Spray type double-layer hydrogel dressing for diabetes wound healing and preparation method of spray type double-layer hydrogel dressing

Abstract

The invention discloses a spray type double-layer hydrogel dressing for healing a diabetic wound and a preparation method of the spray type double-layer hydrogel dressing. The preparation method of the spray type double-layer hydrogel dressing comprises the following steps: spraying a glycyrrhizic acid / natural food-borne nutrient micelle solution, and spraying a calcium ion solution on the surface of the glycyrrhizic acid / natural food-borne nutrient micelle solution to form glycyrrhizic acid / natural food-borne nutrient hydrogel; the glycyrrhizic acid / photosensitizer micelle solution is sprayed on the glycyrrhizic acid / natural food-borne nutrient hydrogel, the calcium ion solution is sprayed on the surface of the glycyrrhizic acid / photosensitizer micelle solution to form glycyrrhizic acid / photosensitizer hydrogel, and the double-layer hydrogel is formed. The spray type hydrogel dressing provided by the invention quickly acts on a wound in a spraying form through a unique double-layer structural design to form an effective wound barrier, and the upper layer and the lower layer are respectively loaded with medicines with different functions to synergistically promote healing of the diabetic wound.

Description

Technical Field

[0001] This invention relates to the field of medical hydrogel dressing technology, and in particular to a spray-type double-layer hydrogel for treating wounds in diabetic patients and its preparation method. Background Technology

[0002] Poor wound healing in diabetes is a common and potentially fatal complication. Traditional dressings, with their limited functionality, cannot meet the complex treatment needs. Hydrogels have attracted attention due to their ability to absorb wound exudate and maintain a moist wound environment. Meanwhile, natural polymer hydrogels and other components show application potential. However, current solutions address issues such as bacterial infection and chronic inflammation associated with this complication. Furthermore, single-layer natural polymer hydrogels suffer from insufficient wound infection barrier function and limited drug loading capacity. Antibiotics, a common antibacterial treatment, are prone to bacterial resistance and toxic side effects. Nanoparticle antibacterial materials, on the other hand, can be stored within the body. Toxicity and stability issues exist in wound dressings. Antimicrobial peptides are costly and unstable, while photosensitizers used in photodynamic therapy suffer from phototoxicity, poor water solubility, and low bioavailability. Natural food-derived nutrients also exhibit poor water solubility and insufficient stability. Existing solutions include developing a multifunctional hydrogel (e.g., published in CN115970046A) made from hyaluronic acid and poly-6-aminocaproic acid; designing a bilayer hydrogel with an antibacterial upper layer and an antioxidant and regenerative lower layer; employing photodynamic therapy as a novel antibacterial method; and introducing glycyrrhizic acid as an active ingredient in wound dressings. However, these solutions have yet to create a diabetic wound dressing that simultaneously provides antibacterial, antioxidant, tissue growth-promoting, and complex wound microenvironments. Summary of the Invention

[0003] To address the shortcomings of single-layer hydrogels, such as insufficient barrier function against wound infection and limited drug loading capacity, making them unsuitable for complex wound microenvironments, this invention aims to provide a sprayable, two-layer hydrogel dressing for diabetic wound healing and its preparation method. This hydrogel dressing possesses highly efficient, broad-spectrum antibacterial properties, reactive oxygen species elimination capabilities, and sprayability, enabling it to address the complex tissue microenvironment of diabetic infected wounds and promote wound healing in diabetic patients.

[0004] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a spray-type double-layer hydrogel dressing for healing diabetic wounds, comprising a lower glycyrrhizic acid / natural food-derived nutrient gel layer and an upper glycyrrhizic acid / photosensitizer gel layer; The glycyrrhizic acid / natural food-derived nutrient gel layer is obtained by cross-linking glycyrrhizic acid / natural food-derived nutrient micelle solution and calcium ion solution; The glycyrrhizic acid / photosensitizer gel layer is obtained by crosslinking glycyrrhizic acid / photosensitizer micelle solution and calcium ion solution.

[0005] Furthermore, the volume ratio of the glycyrrhizic acid / natural food-derived nutrient micelle solution and the glycyrrhizic acid / photosensitizer micelle solution is (20~100):(20~100).

[0006] Furthermore, the concentration of the calcium ion solution is 5~50 mg / ml.

[0007] This invention provides a method for preparing the above-mentioned spray-on double-layer hydrogel dressing for diabetic wound healing, comprising: Glycyrrhizic acid and natural food-derived nutrients are dissolved and reacted in a solvent, and then the first product is obtained by rotary evaporation and vacuum drying. The first product is added to water or other buffer salt solution to obtain glycyrrhizic acid / natural food-derived nutrients micelle solution. Under light-protected conditions, glycyrrhizic acid and photosensitizer are dissolved and reacted in a solvent, and then dried by rotary evaporation and vacuum drying to obtain a second product. The second product is added to water or other buffer salt solution to obtain a glycyrrhizic acid / photosensitizer micelle solution. At the target location, a glycyrrhizic acid / natural food-derived nutrient micelle solution is sprayed, followed by a calcium ion solution to form a glycyrrhizic acid / natural food-derived nutrient hydrogel; then, a glycyrrhizic acid / photosensitizer micelle solution is sprayed onto the glycyrrhizic acid / natural food-derived nutrient hydrogel, followed by a calcium ion solution to form a glycyrrhizic acid / photosensitizer hydrogel.

[0008] Furthermore, glycyrrhizic acid and natural food-derived nutrients were dissolved in a solvent and reacted at 30~100℃ in the dark for 5 min~2 h.

[0009] Furthermore, the mass ratio of glycyrrhizic acid to natural food-derived nutrients is 10: (0.2~2).

[0010] Furthermore, the natural food-derived nutrients include one or more of astaxanthin, curcumin, and lutein.

[0011] Furthermore, glycyrrhizic acid and photosensitizer are dissolved in a solvent and reacted at 30-100°C in the dark for 5 min-2 h.

[0012] Furthermore, the mass ratio of glycyrrhizic acid to photosensitizer is 10:(0.2~50).

