Preparation method and application of GO-coated LDH with functions of removing ROS and regulating tissue microenvironment to improve acute myocardial infarction

Abstract

The invention belongs to the field of biological medicine, and particularly discloses a preparation method and application of GO-coated LDH with the functions of removing ROS, regulating and controlling tissue microenvironment and improving acute myocardial infarction. The material is synthesized by taking graphene oxide (GO) as a core and layered double hydroxide (LDH) as a shell through a coprecipitation method under an alkaline condition. The GO-coated LDH provided by the invention has good biocompatibility and remarkable ROS (reactive oxygen species) removal capability, and can inhibit expression of proinflammatory factors, promote expression of anti-inflammatory factors and promote angiogenesis. In-vitro experiments show that the material can protect myocardial cells under the sugar-oxygen deprivation condition, improve the microenvironment after myocardial infarction and promote functional recovery. The invention also provides application of the composite material in preparation of drugs for treating acute myocardial infarction, and the composite material has a wide clinical transformation prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, and specifically discloses a method for preparing GO@LDH with the functions of ROS scavenging and regulating the tissue microenvironment to improve acute myocardial infarction and its application. Background Technology

[0002] Acute myocardial infarction is a disease with a high clinical mortality rate. Survivors often suffer from heart failure due to myocardial necrosis and poor ventricular remodeling, placing a heavy burden on patients' families and society. Currently, standard clinical treatments include drug therapy, percutaneous coronary intervention, thrombolysis, and surgical bypass. While these methods can improve survival rates, they cannot address the fundamental problems such as the massive loss of myocardial cells and the irreversible decline in cardiac function caused by the deterioration of the cardiac microenvironment.

[0003] Following myocardial infarction, ischemia and hypoxia lead to a massive accumulation of reactive oxygen species (ROS), triggering oxidative stress, inflammatory cell infiltration, and the release of pro-inflammatory factors, thereby creating a harsh microenvironment that inhibits tissue regeneration. This microenvironment not only exacerbates cardiomyocyte apoptosis but also hinders the effectiveness of emerging therapeutic strategies such as stem cell transplantation, because implanted cells have low survival rates in a high ROS and inflammatory environment, making it difficult for them to exert their repair function.

[0004] In recent years, biomaterials have provided new insights into myocardial repair. Layered double hydroxides (LDHs), as two-dimensional nanomaterials, possess good biocompatibility and drug delivery potential, but their antioxidant capacity is limited. While graphene oxide (GO) exhibits excellent antioxidant properties, its biological toxicity (such as inducing inflammatory responses) and potential biosafety issues restrict its direct application. Furthermore, the single function of a single material is insufficient to address the complex pathological microenvironment following myocardial infarction (such as oxidative stress, inflammation, and impaired angiogenesis). Therefore, developing a novel composite material that can synergistically scavenge ROS, regulate the immune microenvironment, and possesses high biosafety is of great significance for promoting cardiac repair after myocardial infarction. Summary of the Invention

[0005] To address the aforementioned issues, this invention discloses a method for preparing GO@LDH, which possesses ROS scavenging and tissue microenvironment-regulating functions to improve acute myocardial infarction, and its applications. Based on layered double hydroxide LDH and graphene oxide (GO), this invention synthesizes a composite material, GO@LDH, in which LDH encapsulates GO. In vitro experiments have demonstrated that this material exhibits effective ROS scavenging, while also regulating the release of inflammatory factors. It promotes in vitro angiogenesis in human umbilical vein endothelial cells by regulating macrophages, and effectively scavenges ROS under glucose-oxygen deprivation conditions, providing good protection for cardiomyocytes. This material has significant application potential for improving the microenvironment and promoting myocardial function recovery after acute myocardial infarction.

