Injectable responsive multifunctional hydrogel and its use

Abstract

The present invention discloses an injectable responsive multifunctional hydrogel and its use, belonging to the field of biomedicine technology, and specifically relates to 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-based 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 including at least one of polyvinyl alcohol, polyethylene glycol, gelatin, and polysucrose, and the polymer aldehyde donor including oxidized dextran or oxidized hyaluronic acid. The injectable responsive multifunctional hydrogel prepared by the present invention has good biocompatibility, excellent drug release performance, and good antibacterial properties, and can be used to prepare drugs for intervertebral disc degeneration, wound healing, bone tissue engineering, postoperative adhesion prevention, or sustained-release repair of inflammatory tissue.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to an injectable responsive multifunctional hydrogel and applications thereof. Background Art

[0002] Injectable hydrogels have been widely used in medical fields such as drug delivery, tissue repair, and anti-inflammatory therapy due to their excellent biocompatibility, localized gelation, and drug-loading properties. In recent years, environmentally responsive hydrogel systems, particularly those that respond to reactive oxygen species (ROS) and acidic microenvironments, have become a research hotspot. Existing technologies graft phenylboronic acid (PBA) groups onto ε-poly-L-lysine (ε-PLL) molecules to form phenylboronic acid-grafted ε-PLL polymers (PBA-g-PLL). This polymer can undergo a reversible Schiff base reaction with the aldehyde groups of oxidized dextran (OD) to construct an injectable hydrogel structure. However, existing technologies have the following problems: (1) It is impossible to form a hydrogel with a stable structure: even if the reaction concentration is increased, 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 the introduction of polyvinyl alcohol (PVA) can promote rapid gelation, the cross-linking structure is uneven, and the mechanical properties after gelation are poor, making it difficult to use for actual injection or long-term delivery; (3) The ability to balance and control "injectability, gelation uniformity, and mechanical stability" is insufficient, resulting in poor applicability in clinical applications. Therefore, the current hydrogel constructed based on the PBA-g-PLL / OD system has technical bottlenecks such as poor stability, uneven gelation structure, and poor injection compatibility. It is urgent to develop new structural regulation mechanisms to achieve performance improvement. Summary of the Invention

[0003] The purpose of the present invention is to provide an injectable responsive multifunctional hydrogel with good biocompatibility, good drug release performance and good antibacterial performance and its use.

[0004] The technical solutions adopted by the present invention to achieve the above-mentioned purpose are:

[0005] The invention relates to 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 the injectable responsive multifunctional hydrogel; the polymer hydrogel skeleton composite liquid is a mixture of a phenylboronic acid-based 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.

[0006] The injectable, responsive, multifunctional hydrogel prepared by the present invention incorporates 2-hydroxyethyl cellulose to form a three-dimensional entangled system with a phenylboronic acid-based polymer. This effectively avoids the uneven crosslinking and structural collapse that occurs when Schiff base crosslinking is formed solely by the phenylboronic acid-based polymer and oxidized dextran system, thereby improving the hydrogel's injectability and gelation uniformity. Its network structure is synergistically constructed through multiple reversible chemical interactions, including Schiff base bonds, borate ester bonds, aldol condensation reactions, and hydrogen bonds, enabling dynamic regulation of microenvironmental pH and reactive oxygen species (ROS) levels. The injectable, responsive, multifunctional hydrogel prepared by the present invention not only exhibits excellent drug loading versatility and compatibility with a variety of dosage forms, including small molecule drugs, biomacromolecules, nanoparticles, and microspheres, demonstrating broad clinical application prospects. Furthermore, its preparation process utilizes an all-aqueous system, eliminating the need for organic solvents or high-temperature reactions. This process offers advantages such as rapid gelation, ease of operation, and safe and reliable raw materials. It also possesses promising clinical translational potential and preliminary antibacterial properties.

[0007] Preferably, in the preparation of the phenylboronic acid-based polymer, the modified raw material is reacted with 3-fluoro-4-carboxyphenylboronic acid to obtain the phenylboronic acid-based polymer.

[0008] More preferably, the modified raw material includes at least one of ε-polylysine, gelatin and polyethyleneimine.

[0009] Preferably, the polymer structure skeleton includes at least one of 2-hydroxyethyl cellulose, sodium carboxymethyl cellulose, gelatin, chitosan and xanthan gum.

[0010] Preferably, the high molecular weight hydroxyl donor further contains a hydroxyl polymer.

[0011] More preferably, in the preparation of the hydroxy polymer, the monomer undergoes a free radical polymerization reaction under the action of an initiator to obtain the hydroxy polymer.

[0012] More preferably, the initiator is azobisisobutyronitrile.

[0013] More preferably, the monomers include at least dihydromyrcenol and dihydrocarveol. The hydroxyl polymer prepared by the present invention contains a large number of hydroxyl groups, which form dynamic covalent bonds to achieve pH / ROS-responsive dissociation, enabling the release of loaded drugs from the hydrogel. Furthermore, the active hydroxyl groups in the polymer chain interact with microbial cell membranes, disrupting their integrity and exerting an antibacterial effect.

