Modified polyglutamic acid-based tissue adhesive as well as preparation method and application thereof

Abstract

The invention provides a modified polyglutamic acid-based tissue adhesive as well as a preparation method and application thereof. The tissue adhesive is composed of the following components: 1-30 wt% of acidic aldehyde polyglutamic acid; 1 to 15 wt% of a linear protein; 1 to 15 wt% of spherical protein; and the balance of water. The tissue adhesive provided by the invention has the advantages of self-healing performance, low underwater swelling rate and stable adhesion to wet tissues in a physiological environment, and can be applied to rapid hemostasis of dynamic wet tissue wounds in the physiological environment and sealing of wound liquid leakage. Experiments show that the tissue adhesive is high in tissue adhesion stability in a wet physiological environment, and the adhesion strength of the tissue adhesive to wet pigskin tissues can reach 37.4 kPa or above after the tissue adhesive is placed for 12 h under the physiological condition.

Description

Technical Field

[0001] The invention belongs to the field of medical tissue adhesives, and in particular relates to a modified polyglutamic acid-based tissue adhesive and a preparation method and application thereof. Background Art

[0002] It is well known that maintaining stable adhesion in a humid environment is a necessary condition for tissue adhesives to be used in the body. When tissue adhesives come into contact with moist tissues, they quickly form hydrogen bond adhesion and then covalent bond adhesion. It can not only close tissue wounds, but also provide a moist microenvironment for wounds, thereby promoting cell migration to accelerate wound healing. However, most tissue adhesives tend to swell in a humid environment, resulting in a significant decrease in their interfacial adhesion strength and bulk mechanical properties, and may even fall off from the tissue surface. Excessive swelling can also cause compression damage to surrounding tissues or nerves. Therefore, tissue adhesives that can maintain stable adhesion in a humid environment will have broader application prospects. In order to maintain the bulk mechanical properties of tissue adhesives in a humid environment, researchers have explored a variety of methods, such as introducing hydrophobic components, adding absorbent fillers, or utilizing electrostatic interactions, but these methods all have an adverse effect on their adhesion properties.

[0003] In nature, aquatic organisms such as mussels and sandcastle worms exhibit excellent adhesion to wet substrate surfaces. Studies have shown that the reason why mussel adhesives have excellent wet adhesion is due to the synergistic effect between the hydrophobic structure and the catechol structure in the adhesive. The hydrophobic structure in the mussel adhesive can exclude interfacial moisture through hydrophobic interactions, form bonds with the substrate through the catechol structure, and use the hydrophobic interactions of the hydrophobic structure and the internal multiple cross-linking mechanism to inhibit its own swelling, thereby achieving strong and stable adhesion to the wet substrate. Similarly, sandcastle worms can secrete anionic and cationic proteins to form coagulants underwater to exclude interfacial moisture, and inhibit their own swelling through electrostatic interactions, which can also achieve stable adhesion to wet substrates. Most tissue adhesives achieve stable adhesion by imitating the adhesion mechanism of mussels or sandcastle worms. However, these tissue adhesives usually have weak bulk mechanical properties, and their interfacial adhesion is easily affected by the physiological environment, resulting in decreased adhesion performance or even failure.

[0004] Therefore, the preparation of tissue adhesives with self-healing properties, low swelling ratio, and stable interfacial adhesion remains a major challenge. Summary of the invention

[0005] In view of this, the object of the present invention is to provide a modified polyglutamic acid-based tissue adhesive and a preparation method and application thereof. The tissue adhesive has excellent adhesion stability and self-healing performance, and has a low swelling rate.

[0006] To achieve this object, the present invention adopts the following technical solutions: In a first aspect, the present invention provides a modified polyglutamic acid-based tissue adhesive, which is composed of the following components by mass fraction: 1-30 wt% of acidic aldehyde-modified polyglutamic acid; 1-15 wt% linear protein; 1-15 wt% globular protein; The balance is water.

[0007] Preferably, the pH of the acidic aldehyded polyglutamic acid is 1-3.

[0008] Preferably, the degree of aldehyde substitution of the acidic aldehyded polyglutamic acid is 2-20%.

[0009] Preferably, the molecular weight of the acidic aldehyde-modified polyglutamic acid is 100-2000 kDa.

[0010] Preferably, the globular protein is selected from any one or more of plant lysozyme, animal lysozyme, microbial lysozyme or egg white lysozyme.

[0011] Preferably, the linear protein is selected from any one or more of reduced plant lysozyme, animal lysozyme, microbial lysozyme or egg white lysozyme.

[0012] Preferably, the plant lysozyme is selected from fig lysozyme.

[0013] Preferably, the animal lysozyme is selected from mammalian lysozyme.

[0014] Preferably, the microbial lysozyme is selected from glucosaminidase-like lysozymes.

[0015] Preferably, the egg white lysozyme is selected from hen egg white lysozyme.

[0016] In a second aspect, the present invention provides a method for preparing the modified polyglutamic acid-based tissue adhesive, comprising the following steps: S1: subjecting sodium polyglutamate to aldehyde treatment and acid treatment to obtain acidic aldehyde-treated polyglutamic acid; subjecting the globular protein to reduction treatment to obtain linear protein; S2: Evenly mix the acidic aldehyde-modified polyglutamic acid, the globular protein and the linear protein in the presence of a solvent to obtain a modified polyglutamic acid-based tissue adhesive.

