An antifouling zwitterionic hydrogel and its preparation method and application

Abstract

The present application relates to the field of hydrogel preparation, and discloses an anti-fouling zwitterionic hydrogel and a preparation method and application thereof. First, a double-hydroxyl polyoxazine is generated by initiating polymerization of an oxazine derivative monomer through a double-end initiator. Then, the side chain of the polymer is removed to obtain a double-hydroxyl polypropylene imine. After that, the double-hydroxyl polypropylene imine is selected for further reaction to generate a zwitterionic precursor polymer, and the zwitterionic precursor polymer is hydrolyzed to obtain a polypropylene imine zwitterionic derivative. Finally, the two end hydroxyl groups are converted to synthesize a zwitterionic macromolecular crosslinking agent, and the zwitterionic macromolecular crosslinking agent is polymerized and crosslinked in an aqueous solution to prepare the anti-fouling zwitterionic hydrogel. The hydrogel formula of the present application is simple, and the hydrogel is composed of a zwitterionic crosslinking agent only, has excellent anti-fouling properties such as anti-protein adsorption, anti-bacterial adsorption and anti-cell adsorption, and is suitable for biological materials, medical devices, implant materials and cell delivery materials.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of hydrogel preparation, in particular to an anti-fouling zwitterionic hydrogel and its preparation method and application. BACKGROUND

[0002] As a kind of high biocompatibility material, hydrogel has shown wide application in biomedical field, such as implant material, tissue engineering, wound dressing, etc. However, the biofouling on the surface of the material limits its wider application. Protein adsorption on the surface of implant material will cause rejection reaction and blood clotting, bacterial adsorption on the surface of the material will lead to the formation of biofilm, which is harmful to human health. For blood contact materials, cell adsorption on the surface of the material will cause thrombosis and lead to material failure. Therefore, it is of great significance to endow hydrogel with anti-fouling properties to resist non-specific adsorption of proteins, bacteria and cells.

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

[0004] Recently, zwitterionic polymers have shown excellent anti-fouling properties due to the strong hydration layer formed by ionic solvation, which can effectively resist the adsorption of proteins, cells and microorganisms. Existing zwitterionic hydrogel preparation techniques mainly involve the polymerization of zwitterionic monomers (such as CBMA, SBMA, CBAA) with hydrophobic crosslinking agents (MBAA) to obtain hydrogels. The introduction of hydrophobic crosslinking agents will reduce the anti-fouling properties. Small molecule zwitterionic crosslinking agents are reported in Biomaterials 2011, 32, 961-968 and Biomaterials 2011, 32, 6893-6899. To date, there has been no related technical report on macromolecular zwitterionic crosslinking agents and their preparation of hydrogels. SUMMARY

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

[0006] The anti-fouling zwitterionic hydrogel provided by the application is obtained by polymerization and cross-linking of a novel structure of macromolecular zwitterionic cross-linking agent, and compared with the hydrogel prepared by traditional polyethylene glycol macromolecular cross-linking agent, the hydrogel has better anti-protein adsorption, anti-bacterial adhesion and anti-cell adhesion anti-fouling performance, and has mechanical properties comparable to the hydrogel prepared by the polyethylene glycol cross-linking agent, and is expected to be applied in the field of implant materials and the like.

[0007] The application provides a macromolecular zwitterionic cross-linking agent, which is selected from one of the following structures:

[0008] , or

[0009]

[0010] wherein R is C1-C 18 alkyl, phenyl, polyethylene glycol group or disulfide bond containing group, and n is a positive integer of 1-1000.

[0011] The application further provides a preparation method of the macromolecular zwitterionic cross-linking agent, comprising the following steps:

[0012] (1) a double-end initiator is used to initiate polymerization of an oxazine derivative monomer to generate a double-hydroxyl polyoxazine, and the structure of the double-hydroxyl polyoxazine is as follows:

[0013] ;

[0014] (2) the side chain of the double-hydroxyl polyoxazine is removed by hydrolysis to obtain a double-hydroxyl polypropylene imine, and the structure of the double-hydroxyl polypropylene imine is as follows:

[0015] ;

[0016] (3) a zwitterionic precursor polymer is generated by using the double-hydroxyl polypropylene imine, and the zwitterionic precursor polymer is hydrolyzed to obtain a polypropylene imine zwitterionic derivative, and the structure of the polypropylene imine zwitterionic derivative is as follows:

[0017] ;

[0018] (4) the two end hydroxyl groups of the polypropylene imine zwitterionic derivative are converted into acrylate groups or methacrylate groups to obtain the macromolecular zwitterionic cross-linking agent.

