An antibacterial peptide composite material and a preparation method and application thereof

Abstract

This invention provides a method for preparing an antimicrobial peptide composite material, comprising: S1 preparing a β-TCP / HyA porous substrate; S2 synthesizing mesoporous polydopamine (MPDA); S3 dispersing the β-TCP / HyA porous scaffold and the mesoporous polydopamine in deionized water for a second crosslinking reaction to form a β-TCP / HyA / MPDA composite suspension; and then adding the antimicrobial peptide KR to the β-TCP / HyA / MPDA composite suspension to induce a binding reaction, thereby obtaining the antimicrobial peptide composite material. This invention crosslinks β-tricalcium phosphate (β-TCP), hyaluronic acid (HyA), and mesoporous polydopamine (MPDA) to construct a multifunctional composite substrate that combines physical support, biocompatibility, and high-efficiency loading. The mesoporous structure of MPDA provides numerous anchoring sites for the antimicrobial peptide KR. Through MPDA adhesion and covalent fixation strategies for the antimicrobial peptide, high-density and high-activity loading of KR molecules is achieved, effectively solving the problems of easy detachment and "fixation leading to inactivation".

Description

Technical Field

[0001] This invention relates to the field of aqueous zinc-ion battery technology, and in particular to an antimicrobial peptide composite material, its preparation method, and its application. Background Technology

[0002] In the field of orthodontics, invisible braces are widely used due to their aesthetic appeal and comfort. However, during wear, braces cover the tooth surface and gum line for extended periods, creating a relatively enclosed space with the oral environment. This can easily lead to a slowdown in saliva flow and an increase in local temperature and humidity, thereby promoting the adhesion of cariogenic bacteria (such as Streptococcus mutans) and the formation of plaque biofilm. The resulting enamel demineralization (chalky spots), dental caries, and gingivitis have become major complications affecting orthodontic treatment outcomes and patients' oral health.

[0003] Currently, plaque control related to braces mainly relies on patients' daily cleaning and auxiliary methods such as fluoride preparations and antibacterial mouthwashes. However, these methods have limitations such as short duration of action and difficulty in sustainably inhibiting plaque reformation. Therefore, endowing braces themselves with long-lasting antibacterial capabilities is of significant clinical importance for improving oral health management during orthodontic treatment. To achieve this goal, existing research mainly focuses on constructing antibacterial coatings for braces materials. Early coating strategies mainly relied on silver-based antibacterial agents (such as silver ions and their nanoparticles) or traditional antibiotics (such as chlorhexidine and tetracycline). Silver ions achieve broad-spectrum bactericidal effects by disrupting bacterial cell membranes, interfering with enzyme systems, and generating reactive oxygen species; while antibiotics exert their effects by specifically inhibiting bacterial protein synthesis or disrupting cell membrane integrity. Although these methods have certain antibacterial effects, silver ions pose potential cytotoxicity and discoloration risks, while long-term use of antibiotics can easily induce bacterial resistance.

[0004] In recent years, antimicrobial peptides (AMPs) have attracted widespread attention as a new generation of antimicrobial strategies. AMPs are small molecule polypeptides produced by the organism's innate immune system. They form pores on the cell membrane of microorganisms through electrostatic interactions, physically disrupting the membrane integrity and causing leakage of cell contents, thus rapidly killing microorganisms. This mechanism of action is less likely to induce bacterial resistance, and AMPs generally have good biocompatibility, thus being considered ideal candidates for antimicrobial agents. However, achieving stable and efficient binding with dental braces materials remains a significant challenge. Current technologies lack effective immobilization methods. Simple physical adsorption or blending results in weak binding forces between AMPs and the substrate, leading to rapid detachment in the dynamic environment of oral cavity, such as saliva rinsing and chewing friction, making it impossible to maintain a long-lasting antimicrobial concentration. On the other hand, overly rigid chemical immobilization easily destroys their active conformation, resulting in "immobilization equals inactivation." Furthermore, as polypeptides, the stability of AMPs in the fluctuating pH environment of the oral cavity still requires systematic verification.

[0005] While existing technologies such as silver-based coatings, antibiotic coatings, and antimicrobial peptide coatings offer preliminary ideas for achieving the antibacterial function of braces, none of these solutions systematically resolve the multiple contradictions between "firm fixation," "activity maintenance," and "long-term biosafety." Therefore, developing an innovative coating technology capable of achieving stable loading, high activity, and long-term safety of antimicrobial peptides on the brace surface has become an urgent and clear technological need in this field. Summary of the Invention

[0006] To address the technical problems in existing technologies, such as the easy induction of drug resistance and poor biocompatibility by silver ions and antibiotic coatings, as well as the common issues of weak binding, easy inactivation, and difficulty in long-term action of antimicrobial peptides when immobilized on device surfaces, this invention provides an antimicrobial peptide composite material, its preparation method, and its application. This invention is the first to crosslink β-tricalcium phosphate (β-TCP), hyaluronic acid (HyA), and mesoporous polydopamine (MPDA) to construct a multifunctional composite substrate that combines physical support, biocompatibility, and high-efficiency loading. The mesoporous structure of MPDA provides a large number of anchoring sites for the antimicrobial peptide KR. Through the MPDA adhesion and antimicrobial peptide covalent fixation strategy, and then through covalent crosslinking, high-density and high-activity loading of KR molecules is achieved, effectively solving the problems of easy detachment and "fixation followed by inactivation".

[0007] The first objective of this invention is to provide a method for preparing an antimicrobial peptide composite material, the method comprising: S1 Preparation of β-TCP / HyA porous substrate: β-tricalcium phosphate β-TCP powder is dispersed in deionized water and ultrasonically treated to form β-TCP slurry; hyaluronic acid HyA powder is dissolved in ultrapure water to obtain HyA solution; β-TCP slurry is slowly added to HyA solution, mixed evenly, and then pre-frozen to obtain uncrosslinked β-TCP / HyA porous scaffold; the porous scaffold is immersed in crosslinking solution to carry out the first crosslinking reaction and then freeze-dried to obtain β-TCP / HyA porous substrate; S2 Synthesis of Mesoporous Polydopamine (MPDA): Dopamine hydrochloride and a template agent are dissolved in an ethanol solution to obtain a first mixed solution; a pore-expanding agent solution is added to the first mixed solution, and the mixture is ultrasonically dispersed to obtain a second mixed solution; Tris-HCl buffer is added to the second mixed solution to carry out a self-polymerization reaction, and the mixture is then centrifuged, washed, and freeze-dried to obtain mesoporous polydopamine (MPDA); S3 disperses the β-TCP / HyA porous scaffold and mesoporous polydopamine together in deionized water to carry out a second crosslinking reaction, forming a β-TCP / HyA / MPDA composite suspension; then, antimicrobial peptide KR is added to the β-TCP / HyA / MPDA composite suspension to carry out a binding reaction, thereby obtaining an antimicrobial peptide composite material.

