Preparation method and application of anti-inflammatory antioxidant hydrogel

Abstract

The application relates to the technical field of hydrogels, in particular to a preparation method and application of an anti-inflammatory and anti-oxidation hydrogel, wherein aldehyde groups are introduced into hyaluronic acid by oxidation modification to obtain oxidized hyaluronic acid, and then pyrrole quinoline quinone is covalently grafted onto epsilon-polylysine to obtain a modified polymer; the two raw materials are respectively prepared into precursor liquids and then uniformly mixed, and the target hydrogel is prepared by in-situ crosslinking through a dynamic Schiff base reaction under physiological conditions; the hydrogel forms a stable three-dimensional crosslinking network, has good biocompatibility and mechanical properties, and can realize anti-inflammatory, anti-oxidation and chronic wound healing promotion effects. The preparation process is simple, the gel forming condition is mild, the obtained hydrogel has excellent mechanical properties and biocompatibility, can long-acting exert anti-inflammatory and anti-oxidation effects, and can effectively promote the healing of a diabetic chronic wound.

Description

Technical Field

[0001] This invention relates to the field of hydrogel technology, specifically to a method for preparing and applying an anti-inflammatory and antioxidant hydrogel. Background Technology

[0002] Chronic skin wounds in diabetic patients are one of the most common and serious complications of diabetes. Long-term hyperglycemia leads to microvascular complications, persistent and uncontrolled inflammatory responses, and excessive accumulation of reactive oxygen species (ROS), severely hindering wound healing. This manifests as persistent wounds, susceptibility to secondary drug-resistant bacterial infections, and even serious consequences such as amputation and sepsis, causing immense physical suffering and financial burden on patients. It remains a challenging problem in clinical wound repair that urgently needs to be addressed. Currently used traditional dressings such as medical gauze, sponges, and polymer bandages only provide basic physical isolation and hemostasis, failing to maintain the moist microenvironment required for wound healing. They are ill-suited to responding to the unique oxidative stress and inflammatory microenvironment of chronic wounds, and their antibacterial and anti-inflammatory functions are limited, failing to break the vicious cycle of "infection-inflammation-oxidative stress-delayed healing" in chronic wounds.

[0003] In recent years, hydrogel materials have become a research hotspot in wound repair due to their three-dimensional network structure, which is highly similar to the extracellular matrix, and their excellent moisturizing and biocompatibility. Among them, hydrogels based on natural polysaccharides and peptides are gradually replacing traditional synthetic polymer hydrogels due to their advantages of degradability and high biosafety. Existing hydrogels based on ε-polylysine (EPL) and hyaluronic acid (HA) can achieve basic moisturizing and antibacterial functions, but they still have obvious shortcomings: HA is easily degraded rapidly by hyaluronidase in vivo, leading to premature disintegration of the hydrogel structure and a short effective duration; pure EPL-based hydrogels have weak mechanical properties and are difficult to adapt to the complex mechanical environment of wounds; moreover, most existing systems only focus on a single antibacterial function and lack synergistic effects such as anti-inflammatory, antioxidant, angiogenesis-promoting, and tissue regeneration-promoting effects, and cannot specifically regulate the core pathological microenvironment of diabetic chronic wounds, resulting in very limited repair effects on infected and refractory diabetic wounds. Furthermore, existing antioxidant active ingredients are mostly introduced into hydrogel systems through physical doping, which leads to problems such as severe burst release, short duration of action, and easy disruption of the gel network structure, further limiting their clinical translational applications. Therefore, developing an in-situ cross-linked hydrogel with mild gelation conditions, excellent biocompatibility, suitable mechanical properties, and long-lasting anti-inflammatory, antioxidant, and regenerative functions has become an urgent need in the field of diabetic chronic wound repair. Summary of the Invention

[0004] (a) Technical problems to be solved To address the shortcomings of existing technologies, this invention provides a method for preparing and applying an anti-inflammatory and antioxidant hydrogel.

[0005] (II) Technical Solution A method for preparing an anti-inflammatory and antioxidant hydrogel, wherein the hydrogel is an ε-polylysine-pyrroloquinoline quinone / oxidized hyaluronic acid EPL-PQQ / OHA in-situ crosslinked hydrogel, comprising the following steps: Preparation of S1 Oxidized Hyaluronic Acid (OHA): Hyaluronic acid (HA) with a weight-average molecular weight of 800-1500 kDa and sodium periodate were added at a molar ratio of 1:(0.8-1.2). Ultrapure water was added and the mixture was stirred at room temperature in the dark for 4-6 hours. Ethylene glycol was added to quench the remaining sodium periodate and the reaction was continued for 1-2 hours. The product was collected by salting out and pre-cooling anhydrous ethanol precipitation. The product was dissolved in ultrapure water and dialyzed for 3-4 days to remove small molecule impurities. After freeze-drying, oxidized hyaluronic acid (OHA) with an aldehyde degree of 30%-50% was obtained. Preparation of S2 EPL-PQQ grafted polymer: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) activation condensation system were used. ε-polylysine (EPL) with a weight-average molecular weight of 2000-5000 Da and pyrroloquinoline quinone (PQQ) were added at a molar ratio of 1:(0.05-0.15). PQQ was first dissolved in MES buffer, and EDC and NHS were added for activation at room temperature in the dark for 20-40 min. The molar ratio of PQQ, EDC, and NHS was 1:(1.2-1.5):(1.5-2.0). Then, EPL was added and the reaction was stirred at room temperature in the dark for 20-28 h. After the reaction, the mixture was purified by dialyzing for 3-4 days and then lyophilized to obtain EPL-PQQ covalently grafted polymer with a PQQ grafting rate of 5%-12%. Preparation of S3 hydrogel precursor solution: The OHA obtained in S1 was aseptically dissolved in PBS buffer at pH 7.2-7.4 to prepare an OHA precursor solution with a mass fraction of 16%-24%; the EPL-PQQ obtained in S2 was aseptically dissolved in the same batch of PBS buffer to prepare an EPL-PQQ precursor solution with a mass fraction of 10%-22%. S4 Physiological Conditions In-situ Crosslinking: OHA precursor solution and EPL-PQQ precursor solution were aseptically mixed at a volume ratio of 1:(0.8~1.2) and crosslinked at a physiological temperature of 35~39℃ through a dynamic Schiff base reaction of aldehyde and amino groups for 5~15 min to obtain the anti-inflammatory and antioxidant hydrogel.

