CN118063366A

CN118063366A - Universal cross-linking agent capable of redox degradation, hydrogel and preparation and application thereof

Info

Publication number: CN118063366A
Application number: CN202410150409.5A
Authority: CN
Inventors: 李思明; 韩子龙; 曲绍兴
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2024-02-02
Filing date: 2024-02-02
Publication date: 2024-05-24

Abstract

The invention discloses a redox degradation general cross-linking agent, hydrogel and preparation and application thereof, wherein the redox degradation general cross-linking agent consists of acrylic acid and selenocysteine dihydrochloride; the cross-linking agent is added into a water-based coagulant monomer and an initiator aqueous solution to realize the preparation of degradable hydrogel by a chemical cross-linking method; the hydrogel monomer comprises an acrylamide monomer, an acrylic acid monomer, an N-isopropyl acrylamide monomer and N, N dimethyl acrylamide monomer; the initiator comprises a thermal initiator and a photoinitiator; the hydrogel can be molded by a mold method and printed and molded by digital light processing by adding a photoinitiator. The hydrogel has responsiveness to redox stimulus, and can adjust the concentration of the cross-linking agent and the concentration of the degradation liquid to realize the adjustable degradation time of 3-48 hours; the cross-linking agent and the hydrogel preparation method are simple, have excellent mechanical properties and biocompatibility, meet the control response of the hydrogel to environmental stimulus, and have wide market prospect.

Description

Universal cross-linking agent capable of redox degradation, hydrogel and preparation and application thereof

Technical Field

The invention relates to the technical field of hydrogels, in particular to a general cross-linking agent capable of redox degradation, a hydrogel, and preparation and application thereof.

Background

The hydrogel is a multifunctional polymer material, has a unique three-dimensional network structure, can absorb and retain a large amount of moisture, and can still retain the solid form. This material can absorb water equivalent to tens to thousands times its own weight, but still maintain structural stability and will not dissolve in water. Hydrogels are widely used in drug delivery systems, wound care, tissue engineering, and the like due to their good biocompatibility and adjustable physicochemical properties, and similar characteristics to biological tissues. For example, hydrogels may be used as drug carrying and sustained release systems, helping drugs to reach target sites more effectively and reducing side effects.

Stimulus degradation hydrogels are a widely studied class of hydrogels that have unique capabilities to change their physical or chemical properties under specific stimuli (e.g., pH changes, temperature changes, light, electromagnetic fields, enzymatic action, etc.), thereby causing degradation of the material. These hydrogels are typically composed of a cross-linkable polymer network capable of absorbing and retaining a large amount of moisture while maintaining their structural integrity when not stimulated. The design of stimulated degradation hydrogels enables them to respond precisely to environmental changes, a property particularly important in the medical and biotechnology fields. For example, in drug delivery systems, such hydrogels may be designed to release drug upon reaching a disease site (e.g., the acidic environment of a tumor microenvironment). Also, in tissue engineering, they can act as scaffolds for cell growth, gradually degrading and being replaced as new tissue is formed. The design and synthesis of such hydrogels involves precise control of their responsiveness, biocompatibility, mechanical strength, and degradation rate. In biomedical applications, these hydrogels must have good biocompatibility to ensure that no immune or toxic reactions are induced. In addition, the degradation products thereof should be non-toxic and capable of being safely absorbed or excluded by the human body.

However, the development and application of stimulated degrading hydrogels presents a number of challenges. First, degradation is too slow and the harsh conditions of the degradation stimulus field limit the use of most stimulus responsive hydrogels in vivo. The degradation rate of the hydrogel must be matched to the therapeutic requirements to ensure efficient drug release and tissue regeneration. Second, maintaining the mechanical stability of hydrogels in vivo is also a challenge, especially in dynamic and complex organisms. In addition, in vivo redox degradation is currently limited to hydrogels built based on disulfide and borate linkages. Therefore, it is important to develop a general cross-linking agent to prepare different functional hydrogels, and to meet the requirements of no toxicity, good biocompatibility, controllable degradation speed, mild degradation conditions, controllable mechanical properties and the like.

