Dihydrophenanthro-modified metal oxide nanozyme diagnosis and treatment system and preparation method and application thereof

Abstract

The application discloses a preparation method and application of a dihydrophenanthrope modified metal oxide nanozyme diagnosis and treatment system. The nanozyme diagnosis and treatment system is mainly composed of bovine serum albumin modified manganese dioxide, dopamine modified ferroferric oxide nanoparticles and photosensitizer dihydrophenanthrope e6. The nanozyme diagnosis and treatment system can be passively targeted to a tumor site through the high permeability and retention effect of a solid tumor. Then, O2 is generated through the peroxidase-like activity of the nanozyme diagnosis and treatment system, and hydroxyl radicals (·OH) are generated through a Fenton reaction, so as to relieve the hypoxia problem of a tumor microenvironment, enhance the curative effect of PDT, and enhance the immunogenic death of tumor cells. In addition, the superparamagnetic property of the MNPT enables the MNPT to be used as a contrast agent for magnetic resonance imaging, so as to provide fluorescence-magnetic resonance dual-mode imaging, and track and feedback the treatment effect in real time.

Description

Technical Field

[0001] This invention belongs to the field of biomedical nanomaterials and technology, specifically relating to the preparation method and application of a dihydroporphyrin-modified metal oxide nanoenzyme diagnostic and therapeutic system. Background Technology

[0002] Photodynamic therapy (PDT), a non-invasive treatment with relatively few toxic side effects, has rapidly developed into a popular cancer treatment method in recent years. It utilizes photosensitizers enriched at the tumor site, which, upon irradiation with a specific wavelength of laser light, convert surrounding oxygen (O2) into cytotoxic reactive oxygen species (ROS), thereby killing tumor cells. Despite significant advancements in cancer treatment, the hypoxic tumor microenvironment (TME) remains a major bottleneck in its clinical application. While O2 is essential for PDT, the malignant proliferation of tumor cells and the irregular growth of intratumoral blood vessels, leading to poor blood supply and vascular obstruction, can cause hypoxia in the tumor microenvironment, severely impacting the effectiveness of PDT. Therefore, addressing the hypoxia problem in the tumor microenvironment is crucial for improving the efficacy of PDT.

[0003] Many methods have been developed to alleviate hypoxia in the tumor microenvironment, among which utilizing nanoparticles to transport or generate O2 is a promising strategy. On one hand, nanoparticles can utilize their enzyme-like activity to generate O2 in situ, thus enhancing the efficacy of photodynamic therapy (PDT). On the other hand, nanoparticles can target tumor sites through enhanced permeability and retention effect (EPR), ensuring efficient O2 transport. In recent years, the application of metal oxide nanozymes in the biological field has seen rapid research and development. Due to their unique enzyme-like activity, they can catalyze the high levels of H2O2 in tumor tissue into O2 through catalase-like activity, enhancing the efficacy of PDT; or they can catalyze the production of ROS from H2O2 and O2 through peroxidase-like (POD) and superoxide dismutase (SOD) activities, inducing tumor cell apoptosis. Simultaneously, nanozymes can also promote the transformation of tumor-associated macrophages, improve the immunosuppression of the tumor microenvironment, and thus enhance the effect of immunotherapy.

[0004] Therefore, to address the hypoxia problem in the tumor microenvironment and enhance the efficacy of tumor cell decomposition therapy (PDT) and immune killing, this experiment proposes a construction scheme for a magnetic nano-platform (MNPT) based on a Fe3O4 / MnO2 composite nanozyme. MNPT consists of bovine albumin (BSA)-modified manganese dioxide (MnO2), dopamine (DPA)-modified Fe3O4 nanoparticles, and the photosensitizer dihydroporphyrin e6 (Chlorin e6, Ce6). As a nanomaterial, MNPT passively targets the tumor site through the high permeability and retention effect of solid tumors. Then, through its catalase-like activity, it generates O2, which in turn produces hydroxyl radicals (·OH) via the Fenton reaction, thereby alleviating the hypoxia problem in the tumor microenvironment, enhancing the efficacy of PDT, and simultaneously increasing the immunogenic death of tumor cells. In addition, the superparamagnetism of MNPT allows it to be used as a contrast agent for magnetic resonance imaging, thereby providing fluorescence-magnetic resonance dual-modality imaging to track and provide feedback on treatment effects in real time. Summary of the Invention

