Antioxidant antibacterial hydrogel as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of antibacterial hydrogel preparation, and particularly relates to antioxidant antibacterial hydrogel as well as a preparation method and application thereof. The method comprises the following steps: dispersing chitosan in water, then adding glacial acetic acid for reaction, adding 2, 3-epoxypropyltrimethylammonium chloride for continuous reaction after the reaction is finished, centrifuging after the reaction is finished to obtain supernate, and then adding a mixed solution of acetone and ethanol for precipitation, suction filtration and freeze drying to obtain chitosan quaternary ammonium salt; the preparation method comprises the following steps: dissolving chitosan quaternary ammonium salt in water, adding Pluronic F-127, stirring until the Pluronic F-127 is completely dissolved, adding the freeze-dried chlorella powder, and uniformly stirring to obtain the antioxidant and antibacterial hydrogel. The characteristics of the wound microenvironment are improved through the freeze-dried chlorella, and the freeze-dried chlorella is cooperated with the antibacterial hydrogel in function, so that an efficient treatment effect is achieved; meanwhile, the preparation process is simple, the raw material source is wide, and the clinical application prospect is good.

Description

Technical Field

[0001] This application belongs to the field of antibacterial hydrogel preparation technology, specifically relating to an antioxidant antibacterial hydrogel, its preparation method, and its application. Background Technology

[0002] Among the various complications caused by diabetes, the difficulty in wound healing is particularly challenging, causing immense suffering for patients and placing a heavy burden on families and society. To address this challenge, various treatment methods have been developed, such as local wound treatments like debridement, topical medications, wound dressings, and adjuvant therapies like oxygen therapy, physical therapy, and stem cell therapy. In recent years, hydrogels, as a highly promising wound dressing material, have become a research hotspot in the field of diabetic wound dressings due to their unique three-dimensional network structure and good biocompatibility. Because the microenvironment of diabetic wounds is complex, addressing hypoxia, clearing excess reactive oxygen species (ROS), and reducing inflammation are key to promoting healing. Recently, the synergistic effect of combining microalgae with hydrogels in promoting diabetic wound healing has shown promising results and has attracted considerable attention. Currently, the commonly used method is the combination of active microalgae and hydrogels. Active algae can improve the hypoxia in diabetic wounds; microalgal polysaccharides can inhibit the release of inflammatory factors in diabetic wounds; the essential amino acids and vitamins contained in microalgae can provide repair materials for repair cells; and the chlorophyll and carotenoids in microalgae can clear local reactive oxygen species (ROS) in the wound. These properties of active algae can effectively improve the wound microenvironment. However, live algae has certain limitations in clinical applications, such as: high storage and transportation costs, requiring a constant temperature of 20℃-25℃, light-proof, aerobic, and sterile water environment; short survival period, usually only a few days to a week; poor stability, as it continues to metabolize during storage, and if nutrition is insufficient or the environment is unsuitable, it is prone to decomposition of active ingredients such as polysaccharides, chlorophyll, and amino acids, and a decrease in photosynthetic oxygen production capacity; and the risk of immune stimulation, as live algae may produce a small amount of metabolic waste (such as organic acids and pigment degradation products) during metabolism, which may cause local irritation to the fragile wound tissues of diabetic patients.

[0003] Therefore, developing a hydrogel with antioxidant and anti-inflammatory properties suitable for clinical scenarios to improve the microenvironment of local wounds and thus play an adjunctive role in enhancing the efficacy of treatment has become an urgent technical problem to be solved. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide an antioxidant and antibacterial hydrogel, its preparation method, and its application. Specifically, the following technical solution is adopted: In a first aspect, the present invention provides a method for preparing an antioxidant and antibacterial hydrogel, comprising the following steps: Chitosan was dispersed in water and then glacial acetic acid was added to react. After the reaction was completed, 2,3-epoxypropyltrimethylammonium chloride was added to continue the reaction. After the reaction was completed, the mixture was centrifuged and the supernatant was added to precipitate the supernatant. The supernatant was then filtered and freeze-dried to obtain chitosan quaternary ammonium salt. After dissolving chitosan quaternary ammonium salt in water, Pluronic F-127 was added and stirred until completely dissolved. Then, freeze-dried Chlorella powder was added and stirred evenly to obtain the antioxidant and antibacterial hydrogel.

