Immunoregulation hydrogel based on ginsenoside Rd, preparation method and application

Abstract

The invention discloses immunoregulation hydrogel based on ginsenoside Rd, a preparation method and application, and relates to the technical field of biomedical materials and hydrogels. The hydrogel is prepared by taking natural polysaccharides modified by phenylboronic acid groups, derivatives of the natural polysaccharides and methacrylic anhydride modified protein biopolymers as biological matrix materials; ginsenoside Rd is used as a bioactive functional component, and polyvinyl alcohol is used as a cross-linking agent; a first dynamic reversible cross-linked network is formed through chemical cross-linking, and is endowed with injectability, self-healing property and glucose responsiveness; ginsenoside Rd is taken as a main raw material and then subjected to addition polymerization reaction under the irradiation of a photoinitiator and light, a second static covalent cross-linked network is formed, mechanical strength and structural stability are given to the system, in addition, ginsenoside Rd is loaded in the system through boric acid ester bonds, and the slow-release effect is achieved. The problems of stability and bioavailability during local application of the Chinese herbal medicine extract are solved, and healing of chronic and serious wounds can be more effectively promoted.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biomedical materials and hydrogels, and particularly relates to an immunomodulatory hydrogel based on ginsenoside Rd, a preparation method and application. BACKGROUND

[0002] Skin, as the largest organ of the human body, is the first line of defense of the human defense system, and bears important physiological functions such as maintaining homeostasis, resisting invasion of external pathogens and perceiving external stimuli. Large-area skin defects caused by severe trauma, burns or chronic ulcers often exceed the self-regeneration capacity of the skin, easily leading to massive loss of body fluids, electrolyte imbalance and bacterial infection, and seriously threatening the life and health of patients.

[0003] Wound healing is a highly coordinated biological process, which includes four consecutive and partially overlapping stages of hemostasis, inflammation, proliferation and remodeling. Inflammatory response is the core link in the healing process, and if the inflammation lasts too long or is too severe, it will hinder the healing process, leading to delayed wound healing or even development into chronic wounds, which not only seriously affects the quality of life of patients, but also brings significant economic burden to the public health system. The current clinically used wound treatment methods have limited effect, and it is urgent to develop more efficient and reliable treatment strategies.

[0004] Hydrogel is a kind of polymer material with three-dimensional network structure, which has high water content and porosity similar to biological tissues, adjustable physical and chemical properties and excellent biocompatibility. At the same time, it is easy to design molecular structure or load active ingredients, so as to endow it with one or more biological functions such as anti-inflammatory, antioxidant, antibacterial and regulation of cell behavior, and realize the controlled release of drugs or active ingredients, and is widely used in the field of biomedicine. Compared with traditional preformed hydrogels, injectable in-situ forming hydrogels have unique advantages such as ideal flexibility, enhanced portability and excellent fit for deep and irregularly shaped wounds, and have become the ideal choice of wound healing hydrogel materials.

[0005] Currently, the immunomodulatory strategies using anti-inflammatory drug formulations (such as dexamethasone, rapamycin), cells and / or cytokines for local delivery have limitations in pharmacokinetics, effective drug concentration, tissue toxicity, high cost and production complexity, and are functionally single. Ginsenoside Rd is a diol-type saponin extracted from natural plants such as ginseng and panax notoginseng. Previous studies have shown that it has anti-inflammatory, antioxidant, cardiovascular protection, neuroprotection and immunomodulatory functions. One of the main challenges in the application of herbal extracts is to ensure their stability and bioavailability. Therefore, protecting the biological activity of ginsenoside Rd by hydrogel and achieving its local sustained release application at the wound site to regulate the microenvironment of the wound, relieve persistent inflammation and high oxidative stress, and improve angiogenesis is an important research value and clinical application prospect for accelerating the healing of chronic wounds and severe trauma. SUMMARY

[0006] In view of the above-mentioned shortcomings and deficiencies of the prior art, the present application provides an immunomodulatory hydrogel based on Chinese herbal extract (ginsenoside Rd), a preparation method and application, to solve the problem of stability and bioavailability of Chinese herbal extract when applied locally, and to more effectively promote the healing of chronic and severe wounds.

[0007] According to one aspect of the present application, an immunomodulatory hydrogel based on ginsenoside Rd is provided, comprising: a benzene boronic acid group modified natural polysaccharide and its derivative, a methacrylic anhydride modified protein-based biological macromolecule, as a biological matrix material; ginsenoside Rd as a bioactive functional component; polyvinyl alcohol as a crosslinking agent; the hydrogel has a double crosslinking structure, first, through the vicinal diol or polyol group on the ginsenoside Rd chain and the vicinal diol structure of polyvinyl alcohol, a covalent chemical crosslinking reaction occurs with the benzene boronic acid group modified natural polysaccharide to form a first heavy dynamic reversible crosslinking network, which gives the hydrogel injectability, self-healing and glucose responsiveness, at the same time, ginsenoside Rd is loaded in the gel network through borate ester bond to achieve a slow release effect; then, the methacrylic anhydride modified protein-based biological macromolecule undergoes polyaddition reaction under the action of a photoinitiator and light irradiation to form a second heavy static covalent crosslinking network, which gives the hydrogel mechanical strength and structural stability.

[0008] Optionally, the benzene boronic acid group modified natural polysaccharide includes benzene boronic acid modified hyaluronic acid, benzene boronic acid modified chitosan, benzene boronic acid modified dextran; the methacrylic anhydride modified protein-based biological macromolecule includes methacrylated silk fibroin, methacrylated gelatin.

[0009] Optionally, the concentration of the natural polysaccharide modified by the phenyl boronic acid group in the hydrogel ranges from 1 wt% to 3 wt%; the concentration of the protein-based bio-macromolecule modified by the methacrylic anhydride in the hydrogel ranges from 4 wt% to 12 wt%; the concentration of the ginsenoside Rd in the hydrogel ranges from 0.1 mg / mL to 5 mg / mL; and the concentration of the polyvinyl alcohol in the hydrogel ranges from 0.25 wt% to 1 wt%.