[0005] The purpose of this invention is to alleviate the problem of hypoxic tumor microenvironment limiting the efficacy of photodynamic therapy. A dihydroporphyrin-modified metal oxide nanoenzyme therapeutic system is designed using nanotechnology to alleviate tumor microenvironment hypoxia, enhance the efficacy of photodynamic therapy, and promote immune killing.

[0006] This invention is achieved through the following technical solution:

[0007] A method for preparing a dihydroporphyrin-modified metal oxide nanozyme diagnostic and therapeutic system includes the following preparation steps:

[0008] Oil-phase nanoparticles OAm@Fe3O4 were prepared by high-temperature pyrolysis.

[0009] Water-soluble magnetic nanoparticles DPA@Fe3O4 were prepared using the nanoparticles OAm@Fe3O4 via a ligand exchange method.

[0010] Preparation of magnetic nanoparticles BSA@MnO2;

[0011] The MNPT nanoenzyme diagnostic and therapeutic system was prepared using the nanoparticles OAm@Fe3O4 and the magnetic nanoparticles BSA@MnO2.

[0012] As a preferred aspect of the present invention, the preparation of oil-phase nanoparticles OAm@Fe3O4 by high-temperature pyrolysis includes:

[0013] Weigh 5.25 g (15 mmol) of ferric acetylacetone (Fe(acac)3) powder into a 500 mL three-necked flask, then add 40 mL (210 mmol) of dibenzyl ether and 60 mL (165 mmol) of oleylamine, mix well, and carry out the reaction under nitrogen protection and reflux with a condenser at a programmed temperature. Before the reaction starts, nitrogen gas is introduced into the reaction vessel to ensure that oxygen in the apparatus is removed; the nitrogen flow rate is 50 mL / min and the duration is about 5 min.

[0014] Then, seal all the connection ports in the temperature control device with sealing film, and set the temperature control device to a two-stage programmed heating process: the first stage is to raise the temperature from room temperature to 220℃ and then maintain the temperature for 1 hour, with a heating rate of 3.3℃ / min; the second stage is to continue to raise the temperature from 220℃ to 290℃ and then maintain the temperature for 5 minutes, with a heating rate of 3.3℃ / min; the temperature control device can be the LC6 programmable temperature controller produced by JULABO GmbH, Germany.

[0015] After the reaction is complete, the heating device is quickly removed to allow the solution temperature in the reaction system to drop sharply to room temperature, thus obtaining OAm@Fe3O4 with a particle size of 5 nm.

[0016] As a preferred aspect of the present invention, the preparation of water-soluble magnetic nanoparticles DPA@Fe3O4 using the nanoparticles OAm@Fe3O4 via ligand exchange includes:

[0017] Weigh 400 mg of dopamine hydrochloride into a 250 mL three-necked flask, add 40 mL of anhydrous dimethyl sulfoxide (DMSO) and 40 mL of chloroform mixture to dissolve it, and mix well;

[0018] Then, the stirrer speed was adjusted to 1000 rpm / min, and 20 mL of OAm@Fe3O4 with a concentration of 10 mg Fe / mL was added dropwise under ultrasonic conditions in a 70℃ water bath. After reacting for 2 hours, the reactants were taken out and placed in an ultracentrifuge tube with 20 mL of chloroform added. The mixture was centrifuged at 5000 rpm / min for 5 minutes, the supernatant was removed, the precipitate was dried with nitrogen, and then 40 mL of ultrapure water was added for ultrasonic dissolution. The pH of the solution was adjusted to 4.0 to obtain dopamine-encapsulated water-soluble ultra-small superparamagnetic Fe3O4 nanoparticles (DPA@Fe3O4).