[0005] This invention utilizes the properties of freeze-dried Chlorella to improve the wound microenvironment. For example, the antioxidant components in the freeze-dried algae alleviate excessive oxidative stress at the wound site, algal polysaccharides act as anti-inflammatory substances to inhibit the release of inflammatory factors in diabetic wounds, and the nutrients (essential amino acids, vitamins, etc.) in the freeze-dried algae can provide repair materials for repair cells. These properties work synergistically with the antibacterial function of the antibacterial hydrogel to achieve highly effective treatment. At the same time, the invention ensures a simple preparation process and a wide range of raw material sources, demonstrating promising clinical application prospects.

[0006] As a further preferred embodiment, the freeze-dried Chlorella powder is prepared by the following steps: Chlorella was inoculated into sterilized BG11 liquid medium and cultured to the logarithmic growth phase. The culture was then washed, concentrated, and freeze-dried to obtain the freeze-dried Chlorella powder.

[0007] As a further preferred embodiment, the mass fraction concentration of the chitosan quaternary ammonium salt after dissolving in water is 0.05%-0.2%.

[0008] As a further preferred embodiment, the concentration of the freeze-dried Chlorella powder is 1×10⁻⁶. 6 Cell count / mL - 1×10 8 Cell count / mL.

[0009] As a further preferred embodiment, the mass fraction concentration of Pluronic F-127 after being stirred until completely dissolved is 18%-22%.

[0010] Secondly, the present invention provides an antioxidant and antibacterial hydrogel, which is prepared by the above-described preparation method.

[0011] Thirdly, the present invention provides the application of the above-mentioned antioxidant and antibacterial hydrogel in the preparation of antibacterial materials.

[0012] Fourthly, the present invention provides the application of the above-mentioned antioxidant and antibacterial hydrogel in the preparation of wound healing materials.

[0013] As a further preferred embodiment, the wound healing material includes materials for healing diabetic wounds.

[0014] Fifthly, the present invention provides a material for promoting the healing of diabetic wounds, the material comprising the aforementioned antioxidant and antibacterial hydrogel.

[0015] The beneficial effects of this invention are as follows: (1) The preparation process of this invention is simple and the raw materials are widely available. The entire process is carried out at low temperature, which avoids the damage of high temperature to the active ingredients of Chlorella. Furthermore, no chemical cross-linking agent is required, which greatly improves the biosafety of the hydrogel.

[0016] (2) Synergistic effect of anti-inflammatory and antioxidant: The chlorophyll and carotenoids of freeze-dried Chlorella can directly remove excess ROS from the wound, while QCS can reduce the release of inflammatory factors (TNF-α, IL-6) by inhibiting the NF-κB pathway. The two work together to form a dual anti-inflammatory mechanism of "ROS removal + inflammatory signal blocking", which reduces the ROS level of the wound and effectively breaks the vicious cycle of "excessive ROS-persistent inflammation" in diabetic wounds. This synergistic effect is far superior to the anti-inflammatory effect of a single component.

[0017] (3) Synergistic protection of antibacterial and immune safety: The quaternary ammonium group of QCS exerts a broad-spectrum antibacterial effect by destroying the bacterial cell membrane (the inhibition rate against Escherichia coli and Staphylococcus aureus is over 95%), while the dormant state of freeze-dried Chlorella (the freeze-drying process eliminates the risk of immune stimulation from live algae) can avoid the local immune response (such as excessive infiltration of neutrophils) that QCS may cause during the antibacterial process. The two work together to achieve a balance of "strong antibacterial effect + low immune stimulation", which solves the contradiction of "strong antibacterial effect and high immune stimulation" in traditional antibacterial hydrogels. This reduces the wound infection rate by 80% while controlling the amount of local immune cell infiltration within the range required for normal repair, creating a safe environment for wound repair.

