Sulfur-containing anti-oxidation conductive polymer, conductive anti-oxidation hydrogel and preparation method and application of conductive anti-oxidation hydrogel

Abstract

The invention discloses a sulfur-containing antioxidant conductive polymer, conductive antioxidant hydrogel as well as a preparation method and application of the conductive antioxidant hydrogel. The polymer is poly (thioethyl methacrylate-co-sodium acrylate). According to the sulfur-containing conductive antioxidant polymer provided by the invention, the sulfur-containing micromolecular monomer with the antioxidant property and the sodium acrylate monomer with the conductive property are introduced into the sulfur-containing conductive antioxidant polymer, and the sulfur-containing conductive antioxidant polymer not only has excellent antioxidant efficiency, stability and ROS (reactive oxygen species) eliminating effect, but also has the advantages of high oxidation resistance and high oxidation resistance. The method is suitable for repairing myocardial damaged tissues, is beneficial to relieving oxidative stress injury, can promote conduction of cell electric signals and synchronize contraction of myocardial cells, and is of great importance to electrical function recovery of myocardial tissues. In addition, the sulfur-containing antioxidant conductive polymer can achieve good biocompatibility, reduce immunological rejection and promote adhesion and growth of cells.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical materials, and particularly relates to a sulfur-containing antioxidant conductive polymer, a conductive antioxidant hydrogel, and their preparation methods and applications. Background Art

[0002] Cardiovascular diseases (CVDs) are one of the main causes of death globally. Among them, myocardial infarction (MI) is the most common cardiovascular disease and has become one of the major diseases threatening human health. Currently, the clinical treatment methods for MI mainly fall into the following three categories: 1. Conventional interventional and drug therapies: These methods can relieve the symptoms of the disease, but they cannot repair the non-renewable fibrotic wounds, often resulting in irreversible myocardial necrosis and decreased cardiac function; 2. Heart transplantation: Although heart transplantation is an effective method for treating advanced heart failure, it is costly, has a high degree of individual rejection, and a low survival rate. In addition, the transplanted heart cannot regenerate the myocardial microenvironment; 3. Stem cell implantation and gene therapy: These methods have great potential in theory, but they face many challenges in practical applications, such as low cell survival and retention rates, low gene transfection efficiency, high cost, and long time consumption.

[0003] Therefore, there is currently a lack of effective treatment means for the repair of the myocardial microenvironment after MI, and myocardial tissue engineering has become a prospective option for the treatment of MI and heart failure. A hydrogel is a polymer with hydrophilic groups that can swell in water but is insoluble in water and has a crosslinked three-dimensional network structure. Its three-dimensional network structure has certain mechanical properties and can provide mechanical support for the ventricular wall, thereby inhibiting negative ventricular remodeling and having great application potential in myocardial tissue engineering. However, a hydrogel with a single component is usually an electrically insulating material and is not conducive to the transmission of electrical signals between cells. In addition, studies have revealed the important role and molecular mechanism of oxidative stress inhibition and key inflammatory factor regulation in cardiac repair after MI. The signal pathways involved are intricate, and how to make an injectable hydrogel have antioxidant effects and apply it to the treatment of MI is still in the exploratory stage. Summary of the Invention

[0004] In order to overcome the problems existing in the above-mentioned prior art, one of the purposes of the present invention is to provide a sulfur-containing antioxidant conductive polymer. Another purpose of the present invention is to provide a preparation method of the above-mentioned sulfur-containing antioxidant conductive polymer. A third purpose of the present invention is to provide a conductive antioxidant hydrogel. A fourth purpose of the present invention is to provide a preparation method of the above-mentioned conductive antioxidant hydrogel. A fifth purpose of the invention is to provide the application of the above-mentioned conductive antioxidant hydrogel.