[0019] As a preferred aspect of the present invention, the preparation of the nanoenzyme diagnostic and therapeutic system MNPT using the nanoparticles OAm@Fe3O4 and the magnetic nanoparticles BSA@MnO2 includes:

[0020] Weigh 31.6 mg of potassium permanganate and dissolve it in 3 mL of ultrapure water for later use. Dissolve 250 mg of bovine serum albumin in 7 mL of ultrapure water and add it dropwise to the potassium permanganate solution. Stir magnetically at 37 °C for 2 h to obtain BSA@MnO2.

[0021] The preparation of the nanozyme diagnostic and therapeutic system MNPT includes:

[0022] Take 0.5 mL of BSA@MnO2, 1.3 mL of ultrapure water and 50 μL of dihydroporphyrin e6 and mix them evenly. Then add 200 μL of EDC with a concentration of 2.5 mg / mL. Place the mixture in a shaker at 37°C for 4 h and store it at 4°C overnight. The next day, add 100 μL of DPA@Fe3O4 while vortexing to obtain the nanozyme diagnostic and therapeutic system MNPT.

[0023] A dihydroporphyrin-modified metal oxide nanozyme diagnostic and therapeutic system based on the above-prepared system is mainly composed of bovine serum albumin-modified manganese dioxide nanoparticles, dopamine-modified iron tetroxide nanoparticles, and the photosensitizer dihydroporphyrin E6.

[0024] As a preferred aspect of the present invention, the mass ratio of the bovine serum albumin-modified manganese dioxide nanoparticles, the dopamine-modified iron tetroxide nanoparticles, and the photosensitizer dihydroporphyrin E6 is 3:1:1.

[0025] As a preferred aspect of the present invention, the core of the metal oxide nanoenzyme diagnostic and therapeutic system is a composite nanoenzyme of iron tetroxide / manganese dioxide; the nanoenzyme diagnostic and therapeutic system exists stably in aqueous solution and has a hydrodynamic size of approximately 25.79±3.45 nm.

[0026] As a preferred aspect of the present invention, the metal oxide nanoenzyme diagnostic and therapeutic system performs fluorescence imaging through the fluorescence of the photosensitizer itself, or the metal oxide nanoenzyme diagnostic and therapeutic system performs magnetic resonance imaging through magnetic nanoparticles.

[0027] As a preferred aspect of the present invention, the metal oxide nanoenzyme diagnostic and therapeutic system passively targets the tumor site through high permeability and retention effect.

[0028] Compared with the prior art, the present invention has the following beneficial effects.

[0029] The nanozyme therapy system of this invention can alleviate the hypoxia problem in the tumor microenvironment, thereby improving the efficacy of photodynamic therapy and enhancing immunotherapy. On one hand, the photosensitizer coupled to this nanozyme therapy system can passively target the tumor site through high permeability and retention effects, enhancing its retention time at the tumor site. On the other hand, the nanozyme therapy system is composed of metal oxide nanozymes, which can exert catalase-like and peroxidase-like activities, thereby generating oxygen and hydroxyl radicals, alleviating the hypoxia problem in tumor photodynamic therapy and improving the tumor-killing effect. Simultaneously, the nanozyme therapy system can also provide fluorescence-magnetic resonance dual-modal imaging for real-time monitoring and feedback of the effects.

[0030] Specific embodiments of the present invention are disclosed in detail with reference to the following description and accompanying drawings, indicating how the principles of the invention can be employed. It should be understood that the embodiments of the present invention are not limited in scope as a result.

