Immunoregulation hydrogel as well as preparation method and application thereof

Abstract

The invention discloses an immunoregulation hydrogel as well as a preparation method and application thereof, and relates to the technical field of medical materials, amino-derived cationic polysaccharide, carboxyl-derived anionic polysaccharide and dopa-modified protein biopolymer materials are used as biological matrix materials, spermidine is used as a bioactive functional component, and the immunoregulation hydrogel is prepared by a one-step method. The preparation method comprises the following steps: by taking a benzaldehyde-terminated high polymer material as a cross-linking agent, forming a first layer of dynamic reversible cross-linked network structure through Schiff base reaction; then calcium ions are introduced, and the calcium ions and carboxylate radicals are complexed and cross-linked to form a second layer of static irreversible cross-linked network structure which can be used for an ESD (Electro-Static Discharge) operation. By adopting a step-by-step cross-linking mode, the first-step cross-linked single-network hydrogel has injectability, self-repairability, tissue adhesiveness and an immune regulation function, and the second-step cross-linking further ensures stable and lasting performance, so that the problems that a submucosal injection material is easy to disperse, short in lasting time and poor in stability are solved. The immunoregulation function effect of the wound repair material is poor.

Description

Technical Field

[0001] The present invention relates to the technical field of medical materials, and particularly relates to an immune-regulating hydrogel and its preparation method and application. Background Art

[0002] Endoscopic submucosal dissection (ESD) is an important operation for resection of early digestive tract tumors and precancerous lesions. It can resect larger lesions en bloc, which is beneficial for accurate assessment of the histopathological stage of the lesions. In addition, it also has the advantages of less trauma and quick recovery. For early digestive tract tumors without the risk of lymph node metastasis, ESD is the preferred treatment option. However, the ESD operation is technically difficult and time-consuming, and the incidence of adverse events such as bleeding and perforation is relatively high.

[0003] During ESD, a liquid needs to be injected into the submucosa to assist the operation. The existing injection liquids can be divided into 3 types: low-viscosity aqueous solutions, mainly including normal saline, hypertonic saline, hypertonic glucose solution, glycerol fructose, etc. The elevation duration is short, and repeated injections are required during the operation. It is suitable for early-stage and general clinical use; viscous polymer solutions, mainly including sodium hyaluronate, sodium alginate, hydroxyethyl starch, fibrinogen, etc. The elevation maintenance effect is good. As the concentration (viscosity) increases, the submucosal persistence performance improves, but the injection pressure also increases, making the injection difficult; hydrogels flow in a liquid state through the injection needle during the injection process, and can maintain a solid state after injection to continuously provide a supporting effect, and can endow other functions besides liquid support according to the different properties and modifications of the matrix materials. However, the clinical operation situation is complex and changeable. Due to the solid property after gelation, it may block the injection needle tube. There are risks of bleeding, perforation, and infection in the wound surface after ESD. Especially for large esophageal lesions, luminal stenosis may be caused by scar fibrotic tissue hyperplasia, affecting the passage of digestive tract contents and seriously affecting the quality of life of patients. The main reasons for poor wound surface repair after ESD are mucosal deficiency, inflammatory reaction, fibrosis, and muscular layer atrophy. For the wound surface after ESD, there are few clinically available drugs for promoting repair and inhibiting scar formation, and there are problems such as poor effects and other adverse complications.

[0004] In recent years, biomaterials have developed rapidly in the fields of regenerative medicine and tissue engineering, providing solutions for various clinical problems. However, for the ESD operation, the currently used materials for submucosal injection and wound surface repair have disadvantages such as single function, easy dispersion, short duration, insufficient repair function, and poor scar inhibition effect. Therefore, optimizing the operation process and reducing postoperative complications are urgent problems to be solved clinically. Developing materials that can be used for both liquid support during ESD and promoting postoperative wound healing and reducing scar formation has important clinical significance and application value. Summary of the Invention

[0005] In view of the above disadvantages and deficiencies of the prior art, the present invention provides an immune-regulating hydrogel, a preparation method and an application thereof, so as to solve the technical problems that the submucosal injection material is easy to disperse, has a short duration, and the immune regulation function of the wound repair material is poor.

[0006] According to one aspect of the present invention, there is provided an immune-regulating hydrogel. The immune-regulating hydrogel is used in ESD surgery and adopts a step-by-step strategy, including: using an amino-derivatized cationic polysaccharide, a carboxyl-derivatized anionic polysaccharide, and a dopamine-modified protein-based biopolymer material as biological matrix materials, using spermidine as a bioactive functional component, and using a polymer material capped with benzaldehyde as a cross-linking agent. Through the Schiff base reaction between amino groups and aldehyde groups, a first-layer dynamic reversible cross-linked network structure is formed to obtain a single-network hydrogel. The single-network hydrogel is used for submucosal injection as a liquid pad for ESD surgery; thereafter, calcium ions are further introduced on the basis of the single-network hydrogel, and the calcium ions are complexed and cross-linked with the carboxylate groups of the carboxyl-derivatized anionic polysaccharide to form a second-layer static irreversible cross-linked network structure, obtaining a double-network hydrogel. The double-network hydrogel is used to close the ESD surgical wound and promote wound repair.

[0007] Optionally, the amino-derivatized cationic polysaccharide includes at least one of deacetylated chitosan, chitin, carboxymethyl chitosan, and hydroxypropyl chitosan.

[0008] Optionally, the carboxyl-derivatized anionic polysaccharide includes at least one of hyaluronic acid, chondroitin sulfate, sodium alginate, and heparin.