Disclosure of Invention

The invention aims to provide a general cross-linking agent capable of redox degradation, and aims to solve the problems that the conventional hydrogel cross-linking agent cannot meet the requirements of universality, good biocompatibility, controllable degradation speed, mild degradation conditions and the like.

Another object of the present invention is to provide a method for preparing the crosslinking agent for degradable hydrogel.

It is another object of the present invention to provide a redox-degradable hydrogel based on the above-mentioned crosslinking agent.

It is another object of the present invention to provide a method for preparing a redox-degradable hydrogel based on the above-mentioned crosslinking agent.

It is another object of the present invention to provide the use of a redox-degradable hydrogel based on the above-mentioned crosslinking agent.

For this purpose, the first technical scheme of the invention is as follows: a redox degradable general cross-linking agent is composed of 0.75-1.5 parts of selenocyamine dihydrochloride and 3 equivalents of acrylic acid.

The second technical scheme of the invention is as follows: the preparation method of the cross-linking agent for the degradable hydrogel comprises the following steps: adding selenocysteine dihydrochloride and acrylic acid into a mixed solution of 1 water, 4-8 Dimethylformamide (DMF); sequentially adding 3-9 equivalents of N, N-diisopropylethylamine and 1-5 equivalents of azodimethoxy triphenylphosphine hydrochloride, and stirring at room temperature for reaction for 30min; after removal of the solvent by rotary evaporation under reduced pressure, dichloromethane was used as a developing solvent: methanol=15-30: and 1, passing through a silica gel column, and removing the solvent by rotary evaporation of eluent to obtain the redox degradation universal crosslinking agent.

The third technical scheme of the invention is as follows: a redox degradable hydrogel based on the cross-linking agent comprises, by weight, 0.2-1 part of hydrogel monomer, 1.8-5 parts of water, 0.002-0.01 part of initiator and 0.002-0.02 part of cross-linking agent.

The above-mentioned scheme is based on and is a preferable scheme of the above-mentioned scheme: the hydrogel monomer comprises one or more of an acrylamide monomer, an acrylic acid monomer, an N-isopropyl acrylamide monomer and N, N dimethyl acrylamide monomer.

The above-mentioned scheme is based on and is a preferable scheme of the above-mentioned scheme: the initiator includes a thermal initiator and a photoinitiator.

The above-mentioned scheme is based on and is a preferable scheme of the above-mentioned scheme: the thermal initiator comprises azo diisobutyl amidine hydrochloride, azo diiso Ding Mi hydrochloride, azo dicyanovaleric acid, azo diisopropyl imidazoline or azo diisobutyronitrile.

The above-mentioned scheme is based on and is a preferable scheme of the above-mentioned scheme: the photoinitiator comprises phenyl (2, 4, 6-trimethyl benzoyl) lithium phosphate (LAP), diphenyl- (2, 4, 6-trimethyl benzoyl) phosphorus oxide (TPO) or dibenzoyl peroxide (BPO).

The fourth technical scheme of the invention is as follows: a preparation method of the redox degradable hydrogel based on the cross-linking agent comprises the following steps: adding the hydrogel monomer into water, stirring uniformly, sequentially adding the initiator and the cross-linking agent, stirring uniformly, and removing bubbles in vacuum; placing the mixed solution at 50-70deg.C for 2-5h, or placing under UV light for 5-10min to obtain redox degradable hydrogel, or printing hydrogel with complex structure by DLP printer.

The fifth technical scheme of the invention is as follows: a redox degradable hydrogel based on the cross-linking agent is used for catheter coating and drug release.

According to the invention, through utilizing selenocyamine dihydrochloride and acrylic acid to synthesize two sections of general cross-linking agents with carbon-carbon double bonds in one step, chemical cross-linking can be realized, and the mechanical properties and degradation time of the final hydrogel are changed by adding cross-linking agents with different concentrations into a gel system.

The crosslinking agent is degraded because the crosslinking agent contains diselenide bond, the bond energy of diselenide bond is 172, and the crosslinking agent is relatively small, can react with hydrogen peroxide to break, and has high reaction speed. According to reaction kinetics, the degradation speed is related to the concentration of reactants, namely the concentration of a cross-linking agent and the concentration of hydrogen peroxide, namely the thicker the reaction is, the faster the reaction is.