[0013] Furthermore, the photosensitizer includes one or more of hematoporphyrin, aluminum phthalocyanine, and aminoporphyrin.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows: The spray-on hydrogel dressing provided by this invention features a unique dual-layer structure design, with the upper and lower layers each carrying drugs with different functions to synergistically promote the healing of diabetic wounds. This invention utilizes the biocompatibility and multiple activities of glycyrrhizic acid, not only improving the in vivo utilization rate of natural dietary nutrients and photosensitizers, but also significantly enhancing the therapeutic effect through its anti-inflammatory and antioxidant properties in synergy with the drugs. The dual-layer structure allows for efficient ROS (reactive oxygen species) generation and sterilization in the upper photosensitizer layer, while the lower layer of natural dietary nutrients balances excess ROS and promotes tissue regeneration. This invention has demonstrated, using a mouse model of full-thickness diabetic skin defects, that this glycyrrhizic acid / photosensitizer / nutrient dual-layer spray-on gel treatment group can effectively promote the healing of diabetic wounds compared to the untreated group and other single-layer hydrogel treatment groups.

[0015] Furthermore, by adjusting the amount of micelle solution used, the thickness of the bilayer hydrogel can be achieved to achieve a similar effect to the Janus membrane, avoiding interference between drugs, ensuring that the drugs exert their effects in the optimal environment, and significantly improving the overall therapeutic effect.

[0016] The preparation method of the present invention is to first prepare two micelle solutions, and then apply the micelle solutions to the wound in sequence by spraying them to form an effective protective barrier in situ. The spray design allows it to easily adapt to wounds of various shapes and depths, achieving full coverage and immediate protection, thus improving the convenience of clinical application.

[0017] In summary, this invention comprehensively improves the clinical efficacy of diabetic wound treatment through its highly efficient antibacterial, anti-inflammatory, and antioxidant properties, layered design, and sprayable application, providing an innovative and efficient solution for the healing of complex wounds. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 This is a synthetic route diagram of the spray-type bilayer hydrogel in this invention; Figure 2 (a) is a schematic diagram of the apparent solubility of glycyrrhizic acid / astaxanthin and free astaxanthin, and (b) is a schematic diagram of the apparent solubility of glycyrrhizic acid / hematoporphyrin and free hematoporphyrin. Figure 3 (a) is a schematic diagram of the sprayability of calcium ion solution, (b) is a schematic diagram of the sprayability of glycyrrhizic acid / astaxanthin micelle solution, and (c) is a schematic diagram of the sprayability of glycyrrhizic acid / hematoporphyrin micelle solution. Figure 4The diagram shows the confocal morphology of the glycyrrhizic acid / astaxanthin / hematoporphyrin bilayer hydrogel. (a) is the front view of the bilayer hydrogel, (b) is the top view of the bilayer hydrogel, and (c) is the side view of the bilayer hydrogel. Figure 5 The therapeutic effects of different spray-on gels on full-thickness skin defects in diabetic mice under laser irradiation were shown in the following groups: (a) glycyrrhizic acid / astaxanthin monolayer gel treatment group, (b) glycyrrhizic acid / hematoporphyrin monolayer gel treatment group, and (c) glycyrrhizic acid / astaxanthin / hematoporphyrin bilayer hydrogel treatment group. Figure 6 These are HE and Masson staining images of a full-thickness skin defect model in diabetic mice under laser irradiation. (a) is the control group, (b) is the glycyrrhizic acid / astaxanthin monolayer gel treatment group, (c) is the glycyrrhizic acid / hematoporphyrin monolayer gel treatment group, and (d) is the glycyrrhizic acid / astaxanthin / hematoporphyrin bilayer hydrogel treatment group. Detailed Implementation

[0020] In this invention, the term "and / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, or B existing alone. A and B can be singular or plural. The character " / " generally indicates that the preceding and following related objects have an "or" relationship.

[0021] In this invention, "at least one" means one or more, and "more than one" means two or more. "At least one of the following" or similar expressions refer to any combination of these items, including any combination of single or multiple items. For example, "at least one of a, b, or c", or "at least one of a, b, and c", can both represent: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, and c can be single or multiple.

[0022] It should be understood that, in various embodiments of the present invention, the sequence numbers of the following processes do not imply the order of execution. Some or all steps may be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present invention.

[0023] The terminology used in the embodiments of this invention is for the purpose of describing particular embodiments only and is not intended to limit the invention. The singular forms “a,” “the,” and “the” as used in the embodiments of this invention and the appended claims are also intended to include the plural forms unless the context clearly indicates otherwise.

[0024] The weights of the relevant components mentioned in the embodiments of this invention can refer not only to the specific content of each component, but also to the proportional relationship between the weights of the components. Therefore, any scaling up or down of the content of the relevant components according to the embodiments of this invention is within the scope disclosed in the embodiments of this invention. Specifically, the mass described in the embodiments of this invention can be a mass unit known in the chemical industry, such as μg, mg, g, or kg.

[0025] The endpoints and any values ​​of the ranges disclosed in this invention are not limited to the precise ranges or values; these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein. In the following, various technical solutions can, in principle, be combined with each other to obtain new technical solutions, which should also be considered as specifically disclosed herein.

[0026] Poor wound healing in diabetes is one of the most common and fatal complications of diabetes, often leading to disability and amputation. Its complexity stems primarily from the combined effects of multiple pathophysiological mechanisms triggered by hyperglycemia, including local susceptibility to bacterial infection, prolonged chronic inflammation, and the generation of excessive reactive oxygen species (ROS). These factors collectively disrupt the normal wound healing process, making treatment extremely complex and difficult. Therefore, how to treat chronic diabetic wounds with bacterial infection has become a crucial and urgent problem to be solved.

[0027] Traditional bandages, sponges, films, and textiles are often single-function and cannot meet the complex needs of diabetic wound healing. New treatment options are needed to improve clinical outcomes and patients' quality of life. Hydrogels, a three-dimensional network structure, can absorb wound exudate and maintain a moist wound environment, thereby promoting wound healing. Both natural and synthetic polymers are widely used in the production of modern hydrogel wound dressings. Natural polymers, with their good biocompatibility, biodegradability, non-toxicity, and low cost, have become a focus in wound dressing applications in recent years. Chinese patent CN115970046A reports a multifunctional hydrogel made from hyaluronic acid and poly6-aminocaproic acid. This gel can promote macrophage polarization, angiogenesis, or cell proliferation, thus regulating angiogenesis and inflammation resolution, thereby treating delayed wound healing in diabetic patients. However, monolayer hydrogels prepared from natural polymers often lack sufficient barrier function against wound infection and have limited drug loading capacity, making them difficult to adapt to the complex wound microenvironment.

