Polylysine-modified tungsten-based heteropoly acid nanocluster and application thereof

Abstract

The application belongs to the field of nanometer material preparation and biological medicine, and discloses a polylysine modified tungsten-based heteropoly acid nanocluster and application thereof. The tungsten-based heteropoly acid nanocluster is synthesized by imitating the Folin phenol detection method, rutin is wrapped on the surface of the tungsten-based heteropoly acid nanocluster as an antioxidant, and the polylysine with positive electricity is further modified on the surface of the tungsten-based heteropoly acid nanocluster. The nanocluster has good stability and biocompatibility, and can be used for treating acetaminophen-induced acute liver failure. The nanocluster specifically removes active oxygen at the liver failure site through oxidation and reduction, and specifically removes extracellular free DNA through electrostatic adsorption, so as to inhibit the inflammatory reaction and effectively relieve the symptoms caused by acute liver failure.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of nanomaterial preparation and biomedicine, and particularly relates to a polylysine modified tungsten-based heteropoly acid nanocluster and application thereof. BACKGROUND

[0002] Acute liver failure (ALF) is a rapid loss of liver function that occurs within days or weeks, usually in patients without pre-existing liver disease. This disease is mainly caused by hepatitis virus or drugs (such as acetaminophen). Acetaminophen (APAP) is a non-prescription drug commonly used to treat fever and pain, which is considered safe at therapeutic doses, but overdose can cause dose-dependent liver injury or liver failure, and even life-threatening. However, even at therapeutic doses, APAP can induce liver injury and acute liver failure under the action of multiple factors such as alcohol intake, chronic liver disease, malnutrition, age and genetics. Liver toxicity caused by overdose of APAP is the main cause of acute liver failure. Therefore, APAP-induced liver injury and liver failure have become a public health problem that cannot be ignored.

[0003] Studies have shown that when APAP is taken in excess, the saturation of phase II metabolic enzymes, excessive N-acetyl-p-benzoquinone imine (NAPQI) will deplete glutathione (GSH) in the cytoplasm and mitochondria, and destroy the normal antioxidant capacity of mitochondria. In addition, excessive NAPQI covalently binds to cellular proteins with sulfhydryl groups (especially mitochondrial proteins) to form acetaminophen protein adducts (APAP-AD), interfere with cellular redox balance, cause mitochondrial dysfunction, and increase reactive oxygen and nitrogen species (RONS). Importantly, NAPQI and RONS can cause mitochondrial DNA damage, activate the stress-activated protein kinase (JNK) signaling pathway. Phosphorylated JNK (p-JNK) translocates to mitochondria, leading to mitochondrial electron transport chain (ETC) dysfunction and increased RONS release. Accumulated RONS continue to induce JNK phosphorylation, and sustained activation of JNK amplifies mitochondrial RONS, forming an activation cycle. In addition, p-JNK can induce the opening of the mitochondrial permeability transition (MPT) pore, increase mitochondrial permeability and pore transition, and initiate the release of apoptosis-inducing factor (AIF) and mitochondrial endonuclease G, leading to nuclear DNA fragmentation, and ultimately causing hepatocyte necrosis. It is currently believed that APAP-induced oxidative stress and mitochondrial dysfunction play a central role in the pathogenesis of acute liver failure (ALF).

[0004] In addition, extracellular free DNA (cfDNA) levels are significantly elevated during acute liver failure. In the setting of liver failure, massive hepatocyte necrosis releases DNA from the nucleus into the blood circulation, leading to a sharp rise in total cfDNA levels. Released cfDNA can be recognized by Toll-like receptor 9 (TLR9) on immune cells, which in turn activates immune cells, triggering the release of a large number of pro-inflammatory factors, exacerbating the inflammatory response. Moreover, cfDNA does not act independently, but forms a vicious cycle with RONS. Excessive RONS cause massive cell death and damage, releasing more cfDNA. Released cfDNA is recognized by immune cells through TLR9, further activating these cells, producing a burst of RONS and more inflammatory factors. More RONS trigger more cell death and cfDNA release, and so on, amplifying inflammation and tissue damage. It has been reported that deoxyribonuclease I (DNase I) degradation of cfDNA can reduce APAP-induced liver injury in animal models. Therefore, by capturing cfDNA to block the link between injury and inflammation, it may be a promising approach to treat acute liver failure (ALF).

