Positioning formed liquid hydrogel as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of material synthesis and molecular biology, and relates to a positioning forming liquid hydrogel as well as a preparation method and application thereof. The raw materials comprise a solution 1 and a solution 2, the solution 1 contains calcium salt, and the solution 2 contains sodium alginate and a small molecule drug; the small molecule drug is formed by oxsalazine, ellagic acid and zinc ions through metal-polyphenol coordination. According to the invention, oxalazine, EA and Zn salt are synthesized into a small molecule drug to remove ROS and relieve inflammation and improve the treatment effect of IBD; according to the present invention, the traditional Chinese medicine extract and the hydrogel raw material can be used to prepare the drinkable liquid hydrogel formed in the stomach so as to overcome the difficult swallowing problem and improve the drug compliance, and further have effects of synergetic anti-inflammation, ROS removing and mitochondrial dysfunction alleviating so as to achieve the colitis in-vivo treatment.

Description

Technical Field

[0001] The invention belongs to the technical field of material synthesis and molecular biology, and relates to a positionally formed liquid hydrogel and a preparation method and application thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not be necessarily regarded as an admission or any form of suggestion that the information constitutes the prior art already known to those skilled in the art.

[0003] Inflammatory bowel disease (IBD) is a chronic, recurrent gastrointestinal inflammatory disease with clinical symptoms including abdominal pain, bloody diarrhea, intestinal mucosal ulcers, fatigue, anemia, and weight loss. It is clinically divided into two subtypes: Crohn's disease and ulcer disease. With the rapid development of the economy and changes in lifestyle, the incidence of IBD has increased rapidly in the past 20 years, especially in the past 10 years, and has tended to be younger. There are three main types of drugs for the clinical treatment of IBD: aminosalicylic acid, glucocorticoids, and immunosuppressants. However, these drugs are limited to relieving symptoms and require long-term drug treatment.

[0004] The inflammatory process of IBD is mainly the formation of reactive oxygen species (ROS) and reactive nitrogen species, which lead to the excessive production of proinflammatory cytokines. Recent studies have shown that the use of antioxidant natural small molecules, proteins or synthetic inorganic polymer nanomaterials to regulate immune balance provides a promising antioxidant therapeutic strategy for the treatment of IBD. Ellagic acid (EA) is a natural polyphenol with antioxidant, antibacterial and anti-inflammatory biological activities. The two aromatic rings in EA lead to its low solubility in water, resulting in its low bioavailability.

[0005] In addition, IBD patients, especially those with dysphagia (such as children), have difficulty swallowing, so their medication compliance is poor, making it difficult to achieve the expected effects of the drugs. Summary of the invention

[0006] In order to overcome the deficiencies of the prior art, the purpose of the present invention is to provide a position-forming liquid hydrogel and a preparation method and application thereof. The present invention first synthesizes olsalazine (Olsa), EA and Zn salt into a small molecule (Olsa / EA / Zn) drug to remove ROS and relieve inflammation, thereby improving the therapeutic effect of IBD; then, it is combined with a hydrogel raw material to not only prepare a drinkable liquid hydrogel formed in the stomach, thereby overcoming the problem of dysphagia and improving drug compliance, but also has the effects of synergistic anti-inflammatory, ROS removal and alleviation of mitochondrial dysfunction, thereby achieving in vivo treatment of colitis.

[0007] In order to achieve the above object, the present invention provides the following technical solutions:

[0008] In the first aspect, a small molecule drug is formed by olsalazine, ellagic acid and zinc ion through metal-polyphenol coordination.

[0009] Low toxicity Zn 2+ It is an essential trace element for the human body and is closely related to anti-inflammatory effects; Olsalazine is a drug for treating intestinal inflammation, and its chemical structure contains a large number of phenolic hydroxyl groups; therefore, the present invention uses Zn 2+ The small molecule Olsa / EA / Zn drug formed by metal-polyphenol coordination with olsalazine and ellagic acid can scavenge ROS and relieve inflammation, thereby improving the therapeutic effect of IBD.

[0010] In a second aspect, a positionally formed liquid hydrogel comprises raw materials including solution 1 and solution 2, wherein solution 1 contains calcium salt, and solution 2 contains sodium alginate (SA) and the above-mentioned small molecule drug.

[0011] When in use, solution 1 and solution 2 are placed in gastric juice in sequence to form a hydrogel directly in the gastric juice.

[0012] The SA (covalent cross-linking) and Ca in the hydrogel provided by the present invention 2+ (Ionic cross-linking) forms a hydrogel dual dynamic cross-linking network that can be quickly formed in gastric juice and has significant stability in the gastric acid environment. After entering the intestine, it has unique low swelling characteristics, reasonable drug release, and good cell compatibility. Because each raw material is in liquid form before use, when applicable, each raw material can be used in sequence to mix in gastric juice to form a hydrogel, thereby avoiding difficulty in swallowing caused by large particle drugs, thereby providing patients with drug compliance.

