Anti-scale zwitterionic hydrogel as well as preparation method and application thereof

Abstract

The invention relates to the field of hydrogel preparation, and discloses anti-scale zwitter-ion hydrogel as well as a preparation method and application thereof. The method comprises the following steps: firstly, initiating an oxazine derivative monomer to polymerize through a double-end initiator to generate dihydroxyl polyoxazine; and then, removing a side chain of the polymer to obtain the dihydroxy polypropylene imine. Then, dihydroxy polypropylene imine is selected for further reaction to generate a zwitter-ion precursor polymer, and the zwitter-ion precursor polymer is hydrolyzed to obtain the polypropylene imine zwitter-ion derivative. And finally, converting hydroxyl groups at two ends, synthesizing a zwitterionic macromolecular cross-linking agent, and carrying out polymerization cross-linking in an aqueous solution to prepare the anti-scale zwitterionic hydrogel. The hydrogel is simple in formula, is completely composed of the zwitterionic cross-linking agent, has excellent anti-scaling performance such as protein adsorption resistance, bacterial adsorption resistance and cell adsorption resistance, and is suitable for biological materials, medical devices, implant materials and cell delivery materials.

Description

Technical Field

[0001] The invention relates to the field of hydrogel preparation, and in particular to an anti-fouling zwitterionic hydrogel and a preparation method and application thereof. Background Art

[0002] As a highly biocompatible material, hydrogel has shown a wide range of applications in the biomedical field, such as implant materials, tissue engineering, and wound dressings. However, biofouling on the surface of the material limits its wider application. Protein adsorption on the surface of implant materials can cause rejection and coagulation, and bacterial adsorption on the surface of materials can lead to the formation of biofilms, which are harmful to human health. For blood-contact materials, cell adsorption on the surface of materials can cause thrombosis and lead to material failure. Therefore, it is of great significance to give hydrogels anti-fouling properties to resist the nonspecific adsorption of proteins, bacteria, and cells.

[0003] Early studies have shown that hydrophilic, electrically neutral polymers can resist protein adsorption through the steric hindrance effect of the hydration layer. Polyethylene glycol (PEG) is a commonly used anti-fouling material, and polyethylene glycol diacrylate (PEGDA) is a commercial cross-linking agent. PEG hydrogels have a wide range of applications. However, PEG is easily oxidized, and studies have reported that PEG materials exhibit mild immunotoxicity. Long-term use may trigger an immune response in the human body, limiting the long-term application of PEG hydrogels in vivo.

[0004] Recently, zwitterionic polymers have shown excellent anti-fouling properties due to the strong hydration layer formed by their ion solvation, and can effectively resist the adsorption of proteins, cells and microorganisms. The existing zwitterionic hydrogel preparation technology mainly obtains hydrogels by polymerizing zwitterionic monomers (such as CBMA, SBMA, CBAA) with hydrophobic crosslinkers (MBAA), wherein the introduction of hydrophobic crosslinkers will reduce the anti-fouling properties. Small molecule zwitterionic crosslinkers were reported in Biomaterials 2011, 32, 961-968 and Biomaterials 2011, 32, 6893-6899. So far, there have been no reports on macromolecular zwitterionic crosslinkers and related technologies for preparing hydrogels. Summary of the invention

[0005] Due to the lack of macromolecular anti-fouling zwitterionic hydrogel in the prior art, the present invention provides an anti-fouling zwitterionic hydrogel and a preparation method and application thereof.

[0006] The anti-fouling zwitterionic hydrogel provided by the present invention is obtained by polymerization cross-linking of a macromolecular zwitterionic cross-linking agent with a new structure. The obtained hydrogel has a simple formula and, compared with the hydrogel prepared by the traditional polyethylene glycol macromolecular cross-linking agent, has better anti-fouling properties of anti-protein adsorption, anti-bacterial adhesion and anti-cell adhesion, and has mechanical properties comparable to those of the hydrogel prepared by the polyethylene glycol cross-linking agent, and is expected to be used as an implant material and the like.

