Intelligent responsive composite hydrogel dressing and preparation method and application thereof

Abstract

The application discloses an intelligent responsive composite hydrogel dressing and a preparation method and application thereof, and belongs to the field of biomedical materials. The preparation method comprises the following steps: preparing arginine grafted quaternary ammonium chitosan LQ; preparing phenylboronic acid functionalized oxidized sodium alginate P-OSA; preparing a multifunctional nanoenzyme complex PMZG; mixing polyvinyl alcohol, LQ, honeysuckle extract and / or PMZG, and then cross-linking with a P-OSA solution to obtain a hydrogel. Through progressive modular design, the hydrogel is endowed with injectability, self-healing, multiple microenvironment responsiveness and cascade catalytic antibacterial, photothermal therapy and pro-angiogenesis capabilities, and can be used for synergistically treating chronic infected wounds, diabetic foot ulcers and drug-resistant bacterial infection wounds, and has a wide clinical application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials and tissue engineering, specifically relating to a smart responsive composite hydrogel dressing, its preparation method, and its application. Background Technology

[0002] Hydrogels are materials with a three-dimensional porous structure composed of a hydrophilic polymer network. Their high water content and extracellular matrix-like physical properties make them ideal candidates for wound dressings. Compared with traditional dressings, hydrogels can not only effectively absorb exudate and maintain a moist wound environment, but also act as a barrier to prevent bacterial invasion and provide a scaffold for cell migration and proliferation. However, traditional hydrogels often have limited functionality and are difficult to synergistically address the complex pathological microenvironment during the healing process of infected wounds, including bacterial colonization and biofilm formation, excessive reactive oxygen species (ROS) accumulation, persistent inflammatory responses, and impaired angiogenesis.

[0003] Polyvinyl alcohol (PVA) is a biocompatible synthetic polymer that can be cross-linked to construct hydrogel networks with excellent mechanical properties, but it lacks inherent biological activity and intelligent responsiveness. Chitosan (CS), as a natural cationic polysaccharide, possesses inherent antibacterial and healing-promoting abilities, but its poor solubility limits its applications. Quaternized chitosan (QCS) retains the advantages of chitosan CS while improving its water solubility, and further enhances its cell adhesion and angiogenesis potential by grafting L-arginine (L-Arg), while also endowing the hydrogel with the ability to generate nitric oxide (NO). Sodium alginate (SA) is a natural anionic polysaccharide, and its oxidation product, oxidized sodium alginate (OSA), can introduce active aldehyde groups onto the molecular chain. Subsequently, phenylboronic acid (PBA) is grafted onto the OSA backbone to construct a smart polymer containing both borate ester bonds and Schiff base bonds—two types of dynamic covalent bonds. These dynamic bonds endow hydrogels with excellent self-healing and injectability, and can selectively break down in the high pH and high glucose concentration microenvironment unique to diabetic wounds, thereby enabling intelligent and on-demand drug release at the site of infection.

[0004] Honeysuckle extract (HPE), as a natural plant-derived active ingredient, possesses broad-spectrum antibacterial, anti-inflammatory, and antioxidant pharmacological effects. Introducing it into hydrogels endows them with mild and sustained biological functions. To enhance the therapeutic effect against drug-resistant bacterial infections, a nanocatalytic system was introduced. Specifically, molybdenum disulfide (MoS2) nanosheets were used as carriers to load glucose oxidase (GOx), and the surface was modified with polydopamine (PDA), followed by loading with nano-zinc oxide (ZnO) to construct a multifunctional nanoplatform (PMZG). This platform can utilize the abundant glucose in wound exudate to initiate a cascade reaction: GOx first catalyzes glucose to generate gluconic acid and hydrogen peroxide (H2O2); subsequently, MoS2, with peroxidase-like activity, converts H2O2 into highly cytotoxic hydroxyl radicals (·OH), achieving highly efficient chemokinetic therapy; simultaneously, the PDA coating endows the material with near-infrared (NIR) thermal conversion capabilities; and ZnO sustainably releases Zn. 2+ It exerts a dual effect of long-lasting antibacterial action and promoting epithelialization.

[0005] Currently, most reported hydrogel dressings fail to organically integrate multiple functions such as injectable self-healing, microenvironment-responsive drug release, catalytic antibacterial activity, photothermal therapy, and angiogenesis promotion. Hydrogels based on natural polymers, in particular, often face problems such as insufficient mechanical properties, slow functional response, and poor adaptability to complex infection environments.

[0006] Therefore, there is a need to develop an integrated hydrogel dressing that is simple in composition, easy to prepare, has excellent injectability and self-healing ability, and can intelligently respond to wound microenvironment (such as ROS and glucose) and external stimuli (such as NIR), thereby synergistically achieving multiple functions such as antibacterial, anti-inflammatory, antioxidant and angiogenesis. Summary of the Invention

[0007] The purpose of this invention is to provide a method for preparing a smart responsive composite hydrogel dressing, so as to solve the technical problems of existing wound dressings having single function, difficulty in intelligently responding to complex infection microenvironments, and insufficient multi-mechanism synergistic ability.