[0010] According to another aspect of the present application, a method for preparing the immunomodulatory hydrogel based on ginsenoside Rd is provided, for preparing the hydrogel as described above, comprising: dissolving polyvinyl alcohol in a PBS buffer solution to obtain a polyvinyl alcohol solution as a crosslinking agent; dissolving a natural polysaccharide bio-macromolecule modified by a phenyl boronic acid group in a PBS buffer to obtain a phenyl boronic acid-polysaccharide solution; dissolving a protein-based bio-macromolecule modified by a methacryl group in a PBS buffer containing a photoinitiator to obtain a methacrylated protein solution; mixing the phenyl boronic acid-polysaccharide solution with the methacrylated protein solution to obtain a hydrogel precursor solution; dissolving ginsenoside Rd in a dimethyl sulfoxide solution to prepare an Rd stock solution, and diluting the Rd stock solution with a PBS buffer; adding the diluted Rd solution into the hydrogel precursor solution, mixing uniformly, and then adding the polyvinyl alcohol solution, to form a first heavy dynamic reversible crosslinking network having injectability, self-healing property and glucose responsiveness, thereby obtaining a primary hydrogel; and then subjecting the primary hydrogel to photo-crosslinking under light irradiation, so that the protein modified by the methacrylic anhydride in the primary hydrogel undergoes further polyaddition reaction, to form a second heavy static covalent crosslinking network having mechanical strength and structural stability, thereby obtaining a final hydrogel, i.e. the immunomodulatory hydrogel based on ginsenoside Rd.

[0011] Optionally, the polyvinyl alcohol is dissolved at a temperature of 80 ℃, and the PBS buffer has a pH ranging from 7.2 to 7.4.

[0012] Optionally, the natural polysaccharide bio-macromolecule modified by the phenyl boronic acid group is dissolved at room temperature at a stirring speed of 600 rpm to 1000 rpm, and the obtained phenyl boronic acid-polysaccharide solution has a concentration ranging from 2 wt% to 6 wt%.

[0013] Optionally, the protein-based bio-macromolecule modified by the methacryl group is dissolved at a temperature ranging from 60 ℃ to 70 ℃, the photoinitiator has a concentration ranging from 0.5 wt% to 1 wt%, and the obtained methacrylated protein solution has a concentration ranging from 8 wt% to 24 wt%.

[0014] Optionally, the concentration of the Rd stock solution is 100 mg / mL, and the concentration of the diluted Rd solution ranges from 1 mg / mL to 50 mg / mL.

[0015] Optionally, the wavelength of the light is 395 nm or 405 nm, and the light irradiation time is 15-60 seconds.

[0016] According to another aspect of the present application, there is provided a use of the ginsenoside Rd-based immunomodulatory hydrogel as described above or the ginsenoside Rd-based immunomodulatory hydrogel prepared by the preparation method as described above in the preparation of a medicament or medical dressing for promoting wound healing.

[0017] The present application has the following advantages: (1) The prior art mostly simply physically embeds a drug in a hydrogel. The present application utilizes the o-diol / polyol group in the molecular structure of ginsenoside Rd to directly participate in a dynamic covalent cross-linking reaction with a phenylboronic acid-modified polysaccharide to become a chemical component for constructing a first heavy network. This not only solves the problems of poor water solubility and easy inactivation of Rd, realizes efficient and stable loading of the drug, but also makes the drug uniformly distributed in the gel network by chemical bonding, thereby laying a solid foundation for realizing sustained release.

[0018] (2) The hydrogel proposed by the present application is synergized and combined by a "phenylboronic ester bond dynamic network" and a "light cross-linking static network", so that the hydrogel has excellent injectability, self-healing ability, perfect fitting to irregular wounds, and enhanced mechanical strength and structural stability. This structural design overcomes the limitation of poor mechanical properties of a single dynamic hydrogel and realizes complementary advantages in performance.

[0019] (3) The hydrogel proposed by the present application realizes multifunctional synergistic regulation of a complex pathological microenvironment. Ginsenoside Rd as a core functional component of the hydrogel can exert multiple biological activities such as anti-inflammatory, antioxidant, immunomodulatory and pro-angiogenic activities, directly targeting the core pathological characteristics of diabetic wounds such as "persistent excessive inflammation, high oxidative stress and vascularization disorders". The hydrogel matrix itself (such as hyaluronic acid and gelatin) can also provide a moist healing environment and simulate the extracellular matrix. The synergistic enhancement effect of "material function" and "drug activity" is expected to more effectively reverse the pathological microenvironment and promote orderly tissue regeneration, solving the limitation of insufficient efficacy of existing single-function preparations.

[0020] (4) The hydrogel proposed by the present application has the ability to release Rd in response to glucose based on the chemical property that the phenylboronic ester bond is more easily broken in a high glucose concentration environment. The hydrogel can accelerate the release of Rd in a high glucose environment of a diabetic wound. This stimulus-responsive release can realize "on-demand drug delivery", further improve the therapeutic effect and reduce potential side effects, and has a significant advantage compared to traditional constant release systems.

[0021] (5) The main matrix (such as hyaluronic acid, gelatin, and polyvinyl alcohol) selected by the hydrogel proposed in this invention are all natural polymers or their derivatives with good biocompatibility. The materials have good biocompatibility, and the preparation conditions are mild and the operation is simple and efficient. Attached Figure Description

[0022] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this invention, illustrate exemplary embodiments of the invention and are used to explain the invention, but do not constitute an undue limitation of the invention. In the drawings: Figure 1 This is a schematic flowchart of the preparation method of the immunomodulatory hydrogel based on ginsenoside Rd according to the present invention. Figure 2 The images show actual pictures of the preparation processes of the Rd-free and Rd-containing hydrogels of this invention. Figure 3 The mechanical properties characterization diagram of the Rd hydrogel of this invention is shown. Figure 4 This is a characterization diagram of the injectability and self-healing properties of the Rd hydrogel of the present invention; Figure 5 This is a characterization diagram of the in vitro cell compatibility of the Rd hydrogel of the present invention; Figure 6 This is a characterization diagram of the in vitro angiogenesis promotion effect of the Rd hydrogel of the present invention; Figure 7 This is a characterization diagram of the in vitro antioxidant properties of the Rd hydrogel of the present invention; Figure 8 This is a characterization diagram of the anti-inflammatory and immunomodulatory properties of the Rd hydrogel of this invention; Figure 9 This is a macroscopic diagram illustrating how the Rd hydrogel of this invention promotes wound healing in mice. Figure 10 HE staining of skin tissue from a mouse wound, showing how the Rd hydrogel of this invention promotes wound healing. Detailed Implementation

[0023] To enable those skilled in the art to better understand the present application, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present application, and not all of them. Based on the embodiments of the present application, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present application. It should be noted that, unless otherwise specified, the embodiments and features in the embodiments of the present application can be combined with each other.

