A pH and ROS dual-responsive double-crosslinked network injectable hydrogel and a preparation method and application thereof

Abstract

The application provides a pH and ROS dual-response double-crosslinking network injectable hydrogel as well as a preparation method and application thereof. The hydrogel comprises solution A and solution B. The solution A contains oxidized polysaccharides, the oxidized polysaccharides have aldehyde groups, and the oxidized polysaccharides are grafted with boronic acid groups. The solution B contains high-molecular materials with amino groups, and the high-molecular materials are grafted with o-diphenol groups. The injectable hydrogel provided by the application is based on high-molecular materials, in-situ gelation in the body after injection, and a double-crosslinking hydrogel network containing pH-sensitive Schiff base bonds and ROS-sensitive borate ester bonds, so that the hydrogel has excellent mechanical properties, responsiveness and stability. Through the synergistic effect of the double-crosslinking network, the balance between the stability of the hydrogel in a normal physiological environment and the rapid response of the hydrogel in a pathological microenvironment is achieved.

Description

Technical Field

[0001] This invention belongs to the field of hydrogel technology, specifically providing a pH and ROS dual-responsive dual cross-linked network injectable hydrogel, its preparation method, and its application. Background Technology

[0002] Injectable hydrogels are fluid solutions that can be injected into the body via a syringe and undergo rapid cross-linking reactions at the target site after injection, forming a gel material with a three-dimensional network structure in situ.

[0003] In the existing technology, in order to improve the targeting and controllability of drug release, researchers have proposed a variety of stimulus-responsive injectable hydrogel schemes, which mainly include the following categories: (1) Single pH-responsive injectable hydrogel schemes, which achieve cross-linking through a single dynamic covalent bond, with a mild gelation process and certain injectability, but its response mechanism mainly depends on pH changes, making it difficult to finely regulate complex pathological microenvironments. (2) Single ROS-responsive hydrogels or material systems, whose main technical means is to utilize the characteristic of increased ROS levels in the lesion area to achieve stimulus response, but most of these schemes rely only on single ROS stimulation, and their stability and gelation controllability in normal physiological environments need further optimization. (3) Multi-component physical or chemical cross-linked hydrogel schemes, which usually improve mechanical properties and gelation speed by adjusting the material ratio or cross-linking density, but their response mode is mostly passive regulation, lacking the ability to accurately respond to specific pathological microenvironments (such as the simultaneous presence of weak acidity and high ROS).

[0004] Existing technologies have been extensively studied in the areas of injectable hydrogels and stimulus-responsive materials. However, most current solutions employ a single response mechanism or a single cross-linking method, leaving room for improvement in their intelligent response capabilities and release regulation precision within complex pathological microenvironments. Even though existing technologies have made some progress in the field of stimulus-responsive injectable hydrogels, improvements are still needed in areas such as adaptability to complex pathological microenvironments, synergistic regulation of gel stability and responsiveness, and ease of fabrication. Summary of the Invention

[0005] This invention aims to at least partially address one of the technical problems in the prior art. Therefore, one objective of this invention is to propose a pH and ROS dual-responsive dual-crosslinked network hydrogel, its preparation method, and its applications, to solve the problems of existing injectable hydrogels, such as limited response to the lesion microenvironment, insufficient precision in drug release, difficulty in balancing gelation performance and stability, and complex preparation processes.

[0006] In a first aspect, the present invention provides an injectable hydrogel with a dual pH and ROS responsive dual crosslinked network, comprising:

[0007] Solution A contains an oxidized polysaccharide, the oxidized polysaccharide having an aldehyde group and a boric acid group grafted onto the oxidized polysaccharide;

[0008] Solution B contains a polymeric material with amino groups grafted onto it.

