Antioxidant hydrogel as well as preparation method and application thereof

Abstract

The invention provides a hydrogel as well as a preparation method and application thereof, and the hydrogel comprises a PVA-TSPBA hydrogel skeleton formed by the reaction of polyvinyl alcohol (PVA) and N1-(4-bromobenzyl)-N3-(4-bromophenyl)-N1, N1, N3, N3-tetramethylpropane-1, 3-diamine (TSPBA), and reduced glutathione modified polydopamine melanin nanoparticles loaded on the skeleton.

Description

Technical Field

[0001] The present application belongs to the field of biomaterial technology, and specifically relates to an antioxidant hydrogel and a preparation method and use thereof. Background Art

[0002] At present, targeting the microenvironmental characteristics of ischemia-reperfusion, the use of injectable strategies for ischemia-reperfusion (I / R) injury repair is a very promising treatment strategy. Currently, injectable hydrogels targeting the reactive oxygen species (ROS) microenvironment are mainly divided into two categories. One category is injectable hydrogels that have the ability to scavenge ROS, which exert functional therapeutic effects by carrying drugs, factors, etc. The main function of this type of hydrogel is to clear the generated ROS, etc., to achieve regulation of the microenvironment. The other category is traditional non-responsive hydrogels, which achieve myocardial infarction repair by carrying nanoparticles with ROS scavenging functions. This type of hydrogel usually mainly plays a mechanical support role and carries factors, nanomaterials, etc. with ROS scavenging functions. However, this type of hydrogel usually leads to low therapeutic efficiency of functional factors and nanomaterials, and lacks the ability to perceive and regulate the surrounding pathological environment.

[0003] Based on this, researchers are currently paying more attention to smart hydrogels, primarily due to their intelligent environmental response and controlled release capabilities. These hydrogels can ensure that the therapeutic drugs and factors they carry are more effectively targeted at the myocardial infarction microenvironment, exerting an effective therapeutic effect. Therefore, how to intelligently respond to the ROS microenvironment of I / R injury while releasing functional materials on demand that effectively correct the early harsh microenvironment and promote repair is key to improving treatment outcomes. Summary of the Invention

[0004] According to the first aspect of the present application, a hydrogel is provided, comprising: a PVA-TSPBA hydrogel skeleton formed by reacting polyvinyl alcohol (PVA) with a crosslinker N1-(4-bromobenzyl)-N3-(4-bromophenyl)-N1,N1,N3,N3-tetramethylpropane-1,3-diamine (TSPBA), and glutathione-modified polydopamine melanin nanoparticles loaded on the PVA-TSPBA hydrogel skeleton.

[0005] In some embodiments, the hydrogel has a porous structure, and optionally, the porosity of the hydrogel is 50-70%;

[0006] The reduced glutathione-modified polydopamine melanin nanoparticles are distributed on the surface of the PVA-TSPBA hydrogel skeleton.

[0007] In some embodiments, the particle size of the reduced glutathione-modified polydopamine melanin nanoparticles is 40-200 nm.

[0008] In some embodiments, the average particle size of the reduced glutathione-modified polydopamine melanin nanoparticles is 90-120 nm.

[0009] In some embodiments, the D90 particle size of the reduced glutathione-modified polydopamine melanin nanoparticles is 60-140 nm.

[0010] In some embodiments, 25-100 μg of the reduced glutathione-modified polydopamine melanin nanoparticles are loaded per milliliter of the hydrogel.

[0011] According to a second aspect of the present application, there is provided a method for preparing a hydrogel, comprising the following steps:

[0012] 1) mixing ammonia water, ethanol, and deionized water, adding dopamine hydrochloride, and stirring to react to obtain a solution containing polydopamine melanin nanoparticles;

[0013] 2) adding reduced glutathione (GSH) to the solution containing polydopamine melanin nanoparticles in step 1), stirring for reaction, and then dialyzing to finally obtain a suspension of reduced glutathione-modified polydopamine melanin (GSH-PDA) nanoparticles; and

[0014] 3) Adding polyvinyl alcohol (PVA) powder to the suspension of GSH-PDA nanoparticles and stirring until the PVA powder is completely dissolved to obtain a PVA solution containing GSH-PDA nanoparticles, stirring and mixing the PVA solution with a crosslinking agent TSPBA solution to react to obtain the hydrogel.

[0015] In some embodiments, in step 1) and step 2), the ratio of ammonia water, ethanol, deionized water, dopamine hydrochloride and GSH is 2-3 mL: 32-48 mL: 72-108 mL: 40-60 mg: 32-48 mg.

[0016] In some embodiments, the stirring reaction temperature in step 1) is room temperature, and the stirring reaction time is 16-32 hours.

[0017] In some embodiments, in step 2), the stirring reaction temperature is 45-55° C., and the stirring reaction time is 6-10 h.