[0013] Furthermore, the raw material also includes polylysine (ε-Poly-l-lysine, ε-PL). ε-PL can form another dynamic cross-linking network with SA, which provides the toughness of the hydrogel and enhances the adhesion of the hydrogel through hydrogen bonds and electrostatic interactions. ε-PL can be placed in solution 1; it can also be made into solution 3. When used, first drink (or gavage) solution 1 and solution 3, and then drink (or gavage) solution 2.

[0014] A third aspect is the use of the above-mentioned small molecule drug or the position-formed liquid hydrogel in the preparation of a drug for treating inflammatory bowel disease.

[0015] The beneficial effects of the present invention are:

[0016] 1. The present invention uses olsalazine and zinc ions to form a small molecule drug through metal-polyphenol coordinated organic-inorganic hybridization with ellagic acid, which can remove ROS and relieve inflammation, thereby improving the therapeutic effect of IBD.

[0017] 2. The liquid hydrogel provided by the present invention is rapidly formed in gastric juice and can be formed in gastric juice by using (drinking) the raw materials separately, thereby overcoming the problem of dysphagia and improving drug compliance.

[0018] 3. The liquid hydrogel provided by the present invention has significant stability in the gastric acid environment, has unique low swelling characteristics after entering the intestine, reasonable drug release, and good cell compatibility. Animal model experiments on mice with colon inflammation showed that the liquid hydrogel provided by the present invention can reduce the disease activity index of mice, significantly increase the colon length of mice and reduce histopathological inflammation, and significantly promote the repair of colon tissue; it has a strong synergistic anti-inflammatory effect. At the same time, the hydrogel added with ε-PL also has the effect of clearing ROS and reducing mitochondrial dysfunction. Therefore, the liquid hydrogel provided by the present invention has the potential for in vivo treatment of colitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0020] Figure 1Figure 1 is a diagram showing the formation and characterization of Olsa / EA / Zn@SA / ε-PL hydrogels in an embodiment of the present invention; A is a photograph of different hydrogel systems in artificial gastric fluid; B is an image of gel formation in the stomach with Olsa / EA / Zn solution loaded, and C is an image of gel formation in the stomach with Olsa / EA / Zn solution loaded; D is a graph showing the formation of Olsa / EA / Zn@SA and Olsa / EA / Zn@SA / ε-PL hydrogels in artificial gastric fluid (AGF), artificial small intestinal fluid (ASF) and artificial colon fluid (ASF). fluid, ACF); E is the swelling ratio of Olsa / EA / Zn@SA hydrogel and Olsa / EA / Zn@SA / ε-PL hydrogel in AGF solution (n=3); F is the swelling ratio of Olsa / EA / Zn@SA hydrogel and Olsa / EA / Zn@SA / ε-PL hydrogel in ASF solution (n=3); G is the swelling ratio of Olsa / EA / Zn@SA hydrogel and Olsa / EA / Zn@SA / ε-PL hydrogel in ACF solution (n=3); H is the collected release medium photos of Olsa / EA / Zn@SA hydrogel and Olsa / EA / Zn@SA / ε-PL hydrogel in AGF solution, and Cumulative release of Olsa / EA / Zn from the hydrogel in AGF solution (n=3); I is the collected release medium photos of Olsa / EA / Zn@SA hydrogel and Olsa / EA / Zn@SA / ε-PL hydrogel in ASF solution, and the cumulative release of Olsa / EA / Zn from Olsa / EA / Zn@SA hydrogel and Olsa / EA / Zn@SA / ε-PL hydrogel in ASF solution (n=3); J is the collected release medium photos of Olsa / EA / Zn@SA hydrogel and Olsa / EA / Zn@SA / ε-PL hydrogel in ACF solution, and the cumulative release of Olsa / EA / Zn from Olsa / EA / Zn@SA hydrogel and Olsa / EA / Zn@SA / ε-PL hydrogel in ACF solution (n=3).

[0021] Figure 2Figure 5 is a characterization diagram of the biocompatibility of Olsa / EA / Zn@SA / ε-PL hydrogel in an embodiment of the present invention; A is a graph showing the cell viability of CCD841 cells treated with different concentrations of Olsa / EA / Zn@SA hydrogel and Olsa / EA / Zn@SA / ε-PL hydrogel for 24h and 48h; B is a graph showing the live cells (green) and dead cells (red) of CCD841 cells treated with Olsa / EA / Zn@SA hydrogel and Olsa / EA / Zn@SA / ε-PL hydrogel (100ng / mL) for 24h and 48h; C is a graph showing the cell migration of CCD841 cells treated with Olsa / EA / Zn@SA hydrogel and Olsa / EA / Zn@SA / ε-PL hydrogel (100ng / mL) for 24h and 48h.