[0031] Features described and / or illustrated for one embodiment may be used in the same or similar manner in one or more other embodiments, combined with features in other embodiments, or substituted for features in other embodiments.

[0032] It should be emphasized that the term "including / comprises" as used herein refers to the presence of a feature, whole, step, or component, but does not exclude the presence or addition of one or more other features, wholes, steps, or components. Attached Figure Description

[0033] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0034] Figure 1 This is a schematic diagram of the structural composition of a dihydroporphyrin-modified metal oxide nanozyme diagnostic and therapeutic system.

[0035] Figure 2 a to Figure 2 d shows transmission electron micrographs of different nanomaterials. Among them, Figure 2 a is dopamine-modified iron oxide nanoparticles (DPA@Fe3O4) prepared by ligand exchange method. Figure 2 b represents bovine serum albumin-modified manganese dioxide nanoparticles (BSA@MnO2). Figure 2 c is BSA@Fe3O4 / MnO2. Figure 2 d is a dihydroporphyrin-modified metal oxide nanozyme therapeutic system (MNPT).

[0036] Figure 3 a to Figure 3 f is the experimental plot of MNPT's functional characterization. Figure 3 a represents the UV absorption spectra of BSA@MnO2, DPA@Fe3O4, BSA@Fe3O4 / MnO2, MNPT, and Ce6. Figure 3 b is the fluorescence spectrum of MNPT and Ce6. Figure 3 c represents the in vitro release of MNPT in buffer systems at pH 5.0, 6.0, and 7.0. Figure 3 d to Figure 3 e is a T1-enhanced magnetic resonance imaging image of MNPT and BSA@MnO2 (manganese content in nanoparticles is 1 mol / mL, 0.5 mol / mL, 0.25 mol / mL, 0.125 mol / mL, and 0.0625 mol / mL). Figure 3 f is a graph showing the ability of BSA@MnO2, DPA@Fe3O4, MNPT, and H2O to consume glutathione.

[0037] Figure 4 a to Figure 4 f represents the study of MNPT-like enzyme activity. Figure 4 a to Figure 4 c) The colorimetric reaction of 3,3',5,5'-tetramethylbenzidine (TMB) in buffer systems at pH 5.0, 6.0, and 7.0 verified that MNPT has peroxidase-like (POD) activity. Figure 4 d to Figure 4 f is to use a dissolved oxygen meter to detect and record the concentration of dissolved oxygen in buffer systems at pH 5.0, 6.0 and 7.0, to prove that MNPT has catalase-like activity.

[0038] Figure 5 a to Figure 5 h represents the in vitro cell experiment of MNPT. Figure 5 a to Figure 5 b is a graph showing the intracellular ROS levels detected using the DCFH-DA fluorescent probe. Figure 5 c to Figure 5 Ce6 and MNPT were co-incubated with cells for 24 h. DAPI solution was added for staining, and cell phagocytosis was observed and recorded using a fluorescence microscope at 0 h, 2 h, 4 h, and 6 h. Figure 5 e to Figure 5 f is a process where cells are co-incubated with BSA@MnO2, DPA@Fe3O4, BSA@Fe3O4 / MnO2, MNPT, and Ce6 for 6 hours, then irradiated with a laser for 15 seconds per well. Cell scratch results are recorded at 0 and 24 hours using an inverted fluorescence microscope. Figure 5g is a graph showing the killing effect of MNPT on tumor cells after co-incubating different concentrations of BSA@Fe3O4 / MnO2, Ce6, and MNPT with cells for 6 hours, followed by laser irradiation for 15 seconds per well for 24 hours, using a CCK-8 kit. Figure 5 h is an apoptosis graph of 4T1 cells after co-incubation with BSA@Fe3O4 / MnO2, Ce6, MNPT and cells for 6 h, followed by laser irradiation for 15 s / well, and co-incubation for 24 h.