[0024] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0025] The terms “comprising” and “having”, and any variations thereof, in the specification and claims of this application are intended to cover non-exclusive inclusion, for example, a process, method, product, or apparatus that includes a series of steps or units is not necessarily limited to those steps or units that are explicitly listed, but may include other steps or units that are not explicitly listed or that are inherent to such process, method, product, or apparatus.

[0026] Example 1: This embodiment provides an immunomodulatory hydrogel based on ginsenoside Rd. The hydrogel uses natural polysaccharides and their derivatives modified with phenylboronic acid groups, and protein-based biopolymers modified with methacrylic anhydride as the biomatrix material; ginsenoside Rd as the bioactive functional component; and polyvinyl alcohol as the crosslinking agent. It possesses a unique dual crosslinking network mechanism. 1. First-layer dynamic reversible cross-linking network: Utilizing the vicinal diol / polyol groups on the ginsenoside Rd chain and the vicinal diol structure of polyvinyl alcohol, a covalent chemical cross-linking reaction is carried out with natural polysaccharides modified with phenylboronic acid groups to form a first-layer dynamic reversible cross-linking network. This dynamic reversible cross-linking network endows the hydrogel with excellent injectability, self-healing properties, and glucose responsiveness. Simultaneously, ginsenoside Rd is stably loaded within the gel network through borate ester bonds, achieving a sustained-release effect.

[0027] 2. Second static covalent cross-linking network: The methacrylic anhydride-modified protein-based biopolymers in the system undergo addition polymerization under photoinitiator and specific wavelength light irradiation, forming a second static covalent cross-linking network. This static covalent cross-linking network enhances the mechanical strength and structural stability of the hydrogel.

[0028] Through the synergistic effect of the above-mentioned "dynamic-static" dual network structure, the immunomodulatory hydrogel based on ginsenoside Rd of this invention simultaneously possesses good injection molding capability, adaptive fit to irregular wound surfaces, and stable mechanical support.

[0029] In a preferred embodiment, the natural polysaccharides modified with the phenylboronic acid group include, but are not limited to: phenylboronic acid-modified hyaluronic acid, phenylboronic acid-modified chitosan, and phenylboronic acid-modified dextran. Phenylboronic acid-modified hyaluronic acid is preferred.

[0030] The aforementioned natural polysaccharide derivatives include, but are not limited to: carboxymethyl chitosan (carboxymethyl chitosan is a derivative of chitosan).

[0031] The aforementioned methacrylic anhydride-modified (i.e., methacrylated) protein biopolymers include, but are not limited to: methacrylated silk fibroin and methacrylated gelatin. Methacrylated gelatin is preferred.

[0032] The concentration ranges of each component in the hydrogel are as follows: Natural polysaccharides and their derivatives modified with phenylboronic acid groups: 1 wt% ~ 3 wt%; Methacrylic anhydride-modified protein-based biopolymers: 4 wt% ~ 12 wt%; Ginsenoside Rd: 0.1 mg / mL ~ 5 mg / mL; Polyvinyl alcohol: 0.25 wt% ~ 1 wt%.

[0033] Example 2: This embodiment provides a method for preparing an immunomodulatory hydrogel based on ginsenoside Rd as described in Example 1, with the steps and procedures as follows. Figure 1 Mainly includes: S1, Prepare a polyvinyl alcohol solution: Polyvinyl alcohol (PVA) was dissolved in PBS buffer (pH=7.2~7.4) at 80 °C to obtain a polyvinyl alcohol solution, which was used as a crosslinking agent.

[0034] S2, Preparation of phenylboronic acid-polysaccharide solution: Natural polysaccharide biopolymers modified with phenylboronic acid groups (such as phenylboronic acid-modified hyaluronic acid, HA-PBA) were dissolved in PBS buffer (pH=7.2~7.4) at room temperature with a stirring speed of 600~1000 r / min to obtain phenylboronic acid-polysaccharide solutions with a concentration range of 2 wt%~6 wt%.

[0035] S3, Prepare a methacrylamide protein solution: Photoinitiators (such as lithium phenyl (2,4,6-trimethylbenzoyl)phosphate, LAP) were dissolved in PBS buffer (pH=7.2~7.4) to prepare photoinitiator solutions with a concentration of 0.5 wt%~1 wt%. Subsequently, methacrylamide-modified protein biopolymers (such as methacrylamide gelatin, GelMA) were added to this photoinitiator solution and stirred and dissolved at 60-70°C to obtain a methacrylamide protein solution with a concentration of 8 wt% to 24 wt%.

[0036] S4, Preparation of hydrogel precursor solution: The phenylboronic acid-polysaccharide solution obtained in S2 and the methacrylamide protein solution obtained in S3 were mixed evenly at a volume ratio of 1:1 to obtain the hydrogel precursor solution.

[0037] S5, Prepare Rd solution: Ginsenoside Rd powder was dissolved in dimethyl sulfoxide (DMSO) to prepare an Rd stock solution with a concentration of 100 mg / mL; The stock solution was then diluted with PBS buffer and heated in a 60 °C water bath to aid dissolution, yielding an Rd solution with a concentration of 1–50 mg / mL.

[0038] S6, Construct the first layer of dynamic cross-linking network: One-tenth of the total volume of the Rd solution prepared in S5 was added to the hydrogel precursor solution obtained in S4, and the mixture was vortexed to achieve a final concentration of ginsenoside Rd of 0.1-5 mg / mL in the hydrogel. At room temperature, a certain amount of polyvinyl alcohol solution prepared in S1 was added and vortexed rapidly. Within a few seconds, dynamic and reversible borate ester bonds were formed between the vicinal diol / polyol structure of ginsenoside Rd, the vicinal diol structure of PVA, and the phenylboronic acid group, thus rapidly constructing the first dynamic cross-linking network and forming a primary hydrogel with injectability, self-healing, and glucose responsiveness.

[0039] S7, Construct the second static crosslinking network: The primary hydrogel (i.e., the uncured / uncured hydrogel) that has formed the first network in step S6 is photocrosslinked under light irradiation at a wavelength of 395 nm or 405 nm for 15-60 s. The light irradiation initiates a photopolymerization addition reaction of the methacrylamide protein in the system, forming a second static covalent crosslinking network, which significantly enhances the final mechanical strength and overall structural stability of the hydrogel, resulting in the final hydrogel product, namely the immunomodulatory hydrogel based on ginsenoside Rd in this invention.