[0018] In some embodiments, in step 2), the size of the dialysis bag is 1000-2000 Da, and the dialysis time is 2-4 days.

[0019] In some embodiments, the method further comprises the step of freeze-drying the suspension of the GSH-PDA nanoparticles obtained in step 2).

[0020] In some embodiments, the concentration of glutathione-modified polydopamine melanin nanoparticles in the suspension of GSH-PDA nanoparticles used in step 3) is 50-200 μg / ml.

[0021] In some embodiments, in step 3), the concentration of PVA in the PVA solution is 40-60 mg / ml.

[0022] In some embodiments, in step 3), the concentration of TSPBA in the cross-linking agent TSPBA solution is 40-60 mg / ml.

[0023] In some embodiments, in step 3), the PVA solution and the cross-linking agent TSPBA solution are mixed in equal volumes.

[0024] In some embodiments, the reaction temperature of the PVA solution and the cross-linking agent TSPBA solution is 60-90°C.

[0025] According to the third aspect of the present application, there is provided use of the hydrogel of the first aspect or the hydrogel prepared according to the method of the second aspect in preparing a medicament for treating ischemia-reperfusion (I / R) injury. Optionally, the medicament is an injectable hydrogel scaffold.

[0026] In some embodiments, the ischemia-reperfusion injury is caused by a disease or condition selected from the group consisting of: myocardial infarction, cerebral infarction, limb arterial embolism, cerebral no-reflow phenomenon after cardiopulmonary resuscitation, stress ulcer, pancreatitis, burns, intestinal ischemia, necrotizing enterocolitis, intermittent claudication, acute tubular necrosis, post-shock liver failure, and multi-system organ failure; optionally, the ischemia-reperfusion injury is myocardial ischemia-reperfusion injury, for example, myocardial ischemia-reperfusion injury caused by myocardial infarction.

[0027] Compared with the prior art, the present application achieves at least one of the following beneficial effects: the present application utilizes glutathione to modify polydopamine melanin nanoparticles and composites them with ROS-responsive hydrogels to obtain a hydrogel that integrates response-ROS scavenging and ROS generation inhibition, providing a good scaffold material for myocardial infarction repair. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The following further describes the embodiments illustrated in this document with reference to the accompanying drawings. It should be understood that the drawings are only intended to enable those skilled in the art to better understand the technical solutions of the present application and are not intended to limit the scope of the technical solutions of the present application.

[0029] Figure 1Characterization diagrams of glutathione-modified polydopamine melanin (GSH-PDA) nanoparticles prepared according to one embodiment of the present application are shown, wherein the left figure is a scanning electron microscope (SEM) image of the GSH-PDA nanoparticles; the right figure is an energy dispersive spectroscopy (EDS) elemental analysis diagram of the GSH-PDA nanoparticles.

[0030] Figure 2 The diagram shows Fourier transform infrared spectroscopy (FT-IR) of dopamine (DA), reduced glutathione (GSH) and glutathione-modified polydopamine melanin (GSH-PDA) nanoparticles prepared according to one embodiment of the present application.

[0031] Figure 3 Scanning electron microscope (SEM) images of GSH-PDA / PVA-TSPBA hydrogels with different GSH-PDA nanoparticle loadings prepared according to the examples of the present application are shown.

[0032] Figure 4 The antioxidant performance test results of glutathione-modified polydopamine melanin (GSH-PDA) nanoparticles with different loading concentrations prepared according to the examples of the present application are shown compared with pure polydopamine melanin (PDA) nanoparticles, where a shows the scavenging rate of H2O2, b shows the scavenging rate of O2·-, and c shows the scavenging rate of ·OH.

[0033] Figure 5 The antioxidant performance test results of the GSH-PDA / PVA-TSPBA hydrogel prepared according to the examples of the present application are shown compared with the non-responsive PVA hydrogel, the responsive PVA-TSPBA hydrogel and the PDA / PVA-TSPBA hydrogel without GSH modification, where a shows the scavenging rate of O2·- and b shows the scavenging rate of ·OH.

[0034] Figure 6 The ROS responsiveness of the GSH-PDA / PVA-TSPBA hydrogel prepared according to the examples of the present application is shown.

[0035] Figure 7 The figure shows the use of Masson staining to analyze cardiac fibrosis and collagen deposition in rats with myocardial ischemia-reperfusion injury (I / R) after 28 days of treatment, where a shows the formation of fibrosis (blue fibroblasts and collagen) in different treatment groups, b shows the infarct area in different treatment groups, and c shows the echocardiogram of different treatment groups. DETAILED DESCRIPTION

[0036] Hereinafter, the inventive concept of the present application will be further elaborated according to specific embodiments. However, the specific embodiments listed are only for illustrative purposes and are not intended to limit the scope of the present application. Those skilled in the art will recognize that the specific features in any of the following embodiments can be used in any other embodiment as long as it does not deviate from the inventive concept described herein.

