Medical tissue adhesive, medical tissue dry adhesive patch and preparation method and application thereof

Abstract

The present application relates to the field of medical tissue adhesive, in particular to a medical tissue adhesive, a medical tissue dry adhesive patch and a preparation method and application thereof. The present application provides a medical tissue adhesive, a medical tissue dry adhesive patch and a preparation method and application thereof. The medical tissue adhesive provided by the present application comprises: 1wt%-30wt% of acidic polyglutamic acid; 1wt%-30wt% of spherical protein; and the balance of water. The medical tissue adhesive provided by the present application has high tissue adhesion strength and fast hemostatic sealing speed in a wet physiological environment, meets the requirements of rapid and efficient tissue hemostatic sealing, and can be applied to rapid hemostasis of tissue wounds in a wet physiological environment, sealing of wound liquid leakage, healing and repair of wound tissue, and antibacterial of wounds. Experiments show that the dry adhesive patch made of the adhesive provided by the present application has high tissue adhesion strength in a wet physiological environment, and the adhesion strength to a wet pigskin tissue can reach more than 60kPa.

Description

Technical Field

[0001] This invention relates to the field of medical tissue adhesives, specifically medical tissue adhesives, medical tissue dry adhesive patches, their preparation methods, and applications. Background Technology

[0002] Statistics show that over 114 million people worldwide undergo wound closure surgery annually due to life-threatening injuries. Currently, surgical sutures or staples are still used to close wounds, but traditional suturing methods are not only cumbersome and laborious, requiring professional operation, but also prone to bleeding and new damage, leading to bacterial infections and causing immense suffering for patients. Therefore, using tissue adhesives to replace or partially replace sutures, and closing surgical incisions or injured areas with adhesives, is currently considered the most ideal repair method. An ideal medical tissue adhesive should be safe, non-toxic, non-irritating, non-teratogenic, have high adhesive strength, good biocompatibility, excellent antibacterial activity, be usable in the presence of blood or tissue fluid, have degradation products or leachates that do not damage tissue, affect tissue regeneration and wound healing processes, and be non-cytotoxic and easy to use. FDA-approved medical tissue adhesives mainly include α-cyanoacrylate and fibrin adhesives. While α-cyanoacrylates offer fast curing and high bonding strength, blood and tissue fluid on the tissue surface severely affect their adhesive properties. Furthermore, they cause significant irritation to the tissue during curing, their high modulus makes them difficult to match with human tissue, and their degradation product, formaldehyde, is highly toxic. Fibrin adhesives offer good biocompatibility, but their low bonding strength and high cost, along with the risk of cross-infection from blood-borne diseases, make them crucial for developing a medical tissue adhesive that is biocompatible, has a modulus similar to that of tissue, strong adhesion, excellent antibacterial properties, and promotes tissue regeneration. Summary of the Invention

[0003] In view of this, the technical problem to be solved by the present invention is to provide a medical tissue adhesive, a medical tissue dry adhesive patch, a preparation method thereof, and an application thereof. The medical tissue adhesive provided by the present invention has high tissue adhesion strength and fast tissue sealing speed under moist physiological environment, and has excellent antibacterial properties, and can promote tissue regeneration, so as to meet the tissue adhesion and repair needs of the diverse and dynamically complex wound environment.

[0004] This invention provides a medical tissue adhesive, comprising:

[0005] 1 wt% to 30 wt% acidic polyglutamic acid;

[0006] 1 wt% to 30 wt% of globular proteins;

[0007] And the remaining water.

[0008] The present inventors have found that co-assembly of acidic polyglutamic acid and globular protein can achieve the synergy of rapid water absorption and strong tissue adhesion of medical tissue adhesive in wet physiological environment, facilitate the formation of interaction between adhesion groups and interface groups of bleeding tissue, endow the polyglutamic acid medical tissue adhesive with strong, stable and durable wet tissue adhesion in wet physiological environment, achieve rapid and efficient tissue hemostatic sealing effect in wet physiological environment, fast hemostatic sealing speed, and less tissue bleeding, and can adapt to dynamic and complex bleeding environment such as large-area wound bleeding and internal organ bleeding. In some embodiments of the present application, the medical tissue adhesive preferably comprises 1wt%-5wt% of acidic polyglutamic acid, 1wt%-5wt% of globular protein, and the balance of water.

[0009] The medical tissue adhesive provided by the present application comprises 1wt%-30wt% of acidic polyglutamic acid. In some embodiments of the present application, the medical tissue adhesive preferably comprises 1wt%-5wt% of acidic polyglutamic acid. In one embodiment, the medical tissue adhesive comprises 1wt% of acidic polyglutamic acid.

