Neural bionic antibacterial hydrogel dressing for diabetes infected wounds and preparation method of neural bionic antibacterial hydrogel dressing

Abstract

The invention discloses a nerve bionic type antibacterial hydrogel dressing for diabetes infected wounds and a preparation method of the nerve bionic type antibacterial hydrogel dressing. The preparation method of hydrogel comprises the following steps: synthesizing phenylboronic acid modified gold-silver alloy nanoparticles (A / M-AuAgNPs); and synthesizing an intelligent hydrogel system composed of aminated gelatin (AGel) and oxidized hyaluronic acid (OHA). And through Schiff bond crosslinking, space-time synergistic release of the calcitonin gene related peptide (CGRP) and A / M-AuAgNPs is realized. According to the hydrogel, wound infection is efficiently removed, and meanwhile, by simulating the dynamic release process of endogenous CGRP, nerve-immune signal interaction of diabetic wound damage is recovered, the wound immune homeostasis is remodeled, and wound repair is accelerated.

Description

Technical Field

[0001] This invention relates to the fields of biomedical materials, tissue engineering, and regenerative medicine, and in particular to a neuro-bionic antibacterial hydrogel dressing for diabetic infected wounds and its preparation method. Background Technology

[0002] Diabetic foot ulcers (DFUs) are among the most serious and difficult-to-treat chronic complications of diabetes, and their pathogenesis involves the complex interaction of multiple pathological factors, including vascular dysfunction, oxidative stress, and hyperglycemia-related chronic inflammation. Recent studies have shown that peripheral neuropathy plays a central role in the occurrence and healing disorders of DFUs, not only leading to decreased pain perception and impaired tissue damage awareness, but also disrupting the fine-tuning of the neuro-immune-vascular network, resulting in persistent local inflammation, insufficient angiogenesis, and delayed tissue regeneration. Simultaneously, DFUs present a microenvironment of hyperglycemia, hypoxia, and immune dysfunction, providing favorable conditions for pathogen colonization and multidrug-resistant infections, further aggravating tissue damage and delaying wound healing. The synergistic effect of neuropathy and infection keeps the wound in a chronic, difficult-to-heal state, significantly increasing the risk of gangrene and lower limb amputation. Therefore, developing comprehensive treatment strategies that can simultaneously inhibit infection and restore local neuromodulation function is of great significance for improving the treatment efficacy of DFUs and reducing the incidence of complications.

[0003] In terms of antibacterial strategies, metallic nanomaterials have attracted widespread attention in infection control and wound treatment due to their unique physicochemical properties and broad-spectrum antibacterial activity. Gold-silver alloy nanoparticles, through an alloying strategy, combine the advantages of gold and silver, exhibiting synergistically enhanced antibacterial activity and superior biocompatibility. The alloy structure not only inhibits silver oxidation and reduces the release of toxic Ag+ to lower cytotoxicity, but also retains the good biocompatibility and surface modifiability of AuNPs. By introducing functional ligands such as phenylboronic acid, AuAgNPs can specifically recognize and bind to polysaccharide structures on bacterial surfaces, thus exhibiting significant antibacterial activity in response to infection, providing a material strategy that combines high efficiency and safety for DFU infection.

[0004] Regarding the recovery of neuromodulation function, Nav1.8 +Calcitonin gene-related peptide (CGRP), released by nociceptive neurons, plays a crucial role in maintaining immune homeostasis and promoting tissue repair. CGRP not only improves local blood flow by promoting vasodilation and enhancing endothelial cell proliferation and angiogenesis, but also regulates macrophage polarization towards the anti-inflammatory M2 phenotype and inhibits excessive neutrophil infiltration, creating a favorable microenvironment for tissue repair. During normal wound healing, CGRP exhibits a highly precise temporal release pattern: rapid release in the early stages of injury to initiate the inflammatory response and blood flow regulation, followed by maintenance release to stabilize immune balance and promote matrix remodeling. However, diabetic neuropathy significantly impairs CGRP secretion, leading to an imbalance in neuro-immune signaling interactions, resulting in a chronic state of persistent inflammation, impaired angiogenesis, and disordered collagen remodeling in the wound. Although exogenous CGRP has shown therapeutic potential in preclinical studies, its clinical application is still limited by key issues such as its short in vivo half-life, rapid enzymatic degradation, insufficient local retention, pain side effects caused by high concentrations, and inability to mimic the endogenous release rhythm, severely restricting its application in the treatment of chronic diabetic wounds.

