CN110964155B - Zwitterionic hydrogel with high anti-fouling performance and preparation method and application thereof - Google Patents

Abstract

The invention discloses a zwitterionic hydrogel with high anti-fouling performance, and a preparation method and application thereof. The preparation method comprises the following steps: forming hydrogel by taking trimethylamine nitroxide containing double bonds as a zwitterionic monomer and hydrophilic molecules containing alkene groups at two ends as a cross-linking agent through free radical polymerization under the action of a photoinitiator; the addition amount of the cross-linking agent is 0.8-10 wt% of the mass fraction of the zwitterion monomer. The hydrogel carrier material designed by the invention has high anti-fouling performance, can efficiently prevent biomolecules, cells and the like from contacting with gel, further shows excellent biocompatibility and stability, and effectively meets the requirements of long-term stability and biocompatibility of fillers after surgical excision of vitreous bodies.

Description

Zwitterionic hydrogel with high anti-fouling performance and preparation method and application thereof

Technical Field

The invention belongs to the technical field of hydrogel preparation, and particularly relates to a zwitterionic hydrogel with high anti-fouling performance, and a preparation method and application thereof.

Background

Vitrectomy procedures for treating retinal diseases often require the injection of fillers into the vitreous cavity to support the retina. Currently, the vitreous cavity filler is generally used in clinic as silicone oil, but silicone oil filling is not suitable for long-term filling because of complications such as emulsification, corneal zonal degeneration and complicated cataract. Hydrogel materials are excellent candidate materials for artificial vitreous bodies, but the conventional hydrogel such as polyethylene glycol, polyvinyl alcohol, polyacrylic acid, polyacrylamide and the like is implanted into eyes, so that the hydrogel lacks high anti-pollution capacity, biomolecules, cells and the like easily contact the surface and extend into the gel, and further irreversible foreign body reaction is caused, a series of complications including inflammation, infection, tissue fibrosis and the like are caused, and meanwhile, the problems of rapid degradation and the like of the hydrogel are caused, and the long-term stable filling of vitreous cavities is difficult to realize. Therefore, the research and development of the artificial vitreous hydrogel with high anti-pollution performance and capable of being filled in eyes for a long time has important clinical significance and market prospect.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides the zwitterionic hydrogel with high antifouling performance, and the preparation method and the application thereof, and can effectively solve the problems of degradation, poor biocompatibility and the like caused by insufficient antifouling performance of the conventional hydrogel.

In order to achieve the purpose, the technical scheme adopted by the invention for solving the technical problems is as follows:

the application of a zwitter-ion hydrogel with high anti-pollution performance in filling or preparing a drug carrier after vitrectomy.

A preparation method of a zwitterionic hydrogel with high anti-pollution performance is characterized in that trimethylamine nitrogen oxide containing double bonds is used as a zwitterionic monomer, hydrophilic molecules containing vinyl groups at two ends are used as a cross-linking agent, and the hydrogel is formed in a free radical polymerization mode under the action of a photoinitiator; the addition amount of the cross-linking agent is 0.8-10 wt% of the mass fraction of the zwitterion monomer.

Further, the crosslinking agent is added in an amount of 1 wt% based on the mass fraction of the zwitterionic monomer.

Further, the preparation method of the zwitterionic monomer is as follows:

mixing a 20-30 mg/mL diethylenetriamine pentaacetic acid solution with a hydrogen peroxide solution, heating to 50-80 ℃, and then filling oxygen; then dropwise adding 1-3 g/mL dimethyl amino alkyl acrylamide solution, reacting at 50-80 ℃ for 4-12 h, concentrating, and precipitating with diethyl ether; the molar ratio of the diethylenetriamine pentaacetic acid solution to the dimethyl amino alkyl acrylamide solution is 1: 30-60.

Further, the dimethylaminoalkylacrylamide is dimethylaminoethylacrylamide, dimethylaminopropylacrylamide, or dimethylaminobutylacrylamide.

The chemical structural formula is as follows:

further, the molar ratio of the diethylenetriamine pentaacetic acid solution to the dimethylamino alkyl acrylamide solution is 1: 30.

