An entanglement-locking unfolding protein-based tissue adhesive, its preparation method and application

Abstract

This invention provides an entanglement-locking unfolded protein-based tissue adhesive, its preparation method, and its application. The invention utilizes a disulfide bond reducing agent to disrupt the protein structure, obtaining unfolded proteins with exposed hydrophobic groups. Subsequently, through strong interactions (electrostatic interactions and hydrogen bonds) between the polyelectrolyte and the unfolded protein, the exposed hydrophobic groups on the surface of the unfolded protein are locked, stabilizing their exposure and locking the chain entanglement between them. This entanglement-locking unfolded protein-based tissue adhesive possesses stable exposed hydrophobic groups and exhibits advantages such as strong hydrophobicity, strong adhesion, stable adhesion performance, excellent bulk properties (high storage modulus), good biocompatibility, and good degradability. Furthermore, when applied to bleeding wounds, this entanglement-locking unfolded protein-based tissue adhesive produces a hemostatic effect, significantly reducing hemostasis time and wound blood loss, demonstrating excellent hemostatic and sealing effects.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, and in particular to an entanglement-locking unfolding protein-based tissue adhesive, its preparation method, and its application. Background Technology

[0002] In modern medicine, blood loss is a major cause of traumatic death, and timely wound treatment and effective hemostasis are crucial for saving lives. Traditional hemostatic materials include gauze, medical sponges, and hemostatic powder. Gauze and medical sponges absorb large amounts of fluid components from the blood, thereby increasing the concentration of clotting factors at the wound site to promote the body's own clotting. However, gauze and medical sponges cannot adhere to biological tissues and require pressure to assist in hemostasis, which is not suitable for internal bleeding or irregular wounds. Hemostatic powder is convenient to carry and easy to use. Existing hemostatic powders mainly achieve rapid hemostasis by absorbing fluid components from the blood, increasing the concentration of clotting factors at the wound site, and attracting red blood cells and platelets. However, they have poor adhesion to tissues, are easily washed away, and can easily remain in the body, leading to inflammatory reactions and affecting wound healing. Therefore, finding a new type of hemostatic material has become an important research direction in the current medical field.

[0003] In recent years, protein-based tissue adhesives have attracted widespread attention due to their advantages such as ease of use, short application time, and minimal tissue damage. Proteins are an important substance widely present in living organisms, possessing good biocompatibility and degradability. Natural proteins (such as bovine serum albumin, hemoglobin, and lactoferrin) inherently possess hydrophobic groups (such as hydrophobic amino acids containing benzene rings) and adhesive groups (such as polar and charged amino acids), with their hydrophobic groups tightly encapsulated in an ordered three-dimensional network. Under specific conditions, the structure of natural proteins can undergo conformational changes, exposing their hydrophobic groups on the surface. When in contact with wound tissue, the exposed hydrophobic groups can repel interfacial liquids (blood or body fluids) through hydrophobic interactions, while the adhesive groups can form stable adhesion with the wound tissue, thereby achieving hemostasis, closure, and tissue adhesion. However, traditional protein-based tissue adhesives still have some problems. For example, existing technologies usually induce the unfolding of the protein structure by breaking the disulfide bonds, thereby exposing hydrophobic groups. However, this method can only achieve temporary exposure of hydrophobic groups (B.Saif, WX Zhang, X. Zhang, Q. Gu, P. Yang. Sn-Triggered Two-DimensionalFast Protein Assembly with Emergent Functions. Acs Nano, 2019, 13(7): 7736-7749.), which makes it difficult to stably expose hydrophobic groups and limits its effectiveness in hemostasis and occlusion.

[0004] In addition, traditional protein-based tissue adhesives have weak bulk properties (such as low energy storage modulus), which limits the application potential of proteins in complex trauma environments.

[0005] Therefore, developing a novel protein-based tissue adhesive that combines stable exposure of hydrophobic groups with excellent bulk properties is an urgent problem to be solved. Summary of the Invention

[0006] This invention provides a method for preparing an entangled-locked unfolded protein-based tissue adhesive. The entangled-locked unfolded protein-based tissue adhesive prepared by this method has stable exposed hydrophobic groups and has the advantages of strong hydrophobicity, strong adhesion, stable adhesion performance, excellent bulk properties (high storage modulus), good biocompatibility and degradability. In addition, when this entangled-locked unfolded protein-based tissue adhesive is applied to bleeding wounds, it produces a hemostatic effect, significantly reducing hemostasis time and wound blood loss, and has excellent hemostatic and sealing effects.

[0007] The present invention also provides an entangled-locked unfolded protein-based tissue adhesive prepared by the above method. Therefore, the entangled-locked unfolded protein-based tissue adhesive has stable exposed hydrophobic groups and has the advantages of strong hydrophobicity, strong adhesion, stable adhesion performance, excellent bulk properties (high storage modulus), good biocompatibility and degradability. In addition, when the entangled-locked unfolded protein-based tissue adhesive is applied to a bleeding wound, it produces a hemostatic effect, significantly reduces the hemostasis time and the amount of blood loss from the wound, and has excellent hemostatic and sealing effects.

[0008] This invention also provides an application of the above-mentioned entangled-locked unfolded protein-based tissue adhesive in the preparation of medical hemostatic materials. The inventors' research shows that when this entangled-locked unfolded protein-based tissue adhesive is applied to a bleeding wound in the liver, it produces a hemostatic effect, significantly reducing hemostasis time and wound blood loss, exhibiting excellent hemostatic and sealing effects. Therefore, it can be used to prepare medical hemostatic materials.

[0009] The first aspect of the present invention provides a method for preparing an entanglement-locking unfolded protein-based tissue adhesive, comprising the following steps: A disulfide bond reducing agent solution was added to a protein solution for incubation to obtain a solution containing unfolded protein. A polyelectrolyte solution is added to the solution containing the unfolded protein and subjected to rapid stirring to obtain the entangled-locked unfolded protein-based tissue adhesive. In the entanglement-locking unfolding protein-based tissue adhesive, the concentration of unfolding protein is 5 w / v% to 15 w / v, and the concentration of polyelectrolyte is 3 w / v% to 15 w / v.

[0010] The method for preparing the entangled-locked unfolded protein-based tissue adhesive as described above, wherein the polyelectrolyte solution is prepared by dissolving a polyelectrolyte in a solvent; The polyelectrolyte is at least one of polyacrylic acid, polyglutamic acid, polystyrene sulfonic acid, polyethyleneimine, and polyallyl.