[0010] The acidic polyglutamic acid described in the present application has a pH of 1-3. In one embodiment, the acidic polyglutamic acid has a pH of 1.5. The lower the pH value, the more carboxyl groups in the polyglutamic acid, and the greater the ability to absorb the blood interfacial hydration layer at the tissue wound or organ damage through hydrogen bonding. In addition, the carboxyl groups of the polyglutamic acid can form hydrogen bonds with the amino groups, carboxyl groups and hydroxyl groups on the surface of the tissue, which can significantly improve the interfacial adhesion between the polyglutamic acid medical tissue adhesive and the wounded tissue.

[0011] The acidic polyglutamic acid described in the present application has a molecular weight of 2kDa-2000kDa. In some embodiments, the acidic polyglutamic acid has a molecular weight of 2kDa, 10kDa, 100kDa, 200kDa, 700kDa, 1100kDa or 2000kDa. The acidic polyglutamic acid described in the present application is obtained by acidification of polyglutamic acid sodium. The molecular weight of the acidic polyglutamic acid depends on the molecular weight of the polyglutamic acid sodium. As the molecular weight increases, the mechanical properties of the medical tissue adhesive obtained are better, and the adhesion strength to soft tissue is greater.

[0012] The medical tissue adhesive provided by the present application further comprises 1wt%-30wt% of a spherical protein. In some embodiments of the present application, the medical tissue adhesive further preferably comprises 1wt%-5wt% of a spherical protein. In one embodiment, the medical tissue adhesive further comprises 1wt% of a spherical protein. The spherical protein of the present application is selected from lysozyme spherical protein (LZM); in some embodiments of the present application, the spherical protein is selected from at least one of plant lysozyme, animal lysozyme, microbial lysozyme or egg white lysozyme; specifically, the plant lysozyme is selected from ficin; the animal lysozyme is selected from mammalian lysozyme; the microbial lysozyme is selected from glucosaminidase-like lysozyme; and the egg white lysozyme is selected from hen egg white lysozyme.

[0013] In some embodiments of the present application, the weight ratio of the acid polyglutamic acid and the spherical protein is 1:1. The medical tissue adhesive provided by the present application, with acid polyglutamic acid and spherical protein as main components, not only has strong adhesion performance in a wet physiological environment, but also has the effect of promoting cell proliferation. In the synergistic effect of the spherical protein, the polyglutamic acid medical tissue adhesive has excellent antibacterial performance, reduces bacterial infection and inflammation, and thus promotes the healing and regeneration repair of wound tissue. Moreover, both components are derived from natural compounds. Therefore, the present application has good biocompatibility and biodegradability, simple preparation process, low cost, and great application potential.

[0014] The present application provides a preparation method of the above medical tissue adhesive, comprising the following steps:

[0015] acidifying polyglutamic acid sodium to obtain acid polyglutamic acid;

[0016] mixing the acid polyglutamic acid solution and the spherical protein solution to obtain the medical tissue adhesive.

[0017] The present application first acidifies polyglutamic acid sodium to obtain acid polyglutamic acid. Specifically, the present application acidifies polyglutamic acid sodium in an acid solution, purifies, dries, and obtains acid polyglutamic acid. In one embodiment, the present application dissolves polyglutamic acid sodium powder in an aqueous solution, then adds an acid solution for acidification, performs dialysis with deionized water, and performs vacuum freeze-drying on the dialyzed solution to obtain acid polyglutamic acid. The acid polyglutamic acid obtained by the present application is sponge-like, also known as acid polyglutamic acid sponge. Dissolving the acid polyglutamic acid sponge in water can obtain an acid polyglutamic acid solution.

[0018] The polyglutamic acid sodium has a molecular weight of 2 kDa to 2000 kDa. In some embodiments, the polyglutamic acid sodium has a molecular weight of 2 kDa, 10 kDa, 100 kDa, 200 kDa, 700 kDa, 1100 kDa or 2000 kDa. With the increase of the molecular weight of the polyglutamic acid sodium, the mechanical property of the polyglutamic acid medical tissue adhesive is better, and the adhesion strength to soft tissue is greater.

[0019] The acidification treatment is performed in an acid liquor, which includes but is not limited to at least one of hydrochloric acid, citric acid, phosphoric acid, oxalic acid or sulfuric acid; and the concentration of the acid liquor is 1 mol / L to 8 mol / L. In one embodiment, the temperature of the acidification treatment is 0 ℃ to 60 ℃, and the time of the acidification treatment is 10 min to 30 min.

[0020] The acidification treatment is performed at a pH of 1 to 3. Maintaining the pH of 1 to 3 can convert the carboxylate in the polyglutamic acid into carboxylic acid sufficiently, increase the number of carboxyl groups in the polyglutamic acid, and enhance the water absorption of the medical tissue adhesive prepared subsequently.