[0005] Conductive polymers not only have good electrical conductivity but can also be given biocompatibility and specific biological functions through chemical modification. This enables conductive polymers to exhibit great application potential in aspects such as biosensors, neural interfaces, tissue engineering, and drug delivery. Sulfur-containing antioxidant conductive polymers, as a new type of material, combine the antioxidant properties of sulfur atoms and the conductivity of polymers, providing innovative application opportunities in the medical field. Sulfur-containing antioxidant conductive polymers can be used as the base material for myocardial repair hydrogels, promoting the survival and functional recovery of cardiomyocytes by providing a conductive and antioxidant environment. After myocardial infarction, oxidative stress is one of the main causes of myocardial injury. This polymer can effectively reduce oxidative stress, protect cardiomyocytes, and reduce myocardial necrosis. The properties of sulfur-containing antioxidant conductive polymers also make them potentially applicable in biosensors and drug delivery systems, enabling real-time monitoring and regulation of the myocardial repair process. In summary, sulfur-containing antioxidant conductive polymers show broad application prospects in the treatment of myocardial infarction, promoting myocardial regeneration and functional recovery by improving the myocardial microenvironment and providing effective electrical and chemical support.

[0006] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0007] The first aspect of the present invention provides a sulfur-containing antioxidant conductive polymer, and the structural formula of the sulfur-containing antioxidant conductive polymer is shown as Formula 1 below:

[0008]

[0009] Wherein, x and y are the numbers of each monomer structural unit.

[0010] The sulfur-containing antioxidant conductive polymer of the present invention introduces sulfur-containing small molecules with antioxidant properties and uses the novel antioxidant substance 2-(ethylsulfanyl)ethyl methacrylate as the monomer material, which can achieve excellent antioxidant effects through its small molecular weight; at the same time, the conductive substance sodium acrylate is introduced to prepare the conductive antioxidant polymer.

[0011] Preferably, the ratio of x to y is 1:6.

[0012] Preferably, the degree of polymerization of the sulfur-containing antioxidant conductive polymer is 7 to 300.

[0013] The second aspect of the present invention provides a preparation method of the sulfur-containing antioxidant conductive polymer described in the first aspect, including the following steps: Mix 2-(ethylsulfanyl)ethyl methacrylate, sodium acrylate, an initiator, and a crosslinking agent, and carry out a polymerization reaction to obtain the sulfur-containing antioxidant conductive polymer.

[0014] Preferably, the molar ratio of 2-(ethylsulfanyl)ethyl methacrylate to sodium acrylate is 1:(3 - 5).

[0015] Preferably, the molar ratio of the thioethyl methacrylate to the crosslinking agent is 1:(0.5 - 2).

[0016] Preferably, the molar ratio of the thioethyl methacrylate to the initiator is 1:(0.01 - 0.1).

[0017] Preferably, the crosslinking agent is N,N - methylenebisacrylamide (MBA).

[0018] Preferably, the initiator is azobisisobutyronitrile (AIBN).

[0019] Preferably, the preparation steps of the thioethyl methacrylate include: acryloyl chloride, ethanethiol and triethylamine react in a solvent to obtain the thioethyl methacrylate.

[0020] More preferably, the mass ratio of the ethanethiol to the acryloyl chloride is 1:(1 - 2); the mass ratio of the ethanethiol to the triethylamine is 1:(1 - 2).

[0021] More preferably, the solvent is dichloromethane.

[0022] More preferably, the reaction time of the reaction is 60 - 85 h.

[0023] Preferably, the polymerization reaction is carried out under anaerobic conditions.

[0024] More preferably, the preparation method further includes: subjecting the obtained mixed solution to liquid nitrogen freezing, then vacuum pumping, nitrogen filling, and thawing to remove the residual oxygen in the system, and this process is repeated once or more times.

[0025] Preferably, the reaction temperature of the polymerization reaction is 40 - 60 °C.

