An immune regulating hydrogel and its preparation method and application

Abstract

The present invention discloses an immunomodulatory hydrogel and its preparation method and application, which relates to the field of medical material technology. It uses amino-derivatized cationic polysaccharides, carboxyl-derivatized anionic polysaccharides, and DOPA-modified protein biopolymer materials as biological matrix materials, spermidine as a biologically active functional component, and a benzaldehyde-terminated polymer material as a cross-linking agent. Through the Schiff base reaction, a first layer of dynamic reversible cross-linked network structure is formed; calcium ions are then introduced and cross-linked by complexation with carboxyl groups to form a second layer of static non-reversible cross-linked network structure, which can be used in ESD surgery. By adopting a step-by-step cross-linking method, the single network hydrogel cross-linked in the first step has injectability, self-repairability, tissue adhesion, and immunomodulatory functions. The second step of cross-linking further ensures stable and long-lasting performance, solving the technical problems of easy dispersion and short duration of submucosal injection materials and poor immunomodulatory effect of wound repair materials.

Description

Technical Field

[0001] The present invention relates to the technical field of medical materials, and in particular to an immunomodulatory hydrogel and a preparation method and application thereof. Background Art

[0002] Endoscopic submucosal dissection (ESD) is an important surgical procedure for resecting early-stage gastrointestinal tumors and precancerous lesions. It can resect larger lesions en bloc, facilitating accurate assessment of their histopathological staging. Furthermore, it offers the advantages of minimal trauma and rapid recovery. For early-stage gastrointestinal tumors without the risk of lymph node metastasis, ESD is the preferred treatment option. However, ESD is technically challenging and time-consuming, and has a relatively high incidence of adverse events such as bleeding and perforation.

[0003] During ESD, fluids must be injected into the submucosal layer to assist the procedure. Available injectable fluids can be categorized into three types: low-viscosity aqueous solutions, primarily including normal saline, hypertonic saline, hypertonic glucose solution, and glycerol-fructose. These solutions offer a short-lasting bulge and require repeated injections during surgery, making them suitable for early and general clinical use. Viscous polymer solutions, primarily including sodium hyaluronate, sodium alginate, hydroxyethyl starch, and fibrinogen, offer excellent bulge maintenance. Increasing concentration (viscosity) improves submucosal susceptibility, but injection pressure also increases, making injection difficult. Hydrogels flow through the needle as a liquid during injection and remain solid after injection, providing continuous support. Furthermore, the matrix material can be modified to provide additional functions beyond liquid support. However, clinical surgical scenarios are complex and varied, and the solid nature of the gel after gel formation can potentially clog the injection needle. ESD wounds carry the risk of bleeding, perforation, and infection. Especially for larger esophageal lesions, scar fibrosis can lead to stenosis, impeding the passage of digestive tract contents and severely impacting patient quality of life. The main reasons for poor wound healing after ESD are mucosal loss, inflammation, fibrosis, and muscle atrophy. Clinically, there are few drugs available to promote wound healing and inhibit scar formation after ESD, and some are ineffective and can cause other adverse complications.

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

[0005] In view of the above-mentioned shortcomings and deficiencies of the prior art, the present invention provides an immunomodulatory hydrogel and its preparation method and application to solve the technical problems that submucosal injection materials are easy to disperse, have a short duration, and have poor immunomodulatory effect of wound repair materials.

[0006] According to one aspect of the present invention, an immunoregulatory hydrogel is provided. The immunoregulatory hydrogel is used for ESD surgery and adopts a step-by-step strategy, including: using amino-derivatized cationic polysaccharides, carboxyl-derivatized anionic polysaccharides, and DOPA-modified protein biopolymer materials as biological matrix materials, spermidine as a biologically active functional component, and a benzaldehyde-terminated polymer material as a cross-linker, and forming a first layer of dynamic reversible cross-linked network structure through Schiff base reaction between amino groups and aldehyde groups to obtain a single network hydrogel. The single network hydrogel is used for submucosal injection as a liquid pad for ESD surgery; then, calcium ions are introduced on the basis of the single network hydrogel, and the calcium ions are used to complex and cross-link with the carboxylate groups of the carboxyl-derivatized anionic polysaccharide to form a second layer of static irreversible cross-linked network structure to obtain a double network hydrogel. The double network hydrogel is used to seal 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 DOPA-modified protein biopolymer material includes DOPA-modified pigskin, gelatin or collagen, and dopamine is grafted onto the protein biopolymer material through an amidation reaction to improve the wet adhesion performance of the hydrogel.

[0010] Optionally, the benzaldehyde-terminated polymer cross-linking agent 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.

