A gel factor loaded with anti-inflammatory drugs, hydrogel, preparation method and application thereof

Abstract

The present invention discloses a gel factor loaded with anti-inflammatory drugs, a hydrogel, and a preparation method and application thereof, which belongs to the field of gel materials and medicine, wherein the structural formula of the gel factor is: The hydrogel prepared by using the gel factor can be used to deliver drugs to the inflamed colon mucosa, and shows a highly effective therapeutic effect after enema in an acute colitis mouse model. With excellent targeting ability and high therapeutic efficiency, the hydrogel provides a powerful platform for the treatment of inflammatory bowel disease while reducing systemic toxicity.

Description

Technical Field

[0001] The invention relates to the field of gel materials and medicine, and in particular to a gel factor loaded with anti-inflammatory drugs, a hydrogel, and a preparation method and application thereof. Background Art

[0002] The American journal Journal of the American Chemical Society (J. AM. CHEM. SOC. 2010, 132, 17707–17709) reported that tripeptide derivatives were coupled with the anti-inflammatory drug olsalazine to produce small molecules. These small molecules self-assembled in water to form supramolecular hydrogels, which underwent a gel-sol phase transition after reduction, thereby allowing the anti-inflammatory drug 5-aminosalicylic acid (5-ASA) to be released in a controlled manner. However, no research has been done on the tracking and therapeutic application of this molecule in animals.

[0003] The American Association for the Advancement of Science's Science Translational Medicine (SCI. TRANSL. MED. 2015, 7(300):300ra128-300ra128.) reported that hydrogels prepared from ascorbyl palmitate were used to encapsulate and target the delivery of the anti-inflammatory corticosteroid dexamethasone prodrug. The drug-loaded hydrogel can preferentially adhere to the inflammatory site after rectal administration, and release dexamethasone under the action of hydrolases to inhibit intestinal inflammation. So far, supramolecular hydrogels formed by gel factors coupled with the anti-inflammatory drug olsalazine have not been seen for targeted treatment of colitis at the animal level. Summary of the invention

[0004] The purpose of the present invention is to provide a gel factor loaded with anti-inflammatory drugs to solve the problems raised in the above background technology.

[0005] To achieve the above objectives, the embodiments of the present invention provide the following technical solutions:

[0006] A gel factor loaded with an anti-inflammatory drug, wherein the structural formula of the gel factor is: .

[0007] Another object of the present invention is to provide a method for preparing the above-mentioned gel factor, which comprises the following steps:

[0008] Compound A was synthesized by solid phase peptide synthesis;

[0009] Dissolving compound A, (S)-(+)-4-nitro-7-(3-aminopyrrolidin-1-yl)-2,1,3-benzoxadiazole, 1-hydroxybenzotriazole, benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate and N,N-diisopropylethylamine in a solvent, and reacting to obtain compound B;

[0010] The N-fluorenylmethoxycarbonyl protecting group of compound B is reacted with a solution containing piperidine, and then trifluoroacetic acid is added to neutralize the base therein to obtain compound C;

[0011] Olsalazine sodium, 1-hydroxybenzotriazole and benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate are mixed and stirred in a solvent containing N,N-diisopropylethylamine, and then compound C is added dropwise thereto for reaction to obtain the gel factor;

[0012] Wherein, the structural formula of compound A is formula A, the structural formula of compound B is formula B, and the structural formula of compound C is formula C: ; ; .

[0013] Preferably, the step of synthesizing compound A by solid phase peptide synthesis specifically comprises:

[0014] After 2-chlorotrityl chloride resin is swollen in a solvent, N-fluorenylmethoxycarbonyl-L-phenylalanine is added, and then N,N-diisopropylethylamine is added to react; then, methanol is used for end-capping; then, the protecting group of phenylalanine is cut off with piperidine, and the activated second amino acid N-fluorenylmethoxycarbonyl-L-phenylalanine is added to react, the protecting group of phenylalanine is cut off, and the activated third amino acid N-fluorenylmethoxycarbonyl-L-phenylalanine is added to react; finally, the synthesized peptide segment is cut off from the resin with a dichloromethane solution of trifluoroacetic acid, precipitated with ether, centrifuged at low temperature and the upper ether is removed by decanting, and the ether is evaporated to dryness, and then separated and purified to obtain the compound A.

