Injectable responsive multifunctional hydrogel and application thereof

Abstract

The invention discloses injectable responsive multifunctional hydrogel and application thereof, belongs to the technical field of biological medicine, and particularly relates to a preparation method of the injectable responsive multifunctional hydrogel, which comprises the following steps: mixing a polymer hydrogel skeleton complex liquid and a polymer active group donor complex liquid to obtain the injectable responsive multifunctional hydrogel; the macromolecular hydrogel skeleton composite liquid is a mixture of a phenylboronic acid-based polymer and a macromolecular structure skeleton; the high-molecular active group donor complex liquid is a mixture of a high-molecular hydroxyl donor and a high-molecular aldehyde donor, the high-molecular hydroxyl donor comprises at least one of polyvinyl alcohol, polyethylene glycol, gelatin and polysucrose, and the high-molecular aldehyde donor comprises oxidized dextran or oxidized hyaluronic acid. The injectable responsive multifunctional hydrogel prepared by the invention is good in biocompatibility, good in drug release performance and good in antibacterial performance, and can be used for preparing drugs for intervertebral disc degeneration, wound healing, bone tissue engineering, postoperative adhesion prevention or slow-release repair of inflammatory tissues.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to injectable responsive multifunctional hydrogels and their uses. Background Art

[0002] Injectable hydrogels have been widely used in medical fields such as drug delivery, tissue repair, and anti-inflammatory treatment due to their excellent biocompatibility, local gel-forming ability, and drug loading performance. In recent years, hydrogel systems with environmental responsiveness, especially hydrogels that can respond to reactive oxygen species (ROS) and acidic microenvironments, have become a current research hotspot. In the prior art, phenylboronic acid groups (PBA) are grafted onto ε-poly-L-lysine (ε-PLL) molecules to form phenylboronic acid-grafted ε-polylysine polymers (PBA-g-PLL), and this polymer can undergo a reversible Schiff base reaction with aldehyde groups in oxidized dextran (OD) to construct an injectable hydrogel structure. However, the prior art has the following problems: (1) It is impossible to form a hydrogel with a stable structure: Even when increasing the reaction concentration, it is still difficult to construct a complete cross-linked network, the gelation time is long, and the colloid is prone to collapse; (2) Although introducing polyvinyl alcohol (PVA) can promote rapid gelation, the cross-linked structure is uneven, and the mechanical properties after gelation are poor, making it difficult to be used for actual injection or long-term delivery; (3) The ability to balance and regulate "injectability, gelation uniformity, and mechanical stability" is insufficient, resulting in poor applicability in clinical applications. Therefore, the hydrogels constructed based on the PBA-g-PLL / OD system currently have technical bottlenecks such as poor stability, uneven gelation structure, and poor injection compatibility, and there is an urgent need to develop a new structure adjustment mechanism to achieve performance improvement. Summary of the Invention

[0003] The purpose of the present invention is to provide injectable responsive multifunctional hydrogels with good biocompatibility, good drug release performance, and good antibacterial performance, and their uses.

[0004] The technical solution adopted by the present invention to achieve the above purpose is as follows: Preparation method of injectable responsive multifunctional hydrogel, comprising: mixing a polymer hydrogel skeleton composite solution and a polymer active group donor composite solution to obtain an injectable responsive multifunctional hydrogel; the polymer hydrogel skeleton composite solution is a mixture of a phenylboronic acid-based polymer and a polymer structural skeleton; the polymer active group donor composite solution is a mixture of a polymer hydroxyl donor and a polymer aldehyde group donor, the polymer hydroxyl donor includes at least one of polyvinyl alcohol, polyethylene glycol, gelatin, and polysucrose, and the polymer aldehyde group donor includes oxidized dextran or oxidized hyaluronic acid; the volume ratio of the polymer hydrogel skeleton composite solution to the polymer active group donor composite solution is 1:1-2.

[0005] The injectable responsive multifunctional hydrogel prepared by the present invention forms a three-dimensional entangled system by introducing 2-hydroxyethyl cellulose and the phenylboronic acid-based polymer, effectively avoiding the problems of uneven cross-linking and structural collapse when only relying on the Schiff base cross-linking of the phenylboronic acid-based polymer and the oxidized dextran system, and improving the injectability and gel-forming uniformity of the hydrogel; its network structure is synergistically constructed by multiple reversible chemical actions such as Schiff base bonds, borate ester bonds, aldol condensation reactions, and hydrogen bonds, realizing the dynamic response regulation of the microenvironment pH value and the level of reactive oxygen species (ROS). The injectable responsive multifunctional hydrogel prepared by the present invention not only has excellent drug loading versatility, can be compatible with various dosage forms such as small molecule drugs, biological macromolecules, nanoparticles, and microspheres, showing broad clinical application prospects, but also its preparation process adopts an all-aqueous phase system, without organic solvents or high-temperature reactions, having the advantages of rapid gel formation, simple operation, safe and reliable raw materials, etc., and at the same time having good clinical transformation potential and preliminary antibacterial ability.

[0006] Preferably, in the preparation of the phenylboronic acid-based polymer, a modified raw material is reacted with 3-fluoro-4-carboxyphenylboronic acid to obtain the phenylboronic acid-based polymer.

[0007] More preferably, the modified raw material includes at least one of ε-polylysine, gelatin, and polyethyleneimine.

[0008] Preferably, the polymer structural skeleton includes at least one of 2-hydroxyethyl cellulose, sodium carboxymethyl cellulose, gelatin, chitosan, and xanthan gum.

[0009] Preferably, the polymer hydroxyl donor further contains a hydroxyl polymer.

[0010] More preferably, in the preparation of the hydroxyl polymer, under the action of an initiator, the monomer undergoes a free radical polymerization reaction to obtain the hydroxyl polymer.

