Preparation method for preparing hydrogel in water phase and application of hydrogel in tumor immune microenvironment assessment ultrasonic detection

Abstract

The invention provides a preparation method for preparing hydrogel in a water phase and application of the hydrogel in tumor immune microenvironment evaluation ultrasonic detection, and relates to the technical field of organic synthesis. The preparation method comprises the following steps that chitosan is dissolved in water, a chitosan solution with a first preset concentration is prepared, and chitosan has carboxyl groups and amino groups; dissolving a condensing agent in water to prepare a reaction solution with a second preset concentration; mixing the chitosan solution with the reaction solution, adding glacial acetic acid, and reacting for preset time at preset temperature and preset pressure to obtain a mixed solution; carrying out dialysis treatment on the mixed solution to obtain hydrogel; wherein the ratio of the first preset concentration to the second preset concentration ranges from 10: 1 to 1: 1. According to the invention, the hydrogel with amido bonds is prepared in a water phase, and the hydrogel has the characteristics of low toxicity, good biocompatibility and good biodegradability.

Description

Technical Field

[0001] The invention relates to the technical field of organic synthesis, and in particular to a method for preparing a hydrogel in an aqueous phase and application of ultrasonic detection in evaluating tumor immune microenvironment of the hydrogel. Background Art

[0002] In recent years, hydrogels have been widely used in the fields of wound dressing, drug delivery, cell culture and differentiation in the medical field due to their unique water absorption, water retention, moisture retention, stability and flexibility. Hydrogels with amide bonds have attracted attention in the fields of biomedicine, environmental engineering and smart wearable technology due to their easy chemical modification, unique network structure and good biocompatibility.

[0003] In the prior art, the preparation of hydrogels is usually carried out in an organic phase, which can prevent the generation of ammonium carboxylate salts and reduce the impact of by-products. At the same time, organic solvents can provide a better solvation environment, which helps to improve the activity of reactants and promote the formation of amide bonds. However, organic reagents usually have certain toxicity, which makes the prepared hydrogels with amide bonds have high toxicity, poor biocompatibility or poor biodegradability, thereby limiting the application of hydrogels in the biomedical field. Summary of the invention

[0004] One object of the first aspect of the present invention is to provide a method for preparing a hydrogel in an aqueous phase, so as to solve the technical problem in the prior art that the hydrogel needs to be prepared in an organic phase solvent, resulting in high toxicity and poor biocompatibility.

[0005] Another object of the first aspect of the present invention is to increase the reaction rate during the preparation of the hydrogel.

[0006] The second aspect of the present invention aims to provide an application of a hydrogel in ultrasound detection for evaluating tumor immune microenvironment, wherein the hydrogel is prepared according to the above-mentioned preparation method.

[0007] According to the purpose of the first aspect of the present invention, the present invention provides a method for preparing a hydrogel in an aqueous phase, comprising the following steps:

[0008] Dissolving chitosan in water to prepare a chitosan solution having a first preset concentration, wherein the chitosan has a carboxyl group and an amino group;

[0009] Dissolving a condensing agent in water to prepare a reaction solution having a second preset concentration;

[0010] The chitosan solution and the reaction solution are mixed, glacial acetic acid is added, and the mixture is reacted at a preset temperature and a preset pressure for a preset time to obtain a mixed solution;

[0011] The mixed solution is dialyzed to obtain the hydrogel; wherein,

[0012] The ratio of the first preset concentration to the second preset concentration is any value in the range of 10:1-1:1.

[0013] Optionally, the chitosan is any one of carboxymethyl chitosan, N-carboxyethyl chitosan or N-succinyl chitosan.

[0014] Optionally, the condensing agent is any one of 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride or 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate.

[0015] Optionally, the mass fraction of the glacial acetic acid is any value in the range of 25%-60%.

[0016] Optionally, the preset temperature is any value in the range of 20°C-60°C, and the preset pressure is any value in the range of 0.03MPa-0.13MPa.