[0015] Preferably, the solvent is N,N-dimethylformamide.

[0016] Another object of an embodiment of the present invention is to provide a hydrogel prepared from the above-mentioned gel factor.

[0017] Another object of the present invention is to provide a method for preparing a hydrogel, which comprises the following steps:

[0018] The gel factor is dispersed in a phosphate buffer, and then a saturated sodium carbonate solution is used to adjust the pH value until the gel factor is completely dissolved to obtain the hydrogel.

[0019] Another object of an embodiment of the present invention is to provide a use of the above-mentioned hydrogel in preparing a drug for treating inflammatory bowel disease.

[0020] The hydrogel formed by the gel factor provided in the embodiment of the present invention is an injectable supramolecular nanomaterial, which has the following significant advantages compared with the existing supramolecular hydrogels: (1) It contains NBD-Apy, which is an environmentally sensitive fluorophore. The higher emission of the nanofibers indicates that the molecules in the nanofibers are more orderly, and the targeting of the hydrogel to the inflammatory site can be traced. (2) The loaded anti-inflammatory drug olsalazine can give the self-assembled nanofibers a negative charge, which can preferentially adhere to the positively charged intestinal inflammatory mucosal surface. (3) The olsalazine loaded in the hydrogel releases 5-ASA in situ in the inflamed colon mucosa. (4) The gel factor can control the gelation of the gel factor by simply adjusting the pH.

[0021] In summary, the supramolecular hydrogel containing olsalazine provided in the embodiments of the present invention can be used to deliver drugs to the inflamed colon mucosa, and shows efficient therapeutic effects after enema in an acute colitis mouse model. The gel factor exhibits excellent self-assembly properties and generates injectable hydrogels. The hydrogel with a strong negative surface charge can preferentially adhere to the inflamed colon and release 5-ASA in situ. Even in the acute colitis mouse model induced by 4% dextran sodium sulfate (DSS), the hydrogel can effectively restore body weight and colon length by rectal administration. With excellent targeting ability and high therapeutic efficiency, the hydrogel provides a powerful platform for the treatment of inflammatory bowel disease while reducing systemic toxicity. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Schematic diagram of Olsa-Hydrogel, a hydrogel formed by self-assembly of the gel factor Olsa-FFF-NBD-Apy, which preferentially binds to the inflamed cationic mucosal surface and releases 5-ASA under the action of azoreductase secreted by the colonic microbiota to treat inflammatory bowel disease.

[0023] Figure 2 This is the high-resolution mass spectrometry (HR-ESI-MS) analysis result of the synthesized gel factor Olsa-FFF-NBD-Apy.

[0024] Figure 3 The hydrogen spectrum of the synthesized gel factor Olsa-FFF-NBD-Apy ( 1 H NMR) analysis results.

[0025] Figure 4 Optical photographs of the transparent solution of Olsa-FFF-NBD-Apy and the hydrogel (20.4 mM, 2 wt%) formed at pH = 8.0.

[0026] Figure 5 This is a cryo-transmission electron microscopy image of the hydrogel Olsa-Hydrogel.

[0027] Figure 6 Zeta potential diagram of hydrogel Olsa-Hydrogel at different pH values ​​in PBS.

[0028] Figure 7 The stress scanning graphs of the dynamic storage modulus (G') and loss modulus (G'') of the hydrogel Olsa-Hydrogel.

[0029] Figure 8 The dynamic frequency scanning graph of the dynamic storage modulus (G') and loss modulus (G'') of the hydrogel Olsa-Hydrogel at 0.1% strain.

[0030] Fig. 9 Fluorescence images and quantification of fluorescence intensity after the distal colon of induced colitis mice and healthy control mice were incubated with hydrogel Olsa-Hydrogel at 37°C for 30 minutes in vitro.

