Injectable hydrogel and preparation method and application thereof

Abstract

The application provides an injectable hydrogel and a preparation method and application thereof, and belongs to the technical field of biomedical material development, and the preparation method of the injectable hydrogel comprises the following steps: respectively dissolving oxidized hyaluronic acid, carboxymethyl chitosan and dopamine-modified hyaluronic acid in a buffer solution to obtain an oxidized hyaluronic acid solution, a carboxymethyl chitosan solution and a dopamine-modified hyaluronic acid solution; mixing the oxidized hyaluronic acid solution, the carboxymethyl chitosan solution and the dopamine-modified hyaluronic acid solution, and standing to obtain the injectable hydrogel. The injectable hydrogel has high safety, good biocompatibility, can be used for heart site drug delivery, and can continuously exert a supporting effect by taking natural polysaccharides as the basis and utilizing some special structures.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials development technology, and particularly relates to an injectable hydrogel, its preparation method and application. Background Technology

[0002] Cardiomyocyte apoptosis is a direct consequence of various acute and chronic cardiac injuries, including but not limited to ischemia (such as myocardial infarction), drug-induced damage (such as doxorubicin cardiomyopathy), and disease-related damage (such as acute and chronic myocarditis). This leads to extracellular matrix (ECM) remodeling or hypertrophy, resulting in scarring. If left uncontrolled, it can ultimately lead to heart failure. Current clinical treatments aim to slow the progression of heart failure through diuresis, vasodilation, or reducing cardiac workload. However, existing treatments suffer from drawbacks such as not being able to reverse the progression of heart failure, low drug bioavailability, and systemic side effects.

[0003] With the development of biomaterials, some biomaterials capable of restoring tissue function are gradually being used to treat damaged cardiac tissue. Strategies such as using hydrogels to provide structural support for damaged hearts, soften scar tissue, and protect cardiomyocytes from oxidative stress and inflammatory damage have been proven beneficial for the structural restoration of the heart.

[0004] Local injection therapy is a practical and highly efficient method of drug delivery. Compared to traditional cardiac surgery, injectable hydrogels allow for minimally invasive treatment via local injection, reducing the risks associated with surgery and secondary damage to local soft tissues. Various methods exist to achieve the injectable effect, such as thermosensitive materials, enzyme-responsive gelling materials, controlled gelation time, and post-injection self-healing. Thermosensitive materials are primarily based on N-isopropylacrylamide with methylenebisacrylamide as the cross-linking structure, constructed through carbon-carbon double bond polymerization. Both N-isopropylacrylamide and methylenebisacrylamide can form hydrogels at 37°C after injection; however, the metabolic issues associated with these synthetic materials significantly limit their clinical translation. Enzyme-responsive gelling materials are mostly derived from biological extracts, such as decellularized extracellular matrix from pigs. These materials can reform into a hydrogel state of the extracellular matrix after injection under the action of various enzymes in the extracellular environment. However, the availability of raw materials is difficult, and ethical restrictions apply.

[0005] Simultaneously, injectable hydrogels need to possess the ability to reside in vivo and adhere to organs. This property can typically be achieved by modifying the hydrogel with N-hydroxymaleimide or using polydopamine. However, N-hydroxymaleimide usually reacts with compounds containing thioether functional groups, thus limiting its application. Furthermore, the reaction of N-hydroxymaleimide is generally reversible, and modification may be unstable. In addition, the instability during the preparation and storage of polydopamine limits its application. Summary of the Invention

[0006] In view of this, the purpose of the present invention is to provide an injectable hydrogel, its preparation method and application, which not only enables injectable drug delivery to the heart, adheres to the heart and provides continuous support, but also has certain free radical scavenging ability and antioxidant stress resistance.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] This invention provides a method for preparing an injectable hydrogel, comprising the following steps:

[0009] Oxidized hyaluronic acid, carboxymethyl chitosan, and dopamine-modified hyaluronic acid were dissolved in buffer solutions to obtain oxidized hyaluronic acid solution, carboxymethyl chitosan solution, and dopamine-modified hyaluronic acid solution, respectively. The oxidized hyaluronic acid solution, carboxymethyl chitosan solution, and dopamine-modified hyaluronic acid solution were mixed and allowed to stand to obtain an injectable hydrogel.