[0007] The object of the present invention is achieved by the following technical solution: The present invention first provides a macromolecular zwitterionic crosslinking agent, which is selected from one of the following structures:

[0008]

[0009] Where R is C1-C 18 Alkyl, phenyl, polyethylene glycol or a compound containing a disulfide bond, wherein n is a positive integer of 1-1000;

[0010] The present invention further provides a method for preparing the macromolecular zwitterionic crosslinking agent, comprising the following steps:

[0011] (1) A double-end initiator initiates polymerization of an oxazine derivative monomer to generate a dihydroxy polyoxazine, the structure of which is shown below:

[0012]

[0013] (2) hydrolyzing and removing the side chain of bishydroxy polyoxazine to obtain bishydroxy polypropylene imine, the structure of which is shown below:

[0014]

[0015] (3) Using dihydroxy polypropylene imine to generate a zwitterion precursor polymer, and hydrolyzing it to obtain a polypropylene imine zwitterion derivative, the structure of the polypropylene imine zwitterion derivative is shown below:

[0016]

[0017] (4) converting the hydroxyl groups at both ends of the polypropylene imine zwitterionic derivative into acrylate groups or methacrylate groups to obtain the macromolecular zwitterionic crosslinking agent;

[0018] Where R is C1-C 18 Alkyl, phenyl, polyethylene glycol or a compound containing a disulfide bond, wherein n is a positive integer of 1-1000.

[0019] In one embodiment of the present invention, in step (1), the double-end initiator is selected from one or more of (E)-1,4-dibromobutyl-2-ene, 1,4-dibromobutane, di-p-toluenesulfonate of C1-C18 diol, α,α'-dibromo-p-xylene, di-p-toluenesulfonate of polyethylene glycol, di-trifluoromethanesulfonate of polyethylene glycol or di-p-toluenesulfonate of 6,6'-dithiobis(1-hexanol).

[0020] In one embodiment of the present invention, in step (1), the oxazine derivative monomer is selected from one or more of 2-methyl-2-oxazine, 2-ethyl-2-oxazine, 2-isopropyl-2-oxazine or 2-phenyl-2-oxazine.

[0021] In one embodiment of the present invention, in step (1), the conditions for initiating polymerization of the double-ended initiator and the oxazine derivative monomer are as follows: the double-ended initiator and the oxazine derivative monomer are added to a microwave reactor at a molar ratio of 1:2-2000, polymerized at 100-160° C. for 30-120 minutes, and 1.0 M potassium hydroxide methanol solution is added at 0° C. to terminate the polymerization, the solvent is dried by spin drying, the solid is dissolved with chloroform, and precipitated in n-hexane three times to obtain the generated dihydroxy polyoxazine.

[0022] In one embodiment of the present invention, in step (2), the conditions for hydrolyzing and removing the side chains of the bishydroxypolyoxazine are: reflux the bishydroxypolyoxazine in 37% concentrated hydrochloric acid for 24-72 hours to remove the side chains of the polymer to obtain bishydroxypolypropyleneimine.

[0023] In one embodiment of the present invention, in step (3), the conditions for generating the zwitterionic precursor polymer using bishydroxypolypropyleneimine are as follows: bishydroxypolypropyleneimine is further reacted with tert-butyl acrylate in a methanol solution at room temperature for 12-48 hours to generate the zwitterionic precursor polymer.

[0024] In one embodiment of the present invention, in step (3), the hydrolysis is carried out under the following conditions: adding the zwitterionic precursor polymer to trifluoroacetic acid and reacting at room temperature for 12 hours to obtain a polypropyleneimine zwitterionic derivative.

[0025] In one embodiment of the present invention, in step (4), 2-isocyanatoethyl acrylate or ethyl isocyanate methacrylate is reacted with a polypropyleneimine zwitterionic derivative, and the hydroxyl groups at both ends of the polypropyleneimine zwitterionic derivative are converted into acrylate groups or methacrylate groups to obtain the macromolecular zwitterionic crosslinking agent.

