Anion-pi-based zwitterionic supramolecular hydrogel as well as preparation method and application thereof

Abstract

The invention discloses zwitter-ion supramolecular hydrogel based on anion-pi as well as a preparation method and application of the zwitter-ion supramolecular hydrogel. The hydrogel is formed by self-assembly of a zwitterionic copolymer and a p-styrenesulfonic acid copolymer. According to the supramolecular hydrogel disclosed by the invention, the supramolecular hydrogel can be rapidly formed only by simply mechanically mixing the zwitterionic polymer copolymer and the p-styrene sulfonate copolymer solution, and the supramolecular hydrogel is endowed with excellent injectability and self-repairing performance due to the dynamic cross-linking characteristic. The hydrogel is composed of zwitterions with excellent immunocompatibility and poly (p-styrene sulfonate) with heparin-imitating performance, so that the hydrogel has excellent immunocompatibility, can play a role in various in-vivo application scenes, and in addition, the hydrogel can be used for preparing an anti-inflammatory drug. The dynamic cross-linked network based on anion-pi interaction and the heparin-like structure are very beneficial to protein preservation, and have wide application prospects in the fields of protein delivery and cell encapsulation.

Description

Technical Field

[0001] The present invention relates to the technical field of medical materials, and specifically relates to an anion-π-based zwitterionic supramolecular hydrogel and its preparation method and application. Background Art

[0002] Hydrogels are a class of polymer materials prepared by chemical or physical cross-linking, with a three-dimensional network structure that can firmly bind a large amount of water molecules to form a gel system with a high water content. This structure endows hydrogels with excellent biocompatibility, so they have great application potential in the biomedical field.

[0003] Due to the stability of their chemical bonds, chemically cross-linked hydrogels exhibit good structural stability. However, this high stability also brings inconvenience in applications. Chemically cross-linked hydrogels with a fixed shape usually need to be implanted through surgery, which not only increases the complexity of the surgery but also limits the choice of the implantation site. It is also easy to cause large-area tissue damage during the surgery, and it lacks flexibility when filling irregularly shaped tissues, severely restricting its application scope.

[0004] In recent years, supramolecular hydrogels based on non-covalent interactions have received extensive attention due to their unique dynamic reversible properties. These hydrogels construct dynamic cross-linked networks through non-covalent bonds such as hydrogen bonds and hydrophobic interactions, and have significant advantages such as injectability and self-healing properties.

[0005] CN119570070A discloses a citric acid-cyclodextrin derivative controllable release composite supramolecular hydrogel and its preparation method, including mixing citric acid and cyclodextrin and fully dissolving them by ultrasonic treatment; adjusting the pH value of the solution to 0.5 - 2.0 to promote the cross-linking reaction, and carrying out the cross-linking reaction at 40 - 60 °C for 3 - 5 h based on the optimized temperature conditions; cooling the reaction solution to room temperature and adding an appropriate amount of cross-linking agent for self-assembly to form a gel; after drying treatment, shaping the gel into the required form and maintaining a stable structure. By optimizing the cross-linking reaction conditions of citric acid and cyclodextrin, a hydrogel with a unique network structure and mechanical strength is obtained. This material has excellent water absorption, biocompatibility and mechanical properties, and is particularly suitable for tissue engineering, biomedicine and environmental protection fields. The preparation process includes multiple steps such as chemical treatment of raw materials, control of reaction conditions, self-assembly and cross-linking reaction, shaping and drying.

[0006] CN110885391A discloses a supramolecular hydrogel based on double hydrogen bonds and a preparation method thereof. Alanylamide hydrochloride and acryloyl chloride are reacted to prepare acryloyl alanylamide (NAAA) monomer. This monomer has a double hydrogen bond structure. A solution with a certain concentration of this monomer and water is polymerized by ultraviolet light irradiation, and a supramolecular hydrogel based on double hydrogen bonds with different solid contents is formed. A comonomer with a double hydrogen bond structure forms a physically hydrogel crosslinked by hydrogen bonds through free radical polymerization. Compared with the gel prepared from acrylamide monomer, this double hydrogen bond monomer has a higher hydrogen bond crosslinking density. Therefore, the double hydrogen bond monomer can form a hydrogel without adding any crosslinking agent, and the prepared supramolecular hydrogel is more stable than the acrylamide hydrogel.