[0008] Another object of the present invention is to provide a smart responsive composite hydrogel dressing prepared by the above preparation method.

[0009] Another object of the present invention is to provide the use of the above-mentioned intelligent responsive composite hydrogel dressing in the preparation of medicaments or medical dressings for treating chronic infected wounds, diabetic foot ulcers, burns or wounds infected by drug-resistant bacteria.

[0010] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides a method for preparing a smart responsive composite hydrogel dressing, comprising the following steps: Step 1: Quaternize chitosan to obtain quaternized chitosan, then graft L-arginine onto it in the presence of EDC·HCl and NHS, and obtain LQ by dialysis and lyophilization. Step 2: Sodium alginate is oxidized to obtain sodium alginate oxide, which is then reacted with 3-aminophenylboronic acid, and P-OSA is obtained by dialysis and freeze-drying. Step 3: Molybdenum disulfide powder is peeled into nanosheets, which are then coated with polydopamine, loaded with nano-zinc oxide and glucose oxidase in sequence. After centrifugation, washing and freeze-drying, PMZG is obtained. Step 4: Mix polyvinyl alcohol aqueous solution and LQ aqueous solution evenly, and selectively add honeysuckle extract and / or PMZG to obtain precursor mixture; Step 5: Mix the precursor mixture from Step 4 with the P-OSA aqueous solution at a volume ratio of 1:1, and form a hydrogel through a cross-linking reaction at room temperature.

[0011] As a preferred embodiment, the preparation of LQ in step one includes: dissolving chitosan in a 1.5% (v / v) aqueous acetic acid solution, adding trimethylammonium chloride, reacting at 60°C for 24 h to obtain quaternized chitosan; after cooling, adding L-arginine, then adding EDC·HCl and NHS, stirring the reaction, dialyzing for 72 h, and lyophilizing.

[0012] As a preferred embodiment, the preparation of P-OSA in step two includes: dissolving sodium alginate in deionized water, slowly adding sodium periodate, stirring at room temperature in the dark for 6 hours, adding ethylene glycol to terminate the reaction, and lyophilizing by dialyzing to obtain oxidized sodium alginate; dissolving oxidized sodium alginate in PBS buffer (pH=7.4), adding 3-aminophenylboronic acid, adjusting the pH to 6.5-7.0, stirring in the dark for 24 hours, and lyophilizing by dialyzing to obtain P-OSA.

[0013] As a preferred embodiment, the preparation of PMZG in step three includes: ultrasonically exfoliating molybdenum disulfide powder in a 40% (v / v) ethanol solution, and collecting molybdenum disulfide nanosheets by centrifugation; dispersing the molybdenum disulfide nanosheets in an ice-water bath, adding Tris-HCl buffer solution of dopamine hydrochloride, stirring in the dark for 12 h, centrifuging, washing, and lyophilizing to obtain MoS2@PDA; dispersing MoS2@PDA in an ice-water bath, adding zinc acetate dihydrate, stirring and adsorbing, adjusting the pH to 10.0, reacting at 60℃ for 4 h, simultaneously adding glucose oxidase, centrifuging, washing, and lyophilizing to obtain PMZG.

[0014] As a preferred embodiment, in step S4, the concentration of the polyvinyl alcohol aqueous solution is 20% (w / v), the concentration of the LQ aqueous solution is 2% (w / v), the final concentration of the honeysuckle extract is 0.5% (w / v), and the final concentration of PMZG is 0.2% (w / v); the mass ratio of honeysuckle extract to PMZG is 5:2.

[0015] As a preferred embodiment, the crosslinking reaction in step five is completed within 10 seconds, and the resulting hydrogel has a dual dynamic covalent crosslinking network of borate ester bonds and Schiff base bonds.

[0016] As a preferred option, honeysuckle extract and / or PMZG may be selectively added or omitted in step four to obtain a basic hydrogel, a drug-loaded hydrogel, or a fully functional hydrogel, respectively.

[0017] Secondly, the present invention provides a smart responsive composite hydrogel dressing prepared by the above-described preparation method.

[0018] As a preferred embodiment, the dynamic covalent cross-linking network of the hydrogel dressing is formed by cross-linking polyvinyl alcohol, arginine-grafted quaternized chitosan and phenylboronic acid-functionalized sodium alginate through dynamic borate ester bonds and Schiff base bonds. The hydrogel dressing also contains honeysuckle extract and multifunctional nanoenzyme complex dispersed in it.

[0019] Thirdly, the present invention provides the application of the above-mentioned intelligent responsive composite hydrogel dressing in the preparation of drugs or medical devices for treating chronic infected wounds, diabetic foot ulcers, burns or wounds infected by drug-resistant bacteria.

[0020] According to the above technical solution, the beneficial effects of the present invention are: 1. This invention achieves modular superposition of functions from "basic smart materials" to "drug delivery systems" and then to "active treatment systems" through a progressive design of PQO → PQO / HPE → PQO / HPE / PMZG. In clinical applications, the required functional level of hydrogel formulation can be flexibly selected or customized according to the degree of wound infection, resulting in flexible application strategies and good clinical adaptability.

