CN113599507B - Preparation method of glucose-triggered active oxygen response injection type composite hydrogel - Google Patents

Preparation method of glucose-triggered active oxygen response injection type composite hydrogel Download PDF

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CN113599507B
CN113599507B CN202110916825.8A CN202110916825A CN113599507B CN 113599507 B CN113599507 B CN 113599507B CN 202110916825 A CN202110916825 A CN 202110916825A CN 113599507 B CN113599507 B CN 113599507B
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CN113599507A (en
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张建军
赵伟
周雨晨
刘捷
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Beijing University of Chemical Technology
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Abstract

The invention provides a preparation method of glucose-triggered active oxygen response injection type composite hydrogel. The preparation method comprises the following steps: preparing hyaluronic acid-aminophenylboronic acid derivative HA-g-PBA with phenylboronic acid in the main chain and glucose oxidase derivative GOx-AC with modified acrylic group; the thiol of mercaptophenylboronic acid and the acrylic ester group of GOx-AC are utilized to carry out Michael addition reaction, so as to prepare the glucose oxidase derivative GOx-g-PBA with phenylboronic acid; the polyvinyl alcohol derivative PVA-g-GOx modified with glucose oxidase is prepared by forming a borate dynamic covalent bond through the hydroxyl of GOx-g-PBA and the hydroxyl of polyvinyl alcohol; and mixing HA-g-PBA and PVA-g-GOx according to a preset proportion, and then forming a borate dynamic covalent bond to prepare the glucose-triggered active oxygen response injection type composite hydrogel. The preparation method is simple and has good application prospect, and the degradation period can be regulated and controlled through glucose and active oxygen dual response.

Description

Preparation method of glucose-triggered active oxygen response injection type composite hydrogel
Technical Field
The invention relates to a preparation method of injection type hydrogel applied to the biomedical field, in particular to a preparation method of glucose-triggered active oxygen response injection type composite hydrogel.
Background
Hydrogels are high molecular polymers with a three-dimensional network structure, whose cross-linked network structure prevents penetration, and these properties make them good drug carriers for surgical implants, local needle injections, and systemic delivery by intravenous injection. Most hydrogels currently studied and used utilize changes in the internal environment of body tissue (temperature, pH, ionic strength, etc.) to initiate the formation of a hydrogel network by physical crosslinking. The temperature-sensitive alginic acid-g-poly (N-isopropyl acrylamide, grafted poly (N-isopropyl acrylamide) and poly (methacrylic acid) are added to cyclic oligosaccharide beta-cyclodextrin to synthesize hydrogel with pH response and temperature response, and can simultaneously release metronidazole, ofloxacin and ion strength sensitized alginate in colon (pH=7.4), so that the synthesis of high molecular materials lays a good foundation for research and application of injectable hydrogel.
Compared with the physical crosslinked gel, the chemically crosslinked gel has the unique advantages of high crosslinking speed, excellent stability and mechanical properties. However, most chemical gel crosslinking processes require additional initiator, which is often highly toxic or requires specific initiation conditions and is not suitable for in situ use in the living body. The hydrogel based on phenylboronic acid forms a borate covalent bond through boric acid and polyvinyl alcohol 1, 2-diol, has the characteristics of both physical crosslinking and chemical crosslinking of the hydrogel, and has the sensitive glucose and active oxygen dual response characteristics. Since small molecule sugars compete with the glycol structure of the hydrogel and form reversible borate bonds with boric acid, the borate bonds of the hydrogel can be broken by competing with glucose present in the tissue environment of the lesion or by oxidation of reactive oxygen species.
Glucose oxidase is an aerobic dehydrogenase which is widely existed in animals and plants, and can specifically catalyze and oxidize beta-D-glucose to generate gluconic acid and hydrogen peroxide in the presence of oxygen. However, similar to most protein drugs, natural GOx has the disadvantages of poor stability, short half-life in vivo, systemic toxicity and the like, and is difficult to be directly used for treating diseases in high-sugar environments. Therefore, the hydrogel material established based on phenylboronic acid and glucose oxidase together has sensitive glucose-induced active oxygen response degradation characteristics, and the hydrogel material has wide clinical application prospect in local sustained release administration of focus tissues with high blood sugar concentration.
Disclosure of Invention
The invention aims to provide a preparation method of a glucose-triggered active oxygen response injection type glucose-excited active oxygen response composite hydrogel, which is based on the reaction of hyaluronic acid-amino phenylboronic acid derivative HA-g-PBA grafted with phenylboronic acid and polyvinyl alcohol derivative PVA-g-GOx modified with glucose oxidase to construct a novel injection type hydrogel material with strong plasticity, strong adhesiveness and controllable biodegradation time.
In particular, the invention provides a preparation method of glucose-triggered active oxygen response injection type composite hydrogel, which comprises the following steps:
Preparing hyaluronic acid-aminophenylboronic acid derivative HA-g-PBA with phenylboronic acid in the main chain and glucose oxidase derivative GOx-AC with modified acrylic group;
the thiol of mercaptophenylboronic acid and the acrylic ester group of GOx-AC are utilized to carry out Michael addition reaction, so as to prepare the glucose oxidase derivative GOx-g-PBA with phenylboronic acid;
the polyvinyl alcohol derivative PVA-g-GOx modified with glucose oxidase is prepared by forming a borate dynamic covalent bond through the hydroxyl of GOx-g-PBA and the hydroxyl of polyvinyl alcohol;
And mixing HA-g-PBA and PVA-g-GOx according to a preset proportion, and then forming a borate dynamic covalent bond to prepare the glucose-triggered active oxygen response injection type composite hydrogel.
Optionally, in the preparation of hyaluronic acid-aminophenylboronic acid derivative HA-g-PBA with phenylboronic acid in the main chain and glucose oxidase derivative GOx-AC modified by acrylic group, the preparation method of GOx-AC comprises the following steps:
dissolving glucose oxidase in a first buffer solution to be configured into a first solution with the mass percent of the glucose oxidase being 0.5% -1.5%;
Dissolving N-acryloyloxy succinimide in dimethyl sulfoxide (DMSO) to obtain a second solution, wherein the molar ratio of the N-acryloyloxy succinimide to the glucose oxidase is 15-25:1;
and (3) applying the second solution to the first solution, stirring at room temperature, and dialyzing and purifying to obtain the GOx-AC.
