A sulfur-containing antioxidant conductive polymer, a conductive antioxidant hydrogel and a preparation method and application thereof

Abstract

The application discloses a kind of sulphur-containing antioxidative conductive polymer, conductive antioxidative hydrogel and its preparation method and application, the polymer is poly (thioethyl methacrylate-co-sodium acrylate).The application provides a kind of sulphur-containing conductive antioxidative polymer, the sulphur-containing conductive antioxidative polymer is introduced with antioxidative characteristic sulphur-containing small molecule monomer, and with conductive characteristic sodium acrylate monomer, the conductive antioxidative sulphur-containing polymer not only has excellent antioxidative efficiency, stability and the effect of eliminating ROS, is suitable for myocardial injury tissue repair, is conducive to reducing oxidative stress injury, can also promote the conduction of cell electric signal, the contraction of synchronous myocardial cell, is crucial for the electrical function recovery of myocardial tissue.In addition, the sulphur-containing antioxidative conductive polymer of the application can realize good biocompatibility, reduce immune rejection response, promote the adhesion and growth of cell.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, specifically relating to a sulfur-containing antioxidant conductive polymer, a conductive antioxidant hydrogel, and their preparation methods and applications. Background Technology

[0002] Cardiovascular disease (CVD) is one of the leading causes of death worldwide. Myocardial infarction (MI) is the most common CVD and has become a major threat to human health. Currently, clinical treatments for MI mainly fall into three categories: 1. Conventional interventional and drug therapy: These methods can alleviate symptoms but cannot repair the irreversible fibrotic damage, often leading to irreversible myocardial necrosis and decreased cardiac function; 2. Heart transplantation: Although heart transplantation is an effective treatment for advanced heart failure, it is costly, has a high rate of individual rejection, and a low survival rate. Furthermore, transplanted hearts cannot regenerate the myocardial microenvironment; 3. Stem cell implantation and gene therapy: These methods have great theoretical potential, but face many challenges in practical application, such as low cell survival and retention rates, low gene transfection efficiency, high cost, and long treatment time.

[0003] Therefore, there are currently no effective treatments for repairing the myocardial microenvironment after myocardial infarction, and myocardial tissue engineering has become a prospective option for the treatment of myocardial infarction and heart failure. Hydrogels are polymers with hydrophilic groups that can swell in water but are insoluble in water, possessing a cross-linked three-dimensional network structure. Their three-dimensional network structure has certain mechanical properties and can provide mechanical support for the ventricular wall, thereby inhibiting negative ventricular remodeling. They have great application potential in myocardial tissue engineering. However, single-component hydrogels are usually electrically insulating materials, which is not conducive to the transmission of intercellular electrical signals. Furthermore, studies have revealed the important role and molecular mechanisms of oxidative stress inhibition and key inflammatory factor regulation in cardiac repair after myocardial infarction (MI). The signaling pathways involved are complex, and how to enable injectable hydrogels to have antioxidant effects and apply them to the treatment of MI remains in the exploratory stage. Summary of the Invention

[0004] To overcome the problems existing in the prior art, one objective of this invention is to provide a sulfur-containing antioxidant conductive polymer. A second objective of this invention is to provide a method for preparing the aforementioned sulfur-containing antioxidant conductive polymer. A third objective of this invention is to provide a conductive antioxidant hydrogel. A fourth objective of this invention is to provide a method for preparing the aforementioned conductive antioxidant hydrogel. A fifth objective of this invention is to provide applications of the aforementioned conductive antioxidant hydrogel.