[0018] (4) Synergistic support of nutrient supply and wound microenvironment improvement: The amino acids and vitamins released by freeze-dried Chlorella can provide nutrition for repair cells (fibroblasts and keratinocytes) and promote cell proliferation; the gel form of Pluronic F-127 can closely adhere to the wound, reduce water evaporation and external pollution, and maintain a moist wound microenvironment; the biodegradability of QCS can slowly release glucosamine (a product of chitosan degradation), further promoting collagen synthesis. The three work together to form a repair microenvironment of "nutrient supply-moisture protection-matrix synthesis promotion", which increases the proliferation rate of fibroblasts in diabetic wounds and increases the amount of collagen deposition compared with single component groups, significantly accelerating the wound healing process. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 The figure shows the hydrogen peroxide scavenging capacity of different freeze-dried Chlorella species. Figure 2 The figure shows the gelation temperature and time of the freeze-dried algae gel. Figure 3 The image shows the FT-IR spectra of different components of the freeze-dried algal gel; Figure 4 The rheological properties of the freeze-dried algal gel are shown. Figure 5 The diagram shows the phase transition of the gel at different temperatures; Figure 6 The image shows the adhesion of the gel at different bending angles; Figure 7 The image shows SEM images of a blank gel (left) and a gel containing algae (right); Figure 8 The image shows the antibacterial effect of the hydrogel on Staphylococcus aureus and Escherichia coli. Figure 9 The image shows the antioxidant capacity of the gel; Figure 10 The image shows the effect of freeze-dried algae gel on wound healing in diabetic mice. Figure 11 The image shows the effects of live algae gel and freeze-dried algae gel on wound healing in diabetic mice. Figure 12 The image shown is a HE staining image of the wound tissue. Detailed Implementation

[0021] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0022] Example 1 A method for preparing an antioxidant and antibacterial hydrogel, specifically comprising the following steps: (1) Preparation of freeze-dried Chlorella powder Chlorella was inoculated into sterilized BG11 liquid medium (the composition and content of which are shown in Table 1) and cultured until the logarithmic growth phase to obtain fresh algal solution. The cell density of the fresh algal solution was then measured, and a cell density of 1×10⁻⁶ was selected. 8 After washing the Cells / mL algal solution three times with deionized water, the algal solution was concentrated by low-speed centrifugation (3000 rpm / min, 10 min). The concentrated algal solution was placed in a freeze dryer and freeze-dried at -80℃ and 0.03 MPa for 18 h to obtain the freeze-dried material. Finally, the obtained freeze-dried material was passed through a 100-mesh sieve to obtain freeze-dried Chlorella powder, which can be stored in a sealed container at 4℃.

[0023] Table 1. Composition and content of BG11 liquid culture medium (2) Preparation of chitosan quaternary ammonium salt First, 5g of chitosan (CS) was suspended in deionized water. Then, 1% glacial acetic acid was added dropwise while stirring to completely dissolve the CS. The reaction was carried out at 55℃ for 10-300 minutes. Then, 60% 2,3-epoxypropyltrimethylammonium chloride (GTA, with a mass ratio of CS to GTA of 1:4) was added dropwise. The reaction was carried out at 40℃-60℃ for 18 hours. The reaction solution was centrifuged at 9000 rpm / min for 10-30 minutes to obtain the supernatant. Pre-cooled acetone / ethanol solution (1:1, v / v) was added to the supernatant to precipitate completely. Finally, the solution was filtered and freeze-dried to obtain quaternized chitosan quaternary ammonium salt (QCS).

[0024] (3) Preparation of QCS-F127 gel Chitosan quaternary ammonium salt (QCS) was dissolved in water to prepare a QCS solution with a mass fraction of 0.05%; then Pluronic F-127 was added, and the solution was stirred at low temperature (4℃) until it was completely dissolved to prepare a QCS-F127 gel containing 18% Pluronic F-127. (4) Preparation of antioxidant and antibacterial hydrogels (lyophilized algae gels) Take the freeze-dried Chlorella powder obtained in step (1) and slowly add it to the QCS-F127 gel prepared in step (3) at a low temperature (4℃) so that the content of freeze-dried algae in the final gel is 1×10 8 Cells / mL, continuously stirred at 4℃ for 1h to uniformly disperse the lyophilized Chlorella powder into the QCS-F127 gel, thus obtaining the lyophilized algal gel.

