Antibacterial nanomaterial and preparation method and application thereof

Abstract

The present application relates to an antibacterial nanomaterial and a preparation method and application thereof, the antibacterial nanomaterial comprising polydopamine nanoparticles and a silver layer loaded on the surface of the polydopamine nanoparticles.The present application relates to a silver-loaded nanomaterial reduced and deposited in situ by polydopamine, and under physiological conditions, polypeptides can be efficiently, rapidly and stably loaded by in-situ silver nanoparticles; meanwhile, the polydopamine and silver nanoparticles are used to realize efficient photothermal antibacterial effect, the material can be universally applied to wound repair materials, and provides a new material with anti-infection and tissue microenvironment regulation functions for large-area and chronic wound repair; the material can effectively improve the mechanical properties and tissue adhesion of wound repair materials by using the rich pi-pi stacking force and hydrogen bond of polydopamine; and provides an excellent carrier for drug loading and responsive release.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to an antibacterial nanomaterial, its preparation method and application, specifically to an antibacterial nanomaterial, a polypeptide-loaded antibacterial nanomaterial, its preparation method and application, and a wound repair hydrogel material and its preparation method. Background Technology

[0002] As the largest organ in the human body, the skin plays a vital protective role, regulating body temperature and resisting the invasion of external pathogens. Although the skin has a certain ability to repair itself, once it is severely damaged, there is a risk of repeated infections, persistent inflammatory reactions, and various complications.

[0003] Currently, skin grafting remains the gold standard for clinical wound repair. However, autologous transplantation carries risks such as limited donors and secondary injury. Allogeneic and xenogeneic transplantation, on the other hand, present challenges in tissue preservation, post-transplant immune rejection, and viral transmission. This has led to a growing clinical demand for wound repair materials. However, existing clinical dressings generally lack anti-infective properties, and long-term antibiotic use has side effects and can easily lead to the development of drug-resistant pathogens. Despite significant advancements in the design and fabrication of functional dressings and hydrogels, the exposed nature of wounds and the high risk of infection, coupled with the crucial role of early inflammatory responses in tissue regeneration and repair, mean that their occurrence, development, and outcome are critical to the quality of wound repair. Therefore, developing materials with anti-infective properties and the ability to regulate the inflammatory microenvironment has significant clinical and market value.

[0004] Peptides, as functional fragments of natural proteins, are composed of amino acids linked by amide bonds and exert their biological activities according to their specific amino acid sequences. Due to their high activity, good biocompatibility, and low immunogenicity, they are widely used in the modification of biomaterials to achieve diverse biological effects and regulate various physiological functions at the cellular level, such as cell adhesion, targeting, immune regulation, and antibacterial activity. Achieving efficient loading of peptide molecules into biomaterials is crucial. Currently, peptide modification methods include: introducing functional groups or adding coupling molecules to achieve covalent linkage through strategies such as Michael addition reactions or amide condensation reactions, or using electrostatic interactions and hydrogen bonding for non-covalent loading. Covalent linkage suffers from cumbersome reaction processes, and reaction conditions directly affect the modification rate and peptide activity; while electrostatic and hydrogen bonding interactions have drawbacks such as instability and insufficient loading efficiency. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide an antibacterial nanomaterial, its preparation method, and its application. Specifically, it provides an antibacterial nanomaterial, its preparation method, and its application; an antibacterial nanomaterial loaded with peptides, its preparation method, and its application; and a wound repair hydrogel material and its preparation method. These solutions address the problems of existing clinical dressings having limited functionality, poor antibacterial effects, and insufficient bioactivity.

[0006] To achieve this objective, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides an antibacterial nanomaterial comprising polydopamine nanoparticles and a silver layer loaded on the surface of the polydopamine nanoparticles.

[0008] This invention relates to a silver-loaded nanomaterial based on in-situ reduction deposition of polydopamine. Under physiological conditions, this material can achieve efficient, rapid, and stable loading of peptides through in-situ silver nanoparticles. Simultaneously, it utilizes polydopamine in synergistically with silver nanoparticles to achieve a highly efficient photothermal antibacterial effect. This material can be widely applied to wound repair materials, providing a new material with anti-infection and tissue microenvironment regulation functions for the repair of large-area and chronic wounds. Furthermore, by utilizing the abundant π-π stacking forces and hydrogen bonds of polydopamine, this material can effectively improve the mechanical properties and tissue adhesion of wound repair materials, and provides an excellent carrier for drug loading and responsive release.

[0009] Preferably, the particle size range of the polydopamine nanoparticles is 60-350nm, such as 90nm, 120nm, 150nm, 170nm, 200nm, 250nm, 300nm, etc.; the particle size range of the antibacterial nanomaterials is 80-400nm, such as 100nm, 120nm, 200nm, 300nm, 350nm, etc. Other specific values ​​within the above ranges can be selected, and will not be elaborated here.

[0010] The particle size ranges of the polydopamine nanoparticles and antibacterial nanomaterials are selected as 60-350nm and 80-400nm, respectively, especially 100-180nm and 200-260nm, which is beneficial to the stability of the nanoparticles and the improvement of peptide loading efficiency.

