Purple phosphorus-manganese dioxide nanocomposite enzyme and preparation method and application thereof

Abstract

The application discloses purple phosphorus-manganese dioxide nanocomposite enzyme and a preparation method and application thereof, and belongs to the technical field of biological medicines. The purple phosphorus-manganese dioxide nanocomposite enzyme is formed by in-situ mineralization of purple phosphorus nanosheets and nanometer manganese dioxide, and is surface-modified with methoxypolyethylene glycol amine. The preparation method comprises the following steps: preparing purple phosphorus nanosheet material through ultrasonic liquid phase stripping; at room temperature, a potassium permanganate aqueous solution is dropped into a purple phosphorus nanosheet material aqueous solution, then a methoxypolyethylene glycol amine aqueous solution is added, stirring is conducted overnight, a precipitate is collected through centrifugation, and the purple phosphorus-manganese dioxide nanocomposite enzyme is obtained. The purple phosphorus-manganese dioxide nanocomposite enzyme provided by the application is composed of manganese dioxide nanomaterials with self-enhanced chemical kinetic catalytic performance and purple phosphorus nanosheets with near-infrared photothermal conversion performance, and can realize the function of NIR-II synergistic chemical kinetic catalysis for killing tumor cells.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to a purple phosphorus-manganese dioxide nanocomposite enzyme as well as a preparation method and application thereof. BACKGROUND

[0002] As an important technical means in the field of tumor treatment, the curative effect of nanocatalytic therapy is mainly derived from the generation of therapeutic products represented by reactive oxygen species (ROS) in specific physiological regions under the regulation of external physical / chemical stimulation or tumor microenvironment (TME), or the destruction of TME homeostasis by eliminating adverse molecules, finally inducing programmed cell death of tumor cells. The generation of ROS can be achieved through various pathways such as chemical dynamics (typical Fenton / Fenton-like reaction), photodynamics, and sonodynamics. The core mechanism is to regulate the electronic structure of the catalyst by taking advantage of the size effect and surface modification characteristics of nanomaterials, so as to respond to the characteristics of TME or external physical stimulation and efficiently produce cytotoxic ROS. Obviously, catalytic efficiency is the key factor to determine the effect of catalytic medical treatment.

[0003] In the treatment of solid tumors, catalytic efficiency is severely limited by TME: first, insufficient substrate supply, the hypoxic state and low concentration of hydrogen peroxide (H2O2) commonly existing in tumor tissues directly restricts the generation efficiency of cytotoxic ROS; second, redox balance interference, the high expression of reduced glutathione (GSH) and peroxidase in tumor cells will consume a large amount of generated ROS, weakening the therapeutic effect; third, reaction conditions do not match, the optimal pH value of Fenton / Fenton-like reaction to maintain low-valence metal concentration is usually 2-4, while the TME is weakly acidic (pH about 6.5), which cannot meet the suitable conditions of the reaction. In addition, the catalytic reaction mediated by external excitation source also has strict requirements on energy intensity and tissue penetration depth, further limiting the clinical application of catalytic therapy. Therefore, improving catalytic efficiency and breaking through TME limitation are the core needs to promote the development of tumor catalytic therapy, and it is imperative to develop new cancer catalytic medical technology.

[0004] As a safe and efficient tumor treatment method, photothermal therapy (PTT) converts external laser energy into heat energy through light-heat conversion agents enriched in tumor sites, causing the temperature of the treatment area to rise, leading to denaturation of biological macromolecules, organelle dysfunction, and ultimately cell death. However, the near-infrared region I (NIR-I, wavelength 700-900 nm) laser used in traditional photothermal therapy has significant drawbacks: its penetration depth in tumor tissue is shallow, usually only about 5 mm, making it difficult to eliminate tumor cells outside the laser irradiation range, and single PTT often cannot achieve complete tumor cure. SUMMARY

[0005] To solve the above technical problems, the present application provides a purple phosphorus-manganese dioxide nanocomposite enzyme and its preparation method and application. Compared with the prior art, the near-infrared region II (NIR-II, wavelength 1000-1700 nm) laser has significant advantages: longer wavelength can effectively reduce the absorption and scattering of light by biological tissues, with a penetration depth of several centimeters, which can cover a larger range of tumor tissues, especially for deep solid tumor treatment; at the same time, NIR-II laser causes less damage to normal tissues, allowing efficient tumor killing while reducing treatment side effects and improving treatment safety. In addition, NIR-II laser can also provide sufficient energy for photocatalytic therapy, ensuring the effective activation of photosensitizers in deep solid tumor tissues. Therefore, the combination of NIR-II photothermal therapy and photocatalytic therapy can achieve complementary advantages of the two technologies, further enhancing the efficacy of nanometer agents in killing and removing tumor cells, and providing a new effective strategy for solid tumor treatment.