[0008] Specifically, in step S1, the concentration of the β-TCP slurry is 20-30 mg / mL, and the concentration of the HyA solution is 100-120 mg / mL; the volume ratio of the β-TCP slurry to the HyA solution is 1:1.

[0009] Specifically, the ultrasonic treatment in step S1 has a power of 100W and a time of 10-20min; the pre-freezing treatment temperature is as follows: the device is placed in a pre-freezing chamber at a temperature of -20℃ for 24h, and then transferred to a freeze dryer for freeze drying at a temperature of -50℃ and a pressure of <10Pa for 48h.

[0010] Specifically, the crosslinking solution in step S1 is any one of an ethanol solution composed of EDC and NHS, an ethanol solution of diimide, and an ethanol solution of genipin; the temperature of the first crosslinking reaction is 25-30℃, and the time is 20-24h.

[0011] Specifically, in step S2, the mass ratio of dopamine hydrochloride to template agent is 1.5:1.9; the ethanol solution is composed of deionized water and anhydrous ethanol in a volume ratio of 1:1.

[0012] Specifically, in step S2, the mass ratio of dopamine hydrochloride to the template agent is 1.5:1.9; the template agent is triblock copolymer F127; the ethanol solution is composed of deionized water and anhydrous ethanol in a volume ratio of 1:1; the concentration of dopamine hydrochloride in the first mixed solution in step S2 is 3.5-4 mg / mL; the pore-expanding agent solution is a 1 mg / mL solution of mesitylene, which is prepared by dissolving mesitylene in a mixed solvent; the mixed solvent is composed of anhydrous ethanol and PBS buffer in a volume ratio of 1:9.

[0013] Specifically, in step S2, the volume ratio of the first mixed solution to the pore-expanding agent solution is (62-65):1; the concentration of the Tris-HCl buffer solution is 10mM and the pH value is 8.5; the temperature of the self-polymerization reaction is 25-30℃ and the time is 2-4h.

[0014] Specifically, in step S3, the mass ratio of the β-TCP / HyA porous scaffold, mesoporous polydopamine, and antimicrobial peptide is 125:75:2; the temperature of the second crosslinking reaction is 25-30℃, and the time is 24h.

[0015] Specifically, the binding reaction in step S3 is the binding of antimicrobial peptide KR with mesoporous polydopamine; the binding reaction can be carried out by any one of covalent bonding, metal ion coordination and electrostatic adsorption; the temperature of the binding reaction is 25-30℃ and the time is 20-24h.

[0016] The second objective of this invention is to provide an antimicrobial peptide composite material prepared by the preparation method described above.

[0017] A third objective of this invention is to provide an application of the aforementioned antimicrobial peptide composite material, specifically: coating the antimicrobial peptide composite material onto medical products or immersing medical products in the antimicrobial peptide composite material; the coating method includes spin coating and spray coating; the medical products include one or more of medical devices, medical materials, and implants; the medical devices include dental braces; the implants include orthopedic screws and joint prostheses; and the medical materials include silicone endotracheal tubes and urinary catheters.

[0018] Compared with the prior art, the beneficial effects of the present invention include: (1) This invention is the first to crosslink β-tricalcium phosphate (β-TCP), hyaluronic acid (HyA), and mesoporous polydopamine (MPDA) to construct a multifunctional composite substrate that combines physical support, biocompatibility, and high loading efficiency. The mesoporous structure of MPDA provides numerous anchoring sites for the antimicrobial peptide KR. Through MPDA adhesion and covalent fixation of the antimicrobial peptide, high-density and high-activity loading of KR molecules is achieved via covalent crosslinking, effectively solving the problems of easy detachment and "fixation leading to inactivation." This forms a structurally stable, high-loading, and firmly bound functional interface. This interface can retain the natural conformation and bactericidal activity of KR to the maximum extent, ensuring its long-term stability in the dynamic oral environment and laying the foundation for achieving a long-lasting and safe antibacterial effect. (2) In this invention, the antimicrobial peptide KR is selected as the core bactericidal unit, which works by non-specifically physically destroying the bacterial cell membrane and cell wall; β-TCP serves as the inorganic framework, and its surface is rich in calcium ions (Ca²⁺). +This not only provides a porous support structure for the coating, but also allows for ionic coordination with the carboxyl and phenolic hydroxyl groups of organic components, enhancing mechanical stability and promoting MPDA dispersion. HyA, as an organic network, with its long-chain polysaccharide structure and numerous carboxyl and hydroxyl groups, can chemically cross-link with MPDA and antimicrobial peptides to form a flexible three-dimensional network, filling inorganic pores and significantly improving the hydrophilicity and biocompatibility of the coating. MPDA, as an interfacial functional center, leverages its high specific surface area and mesoporous structure to strongly adsorb and bind MPDA through π-π stacking, hydrogen bonding, and covalent bonds (such as Michael addition / Schiff base reactions). By immobilizing antimicrobial peptides, high loading and controlled release are achieved. The three elements work synergistically to construct a stable composite layer integrating an inorganic framework, an organic network, and a functional interface, ultimately forming a β-TCP / HyA / MPDA@KR antimicrobial peptide composite material. This significantly improves the loading and distribution uniformity of the antimicrobial peptides, greatly enhancing their stability and long-lasting effect in complex environments. Furthermore, the covalent and non-covalent bonding imparts excellent mechanical stability to the coating. Its stable structure ensures that the antimicrobial peptides can maintain their antimicrobial function stably and over a long period, even under fluctuations in pH, temperature, and salt ion concentration experienced in the oral environment. (3) The composite material of the present invention is prepared in a normal temperature and pressure environment. The process is simple and the conditions are mild, requiring no expensive equipment. In addition, the antibacterial coating is repairable. If the coating wears down and the antibacterial effect decreases due to long-term use, the user only needs to immerse the braces in the composite suspension again to achieve in-situ repair and functional regeneration of the coating, which greatly extends the service life of the product. The composite material prepared by the present invention is not only applicable to invisible dental braces (polymer materials such as PETG), but can also be extended to the surface of other medical implants that urgently need antibacterial function, such as metals (orthopedic nails, joint prostheses) and silicone (endotracheal tubes, urinary catheters), providing a general solution to a wide range of medical device-related infection problems. Attached Figure Description

[0019] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings in the following description are merely exemplary, and those skilled in the art can derive other embodiments based on the provided drawings without creative effort.