The preparation method of the cross-linking agent is simple and easy to control, can realize large-scale production, and the prepared cross-linking agent is nontoxic and has good biocompatibility and biodegradability.

Hydrogels prepared by the above method are redox-responsive. The hydrogel is soaked in hydrogen peroxide or ammonium persulfate solution, and the hydrogel cross-linking agent breaks due to oxidation-reduction reaction, so that the morphology and structure of the hydrogel are changed.

In addition, the hydrogel can be used for efficiently preparing a complex structure through 3D printing, and can be used for loading hydrophilic or hydrophobic drugs as a carrier for drug release. Due to the also anisotropic redox properties of the cross-linking agent, the cross-linking agent can be used as a targeting drug carrier in high redox environments, such as diabetes wounds, psoriasis and cancer cell environments.

Compared with the prior art, the invention has the beneficial effects that:

1. The cross-linking agents for degradable hydrogels are versatile and therefore can impart functionality to hydrogels by adding different monomers.

2. The cross-linking agent can realize the preparation of degradable hydrogel by a chemical cross-linking method, and the concentration of the cross-linking agent and the concentration of the degradation liquid can be adjusted to realize the adjustable degradation time.

3. The cross-linking agent and the hydrogel preparation method are simple, and simultaneously meet the requirements of nontoxic hydrogel, good biocompatibility, controllable degradation speed, mild degradation condition and controllable mechanical property, can be applied to in vivo drug release, wound care and tissue engineering, and has good market application and popularization prospects.

Drawings

FIG. 1 is a stress-strain diagram of hydrogels prepared in examples 1-3.

FIG. 2 is a diagram showing the real life of the hydrogel prepared in example 2 before and after 5 hours of degradation in pure water, 0.01%, 0.1% and 1% hydrogen peroxide; (a) pure water, (b) hydrogen peroxide with concentration of 0.01 percent, (c) hydrogen peroxide with concentration of 0.1 percent and (d) hydrogen peroxide with concentration of 1 percent.

Fig. 3 is a diagram of the 3D printing complex structure hydrogel prepared in example 4 before and after degradation for 2h and 4h in 0.1% hydrogen peroxide.

FIG. 4 is a diagram showing the real life of the hydrogel prepared in example 5 before and after degradation in 0.1% hydrogen peroxide and pure water for 4 hours; pure water (a) and hydrogen peroxide (b) with concentration of 0.1%.

FIG. 5 is a diagram showing the real life of the hydrogel prepared in example 6 before and after degradation for 3h and 8h in 0.1% hydrogen peroxide and pure water; pure water (a) and hydrogen peroxide (b) with concentration of 0.1%.

FIG. 6 is a diagram showing the real life of the hydrogel prepared in example 7 before and after degradation for 3h and 8h in 0.1% hydrogen peroxide and pure water; pure water (a) and hydrogen peroxide (b) with concentration of 0.1%.

FIG. 7 is a graph showing the results of cytotoxicity test on the hydrogel material prepared in example 8.

Detailed Description

Further details are provided below in connection with the drawings and the embodiments of the invention.

Example 1

The preparation of the redox degradation polyacrylamide hydrogel comprises the following steps:

(1) Preparing a redox degradable cross-linking agent: 0.4g of selenocyamine dihydrochloride and 3 equivalents of acrylic acid are added into a mixed solution of 1ml of water and 4ml of dimethylformamide, 6 equivalents of N, N-diisopropylethylamine and 3 equivalents of azodimethoxy triphenylphosphine hydrochloride are sequentially added, the mixture is stirred at room temperature for reaction for 30min, and after the solvent is removed by rotary evaporation under reduced pressure, a developing solvent of dichloromethane is used: methanol 20: and 1, passing through a silica gel column, and removing the solvent by rotary evaporation of eluent to obtain the redox degradation universal crosslinking agent.