[0017] Preferably, the process of obtaining the acidic aldehyde-formyl polyglutamic acid is: The sodium polyglutamate is subjected to hydroxylation treatment, first acidification treatment, oxidation treatment and second acidification treatment; the molecular weight of the sodium polyglutamate is 100-2000 kDa.

[0018] Preferably, the reagent for the hydroxylation treatment includes but is not limited to any one or more of 3-amino-1,2-propanediol, D-glucosamine, 3-amino-1-propanol, ethanolamine or 3-amino-2-methylpropanol, the reagent for the oxidation treatment is sodium periodate, and the oxidation treatment time is 30 to 120 min.

[0019] Preferably, the pH of the first acidification treatment and the second acidification treatment is 1-3, the temperature of the first acidification treatment and the second acidification treatment is 0-60° C., and the time of the first acidification treatment and the second acidification treatment is 24-72 h.

[0020] Preferably, the reduction treatment is: reacting the spherical protein at a pH of 1 to 3, and then quenching the obtained reaction solution in an ice water bath.

[0021] Preferably, the solvent is water.

[0022] In a third aspect, the present invention provides a dynamic tissue wound dressing, which is prepared from the above-mentioned modified polyglutamic acid-based tissue adhesive.

[0023] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a modified polyglutamic acid-based tissue adhesive, which is obtained by co-assembling acidic aldehyde-formyl polyglutamic acid, linear protein and globular protein by forming physical effects (electrostatic interactions) and chemical crosslinking (Schiff base bonds). Among them, the carboxyl group and aldehyde group in the acidic aldehyde-formyl polyglutamic acid can form non-covalent bonds and covalent bonds such as hydrogen bonds, electrostatic interactions and Schiff base bonds with the tissue surface, so that the tissue adhesive can instantly adhere to wet tissues and maintain stable adhesion under moist physiological environments. In addition, for linear proteins, on the one hand, the additional exposed amino groups and other groups can form more chemical crosslinks with the aldehyde groups on the acidic aldehyde-formyl polyglutamic acid, thereby improving the bulk strength of the tissue adhesive. On the other hand, the hydrophobic interaction of hydrophobic amino acids in linear proteins can make the tissue adhesive show a very low swelling rate underwater. The globular protein can increase the dynamic crosslinking points, thereby increasing the toughness and dynamic self-recovery of the adhesive.

[0024] Based on the cross-linking of non-covalent bonds and dynamic covalent bonds, the modified polyglutamic acid-based tissue adhesive provided by the present invention has good self-healing properties, low swelling rate under water, and can still maintain stable adhesion after being placed for 12 hours under physiological conditions, and the adhesion strength can reach 37.4 kPa, which shows that it has excellent adhesion stability. Therefore, based on instantaneous and stable wet adhesion, the tissue adhesive provided by the present invention can be used to prepare dynamic tissue wound dressings, which are expected to be able to quickly close dynamic tissue organ wounds and stably adhere to the wounds, and achieve rapid hemostasis. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The images of the fracture, self-healing and stress tolerance process of the tissue adhesive provided in Example 1; Figure 2 The lap shear adhesion strength images of the tissue adhesive provided in Example 1 at different time points under PBS conditions. DETAILED DESCRIPTION

[0026] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0027] In view of the problems of poor self-healing performance, high swelling rate and poor interface adhesion stability of tissue adhesives in the prior art, the present invention provides a modified polyglutamic acid-based tissue adhesive, which is composed of the following components by mass fraction: 1-30 wt% of acidic aldehyde-modified polyglutamic acid; 1-15 wt% linear protein; 1-15 wt% globular protein; The balance is water.

[0028] In the present invention, the modified polyglutamic acid-based tissue adhesive comprises 1 to 30 wt% of acidic aldehyde-formyl polyglutamic acid. In some embodiments of the present invention, the medical tissue adhesive preferably comprises 1 to 20 wt% of acidic aldehyde-formyl polyglutamic acid, and more preferably comprises 4 to 10 wt% of acidic aldehyde-formyl polyglutamic acid. The present invention uses acidic aldehyde-formyl polyglutamic acid as one of the components of the tissue adhesive, and can form non-covalent and covalent bonds such as hydrogen bonds, electrostatic interactions and Schiff base bonds with the tissue surface by means of the carboxyl and aldehyde groups in its side chains, so that the tissue adhesive can instantly adhere to wet tissues and maintain stable adhesion even in a moist physiological environment.

[0029] It should be noted that the lower the pH value of the acidic aldehyde-modified polyglutamic acid, the more carboxyl groups there are in the polyglutamic acid, and the stronger its ability to absorb the blood interface hydration layer at the tissue trauma or organ damage through hydrogen bonding. At the same time, the carboxyl groups of polyglutamic acid can form hydrogen bonds with amino groups, carboxyl groups and hydroxyl groups on the tissue surface to significantly improve the interfacial adhesion strength between the tissue adhesive and the wounded tissue. Therefore, the present invention is optimized, and preferably the pH of the acidic aldehyde-modified polyglutamic acid is 1 to 3, and more preferably 1.5 to 2.5.