[0005] In recent years, nanomaterials have received much attention from researchers due to their adjustable shape and size, excellent physical and chemical properties, and on-demand synthesis strategies. Among them, polyoxometalates (POMs), as a kind of oxygen-containing polyoxometalates formed by coordination of transition metal elements (such as tungsten, molybdenum, and niobium) and oxygen elements in a specific structure, have been widely used in chemical catalysis, biomedicine, and other fields. With its simple synthesis strategy and controllable physical and chemical properties, polyoxometalates have been applied to the treatment of various diseases such as anti-tumor, alleviating acute kidney injury, and ischemic stroke.

[0006] Previous studies have shown that tannic acid and melanin-modified tungsten-based polyoxometalate nanodrugs (MHT) can effectively reduce neuroinflammation and inhibit neuronal apoptosis by scavenging mitochondrial reactive oxygen species (mtROS) and inhibiting mitochondrial DNA (mtDNA) release, thereby efficiently treating cerebral ischemia-reperfusion injury. In addition, the use of cationic nanomaterials to scavenge cfDNA has been proven to have significant effects on treating cfDNA-related diseases, including sepsis, rheumatic diseases, inflammatory bowel disease, psoriasis, toxic shock, and periodontitis. These cationic nanomaterials are usually modified with positively charged polymers such as polyethyleneimine (PEI) and efficiently capture cfDNA through electrostatic interactions.

[0007] There is no published data on tungsten-based polyoxometalate nanoclusters for treating APAP-induced acute liver failure (ALF). SUMMARY

[0008] To solve the limitations of conventional drugs in the current clinical application scope, medication timing and potential side effects, the present application provides a polylysine modified tungsten-based heteropoly acid nanocluster and its application in treating acetaminophen-induced acute liver failure. By simulating the Folin phenol detection method, a tungsten-based heteropoly acid nanocluster with high RONS scavenging and cfDNA scavenging capacity is synthesized. Due to its unique physicochemical properties, the nanocluster can target enrichment at the liver injury site, specifically scavenge excess RONS, significantly reduce oxidative stress, promote liver cell repair and regeneration, and thus effectively treat APAP-induced ALF, providing a new and efficient treatment strategy for acute liver failure.

[0009] To achieve the above-mentioned purpose, the present application adopts the following technical solutions:

[0010] The present application first discloses a polylysine modified tungsten-based heteropoly acid nanocluster, characterized in that: the polylysine modified tungsten-based heteropoly acid nanocluster (denoted as WRPOM@PLL) is a tungsten-based heteropoly acid nanocluster (WRPOM) core with a positive polylysine (PLL) modified on the surface of the tungsten-based heteropoly acid nanocluster. The preparation method comprises the following steps:

[0011] In an alkaline environment provided by a sodium carbonate solution, rutin and phosphotungstic acid hydrate are sequentially added and stirred for 1-6h to obtain a reaction solution; then polylysine is added to the reaction solution and continues to be stirred for 1-6h, ultrasonic for 1-3h, dialysis for 24-72h, and freeze-drying to obtain the polylysine modified tungsten-based heteropoly acid nanocluster. The polylysine modified tungsten-based heteropoly acid nanocluster prepared by the present application has a diameter of 20-200nm and a positive surface charge, and can target enrichment at the liver failure site through nanoscale size effect.

[0012] As a preferred, the mass ratio of rutin, phosphotungstic acid hydrate and polylysine is 10-150mg:10-80mg:10-80mg.

[0013] As a preferred, the stirring speed is 80-1000rpm, and the ultrasonic power is 80-600W.

[0014] The polylysine modified tungsten-based heteropoly acid nanocluster of the present application has the following characteristics: good biological safety at the cellular and animal levels; the ability to scavenge reactive oxygen and reactive nitrogen species; positive surface charge for specific binding of cfDNA, thereby reducing oxidative stress and protecting cells and tissues from oxidative damage to achieve the effect of treating acetaminophen-induced acute liver failure.

[0015] The beneficial effects of the present application are reflected in:

[0016] 1、 The tungsten-based heteropoly acid nanocluster has good oxidation resistance, biocompatibility and stability through the modification of lysine. The tungsten-based heteropoly acid nanocluster contains mixed valence W elements, and the +5 valence W is oxidized to +6 valence by redox reaction with RONS, thereby efficiently removing RONS. At the same time, the positive polylysine can effectively capture cfDNA through electrostatic interaction, prevent TLR9-mediated pro-inflammatory signals, and further inhibit inflammatory reactions. Based on the above mechanism, the polylysine-modified tungsten-based heteropoly acid nanocluster of the application can achieve effective treatment of acute liver failure. The treatment method of removing RONS and cfDNA effectively overcomes the problems of narrow treatment window, poor targeting and easy drug resistance of traditional antioxidant drugs (such as N-acetylcysteine, NAC).