[0011] According to another aspect of the present invention, a method for preparing an immunomodulatory hydrogel is provided, which is used to prepare the above-mentioned immunomodulatory hydrogel, comprising: step 1, synthesizing a DOPA-modified protein biopolymer material: dissolving the protein biopolymer material in deionized water, sequentially adding EDC, NHS, and dopamine hydrochloride; under the catalytic action of EDC and NHS, the amino group of dopamine hydrochloride undergoes an amidation reaction with the carboxyl group on the protein biopolymer material; after the reaction is completed, dialyzing and freeze-drying are performed to obtain the DOPA-modified protein biopolymer material. Materials, wherein EDC is 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride, and NHS is N-hydroxysuccinimide; Step 2, preparing a polymer matrix material solution; adding amino-derivatized cationic polysaccharide, carboxyl-derivatized anionic polysaccharide, and DOPA-modified protein biopolymer material to physiological saline to prepare amino-derivatized cationic polysaccharide solution, carboxyl-derivatized anionic polysaccharide solution, and DOPA-modified protein biopolymer material solution; Step 3, preparing a crosslinker solution: adding benzaldehyde-terminated polymer material to the Dissolve in physiological saline to prepare a benzaldehyde-terminated polymer crosslinker; add anhydrous calcium chloride powder to physiological saline to prepare a CaCl2 crosslinker; Step 4, prepare a precursor solution: mix the carboxyl-derivatized anionic polysaccharide solution, the benzaldehyde-terminated polymer crosslinker solution and the spermidine solution diluted in a certain proportion to obtain a precursor solution A; mix the amino-derivatized cationic polysaccharide solution and the DOPA-modified protein biopolymer material solution to obtain a precursor solution B; Step 5, prepare a single network hydrogel: at room temperature, mix the precursor solution A with the precursor solution B. The hydrogel is prepared by mixing the hydrogel with the aqueous solution B, so that the amino groups of the amino-derivatized cationic polysaccharide, the amino groups on the spermidine molecular chain, and the amino groups on the DOPA-modified protein biopolymer material undergo Schiff base reaction with the aldehyde groups on the polymer cross-linker terminated with benzaldehyde, thereby forming a first layer of reversible cross-linked network structure to obtain a single network hydrogel; step six, double network cross-linking: adding CaCl2 cross-linker solution by immersion or spraying, so that calcium ions are complexed and cross-linked with the carboxylate radicals of the carboxyl-derivatized anionic polysaccharide to form a second layer of static non-reversible cross-linked network to obtain a double network hydrogel.

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

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

[0014] Optionally, the concentration of the CaCl2 cross-linking agent in step six is 1 wt% to 5 wt%.

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

[0016] The beneficial effects of the present invention are:

[0017] The present invention provides a hydrogel with immunomodulatory function. It adopts a step-by-step (two-step) cross-linking strategy, using amino-derivatized cationic polysaccharides, carboxyl-derivatized anionic polysaccharides, and DOPA-modified protein biopolymer materials as biomatrix materials, spermidine as a bioactive functional component, and a benzaldehyde-terminated polymer material as a cross-linking agent. The amino groups and aldehyde groups undergo a Schiff base reaction to form a first layer of a dynamically reversible cross-linked network structure. This dynamically reversible cross-linking method makes the hydrogel injectable, allowing it to be conveniently applied to various complex areas via a syringe. For example, it can be precisely injected into the desired location during ESD surgery.

[0018] At the same time, due to the reversibility of Schiff base bonds, hydrogels also have self-healing properties. When the hydrogel is damaged to a certain extent, the internal dynamic reversible cross-linking network can reform chemical bonds to achieve self-repair and extend the service life of the hydrogel.

[0019] In addition, inspired by the adhesion mechanism of mussel proteins, DOPA molecules can form covalent and non-covalent bonds with substrates. Modifying protein molecules with DOPA can improve the wet adhesion properties of the material, allowing it to adhere well to the wound surface even in a moist wound environment, providing a stable adhesion foundation.

[0020] Spermidine is introduced as a bioactive ingredient. It can induce autophagy, improve mitochondrial function, and has anti-inflammatory and immune-regulating properties. The amino groups on the spermidine molecular chain react with the aldehyde groups on the benzaldehyde-terminated polymer cross-linker. Spermidine is loaded into the hydrogel through a Schiff base reaction. As the imine bond dissociates, spermidine is gradually released from the hydrogel, exerting its anti-inflammatory effects.

[0021] After forming the first layer of dynamic reversible cross-linked network structure, the present invention introduces calcium ions and utilizes them to complex and cross-link with carboxylate groups to form a second layer of static irreversible cross-linked network. The introduction of the second layer of cross-linked network can enhance the mechanical properties of the hydrogel, enabling it to withstand certain external forces without rupture, thereby ensuring the stability of the hydrogel in various environments.

