A dual crosslinking tissue adhesive and methods of use and applications thereof

Abstract

The present application relates to a kind of double crosslinking tissue adhesive.The adhesive can still achieve fast gelation and high adhesion to wet surface under physiological conditions, and has the scheme of operability, storage stability and low residue;Through double-channel curing (chemical crosslinking+enzymatic crosslinking), introducing auxiliary amine group donor (such as amino hyaluronic acid) and network regulator (such as gamma-polyglutamic acid), and cooperating with A / B split + differential pH reconstitution and freeze-dried dosage form, to realize comprehensive promotion between initial adhesion of second level and solidification of minute level, wet surface washing resistance and final strength / tenacity.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of tissue adhesive, in particular to a double cross-linked tissue adhesive and its use method and application. BACKGROUND

[0002] Medical tissue adhesives have become an important supplement and even a replacement for surgical suturing and stapling due to their advantages such as easy operation, small trauma, immediate sealing and hemostasis, and shortening of operation time. The mechanism of action usually relies on the stable combination of reactive groups in the material and the functional groups on the tissue surface under physiological conditions; common reactive groups include NHS ester / active carbonate (including PEG-NHS, PEG-SC, etc.), cyanoacrylate, aldehyde group, catechol / ortho-phenol, isocyanate, azide, etc. In addition to covalent bonds, physical entanglement, hydrogen bonding, hydrophobic and electrostatic interactions, and other non-covalent interactions are also used to synergistically improve the wet surface initial adhesion and overall bonding strength; on the instrument side, double liquid compatibility and on-site mixing (such as double syringes + static mixers) are also common drug delivery and curing methods in the prior art.

[0003] Existing medical tissue adhesives are mainly divided into four categories: (1) synthetic polymers (such as cyanoacrylate, PEG-based, polyurethane, polyester, etc.); (2) proteins (such as fibrin glue, gelatin, albumin); (3) polysaccharides (such as chitosan, alginate, dextran, hyaluronic acid); (4) biomimetic adhesion (such as systems inspired by mussels, geckos, spiders, or plants). Different routes have their own advantages in terms of curing speed, wet surface adaptability, mechanical strength, biological safety, operability, and storage stability. Public data shows that double liquid systems based on PEG active ester and protein are relatively common, and commercial proteins are mainly albumin or fibrin; while the productization of PEG active ester and mussel adhesive protein (MAPs) is relatively limited, and related schemes are still at the exploration stage of scientific research or patents.

[0004] Although tissue adhesives based on polyethylene glycol (PEG) active esters and proteins have achieved some commercialization, the available public information and commercial information show that there is still a lack of mature commercialization scheme for products compounded with PEG active esters and mussel adhesive proteins (MAPs). The existing commercial products mostly use conventional proteins such as albumin and fibrin, which have rich amino sites, wide sources and low cost, but still have the following common deficiencies in wet surface adhesion, rapid solidification and storage stability: (1) PEG active ester hydrolysis inactivation: under the condition of close to physiological pH and water phase, the active ester is easy to be hydrolyzed, which leads to the shortening of shelf life and the narrowing of on-site operation window; in order to make up for the reaction rate, it is often necessary to increase the solid content; (2) high solid content requirement brings operability decline: when albumin / gelatin is used as the reaction substrate, due to the limitation of the density of reactive lysine and the effective multifunctionality, if the glue is formed in minutes and enough strength is obtained, the solid content of 10-20% (w / v) is usually required, which causes high viscosity, coating and injection difficulty; (3) insufficient anti-interference of wet surface and flushing environment: the system of single chemical condensation or single physical action is easy to be diluted or interrupted under bleeding / flushing, which leads to insufficient initial adhesion, shear / closure strength decline; (4) MAPs stability challenge: although mussel adhesive proteins have innate wet adhesion, the catechol (DOPA) group is sensitive to oxygen and trace metals, and is easy to be oxidized to quinone, which leads to activity decline / color change; if there is no antioxidant and metal chelation strategy, it is difficult to realize stable formulation; (5) poor storage and activation mode: lacking of A / B component split and pH differential reconstitution as well as systematic design of freeze-dried dosage form, it is difficult to balance long-term stability and clinical ready-to-use; (6) single mechanism difficult to balance strength and toughness: the network obtained by active ester chemical crosslinking is usually high in brittleness; and only relying on non-covalent interaction is insufficient in durability, which is difficult to balance between wet surface initial adhesion-structure solidification-long-term stability.

[0005] Therefore, the existing tissue adhesives have the following problems to be solved: 1. insufficient wet surface rapid adhesion: on the surface of wet or slightly bleeding tissue, it is difficult to obtain second-level initial adhesion and minute-level solidification at physiological pH; 2. harsh chemical condensation conditions and potential irritation: when relying on acidic condensation or active chemical system (such as traditional EDC / NHS on-site condensation), the environment is high, and the residual and irritation risk is difficult to balance; it is necessary to complete the crosslinking under the condition close to neutral; 3. single mechanism is interfered by dilution, and it is difficult to balance strength and toughness: only relying on single covalent or non-covalent interaction, it is easy to be weakened by body fluid flushing / dilution, and it is difficult to simultaneously improve shear strength and ductility; 4. insufficient operation and adaptability: the existing system has narrow operation window, and the viscosity / gel time is not adjustable, which is difficult to adapt to different parts and operation pace; it is necessary to mix A / B double liquid on site and adjust the gel time. SUMMARY