Optionally, the thiol group of mercaptophenylboronic acid is subjected to Michael addition reaction with an acrylate group of GOx-AC to prepare the glucose oxidase derivative GOx-g-PBA with phenylboronic acid, which comprises the following steps:
Dissolving 4-mercaptophenylboronic acid in dimethyl sulfoxide to obtain a third solution, wherein the molar ratio of the 4-mercaptophenylboronic acid to the glucose oxidase is 15-25:1;
And (3) applying the third solution to the GOx-AC, stirring at 30-37 ℃, and purifying by dialysis to obtain the GOx-g-PBA.
Optionally, the polyvinyl alcohol derivative PVA-g-GOx modified with glucose oxidase is prepared by forming a borate dynamic covalent bond by hydroxyl groups of GOx-g-PBA and hydroxyl groups of polyvinyl alcohol, and comprises the following steps:
Dissolving polyvinyl alcohol in a second buffer solution to prepare a fourth solution with the mass percent of the polyvinyl alcohol being 3-10%;
and applying the GOx-g-PBA to the fourth solution, stirring at room temperature, and dialyzing and purifying to obtain the PVA-g-GOx.
Optionally, the preparation method comprises the steps of mixing HA-g-PBA and PVA-g-GOx according to a preset proportion, and then forming a borate dynamic covalent bond to prepare the glucose-triggered active oxygen response composite hydrogel, wherein the preparation method comprises the following steps:
dissolving the HA-g-PBA and the PVA-g-GOx with the mass ratio of 0.5-2:1 in a third buffer solution to prepare a fifth solution;
and (3) mixing and injecting the fifth solution according to the volume ratio of 1:1 by a two-component injector to obtain a solution, and reacting and crosslinking the solution at the temperature of 30-37 ℃ to generate the active oxygen response injection type composite hydrogel.
Optionally, the first buffer, the second buffer, and the third buffer are all selected to be phosphate solutions.
Optionally, the preparation method for obtaining hyaluronic acid-aminophenylboronic acid derivative HA-g-PBA with phenylboronic acid in the main chain and acrylic acid group modified glucose oxidase derivative GOx-AC comprises the following steps:
preparing 0.2-2% of hyaluronic acid aqueous solution and 0.5-1.5% of dimethyl sulfoxide solution of 4-aminophenylboric acid;
Mixing an aqueous solution of hyaluronic acid, a dimethyl sulfoxide solution of 4-aminophenylboric acid and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride, and adjusting the pH value of the mixed solution to 4.5-5 to obtain a sixth solution;
And adding N-hydroxysuccinimide into the sixth solution, and freeze-drying and preserving after reaction dialysis purification to obtain the HA-g-PBA.
Optionally, the mass ratio of the hyaluronic acid aqueous solution, the dimethyl sulfoxide solution of the 4-aminophenylboronic acid, the 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride and the N-hydroxysuccinimide is 1:0.3-0.5:0.5-1.5:0.5-1.5.
Optionally, the hyaluronic acid has a molecular weight of 40-200KDA.
Optionally, in the step of obtaining GOx-AC after dialysis purification by applying the second solution to the first solution and stirring at room temperature, a dialysis bag with a molecular weight cutoff of 3500Da is used for dialysis purification;
Optionally, in the step of applying the GOx-g-PBA to the fourth solution and stirring at room temperature to obtain PVA-g-GOx after dialysis purification, a dialysis bag with a molecular weight cut-off of 200Da is used for dialysis purification;
optionally, adding N-hydroxysuccinimide into the sixth solution, performing dialysis purification by reaction, and freeze-drying and preserving to obtain the HA-g-PBA, wherein a dialysis bag with a molecular weight cut-off of 8-14kDa is used for dialysis purification.
According to the scheme of the invention, because HA in the main body material HA-g-PBA for generating the glucose-triggered active oxygen response injection type composite hydrogel is viscous polysaccharide widely distributed on various parts of a human body, the main body material HA-g-PBA HAs excellent biocompatibility and biodegradability and is widely applied to the fields of cosmetics, health care and medicines, and the polyvinyl alcohol in the other main body material PVA-g-GOx is medical grade polyvinyl alcohol, is an extremely safe high molecular organic matter, is nontoxic to the human body, HAs no side effect, HAs good biocompatibility, and particularly HAs wide application in medical treatment such as the aspects of ophthalmic, wound dressing and artificial joint of aqueous gel thereof. Therefore, the glucose-triggered active oxygen response injection type composite hydrogel finally prepared has the advantages. In addition, the preparation method is simple, has good application prospect, and can regulate and control the degradation period through glucose and active oxygen dual response.
And the borate dynamic covalent bond formed by the hydroxyl of GOx-g-PBA and the hydroxyl of polyvinyl alcohol has environmental friendliness and reaction speed controllability, and lays a good foundation for the application of the active oxygen response injection type composite hydrogel in the in-vivo injection type biomedical hydrogel. And compared with the traditional physical association injection type gel, the dynamic covalent crosslinking composite hydrogel of the phenylboronic acid sodium hyaluronate derivative/the polyvinyl alcohol derivative grafted with glucose has excellent stability and mechanical property.
The above, as well as additional objectives, advantages, and features of the present invention will become apparent to those skilled in the art from the following detailed description of a specific embodiment of the present invention when read in conjunction with the accompanying drawings.