[0037] hydrogel

[0038] The hydrogel provided by the present application includes: a PVA-TSPBA hydrogel skeleton formed by the reaction of polyvinyl alcohol (PVA) and a crosslinker N1-(4-bromobenzyl)-N3-(4-bromophenyl)-N1,N1,N3,N3-tetramethylpropane-1,3-diamine (TSPBA), and reduced glutathione (GSH)-modified polydopamine melanin nanoparticles loaded on the PVA-TSPBA hydrogel skeleton.

[0039] The inventors of this application have provided a smart antioxidant hydrogel based on the microenvironmental characteristics of ischemia-reperfusion (I / R) injury. The hydrogel can clear ROS in the extracellular matrix of the infarcted area by responding to the ROS microenvironment. The PVA-TSPBA hydrogel skeleton itself can be cleaved under ROS stimulation, thereby releasing functional nanoparticles, which inhibit the production of ROS in the infarcted area cells by clearing iron ions, thereby providing a new integrated treatment method for I / R injury repair that responds to and clears ROS and inhibits ROS generation.

[0040] In some embodiments, the hydrogel provided herein has a porous structure, and reduced glutathione-modified polydopamine melanin nanoparticles are distributed on the surface of the PVA-TSPBA hydrogel skeleton.

[0041] In some embodiments, the hydrogel provided herein has a uniform porous structure, and the glutathione-modified polydopamine melanin nanoparticles are uniformly distributed on the surface of the PVA-TSPBA hydrogel skeleton.

[0042] In some embodiments, the porosity of the hydrogels provided herein is 50-70%, for example, 50%, 60%, 70%, or any value within a range consisting thereof. In some embodiments, the porosity of the hydrogels provided herein is 60%. As used herein, the term "porosity" refers to the ratio of the internal micropore volume of the hydrogel in the freeze-dried state to the external surface volume of the freeze-dried hydrogel.

[0043] In some embodiments, the particle size of the reduced glutathione-modified polydopamine melanin nanoparticles is 40-200 nm, for example, 40 nm, 50 nm, 60 nm, 80 nm, 100 nm, 150 nm, 200 nm, or any value within a range consisting thereof.

[0044] In some embodiments, the average particle size of the reduced glutathione-modified polydopamine melanin nanoparticles is 90-120 nm, for example, an average particle size of 90 nm, 100 nm, 110 nm, 120 nm, or any value within a range consisting thereof.

[0045] In some embodiments, the D90 particle size of the reduced glutathione-modified polydopamine melanin nanoparticles is 60 nm to 140 nm. The term "particle size D90" as used herein refers to the particle size range of 90% of the nanoparticles extending from the center of the particle size distribution graph to both sides, and can also be understood as the particle size range of the remaining nanoparticles after excluding the 5% of nanoparticles with the smallest particle size and the 5% of nanoparticles with the largest particle size.

[0046] In some embodiments, in the hydrogel provided herein, 25-100 μg of reduced glutathione-modified polydopamine melanin nanoparticles are loaded per milliliter of hydrogel.

[0047] Preparation of hydrogel

[0048] The present application provides a method for preparing the above-mentioned hydrogel, comprising the following steps:

[0049] 1) Mixing appropriate amounts of ammonia water, ethanol, and deionized water, adding dopamine hydrochloride, and stirring to react to obtain a solution containing polydopamine melanin nanoparticles;

[0050] 2) adding reduced glutathione (GSH) to the solution containing polydopamine melanin nanoparticles in step 1), stirring for reaction, and then dialyzing to finally obtain a suspension of reduced glutathione-modified polydopamine melanin (GSH-PDA) nanoparticles; and

[0051] 3) Adding polyvinyl alcohol (PVA) powder to the suspension of GSH-PDA nanoparticles and stirring until the PVA powder is completely dissolved to obtain a PVA solution containing GSH-PDA nanoparticles. Stirring the obtained PVA solution with a crosslinking agent TSPBA solution to react and obtain the hydrogel.

[0052] According to the preparation method of the above-mentioned hydrogel of the present application, polydopamine melanin nanoparticles are prepared by oxidative self-polymerization of dopamine hydrochloride under alkaline conditions, and then reduced glutathione with the function of inhibiting the generation of reactive oxygen species is modified on the polydopamine melanin nanoparticles to obtain glutathione-modified polydopamine melanin (GSH-PDA) nanoparticles, TSPBA is mixed with a PVA solution containing GSH-PDA nanoparticles, and the hydroxyl groups of TSPBA and the hydroxyl groups of PVA are dehydrated and condensed to lock the GSH-PDA nanoparticles, thereby loading the GSH-PDA nanoparticles onto the ROS-responsive hydrogel obtained by the reaction of PVA and the crosslinker TSPBA, thereby obtaining an intelligent response-ROS scavenging and ROS generation inhibition integrated antioxidant hydrogel, namely, GSH-PDA / PVA-TSPBA hydrogel.