Further, the hydrophilic molecule containing vinyl groups at two ends is vinyl polyethylene glycol.

Further, the preparation method of the vinyl polyethylene glycol is as follows:

adding 2-bromoethyl methacrylate into a polyethylene glycol solution with the concentration of 0.1-0.5 g/mL, reacting at 20-40 ℃ for 20-40 h, adding acetic acid for neutralization, filtering, concentrating, and precipitating with diethyl ether; the molar ratio of the 2-bromoethyl methacrylate to the polyethylene glycol is 2-10: 1.

The chemical structural formula is as follows:

further, the concentration of the polyethylene glycol solution was 0.1g/mL, and the solvent was methylene chloride.

Further, the molar ratio of ethyl 2-bromomethacrylate to polyethylene glycol was 3: 1.

Further, the photoinitiator is 2-hydroxy-2-methyl propiophenone, thiopropoxy thioxanthone, alpha-hydroxyalkyl benzophenone, alpha-amine alkyl benzophenone, bis-benzoylphenyl phosphine oxide or benzophenone.

Further, the reaction conditions of the zwitterionic monomer and the cross-linking agent are as follows:

and (3) reacting at room temperature for 30-80 min under the irradiation of 365nm ultraviolet light and the action of a photoinitiator, and dialyzing to remove unreacted components.

The zwitterionic hydrogel prepared by the preparation method is provided.

The invention has the beneficial effects that:

1. the antifouling performance of the zwitterion is closely related to sites of positive charges and negative charges of the zwitterion head group, and the smaller the distance between the positive charges and the negative charges, the stronger the hydration capability is, and the better the antifouling performance is. Compared with the conventional commonly used zwitter-ion hydrogel as the vitreous body filler, the zwitter-ion hydrogel used in the invention has smaller molecular distance between positive charge sites and negative charge sites, and the formed zwitter-ion hydrogel has good hydration capability and anti-pollution performance, so that the contact of biomolecules, cells and the like is prevented, the problems of poor biocompatibility, easy degradation and the like caused by the lack of anti-pollution capability of the conventional vitreous body filler are solved, and the long-term filling in the eyes of the vitreous body is realized.

2. The hydrogel carrier material designed by the invention has high anti-fouling performance, can efficiently prevent biomolecules, cells and the like from contacting with gel, further shows excellent biocompatibility and stability, is similar to a natural vitreous body in performance, and effectively meets the requirements of long-term stability and biocompatibility of a vitreous body filler after clinical operation.

Detailed Description

The following description of the embodiments of the present invention is provided to facilitate the understanding of the present invention by those skilled in the art, but it should be understood that the present invention is not limited to the scope of the embodiments, and it will be apparent to those skilled in the art that various changes may be made without departing from the spirit and scope of the invention as defined and defined in the appended claims, and all matters produced by the invention using the inventive concept are protected.

Example 1

A preparation method of a zwitterionic hydrogel with high anti-fouling performance comprises the following steps:

(1) synthesis of zwitterionic Monomer (MO) of trimethylamine nitroxide: diethylenetriaminepentaacetic acid (800mg) was added to a reaction flask containing 30mL of water and stirred vigorously until the white powder was dissolved. Then, hydrogen peroxide (50% solution, 2.87g) was added slowly, the flask was heated to 60 ℃ and oxygen was slowly charged. Slowly dropping dimethylaminopropylacrylamide (14.4g) dissolved in 10mL of water into a reaction bottle, reacting at 60 ℃ for 6 hours, concentrating the liquid to 3mL after the reaction is finished, and slowly dropping the concentrated liquid into dichloromethane for precipitation to obtain a zwitterionic Monomer (MO) precipitate of trimethylamine nitrogen oxide, wherein the structural formula is shown as (a).

