Polyethylene glycol modified magnesium boride nanosheet and application thereof in treatment of acute liver failure and concurrent hepatic encephalopathy caused by acetaminophen

Abstract

This invention discloses a polyethylene glycol-modified magnesium boride nanosheet and its application in treating acute liver failure and hepatic encephalopathy caused by acetaminophen. The nanosheet uses magnesium boride nanosheets as a core, with polyethylene glycol modified on the surface to improve biocompatibility. The nanosheet of this invention exhibits good antioxidant properties and can be used to prepare nanomedicines for treating acute liver failure and / or hepatic encephalopathy caused by acetaminophen. It generates reducing hydrogen gas through a hydrolysis reaction, effectively scavenging reactive oxygen species at the site of liver failure, thereby inhibiting inflammatory responses and effectively alleviating symptoms caused by acute liver failure.

Description

Technical Field

[0001] This invention belongs to the field of nanomaterial preparation and biomedicine, specifically relating to a polyethylene glycol-modified magnesium boride nanosheet and its application in the treatment of acute liver failure and hepatic encephalopathy caused by acetaminophen. Background Technology

[0002] Acetaminophen (APAP) poisoning is one of the main causes of acute liver failure (ALF). Due to the severity and rapid progression of the disease, patients often experience extensive hepatocellular necrosis, severe coagulation dysfunction, and hepatic encephalopathy within a short period, resulting in an extremely high mortality rate and posing a significant challenge to clinical treatment.

[0003] As a widely used antipyretic and analgesic drug in clinical practice, APAP has a good safety profile at therapeutic doses. Its metabolism is mainly catalyzed by uridine diphosphate glucuronide transferase (UGT) and sulfotransferase (SULT), converting it into non-toxic metabolites before excretion. However, when APAP is ingested in excess, the excess APAP is metabolized by cytochrome P450 enzymes (CY), producing cytotoxic N-acetyl-p-benzoquinone imine (NAPQI). This toxic product further depletes the liver's glutathione (GSH) reserves, disrupting the redox balance and leading to a large accumulation of reactive oxygen species and nitrogenous substances (RONS), mitochondrial dysfunction, and depletion of the antioxidant system. This, in turn, drives various forms of programmed cell death in hepatocytes, including necrosis, apoptosis, and ferroptosis, ultimately resulting in widespread liver tissue damage and the development of alanine fibrosis (ALF). Given the complex pathological mechanisms of APAP-induced ALF, involving multiple processes such as oxidative stress, inflammatory response, and programmed cell death, traditional single-target interventions often fail to achieve ideal therapeutic effects.

[0004] Therefore, developing a multifunctional therapeutic strategy that can simultaneously target and inhibit ferroptosis, alleviate oxidative stress, and effectively promote hepatocyte regeneration has become a key research focus and potential effective pathway in the current field of APAP-induced ALF treatment.

[0005] In recent years, the application of nanomaterials in the biomedical field has become a hot topic in cutting-edge research. Magnesium boride (MgB2), as a novel inorganic material composed of biologically essential elements, has attracted much attention in the biomedical field due to its excellent biocompatibility and controllable degradation characteristics. Magnesium boride can generate hydrogen gas and produce boron hydroxyl derivatives through hydrolysis. Hydrogen gas, due to its excellent antioxidant capacity, can effectively scavenge RONS and alleviate oxidative stress in liver failure sites. In addition, the boron hydroxyl compounds generated in situ can specifically capture excess endotoxins (LPS) in the blood and effectively block the LPS-triggered cascade inflammatory response by forming stable boron ester bonds, thereby significantly reducing hepatocellular damage and achieving dual intervention therapy for liver failure.

[0006] However, MgB2 nanosheets alone suffer from drawbacks such as strong hydrophobicity, poor in vivo dispersibility, and short circulating half-life, making them easily captured by the reticuloendothelial system (RES) and hindering effective accumulation at sites of liver failure, thus greatly limiting their application in in vivo therapy. Therefore, improving the in vivo physicochemical properties of MgB2 nanosheets through surface modification techniques to enhance their biocompatibility, cyclic stability, and targeting ability has become a key technical challenge that urgently needs to be addressed in current research on the application of this type of material in the treatment of liver failure. Summary of the Invention