[0006] To achieve the above purpose, the present application provides the following technical solutions:

[0007] One of the purposes of the present application is to provide a purple phosphorus-manganese dioxide nanocomposite enzyme (MVPs), which is formed by in-situ mineralization of purple phosphorus nanosheets (VPNSs) and nanometer manganese dioxide (MnO2), and is modified with methoxypolyethylene glycol amine on the surface.

[0008] The method provided by the present application uses purple phosphorus nanosheet material as a growth template to form nanometer manganese dioxide in-situ, and modifies methoxypolyethylene glycol amine to improve the pharmacokinetics of nanometer enzymes. The purple phosphorus-manganese dioxide nanocomposite enzyme provided by the present application is composed of manganese dioxide nanometer material with self-enhanced chemical kinetic catalytic performance and purple phosphorus nanosheet with near-infrared photothermal conversion performance, which can realize the function of killing tumor cells by NIR-II synergistic chemical kinetic catalysis.

[0009] The purple phosphorus-manganese dioxide nanocomposite enzyme in the application has a synergistic effect of killing and removing tumor cells between the purple phosphorus nanosheet and the nanomanganese dioxide, and significantly improves the antitumor effect. Specifically embodied in: firstly, manganese dioxide consumes GSH in TME to produce Fenton-like reagent divalent manganese ion (Mn 2+ ) and oxidized glutathione (GSSG), on the one hand, Mn 2+ as a Fenton-like reagent generates a killing ROS using the H2O2 overexpressed in TME, and on the other hand, GSH consumption can prolong the ROS life, self-enhance the chemical kinetics to catalyze the curative effect. Secondly, the purple phosphorus nanosheet has NIR-II photocatalytic activity, on the one hand, it can catalyze the production of killing ROS with H2O and H2O2 as substrates, and on the other hand, the purple phosphorus nanosheet has the ability to consume GSH, forming a NIR-II mediated self-amplification TME redox steady state destruction. Thirdly, the purple phosphorus nanosheet has NIR-II photothermal activity, which can convert light energy into heat energy to kill tumor cells, realizing NIR-II photothermal synergistic nanocatalytic tumor treatment. Fourthly, the purple phosphorus nanosheet has a high specific surface area and rich attachment sites, which can promote the uniform loading and in-situ mineralization of MnO2 on its surface, forming a stable composite structure to avoid the agglomeration of MnO2 nanoparticles.

[0010] Further, the purple phosphorus nanosheet is prepared by ultrasonic liquid phase exfoliation, and the nanomanganese dioxide is in-situ mineralized to form.

[0011] The second purpose of the application is to provide a preparation method of a purple phosphorus-manganese dioxide nanocomposite enzyme, comprising the following steps:

[0012] (1) preparing a purple phosphorus nanosheet material by ultrasonic liquid phase exfoliation;

[0013] (2) at room temperature, drop potassium permanganate aqueous solution into the purple phosphorus nanosheet material aqueous solution, and react to obtain an intermediate product mixture;

[0014] (3) adding methoxypolyethylene glycol amine aqueous solution to the intermediate product mixture, stirring overnight, centrifuging to collect the precipitate, and obtaining the purple phosphorus-manganese dioxide nanocomposite enzyme.

[0015] Further, the preparation method of the purple phosphorus nanosheet material comprises the following steps: dispersing purple phosphorus powder in an organic solvent, performing probe circulation ultrasonic treatment and water bath ultrasonic treatment under ice bath condition, centrifuging at a speed of 2687xg for 20 minutes, collecting the supernatant, centrifuging at a speed of 43000xg for 30 minutes, collecting the precipitate, and washing with anhydrous ethanol and water alternately for multiple times to remove the organic solvent, and obtaining the purple phosphorus nanosheet material.

[0016] Further, the ratio of the amount of the purple phosphorus powder to the amount of the organic solvent is 1 mg:2 mL; the organic solvent is N-methyl pyrrolidone (NMP).

[0017] Further, the power of the probe circulation ultrasonic treatment is 500 W, 2 seconds on / 3 seconds off for one cycle, and the ultrasonic treatment is performed for 12 hours.

[0018] The power of the water bath ultrasonic treatment is 600 W, and the ultrasonic treatment is performed for 8 hours.

[0019] The average particle size of the purple phosphorus nanosheet material prepared by the above preparation method is 197.5 nm, and the thickness is 2.50-2.90 nm.

[0020] Further, the mass ratio of the purple phosphorus nanosheet material, potassium permanganate, and methoxy polyethylene glycol amine is 1:1:10.