[0028] The bilayer hydrogel endows hydrogel dressings with enhanced functionality and adaptability. Specifically, the upper layer of the bilayer dressing can be designed as an antibacterial layer to inhibit the growth and reproduction of pathogenic microorganisms. The lower layer can be designed as a regenerative layer to balance excessive ROS production at the wound site, thereby alleviating inflammation and promoting angiogenesis and cell proliferation. This bilayer dressing design not only enhances its practicality but also provides a new research direction for addressing the interactions between encapsulated drugs, thus significantly improving the healing effect of diabetic wounds.

[0029] In the field of antibacterial hydrogels, most hydrogels utilize components such as antibiotics, nano-antibacterial materials, and antimicrobial peptides, which typically have certain limitations. For example, antibiotic treatment may lead to bacterial resistance and toxic side effects; nano-antibacterial materials may cause toxicity and material stability issues in vivo; and antimicrobial peptides have high production costs and insufficient stability. In recent years, photodynamic therapy, a novel antibacterial method, has received widespread attention due to its advantages such as non-invasiveness, broad-spectrum antibacterial activity, and low drug resistance. Its essence is to generate reactive oxygen species (ROS) through the action of photosensitizers and light sources, thereby killing bacteria. However, problems such as phototoxicity, poor water solubility, and low bioavailability of photosensitizers still need to be addressed. On the other hand, in the field of antioxidants, natural dietary nutrients (such as curcumin and astaxanthin) possess various biological activities, including natural anti-inflammatory and antioxidant activities, but their practical application is also limited by poor water solubility and insufficient stability. Glycyrrhizic acid, due to its multiple biological activities such as anti-inflammatory, antioxidant, antibacterial, cell proliferation promotion, and immunomodulation, has shown significant therapeutic effects in wound healing. Meanwhile, its natural origin, sustained-release properties, multifunctionality, and good biocompatibility make it an ideal active ingredient for wound dressings, especially suitable for the treatment of diabetic wounds.

[0030] To address the above issues, this invention utilizes the antibacterial effect of photodynamic therapy combined with the anti-inflammatory effect of natural dietary nutrients to develop a dual-layer spray-on hydrogel dressing. This spray can quickly and completely cover the wound surface, providing a wound barrier. The upper photodynamic layer generates ROS for sterilization, while the lower nutrient layer balances excess ROS for anti-inflammation. By adjusting the thickness of the upper and lower layers, both antibacterial and anti-inflammatory effects are achieved simultaneously, thereby alleviating inflammation and promoting tissue growth. Currently, there are no publicly available reports on the use of sprayable dual-layer hydrogels for treating diabetic wound healing.

[0031] In this invention, the gel layer that contacts the target location is used as the lower layer.

[0032] The present invention relates to a method for preparing a spray-on bilayer hydrogel for healing diabetic wounds, comprising: S1, Glycyrrhizic acid and natural food-derived nutrients are dissolved and reacted in a solvent, and then the first product is obtained by rotary evaporation and vacuum drying. The first product is added to water or other buffer solution to obtain glycyrrhizic acid / natural food-derived nutrients micelle solution. S2, under light-protected conditions, glycyrrhizic acid and photosensitizer are dissolved and reacted in a solvent, and then dried by rotary evaporation and vacuum drying to obtain a second product. The second product is added to water or other buffer solution to obtain a glycyrrhizic acid / photosensitizer micelle solution. S3. Glycyrrhizic acid / natural food-derived nutrient micelle solution and glycyrrhizic acid / photosensitizer micelle solution are respectively loaded into the lung nebulizer, and the range axis of the nebulizer is set.

[0033] S4. Prepare a calcium ion solution of a certain concentration. At the target location, first spray glycyrrhizic acid / natural food-derived nutrient micelle solution, then spray calcium ion solution to form glycyrrhizic acid / natural food-derived nutrient hydrogel (i.e., the lower anti-inflammatory and growth-promoting layer); then continue to spray micelle solution on the glycyrrhizic acid / natural food-derived nutrient hydrogel, and then spray calcium ion solution to form glycyrrhizic acid / photosensitizer hydrogel (i.e., the upper antibacterial layer), thus obtaining a double-layer hydrogel for treating diabetic wounds.

[0034] This invention provides a spray-type double-layer hydrogel dressing that combines the antibacterial advantages of photodynamic therapy with the anti-inflammatory properties of natural food-derived nutrients. It effectively solves the problems of insufficient antibacterial barrier, limited drug-carrying capacity, and difficulty in adapting to complex wound microenvironments associated with single-layer hydrogels. Its spray form easily adapts to wounds of different shapes and depths, achieving comprehensive coverage and immediate protection. The upper layer of the double-layer hydrogel dressing carries a photosensitizer, which effectively generates reactive oxygen species for sterilization; the lower layer carries natural food-derived nutrients, which balance excess reactive oxygen species, achieving a synergistic effect similar to a Janus membrane, avoiding drug interference, and ensuring that the drugs exert their effects in the optimal environment. The introduced natural herbal extract glycyrrhizic acid has both anti-inflammatory and antioxidant effects, which can reduce inflammatory responses, improve the wound microenvironment, and promote healing. Its good biocompatibility stably loads the photosensitizer and natural food-derived nutrients, improving component utilization and synergistically exerting antibacterial, anti-inflammatory, and regenerative effects. Furthermore, it is biodegradable and has low toxicity, significantly improving treatment efficiency and safety. This unique layered design provides a new research direction for addressing the interactions between encapsulated drugs.