[0009] The hydrogel provided by this invention involves functionalizing natural polymer materials to prepare two polymer solutions capable of forming dynamic covalent bonds. After injection and mixing, the two solutions rapidly form a double-crosslinked hydrogel network structure in situ at the target site in vivo through the synergistic crosslinking effect of Schiff base bonds and borate ester bonds. This hydrogel maintains structural stability in normal physiological environments, while undergoing crosslinking bond breakage or network rearrangement in weakly acidic, high-ROS pathological microenvironments, thereby achieving controllable degradation of the hydrogel and intelligent drug release. Specifically, the aldehyde groups on the oxidized polysaccharide molecular chains in solution A react with the amino groups on the polymer molecular chains in solution B to form Schiff base bonds (–C=N–), constituting the first crosslinked network layer. This crosslinking bond is sensitive to pH changes and is easily hydrolyzed in acidic environments. The borate groups grafted onto the oxidized polysaccharide in solution A react with the catechol groups grafted onto the polymer molecular chains in solution B to form borate ester bonds, constituting the second crosslinked network layer. This crosslinking bond is sensitive to ROS and pH changes and undergoes oxidative breakage under high ROS conditions. The two cross-linking methods mentioned above work synergistically to form a hydrogel network with a double cross-linking structure, thereby endowing the hydrogel with rapid gelation, dual responsiveness, and self-healing ability.

[0010] In some embodiments of the present invention, the oxidized polysaccharide includes one of oxidized konjac polysaccharide, oxidized sodium alginate, oxidized hyaluronic acid, and oxidized starch, preferably oxidized konjac polysaccharide.

[0011] In some embodiments of the present invention, the polymeric material includes one of chitosan, amino-modified hyaluronic acid, and polyethyleneimine-modified material, preferably chitosan.

[0012] Furthermore, the oxidized polysaccharide is selected from oxidized konjac polysaccharide, and the polymer material is selected from chitosan. The main component of solution A is oxidized konjac polysaccharide grafted with phenylboronic acid (OKGM-PBA). Multiple aldehyde structures are introduced into the oxidized konjac polysaccharide molecular chain, and phenylboronic acid is covalently grafted onto the oxidized konjac molecular chain, giving the oxidized polysaccharide both aldehyde and boric acid groups. The main component of solution B is chitosan grafted with caffeic acid (CS-CA). The chitosan molecular chain contains a large number of amino groups, and caffeic acid is grafted onto the chitosan molecular chain via amide bonds, giving this polymer material both amino and catechol structures. For details, please refer to [reference needed]. Figure 1 .

[0013] In some embodiments of the present invention, the concentration of the oxidized polysaccharide in solution A is 30-50 g / L, preferably 40 g / L.

[0014] In some embodiments of the present invention, the concentration of the polymeric material in solution B is 30-50 g / L, preferably 40 g / L.

[0015] In some embodiments of the present invention, the aldehyde group content in the oxidized polysaccharide is 2-3 mmol / g, preferably 2.34 mmol / g, and the boric acid group content is 2-3 mmol / g, preferably 2.7 mmol / g.

[0016] In some embodiments of the present invention, the content of the amino group in the polymer material is 5-6 mmol / g, preferably 5.44 mmol / g, and the content of the catechol group is 1-3 mmol / g, preferably 1.91 mmol / g.

[0017] The concentrations of the main active ingredients and the degree of grafting of active groups in solutions A and B can be adjusted within a certain range to change the gelation rate, mechanical properties, and release behavior, as long as the injectability and dual-response characteristics of the hydrogel are not affected.

[0018] In a second aspect, the present invention provides a method for preparing the above-mentioned pH and ROS dual-responsive dual crosslinked network injectable hydrogel, comprising:

[0019] (1) Dissolve the polysaccharide in water and add an oxidizing agent to oxidize it, thus obtaining oxidized polysaccharide;

[0020] (2) In the presence of a condensing agent, phenylboronic acid is grafted onto the molecular chain of the oxidized polysaccharide in an aqueous solution to obtain product A. Product A is then dissolved in a buffer solution to obtain solution A.

[0021] (3) Under the action of an activator, caffeic acid reacts with the amino groups on the molecular chain of the polymer material in an aqueous solution to obtain product B. Product B is dissolved in a buffer solution to obtain solution B.