[0026] In one embodiment of the present invention, in step (4), the conditions for the reaction of 2-isocyanatoethyl acrylate or ethyl isocyanatoethyl methacrylate with the polypropyleneimine zwitterionic derivative are as follows: the polypropyleneimine zwitterionic derivative is dissolved in chloroform or dimethyl sulfoxide, 2-isocyanatoethyl acrylate or ethyl isocyanatoethyl methacrylate is added at a molar ratio of NCO:OH=5:1, and the reaction is carried out at 40-80°C for 1-3 days to convert the hydroxyl groups at both ends into acrylate groups or methacrylate groups to synthesize a zwitterionic macromolecular crosslinking agent.

[0027] The present invention further provides a method for preparing the anti-fouling zwitterionic hydrogel, comprising the following steps: dissolving a macromolecular zwitterionic crosslinking agent in a solution, adding an initiator, initiating polymerization, and obtaining the anti-fouling zwitterionic hydrogel.

[0028] In one embodiment of the present invention, the initiator is a photoinitiator or a thermal initiator, selected from one or a combination of phenyl-2,4,6-trimethylbenzoylphosphonate, ammonium persulfate, potassium perchlorate, benzophenone carboxylate, azobisisobutylimidazoline hydrochloride, and azobisisobutylamidine hydrochloride.

[0029] In one embodiment of the present invention, the solution used to dissolve the macromolecular zwitterionic crosslinker is selected from distilled water, physiological saline, buffer, decellularized matrix or cell culture medium solution or a combination thereof. Different media can be selected according to different applications.

[0030] In one embodiment of the present invention, the mass fraction of the zwitterionic macromolecular cross-linking agent solution is 10%-40%.

[0031] In one embodiment of the present invention, when a photoinitiator is used, the polymerization is carried out at room temperature and the anti-fouling zwitterionic hydrogel is obtained under ultraviolet light irradiation for 1-3 hours.

[0032] In one embodiment of the present invention, when a thermal initiator is used, the polymerization is carried out at a temperature of 60-80° C. and the reaction time is 4-12 hours to obtain the anti-fouling zwitterionic hydrogel.

[0033] In one embodiment of the present invention, the preparation method of the anti-fouling zwitterionic hydrogel is to dissolve the zwitterionic crosslinker in a 1M NaCl solution to prepare a solution with a mass concentration of 10-40%, add a photoinitiator or a thermal initiator, and then inject it into a closed mold to initiate monomer polymerization by photoinitiation or thermal initiation. After a period of time, demolding and soaking in water for at least 3 days to remove unreacted monomers to obtain the anti-fouling zwitterionic hydrogel.

[0034] The present invention further provides an anti-fouling zwitterionic hydrogel obtained based on the above preparation method.

[0035] The present invention further provides the use of the anti-fouling zwitterionic hydrogel in the preparation of medical devices, implant materials and cell delivery materials.

[0036] The invention uses a double-terminal initiator to initiate the polymerization of an oxazine derivative monomer to generate a dihydroxy polyoxazine. Subsequently, the side chain of the polymer is removed to obtain a dihydroxy polypropylene imine. After that, the dihydroxy polypropylene imine is selected for further reaction to generate a zwitterion precursor polymer, which is hydrolyzed to obtain a polypropylene imine zwitterion derivative. Finally, the hydroxyl groups at both ends are converted to synthesize a zwitterion macromolecular crosslinking agent, which is polymerized and crosslinked in an aqueous solution to prepare an anti-fouling zwitterion hydrogel.

[0037] Compared with the prior art, the advantages and beneficial effects of the present invention are embodied in the following aspects:

[0038] The hydrogel of the invention has a simple formula, is completely composed of a zwitterionic crosslinking agent, has excellent anti-fouling properties such as anti-protein adsorption, anti-bacterial adsorption and anti-cell adsorption, and is suitable for preparing medical devices, implant materials and cell delivery materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 The synthetic route of the zwitterionic crosslinking agent PCBDA prepared by the present invention;

[0040] Figure 2 The zwitterionic crosslinking agent PCBDA prepared by the present invention is 1 H NMR spectrum;

[0041] Figure 3 Characterization of the protein adsorption resistance of the anti-fouling zwitterionic hydrogel prepared by the present invention;

[0042] Figure 4 Characterization of the bacterial adsorption performance of the anti-fouling zwitterionic hydrogel prepared by the present invention;