[0030] Furthermore, the higher the degree of aldehyde substitution of the acidic aldehyde-formyl polyglutamic acid (the degree of aldehyde substitution is the amount of carboxyl groups in the acidic aldehyde-formyl polyglutamic acid replaced by aldehyde groups), the more aldehyde groups there are in the polyglutamic acid, and the aldehyde groups in the polyglutamic acid can form Schiff base bonds with the amino groups on the tissue surface, which can significantly improve the interfacial adhesion stability between the tissue adhesive and the wound tissue. However, the content of aldehyde groups in polyglutamic acid cannot be too high, because the increase in internal aldehyde groups will form more crosslinks with linear lysozyme, making the adhesive brittle, and at the same time making the instantaneous bonding strength of the interface lower, resulting in insufficient adhesion. Therefore, the present invention is optimized, and preferably the degree of aldehyde substitution of the acidic aldehyde-formyl polyglutamic acid is 2% to 20%, and more preferably 4 to 10%.

[0031] In the present invention, the modified polyglutamic acid-based tissue adhesive comprises 1 to 15 wt% of globular protein, preferably 1 to 10 wt% of globular protein, and more preferably 1 to 5 wt% of globular protein. Wherein, the globular protein is selected from lysozyme, such as any one or more of plant lysozyme, animal lysozyme, microbial lysozyme or clear lysozyme. Specifically, the plant lysozyme is selected from fig lysozyme; the animal lysozyme is selected from mammalian lysozyme; the microbial lysozyme is selected from glucosaminidase-like lysozyme; the egg white lysozyme is selected from hen egg white lysozyme. In some embodiments of the present invention, the globular protein selects hen egg white lysozyme.

[0032] The present invention increases the dynamic cross-linking points in the system by introducing spherical proteins, so that the adhesive has excellent self-recovery and toughness.

[0033] In the present invention, the modified polyglutamic acid-based tissue adhesive comprises 1 to 15 wt% of linear protein, preferably 1 to 10 wt% of linear protein, and more preferably 1 to 5 wt% of linear protein. In the present invention, the linear protein is obtained by reducing the globular protein. Therefore, in the present invention, the linear protein is selected from any one or more of reduced plant lysozyme, animal lysozyme, microbial lysozyme or egg white lysozyme.

[0034] The present invention, by introducing linear proteins, can, on the one hand, form a highly cross-linked structure with acidic aldehyde-modified polyglutamic acid through more exposed amino groups, and on the other hand, can endow the tissue adhesive with better anti-swelling properties through the exposure of hydrophobic amino acids.

[0035] The present invention explores the adhesion strength and swelling rate of the modified polyglutamic acid-based tissue adhesive of the above three components at different compositions (the weight ratios of acidic aldehyde-formyl polyglutamic acid, linear protein and globular protein are 10:0:10, 10:1:9, 10:3:7, 10:5:5, 10:7:3, 10:9:1 and 10:10:0, respectively). The results show that as the amount of linear protein increases and the amount of globular protein decreases, the adhesion strength of the obtained tissue adhesive shows a trend of first increasing and then decreasing, while the swelling rate shows a trend of gradually decreasing. Therefore, the present invention believes that when the weight ratios of acidic aldehyde-formyl polyglutamic acid, linear protein and globular protein are 10:1:9, 10:3:7, 10:5:5, 10:7:3 and 10:9:1, respectively, they can meet the use requirements. When the weight ratio of acidic aldehyde-modified polyglutamic acid, linear protein and globular protein is 10:5:5, the obtained tissue adhesive has the highest adhesion strength of 50.8 kPa, and the swelling rate is 34.0%, which can be used as the preferred technical solution.

[0036] The tissue adhesive provided by the present invention has acidic aldehyde-modified polyglutamic acid, linear protein and globular protein as main components, and has self-healing properties through cross-linking of non-covalent bonds and dynamic covalent bonds, so that it has the potential to be used in dynamic tissue wound closure.

[0037] The present invention also provides a method for preparing a modified polyglutamic acid-based tissue adhesive, comprising the following steps: S1: subjecting sodium polyglutamate to aldehyde treatment and acid treatment to obtain acidic aldehyde-treated polyglutamic acid; subjecting the globular protein to reduction treatment to obtain linear protein; S2: Evenly mix the acidic aldehyde-modified polyglutamic acid, the globular protein and the linear protein in the presence of a solvent to obtain a modified polyglutamic acid-based tissue adhesive.

[0038] According to the present invention, sodium polyglutamate is firstly subjected to aldehyde treatment and acidification treatment to obtain acidic aldehyde-formylated polyglutamic acid. Specifically, the present invention firstly performs hydroxylation, a first acidification treatment, an oxidation treatment and a second acidification treatment on sodium polyglutamate, and then purifies and dries to obtain acidic aldehyde-formylated polyglutamic acid.

[0039] In some embodiments of the present invention, it is preferred that sodium polyglutamate powder is dissolved in an aqueous solution, a hydroxylation agent is added thereto for hydroxylation, a first acidification treatment is performed in an acid solution and dialyzed, an oxidizing agent is added for oxidation, and finally an acid solution is added thereto for a second acidification treatment and dialyzed, and the dialyzed solution is subjected to vacuum freeze drying to obtain acidic aldehyde-formyl polyglutamic acid. The acidic aldehyde-formyl polyglutamic acid obtained by the present invention is sponge-like, also known as acidic aldehyde-formyl polyglutamic acid sponge, and it is dissolved in water to obtain an acidic aldehyde-formyl polyglutamic acid solution. The molecular weight cut-off of the above-mentioned dialysis is 8-14 kDa, and the dialysis is performed at room temperature using an ultrapure aqueous solution with a pH of 1-3, and the time is about 3 days, and the water is changed every 3-4 h.

