Manganese dioxide coated arsenic nanosheet material, preparation method thereof and application of manganese dioxide coated arsenic nanosheet material in preparation of medicine for treating hepatocellular carcinoma

Abstract

The invention belongs to the technical field of medicines for treating hepatocellular carcinoma, and particularly discloses a manganese dioxide coated arsenic nanosheet material, a preparation method thereof and application of the manganese dioxide coated arsenic nanosheet material in preparation of medicines for treating hepatocellular carcinoma. The surface of an arsenic nanosheet is coated with a layer of manganese dioxide through an in-situ self-sacrifice reduction method, and the method is simple and convenient to operate; the obtained manganese dioxide coated arsenic nanosheet has good stability and dispersibility; the manganese dioxide coated arsenic nanosheet is controlled to be converted by glutathione and hydrogen peroxide in a tumor microenvironment, so that the leakage of As2O3 is avoided, the biological safety is improved, and clinical use is facilitated; compared with a single arsenic nanosheet, the active oxygen generation and glutathione consumption capacities are further improved, and the liver cancer cell killing capacity is enhanced; meanwhile, the capability of degrading Pin1 in hepatocellular carcinoma cells is achieved, and heterogeneity of hepatocellular carcinoma can be overcome to achieve treatment.

Description

Technical Field

[0001] The invention relates to the technical field of drugs for treating hepatocellular carcinoma, in particular to a manganese dioxide-coated arsenic nanosheet material and a preparation method thereof and application thereof in preparing drugs for treating hepatocellular carcinoma. Background Art

[0002] Hepatocellular carcinoma (HCC) is one of the most common malignant tumors in the world, and its morbidity and mortality rates are increasing year by year. Due to the occult nature and rapid progression of HCC, most patients are not discovered until the middle or late stages, which places a huge burden on the diagnosis and treatment of liver cancer. Although there are currently a variety of treatment options, such as surgical resection, liver transplantation, radiofrequency ablation, and drug therapy, the therapeutic effects of these methods are still unsatisfactory, with a 5-year survival rate of less than 20%. In addition, the various inducing factors of HCC (such as obesity, drinking, and viral infection) lead to a high degree of heterogeneity in genetic composition and phenotype, making clinical treatment drugs for HCC scarce and ineffective. Therefore, how to overcome the high heterogeneity to achieve effective treatment of hepatocellular carcinoma (HCC) is a key problem that needs to be solved urgently.

[0003] Hydroxyproline isomerase (Pin1) is mainly expressed in the cell nucleus and changes the structure and function of the substrate by recognizing and catalyzing two domains. Multiple studies have shown that Pin1 is overexpressed in more than 50% of HCC tissues and causes poor clinical prognosis by activating more than 60 oncoproteins and inhibiting more than 30 tumor suppressors. Therefore, the strategy of inhibiting Pin1 to achieve HCC treatment has attracted much attention. Researchers have found that arsenic trioxide (As2O3), a drug with a long history, can degrade Pin1 by non-covalent binding and apply it to HCC treatment. Although it has achieved certain results, the drug has defects such as significant toxic side effects and poor in vivo stability, which limits its further clinical translation application. Recently, gray arsenic composed of a single arsenic element was found to have a layered buckled honeycomb structure and chemical properties similar to black phosphorus. They have special optical properties and unique ability to induce tumor cell apoptosis. More importantly, compared with As2O3, single element arsenic significantly improves its biocompatibility and does not produce toxicity in normal cells. Under the action of high concentration of hydrogen peroxide in the tumor microenvironment, low-toxic arsenic is converted into highly toxic As2O3, which kills liver cancer cells. Although the selective killing property is of great significance for the safe anti-tumor application of gray arsenic, it is difficult to achieve complex in vivo delivery and effective anti-tumor effects by relying solely on gray arsenic. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides a manganese dioxide-coated arsenic nanosheet material and a preparation method thereof and an application of the manganese dioxide-coated arsenic nanosheet material in preparing a drug for treating hepatocellular carcinoma.

