Preparation method and application of a double-crosslinked ultrasonic response hyaluronic acid hydrogel

Abstract

The application discloses a preparation method of a double-crosslinking ultrasonic response hyaluronic acid hydrogel, and belongs to the field of drug delivery. First, hyaluronic acid is used as raw material to prepare hyaluronic acid dopamine through an amidation reaction; then, a block polymer poly(polyethylene glycol methacrylate-co-4-vinylphenylboronic acid) is synthesized through a copolymerization reaction, and is mixed and dissolved with the hyaluronic acid dopamine in deionized water to obtain a mixed solution; after preparing drug-loaded iron oxide nanomagnetic microbubbles, the drug-loaded iron oxide nanomagnetic microbubbles are added into the mixed solution to obtain a hydrogel solution; after adjusting the pH value to 5-6, the hydrogel solution is left to stand, and the hyaluronic acid hydrogel is obtained. The application solves the problem that traditional single hyaluronic acid hydrogel often has insufficient mechanical strength and elasticity, the prepared hyaluronic acid hydrogel has excellent mechanical strength and structural stability, can release drugs through ultrasonic, has good release effect, has the characteristics of low toxicity and good biocompatibility, and has good application prospect in the field of biological application.

Description

Technical Field

[0001] This invention belongs to the field of drug delivery technology, specifically relating to a method for preparing a double-crosslinked ultrasound-responsive hyaluronic acid hydrogel and its application. Background Technology

[0002] In the fields of tissue engineering and drug delivery, the construction of safe and effective tissue engineering and drug delivery systems is crucial, making efficient, precise, and safe drug delivery systems a research focus. Drug delivery hydrogel systems have attracted considerable attention as a promising drug delivery technology. These systems load drugs into a gel, allowing for control of drug release kinetics through gel degradation, thereby improving drug bioavailability and stability. Therefore, hydrogel design is extremely important. Ideal hydrogel materials need to possess good biocompatibility and degradability, as well as sufficient strength and rigidity to control drug release through various mechanisms such as diffusion, expansion, and degradation.

[0003] In existing technologies, hydrogels are a class of materials with a three-dimensional network structure formed by chemical or physical cross-linking of hydrophilic polymer chains. Their main components include natural polymers (such as hyaluronic acid and chitosan), synthetic polymers (such as polyacrylamide and polyvinyl alcohol), and inorganic materials (such as nanoclay). They can be designed for different types of drug delivery in specific applications. In recent years, hydrogels have been widely used as drug carriers in tissue repair, bone repair, cardiac regeneration, and vascular remodeling. Controlled drug release is a common goal for hydrogels in scaffold-based drug delivery. However, due to the significant variations in the normal human microenvironment and physiological conditions between different disease sites, ordinary hydrogels as local drug release systems may not achieve the desired results in complex pathological microenvironments. Furthermore, ordinary hydrogels generally suffer from poor physical properties, such as low mechanical strength, poor biocompatibility, and unstable drug release, thus limiting their application.

[0004] Among the many hydrogel materials currently used, hyaluronic acid (HA) has become a highly regarded primary material in the field of drug delivery due to its ability to mimic the physiological conditions of the natural tissue microenvironment, its unique biocompatibility, biodegradability, and excellent interaction with cells and tissues, as well as its superior drug release control properties. Hyaluronic acid is a polysaccharide naturally found in human tissues, exhibiting excellent biocompatibility and biodegradability. Hyaluronic acid hydrogels, with their three-dimensional network structure, can effectively encapsulate various drug molecules, providing a stable storage environment for drugs. Their high water content and soft texture ensure good compatibility with biological tissues, reducing the risk of inducing inflammation and immune rejection. During drug delivery, hyaluronic acid hydrogels can achieve precise control of drug release by adjusting their molecular structure, degree of cross-linking, and degradation rate. This is crucial for treatment regimens requiring long-term continuous release or release at specific time points. Currently, HA is widely used in the treatment of various diseases, such as arthritis and eye diseases. However, many challenges remain in practical applications. On the one hand, traditional HA hydrogels often lack the mechanical strength required for precise control, limiting their use in certain applications. On the other hand, controlled drug release within hydrogels presents challenges in controlling the release rate. Existing drug delivery methods, such as physical encapsulation and chemical assembly, struggle to achieve precise, on-demand drug release, resulting in low drug utilization efficiency and limited therapeutic efficacy.

