Positioning shaped liquid hydrogel and preparation method and application thereof

Abstract

The present application belongs to the technical field of material synthesis and molecular biology, and relates to a liquid hydrogel for site forming and a preparation method and application thereof. Raw materials include solution 1 and solution 2, the solution 1 contains calcium salt, and the solution 2 contains sodium alginate and small molecule drugs; the small molecule drugs are formed by metal-polyphenol coordination of oxalate, ellagic acid and zinc ions. The present application first synthesizes small molecule drugs from oxalate, EA and Zn salt to remove ROS and relieve inflammation, and improve the treatment effect of IBD; then the small molecule drugs are combined with hydrogel raw materials to prepare liquid hydrogel which can be drunk and formed in the stomach, overcome the problem of swallowing difficulty, improve drug compliance, and has the effects of synergistic anti-inflammatory, ROS removal and mitochondrial dysfunction reduction, thereby realizing in-vivo treatment of colitis.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of material synthesis and molecular biology, and relates to a positioning formed liquid hydrogel as well as a preparation method and application thereof. BACKGROUND

[0002] The information disclosed in this Background section is for the purpose of increasing the understanding of the general background of the application and does not necessarily constitute an admission or a recognition that any of this information constitutes prior art.

[0003] Inflammatory bowel disease (IBD) is a chronic and recurrent gastrointestinal inflammation disease, and its clinical symptoms include abdominal pain, bloody diarrhea, intestinal mucosal ulcer, fatigue, anemia and weight loss, etc. Clinically, it is divided into two subtypes of Crohn's disease and ulcerative colitis. With the rapid economic development and the change of life style, in the past 20 years, especially in the past 10 years, the incidence of IBD has rapidly increased and tends to be younger. There are mainly three types of drugs for the clinical treatment of IBD: aminosalicylic acid, glucocorticoid and immunosuppressant. However, these drugs are only limited to relieving symptoms, and long-term drug treatment is needed.

[0004] The IBD inflammatory process is mainly the formation of reactive oxygen species (ROS) and reactive nitrogen species, which can lead to the excessive production of pro-inflammatory 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 treatment strategy for the treatment of IBD. Ellagic acid (EA) is a natural polyphenol with antioxidant, antibacterial, anti-inflammatory and other biological activities. The two aromatic rings in EA result in its low solubility in water, which leads to its low bioavailability.

[0005] In addition, IBD patients, especially those with difficulty swallowing (such as children), have problems with difficulty swallowing, so the drug compliance is poor, and it is difficult to achieve the expected effect of the drug. SUMMARY

[0006] In order to solve the problems of the prior art, the present application aims to provide a liquid hydrogel with positioning and shaping and a preparation method and application thereof. The olsalazine (Olsa), EA and Zn salt are first synthesized into a small molecule (Olsa / EA / Zn) drug to remove ROS and relieve inflammation, thereby improving the treatment effect of IBD. Then, the small molecule drug is combined with hydrogel raw materials to prepare a liquid hydrogel with positioning and shaping in the stomach, so as to overcome the swallowing difficulty and improve the drug compliance. In addition, the liquid hydrogel has the effects of synergistic anti-inflammatory, ROS removal and mitochondrial dysfunction alleviation, thereby realizing the in vivo treatment of colitis.

[0007] In order to achieve the above-mentioned purpose, the present application provides the following technical scheme:

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

[0009] Zn has low toxicity 2+ Zn is an essential trace element for human body and is closely related to anti-inflammatory effect. Olsalazine is a drug for treating intestinal inflammation, and a large number of phenolic hydroxyl groups are present in the chemical structure of olsalazine. Therefore, the present application forms a small molecule Olsa / EA / Zn drug by metal-polyphenol coordination of Zn, olsalazine and ellagic acid, which can remove ROS and relieve inflammation, thereby improving the treatment effect of IBD. 2+

[0010] In a second aspect, a liquid hydrogel with positioning and shaping is provided. The raw materials include solution 1 and solution 2. The solution 1 contains a calcium salt, and the solution 2 contains sodium alginate (SA) and the above-mentioned small molecule drug.