[0005] To achieve the above object, the present invention is implemented according to the following technical solutions:

[0006] One of the purposes of the present invention is to provide a method for preparing a manganese dioxide-coated arsenic nanosheet material, comprising the following steps:

[0007] S1. Take PEG-NH2 and gray arsenic powder in a mass ratio of 2:1, dissolve PEG-NH2 in ultrapure water, add gray arsenic powder with a particle size of 0.5-5 μm, and mix well; precool the obtained mixed solution with liquid nitrogen, perform water bath ultrasound, and perform ice water bath ultrasound for 30 minutes each, and repeat several times; centrifuge the mixed solution, wash with water, and freeze-dry to obtain arsenic nanosheet powder;

[0008] S2. Take arsenic nanosheet powder and sodium hydroxide solution in a mass-to-volume ratio of 1 mg:1 mL, dissolve the arsenic nanosheet powder in the sodium hydroxide solution, then add KMnO4 which is one-fourth the mass of the arsenic nanosheet powder, stir at room temperature for 1-2 hours, and then remove the unreacted products by centrifugation and water washing; add the mixed solution to a PEG-NH2 solution of equal volume to the sodium hydroxide solution, and stir for 8-10 hours; after the reaction is completed, centrifuge, dialyze, and freeze-dry to obtain manganese dioxide-coated arsenic nanosheet powder.

[0009] Furthermore, in step S2, the concentration of the sodium hydroxide solution is 0.001-0.01M.

[0010] Furthermore, in step S2, the concentration of the PEG-NH2 solution is 2 mg / mL.

[0011] The second purpose of the present invention is to provide a manganese dioxide coated arsenic nanosheet material prepared by the above method, wherein the manganese dioxide coated arsenic nanosheet material is a sheet-like planar structure with a diameter of 190-230nm and a thickness of 7-10nm. The valence state of the core arsenic nanosheet of the manganese dioxide coated arsenic nanosheet material is 0, and the valence state of the coated manganese dioxide on the surface is +4.

[0012] The third purpose of the present invention is to provide a manganese dioxide coated arsenic nanosheet material for use in the preparation of drugs for treating hepatocellular carcinoma, and its mechanism is: manganese dioxide coated arsenic nanosheets are used to degrade hydroxyproline isomerase to treat hepatocellular carcinoma, and the manganese dioxide coating has a good protective effect, avoiding the premature exposure of the arsenic nanosheet core during the body circulation process, and also enhancing the killing ability of liver cancer cells by consuming glutathione. The arsenic nanosheet core releases As2O3 under the action of hydrogen peroxide and thereby degrades Pin1 overexpressed in hepatocellular carcinoma. At the same time, the release of As2O3 can induce oxidative stress and apoptosis of cells, thereby achieving the purpose of treating hepatocellular carcinoma.

[0013] Compared with the prior art, the present invention has the following beneficial effects:

[0014] 1. The present invention coats a layer of manganese dioxide on the surface of arsenic nanosheets by an in-situ self-sacrificial reduction method. The method is simple and easy to operate. The obtained manganese dioxide-coated arsenic nanosheets have good stability and dispersibility.

[0015] 2. The manganese dioxide-coated arsenic nanosheets of the present invention are transformed under the control of glutathione and hydrogen peroxide in the tumor microenvironment, thereby avoiding the leakage of As2O3 and improving biosafety, which is beneficial for clinical use.

[0016] 3. Compared with the arsenic nanosheets alone, the manganese dioxide-coated arsenic nanosheets of the present invention further improve the ability to produce active oxygen and consume glutathione, thereby enhancing the ability to kill liver cancer cells.

[0017] 4. The manganese dioxide-coated arsenic nanosheets of the present invention have the ability to degrade Pin1 in liver cancer cells, which is beneficial for overcoming the heterogeneity of hepatocellular carcinoma and achieving treatment.

[0018] 5. The manganese dioxide-coated arsenic nanosheets of the present invention significantly inhibited tumor growth in a subcutaneous liver cancer model, showing a good therapeutic effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Schematic diagram of the synthesis of manganese dioxide-coated arsenic nanosheets.

