro degradation (B) diagrams of MPC-Gel and HPB MPC-Gel.

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

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

[0028] Figure 13 Adhesion ability diagrams of HPB and HPB MPC-Gel.

[0029] Figure 14 Standard curve of HPB.

[0030] Figure 15 Drug release curve of HPB MPC-Gel.

[0031] Figure 16 Cytotoxicity of extracts of HPB MPC-Gel at different concentrations.

[0032] Figure 17 After rectal administration, fluorescence images of the colon of mice at different time points for HPB@IR780 and HPB@IR780 MPC-Gel IBD.

[0033] Figure 18 Manifest blood in the stool in mice of each group.

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

[0035] Figure 20 Colon appearance photos of mice in each group.

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

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

[0038] Figure 23 MPO activities in colon tissues of mice in each group (n = 3, ***P < 0.001).

[0039] Figure 24 Colon tissue pathology of mice in each group (scale bar is 100 μm).

[0040] Figure 25 Pathohistological scores of colon tissues of mice in each group.

[0041] Figure 26 Alcian blue-periodic acid Schiff staining results (scale bar is 100 μm).

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

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

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

[0045] Figure 30Hemolysis rates and photos of red blood cell states for each group of mice. From left to right are Water, PBS, HPB, MPC-Gel, and HPB MPC-Gel (n = 3). Detailed implementation manners

[0046] For the technical solutions described in the present invention, unless otherwise specified, they are all conventional solutions in the art. The reagents or raw materials used, unless otherwise specified, are all purchased from commercial channels or are publicly available.

[0047] The embodiments of the present invention provide a method for preparing a mucin thermosensitive hydrogel, which includes the following steps:

[0048] Mix Pluronic F127 dibenzaldehyde, glycidyl chitosan, and mucin solution, add hollow Prussian blue nanozyme to form a mixed solution, and then the mucin thermosensitive hydrogel HPB MPC-Gel can be obtained.

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

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

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

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

[0053] In some specific embodiments, the preparation method of the hollow Prussian blue nanozyme is as follows: Dissolve Prussian blue nanozyme and polyvinylpyrrolidone in hydrochloric acid solution, stir at room temperature, then transfer to a high-pressure reaction kettle, and continue the reaction in an oil bath. After the reaction is completed, centrifuge, wash, and lyophilize in sequence to obtain the hollow Prussian blue nanozyme.

[0054] In some specific embodiments, 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 h;

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

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

[0058] In some specific embodiments, the preparation method of the Prussian blue nanozyme is as follows: Dissolve potassium ferricyanide and polyvinylpyrrolidone in hydrochloric acid solution, stir magnetically, carry out high-temperature and high-pressure reaction, and after the reaction is completed, centrifuge, wash, and freeze-dry in sequence to obtain the Prussian blue nanozyme.

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

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

[0061] The conditions of the high-temperature and high-pressure reaction are: constant temperature reaction at 80 °C for 20 h;

[0062] The conditions of the centrifugation are: 11000 rpm / min, 15 min.

[0063] The present invention also provides a mucin thermosensitive hydrogel prepared by the above preparation method.

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

[0065] The present invention also provides a drug 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 in the dark at room temperature for 24 h, then centrifuge (11000 rpm / min, 10 min), and wash the precipitate with ultrapure water three times 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 of HPB@IR780, add double-distilled water to 10 mL, mix well to obtain an 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] Dissolve 660 mg of potassium ferricyanide and 15 g of polyvinylpyrrolidone (PVP-K30) in 200 mL of hydrochloric acid solution (0.01 M). After magnetic stirring at 200 rpm for 0.5 h, transfer it to a hydrothermal reaction kettle. After reacting at a constant temperature of 80 °C for 20 h, centrifuge (11000 rpm / min, 15 min). Wash the obtained precipitate three times with ultrapure water and freeze-dry to obtain Prussian blue nanozyme (PB).

[0071] 1.2 Synthesis of hollow Prussian blue nanozyme (HPB)

[0072] Dissolve 200 mg of PB and 1 g of PVP in 200 mL of hydrochloric acid solution (1 M). After stirring at room temperature at 200 rpm for 3.5 h, transfer it to a high-pressure reaction kettle. After reacting at a constant temperature of 140 °C for 4 h, centrifuge (11000 rpm / min, 10 min). Wash the obtained precipitate three times with ultrapure water and freeze-dry to obtain hollow mesoporous Prussian blue nanozyme (HPB).

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

[0074] 2 Characterization of nanoparticles

[0075] Use a NanoZS90 laser particle size analyzer to measure the particle size and zeta potential of PB and HPB. Use a JEM-2000EX transmission electron microscope to observe the morphology of PB and HPB and take pictures. Use an ultraviolet-visible spectrophotometer to measure the ultraviolet-visible spectra (Ultraviolet-visible spectroscopy, UV-vis) of PB and HPB solutions. The scanning range is 400 - 1000 nm. Use a Fourier transform infrared spectrometer to measure the Fourier transform infrared spectra (Fourier transforminfraredspectroscopy, FTIR) of PB and HPB. By the potassium bromide tablet pressing method, after grinding potassium bromide, dry it at 120 °C for 12 h. Mix and grind the sample with potassium bromide powder and then press it into a tablet. The scanning wave number range is 4000 - 500 cm -1 , and the resolution is 4 cm -1 . Perform phase characterization of the obtained PB and HPB by X-ray diffraction (X ray diffraction, XRD). Take an appropriate amount of freeze-dried PB and HPB and grind them into a powder without particle sense in a mortar. Add the sample to the sample cell and flatten it, then conduct on-machine detection.