[0011] Even more preferably, the initiator is azobisisobutyronitrile.

[0012] More preferably, the monomer at least includes dihydromyrcenol and dihydrocarveol. The hydroxy polymer prepared by the present invention contains a large number of hydroxy groups, and through the formation of dynamic covalent bonds, pH / ROS-responsive dissociation is achieved, realizing the release of the loaded drug by the hydrogel; moreover, the active hydroxy groups in the polymer chain interact with the microbial cell membrane, destroying the integrity of the cell membrane and exerting an antibacterial effect.

[0013] More preferably, the preparation method of the hydroxy polymer is specifically as follows: Under a nitrogen atmosphere, dissolve the monomer in tetrahydrofuran, add the initiator and stir evenly, react at 60 - 80 °C for 6 - 12 h, after the reaction, wash with deionized water 2 - 5 times, and dry under vacuum to obtain the hydroxy polymer.

[0014] Even more preferably, the monomer includes at least one of dihydromyrcenol, dihydrocarveol, and dimethyl (4-hydroxy-2-butenyl) maleate. In the present invention, the unit of dimethyl (4-hydroxy-2-butenyl) maleate is further introduced into the hydroxy polymer, significantly increasing the hydrophilicity of the polymer main chain, promoting the diffusion of drug molecules, and further improving the drug release performance of the hydrogel; at the same time, active groups are introduced to disrupt the normal metabolic balance of the cell membrane, thereby further enhancing the antibacterial performance of the hydrogel.

[0015] Even more preferably, the dosage ratio of tetrahydrofuran to dihydromyrcenol is 1 mL: 0.01 - 0.05 g.

[0016] Even more preferably, the dosage ratio of tetrahydrofuran to dihydrocarveol is 1 mL: 0.02 - 0.05 g.

[0017] Even more preferably, the dosage ratio of tetrahydrofuran to dimethyl (4-hydroxy-2-butenyl) maleate is 1 mL: 0.01 - 0.02 g.

[0018] Even more preferably, the initiator is azobisisobutyronitrile.

[0019] Even more preferably, the dosage ratio of tetrahydrofuran to the initiator is 1 mL: 0.001 - 0.002 g.

[0020] Preferably, the preparation method of the phenylboronic acid-based polymer is specifically as follows: Dissolve 3-fluoro-4-carboxyphenylboronic acid in dimethyl sulfoxide, add the activator, stir at 20 - 25 °C for 0.5 - 2 h, add the modification raw material and react for 24 - 48 h, dialyze with deionized water for 8 - 24 h, repeat dialysis 2 - 5 times, and dry under vacuum to obtain the phenylboronic acid-based polymer.

[0021] More preferably, the dosage ratio of 3-fluoro-4-carboxyphenylboronic acid to dimethyl sulfoxide is 1 g: 100 - 200 mL.

[0022] More preferably, the activator includes 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide.

[0023] Even more preferably, the mass ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide to N-hydroxysuccinimide is 1:0.2 - 1.

[0024] More preferably, the mass ratio of 3-fluoro-4-carboxyphenylboronic acid to the activator is 1:2 - 10.

[0025] More preferably, the modified raw material includes at least one of ε-polylysine, gelatin, and polyethyleneimine.

[0026] More preferably, the mass ratio of 3-fluoro-4-carboxyphenylboronic acid to the modified raw material is 1:1 - 5.

[0027] Preferably, the preparation method of the polymer hydrogel skeleton composite liquid is specifically as follows: Mix the phenylboronic acid-based polymer and the polymer structure skeleton, add phosphate buffer solution (PBS) and stir evenly to obtain the polymer hydrogel skeleton composite liquid.

[0028] More preferably, the polymer structure skeleton includes at least one of 2-hydroxyethyl cellulose, sodium carboxymethyl cellulose, gelatin, chitosan, and xanthan gum.

[0029] More preferably, the mass ratio of the phenylboronic acid-based polymer to the polymer structure skeleton is 1:0.01 - 0.05.

[0030] More preferably, the dosage ratio of the phenylboronic acid-based polymer to PBS is 1 g:5 - 20 mL.

[0031] Preferably, the preparation method of the polymer active group donor composite liquid is specifically as follows: Mix the polymer hydroxyl donor and the polymer aldehyde donor, add PBS and stir evenly to obtain the polymer active group donor composite liquid.

[0032] More preferably, the polymer hydroxyl donor includes at least one of polyvinyl alcohol, polyethylene glycol, gelatin, polysucrose, and hydroxyl polymer.

[0033] Even more preferably, the polymer hydroxyl donor includes polyvinyl alcohol and hydroxyl polymer. The mass ratio of polyvinyl alcohol to hydroxyl polymer is 1:1 - 2.

[0034] More preferably, the polymer aldehyde donor includes oxidized dextran or oxidized hyaluronic acid.

[0035] More preferably, the mass ratio of the polymer hydroxyl donor to the polymer aldehyde donor is 1:1 - 2.

[0036] More preferably, the dosage ratio of the polymer hydroxyl donor to PBS is 1 g: 10 - 20 mL.

[0037] Preferably, the preparation method of the injectable responsive multifunctional hydrogel is specifically as follows: Mix the polymer hydrogel skeleton composite solution and the polymer active group donor composite solution at room temperature to obtain the injectable responsive multifunctional hydrogel.

[0038] More preferably, the volume ratio of the polymer hydrogel skeleton composite solution to the polymer active group donor composite solution is 1: 1 - 2.

[0039] The present invention also discloses the injectable responsive multifunctional hydrogel prepared by the above preparation method.

