Purple phosphorus-manganese dioxide nano compound enzyme as well as preparation method and application thereof

Abstract

The invention discloses a purple phosphorus-manganese dioxide nano-composite enzyme as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. The purple phosphorus-manganese dioxide nano-composite enzyme is formed by compounding a purple phosphorus nanosheet and nano-manganese dioxide through in-situ mineralization, and the surface of the purple phosphorus-manganese dioxide nano-composite enzyme is modified with methoxy polyethylene glycol amine. The preparation method comprises the following steps: preparing a purple phosphorus nanosheet material through ultrasonic liquid phase stripping; and at room temperature, dropwise adding a potassium permanganate aqueous solution into the purple phosphorus nanosheet material aqueous solution, then adding a methoxy polyethylene glycol amine aqueous solution, stirring overnight, and centrifugally collecting precipitate to obtain the purple phosphorus-manganese dioxide nano compound enzyme. The purple phosphorus-manganese dioxide nano-composite enzyme provided by the invention is composed of a manganese dioxide nano-material with self-enhanced chemical kinetic catalytic performance and a purple phosphorus nanosheet with near-infrared photothermal conversion performance, and the effect of killing tumor cells under NIR-II synergistic chemical kinetic catalysis can be realized.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to a purple phosphorus-manganese dioxide nanocomposite enzyme, its preparation method and application. Background Technology

[0002] Nanocatalysis therapy, as an important technological approach in the field of tumor treatment, relies on the core efficacy of nanocatalysts to precisely generate therapeutic products, such as toxic reactive oxygen species (ROS), in specific physiological regions under the regulation of external physical / chemical stimuli or the tumor microenvironment (TME). Alternatively, it can induce programmed cell death in tumor cells by eliminating harmful molecules that disrupt TME homeostasis. ROS generation can be achieved through various pathways, including chemical kinetics (typically Fenton / Fenton-like reactions), photodynamics, and acoustic kinetics. The core mechanism lies in leveraging the size effect and surface modification characteristics of nanomaterials to regulate the physicochemical properties of catalysts, such as their electronic structure, enabling them to efficiently produce cytotoxic ROS in response to TME characteristics or external physical stimuli. Clearly, catalytic efficiency is a key factor determining the therapeutic effect of catalytic medicine.

[0003] In the treatment of solid tumors, catalytic efficiency faces multiple severe limitations imposed by the tumor microenvironment (TME): First, insufficient substrate supply; the prevalent hypoxia and low concentration of hydrogen peroxide (H2O2) in tumor tissues directly restrict the generation efficiency of cytotoxic reactive oxygen species (ROS). Second, redox balance disruption; the high expression of reduced glutathione (GSH) and peroxidases in tumor cells consumes large amounts of the generated ROS, weakening the therapeutic effect. Third, mismatched reaction conditions; the optimal pH for maintaining low-valence metal concentrations in Fenton / Fenton-like reactions is typically 2-4, while the TME is weakly acidic (pH approximately 6.5), failing to meet suitable reaction conditions. Furthermore, externally induced catalytic reactions also have strict requirements on energy intensity and tissue penetration depth, further limiting the clinical application of catalytic therapy. Therefore, improving catalytic efficiency and overcoming the TME limitations have become core requirements for advancing tumor catalytic therapy, making the development of novel cancer catalytic medical technologies imperative.