[0014] More preferably, the preparation method of the hydroxy polymer is specifically,

[0015] Under nitrogen atmosphere, the monomer is dissolved in tetrahydrofuran, the initiator is added and stirred evenly, and the mixture is reacted at 60-80° C. for 6-12 hours. After the reaction, the mixture is washed with deionized water for 2-5 times and vacuum dried to obtain a hydroxy polymer.

[0016] More preferably, the monomers include at least one of dihydromyrcenol, dihydrocarveol, and (4-hydroxy-2-butenyl) dimethyl maleate. The present invention further introduces (4-hydroxy-2-butenyl) dimethyl maleate units into the hydroxy polymer, significantly increasing the hydrophilicity of the polymer backbone, promoting the diffusion of drug molecules, and further improving the drug release properties of the hydrogel. Simultaneously, the introduction of active groups disrupts the normal metabolic balance of the cell membrane, thereby further enhancing the antibacterial properties of the hydrogel.

[0017] More preferably, the usage ratio of tetrahydrofuran to dihydromyrcenol is 1 mL: 0.01-0.05 g.

[0018] More preferably, the usage ratio of tetrahydrofuran to dihydrocarveol is 1 mL: 0.02-0.05 g.

[0019] More preferably, the usage ratio of tetrahydrofuran to dimethyl (4-hydroxy-2-butenyl)maleate is 1 mL: 0.01-0.02 g.

[0020] More preferably, the initiator is azobisisobutyronitrile.

[0021] More preferably, the usage ratio of tetrahydrofuran to initiator is 1 mL: 0.001-0.002 g.

[0022] Preferably, the preparation method of the phenylboronic acid-based polymer is specifically as follows:

[0023] 3-Fluoro-4-carboxyphenylboronic acid is dissolved in dimethyl sulfoxide, an activator is added, and the mixture is stirred at 20-25° C. for 0.5-2 hours. The modified raw material is added and reacted for 24-48 hours. The mixture is dialyzed with deionized water for 8-24 hours, the dialysis is repeated 2-5 times, and the mixture is dried under vacuum to obtain a phenylboronic acid-based polymer.

[0024] More preferably, the usage ratio of 3-fluoro-4-carboxyphenylboronic acid and dimethyl sulfoxide is 1 g:100-200 mL.

[0025] More preferably, the activating agent includes 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide.

[0026] More preferably, the mass ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide to N-hydroxysuccinimide is 1:0.2-1.

[0027] More preferably, the mass ratio of 3-fluoro-4-carboxyphenylboronic acid to the activator is 1:2-10.

[0028] More preferably, the modified raw material includes at least one of ε-polylysine, gelatin and polyethyleneimine.

[0029] More preferably, the mass ratio of 3-fluoro-4-carboxyphenylboronic acid to the modified raw material is 1:1-5.

[0030] Preferably, the preparation method of the polymer hydrogel skeleton composite liquid is specifically as follows:

[0031] The phenylboronic acid-based polymer and the polymer structure skeleton are mixed, and phosphate buffer solution (PBS) is added and stirred evenly to obtain a polymer hydrogel skeleton composite liquid.

[0032] More preferably, the polymer structure skeleton includes at least one of 2-hydroxyethyl cellulose, sodium carboxymethyl cellulose, gelatin, chitosan and xanthan gum.

[0033] More preferably, the mass ratio of the phenylboronic acid-based polymer to the polymer structure skeleton is 1:0.01-0.05.

[0034] More preferably, the usage ratio of the phenylboronic acid-based polymer to PBS is 1 g:5-20 mL.

[0035] Preferably, the method for preparing the polymer active group donor composite liquid is as follows:

[0036] The polymer hydroxyl donor and the polymer aldehyde donor are mixed, added into PBS and stirred evenly to obtain a polymer active group donor composite solution.

[0037] More preferably, the high molecular weight hydroxyl donor includes at least one of polyvinyl alcohol, polyethylene glycol, gelatin, polysucrose and a hydroxyl polymer.

[0038] More preferably, the high molecular weight hydroxyl donor comprises polyvinyl alcohol and a hydroxyl polymer, and the mass ratio of polyvinyl alcohol to the hydroxyl polymer is 1:1-2.

[0039] More preferably, the high molecular weight aldehyde donor includes oxidized dextran or oxidized hyaluronic acid.

[0040] More preferably, the mass ratio of the polymer hydroxyl donor to the polymer aldehyde donor is 1:1-2.

[0041] More preferably, the usage ratio of the high molecular weight hydroxyl donor to PBS is 1 g:10-20 mL.

[0042] Preferably, the preparation method of the injectable responsive multifunctional hydrogel is specifically as follows:

[0043] The polymer hydrogel skeleton composite liquid and the polymer active group donor composite liquid are mixed at room temperature to obtain an injectable responsive multifunctional hydrogel.

[0044] More preferably, the volume ratio of the polymer hydrogel skeleton composite liquid to the polymer active group donor composite liquid is 1:1-2.

[0045] The invention also discloses an injectable responsive multifunctional hydrogel prepared by the preparation method.