[0043] Figure 5 The present invention is to characterize the anti-cell adsorption performance of the anti-fouling zwitterionic hydrogel prepared by the present invention. DETAILED DESCRIPTION

[0044] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0045] Embodiment 1:

[0046] This example provides a method for synthesizing dihydroxy-terminated poly(2-methyl-2-oxazine). Figure 1 , the detailed steps are as follows:

[0047] 2-Methyl-2-oxazine (81.0 mmol, 8.00 g), (E)-1,4-dibromobutyl-2-ene (1.61 mmol, 0.35 g) and acetonitrile (12.4 mL) were mixed in a microwave vial. The polymerization reaction was carried out at 140 °C for 50 minutes. After the reaction was completed, 1.0 M potassium hydroxide methanol solution (4.1 mL) was added at 0 °C and stirred at room temperature overnight to terminate the polymerization. The solvent was spin-dried and the solid was dissolved in chloroform. The polymer was precipitated three times in cold n-hexane to obtain a viscous yellow solid HO-PMeOZI-OH. HO-PMeOZI-OH (0.7 mmol, 3.5 g) was dissolved in hot water and heated to reflux. 37% hydrochloric acid was added to the mixture and stirred for 48 hours. The residual solid was dissolved in hot water and the pH was adjusted to 9-10 with sodium hydroxide solution. The solid was collected by three centrifugations and dried overnight in a vacuum to finally obtain yellow solid dihydroxy-terminated polypropylene imine (HO-PPI-OH), the hydrogen nuclear magnetic resonance spectrum data of which are as follows: 1 H NMR (600 MHz, methanol-d4): 2.64 (t, 4H), 1.73 (p, 2H).

[0048] In this embodiment,

[0049] The HO-PMeOZI-OH structure is shown below:

[0050]

[0051] The HO-PPI-OH structure is shown below:

[0052]

[0053] Example 2

[0054] This example provides a method for synthesizing polycarboxylic acid betaine dimethacrylate (PCBDA). Figure 1 The detailed steps are as follows:

[0055] HO-PPI-OH (8.0 g, 137.6 mmol) was dissolved in 40 mL of methanol, and then tert-butyl acrylate (35.3 g, 275.2 mmol) was added dropwise. The mixture was stirred at room temperature for 24 hours. 200 mL of n-methane was added for extraction and separation, and the methanol phase was retained and repeated three times. Methanol was removed by rotary evaporation, and the solid was dissolved in 40 mL of chloroform. Ethyl methacrylate isocyanate (2.8 g, 18.0 mmol) was added to the solution, and the reaction was carried out at 60 ° C for 2 days. 4-methoxyphenol (22.2 mg, 0.18 mmol) was added as a free radical inhibitor. After the reaction was completed, the solvent was evaporated, and trifluoroacetic acid (25 mL) was added to remove the tert-butyl protecting group. After the mixture was stirred at room temperature for 12 hours, the mixture was precipitated in 200 mL of ether. The solid was redissolved in 20 mL of methanol and precipitated in 200 mL of ether. This operation was repeated more than 3 times. Finally, the solid was dried in a vacuum oven to obtain yellow powder PCBDA. The H NMR spectrum of PCBDA is as follows: Figure 2 As shown, the peak assignment and integration data confirmed the successful synthesis of PCBDA.

[0056] Example 3

[0057] This embodiment provides a method for preparing an anti-fouling zwitterionic hydrogel, and the detailed steps are as follows:

[0058] PCBDA is dissolved in 1M NaCl solution to prepare a solution with a mass concentration of 20%. 0.1 mol% of a photoinitiator (relative to a crosslinker), namely phenyl-2,4,6-trimethylbenzoylphosphonate (LAP), is added. Polymerization is carried out in a transparent mold with a thickness of 2 mm and a diameter of 8 mm. Using 365 nm, 24 watts of ultraviolet light, each side of the hydrogel is irradiated for 30 minutes. The hydrogel needs to be soaked in a PBS solution for at least 5 days to remove unreacted chemicals. The anti-fouling zwitterionic hydrogel obtained in this embodiment is PCB-20%.