[0022] This invention is based on natural polymer materials. By functionalizing and modifying oxidized polysaccharides and amino-containing polymers separately, a two-component system capable of forming dynamic covalent bonds is constructed. After injection and mixing, the two solutions rapidly gel in situ in vivo, forming a double-crosslinked hydrogel network containing both pH-sensitive Schiff base bonds and ROS-sensitive borate ester bonds. This double-crosslinked network, composed of both Schiff base and borate ester bonds, endows the hydrogel with excellent mechanical properties, responsiveness, and stability. Through the synergistic effect of the double-crosslinked network, a balance is achieved between the hydrogel's stability in normal physiological environments and its rapid response in pathological microenvironments. The weak acidity and high ROS characteristics of pathological microenvironments trigger structural changes in the hydrogel, enabling intelligent, on-demand drug release. The hydrogel system possesses comprehensive properties including rapid gelation, self-healing, and good injectability, making it suitable for minimally invasive drug delivery and local treatment applications. This hydrogel remains stable in normal physiological environments but undergoes structural regulation or degradation in weakly acidic, high-ROS pathological microenvironments, thereby achieving on-demand drug release and exhibiting excellent injectability, biocompatibility, and intelligent responsiveness. The preparation process is mild and the steps are clear, making it suitable for large-scale preparation and conducive to maintaining the activity of the loaded drug.

[0023] In some embodiments of the present invention, the oxidant includes sodium periodate.

[0024] In some embodiments of the present invention, the condensing agent includes a carbodiimide condensing agent, preferably 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC).

[0025] In some embodiments of the present invention, the activator includes N-hydroxysuccinimide (NHS).

[0026] As an example, the oxidized polysaccharide is selected from oxidized konjac polysaccharide, the polymer material is selected from chitosan, and the preparation method of the hydrogel includes the following steps:

[0027] Step S1: Preparation of oxidized konjac polysaccharide: Konjac glucomannan was dissolved in deionized water, and sodium periodate was added to carry out an oxidation reaction to introduce aldehyde groups; after the reaction was completed, the reaction was terminated, dialyzed and dried to obtain oxidized konjac polysaccharide.

[0028] Step S2: Preparation of phenylboronic acid grafted onto konjac polysaccharide: In the presence of a carbodiimide condensing agent, phenylboronic acid was grafted onto the konjac oxidase molecular chain, and OKGM-PBA was obtained after purification.

[0029] Step S3: Preparation of chitosan grafted with caffeic acid: Caffeic acid reacts with the amino groups on the chitosan molecular chain under the action of an activator to form amide bonds, thus obtaining CS-CA.

[0030] Step S4: Solution Preparation and Injection Gel Formation: Dissolve OKGM-PBA and CS-CA in buffer solutions to prepare solutions A and B, respectively. Load both solutions into a dual-tube syringe and inject them into the target site through the mixing head to achieve rapid in-situ gel formation.

[0031] Drug components can be added to solution A and / or solution B to form a drug-loaded solution, in which the drug is embedded in the gel network after hydrogel formation. In a weakly acidic pathological microenvironment with high ROS concentration, Schiff base bonds and borate ester bonds in the hydrogel break or rearrange, and the hydrogel structure gradually loosens or degrades, thereby accelerating drug release; while in a normal physiological environment, the hydrogel structure remains relatively stable, achieving sustained drug release.

[0032] In a third aspect, the present invention proposes the application of the above-mentioned pH and ROS dual-responsive dual crosslinked network injectable hydrogel in material loading.

[0033] The injectable hydrogel provided by this invention is based on polymer materials and forms gel in situ in vivo after injection. It has the advantages of minimally invasive drug delivery, local retention and sustained drug release, and can be widely used in biomedical fields such as tumor treatment, scar repair and tissue engineering.

[0034] In some embodiments of the present invention, the substance includes one or more small molecule drugs, proteins, polypeptides, nucleic acids, and cells.

[0035] In a fourth aspect, the present invention provides a method for using the above-mentioned pH and ROS dual-responsive dual cross-linked network injectable hydrogel, comprising: mixing solution A and solution B during injection, wherein the volume ratio of solution A to solution B is 1:(0.8-1.2), preferably 1:1.

[0036] This invention uses a dual-tube syringe for mixing injection. Without affecting the instant mixing and in-situ gelation of the two solutions, multi-tube syringes, static mixers, or other equivalent mixing devices can be used to achieve solution mixing.