[0019] wherein R is C1-C 18 alkyl, phenyl, polyethylene glycol group or disulfide bond containing group, and n is a positive integer of 1-1000.

[0020] In one embodiment of the present application, in step (1), the double-terminated initiator is selected from one or more of the group consisting of 1,4-dibromobut-2-ene, 1,4-dibromobutane, di-p-toluene sulfonate of C1-C18 diols, a,a'-dibromo-p-xylene, di-p-toluene sulfonate of polyethylene glycol, di-triflate of polyethylene glycol, or di-p-toluene sulfonate of 6,6'-dithiobis(1-hexanol). E)

[0021] In one embodiment of the present application, in step (1), the oxazine derivative monomer is selected from one or more of the group consisting of 2-methyl-2-oxazine, 2-ethyl-2-oxazine, 2-isopropyl-2-oxazine, or 2-phenyl-2-oxazine.

[0022] In one embodiment of the present application, in step (1), the condition for the double-terminated initiator to initiate the polymerization of the oxazine derivative monomer is that the double-terminated initiator and the oxazine derivative monomer are added into a microwave reactor at a molar ratio of 1:2-2000, and the polymerization is carried out at 100-160 °C for 30-120 minutes, and the polymerization is terminated by adding 1.0 M potassium hydroxide methanol solution at 0 °C, and the solvent is rotary evaporated, and the solid is dissolved in chloroform, and the chloroform solution is precipitated in n-hexane for three times to obtain the dihydroxylated polyoxazine.

[0023] In one embodiment of the present application, in step (2), the condition for the hydrolysis to remove the side chains of the dihydroxylated polyoxazine is that the dihydroxylated polyoxazine is refluxed in 37% hydrochloric acid for 24-72 hours to remove the side chains of the polymer to obtain the dihydroxylated polypropylene imine.

[0024] In one embodiment of the present application, in step (3), the condition for the dihydroxylated polypropylene imine to generate the zwitterionic precursor polymer is that the dihydroxylated polypropylene imine is reacted with tert-butyl acrylate in methanol solution at room temperature for 12-48 hours to generate the zwitterionic precursor polymer.

[0025] In one embodiment of the present application, in step (3), the condition for the hydrolysis is that the zwitterionic precursor polymer is added into trifluoroacetic acid, and the reaction is carried out at room temperature for 12 hours to obtain the polypropylene imine zwitterionic derivative.

[0026] In one embodiment of the present application, in step (4), the acrylic acid-2-isocyanate ethyl or the methacrylic acid isocyanate ethyl is reacted with the polypropylene imine zwitterionic derivative to convert the two end hydroxyl groups of the polypropylene imine zwitterionic derivative into acrylate groups or methacrylate groups to obtain the macromolecular zwitterionic crosslinking agent.

[0027] ​In one embodiment of the present application, in step (4), the reaction conditions of acrylic acid-2-isocyanate ethyl or methacrylic acid isocyanate ethyl with polypropylene imine zwitterionic derivative are: dissolving the polypropylene imine zwitterionic derivative in chloroform or dimethyl sulfoxide, adding acrylic acid-2-isocyanate ethyl or methacrylic acid isocyanate ethyl according to the molar ratio NCO:OH = 5:1, reacting at 40-80 °C for 1-3 days to convert both end hydroxyl groups into acrylate groups or methacrylate groups, and synthesizing a macromolecular zwitterionic crosslinking agent.

[0028] The present application further provides a preparation method of an anti-fouling zwitterionic hydrogel, comprising the following steps: dissolving the macromolecular zwitterionic crosslinking agent in a solution or distilled water, adding an initiator, initiating polymerization, and obtaining the anti-fouling zwitterionic hydrogel.

[0029] In one embodiment of the present application, the initiator is a photoinitiator or a thermal initiator, and is selected from one or a combination of several of phenyl-2,4,6-trimethylbenzoyl phosphonic acid salt, ammonium persulfate, potassium perchlorate, benzophenone carboxylate, azobisimidozolinium hydrochloride, and azobisimidoamide hydrochloride.

[0030] In one embodiment of the present application, the solution used to dissolve the macromolecular zwitterionic crosslinking agent is selected from one or a combination of several of physiological saline, buffer, decellularized matrix, or cell culture medium solution. Different media can be selected according to different applications.

