Hydrogel for promoting wound healing of dual-response controlled-release astaxanthin and amino small-molecule medicine as well as preparation method and application of hydrogel

Abstract

The invention discloses a double-response controlled-release hydrogel for promoting wound healing of astaxanthin and an amino small molecule drug as well as a preparation method and application of the hydrogel, and belongs to the field of biomedical materials. The material is composed of a drug-loaded nano-micelle and a self-healing hydrogel network. Hydrophilic modification of astaxanthin and dynamic coupling of the astaxanthin and an amino small molecule drug are achieved through an astaxanthin-polyethylene glycol-benzaldehyde intermediate, and the drug-loaded nano-micelle has the pH response drug release capacity; the hydrogel network is formed by crosslinking phenylboronic acid modified carboxymethyl chitosan and dopamine through dynamic phenylboronic acid ester bonds, and has ROS responsiveness and self-healing characteristics; the multifunctional intelligent wound dressing can intelligently respond to high ROS and acidic pH microenvironment of chronic wounds, controllably release astaxanthin and amino-containing small molecule drugs, synergistically remove ROS, improve cell oxidative stress, inhibit inflammation, promote blood vessel and tissue regeneration and accelerate chronic wound healing, is excellent in self-healing performance and biocompatibility, and is an ideal multifunctional intelligent wound dressing.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, specifically relating to a wound-healing hydrogel with dual-response controlled-release astaxanthin and amino small molecule drugs, its preparation method and application. Background Technology

[0002] Chronic wounds, such as diabetic foot ulcers and pressure ulcers, pose a significant challenge to clinical treatment due to their complex and abnormal pathological microenvironment. The clinical challenge in treating chronic wounds stems from their abnormal pathological microenvironment, which exhibits multiple pathological characteristics, including hypoxia, acidic pH, high levels of reactive oxygen species (ROS), persistent inflammation, and susceptibility to infection. These factors are interconnected and collectively hinder the normal healing process. Effectively regulating this microenvironment is crucial for promoting healing. In recent years, multi-drug combined interventions targeting multiple pathological stages have shown great therapeutic potential. Astaxanthin (AST), as a potent natural antioxidant, can significantly reduce oxidative stress and reduce inflammation; while many amino-containing small molecule drugs (such as metformin and gentamicin) have potential in controlling blood sugar, antibacterial activity, and anti-inflammation. Theoretically, synergistic delivery of astaxanthin and amino-containing drugs is expected to synergistically regulate the wound microenvironment through multiple pathways, promoting wound healing.

[0003] However, translating this combination therapy strategy into clinical application faces multiple technical bottlenecks. First, the strong hydrophobicity and chemical instability of astaxanthin severely restrict its solubility, effective delivery, and bioavailability in aqueous biological systems, which is the core obstacle to its clinical translation. Second, if the two drugs are simply physically mixed or co-loaded, their release behavior is often non-synergistic and uncontrollable, unable to be linked with dynamically changing pathological signals (ROS bursts, pH drops), resulting in insufficient effective drug concentrations at the lesion site and asynchronous effects. This not only limits the therapeutic effect but may also increase the risk of systemic side effects due to non-specific release.

[0004] To overcome the above challenges, the development of nanodelivery systems that can intelligently respond to the wound microenvironment and achieve controlled drug release has become a research focus. Currently, some studies have been dedicated to constructing drug delivery systems with a single response mechanism. For example, Chinese patent CN111437438A discloses a ROS-responsive hydrogel based on phenylboronic acid ester bonds, and Chinese patent CN116637206A discloses a pH-responsive hydrogel based on imine bonds, which can be used to deliver a single type of drug. However, such technical solutions have obvious limitations: 1) They are difficult to effectively load and deliver strongly hydrophobic drugs like astaxanthin, failing to solve the fundamental delivery problem of astaxanthin's poor water solubility and low stability; 2) They usually only have a single ROS response or pH response mechanism, and cannot simultaneously respond to the dual pathological characteristics of acidic pH and high ROS in chronic wounds, resulting in insufficient drug release intelligence and environmental matching; 3) They are mostly used to deliver single drugs or drugs with similar physicochemical properties, lacking an integrated chemical strategy and carrier design that can efficiently and stably couple strongly hydrophobic drugs (such as astaxanthin) with hydrophilic amino small molecule drugs and achieve synergistic release of the two under the trigger of pathological signals. Summary of the Invention

[0005] To address the technical challenges of existing technologies in simultaneously achieving efficient and stable loading of highly hydrophobic astaxanthin, its chemical coupling and synergistic delivery with hydrophilic amino drugs within a single hydrogel system, and the inability to respond to the dual pathological signals of high ROS and acidic pH in chronic wounds to achieve intelligent on-demand drug release, this invention aims to provide a wound-healing hydrogel with dual-response controlled release of astaxanthin and amino small molecule drugs, along with its preparation method and applications. This wound-healing hydrogel is an intelligent hydrogel capable of responding to the microenvironment of chronic inflammatory wounds and synergistically delivering astaxanthin and amino small molecule drugs. As a wound dressing, it can achieve targeted enrichment and microenvironment-responsive release of the two drugs at the wound site, thereby synergistically regulating multiple pathological processes such as oxidative stress, inflammation, infection, and angiogenesis disorders.