[0020] Figure 1 This is a process flow diagram of the composite material prepared in Example 1 of the present invention; Figure 2 This is a schematic diagram of the material composition and structure of the composite material prepared in Example 1 of the present invention; Figure 3 (ac) are SEM images of the composite material prepared in Example 2 of this invention at different resolutions; Figure 4 This is a schematic diagram illustrating the application of the composite material prepared in Example 2 of the present invention in forming a coating on dental braces. Figure 5 These are cryo-electron microscopy images of the composite materials prepared in Examples 1-3 of this invention applied to dental braces. Figure 6 This is a comparison of the bactericidal effects of the composite materials prepared in Examples 1-3 of the present invention and tetracycline in Comparative Example 2; Figure 7 The image shows a comparison of the survival rates of Streptococcus mutans after the composite materials prepared in Examples 1-3 of this invention and the tetracycline applied to dental braces in Comparative Example 2 were treated with the composite material; Figure 8 The graph shows the effect of the composite materials prepared in Examples 1-3 of this invention and Comparative Examples 1 and 3 on the growth curve of Streptococcus mutans after being applied to dental braces. Detailed Implementation

[0021] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by those skilled in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing specific embodiments only and is not intended to limit the present invention.

[0022] Please see Figure 1 and 2 , Figure 1 This is a process flow diagram of the composite material prepared in Example 1 of the present invention; Figure 2 This is a schematic diagram of the material composition and structure of the composite material prepared in Example 1 of the present invention.

[0023] Example 1 Preparation of β-TCP / HyA porous substrate (S1): 0.9 g of β-tricalcium phosphate (β-TCP) powder was dispersed in 40 mL of deionized water and ultrasonically treated at 100 W for 10 min to form a β-TCP slurry; 0.9 g of hyaluronic acid (HyA) powder was dissolved in 8 mL of ultrapure water to obtain a HyA solution; 8 mL of β-TCP slurry was slowly added to the HyA solution, mixed thoroughly, and then pre-frozen at -20 °C for 24 h, before being transferred to a freeze dryer for further processing. Uncrosslinked β-TCP / HyA porous scaffolds were obtained by freeze-drying at -50℃ and pressure <10Pa for 48h. The porous scaffolds were then immersed in 400mL of crosslinking solution (an ethanol crosslinking solution composed of EDC and NHS, wherein the volume ratio of EDC to NHS was 5:2 and the concentration of the ethanol crosslinking solution was 75%) and subjected to a first crosslinking reaction at 25℃ for 24h, followed by freeze-drying at -50℃ for 48h to obtain β-TCP / HyA porous substrates. S2 Synthesis of Mesoporous Polydopamine (MPDA): 0.15 g dopamine hydrochloride and 0.19 g template (triblock copolymer F127) were dissolved in 40 mL of 50% ethanol solution to obtain a first mixed solution. 640 μL of a pore-expanding agent solution (trimethylbenzene (TMB) solution, prepared by dissolving 10 mg TMB in 1 mL of anhydrous ethanol and then diluting with 9 mL of PBS buffer) was added to the first mixed solution, and the mixture was ultrasonically dispersed for 2 min to obtain a second mixed solution. 375 μL of Tris-HCl buffer (10 mM, pH 8.5) was added to the second mixed solution, and the mixture was subjected to self-polymerization at 25 °C with continuous magnetic stirring for 2 h. The mixture was then centrifuged at 13,000 rpm for 10 min, the precipitate was collected, and the precipitate was washed three times alternately with ethanol and deionized water to obtain mesoporous polydopamine (MPDA). S3. The 6 mg β-TCP / HyA porous scaffold and 6 mg mesoporous polydopamine were co-dispersed in 40 mL of deionized water and subjected to a second crosslinking reaction at 25 °C for 24 h to form a 300 μg / mL β-TCP / HyA / MPDA composite suspension. Then, 0.24 mg of antimicrobial peptide KR was added to the β-TCP / HyA / MPDA composite suspension, i.e., the amount of KR added was 6 μg / mL. The mixture was allowed to stand at 25 °C for 24 h in a covalent bonding manner to undergo a binding reaction, thereby obtaining the antimicrobial peptide composite material prepared in Example 1.