[0006] Therefore, it is necessary to provide a double cross-linking tissue adhesive based on the deficiencies of the prior art, which can achieve rapid gelation and high adhesion to wet surface under physiological conditions, and has operability, storage stability and low residue. Through double-channel curing (chemical cross-linking + enzymatic cross-linking), the introduction of auxiliary amine group donors (such as amino hyaluronic acid) and network regulators (such as gamma-polyglutamic acid), and the combination of A / B split + differential pH reconstitution and freeze-dried dosage form, the comprehensive improvement between initial adhesion in seconds and solidification in minutes, wet surface washing resistance and final strength / tenacity is achieved.

[0007] The application provides a double cross-linking tissue adhesive, which is prepared from raw materials including A component and B component.

[0008] In the raw materials, the polyethylene glycol active ester, the recombinant mussel adhesive protein, the amino hyaluronic acid, the gamma-polyglutamic acid, the transglutaminase, the sodium ascorbate and the ethylenediaminetetraacetic acid disodium salt are used in a ratio of (100-200) mg:(10-40) mg:(5.5-15) mg:(5.5-15) mg:(10-50) U:(1-3) mol:(0.1-0.25) mol.

[0009] In one of the embodiments, the polyethylene glycol active ester includes linear polyethylene glycol active ester or multi-arm polyethylene glycol active ester, and the amino substitution degree of the amino hyaluronic acid is 1.0-2.0%.

[0010] In one of the embodiments, the polyethylene glycol active ester includes at least one of four-arm polyethylene glycol succinimidyl succinate (PEG-SS), four-arm polyethylene glycol succinimidyl glutarate, double-end polyethylene glycol succinimidyl succinate and double-end polyethylene glycol succinimidyl glutarate.

[0011] In one of the embodiments, the recombinant mussel adhesive protein can be replaced by recombinant human collagen or recombinant human fibronectin.

[0012] In one of the embodiments, the polyethylene glycol active ester is four-arm polyethylene glycol active ester with a molecular weight of 10-20 kDa, the amino hyaluronic acid has a molecular weight of 50-300 kDa, and the gamma-polyglutamic acid has a molecular weight of 300-1500 kDa.

[0013] In one embodiment, the polyethylene glycol active ester is a four-arm polyethylene glycol active ester with a molecular weight of 20 kDa; the amino-modified hyaluronic acid has a molecular weight of 100 kDa; and the γ-polyglutamic acid has a molecular weight of 500-1000 kDa.

[0014] In one embodiment, the γ-polyglutamic acid has a molecular weight of 700 kDa.

[0015] In one embodiment, the buffer of component A includes 2-morpholinoethanesulfonic acid (MES), the lyophilization protectant of component A includes trehalose and mannitol, the buffer of component B includes 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES) or phosphate buffered saline (PBS), and the lyophilization protectant of component B includes trehalose and mannitol.

[0016] In one embodiment, the buffer of component B also contains sodium chloride.

[0017] In one embodiment, the concentration of MES after dissolving the A component buffer is 50-100 mM; the concentration of HEPES after dissolving the B component buffer is 20-50 mM, and the concentration of sodium chloride is 150 mM.

[0018] 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.

[0019] In one embodiment, the buffer mass of component A is 5-15 mg.

[0020] The present invention also provides a method for using the above-mentioned tissue adhesive, characterized by comprising the following steps:

[0021] Dissolving component A: Dissolve component A buffer, add it to the remaining raw material of component A, and mix well to obtain component A solution;

[0022] Dissolving component B: Dissolve the buffer of component B, add it to the remaining raw material of component B, and mix well to obtain a solution of component B;

[0023] Mixing components A and B: Mix the solutions of components A and B evenly, apply them to the areas to be bonded, and allow to cure.

[0024] In one of the embodiments, the mass concentration of polyethylene glycol active ester in the A component solution is 100-200 mg / mL; the mass concentration of recombinant mussel myoglobin in the B component solution is 10-40 mg / mL, the mass concentration of aminated hyaluronic acid is 5.5-15 mg / mL, the mass concentration of gamma-polyglutamic acid is 5.5-15 mg / mL, the concentration of transglutaminase is 10-50 U / mL, the concentration of sodium ascorbate is 1-3 mM, and the concentration of ethylenediaminetetraacetic acid disodium salt is 0.1-0.25 mM.

[0025] In one of the embodiments, the mass concentration of polyethylene glycol active ester in the A component solution is 120-180 mg / mL; the mass concentration of recombinant mussel myoglobin in the B component solution is 20-30 mg / mL, and the concentration of transglutaminase is 30-40 U / mL.

[0026] In one of the embodiments, the mass concentration of polyethylene glycol active ester in the A component solution is 150 mg / mL.

[0027] In one of the embodiments, the pH of the A component buffer after dissolution is 4.5-6.5; the pH of the B component buffer after dissolution is 6.5-8.5, and the pH after mixing of the A and B components is 6.0-7.8.