[0007] Anion-π interaction is a newly emerging non-covalent force, referring to the electrostatic attraction between electron-rich anions (such as sulfonate, carboxylate) and electron-deficient aromatic rings (such as hexafluorobenzene ring, triazine ring). Although the mechanism of anion-π interaction has been clearly defined in biological processes such as enzyme catalysis and bioadhesion, its application in materials engineering is still relatively scarce. Currently, no supramolecular hydrogel based on anion-π interaction has been reported, which limits the understanding and application of this interaction in material design. Summary of the Invention

[0008] Aiming at the difficulty of supramolecular hydrogels to combine the advantages of structural stability and injectability, the present invention provides an amphiphilic ion supramolecular hydrogel based on anion-π. This hydrogel is obtained by self-assembly of an amphiphilic ion copolymer and a styrene sulfonate copolymer. Its dynamic crosslinking characteristics endow it with excellent injectability and self-healing properties, and have broad application prospects in the medical field.

[0009] To achieve the above object, the technical solution adopted by the present invention is: An amphiphilic ion supramolecular hydrogel based on anion-π, which is self-assembled from a polyamphiphilic ion copolymer and a styrene sulfonic acid copolymer.

[0010] In the present invention, the supramolecular hydrogel can be rapidly formed by simply mechanically mixing the amphiphilic ion copolymer solution and the styrene sulfonate copolymer solution. Its dynamic crosslinking characteristics endow it with excellent injectability and self-healing properties. This hydrogel is composed of amphiphilic ions with excellent immunocompatibility and a styrene sulfonate copolymer with heparin-like properties. Therefore, it has excellent immunocompatibility and can play a role in various in vivo application scenarios. In addition, this dynamic crosslinking network based on anion-π interaction and heparin-like structure is very conducive to the preservation of proteins, and has broad application prospects in the fields of protein delivery and cell encapsulation.

[0011] The zwitterionic copolymer is obtained by copolymerizing raw materials including zwitterionic monomers. The zwitterionic monomers include any one or more of sulfobetaine methacrylate (SBMA), carboxybetaine methacrylate (CBMA), and 2-methacryloyloxyethyl phosphorylcholine (MPC); The styrene sulfonate copolymer is obtained by copolymerizing monomers including styrene sulfonic acid-containing monomers. The styrene sulfonic acid-containing monomers include one or more of styrene sulfonic acid, sodium styrene sulfonate, potassium styrene sulfonate, and lithium styrene sulfonate.

[0012] The weight-average molecular weight of the zwitterionic copolymer is 50,000 to 5,000,000; the weight-average molecular weight of the styrene sulfonate copolymer is 50,000 to 5,000,000. The greater the molecular weight, the higher the gel strength and the better the elasticity.

[0013] The molar ratio of zwitterions to styrene sulfonic acid repeating units in the zwitterionic copolymer and the styrene sulfonate copolymer is 1:0.1 - 10. Preferably, the molar ratio of zwitterions to styrene sulfonic acid repeating units in the zwitterionic copolymer and the styrene sulfonate copolymer is 1:0.25 - 4; preferably, the molar ratio of zwitterions to styrene sulfonic acid repeating units in the zwitterionic copolymer and the styrene sulfonate copolymer is 1:0.5 - 2; when the molar ratio of zwitterions to styrene sulfonate repeating units in the zwitterionic copolymer and the styrene sulfonate is 1:1, the interaction is the strongest, and the elasticity and strength of the hydrogel are more excellent.

[0014] The polymerization monomer raw materials of the zwitterionic copolymer further include a first comonomer; the first comonomer includes one or more monomers of acrylate esters, methacrylate esters, acrylamide, etc.; Preferably, the mass ratio of the zwitterionic monomer in the polymerization monomer raw materials of the zwitterionic copolymer is more than 50% to effectively form a gel. Further preferably, the mass ratio is more than 60%, more than 70%, and more than 80%.

[0015] The polymerization monomer raw materials of the styrene sulfonate copolymer further include a second comonomer; the second comonomer includes one or more of acrylic acid, methacrylic acid, maleic anhydride, styrene or its derivatives, acrylate esters, methacrylate esters, acrylamide, etc.; Preferably, the mass ratio of the styrene sulfonate monomer in the polymerization monomer raw materials of the styrene sulfonate copolymer is 50% to effectively form a gel. Further preferably, the mass ratio is more than 60%, more than 70%, and more than 80%.

