Tissue adhesive as well as preparation method and application thereof

Abstract

The invention discloses a tissue adhesive and a preparation method and application thereof.The tissue adhesive is formed by polymerizing a zwitterionic monomer, a polyphenol compound, ferric ions, a cross-linking agent and an initiator in an aqueous solution through a redox dual-catalysis system, and the preparation method comprises the steps that the zwitterionic monomer, the polyphenol compound and the cross-linking agent are evenly mixed; then adding a ferric ion solution to form a complexing system, and finally adding an initiator to trigger a polymerization reaction so as to form the hydrogel within 60 seconds at room temperature. According to the preparation method, a catalysis-polymerization-adhesion three-function synergistic system is constructed by accurately regulating and controlling the adding sequence and concentration of the components, and the obtained hydrogel has excellent biocompatibility, controllable mechanical property, rapid room-temperature gelling property, high tissue adhesion and potential antibacterial and antioxidant functions. The tissue adhesive can be widely applied to the fields of surgical wound closure, hemostasis, viscera repair, tissue regeneration and the like.

Description

Technical Field

[0001] This invention relates to the field of biomedical engineering materials technology, specifically to a tissue adhesive, its preparation method, and its application. Background Technology

[0002] In surgical procedures, rapid and effective wound closure, fluid sealing, and bleeding control are crucial for surgical success and postoperative recovery. While traditional mechanical methods such as sutures and U-shaped staples are effective, they have limitations in minimally invasive surgeries, hemostasis of fragile tissues (such as the liver and spleen), and fluid sealing. Therefore, tissue adhesives are playing an increasingly important role in clinical applications as an alternative or adjunct method.

[0003] Currently, commercially available tissue adhesives are mainly divided into two categories: chemical adhesives and medical bio-adhesives. Chemical adhesives, represented by cyanoacrylates, use small monomers containing cyano (-CN) and ester (-COO-) groups (such as butyl cyanoacrylate). These monomers rapidly undergo Michael addition reactions with amino groups (-NH2) on the tissue surface, initiating monomer polymerization to form a rigid polymer film. Adhesion is achieved through mechanical intercalation and covalent bonds between the film and the tissue, resulting in strong adhesion. However, the polymerization process is violently exothermic, with local temperatures reaching over 60°C, easily burning tissue. Simultaneously, its degradation products, formaldehyde and cyanoacetate, have certain cytotoxicity and can easily trigger inflammatory reactions, limiting their application in fragile tissues and sensitive areas in vivo. Bio-adhesives, represented by fibrin glue, are composed of fibrinogen (extracted from plasma) and thrombin. Thrombin catalyzes the conversion of fibrinogen into fibrin, forming a network structure through physical cross-linking. This network wraps around tissue cells to achieve adhesion, while simultaneously activating coagulation factors to assist in hemostasis, exhibiting good biocompatibility and hemostatic function. However, the network structure is physically cross-linked (not a strong covalent bond), and the cross-linking density is low, resulting in weak adhesion and poor mechanical strength, which limits its application range.

[0004] Existing tissue adhesives present a contradiction between "high adhesion and low biotoxicity" and "good biocompatibility and low mechanical strength." To resolve this contradiction, researchers have attempted to develop novel hydrogel adhesives, with zwitterionic materials attracting significant attention due to their excellent resistance to protein adsorption and biocompatibility. However, how to construct hydrogels from zwitterionic materials that can be rapidly molded in situ, form strong adhesion to tissues, and possess good mechanical properties and functional extensibility remains a pressing technical challenge in this field. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a tissue adhesive and its preparation method. By precisely controlling the order and concentration of component addition, a synergistic "catalysis-polymerization-adhesion" three-function system is constructed. The resulting hydrogel possesses excellent biocompatibility, controllable mechanical properties, rapid room-temperature gelation characteristics, high tissue adhesion, and potential antibacterial and antioxidant functions. This tissue adhesive can be widely used in surgical wound closure, hemostasis, visceral repair, and tissue regeneration.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: This invention provides a method for preparing a tissue adhesive, comprising the following steps: S1. Dissolve the zwitterionic monomer and polyphenol compound in pure water, and add a cross-linking agent to obtain the first mixture; Among them, the zwitterionic monomer is methacryloyl ethyl sulfobetaine, the polyphenolic compound is one of gallic acid, epigallocatechin gallate, and tannic acid, and the crosslinking agent is polyethylene glycol dimethacrylate. S2, Add Fe to the first mixture 3+ The solutions are mixed thoroughly to obtain a second mixture. S3. Add an initiator to the second mixture, mix well, trigger the polymerization reaction, and form a hydrogel tissue binder at room temperature; The initiator is ammonium persulfate.