[0076] The crystal structure was characterized by X-ray photoelectron spectrometer (XPS). An appropriate amount of freeze-dried PB and HPB was ground into a particle-free powder in a mortar. The sample was added to the sample cell, flattened, and then detected on the machine. The crystal structure was measured using an X-ray photoelectron spectrometer (XPS).

[0077] As shown in Table 1 and Figure 2 as indicated, in this experiment, the prepared nanoparticles had a narrow and single particle size distribution. The diameter of the nanoparticles was all in the range of 180 - 210 nm, and the PDI < 0.3. The lower PDI value indicated that the nanoparticles had a uniform particle size and good dispersibility. The average particle size of PB was 186.13 ± 0.23 nm, and the average particle size of HPB was 208.87 ± 0.60 nm. The particle size of HPB was larger than that of PB because it had a hollow mesoporous structure, which made there be cavities and pores inside the particles, thus increasing the overall size of the particles. The nanoparticles carried charges on their surfaces and there would be electrostatic interactions between them, which affected the stability and biological activity of the nanoparticles. The absolute value of the Zeta potential could be used to reflect the magnitude of the electrostatic repulsion between the nanoparticles. The higher the value, the greater the electrostatic repulsion between the particles and the better the physical stability. In this experiment, both PB and HPB showed electronegativity. The increase in the Zeta potential value of HPB indicated an improvement in its physical stability.

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

[0079]

[0080] The results of the TEM-generated images showed that the morphologies of PB and HPB were cubic and arranged uniformly. The average particle size of HPB was larger than that of PB, which was consistent with the trend of the particle size results.

[0081] The UV-vis absorption spectra of PB and HPB solutions were as Figure 3 shown. It could be seen from the results that PB and HPB had a broad absorption peak at 600 nm to 1000 nm. The maximum absorption peaks of PB and HPB appeared at 710 nm and 730 nm, fully reflecting the presence of Fe 2+ and Fe 3+ .

[0082] The FTIR results were as Figure 4 shown. PB and HPB had an absorption peak at 2060 cm -1 , which was caused by the stretching vibration of the Fe-CN-Fe bond.

[0083] The chemical composition and elemental chemical valence states of PB and HPB were analyzed by XPS. Figure 5For the XPS full spectrum, it can be seen 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, as Figure 6 shown, revealing diffraction peaks at 2θ angles of 17.43°, 24.74°, 35.25°, and 39.58°; 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 Pharmacodynamic Effects of Prussian Blue Nanozyme Mucin Thermosensitive Hydrogel

[0087] 1 Preparation of Mucin Thermosensitive Hydrogel (MPC-Gel)

[0088] Mix different volumes of Pluronic F127 dibenzaldehyde (PF127-CHO), glycidyl chitosan (Gly-CS), and mucin solutions, and heat them in a water bath at 37°C to obtain Mucin / PF127-CHO / Gly-CS Hydrogel (MPC-Gel).

[0089] To determine the gelling concentration for the system to form a stable hydrogel, the selection of the concentrations of each substance in the hydrogel preparation system was analyzed, and the gelling effects of the gel systems were compared through the vial inversion experiment.

[0090] (1) Selection of Gly-CS Concentration (the concentrations described in this invention are mass concentrations unless otherwise specified)

[0091] To determine the concentration of Gly-CS, different concentrations of glycidyl chitosan (Gly-CS) were prepared according to the general steps of hydrogel preparation, and the vial inversion experiment was used to observe whether gelation occurred. As shown in Table 2, during the experiment, the PF127-CHO concentration was 15%, the mucin concentration was 3%, and the Gly-CS concentrations were set to 0, 0.5%, 1%, 1.5%, 2%, and 2.5% respectively. They were placed in water at room temperature and 37°C respectively, and the vial inversion experiment was carried out to observe their gelation situations.

[0092] Table 2 Gelation Situations of Different Concentrations of Gly-CS

[0093]

[0094] The concentration of fixed PF127-CHO was 15%, and the concentration of Mucin was 2% without change. For different concentrations of Gly-CS, the gelation of MPC-Gel at 25 °C and 37 °C was observed. The results showed that when the concentration of Gly-CS was below 2.5%, it was a liquid at both 25 °C and 37 °C. When the concentration of Gly-CS was 2.5%, it was a flowing liquid at 25 °C and transformed into a gel at 37 °C. Therefore, 2.5% was selected as the concentration of Gly-CS.