[0006] Preferably, the molar ratio of HA to sodium periodate in S1 is 1:1, the reaction time at room temperature in the dark is 5 hours, and the molecular weight cutoff of the dialysis bag used for dialysis is 8000~14000 Da.

[0007] Preferably, the molar ratio of EPL to PQQ in S2 is 1:0.1, the molar ratio of PQQ, EDC, and NHS is 1:1.3:1.6, the activation time is 30 min, and the stirring reaction time at room temperature is 24 h.

[0008] Preferably, in S3, the mass fraction of the OHA precursor solution is 20%, and the mass fraction of the EPL-PQQ precursor solution is 12%, 16%, or 20%; in S4, the mixing volume ratio of the OHA precursor solution and the EPL-PQQ precursor solution is 1:1, and the final mass fraction of EPL-PQQ in the mixed system is 6%, 8%, or 10%.

[0009] Preferably, the hydrogel prepared by S4 exhibits a storage modulus recovery rate of ≥65% after three large strain-small strain loading-unloading cycles at a frequency of 1Hz and a temperature of 25℃, demonstrating shear thinning characteristics and rapid self-healing performance.

[0010] Preferably, the extract of the hydrogel at a concentration of 25 mg / mL has a significant protective effect against H2O2-induced oxidative damage to L929 cells, reducing intracellular ROS levels by more than 40%, and downregulating the expression of intracellular Sod2 and Cat antioxidant stress-related genes.

[0011] Preferably, the method for preparing the anti-inflammatory and antioxidant hydrogel yields an anti-inflammatory and antioxidant hydrogel, wherein the hydrogel is formed by cross-linking EPL-PQQ grafted polymer and OHA through dynamic Schiff base covalent bonds to form a three-dimensional porous network structure, and simultaneously possesses moisturizing and water-locking, exudate absorption, self-repair, anti-inflammatory, antioxidant and skin tissue regeneration promotion properties.

[0012] The application of the aforementioned anti-inflammatory and antioxidant hydrogel in the preparation of medical wound repair dressings.

[0013] Preferably, the medical wound repair dressing is a special repair dressing for diabetic chronic skin wounds and bacterial infected skin wounds. The dressing can form gel in situ under the physiological environment of the wound, and simultaneously achieve efficient absorption of wound exudate, regulation of the oxidative stress microenvironment, inhibition of inflammatory response, and promotion of granulation tissue formation, collagen deposition and epithelial regeneration.

[0014] (iii) Beneficial technical effects Compared with existing technologies, the beneficial effects of this invention are: Oxidative modification of hyaluronic acid with aldehyde functionalization effectively solves the problems of easy degradation and poor gel structure stability of natural hyaluronic acid. At the same time, pyrroloquinoline quinone is fixed to the ε-polylysine molecular chain through covalent grafting, and then in-situ cross-linking with oxidized hyaluronic acid is achieved through dynamic Schiff base reaction, constructing a stable and dense three-dimensional network structure. This overcomes the limitation of weak mechanical properties of pure ε-polylysine hydrogels, endowing the hydrogel with excellent mechanical adaptability, good swelling properties and rapid self-healing ability. It can maintain structural stability for a long time in the complex environment of wounds, while efficiently absorbing wound exudate and continuously maintaining the moist microenvironment required for wound healing.

[0015] Stable loading of active ingredients is achieved through covalent grafting, avoiding the burst release problem caused by physical doping. It can exert synergistic biological functions for a long time, achieving broad-spectrum antibacterial effects, avoiding the risk of infection by drug-resistant bacteria, effectively clearing excess reactive oxygen species in the wound, regulating the local oxidative stress microenvironment, significantly inhibiting the overexpression of pro-inflammatory factors, alleviating the persistent inflammatory response of the wound, breaking the vicious cycle of refractory wounds, effectively promoting fibroblast proliferation, granulation tissue formation and epithelial regeneration, and has excellent healing effects on chronic infectious wounds of diabetic patients. Attached Figure Description