[0022] The present invention adopts a two-step cross-linking strategy to construct single and double network hydrogels in stages to achieve adaptation to different functions and application scenarios. The single network hydrogel constructed by the first step of cross-linking can be used as a liquid pad for ESD surgery, providing a long-lasting and stable mucosal support effect; on this basis, the double network hydrogel constructed by the second step of cross-linking with calcium ions can seal the ESD surgical wound, further ensuring the stability and durability of the function, accelerating the healing process of the wound, and reducing fibrosis and scar formation. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of the present invention. The exemplary 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:

[0024] Figure 1 Schematic diagram of the preparation process of the immunomodulatory hydrogel of the present invention;

[0025] Figure 2 This is a physical diagram of the immunomodulatory (single network) hydrogel of the present invention;

[0026] Figure 3 Characterization diagram of the injectability and macroscopic self-healing of the immunomodulatory (single network) hydrogel of the present invention;

[0027] Figure 4 This is a characterization diagram of the tissue adhesion performance and elasticity analysis of the immunomodulatory (double network) hydrogel of the present invention;

[0028] Figure 5 This is a characterization diagram of the effect of the immunomodulatory (double network) hydrogel of the present invention on macrophage morphology in vitro;

[0029] Figure 6 CD86 and CD206 immunofluorescence images of the effects of the immunomodulatory (dual network) hydrogel of the present invention on macrophage phenotypic polarization in vitro;

[0030] Figure 7 This is a characterization diagram of the effect of the immunomodulatory (double network) hydrogel of the present invention on the content of IL-6 and TNF-α in the supernatant of macrophages in vitro;

[0031] Figure 8 This is a characterization diagram of the liquid pad support effect during ESD surgery using the immunomodulatory (single network) hydrogel of the present invention;

[0032] Figure 9 This is a characterization diagram of the immune regulation (dual network) hydrogel of the present invention promoting the healing of pig esophageal wounds. DETAILED DESCRIPTION

[0033] In order 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 in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are only embodiments of a part of the present application, not all embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without making creative work should fall within the scope of protection of this application. It should be noted that, in the absence of conflict, the embodiments in the present application and the features in the embodiments can be combined with each other.

[0034] Secondly, the term "one embodiment" or "embodiment" 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 various places throughout this specification does not necessarily refer to the same embodiment, nor does it necessarily refer to a separate or selective embodiment that is mutually exclusive with other embodiments.

[0035] The terms "comprises" and "having" and any variations thereof in the specification and claims of this application are intended to cover non-exclusive inclusions. For example, a process, method, product or apparatus that includes a series of steps or elements is not necessarily limited to those steps or elements expressly listed, but may include other steps or elements not expressly listed or inherent to such process, method, product or apparatus.

[0036] Example 1: This example provides an immunomodulatory hydrogel, which uses amino-derivatized cationic polysaccharides, carboxyl-derivatized anionic polysaccharides, and DOPA-modified protein biopolymer materials as biological matrix materials (providing extracellular nutrient matrix), spermidine as a biologically active functional component (exerting anti-inflammatory immunomodulatory effects), and a benzaldehyde-terminated polymer material as a cross-linker. The amino groups and aldehyde groups undergo a Schiff base reaction to form a first layer of dynamic reversible cross-linked network structure to produce a single-network hydrogel; calcium ions are then introduced and the calcium ions are used to complex and cross-link with the carboxylate groups of the carboxyl-derivatized anionic polysaccharide to form a second layer of static irreversible cross-linked network structure to produce a double-network hydrogel.

[0037] Through step-by-step (two-step) cross-linking, the immunomodulatory hydrogel of the present invention not only has the function of immunomodulation (its own properties), but is also divided into single and double networks in terms of gel morphology. The two networks have their own focuses in performance and application, and cooperate with each other. Among them, the single-network hydrogel formed in the first step is injectable and self-healing, and has adhesion, which is convenient for injection and adhesion to surgical wounds. The double-network hydrogel formed in the second step is further sealed and solidified on the basis of the single-network hydrogel, which enhances the mechanical strength and stability to maintain stable and long-lasting performance.

[0038] The amino-derivatized cationic polysaccharide includes at least one of deacetylated chitosan, chitin, carboxymethyl chitosan and hydroxypropyl chitosan.

[0039] The carboxyl-derivatized anionic polysaccharide includes at least one of hyaluronic acid, chondroitin sulfate, sodium alginate and heparin.

[0040] DOPA-modified protein biopolymers include DOPA-modified pigskin, gelatin, or collagen. The modification principle involves a chemical reaction. Under an acidic environment, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) catalyze the amidation reaction between the protein biopolymer and dopamine hydrochloride, grafting dopamine onto the protein biopolymer. This maintains the inherent biological activity of the protein biopolymer while improving the wet adhesion properties of the hydrogel.

[0041] The benzaldehyde-terminated polymer cross-linking agent 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.

[0042] like Figure 1 As shown, the preparation method of the immunomodulatory hydrogel comprises the following steps:

[0043] Step 1: Synthesis of DOPA-modified protein biopolymer materials:

[0044] The protein biopolymer material was dissolved in deionized water at 50°C. After complete dissolution, EDC and NHS powders were added sequentially at 37°C to adjust the pH to 5.0-5.5. Dopamine hydrochloride powder was then added to adjust the pH to 5.5-6.0, and the mixture was reacted under nitrogen flow for 24 hours. After the reaction, the resulting aqueous solution was dialyzed for 3 days to completely remove unreacted reagents. The dialyzed solution was then freeze-dried to obtain the DOPA-modified protein biopolymer material.

