Preparation method and application of long-residence hydrogel with antibacterial, anti-inflammatory and repair-promoting functions

Abstract

The invention belongs to the technical field of medical hydrogel, and particularly relates to a preparation method and application of antibacterial, anti-inflammatory and repair-promoting long-residence hydrogel, the method comprises the following steps: step 1, preparation of modified konjac mannan: performing oxidation reaction on konjac mannan and an oxidizing agent in the presence of a solvent to obtain modified konjac mannan; the oxidized konjac mannan is obtained; carrying out acylation reaction on the oxidized konjac mannan and an acylation reagent in the presence of a catalyst to obtain modified konjac mannan; step 2, preparing modified gelatin: reacting gelatin with a compound containing a boric acid group and an aldehyde group, and then reacting with an acylating agent containing a carbon-carbon double bond to obtain the modified gelatin; step three, preparation of long-retention hydrogel: preparing a solution A from the modified konjac mannan obtained in the step one and tannic acid; preparing an antibacterial agent, a photoinitiator and the modified gelatin obtained in the step 2 into a solution B; and mixing the solution A and the solution B, and initiating curing through light irradiation to form the long-residence hydrogel. Compared with the prior art, the hydrogel has the advantages that through the synergistic effect of aldehyde covalent bonding of the modified konjac polysaccharide and multi-element hydrogen bonds of the tannic acid, the uterine cavity adhesion force and the long-acting residence capacity are remarkably enhanced, uterine cavity contraction can be effectively resisted, the positive effect is achieved in the aspect of uterine cavity adhesion prevention, and the application prospect is wide. Particularly, the antibacterial, anti-inflammatory and repair-promoting effects are good.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medical hydrogels, and particularly relates to a preparation method and application of a long-residence hydrogel with antibacterial and anti-inflammatory effects and repair promotion. BACKGROUND

[0002] Intramural uterine adhesion is a disease caused by abnormal repair of the damaged endometrial basal layer and endometrial fibrosis, which leads to partial or complete adhesion in the uterus, and often causes serious consequences such as menstrual abnormalities, infertility or repeated miscarriage; the treatment purpose of intramural uterine adhesion is to remove the adhesion and restore the normal uterine cavity shape and function of the uterus, and at present, transcervical resection of adhesions (TCRA) is the preferred treatment method for IUA. However, due to the damage to the endometrial basal layer, the self-repairing ability is reduced, and even after the transcervical resection of adhesions, the formation of re-adhesion is very frequent, and the heavier the degree of intramural uterine adhesion, the higher the recurrence rate. Therefore, it is necessary to take measures to prevent the formation or recurrence of intramural uterine adhesion after hysteroscopic surgery. At present, the following treatment methods are often taken after surgery: postoperative administration of estrogen and progestin cycle therapy, or use of physical barriers, such as placement of Foley balloon catheter, intrauterine device, etc., but there is no clear evidence that these treatment methods have obvious effect on preventing intramural uterine adhesion.

[0003] Hydrogel is a three-dimensional network structure gel with hydrophilic, which is formed by water-soluble or hydrophilic polymer through certain chemical or physical crosslinking. The injectable in-situ gelation hydrogel has very good research prospects in the treatment of intramural uterine adhesion. The hydrogel is in a liquid state under in vitro conditions, and can be changed into a gel state under the corresponding stimulating factors in the body after minimally invasive injection into the uterine cavity, which is simple and convenient in clinical operation; after injection into the uterine cavity, the hydrogel can directly form a physical barrier in the uterine cavity to prevent the formation of adhesion. Although the size and shape of the uterine cavity of different people are different, this hydrogel can be well covered on the surface of various irregular uterine cavities to achieve individualized treatment; the degradability of the hydrogel enables it to naturally degrade or be absorbed after completing the treatment in the uterine cavity, without the need for surgical removal. However, due to the humid environment in the uterine cavity, the conventional hydrogel is more likely to be discharged out of the body under the compression of uterine contraction, and the residence time in the uterine cavity is relatively short, thereby losing the effect of preventing uterine adhesion. In addition, the conventional hydrogel only has the effect of preventing uterine adhesion, and does not have the effects of antibiosis, anti-inflammation and repair promotion, which is not conducive to the recovery of the uterine cavity.

[0004] Therefore, based on the shortcomings of the current hydrogel for preventing and treating uterine adhesion, the current hydrogel needs to be improved so that it can reside in the uterine cavity for a long time, and has the effects of antibiosis, anti-inflammation and repair promotion. SUMMARY

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method for preparing and applying a long-lasting hydrogel that combines antibacterial, anti-inflammatory, and repair-promoting properties. This hydrogel significantly enhances intrauterine adhesion and long-term retention capacity through the synergistic effect of the aldehyde covalent bonds of modified konjac polysaccharide and the multiple hydrogen bonds of tannic acid, and can effectively resist intrauterine contractions. It loads multiple antibiotics through electrostatic interaction, achieving continuous drug release and long-lasting antibacterial and anti-inflammatory effects. At the same time, oxidized konjac polysaccharide and tannic acid synergistically scavenge free radicals and regulate the inflammatory microenvironment, creating favorable conditions for tissue repair.

