An algal particle-based gel dressing and its preparation method and application

Abstract

The application discloses an algal particle-based gel dressing and a preparation method and application thereof, and belongs to the technical field of biological medicine. The gel dressing is composed of PUE and CS loaded algae and gold nanoparticles. The gold nanoparticles are prepared in situ by biological mineralization using the self advantages of natural active microorganism algae. The local surface plasmon resonance effect of the gold nanoparticles is used to promote the transfer of photoelectrons, enhance the Chlorella vulgaris light hydrogen production performance, and use the colonization function of the hydrogel to direct the delivery of hydrogen generated by the Chlorella vulgaris to the wound site. The synthesis method is simple and green, the raw materials are abundant, and the method has the advantages of low cost, low toxicity and high efficiency. At the same time, the hydrogen generated by the Chlorella vulgaris is expanded to the medical field through the energy direction, and the application of hydrogen is expanded. The gel dressing provided by the application is simple to prepare, can effectively promote the healing of diabetic wounds, has high biological safety, and has a wide application prospect in wound healing.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a gel dressing based on algae particles, its preparation method, and its application. Background Technology

[0002] Currently, 96% of hydrogen (H2) originates from fossil fuels, while only 4% is "biohydrogen" produced by microorganisms such as bacteria and microalgae. As a clean, economical, and low-energy-consumption resource, hydrogen is widely used in various energy fields. In recent years, hydrogen, as a gas with a small molecular weight, has attracted widespread attention in the biomedical field due to its strong diffusivity and high biocompatibility. Studies have shown that hydrogen possesses various biological effects, including antioxidant, anti-inflammatory, and cell-protective properties, and can selectively scavenge hydroxyl radicals (·OH) and peroxynitrite anions (ONOO). - Hydrogen can reduce oxidative stress levels. Simultaneously, it can improve the inflammatory microenvironment by regulating inflammation-related signaling pathways such as NF-κB, inhibiting the expression of inflammatory factors such as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β). Furthermore, hydrogen can regulate the expression of apoptosis-related proteins, promoting the proliferation and migration of fibroblasts, endothelial cells, and keratinocytes, playing a positive role in tissue repair and regeneration. Hydrogen has demonstrated potential applications in clinical and experimental studies of various diseases, including ischemia-reperfusion injury, neurodegenerative diseases, metabolic diseases, and chronic inflammatory diseases.

[0003] Diabetes mellitus, a chronic metabolic disease, has a global prevalence of 11.1%. Approximately one-third of patients experience chronic damage and dysfunction in multiple tissues due to persistently high glucose levels, leading to various chronic complications. At the wound site, the high-glucose microenvironment promotes the accumulation of advanced glycation end-products (AGEs), altering the wound's redox state and immune response, affecting macrophage polarization, continuously secreting pro-inflammatory cytokines, reducing fibroblast proliferation and migration, decreasing angiogenesis, inhibiting fibroblast activity, and further delaying the healing process. Therefore, the continuous accumulation of AGEs, persistent chronic inflammation, and oxidative stress at the wound site are key factors affecting wound healing. Addressing the potential problems of AGE accumulation, chronic inflammation, and oxidative stress provides an effective approach for the treatment of chronic wounds in diabetic patients.

[0004] Gas therapy, as a novel, effective, and safe treatment method, is gradually gaining popularity in the field of diabetic wound care. Common gases such as oxygen (O2), nitric oxide (NO), carbon monoxide (CO), and hydrogen sulfide (H2S) are often unsuitable for providing a safe, continuous, and sufficient gas supply to the wound site due to limitations in their sources or treatment pathways. For example, hyperbaric oxygen therapy (HBOT) and topical oxygen therapy (TOT) fail to achieve significantly effective wound healing because these treatments suffer from low O2 solubility, poor tissue permeability, and difficulty in maintaining effective O2 concentrations. The release of NO from tissues depends on the catalytic reaction of nitric oxide synthases (NOS). However, chronic wounds are often characterized by NOS deficiency, hindering the release process. CO has a high affinity for hemoglobin (Hb), potentially leading to tissue hypoxia. H2S, when used at concentrations exceeding 50 ppm, may cause eye, nervous system, skin, cardiovascular, and respiratory symptoms. Hydrogen, as a gas with relatively high biocompatibility, exhibits unique advantages in the treatment of diabetic wounds. Hydrogen can selectively scavenge cytotoxic hydroxyl radicals, reducing damage caused by oxidative stress. It can also inhibit the expression of RAGE receptors, suppressing the activation of downstream inflammatory pathways, thereby reducing skin cell apoptosis, promoting skin cell proliferation and migration, and accelerating the healing of chronic diabetic wounds. However, as a small molecule gas, hydrogen has high diffusivity and low solubility, making stable, controllable delivery and continuous release difficult, and unable to maintain an effective therapeutic concentration at the wound site, thus limiting its therapeutic effect. Furthermore, current hydrogen supply methods are mostly intermittent or short-term, making long-term stable release difficult, limiting its application in chronic diseases. In recent years, research on Chlorella as a hydrogen donor to promote diabetic wound healing has mainly involved co-culturing Chlorella with bacteria and applying it to the wound site, but this easily leads to bacterial infection at the wound site. Hydrogels, due to their three-dimensional porous network structure and good biocompatibility, have been extensively studied in the repair of diabetic wounds. However, most existing hydrogels are only used as drug carriers and lack hydrogen production capabilities, thus failing to exert anti-inflammatory and antioxidant bioactivities, and the overall therapeutic effect still needs improvement. Addressing this clinical need for diabetic wound repair, using hydrogen gas for intervention is a novel technological approach, and related technical solutions are still relatively scarce, indicating significant room for innovation and application prospects.