[0005] In view of this, this invention proposes a novel dual crosslinking strategy aimed at controlling the mechanical strength of the hydrogel to meet higher requirements, while simultaneously achieving on-demand drug release under controlled external conditions. By introducing specific crosslinking agents to construct a complex and stable crosslinking network, not only are the mechanical properties of the hyaluronic acid hydrogel enhanced, but it also provides the possibility for precise controlled drug release, potentially solving key problems in the current drug delivery field and expanding the application scope of hydrogels in the medical field. Summary of the Invention

[0006] The purpose of this invention is to solve the problems of insufficient mechanical strength, poor biocompatibility, and difficulty in achieving precise on-demand drug release in existing hydrogels. This invention provides a method for preparing a double-crosslinked, ultrasound-responsive hyaluronic acid hydrogel. The prepared hydrogel has excellent biocompatibility and mechanical strength, and can achieve controllable and precise drug release.

[0007] This invention uses hyaluronic acid as the main material. Catechol groups are grafted onto the hyaluronic acid backbone via an amide reaction for functionalization. Boric acid groups are introduced into polyethylene glycol methacrylate to form borate ester bonds with caustic acid, serving as the first crosslinking of the gel. Simultaneously, bubbles carrying drugs and iron oxide nanoparticles are embedded within the gel, utilizing the covalent interaction between the caustic acid groups and the iron oxide nanoparticles as the second crosslinking of the gel. The drug-loaded bubbles encapsulated with iron oxide nanoparticles act as ultrasonic response units. Under ultrasound, the bubbles vibrate stably, releasing the drug and iron oxide nanoparticles from their surface, achieving ultrasound-controlled drug release from the hydrogel scaffold. The specific scheme is as follows:

[0008] A method for preparing a double-crosslinked, ultrasound-responsive hyaluronic acid hydrogel includes the following steps:

[0009] (1) Hyaluronic acid was dissolved in phosphate buffer at pH 5.5, and then 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and N-hydroxysuccinimide ester (NHS) were added. After stirring evenly, dopamine hydrochloride was added. After stirring evenly under nitrogen protection, the pH was adjusted to 5-5.5, and the reaction was carried out overnight at room temperature. The reaction product was collected and dialyzed. Finally, it was freeze-dried under vacuum to obtain hyaluronic acid dopamine.

[0010] (2) 4-vinylphenylboronic acid was copolymerized with polyethylene glycol methacrylate to obtain the block polymer poly(polyethylene glycol methacrylate-co-4-vinylphenylboronic acid);

[0011] (3) Hyaluronic acid dopamine and block polymer poly(polyethylene glycol methacrylate-co-4-vinylphenylboronic acid) were mixed and dissolved in deionized water to obtain a mixture;

[0012] (4) Prepare drug-loaded iron oxide nanomagnetic microbubbles, and then add them to the mixture in step (3), stir evenly, and obtain a hydrogel solution;

[0013] Further, in step (4), the preparation method of the drug-loaded iron oxide magnetic nanobubbles is as follows: prepare a drug solution of 0.6 mg / mL, a sodium dodecyl sulfate aqueous solution of 10 mM concentration, and a Fe3O4 aqueous solution of 2-10 mg / mL, respectively, and then mix them in a volume ratio of 3:3:8. Place them in an ice bath environment, stir them evenly with a homogenizer at a speed of not less than 20,000 rpm, let them stand for more than 12 hours, and then purify and wash them at least three times with a magnet and deionized water to obtain drug-loaded iron oxide magnetic nanobubbles.

[0014] (5) Add alkali solution to the hydrogel solution prepared in step (4) to make the pH value 5-6, mix evenly and let stand to obtain a double cross-linked ultrasound-responsive hyaluronic acid hydrogel.

