A method for preparing Mn3O4 nanomaterials and its application

Abstract

The present invention discloses a Mn 3 O 4 Nanomaterial preparation method and application thereof. The present invention draws on the bionic mineralization method and utilizes organic protein to in situ regulate the crystallization mineralization of soluble manganese salt to obtain Mn with a lamellar structure. 3 O 4 Nanomaterials, prepared Mn 3 O 4 Nanomaterials not only have the catalytic functions of glucose oxidase and catalase, but also have good photothermal conversion properties. They are expected to consume the glucose energy source on which tumors rely for survival through catalytic action, and use starvation therapy in conjunction with photothermal / photodynamic effects to effectively inhibit tumor growth.

Description

Technical Field

[0001] The invention belongs to the field of functional nano materials, and in particular relates to a Mn3O4 nano-mimetic enzyme with cascade catalytic function and a preparation method thereof. Background Art

[0002] As an essential trace element for the human body, manganese plays an important role in maintaining physiological balance. Manganese tetraoxide (Mn3O4) nanoparticles prepared by traditional high-temperature hydrothermal and calcination methods have catalytic functions similar to hydrogen peroxide (H2O2) enzymes, which can catalyze high-concentration H2O2 in tumor sites to produce a large amount of oxygen (O2) and improve the hypoxic environment of tumors. At the same time, Mn3O4 consumes glutathione molecules highly expressed in tumors through the Fenton reaction, and consumes O2 while producing reactive oxygen species (ROS) such as hydroxyl free radicals that are sensitive to tumor cells to kill tumors. However, the reported Mn3O4 nanoparticles themselves do not have glucose-like enzyme catalytic functions and good photothermal properties. Summary of the invention

[0003] In view of the defects or shortcomings of the prior art, the present invention provides a method for preparing a Mn3O4 nanomaterial.

[0004] To this end, the preparation method of the Mn3O4 nanomaterial provided by the present invention comprises:

[0005] The organic protein reacts with a soluble manganese salt aqueous solution at room temperature, and then the pH value of the reaction solution is adjusted to 8-12 and then the reaction is continued at room temperature to prepare a Mn3O4 nanomaterial;

[0006] The organic protein is selected from bovine serum albumin, silk fibroin or statherin.

[0007] Optionally, the soluble manganese salt is selected from manganese acetate, manganese carbonate, manganese chloride or manganese nitrate.

[0008] An optional solution is that the concentration of the soluble manganese salt aqueous solution is 1×10 -3 ~10×10 -3 M.

[0009] Furthermore, the method of the present invention further comprises: collecting the precipitate after the reaction is completed, and washing and drying the precipitate to obtain the Mn3O4 nanomaterial.

[0010] An optional scheme is that the organic protein molecules react with the soluble manganese salt aqueous solution at room temperature for 2 to 5 hours; the reaction is continued for 5 to 12 hours.

[0011] An optional solution is to use sodium hydroxide, potassium hydroxide or ammonia water to adjust the pH value of the reaction solution.

[0012] An optional solution is characterized in that the concentration of the organic protein in the soluble manganese salt aqueous solution is 1 to 10 mg / mL.

[0013] The present invention utilizes a simple and mild biomimetic mineralization strategy to in situ prepare a lamellar Mn3O4 nanomaterial, and obtains a Mn3O4 nanosheet with both cascade catalytic function and photothermal properties. This special structure of Mn3O4 nanosheet not only exhibits tumor acid-responsive glucose oxidase and catalase catalytic functions, but also has good photothermal conversion properties. It is expected that it will consume the glucose energy source on which the tumor depends for survival through catalytic action, and utilize starvation therapy in synergistic with photothermal / photodynamic therapy to effectively inhibit tumor growth.

[0014] Compared with traditional drug treatments for tumors, tumor starvation therapy and photothermal therapy, as two important tumor treatment methods, have shown significant superiority and have received widespread attention. Among them, tumor starvation therapy mainly achieves the function of "starving" tumor cells by using glucose oxidase to consume the glucose energy source required for the survival of tumor cells; near-infrared light (NIR)-triggered photothermal therapy usually relies on inorganic nanophotosensitive materials with photothermal conversion characteristics. Under NIR irradiation, the photosensitive material absorbs the energy of photons and activates from the ground singlet state to the excited singlet state. Subsequently, the energy of the electron excitation undergoes a non-radiative form of decay-vibrational relaxation, and returns to the ground state through collisions between the photosensitive material and the surrounding molecules. The increased kinetic energy heats up the surrounding microenvironment, thereby generating a thermal effect to disinfect tumor cells. However, the tumor inhibition effect of starvation therapy alone is not good, and high temperature can easily damage the surrounding normal tissues while eliminating tumors. Therefore, constructing a nano-tumor treatment platform that combines starvation therapy and photothermal therapy is an effective measure to improve the tumor inhibition effect and make up for the shortcomings of each treatment. Generally, in order to achieve starvation therapy for tumors, natural glucose oxidase (GOx) active molecules are currently more often selected to catalyze the consumption of glucose in the tumor, thereby cutting off the energy supply of the tumor.

