Porous Fe / Cu nanoreactor with double insurance design as well as preparation method and application of porous Fe / Cu nanoreactor

Abstract

The invention provides a porous Fe / Cu nanoreactor with double insurance design and a preparation method and application thereof, and belongs to the technical field of biology. The porous Fe / Cu nano-carrier is firstly prepared and used for reducing toxic and side effects of chemotherapeutic drugs, pyridazinone micromolecule chemotherapeutic drugs IMB are loaded on the carrier, toxicity of IMB is further reduced through wrapping and targeting effects of 3F8 antibodies, targeted controllable slow release of the chemotherapeutic drugs is achieved through acid response of the porous Fe / Cu nano-carrier, and the effect of reducing toxic and side effects of the chemotherapeutic drugs is achieved. The double-insurance effect and the precise chemical kinetics treatment of melanoma are achieved. In the tumor acidic microenvironment, the porous Fe / Cu nano reactor with the double insurance design can specifically respond to the tumor acidic microenvironment, and controllable slow release of small molecule chemotherapeutic drugs is achieved. Meanwhile, through the targeting effect of the 3F8 antibody, targeted delivery and accumulation of small molecule chemotherapeutic drugs at the tumor site are enhanced, and the chemotherapeutic effect is enhanced.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a porous Fe / Cu nanoreactor with a double insurance design and a preparation method and application thereof. Background Art

[0002] Skin melanoma, also known as melanocytoma, pigment disease, gangrene, and gangrene, is a common skin tumor caused by excessive proliferation of abnormal melanocytes. Although only 4% of all skin cancer cases are melanoma, melanoma is the most deadly cancer among malignant skin tumors, accounting for the vast majority of deaths from skin tumors.

[0003] There are many treatments for skin melanoma, including surgical resection, chemotherapy, targeted therapy and radiotherapy. Among them, surgical resection has obvious defects, such as tissue loss, scarring, long healing time and high risk of infection. Therefore, chemotherapy and targeted therapy are the main options for the treatment of skin melanoma. However, chemotherapy, as one of the commonly used treatment strategies, is often accompanied by serious toxic side effects and difficulties in targeted delivery and controlled release during long-term treatment. Targeted therapy is a new treatment technology developed in recent decades. Although targeted therapy has been proven to be effective, some limitations, including high cost, complexity of the treatment process and long-term survival of reprogrammed cells are major problems. Therefore, the development of safe, effective, minimally invasive treatments with few side effects is crucial for the treatment of melanoma. Summary of the invention

[0004] The purpose of the present invention is to provide a porous Fe / Cu nanoreactor with a double insurance design and a preparation method and application thereof. The obtained porous Fe / Cu nanoreactor with a double insurance design is safe, effective and has little side effect.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions: The present invention provides a method for preparing a porous Fe / Cu nanoreactor with a double insurance design, comprising the following steps: (1) Preparation of porous Fe / Cu nanocarrier: dissolving iron salt and copper salt in a first solvent and reacting to obtain a porous Fe / Cu nanocarrier; (2) IMB loading: dispersing the porous Fe / Cu nanocarrier prepared in step (1) in a second solvent, adding the pyridazinone small molecule chemotherapy drug IMB, incubating and loading, and obtaining a porous Fe / Cu nanocarrier solution loaded with IMB; (3) GD2 targeting antibody 3F8 loading: GD2 targeting antibody 3F8 was added to the porous Fe / Cu nanocarrier solution loaded with IMB in step (2), and the loading was incubated to obtain a porous Fe / Cu nanoreactor with a double insurance design.

[0006] Preferably, in step (1), the iron salt includes one of FeCl3.6H2O, anhydrous ferric chloride, ferric nitrate, ferric sulfate, ferric phosphate, ferric acetate, etc.; the copper salt includes one of CuCl2.2H2O, copper sulfate, copper acetate, copper phosphate, copper carbonate; and the first solvent includes one or more of DMF, terephthalic acid, trimesic acid, and biphenyl dicarboxylic acid.