[0005] Conductive polymers not only possess excellent electrical conductivity but can also be chemically modified to acquire biocompatibility and specific biological functions. This makes conductive polymers demonstrate great application potential in biosensors, neural interfaces, tissue engineering, and drug delivery. Sulfur-containing antioxidant conductive polymers, as a novel material, combine the antioxidant properties of sulfur atoms with the conductivity of polymers, offering innovative application opportunities in the medical field. Sulfur-containing antioxidant conductive polymers can serve as the base material for myocardial repair hydrogels, promoting cardiomyocyte survival and functional recovery by providing a conductive and antioxidant environment. After myocardial infarction, oxidative stress is one of the main causes of myocardial damage. This polymer can effectively reduce oxidative stress, protect cardiomyocytes, and reduce myocardial necrosis. The properties of sulfur-containing antioxidant conductive polymers also make them potential applications 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 electrochemical support.

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

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

[0008]

[0009] Where x and y represent the number of structural units in each individual unit.

[0010] The sulfur-containing antioxidant conductive polymer of the present invention introduces sulfur-containing small molecules with antioxidant properties and uses a novel antioxidant, ethyl thioacrylate, as a monomer material, which can achieve excellent antioxidant effect through its small molecular weight; at the same time, sodium acrylate, a conductive material, is introduced to prepare a 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 method for preparing the sulfur-containing antioxidant conductive polymer described in the first aspect, comprising the following steps: mixing ethyl thioacrylate, sodium acrylate, an initiator and a crosslinking agent, and carrying out a polymerization reaction to obtain the sulfur-containing antioxidant conductive polymer.

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

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

[0016] Preferably, the molar ratio of thioethyl methacrylate to the initiator is 1:(0.01 to 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: reacting acryloyl chloride, ethanethiol and triethylamine in a solvent to obtain the thioethyl methacrylate.

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

[0021] More preferably, the solvent is dichloromethane.

[0022] More preferably, the reaction time is 60 to 85 hours.

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

[0024] More preferably, the preparation method further includes: freezing the mixed solution with liquid nitrogen, then evacuating, filling with nitrogen, and thawing to remove residual oxygen in the system, and repeating this process once or multiple times.

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

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

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

[0028] The fourth aspect of the present invention provides a method for preparing the conductive antioxidant hydrogel described in the third aspect, comprising the following steps: mixing the gel matrix, the sulfur-containing antioxidant conductive polymer, the initiator and the 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 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 gel matrix to the sulfur-containing antioxidant conductive polymer is 1:(0.5-1.5).

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

[0035] The beneficial effects of this invention are:

[0036] (I) This invention provides a sulfur-containing conductive antioxidant polymer, which incorporates sulfur-containing small molecule monomers with antioxidant properties and sodium acrylate monomers with conductive properties. This conductive antioxidant sulfur-containing polymer not only possesses excellent antioxidant efficiency, stability, and ROS elimination capabilities, making it suitable for repairing damaged myocardial tissue and helping to reduce oxidative stress damage, but also promotes the transmission of cellular electrical signals and synchronizes myocardial cell contraction, which is crucial for the recovery of myocardial tissue electrical function. Furthermore, the sulfur-containing antioxidant conductive polymer of this invention achieves good biocompatibility, reduces immune rejection reactions, and promotes cell adhesion and growth.

[0037] (II) The present invention also provides a conductive antioxidant hydrogel, which is obtained by crosslinking the above-mentioned sulfur-containing conductive antioxidant polymer with macromolecular compounds. The three-dimensional network structure of the conductive antioxidant hydrogel itself has certain mechanical properties and can provide mechanical support for the ventricular wall. Combined with the conductive and antioxidant modification brought by the polymer, the hydrogel can facilitate the transmission of intercellular electrical signals. In addition, the conductive antioxidant hydrogel of the present invention has good injectability and biocompatibility, and can simulate the tissue microenvironment to promote the growth of cardiomyocytes, as well as promote angiogenesis and repair of damaged tissues. Attached Figure Description

[0038] Figure 1 The hydrogen nuclear magnetic resonance spectra of thioethyl methacrylate and sulfur-containing conductive antioxidant polymers are shown.

[0039] Figure 2 This is a staining illustration of the sulfur-containing conductive antioxidant hydrogel used in Example 2 to show the viability and mortality of H9C2 cells.