[0010] Preferably, the mass ratio of oxidized hyaluronic acid, carboxymethyl chitosan, and dopamine-modified hyaluronic acid is 1-5:2-10:1-5, the buffer solution is PBS buffer with pH 7.2-7.5, and the standing time is 2-3 minutes.

[0011] Preferably, the method for preparing oxidized hyaluronic acid includes the following steps:

[0012] Sodium periodate solution was added dropwise to hyaluronic acid solution, followed by dialyzing and lyophilization to obtain oxidized hyaluronic acid.

[0013] More preferably, the concentration of the hyaluronic acid solution is 0.010–0.015 g / ml, the concentration of the sodium periodate solution is 0.10–0.15 g / ml, the addition is followed by standing for 4.5–5.5 h, the standing temperature is 23–27 °C, the dialysis time is 70–75 h, the lyophilization temperature is -70–-80 °C, and the lyophilization vacuum degree is 15–25 MPa.

[0014] Preferably, the preparation method of the dopamine-modified hyaluronic acid includes the following steps:

[0015] 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide were dissolved in dimethyl sulfoxide solution to obtain a catalytic solution; hyaluronic acid solution was mixed with the catalytic solution to obtain a mixed solution; dopamine hydrochloride solution was added dropwise to the mixed solution, the pH was adjusted, and the solution was dialyzed and lyophilized to obtain dopamine-modified hyaluronic acid.

[0016] More preferably, the concentration of the hyaluronic acid solution is 0.005–0.015 g / ml, and the mixed solution is allowed to stand for 45–50 h after pH adjustment, with the standing temperature being 23–27 °C.

[0017] This invention provides an injectable hydrogel obtained by the preparation method described above.

[0018] The present invention also provides the use of the injectable hydrogel in the preparation of medicaments for treating acute and chronic cardiac injuries.

[0019] Preferably, the acute and chronic cardiac injuries include myocardial infarction, doxorubicin cardiomyopathy, acute and chronic myocarditis, and heart failure.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The principle of preparing injectable hydrogels in this invention is as follows: under the oxidation of sodium periodate, the ortho-dihydroxy group of hyaluronic acid can be cleaved to form an aldehyde group; dopamine hydrochloride can form an amide with the carboxyl group in the hyaluronic acid structure under the catalysis of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide; at the same time, the aldehyde group formed by the cleavage of hyaluronic acid and the amino group in carboxymethyl chitosan undergo a Schiff base reaction in a weakly alkaline environment to form a carbon-nitrogen double bond; then the carboxymethyl chitosan chain crosslinks with the hyaluronic acid chain to form a network structure and finally form a hydrogel; in addition, the catechol structure of dopamine can form hydrogen bonds with the hyaluronic acid sugar chain and the carboxymethyl chitosan sugar chain to further enhance the crosslinking effect of the hydrogel, and the catechol structure also provides a certain tissue adhesion effect for the hydrogel.

[0022] This invention utilizes natural polysaccharides and special structures, such as Schiff bases, phenylboronic acid esters, and ionic bonds, to control gelation time and achieve self-healing effects, while avoiding the problems existing in prior art. The biomaterials based on natural polysaccharides prepared by this invention can not only mimic the natural extracellular matrix, but also achieve multiple functions through modification.

[0023] The injectable hydrogel prepared in this invention enables injectable drug delivery to the heart, adhering to the heart and providing sustained support. This hydrogel not only provides sustained antioxidant effects to protect cardiomyocytes but also serves as a potential platform for drug delivery and release. Furthermore, since the hydrogel's raw materials are all derived from natural polysaccharides, it exhibits excellent biocompatibility. It can also serve as a suitable platform for stem cell delivery.

