Degradable biological adhesive material as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of polymer biomedical materials, and particularly relates to a degradable biological adhesive material as well as a preparation method and application thereof. Specifically, the degradable biological binding material at least comprises a solid material and a liquid material; the solid material comprises multi-arm polyethylene glycol carboxyl activated ester and an amino compound, and the liquid material comprises a first phosphate buffer solution with an acidic pH value and a second phosphate buffer solution with an alkaline pH value; the degradable biological adhesive material can be spontaneously covalently cross-linked through the amino group of the amino compound and the active NHS ester group, so that strong adhesion is formed on various wet tissues under physiological conditions, and meanwhile, rapid self-gelation is realized; and the adhesive has the advantages of controllable gel forming speed, high mechanical strength, strong adhesion to various tissues, capability of bearing ultrahigh bursting pressure, good anti-adhesion effect and the like, so that the adhesive has important application value and market prospect.

Description

Technical Field

[0001] This invention belongs to the field of polymer biomedical materials technology, specifically relating to a biodegradable bioadhesive material, its preparation method, and its application. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Bioadhesive materials play an important role in the medical field and can be widely used in scenarios such as closed hemostasis, skin tissue regeneration, wound closure and postoperative repair. Their stable adhesive properties are the key prerequisite for ensuring the effectiveness of clinical applications. With the continuous deepening of research on the natural bioadhesion mechanism, the medical adhesive products currently used in clinical applications can be mainly divided into the following categories: (1) Natural component sealant (fibrin adhesive): This type of adhesive has high biocompatibility and has been used in clinical practice for a long time. However, it has obvious shortcomings. It has insufficient adhesion to moist tissues and it is difficult to form a stable and effective bond in a moist physiological environment. At the same time, its extensibility is poor and it cannot adapt well to the physiological movement of tissues, which limits its application in some complex surgical scenarios. (2) Semi-synthetic adhesive (gelatin and albumin adhesive): Made with gelatin and albumin as the main raw materials, it has improved some properties to a certain extent, but there is still room for improvement in terms of adhesive strength and stability. (3) Fully synthetic adhesive (acrylate and polyethylene glycol adhesive): Cyanoacrylate adhesive is one of them. It exhibits good adhesion to moist interfaces, which is its significant advantage. However, it also has serious drawbacks. Excessive stiffness can hinder the physiological movement of tissues, affecting the recovery of normal tissue function. Furthermore, its degradation products are cytotoxic and may have adverse effects on the body. While adhesives based on human serum albumin and polyethylene glycol possess high burst strength, the separation of human serum albumin poses a potential risk of disease transmission, preventing their large-scale application. Polyethylene glycol sealants, represented by DuraSeal® products from Intergra Life Sciences Holdings and COSEAL® products from Baxter International, are widely used in neurosurgery, but their high price significantly increases medical costs, hindering widespread adoption.

[0004] In addition, there are related patent documents in the existing technology, such as CN202110829780.0, which discloses a surgical sealant kit and its application in brain and spinal surgery. This sealant is prepared by reacting multi-arm polyethylene glycol active ester with at least one crosslinking agent selected from polylysine, polyethyleneimine, or amino polyethylene glycol. However, the raw material used in this technology is multi-arm polyethylene glycol active ester, which has a high purchase cost, making it difficult to reduce the production cost of the product and limiting its market application.

[0005] In summary, existing bio-adhesive materials have certain shortcomings in terms of performance, safety, and cost. Developing a high-performance, safe, reliable, and low-cost biodegradable bio-adhesive material has significant clinical and market value. Summary of the Invention

[0006] To address the problems of insufficient adhesion, poor extensibility, cytotoxicity, disease transmission risk, high cost, and complex preparation processes in existing bioadhesive materials, this invention provides a biodegradable bioadhesive material, its preparation method, and its applications. Specifically, this biodegradable bioadhesive material can spontaneously covalently crosslink with the amino groups of an amino compound and the active NHS ester groups, forming strong adhesion to various wet tissues under physiological conditions while rapidly self-gelling. It possesses advantages such as controllable gelation rate, high mechanical strength, strong adhesion to various tissues, ability to withstand ultra-high burst pressure, and good anti-adhesion effect. Based on the above research results, this invention was completed.

[0007] To achieve the above-mentioned technical objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides a biodegradable bioadhesive material, said biodegradable bioadhesive material comprising at least a solid material and a liquid material; The solid material comprises a multi-arm polyethylene glycol carboxyl-activated ester and an amino compound, and the liquid material comprises a first phosphate buffer solution with an acidic pH and a second phosphate buffer solution with an alkaline pH.

[0008] The multi-arm polyethylene glycol carboxyl activated ester can be N-hydroxysuccinimide ester modified polyethylene glycol with 4 to 8 arms, including but not limited to four-arm polyethylene glycol succinimide succinate (4-arm-PEG-SS), four-arm polyethylene glycol succinimide glutarate (4-arm-PEG-SG), six-arm polyethylene glycol succinimide succinate (6-arm-PEG-SS), six-arm polyethylene glycol succinimide glutarate (6-arm-PEG-SG), eight-arm polyethylene glycol succinimide succinate (8-arm-PEG-SS), and eight-arm polyethylene glycol succinimide glutarate (8-arm-PEG-SG). The amino compound can be trilysine, ε-polylysine (degree of polymerization 10~50), or amino polyethylene glycol. Further, the amino polyethylene glycol is four-arm polyethylene glycol lysine (4-arm-PEG-Lys).

[0009] A second aspect of the present invention provides a method for preparing the above-mentioned biodegradable bioadhesive material, the method comprising: dissolving the multi-arm polyethylene glycol carboxyl activated ester in a first phosphate buffer solution to obtain a first component, and dissolving the amino compound in a second phosphate buffer solution to obtain a second component; The first and second components are mixed, and the two components undergo a cross-linking reaction to form a bio-adhesive material.

[0010] A third aspect of the present invention provides a sealant kit, the sealant kit comprising at least the above-mentioned biodegradable bioadhesive material; furthermore, the sealant kit may also comprise other commonly used kit materials, such as syringes, instructions, etc., without specific limitations herein.

[0011] A fourth aspect of the present invention provides the application of the above-described bioadhesive materials and sealant kits in the preparation of medical devices.

[0012] The beneficial technical effects of one or more of the above technical solutions are as follows: (1) In the prior art, when synthesizing multi-arm polyethylene glycol, ethylene oxide (flammable and explosive) is used as raw material or polyol is halogenated. The polyol and polyethylene glycol are connected by ether bond. The preparation process is complicated and costly. In contrast, the present invention directly uses polyol and carboxyl-modified polyethylene glycol to undergo esterification reaction. The polyol is connected by ester bond and can be hydrolyzed and degraded in physiological solution.

[0013] (2) In the preparation of 4-arm-PEG-Lys, a new method for synthesizing four-arm polyethylene glycolamine (4-arm-PEG-NH2) is provided. Usually, the existing technology for preparing 4-arm-PEG-NH2 requires first synthesizing four-arm polyethylene glycol and then activating it to 4-arm-PEG-NH2; while the present invention first synthesizes NH2-PEG-NH2 and then synthesizes 4-arm-PEG-NH2, which finally reacts with the carboxyl group of lysine to form an amide bond, which is convenient for synthesis and reduces costs.

