A multifunctional bionic hydrogel and its preparation method and application

Abstract

The present invention relates to a multifunctional bionic hydrogel and its preparation method and application, belonging to the technical field of hydrogel preparation. The present invention discloses a multifunctional bionic hydrogel, which is composed of fibrin (Fn), HA-SH-DA (a product obtained by the amide reaction of thiol, dopamine (DA) and hyaluronic acid (HA)), Ag<supgt;+< / supgt; and thrombin (containing basic fibroblast growth factor and alanyl glutamine). In the multifunctional bionic hydrogel of the present invention, Ag<supgt;+< / supgt>, HA-SH-DA, fibrinogen and thrombin interact with each other to form a hydrogel with a double-network interweaving structure; the multifunctional bionic hydrogel provided by the present invention gradually degrades in the acidic intestinal environment under ulcerative colitis (UC) conditions and continuously releases therapeutic contents, making it have good application prospects in the preparation of drugs for treating ulcerative colitis.

Description

Technical Field

[0001] The present invention belongs to the technical field of the preparation of bionic hydrogels, and relates to a multifunctional bionic hydrogel, a preparation method thereof, and an application thereof. Background Art

[0002] Ulcerative colitis (UC) is a common inflammatory bowel disease with a high incidence and serious consequences. It is one of the intestinal diseases with the highest incidence, seriously affecting people's quality of life and characterized by chronic inflammation of the colonic mucosa. In the normal intestine, pro-inflammatory and anti-inflammatory mediators are maintained in a steady state to enhance the effective defense against invading pathogens while preventing excessive immune responses. However, UC severely disrupts the homeostatic balance between the gut microbiota and the mucosal immune system, ultimately leading to persistent mucosal damage. Although the pathological mechanism of human UC is still unclear, scientists have found that abnormal mucosal immune function and abnormal gut microbiota are two key factors in the onset of UC.

[0003] Typical clinical data show that ulcerative lesions are significantly infiltrated by effector immune cells (such as M1 macrophages), which are characterized by the overactivation of the immune signal of nuclear transcription factor (NF-κB), and can produce excessive pro-inflammatory cytokines, including tumor necrosis factor (TNF-α), interleukin 1β (IL-1β), and interleukin-6 (IL-6), thus driving the autoimmune response. At the same time, there is specific evidence that the gut microbiota of UC patients is usually in an abnormal state, with the proliferation of harmful bacteria, which may cross the damaged gut mucosal barrier and exacerbate local inflammation. Therefore, developing new treatment methods may restore gut immunity and microbial homeostasis to effectively treat UC, which has great clinical interest.

[0004] However, due to the unique pathological characteristics of UC, its treatment remains a major clinical challenge. Specifically, the integrity and function of the intestinal mucosal barrier in ulcerative lesions are in a highly damaged state, and when exposed to abnormal gut microbiota, it will amplify the harmful immune response and cause chronic refractory inflammation. Currently available treatment methods, such as oral anti-inflammatory drugs or enemas, cannot fully relieve the UC symptoms of a considerable number of patients and may also cause serious side effects such as severe diarrhea and systemic immune disorders. Interestingly, hydrogels are a type of scaffold polymer biomaterial that is very similar to living tissues and has become a promising option for UC treatment.

[0005] Overall, the structurally tailored hydrogel can form a biomimetic interface on the ulcer lesion to protect potential healthy cells from the stimulation of toxic substances and harmful bacteria. At the same time, the interconnected porous structure of the hydrogel also enhances the easy incorporation of bioactive agents with multiple therapeutic functions. Therefore, a rationally designed hydrogel system can overcome the current challenges in UC treatment and greatly improve the treatment effect. Summary of the Invention

[0006] In view of this, one of the objectives of the present invention is to provide a multifunctional biomimetic hydrogel; another objective of the present invention is to provide a preparation method of the multifunctional biomimetic hydrogel; and a third objective of the present invention is to provide the application of the multifunctional biomimetic hydrogel in the preparation of drugs for treating ulcerative colitis.

[0007] To achieve the above objectives, the present invention provides the following technical solutions:

[0008] 1. A multifunctional biomimetic hydrogel, which is composed of fibrinogen, HA-SH-DA, Ag + and thrombin;

[0009] The HA-SH-DA is a product obtained by an amide reaction of thiol, dopamine hydrochloride (DA), and hyaluronic acid (HA);

[0010] The thrombin contains basic fibroblast growth factor and alanyl glutamine.

[0011] Preferably, the HA-SH-DA is prepared according to the following method:

[0012] (1) Preparation of HA-SH: Dissolve hyaluronic acid (HA) in 2-(N-morpholino)ethanesulfonic acid (MES) buffer, add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS), stir and activate, then add cysteamine hydrochloride, continue to stir and react for 24 h, and then dialyze in pure water for 2 - 3 days and freeze-dry to obtain HA-SH;

[0013] (2) Preparation of HA-SH-DA: Dissolve the HA-SH prepared in step (1) in 2-(N-morpholino)ethanesulfonic acid (MES) buffer, add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS), stir and activate for 3 h, then add dopamine hydrochloride (DA), stir and react for 24 h under nitrogen protection at 37 °C, dialyze for 3 days, and then freeze-dry to obtain HA-SH-DA.