[0017] 2、 Dual-target inhibition breaks through the limitations of single-mechanism drugs. Compared with traditional drugs that can only remove RONS or can only adsorb cfDNA, WRPOM@PLL can simultaneously remove two key inflammatory mediators, RONS and cfDNA, break the vicious cycle of RONS and cfDNA in inflammation and tissue damage, and more effectively suppress the amplification and spread of inflammatory signals from the source.

[0018] 3、 The nanocluster of the application has good biocompatibility, degradability and in vivo clearance efficiency, can be quickly excreted through the kidney, avoids the accumulation toxicity of nanomaterials in extrahepatic tissues, significantly improves the drug safety, avoids the toxicity caused by the accumulation of nanoclusters in vivo, and improves the narrow treatment window period in NAC treatment.

[0019] 4、 The polylysine and tungsten-based heteropoly acid nanocluster are efficiently compounded through a one-step synthesis process, realizing the “structure-function” integrated design. The method is simple in operation and good in repeatability, and the obtained WRPOM@PLL nanocluster has high antioxidant activity and cfDNA double removal capacity, significantly enhancing the comprehensive treatment effect on APAP-induced acute liver failure. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 The synthesis route of the polylysine-modified tungsten-based heteropoly acid nanocluster of the application is shown in the figure.

[0021] Figure 2 The transmission electron microscope image of the polylysine-modified tungsten-based heteropoly acid nanocluster prepared in Example 1 is shown in the figure.

[0022] Figure 3 The particle size diagram of the polylysine-modified tungsten-based heteropoly acid nanocluster prepared in Example 1 is shown in the figure.

[0023] Figure 4Zeta potential chart of polylysine modified tungsten-based heteropoly acid nanoclusters and unmodified tungsten-based heteropoly acid nanoclusters prepared for Example 1.

[0024] Figure 5 UV chart of polylysine modified tungsten-based heteropoly acid nanoclusters prepared for Example 1.

[0025] Figure 6 In vitro adsorption cfDNA effect chart of polylysine modified tungsten-based heteropoly acid nanoclusters prepared for Example 1.

[0026] Figure 7 In vitro hydroxyl radical scavenging chart of polylysine modified tungsten-based heteropoly acid nanoclusters and unmodified polylysine tungsten-based heteropoly acid nanoclusters prepared for Example 1.

[0027] Figure 8 In vitro DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging chart of polylysine modified tungsten-based heteropoly acid nanoclusters and unmodified polylysine tungsten-based heteropoly acid nanoclusters prepared for Example 1.

[0028] Figure 9 In vitro ABTS (2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid) radical scavenging chart of polylysine modified tungsten-based heteropoly acid nanoclusters and unmodified polylysine tungsten-based heteropoly acid nanoclusters prepared for Example 1.

[0029] Figure 10 Hemolysis experiment chart of polylysine modified tungsten-based heteropoly acid nanoclusters prepared for Example 1.

[0030] Figure 11 MTT experiment chart of polylysine modified tungsten-based heteropoly acid nanoclusters prepared for Example 1.

[0031] Figure 12 Cellular reactive oxygen species staining chart of polylysine modified tungsten-based heteropoly acid nanoclusters prepared for Example 1.

[0032] Figure 13 Cellular AM (calcium acetoxymethyl succinate) / PI (propyl iodide) live and dead staining chart of polylysine modified tungsten-based heteropoly acid nanoclusters and unmodified polylysine tungsten-based heteropoly acid nanoclusters prepared for Example 1.

[0033] Figure 14 Distribution chart of polylysine modified tungsten-based heteropoly acid nanoclusters prepared for Example 1 at different time points in mice, wherein: Figure 14 the (a) chart in is the in vivo distribution chart of WRPOM@PLL@Cy5.5 at different time periods in control group and AILI model group mice; Figure 14 the (b) chart in is the fluorescence intensity chart of heart, liver, spleen, lung and kidney of control group mice at different time periods; Figure 14Figure (c) shows the fluorescence intensity of the heart, liver, spleen, lungs and kidneys in the AILI model group at different time points.

[0034] Figure 15 The polylysine-modified tungsten-based heteropolyacid nanoclusters prepared in Example 1 showed an effect on the reduction of alanine aminotransferase (ALT) in mice with APAP-induced acute liver injury. Figure 15 (a) of the above, aspartate aminotransferase (AST) Figure 15 (b) Statistical chart in the middle.