[0024] Meanwhile, the injectable hydrogel prepared by this invention has the following characteristics: it is stretchable, injectable, and tissue-adhesive; it can responsively degrade in a reactive oxygen species environment (100 μM); it has a certain free radical scavenging ability (DPPH detection); it can resist cellular oxidative stress; and it has excellent biocompatibility. Furthermore, compared with existing technologies, the method provided by this invention is simple to prepare, uses natural materials, and has good safety. Attached Figure Description

[0025] Figure 1 The results were obtained from test example 1;

[0026] Figure 2 This is a schematic diagram of the gelation process of injectable hydrogels;

[0027] Figure 3 The curves showing the changes in storage modulus (G′) and loss modulus (G″) of injectable hydrogel with rotational frequency before and after the addition of dopamine-modified hyaluronic acid (from top to bottom: the curves before and after the addition of dopamine-modified hyaluronic acid).

[0028] Figure 4 The curves showing the changes in storage modulus (G′) and loss modulus (G″) of the injectable hydrogel before and after the addition of dopamine-modified hyaluronic acid are as a function of shear strain (from top to bottom, the curves are the changes measured before and after the addition of dopamine-modified hyaluronic acid).

[0029] Figure 5 The experimental results show the gelation time of the hydrogel;

[0030] Figure 6 The images show the injectability and adhesion properties of the hydrogel, with the images from left to right depicting the injectability, adhesion, and other properties.

[0031] Figure 7 The degradation diagrams of the hydrogel in 100 μM H2O2 solution and PBS buffer solution are shown.

[0032] Figure 8 This is a diagram showing the scavenging effect of the hydrogel on DPPH free radicals.

[0033] Figure 9 The results of the hydrogel on the antioxidant stress of the H9C2 cell line are shown. Control represents the treatment group with "normal culture medium", H2O2 represents the treatment group with "simulated ROS environment", and H2O2+Hydrogel represents the treatment group with "cells loaded on hydrogel under ROS environment".

[0034] Figure 10 The results show the biocompatibility of the hydrogel with RAW 264.7 and H9C2 cell lines. Detailed Implementation

[0035] This invention provides a method for preparing an injectable hydrogel, comprising the following steps:

[0036] Oxidized hyaluronic acid, carboxymethyl chitosan, and dopamine-modified hyaluronic acid were dissolved in buffer solutions to obtain oxidized hyaluronic acid solution, carboxymethyl chitosan solution, and dopamine-modified hyaluronic acid solution, respectively. The oxidized hyaluronic acid solution, carboxymethyl chitosan solution, and dopamine-modified hyaluronic acid solution were mixed and allowed to stand to obtain an injectable hydrogel.

[0037] In this invention, the preferred mass ratio of oxidized hyaluronic acid, carboxymethyl chitosan, and dopamine-modified hyaluronic acid is 1–5:2–10:1–5, more preferably 2–4:4–8:2–4, and even more preferably 3:6:3; the preferred buffer solution is PBS buffer with pH 7.2–7.5, more preferably pH 7.3–7.4; the same volume of buffer solution is used for dissolution; and the preferred standing time is 2–3 min, more preferably 2.5 min.

[0038] In this invention, the preferred method for preparing oxidized hyaluronic acid includes the following steps:

[0039] Sodium periodate solution was added dropwise to hyaluronic acid solution, followed by dialyzing and lyophilization to obtain oxidized hyaluronic acid.