[0021] After obtaining the acid polyglutamic acid, the acid polyglutamic acid solution and the spherical protein are mixed to obtain the medical tissue adhesive. Specifically, the acid polyglutamic acid is mixed with water to obtain an acid polyglutamic acid solution, the spherical protein is mixed with water to obtain a spherical protein solution, and the acid polyglutamic acid solution and the spherical protein solution are mixed to obtain the medical tissue adhesive. In some embodiments of the present application, the acid polyglutamic acid is dissolved in deionized water, and is uniformly dissolved at room temperature to obtain the acid polyglutamic acid solution; the spherical protein is dissolved in deionized water, and is uniformly dissolved by stirring at room temperature to obtain the spherical protein solution; and the acid polyglutamic acid solution and the spherical protein solution are mixed at room temperature to obtain the medical tissue adhesive.

[0022] The concentration of the acid polyglutamic acid solution is 1 wt% to 60 wt%, preferably 1 wt% to 10 wt%, more preferably 2 wt% to 10 wt%, and more preferably 2 wt%; and the concentration of the spherical protein solution is 1 wt% to 60 wt%, preferably 1 wt% to 10 wt%, more preferably 2 wt% to 10 wt%, and more preferably 2 wt%. The acid polyglutamic acid aqueous solution and the spherical protein aqueous solution are mixed at a volume ratio of 1:1. The acid polyglutamic acid and the spherical protein are the same as described above, and will not be described again.

[0023] The application provides a preparation method of a medical tissue adhesive, which comprises the following steps: uniformly mixing polyglutamic acid sponge obtained through strong acidification treatment with spherical protein to prepare the medical tissue adhesive.

[0024] The application further provides a medical tissue dry adhesive patch prepared from the medical tissue adhesive.

[0025] The medical tissue adhesive prepared by the application can safely and quickly seal skin incisions, organ defects, gas and liquid leakage, and has good healing effect.

[0026] The application further provides application of the medical tissue adhesive or the medical tissue adhesive prepared by the method or the medical tissue dry adhesive patch in preparation of a tissue wound hemostasis aid, a tissue wound closure product, a tissue wound repair product and a tissue wound antibacterial product.

[0027] The application provides a medical tissue adhesive, a medical tissue dry adhesive patch and a preparation method and application thereof. The medical tissue adhesive provided by the application comprises 1wt%-30wt% of acidic polyglutamic acid, 1wt%-30wt% of spherical protein and the balance of water. The medical tissue adhesive provided by the application has high tissue adhesion strength and fast hemostatic sealing speed in a wet physiological environment, meets the requirements of fast and efficient tissue hemostatic sealing, and can be applied to fast hemostasis of tissue wounds in a wet physiological environment, sealing of liquid leakage of wounds, healing and repair of wound tissues and antibacterial of wounds. Experiments show that the dry adhesive patch made of the medical tissue adhesive has high tissue adhesion strength in a wet physiological environment, and the adhesion strength to a wet pigskin tissue can reach more than 60kPa. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 It is a tissue adhesion tensile test diagram;

[0029] Figure 2 It is a schematic diagram of adhesion and sealing of a pig stomach;

[0030] Figure 3 It is a comparison diagram of the appearance of the medical tissue adhesive prepared in Example 10 and Comparative Examples 3-5;

[0031] Figure 4 It is a schematic diagram of the adhesive properties of the adhesive prepared by the acidic polyglutamic acid and LZM protein and Plys protein respectively;

[0032] Figure 5 It is a live / dead staining diagram of L929 cells directly contacted with a cell culture plate (control) and the medical tissue adhesive (experimental group) described in the application for 5 days;

[0033] Figure 6 It is a schematic diagram of surviving bacteria of Escherichia coli and Staphylococcus aureus proliferated on an agar plate after being contacted with a d-PGA / LZM adhesive or a polyethylene film (control);

[0034] Figure 7 It is a test result diagram of the antibacterial ability of the d-PGA / LZM adhesive to Escherichia coli and Staphylococcus aureus;

[0035] Figure 8 It is a schematic diagram of an antibacterial activity inhibition zone test;

[0036] Figure 9 It is a diagram of the inhibition zone diameter of the quantitative antibacterial test of PE film and d-PGA / LZM to Escherichia coli and Staphylococcus aureus;

[0037] Figure 10 It is a wound healing diagram of SD rats in the 15th day in the test of promoting healing of infected wounds;

[0038] Figure 11 H&E staining images of the wound tissue after 15 days in the control group and the d-PGA / LZM group in the SD rat infected wound healing promotion test;

[0039] Figure 12 Masson staining images of the wound tissue after 15 days in the control group and the d-PGA / LZM group in the SD rat infected wound healing promotion test. DETAILED DESCRIPTION

[0040] The present application discloses a medical tissue adhesive, a medical tissue dry adhesive patch, and a preparation method and application thereof. Those skilled in the art can refer to the content herein and appropriately improve the process parameters for implementation. It is particularly important to note that all similar substitutions and changes are obvious to those skilled in the art, and they are considered to be included in the present application. The method and application of the present application have been described by preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the method and application herein without departing from the content, spirit and scope of the present application, to realize and apply the present application technology.