[0009] Optionally, the dopamine-modified protein-based biopolymer material includes dopamine-modified pigskin, gelatin or collagen, and dopamine is grafted onto the protein-based biopolymer material through amidation reaction to improve the wet adhesion performance of the hydrogel.

[0010] Optionally, the polymer cross-linking agent capped with benzaldehyde is a multi-arm polyethylene glycol containing at least one benzaldehyde group, including at least one of a two-arm benzaldehyde-capped polyethylene glycol, a four-arm benzaldehyde-capped polyethylene glycol, a six-arm benzaldehyde-capped polyethylene glycol, an eight-arm benzaldehyde-capped polyethylene glycol, and a five-arm benzaldehyde tri-arm triiodobenzoate.

[0011] According to another aspect of the present invention, there is provided a method for preparing an immune-regulating hydrogel for preparing the immune-regulating hydrogel as described above, including: Step 1, synthesizing a dopamine-modified protein-based biopolymer material: dissolving the protein-based biopolymer material in deionized water, and successively adding EDC, NHS, and dopamine hydrochloride. Under the catalytic action of EDC and NHS, the amino group of dopamine hydrochloride reacts with the carboxyl group on the protein-based biopolymer material to undergo an amidation reaction. After the reaction, dialysis and freeze-drying are carried out to obtain the dopamine-modified protein-based biopolymer material, where EDC is 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and NHS is N-hydroxysuccinimide; Step 2, preparing a polymer matrix material solution; adding the amino-derivatized cationic polysaccharide, the carboxyl-derivatized anionic polysaccharide, and the dopamine-modified protein-based biopolymer material into physiological saline respectively to prepare an amino-derivatized cationic polysaccharide solution, a carboxyl-derivatized anionic polysaccharide solution, and a dopamine-modified protein-based biopolymer material solution; Step 3, preparing a cross-linking agent solution: dissolving the polymer material capped with benzaldehyde in physiological saline to prepare a polymer cross-linking agent capped with benzaldehyde; adding anhydrous calcium chloride powder into physiological saline to prepare a CaCl2 cross-linking agent; Step 4, preparing a precursor solution: mixing the carboxyl-derivatized anionic polysaccharide solution, the polymer cross-linking agent solution capped with benzaldehyde, and a spermidine solution diluted in a certain proportion to obtain a precursor solution A; mixing the amino-derivatized cationic polysaccharide solution and the dopamine-modified protein-based biopolymer material solution to obtain a precursor solution B; Step 5, preparing a single-network hydrogel: at room temperature, mixing the precursor solution A and the precursor solution B, so that the amino group of the amino-derivatized cationic polysaccharide, the amino group on the spermidine molecular chain, and the amino group on the dopamine-modified protein-based biopolymer material react with the aldehyde group on the polymer cross-linking agent capped with benzaldehyde to form a first-layer reversible cross-linking network structure to obtain a single-network hydrogel; Step 6, double-network cross-linking: then adding the CaCl2 cross-linking agent solution by means of soaking or spraying, so that calcium ions are complexed and cross-linked with the carboxylate group of the carboxyl-derivatized anionic polysaccharide to form a second-layer static irreversible cross-linking network to obtain a double-network hydrogel.

[0012] Optionally, the reaction process in Step 1 is maintained under acidic conditions and protected by a nitrogen stream.

[0013] Optionally, in the solution after mixing precursor solution A and precursor solution B in step five, the concentration of the amino-derivatized cationic polysaccharide solution is 0.5 wt% to 2 wt%, the concentration of the carboxyl-derivatized anionic polysaccharide solution is 0.2 wt% to 1.5 wt%, the concentration of the dopamine-modified protein-based biopolymer material is 4 wt% to 10 wt%, the concentration of spermidine is 25 to 500 μM, the concentration of the benzaldehyde-capped polymer crosslinker is 0.5 wt% to 1.5 wt%, and the molecular weight of the benzaldehyde-capped polymer crosslinker is 10 to 20 kDa.

[0014] Optionally, in step six, the concentration of the CaCl2 crosslinker is 1 wt% to 5 wt%.

[0015] According to another aspect of the present invention, there is also provided an application of the immunomodulatory hydrogel as described above or the immunomodulatory hydrogel prepared by the preparation method as described above in the ESD operation, wherein the ESD operation includes ESD operations on the esophagus, stomach, and intestine.

[0016] The beneficial effects of the present invention are as follows: The present invention provides a hydrogel with immunomodulatory function, which adopts a stepwise (two-step) crosslinking strategy. Using amino-derivatized cationic polysaccharide, carboxyl-derivatized anionic polysaccharide, and dopamine-modified protein-based biopolymer material as biological matrix materials, spermidine as a bioactive functional component, and benzaldehyde-capped polymer material as a crosslinker, a Schiff base reaction occurs between amino and aldehyde groups to form the first layer of dynamic reversible crosslinking network structure. This dynamic reversible crosslinking method endows the hydrogel with injectability, enabling it to be conveniently applied to various complex sites through a syringe. For example, it can be precisely injected to the required position during the ESD operation; At the same time, due to the reversibility of the Schiff base bond, the hydrogel also has self-healing performance. When the hydrogel is damaged to a certain extent, the internal dynamic reversible crosslinking network can re-form chemical bonds to achieve self-repair and extend the service life of the hydrogel; In addition, inspired by the mussel protein adhesion mechanism, dopamine molecules can form covalent and non-covalent bonds with the substrate. By modifying the protein molecules with dopamine, the wet adhesion performance of the material can be improved, enabling it to fit well with the wound surface in a moist wound environment and providing a stable attachment basis; Spermidine is introduced as a bioactive component. Spermidine can induce autophagy, improve mitochondrial function, and has anti-inflammatory and immunomodulatory functions. The amino group on the spermidine molecular chain reacts with the aldehyde group on the benzaldehyde-capped polymer crosslinker, and spermidine is loaded into the hydrogel through a Schiff base reaction. As the imine bond dissociates, spermidine is gradually released from the hydrogel to exert an anti-inflammatory effect; After the formation of the first - layer dynamic reversible cross - linked network structure, calcium ions are introduced in the present invention. By using the complexation cross - linking between calcium ions and carboxylate groups, a second - layer static irreversible cross - linked network is formed. The introduction of the second - layer cross - linked network can enhance the mechanical properties of the hydrogel, enabling it to withstand a certain external force without rupture, and ensuring the stability of the hydrogel in various environments.