[0021] Further, the concentration of the purple phosphorus nanosheet material aqueous solution is 300 μg / mL.

[0022] The concentration of the potassium permanganate aqueous solution is 300 μg / mL.

[0023] The concentration of the methoxy polyethylene glycol amine aqueous solution is 10 mg / mL.

[0024] The third object of the present application is to provide an application of the purple phosphorus-manganese dioxide nanocomposite enzyme in the preparation of a tumor treatment drug.

[0025] The fourth object of the present application is to provide a tumor treatment drug, wherein the purple phosphorus-manganese dioxide nanocomposite enzyme is used as an active ingredient, and pharmaceutically acceptable adjuvants are added.

[0026] Compared with the prior art, the present application has the following advantages and technical effects:

[0027] The present application provides a purple phosphorus-manganese dioxide nanocomposite enzyme, which has a simple preparation method and controllable size; has good catalytic activity, and at the same time has good photothermal performance; experiments have proved that the purple phosphorus-manganese dioxide nanocomposite enzyme provided by the present application can realize tumor cell oxidative damage and apoptosis through self-enhanced catalytic therapy; at the same time, the purple phosphorus nanosheet component in the nanocomposite enzyme has good NIR-II photothermal performance, which can synergistically kill tumor cells, achieving the effect of NIR-II photothermal synergistic catalytic therapy for treating tumors. BRIEF DESCRIPTION OF DRAWINGS

[0028] The accompanying drawings, which form a part of the present application, are used to provide a further understanding of the present application, and the schematic embodiments of the present application and their descriptions are used to explain the present application, and do not constitute an improper limitation on the present application. In the drawings:

[0029] Figure 1 Structural property figures of the purple phosphorus nanosheet material prepared in Example 1; wherein, A is a transmission electron microscope figure of the purple phosphorus nanosheet material, B is a high-resolution transmission electron microscope figure of the purple phosphorus nanosheet material, C is a simulated interplanar spacing of the purple phosphorus nanosheet material, D is a simulated interplanar spacing of the purple phosphorus nanosheet material, E is an atomic force microscope figure and thickness statistics of the purple phosphorus nanosheet material;

[0030] Figure 2 Hydrodynamic particle size distribution statistics figures of the purple phosphorus nanosheet material and the purple phosphorus-manganese dioxide nanocomposite enzyme prepared in Example 1;

[0031] Figure 3 Structural property figures of the purple phosphorus-manganese dioxide nanocomposite enzyme prepared in Example 1; wherein, A is a transmission electron microscope figure of the purple phosphorus-manganese dioxide nanocomposite enzyme, B is a high-resolution transmission electron microscope figure of the purple phosphorus-manganese dioxide nanocomposite enzyme, C is a simulated interplanar spacing of the purple phosphorus-manganese dioxide nanocomposite enzyme, D is an energy dispersive X-ray element mapping (EDX-mapping) figure of the purple phosphorus-manganese dioxide nanocomposite enzyme, E is an atomic force microscope figure and thickness statistics of the purple phosphorus-manganese dioxide nanocomposite enzyme;

[0032] Figure 4 X-ray photoelectron spectroscopy (XPS) of the purple phosphorus nanosheet material, the purple phosphorus-manganese dioxide nanocomposite enzyme and manganese dioxide (MnO2) prepared in Example 1; wherein, A is an XPS full spectrum, B is a high-resolution XPS of P and Mn elements;

[0033] Figure 5 X-ray diffraction figure (XRD) of the purple phosphorus-manganese dioxide nanocomposite enzyme prepared in Example 1;

[0034] Figure 6 Photothermal performance research results of the purple phosphorus nanosheet material, the purple phosphorus-manganese dioxide nanocomposite enzyme and manganese dioxide prepared in Example 1; wherein, A is a photothermal temperature rise curve, B is the photothermal stability of the purple phosphorus-manganese dioxide nanocomposite enzyme;

[0035] Figure 7 Photothermal performance research results of the purple phosphorus nanosheet material, the purple phosphorus-manganese dioxide nanocomposite enzyme and manganese dioxide and manganese ions (Mn 2+The results of the study of the glutathione-like peroxidase activity of the purple phosphorus nanosheet material, the purple phosphorus-manganese dioxide nanocomposite enzyme, and the manganese dioxide and manganese ion (Mn

[0036] Figure 8 The ROS-producing catalytic activity of the purple phosphorus nanosheet material, the purple phosphorus-manganese dioxide nanocomposite enzyme, and the manganese dioxide and manganese ion (Mn 2+

[0037] Figure 9 The ROS-producing catalytic activity of the purple phosphorus nanosheet material, the purple phosphorus-manganese dioxide nanocomposite enzyme, and the manganese dioxide and manganese ion (Mn DETAILED DESCRIPTION

[0038] The various illustrative logics described herein will be presented in terms of methods that can be implemented as software programs or code on a machine readable medium. Unless otherwise specified, the methods described herein could be implemented as code and / or software programs that are executable on a machine readable medium that contain instructions that, when executed, can perform the methods. The methods described herein can be implemented across various elements, devices, systems, or objects, and in a variety of contexts. The methods described herein can be implemented with hardware elements, software elements, or a combination of both hardware and software elements.