[0039] Figure 6 a to Figure 6 Figure g shows in vivo experimental data from breast cancer mice constructed using 4T1 cells. Figure 6 a to 6c represent tumor changes, survival time, and weight changes in tumor-bearing mice after different treatment regimens (treatment groups were Control+laser, Control, BSA@Fe3O4 / MnO2+laser, BSA@Fe3O4 / MnO2, MNPT+laser, MNPT, Ce6+laser, and Ce6, respectively). The nanoparticles were administered subcutaneously near the tumor site at a dose of 0.5 mg [Fe] / g, administered four times every other day. The laser group received 660 nm laser irradiation (0.5 mg / g) at 4 h and 24 h after injection. W / cm 2 5 min / mouse. All treatments resulted in subcutaneous tumors reaching 60 mm in size in mice. 3 (Start from left and right.) Figure 6 d is a fluorescence imaging image of tumor-bearing mice at different time points after injection of MNPT (the injection method was subcutaneous administration next to the tumor, and the injection dose was 1 μg [Fe] / g); Figure 6 e is a T1-enhanced magnetic resonance imaging image of tumor-bearing mice 4 hours after injection of MNPT (the injection method was subcutaneous administration next to the tumor, and the injection dose was 1 μg [Fe] / g); Figure 6 f represents the signal ratio between the tumor site and the surrounding normal tissue in mice; Figure 6 g is a fluorescence imaging image of the major organs and tumor tissues taken from tumor-bearing mice 24 hours after injection of MNPT (the injection method was subcutaneous administration next to the tumor, and the injection dose was 1 μg [Fe] / g).

[0040] Figure 7 This is a schematic diagram illustrating the construction of a dihydroporphyrin-modified metal oxide nanozyme diagnostic and therapeutic system and its enhancement effect on tumor photodynamic therapy. Detailed Implementation

[0041] To enable those skilled in the art to better understand the technical solutions of this invention, the technical solutions of the embodiments of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of this invention.

[0042] 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 invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0043] Example 1

[0044] The complex described in this invention consists of three parts ( Figure 1 The nanoenzyme therapy system is composed of bovine serum albumin-modified manganese dioxide nanoparticles, dopamine-modified iron tetroxide nanoparticles, and the photosensitizer dihydroporphyrin E6. This nanoenzyme therapy system can alleviate tumor hypoxia, enhance the efficacy of photodynamic therapy, and simultaneously enhance tumor cell immunogenic death. Specific embodiments are as follows.

[0045] Step 1: Prepare oil-phase Fe3O4 nanoparticles by high-temperature pyrolysis.

[0046] Add 5.25 g of ferric acetylacetone, 40 mL of dibenzyl ether, and 60 mL of oleic acid amide (OAm) to a 500 mL three-necked flask and mix thoroughly. Before starting the reaction, purge with nitrogen to remove oxygen from the reaction apparatus. Then, under nitrogen protection and reflux with a condenser, perform a programmed temperature increase. The reaction ends when the system changes from reddish-brown to bright black. After the reaction, remove the heating apparatus and allow the reaction to cool rapidly to room temperature to obtain OAm@Fe3O4. To remove unreacted residue, add 100 mL of anhydrous ethanol and perform four magnetic separation washes. Finally, dissolve the OAm@Fe3O4 precipitate in 100 mL of chloroform for storage.

[0047] Step 2: Preparation of water-soluble magnetic nanoparticles DPA@Fe3O4 by ligand exchange method.