[0017] By adopting a two - step cross - linking strategy, the present invention constructs single - and double - network hydrogels in stages to achieve the adaptation of different functions and application scenarios. The single - network hydrogel constructed by the first - step cross - linking can be used as a liquid pad for ESD surgery, providing a lasting and stable mucosal support effect; on this basis, the double - network hydrogel constructed by the second - step cross - linking with calcium ions can seal the ESD surgical wound, further ensuring the stability and persistence of the function, accelerating the wound - healing process, and reducing fibrosis and scar formation. Brief Description of the Drawings

[0018] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings: Figure 1 It is a schematic flow chart of the preparation method of the immune - regulating hydrogel of the present invention; Figure 2 It is a physical diagram of the gel formation of the immune - regulating (single - network) hydrogel of the present invention; Figure 3 It is a characterization diagram of the injectability and macroscopic self - healing of the immune - regulating (single - network) hydrogel of the present invention; Figure 4 It is a characterization diagram of the tissue adhesion performance and elasticity analysis of the immune - regulating (double - network) hydrogel of the present invention; Figure 5 It is a characterization diagram of the influence of the immune - regulating (double - network) hydrogel of the present invention on the macrophage morphology in vitro; Figure 6 It is an immunofluorescence diagram of CD86 and CD206 showing the influence of the immune - regulating (double - network) hydrogel of the present invention on the macrophage phenotype polarization in vitro; Figure 7 It is a characterization diagram of the influence of the immune - regulating (double - network) hydrogel of the present invention on the contents of IL - 6 and TNF - α in the macrophage supernatant in vitro; Figure 8 It is a characterization diagram of the support effect of the immune - regulating (single - network) hydrogel as a liquid pad during ESD surgery of the present invention; Figure 9 It is a characterization diagram of the immune - regulating (double - network) hydrogel of the present invention promoting the healing of porcine esophageal wounds. Detailed Embodiments

[0019] To enable those skilled in the art to better understand the solution of this application, the technical solutions in the embodiments of this application will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of this application. Obviously, the described embodiments are only a part of the embodiments of this application, rather than all of the embodiments. Based on the embodiments in this application, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of this application. It should be noted that, without conflict, the embodiments in this application and the features in the embodiments may be combined with each other.

[0020] Secondly, the so-called "one embodiment" or "embodiment" herein refers to a specific feature, structure or characteristic that may be included in at least one implementation manner of the present invention. The "in one embodiment" that appears in different places in this specification does not necessarily refer to the same embodiment, nor is it an embodiment that is separate or selectively exclusive of other embodiments.

[0021] The terms "comprising" and "having" and any variations thereof in the description and claims of this application are intended to cover non-exclusive inclusion. For example, a process, method, product or device that comprises a series of steps or units does not necessarily have to be limited to those steps or units clearly listed, but may include other steps or units that are not clearly listed or are inherent to these processes, methods, products or devices.

[0022] Example 1: An immune-regulating hydrogel provided in this example uses amino-derivatized cationic polysaccharide, carboxyl-derivatized anionic polysaccharide, and dopamine-modified protein-based biopolymer material as biological matrix materials (providing extracellular nutrient matrix), spermidine as a bioactive functional component (playing an anti-inflammatory and immune-regulating role), and a polymer material capped with benzaldehyde as a crosslinking agent. Through the Schiff base reaction between amino groups and aldehyde groups, a first-layer dynamic reversible crosslinked network structure is formed to obtain a single-network hydrogel; then calcium ions are introduced, and the calcium ions are complexed and crosslinked with the carboxylate groups of the carboxyl-derivatized anionic polysaccharide to form a second-layer static irreversible crosslinked network structure to obtain a double-network hydrogel.

[0023] Through step-by-step (two-step) crosslinking, the immune-regulating hydrogel in the present invention has the function of immune regulation (its own property) while being phased into single and double networks in terms of gel-forming morphology. The two networks have different emphases in performance and application and cooperate with each other. Among them, the single-network hydrogel formed in the first step has injectability, self-healing property, and adhesiveness, which is convenient for injection and adhesion to the surgical wound. The double-network hydrogel formed in the second step is further sealed and solidified on the basis of the single-network hydrogel, enhancing the mechanical strength and stability to maintain stable and lasting performance.

[0024] Among them, the amino-derivatized cationic polysaccharides include at least one of chitosan, chitin, carboxymethyl chitosan, and hydroxypropyl chitosan.

[0025] The carboxyl-derivatized anionic polysaccharides include at least one of hyaluronic acid, chondroitin sulfate, sodium alginate, and heparin.

