A mussel myoglobin tissue adhesive and methods of use and applications thereof

Abstract

The present application relates to a kind of mussel adhesive. The mussel adhesive has two-component system of two-channel chemical crosslinking+enzymatic secondary crosslinking+catechol wet surface adhesion synergy: component A is PEG active ester, component B is recombinant mussel adhesive protein (MAPs) and catechol grafted chitosan (CS-Cat) and transglutaminase (TGase) are matched. After mixing, under the condition of pH≈6.9, first, by active ester-amine rapid amidation, it is realized that setting and interface covalent anchoring, then TGase forms ε-(γ-glutamyl)-lysine / γ-glutamyl-amide secondary crosslinking reinforcement, while MAPs-DOPA / CS-Cat catechol group provides wet surface multi-point adhesion and energy dissipation. The synergistic mechanism faces the comprehensive goal of "fast curing+high strength / pressure resistance+resistance to washing+good compatibility and stability", overcome the key short board of prior art in wet surface application.

Description

Technical Field

[0001] This invention relates to the field of tissue adhesive technology, and in particular to a mussel adhesive protein tissue adhesive and its application and usage. Background Technology

[0002] Medical tissue adhesives have become an important supplement to surgical sutures / staples due to their advantages such as ease of use, minimal trauma, immediate sealing and hemostasis, and reduced operation time. Adhesives typically form bonds with tissue surface functional groups under physiological conditions through reactive groups in the material. Common reactive groups include: NHS / activated carbonates (activated esters), cyanoacrylates, aldehydes, catechols / catechols, isocyanates, and azides (click reaction). In addition to covalent bonds, non-covalent interactions such as hydrogen bonding, hydrophobic interactions, electrostatic interactions, metal coordination, and physical entanglement are also frequently used to improve initial tack and overall bond strength on wet surfaces.

[0003] According to the main components and curing mechanism, existing tissue adhesives can be roughly divided into four categories: (1) Synthetic polymers: such as cyanoacrylate, PEG, polyurethane, polyester, etc., which rely on rapid polymerization or reaction with tissue to achieve curing; (2) Proteins: such as fibrin glue, gelatin, albumin, etc., which rely on coagulation cascade or chemical cross-linking to achieve curing; (3) Polysaccharides: such as chitosan, alginate, dextran, hyaluronic acid, etc., which often achieve curing through ionic / physical or introduction of reactive groups; (4) Biomimetic adhesives: inspired by mussels, geckos, plant polyphenols, etc., which use groups such as catechol to achieve adhesion on wet surfaces.

[0004] Among them, the technical route most similar to this invention is a two-component system composed of PEG active esters (such as PEG-NHS / PEG-SC) and proteins (commonly albumin, gelatin, collagen, etc.): one side is the active ester, and the other side is the amine-containing protein. After mixing, it is amidated to form a gel network. Some schemes are supplemented with physical thickening or surface activity to improve wet surface compatibility. A few other studies have explored the use of catechol-modified polymers or transglutaminase (TGase) to enhance wet surface adhesion or secondary cross-linking, but most of them are single-channel modifications, and there is still a trade-off between gelation speed, wet surface strength, washability and storage stability.

[0005] Although the above systems have been applied to some extent, the following common problems still exist: (1) Insufficient wet surface compatibility and unstable initial adhesion: Most PEG-protein or polysaccharide systems mainly rely on single chemical cross-linking. Under continuous wetting and rinsing conditions of blood / body fluid, the interfacial anchoring force is insufficient, and adhesion failure or sudden drop in strength is likely to occur; (2) Difficulty in balancing gelation speed and operating window: Although cyanoacrylate and other materials cure quickly, their brittleness, heat release and irritation limit their application in deep or large wet surfaces; fibrin / gelatin types cure slowly and have low strength, limiting intraoperative efficiency; (3) Strength and rinsing resistance are constrained by the density of cross-linking points: In order to obtain usable strength, PEG active ester-protein often requires a high solid content (e.g., ≥10~15% w / v), but this will increase viscosity, reduce injectability, and NHS / SC end groups are easily hydrolyzed and deactivated in aqueous phase, resulting in insufficient effective cross-linking points and increased batch-to-batch differences; (4) A single mechanism cannot simultaneously satisfy "sealing, pressure resistance and rinsing resistance": relying solely on Chemically cross-linked bulk networks often lack sufficient sealing / burst pressure; polysaccharide systems relying solely on physical / ionic interactions lack sufficient mechanical strength and stability; (5) trade-off between biosafety and tissue compatibility: aldehydes / isocyanates are highly reactive, but have a high risk of potential tissue irritation / cytotoxicity and residual reaction byproducts; the brittleness of cyanoacrylates and possible tissue irritation reactions also limit their long-term application in certain scenarios; (6) shelf life and on-site availability issues: aqueous solutions containing active esters have poor storage stability and require cold chain or fresh preparation, making it difficult to balance long-term stability and rapid intraoperative use; catechol materials are prone to self-oxidation and discoloration and activity decay in the presence of metal ions / oxygen, affecting product performance; (7) insufficient synergistic curing design: most publicly available solutions are single-channel (either chemical cross-linking, biomimetic adhesion, or enzymatic), lacking a systematic design that simultaneously optimizes the three aspects of "rapid shaping + secondary reinforcement + wet surface anchoring" and precisely coordinates A / B components and pH programming.

[0006] Existing tissue adhesives generally suffer from problems such as insufficient wet surface compatibility, difficulty in balancing gelation speed and operability, insufficient strength and wash resistance, and trade-offs between biosafety and storage stability. For example: (1) When relying solely on single chemical crosslinking or physical action, the interfacial anchoring force is insufficient under blood / body fluid flushing, which easily leads to adhesion failure; (2) High solids content is often required to obtain usable strength, resulting in injection difficulties / narrow operating window; active esters are easily hydrolyzed and deactivated in aqueous phase, and catechols are easily self-oxidized and discolored, affecting shelf-life and shelf-life performance; (3) Some systems (such as cyanoacrylate, aldehyde / isocyanate crosslinking) may cause tissue irritation or residual reaction byproducts; (4) Naturally derived proteins have batch-to-batch differences and potential pathogen introduction risks, and are still insufficient in terms of wet surface strength and pressure sealing. Summary of the Invention

[0007] Therefore, it is necessary to address the shortcomings of existing technologies by providing a mussel adhesive tissue system. This adhesive features a two-component system with synergistic effects of dual-channel chemical crosslinking, enzymatic secondary crosslinking, and catechol-based wet-surface adhesion: Component A is a PEG-active ester (PEG-SS), and Component B is recombinant mussel adhesive proteins (MAPs) and catechol-grafted chitosan (CS-Cat) combined with transglutaminase (TGase). When using this adhesive, components A and B are mixed and, under pH ≈ 6.9, rapid amidation of the active ester with an amine achieves shaping and interfacial covalent anchoring. Subsequently, TGase forms ε-(γ-glutamyl)-lysine / γ-glutamyl-amine secondary crosslinking reinforcement. Simultaneously, the catechol groups of MAPs-DOPA / CS-Cat provide multi-point adhesion and energy dissipation on the wet surface. This synergistic mechanism aims to achieve a comprehensive goal of "rapid curing + high strength / pressure resistance + washability + good compatibility and stability," overcoming the key shortcomings of existing technologies in wet-surface applications.