[0039] It should be understood that the terms used herein are merely for describing particular embodiments and are not intended to limit the present application. Also, for numerical ranges in the present application, it should be understood that every intermediate value or value between the upper and lower limits of the range is specifically disclosed. Every intermediate value or value between the stated values or intermediate values in the stated ranges is also included in the present application. The upper and lower limits of these smaller ranges can be independently included or excluded from the ranges.

[0040] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, preferred methods and materials are described. All publications mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any reference is not an admission that it is prior art with respect to the present application. All documents mentioned herein are incorporated herein by reference.

[0041] ​Many modifications and variations to the illustrative embodiments described herein will be apparent to those skilled in the art from consideration of the specification and practice of the subject technology. Additional embodiments of the technology will be apparent to those skilled in the art from consideration of the specification and practice of the subject technology. The specification and examples given herein are by way of illustration only and are not intended to limit the scope of the subject technology.

[0042] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having" or variants thereof are open-ended, and specifically do not exclude additional, unrecited elements or method steps.

[0043] The embodiment of the present application provides a preparation method of purple phosphorus-manganese dioxide nanocomposite enzyme, comprising the following steps:

[0044] (1) Preparation of purple phosphorus nanosheet material: disperse purple phosphorus powder in NMP, perform probe cycle ultrasonic treatment and water bath ultrasonic treatment under ice bath conditions, centrifuge at a speed of 2687xg for 20 minutes, collect supernatant, centrifuge at a speed of 43000xg for 30 minutes, collect precipitate, and clean the precipitate with anhydrous ethanol and water alternately twice to remove organic solvents, so as to obtain purple phosphorus nanosheet material; the average particle size of the obtained purple phosphorus nanosheet is 197.5nm, and the average thickness is 2.50-2.90nm;

[0045] (2) Preparation of purple phosphorus-manganese dioxide nanocomposite enzyme: at room temperature, slowly drop potassium permanganate aqueous solution (KMnO4, concentration is 300μg / mL) into the purple phosphorus nanosheet material aqueous solution (VPNS concentration is 300μg / mL) prepared in step (1) by using a peristaltic pump, then add methoxy polyethylene glycol amine aqueous solution (concentration is 10mg / mL) by using the peristaltic pump, stir overnight, centrifuge to collect precipitate (speed is 43000xg, time is 30 minutes), and obtain purple phosphorus-manganese dioxide nanocomposite enzyme.

[0046] In the following preferred embodiments of the present application, in step (1), the use amount ratio of the purple phosphorus powder to NMP is 1mg:2mL.

[0047] In the following preferred embodiments of the present application, in step (1), the power of the probe cycle ultrasonic treatment is 500W, 2 seconds on / 3 seconds off is one cycle, and the ultrasonic treatment is performed for 12 hours.

[0048] In the following preferred embodiments of the present application, in step (1), the power of the water bath ultrasonic treatment is 600W, and the ultrasonic treatment is performed for 8 hours.

[0049] In the following preferred embodiments of the present application, the mass ratio of the purple phosphorus nanosheet material, potassium permanganate and methoxy polyethylene glycol amine is 1:1:10.

[0050] A purple phosphorus-manganese dioxide nanocomposite enzyme can be prepared by using the preparation method.

[0051] The purple phosphorus-manganese dioxide nanocomposite enzyme can be used in the preparation of a tumor drug.

[0052] A tumor treatment drug can be prepared by taking the purple phosphorus-manganese dioxide nanocomposite enzyme as an active ingredient and adding pharmaceutically acceptable adjuvants.

[0053] In the present application, "room temperature" refers to 25±2℃ unless otherwise specified.

[0054] The raw materials used in the present application are commercially available.

[0055] The technical solutions of the present application are further described below through examples.