[0015] Compared with the single function of traditional glucose enzymes and photosensitive particles, the Mn3O4 nanosheets prepared by the present invention not only have tumor acid-responsive glucose oxidase-like and catalase catalytic activities, but also have good photothermal conversion properties, integrating catalytic and photothermal properties, that is, firstly, the glucose oxidase of the Mn3O4 nanosheet is used to catalyze the glucose in the tumor environment, and then the H2O2 produced by glucose catalysis is further catalyzed by Mn3O4 and laser irradiation to generate a large amount of tumor-toxic ROS, and the photothermal / photodynamic synergistic tumor starvation therapy effectively realizes the tumor inhibition function;

[0016] Different from the problems of short half-life and toxicity of traditional glucose oxidase and photosensitizer, the Mn3O4 nanomaterial prepared by the present invention and its degradation product manganese have good biosafety as a trace element for human body; BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a TEM image of the Mn3O4 nanomaterial prepared in an embodiment of the present invention; the image shows that the Mn3O4 nanomaterial prepared in the present invention is composed of rectangular nanoparticles with a major axis size of approximately 40 nm.

[0018] Figure 2 XRD (A) and XPS (B) spectra of the Mn3O4 nanomaterial prepared in the embodiment of the present invention; crystal analysis found that the bionic mineralization method successfully prepared Mn3O4 nanosheets, and the organic components of the mineralization template BSA protein were attached to the surface of the Mn3O4 nanosheets.

[0019] Figure 3 The photothermal performance test results of the Mn3O4 nanomaterial prepared in the embodiment of the present invention; the results show that the Mn3O4 nanomaterial has good photothermal conversion function, showing a trend of increasing with the increase of the Mn3O4 nanomaterial concentration (A) and the NIR power density (B); it means that the photothermal temperature of the Mn3O4 nanomaterial of the present invention can be controllably adjusted by the Mn3O4 nanomaterial concentration and the NIR power.

[0020] Figure 4 This is a time-photothermal temperature change curve of the heating-cooling process of the Mn3O4 nanomaterial prepared in an embodiment of the present invention; through testing and calculation, it was found that the photothermal conversion efficiency of the Mn3O4 nanomaterial was 31.68%.

[0021] Figure 5 UV absorption curves of H2O2 (A) and glucose (B) catalyzed by Mn3O4 nanomaterials prepared in the examples of the present invention; compared with BSA and leaching groups, colorimetric experiments found that the Mn3O4 nanosheets synthesized by BSA showed an obvious UV absorption peak at 650nm, indicating that the Mn3O4 nanosheets exhibited catalytic properties similar to H2O2 and glucose enzymes.

[0022] Figure 6 The relative activity diagrams of the Mn3O4 nanomaterials prepared in the embodiments of the present invention in catalyzing H2O2 (A) and glucose (B) under different pH conditions; the Mn3O4 nanomaterials can catalyze the reaction substrate TMB to show blue, and both show a trend of gradually increasing catalytic efficiency with decreasing pH, indicating that the catalytic efficiency of Mn3O4 has acid-responsive characteristics and can adaptively respond to the acidic microenvironment of the tumor to exert its dual-enzyme catalytic function. DETAILED DESCRIPTION

[0023] Unless otherwise specified, the scientific and technical terms used herein are understood according to the knowledge of ordinary technicians in the relevant fields.

[0024] The present invention draws on the biomimetic mineralization method, uses an organic molecular template to in situ regulate the crystallization and mineralization of manganese ions, and obtains a manganese tetraoxide (Mn3O4) nanomaterial with a lamellar structure. The Mn3O4 nanomaterial exhibits acid-responsive glucose oxidase and catalase cascade catalytic functions and good photothermal conversion properties. After further verification, the Mn3O4 nanomaterial of the present invention not only has acid-responsive glucose oxidase and catalase catalytic functions at the same time. It is expected to consume the glucose energy source on which the tumor depends for survival through catalysis, and use starvation therapy to cooperate with photothermal stimulation to effectively inhibit tumor growth.

[0025] The following specific examples are provided to further explain the present invention. It should be noted that the reaction raw materials, reaction time, pH value and substance concentration involved in the following examples are all preferred examples of the present invention. Those skilled in the art can obtain the Mn3O4 nanomaterial of the present invention by selecting the above factors based on the disclosure of the present invention. The materials used in the following examples are all commercially available products.

