Copper-iron double-metal nano composite material as well as preparation method and application thereof

Abstract

The invention relates to a copper-iron double-metal nano composite material, a copper-iron double-metal organic framework material is used as a carrier to load a drug, DSPE-PEG-iRGD is used to carry out surface functional modification on the copper-iron double-metal organic framework material, the copper-iron double-metal organic framework material is obtained by replacing part of 2-methylimidazole in CuFe-ZIF with 3-amino-1, 2, 4-triazole through ligand exchange, and the copper-iron double-metal organic framework material is a copper-iron double-metal nano composite material. The medicine is berberine. The preparation method comprises the following steps: S1, preparing CuFe-ZIF; s2, CuFe-ZIF-3AT is prepared, and the And S3, the copper and iron bimetal nano composite material CuFe-ZIF-3AT-Ber-PEG is prepared. The copper-iron bimetallic nano composite material is used as a drug carrier to be applied to tumor treatment. According to the invention, multiple anti-tumor effects such as chemical kinetics treatment, ferroptosis / copper death and chemotherapy can be realized.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to a copper-iron bimetallic nanocomposite material, a preparation method thereof, and an application thereof. Background Art

[0002] The harm of cancer has been intensifying globally and has become the number one "killer" threatening human health. Currently, the most commonly used treatment methods in clinics are surgery, chemotherapy, and radiotherapy. Chemotherapy can cause a series of acute and chronic organ toxicities. Since many chemotherapeutic drugs and their metabolites are excreted through renal tubular epithelial cells, the kidneys are easily damaged by chemotherapeutic drugs. Therefore, the use of chemotherapeutic drugs is sometimes limited by their nephrotoxic side effects. Chemotherapy-related kidney damage usually leads to insufficient cancer treatment because renal dysfunction requires clinicians to reduce the chemotherapy dose to avoid further kidney damage. Kidney damage can also cause other adverse complications, such as retention of water and nitrogenous wastes, electrolyte disorders, decreased immunity, etc. Developing effective drug carriers to reduce the inherent adverse reactions of chemotherapeutic drugs and improve the treatment effect has become one of the key issues in cancer treatment. Currently, in order to enhance the enhanced permeability and retention effect, drug nanocarriers have been developed to improve the accumulation of drugs in the tumor region, and the nanocarriers include polymer vesicles, micelles, polymer nanoparticles, inorganic nanoparticles, and hybrid porous solids. Among them, nano-metal-organic frameworks (MOFs) have attracted attention as drug nanocarriers with high drug loading capacity due to their high pore volume, large surface area, and adjustable pore size within the framework.

[0003] As an emerging treatment strategy, CDT usually utilizes the Fenton reaction mediated by Fe(II) ions to convert endogenous hydrogen peroxide (H2O2) into highly reactive and toxic hydroxyl radicals (·OH) with strong oxidation ability. CDT usually functions under acidic conditions and does not require external energy and oxygen, which is a safe and effective tumor treatment method. The efficiency of generating ·OH in a typical Fenton catalytic reaction mainly depends on the type of catalyst and the level of endogenous H2O2. Although the intracellular H2O2 level is higher than that of normal cells due to altered metabolism in tumor cells, the insufficient content of endogenous H2O2 is difficult to achieve satisfactory CDT efficacy.

[0004] Copper death is a newly discovered form of programmed cell death. Imbalance of intracellular copper metabolism, especially copper ion overload, may lead to the occurrence of copper death. Excessive copper accumulation targets and binds to lipoylated tricarboxylic acid cycle proteins, causing abnormal aggregation and triggering cell death. Multiple molecules such as ferredoxin 1 (FDX1) can regulate copper death activity. The homeostasis of copper metabolism in tumor cells is mainly maintained by the interaction of four groups of copper-related proteins, which affects related signaling pathways such as receptor tyrosine kinase (RTK), autophagy, and Notch, and is closely related to the occurrence and development of tumors.

[0005] Berberine is an isoquinoline alkaloid in Coptis chinensis, which can produce anti-tumor effects by inhibiting tumor cell proliferation, promoting tumor cell apoptosis, blocking the cell cycle, inhibiting tumor cell migration and invasion, inducing tumor cell autophagy, anti-inflammatory and antioxidant effects, and immunomodulation. However, the anti-cancer effect of using it alone is poor.

[0006] Based on the above situation, it is necessary to develop a nano-drug delivery complex that can directly deliver berberine to cancer cells and achieve the combined treatment of ferroptosis / copper death, photothermal therapy, and chemotherapy. Summary of the Invention

[0007] Based on this, it is necessary to provide a copper-iron bimetallic nanocomposite material, its preparation method and application in view of the defects and deficiencies of the prior art.

[0008] A copper-iron bimetallic nanocomposite material uses a copper-iron bimetallic organic framework material as a carrier, loads drugs, and is surface-functionalized with DSPE-PEG-iRGD. Among them, the copper-iron bimetallic organic framework material is obtained by replacing part of 2-methylimidazole in CuFe-ZIF with 3-amino-1,2,4-triazole through ligand exchange, and the drug is berberine.

[0009] As a preferred scheme, the CuFe-ZIF is obtained by coordinating copper ions, iron ions and zinc ions as metal sources with 2-methylimidazole.

[0010] A preparation method of the copper-iron bimetallic nanocomposite material as described above includes the following steps:

[0011] S1. Prepare CuFe-ZIF;

[0012] S2. Make the CuFe-ZIF prepared in S1 undergo a ligand exchange reaction with 3-amino-1,2,4-triazole to obtain CuFe-ZIF-3AT;

[0013] S3. React the CuFe-ZIF-3AT prepared in S2 with berberine and DSPE-PEG-iRGD to obtain the copper-iron bimetallic nanocomposite CuFe-ZIF-3AT-Ber-PEG.

[0014] As a preferred embodiment, the method for preparing CuFe-ZIF in S1 includes the following steps:

[0015] S11. Dissolve ferric nitrate nonahydrate and zinc nitrate hexahydrate in methanol to obtain solution A;

[0016] S12. Dissolve 2-methylimidazole in methanol to obtain solution B;

[0017] S13. Ultrasonic the copper foil in the solution A prepared in S11 to obtain solution C;

[0018] S14. Drop the solution C prepared in S13 into the solution B prepared in S12 and stir to react to obtain CuFe-ZIF.

[0019] As a preferred embodiment, the mass ratio of the ferric nitrate nonahydrate, the zinc nitrate hexahydrate, the copper foil and the 2-methylimidazole is 15:28:144:150.

[0020] As a preferred embodiment, the mass ratio of the CuFe-ZIF and the 3-amino-1,2,4-triazole in S2 is 2:1.