[0004] Photothermal therapy (PTT), a safe and highly effective cancer treatment method, converts external laser energy into heat energy through a photothermal conversion agent enriched at the tumor site. This raises the temperature of the treatment area, causing denaturation of tumor cell macromolecules and dysfunction of organelles, thereby promoting cell death. However, the near-infrared I (NIR-I, wavelength 700-900nm) laser used in traditional photothermal therapy has significant drawbacks: its penetration depth in tumor tissue is relatively shallow, typically only about 5 mm. This makes it difficult to eliminate tumor cells outside the laser irradiation range, and PTT alone often cannot achieve a complete cure for the tumor. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention proposes a purple phosphorus-manganese dioxide nanocomposite enzyme, its preparation method, and its applications. Compared with existing technologies, near-infrared II (NIR-II, wavelength 1000-1700nm) lasers have significant advantages: the longer wavelength effectively reduces the absorption and scattering of light by biological tissues, allowing for penetration depths of several centimeters and coverage of a wider range of tumor tissues, making it particularly suitable for the treatment of deep solid tumors; simultaneously, NIR-II lasers cause less damage to normal tissues, achieving efficient tumor killing while reducing treatment side effects and improving treatment safety. Furthermore, NIR-II lasers can provide sufficient energy for photocatalytic therapy, ensuring effective activation of photosensitizers in deep solid tumor tissues. Therefore, combining NIR-II photothermal therapy with photocatalytic therapy can achieve complementary advantages of the two technologies, further enhancing the efficacy of nano-formulations in killing and eliminating tumor cells, and providing a new and effective strategy for the treatment of solid tumors.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] One of the objectives of this invention is to provide a purple phosphorus-manganese dioxide nanocomposite enzyme (MVPs), which is formed by in-situ mineralization of purple phosphorus nanosheets (VPNSs) and nano-manganese dioxide (MnO2), and the surface is modified with methoxy polyethylene glycol amine.

[0008] The method provided by this invention uses purple phosphorus nanosheets as a growth template to form nano-manganese dioxide through in-situ mineralization, and modifies it with methoxy polyethylene glycol amine to improve the pharmacokinetics of the nanoenzyme. The purple phosphorus-manganese dioxide nanocomposite enzyme provided by this invention consists of manganese dioxide nanomaterials with self-enhanced chemokinetic catalytic properties and purple phosphorus nanosheets with near-infrared photothermal conversion properties, enabling NIR-II synergistic chemokinetic catalysis to kill tumor cells.

[0009] The purple phosphorus-manganese dioxide nanocomposite enzyme of this invention exhibits a synergistic effect between purple phosphorus nanosheets and nano-manganese dioxide, killing and eliminating tumor cells, significantly enhancing the anti-tumor efficacy. Specifically, this is manifested in: firstly, manganese dioxide consumes GSH in the TME to generate Fenton-like reagent-like divalent manganese ions (Mn... 2+ ) and oxidized glutathione (GSSG), on the one hand, Mn 2+ As a Fenton-like reagent, it utilizes TME-overexpressed H2O2 to generate cytotoxic ROS. Furthermore, GSH consumption prolongs ROS lifetime, thus enhancing the catalytic efficacy through chemical kinetics. Secondly, purple phosphorus nanosheets possess NIR-II photocatalytic activity. On one hand, they can simultaneously catalyze the generation of cytotoxic ROS using both H2O and H2O2 as substrates, overcoming the limitations on catalytic effects caused by insufficient substrate variety and concentration. On the other hand, purple phosphorus nanosheets also have the ability to consume GSH, forming NIR-II-mediated self-amplified TME redox homeostasis disruption. Thirdly, purple phosphorus nanosheets possess NIR-II photothermal activity, capable of converting light energy into heat energy to kill tumor cells, achieving NIR-II photothermal synergistic nanocatalytic tumor therapy. Fourthly, purple phosphorus nanosheets have a high specific surface area and abundant attachment sites, promoting uniform loading and in-situ mineralization of MnO2 on their surface, forming a stable composite structure and preventing MnO2 nanoparticle aggregation.

[0010] Furthermore, the purple phosphorus nanosheets are prepared by ultrasonic liquid phase exfoliation, and the nano-manganese dioxide is formed by in-situ mineralization using the purple phosphorus nanosheets as a growth template.

[0011] The second objective of this invention is to provide a method for preparing a purple phosphorus-manganese dioxide nanocomposite enzyme, comprising the following steps:

[0012] (1) Purple phosphorus nanosheets were prepared by ultrasonic liquid phase exfoliation;

[0013] (2) At room temperature, potassium permanganate aqueous solution was added dropwise to purple phosphorus nanosheet material aqueous solution to obtain intermediate product mixture;

[0014] (3) Add methoxy polyethylene glycol amine aqueous solution to the intermediate product mixture, stir overnight, centrifuge and collect the precipitate to obtain purple phosphorus-manganese dioxide nanocomposite enzyme.