[0040] Wherein, the hydroxylation treatment is carried out in a hydroxylation agent, and the hydroxylation agent includes but is not limited to any one or more of 3-amino-1,2-propanediol, D-glucosamine, 3-amino-1-propanol, ethanolamine or 3-amino-2-methylpropanol; the concentration of the hydroxylation agent is 0.01~0.1 mol / L, preferably 0.02~0.05 mol / L. The temperature of the hydroxylation treatment is room temperature, specifically 0℃~37℃, preferably 10~30℃; the time of the hydroxylation treatment is 10~60min, preferably 30~50min.

[0041] The first acidification treatment and the second acidification treatment are both carried out in an acid solution, and the acid solution includes but is not limited to any one or more of hydrochloric acid, citric acid, phosphoric acid, oxalic acid or sulfuric acid; the concentration of the acid solution is 1~8 mol / L, preferably 2~6 mol / L, and the pH is 1~3, preferably 1.5~2.5. In some embodiments of the present invention, the temperature of the first acidification treatment and the second acidification treatment is 0℃~60℃, preferably 10~50℃; the time is 24~72 h, preferably 30~48 h. The present invention can fully convert the carboxylate in sodium polyglutamate into carboxylic acid by maintaining the pH of the acid solution at 1~3, preferably 2, thereby increasing the number of carboxyl groups in polyglutamic acid and enhancing the water absorption of the tissue adhesive prepared subsequently.

[0042] The oxidation treatment is carried out in an oxidizing agent, which includes but is not limited to any one or more of perchlorate, permanganate, periodate, dichromate, sodium peroxide, hydrogen peroxide, silver nitrate or hypochlorite; the concentration of the oxidizing agent is 1-10 mol / L, preferably 2-5 mol / L. The temperature of the oxidation treatment is room temperature, specifically 0°C-37°C, preferably 10-30°C; the time of the oxidation treatment is 30-120 min, preferably 60-90 min.

[0043] In the present invention, the spherical protein is subjected to a reduction treatment to obtain a linear protein. The reduction treatment comprises: reacting the spherical protein under a pH condition of 1 to 3, and then quenching the obtained reaction solution in an ice-water bath.

[0044] In some embodiments of the present invention, it is preferred to use 1 mol / L hydrochloric acid solution to adjust the pH of the aqueous solution of the globular protein to 1-3, preferably 2, and then stir the reaction at 80-120°C for 10-24 h, preferably at 90°C for 12 h, and then immediately immerse the reaction solution in an ice-water bath to quench the reaction. After purification, freeze-drying is performed to obtain a linear protein. The purification is performed by dialysis, the molecular weight cutoff of the dialysis is 8-14 kDa, and the dialysis is performed at room temperature using ultrapure water for about 3 days, and the water is changed every 3-4 h.

[0045] After obtaining the acidic aldehyde-formyl polyglutamic acid and the linear protein, the present invention uniformly mixes the acidic aldehyde-formyl polyglutamic acid, the spherical protein and the linear protein in the presence of a solvent to obtain a modified polyglutamic acid-based tissue adhesive, wherein the solvent is preferably water.

[0046] In the present invention, the acidic aldehyde-formyl polyglutamic acid solution, the linear protein solution and the globular protein solution are mixed to obtain a tissue adhesive. Specifically, the present invention mixes the acidic aldehyde-formyl polyglutamic acid and water, preferably stirring at room temperature to dissolve them uniformly, to obtain an acidic polyglutamic acid solution; mixes the linear protein and water, preferably stirring at room temperature to dissolve them uniformly, to obtain a linear protein solution; mixes the globular protein and water, preferably stirring at room temperature to dissolve them uniformly, to obtain a globular protein solution, and then mixes the acidic aldehyde-formyl polyglutamic acid solution, the linear protein solution and the globular protein solution to obtain a tissue adhesive.

[0047] In the present invention, the concentration of the acidic aldehyde-formyl polyglutamic acid solution is 1-60 wt%, preferably 1-10 wt%, more preferably 2-10 wt%, and further preferably 2 wt%; the concentration of the linear protein solution is 1-60 wt%, preferably 1-10 wt%, more preferably 2-10 wt%, and further preferably 2 wt%; the concentration of the spherical protein solution is 1-60 wt%, preferably 1-10 wt%, more preferably 2-10 wt%, and further preferably 2 wt%. The acidic aldehyde-formyl polyglutamic acid aqueous solution, the linear protein aqueous solution and the spherical protein aqueous solution of the present invention are mixed in a volume ratio of 10:0:10, 10:1:9, 10:3:7, 10:5:5, 10:7:3, 10:9:1 and 10:10:0. The acidic aldehyde-formyl polyglutamic acid, the linear protein and the spherical protein of the present invention are the same as above and will not be repeated.

[0048] The preparation method of the tissue adhesive provided by the present invention is simple, convenient, easy to implement, and conducive to industrial production.

[0049] The preparation method of the tissue adhesive provided by the present invention is to use the aldehyde-formylated polyglutamic acid sponge obtained after aldehyde-formylation and strong acid acidification as a raw material, and uniformly mix it with linear protein and globular protein to prepare the tissue adhesive. In the method, sodium polyglutamate is aldehyde-formylated and acidified in sequence using a hydroxylating agent, an oxidizing agent and a strong acid, so that the carboxylate of sodium polyglutamate can be converted into an aldehyde group and a carboxylic acid to obtain acidic aldehyde-formylated polyglutamic acid. The whole process of the preparation method is simple and convenient, easy to implement, and is conducive to realizing industrial production.