[0014] (3) The raw materials used in this invention are polyethylene glycol, polyol, acid anhydride, and trilysine. The buffer solution is phosphate, which avoids the halogenation reaction of polyol. This approach simplifies the preparation process, reduces costs, and minimizes side effects during product use and degradation. Animal experiments have verified that it is safe and effective.

[0015] (4) The bio-adhesive material of the present invention has controllable curing time, low swelling rate, high burst strength (cohesion) and good anti-adhesion effect, which meets the requirements for use. Attached Figure Description

[0016] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0017] Figure 1 The results are from the cell compatibility tests of the bioadhesive materials prepared in Examples 1-6 of this invention.

[0018] Figure 2 The 1H NMR spectrum of the 4-arm-PEG-SG prepared in Example 1 of this invention.

[0019] Figure 3 The infrared spectrum of 4-arm-PEG-SG prepared in Example 1 of this invention.

[0020] Figure 4 The 1H NMR spectrum of 4-arm-PEG-NH2 prepared in Example 1 of this invention.

[0021] Figure 5 The 4-arm-PEG-Lys 1H NMR spectrum prepared in Example 1 of this invention.

[0022] Figure 6 The infrared spectrum of the bio-adhesive material prepared in Example 1 of this invention is shown.

[0023] Figure 7 This is a test diagram of the fracture strength of the bio-adhesive material prepared in Example 5 of the present invention.

[0024] Figure 8 This is a schematic diagram of the gelation process of the bio-adhesive material prepared in Example 1 of the present invention.

[0025] Figure 9 The diagram shows the adhesion and hemostasis process of the bio-adhesive material prepared in Example 1 of the present invention during craniotomy in rats. In the diagram, a is the rat skull hemorrhage wound without the application of bio-adhesive material; b is the rat skull wound after the application of bio-adhesive material; c is the healing status of the skull skin wound after suturing for one week after the application of bio-adhesive material; and d is the healing status of the rat skull skin wound one month after the application of bio-adhesive material.

[0026] Figure 10 The diagram shows the subcutaneous implantation process of the bioadhesive materials prepared in Examples 1-3 of this invention on the back of rats. In the diagram, a is the sequence of sample implantation; b and c are process marking diagrams before and after sample implantation; and d is the tissue structure diagram of the sample implantation site after the rats were sacrificed 6 months later.

[0027] Figure 11 The images shown are scanning electron microscope (SEM) images of the bio-adhesive material prepared in Example 1 of the present invention, where a is a cross-sectional SEM image of the bio-adhesive material; and b is a cross-sectional SEM image of the bio-adhesive material under high magnification.

[0028] Figure 12 Three months after applying the bio-adhesive material prepared in Example 1 to a circular defect with a 3mm diameter opening in the skull of a rat in this invention, the rat was sacrificed. Histological pathological images of the application site are shown.

[0029] Figure 13 The images are histological pathological images of the implantation sites of rats that were euthanized one month after the subcutaneous implantation experiment in this invention. Among them, (1)-(3) are the HE staining results of the experimental group (product of Example 1), and (4)-(6) are the HE staining results of the control group (commercially available products containing polyethyleneimine).

[0030] Figure 14 The diagram shows the experimental process of using the bio-adhesive material prepared in Example 6 of this invention for postoperative repair of anal fistula in mice. In the diagram, ab represents the modeling of the experimental group mice and the wound infection status one week later, while cd represents the modeling of the blank group mice and the wound infection status one week later.

[0031] Figure 15 The diagram shows the experimental process of using the bio-adhesive material prepared in Example 7 of this invention for the healing of mouse skin incisions. In the diagram, a is the skin incision of the experimental group mice in which the bio-adhesive material was applied; b is the back incision of the experimental group mice in which the bio-adhesive material was applied covered with gauze; c is the healing status of the back incision of the experimental group mice two weeks after the application of the bio-adhesive material; d is the skin incision of the control group mice in which the bio-adhesive material was not applied; and e is the healing status of the back incision of the control group mice in which the bio-adhesive material was not applied two weeks later.

[0032] Figure 16 The diagram shows the experimental process of using the bio-adhesive material prepared in Example 8 of the present invention for hemostasis of the abdominal aorta in rats. In the diagram, ab represents the puncture and bleeding of the abdominal aorta of rats with an injection needle; c represents the application of the bio-adhesive material to the bleeding aortic vessel.

[0033] Figure 17 This diagram illustrates the experimental process of using the gentamicin-loaded bioadhesive material prepared in Example 9 of this invention for healing infected wounds on the head of rabbits.

[0034] Figure 18 This diagram illustrates the experimental process of using the bio-adhesive material prepared in Example 10 of the present invention for hemostasis and sealing during sheep dura mater surgery. In this diagram, a represents hemostasis of the dura mater defect area; be represents the experimental group (the defect area is treated with bio-adhesive material); and fi represents the control group (the defect area is covered with autologous fascia).

[0035] Figure 19 The images are MRI images of sheep in the experimental group (a) and control group (b) in Example 9. Detailed Implementation

[0036] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0037] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments according to the present invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0038] As mentioned earlier, existing bio-adhesive materials have certain shortcomings in terms of performance, safety, and cost. Developing a high-performance, safe, reliable, and low-cost biodegradable bio-adhesive material has important clinical significance and market value.

[0039] In view of this, in one specific embodiment of the present invention, a biodegradable bioadhesive material is provided, the biodegradable bioadhesive material comprising at least a solid material and a liquid material; The solid material comprises a multi-arm polyethylene glycol carboxyl-activated ester and an amino compound, and the liquid material comprises a first phosphate buffer solution with an acidic pH and a second phosphate buffer solution with an alkaline pH; further, the first phosphate buffer solution is used to dissolve the multi-arm polyethylene glycol carboxyl-activated ester, and the second phosphate buffer solution is used to dissolve the amino compound.

[0040] The multi-arm polyethylene glycol carboxyl activated ester can be N-hydroxysuccinimide ester modified polyethylene glycol with 4 to 8 arms, including but not limited to four-arm polyethylene glycol succinimide succinate (4-arm-PEG-SS), four-arm polyethylene glycol succinimide glutarate (4-arm-PEG-SG), six-arm polyethylene glycol succinimide succinate (6-arm-PEG-SS), six-arm polyethylene glycol succinimide glutarate (6-arm-PEG-SG), eight-arm polyethylene glycol succinimide succinate (8-arm-PEG-SS), and eight-arm polyethylene glycol succinimide glutarate (8-arm-PEG-SG). Furthermore, the multi-arm polyethylene glycol carboxyl-activated ester has the structure shown in Formula I:

[0041] Formula I In Formula I above, p is 2 or 3; n is a positive integer in the range of 10 to 250; m is a positive integer in the range of 4 to 8, preferably m = 4, 6 or 8; more preferably m = 4 or 6; R1 is selected from pentaerythritol, dipentaerythritol or tripentaerythritol; the molecular weight of the compound shown in Formula I is 10000 to 30000 Da.

[0042] In this invention, a 4-8 arm polyethylene glycol carboxyl-activated ester is selected. On the one hand, it can optimize the spatial network structure. The multi-arm structure (4-8 arms) can provide more terminal active sites (each molecule contains 4-8 NHS ester groups). When reacting with amino compounds, it can form a three-dimensional network structure (rather than a non-linear cross-linked chain structure), which significantly improves the mechanical strength of the gel. On the other hand, it can achieve controllable degradation rate: by adjusting the number of arms, the network porosity can be changed, thereby regulating the degradation rate and adapting to the repair cycle of different tissues.