[0021] 2. Excellent self-healing and mechanical properties. The dynamic borate ester bonds introduced into the basic PQO network endow all derived hydrogels with excellent self-healing capabilities. Among them, PQO / HPE / PMZG can rapidly recover within 30 seconds after macroscopic structural damage. The introduction of the multifunctional nanozyme PMZG significantly improves the storage modulus (G′) and crosslinking density of the hydrogel, enabling it to withstand strains of up to 511% without breaking, providing a stable and damage-resistant physical barrier for friction-prone wounds.

[0022] 3. Multiple intelligent response and synergistic treatment mechanism: The hydrogels constructed in this invention (especially PQO / HPE / PMZG) can respond synchronously to multiple endogenous signals of the wound microenvironment and synergistically with exogenous stimuli to achieve precise controlled release and cascade treatment: (1) pH response: Phenylboronic acid ester bonds selectively break in the weakly alkaline microenvironment of diabetic infected wounds, accelerating the disintegration of the hydrogel network and the targeted release of loaded drugs; (2) Glucose response: Loaded GOx can specifically catalyze excess glucose in wound exudate to generate gluconic acid (lowering local pH and synergistically promoting the breaking of boronic acid ester bonds and drug release) and H2O2 (as a key substrate for chemokinetic therapy); (3) NIR response: PDA components endow the hydrogel with excellent photothermal conversion capabilities, enabling remote thermal activation and enhancing local antibacterial and anti-inflammatory effects. This "internal and external linkage" response mechanism realizes the spatiotemporal controllable release of drugs.

[0023] 4. Highly efficient synergistic antibacterial and healing-promoting capabilities: This hydrogel system integrates multiple functional mechanisms: physical barrier (hydrogel matrix), chemical antibacterial (quaternary ammonium groups and Zn) 2+ The mechanisms involved include natural antibacterial / anti-inflammatory components (HPE), chemokinetic therapy (MoS2 catalyzing the generation of ·OH from H2O2), and NIR photothermal therapy (PDA). These mechanisms exhibit a synergistic effect in inhibiting drug-resistant bacteria. Furthermore, the introduction of L-Arg helps promote cell adhesion and angiogenesis, and Zn... 2+ It can also promote epithelialization, accelerating wound healing from both anti-infection and regeneration perspectives.

[0024] 5. Controlled Degradation and Intelligent Drug Release Kinetics: The in vitro degradation behavior of hydrogels and the release rate of loaded drugs can be controlled by the component ratio. Among them, highly cross-linked PQO / HPE / PMZG hydrogels exhibit slower and more microenvironment-responsive degradation and release characteristics, which can better match the wound healing cycle and ensure the durability of the therapeutic effect.

[0025] In summary, the hydrogel dressing prepared by this invention is highly integrated in function, programmable in performance, and capable of intelligently responding to complex wound microenvironments. Its unique progressive design and multiple synergistic mechanisms give it great clinical application potential in treating refractory wounds such as chronically infected wounds, burns, and diabetic foot ulcers. Attached Figure Description

[0026] Figure 1The images show the preparation and structural characterization of the PQO, PQO / HPE, and PQO / HPE / PMZG series hydrogels, where: a) is a schematic diagram of the preparation and function of PQO / HPE / PMZG; b) is the Fourier transform infrared (FTIR) spectrum of SA, OSA, P-OSA, CS, QCS, L-Arg, and LQ; c) is the scanning electron microscope (SEM) image of the three hydrogels and the mapping elemental distribution of PQO / HPE / PMZG.

[0027] Figure 2 The rheological properties of the hydrogel are shown in the figure, where: a is the frequency scan curve (storage modulus G′ and loss modulus G″); b is the strain scan curve; and c is the alternating strain scan test of the self-healing process.

[0028] Figure 3 The diagram shows the physical properties of the PQO / HPE / PMZG hydrogel, where: a) shows injectability; b) shows self-healing behavior; c) shows macroscopic adhesion; and d) shows quantitative assessment of adhesion strength (n=3).

[0029] Figure 4 The graph shows the swelling and stability of the hydrogel, where: a represents the quantitative assessment of the swelling rate; and b represents the mass residue rate.

[0030] Figure 5 The graph shows the HPE release curves in the hydrogel, where: a is the release curve under different pH environments (without glucose); b is the release curve under pH 7.4 environment containing 10 mM glucose.

[0031] Figure 6 The images show the in vitro cell compatibility evaluation of the hydrogel, where: a is the cytotoxicity of L929 cells detected by CCK-8 assay (n=3; ***p<0.001, *p<0.05); b is the live / dead staining image of L929 cells.

[0032] Figure 7 The figures show the in vitro antibacterial properties of the hydrogel, where: a is a macroscopic view of the antibacterial effect; b is the bactericidal rate against Staphylococcus aureus (n=3; **p<0.01); c is the bactericidal rate against Escherichia coli (n=3; **p<0.01, *p<0.05); and d is the bactericidal rate against methicillin-resistant Staphylococcus aureus (MRSA; n=3; ***p<0.001, *p<0.05).