[0045] Step 2: Prepare a polymer matrix material solution:

[0046] Amino-derivatized cationic polysaccharide, carboxyl-derivatized anionic polysaccharide, and DOPA-modified protein biopolymer powders were dissolved in normal saline (NS) at a temperature of 50-60°C and a rotation speed of 1000-2000 rpm to obtain a 2 wt%-8 wt% amino-derivatized cationic polysaccharide solution, a 0.8 wt%-6 wt% carboxyl-derivatized anionic polysaccharide solution, and a 16 wt%-40 wt% DOPA-modified protein biopolymer solution.

[0047] Step 3: Prepare crosslinker solution:

[0048] Dissolving benzaldehyde-terminated polymer powder in NS solution at room temperature to obtain a benzaldehyde-terminated polymer crosslinker solution;

[0049] Anhydrous calcium chloride (CaCl2) powder was dissolved in NS to obtain a CaCl2 crosslinker solution;

[0050] Step 4: Prepare the precursor solution:

[0051] The benzaldehyde-terminated polymer solution and the spermidine solution are sequentially added to the carboxyl-derivatized anionic polysaccharide solution to obtain a precursor solution A;

[0052] The amino-derivatized cationic polysaccharide and the DOPA-modified protein biopolymer solution were mixed in a volume ratio of 1:1, centrifuged at 1000-2000 rpm and 25-37°C for 10 min to form a uniformly mixed solution as precursor solution B.

[0053] Step 5: Preparation of single network hydrogel:

[0054] Precursor solution A is mixed with precursor solution B (the concentration of the amino-derivatized cationic polysaccharide solution in the mixed solution is 0.5wt%~2wt%, the concentration of the carboxyl-derivatized anionic polysaccharide solution is 0.2wt%~1.5wt%, the concentration of the DOPA-modified protein biopolymer material is 4wt%~10wt%, the concentration of spermidine is 25~500μM, the concentration of the benzaldehyde-terminated polymer cross-linker is 0.5wt%~1.5wt%, and the molecular weight is 10~20kDa) to obtain an injectable hydrogel pre-gel solution, and a Schiff base reaction occurs to form a first layer of cross-linked network. Within 10 minutes, a single-network hydrogel with injectable properties is obtained;

[0055] Step 6: Dual network cross-linking:

[0056] The single network hydrogel is immersed in a CaCl2 crosslinker solution, or the CaCl2 crosslinker solution is sprayed on the surface of the single network hydrogel, so that calcium ions are complexed and cross-linked with the carboxylate groups of the carboxyl-derivatized anionic polysaccharide to form a second layer of irreversible crosslinked network, thereby preparing a double network hydrogel, wherein the concentration of the soaking or spraying CaCl2 crosslinker solution is 1wt%~5wt%.

[0057] Example 2: This example further provides a method for preparing an immunomodulatory hydrogel. The difference from Example 1 is that Example 2 provides more specific materials, ratios and operating conditions, and is equipped with spermidine hydrogels of different concentrations.

[0058] The preparation method in this embodiment includes:

[0059] (1) DOPA modification of protein biopolymer materials:

[0060] 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. Then, slowly add 1.0 g of EDC and adjust the pH of the solution to 5.0-5.5. Activate the solution for 15 minutes. Then, add 0.6 g of NHS and adjust the pH to 5.0-5.5 again. Continue the reaction for 15 minutes.

[0061] 2.0 g of dopamine hydrochloride was added to the reaction system and reacted at 37 °C for 24 h. During the entire reaction, the pH value of the solution was always maintained between 5.5 and 6.0, and a nitrogen flow was used to protect the solution.

[0062] After the reaction, the resulting mixture was dialyzed against a 1 M hydrochloric acid (HCl) aqueous solution adjusted to pH = 5.5 for 3 days with a molecular weight cutoff (MWCO) of 8000–14000 Da to completely remove unreacted reagents;

[0063] After dialysis, the liquid was frozen and freeze-dried using a freeze dryer to obtain an off-white sponge-like GelDA conjugate sample, i.e., dopamine-modified gelatin. The sample was sealed and stored at -20°C until use. Gel represents gelatin and DA represents dopamine.

[0064] (2) Prepare polymer matrix material solution and cross-linking agent solution:

[0065] 0.4 g of carboxymethyl chitosan (OCMCS) powder was added to 10 ml of NS and stirred in a 50 °C water bath for 6 h until the OCMCS solid was completely dissolved to obtain an OCMCS solution with a concentration of 4 wt%;

[0066] 0.1 g of sodium alginate (SA) powder was added to 10 ml of NS and stirred in a 50 °C water bath until completely dissolved to obtain a 1 wt% SA solution;

[0067] 2.4 g of DOPA-modified gelatin (GelDA) was added to 10 ml of NS and stirred in a 50 °C water bath until completely dissolved to obtain a GelDA solution with a concentration of 24 wt%;

[0068] 0.2 g of benzaldehyde-terminated four-arm polyethylene glycol (4aPEG-BA) powder was dissolved in 1 ml of NS to obtain a 20 wt% 4aPEG-BA crosslinker solution;

[0069] 1 g of anhydrous calcium chloride powder was added to 50 ml of NS to obtain a CaCl2 crosslinker solution with a concentration of 2 wt%;

[0070] (3) Preparation of spermidine (SPD) solutions with different concentrations:

[0071] Dilution of SPD solution: add 15.7 μL of SPD stock solution to 1 mL of NS solution and mix thoroughly to obtain a 100 mM SPD solution, designated as SPD-1.