[0043] The multi-armed polyethylene glycol carboxyl-activated ester can be prepared by the following method: by reacting multi-armed carboxylated polyethylene glycol with N-hydroxysuccinimide (NHS) using 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) as a catalyst; wherein the multi-armed carboxylated polyethylene glycol is prepared by reacting carboxylated polyethylene glycol (COOH-PEG-COOH) with a polyol using p-toluenesulfonic acid as a catalyst.

[0044] In this invention, the polyol may be pentaerythritol, dipentaerythritol, or tripentaerythritol; the COOH-PEG-COOH is obtained by modifying PEG with anhydride; and the anhydride may be succinic anhydride or glutaric anhydride.

[0045] Furthermore, the preparation method of the multi-arm polyethylene glycol carboxyl-activated ester includes: S1. Synthesis of polyethylene glycol with carboxyl-terminated groups (COOH-PEG-COOH): PEG (molecular weight 2000~8000 Da), acid anhydride, and p-toluenesulfonic acid were dissolved in an organic solvent; the mixture was refluxed at 50~100℃ for 5~10 h, cooled to room temperature, washed, shaken to separate the layers, and the lower organic phase was collected; the organic phase was purified to obtain COOH-PEG-COOH. The molar ratio of polyethylene glycol (PEG), acid anhydride, and p-toluenesulfonic acid is (1~20):(1~50):1, more preferably (5~15):(25~35):1; the concentration of PEG in the organic solvent is 1~50% (m / v, g / 100mL), more preferably 5~20%; the reaction temperature is preferably 60℃, and the reaction time is preferably 7 h. The acid anhydride is selected from succinic anhydride or glutaric anhydride; the organic solvent can be dichloromethane (CH2Cl2). In this reaction step, carboxyl groups are introduced through the esterification reaction of PEG and acid anhydride. Compared with the traditional chloroacetic anhydride method (which is highly corrosive), the reaction conditions are milder, and the carbon chain length of the acid anhydride can control the ester bond spacing, thereby affecting the subsequent degradation rate.

[0046] S2, Synthesis of multi-arm carboxylated polyethylene glycol The COOH-PEG-COOH and p-toluenesulfonic acid obtained in step S1 were dissolved in an organic solvent. A polyol was added to the solution, and the mixture was refluxed at 50-100℃ for 5-10 h to obtain a dark yellow-brown viscous liquid. After cooling, the liquid solidified into a solid. Water was added to the solid for complete hydrolysis. The hydrolysate was extracted with an organic solvent. The organic phase obtained was dried to remove the organic solvent and then yielded a multi-arm polyethylene glycol with carboxyl groups at the end. Wherein, the molar ratio of COOH-PEG-COOH to p-toluenesulfonic acid is 1:(0.005~1); more preferably 1:(0.01~0.1); the molar ratio of COOH-PEG-COOH to polyol is (2~6):1, preferably 4:1; the concentration of COOH-PEG-COOH in the organic solvent is 1~50% (m / v, g / 100mL), more preferably 5~20%; the organic solvent can be dichloromethane; Furthermore, the polyol is selected from pentaerythritol (corresponding to four-arm polyethylene glycol), bispentaerythritol (corresponding to six-arm polyethylene glycol) or tripentaerythritol (corresponding to eight-arm polyethylene glycol).

[0047] In this step, a multi-arm structure is constructed by esterification reaction of polyol with COOH-PEG-COOH. The ester bond (-COO-) formed can be hydrolyzed into carboxyl and hydroxyl groups under physiological conditions (non-toxic), while the ether bond (-O-) of the existing technology is difficult to degrade, effectively solving the technical problem of "non-degradable multi-arm structure".

[0048] S3, Synthesis of multi-arm polyethylene glycol carboxyl activated ester The multi-arm carboxylated polyethylene glycol, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), and N-hydroxysuccinimide (NHS) obtained in step S2 are dissolved in an organic solvent and reacted at room temperature for 5-10 h. After purification, the product is obtained. The molar ratio of the multi-arm carboxylated polyethylene glycol, EDCI, and NHS is 1:(4~8):(4~8); the concentration of the multi-arm carboxylated polyethylene glycol in the organic solvent is 0.1~50% (m / v, g / 100mL), more preferably 5~20%; The purification process includes steps such as filtration, washing, rotary evaporation, and drying, which will not be described in detail here.

[0049] In this step, the reaction between carboxyl groups and NHS is catalyzed by EDCI to generate highly active NHS ester groups, which can react rapidly with amino groups under physiological conditions, avoiding the limitations of the traditional acyl chloride activation method (which requires anhydrous conditions and produces toxic HCl).

[0050] In another specific embodiment of the present invention, the amino compound may be trilysine, ε-polylysine (degree of polymerization 10~50) and amino polyethylene glycol, and further, the amino polyethylene glycol may be four-arm polyethylene glycol lysine (4-arm-PEG-Lys).

[0051] In this invention, the 4-arm-PEG-Lys has the structure shown in Formula II, where the molecular weight of the compound shown in Formula II is 1000–10000 Da:

[0052] Formula II.

[0053] In another specific embodiment of the present invention, the 4-arm-PEG-Lys is obtained by deprotecting a tetra-armed polyethylene glycolamine (4-arm-PEG-NH2) after reacting it with Boc-protected trilysine (Boc-Tri-Lys) or Boc-L-lysine; the 4-arm-PEG-NH2 is prepared by reacting amino polyethylene glycol with tetrabromoneopentane using tetrabutylammonium bromide (TBAB) as a catalyst; the amino polyethylene glycol is prepared by reacting polyethylene glycol with 2-chloroethylamine under alkaline conditions.

[0054] Furthermore, the 4-arm-PEG-Lys is prepared as follows: S1. Synthesize polyethylene glycolamine (NH2-PEG-NH2) PEG (molecular weight 200~2000 Da) and 2-chloroethylamine were dissolved in an alkaline aqueous solution and reacted at 100~120℃ for 24~36 h. After cooling to room temperature, the mixture was purified to obtain NH2-PEG-NH2. Wherein, the molar ratio of PEG to 2-chloroethylamine is 1:(1~5); The alkaline aqueous solution may be a NaOH aqueous solution with a mass concentration of 10% to 30%; the concentration of PEG in the alkaline aqueous solution is 10% to 20% (m / v, g / 100mL).

[0055] The introduction of amino groups through the nucleophilic substitution reaction of PEG with 2-chloroethylamine offers higher raw material safety and a higher amino substitution rate compared to the traditional azide reduction method (which requires highly toxic azides).

[0056] S2. Synthesis of four-arm polyethylene glycolamine (4-arm-PEG-NH2) The NH2-PEG-NH2 obtained in step S1 was dissolved in acetonitrile, tetrabutylammonium bromide (TBAB) was added as a catalyst, and then anhydrous potassium carbonate and an acetonitrile solution containing tetrabromoneopentane were added. The mixture was refluxed overnight and purified to obtain the final product. The molar amount of tetrabutylammonium bromide added is controlled to be 5-15% of the molar amount of NH2-PEG-NH2; The molar ratio of NH2-PEG-NH2, anhydrous potassium carbonate, and tetrabromoneopentane is (1~5):(5~10):1; preferably 4:8:1. Purification includes processes such as dialysis and drying, which will not be elaborated here.

[0057] In this step, tetrabromo-neopentane is used as a crosslinking agent to connect NH2-PEG-NH2 into a four-arm structure through a halogen substitution reaction. The use of TBAB catalyst can further improve the reaction efficiency.