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

[0012] The hydrogel provided by the present application has a hydrogel double dynamic crosslinking network formed by SA (covalent crosslinking) and Ca 2+ (ion crosslinking), which can quickly form in gastric juice and has significant stability in a gastric acid environment. After entering the intestinal tract, the hydrogel has a unique low swelling property, reasonable drug release and good cell compatibility. Since each raw material is in a liquid state before use, each raw material can be used sequentially to mix in the gastric juice to form a hydrogel, thereby avoiding the swallowing difficulty caused by large particle drugs and improving the drug compliance of patients.

[0013] ​Further, the raw materials further comprise polylysine (epsilon-Poly-l-lysine, epsilon-PL). The epsilon-PL can form another dynamic cross-linking network with the SA, providing the toughness of the hydrogel, and enhancing the adhesion of the hydrogel through hydrogen bonds and electrostatic interactions. The epsilon-PL can be placed in solution 1; or solution 3 can be prepared, and when used, solution 1 and solution 3 are first drunk (or gavaged), and then solution 2 is drunk (or gavaged).

[0014] In a third aspect, the application provides a use of the small molecule drug or the liquid hydrogel prepared by positioning and shaping in the preparation of a drug for treating inflammatory bowel disease.

[0015] The application has the following beneficial effects:

[0016] 1. The application uses olsalazine and zinc ions to form a small molecule drug through organic-inorganic hybridization and complex coordination of ellagic acid by metal-polyphenol coordination, which can scavenge ROS and relieve inflammation, and improve the treatment effect of IBD.

[0017] 2. The liquid hydrogel provided by the application can be quickly formed in gastric juice, and can be used (drunk) by separating the raw materials to form a liquid hydrogel in gastric juice, thereby overcoming the problem of difficulty in swallowing and improving drug compliance.

[0018] 3. The liquid hydrogel provided by the application has significant stability in a gastric acid environment, has unique low-swelling properties after entering the intestinal tract, reasonable drug release, and good cell compatibility. Animal model experiments on colitis mice show that the liquid hydrogel provided by the application 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; has a strong synergistic anti-inflammatory effect. At the same time, the hydrogel added with epsilon-PL also has the effects of scavenging ROS and reducing mitochondrial dysfunction. Therefore, the liquid hydrogel provided by the application has the potential for in vivo treatment of colitis. BRIEF DESCRIPTION OF DRAWINGS

[0019] The drawings constituting a part of the specification of the application are used to provide a further understanding of the application, and the illustrative embodiments of the application and the description thereof are used to explain the application, and do not constitute an improper limitation on the application.

[0020] Figure 1Figure A is a photograph of different hydrogel systems in artificial gastric fluid; B is an image of Olsa / EA / Zn solution loaded in the stomach to form a gel, C is an image of Olsa / EA / Zn solution loaded in the stomach to form a gel; D is a photograph of Olsa / EA / Zn@SA and Olsa / EA / Zn@SA / ε-PL hydrogels in artificial gastric fluid (AGF), artificial small intestine fluid (ASF) and artificial colon fluid (ACF); E is the swelling rate of Olsa / EA / Zn@SA hydrogel and Olsa / EA / Zn@SA / ε-PL hydrogel in AGF solution (n=3); F is the swelling rate of Olsa / EA / Zn@SA hydrogel and Olsa / EA / Zn@SA / ε-PL hydrogel in ASF solution (n=3); G is the swelling rate of Olsa / EA / Zn@SA hydrogel and Olsa / EA / Zn@SA / ε-PL hydrogel in ACF solution (n=3); H is a photograph of the release medium of Olsa / EA / Zn@SA hydrogel and Olsa / EA / Zn@SA / ε-PL hydrogel collected in AGF solution, and the cumulative release of Olsa / EA / Zn in Olsa / EA / Zn@SA hydrogel and Olsa / EA / Zn@SA / ε-PL hydrogel in AGF solution (n=3); I is a photograph of the release medium of Olsa / EA / Zn@SA hydrogel and Olsa / EA / Zn@SA / ε-PL hydrogel collected in ASF solution, and the cumulative release of Olsa / EA / Zn in Olsa / EA / Zn@SA hydrogel and Olsa / EA / Zn@SA / ε-PL hydrogel in ASF solution (n=3); J is a photograph of the release medium of Olsa / EA / Zn@SA hydrogel and Olsa / EA / Zn@SA / ε-PL hydrogel collected in ACF solution, and the cumulative release of Olsa / EA / Zn in Olsa / EA / Zn@SA hydrogel and Olsa / EA / Zn@SA / ε-PL hydrogel in ACF solution (n=3).