[0006] This invention is achieved through the following technical solution: a method for preparing a long-lasting hydrogel that combines antibacterial, anti-inflammatory, and repair-promoting properties, comprising the following steps:

[0007] Step 1, Preparation of modified konjac mannan:

[0008] Konjac mannan was oxidized by reacting it with an oxidizing agent in the presence of a solvent to obtain oxidized konjac mannan.

[0009] The oxidized konjac mannan was subjected to an acylation reaction with an acylation agent in the presence of a catalyst to obtain modified konjac mannan.

[0010] Step 2, Preparation of modified gelatin:

[0011] Gelatin is first reacted with a compound containing boric acid groups and aldehyde groups, and then reacted with an acylating agent containing carbon-carbon double bonds to obtain modified gelatin.

[0012] Step 3, Preparation of long-lasting hydrogel:

[0013] The modified konjac mannan obtained in step one is prepared into solution A with tannic acid; the antibacterial drug, photoinitiator and modified gelatin obtained in step two are prepared into solution B; after mixing solution A and solution B, the mixture is irradiated with light to induce curing and form the long-stay hydrogel.

[0014] Further, in step one, the konjac mannan is first purified. The purification process involves dissolving konjac mannan with a molecular weight of 20-100 kDa in water, precipitating the konjac mannan with ethanol, filtering, and then freeze-drying.

[0015] Further, in step one, the oxidant is sodium periodate, and the mass ratio of konjac mannan to sodium periodate is 1:1 to 5; the solvent is a mixed solution of ethanol and water, wherein the volume ratio of water to ethanol is 1:5 to 10; and the oxidation reaction time is 12-48 h.

[0016] Furthermore, in step one, the solvent for the acylation reaction is one of N,N-dimethylformamide, dimethylacetamide, or dimethylpropionamide.

[0017] Further, in step one, the mass ratio of the catalyst to the oxidized konjac mannan is 1:5-20, and the catalyst is one or two of pyridine, 4-dimethylaminopyridine and triethylamine.

[0018] Further, in step one, the acylating agent is acetyl chloride, the ratio of the oxidized konjac mannan to the acetyl chloride is 1:1-4, the acylation reaction temperature is 45-75℃, and the reaction time is 12-36h.

[0019] Further, in step two, the mass ratio of the compound containing boronic acid group and aldehyde group to the gelatin is 1:2-10, and the reaction time is 6-12h.

[0020] The compound containing boronic acid group and aldehyde group is one or two of 4-fluoro-3-aldehyde benzene boronic acid, 3-fluoro-4-aldehyde benzene boronic acid, 2-formyl benzene boronic acid, 3-formyl benzene boronic acid and 4-formyl benzene boronic acid.

[0021] Further, in step two, the acylating agent containing carbon-carbon double bond is methacrylic anhydride; the mass ratio of the methacrylic anhydride to the gelatin is 1:10-20, the reaction temperature is 40-80℃, and the reaction time is 2-6h.

[0022] Further, in step three, in the A solution, the mass concentration of the modified konjac mannan is 2%-8%, and the mass concentration of the tannin acid is 5%-20%.

[0023] In the B solution, the concentration of the antibacterial drug is 0.1%-2%, the concentration of the photoinitiator is 0.25%-1%, and the concentration of the modified gelatin is 5%-25%.

[0024] The antibacterial drug is one or two of tobramycin, kanamycin and gentamicin.

[0025] Finally, the long residence hydrogel obtained based on the preparation method is applied to the prevention and treatment of intrauterine adhesion.

[0026] The beneficial effects of the present application are:

[0027] The long-residence hydrogel mainly comprises modified konjac mannan, tannic acid, antibacterial and anti-inflammatory drugs and modified gelatin, wherein the modified konjac mannan and the antibacterial and anti-inflammatory drugs have good antibacterial and anti-inflammatory effects, and further play a role in promoting repair; the modified konjac mannan and the tannic acid can play a role in chemical and physical adhesion, so that a longer residence time is achieved, and the failure caused by premature loss of the gel is avoided; the physical and chemical interaction between the modified konjac mannan, the tannic acid and the modified gelatin can form a hydrogel with good strength, and the uterine cavity adhesion caused by uterine wall contraction and contact can be avoided; in addition, the antibacterial drugs can be combined with the gel skeleton part to play a sustained-release role, the burst release of the drugs is avoided, and the utilization rate of the drugs is improved.