[0040] The present invention also discloses the use of the injectable responsive multifunctional hydrogel in the preparation of drugs for intervertebral disc degeneration, wound healing, bone tissue engineering, postoperative anti-adhesion or slow-release repair of inflammatory tissues.

[0041] Since the present invention adopts a dynamic cross-linked hydrogel system composed of a phenylboronic acid-based polymer, a polymer structural skeleton, a polymer hydroxyl donor and a polymer aldehyde donor, the polymer structural skeleton includes at least one of 2-hydroxyethyl cellulose, sodium carboxymethyl cellulose, gelatin, chitosan and xanthan gum, the polymer hydroxyl donor includes at least one of polyvinyl alcohol, polyethylene glycol, gelatin, polysucrose and hydroxyl polymer, and the polymer aldehyde donor includes oxidized dextran or oxidized hyaluronic acid, thus having the following beneficial effects: The injectable responsive multifunctional hydrogel prepared by the present invention has good biocompatibility, good drug release performance and good antibacterial performance. The cell survival rate after being treated with the injectable responsive multifunctional hydrogel prepared by the present invention reaches more than 88%, higher than the minimum value of 70% specified in the national standard GB / T 16886.5 - 2017, and there is no potential cytotoxicity; the drug release rate is 28.1 - 41.4%, and it can be continuously released for more than 2 weeks, and the release behavior has responsive condition characteristics; the antibacterial rate is 75.4 - 95.6%, and it is applicable to clinical transformation. Therefore, the present invention is an injectable responsive multifunctional hydrogel with good biocompatibility, good drug release performance and good antibacterial performance, and can be used to prepare drugs for intervertebral disc degeneration, wound healing, bone tissue engineering, postoperative anti-adhesion or slow-release repair of inflammatory tissues. Description of the Drawings

[0042] Figure 1 It is the cytotoxicity of the injectable responsive multifunctional hydrogel. Detailed Embodiments

[0043] The present invention will be further described in detail below in conjunction with specific embodiments. The provided embodiments are only for clarifying the present invention and not for limiting the scope of the present invention. The following provided embodiments can be used as a guide for those of ordinary skill in the art to make further improvements and do not constitute any limitation to the present invention in any way.

[0044] In the following embodiments, the experimental methods are all conventional methods unless otherwise specified. The materials, reagents, etc. used in the following embodiments can all be obtained from commercial channels unless otherwise specified.

[0045] Example 1: The preparation of the phenylboronic acid-based polymer includes: Dissolve 3-fluoro-4-carboxyphenylboronic acid in dimethyl sulfoxide, add an activator, stir at 20 °C for 1 h, add a modification raw material and react for 24 h, dialyze with deionized water for 12 h, repeat dialysis 3 times, and dry in vacuum to obtain the phenylboronic acid-based polymer. The dosage ratio of 3-fluoro-4-carboxyphenylboronic acid to dimethyl sulfoxide is 1 g: 200 mL; the activator includes 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, and the mass ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide to N-hydroxysuccinimide is 1: 0.5; the mass ratio of 3-fluoro-4-carboxyphenylboronic acid to the activator is 1: 5; the modification raw material is ε-polylysine, and the mass ratio of 3-fluoro-4-carboxyphenylboronic acid to the modification raw material is 1: 3.

[0046] The preparation of the polymer hydrogel skeleton composite liquid includes: Mix the phenylboronic acid-based polymer and the polymer structure skeleton, add phosphate buffer solution (PBS) and stir evenly to obtain the polymer hydrogel skeleton composite liquid. The polymer structure skeleton is 2-hydroxyethyl cellulose, and the mass ratio of the phenylboronic acid-based polymer to the polymer structure skeleton is 1: 0.03; the dosage ratio of the phenylboronic acid-based polymer to PBS is 1 g: 10 mL.

[0047] The preparation of the polymer active group donor composite liquid includes: Mix the polymer hydroxyl donor and the polymer aldehyde donor, add PBS and stir evenly to obtain the polymer active group donor composite liquid. The polymer hydroxyl donor is polyvinyl alcohol, the polymer aldehyde donor is oxidized dextran, the mass ratio of the polymer hydroxyl donor to the polymer aldehyde donor is 1: 1, and the dosage ratio of the polymer hydroxyl donor to PBS is 1 g: 10 mL.

[0048] The preparation of the injectable responsive multifunctional hydrogel includes: Mix the polymer hydrogel skeleton composite liquid and the polymer active group donor composite liquid at room temperature to obtain an injectable responsive multifunctional hydrogel. The volume ratio of the polymer hydrogel skeleton composite liquid to the polymer active group donor composite liquid is 1:1.

[0049] Example 2: The preparation of the phenylboronic acid-based polymer is the same as that in Example 1.

[0050] The preparation of the polymer hydrogel skeleton composite liquid is the same as that in Example 1, except that 2-hydroxyethyl cellulose is replaced with sodium carboxymethyl cellulose.

[0051] The preparation of the polymer active group donor composite liquid is the same as that in Example 1.

[0052] The preparation of the injectable responsive multifunctional hydrogel is the same as that in Example 1, except that the polymer hydrogel skeleton composite liquid is replaced with the polymer hydrogel skeleton composite liquid prepared in this example.

[0053] Example 3: The preparation of the phenylboronic acid-based polymer is the same as that in Example 1.

[0054] The preparation of the polymer hydrogel skeleton composite liquid is the same as that in Example 1, except that 2-hydroxyethyl cellulose is replaced with gelatin.

[0055] The preparation of the polymer active group donor composite liquid is the same as that in Example 1.

[0056] The preparation of the injectable responsive multifunctional hydrogel is the same as that in Example 1, except that the polymer hydrogel skeleton composite liquid is replaced with the polymer hydrogel skeleton composite liquid prepared in this example.