[0021] Figure 2Figure for the biocompatibility characterization of Olsa / EA / Zn@SA / ε-PL hydrogel in the embodiments of the present application; A is a graph of cell viability results after treating CCD841 cells with Olsa / EA / Zn@SA hydrogel and Olsa / EA / Zn@SA / ε-PL hydrogel at different concentrations for 24 h and 48 h; B is a graph of results of detecting live cells (green) and dead cells (red) after treating CCD841 cells with Olsa / EA / Zn@SA hydrogel and Olsa / EA / Zn@SA / ε-PL hydrogel (100 ng / mL) for 24 h and 48 h; C is a graph of results of detecting cell migration after treating CCD841 cells with Olsa / EA / Zn@SA hydrogel and Olsa / EA / Zn@SA / ε-PL hydrogel (100 ng / mL) for 24 h and 48 h.

[0022] Figure 3 Figure for the results of treating DSS-induced chronic colitis mice with Olsa / EA / Zn@SA / ε-PL hydrogel in the embodiments of the present application; A is a schematic diagram of a DSS-induced chronic colitis mouse model, 2.5% DSS was continuously given for 0-7 days, and control materials and Olsa / EA / Zn@SA / ε-PL hydrogel were orally administered by gavage at 1, 3, 5, and 7 days; B is the rate of change of body weight of mice during the treatment; C is the DAI score of mice during the treatment; D is a photograph of colon tissue of mice in different groups; E is a H&E staining graph of colon tissue of mice in different groups (low magnification (top), high magnification (bottom)); F is a statistical graph of colon tissue of mice in different groups; G is a statistical graph of colon injury score; the numerical values represent mean ± SD, *p<0.05, **p<0.01, ***p<0.001.

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

[0024] Figure 5Figure for the effect of Olsa / EA / Zn@SA / epsilon-PL hydrogel on DSS-induced oxidative stress in the embodiments of the present application; A is the representative ROS staining in the colon tissue; B is the representative mtROS staining in the colon tissue; C is the Western blotting detection of the expression level of colon SOD2 and Nrf-2 protein in the colon; D is the statistical chart of the expression level of colon SOD2 and Nrf-2 protein in each group (n=4); the numerical value is the mean value ± SD. *p<0.05, **p<0.01, ***p<0.001. DETAILED DESCRIPTION

[0025] It should be noted that the following detailed description is exemplary in nature and is intended to provide further description of the application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0026] It should be noted that the terms used herein are only for the purpose of describing specific embodiments, and are not intended to limit the exemplary embodiments according to the present application. As used herein, the singular form is intended to include the plural form unless the context clearly indicates otherwise, and furthermore, it should be understood that when the terms "comprise" and / or "include" are used in the specification, there is a reference to the presence of a feature, step, operation, device, component, and / or combinations thereof.

[0027] In view of the low solubility of EA, which leads to low bioavailability, the effect of EA on treating IBD inflammation is poor; at the same time, IBD patients have difficulty swallowing, which makes it difficult for the drug to reach the desired effect; therefore, the present application proposes a positioning shaped liquid hydrogel and a preparation method and application thereof.