[0011] In the preparation method of the entangled-locked unfolding protein-based tissue adhesive described above, the polyelectrolyte has a weight-average molecular weight of 2 kDa to 240 kDa.

[0012] The method for preparing the entangled-locked unfolded protein-based tissue adhesive as described above, wherein the protein solution is prepared by dissolving the protein in a solvent; The protein is any one of bovine serum albumin, hemoglobin, lactoferrin, transferrin, β-lactoglobulin, ovalbumin, α-lactalbumin, insulin, lysozyme, and trypsin.

[0013] In the preparation method of the entangled-locked unfolded protein-based tissue adhesive as described above, the disulfide bond reducing agent solution is prepared by dissolving the disulfide bond reducing agent in a solvent; The disulfide bond reducing agent is at least one of tris(2-carboxyethyl)phosphine, dithiothreitol, 2-mercaptoethanol, glutathione, and cysteine.

[0014] In the preparation method of the entangled-locked unfolded protein-based tissue adhesive as described above, the concentration of the disulfide bond reducing agent solution is 1 mmol / L to 100 mmol / L.

[0015] The preparation method of the entangled-locked unfolded protein-based tissue adhesive as described above, wherein the incubation reaction includes: incubating at room temperature for 10 min to 30 min.

[0016] The method for preparing the entangled-locked unfolded protein-based tissue adhesive as described above, wherein the rapid stirring reaction comprises: performing a rapid stirring reaction at room temperature for 5 s to 30 s.

[0017] A second aspect of the present invention provides an entanglement-locking unfolding protein-based tissue adhesive, which is prepared by the method for preparing the entanglement-locking unfolding protein-based tissue adhesive.

[0018] A third aspect of the present invention provides a method for preparing the entangled-locked unfolded protein-based tissue adhesive, and the application of the entangled-locked unfolded protein-based tissue adhesive in the preparation of medical hemostatic materials.

[0019] The solution of the present invention has at least the following effects: The present invention provides a method for preparing an entangled-locked unfolded protein-based tissue adhesive. This method utilizes a disulfide bond reducing agent to disrupt the protein structure, resulting in unfolded proteins with exposed hydrophobic groups. Subsequently, through strong interactions (electrostatic interactions and hydrogen bonding) between a polyelectrolyte and the unfolded protein, the exposed hydrophobic groups on the surface of the unfolded protein are locked, ensuring stable exposure and locking the chain entanglement between the unfolded protein and the polyelectrolyte. The stable exposed hydrophobic groups repel interfacial liquids, forming a strong and stable interfacial bond. The locked chain entanglement improves bulk properties. This entangled-locked unfolded protein-based tissue adhesive possesses stable exposed hydrophobic groups and exhibits advantages such as strong hydrophobicity, strong adhesion, stable adhesion, excellent bulk properties (high storage modulus), good biocompatibility, and good degradability. Furthermore, when applied to bleeding wounds, this entangled-locked unfolded protein-based tissue adhesive produces a hemostatic effect, significantly reducing hemostasis time and wound blood loss, demonstrating excellent hemostatic and sealing effects. The present invention can prepare this entangled-locked unfolded protein-based tissue adhesive through only two reaction steps. The preparation process is simple, the conditions are easy to control, and it has high economic value and promising market application prospects.

[0020] The entangled-locked unfolded protein-based tissue adhesive provided by this invention has stable exposed hydrophobic groups and also has the advantages of strong adhesion, stable adhesion performance, excellent bulk properties (high storage modulus), good biocompatibility and degradability. In addition, when this entangled-locked unfolded protein-based tissue adhesive is applied to bleeding wounds, it produces a hemostatic effect, significantly reducing hemostasis time and wound blood loss, and has excellent hemostatic and sealing effects, solving the problems of weak adhesion and poor hemostatic and sealing effects of traditional tissue adhesives to tissues covered with blood or body fluids. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 This is a schematic diagram illustrating the reaction principle of the entanglement-locking unfolding protein-based tissue adhesive of the present invention, wherein, Figure 1 A is a schematic diagram illustrating the reaction principle for preparing unfolded proteins according to the present invention. Figure 1 B is a schematic diagram of the reaction principle for preparing the entangled-locking unfolded protein-based tissue adhesive of the present invention; Figure 2 This is a schematic diagram illustrating the principle of using the entanglement-locking unfolded protein-based tissue adhesive of the present invention as a medical hemostatic material. Figure 2A is a schematic diagram of covering wound tissue with the entanglement-locking unfolded protein-based tissue adhesive of the present invention. Figure 2 B is a schematic diagram of the stable exposed hydrophobic groups in the entanglement-locking unfolded protein-based tissue adhesive of the present invention, which repel interfacial liquids (blood). Figure 2 C is a schematic diagram of the adhesion groups forming an adhesion between the adhesive groups and the tissue in the entanglement-locking unfolded protein-based tissue adhesive of the present invention; Figure 3 The results of shear strength tests performed on glass slides for each embodiment and comparative example of the present invention are shown below. Figure 3 A represents the shear strength of the entangled-locked unfolded protein-based tissue adhesive in Examples 1-5. Figure 3 B represents the shear strength of the entangled-locked unfolded protein-based tissue adhesive in Examples 6-10. Figure 3 C represents the shear strength of the entangled-locked unfolded protein-based tissue adhesive in Examples 11-12. Figure 3 D represents the shear strength of the protein-based adhesive (BSA-PAA) in Comparative Example 1, the commercial tissue adhesive Fibrin glue in Comparative Example 2, the unfolded protein solution (rBSA) in Comparative Example 3, and the polyelectrolyte solution (PAA) in Comparative Example 4. Figure 4 The results show the test results of the affinity between proteins and polyelectrolytes in Example 1 of the present invention and the affinity between proteins and polyelectrolytes in Comparative Example 1. Figure 5 The contact angle test results are those of the entangled-locked unfolded protein-based tissue adhesive in Example 1 of the present invention and the protein-based adhesive in Comparative Example 1. Figure 6 The storage modulus of the entangled-locked unfolded protein-based tissue adhesive in Example 1 and the protein-based adhesive in Comparative Example 1; Figure 7 The shear strength of the entangled-locked unfolded protein-based tissue adhesive in Example 1 of the present invention, the protein-based adhesive in Comparative Example 1, and the commercial tissue adhesive Fibrin glue in Comparative Example 2; Figure 8 The interfacial toughness of the entangled-locked unfolded protein-based tissue adhesive in Example 1 of the present invention, the protein-based adhesive in Comparative Example 1, and the commercial tissue adhesive Fibrin glue in Comparative Example 2; Figure 9 The tensile strength of the entangled-locked unfolded protein-based tissue adhesive in Example 1 of the present invention, the protein-based adhesive in Comparative Example 1, and the commercial tissue adhesive Fibrin glue in Comparative Example 2; Figure 10The results of the biodegradability test of the entangled-locked unfolded protein-based tissue adhesive in Example 1 of the present invention; Figure 11 The results show the biocompatibility test results of the entanglement-locking unfolding protein-based tissue adhesive in Example 1 and the commercial tissue adhesive Fibrin glue in Comparative Example 2. Figure 11 A represents the H&E staining results after the adhesive sample was implanted into the back of a rat at 1 and 2 weeks. Figure 11 B represents the degree of inflammation assessed by blinded pathological examination; Figure 12 The results of hemostasis and occlusion tests are as follows: Example 1 shows the entanglement-locking unfolded protein-based tissue adhesive; Comparative Example 2 shows the commercial tissue adhesive Fibrin glue; and the control group shows the results of the tests. Figure 12 Figure A shows the hemostasis time in a rat liver incision bleeding model. Figure 12 Figure B shows the blood loss in a rat liver incision bleeding model. Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions in the embodiments of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention. Where specific techniques or conditions are not specified in the embodiments, they shall be performed in accordance with the techniques or conditions described in the literature in this art or in accordance with the product instructions. Reagents or instruments used without specified manufacturers are all conventional products that can be obtained commercially.