[0028] In one of the embodiments, the pH of the A component buffer after dissolution is 5.0-5.6; the pH of the B component buffer after dissolution is 7.2-7.6, and the ionic strength is 0.10-0.20 M; the pH after mixing of the A and B components is 6.7-7.1, and the use environment temperature is 36-38℃.

[0029] In one of the embodiments, the pH of the A component buffer after dissolution is 5.3-5.5, and the pH of the B component buffer after dissolution is 7.3-7.5; the pH after mixing of the A and B components is 6.9, and the use environment temperature is 37℃.

[0030] In one of the embodiments, the pH of the A component buffer after dissolution is 5.4, and the pH of the B component buffer after dissolution is 7.4.

[0031] In one of the embodiments, in the mixing step of the A and B components, the volume ratio of the A component solution to the B component solution is 1:1; and the mixing mode is double syringe static mixing.

[0032] In one of the embodiments, the A component solution obtained after dissolution of the A component is 2 mL; and the B component solution obtained after dissolution of the B component is 2 mL.

[0033] In addition, the application also provides a use of the double cross-linked tissue adhesive in the above-mentioned double cross-linked tissue adhesive or the use method in the preparation of a medical tissue adhesive.

[0034] Compared with the prior art, the application has the following beneficial effects:

[0035] 1. Double mechanism curing: (1) fast cross-linking mechanism: PEG-activated ester and amine group of MAPs / HA-NH2 form amide bond, structure gel in 0.5-2 min; (2) double cross-linking mechanism: TGase catalyzes Gln-Lys isopeptide bond, strength increases and wash resistance improves in 5-15 min.

[0036] 2. Wet surface synergistic enhancement: MAPs (DOPA) + γ-PGA (anion) + HA-NH2 (flexible chain) form hydrogen bond / electricity / tangle network, improve initial adhesion and toughness.

[0037] 3. pH programming stability / activation: A side acid inhibits activated ester hydrolysis, B side neutral ensures protein conformation and enzyme activity; mixed in near neutral to adapt to the tissue environment.

[0038] 4. Adjustable operation window: through PEG concentration, total amine density (MAPs+HA-NH2), B side pH, A:B volume ratio, fine-tune the initial adhesion interval of 10-60 s, gel 0.5-3 min. DETAILED DESCRIPTION

[0039] In order to facilitate the understanding of the present application, the present application will be described more fully below with reference to relevant examples. However, the present application can be realized in many different forms, and is not limited to the embodiments described herein. On the contrary, the purpose of providing these examples is to make the disclosure of the present application more thorough and comprehensive.

[0040] 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 the present application belongs. The terminology used in the description of the present application herein is only for the purpose of describing the specific embodiments and is not intended to limit the present application. The term "and / or" used herein includes any and all combinations of one or more of the associated listed items.

[0041] The reagents, materials and equipment used in the present examples are commercially available unless otherwise specified. The test methods are conventional test methods in the art unless otherwise specified.

[0042] EXAMPLE

[0043] I. Preparation of preparation

[0044] According to the content of each component in A and B bottles, weigh and sub-pack, add stopper and seal.

[0045] According to the preparation of the reconstituted solution in S-A (for A bottle) and S-B (for B bottle), 0.22 μm sterile filter membrane is used for filtration, filling and sealing.

[0046] Among them, S-A contains MES, and the concentration after reconstitution is 50-100 mM, pH=5.4; S-B contains HEPES and NaCl, and the concentration of HEPES after reconstitution is 20-50 mM, the concentration of NaCl is 150 mM, and the pH is 7.4.

[0047] II. Use and gelation

[0048] 1. Add 2 mL of S-A / S-B reconstituted solution to A and B bottles respectively, and spin to clarify.

[0049] 2. Take equal volumes of A and B liquids and place them into a double syringe, mix through a static mixer, and then uniformly coat.

[0050] The embodiment of the present application provides a double cross-linking tissue adhesive, which is composed of two components: the A component includes polyethylene glycol active ester, and the B component includes recombinant protein combined with functional polysaccharide and enzyme. The recombinant protein includes but is not limited to recombinant mussel adhesive protein, recombinant human collagen, recombinant human fibronectin and the like; the polyethylene glycol active ester includes but is not limited to four-arm polyethylene glycol succinimidyl succinate (PEG-SS), four-arm polyethylene glycol succinimidyl glutarate, double-end polyethylene glycol succinimidyl succinate, double-end polyethylene glycol succinimidyl glutarate and the like.

[0051] The formula of the double cross-linking tissue adhesive of the embodiment of the present application (counted after reconstitution) includes:

[0052] The A component (active ester): the mass concentration of four-arm PEG-SS is 150-180 mg / mL (corresponding to 300-360 mg / 2 mL), and the molecular weight is 10000-20000;

[0053] The B component (protein / polysaccharide / enzyme): the mass concentration of recombinant mussel adhesive protein is 20-30 mg / mL (corresponding to 40-60 mg / 2 mL), the mass concentration of amino hyaluronic acid is 6-8 mg / mL (corresponding to 12-16 mg / 2 mL, the molecular weight is about 100 kDa, and the amino substitution degree is about 1.5%), the mass concentration of gamma-polyglutamic acid is 6 mg / mL (corresponding to 12 mg / 2 mL, the molecular weight is about 700 kDa), and the concentration of transglutaminase is 30-40 U / mL (corresponding to 60-80 U / 2 mL); sodium ascorbate is contained, and the concentration is 2 mM.