[0007] Preferably, the molar volume ratio of the iron salt: the copper salt: the first solvent is 1-2 mmol: 1-2 mmol: 20-60 mL; the reaction temperature is 25-180° C., and the reaction time is 0.5-48 h.

[0008] Preferably, in step (2), the second solvent comprises one of phosphate buffer, ultrapure water and physiological saline; the loading mass ratio of the porous Fe / Cu nanocarrier to IMB is 1:1-1:10; the pH value during the incubation loading is pH 6.5-8.5, the temperature is 2-37°C, and the time is 1-48 h.

[0009] Preferably, in step (3), the mass ratio of the porous Fe / Cu nanocarrier to 3F8 is 1:1-1:10; the pH value during the incubation and loading is pH 6.5-8.5, and the incubation and loading time is 1-48 h.

[0010] The present invention also provides a porous Fe / Cu nanoreactor with a double safety design prepared by a method for preparing the porous Fe / Cu nanoreactor with a double safety design.

[0011] The present invention also provides an application of a porous Fe / Cu nanoreactor with a double insurance design in preparing a drug for treating melanoma.

[0012] The beneficial effects of the present invention compared with the prior art are: (1) The present invention first prepares a porous Fe / Cu nanocarrier for reducing the toxic side effects of chemotherapy drugs, and loads the pyridazinone small molecule chemotherapy drug IMB on the carrier. The toxicity of IMB is further reduced through the encapsulation and targeting effect of the 3F8 antibody, and the targeted and controlled sustained release of chemotherapy drugs is achieved by the acidic response of the porous Fe / Cu nanocarrier, thereby achieving a double insurance effect and precise chemodynamic treatment of melanoma.

[0013] (2) The porous Fe / Cu nanoreactor with double insurance design of the present invention is uniform and stable in a normal physiological environment, which can prevent the leakage of chemotherapy drugs in normal tissues, significantly reduce the toxic side effects of small molecule chemotherapy drugs, and has high safety. In the acidic microenvironment of the tumor, the porous Fe / Cu nanoreactor with double insurance design can specifically respond to the acidic microenvironment of the tumor and achieve controlled sustained release of small molecule chemotherapy drugs. At the same time, the targeting effect of the 3F8 antibody enhances the targeted delivery and accumulation of small molecule chemotherapy drugs in the tumor site, thereby enhancing the efficacy of chemotherapy.