[0011] In a second aspect, the present invention provides a method for preparing the antibacterial nanomaterial according to the first aspect, the method comprising:

[0012] (1) Mix dopamine with a solvent containing alkaline substances, react, and then centrifuge to obtain a precipitate, which is polydopamine nanoparticles.

[0013] (2) Mix the polydopamine nanoparticle dispersion with a silver salt solution containing alkaline substances, react, and then centrifuge to obtain a precipitate, which is the antibacterial nanomaterial.

[0014] The preparation method of the above-mentioned antibacterial nanomaterials involved in this invention is simple and easy to operate, and is very suitable for industrial production.

[0015] Preferably, the alkaline substance in step (1) includes any one or a combination of at least two of ammonia, sodium hydroxide, or tris(hydroxymethyl)aminomethane.

[0016] Preferably, the solvent in step (1) includes any one or a combination of at least two of anhydrous ethanol, anhydrous methanol, an aqueous ethanol solution, or an aqueous methanol solution.

[0017] Preferably, the reaction in step (1) is carried out at 4-40℃ (e.g., 4℃, 10℃, 15℃, 20℃, 25℃, 30℃, 35℃, 40℃, etc.) for 18-30h (e.g., 18h, 20h, 22h, 25h, 28h, 30h, etc.).

[0018] Preferably, the reaction in step (1) is carried out under stirring, and the stirring speed in step (1) is 300-1200 rpm (e.g., 300 rpm, 400 rpm, 500 rpm, 600 rpm, 700 rpm, 800 rpm, 900 rpm, 1000 rpm, 1100 rpm, 1200 rpm, etc.).

[0019] Preferably, the centrifugation in step (1) is carried out at 4-40℃ (e.g., 4℃, 10℃, 15℃, 20℃, 25℃, 30℃, 35℃, 40℃, etc.) at 8000-15000rpm (e.g., 8000rpm, 9000rpm, 10000rpm, 11000rpm, 12000rpm, 13000rpm, 15000rpm, etc.) for 10-30min (e.g., 10min, 15min, 20min, 25min, 30min, etc.).

[0020] The specific operation of mixing dopamine with a solvent containing an alkaline substance can be as follows: slowly inject an aqueous solution of dopamine into the solvent containing an alkaline substance for mixing.

[0021] After centrifugation to obtain a precipitate, the precipitate is further subjected to alcohol washing and water washing, and the washed precipitate is resuspended in ultrapure water and stored at 4°C.

[0022] Preferably, the alkaline substance in step (2) includes any one or a combination of two of ammonia or sodium hydroxide.

[0023] Preferably, the silver salt in step (2) includes any one or a combination of at least two of silver nitrate, silver chloride, silver fluoride or silver perchlorate.

[0024] Preferably, the mass-volume fraction of the polydopamine nanoparticle dispersion in step (2) is 0.005-0.200%, such as 0.005%, 0.010%, 0.020%, 0.030%, 0.050%, 0.080%, 0.100%, 0.150%, 0.200%, etc.

[0025] Preferably, the reaction in step (2) is carried out at 4-40℃ (e.g., 4℃, 10℃, 15℃, 20℃, 25℃, 30℃, 35℃, 40℃, etc.) for 30-180 min (e.g., 30 min, 50 min, 60 min, 80 min, 100 min, 120 min, 140 min, 160 min, 180 min, etc.).

[0026] Preferably, the reaction in step (2) is carried out under stirring at a speed of 200-1000 rpm (e.g., 200 rpm, 300 rpm, 400 rpm, 500 rpm, 600 rpm, 700 rpm, 800 rpm, 900 rpm, 1000 rpm, etc.).

[0027] Preferably, the centrifugation in step (2) is carried out at 4-40℃ (e.g., 4℃, 10℃, 15℃, 20℃, 25℃, 30℃, 35℃, 40℃) at 8000-15000rpm (e.g., 8000rpm, 9000rpm, 10000rpm, 11000rpm, 12000rpm, 13000rpm, 15000rpm, etc.) for 10-30min (e.g., 10min, 15min, 20min, 25min, 30min, etc.).

[0028] After centrifugation to obtain a precipitate, the precipitate is further subjected to alcohol washing and water washing, and the washed precipitate is resuspended in ultrapure water and stored at 4°C.

[0029] Thirdly, the present invention provides the application of the antibacterial nanomaterials according to the first aspect in the preparation of peptide-loaded nanomaterials.

[0030] The present invention also provides a strategy for efficiently loading bioactive peptides onto biomedical materials, namely, using the aforementioned antibacterial nanomaterials as carriers to load bioactive peptides.

[0031] Fourthly, the present invention provides an antibacterial nanomaterial loaded with peptides, wherein the antibacterial nanomaterial loaded with peptides uses the antibacterial nanomaterial described in the first aspect as a carrier, and is loaded with bioactive materials including bioactive peptides.