[0024] Those skilled in the art should understand that, in the following description of the embodiments of the present invention, the sequence of numbers does not imply the order of execution. Some or all steps may be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present invention.

[0025] Those skilled in the art will understand that the numerical ranges in the embodiments of the present invention should be understood to specifically disclose each intermediate value between the upper and lower limits of the range. Each smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this application. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0026] Unless otherwise stated, the technical / scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. While only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0027] Please see Figure 1 , Figure 1 This is a schematic diagram illustrating the reaction principle of the entanglement-locking unfolding protein-based tissue adhesive of the present invention, wherein, Figure 1 A is a schematic diagram illustrating the reaction principle for preparing unfolded proteins according to the present invention. Figure 1 B is a schematic diagram illustrating the reaction principle of the entanglement-locking unfolding protein-based tissue adhesive prepared according to the present invention, as shown in Figure 1. Figure 1 As shown, a first aspect of the present invention provides a method for preparing an entanglement-locking unfolding protein-based tissue adhesive, comprising the following steps: A disulfide bond reducing agent solution was added to a protein solution for incubation to obtain a solution containing unfolded protein. A polyelectrolyte solution is added to the solution containing the unfolded protein and subjected to rapid stirring to obtain the entangled-locked unfolded protein-based tissue adhesive. In the entanglement-locking unfolding protein-based tissue adhesive, the concentration of unfolding protein is 5 w / v% to 15 w / v, and the concentration of polyelectrolyte is 3 w / v% to 15 w / v.

[0028] The object of this invention is an entangled-locked unfolded protein-based tissue adhesive (hereinafter referred to as unfolded protein-based tissue adhesive). Specifically, a disulfide bond reducing agent solution is first added to a protein solution for incubation. The disulfide bond reducing agent cleaves the disulfide bonds of the protein (i.e., the original protein) to destroy the protein structure. After the disulfide bonds are cleaved by the disulfide bond reducing agent, the protein exhibits temporarily exposed hydrophobic groups and disordered unfolded protein, resulting in a solution containing unfolded protein. Figure 1 (A); Subsequently, the polyelectrolyte solution is added to the solution containing the unfolding protein and subjected to rapid stirring reaction. The polyelectrolyte and the unfolding protein have strong interactions (electrostatic interactions and hydrogen bonds). Through strong interactions, the exposed hydrophobic groups on the surface of the unfolding protein are locked to stabilize the exposure of the hydrophobic groups and to lock the entanglement between the unfolding protein chains and the polyelectrolyte chains, ultimately resulting in an entangled-locked unfolding protein-based tissue adhesive. Figure 1 B).

[0029] In this invention, the concentration of the unfolding protein in the entangled-locked unfolding protein-based tissue adhesive is 5 w / v% to 15 w / v%, which can improve the shear strength of the entangled-locked unfolding protein-based tissue adhesive and is beneficial to improving the adhesion performance of the entangled-locked unfolding protein-based tissue adhesive.

[0030] In this invention, the concentration of polyelectrolyte in the entangled-locked unfolded protein-based tissue adhesive is 3 w / v% to 15 w / v%, which can improve the shear strength of the entangled-locked unfolded protein-based tissue adhesive and is beneficial to improving the adhesion performance of the entangled-locked unfolded protein-based tissue adhesive.

[0031] The present invention provides a method for preparing an entangled-locked unfolded protein-based tissue adhesive. This method utilizes a disulfide bond reducing agent to disrupt the protein structure, resulting in unfolded proteins with exposed hydrophobic groups. Subsequently, through strong interactions (electrostatic interactions and hydrogen bonding) between a polyelectrolyte and the unfolded protein, the exposed hydrophobic groups are locked, ensuring stable exposure and locking the chain entanglement between the unfolded protein and the polyelectrolyte. The stable exposed hydrophobic groups repel interfacial liquids, forming a strong and stable interfacial bond. The locked chain entanglement improves bulk properties. This entangled-locked unfolded protein-based tissue adhesive possesses stable exposed hydrophobic groups and exhibits advantages such as strong hydrophobicity, strong adhesion, stable adhesion, excellent bulk properties (high storage modulus), good biocompatibility, and good degradability. Furthermore, when applied to bleeding wounds, this entangled-locked unfolded protein-based tissue adhesive produces a hemostatic effect, significantly reducing hemostasis time and wound blood loss, demonstrating excellent hemostatic and sealing effects.