[0025] Example 2 An antioxidant and antibacterial hydrogel preparation method is similar to that in Example 1. The only difference is that in step (2) of Example 1, "preparing a QCS solution with a mass fraction of 0.05%" is replaced with "preparing a QCS solution with a mass fraction of 0.1%", and a QCS-F127 gel containing 18% Pluronic F-127 is prepared. The rest of the process remains unchanged, and a freeze-dried algae gel is obtained.

[0026] Example 3 An antioxidant and antibacterial hydrogel preparation method is similar to that in Example 1. The only difference is that in step (2) of Example 1, "preparing a QCS solution with a mass fraction of 0.05%" is replaced with "preparing a QCS solution with a mass fraction of 0.2%", and a QCS-F127 gel containing 18% Pluronic F-127 is prepared. The rest of the process remains unchanged, and a freeze-dried algae gel is obtained.

[0027] Example 4 An antioxidant and antibacterial hydrogel preparation method is similar to that in Example 1. The only difference is that in step (2) of Example 1, "preparing QCS-F127 gel containing 18% Pluronic F-127" is replaced with "preparing QCS-F127 gel containing 20% ​​Pluronic F-127". The rest of the process remains unchanged, and freeze-dried algae gel is obtained.

[0028] Example 5 An antioxidant and antibacterial hydrogel preparation method is similar to that in Example 1. The only difference is that in step (2) of Example 1, "preparing QCS-F127 gel containing 18% Pluronic F-127" is replaced with "preparing QCS-F127 gel containing 22% Pluronic F-127". All other processes remain unchanged, and freeze-dried algae gel is obtained.

[0029] Example 6 An antioxidant and antibacterial hydrogel preparation method is similar to that in Example 1. The only difference is that in step (2) of Example 1, "preparing a QCS solution with a mass fraction of 0.05%" is replaced with "preparing a QCS solution with a mass fraction of 0.1%" and "preparing a QCS-F127 gel containing 18% Pluronic F-127" is replaced with "preparing a QCS-F127 gel containing 20% ​​Pluronic F-127"; the rest of the process remains unchanged, and the freeze-dried algae gel is obtained.

[0030] Example 7 An antioxidant and antibacterial hydrogel preparation method is similar to that in Example 1. The only difference is that in step (2) of Example 1, "preparing a QCS solution with a mass fraction of 0.05%" is replaced with "preparing a QCS solution with a mass fraction of 0.1%" and "preparing a QCS-F127 gel containing 18% Pluronic F-127" is replaced with "preparing a QCS-F127 gel containing 22% Pluronic F-127"; the rest of the process remains unchanged, and the freeze-dried algae gel is obtained.

[0031] Example 8 An antioxidant and antibacterial hydrogel preparation method is similar to that in Example 1. The only difference is that in step (2) of Example 1, "preparing a QCS solution with a mass fraction of 0.05%" is replaced with "preparing a QCS solution with a mass fraction of 0.2%" and "preparing a QCS-F127 gel containing 18% Pluronic F-127" is replaced with "preparing a QCS-F127 gel containing 20% ​​Pluronic F-127"; the rest of the process remains unchanged, and the freeze-dried algal gel is obtained.

[0032] Example 9 An antioxidant and antibacterial hydrogel preparation method is similar to that in Example 1. The only difference is that in step (2) of Example 1, "preparing a QCS solution with a mass fraction of 0.05%" is replaced with "preparing a QCS solution with a mass fraction of 0.2%" and "preparing a QCS-F127 gel containing 18% Pluronic F-127" is replaced with "preparing a QCS-F127 gel containing 22% Pluronic F-127"; the rest of the process remains unchanged, and the freeze-dried algal gel is obtained.

[0033] Comparative Example 1 The effects of live algae gel and freeze-dried algae gel (freeze-dried Chlorella gel prepared in Example 5) on the healing of diabetic wounds were investigated, and the specific process is as follows: Preparation of live algae gel: Collect the microalgae suspension, wash it twice with PBS, and collect the microalgae by low-speed centrifugation (3000 rpm / min). After adding it to the QCS / F127 gel, the algae density is 1×10⁻⁶. 8 Cells / mL (density same as lyophilized algae density) yields live algae gel.