[0072] 100 μL of SPD-1 solution was added to 300 μL of NS solution and mixed thoroughly to obtain a 25 mM SPD solution, which was designated as SPD-2.

[0073] 50 μL of SPD-1 solution was added to 350 μL of NS solution and mixed thoroughly to obtain an SPD solution with a concentration of 12.5 mM, which was recorded as SPD-3;

[0074] 20 μL of SPD-1 solution was added to 380 μL of NS solution and mixed thoroughly to obtain a 5 mM SPD solution, which was designated as SPD-4.

[0075] 20 μL of SPD-1 solution was added to 780 μL of NS solution and mixed thoroughly to obtain a 2.5 mM SPD solution, which was designated as SPD-5.

[0076] (4) Preparation of precursor solution:

[0077] 40 µL of 4aPEG-BA crosslinker solution was added to 500 µL of SA solution and vortexed for 15 seconds to mix thoroughly. 20 µL of SPD solution (i.e., SPD-2, SPD-3, SPD-4, SPD-5) and NS were added to the mixed solution respectively and vortexed for 20 seconds to obtain precursor solution A.

[0078] Mix equal volumes of 250 µL of OCMCS solution and 250 µL of GelDA solution to obtain precursor solution B.

[0079] (5) Preparation of single network hydrogel:

[0080] like Figure 2 As shown, the precursor solution A (SA / 4aPEG-BA in the figure) and precursor solution B (OCMCS / GelDA in the figure) were mixed, vortexed, and allowed to 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 reacted with the aldehyde groups on the crosslinker to form the first layer of a dynamically reversible crosslinked network. The gelation time was determined as the time within 60 seconds after the reagent bottle was inverted without sample flow; in this example, the time used was within 5 minutes. The resulting hydrogels were single-network hydrogels with injectable and self-healing properties. The SPD concentrations in the hydrogels were 500, 250, 100, 50, and 0 μM, respectively, and are designated as SN-SPD500, SN-SPD250, SN-SPD100, SN-SPD 50, and SN-SPD50.

[0081] (6) Preparation of double network hydrogel:

[0082] The single network hydrogel prepared according to the above steps was sprayed or soaked with 2% CaCl2 solution for 2 min to obtain the corresponding double network hydrogels DN-SPD500, DN-SPD250, DN-SPD100, DN-SPD50, and DN.

[0083] 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.

[0084] Characterization of injectability: Pour the mixed pre-gel solution into a 1 ml or 2.5 ml syringe, push the syringe after gelation, and observe whether it can be injected smoothly and form a specific pattern. Figure 3 As shown in (a), SN-SPD100 hydrogel can pass through the syringe smoothly, forming a controllable injection path.

[0085] Characterization of self-healing performance: The SN-SPD100 hydrogel prepared in Example 2 was prepared by transferring the pre-gel solution with and without dye added to a "circular" mold to prepare a circular SN-SPD 100 hydrogel sample. After in situ gelation, the sample was cut into two identical semicircles. The semicircular hydrogels with and without dye added were then placed side by side and observed for their healing ability at 37°C. Figure 3As shown in (b), two semicircular hydrogels with dye added (red) and without dye added (brown) can form a complete circular hydrogel within 10 minutes after contact, indicating that the SN-SPD100 hydrogel has self-healing properties.

[0086] The above results indicate that the immunomodulatory hydrogel proposed in the present invention (at the single-network hydrogel stage) has excellent injectability and self-healing properties, can fill irregular or deep wound areas, and has broad clinical application prospects.

[0087] 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.

[0088] According to Example 2, the SN-SPD250 hydrogel pregel solution was injected onto the surface of pig skin and pig esophagus. After the hydrogel solidified, a 2% CaCl2 solution was sprayed on the surface to form the DN-SPD250 hydrogel in situ on the pig skin and pig esophagus. The adhesion of the DN-SPD250 hydrogel to the pig skin and pig esophagus was observed using stretching, torsion, bending, and water flushing tests.

[0089] like Figure 4 As shown in (ab), the DN-SPD250 hydrogel adhered to the surface of pig skin and pig esophagus can withstand stretching, bending, and torsion, indicating that it has good adhesion and toughness and can withstand a certain amount of external force without falling or breaking. Moreover, under the wet condition of running water, the DN-SPD250 hydrogel on the surface of pig esophagus can still maintain its adhesion state, indicating that it has good wet tissue adhesion. In addition, as Figure 4 (c) Through compression and rebound observation, it was found that DN-SPD250 hydrogel has excellent mechanical properties, combining strength and flexibility.