[0005] Therefore, addressing the problems of low hydrogen delivery efficiency, poor sustainability, and limited wound treatment methods in existing technologies, developing a simple and sustainable hydrogen supply strategy is crucial for controlling the accumulation of AGEs, chronic inflammation, and oxidative stress at the wound site. This invention is therefore proposed. Summary of the Invention

[0006] The technical problem to be solved by this invention is how to solve the problems of existing wound healing dressings not having hydrogen production capacity and poor anti-inflammatory properties.

[0007] The present invention solves the above-mentioned technical problems through the following technical means: A first aspect of the present invention provides a gel dressing based on algae particles, comprising a dressing matrix, algae particles and gold nanoparticles, wherein the algae particles and gold nanoparticles are loaded on the dressing matrix, and the dressing matrix comprises puerarin (PUE) and chitosan (CS).

[0008] Preferably, the ratio of puerarin (PUE) to chitosan (CS) is (200~1000) mg: (20~200) mg.

[0009] A second aspect of the present invention provides a method for preparing the above-mentioned gel dressing, comprising the following steps: S1: Chlorella (Ch) solution was mixed with chloroauric acid (HAuCl4) solution and incubated to obtain Chlorella (ChAu) with mineralized gold nanoparticles. S2: Mix puerarin, chitosan, glacial acetic acid solution, and water and heat to obtain the dressing matrix; S3: Add the ChAu obtained in S1 to the dressing matrix in S2 and mix.

[0010] Preferably, in S1, the volume ratio of the Chlorella solution to the chloroauric acid solution is (9~19):(0.5~5), and more preferably 9:1.

[0011] Preferably, in S1, the concentration of Chlorella is (2~5)×10⁻⁶. 7 cells / mL; chloroauric acid concentration of 0.5–4 mM; further preferred concentration of Chlorella is 5 × 10⁻⁶ cells / mL. 7 The concentration of chloroauric acid was 1 mM, with cells / mL.

[0012] Preferably, in S1, the incubation method is to culture in a constant temperature and light shaker for 10-14 h; Further preferred illumination times are 10-14 h, light intensity is 2000-3000 Lux, temperature is 24-26℃, and shaking speed is 80-100 rpm; further preferred times are 12 h, 2500 Lux, 25℃, and 80 rpm.

[0013] Preferably, step S1 further includes centrifugation and washing; the centrifugation conditions are: 4000~6000 rpm, 2~5 min; more preferably 5000 rpm, 3 min.

[0014] Preferably, in S2, the ratio of puerarin, chitosan, glacial acetic acid solution, and water is (200~1000) mg: (20~200) mg: 10 mL: 10 mL, and more preferably, it is 500 mg: 200 mg: 10 mL: 10 mL.

[0015] Preferably, in S2, the glacial acetic acid solution is an aqueous solution of glacial acetic acid with a glacial acetic acid content of 1%.

[0016] Preferably, in S2, the heating method is water bath heating at 100-110 ℃.

[0017] Preferably, in S3, the ratio of ChAu to dressing matrix is ​​2×10. 7 ~5×10 7 cells / mL: 1 mL.

[0018] Preferably, the gel dressing needs to be gelled at 37°C.

[0019] A third aspect of the present invention proposes the application of the above-described gel dressing or the gel dressing prepared by the above-described preparation method in the preparation of a wound-healing drug.

[0020] The application is the use of the above-mentioned gel dressing in the preparation of drugs that promote wound healing.

[0021] The beneficial effects of this invention are as follows: 1. This invention develops a new application for hydrogen produced by microalgae, by covering the wound site with ChAu to achieve targeted hydrogen therapy, thus expanding its application pathways.

[0022] 2. This invention utilizes the biomineralization of Chlorella to synthesize gold nanoparticles, thereby achieving the continuous generation and controllable release of hydrogen under light-driven conditions. This hydrogen production function can be used to realize the application of "biohydrogen" at the wound site, improving its stability and therapeutic effect in complex pathological microenvironments, thus expanding from a single energy source to clinical applications.

[0023] 3. This invention uses puerarin (PUE) and chitosan (CS) as hydrogel matrices to form a self-assembled hydrogel, with algae loaded within the pores of this hydrogel. The algae not only possess the ability to continuously produce hydrogen through photosynthesis, but also contain various reducing cytochromes, lipids, and sugars, serving as host cells for intracellular biomineralization of metal ions. During photosynthesis, they generate electrons, utilizing these electrons to transfer [H₂O → 1] in the photosynthetic chain. 1 / 2O2+ 2H + + 2e - (PSII)], Au 3+ Restore to Au 0 This enables the intracellular mineralization of gold nanoparticles. The intracellular biomineralization of gold nanoparticles also provides additional electrons for photosynthesis. Compared to other metal nanoparticles, gold nanoparticles can release additional electrons through their unique Localized Surface Plasmon Resonance (LSPR), providing more electrons for hydrogenase and thus enhancing the hydrogen production capacity of Chlorella under light conditions.

[0024] 4. The gel dressing containing gold nanoparticles and microalgae prepared in this invention has multiple functions. This invention combines algae and gold nanoparticles to achieve continuous hydrogen production, effectively scavenging reactive oxygen species and reducing oxidative stress. It also promotes angiogenesis and accelerates the healing of diabetic wounds by inhibiting AGE-RAGE expression. Furthermore, algae can lower glucose levels, making it more suitable for treating diabetic wounds. PUE and CS are used as gel carriers; PUE has anti-inflammatory and antioxidant effects, and the addition of CS enhances the mechanical properties of the gel dressing, resulting in high biocompatibility.