[0032] Preferably, the bioactive material includes any one or a combination of at least two of the following: bioactive peptides, natural polymers, drugs, growth factors, or cytokines.

[0033] The aforementioned antibacterial nanomaterials can not only load bioactive peptides, but also be further loaded with natural polymer materials, drugs, etc., according to actual needs, thus endowing the materials with multifunctionality.

[0034] Preferably, the bioactive peptide is a bioactive peptide modified with a thiol group.

[0035] Thiol-modified bioactive peptides can bind to silver in antibacterial nanomaterials through S-Ag coordination interactions, achieving efficient, rapid, and stable loading.

[0036] Fifthly, the present invention provides a method for preparing antibacterial nanomaterials loaded with peptides according to the fourth aspect, the method comprising:

[0037] The bioactive material solution is mixed with the antibacterial nanomaterial dispersion, reacted, and then centrifuged to obtain a precipitate, which is the antibacterial nanomaterial loaded with the peptide.

[0038] Preferably, the reaction is carried out at 4-40°C (e.g., 4°C, 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, etc.) for 30-180 min (e.g., 30 min, 50 min, 60 min, 80 min, 100 min, 120 min, 140 min, 160 min, 180 min, etc.).

[0039] Preferably, the reaction is carried out under stirring at a speed of 200-1200 rpm (e.g., 200 rpm, 300 rpm, 400 rpm, 500 rpm, 600 rpm, 700 rpm, 800 rpm, 900 rpm, 1100 rpm, 1200 rpm, etc.).

[0040] Preferably, the centrifugation is performed at 4-40℃ (e.g., 4℃, 10℃, 15℃, 20℃, 25℃, 30℃, 35℃, 40℃, etc.) at 8000-15000rpm (e.g., 8000rpm, 9000rpm, 10000rpm, 11000rpm, 12000rpm, 13000rpm, 15000rpm, etc.) for 10-30min (e.g., 10min, 15min, 20min, 25min, 30min, etc.).

[0041] After centrifugation to obtain a precipitate, the precipitate is further subjected to alcohol washing and water washing, and the washed precipitate is resuspended in ultrapure water and stored at 4°C.

[0042] In a sixth aspect, the present invention provides the application of the antimicrobial nanomaterial loaded with peptides according to the fifth aspect in the preparation of wound repair materials.

[0043] In a seventh aspect, the present invention provides a wound repair hydrogel material, the wound repair hydrogel material comprising a hydrogel matrix and an antibacterial nanomaterial loaded with polypeptides as described in the fourth aspect dispersed in the hydrogel matrix.

[0044] This wound repair hydrogel material effectively addresses the shortcomings of existing clinical dressings, such as limited functionality, poor antibacterial effect, and insufficient bioactivity. Bioactive peptides are loaded onto antibacterial nanomaterials, ensuring the in-situ retention of the peptides and enabling them to exert long-lasting physiological activity within the hydrogel, thus guaranteeing the long-term effectiveness of the hydrogel material. The selected hydrogel substrate can be any hydrogel material, without limitation, such as methacrylamide chitosan, polyphenol-modified chitosan, methacrylamide gelatin (GelMA), methacrylamide hyaluronic acid (HAMA), chondroitin sulfate, agarose, methacrylamide silk fibroin (Sil-MA), decellularized matrix, collagen, cationic guar gum, etc.

[0045] Eighthly, the present invention provides a method for preparing a wound repair hydrogel material according to the seventh aspect, the method comprising: dispersing an antibacterial nanomaterial loaded with peptides in a hydrogel raw material preparation solution, stirring at 4-40℃ (e.g., 4℃, 10℃, 15℃, 20℃, 25℃, 30℃, 35℃, 40℃, etc.), molding the mixture into a mold, and allowing it to stand to obtain the final product.

[0046] The preparation method of the above-mentioned wound repair hydrogel material involved in this invention is simple and easy to operate, and is very suitable for industrial production. Attached Figure Description

[0047] Figure 1 These are transmission electron microscope images of PDA, Ag@PDA, M2pep-Ag@PDA, and GFLGC-Ag@PDA;

[0048] Figure 2 These are scanning electron microscope images of PDA, Ag@PDA, M2pep-Ag@PDA, and GFLGC-Ag@PDA;

[0049] Figure 3 These are energy spectrum elemental analysis mapping images of PDA, Ag@PDA, and M2pep-Ag@PDA;

[0050] Figure 4 This is the energy dispersive spectroscopy (EDS) elemental analysis spectrum of GFLGC-Ag@PDA;

[0051] Figure 5 These are X-ray diffraction patterns of PDA and Ag@PDA nanoparticles;

[0052] Figure 6 These are near-infrared photothermal effect analysis spectra of PDA and Ag@PDA nanoparticles based on ultraviolet spectroscopy;

[0053] Figure 7 These are the UV spectra of PDA, Ag@PDA, and M2pep-Ag@PDA nanoparticles;

[0054] Figure 8 These are X-ray photoelectron spectra of PDA, Ag@PDA, and M2pep-Ag@PDA nanoparticles;