[0014] (3) The porous Fe / Cu nanoreactor with a double insurance design of the present invention can catalyze hydrogen peroxide and consume glutathione to produce highly toxic reactive oxygen species, which is expected to enhance chemodynamic therapy in tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0016] Figure 1 This is a scanning electron microscope image of the porous Fe / Cu nanocarrier prepared in Example 1 of Experimental Example 1 of the present invention; Figure 2 The transmission electron microscopy image and corresponding element mapping of the porous Fe / Cu nanocarrier prepared in Example 1 of Experimental Example 1 of the present invention, wherein A is the transmission electron microscopy image and B is the corresponding element mapping; Figure 3 is the pore size and area of ​​the porous Fe / Cu nanocarrier prepared in Example 1 of Experimental Example 1 of the present invention; Figure 4The results of porous Fe / Cu nanocarriers (FeCuNPs) catalyzing hydrogen peroxide in Experimental Example 2 of the present invention, wherein a is the reaction principle of porous Fe / Cu nanocarriers (FeCuNPs) catalyzing hydrogen peroxide; b represents the change of ultraviolet-visible absorbance of reactive oxygen generated under different porous Fe / Cu nanocarriers (FeCuNPs) conditions, c represents the ultraviolet absorption spectrum of reactive oxygen generated under different porous Fe / Cu nanocarrier conditions, d represents the ultraviolet absorption spectrum of reactive oxygen generated under different hydrogen peroxide concentrations, e represents the fluorescence spectrum of porous Fe / Cu nanocarriers catalyzing hydrogen peroxide to generate reactive oxygen under different treatment conditions, f represents the ultraviolet absorption spectrum of glutathione consumption catalyzed by porous Fe / Cu nanocarriers at different concentrations, g represents the ultraviolet absorption spectrum of glutathione consumption catalyzed by porous Fe / Cu nanocarriers under different treatment conditions, and h represents the relative consumption of glutathione under different treatment conditions; Figure 5 The ultraviolet and infrared characteristic absorption peaks of the porous Fe / Cu nanocarrier prepared in Example 1 of Experimental Example 3 of the present invention before and after loading the pyridazinone small molecule chemotherapy drug and modifying the 3F8 antibody, wherein A is the ultraviolet characteristic absorption peak and B is the infrared characteristic absorption peak; Figure 6 The UV spectra of different concentrations of IMB in Experimental Example 4 of the present invention, the standard curve and the release amounts incubated for different times under the conditions of pH 5.5 and 7.3 of the IMB buffer system, wherein A is the UV spectra of different concentrations of IMB, B is the standard curve, and C is the release amounts incubated for different times under the conditions of pH 5.5 and 7.3 of the IMB buffer system; Figure 7 Figure 4 is the cytotoxicity in Experimental Example 4 of the present invention, wherein a is the release curve of FeCuNPs@IMB and 3F8@FeCuNPs@IMB over time in different buffer systems, b is the cytotoxicity of IMB to M21 cells, C is the cytotoxicity of IMB to MCF-10A cells, Figure d is the cytotoxicity of FeCuNPs, FeCuNPs@IMB and 3F8@FeCuNPs@IMB to M21 cells under normal physiological solution conditions, Figure e is the cytotoxicity of 3F8@FeCuNPs@IMB under pH 5.5 conditions, and Figure f is the exploration of the uptake pathway of 3F8@FeCuNPs@IMB; Figure 8 The targeted imaging effect of the porous Fe / Cu nanoreactor designed for double insurance in Experimental Example 5 of the present invention in a melanoma animal model; Fig. 9 This is the treatment effect of melanoma under different treatment conditions in Experimental Example 5 of the present invention, wherein A is the relative tumor volume change of melanoma, B is the weight of the peeled tumor at the end of treatment, and C is a photo of the peeled tumor at the end of treatment. DETAILED DESCRIPTION

[0017] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0018] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0019] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0020] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0021] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0022] The present invention provides a method for preparing a porous Fe / Cu nanoreactor with a double insurance design, comprising the following steps: (1) Preparation of porous Fe / Cu nanocarrier: dissolving iron salt and copper salt in a first solvent and reacting to obtain a porous Fe / Cu nanocarrier; (2) IMB loading: dispersing the porous Fe / Cu nanocarrier prepared in step (1) in a second solvent, adding the pyridazinone small molecule chemotherapy drug IMB, incubating and loading, and obtaining a porous Fe / Cu nanocarrier solution loaded with IMB; (3) GD2 targeting antibody 3F8 loading: GD2 targeting antibody 3F8 was added to the porous Fe / Cu nanocarrier solution loaded with IMB in step (2), and the loading was incubated to obtain a porous Fe / Cu nanoreactor with a double insurance design.

[0023] Example 1 The present invention provides a method for preparing a porous Fe / Cu nanoreactor with a double insurance design in Example 1, and the specific steps are as follows: (1) Preparation of porous Fe / Cu nanocarriers: 1 mmol of FeCl3.6H2O, 1 mmol of CuCl2.2H2O and 1 mmol of terephthalic acid were dissolved in 30 mL of DMF and transferred to a high-pressure reactor for reaction at 140 °C for 12 h to obtain porous Fe / Cu nanocarriers (FeCuNPs).

[0024] (2) IMB loading: 10 mg of porous Fe / Cu nanocarriers and 10 mg of pyridazinone small molecule chemotherapy drug IMB were ultrasonically dispersed in 10 mL of PBS. After incubation at 4 °C for 12 h, a porous Fe / Cu nanocarrier solution loaded with IMB (FeCuNPs@IMB) was obtained.

