Water-soluble antioxidant nanosheet as well as preparation method and application thereof in acute liver injury

Abstract

The invention relates to a water-soluble antioxidant nanosheet as well as a preparation method and application thereof in acute liver injury, and relates to the technical field of nano-drugs. The water-soluble antioxidant nanosheet disclosed by the invention is obtained by self-assembling two-dimensional transition metal disulfide and protein, and has proper size, good life compatibility and superstrong antioxidant activity. The water-soluble antioxidant nanosheet disclosed by the invention can be used for effectively treating acute liver injury induced by acetaminophen by inhibiting oxidative stress; damaged liver tissues can be effectively targeted, and a treatment effect is achieved; mitochondrial ROS level can be reduced, mitochondrial membrane potential can be recovered, and mitochondrial damage can be reduced; the level of inflammatory factors of the liver in the acute liver injury process can be remarkably reduced, infiltration of macrophages is reduced, and excessive inflammatory response of the liver is relieved. The preparation method of the water-soluble antioxidant nanosheet is a simple and green synthesis method, and raw materials are low in price and easy to obtain.

Description

Technical Field

[0001] The invention relates to the technical field of nano-medicine, and in particular to a water-soluble antioxidant nano-sheet and a preparation method thereof and application thereof in acute liver injury. Background Art

[0002] Acute liver injury (ALI) refers to liver cell damage caused by various reasons in the absence of chronic hepatitis. Drug-induced, ischemia-reperfusion injury, autoimmunity or viral infection are the main causes of acute liver injury. ALI has a high incidence rate and seriously affects people's health. Its rapid and powerful destructiveness often endangers the lives of patients. Among them, more than 50% of ALI cases are caused by the widely used antipyretic, analgesic and anti-inflammatory drug acetaminophen (APAP), and the incidence rate is increasing year by year. N-acetylcysteine ​​(NAC) is currently the only drug approved clinically for the treatment of acetaminophen-induced ALI. However, due to its poor targeting, low bioavailability, narrow therapeutic window and many side effects, the clinical efficacy is not ideal. Therefore, more effective strategies are urgently needed to prevent and treat ALI.

[0003] The main pathological manifestation of acute liver injury is severe damage to liver function and structure, which is caused by the violent outbreak of reactive oxygen species (ROS) in liver cells. In the process of acute liver injury, mitochondria are the main source of ROS. Excessive ROS damage a variety of biomacromolecules and lead to the destruction of mitochondrial function and structure, causing a large number of liver cell death and the subsequent sharp decline in liver function. Therefore, antioxidants that can play a protective role in mitochondria have great application prospects in the treatment of acute liver injury. Due to the particularity of liver structure, nanomedicines of appropriate size can reach the lesion site through the opening of the liver sinusoids (50-200nm) without being directly cleared by the kidneys, avoiding the damage caused by a large amount of accumulation to healthy tissues and ensuring the good performance of drug efficacy. The safety and targeting of nanomedicines are important indicators for evaluating whether they can be applied in clinical practice. However, most of the nanomedicines currently developed for the treatment of ALI are greatly limited in clinical transformation due to their limited bioavailability and potential toxicity.

[0004] Two-dimensional transition metal dichalcogenides have been widely used in biomedical fields such as clinical implants, artificial joints, stents, and vascular clips in the past decade due to their excellent biocompatibility and physicochemical properties. In particular, tantalum, tungsten, and molybdenum have a variety of valence states, so their redox activity is very strong, making materials such as tantalum sulfide, molybdenum sulfide, and tungsten sulfide have strong ROS scavenging potential and have great prospects in the treatment of ALI. However, two-dimensional transition metal dichalcogenides are limited by particle size or water solubility and cannot target damaged liver tissue. Summary of the invention

[0005] The present invention aims to solve the technical problem in the prior art that two-dimensional transition metal disulfides are limited by particle size or water solubility and cannot target damaged liver tissue, and provides a water-soluble antioxidant nanosheet and a preparation method thereof and an application in acute liver injury.

[0006] In order to solve the above technical problems, the technical solutions of the present invention are as follows:

[0007] A water-soluble antioxidant nanosheet is a nanosheet obtained by self-assembly of two-dimensional transition metal disulfide and protein.