[0021] As a preferred embodiment, the mass ratio of the CuFe-ZIF-3AT, the berberine and the DSPE-PEG-iRGD in S3 is 3:2:2.

[0022] A copper-iron bimetallic nanocomposite prepared by the above preparation method is used as a drug carrier in tumor treatment.

[0023] The beneficial effects of the present invention are as follows: The copper-iron bimetal-doped zeolitic imidazolate framework material (CuFe-ZIF) was successfully prepared by a one-step synthesis method. Based on the structural homology and stronger coordination ability of 3-amino-1,2,4-triazole (3-AT) and the ligand 2-methylimidazole (2-mIm) in CuFe-ZIF, partial substitution of 2-mIm ligands in the CuFe-ZIF framework by 3-AT was achieved through a ligand exchange strategy, resulting in CuFe-ZIF-3AT. The CuFe-ZIF-3AT prepared in the present invention not only maintains the integrity of the framework but also has the largest specific surface area and porosity. Using it as a carrier, the drug berberine (Ber) was efficiently loaded and surface-functionalized with DSPE-PEG-iRGD, successfully constructing CuFe-ZIF-3AT-Ber-PEG with good biocompatibility. Through the synergistic effect of CuFe-ZIF-3AT and berberine, multiple anti-tumor effects such as chemodynamic therapy, ferroptosis / cuproptosis, and chemotherapy were achieved. Description of the Drawings

[0024] Figure 1 1H NMR spectra of 2-methylimidazole (2-mIm), 3-amino-1,2,4-triazole (3-AT), CuFe-ZIF-3AT(25), CuFe-ZIF-3AT(36), CuFe-ZIF-3AT(50), CuFe-ZIF-3AT(74), and CuFe-ZIF-3AT(148) of the present invention;

[0025] Figure 2 X-ray diffraction spectra of CuFe-ZIF-3AT(25), CuFe-ZIF-3AT(36), CuFe-ZIF-3AT(50), CuFe-ZIF-3AT(74), and CuFe-ZIF-3AT(148) of the present invention;

[0026] Figure 3 N2 adsorption-desorption isotherm curves (a) and pore size distribution curves (b) of CuFe-ZIF-3AT(25), CuFe-ZIF-3AT(36), CuFe-ZIF-3AT(50), CuFe-ZIF-3AT(74), and CuFe-ZIF-3AT(148) of the present invention;

[0027] Figure 4 X-ray diffraction spectra of ZIF-8, CuFe-ZIF, CuFe-ZIF-3AT, CuFe-ZIF-3AT-Ber, and CuFe-ZIF-3AT-Ber-PEG of the present invention;

[0028] Figure 5SEM images of CuFe-ZIF (a), CuFe-ZIF-3AT (b), CuFe-ZIF-3AT-Ber (c) and CuFe-ZIF-3AT-Ber-PEG (d) of the present invention, and TEM images of (CuFe-ZIF (e), CuFe-ZIF-3AT (f), CuFe-ZIF-3AT-Ber (g) and CuFe-ZIF-3AT-Ber-PEG (h);

[0029] Figure 6 Hydrodynamic particle size diagrams of ZIF-8, CuFe-ZIF, CuFe-ZIF-3AT, CuFe-ZIF-3AT-Ber and CuFe-ZIF-3AT-Ber-PEG of the present invention;

[0030] Figure 7 Zeta potential diagrams of CuFe-ZIF, CuFe-ZIF-3AT, CuFe-ZIF-3AT-Ber and CuFe-ZIF-3AT-Ber-PEG of the present invention;

[0031] Figure 8 Fourier transform infrared spectra of CuFe-ZIF, CuFe-ZIF-3AT, CuFe-ZIF-3AT-Ber and CuFe-ZIF-3AT-Ber-PEG of the present invention;

[0032] Figure 9 Nitrogen adsorption-desorption isotherm curves (a) and pore size distribution diagrams (b) of CuFe-ZIF, CuFe-ZIF-3AT, CuFe-ZIF-3AT-Ber and CuFe-ZIF-3AT-Ber-PEG of the present invention;

[0033] Figure 10 Content diagrams of Zn, Fe and Cu in CuFe-ZIF of the present invention;

[0034] Figure 11 Berberine release curves of CuFe-ZIF-3AT-Ber-PEG in PBS at pH 5.0, pH 6.5 and pH 7.4;

[0035] Figure 12 TMB colorimetric assay results of Control 1 to Control 7 of the present invention (a), CuFe-ZIF-3AT-2 mM TMB-10 mM H2O2 system at pH 4.0 (b), different H2O2 concentrations (c) and absorbance at 652 nm at different TMB concentrations (d);

[0036] Figure 13CuFe-ZIF and CuFe-ZIF-3AT electron spin resonance spectroscopy detection of ·OH diagram of the present invention;

[0037] Figure 14 Comparison diagram of the uptake of CuFe-ZIF-3AT-RhB-PEG by A549 cells at different times of the present invention (scale bar: 100 μm);

[0038] Figure 15 Cell viability of ZIF-8, Ber, 3-AT, CuFe-ZIF, CuFe-ZIF-3AT, CuFe-ZIF-3AT-Ber and CuFe-ZIF-3AT-Ber-PEG on A549 cells cultured for 24 h of the present invention;

[0039] Figure 16 Cell viability of ZIF-8, Ber, 3-AT, CuFe-ZIF, CuFe-ZIF-3AT, CuFe-ZIF-3AT-Ber and CuFe-ZIF-3AT-Ber-PEG on L929 cells cultured for 24 h of the present invention;

[0040] Figure 17 ROS levels of A549 cells treated with DCFH-DA in each group of the present invention (scale bar: 50 μm);

[0041] Figure 18 Live and dead cells of A549 cells treated with Calcein-AM / PI staining in each group of the present invention (scale bar: 100 μm);

[0042] Figure 19 JC-1 staining detection of mitochondrial membrane potential changes in A549 cells after treatment in each group of the present invention (scale bar: 100 μm);

[0043] Figure 20 For C of the present invention 11 -BODIPY detection of lipid peroxide levels in A549 cells after different treatments (scale bar: 100 μm);

[0044] Figure 21 In vitro chemical kinetics effect diagram of A549 cells after treatment with different groups of the present invention;

[0045] Figure 22 Cell viability diagram of A549 cells after treatment with different concentrations of H2O2 of the present invention. Detailed implementation manners

[0046] The endpoints and any values in the ranges disclosed in this document are not limited to the exact ranges or values. These ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the endpoint values of each range, between the endpoint values of each range and individual point values, and between individual point values can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed in this document.

[0047] The following is a detailed description of the specific embodiments of the present invention. It should be understood that the specific embodiments described herein are only for explaining and illustrating the present invention and are not used to limit the present invention.