[0015] Furthermore, in step (1), the molecular weight of the hyaluronic acid is 200,000 to 400,000.

[0016] Furthermore, in step (1), the dialysis uses a dialysis bag with a molecular weight cutoff of 35KD, and the vacuum freeze-drying temperature is -80℃ and the time is 48 to 96 hours.

[0017] Furthermore, in step (1), the molar ratio of hyaluronic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, N-hydroxysuccinimide ester, and dopamine hydrochloride is 1:1:2:1.

[0018] Further, in step (2), the block polymer poly(polyethylene glycol methacrylate-co-4-vinylphenylboronic acid) is prepared as follows: 191.8 mg of 4-vinylphenylboronic acid, 2.4 mL of polyethylene glycol methacrylate, and 10 mg of initiator are added to a container containing 3.5 mL of 1,4-dioxane. After mixing evenly, a reaction solution is obtained. Nitrogen gas is introduced into the container and the mixture is continuously stirred. The reaction solution is heated to 80°C. After stirring and reacting, the reaction product is precipitated in n-hexane and then dissolved using 1,4-dioxane. The precipitation and dissolution operations are repeated 3 times. Finally, the precipitate is vacuum dried overnight to obtain the block polymer poly(polyethylene glycol methacrylate-co-4-vinylphenylboronic acid).

[0019] Furthermore, in step (3), the mass concentration of hyaluronic acid dopamine in the mixture is 50 mg / mL, and the mass concentration of the block polymer poly(polyethylene glycol methacrylate-co-4-vinylphenylboronic acid) is 75 mg / mL.

[0020] Furthermore, in step (4), the amount of drug-loaded iron oxide nanomagnetic microbubbles added is 50 mg / mL.

[0021] Application of the double cross-linked ultrasound-responsive hyaluronic acid hydrogel prepared by the above method in drug delivery.

[0022] The beneficial effects of this invention are:

[0023] 1) This invention provides a method for preparing a dual-crosslinked, ultrasound-responsive hyaluronic acid hydrogel. Using hyaluronic acid as the main material, catechol groups are grafted onto the HA backbone via an amide reaction for functionalization. Boric acid groups are introduced to form borate ester bonds with catechol as the first crosslinking of the gel. Simultaneously, drug-loaded iron oxide nanoparticles encapsulate air bubbles within the gel, utilizing the covalent interaction between the catechol groups and the iron oxide nanoparticles as the second crosslinking. The drug-loaded air bubbles encapsulated within the iron oxide nanoparticles act as an ultrasound-responsive unit. Under ultrasound, these bubbles undergo stable vibration, releasing the drug and iron oxide nanoparticles from their surface, achieving ultrasound-controlled drug release.

[0024] 2) This invention innovatively proposes a dual crosslinking strategy, which significantly improves the performance of hydrogels. In terms of mechanical strength, traditional single-linked hyaluronic acid hydrogels often suffer from insufficient mechanical strength and elasticity, making them unsuitable for certain applications. However, the dual crosslinking strategy of this invention enhances the mechanical strength of the hydrogel. Under the same testing conditions, the modulus of traditional single-linked hyaluronic acid hydrogels is 100–500 Pa, while the hydrogel prepared using the dual crosslinking strategy of this invention exhibits a significantly increased modulus of 1000–1200 Pa. This significant modulus improvement not only enhances the structural stability of the hydrogel but may also increase its applicability in biomedical applications such as myocardial repair.

[0025] 3) The double cross-linked ultrasound-responsive hyaluronic acid hydrogel of the present invention can release drugs through ultrasound and has a good release effect.

[0026] 4) The double cross-linked ultrasound-responsive hyaluronic acid hydrogel structure of the present invention is simple in design, widely available, and inexpensive.