[0032] Please see Figure 2 , Figure 2 This is a schematic diagram illustrating the principle of using the entanglement-locking unfolded protein-based tissue adhesive of the present invention as a medical hemostatic material. Figure 2 A is a schematic diagram of covering wound tissue with the entanglement-locking unfolded protein-based tissue adhesive of the present invention. Figure 2 B is a schematic diagram of the stable exposed hydrophobic groups in the entanglement-locking unfolded protein-based tissue adhesive of the present invention, which repel interfacial liquids (blood). Figure 2 C is a schematic diagram illustrating the adhesion between the adhesive groups and tissue in the entanglement-locking unfolded protein-based tissue adhesive of the present invention, as shown in Figure 1. Figure 2 As shown, when this entangled-locked unfolded protein-based tissue adhesive is applied to wound tissue, the hydrophobic groups exposed on the surface of the unfolded protein can repel interfacial liquid (blood) through hydrophobic interactions, thereby allowing the adhesive groups to adhere to the bleeding wound interface. The entanglement effect, combined with strong interactions, enhances the bulk properties, achieving tissue adhesion and hemostasis.

[0033] In one specific embodiment, the above-mentioned polyelectrolyte solution is prepared by dissolving a polyelectrolyte in a solvent; the polyelectrolyte is at least one selected from polyacrylic acid, polyglutamic acid, polystyrene sulfonic acid, polyethyleneimine, and polyallyl; the solvent is a conventional type, such as deionized water, phosphate buffer solution (PBS solution), etc.

[0034] In this invention, when the above-mentioned substance is used as a polyelectrolyte, it has strong interactions (electrostatic interactions and hydrogen bonds) with the unfolding protein. It can not only lock the exposed hydrophobic groups in the unfolding protein through strong interactions to stabilize the exposure of the hydrophobic groups and enhance the adhesion performance of the entangled-locked unfolding protein-based tissue adhesive, but also lock the entanglement between the unfolding protein chain and the polyelectrolyte chain through strong interactions to improve the bulk properties of the entangled-locked unfolding protein-based tissue adhesive.

[0035] In one specific embodiment, the weight-average molecular weight of the polyelectrolyte is 2 kDa to 240 kDa, preferably 100 kDa to 240 kDa.

[0036] When the weight-average molecular weight of the polyelectrolyte is within the above range, the polyelectrolyte can better entangle with the unfolded protein chains and lock the exposed hydrophobic groups on their surface, thereby achieving the effects of improved bulk properties and stable exposure of hydrophobic groups.

[0037] In one specific embodiment, the protein solution is prepared by dissolving a protein in a solvent; the protein is any one of bovine serum albumin, hemoglobin, lactoferrin, transferrin, β-lactoglobulin, ovalbumin, α-lactalbumin, insulin, lysozyme, and trypsin; the solvent is a conventional type, such as deionized water, phosphate buffer solution (PBS solution), etc.

[0038] In one specific embodiment, the disulfide bond reducing agent solution is prepared by dissolving a disulfide bond reducing agent in a solvent; the disulfide bond reducing agent is at least one of tris(2-carboxyethyl)phosphine, dithiothreitol, 2-mercaptoethanol, glutathione, and cysteine; the solvent is a conventional type, such as deionized water, phosphate buffer solution (PBS solution), etc.

[0039] In this invention, when the above-mentioned substance is used as a disulfide bond reducing agent, the disulfide bonds of the protein will be cleaved to destroy the protein structure. After the disulfide bonds are cleaved by the disulfide bond reducing agent, the protein exhibits temporary exposure of hydrophobic groups and disordered unfolding of the protein chain, resulting in unfolded protein.

[0040] In one specific embodiment, the concentration of the disulfide bond reducing agent solution is 1 mmol / L to 100 mmol / L, preferably 20 mmol / L to 80 mmol / L.

[0041] When the concentration of the disulfide bond reducing agent solution is within the above range, it can effectively cleave the disulfide bonds of proteins.

[0042] In one specific embodiment, the above-mentioned incubation reaction includes: incubating at room temperature for 10 min to 30 min.

[0043] When an incubation reaction is carried out under the above conditions, the disulfide bonds that stabilize the three-dimensional structure of the protein can be effectively broken, resulting in protein unfolding and temporary exposure of hydrophobic groups.

[0044] In one specific embodiment, the above-mentioned rapid stirring reaction includes: conducting a rapid stirring reaction at room temperature for 5 s to 30 s.

[0045] When a rapid stirring reaction is carried out under the above conditions, the polyelectrolyte and the unfolded protein chains form a stable unfolded protein gel.

[0046] A second aspect of the present invention provides an entangled-locked unfolded protein-based tissue adhesive, prepared by the method described above. This entangled-locked unfolded protein-based tissue adhesive has stably exposed hydrophobic groups and possesses advantages such as strong hydrophobicity, strong adhesion, stable adhesion performance, excellent bulk properties (high storage modulus), good biocompatibility, and good degradability. Furthermore, when applied to bleeding wounds, this entangled-locked unfolded protein-based tissue adhesive produces a hemostatic effect, significantly reducing hemostasis time and wound blood loss, demonstrating excellent hemostatic and sealing effects.

[0047] A third aspect of the present invention provides the application of the described entangled-locked unfolded protein-based tissue adhesive in the preparation of medical hemostatic materials. The inventors' research shows that when this entangled-locked unfolded protein-based tissue adhesive is applied to bleeding wounds, it produces a hemostatic effect, significantly reducing hemostasis time and wound blood loss, exhibiting excellent hemostatic and sealing effects. Therefore, it can be used in the preparation of medical hemostatic materials.

[0048] The present invention will be further described below through specific embodiments.

[0049] In the following examples, bovine serum albumin was purchased from Beyotime (CAS No. 9048-46-8); tris(2-carboxyethyl)phosphine was purchased from Aladdin (CAS No. 51805-45-9); and polyacrylic acid was purchased from Bailingwei Technology (CAS No. 9003-01-4).

[0050] Example 1 The method for preparing the entangled-locking unfolded protein-based tissue adhesive provided in this embodiment includes the following steps: (1) Bovine serum albumin was dissolved in deionized water to prepare a protein solution with an initial concentration of 40 w / v%; tris(2-carboxyethyl)phosphine was dissolved in deionized water to prepare a disulfide bond reducing agent solution with an initial concentration of 50 mmol / L; polyacrylic acid with a weight average molecular weight of 240 kDa was dissolved in deionized water to prepare a polyelectrolyte solution with an initial concentration of 18 w / v%.

