Antibacterial hydrogel adhesive as well as preparation method and application thereof

Abstract

The invention provides an antibacterial hydrogel adhesive as well as a preparation method and application thereof, and belongs to the technical field of composite material preparation. The antibacterial hydrogel adhesive is prepared from silk fibroin, epigallocatechin gallate and gallic acid according to the mass ratio of (2 to 24) to (4 to 24) to 1. Phenolic hydroxyl groups in polyphenol molecules and polar groups of silk fibroin remarkably enhance the intermolecular binding force through the synergistic effect of hydrogen bond-hydrophobic interaction, and the multifunctional silk fibroin polyphenol hydrogel with excellent biocompatibility, high adhesion performance and broad-spectrum antibacterial property is successfully prepared. The antibacterial hydrogel adhesive disclosed by the invention can be firmly adhered to a cesarean delivery wet environment incision, effectively kills group B streptococcus, and prevents formation of uterine diverticulosis due to poor healing caused by incision infection.

Description

Antibacterial hydrogel adhesives, their preparation methods, and applications Technical Field

[0001] This invention relates to the field of composite material preparation technology, and in particular to an antibacterial hydrogel adhesive, its preparation method, and its application. Background Technology

[0002] Cesarean section is a surgical procedure performed by making an incision in the abdominal wall and uterine wall of a woman at or after 28 weeks of gestation to remove the fetus and its appendages. However, while cesarean section resolves the delivery problem, it also poses potential threats to the health of both mother and baby. Uterine scar diverticulum (CSD or PCSD) is a common postoperative complication with a high incidence. CSD forms due to poor healing of the uterine incision, leading to thinning of the myometrium at the scar site, which in turn forms a depression or cavity communicating with the uterine cavity. This can cause a series of clinical symptoms and affect the patient's quality of life.

[0003] Studies show that approximately 22% of women who undergo cesarean section may develop large diverticula, with a depth exceeding 5 mm, which are closely associated with long-term complications. These complications include, but are not limited to, postmenstrual spotting, vaginitis, and pelvic inflammatory disease. In more severe cases, they may affect fertility and even lead to scar rupture or uterine rupture during subsequent pregnancies, which is particularly dangerous. Once a uterine diverticulum forms, it cannot heal, making prevention of CSD (corneal diverticulum stenosis) crucial.

[0004] Tracing the causes of diverticula, CSD formation is inextricably linked to infection, particularly Group B Streptococcus (GBS) infection. Currently, prophylactic intravenous antibiotics are mainly used clinically to reduce the risk of GBS infection and prevent diverticula to some extent. However, this method is limited by the clinical condition of pregnant women and the safety considerations of antibiotic use, and its effectiveness remains insufficient. Therefore, exploring more direct and effective preventive measures is particularly urgent. Summary of the Invention

[0005] In view of this, in order to at least partially solve at least one of the aforementioned technical problems, the present invention provides an antibacterial hydrogel adhesive, its preparation method, and its application.

[0006] According to one aspect of the present invention, an antibacterial hydrogel adhesive is provided, the antibacterial hydrogel adhesive comprising silk fibroin, epigallocatechin gallate and gallic acid in a mass ratio of (2~24):(4~24):1.

[0007] According to another aspect of the present invention, a method for preparing the antibacterial hydrogel adhesive as described above is provided, the method comprising: dissolving epigallocatechin gallate and gallic acid in water at 20-30°C to obtain a polyphenol solution; and mixing a silk fibroin solution with the polyphenol solution in an equal volume ratio to obtain the antibacterial hydrogel adhesive.

[0008] According to another aspect of the invention, the use of the antibacterial hydrogel adhesive as described above in the preparation of a medicament for the prevention of uterine scar diverticulum is provided.

[0009] Based on the above technical solution, the antibacterial hydrogel adhesive and its preparation method provided by the present invention, and its application in the prevention of uterine scar diverticulum, have at least one of the following beneficial effects:

[0010] 1. The antibacterial hydrogel adhesive prepared by this invention is made from a mixture of natural protein polymer silk fibroin and plant polyphenols. Silk fibroin is derived from natural silkworm silk, and its amino acid composition is similar to that of human proteins. It can be slowly degraded into harmless amino acids in the body, posing no risk of immune rejection. Plant polyphenols are extracted from natural plants and also possess low toxicity and high safety. This method does not involve complex chemical cross-linking or toxic cross-linking agents. The raw materials and cross-linking methods exhibit good biocompatibility, which is beneficial for clinical translation.

[0011] 2. The antibacterial hydrogel adhesive prepared by this invention exhibits excellent adhesion and antibacterial properties. Silk fibroin possesses a unique molecular structure and abundant active groups, providing a fundamental support for adhesion; the structures of catechol, gallic acid, and other plant polyphenols mimic the adhesion mechanism of mussel byssal silk proteins in nature, tightly binding to the substrate surface through multiple forces such as hydrogen bonds and π-π interactions. When the two are mixed, the resulting antibacterial hydrogel adhesive can firmly adhere to the surface of moist biological tissues.

[0012] The phenolic hydroxyl structure abundant in plant polyphenols can exert a strong inhibitory effect on group B streptococci through multiple mechanisms such as disrupting bacterial cell membranes, interfering with bacterial metabolism, and inhibiting biofilm formation. At the same time, after binding with silk fibroin, it can form a stable antibacterial network through hydrogen bonds and hydrophobic interactions. Based on the long-term sustained release of polyphenols in the antibacterial network, the antibacterial activity of polyphenols can be fully exerted and is not easily lost.

[0013] 3. The natural antibacterial hydrogel prepared by this invention firmly adheres to cesarean section incisions, effectively kills Group B Streptococcus, and can effectively prevent incision infection leading to poor healing and the formation of uterine diverticula. Furthermore, the antibacterial hydrogel adhesive prepared by this invention has high biocompatibility, providing an important guarantee for its clinical application in preventing uterine scar diverticula. Attached Figure Description

[0014] The above and other objects, features and advantages of the present invention will become more apparent from the following description of embodiments of the invention with reference to the accompanying drawings, in which:

[0015] Figure 1 is a schematic diagram of the gelation of different concentrations of SF and different concentrations of polyphenols (concentration is the input concentration) in Example 1 of the present invention;

[0016] Figure 2 shows the potential of silk fibroin at different pH values ​​in Example 1 of the present invention (n=3).

[0017] Figures 3 to 5 show the effects of different concentrations of EGG and GA on hydrogel formation when the SF solution concentration is 20, 40, and 60 mg / mL in Example 1 of the present invention.

[0018] Figure 6 shows the adhesion characteristics test results of the antibacterial hydrogel adhesives prepared based on different concentrations of EGCG in Example 2 of the present invention;

[0019] Figure 7 shows the adhesion characteristics test results of the antibacterial hydrogel adhesives prepared based on different concentrations of GA in Example 2 of the present invention;

[0020] Figure 8 is a comparison of the adhesion strength of the antibacterial hydrogel adhesives of Example 2 and Comparative Examples 2-1 to 2-2 of the present invention (n=3).