[0008] In the above technical solution, preferably, the two-dimensional transition metal disulfide is tantalum sulfide, molybdenum sulfide or tungsten sulfide; and the protein is serum albumin.

[0009] In the above technical solution, preferably, the particle size of the water-soluble antioxidant nanosheets is between 50 and 200 nm.

[0010] A method for preparing a water-soluble antioxidant nanosheet comprises the following steps:

[0011] The two-dimensional transition metal disulfide powder is peeled off to obtain a single-layer two-dimensional transition metal disulfide, and the obtained single-layer two-dimensional transition metal disulfide and a protein solution are stirred at room temperature in the dark under the protection of argon (Ar) to perform self-assembly to obtain the water-soluble antioxidant nanosheet.

[0012] In the above technical solution, preferably, the exfoliation is liquid phase exfoliation for 1 week, and the two-dimensional transition metal disulfide powder needs to be ground before exfoliation, and the grinding time is 2 to 3 hours.

[0013] In the above technical solution, preferably, the concentration of the protein solution is 10 mg / mL.

[0014] In the above technical solution, preferably, the volume ratio of the monolayer two-dimensional transition metal disulfide and the protein solution is 1:10.

[0015] In the above technical solution, preferably, the stirring self-assembly time is 2 to 3 hours.

[0016] In the above technical solution, it is preferred that:

[0017] After the two-dimensional transition metal disulfide powder was exfoliated, it was centrifuged at 3000 rpm for 10 minutes and the supernatant was collected;

[0018] The water-soluble antioxidant nanosheets are centrifuged and washed with a protein solution to remove unreacted impurities. The centrifugation parameters are: 13,000 rpm, 10 minutes, and the washing times are 3 times.

[0019] Application of a water-soluble antioxidant nanosheet in the preparation of a drug for treating acute liver injury.

[0020] The beneficial effects of the present invention are:

[0021] The water-soluble antioxidant nanosheet provided by the present invention is a nanosheet prepared by self-assembly of two-dimensional transition metal disulfide and protein, and has a suitable size ( Figure 1 ) and strong antioxidant activity ( Figure 2 ).

[0022] The water-soluble antioxidant nanosheet provided by the present invention can effectively treat acetaminophen-induced acute liver injury ( Figure 3 ).

[0023] The water-soluble antioxidant nanosheet provided by the present invention can effectively target damaged liver tissue and exert a therapeutic effect ( Figure 4 ).

[0024] The present invention provides a water-soluble antioxidant nanosheet that can reduce mitochondrial ROS (mtROS) levels, restore mitochondrial membrane potential, and alleviate mitochondrial damage ( Figure 5 ).

[0025] The water-soluble antioxidant nanosheet provided by the present invention can significantly reduce the level of inflammatory factors in the liver during acute liver injury, reduce the infiltration of macrophages, and alleviate the excessive inflammatory response of the liver ( Figure 6 ).

[0026] The water-soluble antioxidant nanosheet provided by the present invention has good life compatibility and is non-toxic to human liver cancer cells (HepG2 cells) at a concentration far exceeding that of the present invention. Figure 7 ), and long-term ultra-high dose administration had no effect on the heart, liver, spleen, lung, and kidney of normal mice ( Figure 8 ).

[0027] The invention provides a method for preparing a water-soluble antioxidant nanosheet, which adopts a simple and green synthesis method to prepare a water-soluble nanosheet with high antioxidant activity, and the raw materials adopted are low-priced and easily available. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments.

[0029] Figure 1 This is a transmission electron microscopy image of the serum albumin-tantalum sulfide nanosheets prepared in Example 1 in water.

[0030] Figure 2The results of the in vitro free radical scavenging ability of the serum albumin-tantalum sulfide nanosheets prepared in Example 1 are shown in FIG. 1 , wherein (A) is a superoxide anion, (B) is a hydroxyl radical, (C) is hydrogen peroxide, and (D) is a peroxynitrite anion.