[0026] Embodiment 1:

[0027] (1) Under magnetic stirring, add 3×10 -3 M manganese acetate aqueous solution, the concentration of fetal bovine serum albumin in the solution was 1 mg / mL after addition, and then reacted at room temperature for 2 hours;

[0028] (2) slowly adjusting the pH value of the mixed solution of step (1) to about 8 using 1 M sodium hydroxide, and continuing the reaction for 5 hours under magnetic stirring;

[0029] (3) The solution in step (2) was centrifuged at 10,000 rpm for 10 minutes to collect the substrate, which was then washed three times with deionized water and once with anhydrous ethanol. The Mn3O4 nanoparticles were collected after being dried in a vacuum oven at 55°C for 24 hours.

[0030] The material prepared in this example was subjected to TEM, XRD and XPS analysis, and the results were as follows: Figure 1 and 2 The photothermal performance, photothermal conversion rate, catalytic H2O2, and catalytic glucose performance of the material prepared in this example were further tested:

[0031] Photothermal performance: The photothermal conversion characteristics of the material were tested using a near-infrared thermal imager (Testo 885-2, Germany). First, aqueous solutions of the material prepared in Example 1 at three different concentrations, 1.0 mg / mL, 2.5 mg / mL, and 5.0 mg / mL, were prepared and exposed to 808 nm laser irradiation (1.25 W / cm 2 ), and the temperature curve of the nanoparticle aqueous solution was obtained as a function of irradiation time; then the laser power density was adjusted to explore the photothermal temperature curve of the 2.5 mg / mL Mn3O4 aqueous solution as a function of time. Figure 3 shown.

[0032] Photothermal conversion rate: The photothermal conversion rate of the material prepared in this embodiment was tested according to the nanoparticle photothermal calculation method reported in the literature (ACS Nano 2011, 5, 12, 9761-9771). Specifically, under NIR light stimulation (power density: 0.5 W / cm 2 ), the temperature rise-cooling curve of 2.5 mg / mL Mn3O4 aqueous solution was tested to obtain the photothermal conversion rate. The results are as follows Figure 4 shown.

[0033] Catalytic performance of H2O2 and glucose:

[0034] Catalase can catalyze H2O2 to oxidize 3,3',5,5'-tetramethylbenzidine (TMB) to produce a color reaction, and the catalytic effect can be evaluated under ultraviolet absorption at a specific wavelength (see ACS Appl. Mater. Interfaces 2022, 14, 20, 22728-22736). In order to detect the catalytic performance of the material prepared in this example on H2O2, the catalytic characteristics were detected using the TMB colorimetric method commonly used in the art: the groups were set as: supernatant leaching group (i.e., the supernatant of the precipitate collected by centrifugation in step (3), which mainly contained BSA and Mn ions that did not participate in the reaction; Leaching group), organic template control group (BSA group) and experimental group (material group prepared in Example 1); first, TMB (1mmol / L) was added to a weakly acidic acetate buffer in the dark, and then the above-mentioned group components and H2O2 (0.1mol / L) were added in turn and shaken evenly. The mixture was incubated at room temperature for 30 minutes, and the absorption was detected using an ultraviolet spectrophotometer. In the corresponding group, the amount of Mn3O4 nanomaterial used was 20mg / L, BSA was 2mg / mL, and the filtrate was 0.5mL. The results are as follows Figure 5 As shown in A.

[0035] Similarly, because glucose generates H2O2 under the catalysis of glucose enzyme, and Mn3O4 nanomaterials have a catalytic effect on H2O2, the glucose enzyme catalytic performance of Mn3O4 nanomaterials can still be detected by TMB colorimetry; glucose (2mmol / L), TMB (0.5mmol / L), Mn3O4 nanomaterials (20mg / L) and horseradish peroxidase (HRP, 500mg / L) were added to acetate buffer in sequence, and absorption detection was performed using an ultraviolet spectrophotometer; the results are as follows Figure 5 As shown in B.

[0036] Furthermore, in order to explore the H2O2 catalytic efficiency of Mn3O4 nanomaterials under different pH conditions, six buffer solutions with different pH values ​​of 4.5, 5.0, 5.5, 6.0, 6.5, and 7.0 were first prepared, and then TMB (1mmol / L), Mn3O4 nanomaterials (20mg / L) and H2O2 (0.1mol / L) were added in sequence, and the mixture was shaken evenly. The corresponding absorbance values ​​were tested under ultraviolet light with a wavelength of 650nm by TMB colorimetry. Then, the OD value of pH=4.5 was used as the benchmark to calculate the catalytic activity ratios of other different pH value groups. The results are shown in the figure below. Figure 6 shown.