[0046] The present invention also discloses the use of the injectable responsive multifunctional hydrogel in preparing medicines for intervertebral disc degeneration, wound healing, bone tissue engineering, postoperative adhesion prevention or sustained-release repair of inflammatory tissue.

[0047] The present invention adopts a phenylboronic acid-based polymer, a polymer structural skeleton, a polymer hydroxyl donor and a polymer aldehyde donor to form a dynamic cross-linked hydrogel system, wherein 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 a hydroxyl polymer, and the polymer aldehyde donor includes oxidized dextran or oxidized hyaluronic acid. Therefore, the present invention has the following beneficial effects: the injectable responsive multifunctional hydrogel prepared by the present invention has good biocompatibility, excellent drug release performance and good antibacterial performance. The cell survival rate after treatment with the injectable responsive multifunctional hydrogel prepared by the present invention reaches more than 88%, which is higher than the minimum value of 70% specified in the national standard GB / T 16886.5-2017 and has no potential cytotoxicity; the drug release rate is 28.1-41.4%, the release can be sustained for more than 2 weeks, and the release behavior has responsive conditional characteristics; the antibacterial rate is 75.4-95.6%, and it is suitable for clinical transformation. Therefore, the present invention is an injectable responsive multifunctional hydrogel with good biocompatibility, excellent drug release performance and good antibacterial performance, which can be used to prepare drugs for intervertebral disc degeneration, wound healing, bone tissue engineering, postoperative adhesion prevention or sustained-release repair of inflammatory tissue. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 Cytotoxicity of Injectable Responsive Multifunctional Hydrogels. DETAILED DESCRIPTION

[0049] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0050] The experimental methods in the following examples are conventional methods unless otherwise specified. The materials and reagents used in the following examples are commercially available unless otherwise specified.

[0051] Example 1:

[0052] Preparation of phenylboronic acid-based polymers, comprising:

[0053] 3-Fluoro-4-carboxyphenylboronic acid was dissolved in dimethyl sulfoxide (DMSO), an activator was added, and the mixture was stirred at 20°C for 1 hour. The modified raw material was then added and allowed to react for 24 hours. The mixture was dialyzed against deionized water for 12 hours, repeated three times, and dried under vacuum to obtain a phenylboronic acid-based polymer. The ratio of 3-Fluoro-4-carboxyphenylboronic acid to DMSO was 1 g:200 mL. The activator consisted of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, with a mass ratio of 1:0.5. The mass ratio of 3-Fluoro-4-carboxyphenylboronic acid to the activator was 1:5. The modified raw material was ε-polylysine, and the mass ratio of 3-Fluoro-4-carboxyphenylboronic acid to the modified raw material was 1:3.

[0054] Preparation of polymer hydrogel skeleton composite liquid, including:

[0055] The phenylboronic acid polymer and the polymer structure framework were mixed, and phosphate buffered saline (PBS) was added and stirred to obtain a polymer hydrogel framework composite solution. The polymer structure framework was 2-hydroxyethyl cellulose, and the mass ratio of the phenylboronic acid polymer to the polymer structure framework was 1:0.03. The amount ratio of the phenylboronic acid polymer to PBS was 1g:10mL.

[0056] The preparation of polymer active group donor complex solution includes:

[0057] A polymeric hydroxyl donor and a polymeric aldehyde donor were mixed, added to PBS, and stirred evenly to obtain a polymeric active group donor complex solution. The polymeric hydroxyl donor was polyvinyl alcohol, the polymeric aldehyde donor was oxidized dextran, the mass ratio of the polymeric hydroxyl donor to the polymeric aldehyde donor was 1:1, and the amount ratio of the polymeric hydroxyl donor to PBS was 1g:10mL.

[0058] Preparation of injectable responsive multifunctional hydrogels, including,

[0059] The polymer hydrogel skeleton composite solution and the polymer active group donor composite solution were mixed at room temperature to obtain an injectable responsive multifunctional hydrogel. The volume ratio of the polymer hydrogel skeleton composite solution to the polymer active group donor composite solution was 1:1.

[0060] Example 2:

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

[0062] The preparation of the polymer hydrogel skeleton composite liquid was performed in the same manner as in Example 1 except that 2-hydroxyethyl cellulose was replaced with sodium carboxymethyl cellulose.

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

[0064] The preparation of the injectable responsive multifunctional hydrogel was carried out in the same manner as in Example 1 except that the polymer hydrogel skeleton composite liquid was replaced with the polymer hydrogel skeleton composite liquid prepared in this example.

[0065] Example 3:

[0066] The preparation of phenylboronic acid-based polymer is the same as in Example 1.

[0067] The preparation of the polymer hydrogel skeleton composite liquid was similar to that of Example 1 except that 2-hydroxyethyl cellulose was replaced by gelatin. Other conditions were the same as those of Example 1.

[0068] The preparation of the polymer active group donor composite solution is the same as in Example 1.

