Polylysine-modified tungsten-based heteropolyacid nano-cluster and application thereof

Abstract

The invention belongs to the crossing field of nano material preparation and biological medicine, and discloses a polylysine modified tungsten-based heteropolyacid nano-cluster and application thereof. A tungsten-based heteropolyacid nano-cluster is synthesized by simulating a folin phenol detection method, rutin is used as an antioxidant to wrap the surface of the tungsten-based heteropolyacid nano-cluster, and electropositive polylysine is further modified on the surface of the tungsten-based heteropolyacid nano-cluster. The nano-cluster disclosed by the invention has good stability and biocompatibility, can be used for treating acetaminophen-induced acute hepatic failure, and can be used for specifically removing active oxygen at a hepatic failure part in a targeted manner through oxidation reduction and specifically removing extracellular free DNA (Deoxyribonucleic Acid) through an electrostatic adsorption effect, so that inflammatory response is inhibited; the symptoms caused by acute hepatic failure are effectively relieved.

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] Currently, 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: 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) as the core, and the surface of the tungsten-based heteropoly acid nanocluster is modified with positively charged polylysine (PLL). The preparation method comprises the following steps: Under the alkaline environment provided by the 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 the stirring reaction is continued for 1-6h, ultrasonic treatment is performed for 1-3h, dialysis is performed for 24-72h, and freeze-drying is performed to obtain the polylysine-modified tungsten-based heteropoly acid nanocluster. The diameter of the polylysine-modified tungsten-based heteropoly acid nanocluster prepared by the present application is 20-200nm, and the surface is positively charged. It can target enrichment at the liver failure site through the nanometer size effect.

[0010] As a preferred, the mass ratio of rutin, phosphotungstic acid hydrate and polylysine is 10-150mg:10-80mg:10-80mg.

[0011] As a preferred, the stirring speed is 80-1000rpm, and the ultrasonic power is 80-600W.

[0012] 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; positively charged surface, specific binding to cfDNA, thereby reducing oxidative stress, protecting cells and tissues from oxidative damage, and achieving the effect of treating acetaminophen-induced acute liver failure.

[0013] The beneficial effects of the present application are reflected in: 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).

[0014] 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.

[0015] 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 at the same time improves the narrow treatment window period in NAC treatment.

[0016] 4、 The polylysine and tungsten-based heteropoly acid nanocluster are efficiently compounded by 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

[0017] Figure 1 The synthesis route of the polylysine-modified tungsten-based heteropoly acid nanocluster of the application is shown in the figure.

[0018] 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.

[0019] Figure 3 The particle size graph of the polylysine-modified tungsten-based heteropoly acid nanocluster prepared in Example 1 is shown in the figure.

[0020] Figure 4Zeta potential graph of polylysine-modified tungsten-based heteropoly acid nanoclusters prepared for Example 1 and unmodified tungsten-based heteropoly acid nanoclusters.

[0021] Figure 5 UV graph of polylysine-modified tungsten-based heteropoly acid nanoclusters prepared for Example 1.

[0022] Figure 6 In vitro adsorption cfDNA effect graph of tungsten-based heteropoly acid nanoclusters modified with polylysine prepared for Example 1.

[0023] Figure 7 In vitro hydroxyl radical scavenging graph of tungsten-based heteropoly acid nanoclusters modified with polylysine and unmodified polylysine prepared for Example 1.

[0024] Figure 8 In vitro DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging graph of tungsten-based heteropoly acid nanoclusters modified with polylysine and unmodified polylysine prepared for Example 1.

[0025] Figure 9 In vitro ABTS (2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid) radical scavenging graph of tungsten-based heteropoly acid nanoclusters modified with polylysine and unmodified polylysine prepared for Example 1.

[0026] Figure 10 Hemolysis experiment graph of polylysine-modified tungsten-based heteropoly acid nanoclusters prepared for Example 1.

[0027] Figure 11 MTT experiment graph of polylysine-modified tungsten-based heteropoly acid nanoclusters prepared for Example 1.

[0028] Figure 12 Cellular reactive oxygen species staining graph of polylysine-modified tungsten-based heteropoly acid nanoclusters prepared for Example 1.

[0029] Figure 13 Cellular AM (calcium acetoxymethyl succinate) / PI (propidium iodide) live and dead staining graph of tungsten-based heteropoly acid nanoclusters modified with polylysine and unmodified polylysine prepared for Example 1.