[0056] Example 1

[0057] A preparation method of a purple phosphorus-manganese dioxide nanocomposite enzyme comprises the following steps:

[0058] (1) Preparation of purple phosphorus nanosheet material: 40 mg of purple phosphorus powder is dispersed in 80 mL of organic solvent NMP, and under the condition of ice bath and 500 W power, the probe is ultrasonically treated for 12 hours with a cycle of 2 seconds on / 3 seconds off, and then ultrasonically treated for 8 hours under the condition of 4℃ water bath and 600 W power. Then, the supernatant is collected by using an ultracentrifuge at 2687xg for 20 minutes at 4℃, and then the precipitate is collected by using an ultracentrifuge at 43000xg for 30 minutes at 4℃. The precipitate is washed twice with anhydrous ethanol / water to remove the organic solvent, and purple phosphorus nanosheet material (VPNSs) is obtained.

[0059] (2) Preparation of purple phosphorus-manganese dioxide nanocomposite enzyme: the mass ratio of purple phosphorus nanosheet material, potassium permanganate and methoxy polyethylene glycol amine is 1:1:10. At room temperature, the potassium permanganate aqueous solution (KMnO4, concentration is 300 μg / mL, volume is 500 μL) is slowly dropped into the purple phosphorus nanosheet material aqueous solution (VPNSs, concentration is 300 μg / mL, volume is 500 μL) prepared in step S1 by using a constant flow pump at a flow rate of 1.5 mL / min, and stirred at a speed of 1000 revolutions per minute by using a magnetic stirrer for 30 minutes. Then, the methoxy polyethylene glycol amine (NH2-PEG-OMe, concentration is 10 mg / mL, volume is 150 μL) is added by using a constant flow pump at a flow rate of 1.5 mL / min, and stirred at a speed of 1000 revolutions per minute overnight. The precipitate is collected by centrifugation at 4℃ (speed is 43000xg, time is 30 minutes), and washed twice with deionized water to obtain purple phosphorus-manganese dioxide nanocomposite enzyme (MVPs).

[0060] Performance test

[0061] 1. Structural property determination of purple phosphorus nanosheet material

[0062] Transmission electron microscopy analysis was performed on the purple phosphorus nanosheet material prepared in Example 1, and the results are shown in Figure 1 . As can be seen from A in Figure 1 , the prepared purple phosphorus nanosheet has a rectangular shape; as can be seen from B in Figure 1 , the purple phosphorus nanosheet has clear lattice diffraction fringes; as can be seen from C and D in Figure 1 , the interplanar spacing fitted according to the B figure is 2.88 Å and 6.27 Å, which corresponds to 2Theta angle of 31.0° and 13.7° on the purple phosphorus PDF #44-0906 card, indicating that the purple phosphorus nanosheet is a purple phosphorus crystal; as can be seen from E in Figure 1 , the thickness of the purple phosphorus nanosheet is between 2.50-2.90 nm. The above results indicate the successful preparation of the purple phosphorus nanosheet.

[0063] Fluid dynamics particle size distribution statistics were performed on the purple phosphorus nanosheet material prepared in Example 1, and the results are shown in Figure 2 . As can be seen from Figure 2 , the particle size of the purple phosphorus nanosheet has a Gaussian distribution, and the average hydrated particle size is 197.5 nm.

[0064] 2. Physicochemical property determination of purple phosphorus-manganese dioxide nanocomposite enzyme

[0065] 1) Fluid dynamics particle size distribution statistics were performed on the purple phosphorus-manganese dioxide nanocomposite enzyme prepared in Example 1, and the results are shown in Figure 2 , from which it can be seen that the particle size of the purple phosphorus-manganese dioxide nanocomposite enzyme has a Gaussian distribution, and the average hydrated particle size is 211.5 nm.

[0066] 2) Transmission electron microscopy analysis was performed on the purple phosphorus-manganese dioxide nanocomposite enzyme prepared in Example 1, and the results are shown in Figure 3 . As can be seen from A in Figure 3 , the purple phosphorus-manganese dioxide nanocomposite enzyme has a rectangular flake structure with a particle size of about 150 nm; as can be seen from B in Figure 3 , the surface of the purple phosphorus-manganese dioxide nanocomposite enzyme has both clear lattice diffraction fringes (rectangular frame) and amorphous substances (circular frame). Fourier transform and inverse Fourier transform were performed on the picture in the rectangular frame in B, and the fitted interplanar spacing was 6.42 Å (see C in Figure 3 ), which corresponds to 2Theta angle of 13.7° on the purple phosphorus PDF #44-0906 card, indicating that the surface of the purple phosphorus-manganese dioxide nanocomposite enzyme has both exposed portions of purple phosphorus crystals and portions coated with MnO2 or PEG. EDS-elemental mapping was performed on the purple phosphorus-manganese dioxide nanocomposite enzyme prepared in Example 1, and the results are shown in Figure 3As can be seen from D, the purple phosphorus-manganese dioxide nanocomposite enzyme contains P, Mn, O, C, and N elements, which preliminarily indicates that the purple phosphorus-manganese dioxide nanocomposite enzyme of the present invention has been successfully prepared. Figure 3 Figure E shows an atomic force microscope image and thickness statistics of the purple phosphorus-manganese dioxide nanocomposite enzyme. As can be seen from Figure E, the thickness of the nanocomposite enzyme is approximately 3.2 nm.