[0050] The tissue adhesive prepared by the present invention is cross-linked by non-covalent bonds and dynamic covalent bonds, and has good self-healing properties. The exposure of the hydrophobic amino acids of the linear protein can reduce the underwater swelling rate of the adhesive, and the carboxyl group and aldehyde group of the acidic aldehyde-formyl polyglutamic acid form hydrogen bonds and Schiff base bonds with the tissue surface, respectively, to enhance the adhesion stability of the interface and achieve adhesion to dynamic wet tissue. After testing, the tissue adhesive has good self-healing properties, and can still maintain stable adhesion after being placed for 12 hours under physiological conditions, and the adhesion strength can reach 37.4 kPa. It can be seen that the tissue adhesive prepared by the present invention can safely, quickly and stably close dynamic tissue wounds.

[0051] Based on this, the present invention also provides a use of the above tissue adhesive in the preparation of medical tissue adhesive.

[0052] Specifically, the present invention provides a dynamic tissue wound dressing, which is prepared from the modified polyglutamic acid-based tissue adhesive provided by the present invention.

[0053] In summary, the tissue adhesive provided by the present invention has stable adhesion to wet tissue under physiological conditions, and can be applied to rapid hemostasis of dynamic wet tissue trauma and sealing of wound liquid leakage under physiological conditions.

[0054] In order to further illustrate the present invention, the following examples are provided for detailed description. The experimental raw materials used in the following examples of the present invention are all common commercially available products.

[0055] Example 1 (1) 4 g of 2000 kDa sodium polyglutamate powder was dissolved in 200 mL of deionized water, and then 0.24 g of 3-amino-1,2-propanediol was added dropwise to the above solution. The mixture was stirred for 30 min, and then 1.25 mL of 0.28 g / mL 1-hydroxybenzotriazole in DMSO was added. The pH of the solution was adjusted to 6 with 1 mol / L hydrochloric acid solution. After 20 min of treatment, 1.25 mL of 0.12 g / mL 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) aqueous solution was added. The reaction was stirred at room temperature for 12 h, and the reaction solution was transferred to a dialysis bag (molecular weight cutoff = 8~14 kDa). It was dialyzed in an aqueous solution with a pH of 2 at room temperature for 3 days, and the water was changed every 3 h. The dialyzed solution was freeze-dried to obtain hydroxylated polyglutamic acid powder.

[0056] 4 g of hydroxylated polyglutamic acid powder was dissolved in 200 mL of deionized water to obtain a hydroxylated polyglutamic acid solution with a concentration of 2 wt%, and a certain amount of 1 mol / L sodium hydroxide solution was added until the solution became transparent, and then 2.5 mL of 0.236 g / mL (1.1 mol / L) NaIO4 solution was added dropwise, and stirred for 1 h at room temperature in the dark, and then 0.8 g of ethylene glycol was added, and stirred for 1 h at room temperature, and the solution after the reaction was transferred to a dialysis bag (molecular weight cutoff = 8~14 kDa), and dialyzed in an aqueous solution with a pH of 2 at room temperature for 3 days, and the water was changed every 3 h. The dialyzed solution was freeze-dried to obtain an acidic aldehyde-modified polyglutamic acid sponge.

[0057] (2) Dissolve 24 g of spherical lysozyme in 500 mL of deionized water to obtain a spherical lysozyme solution with a concentration of 4.8 wt%. Transfer the solution to a dialysis bag (molecular weight cutoff = 8~14 kDa) and dialyze in ultrapure water at room temperature for 3 days, changing the water every 3 hours. The dialyzed solution was freeze-dried to obtain a purified spherical lysozyme powder. Dissolve 10 g of the purified spherical lysozyme powder in 500 mL of deionized water to obtain a spherical lysozyme solution with a concentration of 2 wt%. Adjust the pH of the solution to 2 with 1 mol / L hydrochloric acid solution, and stir the reaction at 90 °C for 12 hours. Then, immediately immerse the reaction solution in an ice water bath to quench the reaction. Transfer the solution to a dialysis bag (molecular weight cutoff = 8~14 kDa) and dialyze in ultrapure water at room temperature for 3 days, changing the water every 3 hours. The dialyzed solution was freeze-dried to obtain a linear lysozyme powder.

[0058] (3) Dissolve 2 g of acidic aldehyde-modified polyglutamic acid sponge in 100 mL of deionized water and stir to make it dissolve evenly to obtain an acidic aldehyde-modified polyglutamic acid solution with a concentration of 2 wt%.

[0059] (4) Dissolve 2 g of hen egg white spherical lysozyme in 100 mL of deionized water and stir to dissolve evenly to obtain a hen egg white spherical lysozyme solution with a concentration of 2 wt%.

[0060] (5) Dissolve 2 g of hen egg white linear lysozyme in 100 mL of deionized water and stir to dissolve evenly to obtain a hen egg white linear lysozyme solution with a concentration of 2 wt%.

[0061] (6) The acidic aldehyde-modified polyglutamic acid, the linear protein and the globular protein are mixed at a weight ratio of 10:0:10 at room temperature, and the mixture is quickly stirred to mix well. The precipitate is poured into a mold and freeze-dried for 20 minutes to obtain a medical tissue adhesive.