[0007] In the preparation method of this invention, the order of addition of each component is crucial. The process can be divided into "premixing - triggering gelation," both completed at room temperature without the need for additional energy (such as UV or high temperature). Preferably, the process involves "monomer + polyphenol + crosslinking agent → Fe..." 3+ The sequence of "→initiator" is particularly important when the polyphenol is tannic acid. This specific sequence enables the formation of hydrogels with a large, interconnected honeycomb microstructure, which facilitates mass exchange and heat diffusion, thereby giving the material better thermal response and more stable adhesion properties.

[0008] Further, in step S1, the concentration of the zwitterionic monomer in the first mixture is 3.5-4.5M; the amount of crosslinking agent added is 0.5-2% of the mass of the zwitterionic monomer.

[0009] Preferably, the concentration of the zwitterionic monomer in the first mixture is 4M, which ensures the density of the polymer chains and is the basis for forming a stable gel network; the amount of crosslinking agent added is 1% of the mass of the zwitterionic monomer.

[0010] Furthermore, when the polyphenolic compound is gallic acid (GA), its concentration in the first mixed aqueous solution is 2-16 mM.

[0011] Furthermore, when the polyphenolic compound is epigallocatechin gallate (EGCG), its concentration in the first mixed aqueous solution is 2-8 mM.

[0012] Furthermore, when the polyphenolic compound is tannic acid (TA), its concentration in the first mixed aqueous solution is 2-8 mM. Most preferably, it is 4 mM.

[0013] These natural polyphenols not only serve as components of catalytic systems, but their abundant phenolic hydroxyl groups also endow materials with adhesion, antioxidant, and potential antibacterial functions.

[0014] Furthermore, in step S2, Fe 3+ The concentration in the second mixture is 2-6 mM.

[0015] Preferably, Fe 3+ The concentration in the second mixture was 3 mM. Experiments showed that at this concentration, Fe... 3+ It can efficiently catalyze polymerization reactions, participate in network construction as a dynamic cross-linking point, and avoid the cytotoxicity that high concentrations of metal ions may cause. Fe 3+ The synergistic effect with polyphenols is the core of this invention. Both are indispensable and together they form a redox pair, effectively activating the initiator.

[0016] Further, in step S3, the concentration of the initiator in the second mixture is 6-10 mg / mL.

[0017] Preferably, the concentration of the initiator in the second mixture is 8 mg / mL. APS in this system is reacted with Fe... 3+ - Polyphenol complexes are highly activated to generate free radicals, thereby initiating polymerization under mild conditions.

[0018] Furthermore, in step S3, the polymerization reaction is completed within 10-60 seconds, and the maximum temperature of the reaction system does not exceed 40°C.

[0019] The present invention also provides a tissue adhesive prepared by the above-described preparation method.

[0020] This tissue adhesive is essentially a hydrogel containing a network of zwitterionic polymers, the network of which consists of zwitterionic polymer chains linked by covalent cross-linking points (from the cross-linking agent) and dynamic coordination bonds (Fe). 3+ It is cross-linked with polyphenols and groups on polymer chains. The working principle of tissue adhesives is dual-catalytic polymerization + multi-functional adhesion. First, the self-polymerization of hydrogels depends on "Fe". 3+The "polyphenol redox dual catalytic system" uses ammonium persulfate (APS) as the initiator to slowly generate free radicals under normal conditions, but in this system, Fe... 3+ It forms a redox pair with polyphenols (GA / EGCG / TA), activating APS through a single-electron transfer process and accelerating free radical generation; the generated free radicals further initiate the opening of the double bond in the zwitterionic monomer methacryloylethyl sulfobetaine (SBMA), undergoing a chain polymerization reaction to form a linear SBMA polymer chain; simultaneously, Fe... 3+ The hydrogel undergoes a coordination reaction with the catechol groups in polyphenol molecules, forming cross-linking points between polymer chains, ultimately constructing a three-dimensional hydrogel network of "linear polymer chains + coordination cross-linking". Furthermore, the adhesion between the hydrogel and biological tissues is not a single action, but rather a multi-faceted binding through "covalent bonds, coordination bonds, hydrogen bonds, and dipole interactions". The polyphenols (GA / EGCG / TA) in Fe... 3+ Catalytic oxidation produces quinones, whose quinone groups can undergo Schiff base reactions or Michael addition reactions with amino (-NH2) and thiol (-SH) groups in proteins on the tissue surface to form stable covalent bonds; Fe2+ that does not participate in catalysis is oxidized to quinones. 3+ It can simultaneously form coordination bonds with polyphenol groups in hydrogels and carboxyl groups (-COOH) on tissue surfaces, further strengthening interfacial bonding; hydrogen bonds can be formed between polyphenol molecules, and the zwitterionic structure of SBMA monomers (containing positively charged quaternary ammonium groups and negatively charged sulfonate groups) can be adsorbed onto tissue surfaces (such as polar groups of cell membranes) through ionic dipole interactions, enhancing initial adhesion; hydrogen bonds and coordination bonds, as "sacrificial bonds," can preferentially break when subjected to external stretching, absorbing energy and preventing direct rupture of the hydrogel, while unbroken covalent bonds maintain basic adhesion, ensuring adhesive stability.

