Biodegradable antibacterial medical adhesive and preparation method and application thereof

Abstract

The application provides a biodegradable antibacterial medical adhesive and a preparation method and application thereof, and belongs to the technical field of biomedical materials and medical devices. The antibacterial medical adhesive is obtained by forming a double cross-linking network hydrogel through Schiff base reaction, electrostatic interaction and hydrogen bond of chitosan guanidine salt, aldehyde-based dextran and tannic acid, and specifically, 0.1-30wt% aldehyde-based dextran aqueous solution and 0.1-25wt% tannic acid aqueous solution are mixed to obtain a mixed solution, then 0.1-30wt% chitosan guanidine salt aqueous solution is added into the mixed solution, and uniform mixing is carried out at room temperature. The tannic acid is introduced on the basis of the chitosan guanidine salt and aldehyde-based dextran to further enhance the adhesion effect, hemostasis, antibacterial and antioxidant properties, and the antibacterial medical adhesive has the advantages of no biological toxicity and can be biodegraded and absorbed.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials and medical device technology, and particularly relates to a biodegradable antibacterial medical adhesive, its preparation method and application. Background Technology

[0002] Currently, commercially available medical adhesives mainly include cyanoacrylates, fibrin adhesives, and protein adhesives. These medical adhesives are used in wound closure, hemostasis, tissue repair, drug delivery, and wound dressing. Among these, cyanoacrylate medical adhesives are the most widely used; however, they suffer from drawbacks such as rapid curing, lack of elasticity, and inherent tissue toxicity. Furthermore, while fibrin adhesives or protein adhesives have good biocompatibility, their bonding strength is low, and their preparation cost is high; non-autologous fibrin also poses a risk of viral infection. While the aforementioned medical adhesives are widely used in minimally invasive and surgical procedures, none possess significant antibacterial properties.

[0003] To address the shortcomings of current clinically used medical adhesives in terms of antibacterial properties, developing an antibacterial medical adhesive to prevent bacterial infections and inflammation is crucial. Recent research on antibacterial medical adhesives typically involves adding antibacterial agents, such as antibiotics (ACS Sustainable Chem. Eng., 2018, 6, 7826–7840; Drug Dev. Ind. Pharm., 2020, 46, 1318–1333.), metal ions (Surf. Coat. Technol., 2020, 397, 126020.), and antimicrobial peptides (RSC Adv., 2016, 6, 8620–8627; Colloids Surf., B, 2021, 202, 111682.), to improve the adhesive's antibacterial properties. However, these methods have drawbacks: antibiotic use can lead to bacterial resistance, metal ions are often cytotoxic, and antimicrobial peptides are relatively expensive. Therefore, this invention provides a novel biodegradable antibacterial medical adhesive. Summary of the Invention

[0004] To better serve practical applications and meet new clinical needs, this invention proposes a biodegradable antibacterial medical adhesive, its preparation method, and its application, by constructing a polysaccharide-based crosslinked hydrogel. The novel biodegradable antibacterial medical adhesive of this invention comprises chitosan guanidine salt and tannic acid. Chitosan guanidine salt is a highly positively charged cationic polymer that can disrupt the negatively charged bacterial outer membrane through electrostatic interactions, leading to osmotic pressure imbalance and electrolyte leakage within the bacteria, thus generating antibacterial activity. Tannic acid is a natural polyphenol compound whose antibacterial mechanism includes inhibiting extracellular microbial enzymes, depriving microorganisms of the substrate required for growth, inhibiting microbial metabolism through oxidative phosphorylation, depriving metal ions, or forming cell membrane complexes with bacteria that cause changes in cell wall morphology and increased membrane permeability. Combining the antibacterial mechanisms of chitosan guanidine salt and tannic acid yields a medical adhesive with high antibacterial activity.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] One of the technical solutions of the present invention:

[0007] A method for preparing a biodegradable antibacterial medical adhesive involves forming a double cross-linked network hydrogel from chitosan guanidine salt, aldehyde-modified dextran, and tannic acid through Schiff base reaction, electrostatic interaction, and hydrogen bonding, thereby obtaining the biodegradable antibacterial medical adhesive.

[0008] Further, the specific steps include: mixing an aqueous solution of aldehyde-modified dextran and an aqueous solution of tannic acid to obtain a mixture, then adding an aqueous solution of chitosan guanidine salt to the mixture, and mixing evenly at room temperature to obtain the biodegradable antibacterial medical adhesive.

[0009] Furthermore, the volume ratio of the aldehyde-modified dextran aqueous solution, the tannic acid aqueous solution, and the chitosan guanidine salt aqueous solution is 1:1:(1-4).

[0010] Furthermore, the concentration of the aldehyde-modified dextran aqueous solution is 0.1–30 wt%, the concentration of the tannic acid aqueous solution is 0.1–25 wt%, and the concentration of the chitosan guanidine salt aqueous solution is 0.1–30 wt%.

[0011] Furthermore, the chitosan guanidine salt includes one or both of chitosan monoguanidine salt and chitosan biguanidine salt.

[0012] Chitosan is a natural polysaccharide obtained by the deacetylation of chitin. It contains a large number of amino and hydroxyl groups and has good biodegradability and antibacterial ability. However, chitosan is difficult to dissolve in water. To overcome this shortcoming, this invention modifies the solution by grafting or changing the pH of the solution. The resulting chitosan guanidine salt is a cationic polymer prepared by modifying the guanidine salt groups of chitosan. Compared with chitosan, chitosan guanidine salt is water-soluble, which facilitates dissolution and handling in the preparation of adhesives.