[0048] Example 1

[0049] Preparation of CuFe-ZIF

[0050] S11. Weigh 150.0 mg of iron nitrate (Fe(NO3)3·9H2O) and 280.0 mg of zinc nitrate (Zn(NO3)2·6H2O) and dissolve them in 40.0 mL of methanol to obtain solution A.

[0051] S12. Weigh 1500.0 mg of 2-methylimidazole (2-MIm) and dissolve it in 40.0 mL of methanol to obtain solution B.

[0052] S13. Take 12 copper foils (size 2×2 cm, weight 120 mg / piece) and place them in solution A prepared in S11 for ultrasonic treatment to obtain solution C.

[0053] S14. Under high-speed stirring, slowly add solution C prepared in S13 dropwise to solution B prepared in S12, stir at room temperature for 1 h, centrifuge to collect the solid, and wash it 3 times with methanol solution. Finally, place the solid in an oven at 60 °C overnight for drying to obtain CuFe-ZIF.

[0054] Example 2

[0055] Preparation of CuFe-ZIF-3AT(25)

[0056] Weigh 3-AT (3-amino-1,2,4-triazole) and dissolve it in 5.0 mL of methanol solution. Take CuFe-ZIF and disperse it in 25.0 mL of methanol solution. Among them, the mass ratio of 3-AT to CuFe-ZIF is 1:2. Under high-speed stirring, mix the two solutions and stir at room temperature for 10 h. Centrifuge to collect the solid and wash it 3 times with methanol solution. Finally, place the solid in an oven at 60 °C overnight for drying to obtain CuFe-ZIF-3AT(25).

[0057] Example 3

[0058] Preparation of Copper-Iron Bimetallic Nanocomposite CuFe-ZIF-3AT-Ber-PEG

[0059] Take 10.0 mg of berberine and dissolve it in 3.0 mL of methanol to obtain Solution 1. Take 10.0 mg of DSPE-PEG-iRGD and dissolve it in 2.0 mL of methanol to obtain Solution 2. Take 15.0 mg of CuFe-ZIF-3AT and disperse it in 8.0 mL of methanol to obtain Solution 3. Under high-speed stirring, slowly drop Solution 1 and Solution 2 into Solution 3 in sequence, and stir at room temperature. Centrifuge to collect the solid, and freeze-dry the solid to obtain the copper-iron bimetallic nanocomposite CuFe-ZIF-3AT-Ber-PEG.

[0060] Comparative Example 1

[0061] Preparation of CuFe-ZIF-3AT(36)

[0062] The mass ratio of the 3-AT to CuFe-ZIF is 1:1.38, and the remaining steps are the same as those described in Example 2.

[0063] Comparative Example 2

[0064] Preparation of CuFe-ZIF-3AT(50)

[0065] The mass ratio of the 3-AT to CuFe-ZIF is 1:1, and the remaining steps are the same as those described in Example 2.

[0066] Comparative Example 3

[0067] Preparation of CuFe-ZIF-3AT(74)

[0068] The mass ratio of the 3-AT to CuFe-ZIF is 1:0.67, and the remaining steps are the same as those described in Example 2.

[0069] Comparative Example 4

[0070] Preparation of CuFe-ZIF-3AT(148)

[0071] The mass ratio of the 3-AT to CuFe-ZIF is 1:0.34, and the remaining steps are the same as those described in Example 2.

[0072] Comparative Example 5

[0073] Preparation of CuFe-ZIF-3AT-Ber

[0074] Dissolve 10.0 mg of berberine in 3.0 mL of methanol to obtain Solution 1. Disperse 15.0 mg of CuFe-ZIF-3AT in 8.0 mL of methanol to obtain Solution 2. Under high-speed stirring, slowly drop Solution 1 into Solution 2 in sequence, and stir at room temperature. Centrifuge to collect the solid, and freeze-dry the solid to obtain CuFe-ZIF-3AT-Ber.

[0075] Test Example 1

[0076] Nuclear magnetic resonance experiment

[0077] Use a nuclear magnetic resonance spectrometer to test commercially available 2-methylimidazole (2-mIm), commercially available 3-amino-1,2,4-triazole (3-AT), CuFe-ZIF-3AT(25) prepared in Example 2, CuFe-ZIF-3AT(36) prepared in Comparative Example 1, CuFe-ZIF-3AT(50) prepared in Comparative Example 2, CuFe-ZIF-3AT(74) prepared in Comparative Example 3, and CuFe-ZIF-3AT(148) prepared in Comparative Example 4. The experimental results are as Figure 1 shown.

[0078] It can be seen from Figure 1 that for CuFe-ZIF-3AT after exchange with different 3-AT ratios (1:2, 1:1.38, 1:1, 1:0.67, 1:0.34), the 3-AT:2-mIm are 0.6:1, 0.8:1, 1.12:1, 1.26:1, 1.35:1 respectively. The calculated 3-AT contents are 27.38%, 32.50%, 38.60%, 40.75%, 42.04% respectively.

[0079] Test Example 2

[0080] X-ray diffraction experiment

[0081] Use an X-ray diffractometer to perform X-ray diffraction tests on CuFe-ZIF-3AT(25) prepared in Example 2, CuFe-ZIF-3AT(36) prepared in Comparative Example 1, CuFe-ZIF-3AT(50) prepared in Comparative Example 2, CuFe-ZIF-3AT(74) prepared in Comparative Example 3, and CuFe-ZIF-3AT(148) prepared in Comparative Example 4. The test results are as Figure 2 shown.

[0082] It can be seen from Figure 2It can be seen that as the input amount of 3-AT increases, the diffraction peak intensity of the synthesized CuFe-ZIF-3AT gradually decreases, indicating that the increase in the input amount of 3-AT has a greater impact on the CuFe-ZIF crystal. Among them, CuFe-ZIF-3AT(25) has the best-preserved crystal structure.

[0083] Test Example 3

[0084] Nitrogen adsorption-desorption experiment

[0085] The specific surface area and pore size of CuFe-ZIF-3AT(25) prepared in Example 2, CuFe-ZIF-3AT(36) prepared in Comparative Example 1, CuFe-ZIF-3AT(50) prepared in Comparative Example 2, CuFe-ZIF-3AT(74) prepared in Comparative Example 3, and CuFe-ZIF-3AT(148) prepared in Comparative Example 4 were measured using a specific surface area adsorption instrument. The experimental results are as Figure 3 shown.