[0021] The present invention also provides the application of the above-mentioned tissue adhesive in the preparation of medical products for surgical wound closure, hemostasis, and tissue repair.

[0022] Compared with the prior art, the present invention has the following beneficial effects: (1) This invention uses non-toxic natural polyphenols and biocompatible zwitterionic monomers as the main components, and optimizes Fe 3+ The concentration avoids the toxic risks of high concentrations of metal ions, fundamentally ensuring the safety of the material. Animal experiments show that the hydrogels of this invention (especially the Fe-ESB group) can significantly promote wound healing and reduce inflammatory responses.

[0023] (2) This invention adjusts Fe 3+The concentration and ratio of polyphenols can be precisely controlled to adjust the adhesion strength and mechanical properties (such as tensile strength and Young's modulus) of the hydrogel, enabling it to adapt to the needs of various tissues, from soft internal organs to active skin. Experimental data show that its adhesion to glass substrates can reach up to approximately 400 kPa, far exceeding that of traditional bioadhesives such as fibrin glue.

[0024] (3) Benefiting from Fe 3+ The high efficiency of the polyphenol dual-catalytic system allows the hydrogel of this invention to be formed within 60 seconds at room temperature, without the need for external stimuli such as ultraviolet light or high temperatures, thus avoiding thermal and photodamage. The maximum temperature of the gelation process is below 40°C, far lower than that of cyanoacrylate adhesives, ensuring safety for clinical use.

[0025] (4) This invention achieves the simultaneous completion of three major functions—catalysis, polymerization, and adhesion—through ingenious component design. Furthermore, Fe... 3+ Polyphenols themselves possess antibacterial and antioxidant potential, providing a feasible technical platform for developing multifunctional (such as antibacterial and healing-promoting) tissue adhesives, overcoming the limitations of existing adhesives with single functions. Attached Figure Description

[0026] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.

[0027] Figure 1 The graph shows the adhesion performance test results of the three hydrogels in Example 3 on different substrates (copper, plastic, glass, wood block, polytetrafluoroethylene mold, weight). Figure 2 The graph shows the adhesion performance of the three hydrogels to different visceral tissues in Example 3. Figure 3 The images show thermal images of the three hydrogels Fe-GSB, Fe-ESB and Fe-TSB in Example 4, as well as n-butyl α-cyanoacrylate after being coated on the wound surface. Figure 4 The images show the apparent healing of three hydrogels (Fe-GSB, Fe-ESB, and Fe-TSB) and α-cyanoacrylate n-butyl acrylate applied to the wound surface in Example 5, along with H&E staining and Masson staining results. Figure 5 The microstructures of the hydrogels obtained by different gelation sequences were observed under a scanning electron microscope (SEM) in Example 6. Figure 6 Violin diagrams of the hydrogels obtained from six different gelation sequences in Example 6 (analyzed from both thermal and adhesive properties). Detailed Implementation

[0028] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0029] Raw materials and testing methods involved in the examples: Raw materials: Methacrylethyl sulfobetaine (SBMA), gallic acid (GA), epigallocatechin gallate (EGCG), tannic acid (TA), ammonium persulfate (APS), polyethylene glycol dimethacrylate (PEGDMA), and ferric chloride hexahydrate (FeCl3·6H2O) were all commercially available analytical grade or reagent grade.

[0030] Gel formation time determination: The inverted vial method is used. The reaction system is added to the vial and mixed evenly. Timing is started. When the contents of the vial do not flow when the vial is inverted, it is considered that the gel has been completely formed. The time at this point is the gel formation time.

[0031] Adhesion test: The ungelled reaction solution was evenly applied to approximately 1 / 4 of the surface of a standard glass plate, and then quickly covered with another glass plate of the same specifications. After complete gelation, it was allowed to stand at room temperature for 1 hour. The test was conducted using a universal testing machine at a tensile speed of 5 mm / min. The effective area of ​​adhesion between the two glass plates and the maximum force during the tensile process were recorded, and the adhesion force of the hydrogel was calculated based on the above data.