[0041] Poly(γ-glutamic acid) sodium (PGNa) with different molecular weights (2kDa, 10kDa, 100kDa, 200kDa, 700kDa, 1100kDa, 2000kDa) was purchased from Nanjing Xuan Kai. Chicken egg white lysozyme was purchased from Saite. Fluorescein isothiocyanate (FITC) was purchased from Aladdin. Other chemical reagents (AR grade) can be used directly. Cell counting kit-8 (CCK-8) was from Boost Biological Technology Co., Ltd., hematoxylin and eosin staining (H&E staining kit) was purchased from Wuhan Service Biological Technology Co., Ltd.

[0042] Staphylococcus aureus (ATCC 6538) and Escherichia coli (ATCC 25922) were from Nanjing Clinical Biological Technology Co., Ltd. L929 mouse fibroblast cell line was from Shanghai Astronautical Cell Resource Center, Chinese Academy of Sciences. Female SD rats (~ 250g) purchased from Liaoning Changsheng Biological Technology Co., Ltd. were used for in vivo experiments. All animal experiments were conducted in accordance with the guidelines of the Animal Care and Ethics Committee of Changchun Institute of Applied Chemistry, Chinese Academy of Sciences (2201042839670).

[0043] The present application is further described in conjunction with the following examples:

[0044] Example 1

[0045] (1) 2 g of 2 kDa poly(γ-glutamic acid) sodium powder was dissolved in 100 mL of deionized water, and then 3.4 mL of 6 mol / L hydrochloric acid solution was added to adjust the pH to 1.5. The acidified solution was loaded into a dialysis bag and dialyzed with deionized water. The dialyzed solution was placed in a freeze dryer at -60°C for vacuum freeze drying for 48 hours to obtain a polyglutamic acid sponge.

[0046] (2) 2 g of polyglutamic acid sponge was dissolved in 100 mL of deionized water, and stirred to dissolve uniformly to obtain a polyglutamic acid solution with a concentration of 2 wt%.

[0047] (3) 2 g of hen egg white lysozyme was dissolved in 100 mL of deionized water, and stirred to dissolve uniformly to obtain a hen egg white lysozyme solution with a concentration of 2 wt%.

[0048] (4) The same volume of the above acidified reaction treated polyglutamic acid solution and hen egg white lysozyme solution were mixed at room temperature, and stirred quickly to mix uniformly. The precipitate was poured into a mold and freeze dried for 20 min to obtain a polyglutamic acid medical tissue adhesive dry adhesive patch.

[0049] The adhesive strength of the adhesive was tested using the 180° tensile test mode of the universal testing machine, with a tensile speed of 50 mm / min, and three parallel samples.

[0050] Example 2

[0051] A polyglutamic acid medical tissue adhesive dry adhesive patch was prepared by the same method as in Example 1, except that the poly(γ-glutamic acid) sodium powder used in this example was 10 kDa poly(γ-glutamic acid) sodium powder.

[0052] The adhesive strength of the adhesive was tested using the 180° tensile test mode of the universal testing machine, with a tensile speed of 50 mm / min, and three parallel samples.

[0053] Example 3

[0054] A polyglutamic acid medical tissue adhesive dry adhesive patch was prepared by the same method as in Example 1, except that the poly(γ-glutamic acid) sodium powder used in this example was 100 kDa poly(γ-glutamic acid) sodium powder.

[0055] The adhesive strength of the adhesive was tested using the 180° tensile test mode of the universal testing machine, with a tensile speed of 50 mm / min, and three parallel samples.

[0056] Example 4

[0057] A polyglutamic acid medical tissue adhesive dry adhesive patch was prepared by the same method as Example 1, except that the poly(γ-glutamic acid) sodium powder used in this example was a 700 kDa poly(γ-glutamic acid) sodium powder.

[0058] The adhesive strength of the adhesive was tested using the 180° tensile test mode of the universal testing machine, with a tensile speed of 50 mm / min, and three parallel samples.

[0059] Example 5

[0060] A polyglutamic acid medical tissue adhesive dry adhesive patch was prepared by the same method as Example 1, except that the poly(γ-glutamic acid) sodium powder used in this example was a 700 kDa poly(γ-glutamic acid) sodium powder.

[0061] The adhesive strength of the adhesive was tested using the 180° tensile test mode of the universal testing machine, with a tensile speed of 50 mm / min, and three parallel samples.

[0062] Example 6

[0063] A polyglutamic acid medical tissue adhesive dry adhesive patch was prepared by the same method as Example 1, except that the poly(γ-glutamic acid) sodium powder used in this example was a 700 kDa poly(γ-glutamic acid) sodium powder.