[0031] Fig.10 The figure shows the therapeutic effect of hydrogel Olsa-Hydrogel on acute colitis (the observation period is 9 days); among them, Figure a shows the daily changes in mouse body weight; Figure b shows the daily changes in disease activity index (DAI) score; Figure c shows the optical photograph of the macroscopic colon appearance of each mouse; Figure d shows the colon length statistics of each group of mice (n=5); the significance between each two groups was evaluated using t-test; ns, not significant; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0032] Fig.11 The figure shows the therapeutic effect of hydrogel Olsa-Hydrogel on acute colitis (the observation period was 12 days); among them, Figure a shows the daily changes in mouse body weight; Figure b shows the daily changes in disease activity index (DAI) score; Figure c shows the optical photograph of the macroscopic colon appearance of each mouse; Figure d shows the colon length statistics of each group of mice (n=5); the significance between each two groups was evaluated using t-test; ns, not significant; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. DETAILED DESCRIPTION

[0033] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0034] In one embodiment of the present invention, a gel factor loaded with an anti-inflammatory drug is provided, wherein the structural formula of the gel factor is: .

[0035] In another embodiment of the present invention, a method for preparing the above-mentioned gel factor is also provided, and the synthesis process thereof is as follows: ;

[0036] The above preparation method specifically comprises the following steps:

[0037] S1. Synthesize compound A by solid phase peptide synthesis; specifically, 1 mmol of 2-chlorotrityl chloride resin was swollen in 2-3 mL of N,N-dimethylformamide for 20-40 minutes, and then 2 mmol of N-fluorenylmethoxycarbonyl-L-phenylalanine was added, and then 2 mmol of N,N-diisopropylethylamine was added, and the reaction was continued for 6-8 hours. End-capping was performed with 200 μL of methanol for 30 minutes. Then, the protecting group of phenylalanine was cut off with 20% piperidine, and 1.6 mmol of the second amino acid N-fluorenylmethoxycarbonyl-L-phenylalanine was added to react for 6-8 hours, and the protecting group of phenylalanine was cut off, and 1.6 mmol of the third amino acid N-fluorenylmethoxycarbonyl-L-phenylalanine was added to react for 6-8 hours. Finally, the synthesized peptide segment was cut from the resin using a dichloromethane solution with a volume concentration of 1% trifluoroacetic acid, precipitated with ether, centrifuged and the upper ether was removed by decantation, and the white solid powder obtained after the ether was evaporated to dryness was the crude product of the oligopeptide sequence of compound A - N-fluorenylmethoxycarbonyl-L-phenylalanine-phenylalanine-phenylalanine, which was separated and purified by high performance liquid chromatography to obtain compound A.

[0038] S2. Dissolve compound A, (S)-(+)-4-nitro-7-(3-aminopyrrolidin-1-yl)-2,1,3-benzoxadiazole ((S)-(+)-NBD-APy), 1-hydroxybenzotriazole (HOBT), benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) and N,N-diisopropylethylamine (DIPEA) in N,N-dimethylformamide (DMF), stir at room temperature for 2-4 hours to react, and obtain compound B.

[0039] S3. The N-fluorenylmethoxycarbonyl (Fmoc) protecting group of compound B is reacted with a DMF solution containing 10% piperidine at 0°C with stirring for 5-15 minutes, and then trifluoroacetic acid is added to neutralize the base therein to obtain compound C.

[0040] S4, mixing olsalazine sodium, 1-hydroxybenzotriazole and benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate in a solvent containing N,N-diisopropylethylamine, and then adding compound C dropwise thereto for reaction to obtain a gel factor named Olsa-FFF-NBD-Apy;

[0041] In another embodiment of the present invention, a method for preparing a hydrogel is provided, comprising the following steps:

[0042] The above-mentioned gel factor Olsa-FFF-NBD-Apy was dispersed in phosphate buffer (pH=7.4), and then the pH value was adjusted to 8.0 using a saturated sodium carbonate solution to completely dissolve the gel factor to obtain a transparent hydrogel named Olsa-Hydrogel.

[0043] Example 1: This example provides a method for synthesizing compounds A, B, C and gel factor Olsa-FFF-NBD-Apy, which specifically includes the following steps:

[0044] S1. Synthesize compound A by solid phase peptide synthesis; specifically, 1 mmol of 2-chlorotrityl chloride resin was swollen in 2.5 mL of N,N-dimethylformamide for 30 minutes, and then 2 mmol of N-fluorenemethoxycarbonyl-L-phenylalanine was added, and then 2 mmol of N,N-diisopropylethylamine was added, and the reaction was continued for 7 hours. Cap with 200 μL of methanol for 30 minutes. Then, the protecting group of phenylalanine was cut off with 20% piperidine, and 1.6 mmol of the second amino acid N-fluorenemethoxycarbonyl-L-phenylalanine was added to react for 7 hours, and the protecting group of phenylalanine was cut off, and 1.6 mmol of the third amino acid N-fluorenemethoxycarbonyl-L-phenylalanine was added to react for 7 hours. Finally, the synthesized peptide segment was cut from the resin using a dichloromethane solution with a volume concentration of 1% trifluoroacetic acid, precipitated with ether, centrifuged and the upper ether was removed by decantation, and the white solid powder obtained after the ether was evaporated to dryness was the crude product of the oligopeptide sequence of compound A - N-fluorenylmethoxycarbonyl-L-phenylalanine-phenylalanine-phenylalanine, which was separated and purified by high performance liquid chromatography to obtain compound A.

[0045] S2. Compound A (410 mg, 0.601 mmol), (S)-(+)-4-nitro-7-(3-aminopyrrolidin-1-yl)-2,1,3-benzoxadiazole ((S)-(+)-NBD-APy, 100 mg, 0.401 mmol), 1-hydroxybenzotriazole (HOBT, 81.2 mg, 0.601 mmol), benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU, 227.9 mg, 0.601 mmol) and N,N-diisopropylethylamine (DIPEA, 77.6 mg, 0.601 mmol) were dissolved in N,N-dimethylformamide (DMF, 3.0 mL), and the mixture was stirred at room temperature for 3 hours. The obtained compound was separated and purified by high performance liquid chromatography and named as compound B.

[0046] S3. The N-fluorenylmethoxycarbonyl (Fmoc) protecting group of compound B was reacted with a DMF solution containing 10% piperidine (0.2 mL piperidine + 1.8 mL DMF) at 0°C with stirring for 10 minutes, and then 180 μL trifluoroacetic acid was added to neutralize the base. The obtained compound was separated and purified by HPLC and named compound C.

[0047] S4. Olsalazine sodium (164 mg, 0.47 mmol), HOBT (64 mg, 0.47 mmol) and HBTU (179.7 mg, 0.47 mmol) were mixed and stirred in DMF (5 mL) containing DIPEA (122.6 mg, 0.948 mmol) for 30 minutes to obtain a mixture. Compound C (163.7 mg, 0.23 mmol, dissolved in 2 mL DMF) was added dropwise to the above mixture and stirred at room temperature for 3 hours. The obtained compound was separated and purified by high performance liquid chromatography to obtain the gel factor Olsa-Phe-Phe-Phe-(S)-(+)-4-(3-Amino-pyrrolidino)-7-nitrobenzofurazan (Olsa-FFF-NBD-Apy).

[0048] The hydrogel Olsa-Hydrogel formed by the self-assembly of the above-mentioned gel factor Olsa-FFF-NBD-Apy can preferentially bind to the inflamed cationic mucosal surface and release 5-aminosalicylic acid (5-ASA) under the action of azoreductase secreted by the colonic microbiota to treat inflammatory bowel disease, specifically Figure 1As shown in the figure, the structure of Olsa-FFF-NBD-Apy contains three components: (1) NBD-Apy is an environmentally sensitive fluorophore, and the higher emission of the nanofibers indicates that the molecules in the nanofibers are more ordered. (2) FFF serves as the self-assembly backbone and the linker of NBD-Apy and Olsa through amide bonds. (3) Olsalazine can not only release 5-ASA as an anti-inflammatory drug, but also endow the self-assembled nanofibers with negative charges, targeting cationic inflammatory mucosal surfaces.