[0032] Example 1 This embodiment aims to investigate the effect of fixing the polyphenol concentration (4mM) and changing the Fe... 3+ Effect of concentration on hydrogel gelation time and adhesion properties.

[0033] S1. Dissolve SBMA monomer (4M, calculated based on the final water volume, e.g., 279.35mg for 250μL of water), polyphenol (4mM), and PEGDMA (1% of the mass of SBMA) in 250μL of deionized water, and vortex until clear and transparent to obtain the first mixture.

[0034] S2. Add different volumes of FeCl3 solution to the first mixture, respectively, so that the final Fe... 3+ The concentrations were 2, 3, 4, 5, and 6 mM, respectively. After thorough mixing, a second mixture was obtained.

[0035] S3. Finally, add 2 mg of APS solution, mix quickly, start timing and record the gelation time. After gelation, test the adhesion strength as described above. The test results are shown in Table 1-3.

[0036] The polyphenols selected were gallic acid (GA), epigallocatechin gallate (EGCG), and tannic acid (TA). Three types without added Fe... 3+ The SBMA monomer solutions were all clear and transparent; when Fe was introduced into the system... 3+ After that, Fe 3+ It can undergo complexation reactions with gallic acid (GA), epigallocatechin gallate (EGCG), and tannic acid (TA) to form complexes with characteristic colors. The Fe-GA, Fe-EGCG, and Fe-TA complexes are light green, pale blue, and dark green, respectively. Upon addition of ammonium persulfate (APS) solution to the above system, a rapid exothermic gelation reaction occurs, and the polymerization of the hydrogel can be completed within 60 seconds. These results demonstrate that the hydrogel system can polymerize at room temperature without the need for external initiation methods such as ultraviolet radiation or heating.

[0037] Table 1 Different Fe 3+ Performance of Fe-GSB hydrogels at different concentrations Table 2 Different Fe 3+ Performance of Fe-ESB hydrogels at different concentrations Table 3 Different Fe 3+ Performance of Fe-TSB hydrogels at different concentrations The results showed that, under the condition of fixed polyphenol concentration, with the increase of Fe... 3+ With increasing Fe concentration, the gelation time of the hydrogel gradually shortened, but the adhesion strength peaked at 3 mM and then decreased. Analysis revealed this was due to an excessively rapid polymerization rate leading to an uneven network structure and an undesirable distribution of crosslinking points. Therefore, 3 mM Fe was selected as the optimal concentration. 3+ This will serve as the preferred concentration for subsequent experiments.

[0038] Example 2 This embodiment fixes Fe 3+ The effects of three polyphenols, GA, EGCG, and TA, at different concentrations (2, 4, 8, 12, and 16 mM) on the hydrogel properties were investigated at a concentration of 3 mM. The preparation method was basically the same as in Example 1, except that the types and amounts of polyphenols were changed.

[0039] The test results are shown in Table 4-6.

[0040] Table 4. Properties of Fe-GSB hydrogels at different GA concentrations Table 5. Properties of Fe-ESB hydrogels at different EGCG concentrations Table 6. Properties of Fe-TSB hydrogels at different TA concentrations The results show that in Fe 3+ Under constant concentration conditions, the gelation time of the hydrogel gradually increased with increasing polyphenol concentration. Specifically, the gelation time of the GA experimental group remained consistently below 60 seconds within the set polyphenol concentration range. Similarly, the gelation time of the EGCG and TA experimental groups also remained relatively stable within 60 seconds when the polyphenol concentration was between 2-8 mM. This phenomenon is related to the differences in the molecular structures of the three polyphenols: GA, EGCG, and TA.

[0041] In summary, Fe 3+ The introduction of Fe can significantly accelerate the gelation reaction rate of hydrogels, but with Fe 3+ With increasing polyphenol concentration, the adhesion performance of the hydrogel decreased, possibly due to insufficient cross-linking of the gel network caused by an excessively rapid gelation rate. However, as the polyphenol concentration increased, the adhesion performance of the hydrogel initially increased and then decreased, while the gelation time gradually increased with increasing polyphenol concentration. Considering key indicators such as gelation efficiency and adhesion performance, subsequent experiments determined that 3mM Fe was the optimal choice. 3+ The optimal reaction concentration combination is 4 mM polyphenols.

[0042] Example 3 This embodiment aims to study the broad-spectrum adhesive properties of hydrogels: According to the preferred formula (Fe) 3+ Three types of hydrogels, Fe-GSB, Fe-ESB, and Fe-TSB, were prepared using a mixture of -3mM polyphenols, -4mM polyphenols, and -4mM SBMA. The reaction mixtures were injected into circular molds with a diameter of 11mm, and the gel thickness was controlled to be 1.5mm. After cross-linking and molding, the gels were demolded. The resulting hydrogel samples were placed on clean, transparent glass plates and subjected to adhesion tests with copper sheets, wood, plastic, glass, a polytetrafluoroethylene mold, and a 50g weight, respectively, to characterize the adhesion performance of the hydrogels to different hard substrates.