[0054] The most preferred formula is: A component uses four-arm PEG-SS (molecular weight is 20000), mass concentration is 150 mg / mL (corresponding to 300 mg / 2 mL); B component uses recombinant mussel adhesive protein, mass concentration is 25 mg / mL (corresponding to 50 mg / 2 mL), HA-NH2 mass concentration is 6 mg / mL (corresponding to 12 mg / 2 mL, molecular weight is about 100 kDa, and aminated substitution degree is about 1.5%), γ-PGA mass concentration is mg / mL (corresponding to 12 mg / 2 mL, molecular weight is about 700 kDa), and TGase concentration is 35 U / mL (corresponding to 70 U / 2 mL); A component is dissolved in MES buffer, the buffer pH is 5.4, B component is dissolved in HEPES buffer, the buffer pH is 7.4, and the pH of the system is about 6.9 after equal volume mixing.

[0055] The preparation and use method of the double crosslinking tissue adhesive in the embodiment of the application comprises the following steps:

[0056] (1) A component: 300 mg of four-arm PEG-SS (molecular weight is 20000) is taken and dissolved in 2 mL of MES buffer (concentration is 50 mM, pH is 5.4) to obtain A solution (mass concentration of four-arm PEG-SS is 150 mg / mL).

[0057] (2) B component: 50 mg of recombinant mussel adhesive protein, 12 mg of aminated hyaluronic acid (molecular weight is about 100 kDa, and aminated substitution degree is about 1.5%), and 12 mg of γ-polyglutamic acid (molecular weight is about 700 kDa) are taken, 70 U of transglutaminase, 2 mM of sodium ascorbate, and 0.20 mM of EDTA-Na2 are added, and the mixture is dissolved in 2 mL of HEPES buffer (concentration is 25 mM, contains 150 mM of NaCl, and pH is 7.4) to obtain B solution (mass concentration of MAPs is 25 mg / mL, mass concentration of HA-NH2 is 6 mg / mL, mass concentration of γ-PGA is 6 mg / mL, and concentration of TGase is 25 U / mL).

[0058] (3) A and B solutions obtained in steps (1) and (2) are uniformly mixed in equal volume, and are quickly and thinly smeared on a position requiring adhesion within 1 minute to complete tissue adhesion.

[0059] Example 1

[0060] The preparation method of the double crosslinking tissue adhesive in the embodiment comprises the following steps:

[0061] (1) A component: 300 mg of four-arm PEG-SS (molecular weight 10000) was dissolved in 2 mL of MES buffer, and the buffer pH was 5.4 (the mass concentration of four-arm PEG-SS was 150 mg / mL).

[0062] (2) B component: 50 mg of MAPs, 12 mg of HA-NH2 (molecular weight about 100 kDa, and the amino substitution degree was about 1.5%), 12 mg of γ-PGA, TGase 70 U, sodium ascorbate (the final concentration was 2 mM), EDTA-Na2 (the final concentration was 0.20 mM) were dissolved in 2 mL of HEPES buffer, and the buffer pH was 7.4 (the buffer contained NaCl, and the concentration was 150 mM).

[0063] (3) The A and B components were mixed in equal volume, and were quickly thin-layer coated on the position to be bonded within 1 minute, and were solidified.

[0064] Example 2

[0065] (1) A component: 300 mg of four-arm PEG-SS (molecular weight 20000) was dissolved in 2 mL of MES buffer, and the buffer pH was 5.4 (the mass concentration of four-arm PEG-SS was 150 mg / mL).

[0066] (2) B component: same as example 1.

[0067] (3) The operation was same as example 1.

[0068] Example 3

[0069] (1) A component: 360 mg of four-arm PEG-SS (molecular weight 20000) was dissolved in 2 mL of MES buffer, and the buffer pH was 5.4 (the mass concentration of four-arm PEG-SS was 180 mg / mL).

[0070] (2) B component: 60 mg of MAPs, 12 mg of HA-NH2, 12 mg of γ-PGA, TGase 80 U, sodium ascorbate (the final concentration was 2 mM), EDTA-Na2 (the final concentration was 0.20 mM) were dissolved in 2 mL of HEPES buffer, and the buffer pH was 7.4.

[0071] (3) The operation was same as example 1.

[0072] Example 4

[0073] (1) A component: 240 mg of four-arm PEG-SS (molecular weight 10000) was dissolved in 2 mL of MES buffer, and the buffer pH was 5.4 (the mass concentration of four-arm PEG-SS was 120 mg / mL).

[0074] (2) B component: 40 mg MAPs, 16 mg HA-NH2, 16 mg γ-PGA, TGase 60 U, sodium ascorbate (final concentration 2 mM), EDTA-Na2 (final concentration 0.20 mM), dissolved in 2 mL HEPES buffer, buffer pH = 7.4.

[0075] (3) The operation is the same as Example 1.

[0076] Comparative Example 1 (no TGase)

[0077] (1) A component: 300 mg four-arm PEG-SS (molecular weight 20000) dissolved in 2 mL MES buffer, buffer pH = 5.4.