[0028] The aminoglycoside drugs (tobramycin / ceftazidime, etc.) loaded by the hydrogel in the application are combined with the gel network through electrostatic interaction, continuously and slowly release to inhibit uterine cavity infection, and the side effects caused by burst release of the drugs are avoided; the oxidative konjac mannan improves the free radical scavenging capacity, and the tannic acid effectively inhibits the key inflammatory factors TNF-α and IL-6, and jointly blocks the inflammatory cascade reaction to achieve antibacterial and anti-inflammatory effects.

[0029] The tannic acid regulates the polarization of macrophages from M1 type (pro-inflammatory) to M2 type (repair), and changes the inflammatory microenvironment into a microenvironment promoting repair; the active aldehyde group of the modified gelatin promotes cell adhesion, and the degradation product accelerates the regeneration of endometrial epithelium, and shortens the repair cycle.

[0030] In the application, the aldehyde group of the modified konjac mannan covalently bonded to the amino group on the tissue surface after double modification of oxidation and acetylation, and the hydrophobic segment enhances the mucosal adhesion force; the tannic acid forms a strong hydrogen bond network through the polyphenol structure to combine with the mucosa, and cooperates with the modified konjac mannan to construct a physical-chemical double adhesion barrier, which significantly resists uterine cavity contraction and flushing in a wet environment, realizes long-term residence, and further plays a good effect in preventing and treating uterine cavity adhesion.

[0031] An A / B double-solution step-by-step crosslinking design is adopted to resist uterine cavity pressure, the A solution forms a physical network through hydrogen bonding and hydrophobic interaction; the B solution forms a covalent crosslinking network under UV irradiation. The structure makes the gel have good flexibility and mechanical strength, can completely fill the irregular uterine cavity, resist the contraction pressure of the uterine wall, and realize physical isolation and anti-adhesion. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 is a schematic diagram of the establishment of an SD rat intrauterine adhesion (IUA) model and the treatment process of the hydrogel according to the application;

[0033] Figure 2 is a comprehensive evaluation diagram of the composition, crosslinking mechanism, functional characteristics and treatment effect of the long-residence hydrogel according to the application. DETAILED DESCRIPTION

[0034] In order to make the objects, technical solutions and advantages of the present application clearer, the present application will be further described in details below with specific examples. It should be understood that the specific examples described herein are only used to explain the present application and not used to limit the present application.

[0035] All other examples obtained by those skilled in the art based on the examples in the present application without making creative efforts are within the scope of protection of the present application. The experimental methods not specified in the following examples are usually carried out according to the conventional conditions or the conditions suggested by the manufacturers.

[0036] The reagents and raw materials used in the examples and comparative examples of the present application can be obtained through commercial channels unless otherwise specified.

[0037] Example 1

[0038] The preparation method of the long-residence hydrogel with antibacterial and anti-inflammatory and repair-promoting functions in the present example is as follows:

[0039] (1) Preparation of modified konjac mannan:

[0040] First, 5 g of konjac mannan with a molecular weight of 20 kDa was dissolved in 100 ml of deionized water, stirred uniformly, and then filtered. Then, the filtrate was gradually added to 500 ml of anhydrous ethanol, precipitated, and the precipitate was suction filtered and freeze-dried to obtain purified konjac mannan;

[0041] 5 g of sodium periodate was dissolved in 50 ml of a mixed solution of ethanol and water with a volume ratio of 1:5, and then 5 g of purified konjac mannan was added to the solution. After stirring for 48 h, the oxidized konjac mannan was purified and dried to obtain the oxidized konjac mannan;

[0042] 5 g of oxidized konjac mannan was added to 50 ml of N,N-dimethylformamide and dissolved at 50°C, and then 1 g of pyridine was added to obtain an oxidized konjac mannan solution. 5 ml of acetyl chloride was added to 5 ml of N,N-dimethylformamide and mixed uniformly to obtain an acetyl chloride mixed solution;

[0043] Then, the acetyl chloride mixed solution was added dropwise to the oxidized konjac mannan solution, and after the addition was completed, the reaction was carried out at 45°C for 48 h. After the reaction was completed, the modified konjac mannan was purified and dried.

[0044] (2) Preparation of modified gelatin: 10 g of gelatin was dissolved in 100 ml of a buffer solution with a pH of 8, and then 1 g of 4-fluoro-3-aldehyde benzene boronic acid was added, and stirred for 12 h of reaction; then 1 g of methacrylic anhydride was added to the solution, and stirred for 6 h of reaction at 80°C, and after the reaction was completed, the modified gelatin was obtained after purification and drying;

[0045] (3) 0.2 g of modified konjac mannan and 0.2 g of tannic acid were dissolved in 10 ml of deionized water to obtain solution A, 0.01 g of tobramycin, 0.025 g of photocuring agent and 0.5 g of modified gelatin were dissolved in 10 ml of deionized water to obtain solution B, and after the solutions A and B were mixed uniformly, the long-residence hydrogel with antibacterial, anti-inflammatory and repair-promoting effects was obtained by irradiation with 365 nm light.