[0040] Figure 3The staining diagram illustrates the effect of reactive oxygen species on the sulfur-containing conductive antioxidant hydrogel of Example 2 and the non-conductive non-antioxidant hydrogel of Comparative Example 1 in the H9C2 cell control group.

[0041] Figure 4 The staining illustration shows the effect of reactive oxygen species on the sulfur-containing conductive antioxidant hydrogel of Example 2 and the non-conductive non-antioxidant hydrogel of Comparative Example 1 in the OGDR group of H9C2 cells.

[0042] Figure 5 Results on cardiomyocyte survival under oxidative stress conditions for ethanethiol, dopamine, and tea polyphenols;

[0043] Figure 6 The conductivity is shown in Example 2 for the sulfur-containing conductive antioxidant hydrogel and in Comparative Example 1 for the hydrogel. Detailed Implementation

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

[0045] Example 1

[0046] This embodiment provides a sulfur-containing antioxidant conductive polymer, the synthesis equation of which is as follows:

[0047]

[0048] The preparation process is as follows:

[0049] Add 100-150 mL of dichloromethane, 10-30 g of triethylamine, and 10-20 g of ethanethiol to a four-necked flask and mix well. Then, add 20-30 g of acryloyl chloride dropwise while stirring. During the addition, the temperature of the reaction mixture will rise; if necessary, add 10-20 mL of dichloromethane to rinse the funnel walls. After stirring the reaction for 3 days, remove the dichloromethane solvent using a rotary evaporator. Add tetrahydrofuran to dissolve the reaction product, simultaneously precipitating triethylamine hydrochloride to form a solid-liquid mixture. Filter the solid-liquid mixture through a suction funnel to separate the solution containing the product and the triethylamine hydrochloride. Remove the tetrahydrofuran from the solution containing the product again using a rotary evaporator to obtain the thioethyl methacrylate monomer.

[0050] The four substances were mixed in a molar ratio of ethyl thioacrylate: sodium acrylate: MBA: AIBN = 1:4:1:0.03 (specific amounts: 1.33 g ethyl thioacrylate, 3.76 g sodium acrylate, 1.54 g MBA, and 49.2 mg AIBN). 10 mL of DMSO and 30 mL of deionized water were then added. The mixture was frozen solid with liquid nitrogen and then evacuated for 30 seconds. The mixture was then thawed in a water bath, and the process of freezing with liquid nitrogen and evacuating was repeated three times to remove oxygen from the tube. Finally, the mixture was placed in an oil bath at 40-60°C to react, and the state of the mixture was observed. When it reached a jelly-like consistency, poly(ethyl thioacrylate-co-sodium acrylate) was successfully synthesized.

[0051] The proton nuclear magnetic resonance spectra of ethyl thioacrylate (S monomer) and sulfur-containing conductive antioxidant polymers are as follows: Figure 1 As shown, through 1 ¹H NMR was used to characterize the sulfur-containing polymer to determine whether the polymer was successfully prepared. A unique peak of 5.3 ppm appeared in the ¹H NMR spectrum of the S monomer, which is attributed to the -C=C- in ethyl thiomethacrylate, while in the polymer... 1 The disappearance of the double bond peaks in the 1H NMR spectrum indicates successful polymer preparation. The degree of polymerization was determined to be 7-300 by gel permeation chromatography (GPC).

[0052] Example 2

[0053] This embodiment provides an antioxidant conductive hydrogel. The preparation process is as follows: 1 mL of 10% (w / v) gelatin aqueous solution is used as the reaction system. Then, 1% (w / v) ammonium persulfate (APS), 0.3% (w / v) N,N'-methylenebisacrylamide (BIS), and 100 μg of the sulfur-containing antioxidant conductive polymer of Example 1 are added to the solution. After thorough mixing, the mixture is poured into a mold and placed in a 60°C water bath for 15 minutes before demolding to obtain the conductive antioxidant hydrogel.