[0017] Optionally, the step of dialyzing the mixed solution further comprises the following steps:

[0018] The mixed solution is replaced with dialysate at preset time intervals.

[0019] Optionally, the specification of the dialysis bag in the dialysis treatment process is any value in the range of 3000 kDa-4000 kDa.

[0020] According to the purpose of the second aspect of the present invention, the present invention provides an application of a hydrogel in ultrasonic detection of tumor immune microenvironment assessment, wherein the hydrogel is prepared according to any of the preparation methods described above.

[0021] The present invention mixes a chitosan solution with a first preset concentration and a reaction solution with a second preset concentration, and performs an amide condensation reaction under the catalytic action of glacial acetic acid to condense the carboxyl groups and amino groups of the chitosan itself to form amide bonds, so that the chitosan itself is cross-linked to form a hydrogel, and impurities in the hydrogel are removed by dialysis treatment to obtain a hydrogel with low toxicity, good biocompatibility and biodegradability.

[0022] Furthermore, the mass fraction of the glacial acetic acid of the present invention is any value in the range of 25%-60%. The glacial acetic acid, as a catalyst, can reduce the activation energy of the reaction, thereby accelerating the reaction process, promoting the amide condensation reaction between the carboxyl group and the amino group in the chitosan, thereby increasing the reaction rate in the hydrogel preparation process.

[0023] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and implement it according to the contents of the specification, the following is a detailed description of the preferred embodiments of the present invention in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Hereinafter, some specific embodiments of the present invention will be described in detail in an exemplary and non-limiting manner with reference to the accompanying drawings. The same reference numerals in the accompanying drawings indicate the same or similar components or parts. It should be understood by those skilled in the art that these drawings are not necessarily drawn to scale. In the accompanying drawings:

[0025] Figure 1 is a schematic flow chart of a method for preparing a hydrogel according to one embodiment of the present invention;

[0026] Figure 2 is the degradation of the hydrogel in water, H2O2 and HCl according to one embodiment of the present invention;

[0027] Figure 3 The degradation of the hydrogel according to one embodiment of the present invention in water, H2O2 and HCl after 0h, 0.25h, 0.5h and 1h of reaction;

[0028] Figure 4 is a SEM image of a hydrogel according to an embodiment of the present invention and the hydrogel after reacting with H2O2;

[0029] Figure 5 is a SEM image of a hydrogel according to an embodiment of the present invention and a hydrogel after reacting with HCl;

[0030] Figure 6 is a rheological diagram of a hydrogel before reacting with H2O2 and HCl according to one embodiment of the present invention;

[0031] Figure 7 is a rheological diagram of a hydrogel after reacting with H2O2 and HCl according to one embodiment of the present invention. DETAILED DESCRIPTION

[0032] The specific implementation of the present invention is further described in detail below in conjunction with the accompanying drawings and examples. The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0033] In order to make the above-mentioned purposes, features and advantages of the present application more obvious and easy to understand, the specific implementation methods of the present application are described in detail below in conjunction with the accompanying drawings. It is to be understood that the specific embodiments described herein are only used to explain the present application, rather than to limit the present application. It should also be noted that, for ease of description, only some structures related to the present application are shown in the accompanying drawings, rather than all structures. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of this application.

[0034] The terms "including" and "having" and any variations thereof in this application are intended to cover non-exclusive inclusions. For example, a process, method, system, product or device comprising a series of steps or units is not limited to the listed steps or units, but may optionally include steps or units not listed, or may optionally include other steps or units inherent to these processes, methods, products or devices.

[0035] Reference to "embodiments" herein means that a particular feature, structure, or characteristic described in conjunction with the embodiments may be included in at least one embodiment of the present application. The appearance of the phrase in various locations in the specification does not necessarily refer to the same embodiment, nor is it an independent or alternative embodiment that is mutually exclusive with other embodiments. It is explicitly and implicitly understood by those skilled in the art that the embodiments described herein may be combined with other embodiments.