[0059] Example 4

[0060] This embodiment provides a method for preparing an anti-fouling zwitterionic hydrogel, and the detailed steps are as follows:

[0061] PCBDA is dissolved in 1M NaCl solution to prepare a solution with a mass concentration of 30%. 0.1 mol% of a photoinitiator (relative to a crosslinker), namely phenyl-2,4,6-trimethylbenzoylphosphonate (LAP), is added. Polymerization is carried out in a transparent mold with a thickness of 2 mm and a diameter of 8 mm. Using 365 nm, 24 watts of ultraviolet light, each side of the hydrogel is irradiated for 30 minutes. The hydrogel needs to be soaked in a PBS solution for at least 5 days to remove unreacted chemicals. The anti-fouling zwitterionic hydrogel obtained in this embodiment is PCB-30%.

[0062] Example 5

[0063] This embodiment provides a method for preparing an anti-fouling zwitterionic hydrogel, and the detailed steps are as follows:

[0064] PCBDA is dissolved in 1M NaCl solution to prepare a solution with a mass concentration of 40%. 0.1 mol% of a photoinitiator (relative to a crosslinker), namely phenyl-2,4,6-trimethylbenzoylphosphonate (LAP), is added. Polymerization is carried out in a transparent mold with a thickness of 2 mm and a diameter of 8 mm. Using 365 nm, 24 watts of ultraviolet light, each side of the hydrogel is irradiated for 30 minutes. The hydrogel needs to be soaked in a PBS solution for at least 5 days to remove unreacted chemicals. The anti-fouling zwitterionic hydrogel obtained in this embodiment is PCB-40%.

[0065] Comparative Example 1

[0066] This comparative example provides a method for preparing polyethylene glycol hydrogel, and the detailed steps are as follows:

[0067] Polyethylene glycol diacrylate (PEGDA) was dissolved in 1M NaCl solution to prepare a solution with a mass concentration of 40%. A photoinitiator (relative to the crosslinker), phenyl-2,4,6-trimethylbenzoylphosphonate (LAP) was added at 0.1 mol%. The polymerization was carried out in a transparent mold with a thickness of 2 mm and a diameter of 8 mm. The hydrogel was irradiated with 365 nm, 24 watts of ultraviolet light for 30 minutes on each side. The hydrogel was soaked in PBS solution for at least 5 days to remove unreacted chemicals.

[0068] Test Example 1

[0069] This test example provides a method for testing the anti-protein adsorption performance of the hydrogels prepared in Example 3, Example 4, Example 5, and Comparative Example 1, and the specific steps are as follows:

[0070] Protein adsorption on the hydrogel was evaluated by fluorescence. The hydrogels were soaked in PBS buffer for more than 3 days. The hydrogels equilibrated with PBS buffer were cut into discs with a diameter of 5 mm and a thickness of 2 mm and then transferred to a 96-well flat-bottom polystyrene (TCPS) plate. 1 mL of a 1 mg / mL rhodamine B-labeled human fibrinogen (RB-Fg) solution was added to each sample. The samples were equilibrated in the RB-Fg solution for 240 minutes to promote protein adsorption. Afterwards, the samples were lifted with sterile tweezers and gently rinsed three times in PBS buffer to remove excess dye solution. The samples were then transferred to a glass slide and analyzed using an inverted laser scanning confocal microscope (LSM900, Zeiss) with a 10× objective. Three samples of each material were measured, and five images were taken at different locations on each sample surface. The fluorescence intensity was analyzed using ImageJ software. As Figure 3 As shown, compared with TCPS (tissue culture polystyrene) well plates and PEGDA hydrogel samples, the red RB-Fg on the surface of PCB hydrogel samples was significantly reduced, indicating that the anti-fouling zwitterionic hydrogel has good anti-protein adsorption performance.