[0055] Figure 9 This is a graph showing the results of the rat tail hemostatic performance evaluation test;

[0056] Figure 10 This is a graph showing the results of the antibacterial performance evaluation test;

[0057] Figure 11 This is a picture showing the result of the repair of a burn wound with full-layer skin loss;

[0058] Figure 12 This is a schematic diagram for evaluating the tissue adhesion properties of hydrogels. Detailed Implementation

[0059] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0060] Example 1

[0061] Preparation of polydopamine nanoparticles (PDA):

[0062] (1) Slowly add 2.0 mL of ammonia water (NH4OH, 28-30%, Sigma 221228) to a mixed solution of 40 mL of ethanol and 90 mL of ultrapure water. Stir the solution at 800 rpm for 30 min in the dark at 20 °C.

[0063] (2) Dissolve 0.5g of dopamine (Sigma H8502) in 10mL of ultrapure water, slowly inject the mixed dopamine solution into the above (1) mixed solution, and react at 20℃ for 24h.

[0064] (3) Centrifuge the solution from (2) after reaction at 12000 rpm at 20°C for 20 min and discard the supernatant. Wash the obtained product several times with ethanol and water. Resuspend the final polydopamine nanoparticles in ultrapure water and store at 4°C.

[0065] Example 2

[0066] Preparation of silver-modified polypamine nanoparticles (Ag@PDA):

[0067] (1) A certain amount of silver nitrate (AgNO3, Aladdin S116265) was slowly added to ultrapure water and stirred for 30 min in the dark to obtain a mixed solution with a concentration of 5% (w / v).

[0068] (2) Slowly add ammonia water to the solution in (1) above, causing the solution to change from a clear solution to a turbid solution and then back to a clear solution, and stir for 30 minutes in the dark.

[0069] (3) The polydopamine nanoparticles prepared in Example 1 were dispersed evenly under ultrasonic conditions and slowly dropped into the mixed solution (2). The mixture was stirred at 500 rpm for 60 min in the dark at 20°C. The concentration of the polydopamine nanoparticles was 0.03% (w / v).

[0070] (4) Centrifuge the solution after reaction (3) at 12000 rpm at 20°C for 20 minutes and discard the supernatant. Wash the obtained product with ethanol and ultrapure water, resuspend it in ultrapure water, and store it at 4°C.

[0071] Example 3

[0072] Preparation of silver-modified polypamine nanoparticles loaded with M2 macrophage-targeting peptides (M2pep-Ag@PDA):

[0073] (1) M2pep (sequence YEQDPWGVKWWYGGGSKKKC, this polypeptide has M2 macrophage targeting activity and can be used for affinity enrichment of M2 macrophages, wherein the C-terminus of the polypeptide sequence on the right is modified with a cysteine ​​residue with a thiol group using the FMOC solid-phase synthesis method). Suspensions of M2pep at concentrations of 10 mg / mL and Ag@PDA at concentrations of 1 mg / mL were prepared respectively.

[0074] (2) Take 0.1 mL of 10 mg / mL M2pep and slowly add it to 3 mL of 1 mg / mL Ag@PDA. Stir at 600 rpm for 120 min in the dark at 20°C.

[0075] (3) Centrifuge the solution after reaction (2) at 12000 rpm at 20℃ for 20 min and discard the supernatant. Wash the product with ethanol and ultrapure water and resuspend it in ultrapure water. Store at 4℃.

[0076] Example 4

[0077] Preparation of silver-modified polypamine nanoparticles (GFLGC-Ag@PDA) loaded with cathepsin-hydrolyzed peptides:

[0078] (1) The cathepsin-hydrolyzed polypeptide (sequence GFLGC, which exhibits cathepsin-responsive hydrolysis, wherein the right C-terminus is modified with a thiol-containing cysteine ​​residue using FMOC solid-phase synthesis). Suspensions of GFLGC at concentrations of 10 mg / mL and Ag@PDA at concentrations of 1 mg / mL were prepared.

[0079] (2) Take 0.1 mL of 10 mg / mL GFLGC and slowly add it to 3 mL of 1 mg / mL Ag@PDA. Stir at 600 rpm for 120 min in the dark at 20°C.

[0080] (3) Centrifuge the solution after reaction (2) at 12000 rpm at 20℃ for 20 min and discard the supernatant. Wash the product with ethanol and ultrapure water and resuspend it in ultrapure water. Store at 4℃.

[0081] Example 5

[0082] Preparation of silver-modified polypamine nanoparticles (YIGSRC-Ag@PDA) loaded with endothelial cell adhesion-promoting peptides:

[0083] (1) Endothelial cell adhesion-promoting peptide (sequence YIGSRC, this peptide has an amino acid sequence derived from laminin, which can promote endothelial cell adhesion, wherein the right C-terminus is modified with a thiol-containing cysteine ​​using FMOC solid-phase synthesis). Suspensions of YIGSRC at concentrations of 10 mg / mL and Ag@PDA at concentrations of 1 mg / mL were prepared respectively.