[0054] Comparative Example 1

[0055] This comparative example provides a hydrogel, the preparation process of which is as follows: First, a 10% gelatin solution is prepared in a 1 mL system. Then, 1% ammonium persulfate (APS), 0.3% N,N'-methylenebisacrylamide (BIS), and 10% sodium acrylic acid are added to the solution and mixed thoroughly. The mixture is then poured into a mold and placed in a 60°C water bath for 15 minutes before demolding to obtain a non-conductive, non-oxidizing hydrogel.

[0056] Biological Experimental Analysis

[0057] 1. Biotoxicity and compatibility tests

[0058] H9C2 cardiomyocytes were seeded at the same cell concentration onto four sulfur-containing conductive antioxidant hydrogels prepared in Example 2. Live / dead cell staining was performed at 1, 3, 5, and 7 days of culture to observe cell viability on the hydrogels. After reaching these time points, the four groups of cell-inoculated hydrogels were removed, washed three times with PBS to remove residual culture medium, and the prepared live / dead staining working solution was added, ensuring complete immersion of the sample. The samples were incubated at room temperature in the dark for approximately 5-10 minutes, and then observed and photographed using a laser confocal microscope. Figure 2 The image shows a staining pattern of H9C2 cells on the sulfur-containing conductive antioxidant hydrogel of Example 2, indicating that the cell survival rate on the hydrogel exceeded 95%, demonstrating that the hydrogel prepared in Example 2 using polymer as raw material is non-toxic and has good biocompatibility.

[0059] 2. Antioxidant Experiment

[0060] Antioxidant experiments were conducted using H9C2 cells to explore and establish a cell (oxygen-glucose deprivation / reoxygenation) injury (OGDR) model. The model was established as follows: H9C2 cardiomyocytes were seeded at the same cell concentration onto hydrogels from Example 2 and Comparative Example 1. After the cells grew to a continuous monolayer, the culture medium was replaced with glucose-free medium, and the cells were transferred to a tri-gas incubator (37°C, 5% CO2, 90% N2, 5% O2) for 4 hours of hypoxic culture. The glucose-free medium was then replaced with AM medium, and the cells were placed in a CO2 incubator for 24 hours for reoxygenation, simulating myocardial ischemia-reperfusion injury. After model establishment, the wells were removed, and the cells were washed three times with PBS. Subsequently, the cells were stained with the DCFH-DA fluorescent probe. The DCFH-DA staining solution was diluted 1:1000 with DMEM medium and incubated at 37°C for 30 minutes. After staining, the staining solution was removed, and the cells were washed with PBS. The cell nuclei were then stained with Hoechst staining solution, and the cells were observed and imaged using a fluorescence microscope. Additionally, a control group is set up, which is not processed in any way.

[0061] Figure 3 The staining diagram illustrates the effects of the sulfur-containing conductive antioxidant polymer of Example 1 and the polymer of Comparative Example 1 on the H9C2 cell control group. Figure 4The staining diagram shows 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, the control group showed a large amount of strong green fluorescence, indicating that the ROS level of H9C2 cells was high. The intensity of green fluorescence in the cells of the antioxidant conductive hydrogel group was reduced, indicating that the conductive antioxidant hydrogel effectively inhibited ROS expression, had excellent anti-inflammatory and antioxidant effects, significantly reduced the persistent damage caused by inflammation and helped repair myocardial tissue damage.