[0035] In some specific embodiments of the present invention, the natural food-derived nutrients include one or more of astaxanthin, curcumin, and lutein. Astaxanthin is a carotenoid, typically extracted from shrimp, crab, and certain algae. Astaxanthin exhibits significant advantages in the treatment of diabetic wounds; its powerful antioxidant capacity can neutralize excessive ROS generated locally in the wound, reduce oxidative stress, and promote tissue repair. As a dressing, astaxanthin has good biocompatibility and can be applied directly to the wound. Photodynamic therapy generates ROS through the synergistic effect of photosensitizers and specific wavelengths of light, thereby achieving highly efficient bactericidal and anti-inflammatory effects. In photodynamic therapy, astaxanthin can act as an adjunct photosensitizer or antioxidant, enhancing the effect of the photodynamic response while preventing damage to normal tissues from excessive ROS generated during photodynamic therapy. This dual effect allows astaxanthin to not only improve bactericidal efficiency in photodynamic therapy but also protect surrounding healthy tissues.

[0036] In some specific embodiments of the present invention, in S1, the mass ratio of glycyrrhizic acid to natural food-derived nutrients is 10:(0.2-2). Glycyrrhizic acid and natural food-derived nutrients are dissolved in a solvent and reacted at 30-100°C in the dark for 5 min to 2 h.

[0037] In some specific embodiments of the present invention, the photosensitizer includes one or more of hematoporphyrin (HP), aluminum phthalocyanine (ALPC), and aminoporphyrin (NH4P). Hematoporphyrin, as a traditional photosensitizer, can effectively absorb light of specific wavelengths (such as 630 nm red light), generating high concentrations of reactive oxygen species (ROS), which have a highly efficient killing effect on pathogenic microorganisms (such as bacteria and fungi). Simultaneously, hematoporphyrin also exhibits selective killing ability against tumor cells, making it play an important role in the treatment of chronic wound infections and refractory wounds.

[0038] In some specific embodiments of the present invention, in S2, the mass ratio of glycyrrhizic acid to photosensitizer is 10:(0.2~50). Glycyrrhizic acid and photosensitizer are dissolved in a solvent and reacted at 30~100°C in the dark for 5 min~2 h.

[0039] In some specific embodiments of the present invention, the solvent of the present invention is an organic solvent, including any one of methanol, acetonitrile and tetrahydrofuran or a mixture thereof in any proportion.

[0040] The present invention also provides a spray-on double-layer hydrogel dressing for healing diabetic wounds, comprising a lower glycyrrhizic acid / natural food-derived nutrient gel layer and an upper glycyrrhizic acid / photosensitizer gel layer; the glycyrrhizic acid / natural food-derived nutrient gel layer is obtained by cross-linking a glycyrrhizic acid / natural food-derived nutrient micelle solution and a calcium ion solution; the glycyrrhizic acid / photosensitizer gel layer is obtained by cross-linking a glycyrrhizic acid / photosensitizer micelle solution and a calcium ion solution.

[0041] In some specific embodiments of the present invention, the volume ratio of glycyrrhizic acid / natural food-derived nutrient micelle solution and glycyrrhizic acid / photosensitizer micelle solution is (20~100):(20~100); the concentration of calcium ion solution is 5-50 mg / ml.

[0042] Taking glycyrrhizic acid, astaxanthin, and hematoporphyrin as examples, the reaction process of this invention is explained as follows: Figure 1 As shown, glycyrrhizic acid and hematoporphyrin were fully dissolved in anhydrous methanol and mixed thoroughly in a certain proportion. Astaxanthin was fully dissolved in tetrahydrofuran. Glycyrrhizic acid and astaxanthin were then mixed thoroughly in a certain proportion. After refluxing in the dark for a period of time, the organic solvents methanol and tetrahydrofuran were removed, and the mixture was redissolved in deionized water to obtain glycyrrhizic acid / hematoporphyrin micelle solution and glycyrrhizic acid / astaxanthin micelle solution. These solutions were then loaded into lung nebulizers, and sprayed sequentially at the wound site with the set range to allow them to fully react with calcium chloride solution, thus obtaining the final glycyrrhizic acid / astaxanthin / hematoporphyrin spray-type bilayer hydrogel.

[0043] To make the technical problem to be solved, the technical solution, and the beneficial effects of the present invention clearer, the present invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present invention and are not intended to limit the present invention.

[0044] The following examples use instruments and equipment conventional in the art. Experimental methods in the following examples, unless otherwise specified, are generally performed under standard conditions or as recommended by the manufacturer. All raw materials used in the following examples are conventional commercially available products with specifications in the art, unless otherwise stated.

[0045] Example 1: (1) Preparation of glycyrrhizic acid / astaxanthin (GA / AST) micelle solution 20 mg of glycyrrhizic acid and 10 mg of astaxanthin were dissolved in 20 ml of methanol and tetrahydrofuran, respectively, and sonicated until fully dissolved. Glycyrrhizic acid and astaxanthin were then thoroughly mixed at a mass ratio of 10:1. After sonication for 30 min, the sample solution showed no precipitate and was clear and bright in color. The reaction was carried out at 45℃ under reflux in the dark for 40 min. The solvent was completely removed by rotary evaporation and vacuum drying. 2 mL of deionized water was added for hydration. The sample was fully dissolved in the reaction flask to obtain the GA / AST micelle solution.

[0046] (2) Preparation of glycyrrhizic acid / hematoporphyrin (GA / HP) micelle solution Under completely dark conditions, 20 mg of glycyrrhizic acid and 10 mg of hematoporphyrin were fully dissolved in 20 ml of methanol and ultrasonically stirred until fully dissolved. The glycyrrhizic acid and hematoporphyrin were then thoroughly mixed at a mass ratio of 10:1 and ultrasonicated for 30 min. After this process, the sample solution showed no precipitate and was clear and bright in color. The reaction was carried out at 45℃ under light-protected reflux for 40 min. The solvent was then completely removed by rotary evaporation and vacuum drying. 2 mL of phosphate buffer was added for hydration. The sample in the reaction flask was fully dissolved, yielding the GA / HP micelle solution.

[0047] (3) Quantitative labeling of two micelle solutions Load 100 μL of GA / AST micelle solution into the lung nebulizer and set the volume to 100 μL; load 100 μL of GA / HP micelle solution into the lung nebulizer and set the volume to 100 μL.