[0031] Figure 3 The results of normal mice and mice with acute liver injury induced by acetaminophen after treatment with serum albumin-tantalum sulfide nanosheets prepared in Example 1 at different concentrations for 22 hours are shown; wherein, (A) is a graph showing the level of serum alanine aminotransferase (ALT), and (B) is a graph showing the level of aspartate aminotransferase (AST).

[0032] Figure 4 The results of liver stereofluorescence at different time points after normal mice and mice with acute kidney injury were injected with serum albumin-tantalum sulfide prepared in Example 1; (A) is a normal mouse, and (B) is a mouse with acute kidney injury.

[0033] Figure 5 These are the staining results of the control group human liver cancer cells, APAP-induced liver injury cells and cells injected with serum albumin-tantalum sulfide nanosheets prepared in Example 1; (A) is MitoSox Red, and (B) is JC-1.

[0034] Figure 6 These are F4 / 80 immunohistochemical staining images of liver sections of normal mice, mice with acute liver injury, and mice with acute liver injury after injection of serum albumin-tantalum sulfide nanosheets prepared in Example 1.

[0035] Figure 7 This is a graph showing the test results of the effect of serum albumin-tantalum sulfide nanosheets prepared in Example 1 at different concentrations on the viability of HepG2 cells.

[0036] Figure 8 HE staining images of the main organs of normal mice after continuous intravenous injection of serum albumin-tantalum sulfide nanosheets prepared in Example 1 for 28 days. DETAILED DESCRIPTION

[0037] The technical solution of the present invention is clearly and completely described below in conjunction with the accompanying drawings and embodiments, but it should be understood that the following embodiments do not limit the protection scope of the present invention.

[0038] Example 1

[0039] (1) Synthesis of nanosheets

[0040] Weigh 0.2g of tantalum sulfide powder, grind it in a clean mortar for 2 hours, transfer it to a glass bottle, measure 20mL of N-methylpyrrolidone and pour it into the glass bottle, mix the two and place them in ultrasonic liquid phase stripping for 1 week. Take out 5mL of liquid from the glass bottle and centrifuge it at 3000 rpm for 10 minutes, take the supernatant and add it dropwise to 50mL of 10mg / mL serum albumin solution. Stir the reaction in a beaker with a magnetic rotor under the protection of argon for 2 hours.

[0041] (2) Purification

[0042] The sample solution obtained in step (1) was transferred to a centrifuge tube and centrifuged at 13,000 rpm for 10 minutes; the sample was washed three times with serum albumin solution to remove unreacted impurities.

[0043] (3)Save

[0044] The sample obtained after purification in step (2) is resuspended in a certain amount of serum albumin solution, filled with argon, protected from light, and stored at 4° C. The prepared serum albumin-tantalum sulfide nanosheets are represented by S-TaS2.

[0045] Figure 1 The prepared serum albumin-tantalum sulfide nanosheets (S-TaS2) were photographed using a transmission electron microscope (TEM), and it can be seen that the particle size of the nanosheets is about 185nm.

[0046] Example 2

[0047] (1) Synthesis of nanosheets

[0048] Weigh 0.13g of molybdenum sulfide powder, grind it in a clean mortar for 3 hours, transfer it to a glass bottle, measure 20mL of N-methylpyrrolidone and pour it into the glass bottle, mix the two and place them in ultrasonic liquid phase stripping for 1 week. Take out 5mL of liquid from the glass bottle and centrifuge it at 3000 rpm for 10 minutes, take the supernatant and add it dropwise to 50mL of 10mg / mL serum albumin solution. Stir the reaction in a beaker with a magnetic rotor under the protection of argon for 3 hours.

[0049] (2) Purification

[0050] Purification was the same as in Example 1.

[0051] (3)Save

[0052] The same conditions as in Example 1 are maintained, and the obtained nanosheet is referred to as Nanosheet 2.

[0053] The nanosheets prepared in this embodiment have a particle size in the range of 50 to 200 nm, good life compatibility and super strong antioxidant activity; can effectively target damaged liver tissue and exert a therapeutic effect; can reduce mitochondrial ROS (mtROS) levels, restore mitochondrial membrane potential, and alleviate mitochondrial damage.