[0014] Further preferably, in step (1), the mass ratio of hyaluronic acid (HA), 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC), N-hydroxysulfosuccinimide (NHS), and cysteamine hydrochloride is 1000:478:288:568.

[0015] Further preferably, in step (2), the mass ratio of HA-SH, 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC), N-hydroxysulfosuccinimide (NHS), and dopamine hydrochloride (DA) is 1000:478:288:1890.

[0016] Preferably, the ratio of fibrinogen, HA-SH-DA, Ag + and thrombin is 20:20:0.12:100, mg:mg:mmol:U.

[0017] Preferably, the concentration of basic fibroblast growth factor in the thrombin is 1 μg / mL, and the concentration of alanyl glutamine is 160 mM.

[0018] 2. The preparation method of the above-mentioned multifunctional bionic hydrogel, and the specific preparation method is as follows:

[0019] Mix fibrinogen, HA-SH-DA and Ag + uniformly, and then add thrombin containing basic fibroblast growth factor and alanyl glutamine. After mixing uniformly, the multifunctional bionic hydrogel (HA-SH-Ag-Fn@bFGF@ALG) can be obtained.

[0020] 3. Application of the above-mentioned multifunctional bionic hydrogel in the preparation of drugs for treating ulcerative colitis.

[0021] The beneficial effects of the present invention are as follows: The present invention discloses a multifunctional bionic hydrogel, which is composed of fibrinogen, HA-SH-DA (a product obtained by the amide reaction of thiol, dopamine hydrochloride (DA) and hyaluronic acid (HA)), Ag + and thrombin (containing basic fibroblast growth factor and alanyl glutamine). In the multifunctional bionic hydrogel of the present invention, Ag + , HA-SH-DA and the thiol structure on fibrinogen interact with each other to form a hydrogel with a double network intertwined; at the same time, the basic fibroblast growth factor (bFGF) and alanyl glutamine (ALG) contained in the thrombin of the multifunctional bionic hydrogel can be loaded inside the loose and porous hydrogel; in addition, due to the inherent degradability of the fibrin hydrogel (Fn) skeleton and Ag +The acidic-triggered decoupling of the thiol coordination bond enables the multifunctional biomimetic hydrogel provided by the present invention to gradually degrade and continuously release therapeutic contents in the acidic intestinal environment under ulcerative colitis (UC) conditions, showing good application prospects in the preparation of drugs for treating ulcerative colitis.

[0022] Other advantages, objectives, and features of the present invention will to some extent be elaborated in the subsequent description, and to some extent, will be obvious to those skilled in the art based on the study of the following text, or can be taught from the practice of the present invention. The objectives and other advantages of the present invention can be achieved and obtained through the following description. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be described in detail with preference in conjunction with the accompanying drawings, where:

[0024] Figure 1 1H NMR spectra of HA-SH (a) and HA-SH-DA (b) prepared in Example 1;

[0025] Figure 2 In 16, a is the preparation flow chart of HA-SH-DA in Example 1, and b is the preparation flow chart of the multifunctional biomimetic hydrogel (HA-SH-DA-Ag-Fn@bFGF@ALG hydrogel) in Example 1;

[0026] Figure 3 Scanning electron microscope images (a), Young's modulus diagrams (b), frequency-dependent rheological measurement diagrams (c), time-dependent rheological measurement diagrams (d), swelling diagrams varying with time in an aqueous environment (e), degradation effect diagrams under the condition of PBS buffer solution with pH = 7.4 (f), and degradation effect diagrams under the condition of PBS buffer solution with pH = 5.5 (g) of different hydrogels prepared in Example 1, where Ⅰ is the fibrin hydrogel (Fn) prepared in Comparative Example 1, Ⅱ is the HA-SH-DA-Ag-Fn hydrogel prepared in Comparative Example 2, Ⅲ is the HA-SH-DA-Ag-Fn@bFGF hydrogel prepared in Comparative Example 3, and Ⅳ is the HA-SH-DA-Ag-Fn@bFGF@ALG hydrogel prepared in Example 1.