[0078] (2) B component: 50 mg MAPs, 12 mg HA-NH2, 12 mg γ-PGA, no TGase, the rest is the same as Example 2; dissolved in 2 mL HEPES buffer, buffer pH = 7.4.

[0079] (3) The operation is the same as Example 1.

[0080] Comparative Example 2 (HA not aminated)

[0081] (1) A component: the same as Example 2.

[0082] (2) B component: replace 12 mg HA-NH2 with 12 mg unmodified HA, the rest is the same as Example 2; dissolved in 2 mL HEPES buffer, buffer pH = 7.4.

[0083] (3) The operation is the same as Example 1.

[0084] Comparative Example 3 (no γ-PGA, 2 mL system)

[0085] (1) A component: the same as Example 2.

[0086] (2) B component: remove γ-PGA, the rest is the same as Example 2; dissolved in 2 mL HEPES buffer, buffer pH = 7.4.

[0087] (3) The operation is the same as Example 1.

[0088] Comparative Example 4

[0089] (1) A component: 200 mg four-arm PEG-SS (molecular weight 10000) dissolved in 2 mL MES buffer, buffer pH = 5.4 (mass concentration of four-arm PEG-SS is 100 mg / mL).

[0090] (2) Group B: 20 mg MAPs, 6 mg HA-NH2, 6 mg γ-PGA, TGase 40 U, the rest is the same as Example 2; dissolved in 2 mL HEPES buffer, buffer pH = 7.4.

[0091] (3) The operation is the same as Example 1.

[0092] Performance test

[0093] 1, Gel time characterization

[0094] Test method: According to the proportion in the implementation, respectively reconstitute A component (four-arm PEG-SS, MES pH = 5.4) and B component (MAPs + HA-NH2 + γ-PGA + TGase, HEPES pH = 7.4), mix equal volume after static mixer, quickly drop onto the rotating rheometer loading table (37℃); With fixed frequency / strain record modulus change curve with time, define the intersection of storage modulus G' and loss modulus G" as gel point, corresponding time is recorded as gel time (n = 5).

[0095] Table 1 Gel time

[0096]

[0097] Results show:

[0098] (1) The gel time of Examples 1-3 is significantly shorter than each of the comparative examples, which reflects the initial solidification advantage brought by PEG-SS rapid amidation + pH programming;

[0099] (2) Example 3 (higher concentration of PEG-SS, TGase concentration is 40 U / mL) is the shortest; Example 4 (lower concentration of PEG-SS, higher concentration of HA / γ-PGA) has a longer operation window;

[0100] (3) Comparative Example 1 (without TGase) and Comparative Example 2 (HA is not aminated) have delayed gelation, which shows that the enzymatic channel and the amine group density of polysaccharide promote the formation of early network; Comparative Example 4 (low solid content) is the slowest.

[0101] 2, Lap-shear tensile strength

[0102] Test method: According to "YY / T 0729.2-2009 Tissue Adhesive Bonding Performance Test Method Part 1: Lap-Shear Tensile Strength". Fresh pigskin is cut according to the standard size and moistened with PBS, and the A / B equal volume mixture prepared according to the implementation is uniformly coated on the lap surface, and is fixed to the curing completion under 1~2N pressure. The test strip is clamped so that the stress direction is consistent with the long axis, and loaded to failure at a speed of 5 mm / min, and the failure load is recorded and converted into bearing strength (n = 6).

[0103] Table 2 Lap-shear tensile strength

[0104]

[0105] Results show:

[0106] (1) Examples 1-4 are significantly higher than Comparative Examples 1-4, indicating that the covalent crosslinking of MAPs+HA-NH2 and PEG-SS and the wet face synergy of γ-PGA significantly improve the wet face shear strength;

[0107] (2) Example 3 is the highest, reflecting the superimposed effect of higher active ester / amine ratio + TGase enhancement;

[0108] (3) Comparative Example 2 (HA not aminated) has a sparse covalent network due to insufficient amine sites; Comparative Example 3 (no γ-PGA) has insufficient wet face initial adhesion and decreased interfacial energy dissipation capacity; Comparative Example 4 (low solid content) has the lowest overall strength.

[0109] 3. T-peel strength

[0110] Test method: Take two fresh tissues (width recorded as W), set the lap length to 25 mm. Mix A / B in equal volumes according to the implementation, and evenly coat the lap area (wet glue amount about 0.15~0.20g) after pressing and fixing with 1~2N pressure until curing (≥10min). Clamping the two ends of the sample to the upper and lower clamps respectively, setting the peel angle to 180° (or 90°), the crossbeam speed to 50mm / min, continuously peeling for ≥50mm, and recording the force-displacement curve. Take the average peeling force F (N) in the stable section, calculate the peeling strength P=F / W (N / m); and record the failure mode (adhesion / cohesion / substrate).

[0111] Failure mode determination index:

[0112] Adhesive failure (Adhesive failure): occurs at the interface between the glue layer and the substrate (tissue).

[0113] Performance: The glue is left on the opposite side or is completely peeled off, and there is almost no residual glue on the tissue surface on one side. This indicates that the interface bonding is weak or the interface is hydrated / polluted.

[0114] Cohesive failure (Cohesive failure): occurs within the glue layer.