[0048] 40 mg of dopamine hydrochloride was weighed into a 250 mL three-necked round-bottom flask, and 4 mL of anhydrous dimethyl sulfoxide and 4 mL of chloroform solution were added to dissolve and mix thoroughly. Under mechanical stirring at 1000 rpm and ultrasonic treatment in a 70 °C water bath, 2 mL of 10 mg Fe / mL OAm@Fe3O4 solution was added dropwise. After reacting for 2 h, the reactant was transferred to an ultracentrifuge tube, 2 mL of chloroform was added, and the mixture was centrifuged at 5000 rpm for 5 min. After removing the supernatant, the precipitate was dried under nitrogen gas, and approximately 4 mL of ultrapure water was added to dissolve it. The pH of the solution was adjusted to 4.0, thus obtaining water-soluble, ultra-small, superparamagnetic Fe3O4 nanoparticles (DPA@Fe3O4) encapsulated with dopamine. The DPA@Fe3O4 was then ultracentrifuged at 1000 rpm for 10 min to remove any larger particles present in the product. Unreacted dopamine hydrochloride was removed from the supernatant using a 30 kDa ultrafiltration tube, and small amounts of aggregates were removed using a 220 nm filter membrane. The purified DPA@Fe3O4 was stored at 4 °C.

[0049] Step 3: Prepare magnetic nanoparticles BSA@MnO2.

[0050] Weigh 31.6 mg of potassium permanganate and dissolve it in 3 mL of ultrapure water for later use. Dissolve 250 mg of bovine serum albumin in 7 mL of ultrapure water and add it dropwise to the potassium permanganate solution. Stir magnetically at 37 °C for 2 h to obtain BSA@MnO2.

[0051] Step 4: Prepare the nanozyme diagnostic and therapeutic system MNPT.

[0052] Mix 0.5 mL of BSA@MnO2, 1.3 mL of ultrapure water, and 50 μL of dihydroporphyrin E6 thoroughly. Then add 200 μL of EDC (2.5 mg / mL). Incubate at 37°C for 4 hours on a shaker, then store at 4°C overnight. The next day, add 100 μL of DPA@Fe3O4 while vortexing to obtain the MNPT nanozyme diagnostic and therapeutic system.

[0053] Example 2

[0054] The in vitro killing effect of nanozyme diagnostic and therapeutic systems.

[0055] Different concentrations of BSA@Fe3O4 / MnO2, Ce6, and MNPT were co-incubated with 4T1 cells, establishing a laser-treated group and a non-laser-treated group. After 6 hours of co-incubation, the laser-treated group was irradiated for 15 seconds. Cell viability of 4T1 cells was measured after 24 hours of co-incubation to verify the killing effect of the nanozyme therapy system on 4T1 cells.

[0056] Example 3

[0057] The in vivo killing effect of nanozyme diagnostic and therapeutic systems.

[0058] Forty mice with uniform body weight and tumor volume (subcutaneous tumors constructed from 4T1 cells) were randomly divided into four groups: control group, Ce6 group, BSA@Fe3O4 / MnO2 group, and MNPT group. Each group was further randomly divided into laser-treated and non-laser-treated groups, with five mice in each group. Seven days after tumor modeling, the mice were injected subcutaneously with 50 μL of nanoparticles (containing 0.2 mg / mL iron) on days 1, 3, 5, and 7. The condition of the tumor-bearing mice was recorded in real time during the treatment. The laser-treated group was irradiated with a 660 nm (0.5 mg / mL) laser at 4 h and 24 h after treatment. W / cm 2 5 min / each.

[0059] like Figure 7 As shown, this embodiment proposes a construction scheme for a magnetic nano-platform (MNPT) based on a composite nanozyme (Fe3O4 / MnO2) of iron(III) oxide and manganese(II) oxide (BSA). MNPT consists of manganese(II) oxide (MnO2) modified with bovine albumin (BSA), iron(III) oxide (Fe3O4) nanoparticles modified with dopamine (DPA), and the photosensitizer dihydroporphyrin e6 (Chlorin e6, Ce6). As a nanomaterial, MNPT can passively target tumor sites through the high permeability and retention effect of solid tumors. Then, it generates O2 through its catalase-like activity, and produces hydroxyl radicals (·OH) through the Fenton reaction, thereby alleviating the hypoxia problem in the tumor microenvironment, enhancing the efficacy of PDT, and simultaneously enhancing the immunogenic death of tumor cells. In addition, the superparamagnetism of MNPT allows it to be used as a contrast agent for magnetic resonance imaging, thereby providing fluorescence-magnetic resonance dual-modality imaging, real-time tracking and feedback of treatment effects.