[0006] To achieve the above objectives, the present invention employs the following technical solution: This invention provides a wound healing hydrogel with dual-response controlled release of astaxanthin and amino small molecule drugs. The wound healing hydrogel is composed of drug-loaded nanomicelles and a self-healing hydrogel network. The drug-loaded nanomicelles are fixed in the hydrogel network through physical embedding and hydrogen bonding interactions. The drug-loaded nanomicelles are formed by self-assembly of an astaxanthin-polyethylene glycol-benzaldehyde (CM-PEG-BA) amphiphilic intermediate and an amino-containing small molecule drug via dynamic imine bond coupling. The self-healing hydrogel network is formed by cross-linking phenylboronic acid-modified carboxymethyl chitosan and dopamine through dynamic phenylboronic acid ester bonds. The first component is phenylboronic acid-modified carboxymethyl chitosan (CMCS-PBA), and the second component is dopamine (DA). The phenylboronic acid groups on CMCS-PBA and the catechol groups on dopamine are reversibly cross-linked through dynamic phenylboronic acid ester bonds to form a hydrogel network with self-healing properties.

[0007] The astaxanthin-polyethylene glycol-benzaldehyde amphiphilic intermediate was prepared by esterification of astaxanthin with carboxymethyl-polyethylene glycol-benzaldehyde under a catalyst. This esterification reaction constructed the "astaxanthin-polyethylene glycol-benzaldehyde" intermediate, a design that effectively addresses the water solubility and stability issues of astaxanthin and provides a universal, efficient, and dynamically reversible coupling platform for various amino-containing small molecule drugs. The resulting amphiphilic polymer, "astaxanthin-polyethylene glycol-amino small molecule drug," can further self-assemble into nanomicelles in an aqueous system.

[0008] The amino-containing small molecule drug is selected from metformin, gentamicin, histamine, or a pharmaceutically acceptable salt thereof.

[0009] In the phenylboronic acid-modified carboxymethyl chitosan, the degree of phenylboronic acid modification is 5% to 20%.

[0010] The mass fraction of the drug-loaded nanomicelles in the hydrogel network is 0.1% to 2%.

[0011] This invention provides a method for preparing a wound-healing hydrogel containing a dual-response controlled-release astaxanthin and an amino small molecule drug, comprising: Step 1: Under the action of a catalyst, astaxanthin is esterified with carboxymethyl-polyethylene glycol-benzaldehyde, and after purification, an amphiphilic intermediate of astaxanthin-polyethylene glycol-benzaldehyde is obtained. Step 2: The intermediate obtained in Step 1 is reacted with an amino-containing small molecule drug and purified to obtain an astaxanthin-polyethylene glycol-amino small molecule drug conjugate; the conjugate is dissolved in water and self-assembled to form a drug-loaded nanomicelle dispersion. Step 3: After activating carboxyphenylboronic acid, it is linked with carboxymethyl chitosan through an amidation reaction, and after purification, carboxymethyl chitosan modified with phenylboronic acid is obtained. Step 4: Mix the drug-loaded nanomicelle dispersion obtained in Step 2, the phenylboronic acid-modified carboxymethyl chitosan solution obtained in Step 3, and the dopamine solution, and perform a cross-linking reaction to form the wound-healing hydrogel.

[0012] In step 1, the molar ratio of astaxanthin to carboxymethyl polyethylene glycol-benzaldehyde is 1:1 to 1:4; the catalyst is 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 4-dimethylaminopyridine; the esterification reaction temperature is 25~50℃; and the time is 10~30 h.

[0013] In step 2, the molar ratio of the intermediate to the amino-containing small molecule drug is 2:1 to 1:2; the reaction temperature is 25 to 50°C; and the reaction time is 10 to 24 h.

[0014] In step 3, the activating agent for carboxyphenylboronic acid is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide in a molar ratio of 1:1; the mass ratio of the activated carboxyphenylboronic acid to carboxymethyl chitosan is 1:5 to 1:10, the amidation reaction is carried out at room temperature for 10 to 14 h, and the pH value is 8.0 to 10.0.

[0015] In step 4, the mass-volume concentration of the phenylboronic acid-modified carboxymethyl chitosan solution is 1%~5%, the mass-volume concentration of the dopamine solution is 1%~5%, and the final concentration of the drug-loaded nanomicelles in the mixed system is 0.01%~1%.

[0016] This invention provides the application of the above-mentioned dual-response controlled-release astaxanthin and amino small molecule drug wound healing hydrogel in the preparation of medical dressings for promoting wound healing, inhibiting inflammation, scavenging reactive oxygen species, improving oxidative stress, or regulating the wound microenvironment.