[0040] In this invention, the preparation method of the sodium periodate solution and hyaluronic acid solution is as follows: hyaluronic acid and sodium periodate are dissolved in buffer solutions to obtain hyaluronic acid solution and sodium periodate solution, respectively. The buffer solution is preferably a PBS buffer with a pH of 7.2–7.5, more preferably 7.3–7.4; the concentration of the hyaluronic acid solution is preferably 0.010–0.015 g / ml, more preferably 0.012–0.014 g / ml, and even more preferably 0.013 g / ml; the concentration of the sodium periodate solution is preferably 0.10–0.15 g / ml, more preferably 0.12–0.14 g / ml, and even more preferably 0.13 g / ml; after addition, the solution is preferably left to stand for 4.5–5.5 h, more preferably 4.8–5.2 h, and even more preferably... The preferred time for dialysis is 5.0 h; the preferred settling temperature is 23–27°C, more preferably 24–26°C, and even more preferably 25°C; the preferred dialysis time is 70–75 h, more preferably 72–74 h, and even more preferably 73 h; the preferred freeze-drying temperature is -70–-80°C, more preferably -72–-78°C, and even more preferably -74–-76°C; the preferred vacuum degree for freeze-drying is 15–25 MPa, more preferably 18–23 MPa, and even more preferably 20–22 MPa; the preferred average molecular weight of the hyaluronic acid is 1.5 × 10⁻⁶. 6 .

[0041] In this invention, the preferred method for preparing dopamine-modified hyaluronic acid includes the following steps:

[0042] 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide were dissolved in dimethyl sulfoxide solution to obtain a catalytic solution; hyaluronic acid solution was mixed with the catalytic solution to obtain a mixed solution; dopamine hydrochloride solution was added dropwise to the mixed solution, the pH was adjusted, and the solution was dialyzed and lyophilized to obtain dopamine-modified hyaluronic acid.

[0043] In this invention, the concentration of the hyaluronic acid solution is preferably 0.005–0.015 g / ml, more preferably 0.008–0.012 g / ml, and even more preferably 0.010 g / ml; after adjusting the pH, the mixed solution is preferably allowed to stand for 45–50 h, more preferably 46–48 h, and even more preferably 47 h; the standing temperature is preferably 23–27 °C, more preferably 24–26 °C, and even more preferably 25 °C.

[0044] This invention provides an injectable hydrogel obtained by the preparation method described above.

[0045] The present invention also provides the use of the injectable hydrogel in the preparation of medicaments for treating acute and chronic cardiac injuries.

[0046] In this invention, the acute and chronic cardiac injuries preferably include myocardial infarction, doxorubicin cardiomyopathy, acute and chronic myocarditis, and heart failure.

[0047] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0048] Example 1

[0049] A method for preparing an injectable hydrogel includes the following steps:

[0050] 2g of material with an average molecular weight of 1.5 × 10⁻⁶ was used. 6 Hyaluronic acid was dissolved in 190 ml of PBS buffer at pH 7.4 to obtain a hyaluronic acid solution. 1.4 g of sodium periodate (1.2 eq) was dissolved in 10 ml of PBS buffer at pH 7.4 to obtain a sodium periodate solution with a concentration of 0.14 g / ml. The obtained sodium periodate solution was added dropwise to the hyaluronic acid solution, and the mixture was allowed to stand at 25 °C for 5 h, dialyzed at Mw 3500 for 72 h, and then lyophilized at -80 °C and 20 MPa to obtain oxidized hyaluronic acid (AHA).

[0051] 1g of material with an average molecular weight of 6×10 5 Hyaluronic acid was dissolved in 100 ml of deionized water to obtain a hyaluronic acid solution with a concentration of 0.010 g / ml; 0.569 g of dopamine hydrochloride was dissolved in 50 ml of dimethyl sulfoxide to obtain a dopamine hydrochloride solution; 690 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 414 mg of N-hydroxysuccinimide were dissolved in 100 ml of dimethyl sulfoxide solution to obtain a catalytic solution; 100 ml of the hyaluronic acid solution with a concentration of 0.010 g / ml was mixed with 100 ml of the catalytic solution to obtain a mixed solution; the dopamine hydrochloride solution was added dropwise to the mixed solution, the pH was adjusted to 5 with 1 M hydrochloric acid, and the mixture was allowed to stand at 25 °C for 48 h. After dialyzing and lyophilization, dopamine-modified hyaluronic acid (HA-Dop) was obtained.