[0021] Figure 9 shows the SEM cross-sectional morphology and mechanical property characterization of SPH in Embodiment 2 of the present invention, where A is the SEM cross-sectional view, B is the storage modulus and loss modulus of SF / E&G / SPH (n = 3), C is the rheological properties of SPH at 25 ºC with amplitude scanning from 0.1% to 1000% strain, and D is the rheological properties of SPH at 25 ºC with frequency scanning from 0.1 to 10 Hz.

[0022] Figure 10 shows the FTIR spectral analysis results of the antibacterial hydrogel adhesive, where A is the FTIR spectrum of SF, EGCG, GA and SPH, B is the quantitative analysis diagram of the fitted secondary structure of SF, C is the quantitative analysis diagram of the fitted secondary structure of SPH, and D is the comparison diagram of the β-sheet content of SF and SPH (n=3).

[0023] Figure 11 shows the degradation of SPH in different solvents in Example 2 of the present invention;

[0024] Figure 12 shows the dynamic and long-term adhesion results of SPH on the substrate in the embodiment of the present invention. A is a schematic diagram of the strong adhesion of SPH on different substrates, and B is the long-term wet adhesion performance of SPH on pig uterine tissue.

[0025] Figure 13 shows fluorescence microscopy and SEM images of the cross-section of SPH adhering to porcine uterine tissue;

[0026] Figure 14 shows the shear strength of SPH in different tissues in Example 2 of the present invention (n = 3).

[0027] Figure 15 shows the interfacial toughness of SPH in different tissues in Example 2 of the present invention (n = 3);

[0028] Figure 16 shows the tensile strength of SPH in different tissues in Example 2 of the present invention (n = 3);

[0029] Figure 17 shows the release curve of total polyphenols in SPH within PBS buffer in Example 2 of the present invention;

[0030] Figure 18 shows the antibacterial effect of SPH on Staphylococcus aureus and multiple clinical group B streptococci in Example 2 of the present invention, where A is the inhibition zone image and B is the quantitative bar chart of antibacterial activity.

[0031] Figure 19 shows SEM and TEM images of bacterial morphology after co-culturing SPH with bacteria in Example 2 of the present invention, where A is Staphylococcus aureus and B is Group B Streptococcus.

[0032] Figure 20 shows the antioxidant activity of SPH in Example 2 of the present invention, where A is the UV absorption spectrum of SPH extracts of different concentrations based on the ABTS method, B is the quantitative result of antioxidant activity based on the ABTS method (n = 3), C is the UV absorption spectrum of SPH extracts of different concentrations based on the DPPH method, and D is the quantitative result of antioxidant activity based on the DPPH method (n = 3).

[0033] Figure 21 shows the animal model creation and experimental group surgical plan in Application Example 1 of the present invention, where Figure A is a schematic diagram of animal model creation and Figure B is a representative image of the treatment plan and the main steps of the experimental group surgery.

[0034] Figure 22 shows the in vivo preventive evaluation of the hydrogel in the rat model of uterine diverticulum in Example 1 of this invention. Figure A shows photographs of the uterus 21 days post-surgery in each group; Figure B shows H&E-stained images of cross-sections of uterine tissue in each group 21 days post-surgery.

[0035] Figure 23 shows the quantitative analysis of the ratio of the thickness of the uterine basal layer at the surgical incision site to the thickness of the adjacent basal layer in hematoxylin-eosin (H&E) staining in Application Example 1 of the present invention (n = 3).

[0036] Figure 24 shows the Masson staining results of cross-sections of uterine tissue in each group 21 days after surgery, where A is the Masson staining image and B is the quantitative analysis of the relative area coverage of collagen in Masson staining.

[0037] Figure 25 shows the H&E staining results of major organs (heart, liver, spleen, lung, and kidney) in each group 21 days after surgery in the application examples of this invention.

[0038] Figure 26 shows the blood sample indicators of each group 21 days after surgery in Application Example 1 of the present invention, where A to H are, in order, white blood cell count, percentage of neutrophils, percentage of lymphocytes, percentage of monocytes, percentage of eosinophils, percentage of basophils, alanine aminotransferase and urea content. Detailed Implementation

[0039] Hereinafter, embodiments of the present invention will be described with reference to the accompanying drawings. However, it should be understood that these descriptions are exemplary only and are not intended to limit the scope of the invention. In the following detailed description, numerous specific details are set forth to provide a thorough understanding of the embodiments of the invention for ease of explanation. However, it will be apparent that one or more embodiments may be practiced without these specific details. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concept of the invention.

[0040] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the invention. The term "comprising" as used herein indicates the presence of features, steps, or operations, but does not exclude the presence or addition of one or more other features.

[0041] When using expressions such as "at least one of A, B and C", they should generally be interpreted in accordance with the meaning that is commonly understood by those skilled in the art (e.g., "a system having at least one of A, B and C" should include, but is not limited to, a system having A alone, a system having B alone, a system having C alone, a system having A and B, a system having A and C, a system having B and C, and / or a system having A, B and C, etc.).

[0042] Post-cesarean section uterine scar diverticulum (CSD), also known as post-cesarean section uterine incision defect (PCSD), or simply "uterine scar diverticulum" or "uterine diverticulum" in this invention, refers to a depression or cavity formed on the uterine cavity surface due to the incomplete healing of the cesarean section incision. Once formed, it is difficult to heal, so preventing the occurrence of CSD is very important.

[0043] Group B streptococcal (GBS) infection is considered a clear risk factor for diverticulum development (CSD). Clinically, prophylactic intravenous antibiotics are mainly used to reduce the risk of GBS infection and prevent diverticulum development to some extent. However, this method is limited by the clinical condition of pregnant women and the safety considerations of antibiotic use, and its effectiveness is still insufficient.

[0044] Against this backdrop, research on the application of antibacterial and anti-inflammatory materials to cesarean section incisions to effectively prevent infection and subsequent poor healing and diverticulum formation has gradually gained attention. However, existing wound adhesives, such as cyanoacrylate glue and fibrin adhesives, while promoting wound healing to some extent, have weak adhesion, lack antibacterial effects, and are difficult to adapt to the significant contractions during uterine repositioning, thus failing to meet clinical needs.

[0045] In view of the above-mentioned shortcomings, this invention was finally obtained after a long period of research and practice. By adding a mixed solution of epigallocatechin gallate (EGCG) and gallic acid (GA) to a silk fibroin (SF) solution for crosslinking, the pH value close to the isoelectric point of SF promotes the layered assembly of SF molecules, triggering β-sheet formation. The phenolic hydroxyl groups in the polyphenol molecules and the polar groups of SF significantly enhance the intermolecular binding force through the synergistic effect of hydrogen bonding and hydrophobic interactions, successfully preparing a multifunctional silk fibroin polyphenol hydrogel, abbreviated as SPH, with excellent biocompatibility, high adhesion performance, and broad-spectrum antibacterial properties. Group B streptococcal infection is inseparable from the formation of uterine diverticula, and plant polyphenols have a strong inhibitory effect on Group B streptococci; at the same time, a stable antibacterial network is formed through hydrogen bonding and hydrophobic interactions with SF, achieving long-term stable release of polyphenols. This hydrogel can firmly adhere to the wet environment of the cesarean section incision, effectively kill Group B Streptococcus, and prevent incision infection that could lead to poor healing and the formation of uterine diverticulum.