[0025] (3) GD2 targeting antibody 3F8 loading: 10 mg of GD2 targeting antibody 3F8 was added to the porous Fe / Cu nanocarrier solution loaded with IMB in step (2), incubated for loading, washed three times with PBS and resuspended in 10 mL of PBS solution to obtain a porous Fe / Cu nanoreactor with a double insurance design (3F8@FeCuNPs@IMB).

[0026] Test Example 1 Experimental Example 1 of the present invention characterized the porous Fe / Cu nanocarrier prepared in step (1) of Example 1, and the specific steps are as follows: (1) The morphology and elemental composition of the porous Fe / Cu nanocarrier were obtained by Hitachi S-4800 SEM and Tecnai G2 F20. Figure 1 , 2 shown.

[0027] Figure 1 , 2 It is shown that the porous Fe / Cu nano-carrier of the present invention has a regular octahedral morphology, an average particle size of 210 nm, good dispersibility and uniform size.

[0028] (2) The pore size and surface area of ​​the porous Fe / Cu nanocarrier were determined by nitrogen desorption experiment using the Quantachrome NOVAtouch. The results are as follows: Figure 3 shown.

[0029] Figure 3 The porous Fe / Cu nanocarrier has a pore size of 2.3 nm and a surface area of ​​804.68 m 2 / g.

[0030] Test Example 2 Experimental Example 2 of the present invention detects the effect of the porous Fe / Cu nanoreactor with double insurance design prepared in Example 1 on catalyzing hydrogen peroxide, and the specific steps are as follows: FeCuNPs nanozymes (different concentrations of 0, 12.5, 25, 50, 100 µg mL -1 The ROS generation ability of porous Fe / Cu nanocarriers (FeCuNPs) was detected by a mixed suspension of 100 mM, H2O2 (100 mM) and 3,3′,5,5′-tetramethylbenzidine (TMB, Beyotime, China, ≥98%, 1 mM). After 10 min of reaction, the absorbance of oxidized TMB at 650 nm was measured to evaluate the generation of ·OH promoted by FeCuNPs nanozyme catalyzed by H2O2. TA was used as a fluorescent probe instead of TMB to detect the fluorescence spectrum of ·OH generation. Different concentrations of FeCuNPs (0, 1.25, 2.5, 5, 10, 20 µg mL -1 The UV-vis absorption spectra of ·OH generation were evaluated in mixed suspensions of 10% ·mL MgCl2 and 10% MgCl2 and different concentrations of H2O2 (0.05, 0.1, 0.5, 1, 1.5, 2 M) and TMB (1 mM). The absorption spectra of the supernatant were recorded at 400-800 nm using a multi-mode microplate reader (BioTek H1 MFG).

[0031] DTNB was used to verify the consumption capacity of FeCuNPs for GSH. 300 μL FeCuNPs nanozymes (different concentrations of 0, 12.5, 25, 50, 100 μg mL -1 ) was mixed with 300 µL GSH (1 mM). After the mixture was reacted at 37 °C for 12 h, 300 μL 1 mM DTNB Tris-HCl buffer solution (pH 8.5) was added to measure GSH consumption. The UV-vis absorption spectrum and the characteristic absorption value at 412 nm were recorded by a multi-mode microplate reader (BioTek H1 MFG). The results are shown in Figure 4 shown.

[0032] Figure 4 Porous Fe / Cu nanocarriers (FeCuNPs) were shown to catalyze hydrogen peroxide and consume glutathione for the production of highly toxic reactive oxygen species, which is expected to enhance chemodynamic therapy within tumors, where GSH consumption and reactive oxygen species generation were significantly increased in the presence of hydrogen peroxide.