[0040] The dual-network structure design in this embodiment of the invention enables the hydrogel to simultaneously possess injectable self-healing properties and enhanced mechanical properties (see Example 4 below for specific performance characterization), achieving complementary advantages in performance. By enabling ginsenoside Rd to directly participate in the formation of the cross-linking network, efficient and stable drug loading and controllable release are achieved, solving the problems of poor water solubility and easy inactivation. This hydrogel can achieve multifunctional synergistic regulation of the complex pathological microenvironment of chronic wounds (such as excessive inflammation and high oxidative stress), combining the biological functions of the material itself with the therapeutic activity of the drug.

[0041] It should be further noted that those skilled in the art will understand that this invention aims to provide a relatively universal overall preparation process or technical principle, and to cover as many different applicable scenarios and conditions as possible (such as different raw material characteristics, production scale, product demand, etc.). The specific operations and parameters listed in the embodiments may be adjusted due to differences in scale or conditions during actual implementation, and are not exhaustive of all implementation methods. Those skilled in the art can make appropriate and flexible adjustments to the process conditions according to the actual situation without departing from the technical principles of this invention, as long as the same or similar technical effects as the embodiments of this invention can be achieved. For example, the "80 ℃ condition" mentioned in step S1 can also achieve the corresponding dissolution effect by using similar temperature conditions such as 81 ℃ or 79 ℃ in actual operation, and the processing time can also be adjusted according to the actual material characteristics and production scale. Other operating conditions (such as stirring speed, reaction time, etc.) are also subject to the same principle and will not be described in detail here.

[0042] Example 3: This embodiment further provides a method for preparing an immunomodulatory hydrogel based on ginsenoside Rd. The difference between Example 3 and Example 2 is that Example 3 explicitly selects phenylboronic acid-modified hyaluronic acid (HA-PBA) and methacrylamide gelatin (GelMA) as the biomatrix materials, and provides complete and detailed process steps from raw material synthesis (such as the preparation of HA-PBA) to the final hydrogel preparation. It also provides a preparation process for hydrogels loaded with different concentrations of ginsenoside Rd (Rd). The preparation process is as follows: S1, Preparation of phenylboronic acid-modified hyaluronic acid: S11, add 1g of hyaluronic acid (HA, 90~100 kDa) to 100 mL of PBS solution, and stir at room temperature and 1000 r / min until the HA powder is completely dissolved; S12, 1.4 g of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine hydrochloride (DMTMM) was added to the HA solution of S11 above, and the reaction was carried out at room temperature for 1 hour; S13: Add 0.114 g of 3-aminophenylboronic acid to 1 mL of DMSO solution and dissolve it completely at room temperature. Then add it dropwise to the above S12 solution and react at room temperature for 24 hours. S14: The solution after the reaction in S13 was placed in a dialysis bag (MWCO=14000 Da) and dialyzed in deionized water at room temperature for 3 days to remove unreacted byproducts and small molecules. The purified solution was then lyophilized to obtain the solid product HA-PBA, which was stored at -20 °C for later use.

[0043] S2, Preparation of the hydrogel precursor solution: S21, 0.4 g of phenylboronic acid-modified hyaluronic acid (i.e., HA-PBA prepared in step S1) was dissolved in 10 mL of PBS solution and completely dissolved at room temperature and 1000 r / min. S22, 0.1 g of photoinitiator lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP) was added to 10 mL of PBS solution and placed in a 50 ℃ water bath until completely dissolved to prepare a 1 wt% LAP solution; S23, 1.6 g of methacrylamide gelatin (GelMA) was added to the LAP solution prepared in S22 above, and the solution was dissolved in a water bath at 65 °C until completely dissolved to obtain a 16 wt% GelMA solution (containing 1 wt% LAP). S24. Mix the HA-PBA solution prepared in S21 with the GelMA solution prepared in S23 at a volume ratio of 1:1, and centrifuge at 3000 rpm for 2 min to obtain a homogeneous hydrogel precursor solution of 4 wt% HA-PBA / 8 wt% GelMA.

[0044] S3, Preparation of PVA solution: S31, 0.3 g of polyvinyl alcohol (PVA) was dissolved in 10 mL of PBS solution under 80 ℃ water bath conditions to obtain a 3 wt% PVA solution.

[0045] S4, Preparation of Rd solutions of different concentrations: S41, 10 mg of ginsenoside Rd (Rd) powder was added to 100 μL of DMSO solution and dissolved completely at room temperature to prepare an Rd stock solution with a concentration of 100 mg / mL;

[0046] S42, take 20 μL of Rd stock solution and add it to 80 μL of PBS solution, and dissolve it in a water bath at 60 ℃ to obtain a 20 mg / mL Rd solution, which is denoted as Rd-1; S43, add 10 μL of Rd stock solution to 90 μL of PBS solution, and dissolve in water at 60 ℃ to obtain a 10 mg / mL Rd solution, denoted as Rd-2.

[0047] S5, Preparation of hydrogels containing ginsenoside Rd: S51, take 10 μL of Rd-1 and Rd-2 solutions respectively, add them to 150 μL of HA-PBA / GelMA precursor solution prepared by S24, vortex for 10 s to mix thoroughly, and place them in a 60 ℃ water bath for 5 min to assist crosslinking for 5 min. S52, then add 50 μL of 2 wt% PVA solution and quickly vortex to form a gel. At this time, the vicinal diol / polyol groups on the Rd chain and the vicinal diol structure in PVA form borate ester dynamic chemical bonds with the phenylboronic acid groups on the HA-PBA chain, thus constructing the first dynamic cross-linking network. S53, the system obtained in S52 is further irradiated with a 405 nm wavelength blue light flashlight for 15 s to allow the internal GelMA to complete photocrosslinking, forming a robust second network, ultimately yielding hydrogels loaded with different concentrations of Rd, which are the immunomodulatory hydrogels based on ginsenoside Rd in this invention. The hydrogel prepared from the Rd-1 solution is labeled Gel@RD 1, and the hydrogel prepared from the Rd-2 solution is labeled Gel@RD 0.5.