[0031] In one embodiment of the present application, the mass fraction of the macromolecular zwitterionic crosslinking agent is 10%-40%.

[0032] In one embodiment of the present application, when a photoinitiator is used, the polymerization is carried out at room temperature, and the anti-fouling zwitterionic hydrogel is obtained after ultraviolet light irradiation for 1-3 hours.

[0033] In one embodiment of the present application, when a thermal initiator is used, the polymerization is carried out at a temperature of 60-80 °C, and the anti-fouling zwitterionic hydrogel is obtained after reaction for 4-12 hours.

[0034] In one embodiment of the present application, the preparation method of the anti-fouling zwitterionic hydrogel is: dissolving the zwitterionic crosslinking agent in a 1M NaCl solution to prepare a solution with a mass concentration of 10-40%, adding a photoinitiator or a thermal initiator, then injecting into a sealed mold, initiating monomer polymerization by photoinitiation or thermal initiation, demolding after a period of time, and soaking in water for at least 3 days to remove unreacted monomers, so as to obtain the anti-fouling zwitterionic hydrogel.

[0035] The present application further provides an anti-fouling zwitterionic hydrogel obtained based on the above preparation method.

[0036] The present application further provides the use of the anti-fouling zwitterionic hydrogel in the preparation of medical devices, implant materials and cell delivery materials.

[0037] The present application polymerizes the monomer of the oxazine derivative by the double-end initiator to generate the double-hydroxyl polyoxazine. Subsequently, the side chain of the polymer is removed to obtain the double-hydroxyl polypropylenimine. Then, the double-hydroxyl polypropylenimine is selected for further reaction to generate the zwitterionic precursor polymer, and the zwitterionic precursor polymer is hydrolyzed to obtain the polypropylenimine zwitterionic derivative. Finally, the two end hydroxyl groups are converted to synthesize the macromolecular zwitterionic crosslinking agent, and the macromolecular zwitterionic crosslinking agent is polymerized and crosslinked in the aqueous solution to prepare the anti-fouling zwitterionic hydrogel.

[0038] Compared with the prior art, the present application has the advantages and beneficial effects as follows:

[0039] The hydrogel formula of the present application is simple, and is composed of the zwitterionic crosslinking agent only, and has excellent anti-fouling properties such as anti-protein adsorption, anti-bacterial adsorption and anti-cell adsorption, and is suitable for the preparation of medical devices, implant materials and cell delivery materials. BRIEF DESCRIPTION OF DRAWINGS

[0040] Figure 1 The synthesis route of the zwitterionic crosslinking agent PCBDA prepared in the present application;

[0041] Figure 2 The anti-protein adsorption performance characterization of the anti-fouling zwitterionic hydrogel prepared in the present application; 1 H NMR spectrum;

[0042] Figure 3 The anti-protein adsorption performance characterization of the anti-fouling zwitterionic hydrogel prepared in the present application;

[0043] Figure 4 The bacterial adsorption performance characterization of the anti-fouling zwitterionic hydrogel prepared in the present application;

[0044] Figure 5 The anti-cell adsorption performance characterization of the anti-fouling zwitterionic hydrogel prepared in the present application. DETAILED DESCRIPTION

[0045] The present application will be described in detail below in combination with the drawings and specific embodiments.

[0046] Example 1

[0047] The present embodiment provides a synthesis method of the dihydroxyl end poly(2-methyl-2-oxazine), and the process is referred to as Figure 1 The detailed steps are as follows:

[0048] 2-methyl-2-oxazine (81.0 mmol, 8.00 g), (E) -1,4-Dibromo-but-2-ene (1.61 mmol, 0.35 g) and acetonitrile (12.4 mL) were mixed in a microwave vial. Polymerization was carried out at 140 °C for 50 min. After the reaction was complete, 1.0 M potassium hydroxide in methanol (4.1 mL) was added at 0 °C and stirred at room temperature overnight to terminate the polymerization. The solvent was spun down and the solid was dissolved in chloroform. The polymer was precipitated in cold n-hexane three times to obtain a sticky 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 h. 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 centrifugation three times and dried in vacuum overnight to obtain a yellow solid dihydroxyl terminated polypropylene imine (HO-PPI-OH) with the following proton nuclear magnetic resonance data: 1 H NMR (600 MHz, Methanol-d4): 2.64 (t, 4H), 1.73 (p, 2H).