[0027] Figure 4 In 24, a, b, and c are the release curves of silver ions (Ag + ), basic fibroblast growth factor (bFGF), and alanyl glutamine (ALG) from the multifunctional biomimetic hydrogel (HA-SH-Ag-Fn@bFGF@ALG) prepared in Example 1 in PBS buffer solutions with pH = 5.5 and pH = 7.4;

[0028] Figure 5 Graph for testing the effects of different drugs in treating ulcerative colitis. Among them, a is the flow chart of induction and treatment of ulcerative colitis in mice, b is the line graph of the change in disease activity index during the induction and hydrogel treatment of ulcerative colitis in mice, c is the line graph of the change in body weight during the induction and hydrogel treatment of ulcerative colitis in mice, d is the physical map of the change in colon length of mice after treating ulcerative colitis, e is the statistical graph of the change in colon length of mice after treating ulcerative colitis, f is the physical map of the change in spleen of mice after treating ulcerative colitis, g is the statistical graph of the change in spleen of mice after treating ulcerative colitis, h is the graph of the change in myeloperoxidase detected in the colon tissue extracted after treating ulcerative colitis, i is the physical map of evaluating the histopathological changes of mouse tissues by HE staining after treating ulcerative colitis, j is the statistics of evaluating the histopathological changes of mouse tissues by HE staining after treating ulcerative colitis. Among them, Ⅰ is the control group (Control) containing only sterile water, Ⅱ is the dextran sulfate sodium (DSS) induction group, Ⅲ is the group of dextran sulfate sodium (DSS) induction + fibrin hydrogel (Fn) prepared in Comparative Example 1, Ⅳ is the group of dextran sulfate sodium (DSS) induction + HA-SH-DA-Ag-Fn prepared in Comparative Example 2, Ⅴ is the group of dextran sulfate sodium (DSS) induction + HA-SH-DA-Ag-Fn@bFGF prepared in Comparative Example 3, Ⅵ is the group of dextran sulfate sodium (DSS) induction + HA-SH-DA-Ag-Fn@bFGF@ALG prepared in Example 1;

[0029] Figure 6Anti-inflammatory, antibacterial, and repair effect diagrams of different drugs for the treatment of ulcerative colitis. Among them, a shows the change in the M2 / M1 macrophage ratio in the colitis site after treatment (which can be used as an indicator of immune component changes), the mRNA levels of inflammation-related markers in the colitis tissue after different treatments detected by qPCR (the marker in b is TNF-α, the marker in c is IL-6, the marker in d is IL-1β, the marker in e is Fizz, the marker in f is IL-10, and the marker in g is Arg-1), the mRNA levels of occludin (h) and ZO-1 (i) in the colitis tissue after treatment detected by qPCR, j shows the expression levels of proteins related to the TLR4-NF-κB pathway in the colitis tissue of mice after treatment detected by Western blot, the changes in bacterial richness (k), Chao index (l), and Shannon index (m) after different treatments detected by 16S rRNA sequencing, n shows the unique and shared OTUs in each group after treatment, indicating the change in the microbial community richness induced by the hydrogel in the background of the intestinal flora affected by ulcerative colitis, o is a community histogram showing the change in microbial composition after treatment at the phylum level, and the relative abundances of the beneficial Lactobacillus (p) and harmful Proteobacteria (q) flora significantly changed at the genus and phylum levels. Among them, I is the control group (Control) containing only sterile water, II is dextran sulfate sodium (DSS), III is treatment with fibrin (Fn) prepared in Comparative Example 1 after induction with dextran sulfate sodium (DSS), IV is treatment with HA-SH-DA-Ag-Fn prepared in Comparative Example 2 after induction with dextran sulfate sodium (DSS), V is treatment with HA-SH-DA-Ag-Fn@bFGF prepared in Comparative Example 3 after induction with dextran sulfate sodium (DSS), and VI is treatment with HA-SH-DA-Ag-Fn@bFGF@ALG prepared in Example 1 after induction with dextran sulfate sodium (DSS);

[0030] Figure 7 This is the mechanism diagram of the multifunctional biomimetic hydrogel (HA-SH-Ag-Fn@bFGF@ALG) of the present invention. Detailed implementation manners

[0031] The following specific examples illustrate the implementation manners of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific implementation manners. Various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that the diagrams provided in the following examples only illustrate the basic concept of the present invention in a schematic manner. Without conflict, the following examples and the features in the examples can be combined with each other.

[0032] Example 1

[0033] A multifunctional bionic hydrogel, and the specific preparation method comprises the following steps:

[0034] (1) Preparation of HA-SH: Dissolve 1 g of hyaluronic acid (HA) in 2-morpholinoethanesulfonic acid (MES) buffer solution, add 478 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 288 mg of N-hydroxysulfosuccinimide (NHS), stir and activate at a speed of 600 rpm for 3 h, then add 568 mg of cysteamine hydrochloride, continue to stir and react for 24 h, and then dialyze in pure water for 2-3 days and freeze-dry to obtain HA-SH, and its structural formula is:

[0035]

[0036] (2) Preparation of HA-SH-DA: Dissolve 1 g of HA-SH prepared in step (1) in 2-morpholinoethanesulfonic acid (MES) buffer solution, add 478 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 288 mg of N-hydroxysulfosuccinimide (NHS), stir and activate for 3 h, then add 1.89 g of dopamine hydrochloride (DA), stir and react at 37 °C under nitrogen protection for 24 h, dialyze for 3 days, and freeze-dry to obtain HA-SH-DA, and its structural formula is:

[0037]