[0025] Of course, implementing any product or method of the present invention does not necessarily require achieving all of the advantages described above at the same time. Attached Figure Description

[0026] Figure 1 This is a schematic diagram of the process of in-situ mineralization of gold nanoparticles (ChAu) from Chlorella in Example 1 of the present invention; Figure 2 The ultraviolet spectra of Chlorella (ChAu) after mixing chloroauric acid (HAuCl4), Chlorella and chloroauric acid (HAuCl4) in Example 1 of the present invention and after mineralizing gold nanoparticles are shown. Figure 3 The potential changes of Ch and ChAu in Embodiment 1 of the present invention; Figure 4 The infrared spectra of Ch and ChAu in Embodiment 1 of the present invention; Figure 5The images are transmission electron microscope images of Ch and ChAu in Example 1 of the present invention. The scale bars are 1 μm and 200 nm. Figure 6 This is a transmission electron microscopy elemental mapping of ChAu in Example 1 of the present invention, with a scale bar of 1 μm; Figure 7 This describes the hydrogen production process of Ch and ChAu under illumination in Example 1 of the present invention. Figure 8 The images are scanning electron microscope images of CAP gel and gel in Example 1 and Comparative Example 1 of the present invention. The scale bars are 100 μm and 10 μm. Figure 9 This is a graph showing the modulus changes of CAP gel and gel in Embodiment 1 and Comparative Example 1 of the present invention; Figure 10 Example 1 of the present invention demonstrates the ability of the gel dressing to consume glucose in vitro. Figure 11 Example 1 demonstrates the in vitro hydrogen peroxide decomposition capability of the gel dressing in this invention. Figure 12 Example 2 demonstrates the in vitro cell compatibility of the gel dressing used in this invention. Figure 13 This relates to the in vitro blood compatibility of the gel dressing in Application Example 2 of the present invention; Figure 14 To demonstrate the in vitro reactive oxygen species scavenging capability of the gel dressing in Example 3 of this invention, the scale bar is 50 μm; Figure 15 shows the inhibitory effect of the gel dressing in Example 4 of the present invention on the AGE-RAGE pathway; Figure 16 Example 4 shows the wound healing of diabetic mice after treatment with the gel dressing according to the present invention. Figure 17 For the in vivo wound healing tissue of diabetic mice treated for 12 days in Example 4 of this invention, hematoxylin and eosin (H&E) staining and Masson trichrome staining were performed. The scale bars are 500 μm, 200 μm, and 20 μm. Figure 18 Immunofluorescence staining of CD31 in wounds in diabetic mice treated for 3, 7, and 14 days in this invention is shown in Example 4. The scale bar is 100 μm. Figure 19 Example 4 of this invention demonstrates the regulatory effect of wound dressing on the expression of AGEs in diabetic mice treated for 14 days. Figure 20 The image shows a transmission electron microscope (TEM) image of ChAu cultured at 60 and 80 rpm in Comparative Example 2 of this invention. The scale bars are 2.0 μm and 500 nm. Figure 21The growth curves of natural Chlorella (Ch0) and 5 mM Chlorella (Ch5) in Comparative Example 3 of this invention are shown. Figure 22 These are images of Chlorella agar plates with no additive (Control), CAP gel, and CS added in Comparative Example 4 of this invention. Figure 23 Images of Gel, CS solution and CAP gel in Comparative Example 5 of this invention; The data in the attached figure were analyzed using a one-way ANOVA test to determine the significance of differences between groups. P < 0.001 indicates a highly significant difference, denoted as ***; 0.001 ≤ P < 0.01 indicates a highly significant difference, denoted as **; 0.01 < P ≤ 0.05 indicates a significant difference, denoted as *; and P > 0.05 indicates no significant difference, denoted as ns. Detailed Implementation

[0027] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise defined, the technical terms used below have the same meaning as understood by those skilled in the art.

[0028] Unless otherwise specified, the test materials and reagents used in the following examples are commercially available or prepared by known methods.

[0029] Unless otherwise specified, all techniques or conditions described in the embodiments can be performed in accordance with the techniques or conditions described in the literature in this field or in the product manual. Unless otherwise specified, the quantitative experiments in the following embodiments are all repeated three times or more, and the results are averaged.

[0030] The Chlorella species mentioned below was purchased from the Freshwater Algae Culture Collection of the Chinese Academy of Sciences.

[0031] Example 1: A method for preparing a wound-healing gel dressing based on algae particles includes the following steps: (1) Synthesis and characterization of ChAu: 9 mL of Chlorella containing kanamycin (5 × 10⁻⁶) was used to synthesize ChAu. 7A mixture of 0.08 mg / mL kanamycin (cells / mL) and 1 mL of chloroauric acid (HAuCl4, 1 mM) was placed in an Erlenmeyer flask and incubated at 25°C and 80 rpm for 12 h. Afterward, the Chlorella solution containing the mineralized gold nanoparticles was transferred to a 10 mL centrifuge tube, centrifuged at 5000 rpm for 3 min, and washed with deionized water, repeated three times. This yielded Chlorella with mineralized gold nanoparticles (ChAu).