[0024] Example 2 S1 Preparation of β-TCP / HyA porous substrate: 0.9 g of β-tricalcium phosphate β-TCP powder was dispersed in 40 mL of deionized water and ultrasonicated at 100 W for 10 min to form β-TCP slurry; 0.9 g of hyaluronic acid HyA powder was dissolved in 8 mL of ultrapure water to obtain HyA solution; 8 mL of β-TCP slurry was slowly added to HyA solution, mixed evenly, and pre-frozen at -20℃ for 24 h to obtain uncrosslinked β-TCP / HyA porous scaffold; the porous scaffold was immersed in 400 mL of crosslinking solution (an ethanol crosslinking solution composed of EDC and NHS, wherein the volume ratio of EDC and NHS is 5:2, and the concentration of ethanol crosslinking solution is 75%) and subjected to the first crosslinking reaction at 25℃ for 24 h, and then freeze-dried at -50℃ for 48 h to obtain β-TCP / HyA porous substrate; S2 Synthesis of Mesoporous Polydopamine (MPDA): 0.15 g dopamine hydrochloride and 0.19 g template (triblock copolymer F127) were dissolved in 40 mL of 50% ethanol solution to obtain a first mixed solution. 640 μL of a pore-expanding agent solution (trimethylbenzene (TMB) solution, prepared by dissolving 10 mg TMB in 1 mL of anhydrous ethanol and then diluting with 9 mL of PBS buffer) was added to the first mixed solution, and the mixture was ultrasonically dispersed for 2 min to obtain a second mixed solution. 375 μL of Tris-HCl buffer (10 mM, pH 8.5) was added to the second mixed solution, and the mixture was subjected to self-polymerization at 25 °C with continuous magnetic stirring for 2 h. The mixture was then centrifuged at 13,000 rpm for 10 min, the precipitate was collected, and the precipitate was washed three times alternately with ethanol and deionized water to obtain mesoporous polydopamine (MPDA). S3. The 6 mg of β-TCP / HyA porous scaffold and 6 mg of mesoporous polydopamine were dispersed together in 40 mL of deionized water and subjected to a second crosslinking reaction at 25 °C for 24 h to form a β-TCP / HyA / MPDA composite suspension. Then, 0.32 mg of antimicrobial peptide KR was added to the β-TCP / HyA / MPDA composite suspension, i.e., the amount of KR added was 8 μg / mL. The mixture was allowed to stand at 25 °C for 24 h in a covalent bonding manner to undergo a binding reaction, thereby obtaining the antimicrobial peptide composite material prepared in Example 2.

[0025] Example 3 S1 Preparation of β-TCP / HyA porous substrate: 0.9 g of β-tricalcium phosphate β-TCP powder was dispersed in 40 mL of deionized water and ultrasonicated at 100 W for 10 min to form β-TCP slurry; 0 g of hyaluronic acid HyA powder was dissolved in 8 mL of ultrapure water to obtain HyA solution; 8 mL of β-TCP slurry was slowly added to HyA solution, mixed evenly, and pre-frozen at -20℃ for 24 h to obtain uncrosslinked β-TCP / HyA porous scaffold; the porous scaffold was immersed in 400 mL of crosslinking solution (an ethanol crosslinking solution composed of EDC and NHS, wherein the volume ratio of EDC and NHS is 5:2, and the concentration of ethanol crosslinking solution is 75%) and subjected to the first crosslinking reaction at 25℃ for 24 h, and then freeze-dried at -50℃ for 48 h to obtain β-TCP / HyA porous substrate; S2 Synthesis of Mesoporous Polydopamine (MPDA): 0.15 g dopamine hydrochloride and 0.19 g template (triblock copolymer F127) were dissolved in 40 mL of 50% ethanol solution to obtain a first mixed solution. 640 μL of a pore-expanding agent solution (trimethylbenzene (TMB) solution, prepared by dissolving 10 mg TMB in 1 mL of anhydrous ethanol and then diluting with 9 mL of PBS buffer) was added to the first mixed solution, and the mixture was ultrasonically dispersed for 2 min to obtain a second mixed solution. 375 μL of Tris-HCl buffer (10 mM, pH 8.5) was added to the second mixed solution, and the mixture was subjected to self-polymerization at 25 °C with continuous magnetic stirring for 2 h. The mixture was then centrifuged at 13,000 rpm for 10 min, the precipitate was collected, and the precipitate was washed three times alternately with ethanol and deionized water to obtain mesoporous polydopamine (MPDA). S3. The 6 mg of β-TCP / HyA porous scaffold and 6 mg of mesoporous polydopamine were dispersed together in 40 mL of deionized water and subjected to a second crosslinking reaction at 25 °C for 24 h to form a β-TCP / HyA / MPDA composite suspension. Then, 0.4 mg of antimicrobial peptide KR was added to the β-TCP / HyA / MPDA composite suspension, i.e., the amount of KR added was 10 μg / mL. The mixture was allowed to stand at 25 °C for 24 h in a covalent bonding manner to undergo a binding reaction, and the antimicrobial peptide composite material prepared in Example 3 was obtained.

[0026] Example 4 S1 Preparation of β-TCP / HyA porous substrate: 0.9 g of β-tricalcium phosphate β-TCP powder was dispersed in 40 mL of deionized water and ultrasonicated at 100 W for 10 min to form β-TCP slurry; 0.9 g of hyaluronic acid HyA powder was dissolved in 8 mL of ultrapure water to obtain HyA solution; 8 mL of β-TCP slurry was slowly added to HyA solution, mixed evenly, and pre-frozen at -20℃ for 24 h to obtain uncrosslinked β-TCP / HyA porous scaffold; the porous scaffold was immersed in 400 mL of crosslinking solution (an ethanol crosslinking solution composed of EDC and NHS, wherein the volume ratio of EDC and NHS is 5:2, and the concentration of ethanol crosslinking solution is 75%) and subjected to the first crosslinking reaction at 25℃ for 24 h, and then freeze-dried at -50℃ for 48 h to obtain β-TCP / HyA porous substrate; S2 Synthesis of Mesoporous Polydopamine (MPDA): 0.15 g dopamine hydrochloride and 0.19 g template (triblock copolymer F127) were dissolved in 40 mL of 50% ethanol solution to obtain a first mixed solution. 640 μL of a pore-expanding agent solution (trimethylbenzene (TMB) solution, prepared by dissolving 10 mg TMB in 1 mL of anhydrous ethanol and then diluting with 9 mL of PBS buffer) was added to the first mixed solution, and the mixture was ultrasonically dispersed for 2 min to obtain a second mixed solution. 375 μL of Tris-HCl buffer (10 mM, pH 8.5) was added to the second mixed solution, and the mixture was subjected to self-polymerization at 25 °C with continuous magnetic stirring for 2 h. The mixture was then centrifuged at 13,000 rpm for 10 min, the precipitate was collected, and the precipitate was washed three times alternately with ethanol and deionized water to obtain mesoporous polydopamine (MPDA). S3. The 6 mg of β-TCP / HyA porous scaffold and 6 mg of mesoporous polydopamine were dispersed together in 40 mL of deionized water and subjected to a second crosslinking reaction at 25 °C for 24 h to form a β-TCP / HyA / MPDA composite suspension. Then, 0.24 mg of antimicrobial peptide KR was added to the β-TCP / HyA / MPDA composite suspension, i.e., the amount of KR added was 6 μg / mL. The mixture was allowed to stand at 25 °C for 24 h in a covalent bonding manner to undergo a binding reaction, and the antimicrobial peptide composite material prepared in Example 4 was obtained.