[0051] (2) Mix 0.25 mL of the above disulfide bond reducing agent solution with 0.25 mL of protein solution and incubate at room temperature for 20 min to obtain a solution containing unfolded protein; (3) Mix 0.5 mL of the above solution containing spreading protein with 0.5 mL of polyelectrolyte solution and stir rapidly at room temperature for 8 s to obtain entangled-locked spreading protein-based tissue adhesive (hereinafter referred to as spreading protein-based tissue adhesive); in the entangled-locked spreading protein-based tissue adhesive, the concentration of spreading protein (BSA) is 10 w / v and the concentration of polyacrylic acid (PAA) is 9 w / v.

[0052] Example 2 (the concentration of the developed protein was 5 w / v%) The preparation method of the entangled-locked unfolded protein-based tissue adhesive provided in this embodiment is basically the same as that in Example 1, except that: Replace the protein solution with an initial concentration of 40 w / v% with a protein solution with an initial concentration of 20 w / v%.

[0053] An entangled-locked unfolded protein-based tissue adhesive was prepared using the protein solution of this embodiment; in the entangled-locked unfolded protein-based tissue adhesive, the concentration of unfolded protein was 5 w / v and the concentration of polyacrylic acid was 9 w / v.

[0054] Example 3 (the concentration of the protein was 7.5 w / v%) The preparation method of the entangled-locked unfolded protein-based tissue adhesive provided in this embodiment is basically the same as that in Example 1, except that: Replace the protein solution with an initial concentration of 40 w / v% with a protein solution with an initial concentration of 30 w / v%.

[0055] An entangled-locked unfolding protein-based tissue adhesive was prepared using the protein solution of this embodiment; in the entangled-locked unfolding protein-based tissue adhesive, the concentration of unfolding protein was 7.5 w / v and the concentration of polyacrylic acid was 9 w / v.

[0056] Example 4 (the concentration of the protein was 12.5 w / v%) The preparation method of the entangled-locked unfolded protein-based tissue adhesive provided in this embodiment is basically the same as that in Example 1, except that: Replace the protein solution with an initial concentration of 40 w / v% with a protein solution with an initial concentration of 50 w / v%.

[0057] An entangled-locked unfolded protein-based tissue adhesive was prepared using the protein solution of this embodiment; in the entangled-locked unfolded protein-based tissue adhesive, the concentration of unfolded protein was 12.5 w / v and the concentration of polyacrylic acid was 9 w / v.

[0058] Example 5 (the concentration of the protein was 15 w / v%) The preparation method of the entangled-locked unfolded protein-based tissue adhesive provided in this embodiment is basically the same as that in Example 1, except that: Replace the protein solution with an initial concentration of 40 w / v% with a protein solution with an initial concentration of 60 w / v%.

[0059] An entangled-locked unfolding protein-based tissue adhesive was prepared using the protein solution of this embodiment; in the entangled-locked unfolding protein-based tissue adhesive, the concentration of unfolding protein was 15 w / v and the concentration of polyacrylic acid was 9 w / v.

[0060] Example 6 (Polyelectrolyte concentration is 3.6 w / v%) The preparation method of the entangled-locked unfolded protein-based tissue adhesive provided in this embodiment is basically the same as that in Example 1, except that: Replace the polyelectrolyte solution with an initial concentration of 18 w / v% with a polyelectrolyte solution with an initial concentration of 7.2 w / v%.

[0061] An entangled-locked unfolding protein-based tissue adhesive was prepared using the polyelectrolyte solution of this embodiment; in the entangled-locked unfolding protein-based tissue adhesive, the concentration of unfolding protein was 10 w / v and the concentration of polyacrylic acid was 3.6 w / v.

[0062] Example 7 (Polyelectrolyte concentration is 5.4 w / v%) The preparation method of the entangled-locked unfolded protein-based tissue adhesive provided in this embodiment is basically the same as that in Example 1, except that: Replace the polyelectrolyte solution with an initial concentration of 18 w / v% with a polyelectrolyte solution with an initial concentration of 10.8 w / v%.

[0063] An entangled-locked unfolding protein-based tissue adhesive was prepared using the polyelectrolyte solution of this embodiment; in the entangled-locked unfolding protein-based tissue adhesive, the concentration of unfolding protein was 10 w / v and the concentration of polyacrylic acid was 5.4 w / v.

[0064] Example 8 (Polyelectrolyte concentration is 7.2 w / v%) The preparation method of the entangled-locked unfolded protein-based tissue adhesive provided in this embodiment is basically the same as that in Example 1, except that: Replace the polyelectrolyte solution with an initial concentration of 18 w / v% with a polyelectrolyte solution with an initial concentration of 14.4 w / v%.

[0065] An entangled-locked unfolding protein-based tissue adhesive was prepared using the polyelectrolyte solution of this embodiment; in the entangled-locked unfolding protein-based tissue adhesive, the concentration of unfolding protein was 10 w / v and the concentration of polyacrylic acid was 7.2 w / v.

[0066] Example 9 (The concentration of the polyelectrolyte solution is 10.8 w / v%) The preparation method of the entangled-locked unfolded protein-based tissue adhesive provided in this embodiment is basically the same as that in Example 1, except that: Replace the polyelectrolyte solution with an initial concentration of 18 w / v% with a polyelectrolyte solution with an initial concentration of 21.6 w / v%.

[0067] An entangled-locked unfolding protein-based tissue adhesive was prepared using the polyelectrolyte solution of this embodiment; in the entangled-locked unfolding protein-based tissue adhesive, the concentration of unfolding protein was 10 w / v and the concentration of polyacrylic acid was 10.8 w / v.

[0068] Example 10 (The concentration of the polyelectrolyte solution was 12.6 w / v%) The preparation method of the entangled-locked unfolded protein-based tissue adhesive provided in this embodiment is basically the same as that in Example 1, except that: Replace the polyelectrolyte solution with an initial concentration of 18 w / v% with a polyelectrolyte solution with an initial concentration of 25.2 w / v%.

[0069] An entangled-locked unfolding protein-based tissue adhesive was prepared using the polyelectrolyte solution of this embodiment; in the entangled-locked unfolding protein-based tissue adhesive, the concentration of unfolding protein was 10 w / v and the concentration of polyacrylic acid was 12.6 w / v.

[0070] Example 11 (The weight-average molecular weight of polyacrylic acid is 2 kDa) The preparation method of the entangled-locked unfolded protein-based tissue adhesive provided in this embodiment is basically the same as that in Example 1, except that: The weight-average molecular weight of polyacrylic acid is 2 kDa.