[0038] (3) Preparation of multifunctional bionic hydrogel (HA-SH-DA-Ag-Fn@bFGF@ALG): In order to load basic fibroblast growth factor (bFGF) and alanyl glutamine (ALG) into the hydrogel, add them to thrombin to obtain a thrombin solution containing basic fibroblast growth factor (bFGF) and alanyl glutamine (ALG) (where the concentration of basic fibroblast growth factor (bFGF) is 1 μg / mL and the concentration of alanyl glutamine (ALG) is 160 mM). Mix the HA-SH-DA aqueous solution with a concentration of 20 mg / mL, the silver ion solution with a concentration of 60 mM, the fibrinogen solution with a concentration of 40 mg / mL, and the thrombin solution with a concentration of 100 U / mL (where the concentration of basic fibroblast growth factor (bFGF) is 1 μg / mL and the concentration of alanyl glutamine (ALG) is 160 mM) according to a volume ratio of 1:1:2:1, and the product is denoted as multifunctional bionic hydrogel (HA-SH-DA-Ag-Fn@bFGF@ALG).

[0039] Comparative Example 1

[0040] Prepare fibrin hydrogel according to the following method:

[0041] Preparation of fibrin hydrogel: Fibrinogen was dissolved in physiological saline to obtain a fibrinogen solution with a concentration of 20 mg / mL, and thrombin was dissolved in a 40 mM calcium chloride solution to form a thrombin solution with a concentration of 100 U / mL. The two solutions were mixed at a volume ratio of 2:1, and a fibrin hydrogel (Fn) was formed after a few seconds.

[0042] Comparative Example 2

[0043] Preparation of hydrogel (HA-SH-DA-Ag+-Fn) was carried out according to the following method:

[0044] The HA-SH-DA prepared in step (2) of Example 1 was formulated into an HA-SH-DA aqueous solution with a concentration of 20 mg / mL, and was mixed with a 60 mM silver ion solution, a fibrinogen solution with a concentration of 40 mg / mL, and a thrombin solution with a concentration of 100 U / mL at a volume ratio of 1:1:2:1 to form a hydrogel (HA-SH-DA-Ag+-Fn);

[0045] Comparative Example 3

[0046] Preparation of HA-SH-DA-Ag-Fn@bFGF was carried out according to the following method:

[0047] Basic fibroblast growth factor (bFGF, 1 μg / mL) was added to a thrombin solution with a concentration of 100 U / mL, and was mixed with an HA-SH-DA aqueous solution with a concentration of 20 mg / mL prepared in step (2) of Example 1, a 60 mM silver ion solution, and a fibrinogen solution with a concentration of 40 mg / mL at a volume ratio of 1:1:1:2 to form a hydrogel (HA-SH-DA-Ag-Fn@bFGF);

[0048] Performance detection:

[0049] Figure 1 1H NMR spectra of HA-SH (a) and HA-SH-DA (b) prepared in Example 1. Figure 2 In figure a is the preparation flow chart of HA-SH-DA in Example 1, and in figure b is the preparation flow chart of the multifunctional biomimetic hydrogel (HA-SH-DA-Ag-Fn@bFGF@ALG hydrogel) in Example 1.

[0050] Figure 3Scanning electron micrographs (a), Young's modulus graphs (b), frequency-dependent rheological measurement graphs (c), time-dependent rheological measurement graphs (d), swelling graphs over time in an aqueous environment (e), degradation effect graphs under PBS buffer conditions at pH = 7.4 (f), and degradation effect graphs under PBS buffer conditions at pH = 5.5 (g) of different hydrogels prepared in Example 1, where I is the fibrin hydrogel (Fn) prepared in Comparative Example 1, II is HA-SH-DA-Ag-Fn prepared in Comparative Example 2, III is HA-SH-DA-Ag-Fn@bFGF prepared in Comparative Example 3, and IV is HA-SH-DA-Ag-Fn@bFGF@ALG prepared in Example 1.

[0051] Figure 4 Among them, a, b, and c are the release curves of silver ions (Ag + ), basic fibroblast growth factor (bFGF), and alanyl glutamine (ALG) from the multifunctional biomimetic hydrogel (HA-SH-Ag-Fn@bFGF@ALG) prepared in Example 1 in PBS buffer at pH = 5.5 and pH = 7.4.

[0052] It can be seen from Figures 1 to 4 that the multifunctional biomimetic hydrogel (HA-SH-Ag-Fn@bFGF@ALG) can indeed be prepared by the method of the present invention, and the multifunctional biomimetic hydrogel (HA-SH-Ag-Fn@bFGF@ALG) has the properties of being porous and biodegradable and releaseable.