[0054] Example 3

[0055] (1) Synthesis of nanosheets

[0056] Weigh 0.2g of tungsten sulfide powder, grind it in a clean mortar for 2 hours, transfer it to a glass bottle, measure 20mL of N-methylpyrrolidone and pour it into the glass bottle, mix the two and place them in ultrasonic liquid phase stripping for 1 week. Take out 5mL of liquid from the glass bottle and centrifuge it at 3000 rpm for 10 minutes, take the supernatant and add it dropwise to 50mL of 10mg / mL serum albumin solution. Stir the reaction in a beaker with a magnetic rotor under the protection of argon for 2 hours.

[0057] (2) Purification

[0058] Purification was the same as in Example 1.

[0059] (3)Save

[0060] The same conditions as in Example 1 are maintained, and the obtained nanosheet is called Nanosheet 3.

[0061] The nanosheets prepared in this embodiment have a particle size in the range of 50 to 200 nm, good life compatibility and super strong antioxidant activity; can effectively target damaged liver tissue and exert a therapeutic effect; can reduce mitochondrial ROS (mtROS) levels, restore mitochondrial membrane potential, and alleviate mitochondrial damage.

[0062] Example 4

[0063] (1) Synthesis of nanosheets

[0064] Weigh 0.2g of tantalum sulfide powder, grind it in a clean mortar for 3 hours, transfer it to a glass bottle, measure 20mL of isopropanol and pour it into the glass bottle, mix the two and place them in ultrasonic liquid phase stripping for 1 week. Take out 5mL of liquid from the glass bottle and centrifuge it at 3000 rpm for 10 minutes, take the supernatant and add it dropwise to 50mL of 10mg / mL serum albumin solution. Stir the reaction in a beaker with a magnetic rotor under the protection of argon for 3 hours.

[0065] (2) Purification

[0066] Purification was the same as in Example 1.

[0067] (3)Save

[0068] The same conditions as in Example 1 are maintained, and the obtained nanosheet is referred to as Nanosheet 4.

[0069] The nanosheets prepared in this embodiment have a particle size in the range of 50 to 200 nm, good life compatibility and super strong antioxidant activity; can effectively target damaged liver tissue and exert a therapeutic effect; can reduce mitochondrial ROS (mtROS) levels, restore mitochondrial membrane potential, and alleviate mitochondrial damage.

[0070] Example 5

[0071] Weigh 0.13g of molybdenum sulfide powder, grind it in a clean mortar for 3 hours, transfer it to a glass bottle, measure 20mL of isopropanol and pour it into the glass bottle, mix the two and place them in ultrasonic liquid phase stripping for 1 week. Take out 5mL of liquid from the glass bottle and centrifuge it at 3000 rpm for 10 minutes, take the supernatant and add it dropwise to 50mL of 10mg / mL serum albumin solution. Stir the reaction in a beaker with a magnetic rotor under the protection of argon for 3 hours.

[0072] (2) Purification

[0073] Purification was the same as in Example 1.

[0074] (3)Save

[0075] The same conditions as in Example 1 are maintained, and the obtained nanosheet is referred to as nanosheet 5.

[0076] The nanosheets prepared in this embodiment have a particle size in the range of 50 to 200 nm, good life compatibility and super strong antioxidant activity; can effectively target damaged liver tissue and exert a therapeutic effect; can reduce mitochondrial ROS (mtROS) levels, restore mitochondrial membrane potential, and alleviate mitochondrial damage.

[0077] Example 6

[0078] Weigh 0.2g of tungsten sulfide powder, grind it in a clean mortar for 3 hours, transfer it to a glass bottle, measure 20mL of isopropanol and pour it into the glass bottle, mix the two and place them in ultrasonic liquid phase stripping for 1 week. Take out 5mL of liquid from the glass bottle and centrifuge it at 3000 rpm for 10 minutes, take the supernatant and add it dropwise to 50mL of 10mg / mL serum albumin solution. Stir the reaction in a beaker with a magnetic rotor under the protection of argon for 3 hours.

[0079] (2) Purification

[0080] Purification was the same as in Example 1.

[0081] (3)Save

[0082] The same conditions as in Example 1 are maintained, and the obtained nanosheet is referred to as Nanosheet 6.