[0005] Hydrogels are widely considered ideal biological carriers due to their highly hydrated network structure resembling the extracellular matrix (ECM), excellent biocompatibility, and tunable mechanical properties. Their three-dimensional network not only supports cell adhesion, migration, proliferation, and angiogenesis, but also enables the spatiotemporal controllable release of bioactive molecules through precise structural design, mimicking the dynamic repair process of wound healing and providing possibilities for precise regulation of the local tissue microenvironment. In recent years, hydrogels have made significant progress as wound dressings in antibacterial, antioxidant, and angiogenesis-promoting effects. However, existing designs mostly focus on surface inflammation regulation or vascularization, lacking precise intervention strategies for neurological deficits in diabetic wounds, thus limiting their therapeutic efficacy.

[0006] Therefore, there is an urgent need to provide a novel neuromimetic antibacterial hydrogel dressing for diabetic infected wounds and its preparation method to address the current clinical challenges. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide a neuro-bionic antibacterial hydrogel dressing for diabetic infected wounds and its preparation method. While achieving efficient and broad-spectrum antibacterial effect, it restores the damaged neuro-immune signal interaction of diabetic wounds by simulating the dynamic release of endogenous CGRP, reshapes the local immune homeostasis of the wound, thereby accelerating wound closure and achieving tissue function regeneration.

[0008] To solve the above-mentioned technical problems, one technical solution adopted by the present invention is: to provide a neuromimetic antibacterial hydrogel dressing for diabetic infected wounds and its preparation method, comprising the following steps:

[0009] S1: Add chloroauric acid solution, silver nitrate solution, and sodium citrate to the reaction vessel and mix thoroughly. Heat until the solution turns brownish-yellow, then stop heating and allow it to cool naturally to room temperature. After centrifugation and washing, dilute to volume with deionized water to obtain gold-silver alloy nanoparticles (AuAgNPs).

[0010] S2: The AuAgNPs solution was washed multiple times with deionized water to remove excess reactants. A mixed solution of phenylboronic acid derivatives was added, and the mixture was stirred continuously at room temperature for a period of time to achieve surface modification of nanoparticles by phenylboronic acid groups. After centrifugation, washing, and dilution with deionized water, phenylboronic acid-modified gold-silver alloy nanoparticles (A / M-AuAgNPs) were obtained.

[0011] S3: Dissolve an appropriate amount of gelatin in deionized water and stir magnetically until completely dissolved. Add a quantitative amount of adipic dihydrazide (ADH), adjust the pH of the system with dilute hydrochloric acid or sodium hydroxide solution, and add a certain amount of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) while continuously stirring. After reacting for a period of time, add more EDC and continue reacting for a period of time. Dialyze the reaction solution with deionized water, freeze, and dry to obtain aminated gelatin (AGel), which is then stored in a desiccator for later use.

[0012] S4: Dissolve an appropriate amount of hyaluronic acid (HA) in deionized water and stir magnetically at room temperature until completely dissolved; add a quantitative amount of sodium periodate (NaIO4) solution, stir continuously at room temperature in the dark for a period of time, add an appropriate amount of ethylene glycol, and terminate the oxidation reaction after a period of time; the reaction solution is dialyzed with deionized water, frozen and dried to obtain oxidized hyaluronic acid (OHA), which is stored in a desiccator for later use.

[0013] S5: Take a certain amount of CGRP powder, prepare a CGRP solution of a certain concentration with sterile PBS, and store it in a low temperature freezer for later use;

[0014] S6: Dissolve appropriate amounts of oxidized hyaluronic acid (OHA) and aminated gelatin (AGel) separately in PBS buffer to prepare polymer solutions of the specified concentrations. Mix the two polymer solutions uniformly with A / M-AuAgNPs and CGRP dispersions. After mixing, the aldehyde groups of OHA and the amino groups of AGel spontaneously undergo a Schiff bond reaction under physiological conditions, thus forming an in-situ A / MO / A@C series hydrogel dressing with time- and temperature-controlled properties.