[0048] (4) Preparation of glycyrrhizic acid / astaxanthin / hematoporphyrin (GA / AST / HP) spray-on bilayer hydrogel Anhydrous calcium chloride was dissolved in water to a concentration of 10 mg / ml. At a distance of 5 cm from the target location, GA / AST micelle solution was sprayed first, followed by calcium ion solution. After sufficient reaction, GA / AST hydrogel was formed. Then, GA / HP micelle solution was sprayed onto the GA / AST hydrogel, followed by calcium ion solution. After sufficient reaction, a GA / AST / HP bilayer hydrogel was formed.

[0049] Example 2: (1) Preparation of glycyrrhizic acid / curcumin (GA / CUR) micelle solution 20 mg of glycyrrhizic acid and 10 mg of curcumin were dissolved separately in 20 ml of methanol and ultrasonically stirred until fully dissolved. The glycyrrhizic acid and curcumin were then thoroughly mixed at a mass ratio of 10:2. After ultrasonication for 30 min, the sample solution showed no precipitate and was clear and bright in color. The reaction was carried out at 45℃ under reflux in the dark for 40 min. The solvent was completely removed by rotary evaporation and vacuum drying. 2 mL of deionized water was added for hydration. The sample in the reaction flask was fully dissolved, yielding the GA / CUR micelle solution.

[0050] (2) Preparation of glycyrrhizic acid / aluminum phthalocyanine (GA / ALPC) micelle solution Under completely dark conditions, 20 mg of glycyrrhizic acid and 10 mg of aluminum phthalocyanine were fully dissolved in 20 ml of methanol and ultrasonically stirred until fully dissolved. The glycyrrhizic acid and aluminum phthalocyanine were then thoroughly mixed at a mass ratio of 10:1. After ultrasonication for 30 min, the sample solution showed no precipitate and was clear and bright in color. The reaction was carried out at 30℃, refluxed in the dark for 20 min, and the solvent was completely removed by rotary evaporation and vacuum drying. 2 mL of phosphate buffer was added for hydration. The sample in the reaction flask was fully dissolved, yielding the GA / ALPC micelle solution.

[0051] (3) Quantitative labeling of two micelle solutions Load 100 μL of GA / CUR micelle solution into the nebulizer and set the volume to 50 μL; load 100 μL of GA / ALPC micelle solution into the nebulizer and set the volume to 80 μL. (4) Preparation of glycyrrhizic acid / curcumin / aluminum phthalocyanine (GA / CUR / ALPC) spray-on bilayer hydrogel Anhydrous calcium chloride was dissolved in water to a concentration of 50 mg / ml. At a distance of 5 cm from the target location, GA / CUR micelle solution was sprayed first, followed by calcium ion solution. After sufficient reaction, GA / CUR hydrogel was formed. Then, GA / ALPC micelle solution was sprayed onto the GA / CUR hydrogel, followed by calcium ion solution. After sufficient reaction, a GA / CUR / ALPC bilayer hydrogel was formed.

[0052] Example 3: (1) Preparation of glycyrrhizic acid / lutein (GA / LUT) micelle solution 20 mg of glycyrrhizic acid and 10 mg of lutein were dissolved in 20 mL of methanol and acetonitrile, respectively, and sonicated until fully dissolved. The glycyrrhizic acid and lutein were then thoroughly mixed at a mass ratio of 10:0.5. After sonication for 30 min, the sample solution showed no precipitate and was clear and bright in color. The reaction was carried out at 45°C under reflux in the dark for 40 min. The solvent was completely removed by rotary evaporation and vacuum drying. 2 mL of deionized water was added for hydration. The sample in the reaction flask was fully dissolved, yielding the GA / LUT micelle solution.

[0053] (2) Preparation of glycyrrhizic acid / aminoporphyrin (GA / NH4P) micelle solution Under completely dark conditions, 20 mg of glycyrrhizic acid and 10 mg of aminoporphyrin were fully dissolved in 20 ml of methanol and sonicated until fully dissolved. The glycyrrhizic acid and aminoporphyrin were then thoroughly mixed at a mass ratio of 10:2. After sonication for 30 min, the sample solution showed no precipitate and was clear and bright in color. The reaction was carried out at 60℃ and refluxed in the dark for 45 min. The solvent was then completely removed by rotary evaporation and vacuum drying. 2 mL of phosphate buffer was added for hydration. The sample in the reaction flask was fully dissolved, yielding the GA / HP micelle solution.

[0054] (3) Quantitative labeling of two micelle solutions Load 100 μL of GA / LUT micelle solution into the nebulizer and set the volume to 25 μL; load 100 μL of GA / NH4P micelle solution into the nebulizer and set the volume to 100 μL.

[0055] (4) Preparation of glycyrrhizic acid / lutein / aminoporphyrin (GA / LUT / NH4P) spray-on bilayer hydrogel Anhydrous calcium chloride was dissolved in water to a concentration of 5 mg / ml. At a distance of 5 cm from the target location, GA / LUT micelle solution was sprayed first, followed by calcium ion solution. After sufficient reaction, GA / LUT hydrogel was formed. Then, GA / NH4P micelle solution was sprayed on the GA / LUT hydrogel, followed by calcium ion solution. After sufficient reaction, GA / LUT / NH4P bilayer hydrogel was formed.

[0056] Example 4: (1) Preparation of glycyrrhizic acid / astaxanthin (GA / AST) micelle solution 20 mg of glycyrrhizic acid and 10 mg of astaxanthin were dissolved separately in 20 mL of tetrahydrofuran and sonicated until fully dissolved. The glycyrrhizic acid and astaxanthin were then thoroughly mixed at a mass ratio of 10:1.5. After sonication for 30 min, the sample solution showed no precipitate and was clear and bright in color. The reaction was carried out at 45 °C under reflux in the dark for 40 min. The solvent was completely removed by rotary evaporation and vacuum drying. 2 mL of deionized water was added for hydration. The sample in the reaction flask was fully dissolved, yielding the GA / AST micelle solution.