[0086] It can be seen from Figure 3 that as the input amount of 3-AT increases, the specific surface area and pore volume of CuFe-ZIF-3AT show a gradually decreasing trend. The specific surface area of CuFe-ZIF-3AT(25) is 920.9 m 2 / g, and the pore volume is 0.27 cm 3 / g; the specific surface area of CuFe-ZIF-3AT(36) is 673.8 m 2 / g, and the pore volume is 0.19 cm 3 / g; the specific surface area of CuFe-ZIF-3AT(50) is 675.0 m 2 / g, and the pore volume is 0.19 cm 3 / g; the specific surface area of CuFe-ZIF-3AT(74) is 593.8 m 2 / g, and the pore volume is 0.17 cm 3 / g; the specific surface area of CuFe-ZIF-3AT(148) is 583.2 m 2 / g, and the pore volume is 0.19 cm 3 / g. It can be seen that CuFe-ZIF-3AT(25) has the greatest drug-loading potential. Therefore, CuFe-ZIF-3AT(25) with the largest specific surface area and the highest porosity was selected for subsequent experiments. Subsequently, CuFe-ZIF-3AT(25) was uniformly named CuFe-ZIF-3AT.

[0087] Test Example 4

[0088] X-ray diffraction experiment

[0089] X-ray diffraction tests were carried out on commercially available ZIF-8, CuFe-ZIF prepared in Example 1, CuFe-ZIF-3AT prepared in Example 2, CuFe-ZIF-3AT-Ber prepared in Comparative Example 5, and CuFe-ZIF-3AT-Ber-PEG prepared in Example 3. The test results are as Figure 4 shown.

[0090] It can be Figure 4 seen that the CuFe-ZIF sample after Cu / Fe doping exhibits diffraction peaks similar to those of ZIF-8, indicating that the complete ZIF-8 crystal structure is retained after Cu / Fe doping. The diffraction peaks of CuFe-ZIF-3AT, CuFe-ZIF-3AT-Ber, and CuFe-ZIF-3AT-Ber-PEG are still basically the same as those of ZIF-8, indicating that whether it is 3-AT exchange, berberine loading, or surface PEG modification, the basic crystal structure is completely retained.

[0091] Test Example 5

[0092] Scanning electron microscopy and transmission electron microscopy experiments

[0093] Scanning electron microscopy and transmission electron microscopy were used to test CuFe-ZIF prepared in Example 1, CuFe-ZIF-3AT prepared in Example 2, CuFe-ZIF-3AT-Ber prepared in Comparative Example 5, and CuFe-ZIF-3AT-Ber-PEG prepared in Example 3 respectively. The experimental results are as Figure 5 shown.

[0094] It can be Figure 5 seen that CuFe-ZIF basically maintains the rhombic dodecahedron structure of ZIF-8. Due to the addition of 3-AT, the surface morphology of CuFe-ZIF-3AT is affected, the edges and corners are relatively blurred, and the particles increase. After loading berberine, it can be clearly seen that the morphology of CuFe-ZIF-3AT-Ber begins to change slightly, and the particle size of CuFe-ZIF-3AT increases slightly. After PEG surface modification, the surface of CuFe-ZIF-3AT-Ber-PEG changes further and the particle size increases further ( Figure 5 d), and the morphology is observed to be close to circular under TEM.

[0095] Test Example 6

[0096] Particle size distribution experiment

[0097] The particle size tests of commercially available ZIF-8, CuFe-ZIF prepared in Example 1, CuFe-ZIF-3AT prepared in Example 2, CuFe-ZIF-3AT-Ber prepared in Comparative Example 5, and CuFe-ZIF-3AT-Ber-PEG prepared in Example 3 were carried out by a particle size analyzer, and the experimental results are as Figure 6 shown.

[0098] It can be Figure 6 seen that the particle size of ZIF-8 is about 116 nm, and it slightly increases to about 120 nm after doping with metal ions. Due to the addition of 3-AT, the surface morphology of CuFe-ZIF-3AT is affected and the particle size increases to about 133 nm, which is consistent with the SEM results. After loading berberine, the particles partially aggregate, and the measured particle size of CuFe-ZIF-3AT-Ber is about 155 nm. Since PEG is hydrophilic and will fully expand in aqueous solution, the hydrated particle size of CuFe-ZIF-3AT-Ber-PEG is much larger than that of CuFe-ZIF-3AT-Ber, reaching about 181 nm.

[0099] Test Example 7

[0100] Zeta potential experiment

[0101] The Zeta potential tests of CuFe-ZIF prepared in Example 1, CuFe-ZIF-3AT prepared in Example 2, CuFe-ZIF-3AT-Ber prepared in Comparative Example 5, and CuFe-ZIF-3AT-Ber-PEG prepared in Example 3 were carried out by a Zeta potential tester, and the experimental results are as Figure 7 shown.

[0102] It can be Figure 7 seen that the Zeta potential of CuFe-ZIF is +23.5 mV. 3-AT is a triazole and itself has a negative charge, so the potential of CuFe-ZIF-3AT decreases to +11.7 mV. Berberine is a quaternary ammonium alkaloid and itself has a positive charge, so the potential increases to +14.5 mV after loading berberine. PEG has strong water solubility, and in the liquid phase medium, the ether bonds on the molecular surface of PEG have a weak negative charge, so the potential of CuFe-ZIF-3AT-Ber-PEG is 5.1 mV. The successful preparation of CuFe-ZIF-3AT-Ber-PEG is indirectly proved by the change of Zeta potential.

[0103] Test Example 8

[0104] Fourier transform infrared spectroscopy determination

[0105] The CuFe-ZIF prepared in Example 1, commercially available 3-amino-1,2,4-triazole (3-AT), CuFe-ZIF-3AT prepared in Example 2, commercially available berberine, CuFe-ZIF-3AT-Ber prepared in Comparative Example 5, and CuFe-ZIF-3AT-Ber-PEG prepared in Example 3 were tested with a Fourier transform infrared spectrometer, and the experimental results are as Figure 8 shown.

[0106] As Figure 8 can be seen, the small peaks in the ranges of 3135 cm -1 and 3000 - 2850 cm -1 can be attributed to the C-H stretching vibration modes of the imidazole ring and methyl group present in the linker, respectively. The peak at 1578 cm -1 may be due to the C=N stretching mode, while the strong peaks at 1454 and 1378 cm -1 correspond to the stretching of the entire ring. The several spectral bands observed in the range of 1350 to 900 cm -1 can be attributed to the in-plane bending of the ring, and the peaks at 760 and 690 cm -1 are related to the aromatic sp 2 C-H bending. Finally, a strong band is observed at 424 cm -1 which corresponds to the Zn-N stretching when the zinc atom in the ZIF-8 structure is connected to the nitrogen atom of the 2-mIm linker during the formation of ZIF-8. The C-N stretching vibration and N-H bending vibration of 3-AT correspond to 1212 cm -1 and 1633 cm -1 respectively; between 3000 cm -1 -2800 cm -1 are the bending vibration and resonance vibration of the aromatic C=N double bond. The above indicates the successful preparation of CuFe-ZIF and the successful exchange of 3-AT. In the infrared spectrum of CuFe-ZIF-3AT-Ber, the peak at 1493 cm -1 may be the stretching vibration of the aromatic C=N of berberine; 2844 cm -1 and 961 cm -1 are the absorption peaks of the substituents (methoxy groups) of berberine, indicating the successful loading of berberine. In the infrared spectrum of CuFe-ZIF-3AT-Ber-PEG, the broad peak at 1095 - 1060 cm -1 corresponds to the C-O stretching vibration of the PEG molecule, and the small peak at 1350 cm -1 can be attributed to the bending vibration of the methyl group in PEG. The above can illustrate the successful modification of PEG and the successful preparation of CuFe-ZIF-3AT-Ber-PEG.