[0057] Example 4: The preparation of the phenylboronic acid-based polymer is the same as that in Example 1.

[0058] The preparation of the polymer hydrogel skeleton composite liquid is the same as that in Example 1, except that 2-hydroxyethyl cellulose is replaced with chitosan.

[0059] The preparation of the polymer active group donor composite liquid is the same as that in Example 1.

[0060] The preparation of the injectable responsive multifunctional hydrogel is the same as that in Example 1, except that the polymer hydrogel skeleton composite liquid is replaced with the polymer hydrogel skeleton composite liquid prepared in this example.

[0061] Example 5: The preparation of the phenylboronic acid-based polymer is the same as that in Example 1.

[0062] The preparation of the polymer hydrogel skeleton composite solution is the same as that in Example 1, except that 2-hydroxyethyl cellulose is replaced by xanthan gum.

[0063] The preparation of the polymer active group donor composite solution is the same as that in Example 1.

[0064] The preparation of the injectable responsive multifunctional hydrogel is the same as that in Example 1, except that the polymer hydrogel skeleton composite solution is replaced by the polymer hydrogel skeleton composite solution prepared in this example.

[0065] Example 6: The preparation of the phenylboronic acid-based polymer is the same as that in Example 1.

[0066] The preparation of the polymer hydrogel skeleton composite solution is the same as that in Example 1.

[0067] The preparation of the polymer active group donor composite solution is the same as that in Example 1, except that polyvinyl alcohol is replaced by polyethylene glycol.

[0068] The preparation of the injectable responsive multifunctional hydrogel is the same as that in Example 1, except that the polymer active group donor composite solution is replaced by the polymer active group donor composite solution prepared in this example.

[0069] Example 7: The preparation of the phenylboronic acid-based polymer is the same as that in Example 1.

[0070] The preparation of the polymer hydrogel skeleton composite solution is the same as that in Example 1.

[0071] The preparation of the polymer active group donor composite solution is the same as that in Example 1, except that polyvinyl alcohol is replaced by gelatin.

[0072] The preparation of the injectable responsive multifunctional hydrogel is the same as that in Example 1, except that the polymer active group donor composite solution is replaced by the polymer active group donor composite solution prepared in this example.

[0073] Example 8: The preparation of the phenylboronic acid-based polymer is the same as that in Example 1.

[0074] The preparation of the polymer hydrogel skeleton composite solution is the same as that in Example 1.

[0075] The preparation of the polymer active group donor composite solution is the same as that in Example 1, except that polyvinyl alcohol is replaced by polysucrose.

[0076] Compared with Example 1, the preparation of the injectable responsive multifunctional hydrogel is the same as Example 1 except that the polymer active group donor composite liquid is replaced by the polymer active group donor composite liquid prepared in this example. Other conditions are the same as Example 1.

[0077] Embodiment 9: The preparation of phenylboronic acid-based polymer is the same as in Example 1.

[0078] The preparation of the polymer hydrogel skeleton composite liquid is the same as in Example 1.

[0079] The preparation of the polymer active group donor composite solution is similar to that of Example 1 except that the oxidized dextran is replaced by oxidized hyaluronic acid. The oxidized hyaluronic acid is purchased from Xi'an Qiyue Biotechnology Co., Ltd.

[0080] Compared with Example 1, the preparation of the injectable responsive multifunctional hydrogel is the same as Example 1 except that the polymer active group donor composite liquid is replaced by the polymer active group donor composite liquid prepared in this example. Other conditions are the same as Example 1.

[0081] Embodiment 10: Preparation of phenylboronic acid-based polymers, comprising: 3-Fluoro-4-carboxyphenylboronic acid was dissolved in dimethyl sulfoxide, an activator was added, the mixture was stirred at 25°C for 1 hour, a modified raw material was added and reacted for 48 hours, the mixture was dialyzed with deionized water for 12 hours, the dialysis was repeated 3 times, and the mixture was dried under vacuum to obtain a phenylboronic acid-based polymer. The dosage ratio of 3-fluoro-4-carboxyphenylboronic acid to dimethyl sulfoxide was 1 g: 200 mL; the activator included 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, and the mass ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide to N-hydroxysuccinimide was 1: 0.5; the mass ratio of 3-fluoro-4-carboxyphenylboronic acid to the activator was 1: 5; the modified raw material was polyethyleneimine; and the mass ratio of 3-fluoro-4-carboxyphenylboronic acid to the modified raw material was 1: 3.

[0082] The preparation of the polymer hydrogel skeleton composite liquid is similar to that of Example 1 except that the phenylboronic acid-based polymer is replaced by the phenylboronic acid-based polymer prepared in this example. Other conditions are the same as those of Example 1.

[0083] The preparation of the polymer active group donor composite liquid is the same as in Example 1.

[0084] The preparation of the injectable responsive multifunctional hydrogel is similar to that of Example 1 except that the polymer hydrogel skeleton composite liquid is replaced by the polymer hydrogel skeleton composite liquid prepared in this example. Other conditions are the same as those of Example 1.

[0085] Embodiment 11: Preparation of phenylboronic acid-based polymer, including Dissolve 3-fluoro-4-carboxyphenylboronic acid in dimethyl sulfoxide, add an activator, stir at 25 °C for 1 h, add a modification raw material and react for 48 h, dialyze with deionized water for 12 h, repeat dialysis 3 times, and dry in vacuum to obtain the phenylboronic acid-based polymer. The dosage ratio of 3-fluoro-4-carboxyphenylboronic acid to dimethyl sulfoxide is 1 g:200 mL; the activator includes 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, and the mass ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide to N-hydroxysuccinimide is 1:0.5; the mass ratio of 3-fluoro-4-carboxyphenylboronic acid to the activator is 1:5; the modification raw material is gelatin; the mass ratio of 3-fluoro-4-carboxyphenylboronic acid to the modification raw material is 1:3.