[0017] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a wound-healing hydrogel with dual-response controlled-release astaxanthin and amino small molecule drugs. By coupling astaxanthin with strongly hydrophilic carboxymethyl-polyethylene glycol-benzaldehyde and amino small molecule drugs, a drug-loaded nanomicelle with a "hydrophilic-hydrophobic" core-shell structure is successfully constructed, encapsulating astaxanthin within it. This fundamentally overcomes the inherent strong hydrophobicity and instability of astaxanthin, greatly facilitating its loading in the hydrogel system and laying the foundation for its effective delivery and action in vivo. The coupling of the amino drug with dynamic imine bonds introduces a pH-responsive mechanism, allowing the drug to be released under acidic wound conditions. The self-healing hydrogel network is composed of phenylboronic acid-modified carboxymethyl chitosan and dopamine cross-linked through dynamic phenylboronic acid ester bonds. Phenylboronic acid modification endows carboxymethyl chitosan with ROS responsiveness, and the dynamic phenylboronic acid ester bonds break at high ROS levels to release the drug. Furthermore, the reversibility of these dynamic bonds provides self-healing capability, ensuring the hydrogel can self-repair after damage. This hydrogel can simultaneously respond to the pathological characteristics of high ROS and acidic pH at the wound site, enabling targeted and intelligent drug release, improving treatment efficacy and reducing side effects. Based on dynamic phenylboronic acid ester bonds, the hydrogel can automatically repair itself after being damaged by external forces, extending the lifespan of the dressing on the wound and avoiding secondary damage caused by frequent changes. Excellent biocompatibility and safety: Made from biocompatible carboxymethyl chitosan, polyethylene glycol, and other materials, the degradation products are non-toxic and have no side effects, ensuring the biosafety of this hydrogel as a wound dressing.

[0018] Furthermore, astaxanthin is linked to polyethylene glycol chains via esterification, forming stable chemical bonds that effectively combine the strongly hydrophobic astaxanthin with the strongly hydrophilic polyethylene glycol chains, constructing an amphiphilic structure. This improves the solubility of astaxanthin and its embedding stability within micelles. Benzaldehyde groups are modified at the ends of the polyethylene glycol chains, providing specific reaction sites that facilitate efficient coupling with amino-containing small molecule drugs via dynamic imine bonds, ensuring precise drug loading and responsive release. All drugs contain active amino groups that efficiently form dynamic imine bonds with the benzaldehyde groups at the ends of the astaxanthin-polyethylene glycol-benzaldehyde amphiphilic intermediate, ensuring stable construction and controlled release of the drug-loaded nanomicelles. Metformin is suitable for diabetic wounds, regulating blood glucose levels and reducing inflammation; gentamicin provides potent antibacterial activity against bacterially infected wounds; and histamine is used to regulate the inflammatory microenvironment of wounds. Pharmaceutically acceptable salt forms enhance drug solubility and biocompatibility, preventing precipitation or inactivation during coupling.

[0019] The preparation method provided by this invention utilizes the hydrophilicity of carboxymethyl-polyethylene glycol-benzaldehyde to overcome the strong hydrophobicity of astaxanthin through esterification, forming a stable amphiphilic structure that facilitates subsequent self-assembly. Based on dynamic imine bond coupling, hydrophobic astaxanthin and hydrophilic amino drugs are synergistically loaded into the core-shell structure, ensuring intelligent drug release under pathological signals. Phenylboronic acid groups are introduced through chemical modification to provide cross-linking sites for the hydrogel network, supporting dynamic bond formation. Cross-linking is achieved using dynamic phenylboronic acid ester bonds, integrating drug-loaded micelles into the hydrogel network, ensuring structural self-healing and dual responsiveness. This method efficiently constructs a stable and intelligent hydrogel system through sequential synthesis and assembly.

[0020] The application provided by this invention allows the dressing to simultaneously promote wound healing through the synergistic stimulation of cell regeneration and angiogenesis by astaxanthin and amino-based small molecule drugs released from the hydrogel; inhibit inflammation by reducing the release of inflammatory factors using the anti-inflammatory properties of astaxanthin and the immunomodulatory effects of amino-based drugs; regulate oxidative stress by balancing the redox state based on the potent antioxidant capacity of astaxanthin; scavenge ROS by triggering drug release in response to the high ROS environment of the wound to directly neutralize ROS; and regulate pH by adjusting the microenvironment's acidity and alkalinity to the physiological range using the pH-responsive dynamic bonding mechanism of the hydrogel. These features work synergistically to ensure that the dressing can simultaneously address multiple pathological aspects of chronic wounds during application.

[0021] Furthermore, diabetic wounds are often accompanied by hyperglycemia, susceptibility to infection, and impaired angiogenesis. The antioxidant, anti-inflammatory, and antibacterial properties of hydrogels can be maximized in such wounds, enabling targeted treatment optimization. Attached Figure Description