[0046] Specifically, according to one aspect of the present invention, an antibacterial hydrogel adhesive SPH is provided, comprising SF, EGCG and GA in a mass ratio of (2~24):(4~24):1.

[0047] According to some embodiments of the present invention, SF, as the main hydrogel network forming agent, is rich in amino acid residues. Its amino acid composition is similar to that of human proteins, enabling it to form various interactions with proteins on the tissue surface. It can be degraded into harmless amino acids in vivo, posing no risk of immune rejection. The addition of GA is suitable for adjusting pH to regulate the layered assembly of the natural polymer SF, triggering its conformational transition from random coils to β-sheets, thereby inducing rapid gelation. However, GA has a relatively small molecular weight and few phenolic hydroxyl groups, insufficient to provide adhesive force. The addition of EGCG, through stronger hydrogen bonding-hydrophobic interactions, forms a denser network, significantly improving the adhesive strength of the antibacterial hydrogel adhesive and also enhancing structural stability. GA and EGCG, as polyphenolic compounds, can provide antibacterial and antioxidant effects, especially against Group B Streptococcus. This endows the complex with strong antibacterial and antioxidant activities, and a multifunctional hydrogel with excellent biocompatibility, high adhesion performance and broad-spectrum antibacterial properties, abbreviated as SPH, has been successfully prepared. This hydrogel can firmly adhere to the cesarean section incision, effectively kill Group B Streptococcus, and prevent incision infection leading to poor healing and the formation of uterine diverticulum.

[0048] According to some embodiments of the present invention, the mass ratio of SF, EGCG, and GA can be (2~24):(4~24):1, for example, it can be 2:4:1, 4:4:1, 6:4:1, 8:4:1, 12:4:1, 16:4:1, 20:4:1, 24:4:1, 4:8:1, 4:12:1, 4:16:1, 4:20:1, 4:24:1, 8:8:1, 8:12:1, 8:16:1, 8:20:1, 8:24 :1, 12:8:1, 12:10:1, 12:12:1, 12:16:1, 12:20:1, 12:24:1, 16:8:1, 16:12:1, 16:16:1, 16:20:1, 16:24:1, 20:8:1, 20:12:1, 20:16:1, 20:20:1, 20:24:1, 24:8:1, 24:12:1, 24:16:1, 24:20:1, 24:24:1, etc.

[0049] Optionally, the mass ratio of SF, EGCG, and GA can be (4~24):(8~16):1. SF and EGCG are the main active crosslinking and functional components, and are directly related to the adhesion strength of the hydrogel. If the amount of SF is too small, it is difficult to form a sufficient and stable amount of gel, making it difficult to achieve strong adhesion to the matrix through intermolecular forces. If the amount of SF is too large, it is difficult to effectively release polyphenols and exert their antibacterial and antioxidant effects. If the amount of EGCG is too small, the limited number of phenolic hydroxyl groups will result in limited improvement in adhesion. If the amount of EGCG is too large, it may cause intermolecular self-aggregation, forming polyphenol aggregates, which will hinder sufficient contact with the substrate, such as tissues, and will also lead to a decrease in adhesion strength. If the amount of GA is too small, it will be difficult for SF and EGCG to form a gel. If the amount of GA is too large, it is easy to cause excessive crosslinking reaction, making the hydrogel network dense and reducing flexibility, which is also not conducive to improving adhesion strength.

[0050] According to some embodiments of the present invention, the antibacterial hydrogel adhesive has a three-dimensional porous network structure. The antibacterial hydrogel crosslinking agent of the present invention has a complete and dense three-dimensional porous network structure, exhibiting excellent structural stability and the ability to withstand large elastic deformation, providing mechanical support for its application in dynamic physiological environments.

[0051] According to another embodiment of the present invention, a method for preparing the antibacterial hydrogel adhesive as described above is provided, the method comprising operations S11 to S14.

[0052] In operation S11, EGCG and GA are dissolved in water at 20~30℃ to obtain a polyphenol solution;

[0053] In operation S12, the SF solution and the polyphenol solution are mixed in equal volume ratio to obtain an antibacterial hydrogel adhesive.

[0054] According to some embodiments of the present invention, the equal-volume mixing method can maintain a preset ratio after mixing the SF solution and the polyphenol solution, which is a reliable method to achieve the preset adhesion strength and initiate rapid gelation. In this case, mixing the slightly neutral or weakly alkaline SF solution and the polyphenolic acid solution allows the SF molecular chains to fully contact a large number of polyphenols EGCG and GA, triggering hydrogen bonding-hydrophobic interactions, and enabling the formation of a uniform and stable three-dimensional network hydrogel in a relatively short time.

[0055] According to some embodiments of the present invention, SF solution can be prepared by lithium salt dissolution method. Specifically, the preparation method of SF is as follows: operation S21 to operation S23.

[0056] In operation S21, the silkworm cocoons are degummed and washed sequentially to obtain degummed silk.

[0057] In operation S22, the degummed silk is dissolved in lithium bromide solution and dialyzed to obtain the dialyzed solution.

[0058] In operation S23, the dialysis solution is centrifuged to obtain a silk fibroin solution.

[0059] According to some embodiments of the present invention, natural silk can be transformed into a biocompatible biopolymer through lithium salt dissolution. Degumming and washing remove the sericin surrounding the silk, preventing inflammatory reactions caused by its immunogenicity in vivo. Lithium bromide solution disrupts the highly crystalline structure of the silk fibroin fibers, dissolving them into a clear, transparent golden-yellow solution. Dialysis and centrifugation remove impurities such as small-molecule salt ions and insoluble particles, thereby obtaining high-molecular-weight, complete SF molecular chains.

[0060] For example, operation S21 may specifically include: adding silkworm cocoons to a boiling sodium carbonate solution, heating and maintaining boiling for a period of time, such as 30 minutes, while stirring, to achieve complete degumming. After the reaction is complete, the silk is transferred to deionized water, stirred and washed multiple times, such as 3 times, and then dried to obtain degummed silk.

[0061] For example, operation S22 may specifically include: placing the degummed silk into a lithium bromide solution with a concentration of, for example, 9.3 mol / L, heating it at 60°C for a period of time, for example, 4 hours, dialyzing the dissolved and cooled solution using a dialysis membrane with a molecular weight of 3.5K, changing the deionized water regularly, and the dialysis time can be 3 to 7 days to ensure that the salt is completely removed.