[0069] The preparation of the injectable responsive multifunctional hydrogel was carried out in the same manner as in Example 1 except that the polymer hydrogel skeleton composite liquid was replaced with the polymer hydrogel skeleton composite liquid prepared in this example.

[0070] Example 4:

[0071] The preparation of phenylboronic acid-based polymer is the same as in Example 1.

[0072] The preparation of the polymer hydrogel skeleton composite liquid was similar to that of Example 1 except that 2-hydroxyethyl cellulose was replaced by chitosan. Other conditions were the same as those of Example 1.

[0073] The preparation of the polymer active group donor composite solution is the same as in Example 1.

[0074] The preparation of the injectable responsive multifunctional hydrogel was carried out in the same manner as in Example 1 except that the polymer hydrogel skeleton composite liquid was replaced with the polymer hydrogel skeleton composite liquid prepared in this example.

[0075] Example 5:

[0076] The preparation of phenylboronic acid-based polymer is the same as in Example 1.

[0077] The preparation of the polymer hydrogel skeleton composite liquid was performed in the same manner as in Example 1 except that 2-hydroxyethyl cellulose was replaced with xanthan gum.

[0078] The preparation of the polymer active group donor composite solution is the same as in Example 1.

[0079] The preparation of the injectable responsive multifunctional hydrogel was carried out in the same manner as in Example 1 except that the polymer hydrogel skeleton composite liquid was replaced with the polymer hydrogel skeleton composite liquid prepared in this example.

[0080] Example 6:

[0081] The preparation of phenylboronic acid-based polymer is the same as in Example 1.

[0082] The preparation of the polymer hydrogel skeleton composite liquid is the same as that in Example 1.

[0083] The preparation of the polymer active group donor composite solution was similar to that in Example 1 except that polyvinyl alcohol was replaced by polyethylene glycol. Other conditions were the same as those in Example 1.

[0084] The preparation of the injectable responsive multifunctional hydrogel was carried out in the same manner as in Example 1 except that the polymer active group donor complex solution was replaced with the polymer active group donor complex solution prepared in this example.

[0085] Example 7:

[0086] The preparation of phenylboronic acid-based polymer is the same as in Example 1.

[0087] The preparation of the polymer hydrogel skeleton composite liquid is the same as that in Example 1.

[0088] The preparation of the polymer active group donor composite solution was similar to that of Example 1 except that polyvinyl alcohol was replaced by gelatin. Other conditions were the same as those of Example 1.

[0089] The preparation of the injectable responsive multifunctional hydrogel was carried out in the same manner as in Example 1 except that the polymer active group donor complex solution was replaced with the polymer active group donor complex solution prepared in this example.

[0090] Example 8:

[0091] The preparation of phenylboronic acid-based polymer is the same as in Example 1.

[0092] The preparation of the polymer hydrogel skeleton composite liquid is the same as that in Example 1.

[0093] The preparation of the polymer active group donor composite solution was similar to that of Example 1 except that polyvinyl alcohol was replaced with polysucrose. Other conditions were the same as those of Example 1.

[0094] The preparation of the injectable responsive multifunctional hydrogel was carried out in the same manner as in Example 1 except that the polymer active group donor complex solution was replaced with the polymer active group donor complex solution prepared in this example.

[0095] Example 9:

[0096] The preparation of phenylboronic acid-based polymer is the same as in Example 1.

[0097] The preparation of the polymer hydrogel skeleton composite liquid is the same as that in Example 1.

[0098] The preparation of the polymer active group donor complex solution was similar to that in Example 1 except that oxidized dextran was replaced by oxidized hyaluronic acid. Oxidized hyaluronic acid was purchased from Xi'an Qiyue Biotechnology Co., Ltd.

[0099] The preparation of the injectable responsive multifunctional hydrogel was carried out in the same manner as in Example 1 except that the polymer active group donor complex solution was replaced with the polymer active group donor complex solution prepared in this example.

[0100] Example 10:

[0101] Preparation of phenylboronic acid-based polymers, comprising:

[0102] 3-Fluoro-4-carboxyphenylboronic acid was dissolved in dimethyl sulfoxide (DMSO), an activator was added, and the mixture was stirred at 25°C for 1 hour. The modified raw material was then added and allowed to react for 48 hours. The mixture was dialyzed against deionized water for 12 hours, repeated three times, and dried under vacuum to obtain a phenylboronic acid-based polymer. The ratio of 3-Fluoro-4-carboxyphenylboronic acid to DMSO was 1 g:200 mL. The activator consisted of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, with a mass ratio of 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.

[0103] 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.

[0104] The preparation of the polymer active group donor composite solution is the same as in Example 1.

[0105] The preparation of the injectable responsive multifunctional hydrogel was carried out in the same manner as in Example 1 except that the polymer hydrogel skeleton composite liquid was replaced with the polymer hydrogel skeleton composite liquid prepared in this example.