[0030] Figure 14 Distribution graph of polylysine-modified tungsten-based heteropoly acid nanoclusters prepared for Example 1 at different time points in mice, wherein: Figure 14 the (a) graph in is the in vivo distribution graph of WRPOM@PLL@Cy5.5 at different time periods in control group and AILI model group mice; Figure 14 the (b) graph in is the fluorescence intensity graph of heart, liver, spleen, lung and kidney of control group mice at different time periods; Figure 14The (c) figure in (b) is the fluorescence intensity diagram of the heart, liver, spleen, lung and kidney of the AILI model group at different time periods.

[0031] Figure 15 The graph of the survival rate of the acute liver failure mice induced by APAP treated by the polylysine modified tungsten-based heteropoly acid nanocluster prepared in Example 1. Figure 15 The (a) graph in (b) is the graph of alanine aminotransferase (ALT) of the acute liver failure mice induced by APAP treated by the polylysine modified tungsten-based heteropoly acid nanocluster prepared in Example 1. Figure 15 The (b) graph in (b) is the graph of aspartate aminotransferase (AST) of the acute liver failure mice induced by APAP treated by the polylysine modified tungsten-based heteropoly acid nanocluster prepared in Example 1.

[0032] Figure 16 The graph of the survival rate of the acute liver failure mice induced by APAP treated by the polylysine modified tungsten-based heteropoly acid nanocluster prepared in Example 1. DETAILED DESCRIPTION

[0033] In order to make the above-mentioned purposes, features and advantages of the present application more obvious and easy to understand, the specific embodiments of the present application will be described in detail below. The following content is only an example and description of the concept of the present application, and various modifications or supplements or similar replacements of the described specific embodiments can be made by those skilled in the art without deviating from the concept of the present application, which should belong to the protection scope of the present application.

[0034] Example 1 The polylysine modified tungsten-based heteropoly acid nanocluster in this embodiment is prepared by the following method: First, 9 mL of 25 mg / mL sodium carbonate solution is prepared, and then 100 mg of rutin is dissolved. After the solution becomes clear and transparent, 1 mL of 60 mg / mL phosphotungstic acid hydrate aqueous solution is added to the solution, and the reaction is continuously stirred at a speed of 350 rpm for 3 h. Then, 70 mg of polylysine is added, and the solution is continuously stirred at a speed of 350 rpm for 2 h. Then, the solution is ultrasonically treated at a power of 250 W for 2 h. Finally, the solution is dialyzed (MW=3500 Da) with pure water for 72 h, and then freeze-dried to obtain the polylysine modified tungsten-based heteropoly acid nanocluster, which is denoted as WRPOM@PLL and stored at 4°C.

[0035] For comparison, the tungsten-based heteropoly acid nanocluster without polylysine modification is also prepared in this embodiment by the following method: 9 mL of 25 mg / mL sodium carbonate solution is prepared, and then 100 mg of rutin is dissolved. After the solution becomes clear and transparent, 1 mL of 60 mg / mL phosphotungstic acid hydrate aqueous solution is added to the solution, and the reaction is continuously stirred for 3 h. Then, the solution is dialyzed (MW=3500 Da) with pure water for 72 h, and then freeze-dried to obtain the tungsten-based heteropoly acid nanocluster without polylysine modification, which is denoted as WRPOM and stored at 4°C.

[0036] 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.

[0037] 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 thereof 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.

[0038] 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.

[0039] 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 attributed to the electronic relaxation polarization caused by the charge transfer between the reduced W 5+ and oxidized W 6+ through the bridging oxygen bond, and the coloring effect in tungsten oxide and phosphate containing W 5+ .

[0040] Figure 6The figure shows the in vitro cfDNA adsorption effect of WRPOM@PLL nanoclusters obtained in this 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, and 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%.

[0041] Figure 7 The figure shows the in vitro hydroxyl radical clearance effect of WRPOM@PLL nanoclusters and WRPOM nanoclusters obtained in this embodiment. H2O2 and Fe 2+ Hydroxyl radicals (·OH) are generated, which react with salicylic acid to generate 2,3-dihydroxybenzoic acid and 2,5-dihydroxybenzoic acid, which have a characteristic absorption peak at 510 nm. 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), and react for 1 h. After the reaction is complete, add 9 mmol / L salicylic acid solution to develop the 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. The clearance effect of WRPOM@PLL is significantly higher than that of WRPOM, and when the concentration of WRPOM@PLL is 400 μg / mL, the clearance rate of ·OH reaches 70%.

[0042] 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%.