[0027] 5) The material used in the double cross-linked ultrasound-responsive hyaluronic acid hydrogel of the present invention has the characteristics of low toxicity and good biocompatibility, and has a good application prospect in the field of biological applications. Attached Figure Description

[0028] Figure 1 The ultraviolet spectrum of the hyaluronic acid dopamine prepared in Example 1;

[0029] Figure 2 Raman spectra of the hydrogels prepared in Example 1 and Comparative Examples 1-2;

[0030] Figure 3 Scanning electron microscope image of the double crosslinked ultrasound-responsive hyaluronic acid hydrogel prepared in Example 1;

[0031] Figure 4The rheological property curves of the hydrogels prepared in Example 1 and Comparative Examples 1-2 are shown.

[0032] Figure 5 The results of the drug release rate test for the double crosslinked ultrasound-responsive hyaluronic acid hydrogel prepared in Example 1;

[0033] Figure 6 Cell adhesion fluorescence confocal three-dimensional imaging of the double cross-linked ultrasound-responsive hyaluronic acid hydrogel prepared in Example 1;

[0034] Figure 7 The results of the cytotoxicity test for the double crosslinked ultrasound-responsive hyaluronic acid hydrogel prepared in Example 1 are shown. Detailed Implementation

[0035] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0036] Example 1

[0037] A method for preparing a double-crosslinked, ultrasound-responsive hyaluronic acid hydrogel includes the following steps:

[0038] (1) Dissolve 1g of hyaluronic acid (molecular weight of 300,000) in 100mL of phosphate buffer with pH 5.5, then add 338mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and 405mg of N-hydroxysuccinimide ester (NHS), stir well, and then add 2.358g of dopamine hydrochloride. The molar ratio of hyaluronic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, N-hydroxysuccinimide ester and dopamine hydrochloride is 1:1:2:1. After stirring well under nitrogen protection, adjust the pH to 5-5.5 with hydrochloric acid or sodium hydroxide, maintain the pH value unchanged and react for half an hour, then continue to react overnight at room temperature. Collect the reaction product, dialyze it using a dialysis bag with a molecular weight cutoff of 35KD, and finally freeze-dry it under vacuum at -80℃ for 72h to obtain hyaluronic acid dopamine.

[0039] (2) 191.8 mg of 4-aminostyrene-1-carboxylic acid ester, 2.4 mL of polyethylene glycol methacrylate, and 10 mg of initiator (azobisisobutyronitrile) were added to a flask containing 3.5 mL of 1,4-dioxane. The mixture was sonicated for 2 min to obtain a reaction solution. Nitrogen gas was introduced into the container for 30 min while stirring continuously. The reaction solution was heated to 80 °C. After stirring, the reaction product was precipitated in n-hexane and then dissolved using 1,4-dioxane. The precipitation and dissolution operations were repeated 3 times. Finally, the precipitate was vacuum dried overnight to obtain the block polymer poly(polyethylene glycol methacrylate-co-4-vinylphenylboronic acid).

[0040] (3) Mix 50 mg of hyaluronic acid dopamine with 75 mg of block polymer poly(polyethylene glycol methacrylate-co-4-vinylphenylboronic acid) and dissolve in 1 mL of deionized water to obtain a mixture;

[0041] (4) First, prepare a 0.6 mg / mL solution of the drug to be loaded (modified Cy5.5 HSA human serum albumin), a 10 mM sodium dodecyl sulfate aqueous solution and a 6 mg / mL Fe3O4 aqueous solution, then mix them in a volume ratio of 3:3:8 and place them in an ice bath environment. Use a homogenizer to stir at a speed of not less than 20,000 rpm for 1-3 min, then let it stand for more than 12 h, and then purify and wash it at least three times with a magnet and deionized water to obtain drug-loaded iron oxide nanomagnetic microbubbles; add the drug-loaded iron oxide nanomagnetic microbubbles to the mixture in step (3) at an addition amount of 50 mg / mL, stir evenly to obtain a hydrogel solution;

[0042] (5) Add 1 mol / L sodium hydroxide solution to the hydrogel solution prepared in step (4) until the pH of the system is 5-6. After mixing evenly, let stand to obtain a double cross-linked ultrasonic-responsive hyaluronic acid hydrogel.