[0028] In a typical embodiment of the present application, a small molecule drug is formed by metal-polyphenol coordination of olsalazine, ellagic acid and zinc ions.

[0029] In some embodiments, the preparation process is: dissolving ellagic acid in ethanol, dissolving olsalazine in water, dissolving zinc salt in water, mixing the ellagic acid solution, olsalazine solution and zinc salt solution, and then sequentially performing ultrasonic treatment and stirring, and then freeze-drying after filtration. The present application dissolves ellagic acid by ethanol, and uses the mutual solubility of ethanol and water to complex and coordinate olsalazine, ellagic acid and zinc ions. Freeze-drying is used to avoid the destruction of the coordination structure caused by high temperature in ordinary drying. In order to avoid the introduction of impurities, the water used in the embodiments of the present application is ddH2O, also known as double deionized water, double distilled water, ultrapure water, etc.

[0030] The zinc salt according to the present application refers to a compound with zinc ions as the cation, 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 10-30 min.

[0033] Specifically, the stirring time is 24-48 h.

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

[0035] Another embodiment of the present application provides a positioning shaped liquid hydrogel, raw materials of which include solution 1 containing calcium salt and solution 2 containing sodium alginate and the above-mentioned small molecule drug.

[0036] The calcium salt in the present application is a compound with calcium ion as cation, such as calcium chloride, calcium nitrate, etc.

[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. Unit mM refers to mmol / L, and 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 materials further include 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] The third embodiment of the present application provides an application of the above-mentioned small molecule drug or positioning shaped liquid hydrogel in preparing a drug for treating inflammatory bowel disease.

[0044] In some embodiments, the administration mode of the drug is oral or intragastric.

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

[0046] Embodiments

[0047] Experimental methods:

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

[0049] Olsa / EA / Zn solution was prepared by dissolving 10 mg EA in 2 mL ethanol, 30 mg Olsa in 10 mL ddH2O, and 200 mg ZnCl2 in 10 mL ddH2O. After 20 min of sonication and 36 h of stirring at room temperature, the solution was filtered with 0.45 μm membrane and freeze-dried to obtain the compound powder (Olsa / EA / Zn). The Olsa / EA / Zn was resuspended in deionized water to obtain the Olsa / EA / Zn solution.

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

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

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

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

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

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

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

[0057] The gel formation step includes: (1) adding 150 μL of CaCl2(200 mM) crosslinking agent solution, (2) then adding 20 μL of ε-PL solution; (3) finally adding 30 μL of Olsa drug-containing (Olsa, 8 mg / mL) sodium alginate solution; after mixing in the gastric acid environment, the polymer solution is network crosslinked to form Olsa@SA / ε-PL hydrogel.

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

[0059] The gel formation step includes: (1) adding 150 μL of CaCl2(200 mM) crosslinking agent solution, (2) finally adding 30 μL of Olsa drug-containing sodium alginate solution; after mixing in the gastric acid environment, the polymer solution is network crosslinked to form Olsa / EA / Zn@SA hydrogel.

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

[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 collected spectral range was 3600-500 cm -1

[0062] Swelling rate of Olsa / EA / Zn@SA / ε-PL hydrogel: The hydrogel was immersed in artificial gastric fluid (AGF, pH = 1.2), artificial small intestinal fluid (ASF, pH = 6.8) and artificial colon fluid (ACF, pH = 7.8), respectively, and the initial weight was recorded as W1. Then the moisture of the hydrogel was absorbed with filter paper, and the weight of the hydrogel at different times (0, 1, 2, 4, 8, 12, 24, 48 h) was recorded as W2, and the swelling ratio (%) = (W2-W1) / W1 x 100%.

[0063] Drug release rate of Olsa / EA / Zn@SA / ε-PL hydrogel:

[0064] 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 48 h), 100 μL of supernatant was taken out, and the absorbance of the release medium was determined by a microplate reader. By analyzing the absorbance of the release medium at 360 nm, combined with the standard curve of Olsa / EA / Zn in water, the release rate of Olsa / EA / Zn was determined. Olsa / EA / Zn release rate (%) = [total amount of Olsa / EA / Zn released at predetermined time / Olsa / EA / Zn amount initially loaded] x 100.