[0022] Figure 3 The results of Olsa / EA / Zn@SA / ε-PL hydrogel treating DSS-induced chronic colitis mice in an embodiment of the present invention; A is a schematic diagram of the DSS-induced chronic colitis mouse model, in which 2.5% DSS was continuously administered for 0-7 days, and control materials and Olsa / EA / Zn@SA / ε-PL hydrogel were orally administered by gavage on days 1, 3, 5, and 7; B is the weight change rate of mice during the treatment period; C is the DAI score of mice during the treatment period; D is a photo of colon tissue of mice in different groups; E is a low-magnification (upper) and high-magnification (lower) H&E staining image of colon tissue of mice in each group; F is a statistical graph of colon tissue of mice in different groups; G is a statistical graph of colon injury score; the values ​​represent mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001.

[0023] Figure 4 The results of the Olsa / EA / Zn@SA / ε-PL hydrogel in the present invention on DSS-induced inflammation in mice; A is a representative immunofluorescence staining of CD68 or F4 / 80 positive cells; B is the CD68 in the colon tissue of each group + or F4 / 80 + The percentage of the area (n = 6); C is the representative immunofluorescence staining of CD206 or F4 / 80 positive cells in colon tissue; D is the CD206 + or F4 / 80 + The percentage of the area (n=6); E is the expression level of IL-1β and Arg-1 protein in colon tissue; F is the statistical graph of the expression level of IL-1β and Arg-1 protein in colon tissue (n=4); the values ​​represent the mean±SD, *p<0.05, **p<0.01, ***p<0.001.

[0024] Figure 5The results of the Olsa / EA / Zn@SA / ε-PL hydrogel on the DSS-induced oxidative stress response in the present invention are shown in Figure 1. A is a representative ROS staining in colon tissue; B is a representative mtROS staining in colon tissue; C is the expression level of colon SOD2 and Nrf-2 protein in the colon detected by Western blotting; D is a statistical chart of the expression level of colon SOD2 and Nrf-2 protein in each group (n=4); the values ​​are mean ± SD. *p<0.05, **p<0.01, ***p<0.001. DETAILED DESCRIPTION

[0025] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0026] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0027] Since EA has a low solubility and thus a low bioavailability, its effect in treating IBD inflammation is poor. At the same time, IBD patients have difficulty swallowing, which makes it difficult for the drug to achieve the expected effect. Therefore, the present invention proposes a position-forming liquid hydrogel, a preparation method and an application thereof.

[0028] A typical embodiment of the present invention provides a small molecule drug formed by olsalazine, ellagic acid and zinc ion through metal-polyphenol coordination.

[0029] In certain embodiments, the preparation process is: ellagic acid is dissolved in ethanol, olsalazine is dissolved in water, zinc salt is dissolved in water, ellagic acid solution, olsalazine solution and zinc salt solution are mixed, ultrasonic treatment and stirring are carried out successively, and lyophilization is carried out after filtering. The present invention dissolves ellagic acid by ethanol, and utilizes the mutual solubility of ethanol and water to carry out complex coordination of olsalazine, ellagic acid and zinc ion. It is avoided that the temperature in ordinary drying is too high to destroy its coordination structure by freeze drying. In order to avoid introducing impurities, the water used in the present invention in the embodiment is ddH2O, which is also called double deionized water, double distilled water, ultrapure water, etc.

[0030] The zinc salt described in the present invention refers to a compound whose cation is a zinc ion, such as zinc chloride, zinc nitrate, zinc sulfate, etc.

[0031] Specifically, the mass ratio of ellagic acid, olsalazine and zinc salt is 1:1.5-2.5:15-25.

[0032] Specifically, the ultrasonic treatment time is 10 to 30 minutes.

[0033] Specifically, the stirring time is 24 to 48 hours.

[0034] Specifically, a microporous membrane is used for filtration. More specifically, the pore size of the microporous membrane is 0.40-0.50 μm.

[0035] Another embodiment of the present invention provides a positionally formed liquid hydrogel, the raw materials of which include solution 1 and solution 2, wherein solution 1 contains calcium salt, and solution 2 contains sodium alginate and the above-mentioned small molecule drug.

[0036] The calcium salt described in the present invention is a compound whose cation is calcium ion, such as calcium chloride, calcium nitrate and the like.

[0037] In some embodiments, the mass ratio of sodium alginate, calcium salt, and small molecule drug is 1-2:30-35:1.

[0038] In some embodiments, the concentration of calcium salt in solution 1 is 100-300 mM. The unit mM refers to mmol / L, and the unit M refers to mol / L.

[0039] In some embodiments, the concentration of sodium alginate in solution 2 is 1-5% w / v, wherein 1% w / v represents 10 mg / mL.