[0026] The dopamine-modified protein-based biopolymer materials include dopamine-modified pigskin, gelatin, or collagen. Among them, the modification principle is that by means of a chemical reaction, under acidic conditions, through the catalysis of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), an amidation reaction occurs between the protein-based biopolymer material and dopamine hydrochloride, grafting dopamine onto the protein-based biopolymer material, while maintaining the inherent biological activity of the protein-based biopolymer material and improving the wet adhesion performance of the hydrogel.

[0027] The benzaldehyde-terminated polymer crosslinker is a multi-arm polyethylene glycol containing at least one benzaldehyde group, including at least one of two-arm benzaldehyde-terminated polyethylene glycol, four-arm benzaldehyde-terminated polyethylene glycol, six-arm benzaldehyde-terminated polyethylene glycol, eight-arm benzaldehyde-terminated polyethylene glycol, and five-arm benzaldehyde three-arm triiodobenzoate.

[0028] As Figure 1 shown, the preparation method of the immunomodulatory hydrogel includes the following steps: Step 1, synthesize the dopamine-modified protein-based biopolymer material: The protein-based biopolymer material is dissolved in deionized water at 50 °C. After complete dissolution, under the reaction conditions of 37 °C, EDC and NHS powders are added sequentially, and the pH is adjusted to 5.0 - 5.5; then dopamine hydrochloride powder is added, and the pH is adjusted to 5.5 - 6.0, and the reaction is carried out for 24 h under the protection of nitrogen gas flow; after the reaction is completed, the obtained mixture aqueous solution is dialyzed for 3 days to completely remove the unreacted reagents; the dialyzed liquid is freeze-dried to obtain the dopamine-modified protein-based biopolymer material; Step 2, prepare the polymer matrix material solution: The amino-derivatized cationic polysaccharide, carboxyl-derivatized anionic polysaccharide, and dopamine-modified protein-based biopolymer material powders are dissolved in normal saline (NS), and completely dissolved at a temperature of 50 - 60 °C and a rotation speed of 1000 - 2000 revolutions per minute to obtain an amino-derivatized cationic polysaccharide solution with a concentration of 2 wt% - 8 wt%, a carboxyl-derivatized anionic polysaccharide solution with a concentration of 0.8 wt% - 6 wt%, and a dopamine-modified protein-based biopolymer material solution with a concentration of 16 wt% - 40 wt%; Step 3, prepare the crosslinker solution: The polymer material powder capped with benzaldehyde is dissolved in NS solution at room temperature to obtain a crosslinking agent solution of the polymer material capped with benzaldehyde; Anhydrous calcium chloride (CaCl2) powder is dissolved in NS to obtain a CaCl2 crosslinking agent solution; Step 4, preparing a precursor solution: The polymer material solution capped with benzaldehyde and spermidine solution are successively added to the carboxyl-derivatized anionic polysaccharide solution to obtain a precursor solution A; The amino-derivatized cationic polysaccharide and the protein-based biopolymer material solution modified with dopamine are mixed at a volume ratio of 1:1, centrifuged at a speed of 1000 - 2000 rpm and a temperature of 25 - 37 °C for 10 min to form a uniformly mixed solution as precursor solution B; Step 5, preparing a single-network hydrogel: The precursor solution A and the precursor solution B are mixed (in the mixed solution, the concentration of the amino-derivatized cationic polysaccharide solution is 0.5 wt% - 2 wt%, the concentration of the carboxyl-derivatized anionic polysaccharide solution is 0.2 wt% - 1.5 wt%, the concentration of the protein-based biopolymer material modified with dopamine is 4 wt% - 10 wt%, the concentration of spermidine is 25 - 500 μM, the concentration of the polymer crosslinking agent capped with benzaldehyde is 0.5 wt% - 1.5 wt%, and the molecular weight is 10 - 20 kDa) to obtain an injectable hydrogel pre-gel solution, and a Schiff base reaction occurs to form the first crosslinking network. Within 10 min, a single-network hydrogel with injectable properties is obtained; Step 6, double-network crosslinking: The single-network hydrogel is soaked in the CaCl2 crosslinking agent solution or the CaCl2 crosslinking agent solution is sprayed on the surface of the single-network hydrogel to complex and crosslink calcium ions with the carboxylate groups of the carboxyl-derivatized anionic polysaccharide to form a second irreversible crosslinking network, and a double-network hydrogel is prepared. Among them, the concentration of the CaCl2 crosslinking agent solution for soaking or spraying is 1 wt% - 5 wt%.

[0029] Example 2: This example further provides a preparation method of an immunomodulatory hydrogel. The difference from Example 1 is that Example 2 provides more specific materials, ratios, and operating conditions, and is equipped with hydrogels with different concentrations of spermidine.