[0036] In particular, the present invention provides a method for preparing a hydrogel in an aqueous phase, which utilizes the self-polymerization of the carboxyl groups and amino groups of chitosan itself to form a hydrogel with low toxicity, good biocompatibility and good biodegradability. It should be noted here that the preparation of the hydrogel is usually carried out in an organic phase, because the carboxyl groups in the carboxylic acid are easy to form carboxylic acid ammonium salts with the amino groups in the amine, and the carboxylic acid ammonium salts, as a by-product of the amide condensation reaction, will reduce the reaction rate and reduce the yield of the target amide. In addition, the organic solvent can provide a better solvation environment, which helps to improve the activity of the reactants, thereby promoting the formation of amide bonds. At the same time, since the amide condensation reaction requires the use of condensing agents and catalysts, and these reagents have better solubility and reactivity in organic solvents, they can more efficiently catalyze the amide condensation reaction, reduce the occurrence of side reactions, and improve the selectivity of the target product. However, since most of the organic phase solvents are toxic, even if the prepared hydrogel is removed from the organic phase by dialysis, extraction or crystallization, the hydrogel itself will have slight toxicity.

[0037] In order to prepare hydrogels with low toxicity and good biocompatibility and biodegradability, the inventors have made a lot of attempts. For example, low-toxic organic reagents were used to prepare hydrogels, but the prepared hydrogels had poor biodegradability in the body. Condensation agents were used to directly catalyze amide condensation reactions in the aqueous phase, but the reaction rate was slow during the preparation of the hydrogels and the cross-linking of the hydrogels was poor.

[0038] By chance, the inventors reacted chitosan having amino and carboxyl groups with a condensation agent and synthesized a hydrogel with good biodegradability, high degree of cross-linking, non-toxicity and good biocompatibility under the catalysis of glacial acetic acid.

[0039] Figure 1 is a schematic flow chart of a method for preparing a hydrogel according to an embodiment of the present invention.

[0040] like Figure 1 As shown, the present invention provides a method for preparing a hydrogel in an aqueous phase, comprising the following steps:

[0041] Step S100: dissolving chitosan in water to prepare a chitosan solution having a first preset concentration, wherein the chitosan has a carboxyl group and an amino group;

[0042] Step S200: dissolving a condensing agent in water to prepare a reaction solution having a second preset concentration;

[0043] Step S300: mixing the chitosan solution and the reaction solution, adding glacial acetic acid, and reacting at a preset temperature and a preset pressure for a preset time to obtain a mixed solution;

[0044] Step S400: performing dialysis treatment on the mixed solution to obtain a hydrogel; wherein the ratio of the first preset concentration to the second preset concentration is any value in the range of 10:1-1:1.

[0045] In this embodiment, chitosan is first dissolved in water to prepare a chitosan solution with a first preset concentration, a condensing agent is dissolved in water to prepare a reaction solution with a second preset concentration, the chitosan solution and the reaction solution are mixed, glacial acetic acid is added, and the mixture is reacted at a preset temperature and a preset pressure for a preset time to obtain a mixed solution, and then the mixed solution is dialyzed to obtain a hydrogel.

[0046] In this embodiment, a chitosan solution having a first preset concentration and a reaction solution having a second preset concentration are mixed, and an amide condensation reaction is carried out under the catalysis of glacial acetic acid to condense the carboxyl groups and amino groups of the chitosan itself to form amide bonds, so that the chitosan itself is cross-linked to form a hydrogel, and impurities in the hydrogel are removed by dialysis treatment to obtain a hydrogel with low toxicity, good biocompatibility and biodegradability. Here, the ratio of the first preset concentration to the second preset concentration is any value in the range of 10:1-1:1, that is, the ratio of the first preset concentration to the second preset concentration can be 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1 or 1:1, or it can be any value in the range of 10:1-1:1. When the concentration of the chitosan solution and the concentration of the reaction solution are within the above range, the solubility of chitosan and the condensation agent in water is high and the reaction activity is high, so that the carboxyl group of chitosan can react completely with the amino group to form a hydrogel with a certain degree of cross-linking, thereby reducing the occurrence of side reactions.