[0016] Figure 1 This is a flowchart of a method for preparing an anti-inflammatory and antioxidant hydrogel disclosed in this invention; Figure 2 These are the Fourier transform infrared spectroscopy test results of the OHA sample of this invention; Figure 3 These are the Fourier transform infrared spectroscopy test results of the EPL-PQQ sample of this invention; Figure 4 These are the angular frequency scan test results from Examples 1-3; Figure 5 These are the results of the three-loop loading-unloading self-repair test in Examples 1-3; Figure 6 The results show the protective effect of the hydrogel in Example 1 against H2O2-induced oxidative damage in L929 cells. Figure 7 This is the result of the test on the protective effect of the hydrogel against H2O2-induced oxidative damage in L929 cells in Example 2; Figure 8 This is the result of the test on the protective effect of the hydrogel against H2O2-induced oxidative damage in L929 cells in Example 3; Figure 9 The results of the hydrogel extracts from Examples 1-3 inhibiting LPS-induced TNF-α pro-inflammatory factors in macrophages; Figure 10 The results of the hydrogel extracts from Examples 1-3 inhibiting LPS-induced IL-6 pro-inflammatory factor in macrophages; Figure 11 The results of the hydrogel in Examples 1-3 show the regulation of the mRNA expression level of antioxidant-related genes. Detailed Implementation

[0017] according to Figures 1 to 4 The specific embodiments of the present invention are as follows: The present invention will be further described in detail below with reference to specific embodiments. The following embodiments are explanations of the present invention, but the present invention is not limited to the following embodiments. Unless otherwise specified, all operations in this embodiment are performed under normal temperature and pressure and in a sterile environment. Unless otherwise specified, all reagents used are of analytical grade or higher, and all cells and experimental animals used comply with relevant ethical requirements.

[0018] I. Main Experimental Materials and Instruments 1. Main experimental materials Hyaluronic acid (HA): weight average molecular weights of 800kDa, 1000kDa, and 1500kDa, pharmaceutical grade, purchased from Bloomage Biotechnology Co., Ltd. ε-polylysine (EPL): weight average molecular weights of 2000 Da, 3000 Da, and 5000 Da, with a purity of ≥95%, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. Pyrroloquinoline quinone (PQQ): Purity ≥98%, purchased from Shanghai Yuanye Biotechnology Co., Ltd.; Sodium periodate, ethylene glycol, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), 2-(N-morpholino)ethanesulfonic acid (MES), anhydrous ethanol, and sodium chloride were all of analytical grade and purchased from Sinopharm Chemical Reagent Co., Ltd. PBS buffer (pH 7.2, pH 7.4), DMEM complete medium, fetal bovine serum, trypsin, CCK-8 kit, Calcein-AM / PI live / dead cell staining kit, ELISA kit (TNF-α, IL-6), RNA extraction kit, reverse transcription kit, and RT-qPCR kit were all purchased from Thermo Fisher Scientific (China) Co., Ltd. L929 mouse fibroblasts, RAW264.7 mouse macrophages, and Staphylococcus aureus were all purchased from the Cell Bank of the Chinese Academy of Sciences Type Culture Collection Committee. SPF-grade GK rats (type 2 diabetes model), weighing 200-250g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., and the animal experiments were approved by the ethics committee.

[0019] 2. Main experimental instruments Freeze dryer (SCIENTZ-18N, Ningbo Xinzhi Biotechnology Co., Ltd.); Fourier transform infrared spectrometer (FT-IR, Nicolet iS50, Thermo Fisher Scientific); Nuclear magnetic resonance spectrometer (1H-NMR, AVANCE III 400MHz, Bruker (Beijing) Technology Co., Ltd.); Rotational rheometer (MCR302, Anton Paar (Shanghai) Trading Co., Ltd.); UV-2600 UV-Vis spectrophotometer (Shimadzu Enterprise Management (China) Co., Ltd.); Microplate reader (Multiskan FC, Thermo Fisher Scientific); Fluorescence inverted microscope (IX73, Olympus (China) Co., Ltd.); Real-time quantitative PCR instrument (QuantStudio 5, Thermo Fisher Scientific); Thermostatic Shaker (THZ-300, Shanghai Yiheng Scientific Instruments Co., Ltd.); Carbon dioxide cell incubator (HERAcell 150i, Thermo Fisher Scientific).

[0020] II. Preparation of core functional raw materials This section describes the reaction principles and preparation process of OHA synthesis, EPL-PQQ grafting modification, and subsequent in-situ hydrogel crosslinking; the schematic diagram of the OHA oxidative synthesis reaction is shown below: (I) Preparation of Oxidized Hyaluronic Acid (OHA) The degree of aldehyde grouping of the OHA prepared in this section can be controlled within the range of 30%-50%. The specific preparation steps are as follows: S1 Weigh 5g of HA with a weight average molecular weight of 1000kDa, add it to 500mL of ultrapure water, stir at room temperature in the dark for 12h until the HA is completely dissolved, and obtain a homogeneous and transparent HA aqueous solution. S2 weigh out sodium periodate solid according to the molar ratio of HA to sodium periodate 1:1, slowly add it to the HA aqueous solution, seal it in the dark, and stir continuously at 300 rpm for 5 hours at room temperature. After the S3 reaction is completed, 5 mL of ethylene glycol is added to the reaction system, and the reaction is stirred for another 1.5 h to quench any unreacted sodium periodate in the system and terminate the oxidation reaction. S4 Add 5g of sodium chloride to the reaction system and stir until it is completely dissolved and salting out is completed. Then slowly add 4°C pre-cooled anhydrous ethanol to the system until a large amount of white flocculent precipitate appears in the system. After standing for 30 minutes, filter and collect the precipitate. S5 The collected precipitate was redissolved in 500 mL of ultrapure water, placed in a dialysis bag with a molecular weight cutoff of 8000-14000 Da, and dialyzed in ultrapure water for 4 days. The dialysis solution was changed every 6 hours to completely remove residual small molecule impurities such as sodium chloride, ethylene glycol, and iodate from the system. After S6 dialysis is completed, the solution in the dialysis bag is collected, pre-frozen, and then freeze-dried in a freeze dryer for 48 hours to obtain white flocculent OHA solid. The solid is sealed and stored in a dry environment at -20℃ for later use.