[0027] Example 5 S1 Preparation of β-TCP / HyA porous substrate: 1.2g of β-tricalcium phosphate β-TCP powder was dispersed in 40mL of deionized water and ultrasonicated at 100W for 20min to form β-TCP slurry; 0.8g of hyaluronic acid HyA powder was dissolved in 8mL of ultrapure water to obtain HyA solution; 8mL of β-TCP slurry was slowly added to HyA solution, mixed evenly, and pre-frozen at -20℃ for 24h to obtain uncrosslinked β-TCP / HyA porous scaffold; the porous scaffold was immersed in 400mL of diimide ethanol solution, and the first crosslinking reaction was carried out at 30℃ for 22h, followed by freeze-drying at -50℃ for 20h to obtain β-TCP / HyA porous substrate; S2 Synthesis of Mesoporous Polydopamine (MPDA): 0.15 g dopamine hydrochloride and 0.19 g template (triblock copolymer F127) were dissolved in 40 mL of 50% ethanol solution to obtain a first mixed solution. 640 μL of a pore-expanding agent solution (trimethylbenzene (TMB) solution, prepared by dissolving 10 mg TMB in 1 mL of anhydrous ethanol and then diluting with 9 mL of PBS buffer) was added to the first mixed solution, and the mixture was ultrasonically dispersed for 2 min to obtain a second mixed solution. 375 μL of Tris-HCl buffer (10 mM, pH 8.5) was added to the second mixed solution, and the mixture was subjected to self-polymerization at 30 °C with continuous magnetic stirring for 4 h. The mixture was then centrifuged at 13,000 rpm for 10 min, the precipitate was collected, and the precipitate was washed three times alternately with ethanol and deionized water to obtain mesoporous polydopamine (MPDA). S3 dispersed the 6 mg β-TCP / HyA porous scaffold and 6 mg mesoporous polydopamine in 40 mL of deionized water and carried out a second crosslinking reaction at 30 °C for 24 h to form a β-TCP / HyA / MPDA composite suspension; then, 0.32 mg of antimicrobial peptide KR was added to the β-TCP / HyA / MPDA composite suspension and a binding reaction was carried out at 25 °C for 24 h via metal ion coordination to obtain the antimicrobial peptide composite material prepared in Example 5.

[0028] Example 6 S1 Preparation of β-TCP / HyA porous substrate: 0.9 g of β-tricalcium phosphate β-TCP powder was dispersed in 40 mL of deionized water and ultrasonicated at 100 W for 10 min to form β-TCP slurry; 0.9 g of hyaluronic acid HyA powder was dissolved in 8 mL of ultrapure water to obtain HyA solution; 8 mL of β-TCP slurry was slowly added to HyA solution, mixed evenly, and pre-frozen at -20℃ for 24 h to obtain uncrosslinked β-TCP / HyA porous scaffold; the porous scaffold was immersed in 400 mL of crosslinking solution (an ethanol crosslinking solution composed of EDC and NHS, wherein the volume ratio of EDC and NHS is 5:1, and the concentration of the crosslinking solution is 75%) and subjected to the first crosslinking reaction at 25℃ for 24 h, and then freeze-dried at -50℃ for 24 h to obtain β-TCP / HyA porous substrate; S2 Synthesis of Mesoporous Polydopamine (MPDA): 0.15 g dopamine hydrochloride and 0.19 g template (triblock copolymer F127) were dissolved in 40 mL of 50% ethanol solution to obtain a first mixed solution. 640 μL of a pore-expanding agent solution (trimethylbenzene (TMB) solution, prepared by dissolving 10 mg TMB in 1 mL of anhydrous ethanol and then diluting with 9 mL of PBS buffer) was added to the first mixed solution, and the mixture was ultrasonically dispersed for 2 min to obtain a second mixed solution. 375 μL of Tris-HCl buffer (10 mM, pH 8.5) was added to the second mixed solution, and the mixture was subjected to self-polymerization at 25 °C with continuous magnetic stirring for 2 h. The mixture was then centrifuged at 13,000 rpm for 10 min, the precipitate was collected, and the precipitate was washed three times alternately with ethanol and deionized water to obtain mesoporous polydopamine (MPDA). S3 dispersed 6 mg of the β-TCP / HyA porous scaffold and 6 mg of mesoporous polydopamine in 40 mL of deionized water and carried out a second crosslinking reaction at 25 °C for 22 h to form a β-TCP / HyA / MPDA composite suspension; then added 0.32 mg of antimicrobial peptide KR to the β-TCP / HyA / MPDA composite suspension for electrostatic adsorption (using a barrel plate model as the mechanism, under neutral pH, with positive electrostatic adsorption, a binding reaction occurred at 25 °C for 22 h) to obtain the antimicrobial peptide composite material prepared in Example 6.