[0071] Test Example 2

[0072] This test example provides a method for testing the antibacterial adsorption performance of the hydrogels prepared in Example 3, Example 4, Example 5, and Comparative Example 1, and the specific steps are as follows:

[0073] Escherichia coli O157 was cultured in Luria–Bertani (LB) medium (20 g L -1 ) until the absorbance at 600 nm reached 0.7. Subsequently, the bacterial solution was diluted with LB medium to an absorbance of 0.4 at 600 nm. After the hydrogel was soaked in PBS, it was cut into cylinders with a diameter of 5 mm and a thickness of 2 mm using a biopsy punch. Next, the hydrogel was immersed in the bacterial solution and cultured for 24 hours. In order to analyze the bacterial density on the surface of the hydrogel, the sample was gently rinsed with clean water and stained with a bacterial live and dead cell viability / cytotoxicity detection kit. After staining, the number of live and dead cells was determined by a Zeiss (Germany) LSM 980Airyscan fluorescence microscope using a 10× objective lens and FITC and Cy5 filters. Figure 4 As shown, compared with the TCPS well plate, there is basically no green bacterial fluorescence on the surface of PCB hydrogel and PEG hydrogel, indicating that the anti-fouling zwitterionic hydrogel has good anti-bacterial adsorption performance.

[0074] Test Example 3

[0075] This test example provides a method for testing the anti-cell adhesion performance of the hydrogels prepared in Example 3, Example 4, Example 5, and Comparative Example 1, and the specific steps are as follows:

[0076] Mouse fibroblasts (NIH-3T3) expressing FLuc+GFP were selected for cell adhesion experiments on hydrogels. These cells were cultured in DMEM medium containing 10% FBS and 0.8 μg / mL puromycin. Hydrogels (5 mm in diameter, 2 mm in thickness) were pre-equilibrated in PBS for 24 hours and sterilized under UV irradiation for at least 30 minutes before being placed in 96-well plates. NIH / 3T3 cells were plated at 8×10 5 The cells were seeded onto the hydrogel discs at a density of 100 cells / mL and incubated at 37°C and 5% CO2 for 24 hours. Finally, the culture medium was removed and the hydrogels were gently rinsed three times with PBS. An inverted fluorescence microscope (Olympus IX83) was used to observe the cell adhesion on the hydrogel surface. Figure 5 As shown, compared with the TCPS well plate and PEGDA hydrogel samples, there is no green cell adsorption on the PCB hydrogel sample, indicating that the anti-fouling zwitterionic hydrogel has good anti-cell adhesion function.

[0077] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. A macromolecular zwitterionic crosslinker, characterized in that: Select one of the following structures: Where R is C1-C 18 Alkyl, phenyl, polyethylene glycol or a compound containing a disulfide bond, wherein n is a positive integer of 1-1000.

2. The method for preparing the macromolecular zwitterionic crosslinking agent according to claim 1, characterized in that: The following steps are involved: (1) A double-end initiator initiates polymerization of an oxazine derivative monomer to generate a dihydroxy polyoxazine, the structure of which is shown below: (2) hydrolyzing and removing the side chain of bishydroxy polyoxazine to obtain bishydroxy polypropylene imine, the structure of which is shown below: (3) Using dihydroxy polypropylene imine to generate a zwitterion precursor polymer, and hydrolyzing it to obtain a polypropylene imine zwitterion derivative, the structure of the polypropylene imine zwitterion derivative is shown below: (4) converting the hydroxyl groups at both ends of the polypropylene imine zwitterionic derivative into acrylate groups or methacrylate groups to obtain the macromolecular zwitterionic crosslinking agent; Where R is C1-C 18 Alkyl, phenyl, polyethylene glycol or a compound containing a disulfide bond, wherein n is a positive integer of 1-1000.

3. The method for preparing the macromolecular zwitterionic crosslinking agent according to claim 2, characterized in that: In step (1), the double-end initiator is selected from one or more of (E)-1,4-dibromobutyl-2-ene, 1,4-dibromobutane, di-p-toluenesulfonate of C1-C18 diol, α,α'-dibromo-p-xylene, di-p-toluenesulfonate of polyethylene glycol, di-trifluoromethanesulfonate of polyethylene glycol or di-p-toluenesulfonate of 6,6'-dithiobis(1-hexanol); In step (1), the oxazine derivative monomer is selected from one or more of 2-methyl-2-oxazine, 2-ethyl-2-oxazine, 2-isopropyl-2-oxazine or 2-phenyl-2-oxazine; In step (1), the conditions for initiating polymerization of the double-ended initiator and the oxazine derivative monomer are as follows: the double-ended initiator and the oxazine derivative monomer are added to a microwave reactor at a molar ratio of 1:2-2000, polymerized at 100-160° C. for 30-120 minutes, and 1.0 M potassium hydroxide methanol solution is added at 0° C. to terminate the polymerization, the solvent is spin-dried, the solid is dissolved with chloroform, and precipitated in n-hexane three times to obtain the generated dihydroxy polyoxazine.