[0033] Test Example 3 In Experimental Example 3 of the present invention, the porous Fe / Cu nanoreactor with a double insurance design prepared in Example 1 was characterized. The ultraviolet and infrared characteristic absorption peaks and labeling rates of the porous Fe / Cu nanocarrier before and after loading the pyridazinone small molecule chemotherapy drug and modifying the 3F8 antibody were measured by Multimode Microplate Reader (BioTek H1 MFG) and Nicolet 6700 FT-IR spectrometer of Nicolet, USA. The results are as follows: Figure 5 The UV-visible absorption spectra and IR spectra of FeCuNPs, IMB, FeCuNPs@IMB and 3F8@FeCuNPs@IMB show that 3F8@FeCuNPs@IMB has -1 Two characteristic peaks of IMB appeared at , and a blue shift occurred, indicating that IMB was effectively loaded into FeCuNPs. Compared with the free 3F8 antibody, the characteristic peaks of 3F8@FeCuNPs@IMB were significantly red-shifted, indicating that the 3F8 antibody was successfully modified.

[0034] Test Example 4 Experimental Example 4 of the present invention detects the sustained release effect and cytotoxicity of the porous Fe / Cu nanoreactor with double insurance design prepared in Example 1, and the specific steps are as follows: (1) Sustained release effect testing The absorbance of IMB at different concentrations was detected to obtain the IMB absorbance standard curve.

[0035] 3 mg of the porous Fe / Cu nanoreactor with double insurance design was dispersed in phosphate buffer with pH values ​​of 5.5 and 7.3 by ultrasonication to a final concentration of 1 mg mL -1 The cells were incubated at room temperature for 0, 10, 30, 60, 360, 720, 1440, and 2880 minutes, respectively. The supernatant was collected after centrifugation to detect the content of the pyridazinone small molecule chemotherapy drug released in the supernatant. The release amount of tanshinone was calculated using the absorbance standard curve of the pyridazinone small molecule chemotherapy drug. The results are as follows: Figure 7 As shown in a.

[0036] In order to explore the uptake pathway of the double-insurance nanoreactor, the present invention seeded M21 cells at a density of 300,000 / well in a 6-well plate and cultured for 24 hours. The cells were incubated with chlorpromazine (50 µM), cytochalasin D (5 µM), wortmannin (5 µM), filipin III (7.5 µM) or 4 ºϹ treated cells (as a positive control group) for 30 minutes. Then 20 µg mL-1 FITC-labeled 3F8@FeCuNPs@IMB was added and incubated for another 2 hours. The treated cells were washed with PBS and analyzed by flow cytometry (BD, USA).

[0037] Figure 7 a shows that the porous Fe / Cu nanoreactor with a double insurance design loaded with IMB can achieve controlled sustained release of small molecule chemotherapeutic drugs in an acidic environment.

[0038] (2) Cytotoxicity assay Under normal physiological conditions, logarithmically growing human melanoma cells M21 and MCF7-10A cells were incubated with different concentrations of porous Fe / Cu nanocarriers (FeCuNPs), pyridazinone small molecule chemotherapy drugs (IMB), porous Fe / Cu nanocarriers loaded with IMB (FeCuNPs@IMB), and double-insurance porous Fe / Cu nanoreactors (3F8@FeCuNPs@IMB) for 24 hours, and the cytotoxicity was detected by CCK-8. The experimental results are shown in Figure 7 shown.

[0039] Figure 7 It was shown that under normal physiological conditions, the loading of porous Fe / Cu nanocarriers significantly reduced the toxic side effects of pyridazinone small molecule chemotherapy drugs. Co-loading of IMB and 3F8 showed lower cytotoxicity than IMB and FeCuNPs@IMB. Since the porous Fe / Cu nanocarriers are acid-responsive nanocarriers, they can prevent the leakage of IMB in normal physiological solutions, thereby reducing damage to normal tissues. In addition, 3F8 antibody, as a non-toxic protein, is wrapped on the surface of FeCuNPs@IMB, forming a second layer of insurance to prevent the leakage of IMB. At the same time, it has a tumor targeting effect, playing a double insurance role. Figure 7 As shown, the LC50 of IMB is 3.73 µg ml -1 , the half lethal concentration of FeCuNPs@IMB is 17.15 µg ml -1 , the LC50 of 3F8@FeCuNPs@IMB was 59.95µg ml -1However, under acidic conditions, the LC50 of 3F8@FeCuNPs@IMB was 7.46 µg ml -1 Therefore, at the cellular level, it was shown that the cytotoxicity of 3F8@FeCuNPs@IMB under normal physiological environment was significantly less than that under acidic environment.