[0058] S3. Synthesis of four-arm polyethylene glycol lysine (4-arm-PEG-Lys) The 4-arm-PEG-NH2, Boc-protected trilysine (Boc-Tri-Lys), or Boc-L-lysine obtained in step S2 were dissolved in an organic solvent, and EDCI was added as a catalyst. The reaction was carried out at room temperature for 16-24 h. After cooling to room temperature, trifluoroacetic acid (TFA) was added, and the reaction was carried out at room temperature overnight. The product was then purified.

[0059] The molar ratio of 4-arm-PEG-NH2, Boc-protected trilysine (Boc-Tri-Lys), or Boc-L-lysine is 1:(1~10), and the organic solvent can be dichloromethane; the concentration of 4-arm-PEG-NH2 in the organic solvent is 10%~20% (m / v, g / 100mL). The purification process includes steps such as filtration, washing, rotary evaporation, and drying, which will not be described in detail here.

[0060] In this step, the precise amidation of lysine and 4-arm-PEG-NH2 is achieved by introducing and removing Boc protecting groups, ensuring that lysine is grafted onto each arm, thus solving the problem of uneven lysine distribution in the physical mixing system.

[0061] In another specific embodiment of the present invention, a method for preparing the above-mentioned biodegradable bioadhesive material is provided, the method comprising: dissolving the multi-arm polyethylene glycol carboxyl activated ester in a first phosphate buffer solution to obtain a first component, and dissolving the amino compound in a second phosphate buffer solution to obtain a second component; The first and second components are mixed, and the two components undergo a cross-linking reaction to form a bio-adhesive material.

[0062] The concentration of the multi-arm polyethylene glycol carboxyl activated ester in the first component is 10~400 mg / mL, and the concentration of the amino compound in the second component is 1~200 mg / mL; furthermore, the molar ratio of the N-hydroxysuccinimide group in the first component to the amino group in the second component is 1:(0.1~10).

[0063] The bio-adhesive material prepared by this invention uses high-strength amide bonds as the crosslinking core and combines network density regulation to overcome the limitations of traditional physical gels that are "low in strength and poor in stability". Specifically, by regulating the ratio of polyethylene glycol carboxyl-activated ester and amino compound, a nucleophilic substitution reaction occurs rapidly in phosphate buffer to form an adhesive hydrogel with a three-dimensional network structure.

[0064] This invention demonstrates through experiments that the bio-adhesive material has a dense, porous internal structure and a smooth, non-porous outer surface. When sprayed onto damaged tissue, it adheres to the wound surface, sealing bleeding points and stopping bleeding. Its internal pores can hold blood, promoting wound healing. Simultaneously, the bio-adhesive material acts as a physical barrier, preventing direct contact between the wound and surrounding tissues, while its smooth, non-porous outer surface effectively prevents fiber adhesion. Furthermore, the bio-adhesive material is safe and non-irritating, thus also exhibiting excellent anti-adhesion properties.

[0065] In another specific embodiment of the present invention, a sealant kit is provided, the sealant kit comprising at least the above-mentioned biodegradable bioadhesive material; furthermore, the surgical sealant kit may also comprise other commonly used kit materials, such as syringes, instructions, etc., which are not specifically limited here.

[0066] In another specific embodiment of the present invention, the application of the above-mentioned bio-adhesive material and sealant kit in the preparation of medical devices is provided. The medical device may be a drug-device combination product, which refers to a product composed of a drug and a medical device, and manufactured as a single entity. One specific embodiment of the drug-device combination product may contain the above-mentioned bio-adhesive material, and the bio-adhesive material may also load a drug.

[0067] The active ingredient of the drug may be a nucleic acid, antibiotic, anti-inflammatory agent, antibody or antibody fragment thereof, growth factor, cytokine, enzyme, peptide, protein, fusion protein, synthetic molecule, organic molecule, carbohydrate or analogue, lipid, hormone, microsome, derivative or variant thereof, and any combination thereof.

[0068] Furthermore, the medical device has advantages such as controllable curing time, low swelling rate, high burst strength (cohesion), and good anti-adhesion effect. Therefore, it can be used in a variety of clinical treatment scenarios such as closed hemostasis, tissue fixation, tissue regeneration, anti-adhesion, anti-leakage, tissue filling, tumor embolization, and postoperative repair.

[0069] The aforementioned closed hemostasis includes skin incision sealing, vascular hemostasis, visceral hemostasis, and intraoperative hemostasis of the dura mater; the leakage prevention includes dura mater sealing; the postoperative repair includes postoperative repair of anal fistula; and the anti-adhesion refers to postoperative anti-adhesion after surgery.

[0070] The following examples further illustrate the present invention, but do not constitute a limitation thereof. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the invention.

[0071] Example 1 1. Preparation of four-arm polyethylene glycol carboxyl-activated ester (1) Synthesis of polyethylene glycol with carboxyl end groups (COOH-PEG-COOH): Weigh 10 g of PEG 5000 and 0.685 g of glutaric anhydride and dissolve them in 100 mL of CH2Cl2. Add 0.035 g of p-toluenesulfonic acid and reflux at 60 °C for 7 h. Cool to room temperature, wash twice with water, shake to separate the layers, collect the lower layer, rotary evaporate to constant weight in the flask, and dry under vacuum at 40 °C to obtain 9.51 g of COOH-PEG-COOH.

[0072]

[0073] (2) Synthesis of four-arm carboxylated polyethylene glycol (4-arm-PEG-COOH): 10 g of carboxylated PEG (COOH-PEG-COOH) was dissolved in 100 mL of CH2Cl2. 0.01 g of p-toluenesulfonic acid was added, and 0.068 g of pentaerythritol was dissolved in 10 mL of N,N-dimethylformamide (DMF). The pentaerythritol solution was added dropwise to the solution, and the mixture was refluxed at 60 °C for 8 h to obtain a dark yellow-brown viscous liquid, which solidified upon cooling. Water was added to the solid, and after complete hydrolysis, the mixture was extracted with CH2Cl2, dried over anhydrous sodium sulfate, and the CH2Cl2 was removed by rotary evaporation to obtain 8.04 g of 4-arm-PEG-COOH.

[0074] (3) Synthesis of four-arm polyethylene glycol (4-arm-PEG-SG) with carboxyl-activated ester end groups: Weigh 10 g of 4-arm-PEG-COOH, 0.575 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), and 0.345 g of N-hydroxysuccinimide (NHS) and dissolve them in 100 mL of CH2Cl2. React at room temperature for 8 h. Filter the reaction solution and wash the residue three times with CH2Cl2. Rotary evaporate the filtrate and dry it under vacuum at 40 °C for more than 12 h to obtain 9.47 g of the product, four-arm polyethylene glycol carboxyl activated ester (4-arm-PEG-SG).

[0075] Through analysis 1 Both the H-NMR spectrum and the infrared spectrum showed the introduction of glutaric acid and NHS groups. Figure 2 As shown, a singlet occurs at the shift of δ = 2.75–2.90 ppm (peak a), which is attributed to the proton peak at the a-position of the NHS modification group (-C(O)CH2CH2C(O)-). The three sets of peaks at shifts of δ = 2.60–2.75 ppm (peak b), δ = 2.00–2.20 ppm (peak c), and δ = 2.40–2.60 ppm (peak d) are the proton peaks of the glutaric acid modification group (-C(O)CH2CH2CH2C(O)-). Figure 2 The terminal substitution rate can be calculated to be 100%.

