Preparation method and application of two-dimensional sheet-like manganese copper ore nanozyme

Abstract

This invention belongs to the field of nanomaterials and tumor treatment technologies, specifically to a method for preparing and applying a two-dimensional sheet-like manganese-copper nanozyme. The sheet-like manganese-copper nanozyme is prepared by a hydrothermal reaction between a divalent copper salt and a divalent manganese salt. The nanozyme exhibits excellent catalase- and peroxidase-like activities, catalyzing H₂O₂ to produce O₂ or ·OH, and exhibits rapid and specific glutathione consumption, while also exhibiting strong anti-tumor cell cytotoxicity.

Description

Technical Field

[0001] The present invention belongs to the technical field of nanomaterials and tumor treatment, and particularly relates to a preparation method and application of a two-dimensional sheet-shaped manganese copper ore nanozyme. Background Art

[0002] Cancer has always been a threat to human life. Inducing apoptosis in tumor cells is currently the most important treatment for cancer. However, some tumor cells develop immune resistance to apoptosis, and the effect of apoptosis-based cancer treatment is unsatisfactory. Therefore, combining apoptosis with non-apoptotic tumor treatment methods will increase the chance of anti-tumor. Ferroptosis is a new, iron-dependent non-apoptotic cell death method characterized by the accumulation of lipid peroxides. Increased intracellular iron ion concentration, the production of ·OH, or decreased glutathione leading to the inactivation of GPX4 protein can lead to the accumulation of lipid peroxides and thus induce ferroptosis. Since the death pathway of ferroptosis is different from apoptosis, it can avoid the problem of tumor cell resistance, thereby increasing the chance of successful anti-tumor treatment. However, there are relatively few studies in this area.

[0003] Nanozymes are a type of nanomaterials with the catalytic activity of natural enzymes. They combine the advantages of natural enzymes and nanomaterials, with the advantages of high activity, good stability and good controllability. In recent years, they have become an emerging research field. Unlike biological enzymes that can only tolerate mild environments, metal-based nanozymes can still maintain robust catalytic performance in harsh environments, showing great biomedical applications. It is reported that various nanozymes can catalyze H2O2 or O2 to produce different toxic free radicals (·OH or ·O2 - Nanocatalytic agents, such as glutathione (GSH), have great potential in this type of reactive oxygen species (ROS)-mediated tumor therapy. However, the abundant glutathione (GSH) in the tumor microenvironment also consumes some ROS, such as H2O2 and ·OH, leading to a decrease in ROS-mediated anti-tumor efficacy. Therefore, consuming excess GSH and in situ catalyzing H2O2 to produce species such as O2 and ·OH has significant practical significance. Summary of the Invention

[0004] To achieve the above technical objectives, the present invention provides a preparation method and application of a two-dimensional sheet-like manganese copper ore nanozyme, which has the ability to rapidly and specifically consume glutathione and can catalyze H2O2 to produce ·OH.

[0005] The first aspect of the present invention provides a method for preparing a two-dimensional sheet-like manganese copper ore nanozyme, comprising the following steps:

[0006] (1) dissolving a divalent copper salt and a divalent manganese salt in deionized water, stirring for 5-30 minutes until completely dissolved, to obtain a mixed aqueous solution of the divalent copper salt and the divalent manganese salt, and then adjusting the pH value of the mixed solution to 13. This alkaline environment can greatly ensure the nanosheet morphology;

[0007] (2) placing the mixed solution after treatment in step (1) into a tetrafluoroethylene-lined reactor, heating it in a high-temperature oven for reaction, then taking it out and aging it at 20-30° C. for 3-5 hours to make the surface morphology of the nanozyme more uniform;

[0008] (3) The mixture obtained in step (2) is washed and centrifuged, the supernatant is removed, and the mixture is dried to obtain the desired product.

[0009] Preferably, the divalent copper salt in step (1) is Cu(NO3)2·3H2O or CuCl2·2H2O; the divalent manganese salt is any one of Mn(NO3)2·4H2O, MnCl2·4H2O or MnCl2·2H2O; the anions carried by these metal salts are easy to remove, which facilitates experimental operation; the atomic ratio of Mn to Cu in the prepared manganese copper ore is 1:1.