[0007] Current conventional drugs for treating acetaminophen-induced acute liver failure (ALF) suffer from limitations such as narrow applicability, strict timing of administration, significant potential side effects, and difficulty in simultaneously addressing multiple pathological issues in ALF development, including oxidative stress, inflammatory response, and hepatocellular damage. Furthermore, existing magnesium boride nanomaterials suffer from drawbacks such as strong hydrophobicity, poor in vivo dispersibility, and insufficient targeting, further restricting their clinical application. This invention provides polyethylene glycol-modified magnesium boride nanosheets and their application in treating acetaminophen-induced acute liver failure and its complication, hepatic encephalopathy. By designing and synthesizing polyethylene glycol-modified magnesium boride nanosheets with highly efficient reactive oxygen species (ROS) scavenging, inflammation suppression, and hepatocellular protection functions, this invention can effectively remove excessive ROS in liver tissue after APAP poisoning, significantly reduce hepatic oxidative stress, effectively promote hepatocellular repair and regeneration, and significantly reduce brain ammonia levels, effectively alleviating the severe complication of ALF-induced hepatic encephalopathy. This achieves highly effective treatment for APAP-induced ALF, providing a novel and efficient treatment strategy for acute liver failure.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: This invention first discloses a polyethylene glycol-modified magnesium boride nanosheet for treating acute liver failure and hepatic encephalopathy caused by acetaminophen. The key feature is that the polyethylene glycol-modified magnesium boride nanosheet uses magnesium boride nanosheets as a core, with hydrophilic polyethylene glycol modified on the surface of the magnesium boride nanosheets. The preparation method includes the following steps: Magnesium boride powder was added to anhydrous ethanol solution and ultrasonically crushed. After centrifugation, the supernatant was collected. Polyethylene glycol was added to the supernatant and ultrasonically treated. After centrifugation, washing, and freeze-drying, polyethylene glycol-modified magnesium boride nanosheets with a thickness of 1–2 nm and a diameter of 150–250 nm were obtained.

[0009] Furthermore, the mass ratio of polyethylene glycol to magnesium boride nanosheets in the supernatant is 1–9:1.

[0010] Furthermore, the ultrasound is performed alternately with probe ultrasound and room temperature water bath ultrasound. The power of probe ultrasound is 80~600 W and the total time is 1~5 h, while the power of room temperature water bath ultrasound is 10~200 W and the total time is 1~5 h.

[0011] In the technical solution of this invention, to address the shortcomings of simple magnesium boride (MgB2) nanosheets, such as strong hydrophobicity, poor in vivo dispersibility, easy capture by the reticuloendothelial system, and insufficient targeting, a key strategy is to construct nano-formulations suitable for in vivo treatment by modifying the surface of MgB2 nanosheets with polyethylene glycol (PEG). PEG, as a hydrophilic polymer with good biocompatibility, can form a stable steric hindrance and hydrophilic canopy on the surface of MgB2 nanosheets. This structure effectively shields the hydrophobic properties of the MgB2 nanosheet surface, significantly inhibits the non-specific adsorption of plasma proteins, thereby prolonging the circulating half-life of MgB2 nanosheets in the blood, effectively avoiding capture by the reticuloendothelial system (RES), and achieving efficient passive targeted enrichment at sites of liver failure through high permeability and nanosize effect, laying the foundation for improving therapeutic efficacy.

[0012] The polyethylene glycol-modified magnesium boride nanosheets of this invention have well-defined characteristics and mechanisms of action, and can specifically address multiple pathological problems of APAP-induced acute liver failure: they exhibit good biocompatibility at both cellular and animal levels; they possess highly efficient RONS scavenging capabilities, significantly reducing hepatic oxidative stress and protecting cells and liver tissue from oxidative damage; they can inhibit the production of lipid peroxides in liver failure sites and block hepatocyte ferroptosis pathways; simultaneously, they can specifically capture upregulated endotoxins in the blood, alleviate LPS-induced inflammatory cascade reactions, significantly reduce brain ammonia levels, effectively alleviate hepatic encephalopathy, a serious complication of ALF, and ultimately achieve highly effective treatment for APAP-induced ALF.