[0016] The present invention also provides a preparation method of the anion-π-based zwitterionic supramolecular hydrogel according to the above, including the steps: Step 1: Copolymerize the raw materials containing zwitterionic monomers to obtain a zwitterionic copolymer; copolymerize the raw materials including monomers containing styrenesulfonic acid to obtain a styrenesulfonate copolymer. Step 2: Dissolve the zwitterionic copolymer and the styrenesulfonate copolymer in an aqueous solution respectively, and after mixing, self-assemble to obtain the zwitterionic supramolecular hydrogel.

[0017] In Step 2, the total concentration of the zwitterionic copolymer and the styrenesulfonate copolymer in the aqueous solution is 1 - 60 wt%. Preferably, the mass concentration of the zwitterionic copolymer and the styrenesulfonate copolymer in the aqueous solution is 0.1 - 50 wt%; more preferably 0.1 - 20 wt%, and even more preferably 0.1 - 15 wt%.

[0018] The aqueous solution includes one or more of water, PBS buffer solution, physiological saline, Hanks buffer solution, and Tris buffer solution.

[0019] In Step 1, the copolymerization contains an initiator, the copolymerization temperature is 60 - 90 °C, the reaction is for 6 - 18 h, and the product is dialyzed and freeze-dried to obtain the copolymer.

[0020] The initiator includes one or more of sodium persulfate, ammonium persulfate, and potassium persulfate, and the mass fraction of the initiator in the aqueous solution is 0.1 - 5%; The present invention also provides the application of the anion-π zwitterionic supramolecular hydrogel in the preparation of immunocompatible drugs or medical materials. For example, it is used for diabetic wound dressings: The supramolecular hydrogel based on anion-π interaction, because it is composed of zwitterions with super hydrophilicity and styrenesulfonate with a heparin-mimicking structure, so it has a stabilizing effect on basic fibroblast growth factor with the ability to promote wound healing. At the same time, the zwitterionic structure can promote angiogenesis at the wound site. Therefore, this hydrogel can be used for the loading of basic fibroblast growth factor and applied to the healing dressings of diabetic wounds. The data is as follows: Another example is for cell culture scaffolds: The supramolecular hydrogel based on anion-π interaction is composed of zwitterionic polymers with excellent anti-fouling properties. Therefore, this hydrogel has excellent anti-cell and anti-bacterial adhesion properties. At the same time, this highly immunocompatible hydrogel based on this dynamic covalent bond structure can be used for cell culture scaffolds.

[0021] Compared with the prior art, the present invention has the following beneficial effects: (1) The present invention prepares an anion-π zwitterionic supramolecular hydrogel, breaking through the technical prejudice that "anion-π interaction is difficult to support the gel network" in the traditional view, and broadening the preparation principle of supramolecular hydrogels; (2) The prepared supramolecular hydrogel does not require sophisticated structural design and can be rapidly and simply prepared by using commercially available zwitterionic polymers and styrene sulfonate copolymers, avoiding the introduction of other chemical substances such as cross-linking agents. The process is simple and easy to popularize. (3) The anion-π supramolecular hydrogel prepared in the present invention has excellent immunocompatibility and shows broad application prospects in the field of biomedicine. Description of the Drawings

[0022] To more clearly illustrate the technical solutions of the embodiments of the present application, the following will briefly introduce the drawings required to be used in the embodiments of the present application. It should be understood that the following drawings only show some embodiments of the present application and should not be regarded as limiting the scope. For those of ordinary skill in the art, other related drawings can be obtained based on these drawings without creative efforts.

[0023] Figure 1 Strain sweep ( Figure 1 in a) and angular frequency sweep test ( Figure 1 in b) results of the anion-π supramolecular hydrogel prepared in Example 1.

[0024] Figure 2 UV-visible absorption spectra of the anion-π supramolecular hydrogel and two copolymer raw materials.

[0025] Figure 3 Heat released by the PSBMA solution titrated with the PSSNa solution measured by a nano-drop calorimeter and the Gibbs free energy, enthalpy change and entropy change obtained after fitting calculation.

[0026] Figure 4 Schematic diagram of the action principle of the anion-π supramolecular hydrogel prepared in Example 1.

[0027] Figure 5 Visualization diagram of the formation mechanism of the anion-π supramolecular hydrogel proved by molecular simulation calculation: no sodium ions (left), with sodium ions (right).

[0028] Figure 6 Statistical chart of the elasticity and strength of the gels obtained by mixing PSBMA and PSSNa at different molar ratios of repeating units in Example 4.