[0008] This invention provides a mussel adhesive tissue binder, wherein the raw materials for preparing the mussel adhesive tissue binder include component A and component B: component A includes polyethylene glycol active ester, and component B includes recombinant mussel adhesive protein, catechol-grafted chitosan, and transglutaminase; the recombinant mussel adhesive protein contains 3,4-dihydroxyphenylalanine (DOPA).

[0009] In the preparation of the raw materials, the ratio of polyethylene glycol active ester, recombinant mussel adhesive protein, catechol-grafted chitosan and transglutaminase is (100~200) mg: (10~40) mg: (3~15) mg: (10~50) U.

[0010] In one embodiment, the polyethylene glycol active ester includes linear polyethylene glycol active ester or multi-arm polyethylene glycol active ester, wherein the degree of deacetylation of the catechol-grafted chitosan is 80-95%, and the degree of substitution of catechol is 5-15 mol.

[0011] In one embodiment, the catechol-grafted chitosan can be dissolved into a salt form to increase solubility, the salt form including free amine, lactate, hydrochloride or carboxymethyl chitosan-Cat.

[0012] In one embodiment, the degree of deacetylation of the catechol-grafted chitosan is 90%, and the degree of substitution (DS) of catechol is 8-12 mol.

[0013] In one embodiment, the polyethylene glycol active ester has a molecular weight of 5-40 kDa, and the catechol-grafted chitosan has a molecular weight of 50-200 kDa.

[0014] In one embodiment, the polyethylene glycol active ester comprises a four-arm polyethylene glycol active ester with a molecular weight of 10-20 kDa.

[0015] In one embodiment, the polyethylene glycol active ester comprises a four-arm polyethylene glycol active ester with a molecular weight of 10 kDa.

[0016] In one embodiment, the catechol-grafted chitosan has a molecular weight of 100 kDa.

[0017] In one embodiment, component A further includes a buffer and a lyophilization protectant; the buffer of component A includes 2-morpholinoethanesulfonic acid (MES), and the lyophilization protectant of component A includes trehalose and mannitol; component B further includes sodium ascorbate, disodium ethylenediaminetetraacetate (EDTA-Na2), a buffer and a lyophilization protectant; the buffer of component B includes 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES) and sodium chloride, and the lyophilization protectant of component B includes trehalose and mannitol.

[0018] In one embodiment, the polyethylene glycol active ester end group includes N-hydroxythiosuccinimide (NHS) or succinic carbonate (SC), which are interchangeable in linear or multi-arm form, and whose molecular weight can be equivalently adjusted in the range of 5 to 40 kDa to match viscosity / strength.

[0019] In one embodiment, the catechol-grafted chitosan (CS-Cat) may be replaced with carboxymethyl chitosan-Cat.

[0020] In one embodiment, the catechol amino polymeric group provided includes at least one of recombinant mussel adhesive protein or catechol-grafted chitosan.

[0021] In one embodiment, the ascorbic acid may be replaced with glutathione, or used in combination with glutathione, wherein the concentration of glutathione is 1-2 mM.

[0022] In one embodiment, the disodium ethylenediaminetetraacetate can be replaced with pentasodium diethylenetriaminepentaacetate (DTPA·5Na), wherein the concentration of the pentasodium diethylenetriaminepentaacetate is 0.05~0.2mM.

[0023] In one embodiment, the 4-hydroxyethylpiperazine ethanesulfonic acid may be replaced with phosphate-buffered saline (PBS) or tris(hydroxymethyl)aminomethane (Tris).

[0024] In one embodiment, the mass of trehalose, the freeze-drying protectant in component A, is 50-100 mg, and the mass of mannitol is 20-50 mg; the mass of trehalose, the freeze-drying protectant in component B, is 50-100 mg, and the mass of mannitol is 20-50 mg.

[0025] The present invention also provides a method for using the above-mentioned mussel adhesive protein tissue binder, comprising the following steps:

[0026] Dissolving component A: Take component A raw material, dissolve it, mix it evenly, and obtain a solution of component A;

[0027] Dissolving component B: Take the raw material of component B, dissolve it, mix it evenly, and obtain a solution of component B;

[0028] Mixing components A and B: Mix the solutions of components A and B evenly, apply to the areas to be bonded, and allow to cure.

[0029] In one embodiment, the A component solution contains polyethylene glycol active ester at a mass concentration of 100-200 mg / mL; the B component solution contains recombinant mussel adhesive protein at a mass concentration of 10-40 mg / mL, catechol-grafted chitosan at a mass concentration of 3-15 mg / mL, transglutaminase at a concentration of 10-50 U / mL, sodium ascorbate at a concentration of 1-3 mM, and disodium ethylenediaminetetraacetate at a concentration of 0.1-0.25 mM.

[0030] In one embodiment, the A component solution contains polyethylene glycol active ester at a mass concentration of 120-180 mg / mL; the B component solution contains recombinant mussel adhesive protein at a mass concentration of 20-30 mg / mL, catechol-grafted chitosan at a mass concentration of 4-8 mg / mL, transglutaminase at a concentration of 30-40 U / mL, sodium ascorbate at a concentration of 2 mM, and disodium ethylenediaminetetraacetate at a concentration of 0.20 mM.

[0031] In one embodiment, the A component solution contains polyethylene glycol active ester at a mass concentration of 150 mg / mL, the B component solution contains recombinant mussel adhesive protein at a mass concentration of 12.5 mg / mL, catechol-grafted chitosan at a mass concentration of 4 mg / mL, and transglutaminase at a concentration of 17.5 U / mL.

[0032] In one embodiment, the pH of the A component buffer after dissolution is 5-6; the pH of the B component buffer after dissolution is 7-8; and the pH of the mixture of components A and B is 6.5-7.5.

[0033] In one embodiment, the concentration of component A buffer after dissolution is 50-100 mM; the concentration of 4-hydroxyethylpiperazine ethanesulfonic acid after dissolution of component B buffer is 20-50 mM, and the concentration of sodium chloride is 120-160 mM.

[0034] In one embodiment, the sodium chloride concentration of the B component buffer after dissolution is 150 mM.

[0035] In one embodiment, the pH of the A component buffer after dissolution is 5.3-5.5; the pH of the B component buffer after dissolution is 7.2-7.6; the pH of the mixture of components A and B is 6.7-7.1; and the ambient temperature is 36-38°C.

[0036] In one embodiment, the pH of the A component buffer after dissolution is 5.4; the pH of the B component buffer after dissolution is 7.3~7.5; the pH of the mixture of components A and B is 6.9; and the ambient temperature for use is 37°C.

[0037] In one embodiment, the pH of the B component buffer after dissolution is 7.4.