[0115] Performance: Both sides of the tissue surface have residual glue, and the glue layer can be seen to be torn / stranded; the interface is still sticky. This indicates that the interface is strong, but the body strength or toughness of the glue is insufficient (or the load is too large).

[0116] Substrate failure: Occurred in the tissue itself (internal tearing, delamination).

[0117] Performance: The adhesive layer is still intact on the opposite side or both sides, while the tissue fibers are pulled apart. It indicates that the adhesion is higher than the substrate strength.

[0118] Mixed failure: Two or more modes coexist in the same sample (e.g., "cohesion + adhesion").

[0119] Report the percentage of each mode.

[0120] Table 3: Peel and failure mode evaluation

[0121]

[0122] The results show that:

[0123] Compared with the comparative examples, the peel strength of examples 1-4 is higher overall (about 320-410 N / m, with the highest in example 3), and the failure mode changes from adhesion-based to cohesion-based with a small amount of substrate failure, indicating that the interface bonding and gel network are more stable. Removing TGase (comparative example 1), not aminating HA (comparative example 2), or removing γ-PGA (comparative example 3) all reduce the strength and increase the proportion of adhesion failure, with the lowest solid content (comparative example 4) performing the worst. This shows that the "chemical + enzymatic" dual solidification combined with HA-NH2 / γ-PGA can significantly improve the wet surface peel resistance and interface stability.

[0124] 4. Wound closure strength (wound edge approximation)

[0125] Test method: Take the pig mesentery and cut it into 20mm x 60mm strip samples; cut a standard slit 20mm long in the middle of the sample. Approximate the two sides of the wound edge and evenly apply glue along the wound edge (coating width 5-8mm), press and fix with 1-2N pressure, and solidify for 10-15min. Clamp the two ends of the sample in the universal testing machine clamp, ensuring that the slit is in the middle of the clamp; stretch to failure at a crosshead speed of 5mm / min, record the maximum load F_max (N) and displacement (mm). Calculate the closure strength σ = F_max / adhesive width (kPa). At the same time, record the failure mode (adhesion / cohesion / substrate or mixed).

[0126] Table 4: Wound closure strength

[0127]

[0128] The results show that:

[0129] The wound closure strength of Examples 1-4 is significantly higher than that of the comparative examples (85-120 kPa vs 30-52 kPa), the fracture displacement is larger (2.8-3.2 mm vs 1.7-2.2 mm), and the failure mode is changed from adhesion in the comparative examples to mainly cohesion, and even cohesive-substrate failure in Example 3, indicating that the interface is stronger than the tissue body. Removing TGase, non-aminoation of HA, or removing γ-PGA all reduce the strength and displacement; low solid content (Comparative Example 4) is the worst. In summary, the "chemical + enzymatic" dual solidification of the application, combined with HA-NH2 / γ-PGA, significantly improves the bearing capacity and toughness of the wound edges.

[0130] 5. Leak / burst pressure (sealing ability)

[0131] Experimental method: Take the isolated pig small intestine, unfold it to cause a circular perforation with a diameter of 2 mm, then uniformly coat the adhesive on the surface and surrounding area of the circular perforation, and after solidification for 10-15 minutes, apply pressure at a speed of 10 mmHg / s through a constant flow pump, and record the first leakage and burst pressure.

[0132] Table 5 Leak pressure and burst pressure

[0133]

[0134] The results show that:

[0135] The leak initiation pressure and burst pressure of Examples 1-4 in the isolated small intestine model are significantly higher than those of the comparative examples (leakage about 110-160 mmHg, burst about 185-250 mmHg; control about 50-80 / 85-120 mmHg), among which Example 3 is the highest (160±16 / 250±20 mmHg), showing the best sealing and pressure resistance limit. Removing TGase, non-aminoation of HA, or removing γ-PGA all significantly reduces the two indicators, and low solid content (Comparative Example 4) is the worst, indicating that the synergy of "chemical + enzymatic" dual solidification combined with HA-NH2 / γ-PGA is the key to improving the anti-leakage and pressure resistance performance.

[0136] 6. Resistance to washing retention rate

[0137] Experimental method: After the "lap-shear" sample is prepared, the shear strength before washing is first measured, and then the sample is washed. The sample is placed in a 37°C constant temperature flow tank, and under the action of circulating water, it is washed for 10 minutes, then the sample is taken out and the shear strength after washing is detected.

[0138] Retention rate R = (strength after washing / strength before washing) x 100%.

[0139] Table 6 Lap-shear tensile strength before and after washing and retention rate

[0140]

[0141] Results show:

[0142] Examples 1-4 still maintain high lap strength and stability after rinsing, with a retention rate of about 83-93% (of which Example 3 is 0.30→0.28 MPa, 93% being the best), significantly better than 50-64% of the comparative examples (and lower initial strength). It shows that the "chemical + enzymatic" dual curing of the application, combined with MAPs / HA-NH2 / γ-PGA, can effectively resist dilution and shear flushing, maintain wet surface adhesion and structural integrity.

[0143] 7. Swelling rate in body fluid

[0144] Experimental method: mix the tissue adhesive according to the operation steps, stand for 30 minutes at room temperature, cut a 10mm diameter, 2mm thick disc, weigh the initial mass m0; place in normal saline, 37℃, light shaking; record the wet mass m after 48 hours t , calculate the swelling rate S= (m t -m0) / m0x100%;

[0145] Table 7 Swelling rate of each component in normal saline

[0146]

[0147] Results show:

[0148] The 24h swelling rate of Examples 1-4 is significantly lower than that of the comparative examples, controlled at below 200%, which will not cause compression of the surrounding tissue.