[0084] (2) Take 0.1 mL of 10 mg / mL YIGSRC and slowly add it to 3 mL of 1 mg / mL Ag@PDA. Stir at 600 rpm for 120 min in the dark at 20°C.

[0085] (3) Centrifuge the solution after reaction (2) at 12000 rpm at 20℃ for 20 min and discard the supernatant. Wash the product with ethanol and ultrapure water and resuspend it in ultrapure water. Store at 4℃.

[0086] Example 6

[0087] Preparation of silver-modified polypamine nanoparticles loaded with M2 macrophage-targeting peptides (M2pep-Ag@PDA):

[0088] (1) Same as Example 3

[0089] (2) Take 0.1 mL of 10 mg / mL M2pep and slowly add it to 0.4 mL of 1 mg / mL Ag@PDA. Stir at 600 rpm for 120 min in the dark at 20°C.

[0090] (3) Same as Example 3.

[0091] Test Example 1

[0092] The PDA, Ag@PDA, M2pep-Ag@PDA, GFLGC-Ag@PDA, and nanoparticles prepared in the above embodiments were analyzed by field emission scanning electron microscopy (FESEM, ZEISS). 55) Transmission electron microscopy (TEM, 200kV, JEM-3200FS JEOL) was used to characterize the morphology of the nanoparticles.

[0093] The results are as follows Figure 1 (The scale bars in the images from left to right represent 0.5μm, 200nm, 100nm, and 50nm, respectively.) Figure 2 As shown in the images (the scale bars from left to right are 1 μm, 200 nm, and 100 nm), electron micrographs show that the prepared PDA, Ag@PDA, M2pep-Ag@PDA, and GFLGC-Ag@PDA nanoparticles all have uniform particle size, and the in-situ reduced Ag nanoparticles are uniformly loaded on the PDA surface.

[0094] Test Example 2

[0095] The chemical elements of the PDA, Ag@PDA, M2pep-Ag@PDA, and GFLGC-Ag@PDA nanoparticles prepared in the above embodiments were analyzed by EDS energy dispersive spectroscopy.

[0096] The mapping results for PDA, Ag@PDA, and M2pep-Ag@PDA are as follows: Figure 3 As shown, the elemental analysis spectrum of GFLGC-Ag@PDA is as follows: Figure 4 As shown, energy dispersive spectroscopy analysis results indicate that Ag@PDA has the characteristic distribution of Ag, while M2pep-Ag@PDA and GFLGC-Ag@PDA nanoparticles have the characteristic elements of Ag and S in peptides, respectively.

[0097] Test Example 3

[0098] The chemical composition of the PDA and Ag@PDA nanoparticles prepared in the above embodiments was further analyzed by X-ray diffraction (XRD, SmartLab 3KW, Japan Rigaku).

[0099] like Figure 5 As shown, the PDA nanoparticles exhibit a peak at 2θ24.3°, indicating their amorphous nature. With the in-situ growth of silver nanoparticles on the PDA surface, the Ag@PDA nanoparticles show characteristic peaks of metallic silver at Ag(111) 38.1°, Ag(200) 44.2°, Ag(220) 64.5°, and Ag(311) 77.4°, respectively, indicating the successful synthesis of silver-loaded Ag@PDA nanoparticles.

[0100] Test Example 4

[0101] The absorbance values ​​of PDA and Ag@PDA nanoparticles prepared in the above embodiments were detected by ultraviolet spectrophotometer at different concentrations in the near-infrared range of 750-900 nm.

[0102] Figure 6 Experimental results show that the absorbance of both PDA and Ag@PDA nanoparticles increases with increasing concentration, exhibiting a concentration-dependent effect. Furthermore, at the same concentration, Ag@PDA nanoparticles show a higher absorbance than PDA nanoparticles. This phenomenon indicates that at the same concentration, Ag@PDA nanoparticles have a stronger near-infrared absorption effect than PDA nanoparticles.

[0103] Test Example 5

[0104] The PDA, Ag@PDA, and M2pep-Ag@PDA nanoparticles prepared in the above embodiments were tested using a UV spectrophotometer, and solutions of PDA, Ag@PDA, and M2pep-Ag@PDA nanoparticles, as well as a 1 mg / mL M2pep solution, were prepared.

[0105] like Figure 7 As shown, both M2pep and M2pep-Ag@PDA exhibit characteristic absorption peaks of peptides at approximately 280 nm. With the loading of Ag nanoparticles, characteristic peaks of silver nanoparticles appear at 440 nm, while the peak position of M2pep-Ag@PDA shows a red shift due to the interaction between its thiol groups and silver nanoparticles.

[0106] Test Example 6

[0107] The elemental composition and valence state changes of the PDA, Ag@PDA and M2pep-Ag@PDA nanoparticles prepared in the above embodiments were determined by X-ray photoelectron spectroscopy (XPS, ThermoFisher ESCALAB XI).