[0035] Figure 16 The survival rate of mice with APAP-induced acute liver failure treated with polylysine-modified tungsten-based heteropolyacid nanoclusters prepared in Example 1 is shown in the figure. Detailed Implementation

[0036] 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 concept of the invention, they should all fall within the protection scope of the present invention.

[0037] Example 1

[0038] In this embodiment, polylysine-modified tungsten-based heteropolyacid nanoclusters were prepared using the following method:

[0039] First, prepare 9 mL of 25 mg / mL sodium carbonate solution, then add 100 mg of rutin to dissolve it. After the solution becomes clear and transparent, add 1 mL of 60 mg / mL phosphotungstic acid hydrate aqueous solution to the solution and stir continuously at 350 rpm for 3 h. Then add 70 mg of polylysine, stir continuously at 350 rpm for 2 h, sonicate at 250 W for 2 h, and finally dialyze with pure water (MW=3500 Da) for 72 h. Freeze-dry to obtain polylysine-modified tungsten-based heteropolyacid nanoclusters, denoted as WRPOM@PLL, and store at 4 °C.

[0040] For comparison, this embodiment also prepared unmodified polylysine-based tungsten-based heteropolyacid nanoclusters. The method was as follows: 9 mL of 25 mg / mL sodium carbonate solution was prepared, and 100 mg of rutin was added to dissolve it. After the solution became clear and transparent, 1 mL of 60 mg / mL phosphotungstic acid hydrate aqueous solution was added to the solution. The reaction was stirred continuously for 3 h, dialyzed with pure water (MW=3500Da) for 72 h, and freeze-dried to obtain unmodified polylysine-based tungsten-based heteropolyacid nanoclusters, denoted as WRPOM, and stored at 4 °C.

[0041] Figure 2The transmission electron microscope image of the WRPOM@PLL nanoclusters obtained in the example is shown in the figure. The characterization method is as follows: the water dispersion of the WRPOM@PLL nanoclusters is added dropwise to a transmission electron microscope copper net, dried, and placed in a transmission electron microscope for observation. It can be seen from the figure that the tungsten-based heteropoly acid nanoclusters have a diameter of 100-200 nm.

[0042] Figure 3 The hydration particle size diagram of the WRPOM@PLL nanoclusters obtained in the example is shown in the figure. The characterization method is as follows: the synthesized WRPOM@PLL nanoclusters are dispersed in an aqueous solution, and the particle size is measured by a Malvern laser particle size analyzer. It can be seen from the figure that the diameter of the nanoclusters is about 130 nm.

[0043] Figure 4 The Zeta potential diagram of the WRPOM@PLL nanoclusters and WRPOM nanoclusters obtained in the example is shown in the figure. The characterization method is as follows: 500 μL of the water dispersion of the nanoclusters is added to a Zeta potential sample cell, and a NanoZS90 Malvern particle size analyzer is used for testing. It can be seen from the figure that the Zeta potential of the WRPOM nanoclusters without modification of polylysine is -29 mV, and the Zeta potential of the WRPOM@PLL nanoclusters after modification of polylysine is +9 mV, indicating that the polylysine is successfully modified to the surface of the tungsten-based heteropoly acid nanoclusters.

[0044] Figure 5 The ultraviolet spectrum diagram of the WRPOM@PLL nanoclusters obtained in the example is shown in the figure. The characterization method is as follows: the water dispersion of the WRPOM@PLL nanoclusters with different concentrations (the final concentration of the nanoclusters in the system is 12.5, 25, 50, 75, 100, 200, and 400 μg / mL, respectively) is tested by an ultraviolet spectrophotometer. It can be seen from the figure that the WRPOM@PLL nanoclusters have a maximum absorption peak at 250 nm. It is worth noting that the relatively wide absorption of the WRPOM@PLL in the entire UV-vis-NIR region is due to the electronic relaxation polarization caused by the charge transfer between the reduced W 5+ and oxidized W 6+ , and the coloring effect of tungsten oxide and phosphate containing W 5+ .