Drawings
Some specific embodiments of the invention will be described in detail hereinafter by way of example and not by way of limitation with reference to the accompanying drawings. The same reference numbers will be used throughout the drawings to refer to the same or like parts or portions. It will be appreciated by those skilled in the art that the drawings are not necessarily drawn to scale. In the accompanying drawings:
FIG. 1 shows a schematic flow chart of a method for preparing a glucose-triggered reactive oxygen species injection type composite hydrogel in accordance with one embodiment of the present invention;
FIG. 2 shows a schematic flow chart of a preparation method of hyaluronic acid-aminophenylboronic acid derivative HA-g-PBA having a phenylboronic acid in its main chain according to one embodiment of the present invention;
FIG. 3 shows a schematic flow chart of a method for preparing an acrylic-based modified glucose oxidase derivative GOx-AC according to one embodiment of the invention;
FIG. 4 shows a schematic flow chart of a process for the preparation of GOx-g-PBA with phenylboronic acid glucose oxidase derivatives according to one embodiment of the present invention;
FIG. 5 shows a schematic flow chart of a method for preparing a polyvinyl alcohol derivative PVA-g-GOx modified with glucose oxidase according to an embodiment of the invention;
FIG. 6 shows a schematic flow chart of a method of preparing a glucose-triggered reactive oxygen species responsive composite hydrogel in accordance with one embodiment of the invention;
FIG. 7 shows a reaction scheme for preparing the obtained HA-g-PBA according to one embodiment of the present invention;
FIG. 8 shows a reaction scheme for preparing PVA-g-GOx according to one embodiment of the present invention;
FIG. 9 shows a schematic mechanism diagram of the preparation of glucose-triggered forming active oxygen responsive injectable composite hydrogels by HA-g-PBA and PVA-g-GOx according to one embodiment of the present invention;
FIG. 10 shows a mass change plot of active oxygen responsive injection type composite hydrogel degradation according to an embodiment of the present invention;
FIG. 11 is a graph showing the change in pH of a solution during degradation of an active oxygen responsive injectable composite hydrogel in accordance with an embodiment of the present invention;
FIG. 12 is a graph showing the characterization of the mechanical properties of an active oxygen responsive injectable composite hydrogel according to an embodiment one of the present invention;
FIG. 13 is a scanning electron microscope image of an active oxygen responsive injection type composite hydrogel according to an embodiment of the present invention;
fig. 14 shows fourier infrared spectra of HA-g-PBA and HA according to the first embodiment of the present invention.
FIG. 15 shows Fourier infrared spectra of PVA-g-GO x and GO x -AC according to example one of the present invention.
Detailed Description
Other advantages and effects of the present application will become apparent to those skilled in the art from the following disclosure, which describes the embodiments of the present application with reference to specific examples. It will be apparent that the described embodiments are only some, but not all, embodiments of the application. The application may be practiced or carried out in other embodiments that depart from the specific details, and the details of the present description may be modified or varied from the spirit and scope of the present application. It should be noted that the following embodiments and features in the embodiments may be combined with each other without conflict. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, are intended to be within the scope of the application.
FIG. 1 shows a schematic flow chart of a method for preparing a glucose-triggered reactive oxygen species-responsive composite hydrogel in accordance with one embodiment of the invention. As shown in FIG. 1, the preparation method of the active oxygen response injection type composite hydrogel comprises the following steps:
step S100, preparing hyaluronic acid-aminophenylboronic acid derivative HA-g-PBA with phenylboronic acid in the main chain and glucose oxidase derivative GOx-AC modified by acrylic acid group;
step S200, performing Michael addition reaction on sulfhydryl groups of mercaptophenylboronic acid and acrylate groups of GOx-AC to prepare a glucose oxidase derivative GOx-g-PBA with phenylboronic acid;
Step S300, a borate dynamic covalent bond is formed by hydroxyl groups of GOx-g-PBA and hydroxyl groups of polyvinyl alcohol, so that a polyvinyl alcohol derivative PVA-g-GOx modified with glucose oxidase is prepared;
Step S400, mixing HA-g-PBA and PVA-g-GOx according to a preset proportion, and then forming a borate dynamic covalent bond to prepare the glucose-triggered active oxygen response injection type composite hydrogel.
According to the scheme of the invention, because HA in the main body material HA-g-PBA for generating the glucose-triggered active oxygen response injection type composite hydrogel is viscous polysaccharide widely distributed on various parts of a human body, the main body material HA-g-PBA HAs excellent biocompatibility and biodegradability and is widely applied to the fields of cosmetics, health care and medicines, and the polyvinyl alcohol in the other main body material PVA-g-GOx is medical grade polyvinyl alcohol, is an extremely safe high molecular organic matter, is nontoxic to the human body, HAs no side effect, HAs good biocompatibility, and particularly HAs wide application in medical treatment such as the aspects of ophthalmic, wound dressing and artificial joint of aqueous gel thereof. Therefore, the glucose-triggered active oxygen response injection type composite hydrogel finally prepared has the advantages. In addition, the preparation method is simple, has good application prospect, and can regulate and control the degradation period through glucose and active oxygen dual response.
And the borate dynamic covalent bond formed by the hydroxyl of GOx-g-PBA and the hydroxyl of polyvinyl alcohol has environmental friendliness and reaction speed controllability, and lays a good foundation for the application of the active oxygen response injection type composite hydrogel in the in-vivo injection type biomedical hydrogel. And compared with the traditional physical association injection type gel, the dynamic covalent crosslinking composite hydrogel of the phenylboronic acid sodium hyaluronate derivative/the polyvinyl alcohol derivative grafted with glucose has excellent stability and mechanical property.
Fig. 2 shows a schematic flow chart of a preparation method of hyaluronic acid-aminophenylboronic acid derivative HA-g-PBA having a phenylboronic acid in a main chain according to an embodiment of the present invention. As shown in fig. 2, the method for preparing hyaluronic acid-aminophenylboronic acid derivative HA-g-PBA having phenylboronic acid in the main chain in this step S100 comprises:
Step S110, preparing 0.2-2% of hyaluronic acid aqueous solution and 0.5-1.5% of dimethyl sulfoxide solution of 4-aminophenylboric acid;
Step S120, mixing an aqueous solution of hyaluronic acid, a dimethyl sulfoxide solution of 4-aminophenylboronic acid and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride, and adjusting the pH value of the mixed solution to 4.5-5 to obtain a sixth solution;
And step S130, adding N-hydroxysuccinimide into the sixth solution, and freeze-drying and preserving after reaction dialysis purification to obtain HA-g-PBA.
In the step S110, the molecular weight of the hyaluronic acid is any of 40-200kDa, for example, 40kDa, 100kDa or 200kDa. The aqueous solution of hyaluronic acid is prepared to have any value of 0.2% -2% by mass, for example, 0.2%, 0.5%, 1% or 2%. The hyaluronic acid aqueous solution has the advantages that the hyaluronic acid aqueous solution has a small mass percentage value, so that the products prepared later cannot be gelled, and the hyaluronic acid aqueous solution has a large mass percentage value, so that the reaction is affected. In this step S120, the pH of the mixed solution is adjusted to 4.5 to 5 by adding sodium hydroxide and hydrochloric acid to the solution. In this step S130, dialysis purification uses a dialysis bag with a molecular weight cut-off of 8-14 kDa.