[0062] For example, operation S23 may specifically include: centrifuging the dialysis solution at, for example, 9000 rpm for 20 minutes, and repeating this operation multiple times to obtain a supernatant containing silk fibroin. Further, the concentration of the supernatant can be adjusted to obtain a silk fibroin solution.

[0063] According to some embodiments of the present invention, in the polyphenol solution, the concentration of EGCG can be 40-60 mg / mL, for example, 40 mg / mL, 42 mg / mL, 44 mg / mL, 46 mg / mL, 50 mg / mL, 52 mg / mL, 54 mg / mL, 56 mg / mL, 58 mg / mL, 60 mg / mL, etc.; the concentration of GA is 2.5-7.5 mg / mL, for example, 2.5 mg / mL, 3 mg / mL, 3.5 mg / mL, 4 mg / mL, 4.5 mg / mL, 5 mg / mL, 5.5 mg / mL, 6 mg / mL, 6.5 mg / mL, 7 mg / mL, 7.5 mg / mL, etc.; in the silk fibroin solution, the concentration of SF can be 40-60 mg / mL, for example, 40 mg / mL, 42 mg / mL, 44 mg / mL, 46 mg / mL, 50 mg / mL, 52 mg / mL, 54 mg / mL, 56 mg / mL, 58 mg / mL, etc. mg / mL, 60 mg / mL, etc. Appropriate concentrations of SF, EGCG, and GA can optimize the pH environment for cross-linking, regulate the cross-linking network and speed between SF and EGCG through hydrogen bonding-hydrophobic interactions, form a stable hydrogel cross-linking network, and synergistically enhance antibacterial effects and adhesion strength.

[0064] According to some embodiments of the present invention, the pH of the polyphenol solution is 3-4, for example, it can be 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, etc., more preferably 3.2-3.4. In this way, the pH value of the polyphenol solution is close to the isoelectric point of EGCG, thereby promoting the layered assembly of SF molecules, triggering the conformational transition of SF molecules from random coils to β-sheets, and achieving gelation.

[0065] According to another aspect of the present invention, the use of the antibacterial hydrogel adhesive as described above in the preparation of a medicament for preventing uterine scar diverticulum is also provided.

[0066] According to some embodiments of the present invention, the antibacterial hydrogel adhesive can be applied in gel form, for example, by injection into the target tissue, such as a uterine wound site, but is not limited thereto. In this case, the antibacterial hydrogel adhesive comes into contact with the target tissue, and the local antibacterial and antioxidant effects persist and act on the target tissue, thereby preventing uterine scar diverticula.

[0067] For example, antibacterial hydrogel adhesives can also be applied in situ, specifically by applying polyphenol solutions and silk fibroin solutions separately to the target tissue site, such as a uterine wound, to create an antibacterial hydrogel in situ. In this case, the in-situ formed antibacterial hydrogel adhesive exhibits excellent tissue adhesion properties and can stably adhere to the target tissue surface.

[0068] According to some embodiments of the present invention, the antibacterial hydrogel adhesive provided by the present invention exhibits antibacterial activity against Gram-positive Staphylococcus aureus and various Group B Streptococci, and in particular, can prevent uterine scar diverticulum by inhibiting Group B Streptococcus infection.

[0069] The following exemplifies the designed antibacterial hydrogel adhesive, its preparation method, and its application in preventing uterine scar diverticulum. It should be noted that this exemplification is merely a specific embodiment of the present invention and does not limit the scope of protection of the present invention. Unless otherwise specified, all reagents used are commercially available, and all experimental methods used are conventional experimental methods in the art. In the following experiments, each experimental group has n=3 parallel replicates.

[0070] Preparation example: Silk fibroin solution

[0071] Silk fibroin solution is prepared by the following steps:

[0072] (1) Add 7.5 g of silkworm cocoons to boiling sodium carbonate solution. Heat the cocoons while stirring and keep them boiling for 30 min to achieve complete degumming. After the reaction is complete, transfer the silk to deionized water and wash it three times with stirring, each time for 20 min. After washing, transfer the silk to an oven to dry.

[0073] (2) The completely dried silk was placed in a 9.3 M lithium bromide solution. It was heated in a 60 ℃ water bath for 4 h to dissolve. Then the dissolved and cooled solution was dialyzed through a dialysis membrane with a molecular weight of 3.5 K, and the deionized water was replaced periodically for 3 days.

[0074] (3) Centrifuge the dialysis solution at 9000 r for 20 min, repeat the operation twice, store the supernatant at low temperature, take 3 mL of the solution, dry it and weigh it to calculate the concentration. At this time, the mass concentration of the silk fibroin solution is about 60-70 mg / mL. Prepare silk fibroin solutions of different concentrations (20 mg / mL, 40 mg / mL and 60 mg / mL) for later use.

[0075] Example 1: Pre-gelation test

[0076] Example 1-1:

[0077] 1. Prepare the polyphenol solution using the following steps:

[0078] EGCG and GA were dissolved in deionized water at a mass ratio of 8:1, resulting in a EGCG concentration of 20 mg / mL and a gallic acid concentration of 5 mg / mL. The solution was dissolved by sonication at room temperature to obtain a mixed polyphenol solution, denoted as E&G, with a pH of 3.3.

[0079] 2. Preparation of antibacterial hydrogel adhesive, the steps are as follows:

[0080] The 20 mg / mL silk fibroin solution obtained in the preparation example was mixed with the polyphenol solution in a centrifuge tube at a volume ratio of 1:1 to obtain a hydrogel adhesive, abbreviated as SPH.

[0081] Examples 1-2 to 1-18:

[0082] Similar to the steps in Example 1-1, the main difference is that the concentrations of EGCG, GA and SF are changed according to Table 1 below to observe the gelation process.

[0083] Analysis of preliminary test results:

[0084] Figure 1 is a schematic diagram of the gelation of different concentrations of SF and different concentrations of polyphenols in Example 1 of the present invention. As shown in Figure 1, after SF is mixed with pure EGCG, it cannot form a gel and presents a solution state; after SF is mixed with pure GA, it forms a gel by vortexing, but the hydrogel has no adhesiveness; after SF is mixed with polyphenol solution E&G, it forms a gel immediately and the hydrogel has strong adhesiveness.

[0085] This is because the pH of the EGCG solution alone is too high, failing to reach the isoelectric point of SF, and the molecular weight of GA alone is too small, with too few phenolic hydroxyl groups to provide sufficient adhesion. Specifically, Figure 2 shows the potential of silk fibroin at different pH values ​​(n=3) in Example 1 of this invention. As shown in Figure 2, the average value of three Zeta potential tests is obtained. By adjusting the pH of the EGCG solution to 3.3 with GA, which is closer to the isoelectric point of SF, electrostatic repulsion between adjacent SF molecules can be suppressed, promoting the layered assembly of silk fibroin molecules and triggering the conformational transition from random coil to β-sheet.