[0043] Figure 1 The image shows the ultraviolet spectrum of the hyaluronic acid dopamine prepared in Example 1. It can be seen that, compared with dopamine, hyaluronic acid dopamine has a peak at a wavelength of 276 nm near the characteristic peak of the catechol group of dopamine at 280 nm. Therefore, it is determined that the synthesis of hyaluronic acid dopamine was successful.

[0044] Comparative Example 1

[0045] A method for preparing hyaluronic acid-dopamine hydrogel: 1g of hyaluronic acid (molecular weight 300,000) is dissolved in 100mL of phosphate buffer with pH 5.5, then 338mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and 405mg of N-hydroxysuccinimide ester (NHS) are added. After stirring evenly, 2.358g of dopamine hydrochloride is added. After stirring evenly under nitrogen protection, the pH is adjusted to 5-5.5 with hydrochloric acid or sodium hydroxide. The pH is maintained at a constant value for half an hour, and then the reaction is continued overnight at room temperature. The reaction product is collected and dialyzed using a dialysis bag with a molecular weight cutoff of 35KD to obtain hyaluronic acid-dopamine hydrogel.

[0046] Comparative Example 2

[0047] A method for preparing a single crosslinked hyaluronic acid hydrogel: The preparation of hyaluronic acid dopamine and block polymer poly(polyethylene glycol methacrylate-co-4-vinylphenylboronic acid) is the same as in Example 1. 50 mg of hyaluronic acid dopamine and 75 mg of block polymer poly(polyethylene glycol methacrylate-co-4-vinylphenylboronic acid) are mixed and dissolved in 1 mL of deionized water to obtain a mixture. 1 mol / L sodium hydroxide solution is added until the pH of the system is 5-6. After mixing evenly, the mixture is allowed to stand to obtain the final product.

[0048] Figure 2 The Raman spectra of the hydrogels prepared in Example 1 and Comparative Examples 1-2 are shown in the figures. It can be seen from the figures that the Raman spectra of the hyaluronic acid-dopamine hydrogel, the single-crosslinked hyaluronic acid hydrogel, and the double-crosslinked ultrasound-responsive hyaluronic acid hydrogel prepared in Example 1 are all in the range of 1250–1500 cm⁻¹. -1 The position exhibits characteristic peaks of benzene ring vibration, observed in the double-crosslinked ultrasonically responsive hyaluronic acid hydrogel prepared in Example 1 at 550–650 cm⁻¹. -1 Catechol-iron coordination.

[0049] Figure 3 This is a scanning electron microscope image of the double-crosslinked, ultrasound-responsive hyaluronic acid hydrogel prepared in Example 1. Figure 3 It can be seen that the hydrogel is filled with a large number of interconnected irregular pores, exhibiting a porous structure.

[0050] 1. The rheological properties of the hydrogels prepared in Example 1 and Comparative Examples 1-2 were tested using a rheometer. The dynamic frequency scanning test was performed at 25°C using a 25 mm parallel plate in a frequency range of 0.1 to 100 rad / s and a strain of 1%.

[0051] Figure 4 The rheological property curves of the hydrogels prepared in Example 1 and Comparative Examples 1-2 are shown below. Figure 4 It can be seen that, under the same test conditions, the hydrogels prepared by Comparative Example 1 and Comparative Example 2 have a modulus of 100 to 500 Pa, while the hydrogel prepared by Example 1 of the present invention has a modulus that is significantly increased to 1000 Pa to 1200 Pa. This significant increase in modulus not only improves the structural stability of the hydrogel, but may also enhance its applicability in biomedical applications such as myocardial repair.

[0052] 2. Test the drug release rate of the double cross-linked ultrasound-responsive hyaluronic acid hydrogel prepared in Example 1.

[0053] Step 1: The double cross-linked ultrasound-responsive hyaluronic acid hydrogel (loaded with HSA human serum albumin) prepared in Example 1 was placed into a glass bottle. Then, ultrasound was applied using a focused ultrasound transducer. The ultrasound frequency was adjusted to the range of 400-800 kHz using a function generator, and the amplitude was set to 20 Vpp. Ultrasound was applied using a focused ultrasound transducer. The time of each ultrasound cycle was set to 4 seconds, and the ultrasound cycle was 50 cycles.