[0006] Based on the above research, the present invention includes the following technical solutions:

[0007] A method for preparing a GO@LDH composite material includes the following steps:

[0008] (a) Provide a graphene oxide (GO) dispersion, said dispersion being prepared by sonicating graphene oxide in an alkaline aqueous solution;

[0009] (b) Under an inert atmosphere and with mechanical stirring, a salt solution containing divalent metal ions and trivalent metal ions is slowly added to the dispersion obtained in step (a), wherein the molar ratio of divalent metal ions to trivalent metal ions is (2:1) to (5:1).

[0010] (c) The mixture from step (b) is continuously stirred at 50-80°C to allow layered double hydroxides (LDHs) to grow and recombine on the GO surface, thereby obtaining the reaction product;

[0011] (d) The reaction product obtained in step (c) is centrifuged, washed and collected to obtain the GO@LDH composite material.

[0012] Furthermore, in the above preparation method, the alkaline aqueous solution in step (a) is a sodium hydroxide solution, and the pH of the final mixed system in step (b) is adjusted to 8-12 using a sodium hydroxide solution.

[0013] Furthermore, in the above preparation method, the divalent metal ion in step (b) is selected from Mg. 2+ Zn 2+ Co 2+ At least one of the following, wherein the trivalent metal ion is selected from Fe 3+ At least one of Al3+.

[0014] Furthermore, in the above preparation method, the molar ratio of divalent metal ions to trivalent metal ions in step (b) is (3:1) to (4:1).

[0015] This invention discloses a GO@LDH composite material prepared by the above preparation method. The composite material has a core-shell structure with GO as the core and LDH as the shell, and its Zeta potential is positive.

[0016] The present invention also discloses the application of the above-mentioned GO@LDH composite material in the preparation of reactive oxygen species (ROS) scavengers.

[0017] The present invention also discloses the application of the above-mentioned GO@LDH composite material in the preparation of formulations for regulating the immune microenvironment, characterized in that the regulation of the immune microenvironment includes inhibiting the expression of pro-inflammatory cytokines and / or promoting the expression of anti-inflammatory cytokines.

[0018] The present invention also discloses the application of the above-mentioned GO@LDH composite material in the preparation of formulations for promoting angiogenesis.

[0019] The present invention also discloses the application of the above-mentioned GO@LDH composite material in the preparation of a drug for treating acute myocardial infarction, characterized in that the drug improves myocardial function recovery through one or more pathways, such as scavenging ROS, regulating the immune microenvironment, or promoting angiogenesis.

[0020] The present invention also discloses a pharmaceutical composition for treating acute myocardial infarction, comprising an effective amount of GO@LDH composite material and a pharmaceutically acceptable carrier.

[0021] Compared with the prior art, the present invention has the following outstanding advantages:

[0022] 1. The preparation method is simple and efficient, and the material properties are stable: The one-step coprecipitation method provided by this invention is simple, mild, and reproducible, and can stably prepare GO@LDH composite materials with core-shell structure. The Zeta potential indicates that LDH is successfully modified on the GO surface, which solves the problems of easy aggregation of GO, high biotoxicity, and single function of LDH.

[0023] 2. Significantly improved biocompatibility: CCK-8 cytotoxicity assay showed that the prepared GO@LDH composite material did not exhibit significant toxicity after being co-cultured with H9C2 cardiomyocytes and RAW264.7 macrophages for 48 hours at a concentration as high as 80 μg / mL, demonstrating better biocompatibility than pure GO and laying a safe foundation for its in vivo application.

[0024] 3. Possesses synergistically enhanced ROS scavenging ability: DCFH-DA experiments have demonstrated that GO@LDH at a concentration of 10 μg / mL exhibits the highest ROS scavenging efficiency, which is superior to that of LDH or GO components alone. It can effectively alleviate oxidative stress and provide a key mechanism for protecting ischemic and hypoxic cardiomyocytes.

[0025] 4. Highly effective regulation of the immune microenvironment: Through a macrophage inflammation model, GO@LDH has been shown to significantly inhibit the expression of pro-inflammatory factors (IL-1β, IL-12) while promoting the expression of anti-inflammatory factors (YM1, CD206), effectively inducing macrophages to reparative M2 polarization, thereby improving the inflammatory microenvironment after myocardial infarction.