[0065] Induction of colitis and drug management in animals:

[0066] C57BL / 6J male mice (21-23 g) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. and were raised in the SPF conditions of the Experimental Animal Center of Shandong University. All mouse experiments were approved by the Animal Ethics and Welfare Committee of Shandong University (Approval No.: ECSBMSSDU2024-02-310). After the mice arrived, they were adapted to the environment for 7 days, and then divided into 8 groups. Each group was: 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), DSS+control V group (Olsa / EA / Zn@SA hydrogel). The control group was fed with mouse feed + water, and the remaining 7 groups were fed with mouse feed + 2.5% DSS drinking water for 7 consecutive days. At the same time, the mice were given the above hydrogels by gavage on the 1st, 3rd, 5th, and 7th days. Body weight, stool consistency, and fecal bleeding were observed and recorded daily, and the mice were sacrificed on the 8th day. Figure 3 A.

[0067] Cell survival rate:

[0068] Cell viability was evaluated using the Cell Counting Kit CCK-8 according to the manufacturer's instructions. CCD841 cells and PC12 cells were seeded in 96-well plates, then treated with Olsa / EA / Zn@SA and

[0069] 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 the 96-well plates for 2 h. Cell viability was determined at 450 nm using a microplate reader.

[0070] Cell scratch test:

[0071] 4 x 10 5 Cells were seeded at 4 x 10

[0072] Live / dead cell assay:

[0073] Cell viability of Olsa / EA / Zn@SA and Olsa / EA / Zn@SA / ε-PL treated cells was evaluated according to the live / dead staining kit instruction, counted after 24h and 48h incubation and observed using fluorescence microscope.

[0074] Immunofluorescence staining:

[0075] Colonic tissues were embedded and cut into 10 pm sections. Next, colonic 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). Image acquisition was performed using a fluorescence microscope and the fluorescence intensity of each signal was quantified by Image J.

[0076] Disease activity index score:

[0077] DAI was used to evaluate the grading of intestinal inflammation in mice of each group. The score of each category (from 0 to 4) was calculated and then averaged. Briefly, body weight: 0 points, <1%; 1 point, 1-5%; 2 points, 5-10%; 3 points, 10-15%; 4 points, >15%. Stool consistency: 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.

[0078] Detection of reactive oxygen species:

[0079] ROS levels were detected by ROS-green and MitoSOX-Red. Colonic sections were incubated with ROS (1:1000) and MitoSOX (5 mM) at 37 °C for 30 min and images were captured under fluorescence microscope.

[0080] Western blot detection of inflammation and antioxidant-related protein expression

[0081] Colon tissue was extracted and frozen at -80°C. Homogenized with RIPA containing PMSF and a protease / phosphatase inhibitor, centrifuged at 12,000 rpm for 10 min at 4°C, and then 5× or 2× loading buffer was added to the protein supernatant. Western blotting was performed using 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), and anti-β-actin (1:1000, Zhongshan Golden Bridge Biotechnology) incubated overnight at 4°C. Secondary antibody incubation was performed for 1 hour. Chemiluminescent signals were developed using ECL kit reagents (MILLIPORE, USA) and then imaged using a Tanon imaging system (Tanon-4600).