[0013] The preparation method of the chitosan biguanide salt includes the following steps:

[0014] Chitosan is dissolved in an aqueous HCl solution with a concentration of 1-2 wt%. After complete dissolution, a chitosan solution is obtained. The chitosan solution is reacted at 50-60°C for 3-4 hours. Dicyandiamide is dissolved in water to obtain a dicyandiamide solution. The dicyandiamide solution is added dropwise to the chitosan solution and reacted at 75-120°C for 3-24 hours. After the reaction is completed, the mixture is cooled to room temperature, and excess ethanol is added to generate a precipitate. The precipitate is separated by centrifugation or filtration. The precipitate is dissolved in water, dialyzed at room temperature for 3-5 days, and then freeze-dried to obtain chitosan biguanide salt. The molar ratio of chitosan to dicyandiamide is 1:(0.1-50), and the molecular weight of chitosan is 10,000-2,000,000.

[0015] The preparation method of the chitosan monoguanidine salt includes the following steps:

[0016] Hydrogen peroxide was placed in a beaker, and concentrated sulfuric acid was added and stirred to prepare an acidic hydrogen peroxide solution. A portion of the acidic hydrogen peroxide solution was first added to an Erlenmeyer flask, followed by alternating addition of thiourea dioxide and acidic hydrogen peroxide solutions. The addition was completed within 80 minutes at 50–55°C, followed by stirring for another 15 minutes, resulting in a large amount of colorless granular crystals. After standing at below 10°C for 2 hours, the mixture was filtered to obtain thiourea trioxide hydrate crystals. Chitosan was dissolved in a 0.5–2 wt% HCl aqueous solution, and the pH was adjusted with 5% Na₂CO₃ solution. The pH of the precipitate was adjusted to 7.5–10. The precipitate was washed with distilled water until the pH reached 7.0–7.5, removing most of the water to obtain concentrated chitosan solution. Thiourea trioxide hydrate crystals were slowly added to the concentrated chitosan solution with stirring at 30–70°C. The reaction was carried out at 30–70°C for 15–100 minutes, then cooled to room temperature. Ethanol was poured in to precipitate the precipitate. The solution was washed several times with an ethanol-water solution and then vacuum dried to obtain chitosan monoguanidine salt. The molar ratio of chitosan to thiourea trioxide was 1:(0.1–50), and the molecular weight of chitosan was 10,000–2,000,000.

[0017] Dextran is a polysaccharide derived from microorganisms and composed of glucose as a monosaccharide. It possesses excellent swelling and degradation properties, and as a polysaccharide, it has no biological toxicity. After aldehyde-modification of dextran, the aldehyde group reacts rapidly with the amino group in chitosan guanidine salt to form a Schiff base covalent bond, resulting in a short gelation time (1-2 minutes), facilitating clinical use and operation. Furthermore, the preparation method of the aldehyde-modified dextran includes the following steps:

[0018] Dextran was dissolved in water to obtain a dextran solution, and sodium periodate was dissolved in water to obtain a sodium periodate solution. The sodium periodate solution was added dropwise to the dextran solution under light-protected conditions, and the reaction was carried out at room temperature for 2 to 24 hours. Then, ethylene glycol was added to terminate the reaction for 1 to 2 hours. The reaction was dialyzed at room temperature for 3 to 5 days, and then freeze-dried to obtain aldehyde-modified dextran. The molar ratio of dextran to sodium periodate was 1:0.1 to 1:20, and the molecular weight of dextran was 10,000 to 2,000,000.

[0019] The second technical solution of the present invention:

[0020] A biodegradable antibacterial medical adhesive prepared by the preparation method described above.

[0021] The third technical solution of the present invention:

[0022] The application of the aforementioned biodegradable antibacterial medical adhesive in the preparation of hemostatic, antibacterial, and repair materials.

[0023] Compared with the prior art, the present invention has the following advantages and technical effects:

[0024] (1) The antibacterial mechanism of the medical adhesive of the present invention is as follows: Chitosan guanidine salt has strong antibacterial properties and can destroy the negatively charged cell membrane of bacteria, thereby producing significant contact antibacterial activity. In addition, tannic acid is a natural polyphenol compound with hemostatic, antioxidant, antibacterial and anti-inflammatory effects. Adding tannic acid to the chitosan guanidine salt solution can crosslink the polysaccharide polymer with the polysaccharide through electrostatic and hydrogen bonding, and can also improve the adhesion between the medical adhesive and tissue through covalent bonds, electrostatic, hydrogen bonding and hydrophobic interactions; in addition, tannic acid has sustained-release properties, and its release from the medical adhesive can produce antibacterial and antioxidant effects.

[0025] (2) The present invention uses chitosan guanidine salt and aldehyde-modified dextran to form a hydrogel, and introduces tannic acid on the basis of the hydrogel composed of chitosan guanidine salt and aldehyde-modified dextran to further enhance its adhesion, hemostasis, antibacterial and antioxidant properties. It also has the advantages of being non-biotoxic, biodegradable and absorbable, and inexpensive. Attached Figure Description

[0026] The accompanying drawings, which form part of this application, are used to provide a further understanding of this application. The illustrative embodiments and descriptions of this application are used to explain this application and do not constitute an undue limitation of this application. In the drawings:

[0027] Figure 1 Adhesion images of CSG / ODex-TA3 prepared in Example 3 to different material interfaces and different organs of mice;

[0028] Figure 2 The adhesion strength curves are for different adhesive products in Examples 1-3 and Comparative Examples 1-2 and 6.

[0029] Figure 3 The results show the colony growth of different groups of adhesives after co-culturing with Escherichia coli and Staphylococcus aureus for 12 hours, followed by dilution of the culture medium and even spreading it on agar plates for 24 hours of incubation.