[0053] In some embodiments, in step 1) and step 2), the ratio of ammonia water, ethanol, deionized water, dopamine hydrochloride and GSH is 2-3 mL: 32-48 mL: 72-108 mL: 40-60 mg: 32-48 mg.

[0054] In some embodiments, in step 1) and step 2), the ratio of ammonia water, ethanol, deionized water, dopamine hydrochloride and GSH is 2.5 mL:40 mL:90 mL:50 mg:40 mg.

[0055] In some embodiments, the stirring reaction time in step 1) is 16-32 hours, for example, 24 hours.

[0056] In some embodiments, the temperature of the stirring reaction in step 1) is room temperature.

[0057] In some embodiments, in step 2), the stirring reaction temperature is 45-55°C, for example 50°C.

[0058] In some embodiments, in step 2), the stirring reaction time is 6-10 h, for example, 7 h.

[0059] In some embodiments, in step 2), the size of the dialysis bag is 1000-2000 Da, for example, 1000 Da.

[0060] In some embodiments, in step 2), the dialysis time is 2-4 days, for example, 3 days.

[0061] In some embodiments, the preparation method further comprises freeze-drying the suspension of GSH-PDA nanoparticles obtained in step 2). Depending on the reaction requirements, the suspension of GSH-PDA nanoparticles obtained in step 2) can be used directly in step 3), or a suspension of GSH-PDA nanoparticles can be prepared by adding water to the freeze-dried powder.

[0062] In some embodiments, in the suspension of glutathione-modified polydopamine melanin (GSH-PDA) nanoparticles used in step 3), the concentration of glutathione-modified polydopamine melanin nanoparticles is 50-200 μg / ml, for example, 100 μg / ml.

[0063] In some embodiments, in step 3), the concentration of PVA in the PVA solution containing GSH-PDA nanoparticles is 40-60 mg / ml, for example, 50 mg / ml.

[0064] In some embodiments, in step 3), the concentration of TSPBA in the cross-linking agent TSPBA solution is 40-60 mg / ml, for example, 50 mg / ml.

[0065] In some embodiments, in step 3), the PVA solution containing GSH-PDA nanoparticles and the cross-linking agent TSPBA solution are mixed in equal volumes.

[0066] In some embodiments, the reaction temperature of the PVA solution and the cross-linking agent TSPBA solution in step 3) is 60-90°C, for example 60°C.

[0067] In some embodiments, in step 3), the cross-linking agent TSPBA solution is obtained by dissolving solid TSPBA in deionized water.

[0068] In some embodiments, solid TSPBA is prepared by the following method: dispersing 4-(bromomethyl)phenylboronic acid in N',N-dimethylformamide and stirring to dissolve, then adding N,N,N',N'-tetramethyl-1,3-propanediamine, reacting overnight, washing with tetrahydrofuran (THF), and vacuum drying to remove residual solvent to obtain solid TSPBA.

[0069] In some embodiments, the molar ratio of 4-(bromomethyl)phenylboronic acid to N,N,N',N'-tetramethyl-1,3-propanediamine is 1:2.

[0070] According to a third aspect of the present application, there is provided use of the hydrogel according to any one of the above embodiments in preparing a medicament for treating ischemia-reperfusion (I / R) injury.

[0071] In some embodiments, there is provided a use of the hydrogel according to any of the above embodiments for preparing an injectable hydrogel scaffold material for repairing ischemia-reperfusion (I / R) injury.

[0072] In some embodiments, the present application also provides a method for repairing ischemia-reperfusion (I / R) injury, comprising using the hydrogel according to any one of the above embodiments.

[0073] The ischemia-reperfusion (I / R) injury mentioned in the present application is caused by a disease or condition selected from the following: myocardial infarction, cerebral infarction, limb arterial embolism, cerebral no-reflow phenomenon after cardiopulmonary resuscitation, stress ulcer, pancreatitis, burns, intestinal ischemia, necrotizing enterocolitis, intermittent claudication, acute tubular necrosis, post-shock liver failure and multi-system organ failure. In particular, the ischemia-reperfusion injury is the ischemia-reperfusion (I / R) injury of the myocardial infarction site (such as the myocardium), for example, the myocardial ischemia-reperfusion injury caused by myocardial infarction.

[0074] Example

[0075] The following embodiments are provided to facilitate a better understanding of the embodiments of the present application, but are not intended to limit them in any way. The experimental methods used in the following embodiments are conventional methods unless otherwise specified, and the materials, reagents, etc. used are all commercially available unless otherwise specified.