[0062] To verify the superior performance of the sulfur-containing antioxidant ethanethiol in this invention, two sulfur-free but antioxidant active substances, namely dopamine and tea polyphenols, were selected as comparisons. Dopamine is a small molecule compound whose phenolic hydroxyl groups provide antioxidant activity; tea polyphenols are natural polyphenolic compounds that scavenge free radicals through a hydrogen donor mechanism. Figure 5 Analysis of CCK8 data for three drugs under oxidative stress conditions in cardiomyocytes showed that ethanethiol exhibited better stability and a certain degree of cell regeneration. The sulfur-containing antioxidant of this invention significantly outperformed the selected contrast agent in terms of antioxidant efficiency, stability, and bioprotective properties. The sulfur-containing antioxidant possesses higher free radical scavenging efficiency. Under oxidative stress conditions (high temperature, pH fluctuations, etc.), the sulfur-containing antioxidant exhibited better stability. In cell experiments, the sulfur-containing antioxidant significantly reduced reactive oxygen species (ROS) levels.

[0063] 3. Conductivity test

[0064] The conductivity of the hydrogels in Example 2 and Comparative Example 1 was tested using a four-probe method in an electrochemical workstation. The hydrogel samples were immersed in PBS buffer for 72 hours, and the resistivity Rp of the hydrogel samples was measured using a linear voltammetric cycle method. Since conductivity (σ) and resistivity (Rp) are reciprocals, the conductivity can be calculated.

[0065] σ = 1 / ρ = L / Rp;

[0066] Test results are as follows Figure 5 As shown, the conductivity of the hydrogel using conductive antioxidant polymers as raw materials is significantly improved. This hydrogel's conductivity is within the range of myocardial conductivity (5 x 10⁻⁶). -3 -1.6x10 -1 The S / m ratio indicates that the hydrogel of Example 2 is more effective than that of Comparative Example 1 in promoting the transmission of cellular electrical signals and synchronizing the contraction of cardiomyocytes, which is crucial for the recovery of electrical function of myocardial tissue.

[0067] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications are also considered to be within the scope of protection 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: Formula 1; Where x and y are the number of each individual structural unit; the ratio of x to y is 1:6; The degree of polymerization of the sulfur-containing antioxidant conductive polymer is 7 to 300.

2. The method for preparing the sulfur-containing antioxidant conductive polymer according to claim 1, characterized in that, The process includes the following steps: mixing ethyl thioacrylate, sodium acrylate, an initiator, and a crosslinking agent, and carrying out a polymerization reaction to obtain the sulfur-containing antioxidant conductive polymer; The molar ratio of thioethyl methacrylate to sodium acrylate is 1:(3~5); The preparation steps of the thioethyl methacrylate include: reacting acryloyl chloride, ethanethiol and triethylamine in a solvent to obtain the thioethyl methacrylate.

3. The method for preparing the sulfur-containing antioxidant conductive polymer according to claim 2, characterized in that, The molar ratio of thioethyl methacrylate to the crosslinking agent is 1:(0.5~2); And / or, the molar ratio of the thioethyl methacrylate to the initiator is 1:(0.01~0.1).

4. The method for preparing the sulfur-containing antioxidant conductive polymer according to claim 2, characterized in that, The polymerization reaction is carried out under anaerobic conditions; The polymerization reaction is carried out at a temperature of 40~60℃.

5. A conductive antioxidant hydrogel, characterized in that, The raw materials for preparing the conductive antioxidant hydrogel include a gel matrix and the sulfur-containing antioxidant conductive polymer as described in claim 1; The gelling matrix is ​​selected from at least one of gelatin, polyvinyl alcohol, polyacrylamide, and chitosan.

6. The method for preparing the conductive antioxidant hydrogel according to claim 5, characterized in that, The process includes the following steps: mixing the gel matrix, the sulfur-containing antioxidant conductive polymer, the initiator, and the crosslinking agent in water, and carrying out a crosslinking and curing reaction to obtain the conductive antioxidant hydrogel.

7. The method for preparing the conductive antioxidant hydrogel according to claim 6, characterized in that, The crosslinking curing reaction is carried out at a temperature of 50-70°C; and / or the crosslinking curing reaction is carried out for a time of 10-30 minutes.

8. The use of the conductive antioxidant hydrogel of claim 5 in the preparation of products for myocardial repair.

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