[0057] (2) Preparation of glycyrrhizic acid / aminoporphyrin (GA / NH4P) micelle solution Under completely dark conditions, 20 mg of glycyrrhizic acid and 10 mg of aminoporphyrin were fully dissolved in 20 ml of methanol and sonicated until fully dissolved. The glycyrrhizic acid and aminoporphyrin were then thoroughly mixed at a mass ratio of 10:0.5 and sonicated for 30 min. The sample solution showed no precipitate and was clear and bright in color. The reaction was carried out at 100℃ and refluxed in the dark for 55 min. The solvent was then completely removed by rotary evaporation and vacuum drying. 2 mL of phosphate buffer was added for hydration. The sample in the reaction flask was fully dissolved, yielding the GA / HP micelle solution.

[0058] (3) Quantitative labeling of two micelle solutions 100 μL of GA / AST micelle solution was loaded into the lung nebulizer, and the volume was set to 30 μL; 100 μL of GA / NH4P micelle solution was loaded into the lung nebulizer, and the volume was set to 50 μL.

[0059] (4) Preparation of glycyrrhizic acid / astaxanthin / aminoporphyrin (GA / AST / NH4P) spray-on bilayer hydrogel Anhydrous calcium chloride was dissolved in water to a concentration of 20 mg / ml. The solution was sprayed onto the wound at a distance of 5 cm from the target location. First, GA / AST micelle solution was sprayed, followed by calcium ion solution. After sufficient reaction, GA / AST hydrogel was formed. Then, GA / NH4P micelle solution was sprayed onto the GA / AST hydrogel, followed by calcium ion solution. After sufficient reaction, a GA / AST / NH4P bilayer hydrogel was formed.

[0060] Example 5: (1) Preparation of glycyrrhizic acid / curcumin (GA / CUR) micelle solution 20 mg of glycyrrhizic acid and 10 mg of curcumin were dissolved separately in 20 ml of acetonitrile and sonicated until fully dissolved. The glycyrrhizic acid and curcumin were then thoroughly mixed at a mass ratio of 10:1. After sonication for 30 min, the sample solution showed no precipitate and was clear and bright in color. The reaction was carried out at 45℃ under reflux in the dark for 40 min. The solvent was completely removed by rotary evaporation and vacuum drying. 2 mL of deionized water was added for hydration. The sample in the reaction flask was fully dissolved to obtain the GA / CUR micelle solution.

[0061] (2) Preparation of glycyrrhizic acid / hematoporphyrin (GA / HP) micelle solution Under completely dark conditions, 20 mg of glycyrrhizic acid and 10 mg of hematoporphyrin were fully dissolved in 20 ml of methanol and sonicated until fully dissolved. Glycyrrhizic acid and astaxanthin were then thoroughly mixed at a mass ratio of 10:2. After sonication for 30 min, the sample solution showed no precipitate and was clear and bright in color. The reaction was carried out at 80℃ under light-protected reflux for 30 min, and the solvent was completely removed by rotary evaporation and vacuum drying. 2 mL of phosphate buffer was added for hydration. The sample in the reaction flask was fully dissolved, yielding the GA / HP micelle solution.

[0062] (3) Quantitative labeling of two micelle solutions Load 100 μL of GA / CUR micelle solution into the nebulizer and set the volume to 60 μL; load 100 μL of GA / HP micelle solution into the nebulizer and set the volume to 80 μL.

[0063] (4) Preparation of glycyrrhizic acid / curcumin / hematoporphyrin (GA / CUR / HP) spray-on bilayer hydrogel Anhydrous calcium chloride was dissolved in water to a concentration of 30 mg / ml. At a distance of 5 cm from the target location, GA / CUR micelle solution was sprayed first, followed by calcium ion solution. After sufficient reaction, GA / CUR hydrogel was formed. Then, GA / HP micelle solution was sprayed onto the GA / CUR hydrogel, followed by calcium ion solution. After sufficient reaction, a GA / CUR / HP bilayer hydrogel was formed.

[0064] Example 6: (1) Preparation of glycyrrhizic acid / lutein (GA / LUT) micelle solution 20 mg of glycyrrhizic acid and 10 mg of lutein were dissolved in 20 mL of acetonitrile and tetrahydrofuran, respectively, and sonicated until fully dissolved. The glycyrrhizic acid and lutein were then thoroughly mixed at a mass ratio of 10:0.5. After sonication for 30 min, the sample solution showed no precipitate and was clear and bright in color. The reaction was carried out at 45°C under reflux in the dark for 40 min. The solvent was completely removed by rotary evaporation and vacuum drying. 2 mL of deionized water was added for hydration. The sample in the reaction flask was fully dissolved, yielding the GA / LUT micelle solution.

[0065] (2) Preparation of glycyrrhizic acid / aluminum phthalocyanine (GA / ALPC) micelle solution Under completely dark conditions, 20 mg of glycyrrhizic acid and 10 mg of aluminum phthalocyanine were fully dissolved in 20 ml of methanol and ultrasonically stirred until fully dissolved. The glycyrrhizic acid and aluminum phthalocyanine were then thoroughly mixed at a mass ratio of 10:1.5. After ultrasonication for 30 min, the sample solution showed no precipitate and was clear and bright in color. The reaction was carried out at 70℃ and refluxed in the dark for 120 min. The solvent was then completely removed by rotary evaporation and vacuum drying. 2 mL of phosphate buffer was added for hydration. The sample in the reaction flask was fully dissolved, yielding the GA / ALPC micelle solution.

[0066] (3) Quantitative labeling of two micelle solutions Load 100 μL of GA / LUT micelle solution into the nebulizer and set the volume to 80 μL; load 100 μL of GA / ALPC micelle solution into the nebulizer and set the volume to 100 μL.

[0067] (4) Preparation of glycyrrhizic acid / lutein / aluminum phthalocyanine (GA / LUT / ALPC) spray-on bilayer hydrogel Anhydrous calcium chloride was dissolved in water to a concentration of 40 mg / ml. At a distance of 5 cm from the target location, GA / LUT micelle solution was sprayed first, followed by calcium ion solution. After sufficient reaction, GA / LUT hydrogel was formed. Then, GA / ALPC micelle solution was sprayed onto the GA / LUT hydrogel, followed by calcium ion solution. After sufficient reaction, a GA / LUT / ALPC bilayer hydrogel was formed.