[0106] Example 11:

[0107] Preparation of phenylboronic acid-based polymers, comprising:

[0108] 3-Fluoro-4-carboxyphenylboronic acid was dissolved in dimethyl sulfoxide (DMSO), an activator was added, and the mixture was stirred at 25°C for 1 hour. The modified raw material was then added and allowed to react for 48 hours. The mixture was dialyzed against deionized water for 12 hours, repeated three times, and dried under vacuum to obtain a phenylboronic acid-based polymer. The ratio of 3-Fluoro-4-carboxyphenylboronic acid to DMSO was 1 g:200 mL. The activator consisted of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, with a mass ratio of 1:0.5. The mass ratio of 3-Fluoro-4-carboxyphenylboronic acid to the activator was 1:5. The modified raw material was gelatin, and the mass ratio of 3-Fluoro-4-carboxyphenylboronic acid to the modified raw material was 1:3.

[0109] 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.

[0110] The preparation of the polymer active group donor composite solution is the same as in Example 1.

[0111] The preparation of the injectable responsive multifunctional hydrogel was carried out in the same manner as in Example 1 except that the polymer hydrogel skeleton composite liquid was replaced with the polymer hydrogel skeleton composite liquid prepared in this example.

[0112] Example 12:

[0113] The preparation of phenylboronic acid-based polymer is the same as in Example 1.

[0114] Preparation of hydroxy polymers, comprising,

[0115] Under a nitrogen atmosphere, dihydromyrcenol and dihydrocarveol were dissolved in tetrahydrofuran, and an initiator was added, stirred, and reacted at 70°C for 8 hours. After completion of the reaction, the mixture was washed three times with deionized water and dried under vacuum to obtain a hydroxy polymer. The ratio of tetrahydrofuran to dihydromyrcenol was 1 mL:0.05 g, and the ratio of tetrahydrofuran to dihydrocarveol was 1 mL:0.05 g. The initiator was azobisisobutyronitrile, and the ratio of tetrahydrofuran to initiator was 1 mL:0.001 g.

[0116] The preparation of the polymer hydrogel skeleton composite liquid is the same as that in Example 1.

[0117] The preparation of the polymer active group donor composite liquid is similar to that of Example 1 except that the polymer hydroxyl donor is changed to a mixture of polyvinyl alcohol and a hydroxyl polymer, and the mass ratio of polyvinyl alcohol to the hydroxyl polymer is changed to 1:1. Other conditions are the same as those of Example 1.

[0118] The preparation of the injectable responsive multifunctional hydrogel was carried out in the same manner as in Example 1 except that the polymer active group donor complex solution was replaced with the polymer active group donor complex solution prepared in this example.

[0119] Example 13:

[0120] The preparation of phenylboronic acid-based polymer is the same as in Example 1.

[0121] The preparation of the hydroxy polymer was carried out under the same conditions as in Example 12 except that the ratio of tetrahydrofuran to dihydromyrcenol was changed to 1 mL:0.01 g.

[0122] The preparation of the polymer hydrogel skeleton composite liquid is the same as that in Example 1.

[0123] The preparation of the polymer active group donor composite liquid is similar to that of Example 1 except that the polymer hydroxyl donor is changed to a mixture of polyvinyl alcohol and a hydroxyl polymer, and the mass ratio of polyvinyl alcohol to the hydroxyl polymer is changed to 1:1. Other conditions are the same as those of Example 1.

[0124] The preparation of the injectable responsive multifunctional hydrogel was carried out in the same manner as in Example 1 except that the polymer active group donor complex solution was replaced with the polymer active group donor complex solution prepared in this example.

[0125] Example 14:

[0126] The preparation of phenylboronic acid-based polymer is the same as in Example 1.

[0127] The preparation of the hydroxy polymer was carried out under the same conditions as in Example 12 except that the ratio of tetrahydrofuran to dihydrocarveol was changed to 1 mL:0.02 g.

[0128] The preparation of the polymer hydrogel skeleton composite liquid is the same as that in Example 1.

[0129] The preparation of the polymer active group donor composite liquid is similar to that of Example 1 except that the polymer hydroxyl donor is changed to a mixture of polyvinyl alcohol and a hydroxyl polymer, and the mass ratio of polyvinyl alcohol to the hydroxyl polymer is changed to 1:1. Other conditions are the same as those of Example 1.

[0130] The preparation of the injectable responsive multifunctional hydrogel was carried out in the same manner as in Example 1 except that the polymer active group donor complex solution was replaced with the polymer active group donor complex solution prepared in this example.

[0131] Example 15:

[0132] The preparation of phenylboronic acid-based polymer is the same as in Example 1.

[0133] Preparation of hydroxy polymers, comprising,

[0134] Under a nitrogen atmosphere, dihydromyrcenol, dihydrocarveol, and dimethyl (4-hydroxy-2-butenyl) maleate were dissolved in tetrahydrofuran, and the initiator was added and stirred. The mixture was reacted at 70°C for 8 hours. After completion of the reaction, the mixture was washed three times with deionized water and dried under vacuum to obtain a hydroxy polymer. The ratio of tetrahydrofuran to dihydromyrcenol was 1 mL:0.05 g; the ratio of tetrahydrofuran to dihydrocarveol was 1 mL:0.05 g; and the ratio of tetrahydrofuran to dimethyl (4-hydroxy-2-butenyl) maleate was 1 mL:0.02 g. The initiator was azobisisobutyronitrile, and the ratio of tetrahydrofuran to initiator was 1 mL:0.001 g.