[0064] The adhesive strength of the adhesive was tested using the 180° tensile test mode of the universal testing machine, with a tensile speed of 50 mm / min, and three parallel samples.

[0065] Example 7

[0066] A polyglutamic acid medical tissue adhesive dry adhesive patch was prepared by the same method as Example 1, except that the poly(γ-glutamic acid) sodium powder used in this example was a 700 kDa poly(γ-glutamic acid) sodium powder.

[0067] The adhesive strength of the adhesive was tested using the 180° tensile test mode of the universal testing machine, with a tensile speed of 50 mm / min, and three parallel samples.

[0068] The specific test results of the above Examples 1-7 are shown in Table 1 below

[0069] Table 1 Adhesive strength values of the adhesive in each example

[0070]

[0071] Example 8

[0072] (1) 2 g of 2000 kDa poly (γ-glutamic acid) sodium powder was dissolved in 100 mL of deionized water, then 3.4 mL of 6 mol / L hydrochloric acid solution was added to adjust the pH to 1.5, the acidified solution was loaded into a dialysis bag, dialyzed with deionized water, and the dialyzed solution was placed in a freeze dryer at -60°C for vacuum freeze drying for 48 hours to obtain a polyglutamic acid sponge.

[0073] (2) 2 g of polyglutamic acid sponge was dissolved in 100 mL of deionized water, and stirred to dissolve uniformly to obtain a polyglutamic acid solution with a concentration of 2 wt%.

[0074] (3) 2 g of hen egg white lysozyme was dissolved in 100 mL of deionized water, and stirred to dissolve uniformly to obtain a hen egg white lysozyme solution with a concentration of 2 wt%.

[0075] (4) The same volume of the above acidification reaction treated polyglutamic acid solution and hen egg white lysozyme solution were mixed at room temperature, quickly stirred to mix uniformly, and the precipitate was poured into a mold and freeze-dried for 20 min to obtain a polyglutamic acid medical tissue adhesive dry adhesive patch.

[0076] The polyglutamic acid medical tissue adhesive of this example had a wet-state pig skin tissue adhesion strength of 66 kPa in a wet-state physiological environment.

[0077] Comparative Example 1

[0078] (1) 2 g of 2000 kDa poly (γ-glutamic acid) sodium powder was dissolved in 100 mL of deionized water, then 3.4 mL of 6 mol / L hydrochloric acid solution was added to adjust the pH to 1.5, the acidified solution was loaded into a dialysis bag, dialyzed with deionized water, and the dialyzed solution was placed in a freeze dryer at -60°C for vacuum freeze drying for 48 hours to obtain a polyglutamic acid sponge.

[0079] (2) 2 g of polyglutamic acid sponge was dissolved in 100 mL of deionized water, and stirred to dissolve uniformly to obtain a polyglutamic acid solution with a concentration of 2 wt%.

[0080] (3) 2 g of hen egg white lysozyme was dissolved in 100 mL of deionized water, and stirred to dissolve uniformly to obtain a hen egg white lysozyme solution with a concentration of 2 wt%.

[0081] (4) The same volume of the above acidification reaction treated polyglutamic acid solution and hen egg white lysozyme solution were mixed at room temperature, quickly stirred to mix uniformly, and the precipitate was poured into a mold and freeze-dried for 20 min to obtain a polyglutamic acid medical tissue adhesive dry adhesive patch.

[0082] The polyglutamic acid medical tissue adhesive of this example had a wet-state pig skin tissue adhesion strength of 66 kPa in a wet-state physiological environment.

[0083] Comparative Example 2

[0084] (1) 2 g of 2000 kDa poly(γ-glutamic acid) sodium powder was dissolved in 100 mL of deionized water, the dissolved solution was loaded into a dialysis bag, dialyzed with deionized water, and the dialyzed solution was placed in a freeze dryer and vacuum freeze-dried at -60°C for 48 hours to obtain a polyglutamic acid sodium sponge.

[0085] (2) 2 g of polyglutamic acid sodium sponge was dissolved in 100 mL of deionized water, and stirred to dissolve uniformly to obtain a polyglutamic acid sodium solution with a concentration of 2 wt%.

[0086] (3) 2 g of hen egg white lysozyme was dissolved in 100 mL of deionized water, and stirred to dissolve uniformly to obtain a hen egg white lysozyme solution with a concentration of 2 wt%.

[0087] (4) The same volume of the above polyglutamic acid sodium solution and hen egg white lysozyme solution were mixed at room temperature, quickly stirred to mix uniformly, and the precipitate was poured into a mold and freeze-dried for 20 min to obtain a polyglutamic acid medical tissue adhesive dry adhesive patch.