[0040] In some embodiments, the raw material further comprises polylysine.

[0041] Specifically, polylysine is prepared into solution 3, and the concentration of polylysine in solution 3 is 5-15% w / v.

[0042] Specifically, the mass ratio of sodium alginate, polylysine, calcium salt and small molecule drug is 1-2:20-25:30-35:1.

[0043] A third embodiment of the present invention provides a use of the above-mentioned small molecule drug or the position-shaped liquid hydrogel in the preparation of a drug for treating inflammatory bowel disease.

[0044] In some embodiments, the drug is administered orally or intragastricly.

[0045] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0046] Example

[0047] Experimental methods:

[0048] Preparation of Olsa / EA / Zn solution:

[0049] Weigh 10 mg EA and dissolve it in 2 mL ethanol; weigh 30 mg Olsa and dissolve it in 10 mL ddH2O; weigh 200 mg ZnCl2 and dissolve it in 10 mL ddH2O. Ultrasonic treatment for 20 min and stirring at room temperature for 36 h. Then filter the solution with a 0.45 μm microporous membrane and freeze-dry to obtain a compound powder (abbreviated as Olsa / EA / Zn). Olsa / EA / Zn is resuspended with deionized water to obtain an Olsa / EA / Zn solution.

[0050] Preparation of Olsa / EA / Zn@SA / ε-PL hydrogel:

[0051] 10 mg / mL (1% w / v) SA, 100 mg / mL ε-PL, and 200 mM CaCl2 solutions were prepared using ddH2O. The SA drug-containing solution was a 1:1 volume mixture of SA and Olsa / EA / Zn solution (6.5 mg / mL). The gel formation steps included: (1) adding 150 μL CaCl2 (200 mM) of cross-linker solution, (2) then adding 20 μL ε-PL solution; (3) finally adding 30 μL sodium alginate drug-containing solution; after mixing in a gastric acid environment, the polymer solution underwent network cross-linking to form Olsa / EA / Zn@SA / ε-PL hydrogel.

[0052] Preparation of SA / ε-PL hydrogel:

[0053] The gel formation steps include: (1) adding 150 μL CaCl2 (200 mM) crosslinker solution, (2) then adding 20 μL ε-PL solution; (3) finally adding 30 μL sodium alginate solution; after mixing in a gastric acid environment, the polymer solution undergoes network crosslinking to form SA / ε-PL hydrogel.

[0054] Preparation of EA / Zn@SA / ε-PL hydrogel:

[0055] The gel formation steps include: (1) adding 150 μL CaCl2 (200 mM) cross-linker solution, (2) then adding 20 μL ε-PL solution; (3) finally adding 30 μL sodium alginate drug-containing (EA / Zn, 6.5 mg / mL) solution; after mixing in a gastric acid environment, the polymer solution undergoes network cross-linking to form EA / Zn@SA / ε-PL hydrogel.

[0056] Preparation of Olsa@SA / ε-PL hydrogel:

[0057] The gel formation steps include: (1) adding 150 μL CaCl2 (200 mM) cross-linker solution, (2) then adding 20 μL ε-PL solution; (3) finally adding 30 μL sodium alginate drug-containing (Olsa, 8 mg / mL) solution; after mixing in a gastric acid environment, the polymer solution undergoes network cross-linking to form Olsa@SA / ε-PL hydrogel.

[0058] Preparation of Olsa / EA / Zn@SA hydrogel:

[0059] The gel formation steps include: (1) adding 150 μL of CaCl2 (200 mM) cross-linker solution, and (2) finally adding 30 μL of sodium alginate drug solution; after mixing in a gastric acid environment, the polymer solution undergoes network cross-linking to form Olsa / EA / Zn@SA hydrogel.

[0060] Olsa / EA / Zn@SA / ε-PL hydrogel composition analysis:

[0061] Fourier transform infrared spectroscopy was used to analyze the chemical composition of the hydrogels (Olsa / EA / Zn@SA / ε-PL, SA / ε-PL and Olsa / EA / Zn@SA hydrogels). The spectrum collected ranged from 3600 to 500 cm -1 .

[0064] Olsa / EA / Zn@SA / ε-PL hydrogel swelling rate: The hydrogel was immersed in artificial gastric juice (AGF, pH = 1.2), artificial small intestinal fluid (ASF, pH = 6.8) and artificial colonic fluid (ACF, pH = 7.8), and the initial weight was recorded as W1. Then the water of the hydrogel was adsorbed with filter paper, and the weight of the hydrogel at different times (0, 1, 2, 4, 8, 12, 24, 48h) was recorded as W2, and the swelling ratio (%) = (W2-W1) / W1×100%.