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

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

[0097] Table 3 Gelation of PF127-CHO with different concentrations

[0098]

[0099] The concentration of fixed Mucin was 2%, and the concentration of Gly-CS was 2.5% without change. For different concentrations of PF127-CHO, the gelation of HPB MPC-Gel at 25 °C and 37 °C was observed. The results showed that when the concentration of PF127-CHO was below 10%, it was a liquid at both 25 °C and 37 °C. When the concentration of PF127-CHO was 10%, it was a flowing liquid at 25 °C and gelation occurred at 37 °C. As the concentration increased, the gelation time gradually shortened. To ensure smooth conversion into a gel in vivo, 15% was selected as the subsequent experimental concentration of PF127-CHO.

[0100] (3) Selection of the concentration of mucin

[0101] To determine the concentration of mucin, according to the general steps for preparing hydrogels, mucins with different concentrations were prepared, and the gelation effect and stability were observed through the vial inversion experiment. As shown in Table 4, during the experiment, the concentration of Gly-CS was 2.5% and the concentration of PF127-CHO was 15%. The concentrations of mucin were set at 0, 1%, 2%, 3%, 4% and 5% respectively, and they were placed in water at room temperature and 37 °C respectively for the vial inversion experiment to observe their gelation.

[0102] Table 4 Different concentrations of Mucin

[0103]

[0104] The concentration of Gly-CS was fixed at 2.5% and that of PF127-CHO at 15%. With different concentrations of Mucin, the gelation of MPC-Gel at 25 °C and 37 °C was observed. The results showed that when the concentration of Mucin was above 4%, it was liquid at both 25 °C and 37 °C. When the concentration of Mucin was below 4%, it was a flowing liquid at 25 °C and gelation occurred at 37 °C. As the concentration increased, the gelation time gradually increased and the gel temperature gradually rose. To ensure smooth conversion into a gel in vivo, 3% was selected as the subsequent experimental concentration of Mucin.

[0105] 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, and add double-distilled water to 10 mL. Mix well to obtain 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 mixing system, the mass concentration of Pluronic F127 dibenzaldehyde is 15%; the mass concentration of glycidyl chitosan is 2.5%; the mass concentration of mucin is 3%; the concentration of hollow Prussian blue nanozyme is 1 mg / mL.

[0107] The synthesis process of the hydrogel is as Figure 7 shown. After MPC-Gel and HPB MPC-Gel were synthesized into pre-gels, they were converted into homogeneous gels after a 5-min water bath at 37 °C.

[0108] 3 Characterization of HPB MPC-Gel

[0109] 3.1 Microscopic morphology detection: Appropriate amounts of freeze-dried samples of HPB MPC-Gel and MPC-Gel were taken respectively, and the morphology of the gels was observed by scanning electron microscopy (SEM).

[0110] The cross-sectional morphologies of MPC-Gel and HPB MPC-Gel were observed by SEM. The results are as Figure 8 shown. Both MPC-Gel and HPB MPC-Gel presented a porous three-dimensional structure with a uniform and closely interconnected network. The added HPB was distributed in the honeycomb-like MPC-Gel. This network structure endows the hydrogel with a large specific surface area, which is more conducive to the diffusion of drugs.

[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 and dried at a temperature of 60 °C for 48 h. The dried samples were ground into fine powders, and the infrared spectra of the samples were recorded by the KBr tablet method. The detection range was 4000 - 500 cm -1 .

[0112] The FTIR of HPB, Mucin, PF127-CHO, Gly-CS, MPC-Gel, and HPB MPC-Gel is as Figure 9 shown: In the PF127-CHO spectrum, the stretching vibrations of the C=O and C-H bonds on the aldehyde group are at 1717 cm -1 and 2737 cm -1 . In the Mucin spectrum, the symmetric stretching vibration peak of the amino group of the amide bond is at 3290 cm -1 , and the stretching vibration band of the carbonyl group (amide absorption band I) is at 1632 cm -1 . In the Gly-CS spectrum, the stretching vibration peaks of O-H on the hydroxyl group and N-H on the amino group are at 3700 - 3000 cm -1 . In the HPB spectrum, the stretching vibration peak of the carbonyl group (C=O) is at 1644 cm -1 . In the MPC-Gel spectrum, there are absorption peaks similar to those of PF127-CHO, Mucin, and Gly-CS near 1700 cm -1 , 2700 cm -1 , and 3000 - 3700 cm -1 , indicating the successful synthesis of MPC-Gel. In the HPB MPC-Gel spectrum, there is a peak similar to that of HPB near 1600, indicating the successful synthesis of HPB MPC-Gel.

[0113] 3.3 Swelling and in vitro Degradation Behaviors:

[0114] The swelling properties of the hydrogels and nanocomposite hydrogels were evaluated by the 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) respectively, and incubated under gentle shaking (37 °C, 55 rpm) to reach swelling equilibrium, and the samples were taken out and weighed (W s ) at 0.5 h, 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, and 7 h. The swelling ratio (Swelling percentage, SP) was calculated according to the following formula:

[0115]

[0116] Where: Ws is the mass of the swollen sample, and W0 is the mass of the sample before swelling.