In the embodiment, two sections of general cross-linking agents with carbon-carbon double bonds are synthesized by utilizing selenocyamine dihydrochloride and acrylic acid (AAC+) in one step, so that chemical cross-linking can be realized, and the mechanical properties and degradation time of the final hydrogel can be changed by adding cross-linking agents with different concentrations into a gel system. The degradation of the cross-linking agent is that the cross-linking agent contains diselenide bond, the bond energy of diselenide bond is 172kJ/mol, and the cross-linking agent is relatively small, can react with hydrogen peroxide to break, and has high reaction speed. According to reaction kinetics, the degradation speed is related to the concentration of reactants, namely the concentration of a cross-linking agent and the concentration of hydrogen peroxide, namely the thicker the reaction is, the faster the reaction is. The following is the principle of synthesis and degradation of the crosslinking agent:

Se-Se+H₂O₂→-SeOOH+H₂O

(2) Preparation of redox hydrogels: adding 1.42g of acrylamide monomer into 5ml of water, stirring uniformly, adding 200 mu l of 2.5% LAP solution and 5mg of the crosslinking agent prepared in the step (1) in sequence, stirring uniformly, removing bubbles in vacuum, and irradiating for 5min by using 385nm ultraviolet light to obtain the hydrogel with oxidation-reduction stimulation degradation. Tensile properties were tested as in curve 1 of figure 1.

Example 2

The preparation and degradation of the redox degradation polyacrylamide hydrogel comprise the following steps:

(1) The same as in step (1) of example 1.

(2) Adding 1.42g of acrylamide monomer into 5ml of water, stirring uniformly, adding 200 mu l of 2.5% LAP solution and 10mg of the crosslinking agent prepared in the step (1) in sequence, stirring uniformly, removing bubbles in vacuum, and irradiating for 5min by using 385nm ultraviolet light to obtain the hydrogel with oxidation-reduction stimulation degradation. Tensile properties were tested as in curve 2 of figure 1.

Redox degradation test of hydrogel materials: 5ml of 0, 0.01%, 0.1% and 1% hydrogen peroxide solution was prepared, and the mixture was added to a bottle containing the above hydrogel, and after reacting at room temperature of 20℃for 5 hours, the bottle was inverted to observe the degradation of the hydrogel. As shown in fig. 2, it can be seen that only the hydrogel swells in pure water (a), but the hydrogel degrades under the oxidation of hydrogen peroxide, and the degradation degree of the hydrogel increases as the concentration of hydrogen peroxide increases. Therefore, the hydrogel prepared by the method has the characteristic of redox stimulus degradation.

Example 3

(1) The same as in step (1) of example 1.

(2) Adding 1.42g of acrylamide monomer into 5ml of water, stirring uniformly, adding 200 mu l of 2.5% LAP solution and 20mg of the crosslinking agent prepared in the step (1) in sequence, stirring uniformly, removing bubbles in vacuum, and irradiating for 5min by using 385nm ultraviolet light to obtain the hydrogel with redox stimulation degradation. Tensile properties were tested as in curve 3 of figure 1.

Example 4

The preparation and degradation of the 3D printing redox degradation polyacrylamide hydrogel comprise the following steps:

(1) The same as in step (1) of example 1.

(2) 3D printing precursor liquid preparation: adding 2g of acrylamide monomer into 5ml of water, stirring uniformly, sequentially adding 800 μl of 2.5% LAP solution and 20mg of the crosslinking agent, stirring uniformly, removing bubbles in vacuum, and printing the hydrogel with a complex structure by using a DLP printer to obtain the hydrogel with oxidation-reduction stimulation degradation.

(3) Redox degradation test of 3D printed hydrogel material: placing the 3D printing hydrogel in a glass dish, adding 10ml of 0.1% hydrogen peroxide, and observing the degradation condition of the hydrogel at intervals of 0h,2h and 4h. Fig. 3 shows that after 4 hours the hydrogel has failed to support its shape, collapsing and degrading.

Example 5

The preparation of the redox degradation polyacrylic acid hydrogel comprises the following steps:

(1) The same as in step (1) of example 1.