[0048] This invention also provides a control group—the preparation process of a hydrogel without ginsenoside Rd, as follows: S6, Preparation of hydrogel without ginsenoside Rd (control group): S61, 50 μL of PVA solution was added to 150 μL of HA-PBA / GelMA precursor solution prepared in S24 and rapidly vortexed to form a gel. During this process, the phenylboronic acid groups on HA-PBA and the cis-diol units on PVA formed dynamic covalent bonds of borate esters. In addition, a large number of hydrogen bonds were formed between HA-PBA, GelMA and PVA, constructing the first dynamic reversible cross-linking network. S62. Subsequently, the hydrogel obtained in S61 was irradiated with a 405 nm wavelength blue light flashlight for 15 s. The light irradiation triggered an addition polymerization reaction of GelMA, which further formed a stable second cross-linked network, improving the overall mechanical properties and structural stability of the hydrogel. This hydrogel without ginsenoside Rd was labeled as Gel.

[0049] The preparation processes of the above-mentioned hydrogels containing ginsenoside Rd (Gel@RD) and hydrogels without ginsenoside Rd (Gel) are shown in the following physical examples. Figure 2 As shown. It should be noted that, Figure 2 In this context, HA-PBA+GelMA means that Rd is not added after the precursor solution is prepared by HA-PBA+GelMA, while HA-PBA+GelMA+Rd means that Rd is added after the precursor solution is prepared by HA-PBA+GelMA.

[0050] Example 4: The present invention provides performance characterization of the above-mentioned immunomodulatory hydrogel based on ginsenoside Rd (hereinafter referred to as Rd hydrogel), including: (1) Mechanical properties: Mechanical properties were tested using a universal testing machine. The specific experimental steps and results analysis are as follows: Take 300 μL of HA-PBA / GelMA hydrogel precursor solution into a 1.5 mL flat-bottomed EP tube, add 100 μL of PVA solution, and quickly vortex to form a gel, resulting in a cylindrical uncured gel with a diameter of 10 mm.

[0051] Take 300 μL of HA-PBA / GelMA hydrogel precursor solution into a 1.5 mL flat-bottomed EP tube, then add 10 μL of Rd solution with concentrations of 20 mg / mL and 40 mg / mL respectively, followed by 100 μL of PVA solution, and quickly vortex to form a gel, obtaining cylindrical uncured Gel@RD 0.5 and Gel@RD 1 hydrogels with a diameter of 10 mm.

[0052] Take 300 μL of HA-PBA / GelMA hydrogel precursor solution into a 1.5 mL flat-bottomed EP tube, then add 100 μL of LPVA solution, quickly vortex to form a gel, and then irradiate under a curing lamp at a wavelength of 405 nm for 15 s to prepare a cylindrical gel hydrogel with a diameter of 10 mm.

[0053] Take 300 μL of HA-PBA / GelMA hydrogel precursor solution into a 1.5 mL flat-bottomed EP tube, then add 10 μL of Rd solution with concentrations of 20 mg / mL and 40 mg / mL respectively, followed by 100 μL of PVA solution. Vortex rapidly to form a gel, and then irradiate under a curing lamp at a wavelength of 405 nm for 15 s to prepare cylindrical Gel@RD 0.5 and Gel@RD 1 hydrogels with a diameter of 10 mm.

[0054] Using a universal testing machine (SHIMADZU), the uncured Gel, Gel@RD 0.5, Gel@RD 1 and cured Gel, Gel@RD 0.5, Gel@RD 1 hydrogel samples were compressed to 70% of their maximum strain at a compression rate of 0.5 mm / min. Stress-strain curves were plotted for each hydrogel sample, and the compressive modulus of each hydrogel was obtained by calculating the slope of the linear region of the stress-strain curve from 0 to 20%.

[0055] Test results are as follows Figure 3As shown, the test results indicate that the uncured hydrogel (i.e., before photocuring and crosslinking) exhibits weak mechanical properties, while photocuring and crosslinking significantly improve the mechanical properties of the hydrogel. The compressive modulus of the uncured gel sample was 3.03 kPa, which increased to 18.65 kPa after photocuring. The addition of ginsenoside Rd reduced the compressive modulus of the hydrogel. This is attributed to the competitive binding of phenylboronic acid groups on the Rd chain with PVA, forming crosslinking termination points. This reduces the number of crosslinking points for phenylboronic acid groups that can form a polymer network with PVA, resulting in a looser crosslinking network. Furthermore, the steric hindrance effect of Rd may have affected the photopolymerization efficiency of GelMA, reducing the crosslinking density and thus leading to a decrease in the mechanical properties of the Rd hydrogel. The compressive moduli of the uncured Gel@Rd 0.5 and Gel@Rd 1 hydrogels were 2.32 kPa and 2.18 kPa, respectively, while the compressive moduli of the cured Gel@Rd 0.5 and Gel@Rd 1 hydrogels reached 14.2 and 13.7 kPa, respectively. The experimental results show that the Rd hydrogel proposed in this invention can improve the mechanical properties of the hydrogel through a double crosslinking strategy, thus solving the problems of weak mechanical properties and poor structural stability of traditional injectable hydrogels.

[0056] (2) Injectability and self-healing properties: Figure 4 This is a macroscopic characterization diagram of the injectability and self-healing properties of the Rd hydrogel proposed in this invention.

[0057] like Figure 4 As shown, when two parts of Gel@Rd 1 hydrogel of different colors are cut and then brought into close contact, the two parts of hydrogel can quickly re-heal into a whole without external force intervention. No cracks are generated at the healed area under external force stretching, which proves its excellent dynamic reversible cross-linking and self-healing ability.

[0058] also, Figure 4 In this study, Gel@Rd 1 hydrogel was continuously injected into a "heart-shaped" mold without clogging the needle. It conformed to the mold and formed the corresponding shape, and could be picked up immediately with tweezers. This indicates that the hydrogel has good injectability and adaptability to irregular shapes, and has the ability to fill irregular wounds. The rapid recovery of the hydrogel's state and strength after injection further confirms its excellent self-healing ability.

[0059] The above results demonstrate that the Rd hydrogel proposed in this invention has excellent injectability and self-healing properties, and can fill irregular or deep wound areas, showing broad clinical application prospects.

[0060] (3) In vitro cell compatibility: The effect of hydrogel extract on L929 cell viability was evaluated using the CCK-8 assay. Results are as follows:Figure 5 As shown, Figure 5 This is a characterization diagram of the in vitro cell compatibility of the Rd hydrogel provided by the present invention.