[0117] The in vitro degradation behaviors of the hydrogel and nanocomposite hydrogel were evaluated by gravimetry. 2 mL of HPB MPC-Gel and MPC-Gel were taken, and the gels were stabilized in a 37 °C constant temperature water bath for 30 min. The weight of the sample was recorded as W0, and then an equal volume of PBS was added to the surface of the hydrogel. It was placed in a constant temperature shaker (37 °C, 55 rpm). Weighed and recorded the remaining mass (Wt) at 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, 48 h, and 72 h respectively. The weight retention rate (Weight remaining, WR) was calculated according to the following formula:

[0118]

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

[0120] As Figure 10 shown in A, both MPC-Gel and HPB MPC-Gel showed water absorption in PBS solution with pH = 7.4, rapidly swelled within 3 h, and the equilibrium swelling ratios of MPC-Gel and HPB MPC-Gel reached 337.18% and 398.87% respectively, and reached swelling equilibrium after 6 h, showing good water absorption ability. The swelling ratio of HPB MPC-Gel was lower than that of MPC-Gel, and then the degradation properties of MPC-Gel and HPB MPC-Gel were evaluated. As Figure 10 shown in B, when MPC-Gel and HPB MPC-Gel were in PBS solution, the weight retention rates at 48 h were 53.33% and 39.42% respectively, and both underwent rapid degradation. The weight retention rates at 72 h were 32.85% and 29.57% respectively, and the degradation rate of the gel was slower. This ensured that the gel maintained sufficient colloidal mass in the first few hours, enabling the full release of HPB, and at the same time allowing other intestinal contents to pass through smoothly until a new gel was injected.

[0121] 3.4 Rheological property detection: To study the mechanical properties of the hydrogel, a rotational rheometer was used for testing. HPB MPC-Gel and MPC-Gel were prepared, and after the nanocomposite hydrogel was formed, it was placed on the sample stage. The test rotor model was a parallel plate of 50 mm, and the measurement gap was set to 1 mm. The angular frequency change range was set from 0.1 - 200 rad / s at 25 °C and 37 °C respectively, and the corresponding curves of the storage modulus (G') and loss modulus (G") were obtained.

[0122] The MPC-Gel and HPB MPC-Gel were scanned and tested by a rheometer at 25 °C and 37 °C. As Figure 11As shown, at 25 °C, the values of G' and G'' are both small, and G' is lower than G'', showing a sol state; when at 37 °C, G'' > G', the value of G' is about 71480 Pa, and the value of G'' is about 10930 Pa, and the difference between the two values is nearly 60000 Pa. This indicates that the hydrogel transforms into a high-strength and stable gel state, and this temperature can also meet the requirements of intraperitoneal injection for drug administration.

[0123] 3.5 Injectability detection: Use a 1 mL syringe to suck in the newly prepared hydrogel, and inject it into a 5 mL sample bottle filled with ultrapure water at 37 °C, and observe and take pictures for recording.

[0124] By loading MPC-Gel and HPB MPC-Gel into a 1 mL syringe, the injectability of MPC-Gel and HPB MPC-Gel in water at 37 °C was explored by observing their states during injection. As Figure 12 shown, both MPC-Gel and HPB MPC-Gel are filamentous and have good fluidity in water at 37 °C, indicating that they can be applied to rectal drug administration.

[0125] 3.6 Adhesion evaluation: Place the composite hydrogel HPB MPC-Gel directly on the colon segment between the cecum and rectum of SD rats for 10 min, and inject 30 mL of PBS solution (pH = 7.4) within 30 s to continuously impact the nano-composite hydrogel to determine whether HPB MPC-Gel will shift.

[0126] The strong adhesion of the hydrogel can improve the drug treatment effect and increase the drug retention effect. To further explore the adhesion of HPB MPC-Gel to biological tissues, a study was carried out using rat colon tissue as a carrier. The results are as Figure 13 shown, the adhesion schematic diagram of HPB and HPB MPC-Gel in the intestine. Inject 30 mL of PBS solution continuously within 30 s to impact the gel, and it is found that HPB is easily washed away, while HPB-hydrogel does not move. This indicates that despite the continuous impact, HPB MPC-Gel can still remain in the intestine for a certain period of time, increasing the drug retention time.

[0127] 4 In vitro drug release performance evaluation

[0128] 4.1 Establishment of the analysis method for HPB content determination

[0129] The content of HPB hydrogel was determined by ultraviolet-visible spectrophotometry. Standard curve of HPB: HPB was diluted 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) with PBS, and the absorbance of the solution was measured at 720 nm. The standard curve was plotted with absorbance as the ordinate and the corresponding drug concentration as the abscissa.

[0130] Select 720 nm as the measurement wavelength, establish a linear relationship between the absorbance (A) and concentration (C) of HPB, and plot the standard curve ( Figure 14 ), and obtain the linear regression equation A = 0.0032C - 0.0041, R 2 = 0.9997. The linear relationship between the absorbance and concentration of HPB is good, and the linear range is: 15.625 μg / mL - 1 mg / mL.