[0083] The nanosheets prepared in this embodiment have a particle size in the range of 50 to 200 nm, good life compatibility and super strong antioxidant activity; can effectively target damaged liver tissue and exert a therapeutic effect; can reduce mitochondrial ROS (mtROS) levels, restore mitochondrial membrane potential, and alleviate mitochondrial damage.

[0084] Example 7

[0085] Taking the serum albumin-tantalum sulfide nanosheets synthesized in Example 1 as an example, the particle size and in vitro free radical scavenging activity of the nanosheets were investigated. The specific steps are as follows:

[0086] (1) Transmission electron microscopy characterization: Transmission electron microscopy images of serum albumin-tantalum sulfide nanosheets were taken with a TECNAI G2 transmission electron microscope at normal resolution and high resolution. The results are shown in Figure 1 As shown, the serum albumin-tantalum sulfide nanosheets have clear outlines, and the lattice is clearly visible under high resolution. They have a suitable size (185nm), a crystal plane spacing of 0.590nm, and still maintain a two-dimensional layered structure.

[0087] (2) Free radical scavenging ability assessment:

[0088] The NBT method was used to detect the effect of serum albumin-tantalum sulfide nanosheets on superoxide anions (O2 ·- ) scavenging ability. Methionine (390 μL, 20 μmol / L), riboflavin (6 μL, 0.01 mol / L), tetrazolium blue (22.5 μL, 0.01 mol / L), serum albumin-tantalum sulfide nanosheets of different concentrations (0 ng / mL, 150 ng / mL, 300 ng / mL, 600 ng / mL, 1200 ng / mL, 2400 ng / mL) and PBS (1.5 mL, 0.01 mol / L, pH = 7.4) were mixed with deionized water to fix the final volume of the reaction system to 3 mL. After mixing, the cuvette was exposed to ultraviolet light for 5 minutes, and the absorbance was measured at a wavelength of 560 nm.

[0089] The 3,3',5,5'-tetramethylbenzidine (TMB) method was used to detect the scavenging ability of serum albumin-tantalum sulfide nanosheets on hydroxyl radicals (·OH). 3,3',5,5'-tetramethylbenzidine (600μL, 5mM), ferrous sulfate (600μL, 5mM), hydrogen peroxide (60μL, 100mM) and serum albumin-tantalum sulfide nanosheets of different concentrations (0ng / mL, 40ng / mL, 80ng / mL, 160ng / mL, 320ng / mL, 640ng / mL) were prepared, and PBS (0.01mol / L, pH=7.4) solution was added to fix the final volume of the reaction system to 3mL. After mixing, it was placed for 4 minutes, transferred to a cuvette, and the absorbance was measured at a wavelength of 652nm.

[0090] The scavenging ability of serum albumin-tantalum sulfide nanosheets on hydrogen peroxide (H2O2) was detected by UV-visible spectrophotometry. Hydrogen peroxide of different concentrations (0mM, 5mM, 10mM, 20mM, 40mM, 80mM) was mixed with serum albumin-tantalum sulfide nanosheets (200μg / mL) and incubated in the dark for 12 hours. The ultraviolet light absorption at 350-850nm was detected.

[0091] The effect of serum albumin-tantalum sulfide nanosheets on peroxynitrite anion (ONOO - ) was removed by mixing pyrogallol red (5 mM), peroxynitrite and anions with serum albumin-tantalum sulfide nanosheets of different concentrations (0 μg / mL, 15 μg / mL, 30 μg / mL, 60 μg / mL, 120 μg / mL) for 15 minutes. The UV absorption spectrum was scanned to determine the removal rate of peroxynitrite anions.

[0092] The results of in vitro free radical scavenging experiments are as follows Figure 2 As shown in Figure 2, serum albumin-tantalum sulfide nanosheets can efficiently remove various reactive oxygen species, such as superoxide anions ( Figure 2 A), hydroxyl radical ( Figure 2 B), hydrogen peroxide ( Figure 2 C) and peroxynitrite anion ( Figure 2 D).