[0040] Test Example 5 Experimental Example 5 of the present invention detected the in vivo targeted imaging effect and therapeutic effect of the porous Fe / Cu nanoreactor with double insurance design prepared in Example 1 in a melanoma animal model, and the specific method was as follows: (1) Targeted imaging Five million M21 cells were injected subcutaneously into the right lower abdomen of 8-week-old female BALB / c nude mice to establish a melanoma tumor model.

[0041] After about 2 weeks of growth, the volume of the small melanoma tumor reached 100 mm 3 At the same time, 50 µg of the double-insurance porous Fe / Cu nanoreactor was injected into the tumor-bearing mice by tail vein injection. Targeted fluorescence imaging of the small animals was performed by a small animal fluorescence imager 1.0 h, 6h, 12h, 24h, and 48h after injection. The results are shown in Figure 8 shown.

[0042] Figure 8 The results showed that the porous Fe / Cu nanoreactor with double insurance design can target melanoma tumor cells, and the fluorescence signal quickly accumulated at the edge of the tumor in M21 tumor-bearing nude mice at 1, 6, 12, 24, and 48 hours after injection. Subsequently, the accumulation of 3F8@FeCuNPs@IMB at the tumor site was observed to increase steadily, reaching a peak at 24 hours after injection. It is worth noting that there was no significant decrease in the fluorescence intensity at the tumor site after 48 hours. These results indicate that 3F8@FeCuNPs@IMB exhibits strong targeting to melanoma and can prolong the retention time of drugs at the tumor site, which is suitable for continuous chemotherapy and CDT.

[0043] (2) Treatment effect The mice were divided into 6 groups, 5 in each group, and 50 μL of saline (control group), porous Fe / Cu nanocarriers (FeCuNPs, 200 μg), IMB (200 μg), 3F8 (200 μg), porous Fe / Cu nanocarriers loaded with IMB (FeCuNPs@IMB, 200 μg), and porous Fe / Cu nanoreactors with double insurance design (3F8@FeCuNPs@IMB, 200 μg) were injected into the tumor-bearing mice by tail vein injection. The changes in the tumor volume of the mice were observed and measured 28 days after injection. After the treatment, the tumor growth curve was drawn, the tumor was peeled off and photographed, and the results were as follows: Fig. 9 shown.