[0015] Furthermore, the preparation method of the purple phosphorus nanosheet material includes the following steps: dispersing purple phosphorus powder in an organic solvent, performing probe cyclic ultrasonic treatment and water bath ultrasonic treatment under ice bath conditions, centrifuging at 2687×g for 20 minutes, collecting the supernatant, centrifuging at 43000×g for 30 minutes, collecting the precipitate, and washing repeatedly with anhydrous ethanol and water to remove the organic solvent, thereby obtaining the purple phosphorus nanosheet material.

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

[0017] Furthermore, the power of the probe for cyclic ultrasonic treatment is 500W, with 2 seconds on and 3 seconds off constituting one cycle, and the ultrasonic treatment lasts for 12 hours;

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

[0019] The purple phosphorus nanosheets prepared by the above method have an average particle size of 197.5 nm and a thickness of 2.50-2.90 nm.

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

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

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

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

[0024] The third objective of this invention is to provide an application of purple phosphorus-manganese dioxide nanocomposite enzyme in the preparation of drugs for treating tumors.

[0025] The fourth objective of this invention is to provide a tumor treatment drug, which uses the aforementioned purple phosphorus-manganese dioxide nanocomposite enzyme as the active ingredient and is formulated with pharmaceutically acceptable excipients.

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

[0027] This invention provides a purple phosphorus-manganese dioxide nanocomposite enzyme, which has a simple preparation method and controllable size; it has good catalytic activity and good photothermal properties. Experiments have shown that the purple phosphorus-manganese dioxide nanocomposite enzyme provided by this invention can achieve oxidative damage and apoptosis of tumor cells through self-enhanced catalytic therapy; at the same time, the good NIR-II photothermal properties of the purple phosphorus nanosheet component in this nanoenzyme synergistically kill tumor cells, achieving the effect of NIR-II photothermal synergistic catalytic therapy for tumor treatment. Attached Figure Description

[0028] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:

[0029] Figure 1 The diagram shows the structural properties of the purple phosphorus nanosheets prepared in Example 1; where A is a transmission electron microscope image of the purple phosphorus nanosheets, B is a high-resolution transmission electron microscope image of the purple phosphorus nanosheets, C is a simulated interplanar spacing of the purple phosphorus nanosheets, D is a simulated interplanar spacing of the purple phosphorus nanosheets, and E is an atomic force microscope image and thickness statistics of the purple phosphorus nanosheets.

[0030] Figure 2 The hydrodynamic particle size distribution of the purple phosphorus nanosheet material and the purple phosphorus-manganese dioxide nanocomposite enzyme prepared in Example 1 is shown in the statistical diagram.

[0031] Figure 3 The diagram shows the structural properties of the purple phosphorus-manganese dioxide nanocomposite enzyme prepared in Example 1; where A is a transmission electron microscope image of the purple phosphorus-manganese dioxide nanocomposite enzyme, B is a high-resolution transmission electron microscope image of the purple phosphorus-manganese dioxide nanocomposite enzyme, C is the simulated interplanar spacing of the purple phosphorus-manganese dioxide nanocomposite enzyme, D is the energy dispersive X-ray elemental mapping (EDX-mapping) spectrum of the purple phosphorus-manganese dioxide nanocomposite enzyme, and E is an atomic force microscope image 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, purple phosphorus-manganese dioxide nanocomposite enzyme, and manganese dioxide (MnO2) prepared in Example 1; wherein, A is the full XPS spectrum, and B is the high-resolution XPS of P and Mn elements;

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

[0034] Figure 6 The results of the photothermal properties study of the purple phosphorus nanosheet material, purple phosphorus-manganese dioxide nanocomposite enzyme, and manganese dioxide prepared in Example 1 are shown; where A is the photothermal heating curve and B is the photothermal stability of the purple phosphorus-manganese dioxide nanocomposite enzyme.

[0035] Figure 7 The purple phosphorus nanosheets, purple phosphorus-manganese dioxide nanocomposite enzyme, and manganese dioxide and manganese ions (Mn) prepared in Example 1 are described. 2+The results of the study on the glutathione peroxidase activity of different materials are as follows: A represents the glutathione peroxidase activity of different materials under pH 7.4 without 1064nm light; B represents the glutathione peroxidase activity of different materials under pH 7.4 with 1064nm light; C represents the glutathione peroxidase activity of different materials under pH 6.5 without 1064nm light; and D represents the glutathione peroxidase activity of different materials under pH 6.5 with 1064nm light.