[0090] The above results show that the immunomodulatory hydrogel proposed in the present invention (in the double-network hydrogel stage) has stable adhesion and mechanical properties, and can fit well and adhere stably in a humid environment, ensuring the stability and durability of its function.

[0091] Application Example 3: This application example studies the effects of DN-SPD hydrogel (i.e., the double-network hydrogel of the present invention) on the secretion of inflammatory factors and cell phenotype polarization of macrophages at the in vitro cellular level, and clarifies the mechanism of action of different hydrogels DN, DN-SPD50, DN-SPD100, and DN-DETA100 (DN-DETA-modified hydrogel prepared by replacing spermidine with diethylenetriamine (DETA), which has a similar structure to SPD) on macrophages under lipopolysaccharide (LPS)-induced inflammatory conditions.

[0092] RAW 264.7 macrophages were cultured at a volume of 5 × 10 4 The cells were seeded at a density of 100 cells / well in a 24-well plate. After the cells adhered to the wall, the original culture medium was aspirated and the cells were divided into groups for intervention:

[0093] Positive control group: supplemented with complete culture medium containing 1 μg / mL LPS;

[0094] Experimental group: Complete medium containing 1 μg / mL LPS was supplemented, and then a Transwell chamber was placed in a well plate. A 150 μL cylindrical hydrogel (DN, DN-SPD50, DN-SPD100, DN-DETA100) prepared under sterile conditions according to Example 2 was placed in the upper chamber of the Transwell.

[0095] Blank control group: replace with fresh complete culture medium.

[0096] After cells in each group were cultured for 48 h, the cell morphology was observed and photographed, and the supernatant was recovered. The levels of IL-6 and TNF-α in the supernatant were detected by ELISA, and the macrophage-specific protein markers CD86 (M1 type) and CD206 (M2 type) were immunofluorescently stained.

[0097] like Figure 5 As shown, microscopic observation of cell morphology revealed that RAW 264.7 macrophages in the blank control group (Blank) were uniformly sized, round, and clustered. Under LPS-induced conditions (LPS in the figure), cells varied in size and exhibited multiple pseudopodia or irregular morphology. Under LPS-induced inflammation, the number of cells with irregular morphology or multiple pseudopodia in the DN-SPD hydrogel-treated groups (DN-SPD50 and DN-SPD100 in the figure) significantly decreased, while numerous M2 cells with a "spindle-shaped" morphology, characterized by two elongated pseudopodia, appeared. The number of spindle-shaped cells increased with increasing SPD concentration. In contrast, macrophages in the DN and DN-DETA hydrogel groups exhibited irregular morphology or multiple pseudopodia, similar to those in the LPS-induced group. This preliminary suggests that DN-SPD hydrogels can influence macrophage phenotypic polarization.

[0098] like Figure 6 As shown, immunofluorescence results showed that, compared with the blank control group, LPS induction alone showed strong positive expression of CD86, a marker for M1 macrophages (red fluorescence). In contrast, under LPS-induced inflammatory conditions, CD86 fluorescence expression intensity decreased in the DN-SPD hydrogel-treated group, while more positive expression of CD206, a marker for M2 macrophages (red fluorescence), was observed. However, no significant differences were observed between the DN and DN-DETA hydrogel-treated groups and the LPS-only induction group.

[0099] Enzyme-linked immunosorbent assay (ELISA) results Figure 7 As shown in the results, the IL-6 content in the supernatant of the DN-SPD50 and DN-SPD100 hydrogel-treated groups decreased by 34.93% and 54.85%, and the TNF-α content decreased by 24.54% and 40.85%. In contrast, the DN and DN-DETA hydrogel-treated groups had no significant effect on the IL-6 and TNF-α content in the supernatant.

[0100] The above results indicate that DN-SPD hydrogel inhibits the secretion of inflammatory factors and can effectively inhibit the polarization of macrophages to the M1 pro-inflammatory phenotype and promote their polarization to the M2 anti-inflammatory and pro-repair phenotype.

[0101] Application Example 4: Given that DN-SPD hydrogel exhibits significant inflammatory response reduction and immune regulation, this application example further established a Bama Xiang pig esophageal ESD surgical model (n=3) to evaluate the support effect of SN-SPD hydrogel as an intraoperative liquid cushion and the effect of DN-SPD hydrogel on the healing rate and scar formation of the mucosal wound after esophageal ESD. The specific steps are as follows:

[0102] (1) Preoperative preparation and anesthesia: Female Bama Xiang pigs weighing 20-25 kg were fasted for 24 hours and deprived of water for 6 hours before surgery. After isoflurane inhalation anesthesia, propofol was injected into the ear vein to achieve deep anesthesia. Subsequently, with the assistance of a laryngoscope, a tracheal tube was inserted. Low-flow isoflurane was continuously inhaled during the operation to maintain anesthesia.