[0046] Example 2

[0047] The preparation method of the long-residence hydrogel with antibacterial, anti-inflammatory and repair-promoting effects in this example is as follows:

[0048] (1) Preparation of modified konjac mannan:

[0049] First, 5 g of konjac mannan with a molecular weight of 100 kDa was dissolved in 100 ml of deionized water, stirred uniformly, filtered, and then the filtrate was gradually added to 500 ml of anhydrous ethanol, precipitated, and the precipitate was suction filtered and freeze-dried to obtain purified konjac mannan;

[0050] 15 g of sodium periodate was dissolved in 50 ml of a mixed solution of ethanol and water with a volume ratio of 1:5, and then 5 g of purified konjac mannan was added to the solution, stirred for 48 h of reaction, and then purified and dried to obtain oxidized konjac mannan;

[0051] 5 g of oxidized konjac mannan was added to 50 ml of dimethylacetamide and dissolved at 80°C, and then 0.25 g of 4-dimethylaminopyridine was added to obtain an oxidized konjac mannan solution; 2.5 ml of acetyl chloride was added to 5 ml of dimethylacetamide and mixed uniformly to obtain an acetyl chloride mixed solution;

[0052] Then, the acetyl chloride mixed solution was added dropwise to the oxidized konjac mannan solution, and after the addition was completed, the reaction was carried out at 60°C for 24 h, and after the reaction was completed, the modified konjac mannan was obtained after purification and drying;

[0053] (2) Preparation of modified gelatin: 10 g of gelatin was dissolved in 100 ml of a buffer solution with a pH of 8, and then 1 g of 4-fluoro-3-aldehyde benzene boronic acid was added, and stirred for 12 h of reaction; then 1 g of methacrylic anhydride was added to the solution, and stirred for 6 h of reaction at 80°C, and after the reaction was completed, the modified gelatin was obtained after purification and drying;

[0054] (3) 0.5 g modified konjac mannan and 1.0 g tannic acid are dissolved in 10 ml deionized water to obtain solution A, 0.1 g gentamicin, 0.05 g photocuring agent and 1.0 g modified gelatin are dissolved in 10 ml deionized water to obtain solution B, after mixing solution A and solution B uniformly, the long-residence hydrogel with antibacterial and anti-inflammatory effects and promoting repair effect can be obtained by irradiation with 385 nm light.

[0055] Example 3

[0056] The preparation method of the long-residence hydrogel with antibacterial and anti-inflammatory effects and promoting repair effect of the present embodiment is as follows:

[0057] (1) Preparation of modified konjac mannan:

[0058] First, 5 g of konjac mannan with a molecular weight of 200 kDa is dissolved in 200 ml of deionized water, stirred uniformly, filtered, then the filtrate is gradually added to 1000 ml of anhydrous ethanol, precipitated, and the precipitate is suction filtered and freeze-dried to obtain purified konjac mannan;

[0059] 50 g of sodium periodate is dissolved in 50 ml of a mixed solution of ethanol and water with a volume ratio of 1:10, then 5 g of purified konjac mannan is added to the solution, stirred for 12 h, and then purified and dried to obtain oxidized konjac mannan;

[0060] 5 g of oxidized konjac mannan is added to 50 ml of dimethylpropylamide and dissolved at 60°C, then 0.5 g of triethylamine is added to obtain an oxidized konjac mannan solution; 1.25 ml of acetyl chloride is added to 5 ml of dimethylacetamide and mixed uniformly to obtain an acetyl chloride mixed solution;

[0061] Then, the acetyl chloride mixed solution is added dropwise to the oxidized konjac mannan solution, and after the addition is completed, it is reacted at 75°C for 12 h, and after the reaction is completed, purification and drying are performed to obtain modified konjac mannan;

[0062] (2) Preparation of modified gelatin: 10 g of gelatin is dissolved in 100 ml of a buffer solution with a pH of 8, then 2 g of 2-formylphenylboronic acid is added, and stirred for 9 h; then 0.5 g of methacrylic anhydride is added to the solution, and stirred at 60°C for 6 h, and after the reaction is completed, purification and drying are performed to obtain modified gelatin;

[0063] (3) 0.8 g modified konjac mannan and 2.0 g tannic acid are dissolved in 10 ml deionized water to obtain solution A, 0.2 g kanamycin, 0.10 g photocuring agent and 2.0 g modified gelatin are dissolved in 10 ml deionized water to obtain solution B, after mixing solution A and solution B uniformly, the long-residence hydrogel with antibacterial, anti-inflammatory and repair-promoting effects can be obtained by irradiation with 405 nm light.