[0049] In this example,

[0050] The structure of HO-PMeOZI-OH is shown below:

[0051]

[0052] The structure of HO-PPI-OH is shown below:

[0053]

[0054] Example 2

[0055] This example provides a method for synthesizing polycarboxybetaine dimethyl acrylate (PCBDA), the process is shown in Figure 1 The detailed steps are as follows:

[0056] HO-PPI-OH (8.0 g, 137.6 mmol) was dissolved in 40 mL of methanol, then t-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 liquid-liquid extraction, and the methanol phase was retained and repeated three times. The methanol was removed by rotary evaporation, and the solid was dissolved in 40 mL of chloroform. To the solution, isocyanate methyl methacrylate (2.8 g, 18.0 mmol) was added, and the reaction was carried out at 60 °C for 2 days, with 4-methoxyphenol (22.2 mg, 0.18 mmol) 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 t-butyl protecting group. After stirring the mixture at room temperature for 12 hours, the mixture was precipitated in 200 mL of ethyl ether. The solid was redissolved in 20 mL of methanol and precipitated in 200 mL of ethyl ether, and this operation was repeated 3 times or more. Finally, the solid was dried in a vacuum oven to obtain a yellow powder of PCBDA. The proton nuclear magnetic resonance spectrum of PCBDA is shown in Figure 2

[0057] Example 3

[0058] This example provides a method for preparing an anti-fouling zwitterionic hydrogel, the detailed steps of which are as follows:

[0059] PCBDA was dissolved in a 1M NaCl solution to prepare a solution with a mass concentration of 20%. 0.1 mol% of a photoinitiator (relative to the crosslinking agent), i.e., phenyl-2,4,6-trimethylbenzoyl phosphonate (LAP), was added. The polymerization was carried out in a transparent mold with a thickness of 2 mm and a diameter of 8 mm. A 365 nm, 24 watt ultraviolet light was used for irradiation, with each side of the hydrogel irradiated for 30 minutes. The hydrogel was soaked in a PBS solution for at least 5 days to remove unreacted chemicals. The anti-fouling zwitterionic hydrogel obtained in this example is PCB-20%.

[0060] Example 4

[0061] This example provides a method for preparing an anti-fouling zwitterionic hydrogel, the detailed steps of which are as follows:

[0062] ​PCBDA was dissolved in 1 M NaCl solution to make a 30% mass concentration solution. 0.1 mol% of a photoinitiator (relative to the crosslinking agent), namely phenyl-2,4,6-trimethylbenzoyl phosphinate (LAP), was added. Polymerization was carried out in a 2 mm thick, 8 mm diameter transparent mold. UV irradiation was performed using a 365 nm, 24 watt UV lamp, with each side of the hydrogel being irradiated for 30 minutes. The hydrogel was soaked in PBS solution for at least 5 days to remove unreacted chemicals. The anti-fouling zwitterionic hydrogel obtained in this example is PCB-30%.

[0063] Example 5

[0064] This example provides a method for preparing an anti-fouling zwitterionic hydrogel, the detailed steps of which are as follows:

[0065] PCBDA was dissolved in 1 M NaCl solution to make a 40% mass concentration solution. 0.1 mol% of a photoinitiator (relative to the crosslinking agent), namely phenyl-2,4,6-trimethylbenzoyl phosphinate (LAP), was added. Polymerization was carried out in a 2 mm thick, 8 mm diameter transparent mold. UV irradiation was performed using a 365 nm, 24 watt UV lamp, with each side of the hydrogel being irradiated for 30 minutes. The hydrogel was soaked in PBS solution for at least 5 days to remove unreacted chemicals. The anti-fouling zwitterionic hydrogel obtained in this example is PCB-40%.

[0066] Comparative Example 1

[0067] This comparative example provides a method for preparing a polyethylene glycol hydrogel, the detailed steps of which are as follows:

[0068] Polyethylene glycol diacrylate (PEGDA) was dissolved in 1 M NaCl solution to make a 40% mass concentration solution. 0.1 mol% of a photoinitiator (relative to the crosslinking agent), namely phenyl-2,4,6-trimethylbenzoyl phosphinate (LAP), was added. Polymerization was carried out in a 2 mm thick, 8 mm diameter transparent mold. UV irradiation was performed using a 365 nm, 24 watt UV lamp, with each side of the hydrogel being irradiated for 30 minutes. The hydrogel was soaked in PBS solution for at least 5 days to remove unreacted chemicals.