[0038] In one embodiment, in the mixing step of components A and B, the volume ratio of component A solution to component B solution is 1:1; the mixing method is static mixing with a dual syringe.

[0039] In one embodiment, the A component solution obtained after dissolving the A component is 2 mL; the B component solution obtained after dissolving the B component is 2 mL.

[0040] In addition, the present invention also provides the use of mussel adhesive tissue adhesive as described above, or the mussel adhesive tissue adhesive used in the above method of use, in the preparation of medical tissue adhesives.

[0041] Compared with the prior art, the present invention has the following beneficial effects: (1) fast curing and easy operation; (2) high-strength adhesion to wet surfaces; (3) excellent sealing and pressure resistance; (4) strong resistance to rinsing and structural retention; (5) dimensional stability in body fluids; (6) hemostasis and healing promotion; (7) good biosafety; (8) advanced mechanism and clear synergistic effect; (9) stable dosage form and clinically friendly; (10) simplified system and strong scalability. Detailed Implementation

[0042] To facilitate understanding of the present invention, a more complete description will be given below with reference to relevant embodiments. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0043] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0044] Source: Unless otherwise specified, all reagents, materials and equipment used in this embodiment are commercially available; unless otherwise specified, all experimental methods are conventional experimental methods in this field.

[0045] Example

[0046] I. Composition and Administration

[0047] The mussel adhesive tissue binder of this invention consists of two components: Component A is polyethylene glycol active ester (PEG-SS); Component B consists of recombinant mussel adhesive proteins (MAPs), catechol-grafted chitosan (CS-Cat), and transglutaminase (TGase), supplemented with sodium ascorbate and EDTA-Na2. Both components are lyophilized separately in sterile vials. Before use, each component is reconstituted with its corresponding rehydration solution. 2 mL of each component is simultaneously extruded and mixed using a double syringe and a static mixer in equal volumes before use.

[0048] II. Matching Reconstitution Solution

[0049] (1) SA (for bottle A): The concentration of MES buffer is 50~100mM, pH=5.4;

[0050] (2) SB (for bottle B): The concentration of HEPES buffer is 20~50mM, and it also contains NaCl with a concentration of 150mM and pH=7.4.

[0051] The pH of the system after mixing solutions A and B is approximately 6.9 (37℃).

[0052] III. Preparation of Components A / B (lyophilized powder)

[0053] The present invention also provides a method for preparing the freeze-dried powders of components A and B, comprising the following steps:

[0054] (1) Component A (PEG-SS lyophilized powder): Weigh the target amount of four-arm PEG-SS (molecular weight 10000) and dissolve it in SA; add 50~100mg / bottle of trehalose and 20~50mg / bottle of mannitol as lyophilization protectant; adjust the pH to 5.3~5.5. Dispense (each bottle is reconstituted to a target volume of 2mL), pre-freeze at -40℃, perform gradient sublimation drying, then stopper and seal, and store in the dark at 2~8℃.

[0055] (2) Component B (MAPs / CS-Cat / TGase lyophilized powder): Weigh out MAPs, CS-Cat and TGase and dissolve them in SB; add sodium ascorbate (final concentration 2mM) and EDTA-Na2 (final concentration 0.20mM); similarly add trehalose 50~100mg / bottle and mannitol 20~50mg / bottle; adjust pH to 7.3~7.5. Dispense, pre-freeze and lyophilize under the same conditions as component A, and store in the dark at 2~8℃.

[0056] IV. Usage Steps

[0057] (1) Add 2 mL of the corresponding reconstitution solution (SA / SB) to each of bottles A and B respectively, and shake gently until completely dissolved;

[0058] (2) Load the dual syringe and connect the static mixer;

[0059] (3) Apply adhesive evenly to the wound edge or the area that needs to be sealed, press lightly with 1~2N to fix, initial curing in 90~120s, and further increase strength in 5~15min;

[0060] (4) During the procedure, physiological saline can be used to gently rinse and verify adhesion stability.

[0061] Among them, after sizing, the acidity of the A side formed by component A helps to inhibit the hydrolysis of active esters, ensuring the reactivity and shelf life at the time of use; the near-neutral B side formed by component B maintains the nucleophilicity of amines and TGase activity; CS-Cat provides "amine + catechol" bifunctional sites to enhance wet surface initial tack and bulk crosslinking.

[0062] All the following examples are based on the principle that "each bottle has a reconstitution volume of 2 mL and the volume ratio of component A to component B is 1:1".

[0063] Example 1 (Standard Type)

[0064] (1) Component A: Four-arm PEG-SS (molecular weight 10000), concentration after dissolution is 300mg / 2mL (SA, pH=5.4; contains trehalose / mannitol).

[0065] (2) Component B: MAPs, with a concentration of 25 mg / 2 mL after dissolution; CS-Cat, with a concentration of 8 mg / 2 mL after dissolution (degree of deacetylation ≈ 90%, molecular weight ≈ 100 kDa, degree of catechol substitution ≈ 10%); TGase, with a concentration of 35 U / 2 mL after dissolution, and containing sodium ascorbate (concentration of 2 mM) and EDTA-Na2 (concentration of 0.20 mM) (SB, pH=7.4; containing trehalose / mannitol).

[0066] (3) Application: Mix components A and B in equal volumes and apply to the target area quickly. Apply pressure of 1~2N and wait for curing to complete.

[0067] Example 2 (Enhanced Catechol / Primary Viscosity)

[0068] (1) Component A: PEG-SS, the concentration after dissolution is 300mg / 2 mL (SA, pH=5.4).

[0069] (2) Component B: MAPs, with a concentration of 25 mg / 2 mL after dissolution, CS-Cat, with a concentration of 12 mg / 2 mL after dissolution, TGase, with a concentration of 35 U / 2 mL after dissolution, and containing sodium ascorbate (concentration of 2 mM) and EDTA-Na2 (concentration of 0.20 mM) (SB, pH=7.4).

[0070] (3) Use the same method as in Example 1.

[0071] Example 3 (High Strength / Fast)

[0072] (1) Component A: PEG-SS, the concentration after dissolution is 360mg / 2mL (SA, pH=5.4).

[0073] (2) Component B: MAPs, with a concentration of 30 mg / 2 mL after dissolution, CS-Cat, with a concentration of 10 mg / 2 mL after dissolution, TGase, with a concentration of 40 U / 2 mL after dissolution, and containing sodium ascorbate (concentration of 2 mM) and EDTA-Na2 (concentration of 0.20 mM) (SB, pH=7.4).

[0074] (3) Use the same method as in Example 1.

[0075] Example 4 (Longer operating window / More flexible)

[0076] (1) Component A: PEG-SS, the concentration after dissolution is 240mg / 2mL (SA, pH=5.4).

[0077] (2) Component B: MAPs, with a concentration of 20 mg / 2 mL after dissolution, CS-Cat, with a concentration of 14 mg / 2 mL after dissolution, TGase, with a concentration of 30 U / 2 mL after dissolution, and containing sodium ascorbate (concentration of 2 mM) and EDTA-Na2 (concentration of 0.20 mM) (SB, pH=7.4).