[0021] Following the same steps described above, the molar ratios of HA and sodium periodate were adjusted to 1:0.8 and 1:1.2, respectively, and the reaction times were 4 h and 6 h, respectively. The molecular weights of HA were 800 kDa and 1500 kDa, respectively, to prepare OHA with different degrees of aldehyde sizing and different molecular weights. The degrees of aldehyde sizing of the obtained OHA were 32%, 45%, and 48%, respectively, as determined by hydroxylamine titration.

[0022] (II) Preparation of EPL-PQQ covalently grafted polymer The PQQ grafting rate of the EPL-PQQ prepared in this section can be controlled within the range of 5%-12%. The specific preparation steps are as follows: S1 Weigh 1 mmol of PQQ and add it to 100 mL of 0.1 mol / L PBS buffer (Ph 7.0). Stir at room temperature in the dark until completely dissolved to obtain a PQQ solution. S2 weighed out solid EDC and solid NHS according to the molar ratio of PQQ, EDC and NHS of 1:1.3:1.5, added them to the PQQ solution, sealed in the dark, and stirred at room temperature for 30 min to obtain the activated PQQ reaction solution. S3 Weigh 10 mmol of EPL with a weight-average molecular weight of 3000 Da according to the molar ratio of EPL to PQQ of 1:0.1, add it to the activated PQQ reaction solution, seal it in the dark, and stir continuously at 300 rpm for 24 h at room temperature. After the S4 reaction is completed, the reaction solution is put into a dialysis bag with a molecular weight cutoff of 3000 Da and dialyzed in ultrapure water for 4 days. The dialysis solution is changed every 6 hours to completely remove unreacted small molecule impurities such as PQQ, EDC, and NHS. After S5 dialysis is completed, the solution in the dialysis bag is collected, pre-frozen, and then freeze-dried in a freeze dryer for 48 hours to obtain a brownish-yellow flocculent EPL-PQQ grafted polymer, which is then sealed and stored in a desiccator for later use.

[0023] Following the same steps described above, the molar ratios of EPL and PQQ were adjusted to 1:0.05 and 1:0.15, respectively, and the molar ratios of PQQ, EDC, and NHS were adjusted to 1:1.2:1.5 and 1:1.5:2.0, respectively. The activation times were 20 min and 40 min, and the reaction times were 20 h and 28 h, respectively. The molecular weights of EPL were 2000 Da and 5000 Da, respectively. EPL-PQQ polymers with different grafting rates were prepared. The PQQ grafting rates of the obtained EPL-PQQ were 5.2%, 8.7%, and 11.6%, respectively, as determined by ultraviolet spectrophotometry.

[0024] III. Preparation Examples of Anti-inflammatory and Antioxidant Hydrogels This section describes a series of hydrogels prepared using precursor solutions of different concentrations. The core principle of their cross-linking and gelation is the dynamic Schiff base reaction between OHA aldehyde groups and EPL-PQQ amino groups, which enables rapid in-situ gelation at physiological temperatures.

[0025] Example 1 The anti-inflammatory and antioxidant hydrogel prepared in this embodiment is denoted as EOP1. The specific preparation steps are as follows: Preparation of S1 precursor solution: Under aseptic conditions, weigh the OHA solid with 45% aldehyde degree prepared above, aseptically dissolve it in PBS buffer at pH 7.4, stir at room temperature until completely dissolved, and prepare a 20% OHA precursor solution. Filter the solution through a 0.22μm sterile membrane for sterilization and use. Under aseptic conditions, weigh the EPL-PQQ solid with 8.7% PQQ grafting rate prepared above, aseptically dissolve it in the same batch of PBS buffer at pH 7.4, stir at room temperature until completely dissolved, and prepare a 12% EPL-PQQ precursor solution. Filter the solution through a 0.22μm sterile membrane for sterilization and use. S2 In-situ Crosslinking: Under sterile conditions, 20% of the above-mentioned OHA precursor solution and 12% of the EPL-PQQ precursor solution were added to a sterile container at a volume ratio of 1:1. The mixture was quickly vortexed for 30 seconds until homogeneous, and then placed in a 37°C constant temperature incubator for static crosslinking for 10 minutes to obtain a transparent and homogeneous anti-inflammatory and antioxidant hydrogel. The final mass fraction of EPL-PQQ in the mixed system was 6%.

[0026] Example 2 The anti-inflammatory and antioxidant hydrogel prepared in this embodiment is denoted as EOP2. The specific preparation steps are as follows: Preparation of S1 precursor solution: Under aseptic conditions, weigh the OHA solid with 45% aldehyde degree prepared above, aseptically dissolve it in PBS buffer at pH 7.4, stir at room temperature until completely dissolved, and prepare an OHA precursor solution with a mass fraction of 20%. Filter the solution through a 0.22μm sterile filter membrane for sterilization and use. Under aseptic conditions, weigh the EPL-PQQ solid with 8.7% PQQ grafting rate prepared above, aseptically dissolve it in PBS buffer at pH 7.4 of the same batch, stir at room temperature until completely dissolved, and prepare an EPL-PQQ precursor solution with a mass fraction of 16%. Filter the solution through a 0.22μm sterile filter membrane for sterilization and use. S2 In-situ Crosslinking: Under sterile conditions, 20% of the above-mentioned OHA precursor solution and 16% of the EPL-PQQ precursor solution were added to a sterile container at a volume ratio of 1:1. The mixture was quickly vortexed for 30 seconds until homogeneous, and then placed in a 37°C constant temperature incubator for static crosslinking for 8 minutes to obtain a transparent and homogeneous anti-inflammatory and antioxidant hydrogel. The final mass fraction of EPL-PQQ in the mixed system was 8%.