[0131] 4.2 Investigation of in vitro release behavior: Take 2 mL of HPB MPC-Gel and let it completely gel at 37 °C, then add it to 20 mL of PBS buffer. Place it in a constant temperature shaker and continuously shake at a speed of 55 rpm at 37 °C. Take 2 mL of the supernatant for content determination at 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, 48 h and 72 h respectively, and add an equal amount of PBS buffer to keep the volume of the release system constant. The experiment was repeated in parallel 3 times. The release solution was injected and detected according to the conditions described in "2.4.1". The cumulative release percentage of the drug was calculated according to the following formula:

[0132]

[0133] Where: Cn is the concentration of HPB in the release solution at the nth sampling, V0 is the volume of the release medium, and V is the volume of the release solution taken each time.

[0134] The SEM images and in vitro degradation behavior of HPB-hydrogel show that it has appropriate pore sizes and certain in vitro degradation ability. This experiment further investigated the drug release process of HPB MPC-Gel in PBS solution at pH = 7.4. As Figure 15 shown, with the increase of time, the release of HPB in HPB MPC-Gel. Within 12 h, there was a burst release behavior of HPB, and the cumulative release percentage of the drug reached 81.14%. After 24 h, the release gradually flattened. At 72 h, the drug release percentage was 94.27%. The drug release was continuous throughout the process, indicating that the hydrogel is an ideal drug controlled-release carrier.

[0135] 5 Investigation of cell compatibility

[0136] The cytotoxicity of HPB MPC-Gel was analyzed by the CCK-8 method. RAW 264.7 cells were cultured in vitro with the extract of HPB MPC-Gel to evaluate the cytotoxicity of HPB MPC-Gel. The gel HPB MPC-Gel was irradiated with ultraviolet light for 2 h to kill bacteria, and then extracted with 50 mL of complete DMEM medium at 37 °C for 24 h. After extraction, the extract was filtered through a 0.22 μm microporous filter membrane and stored in a refrigerator at 4 °C. Before use, the extract was diluted with the culture medium to a concentration range of 0.34 - 5.4 mg / mL. When changing the medium for the experimental group cells, different concentrations of the HPB MPC-Gel extract were added respectively, and the control group was added with an equal amount of culture medium. After culturing for 24 h or 48 h, the culture medium and the extract were removed, 100 μL of complete DMEM medium containing 10% CCK-8 was added to each well, and incubated at 5% CO2 and 37 °C for 30 min. The absorbance at 450 nm was detected by an enzyme-linked immunosorbent assay instrument. The cell survival rate was calculated.

[0137] The cytotoxicity of HPB MPC-Gel against RAW 264.7 cells was detected by the CCK-8 method. The results are as Figure 16 shown. After incubating RAW 264.7 cells with different concentrations of the HPB MPC-Gel extract for 24 h and 48 h respectively, the cell survival rate was above 90%, indicating that HPB MPC-Gel has good biocompatibility with cells.

[0138] 6 Pharmacodynamic evaluation of HPB MPC-Gel in vivo

[0139] 6.1 Establishment of IBD mouse model: 8-week-old male C57BL / 6J mice weighing 20 - 22 g were used, and the breeding environment temperature was 20 - 22 °C, with a relative humidity of about 55%. The light and dark times alternated for 12 hours each. The content of the animal experiment complied with international ethical standards. Weighed a certain mass of DSS and configured it with double-distilled water into a 3% DSS solution. The mice drank water freely, and the freshly prepared DSS solution was changed every day. The modeling period was 7 days to establish a mouse enteritis model. During this period, the body weight, stool characteristics, and stool blood in the mice were recorded daily, the body weight change rate of the mice was calculated, and the disease activity index (DAI) score of the IBD model was evaluated according to Table 5, and the DAI score was calculated to evaluate the severity of the model lesions.

[0140]

[0141] DAI = body weight loss score + fecal shape score + fecal bleeding score;

[0142] Table 5 DAI scoring criteria

[0143]

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

[0145] To determine whether HPB MPC-Gel can prolong the retention of HPB in the colonic lumen, HPB@IR780 and HPB@IR780 MPC-Gel were prepared, and the retention after rectal perfusion was evaluated by in vivo imaging. As Figure 17 shown, strong fluorescence was present in the colon for HPB@IR780 and HPB@IR780 MPC-Gel, and the fluorescence intensity of HPB@IR780 and HPB@IR780 MPC-Gel decreased with time. However, compared with HPB@IR780 MPC-Gel, the signal of HPB@IR780 decreased rapidly. Only a weak fluorescence signal was visible for HPB@IR780 at 12 h after rectal injection, and no fluorescence signal was detected at 24 h. In contrast, at 12 h after rectal perfusion, HPB@IR780 MPC-Gel had a strong fluorescence signal. Even at 24 h, a fluorescence signal was still present for HPB@IR780 MPC-Gel. HPB@IR780 MPC-Gel can prolong the retention time of HPB and better play an anti-inflammatory role.