[0037] Embodiment 2:

[0038] This embodiment is different from embodiment 1 in that the concentration of fetal bovine serum albumin in step (1) is 2 mg / mL, followed by reaction at room temperature for 3 hours; and 2M sodium hydroxide is used to adjust the pH value in step (2).

[0039] Embodiment 3:

[0040] This embodiment is different from embodiment 1 in that the concentration of fetal bovine serum albumin in step (1) is 5 mg / mL, followed by reaction at room temperature for 5 hours; and in step (2) the pH value is adjusted with 3 M sodium hydroxide, followed by reaction for 6 hours.

[0041] Embodiment 4:

[0042] This embodiment is different from embodiment 1 in that the concentration of fetal bovine serum albumin in step (1) is 8 mg / mL, followed by reaction at room temperature for 5 hours; and in step (2) the pH value is adjusted with 2M sodium hydroxide, followed by reaction for 8 hours.

[0043] Embodiment 5:

[0044] This embodiment is different from embodiment 1 in that the concentration of fetal bovine serum albumin in step (1) is 10 mg / mL; in step (2), 5 M sodium hydroxide is used to adjust the pH value, and the reaction is continued for 5 hours.

[0045] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims

1. Application of Mn3O4 nanomaterials as catalase and glucose oxidase; the preparation method of the Mn3O4 nanomaterials comprises: The organic protein reacts with a soluble manganese salt aqueous solution at room temperature, and then the pH value of the reaction solution is adjusted to 8 to 12 and then the reaction is continued at room temperature to prepare the Mn3O4 nanomaterial; the organic protein molecule reacts with the soluble manganese salt aqueous solution at room temperature for 2 to 5 hours; the reaction is continued for 5 to 12 hours; The organic protein is selected from bovine serum albumin, silk fibroin or statherin; the soluble manganese salt is selected from manganese acetate, manganese carbonate, manganese chloride or manganese nitrate; The concentration of the soluble manganese salt aqueous solution is 1×10 -3 ~10 ×10 -3 M; the concentration of the organic protein in the soluble manganese salt aqueous solution is 1 to 10 mg / mL; The Mn3O4 nanomaterial is a Mn3O4 nanosheet, which is composed of rectangular nanoparticles with a major axis size of 40 nm.

2. The use of the Mn3O4 nanomaterial according to claim 1, characterized in that: The method also includes: collecting precipitates after the reaction is completed, and washing and drying the precipitates to obtain Mn3O4 nanometer materials.

3. The use of the Mn3O4 nanomaterial according to claim 1, characterized in that: The pH value of the reaction solution is adjusted using sodium hydroxide, potassium hydroxide or ammonia water.

4. Application of Mn3O4 nanomaterials as catalase and glucose oxidase under near-infrared laser irradiation conditions; the preparation method of the Mn3O4 nanomaterials comprises: The organic protein reacts with a soluble manganese salt aqueous solution at room temperature, and then the pH value of the reaction solution is adjusted to 8 to 12 and then the reaction is continued at room temperature to prepare the Mn3O4 nanomaterial; the organic protein molecule reacts with the soluble manganese salt aqueous solution at room temperature for 2 to 5 hours; the reaction is continued for 5 to 12 hours; The organic protein is selected from bovine serum albumin, silk fibroin or statherin; the soluble manganese salt is selected from manganese acetate, manganese carbonate, manganese chloride or manganese nitrate; The concentration of the soluble manganese salt aqueous solution is 1×10 -3 ~10 ×10 -3 M; the concentration of the organic protein in the soluble manganese salt aqueous solution is 1 to 10 mg / mL; The Mn3O4 nanomaterial is a Mn3O4 nanosheet, which is composed of rectangular nanoparticles with a major axis size of 40 nm.

5. Application of Mn3O4 nanomaterials in the preparation of tumor starvation therapy drugs; the drugs treat tumors by catalytic degradation of glucose and hydrogen peroxide; The preparation method of the Mn3O4 nanomaterial comprises: The organic protein reacts with a soluble manganese salt aqueous solution at room temperature, and then the pH value of the reaction solution is adjusted to 8 to 12 and then the reaction is continued at room temperature to prepare the Mn3O4 nanomaterial; the organic protein molecule reacts with the soluble manganese salt aqueous solution at room temperature for 2 to 5 hours; the reaction is continued for 5 to 12 hours; The organic protein is selected from bovine serum albumin, silk fibroin or statherin; the soluble manganese salt is selected from manganese acetate, manganese carbonate, manganese chloride or manganese nitrate; The concentration of the soluble manganese salt aqueous solution is 1×10 -3 ~10 ×10 -3 M; the concentration of the organic protein in the soluble manganese salt aqueous solution is 1 to 10 mg / mL; The Mn3O4 nanomaterial is a Mn3O4 nanosheet, which is composed of rectangular nanoparticles with a major axis size of 40 nm.

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