[0093] Example 8

[0094] Taking the serum albumin-tantalum sulfide nanosheet synthesized in Example 1 as an example, KM mice (6-7 weeks, 20-22g) were intraperitoneally injected with 250mg / kg acetaminophen to induce an acute liver injury model. After 2 hours of acetaminophen administration, normal saline (equal volume), 1mg / kg or 2mg / kg serum albumin-tantalum sulfide nanosheets, and 2mg / kg N-acetylcysteine ​​were injected into the tail vein. The mice were euthanized 24 hours after modeling, blood was collected, and serum was separated by centrifugation at a speed of 3000 rpm for 10 minutes. The levels of alanine aminotransferase and aspartate aminotransferase in serum were analyzed using an automatic biochemical analyzer BS-2000M.

[0095] The results are as follows Figure 3 As shown, 1 mg / kg and 2 mg / kg of the water-soluble antioxidant nanosheets prepared by the present invention can effectively restore the liver function of mice with acute liver injury induced by acetaminophen.

[0096] Example 9

[0097] Taking the serum albumin-tantalum sulfide nanosheets synthesized in Example 1 as an example, stereoscopic fluorescence results of livers of normal mice, mice with acute kidney injury and mice injected with serum albumin-tantalum sulfide nanosheets synthesized in Example 1 at different time points were taken to observe the targeting of the nanosheets to the damaged liver. The specific steps are as follows:

[0098] First, prepare serum albumin-tantalum sulfide nanosheets connected with fluorescein isothiocyanate (FITC), weigh 0.2g of fluorescein isothiocyanate and dissolve it in 2mL of DMSO, measure the solution containing 10mg of serum albumin-tantalum sulfide nanosheets, adjust the final volume to 10mL with ultrapure water, mix well in an EP tube, protect from light, and stir to react for 5 to 6 hours. The sample obtained after the reaction is placed in a dialysis bag with a molecular weight cutoff of 3.5kD, and the dialysis bag is placed in 2L ultrapure water. Dialysis is performed under magnetic stirring, and ultrapure water is replaced every 4 hours for a total of 24 hours to remove unreacted impurities. After the dialysis is completed, the drug solution is placed at 4°C and stored in the dark.

[0099] The mouse modeling conditions were the same as those in Example 8. Two hours after the administration of acetaminophen, serum albumin-tantalum sulfide nanosheets connected with fluorescein isothiocyanate (FITC) were injected through the tail vein. The livers of mice in each group were collected at 0 h, 6 h, and 22 h and observed under a Leica M205 FA stereo fluorescence microscope.

[0100] The results are as follows Figure 4 As shown in the figure, the fluorescence intensity of the liver of each group of mice increased over time. More importantly, at the same time point, the fluorescence intensity of the liver of mice with acute liver injury was significantly higher than that of normal mice, and the liver injury area continued to shrink over time. The above results show that after intravenous administration, serum albumin-tantalum sulfide nanosheets can preferentially accumulate in the liver injury area and play a therapeutic role.

[0101] Example 10

[0102] Taking the serum albumin-tantalum sulfide nanosheets synthesized in Example 1 as an example, the protective effect of the nanosheets on mitochondria in HepG2 cells was evaluated. The specific steps are as follows:

[0103] HepG2 cells were cultured at 1×10 5 / well density was seeded into 6-well plates and incubated for 24 hours. The cells were dispersed in culture medium with 1×PBS (equal volume), acetaminophen (10mM) and serum albumin-tantalum sulfide nanosheets (1.25μg / mL and 2.5μg / mL) to prepare different cell culture solutions, and the above cell culture solutions were added to HepG2 cells and incubated for another 24 hours. MitoSox Red (a new type of fluorescent probe for detecting superoxide in mitochondria, which can quickly and specifically target mitochondria, thereby selectively detecting superoxide in mitochondria.) and JC-1 (JC-1 is an ideal fluorescent probe widely used to detect mitochondrial membrane potential ΔΨm. It can detect the mitochondrial membrane potential of cells, tissues or purified mitochondria. When the mitochondrial membrane potential is high, JC-1 aggregates in the matrix of mitochondria to form polymers, which can produce red fluorescence; when the mitochondrial membrane potential is low, JC-1 cannot aggregate in the matrix of mitochondria. At this time, JC-1 is a monomer and can produce green fluorescence; the change of mitochondrial membrane potential can be detected by the change of fluorescence color.) probes were used to stain HepG2 cells according to the instructions and observed and photographed under an inverted fluorescence microscope.