[0013] The specific therapeutic mechanism is as follows: Polyethylene glycol-modified magnesium boride nanosheets hydrolyze under physiological conditions in vivo, simultaneously generating reducing hydrogen and boron dihydroxyl groups. Hydrogen effectively removes excess RONS from liver tissue, exerting a strong antioxidant effect while inhibiting lipid peroxide accumulation, thus effectively suppressing the hepatocyte ferroptosis pathway. Boron dihydroxyl groups specifically capture excess LPS in the blood through esterification, forming stable boron ester bonds to block LPS activity and inhibit the inflammatory cascade triggered by it, thereby alleviating oxidative stress damage and reducing hepatocyte apoptosis. After tail vein injection, the nanosheets can target and accumulate at the site of liver injury, significantly improving the symptoms of APAP-induced acute liver failure.

[0014] The beneficial effects of this invention are specifically reflected in the following aspects: 1. The polyethylene glycol-modified magnesium boride nanosheets of the present invention possess excellent antioxidant properties, biocompatibility, and in vivo stability. The reducing hydrogen gas generated by its hydrolysis can efficiently scavenge RONS and protect cells from oxidative stress damage, while inhibiting the production of lipid peroxides and blocking the ferroptosis pathway. The boron hydroxyl compounds generated in situ can specifically capture LPS in the blood through borate ester bonds, avoiding the amplification of inflammatory responses, while reducing brain ammonia levels, effectively alleviating hepatic encephalopathy, a complication of liver failure. This achieves dual synergistic treatment of oxidative stress and inflammatory response in liver failure, with efficacy superior to traditional single-target formulations.

[0015] 2. The two-dimensional polyethylene glycol-modified magnesium boride nanosheets of the present invention significantly improve the anti-inflammatory, antioxidant and targeted therapeutic properties of nanomedicines compared with unmodified MgB2 nanosheets and conventional drugs, while effectively reducing the clinical dosage and reducing potential drug risks.

[0016] 3. The preparation process of the polyethylene glycol modified magnesium boride nanosheets of the present invention is simple, the reaction conditions are mild, no complex and expensive equipment is required, the parameters are easy to control, and large-scale production can be achieved, which has significant prospects for industrial application.

[0017] 4. All raw materials used in this invention have excellent biocompatibility. In vivo safety evaluation shows that the nanosheets have no direct or indirect toxic side effects on mice and have no potential toxicity.

[0018] 5. The two-dimensional polyethylene glycol-modified magnesium boride nanosheets prepared by this invention have good dispersibility and stable physicochemical properties, which can effectively meet the stringent requirements for the stability of nano-formulations in clinical applications. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of the synthesis of the present invention.

[0020] Figure 2 Transmission electron microscopy (TEM) image of the MgB2@PEG nanosheets prepared in Example 1.

[0021] Figure 3 The atomic force microscopy characterization results of the MgB2@PEG nanosheets prepared in Example 1 are shown in (a) as a two-dimensional morphology image of the nanosheets and (b) as a height distribution curve measured along the white line in Figure (a).

[0022] Figure 4 The image shows the XRD pattern of the MgB2@PEG nanosheets prepared in Example 1.

[0023] Figure 5 Fourier transform infrared absorption spectra of MgB2@PEG nanosheets, magnesium boride nanosheets, and polyethylene glycol raw materials prepared in Example 1.

[0024] Figure 6The image shows the hydrogen generation of the MgB2@PEG nanosheets prepared in Example 1.

[0025] Figure 7 The diagram shows the in vitro ABTS (2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid) radical scavenging of MgB2@PEG nanosheets and pure MgB2 nanosheets prepared in Example 1.

[0026] Figure 8 The in vitro scavenging diagrams of MgB2@PEG nanosheets and pure MgB2 nanosheets prepared in Example 1 are shown.

[0027] Figure 9 This is a biocompatibility diagram of MgB2@PEG nanosheets prepared in Example 1 at different concentrations.

[0028] Figure 10 The MTT assay results are shown for MgB2@PEG nanosheets prepared in Example 1 and pure MgB2 nanosheets.

[0029] Figure 11 Cellular reactive oxygen species staining images of MgB2@PEG nanosheets prepared in Example 1 and pure MgB2 nanosheets.

[0030] Figure 12 The images show AM / PI staining of cells with MgB2@PEG nanosheets prepared in Example 1 and pure MgB2 nanosheets.

[0031] Figure 13 The distribution of MgB2@PEG nanosheets prepared in Example 1 in mice at different time points.

[0032] Figure 14 The statistical graph shows the serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities of MgB2@PEG nanosheets prepared in Example 1 after treatment with APAP-induced acute liver injury in mice.