In the step S120 and the step S130, the mass ratio of the aqueous solution of hyaluronic acid, the solution of 4-aminophenylboronic acid in dimethyl sulfoxide, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide is any one of 1:0.3-0.5:0.5-1.5:0.5-1.5, for example, 1:0.3:0.5:0.5, 1:0.5: 1.5:1.5 or 1:0.4:1:1. In the steps S110 to S130, the condensation reaction is carried out between the amino group of 4-aminophenylboronic acid initiated by 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride and the main chain carboxyl group of HA initiated by N-hydroxysuccinimide, so as to obtain HA-g-PBA.
FIG. 3 shows a schematic flow chart of a method for preparing an acrylic group modified glucose oxidase derivative GOx-AC according to an embodiment of the present invention. As shown in fig. 3, the preparation method for obtaining GOx-AC in this step S100 includes:
step S101, glucose oxidase is dissolved in a first buffer solution to be configured into a first solution with the mass percent of the glucose oxidase being 0.5% -1.5%;
Step S102, dissolving N-acryloyloxy succinimide in dimethyl sulfoxide to obtain a second solution, wherein the molar ratio of the N-acryloyloxy succinimide to the glucose oxidase is 15-25:1;
Step S103, the second solution is applied to the first solution, and stirred at room temperature, and GOx-AC is obtained after dialysis and purification.
In step S103, the stirring time is 4 hours or more and 8 hours or less, and a dialysis bag having a molecular weight cut-off of 3500Da is used for dialysis purification.
In step S101, an excessive mass percentage of glucose oxidase in the first solution affects the reaction with N-acryloyloxy succinimide, and thus, the intended product cannot be produced.
FIG. 4 shows a schematic flow chart of a preparation method of glucose oxidase derivative GOx-g-PBA with phenylboronic acid according to one embodiment of the present invention. As shown in fig. 4, the step S200 includes:
step S210, dissolving 4-mercaptophenylboronic acid in dimethyl sulfoxide to obtain a third solution, wherein the molar ratio of the 4-mercaptophenylboronic acid to the glucose oxidase is 15-25:1;
Step S220, the third solution is applied to GOx-AC, stirred at 30-37 ℃ and purified by dialysis to obtain GOx-g-PBA.
In step S220, the stirring time is 2h or more and 6h or less.
FIG. 5 shows a schematic flow chart of a preparation method of a polyvinyl alcohol derivative PVA-g-GOx modified with glucose oxidase according to an embodiment of the invention. As shown in FIG. 5, the method for preparing the polyvinyl alcohol derivative PVA-g-GOx modified with glucose oxidase in the step S300 comprises the following steps:
step S310, dissolving polyvinyl alcohol in a second buffer solution to prepare a fourth solution with the mass percent of the polyvinyl alcohol being 3% -10%;
In step S320, GOx-g-PBA is applied to the fourth solution, and stirred at room temperature, and PVA-g-GOx is obtained after dialysis and purification.
In the step S320, the stirring time is 12h or more and 24h or less, and a dialysis bag with a molecular weight cut-off of 200Da is used for dialysis purification.
FIG. 6 shows a schematic flow chart of a method of preparing a glucose-triggered reactive oxygen species responsive composite hydrogel in accordance with one embodiment of the invention. As shown in fig. 6, the preparation method of the glucose-triggered reactive oxygen species-responsive composite hydrogel in step S400 includes:
Step S410, HA-g-PBA and PVA-g-GOx with the mass ratio of 0.5-2:1 are dissolved in a third buffer solution to prepare a fifth solution;
Step S420, mixing and injecting the fifth solution according to the volume ratio of 1:1 by a two-component injector to obtain a solution, and reacting and crosslinking the solution at the temperature of 30-37 ℃ to generate the active oxygen response injection type composite hydrogel.
FIG. 7 shows a reaction scheme for preparing the obtained HA-g-PBA according to one embodiment of the present invention. FIG. 8 shows a reaction mechanism diagram for preparing PVA-g-GOx according to one embodiment of the present invention. FIG. 9 shows a schematic mechanism diagram of the preparation of glucose-triggered reactive oxygen species-responsive injectable composite hydrogels by HA-g-PBA and PVA-g-GOx according to one embodiment of the present invention. The chemical structure of each product can be seen from fig. 7 to 9.
FIG. 10 shows a mass change graph of active oxygen responsive injection type composite hydrogel degradation according to an embodiment of the present invention. In FIG. 10, 1 represents the HA-g-PBA and PVA-g-GOx mass ratio of 18:10,2 represents the HA-g-PBA/PVA-g-GOx mass ratio of 15:8,3 represents the HA-g-PBA/PVA-g-GOx mass ratio of 18:8, and 4 represents the HA-g-PBA/PVA-g-GOx mass ratio of 15:10. As can be seen from fig. 10, the hydrogel degradation was almost completed in 12 hours and was completely degraded in 24 hours.
FIG. 11 shows a graph of the change in pH of a solution during degradation of an active oxygen responsive injectable composite hydrogel in accordance with an embodiment of the invention. In FIG. 11, 1 represents the HA-g-PBA and PVA-g-GOx mass ratio of 18:10,2 represents the HA-g-PBA and PVA-g-GOx mass ratio of 15:8,3 represents the HA-g-PBA and PVA-g-GOx mass ratio of 18:8, and 4 represents the HA-g-PBA and PVA-g-GOx mass ratio of 15:10. From fig. 11, it can be seen that PVA successfully bridges glucose oxidase with a mass ratio of 18:10 is most pronounced.
FIG. 12 is a graph showing the mechanical properties of an active oxygen responsive injectable composite hydrogel according to an embodiment of the invention, wherein E' represents the storage modulus and E "represents the loss modulus. In FIG. 12, the HA-g-PBA and PVA-g-GOx mass ratio is 18:10. as can be seen from fig. 12, the active oxygen responsive injection type composite hydrogel prepared by the above method has good mechanical properties.