[0045] Figure 6Figure of in vitro cfDNA adsorption effect of WRPOM@PLL nanoclusters obtained in the present embodiment, the specific steps are as follows: first, mix 12 μL of 1 mg / mL ctDNA PBS solution and 12 μL of 1 mg / mL EtBr PBS solution, then add different volumes of 1 mg / mL WRPOM@PLL nanocluster aqueous dispersion to 48 μL of fetal bovine serum, and adjust the final volume to 480 μL with PBS (the final mass ratio of nanoclusters to DNA is 0.3, 0.6, 1.2, 2.5, 5, 10, 20, 40, 80), incubate at 37°C for 24 h, then take 100 μL of supernatant and place it in a 96-well plate, pay attention to avoid precipitation when harvesting, excite at a wavelength of 485 nm, and detect the fluorescence intensity of the complex at 590 nm. As can be seen from the figure, the cfDNA adsorption effect of WRPOM@PLL is significant, and the adsorption efficiency gradually increases with the increase of the “material to DNA mass ratio”. When the mass ratio of material to DNA is 80, the cfDNA clearance rate is as high as 95%.

[0046] Figure 7 Figure of in vitro hydroxyl radical clearance of WRPOM@PLL nanoclusters and WRPOM nanoclusters obtained in the present embodiment. H2O2 and Fe 2+ generate hydroxyl radicals (•OH), •OH reacts with salicylic acid to generate 2,3-dihydroxybenzoic acid and 2,5-dihydroxybenzoic acid, which has a characteristic absorption peak at 510 nm, and the sample's ability to scavenge hydroxyl radicals can be evaluated by the degree of inhibition of the above reaction. The specific steps are as follows: first, prepare 9 mmol / L FeSO4 solution and 8.8 mmol / L H2O2 solution, mix them in a volume ratio of 1:1 to generate hydroxyl radicals for 10 min, then add different concentrations of WRPOM@PLL or WRPOM nanocluster aqueous dispersion to the system (the final concentration of nanoclusters in the system is 12.5, 25, 50, 100, 200, 400 μg / mL), react for 1 h, then add 9 mmol / L salicylic acid solution to develop color, and measure the wavelength at 540 nm. As can be seen from the figure, the clearance rate of •OH is positively correlated with the concentration of nanoclusters, and the clearance rate gradually increases with the increase of concentration, and the clearance effect of WRPOM@PLL is significantly higher than that of WRPOM, when the concentration of WRPOM@PLL is 400 μg / mL, the clearance rate of •OH reaches 70%.

[0047] Figure 8Figure 6 shows the DPPH radical scavenging effect of the WRPOM@PLL nanoclusters and WRPOM nanoclusters obtained in this example. DPPH is a stable nitrogen-centered free radical that can exist stably in organic solvents. Its alcohol solution is purple, has a single electron, and can accept an electron or hydrogen ion. It has a maximum absorption at a wavelength of 517 nm. When a free radical scavenger is present, the single electron of DPPH is captured, causing the color to become lighter, and the absorbance at the maximum absorption wavelength decreases in a linear relationship. The decrease in absorbance level indicates an increase in antioxidant activity, thereby evaluating the antioxidant capacity of the test sample. The effect of DPPH scavenging is detected by detecting the ultraviolet absorption peak at 519 nm after the material is incubated with DPPH. The specific experimental steps are as follows: 1.15 mg of DPPH was dissolved in 5 mL of absolute ethanol and ultrasonicated to completely dissolve. 100 μL of DPPH solution was added to each well of a 96-well plate, and 100 μL of water dispersion of WRPOM@PLL or WRPOM nanoclusters at different concentrations (the final concentration of nanoclusters in the system was 12.5, 25, 50, 100, and 200 μg / mL, respectively) was added. The "DPPH + water" was set as a positive control. After incubation for 30 min, the ultraviolet absorption at 519 nm was measured. As can be seen from the figure, the DPPH scavenging rate is positively correlated with the concentration of nanoclusters, and the scavenging effect of WRPOM@PLL is significantly higher than that of WRPOM. When the concentration of WRPOM@PLL is 200 μg / mL, the DPPH scavenging rate reaches 70%.