[0033] Figure 8 The graphs show the evaluation effect of PQO / HPE / PMZG hydrogel on wound healing in diabetic MRSA infection (mouse model), where: a is a wound photograph at different time points; b is a statistical graph of wound closure area; and c is a statistical graph of wound healing rate.

[0034] Figure 9 The images show hematoxylin-eosin (H&E) staining of tissue sections on postoperative day 7, where: a is the H&E staining image; b is the quantitative analysis of epidermal thickness (n=3; ***p<0.001, **p<0.01); c is the quantitative assessment of inflammatory cell infiltration (n=3; ***p<0.001).

[0035] Figure 10 Masson staining images of tissue sections on postoperative day 7, where: a is Masson staining image; b is collagen deposition assessment image (n=3; ***p<0.001, **p<0.01). Detailed Implementation

[0036] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings. Unless otherwise specified, the raw materials used in the following embodiments are commercially available; the experimental methods described are conventional methods in the art. Example 1

[0037] This embodiment provides a method for preparing a basic smart responsive composite hydrogel dressing (PQO), specifically including the following steps: S1, 1.00 g of chitosan (CS) was dissolved in 100 mL of 1.5% (v / v) acetic acid aqueous solution and stirred until completely dissolved. Then, 0.38 g of trimethylammonium chloride (GTMAC) was added, and the mixture was stirred continuously at 60 °C for 24 h to obtain a quaternized chitosan (QCS) solution. After the solution cooled to room temperature, 0.43 g of L-arginine (L-Arg) was added, followed by 0.57 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) and 0.34 g of N-hydroxysuccinimide (NHS), and the mixture was stirred thoroughly. After the reaction was complete, the mixture was placed in a dialysis bag with a molecular weight cutoff (MWCO) of 3500 Da and dialyzed in deionized water for 72 h, with the dialysate changed every 8 h. After dialysis, the product was freeze-dried to obtain a grayish-white solid, namely L-Arg-grafted QCS (LQ).

[0038] S2, 1.00 g of sodium alginate (SA) was completely dissolved in 100 mL of deionized water. Separately, 0.86 g of sodium periodate (NaIO4) was dissolved in 10 mL of deionized water and slowly added dropwise to the SA solution. The reaction system was magnetically stirred for 6 h at room temperature (25℃) in the dark. After the reaction was complete, 4 mL of ethylene glycol was added to terminate the reaction, and stirring was continued for 30 min. The resulting reaction solution was dialyzed in deionized water for 72 h using a dialysis bag with a MWCO of 3500 Da (water changed every 8 h). The dialysate was freeze-dried to obtain OSA.

[0039] S3, dissolve 0.50 g of the above OSA in 50 mL of PBS buffer (pH=7.4); separately dissolve 0.68 g of 3-aminophenylboronic acid (3-APBA) in 10 mL of deionized water, and slowly add the solution to the OSA solution while stirring. Adjust the pH of the mixture to 6.5-7.0, and react continuously with stirring at room temperature in the dark for 24 h. After the reaction is complete, transfer the system to a dialysis bag with a MWCO of 3500 Da, and dialyze in deionized water for 72 h (changing the water every 8 h). The dialysate is freeze-dried to obtain a grayish-white solid, namely phenylboronic acid-functionalized OSA (P-OSA).

[0040] S4. Weigh 10 g of polyvinyl alcohol (PVA) powder and add it to 50 mL of deionized water. Stir in a 90℃ water bath until completely dissolved. Separately, dissolve 1 g of LQ in 50 mL of deionized water. Mix the two solutions in equal volumes and stir thoroughly until homogeneous to obtain a PQ solution. Separately, dissolve 10 g of P-OSA in 100 mL of deionized water. Mix the P-OSA solution and PQ solution at a volume ratio of 1:1 and stir for a few seconds (within 10 seconds). The system rapidly cross-links to obtain hydrogel PQO. Example 2

[0041] This embodiment provides a method for preparing a drug-loaded smart responsive composite hydrogel dressing (PQO / HPE), specifically including the following steps: Steps S1 to S3 are the same as steps S1 to S3 in Embodiment 1.

[0042] S4. Weigh 10 g of PVA powder and add it to 50 mL of deionized water. Stir in a 90℃ water bath until completely dissolved. Separately, dissolve 1 g of LQ in 50 mL of deionized water. Mix the two solutions in equal volumes and stir thoroughly until homogeneous to obtain a PQ solution. Add 0.5 g of honeysuckle extract (HPE) to the PQ solution and mix thoroughly to obtain a PQ / HPE solution. Separately, dissolve 10 g of P-OSA in 100 mL of deionized water. Mix the P-OSA solution and the PQ / HPE solution at a volume ratio of 1:1. After stirring for a few seconds (within 10 seconds), the system rapidly cross-links to obtain the hydrogel PQO / HPE. Example 3

[0043] This embodiment provides a method for preparing a fully functional intelligent responsive composite hydrogel dressing (PQO / HPE / PMZG), specifically including the following steps: Steps S1 to S3 are the same as steps S1 to S3 in Embodiment 1.