[0033] Figure 15 The survival rate of mice with APAP-induced acute liver failure treated with MgB2@PEG nanosheets prepared in Example 1 is shown in the figure.

[0034] Figure 16 Evaluation of the ameliorative effect of MgB2@PEG nanosheets prepared in Example 1 on hepatic encephalopathy in mice with APAP-induced acute liver failure: (a) Trajectory graphs (top row) and heat maps (bottom row, colors from blue to red / yellow represent dwell time from short to long, 0s~5s) of mice in each group; (b) Statistical analysis of the total movement distance of mice in each group; (c) Statistical analysis of the dwell time of mice in the central area of ​​the open field experiment in each group.

[0035] Figure 17The graph shows the statistical content of LPS in the blood of mice with APAP-induced acute liver failure after treatment with MgB2@PEG nanosheets prepared in Example 1.

[0036] Figure 18 The graph shows the brain ammonia content of MgB2@PEG nanosheets prepared in Example 1 after treatment with APAP-induced acute liver failure mice. Detailed Implementation

[0037] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to examples. The following content is merely an example and illustration of the concept of the present invention. Those skilled in the art can make various modifications or additions to the described specific embodiments or use similar methods to replace them, as long as they do not depart from the inventive concept or exceed the scope defined by the claims, all of which should fall within the protection scope of the present invention.

[0038] Example 1 like Figure 1 As shown, in this embodiment, polyethylene glycol-modified magnesium boride nanosheets were prepared according to the following method: 30 mL of anhydrous ethanol was placed in a reaction vessel, and 0.3 g of magnesium boride powder (MgB2) was added. The fragmentation and peeling process was carried out by alternating between probe ultrasound and room temperature water bath ultrasound. The probe ultrasound power was 400 W and the duration of a single ultrasound was 0.5 h. The room temperature water bath ultrasound power was 150 W and the duration of a single ultrasound was 0.5 h. The ultrasound was alternated and cycled, and the total ultrasound treatment time was 4 h.

[0039] After ultrasonic treatment, the reaction system was centrifuged at 3000 rpm for 20 min. After centrifugation, the supernatant (i.e., magnesium boride nanosheet dispersion) was collected, and the coarse particles that were not peeled off at the bottom were discarded.

[0040] Add 20 mg of polyethylene glycol (Mw=5000Da) to the collected supernatant (containing 8-10 mg of MgB2 nanosheets), and use 480 W power ultrasonication to break down and modify the PEG for 30 min, so that the PEG can be fully adsorbed and modified on the surface of magnesium boride nanosheets.

[0041] After modification, the reaction system was centrifuged at 12,000 rpm for 15 min at room temperature, and the precipitate was collected. After washing the precipitate to remove unreacted PEG and impurities, it was freeze-dried to obtain polyethylene glycol modified magnesium boride nanosheets, denoted as MgB2@PEG.

[0042] Figure 2The image shows a transmission electron microscope (TEM) image of the MgB2@PEG nanosheets obtained in this embodiment. The characterization method was as follows: an ethanol dispersion of MgB2@PEG nanosheets was dropped onto a copper grid of a TEM, dried, and then observed in the TEM. The image shows that the nanosheets have a diameter of 150–250 nm.

[0043] Figure 3 The image shows an atomic force microscope (AFM) image of the MgB2@PEG nanosheets obtained in this embodiment. The characterization method was as follows: an aqueous dispersion of MgB2@PEG nanosheets was dropped onto a mica sheet, dried, and then observed under an AFM. The image shows that the thickness of the nanosheets is 1–2 nm.

[0044] Figure 4 The image shows the XRD spectrum of the MgB2@PEG nanosheets obtained in this embodiment. The characterization method was as follows: the nanosheet solution was freeze-dried into powder using a freeze dryer, and its spectrum was measured using an X-ray diffractometer and compared with a standard magnesium boride XRD card. The image shows that the synthesized MgB2@PEG nanosheets still retain the crystalline characteristics of magnesium boride.

[0045] Figure 5 To compare the Fourier transform infrared (FTIR) spectra of the MgB2@PEG nanosheets, MgB2 nanosheets, and PEG powder obtained in this embodiment, the characterization method was as follows: MgB2@PEG nanosheets, MgB2 nanosheets, and PEG mixed with KBr were ground separately, and the samples were analyzed using the pellet compression method. The figures show that polyethylene glycol and MgB2@PEG nanosheets exhibit similar characteristics at 1104 cm⁻¹. -1 The formation of a peak at this location indicates successful modification of polyethylene glycol.