The following is a detailed description of specific embodiments:
Example 1:
A preparation method of glucose-triggered active oxygen response injection type composite hydrogel comprises the following steps:
1) Preparation of hyaluronic acid-aminophenylboronic acid derivatives (HA-g-PBA)
2G of HA (molecular weight 40 kDa) was dissolved in 800mL of ultrapure water, then 690mg of 4-aminobenzene boric acid was added to dissolve in 100mL of DMSO solution, then 1530mg of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride was added, and the pH of the solution was adjusted to 4.5-5.0 by hydrochloric acid, and then 920mg of N-hydroxysuccinimide was added to react for 6 hours; the product HA-g-PBA solution is subjected to dialysis purification (using a dialysis bag with a molecular weight cut-off of 8-10 kDa) and then freeze-dried for storage.
2) Preparation of polyvinyl alcohol derivatives modified with glucose oxidase
Dissolving 50mg of glucose oxidase in 5mL of buffer solution (PBS, pH=7.4-8.0, 150 mM), dripping 10 mu L N-acryloyloxysuccinimide (w/v) 10% DMSO solution into the glucose oxidase solution, stirring at room temperature for 4 hours, and dialyzing and purifying the product GOx-AC at 4 ℃ (molecular weight cut-off 3500 dialysis bag); then 20 mu L of 1% (w/v) DMSO solution of 4-mercaptophenylboronic acid is dripped into the solution purified in the previous step, the mixture is stirred for 2 hours at 37 ℃, and the product (GOx-g-MPBA) is dialyzed and purified at 4 ℃ (molecular weight cut-off 3500 dialysis bag); 10mL of 10% (w/v) buffer solution of polyvinyl alcohol (PBS, pH=7.4-8.0, 150 mM) is taken, diluted by 2 times, the solution after dialysis purification is dripped into the polyvinyl alcohol solution, stirred for 12 hours at room temperature, and the product PVA-g-GOx is obtained after dialysis purification (dialysis bag with molecular weight cut-off of 200 kDa).
3) Preparation of hydrogels
Dissolving 18mg of HA-g-PBA prepared in step 1) in 100. Mu.L of 150mM buffer, wherein the buffer is selected to be Phosphate Buffered Saline (PBS), and the pH of the PBS buffer is 7.4-8; 10mg of PVA-g-GOx prepared in step 2) is dissolved in 100. Mu.L of 150mM PBS buffer, and the pH of the PBS buffer is 7.4-8; after the two phases are completely dissolved, mixing the two phases in equal volume to obtain a hydrogel precursor solution, and then crosslinking and solidifying the hydrogel precursor solution in a baking oven at 37 ℃ for 40-60min to form the glucose-triggered active oxygen response injection type composite hydrogel.
Fig. 13 shows a scanning electron microscope image of an active oxygen responsive injection type composite hydrogel according to the first embodiment of the present invention. As shown in fig. 13, the hydrogel cross-section SEM presents clear pore channels, is in a three-dimensional network structure, and shows that the structure is good, and is a good drug carrier.
Fig. 14 shows fourier infrared spectra of HA-g-PBA and HA according to the first embodiment of the present invention. As shown in FIG. 14, the successful condensation of the amino phenylboronic acid on the HA backbone can be seen by the appearance of a benzene ring peak around 1500-1600cm -1. FIG. 15 shows Fourier infrared spectra of PVA-g-GOx and GOx-AC according to an embodiment of the present invention. As shown in FIG. 15, benzene ring peaks also appear near 1500-1600cm -1, while the enhancement of O-H peaks at 3200-3650cm -1 indicates that mercaptophenylboronic acid-grafted glucose oxidase successfully forms a borate dynamic covalent bond with polyvinyl alcohol.
Example 2:
the preparation method of the glucose-triggered active oxygen response injection type composite hydrogel comprises the following steps:
1) Preparation of hyaluronic acid-aminophenylboronic acid derivatives (HA-g-PBA)
2G of HA (molecular weight 20 kDa) was dissolved in 800mL of ultrapure water, then 1380mg of 4-aminobenzene boric acid was added to dissolve in 100mL of DMSO solution, then 3060mg of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride was added, the pH of the solution was adjusted to 4.5-5.0 by hydrochloric acid, and then 1920mg of N-hydroxysuccinimide was added to react for 6 hours; the product HA-APBA solution is subjected to dialysis purification (a dialysis bag with a molecular weight cut-off of 8-10 kDa) and then is freeze-dried for storage.
2) Preparation of polyvinyl alcohol derivatives modified with glucose oxidase
Dissolving 50mg of glucose oxidase in 5mL of buffer solution (PBS, pH=7.4-8.0, 150 mM), dripping 10 mu L N-acryloyloxysuccinimide (w/v) 10% DMSO solution into the glucose oxidase solution, stirring at room temperature for 4 hours, and dialyzing and purifying the product GOx-AC at 4 ℃ (molecular weight cut-off 3500 dialysis bag); then 20 mu L of 1% (w/v) DMSO solution of 4-mercaptophenylboronic acid is dripped into the solution purified in the previous step, the mixture is stirred for 2 hours at 37 ℃, and the product (GOx-g-MPBA) is dialyzed and purified at 4 ℃ (molecular weight cut-off 3500 dialysis bag); 10mL of 10% (w/v) buffer solution of polyvinyl alcohol (PBS, pH=7.4-8.0, 150 mM) is taken, diluted 3 times, the solution after dialysis purification is dripped into the polyvinyl alcohol solution, stirred for 12 hours at room temperature, and the product PVA-g-GOx is obtained after dialysis purification (dialysis bag with molecular weight cut-off of 200 kDa).
3) Preparation of hydrogels
18Mg of HA-g-PBA prepared in step 1) of example 2 was dissolved in 100. Mu.L of buffer (PBS, pH=7.4-8.0, 150 mM); 10mg of PVA-g-GOx produced in step 2) was dissolved in 100. Mu.L of buffer (PBS, pH=7.4-8.0, 150 mM); after the two phases are completely dissolved, mixing the two phases in equal volume to obtain a hydrogel precursor solution, and then crosslinking and curing the hydrogel precursor solution in a baking oven at 37 ℃ for 40-60min to form the glucose-triggered active oxygen response composite hydrogel.