[0069] Test Example 1

[0070] 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, the specific steps of which are as follows:

[0071] The protein adsorption on the hydrogels was evaluated by fluorescence method. The hydrogels were soaked in PBS buffer for more than 3 days, and the PBS equilibrated hydrogels were cut into 5 mm in diameter and 2 mm in thickness discs, which were then transferred to 96-well flat bottom polystyrene (TCPS) plates. 1 mL of 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 min to promote protein adsorption. After that, the samples were lifted up with sterile forceps and rinsed gently in PBS buffer for three times to remove the excess dye solution. Then the samples were transferred to glass slides and analyzed using an inverted laser scanning confocal microscope (LSM900, Zeiss) with a 10x objective. Three samples were measured for each material, and five images were taken at different locations on the surface of each sample. The fluorescence intensity was analyzed using ImageJ software. As shown in FIG. 7, compared with the TCPS (tissue culture polystyrene) well plate and PEGDA hydrogel samples, the PCB hydrogel sample showed significantly reduced red RB-Fg on the surface, indicating that the antifouling zwitterionic hydrogel has good anti-protein adsorption performance. Figure 3

[0072] Test Example 2

[0073] This test example provides a method for testing the anti-bacterial adsorption performance of the hydrogels prepared in Example 3, Example 4, Example 5, and Comparative Example 1, and the specific steps are as follows:

[0074] Escherichia coli O157 was cultured in Luria-Bertani (LB) medium (20 g L −1 ) at 37 °C until the absorbance at 600 nm reached 0.7. Subsequently, the bacterial solution was diluted to an absorbance of 0.4 at 600 nm using LB medium. After the hydrogels were soaked in PBS, they were cut into 5 mm in diameter and 2 mm in thickness cylinders using a biopsy punch. Then, the hydrogels were immersed in the bacterial solution and incubated for 24 hours. To analyze the bacterial density on the surface of the hydrogels, the samples were gently rinsed with water and stained with a bacterial live / dead cell viability / cytotoxicity assay kit. After staining, the number of live and dead cells was determined by a LSM 980 Airyscan fluorescence microscope of Zeiss (Germany) using a 10x objective and FITC and Cy5 filters. As shown in FIG. 8, compared with the TCPS well plate, the PCB hydrogel and PEG hydrogel showed almost no green bacterial fluorescence on the surface, indicating that the antifouling zwitterionic hydrogel has good anti-bacterial adsorption performance. Figure 4

[0075] Test Example 3

[0076] ​​The 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:

[0077] 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, and then placed in a 96-well plate. NIH / 3T3 cells were seeded onto the hydrogel discs at a density of 8 x 10 5 Cells / mL and incubated at 37 °C, 5% CO2 for 24 hours. Finally, the culture medium was removed and the hydrogels were gently washed with PBS for 3 times. The cell adhesion on the surface of the hydrogels was observed using an inverted fluorescence microscope (Olympus IX83). As shown in FIG. 6, compared with the TCPS well plate and the PEGDA hydrogel sample, there was no green cell adhesion on the PCB hydrogel sample, indicating that the anti-fouling zwitterionic hydrogel has good anti-cell adhesion function. Figure 5

[0078] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. Those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without having to go through creative labor. 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 crosslinking agent, characterized in that, selected from one of the following structures: or wherein R is a C1-C 18 alkyl, phenyl, polyethylene glycol group or disulfide-containing group, and n is a positive integer from 1 to 1000.

2. A process for the preparation of the macromolecular zwitterionic crosslinking agent of claim 1, characterized in that, comprising the following steps: (1) a double-end initiator initiates polymerization of the oxazine derivative monomer to generate a double-hydroxyl polyoxazine, the structure of which is shown as follows: ； (2) the side chain of the double-hydroxyl polyoxazine is removed by hydrolysis to obtain a double-hydroxyl polypropylene imine, the structure of which is shown as follows: ； (3) the double-hydroxyl polypropylene imine is used to generate a zwitterionic precursor polymer, and the zwitterionic precursor polymer is hydrolyzed to obtain a polypropylene imine zwitterionic derivative, the structure of which is shown as follows: ； (4) the terminal hydroxyl groups of the polypropylene imine zwitterionic derivative are converted into acrylate groups or methacrylate groups to obtain the macromolecular zwitterionic crosslinking agent; wherein R is a C1-C 18 alkyl, phenyl, polyethylene glycol group or disulfide-containing group, and n is a positive integer from 1 to 1000.