[0037] Compared with the prior art, the present invention can achieve at least one of the following beneficial effects:

[0038] (1) The dual-response mechanism significantly enhances environmental adaptability and release precision: By simultaneously introducing pH-sensitive Schiff base bonds and ROS-sensitive borate ester bonds into the same hydrogel system, the hydrogel can respond to both weak acidity and high ROS levels in the pathological microenvironment. Under simulated pathological microenvironment conditions (pH approximately 5.0, hydrogen peroxide concentration 1-10 mM), the cross-linking bonds in the hydrogel network undergo synergistic breakage or rearrangement, the gel structure gradually loosens or degrades, and the drug release rate is significantly increased; while under simulated normal physiological conditions (pH 7.4, low ROS), the hydrogel structure remains stable, and the drug release rate is significantly reduced. Compared to hydrogel systems with only a single pH response or a single ROS response, this invention can more accurately distinguish between the lesion environment and the normal tissue environment, improving drug utilization efficiency.

[0039] (2) Fast gelation speed, meeting the requirements for injectable and in-situ gelation: This invention adopts a dual-tube injection mixing gelation method, in which the two solutions can form a stable hydrogel in a short time after mixing. Measurements using the inverted test tube method show that, under normal temperature and neutral conditions, the gelation time of the hydrogel can typically be controlled within a few seconds to one minute. This gelation speed avoids premature gelation before injection and prevents material spillage after injection, which is beneficial for improving the safety and controllability of clinical operations and is superior to some existing technical solutions with excessively long or uncontrollable gelation times.

[0040] (3) Good structural stability under normal physiological conditions: Because the present invention uses a dual dynamic covalent bond to construct a cross-linked network structure, the two cross-linked bonds can synergistically maintain the overall structural stability of the hydrogel under normal physiological conditions. Experimental observations show that under pH 7.4 and no or low ROS conditions, the hydrogel can maintain its intact morphology for a relatively long time, only undergoing slow swelling or degradation. This characteristic effectively avoids the problem of premature release or structural failure in non-lesion areas of existing partially responsive hydrogels, and helps to reduce potential side effects.

[0041] (4) The drug release behavior is adjustable and suitable for various dosing needs.

[0042] By adjusting the concentration of the polymer solution, the degree of grafting, and the ratio of the two solutions, the crosslinking density and network structure of the hydrogel of this invention can be controlled within a certain range, thereby achieving regulation of the drug release rate and release cycle. Taking a model drug as an example, under simulated pathological microenvironment conditions, the cumulative release rate of the hydrogel within 24 hours can be significantly higher than the release level under normal physiological conditions; while under normal conditions, the release process is more gradual, which is conducive to achieving long-acting drug delivery.

[0043] (5) Good biocompatibility and high safety: The main raw materials used in this invention include natural or near-natural substances such as konjac glucomannan, chitosan and caffeic acid, which have good biocompatibility and biodegradability. The hydrogel formation process does not require high temperature, strong acid or alkali or toxic crosslinking agents. The reaction conditions are mild and have little impact on the activity of the loaded drug, making it suitable for a variety of applications in the biomedical field.

[0044] (6) The process is simple and has good prospects for practical application: The preparation method of the present invention has clear steps, mild reaction conditions, wide availability of raw materials and low cost. The preparation process does not rely on complex equipment or harsh conditions and has good repeatability and scalability. Compared with hydrogel solutions that involve partial synthesis of polymers or complex multi-step reactions, the present invention significantly reduces the complexity of the process while ensuring functionality, which is conducive to industrialization and clinical promotion. Attached Figure Description

[0045] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0046] Figure 1 This is a schematic diagram illustrating the preparation mechanism of a hydrogel provided by the present invention. Detailed Implementation

[0047] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below in conjunction with specific embodiments and corresponding drawings. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0048] Example 1

[0049] This embodiment provides an injectable hydrogel with a dual pH and ROS responsive, dual cross-linked network, and the specific preparation process is as follows:

[0050] Step S1: Preparation of oxidized konjac polysaccharide: Konjac glucomannan was dissolved in deionized water, and sodium periodate was added to carry out an oxidation reaction to introduce aldehyde groups; after the reaction was completed, the reaction was terminated, dialyzed and dried to obtain oxidized konjac polysaccharide.