[0076] Material Description

[0077] Dopamine hydrochloride: Aladdin (Product No.: D103111)

[0078] Reduced glutathione (GSH): Macklin (Cat. No. G6268)

[0079] Dialysis bag (1000Da): Beijing Solebeau Technology Co., Ltd. (Cat. No.: YA1049)

[0080] 4-(Bromomethyl)phenylboronic acid:Sigma (Cat. No. 679437)

[0081] N',N-Dimethylformamide: MacLean (Product No.: N6259)

[0082] N,N,N',N'-Tetramethyl-1,3-propanediamine: Sigma (Cat. No. 549983)

[0083] Polypropylene alcohol (PVA) powder: Aladdin (Product No.: P434368)

[0084] Example 1: Preparation of polydopamine melanin nanoparticles

[0085] Take 2.5 ml of ammonia water, 40 ml of ethanol and 90 ml of deionized water, stir for 30 minutes to mix evenly; then add 50 mg of dopamine hydrochloride to the above mixed solution and stir for 24 hours to obtain polydopamine melanin (PDA) nanoparticles.

[0086] Example 2: Preparation of glutathione-modified polydopamine melanin nanoparticles

[0087] Take 2.5ml of ammonia water, 40ml of ethanol, and 90ml of deionized water and stir for 30 minutes to mix thoroughly. Then, add 50mg of dopamine hydrochloride to the mixed solution and stir at room temperature for 24 hours. Then add 40mg of reduced glutathione (GSH) to the solution and stir at 50°C for 7 hours. Finally, use a 1000Da dialysis bag to dialyze the resulting mixed solution for 3 days to obtain the final suspension of reduced glutathione-modified polydopamine melanin (GSH-PDA) nanoparticles. The solid content of GSH-PDA nanoparticles in this suspension is 100μg / ml. It is then freeze-dried for subsequent experimental testing.

[0088] The prepared glutathione-modified polydopamine melanin nanoparticles were freeze-dried, and the sample surface was sprayed with gold. The sample was placed under an electron gun, and the morphology of the nanoparticles was observed using a scanning electron microscope (Zeiss Supra55). Figure 1 As shown in the middle left figure, the formed particles are uniformly dispersed GSH-PDA nanospheres with a diameter of about 40-200nm, an average particle size of 100nm, and a D90 particle size of 60-140nm. The elements on the surface of GSH-PDA nanoparticles were further analyzed using energy dispersive spectroscopy (EDS). The results are as follows: Figure 1 As shown in the middle right picture, GSH-PDA nanoparticles are not only rich in N, C, and O, which are common elements in the formation process of polydopamine melanin, but also S element is detected, proving that GSH is successfully repaired to polydopamine melanin nanoparticles.

[0089] In addition, in order to investigate the changes in the chemical bonds on the surface of GSH-PDA nanoparticles, Fourier transform infrared spectroscopy (FT-IR) measurements were performed on dopamine (DA), reduced glutathione (GSH) and prepared GSH-PDA nanoparticles. Figure 2 As shown, the 1616cm -1 The absorption characteristic peak at 3225 cm represents the stretching vibration of the C=C double bond in the aromatic ring; -1 The strong absorption characteristic peak at represents the stretching vibration of OH and NH in GSH-PDA, proving that GSH is modified on PDA.

[0090] Example 3: Preparation of GSH-PDA / PVA-TSPBA hydrogel

[0091] 2.5 ml of ammonia water, 40 ml of ethanol, and 90 ml of deionized water were mixed and stirred for 30 minutes. 50 mg of dopamine hydrochloride was then added to the mixed solution and stirred at room temperature for 24 hours. 40 mg of reduced glutathione (GSH) was then added to the solution and stirred at 50°C for 7 hours. Finally, the resulting mixed solution was dialyzed for 3 days using a 1000 Da dialysis bag to obtain a suspension of glutathione-modified polydopamine melanin (GSH-PDA) nanoparticles with a solids content of 100 μg / ml.

[0092] Disperse 0.5 g of 4-(bromomethyl)phenylboronic acid in 10 ml of N',N-dimethylformamide at 60°C and stir to dissolve. Then, add 800 μl of N,N,N',N'-tetramethyl-1,3-propanediamine and allow to react overnight. After washing three times with tetrahydrofuran (THF), vacuum drying to remove residual solvent yields solid N1-(4-bromobenzyl)-N3-(4-bromophenyl)-N1,N1,N3,N3-tetramethylpropane-1,3-diamine (TSPBA).