[0027] Example 3 The anti-inflammatory and antioxidant hydrogel prepared in this embodiment is denoted as EOP3. The specific preparation steps are as follows: Preparation of S1 precursor solution: Under aseptic conditions, weigh the OHA solid with 45% aldehyde degree prepared above, aseptically dissolve it in PBS buffer at pH 7.4, stir at room temperature until completely dissolved, and prepare a 20% OHA precursor solution. Filter the solution through a 0.22μm sterile membrane for sterilization and use. Under aseptic conditions, weigh the EPL-PQQ solid with 8.7% PQQ grafting rate prepared above, aseptically dissolve it in the same batch of PBS buffer at pH 7.4, stir at room temperature until completely dissolved, and prepare a 20% EPL-PQQ precursor solution. Filter the solution through a 0.22μm sterile membrane for sterilization and use. S2 In-situ Crosslinking: Under sterile conditions, 20% of the above-mentioned OHA precursor solution and 20% of the EPL-PQQ precursor solution were added to a sterile container at a volume ratio of 1:1. The mixture was quickly vortexed for 30 seconds until homogeneous, and then placed in a 37°C constant temperature incubator for static crosslinking for 5 minutes to obtain a transparent and homogeneous anti-inflammatory and antioxidant hydrogel. The final mass fraction of EPL-PQQ in the mixed system was 10%.

[0028] Example 4 The anti-inflammatory and antioxidant hydrogel prepared in this embodiment is prepared by the following specific steps: Preparation of S1 precursor solution: Under aseptic conditions, weigh the OHA solid with an aldehyde degree of 32% prepared above, aseptically dissolve it in PBS buffer at pH 7.2, stir at room temperature until completely dissolved, and prepare an OHA precursor solution with a mass fraction of 16%. Filter the solution through a 0.22μm sterile membrane for sterilization and use. Under aseptic conditions, weigh the EPL-PQQ solid with a PQQ grafting rate of 5.2% prepared above, aseptically dissolve it in PBS buffer at pH 7.2 of the same batch, stir at room temperature until completely dissolved, and prepare an EPL-PQQ precursor solution with a mass fraction of 10%. Filter the solution through a 0.22μm sterile membrane for sterilization and use. S2 In-situ Crosslinking: Under sterile conditions, take 16% of the above-mentioned OHA precursor solution and 10% of the EPL-PQQ precursor solution and add them to a sterile container at a volume ratio of 1:0.8. Vortex mix quickly for 30 seconds until homogeneous, and then place it in a constant temperature environment of 35℃ and let it stand for crosslinking for 15 minutes to obtain a transparent and homogeneous anti-inflammatory and antioxidant hydrogel.

[0029] Example 5 The anti-inflammatory and antioxidant hydrogel prepared in this embodiment is prepared by the following specific steps: Preparation of S1 precursor solution: Under aseptic conditions, weigh the OHA solid with an aldehyde degree of 48% prepared above, aseptically dissolve it in PBS buffer at pH 7.4, stir at room temperature until completely dissolved, and prepare an OHA precursor solution with a mass fraction of 24%. Filter the solution through a 0.22μm sterile filter membrane for sterilization and use. Under aseptic conditions, weigh the EPL-PQQ solid with a PQQ grafting rate of 11.6% prepared above, aseptically dissolve it in PBS buffer at pH 7.4 of the same batch, stir at room temperature until completely dissolved, and prepare an EPL-PQQ precursor solution with a mass fraction of 22%. Filter the solution through a 0.22μm sterile filter membrane for sterilization and use. S2 In-situ Crosslinking: Under sterile conditions, take 24% OHA precursor solution and 22% EPL-PQQ precursor solution and add them to a sterile container at a volume ratio of 1:1.2. Vortex mix quickly for 30 seconds until homogeneous, then place in a constant temperature environment of 39℃ and let stand for 5 minutes to crosslink, to obtain a transparent and homogeneous anti-inflammatory and antioxidant hydrogel.

[0030] IV. Comparative Preparation Comparative Example 1 This comparative example does not introduce PQQ; instead, it directly prepares hydrogels by crosslinking EPL and OHA. The specific steps are as follows: Preparation of S1 precursor solution: Under aseptic conditions, weigh out the same batch of OHA solid with 45% aldehyde degree as in Example 2, aseptically dissolve it in PBS buffer at pH 7.4 to prepare a 20% OHA precursor solution, filter and sterilize for later use; Under aseptic conditions, weigh out the same batch of pure EPL with a weight average molecular weight of 3000 Da as in Example 2, aseptically dissolve it in the same batch of PBS buffer to prepare a 16% EPL precursor solution, filter and sterilize for later use; S2 Crosslinking: Under sterile conditions, 20% OHA precursor solution and 16% EPL precursor solution were mixed at a volume ratio of 1:1. After vortexing to homogenize, the mixture was placed in a 37℃ incubator for crosslinking for 10 min to obtain EPL / OHA hydrogel, which was labeled as the control group EO.