[0034] The experimental mice were 6-8 week old SPF-grade male C57BL / 6J mice. After one week of acclimatization, the mice were intraperitoneally injected with 120 mg / kg of 1% STZ (streptozotocin) citrate buffer. Blood glucose levels were measured one week after the injection. Successful modeling was considered achieved if the random blood glucose level was greater than 16.7 mmol / L for three consecutive days. All animal experiments were conducted in accordance with the Chinese Animal Research Guidelines and were approved by the Ethics Review Committee of Jiangxi University of Science and Technology.

[0035] Before creating the diabetic wound model, mice were anesthetized with isoflurane. The back area of ​​the mice was shaved using a shaver, and then a full-thickness wound was created in the diabetic mice using an 8mm diameter skin punch. The mice were divided into five groups with different treatments: control group (non-diabetic mice), model group, blank gel group (QCS / F127 gel), live algae gel group, and freeze-dried algae gel group (n=8). The control and model groups received no treatment. The blank gel group, live algae gel group, and freeze-dried algae gel were applied to the wound site and changed daily.

[0036] Photos of the wound were taken on days 4, 7, and 12 to observe the wound healing process, and ImageJ software was used to assess the wound healing.

[0037] from Figure 11 It can be seen that on the 4th day of wound healing, the live algae gel and freeze-dried algae gel groups showed reduced redness and swelling, and a significantly higher healing rate (approximately 65%-70%) than the model group (statistics show * P<0.05), indicating that both live algae gel and freeze-dried algae gel groups can alleviate wound inflammation and initiate repair in the early stages of healing. On the 7th day, the core repair stage, the healing rate of the model group was only about 70%, and the wound still had obvious defects; the healing rate of the live algae gel and freeze-dried algae gel groups increased to approximately 90%, and the healing rate of the live algae group was significantly higher than that of the model group (***P<0.001), while the freeze-dried algae group was also significantly better than the model group (*P<0.05); the wound appearance of the live algae / freeze-dried algae gel groups was closer to the healed state (the defect area was reduced), indicating that the live algae gel and freeze-dried algae gel can accelerate granulation tissue formation and wound contraction during the proliferation period, which is far superior to the untreated model group. On day 12, the healing rate of the model group was about 98%, but there were still a few residual defects in the wound; the healing rate of the live algae gel and freeze-dried algae gel groups was close to 100%, the wound was basically completely closed, and the effect was significantly better than that of the model group (*P<0.05); the wound healing quality of the algae gel group was higher (the appearance was smoother), indicating that the algae gel can promote the completion of late remodeling of diabetic wounds and avoid the healing delay of the model group.

[0038] Both live algae gel group and freeze-dried algae gel group can effectively improve the healing status of diabetic wounds. The therapeutic effect of freeze-dried algae gel group is not significantly different from that of live algae gel group. The live algae group has a slightly better mid-term healing rate, but the application value of freeze-dried algae gel group is more prominent.

[0039] Example 10 The materials prepared above were subjected to performance testing. (1) To investigate the in vitro hydrogen peroxide depletion capacity of freeze-dried Chlorella powder at different concentrations: To determine the H2O2 depletion efficiency of different concentrations of lyophilized Chlorella powder, the lyophilized Chlorella powder was added to PBS containing 16.7 mmol / L H2O2 and incubated for 20 min. After centrifugation at 3000 rpm / min for 5 min, the concentration of H2O2 in the supernatant was determined according to the instructions of the H2O2 content determination kit (Solarbio BC3595). We conducted in vitro tests on the ability of freeze-dried Chlorella powder to remove ROS: from Figure 1 As shown, the H2O2 removal ability of freeze-dried Chlorella powder exhibits a concentration-dependent effect; the removal effect is better with increasing concentration of freeze-dried Chlorella powder, especially at a microalgae density of 1×10⁻⁶. 8 At a concentration of Cells / mL, it exhibits good elimination of hydrogen peroxide.

[0040] (2) Investigating the gelation temperature and time of freeze-dried algae gel Gelation temperature: Pour 2 mL of thermosensitive gel into a test tube, insert the test tube into a constant temperature water bath, keeping the gel level 2 cm lower than the water bath level. Insert a thermometer with an accuracy of 0.1℃ into the water, level with the test tube. Set the initial temperature to 25℃. For every 0.5℃ increase in temperature, remove the test tube, invert it, and observe. Record the temperature after no flow is observed 30 seconds after the test tube is inverted.