[0108] Figure 8 Experimental results show that PDA, Ag@PDA, and M2pep-Ag@PDA nanoparticles all contain characteristic peaks of C 1s, N 1s, and O 1s. The XPS spectrum of Ag@PDA shows peaks at 368 eV and 374 eV, while these peaks are not present in the spectrum of PDA nanoparticles, indicating that silver nanoparticles are synthesized in situ on the PDA surface. For M2pep-Ag@PDA nanoparticles, not only are the peaks of silver nanoparticles retained in their spectrum, but the XPS spectrum also shows a characteristic S 2p peak, indicating successful loading of M2pep.

[0109] Test Example 7

[0110] This test example evaluates the peptide loading efficiency. A UV spectrophotometer was used to detect the characteristic peaks of the uncentrifuged mixed solution, the centrifuged supernatant, and the precipitate in step (3) of Examples 3-6. Characteristic absorption peaks of the modified peptide were observed in both the uncentrifuged mixed solution and the precipitate, while no characteristic absorption peaks of the modified peptide were detected in the centrifuged supernatant. The experimental results show that, according to the feed ratios in Examples 3-6, the peptide can be 100.0% loaded into Ag@PDA nanoparticles.

[0111] Application Example 1

[0112] Preparation of M2pep-Ag@PDA-loaded hydrogel:

[0113] (1) Preparation of catechol-modified chitosan (C-CS): 1.0 g of chitosan (CS, Chitosan, MW = 190000-310000 Da, 75%-85% degree of deacetylation) was fully dissolved in 100 mL of 0.5% (v / v) glacial acetic acid solution to obtain a 1% (w / v) chitosan solution, which was adjusted to pH = 5.0 with 2M hydrochloric acid solution; 0.98 g of 3,4-dihydroxyphenylpropionic acid (HCA) was added and stirred continuously; 1.03 g of 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide Hydrochloride (EDC) and 0.62 g of... N-Hydroxysuccinimide (NHS) was dissolved in 30 mL of anhydrous ethanol / ultrapure water at a volume ratio of 1:1 and slowly added to the above solution. The pH was adjusted to 5.0 with 3M sodium hydroxide, and the reaction was carried out with vigorous stirring for 10 h in an anaerobic atmosphere. Then, the mixture was dialyzed in an acidic solution at pH 3.5 for 3 days and in ultrapure water for 1 day using a dialysis bag, and then freeze-dried.

[0114] (2) Prepare PBS solutions of 4% (w / v) C-CS, 20% (w / v) gelatin and 20% (w / v) α,ω-bis(4-formyl-benzamide) polyethylene glycol (DF-PEG-DF, Mw=2000, Xibao Biotechnology (Shanghai) Co., Ltd.);

[0115] (3) Disperse 1 mg of M2pep-Ag@PDA prepared in Example 3 in 1 mL of gelatin PBS solution to make the concentration of M2pep-Ag@PDA 1 mg / mL;

[0116] (4) Mix 0.5 mL of C-CS PBS solution with 0.5 mL of gelatin PBS solution containing M2pep-Ag@PDA prepared in step (3) evenly;

[0117] (5) Add 50 μL of DF-PEG-DF PBS solution, vortex at 25°C for 10 s, then add to the mold and let stand at 37°C to form a gel.

[0118] Meanwhile, similar methods were used to prepare a simple hydrogel group without M2pep-Ag@PDA (step (3) omitted), an Ag@PDA-loaded hydrogel group (M2pep-Ag@PDA was replaced with Ag@PDA, and the amount of PDA was kept the same), and a PDA-loaded hydrogel group (M2pep-Ag@PDA was replaced with PDA, and the amount of PDA was kept the same).

[0119] Application Example 2

[0120] Preparation of GFLGC-Ag@PDA-loaded hydrogel:

[0121] The preparation method is the same as in Application Example 1, except that the M2pep-Ag@PDA prepared in Example 3 is replaced with the GFLGC-Ag@PDA prepared in Example 4.

[0122] Application Example 3

[0123] Preparation of YIGSRC-Ag@PDA-loaded hydrogel:

[0124] The preparation method is the same as in Application Example 1, except that the M2pep-Ag@PDA prepared in Example 3 is replaced with the YIGSRC-Ag@PDA prepared in Example 5.

[0125] Test Example 8

[0126] This test case evaluates the biocompatibility of the hydrogels prepared in Examples 1-3. The experimental setup included: a control group (plate control), an M2pep-Ag@PDA-loaded hydrogel group, a GFLGC-Ag@PDA-loaded hydrogel group, and a YIGSRC-Ag@PDA-loaded hydrogel group. The specific procedures were as follows: L929 cells (mouse fibroblasts, derived from the Chinese Academy of Sciences Cell Bank) were digested, and cell counting was performed using a cell counting chamber at a density of 3 × 10⁻⁶ cells / well. 4 Cells were seeded at a density of [number] cells / well (24-well plate). After 24 hours of cell adhesion and growth, 50 μL of hydrogel was added to form a gel. The cells were then incubated for 24 hours in a 5% CO2, 37°C, 100% humidity incubator before measurement. The CCK-8 assay kit was used for detection. The results showed that the cell viability in each group was higher than 98±4%, with no significant difference compared to the blank plate group. This result fully demonstrates the good biocompatibility of the hydrogel prepared in this invention.