[0010] Preferably, in step (1), NaOH solution is used to adjust the pH value of the solution, wherein the concentration of the NaOH solution is 0.5-5.0M.

[0011] Preferably, the heating temperature in step (2) is 80° C. and the reaction time is 2 h.

[0012] Preferably, the specific operations of washing and centrifuging in step (3) are: washing with water and ethanol three times respectively, and then centrifuging at a speed of 8000-10000 rpm.

[0013] The obtained manganese copper ore nanozyme has a uniform flower-like nanosheet structure with a length and width of 200-300nm and a thickness of 20-30nm.

[0014] The second aspect of the present invention provides the use of the above-mentioned sheet-like manganese copper nanozyme in anti-tumor.

[0015] Under ultrasonic conditions, methoxypolyethylene glycol phospholipid (DSPE-PEG) powder was added to the aqueous solution of sheet-like manganese copper nanozyme to prepare a stable manganese copper nanosheet aqueous solution.

[0016] Experiments have confirmed that this manganese copper nanozyme has excellent glutathione peroxidase-like activity, rapidly consuming glutathione in the environment at low concentrations. Furthermore, the nanozyme also exhibits catalase- and peroxidase-like activities, catalyzing H2O2 to produce O2 and ·OH, respectively, alleviating the hypoxic state of the tumor environment and generating ROS for anti-tumor effects.

[0017] The beneficial effects of the present invention are:

[0018] 1. The sheet-like manganese copper ore nanozyme of the present invention is prepared by a hydrothermal method. The raw materials used are non-toxic, environmentally friendly, low-cost, simple in process, easy to operate and control, suitable for large-scale production, and the preparation process is green and environmentally friendly.

[0019] 2. The manganese copper ore nanozyme provided by the present invention is a nanoscale material that can enter cells well and can be slowly degraded into corresponding copper ions and manganese ions under acidic conditions. It will not be deposited in the body. Both are essential elements for the human body and have high biocompatibility.

[0020] 3. Compared with existing nanozymes, the manganese copper nanozyme provided by the present invention mainly targets the highly selective and efficient consumption of glutathione in the tumor environment, and also exhibits an excellent glutathione consumption rate under hypoxic conditions. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the SEM image of the sheet-like manganese copper ore nanozyme of Example 1;

[0022] Figure 2 This is a TEM image of the sheet-like manganese copper ore nanozyme of Example 1;

[0023] Figure 3 This is the XRD pattern of the sheet-like manganese copper ore nanozyme of Example 1;

[0024] Figure 4 This is a graph for evaluating the catalase-like activity of the sheet-like manganese copper ore nanozyme of Example 1;

[0025] Figure 5 This is a graph evaluating the peroxidase-like activity of the sheet-like manganese copper ore nanozyme in Example 1 under different pH environments;

[0026] Figure 6 This is a graph showing the change in glutathione consumption over time by sheet-like manganese copper nanozymes at different concentrations;

[0027] Figure 7 This is a comparison chart of the selectivity of the sheet-like manganese copper nanozyme for glutathione in Example 1;

[0028] Figure 8 This is a comparison chart of glutathione consumption by the sheet-like manganese copper nanozyme of Example 1 in different atmosphere environments;

[0029] Figure 9 This is a cytotoxicity test diagram of the sheet-like manganese copper ore nanozyme in Example 1. DETAILED DESCRIPTION

[0030] The present invention will be further described below with reference to the accompanying drawings and specific embodiments using examples.

[0031] Example 1

[0032] Preparation of sheet-like manganese copper ore nanozyme CuMnO2:

[0033] 0.0724g of Cu(NO3)2·3H2O and 0.0592g of MnCl2·4H2O were dissolved in 20mL of deionized water and stirred for 30 minutes to obtain mixed solution I. 0.2g of solid sodium hydroxide was weighed and added to 5mL of deionized water, dissolving completely to obtain a 1.0M NaOH solution. The pH of mixed solution I was adjusted to 13 using the prepared NaOH solution. The mixture was then hydrothermally reacted at 80°C for 2 hours and aged at 25°C for 4 hours. The resulting dark brown precipitate was washed three times with deionized water and ethanol, respectively. After each wash, the precipitate was centrifuged at 8000 rpm, the supernatant removed, and finally dried to obtain the flaky manganese copper nanozyme. Figure 1 This is the SEM image of the sheet-like manganese copper ore nanozyme of this embodiment; Figure 2 TEM image of the sheet-like manganese copper nanozyme of this example. As can be seen from the figure, manganese copper nanozyme has a uniform flower-like nanosheet structure with a length and width of 245nm and a thickness of 27nm. Figure 3 This is the XRD pattern of the sheet-like manganese copper nanozyme of this embodiment, which shows that the structure of manganese copper nanozyme is CuMnO2.