[0088] The polyglutamic acid medical tissue adhesive of this example had an adhesion strength to wet pig skin tissue of 3 kPa in a wet physiological environment.

[0089] Example 9

[0090] A polyglutamic acid medical tissue adhesive was tested for its adhesion to pig organs:

[0091] (1) Preparation: The patch adhesive obtained in Example 1 was placed on the two ends of the pig tissue and overlapped together to allow it to fully contact the pig tissue under certain stress, as shown in Figure 1 , which is a tissue adhesion tensile test diagram. Figure 1

[0092] (2) Adhesion test: The adhesion 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. The adhesion strength value was obtained by dividing the maximum tensile force tested by the contact area of the adhesive and the pig tissue. The adhesion strength values for the pig heart, pig liver, and pig stomach were 8.4 kPa, 9.1 kPa, and 25.1 kPa, respectively.

[0093] Example 10

[0094] A polyglutamic acid medical tissue adhesive was tested for its occlusion performance on a pig stomach:

[0095] ​(1) Sample preparation: take a pig stomach, squeeze out the contents of the stomach, drill a hole with a diameter of 4 mm at the middle position of the stomach, fill the stomach with deionized water, then adhere the 5 cm diameter circular patch in example 1 to the hole and around it to block the water outlet; as shown in Figure 2 Figure 2 is a schematic diagram for adhering and blocking the pig stomach.

[0096] Example 11

[0097] (1) 2 g of 2000 kDa poly (γ-glutamic acid) sodium powder was dissolved in 100 mL of deionized water, then 3.4 mL of 6 mol / L hydrochloric acid solution was added to adjust the pH to 1.5, the acidified solution was loaded into a dialysis bag, and deionized water was used for dialysis. The dialyzed solution was placed in a freeze dryer at -60°C for vacuum freeze drying for 48 hours to obtain a polyglutamic acid sponge.

[0098] (2) 2 g of polyglutamic acid sponge was dissolved in 100 mL of deionized water, and stirred to dissolve uniformly to obtain a polyglutamic acid solution with a concentration of 2 wt%.

[0099] (3) 2 g of hen egg white lysozyme was dissolved in 100 mL of deionized water, and stirred to dissolve uniformly to obtain a hen egg white lysozyme solution with a concentration of 2 wt%.

[0100] (4) The same volume of the above acidified reaction treated polyglutamic acid solution and hen egg white lysozyme solution were mixed at room temperature, and quickly stirred to obtain a polyglutamic acid medical tissue adhesive.

[0101] Comparative Example 3

[0102] The preparation of polyglutamic acid medical tissue adhesive was carried out according to the method of example 11, except that hen egg white lysozyme was replaced by casein (Try protein).

[0103] Comparative Example 4

[0104] The preparation of polyglutamic acid medical tissue adhesive was carried out according to the method of example 11, except that hen egg white lysozyme was replaced by bovine serum albumin (BSA protein).

[0105] Comparative Example 5

[0106] The preparation of polyglutamic acid medical tissue adhesive was carried out according to the method of example 11, except that hen egg white lysozyme was replaced by polylysine protein (Plys protein).

[0107] ​The adhesion properties of the medical tissue adhesives prepared in Example 11 and Comparative Examples 3-5 were compared. Only lysozyme from egg white could form a gel with acidic polyglutamic acid and had excellent adhesion properties. This is because the surface of lysozyme (LZM) is rich in polar amino acids, which can form hydrogen bonds with the carboxyl groups of polyglutamic acid. In addition, lysozyme as a whole is positively charged and can form electrostatic interactions with the remaining carboxyl groups of polyglutamic acid. Finally, lysozyme is a spherical nanoparticle protein that can serve as a cross-linking center for polyglutamic acid chains.

[0108] like Figure 3 As shown, Figure 3 The images show a comparison of the appearance of the medical tissue adhesives prepared in Example 11 and Comparative Examples 3-5. BSA protein carries a negative charge and cannot form electrostatic interactions with polyglutamic acid. Although Try protein carries a positive charge and forms electrostatic interactions with polyglutamic acid, its surface is composed of hydrophobic amino acids, preventing it from forming hydrogen bonds with polyglutamic acid. Therefore, neither of these proteins exhibits significant gelation properties. While Plys polypeptide carries both a negative charge and polar amino acids, it is a linear polypeptide and cannot serve as a cross-linking center for polyglutamic acid chains. Consequently, the adhesive formed by acidic polyglutamic acid and Plys polypeptide exhibits very poor mechanical properties. Figure 4 As shown, Figure 4 This diagram illustrates the adhesive properties of adhesives made from acidic polyglutamic acid with lysozyme from egg white and Plys protein, respectively. Therefore, acidic polyglutamic acid cannot form high-performance adhesives with BSA protein, Try protein, or Plys protein.