[0060] Any numerical values ​​cited herein include all values ​​ranging from a lower limit to an upper limit, increasing by one unit, with at least two units between any lower and any higher value. For example, if the quantity of a component or the value of a process variable (e.g., temperature, pressure, time, etc.) is described as being from 1 to 90, preferably from 20 to 80, more preferably from 30 to 70, the purpose is to illustrate that values ​​such as 15 to 85, 22 to 68, 43 to 51, 30 to 32 are also explicitly listed in this specification. For values ​​less than 1, a unit is appropriately considered to be 0.0001, 0.001, 0.01, 0.1, etc. These are merely examples intended for explicit expression, and it can be assumed that all possible combinations of values ​​listed between the minimum and maximum values ​​are similarly explicitly stated in this specification.

[0061] Unless otherwise stated, all ranges include the endpoints and all numbers between them. The terms "approximately" or "about" used with ranges apply to both endpoints of the range. Thus, "approximately 20 to 30" is intended to cover "approximately 20 to approximately 30," including at least the specified endpoints.

[0062] All articles and references disclosed herein, including patent applications and publications, are incorporated herein by reference for various purposes. The term “substantially constitutes…” used to describe a combination should include the identified elements, components, parts, or steps, as well as other elements, components, parts, or steps that do not substantially affect the essential novelty of the combination. The use of the terms “comprising” or “including” to describe combinations of elements, components, parts, or steps herein also contemplates embodiments substantially constituted by such elements, components, parts, or steps. The use of the term “may” herein is intended to indicate that any described attribute included by “may” is optional.

[0063] Multiple elements, components, parts, or steps can be provided by a single integrated element, component, part, or step. Alternatively, a single integrated element, component, part, or step can be divided into multiple separate elements, components, parts, or steps. The use of "a" or "an" to describe an element, component, part, or step does not imply the exclusion of other elements, components, parts, or steps.

[0064] It should be understood that the above description is for illustrative purposes and not for limitation. Many embodiments and applications beyond the provided examples will be apparent to those skilled in the art upon reading the above description. Therefore, the scope of this teaching should not be determined by reference to the above description, but rather by reference to the appended claims and the full scope of their equivalents. For purposes of completeness, all articles and references, including patent applications and publications, are incorporated herein by reference. The omission of any aspect of the subject matter disclosed herein in the preceding claims is not intended as a waiver of that subject matter, nor should it be construed as an indication that the inventors have not considered that subject matter as part of the disclosed inventive subject matter.

Claims

1. A method for preparing a dihydroporphyrin-modified metal oxide nanoenzyme diagnostic and therapeutic system, characterized in that, The preparation steps include the following: Oil-phase nanoparticles OAm@Fe3O4 were prepared by high-temperature pyrolysis. Water-soluble magnetic nanoparticles DPA@Fe3O4 were prepared using the nanoparticles OAm@Fe3O4 via a ligand exchange method. Preparation of magnetic nanoparticles BSA@MnO2; The MNPT nanoenzyme diagnostic and therapeutic system was prepared using the DPA@Fe3O4 nanoparticles and the BSA@MnO2 magnetic nanoparticles; the preparation of the MNPT nanoenzyme diagnostic and therapeutic system includes: Take 0.5 mL of BSA@MnO2, 1.3 mL of ultrapure water and 50 μL of dihydroporphyrin e6 and mix them evenly. Then add 200 μL of EDC with a concentration of 2.5 mg / mL. Place the mixture in a shaker at 37 °C for 4 h and store it at 4 °C overnight. The next day, add 100 μL of DPA@Fe3O4 while vortexing to obtain the nanozyme diagnostic and therapeutic system MNPT.