[0015] In a preferred embodiment of the present invention, in step S1, the preparation method of AuAgNPs is as follows: chloroauric acid solution, silver nitrate solution and sodium citrate are added to a reaction vessel in a molar ratio of 1:(1-2):6. After being thoroughly mixed, the mixture is heated at 120-130 °C until the solution color changes from colorless to brownish-yellow. Heating is then stopped and the mixture is allowed to cool naturally to room temperature. After multiple centrifugations and washings, deionized water is added to make up the volume to obtain the AuAgNPs solution.

[0016] In step S1, the amount of chloroauric acid solution is 0.0002 mmol and the volume is 10 mL, and the concentrations of silver nitrate and sodium citrate solutions are calculated accordingly.

[0017] In a preferred embodiment of the present invention, the preparation method of A / M-AuAgNPs in step S2 is as follows: the AuAgNPs solution is washed repeatedly with deionized water to remove excess reactants, and then a mixed solution of phenylboronic acid derivatives (wherein the concentration of 3-aminophenylboronic acid is 4.6-9.1 mg / mL and the concentration of 4-mercaptophenylboronic acid is 2.5-5.1 mg / mL) is added, and the reaction is continuously stirred at room temperature for 2 h to achieve surface modification of nanoparticles by phenylboronic acid groups; after centrifugation and washing, the solution is diluted with deionized water to obtain A / M-AuAgNPs.

[0018] In a preferred embodiment of the present invention, in step S3, the preparation method of AGel is as follows: Take an appropriate amount of gel and dissolve it in deionized water to prepare a solution with a concentration of 0.01-0.1 g / mL, and stir magnetically until completely dissolved; add 0.696-2.09 g of ADH, adjust the pH of the system to 4.75-6.25 with dilute hydrochloric acid or sodium hydroxide solution, add 0.19-0.57 g of EDC while stirring continuously, and after reacting for 1 h, add an equal amount of EDC and continue reacting for 2 h; after dialysis with deionized water and freeze-drying, aminated gelatin (AGel) is obtained and stored in a desiccator for later use.

[0019] In a preferred embodiment of the present invention, in step S4, the preparation method of OHA is as follows: Take an appropriate amount of HA and dissolve it in deionized water to prepare a solution with a concentration of 0.01-0.1 g / mL, and stir magnetically at room temperature until completely dissolved; add a quantitative concentration of NaIO4 solution with a concentration of 0.13-0.39 g / mL, stir continuously at room temperature in the dark for 2 h, add 1 mL of ethylene glycol after the reaction is completed, continue stirring for 2 h to terminate the oxidation reaction; the reaction solution is dialyzed with deionized water and freeze-dried to obtain OHA, which is stored in a desiccator for later use.

[0020] In a preferred embodiment of the present invention, in step S6, the hydrogel dressing is a combination of two or more of AGel, OHA, A / M-AuAgNPs, and CGRP. The preparation method is as follows: Take appropriate amounts of OHA and AGel and dissolve them in PBS buffer, then mix the two polymer solutions with the dispersions of A / M-AuAgNPs and CGRP to make the concentration of A / M-AuAgNPs in the system 25-100 μg / mL and the concentration of CGRP 2-8 μg / mL. After mixing, the aldehyde group of OHA and the amino group of AGel spontaneously undergo a Schiff bond reaction to form an A / MO / A@C series hydrogel dressing with time- and temperature-controlled function in situ.

[0021] To solve the above-mentioned technical problems, another technical solution adopted by the present invention is to provide a neuro-bionic antibacterial hydrogel dressing for diabetic infected wounds as described in any of the above claims and a method for preparing the same.