[0030] Figure 4 The statistical results of plate counts were obtained after different groups of adhesives were co-cultured with Escherichia coli and Staphylococcus aureus for 12 hours, the culture medium was diluted and evenly spread on agar plates and incubated for 24 hours.

[0031] Figure 5 The results of the inhibition zone experiment after co-culturing different groups of adhesives with methicillin-resistant Staphylococcus aureus (MRSA) for 24 hours;

[0032] Figure 6 The results of statistical analysis of the diameter of inhibition zones after co-culturing different groups of adhesives with methicillin-resistant Staphylococcus aureus (MRSA) for 24 hours.

[0033] Figure 7 The results of a comparative experiment on the inhibition zones of CSG / ODex-TA2 adhesive and fibrin glue against methicillin-resistant Staphylococcus aureus (MRSA);

[0034] Figure 8 Statistical results of the inhibition zone diameter of CSG / ODex-TA2 adhesive and fibrin glue against methicillin-resistant Staphylococcus aureus (MRSA);

[0035] Figure 9 The results show the cell viability of mouse embryonic fibroblast L929 cells after 24 hours of co-culturing with different groups of adhesives in Comparative Examples 1, 6 and Examples 1-3.

[0036] Figure 10 The results show the nitrogen free radical scavenging rates of different groups of adhesives in Comparative Examples 1, 6, and Examples 1-3;

[0037] Figure 11 The images show the healing process of infected skin wounds in mice after different treatments. The experimental treatment groups included: I: blank group (wounds without bacterial infection), II: control group (wounds with bacterial infection, without sample treatment), III: infected wounds treated with fibrin glue, IV: infected wounds treated with CSG / ODex, and V: infected wounds treated with CSG / ODex-TA2.

[0038] Figure 12 The statistical results of the healing area of ​​infected skin wounds in mice after different treatments were presented. The experimental treatment groups included: I: blank group (wounds without bacterial infection), II: control group (wounds with bacterial infection, without sample treatment), III: infected wound group treated with fibrin glue, IV: infected wound group treated with CSG / ODex, and V: infected wound group treated with CSG / ODex-TA2.

[0039] Figure 13 The images show the bacterial smears taken from infected wounds of mice on days 3 and 9 after different treatments. The experimental treatment groups included: I: blank group (wounds without bacterial infection), II: control group (wounds with bacterial infection, no sample treatment), III: infected wounds treated with fibrin glue, IV: infected wounds treated with CSG / ODex, and V: infected wounds treated with CSG / ODex-TA2.

[0040] Figure 14 To obtain the bacterial colony quantification results of bacteria collected from infected wounds in mice on days 3 and 9 after different treatments, the experimental treatment groups included: I: blank group (wounds without bacterial infection), II: control group (wounds with bacterial infection, no sample treatment), III: infected wounds treated with fibrin glue, IV: infected wounds treated with CSG / ODex, and V: infected wounds treated with CSG / ODex-TA2.

[0041] Figure 15 Photographs of experimental results for different groups of adhesives used for liver hemostasis;

[0042] Figure 16 Statistical results of bleeding volume after different groups of adhesives were used for hemostasis of the liver;

[0043] Figure 17 The statistical results of degradation experiments of different groups of adhesives in phosphate buffer solution (PBS, pH=7.4) at 37℃ are presented. Detailed Implementation

[0044] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0045] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0046] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0047] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0048] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0049] This invention provides a method for preparing a biodegradable antibacterial medical adhesive, wherein chitosan guanidine salt, aldehyde-modified dextran, and tannic acid form a double cross-linked network hydrogel through Schiff base reaction, electrostatic interaction, and hydrogen bonding to obtain the biodegradable antibacterial medical adhesive.

[0050] In this embodiment of the invention, the specific steps include: mixing an aqueous solution of aldehyde-modified dextran and an aqueous solution of tannic acid to obtain a mixture, then adding an aqueous solution of chitosan guanidine salt to the mixture, and mixing evenly at room temperature to obtain the biodegradable antibacterial medical adhesive.

[0051] In a preferred embodiment of the present invention, the volume ratio of the aldehyde-modified dextran aqueous solution, the tannic acid aqueous solution, and the chitosan guanidine salt aqueous solution is 1:1:(1-4).

[0052] In a preferred embodiment of the present invention, the concentration of the aldehyde-modified dextran aqueous solution is 0.1–30 wt%, the concentration of the tannic acid aqueous solution is 0.1–25%, and the concentration of the chitosan guanidine salt aqueous solution is 0.1–30 wt%.

[0053] In a preferred embodiment of the present invention, the chitosan guanidine salt includes one or both of chitosan monoguanidine salt and chitosan biguanidine salt.

[0054] In this embodiment of the invention, the preparation method of the chitosan biguanide salt includes the following steps:

[0055] Chitosan is dissolved in an aqueous HCl solution with a concentration of 1-2 wt% (preferably 1 wt%) until completely dissolved to obtain a chitosan solution. The chitosan solution is reacted at 50-60°C for 3-4 hours, preferably at 60°C for 3 hours. Dicyandiamide is dissolved in water to obtain a dicyandiamide solution. The dicyandiamide solution is added dropwise to the chitosan solution and reacted at 75-120°C for 3-24 hours, preferably at 95°C for 3 hours. After the reaction is completed, the mixture is cooled to room temperature, and excess ethanol is added to generate a precipitate. The precipitate is separated by centrifugation or filtration. The precipitate is dissolved in water and dialyzed at room temperature for 3-5 days, preferably 3 days. After freeze-drying, chitosan biguanide salt is obtained. The molar ratio of chitosan to dicyandiamide is 1:(0.1-50), and the molecular weight of chitosan is 10,000-2,000,000.