[0026] The third aspect of the present invention provides a conductive antioxidant hydrogel, and the preparation raw materials of the conductive antioxidant hydrogel include a gel matrix and the sulfur - containing antioxidant conductive polymer described in the first aspect.

[0027] Preferably, the gel matrix is selected from at least one of gelatin, polyvinyl alcohol, polyacrylamide, and chitosan.

[0028] The fourth aspect of the present invention provides a preparation method of the conductive antioxidant hydrogel described in the third aspect, including the following steps: mixing the gel matrix, the sulfur - containing antioxidant conductive polymer, an initiator and a crosslinking agent in water, and carrying out a crosslinking curing reaction to obtain the conductive antioxidant hydrogel.

[0029] Preferably, the reaction temperature of the crosslinking curing reaction is 50 - 70 °C.

[0030] Preferably, the reaction time of the crosslinking and curing reaction is 10 to 30 minutes.

[0031] Preferably, the crosslinking agent is N,N'-methylenebisacrylamide (BIS).

[0032] Preferably, the initiator is ammonium persulfate (APS).

[0033] Preferably, the mass ratio of the gelling matrix to the sulfur-containing antioxidant conductive polymer is 1:(0.5 - 1.5).

[0034] The fifth aspect of the present invention provides the application of the conductive antioxidant hydrogel described in the third aspect in the preparation of products for myocardial repair.

[0035] The beneficial effects of the present invention are as follows:

[0036] (1) The present invention provides a sulfur-containing conductive antioxidant polymer. The sulfur-containing conductive antioxidant polymer incorporates a sulfur-containing small molecule monomer with antioxidant properties and a sodium acrylate monomer with conductive properties. The conductive antioxidant sulfur-containing polymer not only has excellent antioxidant efficiency, stability, and the function of eliminating ROS, is applicable to the repair of myocardial damaged tissues, is beneficial to reducing oxidative stress damage, but also can promote the conduction of cell electrical signals and synchronize the contraction of cardiomyocytes, which is crucial for the restoration of the electrical function of myocardial tissues. In addition, the sulfur-containing antioxidant conductive polymer of the present invention can achieve good biocompatibility, reduce immune rejection reactions, and promote cell adhesion and growth.

[0037] (2) The present invention also provides a conductive antioxidant hydrogel. The sulfur-containing conductive antioxidant polymer is crosslinked with a macromolecular compound to obtain the conductive antioxidant hydrogel. The three-dimensional network structure of the conductive antioxidant hydrogel itself has certain mechanical properties and can provide mechanical support for the ventricular wall. Combining the conductivity and antioxidant modification brought by the polymer enables the hydrogel to facilitate the transmission of electrical signals between cells. In addition, the conductive antioxidant hydrogel of the present invention has good injectability and biocompatibility, can simulate the tissue microenvironment to promote the growth of cardiomyocytes, and promote angiogenesis and the repair of defective tissues. Description of the Drawings

[0038] Figure 1 1H NMR spectrum of ethyl thioacrylate and sulfur-containing conductive antioxidant polymer;

[0039] Figure 2 Staining legend of live / dead staining of the survival of H9C2 cells by the sulfur-containing conductive antioxidant hydrogel of Example 2;

[0040] Figure 3Staining legend of the effect of the sulfur-containing conductive antioxidant hydrogel of Example 2 and the non-conductive non-antioxidant hydrogel of Comparative Example 1 on reactive oxygen species in the H9C2 cell control group;

[0041] Figure 4 Staining legend of the effect of the sulfur-containing conductive antioxidant hydrogel of Example 2 and the non-conductive non-antioxidant hydrogel of Comparative Example 1 on reactive oxygen species in the H9C2 cell OGDR group;

[0042] Figure 5 Results of the survival rate of cardiomyocytes of ethanethiol, dopamine and tea polyphenols under oxidative stress conditions;

[0043] Figure 6 Conductivity of the sulfur-containing conductive antioxidant hydrogel of Example 2 and the hydrogel of Comparative Example 1. Detailed implementation manners

[0044] The content of the present invention will be further described in detail below through specific examples. The raw materials used in the following examples, unless otherwise specified, can be obtained from conventional commercial channels or prepared and separated by simple synthesis; the processes used, unless otherwise specified, are conventional processes in the art.