[0054] Step 2: After the ultrasonic cycle is completed, soak in PBS. After 2 hours, collect the supernatant to measure the fluorescence value of the released solution, and use the corresponding protein detection kit to determine the quantitative protein content.

[0055] Figure 5 The drug release rate test results for the double cross-linked ultrasound-responsive hyaluronic acid hydrogel prepared in Example 1 show that the drug release is excellent, reaching 83%.

[0056] 3. Test the biocompatibility of the double cross-linked ultrasound-responsive hyaluronic acid hydrogel prepared in Example 1.

[0057] Detection method: Human umbilical vein endothelial cells (HUVECs) were cultured in DMEM medium containing 10% FBS until they reached a good condition. The double cross-linked ultrasound-responsive hyaluronic acid hydrogel prepared in Example 1 was sterilized. Then, cells in the logarithmic growth phase were seeded at a density of 10,000 / mL on the surface of the sterilized hydrogel and placed in an incubator at 37°C with 5% CO2. After 24 hours, live cells were labeled with fluorescent dye am. The sample was removed, rinsed with PBS, and three-dimensional distribution images of cells in the hydrogel were obtained by confocal microscopy to observe cell distribution and adhesion in order to evaluate the compatibility between the hydrogel and cells.

[0058] Figure 6 The image shows a three-dimensional confocal fluorescence image of cell adhesion in the double-crosslinked ultrasound-responsive hyaluronic acid hydrogel prepared in Example 1. It can be seen that the cells are evenly distributed in the hydrogel and are tightly adhered to the surface and internal structure of the hydrogel, showing good cell compatibility.

[0059] 4. Test the cytotoxicity of the double cross-linked ultrasound-responsive hyaluronic acid hydrogel prepared in Example 1.

[0060] Detection Method: Human umbilical vein endothelial cells (HUVECs) were cultured to the logarithmic growth phase in DMEM high-glucose medium (containing both antibiotics and serum-containing media). The hydrogel samples prepared in Example 1 and Comparative Example 2 were sterilized and soaked in serum-free medium to prepare two hydrogel degradation solutions. HUVECs were seeded at a specific density in 96-well plates, with an appropriate amount of cell suspension added to each well. A blank control group and two experimental groups were established: a blank control group (normal medium without hydrogel degradation solution), experimental group 1 (medium medium with Comparative Example 2 hydrogel degradation solution), and experimental group 2 (medium medium with Example 1 hydrogel degradation solution). The 96-well plates were incubated for 48 hours. After incubation, CCK8 detection solution was added and incubated for an appropriate time. Finally, the absorbance was measured using a microplate reader, and cell viability was calculated to assess the cytotoxicity of the hydrogels.

[0061] Figure 7 The cytotoxicity test results of the double cross-linked ultrasound-responsive hyaluronic acid hydrogel prepared in Example 1 show that the double cross-linked ultrasound-responsive hyaluronic acid hydrogel prepared in Example 1 has low cytotoxicity.

[0062] The above figures and test data fully demonstrate that the hydrogel of this invention possesses excellent mechanical properties, biocompatibility, and superior drug release performance. Furthermore, the preparation process is simple, low-cost, and energy-saving, facilitating large-scale research and application. In addition, it provides strong support for its wide application in the biomedical field and has significant research value in the field of drug delivery.