[0109] The polyglutamic acid medical tissue adhesive of Example 11 was subjected to cell proliferation promotion test, antibacterial activity test, and infected wound healing promotion test, as detailed below:

[0110] Cell proliferation promotion assay

[0111] The optimal bioadhesive for wound repair should be biocompatible and biodegradable. To evaluate the cell compatibility of the medical tissue adhesive (d-PGA / LZM adhesive) described in this invention, L929 cells were cultured in Durbeco Modified Eagle Medium (DEME) with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin solution (P / S). Complete culture medium (30 mg / mL) was used. -1 L929 cells were cultured at 37°C for 24 hours. 2000 cells per well were placed in a 96-well plate. After cell adhesion, 100 μL of polyglutamic acid medical tissue adhesive was added. Complete culture medium was used as a control. Cell proliferation was assessed by CCK-8 assay on days 1, 3, and 5.

[0112] To determine the survival and status of L929 cells, the medical tissue adhesive described in the present application was co-cultured with a live / dead cell double staining kit, and live and dead cells were stained green and red with calcein and PI, respectively, and observed under an inverted fluorescence microscope; as shown in Figure 5 , Figure 5 The live / dead staining images of L929 cells directly contacted with a cell culture plate (control) and the medical tissue adhesive described in the present application (experimental group) for 5 days are shown in FIG. 5, wherein Figure 5 the scale of the control group and the d-PGA / LZM group images of Day 5 is 40 μm, and the Inset is a magnified image of the scale of 10 μm at the box of the Day 5 image.

[0113] As can be seen from Figure 5 , most of the L929 cells grew well after being co-cultured with the d-PGA / LZM adhesive for 5 days, and almost no dead cells were observed in the live / dead test during the culture period. The low cytotoxicity of this adhesive is due to the high biocompatibility inherent in PGA and LZM. This result shows that the extracellular matrix component has good biological activity in promoting cell proliferation by simulating the microenvironment of cell life. In general, the medical tissue adhesive described in the present application has good cell compatibility and can be further applied as an effective wound hemostatic agent in vivo.

[0114] Antibacterial activity test

[0115] Bacterial infection generally prolongs the wound healing time, and therefore a hemostatic adhesive with antibacterial activity has important value in promoting wound healing by inhibiting pathogenic bacterial contamination. Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) were used as gram-positive and gram-negative bacterial models, respectively, to evaluate the antibacterial ability of the medical tissue adhesive (d-PGA / LZM adhesive) described in the present application. The bacterial suspension was incubated with the d-PGA / LZM adhesive or polyethylene film (control group) in a 24-well plate for 24 hours in a humidified incubator at 37°C, and then the images of the bacterial suspension spread on nutrient agar for 24 hours were observed, and the antibacterial ability of the d-PGA / LZM adhesive was evaluated by counting the colony forming units (CFUs), as follows: The d-PGA / LZM adhesive and 1.0 x 1.0 cm 2 polyethylene film (control group) were placed in a 24-well plate, and then 15 μL of E. coli or S. aureus suspension (10 6 CFU / mL) was added to the d-PGA / LZM adhesive and polyethylene film. After incubation at 37°C for 24 h, the surviving bacteria were collected by ultrasonic mixing with 1.9 mL of phosphate buffered saline (PBS) buffer. The colony counting test was performed by plating the bacterial suspension at 37°C for 24 h.

[0116] As shown in Figures 6-7 , Figure 6 The schematic diagram of surviving bacteria of E. coli and S. aureus proliferated on agar plates after contact with d-PGA / LZM adhesive or polyethylene film (control), wherein the scale is 3 cm; Figure 7 The test results of the bacteriostatic ability of d-PGA / LZM adhesive on E. coli and S. aureus are shown in the figure. Figure 6 It can be seen that almost no bacterial clones are formed in the d-PGA / LZM adhesive group, which indicates that the CFU count of S. aureus and E. coli is significantly lower than that of the control group. Figure 7 It can be seen that the killing rate of d-PGA / LZM adhesive on S. aureus and E. coli is 99.84% and 99.22%, respectively, and the data of S. aureus and E. coli are significantly different from the blank group.

[0117] Subsequently, we performed inhibition zone experiments on S. aureus and E. coli. As shown in Figures 8-9 , Figure 8 The schematic diagram of antibacterial activity inhibition zone test, Figure 9 The inhibition zone diameter diagram of PE film and d-PGA / LZM for quantitative bacteriostatic test on E. coli and S. aureus is shown in the figure. Figure 8 It can be seen that there is an obvious inhibition zone around D-PGA / LZM, while the PE film has no obvious antibacterial activity; by Figure 9 It can be seen that the average inhibition zone diameter of D-PGA / LZM group on S. aureus and E. coli is about 10.5 mm and 10.9 mm, respectively. These results show that D-PGA / LZM has good antibacterial activity by releasing LZM.