[0086] Figures 3 to 5 show the effects of different concentrations of EGCG and GA (20, 40, and 60 mg / mL SF solution) on the hydrogel formation in Example 1 of this invention. As shown in Figures 3 to 5, the hydrogel formation is consistent with Figure 1. When the GA concentration is 0 mg / mL, no gel is formed when SF and EGCG solutions of different concentrations are mixed. When the SF and EGCG solutions are at a low concentration of 20 mg / mL, they cannot quickly and immediately form a gel to achieve an adhesive state; vortexing is required for gel formation. Comparing the gel content of different concentrations of SF solution, the gel amount is significantly increased when the concentration is 60 mg / mL. This is because the high concentration of silk fibroin molecules forms a dense dispersion system in the solution, and the cross-linking reaction can form a complete and dense three-dimensional network structure in a shorter time, such as a few seconds, resulting in a significant increase in the gel amount.

[0087] Based on the comprehensive preliminary test results, a SF concentration of 60 mg / mL can ensure sufficient and stable gelation of the hydrogel, and can also achieve strong adhesion to the matrix by enhancing intermolecular forces. Therefore, it was determined to be the optimal preparation concentration.

[0088] Table 1

[0089]

[0090] Note: The concentrations mentioned in the table are the concentrations used when synthesizing hydrogels. The hydrogels were prepared by combining SF solution with EGCG and GA mixed solution at a volume ratio of 1:1.

[0091] Example 2

[0092] Example 2-1:

[0093] 1. Prepare the polyphenol solution using the following steps:

[0094] EGCG and GA were dissolved in deionized water at a mass ratio of 8:1, resulting in a concentration of 40 mg / mL for EGCG and 5 mg / mL for gallic acid. The solution was dissolved by sonication at room temperature to obtain a polyphenol solution, denoted as E&G, with a pH of 3.3.

[0095] 2. Preparation of antibacterial hydrogel adhesive, the steps are as follows:

[0096] The 60 mg / mL silk fibroin solution obtained in the preparation example was mixed with the polyphenol solution at a volume ratio of 1:1 to obtain an antibacterial hydrogel adhesive, abbreviated as SPH.

[0097] Examples 2-2 to 2-5:

[0098] Similar to the steps in Example 2-1, the main difference lies in changing the concentration of EGCG in the polyphenol solution to 20 mg / mL, 30 mg / mL, 40 mg / mL, 50 mg / mL, and 60 mg / mL, respectively, to obtain antibacterial hydrogel adhesives, which are abbreviated as S in turn. 60 E 20 G5, S 60 E 30 G5, S 60 E 40 G5, S 60 E 50 G5, S 60 E 60 G5.

[0099] Examples 2-6 to 2-8:

[0100] Similar to the steps in Example 2-1, the main difference lies in changing the concentration of GA in the polyphenol solution to 2.5 mg / mL, 7.5 mg / mL, and 10 mg / mL, respectively, to obtain antibacterial hydrogel adhesives, which are abbreviated as S in turn. 60 E 40 G 2.5 S 60 E 40 G 7.5 S 60 E 40 G 10 .

[0101] Comparative Example 2-1:

[0102] Similar to the steps in Example 2-1, the main difference is that the concentration of GA in the polyphenol solution is 0 mg / mL, resulting in an antibacterial hydrogel adhesive, abbreviated as S. 60 E 40 .

[0103] Comparative Example 2-2:

[0104] Similar to the steps in Example 2-1, the main difference is that the concentration of GA in the polyphenol solution is 0 mg / mL, and the pH of the polyphenol solution is adjusted to 3.3 using acetic acid, abbreviated as S. 60 E 40 -HAc.

[0105] Result characterization and testing:

[0106] 2.1 Adhesion strength:

[0107] 2.1.1 The results of the pre-gelation test show that the gelation rate is faster when the GA concentration is 5 mg / mL. Therefore, when the SF solution concentration is 60 mg / mL and the GA solution concentration is 5 mg / mL, the effects of different concentrations of EGCG solution on the adhesion strength of hydrogel will be investigated through Examples 2-1 to 2-5.

[0108] The adhesion strength of different hydrogels was evaluated by lap shear test. A glass slide was used as the adhesion matrix. The adhesion area of ​​the hydrogel was 25 mm × 20 mm. Tensile tests were performed on the samples using a universal testing machine. All tests were completed at a constant tensile rate of 50 mm / min.

[0109] Figure 6 shows the adhesion characteristics test results of the antibacterial hydrogel adhesives prepared based on different concentrations of EGCG in Example 2 of this invention. As shown in Figure 6, the adhesion strength of the antibacterial hydrogel adhesive significantly increases with the increase of EGCG concentration. When the EGCG concentration is 40 mg / mL, the adhesion strength reaches a stable state. This may be because at low concentrations, the number of phenolic hydroxyl groups is limited, resulting in insufficient interaction sites with the substrate and thus weak adhesion. As the concentration increases, the phenolic hydroxyl groups can form a denser interaction network, and the adhesion strength shows a significant upward trend. At the same time, excessively high EGCG concentrations may induce intermolecular self-aggregation, forming polyphenol aggregates, which hinders sufficient contact with the substrate and leads to a decrease in adhesion strength. Therefore, the EGCG concentration of 40 mg / mL was selected for subsequent experiments.

[0110] 2.1.2 To further verify the effect of GA on the gelation of the SF-EGCG system, the effect of different concentrations of GA solution on the adhesion strength of the hydrogel was tested when the SF solution concentration was 60 mg / mL and the EGCG concentration in the polyphenol solution was 40 mg / mL.

[0111] Figure 7 shows the adhesion characteristics test results of the antibacterial hydrogel adhesives prepared based on different concentrations of GA in Example 2 of this invention. As shown in Figure 7, as the GA concentration increases from 2.5 mg / mL to 10 mg / mL, the hydrogel adhesion strength shows a trend of first increasing and then decreasing. In the low concentration stage, the number of phenolic hydroxyl groups in GA molecules increases with increasing concentration, forming a denser hydrogen bond network, which significantly improves the adhesion strength with increasing concentration. However, at high concentrations, GA molecules will induce excessive cross-linking reaction, making the hydrogel network structure dense and rigid, reducing flexibility, and making it difficult to adapt to deformation when adhering to the substrate surface, thus causing the adhesion strength to decrease with increasing concentration. Therefore, the adhesion performance reaches its maximum when the GA concentration is 5 mg / mL. Therefore, a GA concentration of 5 mg / mL was selected for subsequent experiments.

[0112] Figure 8 is a comparison of the adhesion strength of the antibacterial hydrogel adhesives of Example 2 and Comparative Examples 2-1 to 2-2 of the present invention (n=3). As shown in Figure 8, in Comparative Example 2-1, no gel could be formed and there was no adhesion when the pH was not adjusted; in Comparative Example 2-2, when the pH of the EGCG solution was adjusted to 3.3 by acetic acid, a hydrogel was formed but the gel adhesion was weak; while in Example 2-1, when the pH was adjusted to 3.3 by GA, the gel adhesion was significantly improved, which was 3-4 times that of the acetic acid adjustment (at which point the solution pH was the same), showing the excellent adhesion performance of the material.