(2) Preparation of redox hydrogels: adding 1.44g of acrylic acid monomer into 3.5ml of water, stirring uniformly, sequentially adding 200 mu l of 2.5% LAP solution and 5mg of the crosslinking agent prepared in the step (1), wherein the molar ratio of the crosslinking agent to the monomer is 0.0007, stirring uniformly, removing bubbles in vacuum, and irradiating for 5min by using 385nm ultraviolet light to obtain the hydrogel with redox stimulation degradation.

Redox degradation test of hydrogel materials: 3ml of hydrogen peroxide solution with concentration of 0 and 0.1% is prepared and added into a bottle with hydrogel, and after reaction is carried out for 4 hours at room temperature of 20 ℃, the bottle is inverted to observe the degradation condition of the hydrogel. As can be seen from fig. 4, the hydrogel only swells in pure water, but the hydrogel degrades under the oxidation of hydrogen peroxide. The versatility and oxidative degradation stimulus responsiveness of the crosslinking agent are described.

Example 6

The preparation and degradation of the redox degradation poly N-isopropyl acrylamide hydrogel comprise the following steps:

(1) The same as in example 1 (1).

(2) Preparation of redox hydrogels: adding 0.565g N-isopropyl acrylamide monomer into 5ml of water, stirring uniformly, sequentially adding 200 μl of 2.5% LAP solution and 10mg of the cross-linking agent, stirring uniformly, removing bubbles in vacuum, and irradiating with 385nm ultraviolet light for 5min to obtain hydrogel with oxidation-reduction stimulation degradation.

(3) Redox degradation test of hydrogel materials: 3ml of hydrogen peroxide solution with concentration of 0 and 0.1% is prepared and added into a bottle with hydrogel, and after reaction is carried out for 4 hours at room temperature of 20 ℃, the bottle is inverted to observe the degradation condition of the hydrogel. As can be seen from fig. 5, the hydrogel swelled only in pure water, but the hydrogel degraded under the oxidation of hydrogen peroxide. The versatility of the crosslinking agent and the oxidative degradation stimulus responsiveness of the poly-N-isopropyl acrylamide hydrogel are demonstrated.

Example 7

Preparation and degradation of thermal polymerization redox degradation polyacrylamide hydrogel

(1) The same as in example 1 (1).

(2) Preparation of a thermally polymerized hydrogel: 1.42g of acrylamide monomer is added into 5ml of water and stirred uniformly, 7mg of azodicarbonyl valeric acid and 10mg of the cross-linking agent are added in turn, stirred uniformly, bubbles are removed in vacuum, and then the mixture is thermally polymerized in an oven at 70 ℃ for 4 hours, so that the hydrogel with oxidation-reduction stimulation degradation is obtained.

(3) Redox degradation test of hydrogel materials: 5ml of hydrogen peroxide solution with concentration of 0 and 0.1% is prepared and added into a bottle with hydrogel, and after reaction is carried out for 4 hours at room temperature of 20 ℃, the bottle is inverted to observe the degradation condition of the hydrogel. As can be seen from fig. 6, the hydrogel swelled only in pure water, but the hydrogel degraded under the oxidation of hydrogen peroxide. The cross-linking agent can not only photo-polymerize hydrogel but also thermally polymerize hydrogel, and the obtained hydrogel has good mechanical properties and degradability.

Example 8

Cytotoxicity test of hydrogel materials

(1) Preparation of hydrogel network: 5ml of an aqueous solution of 15% acrylic acid and 1% acrylic acid succinimidyl ester in mass fraction was prepared, 150. Mu.l of a 2.5% LAP solution and 20mg of a crosslinking agent were added, stirred uniformly, air bubbles were removed in vacuo, and then a single network hydrogel was irradiated with 385nm ultraviolet light for 8 min. 100ml of an aqueous solution of 30% acrylic acid and 1% succinic imido acrylate by mass fraction was prepared, 3000. Mu.l of a 2.5% LAP solution and 80mg of a crosslinking agent were added, and the mixture was stirred uniformly, and air bubbles were removed in vacuo. Then soaking the single-network hydrogel in the solution for 24 hours to fully swell, and then irradiating for 8 minutes by using 385nm ultraviolet light to obtain the double-network hydrogel. The double network hydrogel was immersed in pure water for 3 days to remove unreacted monomers, with water being changed every 24 hours.