[0104] like Figure 5 As shown, APAP-treated HepG2 cells showed high levels of mitochondrial superoxide and a significant decrease in mitochondrial membrane potential. In S-TaS2-treated HepG2 cells, the MitoSOX Red fluorescence intensity was significantly reduced, while the JC-1 red fluorescence ratio was significantly increased, indicating that S-TaS2 effectively reduced mitochondrial superoxide levels, significantly restored mitochondrial membrane potential, and alleviated APAP-induced mitochondrial damage.

[0105] Embodiment 11

[0106] Taking the serum albumin-tantalum sulfide nanosheets synthesized in Example 1 as an example, the effect of the nanosheets on the infiltration of liver macrophages in mice with acute liver injury was evaluated. The specific steps are as follows:

[0107] The mouse modeling, medication and material collection process are the same as in Example 8. Subsequently, the liver tissue of each group of mice (3 mice in each group) is cut into small pieces and placed in a 4% paraformaldehyde solution for fixation. The liver tissue is then placed in an embedding box and flushed with running water to remove the fixative remaining in the tissue. The embedding box is immersed in gradient ethanol for dehydration, and then immersed in xylene to dissolve the tissue and embedding medium and infiltrate. The transparent tissue block is placed in the melted paraffin and placed in a wax dissolving box for overnight insulation. The wax-impregnated tissue material is placed in the center of a metal embedding frame containing wax liquid, and transplanted on ice after dripping wax. When the surface of the wax liquid solidifies, tweezers are used to clamp the pre-embedded tissue, adjust the direction of the tissue, insert it into the wax liquid, and place the uncovered embedding box flat on the surface of the mold to cover it, drip wax liquid to cover the mold, press gently, and place it in the solidification area until the wax block is completely solidified. Cool at 0°C for about 30 minutes, fix the embedded wax block on a slicer, and cut it into 4 micron thick slices. The slices were ironed flat in heated water, then attached to a glass slide and dried in a 65°C thermostat. The slices were then placed in xylene for dewaxing, and then soaked in 1×EDTA repair solution, cooled naturally after a water bath for 15 minutes, washed three times with TBST, and an appropriate amount of endogenous catalase blocker was added to the center of the tissue, incubated at 37°C for 15 minutes, and washed three times with TBST. 5% BSA was blocked at 37°C for 1 hour and washed. After the primary antibody (F4 / 80 antibody) was incubated at 4°C for 16 hours, an appropriate amount of reaction enhancement solution was added, incubated at 37°C for 20 minutes, and washed three times with TBST; an appropriate amount of enzyme-labeled sheep anti-mouse and rabbit IgG polymer was added and incubated at 37°C for 30 minutes. Concentrated DAB solution was incubated at room temperature for 5-10 minutes, and the reaction was terminated by soaking in tap water. Hematoxylin was counterstained for 20 seconds, rinsed with tap water for 2 minutes, differentiated with 1% hydrochloric acid ethanol for 1 second, and soaked in tap water for 3 minutes to return to blue. Finally, the slides were placed in ethanol for dehydration, placed in xylene to make them transparent, air-dried, and sealed with neutral resin.

[0108] The results are as follows Figure 6 As shown, after treatment with serum albumin-tantalum sulfide nanosheets, macrophage infiltration in the liver area of ​​mice with acute liver injury was significantly reduced, indicating that serum albumin-tantalum sulfide nanosheets can effectively inhibit excessive inflammation during acute liver injury.