(2) Synthesis of vinyl polyethylene glycol: dissolving polyethylene glycol (Mn ═ 500, 1g, 2mmol) in 10mL dichloromethane, adding 2-bromoethyl methacrylate (0.924g, 6mmol), reacting at 25 ℃ for 24h, neutralizing with acetic acid, filtering, concentrating the liquid to 3mL, slowly dropwise adding into ether to precipitate to obtain vinyl polyethylene glycol precipitate, wherein the structural formula is shown in (b).

(3) Preparation of the high stain resistance zwitterionic hydrogel: dissolving 330mg of the zwitterionic monomer synthesized in the step (1) in 0.67mL of water, adding 1 wt% (3.3mg) of vinyl polyethylene glycol and 0.33mg of 2-hydroxy-2-methyl propiophenone in mass fraction of the zwitterionic monomer, reacting at room temperature for 30min under the irradiation of ultraviolet light 365nm, adding the prepared hydrogel into distilled water, soaking at room temperature for 72h to remove unreacted zwitterionic monomer and 2-hydroxy-2-methyl propiophenone, and changing water every 6h to obtain the zwitterionic hydrogel with high antifouling property.

Example 2

A preparation method of a zwitterionic hydrogel with high anti-fouling performance comprises the following steps:

(1) synthesis of zwitterionic monomer of trimethylamine nitroxide (MO 1): diethylenetriaminepentaacetic acid (800mg) was added to a reaction flask containing 30mL of water and stirred vigorously until the white powder was dissolved. Then, hydrogen peroxide (50% solution, 2.87g) was added slowly, the flask was heated to 60 ℃ and oxygen was slowly charged. Slowly dropping dimethylaminoethylacrylamide (12.4g) dissolved in 10mL of water into a reaction bottle, reacting at 60 ℃ for 6 hours, concentrating the liquid to 3mL after the reaction is finished, and slowly dropping the liquid into dichloromethane to precipitate to obtain the zwitterionic Monomer (MO) precipitate of trimethylamine nitrogen oxide, wherein the structural formula is shown as (a').

(2) Synthesis of vinyl polyethylene glycol: dissolving polyethylene glycol (Mn ═ 1000, 1g, 2mmol) in 10mL dichloromethane, adding 2-bromoethyl methacrylate (0.467g, 6mmol), reacting at 25 ℃ for 24h, neutralizing with acetic acid, filtering, concentrating the liquid to 3mL, slowly dropping into ether to precipitate to obtain vinyl polyethylene glycol precipitate, wherein the structural formula is shown as (b).

(3) Preparation of the high stain resistance zwitterionic hydrogel: dissolving 220mg of the zwitterionic monomer synthesized in the step (1) in 0.67mL of water, adding 1 wt% (2.2mg) of vinyl polyethylene glycol and 0.22mg of 2-hydroxy-2-methyl propiophenone in mass fraction of the zwitterionic monomer, reacting at room temperature for 30min under the irradiation of ultraviolet light 365nm, adding the prepared hydrogel into distilled water, soaking at room temperature for 72h to remove unreacted zwitterionic monomer and 2-hydroxy-2-methyl propiophenone, and changing water every 6h to obtain the zwitterionic hydrogel with high antifouling property.

Example 3

A preparation method of a zwitterionic hydrogel with high anti-fouling performance comprises the following steps:

(1) synthesis of zwitterionic monomer of trimethylamine nitroxide (MO 2): diethylenetriaminepentaacetic acid (600mg) was added to a reaction flask containing 20mL of water and stirred vigorously until the white powder was dissolved. Then, hydrogen peroxide (50% solution, 2.45g) was added slowly, the reaction flask was heated to 50 ℃ and oxygen was slowly charged. Slowly dropping dimethylaminopropylacrylamide (11.4g) dissolved in 10mL of water into a reaction bottle, reacting at 70 ℃ for 6 hours, concentrating the liquid to 3mL after the reaction is finished, and slowly dropping the concentrated liquid into dichloromethane to precipitate to obtain a zwitterionic Monomer (MO) precipitate of trimethylamine nitrogen oxide, wherein the structural formula is shown as (a ").