[0056] In this embodiment of the invention, the preparation method of the chitosan monoguanidine salt includes the following steps:

[0057] Hydrogen peroxide was placed in a beaker, and concentrated sulfuric acid was added and stirred to prepare an acidic hydrogen peroxide solution. A portion of the acidic hydrogen peroxide solution was first added to an Erlenmeyer flask, followed by alternating addition of thiourea dioxide and acidic hydrogen peroxide solution. The addition was carried out within 80 minutes at a temperature of 50–55°C, preferably 50°C, followed by stirring for 15 minutes to generate a large amount of colorless granular crystals. After standing at below 10°C for 2 hours, the mixture was filtered to obtain thiourea trioxide hydrate crystals. Chitosan was dissolved in a 0.5–2 wt% (preferably 0.5 wt%) HCl aqueous solution, and 5%... Adjust the pH of the Na2CO3 solution to 7.5–10, preferably to 8–9. Wash the precipitate with distilled water until the pH reaches 7.0–7.5 to remove most of the water, thus obtaining concentrated chitosan. Slowly add thiourea trioxide hydrate crystals to the concentrated chitosan solution while stirring at 30–70°C. React at 30–70°C for 15–100 minutes, preferably at 50°C for 15 minutes. Then cool to room temperature, pour in ethanol to precipitate, wash several times with an ethanol-water solution, and then vacuum dry to obtain chitosan monoguanidine salt. The molar ratio of chitosan to thiourea trioxide is 1:(0.1–50), and the molecular weight of chitosan is 10,000–2,000,000.

[0058] In this embodiment of the invention, the preparation method of the aldehyde-modified dextran includes the following steps:

[0059] Dextran is dissolved in water to obtain a dextran solution, and sodium periodate is dissolved in water to obtain a sodium periodate solution. The sodium periodate solution is added dropwise to the dextran solution under light-protected conditions, and the reaction is carried out at room temperature for 2 to 24 hours, preferably 3 hours. Then, ethylene glycol is added to terminate the reaction for 1 to 2 hours, preferably 1 hour. The reaction is dialyzed at room temperature for 3 to 5 days, preferably 3 days, and then freeze-dried to obtain aldehyde-modified dextran. The molar ratio of dextran to sodium periodate is 1:0.1 to 1:20, and the molecular weight of dextran is 10,000 to 2,000,000.

[0060] This invention also proposes a biodegradable antibacterial medical adhesive prepared by the aforementioned preparation method.

[0061] This invention also proposes the application of the aforementioned biodegradable antibacterial medical adhesive in hemostatic and antibacterial repair materials.

[0062] All raw materials used in the embodiments of this invention were obtained through commercial purchase.

[0063] In this embodiment of the invention, room temperature refers to 25±2℃.

[0064] The technical solution of the present invention will be further illustrated by the following embodiments.

[0065] Example 1

[0066] (1) Preparation of aldehyde-modified dextran (ODex)

[0067] 2g of dextran (molecular weight 70,000, purchased from Maclean's, catalog number D806715) was dissolved in 40mL of water and stirred until completely dissolved to obtain a dextran solution. 1.98g of sodium periodate (molar ratio of dextran to sodium periodate was 1.5:1) was dissolved in 18mL of water to obtain a sodium periodate solution. The sodium periodate solution was added dropwise to the dextran solution under light-protected conditions, and the reaction was continued at room temperature for 3 hours in the dark. Then, 1mL of ethylene glycol was added to terminate the reaction for 1 hour. The reaction was dialyzed at room temperature for 3 days, and then freeze-dried to obtain aldehyde-modified dextran.

[0068] (2) Preparation of chitosan biguanide salt (CSG)

[0069] 2g of chitosan (molecular weight 100,000-300,000, purchased from Maclean, catalog number C804726) was dissolved in 200mL of 1wt% HCl aqueous solution and stirred until completely dissolved to obtain a chitosan solution. The chitosan solution was placed at 60℃ and reacted for 3 hours. 5.2g of dicyandiamide (molar ratio of chitosan to dicyandiamide was 1:5) was dissolved in 100mL of water to obtain a dicyandiamide solution. The dicyandiamide solution was added dropwise to the chitosan solution and reacted at 95℃ for 3 hours. After the reaction was completed, the mixture was cooled to room temperature, and excess ethanol was added to generate a precipitate. The precipitate was separated by centrifugation, dissolved in water, dialyzed at room temperature for 3 days, and then freeze-dried to obtain chitosan biguanide salt.

[0070] (3) Preparation of antibacterial medical adhesives

[0071] The aldehyde-modified dextran obtained in step (1) was dissolved in water to obtain an aqueous solution of aldehyde-modified dextran with a concentration of 6 wt%.

[0072] The chitosan biguanide salt obtained in step (2) was dissolved in water to obtain a chitosan biguanide salt aqueous solution with a concentration of 3 wt%.

[0073] Tannic acid was dissolved in water to obtain a 4 wt% tannic acid (TA) aqueous solution;

[0074] Mix 150 μL of aldehyde-modified dextran aqueous solution and 150 μL of tannic acid aqueous solution (volume ratio 1:1), then add 300 μL of chitosan biguanide salt aqueous solution to the mixture and mix at room temperature for 1 minute to obtain an antibacterial medical adhesive, denoted as CSG / ODex-TA1.

[0075] Example 2

[0076] Tannic acid was dissolved in water to obtain an 8 wt% tannic acid (TA) aqueous solution. The remaining steps were the same as in Example 1. The antibacterial medical adhesive prepared in Example 2 was designated as CSG / ODex-TA2.

[0077] Example 3

[0078] Tannic acid was dissolved in water to obtain a 12 wt% tannic acid (TA) aqueous solution. The remaining steps were the same as in Example 1. The antibacterial medical adhesive prepared in Example 3 was designated as CSG / ODex-TA3.