[0036] Figure 8 The purple phosphorus nanosheets, purple phosphorus-manganese dioxide nanocomposite enzyme, and manganese dioxide and manganese ions (Mn) prepared in Example 1 are described. 2+ The catalytic activity of ROS production;

[0037] Figure 9 This is a statistical chart showing the purple phosphorus nanosheet material, purple phosphorus-manganese dioxide nanocomposite enzyme, and manganese dioxide tumor cell toxicity test results prepared in Example 1. Detailed Implementation

[0038] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0039] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0040] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0041] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0042] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0043] This invention provides a method for preparing a purple phosphorus-manganese dioxide nanocomposite enzyme, comprising the following steps:

[0044] (1) Preparation of purple phosphorus nanosheet material: Purple phosphorus powder was dispersed in NMP, and subjected to probe cyclic ultrasonic treatment and water bath ultrasonic treatment under ice bath conditions. The supernatant was collected after centrifugation at 2687×g for 20 minutes, and then centrifuged at 43000×g for 30 minutes. The precipitate was collected and washed twice with anhydrous ethanol and water to remove organic solvent, and purple phosphorus nanosheet material was obtained. The average particle size of the obtained purple phosphorus nanosheets was 197.5 nm and the average thickness was 2.50-2.90 nm.

[0045] (2) Preparation of purple phosphorus-manganese dioxide nanocomposite enzyme: At room temperature, potassium permanganate aqueous solution (KMnO4, concentration of 300 μg / mL) was slowly added dropwise to the purple phosphorus nanosheet material aqueous solution (VPNSs concentration of 300 μg / mL) prepared in step (1) using a peristaltic pump. Then, methoxy polyethylene glycol amine aqueous solution (concentration of 10 mg / mL) was added using a peristaltic pump. The mixture was stirred overnight, and the precipitate was collected by centrifugation (speed of 43000×g, time of 30 minutes) to obtain purple phosphorus-manganese dioxide nanocomposite enzyme.

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

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

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

[0049] In the following preferred embodiments of the present invention, 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 using the above preparation method.

[0051] The purple phosphorus-manganese dioxide nanocomposite enzyme can be used in the preparation of drugs for treating tumors.

[0052] A tumor treatment drug can be prepared by using the aforementioned purple phosphorus-manganese dioxide nanocomposite enzyme as the active ingredient and supplementing it with pharmaceutically acceptable excipients.

[0053] Unless otherwise specified, "room temperature" in this invention refers to 25±2℃.

[0054] All raw materials used in this invention were purchased from the market.

[0055] The technical solution of the present invention will be further illustrated by the following embodiments.

[0056] Example 1

[0057] A method for preparing a purple phosphorus-manganese dioxide nanocomposite enzyme includes the following steps:

[0058] (1) Preparation of purple phosphorus nanosheet material: 40 mg of purple phosphorus powder was weighed and dispersed in 80 mL of organic solvent NMP. The probe was ultrasonically treated for 12 hours under ice bath and 500 W power conditions with a cycle of 2 seconds on and 3 seconds off. Then, it was ultrasonically treated in a water bath at 4 °C for 8 hours under 600 W power conditions. Then, it was centrifuged at 2687 × g speed for 20 minutes at 4 °C. The supernatant was collected. Then, it was centrifuged at 43000 × g speed for 30 minutes at 4 °C. The precipitate was collected and washed twice with anhydrous ethanol / water alternately to remove organic solvent, thus obtaining purple phosphorus nanosheet material (VPNSs).

[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, potassium permanganate aqueous solution (KMnO4, concentration 300 μg / mL, volume 500 μL) was slowly added dropwise to the purple phosphorus nanosheet material aqueous solution (VPNSs, concentration 300 μg / mL, volume 500 μL) prepared in step S1 at a flow rate of 1.5 mL / min using a constant flow pump. The mixture was stirred at 1000 rpm for 30 minutes using a magnetic stirrer. Subsequently, methoxy polyethylene glycol amine (NH2-PEG-OMe, concentration 10 mg / mL, volume 150 μL) was added at a flow rate of 1.5 mL / min using a constant flow pump. The mixture was stirred overnight at 1000 rpm. The precipitate was collected by centrifugation at 4°C (43000 × g, time 30 minutes) and washed twice with deionized water to obtain purple phosphorus-manganese dioxide nanocomposite enzymes (MVPs).