[0149] 8. Blood compatibility and hemostatic performance

[0150] Hemolysis experiment Experimental method: take the same donor's anticoagulant whole blood (sodium citrate), prepare a 2% red blood cell suspension (0.9% NaCl wash 3 times to prepare). The gel prepared according to the operation of the application is completely cured at 37℃, and the extraction liquid is obtained by extracting 0.9% NaCl for 24h at a surface / volume ratio of 3cm 2 / mL (and a material blank is used to deduct background). Take 0.5mL of the extraction liquid and 0.5mL of the red blood cell suspension, mix well, and incubate at 37℃ for 60min; centrifuge (1000g, 5min) and take the supernatant to measure the absorbance at 540nm.

[0151] Hemolysis rate = (OD 试验管 -OD 阴性对照管 ) / (OD 阳性对照管 -OD 阴性对照管 )%

[0152] The negative control is normal saline, and the positive control is pure water.

[0153] Table 8 Hemolysis rate of each group

[0154]

[0155] The results show that:

[0156] The hemolysis rate of all groups is ≤5% threshold, indicating good blood compatibility.

[0157] Whole blood clotting index (WBCI) experimental method: the sample disc pre-warmed at 37℃ is placed at the bottom of the transparent well plate (PBS is washed 3 times, and the free liquid is removed), 200 μL of sodium citrate anticoagulated whole blood and 20 μL of 0.2M CaCl2 are added to each well to start recalcification, and after incubation at the preset time point (5 / 10 / 20min), 2.0mL of deionized water is quickly added and shaken for 3min to dissolve the red blood cells not embedded in the clot, and the supernatant is measured at 540nm; taking the blank well (without sample surface) as the reference to calculate WBCI (%), the lower the value, the more sufficient the coagulation at the time point (the less residual soluble hemoglobin).

[0158] Table 9 Whole blood clotting index of each group

[0159]

[0160] The results show that:

[0161] The WBCI of Examples 1-4 at 5 / 10 / 20min is significantly lower than that of the comparative example (p<0.05), indicating that the system of the present application is more conducive to the rapid formation of stable clots and embedding of red blood cells; Example 3 is the lowest, and at 10-20min, it has approached the "sufficient coagulation" platform, suggesting that sufficient chemical crosslinking density, TGase secondary curing, and surface interaction of HA-NH2 / γ-PGA and MAPs together accelerate the formation of fibrin network and improve the thrombus density, which is consistent with the trend of the aforementioned "closure strength, resistance to leakage / explosion, and resistance to washing retention rate".

[0162] 9. In vitro cytotoxicity test

[0163] Test method: according to "GB / T16886.5-2017 Medical Devices Biological Evaluation Part 5: In Vitro Cytotoxicity Test". After the colloidal complete curing according to the implementation mode, the medium is extracted for 24h according to the standard method; the extracted liquid is used on L929 cells for 24h, and the absorbance is detected by CCK-8 method and the survival rate is calculated. Taking the normally cultured cells as the control; if the survival rate is <70%, it is determined to have potential cytotoxicity (n=3).

[0164] Table 10 Potential cytotoxicity

[0165]

[0166] Results show:

[0167] The cell survival rate of each group is ≥90%, without potential cytotoxicity, proving that the system has good cell compatibility.

[0168] 10. Skin sensitization test

[0169] Test method: According to GB / T 16886.10-2017 Medical devices-Biological evaluation of medical devices-Part 10: Sensitization and Irritation Tests. Healthy New Zealand rabbits were selected; the back was shaved 10x15cm as the contact and observation site. A / B colloids were prepared according to the embodiments, mixed in equal volume and coated on a 2.5x2.5cm area until complete gelation; after 4h contact, remove with a wet gauze. Record skin reactions and score at 1, 24, 48, 72h respectively.

[0170] Table 11 Skin reaction score and rabbit primary irritation index type

[0171]

[0172] Results show:

[0173] There is no skin sensitization reaction in all examples and comparative examples, and the biological safety is good.

[0174] 11. Skin wound healing experiment

[0175] Test method: Healthy SD rats were selected; the back was shaved 4x6cm as the observation area. After iodophor disinfection, a 2cm full-thickness skin incision was prepared with a scalpel. A / B colloids were prepared according to the embodiments, mixed in equal volume and coated on the middle and periphery of the incision 2.5x0.5cm area until complete gelation; record the time required for complete closure of the wound (n=6).

[0176] Table 12 Evaluation of adhesive healing time on rat wounds

[0177]

[0178] Results show:

[0179] (1) The healing time of examples 1-4 is significantly shorter than that of the comparative examples, and example 3 (high active ester / moderate amine ratio + TGase enhancement) is the fastest;

[0180] (2) Comparative example 2 (HA not aminated) and comparative example 4 (low solid content) heal slower, suggesting that the density of covalent network and solid content are crucial for early stable closure;

[0181] (3) Overall, the present application performs better than the controls in terms of rapid solidification of wet surface, improvement of shear bearing strength and promotion of tissue healing.