[0079] Example 4

[0080] (1) Preparation of aldehyde-modified dextran (ODex)

[0081] 2g of dextran (molecular weight 70,000, purchased from Maclean's, catalog number D806715) was dissolved in 40mL of water and stirred until completely dissolved to obtain a chitosan solution. 1.98g of sodium periodate (molar ratio of dextran to sodium periodate was 1.5:1) was dissolved in 18mL of water to obtain a sodium periodate solution. The sodium periodate solution was added dropwise to the dextran solution under light-protected conditions, and the reaction was continued at room temperature for 3 hours under light-protected conditions. Then, 1mL of ethylene glycol was added to terminate the reaction for 1 hour. The reaction was dialyzed at room temperature for 3 days, and then freeze-dried to obtain aldehyde-modified dextran.

[0082] (2) Preparation of chitosan monoguanidine salt (CSMG)

[0083] Take 65 mL of hydrogen peroxide in a beaker, add 1.7 mL of concentrated sulfuric acid and stir to prepare an acidic hydrogen peroxide solution. First, add 10 mL of the acidic hydrogen peroxide solution to an Erlenmeyer flask, then alternately add thiourea dioxide and acidic hydrogen peroxide solution, adding a total of 54 g of thiourea dioxide. Add the solution over 80 minutes at 50°C, then stir for another 15 minutes to generate a large amount of granular colorless crystals. After standing at below 10°C for 2 hours, filter to obtain thiourea trioxide hydrate crystals. Add 2 g of chitosan (molecular weight 100,000-300,000, purchased from Maclean's, catalog number C)... 804726) was dissolved in a 0.5 wt% HCl aqueous solution, and the pH value was adjusted to 8-9 with 5% Na2CO3 solution. The precipitate was washed with distilled water until the pH value was 7.0-7.5 to remove most of the water, thus obtaining chitosan concentrate. 1.53 g of thiourea trioxide (the molar ratio of chitosan to thiourea trioxide was 1:1) hydrate crystals were slowly added to the chitosan concentrate under stirring at 50°C. The reaction was carried out at 50°C for 15 minutes, then cooled to room temperature, and ethanol was poured in to precipitate. The mixture was washed several times with an ethanol aqueous solution and then vacuum dried to obtain chitosan monoguanidine salt.

[0084] (3) Preparation of antibacterial medical adhesives

[0085] The aldehyde-modified dextran obtained in step (1) was dissolved in water to obtain an aqueous solution of aldehyde-modified dextran with a concentration of 6 wt%.

[0086] The chitosan monoguanidine salt obtained in step (2) was dissolved in water to obtain a chitosan monoguanidine salt aqueous solution with a concentration of 3 wt%.

[0087] Tannic acid was dissolved in water to obtain a 12 wt% tannic acid (TA) aqueous solution;

[0088] An aqueous solution of aldehyde-modified dextran and an aqueous solution of tannic acid were mixed at a volume ratio of 1:1 to obtain a mixture. Then, an equal volume of chitosan monoguanidine salt aqueous solution was added to the mixture, and the mixture was stirred evenly at room temperature to obtain an antibacterial medical adhesive, denoted as CSMG / ODex-TA3.

[0089] Example 5

[0090] Same as Example 4, except that the molecular weight of the dextran used in step (1) is 2 million (Maclean's product number: D769364), the molecular weight of the chitosan used in step (2) is 30,000 (Maclean's product number: C850346), and the preparation of the antibacterial medical adhesive in step (3) is different, specifically:

[0091] The obtained aldehyde-modified dextran was dissolved in water to obtain an aqueous solution of aldehyde-modified dextran with a concentration of 0.1 wt%.

[0092] The chitosan monoguanidine salt obtained in step (2) was dissolved in water to obtain a chitosan monoguanidine salt aqueous solution with a concentration of 30 wt%.

[0093] Tannic acid was dissolved in water to obtain a 25 wt% tannic acid (TA) aqueous solution;

[0094] An aqueous solution of aldehyde-modified dextran and an aqueous solution of tannic acid were mixed at a volume ratio of 1:1 to obtain a mixture. Then, an equal volume of chitosan monoguanidine salt aqueous solution was added to the mixture, and the mixture was stirred evenly at room temperature to obtain an antibacterial medical adhesive, denoted as CSMG / ODex-TA4.

[0095] Example 6

[0096] Same as Example 4, except that the molecular weight of the dextran used in step (1) is 10,000 (Maclean's product number: D992645), the molecular weight of the chitosan used in step (2) is 1-2 million (Maclean's product number: C804729), and the preparation of the antibacterial medical adhesive in step (3) is different, specifically:

[0097] The aldehyde-modified dextran obtained in step (1) was dissolved in water to obtain an aqueous solution of aldehyde-modified dextran with a concentration of 30 wt%.

[0098] The chitosan monoguanidine salt obtained in step (2) was dissolved in water to obtain a chitosan monoguanidine salt aqueous solution with a concentration of 0.1 wt%.

[0099] Tannic acid was dissolved in water to obtain a 0.1 wt% aqueous solution of tannic acid (TA);

[0100] An aqueous solution of aldehyde-modified dextran and an aqueous solution of tannic acid were mixed at a volume ratio of 1:1 to obtain a mixture. Then, an equal volume of chitosan monoguanidine salt aqueous solution was added to the mixture, and the mixture was stirred evenly at room temperature to obtain an antibacterial medical adhesive, denoted as CSMG / ODex-TA5.

[0101] Example 7

[0102] Same as Example 1, except that the aldehyde-modified dextran aqueous solution, tannic acid aqueous solution and chitosan biguanide salt aqueous solution are mixed in a volume ratio of 1:1:4 and then mixed evenly at room temperature to obtain an adhesive, denoted as CSG / ODex-TA6.