[0064] Example 4

[0065] The preparation method of the long-residence hydrogel with antibacterial, anti-inflammatory and repair-promoting effects of the present embodiment is as follows:

[0066] (1) Preparation of modified konjac mannan:

[0067] First, 5 g of konjac mannan with a molecular weight of 50 kDa is dissolved in 100 ml of deionized water, stirred uniformly, filtered, then the filtrate is gradually added to 500 ml of anhydrous ethanol, precipitated, and the precipitate is suction filtered and freeze-dried to obtain purified konjac mannan;

[0068] 10 g of sodium periodate is dissolved in 50 ml of a mixed solution of ethanol and water in a volume ratio of 1:8, then 5 g of purified konjac mannan is added to the solution, stirred for 12 h, and then purified and dried to obtain oxidized konjac mannan;

[0069] 5 g of oxidized konjac mannan is added to 50 ml of N,N-dimethylformamide and dissolved at 50°C, then 0.25 g of triethylamine and 0.25 g of pyridine are added to obtain an oxidized konjac mannan solution; 5 ml of acetyl chloride is added to 5 ml of N,N-dimethylformamide and mixed uniformly to obtain an acetyl chloride mixed solution;

[0070] Then, the acetyl chloride mixed solution is added dropwise to the oxidized konjac mannan solution, and after the addition is completed, it is reacted at 60°C for 36 h, and after the reaction is completed, purification and drying are performed to obtain modified konjac mannan;

[0071] (2) Preparation of modified gelatin: 10 g of gelatin is dissolved in 100 ml of a buffer solution with a pH of 8, then 5 g of 3-formylphenylboronic acid is added, and stirred for 12 h; then 1.0 g of methacrylic anhydride is added to the solution, and stirred at 80°C for 2 h, and after the reaction is completed, purification and drying are performed to obtain modified gelatin;

[0072] (3) 0.2 g modified konjac mannan and 2.0 g tannic acid are dissolved in 10 ml deionized water to obtain solution A, 0.05 g kanamycin and 0.05 g tobramycin, 0.075 g photocuring agent and 1.0 g modified gelatin are dissolved in 10 ml deionized water to obtain solution B, after mixing solution A and solution B uniformly, the long-residence water gel with antibacterial, anti-inflammatory and repair-promoting effects can be obtained by irradiation with 365 nm light.

[0073] Example 5

[0074] The preparation method of the long-residence water gel with antibacterial, anti-inflammatory and repair-promoting effects of the present example is as follows:

[0075] (1) Preparation of modified konjac mannan:

[0076] First, 5 g of konjac mannan with a molecular weight of 20 kDa is dissolved in 100 ml of deionized water, stirred uniformly, filtered, then the filtrate is gradually added to 500 ml of anhydrous ethanol, precipitated, and the precipitate is suction filtered and freeze-dried to obtain purified konjac mannan;

[0077] 30 g of sodium periodate is dissolved in 50 ml of a mixed solution of ethanol and water with a volume ratio of 1:5, then 5 g of purified konjac mannan is added to the solution, stirred for 12 h, and then purified and dried to obtain oxidized konjac mannan;

[0078] 5 g of oxidized konjac mannan is added to 50 ml of dimethylacetamide and dissolved at 80°C, then 0.125 g of triethylamine and 0.125 g of 4-dimethylaminopyridine are added to obtain an oxidized konjac mannan solution; 2.5 ml of acetyl chloride is added to 5 ml of dimethylacetamide and mixed uniformly to obtain an acetyl chloride mixed solution;

[0079] Then, the acetyl chloride mixed solution is added dropwise to the oxidized konjac mannan solution, and after the addition is completed, it is reacted at 75°C for 12 h, and then purified and dried to obtain modified konjac mannan;

[0080] (2) Preparation of modified gelatin: 10 g of gelatin is dissolved in 100 ml of a buffer solution with a pH of 8, then 0.5 g of 3-formylphenylboronic acid and 0.5 g of 4-formylphenylboronic acid are added, stirred for 6 h; then 0.5 g of methacrylic anhydride is added to the solution, and stirred at 60°C for 4 h, and then purified and dried to obtain modified gelatin;

[0081] (3) 0.5 g modified konjac mannan and 1.0 g tannic acid were dissolved in 10 ml of deionized water to obtain solution A, 0.1 g kanamycin and 0.1 g gentamicin, 0.05 g photocuring agent and 2.0 g modified gelatin were dissolved in 10 ml of deionized water to obtain solution B, and the long-residence water gel with antibacterial and anti-inflammatory effects and promoting repair effect was obtained by mixing solution A and solution B and then irradiating with 405 nm light.