[0067] 3) X-ray photoelectron spectroscopy was performed on the purple phosphorus nanosheets, purple phosphorus-manganese dioxide nanocomposite enzyme, and manganese dioxide prepared in Example 1. The results are as follows: Figure 4 As shown. By Figure 4 As shown in Figure A, both the purple phosphorus nanosheet material and the purple phosphorus-manganese dioxide nanocomposite enzyme contain P, C, and O elements, and both manganese dioxide and the purple phosphorus-manganese dioxide nanocomposite enzyme also contain Mn element. High-resolution XPS images of P and Mn elements are shown in (see Figure A). Figure 4 B) indicates that in VPNSs, the strong singlets at 129.7 eV and 130.5 eV correspond to P 2p, respectively. 1 / 2 and P 2p 3 / 2 The characteristic peak at 133.7 eV represents crystalline P; while the broad peak near 133.7 eV indicates that partial oxidation occurred on the VPNSs surface during preparation. The characteristic peaks of MVPs are highly consistent with those of VPNSs, and the peak area ratio of crystalline P to oxidized P did not change significantly, indicating that the synthesis process of MVPs did not alter the composition of VPNSs. Except for Mn 2+ In addition to the characteristic peaks (652.5 eV and 640.1 eV), Mn in MVPs 4+ and Mn 7+ The signal distribution is similar to that of MnO2. In the prepared MVPs, Mn... 4+ The characteristic signals appear at 654.0 eV and 642.1 eV, and their intensity is much higher than that of Mn. 7+ The signals (656.0 eV and 644.1 eV) indicate that the Mn mineralized on the VPNSs surface mainly exists in the form of MnO2. High-resolution XPS results further confirm the successful binding of VPNSs and MnO2 in MVPs. These results show that the characteristic absorption peaks of the composite nanozyme perfectly match the characteristic peaks of purple phosphorus crystals and manganese dioxide crystals, indicating that the purple phosphorus-manganese dioxide nanocomposite enzyme of this invention was successfully prepared without altering its crystal structure.

[0068] X-ray diffraction pattern analysis was performed on the purple phosphorus-manganese dioxide nanocomposite enzyme prepared in Example 1, and the results are as follows: Figure 5 As shown. From Figure 5As can be seen, the prepared purple phosphorus-manganese dioxide nanocomposite enzyme exhibited diffraction peaks attributed to purple phosphorus nanosheets (corresponding to standard card PDF#44-0906), such as a single peak at a diffraction angle of 16.4° and multiple peaks in the 28.0°-35.3° range. This indicates that the structural integrity of the purple phosphorus nanosheets was preserved during the preparation of the purple phosphorus-manganese dioxide nanocomposite enzyme. Furthermore, diffraction peaks corresponding to manganese dioxide were observed at diffraction angles of 37.1° and 56.1° (corresponding to standard card PDF#30-0820); and the strong signal at 40.3° can be attributed to the superposition of VPNSs and manganese dioxide characteristic peaks. This result indicates that manganese dioxide was successfully loaded into the prepared purple phosphorus-manganese dioxide nanocomposite enzyme.

[0069] 3. Determination of the photothermal properties of purple phosphorus-manganese dioxide nanocomposite enzyme

[0070] The photothermal properties of the purple phosphorus-manganese dioxide nanocomposite enzyme prepared in Example 1 were tested using a power density of 1 W / cm². 2 The purple phosphorus-manganese dioxide nanocomposite enzyme, the purple phosphorus nanosheet material, and the manganese dioxide nanoparticle (MnO2) suspension prepared in Example 1 were irradiated with a 1064 nm NIR-II laser. The concentrations of the purple phosphorus-manganese dioxide nanocomposite enzyme and the purple phosphorus nanosheet material were 30 μg / mL (based on phosphorus); the concentration of MnO2 was 4.16 μg / mL (based on Mn). Water was used as a control group. The irradiation time was 10 minutes. The temperature of the system was monitored using a thermal infrared camera during the irradiation process. Furthermore, a power density of 1 W / cm² was used. 2 A 1064nm NIR-II laser was used to irradiate the purple phosphorus-manganese dioxide nanocomposite enzyme at a 10-minute on / 10-minute off frequency for a total of 5 cycles. During irradiation, the sample temperature was monitored using a thermal infrared camera. Figure 6 As shown in Figure A, the purple phosphorus-manganese dioxide nanocomposite enzyme exhibits a higher temperature rise under 1064 nm laser excitation, exceeding that of individual purple phosphorus nanosheets and manganese dioxide nanoparticles, demonstrating excellent photothermal properties; from Figure 6 As shown in section B, the purple phosphorus-manganese dioxide nanocomposite enzyme maintained a temperature increase of approximately 27°C after five heating / cooling cycles, indicating good photothermal stability. Therefore, the purple phosphorus-manganese dioxide nanocomposite enzyme prepared in this invention, when applied to the human body (at a 37°C environment), can raise the local temperature to above 60°C, meeting the temperature requirements of above 45°C required for photothermal therapy to cauterize tumors.