[0076] like Figure 3 The figure shows that at 1740.28 cm -1 A strong stretching vibration peak of the ester carbonyl group (C=O) appeared at 1558.46 cm⁻¹. -1 The characteristic absorption peak of amide (NH) is at 1653.03 cm⁻¹. -1 The characteristic absorption peak at that point is attributed to the (C=O) stretching vibration peak of the amide I band.

[0077] 2. Preparation of four-arm polyethylene glycol lysine (4-arm-PEG-Lys) (1) Preparation of polyethylene glycolamine (NH2-PEG-NH2) Weigh 8 g of PEG 2000 and 0.636 g of 2-chloroethylamine and dissolve them in 80 mL of 20% NaOH aqueous solution. React at 110 °C for 30 h. After cooling to room temperature, wash twice with 0.5 mol / L hydrochloric acid. After rotary evaporation to constant weight in the flask, dry under vacuum at 40 °C to obtain 7.54 g of NH2-PEG-NH2.

[0078] (2) Preparation of four-arm polyethylene glycolamine (4-arm-PEG-NH2) 4.66 g of NH2-PEG-NH2 was weighed and dissolved in 10 mL of acetonitrile. 0.075 g of tetrabutylammonium bromide (TBAB) was added as a catalyst. 0.644 g of anhydrous potassium carbonate was added to neutralize hydrogen bromide. 0.225 g of tetrabromoneopentane was weighed and dissolved in 10 mL of acetonitrile. The solution was poured into a constant-pressure dropping funnel and added dropwise to the reaction system. The mixture was refluxed overnight. The solution was dialyzed against pure water for 48 h, evaporated to dryness under vacuum, and dried in a vacuum drying oven at 50 °C for 6 h to obtain 3.85 g of 4-arm-PEG-NH2.

[0079] 1H-NMR spectrum as shown Figure 4 As shown, the multiplet at the δ=3.45~3.80 ppm (peak f) shift is attributed to hydrogen on the repeating unit (-O-CH2-CH2-) of the PEG chain, the singlet at the δ=3.35~3.45 ppm shift is attributed to -CH2O- at the g-position of the four-arm intermediate connecting structure, and the singlet at the δ=2.15~2.17 ppm shift is attributed to hydrogen on the terminal amino group.

[0080] (3) Preparation of four-arm polyethylene glycol lysine (4-arm-PEG-Lys) Weigh 8.0 g of 4-arm-PEG-NH2 and 2.27 g of Boc-Tri-Lys and dissolve them in 80 mL of CH2Cl2. Add 0.80 g of EDCI and react at room temperature for 20 h using EDCI as a catalyst. Cool to room temperature and add 90 mL of trifluoroacetic acid (TFA). React at room temperature overnight. Filter the reaction solution and wash the residue three times with CH2Cl2. Rotary evaporate the filtrate and dry it under vacuum at 40 °C for more than 12 h to obtain 6.48 g of 4-arm-PEG-Lys.

[0081] Analysis of the 1H-NMR spectrum revealed the introduction of amino and amide bonds. Figure 5As shown, the peak at a shift of δ = 2.50 ~ 3.00 ppm is the hydrogen on the α carbon of lysine, and the peak at a shift of δ = 7.10 ~ 7.50 ppm is the hydrogen on the amide bond (-CO-NH-).

[0082] 3. Preparation of biodegradable bioadhesive materials The first component was obtained by dissolving 4-arm-PEG-SG in 2.5 mL of phosphate buffer solution at pH 5.0, and the second component was obtained by dissolving 4-arm-PEG-Lys in 2.0 mL of phosphate buffer solution at pH 9.0. The first and second components were then mixed, and a cross-linking reaction occurred between the two components to form a bioadhesive material. A schematic diagram of the gelation process of the bioadhesive material is shown below. Figure 8 As shown. The concentration of 4-arm-PEG-SG in the first component is 200 mg / mL, and the concentration of 4-arm-PEG-Lys in the second component is 100 mg / mL. The molar ratio of N-hydroxysuccinimide groups in the first component to amino groups in the second component is 1:3.

[0083] like Figure 6 The infrared spectrum of the four-arm polyethylene glycol carboxyl-activated ester is shown. Figure 3 In comparison, the infrared spectrum of the bio-adhesive material prepared in this embodiment is at 1653.44 cm⁻¹. -1 A stretching vibration peak of the amide carbonyl group (C=O) appeared at 1544.39 cm⁻¹. -1 The peak at this point represents the bending vibration of the NH bond, indicating that the reaction between the carboxyl-activated polyethylene glycol ester and the amino compound forms a new amide bond.

[0084] Example 2 In this embodiment, another biodegradable bioadhesive material is provided, which differs from Example 1 in that the multi-arm polyethylene glycol carboxyl activated ester is a six-arm polyethylene glycol succinimide glutarate (6-arm-PEG-SG).

[0085] Example 3 In this embodiment, another biodegradable bioadhesive material is provided, which differs from Example 1 in that the multi-arm polyethylene glycol carboxyl activated ester is an eight-arm polyethylene glycol succinimide glutarate (8-arm-PEG-SG).

[0086] Example 4 In this embodiment, a biodegradable bioadhesive material is provided. The difference from Example 1 lies in the preparation of the four-armed polyethylene glycol lysine, which is carried out using the following specific steps: Weigh 8.0 g of 4-arm-PEG-NH2 and 1.03 g of Boc-L-lysine and dissolve them in 100 mL of CH2Cl2. Add 0.80 g of EDCI and react at room temperature for 20 h using EDCI as a catalyst. Cool to room temperature and add 90 mL of trifluoroacetic acid (TFA). React at room temperature overnight. Filter the reaction solution and wash the residue three times with CH2Cl2. Rotary evaporate the filtrate and dry it under vacuum at 40 °C for more than 12 h to obtain 4-arm-PEG-Lys.

[0087] Example 5 In this embodiment, a biodegradable bioadhesive material is provided, which differs from Example 1 in that the amino compound is ε-polylysine (degree of polymerization 30).

[0088] Example 6 In this embodiment, a biodegradable bioadhesive material for postoperative repair of anal fistula is provided, and its preparation method is the same as that in Example 5.

[0089] A fistula model was constructed using mice as experimental subjects, and experimental groups were set up ( Figure 14 a) and blank group ( Figure 14 c) The bio-adhesive material of this embodiment was injected into the fistula of the experimental group mice. The control group mice received no treatment. The wound condition was observed one week later. Figure 14 As shown in b, in the experimental group of mice injected with the bio-adhesive material of this embodiment into the fistula, the wound diameter decreased after one week, and there was no infection; while as Figure 14 As shown in d, the control group mice that did not have the bio-adhesive material of this embodiment injected into the fistula showed that the wound infection expanded and obvious infection appeared one week later.

[0090] Example 7 In this embodiment, a biodegradable bioadhesive material for sealing skin incisions is provided, and its preparation method is the same as that in Example 1.

[0091] Mice were used as experimental subjects, forming the experimental group. After anesthetizing them, the hair on their backs was shaved, and a 2-3 cm long incision was made in the skin on their backs with a scalpel. The product prepared in this embodiment was then applied to the incision site. Figure 15 a), then cover the wound with medical gauze ( Figure 15 (b) Remove the gauze after two weeks and observe the healing of the wound on the mouse's back. Figure 15 As shown in Figure c, the skin wound on the back of the mouse treated with the bio-adhesive material of this embodiment healed well after two weeks.