[0043] The method for testing the apparent adhesion properties of hydrogels to biological visceral tissues is as follows: Take the above-mentioned molded hydrogel sample and place it on the surface of a transparent glass plate. Then, conduct adhesion experiments with the heart, liver, spleen, lung, and kidney tissues of mice to evaluate the adhesion effect of hydrogels on different organ tissues.

[0044] The experimental results are shown in Figure 1 and Figure 2The scale bar of all experimental characterization images was set to 1 cm.

[0045] The three hydrogels all exhibited good adhesion to different substrates (copper, plastic, glass, wood blocks, PTFE molds, weights, and internal organs), fully demonstrating that this series of hydrogels possesses excellent adhesion properties.

[0046] Example 4 This embodiment aims to study the mild gelation properties of hydrogels: Experimental Methods: A dorsal skin wound model was constructed using rats. A 2cm linear wound was made on the rat's back using a sterile scalpel. Thermal images of the wound at its initial state (0s) were acquired using a thermal imager (model: FLIR-E6390) as a blank control. Subsequently, the wound was treated according to the preferred formulation (Fe... 3+ Three hydrogels—Fe-GSB, Fe-ESB, and Fe-TSB—prepared with 3mM polyphenols, 4mM polyphenols, and 4mM SBMA, along with butyl α-cyanoacrylate (a commercial medical adhesive), were applied to the wound surface for sealing. Thermal images of the wound area were continuously acquired at 10-second intervals within 1 minute after sealing to dynamically monitor temperature changes. The obtained thermal imaging data were analyzed using FLIR Tools software, with the temperature monitoring range set between 25-40℃ during the experiment.

[0047] Test results are available Figure 3 During the entire wound closure process, the system temperature of Fe-GSB, Fe-ESB, and Fe-TSB hydrogels did not exceed 40℃, and the heat release process was more uniform and gradual. In contrast, commercial medical adhesive α-cyanoacrylate n-butyl ester, when applied to the wound, rapidly releases a large amount of heat, with local temperature peaks reaching as high as 60.9℃. This intense heat release effect can easily cause burns to the wound tissue, thereby inducing a more severe inflammatory response.

[0048] Example 5 Evaluation of wound healing effect in animals: 1. Experimental Methods: A 2wt% sodium pentobarbital solution was prepared using physiological saline and used to anesthetize rats at a ratio of 0.002 mL / g. After anesthesia, the rats were dehaired, and the skin surface was cleaned and disinfected with povidone-iodine. The incision sites were marked with a marker after disinfection, and then two incisions were made using a scalpel to create a surgical incision model. The wounds were photographed and recorded. The rats were divided into four groups: a blank control group (no treatment), Fe-GSB group, Fe-ESB group, Fe-TSB group, and α-butyl cyanoacrylate group. Gel was administered according to the groupings, and the administration was photographed. The wounds were covered with bandages to prevent biting. The wound condition was observed and photographed on Day 3 and Day 7. On Day 10, the wound condition was photographed, and tissue samples were collected for dehydration, embedding, sectioning, and staining.

[0049] After H&E staining and sectioning, the slides were dried in a 65°C oven for 6 hours for dewaxing and preservation. Before staining, the paraffin was melted in a 65°C oven for 30 minutes. Then, the tissue sections were immersed in xylene twice for 15 minutes each time to remove the paraffin from the tissue surface. The tissue sections were then dehydrated in a gradient of ethanol: anhydrous ethanol for 5 minutes, 95% ethanol for 5 minutes, and 80% ethanol for 5 minutes. The tissue sections were then immersed in pure water for 5 minutes to wash away excess ethanol. After removing the tissue sections from the pure water, excess water on the slides was wiped off with paper towels. Hematoxylin dye was added to the tissue using a dropper, and after 1 minute, excess hematoxylin dye was washed off with tap water. The tissue sections were then immersed in PBS solution for 3 minutes to achieve blue reversion. Excess water on the slides was wiped off with paper towels, eosin dye was added, and after 35 seconds, excess dye was washed off with tap water. Tissue sections were dehydrated in a gradient of ethanol, in the following order: 80% ethanol solution for 10 seconds, 95% ethanol solution for 5 minutes, and 100% ethanol solution for 5 minutes. The tissue sections were then cleared in xylene twice, for 15 minutes each time. Excess xylene was wiped off the slide with a paper towel. A drop of neutral resin was placed on the tissue using a dropper, a coverslip was placed on top, and any excess air bubbles were removed using a pipette tip. The slides were then placed in a fume hood to air dry. Images were acquired using a Leica upright optical microscope (MD750) after drying.