[0093] The prepared solid TSPBA was dissolved in deionized water to obtain a TSPBA crosslinker solution (TSPBA concentration 50 mg / ml). 1 g of PVA was added to 20 ml of the suspension of GSH-PDA nanoparticles synthesized above and stirred at 90°C until the PVA powder was completely dissolved, yielding a PVA solution containing GSH-PDA nanoparticles. Finally, equal volumes of the TSPBA crosslinker solution and the PVA solution containing GSH-PDA nanoparticles were stirred and reacted at 60°C to yield a GSH-PDA / PVA-TSPBA hydrogel, a composite hydrogel structure composed of GSH-PDA nanoparticles loaded on a PVA-TSPBA hydrogel backbone formed by the reaction of PVA and TSPBA.

[0094] In addition, by changing the solid content of the suspension of polydopamine melanin (GSH-PDA) nanoparticles (50 μg / ml and 200 μg / ml) and keeping other conditions unchanged, GSH-PDA / PVA-TSPBA hydrogels with different GSH-PDA nanoparticle loadings were prepared.

[0095] After freeze-drying, the surface of the prepared GSH-PDA / PVA-TSPBA hydrogel was gold-sprayed and the sample was placed under an electron gun. The morphology of the hydrogel and the nanoparticles loaded thereon was observed using a scanning electron microscope (Zeiss Supra55). Figure 3 As shown, the hydrogel exhibits a uniform porous structure. The structural properties of the hydrogel do not change significantly with changes in the GSH-PDA loading, and GSH-PDA nanoparticles are uniformly dispersed on the hydrogel surface. Calculations show that for a suspension of polydopamine melanin (GSH-PDA) nanoparticles with a solid content of 100 μg / ml, the hydrogel porosity is 60%, and 50 μg of GSH-PDA nanoparticles are loaded per milliliter of GSH-PDA / PVA-TSPBA hydrogel.

[0096] Performance testing

[0097] 1. Detection of the antioxidant properties of GSH-PDA nanoparticles

[0098] PDA nanoparticles were prepared according to the method of Example 1, freeze-dried, and then water was added to reconstitute a suspension containing PDA nanoparticles, wherein the solid contents of the PDA nanoparticles were 100 μg / ml, 150 μg / ml, and 200 μg / ml, respectively. Freeze-dried samples of GSH-PDA nanoparticles were prepared according to the method of Example 2, and then the freeze-dried samples were reconstituted with water until the solid contents of the GSH-PDA nanoparticles were 50 μg / ml, 100 μg / ml, and 200 μg / ml, respectively.

[0099] Then the antioxidant performance was tested. O2·-, ·OH and H2O2 are the main components of reactive oxygen species (ROS). O2·- detection kit, hydroxyl radical (·OH) detection kit and H2O2 detection kit were used to detect O2·-, hydroxyl radical and H2O2 respectively. The antioxidant performance of pure PDA and GSH-PDA nanoparticles with different concentrations (50, 100 and 200 μg / mL) was verified. Figure 4 b), ·OH( Figure 4 c) and H2O2( Figure 4 The scavenging effect of a).

[0100] The results showed that PDA melanin nanoparticles alone exhibited significant antioxidant activity, with the scavenging efficiency for O2·-, ·OH, and H2O2 increasing significantly with increasing concentration. At 100 μg / mL, PDA achieved a scavenging rate of 48% for O2·-, 60% for hydroxyl radicals, and 47.3% for H2O2. Glutathione (GSH) modification significantly enhanced the antioxidant properties of PDA. At the same concentration, GSH-PDA achieved a scavenging rate of 64% for O2·-, 78% for hydroxyl radicals, and 73% for H2O2.

[0101] 2. Detection of the antioxidant properties of GSH-PDA / PVA-TSPBA hydrogel

[0102] To prepare a pure PVA hydrogel, dissolve 1g of PVA in 20ml of water and stir at 90°C until the PVA powder is completely dissolved. Freeze the mixture at -20°C, then bring it to room temperature. Once dissolved, freeze it again at 20°C. Repeat this freeze-thaw cycle twice to prepare the PVA hydrogel.

[0103] Preparation of pure PVA-TSPBA hydrogel: Prepare TSPBA crosslinker using the same method as in Example 3. Dissolve the prepared solid TSPBA in deionized water to obtain a 50 mg / ml crosslinker solution. Add 1 g of PVA to 20 ml of water and stir at 90°C until the PVA powder is completely dissolved, thereby obtaining a PVA solution. Finally, equal volumes of the TSPBA crosslinker solution and the PVA solution were mixed to obtain a PVA-TSPBA hydrogel.

[0104] Preparation of PDA / PVA-TSPBA hydrogel: Except for omitting the step of “adding 40 mg of reduced glutathione (GSH) to the solution and stirring the reaction at 50°C for 7 hours”, a PDA / PVA-TSPBA composite hydrogel (PDA / Gel) loaded with polydopamine melanin (PDA) nanoparticles on the PVA-TSPBA hydrogel skeleton was prepared in the same manner as in Example 3.