[0182] In summary, the double cross-linking tissue adhesive provided by the present application has faster adhesion than existing adhesives: after A / B equal-volume mixing, the initial fixation / gelation is completed within 90-120s, meeting the requirement for rapid operation during surgery; stronger bonding: the lap-shear bearing strength reaches 0.20-0.25MPa (and the retention rate after flushing is ≥80%), and the indicators such as closure and pressure resistance are simultaneously improved; good biological safety: no cytotoxicity, no skin irritation and sensitization, hemolysis rate ≤5%, whole blood clotting index (WBCI) is better than the blank control, and is suitable for application under physiological conditions.

[0183] The technical features of the above-described embodiments can be combined in any manner. To make the description concise, all possible combinations of the technical features in the above-described embodiments are not described, but as long as the combinations of the technical features do not exist contradictory, they should be considered as falling within the scope of the present disclosure.

[0184] The above-described embodiments only express several implementation manners of the present application, and the description is more specific and detailed, but it should not be understood as a limitation on the scope of the patent. It should be pointed out that for those skilled in the art, without departing from the concept of the present application, a number of modifications and improvements can be made, which all belong to the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the appended claims.

Claims

1. A dual crosslinking tissue adhesive, characterized by, The preparation raw materials include A component and B component: the A component includes polyethylene glycol active ester, buffer and freeze-drying protective agent; The B component includes recombinant mussel myeloplasmin, amino hyaluronic acid, gamma-polyglutamic acid, transglutaminase, sodium ascorbate, ethylenediaminetetraacetic acid disodium salt, buffer and freeze-drying protective agent; The buffer of the A component includes 2-morpholinoethanesulfonic acid, the freeze-drying protective agent of the A component includes trehalose and mannitol, the buffer of the B component includes 4-hydroxyethylpiperazine ethanesulfonic acid or phosphate buffer, and the freeze-drying protective agent of the B component includes trehalose and mannitol; The recombinant mussel myeloplasmin contains 3,4-dihydroxyphenylalanine; In the preparation raw materials, the mass concentration of polyethylene glycol active ester in the A component solution is 120-180 mg / mL; in the B component solution, the mass concentration of recombinant mussel myeloplasmin is 20-30 mg / mL, the mass concentration of amino hyaluronic acid is 5.5-15 mg / mL, the mass concentration of gamma-polyglutamic acid is 5.5-15 mg / mL, the concentration of transglutaminase is 30-40 U / mL, the concentration of sodium ascorbate is 1-3 mM, and the concentration of ethylenediaminetetraacetic acid disodium salt is 0.1-0.25 mM; the volume ratio of the A component solution to the B component solution is 1:

1.

2. The dual crosslinking tissue adhesive of claim 1, wherein, The polyethylene glycol active ester includes linear polyethylene glycol active ester or multi-arm polyethylene glycol active ester, and the amino substitution degree of the amino hyaluronic acid is 1.0-2.0%.

3. The dual crosslinking tissue adhesive of claim 2, wherein, The polyethylene glycol active ester includes at least one of four-arm polyethylene glycol succinimidyl succinate, four-arm polyethylene glycol succinimidyl glutarate, double-end polyethylene glycol succinimidyl succinate and double-end polyethylene glycol succinimidyl glutarate.

4. The dual crosslinking tissue adhesive of claim 3, wherein, The polyethylene glycol active ester is four-arm polyethylene glycol active ester with a molecular weight of 10-20 kDa; the molecular weight of the amino hyaluronic acid is 50-300 kDa; and the molecular weight of the gamma-polyglutamic acid is 300-1500 kDa.

5. The method of making a dual cross-linking tissue adhesive according to any one of claims 1-4, wherein, The method comprises the following steps: A component dissolution: the A component buffer is dissolved, and then added to the remaining raw materials of the A component to dissolve and mix uniformly to obtain an A component solution; B component dissolution: the B component buffer is dissolved, and then added to the remaining raw materials of the B component to dissolve and mix uniformly to obtain a B component solution; A and B component mixing: the A and B component solutions are mixed uniformly.

6. The preparation method according to claim 5, characterized in that, In the A component solution, the mass concentration of polyethylene glycol active ester is 120-180 mg / mL; In the B component solution, the mass concentration of recombinant mussel myeloplasmin is 20-30 mg / mL, the mass concentration of amino hyaluronic acid is 5.5-15 mg / mL, the mass concentration of gamma-polyglutamic acid is 5.5-15 mg / mL, the concentration of transglutaminase is 30-40 U / mL, the concentration of sodium ascorbate is 1-3 mM, and the concentration of ethylenediaminetetraacetic acid disodium salt is 0.1-0.25 mM.

7. The preparation method according to claim 5, characterized in that, After the A component buffer is dissolved, the pH is 4.5-6.5; after the B component buffer is dissolved, the pH is 6.5-8.5, and after the A and B components are mixed, the pH is 6.0-7.

8.

8. The preparation method according to claim 5, characterized in that, The volume ratio of the A component solution to the B component solution in the mixing step of the A, B components is 1:

1.

9. Use of the dual cross-linking tissue adhesive according to any one of claims 1 to 4 for the preparation of a medical tissue adhesive.

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