[0043] 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 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 734In the range of 0.7-0.8. Add different concentrations of WRPOM@PLL or WRPOM nanoclusters aqueous dispersion (the final concentration of nanoclusters in the system is 6.25, 12.5, 25, 50, 100 respectively) to ABTS working solution, set "ABTS + water" as positive control, incubate for 30 min. Finally, centrifuge at 15000 rpm for 10 min, take the supernatant 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%.

[0044] 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 sediment 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, take the supernatant and measure the OD value at 540 nm. Calculate the hemolysis rate. As can be seen from the figure, the hemolysis rate of different concentrations of WRPOM@PLL nanoclusters is less than 1%, indicating that the material has good biocompatibility.

[0045] 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 do not have this 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 OD value is measured at 490 nm, and 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 nanoclusters themselves do not produce significant toxic side effects on cells.

[0046] 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 configured 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; 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.

[0047] Figure 13 Cell AM / PI live and dead staining diagram of WRPOM@PLL nanoclusters prepared in 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.

[0048] 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 mice 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 mice 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.

[0049] 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 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): Control group (Control): PBS + water; Model group (APAP): PBS + 300 mg / kg APAP; Positive drug group (NAC): 300 mg / kg NAC + 300 mg / kg APAP; WRPOM@PLL group: 5 mg / kg WRPOM@PLL + 300 mg / kg APAP; WRPOM group: 5 mg / kg WRPOM + 300 mg / kg APAP; Rutin group (Rutin): 5 mg / kg Rutin + 300 mg / kg APAP.

[0050] C57 mice were fasted for 16 h, then APAP (300 mg / kg) was injected intraperitoneally to establish the AILI model, and 3 h later, tail vein administration was performed. After 24 h of APAP treatment, the mice were euthanized, blood was collected, and after standing at room temperature for 2 h, the serum was collected by centrifugation at 3000 rpm for 20 min. The contents of ALT and AST in the serum were measured, Figure 15 (a) in FIG. 1 is a graph of the content of alanine aminotransferase (ALT) in serum; Figure 15 (b) in FIG. 1 is a graph of the content of aspartate aminotransferase (AST) in serum. As can be seen from the figure, compared with the control group, the serum ALT and AST of the model group mice increased significantly, indicating that the liver injury modeling was successful, the positive drug NAC could reduce ALT and AST to a certain extent, but the effect was limited, the ALT and AST contents of the mice treated with 5 mg / kg WRPOM@PLL were significantly lower than those of the APAP group, proving that WRPOM@PLL had excellent therapeutic effect on APAP-induced acute liver injury.

[0051] Figure 16 The survival rate graph of WRPOM@PLL nanoclusters prepared in Example 1 for the treatment of APAP-induced acute liver failure mice. The specific experimental steps are as follows: healthy mice were randomly divided into 4 groups (n = 6): Model group (APAP): PBS + 600 mg / kg APAP; Positive drug group (NAC): 300 mg / kg NAC + 600 mg / kg APAP; WRPOM@PLL group: 5 mg / kg WRPOM@PLL + 600 mg / kg APAP; WRPOM group: 5 mg / kg WRPOM + 600 mg / kg APAP.

[0052] C57 mice were fasted for 16 h, then APAP (300 mg / kg) was injected intraperitoneally to establish the AILI model, and 3 h later, tail vein administration was performed. After 24 h of APAP treatment, the mice were euthanized, blood was collected, and after standing at room temperature for 2 h, the serum was collected by centrifugation at 3000 rpm for 20 min. The contents of ALT and AST in the serum were measured,

[0053] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement and improvement made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A polylysine-modified tungsten-based heteropolyacid nanocluster, characterized in that: The polylysine-modified tungsten-based heteropolyacid nanoclusters have tungsten-based heteropolyacid nanoclusters as the core, and positively charged polylysine is modified on the surface of the tungsten-based heteropolyacid nanoclusters.

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

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

4. A method for preparing polylysine-modified tungsten-based heteropolyacid nanoclusters according to any one of claims 1 to 3, 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.

5. The preparation method according to claim 4, characterized in that: The mass ratio of rutin, phosphotungstic acid hydrate, and polylysine is 10~150mg:10~80mg:10~80mg.

6. The preparation method according to claim 4, characterized in that: The stirring speed is 80-1000 rpm, and the ultrasonic power is 80-600W.

7. The application of the polylysine-modified tungsten-based heteropolyacid nanoclusters according to any one of claims 1 to 3, characterized in that: Nanomedicines for the preparation of acetaminophen-induced acute liver failure.

Images