[0026] 5. Significantly promotes angiogenesis: In vitro tube formation experiments show that macrophage supernatant treated with GO@LDH can effectively promote the formation of more luminal structures by human umbilical vein endothelial cells (HUVECs), proving that it can indirectly promote angiogenesis through immune regulation, providing a new strategy for solving blood supply reconstruction after myocardial infarction.

[0027] 6. Demonstrates strong comprehensive protective effects under simulated ischemic and hypoxic conditions: In the oxygen deprivation (OGD) model, GO@LDH can most effectively remove excess ROS in cells and significantly improve cardiomyocyte survival rate, comprehensively demonstrating its multifunctional synergistic effect of "ROS removal-immune regulation-cell protection", which has great application potential in the treatment of acute myocardial infarction. Attached Figure Description

[0028] Figure 1 SEM images of A. LDH, B. GO, C. GO@LDH, and zeta potentials of D. LDH, GO, and GO@LDH;

[0029] Figure 2 CCK8 analysis of the biocompatibility of LDH, GO and GO@LDH; A, B. 24h and 48h cytotoxicity analysis of materials against H9C2; C, D. 24h and 48h cytotoxicity analysis of materials against RAW264.7.

[0030] Figure 3 Functional analysis of ROS scavenging by different concentrations of LDH, GO, and GO@LDH: A. Fluorescence images of materials scavenging intracellular ROS; B. Quantitative fluorescence analysis diagram.

[0031] Figure 4 Analysis of the inflammatory regulatory capacity of different concentrations of LDH, GO and GO@LDH, where A and B are the results of inflammatory factor expression analysis, and C and D are the results of anti-inflammatory factor expression analysis;

[0032] Figure 5 The material was analyzed by examining its effect on macrophage angiogenesis. A. Microscopic images of angiogenesis, B. Quantitative analysis results;

[0033] Figure 6 Analysis of the ROS scavenging and protective effects of the materials on cells under glucose-oxygen deprivation conditions: A. Fluorescence microscopy analysis of the material's effect on intracellular ROS scavenging under glucose-oxygen deprivation conditions; B. Quantitative fluorescence analysis results; C. Alamar Blue analysis results of the material's effect on cell viability under glucose-oxygen deprivation conditions. Detailed Implementation

[0034] The present invention will be further illustrated below with reference to specific embodiments. Unless otherwise specified, all reagents used in the present invention are commercially available reagents.

[0035] Example 1

[0036] Synthesis of LDH and GO@LDH

[0037] LDH Synthesis: Boil double-distilled water for about 30 minutes, then cool and set aside. Weigh 0.544 g NaOH, dissolve it in 80 mL of carbon dioxide-free double-distilled water, and transfer the solution to a 250 mL three-necked flask. Place the flask in a pre-set 60°C constant temperature water bath, purge with nitrogen, and stir. Weigh 1.538 g Mg(NO3)2·6H2O and 0.606 g Fe(NO3)3·9H2O, and dissolve them in 20 mL of carbon dioxide-free double-distilled water. Add the above solution dropwise to the three-necked flask, purge with nitrogen at 500 rpm at 60°C, and stir for 30 minutes. Centrifuge at 8500 rpm to collect the precipitate and wash it three times with carbon dioxide-free double-distilled water. Resuspend the precipitate in 80 mL of carbon dioxide-free double-distilled water and transfer it to a hydrothermal reactor. React at 100°C for 16 hours. After cooling, centrifuge at 8500 rpm to collect the precipitate and wash it three times with carbon dioxide-free double-distilled water. Measure the wet-to-dry ratio and set aside.