[0029] Example 7 S1 Preparation of β-TCP / HyA porous substrate: 1.2g of β-tricalcium phosphate β-TCP powder was dispersed in 40mL of deionized water and ultrasonicated at 100W for 10min to form β-TCP slurry; 0.85g of hyaluronic acid HyA powder was dissolved in 8mL of ultrapure water to obtain HyA solution; 8mL of β-TCP slurry was slowly added to HyA solution, mixed evenly, and pre-frozen at -20℃ for 24h, then transferred to a freeze dryer and freeze-dried at -50℃ and pressure <10Pa for 48h to obtain uncrosslinked β-TCP / HyA porous scaffold; the porous scaffold was immersed in 400mL of diimide ethanol solution, and the first crosslinking reaction was carried out at 30℃ for 24h, followed by freeze-drying at -50℃ for 24h to obtain β-TCP / HyA porous substrate; S2 Synthesis of Mesoporous Polydopamine (MPDA): 0.14 g dopamine hydrochloride and 0.19 g template (triblock copolymer F127) were dissolved in 40 mL of 50% ethanol solution to obtain a first mixed solution. 640 μL of a pore-expanding agent solution (trimethylbenzene (TMB) solution, prepared by dissolving 10 mg TMB in 1 mL of anhydrous ethanol and then diluting with 9 mL of PBS buffer) was added to the first mixed solution, and the mixture was ultrasonically dispersed for 2 min to obtain a second mixed solution. 375 μL of Tris-HCl buffer (10 mM, pH 8.5) was added to the second mixed solution, and the mixture was subjected to self-polymerization at 30 °C with continuous magnetic stirring for 2 h. The mixture was then centrifuged at 13,000 rpm for 10 min, the precipitate was collected, and the precipitate was washed three times alternately with ethanol and deionized water to obtain mesoporous polydopamine (MPDA). S3. The 6 mg of β-TCP / HyA porous scaffold and 6 mg of mesoporous polydopamine were co-dispersed in 40 mL of deionized water and subjected to a second cross-linking reaction at 30 °C for 24 h to form a β-TCP / HyA / MPDA composite suspension; then 0.3 mg of antimicrobial peptide KR was added to the β-TCP / HyA / MPDA composite suspension via metal ion coordination (Ca in β-TCP...). 2+ The ions undergo ion coordination reactions with the carboxyl and phenolic hydroxyl groups in KR to form coordination bonds. After standing at 25°C for 24 hours, a binding reaction occurs, yielding the antimicrobial peptide composite material prepared in Example 7.

[0030] Comparative Example 1 Prepare a PBS control group: without adding any antibacterial materials.

[0031] Comparative Example 2 Preparation of tetracycline solution: The tetracycline stock solution was purchased from Thermo Fisher Scientific. The stock solution was diluted to 8 and 10 μg / mL using a concentration gradient dilution method to obtain the tetracycline solution.

[0032] Comparative Example 3 Prepare a pure antimicrobial peptide KR solution.

[0033] Performance testing Minimum inhibitory concentration (MIC): The antimicrobial peptide composites with different KR concentrations prepared in Examples 1-3 were added sequentially to a 96-well plate. 10 μl was added to each well. 6 A CFU / mL Streptococcus mutans culture was incubated at 37°C for 6 hours, with the OD600 value measured hourly using a microplate reader. The minimum drug concentration at which no bacterial growth was observed was set as the MIC, which was 8 μg / mL.

[0034] Minimum bactericidal concentration (MBC): Add 50 μl of a 1×10⁻⁶ solution to a 96-well plate. 4 CFU / mL *S. mutans* bacterial culture was used. In the experimental group, 50 μL of antimicrobial peptide composites with different KR concentrations prepared in Examples 1-3 were added. After mixing and incubation for 3 min, 100 μL of the mixture was spread onto LB agar plates and incubated at 37 °C for 36 h. The lowest KR concentration at which no colonies grew was determined as the minimum bactericidal concentration (MBC), which was measured to be 8 μg / mL.

[0035] Stability: The antimicrobial peptide composites prepared in Examples 1-3 with different KR concentrations were treated in simulated oral environments with different pH (4.5, 6, 7.5), temperatures (4-34℃), and salt ion concentrations (50-150 mmol / L NaCl). The antibacterial rate against *S. mutans* was then detected by plate counting. The results showed that under various harsh conditions, the antibacterial rate of KR in each concentration group remained stable above 93.6%, demonstrating that KR has excellent environmental stability. Table 1 shows the antimicrobial peptide composites prepared in Examples 1-3 and the solutions prepared in Comparative Examples 1-2, when added to a concentration of 1×10⁻⁶ mmol / L. 4 Colony statistics were collected after incubating S. mutans culture at CFU / mL on LB agar plates at 37°C for 6 hours.

[0036] Table 1

[0037] As shown in Table 1, in Comparative Example 1 without added antibacterial components, the colony count on LB agar plates was extremely high (568, 584 CFU), indicating normal bacterial growth. In Comparative Example 2, at tetracycline solution concentrations of 6–10 μg / mL, the colony count on LB agar plates remained high (432–527 CFU), indicating that traditional antibiotics have limited inhibitory effects on *S. mutans*. However, in the antimicrobial peptide composite materials of Examples 1-3 of this invention, at a concentration of 6 μg / mL, the colony count on LB agar plates was only 6–7 CFU, with an inhibition rate of approximately 99%. At a concentration of 8 μg / mL, the colony count on LB agar plates decreased to 2–3 CFU; and at a concentration of 10 μg / mL, almost no colony growth occurred on LB agar plates (0–2 CFU). Therefore, in these examples of the invention, KR can achieve near-complete inhibition at extremely low concentrations (6 μg / mL), with significantly better effects than tetracycline at the same concentration.

[0038] Table 2 shows the antimicrobial peptide composite materials prepared in Examples 1-3 of this invention and the solutions prepared in Comparative Examples 1-2, when added dropwise to a concentration of 1×10⁻⁵ μL. 4 The percentage of surviving colonies on LB agar plates containing CFU / mL S. mutans culture was incubated at 37°C for 6 hours.

[0039] Table 1 shows the colony count results, which most intuitively demonstrate the highly efficient bactericidal ability of the antimicrobial peptide composite material of this invention. Data shows that even at the lowest test concentration (6 μg / mL), the material of this invention can drastically reduce the bacterial count from approximately 576 CFU in the control group to single digits (6-7 CFU), achieving an inhibition rate of approximately 99%. This result directly confirms that the bioactivity of the antimicrobial peptide KR is perfectly preserved through loading onto the β-TCP / HyA / MPDA composite substrate, successfully solving the common problem of "immobilization and inactivation" of antimicrobial peptides mentioned in the background art. Simultaneously, at the same concentration gradient, the antimicrobial effect of the traditional antibiotic tetracycline (colony count 432-527 CFU) is far inferior to that of this invention. This highlights the significant advantage of the antimicrobial peptide strategy employed in this invention in terms of antimicrobial efficiency, providing experimental evidence for replacing traditional antibiotics that easily induce drug resistance.