[0044] S4. Weigh 2 g of MoS2 powder and rapidly disperse it in 200 mL of 40% (v / v) ethanol solution. Sonicate for 2 h to achieve initial exfoliation. Then centrifuge at 10000 rpm for 15 min, collect the precipitate, which is the exfoliated MoS2 nanosheets, and freeze-dry for later use.

[0045] S5, 500 mg of the above MoS2 nanosheets were placed in an ice-water bath and ultrasonically dispersed in an appropriate amount of deionized water to form a uniform dispersion. Separately, 1 g of dopamine hydrochloride (DA·HCl) was dissolved in 10 mL of Tris-HCl buffer. Under continuous vigorous stirring and in the dark, the DA solution was added dropwise to the MoS2 nanosheet dispersion. The reaction was then continued for 12 h at room temperature in the dark. During this period, the solution gradually turned dark black, indicating that PDA had successfully polymerized in situ and coated the surface of the MoS2 nanosheets. After the reaction was complete, the precipitate was collected by centrifugation at 10000 rpm for 20 min and washed three times with deionized water. The obtained product was freeze-dried to obtain MoS2@PDA.

[0046] S6. Disperse 800 mg of the above MoS2@PDA in an ice-water bath. Separately, dissolve 1.76 g of zinc acetate dihydrate in 10 mL of deionized water and slowly add it dropwise to the MoS2@PDA dispersion under vigorous stirring. Continue stirring in the dark for 2 h to allow the Zn to settle. 2+ The Zn nanoparticles were fully adsorbed onto the PDA surface. Subsequently, the pH of the mixture was slowly adjusted to 10.0, and the mixture was kept at a constant temperature of 60°C for 4 hours to promote Zn adsorption. 2+ZnO nanoparticles were generated by hydrolysis under alkaline conditions and anchored on the MoS2@PDA surface. 1 g of glucose oxidase (GOx) was added simultaneously during the reaction. After the reaction, the black precipitate was collected by centrifugation at 10000 rpm for 15 min and washed three times alternately with deionized water and anhydrous ethanol. The final product was freeze-dried to obtain PMZG.

[0047] S7. Weigh 10 g of PVA powder and add it to 50 mL of deionized water. Stir in a 90℃ water bath until completely dissolved. Separately, dissolve 1 g of LQ in 50 mL of deionized water. Mix the two solutions in equal volumes and stir thoroughly until homogeneous to obtain a white PQ solution. Add 0.5 g of HPE to the PQ solution and mix thoroughly to obtain a brown PQ / HPE solution. Add 0.2 g of PMZG to the PQ / HPE solution and mix thoroughly to obtain a black PQ / HPE / PMZG solution. Separately, dissolve 10 g of P-OSA in 100 mL of deionized water. Mix the P-OSA solution with the PQ / HPE / PMZG solution at a volume ratio of 1:1. After stirring for a few seconds (within 10 seconds), the system rapidly cross-links to obtain a hydrogel PQO / HPE / PMZG, as shown below. Figure 1 As shown in a.

[0048] The hydrogels prepared in Examples 1-3 above were subjected to the following performance tests.

[0049] 1. Structural characterization of synthetic materials and hydrogels The structures of SA, OSA, P-OSA, CS, QCS, L-Arg, and LQ were confirmed using Fourier transform infrared spectroscopy (FTIR). The microstructure and porous structure of lyophilized PQO, PQO / HPE, and PQO / HPE / PMZG hydrogels were observed under a scanning electron microscope (SEM) after gold sputtering.

[0050] The results showed that, compared with SA, OSA was at 1730 cm -1 The characteristic absorption peak of the aldehyde group (-CHO) appears at ( ). Figure 1 b) The intensity of this peak decreased in the P-OSA spectrum and reached 1360 cm⁻¹. -1 The characteristic absorption peak of borate ester bond appears at 1630 cm⁻¹. -1 The presence of a C=C stretching vibration peak on the benzene ring indicates that phenylboronic acid has been successfully grafted into the OSA framework. The QCS peak is at 1565 cm⁻¹. -1 Quaternary ammonium groups (-N) are present at the position. + Typical absorption peaks of (CH3)3); LQ spectrum at 1650 cm⁻¹ -1The presence of a newly added characteristic absorption peak of amide bonds (-CO-NH-) confirms that L-Arg was successfully grafted onto QCS via amidation. SEM images show that all three hydrogels possess a highly interconnected three-dimensional porous network structure. With the introduction of PMZG nanosheets, the crosslinking density of the hydrogels increases from PQO→PQO / HPE→PQO / HPE / PMZG, manifested as a decrease in average pore size, thickening of pore walls, and a more compact overall structure. Furthermore, elemental distribution analysis of PQO / HPE / PMZG shows that C, Mo, S, and Zn elements are uniformly distributed in the hydrogel network, confirming the successful incorporation of PMZG. Figure 1 c).