[0047] In this embodiment, the dialysis treatment can remove small molecule impurities generated during the preparation of the hydrogel, and improve the mechanical strength, conductivity, biocompatibility, etc. of the hydrogel. Through the dialysis treatment, these small molecule impurities can be effectively removed from the hydrogel, thereby improving the purity and stability of the hydrogel.

[0048] In this embodiment, in the process of dissolving chitosan in water to form a chitosan solution, ultrasonic treatment or heating treatment is required to enable the chitosan to be evenly dispersed and completely dissolved in pure water.

[0049] In a further embodiment, chitosan is any one of carboxymethyl chitosan, N-carboxyethyl chitosan or N-succinyl chitosan. In this embodiment, chitosan can be any one of carboxymethyl chitosan, N-carboxyethyl chitosan or N-succinyl chitosan, or other chitosans containing both carboxyl groups and amino groups, so that an amide condensation reaction occurs under the action of a condensing agent and glacial acetic acid, so that chitosan undergoes a self-crosslinking reaction to form a hydrogel having amide bonds.

[0050] In a further embodiment, the condensing agent is any one of 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride or 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate. In this embodiment, the condensing agent is any one of 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride or 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate. The above condensing agents are all easily soluble in water, that is, the above condensing agents are dissolved in water, and the formed reaction solution has a certain reactivity with the chitosan solution in the aqueous phase, and can catalyze the amino group and carboxyl group of chitosan in the chitosan solution to undergo an amide condensation reaction, thereby increasing the reaction rate of the hydrogel, and then increasing the cross-linking degree of the hydrogel, so that the hydrogel has certain mechanical properties.

[0051] In the present embodiment, 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (DMTMM) is an efficient amide condensation agent, and its reaction mechanism for catalyzing the amide condensation reaction of chitosan is as follows: DMTMM first reacts with the carboxyl group on chitosan to form an active intermediate, which has a higher reactivity and can react more easily with the amino group. At the same time, the amino group on chitosan acts as a nucleophilic agent to attack the activated carboxyl intermediate to form an amide bond. While forming the amide bond, the leaving group (such as methoxy, etc.) in DMTMM is released to complete the condensation reaction. DMTMM has the advantages of high efficiency, low toxicity, and few side effects, and shows good performance when preparing amide bond hydrogels.

[0052] In this embodiment, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) is used as a condensing agent to catalyze the amide condensation reaction of chitosan. The reaction mechanism is as follows: EDC first reacts with the carboxyl group on chitosan to generate an unstable active ester intermediate. The amino group on chitosan acts as a nucleophilic agent to attack the active ester intermediate to form an amide bond. The formed amide bond cross-links the chitosan molecules, thereby stabilizing and forming a hydrogel. EDC has high reactivity and selectivity in the amide condensation reaction, and exhibits good performance in the preparation of amide bond hydrogels.

[0053] In this embodiment, 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), HATU is a highly efficient nitrogen onium salt condensing agent, and its reaction mechanism for catalyzing the amide condensation reaction of chitosan is as follows: HATU first reacts with the carboxyl group on chitosan to form a highly activated ester intermediate, the intermediate has a high reactivity and can react quickly with the amino group. The amino group on chitosan acts as a nucleophilic agent to attack the activated ester intermediate to form an amide bond and a leaving group. While the amide bond is formed, the leaving group is released into the solution to complete the condensation reaction. HATU has extremely high reactivity, so it exhibits a faster reaction speed and higher yield when preparing amide bond hydrogels.