[0078] (3) Use the same method as in Example 1.

[0079] Comparative Example 1 (without TGase)

[0080] (1) Component A: PEG-SS, the concentration after dissolution is 300mg / 2mL (SA, pH=5.4).

[0081] (2) Component B: MAPs, with a concentration of 25 mg / 2 mL after dissolution, and CS-Cat, with a concentration of 8 mg / 2 mL after dissolution, without adding TGase; the rest is the same as in Example 1 (SB, pH=7.4).

[0082] (3) Use the same method as in Example 1.

[0083] Comparative Example 2 (Chitosan without Catechol grafting replaces CS-Cat)

[0084] (1) Component A: Same as in Example 1.

[0085] (2) Component B: MAPs, with a concentration of 25 mg / 2 mL after dissolution; CS-Cat and TGase are replaced by chitosan without grafting catechol (with a concentration of 8 mg / 2 mL after dissolution), with a concentration of 35 U / 2 mL after dissolution; the rest is the same as in Example 1.

[0086] (3) Use the same method as in Example 1.

[0087] Comparative Example 3 (No MAPs, only CS-Cat involved)

[0088] (1) Component A: Same as in Example 1.

[0089] (2) Component B: without MAPs, retaining CS-Cat (concentration of 12mg / 2mL after dissolution) and TGase (concentration of 35U / 2mL after dissolution), and containing sodium ascorbate (concentration of 2mM) and EDTA-Na2 (concentration of 0.20mM) (SB, pH=7.4).

[0090] (3) Use the same method as in Example 2.

[0091] Comparative Example 4 (Low Solids Content)

[0092] (1) Component A: PEG-SS, the concentration after dissolution is 200mg / 2mL (SA, pH=5.4).

[0093] (2) Component B: MAPs, with a concentration of 10 mg / 2 mL after dissolution, CS-Cat, with a concentration of 6 mg / 2 mL after dissolution, TGase, with a concentration of 20 U / 2 mL after dissolution, and containing sodium ascorbate (concentration of 2 mM) and EDTA-Na2 (concentration of 0.20 mM) (SB, pH=7.4).

[0094] (3) Use the same method as in Example 1.

[0095] Performance testing

[0096] 1. Gel time characterization

[0097] Test method: Component A (four-arm PEG-SS, MES buffer pH=5.4) and component B (MAPs+CS-Cat+TGase, HEPES buffer pH=7.4) were reconstituted according to the proportions described in the embodiment. After mixing in equal volumes using a dual syringe and a static mixer, the mixture was rapidly added dropwise to the stage of a rotational rheometer (37°C). The curves of G′ / G″ versus time were recorded at a fixed frequency / strain. The gel point was defined as the intersection of the storage modulus G′ and the loss modulus G″, and the corresponding time was the gel time (n=5).

[0098] Table 1. Gel Time

[0099]

[0100] Results Explanation:

[0101] The gelation times of Examples 1-3 were significantly shorter than those of the comparative examples, demonstrating the initial curing advantage brought by rapid amidation of PEG-SS and pH programming; Example 3 (higher PEG-SS concentration, TGase concentration of 40 U / 2 mL) was the fastest. Example 4 (lower PEG-SS concentration, higher CS-Cat concentration) had a correspondingly longer operating window. Comparative Example 1 (no TGase) and Comparative Example 2 (chitosan without catechol grafting replaced CS-Cat) both showed delayed gelation, indicating that enzymatic secondary network formation and the effective amine / catechol sites provided by CS-Cat can promote early network construction; Comparative Example 3 (no MAPs, only CS-Cat involved) also showed slower gelation due to the lack of Gln / Lys corresponding sites at the protein ends; Comparative Example 4 (low solids content) was the slowest, consistent with the expectation of insufficient reactive site density.

[0102] 2. Overlap-shear tensile bearing strength

[0103] Test method: The test was conducted according to YY / T0729.2-2009 Test Methods for Adhesive Bonding Performance of Tissue Adhesives Part 1: Overlap-Shear Tensile Bearing Strength. Fresh pigskin was cut to standard dimensions and moistened with PBS. Component A (four-arm PEG-SS, MES buffer pH=5.4) and component B (MAPs+CS-Cat+TGase, HEPES buffer pH=7.4) were reconstituted according to the implementation method. Equal volumes were mixed using a dual-syringe and a static mixer, and the mixture was evenly applied to the overlap surface. The sample was then fixed with 1-2 N of pressure until curing was complete. The test strip was clamped with the force direction aligned with the long axis and loaded at a speed of 5 mm / min until failure. The failure load was recorded and the bearing strength was calculated (n=6).

[0104] Table 2. Lap-shear tensile bearing strength

[0105]

[0106] Results Explanation:

[0107] (1) Examples 1-4 were significantly better than the comparative examples, which was due to the rapid amidation of PEG-SS and MAPs / CS-Cat amines superimposed with TGase secondary crosslinking, and the wet surface thickening and interface anchoring provided by catechols (MAPs-DOPA and CS-Cat).

[0108] (2) Example 3 showed the highest activity, demonstrating the synergistic enhancement of a higher ester / amine ratio and TGase (40U / 2mL).

[0109] (3) Comparative Example 1 (without TGase) showed a significant decrease in strength due to the lack of isopeptide / γ-glutamyl-amine secondary network; Comparative Example 2 (chitosan without catechol grafting replaced CS-Cat) had limited interfacial adhesion and bulk cross-linking due to the lack of catechol sites and the reduced density of effective amine sites; Comparative Example 3 (without MAPs, only CS-Cat involved) lacked protein-side Gln / Lys supply, limiting TGase cross-linking and resulting in reduced strength; Comparative Example 4 (low solids content) had the lowest strength due to insufficient cross-linking point density.

[0110] 3. T-Peel Strength

[0111] Test Method: Take two fresh tissue samples (width denoted as W), and set an overlap length of 25 mm. Redissolve component A (four-arm PEG-SS, MES buffer pH=5.4) and component B (MAPs+CS-Cat+TGase, HEPES buffer pH=7.4) separately according to the implementation method. Mix them in equal volumes using a dual-syringe and a static mixer, and then evenly apply the mixture to the overlap area (wet adhesive amount approximately 0.15~0.20 g). Apply pressure of 1~2 N to fix the sample until curing (≥10 min). Clamp both ends of the sample to the upper and lower clamps respectively, set the peel angle to 180° (or 90°), and the beam speed to 50 mm / min. Peel continuously for ≥50 mm and record the force-displacement curve. Take the average peel force F (N) in the stable section and calculate the peel strength P=F / W (N / m); simultaneously record the failure mode (adhesion / cohesion / substrate).

[0112] Failure Mode and Effects Indicators:

[0113] Adhesion failure: occurs at the interface between the adhesive layer and the substrate; there is almost no adhesive residue on one side of the surface.

[0114] Cohesive failure: occurs inside the adhesive layer; residual adhesive / stringing is present on both sides of the surface.