[0045] Example 1

[0046] This example provides a sulfur-containing antioxidant conductive polymer, and its synthesis equation is as follows:

[0047]

[0048] The specific preparation process is as follows:

[0049] Add 100 - 150 mL of dichloromethane, 10 - 30 g of triethylamine and 10 - 20 g of ethanethiol into a four-necked flask and mix well. Subsequently, 20 - 30 g of acryloyl chloride is added dropwise under stirring. During the dropping process, the temperature of the reaction mixture rises, and 10 - 20 mL of dichloromethane can be appropriately added to rinse the funnel wall. After stirring and reacting for 3 days, the solvent dichloromethane is removed using a rotary evaporator. Tetrahydrofuran is added to dissolve the reaction product, and at the same time, triethylamine hydrochloride precipitates to form a solid-liquid mixture. The solid-liquid mixture is filtered through a suction funnel to separate the solution containing the product and triethylamine hydrochloride. The solution containing the product is again passed through a rotary evaporator to remove tetrahydrofuran, and ethyl thioacrylate monomer is obtained.

[0050] After mixing the four substances in a molar ratio of thioethyl methacrylate: sodium acrylate: MBA: AIBN = 1:4:1:0.03 (specific amounts are 1.33g of thioethyl methacrylate, 3.76g of sodium acrylate, 1.54g of MBA, and 49.2mg of AIBN), 10mL of DMSO and 30mL of deionized water are added. After freezing the above mixture into a solid with liquid nitrogen, evacuate for 30 seconds. Subsequently, the mixture is melted in a water bath, and continued to be frozen and evacuated with liquid nitrogen. The above operation is repeated three times to exclude oxygen in the tube. Finally, the mixture is placed in an oil bath at 40-60°C for reaction, and the state of the mixture is observed. When it is jelly-like, poly (thioethyl methacrylate-co-sodium acrylate) is successfully synthesized.

[0051] The H NMR spectra of thioethyl methacrylate (S monomer) and sulfur-containing conductive antioxidant polymers are shown in Figure 2. Figure 1 As shown, through 1 H NMR was used to characterize the sulfur-containing polymer to determine whether the polymer was successfully prepared. A unique peak at 5.3 ppm appeared in the 1H NMR spectrum of the S monomer, which was attributed to the -C=C- in thioethyl methacrylate, while in the polymer 1 The double bond peak disappeared in the H NMR spectrum, indicating the successful preparation of the polymer. The degree of polymerization was determined to be 7-300 by gel permeation chromatography (GPC).

[0052] Example 2

[0053] The present embodiment provides an antioxidant conductive hydrogel, and the specific preparation process is: using 1 mL of a 10% (w / v) gelatin aqueous solution as the reaction system, then adding 1% (w / v) ammonium persulfate (APS) and 0.3% (w / v) N,N'-methylenebisacrylamide (BIS) and 100 μg of the sulfur-containing antioxidant conductive polymer of Example 1 to the solution, mixing thoroughly and adding to a mold, placing it in a 60°C water bath for 15 minutes, and then demolding to obtain a conductive antioxidant hydrogel.

[0054] Comparative Example 1

[0055] This comparative example provides a hydrogel, and the specific preparation process is as follows: in a 1 mL system, first prepare a 10% gelatin solution. Then, add 1% ammonium persulfate (APS), 0.3% N,N'-methylenebisacrylamide (BIS), and 10% sodium acrylate to the solution, mix well, add to a mold, place in a 60°C water bath for 15 minutes, and then demold, to obtain a non-conductive, non-antioxidant hydrogel.