[0107] Test Example 9

[0108] Nitrogen adsorption - desorption experiment

[0109] The specific surface area and pore size of commercially available ZIF - 8, CuFe - ZIF prepared in Example 1, CuFe - ZIF - 3AT prepared in Example 2, CuFe - ZIF - 3AT - Ber prepared in Comparative Example 5, and CuFe - ZIF - 3AT - Ber - PEG prepared in Example 3 were measured using a specific surface area adsorption instrument. The experimental results are as Figure 9 shown.

[0110] It can be seen from Figure 9 that the specific surface area of ZIF - 8 is as high as 1875.6 m 2 / g. After doping with copper and iron ions, the specific surface area of CuFe - ZIF is 1306.0 m 2 / g, and the pore volume is 0.35 cm 3 / g. After 3 - AT exchange, the specific surface area of CuFe - ZIF - 3AT decreased to 920.9 m 2 / g. This is because 3 - AT has an impact on the morphology of CuFe - ZIF, but it still has a relatively high specific surface area. As a guest molecule, berberine is loaded in CuFe - ZIF - 3AT - Ber, occupying most of the pores, and the specific surface area drops sharply to 177.1 m 2 / g, and the pore volume is 0.06 cm 3 / g, which confirms the effective loading of berberine. After surface coating with PEG, the pores are almost blocked, and the measured specific surface area of CuFe - ZIF - 3AT - Ber - PEG is only 35.9 m 2 / g. The above results indirectly prove the successful preparation of CuFe - ZIF - 3AT - Ber - PEG.

[0111] Test Example 10

[0112] Inductively coupled plasma emission spectrometry

[0113] The qualitative and quantitative analysis of elements in CuFe - ZIF prepared in Example 1 was carried out using an inductively coupled plasma mass spectrometer. The experimental results are as Figure 10 shown.

[0114] It can be seen from Figure 10 that the contents of Zn, Fe, and Cu in CuFe - ZIF are 22.26%, 1.44%, and 4.02% respectively. These results indicate the successful doping of Cu and Fe ions.

[0115] Test Example 11

[0116] In vitro release experiment of berberine at different pH values

[0117] To study the drug release behavior of CuFe-ZIF-3AT-Ber-PEG at different pH values, PBS buffer solutions with pH values of 7.4, 6.5, and 5.0 were used to simulate different physiological environments. 6.0 mg of CuFe-ZIF-3AT-Ber-PEG prepared in Example 3 was dispersed in 2.0 mL of PBS solutions with pH values of 5.0, 6.5, and 7.4, and then transferred into a dialysis bag and immersed in 100.0 mL of PBS solution with the corresponding pH value, and reacted with shaking in a water bath at 37 °C. At specific time intervals, 3.0 mL of buffer solution was taken out from the release medium for analysis, and an equal amount of fresh buffer solution was added, and dialysis continued for a period of time. The absorbance of berberine in the medium was detected at 263 nm using a UV-visible spectrophotometer, and the concentration of berberine was calculated through a standard working curve. All samples were repeatedly detected three times, and the results were the average of the three times. The cumulative drug release was calculated by Equation 1.

[0118] Cumulative release=(100C n +3∑C n-1 ) / (m0-cV)(Equation 1)

[0119] where C n and C n-1 are the concentrations of berberine released in the system at the nth and (n - 1)th times, respectively, and n is the time for extracting the buffer solution (n > 0).

[0120] As shown by Figure 11 , when the release time was 7 h, the release rate of berberine began to slow down, but CuFe-ZIF-3AT-Ber-PEG was quite stable at pH 7.4 and 6.5. By 24 h, the release of berberine was 17.21% at pH 7.4, 38.29% at pH 6.5, while there was obvious release of berberine (80.39%) under the condition of pH 5.0, and the release rate was still increasing with time. By 72 h, the release amount of berberine could reach 82.37%. This indicates that CuFe-ZIF-3AT-Ber-PEG has a tumor microenvironment response and can effectively decompose and release drugs.

[0121] Test Example 12

[0122] 3,3',5,5'-Tetramethylbenzidine color development experiment

[0123] First, to confirm the Fenton based on Fe 2+ and Cu +For the Fenton-Like reaction, at pH = 4.0 with other conditions being the same, control groups were set up: Control Group 1 (CuFe-ZIF-3AT alone); Control Group 2 (TMB + H2O2); Control Group 3 (CuFe-ZIF-3AT + TMB); Control Group 4 (CuFe-ZIF-3AT + TMB + H2O2); Control Group 5 (CuFe-ZIF + TMB + H2O2); Control Group 6 (ZIF-8 + TMB + H2O2); Control Group 7 (TMB alone).

[0124] Using TMB as the substrate to detect the peroxidase-like activity of CuFe-ZIF-3AT: At room temperature, add TMB solution (200 μL, 2 mM), 0.5 mL of CuFe-ZIF-3AT solution (10 μL, 1 mg / mL), acetic acid-sodium acetate buffer solution (1.59 mL, 0.1 M, pH = 4.0), and H2O2 solution (200 μL, 10 mM). Starting from 0 min, samples were collected every 3 min until 30 min. In addition, single-factor experiments were designed to explore the effects of different H2O2 concentrations and TMB concentrations on the POD-like catalytic activity. The experimental results are as Figure 12 shown.