[0048] Figure 9 Figure 7 shows the ABTS radical scavenging effect of the WRPOM@PLL nanoclusters and WRPOM nanoclusters obtained in this example. ABTS generates stable blue-green cationic free radicals ABTS ·+ under the action of oxidants, which have a maximum absorbance at 734 nm. When an antioxidant is added, ABTS ·+ is reduced to colorless ABTS, causing the solution color to become lighter and the absorbance to decrease. By comparing the absorbance change before and after the addition of the antioxidant, the ABTS free radical scavenging rate of the sample can be calculated. The specific experimental steps are as follows: 4.5 mg of ABTS was weighed and dissolved in 1.2 mL of ultrapure water and shaken well by a vortex mixer until completely dissolved. 1.5 mg of potassium persulfate was dissolved in 2.2 mL of ultrapure water and shaken well. The two solutions were mixed and reacted at room temperature for 12 hours in the dark to generate stable ABTS ·+ free radicals. After the reaction was completed, the working solution was diluted by 30-40 times, and the A 734The ABTS working solution was added with water dispersions of WRPOM@PLL or WRPOM nanoclusters at different concentrations (the final concentrations of the nanoclusters in the system were 6.25, 12.5, 25, 50, and 100, respectively), and "ABTS + water" was set as a positive control, and incubated for 30 min. Finally, centrifugation was performed at 15000 rpm for 10 min, and the supernatant was taken to measure the ultraviolet absorption at 734 nm. As can be seen from the figure, the clearance rate of ABTS is positively correlated with the concentration of nanoclusters, and the clearance effect of WRPOM@PLL is significantly higher than that of WRPOM. When the concentration of WRPOM@PLL is 100 μg / mL, the clearance rate of DPPH reaches 90%.

[0049] Figure 10 The hemolysis experiment graph of WRPOM@PLL nanoclusters prepared for Example 1. The principle of hemolysis test is that when biomaterials are incubated with blood, the presence of hemolytic components can cause red blood cell destruction and hemoglobin release. After incubation, by measuring the OD value of the supernatant at 540 nm, it can be judged whether hemolysis occurs. The specific steps are as follows: take a small amount of fresh blood and dilute it 10 times in 0.9% NaCl solution, centrifuge at 3000 r / min for 10 min, discard the supernatant, and wash the precipitate with 0.9% NaCl according to the above method for 3-4 times until the supernatant is colorless. Mix different concentrations of samples and blood and incubate for 4 h, and set up positive and negative controls. Then centrifuge at 3000 r / min for 10 min, and measure the OD value at 540 nm. Calculate the hemolysis rate. As can be seen from the figure, the hemolysis rate of WRPOM@PLL nanoclusters at different concentrations is less than 1%, indicating that the material has good biocompatibility.

[0050] Figure 11MTT assay graph of WRPOM@PLL nanoclusters prepared in Example 1. MTT stands for 3-(4,5-dimethylthiazol-2)-2,5-diphenyltetrazolium bromide. MTT colorimetric method is a method for detecting cell survival and growth. Its detection principle is that succinate dehydrogenase in the mitochondria of living cells can reduce exogenous MTT to water-insoluble blue-purple formazan and deposit in the cells, while dead cells have no such function. Dimethyl sulfoxide (DMSO) can dissolve formazan in cells, and its light absorption value is measured at 490 nm wavelength by an enzyme-labeled instrument, which can indirectly reflect the number of living cells. The specific steps are as follows: after AML12 cells are inoculated in a 96-well plate, the cells are grown to 70%, and then different concentrations of water dispersion of WRPOM@PLL nanoclusters are added, incubated for 24 h, and then MTT dye is added, incubated for 4 h, and then DMSO is added, and the OD value is measured at 490 nm, and the cell viability is calculated. The results show that different concentrations of W-POM@PLL nanoclusters all show good biocompatibility and low toxicity, and even if the concentration of nanoclusters is as high as 200 µg / mL, the relative viability of cells still maintains above 80%. This experimental result shows that the nanoclusters themselves do not produce significant toxic side effects on cells.

[0051] Figure 12 DCFH-DA cell reactive oxygen species staining graph of WRPOM@PLL nanoclusters prepared in Example 1 and WRPOM nanoclusters. DCFH-DA stands for 2'-7'-dichlorodihydrofluorescein diacetate, which is one of the most widely used intracellular reactive oxygen species detection probes at present. Its principle is that after cells uptake DCFH-DA, DCFH-DA is deacetylated to a non-fluorescent compound by cell esterase, and then is oxidized to 2'-7'dichlorofluorescein (DCF) by RONS. DCF is a fluorescent compound that can be detected by fluorescence microscopy. The specific experimental steps are as follows: the following different groups of materials are prepared with serum-free 1640 medium: Control (fresh culture medium); 1 mM H2O2; 200 μg / mL WRPOM@PLL; 1 mM H2O2+200 μg / mL WRPOM@PLL; 4 μg / mL NAC (clinical drug N-acetylcysteine); 1 mM H2O2+4 μg / mL NAC;