[0082] Effect Example 1

[0083] Test instrument:

[0084] Tensile tester (model: TA.XTplus Texture Analyzer, Stable Micro Systems, UK) In vitro flushing simulation device (self-made fluid circulation system, adjustable flow rate); light irradiation device: ultraviolet light curing instrument (model: UVP CL-1000, wavelength range: 365-405 nm).

[0085] Test process and test results:

[0086] The water gel samples prepared in Examples 1-5 were selected for the following test items:

[0087] ① Residency (in vitro simulation of uterine fluid flushing residence time), including the following in vitro residence test procedure:

[0088] The water gel sample was coated on the surface of the simulated uterine cavity model (silicone sheet or pig uterine tissue sheet); it was placed in the fluid circulation system and flushed with simulated uterine fluid (PBS buffer, pH 7.4) at a flow rate of 1 ml / min; the residual area of the water gel was recorded every 1 hour until it was completely removed; the residence time (hours) was recorded.

[0089] ② Adhesion (uterine tissue adhesion), including the following adhesion test procedure:

[0090] The water gel sample was contacted and pressed with a fresh pig uterine tissue sheet using a tensile tester, and the pressing time was set to 5 minutes, followed by a tensile test, and the maximum peeling force (unit: N / cm 2 ) was recorded.

[0091] Example number Residence time (h) Intrauterine tissue adhesion force (N / cm 2 )]]> Example 1 12 0.85 Example 2 18 1.12 Example 3 24 1.35 Example 4 20 1.28 Example 5 26 1.42

[0092] From the above table, it can be seen that the hydrogels prepared in all examples exhibit better uterine cavity residence and tissue adhesion than conventional hydrogels. With the optimization of the formula (such as increasing the proportion of tannic acid, optimizing the modification degree, etc., see examples 2 to 5), the residence time and adhesion are significantly improved. Among them, the residence time of example 5 is the longest, and the adhesion is the largest, indicating that it has the best uterine cavity adhesion performance and long-term residence ability, examples 3 and 4 also show good performance. This is due to the covalent bonding of aldehyde groups of modified konjac mannan with amino groups on the tissue surface and the synergistic effect of multiple hydrogen bonds of tannic acid, which enhances the adhesion of the hydrogel. With the optimization of the proportion of tannic acid and the modification degree, the residence time of the hydrogel is gradually prolonged, and the adhesion is enhanced, thereby effectively resisting the contraction of the uterine cavity and the flushing of the wet environment.

[0093] Effect Example 2

[0094] Test instrument:

[0095] UV spectrophotometer (model: UV-2600, Shimadzu Corporation) was used for drug release amount detection; standard petri dish and vernier caliper were used for inhibition zone measurement; microdilution method used enzyme marker (model: SpectraMax iD3, Molecular Devices Corporation, USA).

[0096] Source of raw materials:

[0097] Antibiotics such as tobramycin, gentamicin, kanamycin, etc. are commercially available (Araladin reagent); LB medium, agar powder, PBS buffer, etc. are purchased from China National Pharmaceutical Group or Sigma-Aldrich.

[0098] Test process and test results:

[0099] Drug release test: the hydrogel sample was placed in PBS buffer, incubated in a 37℃ constant temperature shaker, and the drug concentration was determined at regular intervals to calculate the 24h cumulative release rate.

[0100] Inhibition zone test: paper disc diffusion method was used, the hydrogel sample was placed on the agar plate inoculated with bacteria (such as escherichia coli, staphylococcus aureus), and the inhibition zone diameter was measured after 24h incubation.

[0101] Minimum inhibitory concentration (MIC) test: microdilution method was used to observe the growth inhibition of bacteria at the lowest concentration.

[0102] Test reference standards include "GB / T 27894.3-2011 Antibacterial Performance Test Method; Part 3: Inhibition Zone Method" and "CLSIM07-A10 Microbial Drug Sensitivity Test Standard".

[0103]

[0104] From the above table, it can be seen that the hydrogel of the present application can effectively load antibiotics (loading capacity 12.5-22.7 μg / mg) and achieve sustained release (cumulative release rate 68.2-81.3%) within 24 hours without burst release. The drug loading test shows that Example 3 has the highest value, indicating that it has the highest drug loading capacity. All samples show obvious inhibition zone (diameter 14.2-19.5 mm) and low MIC value (1.0-2.0 μg / ml), proving that they have long-acting and high-efficiency antibacterial capacity. Since the larger the diameter of the inhibition zone, the stronger the antibacterial effect, the inhibition zone diameter of Example 3 is the largest, and the minimum inhibitory concentration is the lowest, indicating that it has the highest antibacterial efficiency. This is due to the fact that the hydrogel loads antibiotics through electrostatic interaction and achieves slow release, while the synergistic effect of oxidized konjac mannan and tannic acid enhances the antibacterial and anti-inflammatory effects.