[0054] In a further embodiment, the mass fraction of glacial acetic acid is any value in the range of 25%-60%. In this embodiment, the mass fraction of glacial acetic acid can be 25%, 30%, 35%, 40%, 45%, 50%, 55% or 60%, or any value in the range of 25%-60%. As a catalyst, glacial acetic acid can reduce the activation energy of the reaction, thereby accelerating the reaction process and promoting the amide condensation reaction between the carboxyl group and the amino group in chitosan. When the mass fraction of glacial acetic acid is within the above range, it has sufficient catalytic activity, effectively accelerates the amide condensation reaction, and makes chitosan cross-linked to form a hydrogel faster, while avoiding the adverse effects of excessive concentration, which helps to form a stable hydrogel with a certain mechanical strength. When the mass fraction of glacial acetic acid is less than 25%, the catalytic activity is low, resulting in insufficient catalytic action, slowing down the reaction rate, and resulting in insufficient cross-linking degree or reduced mechanical strength of the hydrogel. When the mass fraction of glacial acetic acid is higher than 60%, it may induce other side reactions or affect the properties of the product.

[0055] In a further embodiment, the preset temperature is any value in the range of 20°C-60°C, and the preset pressure is any value in the range of 0.03MPa-0.13MPa. In this embodiment, the preset temperature can be 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C or 60°C, or any value in the range of 20°C-60°C, and the preset pressure can be 0.03MPa, 0.05MPa, 0.07MPa, 0.09MPa, 0.11MPa or 0.13MPa, or any value in the range of 0.03MPa-0.13MPa. Chitosan, a condensing agent and glacial acetic acid undergo an amide condensation reaction under the above temperature and vacuum conditions to prepare a hydrogel with a stable cross-linked structure, reduce the occurrence of side reactions, and improve the purity of the product. That is, the preset temperature is controlled at 40℃-60℃, the reaction is relatively mild, which is conducive to controlling the reaction rate and product selectivity, and the mobility of the chitosan molecular chain is moderate, which is conducive to forming a stable cross-linked structure. When the preset temperature is controlled at 40℃-60℃, as the temperature increases, the reaction rate accelerates, the reaction time is shortened, and the mobility of the chitosan molecular chain is enhanced, which is conducive to a more complete cross-linking reaction. When the preset temperature is higher than 60℃, it may cause degradation of the chitosan molecular chain or increase of side reactions, affecting the properties of the product.

[0056] In a further embodiment, the step of dialyzing the mixed solution further comprises the following steps:

[0057] The mixed solution is replaced with dialysate at preset intervals.

[0058] In this embodiment, during the dialysis treatment of the mixed solution, the dialysis fluid of the mixed solution is replaced at preset intervals to ensure the dialysis effect and prevent the accumulation of solute concentration in the dialysis fluid from causing a decrease in dialysis efficiency. Here, the dialysis fluid is pure water, the preset time is any value in the range of 3h-5h, and the dialysis treatment time is 1.5 days-2.5 days, that is, the preset time can be 3h, 3.5h, 4h, 4.5h or 5h, or any value in the range of 3h-5h, and the dialysis treatment time can be 1.5 days, 2.0 days or 2.5 days, or any value in the range of 1.5 days-2.5 days.

[0059] In a further embodiment, the specification of the dialysis bag in the dialysis treatment process is any value in the range of 3000kDa-4000kDa. In this embodiment, the specification of the dialysis bag in the dialysis treatment process can be 3000kDa, 3200kDa, 3400kDa, 3600kDa, 3800kDa or 4000kDa, or any value in the range of 3000kDa-4000kDa, so as to filter out most of the solutes with a molecular weight smaller than the membrane pore size, achieve separation and purification of the solution, and adjust the internal structure and composition of the hydrogel, so that it has better mechanical properties and conductive properties.