Example 3
The preparation method of the glucose-triggered active oxygen response injection type composite hydrogel comprises the following steps:
1) Preparation of hyaluronic acid-aminophenylboronic acid derivatives (HA-g-PBA)
2G of HA (molecular weight 80 kDa) was dissolved in 800mL of ultrapure water, then 345mg of 4-aminobenzene boric acid was added to dissolve in 100mL of DMSO solution, then 760mg of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride was added, the pH of the solution was adjusted to 4.5-5.0 by hydrochloric acid, and then 480mg of N-hydroxysuccinimide was added to react for 6 hours; the product HA-g-PBA solution is subjected to dialysis purification (a dialysis bag with a molecular weight cut-off of 8-10 kDa) and then is subjected to freeze-drying preservation, so that the HA-g-PBA is obtained.
2) Preparation of polyvinyl alcohol derivatives modified with glucose oxidase
Dissolving 50mg of glucose oxidase in 5mL of buffer solution (PBS, pH=7.4-8.0, 150 mM), dripping 20 mu L N-acryloyloxysuccinimide 10% (w/v) DMSO solution into the glucose oxidase solution, stirring at room temperature for 4 hours, and dialyzing and purifying the product GOx-AC at 4 ℃ (molecular weight cut-off 3500 dialysis bag); then 60 mu L of 1% (w/v) DMSO solution of 4-mercaptophenylboronic acid is dripped into the solution purified in the previous step, the mixture is stirred for 2 hours at 37 ℃, and the product (GOx-g-MPBA) is dialyzed and purified at 4 ℃ (molecular weight cut-off 3500 dialysis bag); 10mL of 10% (w/v) buffer solution of polyvinyl alcohol (PBS, pH=7.4-8.0, 150 mM) is taken, diluted 3 times, the solution after dialysis purification is dripped into the polyvinyl alcohol solution, stirred for 12 hours at room temperature, and the product PVA-g-GOx is obtained after dialysis purification (dialysis bag with molecular weight cut-off of 200 kDa).
3) Preparation of hydrogels
18Mg of HA-g-PBA prepared in step 1) of example 3 was dissolved in 100. Mu.L of buffer (PBS, pH=7.4-8.0, 150 mM); 10mg of PVA-g-GOx produced in step 2) was dissolved in 100. Mu.L of buffer (PBS, pH=7.4-8.0, 150 mM); after the two phases are completely dissolved, mixing the two phases in equal volume to obtain a hydrogel precursor solution, and then crosslinking and curing the hydrogel precursor solution in a baking oven at 37 ℃ for 40-60min to form the glucose-triggered active oxygen response composite hydrogel.
Example 4
The preparation method of the glucose-triggered active oxygen response injection type composite hydrogel comprises the following steps:
1) Preparation of hyaluronic acid-aminophenylboronic acid derivatives (HA-g-PBA)
2G of HA (molecular weight 40 kDa) was dissolved in 800mL of ultrapure water, then 690mg of 4-aminobenzene boric acid was added to dissolve in 100mL of DMSO solution, then 920mg of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride was added, the pH of the solution was adjusted to 4.5-5.0 by hydrochloric acid, and then 1530mg of N-hydroxysuccinimide was added to react for 6 hours; the product HA-APBA solution is subjected to dialysis purification (a dialysis bag with a molecular weight cut-off of 8-10 kDa) and then is freeze-dried for storage.
2) Preparation of polyvinyl alcohol derivatives modified with glucose oxidase
Dissolving 50mg of glucose oxidase in 5mL of buffer solution (PBS, pH=7.4-8.0, 150 mM), dripping 20 mu L N-acryloyloxysuccinimide 10% (w/v) DMSO solution into the glucose oxidase solution, stirring at room temperature for 4 hours, and dialyzing and purifying the product GOx-AC at 4 ℃ (molecular weight cut-off 3500 dialysis bag); then, 40 mu L of 1% (w/v) DMSO solution of 4-mercaptophenylboronic acid is dripped into the solution purified in the previous step, the mixture is stirred for 2 hours at 37 ℃, and the product (GOx-g-MPBA) is dialyzed and purified at 4 ℃ (molecular weight cut-off 3500 dialysis bag); 15mL of 10% (w/v) buffer solution of polyvinyl alcohol (PBS, pH=7.4-8.0, 150 mM) is taken, diluted 3 times, the solution after dialysis purification is dripped into the polyvinyl alcohol solution, stirred for 12 hours at room temperature, and the product PVA-g-GOx is obtained after dialysis purification (dialysis bag with a molecular weight cutoff of 200 kDa).
3) Preparation of hydrogels
18Mg of HA-g-PBA prepared in step 1) of example 4 was dissolved in 100. Mu.L of buffer (PBS, pH=7.4-8.0, 150 mM); 10mg of PVA-g-GOx produced in step 2) was dissolved in 100. Mu.L of buffer (PBS, pH=7.4-8.0, 150 mM); after the two phases are completely dissolved, mixing the two phases in equal volume to obtain a hydrogel precursor solution, and then crosslinking and curing the hydrogel precursor solution in a baking oven at 37 ℃ for 40-60min to form the glucose-triggered active oxygen response composite hydrogel.
Example 5
The preparation method of the glucose-triggered active oxygen response injection type composite hydrogel comprises the following steps:
1) Preparation of hyaluronic acid-aminophenylboronic acid derivatives (HA-g-PBA)
2G of HA (molecular weight 40 kDa) was dissolved in 800mL of ultrapure water, then 690mg of 4-aminobenzene boric acid was added to dissolve in 100mL of DMSO solution, then 920mg of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride was added, the pH of the solution was adjusted to 4.5-5.0 by hydrochloric acid, and then 1320mg of N-hydroxysuccinimide was added to react for 6 hours; the product HA-APBA solution is subjected to dialysis purification (a dialysis bag with a molecular weight cut-off of 8-10 kDa) and then is freeze-dried for storage.