[0022] Figure 1 The synthetic route of the drug-loaded nanomicelle precursor AST-PEG-Met in Example 1 of this invention; Figure 2 This is a schematic diagram illustrating the synthesis and cross-linking principle of the self-healing hydrogel network cross-linking component in Example 1 of the present invention. Figure 3 The free radical scavenging ability of AST-PEG-Met obtained in Example 1 of this invention was detected; Figure 4 The anti-inflammatory activity results of AST-PEG-Met obtained in Example 1 of this invention; Figure 5 The responsive degradation behavior of the wound healing hydrogel containing dual-response controlled-release astaxanthin and amino small molecule drugs obtained in Example 1 of the present invention. Figure 6This is a characterization of the self-healing ability and dynamic reversibility of the wound-healing hydrogel with dual-response controlled-release astaxanthin and amino small molecule drugs obtained in Example 1 of the present invention. In this figure, A is a macroscopic diagram of the self-healing process of the hydrogel, and B is a diagram of the cyclic strain scanning test results of the hydrogel. The horizontal axis is time (s), and the vertical axis is modulus (G'G'', Pa). Different colors represent the storage modulus G' (red, yellow). Figure 7 This invention presents the effect of the dual-response controlled-release astaxanthin and amino small molecule drug hydrogel obtained in Example 1 of this invention on promoting the healing of full-layer dermal wounds on the back of diabetic rats. In this figure, A shows photographs of the wound morphology of rats in different experimental groups at different time points and corresponding wound area analysis diagrams; B shows the curves of wound healing rate of rats in different experimental groups over time. Detailed Implementation

[0023] To enable those skilled in the art to understand the features and effects of the present invention, the following descriptions and definitions are only general descriptions of the terms and expressions mentioned in the specification and claims. Unless otherwise specified, all technical and scientific terms used herein have the ordinary meaning understood by those skilled in the art regarding the present invention, and in the event of any conflict, the definitions in this specification shall prevail.

[0024] Unless otherwise defined, the technical terms used in the following embodiments have the same meanings as commonly understood by those skilled in the art to which this invention pertains. Unless otherwise specified, the experimental reagents used in the following embodiments are conventional biochemical reagents; and the experimental methods described are conventional methods.

[0025] The construction principle of the dual-response hydrogel of this invention is shown in the attached figure. Figure 1 The synthetic route of the drug-loaded nanomicelle precursor AST-PEG-Met is shown. The obtained AST-PEG-Met is an amphiphilic molecule that can be further self-assembled in aqueous solution to form nanomicelles (PEG molecular weight is 1000, n is 22). Figure 2 The synthesis and cross-linking principle of the cross-linking components of the self-healing hydrogel network were demonstrated. Finally, [the following was achieved / concluded / etc.]. Figure 1 The obtained drug-loaded nanomicelle dispersion and Figure 2 The obtained CMCS-PBA solution and dopamine solution are mixed, and the drug-loaded nanomicelles are fixed in a hydrogel network formed by dynamic phenylboronic acid ester crosslinking through physical embedding and hydrogen bonding interaction, thereby obtaining the composite hydrogel described in this invention.

[0026] Example 1 This embodiment provides a wound-healing hydrogel with dual-response controlled-release astaxanthin and amino small molecule drugs. The specific preparation steps are as follows: (1) Preparation of water-soluble astaxanthin derivative (AST-PEG) 24 mg of astaxanthin and 45 mg of carboxymethyl polyethylene glycol benzaldehyde (PEG molecular weight 1000, n=22) were weighed and dissolved in 18 mL of dichloromethane and stirred until fully dissolved. 300 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI·HCl) and 300 mg of 4-dimethylaminopyridine (DMAP) were added to the above solution as catalysts, and stirring was continued to ensure uniform dispersion of the catalysts. The reaction was carried out at 37 ℃ and 1000 rpm in the dark for 12 h. After the reaction, the entire reaction system was transferred to a dialysis bag with a molecular weight cutoff of 1000 Da and dialyzed in the dark for 3 days. After dialysis, the solution in the dialysis bag was transferred to a freeze dryer and freeze-dried at -80 ℃ to obtain a red solid product, a water-soluble astaxanthin derivative (AST-PEG), which was stored in a -20 ℃ freezer in the dark.

[0027] (2) Preparation of astaxanthin-polyethylene glycol-bismethyl guanidine nanomicelles 45 mg of AST-PEG obtained in step (1) was dissolved in 9 mL of methanol and stirred until completely dissolved. 6 mg of metformin (Met) was added to the above solution, and the reaction was carried out at room temperature for 12 h under light-protected conditions with a stirring speed of 1000 rpm. After the reaction was completed, the reaction system was transferred to a rotary evaporator, and the solvent was removed by rotary evaporation under reduced pressure at 37 °C to obtain the final product AST-PEG-Met (APM). APM was dissolved in water. Due to the hydrophilicity of the polyethylene glycol segments and the hydrophobicity of the astaxanthin moiety, APM could self-assemble to form water-soluble nanomicelles loaded with astaxanthin and metformin.