[0029] Figure 7 Control chart of the fibrosis degree of Masson trichrome immunohistochemical sections after one month of subcutaneous implantation in the back of mice with injectable PEGDA, sodium alginate hydrogel and the PSBMA-PSSNa supramolecular hydrogel prepared in Example 1 in Application Example 1.

[0030] Figure 8Control chart of the PSBMA-PSSNa anion-π supramolecular hydrogel prepared in Application Example 2 for preserving the protein activity of basic fibroblast growth factor (bFGF).

[0031] Figure 9 Control chart of the wound healing area on days 0, 4, 7, and 14 after using the SSH hydrogel loaded with bFGF, the SSH hydrogel without bFGF, a commercial sodium alginate wound dressing, and gauze as wound dressings for diabetic mice in Application Example 3.

[0032] Figure 10 Digital physical control chart of the wound healing condition on days 0, 4, 7, and 14 after using the SSH hydrogel loaded with bFGF, the SSH hydrogel without bFGF, a commercial sodium alginate wound dressing, and gauze as wound dressings for diabetic mice in Application Example 3. Detailed implementation manners

[0033] In order to make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention. Those skilled in the art make modifications or equivalent replacements based on the understanding of the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all should be covered within the protection scope of the present invention.

[0034] The raw materials used in the following detailed implementation manners are all purchased from the market.

[0035] Example 1 Step 1: Dissolve 10% by mass of sulfobetaine monomer (SBMA) in water, then add 1 wt% of sodium persulfate based on the mass of the reaction monomers, and bubble nitrogen for 20 min. Then place it in an oil bath at 70°C and stir for 12 h. Then dialyze and freeze-dry to obtain a PSBMA polymer powder with a molecular weight of 170,000; dissolve 10% by mass of sodium p-styrenesulfonate (SSNa) in water, then add, then add 1 wt% of sodium persulfate based on the mass of the reaction monomers, and bubble nitrogen for 20 min. Then place it in an oil bath at 70°C and stir for 12 h. Then dialyze and freeze-dry to obtain a PSSNa polymer powder with a molecular weight of 200,000; Step 2: Dissolve 10% by mass of PSBMA in PBS buffer and 30% by mass of PSSNa in aqueous solution. Then, take 1 mL of the PSBMA solution and mix it thoroughly with 0.25 mL of the PSSNa solution (the molar ratio of zwitterions to styrene sulfonic acid repeat units in the copolymer is 1:1). Place the resulting hydrogel in an oven at 80 °C for 2 h to equilibrate, and the PSBMA-PSSNa anion-π supramolecular hydrogel is obtained.

[0036] The rheological properties of the anion-π supramolecular hydrogel prepared in Example 1 were characterized. All rheological tests were carried out on a rotational rheometer (TA Instruments, Discovery HR-20) using a parallel plate fixture with a diameter of 20 mm and a gap of 1500 μm at a test temperature of 37 °C. The sample was allowed to stand for 1 minute before performing an oscillatory strain sweep (strain range: 0.1–2000%, fixed frequency of 10 rad / s) and an oscillatory frequency sweep (frequency range: 0.1–100 rad / s, fixed strain of 1%). The results are as Figure 1 shown. These curves indicate that the elastic and viscous properties of the material change with the strain ( Figure 1 a in Figure 1 ) and the angular frequency ( b in

[0037] ), and the elastic property is dominant, further indicating that the material exhibits solid-like behavior. Figure 2

[0038] To confirm the gelation mechanism of the hydrogel, ultraviolet spectroscopy was used to analyze the PSSNa solution, the PSBMA solution, and the mixed and diluted solution of PSSNa and PSBMA. The results are as Figure 3 shown. It can be seen that in the presence of PSBMA, the ultraviolet characteristic absorption peak of the benzene ring in the PSSNa molecule shows a red shift, indicating that its electron cloud distribution is affected. It can be speculated that non-covalent interactions related to the aromatic ring occur in the system.

[0039] Furthermore, as Figure 4The shown anion-π interaction model has two main interactions between PSBMA and PSSNa. First, the sulfonate anions in the PSBMA molecule form anion-π-cation interactions (hereinafter simply referred to as anion-π interactions) with the benzene rings on the side chains of PSSNa and the cations in the system through electron cloud interactions. Second, ionic interactions occur between the quaternary ammonium cations in PSBMA and the sulfonate anions of PSSNa. The synergistic effect of these two interactions promotes the formation of a tight interlocked structure of the side chains of PSBMA and PSSNa, thus significantly affecting the microstructure and macroscopic properties of the mixed system.