[0022] The beneficial effects of this invention are as follows: This invention provides a neuromimetic antibacterial hydrogel dressing for diabetic infected wounds prepared by the above method. This hydrogel, through multidimensional synergistic effects, not only inhibits bacterial infection of the wound but also restores local neuro-immune interactions, achieving antibacterial activity, remodeling immune homeostasis, and promoting tissue repair, thereby accelerating the healing of diabetic wounds. It has the following advantages:

[0023] (1) This invention constructs dual-ligand modified gold-silver alloy nanoparticles (A / M-AuAgNPs), which have regular morphology, controllable composition, and excellent physicochemical properties. Through surface functionalization modification, precise targeting of bacteria is achieved, which can effectively exert a broad-spectrum antibacterial effect and effectively overcome the limitations of traditional antibacterial alloys in terms of targeting and efficacy.

[0024] (2) The hydrogel prepared by the present invention has a loose and porous three-dimensional network structure, which has high flexibility, stable viscoelasticity and typical shear thinning and self-healing ability. It can be molded by in-situ injection with a syringe, which perfectly meets the needs of complex and irregular wound surfaces of diabetic wounds.

[0025] (3) The hydrogel prepared by the present invention can respond to the acidic microenvironment of the wound and undergo structural rearrangement to realize the intelligent release of neuropeptide CGRP, which simulates the spatiotemporal secretion characteristics of sensory neurons for CGRP during wound repair, highlighting its application potential as a biomimetic neuropeptide delivery platform in wound treatment.

[0026] (4) The hydrogel prepared by this invention exhibits multiple synergistic effects in inhibiting infection, relieving inflammation, promoting angiogenesis and tissue remodeling. By integrating antibacterial and neuro-immune signal regulation, this invention provides a systematic experimental basis and technical support for the precise treatment of diabetic infected wounds. Attached Figure Description

[0027] Figure 1 These are the TEM (1A), XRD (1B), and SAED (1C) images of AuAgNPs prepared in the embodiments of the present invention, and the UV-vis (1D) images of A / M-AuNPs, AgNPs, AuAgNPs, and AuAgNPs.

[0028] Figure 2 This describes the killing effect of AuAgNPs and A / M-AuAgNPs prepared in the embodiments of the present invention on Escherichia coli;

[0029] Figure 3 The following diagrams illustrate the process (3A), self-healing, and injectability (3B) of preparing the hydrogel in the examples;

[0030] Figure 4 These are the degradation and swelling curves of the hydrogel prepared in the examples;

[0031] Figure 5 This is the cumulative release curve of the CGRP hydrogel prepared in the examples;

[0032] Figure 6 This is a representative image and quantitative analysis of the hydrogel prepared in the examples used to treat infected wounds in diabetic rats;

[0033] Figure 7 The images shown are immunofluorescence staining images (7A) and semi-quantitative statistical average fluorescence intensity (7B) of the hydrogel prepared in the examples for wound treatment. Figure 8 This is a schematic diagram illustrating the mechanism by which the hydrogel prepared in this embodiment treats diabetic infected wounds. Detailed Implementation

[0034] The preferred embodiments of the present invention will now be described in detail with reference to the accompanying drawings, so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby providing a clearer and more explicit definition of the scope of protection of the present invention.

[0035] Example:

[0036] A neuromimetic antibacterial hydrogel dressing for diabetic infected wounds and its preparation method, comprising the following steps:

[0037] 1) Take 10 mL of 0.002 mmol chloroauric acid solution, 10 mL of 0.002 mmol silver nitrate solution, and 0.012 mmol sodium citrate solution, respectively, and add them to the reaction vessel. Mix thoroughly. Then heat the reaction vessel at 120-130 °C until the solution color gradually changes from colorless to brownish-yellow, indicating the formation of gold-silver alloy nanoparticles. Then stop heating and allow it to cool naturally to room temperature. After centrifugation and washing, dilute to a final volume to obtain AuAgNPs.