[0103] Example 8

[0104] Same as Example 1, except that an aqueous solution of aldehyde-modified dextran and an aqueous solution of tannic acid are mixed at a volume ratio of 1:1 to obtain a mixture. Then, an aqueous solution of chitosan biguanide salt with an equal volume to the aqueous solution of aldehyde-modified dextran is added to the mixture. The mixture is then mixed evenly at room temperature to obtain an adhesive, denoted as CSG / ODex-TA7.

[0105] Comparative Example 1

[0106] (1) The preparation of aldehyde-modified dextran (ODex) is the same as in Example 1.

[0107] (2) The preparation of chitosan biguanide salt (CSG) is the same as in Example 1.

[0108] (3) Preparation of adhesive:

[0109] The aldehyde-modified dextran obtained in step (1) was dissolved in water to obtain an aqueous solution of aldehyde-modified dextran with a concentration of 3 wt%.

[0110] The chitosan biguanide salt obtained in step (2) was dissolved in water to obtain a chitosan biguanide salt aqueous solution with a concentration of 3 wt%.

[0111] An adhesive, denoted as CSG / ODex, is obtained by mixing an aqueous solution of aldehyde-modified dextran and an aqueous solution of chitosan guanidine salt at a volume ratio of 1:1 at room temperature until homogeneous.

[0112] Comparative Example 2

[0113] Commercially available porcine fibrin glue (purchased from Guangzhou Beixiu Biotechnology Co., Ltd.)

[0114] Comparative Example 3

[0115] (1) The preparation of aldehyde-modified dextran (ODex) is the same as in Example 4.

[0116] (2) Preparation of adhesive

[0117] The aldehyde-modified dextran obtained in step (1) was dissolved in water to obtain an aqueous solution of aldehyde-modified dextran with a concentration of 6 wt%.

[0118] Chitosan was dissolved in a 1% acetic acid solution to obtain a 3 wt% chitosan (CS) aqueous solution;

[0119] Tannic acid was dissolved in water to obtain a 12 wt% tannic acid (TA) aqueous solution;

[0120] An aqueous solution of aldehyde-modified dextran and an aqueous solution of tannic acid were mixed at a volume ratio of 1:1 to obtain a mixture. Then, an equal volume of chitosan aqueous solution was added to the mixture, and the mixture was stirred evenly at room temperature to obtain an adhesive, denoted as CS / ODex-TA3.

[0121] Comparative Example 4

[0122] (1) The preparation of chitosan biguanide salt (CSG) is the same as in Example 1.

[0123] (2) Preparation of adhesive

[0124] Dextran was dissolved in water to obtain a 6 wt% dextran (Dex) aqueous solution;

[0125] The chitosan biguanide salt obtained in step (1) was dissolved in water to obtain a chitosan biguanide salt aqueous solution with a concentration of 3 wt%.

[0126] Tannic acid was dissolved in water to obtain a 4 wt% tannic acid (TA) aqueous solution;

[0127] A mixture of dextran aqueous solution and tannic acid aqueous solution was prepared by mixing them at a volume ratio of 1:1. Then, an equal volume of chitosan biguanide salt aqueous solution was added to the mixture and mixed evenly at room temperature. As a result, the mixture did not form a gel adhesive.

[0128] Comparative Example 5

[0129] The two-component medical adhesive prepared according to the method disclosed in Example 1 of CN 107496974 A is denoted as ACMC / ODex.

[0130] Comparative Example 6

[0131] Tannic acid was dissolved in water to obtain a 30 wt% tannic acid (TA) aqueous solution. The remaining steps were the same as in Example 1. The antibacterial medical adhesive prepared in Comparative Example 6 was designated as CSG / ODex-TA5.

[0132] Performance testing

[0133] I. Adhesion Performance Test

[0134] In Example 3, the CSG / ODex-TA3 prepared was bonded to the surfaces of materials with different interfaces and different mouse organs. Adhesion photographs of the materials with different interfaces and different mouse organs are shown below. Figure 1 ,Depend on Figure 1 It can be seen that the CSG / ODex-TA3 adhesive has good adhesion to different material interfaces and to different mouse organs.

[0135] Different adhesive products from Examples 1-3 and Comparative Examples 1-2 and 6 were placed between two pieces of pigskin, and tensile adhesion strength was tested using a universal testing machine. The adhesion strength curves are shown in the figure. Figure 2 ,Depend on Figure 2 It can be seen that the maximum adhesion strength is 20.35 kPa (CSG / ODex-TA3).

[0136] II. Antibacterial Performance Test

[0137] The adhesives prepared in Examples 1-3, Comparative Examples 1 and 6 were sterilized and placed in 48-well plates. An equal volume of bacterial suspension was added, and the plates were co-cultured in a shaking incubator at 37°C for 12 hours. After diluting the culture, 100 μL of the culture was taken and evenly spread on an agar plate. The plates were then incubated in a shaking incubator at 37°C for 24 hours. The colony count was calculated to obtain the antibacterial rate, and a control group without adhesive was set up.

[0138] After co-culturing different groups of adhesives with *Escherichia coli* and *Staphylococcus aureus* for 12 hours, the culture medium was diluted and evenly spread onto agar plates. The colony growth results after incubation for 24 hours are shown in the figure. Figure 3 The statistical results of Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) plate counts after treatment with medical adhesive are shown in [the table below]. Figure 4 .