[0031] Comparative Example 2 This comparative example introduces PQQ through physical doping rather than covalent grafting. The specific steps are as follows: Preparation of S1 precursor solution: Under aseptic conditions, weigh out the same batch of OHA solid with 45% aldehyde degree as in Example 2, aseptically dissolve it in PBS buffer at pH 7.4 to prepare a 20% OHA precursor solution, filter and sterilize for later use; Under aseptic conditions, weigh out the same batch of pure EPL with a weight average molecular weight of 3000 Da as in Example 2, and an equimolar amount of PQQ as in EPL-PQQ in Example 2, dissolve them together in the same batch of PBS buffer to prepare a 16% EPL+PQQ mixed precursor solution, filter and sterilize for later use; S2 Crosslinking: Under sterile conditions, 20% of the above-mentioned OHA precursor solution and 16% of the EPL+PQQ mixed precursor solution were mixed at a volume ratio of 1:1. After vortexing and homogenization, the mixture was placed in a 37℃ incubator for crosslinking for 10 min to obtain physically doped PQQ EPL / OHA hydrogel, which was labeled as the control group EO-P.

[0032] Comparative Example 3 This comparative example uses a low-concentration precursor solution, which exceeds the scope of the claims, to prepare the hydrogel. The specific steps are as follows: S1 Precursor Solution Preparation: Under aseptic conditions, weigh out OHA solid and dissolve it in PBS buffer at pH 7.4 to prepare a 10% OHA precursor solution. Filter and sterilize for later use. Under aseptic conditions, weigh out EPL-PQQ solid and dissolve it in the same batch of PBS buffer to prepare a 6% EPL-PQQ precursor solution. Filter and sterilize for later use. S2 cross-linking gel: Under sterile conditions, 10% of the above-mentioned OHA precursor solution and 6% of EPL-PQQ precursor solution were mixed at a volume ratio of 1:1. After vortexing and homogenization, the mixture was placed in a 37°C incubator. After standing for 30 minutes, a stable gel structure could not be formed, and only a viscous liquid was obtained, which could not meet the requirements for use as wound dressing.

[0033] V. Structural Characterization and Performance Testing of Hydrogels This section details the characterization results of the structure, mechanics, and swelling properties of the hydrogels in the examples and comparative examples. See the appendix for details. Figure 2 ; (I) Chemical structure characterization 1. Fourier Transform Infrared Spectroscopy (FT-IR) Measurement Samples of HA, OHA, EPL, and EPL-PQQ were prepared using the potassium bromide pellet method and placed in an FT-IR spectrometer for testing. The scanning range was 4000-400 cm⁻¹, the resolution was 4 cm⁻¹, and the number of scans was 32. The test results are as follows: Figure 2 , 3 The results show that the spectrum of OHA exhibits a characteristic stretching vibration peak of the aldehyde group at 1735 cm⁻¹, proving that HA was successfully oxidized to introduce an aldehyde group; in the spectrum of EPL-PQQ, the characteristic absorption peak of the primary amine at 3400-3300 cm⁻¹ is significantly weakened in intensity and narrowed in shape, and the characteristic peaks of amide I and amide II in the 1650-1500 cm⁻¹ region show obvious shifts and intensity changes, proving that PQQ was successfully covalently grafted onto the EPL molecular chain through an amidation reaction.

[0034] (ii) Rheological property testing The rheological properties of the hydrogels prepared in Examples 1-3 were tested using a rotational rheometer. The test was conducted using a parallel plate fixture with a diameter of 20 mm and a plate spacing of 1 mm, at a test temperature of 25 °C.

[0035] 1. Angular frequency scan test The scanning angular frequency range was set to 0.1-100 rad / s, and the strain was 1%. The storage modulus (G') and loss modulus (G'') of the hydrogels were tested as a function of angular frequency. The test results showed that, for the hydrogels prepared in Examples 1-3, the storage modulus G' was consistently significantly higher than the loss modulus G'' across the entire tested angular frequency range, and both showed an increasing trend with increasing angular frequency. This demonstrates that all three hydrogels successfully formed a continuous and dense three-dimensional cross-linked network structure, possessing stable solid-like elastic characteristics, overcoming the limitation of weak mechanical properties in pure EPL-based hydrogels. Furthermore, the modulus of the hydrogels increased with increasing EPL-PQQ concentration, exhibiting a significant concentration dependence. Example 3 showed the highest G' value and the best structural stability. Specifically... Figure 4 As shown.

[0036] 2. Three-cycle loading-unloading self-healing test Further, a three-cycle rheological test is used to detect the dynamic cross-linked structure, the test adopts a dynamic time scanning mode, and 3 complete loading-unloading cycles are performed continuously, with unified parameters of 1 Hz frequency and 25°C temperature. As shown in Figure 5 , with the alternation of large and small strains, the dynamic covalent bonds enable the modulus to recover multiple times. In the small strain state, G'>G'' in each group indicates a gel state, and G'<G'' in large strain indicates a sol state. After the gel structure is destroyed, it can recover to the gel state. The initial modulus (G') of EOP hydrogel increases significantly with the increase of concentration, showing the order of EOP3>EOP2>EOP1, indicating that the increase of concentration directly strengthens the network structure of the hydrogel. Among them, EOP1 has the best fluidity, G' rises back to about 400 Pa in the recovery stage, and the recovery rate is about 67%. Both EOP2 and EOP3 have better elasticity. The above groups of hydrogels have their own dynamic cross-linking characteristics, which are conducive to their long-term stable existence in complex wound environments. The mechanical properties of the hydrogel show significant concentration dependence, and samples with different final concentrations show differentiated performance advantages, which provide a performance basis for further research on the function of the hydrogel.