[0103] (2) ESD surgery: used to construct mucosal and submucosal defect wounds and observe the intraoperative liquid pad support effect. It mainly includes the following specific steps: ① Marking range: insert a gastroscope through the mouth, clean the esophageal cavity, select three anatomical sites in the middle and lower esophagus (spacing ≥ 2 cm), and use a transparent cap with scale to assist in positioning and establish a 2 × 3 cm rectangular mucosal marking area. ② Submucosal injection: The marked area was divided from the oral to anal side into the normal saline group (NS), the DN-SPD 250 group, and the DN-SPD 500 group. The normal saline group received a submucosal injection of normal saline as a liquid cushion, while the DN-SPD 250 and DN-SPD 500 groups received injections of SN-SPD 250 and SN-SPD 500 hydrogels, respectively. Hydrogel precursor solution A and precursor solution B were prepared according to Example 2, mixed through a three-way valve, and then injected into the submucosal layer as liquids via a 22 G needle to form a single-network hydrogel in situ. (One pig was randomly selected, and the height maintenance of the liquid cushion was observed 15, 30, and 60 minutes after injection.) ③ Excision: An electrosurgical scalpel was used to excise the mucosa and submucosal tissue along the marked area.

[0104] (3) Wound treatment: After the excision was completed, the corresponding SN hydrogel precursor solution was injected into the wound of the experimental group. After gelation, 2% calcium chloride was sprayed on the wound to further cross-link and form the corresponding DN-SPD250 and DN-SPD500 hydrogels.

[0105] (4) Postoperative observation and sampling: The pigs were fasted for 24 hours after surgery, and a liquid diet was given on the second day. Solid feed was resumed on the third day. Gastroscopy was performed on the 7th, 14th, 21st, and 28th days after surgery to observe wound healing. Subsequently, on the 28th day after surgery, the experimental pigs were killed by overdose of anesthesia, and esophageal tissue samples were obtained. The samples were fixed in 4% paraformaldehyde solution and then stained for tissue section analysis.

[0106] The results of the supporting effect of single network hydrogel as a liquid cushion in ESD surgery, such as Figure 8 As shown in the data, normal saline (NS), SN-SPD250, and SN-SPD500 all achieved good lifting effects after submucosal injection. 15 minutes after injection, the lifting effect of the NS group disappeared. In contrast, the SN-SPD250 and SN-SPD500 hydrogels were able to maintain a good lifting effect, with no significant change from the initial height. Furthermore, even after continuous observation for 60 minutes after injection, the degree of lifting did not significantly decrease.

[0107] The above results indicate that SN-SPD hydrogel has a long-lasting supporting effect as a liquid cushion during ESD surgery.

[0108] Effects of double-network hydrogel on wound healing after esophageal ESD. Figure 9 As shown, endoscopic observation on the 7th day after surgery revealed that new granulation tissue filled the ulcer defect in each group, with regenerated epithelium visible at the edge. The DN-SPD500 wound surface was significantly smaller than that in the NS group. On the 14th day after surgery, endoscopic observation showed that the ulcers in each group had healed, with the surface re-epithelialized to a light red, translucent "film" that gradually merged with the surrounding normal mucosa. Compared with the NS group, the light red area on the mucosal surface in the DN-SPD hydrogel group was significantly reduced, replaced by a pinkish-white epithelium with the same color as the surrounding normal mucosa. The light red area was further reduced in the DN-SPD500 group compared with the DN-SPD 250 group. On the 21st and 28th days after surgery, the light red epithelial area in each group further decreased, especially in the DN-SPD500 group, which was almost consistent with the surrounding normal mucosa.

[0109] The above results show that DN-SPD hydrogel can effectively promote the healing of wounds after esophageal ESD surgery.

[0110] In summary, the present invention proposes an immunomodulatory hydrogel and a preparation method thereof, which adopts a step-by-step strategy and has excellent injectability, self-repairability, tissue adhesion and immunomodulatory functions. It can effectively promote the healing of postoperative wounds and has broad application prospects in ESD surgery in the esophagus, stomach, intestines and other parts.

[0111] The serial numbers of the above embodiments of the present invention are for description only and do not represent the advantages or disadvantages of the embodiments.

[0112] Those skilled in the art should understand that the purpose of the present invention is to provide a more 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 scales, product requirements, etc.). Therefore, some specific operations can be flexibly adjusted according to the situation during actual implementation, as long as the expected technical effect or the same or similar technical effect as in the embodiments of the present invention can be achieved.

[0113] In the above-mentioned embodiments of the present invention, the description of each embodiment has its own emphasis. For parts not described in detail in a certain embodiment, please refer to the relevant description of other embodiments. The steps in the method of the embodiment of the present invention can be adjusted in order, merged and deleted according to actual needs. The various technical features can be combined in any way. In order to make the description concise, not all possible combinations of the various technical features in the embodiment are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of the present invention.