[0040] Figure 5 The shown molecular simulation results further confirm the rationality of the anion-π interaction model at the molecular level. Visualization analysis shows that in the presence of cations, there are non-covalent interactions (anion-π interactions) between the sulfonate groups and the benzene rings, while in the cation-free system, this interaction completely disappears.

[0041] Example 2 Step 1: Dissolve 10% by mass of carboxybetaine monomer (CBAA) in water, then add 1 wt% of sodium persulfate based on the mass of the reaction monomers, and bubble nitrogen for 20 min. Then place it in a 70 °C oil bath and stir for 12 h. Then dialyze and freeze-dry to obtain PCBAA polymer powder; dissolve 10% by mass of sodium p-styrenesulfonate (SSNa) in water, then add 1 wt% of sodium persulfate based on the mass of the reaction monomers, and bubble nitrogen for 20 min. Then place it in a 70 °C oil bath and stir for 12 h. Then dialyze and freeze-dry to obtain PSSNa polymer powder; Step 2: Dissolve 10% by mass of PCBMA in PBS buffer solution and 30% by mass of PSSNa in aqueous solution. Then take 1 mL of PCBAA solution and 0.25 mL of PSSNa solution and mix them well (the molar ratio of zwitterions to p-styrene sulfonic acid repeating units in the copolymer is 1.2:1). Place the obtained hydrogel in an 80 °C oven to equilibrate for 2 h to obtain the CBAA-PSSNa anion-π supramolecular hydrogel.

[0042] Example 3 Step 1: Dissolve 8% by mass of sulfobetaine monomer (SBMA) and 2% by mass of acrylamide (AAM) in water. Subsequently, add 1 wt% of sodium persulfate based on the mass of the reaction monomers, and bubble nitrogen for 20 min. Then, place it in a 70 °C oil bath and stir for 12 h. Subsequently, dialyze and freeze-dry to obtain a PSBMA-AAM polymer powder with a molecular weight of 210,000; dissolve 10% by mass of sodium p-styrenesulfonate (SSNa) in water. Subsequently, add 1 wt% of sodium persulfate based on the mass of the reaction monomers, and bubble nitrogen for 20 min. Then, place it in a 70 °C oil bath and stir for 12 h. Subsequently, dialyze and freeze-dry to obtain a PSSNa polymer powder with a molecular weight of 200,000. Step 2: Dissolve 10% by mass of PCBMA in PBS buffer solution, and dissolve 30% by mass of PSSNa in aqueous solution. Subsequently, take 1 mL of PCBAA solution and mix it thoroughly with 0.25 mL of PSSNa solution. Place the obtained hydrogel in an 80 °C oven and equilibrate for 2 h to obtain the PSBMA-AAM-PSSNa anion-π supramolecular hydrogel.

[0043] Example 4 According to the preparation process of Example 1, change the ratio when mixing the PSBMA solution and the PSSNa solution in Step 2, so that the molar ratio of p-styrenesulfonic acid and zwitterionic repeating units in PSSNa and PSBMA is 1:4, 1:2, 2:1, 3:1, to obtain different hydrogels. The test results of the elasticity and strength of the gels obtained by mixing poly(sulfobetaine methacrylate) (PSBMA) with a molecular weight of 170,000 and sodium p-styrenesulfonate with a molecular weight of 200,000 at different molar ratios of repeating units are as Figure 6 shown. It can be seen that the proportion of the two has an impact on the mechanical properties of the hydrogel. Among them, when the molar ratio of the repeating units of zwitterion and p-styrenesulfonic acid is in the range of 1:0.5 - 2, the hydrogel has more excellent mechanical strength and elasticity.

[0044] Application Example 1: Fibrosis Reaction of Representative Anion-Π Injectable Hydrogel in Vivo 1. Animal surgery procedure In this application example, the fibrosis reaction of the PSBMA-PSSNa anion-π supramolecular hydrogel in a mouse subcutaneous implantation model is described. Inject the PSBMA-PSSNa hydrogel (denoted as SSH in the figure) prepared as in Example 1, PEGDA, and sodium alginate hydrogel (Alg) into the subcutaneous tissue of the back of C57BL / 6 female mice through a syringe, inject 100 μL for each mouse, and evaluate its fibrosis reaction on the 28th day.