[0037] (V) In vitro antioxidant performance test This section presents the in vitro antioxidant performance test results, wherein Figure 6-8 is the test result of the protective effect of the hydrogel on H2O2-induced oxidative damage of L929 cells, Figure 9-10 is the test result of the inhibitory effect of the hydrogel on the expression of pro-inflammatory factors in LPS-induced macrophage RAW 264.7, Figure 11 is the regulation result of the hydrogel on the mRNA expression level of antioxidant-related genes.

[0038] The H2O2-induced oxidative damage model of L929 cells was used to test the antioxidant protection performance of the hydrogel, and the specific steps are as follows: L929 cells in logarithmic growth phase were seeded at a density of 8000 cells / well in 96-well plates and cultured for 12 h until cell adhesion. The original culture medium was then aspirated, and hydrogel extraction medium containing 400 μmol / L H2O2 was added to each well. Pure DMEM medium containing 400 μmol / L H2O2 was used as a model control group. Each group was configured with 6 replicates. After culturing for another 24 h, the intracellular ROS level was detected using the DCFH-DA reactive oxygen species detection kit, followed by microplate reader and fluorescence microscopy. At the same time, the mRNA expression levels of antioxidant-related genes Sod1, Sod2, and Cat were detected by RT-qPCR. The test results showed that, compared with the model control group, the hydrogel extracts of Examples 1-3 could significantly reduce the intracellular ROS level induced by H2O2, and the effect increased with the increase of EPL-PQQ concentration, showing a clear concentration dependence. At the same time, the expression levels of Sod2 and Cat genes in the cells of the hydrogel-treated group decreased significantly, proving that the hydrogel of the present invention can reduce the oxidative stress load of cells by efficiently clearing excess ROS, rather than simply inducing the expression of antioxidant enzymes, and has excellent protective effect on oxidatively damaged cells. It can also specifically regulate the oxidative stress microenvironment of chronic wounds.

[0039] (vi) In vitro anti-inflammatory performance test The anti-inflammatory properties of the hydrogel were tested using an LPS-induced RAW 264.7 macrophage inflammation model. The specific steps are as follows: RAW 264.7 cells in the logarithmic growth phase were harvested and cultured at a concentration of 5 × 10⁻⁶ cells / year. 4 Cells were seeded at a density of 1 cells / well in 12-well plates and cultured for 12 hours until adherence. The original culture medium was then aspirated, and 25 mg / mL hydrogel extract containing 1 μg / mL LPS was added to each well. Pure DMEM medium containing 1 μg / mL LPS was used as the inflammation model group, with three replicates per group. After culturing for another 24 hours, the cell supernatant was collected, centrifuged at 3000 rpm for 5 min to remove cell debris, and the concentrations of TNF-α and IL-6 pro-inflammatory factors in the supernatant were measured using an ELISA kit. The results showed that, compared with the inflammation model group, the hydrogel extracts of Examples 1-3 significantly inhibited the overexpression of TNF-α and IL-6 pro-inflammatory factors induced by LPS in macrophages, and the inhibitory effect increased with increasing EPL-PQQ concentration. Example 3 showed the best inhibitory effect, demonstrating that the hydrogel of this invention possesses excellent anti-inflammatory properties, effectively alleviating persistent inflammatory responses in wounds and breaking the vicious cycle of chronic wound inflammation. The in vitro anti-inflammatory and antioxidant test results in this section are consistent with those in the appendix. Figure 4 The results were consistent, confirming that the hydrogel of the present invention can simultaneously achieve excellent anti-inflammatory and antioxidant effects, and specifically regulate the pathological microenvironment of chronic wounds.

[0040] VI. Examples of Hydrogel Applications In this embodiment, the anti-inflammatory and antioxidant hydrogel prepared according to the present invention is used as a dressing for repairing chronic skin wounds in diabetic patients. The specific application method is as follows: For chronic skin wounds in diabetic patients, routine debridement is performed to remove necrotic tissue and exudate. The wound is rinsed with sterile saline and excess surface moisture is absorbed. Under sterile conditions, freshly prepared OHA precursor solution and EPL-PQQ precursor solution are mixed evenly in proportion and applied uniformly to the wound. At 37°C, the gel forms in situ within 5-10 minutes, perfectly conforming to the wound shape and filling irregular wound defects. The gel surface is covered with sterile gauze for fixation and changed every 2-3 days, adjusting the frequency according to wound exudation. This dressing provides long-lasting moisturizing, antibacterial, anti-inflammatory, and antioxidant effects at the wound site, continuously regulating the wound microenvironment and promoting the healing of chronic diabetic wounds. It also exhibits good biocompatibility, being non-irritating and non-sensitizing, and is suitable for clinical chronic wound repair needs.

[0041] The basic performance comparison between the examples and the comparative examples is shown in the table below: Table 1 The anti-inflammatory and antioxidant properties and in vivo wound healing effects of the examples and comparative examples are compared in the table below: Table 2 Based on the test data in Tables 1 and 2, it can be concluded that the hydrogels prepared in the examples of this invention exhibit significantly superior basic physicochemical properties, biocompatibility, and biological functions compared to the comparative examples and the blank control group. Compared to Comparative Example 1 (without PQQ grafting) and Comparative Example 2 (with physically doped PQQ), the hydrogels in the examples demonstrate more efficient gelation, superior swelling and mechanical properties, and more prominent self-healing ability and cell compatibility. Simultaneously, their antioxidant and anti-inflammatory effects are significantly enhanced, and they show remarkable healing-promoting effects on diabetic chronic wounds. This fully demonstrates the core advantages of the covalent grafting modification and in-situ cross-linking system, precisely meeting the clinical needs of chronic wound repair.