[0065] Olsa / EA / Zn@SA / ε-PL hydrogel drug release rate:

[0066] The release curves of Olsa / EA / Zn in AGF, ASF and ACF solutions of Olsa / EA / Zn@SA hydrogel and Olsa / EA / Zn@SA / ε-PL hydrogel at 37°C were studied. At predetermined time points (0, 1, 2, 4, 8, 12, 24 and 48h), 100μL of supernatant was taken out and the absorbance of the release medium was measured by a microplate reader. The release rate of Olsa / EA / Zn was determined by analyzing the absorbance of the release medium at 360nm and combining it with the standard curve of Olsa / EA / Zn in water. Olsa / EA / Zn release rate (%) = [total amount of Olsa / EA / Zn released at predetermined time / initial amount of Olsa / EA / Zn loaded] × 100.

[0067] Animals and Colitis Induction and Drug Administration:

[0068] C57BL / 6J male mice (21-23 g) were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd. and were kept under SPF conditions in the Experimental Animal Center of Shandong University. All mouse experiments were approved by the Animal Ethics and Welfare Committee of Shandong University (approval number: ECSBMSSDU2024-02-310). The mice were adapted to the environment for 7 days after arrival and then divided into 8 groups. The groups were: control group (Sham), model group (DSS), DSS+Olsa / EA / Zn@SA / ε-PL hydrogel group, DSS+control I group (DSS+SA / ε-PL hydrogel), DSS+control II group (DSS+Olsa / EA / Zn), DSS+control III group (DSS+EA / Zn@SA hydrogel), DSS+control IV group (DSS+Olsa@SA / ε-PL hydrogel), and DSS+control V group (Olsa / EA / Zn@SA hydrogel). The control group was fed with mouse diet + water, and the other 7 groups were fed with mouse diet + 2.5% DSS drinking water for 7 consecutive days. At the same time, the mice were gavaged with the above hydrogels on the 1st, 3rd, 5th, and 7th days. The body weight, stool viscosity, and fecal bleeding were observed and recorded every day. Figure 3 As shown in A.

[0069] Cell Viability:

[0070] Cell viability was assessed using a cell counting kit (CCK-8) according to the manufacturer’s instructions. CCD841 cells and PC12 cells were seeded in 96-well plates and then stained with Olsa / EA / Zn@SA and

[0071] The cells were treated with Olsa / EA / Zn@SA / ε-PL (100 ng / mL) for 24 h and 48 h, and CCK-8 solution was added to 96-well plates and incubated for 2 h. The cell viability was measured at 450 nm using a microplate reader.

[0072] Cell scratch assay:

[0073] 4×10 5 Cells / well were seeded in 6-well plates and cultured for 24 hours. The cells were scraped with a pipette tip and washed with PBS to remove floating cells and debris. Serum-free fresh medium with or without Olsa / EA / Zn@SA and Olsa / EA / Zn@SA / ε-PL was added to 6-well plates and cultured for 24h and 48h. The scratch distance of the cells was photographed and recorded under a microscope at 0h, 24h and 48h to measure the migration distance.

[0074] Live / dead cell analysis:

[0075] The cell viability treated with Olsa / EA / Zn@SA and Olsa / EA / Zn@SA / ε-PL was evaluated according to the instructions of the live / dead staining kit, and the cells were counted using a fluorescence microscope after 24 and 48 h of culture.

[0076] Immunofluorescence staining:

[0077] The colon tissue was embedded and cut into 10 μm sections. Next, the colon sections were incubated overnight at 4°C with primary antibodies: anti-CD206 (1:200, YM3050, Immunoway), anti-CD68 (1:200, YM4678, Immunoway), anti-F4 / 80 (1:100, 29414-1-AP, Proteintech). Images were collected using a fluorescence microscope, and the fluorescence intensity of each signal was quantified using Image J.

[0078] Disease Activity Index Score:

[0079] DAI was used to evaluate the grading of the degree of intestinal inflammation in each group of mice. The score for each category (from 0 to 4) was calculated and then the average was taken. In short, body weight: 0 points, <1%; 1 point, 1-5%; 2 points, 5-10%; 3 points, 10-15%; 4 points, >15%. Stool characteristics: 0 points, normal stool; 1-2 points, soft and shaped; 3 points, soft stool; 4 points, diarrhea); rectal bleeding: 0 points, none; 2 points, occult blood; 4 points, bleeding.

[0080] Detection of active oxygen:

[0081] ROS levels were detected using ROS-green and MitoSOX-Red. Colon sections were incubated with ROS (1:1000) and MitoSOX (5 μM) at 37°C for 30 min, and images were captured under a fluorescence microscope.