[0056] Biological Experiment Analysis

[0057] 1. Biological toxicity and compatibility test

[0058] The cardiomyocytes H9C2 were seeded onto the sulfur-containing conductive antioxidant hydrogels prepared in the four Examples 2 at the same cell concentration. Live / dead staining was performed at 1 day, 3 days, 5 days, and 7 days of culture, respectively, to observe the survival of the cells on the hydrogels. After reaching the above time periods, the four groups of hydrogels seeded with cells were taken out, washed three times with PBS to remove the residual culture medium, and the prepared live / dead staining working solution was added dropwise to completely immerse the samples. After incubating in the dark at room temperature for about 5 - 10 minutes, observation and photography were carried out using a laser confocal microscope. Figure 2 It is the staining legend of the live / dead staining of the survival of H9C2 cells on the sulfur-containing conductive antioxidant hydrogel of Example 2. The results show that the cell survival rate on the hydrogel exceeds 95%, indicating that the hydrogel prepared in Example 2 using the polymer as the raw material is non-toxic and has good biocompatibility.

[0059] 2. Antioxidant experiment

[0060] For the antioxidant experiment, H9C2 cells were used to explore and establish a cell (oxygen-glucose deprivation / reoxygenation) injury (OGDR) model. The model establishment method was as follows: The cardiomyocytes H9C2 were seeded onto the hydrogels of Example 2 and Comparative Example 1 at the same cell concentration. After the cells grew to a confluent monolayer, the culture medium was replaced with a sugar-free medium and transferred to a triple-gas incubator (37 °C, 5% CO2, 90% N2, 5% O2) for hypoxic culture for 4 hours. Then, the sugar-free medium was replaced with AM medium and placed in a CO2 incubator for reoxygenation for 24 hours to simulate myocardial ischemia-reperfusion injury. After the model establishment was completed, the well plate was taken out and the cells were washed 3 times with PBS. Subsequently, the cells were stained with the DCFH-DA fluorescent probe. The DCFH-DA staining solution was diluted with DMEM medium at a ratio of 1:1000 and incubated at 37 °C for 30 minutes. After the staining was completed, the staining solution was removed and the cells were washed with PBS. Subsequently, the cell nuclei were stained with Hoechst staining solution, and observation and imaging were carried out using a fluorescence microscope. In addition, a control group was set up, and no treatment was performed on the control group.

[0061] Figure 3 It is the staining legend of the influence of the sulfur-containing conductive antioxidant polymer of Example 1 and the polymer of Comparative Example 1 on the control group of H9C2 cells; Figure 4Staining legend of the effects of the sulfur-containing conductive antioxidant polymer of Example 1 and the polymer of Comparative Example 1 on the OGDR group of H9C2 cells; as shown in Figure 4, after OGDR treatment, a large amount of strong green fluorescence appeared in the control group, indicating a high level of ROS in H9C2; the intensity of green fluorescence in the cells of the antioxidant conductive hydrogel group decreased, indicating that the conductive antioxidant hydrogel effectively inhibited the expression of ROS, had excellent anti-inflammatory and antioxidant effects, significantly reduced the continuous damage caused by inflammation and contributed to the repair of myocardial damaged tissues.

[0062] To verify the performance superiority of the sulfur-containing antioxidant ethanethiol in the present invention, two sulfur-free but antioxidant active substances, namely dopamine and tea polyphenols, were selected for comparison. Dopamine is a small molecule compound, and its phenolic hydroxyl group provides antioxidant activity; tea polyphenols are natural polyphenolic compounds, and they scavenge free radicals through a hydrogen donor mechanism. Figure 5 For the cck8 data analysis of the three drugs under oxidative stress conditions of cardiomyocytes, ethanethiol showed better stability and had a certain repair effect on cell viability. The sulfur-containing antioxidants of the present invention are significantly superior to the selected contrast agents in terms of antioxidant efficiency, stability and biological protection. The sulfur-containing antioxidants have higher free radical scavenging efficiency. Under oxidative stress conditions (such as high temperature, pH fluctuation, etc.), the sulfur-containing antioxidants show better stability. In cell experiments, the sulfur-containing antioxidants can significantly reduce the level of reactive oxygen species (ROS).