2) Preparation of polyvinyl alcohol derivatives modified with glucose oxidase
Dissolving 50mg of glucose oxidase in 5mL of buffer solution (PBS, pH=7.4-8.0, 150 mM), dripping 10 mu L N-acryloyloxysuccinimide (w/v) 10% DMSO solution into the glucose oxidase solution, stirring at room temperature for 4 hours, and dialyzing and purifying the product GOx-AC at 4 ℃ (molecular weight cut-off 3500 dialysis bag); then, 40 mu L of 1% (w/v) DMSO solution of 4-mercaptophenylboronic acid is dripped into the solution purified in the previous step, the mixture is stirred for 2 hours at 37 ℃, and the product (GOx-g-MPBA) is dialyzed and purified at 4 ℃ (molecular weight cut-off 3500 dialysis bag); 5mL of 10% (w/v) buffer solution of polyvinyl alcohol (PBS, pH=7.4-8.0, 150 mM) is taken, diluted 3 times, the solution after dialysis purification is dripped into the polyvinyl alcohol solution, stirred for 12 hours at room temperature, and the product PVA-g-GOx is obtained after dialysis purification (dialysis bag with molecular weight cut-off of 200 kDa).
3) Preparation of hydrogels
18Mg of HA-g-PBA prepared in step 1) of example 5 was dissolved in 100. Mu.L of buffer (PBS, pH=7.4-8.0, 150 mM); 10mg of PVA-g-GOx produced in step 2) was dissolved in 100. Mu.L of buffer (PBS, pH=7.4-8.0, 150 mM); after the two phases are completely dissolved, mixing the two phases in equal volume to obtain a hydrogel precursor solution, and then crosslinking and curing the hydrogel precursor solution in a baking oven at 37 ℃ for 40-60min to form the glucose-triggered active oxygen response composite hydrogel.
Example 6
The preparation method of the glucose-triggered active oxygen response injection type composite hydrogel comprises the following steps:
1) Preparation of hyaluronic acid-aminophenylboronic acid derivatives (HA-g-PBA)
2G of HA (molecular weight 40 kDa) was dissolved in 800mL of ultrapure water, then 690mg of 4-aminobenzene boric acid was added to dissolve in 100mL of DMSO solution, then 1530mg of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride was added, and the pH of the solution was adjusted to 4.5-5.0 by hydrochloric acid, and then 920mg of N-hydroxysuccinimide was added to react for 6 hours; the product HA-g-PBA solution is subjected to dialysis purification (a dialysis bag with a molecular weight cut-off of 8-10 kDa) and then is subjected to freeze-drying preservation.
2) Preparation of polyvinyl alcohol derivatives modified with glucose oxidase
Dissolving 50mg of glucose oxidase in 5mL of buffer solution (PBS, pH=7.4-8.0, 150 mM), dripping 10 mu L N-acryloyloxysuccinimide (w/v) 10% DMSO solution into the glucose oxidase solution, stirring at room temperature for 4 hours, and dialyzing and purifying the product GOx-AC at 4 ℃ (molecular weight cut-off 3500 dialysis bag); then 20 mu L of 1% (w/v) DMSO solution of 4-mercaptophenylboronic acid is dripped into the solution purified in the previous step, the mixture is stirred for 2 hours at 37 ℃, and the product (GOx-g-MPBA) is dialyzed and purified at 4 ℃ (molecular weight cut-off 3500 dialysis bag); 10mL of 10% (w/v) buffer solution of polyvinyl alcohol (PBS, pH=7.4-8.0, 150 mM) is taken, diluted 3 times, the solution after dialysis purification is dripped into the polyvinyl alcohol solution, stirred for 12 hours at room temperature, and the product PVA-g-GOx is obtained after dialysis purification (dialysis bag with molecular weight cut-off of 200 kDa).
3) Preparation of hydrogels
15Mg of HA-g-PBA prepared in step 1) of example 6 was dissolved in 100. Mu.L of buffer (PBS, pH=7.4-8.0, 150 mM); 8mg of PVA-g-GOx prepared in step 2) was dissolved in 100. Mu.L of buffer (PBS, pH=7.4-8.0, 150 mM); after the two phases are completely dissolved, mixing the two phases in equal volume to obtain a hydrogel precursor solution, and then crosslinking and curing the hydrogel precursor solution in a baking oven at 37 ℃ for 40-60min to form the glucose-triggered active oxygen response composite hydrogel.
Example 7
The preparation method of the glucose-triggered active oxygen response injection type composite hydrogel comprises the following steps:
1) Preparation of hyaluronic acid-aminophenylboronic acid derivatives (HA-g-PBA)
2G of HA (molecular weight 40 kDa) was dissolved in 800mL of ultrapure water, then 690mg of 4-aminobenzene boric acid was added to dissolve in 100mL of DMSO solution, then 1530mg of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride was added, and the pH of the solution was adjusted to 4.5-5.0 by hydrochloric acid, and then 920mg of N-hydroxysuccinimide was added to react for 6 hours; the product HA-g-PBA solution is subjected to dialysis purification (a dialysis bag with a molecular weight cut-off of 8-10 kDa) and then is subjected to freeze-drying preservation.
2) Preparation of polyvinyl alcohol derivatives modified with glucose oxidase
Dissolving 50mg of glucose oxidase in 5mL of buffer solution (PBS, pH=7.4-8.0, 150 mM), dripping 10 mu L N-acryloyloxysuccinimide (w/v) 10% DMSO solution into the glucose oxidase solution, stirring at room temperature for 4 hours, and dialyzing and purifying the product GOx-AC at 4 ℃ (molecular weight cut-off 3500 dialysis bag); then 20 mu L of 1% (w/v) DMSO solution of 4-mercaptophenylboronic acid is dripped into the solution purified in the previous step, the mixture is stirred for 2 hours at 37 ℃, and the product (GOx-g-MPBA) is dialyzed and purified at 4 ℃ (molecular weight cut-off 3500 dialysis bag); 10mL of 10% (w/v) buffer solution of polyvinyl alcohol (PBS, pH=7.4-8.0, 150 mM) is taken, diluted by 2 times, the solution after dialysis purification is dripped into the polyvinyl alcohol solution, stirred for 12 hours at room temperature, and the product PVA-g-GOx is obtained after dialysis purification (dialysis bag with molecular weight cut-off of 200 kDa).
3) Preparation of hydrogels
18Mg of HA-g-PBA prepared in step 1) of example 7 was dissolved in 100. Mu.L of buffer (PBS, pH=7.4-8.0, 150 mM); 8mg of PVA-g-GOx prepared in step 2) was dissolved in 100. Mu.L of buffer (PBS, pH=7.4-8.0, 150 mM); after the two phases are completely dissolved, mixing the two phases in equal volume to obtain a hydrogel precursor solution, and then crosslinking and curing the hydrogel precursor solution in a baking oven at 37 ℃ for 40-60min to form the glucose-triggered active oxygen response composite hydrogel.