[0038] 2) 700 μL of a mixed solution of phenylboronic acid derivatives was added to the obtained AuAgNPs. The concentrations of 3-aminophenylboronic acid and 4-mercaptophenylboronic acid in this mixed solution were 9.1 mg / mL and 5.1 mg / mL, respectively. The reaction was carried out with stirring at room temperature for 2 h to achieve functional modification of the nanoparticle surface by phenylboronic acid groups. After the reaction was completed, unbound small molecules were removed by centrifugation and washing. Finally, the solution was diluted with deionized water and stored at 4 °C for later use.

[0039] 3) Weigh 1 g of Gel and dissolve it in 100 mL of deionized water, stirring magnetically until completely dissolved. Then add 0.696 g of ADH and adjust the pH of the system to 4.75 using dilute hydrochloric acid or sodium hydroxide solution. Under continuous stirring, add 0.19 g of EDC to activate the carboxyl groups. After reacting for 1 h, add another 0.19 g of EDC and continue the reaction for 2 h. After the reaction is complete, dialyze the resulting solution in deionized water for 3 days to remove unreacted small molecules. Finally, freeze-dry the dialyzed solution to obtain AGel.

[0040] 4) Weigh 0.5 g of HA and dissolve it in 50 mL of deionized water. Stir magnetically at room temperature until completely dissolved. Then add 2.5 mL of sodium periodate solution (0.13 g / mL) to the solution and stir continuously at room temperature for 2 h under light-protected conditions to achieve selective oxidation of hyaluronic acid. After the reaction is complete, add 1 mL of ethylene glycol and continue stirring for 1 h to terminate the oxidation reaction. Then dialyze the reaction solution in deionized water for 3 days to remove unreacted small molecules. Finally, freeze-dry the dialyzed solution to obtain OHA.

[0041] 5) Take 1 mg of CGRP powder and prepare a 1 mM CGRP solution with sterile PBS buffer. Store at -80 ℃ for later use.

[0042] 6) OHA and AGel were dissolved in PBS buffer at concentrations of 1% (w / v) and 2% (w / v), respectively. The two polymer solutions were then mixed with A / M-AuAgNPs and CGRP dispersions to achieve a final concentration of 50 μg / mL for A / M-AuAgNPs and 6.7 μg / mL for CGRP. After mixing, the aldehyde group of OHA and the amino group of AGel spontaneously under physiological conditions undergo a Schiff bond reaction, forming an A / MO / A@C hydrogel in situ.

[0043] The morphology and dimensions of the A / M-AuAgNPs prepared in this example are as follows: Figure 1 As shown in the image. TEM image (1A) shows that the prepared AuAgNPs exhibit a regular spherical structure with uniform particle size distribution and good monodispersity, with a nanoparticle size of approximately 30-40 nm. The XRD diffraction pattern is shown in the image. Figure 1 As shown in Figure B, the characteristic diffraction peaks appearing at 2θ = 38.13°, 44.50°, 64.48°, and 77.52° are attributed to the (111), (200), (220), and (311) crystal plane diffraction of the gold / silver face-centered cubic (FCC) structure, respectively. SAED results are as follows... Figure 1 As shown in Figure C, multiple clear and continuous concentric diffraction rings can be observed in the SAED pattern, corresponding to the (111), (200), (220), and (311) crystal plane diffractions of the FCC structure, respectively. This result corroborates the XRD diffraction results, further confirming the crystal structure characteristics of AuAgNPs and providing a reliable structural basis for its subsequent surface modification and functional applications. UV-Vis... Figure 1 As shown in Figure D, AuNPs exhibit single surface plasmon resonance (SPR) peaks at 520-540 nm, and AgNPs at 410-430 nm. However, the SPR peak of AuAgNPs, located at 470-490 nm, is not a simple superposition of single-metal peaks but rather presents a new characteristic peak. Upon introduction of 3-aminophenylboronic acid and 4-mercaptophenylboronic acid, the SPR peak of A / M-AuAgNPs redshifts to 500-520 nm. This change suggests that the interaction between the ligands and the nanoparticle surface may modulate the surface electron cloud density through electronic effects, thereby influencing its plasmon optical properties.