[0046] Figure 6 The hydrogen production graph of the MgB2@PEG nanosheets obtained in this embodiment is shown. The characterization method is as follows: 3 mg of MgB2@PEG was added to 3 mL of methylene blue solution with a concentration of 10 μg / mL, and the reaction was carried out at room temperature. The ultraviolet spectrum was measured at different time points, and the amount of hydrogen produced was determined by ultraviolet absorption. It can be seen from the graph that the MgB2@PEG nanosheets can continuously produce hydrogen.

[0047] Figure 7 The diagram shows the in vitro ABTS (2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid) radical scavenging activity of MgB2@PEG nanosheets and pure MgB2 nanosheets obtained in this embodiment. ABTS is oxidized by an oxidizing agent (such as potassium persulfate) to generate stable blue-green ABTS. ·+ Free radicals. After the sample is added, the antioxidants in the sample will react with ABTS. ·+The free radicals react, causing the solution to decolorize. The effect of ABTS removal can be detected by measuring the change in absorbance at 734 nm using a spectrophotometer. The specific experimental procedure is as follows: Weigh 4.5 mg of ABTS and dissolve it in 1.2 mL of ultrapure water, then vortex thoroughly until completely dissolved. Separately, dissolve 1.5 mg of potassium persulfate in 2.2 mL of ultrapure water, and similarly shake thoroughly. Mix the two solutions and react at room temperature in the dark for 12 hours to generate stable ABTS. ·+ Free radicals. After the reaction is complete, dilute the working solution 30-40 times and adjust to A. 734 Within the range of 0.7-0.8, add different concentrations of MgB2@PEG or pure MgB2 nanosheets (the concentration is adjusted during preparation by adjusting the mass of the nanosheets) and react with ABTS working solution for 30 min (the final concentration of nanosheets in the system is 2.5, 5, 10, 20, or 40 μg / mL, respectively), and set ABTS... + Water served as a positive control. Finally, the sample was centrifuged at 15000 rpm for 10 min, and the supernatant was measured for UV absorption at 734 nm. The figure shows that MgB2@PEG significantly outperformed pure MgB2 nanosheets in scavenging ABTS radicals; the system containing 40 μg / mL MgB2@PEG exhibited approximately 97% ABTS scavenging ability.

[0048] Figure 8 The images show the in vitro hydroxyl radical scavenging activity of MgB2@PEG nanosheets and pure MgB2 nanosheets prepared in Example 1. Hydroxyl radicals were generated via the Fenton reaction using salicylic acid. These hydroxyl radicals reacted with salicylic acid to produce 2,3-dihydroxybenzoic acid and 2,5-dihydroxybenzoic acid, which exhibit characteristic absorption peaks at 510 nm. The hydroxyl radical scavenging ability of the samples was assessed by inhibiting the above reactions. The specific experimental steps were as follows: 0.1 mol / L ferrous sulfate and hydrogen peroxide aqueous solutions were prepared, along with a 0.1 mol / L salicylic acid ethanol solution. The ferrous sulfate and hydrogen peroxide solutions were mixed thoroughly and reacted for 10 min to generate hydroxyl radicals. Different concentrations of MgB2@PEG nanosheets or pure MgB2 nanosheets (the concentration was adjusted during preparation by adjusting the mass of the nanosheets) were added and reacted with the hydroxyl radicals for 1 h (the final concentrations of the nanosheets in the system were 12.5, 25, 50, 100, or 200 μg / mL). After the reaction was complete, salicylic acid was added to the reaction system, mixed thoroughly, and reacted for 10 min. Finally, the mixture was centrifuged at 15000 rpm for 10 min, and the supernatant was taken to measure the UV absorption at 510 nm. The figure shows that MgB2@PEG nanosheets can significantly scavenge hydroxyl radicals; the system containing 200 μg / mL MgB2@PEG nanosheets has a hydroxyl radical scavenging capacity of approximately 90%.