[0032] (2) Preparation and characterization of the gel dressing: 500 mg of PUE powder was added to a 15 mL centrifuge tube, 10 mL of deionized water was added, and then 200 mg of CS was added to a 15 mL centrifuge tube. 10 mL of 1% glacial acetic acid aqueous solution was added and stirred until well mixed. The mixture was placed in a water bath and heated to 100 °C. The mixture was then cooled to room temperature to obtain the hydrogel precursor solution.

[0033] (3) Add the ChAu obtained in step (1) to the above precursor solution, mix well, and let stand to obtain a gel dressing (CAP gel).

[0034] A schematic diagram of the in-situ mineralization process of gold nanoparticles from Chlorella is shown below. Figure 1 As shown; The UV spectra of Chlorella (Ch), chloroauric acid (HAuCl4), a mixture of Chlorella and chloroauric acid, and Chlorella (ChAu) after mineralization of gold nanoparticles are as follows: Figure 2 As shown, the absorption peak at 261 nm of *Chlorella vulgaris* after mineralization with gold nanoparticles disappeared, proving that the form of gold ions changed; the potential diagrams of *Chlorella vulgaris* and *Chlorella vulgaris* after mineralization with gold nanoparticles are shown below. Figure 3 As shown, the potential of Chlorella decreases after mineralization, which is due to the negative charge of the gold nanoparticles.

[0035] The infrared spectra of Ch and ChAu are as follows Figure 4 As shown, the infrared spectra of Ch and ChAu exhibit high similarity, indicating that the carbohydrates, lipids, and proteins of Chlorella have not been damaged.

[0036] Transmission electron micrographs of Chlorella before and after mineralization are shown below. Figure 5 As shown, compared to Chlorella alone, Chlorella after being treated with mineralized gold nanoparticles has black particles.

[0037] ChAu transmission electron microscopy elemental mapping diagram as follows Figure 6 As shown in the mapping diagram, gold nanoparticles are present. This demonstrates that Chlorella successfully mineralized gold nanoparticles in situ.

[0038] Hydrogen production capacity of Chlorella before and after mineralization Figure 7As shown, the light-induced hydrogen production performance of Chlorella vulgaris (ChAu) after mineralizing gold nanoparticles is improved compared with that of Chlorella vulgaris alone (Ch).

[0039] Scanning electron micrographs of blank gel and gel dressing (CAP gel) are shown below. Figure 8 As shown in the figure, the gel and CAP gel have a mesh-like porous structure, and small round particles can be seen in the CAP gel group. This indicates that the gel dressing was successfully prepared.

[0040] Comparative Example 1: A method for preparing a wound dressing includes the following steps: (The difference from Example 1 is that it does not contain ChAu) Preparation and characterization of the gel dressing: 500 mg of PUE powder was added to a 15 mL centrifuge tube, 10 mL of deionized water was added, and then 200 mg of CS was added to a 15 mL centrifuge tube. 10 mL of 1% glacial acetic acid aqueous solution was added and stirred until well mixed. The mixture was placed in a water bath and heated to 100 °C. After cooling to room temperature, the hydrogel precursor solution (Gel) was obtained.

[0041] The modulus change graphs of gel and CAP gel are shown below. Figure 9 As shown, compared with gel, the storage modulus and loss modulus of CAP gel are basically the same, that is, the addition of mineralized gold nanoparticles to Chlorella has little effect on the gel performance.

[0042] Example 2: A method for preparing a wound dressing includes the following steps: (1) Synthesis of ChAu: 10 mL of Chlorella containing kanamycin (2 × 10⁻⁶) was added. 7 A mixture of 0.08 mg / mL kanamycin (cells / mL) and 2 mL of chloroauric acid (HAuCl4, 2 mM) was placed in an Erlenmeyer flask and incubated at 25°C and 80 rpm for 12 h. Afterward, the Chlorella solution containing the mineralized gold nanoparticles was transferred to a 10 mL centrifuge tube, centrifuged at 5000 rpm for 3 min, and washed with deionized water, repeated three times. This yielded Chlorella (ChAu) containing the mineralized gold nanoparticles.

[0043] (2) Preparation and characterization of the gel dressing: 400 mg of PUE powder was added to a 15 mL centrifuge tube, followed by 10 mL of deionized water. Then, 100 mg of CS was added to the 15 mL centrifuge tube, and 10 mL of 1% glacial acetic acid aqueous solution was added. The mixture was stirred and mixed, and then heated in a water bath at 100°C. After cooling to room temperature, a hydrogel precursor solution was obtained. The ChAu obtained in step (1) was added to the above hydrogel precursor solution, mixed evenly, and allowed to stand to obtain the gel dressing (CAP gel).

[0044] Example 3: A method for preparing a wound dressing includes the following steps: (1) Synthesis of ChAu: 9 mL of Chlorella containing kanamycin (2.5 × 10⁻⁶) was added. 7 A mixture of 0.08 mg / mL kanamycin (cells / mL) and 1 mL of chloroauric acid (HAuCl4, 0.5 mM) was placed in an Erlenmeyer flask and incubated at 25°C and 90 rpm for 14 h. Afterward, the Chlorella solution containing the mineralized gold nanoparticles was transferred to a 10 mL centrifuge tube, centrifuged at 5000 rpm for 3 min, and washed with deionized water, repeated three times. This yielded Chlorella (ChAu) containing the mineralized gold nanoparticles.