[0044] The antibacterial properties of AuAgNPs and A / M-AuAgNPs prepared in this example are as follows: Figure 2As shown in the figure, after co-incubation of the two types of nanoparticles and *E. coli* for 18 h, bacterial colonies were widely formed in the control group culture dishes. At a concentration of 50 μg / mL, AuAgNPs showed a significant antibacterial effect against *E. coli*, with a bacterial survival rate of less than 50%, at which point there was no significant difference in antibacterial effect compared to A / M-AuAgNPs (p > 0.05). When the concentration was increased to 100 μg / mL, the survival rate of *E. coli* in the A / M-AuAgNPs treatment group dropped to below 10%, and its antibacterial effect was significantly better than that of AuAgNPs (p < 0.01), indicating that the surface modification with phenylboronic acid functional groups further enhanced the antibacterial properties of the nanoparticles.

[0045] The preparation process of the hydrogel in this example (3A), its self-healing and injectable properties (3B) are as follows: Figure 3 As shown in the figure. This hydrogel can respond to changes in the pH of the wound microenvironment, enabling the controlled release of A / M-AuAgNPs and CGRP, thereby synergistically exerting antibacterial, anti-inflammatory, and healing-promoting effects. The gelation process of the O / A hydrogel is as follows. Figure 3 As shown in Figure A, after mixing equal volumes of OHA solution and AGel solution, a stable gel can be formed in situ within the bottle. Furthermore, this hydrogel exhibits excellent self-healing properties, such as… Figure 3 As shown in Figure B, the two cut pieces of hydrogel can re-adhere after being in contact for a period of time, and the red and yellow dyes diffuse into each other at the interface, indicating that the internal dynamic network structure can be effectively reconstructed, verifying the self-healing ability of the O / A hydrogel. Simultaneously, the hydrogel exhibits excellent injectability, being continuously extruded through a 10 mL syringe without clogging, and can accurately draw complex patterns such as "HIT," demonstrating its good deformation adaptability and ease of operation, making it suitable for in-situ filling needs of irregular diabetic wounds.

[0046] The degradation and swelling of the hydrogel prepared in this example are as follows: Figure 4As shown in the figure, due to the high pH sensitivity of Schiff bonds, the degradation rates of O / A and A / MO / A@C hydrogels in acidic phosphate buffer solution (pH 6.0) were significantly faster than in neutral pH 7.4, exhibiting good pH-responsive degradation characteristics. This pH sensitivity allows the hydrogels to accelerate degradation in the acidic environment of diabetic wounds, thereby continuously releasing the loaded drug and achieving precise controlled drug release. As the pH of the wound microenvironment gradually approaches neutral, the A / MO / A@C hydrogel showed a relatively higher residual mass on day 10, suggesting that the loaded active substance may be more sustained-release, which is beneficial for maintaining the effective concentration of the drug at the wound site. In PBS buffer at pH 7.4, both hydrogels exhibited rapid swelling behavior and reached swelling equilibrium within 48 h. However, the swelling rate of A / MO / A@C hydrogel was significantly slower than that of O / A hydrogel. This phenomenon can be attributed to the steric hindrance and hydrophobic interactions generated by the nanoparticle loading, which restricts water molecules from entering the hydrogel network, thereby reducing the swelling rate. This moderate swelling behavior not only helps the hydrogel maintain its three-dimensional structure and avoids the decline in mechanical properties caused by excessive swelling, but also regulates the drug release rate by controlling the entry and exit of water molecules, further optimizing its application effect in the treatment of diabetic wounds.

[0047] The hydrogel prepared in this example exhibits responsive CGRP release as follows: Figure 5 As shown, under neutral conditions (pH 7.4) simulating the microenvironment of normal tissue, CGRP exhibits a continuous but incomplete release characteristic, with a cumulative release rate of only about 60% after 10 days, indicating that the hydrogel network has a strong binding and retention capacity for peptides. In contrast, under slightly acidic conditions (pH 6.0) simulating the inflammatory microenvironment of diabetic foot ulcers, both the release rate and cumulative release amount of CGRP are significantly increased, approaching 80% within the same time period. This significant pH-dependent release behavior indicates that the A / MO / A@C hydrogel can intelligently release CGRP in response to local microenvironmental stimuli through structural changes, simulating the spatiotemporal secretion dynamics of CGRP by sensory neurons during wound repair, highlighting its important application potential as a biomimetic neuropeptide delivery platform in wound treatment.