[0105] Effect Example 3

[0106] Test instruments:

[0107] Compression performance testing instrument: electronic universal material testing machine (model: Instron 5943, Instron Corporation, USA), equipped with a 10N load sensor; Swelling rate testing equipment: precision electronic balance (model: ME204, Mettler-Toledo Corporation), constant temperature water bath (37°C).

[0108] Sample source: long-residence hydrogel samples prepared in Examples 1-5, all in the form of cylindrical hydrogels (diameter 10 mm, height 10 mm) after light curing molding;

[0109] Test process and test results:

[0110] ① Compression modulus test steps:

[0111] Place the hydrogel sample on the testing machine platform; compress at a speed of 1 mm / min, and record the stress-strain curve; linearly fit within the strain range of 5-15%, and calculate the compression modulus (unit: kPa); repeat the test for each group of samples 3 times, and take the average value.

[0112] ② Swelling rate test steps:

[0113] Weigh the dried hydrogel sample and record the initial mass W0; immerse the sample in 37°C deionized water; take out the sample every 1 hour, weigh after absorbing the surface moisture, and record the mass Wt;

[0114] Calculate the swelling rate: Swelling rate (%) = (Wt-W0) / W0 x 100%; record the maximum swelling rate after 24 hours:

[0115]

[0116] In combination with the above table and Figure 2 It can be seen that based on mechanical testing, the compression modulus of the hydrogel is in the range of 18.2 to 31.7 kPa, indicating that it has good mechanical strength to resist uterine contraction pressure, among which the compression modulus of Example 3 is the highest. The swelling rate is between 160% and 220%, indicating that the hydrogel can absorb water moderately to maintain a moist environment without excessive swelling. The swelling rate of Example 1 is the highest, but the swelling rate of Example 3 is lower, which may be due to the lower water absorption capacity caused by the higher crosslinking density, but this also means that the gel structure is more stable. In summary, Example 5 achieves a good balance between compression modulus and swelling rate, indicating that the double network structure provides good mechanical properties and stability.

[0117] Effect Example 4

[0118] A rat model of intrauterine adhesion was constructed, and the long-resident hydrogel prepared in Examples 1-5, which has both antibacterial and anti-inflammatory effects and promotes repair, was injected into the uterine cavity to observe the preventive and therapeutic effects of the long-resident hydrogel on intrauterine adhesion.

[0119] Operation process: After the female Sprague-Dawley rats (6-8 weeks old) were anesthetized, a 1.5 cm incision was made along the midline of the rat's abdomen to expose the uterus. As shown in Figure 1 , the modeling group injected 0.3 ml of 95% ethanol into the uterus and temporarily closed the proximal end of the uterus with surgical sutures. The prepared hydrogel was injected into the uterine cavity to achieve the therapeutic effect. Immediately after modeling, the hydrogel was injected into the uterine cavity, and the ligation suture was removed. The same operation was performed on the contralateral uterus. As shown in Figure 2 , the rats were randomly divided into four groups: undamaged group (sham operation group), endometrial injury group (model group), hydrogel injection group (treatment group). Finally, the incision and abdominal cavity were closed with surgical sutures.

[0120] In summary, as shown in Figure 2 , the long-resident hydrogel prepared in Example 1 significantly improves the intrauterine adhesion force compared to conventional hydrogels by modifying the aldehyde group of konjac glucomannan through covalent bonding and the multi-hydrogen bond of tannic acid, achieving long-term residence; The tobramycin loaded in it is combined with the gel network through electrostatic force, forming a sustained antibacterial and anti-inflammatory system; At the same time, the oxidative konjac polysaccharide enhances the free radical scavenging ability, and cooperates with tannic acid to regulate the inflammatory microenvironment and promote endometrial regeneration. Comprehensive confirmation of the overall progress of the gel in residence, antibacterial and anti-inflammatory, and repair functions.

[0121] The long-residence hydrogel prepared in Example 2 is physically adhered to the network by a high proportion of tannic acid, which makes the gel resist the uterine contraction better than the prior art; the electrostatic combination of gentamicin and the negatively charged gel matrix prolongs the drug release period and avoids the risk of burst release; the light cross-linked network of modified gelatin provides mechanical support, and the immunomodulatory properties of tannic acid work together to create a pro-repair microenvironment. The enhanced effect of high-tannic acid formulation on multifunctional synergy is verified.

[0122] The long-residence hydrogel prepared in Example 3 effectively inhibits gram-negative bacteria by high-concentration kanamycin; the active aldehyde groups of oxidized konjac polysaccharide cross-link with histamine groups to accelerate the repair of the injury interface; the double-network structure (physical cross-linking A liquid + chemical cross-linking B liquid) improves the gel's compressive strength, completely adheres to the irregular uterine cavity shape, and has a significantly better physical barrier effect than traditional barrier materials. It is confirmed that it accelerates uterine regeneration through broad-spectrum antibacterial and repair factor activation.