[0115] Substrate failure: Occurs within the tissue itself; the adhesive layer remains attached while the tissue is torn.

[0116] Mixed failure: Two or more modes coexist in the same sample (reported percentage %).

[0117] Table 3. Evaluation of peeling and failure modes

[0118]

[0119] Results Explanation:

[0120] Compared with the comparative examples, the peel strength of Examples 1-4 was generally higher (approximately 320~410 N / m; Example 3 showed the highest), and the failure mode changed from mainly adhesive to mainly cohesive with a small amount of substrate failure, indicating a more stable interfacial bond and colloidal network. Removing TGase (Comparative Example 1) significantly reduced the strength; replacing CS-Cat with ungrafted catechol chitosan (Comparative Example 2) resulted in limited interfacial adhesion and bulk crosslinking due to the lack of catechol sites and a decrease in effective amine density; without MAPs and relying solely on CS-Cat (Comparative Example 3), the lack of enzymatic crosslinking sites provided by Gln / Lys on the protein side led to a decline in strength; low solids content (Comparative Example 4) performed the worst due to insufficient crosslinking point density. The results indicate that the present invention, relying on rapid amidation of PEG-SS + secondary crosslinking with TGase and superimposed catechol wet surface adhesion of MAPs and CS-Cat, can significantly improve wet surface peel resistance and interfacial stability.

[0121] 4. Wound closure strength (wound edge closure)

[0122] Test Method: Porcine small intestinal mesentery was taken and cut into 20mm × 60mm strips. A standard tear was created by cutting a 20mm incision along the midline of the strip. Component A (four-arm PEG-SS, MES buffer pH=5.4) and component B (MAPs+CS-Cat+TGase, HEPES buffer pH=7.4) were reconstituted according to the implementation method. After equal-volume mixing using a dual-syringe and a static mixer, the mixture was applied evenly along the tear edge (coating width 5-8mm), and fixed with 1-2N pressure, curing for 10-15 minutes. The two ends of the sample were clamped in the universal testing machine fixture, ensuring the tear was located in the center of the fixture. The sample was stretched at a beam speed of 5mm / min until failure, and the maximum load F_max (N) and displacement (mm) were recorded. The closure strength σ was calculated based on the adhesive width: σ = F_max / adhesive width (kPa). The failure mode (adhesion / cohesion / substrate or mixing) was also recorded.

[0123] Table 4 Wound Closure Strength

[0124]

[0125] Results Explanation:

[0126] The closure strength and fracture displacement of Examples 1-4 were significantly higher than those of the comparative examples (approximately 85-120 kPa vs 30-52 kPa; 3.0-3.2 mm vs 1.7-2.2 mm), and the failure mode changed from adhesion in the comparative examples to cohesion as the main factor. Example 3 even showed cohesion → substrate failure, indicating that the interface was stronger than the tissue bulk. Removing TGase (Comparative Example 1) significantly reduced strength; replacing CS-Cat with chitosan without grafted catechol (Comparative Example 2) resulted in limited interface anchoring and bulk crosslinking due to the lack of catechol sites and the decrease in effective amine density; the absence of MAPs and only CS-Cat participation (Comparative Example 3) resulted in a decrease in strength due to the lack of enzymatic crosslinking sites provided by Gln / Lys on the protein side; low solids content (Comparative Example 4) resulted in the worst performance due to insufficient crosslinking point density. In summary, the synergistic mechanism of "rapid crosslinking of active esters + secondary crosslinking of TGase + wet adhesion of catechol" of the present invention can significantly improve the wound edge bearing capacity and toughness.

[0127] 5. Leakage / burst pressure resistance (sealing capability)

[0128] Experimental Method: Isolated porcine small intestine was taken and expanded to prepare 2 mm diameter circular holes. Component A (four-arm PEG-SS, MES buffer pH=5.4) and component B (MAPs+CS-Cat+TGase, HEPES buffer pH=7.4) were reconstituted according to the prescribed method. They were mixed in equal volumes using a dual-syringe syringe and a static mixer, and then uniformly applied to the perforated surface and surrounding area (covering a diameter of approximately 12-15 mm). The mixture was allowed to cure for 10-15 min. A constant flow pump and a pressure sensor were connected, and the pressure was increased at a rate of 10 mmHg / s. The initial leakage pressure and burst pressure (n≥5) were recorded.

[0129] Table 5 Leakage Pressure and Burst Pressure

[0130]

[0131] Results Explanation:

[0132] In the isolated small intestine model, the leakage initiation pressure and burst pressure of Examples 1-4 were significantly higher than those of the comparative examples (leakage approximately 110-160 mmHg, burst pressure approximately 185-250 mmHg; controls were 50-80 / 85-120 mmHg), with Example 3 showing the highest (160±16 / 250±20 mmHg), exhibiting the best sealing performance and pressure resistance limit. Removal of TGase (Comparative Example 1) significantly reduced both indicators; replacing CS-Cat with ungrafted catechol chitosan (Comparative Example 2) resulted in insufficient wet surface anchoring and bulk cross-linking due to the lack of catechol sites and reduced effective amine density; the absence of MAPs and the participation of only CS-Cat (Comparative Example 3) resulted in a lack of protein-side Gln / Lys supply to form an enzymatic cross-linking network, leading to decreased pressure resistance; and low solids content (Comparative Example 4) resulted in the worst performance due to insufficient cross-linking point density. The results show that the present invention relies on rapid film formation and sealing of PEG-SS, secondary covalent reinforcement of TGase, and the wet surface adhesion of MAPs and CS-Cat to catechol, which can significantly improve the anti-leakage and pressure resistance of perforations in cavity tissues.

[0133] 6. Rinse-resistant retention rate

[0134] Experimental Methods: Following the "overlap-shear" sample preparation procedure, component A (four-arm PEG-SS, MES buffer pH=5.4) and component B (MAPs+CS-Cat+TGase, HEPES buffer pH=7.4) were reconstituted separately, mixed in equal volumes using a dual-syringe, coated, and cured. The shear strength before rinsing was measured first; then, the sample was placed in a 37℃ constant-temperature flow bath and rinsed for 10 min under circulating water. The shear strength after rinsing was then measured again. Retention rate was calculated as follows: Retention rate R = (Strength after rinsing / Strength before rinsing) × 100%.

[0135] Table 6. Overlap Tensile Strength and Retention Rate Before and After Washing

[0136]

[0137] Results Explanation:

[0138] Examples 1-4 maintained high overlap strength and stability after rinsing, with a retention rate of approximately 83-93% (Example 3 showed the best retention rate of 93% from 0.30 to 0.28 MPa), significantly better than the 50-64% of the comparative examples (and with lower initial strength). Removing TGase (Comparative Example 1) significantly reduced the retention rate; replacing CS-Cat with ungrafted catechol chitosan (Comparative Example 2) weakened rinsing resistance due to the lack of catechol sites and decreased effective amine density; CS-Cat without MAPs (Comparative Example 3) lacked protein-side Gln / Lys supply, resulting in insufficient secondary crosslinking; low solids content (Comparative Example 4) was the worst due to insufficient crosslinking point density. The results indicate that the present invention, relying on rapid amidation of PEG-SS + secondary covalent reinforcement with TGase and superimposed catechol wet-surface adhesion of MAPs / CS-Cat, can effectively resist rinsing dilution and shear erosion, maintaining wet-surface adhesion and structural integrity.