[0061] Cytotoxicity assay: L929 cells were inoculated at 5 × 10⁻⁶ cells per cell line. 4 Cells were seeded at a density of cells / well in the lower chamber of a 24-well Transwell incubator and cultured overnight. Different concentrations of Rd hydrogel (150 μL) prepared under aseptic conditions were added to the upper chamber of the Transwell incubator, with wells without hydrogel serving as blank controls (as shown in the figure). Cells were then cultured for another 24 h. After 24 h, the upper chamber was removed and the original culture medium was discarded. Cells were washed with PBS, and fresh culture medium containing 10% CCK-8 reagent was added. Cells were incubated until full color development was observed. The absorbance (OD) value at 450 nm was measured using a microplate reader, and cell viability was calculated using the following formula to determine hydrogel-induced cytotoxicity: ; Cell Viability represents cell activity, and OD Sample The absorbance value (OD) of the cell supernatant in the hydrogel group is expressed as 450 nm. Blank This indicates the absorbance value of the cell supernatant in the blank group (excluding the hydrogel group) at a wavelength of 450 nm.

[0062] like Figure 5 As shown, when the Rd concentration in the hydrogel was ≤1 mg / mL, the viability of L929 cells was above 90%, with the cell viability of the Gel@Rd 0.5 and Gel@Rd 1 groups being 96.76% and 109.12%, respectively. When the Rd concentration increased to 5 mg / mL, the cell viability decreased to 31.20%, indicating that this high concentration inhibited cell proliferation.

[0063] The above results indicate that, within a reasonable Rd concentration range, the immunomodulatory hydrogel based on ginsenoside Rd of this invention exhibits good cell compatibility.

[0064] (4) In vitro angiogenesis-promoting capacity: The pro-angiogenic potential of the hydrogel was evaluated using an in vitro tube-forming experiment with HUVECs cells. The results are as follows: Figure 6 As shown, Figure 6 This is a characterization diagram of the in vitro angiogenesis promotion effect of the Rd hydrogel of the present invention.

[0065] The specific experimental steps were as follows: Matrigel and DMEM culture medium were mixed at a 1:1 ratio, and 200 μL was added to each well of a 24-well plate. The plate was then incubated at 37 °C for 30 minutes. HUVECs cells were cultured at a rate of 1 × 10⁻⁶. 5 Cells were seeded at a density of 100 cells / well onto the surface of a 24-well Matrigel plate and then co-cultured with different hydrogel extracts for 6 hours before being observed and imaged using a microscope.

[0066] Enhanced vascularization provides more nutrients and oxygen for tissue regeneration, thereby promoting the wound healing process. For example... Figure 6 As shown, the number of HUVECs cells forming tubes in the Gel@Rd hydrogel extract treatment group (Gel@Rd 0.5 and Gel@Rd 1 in the figure) was significantly higher than that in the blank group (Blank in the figure, i.e., the group without hydrogel / untreated group) and the Gel group, indicating that the Rd-loaded hydrogel (i.e., the immunomodulatory hydrogel based on ginsenoside Rd in this invention) has a significant angiogenesis-promoting ability.

[0067] (5) In vitro antioxidant properties: Intracellular reactive oxygen species (ROS) levels were detected using a 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) fluorescent probe kit to assess the antioxidant activity of the hydrogel. Results are as follows: Figure 7 As shown, Figure 7 This is a characterization diagram of the in vitro antioxidant properties of the Rd hydrogel provided by the present invention.

[0068] The specific experimental steps are as follows: L929 cells are inoculated at a rate of 1×10⁻⁶. 5 Cells were seeded at a density of cells / well in the lower chamber of a 12-well Transwell cell culture chamber and cultured overnight. For the experimental group, sterile hydrogel (400 μL) was added to the upper chamber of the Transwell for pretreatment for 12 h, followed by stimulation with 200 μM H2O2 in the lower chamber for 6 h. The culture medium was discarded, and the cells were washed three times with PBS and stained with 10 μM DCFH-DA fluorescent probe for 20 min. Images were acquired using a fluorescence microscope. Cells treated with H2O2 alone served as a positive control (+H2O2 in the image), while untreated cells served as a negative control (Blank in the image).

[0069] Excessive accumulation of reactive oxygen species (ROS) at the wound site can trigger severe oxidative stress and inflammatory damage, thereby hindering wound healing. For example... Figure 7As shown, under 200 μM H2O2 stimulation, the positive control group exhibited strong green (ROS) fluorescence expression, indicating that L929 cells underwent significant oxidative stress. After treatment with Gel hydrogel (Gel in the figure), the intensity of green fluorescence expression decreased, but the change was small. However, after treatment with Gel@Rd hydrogel (+H2O2+Gel@Rd 0.5, +H2O2+Gel@Rd 1 in the figure), the intensity of green fluorescence expression decreased significantly, showing a trend dependent on Rd concentration. These results indicate that the Rd hydrogel proposed in this invention has good antioxidant activity, can effectively reduce the level of intracellular ROS production, and thus protect cells from oxidative stress damage.

[0070] (6) In vitro immunomodulatory effects: The effects of hydrogel on LPS-induced macrophage polarization and inflammatory factor secretion were investigated to assess its immunomodulatory function. Results are as follows: Figure 8 As shown, Figure 8 This is an in vitro immunomodulatory characterization diagram of the Rd hydrogel provided in this invention.

[0071] The specific experimental steps were as follows: Raw 264.7 mouse mononuclear macrophage cells were cultured at a rate of 5 × 10⁻⁶ cells / year. 4 Cells were seeded at a density in the lower chamber of a 12-well Transwell plate containing 1 mL of complete DMEM medium and cultured overnight. Raw 264.7 cells were then stimulated with 100 ng / mL LPS to establish inflammatory conditions. Sterile gels, Gel@Rd 0.5, and Gel@Rd 1 (150 μL) were added to the lower chamber of the Transwell plate to treat Raw 264.7 cells for 24 hours. Wells without any treatment served as blank controls (Figure: Blank), and wells stimulated with only 100 ng / mL LPS served as positive controls for M1 macrophages (Figure: LPS). After 24 hours of treatment, the morphology of Raw 264.7 cells was observed and photographed using an inverted fluorescence microscope. Simultaneously, the supernatant from each well was collected, and the levels of pro-inflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in the supernatant were detected by enzyme-linked immunosorbent assay (ELISA).