[0034] Example 2

[0035] Study on the enzyme-like activity of manganese copper nanozymes:

[0036] Under ultrasonic conditions, DSPE-PEG powder and manganese copper nanosheet powder were added to deionized water in a mass ratio of 1:1 to prepare a stable manganese copper nanosheet concentrated stock solution with a concentration of 1 mg / mL. DSPE-PEG can make manganese copper nanozyme more stable and can be stably dispersed in water for at least one week, ensuring the stable manifestation of its enzyme-like activity.

[0037] The catalase-like activity of manganese-copper nanozymes was determined using a dissolved oxygen meter. A 1 mg / mL stock solution of manganese-copper nanosheets was added to a mixture of H2O2 and PBS to prepare a 20 μg / mL manganese-copper nanozyme solution (pH = 7.4). Figure 4 This is a graph evaluating the catalase-like activity of the sheet-like manganese-copper ore nanozyme of Example 1. As shown in the graph, adding manganese-copper ore nanozyme to H2O2 can produce more O2 and has good catalase-like activity.

[0038] The peroxidase-like activity of manganese copper nanozyme was characterized by UV absorption spectroscopy after the reaction of manganese copper nanozyme with 3,3',5,5'-tetramethylbenzidine (TMB). 10 μg / mL manganese copper nanozyme, 6 mM TMB, and 0.66 mM H2O2 were prepared in PBS buffer at pH 4, 5, 6, and 7, respectively. Figure 5 This is a graph evaluating the peroxidase-like activity of the sheet-like manganese copper ore nanozyme in Example 1 under different pH environments. It can be seen from the figure that as the pH value of the solution decreases, more ·OH is produced in the solution, and the solution has better peroxidase-like activity.

[0039] Example 3

[0040] Study on the glutathione peroxidase-like activity of manganese copper nanozyme:

[0041] The glutathione peroxidase-like activity of manganese copper nanozyme was investigated according to the DTNB (5,5'-dithiobis(2-nitrobenzoic acid)) method. At pH = 4, 1, 10, and 20 μg / mL manganese copper nanozyme was mixed with glutathione (0.16 mM). DTNB (0.08 mM) was added at regular intervals and incubated for 15 minutes. The UV absorption spectrum of the solution was then tested, and the absorbance value at 412 nm was used to represent the glutathione content in the solution. As the concentration of manganese copper nanozyme increased, the time required to consume the same amount of glutathione was significantly shortened, as shown in Figure 2. Figure 6 As shown in Figure 3, it can be seen that manganese copper nanozyme has good catalytic activity towards glutathione.

[0042] Due to the complex environment of tumor cells, a variety of glutathione-like biomolecules can easily interfere with the reaction between drugs and glutathione, thereby interfering with anti-tumor therapy. To confirm that manganese copper nanozyme specifically consumes glutathione, the selectivity of the nanozyme for glutathione in the presence of other amino acids was determined. 0.26mM arginine, 0.17mM histidine, 2mM glucose, 0.48mM lysine, 1.6mM ascorbic acid, and 0.16mM glutathione were mixed, and then 20μg / mL manganese copper nanozyme was added. The reaction was allowed to proceed for 0.5h, and 0.08mM DTNB was added and incubated for 15min. The UV absorption spectrum of the solution was then measured, and the ratio of the absorbance change at 412nm to the absorbance without the addition of biomolecules was used to represent the anti-interference ability of glutathione peroxidase-like activity. Figure 7 This is a comparison chart of the selectivity of the sheet-like manganese copper nanozyme for glutathione in Example 1. As can be seen from the figure, the addition of other amino acid biomolecules does not interfere with the reaction between manganese copper nanozyme and glutathione.