[0050] Masson staining was performed by melting paraffin in a 65°C oven for 30 minutes. The tissue sections were then immersed in xylene twice for 15 minutes each time to remove the paraffin from the tissue surface. The tissue sections were then dehydrated in a gradient of ethanol: anhydrous ethanol for 5 minutes, 95% ethanol for 5 minutes, and 80% ethanol for 5 minutes. The tissue sections were then immersed in pure water for 5 minutes to wash away excess ethanol. After removing the tissue sections from the pure water, excess water was wiped off the slides with paper towels. Ponceau S dye was applied to the tissue using a dropper, and after 1 minute, excess Ponceau S dye was washed off the tissue surface with tap water. The tissue sections were then immersed in 0.2% acetic acid solution for 1 minute, and excess water was wiped off the slides with paper towels. 1% phosphomolybdic acid solution was applied to the tissue for differentiation for 30 seconds, observing whether the red color faded to pink. The tissue sections were then immersed in 0.2% acetic acid solution for 1 minute. One tissue section was selected, and toluidine blue dye was applied, with the time recorded. Appropriate colors were selected, and staining times were recorded for uniform staining. Tissue sections were dehydrated in a gradient of ethanol, in the following order: 80% ethanol solution for 10 seconds, 95% ethanol solution for 5 minutes, and 100% ethanol solution for 5 minutes. The tissue sections were then cleared in xylene twice, for 15 minutes each time. Excess xylene was wiped off the slide with a paper towel. A drop of neutral resin was placed on the tissue using a dropper, a coverslip was placed on top, and any excess air bubbles were removed using a pipette tip. The slides were then placed in a fume hood to air dry. Images were acquired using a Leica upright optical microscope (MD750) after drying.

[0051] from Figure 4 From the perspective of apparent healing photographs, the control group showed persistent and significant exudation, crusting, and inflammatory reactions within 0-10 days, with a significantly slower healing rate than the experimental groups. The Fe-ESB group exhibited the most ideal wound healing process, entering the rapid epithelialization stage on the 3rd postoperative day and achieving near-complete epidermal closure by days 7-10, with no significant scar hyperplasia after healing, making it the group with the best macroscopic healing effect. The Fe-GSB group showed a similar wound healing process to the Fe-ESB group, with a significant reduction in wound area observed on the 7th postoperative day, and the epidermis being essentially repaired by the 10th day. The Fe-TSB group showed a slightly slower healing rate than the above two groups, with slight pigmentation still visible on the wound on the 10th postoperative day, but its overall healing effect was better than the control group and the α-cyanoacrylate group. The α-cyanoacrylate group showed significant exudation and crusting in the early stages of wound healing, and although its healing rate was better than the control group, it was significantly slower than the Fe-GSB, Fe-ESB, and Fe-TSB hydrogel experimental groups.

[0052] Hematoxylin-eosin (H&E) staining results showed that the Fe-ESB group exhibited the fastest epidermal continuity recovery rate, the mildest inflammatory cell infiltration in the dermis, and the most regular and orderly collagen fiber arrangement. In the control group, epidermal regeneration was slow, with a large number of densely distributed purple inflammatory cells infiltrating the dermis, and pink collagen fibers showing a loose and disordered arrangement, indicating a severe inflammatory response and poor tissue repair quality. The α-cyanoacrylate n-butyl ester group showed a significantly reduced degree of inflammatory cell infiltration compared to the control group, but still higher than the Fe-ESB group; its collagen fiber density was at a moderate level, while the fiber arrangement was less regular than that of the Fe-ESB group. Considering the overall tissue repair effects of each experimental group, the ranking from best to worst was: Fe-ESB > Fe-GSB > Fe-TSB > α-cyanoacrylate n-butyl ester > control group.

[0053] Masson staining results showed that the Fe-ESB group had the highest collagen fiber density in the wound tissue, and its arrangement was most similar to the reticular structure of normal skin. The Fe-GSB group had relatively regular collagen fiber arrangement, but its density was slightly lower than that of the Fe-ESB group. The Fe-TSB group showed a disordered, bundle-like distribution of collagen fibers, suggesting a higher risk of scarring in this group. In the control group, a large number of dense purple inflammatory cells were observed infiltrating the dermis, and the pink collagen fibers were loose and disordered, indicating a severe inflammatory response and poor tissue repair quality. The α-butyl cyanoacrylate group showed significantly reduced inflammatory cell infiltration compared to the control group, but it was still higher than that of the Fe-ESB group; the collagen fiber density in this group was at a moderate level, and its arrangement was also less regular than that of the Fe-ESB group.