[0045] 2. H&E and Masson's trichrome staining immunohistochemical sections Mice were sacrificed at the 4th week, the implants and surrounding tissues were excised, fixed with 10% formaldehyde, embedded in paraffin, sectioned, and then subjected to hematoxylin-eosin (H&E) / Masson's trichrome staining and immunohistochemical staining. Images were acquired using a Nikon intensilight CHGFI microscope equipped with NIS-Elements AR software and an Olympus VS120-S6-W virtual slide microscope. Collagen density was measured by calculating the coverage percentage of blue pixels every 50 μm in the images of Masson's trichrome-stained tissues.

[0046] As Figure 7 shown, it can be clearly seen from the images that a dense collagen capsule was formed in the tissues around the PEG hydrogel and alginate hydrogel. Notably, almost no fibrotic reaction was observed around the SSH hydrogel, indicating its excellent ability to resist foreign body reactions.

[0047] Application Example 2 Representative Anion-π Injectable Hydrogel for Protein Encapsulation and Maintenance of Protein Activity 1. Experimental Procedure This experiment was used to evaluate the ability of the anion-π injectable hydrogel to preserve protein activity. First, 50 μL of a PSBMA solution with a concentration of 10% and a molecular weight of approximately 1,210,000 was thoroughly mixed with 150 μL of a PBS solution containing 2.5 mg / mL basic fibroblast growth factor (bFGF). Then, the resulting solution was mixed with a PSSNa solution with a concentration of 3.75% and a molecular weight of approximately 200,000 at a volume ratio of 4:1, and gently stirred to form a gel, thereby preparing a gel loaded with bFGF (bFGF@SSH).

[0048] Subsequently, the bFGF@SSH gel and the free bFGF solution were treated separately: a part was incubated at 56 °C for 30 min, and another part was stored in an environment at 37 °C for 3 days, thus constructing four groups of experimental samples, namely bFGF@SSH (heating group), bFGF (heating group), bFGF@SSH (storage group), and bFGF (storage group). Before the experiment, fresh bFGF@SSH, free bFGF solutions, and protein-free SSH gels prepared according to the above preparation process were re-prepared as controls. 4 μL of each sample was taken and added to the culture wells seeded with cells, and the culture plates were incubated under appropriate conditions for 24 h. After the incubation, the cell viability was measured by the MTT method, and the PBS solution group was used as a blank control for subsequent data comparison and analysis.

[0049] 2. Result Analysis The results of the cell proliferation experiment are as Figure 8 shown. After treatment, free bFGF basically lost its activity. In contrast, compared with the fresh bFGF solution, bFGF@SSH still retained about 80% of its activity after heat treatment and about 60% of its activity after storage at 37°C. At the same time, there was no significant difference in the efficiency of promoting cell proliferation between the fresh free bFGF solution and the fresh bFGF@SSH solution. The above results indicate that encapsulating bFGF in SSH does not cause loss of its activity. In addition, due to the stabilizing effect of SSH on proteins, SSH can effectively maintain the activity of proteins in the face of environmental challenges.

[0050] Application Example 3 The SSH hydrogel loaded with basic fibroblast growth factor (bFGF) for promoting wound healing ability 1. Animal surgical procedure To induce a diabetes model, male C57BL / 6 mice (weighing about 20 g) were selected and intraperitoneally injected with 150 mg / kg of streptozotocin (STZ) after 16 h of fasting. When the blood glucose level of the mice ≥13.88 mmol / L (250 mg / dL), the modeling was considered successful. After successful modeling, the mice showed typical diabetic symptoms such as polydipsia, polyphagia, polyuria, and significant weight loss. Before establishing the wound model, the mice were anesthetized, shaved, and disinfected, and then the full-thickness skin was excised in the back and hip regions to form a standardized wound surface. The mice were randomly divided into 4 groups: control group (medical gauze), alginate dressing group (AD), bFGF@SSH hydrogel dressing group (prepared in Application Example 1), and SSH hydrogel dressing group. The wounds were photographed and recorded on days 0, 2, 4, 7, and 14 to evaluate the change in wound area. On day 14, the wound tissue was collected for histological analysis, including hematoxylin-eosin (H&E) staining, Masson's trichrome staining, and immunofluorescence staining. ImageJ and Adobe Photoshop 2025 were used for quantitative analysis of the dermis and epidermis thickness, wound healing area, and collagen volume fraction.