[0139] 7. Swelling rate in body fluids

[0140] Experimental Method: Following the steps of this invention, equal volumes of component A (four-arm PEG-SS, MES buffer pH=5.4) and component B (MAPs+CS-Cat+TGase, HEPES buffer pH=7.4) were mixed. After standing at room temperature for 30 minutes until complete gelation, circular slices with a diameter of 10 mm and a thickness of 2 mm were cut and the initial mass m0 was recorded. The slices were then placed in physiological saline and gently shaken at 37°C for 48 hours. After removing the slices and wiping off the surface moisture, the wet mass mt was measured. The swelling rate was calculated as follows: S = (mt – m0) / m0 × 100%;

[0141] Table 7. Swelling rate of each component in physiological saline

[0142]

[0143] Results Explanation:

[0144] The swelling rates of Examples 1-4 at 48 h were significantly lower than those of the comparative examples, with Example 3 showing the lowest (120±10%). This indicates that rapid amidation of PEG-SS combined with secondary cross-linking by TGase, along with the superposition of multiple sites provided by MAPs / CS-Cat, forms a denser network, reduces water absorption and swelling, and improves dimensional and mechanical stability. The absence of TGase (Comparative Example 1) significantly increased swelling due to the lack of isopeptide / γ-glutamyl-amine secondary cross-linking. Replacing CS-Cat with ungrafted catechol chitosan (Comparative Example 2) resulted in decreased network density due to the absence of catechol sites and reduced effective amine density. The absence of MAPs and the participation of only CS-Cat (Comparative Example 3) limited enzymatic cross-linking due to insufficient Gln / Lys supply on the protein side. The low-solids content (Comparative Example 4) resulted in the lowest cross-linking site density and the largest swelling.

[0145] 8. Blood compatibility and hemostatic properties

[0146] Hemolysis test method: Anticoagulated whole blood (sodium citrate) from the same donor was used to prepare a 2% red blood cell suspension (prepared after washing three times with 0.9% NaCl); the colloid that had been completely solidified at 37℃ was then subjected to a surface area / volume ratio of 3 cm³. 2 Extract the sample with 0.9% NaCl for 24 hours (and prepare a material blank for background subtraction). Mix 0.5 mL of the extract with 0.5 mL of erythrocyte suspension and incubate at 37°C for 60 min. Centrifuge (1000 g, 5 min) and measure the absorbance of the supernatant at 540 nm. Hemolysis rate = (OD test tube - OD negative control tube) / (OD positive control tube - OD negative control tube)%

[0147] The negative control was physiological saline, and the positive control was pure water.

[0148] Table 8 Hemolysis rate in each group

[0149]

[0150] Results Explanation:

[0151] All groups showed hemolysis rates ≤5% of the threshold, indicating good blood compatibility. The overall hemolysis rate of the Example groups was lower than that of the Comparative Groups, with Example 3 showing the lowest rate. Comparative Groups 2 and 4 showed slightly higher rates but still met the requirements, suggesting that the interaction between the extract and the erythrocyte membrane was more pronounced when chitosan was not grafted or the solid content was insufficient. Under neutral buffering, fully solidified, and low soluble matter precipitation conditions, the present invention showed good overall blood compatibility.

[0152] Whole blood coagulation index (WBCI) assay method: Place sample discs pre-warmed to 37℃ at the bottom of a transparent plate (wash 3 times with PBS to remove surface free liquid). Add 200 μL of sodium citrate-anticoagulated whole blood and 20 μL of 0.2M CaCl2 to each well to initiate recalcification. After incubation at the preset time points (5 / 10 / 20 min), quickly add 2.0 mL of deionized water and gently shake for 3 min to dissolve red blood cells not embedded in the clot. Take the supernatant and measure the absorbance at 540 nm. Calculate WBCI (%) based on the blank wells (no sample surface). The lower the value, the more complete the coagulation at that time point (less residual soluble hemoglobin).

[0153] Table 9 Whole blood coagulation index of each group

[0154]

[0155] Results Explanation:

[0156] The WBCI values ​​of Examples 1-4 at all time points (5 / 10 / 20 min) were significantly lower than those of the comparative example (p<0.05), with Example 3 showing the lowest value, and approaching the "fully solidified" plateau at 10-20 min. The reason is:

[0157] 1. Rapid amidation of PEG-SS forms a dense barrier and fixes the wound edges;

[0158] 2. The secondary cross-linking of TGase further densifies the colloidal network within minutes, improving its resistance to percolation and shearing.

[0159] 3. The catechol groups of MAPs and CS-Cat are beneficial for adsorbing fibrinogen / plasma proteins and promoting platelet adhesion and embolism stability, thereby accelerating clot formation and erythrocyte encapsulation.

[0160] In the absence of TGase (Comparative Example 1) or when chitosan without catechol was used to replace CS-Cat (Comparative Example 2), the secondary cross-linking and catechol interfacial interaction were insufficient, resulting in a significantly higher WBCI. In the absence of MAPs and with only CS-Cat involved (Comparative Example 3), the lack of enzymatic cross-linking sites provided by Gln / Lys on the protein side led to decreased clot density. The lowest solids content (Comparative Example 4) was the worst due to insufficient cross-linking point density. These results are consistent with the trends in closure strength, leak / burst resistance, and washout retention, further validating the synergistic hemostasis and sealing mechanism of this invention: "rapid setting of active ester + TGase reinforcement + wet-surface anchoring of catechol."

[0161] 9. In vitro cytotoxicity test

[0162] Test method: According to GB / T16886.5-2017 Biological evaluation of medical devices - Part 5: In vitro cytotoxicity test. After the colloid was prepared and completely cured according to the implementation method, it was extracted with culture medium for 24 hours according to the standard method; the extract was then used to treat L929 cells for 24 hours, and the absorbance was detected by CCK-8 assay and the viability was calculated. Normal cultured cells were used as controls; if the viability was <70%, it was considered to have potential cytotoxicity (n=3).

[0163] Table 10 Potential Cytotoxicity

[0164]

[0165] Results Explanation:

[0166] The cell survival rate of each group was ≥90%, with no potential cytotoxicity, proving that the system of the present invention has good cell compatibility.

[0167] 10. Irritation and Skin Sensitization Test

[0168] Test Method: Following GB / T16886.10-2017 Biological Evaluation of Medical Devices Part 10: Irritation and Skin Sensitization Tests. Healthy New Zealand rabbits were selected; a 10×15cm area on the back was shaved as the contact and observation site. A / B colloids were prepared according to the implementation method, mixed in equal volumes, and applied to a 2.5×2.5cm area until completely gelled; after 4 hours of contact, the gel was removed with a damp gauze. Skin reactions were recorded and scored at 1, 24, 48, and 72 hours.