[0071] 4. Determination of the enzyme catalytic performance of purple phosphorus-manganese dioxide nanocomposite enzyme

[0072] 1) The glutathione peroxidase-like activity of the purple phosphorus-manganese dioxide nanocomposite enzyme prepared in Example 1 was tested. The test method was as follows: purple phosphorus-manganese dioxide nanocomposite enzyme (MVPs, concentration 30 μg / mL, based on P element), purple phosphorus nanosheet material (VPNSs, concentration 30 μg / mL, based on P element), manganese dioxide nanoparticles (MnO2, concentration 4.16 μg / mL, based on Mn element), and manganese ions (Mn 2+ The concentration of 4.16 μg / mL (based on Mn elemental) was dispersed in phosphate buffers of different pH values ​​(pH=6.5, 7.4), and then reduced glutathione (GSH, final concentration 1 mM) was added. Subsequently, a power density of 1 W / cm² was used / not used. 2 The mixture was continuously irradiated with a 1064 nm NIR-II laser for 10 minutes. Then, it was centrifuged at 43000 × g for 15 minutes at 4 °C. The supernatant was diluted 10-fold, and then 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) was added as a probe to detect GSH consumption, with water as a control group. Results are shown below. Figure 7 .

[0073] from Figure 7 It can be seen that the purple phosphorus-manganese dioxide nanocomposite enzyme has a significant GSH-consuming capacity, and its effect is stronger than that of using single purple phosphorus nanosheets, manganese dioxide nanoparticles and Mn. 2+ The experimental group showed that under pH=6.5 and 1064nm laser irradiation conditions, the purple phosphorus-manganese dioxide nanocomposite enzyme exhibited the strongest glutathionease-like activity, indicating that the purple phosphorus-manganese dioxide nanocomposite enzyme can reduce the antioxidant capacity of tumor sites (pH=6.5) by oxidizing GSH, and has little effect on cells under normal physiological conditions (pH=7.4).

[0074] 2) The catalytic activity of the purple phosphorus-manganese dioxide nanocomposite enzyme prepared in Example 1 for reactive oxygen species (ROS) production was tested. The test method was as follows: Purple phosphorus-manganese dioxide nanocomposite enzyme (MVPs, concentration 10 μg / mL, based on phosphorus), purple phosphorus nanosheets (VPNSs, concentration 10 μg / mL, based on phosphorus), manganese dioxide nanoparticles (MnO2, concentration 1.39 μg / mL, based on Mn), and deionized water (H2O, this group served as the blank control group) were dispersed in phosphate buffer (pH=6.5), then 400 μg / mL of 1,3-diphenylisobenzofuran (DPBF) was added, and finally H2O2 (concentration 100 μM) was added. Subsequently, a power density of 1 W / cm² was used / not used. 2 The suspension was continuously irradiated with a NIR-II laser at a wavelength of 1064 nm for 10 minutes. The UV absorption of the suspension was measured using a UV-Vis spectrometer at 0 and 10 minutes. The results are as follows:Figure 8 The results are shown in the following table.

[0075] From Figure 8 It can be seen that under dark conditions, the effect of active oxygen production in each experimental group is not obvious. After 10 minutes of 1064 nm laser irradiation, compared with the experimental groups using single purple phosphorus nanosheet material and manganese dioxide nanoparticles, the purple phosphorus-manganese dioxide nanocomposite enzyme has strong killing active oxygen production capacity in the hydrogen peroxide environment, and shows strong catalytic activity of active oxygen production. In summary, the purple phosphorus-manganese dioxide nanocomposite enzyme has the catalytic ability of glutathione-like enzyme and active oxygen production, and has the ability to regulate tumor microenvironment to kill tumors and prevent tumor recurrence and metastasis, while causing less damage to normal cells.