[0139] Figure 3 and Figure 4 The results showed that the CSG / ODex-TA series adhesives prepared in the examples exhibited good antibacterial effects against Escherichia coli and Staphylococcus aureus. The antibacterial effect of CSG / ODex in Comparative Example 1 against Escherichia coli was relatively weaker, with an antibacterial rate of 93%. However, the adhesives obtained after adding tannic acid showed antibacterial efficiencies of over 99.9% against Escherichia coli. The experimental results indicate that introducing tannic acid into the hydrogel composed of chitosan guanidine salt and aldehyde-modified dextran further enhances the antibacterial properties of the adhesive.

[0140] The adhesives prepared in Examples 1-3 and Comparative Examples 1 and 6 were sterilized and then placed on nutrient agar plates coated with methicillin-resistant Staphylococcus aureus (MRSA). After 24 hours, the diameter of the inhibition zone was observed and measured. Nutrient agar plates coated with MRSA without adhesive were used as a control group. The results are shown in [Figure 1]. Figure 5 and Figure 6 .

[0141] Depend on Figure 5 and Figure 6It can be seen that the diameter of the inhibition zone gradually increases with the increase of tannic acid content, indicating that tannic acid in the CSG / ODex-TA series adhesives is released from the adhesive and kills the surrounding bacteria. The higher the tannic acid content, the greater the release, which leads to a larger diameter of the inhibition zone.

[0142] The results of the inhibition zone comparison experiment and the statistical results of inhibition zone diameter of Example 2, Comparative Example 2 and Control Group (filter paper disc) against methicillin-resistant Staphylococcus aureus (MRSA) are shown in the figure below. Figure 7 and Figure 8 ,Depend on Figure 7 and Figure 8 The inhibition zone diameter of CSG / ODex-TA2 adhesive (22.42 mm) was found to be significantly higher than that of fibrin glue (0 mm), indicating that CSG / ODex-TA2 adhesive has a better antibacterial effect on surrounding bacteria.

[0143] III. Cytotoxicity Test

[0144] The cytotoxicity of CSG / ODex prepared in Comparative Example 1 and the CSG / ODex-TA series adhesives prepared in the Examples to mouse embryonic fibroblast L929 cells was determined using the MTT assay. First, L929 cells were cultured at 1.5 × 10⁶ cells per well. 4 Cells were seeded at a density in 96-well plates and incubated at 37°C with 5% CO2 for 24 hours. Then, the adhesives from Comparative Examples 1 and 6, and Examples 1-3 were placed in culture medium (0.1 g / mL) for 24 hours of extraction. The extract was then co-cultured with the cells for another 24 hours, with 20 μL of MTT solution added to each well. After incubation at 37°C for 4 hours, the absorbance of each well was measured at 490 nm using a microplate reader. Six parallel experiments were performed for each sample concentration. The control group consisted of untreated cells. Cell viability was calculated as follows:

[0145] Cell viability % = (A s -A b ) / (A c -A b )×100%

[0146] In the formula, A s A is the absorbance value of the experimental sample group. c This is the absorbance value of the control group, A. b It is the absorbance value of the background.

[0147] The results of cell viability determination of mouse embryonic fibroblast L929 cells after co-culturing with different groups of adhesives for 24 hours are shown in the figure. Figure 9It can be seen that the CSG / ODex, CSG / ODex-TA1, CSG / ODex-TA2, and CSG / ODex-TA3 adhesives all had a cell survival rate of over 80% after 24 hours, indicating that they have low toxicity and good biocompatibility.

[0148] IV. Antioxidant Test

[0149] The antioxidant activity of different adhesives was evaluated by monitoring their ability to scavenge nitrogen free radicals using 1,1-diphenyl-2-picrylhydrazine (DPPH) as an indicator. DPPH was dissolved in ethanol to obtain a 100 μM solution. 300 μL of each of the adhesives prepared in Example 1 and Comparative Example 1 were crushed, and then 3 mL of the DPPH ethanol solution was mixed with the adhesive. The control group consisted of 300 μL of water. After incubation in the dark for 1 hour, the absorbance of the mixture at 517 nm was measured using a UV spectrophotometer. The nitrogen free radical scavenging rate was calculated using the following formula:

[0150] Nitrogen free radical scavenging rate % = (A s -A b ) / (A c -A b )×100%

[0151] In the formula, A s A is the absorbance value of the experimental sample group. c This is the absorbance value of the control group, A. b It is the absorbance value of the background.

[0152] The results of nitrogen free radical scavenging rates of different groups of adhesives in Comparative Examples 1, 6, and Examples 1-3 are shown in the figure. Figure 10 It can be seen that the adhesive obtained in the examples has a stronger antioxidant capacity than the comparative example.

[0153] V. Bacterial Infection Wound Healing Experiment

[0154] Hair was removed from the backs of 6-8 week old mice (BALB / C strain) weighing 16-22 grams to create a circular full-thickness skin defect with a diameter of approximately 10 mm. 10 μL of a 10% concentration of [unspecified substance] was applied to the wound site of each mouse. 8 CFU / mL methicillin-resistant Staphylococcus aureus bacterial suspension. Mice were randomly divided into 5 groups (n=5 per group), and 300 μL of different samples (CSG / ODex adhesive of Comparative Example 1, porcine fibrin glue of Comparative Example 2, and CSG / ODex-TA2 adhesive of Example 2) were applied to the infected wounds of the mice, and the wound healing was monitored at different time points.

[0155] Photos of mouse skin infection wound healing after different sample treatments are shown below. Figure 11The results of wound healing area of ​​infected mouse skin after different sample treatments are shown in the figure. Figure 12 The experimental treatment groups included: I: blank group (uninfected wound), II: control group (infected wound, no sample treatment), III: infected wound treated with fibrin glue, IV: infected wound treated with CSG / ODex, and V: infected wound treated with CSG / ODex-TA2. Each group was repeated 4 times. The significance test results showed that **p<0.01, ***p<0.001, and ****p<0.0001, indicating that CSG / ODex-TA2 adhesive significantly promoted wound healing.