[0082] Western blot analysis of inflammation and antioxidant related protein expressions

[0083] Colon tissue was extracted and frozen at -80℃. The cells were homogenized with RIPA containing PMSF and protease / phosphatase inhibitors, centrifuged at 4℃, 12000rpm for 10min, and then 5× or 2× loading buffer was added to the protein supernatant. Western Blots were detected with the following primary antibodies: anti-IL-1β (1:1000, 66737-1-ig, Proteintech), anti-Arg-1 (1:1000, 16001-1-AP, Proteintech), anti-SOD2 (1:1000, 66474-1-ig, Proteintech), anti-Nrf2 (1:1000, 16396-1-AP, Proteintech), anti-β-actin (1:1000, Zhongshan Golden Bridge Biotechnology) at 4℃ overnight. Secondary antibodies were incubated for 1 hour. The chemiluminescent signal was developed using ECL kit reagents (MILLIPORE, USA) and then imaged using the Tanon imaging system (Tanon-4600).

[0084] Experimental results and analysis

[0085] Formation and characterization of Olsa / EA / Zn@SA / ε-PL hydrogel

[0086] First, the in vivo and in vitro performance tests of Olsa / EA / Zn@SA / ε-PL hydrogel were carried out. Figure 1 As shown in A, SA / ε-PL hydrogel, Olsa / EA / Zn@SA hydrogel, and Olsa / EA / Zn@SA / ε-PL hydrogel were rapidly formed in simulated gastric fluid. After 4 h of in vivo administration, the formation of Olsa / EA / Zn@SA / ε-PL hydrogel in the stomach was observed ( Figure 1 B). Next, FTIR spectroscopy results further demonstrated the successful preparation of SA / ε-PL hydrogel, Olsa / EA / Zn@SA hydrogel, and Olsa / EA / Zn@SA / ε-PL hydrogel ( Figure 1 C). To determine whether Olsa / EA / Zn@SA or Olsa / EA / Zn@SA / ε-PL hydrogels can protect Olsa / EA / Zn from degradation in the harsh environment of the stomach and release Olsa / EA / Zn in the small intestine and colon, the stability of Olsa / EA / Zn@SA or Olsa / EA / Zn@SA / ε-PL hydrogels in AGF (pH = 1.2), ASF (pH = 6.8), and ACF (pH = 7.8) was evaluated ( Figure 1D). The images show that the morphology of Olsa / EA / Zn@SA or Olsa / EA / Zn@SA / ε-PL hydrogel in AGF solution is relatively complete, indicating that it is stable in AGF solution. It is worth noting that after incubation in ASF and ACF solutions for 2 h, cracking and incomplete morphology can be observed in the images of Olsa / EA / Zn@SA hydrogel. Over time, compared with Olsa / EA / Zn@SA hydrogel, the stability of Olsa / EA / Zn@SA / ε-PL hydrogel in ASF and ACF solutions is more obvious ( Figure 1 D), which may be due to the toughness of Olsa / EA / Zn@SA / ε-PL hydrogel after the introduction of ε-PL. The swelling properties of hydrogels are crucial to their usability as drug carriers. Therefore, the swelling ability of hydrogels in AGF, ASF, and ACF solutions was studied. Olsa / EA / Zn@SA hydrogel and Olsa / EA / Zn@SA / ε-PL hydrogel did not show obvious swelling in the three solutions ( Figure 1 E). In ASF, Olsa / EA / Zn@SA and Olsa / EA / Zn@SA / ε-PL hydrogels reached maximum swelling at 1 h (110%) and 4 h (112%), respectively, and no more swelling occurred thereafter ( Figure 1 F). In ACF, Olsa / EA / Zn@SA and Olsa / EA / Zn@SA / ε-PL hydrogels reached their maximum swelling capacity in 1 h (251%) and 2 h (138%), respectively. Figure 1 G). These results indicate that Olsa / EA / Zn@SA and Olsa / EA / Zn@SA / ε-PL hydrogels have minimal swelling in the acidic stomach, allowing the drug to be physically protected and encapsulated. When the hydrogels pass through the pH-neutral intestine, Olsa / EA / Zn@SA and Olsa / EA / Zn@SA / ε-PL hydrogels can swell dramatically, allowing for rapid drug release. Olsa / EA / Zn@SA / ε-PL hydrogels have low swelling properties, which is beneficial for reducing pressure in the intestine and colon.