[0040] Table 2

[0041] As shown in Table 2, in Comparative Example 2, the tetracycline group, even when its concentration increased from 6 μg / mL to 10 μg / mL, still exhibited a bacterial survival rate of 79.17%-90.54% on the agar plate, indicating a weak antibacterial effect. In Examples 1-3 of this invention, the KR group showed a bacterial survival rate of only 1.13% at a concentration of 6 μg / mL, 0.43% at 8 μg / mL, and a mere 0.17% at 10 μg / mL. This demonstrates that when the concentration of the antimicrobial peptide composite material is in the range of 6–10 μg / mL, its bactericidal rate approaches 99% or higher and exhibits a clear dose-dependent effect, while tetracycline, under the same conditions, shows an antibacterial effect of less than 21%.

[0042] Table 2 shows the survival rate data, further quantifying the superior performance of this invention compared to existing technologies. The material of this invention, at a concentration range of 6-10 μg / mL, can reduce bacterial survival to 1.13% to 0.17%, achieving near-complete eradication. In contrast, tetracycline at the same concentration only provides less than 21% antibacterial effect (bacterial survival rate 79.17%-90.54%). This comparison strongly supports the effects described in the claims of this invention: that the composite material can achieve a high-activity loading of antimicrobial peptides, and due to its unique mechanism of action (physical disruption of the cell membrane), it effectively avoids the drug resistance problem easily generated by traditional antibiotics, thus exhibiting a significant antibacterial effect. The clear dose-dependency data also indicate that the antibacterial effect of this invention is controllable and highly efficient. Table 3 shows the antimicrobial peptide composite materials prepared in Examples 1-3 of this invention, added to a solution with a concentration of 1×10⁻⁶ μl. 4 CFU / mL S. mutans bacterial suspension was placed on LB agar plates, and bacterial growth curves were obtained on each plate after incubation at 37°C for 6 hours, as in Comparative Example 1 without the addition of any antibacterial solution.

[0043] Table 3

[0044] As shown in Table 3, in the absence of KR, the OD value rapidly increased from 0.053 to 1.017, indicating normal bacterial growth. In Examples 1-3, when the concentration of the antimicrobial peptide composite material was 8 μg / mL, the OD value slowly increased from 0.055 to 0.076 (48 hours); when the concentration of the antimicrobial peptide composite material was 16 μg / mL with KR, the OD value remained stable in the range of 0.055-0.061. This shows that in the absence of KR, the OD value rapidly increased from 0.054 to 0.873, indicating normal bacterial growth. In Comparative Example 3, when the concentration of pure KR was 8 μg / mL, the OD value slowly increased from 0.054 to 0.085 (48 hours); when the concentration of pure KR was 16 μg / mL, the OD value remained stable in the range of 0.055-0.061. The two sets of data are almost identical, confirming that the presence of the material does not affect the potent and long-lasting antibacterial ability of KR at high concentrations.

[0045] The growth curve data in Table 3 confirm the long-lasting antibacterial ability of the composite material of the present invention and provide key evidence that the carrier does not affect the activity. In the "Examples 1-3" group, the OD600 value of the sample without KR (0 μg / mL) increased sharply from 0.053 to 1.017 within 48 hours, proving that the bacteria proliferated normally and entered the logarithmic growth phase under antibacterial conditions. In stark contrast, the OD values ​​of the samples containing the composite material of the present invention and loaded with KR (8 μg / mL and 16 μg / mL) were consistently suppressed to extremely low levels (approximately 0.076 and 0.061, respectively) throughout the monitoring period, which intuitively demonstrates that the composite material has a strong and long-lasting antibacterial function. More importantly, the antibacterial curves of the "Examples 1-3" group (composite material loaded with KR) and the "Comparative Example 1" group (should be pure KR solution, see Comparative Example 3 above) at the same KR concentration (8 μg / mL and 16 μg / mL) almost completely overlap. This data overlap provides conclusive evidence that the β-TCP / HyA / MPDA composite substrate, as a carrier, does not impair the bioactivity of the antimicrobial peptide KR at all, successfully achieving a balance between "high loading" and "high activity," fundamentally solving the core problem of "immobilization leading to inactivation" in the field of antimicrobial peptide immobilization. Figure 3 (ac) shows SEM images of the composite material prepared in Example 2 of this invention at different resolutions; as shown Figure 3 As shown, the composite material has a dense and continuous internal structure, with no obvious pores or sponge-like structures. This indicates that the antimicrobial peptides are loaded and immobilized in the composite material with high density and uniformity. This dense, non-porous structure is conducive to the stable storage of the antimicrobial peptides and provides a basis for their efficient contact with bacteria during use. Figure 4 This is a schematic diagram illustrating the application of the composite material prepared in Example 1 of the present invention in forming a coating on dental braces; as shown. Figure 4As shown, the coating is transparent and evenly applied, without altering the inherent color and shape of the braces, ensuring aesthetics during wear and supporting the technical effect that "the coating has good aesthetics and concealment". Figure 5 These are cryo-electron microscopy images of the composite materials prepared in Examples 1-3 of this invention applied to dental braces; as shown. Figure 5 As shown, the coating exhibits good compatibility with the substrate interface, adheres tightly, and forms a continuous surface coating layer, displaying an overall dense and smooth morphological characteristic adapted to its function. This indicates that the coating material can adapt to complex substrate shapes, constructing a stable and integrated functional interface, providing a foundation for achieving its long-term and reliable antibacterial function on the surface of dental braces. Figure 6 This is a comparison of the bactericidal effects of the composite materials prepared in Examples 1-3 of the present invention and tetracycline in Comparative Example 2; Figure 6 The antibacterial effects of the traditional antibiotic tetracycline and the antimicrobial peptide composite material of this invention against *S. mutans* were visually compared using the bacterial plating method. It was observed that within the concentration range of 6-10 μg / mL, the tetracycline-containing plates consistently showed a large number of densely distributed white colonies, indicating limited antibacterial effect. In contrast, at the same concentration gradient, bacterial growth was almost completely inhibited on plates containing the antimicrobial peptide composite material, with only a few colonies or no colonies visible. This visual result is highly consistent with the colony count data in Table 1, directly confirming that the antimicrobial peptide composite material is significantly more effective than the traditional antibiotic tetracycline in killing *S. mutans* at low concentrations. Figure 7 The image shows a comparison of the survival rates of Streptococcus mutans after the composite materials prepared in Examples 1-3 of this invention and the tetracycline applied to dental braces in Comparative Example 2 were treated with the composite material; Figure 7 The bar chart demonstrates that the antimicrobial peptide composite material prepared in this invention exhibits a far more potent bactericidal effect against S. mutans than the traditional antibiotic tetracycline, even at concentrations as low as 6 μg / mL. Furthermore, the effect increases with increasing concentration of the antimicrobial peptide, which supports the technical efficacy of the material of this invention in achieving highly efficient antibacterial activity. Figure 8 The graph shows the effect of the composite materials prepared in Examples 1-3 of this invention and Comparative Examples 1 and 3 on the growth of Streptococcus mutans after being treated on dental braces. Figure 8 As shown, the OD value curves with and without the material (left) highly overlap at the same concentration of the antimicrobial peptide KR. This indicates that loading the antimicrobial peptide KR onto the β-TCP / HyA / MPDA composite suspension prepared in this invention does not significantly differ from the antibacterial effect and final effect of free KR. This supports the technical effect that "the β-TCP / HyA / MPDA composite suspension carrier does not affect the antimicrobial activity of KR," proving the success of the composite material design.