[0146] 6.3 In vivo efficacy experiment: Twenty-five SPF-grade male C57BL / 6J healthy mice were randomly divided into 5 groups (n = 5 in each group). The experimental groups were: Control group, 3% DSS model group, HPB group, MPC-Gel group, and HPB MPC-Gel group. Rectal administration was performed, with a dosing volume of 200 μL, once every two days for 8 days, and the mice were sacrificed 24 h after the last dose. The first day of dosing was recorded as day 0. Mice in each group were rectally administered HPB, MPC-Gel, and HPB MPC-Gel on days 0, 2, 4, 6, and 8 respectively. The dosing dose of HPB was 10 mg / kg. The normal control group and the DSS model group were given the same volume of normal saline daily. The body weight, stool characteristics, and blood in the stool of the mice were observed and recorded every day. On the eighth day of the experiment, the mice were sacrificed by cervical dislocation. The entire intestinal segment was removed from the cecum to the anus. After measuring the colon length and taking pictures, the colon segment was weighed, and about 0.5 cm of tissue 1 cm above the anus was cut and fixed in 4% paraformaldehyde for H&E staining. The remaining colon segments were collected and quickly stored in a -80 °C ultra-low temperature freezer.

[0147] On the third day of the experiment, the feces of the mice in the DSS group became sparse. On the fifth day, the body weight of the mice in the DSS group decreased significantly, and blood in the stool could be observed, as Figure 18 , indicating that a DSS-induced IBD model was successfully established. As shown in B of Figure 19 , compared with the DSS group, the body weights of the HPB group, MPC-Gel group, and HPB MPC-Gel group showed a slower downward trend, indicating a certain therapeutic effect. According to the body weight loss, changes in stool characteristics, and stool bleeding of the mice, the DAI score was calculated. The results are shown in A of Figure 19 . The DAI score of the DSS group increased significantly over time and reached 11.8 on the eighth day. Although the DAI scores of the mice in the HPB group, MPC-Gel group, and HPB MPC-Gel group also increased to a certain extent, the upward trend was relatively gentle. Among them, the HPB MPC-Gel group had the lowest score, with a value of 5.8, indicating that HPB MPC-Gel has a certain therapeutic effect on treating colitis.

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

[0149] The mouse intestinal weight index and spleen index were calculated according to the following formulas respectively:

[0150]

[0151] As shown in Figure 20 and 21As shown in Figure A, the pictures of the colon appearance and the statistical results of the colon length of mice in each group are presented. The colons of the mice in the normal group and the control group are smooth on the surface, without edema or congestion, and the internal feces are formed, with the colon length reaching 6.44 cm. The colon length of the DSS group is significantly shortened, there is no formed feces, and the congestion is obvious, with the intestine length only 3.02 cm. In the HPB group, the MPC-Gel group, and the HPB MPC-Gel group, the degree of colon shrinkage is reduced, bloody contents can be seen in the intestine, the feces are relatively formed, and loose stools and slight edema can be seen in some cecum segments, but the degree is lighter than that of the DSS group. The colon length of the HPB MPC-Gel group is 6.06 cm. Compared with the Blank-hydrogel group and the HPB-hydrogel group, the increase in colon length is more obvious, indicating that the HPB-hydrogel group has a better effect on treating colitis. As Figure 21 As shown in Figures B and C, the intestinal weight index and spleen weight index of the DSS group are 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 show a downward trend to varying degrees, and the values of the intestinal weight index and spleen weight index of the HPB MPC-Gel group are the lowest, 25.41 and 3.25 respectively, indicating that the HPB MPC-Gel group has the best therapeutic effect on colitis. In summary, HPB MPC-Gel can inhibit colon shortening, reduce the intestinal weight index and spleen weight index, and effectively improve the symptoms of DSS-induced IBD.

[0152] 6.5 Detection of cytokines in colon tissue: Weigh the colon tissue samples, add pre-cooled PBS at a weight-to-volume ratio of 1:9, add 3 grinding beads of 3 mm, grind them using a low-temperature grinder, centrifuge at 5000 g for 10 min, and take the supernatant. The subsequent experimental steps are strictly carried out according to the operating procedures of the TNF-α, IL-6, and IL-1β instruction manuals.

[0153] The content of inflammatory factors was measured, and the results are as Figure 22 shown. The content of IL-1β in the blank control group is 49.15 pg / mL, but it increases to 308.85 ρg / mL in the DSS model group. After treatment with different drugs, the contents in the HPN-NPs group, the MPC-Gel group, and the HPB MPC-Gel group are 128.20, 138.40, and 78.60 pg / mL respectively, all showing a downward trend to varying degrees. Similarly, colitis can increase the contents of the two inflammatory factors IL-6 and TNF-α, and after drug treatment, they all show a downward trend to varying degrees. It should be noted that among the three inflammatory factors, the HPB MPC-Gel group is significantly lower than the DSS model group and is 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 were as Figure 23 shown. The MPO values of the normal control group, DSS-induced 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 after drug treatment, the MPO values all decreased. Among them, the value of the HPB MPC-Gel group was closest to the control group, indicating that it had basically returned to the normal value. This shows that HPB MPC-Gel can effectively reduce the MPO activity in the mouse colon.