[0113] 2.2 Mechanical Properties

[0114] The antibacterial hydrogel adhesive SPH prepared in Example 2-1 was observed for its cross-sectional morphology by scanning electron microscopy (SEM), and its mechanical properties were tested. Figure 9 shows the SEM cross-sectional morphology and mechanical property characterization of SPH in Example 2 of this invention, where Figure A is the SEM cross-sectional view, Figure B shows the storage modulus and loss modulus of SF / E&G / SPH (n=3), Figure C shows the rheological properties of SPH at 25 ºC with amplitude scanning from 0.1% to 1000% strain, and Figure D shows the rheological properties of SPH at 25 ºC with frequency scanning from 0.1 to 10 Hz.

[0115] As shown in Figure 9, compared with pure SF solution and E&G mixed solution, the modulus of SPH is significantly improved, and the storage modulus (G') is consistently higher than the loss modulus (G'), confirming that the system has successfully achieved gelation transition (Figure 9B). Further analysis of the viscoelastic behavior of SPH using dynamic strain scanning and frequency scanning tests revealed that G' is consistently higher than G'' within the test range (Figures 9C and D). This indicates that SPH forms a stable elastic cross-linked network with excellent structural stability and can withstand relatively large elastic deformation, providing mechanical support for its application in dynamic physiological environments.

[0116] 2.3 Formation and Interaction Mechanisms of SPH

[0117] The formation mechanism of SPH was explored based on Fourier transform infrared spectroscopy (FTIR). Figure 10 shows the FTIR spectral analysis results of the antibacterial hydrogel adhesive. Figure A shows the FTIR spectra of SF, EGCG, GA, and SPH. Figure B shows the quantitative analysis diagram of the fitted secondary structure of SF. Figure C shows the quantitative analysis diagram of the fitted secondary structure of SPH. Figure D shows the comparison of β-sheet content of SF and SPH (n=3).

[0118] As shown in Figure 10, the spectrum of SPH only shows the typical characteristic peaks of SF and polyphenols, with no new characteristic peaks observed. Only peak shifts are observed, indicating that the components are bonded by non-covalent interactions and no new chemical bonds are formed. (3285 cm⁻¹) −1 The broad absorption peaks observed at [insert value here] confirm the formation of numerous intermolecular hydrogen bonds between SF and polyphenol molecules. This is because hydrogen bonding broadens the vibrational absorption peaks of hydrogen-containing functional groups (-OH, -NH), providing direct spectroscopic evidence for non-covalent intermolecular cross-linking. In the pure SF spectrum, the characteristic peaks of amide I and amide II bands are located at 1648 cm⁻¹, respectively. −1 and 1534 cm −1 In SPH, the frequencies shifted to lower frequencies up to 1635 cm⁻¹. −1 and 1515 cm −1 This phenomenon originates from the fact that polyphenols bind to the hydrophobic segment of SF6 molecules through a hydrophobic effect, inducing conformational rearrangement of the SF6 molecule. This is observed in the amide I band (1600-1700 cm⁻¹). −1 Deconvolution analysis was performed, and the results showed that the relative content of β-sheets in SPH was significantly higher than that in pure SF hydrogel, and the increase in β-sheet structure further enhanced the cohesion and structural stability of the system.

[0119] To further investigate the intermolecular interaction mechanism of SPH, this study used specific interaction disruptors for verification: 100 mM NaCl (electrostatic interaction disruptor), 100 mM Tween 20 (hydrophobic interaction disruptor), and dimethyl sulfoxide (DMSO, hydrogen bond disruptor), with deionized water as a blank control. Specifically, SPH was added to deionized water, NaCl solution, Tween-20, and DMSO, respectively, and the changes in the structure of SPH over time were observed.

[0120] Figure 11 illustrates the disintegration of SPH in different solvents in Example 2 of this invention. As shown in Figure 11, SPH rapidly disintegrates its gel structure in DMSO, exhibits gradual degradation in Tween 20 solution, while its condensed gel structure remains stable for over 72 hours in NaCl solution and deionized water. This phenomenon indicates that hydrogen bonding is the dominant force maintaining the formation and stability of SPH, while hydrophobic interactions play a secondary supporting role.

[0121] 2.4 System Characterization of Adhesion Properties

[0122] Strong adhesion properties are a key prerequisite for hydrogels to maintain long-lasting antibacterial effects and achieve rapid wound healing. This invention further systematically characterizes the adhesion properties of SPH. Specifically, the antibacterial hydrogel adhesive was uniformly applied to different substrates, such as glass, plastic, metal, and porcine uterine tissue. The adhesion properties of the hydrogel were qualitatively judged based on the ease of detachment from these substrates. Using porcine uterine tissue as a substrate, after applying the antibacterial hydrogel adhesive to the surface of the porcine uterine tissue, it was stretched, twisted, completely removed, and rinsed to observe the adhesion state of the hydrogel on the porcine uterine tissue. Furthermore, using porcine uterine tissue as a substrate, after applying the antibacterial hydrogel adhesive to the surface of the porcine uterine tissue, it was immersed in PBS buffer under anaerobic conditions for 21 days to observe the changes in the adhesion state of the hydrogel on the porcine uterine tissue over time.

[0123] Figure 12 shows the dynamic and long-term adhesion results of SPH on the substrate in the embodiments of the present invention. A is a schematic diagram of the strong adhesion of SPH on different substrates, and B is the long-term wet adhesion performance of SPH on porcine uterine tissue. SPH has excellent adhesion ability and can adhere tightly to the surfaces of various materials such as glass, metal, plastic and pigskin. This strong adhesion characteristic mainly comes from the covalent and non-covalent interactions formed between the hydrogel and the substrate. More importantly, SPH has both dynamic adhesion and long-term wet adhesion capabilities. Even after stretching, twisting, bending and water rinsing, it can still adhere tightly to the surface of porcine uterine tissue, showing adhesion stability adapted to the moist physiological environment, as shown in Figure 12, A. Even after immersion in PBS solution under anaerobic conditions for 21 days, the SPH hydrogel continues to maintain tight adhesion to the porcine uterine tissue, as shown in Figure 12, B.

[0124] The adhesion morphology of SPH in porcine uterine tissue was observed by examining the cross-sectional area of ​​the SPH on the tissue using fluorescence microscopy and scanning electron microscopy. Specifically, SPH was formed in situ on the surface of porcine uterine tissue and then lyophilized. The cross-sectional morphology of the lyophilized sample was observed using fluorescence microscopy and scanning electron microscopy. Figure 13 shows fluorescence microscopy and SEM images of the cross-section of SPH adhering to porcine uterine tissue. As shown in Figure 13, both fluorescence imaging and SEM characterization results clearly show that the SPH and porcine uterine tissue interface are tightly adhered without obvious gaps. This characteristic confirms that in situ formed SPH has excellent tissue adhesion properties and can stably adhere to irregular uterine wounds after cesarean section, providing key performance support for its application in the prevention of uterine diverticulum.