[0052] 200 μg / mL WRPOM, 1 mM H2O2 + 200 μg / mL WRPOM. To verify the effect of WRPOM@PLL nanoclusters on the clearance of intracellular reactive oxygen species, 200 μL of AML12 cells were inoculated in each well of a 24-well plate and incubated for 24 h. The supernatant was aspirated, 100 μL of the above prepared solution was added to each well, and incubation was performed for 4 h. After incubation, the supernatant was aspirated, washed with PBS three times, 10 μM of DCFH-DA dyeing solution prepared with PBS was added to each well, and incubation was performed at 37°C in the dark for 30 min. The cells were washed with PBS three times, and fluorescence microscopy was performed. As can be seen from the figure, the cells in the 1 mM H2O2 oxidative stress model showed strong green fluorescence, indicating that ROS accumulated in large amounts. After the addition of 200 μg / mL WRPOM@PLL nanoclusters, the green fluorescence almost disappeared, indicating that WRPOM@PLL eliminated the intracellular reactive oxygen species and thus weakened the green fluorescence. The clearance effect was significantly higher than that of NAC and WRPOM, verifying that WRPOM@PLL nanoclusters can efficiently clear excess RONS in cells.

[0053] Figure 13 Cell AM / PI live and dead staining diagram of WRPOM@PLL nanoclusters prepared for Example 1 and WRPOM nanoclusters. The specific experimental steps are as follows: the following different groups of materials were prepared with serum-free 1640 medium: Control (fresh culture medium); 1 mM H2O2; 200 μg / mL WRPOM@PLL; 1 mM H2O2 + 200 μg / mL WRPOM@PLL; 200 μg / mL WRPOM; 1 mM H2O2 + 200 μg / mL WRPOM. After the AML12 cells were incubated for 24 h, the supernatant was aspirated, washed once with PBS, and the above prepared solution was sequentially added to the wells, and incubation was performed for 24 h. After incubation, the culture solution was aspirated, the cells were washed with PBS once, 200 μL of dye (7.5 μL of Calcein AM (calcein acetoxy methyl) and 5 μL of PI (propyl iodide) were mixed together, and then 5 mL of PBS was added) was added, and incubation was performed at 37°C in the dark for 20 min. Fluorescence microscopy was performed. As can be seen from the figure, compared with 1 mM H2O2, the red fluorescence of the 1 mM H2O2 + 200 μg / mL WRPOM@PLL group was almost invisible, and the proportion of cell death was greatly reduced, indicating that WRPOM@PLL nanoclusters can effectively protect cells from attack by reactive oxygen species, thereby reducing cell death.

[0054] Figure 14Distribution diagram of WRPOM@PLL nanoclusters prepared in Example 1 in mice at different time points. The specific experimental steps are as follows: 100 μL of 0.2 mg / mL Cy5.5 dye was mixed with 5 mg of WRPOM@PLL in the dark for 12 hours, dialyzed (3500 Da) for 72 hours, freeze-dried, and synthesized Cy5.5-labeled WRPOM@PLL, denoted as WRPOM@PLL@Cy5.5. After fasting for 16 h, C57 mice were intraperitoneally injected with acetaminophen (APAP) (300 mg / kg) to establish an AILI model, and healthy mice were used as a control group. WRPOM@PLL@Cy5.5 was administered to AILI and healthy mice by intravenous injection. The heart, liver, spleen, lung, and kidney were collected at 2, 6, 12, and 24 h, and the fluorescence signal was observed under dark conditions. Figure 14 Figure (a) in FIG. 1 is a distribution diagram of WRPOM@PLL@Cy5.5 in control and AILI model mice at different time periods; Figure 14 Figure (b) in FIG. 1 is a fluorescence intensity diagram of the heart, liver, spleen, lung, and kidney of the control group at different time periods; Figure 14 Figure (c) in FIG. 1 is a fluorescence intensity diagram of the heart, liver, spleen, lung, and kidney of the AILI model group at different time periods. As can be seen from the figure, compared with the control group, WRPOM@PLL nanoclusters are more obviously enriched in the liver of AILI mice, indicating that WRPOM@PLL nanoclusters have a therapeutic effect of targeted enrichment in liver injury mice, providing a basis for the pharmacokinetics of its treatment of acute liver failure.