[0051] 2. Result Analysis The results showed that the wound healing rate of the bFGF@SSH-treated group was significantly faster than that of all other groups. Quantitative analysis of the wound closure area ( Figure 9). On the 4th day, the wound closure rate of the bFGF@SSH group was approximately 55%, while those of the medical gauze group and the commercial dressing group were only 25% and 35% respectively. Notably, the healing effect of the SSH hydrogel alone was comparable to that of the commercial sodium alginate dressing. By the 14th day, the wounds in the bFGF@SSH group were almost completely healed, while there were still obvious unclosed wounds in the remaining groups ( Figure 10 ). This demonstrated the application potential of the bFGF-loaded SSH hydrogel in the diabetic wound healing model.

Claims

1. An anion-π-based zwitterionic supramolecular hydrogel, characterized in that, It is self-assembled from an amphoteric ion copolymer and a styrene sulfonate copolymer; the amphoteric ion copolymer is obtained by copolymerizing raw materials including amphoteric ion monomers, and the styrene sulfonate copolymer is obtained by copolymerizing raw materials including monomers containing styrene sulfonic acid.

2. The zwitterionic supramolecular hydrogel based on anion-π according to claim 1, characterized in that, The amphoteric ion monomers include any one or more of sulfobetaine methacrylate, carboxybetaine methacrylate, and methacryloyloxyethyl phosphorylcholine; The monomers containing styrene sulfonic acid include one or more of styrene sulfonic acid, sodium styrene sulfonate, potassium styrene sulfonate, and lithium styrene sulfonate.

3. The zwitterionic supramolecular hydrogel based on anion-π according to claim 1, wherein The weight-average molecular weight of the amphoteric ion copolymer is 50,000 to 5,000,000; the weight-average molecular weight of the styrene sulfonate copolymer is 50,000 to 5,000,000.

4. The zwitterionic supramolecular hydrogel based on anion-π according to claim 1, wherein The molar ratio of the amphoteric ions and styrene sulfonic acid repeating units of the amphoteric ion copolymer and the styrene sulfonate copolymer is 1:0.1 to 10.

5. The zwitterionic supramolecular hydrogel based on anion-π according to claim 1, wherein The polymerization monomer raw materials of the amphoteric ion copolymer further include a first comonomer; the first comonomer includes one or more monomers of acrylate esters, methacrylate esters, acrylamide, etc.; the mass percentage of the amphoteric ion monomers in the polymerization monomer raw materials of the amphoteric ion copolymer is more than 50%.

6. The zwitterionic supramolecular hydrogel based on anion-π according to claim 1, wherein, The polymerization monomer raw materials of the styrene sulfonate copolymer further include a second comonomer; the second comonomer includes one or more of acrylic acid, methacrylic acid, maleic anhydride, styrene or its derivatives, acrylate esters, methacrylate esters, acrylamide, etc.; The mass percentage of the monomers containing styrene sulfonic acid in the polymerization monomer raw materials of the styrene sulfonate copolymer is more than 50%.

7. A method for preparing an anion-π-based zwitterionic supramolecular hydrogel according to any one of claims 1-6, characterized in that, It includes the steps: Step 1, copolymerize the raw materials containing amphoteric ion monomers to obtain an amphoteric ion copolymer; copolymerize the raw materials including monomers containing styrene sulfonic acid to obtain a styrene sulfonate copolymer; Step 2, dissolve the amphoteric ion copolymer and the styrene sulfonate copolymer in an aqueous solution respectively, and obtain the amphoteric ion supramolecular hydrogel after self-assembly by mixing.

8. The preparation method of the zwitterionic supramolecular hydrogel based on anion-π according to claim 7, characterized in that, In Step 2, the total concentration of the amphoteric ion copolymer and the styrene sulfonate copolymer in the aqueous solution is 1 to 60 wt%.

9. The preparation method of the zwitterionic supramolecular hydrogel based on anion-π according to claim 7, characterized in that, In Step 1, the copolymerization contains an initiator, the copolymerization temperature is 60 - 90 °C, the reaction is for 6 - 18 h, and the product is dialyzed and freeze-dried to obtain the copolymer.

10. Use of the amphoteric ion supramolecular hydrogel based on anion-π according to any one of claims 1 - 6 in the preparation of immunocompatible drugs or medical materials.

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