[0028] (3) Preparation of carboxymethyl chitosan-phenylboronic acid (CMCS-PBA) copolymer Accurately weigh 0.83 g of 4-carboxyphenylboronic acid and add it to 60 mL of dimethyl sulfoxide (DMSO). Place the solution on a magnetic stirrer and stir until dissolved. Add 0.96 g of EDCI·HCl and 0.58 g of N-hydroxysuccinimide (NHS), and stir at 1000 rpm for 4 h at room temperature to activate C-PBA and obtain the active ester intermediate. Separately, 6 g of carboxymethyl chitosan was added to 800 mL of deionized water and stirred at 60 ℃ and 1200 rpm for 2 h until dissolved. After the solution cooled to room temperature, a 0.75% wt carboxymethyl chitosan aqueous solution was obtained. The system containing the active ester intermediate was slowly added dropwise to the carboxymethyl chitosan aqueous solution using a syringe, and the reaction was continued at 1000 rpm for 12 h at room temperature to allow C-PBA and CMCS to undergo an amidation reaction. After the reaction was completed, the pH was adjusted to 9.0 with 0.1 M sodium hydroxide solution to ensure a suitable alkaline environment. The reaction solution was transferred to a dialysis bag with a molecular weight cutoff of 8000-14000 Da, and the dialysis bag was placed in a large amount of deionized water for dialysis for 7 days. After dialysis, the solution in the dialysis bag was transferred to a freeze dryer for freeze drying to obtain 4.8 g of white powder CMCS-PBA. The obtained CMCS-PBA was dried and stored at room temperature.

[0029] (4) Preparation of wound healing hydrogels with dual-response controlled-release astaxanthin and amino small molecule drugs Weigh 0.4 g of CMCS-PBA obtained in step (3) and dissolve it completely in 10 mL of PBS (pH=7.4) to prepare a 4% wt CMCS-PBA hydrogel precursor solution.

[0030] Weigh 0.4 mg of APM obtained in step (2), dissolve it completely in 0.4 mL of PBS to obtain a drug-loaded nanomicelle solution, add the drug-loaded nanomicelle solution to the above CMCS-PBA hydrogel precursor solution, and disperse it evenly by ultrasonication. This solution is denoted as solution A. Weigh 0.03 g of dopamine (DA), dissolve it in 1 mL of PBS, and prepare a DA solution with a concentration of 3% wt. This solution is denoted as solution B. Take 1 mL of solution A and 50 μL of solution B (volume ratio 20:1), mix them thoroughly, and inject the mixture into a culture dish with a syringe. A stable drug-loaded hydrogel can be formed at room temperature, namely a wound healing hydrogel with dual-response controlled release of astaxanthin and amino small molecule drugs.

[0031] Example 2 This embodiment provides a wound-healing hydrogel with dual-response controlled-release astaxanthin and amino small molecule drugs. The specific preparation steps are as follows: (1) Preparation of water-soluble astaxanthin derivative (AST-PEG) 24 mg of astaxanthin and 180 mg of carboxymethyl-polyethylene glycol-benzaldehyde were weighed and dissolved in 36 mL of dichloromethane and stirred until fully dissolved. 900 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI·HCl) and 900 mg of 4-dimethylaminopyridine (DMAP) were added to the above solution as catalysts, and stirring was continued to ensure uniform dispersion of the catalysts. The reaction was carried out at 37 ℃ and 1000 rpm in the dark for 24 h. After the reaction, the entire reaction system was transferred to a dialysis bag with a molecular weight cutoff of 1000 Da and dialyzed in the dark for 3 days. After dialysis, the solution in the dialysis bag was transferred to a freeze dryer and freeze-dried at -80 ℃ to obtain a red solid product, a water-soluble astaxanthin derivative (AST-PEG), which was stored in a -20 ℃ freezer in the dark.

[0032] (2) Preparation of astaxanthin-polyethylene glycol-histamine nanomicelles 90 mg of AST-PEG obtained in step (1) was dissolved in 18 mL of methanol and stirred until completely dissolved. 11 mg of histamine (His) was added to the above solution, and the reaction was carried out at room temperature for 12 h under light-protected conditions with a stirring speed of 1000 rpm. After the reaction was completed, the reaction system was transferred to a rotary evaporator, and the solvent was removed by rotary evaporation under reduced pressure at 37 °C to obtain the final product AST-PEG-His (APH). When APH was dissolved in water, due to the hydrophilicity of the polyethylene glycol segments and the hydrophobicity of the astaxanthin moiety, APH could self-assemble to form water-soluble nanomicelles loaded with astaxanthin and histamine.

[0033] (3) Preparation of carboxymethyl chitosan-phenylboronic acid (CMCS-PBA) copolymer Accurately weigh 0.42 g of 4-carboxyphenylboronic acid and add it to 30 mL of dimethyl sulfoxide (DMSO). Stir the mixture on a magnetic stirrer until dissolved. Add 0.48 g of EDCI·HCl and 0.26 g of N-hydroxysuccinimide (NHS). Stir the mixture at 1000 rpm for 2 h at room temperature to activate C-PBA and obtain the active ester intermediate. Separately, 5 g of carboxymethyl chitosan was added to 1000 mL of deionized water and stirred at 60 ℃ and 1200 rpm for 2 h until dissolved. After the solution cooled to room temperature, a 0.5% wt carboxymethyl chitosan aqueous solution was obtained. The system containing the active ester intermediate was slowly added dropwise to the carboxymethyl chitosan aqueous solution using a syringe, and the reaction was continued at 1000 rpm for 12 h at room temperature to allow C-PBA and CMCS to undergo an amidation reaction. After the reaction was completed, the pH was adjusted to 9.0 with 0.1 M sodium hydroxide solution to ensure a suitable alkaline environment. The reaction solution was transferred to a dialysis bag with a molecular weight cutoff of 8000-14000 Da, and the dialysis bag was placed in a large amount of deionized water for dialysis for 7 days. After dialysis, the solution in the dialysis bag was transferred to a freeze dryer for freeze drying to obtain 4 g of white powder CMCS-PBA. The obtained CMCS-PBA was dried and stored at room temperature.