[0043] The hypoxic environment in solid tumors can greatly interfere with the anti-tumor activity of nanozyme materials. The actual effect of manganese copper nanozyme on solid tumors was evaluated by consuming glutathione in different gas atmospheres. The PBS buffer solution was aerated with air, nitrogen, and oxygen for 20 minutes, and then 1 μg / mL manganese copper nanozyme and 0.16 mM glutathione were quickly added and sealed to allow the mixture to react. After 1 hour and 2 hours of reaction, DTNB0.08 mM was added and incubated for 15 minutes. The ultraviolet absorption spectrum of the solution was then tested, and the glutathione consumption capacity was expressed as a percentage of the absorbance change at 412 nm to the absorbance before the reaction. The glutathione consumption capacity (C) was calculated using the following formula:

[0044] C=(A m0 -A mt ) / (A n0 -A nt )*100%

[0045] Among them, A m0 and A mt Respectively represent the absorbance at 412 nm at the start of the reaction and at the reaction time t under the required atmosphere; A n0 and A nt They represent the absorbance at 412 nm at the start of the reaction and at reaction time t under no gas condition, respectively.

[0046] Figure 8 This is a comparison chart of the glutathione consumption of the sheet-like manganese copper nanozyme in Example 1 under different atmospheric environments. It can be seen from the figure that the glutathione consumption is similar when air and nitrogen are passed through, but the glutathione consumption is higher when oxygen is passed through the former two, indicating that oxygen-rich conditions can promote the consumption of glutathione, while hypoxic conditions will not significantly interfere with the consumption of glutathione by manganese copper nanozyme.

[0047] Example 4

[0048] Study on the anti-tumor activity of manganese copper nanozymes:

[0049] The cell activity test of manganese copper nanozyme was carried out in 4T1 cells. 8000 4T1 cells with good growth status were plated in a 96-well plate and left to stand overnight to wait for adhesion; then different concentrations of manganese copper nanozyme were added to incubate with the cells, with four sub-wells for each concentration, and incubated for 24 hours; then 20μL of 3-(4,5-dimethylthiazol-2)-2,5-diphenyltetrazolium bromide (MTT) solution was added to each well, and after incubation for 4 hours, DMSO was added to dissolve the formazan, and after incubation on a constant temperature shaker for 10 minutes, the absorption of each well was measured on a microplate reader, and the absorption at 490nm was used to indicate the size of the cell activity. Figure 9 It can be seen that manganese copper ore nanozymes have a good killing effect on tumor cells.

Claims

1. Application of a two-dimensional sheet-like manganese copper ore nanozyme in the preparation of anti-tumor drugs, characterized in that: The preparation method of the two-dimensional sheet-like manganese copper ore nanozyme comprises the following steps: (1) dissolving a divalent copper salt and a divalent manganese salt in deionized water, stirring for 5-30 minutes to obtain a mixed aqueous solution of the divalent copper salt and the divalent manganese salt, and then adjusting the pH value of the mixed solution to 13; (2) placing the mixed solution after treatment in step (1) into a tetrafluoroethylene-lined reactor, heating it in a high-temperature oven for reaction, then taking it out and aging it at 20-30° C. for 3-5 hours; (3) Washing and centrifuging the mixture obtained in step (2), removing the supernatant, and drying to obtain the desired dark brown solid product; the obtained manganese copper ore nanozyme has a uniform flower-like nanosheet structure with a length and width of 200-300 nm and a thickness of 20-30 nm.

2. The use according to claim 1, characterized in that The divalent copper salt in step (1) is any one of Cu(NO3)2·3H2O or CuCl2·2H2O; the divalent manganese salt is any one of Mn(NO3)2·4H2O, MnCl2·4H2O or MnCl2·2H2O; and the atomic ratio of Mn to Cu in the prepared manganese copper ore is 1:

1.

3. The use according to claim 1, characterized in that In step (1), a NaOH solution is used to adjust the pH value of the solution, wherein the concentration of the NaOH solution is 0.5-5.0M.

4. The use according to claim 1, characterized in that In step (2), the heating temperature is 80° C. and the reaction time is 2 h.

5. The use according to claim 1, characterized in that The specific operations of washing and centrifugation in step (3) are: washing with water and ethanol three times respectively, and then centrifuging at a speed of 8000-10000 rpm.

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