[0060] Performance testing

[0061] 1. Determination of the structural properties of purple phosphorus nanosheet materials

[0062] The purple phosphorus nanosheets prepared in Example 1 were analyzed by transmission electron microscopy. The results are shown in [link to relevant documentation]. Figure 1 .Depend on Figure 1 As can be seen from A in the diagram, the prepared purple phosphorus nanosheets are rectangular; from Figure 1 As can be seen from B, the diffraction fringes of the purple phosphorus nanosheet lattice are clear; from Figure 1 From C and D in the diagram, the interplanar spacings fitted according to Figure B are 2.88 Å and 6.27 Å, corresponding to 2Theta angles of 31.0° and 13.7° on the purple phosphorus PDF#44-0906 card, indicating that the purple phosphorus nanosheets are purple phosphorus crystals; Figure 1 As indicated by E, the thickness of the purple phosphorus nanosheets is between 2.50 and 2.90 nm. These results demonstrate the successful preparation of the purple phosphorus nanosheets.

[0063] The hydrodynamic particle size distribution of the purple phosphorus nanosheets prepared in Example 1 was statistically analyzed, and the results are shown in [reference needed]. Figure 2 .Depend on Figure 2 It can be seen that the particle size of the purple phosphorus nanosheets exhibits a Gaussian distribution, with an average hydrated particle size of 197.5 nm.

[0064] 2. Determination of the physicochemical properties of purple phosphorus-manganese dioxide nanocomposite enzyme

[0065] 1) The hydrodynamic particle size distribution of the purple phosphorus-manganese dioxide nanocomposite enzyme prepared in Example 1 was statistically analyzed, and the results are as follows: Figure 2 As shown, the particle size of the purple phosphorus-manganese dioxide nanocomposite enzyme exhibits a Gaussian distribution, with an average hydrated particle size of 211.5 nm.

[0066] 2) The purple phosphorus-manganese dioxide nanocomposite enzyme prepared in Example 1 was analyzed by transmission electron microscopy. The results are shown in [reference needed]. Figure 3 .Depend on Figure 3 As indicated by A in the diagram, the purple phosphorus-manganese dioxide nanocomposite enzyme has a rectangular sheet-like structure with a particle size of approximately 150 nm; Figure 3 As shown in B in the figure, the surface of the purple phosphorus-manganese dioxide nanocomposite enzyme exhibits both clear lattice diffraction fringes (rectangular frames) and amorphous material (circular frames). Fourier transform and inverse Fourier transform of the images within the rectangular frames in figure B yielded a fitted interplanar spacing of 6.42 Å (see Figure B). Figure 3 The C in the figure corresponds to a 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 purple phosphorus crystals and portions coated with MnO2 or PEG. EDS-elemental surface analysis was performed on the purple phosphorus-manganese dioxide nanocomposite enzyme prepared in Example 1. 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 As shown.

[0075] from Figure 8 It can be seen that under dark conditions, the production of reactive oxygen species (ROS) in each experimental group was not significant. After 10 minutes of 1064nm laser irradiation, compared with the experimental group using only purple phosphorus nanosheets and manganese dioxide nanoparticles, the purple phosphorus-manganese dioxide nanocomposite enzyme exhibited a strong ability to generate cytotoxic ROS in a hydrogen peroxide environment, demonstrating strong catalytic activity for ROS production. In summary, the purple phosphorus-manganese dioxide nanocomposite enzyme possesses glutathionease-like catalytic ability to produce ROS, and has the capacity to regulate the tumor microenvironment to kill tumors and prevent tumor recurrence and metastasis, while causing minimal damage to normal cells.