[0048] The hydrogel prepared in this example was used to treat infected wounds in diabetic rats. Figure 6As shown, the efficacy of hydrogel in vivo treatment was evaluated using a diabetic SD rat model of infected wounds. On day 3 of treatment, all wounds showed a significant shrinkage trend, with the A / MO / A@C group exhibiting a wound area of ​​less than 50%, indicating a faster healing trend. On day 7, the wounds in the A / MO / A@C group showed a significant change, with the tissue color returning to a healthy red, indicating active angiogenesis. Quantitative analysis of the wounds revealed that the wound area in the A / MO / A@C group shrank to approximately 13.84% of the original wound (p < 0.0001), while the control group (gauze dressing) and the single-component hydrogel treatment groups (O / A, A / MO / A, O / A@C) showed slower healing rates, with remaining wound areas exceeding 45%. Statistical analysis on day 14 showed no significant difference in wound area among the gauze group, O / A group, A / MO / A group, and O / A@C group (p > 0.05). Conversely, the wounds in the A / MO / A@C group were almost completely closed, with remaining areas less than 5%. This excellent wound repair effect can be attributed to the synergistic effect of A / M-AuAgNPs and CGRP, which significantly accelerates the wound healing process and improves the closure effect.

[0049] The effect of the hydrogel prepared in this example on inflammation and vascularization at the wound site is as follows: Figure 7 As shown in Figure 7A. Immunofluorescence staining was used to spatially locate (7A) and semi-quantitatively analyze key inflammatory and repair-related biomarkers in wound tissue. On day 7 of treatment, the mean fluorescence intensity of the inflammatory cell biomarker CD11b in the peri-wound area of ​​the A / MO / A@C group was significantly lower than that of the gauze group (p < 0.05), and the mean fluorescence intensity of the pro-inflammatory factor TNF-α was significantly lower (p < 0.0001). At this time, the degree of inflammatory cell infiltration in the wound of this group was significantly reduced, the level of inflammatory signal was significantly decreased, and the overall inflammatory response was effectively relieved. Further analysis of the expression characteristics of CD206, a specific biomarker of M2 macrophages, revealed that the A / MO / A@C group showed a stronger red fluorescence signal than the gauze group (p < 0.01), demonstrating that the A / MO / A@C hydrogel can enrich M2 macrophages with anti-inflammatory and pro-repair phenotypes in the wound microenvironment. To assess the angiogenesis capacity, a key component of wound repair, on day 14 of treatment, the expression of CD31, a vascular endothelial cell-specific marker, was measured. Results showed that the A / MO / A@C group exhibited a stronger green fluorescence signal in the periphery of the wound (p < 0.001), indicating a significantly increased angiogenesis density and more active angiogenesis capacity. In conclusion, the A / MO / A@C hydrogel can regulate the microenvironment of diabetic wounds, promoting wound repair through multiple pathways including inhibiting excessive inflammatory responses, promoting M2 macrophage polarization, and accelerating angiogenesis.