(2) Synthesis of vinyl polyethylene glycol: dissolving polyethylene glycol (Mn is 3000, 1g and 2mmol) in 10mL dichloromethane, adding 2-bromoethyl methacrylate (0.467g and 6mmol), reacting at 25 ℃ for 24h, neutralizing with acetic acid, filtering, concentrating the liquid to 3mL, slowly dropwise adding the liquid into diethyl ether to precipitate to obtain vinyl polyethylene glycol precipitate, wherein the structural formula is shown as (b).

(3) Preparation of the high stain resistance zwitterionic hydrogel: dissolving 400mg of the zwitterionic monomer synthesized in the step (1) in 0.82mL of water, adding 2 wt% (8.0mg) of vinyl polyethylene glycol and 0.4mg of 2-hydroxy-2-methyl propiophenone in mass fraction of the zwitterionic monomer, reacting at room temperature for 30min under the irradiation of ultraviolet light 365nm, adding the prepared hydrogel into distilled water, soaking at room temperature for 72h to remove unreacted zwitterionic monomer and 2-hydroxy-2-methyl propiophenone, and changing water every 6h to obtain the zwitterionic hydrogel with high antifouling property.

Example 4 protein and cell adsorption Performance testing

1. Protein adsorption Performance test

The hydrogel prepared in examples 1 to 3, the methacrylamide zwitterionic hydrogel (control group 1) and the gel formed only by PEG (control group 2) were placed in PNS solution with BSA-FITC concentration of 0.5mg/mL respectively by using BSA-FITC as a mimic protein, then placed in an SHA-C type digital display water bath constant temperature oscillator, oscillated at a constant speed of 37 ℃ for 6 hours, and then various hydrogel samples were taken out and continuously washed with 5X 1mL of PBS (pH 7.4,0.1M) to remove BSA-FITC not adsorbed on the hydrogel surface, and then the protein adsorption amount on the hydrogel surface per unit area was calculated, and the results are shown in Table 1.

TABLE 1 protein adsorption on hydrogel surface

Test item	Example 1	Example 2	Example 3	Control group 1	Control group 2
						BSA adsorption amount (ng/cm)2)	2	1.5	1.8	150	80

2. Cell adsorption Performance test

The hydrogels prepared in examples 1 to 3, control 1 and control 2 were prepared to have a diameter × height of 15mm × 2mm, respectively, and then soaked in alcohol and sterilized under an ultraviolet lamp for 0.5h, the hydrogels were washed twice with PBS (pH 7.4,0.1M) and placed in a 24-well plate, after the cells were recovered, they were digested and blown off with 0.25% pancreatin, and then the cells were counted, and 1mL of hydrogel containing 3 × 10 was dropped into each well containing hydrogel⁴DMEM medium of individual cells, then the hydrogel was incubated at 37 deg.CAt warm environment in CO₂Culturing for 72h in an incubator, and observing the cell adhesion condition of the hydrogel surface under a BM-37XC binocular biomicroscope, wherein the hydrogel surfaces prepared in examples 1-3 have no cell adhesion basically, the control group 1 and the control group 2 both have cells adhered to the surfaces thereof, and the cell adhesion amount in the control group 2 is higher than that in the control group 1.

Therefore, according to the adsorption conditions of the hydrogel prepared in the embodiments 1 to 3 on proteins and cells, the hydrogel prepared in the application has excellent anti-fouling and anti-cell adhesion performances.

Example 5 in vivo gel stability testing in animals

Twenty-five female rabbits with the weight of 3-5 kg are equally divided into five groups, which are respectively marked as A, B, C, D, E, the left eye of each female rabbit is an experimental eye for operation, the right eye is a control, and slit lamp, fundus and intraocular pressure examination is performed before the operation to determine that no eye abnormality exists.

Performing three-channel closed type vitrectomy on the left eyes of all female rabbits, performing gas-liquid exchange after the vitrectomy, gelling the hydrogel prepared in the embodiment 1-3, the methacrylamide hydrogel and the PEG gel in vitro, injecting the hydrogel, the methacrylamide hydrogel and the PEG gel into the vitreous cavities of the left eyes of five groups of female rabbits with the label of A, B, C, D, E, and closing scleral incisions. After operation, the dianbizhi eye drops are dropped into the eye for 3 times every day, and the eye drops are observed by a slit lamp at regular intervals.