[0052] Take 125 mg of the oxidized hyaluronic acid, 250 mg of carboxymethyl chitosan and 125 mg of the dopamine-modified hyaluronic acid obtained above and dissolve them in PBS buffer at pH 7.4 until 10 ml of oxidized hyaluronic acid solution, 10 ml of carboxymethyl chitosan solution and 10 ml of dopamine-modified hyaluronic acid solution are obtained. Mix the obtained solutions in equal volumes and let stand for 2 min to obtain injectable hydrogel.

[0053] Example 2

[0054] A method for preparing an injectable hydrogel includes the following steps:

[0055] 1.9g of an average molecular weight of 1.5×10⁻⁶ was used. 6 Hyaluronic acid was dissolved in 190 ml of PBS buffer at pH 7.2 to obtain a hyaluronic acid solution. 1 g of sodium periodate (1.2 eq) was dissolved in 10 ml of PBS buffer at pH 7.2 to obtain a sodium periodate solution with a concentration of 0.10 g / ml. The obtained sodium periodate solution was added dropwise to the hyaluronic acid solution, and the mixture was allowed to stand at 23 °C for 5.5 h, dialyzed at Mw 3400 for 75 h, and then lyophilized at -70 °C and 25 MPa to obtain oxidized hyaluronic acid (AHA).

[0056] 0.5g of material with an average molecular weight of 4×10⁻⁶ was used. 5 Hyaluronic acid was dissolved in 100 ml of deionized water to obtain a hyaluronic acid solution with a concentration of 0.005 g / ml; 0.569 g of dopamine hydrochloride was dissolved in 50 ml of dimethyl sulfoxide to obtain a dopamine hydrochloride solution; 680 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 424 mg of N-hydroxysuccinimide were dissolved in 100 ml of dimethyl sulfoxide solution to obtain a catalytic solution; 100 ml of the hyaluronic acid solution with a concentration of 0.005 g / ml was mixed with 100 ml of the catalytic solution to obtain a mixed solution; the dopamine hydrochloride solution was added dropwise to the mixed solution, the pH was adjusted to 5 with 1 M hydrochloric acid, and the mixture was allowed to stand at 23 °C for 50 h. After dialyzing and lyophilization, dopamine-modified hyaluronic acid (HA-Dop) was obtained.

[0057] Take 120 mg of the oxidized hyaluronic acid, 240 mg of carboxymethyl chitosan and 120 mg of the dopamine-modified hyaluronic acid obtained above and dissolve them in PBS buffer at pH 7.2 until 10 ml of oxidized hyaluronic acid solution, 10 ml of carboxymethyl chitosan solution and 10 ml of dopamine-modified hyaluronic acid solution are obtained. Mix the obtained solutions in equal volumes and let stand for 2-3 minutes to obtain an injectable hydrogel.

[0058] Example 3

[0059] A method for preparing an injectable hydrogel includes the following steps:

[0060] 2.85g of a substance with an average molecular weight of 1.5×10⁻⁶ was used. 6Hyaluronic acid was dissolved in 190 ml of PBS buffer at pH 7.5 to obtain a hyaluronic acid solution. 1.5 g of sodium periodate (1.2 eq) was dissolved in 10 ml of PBS buffer at pH 7.2–7.5 to obtain a sodium periodate solution with a concentration of 0.15 g / ml. The obtained sodium periodate solution was added dropwise to the hyaluronic acid solution, and the mixture was allowed to stand at 27 °C for 4.5 h, dialyzed at Mw 3600 for 75 h, and then lyophilized at -75 °C and 15 MPa to obtain oxidized hyaluronic acid (AHA).