[0051] Step S2: Preparation of phenylboronic acid grafted onto konjac polysaccharide: In the presence of a carbodiimide condensing agent (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)), phenylboronic acid was grafted onto the konjac oxidase molecular chain in an aqueous solution, and OKGM-PBA was obtained after purification.

[0052] Step S3: Preparation of chitosan grafted with caffeic acid: In an aqueous solution, caffeic acid reacts with the amino groups on the chitosan molecular chain under the action of an activator (N-hydroxysuccinimide (NHS)) to form amide bonds, yielding CS-CA.

[0053] Step S4: Solution preparation and injection gelation: Dissolve OKGM-PBA and CS-CA in buffer solutions to prepare solutions A and B respectively.

[0054] In the hydrogel prepared in Example 1, solution A contained 40 g / L OKGM-PBA, with an aldehyde group content of 2.34 mmol / g and a boric acid group content of 2.7 mmol / g. Solution B contained 40 g / L CS-CA, with an amino group content of 5.44 mmol / g and a catechol group content of 1.91 mmol / g.

[0055] Example 2

[0056] Step S1: Preparation of oxidized hyaluronic acid: Hyaluronic acid is dissolved in deionized water, and sodium periodate is added to carry out an oxidation reaction, introducing aldehyde groups onto the molecular chain; after the reaction is completed, a terminator is added to terminate the reaction, and oxidized hyaluronic acid is obtained after dialysis and drying.

[0057] Step S2: Preparation of oxidized hyaluronic acid derivatives containing boric acid groups: A functional molecule with boric acid groups is introduced into the oxidized hyaluronic acid molecular chain, and after purification, an oxidized hyaluronic acid derivative containing boric acid groups is obtained.

[0058] Step S3: Preparation of chitosan grafted with caffeic acid: Caffeic acid is reacted with amino groups on the chitosan molecular chain under the action of condensing agent and activator to form amide bonds. After purification, the chitosan grafted with caffeic acid product is obtained.

[0059] Step S4: Solution preparation and injection gel formation: Dissolve the above-mentioned oxidized hyaluronic acid derivative containing boric acid groups in a buffer solution to obtain solution A; dissolve chitosan grafted with caffeic acid in a buffer solution to obtain solution B; load solutions A and B into a dual-tube syringe respectively, and inject them together through the mixing head at a volume ratio of 1:1 to form a hydrogel.

[0060] In this embodiment, hyaluronic acid has good biocompatibility and tissue affinity; the aldehyde group introduced after oxidation can form Schiff base bonds with the amino groups on chitosan, while the boric acid group can form borate ester bonds with the catechol group, thereby constructing a pH and ROS dual-responsive double crosslinked network.

[0061] Comparative Example 1

[0062] Comparative Example 1 provides a single-crosslinked network injectable hydrogel. Its preparation method is basically the same as in Example 1, except that: solution A uses only oxidized polysaccharide without introducing boric acid groups; solution B uses chitosan grafted with caffeic acid. Solutions A and B are separately prepared in buffer solutions and mixed and injected using a dual-tube syringe at a 1:1 volume ratio. After mixing, the system mainly forms a single crosslinked network through a Schiff base reaction between the aldehyde groups on the oxidized polysaccharide molecular chain and the amino groups on the chitosan molecular chain, thus obtaining the single-crosslinked injectable hydrogel.

[0063] The hydrogel obtained in this comparative example does not contain the borate ester crosslinking structure formed by boric acid groups and catechol groups, and therefore does not possess the ROS-responsive properties imparted by borate ester bonds; its network structure mainly relies on Schiff base bonds for maintenance. This type of injectable hydrogel relies solely on the aldehyde-amino Schiff base reaction for formation.

[0064] Comparative Example 2

[0065] Comparative Example 2 provides a single-response, single-crosslinked network injectable hydrogel. Its preparation method is basically the same as in Example 1, except that: solution A uses konjac polysaccharide containing boric acid groups, but no aldehyde groups are introduced; solution B uses chitosan containing catechol groups, but no additional aldehyde-amino crosslinking pairs that can participate in the Schiff base reaction are introduced. Solutions A and B are separately prepared in buffer solutions and mixed at a volume ratio of 1:1. After mixing, the system mainly constructs a single-crosslinked network hydrogel through the formation of reversible borate ester bonds between boric acid groups and catechol groups.