[0045] (2) Preparation and characterization of the gel dressing: 200 mg of PUE powder was added to a 15 mL centrifuge tube, followed by 10 mL of deionized water. Then, 20 mg of CS was added to the 15 mL centrifuge tube, and 10 mL of 1% glacial acetic acid aqueous solution was added and stirred until well mixed. The mixture was then placed in a water bath and heated at 110 °C. After cooling to room temperature, a hydrogel precursor solution was obtained. The ChAu obtained in step (1) was added to the above hydrogel precursor solution, mixed evenly, and allowed to stand to obtain the gel dressing (CAP gel).

[0046] Example 4: A method for preparing a wound dressing includes the following steps: (1) Synthesis of ChAu: 9 mL of Chlorella containing kanamycin (3.5 × 10⁻⁶) was added. 7 A mixture of 0.08 mg / mL kanamycin (cells / mL) and 1 mL of chloroauric acid (HAuCl4, 1 mM) was placed in an Erlenmeyer flask and incubated at 26°C and 100 rpm in a constant temperature and light shaker for 10 h. Afterward, the Chlorella solution containing the mineralized gold nanoparticles was transferred to a 10 mL centrifuge tube, centrifuged at 6000 rpm for 5 min, washed with deionized water, and repeated three times. This yielded Chlorella (ChAu) containing the mineralized gold nanoparticles.

[0047] (2) Preparation and characterization of the gel dressing: 1000 mg of PUE powder was added to a 15 mL centrifuge tube, followed by 10 mL of deionized water. Then, 180 mg of CS was added to the 15 mL centrifuge tube, and 10 mL of 1% glacial acetic acid aqueous solution was added. The mixture was stirred and mixed, and then heated in a water bath at 108 °C. After cooling to room temperature, a hydrogel precursor solution was obtained. The ChAu obtained in step (1) was added to the above hydrogel precursor solution, mixed evenly, and allowed to stand to obtain the gel dressing (CAP gel).

[0048] Application Example 1: This application example tests the ability of the gel dressings prepared in Example 1 and Comparative Example 1 to reduce glucose and decompose hydrogen peroxide according to the following steps.

[0049] The samples were divided into three groups: PBS, gel, and CAP gel, with five replicates in each group, for a total of 15 samples. The gel dressings prepared in Example 1 and Comparative Example 1 were co-cultured with BG11 medium containing 33 mM glucose under light conditions for 12 h. Subsequently, 5 μL of the supernatant was taken and the glucose content was determined using a glucose assay kit.

[0050] The samples were divided into three groups: PBS, gel, and CAP gel, with five replicates in each group, for a total of 15 samples. The gel dressings prepared in Example 1 and Comparative Example 1 were co-cultured with BG11 medium containing 16.3 mM H2O2 under light conditions for 1 h. Subsequently, 25 μL of the supernatant was taken and the hydrogen peroxide content was detected using a hydrogen peroxide content detection kit.

[0051] The results of the in vitro glucose-lowering experiment of the gel dressing are as follows: Figure 10 As shown in the figure, the in vitro hydrogen peroxide reduction capacity of the gel dressing is experimentally tested. Figure 11 As shown. From Figure 10 , 11 As can be seen, compared with the PBS and Gel groups, the glucose content and hydrogen peroxide content were significantly reduced after co-culturing with CAP gel. This indicates that CAP gel has the ability to metabolize glucose and has good anti-inflammatory properties.

[0052] Application Example 2: This application example tests the biocompatibility of the gel dressings prepared in Example 1 and Comparative Example 1 according to the following steps.

[0053] MTT assay for cell compatibility: The gel dressings prepared in Example 1 and Comparative Example 1 were co-incubated with 1640 medium (10% serum + 1% penicillin-streptomycin) at 37°C for 24 h, and the supernatant was collected for later use. HUVEC cells were cultured in 1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C and 5% CO2. The relative cell viability of the gel dressing was determined by the MTT assay. In short, 100 μL (5 × 10⁻⁶ cells / mL) of the gel dressing was used. 4 HUVEC cell suspension ( / mL) was seeded into 96-well plates and cultured for 24 h until adherence covered the bottom of the wells. Four groups were formed based on different material components: a blank control group (Control), Gel, CP gel, and CAP gel, with 5 replicates per group. After 24 h, the supernatant of the HUVEC cell suspension was discarded. The above-mentioned dressing co-incubation medium was then co-cultured with the adhered HUVEC cells. The Control group received only 1640 medium as a control. Cultured for another 24 h, then MTT assay solution (medium:MTT = 10:1) was added, and the cells were incubated at 37°C in the dark for 4 h. After 4 h, the supernatant was discarded, and 150 μL of DMSO was added. After gentle dissolution, the OD value was measured at 570 nm using a microplate reader to analyze cytotoxicity. The calculation formula is as follows: Relative cell viability (%) = (OD0 / mL) / (Cellular β-Cell ... 样本 OD 空白 ) / (OD) 对照 OD 空白 ) × 100%. Where OD 样本 The absorbance value of the experimental group sample is OD. 空白 The absorbance value of the blank sample, OD 对照 The absorbance value is for the control sample. The blank sample is a pure cell group without any drug.

[0054] Hemolysis test to detect blood compatibility: Blood from the eyeballs of healthy Balb / c mice was collected, placed in an anticoagulant tube, centrifuged at 5000 rpm for 5 min, washed 5 times with 0.9% NaCl solution to obtain red blood cells (RBCs), and dispersed in 0.9% NaCl solution. Red blood cells incubated with PBS and Triton X-100 were used as negative and positive controls, respectively. Red blood cells were incubated with ChAu prepared in Example 1 (1) at 37°C for 30 min and shaken at 200 rpm. After incubation, the cells were centrifuged at 5000 rpm for 5 min, and the supernatant was collected into a 96-well plate. The absorbance was measured at 540 nm using an ELISA reader. The hemolysis rate was calculated using the following formula: Hemolysis rate (%) = (OD 样本 OD 0.9%NaCl ) / (OD ddH2O OD0.9%NaCl ) × 100%. Where OD 样本 The absorbance value of the experimental group sample is OD. 0.9%NaCl The absorbance value of the 0.9% NaCl sample, OD ddH2O The absorbance value is for the ddH2O sample.