[0156] 6.7 Histopathological experiment of colon tissue: The fluorescence upright microscope was used to observe whether there were pathological changes in the colon tissue. Referring to Table 6, the morphological changes of the mouse intestinal tissue were observed and histopathological scoring was performed. This scoring consisted of the severity of inflammation, the degree of lesion, the degree of crypt destruction, and the scope of the lesion.

[0157] Table 6 Histopathological scoring

[0158]

[0159] To observe the intestinal injury, the colon was stained with H&E and the pathological conditions of the colon tissue of each group of mice were observed. The results were as Figure 24 shown. It can be seen that the colon tissue structure of the normal control group mice was intact, the mucosal structure was intact, the mucosa was rich in goblet cells, without edema and inflammatory cell infiltration. There were no obvious ulcers or inflammatory manifestations. The integrity of the colon mucosa in the DSS model group was damaged, the crypts disappeared, the glands decreased, and a large number of inflammatory cells infiltrated diffusely. Compared with the DSS group, the integrity of the colon tissue of the HPB group and the MPC-Gel group mice was better, and the infiltration of inflammatory cells decreased, indicating that the HPB group and the MPC-Gel group could relieve colitis. By comparing the pathological sections of each group, it was found that the internal structure of the colon in the HPB MPC-Gel group was more substantial, denser, and more complete than that in the HPB group and the MPC-Gel group. Its histopathological score ( Figure 25 ) was significantly lower than that of the DSS group. The above results indicate that HPB-hydrogel has a better effect on treating colitis in mice.

[0160] 6.8 Alcian blue-periodic acid Schiff staining was used to detect the expression of mucin.

[0161] The mucus layer is the first line of defense of the colon against harmful substances and microorganisms. This layer of mucus is produced by goblet cells, which can prevent the invasion of microorganisms into the intestine, separate commensal bacteria from intestinal epithelial cells, protect the integrity of the intestinal barrier, and play an important role in maintaining intestinal homeostasis. The inner mucus layer of the colon is mainly composed of mucin (MUC2), a type of glycosylated protein produced by intestinal goblet cells. Therefore, Alcian blue staining was used to analyze the distribution of mucin MUC2, detect the number of goblet cells, observe the number of goblet cells in the intestinal mucosa and the secretion of mucin, and further judge the degree of damage to the intestinal barrier. Figure 26 The results of Alcian blue-periodic acid Schiff staining for each group are shown. It can be seen that the goblet cells in the colon of normal mice are abundant, arranged neatly, and have a tight structure. The outside of the goblet cells is wrapped by the mucus layer. Compared with the control group, the goblet cells in the colon of the DSS model group mice almost disappeared, the crypt structure was damaged, and the mucus layer was significantly damaged; the number of goblet cells increased in the HPB group and the MPC-Gel group, and the mucus layer was relatively intact; the number of goblet cells in the HPB MPC-Gel group increased significantly, and the mucus layer was similar to that of the control group, indicating that HPB MPC-Gell can provide good protection for intestinal epithelial cells, increase the number of goblet cells, secrete more mucin to improve the intestinal mucus layer, and relieve IBD.

[0162] 6.9 Immunohistochemical experiment.

[0163] The intestinal mucosal barrier includes a mechanical barrier, a chemical barrier, an immune barrier, and a biological barrier. It is a highly selective dynamic barrier that prevents the invasion of pathogenic antigens. Among them, cell junctions are also an important intestinal mechanical barrier, and tight junction proteins are an important part of it. They connect epithelial cells, regulate epithelial polarity and the paracellular movement of solutes and fluids in the cell gap, and prevent the invasion of microorganisms through the paracellular pathway. Abnormal expression will cause an increase in intestinal epithelial permeability, leading to the invasion of foreign antigens and mucosal damage. Therefore, immunohistochemistry was used to detect the expression of tight junction proteins Occludin and ZO-1 in the colon tissues of mice in each group to judge the integrity of the mucosal barrier. The results are as Figure 27 shown. In the DSS model group, Occludin and ZO-1 were hardly expressed, reflecting the impairment of intestinal barrier function and the disruption of intercellular junctions, resulting in increased intestinal permeability, which in turn promoted the development of inflammation and the aggravation of mucosal damage. Compared with the DSS-induced model group, the expression of Occludin and ZO-1 was upregulated in the HPB group, the MPC-Gel group, and the HPB MPC-Gel group. Among them, the expression of Occludin and ZO-1 in the HPB MPC-Gel group was significant, indicating 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] Fix the heart, liver, spleen, lungs, and kidneys of mice with 4% paraformaldehyde fixative and observe whether there are pathological changes under a fluorescence upright microscope.

[0166] Collect the plasma samples of each mouse and detect the contents of AST, ALT, LDH, BUN, Cre, and UREA in the mouse plasma. Detection is carried out according to the instructions of the corresponding kits.