[0061] 1.5g of material with an average molecular weight of 8×10⁻⁶ was used. 5 Hyaluronic acid was dissolved in 100 ml of deionized water to obtain a hyaluronic acid solution with a concentration of 0.015 g / ml; 0.56 g of dopamine hydrochloride was dissolved in 50 ml of dimethyl sulfoxide to obtain a dopamine hydrochloride solution; 700 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 404 mg of N-hydroxysuccinimide were dissolved in 100 ml of dimethyl sulfoxide solution to obtain a catalytic solution; 100 ml of the hyaluronic acid solution with a concentration of 0.015 g / ml was mixed with 100 ml of the catalytic solution to obtain a mixed solution; the dopamine hydrochloride solution was added dropwise to the mixed solution, the pH was adjusted to 5 with 1 M hydrochloric acid, and the mixture was allowed to stand at 27 °C for 45 h. After dialyzing and lyophilization, dopamine-modified hyaluronic acid (HA-Dop) was obtained.

[0062] Take 130 mg of the oxidized hyaluronic acid, 260 mg of carboxymethyl chitosan and 173 mg of the dopamine-modified hyaluronic acid obtained above and dissolve them in PBS buffer at pH 7.5 until 10 ml of oxidized hyaluronic acid solution, 10 ml of carboxymethyl chitosan solution and 10 ml of dopamine-modified hyaluronic acid solution are obtained. Mix the obtained solutions in equal volumes and let stand for 2-3 minutes to obtain an injectable hydrogel.

[0063] Experimental Example 1

[0064] Injectable hydrogels were prepared according to the method in Example 1. The difference was that 1%, 3%, 5%, and 7% carboxymethyl chitosan (CMCs) and 1.5%, 2%, 2.5%, 3%, and 3.5% oxidized hyaluronic acid (AHA) were added to prepare injectable hydrogels with different CMC and AHA contents. The effect of different CMC contents on the injectable hydrogels was investigated. The results are as follows... Figure 1 As shown in the figure. (In the figure, A, B, C, and D are the morphological diagrams of injectable hydrogels prepared by adding 1%, 3%, 5%, and 7% carboxymethyl chitosan, respectively).

[0065] Depend on Figure 1It can be seen that the treatment group that meets both the requirements for injectability and has a certain gel strength is the injectable hydrogel prepared by adding 2.5% oxidized hyaluronic acid and 5% carboxymethyl chitosan.

[0066] Experimental Example 2

[0067] Injectable hydrogels were prepared according to the method in Example 1, wherein, during equal-volume mixing, oxidized hyaluronic acid solution and carboxymethyl chitosan solution were mixed first, and then dopamine-modified hyaluronic acid solution was added. A HAAKE MARSII rotational rheometer was used with a flat plate clamp, the test temperature was set to 25°C, the zero gap was set to 1.5 mm, and the circular hydrogel sample was placed on the rheometer. The oscillation mode-time scan was selected for testing.

[0068] Rotational rheometer was used to investigate the changes in storage modulus (G′) and loss modulus (G″) of the injectable hydrogel with rotational frequency and with shear strain before and after the addition of dopamine-modified hyaluronic acid. The results are as follows: Figure 3 and Figure 4 As shown.

[0069] The results showed that the hydrogel could maintain its gel state even under shaking and peristalsis, which met the requirements for injection in the heart area.

[0070] Experimental Example 3

[0071] Injectable hydrogels were prepared according to the method of Example 2, and the gelation time of the hydrogels was recorded after equal volumes of the obtained solutions were mixed. The results are as follows: Figure 5 As shown.

[0072] Depend on Figure 5 It can be seen that the gelation time of hydrogel is between 110s and 130s, which can meet the gelation requirements after injection.

[0073] Test Example 4

[0074] The injectable hydrogel prepared in Example 2 was stained with Rhodamine B (purchased from Adamas). The stained injectable hydrogel was injected into a dry plate and PBS buffer (pH=7.4) using a 25G needle, and the gel morphology of the injectable hydrogel was observed.