[0123] The long-residence hydrogel prepared in Example 4 overcomes the limitations of single drugs by producing an antibacterial synergistic effect with the combination of tobramycin and kanamycin; the hydrophobic segment of acetylated konjac polysaccharide enhances mucosal retention, while the antioxidant synergistic effect of tannic acid and oxidized konjac polysaccharide effectively blocks the free radical damage chain reaction, creating a low oxidative stress environment for tissue repair. It embodies the advantages of dual-drug synergy and antioxidant damage repair.

[0124] The long-residence hydrogel prepared in Example 5 has a degradation rate that matches the tissue repair process through the combined application of dual-antibacterial drugs, dual-modified gelatin ligands, and optimized konjac polysaccharide / tannic acid ratios, and has both biocompatibility and functional activity. Its anti-adhesion mechanism covers four aspects: physical barrier, antibacterial control, inflammation regulation, and active repair, and the overall therapeutic effect is better.

[0125] Finally, it should be noted that the above-described only for the preferred embodiments of the present application, and not for limiting the present application, although the present application has been described in detail with reference to the foregoing embodiments, for those skilled in the art, it still can modify the technical solutions recorded in the foregoing embodiments, or make equivalent replacement for part of the technical features, any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application, shall be included in the protection scope of the present application.

Claims

1. A method for preparing a long-resident hydrogel with both anti-bacterial and anti-inflammatory and pro-repairing properties, characterized in that, The method comprises the following steps: Step one, preparation of modified konjac mannan: Konjac mannan is oxidized with an oxidizing agent in the presence of a solvent to obtain oxidized konjac mannan; The oxidized konjac mannan is acylated with an acylating agent in the presence of a catalyst to obtain modified konjac mannan; Step two, preparation of modified gelatin: The gelatin is first reacted with a compound containing a boronic acid group and an aldehyde group, and then reacted with an acylating agent containing a carbon-carbon double bond to obtain modified gelatin; Step three, preparation of long residence hydrogel: The modified konjac mannan obtained in step one is formulated into an A solution with tannic acid; an antibacterial drug, a photoinitiator and the modified gelatin obtained in step two are formulated into a B solution; the A solution and the B solution are mixed and then cured by light irradiation to form the long residence hydrogel.

2. The production method according to claim 1, characterized by, In step one, the konjac mannan is first purified, and the purification process is that the konjac mannan with a molecular weight of 20-100 kDa is dissolved in water, and then the konjac mannan is precipitated by ethanol, and then filtered and freeze-dried.

3. The preparation method according to claim 1, characterized in that, In step one, the oxidizing agent is sodium periodate, and the mass ratio of konjac mannan to sodium periodate is 1:1-5; the solvent is a mixed solution of ethanol and water, and the volume ratio of water to ethanol is 1:5-10; the oxidation reaction time is 12-48 h.

4. The method of claim 1, wherein, In step one, the solvent for the acylation reaction is one of N,N-dimethylformamide, dimethylacetamide or dimethylpropionamide.

5. The preparation method according to claim 1, characterized in that, In step one, the mass ratio of the catalyst to the oxidized konjac mannan is 1:5-20, and the catalyst is one or two of pyridine, 4-dimethylaminopyridine and triethylamine.

6. The method of claim 1, wherein, In step one, the acylating agent is acetyl chloride, and the ratio of the oxidized konjac mannan to acetyl chloride is 1:1-4, the acylation reaction temperature is 45-75℃, and the reaction time is 12-36 h.

7. The preparation method according to claim 1, characterized in that, In step two, the mass ratio of the compound containing a boronic acid group and an aldehyde group to the gelatin is 1:2-10, and the reaction time is 6-12 h; The compound containing a boronic acid group and an aldehyde group is one or two of 4-fluoro-3-aldehyde benzene boronic acid, 3-fluoro-4-aldehyde benzene boronic acid, 2-formyl benzene boronic acid, 3-formyl benzene boronic acid and 4-formyl benzene boronic acid.

8. The method of claim 1, wherein, In step two, the acylating agent containing a carbon-carbon double bond is methacrylic anhydride; the mass ratio of methacrylic anhydride to the gelatin is 1:10-20, the reaction temperature is 40-80℃, and the reaction time is 2-6 h.

9. The method of claim 1, wherein, In step three, in the A solution, the mass concentration of the modified konjac mannan is 2%-8%, and the mass concentration of tannic acid is 5%-20%; In the B solution, the concentration of the antibacterial drug is 0.1%-2%, the concentration of the photoinitiator is 0.25%-1%, and the concentration of the modified gelatin is 5%-25%; The antibacterial drug is one or two of tobramycin, kanamycin and gentamicin.

10. The long residence hydrogel prepared by the preparation method of claim 1 is applied to the prevention and treatment of intrauterine adhesion.

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