[0125] As Figure 12 shown in Fig. a, Control Group 1 only contains CuFe-ZIF-3AT, Control Group 2 only contains TMB + H2O2, and Control Group 7 only contains TMB. The three groups have almost no absorption at 652 nm. Control Group 3 is CuFe-ZIF-3AT + TMB, and there is only a weak absorption at 652 nm without the addition of H2O2. Control Group 6 is ZIF-3AT + TMB + H2O2, and there is also only a weak absorption at 652 nm without iron / copper ions. Control Group 4 and Control Group 5 are CuFe-ZIF-3AT + TMB + H2O2 and CuFe-ZIF + TMB + H2O2 respectively, and both groups have strong absorption at 652 nm. The above results indicate that iron / copper metal ions and H2O2 are necessary conditions for initiating the Fenton / Fenton-Like reaction. Both CuFe-ZIF and CuFe-ZIF-3AT have the ability to generate ·OH, and the generation ability of CuFe-ZIF-3AT is stronger. Secondly, the ·OH generation performance of CuFe-ZIF-3AT was evaluated. Under the conditions of pH 4.0, 2 mM TMB, and 10 mM H2O2, the characteristic absorption peak of ox-TMB at 652 nm after the reaction was measured by ultraviolet-visible spectroscopy. The results are as Figure 12 shown in Fig. b. At 30 min of the reaction, a large absorbance can be measured at a wavelength of 652 nm, indicating that CuFe-ZIF-3AT has good ·OH generation performance. Finally, through single-factor experiments, the reaction rates under different TMB concentrations and different H2O2 concentrations were explored. The results are asFigure 12 c and Figure 12 as shown in d, the absorbance increases with the increase of TMB or H2O2 concentration.

[0126] Test Example 13

[0127] Electron spin resonance spectroscopy determination experiment

[0128] In the electron spin resonance (ESR) experiment, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO, 100 μM), 1 mM H2O2 and 2 mg / mL of the material were mixed in PBS (pH 5.5) and stirred for 1 h, and then the ESR spectrum in the X-band was measured. The experimental groups were as follows: 100 μL of CuFe-ZIF (pH 5.5, 1 mg / mL) + 100 μL of H2O2 (0.5 mM) + 500 μL of DMPO (50 mM); 100 μL of CuFe-ZIF-3AT (pH 5.5, 1 mg / mL) + 100 μL of H2O2 (0.5 mM) + 500 μL of DMPO (50 mM). The experimental results are as Figure 13 shown.

[0129] It can be seen from Figure 13 that CuFe-ZIF and CuFe-ZIF-3AT have a strong ability to react with H2O2 to generate ·OH in an acidic environment. This is consistent with the results of the TMB colorimetric experiment.

[0130] Test Example 14

[0131] A549 cell uptake experiment

[0132] A549 cells were seeded into 6-well plates at a density of 1×10 5 and incubated for 24 h. The groups were set up, including a drug administration group and a blank control group. Among them, the drug administration group was added with 50 μg / mL (1 mL per well) of CuFe-ZIF-3AT-RhB-PEG loaded with Rhodamine B (RhB); the blank control group was given 1 mL of 1640 complete medium. After administration, they were incubated for 1, 3, 6, 9, and 12 h respectively. After the administration time was reached, they were washed 3 times with PBS, and then the uptake of the material by A549 cells was observed and photographed under an inverted fluorescence microscope. The results are as Figure 14 shown.

[0133] It can be seen from Figure 14It can be seen that as the uptake time prolongs, the intracellular red fluorescence intensity gradually increases, indicating that the uptake of CuFe-ZIF-3AT-RhB-PEG by cells is time-dependent. When the uptake time is 1 h, only faint sporadic red color is shown, indicating that only a small amount of the material is taken up; when the uptake time is 3 h, the intracellular red fluorescence increases; when the uptake time reaches 6 h, almost all cells show red fluorescence, indicating that the material enters almost all cells at this time; when the uptake time reaches 9 h, the red fluorescence is further enhanced; when the uptake time reaches 12 h, the intracellular red fluorescence is not significantly enhanced, indicating that the uptake of the material by cells tends to be saturated.

[0134] Test Example 15

[0135] Cytotoxicity experiment

[0136] A549 cells or L929 cells were seeded into 96-well plates at a density of 8×10 3 and incubated for 24 h. Grouping was set up, including: ZIF-8, Ber, 3-AT, CuFe-ZIF, CuFe-ZIF-3AT, CuFe-ZIF-3AT-Ber, CuFe-ZIF-3AT-Ber-PEG. Each group was used to treat the cells in the 96-well plates with different concentrations: 25, 50, 100, 150, and 200 μg / mL (100 μL per well), and the cells were incubated for 24 h and 48 h after drug administration. Another blank control group was set up, which was given 100 μL of complete medium and incubated for 24 h and 48 h as well. After the incubation time was reached, the cells were washed twice with PBS, and then 100 μL of 1640 / DMEM medium (containing 5 μg / mL of MTT) was added to each well and incubated for 4 h. After the incubation ended, the medium was aspirated, 100 μL of DMSO was added, and the plate was slowly shaken in the dark for 15 min. Subsequently, the absorbance was measured at a wavelength of 490 nm with an enzyme-linked immunosorbent assay (ELISA) reader, and the cell viability was calculated. The calculation of cell viability is shown by Formula 2:

[0137] Cell viability (%) = (A1 / (A0) × 100% (Formula 2)

[0138] A1 and A0 represent the absorbances of the drug groups with different concentrations and the blank control group, respectively.

[0139] The above data were sorted out and charts were drawn, and the results are as shown in Figure 15 and 16 ;

[0140] All data were expressed as mean ± standard deviation (x±s). Inter-group differences were compared by one-way analysis of variance, and LSD-t test was continued after analysis of variance. GraphPad Prism 8.0 software was used for analysis. The test level was α = 0.05. When P < 0.05, the difference was statistically significant, and when P > 0.05, the difference was not statistically significant.

[0141] As Figure 15 shown, when the administration time was 24 h, the ZIF-8 group had no obvious inhibitory effect on A549 tumor cells, while CuFe-ZIF showed a higher inhibitory effect due to the presence of Cu and Fe ions. In addition, neither the Ber group nor the 3-AT group had a strong inhibitory effect on A549 cells, while CuFe-ZIF-3AT-Ber loaded with both showed a higher cell inhibitory effect than the four groups of Ber, 3-AT, CuFe-ZIF, and CuFe-ZIF-3AT, indicating the synergistic inhibitory effect of CuFe-ZIF with Ber and 3-AT on A549 cells. In addition, due to the surface modification of PEG, the CuFe-ZIF-3AT-Ber-PEG group had enhanced tumor cell targeting and showed the strongest cell inhibition. When the administration concentration was 200 μg / mL, the survival rate of A549 cells was only 24.4%. CuFe-ZIF-3AT-Ber-PEG showed good selectivity for A549 cells. As Figure 16 shown, the survival rate of L929 cells after treatment with each group was higher than that of the corresponding A549 cells. When the concentration of CuFe-ZIF-3AT-Ber-PEG was 200 μg / mL, the survival rate of L929 cells was 64.3%. The above experimental results showed that CuFe-ZIF-3AT-Ber-PEG could effectively and selectively inhibit A549 tumor cells and had high anti-tumor activity.