[0075] Simultaneously, the stained injectable hydrogel was applied to the surface of pigskin, and its adhesion was observed. The results are as follows: Figure 6 As shown.

[0076] Depend on Figure 6It is known that the injectable hydrogel prepared by this invention can be injected with a 25G needle and forms a gel, maintaining its complete gel-like morphology in PBS (pH=7.4). Furthermore, the hydrogel adheres to the tissue surface without falling off, achieving satisfactory adhesion after injection.

[0077] Experimental Example 5

[0078] The injectable hydrogel prepared in Example 3 was placed in PBS (pH=7.4) buffer solution (Control group) and PBS solution containing 200 μmol / mL hydrogen peroxide (pH=7.4) (H2O2 treatment group), respectively. The degradation of the hydrogel under different environments was observed and recorded (1-12 days). The results are as follows: Figure 7 As shown.

[0079] Depend on Figure 7 It is known that hydrogels degrade slowly in the absence of hydrogen peroxide, but degrade more rapidly in the presence of hydrogen peroxide (i.e., in a simulated high ROS environment after injury), which is beneficial for the accelerated release of drugs encapsulated in hydrogels.

[0080] Experimental Example 6

[0081] Injectable hydrogels were prepared according to the method in Example 3, and the mixtures were diluted to 25 μg / mL, 50 μg / mL, 100 μg / mL, and 200 μg / mL, respectively, after equal-volume mixing and before gelation. The free radical scavenging ability of the injectable hydrogels at different concentrations was detected using the DPPH (1,1-diphenyl-2-trinitrophenylhydrazine, purchased from Shanghai Maclean Biochemical Technology Co., Ltd.) method described in "Determination of Antioxidant Activity of Typical Vegetable Oils by DPPH Method" (Ji Lulu, Zhang Li, Zhang Sifang, et al. Determination of Antioxidant Activity of Typical Vegetable Oils by DPPH Method [J]. Guangdong Chemical Industry, 2022, 49(22):220-222.). Specifically:

[0082] 7.89 mg of dibenzopicrylamide (DPPH) free radical was dissolved in 200 mL of 80% (v / v) methanol solution. The stock solution was then diluted with 80% methanol solution to an absorbance of 0.7 at 527 nm. Injectable hydrogel samples of different concentrations were added to 2.95 mL of DPPH diluent and incubated at 25 °C for 1 h. The absorbance at 527 nm was measured using a microplate reader, and the free radical scavenging ratio was calculated. Results are as follows: Figure 8 As shown.

[0083] Depend on Figure 8 It is known that injectable hydrogels still have good free radical scavenging ability after dilution, and the free radical scavenging ability increases with increasing concentration, which is beneficial to improving the high oxidative stress environment after injury after injection.

[0084] Subsequently, H9C2 cells (purchased from BNCC Biotechnology) were used at a rate of 1×10⁻⁶. 6 Cells were seeded per well in 6-well plates and cultured in 200 μmol / mL hydrogen peroxide complete medium to simulate a high ROS environment for 24 h. Cells were washed three times with PBS (pH 7.4), 100 μL each time for 1 min. ROS in the cells were stained with 100 μL of 10 μg / mL DCFH-DA (2',7'-dichlorofluorescein diacetate, purchased from Shanghai Maclean Biochemical Technology Co., Ltd., wherein 10 μg / mL DCFH-DA was prepared using PBS solution at pH 7.4 as a solvent) (DCFH-DA solution and washed cells were co-incubated for 15 min at 37 °C). The staining was performed using the FITC fluorescence channel.

[0085] Depend on Figure 9 It can be seen that there is significant ROS accumulation in cells in the ROS environment, and the green fluorescence of the group co-cultured with the injectable hydrogel diluent is almost the same as that of the control group, indicating that the injectable hydrogel has the effect of clearing ROS in the microenvironment and reducing ROS accumulation in cells.