[0071] Example 12 (The weight-average molecular weight of polyacrylic acid is 100 kDa) The preparation method of the entangled-locked unfolded protein-based tissue adhesive provided in this embodiment is basically the same as that in Example 1, except that: The weight-average molecular weight of polyacrylic acid is 100 kDa.

[0072] Comparative Example 1 (BSA-PAA) The preparation method of the protein-based adhesive provided in this comparative example includes the following steps: (1) Bovine serum albumin was dissolved in deionized water to prepare a protein solution with an initial concentration of 20 w / v%, and polyacrylic acid with a weight average molecular weight of 240 kDa was dissolved in deionized water to prepare a polyelectrolyte solution with an initial concentration of 18 w / v%.

[0073] (2) Mix 0.5 mL of the above polyelectrolyte solution with 0.5 mL of protein solution and stir rapidly at room temperature for 8 s to obtain a protein-based adhesive; in the protein-based adhesive, the protein concentration is 10 w / v and the polyacrylic acid concentration is 9 w / v.

[0074] Comparative Example 2 (Fibrin glue) This comparative example provides a commercially available organizational adhesive, Fibrin glue.

[0075] Comparative Example 3 (rBSA) The method for preparing the protein-based tissue adhesive provided in this comparative example includes the following steps: (1) Bovine serum albumin was dissolved in deionized water to prepare a protein solution with an initial concentration of 20 w / v%, and tris(2-carboxyethyl)phosphine was dissolved in deionized water to prepare a disulfide bond reducing agent solution with an initial concentration of 50 mmol / L.

[0076] (2) Mix 0.5 mL of the above disulfide bond reducing agent solution with 0.5 mL of protein solution and incubate at room temperature for 20 min to obtain the unfolded protein solution; the concentration of the unfolded protein in the unfolded protein solution is 10 w / v.

[0077] Comparative Example 4 (PAA) This comparative example provides a polyelectrolyte solution with a concentration of 9 w / v%, prepared by dissolving polyacrylic acid with a weight-average molecular weight of 240 kDa in deionized water.

[0078] Performance testing 1. The shear strength of the entangled-locked unfolded protein-based tissue adhesive in Examples 1-12 of the present invention, the protein-based adhesive in Comparative Example 1, the commercial tissue adhesive Fibrin glue in Comparative Example 2, the protein-based tissue adhesive in Comparative Example 3, and the polyelectrolyte solution in Comparative Example 4 were tested on glass slides, and the test results are summarized in Table 1 and Table 2. Figure 3 The results of shear strength tests performed on glass slides for each embodiment and comparative example of the present invention are shown below. Figure 3 A represents the shear strength of the entangled-locked unfolded protein-based tissue adhesive in Examples 1-5. Figure 3 B represents the shear strength of the entangled-locked unfolded protein-based tissue adhesive in Examples 6-10. Figure 3 C represents the shear strength of the entangled-locked unfolded protein-based tissue adhesive in Examples 11-12. Figure 3 D represents the shear strength of the protein-based adhesive (BSA-PAA) in Comparative Example 1, the commercial tissue adhesive Fibrin glue in Comparative Example 2, the unfolded protein solution (rBSA) in Comparative Example 3, and the polyelectrolyte solution (PAA) in Comparative Example 4.

[0079] Table 1 Shear strength test results

[0080] Table 2 Shear strength test results

[0081] Depend on Figure 3 As shown in Tables 1 and 2, the entangled-locked unfolded protein-based tissue adhesives in Examples 1-12 exhibit strong adhesion, with shear strengths ranging from 55.92 to 124.91 kPa, significantly higher than the shear strength (5.00 kPa) of the commercial tissue adhesive Fibrin glue in Comparative Example 2. Figure 3 As can be seen from the AC results, the optimal feed group obtained by adjusting the concentration and molecular weight of PAA and the concentration of BSA, namely Example 1 (BSA concentration of 10 w / v%, PAA concentration of 9 w / v%, PAA molecular weight of 240 kDa), has sufficiently strong adhesion strength and was used in a series of subsequent experiments. Figure 3As can be seen from D, the shear strength of the entangled-locked unfolded protein-based tissue adhesive in Example 1 is 114.75 kPa, which is significantly higher than the shear strength of the protein-based adhesive in Comparative Example 1 (56.11 kPa), the shear strength of the commercial tissue adhesive Fibrin glue in Comparative Example 2 (5.00 kPa), the shear strength of the unfolded protein solution in Comparative Example 3 (15.92 kPa), and the shear strength of the polyelectrolyte solution in Comparative Example 4 (7.73 kPa). This indicates that the adhesion of the entangled-locked unfolded protein-based tissue adhesive provided by the present invention is significantly improved.

[0082] 2. The affinity of the protein or developing protein for the polyelectrolyte was determined using a Biacore™ 8K assay. The protein solution containing the developing protein from Example 1 or the protein solution from Comparative Example 1 was covalently coupled to the CM5 chip as a ligand. The running buffer was PBS solution, and the flow rate was 30 μl / min. The polyelectrolyte solution was used as the mobile phase, diluted to different concentration gradients and sequentially flowed through the chip. The data were processed and analyzed using the accompanying BIA evaluation 1.1 software. The results are as follows: Figure 4 As shown; Figure 4 The results show the test results of the affinity between proteins and polyelectrolytes in Example 1 of the present invention and the affinity between proteins and polyelectrolytes in Comparative Example 1.

[0083] A higher reciprocal of the equilibrium dissociation constant (KD) indicates a stronger bond. Figure 4 It can be seen that, compared to the equilibrium dissociation constants of proteins and polyelectrolytes in Comparative Example 1 (KD = 1.02 × 10⁻⁶), -7 M), the proteins and polyelectrolytes developed in Example 1 exhibited lower equilibrium dissociation constants (KD = 2.03 × 10⁻⁶). -8 M) indicates that the protein has a higher binding strength with the polyelectrolyte after unfolding.

[0084] 3. The contact angles of the entangled-locked unfolded protein-based tissue adhesive in Example 1 and the protein-based adhesive in Comparative Example 1 on the glass slides were measured using a DSA 100 contact angle meter. The results are as follows: Figure 5 As shown; Figure 5 The contact angle test results are those of the entangled-locked unfolded protein-based tissue adhesive in Example 1 and the protein-based adhesive in Comparative Example 1.