[0142] Test Example 16

[0143] Experiment on the generation of reactive oxygen species (ROS) in A549 cells

[0144] A549 cells were seeded at 6×10 per well 4Inoculate at a density into 12-well plates and incubate for 24 h. Set up groups, including the drug administration groups: ZIF-8, Ber, 3-AT, CuFe-ZIF, CuFe-ZIF-3AT, CuFe-ZIF-3AT-Ber, CuFe-ZIF-3AT-Ber-PEG, and the blank control group. Among them, the concentration of the drug administration groups is 60 μg / mL (1 mL per well); the blank control group is given 1 mL of 1640 complete medium. Incubate for 12 h after drug administration. After incubation, wash twice with PBS, and then add 2',7'-dichlorofluorescein diacetate (DCFH-DA) at a concentration of 10 μmol / L to each well and incubate for 30 min. After incubation, wash twice with PBS to remove the DCFH-DA that has not entered the cells, and then place it under an inverted fluorescence microscope to observe the generation of intracellular reactive oxygen species and take pictures. The results are as Figure 17 shown.

[0145] As Figure 17 can be seen, the blank control group does not undergo the Fenton / Fenton-Like reaction, so no fluorescence signal can be observed. The ZIF-8 group does not contain transition metals and only produces extremely weak green fluorescence. For CuFe-ZIF doped with copper and iron ions, due to the Fenton reaction induced by Fe 2+ and the Fenton-Like reaction induced by Cu + , it shows more green fluorescence. In addition, the Ber group and the 3-AT group also show weak green fluorescence. This is because berberine increases the intracellular reactive oxygen species level by interfering with the respiratory chain of tumor cells and affecting the mitochondrial function of cells; 3-AT may produce reactive oxygen species in cells by interfering with the antioxidant system, affecting metabolic pathways, and inducing oxidative stress. The components in the CuFe-ZIF-3AT-Ber group and the CuFe-ZIF-3AT-Ber-PEG group containing both of them produce a synergistic effect, further increasing the generation of reactive oxygen species and enhancing the green fluorescence. In addition, the level of reactive oxygen species in the CuFe-ZIF-3AT group is higher than that in the CuFe-ZIF group, which indirectly confirms the enhancing effect of 3-AT on CDT. The results show that CuFe-ZIF doped with Cu and Fe metals can produce a large amount of reactive oxygen species in cells, and after synergistic action with Ber and 3-AT, the generation of reactive oxygen species is further increased.

[0146] Test Example 17

[0147] Calcein-AM / PI double staining experiment for living and dead cells

[0148] Seed A549 cells at a density of 1×10 per well 5Inoculate at a density into 6-well plates and incubate for 24 h. Set up groups, including the drug administration groups: ZIF-8, Ber, 3-AT, CuFe-ZIF, CuFe-ZIF-3AT, CuFe-ZIF-3AT-Ber, CuFe-ZIF-3AT-Ber-PEG, and the blank control group. Among them, the concentration of the drug administration groups is 150 μg / mL (1 mL per well); the blank control group is given 1 mL of 1640 complete medium. Incubate for 4 h after drug administration. After incubation, wash the materials that have not entered the cells with PBS. Subsequently, in 1640 medium without FBS, stain the cells with a 100 μL mixed solution of Calcein-AM (2 μM) and PI (4.5 μM) for 30 min. After staining, aspirate the medium and wash 3 times with PBS, and then place it under an inverted fluorescence microscope to observe the live / dead cell staining and take pictures. The experimental results are as Figure 18 shown.

[0149] As Figure 18 can be seen, after incubation with A549 cells in each group, there are only individual dead cells (red fluorescence) in the blank control group and the ZIF-8 group. There are also only a small number of dead cells in the Ber group and the 3-AT group. The red fluorescence increases in the CuFe-ZIF group. The red fluorescence further increases in the CuFe-ZIF-3AT group after exchanging with 3-AT, indicating that the cytotoxicity of CuFe-ZIF-3AT is further enhanced. While there are only a small number of live cells (green fluorescence) in the CuFe-ZIF-3AT-Ber group and the CuFe-ZIF-3AT-Ber-PEG group. The experimental results show that ferroptosis and cuproptosis induced by CuFe-ZIF can effectively kill A549 cells. The increase in red fluorescence in the CuFe-ZIF-3AT group proves the enhancing effect of 3-AT on ferroptosis. The fact that there are only a small number of live cells left in the CuFe-ZIF-3AT-Ber-PEG group reflects the combined killing effect of CDT, ferroptosis / cuproptosis and the chemotherapy of berberine on tumor cells.

[0150] Test Example 18

[0151] Experiment on detecting the change of mitochondrial membrane potential in A549 cells with JC-1 probe

[0152] Seed A549 cells at a density of 8×10 per dish 4Inoculate at a density into confocal dishes and incubate for 24 h. Set up groups, including the drug administration groups: ZIF-8; Ber; 3-AT; CuFe-ZIF; CuFe-ZIF-3AT; CuFe-ZIF-3AT-Ber; CuFe-ZIF-3AT-Ber-PEG, and the blank control group. Among them, the concentration of the drug administration groups is 50 μg / mL (1 mL per dish); the blank control group is given 1 mL of 1640 complete medium. Incubate for 4 h after drug administration. After incubation, wash the materials that did not enter the cells with PBS. Then add the JC-1 staining working solution and incubate for 20 min. After incubation, aspirate the working solution and wash twice with JC-1 buffer, and then observe the changes in the mitochondrial membrane potential of the cells and take pictures under a laser scanning confocal microscope (CLSM). The experimental results are as Figure 19 shown.

[0153] It can be Figure 19 seen that there is almost no green fluorescence in the control group, and the small amount of fluorescence in the ZIF-8 group is due to the change in mitochondrial membrane potential caused by normal apoptosis of cells. Since berberine can affect the mitochondrial function of tumor cells and thus induce apoptosis; 3-AT may cause apoptosis of tumor cells by inducing oxidative stress, so both of these two groups show weak green fluorescence. The green fluorescence in the CuFe-ZIF group is enhanced, which is due to the entry of a large amount of iron / copper ions into the cells, triggering a highly efficient Fenton / Fenton-Like reaction, and ultimately leading to ferroptosis / cuprotosis. And because 3-AT can promote ferroptosis and the combined treatment effect after loading berberine, the green fluorescence in the CuFe-ZIF-3AT group, CuFe-ZIF-3AT-Ber group and CuFe-ZIF-3AT-Ber-PEG group is enhanced. The above shows that CuFe-ZIF-3AT-Ber-PEG can cause serious damage to mitochondria, and at the same time proves indirectly that CuFe-ZIF-3AT-Ber-PEG triggers ferroptosis / cuprotosis.