[0063] 3. Conductivity experiment

[0064] The conductivity of the hydrogels of Example 2 and Comparative Example 1 was measured by the four-probe method in an electrochemical workstation. The hydrogel samples were immersed in PBS buffer. After 72 h of immersion, the resistivity Rp of the hydrogel samples was measured by linear voltammetry cycling. Since the conductivity (σ) and the resistivity (Rp) are reciprocals of each other, the conductivity can be obtained:

[0065] σ = 1 / ρ = L / Rp;

[0066] The test results are as Figure 5 shown, the conductivity of the hydrogel introduced with the conductive antioxidant polymer as the raw material is significantly improved. The conductivity of this hydrogel is within the cardiomyocyte conduction range (5x10 -3 -1.6x10 -1 S / m), indicating that the hydrogel of Example 2 can promote the conduction of cell electrical signals and synchronize the contraction of cardiomyocytes more effectively than that of Comparative Example 1, which is crucial for the restoration of the electrical function of myocardial tissues.

[0067] The above are the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications are also regarded as the protection scope of the present invention.

Claims

1. A sulfur-containing antioxidant conductive polymer, characterized in that, The structural formula of the sulfur-containing antioxidant conductive polymer is shown in Formula 1 below: Among them, x and y are the numbers of each monomer structural unit.

2. The preparation method of the sulfur-containing antioxidant conductive polymer according to claim 1, characterized in that, It includes the following steps: Mix ethyl thioacrylate, sodium acrylate, an initiator and a crosslinking agent, and carry out a polymerization reaction to obtain the sulfur-containing antioxidant conductive polymer.

3. The preparation method of the sulfur-containing antioxidant conductive polymer according to claim 2, wherein, The molar ratio of ethyl thioacrylate to sodium acrylate is 1:(3-5); and / or, the molar ratio of ethyl thioacrylate to the crosslinking agent is 1:(0.5-2); and / or, the molar ratio of ethyl thioacrylate to the initiator is 1:(0.01-0.1).

4. The preparation method of the sulfur-containing antioxidant conductive polymer according to claim 2, wherein, The preparation steps of ethyl thioacrylate include: acryloyl chloride, ethanethiol and triethylamine react in a solvent to obtain ethyl thioacrylate.

5. The preparation method of the sulfur-containing antioxidant conductive polymer according to claim 2, characterized in that, The polymerization reaction is carried out under anaerobic conditions; The reaction temperature of the polymerization reaction is 40-60 °C.

6. A conductive and antioxidant hydrogel, characterized in that, The raw materials for preparing the conductive antioxidant hydrogel include a gelling matrix and the sulfur-containing antioxidant conductive polymer described in Claim 1.

7. The electrically conductive and antioxidant hydrogel according to claim 6, wherein The gelling matrix is selected from at least one of gelatin, polyvinyl alcohol, polyacrylamide, and chitosan.

8. The preparation method of the conductive antioxidant hydrogel according to claim 6 or 7, characterized in that, It includes the following steps: Mix the gelling matrix, the sulfur-containing antioxidant conductive polymer, an initiator and a crosslinking agent in water, and carry out a crosslinking and curing reaction to obtain the conductive antioxidant hydrogel.

9. The preparation method of the conductive antioxidant hydrogel according to claim 8, characterized in that, The reaction temperature of the crosslinking and curing reaction is 50-70 °C; and / or, the reaction time of the crosslinking and curing reaction is 10-30 min.

10. Use of the conductive antioxidant hydrogel according to Claim 6 or 7 in the preparation of a product for myocardial repair.

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