[0041] Gelation time: Place the test tube in a 25°C water bath for 10 minutes, then place it in a 37°C water bath and start timing immediately. Tilt the test tube every 10 seconds until no flow is observed and stop timing.

[0042] Figure 2 The figure shows the gelation temperature (°C) and gelation time (s) of QCS / F127 gel under different addition conditions.

[0043] (3) The infrared spectra (FT-IR) of each component of the freeze-dried algal gel were investigated, as follows: The infrared spectra of chitosan quaternary ammonium salt (QCS), Pluronic F127, blank gel (QCS-F127), and lyophilized algal gel were determined using Fourier transform infrared spectroscopy (FTIR), with a focal length of 4 cm⁻¹. -1Spectral resolution measurement, wavenumber range: 400 cm⁻¹ -1 -4000cm -1 .

[0044] Figure 3 Infrared spectra of the various components that make up the gel are shown, with the 3291.2 cm⁻¹ spectrum being the largest. -1 1654.73 cm -1 and 1545.52 cm -1 The characteristic absorption peaks of microalgae are the stretching vibrations of OH and CH, and the stretching vibrations of C=C, C=N, N=N, and N=O double bonds, respectively. Small peaks were observed near these three locations in the gel containing microalgae, confirming the presence of microalgae in the gel. The peak at 3291.2 cm⁻¹ is also significant. -1 A blue shift was observed in the peak, which may be related to the disruption of the conjugated structure of QCS and Pluronic F127 when they interact with microalgae; while the characteristic peak of QCS in the QCS-127 gel was not obvious, which may be due to the low concentration of QCS added.

[0045] (4) Investigate the Tg and rheological properties of the gel. Pluronic F-127 exhibits good biocompatibility, biosafety, and thermal reversibility. When heated above its gelation temperature (Tg), the aqueous solution of Pluronic F-127 undergoes a phase transition, transforming into an elastic gel. By cooling the gel below its Tg, it can revert to the liquid phase. To investigate the Tg and rheological properties of this gel, we tested it using a rotational rheometer, as detailed below: The rheological properties of temperature-sensitive hydrogels were determined using a rotational rheometer. The test conditions were as follows: at a constant frequency of 10 rad / s, the G' (storage modulus) and G' (loss modulus) of the hydrogel were measured at a heating rate of 5 °C / min from 0 °C to 40 °C. The viscosity of the hydrogel was also measured at the same time.

[0046] Figure 4 The figure shows the rheological properties of a freeze-dried algal gel composed of 0.05% QCS, 22% Pluronic F-127 and freeze-dried Chlorella powder. The figure shows the changes in storage modulus (G') and loss modulus (G") with temperature. It can be seen that the gel underwent a solution-gel phase transition at 26.1℃ and tended to stabilize at 28.5℃ (G'>G"), exhibiting a gel state and good viscosity.

[0047] (5) To observe the phase transition behavior of temperature-sensitive hydrogels, the hydrogels were placed at two temperatures of 25°C and 37°C respectively, and the state changes of the gels under these two environments were observed and recorded.

[0048] Figure 5 The figure shows the changes in state of the gel without added freeze-dried algae gel and the freeze-dried algae gel under two conditions: room temperature and body temperature. It can be seen that the gel also undergoes a phase transition during the transition from room temperature to body temperature, changing from the initial liquid state to a gel state.

[0049] (6) The gel stored at low temperature was slowly injected into the finger joints, and the adhesion and fit of the gel when the finger was bent and inverted were observed after the gel solidified.

[0050] Figure 6 The figure shows the adhesion of the freeze-dried algae gel under different bending conditions (0°, 90°) with both upright and inverted orientations. This figure illustrates that the gel can stably adhere to the skin and change shape with movement during joint bending, confirming the practical application of this hydrogel as a dressing for joint skin wounds.

[0051] (7) Scanning electron microscope Figure 7 The image shows the microscopic morphology of the blank gel and the lyophilized algal gel under a scanning electron microscope. Observation of the hydrogel's microstructure reveals that it can form a dense three-dimensional network, with the algal-containing gel exhibiting a larger surface area and porosity. This structure provides more opportunities for cell infiltration and substance exchange, while also facilitating drug penetration and exudate drainage at the wound site.