[0167] Use mouse red blood cells to evaluate the hemolysis of each group of samples. Take 1 mL of blood from the mouse eyeballs, collect the blood using a sodium citrate anticoagulant tube, centrifuge the blood at 5000 r / min at 4 °C for 15 min, and discard the supernatant. Add 0.6 mL of H2O, PBS, HPB, MPC-Gel, and HPB MPC-Gel to the red blood cells (2%, 0.6 mL), incubate the mixture at 37 °C for 30 min, and then centrifuge to take the supernatant. Measure the absorbance at 545 nm using an enzyme-linked immunosorbent assay (ELISA) reader and calculate the hemolysis rate:

[0168]

[0169] To explore the biosafety of HPB, MPC-Gel, and HPB MPC-Gel, first take the heart, liver, spleen, lungs, and kidneys of mice, section and perform H&E staining. The results are as Figure 28 shown. The organ tissue structures are intact among each group, and no abnormal conditions such as inflammation, necrosis, congestion, and bleeding are observed. It shows that HPB, MPC-Gel, and HPB MPC-Gel have good tissue safety and no toxic or side effects.

[0170] To more comprehensively evaluate the biosafety, collect the blood of each group of mice at the end of the experiment and measure the contents of ALT, AST, LDH, BUN, UREA, and Cre biochemical index contents. The results of blood biochemical analysis are as Figure 29 shown. The ALT results of each group are 44.37, 43.07, 41.87, 40.90, and 41.30 U / L respectively, the AST results are 153.73, 161.07, 157.50, 156.10, and 155.13 U / L respectively, the LDH results are 328.00, 267.33, 409.00, 348.67, and 320.33 g / mL respectively, the BUN results are 6.64, 6.66, 6.40, 6.72, and 5.98 mM / L respectively, and the Cre results are 21.10, 21.47, 21.00, 21.60, and 20.63 μM / L respectively, and there are no statistical differences. In addition, through the hemolysis test, the hemolysis rate under physiological conditions was further studied. As Figure 30As shown, the blood sample incubated with deionized water was a red transparent solution, indicating that red blood cells ruptured and lysed, resulting in hemolysis. However, no visible hemolysis occurred in the blood samples incubated with HPB, MPC-Gel, and HPB MPC-Gel, and the relative hemolysis rates of each group were less than 1%, indicating that HPB, MPC-Gel, and HPB-hydrogel all have good blood compatibility.

[0171] In summary, the present invention prepared a mucin hydrogel preparation loaded with hollow Prussian blue nanozyme. The in vitro and in vivo evaluation results of HPB MPC-Gel showed that the hydrogel played a certain role in improving IBD symptoms and is expected to become a means for treating IBD.

[0172] Obviously, the above embodiments of the present invention are merely examples for clearly explaining the present invention and are not intended to limit the implementation manners of the present invention. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to enumerate all the implementation manners here. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention shall be included in the protection scope of the claims of the present invention.

Claims

1. A method for preparing a mucin thermosensitive hydrogel, characterized in that: The following steps are involved: The Pluronic F127 dibenzaldehyde, glycidyl chitosan and mucin solution are mixed, and the hollow Prussian blue nanozyme is added to form a mixed solution to obtain the mucin thermosensitive hydrogel HPB MPC-Gel.

2. The preparation method according to claim 1, characterized in that: In the mixed solution, the mass concentration of Pluronic F127 dibenzaldehyde 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 nanozyme was 1 mg / mL.

3. The preparation method according to claim 1, characterized in that: The preparation method of the hollow Prussian blue nanozyme is as follows: Prussian blue nanozyme and polyvinyl pyrrolidone are dissolved in a hydrochloric acid solution, stirred at room temperature, transferred to a high-pressure reactor, and continued to react in an oil bath. After the reaction is completed, the hollow Prussian blue nanozyme is obtained by centrifugation, washing, and freeze-drying.

4. The preparation method according to claim 3, characterized in that: The mass ratio of the Prussian blue nanozyme to polyvinyl pyrrolidone is 1:5; The stirring speed at room temperature is 50-200 rpm, and the time is 3.5 h; The conditions for continuing the reaction in the oil bath are: constant temperature reaction at 140° C. for 4 hours; The centrifugal conditions are: 11000 rpm / min, 10 min.

5. The preparation method according to claim 4, characterized in that: The preparation method of the Prussian blue nanozyme is as follows: potassium ferrocyanide and polyvinyl pyrrolidone are dissolved in a hydrochloric acid solution, magnetically stirred, transferred into a hydrothermal reactor for high temperature and high pressure reaction, and after the reaction is completed, centrifuged, washed, and freeze-dried in sequence to obtain the Prussian blue nanozyme.

6. The preparation method according to claim 5, characterized in that: The mass ratio of potassium ferrocyanide to polyvinyl pyrrolidone is 22:500; The speed of the magnetic stirring is 50-200 rpm, and the time is 0.5 h; The conditions of the high temperature and high pressure reaction in the hydrothermal reactor are: constant temperature of 80°C and reaction time of 20h; The centrifugal conditions are: 11000 rpm / min, 15 min.

7. The mucin thermosensitive hydrogel prepared by the preparation method according to any one of claims 1 to 6.

8. Use of the mucin thermosensitive hydrogel according to claim 7 in the preparation of a medicament for treating inflammatory bowel disease.

9. A drug for treating inflammatory bowel disease, characterized in that: Comprising the mucin thermosensitive hydrogel as described in claim 7.

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