[0042] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for preparing an anti-inflammatory and antioxidant hydrogel, characterized in that, The hydrogel is an ε-polylysine-pyrroloquinolinequinone / oxidized hyaluronic acid EPL-PQQ / OHA in-situ crosslinked hydrogel, comprising the following steps: Preparation of S1 Oxidized Hyaluronic Acid (OHA): Hyaluronic acid (HA) with a weight-average molecular weight of 800-1500 kDa and sodium periodate were added at a molar ratio of 1:(0.8-1.2). Ultrapure water was added and the mixture was stirred at room temperature in the dark for 4-6 hours. Ethylene glycol was added to quench the remaining sodium periodate and the reaction was continued for 1-2 hours. The product was collected by salting out and pre-cooling anhydrous ethanol precipitation. The product was dissolved in ultrapure water and dialyzed for 3-4 days to remove small molecule impurities. After freeze-drying, oxidized hyaluronic acid (OHA) with an aldehyde degree of 30%-50% was obtained. Preparation of S2 EPL-PQQ grafted polymer: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) activation condensation system were used. ε-polylysine (EPL) with a weight-average molecular weight of 2000-5000 Da and pyrroloquinoline quinone (PQQ) were added at a molar ratio of 1:(0.05-0.15). PQQ was first dissolved in PBS buffer, and EDC and NHS were added for activation at room temperature in the dark for 20-40 min. The molar ratio of PQQ, EDC, and NHS was 1:(1.2-1.5):(1.5-2.0). Then, EPL was added and the reaction was stirred at room temperature in the dark for 20-28 h. After the reaction, the mixture was purified by dialyzing for 3-4 days and then lyophilized to obtain EPL-PQQ covalently grafted polymer with a PQQ grafting rate of 5%-12%. Preparation of S3 hydrogel precursor solution: The OHA obtained in S1 was aseptically dissolved in PBS buffer at pH 7.2-7.4 to prepare an OHA precursor solution with a mass fraction of 16%-24%; the EPL-PQQ obtained in S2 was aseptically dissolved in the same batch of PBS buffer to prepare an EPL-PQQ precursor solution with a mass fraction of 10%-22%. S4 Physiological Conditions In-situ Crosslinking: OHA precursor solution and EPL-PQQ precursor solution were aseptically mixed at a volume ratio of 1:(0.8~1.2) and crosslinked at a physiological temperature of 35~39℃ through a dynamic Schiff base reaction of aldehyde and amino groups for 5~15 min to obtain the anti-inflammatory and antioxidant hydrogel.

2. The method for preparing the anti-inflammatory and antioxidant hydrogel according to claim 1, characterized in that, In S1, the molar ratio of HA to sodium periodate is 1:1, the reaction time is 5 hours at room temperature in the dark, and the molecular weight cutoff of the dialysis bag used for dialysis is 8000~14000 Da.

3. The method for preparing the anti-inflammatory and antioxidant hydrogel according to claim 1, characterized in that, In S2, the molar ratio of EPL to PQQ is 1:0.1, the molar ratio of PQQ, EDC, and NHS is 1:1.3:1.6, the activation time is 30 min, and the reaction time at room temperature with stirring is 24 h.

4. The method for preparing the anti-inflammatory and antioxidant hydrogel according to claim 1, characterized in that, In S3, the mass fraction of OHA precursor solution is 20%, and the mass fraction of EPL-PQQ precursor solution is 12%, 16%, or 20%. In S4, the mixing volume ratio of OHA precursor solution to EPL-PQQ precursor solution is 1:1, and the final mass fraction of EPL-PQQ in the mixed system is 6%, 8%, or 10%.

5. The method for preparing the anti-inflammatory and antioxidant hydrogel according to claim 1, characterized in that, The hydrogel prepared by S4 exhibits a storage modulus recovery rate of ≥65% after three large strain-small strain loading-unloading cycles at 1 Hz frequency and 25 ℃, demonstrating shear thinning characteristics and rapid self-healing performance.

6. The method for preparing the anti-inflammatory and antioxidant hydrogel according to claim 1, characterized in that, The extract of the hydrogel at a concentration of 25 mg / mL has a significant protective effect against H2O2-induced oxidative damage in L929 cells, reducing intracellular ROS levels by more than 40% and downregulating the expression of intracellular Sod2 and Cat genes related to antioxidant stress.

7. An anti-inflammatory and antioxidant hydrogel prepared by the method described in any one of claims 1-6, characterized in that, The hydrogel is a three-dimensional porous network structure formed by cross-linking EPL-PQQ grafted polymer and OHA through dynamic Schiff base covalent bonds. It also has the properties of moisturizing and water-locking, exudate absorption, self-repair, anti-inflammation, anti-oxidation and promoting skin tissue regeneration.

8. The application of the anti-inflammatory and antioxidant hydrogel as described in claim 8 in the preparation of medical wound repair dressings.

9. The application of the anti-inflammatory and antioxidant hydrogel according to claim 8 in the preparation of medical wound repair dressings, characterized in that, The medical wound repair dressing is a special repair dressing for diabetic chronic skin wounds and bacterial infected skin wounds. The dressing can form gel in situ under the physiological environment of the wound, and simultaneously achieve efficient absorption of wound exudate, regulation of the oxidative stress microenvironment, inhibition of inflammatory response, and promotion of granulation tissue formation, collagen deposition and epithelial regeneration.

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