[0052] (8) Antibacterial ability test, the specific process is as follows: This embodiment uses *Escherichia coli* (ATCC 8739) and *Staphylococcus aureus* (ATCC 6538) to evaluate the antibacterial effect of hydrogels containing different concentrations of QCS (0.05 w / v%, 0.1 w / v%, 0.2 w / v%). First, single colonies of both bacteria were picked from solid culture media and transferred to liquid culture media, incubated at 37°C for 6-8 hours until the absorbance reached approximately 0.5. Then, the bacteria were diluted with PBS to a concentration of 1×10⁻⁶. 6 CFU / mL. After mixing equal volumes of gel and bacterial suspension and incubating for 10 h, the bacterial suspension was diluted appropriately and spread onto solid culture medium. After incubation at 37℃ for 12 h, the plates were observed and the colony count was recorded. Simultaneously, the absorbance at OD600 of the incubated bacterial suspension was measured, and the inhibition rate of different concentrations of QCS was determined.

[0053] Figure 8 The figure shows the antibacterial effect of the gels at different QCS concentrations (0.05%, 0.1%, and 0.2%) against Escherichia coli and Staphylococcus aureus. The figure shows that when QCS ≥ 0.05%, the gel groups can effectively kill bacteria on their surfaces, including Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus, indicating that the hydrogel QCS / F127 has excellent antibacterial ability.

[0054] (9) Antioxidant capacity determination The antioxidant and free radical scavenging capabilities of hydrogels can directly or indirectly repair the damaged wound healing process by scavenging excess reactive oxygen species (ROS) and improving the oxidative microenvironment of wounds. The specific measurement process is as follows: Based on the fact that antioxidants can reduce Fe under acidic conditions 3+ Based on the principle that tripyridine triazine produces substances that absorb at 593 nm (FRAP method), the total antioxidant capacity of blank gel (QCS / F127 gel), lyophilized algal powder, and lyophilized algal gel were determined using a total antioxidant capacity kit. Based on the principle that nitrogen free radicals contain a single electron and turn purple after dissolving in ethanol, exhibiting absorption at 517 nm, the DPPH free radical scavenging capacity of pure gel, lyophilized algal powder, and lyophilized algal gel was determined.

[0055] Figure 9 The total antioxidant capacity and DPPH free radical scavenging capacity of the blank gel (QCS / F127), lyophilized Chlorella powder, and lyophilized algal gel were determined. The figures show that the antioxidant performance of the lyophilized algal gel was stronger than that of the blank gel and the same concentration of lyophilized microalgae. The lyophilized Chlorella powder possesses antioxidant properties, can scavenge free radicals, significantly improve wound healing, and protect tissues from oxidation.

[0056] (10) The freeze-dried Chlorella gel was used to determine the wound healing ability of diabetic patients. The specific process is as follows: The experimental mice were 6-8 week old SPF-grade male C57BL / 6J mice. After one week of acclimatization, the mice were intraperitoneally injected with 120 mg / kg of 1% STZ (streptozotocin) citrate buffer. Blood glucose levels were measured one week after the injection. Successful modeling was considered achieved if the random blood glucose level was greater than 16.7 mmol / L for three consecutive days. All animal experiments were conducted in accordance with the Chinese Animal Research Guidelines and were approved by the Ethics Review Committee of Jiangxi University of Science and Technology.

[0057] Before creating the diabetic wound model, mice were anesthetized with isoflurane. The back area of ​​the mice was shaved using a shaver, and then a full-thickness dermal wound was created in the diabetic mice using an 8mm diameter mouse skin punch. The mice were divided into five groups with different treatments: control group (non-diabetic mice), model group, blank gel group (QCS / F127), and lyophilized algae gel group (n=8). The control and model groups received no treatment; the gels in the blank gel group and the lyophilized algae gel group were applied to the wound site and changed daily.

[0058] Wound photos were taken on days 4, 7, and 12 to observe wound healing. Image J software was used to assess wound healing, and skin tissue samples were collected on days 4 and 12 for eosin (H&E) and Masson staining.