[0086] Preparation of the polymer hydrogel skeleton composite liquid, compared with Example 1, except that the phenylboronic acid-based polymer is replaced with the phenylboronic acid-based polymer prepared in this example, and other conditions are the same as in Example 1.

[0087] Preparation of the polymer active group donor composite liquid, the same as in Example 1.

[0088] Preparation of the injectable responsive multifunctional hydrogel, compared with Example 1, except that the polymer hydrogel skeleton composite liquid is replaced with the polymer hydrogel skeleton composite liquid prepared in this example, and other conditions are the same as in Example 1.

[0089] Example 12: Preparation of the phenylboronic acid-based polymer, the same as in Example 1.

[0090] Preparation of the hydroxyl polymer, including Under a nitrogen atmosphere, dissolve dihydromyrcenol and dihydrocarvyl alcohol in tetrahydrofuran, add an initiator and stir evenly, react at 70 °C for 8 h, wash 3 times with deionized water after the reaction, and dry in vacuum to obtain the hydroxyl polymer. The dosage ratio of tetrahydrofuran to dihydromyrcenol is 1 mL:0.05 g; the dosage ratio of tetrahydrofuran to dihydrocarvyl alcohol is 1 mL:0.05 g; the initiator is azobisisobutyronitrile, and the dosage ratio of tetrahydrofuran to the initiator is 1 mL:0.001 g.

[0091] Preparation of the polymer hydrogel skeleton composite liquid, the same as in Example 1.

[0092] Preparation of the polymer active group donor composite liquid, compared with Example 1, except that the polymer hydroxyl donor is changed to a mixture of polyvinyl alcohol and the hydroxyl polymer, and the mass ratio of polyvinyl alcohol to the hydroxyl polymer is changed to 1:1, and other conditions are the same as in Example 1.

[0093] Preparation of injectable responsive multifunctional hydrogel. Compared with Example 1, except that the polymer active group donor composite solution was replaced with the polymer active group donor composite solution prepared in this example, other conditions were the same as those in Example 1.

[0094] Example 13: Preparation of phenylboronic acid-based polymer was the same as that in Example 1.

[0095] Preparation of hydroxy polymer. Compared with Example 12, except that the dosage ratio of tetrahydrofuran to dihydromyrcenol was changed to 1 mL: 0.01 g, other conditions were the same as those in Example 12.

[0096] Preparation of polymer hydrogel skeleton composite solution was the same as that in Example 1.

[0097] Preparation of polymer active group donor composite solution. Compared with Example 1, except that the polymer hydroxy donor was changed to a mixture of polyvinyl alcohol and hydroxy polymer, and the mass ratio of polyvinyl alcohol to hydroxy polymer was changed to 1:1, other conditions were the same as those in Example 1.

[0098] Preparation of injectable responsive multifunctional hydrogel. Compared with Example 1, except that the polymer active group donor composite solution was replaced with the polymer active group donor composite solution prepared in this example, other conditions were the same as those in Example 1.

[0099] Example 14: Preparation of phenylboronic acid-based polymer was the same as that in Example 1.

[0100] Preparation of hydroxy polymer. Compared with Example 12, except that the dosage ratio of tetrahydrofuran to dihydrocarvyl alcohol was changed to 1 mL: 0.02 g, other conditions were the same as those in Example 12.

[0101] Preparation of polymer hydrogel skeleton composite solution was the same as that in Example 1.

[0102] Preparation of polymer active group donor composite solution. Compared with Example 1, except that the polymer hydroxy donor was changed to a mixture of polyvinyl alcohol and hydroxy polymer, and the mass ratio of polyvinyl alcohol to hydroxy polymer was changed to 1:1, other conditions were the same as those in Example 1.

[0103] Preparation of injectable responsive multifunctional hydrogel. Compared with Example 1, except that the polymer active group donor composite solution was replaced with the polymer active group donor composite solution prepared in this example, other conditions were the same as those in Example 1.

[0104] Example 15: Preparation of phenylboronic acid-based polymer was the same as that in Example 1.

[0105] Preparation of hydroxy polymer includes, Under a nitrogen atmosphere, dihydromyrcenol, dihydrocarveol, and dimethyl (4-hydroxy-2-butenyl) maleate were dissolved in tetrahydrofuran. An initiator was added and stirred evenly. The reaction was carried out at 70 °C for 8 h. After the reaction, it was washed 3 times with deionized water, dried under vacuum, and a hydroxyl polymer was obtained. The dosage ratio of tetrahydrofuran to dihydromyrcenol was 1 mL: 0.05 g; the dosage ratio of tetrahydrofuran to dihydrocarveol was 1 mL: 0.05 g; the dosage ratio of tetrahydrofuran to dimethyl (4-hydroxy-2-butenyl) maleate was 1 mL: 0.02 g; the initiator was azobisisobutyronitrile, and the dosage ratio of tetrahydrofuran to the initiator was 1 mL: 0.001 g.

[0106] The preparation of the polymer hydrogel skeleton composite liquid was the same as that in Example 1.

[0107] The preparation of the polymer active group donor composite liquid was the same as that in Example 1, except that the polymer hydroxyl donor was changed to a mixture of polyvinyl alcohol and the hydroxyl polymer, and the mass ratio of polyvinyl alcohol to the hydroxyl polymer was changed to 1:1.

[0108] The preparation of the injectable responsive multifunctional hydrogel was the same as that in Example 1, except that the polymer active group donor composite liquid was replaced with the polymer active group donor composite liquid prepared in this example.

[0109] Example 16: The preparation of the phenylboronic acid-based polymer was the same as that in Example 1.