(2) The cytotoxicity experiment of NIH3T3 mouse fibroblasts is carried out on the double-network hydrogel material, and the experimental method is as follows:

experiments were divided into control and experimental groups, 5 duplicate wells were set, and experiments were performed in 24 well plates. The toxic effect of the material on NIH3T3 cells was determined using the CCK-8 method. The material sample is sterilized by ultraviolet irradiation, placed in a 24-well plate, NIH3T3 cells in the logarithmic phase are taken, counted, the cell concentration is adjusted, and inoculated onto the material according to 5X 10 ⁴ cells/well, 5% CO ₂ is cultured in a constant temperature incubator at 37 ℃ for overnight. The culture was performed for 24h according to the above grouping treatment, and the medium was removed. The wells were washed three times with PBS, 10% CCK-8 medium was added at 500. Mu.L/well, incubated in a 37℃incubator at 5% CO ₂ for 2 hours, and after removal of the material, the absorbance at 450nm was detected by a microplate reader and the toxic effect of the material on NIH3T3 cells was analyzed.

As shown in figure 7, the hydrogel has good biocompatibility, and the cell survival rate can reach more than 98% after 24 hours of cell co-culture, which shows that the hydrogel has great application prospect in organisms.

The above description is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above examples, and all technical solutions belonging to the concept of the present invention belong to the protection scope of the present invention. It should be noted that modifications and adaptations to the present invention may occur to one skilled in the art without departing from the principles of the present invention and are intended to be within the scope of the present invention.

Claims

1. A redox-degradable universal crosslinking agent, characterized in that: consists of 0.75-1.5 parts of selenocysteine dihydrochloride and 3 equivalents of acrylic acid.

2. A method of preparing the cross-linking agent for a degradable hydrogel of claim 1, wherein: adding selenocysteine dihydrochloride and acrylic acid into a mixed solution of 1 water, 4-8 dimethylformamide; sequentially adding 3-9 equivalents of N, N-diisopropylethylamine and 1-5 equivalents of azodimethoxy triphenylphosphine hydrochloride, and stirring at room temperature for reaction for 30min; after removal of the solvent by rotary evaporation under reduced pressure, dichloromethane was used as a developing solvent: methanol=15-30: and 1, passing through a silica gel column, and removing the solvent by rotary evaporation of eluent to obtain the redox degradation universal crosslinking agent.

3. A redox degradable hydrogel based on the cross-linking agent of claim 1, characterized in that: comprises, by weight, 0.2-1 part of hydrogel monomer, 1.8-5 parts of water, 0.002-0.01 part of initiator and 0.002-0.02 part of cross-linking agent.

4. A redox degradable hydrogel according to claim 3, wherein: the hydrogel monomer comprises one or more of an acrylamide monomer, an acrylic acid monomer, an N-isopropyl acrylamide monomer and N, N dimethyl acrylamide monomer.

5. A redox degradable hydrogel according to claim 3, wherein: the initiator includes a thermal initiator and a photoinitiator.

6. The redox degradable hydrogel of claim 5, wherein: the thermal initiator comprises azo diisobutyl amidine hydrochloride, azo diiso Ding Mi hydrochloride, azo dicyanovaleric acid, azo diisopropyl imidazoline or azo diisobutyronitrile.

7. The redox degradable hydrogel of claim 5, wherein: the photoinitiator comprises phenyl (2, 4, 6-trimethyl benzoyl) lithium phosphate and diphenyl- (2, 4, 6-trimethyl benzoyl) phosphorus oxide or dibenzoyl peroxide.

8. A method for preparing a redox-degradable hydrogel based on the crosslinking agent of claim 1, characterized in that: adding the hydrogel monomer into water, stirring uniformly, sequentially adding the initiator and the cross-linking agent, stirring uniformly, and removing bubbles in vacuum; placing the mixed solution at 50-70deg.C for 2-5h, or placing under UV light for 5-10min to obtain redox degradable hydrogel, or printing hydrogel with complex structure by DLP printer.

9. A redox degradable hydrogel based on the cross-linking agent of claim 1 for catheter coating and drug release.