[0049] 1 H NMR was obtained on a 400 MHz Bruker AV 400. High-resolution electrospray ionization mass spectra (HR-ESI-MS) were obtained on a Finnigan LCQ Advantage ion trap mass spectrometer (Thermo Fisher Corporation) equipped with a standard ESI source. The results of high-resolution mass spectrometry (HR-ESI-MS) analysis of the above-synthesized gel factor Olsa-FFF-NBD-Apy are shown in Figure 2 As shown. Figure 2 It can be seen that the mass spectrum result of Olsa-FFF-NBD-Apy is obsvd. ESI-MS: 975.34198. The hydrogen spectrum of the above-synthesized gel factor Olsa-FFF-NBD-Apy ( 1 H NMR) analysis results are as follows Figure 3 As shown. Figure 3 It can be seen that the nuclear magnetic resonance hydrogen spectrum of Olsa-FFF-NBD-Apy (d6-dimethyl sulfoxide, 400MHz) δ (ppm): 1 H NMR (400MHz, DMSO- d 6) δ 12.51 (s, 1H), 8.86 (dd, J = 22.6, 7.0 Hz, 1H), 8.53 (dd, J =21.1, 7.3 Hz, 1H), 8.44 – 8.27 (m, 4H), 8.00 (d, J = 8.9 Hz, 1H), 7.84 (d, J = 8.9 Hz, 1H), 7.27 – 7.10 (m, 15H), 7.06 – 6.99 (m, 3H), 6.15 (dd, J = 14.4,7.4 Hz, 1H), 4.68 – 4.07 (m, 8H), 3.07 (dd, J= 13.6, 5.1 Hz, 2H), 2.87 (s,2H), 2.75 – 2.63 (m, 2H), 1.85 (d, J = 74.0 Hz, 2H).

[0050] Example 2: This example provides a method for characterizing the formation, morphology and potential of the supramolecular hydrogel Olsa-Hydrogel, as follows:

[0051] Preparation of hydrogel: The synthesized gel factor Olsa-FFF-NBD-Apy (2 mg, dissolved in dimethyl sulfoxide) was dispersed in 10 mM phosphate buffer (PBS, 200 μL, pH = 7.4); then, a saturated solution of sodium carbonate (Na2CO3) was used to adjust the pH value to 8.0 to completely dissolve the compound, and a transparent orange gel was formed within 20 minutes, which was the hydrogel Olsa-Hydrogel (1 wt%).

[0052] Zeta potential measurement: 200 μL of hydrogel (2 wt%) was dispersed in 1 mL of PBS. The dispersion was further diluted 100 times in PBS with different pH values ​​(6, 6.5, 7, 7.4, 8, 9, 10) and transferred to a cuvette for Zeta potential measurement. Each sample was measured 3 times, and 3 cycles were run each time. The measurement temperature was set to 25 °C.

[0053] Zeta potential was measured on a Zeta Sizer Nano-ZS90 (Malvern, UK). Cryo-transmission electron micrograph (Cryo-TEM) images were acquired on a FEI-Tecnai field emission transmission electron microscope at an accelerating voltage of 200 KV. Figure 4 Optical photographs of the transparent solution of Olsa-FFF-NBD-Apy and the hydrogel (20.4 mM, 2 wt%) formed at pH = 8.0. Figure 5 This is a cryo-TEM image of Olsa-Hydrogel. Figure 6 is the Zeta potential of Olsa-Hydrogel at different pH values ​​in PBS. Figure 4 It can be seen that after Olsa-FFF-NBD-Apy was dispersed in phosphate buffered saline (PBS, pH = 7.4) at a concentration of 20 mg / mL and the pH value was adjusted to 8.0, the solution became clear and then formed an orange gel (20.4 mM, 2 wt%) within 20 minutes. Figure 5 It can be seen that Olsa-Hydrogel showed long and loose fibers in the cryo-TEM images, with an average diameter of 3.0 ± 0.4 nm. Figure 6It can be seen that the zeta potential of Olsa-Hydrogel solution in the pH range of 6 to 10 showed a strong negative surface charge (~-45 mV), which was due to the acidic carboxyl groups provided by olsalazine in the hydrophilic outer layer, indicating that the hydrogel Olsa-Hydrogel has the potential to adhere to cationic inflamed mucosal surfaces.

[0054] Example 3: This example provides an experiment to detect the mechanical properties of the supramolecular hydrogel Olsa-Hydrogel.

[0055] Rheological analysis: Oscillatory rheological experiments were performed using a Haake AR-G2 shear rheometer (TA Instruments). 1 mL of Olsa-Hydrogel (1 wt%) was placed on a 4 cm heated horizontal plate at 37 °C. The sample was subjected to the following process: frequency sweep test from 300.0 to 0.1000, 20 points per decade, 0.10000% strain, and strain sweep test from 0.010000 to 500, 20 points per decade, 6.283 rad / s angular frequency.