[0110] The preparation of the hydroxyl polymer was the same as that in Example 15, except that the dosage ratio of tetrahydrofuran to dimethyl (4-hydroxy-2-butenyl) maleate was changed to 1 mL: 0.01 g.

[0111] The preparation of the polymer hydrogel skeleton composite liquid was the same as that in Example 1.

[0112] The preparation of the polymer active group donor composite liquid was the same as that in Example 1, except that the polymer hydroxyl donor was changed to a mixture of polyvinyl alcohol and the hydroxyl polymer, and the mass ratio of polyvinyl alcohol to the hydroxyl polymer was changed to 1:1.

[0113] The preparation of the injectable responsive multifunctional hydrogel was the same as that in Example 1, except that the polymer active group donor composite liquid was replaced with the polymer active group donor composite liquid prepared in this example.

[0114] Comparative Example 1: The preparation of the phenylboronic acid-based polymer was the same as that in Example 1.

[0115] Preparation of hydroxy polymer. Compared with Example 12, all other conditions are the same as those in Example 12 except that dihydrocarveol is not used.

[0116] Preparation of high molecular weight hydrogel skeleton composite liquid is the same as that in Example 1.

[0117] Preparation of high molecular weight active group donor composite liquid. Compared with Example 1, all other conditions are the same as those in Example 1 except that the high molecular weight hydroxy donor is changed to a mixture of polyvinyl alcohol and hydroxy polymer, and the mass ratio of polyvinyl alcohol to hydroxy polymer is changed to 1:1.

[0118] Preparation of injectable responsive multifunctional hydrogel. Compared with Example 1, all other conditions are the same as those in Example 1 except that the high molecular weight active group donor composite liquid is replaced with the high molecular weight active group donor composite liquid prepared in this example.

[0119] Comparative Example 2: Preparation of phenylboronic acid-based polymer is the same as that in Example 1.

[0120] Preparation of hydroxy polymer. Compared with Example 12, all other conditions are the same as those in Example 12 except that dihydromyrcenol is not used.

[0121] Preparation of high molecular weight hydrogel skeleton composite liquid is the same as that in Example 1.

[0122] Preparation of high molecular weight active group donor composite liquid. Compared with Example 1, all other conditions are the same as those in Example 1 except that the high molecular weight hydroxy donor is changed to a mixture of polyvinyl alcohol and hydroxy polymer, and the mass ratio of polyvinyl alcohol to hydroxy polymer is changed to 1:1.

[0123] Preparation of injectable responsive multifunctional hydrogel. Compared with Example 1, all other conditions are the same as those in Example 1 except that the high molecular weight active group donor composite liquid is replaced with the high molecular weight active group donor composite liquid prepared in this example.

[0124] Comparative Example 3: Preparation of phenylboronic acid-based polymer is the same as that in Example 1.

[0125] Preparation of hydroxy polymer. Compared with Example 15, all other conditions are the same as those in Example 15 except that dihydromyrcenol and dihydrocarveol are not used.

[0126] Preparation of high molecular weight hydrogel skeleton composite liquid is the same as that in Example 1.

[0127] Preparation of high molecular weight active group donor composite liquid. Compared with Example 1, all other conditions are the same as those in Example 1 except that the high molecular weight hydroxy donor is changed to a mixture of polyvinyl alcohol and hydroxy polymer, and the mass ratio of polyvinyl alcohol to hydroxy polymer is changed to 1:1.

[0128] Preparation of injectable responsive multifunctional hydrogel. Compared with Example 1, except that the polymer active group donor composite solution was replaced with the polymer active group donor composite solution prepared in this example, other conditions were the same as those in Example 1.

[0129] Experimental Example: 1. Cytotoxicity Test The cytotoxicity of the injectable responsive multifunctional hydrogels prepared in Examples 1-16 and Comparative Examples 1-3 was characterized by MTT assay. The specific steps were as follows: After ultraviolet sterilization, the injectable responsive multifunctional hydrogels prepared in Examples 1-16 and Comparative Examples 1-3 were added to 8 mL of DMEM medium and soaked at 37 °C for 24 h. After centrifugation, the supernatant was collected. The supernatant was diluted with DMEM medium to a final concentration of 5 mg / mL to obtain the supernatant after treatment of each sample. A control group and 20 experimental groups were set up. In the control group, 100 μL of DMEM medium was added; in the 20 experimental groups, 100 μL of the supernatants of Examples 1-16 and Comparative Examples 1-3 were added in sequence. 100 μL of a rat nucleus pulposus cell suspension with a cell density of 1×10 5 cells / mL was added to each group and cultured at 37 °C for 24 h. All the liquid was discarded, and then 50 μL of thiazolyl blue (MTT) was added and cultured for 2 h. All the liquid was discarded, and 100 μL of isopropanol was added. The absorbance value at 570 nm was measured. Cell survival rate (%) = absorbance value of the experimental group / absorbance value of the control group × 100%.

[0130] Figure 1 For the cytotoxicity of the injectable responsive multifunctional hydrogel, S1 to S16 corresponded to the cytotoxicity of the injectable responsive multifunctional hydrogels prepared in Examples 1-16 in sequence, and S17 to S19 corresponded to the cytotoxicity of the injectable responsive multifunctional hydrogels prepared in Comparative Examples 1-3 in sequence. It can be seen from Figure 1 the results that after the rat nucleus pulposus cells were treated with the injectable responsive multifunctional hydrogels prepared in Examples 1-16 and Comparative Examples 1-3 of the present invention, the cell survival rate was above 88%, higher than the minimum value of 70% specified in the national standard "GB / T 16886.5-2017 Biological evaluation of medical devices - Part 5: Tests for in vitro cytotoxicity". This indicates that the injectable responsive multifunctional hydrogels prepared in Examples 1-16 and Comparative Examples 1-3 of the present invention have no potential cytotoxicity.