[0055] Results of in vitro cell compatibility experiments of the gel dressing are as follows: Figure 12 As shown, compared with the blank control group (Control), the cell viability of the Gel and CAP gel groups reached 90-100%, with no significant difference, indicating that they have high cell compatibility.

[0056] The results of the in vitro blood compatibility test of CAP gel are as follows: Figure 13 As shown, the hemolysis rate of CAP gel is approximately 1%, less than 5%, indicating that the material has high blood compatibility.

[0057] Application Example 3: This application example tests the antioxidant properties of the gel dressings prepared in Example 1 and Comparative Example 1 according to the following steps.

[0058] The reactive oxygen species (ROS) detection kit (DCFH-DA) was used to detect intracellular ROS levels. The gel dressings prepared in Example 1 and Comparative Example 1 were co-incubated with 1640 medium (10% serum + 1% penicillin antibody) at 37°C for 24 h, and the supernatant was collected. HUVEC cells were seeded into 24-well plates and cultured at 37°C with 5% CO2 for 24 h to induce cell adhesion. The supernatant was discarded, and 1640 medium containing 100 μM H2O2 was added to the cells for 12 h to induce high expression of intracellular ROS. The control group did not receive H2O2. After 24 h, the supernatant was discarded, and the dressing co-incubation medium was added. The control group served as a negative control. After 6 h of co-culture, the supernatant was discarded, and DCFH-DA was diluted with serum-free culture medium at a volume ratio of 1:1000 to a final concentration of 10 μmol / mL. Add 500 μL of diluted DCFH-DA to each group and incubate in a 37°C cell culture incubator in the dark for 20 min. Wash the cells three times with PBS to thoroughly remove any untreated DCFH-DA. Observe the stained cells using an inverted fluorescence microscope.

[0059] The results of the in vitro reactive oxygen species scavenging experiment of the gel dressing are as follows: Figure 14 As shown, compared with the positive control H2O2 group, the fluorescence intensity of reactive oxygen species was significantly reduced after the addition of CAP gel dressing, appearing to be comparable to the control group. This is because hydrogen and algae played a role in resisting oxidative stress. This indicates that CAP gel dressing has a high capacity to reduce oxidative stress.

[0060] Application Example 4: This application example tests the inhibitory effect of the gel dressings prepared in Example 1 and Comparative Example 1 on the AGE-RAGE pathway according to the following steps.

[0061] The gel dressings prepared in Example 1 and Comparative Example 1 were incubated with 1640 medium (10% serum + 1% penicillin-streptomycin) at 37°C for 24 h, and the supernatant was collected for later use. HUVEC cells were cultured in 1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C and 5% CO2. ElaBoX was used. TM The human advanced glycation end products receptor (AGE) assay kit was used to detect decreased RAGE expression. In simple terms, HVUEC cells were seeded into 24-well plates and cultured at 37°C with 5% CO2 for 24 h to induce cell adhesion. The supernatant was discarded, and the cells were cultured in 1640 medium containing 33 mM glucose for 12 h to induce high intracellular RAGE expression. The cell culture medium was then transferred to sterile centrifuge tubes and centrifuged at 1000×g for 10 min at 4°C. The supernatant was collected. Subsequent experiments were conducted according to the ElaBoX (ElaBoX) protocol. TM Follow the instructions in the Human Advanced Glycation End Products Receptor Detection Kit (Solarbio|ElaBoX, SEKH-0302). The experimental results of the gel dressing inhibiting RAGE expression are as follows: Figure 15 As shown, compared with the positive control group (high glucose control group), the expression of RAGE protein was reduced after the addition of CAP gel dressing. This is because hydrogen gas played an inhibitory role in AGE-RAGE, indicating that CAP gel dressing has a high ability to inhibit the expression of the AGE-RAGE pathway.

[0062] Application Example 5: This application example tests the therapeutic effect of the gel dressings prepared in Example 1 and Comparative Example 1 in a diabetic wound model according to the following steps.

[0063] C57 mice (6-8 weeks old) were randomly divided into three groups: diabetic mice (DM), blank gel dressing group (Gel), and drug-loaded gel dressing group (CAP gel), with 6 mice in each group. The diabetic mouse model was established as follows: all mice were fed a high-sugar, high-fat diet and 12 hours of normal water / 12 hours of 10% sucrose solution for one week. Then, for five consecutive days, they were intraperitoneally injected with streptozotocin (STZ) at a dose of 60 mg / kg. Fasting was required before each injection, and the mice were fed a high-sugar, high-fat diet with sucrose solution throughout the treatment. One week after STZ injection, blood glucose levels were relatively stable. Mice with a fasting blood glucose level higher than 11.1 mmol / L were defined as diabetic mice for further experiments. Subsequently, the mice underwent hair removal on their backs, and after isoflurane inhalation anesthesia, a circular wound with a diameter of 10 mm was created on their backs, successfully establishing the diabetic model.

[0064] Diabetic mouse group: only wounds were created and no treatment was performed.