[0127] Test Example 9

[0128] This test case evaluates the hemostatic performance of the hydrogel prepared in Example 1 on rat tails. The experimental setup included: gauze treatment group, hydrogel alone group, Ag@PDA loaded hydrogel group, and M2pep-Ag@PDA loaded hydrogel group. The specific procedures were as follows: SD rats (4 weeks old) were anesthetized with isoflurane, and the tail was cut at the middle with surgical scissors and left exposed to air for 15 seconds to ensure normal bleeding. The bleeding sites were then treated as described in the above groups. The volume of hydrogel used was 50 μL. Hemostasis was photographed for each group after 2 minutes.

[0129] The results are as follows Figure 9 As shown, compared to the gauze pressure group, the hydrogel group exhibited less bleeding in the rat tails, demonstrating a significant hemostatic effect. Compared to the simple hydrogel group without M2pep-Ag@PDA, the M2pep-Ag@PDA-loaded hydrogel group and the Ag@PDA-loaded hydrogel group showed better hemostatic effects, with the M2pep-Ag@PDA-loaded hydrogel group exhibiting the most superior hemostatic effect. These experimental results fully demonstrate that the nanoparticle-doped hydrogel prepared in this invention possesses excellent hemostatic properties.

[0130] Test Case 10

[0131] This test case evaluates the antibacterial properties of the hydrogel prepared in Example 1. Experimental setup: PBS group, M2pep-Ag@PDA-loaded hydrogel group, and PDA-loaded hydrogel + near-infrared light irradiation group (NIR, 808nm, 1W / cm²). 2 Irradiation for 5 minutes), M2pep-Ag@PDA loaded hydrogel + near-infrared light irradiation group (NIR, 808nm, 1W / cm). 2 Irradiate for 5 minutes). The specific procedure is as follows: Using *Escherichia coli* (E. coli, ATCC25922) and *Staphylococcus aureus* (S. aureus, ATCC29213) as models, 10 μL of bacterial suspension (1×10⁻⁶) was irradiated. 8 CFU / mL was added to each of the above groups in a 48-well plate (hydrogel volume 0.2 mL). After co-incubating with bacteria at 37°C for 2 h, 1 mL of PBS was added to each group and mixed thoroughly. Then, 100 μL of the bacterial suspension was transferred to an agarose culture plate and incubated at 37°C for 12 h. The bacterial growth was recorded by taking pictures.

[0132] The results are as follows Figure 10As shown, compared to the control group, bacterial growth was significantly inhibited in the M2pep-Ag@PDA-loaded hydrogel, exhibiting a significant antibacterial effect. Furthermore, irradiation with near-infrared light achieved 100% bactericidal effect, which was superior to the PDA-loaded hydrogel + NIR irradiation group. These experimental results fully demonstrate that the hydrogel prepared in this invention possesses excellent antibacterial properties.

[0133] Test Example 11

[0134] This test case evaluates the wound-healing performance of the hydrogel prepared in Example 1. Experimental setup: PBS group, clinical product 3M Tegaderm. TM Film group, M2pep-Ag@PDA loaded hydrogel group, M2pepAg@PDA hydrogel + near-infrared light irradiation group (NIR, 808nm, 1W / cm) 2 (5 min, for 5 consecutive days). The specific procedure is as follows: After anesthetizing SD rats (4 weeks old) with isoflurane, the back was shaved and the skin was prepared. A Φ10mm 100℃ aluminum rod (49.2g) was used to treat the skin on the back for 20 seconds to form a burn wound. After rinsing with physiological saline, the rats were housed individually. 24 hours later, necrotic tissue was removed, and a full-thickness skin defect was created in the burn wound area using a Φ10-mm biopsy needle. Subsequently, 200μL of sterile hydrogel, clinical product 3M group, or PBS (200μL) was applied to the wound, and the wound healing was photographed at specific time points.

[0135] Figure 11 Experimental results showed that compared with the PBS and 3M groups, the M2pep-Ag@PDA and M2pep-Ag@PDA+NIR hydrogel groups had larger healing areas and better healing, exhibiting more ideal repair effects. On the other hand, compared with the M2pep-Ag@PDA group without near-infrared light irradiation, the M2pep-Ag@PDA+NIR group showed better wound healing, with the damaged area basically healed 14 days post-operation. These experimental results fully demonstrate that the hydrogel prepared in this invention possesses excellent wound repair effects.

[0136] Test Example 12

[0137] This test case evaluates the mechanical properties of the hydrogel prepared in Example 1. The experimental setup includes: a simple hydrogel group without M2pep-Ag@PDA and a hydrogel group loaded with M2pep-Ag@PDA. The specific operation is as follows: (1) Prepare a piece of pig skin tissue with a size of 7cm long × 2cm wide, remove the subcutaneous fat tissue, take 0.4mL of hydrogel sample between two pieces of skin, adhere them for 5min, and then suspend a 100g weight to observe the tissue adhesion of the hydrogel. The schematic diagram is shown in the figure. Figure 12As shown; (2) In addition, a Φ10mm×1cm hydrogel was prepared by mold and subjected to a compressive strength test by a universal mechanical testing instrument.