[0086] Experimental Example 7

[0087] 800 mg of the injectable hydrogel prepared in Example 3 was immersed in 4 ml of complete culture medium (DMEM high glucose medium + 10% fetal bovine serum + 1% penicillin and streptomycin, all purchased from Wuhan Pronosai Life Science Technology Co., Ltd.) and cultured for 24 h. The hydrogel extracts were then diluted to prepare hydrogel extracts of 200 μg / mL, 100 μg / mL, 50 μg / mL, 25 μg / mL, and 10 μg / mL.

[0088] RAW 264.7 cells (purchased from BNCC) and H9C2 cell line (purchased from BNCC) were both injected at a dose of 1×10⁻⁶. 6 Cells were seeded per well in 6-well plates, and different concentrations of hydrogel extract were added. Cells were co-cultured for 48 hours. Cells were washed three times with 100 μL of PBS (pH 7.4) for 1 minute each time. Live / dead cells were stained using a 10 μg / mL PBS (pH 7.4, AM and PI both 10 μg / mL) mixture prepared with calcein (AM) / propidium iodide (PI) (purchased from Beyotime Biotechnology) for 20 minutes. Results are as follows: Figure 10 As shown.

[0089] Depend on Figure 10 It can be seen that no obvious cell death was observed in RAW 264.7 and H9C2 cell lines at different concentrations, indicating that the injectable hydrogel has excellent biocompatibility and can meet the needs of in vivo injection.

[0090] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing an injectable hydrogel, characterized in that, Includes the following steps: Oxidized hyaluronic acid, carboxymethyl chitosan, and dopamine-modified hyaluronic acid were dissolved in buffer solution to obtain oxidized hyaluronic acid solution, carboxymethyl chitosan solution, and dopamine-modified hyaluronic acid solution, respectively. The oxidized hyaluronic acid solution, carboxymethyl chitosan solution, and dopamine-modified hyaluronic acid solution were mixed and allowed to stand to obtain injectable hydrogel. The mass ratio of oxidized hyaluronic acid, carboxymethyl chitosan, and dopamine-modified hyaluronic acid is 3:6:

3. The preparation method of the dopamine-modified hyaluronic acid includes the following steps: 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide were dissolved in dimethyl sulfoxide solution to obtain a catalytic solution; hyaluronic acid solution was mixed with the catalytic solution to obtain a mixed solution; Dopamine hydrochloride solution was added dropwise to the mixed solution, the pH was adjusted, and the solution was dialyzed and lyophilized to obtain dopamine-modified hyaluronic acid. The concentration of the hyaluronic acid solution is 0.005~0.015g / ml. After adjusting the pH, the mixed solution is allowed to stand for 45~50h at a temperature of 23~27℃.

2. The preparation method according to claim 1, characterized in that, The buffer solution is a PBS buffer with a pH of 7.2 to 7.5, and the standing time is 2 to 3 minutes.

3. The preparation method according to claim 1, characterized in that, The preparation method of the oxidized hyaluronic acid includes the following steps: Sodium periodate solution was added dropwise to hyaluronic acid solution, followed by dialyzing and lyophilization to obtain oxidized hyaluronic acid.

4. The preparation method according to claim 3, characterized in that, In the method for preparing oxidized hyaluronic acid, the concentration of the hyaluronic acid solution is 0.010~0.015 g / ml, the concentration of the sodium periodate solution is 0.10~0.15 g / ml, the addition is followed by standing for 4.5~5.5 h, the standing temperature is 23~27℃, the dialysis time is 70~75 h, and the freeze-drying temperature is -70~-80℃.

5. The injectable hydrogel obtained by the preparation method according to any one of claims 1 to 4.

6. The use of the injectable hydrogel as described in claim 5 in the preparation of medicaments for treating acute and chronic cardiac injuries.

7. The application according to claim 6, characterized in that, The acute and chronic cardiac injuries mentioned include myocardial infarction, doxorubicin cardiomyopathy, acute and chronic myocarditis, and heart failure.

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