[0049] Figure 9 This image shows the biocompatibility of MgB2@PEG nanosheets prepared in Example 1 at different concentrations. The specific experimental steps were as follows: MgB2@PEG nanosheet dispersions were diluted to 12.5 μg / mL, 25 μg / mL, 50 μg / mL, 100 μg / mL, and 200 μg / mL. 0.2 mL of each of these solutions was mixed with 0.2 mL of treated blood (500 μL of fresh blood was added to 4.5 mL of physiological saline and centrifuged 5–8 times at 3000 rpm for 10 min until the blood supernatant was clear and transparent; the supernatant was then discarded, and the volume was adjusted to 5 mL with physiological saline) and 0.6 mL of physiological saline, respectively. The mixture was incubated at 37°C for 4 h, followed by centrifugation at 3000 rpm for 10 min. The supernatant was then collected to measure the OD (oxidative stress). 541 nm The absorbance at the specified point was used to calculate the hemolysis rate. The figure shows that the hemolysis rate of MgB2@PEG nanosheets at different concentrations is less than 5%, indicating good biocompatibility of the material.

[0050] Figure 10 This image shows the MTT assay results for the MgB2@PEG nanosheets prepared in Example 1. MTT stands for 3-(4,5-dimethylthiazol-2)-2,5-diphenyltetrazolium bromide. The MTT assay is a method for detecting cell viability and growth. Its principle is that succinate dehydrogenase in the mitochondria of living cells can reduce exogenous MTT to water-insoluble blue-purple formazan crystals, which are deposited in the cells. Dead cells lack this function. Dimethyl sulfoxide (DMSO) can dissolve the formazan in the cells. The absorbance value is measured at 490 nm using a microplate reader, which indirectly reflects the number of viable cells. The specific experimental steps were as follows: AML12 cells were seeded in 96-well plates. When the cells reached 70% confluence, different concentrations of MgB2@PEG nanosheets in PBS dispersion were added and incubated for 24 h. After adding MTT dye, the cells were incubated for 4 h, and then DMSO was added. The OD value was measured at 490 nm to calculate cell viability. The results showed that MgB2@PEG nanosheets at different concentrations exhibited good biocompatibility and low toxicity. Even at a concentration as high as 200 µg / mL, the relative cell viability remained above 80%. This experimental result indicates that MgB2@PEG nanosheets themselves do not produce significant toxic side effects on cells.

[0051] Figure 11The images show the cellular reactive oxygen species (ROS) staining of MgB2@PEG nanosheets prepared in Example 1 and pure MgB2 nanosheets. The specific experimental steps were as follows: The following materials were prepared using serum-free 1640 medium: Control (fresh medium); 1 mM H2O2; 200 μg / mL MgB2@PEG; 1 mM H2O2 + 200 μg / mL MgB2@PEG; 200 μg / mL MgB2; 1 mM H2O2 + 200 μg / mL MgB2; 4 μg / mL NAC (clinical drug N-acetylcysteine); 1 mM H2O2 + 4 μg / mL NAC. To verify the scavenging effect of two-dimensional MgB2@PEG nanosheets on intracellular ROS, 100 μL (1 × 10⁻⁶) was seeded in each well of a 96-well plate. 5 AML12 cells were incubated for 24 h. The supernatant was aspirated, and 100 μL of the prepared solution was added to each well, followed by incubation for 4 h. After incubation, the supernatant was aspirated, and the cells were washed three times with PBS. 10 μM DCFH-DA staining solution prepared with PBS was added to each well, and the cells were incubated at 37°C for 30 min in the dark. The cells were then washed three times with PBS, and images were taken using a fluorescence microscope. The images show that the H2O2 + 200 μg / mL MgB2@PEG group showed no obvious green fluorescence, indicating a good ability to scavenge intracellular reactive oxygen species.

[0052] Figure 12 The images show AM / PI staining results for cells containing MgB2@PEG nanosheets prepared in Example 1 and pure MgB2 nanosheets. The specific experimental steps were as follows: The following different groups of materials were prepared using serum-free 1640 medium: Control (fresh medium); 3mM H2O2; 25 μg / mL MgB2; 3mM H2O2 + 25 μg / mL MgB2; 25 μg / mL MgB2@PEG; 3mM H2O2 + 25 μg / mL MgB2@PEG. After incubating AML12 cells for 24 h, the supernatant was aspirated, and the cells were washed once with PBS. The prepared solutions were then added sequentially to the wells of the plate, and incubated for another 24 h. After incubation, the culture medium was aspirated, the cells were washed once with PBS, and 300 μL of dye (6 μL of Calcein AM and 4 μL of PI were mixed and added to 4 mL of PBS) was added. The cells were incubated at 37°C for 20 min in the dark and photographed using a fluorescence microscope. As can be seen from the figure, the red fluorescence of the 3 mM H2O2 + 25 μg / mL MgB2@PEG treatment group was significantly reduced compared with the 3 mM H2O2 treatment group, indicating that MgB2@PEG can protect normal hepatocytes from the threat of H2O2.