[0038] Synthesis of GO@LDH: 0.544 g of sodium hydroxide was dissolved in 80 mL of double-distilled water (with carbon dioxide removed), and 0.066 g of 20-mesh graphene oxide was added. After ultrasonic dispersion, the solution was placed in a three-necked flask. Nitrogen gas was introduced, and the solution was mechanically stirred to continue dispersion. At the same time, 1.538 g of magnesium nitrate hexahydrate and 0.606 g of ferric nitrate nonahydrate were dissolved in 20 mL of double-distilled water (with carbon dioxide removed) and added dropwise to the solution in the three-necked flask. The solution was stirred at 500 rpm for 30 min at 60 °C in the presence of nitrogen. After centrifugation, the solution was washed three times with double-distilled water (with carbon dioxide removed).

[0039] The synthesized material was characterized by SEM morphology and zeta potential, such as... Figure 1 As shown, GO has an irregular lamellar structure, while LDH has a hexagonal lamellar structure. The composite GO@LDH exhibits a core-shell structure modified by LDH on the GO surface. Simultaneously, the zeta potential shows that LDH carries a positive charge of approximately 38.2 ± 0.5 mV, GO carries a negative charge of approximately -22.3 ± 0.4 mV, and the composite GO@LDH carries a positive charge of approximately 24.1 ± 0.4 mV, indicating that LDH effectively modifies the GO surface.

[0040] Example 2

[0041] Biosafety assessment of GO@LDH

[0042] CCK8 cytotoxicity assay: H9C2 and RAW264.7 cells were cultured to approximately 90% confluence. After trypsin digestion and cell counting, 8000 cells per well were seeded into 96-well plates and cultured at 37°C in a 5% CO2 incubator until the cell density reached approximately 70%. Concentration gradients of 1 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL, 40 μg / mL, and 80 μg / mL LDH, GO, and GO@LDH were prepared and cultured for 24 h and 48 h. CCK8 solution was then added to a concentration of 10%, and the cells were incubated for 1-4 h. The absorbance at 450 nm was then measured using a microplate reader.

[0043] The results are as follows Figure 2 As shown, after co-incubation with H9C2 and RAW264.7 cells for 24 h and 48 h, different materials did not show significant inhibitory effects on cell viability, indicating that the materials have good biosafety within a certain concentration range and treatment time.

[0044] Example 3

[0045] ROS removal capability assessment of GO@LDH

[0046] DCFH-DA Experiment: H9C2 cells were cultured to approximately 90% confluence. After trypsin digestion and cell counting, 80,000 cells were seeded per well in 24-well plates. Blank control, positive control, and experimental groups were set up. Cells were cultured at 37°C in a 5% CO2 incubator to approximately 70% confluence. Concentration gradients of 1 μg / mL, 5 μg / mL, and 10 μg / mL LDH, GO, and GO@LDH were prepared. Except for the blank control group, H2O2 at a concentration of 500 μM was added to all other groups, and the cells were cultured for another 24 h. The original culture medium was removed, and a suitable concentration of DCFH-DA solution (diluted with serum-free culture medium) was added. The cells were incubated for 20 min, washed three times with PBS, and analyzed using a fluorescence microscope.

[0047] The experimental results of DCFH-DA are as follows: Figure 3 As shown, LDH, GO, and LDH@GO all have ROS scavenging capabilities to some extent. Compared to the single materials LDH and GO, the composite material GO@LDH has the highest ROS scavenging efficiency at 10 μg / mL.

[0048] Example 4

[0049] Immunomodulatory capacity assessment of GO@LDH

[0050] RAW264.7 cells were cultured to approximately 90% confluence. After trypsin digestion and cell counting, 400,000 cells were seeded per well in 6-well plates. A blank control, an inflammatory stimulation positive control, and an experimental group were set up. The cells were cultured to approximately 70% confluence. Concentration gradients of 1 μg / mL, 5 μg / mL, and 10 μg / mL LDH, GO, and GO@LDH were prepared. Except for the blank control group, 100 ng / mL LPS was added to all other groups. The cells were cultured for another 24 h, and Trizol was added. mRNA was extracted, and the expression of inflammatory factors IL1β and IL12 and anti-inflammatory factors YM1 and CD206 was detected by RT-qPCR.