[0030] The preparation method in this example includes: (1) Dopamine modification of the protein-based biopolymer material: Weigh 2.0 g of gelatin and dissolve it in 100 mL of deionized water at 50 °C. After complete dissolution, transfer the solution to a 250 ml three-necked flask. Subsequently, slowly add 1.0 g of EDC, adjust the solution pH to 5.0 - 5.5, activate for 15 minutes, then add 0.6 g of NHS, adjust the pH to 5.0 - 5.5 again, and continue the reaction for 15 min; Add 2.0 g of dopamine hydrochloride to the reaction system and react at 37 °C for 24 h. Throughout the reaction process, the pH value of the solution is always maintained between 5.5 - 6.0, and the solution is protected by a nitrogen stream; After the reaction, the resulting mixture is dialyzed against an aqueous solution with pH = 5.5 adjusted by 1 M hydrochloric acid (HCl) for 3 days. The dialysis molecular weight cut-off (MWCO) is 8000 - 14000 Da to completely remove unreacted reagents; The dialyzed liquid is frozen and lyophilized using a freeze dryer to obtain an off-white spongy GelDA conjugate sample, namely dopamine-modified gelatin, which is sealed and stored at -20 °C for later use. Here, Gel represents gelatin and DA represents dopamine; (2) Prepare polymer matrix material solutions and cross-linker solutions: Add 0.4 g of carboxymethyl chitosan (OCMCS) powder to 10 ml of NS and stir in a 50 °C water bath for 6 hours until the OCMCS solid is completely dissolved to obtain a 4 wt% OCMCS solution; Add 0.1 g of sodium alginate (SA) powder to 10 ml of NS and stir in a 50 °C water bath until completely dissolved to obtain a 1 wt% SA solution; Add 2.4 g of dopamine-modified gelatin (GelDA) to 10 ml of NS and stir in a 50 °C water bath until completely dissolved to obtain a 24 wt% GelDA solution; Dissolve 0.2 g of benzaldehyde-capped tetra-arm polyethylene glycol (4aPEG-BA) powder in 1 ml of NS to obtain a 20 wt% 4aPEG-BA cross-linker solution; Add 1 g of anhydrous calcium chloride powder to 50 ml of NS to obtain a 2 wt% CaCl2 cross-linker solution; (3)Prepare spermidine (SPD) solutions with different concentrations: Dilution of SPD solution: Add 15.7 µL of SPD stock solution to 1 mL of NS solution, mix well to obtain a 100 mM SPD solution, denoted as SPD-1; Add 100 µL of SPD-1 solution to 300 µL of NS solution and mix well to obtain an SPD solution with a concentration of 25 mM, denoted as SPD-2. Add 50 µL of SPD-1 solution to 350 µL of NS solution and mix well to obtain an SPD solution with a concentration of 12.5 mM, denoted as SPD-3. Add 20 µL of SPD-1 solution to 380 µL of NS solution and mix well to obtain an SPD solution with a concentration of 5 mM, denoted as SPD-4. Add 20 µL of SPD-1 solution to 780 µL of NS solution and mix well to obtain an SPD solution with a concentration of 2.5 mM, denoted as SPD-5. (4) Prepare the precursor solution: Add 40 µL of 4aPEG-BA crosslinker solution to 500 µL of SA solution, vortex for 15 seconds, and mix well. Then add 20 µL of SPD solution (i.e., SPD-2, SPD-3, SPD-4, SPD-5) and NS to the mixed solution respectively, and vortex for 20 seconds to obtain precursor solution A.

[0031] Mix 250 µL of OCMCS solution and 250 µL of GelDA solution in equal volume to obtain precursor solution B.

[0032] (5) Prepare the single-network hydrogel: As Figure 2 shown, after mixing the above precursor solution A (SA / 4aPEG-BA in the figure) and precursor solution B (OCMCS / GelDA in the figure), vortex evenly and let it stand to form a gel. The amino groups on OCMCS, some of the amino groups on GelDA, and the amino groups on SPD in the precursor solution react with the aldehyde groups on the crosslinker to form the first layer of dynamic reversible crosslinking network. The gelation time is determined as the time when the sample does not flow within 60 seconds after inverting the reagent bottle. The time used in this example is within 5 minutes. The hydrogel obtained at this time is a single-network hydrogel with injectable and self-healing properties. The concentrations of SPD in the hydrogel are 500, 250, 100, 50, 0 μM respectively, denoted as SN-SPD500, SN-SPD250, SN-SPD100, SN-SPD50, SN.

[0033] (6) Prepare the double-network hydrogel: Spray or soak the single-network hydrogel prepared according to the above steps with 2% CaCl2 solution for 2 minutes to obtain the corresponding double-network hydrogels DN-SPD500, DN-SPD250, DN-SPD100, DN-SPD50, DN.

[0034] Application Example 1: The purpose of this application example is to characterize the injectability and self-healing properties of the single-network hydrogel SN-SPD100 prepared in Example 2 under macroscopic conditions.

[0035] Characterization of injectability: Pour the mixed pre-gel solution into a 1 ml or 2.5 ml syringe. After gelation, push the syringe and observe whether it can be injected smoothly and form a specific pattern. As Figure 3 shown in (a), the SN-SPD100 hydrogel can pass through the syringe smoothly and form a controllable injection path.

[0036] Characterization of self-healing properties: For the SN-SPD100 hydrogel prepared according to Example 2, transfer the pre-gel solution with and without added dye to a "circular" mold to prepare circular SN-SPD 100 hydrogel samples. After in-situ gelation, cut them into two identical semi-circular shapes, and then place the semi-circular hydrogels with and without added dye side by side. Observe whether they have the ability to heal at 37 °C. As Figure 3 shown in (b), the two semi-circular hydrogels with added dye (red) and without added dye (brown) can form a complete circular hydrogel within 10 minutes after contact, indicating that the SN-SPD100 hydrogel has self-healing properties.

[0037] The above results show that the immune-regulating hydrogel proposed by the present invention (at the single-network hydrogel stage) has excellent injectability and self-repairability, can fill irregular or deep wound areas, and has broad clinical application prospects.

[0038] Application Example 2: The purpose of this application example is to evaluate the tissue adhesion of the double-network hydrogel DN-SPD250 using ex vivo pig skin and pig esophagus.