[0087] Next, the drug release of Olsa / EA / Zn@SA or Olsa / EA / Zn@SA / ε-PL hydrogels was observed. The results showed that in the early stage, the Olsa / EA / Zn release ratio of the two hydrogels was relatively fast. As time went on, the drug release ratio of the two hydrogels slowed down ( Figure 1 HJ). This phenomenon may be due to the relatively loose network structure of the hydrogel at the beginning. As the immersion time in AGF, ASF and ACF solutions increases, the network structure becomes more solid, thus slowing down the release of Olsa / EA / Zn. Figure 1In HJ, Olsa / EA / Zn@SA and Olsa / EA / Zn@SA / ε-PL hydrogels showed more total drug release in ASF and ACF solutions compared with AGF solution. The release curves of Olsa / EA / Zn@SA and Olsa / EA / Zn@SA / ε-PL hydrogels in ASF solution showed that the release amounts in the first 1 h were 45.6% and 13.5%, respectively. Figure 1 I). Meanwhile, the Olsa / EA / Zn release curves of Olsa / EA / Zn@SA / ε-PL hydrogel in ACF solution showed that the burst release rates in the first hour were 38.7% and 22.4%, respectively ( Figure 1 J). After incubation in ASF solution for 48 h, approximately 53.7% and 32.8% of Olsa / EA / Zn@SA and Olsa / EA / Zn@SA / ε-PL hydrogels were released, respectively. Figure 1 I). After incubation in ACF solution for 48 h, about 53.7% and 51.3% of Olsa / EA / Zn@SA and Olsa / EA / Zn@SA / ε-PL hydrogels were released, respectively. Figure 1 J) In summary, SA and Ca 2+ The formed cross-linked network provides physical support for the hydrogel, and the introduction of ε-PL enhances the mechanical properties of the hydrogel. After oral administration, Olsa / EA / Zn@SA / ε-PL hydrogel can be quickly formed in gastric juice and reasonably release Olsa / EA / Zn in the gastrointestinal tract, thereby prolonging the treatment time.

[0088] Biocompatibility of Olsa / EA / Zn@SA / ε-PL hydrogel

[0089] The biocompatibility of hydrogels is a key factor in clinical applications. Figure 2 As shown in A, CCK-8 results showed that compared with the control group, Olsa / EA / Zn@SA and Olsa / EA / Zn@SA / ε-PL hydrogels had no obvious cytotoxic effect on CCD841 cells when incubated with cells at concentrations of 0-100 μg / mL for 24 and 48 h, respectively ( Figure 2 A). Live / dead cell staining results showed that when cells were incubated with Olsa / EA / Zn@SA and Olsa / EA / Zn@SA / ε-PL hydrogels at concentrations of 0-100 μg / mL for 24 and 48 h, respectively, the cells grew normally and a small number of dead cells appeared in the culture medium, indicating that Olsa / EA / Zn@SA / ε-PL hydrogels had good biocompatibility ( Figure 2B). The results of wound healing experiments showed that compared with the control group, Olsa / EA / Zn@SA and Olsa / EA / Zn@SA / ε-PL hydrogels at a concentration of 100 μg / mL had no significant cytotoxic effect on CCD841 cells at 24 and 48 h ( Figure 2 C). This indicates that Olsa / EA / Zn@SA / ε-PL hydrogel has good biocompatibility.

[0090] Olsa / EA / Zn@SA / ε-PL hydrogel alleviates DSS-induced colitis in mice

[0091] like Figure 3 B- Figure 3 As shown in C, DSS treatment can cause weight loss in mice, increased disease activity index (combined score of weight loss, stool consistency and rectal bleeding) score, and shortened colon. Compared with the DSS group, control I (SA / ε-PL hydrogel), control II (Olsa / EA / Zn solution) and control IV (Olsa@SA / ε-PL hydrogel) had no effect on colon improvement, while intervention with EA / Zn@SA / ε-PL, Olsa / EA / Zn@SA or Olsa / EA / Zn@SA / ε-PL hydrogel significantly alleviated DSS-induced colon shortening ( Figure 3 D. Figure 3 F). H&E staining images showed that the DSS-treated group exhibited multiple IBD symptoms, including immune cell infiltration, goblet cell depletion, and colonic crypt loss. The results showed that the histopathological scores of the DSS-treated group were significantly higher than those of the healthy control group, indicating increased inflammatory effects. However, the histopathological scores of mice given Olsa / EA / Zn@SA / ε-PL or Olsa / EA / Zn@SA hydrogels were significantly increased ( Figure 3 E. Figure 3 G), indicating that Olsa / EA / Zn@SA / ε-PL or Olsa / EA / Zn@SA hydrogel can significantly promote the repair of colon tissue. Compared with the Olsa / EA / Zn@SA hydrogel treatment group, the Olsa / EA / Zn@SA / ε-PL hydrogel treatment group showed a good therapeutic effect in alleviating histopathological inflammation, but there was no statistical difference between the two ( Figure 3 E. Figure 3 G). Therefore, Olsa / EA / Zn@SA / ε-PL and Olsa / EA / Zn@SA hydrogels were used for further study.