[0051] RT-qPCR results are as follows Figure 4 The results showed that, compared with LDH and GO alone, 10 μg / mL GO@LDH significantly inhibited the expression of inflammatory factors IL1β and IL12, and promoted the expression of anti-inflammatory factors YM1 and CD206, indicating that GO@LDH has a significant effect on regulating macrophage M2 polarization.

[0052] Example 5

[0053] GO@LDH promotes angiogenesis by regulating macrophages

[0054] RAW264.7 cells were cultured to approximately 90% confluence. After trypsin digestion and cell counting, 80,000 cells were seeded per well in 24-well plates. A blank control, an inflammatory stimulation positive control, and an experimental group were set up. The cells were cultured to approximately 70% confluence. 10 μg / mL LDH, GO, and GO@LDH were prepared. Except for the blank control group, 100 ng / mL LPS was added to the other groups. The cells were cultured for another 24 h, and the supernatant was collected for later use.

[0055] Angiogenesis: Pre-cool the pipette tips, 96-well plates, and thaw Matrigel. Place the 96-well plates on ice and add 75 μL of Matrigel to each well (avoiding air bubbles). Incubate at 37°C for 30 min to solidify. Seed HUVECs with a cell density of approximately 90% at a rate of 50,000 cells per well into the Matrigel-treated 96-well plates. Add the previously collected supernatant at a volume ratio of 1:1 and continue culturing for 6 h. Take pictures and analyze.

[0056] Results of in vitro angiogenesis experiments as follows Figure 5 As shown, compared with GO, HUVECs incubated with macrophage supernatant treated with LDH and GO@LDH were able to form more luminal nodes in vitro, indicating that LDH compounding can improve the in vitro tube-forming ability of GO and promote angiogenesis.

[0057] Example 6

[0058] Evaluation of ROS scavenging and cell protection effects of GO@LDH under hypoxic conditions

[0059] Alamar Blue cell viability analysis: H9C2 cells were cultured to approximately 90% confluence, and after trypsin digestion and cell counting, 8000 cells per well were seeded into 96-well plates. Hypoxia control, hypoxia experimental group, normal control, and normal blank were set up. Cell density was cultured to approximately 70%, and 10 μg / mL LDH, GO, and GO@LDH (prepared in glucose-free and serum-free culture medium) were added. The cells were then subjected to hypoxia for 6 h. After treatment, 100 μL of 10% Alamar Blue solution was added to each well (glucose-free and serum-free culture medium for the hypoxia group, and normal culture medium for the normal group). The cells were incubated for 2 h, and OD values ​​were measured for analysis.

[0060] DCFH-DA Experiment: H9C2 cells were cultured to a cell density of approximately 90%. After trypsin digestion and cell counting, 80,000 cells were seeded per well in a 24-well plate. Hypoxia control, hypoxia experimental group, and normal control group were set up. When the cell density reached approximately 70%, 10 μg / mL LDH, GO, and GO@LDH (prepared with glucose-free and serum-free culture medium) were added, followed by 6 h of hypoxia treatment. Then, the original culture medium was removed, and a suitable concentration of DCFH-DA solution (diluted with serum-free culture medium) was added. The cells were incubated in an incubator for 20 min, washed three times with PBS, and analyzed using a fluorescence microscope.

[0061] Experimental results are as follows Figure 6 As shown, after glucose-oxygen deprivation, more ROS were produced intracellularly. The intracellular ROS content of cells co-incubated with LDH, GO, and GO@LDH was significantly reduced compared to the control, among which GO@LDH had the strongest ROS scavenging ability. Figure 6 A); meanwhile, cell viability assays also showed that GO@LDH could better protect cell viability under glucose-oxygen deprivation conditions. Figure 6 B).