[0056] Figure 7 Stress scan graphs of the dynamic storage modulus (G') and loss modulus (G'') of Olsa-Hydrogel. Figure 8 The dynamic frequency scanning spectrum of the dynamic storage modulus (G') and loss modulus (G'') of Olsa-Hydrogel at 0.1% strain. Figure 7 As shown, the values ​​of the storage modulus (G') and loss modulus (G'') of the sample show a weak dependence in the strain range of 0.01% to 1.00% (G'>G''), indicating that the sample is in a gel-like state. When the strain increases, G' decreases significantly and becomes less than G'' (G'<G''), indicating that the material will exist in an injectable state. Figure 8 It can be seen that after setting the strain amplitude to 0.10% (within the linear response range of the strain amplitude), the value of G' is approximately 8 to 12 times that of G'' in the range of 0.1 to 100 Hz, indicating that the hydrogel has considerable tolerance to external shear force.

[0057] Example 4: This example provides an experiment on the targeting of supramolecular hydrogel Olsa-Hydrogel to inflammatory colon.

[0058] Inflammatory intestinal adhesion test: Eight-week-old male Balb / c mice were divided into: (i) normal control group (healthy mice given normal water) and (ii) DSS-treated group (mice induced with 4% DSS water for 7 days). The mice were sacrificed, and the distal 1.5 cm colon tissue excluding the anus was obtained from each mouse. 200 μL of 2 wt% hydrogel Olsa-Hydrogel was suspended in 10 mL PBS. The colon was cut longitudinally, immersed in 0.5 mL of gel suspension, and incubated at 37°C with gentle shaking for 30 min. After washing with PBS three times, the colon was imaged using an IVIS fluorescent imager (IVIS Spectrum, PerkinElmer), and the fluorescence signal intensity was quantified using Living Image software (version 4.3.1, PerkinElmer). Data are mean ± SEM; p values ​​were determined by Student's t test.

[0059] Fig. 9 Fluorescence images and fluorescence quantification of the distal colon of colitis mice and healthy controls after incubation with Olsa-Hydrogel at 37°C for 30 minutes. Fig. 9 It can be seen that Olsa-Hydrogel preferentially adheres to the inflamed colon, and the fluorescence signal intensity of the inflamed colon group is 1.5 times that of the healthy control group, proving that the supramolecular hydrogel Olsa-Hydrogel has good targeting to the inflammatory colon.

[0060] Example 5: This example provides an experiment on the therapeutic effect of supramolecular hydrogel Olsa-Hydrogel on colitis.

[0061] Animal experiment of Olsa-Hydrogel in the treatment of colitis: To study the therapeutic effect of Olsa-Hydrogel enema on DSS-induced acute colitis model mice, 8-week-old male Balb / c mice were divided into the following groups: (i) normal control group (NC group), healthy mice were given normal water; (ii) PBS treatment group, mice were induced with 4% DSS for 7 days and then given PBS enema; (iii) Olsa treatment group, mice were induced with 4% DSS for 7 days and then given free olsalazine solution enema; (iv) Olsa-Hydrogel treatment group, mice were induced with 4% DSS for 7 days and given 1.0 wt% Olsa-Hydroge enema on the 5th and 7th days, respectively.

[0062] Rectal bleeding, stool consistency, and body weight of mice were recorded daily as daily evaluation indicators. On day 9, feces of the Olsa-Hydrogel-treated group and NC were collected and stored at -80°C until analysis. At the end of the experiment, the intestinal segment from the cecum to the anus was removed and measured in length, and the distal colon was fixed with 4% paraformaldehyde for histological examination.

[0063] Disease Activity Index: The blood in stool, stool consistency, and weight loss of mice were summed up and calculated as DAI. Blood in stool score: 0 points, no blood; 1 point, trace blood; 2 points, slight blood in stool; 3 points, obvious blood; 4 points, severe blood in stool. Stool consistency: 0 points, normal; 1 point, loose stool; 2 points, mild diarrhea; 3 points, diarrhea; 4 points, severe diarrhea. The percentage change in body weight from day 0 to the end of the experiment was calculated to assess weight loss: 0 points, no weight loss; 1 point, 1-5%; 2 points, 5-10%; 3 points, 10-20%; 4 points, >20%).