[0034] (4) Preparation of wound healing hydrogels with dual-response controlled-release astaxanthin and amino small molecule drugs Weigh 0.5 g of CMCS-PBA obtained in step (3), dissolve it completely in 10 mL of PBS (pH=7.4), and prepare a 5% wt CMCS-PBA hydrogel precursor solution.

[0035] Weigh 0.2 mg of the APH obtained in step (2), dissolve it completely in 0.2 mL of PBS to obtain a drug-loaded nanomicelle solution, add the drug-loaded nanomicelle solution to the above CMCS-PBA hydrogel precursor solution, and disperse it evenly by ultrasonication. This solution is denoted as solution A. Weigh 0.03 g of dopamine (DA), dissolve it in 1 mL of PBS, and prepare a DA solution with a concentration of 3% wt. This solution is denoted as solution B. Take 1 mL of solution A and 50 μL of solution B (volume ratio 20:1), mix them thoroughly, and inject the mixture into a culture dish with a syringe. A stable drug-loaded hydrogel can be formed at room temperature, namely a wound-healing hydrogel with dual-response controlled release of astaxanthin and amino small molecule drugs.

[0036] Example 3 The properties of the astaxanthin and amino small molecule drug-loaded nanomicelles prepared in this invention were tested.

[0037] (1) Antioxidant activity For the DPPH free radical scavenging experiment, astaxanthin standard and APM nanomicelles obtained in Example 1 were fully dissolved in DMSO to prepare a 20 μg / mL solution. These solutions were then mixed with an equal volume of 0.1 mM DPPH ethanol solution. After reacting at room temperature in the dark for 30 min, the absorbance was measured at 517 nm. An equal volume of DMSO was used as a blank control instead of the sample solution, and 0.1 mM quinoline dimethacrylate (Trolox) was used as a positive control. The results are as follows: Figure 3 As shown in Figure A. For the ABTS free radical scavenging experiment, ABTS is first generated by reacting ABTS with potassium persulfate. + • Stock solution, diluted with PBS (pH=7.4) to an appropriate absorbance before use. Dissolve the APM nanomicelles obtained in Example 1 thoroughly in PBS (pH=7.4) to prepare sample solutions of 0, 5, 10, 15, 20, and 25 μg / mL. Then, use the diluted ABTS... + • The working solution and the sample to be tested were mixed in equal volumes and reacted at room temperature for 10 min. The absorbance was then measured at 734 nm. PBS (pH=7.4) was used as a blank control instead of the sample solution, and 0.1 mM quinoline dimethacrylate (Trolox) was used as a positive control. The results are as follows: Figure 3 As shown in B.

[0038] From the appendix Figure 3 Data show that APM-loaded drug-coated nanomicelles possess excellent free radical scavenging capabilities: their DPPH free radical scavenging ability is not significantly different from that of AST and the positive control drug Trolox; their ABTS free radical scavenging ability is concentration-dependent, with 25 μg / mL APM exhibiting comparable ABTS free radical scavenging ability to 0.1 mM Trolox. This indicates that the modification of the polyethylene glycol chain and the coupling of amino drugs do not affect the antioxidant capacity of astaxanthin, providing experimental evidence for subsequent regulation of oxidative stress to promote wound healing.

[0039] (2) Anti-inflammatory activity Mouse mononuclear macrophages (RAW 264.7, from Haixing Biotechnology Co., Ltd.) were revived and cultured in DMEM complete medium (containing 10% FBS and 1% penicillin-streptomycin) and then seeded into 6-well plates. The cells were incubated at 37℃ and 5% CO2. A control group (containing 100 ng / mL LPS) and drug treatment groups (each containing 100 ng / mL LPS and a final concentration of 4 μM of the corresponding drug) were set up, with three replicates per group. After incubation for another 24 h, the supernatant was collected, and the results were detected using a TNF-α (EK0527) and IL-10 (EK0417) ELISA kit from Wuhan Boster Biological Engineering Co., Ltd. The results are shown below. Figure 4 As shown.

[0040] From the appendix Figure 4 Data show that the Met, AST, and APM groups all significantly reduced the level of the pro-inflammatory factor TNF-α and increased the level of the anti-inflammatory factor IL-10 in the supernatant, with the APM group showing a more significant regulatory effect. This experiment confirms that the self-assembled nanomicelles of this invention have good anti-inflammatory activity.

[0041] Example 4 The properties of the wound healing hydrogel prepared by this invention, which combines dual-response controlled-release astaxanthin and amino small molecule drugs, were tested.