[0054] Example 6 Key preparation process - optimization of component addition order: This example uses Fe-TSB hydrogel as an example, fixing the final concentration (SBMA-4M, TA-4mM, Fe... 3+ (Fe-3mM, APS-2mg, PEGDMA-1wt%), by changing the order of addition of Fe, TA, and PEGDMA, 6 different addition orders were designed (corresponding to numbers 1-6), and the effects of different addition orders on the maximum temperature rise (Tmax) and the time to reach the maximum temperature (Tmax time) of the hydrogel were investigated.

[0055] The experimental results in Table 7 show that the Tmax value of No. 4 (gelation sequence: TA→PEGDMA→Fe→APS) is 44.37℃, which is significantly higher than the corresponding values ​​of No. 2 and No. 6. In terms of the time to reach the highest temperature, No. 4 (109±5.568s) is comparable to No. 3 and No. 5, and significantly shorter than No. 6. This indicates that this addition sequence can enable the hydrogel to obtain better heating performance and faster thermal response rate.

[0056] Table 7 Properties of hydrogels prepared by different component addition orders SEM experimental method: Fe-TSB hydrogels were prepared according to different addition orders (1-6), and were prepared into cube shapes. These cubes were then broken down using liquid nitrogen to expose their cross-sections. The gels were freeze-dried for 36 hours. After freeze-drying, suitable cross-sections were sputter-coated with gold and then imaged using SEM (SEM model: SU8600, 5.00kV, 5.00k magnification).

[0057] Figure 5 The images show the microstructures of six hydrogels obtained from different gelation sequences observed under a scanning electron microscope (SEM). Sample number 4 represents the gelation sequence protected in this invention, exhibiting a large-pore, interconnected honeycomb structure. This structure facilitates water transport and heat diffusion, forming the microscopic basis for its superior thermal response performance. The microstructural characteristics of the other numbered samples are as follows: Sample number 1: Interwoven fibrous structure with small and unevenly distributed pores; Sample number 2: Uniform porous structure; Sample number 3: Dense fibrous network with poor pore connectivity; Sample number 5: Dense layered fibrous structure with low porosity; Sample number 6: Uniform small-pore structure with pore sizes of approximately 1–2 μm.

[0058] Add the corresponding substances in the order described above. Immediately after adding APS, use a thermal imager (FLIR-E6390) to record the highest temperature and the time required to reach it. For the adhesion test, pour the adhesive onto a glass plate before it forms a gel, and quickly cover it with another glass plate. After the gel forms and stands for 1 hour, measure the adhesion using a universal testing machine. Record the area bonded between the two glass plates, the maximum force under tension, and calculate the adhesion force.

[0059] Figure 6 The violin diagrams visually illustrate the impact of six different gelation sequences (numbered 1-6) on hydrogel properties from two dimensions: thermal and adhesive properties. The width of each "violin" represents the frequency of occurrence of the corresponding property value; a wider violin indicates a larger sample size for that value, reflecting the distribution characteristics and stability of the data. This invention solves the problems of "unstable thermal properties and large fluctuations in adhesive force" in traditional gelation processes by using a specific raw material addition sequence (TA→PEGDMA→Fe→APS).

[0060] The Fe-GSB, Fe-ESB, and Fe-TSB zwitterionic hydrogel tissue adhesives prepared in this invention use methacryloyl ethyl sulfobetaine (SBMA) as the zwitterionic monomer, combined with Fe... 3+A dual-catalytic system was constructed with polyphenols (gallic acid GA, epigallocatechin gallate EGCG, and tannic acid TA) to provide targeted solutions to the core problems of existing tissue adhesives (such as cyanoacrylates and fibrous materials), including poor biocompatibility, insufficient mechanical properties, stringent gelation conditions, and limited functionality. The specific solutions are as follows: 1. Zwitterionic hydrogels improve biocompatibility from the root through "zwitterionic structure + selection of low-toxicity components." GA, EGCG, and TA are all natural polyphenols (derived from plant extracts), SBMA is a zwitterionic monomer (non-toxic side chain), and Fe... 3+ Choose an appropriate concentration (3mM, which is the result of subsequent experimental optimization) to avoid oxidative damage caused by high concentrations of metal ions.