[0051] 2. Rheological properties and gel dynamics The rheological properties of the hydrogel were evaluated using a rotational rheometer. The evolution of the storage modulus G′ and loss modulus G″ over 0–1200 s was monitored using time-scan tests (fixed strain 1%, angular frequency 10 rad / s) to determine the gelation time. The linear viscoelastic region and critical failure strain were determined using strain-scan tests (strain range 0.1%–1000% at a constant angular frequency). The recoverability of the structure was further evaluated using a three-stage oscillatory strain-cycle test: alternating small strain (1%) and large strain (1000%) for 30 s induced reversible network failure and its recovery ability was monitored; subsequently, the recovery of G′ over time (within 700 s) was continuously monitored at small strain (1%) to quantitatively evaluate the self-healing kinetics of the hydrogel.

[0052] Rheological results show that G′ is greater than G″ in all hydrogels, and the two show a clear tendency to separate, which is consistent with the behavior of typical elastic solids. Figure 2 a). From PQO to PQO / HPE / PMZG, the G′ value increases, confirming that PMZG nanosheets, as highly efficient crosslinking nodes, effectively enhance network rigidity and crosslinking density. The PQO / HPE / PMZG hydrogel exhibits excellent strain tolerance, and its gel structure remains intact even under strains up to 511%. Figure 2 b). In alternating strain scanning, the hydrogel rapidly recovered its initial modulus within 30 s after experiencing a large strain failure of 1000%. Figure 2 c), which is attributed to the reversible breaking and recombination of dynamic borate ester bonds.

[0053] 3. Self-healing, injectable, and adhesive properties Qualitative evaluation was conducted through macroscopic experiments. Self-healing test: PQO / HPE / PMZG was cut vertically along its center, and the fresh cut surfaces were immediately and tightly fitted together. After standing for 30 seconds, a tensile test was performed to assess the recovery of mechanical strength at the interface. Injectability test: The PQO / HPE / PMZG precursor solution was loaded into a 5 mL medical syringe and extruded through a needle immersed in PBS at a constant rate of 0.5 mL / min. The ability to be successfully extruded while maintaining structural integrity was observed.

[0054] Two methods were used to evaluate the adhesion strength: (1) PQO / HPE / PMZG was applied to the metacarpophalangeal joint of the index finger, and the interface debonding behavior was recorded by camera during repeated flexion and extension movements; (2) Two pieces of pretreated pigskin with a size of 25×75 mm were taken, and the hydrogel was sandwiched to form a 10×10 mm bonding area. After pre-compression, a tensile testing machine was used to stretch the area at a speed of 10 mm / min, and the maximum shear stress was used as the adhesion strength.

[0055] The results are as follows Figure 3 As shown, PQO / HPE / PMZG, after being cut, achieves complete interfacial fusion within 30 seconds at room temperature and can withstand significant stretching without breaking, attributed to the rapid recoordination of dynamic borate ester bonds between the cross-sections. All precursor solutions can be smoothly extruded through a needle and immediately recover their gel morphology in the receiver dish, demonstrating their excellent injectability. Figure 3 (b) Suitable for filling irregular wounds. When PQO / HPE / PMZG is applied to finger joints, it maintains firm adhesion during repeated flexion and extension. Figure 3 c), and its adhesion strength can reach 28.8 kPa, which is much higher than that of PQO and PQO / HPE (p<0.001); Figure 3 d) 4. Swelling properties and in vitro degradation behavior The precisely weighed lyophilized hydrogel (W0) was immersed in PBS (37°C), removed at a predetermined time point, and its surface moisture was gently wiped off with filter paper before weighing (W). t The swelling ratio is calculated according to formula (1): (1) The sample with an initial dry weight of W0 was incubated in PBS, removed at a predetermined time point, thoroughly rinsed with PBS, and then freeze-dried. The residual dry weight (W0) was then measured. t The residual mass rate is calculated according to formula (2): (2) The swelling results showed that the three groups of hydrogels rapidly absorbed water, which helped maintain structural stability while absorbing exudate. Figure 4a). In vitro degradation curves showed that the introduction of PMZG slowed down the degradation rate, which is beneficial for the dressing to provide long-term protection during the critical period of wound healing. Figure 4 b).

[0056] 5. Drug release behavior Using HPE as a drug model, in vitro drug release studies were conducted using the dialysis bag method. A hydrogel containing a known amount of HPE (50 mg) was placed in a pretreated dialysis bag (MWCO: 3500 Da). The dialysis bag was then immersed in 50 mL of different release media, including glucose-free PBS, glucose-free acetate buffer (pH 5.0), and PBS containing 10 mM glucose, and incubated at 37°C and 100 rpm. For the photothermal group, 808 nm near-infrared light (NIR, power density 1.0 W / cm²) was applied before predetermined time points. 2 Irradiate the sample for 10 min. At each of the set time points (0, 2, 6, 12, and 24 h), take 1 mL of release medium and immediately replenish with an equal volume of fresh release medium preheated to 37°C to maintain a constant volume. Measure the absorbance (OD) at 327 nm using a UV-Vis spectrophotometer. 327 The cumulative drug release is calculated based on a pre-constructed standard curve.