[0092] Olsa / EA / Zn@SA / ε-PL hydrogel can alleviate DSS-induced inflammatory response in mice

[0093] Inflammatory factors are closely related to the pathogenesis of ulcerative colitis, among which macrophages are the main immune cells that secrete inflammatory factors. Macrophages have two phenotypes: inflammation-related M1 macrophages and anti-inflammatory M2 macrophages. M1 macrophages induce inflammatory responses by releasing proinflammatory cytokines, while M2 macrophages secrete anti-inflammatory cytokines to alleviate inflammatory responses. The phenotypic change from M1 to M2 is an important factor affecting IBD inflammation. CD68 immunofluorescence results showed that Olsa / EA / Zn@SA or Olsa / EA / Zn@SA / ε-PL hydrogel treatment significantly reduced the polarization of macrophage M1 in DSS-induced colitis in mice ( Figure 4 A and Figure 4 B), while Olsa / EA / Zn@SA / ε-PL hydrogel treatment significantly enhanced the CD206 positive cells in DSS-induced colitis in mice ( Figure 4 C and Figure 4 D). Olsa / EA / Zn@SA / ε-PL hydrogel significantly reduced the number of macrophages in DSS-induced colitis mice ( Figure 4 B and Figure 4 D), suggesting that Olsa / EA / Zn@SA / ε-PL hydrogel can promote the polarization of macrophages from M1 phenotype to M2 phenotype. In order to study the potential therapeutic mechanism of hydrogel in vivo, the changes of inflammatory factors were detected. Figure 4 E- Figure 4 As shown in Figure F, Olsa / EA / Zn@SA or Olsa / EA / Zn@SA / ε-PL hydrogel treatment significantly reduced the IL-1β level and increased the Arg-1 level in the colon tissue of colitis mice. The results showed that Olsa / EA / Zn@SA or Olsa / EA / Zn@SA / ε-PL hydrogel treatment had a strong synergistic anti-inflammatory effect.

[0094] Olsa / EA / Zn@SA / ε-PL hydrogel can inhibit DSS-induced oxidative stress

[0095] The basic mechanism of most inflammatory diseases, including IBD, is oxidative damage. Reactive oxygen species are essential factors for inducing the inflammatory environment. They are produced in the cytoplasm and mitochondria. Mitochondria are membrane-bound organelles that are necessary for maintaining energy through oxidative phosphorylation and other metabolic functions. Mitochondria are also the main source of ROS. Mitochondrial ROS (mtROS) regulate inflammatory signaling pathways. Figure 5 A- Figure 5As shown in Figure B, Olsa / EA / Zn@SA or Olsa / EA / Zn@SA / ε-PL hydrogel treatment not only inhibited the level of cellular ROS induced by DSS, but also inhibited the level of mtROS in colon tissue. Similarly, it was observed that Olsa / EA / Zn@SA / ε-PL hydrogel treatment increased the protein levels of Nrf2 and SOD2 in the colon tissue of DSS mice ( Figure 5 C- Figure 5 D). In summary, Olsa / EA / Zn@SA / ε-PL hydrogel has the effects of scavenging ROS and alleviating mitochondrial dysfunction.

[0096] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A small molecule drug, characterized in that: It is formed by olsalazine, ellagic acid and zinc ion through metal-polyphenol coordination.

2. The small molecule drug according to claim 1, characterized in that: The preparation process is as follows: dissolving ellagic acid in ethanol, dissolving olsalazine in water, dissolving zinc salt in water, mixing the ellagic acid solution, the olsalazine solution and the zinc salt solution, sequentially performing ultrasonic treatment and stirring, filtering and freeze-drying.

3. The small molecule drug according to claim 2, characterized in that: The mass ratio of ellagic acid, olsalazine and zinc salt is 1:1.5-2.5:15-25; Alternatively, the ultrasonic treatment time is 10 to 30 min; Or, the stirring time is 24 to 48 hours; Alternatively, filtering may be performed using a microporous membrane.

4. A position-shaped liquid hydrogel, characterized in that: The raw materials include solution 1 and solution 2, wherein the solution 1 contains calcium salt, and the solution 2 contains sodium alginate and the small molecule drug according to any one of claims 1 to 3.

5. The liquid hydrogel according to claim 4, characterized in that: The mass ratio of sodium alginate, calcium salt and small molecule drug is 1-2:30-35:

1.

6. The liquid hydrogel according to claim 4, characterized in that: The concentration of calcium salt in solution 1 is 100-300 mM.

7. The liquid hydrogel according to claim 4, characterized in that: The concentration of sodium alginate in solution 2 is 1-5% w / v.

8. The liquid hydrogel according to claim 4, characterized in that: The raw materials also include polylysine.

9. The liquid hydrogel according to claim 8, characterized in that: The polylysine is prepared into a solution 3, wherein the concentration of the polylysine in the solution 3 is 5 to 15% w / v; Or, the mass ratio of sodium alginate, polylysine, calcium salt and small molecule drug is 1-2:20-25:30-35:

1.

10. Use of the small molecule drug according to any one of claims 1 to 3 or the liquid hydrogel according to any one of claims 4 to 9 in the preparation of a drug for treating inflammatory bowel disease.

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