[0065] Dressing Group: The gel dressings prepared in Example 1 and Comparative Example 1 were applied to the wounds on the backs of mice, respectively. The dressings were changed daily for treatment. Wound condition was recorded and observed every other day. At day 14, circular skin tissue was harvested, fixed with 4% paraformaldehyde, embedded in paraffin blocks, and sectioned using a paraffin microtome. The tissues were stained with hematoxylin and eosin (H&E), and the stained sections were observed using a Leica microscope. Masson's trichrome staining method was used to evaluate collagen deposition activity during wound healing. Endothelial cell adhesion molecule-1 (CD31) was used to detect neovascularization in the wound tissue at different stages, and tissues from days 3, 7, and 14 were selected for analysis.

[0066] The wound healing assessment results of diabetic mice 0-14 days after gel dressing treatment are as follows: Figure 16 As shown in the figure, the healing speed of the drug-loaded gel dressing group was the fastest compared to the other two groups, while the healing speed of the DM group was the slowest. This indicates that the gel dressing prepared with Chlorella and gold nanoparticles has the ability to promote the healing of diabetic wounds.

[0067] Images of H&E staining and Masson staining of wound healing tissue in diabetic mice after 14 days of treatment are shown below. Figure 17 As shown, compared with the other two groups, the H&E staining pattern reveals that the drug-loaded gel dressing group has more new blood vessels and fewer inflammatory cells; the Masson staining pattern shows that the drug-loaded gel dressing group has more collagen deposition. These findings indicate that the wound healing effect after treatment with the drug-loaded gel dressing is better, demonstrating that the gel dressing has the ability to promote angiogenesis and collagen deposition.

[0068] Immunofluorescence images of CD31 in diabetic mice at 3, 7, and 14 days after treatment are shown below. Figure 18As shown in the figure. Analysis results showed that, compared with the untreated diabetic mice (DM) group and the blank gel group, the drug-loaded gel dressing group significantly promoted CD31 positive expression; the higher the CD31 expression, the stronger the angiogenesis-promoting ability. The data indicate that CAP gel can promote wound healing in diabetic patients by reducing inflammation, accelerating collagen deposition, and promoting angiogenesis.

[0069] Western blot (WB) expression of AGEs in diabetic mice after 14 days of treatment is as follows: Figure 19 As shown in the figure. Analysis results showed that, compared with the untreated diabetic mouse (DM) group, the drug-loaded gel dressing group significantly inhibited the expression of AGEs, indicating inhibition of the AGE-RAGE pathway. The data suggest that CAP gel can accelerate wound healing in diabetic patients by inhibiting AGE-RAGE expression.

[0070] Example 5: A method for preparing a wound dressing includes the following steps: (1) Synthesis of ChAu: 19 mL of Chlorella containing kanamycin (5 × 10⁻⁶) was used to synthesize ChAu. 7 A mixture of 0.08 mg / mL kanamycin (cells / mL) and 5 mL of chloroauric acid (HAuCl4, 4 mM) was placed in an Erlenmeyer flask and incubated at 24°C and 100 rpm in a constant temperature, light-controlled shaker for 10 h. Afterward, the Chlorella solution containing the mineralized gold nanoparticles was transferred to a 10 mL centrifuge tube, centrifuged at 6000 rpm for 2 min, and washed with deionized water, repeated three times. This yielded Chlorella with mineralized gold nanoparticles (ChAu).

[0071] (2) Preparation and characterization of the gel dressing: 400 mg of PUE powder was added to a 15 mL centrifuge tube, 10 mL of deionized water was added, and then 100 mg of CS was added to a 15 mL centrifuge tube. 10 mL of 1% glacial acetic acid aqueous solution was added and stirred until well mixed. The mixture was placed in a water bath and heated to 100 °C. After cooling to room temperature, the hydrogel precursor solution was obtained.

[0072] Example 6: A method for preparing a wound dressing includes the following steps: (1) Synthesis of ChAu: 19 mL of Chlorella containing kanamycin (4 × 10⁻⁶) was used to synthesize ChAu. 7A mixture of 0.08 mg / mL kanamycin (cells / mL) and 3 mL of chloroauric acid (HAuCl4, 3 mM) was placed in an Erlenmeyer flask and incubated at 26°C and 90 rpm for 14 h. Afterward, the Chlorella solution containing the mineralized gold nanoparticles was transferred to a 10 mL centrifuge tube, centrifuged at 4000 rpm for 5 min, and washed with deionized water, repeated three times. This yielded Chlorella (ChAu) containing the mineralized gold nanoparticles.

[0073] (2) Preparation and characterization of gel dressing: 300 mg of PUE powder was added to a 15 mL centrifuge tube, 10 mL of deionized water was added, and then 150 mg of CS was added to a 15 mL centrifuge tube. 10 mL of 1% glacial acetic acid aqueous solution was added and stirred until well mixed. The mixture was placed in a water bath and heated to 100 °C. After cooling to room temperature, the hydrogel precursor solution was obtained.

[0074] Comparative Example 2: The difference between this comparative example and Example 1 is that in step (1), the constant temperature and light shaking incubation conditions are 25℃, 60rpm, and 12h. The remaining steps are the same as in Example 1.