[0044] Fig. 9 The results showed that the tumor volume of mice treated with the porous Fe / Cu nanoreactor with a double insurance design was the smallest and the treatment effect was the best compared with other groups. The FeCuNPs@IMB group loaded with chemotherapeutic drugs showed significantly enhanced antitumor effects compared with the control group and the single treatment group IMB or FeCuNPs group. This indicates that the combined CDT and chemotherapy treatment using FeCuNPs nanozyme carriers is an effective strategy for the treatment of melanoma. This significantly enhanced therapeutic effect may be attributed not only to the conversion of high concentrations of H2O2 into ROS, which is highly toxic to CDT, catalyzed by nanozymes, but also to the consumption of elevated GSH levels, thereby disrupting redox homeostasis and enhancing CDT efficacy. In addition, nanozymes can serve as the first line of insurance to improve IMB accumulation and achieve controlled release at the tumor site to further enhance chemotherapy. The tumor inhibition effect of the single chemotherapy drug treatment group was very limited, which may be due to the short half-life of the drug and rapid metabolism, making it difficult to effectively inhibit tumor growth. Benefiting from the enhanced permeability retention effect and CDT therapeutic ability of FeCuNPs nanozymes, the tumor inhibition effect of the FeCuNPs treatment group was significantly higher than that of the single chemotherapy group. The nanoreactor encapsulated with 3F8 antibody has tumor targeting ability. By binding to the GD2 antigen overexpressed on melanoma cells, it effectively improves the targeting efficiency of melanoma tissue, while taking into account the effectiveness of chemotherapy and the combination of precision chemodynamic therapy. The 3F8@FeCuNPs@IMB treatment group has the highest tumor inhibition efficiency. The encapsulated 3F8 antibody can also serve as a second insurance to reduce the leakage of chemotherapy drugs in the blood circulation, thereby reducing toxic side effects and improving biosafety. The above synergistic treatment strategy ultimately effectively inhibited the proliferation of melanoma. In addition, the tumor tissues removed from different treatment groups were photographed and weighed after treatment. Consistent with the tumor volume measurement results, the 3F8@FeCuNPs@IMB group had the strongest tumor inhibition effect and the smallest tumor weight. These results demonstrate the effectiveness of 3F8@FeCuNPs@IMB-mediated combined precision chemotherapy and chemodynamic therapy in inhibiting melanoma growth.

[0045] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing a porous Fe / Cu nanoreactor with a double insurance design, characterized in that: The steps include: (1) Preparation of porous Fe / Cu nanocarrier: dissolving iron salt and copper salt in a first solvent and reacting to obtain a porous Fe / Cu nanocarrier; (2) IMB loading: dispersing the porous Fe / Cu nanocarrier prepared in step (1) in a second solvent, adding the pyridazinone small molecule chemotherapy drug IMB, incubating and loading, and obtaining a porous Fe / Cu nanocarrier solution loaded with IMB; (3) GD2 targeting antibody 3F8 loading: GD2 targeting antibody 3F8 was added to the porous Fe / Cu nanocarrier solution loaded with IMB in step (2), and the loading was incubated to obtain a porous Fe / Cu nanoreactor with a double insurance design.

2. The method for preparing the porous Fe / Cu nanoreactor with double insurance design according to claim 1, characterized in that: In step (1), the iron salt includes one of FeCl3.6H2O, anhydrous ferric chloride, ferric nitrate, ferric sulfate, ferric phosphate, and ferric acetate; the copper salt includes one of CuCl2.2H2O, copper sulfate, copper acetate, copper phosphate, and copper carbonate; and the first solvent includes one or more of DMF, terephthalic acid, trimesic acid, and biphenyl dicarboxylic acid.

3. The method for preparing the porous Fe / Cu nanoreactor with double insurance design according to claim 2, characterized in that: The molar volume ratio of the iron salt: the copper salt: the first solvent is 1-2 mmol: 1-2 mmol: 20-60 mL; the reaction temperature is 25-180° C., and the reaction time is 0.5-48 h.

4. The method for preparing the porous Fe / Cu nanoreactor with double insurance design according to claim 1, characterized in that: In step (2), the second solvent includes one or more of phosphate buffer, ultrapure water, and physiological saline; the loading mass ratio of the porous Fe / Cu nanocarrier to IMB is 1:1-1:10; the pH value during the incubation loading is pH 6.5-8.5, the temperature is 2-37°C, and the time is 1-48h.

5. The method for preparing the porous Fe / Cu nanoreactor with double insurance design according to claim 1, characterized in that: In step (3), the mass ratio of the porous Fe / Cu nanocarrier to 3F8 is 1:1-1:10; the pH value during the incubation and loading is pH 6.5-8.5, and the incubation and loading time is 1-48 hours.

6. A porous Fe / Cu nanoreactor with double insurance design prepared by the method for preparing a porous Fe / Cu nanoreactor with double insurance design as claimed in any one of claims 1 to 5.

7. Use of the porous Fe / Cu nanoreactor with double insurance design as claimed in claim 6 in preparing drugs for treating melanoma.

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