[0092] Mice were used as experimental subjects, serving as a control group. After anesthetizing them, the hair on their backs was shaved, and a 1-2 cm long incision was made in the skin on their backs with a scalpel. Figure 15d) No treatment was given, and the healing of the wound on the mouse's back was observed two weeks later. Figure 15 As shown in Figure e, the blank mice that were not treated with the bio-adhesive material of this embodiment showed that the skin wounds on their backs were not completely closed after two weeks.

[0093] Example 8 In this embodiment, a biodegradable bioadhesive material for hemostasis of the abdominal aorta is provided, and its preparation method is the same as that in Example 1.

[0094] Using rats as experimental subjects, after anesthetizing them, the skin was incised along the midline of the abdomen (approximately 4-5 cm in length) with tissue scissors, and the subcutaneous fat was bluntly dissected; the linea alba of the rectus abdominis muscle was located, the peritoneum was lifted with forceps, a small incision was made, and then the incision was extended upwards and downwards along the linea alba to open the abdominal cavity and expose the abdominal aorta. Figure 16 As shown, the abdominal aorta was punctured with a 20G needle. Figure 16 a), and clamp both ends of the damaged artery with hemostatic forceps ( Figure 16 (b) Quickly spray the bio-adhesive material of this embodiment onto the aorta, remove the hemostatic clamps at both ends, and no bleeding occurs at the damaged area after 5 seconds. Figure 16 c).

[0095] Example 9 In this embodiment, a biodegradable sustained-release bioadhesive material loaded with a drug is provided. The preparation method is the same as in Example 1, except that in the preparation process of the biodegradable sustained-release bioadhesive material, the first component, the second component and gentamicin (0.02g, about 20,000 units) are mixed and dissolved in 1.0mL of phosphate buffer solution with pH=7.5 to obtain the biodegradable sustained-release bioadhesive material loaded with gentamicin.

[0096] Modeling: Using rabbits as experimental subjects, a chronic bacterial infection model was established on the skin of the rabbit's head after anesthesia, inducing purulent infection in the wound, i.e., redness and swelling of the wound accompanied by purulent discharge. Figure 17 a).

[0097] First, the infected wound on the rabbit's head was cleaned daily with saline, hydrogen peroxide, and povidone-iodine, and gentamicin was routinely applied topically for anti-inflammatory treatment. After two weeks, the infected wound on the rabbit's head was still not under control; observation continued for another week. Figure 17 As shown in b, the infected incision on the rabbit's head has closed, but a local abscess still exists under the skin. After incising the head skin, purulent infected material can be seen inside. Figure 17 c).

[0098] Then, after rinsing the infected wound on the rabbit's head with saline, disinfecting with hydrogen peroxide, and wiping with iodine, the gentamicin-loaded bioadhesive material prepared in this embodiment was applied to the wound surface. Figure 17 d) Hold the rabbit still with both hands for 2-5 minutes to allow the adhesive material to solidify and close the wound. Figure 17 e), and then observe the healing of the rabbit's head wound daily. It can be seen that the local infection is completely under control, such as... Figure 17 As shown in f, the rabbit's head wound completely healed and the infection subsided after two weeks. This demonstrates that the gentamicin-containing bio-adhesive material in this embodiment can not only seal the wound and provide a barrier for tissue repair, but also achieve local sustained release of gentamicin, exert antibacterial effects, and effectively inhibit purulent infection of the wound.

[0099] Example 10 In this embodiment, a biodegradable bioadhesive material for hemostasis and sealing during dural anesthesia is provided, and its preparation method is the same as that in Example 5.

[0100] (1) Intraoperative hemostasis of the dura mater: Using sheep as experimental subjects, after anesthesia, an 8cm midline longitudinal incision was made with the L4 vertebra on the back as the center. The skin and subcutaneous tissue were incised in sequence. After a series of treatments including peeling, biting, and removal, the surgical area of ​​the dura mater was exposed. The bio-adhesive material prepared in this embodiment was sprayed onto the bleeding sites in the surgical area or rapid hemostasis was performed, such as... Figure 18 As shown in Figure a, the adhesive material exhibits excellent hemostatic effect, with a hemostasis time of approximately 3 seconds. Typically, bleeding areas during spinal dural surgery are structurally complex and adjacent to important nerves and blood vessels. Compared to conventional electrocoagulation hemostasis methods, using the bio-adhesive material of this embodiment for hemostasis offers high safety, low risk, and no damage to adjacent important nerves and blood vessels.

[0101] The criteria for judging the hemostatic effect are as follows: ≤3min is considered excellent immediate hemostatic effect, 3-5min is considered good, and >5min is considered poor hemostatic effect.

[0102] (2) Closure of dural defects: An experimental group and a control group were set up to construct dural defects. A 1.5-2cm linear defect incision was made on the dura mater using meningeal hooks. Figure 18 b, f), standardized suturing is performed using non-absorbable sutures ( Figure 18 c,g).

[0103] Experimental group: The bio-adhesive material prepared in this embodiment was sprayed onto the incision ( Figure 18 d), with a thickness of 1mm~2mm, observe for 5 minutes, and after confirming no cerebrospinal fluid leakage, suture the surgical incision. Figure 18 e).

[0104] Control group: Autologous fascia was used to close the incision. First, fascia tissue adapted to the dura mater defect area was harvested from the sheep's autologous dorsal fascia and pre-treated. Then, the autologous fascia was flatly applied to the periphery of the dura mater suture incision, so that the fascia completely covered the suture site and the surrounding 0.5 cm of dura mater tissue. Figure 18 h); After fascia fixation, gently press the application area with sterile microsurgical instruments to promote initial adhesion between the fascia and dura mater; observe for 5 minutes, and continuous cerebrospinal fluid leakage can be observed. Suture the surgical incision. Figure 18 i).

[0105] One week post-surgery, sheep in both the experimental and control groups underwent MRI scans to observe cerebrospinal fluid leakage at the site of the dura mater defect, in order to evaluate the effectiveness of the two sealing methods. Figure 19 As shown, the MRI results of the experimental group showed no signs of cerebrospinal fluid leakage in the dura mater defect area, indicating that the bio-adhesive material of this embodiment has a good sealing effect on the defect area. Figure 19 a); while the MRI results of the control group showed obvious cerebrospinal fluid leakage in the dural defect area, indicating that the autologous fascia was not effective in sealing the defect area ( Figure 19 b).

[0106] Comparative Example 1 In this comparative example, a biodegradable bioadhesive material is provided. The difference from Example 5 is that the polyethylene glycol carboxyl activated ester is a commercially available four-arm polyethylene glycol succinimide glutarate (molecular weight 20K), and its structural formula is shown in Formula III below.

[0107]

[0108] Formula III Effect verification 1. In vitro cytotoxicity test Rat fibroblasts (L929) were divided into groups of 1×10⁻⁶. 4 Cells were seeded at a density of 100 μL per well in 96-well plates and incubated at 37°C in a 5% CO2 humidified incubator for 24 h. The culture medium was then replaced with the extracts from the bioadhesive materials of each example and comparative example, and the control group's reagent, and incubated for another 24 h. The sample solution was removed and cultured in phosphate-buffered saline (PBS buffer, pH=7.4) containing 1 mg / mL MTT for 2 h. Finally, the PBS buffer (pH=7.4) was replaced with 100 μL of DMSO solution to dissolve the sample, and the absorbance was measured at 570 nm. Results were calculated as the ratio of the average absorbance of the experimental group to the average absorbance of the control group; a ratio less than 70% was considered cytotoxic. Five independent cultures were prepared for each sample, and the cytotoxicity test was repeated three times.