[0066] The hydrogel obtained in this comparative example mainly exhibits pH-dependent dynamic reversible cross-linking characteristics, without a second network structure contributed by Schiff base bonds. This type of pH-responsive self-healing hydrogel is based on boronic acid-catechol complexation.

[0067] The properties of the hydrogels in the examples and comparative examples were determined.

[0068] The gelation time was determined using the inverted test tube method. Solution A and solution B were rapidly mixed at room temperature in a 1:1 volume ratio. The test tubes were inverted every 2 seconds, and the shortest time when the system did not flow when inverted within 30 seconds was recorded as the gelation time. Each group was tested in triplicate, and the average value was taken.

[0069] The results showed that the gelation time for Example 1 was 18±2 s, and for Example 2 it was 22±3 s. The gelation time for Comparative Example 1 was 47±4 s, and for Comparative Example 2 it was 71±6 s. Furthermore, when the system containing only solution B was injected alone as a supplementary control, it did not form a self-supporting gel within 5 minutes, exhibiting only a viscous solution state. These results indicate that the dual-crosslinked system, after mixing, can achieve faster in-situ gelation, significantly superior to the single-crosslinked system.

[0070] The above description is merely an embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principle of the present invention should be included within the scope of the claims of the present invention.

Claims

1. An injectable hydrogel with a dual pH and ROS responsive dual crosslinked network, characterized in that, include: Solution A contains an oxidized polysaccharide, the oxidized polysaccharide having an aldehyde group and a boric acid group grafted onto the oxidized polysaccharide; Solution B contains a polymeric material with amino groups grafted onto it.

2. The hydrogel according to claim 1, characterized in that, The oxidized polysaccharide includes one of oxidized konjac polysaccharide, oxidized sodium alginate, oxidized hyaluronic acid, and oxidized starch, with oxidized konjac polysaccharide being preferred.

3. The hydrogel according to claim 1, characterized in that, The polymeric material includes one of chitosan, amino-modified hyaluronic acid, and polyethyleneimine-modified materials, with chitosan being preferred.

4. The hydrogel according to any one of claims 1-3, characterized in that, In solution A, the concentration of the oxidized polysaccharide is 30-50 g / L; And / or, in solution B, the concentration of the polymer material is 30-50 g / L.

5. The hydrogel according to any one of claims 1-3, characterized in that, In the oxidized polysaccharide, the content of the aldehyde group is 2-3 mmol / g, and the content of the boric acid group is 2-3 mmol / g; And / or, in the polymer material, the content of the amino group is 5-6 mmol / g, and the content of the catechol group is 1-3 mmol / g.

6. A method for preparing the pH and ROS dual-responsive dual crosslinked network injectable hydrogel according to any one of claims 1-5, characterized in that, include: (1) Dissolve the polysaccharide in water and add an oxidizing agent to oxidize it, thus obtaining oxidized polysaccharide; (2) In the presence of a condensing agent, phenylboronic acid is grafted onto the molecular chain of the oxidized polysaccharide in an aqueous solution to obtain product A. Product A is then dissolved in a buffer solution to obtain solution A. (3) Under the action of an activator, caffeic acid reacts with the amino groups on the molecular chain of the polymer material in an aqueous solution to obtain product B. Product B is dissolved in a buffer solution to obtain solution B.

7. The method according to claim 6, characterized in that, The oxidant includes sodium periodate; And / or, the condensing agent includes a carbodiimide condensing agent, preferably 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; And / or, the activator includes N-hydroxysuccinimide.

8. The application of the pH and ROS dual-responsive dual crosslinked network injectable hydrogel according to any one of claims 1-5 in material loading.

9. The application according to claim 8, characterized in that, The substances include one or more small molecule drugs, proteins, polypeptides, nucleic acids, and substances found in cells.

10. A method of using the pH and ROS dual-responsive dual cross-linked network injectable hydrogel according to any one of claims 1-5, characterized in that, Solution A and solution B are mixed during injection, wherein the volume ratio of solution A to solution B is 1:(0.8-1.2), preferably 1:1.

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