[0169] Table 11 Skin Reaction Scores and Rabbit Primary Irritation Index Types

[0170]

[0171] Results Explanation:

[0172] All examples and comparative examples showed no skin sensitization reactions, indicating good biosafety.

[0173] 11. Skin damage healing experiment

[0174] Test method: Healthy SD rats were selected, and a 4×6cm area was shaved on the back as the observation area. After disinfection with povidone-iodine, a 2cm full-thickness skin incision was prepared using a scalpel. Component A (four-arm PEG-SS, MES buffer pH=5.4) and component B (MAPs+CS-Cat+TGase, HEPES buffer pH=7.4) were reconstituted separately according to the implementation method. After mixing in equal volumes using a dual syringe, the mixture was applied to the center and perimeter of the incision in a 2.5×0.5cm area until complete gelation. The time for complete wound closure was recorded.

[0175] Table 12 Evaluation of the effect of adhesive on wound healing time in rats

[0176]

[0177] Results Explanation:

[0178] The healing time of Examples 1-4 was generally shorter than that of the comparative examples (6-7 days vs. 9-11 days), with Example 3 being the fastest. The advantage stemmed from the rapid amidation and shaping of PEG-SS and the secondary covalent reinforcement of TGase, combined with the catechol wet-surface adhesion of MAPs and CS-Cat, which allowed for rapid and stable closure, reduced leakage and micromovement, thus promoting coagulation / fibrin network formation and early epithelialization. Without TGase (Comparative Example 1), the later-stage strength was insufficient and closure maintenance was poor; replacing CS-Cat with ungrafted catechol chitosan (Comparative Example 2) resulted in the loss of catechol sites and a decrease in effective amine density, weakening both interfacial anchoring and bulk cross-linking; without MAPs and with only CS-Cat involved (Comparative Example 3), the stability was insufficient due to the lack of enzymatic cross-linking sites for Gln / Lys on the protein side; low solids content (Comparative Example 4) resulted in the slowest healing due to insufficient cross-linking point density. The overall results were consistent with the aforementioned trends of improved closure strength, leakage / burst resistance, and flushing resistance.

[0179] This invention provides a two-component medical tissue adhesive: component A is polyethylene glycol active ester (PEG-SS), and component B is recombinant mussel adhesive protein (MAPs) + catechol-grafted chitosan (CS-Cat) + transglutaminase (TGase). The optimal formulation is as follows: Component A consists of four-arm PEG-SS (molecular weight 10000) at a concentration of 150 mg / 2 mL, reconstituted with MES (concentration 50 mM, pH 5.3~5.5); Component B consists of MAPs (concentration 25 mg / 2 mL), CS-Cat (concentration 8 mg / 2 mL, degree of deacetylation ≈90%, molecular weight ≈100 kDa, degree of catechol substitution ≈10%), TGase (concentration 35 U / 2 mL), and contains sodium ascorbate (concentration 2 mM) and EDTA-Na2 (concentration 0.20 mM), reconstituted with HEPES (concentration 20~50 mM) + NaCl (concentration 150 mM), and pH 7.3~7.5; 2 mL of each of components A and B are dispensed using a static mixer via a dual-syringe before use, and the pH of the mixed system is approximately 6.9. Under these conditions, a hierarchical network of "rapid setting + slow reinforcement + interfacial anchoring" is formed: Within 1-3 minutes after mixing, PEG-SS in component A rapidly amidates the lysine ε-NH2 and CS-Cat main chain primary amine of MAPs in component B, establishing an initial covalent network in the bulk phase and achieving interfacial covalent anchoring with histamine / thiol groups (explaining the initial solidification of 90-120 s); subsequently, within 5-15 minutes, TGase uses glutamine (Gln) of MAPs as an acyl donor, reacting with the lysine ε-NH2 of MAPs... The primary amines of 2 or CS-Cat generate two types of secondary covalent crosslinks: ε-(γ-glutamyl)-lysine (protein-protein) and γ-glutamyl-amine (protein-polysaccharide), which significantly improve cohesive strength and wash resistance. At the same time, DOPA of MAPs and catechol of CS-Cat provide multi-point hydrogen bonds / hydrophobic-π–π / coordination and other non-covalent adhesions on the wet surface. After being mildly oxidized to ortho-quinone, they undergo Schiff base / Michael addition and catechol-catechin coupling with histamine / thiol groups, further enhancing interfacial anchoring and energy dissipation. The formulation contains ascorbic acid / EDTA to inhibit catechol autoxidation and metal-induced side reactions, ensuring activity and color stability during storage and use. The four-arm PEG-SS (molecular weight of 10kDa, mass concentration of 150mg / 2mL) achieves a balance between reaction rate, crosslinking density and injection operability. MAPs (mass concentration of 25mg / 2mL) provide bifunctional sites of Gln / Lys and DOPA. CS-Cat (mass concentration of 8mg / 2mL) contains both primary amines and catechols to compensate for wet surface initial adhesion and coating under conditions without rheology modifiers. TGase (concentration of 35U / 2mL) covers the 5-15min reinforcement window during the procedure.The aforementioned synergistic mechanism enables the present invention to achieve both rapid gelation and high strength / wash resistance (e.g., gelation / bonding time 90~120s, overlap strength 0.20~0.25MPa, closure strength ≥90kPa, wash retention rate ≥80%, etc.) under physiological wet conditions. Furthermore, its rationality and feasibility can be verified by comparing it with comparative examples of TGase-free, ungrafted chitosan, and low solids content, as well as by SDS-PAGE / fluorescent receptor verification.

[0180] In summary, in view of the common problems of insufficient wet surface compatibility, difficulty in balancing gelation speed and operability, insufficient strength and rinsing resistance, and trade-offs between biosafety and storage stability of existing tissue adhesives, the problems solved by this invention include: (1) achieving rapid curing and stable anchoring in a physiological wet surface environment: achieving initial curing in 90~120s and maintaining interfacial bonding and shape stability under intraoperative rinsing / infiltration conditions; (2) obtaining high wet surface adhesion and load-bearing capacity: improving overlap-shear strength, T-peel strength, wound edge closure strength and resistance to leakage / burst pressure; (3) improving rinsing resistance and body fluid stability: maintaining high resistance under continuous rinsing and long-term immersion. (4) Ensure biosafety and compatibility: meet medical requirements in evaluations of hemolysis, cytotoxicity, irritation / sensitization, etc., and avoid risks from strong irritant / potentially toxic groups; (5) Improve storage and on-site use stability: by using A / B double-bottle freeze-drying + pH programming to balance the preservation of active esters and the antioxidant properties of catechols, it achieves ready-to-use and controllable operation window; (6) Reduce the risk of raw material sources and improve consistency: use recombinant mussel adhesive proteins (MAPs) obtained by fermentation to replace natural extraction, reduce immunogenicity and pathogen-related risks, and at the same time use CS-Cat to provide controllable amine / catechol site density and rheological support.