[0060] In this embodiment, the hydrogel obtained after dialysis is frozen in a freeze dryer at -70°C for a set time, the preset time is any value in the range of 48h-72h, and ground into powder to prepare a hydrogel in a powder state for long-term storage. Here, the hydrogel in a powder state can be reconstituted with water before use. Here, the freezing setting time can be 48h, 50h, 55h, 60h, 65h, 70h or 72h, or any value in the range of 48h-72h.

[0061] According to the second aspect of the present invention, the present invention provides an application of a hydrogel in ultrasound detection of tumor immune microenvironment assessment, wherein the hydrogel is prepared according to the above preparation method. The preparation method of the hydrogel prepared in the aqueous phase will not be described in detail here.

[0062] It should be noted here that clinically, the use of ultrasound to determine whether a tumor is benign or malignant is mainly based on the ultrasound diagnosis physician's analysis of the sonographic features of the patient's lesions, combined with his own experience and comparison with international ultrasound diagnostic standards, to make a comprehensive judgment. That is, the final diagnosis result is based on the physician's subjective judgment, which has certain limitations and is prone to misjudgment. Since ultrasound physicians are usually proficient in the actual operation of ultrasound diagnostic technology and the analysis of clinical imaging features, they have a shallow understanding of the design and development of medical materials, and the basic researchers of related chemical synthesis do not understand the needs of frontline clinical diagnostic physicians and the clinical problems that need to be solved urgently, it is difficult to achieve the combination of medicine and engineering, and it is difficult to design medical materials or new medical devices that can significantly improve the effectiveness of ultrasound diagnosis.

[0063] In this embodiment, the hydrogel has reactive oxygen species (ROS) responsiveness and pH responsiveness, and can react with excess ROS and hydrogen ions in the tumor microenvironment, consume hydrogen ions to improve the acidic pH, and regulate the level of ROS in the tumor microenvironment. The hydrogel itself becomes soft or even degrades, that is, the hardness of the hydrogel is negatively correlated with the degree of acidification in the tumor microenvironment. It can assist in the diagnosis and differentiation of benign and malignant tumors according to the ultrasonic imaging characteristics in the above different modes, and even infer the immune status in malignant tumors. Here, the hydrogel enters the tumor by injection.

[0064] In this embodiment, the amide bonds in the hydrogel can consume ROS and hydrogen ions accumulated in the local tumor, improve the immunosuppressive microenvironment and the local hypoxic conditions of the tumor, and use this treatment system in combination with other tumor treatment methods to enhance tumor immunotherapy.

[0065] In this embodiment, chitosan and the condensation agent have good biocompatibility, enter the body by injection, and can degrade by themselves after the ultrasonic detection is completed, and will not degrade to produce substances harmful to the human body. Chitosan is a monosaccharide structure but not glucose, and has no effect on blood sugar after degradation. In addition, chitosan itself has the function of astringency and hemostasis, and its amino protonation can also play a natural antibacterial role, reducing the trauma caused to the human body by the implantation operation.

[0066] The present application will be further described in detail below with reference to specific embodiments.

[0067] First, 100 mg of carboxymethyl chitosan was dissolved in 10 mL of ultrapure water to prepare a 10 mg / mL chitosan solution, labeled as solution A; then, 150 mg of DMTMM was dissolved in 10 mL of ultrapure water to prepare a 1.5 mg / mL solution, labeled as solution B. We mixed 5 mL of solution A with 1 mL of solution B, added 100 uL of 40% glacial acetic acid, stirred for 10 h at 40 ° C and 0.10 MPa to obtain a mixed solution, transferred the mixed solution to a 3500 kDa dialysis bag, and placed the dialysis bag containing the mixed solution in ultrapure water for 2 days. Ultrapure water was changed every 4 hours. After dialysis, wet CMCS hydrogel was obtained.

[0068] Firstly, the degradation of hydrogel in H2O2 solution environment was characterized. Figure 2 is the degradation of hydrogel in water, H2O2 and HCl, Figure 3 It is the degradation of hydrogel in water, H2O2 and HCl after 0h, 0.25h, 0.5h and 1h of reaction.