[0076] 5. Assay of the activity of purple phosphorus-manganese dioxide nanocomposite enzyme against tumor cells

[0077] The toxicity of the purple phosphorus-manganese dioxide nanocomposite enzymes (MVPs) prepared in Example 1 to mouse breast cancer cells 4T1 was tested by enzyme catalysis-photothermal synergistic toxicity. The test method was as follows: 4T1 cells were cultured at 5 × 10⁶ cells per well. 3 Cells were seeded at a density of 1000 cells / well in 96-well plates and cultured for 24 hours. After washing once with PBS, culture medium containing purple phosphorus-manganese dioxide nanocomposite enzymes (MVPs, 30 μg / mL, based on phosphorus), purple phosphorus nanosheets (VPNSs, 30 μg / mL, based on phosphorus), and manganese dioxide nanoparticles (MnO2, 4.16 μg / mL, based on Mn) was added, and the cells were incubated for another 6 hours. Then, the cells were subjected to continuous incubation at a power density of 1 W / cm² for 10 minutes. 2 Cells were either exposed to laser light at a wavelength of 1064 nm or treated in the dark, followed by incubation for 18 hours. After washing with PBS, cells were incubated for 2 hours using a Cell Counting Kit-8 (CCK-8). Absorbance at 450 nm was measured using a microplate reader to determine relative cell viability using standard cell viability assays. Results are shown below. Figure 9 .

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

[0079] The above are merely preferred embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A purple phosphorus-manganese dioxide nanocomposite enzyme, characterized in that, It is formed by in-situ mineralization of purple phosphorus nanosheets and nano manganese dioxide, and its surface is modified with methoxy polyethylene glycol amine.

2. The purple phosphorus-manganese dioxide nanocomposite enzyme according to claim 1, characterized in that, The purple phosphorus nanosheets were prepared by ultrasonic liquid phase exfoliation, and the nano-manganese dioxide was formed by in-situ mineralization using the purple phosphorus nanosheets as a growth template.

3. A method for preparing the purple phosphorus-manganese dioxide nanocomposite enzyme as described in any one of claims 1-2, characterized in that, Includes the following steps: Purple phosphorus nanosheets were prepared by ultrasonic liquid phase exfoliation. At room temperature, potassium permanganate aqueous solution was added dropwise to purple phosphorus nanosheet material aqueous solution to obtain intermediate product mixture; An aqueous solution of methoxy polyethylene glycolamine was added to the intermediate product mixture, stirred overnight, and the precipitate was collected by centrifugation to obtain purple phosphorus-manganese dioxide nanocomposite enzyme.

4. The preparation method according to claim 3, characterized in that, The preparation method of the purple phosphorus nanosheet material includes the following steps: dispersing purple phosphorus powder in an organic solvent, performing probe cyclic ultrasonic treatment and water bath ultrasonic treatment under ice bath conditions, collecting the supernatant by centrifugation, collecting the precipitate by centrifugation again, and washing with anhydrous ethanol and water alternately to obtain the purple phosphorus nanosheet material.

5. The preparation method according to claim 4, characterized in that, The ratio of purple phosphorus powder to organic solvent is 1 mg: 2 mL; the organic solvent is N-methylpyrrolidone.

6. The preparation method according to claim 4, characterized in that, The probe has a cyclic ultrasonic power of 500W, with 2 seconds on and 3 seconds off as one cycle, and the ultrasonic treatment lasts for 12 hours. The water bath ultrasonic treatment has a power of 600W and lasts for 8 hours.

7. The preparation method according to claim 3, characterized in that, The mass ratio of the purple phosphorus nanosheet material, potassium permanganate, and methoxy polyethylene glycol amine is 1:1:

10.

8. The preparation method according to claim 3, characterized in that, The concentration of the aqueous solution of the purple phosphorus nanosheet material is 300 μg / mL; The concentration of the potassium permanganate aqueous solution is 300 μg / mL; The concentration of the methoxy polyethylene glycolamine aqueous solution is 10 mg / mL.

9. The use of the purple phosphorus-manganese dioxide nanocomposite enzyme as described in any one of claims 1-2 in the preparation of a tumor-treating drug.

10. A tumor treatment drug, characterized in that, It is prepared using the purple phosphorus-manganese dioxide nanocomposite enzyme as described in any one of claims 1-2 as the active ingredient, supplemented with pharmaceutically acceptable excipients.

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