Observing the situation by using a postoperative slit lamp, wherein the inflammatory reaction does not appear in the left eye of A, B, C three groups of experimental rabbits after the operation, and the inflammatory reaction appears in the experimental rabbits marked as groups D and E; meanwhile, the vitreous bodies in the left eyes of A, B, C three groups of experimental rabbits are kept transparent within 15 weeks, and then start to be gradually degraded in the eyes, wherein the degradation time is more than 12 weeks; the vitreous body in the left eye of the experimental rabbit marked as group D keeps transparent within 10 weeks, then starts to degrade in the eye, and completely degrades within 3-5 weeks; the vitreous in the left eye of experimental rabbits designated as group E remained clear for 5-6 weeks, thereafter began to degrade in the eye and was completely degraded within 2 weeks.

Meanwhile, in the case of B-ultrasonic examination of eyes of A, B, C, D, E five groups of experimental rabbits, acoustic images such as vitreous opacity, hemorrhage, vitreous retinal fibroplasia, and retinal detachment were not detected.

The experimental results show that the zwitterionic hydrogel prepared by the invention is used as an intraocular filler to be filled into a vitreous cavity, has good compatibility, slow degradation speed, no obvious inflammatory reaction and good filling effect.

Claims (9)

1. A preparation method of a zwitterionic hydrogel with high stain resistance is characterized in that trimethylamine nitroxide containing double bonds is used as a zwitterionic monomer, vinyl polyethylene glycol is used as a cross-linking agent, and the hydrogel is formed in a free radical polymerization mode under the action of a photoinitiator; the addition amount of the cross-linking agent is 0.8-10 wt% of the mass fraction of the zwitterion monomer.

2. The method of claim 1 wherein the cross-linking agent is added in an amount of 1 wt% based on the weight of the zwitterionic monomer.

3. The method for preparing the zwitterionic hydrogel with high anti-fouling performance according to claim 1 or 2, wherein the zwitterionic monomer is prepared as follows:

mixing a 20-30 mg/mL diethylenetriamine pentaacetic acid solution with a hydrogen peroxide solution, heating to 50-80 ℃, and then filling oxygen; then dropwise adding 1-3 g/mL dimethyl amino alkyl acrylamide solution, reacting at 50-80 ℃ for 4-12 h, concentrating, and precipitating with diethyl ether; the molar ratio of the diethylenetriamine pentaacetic acid solution to the dimethyl amino alkyl acrylamide solution is 1: 30-60.

4. The method for preparing the zwitterionic hydrogel with high stain resistance according to claim 3, wherein the molar ratio of the diethylenetriamine pentaacetic acid solution to the dimethylamino alkyl acrylamide solution is 1: 30.

5. The method for preparing the zwitterionic hydrogel with high stain resistance according to claim 1, wherein the vinyl polyethylene glycol is prepared as follows:

adding 2-bromoethyl methacrylate into a polyethylene glycol solution with the concentration of 0.01-0.1 g/mL, reacting at 20-40 ℃ for 20-40 h, adding acetic acid for neutralization, filtering, concentrating, and precipitating with diethyl ether; the molar ratio of the 2-bromoethyl methacrylate to the polyethylene glycol is 2-10: 1.

6. The method of claim 5, wherein the molar ratio of ethyl 2-bromomethacrylate to polyethylene glycol is 3: 1.

7. The method of claim 1, wherein the reaction conditions of the zwitterionic monomer and the cross-linking agent are as follows:

and (3) reacting at room temperature for 30-80 min under the irradiation of 365nm ultraviolet light and the action of a photoinitiator, and dialyzing to remove unreacted components.

8. The preparation method of any one of claims 2 to 7 is used for preparing the zwitterionic hydrogel with high stain resistance.

9. Use of the zwitterionic hydrogel with high stain resistance of claim 8 in the preparation of a post-operative filling material for vitrectomy.