[0039] According to Example 2, inject the pre-gel solution of the SN-SPD250 hydrogel onto the surfaces of pig skin and pig esophagus. After the hydrogel solidifies, spray 2% CaCl2 solution on the surface to form the DN-SPD250 hydrogel in situ on the surfaces of pig skin and pig esophagus. Through tensile, torsional, bending, and water flow flushing tests, observe the adhesion state of the DN-SPD250 hydrogel to pig skin and pig esophagus.

[0040] As Figure 4As shown in (a - b), the DN - SPD250 hydrogel adhered to the surfaces of porcine skin and porcine esophagus can withstand stretching, bending, and torsion, indicating its good adhesion performance and toughness, and being able to withstand a certain amount of external force without falling off or breaking. Moreover, under the wet condition of running - water rinsing, the DN - SPD250 hydrogel on the surface of the porcine esophagus can still maintain the adhesion state, indicating its good wet - tissue adhesiveness. In addition, as Figure 4 Observed by pressing and rebounding as shown in (c), it was found that the DN - SPD250 hydrogel has good mechanical properties, combining strength and flexibility.

[0041] The above results indicate that the immune - regulating hydrogel proposed in the present invention (at the double - network hydrogel stage) has stable adhesion performance and mechanical properties, can fit well and adhere stably in a wet environment, ensuring the stability and durability of its functions.

[0042] Application Example 3: In this application example, the effects of the DN - SPD hydrogel (i.e., the double - network hydrogel of the present invention) on the secretion of macrophage inflammatory factors and cell - phenotype polarization were studied at the in - vitro cell level, and the action mechanisms of different hydrogels, DN, DN - SPD50, DN - SPD100, DN - DETA100 (a DETA - modified DN - DETA hydrogel prepared by replacing spermidine with diethylenetriamine (DETA) similar in structure to SPD) on macrophages under lipopolysaccharide (LPS) - induced inflammatory conditions were clarified.

[0043] RAW 264.7 macrophages were seeded in 24 - well plates at a density of 5×10 4 cells / well. After the cells adhered, the original culture medium was aspirated and the groups were intervened as follows: Positive control group: Supplemented with complete medium containing 1 μg / mL LPS; Experimental group: Supplemented with complete medium containing 1 μg / mL LPS, then the Transwell chamber was placed into the well plate, and 150 μL (DN, DN - SPD50, DN - SPD100, DN - DETA100) cylindrical hydrogels prepared under sterile conditions according to Example 2 were placed in the upper chamber of the Transwell; Blank control group: Replaced with fresh complete medium.

[0044] After culturing the cells in each group for another 48 h, the cell morphology was observed and photographed, the supernatant was collected, the contents of IL - 6 and TNF - α in the supernatant were detected by ELISA method, and the protein markers CD86 (M1 type) and CD206 (M2 type) specifically expressed by macrophages were immunofluorescently stained.

[0045] As Figure 5As shown, under microscopic observation of cell morphology, RAW 264.7 macrophages in the blank control group (Blank in the figure) were round with uniform size and aggregated in clusters. Under the condition of LPS induction only (LPS in the figure), the cell sizes were different, presenting a multi-pseudopod or irregular cell morphology. Under the inflammatory condition induced by LPS, in the DN-SPD hydrogel treatment group (DN-SPD50 and DN-SPD100 in the figure), the number of cells with irregular morphology or multiple pseudopods decreased significantly, and there appeared multiple "fusiform" M2-shaped cells with two slender pseudopods. Moreover, with the increase in the SPD concentration, the number of "fusiform" cells increased. In contrast, in the DN and DN-DETA hydrogel groups, the macrophages showed an irregular morphology or had multiple pseudopods, the same as the group induced by LPS only. This preliminarily indicates that the DN-SPD hydrogel can affect macrophage phenotype polarization.

[0046] As Figure 6 shown, the immunofluorescence results showed that compared with the blank group, only LPS induction showed strong positive expression of the M1 macrophage marker protein CD86 (red fluorescence). In contrast, under the inflammatory condition induced by LPS, the fluorescence expression intensity of CD86 decreased in the DN-SPD hydrogel treatment group, and more positive expression of the M2 macrophage marker protein CD206 (red fluorescence) was visible. However, there was no significant difference between the DN and DN-DETA hydrogel treatment groups and the group induced by LPS only.

[0047] The results of enzyme-linked immunosorbent assay (ELISA) were as Figure 7 shown. The content of IL-6 in the supernatant of the DN-SPD50 and DN-SPD100 hydrogel treatment groups decreased by 34.93% and 54.85%, and the content of TNF-α decreased by 24.54% and 40.85%. In contrast, the content of IL-6 and TNF-α in the supernatant of the DN and DN-DETA hydrogel treatment groups had no significant effect.

[0048] The above results indicate that the DN-SPD hydrogel inhibits the secretion of inflammatory factors and can effectively inhibit the polarization of macrophages into the M1 pro-inflammatory phenotype, and promote their polarization into the M2 anti-inflammatory and pro-repair phenotype.

[0049] Application Example 4: Given that the DN-SPD hydrogel exhibits significant anti-inflammatory and immune regulatory functions, this application example further established a Bama miniature pig esophageal ESD surgery model (n = 3) to evaluate the supporting effect of the SN-SPD hydrogel as an intraoperative fluid pad, and the effect of the DN-SPD hydrogel on the healing rate and scar formation of the mucosal wound surface after esophageal ESD. The specific steps are as follows: (1)Preoperative preparation and anesthesia: Female Bama minipigs weighing 20 - 25 kg were fasted for 24 h and water-deprived for 6 h before surgery. After inhaled isoflurane anesthesia, propofol was injected via the ear vein for deep anesthesia. Subsequently, under the assistance of a laryngoscope, an endotracheal tube was inserted, and low-flow inhaled isoflurane was continuously administered during the operation to maintain the anesthetic state.