[0072] like Figure 8As shown in the microscopic images of Raw 264.7 macrophages, the control group Raw 264.7 cells exhibited a round, clustered morphology. After stimulation with 100 ng / mL LPS, Raw 264.7 cells exhibited the M1 pro-inflammatory macrophage phenotype with slender or multiple pseudopodia. After stimulation with 100 ng / mL LPS and treatment with Gel@Rd 0.5 and Gel@Rd 1 hydrogels for 24 hours, the number of cells with slender pseudopodia decreased. The effect of Gel@Rd 1 hydrogel treatment was the most significant. Most Raw 264.7 cells showed a round shape similar to the control group cells, while most cells treated with Gel hydrogel showed an M1 phenotype similar to those stimulated with LPS only. ELSA analysis of inflammatory factors in the supernatant showed that the concentrations of IL-6 and TNF-α in the cell supernatant treated with Gel@Rd 1 hydrogel were the lowest, at 123.24 pg / mL and 176.50 pg / mL, respectively, which were reduced by 59.4% and 63.25% compared with the LPS control group.

[0073] The above results indicate that the Rd hydrogel provided by this invention can significantly reduce macrophage polarization towards the M1 pro-inflammatory phenotype, inhibit excessive secretion of inflammatory factors, and has excellent anti-inflammatory and immunomodulatory effects.

[0074] (7) In vivo wound healing effect: Based on the good immunomodulatory function exhibited by the hydrogel in vitro, its in vivo healing-promoting ability was further evaluated using a mouse full-thickness skin defect model. The results are as follows: Figure 9 and Figure 10 As shown.

[0075] Figure 9 This is a characterization diagram of how the Rd hydrogel proposed in this invention promotes wound healing in mice.

[0076] Given the immunomodulatory effects of the Rd hydrogel proposed in this invention on macrophage phenotype and inflammatory factor secretion in vitro, in order to explore whether the anti-inflammatory function of the hydrogel can achieve better wound healing, this invention first established a full-thickness skin defect model in normal mice.

[0077] Specific experimental procedures: Male C57BL / 6 mice (7 weeks old) were anesthetized with isoflurane inhalation. After shaving their backs, two symmetrical circular marks were imprinted on the prepared skin area of ​​the mouse's back using an 8 mm diameter skin perforator dipped in ink. Then, two full-thickness skin defects (Φ = 8 mm) were cut according to the marks using ophthalmic surgical scissors. Disc-shaped gels (Φ = 8 mm, h = 1.5 mm) prepared under sterile conditions were placed on the wounds, and then covered with a 3M Tegaderm transparent membrane. These membranes were removed after two days. A control group (Blank in the figure) was used, with the wound treated with PBS and covered with a 3M Tegaderm transparent membrane. Wounds were photographed at different time points during treatment, and the wound area was calculated using image J (n≥6 per group). Wound healing rate was calculated using the following formula: ; Where A0 and At represent the wound area on day 0 and day t, respectively.

[0078] Wound healing rate is an important indicator for evaluating wound healing effectiveness. Macroscopic photographs of the wound at different time points and schematic diagrams of the dynamic healing process are shown below. Figure 9 As shown, the wound area in each group decreased over time, and no infection or necrosis occurred. Further measurements of the wound area in each group were taken, and the wound healing rate was calculated. Results showed that, compared to the control group, the wounds in the hydrogel-treated groups healed faster. The Gel@Rd 1 hydrogel group had a wound healing rate exceeding 60% by the third day post-operation, followed by the Gel@Rd 0.5 hydrogel group. By the seventh day post-operation, the wound area in all groups had significantly decreased, with the Rd hydrogel groups still exhibiting a faster healing rate. The wound healing rates for Gel@Rd 0.5 and Gel@Rd 1 hydrogels were 87.75 ± 2.46% and 91.57 ± 2.12%, respectively, while the healing rates for the control group and the Gel hydrogel group were 73.03 ± 2.31% and 83.46 ± 2.42%, respectively. On postoperative day 12, the skin tissue at the wound site in the Gel@Rd 1 hydrogel treatment group appeared no different from the surrounding healthy skin tissue, while the skin tissue at the wound site in the blank group and the Gel hydrogel group was light pink, possibly indicating that the mature tissue remodeling process had not yet been completed. H&E staining results of the skin tissue at the wound site on postoperative days 7 and 9 (…). Figure 10The results showed that the wound in the Gel@Rd1 hydrogel treatment group had achieved epithelialization by day 7, while no epithelial tissue formation was observed in the other groups. HE staining results on day 12 postoperatively showed that the wound tissue had achieved re-epithelialization, but the area of ​​immature skin tissue was the smallest in the Gel@Rd1 hydrogel treatment group, followed by the Gel@Rd0.5 hydrogel treatment group, and the largest area of ​​immature skin tissue in the blank group.

[0079] The above results indicate that the Rd hydrogel proposed in this invention can significantly accelerate wound healing and achieve tissue remodeling effects that are closer to those of healthy skin.

[0080] In summary, the Rd hydrogel provided by this invention constructs a dual-network hydrogel through the synergistic combination of dynamic reversible crosslinking of borate ester bonds and photocrosslinking static covalent networks, giving it excellent injectability and self-healing properties, as well as enhanced mechanical strength and stability. Furthermore, this hydrogel exhibits excellent antioxidant activity: effectively scavenging excess ROS, protecting cells from oxidative stress damage, and creating favorable conditions for tissue regeneration; powerful anti-inflammatory and immunomodulatory functions: significantly inhibiting macrophage polarization towards the M1 pro-inflammatory phenotype and effectively reducing the secretion of key pro-inflammatory factors (IL-6, TNF-α), fundamentally alleviating persistent inflammation in chronic wounds; significant pro-angiogenic capacity: effectively promoting HUVECs cell tube formation, improving wound blood supply, and delivering necessary oxygen and nutrients for tissue repair; good cell compatibility: maintaining cell viability above 90% at effective concentrations, ensuring safety for clinical application; and significantly accelerating the closure speed of full-thickness skin defects, promoting higher-quality tissue regeneration and remodeling (such as earlier epithelialization and smaller immature tissue areas).

[0081] The Rd hydrogel provided by this invention is a multifunctional hydrogel dressing that integrates injectable self-healing, mechanical enhancement, intelligent drug release, anti-oxidation, anti-inflammatory immunomodulation, and angiogenesis promotion. It achieves a perfect fusion of material properties and biological functions, directly targeting the core obstacles in the healing process of chronic and severe wounds (persistent inflammation, high oxidative stress, and inhibited angiogenesis), showing great clinical translation potential and application prospects. It can be used in the preparation of drugs or medical dressings to promote wound healing.