[0057] Release kinetic results ( Figure 5 The results show that the hydrogel system possesses multiple intelligent response characteristics: it exhibits sustained-release behavior under physiological conditions (pH 7.4); the release rate is accelerated in a slightly acidic environment (pH 5.0); when glucose is present in the system, GOx catalyzes the formation of gluconic acid and H2O2, triggering the breaking of ROS responsive chemical bonds, thereby accelerating drug release; under NIR irradiation, the photothermal effect further promotes network structure relaxation and drug diffusion, confirming its spatiotemporally controllable on-demand release capability.

[0058] 6. Cell compatibility evaluation The cell compatibility of the hydrogel was assessed using L929 mouse fibroblasts. After co-culturing the hydrogel extract with L929 cells for 24 h, cell viability was detected using the CCK-8 assay, and OD was measured using a microplate reader. 450 Cell viability was calculated. Simultaneously, dual fluorescence staining of live and dead cells was performed: live cells were labeled with calcein-AM (green fluorescence), and dead cells were labeled with propidium iodide (PI) (red fluorescence). Cell morphology, adhesion, and viability were observed using a fluorescence microscope.

[0059] CCK-8 quantitative analysis showed that the cell viability in each experimental group was higher than 95%. Figure 6 a) indicates that the hydrogel extract has no significant cytotoxicity. Live / dead staining results further confirm this.Figure 6 (b) In the culture system containing hydrogel extract, the cells adhered evenly, stretched out in shape, and had clear outlines. The vast majority of cells showed green fluorescence (live cells), with only occasional sporadic red fluorescence signals (dead cells), which fully verified the excellent cell compatibility of the material.

[0060] 7. In vitro antibacterial test Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA), and Escherichia coli were selected as representative strains to evaluate the antibacterial properties of the hydrogel.

[0061] Hydrogel samples were mixed with bacterial suspension (1×10⁻⁶) 8 CFU / mL was placed together with PBS containing 10 mM glucose and incubated at 37°C and 100 rpm for 4 h. After incubation, the mixture was serially diluted 10-fold, and the appropriate dilutions were plated onto nutrient agar plates. After incubation at 37°C upside down for 24 h, the viable bacterial count was determined by plate count. To investigate the synergistic enhancement of antibacterial effect by NIR irradiation, laser (1.0 W / cm²) was applied at the beginning of co-incubation. 2 Irradiate for 10 minutes. The bacterial sterilization rate is calculated according to formula (3): (3) Where M0 represents the number of bacterial colonies in the blank group bacterial suspension, and M1 represents the number of bacterial colonies in the hydrogel-treated group.

[0062] like Figure 7 As shown, the results indicate that PQO / HPE / PMZG achieved a bactericidal rate exceeding 99.7% against all three bacteria. With NIR synergy, the bactericidal rate reached 100%, highlighting the synergistic advantage of the dual-modal antibacterial mechanism of "cascade enzyme catalysis of reactive oxygen species + photothermal effect".

[0063] 8. Evaluation of wound healing in vivo due to infection A diabetic mouse model of full-thickness skin defect complicated with MRSA infection was established and divided into five groups: commercial dressing control group, PQO group, PQO / HPE group, PQO / HPE / PMZG group, and PQO / HPE / PMZG+NIR group (1.0 W / cm²). 2 Irradiation lasted 10 min. Wound changes were photographed and recorded on days 0, 3, 7, and 14, and the wound closure rate was calculated. Animals were sacrificed on postoperative day 7, and wound tissue samples were collected. Hematoxylin-eosin (H&E) staining was used to assess tissue morphology, inflammatory cell infiltration, granulation tissue formation, and epithelial regeneration; Masson staining was used to observe collagen deposition and arrangement.

[0064] like Figure 8As shown, in vivo experimental results indicate that all hydrogel groups are superior to the commercial dressing group (Tegaderm). TM This treatment effectively promoted the repair process of infectious diabetic wounds. Wounds treated with PQO / HPE / PMZG+NIR achieved near-complete closure by day 14. H&E staining results on day 7 post-treatment showed that, compared to the commercial dressing group, the residual wound area in the PQO / HPE / PMZG+NIR group was reduced by 40.1% (p<0.001). Figure 9 (a and b) indicate that the hydrogel can achieve efficient epithelial regeneration. Furthermore, the inflammatory cell infiltration in the dermis of this group was significantly reduced, only 15.7% of that in the control group (p<0.001). Figure 9 c), suggesting a regulatory effect on inflammation. Similarly, Masson staining on day 7 showed that PQO / HPE / PMZG+NIR had the highest collagen deposition rate (292.0%, p<0.001); Figure 10 Furthermore, the collagen fibers are arranged neatly and densely.