[0082] Experimental Results and Analysis

[0083] Formation and characterization of Olsa / EA / Zn@SA / ε-PL hydrogel

[0084] First, in vitro and in vivo performance tests were performed on the Olsa / EA / Zn@SA / ε-PL hydrogel. For example... Figure 1 As shown in Figure A, SA / ε-PL hydrogel, Olsa / EA / Zn@SA hydrogel, and Olsa / EA / Zn@SA / ε-PL hydrogel rapidly formed in simulated gastric juice. The formation of Olsa / EA / Zn@SA / ε-PL hydrogel in the stomach was observed 4 hours after in vivo administration. Figure 1 B). Next, FTIR spectroscopy results further confirmed the successful preparation of SA / ε-PL hydrogels, Olsa / EA / Zn@SA hydrogels, and Olsa / EA / Zn@SA / ε-PL hydrogels. Figure 1 C). To determine whether the Olsa / EA / Zn@SA or Olsa / EA / Zn@SA / ε-PL hydrogels could protect Olsa / EA / Zn from degradation by the harsh environment of the stomach and release Olsa / EA / Zn in the small and colon, we evaluated the stability of the 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). Figure 1D). Images show that the Olsa / EA / Zn@SA or Olsa / EA / Zn@SA / ε-PL hydrogels have relatively intact morphology in AGF solution, indicating their stability in AGF solution. Notably, after 2 hours of incubation in ASF and ACF solutions, cracking and incomplete morphology were observed in the images of the Olsa / EA / Zn@SA hydrogel. Over time, the Olsa / EA / Zn@SA / ε-PL hydrogel showed significantly greater stability in ASF and ACF solutions compared to the Olsa / EA / Zn@SA hydrogel. Figure 1 (D) This may be due to the increased toughness of the Olsa / EA / Zn@SA / ε-PL hydrogel after the introduction of ε-PL. The swelling properties of the hydrogel are crucial to its usability as a drug carrier. Therefore, the swelling capacity of the hydrogel in AGF, ASF, and ACF solutions was investigated. Neither the Olsa / EA / Zn@SA hydrogel nor the Olsa / EA / Zn@SA / ε-PL hydrogel showed significant swelling in any of the three solutions. Figure 1 E). In ASF, Olsa / EA / Zn@SA and Olsa / EA / Zn@SA / ε-PL hydrogels reached their maximum swelling at 1 h (110%) and 4 h (112%), respectively, and then no further swelling occurred. Figure 1 F). In ACF, the Olsa / EA / Zn@SA and Olsa / EA / Zn@SA / ε-PL hydrogels reached their maximum expansion capacity at 1 h (251%) and 2 h (138%), respectively. Figure 1 (G). These results demonstrate that Olsa / EA / Zn@SA and Olsa / EA / Zn@SA / ε-PL hydrogels exhibit minimal swelling in the acidic stomach, thus providing physical protection and encapsulation of the drug. When the hydrogels pass through the neutral pH intestine, Olsa / EA / Zn@SA and Olsa / EA / Zn@SA / ε-PL hydrogels swell rapidly, allowing for rapid drug release. The low swelling properties of Olsa / EA / Zn@SA / ε-PL hydrogel are beneficial in reducing pressure on the intestine and colon.

[0085] Next, drug release from Olsa / EA / Zn@SA or Olsa / EA / Zn@SA / ε-PL hydrogels was observed. The results showed that initially, the Olsa / EA / Zn release ratio of both hydrogels was relatively fast; however, as time increased, the drug release ratio of both hydrogels slowed down. Figure 1 HJ). This phenomenon may be due to the relatively loose network structure of the hydrogel initially, which becomes more robust with increasing immersion time in AGF, ASF, and ACF solutions, thus slowing the release of Olsa / EA / Zn. Figure 1H-J. Compared with AGF solution, Olsa / EA / Zn@SA and Olsa / EA / Zn@SA / ε-PL hydrogels showed more total drug release in ASF and ACF solutions. The release curves of Olsa / EA / Zn@SA and Olsa / EA / Zn@SA / ε-PL hydrogels in ASF solution showed that the release amount in the first 1 h was 45.6% and 13.5%( Figure 1 I), respectively. Meanwhile, the Olsa / EA / Zn release curves of Olsa / EA / Zn@SA / ε-PL hydrogels in ACF solution showed that the burst release rate in the first 1 h was 38.7% and 22.4%( Figure 1 J), respectively. After incubation in ASF solution for 48 h, about 53.7% and 32.8% of Olsa / EA / Zn in Olsa / EA / Zn@SA and Olsa / EA / Zn@SA / ε-PL hydrogels were released, respectively( Figure 1 I). While in ACF solution for 48 h, about 53.7% and 51.3% of Olsa / EA / Zn in Olsa / EA / Zn@SA and Olsa / EA / Zn@SA / ε-PL hydrogels were released, respectively( Figure 2 J). In summary, SA and Ca 2+ The cross-linked network formed by SA and Ca