[0068] Comparative Example 1: (1) Preparation of glycyrrhizic acid / astaxanthin (GA / AST) micelle solution 20 mg of glycyrrhizic acid and 10 mg of astaxanthin were dissolved in 20 ml of methanol and tetrahydrofuran, respectively, and sonicated until fully dissolved. The glycyrrhizic acid and astaxanthin were then thoroughly mixed at a mass ratio of 10:1. After sonication for 30 min, the sample solution showed no precipitate and was clear and bright in color. The reaction was carried out at 45℃ under reflux in the dark for 40 min. The solvent was completely removed by rotary evaporation and vacuum drying. 2 mL of deionized water was added for hydration. The sample in the reaction flask was fully dissolved, yielding the GA / AST micelle solution.

[0069] (2) Preparation of glycyrrhizic acid / astaxanthin (GA / AST) monolayer sprayable hydrogel 100 μL of GA / AST micelle solution is loaded into a lung nebulizer, the volume is set to 50 μL, and sprayed 5 cm vertically at the wound. The micelles react fully with the calcium ion solution at a concentration of 8 mg / ml to obtain a sprayable monolayer hydrogel GA / AST.

[0070] Comparative Example 2: (1) Preparation of glycyrrhizic acid / hematoporphyrin (GA / HP) micelle solution Under completely dark conditions, 20 mg of glycyrrhizic acid and 10 mg of hematoporphyrin were fully dissolved in 20 ml of methanol and sonicated until fully dissolved. Glycyrrhizic acid and astaxanthin were then thoroughly mixed at a mass ratio of 10:2. After sonication for 30 min, the sample solution showed no precipitate and was clear and bright in color. The reaction was carried out at 45℃ under light-protected reflux for 60 min, and the solvent was completely removed by rotary evaporation and vacuum drying. 2 mL of phosphate buffer was added for hydration. The sample in the reaction flask was fully dissolved, yielding the GA / HP micelle solution.

[0071] (2) Preparation of glycyrrhizic acid / hematoporphyrin (GA / HP) monolayer sprayable hydrogel 100 μL of GA / HP micelle solution is loaded into a lung nebulizer, the volume is set to 80 μL, and sprayed 5 cm vertically at the wound to form micelles that fully react with a calcium ion solution with a concentration of 15 mg / ml to obtain a sprayable monolayer hydrogel GA / HP.

[0072] Example 7 (taking Example 1 as an example) Changes in apparent solubility of natural food-derived nutrients and photosensitizers Prepare 4 mg / mL GA / Ast and GA / HP solutions, filter using a 0.22 μm aqueous filter membrane, and obtain the filtrate as an aqueous solution containing free astaxanthin and hematoporphyrin. Measure the absorbance at 472 nm and 670 nm in the aqueous solution before and after filtration using a UV spectrophotometer, and substitute the values ​​into a standard curve to determine the astaxanthin and hematoporphyrin content in the aqueous solution before and after filtration. Figure 2 As shown, glycyrrhizic acid can effectively improve the solubility of astaxanthin and hematoporphyrin.

[0073] Example 8 (taking Example 1 as an example): Calcium ion solution, glycyrrhizic acid / astaxanthin micelle solution, and glycyrrhizic acid / hematoporphyrin micelle solution were placed into spray bottles of the same specification for sprayability testing. For example... Figure 3 As shown, all three solutions can be sprayed at a spray angle greater than 45°, which means that the spray system can evenly and completely cover the surface of diabetic wounds, ensuring rapid coverage at the wound site.

[0074] Example 9 (taking Example 1 as an example): The glycyrrhizic acid / astaxanthin / hematoporphyrin gel was observed using laser confocal scanning electron microscopy (LSCM). Figure 4 As shown, the LSCM image clearly shows the cross-section of the double-layer scaffold, confirming that both layers remain intact, and the interface between the two layers is clearly visible in the double-layer hydrogel.

[0075] Example 10 (taking Example 1 as an example): (1) In vivo therapeutic effect of glycyrrhizic acid / astaxanthin / hematoporphyrin spray-type double-layer hydrogel laser irradiation Six- to eight-week-old male C57 mice, weighing approximately 30-35g, were used as experimental subjects. The development of diabetes symptoms in mice was induced using the common streptozotocin (STZ) intraperitoneal injection method. Successful modeling was defined as a tail vein blood glucose level consistently above 16.7 mmol / L for one week after induction.

[0076] In diabetic model mice, a full-thickness skin lesion with a diameter of approximately 8 mm was created on the back after hair removal. A silicone ring was then attached and sutured around the wound to inhibit skin contraction. Mice with skin injuries were treated with different dressings covering the wounds. Dressings were changed daily, and the mice were irradiated with a 630 nm laser. During dressing changes, the old dressing was thoroughly rinsed off with sterile saline, and the surface moisture was wiped dry with sterile gauze before applying a new dressing. The wound healing process was observed, and the wound area was recorded. A photograph of the wound on day 11 of treatment is shown below. Figure 5 As shown, Figure 5 (a) represents the glycyrrhizic acid / astaxanthin treatment group. Figure 5 (b) represents the glycyrrhizic acid / hematoporphyrin treatment group. Figure 5 The middle (c) group represents the glycyrrhizic acid / astaxanthin / hematoporphyrin treatment group (the left side of the same group is the control group, and the right side is the treatment group). The results showed that on day 11 of treatment, the wound contraction of the glycyrrhizic acid / astaxanthin / hematoporphyrin (GA / AST / HP) spray-on double-layer hydrogel was significantly higher than that of the control and treatment groups, indicating that the spray-on double-layer hydrogel has a good healing effect.

[0077] (2) Healing effect of inflamed areas at the wound site After the treatment cycle, mice were euthanized and dissected, and wound tissue was collected for comparison. The wound tissue was embedded in paraffin, cut into 5 μm sections, and stained with hematoxylin for 5 min. The tissue was then washed with PBS buffer, differentiated with 1% hydrochloric acid-ethanol solution, stained with eosin solution, dehydrated in ethanol, and sealed with a neutral gel. After sectioning, the tissue was stained with Masson's stain to remove excess stain, and then examined under an optical microscope.