[0062] The bonding strength of the medical tissue adhesive was tested using the 180° tensile test mode of a universal testing machine with a tensile speed of 50 mm / min and three parallel samples.

[0063] Immerse the medical tissue adhesive in PBS solution. At specific time intervals (e.g., 12 h), take the adhesive out of the solution and gently wipe off the residual moisture on the surface with filter paper. Then weigh the adhesive and put it back into the solution until the weight of the adhesive no longer changes. Measure the effect of the solution on the bonding strength of the adhesive with three parallel samples.

[0064] Figure 1 Images of the fracture, self-healing and stress tolerance processes of the tissue adhesive provided for this embodiment; Figure 2 The lap shear adhesion strength images of the tissue adhesive provided in this embodiment at different time points under PBS conditions. Figure 1 It can be seen that the adhesive has good self-healing properties. Figure 2 It can be seen that the aldehyde modification makes the adhesive have good underwater adhesion.

[0065] Example 2 The medical tissue adhesive was prepared by the same method as in Example 1, except that in this example, the acidic aldehyde-modified polyglutamic acid, the linear protein and the globular protein were mixed at a weight ratio of 10:1:9 at room temperature.

[0066] The bonding strength of the adhesive was tested using the 180° tensile test mode of a universal testing machine with a tensile speed of 50 mm / min and three parallel samples.

[0067] The adhesive was soaked in PBS solution. At specific time intervals, the adhesive was taken out from the above solution and the residual moisture on the surface was gently wiped off with filter paper. The adhesive was then weighed and then placed back into the above solution until the weight of the adhesive no longer changed. Three parallel samples were used.

[0068] Example 3 The medical tissue adhesive was prepared by the same method as in Example 1, except that in this example, the acidic aldehyde-modified polyglutamic acid, the linear protein and the globular protein were mixed at a weight ratio of 10:3:7 at room temperature.

[0069] The bonding strength of the adhesive was tested using the 180° tensile test mode of a universal testing machine with a tensile speed of 50 mm / min and three parallel samples.

[0070] The adhesive was soaked in PBS solution. At specific time intervals, the adhesive was taken out from the above solution and the residual moisture on the surface was gently wiped off with filter paper. The adhesive was then weighed and then placed back into the above solution until the weight of the adhesive no longer changed. Three parallel samples were used.

[0071] Example 4 The medical tissue adhesive was prepared by the same method as in Example 1, except that in this example, the acidic aldehyde-modified polyglutamic acid, the linear protein and the globular protein were mixed at a weight ratio of 10:5:5 at room temperature.

[0072] The bonding strength of the adhesive was tested using the 180° tensile test mode of a universal testing machine with a tensile speed of 50 mm / min and three parallel samples.

[0073] The adhesive was soaked in PBS solution. At specific time intervals, the adhesive was taken out from the above solution and the residual moisture on the surface was gently wiped off with filter paper. The adhesive was then weighed and then placed back into the above solution until the weight of the adhesive no longer changed. Three parallel samples were used.

[0074] Example 5 The medical tissue adhesive was prepared by the same method as in Example 1, except that in this example, the acidic aldehyde-modified polyglutamic acid, the linear protein and the globular protein were mixed at a weight ratio of 10:7:3 at room temperature.

[0075] The bonding strength of the adhesive was tested using the 180° tensile test mode of a universal testing machine with a tensile speed of 50 mm / min and three parallel samples.

[0076] The adhesive was soaked in PBS solution. At specific time intervals, the adhesive was taken out from the above solution and the residual moisture on the surface was gently wiped off with filter paper. The adhesive was then weighed and then placed back into the above solution until the weight of the adhesive no longer changed. Three parallel samples were used.

[0077] Example 6 The medical tissue adhesive was prepared by the same method as in Example 1, except that in this example, the acidic aldehyde-modified polyglutamic acid, the linear protein and the globular protein were mixed at a weight ratio of 10:9:1 at room temperature.

[0078] The bonding strength of the adhesive was tested using the 180° tensile test mode of a universal testing machine with a tensile speed of 50 mm / min and three parallel samples.

[0079] The adhesive was soaked in PBS solution. At specific time intervals, the adhesive was taken out from the above solution and the residual moisture on the surface was gently wiped off with filter paper. The adhesive was then weighed and then placed back into the above solution until the weight of the adhesive no longer changed. Three parallel samples were used.

[0080] Example 7 The medical tissue adhesive was prepared by the same method as in Example 1, except that in this example, the acidic aldehyde-modified polyglutamic acid, the linear protein and the globular protein were mixed at a weight ratio of 10:10:0 at room temperature.

[0081] The bonding strength of the adhesive was tested using the 180° tensile test mode of a universal testing machine with a tensile speed of 50 mm / min and three parallel samples.

[0082] The adhesive was soaked in PBS solution. At specific time intervals, the adhesive was taken out from the above solution and the residual moisture on the surface was gently wiped off with filter paper. The adhesive was then weighed and then placed back into the above solution until the weight of the adhesive no longer changed. Three parallel samples were used.