[0135] The preparation of the polymer hydrogel skeleton composite liquid is the same as that in Example 1.

[0136] The preparation of the polymer active group donor composite liquid is similar to that of Example 1 except that the polymer hydroxyl donor is changed to a mixture of polyvinyl alcohol and a hydroxyl polymer, and the mass ratio of polyvinyl alcohol to the hydroxyl polymer is changed to 1:1. Other conditions are the same as those of Example 1.

[0137] The preparation of the injectable responsive multifunctional hydrogel was carried out in the same manner as in Example 1 except that the polymer active group donor complex solution was replaced with the polymer active group donor complex solution prepared in this example.

[0138] Example 16:

[0139] The preparation of phenylboronic acid-based polymer is the same as in Example 1.

[0140] The preparation of the hydroxy polymer was performed under the same conditions as in Example 15 except that the ratio of tetrahydrofuran to dimethyl (4-hydroxy-2-butenyl) maleate was changed to 1 mL:0.01 g.

[0141] The preparation of the polymer hydrogel skeleton composite liquid is the same as that in Example 1.

[0142] The preparation of the polymer active group donor composite liquid is similar to that of Example 1 except that the polymer hydroxyl donor is changed to a mixture of polyvinyl alcohol and a hydroxyl polymer, and the mass ratio of polyvinyl alcohol to the hydroxyl polymer is changed to 1:1. Other conditions are the same as those of Example 1.

[0143] The preparation of the injectable responsive multifunctional hydrogel was carried out in the same manner as in Example 1 except that the polymer active group donor complex solution was replaced with the polymer active group donor complex solution prepared in this example.

[0144] Comparative Example 1:

[0145] The preparation of phenylboronic acid-based polymer is the same as in Example 1.

[0146] The preparation of the hydroxy polymer was carried out under the same conditions as in Example 12 except that dihydrocarveol was not used.

[0147] The preparation of the polymer hydrogel skeleton composite liquid is the same as that in Example 1.

[0148] The preparation of the polymer active group donor composite liquid is similar to that of Example 1 except that the polymer hydroxyl donor is changed to a mixture of polyvinyl alcohol and a hydroxyl polymer, and the mass ratio of polyvinyl alcohol to the hydroxyl polymer is changed to 1:1. Other conditions are the same as those of Example 1.

[0149] The preparation of the injectable responsive multifunctional hydrogel was carried out in the same manner as in Example 1 except that the polymer active group donor complex solution was replaced with the polymer active group donor complex solution prepared in this example.

[0150] Comparative Example 2:

[0151] The preparation of phenylboronic acid-based polymer is the same as in Example 1.

[0152] The preparation of the hydroxy polymer was carried out under the same conditions as in Example 12 except that dihydromyrcenol was not used.

[0153] The preparation of the polymer hydrogel skeleton composite liquid is the same as that in Example 1.

[0154] The preparation of the polymer active group donor composite liquid is similar to that of Example 1 except that the polymer hydroxyl donor is changed to a mixture of polyvinyl alcohol and a hydroxyl polymer, and the mass ratio of polyvinyl alcohol to the hydroxyl polymer is changed to 1:1. Other conditions are the same as those of Example 1.

[0155] The preparation of the injectable responsive multifunctional hydrogel was carried out in the same manner as in Example 1 except that the polymer active group donor complex solution was replaced with the polymer active group donor complex solution prepared in this example.

[0156] Comparative Example 3:

[0157] The preparation of phenylboronic acid-based polymer is the same as in Example 1.

[0158] The preparation of the hydroxy polymer was carried out under the same conditions as in Example 15 except that dihydromyrcenol and dihydrocarveol were not used.

[0159] The preparation of the polymer hydrogel skeleton composite liquid is the same as that in Example 1.

[0160] The preparation of the polymer active group donor composite liquid is similar to that of Example 1 except that the polymer hydroxyl donor is changed to a mixture of polyvinyl alcohol and a hydroxyl polymer, and the mass ratio of polyvinyl alcohol to the hydroxyl polymer is changed to 1:1. Other conditions are the same as those of Example 1.

[0161] The preparation of the injectable responsive multifunctional hydrogel was carried out in the same manner as in Example 1 except that the polymer active group donor complex solution was replaced with the polymer active group donor complex solution prepared in this example.