[0053] Figure 13The distribution of MgB2@PEG nanosheets prepared in Example 1 in mice at different time points is shown. The specific experimental steps were as follows: 0.01 mg of Cy5.5 dye was mixed with 1 mg of MgB2@PEG and stirred for 12 hours. The mixture was centrifuged at 13000 rpm for 20 min, the precipitate was collected, washed twice with water, and centrifuged at 15000 rpm for 10 min to synthesize Cy5.5-labeled MgB2@PEG. C57 mice were fasted for 16 h and then intraperitoneally injected with APAP (300 mg / kg) to establish an AILI model. Healthy mice served as the control group. MgB2@PEG was administered intravenously to both AILI and healthy mice. Heart, liver, spleen, lung, and kidney samples were collected at 2, 6, 12, and 24 h, and fluorescence signals were observed in the dark. The figure shows that after intravenous injection, the concentration of MgB2@PEG was highest in the liver, demonstrating a significant targeted enrichment effect on the liver. After 24 hours, the in vivo concentration of MgB2@PEG decreased significantly, indicating good in vivo clearance characteristics.

[0054] Figure 14 The ALT and AST levels of MgB2@PEG nanosheets prepared in Example 1 were statistically analyzed in mice with APAP-induced acute liver injury. The specific experimental steps were as follows: Healthy mice were randomly divided into 5 groups (n = 5): (1) PBS, (2) PBS + APAP, (3) 300 mg / kg NAC + APAP, (4) 25 mg / kg MgB2 + APAP, and (5) 25 mg / kg MgB2@PEG + APAP. C57 mice were fasted for 16 h, and an AILI model was established by intraperitoneal injection of APAP (300 mg / kg). Three h later, the drug was administered via tail vein. Twenty-four h after APAP poisoning, the mice were euthanized, blood was collected, and after standing at room temperature for 2 h, serum was collected by centrifugation at 3000 rpm for 20 min. The ALT and AST levels in the serum were measured. As can be seen from the figure, the ALT and AST levels in mice treated with 25 mg / kg MgB2@PEG were lower than those in the clinical drug NAC group, demonstrating excellent therapeutic effects on acute liver injury caused by APAP.

[0055] Figure 15The survival rate of mice with APAP-induced acute liver failure treated with MgB2@PEG nanosheets prepared in Example 1 is shown in the figure. The specific experimental steps were as follows: Healthy mice were randomly divided into 3 groups (n=6): (1) PBS + APAP, (2) 300 mg / kg NAC + APAP, and (3) 25 mg / kg MgB2@PEG + APAP. After fasting for 16 h, C57 mice were injected intraperitoneally with a high dose of APAP (600 mg / kg) to establish a mouse model. The drug was administered via tail vein 3 h later. The survival of the mice was recorded every 8 h for 120 h. As can be seen from the figure, the survival rate of mice treated with 25 mg / kg MgB2 was significantly higher than that of the control group treated with APAP.

[0056] Figure 16 To evaluate the ameliorative effect of the MgB2@PEG nanosheets prepared in Example 1 on hepatic encephalopathy in mice with APAP-induced acute liver failure, the specific experimental steps were as follows: Healthy mice were randomly divided into 4 groups (n=3): (1) PBS, (2) PBS + APAP, (3) 300 mg / kg NAC + APAP, and (4) 25 mg / kg MgB2@PEG + APAP. After fasting for 16 h, C57 mice were intraperitoneally injected with a high dose of APAP (600 mg / kg) to establish a mouse model, and the drug was administered via tail vein 3 h later. Animal behavior tests were performed on the mice 24 h after APAP poisoning. As can be seen from the figure, the motor ability of the mice treated with 25 mg / kg MgB2@PEG was significantly restored, the total distance of movement of the mice was significantly increased, and the time spent in the central region of the mice was also significantly higher than that of the APAP group, indicating that the behavioral and cognitive abilities of the mice after treatment were significantly improved compared with the APAP group. The results in summary indicate that the MgB2@PEG nanosheets obtained in this example can effectively alleviate hepatic encephalopathy caused by APAP-induced acute liver failure in mice.