[0085] Depend on Figure 5It can be seen that the contact angle of the entangled-locked unfolding protein-based tissue adhesive in Example 1 is 91.9°, which is significantly higher than the contact angle of the protein-based adhesive in Comparative Example 1 (17.71°). This indicates that the hydrophobic properties of the entangled-locked unfolding protein-based tissue adhesive provided by the present invention are significantly improved. The reason is that the strong interaction between the polyelectrolyte and the unfolding protein locks the exposed hydrophobic groups on the surface of the unfolding protein, so that the hydrophobic groups are stably exposed, thereby improving the hydrophobic properties.

[0086] 4. Rheological tests were performed on the entangled-locked unfolded protein-based tissue adhesive in Example 1 and the protein-based adhesive in Comparative Example 1 using a HAAKE MARS 60 to obtain the storage modulus. The results are as follows: Figure 6 As shown; Figure 6 The storage modulus is the entanglement-locking unfolded protein-based tissue adhesive in Example 1 of the present invention and the protein-based adhesive in Comparative Example 1.

[0087] Depend on Figure 6 It can be seen that the storage modulus of the entangled-locked unfolded protein-based tissue adhesive in Example 1 is 10869.6 Pa, which is significantly higher than the storage modulus of the protein-based adhesive in Comparative Example 1 (5560.35 Pa). This indicates that the bulk properties of the entangled-locked unfolded protein-based tissue adhesive provided by the present invention are significantly improved. The reason is that the strong interaction between polyacrylic acid and protein (electrostatic interaction and hydrogen bonding) can lock the entanglement between the polyacrylic acid chain and the unfolded protein chain (i.e., the chain entanglement between the two), thereby improving the bulk properties of the entangled-locked unfolded protein-based tissue adhesive.

[0088] 5. To further evaluate the adhesive's adhesion performance, three adhesion test methods were used to assess its adhesion performance on wet pigskin. The entanglement-locking unfolded protein-based tissue adhesive from Example 1, the protein-based adhesive from Comparative Example 1, and the commercial tissue adhesive Fibrin glue from Comparative Example 2 were used as adhesive samples for lap shear test, T-peel test, and tensile strength test, as detailed below: (1) Overlap shear test Adhesive samples were adhered to wet pigskin to prepare adhesive samples with an adhesive area of ​​1 cm wide and 1 cm long, and tested according to the standard lap shear test (ASTM F2255-05). All tests were performed within 5 mm min. -1 The strain rate was measured. Shear strength was determined by dividing the maximum force by the bonded area. A glass slide was used as a rigid backing for the tissue. Results are shown in […]. Figure 7 .

[0089] Depend on Figure 7It can be seen that the shear strength of the entangled-locked unfolded protein-based tissue adhesive in Example 1 (63.07 kPa) is significantly higher than that of the protein-based adhesive in Comparative Example 1 (18.43 kPa) and the shear strength of the commercial tissue adhesive Fibrin glue in Comparative Example 2 (16.87 kPa).

[0090] (2) T-type peel test Adhesive samples were adhered to wet pigskin to prepare adhesive samples with an adhesive area of ​​1 cm wide and 4 cm long, and tested according to the standard lap shear test (ASTM F2256-05). All tests were conducted at 50 mm min. -1 The strain rate was measured. Interfacial toughness (for 180° peel tests) was obtained by dividing twice the plateau force by the width of the bonded sample. A glass slide was used as a rigid backing for the tissue. Results are shown in [Figure number missing]. Figure 8 .

[0091] Depend on Figure 8 It can be seen that the interfacial toughness of the entangled-locked unfolded protein-based tissue adhesive in Example 1 is 86.73 Jm. -2 The interfacial toughness of the protein-based adhesive in Comparative Example 1 was significantly higher than that in Comparative Example 1 (20 J / m). -2 The interfacial toughness (24.8 J / m) of the commercial adhesive Fibrin glue in Comparative Example 2 and Comparative Example 2. -2 ).

[0092] (3) Tensile strength test Adhesive samples were adhered to wet pigskin to prepare adhesive samples with an adhesive area of ​​1 cm wide and 1 cm long, and tested according to the standard lap shear test (ASTM F2258-05). All tests were performed within 5 mm min. -1 The tensile test was conducted at a strain rate of [value missing]. Shear strength was determined by dividing the maximum force by the bonded area. PTFE clamps were used to provide grip for the tensile test. Results are shown in [reference missing]. Figure 9 .

[0093] Depend on Figure 9 It can be seen that the tensile strength of the entangled-locked unfolded protein-based tissue adhesive in Example 1 (104.5 kPa) is significantly higher than that of the protein-based adhesive in Comparative Example 1 (16.8 kPa) and the tensile strength of the commercial tissue adhesive Fibrin glue in Comparative Example 2 (21.87 kPa).

[0094] 6. Biocompatibility and biodegradability test In vivo biocompatibility and biodegradability tests were conducted on the entanglement-locking unfolded protein-based tissue adhesive in Example 1 and the commercial tissue adhesive Fibrin glue in Comparative Example 2 using a rat dorsal implantation model.

[0095] Male SLAC rats (SD, 220-250 g) were used in this study. All animal surgeries were approved by the Animal Conservation and Use Committee of the Experimental Animal Center of Zhejiang University.

[0096] Experimental Methods: Adhesive samples were prepared into discs (10 mm in diameter, 2 mm thick) under aseptic conditions. Rats were anesthetized with 2% sodium pentobarbital. During the procedure, the rats' hair was removed, and they were placed on a heating pad. Each adhesive sample was implanted into the subcutaneous space on the rat's back through a 1 cm skin incision. Four adhesive samples were placed in each rat. The skin incision was sutured with absorbable sutures. To assess the biodegradability of the adhesive samples, rats were euthanized at weeks 1, 2, 3, 5, 7, and 8, and the implanted adhesive samples were removed and weighed. To assess the biocompatibility of the adhesive samples, the implanted subcutaneous areas were excised at weeks 1 and 2 and fixed in 4% paraformaldehyde solution for 24 hours for histological and immunofluorescence analysis.

[0097] The entangled-locked unfolded protein-based tissue adhesive from Example 1 was used as the adhesive sample, and its biodegradability was tested according to the above method. The results are as follows: Figure 10 The entanglement-locking unfolding protein-based tissue adhesive from Example 1 and the commercial tissue adhesive Fibrin glue from Comparative Example 2 were used as adhesive samples, and biocompatibility tests were performed according to the above method. The results are as follows: Figure 11 .