4. The method for preparing the macromolecular zwitterionic crosslinking agent according to claim 2, characterized in that: In step (2), the conditions for hydrolyzing and removing the side chains of the bishydroxypolyoxazine are as follows: reflux the bishydroxypolyoxazine in 37% concentrated hydrochloric acid for 24-72 hours to remove the side chains of the polymer, thereby obtaining bishydroxypolypropyleneimine. In step (3), the conditions for generating the zwitterionic precursor polymer using bishydroxypolypropyleneimine are as follows: bishydroxypolypropyleneimine is further reacted with tert-butyl acrylate in a methanol solution at room temperature for 12-48 hours to generate the zwitterionic precursor polymer; In step (3), the hydrolysis is carried out under the following conditions: adding the zwitterionic precursor polymer into trifluoroacetic acid and reacting at room temperature for 12 hours to obtain a polypropyleneimine zwitterionic derivative.

5. The method for preparing the macromolecular zwitterionic crosslinking agent according to claim 2, characterized in that: In step (4), 2-isocyanatoethyl acrylate or ethyl isocyanate methacrylate is reacted with a polypropyleneimine zwitterionic derivative to convert the hydroxyl groups at both ends of the polypropyleneimine zwitterionic derivative into acrylate groups or methacrylate groups to obtain the macromolecular zwitterionic crosslinking agent; In step (4), the conditions for the reaction of 2-isocyanatoethyl acrylate or ethyl isocyanatoethyl methacrylate with the polypropyleneimine zwitterionic derivative are as follows: the polypropyleneimine zwitterionic derivative is dissolved in chloroform or dimethyl sulfoxide, 2-isocyanatoethyl acrylate or ethyl isocyanatoethyl methacrylate is added at a molar ratio of NCO:OH=5:1, and the reaction is carried out at 40-80°C for 1-3 days to convert the hydroxyl groups at both ends into acrylate groups or methacrylate groups to synthesize a zwitterionic macromolecular crosslinking agent.

6. A method for preparing an anti-fouling zwitterionic hydrogel, characterized in that: The method comprises the following steps: dissolving the macromolecular zwitterionic crosslinking agent according to claim 1 in a solution, adding an initiator, initiating polymerization, and obtaining an anti-fouling zwitterionic hydrogel.

7. The method for preparing an anti-fouling zwitterionic hydrogel according to claim 6, characterized in that: The initiator is a photoinitiator or a thermal initiator, selected from one or a combination of phenyl-2,4,6-trimethylbenzoylphosphonate, ammonium persulfate, potassium perchlorate, benzophenone carboxylate, azobisisobutylimidazoline hydrochloride, and azobisisobutylamidine hydrochloride; The solution used to dissolve the macromolecular zwitterionic crosslinker is selected from one or a combination of distilled water, physiological saline, buffer, decellularized matrix or cell culture medium solution; The mass fraction of the zwitterionic macromolecular cross-linking agent solution is 10%-40%.

8. The method for preparing an anti-fouling zwitterionic hydrogel according to claim 6, characterized in that: When a photoinitiator is used, the polymerization is carried out at room temperature, and the anti-fouling zwitterionic hydrogel is obtained under ultraviolet light irradiation for 1-3 hours; When a thermal initiator is used, the polymerization is carried out at a temperature of 60-80° C. and the reaction time is 4-12 hours to obtain the anti-fouling zwitterionic hydrogel.

9. An anti-fouling zwitterionic hydrogel prepared by the preparation method according to claim 6, 7 or 8.

10. An application of the anti-fouling zwitterionic hydrogel as claimed in claim 9, characterized in that: The anti-fouling zwitterionic hydrogel is used in preparing medical devices, implant materials and cell delivery materials.

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