Claims

1. A method for preparing a double-crosslinked, ultrasound-responsive hyaluronic acid hydrogel, characterized in that, Includes the following steps: (1) Hyaluronic acid was dissolved in phosphate buffer at pH 5.5, and then 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide ester were added. After stirring evenly, dopamine hydrochloride was added. After stirring evenly under nitrogen protection, the pH was adjusted to 5-5.5, and the reaction was carried out overnight at room temperature. The reaction product was collected and dialyzed. Finally, it was freeze-dried under vacuum to obtain hyaluronic acid dopamine. (2) 4-vinylphenylboronic acid was copolymerized with polyethylene glycol methacrylate to obtain the block polymer poly(polyethylene glycol methacrylate-co-4-vinylphenylboronic acid); (3) Hyaluronic acid dopamine and block polymer poly(polyethylene glycol methacrylate-co-4-vinylphenylboronic acid) were mixed and dissolved in deionized water to obtain a mixture; (4) Prepare drug-loaded iron oxide nanomagnetic microbubbles, and then add them to the mixture in step (3), stir evenly, and obtain a hydrogel solution; (5) Add alkali solution to the hydrogel solution prepared in step (4) to make the pH value 5-6, mix evenly and let stand to obtain a double cross-linked ultrasound-responsive hyaluronic acid hydrogel.

2. The method for preparing the double-crosslinked ultrasound-responsive hyaluronic acid hydrogel as described in claim 1, characterized in that, In step (1), the molecular weight of the hyaluronic acid is 200,000 to 400,000.

3. The method for preparing the double-crosslinked ultrasound-responsive hyaluronic acid hydrogel as described in claim 1, characterized in that, In step (1), the dialysis uses a dialysis bag with a molecular weight cutoff of 35KD, and the vacuum freeze-drying temperature is -80℃ and the time is 48 to 96 hours.

4. The method for preparing the double-crosslinked ultrasound-responsive hyaluronic acid hydrogel as described in claim 1, characterized in that, In step (1), the molar ratio of hyaluronic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, N-hydroxysuccinimide ester, and dopamine hydrochloride is 1:1:2:

1.

5. The method for preparing the double-crosslinked ultrasound-responsive hyaluronic acid hydrogel as described in claim 1, characterized in that, In step (2), the block polymer poly(polyethylene glycol methacrylate-co-4-vinylphenylboronic acid) is prepared as follows: 191.8 mg of 4-vinylphenylboronic acid, 2.4 mL of polyethylene glycol methacrylate, and 10 mg of initiator are added to a container containing 3.5 mL of 1,4-dioxane. After mixing evenly, a reaction solution is obtained. Nitrogen gas is introduced into the container and the mixture is continuously stirred. The reaction solution is heated to 80°C. After stirring and reacting, the reaction product is precipitated in n-hexane and then dissolved using 1,4-dioxane. The precipitation and dissolution operations are repeated 3 times. Finally, the precipitate is vacuum dried overnight to obtain the block polymer poly(polyethylene glycol methacrylate-co-4-vinylphenylboronic acid).

6. The method for preparing the double-crosslinked ultrasound-responsive hyaluronic acid hydrogel as described in claim 1, characterized in that, In step (3), the mass concentration of hyaluronic acid dopamine in the mixture is 50 mg / mL, and the mass concentration of block polymer poly(polyethylene glycol methacrylate-co-4-vinylphenylboronic acid) is 75 mg / mL.

7. The method for preparing the double-crosslinked ultrasound-responsive hyaluronic acid hydrogel as described in claim 1, characterized in that, In step (4), the preparation method of the drug-loaded iron oxide magnetic nanobubbles is as follows: prepare a drug solution of 0.6 mg / mL, a sodium dodecyl sulfate aqueous solution of 10 mM and a Fe3O4 aqueous solution of 2-10 mg / mL respectively, mix them in a volume ratio of 3:3:8, place them in an ice bath environment, stir them evenly with a homogenizer at a speed of not less than 20,000 rpm, let them stand for more than 12 hours, and then purify and wash them at least three times with a magnet and deionized water to obtain drug-loaded iron oxide magnetic nanobubbles.

8. The method for preparing the double-crosslinked ultrasound-responsive hyaluronic acid hydrogel as described in claim 1, characterized in that, In step (4), the amount of drug-loaded iron oxide nanomagnetic microbubbles added is 50 mg / mL.

9. The use of the double-crosslinked ultrasound-responsive hyaluronic acid hydrogel prepared by the method of any one of claims 1 to 8 in the preparation of a drug delivery carrier.

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