[0050] (2)ESD surgery: To construct mucosal and submucosal defect wounds and observe the supporting effect of the liquid pad during the operation, the following specific steps were mainly included: ① Marking the scope: Insert a gastroscope through the mouth, clean the esophageal cavity, select three anatomical sites (spacing ≥ 2 cm) in the middle and lower esophagus, and establish a 2 × 3 cm rectangular mucosal marking area with the assistance of a graduated transparent cap for positioning. ② Submucosal injection: The marked area was divided into a normal saline group (NS), a DN-SPD 250 group, and a DN-SPD 500 group from the oral side to the anal side. Normal saline was injected subcutaneously in the normal saline group as the liquid pad, and SN-SPD250 and SN-SPD500 hydrogels were injected in the DN-SPD250 and DN-SPD500 groups respectively as the liquid pad. According to Example 2, precursor solution A and precursor solution B of the hydrogel were prepared, and after mixing through a three-way valve, they were injected into the submucosa in liquid form through a 22 G injection needle to form a single-network hydrogel in situ (randomly select one pig, and observe the change in the height maintenance of the liquid pad at 15 min, 30 min, and 60 min after injection). ③ Dissection and resection: Use an electric knife to excise the mucosal and submucosal tissues according to the marked scope.

[0051] (3)Wound treatment: After resection, the corresponding SN hydrogel precursor solution was injected into the wounds in the experimental group. After gelation, 2% calcium chloride was sprayed for further cross-linking to form the corresponding DN-SPD250 and DN-SPD500 hydrogels.

[0052] (4)Postoperative observation and specimen collection: The pigs were fasted for 24 h after surgery, given liquid diet on the second day, and started to resume solid feed on the third day. Gastroscopy was performed on the 7th, 14th, 21st, and 28th days after surgery to observe the wound healing. Subsequently, on the 28th day after surgery, the experimental pigs were sacrificed by excessive anesthesia, esophageal tissue samples were obtained, and fixed in 4% paraformaldehyde solution for histological section staining analysis.

[0053] The results of the supporting effect of the single-network hydrogel as a liquid pad in ESD surgery are as Figure 8As shown, good bulging effects were achieved with normal saline (NS), SN-SPD250, and SN-SPD500 after submucosal injection. After 15 minutes of injection, the bulge in the NS group disappeared. In contrast, the SN-SPD250 and SN-SPD500 hydrogels were able to maintain a good bulging effect, with no significant change from the initial height. And when continuously observed until 60 minutes after injection, the degree of bulge still did not significantly shrink.

[0054] The above results indicate that the SN-SPD hydrogel, as a liquid pad during ESD, has a lasting supporting effect.

[0055] The effect of the double-network hydrogel on the wound healing after esophageal ESD is as Figure 9 shown. Gastroscopy observations on the 7th day after the operation showed that in each group, the ulcer defects were filled with newly formed granulation tissue at the wound surface, and regenerated epithelium could be seen at the edges. The wound area of DN-SPD500 was significantly smaller than that of the NS group. Gastroscopy observations on the 14th day after the operation showed that the ulcers in each group healed, the surface was re-epithelialized, presenting a light red translucent "film" appearance, and gradually fused with the surrounding normal mucosa. Compared with the NS group, the range of the light red area on the mucosal surface in the DN-SPD hydrogel group was significantly reduced, replaced by pinkish-white epithelium with the same color as the surrounding normal mucosa, and the light red area of DN-SPD500 was further reduced compared with the DN-SPD 250 group. On the 21st and 28th days after the operation, the light red epithelial areas in each group were further reduced, especially in the DN-SPD500 group, which was almost the same as the surrounding normal mucosa.

[0056] The above results indicate that the DN-SPD hydrogel can effectively promote the wound healing after esophageal ESD.

[0057] In summary, the present invention proposes an immune-regulating hydrogel and its preparation method. Using a step-by-step strategy, it has excellent injectability, self-repairability, tissue adhesiveness, and immune-regulating function, can effectively promote the wound healing after the operation, and has broad application prospects in ESD operations in the esophagus, stomach, intestine and other parts.

[0058] The serial numbers of the above embodiments of the present invention are only for description and do not represent the advantages or disadvantages of the embodiments.

[0059] Those skilled in the art should understand that the purpose of the present invention is to provide a relatively general overall preparation process or technical principle, and cover as many different applicable scenarios and conditions as possible (such as different raw material characteristics, production scales, product requirements, etc.). Therefore, some specific operations can be flexibly adjusted according to the situation during actual implementation, as long as the expected or the same or similar technical effects as those in the embodiments of the present invention can be achieved.

[0060] In the above embodiments of the present invention, the descriptions of the various embodiments have their own emphases. For the parts not detailed in a certain embodiment, reference may be made to the relevant descriptions of other embodiments. The steps in the method of the embodiments of the present invention can be adjusted, combined, and deleted according to actual needs. The various technical features can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the various technical features in the embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as within the scope recorded in the present invention.