[0075] By comparing the quantity and distribution of mineralized gold nanoparticles within Chlorella under conditions of 60 rpm and 80 rpm, the results are as follows: Figure 20 As shown, when the stirring speed was reduced to 60 rpm, the number of gold nanoparticles formed by intracellular biomineralization in Chlorella decreased and their distribution became uneven, indicating that a lower stirring speed is not conducive to the uniform transfer of gold ions and sufficient cell contact. This may be because the low stirring speed reduces the mixing efficiency of the culture medium, making Chlorella cells prone to sedimentation or aggregation, preventing some cells from fully uptakeing chloroauric acid, and simultaneously reducing the photosynthetic electron transfer and the intracellular reduction system's response to Au³⁺. + The reducing power of gold nanoparticles is enhanced. Therefore, 80 rpm provides a better mass transfer environment and cell dispersion, which is beneficial for promoting efficient and uniform biomineralization of gold nanoparticles.

[0076] Comparative Example 3: The difference between this comparative example and Example 1 is that in step (1), the concentration of chloroauric acid added is 5 mM.

[0077] The growth curves of Chlorella vulgaris and Chlorella vulgaris after culturing in 5 mM chloroauric acid solution were analyzed, and the results are as follows: Figure 21 When the concentration of chloroauric acid increased to 5 mM, the number of Chlorella decreased significantly, indicating that excessively high concentrations of gold ions can inhibit the growth of Chlorella. This may be due to excessive Au³⁺. +Upon entering cells, chloroauric acid induces strong oxidative stress, leading to the accumulation of reactive oxygen species (ROS), which damages cell membrane structure and the photosynthetic system. Simultaneously, the rapid formation of large numbers of gold nanoparticles increases the metabolic burden on cells, affecting normal cell proliferation. Therefore, an appropriate concentration of chloroauric acid can induce effective mineralization in Chlorella, while excessively high concentrations can cause cytotoxicity and reduce the stability of the biomineralization system.

[0078] Comparative Example 4: The difference between this comparative example and Example 1 is that puerarin is not added in step (2).

[0079] Agar plate coating experiment was conducted on Chlorella co-cultured in CAP gel and CS solution, and the results are as follows: Figure 22 As shown, co-incubation of Chlorella with chitosan alone resulted in the death of the algae, indicating that while chitosan possesses good antibacterial properties, direct contact with Chlorella can cause cytotoxicity. This may be because the positively charged amino groups in the chitosan molecule can interact electrostatically with the negatively charged structures on the surface of Chlorella cells, disrupting cell membrane integrity. Simultaneously, chitosan covering the cell surface hinders nutrient exchange and photosynthesis, ultimately leading to decreased cell viability. Therefore, adding a small amount of chitosan solution to the puerarin hydrogel can not only reduce the direct damaging effect of chitosan on Chlorella but also enhance the mechanical strength of the hydrogel. Comparative Example 5: The difference between this comparative example and Example 1 is that chitosan is not added in step (2).

[0080] The results are as follows Figure 23 As shown, chitosan solution alone cannot form a hydrogel. When either puerarin or chitosan is absent from the system, the properties of the resulting material deteriorate, indicating that the two components play an irreplaceable synergistic role in the composite system. Puerarin possesses antioxidant, anti-inflammatory, and tissue-repair-promoting effects; its absence reduces the system's ability to regulate the inflammatory microenvironment and oxidative stress. Chitosan, as a crucial structural component of the hydrogel network, provides a three-dimensional support structure and enhances material stability; its absence leads to a decrease in the ability to form a gel network. Furthermore, chitosan solution alone cannot form a stable hydrogel because its linear molecular chains lack effective cross-linking, preventing the construction of a three-dimensional network structure. Therefore, the combined participation of puerarin and chitosan is necessary to achieve stable hydrogel construction and multifunctional therapeutic effects.

[0081] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A gel dressing based on algae particles, characterized in that, It includes a dressing matrix, algae particles, and gold nanoparticles, wherein the algae particles and gold nanoparticles are loaded on the dressing matrix, and the dressing matrix comprises puerarin and chitosan.

2. The method for preparing the gel dressing according to claim 1, characterized in that, Includes the following steps: S1: Chlorella solution was mixed with chloroauric acid solution and incubated to obtain Chlorella (ChAu) with mineralized gold nanoparticles. S2: Mix and heat the puerarin solution and the chitosan solution containing glacial acetic acid to obtain the dressing matrix; S3: Add the ChAu obtained in S1 to the dressing matrix in S2 and mix.

3. The preparation method according to claim 2, characterized in that, In S1, the volume ratio of Chlorella solution to chloroauric acid solution is (9~19):(0.5~5).

4. The preparation method according to claim 2, characterized in that, In S1, the concentration of Chlorella was (2~5)×10 7 cells / mL; chloroauric acid concentration is 0.5~4 mM.

5. The preparation method according to claim 2, characterized in that, In S1, the incubation method is to culture in a constant temperature and light shaker for 10-14 h.

6. The preparation method according to claim 3, characterized in that, In S1, the illumination time is 10~14 h, the light intensity is 2000-3000 Lux, the temperature is 24~26℃, and the shaking speed is 80~100 rpm.

7. The preparation method according to claim 2, characterized in that, In S2, the ratio of puerarin, chitosan, glacial acetic acid solution, and water is (200~1000) mg: (20~200) mg: 10 mL: 10 mL.

8. The preparation method according to claim 2, characterized in that, In S2, the heating method is water bath heating at 100-110 ℃.

9. The preparation method according to claim 2, characterized in that, The ratio of ChAu to dressing matrix is ​​2×10. 7 ~5×10 7 cells / mL: 1 mL.

10. The use of the gel dressing according to claim 1 or the gel dressing prepared by the preparation method according to any one of claims 2 to 9 in the preparation of a wound healing drug.

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