[0109] Example 12

[0110] Taking the serum albumin-tantalum sulfide nanosheets synthesized in Example 1 as an example, the biocompatibility of the nanosheets was evaluated at the cell level and the animal level. The specific steps are as follows:

[0111] Cell level: HepG2 cells were cultured at 1×10 4 / well was inoculated into a 96-well plate and incubated for 24 hours. Serum albumin-tantalum sulfide nanosheets were dispersed in the culture medium to prepare cell culture fluids of different concentrations (0, 0.625, 1.25, 2.5, 5, 10, 20, 40, 80 and 160 μg / mL, respectively). The above cell culture fluid was added to HepG2 cells and incubated for another 24 hours, and then 10 microliters of CCK-8 reagent was added to each well and incubated for half an hour, and cell viability was detected by measuring absorbance at 450nm.

[0112] Animal level: Healthy KM mice were given 20 mg / kg serum albumin-tantalum sulfide nanosheets (dissolved in saline) by tail vein injection for 28 consecutive days, and then the mice were euthanized and the main organs (heart, liver, spleen, lungs and kidneys) were collected. The tissue fixation, embedding, sectioning and dewaxing process are the same as in Example 6. After the sections are dewaxed, the hematoxylin stain solution is added to the tissue to ensure that the tissue is completely covered and dyed for 10-15 minutes. Rinse with running water to wash away the excess stain. Differentiate with 1% hydrochloric acid alcohol solution, soak in water to turn blue for 2 minutes, add eosin stain solution to the tissue and dye for about 10 seconds. After eosin staining is completed, immediately immerse in anhydrous ethanol for dehydration twice (2 minutes each time). Soak in xylene twice (2 minutes each time), dry naturally in a fume hood for 10-30 minutes, and seal with a neutral resin.

[0113] The results are as follows Figure 7 As shown in the figure, serum albumin-tantalum sulfide nanosheets have no obvious toxicity to HepG2 cells, and have a slight pro-proliferation effect at certain concentrations. In addition, HE staining results show that normal mice have no obvious damage to the heart, liver, spleen, lung, and kidney after long-term intravenous injection of serum albumin-tantalum sulfide nanosheets ( Figure 8 ).

[0114] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A water-soluble antioxidant nanosheet, characterized in that: It is a nanosheet obtained by self-assembly of two-dimensional transition metal disulfide and protein.

2. The water-soluble antioxidant nanosheet according to claim 1, characterized in that: The two-dimensional transition metal disulfide is tantalum sulfide, molybdenum sulfide or tungsten sulfide; and the protein is serum albumin.

3. The water-soluble antioxidant nanosheet according to claim 1, characterized in that: The particle size of the water-soluble antioxidant nanosheet is between 50 and 200 nm.

4. A method for preparing the water-soluble antioxidant nanosheet according to any one of claims 1 to 3, characterized in that: The following steps are involved: The two-dimensional transition metal disulfide powder is peeled off to obtain a single-layer two-dimensional transition metal disulfide, and the obtained single-layer two-dimensional transition metal disulfide and a protein solution are stirred at room temperature in the dark under argon protection to perform self-assembly to obtain the water-soluble antioxidant nanosheet.

5. The method for preparing the water-soluble antioxidant nanosheet according to claim 4, characterized in that: The stripping is liquid phase stripping for 1 week, and the two-dimensional transition metal disulfide powder needs to be ground before stripping, and the grinding time is 2 to 3 hours.

6. The method for preparing the water-soluble antioxidant nanosheet according to claim 4, characterized in that: The concentration of the protein solution is 10 mg / mL.

7. The method for preparing the water-soluble antioxidant nanosheet according to claim 6, characterized in that: The volume ratio of the monolayer two-dimensional transition metal disulfide to the protein solution is 1:

10.

8. The method for preparing the water-soluble antioxidant nanosheet according to claim 4, characterized in that: The stirring self-assembly time is 2 to 3 hours.

9. The method for preparing the water-soluble antioxidant nanosheet according to claim 4, characterized in that: After the two-dimensional transition metal disulfide powder was exfoliated, it was centrifuged at 3000 rpm for 10 minutes and the supernatant was collected; After obtaining the water-soluble antioxidant nanosheets, centrifugation and protein solution washing are performed to remove unreacted impurities. The centrifugation parameters are: 13000 rpm, 10 minutes, and the washing times are 3 times.

10. Use of the water-soluble antioxidant nanosheet according to any one of claims 1 to 3 in the preparation of a drug for treating acute liver injury.

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