[0114] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. An immunomodulatory hydrogel, characterized in that: The immunomodulatory hydrogel is used in ESD surgery using a step-by-step strategy, including: Using amino-derivatized cationic polysaccharides, carboxyl-derivatized anionic polysaccharides, and DOPA-modified protein biopolymers as biomatrix materials, spermidine as a bioactive functional component, and a benzaldehyde-terminated polymer as a cross-linking agent, a Schiff base reaction between amino groups and aldehyde groups forms a first layer of a dynamically reversible cross-linked network structure to obtain a single-network hydrogel. The single-network hydrogel is used for submucosal injection as a liquid pad for ESD surgery. Afterwards, calcium ions were introduced on the basis of the single-network hydrogel, and the calcium ions were used to complex and cross-link with the carboxylate groups of the carboxyl-derivatized anionic polysaccharide to form a second layer of static non-reversible cross-linked network structure, thereby obtaining a double-network hydrogel. The double-network hydrogel was used to seal the ESD surgical wound and promote wound repair.

2. The immunomodulatory hydrogel according to claim 1, characterized in that The amino-derivatized cationic polysaccharide includes at least one of deacetylated chitosan, chitin, carboxymethyl chitosan, and hydroxypropyl chitosan.

3. The immunomodulatory hydrogel according to claim 1, characterized in that The carboxyl-derivatized anionic polysaccharide includes at least one of hyaluronic acid, chondroitin sulfate, sodium alginate, and heparin.

4. The immunomodulatory hydrogel according to claim 1, characterized in that The DOPA-modified protein biopolymer material includes DOPA-modified pigskin, gelatin or collagen. Dopamine is grafted onto the protein biopolymer material through an amidation reaction to improve the wet adhesion performance of the hydrogel.

5. The immunomodulatory hydrogel according to claim 1, characterized in that The benzaldehyde-terminated polymer crosslinking agent 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.

6. A method for preparing an immunomodulatory hydrogel, for preparing the immunomodulatory hydrogel according to claim 1, characterized in that: include: Step 1: Synthesis of DOPA-modified protein biopolymer materials: The protein biopolymer material is dissolved in deionized water, and EDC, NHS and dopamine hydrochloride are added in sequence. Under the catalytic action of EDC and NHS, the amino group of dopamine hydrochloride undergoes an amidation reaction with the carboxyl group on the protein biopolymer material. After the reaction is completed, the material is dialyzed and freeze-dried to obtain a dopamine-modified protein biopolymer material, wherein EDC is 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and NHS is N-hydroxysuccinimide. Step 2: Prepare a polymer matrix material solution: Adding amino-derivatized cationic polysaccharide, carboxyl-derivatized anionic polysaccharide and DOPA-modified protein biopolymer material into normal saline respectively to prepare amino-derivatized cationic polysaccharide solution, carboxyl-derivatized anionic polysaccharide solution and DOPA-modified protein biopolymer material solution; Step 3: Prepare crosslinker solution: Dissolving a benzaldehyde-terminated polymer material in physiological saline to prepare a benzaldehyde-terminated polymer cross-linking agent; Anhydrous calcium chloride powder was added to normal saline to prepare CaCl2 cross-linking agent; Step 4: Prepare the precursor solution: Mixing a carboxyl-derivatized anionic polysaccharide solution, a benzaldehyde-terminated polymer crosslinker solution, and a spermidine solution diluted in a certain proportion to obtain a precursor solution A; Mixing an amino-derivatized cationic polysaccharide solution and a DOPA-modified protein biopolymer solution to obtain a precursor solution B; Step 5: Preparation of single network hydrogel: At room temperature, precursor solution A and precursor solution B are mixed to allow the amino groups of the amino-derivatized cationic polysaccharide, the amino groups on the spermidine molecular chain, and the amino groups on the DOPA-modified protein biopolymer material to undergo a Schiff base reaction with the aldehyde groups on the benzaldehyde-terminated polymer cross-linker to form a first layer of reversibly cross-linked network structure, thereby obtaining a single network hydrogel; Step 6: Dual network cross-linking: Then, a CaCl2 crosslinker solution is added by soaking or spraying to allow calcium ions to complex and crosslink with the carboxylate groups of the carboxyl-derivatized anionic polysaccharide to form a second layer of static non-reversible crosslinked network to obtain a double-network hydrogel.

7. The method for preparing the immunomodulatory hydrogel according to claim 6, characterized in that: The reaction process in step 1 is maintained under acidic conditions and protected by nitrogen flow.

8. The method for preparing the immunomodulatory hydrogel according to claim 6, characterized in that: In the solution obtained by mixing the precursor solution A and the precursor solution B in step five, the concentration of the amino-derivatized cationic polysaccharide solution is 0.5wt%~2wt%, the concentration of the carboxyl-derivatized anionic polysaccharide solution is 0.2wt%~1.5wt%, the concentration of the DOPA-modified protein biopolymer material is 4wt%~10wt%, the concentration of spermidine is 25~500μM, the concentration of the benzaldehyde-terminated polymer cross-linker is 0.5wt%~1.5wt%, and the molecular weight of the benzaldehyde-terminated polymer cross-linker is 10~20kDa.

9. The method for preparing the immunomodulatory hydrogel according to claim 6, characterized in that: The concentration of the CaCl2 cross-linking agent in step six is 1 wt% to 5 wt%.

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