[0069] like Figure 2As shown, the hydrogel basically does not undergo degradation reaction in water, and fluidized hydrogel is formed after the reaction with H2O2 and HCl, indicating that the hydrogel will undergo degradation reaction in H2O2 and HCl.

[0070] like Figure 3 As shown, the hydrogel reacted in water for 0h, 0.25h, 0.5h, and 1h. As time went by, the state of the hydrogel at the bottom of the reaction flask remained basically unchanged. The hydrogel reacted in H2O2 for 0h, 0.25h, 0.5h, and 1h. As time went by, the hydrogel at the bottom of the reaction flask was basically degraded and completely formed into a fluidized hydrogel. The hydrogel reacted in HCl for 0h, 0.25h, 0.5h, and 1h. As time went by, the hydrogel at the bottom of the reaction flask was basically degraded and completely formed into a fluidized hydrogel, indicating that the hydrogel can degrade to form a fluidized hydrogel under acidic conditions.

[0071] Then, the hydrogel and the hydrogel after reaction with H2O2 and HCl were characterized by SEM. Figure 4 This is the SEM image of the hydrogel and the hydrogel after reaction with H2O2. Figure 5 are SEM images of the hydrogel and the hydrogel after reaction with HCl.

[0072] like Figure 4 and Figure 5 As shown, the hydrogel has a porous network structure with uniform pore distribution and a clearly visible skeleton structure. After reacting with H2O2 and HCl, the hydrogel has a porous network structure with larger pore distribution and a blurred or broken skeleton, indicating that degradation occurs after the hydrogel reacts with H2O2 and HCl.

[0073] The rheological behavior of the hydrogel under dynamic stress before and after reaction with H2O2 and HCl was further characterized. Figure 6 is the rheological diagram of the hydrogel before reacting with H2O2 and HCl. Figure 7 is the rheological diagram of the hydrogel after reaction with H2O2 and HCl.

[0074] like Figure 6 and Figure 7 As shown, the hydrogel after reaction with H2O2 and HCl has a certain hexagonal shape, indicating that the hydrogel becomes softer, has poorer viscoelasticity, and has lower mechanical stress after degradation.

[0075] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0076] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A method for preparing a hydrogel in an aqueous phase, characterized in that: The steps include: Dissolving chitosan in water to prepare a chitosan solution having a first preset concentration, wherein the chitosan has a carboxyl group and an amino group; Dissolving a condensing agent in water to prepare a reaction solution having a second preset concentration; The chitosan solution and the reaction solution are mixed, glacial acetic acid is added, and the mixture is reacted at a preset temperature and a preset pressure for a preset time to obtain a mixed solution; The mixed solution is dialyzed to obtain the hydrogel; wherein, The ratio of the first preset concentration to the second preset concentration is any value in the range of 10:1-1:

1.

2. The preparation method according to claim 1, characterized in that: The chitosan is any one of carboxymethyl chitosan, N-carboxyethyl chitosan or N-succinyl chitosan.

3. The preparation method according to claim 2, characterized in that: The condensing agent is any one of 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride or 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate.

4. The preparation method according to claim 3, characterized in that: The mass fraction of the glacial acetic acid is any value in the range of 25%-60%.

5. The preparation method according to claim 4, characterized in that: The preset temperature is any value in the range of 20° C.-60° C., and the preset pressure is any value in the range of 0.03 MPa-0.13 MPa.

6. The preparation method according to claim 5, characterized in that: The step of dialyzing the mixed solution further comprises the following steps: The mixed solution is replaced with dialysate at preset time intervals.

7. The preparation method according to any one of claims 1 to 6, characterized in that: The specification of the dialysis bag in the dialysis treatment process is any value in the range of 3000 kDa-4000 kDa.

8. An application of a hydrogel in ultrasound detection for evaluating tumor immune microenvironment, characterized in that: The hydrogel is prepared according to the preparation method described in any one of claims 1-7.

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