Example 8
The preparation method of the glucose-triggered active oxygen response injection type composite hydrogel comprises the following steps:
1) Preparation of hyaluronic acid-aminophenylboronic acid derivatives (HA-g-PBA)
2G of HA (molecular weight 40 kDa) was dissolved in 800mL of ultrapure water, then 690mg of 4-aminobenzene boric acid was added to dissolve in 100mL of DMSO solution, then 1530mg of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride was added, and the pH of the solution was adjusted to 4.5-5.0 by hydrochloric acid, and then 920mg of N-hydroxysuccinimide was added to react for 6 hours; the product HA-g-PBA solution is subjected to dialysis purification (a dialysis bag with a molecular weight cut-off of 8-10 kDa) and then is subjected to freeze-drying preservation.
2) Preparation of polyvinyl alcohol derivatives modified with glucose oxidase
Dissolving 50mg of glucose oxidase in 5mL of buffer solution (PBS, pH=7.4-8.0, 150 mM), dripping 10 mu L N-acryloyloxysuccinimide (w/v) 10% DMSO solution into the glucose oxidase solution, stirring at room temperature for 4 hours, and dialyzing and purifying the product GOx-AC at 4 ℃ (molecular weight cut-off 3500 dialysis bag); then 20 mu L of 1% (w/v) DMSO solution of 4-mercaptophenylboronic acid is dripped into the solution purified in the previous step, the mixture is stirred for 2 hours at 37 ℃, and the product (GOx-g-MPBA) is dialyzed and purified at 4 ℃ (molecular weight cut-off 3500 dialysis bag); 10mL of 10% (w/v) buffer solution of polyvinyl alcohol (PBS, pH=7.4-8.0, 150 mM) is taken, diluted by 2 times, the solution after dialysis purification is dripped into the polyvinyl alcohol solution, stirred for 12 hours at room temperature, and the product PVA-g-GOx is obtained after dialysis purification (dialysis bag with molecular weight cut-off of 200 kDa).
3) Preparation of hydrogels
15Mg of HA-g-PBA prepared in step 1) of example 8 was dissolved in 100. Mu.L of buffer (PBS, pH=7.4-8.0, 150 mM); 10mg of PVA-g-GOx produced in step 2) was dissolved in 100. Mu.L of buffer (PBS, pH=7.4-8.0, 150 mM); after the two phases are completely dissolved, mixing the two phases in equal volume to obtain a hydrogel precursor solution, and then crosslinking and curing the hydrogel precursor solution in a baking oven at 37 ℃ for 40-60min to form the glucose-triggered active oxygen response composite hydrogel.
Example 9
Application of injectable biodegradable hydrogel material: the injectable biodegradable hydrogel material prepared in example 1 is uniformly mixed with drugs, proteins, cells, inorganic particles and the like, and then the mixed solution is poured into a container, a model or injected into tissues, organs and body cavities of animals or human bodies, so that the solution is crosslinked and solidified at 37 ℃.
By now it should be appreciated by those skilled in the art that while a number of exemplary embodiments of the invention have been shown and described in detail herein, many other variations or modifications that are consistent with the general principles of the invention may be directly determined or derived from the disclosure of the invention without departing from the spirit and scope of the invention. Accordingly, the scope of the present invention should be understood and deemed to cover all such other variations or modifications.

Claims (1)

1. The preparation method of the glucose-triggered active oxygen response injection type composite hydrogel is characterized by comprising the following steps of:
step 1) preparation of hyaluronic acid-aminophenylboronic acid derivative HA-g-PBA
2G of HA with the molecular weight of 40Kda is dissolved in 800mL of ultrapure water, then 690mg of 4-aminobenzene boric acid is added to be dissolved in 100mL of DMSO solution, 1530mg of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride is added, the pH of the solution is adjusted to 4.5-5.0 by hydrochloric acid, and 920mg of N-hydroxysuccinimide is added to react for 6 hours; the product HA-g-PBA solution is subjected to dialysis purification and freeze-drying preservation by using a dialysis bag with the molecular weight cut-off of 8-10 kDa;
Step 2) preparation of polyvinyl alcohol derivatives modified with glucose oxidase
Dissolving 50mg of glucose oxidase in 5mL buffer solution with pH=7.4-8.0 and 150mMPBS, dripping 10 mu L N-acryloyloxy succinimide 10% (w/v) DMSO solution into the glucose oxidase solution, stirring at room temperature for 4 hours, and dialyzing and purifying the product GOx-AC at 4 ℃ by using a molecular weight cut-off 3500 dialysis bag; then 20 mu L of 1% (w/v) DMSO solution of 4-mercaptophenylboronic acid is dripped into the solution purified in the previous step, the mixture is stirred for 2 hours at 37 ℃, and the GOx-g-MPBA product is dialyzed and purified by using a dialysis bag with molecular weight cutoff of 3500 at 4 ℃; 10mL of 10% (w/v) buffer solution of polyvinyl alcohol dissolved by pH=7.4-8.0 and 150mMPBS is taken, diluted by 2 times, the solution after dialysis and purification is dripped into the polyvinyl alcohol solution, stirred at room temperature for 12 hours, and the product PVA-g-GOx is obtained after dialysis and purification by using a dialysis bag with a molecular weight cut-off of 200 kDa;
3) Preparation of hydrogels
Dissolving 18mg of HA-g-PBA prepared in step 1) in 100. Mu.L of 150mM buffer, wherein the buffer is selected as phosphate buffered saline PBS, and the pH value of the PBS buffer is 7.4-8; 10mg of PVA-g-GOx prepared in step 2) is dissolved in 100. Mu.L of 150mM PBS buffer, and the pH of the PBS buffer is 7.4-8; after the two phases are completely dissolved, mixing the two phases in equal volume to obtain a hydrogel precursor solution, and then crosslinking and solidifying the hydrogel precursor solution in a baking oven at 37 ℃ for 40-60 min to form the glucose-triggered active oxygen response injection type composite hydrogel.
CN202110916825.8A 2021-08-11 2021-08-11 Preparation method of glucose-triggered active oxygen response injection type composite hydrogel Active CN113599507B (en)

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