[0162] Experimental example:

[0163] 1. Cytotoxicity test

[0164] The cytotoxicity of the injectable responsive multifunctional hydrogel prepared in Example 1-16 and Comparative Example 1-3 was characterized by the MTT experiment. The specific steps are as follows: the injectable responsive multifunctional hydrogel prepared in Example 1-16 and Comparative Example 1-3 was sterilized by UV and then added to 8 mL of DMEM culture medium. The mixture was immersed at 37°C for 24 hours, and the supernatant was collected after centrifugation. The supernatant was diluted with DMEM culture medium to a final concentration of 5 mg / mL to obtain the supernatant after each sample treatment. A control group and 20 experimental groups were set up. In the control group, 100 μL of DMEM culture medium was added; in the 20 experimental groups, 100 μL of the supernatant of Example 1-16 and Comparative Example 1-3 was added in turn. 100 μL of cells with a density of 1×10 5A rat nucleus pulposus cell suspension (cells / mL) was incubated at 37°C for 24 hours. The liquid was discarded, and 50 μL of MTT was added for 2 hours. The liquid was discarded, and 100 μL of isopropanol was added. The absorbance at 570 nm was measured. Cell viability (%) = absorbance of the experimental group / absorbance of the control group × 100%.

[0165] Figure 1 is the cytotoxicity of the injectable responsive multifunctional hydrogel, S1 to S16 correspond to the cytotoxicity of the injectable responsive multifunctional hydrogel prepared in Examples 1-16, and S17 to S19 correspond to the cytotoxicity of the injectable responsive multifunctional hydrogel prepared in Examples 1-3. Figure 1 As shown, the cell viability of rat nucleus pulposus cells treated with the injectable, responsive, multifunctional hydrogels prepared in Examples 1-16 and Comparative Examples 1-3 was above 88%, exceeding the minimum value of 70% specified in the national standard "GB / T 16886.5-2017 Biological Evaluation of Medical Devices - Part 5: In Vitro Cytotoxicity Test." This indicates that the injectable, responsive, multifunctional hydrogels prepared in Examples 1-16 and Comparative Examples 1-3 have no potential cytotoxicity.

[0166] 2. Drug release performance

[0167] The specific steps for testing the drug release performance of the injectable responsive multifunctional hydrogels prepared in Example 1, Examples 12-16, and Comparative Examples 1-3 are as follows: PLGA-PEG nanoparticles co-loaded with metformin and silymarin were used as model drugs and compounded with the injectable responsive multifunctional hydrogel. The mass ratio of metformin and silymarin co-loaded with PLGA-PEG nanoparticles to the injectable responsive multifunctional hydrogel was 1:10 to obtain the drug-loaded hydrogel. The PLGA-PEG nanoparticles co-loaded with metformin and silymarin were purchased from Xi'an Qiyue Biotechnology Co., Ltd. A control group and an experimental group were set up. 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. Then, 5 mL of the drug-loaded hydrogel was added and incubated at 37°C for 48 hours. 400 μL of the release medium was removed, and the release of metformin and silymarin co-loaded PLGA-PEG nanoparticles was measured by HPLC. The drug release rate was calculated as follows: Drug release rate (%) = Released amount / Initial amount × 100%. The drug release properties of the injectable responsive multifunctional hydrogels prepared in Example 1, Examples 12-16, and Comparative Examples 1-3 are shown in Table 1.

[0168] 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 the cells were incubated at 37° C. 400 μL of the release medium was removed every 24 h. 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. The sustained drug release time was recorded.

[0169] The injectable responsive multifunctional hydrogel prepared in Example 1 of the present invention exhibited sustained drug release at pH 7.4 for over 2 weeks, and a 48-hour drug release rate of 11.6%, significantly lower than the drug release rate of the injectable responsive multifunctional hydrogel prepared in Example 1 at pH 5.5. This demonstrates that the injectable responsive multifunctional hydrogel prepared in the present invention exhibits environmentally responsive release properties, releasing toxin A under environmental stimuli, and its release behavior exhibits responsive conditional characteristics.

[0170] Table 1 Drug release rate (%)

[0171]

[0172] As shown in Table 1, the drug release rates of the injectable responsive multifunctional hydrogels prepared in Examples 12-14 of the present invention were higher than those in Example 1 because Examples 12-14 additionally used a hydroxyl polymer composed of dihydromyrcenol and dihydrocarveol units in the preparation of the injectable responsive multifunctional hydrogels. The drug release rate of the injectable responsive multifunctional hydrogel prepared in Example 12 was higher than those in Examples 13 and 14 because the amounts of dihydromyrcenol and dihydrocarveol used in the preparation of the hydroxyl polymers were different. The drug release rates of the injectable responsive multifunctional hydrogels prepared in Examples 12-14 were higher than those in Comparative Examples 1 and 2 because Comparative Example 1 used only dihydromyrcenol alone, while Comparative Example 2 used only dihydrocarveol alone in the preparation of the hydroxyl polymers. This indicates that, compared to using dihydromyrcenol and dihydrocarveol alone, using appropriate amounts of dihydromyrcenol and dihydrocarveol to prepare the hydroxyl polymers and then using the hydroxyl polymers to prepare the injectable responsive multifunctional hydrogels can improve the drug release properties of the injectable responsive multifunctional hydrogels.