[0057] Figure 17The image shows the statistical diagram of LPS content in the blood of mice with APAP-induced acute liver failure after treatment with MgB2@PEG nanosheets prepared in Example 1. The specific experimental steps were as follows: Healthy mice were randomly divided into 4 groups (n=3): (1) PBS, (2) PBS + APAP, (3) 300 mg / kg NAC + APAP, and (4) 25 mg / kg MgB2@PEG + APAP. C57 mice were fasted for 16 h, and a mouse model was established by intraperitoneal injection of a high dose of APAP (600 mg / kg). The drug was administered via tail vein 3 h later. 24 h after APAP poisoning, the mice were euthanized, blood was collected, and after standing at room temperature for 2 h, serum was collected by centrifugation at 3000 rpm for 20 min. The LPS content in the serum was measured. As can be seen from the figure, the LPS content in the blood of mice treated with 25 mg / kg MgB2@PEG was significantly lower than that in the control group treated with APAP, indicating that MgB2@PEG can effectively capture excess LPS in the blood and effectively block the cascade inflammatory response triggered by LPS.

[0058] Figure 18 The figure shows the statistical effect of MgB2@PEG nanosheets prepared in Example 1 on brain ammonia content in mice with APAP-induced acute liver failure. The specific experimental steps were as follows: Healthy mice were randomly divided into 4 groups (n=3): (1) PBS, (2) PBS + APAP, (3) 300 mg / kg NAC + APAP, (4) 25 mg / kg MgB2@PEG + APAP. After fasting for 16 h, C57 mice were injected intraperitoneally with a high dose of APAP (600 mg / kg) to establish a mouse model, and the drug was administered via tail vein 3 h later. After 24 h of APAP poisoning, the mice were euthanized, brain tissue was collected, tissue homogenate was prepared, and the brain ammonia content in the homogenate was tested. As can be seen from the figure, the brain ammonia content of mice treated with 25 mg / kg MgB2@PEG was significantly lower than that of the control group treated with APAP, indicating that MgB2@PEG alleviates hepatic encephalopathy caused by APAP-induced acute liver failure in mice.

[0059] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A polyethylene glycol-modified magnesium boride nanosheet for treating acute liver failure and hepatic encephalopathy caused by acetaminophen, characterized in that: The aforementioned polyethylene glycol-modified magnesium boride nanosheets consist of magnesium boride nanosheets as the core, with polyethylene glycol modified on the surface of the magnesium boride nanosheets.

2. The polyethylene glycol-modified magnesium boride nanosheets according to claim 1, characterized in that: The polyethylene glycol-modified magnesium boride nanosheets have a thickness of 1–2 nm and a diameter of 150–250 nm.

3. The polyethylene glycol-modified magnesium boride nanosheets according to claim 1, characterized in that: The polyethylene glycol-modified magnesium boride nanosheets generate reducing hydrogen gas through hydrolysis, which removes reactive oxygen species from areas of liver failure.

4. A method for preparing polyethylene glycol-modified magnesium boride nanosheets according to any one of claims 1 to 3, characterized in that: Magnesium boride powder was added to anhydrous ethanol solution and ultrasonically crushed. After centrifugation, the supernatant was collected. Polyethylene glycol was added to the supernatant and ultrasonically treated. After centrifugation, washing, and freeze-drying, polyethylene glycol-modified magnesium boride nanosheets were obtained.

5. The preparation method according to claim 4, characterized in that: The mass ratio of polyethylene glycol to magnesium boride nanosheets in the supernatant is 1–9:

1.

6. The preparation method according to claim 4, characterized in that: The ultrasound is performed alternately with probe ultrasound and room temperature water bath ultrasound. The power of probe ultrasound is 80~600 W and the total time is 1~5 h. The power of room temperature water bath ultrasound is 10~200 W and the total time is 1~5 h.

7. An application of the polyethylene glycol-modified magnesium boride nanosheets according to any one of claims 1 to 3, characterized in that: Nanomedicines for the preparation of treatments for acute liver failure and / or hepatic encephalopathy caused by acetaminophen.

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