[0061] 2. Zwitterionic hydrogels achieve the dual advantages of "high adhesion and high biocompatibility" through a "multi-component bonding system + structural design," by adjusting the Fe... 3+ With regard to polyphenol concentration, the adhesion can be optimized. The adhesion of the GA and EGCG groups shows a "first increase and then decrease" trend, while the adhesion of the TA group is the best at the same ratio (higher than the GA and EGCG groups). It can be adjusted according to the adhesion requirements of different tissues (such as skin and internal organs), avoiding the limitation of existing adhesives that have "fixed adhesion and cannot adapt to different tissues". Tensile test shows that all three gels can undergo elastic deformation (stress increases steadily with increasing strain). Among them, Fe-EGCG@SBMA has the highest tensile strength, and Fe-GA@SBMA has the best Young's modulus (about 10 kPa). It can adapt to the activities of soft tissues such as skin and internal organs (such as joint stretching and intestinal peristalsis), avoiding the defects of existing cyanoacrylates that are "rigid and easy to break".

[0062] 3. Zwitterionic hydrogels via Fe 3+ - A dual-catalytic system for catechols enables rapid, room-temperature, and reagent-free gelation. Polymerization is completed within 60 seconds: After the addition of APS (ammonium persulfate), Fe... 3+ The dual-catalytic system formed with polyphenols (GA / EGCG / TA) can activate free radicals, promoting the rapid polymerization of SBMA monomers, with gelation time controlled within 60 seconds (fixed Fe). 3+ At 3mM and 4mM polyphenols, it meets the surgical requirements of "rapid hemostasis and rapid wound closure," avoiding the risk of blood loss due to prolonged waiting; room temperature gelation: the gel can spontaneously gel at room temperature, without relying on ultraviolet radiation (avoiding skin burns) or high temperatures (avoiding tissue thermal damage), making it suitable for complex clinical surgical environments (such as deep wounds); no toxic activating reagents required: in the dual-catalytic system, Fe... 3+ It acts as both a catalyst and a cross-linking agent, eliminating the need for additional toxic / easily deactivated reagents such as H2O2 and horseradish peroxidase, thus avoiding tissue inflammation caused by residual activating agents.

[0063] 4. Zwitterionic hydrogels possess potential for functional expansion through component design: Fe 3+ Metal ions themselves have antibacterial activity (can inhibit bacterial cell membrane synthesis), and TA and EGCG are natural polyphenols (reported in the literature to have antibacterial effects), which solves the problem that existing adhesives "have no antibacterial function and require the use of additional antibiotics"; polyphenols (GA, EGCG, TA) contain a large number of phenolic hydroxyl groups, which can remove DPPH free radicals and ABTS free radicals at the wound site, reduce the damage of oxidative stress to healing cells (such as fibroblasts), promote wound repair, and make up for the defect of existing adhesives "having no antioxidant function".

[0064] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a tissue adhesive, characterized in that, Includes the following steps: S1. Dissolve the zwitterionic monomer and polyphenol compound in pure water, and add a cross-linking agent to obtain the first mixture; Among them, the zwitterionic monomer is methacryloyl ethyl sulfobetaine, the polyphenolic compound is one of gallic acid, epigallocatechin gallate, and tannic acid, and the crosslinking agent is polyethylene glycol dimethacrylate. S2, Add Fe to the first mixture 3+ The solutions are mixed thoroughly to obtain a second mixture. S3. Add an initiator to the second mixture, mix well, trigger the polymerization reaction, and form a hydrogel tissue binder at room temperature; The initiator is ammonium persulfate.

2. The preparation method according to claim 1, characterized in that, In step S1, the concentration of the zwitterionic monomer in the first mixture is 3.5-4.5M; the amount of crosslinking agent added is 0.5-2% of the mass of the zwitterionic monomer.

3. The preparation method according to claim 2, characterized in that, When the polyphenolic compound is gallic acid, its concentration in the first mixed aqueous solution is 2-16 mM.

4. The preparation method according to claim 2, characterized in that, When the polyphenolic compound is epigallocatechin gallate, its concentration in the first mixed aqueous solution is 2-8 mM.

5. The preparation method according to claim 2, characterized in that, When the polyphenolic compound is tannic acid, its concentration in the first mixed aqueous solution is 2-8 mM.

6. The preparation method according to claim 1, characterized in that, In step S2, Fe 3+ The concentration in the second mixture is 2-6 mM.

7. The preparation method according to claim 1, characterized in that, In step S3, the concentration of the initiator in the second mixture is 6-10 mg / mL.

8. The preparation method according to claim 1, characterized in that, In step S3, the polymerization reaction is completed within 10-60 seconds, and the maximum temperature of the reaction system does not exceed 40°C.

9. A tissue adhesive, characterized in that, It is prepared by any one of claims 1-8.

10. The use of the tissue adhesive as described in claim 9 in the preparation of medical products for surgical wound closure, hemostasis, and tissue repair.

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