[0131] 2. Drug Release Performance The specific steps of the drug release performance of the injectable responsive multifunctional hydrogel prepared in Example 1, Examples 12-16 and Comparative Examples 1-3 are as follows: PLGA-PEG nanoparticles co-loaded with metformin and silymarin are used as model drugs and compounded with the injectable responsive multifunctional hydrogel, the mass ratio of the PLGA-PEG nanoparticles co-loaded with metformin and silymarin to the injectable responsive multifunctional hydrogel is 1:10, and the drug-loaded hydrogel is obtained, and the PLGA-PEG nanoparticles co-loaded with metformin and silymarin are purchased from Xi'an Qiyue Biotechnology Co., Ltd. A control group and an experimental group are set. In the experimental group, hydrogen peroxide was diluted with phosphate buffer to a final concentration of 200 μmol / L, and the pH was adjusted to 5.5, 5 mL of drug-loaded hydrogel was added, and cultured at 37°C for 48 h. 400 μL of the release medium was taken out, and the release amount of metformin and silymarin co-loaded PLGA-PEG nanoparticles was detected by high performance liquid chromatography. The calculation formula of drug release rate is: drug release rate (%) = release amount / initial amount × 100%. The drug release performance of the injectable responsive multifunctional hydrogel prepared in Example 1, Examples 12-16 and Comparative Examples 1-3 of the present invention is shown in Table 1.

[0132] In the control group, hydrogen peroxide was diluted with phosphate buffer to a final concentration of 200 μmol / L, and the pH was adjusted to 7.4, 5 mL of the drug-loaded hydrogel prepared in Example 1 was added, and cultured at 37° C. 400 μL of the release medium was taken out every 24 h, and the release amount of metformin and silymarin co-loaded PLGA-PEG nanoparticles was detected by high performance liquid chromatography until the release amount no longer changed, and the sustainable release time of the drug was recorded.

[0133] The injectable responsive multifunctional hydrogel prepared in Example 1 of the present invention has a sustained drug release time of more than 2 weeks under pH 7.4, and the drug release rate at 48h is 11.6%, which is significantly lower than the drug release rate of the injectable responsive multifunctional hydrogel prepared in Example 1 under pH 5.5. This shows that the injectable responsive multifunctional hydrogel prepared in the present invention has environmental responsive release performance, and the release of methylamine under environmental stimulation has the characteristics of responsive conditions.

[0134] Table 1 Drug release rate (%)

[0135] As can be seen from Table 1, the drug release rate of the injectable responsive multifunctional hydrogels prepared in Examples 12-14 of the present invention is higher than that of Example 1. This is because in the preparation of the injectable responsive multifunctional hydrogels, hydroxy polymers composed of dihydromyrcenol and dihydrocarvyl alcohol units were additionally used in Examples 12-14; the drug release rate of the injectable responsive multifunctional hydrogel prepared in Example 12 is higher than that of Example 13 and Example 14 because the usage amounts of dihydromyrcenol and dihydrocarvyl alcohol are different in the preparation of the hydroxy polymer; the drug release rate of the injectable responsive multifunctional hydrogels prepared in Examples 12-14 is higher than that of Comparative Example 1 and Comparative Example 2 because in the preparation of the hydroxy polymer, only dihydromyrcenol was used alone in Comparative Example 1 and only dihydrocarvyl alcohol was used alone in Comparative Example 2. This shows that compared with using dihydromyrcenol and dihydrocarvyl alcohol alone, synergistically using appropriate amounts of dihydromyrcenol and dihydrocarvyl alcohol to prepare a hydroxy polymer and then using the hydroxy polymer to prepare an injectable responsive multifunctional hydrogel can improve the drug release performance of the injectable responsive multifunctional hydrogel.

[0136] The drug release rate of the injectable responsive multifunctional hydrogels prepared in Examples 15-16 of the present invention is higher than that of Example 12 because in the preparation of the hydroxy polymer, dimethyl (4-hydroxy-2-butenyl) maleate was further used in Examples 15-16; the drug release rate of the injectable responsive multifunctional hydrogel prepared in Example 15 is higher than that of Example 16 because the usage amount of dimethyl (4-hydroxy-2-butenyl) maleate is different in the preparation of the hydroxy polymer; the drug release rate of the injectable responsive multifunctional hydrogels prepared in Examples 15-16 is higher than that of Comparative Example 3 because in the preparation of the hydroxy polymer, only dimethyl (4-hydroxy-2-butenyl) maleate was used alone in Comparative Example 3 without using dihydromyrcenol and dihydrocarvyl alcohol. This shows that using a hydroxy polymer composed of dihydromyrcenol, dihydrocarvyl alcohol and dimethyl (4-hydroxy-2-butenyl) maleate units to prepare an injectable responsive multifunctional hydrogel helps to further improve the drug release performance of the injectable responsive multifunctional hydrogel.

[0137] 3. Antibacterial property The injectable responsive multifunctional hydrogels prepared in Example 1, Example 12-16-5 and Comparative Examples 1-3 were sterilized and then their antibacterial properties against Staphylococcus aureus were evaluated. The specific steps were as follows: 25 μL of Staphylococcus aureus bacterial solution was dropped at the center of a petri dish containing LB medium. A control group and 8 experimental groups were set up. In the control group, a sterile membrane was covered; in the 8 experimental groups, the injectable responsive multifunctional hydrogels prepared in Example 1-5 and Comparative Examples 1-3 were covered in sequence. After 1 h of contact for each group, the sterile membrane covered in the control group and the injectable responsive multifunctional hydrogels prepared in Example 1-5 and Comparative Examples 1-3 covered in the 8 experimental groups were collected, 5 mL of phosphate buffer was added respectively, vortexed for 2 min, 10 μL of the vortexed solution was taken and added to 4 mL of LB medium, cultured at 37 °C for 24 h, 200 μL of the bacterial suspension was taken to measure the OD value at 600 nm, and the antibacterial rate (%) = (OD value of the control group - OD value of the experimental group) / OD value of the control group.