[0053] 1. C57BL / 6 mice (male, 7-week-old mice) were randomly divided into six groups, and the drugs used in each group were as follows: Group I was the control group (Control) containing only sterile water, Group II was the dextran sulfate sodium (DSS) induction group, Group III was the group treated with fibrin hydrogel (Fn) prepared in Comparative Example 1 after dextran sulfate sodium (DSS) induction, Group IV was the group treated with HA-SH-DA-Ag-Fn prepared in Comparative Example 2 after dextran sulfate sodium (DSS) induction, Group V was the group treated with HA-SH-DA-Ag-Fn@bFGF prepared in Comparative Example 3 after dextran sulfate sodium (DSS) induction, and Group VI was the group treated with HA-SH-DA-Ag-Fn@bFGF@ALG prepared in Example 1 after dextran sulfate sodium (DSS) induction. The specific method was as follows: A mouse ulcerative colitis model was induced with a 2% dextran sulfate sodium (DSS) aqueous solution for a total of 7 days; after successfully inducing colitis in mice, 50 μL of hydrogel samples from different groups were injected into the colon of ulcerative colitis mice through rectal enema (this is a commonly used enteral administration method in clinical practice); in addition, during the induction and treatment periods, the fecal consistency, blood in the stool, and weight loss of the mice were recorded daily to determine the changes in the DAI index. After 5 days of treatment, all mice were euthanized, and the colon and spleen were taken for further analysis.

[0054] The myeloperoxidase kit (colorimetric method, Nanjing Jiancheng) was used to detect the MPO activity in mouse intestinal tissues. 200 mg of colon tissue was taken from each group, pulverized after being treated with liquid nitrogen, and after being treated with the detection kit, the absorbance value of the sample at 460 nm was detected on an enzyme-labeled instrument. The calculation formula for MPO activity was: MPO activity = (sample group - control group) / (11.3 * 0.1 * 0.18).

[0055] After treatment with disuccinimidyl suberate (DSS), a series of symptoms such as bloody stools, mucosal damage, and colon contraction occurred, and the disease activity index (DAI) increased significantly from 0 (normal mice) to 9.78, clearly demonstrating the inducing effect of DSS-mediated ulcerative colitis in the mouse model. In contrast, the biomimetic hydrogel delivered by enema significantly alleviated the UC symptoms of mice treated with disuccinimidyl suberate (DSS). The test chart of the effects of different drugs in treating ulcerative colitis is as Figure 5As shown, where a is the flow chart of induction and treatment of murine ulcerative colitis, b is the line graph of the change in disease activity index during the induction of murine ulcerative colitis and hydrogel treatment, c is the line graph of the change in body weight during the induction of murine ulcerative colitis and hydrogel treatment, d is the physical map of the change in colon length of mice after treating ulcerative colitis, e is the statistical graph of the change in colon length of mice after treating ulcerative colitis, f is the physical map of the change in spleen of mice after treating ulcerative colitis, g is the statistical graph of the change in spleen of mice after treating ulcerative colitis, h is the change graph of detecting myeloperoxidase in the colon tissue extracted after treating ulcerative colitis, i is the physical map of evaluating the histopathological changes of mouse tissues by HE staining after treating ulcerative colitis, j is the statistics of evaluating the histopathological changes of mouse tissues by HE staining after treating ulcerative colitis, where I is the control group (Control) containing only sterile water, II is dextran sulfate sodium (DSS), III is treatment with fibrin (Fn) prepared in Comparative Example 1 after induction with dextran sulfate sodium (DSS), IV is treatment with HA-SH-DA-Ag-Fn prepared in Comparative Example 2 after induction with dextran sulfate sodium (DSS), V is treatment with HA-SH-DA-Ag-Fn@bFGF prepared in Comparative Example 3 after induction with dextran sulfate sodium (DSS), VI is treatment with HA-SH-DA-Ag-Fn@bFGF@ALG prepared in Example 1 after induction with dextran sulfate sodium (DSS). Colonic tissues of each group were fixed with 4% paraformaldehyde, embedded in paraffin, and cut into 5-μm thick sections. After the colon sections were exposed to xylene and graded ethanol, they were stained with hematoxylin and eosin (HE), and the histological features of the colon specimens were observed under a bright-field microscope to evaluate the histological score of the colon to show the severity of inflammation, the number of crypts, and the degree of ulcers. Under the condition of ulcerative colitis, the characteristics of the inflamed mucosa are enhanced neutrophil infiltration, mucosal erosion, and crypt atrophy. It is worth noting that the treatment with HA-SH-DA-Ag-Fn@bFGF@ALG hydrogel effectively eliminated these inflammatory histopathological features, where the mucosal samples showed continuous epithelium, normal crypt structure restored, and reduced neutrophil infiltration, while the mucosa of other groups was still in a highly inflamed state (as shown in i and j). Consistent with the disease conditions of each group, the spleen weight of the HA-SH-DA-Ag-Fn@bFGF@ALG group was the lowest (58 mg, as shown in f and g), and the activity of colon myeloperoxidase was the lowest (as shown in h). In summary, the in-situ formed hydrogel artificial mucosa can significantly relieve ulcerative-related inflammatory symptoms and restore the function of the colonic mucosa. Figure 5 as shown in i and j. Consistent with the disease conditions of each group, the spleen weight of the HA-SH-DA-Ag-Fn@bFGF@ALG group was the lowest (58 mg, Figure 5 as shown in f and g), and the activity of colon myeloperoxidase was the lowest ( Figure 5 as shown in h). In summary, the in-situ formed hydrogel artificial mucosa can significantly relieve ulcerative-related inflammatory symptoms and restore the function of the colonic mucosa.