[0062] Summary of Examples: This invention systematically validates the preparation method, biosafety, and functional properties of the GO@LDH composite material through six examples. Example 1 details the synthesis process of GO@LDH and confirms its core-shell structure and surface charge characteristics using SEM and Zeta potential. Example 2 demonstrates, through CCK8 assay, that the material exhibits no significant toxicity to H9C2 and RAW264.7 cells within a concentration range of 1–80 μg / mL, demonstrating good biocompatibility. Example 3 shows, using the DCFH-DA method, that GO@LDH achieves the highest ROS scavenging efficiency at 10 μg / mL. Example 4, through qPCR analysis, shows that GO@LDH can significantly inhibit pro-inflammatory factors such as IL1β and IL12, and promote the expression of anti-inflammatory factors such as YM1 and CD206, exhibiting immunomodulatory functions. Example 5 demonstrates, through angiogenesis assay, that it can promote HUVEC tube formation by regulating macrophages. Example 6 further validates the ROS scavenging ability and cytoprotective effect of GO@LDH in a glucose-oxygen deprivation model. In summary, the GO@LDH composite material provided by this invention has the potential for multi-mechanism synergistic treatment of acute myocardial infarction.

[0063] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the above embodiments do not limit the present invention in any way, and all technical solutions obtained by equivalent substitution or equivalent transformation fall within the protection scope of the present invention.

Claims

1. A method for preparing a GO@LDH composite material, characterized in that, Includes the following steps: (a) Provide a graphene oxide (GO) dispersion, said dispersion being prepared by sonicating graphene oxide in an alkaline aqueous solution; (b) Under an inert atmosphere and with mechanical stirring, a salt solution containing divalent metal ions and trivalent metal ions is slowly added to the dispersion obtained in step (a), wherein the molar ratio of divalent metal ions to trivalent metal ions is (2:1) to (5:1). (c) The mixture from step (b) is continuously stirred at 50-80°C to allow layered double hydroxides (LDHs) to grow and recombine on the GO surface, thereby obtaining the reaction product; (d) The reaction product obtained in step (c) is centrifuged, washed and collected to obtain the GO@LDH composite material.

2. The preparation method according to claim 1, characterized in that, The alkaline aqueous solution mentioned in step (a) is a sodium hydroxide solution, and the pH of the final mixed system in step (b) is adjusted to 8-12 using a sodium hydroxide solution.

3. The preparation method according to claim 1, characterized in that, The divalent metal ions mentioned in step (b) are selected from Mg 2 + Zn 2+ Co 2+ At least one of the following, wherein the trivalent metal ion is selected from Fe 3+ At least one of Al3+.

4. The preparation method according to claim 1, characterized in that, The molar ratio of divalent metal ions to trivalent metal ions in step (b) is (3:1) to (4:1).

5. A GO@LDH composite material prepared by the method according to any one of claims 1-4, characterized in that, The composite material has a core-shell structure with GO as the core and LDH as the shell, and its Zeta potential is positive.

6. The application of the GO@LDH composite material as described in claim 5 in the preparation of reactive oxygen species (ROS) scavengers.

7. The application of the GO@LDH composite material as described in claim 5 in the preparation of formulations for modulating the immune microenvironment, characterized in that, The regulation of the immune microenvironment includes inhibiting the expression of pro-inflammatory cytokines and / or promoting the expression of anti-inflammatory cytokines.

8. The use of the GO@LDH composite material as described in claim 5 in the preparation of formulations for promoting angiogenesis.

9. The use of the GO@LDH composite material as described in claim 5 in the preparation of a medicament for treating acute myocardial infarction, characterized in that, The drug improves myocardial function recovery through one or more pathways, such as clearing ROS, regulating the immune microenvironment, or promoting angiogenesis.

10. A pharmaceutical composition for treating acute myocardial infarction, characterized in that, The GO@LDH composite material of claim 5 comprises an effective amount of the composite material and a pharmaceutically acceptable carrier.

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