2. The preparation method according to claim 1, characterized in that, The oil-phase nanoparticles OAm@Fe3O4 prepared by high-temperature pyrolysis include: Weigh 15 mmol of iron acetylacetone powder into a 500 mL three-necked flask, then add 210 mmol of dibenzyl ether and 165 mmol of oleylamine, mix well, and carry out the reaction under nitrogen protection and reflux with a condenser at a programmed temperature. Before the reaction starts, nitrogen gas is introduced into the reaction vessel to ensure that oxygen in the apparatus is removed; the nitrogen flow rate is 50 mL / min and the duration is about 5 min. Then, the temperature control device was set to a two-stage programmed heating process: the first stage was to raise the temperature from room temperature to 220℃ and then maintain the temperature constant for 1 hour, with a heating rate of 3.3℃ / min; the second stage was to continue to raise the temperature from 220℃ to 290℃ and then maintain the constant temperature for 5 minutes, with a heating rate of 3.3℃ / min. After the reaction is complete, the heating device is quickly removed to allow the solution temperature in the reaction system to drop sharply to room temperature, thus obtaining OAm@Fe3O4 with a particle size of 5 nm.

3. The preparation method according to claim 1, characterized in that, The preparation of water-soluble magnetic nanoparticles DPA@Fe3O4 using the nanoparticles OAm@Fe3O4 via ligand exchange method includes: Weigh 400 mg of dopamine hydrochloride into a 250 mL three-necked flask, add 40 mL of anhydrous dimethyl sulfoxide and 40 mL of chloroform mixture to dissolve it, and mix well; Then, the stirrer speed was adjusted to 1000 rpm / min, and 20 mL of OAm@Fe3O4 with a concentration of 10 mg Fe / mL was added dropwise under ultrasonic conditions in a 70 ℃ water bath. After reacting for 2 h, the reactants were taken out and placed in an ultracentrifuge tube, and 20 mL of chloroform was added. The mixture was centrifuged at 5000 rpm / min for 5 min, the supernatant was removed, the precipitate was dried with nitrogen, and then 40 mL of ultrapure water was added for ultrasonic dissolution. The pH of the solution was adjusted to 4.0 to obtain dopamine-encapsulated water-soluble ultra-small superparamagnetic Fe3O4 nanoparticles.

4. The preparation method according to claim 1, characterized in that, The preparation of magnetic nanoparticles BSA@MnO2 includes: weighing 31.6 mg of potassium permanganate and dissolving it in 3 mL of ultrapure water for later use; dissolving 250 mg of bovine serum albumin in 7 mL of ultrapure water and then adding it dropwise to the potassium permanganate solution; and stirring magnetically at 37 °C for 2 h to obtain BSA@MnO2.

5. A dihydroporphyrin-modified metal oxide nanoenzyme diagnostic and therapeutic system based on the preparation method of claim 1, wherein the nanoenzyme diagnostic and therapeutic system is mainly composed of bovine serum albumin-modified manganese dioxide nanoparticles, dopamine-modified iron tetroxide nanoparticles, and photosensitizer dihydroporphyrin E6.

6. The dihydroporphyrin-modified metal oxide nanoenzyme diagnostic and therapeutic system according to claim 5, characterized in that, The mass ratio of the bovine serum albumin-modified manganese dioxide nanoparticles, the dopamine-modified iron tetroxide nanoparticles, and the photosensitizer dihydroporphyrin E6 is 3:1:

1.

7. A dihydroporphyrin-modified metal oxide nanoenzyme diagnostic and therapeutic system according to claim 5, characterized in that, The core of the metal oxide nanozyme diagnostic and therapeutic system is a composite nanozyme of iron tetroxide / manganese dioxide; this nanozyme diagnostic and therapeutic system is stable in aqueous solution and has a hydrodynamic size of approximately 25.79±3.45 nm.

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