[0138] The results are shown in Table 1:

[0139] Table 1

[0140]

[0141]

[0142] As shown in Table 1, the nanomaterials involved in this invention can enhance the adhesion and mechanical properties of hydrogel networks by utilizing the abundant π-π stacking forces and hydrogen bonds of polydopamine, as well as the rapid reaction characteristics between silver nanoparticles and thiol groups.

[0143] The applicant declares that the above embodiments illustrate an antibacterial nanomaterial, its preparation method, and its application. However, the present invention is not limited to the above embodiments, meaning that the present invention must rely on the above embodiments to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of raw materials, additions of auxiliary components, and selection of specific methods, etc., all fall within the protection and disclosure scope of the present invention.

[0144] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details of the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0145] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable way without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

Claims

1. A wound repair hydrogel material, characterized in that, The wound repair hydrogel material includes a hydrogel matrix and an antibacterial nanomaterial loaded with peptides dispersed in the hydrogel matrix. The antibacterial nanomaterial loaded with peptides uses antibacterial nanomaterials as a carrier and is loaded with bioactive materials including bioactive peptides. The antibacterial nanomaterials include polydopamine nanoparticles and a silver layer loaded on the surface of the polydopamine nanoparticles. The bioactive peptide is an M2 macrophage-targeting peptide modified with thiol groups or a cathepsin hydrolysate polypeptide modified with thiol groups. The sequence of the M2 macrophage-targeting peptide is YEQDPWGVKWWYGGGSKKKC; The sequence of the cathepsin hydrolysate polypeptide is GFLGC; The method for preparing the antibacterial nanomaterials includes: (1) Mix dopamine with a solvent containing alkaline substances, react, and then centrifuge to obtain a precipitate, which is polydopamine nanoparticles; (2) Mix the polydopamine nanoparticle dispersion with a silver salt solution containing alkaline substances, react, and then centrifuge to obtain a precipitate, which is the antibacterial nanomaterial. The mass-volume fraction of the polydopamine nanoparticle dispersion in step (2) is 0.005-0.2%; The wound repair hydrogel material is prepared by the following steps: dispersing antibacterial nanomaterials loaded with peptides in a hydrogel raw material preparation solution, stirring at 4-40℃, molding, and allowing to stand to obtain the final product.

2. The wound healing hydrogel material according to claim 1, wherein, The particle size range of the polydopamine nanoparticles is 60-350 nm; the particle size range of the antibacterial nanomaterials is 80-400 nm.

3. The wound healing hydrogel material of claim 1, wherein, The alkaline substance in step (1) includes any one or a combination of at least two of ammonia, sodium hydroxide, or tris(hydroxymethyl)aminomethane.

4. The wound healing hydrogel material of claim 1, wherein, The solvent in step (1) includes any one or a combination of at least two of anhydrous ethanol, anhydrous methanol, an aqueous ethanol solution, or an aqueous methanol solution.

5. The wound healing hydrogel material of claim 1, wherein, The reaction described in step (1) is carried out at 4-40°C for 18-30 h.

6. The wound healing hydrogel material of claim 1, wherein, The reaction in step (1) is carried out under stirring at a speed of 300-1200 rpm.

7. The wound healing hydrogel material of claim 1, wherein, The centrifugation in step (1) is carried out at 4-40℃ and 8000-15000 rpm for 10-30 min.

8. The wound healing hydrogel material of claim 1, wherein, The alkaline substance in step (2) includes any one or a combination of two of ammonia or sodium hydroxide.

9. The wound repair hydrogel material according to claim 1, characterized in that, The silver salt in step (2) includes any one or a combination of at least two of silver nitrate, silver fluoride, or silver perchlorate.

10. The wound healing hydrogel material of claim 1, wherein, The reaction described in step (2) is carried out at 4-40°C for 30-180 min.

11. The wound healing hydrogel material of claim 1, wherein, The reaction in step (2) is carried out under stirring at a speed of 200-1000 rpm.

12. The wound healing hydrogel material of claim 1, wherein, The centrifugation in step (2) is carried out at 4-40℃ and 8000-15000 rpm for 10-30 min.

13. The wound healing hydrogel material according to any one of claims 1 to 12, characterized in that, The antibacterial nanomaterial loaded with the polypeptide is prepared by the following steps, the preparation steps including: The bioactive peptide solution is mixed with the antibacterial nanomaterial dispersion, reacted, and then centrifuged to obtain a precipitate, which is the antibacterial nanomaterial loaded with the peptide. The reaction was carried out at 4-40°C for 30-180 min; The reaction is carried out under stirring at a speed of 200-1200 rpm; The centrifugation was carried out at 4-40℃ and 8000-15000 rpm for 10-30 min.

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