[0125] To quantitatively evaluate the adhesive properties of SPH, lap shear tests were used to measure shear strength, 180° peel tests were used to assess interfacial toughness, and tensile tests were used to determine tensile strength. In the lap shear and 180° peel tests, porcine tissues such as pigskin, liver, heart, and uterus were cut into strips (40 mm long, 10 mm wide); the hydrogel was adhered between two pieces of pigskin, with an adhesion area of ​​10 mm × 10 mm. In the tensile test, the tissues were cut into squares with sides of 20 mm, and the hydrogel adhesion area was 20 mm × 20 mm. All tissue samples were cleaned with phosphate-buffered saline (PBS) before SPH adhesion treatment. After standing for 10 minutes, tensile tests were performed on the samples using a universal testing machine. All tests were conducted at a constant tensile rate of 50 mm / min.

[0126] Figure 14 shows the shear strength of SPH in different tissues in Example 2 of the present invention (n = 3); Figure 15 shows the interfacial toughness of SPH in different tissues in Example 2 of the present invention (n = 3); Figure 16 shows the tensile strength of SPH in different tissues in Example 2 of the present invention (n = 3). As shown in Figures 14 to 16, the shear strength of SPH on pig skin, liver, heart, and uterus tissues are 144.9 ± 1.3, 158.2 ± 6.7, 112.4 ± 3.8, and 99.85 ± 2.3 kPa, respectively; the adhesive toughness are 497.6 ± 52.2, 592.4 ± 53.9, 414.96 ± 32.5, and 310.3 ± 28.5 J / m, respectively. −2 The tensile adhesion strengths were 226.8 ± 13.4, 330.7 ± 14.2, 272.3 ± 6.3, and 231.1 ± 10.8 kPa, respectively. The series of quantitative data fully confirms that SPH has a strong bioadhesion ability on the surface of moist biological tissues.

[0127] 2.5 Sustained-release performance

[0128] The bioactivity of SPH mainly stems from the release of polyphenols. To clarify the polyphenol content and release characteristics in SPH, the total polyphenol content was first determined using the Folin-Ciocalteu method in this embodiment of the invention. The results showed that each 1 mg of SPH contained 0.249 ± 0.0048 mg of polyphenols (using gallic acid as a standard, the results are expressed as gallic acid equivalent (GAE) mg / mg). Subsequently, the release rate of E&G polyphenols was evaluated in PBS buffer, and a release curve was plotted as the percentage of released polyphenols relative to the initial total amount. Figure 17 shows the release curve of total polyphenols in SPH in PBS buffer in Example 2 of this invention; as shown in Figure 17, the polyphenols in SPH were rapidly released on the first day, and then the release rate tended to plateau. After 7 days, the cumulative release amount reached 35.1 ± 2%. This slow release characteristic may be related to the dense network structure and interfacial adhesion of SPH gel.

[0129] 2.6 Antibacterial and antioxidant properties

[0130] Group B streptococcal infection is an integral factor in the formation of uterine scar diverticula. Therefore, this invention focuses on exploring the antibacterial properties of SPH as a diverticulum prevention material. Gram-positive Staphylococcus aureus and several clinical Group B streptococci were used as model bacteria, and their antibacterial activity was evaluated through an inhibition zone test. Figure 18 shows the antibacterial effect of SPH against Staphylococcus aureus and several clinical Group B streptococci in Example 2 of this invention, where Figure A is the inhibition zone image and Figure B is the quantitative inhibition bar chart. As shown in Figure 18, using Staphylococcus aureus and several clinical Group B streptococci grown on the culture medium in the control group as bacterial controls, the size of the inhibition zone shows that SPH exhibits significant antibacterial effects against all of the above strains. The blank SFH (silk fibroin hydrogel without polyphenols) did not show significant antibacterial activity. Combined with in vitro release experiments, it can be seen that the antibacterial activity of SPH originates from the long-acting sustained-release characteristics of polyphenols, ensuring the long-term maintenance of antibacterial efficacy.

[0131] To clarify the antibacterial mechanism, morphological changes of bacteria before and after co-culturing with SPH were observed using SEM and TEM. Figure 19 shows SEM and TEM images of bacterial morphology after co-culturing with SPH in Example 2 of this invention, where Figure A shows Staphylococcus aureus and Figure B shows Group B Streptococcus. As shown in Figure 19, untreated Staphylococcus aureus and Group B Streptococcus maintained their intact structures, with smooth and dense surfaces and good proliferation capacity; however, after incubation with SPH, the bacterial cell membranes showed permanent damage, manifested as rupture, twisting, wrinkling, and shrinkage, with some strains even completely lysing. This indicates that SPH may kill bacteria by disrupting the integrity of the bacterial cell wall and cell membrane permeability, leading to leakage of cell contents.

[0132] Bacterial infection of wounds can trigger a persistent inflammatory response. Excessive inflammation leads to increased oxidative stress, resulting in the accumulation of reactive oxygen species (ROS) such as superoxide anions and hydrogen peroxide. These ROS damage cell structure, thereby delaying wound healing. Therefore, hydrogels with antioxidant activity are of great significance for promoting wound healing and preventing uterine diverticulum. In this invention, the antioxidant capacity of SPH extracts at different concentrations was evaluated using the free radical scavenging methods of 2,2′-adiazonium-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) and 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH). Figure 20 shows the antioxidant activity of SPH in Example 2 of this invention, where Figure A shows the UV absorption spectra of SPH extracts at different concentrations based on the ABTS method, and Figure B shows the quantitative results of antioxidant activity based on the ABTS method (n = 3); Figure B shows the UV absorption spectra of SPH extracts at different concentrations based on the DPPH method, and Figure D shows the quantitative results of antioxidant activity based on the DPPH method (n = 3).

[0133] As shown in Figure 20, in the presence of SPH, the ABTS solution changed from blue to white, and the DPPH solution changed from dark purple to yellow, visually demonstrating their scavenging effect on both free radicals. Quantitative results showed that 20 μg·mL⁻¹… −1 E&G polyphenols on ABTS + The scavenging rate exceeded 90%, and the scavenging rate of DPPH• showed a similar trend, fully demonstrating that SPH has excellent antioxidant activity.