[0055] Figure 15 Statistical diagram of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) of WRPOM@PLL prepared in Example 1 for treatment of APAP-induced acute liver injury in mice. ALT and AST are important indicators of liver function. When the levels of AST and ALT in the blood increase, it indicates that the liver is damaged. The specific experimental steps are as follows: healthy mice were randomly divided into 6 groups (n = 5):

[0056] Control group (Control): PBS + water;

[0057] Model group (APAP): PBS + 300 mg / kg APAP;

[0058] Positive drug group (NAC): 300 mg / kg NAC + 300 mg / kg APAP;

[0059] WRPOM@PLL group: 5 mg / kg WRPOM@PLL + 300 mg / kg APAP;

[0060] WRPOM group: 5 mg / kg WRPOM + 300 mg / kg APAP;

[0061] Rutin group: 5 mg / kg rutin + 300 mg / kg APAP.

[0062] C57 mice were fasted for 16 hours before an AILI model was established by intraperitoneal injection of APAP (300 mg / kg). Three hours later, APAP was administered via tail vein. Twenty-four hours after APAP treatment, the mice were euthanized, and blood was collected. After standing at room temperature for 2 hours, serum was collected by centrifugation at 3000 rpm for 20 minutes. The levels of ALT and AST in the serum were measured. Figure 15 (a) in the figure is a graph showing the serum alanine aminotransferase (ALT) content; Figure 15 (b) shows the serum aspartate aminotransferase (AST) levels. As can be seen from the figure, compared to the control group, the serum ALT and AST levels in the model group mice were significantly increased, indicating successful modeling of liver injury. The positive control drug NAC can reduce ALT and AST to some extent, but the effect is limited. Mice treated with 5 mg / kg WRPOM@PLL had significantly lower ALT and AST levels than the APAP group, demonstrating that WRPOM@PLL has excellent therapeutic effects on APAP-induced acute liver injury.

[0063] Figure 16 The survival rate of mice with APAP-induced acute liver failure treated with WRPOM@PLL nanoclusters prepared in Example 1 is shown in the figure. The specific experimental steps were as follows: Healthy mice were randomly divided into 4 groups (n=6):

[0064] Model group (APAP): PBS + 600 mg / kg APAP;

[0065] Positive drug group (NAC): 300 mg / kg NAC + 600 mg / kg APAP;

[0066] WRPOM@PLL group: 5mg / kgWRPOM@PLL+600mg / kg APAP;

[0067] WRPOM group: 5mg / kgWRPOM+600mg / kg APAP.

[0068] C57 mice were fasted for 16 hours before a mouse model of liver failure was established by intraperitoneal injection of a high dose of APAP (600 mg / kg), followed by tail vein administration 3 hours later. Mice survival was recorded every 8 hours for 192 hours. The figures show that all mice in the model group died within 160 hours, indicating that high-dose APAP-induced liver failure is extremely lethal. Compared to the model group, the survival rate of mice in the 5 mg / kg WRPOM@PLL group was significantly higher (survival rate > 75%), and it showed better therapeutic efficacy than the first-line clinical treatment drug NAC.

[0069] The above merely describes preferred embodiments of the present application, and is not used to limit the present application, any modification, equivalent replacement and improvement within the spirit and principle of the present application should be included in the protection scope of the present application.

Claims

1. A method for preparing polylysine-modified tungsten-based heteropolyacid nanoclusters, characterized in that: In an alkaline environment provided by sodium carbonate solution, rutin and phosphotungstic acid hydrate are added sequentially and stirred for 1-6 hours to obtain a reaction solution. Then, polylysine is added to the reaction solution, stirred for 1-6 hours, sonicated for 1-3 hours, dialyzed for 24-72 hours, and freeze-dried to obtain polylysine-modified tungsten-based heteropolyacid nanoclusters. The polylysine-modified tungsten-based heteropolyacid nanoclusters are composed of tungsten-based heteropolyacid nanoclusters as the core, with positively charged polylysine modified on the surface of the tungsten-based heteropolyacid nanoclusters.

2. The preparation method according to claim 1, characterized in that: The mass ratio of rutin, phosphotungstic acid hydrate, and polylysine is 10~150mg:10~80mg:10~80mg.

3. The preparation method according to claim 1, characterized in that: The stirring speed is 80~1000 rpm, and the ultrasonic power is 80~600W.

4. The preparation method according to claim 1, characterized in that: The diameter of the polylysine-modified tungsten-based heteropolyacid nanoclusters is 20~200 nm.

5. The preparation method according to claim 1, characterized in that: The polylysine-modified tungsten-based heteropolyacid nanoclusters have a positively charged surface.

6. The application of the polylysine-modified tungsten-based heteropolyacid nanoclusters prepared by the preparation method according to any one of claims 1 to 5, characterized in that: Nanomedicines for the preparation of acetaminophen-induced acute liver failure.

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