[0086] Biocompatibility of Olsa / EA / Zn@SA / ε-PL hydrogels

[0087] Biocompatibility of hydrogels is a key factor for clinical application. For example, Figure 2 A), CCK-8 results showed that Olsa / EA / Zn@SA and Olsa / EA / Zn@SA / ε-PL hydrogels had no obvious cytotoxicity to CCD841 cells at 0-100 μg / mL concentration when incubated with cells for 24 and 48 h, respectively( Figure 2 A). Live / dead cell staining results showed that Olsa / EA / Zn@SA and Olsa / EA / Zn@SA / ε-PL hydrogels incubated with cells for 24 and 48 h at 0-100 μg / mL concentration, respectively, the cells grew normally, and a small amount of dead cells appeared in the culture medium, indicating that Olsa / EA / Zn@SA / ε-PL hydrogels had good biocompatibility( Figure 2B). Wound healing experiment results showed that 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 compared to the control group Figure 3 C). It is shown that Olsa / EA / Zn@SA / ε-PL hydrogels have good biocompatibility.

[0088] Olsa / EA / Zn@SA / ε-PL hydrogels can alleviate DSS-induced colitis in mice

[0089] As Figure 3 B- Figure 3 C shows that DSS treatment can cause weight loss in mice, increase in disease activity index (combined score of weight loss, stool consistency and rectal bleeding), and colon shortening. 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, and EA / Zn@SA / ε-PL, Olsa / EA / Zn@SA or Olsa / EA / Zn@SA / ε-PL hydrogel intervention significantly alleviated DSS-induced colon shortening Figure 3 D、 Figure 3 F). H&E staining images show that the DSS treatment group exhibits various IBD symptoms, including immune cell infiltration, goblet cell depletion and loss of colonic crypts. The results show that the histopathological score of the DSS treatment group is significantly higher than that of the healthy control group, indicating an increase in inflammatory effects. However, the histopathological score of mice given Olsa / EA / Zn@SA / ε-PL or Olsa / EA / Zn@SA hydrogel increased significantly 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 colonic tissue. Compared with the Olsa / EA / Zn@SA hydrogel treatment group, the Olsa / EA / Zn@SA / ε-PL hydrogel treatment group showed good therapeutic effect in alleviating histopathological inflammation, but there was no statistical difference between the two groups Figure 3 E、 Figure 4 G). Therefore, further studies were carried out using Olsa / EA / Zn@SA / ε-PL and Olsa / EA / Zn@SA hydrogels.

[0090] Olsa / EA / Zn@SA / ε-PL hydrogels can alleviate DSS-induced inflammatory response in mice

[0091] Inflammatory factors are closely related to the pathogenesis of ulcerative colitis, and 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 pro-inflammatory cytokines, and M2 macrophages secrete anti-inflammatory cytokines to reduce inflammatory responses. The change in phenotype from M1 to M2 is an important factor affecting IBD inflammation. The results of CD68 immunofluorescence showed that Olsa / EA / Zn@SA or Olsa / EA / Zn@SA / ε-PL hydrogel treatment can significantly reduce the M1 polarization of macrophages in DSS-induced colitis mice Figure 4 A and Figure 4 B), while Olsa / EA / Zn@SA / ε-PL hydrogel treatment can significantly enhance CD206 positive cells in DSS-induced colitis mice Figure 4 C and Figure 4 D). Olsa / EA / Zn@SA / ε-PL hydrogel significantly reduces DSS-induced colitis mouse macrophages 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. As shown in Figure 5 E- Figure 5 F, Olsa / EA / Zn@SA or Olsa / EA / Zn@SA / ε-PL hydrogel treatment can significantly reduce the level of IL-1β in the colon tissue of colitis mice and increase the level of Arg-1. The results show that Olsa / EA / Zn@SA or Olsa / EA / Zn@SA / ε-PL hydrogel treatment has strong synergistic anti-inflammatory effect.