[0078] Figure 6 (a) is the control group, (b) is the glycyrrhizic acid / astaxanthin monolayer gel treatment group, (c) is the glycyrrhizic acid / hematoporphyrin monolayer gel treatment group, and (d) is the glycyrrhizic acid / astaxanthin / hematoporphyrin bilayer hydrogel treatment group. According to... Figure 6As can be seen, the control group exhibited severe inflammatory response and significant skin defects. Compared to the control group, the treatment groups showed more complete epithelial recovery and thinner scars. Particularly noteworthy is the superior repair effect demonstrated by the bilayer hydrogel treatment group: more complete reepithelialization, and the growth of more skin appendages such as hair follicles under the epidermis, along with a large number of newly formed capillaries, indicating a significantly enhanced tissue regeneration capacity. Masson staining results showed that the bilayer hydrogel group had richer collagen deposition, and the collagen fibers in the skin tissue were more densely packed and thicker.

[0079] This invention is the first to combine the antibacterial advantages of photodynamic therapy with the anti-inflammatory properties of natural food-derived nutrients, successfully developing a double-layered spray-on hydrogel dressing. By introducing glycyrrhizic acid, a natural herbal extract, the bioavailability of astaxanthin and hematoporphyrin is significantly improved. Applied to wounds in spray form, this dressing easily adapts to wounds of different shapes and depths, achieving comprehensive coverage and immediate protection. Its ingenious double-layer structure features a photosensitizer loaded on the upper layer, effectively generating reactive oxygen species (ROS) for sterilization; the lower layer loads natural food-derived nutrients to balance excess ROS, achieving a synergistic effect similar to a Janus membrane, avoiding drug interference and ensuring the drugs function optimally. Experimental results further verify that even under ordinary flashlight light, the dressing maintains a highly effective antibacterial effect, greatly enhancing its practicality and potential for widespread application. This double-layered hydrogel, through optimized antibacterial, antioxidant, and regenerative functions, significantly improves the healing effect of diabetic wounds. Its sprayability, high efficiency, and biocompatibility offer great potential for the clinical application of diabetic wound dressings, demonstrating a broad prospect in the field of complex wound repair.

[0080] The above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art can still make modifications or equivalent substitutions to the specific implementation of the present invention. Any modifications or equivalent substitutions that do not depart from the spirit and scope of the present invention are within the protection scope of the claims of the present invention pending approval.

Claims

1. A sprayable bilayer hydrogel dressing for diabetic wound healing, characterized in that, The gel layer of glycyrrhizinic acid / natural food-derived nutrient and the gel layer of glycyrrhizinic acid / photodynamic agent are included in the lower layer and the upper layer respectively. The gel layer of glycyrrhizinic acid / natural food-derived nutrient is cross-linked by a glycyrrhizinic acid / natural food-derived nutrient micellar solution and a calcium ion solution. The gel layer of glycyrrhizinic acid / photodynamic agent is cross-linked by a glycyrrhizinic acid / photodynamic agent micellar solution and a calcium ion solution.

2. The sprayable bilayer hydrogel dressing for diabetic wound healing as claimed in claim 1, wherein, The volume ratio of the glycyrrhizinic acid / natural food-derived nutrient micellar solution and the glycyrrhizinic acid / photodynamic agent micellar solution is (20-100):(20-100).

3. The sprayable bilayer hydrogel dressing for diabetic wound healing as claimed in claim 1, wherein, The concentration of the calcium ion solution is 5-50 mg / ml.

4. A method for preparing the sprayable bilayer hydrogel dressing for diabetic wound healing according to any one of claims 1 to 3, characterized in that, The gel layer of glycyrrhizinic acid / natural food-derived nutrient and the gel layer of glycyrrhizinic acid / photodynamic agent are included in the lower layer and the upper layer respectively. The glycyrrhizinic acid / natural food-derived nutrient micellar solution is prepared by dissolving and reacting glycyrrhizinic acid and natural food-derived nutrient in a solvent, and then by rotary evaporation and vacuum drying to obtain a first product, and then by adding the first product into water or other buffer salt solution. The glycyrrhizinic acid / photodynamic agent micellar solution is prepared by dissolving and reacting glycyrrhizinic acid and photodynamic agent in a solvent under light shielding condition, and then by rotary evaporation and vacuum drying to obtain a second product, and then by adding the second product into water or other buffer salt solution. At the target position, the glycyrrhizinic acid / natural food-derived nutrient micellar solution is sprayed, and then the calcium ion solution is sprayed to form a glycyrrhizinic acid / natural food-derived nutrient hydrogel; then the glycyrrhizinic acid / photodynamic agent micellar solution is sprayed on the glycyrrhizinic acid / natural food-derived nutrient hydrogel, and then the calcium ion solution is sprayed to form a glycyrrhizinic acid / photodynamic agent hydrogel.

5. The method of claim 4, wherein the sprayable bilayer hydrogel dressing for diabetic wound healing is prepared by the steps of: The glycyrrhizinic acid and the natural food-derived nutrient are dissolved in a solvent and reacted under light shielding condition at 30-100℃ for 5 min-2 h.

6. The method of claim 4, wherein the sprayable bilayer hydrogel dressing for diabetic wound healing is prepared by the steps of: The mass ratio of the glycyrrhizinic acid to the natural food-derived nutrient is 10:(0.2-2).

7. The method of claim 4, wherein the sprayable bilayer hydrogel dressing for diabetic wound healing is prepared by the steps of: The natural food-derived nutrient includes one or more of astaxanthin, curcumin, and lutein.

8. The method for the preparation of sprayable bilayer hydrogel dressing for diabetic wound healing as claimed in claim 4, wherein, The glycyrrhizinic acid and the photodynamic agent are dissolved in a solvent and reacted under light shielding condition at 30-100℃ for 5 min-2 h.

9. The method of claim 4, wherein the sprayable bilayer hydrogel dressing for diabetic wound healing is prepared by the steps of: The mass ratio of the glycyrrhizinic acid to the photodynamic agent is 10:(0.2-50).

10. The method of claim 4, wherein the sprayable bilayer hydrogel dressing for diabetic wound healing is prepared by the steps of: The photodynamic agent includes one or more of hematoporphyrin, aluminum phthalocyanine, and amioptophyrin.

Images