[0173] The drug release 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 (4-hydroxy-2-butenyl) dimethyl maleate; the drug release rate of the injectable responsive multifunctional hydrogel prepared in Example 15 is higher than that of Example 16 because the amount of (4-hydroxy-2-butenyl) dimethyl maleate used in the preparation of the hydroxyl polymer is different; the drug release 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 (4-hydroxy-2-butenyl) dimethyl maleate alone, without using dihydromyrcenol and dihydrocarveol. This shows that the use of a hydroxyl polymer composed of dihydromyrcenol, dihydrocarveol, and (4-hydroxy-2-butenyl) dimethyl maleate units to prepare the injectable responsive multifunctional hydrogel helps to further improve the drug release performance of the injectable responsive multifunctional hydrogel.

[0174] 3. Antibacterial properties

[0175] The injectable, responsive, multifunctional hydrogels prepared in Example 1, Examples 12-16-5, and Comparative Examples 1-3 were sterilized and then evaluated for their antibacterial activity against Staphylococcus aureus. Specifically, 25 μL of Staphylococcus aureus culture was dripped into the center of a Petri dish containing LB medium. A control group and eight experimental groups were set up. The control group was covered with sterile film; the eight experimental groups were covered with the injectable, responsive, multifunctional hydrogels prepared in Examples 1-5 and Comparative Examples 1-3, respectively. After 1 hour of contact for each group, the sterile film covered in the control group and the injectable responsive multifunctional hydrogels prepared in Examples 1-5 and Comparative Examples 1-3 covered in the 8 experimental groups were collected, and 5 mL of phosphate buffer was added to each of them. The mixture was vortexed for 2 minutes, and 10 μL of the vortexed liquid was added to 4 mL of LB medium. The mixture was cultured at 37°C for 24 hours, and 200 μL of the bacterial suspension was taken to measure the OD value at 600 nm. The antibacterial rate (%) = (OD value of the control group - OD value of the experimental group) / OD value of the control group.

[0176] Table 2 Antibacterial rate (%)

[0177]

[0178] As shown in Table 2, the antibacterial rates of the injectable responsive multifunctional hydrogels prepared in Examples 12-14 of the present invention were higher than those in Example 1 because Examples 12-14 additionally used a hydroxyl polymer composed of dihydromyrcenol and dihydrocarveol units in the preparation of the injectable responsive multifunctional hydrogels. The antibacterial rate of the injectable responsive multifunctional hydrogel prepared in Example 12 was higher than those in Examples 13 and 14 because the amounts of dihydromyrcenol and dihydrocarveol used in the preparation of the hydroxyl polymers were different. The antibacterial rates of the injectable responsive multifunctional hydrogels prepared in Examples 12-14 were higher than those in Comparative Examples 1 and 2 because Comparative Example 1 used only dihydromyrcenol and Comparative Example 2 used only dihydrocarveol in the preparation of the hydroxyl polymers. This indicates that the antibacterial properties of the injectable responsive multifunctional hydrogels can be improved by using appropriate amounts of dihydromyrcenol and dihydrocarveol to prepare the hydroxyl polymers, compared to using dihydromyrcenol and dihydrocarveol alone.

[0179] 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 (4-hydroxy-2-butenyl) dimethyl maleate; the antibacterial rate of the injectable responsive multifunctional hydrogel prepared in Example 15 is higher than that of Example 16 because the amount of (4-hydroxy-2-butenyl) dimethyl maleate used in the preparation of the hydroxyl polymer 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 (4-hydroxy-2-butenyl) dimethyl maleate alone without using dihydromyrcenol and dihydrocarveol. This shows that the use of a hydroxyl polymer composed of dihydromyrcenol, dihydrocarveol and (4-hydroxy-2-butenyl) dimethyl maleate units to prepare the injectable responsive multifunctional hydrogel helps to further improve the antibacterial properties of the injectable responsive multifunctional hydrogel.

[0180] The conventional operations in the operating steps of the present invention are well known to those skilled in the art and will not be described in detail here.

[0181] The embodiments described above provide a detailed description of the technical solutions of the present invention. It should be understood that the above are only specific embodiments of the present invention and are not intended to limit the present invention. Any changes and modifications made within the scope of the principles of the present invention should be included in the scope of protection 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-based 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 is a mixture of polyvinyl alcohol and a hydroxyl polymer, and the polymer aldehyde donor is oxidized glucose. polysaccharide; the volume ratio of the polymer hydrogel skeleton composite liquid and the polymer active group donor composite liquid is 1:1-2; the phenylboronic acid-based polymer is a phenylboronic acid-grafted ε-polylysine polymer, and the polymer structural skeleton is 2-hydroxyethyl cellulose; 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; the monomers are dihydromyrcenol and dihydrocarveol, or dihydromyrcenol, dihydrocarveol and (4-hydroxy-2-butenyl) dimethyl maleate.

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, a modified raw material is reacted with 3-fluoro-4-carboxylphenylboronic acid to obtain the phenylboronic acid-based polymer; the modified raw material is ε-polylysine.

3. The method for preparing the injectable responsive multifunctional hydrogel according to claim 1, characterized in that: The initiator is azobisisobutyronitrile.

4. The injectable responsive multifunctional hydrogel prepared by the preparation method according to any one of claims 1 to 3.

5. Use of the injectable responsive multifunctional hydrogel according to claim 4 in the preparation of a drug for use against Staphylococcus aureus.

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