[0156] On days 3 and 9, bacteria were collected from the wounds of different groups using cotton swabs and placed in culture medium. After incubation at 37°C for 4 hours, the culture was diluted 10⁻⁶. 4 After dilution, take 100 μL and spread it onto a plate. Incubate at 37°C for 24 hours, then determine the colony growth. Figure 13 And count the number of colonies (see below) Figure 14 The experimental treatment groups included: I: blank group (uninfected wound), II: control group (infected wound, no sample treatment), III: infected wound treated with fibrin glue, IV: infected wound treated with CSG / ODex, and V: infected wound treated with CSG / ODex-TA2. Each group was repeated three times. The significance test showed that ***p<0.001 and ****p<0.0001. It can be seen that the CSG / ODex-TA2 adhesive has a more obvious antibacterial effect on the wound.

[0157] VI. Hemostasis Test

[0158] SD rats weighing 230-260 grams were anesthetized and fixed to a surgical cork board. The liver was exposed through abdominal dissection. The tissue fluid around the liver was then removed with gauze, and weighed filter paper was placed directly beneath the liver. The cork board was tilted at a 30-degree angle, and a 1 cm long and 0.4 cm deep incision was made in the liver using an 18G needle to construct a liver hemorrhage model. Fibrin glue (from Example 2) and Comparative Example 2, and CSG / ODex-TA2 medical adhesive were injected into the incision using a syringe. The control group represented liver hemorrhage without medical adhesive hemostasis. Liver hemorrhage was observed by taking photographs at 10, 30, 60, and 120 seconds. After 120 seconds, the weight of the filter paper was measured to determine the hemorrhage mass. The experimental results of different adhesives used for liver hemostasis are shown in the attached images. Figure 15 The statistical results of bleeding volume after different groups of adhesives were used for liver hemostasis are shown in the figure. Figure 16 ,Depend on Figure 15 and 16 It can be seen that both fibrin glue and CSG / ODex-TA2 adhesive have certain hemostatic effects, and CSG / ODex-TA2 adhesive has a better hemostatic effect.

[0159] VII. Degradation Experiment

[0160] The adhesives prepared in Examples 1-3, Comparative Examples 1 and 6 were placed in PBS (pH=7.4) solution and incubated in a 37°C constant temperature water bath. The weight of the adhesive was monitored at different time points, and the percentage of remaining mass was calculated. Each group was repeated three times. The experimental results are shown in the figure. Figure 17 .Depend on Figure 17 It can be seen that the adhesive prepared in the embodiments of the present invention has excellent biodegradability.

[0161] The test results of adhesion strength and MRSA inhibition zone diameter in the examples and comparative examples are shown in Table 1.

[0162] Table 1

[0163] Example 1 CSG / ODex-TA1 10.15 7.25 Example 2 CSG / ODex-TA2 11.84 22.42 Example 3 CSG / ODex-TA3 20.35 25.64 Example 4 CSMG / ODex-TA3 16.62 24.15 Example 5 CSMG / ODex-TA4 12.53 23.86 Example 6 CSMG / ODex-TA5 10.02 6.89 Example 7 CSG / ODex-TA6 8.53 7.19 Example 8 CSG / ODex-TA7 10.21 7.42 Comparative Example 1 CSG / ODex 5.09 1.16 Comparative Example 2 Fibrin glue 3.36 0 Comparative Example 3 CS / ODex-TA3 8.34 16.80 Comparative Example 4 No gel adhesive - - Comparative Example 5 ACMC / ODex <![CDATA[3.92(40g f / cm 2 )]]> 1.04 Comparative Example 6 CSG / ODex-TA5 5.32 29.68

[0164] Based on Table 1, the following conclusions can be drawn:

[0165] (1) Adding TA to the adhesive can increase its adhesion strength, and the adhesion strength gradually increases with the increase of TA content, with the adhesion strength of CSG / ODex-TA3 reaching the maximum value. The adhesion strength of the examples is higher than that of the comparative examples.

[0166] (2) Adding TA to the adhesive can significantly enhance its antibacterial properties. As the TA content increases, the diameter of its MRSA inhibition zone gradually increases. The adhesive prepared in the example has excellent antibacterial properties.

[0167] The above are merely preferred embodiments of this application, but the scope of protection of this application is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in this application should be included within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.

Claims

1. A method for preparing a biodegradable antibacterial medical adhesive, characterized in that, The biodegradable antibacterial medical adhesive is obtained by forming a double cross-linked network hydrogel from chitosan guanidine salt, aldehyde-modified dextran, and tannic acid through Schiff base reaction, electrostatic interaction, and hydrogen bonding. The specific steps include: mixing an aqueous solution of aldehyde-modified dextran and an aqueous solution of tannic acid to obtain a mixture, then adding an aqueous solution of chitosan guanidine salt to the mixture, and mixing evenly at room temperature to obtain the biodegradable antibacterial medical adhesive. The volume ratio of the aldehyde-modified dextran aqueous solution, the tannic acid aqueous solution, and the chitosan guanidine salt aqueous solution is 1:1:(1-4); The concentration of the aldehyde-modified dextran aqueous solution is 0.1–30 wt%, the concentration of the tannic acid aqueous solution is 0.1–25 wt%, and the concentration of the chitosan guanidine salt aqueous solution is 0.1–30 wt%. The chitosan guanidine salt includes one or both of chitosan monoguanidine salt and chitosan biguanidine salt.

2. A biodegradable antibacterial medical adhesive prepared by the preparation method of claim 1.

3. The application of the biodegradable antibacterial medical adhesive as described in claim 2 in the preparation of hemostatic, antibacterial, and repair materials.

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