[0020] Figure 2 This is a transmission electron microscopy image of the manganese dioxide-coated arsenic nanosheets prepared in Example 3.

[0021] Figure 3 This is an atomic force microscope image of the manganese dioxide-coated arsenic nanosheets prepared in Example 3.

[0022] Figure 4 This is the X-ray photoelectron spectrum of the manganese dioxide-coated arsenic nanosheets prepared in Example 3.

[0023] Figure 5 This is the in vitro simulated arsenic ion release graph of the manganese dioxide-coated arsenic nanosheets prepared in Example 3.

[0024] Figure 6 This is the cell active oxygen staining image of the manganese dioxide-coated arsenic nanosheets prepared in Example 3.

[0025] Figure 7 This is a graph of cellular glutathione consumption of manganese dioxide-coated arsenic nanosheets prepared in Example 3.

[0026] Figure 8 This is the Pin1 degradation diagram of cells coated with arsenic nanosheets coated with manganese dioxide prepared in Example 3.

[0027] Fig. 9This is a graph of cell viability after incubation of the manganese dioxide-coated arsenic nanosheets prepared in Example 3 with normal liver cells.

[0028] Fig.10 This is a graph showing cell viability after incubation of liver cancer cells with arsenic-coated manganese dioxide prepared in Example 3.

[0029] Fig.11 This is a diagram of the hemolysis experiment of the manganese dioxide-coated arsenic nanosheets prepared in Example 3.

[0030] Fig.12 This is a graph showing the changes in tumor volume in mice bearing liver cancer treated with manganese dioxide-coated arsenic nanosheets prepared in Example 3. DETAILED DESCRIPTION

[0031] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with embodiments. The specific embodiments described herein are only used to explain the present invention and are not used to limit the invention.

[0032] Unless otherwise specified, the raw materials and reagents used in the following examples were commercially available.

[0033] Example 1

[0034] Reference Figure 1 , preparation of manganese dioxide coated arsenic nanosheets:

[0035] (1) 80 mg of PEG-NH2 was dissolved in 50 mL of ultrapure water, and then 40 mg of gray arsenic powder with a particle size of 2 μm was added and mixed evenly; the resulting mixed solution was pre-cooled with liquid nitrogen, ultrasonicated in a water bath, and ultrasonicated in an ice water bath for 30 minutes each, and repeated six times; the mixed solution was centrifuged, washed with water, and freeze-dried to obtain arsenic nanosheet powder ANs;

[0036] (2) Dissolve 20 mg of arsenic nanosheet powder in 20 mL of 0.001 M sodium hydroxide solution, add 5 mg of KMnO4, stir at room temperature for 1 hour, and then remove the unreacted products by centrifugation and water washing; add the mixed solution into 30 mL of 2 mg / mL PEG-NH2 solution and stir for 10 hours; after the reaction is completed, centrifuge, dialyze, and freeze-dry to obtain manganese dioxide-coated arsenic nanosheet powder AMPNs.

[0037] Example 2

[0038] Reference Figure 1 , preparation of manganese dioxide coated arsenic nanosheets:

[0039] (1) 120 mg of PEG-NH2 was dissolved in 50 mL of ultrapure water, and then 60 mg of gray arsenic powder with a particle size of 0.5 μm was added and mixed evenly; the resulting mixed solution was pre-cooled with liquid nitrogen, ultrasonicated in a water bath, and ultrasonicated in an ice water bath for 30 minutes each, and repeated six times; the mixed solution was centrifuged, washed with water, and freeze-dried to obtain arsenic nanosheet powder ANs;

[0040] (2) 30 mg of arsenic nanosheet powder was dissolved in 30 mL of 0.001 M sodium hydroxide solution, and then 7.5 mg of KMnO4 was added. The mixture was stirred at room temperature for 1 hour, and then the unreacted products were removed by centrifugation and water washing. The mixture was added into 30 mL of 2 mg / mL PEG-NH2 solution and stirred for 10 hours. After the reaction was completed, the mixture was centrifuged, dialyzed, and freeze-dried to obtain manganese dioxide-coated arsenic nanosheet powder AMPNs.