[0154] Test Example 19

[0155] Experiment for detecting lipid peroxidation (LPO) in A549 cells

[0156] Seed A549 cells at 8×10 per dish 4Inoculate at a density into confocal dishes and incubate for 24 h. Set up groups, including the drug administration group: ZIF-8, Ber, 3-AT, CuFe-ZIF, CuFe-ZIF-3AT, CuFe-ZIF-3AT-Ber, CuFe-ZIF-3AT-Ber-PEG, and the blank control group. Among them, the concentration of the drug administration group is 50 μg / mL (1 mL per dish); the blank control group is given 1 mL of 1640 complete medium. Incubate for 4 h after drug administration. After incubation, wash the materials that have not entered the cells with PBS. Then add 2 μM of BODIPY 581 to the dishes and incubate for 30 min. After incubation, aspirate and wash 3 times with PBS. Add 100 μL of Hoechst 33342 for staining for 10 min, and then observe the lipid peroxidation of cells and take pictures under a confocal microscope. The experimental results are as Figure 20 shown.

[0157] As Figure 20 can be seen, there is almost no green fluorescence in the control group and the ZIF-8 group, weak green fluorescence in the berberine group and the 3-AT group, and the green fluorescence of CuFe-ZIF begins to increase, indicating that Fe 2+ / Cu 2+ accumulates ROS through Fenton / Fenton-Like to induce lipid peroxidation in tumor cells. And the green fluorescence of CuFe-ZIF-3AT-Ber-PEG is the strongest, further confirming severe lipid peroxidation. These results are highly consistent with the analysis of reactive oxygen species and clearly prove that CuFe-ZIF-3AT-Ber-PEG induces ferroptosis.

[0158] Test Example 20

[0159] In vitro chemical kinetics experiment

[0160] Seed A549 cells at 8×10 per well 3Inoculate at a density into a 96-well plate and incubate for 24 h. Set up groups, including ZIF-8, Ber, 3-AT, CuFe-ZIF, CuFe-ZIF-3AT, CuFe-ZIF-3AT-Ber, and CuFe-ZIF-3AT-Ber-PEG. Then add each group of materials at concentrations of 0, 25, 50, 100, 150, 200 μg / mL (100 μL per well) respectively. After administration, incubate for 4 h. Set up another blank control group and give 100 μL of 1640 complete medium, and incubate for 4 h as well. After incubation, wash the materials that did not enter the cells with PBS. Then add 100 μL of complete medium containing 100 μM H2O2 to the 96-well plate and incubate in the incubator for 20 h. After the incubation time, wash twice with PBS, and then add 100 μL of complete medium (containing 5 μg / mL MTT) to each well and incubate for 4 h. After the incubation ends, aspirate the medium, add 100 μL of DMSO, shield from light, and shake slowly for 15 min. Then measure the absorbance at a wavelength of 490 nm with an enzyme-linked immunosorbent assay (ELISA) reader and calculate the cell survival rate. The cell survival rate is calculated by the following formula:

[0161] Cell survival rate (%) = (A1 / (A0)×100%

[0162] A1 and A0 represent the absorbances of the drug groups with different concentrations and the blank control group respectively. The experimental results are as Figure 21 and Figure 22 shown.

[0163] It can be seen from Figure 21 that in the ZIF-8 group, Ber group, and 3-AT group, since they do not contain transition metals, the killing effect on A549 is not significantly enhanced after adding H2O2. However, in each treatment group containing CuFe-ZIF in the structure, the killing effect on cells is significantly enhanced after adding H2O2, which proves that CuFe-ZIF-3AT-Ber-PEG has excellent chemodynamic effects. In addition, it can be seen from Figure 22 that alone H2O2 (100 μM) has no obvious inhibition on A549 cells.

[0164] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the above embodiments are described. However, as long as the combinations of these technical features do not conflict, they should all be considered as the scope recorded in this specification.

[0165] The above-described embodiments merely represent several implementation manners of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all fall within the protection scope of the present invention. Therefore, the protection scope of the present invention patent shall be subject to the appended claims.

Claims

1. A copper-iron bimetallic nanocomposite, characterized in that, Using a copper-iron bimetallic organic framework material as a carrier, loading a drug, and performing surface functional modification on it with DSPE-PEG-iRGD. Among them, the copper-iron bimetallic organic framework material is obtained by replacing part of 2-methylimidazole in CuFe-ZIF through ligand exchange with 3-amino-1,2,4-triazole, and the drug is berberine.

2. The copper-iron bimetallic nanocomposite material according to claim 1, wherein The CuFe-ZIF is obtained by coordinating copper ions, iron ions and zinc ions as metal sources with 2-methylimidazole.

3. A method for preparing the copper-iron bimetallic nanocomposite material according to any one of claims 1-2, characterized in that, It includes the following steps: S1. Prepare CuFe-ZIF; S2. Make the CuFe-ZIF prepared in S1 undergo a ligand exchange reaction with 3-amino-1,2,4-triazole to obtain CuFe-ZIF-3AT; S3. Make the CuFe-ZIF-3AT prepared in S2 react with berberine and DSPE-PEG-iRGD to obtain a copper-iron bimetallic nanocomposite CuFe-ZIF-3AT-Ber-PEG.

4. The preparation method of the copper-iron bimetallic nanocomposite material according to claim 3, wherein, The method for preparing CuFe-ZIF in S1 includes the following steps: S11. Dissolve ferric nitrate nonahydrate and zinc nitrate hexahydrate in methanol to obtain solution A; S12. Dissolve 2-methylimidazole in methanol to obtain solution B; S13. Place a copper foil in solution A prepared in S11 and ultrasonicate to obtain solution C; S14. Drop solution C prepared in S13 into solution B prepared in S12 and stir to react to obtain CuFe-ZIF.

5. The preparation method of the copper-iron bimetallic nanocomposite according to claim 4, characterized in that, The mass ratio of the ferric nitrate nonahydrate, the zinc nitrate hexahydrate, the copper foil and the 2-methylimidazole is 15:28:144:

150.

6. The preparation method of the copper-iron bimetallic nanocomposite material according to claim 3, wherein, The mass ratio of the CuFe-ZIF and the 3-amino-1,2,4-triazole in S2 is 2:

1.

7. The preparation method of the copper-iron bimetallic nanocomposite according to claim 3, characterized in that, The mass ratio of the CuFe-ZIF-3AT, the berberine and the DSPE-PEG-iRGD in S3 is 3:2:

2.

8. A copper-iron bimetallic nanocomposite prepared by the preparation method according to any one of claims 3-7 is used as a drug carrier in tumor treatment.

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