[0056] 2. Anti-inflammatory, antibacterial, and repair effects of HA-SH-DA-Ag-Fn@bFGF@ALG hydrogel in the treatment of ulcerative colitis:

[0057] To clarify the therapeutic mechanism of the HA-SH-DA-Ag-Fn@bFGF@ALG hydrogel in vivo, the changes in infected tissues after treating colitis with hydrogels of different groups were further studied. The specific results of the anti-inflammatory, antibacterial, and repair effect diagrams of different drugs for treating ulcerative colitis are as follows Figure 6As shown, where a is the change in the M2 / M1 macrophage ratio in the colitis site after treatment (which can be used as an indicator of changes in immune components), qPCR was used to detect the mRNA levels of inflammation-related markers in colitis tissues after different treatments (the marker in b is TNF-α, the marker in c is IL-6, IL-1β in d, Fizz in e, the marker in f is IL-10, and the marker in g is Arg-1), the mRNA levels of occludin (h) and ZO-1 (i) in colitis tissues after treatment were detected by qPCR, j is the expression level of proteins related to the TLR4-NF-κB pathway in the colitis tissues of mice after treatment detected by Western blot, the changes in bacterial richness (k), Chao index (l), and Shannon index (m) after different treatments were detected by 16S rRNA sequencing, n shows the unique and shared OTUs in each group after treatment, indicating the changes in the microbial community richness induced by the hydrogel in the context of the gut microbiota affected by ulcerative colitis, o is a community histogram showing the changes in microbial composition after treatment at the phylum level, and the relative abundances of beneficial Lactobacillus (p) and harmful Proteobacteria (q) flora that were significantly changed at the genus and phylum levels. Among them, I is the control group (Control) containing only sterile water, II is dextran sulfate sodium (DSS), III is treatment with fibrin (Fn) prepared according to Comparative Example 1 after induction with dextran sulfate sodium (DSS), IV is treatment with HA-SH-DA-Ag-Fn prepared according to Comparative Example 2 after induction with dextran sulfate sodium (DSS), V is treatment with HA-SH-DA-Ag-Fn@bFGF prepared according to Comparative Example 3 after induction with dextran sulfate sodium (DSS), and VI is treatment with HA-SH-DA-Ag-Fn@bFGF@ALG prepared according to Example 1 after induction with dextran sulfate sodium (DSS). The specific method is as follows: First, the biochemical changes of the main inflammation-related immune cell populations in the affected colon tissue samples after rectal enema delivery of various hydrogels were monitored. Intestinal tissues from different treatment groups were extracted and incubated with a digestive solution composed of collagen deoxyribonuclease (0.3 mg / mL), nuclease I (0.25 mg / mL), and neutral protease II (3 mg / mL). Then, intestinal cells were separated by the percoll method. The cell pellet was collected by centrifugation at 1800 rpm for 10 min, resuspended in PBS, and counted. APC-F4 / 80+, PE-CD206, or FITC-iNOS antibodies were added sequentially, and the cells were incubated for 2 h. The cell samples were detected using a flow cytometer (Beckman Coulter). Flow cytometry analysis showed that the expression level of iNOS (a marker of m1-like macrophages) in the colon tissues of DSS-treated mice was enhanced, and the expression level of CD206 (a marker of m2-like macrophages) was decreased, indicating that the local immune components were in a pro-inflammatory state.In contrast, the treatment with HA-SH-DA-Ag-Fn@bFGF@ALG hydrogel decreased the expression level of iNOS and increased the expression level of CD206 (as shown in Figure 6 a). The hydrogel could reshape the local immune components into an anti-inflammatory state. After treating the colon tissues with liquid nitrogen, they were crushed, and total RNA was extracted from the colon tissues using the Trizol method and reverse-transcribed into cDNA using the oligo method. Finally, the expression levels of the key marker mRNAs were quantitatively detected by quantitative real-time PCR. Due to the changes in local immune components caused by the treatment, the mRNA levels of pro-inflammatory cytokines in the colon tissue samples of the HA-SH-DA-Ag-Fn@bFGF@ALG group were significantly lower than those in the DSS group, including TNF-α (75.71%), IL-6 (85.34%), and IL-1β (61.29%), while the mRNA levels of anti-inflammatory cytokines including Fizz (425%), IL-10 (300%), and Arg-1 (408.7%) were significantly upregulated (as shown in Figure 6 b-g). The secretion levels of these cytokines also showed the same trend, with enhanced expression of anti-inflammatory cytokines and inhibited secretion of pro-inflammatory cytokines in the HA-SH-DA-Ag-Fn@bFGF@ALG group.