[0042] (1) Response to degradation behavior Three portions of the wound-healing hydrogel prepared according to the method in Example 1, each 1 mL, were placed in 20 mL of different solutions, including PBS (pH=7.4, simulating normal tissue), PBS (pH=6.5, simulating the acidic microenvironment of a wound), and PBS (pH=6.5) containing 1 mM H2O2 (simulating a high ROS environment in a wound). The degradation kinetics were observed and recorded under a constant temperature of 37 ℃ to obtain the time-to-degree degradation information of the hydrogel under different environments. The results are as follows: Figure 5 As shown.

[0043] From the appendix Figure 5 Data shows that by constructing different pH and ROS environments in vitro, degradation of the constructed drug-loaded hydrogel was observed in various systems. In a physiological environment of pH 7.4, the hydrogel structure was most stable, with slow degradation; while under acidic conditions of pH 6.5, the degradation rate significantly accelerated; and in an environment where pH 6.5 and H₂O₂ coexisted, the hydrogel exhibited the fastest degradation behavior. This experiment confirms that the hydrogel of this invention can simultaneously respond to the acidic pH and high ROS levels of wounds, and its degradation behavior exhibits a significant microenvironment dependence, providing a theoretical basis and experimental foundation for the controlled release of drugs in the wound microenvironment.

[0044] (2) Self-healing properties The self-healing ability of the wound-healing hydrogel containing dual-response controlled-release astaxanthin and amino small molecule drugs prepared in this invention was characterized macroscopically and rheologically.

[0045] A cylindrical hydrogel (Example 1) was cut open, and different cut surfaces were stained and brought into close contact. For mechanical properties, cyclic strain scanning tests were performed on the cylindrical drug-loaded hydrogel (25 mm in diameter, 1 mm in height) using a rheometer (Anton Paar MCR102e rotational rheometer). The strain alternated between 1% and 500%, with each stage lasting 120 s. The low strain of 1% was in the linear viscoelastic region, where the hydrogel structure was relatively stable, and the rheological parameters reflected its initial mechanical properties. The high strain of 500% caused significant damage to the hydrogel's network structure. By alternating strains for a certain period, the mechanical response and structural reversibility of the hydrogel under different strain conditions could be studied. The results are as follows: Figure 6 As shown.

[0046] From the appendix Figure 6 Macroscopic observation revealed that the hydrogel re-healed within 30 minutes and could withstand its own weight without breaking, indicating the existence of dynamic interactions within the hydrogel that enable rapid reconstruction of connections after damage, restoring overall continuity and mechanical strength. Cyclic strain scanning tests showed that the hydrogel's modulus decreased sharply under 500% strain due to network structure disruption, but when the strain recovered to 1%, its modulus rapidly recovered to over 90% of its initial value, and this process could be repeated multiple times.

[0047] Through macroscopic observation and rheological testing, the hydrogel network constructed based on dynamic phenylboronic acid ester bonds was demonstrated from different perspectives to possess rapid and efficient self-healing capabilities and excellent dynamic reversibility. Macroscopically, the cross-section can heal rapidly and bear weight, while microscopically, rheological testing shows that the modulus can recover rapidly after high strain failure and can be cycled multiple times. This provides strong experimental support for the application of this hydrogel in fields that require self-healing and dynamic response characteristics.

[0048] (3) Effect on the healing of diabetic wounds A full-layer skin wound model on the back of diabetic rats was constructed, and the drug-loaded hydrogel obtained in Example 1 was used to verify its effect on promoting skin wound healing in rats.

[0049] Four-week-old male rats were selected and, after acclimatization, were induced to have diabetes by a single intraperitoneal injection of 45 mg / kg streptozotocin (STZ). One week later, rats with a persistent blood glucose level ≥16.7 mmol / L were selected for the experiment. The qualified diabetic rats were randomly divided into three groups: the Blank group (no wound treatment), the blank hydrogel group (wound covered with a blank hydrogel without APM nanomicelles), and the drug-loaded hydrogel group (wound covered with the wound-healing hydrogel prepared in Example 1). After anesthesia, a 10 mm diameter full-skin wound was prepared on the back of the rats, and they were treated according to their groups and then fed for another 14 days. During the feeding period, the rats were kept in a stable environment with sufficient food and water, and suitable temperature, humidity, and light conditions were maintained to promote the normal wound healing process. Wound morphology was photographed regularly, and the wound healing rate was quantified using ImageJ software. The results are shown below. Figure 7 As shown.

[0050] From the appendix Figure 7 Data shows that, compared with the Blank group and the blank hydrogel group, the drug-loaded hydrogel treatment group exhibited the fastest wound closure rate throughout the observation period, indicating that the drug-loaded hydrogel has a positive promoting effect on wound healing in diabetic rats. The drug-loaded hydrogel treatment group showed significant wound contraction, an important indicator of wound healing, suggesting that the drug-loaded hydrogel can promote the contraction of wound edge tissues and accelerate wound closure. By day 14, the healing rate of the drug-loaded hydrogel treatment group was significantly higher than that of the control group (Blank group and blank hydrogel group), further confirming that the wound-healing hydrogel of this invention has a significant promoting effect on the repair and regeneration of refractory diabetic wounds. The high healing rate indicates that the wound-healing hydrogel of this invention can effectively improve the healing environment of diabetic wounds and accelerate the wound healing process, possessing potential clinical application value.