[0050] The embodiments of the present invention are merely examples for clearly illustrating the invention and are not intended to limit the implementation of the invention. Those skilled in the art will recognize that other variations or modifications can be made based on the above embodiments, and it is neither necessary nor possible to exhaustively describe all possible implementations here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A nerve-bionic antibacterial hydrogel dressing for diabetic infected wounds and a method for preparing the same, characterized in that, Includes the following steps: S1: Add chloroauric acid solution, silver nitrate solution, and sodium citrate to the reaction vessel and mix thoroughly. Heat until the solution turns brownish-yellow, then stop heating and allow it to cool naturally to room temperature. After centrifugation and washing, dilute to volume with deionized water to obtain gold-silver alloy nanoparticles (AuAgNPs). S2: The AuAgNPs solution was washed multiple times with deionized water to remove excess reactants. A mixed solution of phenylboronic acid derivatives was added, and the mixture was stirred continuously at room temperature for a period of time to achieve surface modification of nanoparticles by phenylboronic acid groups. After centrifugation, washing, and dilution with deionized water, phenylboronic acid-modified gold-silver alloy nanoparticles (A / M-AuAgNPs) were obtained. S3: Dissolve an appropriate amount of gelatin in deionized water and stir magnetically until completely dissolved. Add a certain amount of adipic dihydrazide (ADH), adjust the pH of the system with dilute hydrochloric acid or sodium hydroxide solution, and add a certain amount of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) while stirring continuously. After reacting for a period of time, add more EDC and continue reacting for a period of time. Dialyze the reaction solution with deionized water, freeze, and dry to obtain aminated gelatin (AGel), and store it in a desiccator for later use. S4: Dissolve an appropriate amount of hyaluronic acid (HA) in deionized water and stir magnetically at room temperature until completely dissolved; add a certain amount of sodium periodate (NaIO4) solution, stir continuously at room temperature in the dark for a period of time, add an appropriate amount of ethylene glycol, and terminate the oxidation reaction after a period of time; the reaction solution is dialyzed with deionized water, frozen and dried to obtain oxidized hyaluronic acid (OHA), which is stored in a desiccator for later use. S5: Take a certain amount of CGRP powder, prepare a CGRP solution of a certain concentration with sterile PBS, and store it in a low temperature freezer for later use; S6: Dissolve appropriate amounts of oxidized hyaluronic acid (OHA) and aminated gelatin (AGel) separately in PBS buffer to prepare polymer solutions of the specified concentrations. Mix the two polymer solutions uniformly with A / M-AuAgNPs and CGRP dispersions. After mixing, the aldehyde groups of OHA and the amino groups of AGel spontaneously undergo a Schiff bond reaction under physiological conditions, thus forming an in-situ A / MO / A@C series hydrogel dressing with time- and temperature-controlled properties.

2. The process for the preparation of neurogenically inspired antibacterial hydrogel dressing for diabetic infected wounds as claimed in claim 1 wherein, In step S1, the ratio of chloroauric acid solution, silver nitrate solution and sodium citrate is 1:(1-2):6; the heating temperature is 120-130 ℃.

3. The process for the preparation of neurogenically inspired antibacterial hydrogel dressing for diabetic infected wounds as claimed in claim 1 wherein, In step S2, the concentration of 3-aminophenylboronic acid in the phenylboronic acid derivative mixed solution is 4.6-9.1 mg / mL, and the concentration of 4-mercaptophenylboronic acid is 2.5-5.1 mg / mL; the reaction is carried out with stirring at room temperature for 2 h.

4. The process for the preparation of neurogenically inspired antibacterial hydrogel dressing for diabetic infected wounds as claimed in claim 1 wherein, In step S3, the concentration of gel is 0.01-0.1 g / mL, the mass of ADH is 0.696-2.09 g, the pH of the system is 4.75-6.25, and the mass of EDC is 0.19-0.57 g.

5. The process for the preparation of neurogenically inspired antibacterial hydrogel dressing for diabetic infected wounds as claimed in claim 1 wherein, In step S4, the concentration of HA is 0.01-0.1 g / mL and the concentration of NaIO4 is 0.13-0.39 g / mL.

6. The process for the preparation of neurogenically inspired antibacterial hydrogel dressing for diabetic infected wounds as claimed in claim 1 wherein, In step S6, the hydrogel dressing is a combination of two or more of AGel, OHA, A / M-AuAgNPs, and CGRP.

7. The process for the preparation of neurogenically inspired antibacterial hydrogel dressing for diabetic infected wounds as claimed in claim 1 wherein, In step S6, the concentration of A / M-AuAgNPs is 25-100 μg / mL, and the concentration of CGRP is 2-8 μg / mL.

8. A neuro-bionic antibacterial hydrogel dressing for diabetic infected wounds prepared by the method of preparing the neuro-bionic antibacterial hydrogel dressing for diabetic infected wounds according to any one of claims 1 to 7.

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