[0064] Fig.10 Results of the 9-day observation period for the evaluation of the therapeutic effect of colitis: Fig.10 a is the daily weight change of mice; Fig.10 b is the change of daily disease activity index (DAI) score of mice; Fig.10 c is the macroscopic colon appearance of each mouse; Fig.10 The d is the colon length statistics of each group of mice. The significance between each two groups was evaluated using the t test (and nonparametric test); ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0065] Fig.11 Results of the 12-day observation period for the evaluation of the therapeutic effect of colitis: Fig.11 a is the daily weight change of mice; Fig.11 b is the change of daily disease activity index (DAI) score of mice; Fig.11 c is the macroscopic colon appearance of each mouse; Fig.11 The d is the colon length statistics of each group of mice. The significance between each two groups was evaluated using the t test (and nonparametric test); ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0066] like Fig.10 As shown in a, 5 days after induction with DSS, the body weight of all mice began to decrease rapidly relative to the normal group. However, after treatment with Olsa-Hydrogel on the 7th day, the body weight of mice gradually recovered. Fig.10 As can be seen in b, mice treated with Olsa-Hydrogel showed the lowest disease activity index among all mice with colitis. Fig.10As shown in c and d, the colon length of the Olsa-Hydrogel-treated group was significantly longer than that of the PBS and olsalazine-treated groups on day 9, indicating that Olsa-Hydrogel has a protective effect on DSS-induced reduction in colon length. However, compared with the normal control group, the colon length of the Olsa-Hydrogel-treated group was still slightly shorter on day 9. Fig.11 It can be seen that the observation time of the Olsa-Hydrogel-treated group and the normal group was further extended to day 12, and the colon of the DSS-treated mouse group recovered to a length similar to that of the colon of normal mice after Olsa-Hydrogel administration. In addition, the body weight showed a recovery trend, and the DAI score quickly dropped to normal after treatment.

[0067] Based on the above-mentioned ideal embodiments of the present invention, the relevant staff can make various changes and modifications without departing from the technical concept of the present invention through the above-mentioned description. The technical scope of the present invention is not limited to the contents of the specification.

Claims

1. A hydrogel, characterized in that: The preparation method of the hydrogel comprises the following steps: Dispersing the gel factor in a phosphate buffer, and then adjusting the pH value using a saturated sodium carbonate solution until the gel factor is completely dissolved, thereby obtaining the hydrogel; The structural formula of the gel factor is: ； The preparation method of the gel factor comprises the following steps: Compound A was synthesized by solid phase peptide synthesis; Dissolving compound A, (S)-(+)-4-nitro-7-(3-aminopyrrolidin-1-yl)-2,1,3-benzoxadiazole, 1-hydroxybenzotriazole, benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate and N,N-diisopropylethylamine in a solvent, and reacting to obtain compound B; The N-fluorenylmethoxycarbonyl protecting group of compound B is reacted with a solution containing piperidine, and then trifluoroacetic acid is added to neutralize the base therein to obtain compound C; Olsalazine sodium, 1-hydroxybenzotriazole and benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate are mixed and stirred in a solvent containing N,N-diisopropylethylamine, and then compound C is added dropwise thereto for reaction to obtain the gel factor; Wherein, the structural formula of compound A is formula A, the structural formula of compound B is formula B, and the structural formula of compound C is formula C: ； ； ； The steps of synthesizing compound A by solid phase peptide synthesis specifically include: After 2-chlorotrityl chloride resin is swollen in a solvent, N-fluorenylmethoxycarbonyl-L-phenylalanine is added, and then N,N-diisopropylethylamine is added to react; then, methanol is used for end-capping; then, the protecting group of phenylalanine is cut off with piperidine, and the activated second amino acid N-fluorenylmethoxycarbonyl-L-phenylalanine is added to react, the protecting group of phenylalanine is cut off, and the activated third amino acid N-fluorenylmethoxycarbonyl-L-phenylalanine is added to react; finally, the synthesized peptide segment is cut off from the resin with a dichloromethane solution of trifluoroacetic acid, precipitated with ether, centrifuged at low temperature and the upper ether is removed by decanting, and the ether is evaporated to dryness, and then separated and purified to obtain the compound A.

2. The hydrogel according to claim 1, characterized in that The solvent is N,N-dimethylformamide.

3. Use of the hydrogel according to claim 1 or 2 in the preparation of a drug for treating inflammatory bowel disease.

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