[0076] 5. Tumor cell activity determination of purple phosphorus-manganese dioxide nanocomposite enzyme

[0077] The toxicity of the purple phosphorus-manganese dioxide nanocomposite enzyme (MVPs) prepared in Example 1 to mouse breast cancer cells 4T1 was tested by enzyme catalysis-photothermal synergistic induction. The test method is as follows: 4T1 cells were inoculated into a 96-well plate at a density of 5×10 3 cells per well and cultured for 24 hours. After washing once with PBS, the culture medium containing the purple phosphorus-manganese dioxide nanocomposite enzyme (MVPs, concentration of 30 μg / mL, calculated based on P element), purple phosphorus nanosheet material (VPNSs, concentration of 30 μg / mL, calculated based on P element), and manganese dioxide nanoparticles (MnO2, concentration of 4.16 μg / mL, calculated based on Mn element) was added, and the cells were incubated for 6 hours. Then, the cells were treated with 1064 nm laser irradiation at a power density of 1 W / cm 2 for 10 minutes or in the dark, and then incubated for another 18 hours. After washing with PBS, the cells were incubated with Cell Counting Kit-8 (CCK-8) for 2 hours, and the absorbance at 450 nm wavelength was measured using a microplate reader to determine the relative cell viability by standard cell viability determination. The results are shown in the following table. Figure 9 .

[0078] From Figure 9 It can be seen that, under dark conditions, compared with the control group (Saline), the single use of manganese dioxide nanoparticles (MnO2) and the single use of purple phosphorus nanosheet material (VPNSs), the use of purple phosphorus-manganese dioxide nanocomposite enzyme showed stronger tumor cell toxicity. After 1064 nm laser irradiation, under the synergistic treatment of photothermal and enzyme catalysis, the toxicity of the purple phosphorus-manganese dioxide nanocomposite enzyme to tumor cells was greatly enhanced, achieving a significant tumor killing effect.

[0079] The above merely describes the preferred embodiments of the present application, but the protection scope of the present application is not limited thereto, and any person skilled in the art can easily think of changes or replacements within the technical scope disclosed by the present application, which should be covered in the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the protection scope of the claims.

Claims

1. A method for preparing a purple phosphorus-manganese dioxide nanocomposite enzyme, characterized in that, Comprise the following steps: (1) Preparation of purple phosphorus nanosheet material: disperse purple phosphorus powder in NMP, carry out probe cycle ultrasonic treatment and water bath ultrasonic treatment under ice bath condition, centrifuge at a speed of 2687xg for 20 minutes, collect supernatant, centrifuge at a speed of 43000xg for 30 minutes, collect precipitate, clean twice with anhydrous ethanol and water alternately to remove organic solvent, and obtain purple phosphorus nanosheet material; (2) Preparation of purple phosphorus-manganese dioxide nanocomposite enzyme: slowly drop potassium permanganate aqueous solution into the purple phosphorus nanosheet material aqueous solution prepared in step (1) at room temperature using a peristaltic pump, then add methoxypolyethylene glycol amine aqueous solution using a peristaltic pump, stir overnight, centrifuge to collect precipitate, and obtain purple phosphorus-manganese dioxide nanocomposite enzyme; The purple phosphorus-manganese dioxide nanocomposite enzyme is formed by in-situ mineralization of purple phosphorus nanosheet and nanometer manganese dioxide, and is modified with methoxypolyethylene glycol amine on the surface.

2. The production method according to claim 1, characterized by, The use ratio of the purple phosphorus powder and NMP is 1mg:2mL.

3. The production method according to claim 1, characterized by, The power of the probe cycle ultrasonic treatment is 500W, 2 seconds on / 3 seconds off for one cycle, and ultrasonic treatment is performed for 12 hours; The power of the water bath ultrasonic treatment is 600W, and ultrasonic treatment is performed for 8 hours.

4. The method of claim 1, wherein, The mass ratio of the purple phosphorus nanosheet material, potassium permanganate and methoxypolyethylene glycol amine is 1:1:

10.

5. The preparation method according to claim 1, characterized in that, The concentration of the purple phosphorus nanosheet material aqueous solution is 300μg / mL; The concentration of the potassium permanganate aqueous solution is 300μg / mL; The concentration of the methoxypolyethylene glycol amine aqueous solution is 10mg / mL.

6. Use of the purple phosphorus-manganese dioxide nanocomposite enzyme prepared by the preparation method of any one of claims 1-5 in preparation of a tumor treatment drug.

7. A medicament for treating a tumor, characterized by comprising the compound according to claim 1. The purple phosphorus-manganese dioxide nanocomposite enzyme prepared by the preparation method of any one of claims 1-5 is used as an active ingredient, and is prepared together with a pharmaceutically acceptable adjuvant.

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