[0041] Example 3

[0042] Reference Figure 1 , preparation of manganese dioxide coated arsenic nanosheets:

[0043] (1) 100 mg of PEG-NH2 was dissolved in 50 mL of ultrapure water, and then 50 mg of gray arsenic powder with a particle size of 5 μm was added and mixed evenly; the resulting mixed solution was pre-cooled with liquid nitrogen, ultrasonicated in a water bath, and ultrasonicated in an ice water bath for 30 minutes each, and repeated six times; the mixed solution was centrifuged, washed with water, and freeze-dried to obtain arsenic nanosheet powder ANs;

[0044] (2) Dissolve 20 mg of arsenic nanosheet powder in 20 mL of 0.001 M sodium hydroxide solution, add 5 mg of KMnO4, stir at room temperature for 1 hour, and then remove the unreacted products by centrifugation and water washing; add the mixed solution into 30 mL of 2 mg / mL PEG-NH2 solution and stir for 10 hours; after the reaction is completed, centrifuge, dialyze, and freeze-dry to obtain manganese dioxide-coated arsenic nanosheet powder AMPNs.

[0045] Taking the manganese dioxide-coated arsenic nanosheets prepared in Example 3 as an example, their physical properties were tested and characterized in turn. Figure 2 This is a transmission electron microscope image of the manganese dioxide-coated arsenic nanosheet obtained in Example 3. Figure 2 It can be seen that the nanosheet has a sheet-like planar structure with a diameter of 190 to 230 nm. Figure 3 This is an atomic force microscope image of the manganese dioxide-coated arsenic nanosheet obtained in Example 3. Figure 3 It can be seen that the thickness of the nanosheet is 7 to 10 nm. Figure 4 This is the X-ray photoelectron spectrum of the manganese dioxide coated arsenic nanosheets obtained in Example 3. Figure 4 It can be seen that the valence state of the arsenic element contained in the nanozyme is 0.

[0046] In order to verify the effect of the obtained manganese dioxide coated arsenic nanosheets on treating hepatocellular carcinoma, in vitro experiments and in vivo animal experiments were carried out using the manganese dioxide coated arsenic nanosheets prepared in Example 3:

[0047] 1. In vitro experiments

[0048] 1) 3 mL of manganese dioxide-coated arsenic nanosheet solution containing 500 μg of arsenic was placed in a dialysis bag with a molecular weight cutoff of 7000 Da, immersed in 27 mL of PBS buffer with or without glutathione for 6 h, and then transferred to a buffer with or without hydrogen peroxide. At predetermined time intervals, 3 mL of buffer was drawn and added for determination of arsenic ion concentration. Figure 5 The in vitro simulated arsenic ion release diagram of the manganese dioxide coated arsenic nanosheets obtained in Example 3 is shown in FIG. Figure 5 It can be seen that under the dual action of glutathione and hydrogen peroxide, the release of arsenic ions is greatly increased.

[0049] 2) In order to verify the effect of manganese dioxide-coated arsenic nanosheets on inducing the production of intracellular reactive oxygen species, 1×10 6 Hepa1-6 cells (i.e., mouse liver cancer cells, purchased from the Cell Bank of the Chinese Academy of Sciences) were cultured for 24 hours and co-incubated with cells for 12 hours according to the following groups: control group (fresh DMEM medium), ANs (5 μg / mL (concentration after dispersion in DMEM medium)), AMPNs (5 μg / mL (concentration after dispersion in DMEM medium)), ANs (10 μg / mL (concentration after dispersion in DMEM medium)), AMPNs (10 μg / mL (concentration after dispersion in DMEM medium)). After discarding the old medium and washing, the intracellular reactive oxygen species were stained with DCFH-DA dye and observed by fluorescence microscopy. Figure 6 This is the cell active oxygen staining image of the manganese dioxide coated arsenic nanosheet obtained in this example. Figure 6 It can be seen that the AMPNs (10 μg / mL) group showed the strongest green fluorescence, indicating that it has a good ability to induce the production of reactive oxygen species in cells. After incubation for 12 hours in the above experiment, Hepa1-6 cells were collected by washing, digestion and centrifugation. The cells were frozen in liquid nitrogen three times and thawed at 37 degrees, and the supernatant was collected to determine the GSH content. Figure 7 The cellular glutathione content of the manganese dioxide-coated arsenic nanosheets obtained in this example is shown in FIG. Figure 7 As can be seen, the relative content of GSH in the AMPNs (10 μg / mL) group was the lowest (15.3%), indicating its effective depletion of intracellular GSH.