[0059] Figure 10 The image shows the wound healing process of a full-thickness dermal wound treated with lyophilized Chlorella gel on days 0, 4, 7, and 12 in diabetic mice. Except for the control group (non-diabetic mice), the other three groups consisted of diabetic mice with blood glucose levels ≥16.7 mmol / mL. Results showed that in the first four days of treatment, the lyophilized Chlorella gel group exhibited better healing than the other diabetic groups (P≤0.001), with an average wound healing rate of 77.6%, compared to 56.8% in the model group and 59% in the control group. By day 12, the wounds in all groups were close to healing, but the model group showed the worst healing, followed by the control group. The lyophilized Chlorella gel group showed the best results, with the wound essentially healed and beginning to re-epithelialize.

[0060] Figure 12 To assess wound healing progress at different stages, HE staining was performed on mouse wound tissues on days 4 and 12. The healing effect of the hydrogel on wound tissue was further evaluated using HE staining. As shown in the staining results on day 4, significant inflammatory infiltration was observed in the wound tissues of the model group and the blank gel group, while the inflammation was not obvious in the blank group and the freeze-dried algae gel group. This may be attributed to the antioxidants and anti-inflammatory polysaccharides contained in the freeze-dried algae. The HE staining images on day 12 showed significant differences in wound gaps between groups (indicated by the size of the arrows). The epidermal tissues of the blank group and the freeze-dried algae gel group were intact, with smaller wound gaps. The thickness of the regenerated epidermis is an important indicator of wound healing effectiveness; the regenerated epidermis in the model group was thinner than in the other hydrogel groups. Among the hydrogel groups, the regenerated epidermis produced by the freeze-dried algae gel group tended to be thicker than the other two groups and had more hair follicles. In conclusion, the freeze-dried algae gel can promote wound repair by reducing inflammatory cell infiltration and accelerating wound contraction.

[0061] The embodiments of this application have been described above with reference to the accompanying drawings. Specific examples have been used to illustrate the principles and implementation methods of this application. The description of the above embodiments is only for the purpose of helping to understand the core ideas of this application. However, this application is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of this application without departing from the spirit and scope of the claims, and all of these forms are within the protection scope of this application.

Claims

1. A method for preparing an antioxidant antibacterial hydrogel, characterized by, The method comprises the following steps: The chitosan is dispersed in water and then reacted with ice acetic acid. After the reaction, 2,3-epoxypropyl trimethyl ammonium chloride is added for further reaction. After the reaction, centrifugation is performed, and the supernatant is obtained. Then, a mixed solution of acetone and ethanol is added to precipitate the supernatant. After suction filtration, freeze-drying is performed to obtain the chitosan quaternary ammonium salt. The chitosan quaternary ammonium salt is dissolved in water, and then Pluronic F-127 is added and stirred until completely dissolved. Then, freeze-dried Chlorella powder is added and stirred until uniform to obtain the antioxidant and antibacterial hydrogel.

2. The production method according to claim 1, characterized by, The freeze-dried Chlorella powder is prepared by the following steps: Chlorella is inoculated into sterilized BG11 liquid medium and cultured to the logarithmic growth phase. After washing, concentration and freeze-drying, the freeze-dried Chlorella powder is obtained.

3. The preparation method according to claim 1, characterized in that, After the chitosan quaternary ammonium salt is dissolved in water, the mass fraction concentration of the chitosan quaternary ammonium salt is 0.05%-0.2%.

4. The method of claim 1, wherein, The concentration of the freeze-dried chlorella powder to be added is 1 x 10 6 Cell number / ml - 1 x 10 8 Cell number / ml.

5. The preparation method according to claim 1, characterized in that, After the Pluronic F-127 is stirred until completely dissolved, the mass fraction concentration of the Pluronic F-127 is 18%-22%.

6. An antioxidant antibacterial hydrogel, characterized by, The antioxidant and antibacterial hydrogel is prepared by the preparation method of any one of claims 1-5.

7. Use of the antioxidant and antibacterial hydrogel of claim 6 in the preparation of antibacterial materials.

8. Use of the antioxidant and antibacterial hydrogel of claim 6 in the preparation of wound healing materials.

9. Use according to claim 8, characterized in that, The wound healing materials include materials for diabetic wound healing.

10. A material for promoting healing of diabetic wounds, characterized in that, The materials include the antioxidant and antibacterial hydrogel of claim 6.

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