[0109] Depend on Figure 1As can be seen, after 24 h of culture, the ratio of the average absorbance value obtained by the bio-adhesive materials prepared in each embodiment and comparative example to the average absorbance value in the culture medium is greater than 70%, indicating that the bio-adhesive materials prepared in each embodiment and comparative example have no obvious cytotoxicity and have good biocompatibility.

[0110] 2. Curing time Place a 3 mL round-bottom centrifuge tube equipped with a 6*3 mm micro magnetic stirring rod in the center of a magnetic stirrer. Rotate the stirring rod at a constant speed of 1000 rpm. Take 0.2 mL of the uncured bio-adhesive material and push it to the bottom of the round-bottom centrifuge tube. Use a calibrated stopwatch to record the material curing time. Start timing from the time the material component is pushed in until the stirring rod stops rotating, and record the time it takes for the material to solidify into a gel.

[0111] 3. Swelling rate Take about 1 g of the prepared bioadhesive material sample and shape it into a cylinder with a diameter of about 15.3 mm and a height of about 5.5 mm (prepared in a 10 mL syringe and then removed). Place the cylinder into a 50 mL beaker and add physiological saline buffer that has been preheated to 37 ± 1℃. The mass of the buffer is 40 times that of the test sample. Seal the beaker and place it in an incubator at 37 ± 1℃. After 24 hours, take out the sample, absorb the surface moisture with filter paper, weigh it accurately, and calculate the swelling rate of the material according to the following formula.

[0112]

[0113] 4. Bursting strength A flexible pig casing was fixed to the testing device, and a hole was punched using a needle with an outer diameter of 3 mm. 0.5 mL of the bio-adhesive material was sprayed onto the hole (thickness controlled at 1.6 mm ± 0.4 mm) to completely seal the hole. After waiting 5 minutes, pressure was applied to the bio-adhesive material from below the hole at a rate of 2 mL / min until the bio-adhesive material broke. The pressure at this point was recorded. The breaking strength test diagram of the bio-adhesive material prepared in Example 5 is shown below. Figure 7 As shown.

[0114] 5. In vitro degradation time The prepared bioadhesive material was placed in a phosphate buffer solution with pH 7.4 and isotonic with blood at 37±1℃, and observed daily until it was no longer visible to the naked eye. This time was recorded as the in vitro degradation time of the material.

[0115] Table 1 Performance test results of each embodiment and comparative example

[0116] As shown in Table 1, all examples and comparative examples meet the clinical use standards in terms of physical properties such as curing time, swelling ratio, burst strength, and in vitro degradation time. However, compared to the commercially available four-arm polyethylene glycol succinimide glutarate used in Comparative Example 1, the four-arm polyethylene glycol carboxyl-activated ester prepared in Example 1 of this invention has a simpler synthesis process and lower cost, exhibits a lower swelling ratio, higher burst strength, and is more easily degraded in physiological solutions.

[0117] 6. Animal experiments (1) To verify the actual adhesion effect of the bio-adhesive material prepared in this invention on tissues, rats were used as test subjects. After anesthetizing them, the hair on their heads was shaved. A wound about 2.5 cm long was first cut on the epidermis of their heads with a scalpel. Then, the periosteum was separated with a scalpel and gently pulled apart to expose the skull. Physiological saline was dripped in to soak the skull. The size of the opening was pre-marked. The diameter of the opening was about 3 mm. A brushless grinder with a 3 mm drill bit was used to make an opening in the rat skull. Figure 9 a); The product prepared in Example 1 was applied to a 1 mm perforated dura mater for adhesion and hemostasis; after the experiment, the rat head skin was sutured with 4-0 absorbable sutures ( Figure 9 c) Wipe the surface with iodine solution. Adhesion and hemostasis effects are as follows: Figure 9 As shown in b, it can be seen that the product can form a gel in situ at the wound site, and adhere tightly to the tissue, sealing the site of dural hemorrhage, quickly stopping bleeding and preventing cerebrospinal fluid leakage; after one month, the rat's head wound healed well. Figure 9 d).

[0118] (2) To verify the skin degradation and anti-adhesion effect of the bio-adhesive material prepared in this invention, rats were used as experimental subjects. After anesthetizing them, the hair on their backs was shaved, and a small wound was made on their backs with a scalpel. An opening was made at the position where a 10 mm diameter silicone rubber ring was placed. Figure 10 b), then the products prepared in Examples 1-3 and commercially available products containing polyethyleneimine (PEI) were sequentially processed according to... Figure 10 The implantation sequence of a is applied to the opening and mixes within the silicone rubber ring to form a gel. The silicone rubber ring is then removed. Figure 10 c) After the experiment, the rat's dorsal wound was sutured with 4-0 absorbable sutures, and iodine was applied to the surface. The rats were sacrificed after 6 months, and the degradation of the product implantation site and changes in tissue structure were observed. The results are as follows: Figure 10 As shown in d, it can be seen that the bio-adhesive material at the implantation site (dashed line) is completely degraded and has a smooth surface, without forming adhesion with adjacent tissues, indicating that the product of the present invention has excellent skin degradation and anti-adhesion effects; while adhesion is visible at the animal tissue site (solid line) where commercially available control bio-adhesive material is implanted, which may be because the PEI component irritates the surrounding tissue, leading to adhesion.

[0119] Cross-sectional scanning electron microscope image of the bio-adhesive material prepared by this invention is shown below. Figure 11 As shown in the figure, the material has a dense porous structure inside and a smooth, non-porous outer surface. When sprayed onto the surface of damaged tissue, it adheres to the tissue surface and seals and stops bleeding or leakage of tissue fluid. The dense porous structure inside the material confines blood within the pores, promoting wound healing. Furthermore, as a physical barrier, the bio-adhesive material not only prevents direct contact between the wound and surrounding tissue, but its smooth, non-porous outer surface, which does not come into contact with wet tissue, also effectively blocks fiber adhesion. In addition, the bio-adhesive material of this invention is safe and effective, and does not irritate surrounding tissues; therefore, it exhibits excellent anti-adhesion effects.

[0120] In this experiment, rats were euthanized after 3 months of treatment with the bio-adhesive material prepared in Example 1 at a circular defect in the skull with a 3mm diameter. The histopathological results of the application site are as follows: Figure 12 As shown, no obvious hydrogel residue structure was observed in the sections, indicating that the adhesive material had completely degraded. The skull defect area was filled with new bone tissue, forming mature lamellar bone with a morphology similar to that of a normal skull, indicating good repair of the 3mm non-critical skull defect in rats. Dense fibrous tissue proliferation was visible beneath the skull at the defect site, indicating that the damaged dura mater had been repaired. In this experiment, rats were sacrificed one month after subcutaneous implantation. HE staining results at the implantation site are shown below. Figure 13 As shown, the implantation site ( The capsule is seen around the ) As can be seen, the histopathological changes of experimental groups (1)-(3) (products of Example 1) were similar, and macrophages ① could be observed; the histopathological changes of control groups (4)-(6) (commercially available products containing polyethyleneimine) were similar, and in addition to macrophages ①, lymphocytes ② and plasma cells ③ could also be observed. According to GB / T 16886.6-2022 "Biological Evaluation of Medical Devices, Part VI: Local Reaction Test after Implantation", the tissue compatibility of the materials of the experimental group and the control group after local implantation in the subcutaneous tissue was evaluated. Compared with the control group, the experimental group products had less irritation to the implantation site tissue.