[0083] The specific test results of the above embodiments 1 to 7 are shown in Table 1 below: Table 1 Detection indicators Example 1 (10:0:10) Example 2 (10:1:9) Example 3 (10:3:7) Example 4 (10:5:5) Example 5 (10:7:3) Example 6 (10:9:1) Example 7 (10:10:0) Adhesion strength 23.0 kPa 33.4 kPa 41.5 kPa 50.8 kPa 35.7 kPa 11.1 kPa 5.4 kPa Swelling rate 87.2% 70.0% 55.9% 34.0% 24.8% 10.5% 5.5% From the data in Table 1, it can be seen that, as the content of fixed aldehyde-modified polyglutamic acid increases with the increase of the linear lysozyme content in the system, the bonding strength of the adhesive to the interface first increases and then weakens, while the swelling rate decreases with the increase of linear lysozyme.

[0084] Example 8 (1) Dissolve 4 g of 2000 kDa sodium polyglutamate powder in 200 mL of deionized water, then add 0.24 g of 3-amino-1,2-propanediol dropwise to the solution, stir for 30 min, then add 1.25 mL of 0.28 g / mL 1-hydroxybenzotriazole DMSO solution, adjust the solution pH to 6 with 1 mol / L hydrochloric acid solution, and add 1.25 mL of 0.12 g / mL 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) aqueous solution. Stir the reaction at room temperature for 12 h, transfer the reaction solution to a dialysis bag (molecular weight cutoff = 8~14 kDa), and dialyze in a pH 2 aqueous solution at room temperature for 3 days, changing the water every 3 h. The dialyzed solution is freeze-dried to obtain hydroxylated polyglutamic acid powder. 4 g of hydroxylated polyglutamic acid powder was dissolved in 200 mL of deionized water to obtain a hydroxylated polyglutamic acid solution with a concentration of 2 wt%, and a certain amount of 1 mol / L sodium hydroxide solution was added until the solution became transparent, and then 2.5 mL of 0.236 g / mL NaIO4 solution was added dropwise, and stirred for 1 h at room temperature in the dark, and then 0.8 g of ethylene glycol was added, and stirred for 1 h at room temperature, and the solution after the reaction was transferred to a dialysis bag (molecular weight cutoff = 8~14 kDa), and dialyzed in an aqueous solution with a pH of 2 at room temperature for 3 days, and the water was changed every 3 h. The dialyzed solution was freeze-dried to obtain an acidic aldehyde-modified polyglutamic acid sponge.

[0085] (2) Dissolve 24 g of spherical lysozyme in 500 mL of deionized water to obtain a spherical lysozyme solution with a concentration of 4.8 wt%. Transfer the solution to a dialysis bag (molecular weight cutoff = 8~14 kDa) and dialyze in ultrapure water at room temperature for 3 days, changing the water every 3 hours. The dialyzed solution was freeze-dried to obtain a purified spherical lysozyme powder. Dissolve 10 g of the purified spherical lysozyme powder in 500 mL of deionized water to obtain a spherical lysozyme solution with a concentration of 2 wt%. Adjust the pH of the solution to 2 with 1 mol / L hydrochloric acid solution, and stir the reaction at 90 °C for 12 hours. Then, immediately immerse the reaction solution in an ice-water bath to quench the reaction. Transfer the solution to a dialysis bag (molecular weight cutoff = 8~14 kDa) and dialyze in ultrapure water at room temperature for 3 days, changing the water every 3 hours. The dialyzed solution was freeze-dried to obtain a linear lysozyme powder.

[0086] (3) Dissolve 2 g of acidic aldehyde-modified polyglutamic acid sponge in 100 mL of deionized water and stir to make it dissolve evenly to obtain an acidic aldehyde-modified polyglutamic acid solution with a concentration of 2 wt%.

[0087] (4) Dissolve 2 g of hen egg white spherical lysozyme in 100 mL of deionized water and stir to dissolve evenly to obtain a hen egg white spherical lysozyme solution with a concentration of 2 wt%.

[0088] (5) Dissolve 2 g of hen egg white linear lysozyme in 100 mL of deionized water and stir to dissolve evenly to obtain a hen egg white linear lysozyme solution with a concentration of 2 wt%.

[0089] (6) The acidic aldehyde-modified polyglutamic acid, the globular protein and the linear protein are mixed at a weight ratio of 10:0:10 at room temperature, and the mixture is quickly stirred to mix well. The precipitate is poured into a mold and freeze-dried for 20 minutes to obtain a medical tissue adhesive.

[0090] The bonding strength of the medical tissue adhesive was tested using the 180° tensile test mode of the universal testing machine, with a waiting time of 12 h, a tensile speed of 50 mm / min, and three parallel samples.

[0091] The adhesion strength of the medical tissue adhesive of this embodiment to wet pig skin tissue under a wet physiological environment is 37.4 kPa, indicating that the medical tissue adhesive provided by the present invention has excellent adhesion stability.

[0092] Comparative Example 1 (1) Dissolve 4 g of 2000 kDa sodium polyglutamate powder in 200 mL of deionized water and dialyze it in a pH 2 aqueous solution at room temperature for 3 days, changing the water every 3 hours. The dialyzed solution is freeze-dried to obtain an acidic polyglutamate sponge.