[0058] Both occludin and ZO-1 were significantly downregulated in the ulcerative colitis-infected mice (as shown in Figure 6as shown in h and i), indicating the erosive state of the intestinal mucosa under ulcerative colitis conditions. Treatment of UC mice with fibrin and HA-SH-DA-Ag-Fn hydrogel can moderately restore the expression of occludin-1 and ZO-1, which is due to their barrier-like function that can prevent repeated stimulation of the damaged mucosa by foreign antigens and intestinal flora. Notably, the expression levels of occludin and ZO-1 in the HA-SH-DA-Ag-Fn@bFGF@ALG group were restored to the greatest extent, almost comparable to those of the mucosal layer of healthy mice. This result clearly confirmed that bFGF and ALG delivered by the hydrogel can promote the restoration of the epithelial mucosal barrier after enema, contributing to the treatment of ulcerative colitis. Protein extraction and quantification of the colon tissues of rats in each group were performed using a BCA protein assay kit. Then the proteins were boiled in loading buffer, separated by 10% SDS-PAGE, and transferred onto PVDF membranes. The proteins were blocked with 5% bovine serum albumin at room temperature for 2 h, and incubated with primary antibodies (TLR4, 1:2000; IKKα, 1:2000; IKKβ, 1:2000; p-IKKα / β, 1:1000; IκBα, 1:2000; p65 1:1000; p-p65 1:1000; β-actin, 1:5000; ZO-1 1:5000; occludin, 1:5000) overnight at 4 °C, and then incubated with secondary antibodies for 2 h at room temperature. The final results were imaged on a Molecular Imager Versa doc MP 4000 system (Bio-Rad). Western blot analysis of the colon tissues further showed that treatment with fibrin-containing hydrogel could effectively inhibit the expression of local macrophage TLR4 and inhibit the downstream NF-κB immune signaling pathway through the TLR4-p-IKKα / β-p-IκBα-p65 axis (as Figure 6 shown in j), and these results support the anti-inflammatory ability of the hydrogel in vivo.

[0059] Fecal samples of mice were collected 5 days after treatment, stored in sterile EP tubes at -80 °C, and then sent to Novogene for 16S rRNA intestinal flora sequencing. DNA was extracted and detected, PCR amplification, product purification, library preparation, library inspection were carried out, and finally on-board sequencing was performed using NovaSeq6000. We detected that DSS treatment led to a significant decrease in the operational taxonomic units (OTUs, as Figure 6 shown in k and n) and α-diversity (Chao and Shannon indices, as Figure 6 shown in l and m) of the microorganisms in the feces of mice, indicating that there was microbial metabolic disorder after DSS-induced UC. Interestingly, treatment of mice infected with UC with HA-SH-DA-Ag-Fn@bFGF@ALG hydrogel led to a significant restoration of community richness and diversity.

[0060] Further analyze the flora structure of different groups at the genus level to clarify how the enema hydrogel affects the intestinal flora to relieve UC in vivo (as shown in Figure 6 o). By analyzing the relative abundances of the four major bacterial phyla in the mouse intestine, Bacteroides, Firmicutes, Campilobacterota, Actinobacteria, and Proteobacteria, we found that DSS-induced UC significantly reduced the abundance of Firmicutes. In contrast, treatment with the HA-SH-DA-Ag-Fn@bFGF@ALG hydrogel effectively increased the relative abundance of Firmicutes, comparable to the level in healthy mice. In addition, we further analyzed the changes in the abundances of beneficial and harmful flora in each group (as shown in Figure 6 p and q). Typical consequences of DSS-induced UC in mice include a decrease in beneficial Lactobacilli and an increase in harmful Proteobacteria, while treating mice with the HA-SH-DA-Ag-Fn@bFGF@ALG hydrogel reversed these trends. These observations can be easily explained by the selective toxicity of Ag+ to harmful intestinal flora, which is beneficial for optimizing the intestinal flora to improve immune imbalance.