[0105] Preparation of GSH-PDA / PVA-TSPBA hydrogel: GSH-PDA / PVA-TSPBA hydrogel (GSH-PDA / Gel, the solid content of the suspension of GSH-PDA nanoparticles was 100 μg / ml) was prepared according to the same method as in Example 3.

[0106] Then, the antioxidant properties of PVA hydrogel (PVA), PVA-TSPBA hydrogel (Gel), PDA / PVA-TSPBA hydrogel (PDA / Gel) and GSH-PDA / PVA-TSPBA hydrogel (GSH-PDA / Gel) were tested, and their scavenging effects on O2·- and hydroxyl radicals were tested using an O2·- detection kit and a hydroxyl radical (·OH) detection kit, respectively. The results are shown in Figure 2. Figure 5 As shown in the figure, it can be seen that pure PVA hydrogel has no antioxidant properties, and the non-responsive PVA hydrogel has no obvious scavenging ability for O2·- and ·OH. However, the ROS-responsive gel can effectively scavenge O2·- and ·OH, and the scavenging rate of the gel carrying GSH-PDA nanoparticles is the highest.

[0107] In order to verify whether GSH-PDA / Gel can effectively respond to ROS, GSH-PDA / Gel was first placed in a 500 μM H2O2 solution. Figure 6 As shown, the degradation of the hydrogel was observed over time, and it was found that the hydrogel was significantly degraded after 12 hours and GSH-PDA nanoparticles were released. A large amount of GSH-PDA nanoparticles were released after 24 hours, and the GSH-PDA nanoparticles were almost completely released after 72 hours. Figure 6 The reaction principle of PDA melanin nanoparticles and GSH and the principle of ROS scavenging process of GSH-PDA / Gel are also shown: the phenolic hydroxyl groups in polydopamine can undergo redox reaction with ROS, converting ROS into more stable molecules, such as water.

[0108] 3. Animal experiments

[0109] PVA-TSPBA hydrogel (Gel), PDA / PVA-TSPBA hydrogel (PDA / Gel), and GSH-PDA / PVA-TSPBA hydrogel (GSH-PDA / Gel) were prepared according to the same method as above.

[0110] Forty male Sprague Dawley rats (220 ± 20 g) were anesthetized with 2% sodium pentobarbital. Myocardial ischemia-reperfusion injury (I / R) was modeled: After rib stripping, the left anterior descending coronary artery (LAD) was ligated to expose the heart, and the ligature was tightened with a suture ring to ensure LAD occlusion, which was then removed 60 minutes later to allow reperfusion. The rats were then randomly divided into four groups: (1) I / R + PBS group; (2) I / R + Gel group; (3) I / R + PDA / Gel (100 μg / ml); (4) I / R + GSH-PDA / Gel (100 μg / ml). After 20 minutes of reperfusion, a total of 100 μL of hydrogel was injected into three points in the infarct edge area, while the PBS group was directly injected with 100 μL of PBS. The chest cavity was then closed and sutured, and the vital signs were observed and fed for 4 weeks. The cardiac function was detected by ultrasound and the infarct size was determined by Masson staining after the animals were sacrificed.

[0111] After 28 days of treatment, if Figure 7 As shown in a and b, fibrosis (blue fibroblasts and collagen) can be observed in the base, mid, and Apex parts of the PBS group, and the fibrosis in the mid and Apex parts is obvious, and the infarct area is significant. In the group injected with ROS-responsive hydrogel alone (Gel), fibrosis is mainly distributed in the mid and Apex parts. Compared with the PBS group and the ROS-responsive hydrogel alone group, the group injected with ROS-responsive hydrogel loaded with GSH-PDA (GSH-PDA / Gel) can observe the least fibrosis (blue fibroblasts and collagen) in the base, mid, and Apex parts, indicating that the lesion area is the smallest and the thickness of the left ventricular (LV) wall is significantly increased. The infarct area is also greatly reduced, and most of the fibrotic tissue returns to normal red myocardium.

[0112] Cardiac function was assessed by echocardiography 28 days after I / R ( Figure 7 (C) Compared with normal hearts, infarcted hearts treated with PBS exhibited typical features of myocardial infarction, with significant decreases in left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS), and a significant increase in left ventricular internal diameter at end-systole. Left ventricular end-systolic diameter (LVIDS) and left ventricular end-diastolic diameter (LVIDD) were significantly increased. ROS hydrogel alone had limited effect on cardiac repair, with an increase in LVEF and a decrease in LVIDS. ROS hydrogel increased LVEF by approximately 10%, which is comparable to previous reports. Notably, a significant increase in LVEF and LVFS was found in the ROS hydrogel-loaded GSH-PDA (GSH-PDA / Gel) group, indicating that pumping function and ventricular filling improved to approximately 60% of LVEF, demonstrating a significant improvement in myocardial repair performance.