[0121] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A biodegradable bioadhesive material, characterized in that, The biodegradable bioadhesive material comprises at least solid and liquid materials; The solid material comprises a multi-arm polyethylene glycol carboxyl-activated ester and an amino compound, and the liquid material comprises a first phosphate buffer solution with an acidic pH and a second phosphate buffer solution with an alkaline pH.

2. The biodegradable bioadhesive material as described in claim 1, characterized in that, The multi-arm polyethylene glycol carboxyl activated ester is a 4- to 8-arm N-hydroxysuccinimide ester modified polyethylene glycol, including four-arm polyethylene glycol succinimide succinate, four-arm polyethylene glycol succinimide glutarate, six-arm polyethylene glycol succinimide succinate, six-arm polyethylene glycol succinimide glutarate, eight-arm polyethylene glycol succinimide succinate, and eight-arm polyethylene glycol succinimide glutarate. Furthermore, the multi-arm polyethylene glycol carboxyl-activated ester has the structure shown in Formula I: Formula I In Formula I above, p is 2 or 3; n is a positive integer in the range of 10 to 250; m is a positive integer in the range of 4 to 8, preferably m = 4, 6 or 8; more preferably m = 4 or 6; R1 is selected from pentaerythritol, dipentaerythritol, and tripentaerythritol; the molecular weight of the compound shown in Formula I is 10000 to 30000 Da.

3. The biodegradable bioadhesive material as described in claim 2, characterized in that, The multi-armed polyethylene glycol carboxyl-activated ester is prepared by the following method: multi-armed carboxylated polyethylene glycol is prepared by reacting N-hydroxysuccinimide (NHS) with 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) as a catalyst; wherein, the multi-armed carboxylated polyethylene glycol is prepared by reacting carboxylated polyethylene glycol (COOH-PEG-COOH) with a polyol using p-toluenesulfonic acid as a catalyst. Furthermore, the preparation method of the multi-arm polyethylene glycol carboxyl-activated ester includes: S1. Synthesis of polyethylene glycol with carboxyl-terminated groups (COOH-PEG-COOH): PEG (molecular weight 2000~8000 Da), acid anhydride, and p-toluenesulfonic acid were dissolved in an organic solvent; the mixture was refluxed at 50~100℃ for 5~10 h, cooled to room temperature, washed, shaken to separate the layers, and the lower organic phase was collected; the organic phase was purified to obtain COOH-PEG-COOH. S2, Synthesis of multi-arm carboxylated polyethylene glycol The COOH-PEG-COOH and p-toluenesulfonic acid obtained in step S1 were dissolved in an organic solvent. A polyol was added to the solution, and the mixture was refluxed at 50-100℃ for 5-10 h to obtain a dark yellow-brown viscous liquid. After cooling, the liquid solidified into a solid. The solid was hydrolyzed with water, and the hydrolysate was extracted with an organic solvent. The organic phase obtained was dried to remove the organic solvent and then yielded a multi-arm polyethylene glycol with carboxyl groups at the end. S3, Synthesis of multi-arm polyethylene glycol carboxyl activated ester The multi-arm carboxylated polyethylene glycol, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), and N-hydroxysuccinimide (NHS) obtained in step S2 are dissolved in an organic solvent and reacted at room temperature for 5-10 hours. After purification, the product is obtained.

4. The biodegradable bioadhesive material as described in claim 1, characterized in that, The amino compound is trilysine, ε-polylysine (degree of polymerization 10~50) and amino polyethylene glycol, and further, the amino polyethylene glycol is tetra-arm polyethylene glycol lysine.

5. The biodegradable bioadhesive material as described in claim 4, characterized in that, The four-armed polyethylene glycol lysine has the structure shown in Formula II, where the molecular weight of the compound shown in Formula II is 1000–10000 Da: Formula II.

6. The biodegradable bioadhesive material as described in claim 5, characterized in that, The four-arm polyethylene glycol lysine is obtained by deprotecting four-arm polyethylene glycol amine (4-arm-PEG-NH2) after reacting with Boc-protected trilysine (Boc-Tri-Lys) or Boc-L-lysine; the four-arm polyethylene glycol amine is prepared by reacting amino polyethylene glycol with tetrabromoneopentane using tetrabutylammonium bromide (TBAB) as a catalyst; the amino polyethylene glycol is prepared by reacting polyethylene glycol with 2-chloroethylamine under alkaline conditions. Furthermore, the four-arm polyethylene glycol lysine (4-arm-PEG-Lys) is prepared as follows: S1. Synthesize polyethylene glycolamine (NH2-PEG-NH2) PEG (molecular weight 200~2000 Da) and 2-chloroethylamine were dissolved in an alkaline aqueous solution and reacted at 100~120℃ for 24~36 h. After cooling to room temperature, the mixture was purified to obtain NH2-PEG-NH2. S2. Synthesis of four-arm polyethylene glycolamine (4-arm-PEG-NH2) The NH2-PEG-NH2 obtained in step S1 was dissolved in acetonitrile, tetrabutylammonium bromide (TBAB) was added as a catalyst, and then anhydrous potassium carbonate and an acetonitrile solution containing tetrabromoneopentane were added. The mixture was refluxed overnight and purified to obtain the final product. S3. Synthesis of four-arm polyethylene glycol lysine (4-arm-PEG-Lys) The 4-arm-PEG-NH2, Boc-protected trilysine (Boc-Tri-Lys), or Boc-L-lysine obtained in step S2 were dissolved in an organic solvent, and EDCI was added as a catalyst. The reaction was carried out at room temperature for 16-24 h. After cooling to room temperature, trifluoroacetic acid (TFA) was added, and the reaction was carried out at room temperature overnight. The product was then purified.

7. A method for preparing the biodegradable bioadhesive material according to any one of claims 1-6, characterized in that, The preparation method includes: dissolving the multi-arm polyethylene glycol carboxyl activated ester in a first phosphate buffer solution to obtain a first component, and dissolving the amino compound in a second phosphate buffer solution to obtain a second component; The first and second components are mixed, and the two components undergo a cross-linking reaction to form a bio-adhesive material.

8. The preparation method according to claim 7, characterized in that, The concentration of the multi-arm polyethylene glycol carboxyl activated ester in the first component is 10~400 mg / mL, and the concentration of the amino compound in the second component is 1~200 mg / mL; furthermore, the molar ratio of the N-hydroxysuccinimide group in the first component to the amino group in the second component is 1:(0.1~10).

9. A sealant kit, characterized in that, The sealant kit contains at least the biodegradable bioadhesive material as described in any one of claims 1-6.

10. The use of the biodegradable bioadhesive material according to any one of claims 1-6 and the sealant kit according to claim 9 in the preparation of medical devices; Furthermore, the medical device is a combination of drug and medical device; the medical device has the functions of controllable curing time, low swelling rate, high burst strength and anti-adhesion. Furthermore, the medical device is used for closed hemostasis, tissue fixation, tissue regeneration, anti-adhesion, anti-leakage, tissue filling, tumor embolization, and postoperative repair. Furthermore, the closed hemostasis includes skin incision sealing, vascular hemostasis, visceral hemostasis, and intraoperative hemostasis of the dura mater; the leakage prevention includes dura mater sealing; the postoperative repair includes postoperative repair of anal fistula; and the anti-adhesion is postoperative anti-adhesion in surgery.

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