[0098] Figure 10 The results show the biodegradability test results of the entangled-locked unfolded protein-based tissue adhesive in Example 1 of this invention. Figure 10 It can be seen that the entangled-locked unfolded protein-based tissue adhesive in Example 1 was basically completely degraded after 8 weeks of implantation, indicating that the entangled-locked unfolded protein-based tissue adhesive in Example 1 has good biodegradability.

[0099] Figure 11 The results show the biocompatibility test results of the entanglement-locking unfolding protein-based tissue adhesive in Example 1 and the commercial tissue adhesive Fibrin glue in Comparative Example 2. Figure 11 A represents the H&E staining results after the adhesive sample was implanted into the back of a rat at 1 and 2 weeks. Figure 11 B represents the degree of inflammation assessed by blinded pathological examination. Figure 11It can be seen that there is no significant difference in the degree of inflammation between the entanglement-locking unfolded protein-based tissue adhesive of Example 1 and the commercial tissue adhesive Fibrin glue of Comparative Example 2, indicating that the entanglement-locking unfolded protein-based tissue adhesive of Example 1 has good biocompatibility.

[0100] 7. The hemostatic and sealing effects of the entangled-locked unfolded protein-based tissue adhesive in Example 1 and the commercial tissue adhesive Fibrin glue in Comparative Example 2 were tested using a rat liver incision model.

[0101] Male SLAC rats (SD, 220-250 g) were used in this study. All animal surgeries were approved by the Animal Conservation and Use Committee of the Experimental Animal Center of Zhejiang University.

[0102] Experimental Methods: Rats were anesthetized with 2% sodium pentobarbital, and abdominal hair was removed to expose the liver through an abdominal incision. Pre-weighed filter paper was placed under the liver. A 10 mm long, 4 mm deep incision was made in the liver with a blade. Hemostatic material was applied to the wound, and the bleeding time was recorded. After 180 seconds, the filter paper was weighed, and the blood loss was calculated. The control group received no treatment.

[0103] The entanglement-locking unfolded protein-based tissue adhesive from Example 1 and the commercial tissue adhesive Fibrin glue from Comparative Example 2 were used as hemostatic materials, and their hemostatic and sealing effects were tested according to the above method. The results are as follows: Figure 12 .

[0104] Figure 12 The results of hemostasis and occlusion tests are as follows: Example 1 shows the entanglement-locking unfolded protein-based tissue adhesive; Comparative Example 2 shows the commercial tissue adhesive Fibrin glue; and the control group shows the results of the tests. Figure 12 Figure A shows the hemostasis time in a rat liver incision bleeding model. Figure 12 Figure B shows the blood loss in a rat liver incision bleeding model.

[0105] Depend on Figure 12 As shown in A, the hemostasis time of the entanglement-locking unfolded protein-based tissue adhesive in Example 1 (9.2 s) was significantly lower than that of the commercial tissue adhesive Fibrin glue in Comparative Example 2 (71.8 s) and the control group (178 s). Figure 12As shown in section B, the blood loss using the entangled-locked unfolded protein-based tissue adhesive in Example 1 was 43.7 mg, significantly lower than that of Comparative Example 1 (900.96 mg) and the blank group (3045.9 mg). These results indicate that when the entangled-locked unfolded protein-based tissue adhesive provided in this invention is applied to bleeding wounds, it produces a hemostatic effect, significantly reducing hemostasis time and wound blood loss, demonstrating excellent hemostatic and sealing effects. This is because: the polyelectrolyte and the unfolded protein generate strong interactions (electrostatic interactions and hydrogen bonds). The hydrophobic groups exposed on the surface of the unfolded protein, locked by these strong interactions, are stably exposed, repelling interfacial liquid (blood), thereby allowing the adhesive groups to form a stable interfacial adhesion with the tissue; the entanglement effect of the strongly interacting and locked polyelectrolyte chains and unfolded protein chains enhances the bulk properties of the adhesive, achieving excellent hemostatic and sealing effects through the synergistic effect of interfacial adhesion and bulk properties.

[0106] In summary, the entangled-locked unfolded protein-based tissue adhesive prepared by the method provided in this invention has stable exposed hydrophobic groups and possesses advantages such as strong hydrophobicity, strong adhesion, stable adhesion performance, excellent bulk properties (high storage modulus), good biocompatibility, and good degradability. When this entangled-locked unfolded protein-based tissue adhesive is applied to bleeding wounds, it produces a hemostatic effect, significantly reducing hemostasis time and wound blood loss, and exhibits excellent hemostatic and sealing effects.

[0107] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing an entanglement-locking unfolding protein-based tissue adhesive, characterized in that, Includes the following steps: A disulfide bond reducing agent solution is added to a protein solution for incubation to obtain a solution containing unfolded protein; wherein, the disulfide bond reducing agent solution is prepared by dissolving a disulfide bond reducing agent in a solvent, and the disulfide bond reducing agent is at least one of tris(2-carboxyethyl)phosphine, dithiothreitol, 2-mercaptoethanol, glutathione, and cysteine. A polyelectrolyte solution is added to the solution containing the unfolding protein and subjected to rapid stirring to obtain the entangled-locked unfolding protein-based tissue adhesive; wherein the polyelectrolyte solution is prepared by dissolving a polyelectrolyte in a solvent, and the polyelectrolyte is at least one of polyacrylic acid, polyglutamic acid, polystyrene sulfonic acid, and polyethyleneimine, and the weight-average molecular weight of the polyelectrolyte is 2kDa to 240kDa. In the entanglement-locking unfolding protein-based tissue adhesive, the concentration of the unfolding protein is 5 w / v% to 15 w / v, and the concentration of the polyelectrolyte is 3 w / v% to 15 w / v%. The protein solution is prepared by dissolving the protein in a solvent; The protein is any one of bovine serum albumin, hemoglobin, lactoferrin, transferrin, β-lactoglobulin, ovalbumin, α-lactalbumin, insulin, lysozyme, and trypsin; The concentration of the disulfide bond reducing agent solution is 1 mmol / L to 100 mmol / L; The incubation reaction includes: incubation at room temperature for 10 min to 30 min; The rapid stirring reaction includes: a rapid stirring reaction at room temperature for 5 to 30 seconds.

2. An entanglement-locking unfolding protein-based tissue adhesive, characterized in that, It is prepared by the preparation method described in claim 1.

3. The application of an entangled-locked unfolded protein-based tissue adhesive prepared by the preparation method of claim 1 or the entangled-locked unfolded protein-based tissue adhesive of claim 2 in the preparation of medical hemostatic materials.

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