[0061] Thus, it can be seen that the mechanism of action of the multifunctional biomimetic hydrogel (HA-SH-Ag-Fn@bFGF@ALG) prepared by the present invention is as shown in Figure 7 and can adhere to the ulcer lesion after rectal enema to form a biofunctional mucosal-like barrier, restoring the homeostatic balance among the intestinal flora, mucosal immune system, and epithelial barrier integrity. Mixing precursors such as fibrinogen, thrombin, Ag + , HA-SH-DA, bFGF, and ALG can easily prepare the hydrogel. These precursors can spontaneously and orderly assemble. The prepared multifunctional biomimetic hydrogel (HA-SH-Ag-Fn@bFGF@ALG) has high tissue adhesiveness, is basically stable at physiological pH, but can accelerate degradation under acidic conditions similar to the UC environment. The fibrin scaffold can normalize mucosal immune function by inhibiting the activity of the M1 macrophage TLR4-NF-κB immune signaling pathway and polarizing it back to the M2 phenotype. At the same time, the released Ag +It can effectively eliminate harmful bacteria in the intestinal flora changed by UC and improve the related immune imbalance. In addition, the hydrogel can protect the damaged mucosa from further irritation, while continuously releasing bFGF and ALG to promote the restoration of the mucosal structure and function. These advantages can significantly reduce the severity of UC in the body in a cooperative manner. This work provides a paradigm for promoting the therapeutic effect of UC without obvious side effects and may provide a promising alternative for clinical UC management. In addition, the Fn network can inhibit the expression of Toll-like receptor 4 (TLR4) by suppressing the downstream NF-κB signal, induce its repolarization into an anti-inflammatory M2 phenotype, and the released Ag+ can eliminate local harmful bacteria, normalize the composition of the intestinal flora, synergistically restore the homeostatic balance at the ulcer site, and improve the inflammatory symptoms. In addition, both bFGF and ALG are clinically approved drugs for wound treatment and can accelerate the healing rate of ulcerated mucosa. The multifunctional biomimetic hydrogel provided by the present invention can significantly reduce the disease severity of the colitis mouse model without obvious side effects. The present invention provides a proof of concept for the injectable biomimetic hydrogel as an artificial intestinal mucosa for effective and safe treatment of UC clinically.

[0062] In summary, the present invention discloses a multifunctional biomimetic hydrogel, which is composed of fibrinogen, HA-SH-DA (a product obtained by the amide reaction of thiol, dopamine hydrochloride (DA) and hyaluronic acid (HA)), Ag + and thrombin (containing basic fibroblast growth factor and alanyl glutamine). In the multifunctional biomimetic hydrogel of the present invention, the thiol structures on Ag + , HA-SH-DA and fibrinogen interact with each other to form a hydrogel with a double-network interweaving; at the same time, the basic fibroblast growth factor (bFGF) and alanyl glutamine (ALG) contained in the thrombin of the multifunctional biomimetic hydrogel can be loaded inside the loose and porous hydrogel; in addition, due to the inherent degradability of the Fn skeleton and the acid-triggered decoupling of the Ag + -thiol coordination bond, the multifunctional biomimetic hydrogel provided by the present invention gradually degrades and continuously releases therapeutic contents in the acidic intestinal environment under ulcerative colitis (UC) conditions, making it have good application prospects in the preparation of drugs for treating ulcerative colitis.

[0063] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the purpose and scope of the present technical solution, and they should all be covered by the scope of the claims of the present invention.

Claims

1. A multifunctional biomimetic hydrogel, characterized in that, The bionic hydrogel is composed of fibrinogen, HA-SH-DA, Ag + and thrombin; the HA-SH-DA is a product obtained by an amide reaction of thiol, dopamine hydrochloride and hyaluronic acid; the thrombin contains basic fibroblast growth factor and alanyl glutamine; the HA-SH-DA is prepared according to the following method: (1) Preparation of HA-SH: Dissolve hyaluronic acid in 2-morpholinoethanesulfonic acid buffer solution, add 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysulfosuccinimide, stir and activate, then add cysteamine hydrochloride, continue to stir and react for 24 h, and then dialyze in pure water for 2-3 days and freeze-dry to obtain HA-SH; (2) Preparation of HA-SH-DA: Dissolve the HA-SH prepared in step (1) in 2-morpholinoethanesulfonic acid buffer solution, add 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysulfosuccinimide, stir and activate for 3 h, then add dopamine hydrochloride, and stir and react for 24 h under nitrogen protection at 37 °C. After dialysis for 3 days, freeze-dry to obtain HA-SH-DA.

2. The biomimetic hydrogel according to claim 1, characterized in that, in step (1), the mass ratio of hyaluronic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, N-hydroxysulfosuccinimide and cysteamine hydrochloride is 1000:478:288:

568.

3. The biomimetic hydrogel according to claim 2, characterized in that, in step (2), the mass ratio of HA-SH, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, N-hydroxysulfosuccinimide and dopamine hydrochloride is 1000:478:288:1890.

4. The biomimetic hydrogel according to claim 1, characterized in that, The fibrinogen, HA-SH-DA, Ag + and thrombin are in a ratio of 20:20:0.12:100, mg:mg:mmol:U.

5. The biomimetic hydrogel according to claim 1, characterized in that, the concentration of basic fibroblast growth factor in the thrombin is 1 μg / mL, and the concentration of alanyl glutamine is 160 mM.

6. The multifunctional biomimetic hydrogel according to any one of claims 1-5, characterized in that, the preparation method is specifically as follows: Mix fibrinogen, HA-SH-DA and Ag + uniformly, and then add thrombin containing basic fibroblast growth factor and alanyl glutamine. After mixing uniformly, a multifunctional biomimetic hydrogel can be obtained.

7. Use of the multifunctional biomimetic hydrogel according to claim 6 in the preparation of a drug for treating ulcerative colitis.

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