[0050] 3) In order to verify the effect of manganese dioxide-coated arsenic nanosheets on degrading intracellular Pin1, 1×10 4 Hepa1-6 cells were cultured for 24 hours. The cells were incubated for 24 hours according to the following groups: control group (fresh culture medium), ANs (2.5 μg / mL), AMPNs (2.5 μg / mL), ANs (5 μg / mL), AMPNs (5 μg / mL). After discarding the old culture medium and washing, the cells were lysed and the protein was collected to detect the Pin1 content by enzyme-linked immunosorbent assay. Figure 8 Pin 1 is a picture of the degradation cell of arsenic nanosheets coated with manganese dioxide obtained in this example. Figure 8 It can be seen that the expression level of Pin1 decreased with the increase of arsenic content, and the lowest level was only 23.9% of the control group, which indicates that AMPNs effectively degrade Pin1.

[0051] 4) Inoculate 1×10 per well of a 96-well plate 4 AML12 cells (i.e. normal mouse liver cells, purchased from the cell bank of the Chinese Academy of Sciences) were added and incubated for 24 hours. After aspirating the supernatant, 200 μL DMEM was added to each well to prepare As2O3 (0, 0.5, 1, 3, 5, 10 μg / mL) and AMPNs (0, 5, 10, 15, 20, 30 μg / mL) and incubated for 24 hours. After the incubation, 20 μL CCK8 was added to each well, and the absorbance at 490 nm was measured after culturing for 2 hours. Fig. 9 This is a biocompatibility test diagram of the manganese dioxide coated arsenic nanosheets obtained in this example. Fig. 9 It can be seen that the cell viability was less than 30% after incubation with 10 μg / mL As2O3, while the cell viability in the AMPNs (30 μg / mL) group was above 85%, indicating the excellent biosafety of AMPNs.

[0052] 5) Inoculate 1×10 per well of a 96-well plate 4 After culturing Hepa1-6 cells for 24 hours, 200 μL of AMPNs dispersed in DMEM (AMPNs concentrations were 0, 1, 2, 5, 10, 15, 20 μg / mL) were added to each well. After incubation for another 24 hours, 20 μL of CCK8 was added to each well, and the absorbance at 490 nm was measured after culturing for 2 hours. Fig.10 This is a cell viability diagram of the manganese dioxide-coated arsenic nanosheets obtained in this example after incubation with liver cancer cells. Fig.10 The results showed that the viability of Hepa1-6 cells decreased with the increase of concentration, indicating that AMPNs have a good killing ability on liver cancer cells.

[0053] 6) The AMPNs dispersion was diluted with physiological saline to 5 μg / mL, 10 μg / mL, 15 μg / mL, 20 μg / mL, 30 μg / mL and 40 μg / mL, and 0.8 mL of the above solution and 0.2 mL of treated blood (500 μL of fresh blood was added to 4.5 mL of physiological saline and centrifuged for 5 to 8 times at a centrifugal speed of 3000 rpm for 10 min until the blood supernatant was clear and transparent, then the supernatant was discarded and the volume was fixed to 5 mL with physiological saline) were mixed and incubated at 37°C for 4 h, and then centrifuged at 3000 rpm for 10 min. The supernatant was aspirated to measure the absorbance at 541 nm and the hemolysis rate was calculated. Fig.11 This is a hemolysis experiment diagram of the manganese dioxide coated arsenic nanosheet obtained in this example. Fig.11 It can be seen that the hemolysis rate of manganese dioxide coated arsenic nanosheets is less than 5%, indicating that the material has good biocompatibility.