[0134] Application Example 1

[0135] 1. Evaluation of the preventive effect against uterine diverticulum

[0136] To evaluate the preventive effect of SPH on uterine diverticulum formation, a rat model of uterine diverticulum induced by Group B Streptococcus infection was established in this application example. Figure 21 shows the animal model creation and surgical procedure of the experimental group in Application Example 1 of this invention, where Figure A is a schematic diagram of animal model creation, and Figure B is a representative image of the treatment plan and the main surgical steps of the experimental group. As shown in Figure 21, the experimental rats were first injected with progesterone to simulate the state of the pregnant rat uterus. After 23 days, they were randomly divided into 4 groups: sterile wound group, bacterial wound group, bacterial-polyphenol solution group, and bacterial-SPH group. In the sterile wound group, a wound was created on one side of the rat's uterus, and sterile sutures were implanted and surgically closed. In the bacterial wound group, after the same wound treatment, sutures soaked in Group B Streptococcus solution (GBS) were implanted in one side of the uterus (shown in the box) and closed. In the bacterial-polyphenol solution group, in addition to the treatment of the bacterial wound group, polyphenol solution was injected into the wound in situ after suturing. In the bacterial-SPH group, SPH was injected into the wound in situ after the bacterial wound was closed. The contralateral uterus of all rats was not treated in any way.

[0137] Twenty-one days post-surgery, rat uterine tissue was collected for observation and gross morphological measurements. Figure 22 shows the in vivo preventive evaluation of the hydrogel used in Example 1 of this invention in a rat model of uterine diverticulum. Figure A shows photographs of the uterus in each group 21 days post-surgery; Figure B shows H&E stained images of cross-sections of uterine tissue in each group 21 days post-surgery (a pair of arrows indicates the endometrial thickness at the incision site and the thickness of the adjacent basal layer endometrium, respectively). Figure 23 shows the quantitative analysis of the ratio of the basal layer thickness at the surgical incision site to the thickness of the adjacent basal layer in hematoxylin-eosin (H&E) staining in Example 1 of this invention (n = 3).

[0138] As shown in Figure 22A, the sterile wound group and the sterile-polyphenol solution group exhibited uneven uterine thickness, swelling, and purulent discharge or inflammatory symptoms. The sterile-SPH group, however, demonstrated excellent anti-Group B Streptococcus infection effects, with faster wound healing and significantly better results than the control group. 21 days post-surgery, the uterus was light pink and elastic, similar to a normal sterile uterus in thickness, shape, and elasticity. As shown in Figures 22B and 23, the sterile wound group, due to the implantation of sterile sutures, showed good uterine incision healing with uniform myometrial thickness, and the ratio of the incision thickness to the adjacent myometrial thickness was close to 1. In contrast, the sterile wound group with implanted sterile sutures and the sterile-polyphenol solution group showed severely poor uterine wound healing, with thinner myometrial thickness at the incision site and a significant difference in thickness compared to the adjacent myometrial layers (ratio approximately 0.32), indicating a very high risk of uterine diverticulum formation. The sterile-SPH treatment group, on the other hand, showed good incision healing with uniform myometrial thickness, and the ratio of the incision thickness to the adjacent myometrial thickness was close to 1, similar to the condition of a healthy uterus. This indicates that SPH can effectively kill Group B Streptococcus, reduce the inflammatory response caused by infection, and promote wound healing, thereby achieving the preventive effect of uterine diverticulum.

[0139] Masson staining was used to further monitor collagen deposition during wound healing, and the collagen area fraction was calculated for quantitative analysis. Figure 24 shows the Masson staining results of uterine tissue cross-sections 21 days post-surgery in each group, where A is the Masson staining image and B is the quantitative analysis of the relative area coverage of collagen in Masson staining. As shown in Figure 24, at 21 days post-surgery, the amount of collagen deposition in the sterile-SPH group was significantly higher than that in the sterile wound group and the sterile-polyphenol solution group, and similar to that in the sterile wound group. Meanwhile, the SPH group exhibited a more regular collagen fiber structure. This indicates that SPH can effectively regulate the wound microenvironment, promote collagen deposition, and thus accelerate the healing process of infected wounds.

[0140] 2. Biosafety assessment

[0141] Biosafety is a crucial prerequisite for the use of SPH in the prevention of uterine diverticulum. Therefore, this application example assesses its biosafety through H&E staining of major organs (heart, liver, spleen, lung, and kidney) and detection of blood chemical indicators in rats. Figure 25 shows the H&E staining results of major organs (heart, liver, spleen, lung, and kidney) in each group 21 days post-surgery in this application example. Figure 26 shows the blood indicators of each group 21 days post-surgery in application example 1 of this invention, where A to H represent white blood cell count, neutrophil percentage, lymphocyte percentage, monocyte percentage, eosinophil percentage, basophil percentage, alanine aminotransferase (ALT), and urea content, respectively. As shown in Figure 25, compared with the sterile wound group, no significant adverse reactions were observed in the H&E staining of major organs in all treatment groups. As shown in Figure 26, compared with the sterile wound group, no significant abnormalities were found in the blood chemical indicators of all treatment groups. These results confirm the excellent in vivo biosafety of SPH, providing important assurance for its potential clinical application in the prevention of uterine diverticulum.

[0142] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An antibacterial hydrogel adhesive, characterized in that, The antibacterial hydrogel adhesive comprises silk fibroin, epigallocatechin gallate, and gallic acid in a mass ratio of (2~24):(4~24):

1.

2. The antibacterial hydrogel adhesive according to claim 1, characterized in that, The mass ratio of silk fibroin, epigallocatechin gallate and gallic acid is (4~24):(8~16):

1.

3. The antibacterial hydrogel adhesive according to claim 1, characterized in that, The mass ratio of silk fibroin, epigallocatechin gallate and gallic acid is 12:8:

1.

4. The antibacterial hydrogel adhesive according to any one of claims 1 to 3, characterized in that, The antibacterial hydrogel adhesive has a three-dimensional porous network structure.

5. A method for preparing an antibacterial hydrogel adhesive as described in any one of claims 1 to 4, characterized in that, The preparation method includes: dissolving epigallocatechin gallate and gallic acid in water at 20-30°C to obtain a polyphenol solution; and mixing silk fibroin solution and the polyphenol solution in an equal volume ratio to obtain the antibacterial hydrogel adhesive.

6. The preparation method according to claim 5, characterized in that, In the silk fibroin solution, the concentration of silk fibroin is 40-60 mg / mL; in the polyphenol solution, the concentration of epigallocatechin gallate is 40-60 mg / mL, and the concentration of gallic acid is 2.5-7.5 mg / mL.

7. The preparation method according to claim 5 or 6, characterized in that, The silk fibroin is prepared by the following method: silkworm cocoons are degummed and washed sequentially to obtain degummed silk; the degummed silk is dissolved in lithium bromide solution and dialyzed to obtain a dialyzed solution. The dialysis solution was centrifuged to obtain a silk fibroin solution.

8. The preparation method according to claim 5 or 6, characterized in that, The pH of the polyphenol solution is 3-4, more preferably 3.2-3.

4.

9. The use of an antibacterial hydrogel adhesive as described in any one of claims 1 to 4 in the preparation of a medicament for the prevention of uterine scar diverticulum.

10. The application according to claim 9, characterized in that, The antibacterial hydrogel adhesive prevents the uterine scar diverticulum by inhibiting Group B Streptococcus infection; and / or, the antibacterial hydrogel adhesive is applied to the target tissue site by in-situ molding.

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