[0093] (2) Dissolve 24 g of spherical lysozyme in 500 mL of deionized water to obtain a spherical lysozyme solution with a concentration of 4.8 wt%. Transfer the solution to a dialysis bag (molecular weight cutoff = 8~14 kDa) and dialyze in ultrapure water at room temperature for 3 days, changing the water every 3 hours. The dialyzed solution was freeze-dried to obtain a purified spherical lysozyme sponge. Dissolve 10 g of the purified spherical lysozyme sponge in 500 mL of deionized water to obtain a spherical lysozyme solution with a concentration of 2 wt%. Adjust the pH of the solution to 2 with 1 mol / L hydrochloric acid solution, and stir the reaction at 90 °C for 12 hours. Then, immediately immerse the reaction solution in an ice-water bath to quench the reaction. Transfer the solution to a dialysis bag (molecular weight cutoff = 8~14 kDa) and dialyze in ultrapure water at room temperature for 3 days, changing the water every 3 hours. The dialyzed solution was freeze-dried to obtain a linear lysozyme sponge.

[0094] (3) Dissolve 2 g of acidic polyglutamic acid sponge in 100 mL of deionized water and stir to make it dissolve evenly to obtain an acidic polyglutamic acid solution with a concentration of 2 wt%.

[0095] (4) Dissolve 2 g of spherical lysozyme in 100 mL of deionized water and stir to obtain a spherical lysozyme solution with a concentration of 2 wt%.

[0096] (5) Dissolve 2 g of linear lysozyme in 100 mL of deionized water and stir to obtain a linear lysozyme solution with a concentration of 2 wt%.

[0097] (6) Mixing acidic polyglutamic acid, the linear protein and the globular protein at a weight ratio of 10:5:5 at room temperature, stirring rapidly to mix, pouring the precipitate into a mold, and freeze-drying for 20 minutes to obtain a medical tissue adhesive.

[0098] The bonding strength of the adhesive was tested using the 180° tensile test mode of the universal testing machine, waiting for 12 h, with a tensile speed of 50 mm / min and three parallel samples.

[0099] The adhesion strength of the medical tissue adhesive of this embodiment to wet pig skin tissue under a wet physiological environment is 9.8 kPa.

[0100] It can be seen from the comparison between the examples and comparative example 1 that the use of acidic polyglutamic acid to replace the acidic aldehyde-modified polyglutamic acid in the present application will reduce the adhesion strength of the obtained tissue adhesive.

[0101] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A modified polyglutamic acid-based tissue adhesive, characterized in that: The components by mass fraction are: 1-30 wt% of acidic aldehyde-modified polyglutamic acid; 1-15 wt% linear protein; 1-15 wt% globular protein; The balance is water.

2. The modified polyglutamic acid-based tissue adhesive according to claim 1, characterized in that: The pH of the acidic aldehyde-formyl polyglutamic acid is 1 to 3; The degree of substitution of the aldehyde group of the acidic aldehyde-formyl polyglutamic acid is 2-20%; The molecular weight of the acidic aldehyde-modified polyglutamic acid is 100-2000 kDa.

3. The modified polyglutamic acid-based tissue adhesive according to claim 1 or 2, characterized in that: The globular protein is selected from any one or more of plant lysozyme, animal lysozyme, microbial lysozyme or egg white lysozyme.

4. The modified polyglutamic acid-based tissue adhesive according to any one of claims 1 to 3, characterized in that: The linear protein is selected from any one or more of reduced plant lysozyme, animal lysozyme, microbial lysozyme or egg white lysozyme.

5. The modified polyglutamic acid-based tissue adhesive according to claim 3 or 4, characterized in that: The plant lysozyme is selected from fig lysozyme; The animal lysozyme is selected from mammalian lysozyme; The microbial lysozyme is selected from glucosaminidase-like lysozyme; The egg white lysozyme is selected from hen egg white lysozyme.

6. The method for preparing the modified polyglutamic acid-based tissue adhesive according to any one of claims 1 to 5, characterized in that: The following steps are involved: S1: subjecting sodium polyglutamate to aldehyde treatment and acid treatment to obtain acidic aldehyde-treated polyglutamic acid; The spherical protein is reduced to obtain the linear protein; S2: Evenly mix the acidic aldehyde-modified polyglutamic acid, the globular protein and the linear protein in the presence of a solvent to obtain a modified polyglutamic acid-based tissue adhesive.

7. The preparation method according to claim 6, characterized in that: The process of obtaining the acidic aldehyde-formyl polyglutamic acid is: The sodium polyglutamate is subjected to hydroxylation treatment, first acidification treatment, oxidation treatment and second acidification treatment; the molecular weight of the sodium polyglutamate is 100-2000 kDa.

8. The preparation method according to claim 6 or 7, characterized in that: The reagent for the hydroxylation treatment is, but is not limited to, any one or more of 3-amino-1,2-propanediol, D-glucosamine, 3-amino-1-propanol, ethanolamine, or 3-amino-2-methylpropanol; the reagent for the oxidation treatment is sodium periodate; and the oxidation treatment time is 30 to 120 min; The pH of the first acidification treatment and the second acidification treatment is 1-3, the temperature of the first acidification treatment and the second acidification treatment is 0-60° C., and the time of the first acidification treatment and the second acidification treatment is 24-72 h.

9. The preparation method according to any one of claims 6 to 8, characterized in that The reduction treatment comprises: reacting the spherical protein at a pH of 1 to 3, and then quenching the obtained reaction solution in an ice water bath; The solvent is water.

10. A dynamic tissue wound dressing, characterized in that: The adhesive is prepared from the modified polyglutamate-based tissue adhesive according to any one of claims 1 to 5 or the modified polyglutamate-based tissue adhesive prepared by the preparation method according to any one of claims 6 to 9.

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