[0181] The double cross-linked tissue adhesive provided by this invention can be used for closure and sealing of soft tissues on the body surface / internal cavity, hemostasis, leakage prevention / burst prevention; rapid sealing of perforations in hollow organs or blood vessels; tissue adhesion and wound edge apposition in wet environments. It also has the following advantages:

[0182] 1. Fast curing and easy operation: After mixing A and B in equal volumes, the initial curing is completed in 90~120s (80~165s in the example), and it can be shaped by light pressure of 1~2N, which meets the operational requirements of rapid leakage stop / closure during operation.

[0183] 2. High-strength adhesion on wet surfaces: Overlap-shear strength 0.24~0.30MPa, T-peel strength 320~410N / m. The failure mode changed from the adhesion failure of the control to cohesion-based failure with a small amount of substrate failure. The interfacial bonding and colloidal network are more stable.

[0184] 3. Excellent sealing and pressure resistance: In the isolated small intestine perforation model, the leakage initiation pressure is 110~160 mmHg and the burst pressure is 185~250 mmHg, which is significantly better than the control group (50~80 / 85~120 mmHg), making it suitable for sealing hollow organs / vascular wounds.

[0185] 4. Strong resistance to rinsing and structural retention: After rinsing with 37°C running water for 10 minutes, the strength retention rate is 83~93% (compared to 50~64% in the control group), which can effectively resist surgical field rinsing and body fluid shearing.

[0186] 5. Size stability in body fluids: The swelling rate of physiological saline after 48 hours was 120-180% (Example), which was significantly lower than that of the control (170-224%), indicating that the cross-linked network was denser and had better size and mechanical stability.

[0187] 6. Hemostasis and healing promotion: The whole blood coagulation index (WBCI) was significantly lower than that of the control at all time points of 5 / 10 / 20 min, indicating that a dense clot was formed more quickly; the healing time of the 2cm skin incision model in rats was 6-7 days (9-11 days in the control), which showed better early closure maintenance and tissue healing promotion.

[0188] 7. Good biocompatibility: hemolysis rate ≤5%, no cytotoxicity, no irritation / sensitization in vitro; buffer and ionic strength close to physiological conditions, system-friendly.

[0189] 8. Advanced mechanism and clear synergy: The three-channel synergy of active ester-amine fast cross-linking (PEG-SS) + enzymatic secondary cross-linking (TGase forming ε-(γ-glutamyl)-lysine / γ-glutamyl-amine) + catechol wet surface adhesion (MAPs-DOPA and CS-Cat) ensures rapid shaping and provides post-reinforcement and interface anchoring.

[0190] 9. Stable dosage form and clinically friendly: The A / B dual-bottle lyophilized solution with differentiated pH reconstitution (Component A: MES buffer pH=5.3~5.5; Component B: HEPES buffer pH=7.3~7.5; pH≈6.9 after mixing) balances storage stability, operating window and reaction efficiency; ascorbic acid / EDTA inhibits uncontrolled oxidation of catechols and metal-induced side reactions.

[0191] 10. Simplified system and strong scalability: The above performance can still be achieved by relying on the "amine + catechol" dual function of CS-Cat without using tannic acid and γ-PGA; the PEG active ester end group, CS-Cat salt type / substitution degree, molecular weight and concentration can all be adjusted within a reasonable range, and it has the potential for large-scale and scenario-specific adaptation.

[0192] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0193] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A mussel myoglobin tissue adhesive, characterized in that, The preparation raw materials include A component and B component: the A component includes polyethylene glycol active ester, the B component includes recombinant Mytilus edulis mucin, catechol grafted chitosan and transglutaminase; The recombinant Mytilus edulis mucin contains 3,4-dihydroxyphenylalanine; In the preparation raw materials, the use amount ratio of polyethylene glycol active ester, recombinant Mytilus edulis mucin, catechol grafted chitosan and transglutaminase is (100-200) mg:(10-40) mg:(3-15) mg:(10-50) U; The polyethylene glycol active ester includes linear polyethylene glycol active ester or multi-arm polyethylene glycol active ester, and the deacetylation degree of the catechol grafted chitosan is 80-95%, and the catechol substitution degree is 5-15 mol%.

2. The mussel adhesive protein tissue adhesive according to claim 1, characterized in that, The molecular weight of the polyethylene glycol active ester is 5-40 kDa, and the molecular weight of the catechol grafted chitosan is 50-200 kDa.

3. The mussel-mucin tissue adhesive according to any one of claims 1-2, characterized in that, The A component further includes a buffer and a freeze-drying protective agent; the buffer of the A component includes 2-morpholinoethanesulfonic acid, and the freeze-drying protective agent of the A component includes trehalose and mannitol; The B component further includes sodium ascorbate, ethylenediaminetetraacetic acid disodium salt, a buffer and a freeze-drying protective agent; the buffer of the B component includes 4-hydroxyethylpiperazine ethanesulfonic acid and sodium chloride, and the freeze-drying protective agent of the B component includes trehalose and mannitol.

4. The method of claim 1, wherein the mussel adhesive protein tissue adhesive is prepared by the steps of: The method comprises the following steps: A component dissolution: taking the A component raw material, dissolving and uniformly mixing to obtain an A component solution; B component dissolution: taking the B component raw material, dissolving and uniformly mixing to obtain a B component solution; A and B component mixing: uniformly mixing the A component solution and the B component solution to obtain the Mytilus edulis mucin tissue adhesive.

5. The preparation method according to claim 4, characterized in that, In the A component solution, the mass concentration of the polyethylene glycol active ester is 100-200 mg / mL; In the B component solution, the mass concentration of the recombinant Mytilus edulis mucin is 10-40 mg / mL, the mass concentration of the catechol grafted chitosan is 3-15 mg / mL, the concentration of the transglutaminase is 10-50 U / mL, the concentration of the sodium ascorbate is 1-3 mM, and the concentration of the ethylenediaminetetraacetic acid disodium salt is 0.1-0.25 mM.

6. The preparation method according to claim 4, characterized in that, In the A component solution, the mass concentration of the polyethylene glycol active ester is 120-180 mg / mL; In the B component solution, the mass concentration of the recombinant Mytilus edulis mucin is 20-30 mg / mL, the mass concentration of the catechol grafted chitosan is 4-8 mg / mL, the concentration of the transglutaminase is 30-40 U / mL, the concentration of the sodium ascorbate is 2 mM, and the concentration of the ethylenediaminetetraacetic acid disodium salt is 0.20 mM.

7. The preparation method according to claim 4, characterized in that, The pH of the A component buffer after dissolution is 5-6, the pH of the B component buffer after dissolution is 7-8, and the pH of the mixture of the A component and the B component is 6.5-7.

5.

8. The preparation method according to claim 4, characterized in that, In the A and B component mixing step, the volume ratio of the A component solution to the B component solution is 1:

1.

9. Use of the Mytilus edulis mucin tissue adhesive according to claim 1 in the preparation of a medical tissue adhesive.

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