[0118] Promoting infected wound healing test

[0119] An ideal wound dressing should have the advantages of promoting healing, anti-infection, and less irritation. Considering that the medical tissue adhesive (d-PGA / LZM adhesive) described in the present application has good wound adhesion and promotes wound healing, a customized S. aureus-induced infected rat model was used to study its effect on infected wounds. The specific steps of the promoting infected wound healing test are as follows: after anesthesia, four 10 mm circular full-thickness skin defects are cut on the back of the rat, and on the 5th, 10th, and 15th day, the rat wound is photographed, the wound area is measured using Image J, the pathological sections are stained with H&E, Masson, and immunohistochemistry, and the images are collected using a scanner. The immunohistochemical sections are scanned using a digital tissue section scanner.

[0120] In the trial promoting wound healing of infected wounds, the d-PGA / LZM group consisted of patients whose wounds were treated with d-PGA / LZM adhesive, while the Control group consisted of patients who were not treated with d-PGA / LZM adhesive. Figure 10 As shown, Figure 10 Image showing wound healing in SD rats on day 15 during an assay to promote infected wound healing. (Source: [Original Text]) Figure 10 It can be seen that the wound size of rats in the d-PGA / LZM group gradually decreased significantly within 15 days, suggesting that it can effectively promote the healing of chronic infected wounds.

[0121] To further observe wound healing, we also performed histomorphological analysis on the regenerated skin tissue to assess the treatment efficacy of these samples during the healing phase. Figures 11-12 As shown, Figure 11 H&E staining images of wound tissue from SD rats on days 5 and 15 in an assay to promote wound healing of infected animals. Figure 12 To promote wound healing in SD rats, images of wound tissue stained with masson dye on days 5 and 15 are shown. The scale of the Day 5 and Day 15 images is 500 μm. The Inset images are magnified views of the boxes in the Day 5 and Day 15 images, with a scale of 50 μm.

[0122] Depend on Figures 11-12 It was observed that on day 5 of treatment, both the control group and the d-PGA / LZM group of SD rats showed significant defects in the epidermal and dermal structures of their skin, with the d-PGA / LZM group exhibiting less inflammatory cell infiltration than the control group. Granulation tissue regeneration is crucial for wound healing. Figure 11 It was also shown that, compared with the control group, the d-PGA / LZM adhesive group significantly increased the thickness of granulation tissue. Figures 11-12 It was also shown that after 15 days of treatment, each group showed intact epithelial and dermal structures. The epidermis in the d-PGA / LZM adhesive group was the thinnest, with the most newly formed blood vessels and hair follicles, and it was the most normal tissue-like structure in the remodeled tissue. In addition, wounds treated with d-PGA / LZM adhesive produced more epithelial tissue, hair follicles, and sebaceous glands on day 15.

[0123] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A medical tissue adhesive, characterized by, It comprises: 1 wt%~30 wt% of acidic polyglutamic acid; 1 wt%~30 wt% of globular protein; The globular protein is selected from at least one of plant lysozyme, animal lysozyme, microbial lysozyme or egg white lysozyme; and the balance of water; The medical tissue adhesive is prepared by the following steps: acidifying sodium polyglutamate to obtain acidic polyglutamic acid; mixing the acidic polyglutamic acid solution and the globular protein solution to obtain a medical tissue adhesive.

2. The medical tissue adhesive according to claim 1, characterized in that It comprises: 1 wt%~5 wt% of acidic polyglutamic acid; 1 wt%~5 wt% of globular protein; and the balance of water.

3. The medical tissue adhesive according to claim 1, wherein The pH of the acidic polyglutamic acid is 1~3.

4. The medical tissue adhesive of claim 1, wherein The molecular weight of the acidic polyglutamic acid is 2 kDa~2000 kDa.

5. The medical tissue adhesive of claim 1, wherein The plant lysozyme is selected from ficin; 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. A method for producing the medical tissue adhesive according to any one of claims 1 to 5, characterized by, It comprises the following steps: acidifying sodium polyglutamate to obtain acidic polyglutamic acid; mixing the acidic polyglutamic acid solution and the globular protein solution to obtain a medical tissue adhesive.

7. The production method according to claim 6, characterized by, The temperature of the acidification treatment is 0℃~60℃, and the time of the acidification treatment is 10 min~30 min.

8. A medical tissue dry adhesive patch, characterized by, It is prepared from the medical tissue adhesive of any one of claims 1~5 or the medical tissue adhesive prepared by the method of any one of claims 6~7.

9. Use of the medical tissue adhesive of any one of claims 1~5 or the medical tissue adhesive prepared by the method of any one of claims 6~7 or the medical tissue dry adhesive patch of claim 8 in the preparation of a tissue wound hemostasis aid, a tissue wound closure product, a tissue wound repair product and a tissue wound antibacterial product.

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