[0054] 2. Animal in vivo experiments

[0055] In order to verify the ability of the manganese dioxide-coated arsenic nanosheets obtained in this example to treat hepatocellular carcinoma, the following tests were performed:

[0056] Fifteen 6- to 8-week-old female C57BL / 6 mice (purchased from the Experimental Animal Center of Sun Yat-sen University) were subcutaneously inoculated with 1×10 6 Hepa1-6 cells were used to establish a Hepa1-6 tumor-bearing mouse model; when the mouse tumor reached 50 mm 3 Mice were randomly divided into 3 groups and intravenously injected with (1) PBS, (2) 5 mg / kg ANs (1 mg / mL, diluted with saline), and (3) 5 mg / kg AMPNs (1 mg / mL, diluted with saline) on days 0, 3, 6, and 9. During the 14-day experimental period, the long and short diameters of the tumors were measured every 2 days and the tumor volume was calculated based on the ratio (1 / 2 × long diameter × short diameter). 2 ).from Fig.12 It can be seen that the AMPNs treatment group not only inhibited tumor growth in the early stage, but also achieved an inhibition rate of 97.5% on the 14th day.

[0057] In summary, the preparation of the present invention can be used to prepare a drug for treating hepatocellular carcinoma, so as to be used for treating hepatocellular carcinoma.

[0058] The technical solution of the present invention is not limited to the above-mentioned specific embodiments. All technical variations made according to the technical solution of the present invention fall within the protection scope of the present invention.

Claims

1. A method for preparing a manganese dioxide-coated arsenic nanosheet material, characterized in that: The following steps are involved: S1. Take PEG-NH2 and gray arsenic powder in a mass ratio of 2:1, dissolve PEG-NH2 in ultrapure water, add gray arsenic powder with a particle size of 0.5-5 μm, and mix well; precool the obtained mixed solution with liquid nitrogen, perform water bath ultrasound, and perform ice water bath ultrasound for 30 minutes each, and repeat several times; centrifuge the mixed solution, wash with water, and freeze-dry to obtain arsenic nanosheet powder; S2. Take arsenic nanosheet powder and sodium hydroxide solution in a mass-to-volume ratio of 1 mg:1 mL, dissolve the arsenic nanosheet powder in the sodium hydroxide solution, then add KMnO4 which is one-fourth the mass of the arsenic nanosheet powder, stir at room temperature for 1-2 hours, and then remove the unreacted products by centrifugation and water washing; add the mixed solution to a PEG-NH2 solution of equal volume to the sodium hydroxide solution, and stir for 8-10 hours; after the reaction is completed, centrifuge, dialyze, and freeze-dry to obtain manganese dioxide-coated arsenic nanosheet powder.

2. The method for preparing the manganese dioxide-coated arsenic nanosheet material according to claim 1, characterized in that: In step S2, the concentration of the sodium hydroxide solution is 0.001-0.01M.

3. The method for preparing the manganese dioxide-coated arsenic nanosheet material according to claim 1, characterized in that: In step S2, the concentration of the PEG-NH2 solution is 2 mg / mL.

4. A manganese dioxide coated arsenic nanosheet material prepared by the method according to any one of claims 1 to 3, characterized in that: The manganese dioxide coated arsenic nanosheet material is a sheet-like planar structure with a diameter of 190-230 nm and a thickness of 7-10 nm. The valence state of the core arsenic nanosheet of the manganese dioxide coated arsenic nanosheet material is 0, and the valence state of the coated manganese dioxide on the surface is +4.

5. Use of the manganese dioxide-coated arsenic nanosheet material as claimed in claim 4 in the preparation of a drug for treating hepatocellular carcinoma.

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