[0065] Examples 1-3 above correspond to different functional levels of the progressive design of this invention. Example 1 provides a basic dynamic covalent network (PQO), endowing the hydrogel with injectability and self-healing capabilities; Example 2 introduces honeysuckle extract (HPE) into the basic network to enhance anti-inflammatory and antioxidant functions; Example 3 further integrates a multifunctional nanoenzyme complex (PMZG) to achieve active therapeutic capabilities such as cascade catalytic antibacterial activity, photothermal response, and angiogenesis promotion. In clinical applications, the hydrogel formulation of the required functional level can be flexibly selected or customized according to the degree of wound infection.

[0066] It should be noted that the above embodiments are only used to illustrate the present invention, but the present invention is not limited to the above embodiments. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention shall fall within the protection scope of the present invention.

Claims

1. A method for preparing a smart responsive composite hydrogel dressing, characterized in that: Includes the following steps: Step 1: Quaternize chitosan to obtain quaternized chitosan, then graft L-arginine onto it in the presence of EDC·HCl and NHS, and obtain LQ by dialysis and lyophilization. Step 2: Sodium alginate is oxidized to obtain sodium alginate oxide, which is then reacted with 3-aminophenylboronic acid, and P-OSA is obtained by dialysis and freeze-drying. Step 3: Molybdenum disulfide powder is peeled into nanosheets, which are then coated with polydopamine, loaded with nano-zinc oxide and glucose oxidase in sequence. After centrifugation, washing and freeze-drying, PMZG is obtained. Step 4: Mix polyvinyl alcohol aqueous solution and LQ aqueous solution evenly, and selectively add honeysuckle extract and / or PMZG to obtain precursor mixture; Step 5: Mix the precursor mixture from Step 4 with the P-OSA aqueous solution at a volume ratio of 1:1, and form a hydrogel through a cross-linking reaction at room temperature.

2. The preparation method according to claim 1, characterized in that: The preparation of LQ in step one includes: dissolving chitosan in a 1.5% (v / v) aqueous acetic acid solution, adding trimethylammonium chloride, reacting at 60°C for 24 h to obtain quaternized chitosan; after cooling, adding L-arginine, then adding EDC·HCl and NHS, stirring the reaction, dialyzing for 72 h, and lyophilizing.

3. The preparation method according to claim 1, characterized in that: The preparation of P-OSA in step two includes: dissolving sodium alginate in deionized water, slowly adding sodium periodate, stirring at room temperature in the dark for 6 hours, adding ethylene glycol to terminate the reaction, dialyzing and lyophilizing to obtain oxidized sodium alginate; dissolving oxidized sodium alginate in PBS buffer (pH=7.4), adding 3-aminophenylboronic acid, adjusting the pH to 6.5-7.0, stirring in the dark for 24 hours, dialyzing and lyophilizing to obtain P-OSA.

4. The preparation method according to claim 1, characterized in that: The preparation of PMZG in step three includes: ultrasonically exfoliating molybdenum disulfide powder in a 40% (v / v) ethanol solution, collecting molybdenum disulfide nanosheets by centrifugation; dispersing the molybdenum disulfide nanosheets in an ice-water bath, adding Tris-HCl buffer solution of dopamine hydrochloride, stirring in the dark for 12 h, centrifuging, washing and lyophilizing to obtain MoS2@PDA; dispersing MoS2@PDA in an ice-water bath, adding zinc acetate dihydrate, stirring and adsorbing, adjusting the pH to 10.0, reacting at 60℃ for 4 h, simultaneously adding glucose oxidase, centrifuging, washing and lyophilizing to obtain PMZG.

5. The preparation method according to claim 1, characterized in that: In step four, the concentration of the polyvinyl alcohol aqueous solution is 20% (w / v), the concentration of the LQ aqueous solution is 2% (w / v), the final concentration of the honeysuckle extract is 0.5% (w / v), and the final concentration of PMZG is 0.2% (w / v); the mass ratio of honeysuckle extract to PMZG is 5:

2.

6. The preparation method according to claim 1, characterized in that: The cross-linking reaction in step five is completed within 10 seconds, and the resulting hydrogel has a dual dynamic covalent cross-linking network of borate ester bonds and Schiff base bonds.

7. The preparation method according to claim 1, characterized in that: In step four, honeysuckle extract and / or PMZG may be selectively added or not added to obtain basic hydrogels, drug-loaded hydrogels, or multifunctional hydrogels, respectively.

8. A smart responsive composite hydrogel dressing prepared by the preparation method according to any one of claims 1-7.

9. The intelligent responsive composite hydrogel dressing according to claim 8, characterized in that: The dynamic covalent cross-linked network of the hydrogel dressing is formed by cross-linking polyvinyl alcohol, arginine-grafted quaternized chitosan and phenylboronic acid-functionalized sodium alginate through dynamic borate ester bonds and Schiff base bonds. The hydrogel dressing also contains honeysuckle extract and multifunctional nanoenzyme complex dispersed in it.

10. The use of the smart responsive composite hydrogel dressing of claim 8 in the preparation of a medicament or medical device for treating chronically infected wounds, diabetic foot ulcers, burns, or wounds infected by drug-resistant bacteria.

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