[0082] The sequence numbers of the above embodiments of the present invention are for descriptive purposes only and do not represent the superiority or inferiority of the embodiments.

[0083] Those skilled in the art will understand that the purpose of this invention is to provide a relatively universal overall preparation process or technical principle, and to cover as many different applicable scenarios and conditions as possible (such as different raw material characteristics, production scale, product demand, etc.). Therefore, some specific operations can be flexibly adjusted according to the situation in actual implementation, as long as the expected or the same or similar technical effects as those in the embodiments of this invention can be achieved.

[0084] In the above embodiments of the present invention, the descriptions of each embodiment have their own emphasis. Parts not described in detail in a certain embodiment can be referred to in the relevant descriptions of other embodiments. The steps in the method of the embodiments of the present invention can be adjusted, combined, or deleted according to actual needs. The technical features can be combined arbitrarily. For the sake of brevity, not all possible combinations of the technical features in the embodiments are described. However, as long as the combination of these technical features does not contradict each other, it should be considered within the scope of the present invention.

[0085] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An immunomodulatory hydrogel based on ginsenoside Rd, characterized in that, include: Natural polysaccharides and their derivatives modified with phenylboronic acid groups, and protein-based biopolymers modified with methacrylic anhydride, are used as biomatrix materials. Ginsenoside Rd, as a bioactive functional component; Polyvinyl alcohol, as a crosslinking agent; The hydrogel has a dual cross-linking structure. First, through the vicinal diol or polyol groups on the ginsenoside Rd chain and the vicinal diol structure of polyvinyl alcohol, a covalent chemical cross-linking reaction occurs with natural polysaccharides modified with phenylboronic acid groups to form a first dynamic reversible cross-linking network, which endows the hydrogel with injectability, self-healing and glucose responsiveness. At the same time, ginsenoside Rd is loaded in the gel network through borate ester bonds to achieve a sustained-release effect. Subsequently, the protein-based biopolymer modified with methacrylic anhydride undergoes an addition polymerization reaction under photoinitiator and light irradiation to form a second static covalent cross-linked network, which endows the hydrogel with mechanical strength and structural stability.

2. The immunomodulatory hydrogel based on ginsenoside Rd according to claim 1, characterized in that, The natural polysaccharides modified with phenylboronic acid groups include hyaluronic acid modified with phenylboronic acid, chitosan modified with phenylboronic acid, and dextran modified with phenylboronic acid. The methacrylic anhydride-modified protein biopolymers include methacrylamide silk fibroin and methacrylamide gelatin.

3. The immunomodulatory hydrogel based on ginsenoside Rd according to claim 1, characterized in that, The concentration range of the natural polysaccharides and their derivatives modified with phenylboronic acid groups in the hydrogel is 1 wt% to 3 wt%. The concentration range of the methacrylic anhydride-modified protein-based biopolymer in the hydrogel is 4 wt% to 12 wt%. The concentration range of ginsenoside Rd in the hydrogel is 0.1~5 mg / mL; The concentration of polyvinyl alcohol in the hydrogel ranges from 0.25 wt% to 1 wt%.

4. A method for preparing an immunomodulatory hydrogel based on ginsenoside Rd, used to prepare the hydrogel as described in claim 1, characterized in that, include: Polyvinyl alcohol was dissolved in PBS buffer solution to obtain a polyvinyl alcohol solution, which was used as a crosslinking agent. A phenylboronic acid-modified natural polysaccharide biopolymer was dissolved in PBS buffer to obtain a phenylboronic acid-polysaccharide solution. Methacrylamide-modified protein biopolymers were dissolved in PBS buffer containing a photoinitiator to obtain a methacrylamide-modified protein solution. The phenylboronic acid-polysaccharide solution was mixed with the methacrylamide protein solution to obtain a hydrogel precursor solution; Ginsenoside Rd was dissolved in dimethyl sulfoxide solution to prepare Rd stock solution, and then diluted with PBS buffer. The diluted Rd solution was added to the hydrogel precursor solution and mixed evenly. Then the polyvinyl alcohol solution was added and mixed to form a first dynamic reversible cross-linked network with injectability, self-healing and glucose responsiveness, thus obtaining the first-generation hydrogel. The primary hydrogel was then subjected to photocrosslinking under light, causing the methacrylic anhydride-modified protein to undergo further addition polymerization, forming a second static covalent crosslinking network with mechanical strength and structural stability, thus obtaining the final hydrogel product, namely the immunomodulatory hydrogel based on ginsenoside Rd.

5. The method for preparing the immunomodulatory hydrogel based on ginsenoside Rd according to claim 4, characterized in that, The polyvinyl alcohol has a dissolution temperature of 80 °C, and the pH of the PBS buffer is 7.2~7.

4.

6. The method for preparing the immunomodulatory hydrogel based on ginsenoside Rd according to claim 4, characterized in that, The phenylboronic acid-modified natural polysaccharide biopolymer was dissolved at room temperature with a stirring speed of 600-1000 r / min, and the concentration of the resulting phenylboronic acid-polysaccharide solution ranged from 2 wt% to 6 wt%.

7. The method for preparing the immunomodulatory hydrogel based on ginsenoside Rd according to claim 4, characterized in that, The solubility temperature of the methacrylamide protein biopolymer is 60-70°C, the concentration range of the photoinitiator is 0.5 wt%-1 wt%, and the concentration range of the resulting methacrylamide protein solution is 8 wt%-24 wt%.

8. The method for preparing the immunomodulatory hydrogel based on ginsenoside Rd according to claim 4, characterized in that, The concentration of the Rd stock solution is 100 mg / mL, and the concentration of the diluted Rd solution is 1~50 mg / mL.

9. The method for preparing the immunomodulatory hydrogel based on ginsenoside Rd according to claim 4, characterized in that, The wavelength of the illumination is 395 nm or 405 nm, and the illumination time is 15 to 60 seconds.

10. The use of an immunomodulatory hydrogel based on ginsenoside Rd as described in any one of claims 1 to 3, or an immunomodulatory hydrogel based on ginsenoside Rd prepared by the preparation method described in any one of claims 4 to 9, in the preparation of a medicament or medical dressing for promoting wound healing.

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