[0061] The above is only the preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. An immune-regulating hydrogel, characterized in that, The immune-regulating hydrogel is used in ESD surgery and adopts a step-by-step strategy, including: Using amino-derivatized cationic polysaccharides, carboxyl-derivatized anionic polysaccharides, and dopamine-modified protein-based biopolymer materials as biological matrix materials, spermidine as a bioactive functional component, and a polymer material capped with benzaldehyde as a crosslinking agent. Through the Schiff base reaction between amino and aldehyde groups, a first-layer dynamic reversible crosslinked network structure is formed to obtain a single-network hydrogel. The single-network hydrogel is used for submucosal injection as a liquid pad for ESD surgery. After that, calcium ions are further introduced on the basis of the single-network hydrogel, and the calcium ions are complexed and crosslinked with the carboxylate groups of the carboxyl-derivatized anionic polysaccharides to form a second-layer static irreversible crosslinked network structure, obtaining a double-network hydrogel. The double-network hydrogel is used to close the ESD surgical wound and promote wound repair.

2. The immunomodulatory hydrogel according to claim 1, wherein The amino-derivatized cationic polysaccharides include at least one of deacetylated chitosan, chitin, carboxymethyl chitosan, and hydroxypropyl chitosan.

3. The immunomodulatory hydrogel according to claim 1, wherein The carboxyl-derivatized anionic polysaccharides include at least one of hyaluronic acid, chondroitin sulfate, sodium alginate, and heparin.

4. The immunomodulatory hydrogel according to claim 1, wherein The dopamine-modified protein-based biopolymer materials include dopamine-modified pigskin, gelatin, or collagen. Dopamine is grafted onto the protein-based biopolymer materials through amidation reaction to improve the wet adhesion performance of the hydrogel.

5. The immunomodulatory hydrogel according to claim 1, wherein The polymer crosslinking agent capped with benzaldehyde is a multi-arm polyethylene glycol containing at least one benzaldehyde group, including at least one of two-arm benzaldehyde-capped polyethylene glycol, four-arm benzaldehyde-capped polyethylene glycol, six-arm benzaldehyde-capped polyethylene glycol, eight-arm benzaldehyde-capped polyethylene glycol, and five-arm benzaldehyde three-arm triiodobenzoate.

6. A method for preparing an immune-regulating hydrogel for preparing the immune-regulating hydrogel according to claim 1, characterized in that, Including: Step 1, synthesize dopamine-modified protein-based biopolymer materials: Dissolve the protein-based biopolymer materials in deionized water, and successively add EDC, NHS, and dopamine hydrochloride. Under the catalytic action of EDC and NHS, the amino group of dopamine hydrochloride reacts with the carboxyl group on the protein-based biopolymer materials through amidation reaction. After the reaction, dialysis and freeze-drying are carried out to obtain dopamine-modified protein-based biopolymer materials, where EDC is 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and NHS is N-hydroxysuccinimide. Step 2, prepare a polymer matrix material solution: Respectively add the amino-derivatized cationic polysaccharides, carboxyl-derivatized anionic polysaccharides, and dopamine-modified protein-based biopolymer materials into physiological saline to prepare an amino-derivatized cationic polysaccharide solution, a carboxyl-derivatized anionic polysaccharide solution, and a dopamine-modified protein-based biopolymer material solution. Step 3, prepare a crosslinking agent solution: Dissolve the polymer material capped with benzaldehyde in physiological saline to prepare a polymer crosslinking agent capped with benzaldehyde. Add anhydrous calcium chloride powder into physiological saline to prepare a CaCl2 crosslinking agent. Step 4, prepare a precursor solution: Mix the carboxyl-derivatized anionic polysaccharide solution, the polymer crosslinking agent solution capped with benzaldehyde, and a spermidine solution diluted in a certain proportion to obtain a precursor solution A. Mix the amino-derivatized cationic polysaccharide solution and the dopamine-modified protein-based biopolymer material solution to obtain precursor solution B; Step Five: Prepare a single-network hydrogel: At room temperature, mix precursor solution A and precursor solution B to cause the amino groups of the amino-derivatized cationic polysaccharide, the amino groups on the spermidine molecular chain, and the amino groups on the dopamine-modified protein-based biopolymer material to react with the aldehyde groups on the benzaldehyde-capped polymer crosslinker to form a first-layer reversible crosslinked network structure, thereby obtaining a single-network hydrogel; Step Six: Double-network crosslinking: Then add the CaCl2 crosslinker solution by soaking or spraying to cause the calcium ions to complex and crosslink with the carboxylate groups of the carboxyl-derivatized anionic polysaccharide to form a second-layer static irreversible crosslinked network, thereby obtaining a double-network hydrogel.

7. The preparation method of the immune-regulating hydrogel according to claim 6, wherein The reaction process in Step One is maintained under acidic conditions and protected by a nitrogen stream.

8. The preparation method of the immune-regulating hydrogel according to claim 6, wherein, In the solution after mixing precursor solution A and precursor solution B in Step Five, the concentration of the amino-derivatized cationic polysaccharide solution is 0.5 wt% - 2 wt%, the concentration of the carboxyl-derivatized anionic polysaccharide solution is 0.2 wt% - 1.5 wt%, the concentration of the dopamine-modified protein-based biopolymer material is 4 wt% - 10 wt%, the concentration of spermidine is 25 - 500 μM, the concentration of the benzaldehyde-capped polymer crosslinker is 0.5 wt% - 1.5 wt%, and the molecular weight of the benzaldehyde-capped polymer crosslinker is 10 - 20 kDa.

9. The preparation method of the immune-regulating hydrogel according to claim 6, wherein The concentration of the CaCl2 crosslinker in Step Six is 1 wt% - 5 wt%.

10. Use of an immune-regulating hydrogel as described in any one of claims 1 to 5 or an immune-regulating hydrogel prepared by the preparation method as described in any one of claims 6 to 9 in an ESD operation, wherein, The ESD surgery includes ESD surgeries on the esophagus, stomach, and intestine.

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