3. The preparation method of the macromolecular zwitterionic crosslinking agent according to claim 2, characterized in that, in step (1), the double-end initiator is selected as one or more of 1,4-dibromobutane, di-p-toluenesulfonate of C1-C18 diols, α,α'-dibromo-p-xylene, di-p-toluenesulfonate of polyethylene glycol, di-triflate of polyethylene glycol, or di-p-toluenesulfonate of 6,6'-dithiobis(1-hexanol); in step (1), the oxazine derivative monomer is selected as 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 the double-end initiator to initiate polymerization of the oxazine derivative monomer are as follows: the double-end initiator and the oxazine derivative monomer are added into a microwave reactor at a molar ratio of 1:2-2000, polymerization is performed at 100-160 °C for 30-120 minutes, 1.0 M potassium hydroxide methanol solution is added at 0 °C to terminate the polymerization, the solvent is spun dry, the solid is dissolved in chloroform, and the solution is precipitated in n-hexane three times to obtain the double-hydroxyl polyoxazine.

4. The preparation method of the macromolecular zwitterionic crosslinking agent according to claim 2, characterized in that, in step (2), the conditions for the side chain of the double-hydroxyl polyoxazine to be removed by hydrolysis are as follows: the double-hydroxyl polyoxazine is refluxed in 37% concentrated hydrochloric acid for 24-72 hours to remove the side chain of the polymer to obtain the double-hydroxyl polypropylene imine; in step (3), the conditions for the double-hydroxyl polypropylene imine to generate the zwitterionic precursor polymer are as follows: the double-hydroxyl polypropylene imine and tert-butyl acrylate are reacted in a methanol solution at room temperature for 12-48 hours to generate the zwitterionic precursor polymer; in step (3), the conditions for the hydrolysis are as follows: the zwitterionic precursor polymer is added into trifluoroacetic acid, and the reaction is performed at room temperature for 12 hours to obtain the polypropylene imine zwitterionic derivative.

5. The method for preparing the macromolecular zwitterionic crosslinking agent according to claim 2, characterized in that, in step (4), acrylate-2-isocyanate ethyl or methacrylate isocyanate ethyl is reacted with the polypropylene imine zwitterionic derivative to convert the terminal hydroxyl groups of the polypropylene imine zwitterionic derivative into acrylate groups or methacrylate groups to obtain the macromolecular zwitterionic crosslinking agent; In step (4), the reaction conditions are: dissolving the polypropylenimine amphoteric ion derivative in chloroform or dimethyl sulfoxide, adding 2-isocyanatoethyl acrylate or isocyanatoethyl methacrylate in a molar ratio of NCO:OH=5:1, reacting at 40-80°C for 1-3 days to convert both end hydroxyl groups into acrylate groups or methacrylate groups, and synthesizing a macromolecular amphoteric ion crosslinking agent.

6. A method of preparing an anti-fouling zwitterionic hydrogel, characterized in that, The method comprises the following steps: dissolving the macromolecular amphoteric ion crosslinking agent of claim 1 in a solution or distilled water, adding an initiator, and initiating polymerization to obtain an anti-fouling amphoteric ion hydrogel.

7. A method of preparing an anti-fouling zwitterionic hydrogel according to claim 6, wherein, The initiator is a photo initiator or a thermal initiator, and is selected from one or a combination of several of phenyl-2,4,6-trimethylbenzoyl phosphonic acid salt, ammonium persulfate, potassium perchlorate, benzophenone carboxylate, azobisimidozolinium hydrochloride, and azobisimidoamidine hydrochloride; The solution used to dissolve the macromolecular amphoteric ion crosslinking agent is selected from one or a combination of several of physiological saline, buffer, decellularized matrix, or cell culture medium solution; The mass fraction of the macromolecular amphoteric ion crosslinking agent is 10%-40%.

8. A method of preparing an anti-fouling zwitterionic hydrogel according to claim 7, wherein, When a photo initiator is used, the polymerization is carried out at room temperature, and the anti-fouling amphoteric ion hydrogel is obtained after 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 anti-fouling amphoteric ion hydrogel is obtained after reacting for 4-12 hours.

9. An anti-fouling amphoteric ion hydrogel prepared by the preparation method of claim 6 or 7 or 8.

10. Use of the antifouling zwitterionic hydrogel according to claim 9, characterized in that, The anti-fouling amphoteric ion hydrogel is used in the preparation of medical devices, implant materials, and cell delivery materials.

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