[0046] Therefore, it can be seen that no bacteria grew on the surface of the dental braces coated with the antimicrobial peptide composite materials prepared in Examples 1-3 of this invention, and the surface morphology was similar, indicating that the antimicrobial coating has a stable antimicrobial effect. Its stable structure ensures that the antimicrobial peptide can still exert its antimicrobial function stably and for a long time under the fluctuations of pH, temperature and salt ion concentration experienced in the oral environment.

[0047] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for preparing an antimicrobial peptide composite material, characterized in that, The preparation method includes: S1 Preparation of β-TCP / HyA porous substrate: β-tricalcium phosphate β-TCP powder is dispersed in deionized water and ultrasonically treated to form β-TCP slurry; hyaluronic acid HyA powder is dissolved in ultrapure water to obtain HyA solution; β-TCP slurry is slowly added to HyA solution, mixed evenly, and then pre-frozen to obtain uncrosslinked β-TCP / HyA porous scaffold; the porous scaffold is immersed in crosslinking solution to carry out the first crosslinking reaction and then freeze-dried to obtain β-TCP / HyA porous substrate; S2 Synthesis of Mesoporous Polydopamine (MPDA): Dopamine hydrochloride and a template agent are dissolved in an ethanol solution to obtain a first mixed solution; a pore-expanding agent solution is added to the first mixed solution, and the mixture is ultrasonically dispersed to obtain a second mixed solution; Tris-HCl buffer is added to the second mixed solution to carry out a self-polymerization reaction, and the mixture is then centrifuged, washed, and freeze-dried to obtain mesoporous polydopamine (MPDA); S3 disperses the β-TCP / HyA porous scaffold and mesoporous polydopamine together in deionized water to carry out a second crosslinking reaction, forming a β-TCP / HyA / MPDA composite suspension; then, antimicrobial peptide KR is added to the β-TCP / HyA / MPDA composite suspension to carry out a binding reaction, thereby obtaining an antimicrobial peptide composite material.

2. The preparation method according to claim 1, characterized in that, In step S1, the concentration of the β-TCP slurry is 20-30 mg / mL, and the concentration of the HyA solution is 100-120 mg / mL; the volume ratio of the β-TCP slurry to the HyA solution is 1:

1.

3. The preparation method according to claim 1, characterized in that, The ultrasonic treatment in step S1 has a power of 100W and a time of 10-20min; the pre-freezing treatment temperature is as follows: place it in a pre-freezing chamber at a temperature of -20℃ for 24h, and then transfer it to a freeze dryer for freeze drying at a temperature of -50℃ and a pressure of <10Pa for 48h.

4. The preparation method according to claim 1, characterized in that, The crosslinking solution in step S1 is any one of the following: an ethanol solution composed of EDC and NHS, an ethanol solution of diimide, and an ethanol solution of genipin; the temperature of the first crosslinking reaction is 25-30℃, and the time is 20-24h.

5. The preparation method according to claim 1, characterized in that, In step S2, the mass ratio of dopamine hydrochloride to the template agent is 1.5:1.9; the template agent is triblock copolymer F127; the ethanol solution is composed of deionized water and anhydrous ethanol in a volume ratio of 1:1; the concentration of dopamine hydrochloride in the first mixed solution in step S2 is 3.5-4 mg / mL; the pore-expanding agent solution is a 1 mg / mL solution of mesitylene, which is prepared by dissolving mesitylene in a mixed solvent; the mixed solvent is composed of anhydrous ethanol and PBS buffer in a volume ratio of 1:

9.

6. The preparation method according to claim 1, characterized in that, In step S2, the volume ratio of the first mixed solution to the pore-expanding agent solution is (62-65):1; the concentration of the Tris-HCl buffer solution is 10mM and the pH value is 8.5; the temperature of the self-polymerization reaction is 25-30℃ and the time is 2-4h.

7. The preparation method according to claim 1, characterized in that, In step S3, the mass ratio of the β-TCP / HyA porous scaffold to the mesoporous polydopamine is 1:1; the amount of antimicrobial peptide KR added to the β-TCP / HyA / MPDA composite suspension is 6-16 μg / mL; the temperature of the second crosslinking reaction is 25-30℃, and the time is 24h.

8. The preparation method according to claim 1, characterized in that, The binding reaction described in step S3 specifically involves the binding of antimicrobial peptide KR with mesoporous polydopamine; the binding reaction can be carried out through any one of covalent bonding, metal ion coordination, and electrostatic adsorption; the temperature of the binding reaction is 25-30℃, and the time is 20-24h.

9. An antimicrobial peptide composite material prepared by the preparation method according to any one of claims 1 to 8, characterized in that, The antimicrobial peptide composite material includes a β-TCP / HyA / MPDA composite mediating layer and an antimicrobial peptide KR functional layer loaded thereon.

10. The application of the antimicrobial peptide composite material as described in claim 9, characterized in that, The antimicrobial peptide composite material is coated onto medical products or the medical products are immersed in the antimicrobial peptide composite material; the coating method includes spin coating and spray coating; the medical products include one or more of medical devices, medical materials and implants; the medical devices include dental braces; the implants include orthopedic screws and joint prostheses; the medical materials include silicone endotracheal tubes and urinary catheters.

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