[0051] This in vivo experiment, using a full-scale dorsal skin wound model of diabetic rats and comparing different experimental groups, fully demonstrates that the wound-healing hydrogel provided by this invention can effectively promote the repair and regeneration of refractory diabetic wounds. The synergistic effect of the drug components in the wound-healing hydrogel and the properties of the hydrogel itself provides a new and effective method for the treatment of diabetic wounds, with significant clinical application potential.

[0052] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of the claims of this invention.

Claims

1. A wound-healing hydrogel with dual-response controlled-release of astaxanthin and amino small molecule drugs, characterized in that, The wound healing hydrogel is composed of drug-loaded nanomicelles and a self-healing hydrogel network. The drug-loaded nanomicelles are fixed in the hydrogel network through physical embedding and hydrogen bonding interactions. The drug-loaded nanomicelles are formed by self-assembly of an astaxanthin-polyethylene glycol-benzaldehyde amphiphilic intermediate and an amino-containing small molecule drug via dynamic imine bond coupling. The self-healing hydrogel network is formed by cross-linking phenylboronic acid-modified carboxymethyl chitosan and dopamine through dynamic phenylboronic acid ester bonds.

2. The wound-healing hydrogel with dual-response controlled-release astaxanthin and amino small molecule drugs according to claim 1, characterized in that, The astaxanthin-polyethylene glycol-benzaldehyde amphiphilic intermediate is prepared by esterification of astaxanthin with carboxymethyl-polyethylene glycol-benzaldehyde under the action of a catalyst.

3. The wound-healing hydrogel with dual-response controlled-release astaxanthin and amino small molecule drugs according to claim 1, characterized in that, The amino-containing small molecule drug is selected from metformin, gentamicin, histamine, or a pharmaceutically acceptable salt thereof.

4. A method for preparing a wound-healing hydrogel with dual-response controlled-release astaxanthin and amino small molecule drugs as described in any one of claims 1 to 3, characterized in that, include: Step 1: Under the action of a catalyst, astaxanthin is esterified with carboxymethyl-polyethylene glycol-benzaldehyde, and after purification, an amphiphilic intermediate of astaxanthin-polyethylene glycol-benzaldehyde is obtained. Step 2: The intermediate obtained in Step 1 is reacted with an amino-containing small molecule drug and purified to obtain an astaxanthin-polyethylene glycol-amino small molecule drug conjugate; the conjugate is dissolved in water and self-assembled to form a drug-loaded nanomicelle dispersion. Step 3: After activating carboxyphenylboronic acid, it is linked with carboxymethyl chitosan through an amidation reaction, and after purification, carboxymethyl chitosan modified with phenylboronic acid is obtained. Step 4: Mix the drug-loaded nanomicelle dispersion obtained in Step 2, the phenylboronic acid-modified carboxymethyl chitosan solution obtained in Step 3, and the dopamine solution, and perform a cross-linking reaction to form the wound-healing hydrogel.

5. The method for preparing a wound-healing hydrogel with dual-response controlled-release astaxanthin and amino small molecule drugs according to claim 4, characterized in that, In step 1, the molar ratio of astaxanthin to carboxymethyl polyethylene glycol-benzaldehyde is 1:1 to 1:4; the catalyst is 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 4-dimethylaminopyridine; the esterification reaction temperature is 25~50 °C; and the time is 10~30 h.

6. The method for preparing a wound-healing hydrogel with dual-response controlled-release astaxanthin and amino small molecule drugs according to claim 4, characterized in that, In step 2, the molar ratio of the intermediate to the amino-containing small molecule drug is 2:1 to 1:2; the reaction temperature is 25 to 50 °C; and the reaction time is 10 to 24 h.

7. The method for preparing a wound-healing hydrogel with dual-response controlled-release astaxanthin and amino small molecule drugs according to claim 4, characterized in that, In step 3, the activating agent for carboxyphenylboronic acid is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide in a molar ratio of 1:1; the mass ratio of the activated carboxyphenylboronic acid to carboxymethyl chitosan is 1:5 to 1:10, the amidation reaction is carried out at room temperature for 10 to 14 h, and the pH value is 8.0 to 10.

0.

8. The method for preparing a wound-healing hydrogel with dual-response controlled-release astaxanthin and amino small molecule drugs according to claim 4, characterized in that, In step 4, the mass-volume concentration of the phenylboronic acid-modified carboxymethyl chitosan solution is 1%~5%, the mass-volume concentration of the dopamine solution is 1%~5%, and the final concentration of the drug-loaded nanomicelles in the mixed system is 0.01%~1%.

9. The use of a wound-healing hydrogel of a dual-response controlled-release astaxanthin and amino small molecule drug as described in any one of claims 1-3 in the preparation of medical dressings for promoting wound healing, inhibiting inflammation, scavenging reactive oxygen species, improving oxidative stress, or regulating the wound microenvironment.

10. The application according to claim 9, characterized in that, The medical dressing is used to promote the healing of chronic diabetic wounds.

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