[0113] The above description is merely a specific embodiment of the invention covered by this application, but the scope of protection of this application is not limited thereto. Any changes or substitutions that can be easily conceived by a person skilled in the art within the technical scope disclosed in this application should be included in the scope of protection of this application. Therefore, the scope of protection of this application should be based on the scope of protection of the claims.

Claims

1. Hydrogel, comprising: The PVA-TSPBA hydrogel skeleton is formed by the reaction of polyvinyl alcohol (PVA) with the crosslinker N1-(4-bromobenzyl)-N3-(4-bromophenyl)-N1,N1,N3,N3-tetramethylpropane-1,3-diamine (TSPBA), and Reduced glutathione-modified polydopamine melanin nanoparticles loaded on the PVA-TSPBA hydrogel skeleton.

2. The hydrogel according to claim 1, wherein: The hydrogel has a porous structure, and optionally, the porosity of the hydrogel is 50-70%; The reduced glutathione-modified polydopamine melanin nanoparticles are distributed on the surface of the PVA-TSPBA hydrogel skeleton.

3. The hydrogel according to claim 1 or 2, wherein the particle size of the reduced glutathione-modified polydopamine melanin nanoparticles is 40-200 nm, Optionally, the average particle size of the reduced glutathione-modified polydopamine melanin nanoparticles is 90-120 nm. Optionally, the D90 particle size of the reduced glutathione-modified polydopamine melanin nanoparticles is 60-140 nm.

4. The hydrogel according to any one of claims 1 to 3, wherein Each milliliter of the hydrogel is loaded with 25-100 μg of the reduced glutathione-modified polydopamine melanin nanoparticles.

5. A method for preparing a hydrogel, comprising the following steps: 1) mixing ammonia water, ethanol, and deionized water, adding dopamine hydrochloride, and stirring to react to obtain a solution containing polydopamine melanin nanoparticles; 2) adding reduced glutathione (GSH) to the solution containing polydopamine melanin nanoparticles in step 1), stirring for reaction, and then dialyzing to finally obtain a suspension of reduced glutathione-modified polydopamine melanin (GSH-PDA) nanoparticles; as well as 3) Adding polyvinyl alcohol (PVA) powder to the suspension of GSH-PDA nanoparticles and stirring until the PVA powder is completely dissolved to obtain a PVA solution containing GSH-PDA nanoparticles, stirring and mixing the PVA solution with a crosslinking agent TSPBA solution to react to obtain the hydrogel.

6. The method according to claim 5, wherein: In step 1) and step 2), the ratio of ammonia water, ethanol, deionized water, dopamine hydrochloride and GSH is 2-3 mL: 32-48 mL: 72-108 mL: 40-60 mg: 32-48 mg; Optionally, the stirring reaction temperature in step 1) is room temperature, and the stirring reaction time is 16-32h; Optionally, in step 2), the stirring reaction temperature is 45-55° C., and the stirring reaction time is 6-10 h; Optionally, in step 2), the size of the dialysis bag is 1000-2000 Da, and the dialysis time is 2-4 days. 7 . The method according to claim 5 , wherein the method further comprises the step of freeze-drying the suspension of GSH-PDA nanoparticles obtained in step 2).

8. The method according to any one of claims 5 to 7, wherein: In the suspension of the GSH-PDA nanoparticles used in step 3), the concentration of the glutathione-modified polydopamine melanin nanoparticles is 50-200 μg / ml; or In step 3), the concentration of PVA in the PVA solution is 40-60 mg / ml; or In step 3), the concentration of TSPBA in the cross-linking agent TSPBA solution is 40-60 mg / ml; or In step 3), the PVA solution and the cross-linking agent TSPBA solution are mixed in equal volumes. Optionally, the reaction temperature of the PVA solution and the cross-linking agent TSPBA solution is 60-90°C.

9. Use of the hydrogel according to any one of claims 1 to 4 or the hydrogel prepared according to the method according to any one of claims 5 to 8 in preparing a medicament for treating ischemia-reperfusion (I / R) injury, optionally wherein the medicament is an injectable hydrogel scaffold.

10. The use according to claim 9, wherein the ischemia-reperfusion injury is caused by a disease or condition selected from the group consisting of: myocardial infarction, cerebral infarction, limb arterial embolism, cerebral no-reflow phenomenon after cardiopulmonary resuscitation, stress ulcer, pancreatitis, burns, intestinal ischemia, necrotizing enterocolitis, intermittent claudication, acute tubular necrosis, post-shock liver failure, and multiple organ failure; optionally, the ischemia-reperfusion injury is myocardial ischemia-reperfusion injury, for example, myocardial ischemia-reperfusion injury caused by myocardial infarction.

Images