[0092] Olsa / EA / Zn@SA / ε-PL hydrogel can inhibit DSS-induced oxidative stress

[0093] The basic mechanism of most inflammatory diseases, including IBD, is oxidative damage. Reactive oxygen species are essential factors for inducing inflammatory environments, which are produced in the cytoplasm and mitochondria. Mitochondria are membrane-bound organelles necessary for maintaining energy through oxidative phosphorylation and other metabolic functions. Mitochondria are also the main source of ROS. Mitochondrial ROS (mtROS) regulates inflammatory signaling pathways. As shown in Figure 5 A- Figure 5As shown in FIG. 8B, Olsa / EA / Zn@SA or Olsa / EA / Zn@SA / ε-PL hydrogel treatment not only inhibited DSS-induced cellular ROS levels, but also inhibited mtROS levels in colonic tissue. Likewise, Olsa / EA / Zn@SA / ε-PL hydrogel treatment was observed to increase protein levels of Nrf2 and SOD2 in colonic tissue of DSS mice ​ C- ​ D). In summary, Olsa / EA / Zn@SA / ε-PL hydrogel has the effect of scavenging ROS and alleviating mitochondrial dysfunction.

[0094] The preferred embodiments of the present application have been described above with the aid of drawing only for purpose of illustration, and the present application should in no way be restricted to those preferred embodiments, and can be variously modified and altered without departing from the spirit and scope of the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the scope of the present application.

Claims

1. A shaped liquid hydrogel positioned to form a shape, characterized by, The raw materials include solution 1 containing calcium salt and solution 2 containing sodium alginate and small molecule drug; The small molecule drug is formed by metal-polyphenol coordination of oxalate, ellagic acid and zinc ion.

2. The liquid hydrogel as described in claim 1, characterized in that, The preparation process of the small molecule drug is as follows: dissolving ellagic acid in ethanol, dissolving oxalate in water, dissolving zinc salt in water, mixing the solutions of ellagic acid, oxalate and zinc salt, and then sequentially performing ultrasonic treatment and stirring, filtering and freeze-drying.

3. The liquid hydrogel of claim 2, wherein the hydrogel is a liquid at 25°C and a gel at 37°C. The mass ratio of ellagic acid, oxalate and zinc salt in the small molecule drug is 1:1.5-2.5:15-25. Or, the ultrasonic treatment time is 10-30 min. Or, the stirring time is 24-48 h. Or, the filtering is performed by using microporous membrane.

4. The liquid hydrogel of claim 1, wherein the hydrogel is a liquid at room temperature and a gel at body temperature. The mass ratio of sodium alginate, calcium salt and small molecule drug is 1-2:30-35:

1.

5. The liquid hydrogel of claim 1 wherein the hydrogel is a liquid at 25°C and a gel at 37°C. The concentration of calcium salt in solution 1 is 100-300 mM.

6. The liquid hydrogel of claim 1 wherein the hydrogel is a liquid at 25°C and a gel at 37°C. The concentration of sodium alginate in solution 2 is 1-5% w / v.

7. The liquid hydrogel of claim 1 wherein, The raw materials further include polylysine.

8. The liquid hydrogel of claim 7, wherein the hydrogel is a solution of the polymer in water. The polylysine is prepared into solution 3, and the concentration of polylysine in solution 3 is 5-15% w / v. Or, the mass ratio of sodium alginate, polylysine, calcium salt and small molecule drug is 1-2:20-25:30-35:

1.

9. Use of the liquid hydrogel according to any one of claims 1-8 in the preparation of a medicament for treating inflammatory bowel disease.

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