[0138] Table 2 Antibacterial rate (%)

[0139] As can be seen from Table 2, the antibacterial rates of the injectable responsive multifunctional hydrogels prepared in Examples 12-14 of the present invention are higher than that of Example 1 because in the preparation of the injectable responsive multifunctional hydrogel, Examples 12-14 additionally used a hydroxyl polymer composed of dihydromyrcenol and dihydrocarveol units; the antibacterial rate of the injectable responsive multifunctional hydrogel prepared in Example 12 is higher than that of Example 13 and Example 14 because in the preparation of the hydroxyl polymer, the usage amounts of dihydromyrcenol and dihydrocarveol are different; the antibacterial rates of the injectable responsive multifunctional hydrogels prepared in Examples 12-14 are higher than those of Comparative Example 1 and Comparative Example 2 because in the preparation of the hydroxyl polymer, Comparative Example 1 only used dihydromyrcenol alone and Comparative Example 2 only used dihydrocarveol alone. This shows that compared with the individual use of dihydromyrcenol and dihydrocarveol, the synergistic use of appropriate amounts of dihydromyrcenol and dihydrocarveol to prepare a hydroxyl polymer and then using the hydroxyl polymer to prepare an injectable responsive multifunctional hydrogel can improve the antibacterial performance of the injectable responsive multifunctional hydrogel.

[0140] The antibacterial rate of the injectable responsive multifunctional hydrogel prepared in Examples 15-16 of the present invention is higher than that of Example 12 because in the preparation of the hydroxyl polymer, Examples 15-16 further use dimethyl (4-hydroxy-2-butenyl) maleate; the antibacterial rate of the injectable responsive multifunctional hydrogel prepared in Example 15 is higher than that of Example 16 because in the preparation of the hydroxyl polymer, the usage amount of dimethyl (4-hydroxy-2-butenyl) maleate is different; the antibacterial rate of the injectable responsive multifunctional hydrogel prepared in Examples 15-16 is higher than that of Comparative Example 3 because in the preparation of the hydroxyl polymer, Comparative Example 3 only uses dimethyl (4-hydroxy-2-butenyl) maleate alone and does not use dihydromyrcenol and dihydrocarveol. This shows that using a hydroxyl polymer composed of dihydromyrcenol, dihydrocarveol and dimethyl (4-hydroxy-2-butenyl) maleate units to prepare an injectable responsive multifunctional hydrogel helps to further improve the antibacterial performance of the injectable responsive multifunctional hydrogel.

[0141] The conventional operations in the operation steps of the present invention are well known to those skilled in the art and will not be elaborated here.

[0142] The above-described embodiments have described the technical solutions of the present invention in detail. It should be understood that the above are only specific embodiments of the present invention and are not used to limit the present invention. Any changes and variations made within the scope of the principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing an injectable responsive multifunctional hydrogel, comprising: mixing a polymer hydrogel skeleton composite liquid and a polymer active group donor composite liquid to obtain an injectable responsive multifunctional hydrogel; the polymer hydrogel skeleton composite liquid is a mixture of a phenylboronic acid polymer and a polymer structural skeleton; the polymer active group donor composite liquid is a mixture of a polymer hydroxyl donor and a polymer aldehyde donor, the polymer hydroxyl donor includes at least one of polyvinyl alcohol, polyethylene glycol, gelatin and polysucrose, and the polymer aldehyde donor includes oxidized dextran or oxidized hyaluronic acid; the volume ratio of the polymer hydrogel skeleton composite liquid to the polymer active group donor composite liquid is 1:1-2.

2. The method for preparing the injectable responsive multifunctional hydrogel according to claim 1, characterized in that: In the preparation of the phenylboronic acid-based polymer, the modified raw material and 3-fluoro-4-carboxyphenylboronic acid are reacted to obtain the phenylboronic acid-based polymer.

3. The method for preparing the injectable responsive multifunctional hydrogel according to claim 2, characterized in that: The modified raw material includes at least one of ε-polylysine, gelatin and polyethyleneimine.

4. The method for preparing the injectable responsive multifunctional hydrogel according to claim 1, characterized in that: The polymer structure skeleton includes at least one of 2-hydroxyethyl cellulose, sodium carboxymethyl cellulose, gelatin, chitosan and xanthan gum.

5. The method for preparing the injectable responsive multifunctional hydrogel according to claim 1, characterized in that: The high molecular weight hydroxyl donor further contains a hydroxyl polymer.

6. The method for preparing the injectable responsive multifunctional hydrogel according to claim 5, characterized in that: In the preparation of the hydroxy polymer, under the action of an initiator, the monomer undergoes a free radical polymerization reaction to obtain the hydroxy polymer.

7. The method for preparing the injectable responsive multifunctional hydrogel according to claim 6, characterized in that: The initiator is azobisisobutyronitrile.

8. The method for preparing the injectable responsive multifunctional hydrogel according to claim 6, characterized in that: The monomers include at least dihydromyrcenol and dihydrocarveol.

9. The injectable responsive multifunctional hydrogel prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the injectable responsive multifunctional hydrogel according to claim 9 in the preparation of drugs for intervertebral disc degeneration, wound healing, bone tissue engineering, postoperative adhesion prevention or sustained-release repair of inflammatory tissue.

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