An anticancer drug complex and its preparation method and application

Abstract

The present invention discloses an anticancer drug complex, which is composed of zeolite imidazolate skeleton structure material as a base frame, composite ferrous sulfate and anticancer active components. The present invention also discloses a preparation method of the anticancer drug complex, comprising: 1) dissolving a metal salt in a solvent to form a solution A; 2) dissolving the anticancer active component in a solvent to form a solution B; 3) dropping solution B into solution A and stirring evenly to form a solution C; 4) adding an imidazole source solution to a solvent to form a solution D; 5) dropping solution D into solution C, stirring evenly, and reacting for 24 hours. The anticancer drug complex of the present invention is used in the preparation of drugs for treating cancer. The anticancer drug complex of the present invention has good anticancer effect and good in vivo safety. The raw materials of the preparation method are easily available, and the anticancer drug complex can be stably and reliably prepared with simple operations and processes.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to an anticancer drug complex and a preparation method and application thereof. Background Art

[0002] Metal-organic frameworks are porous, crystalline materials formed by the self-assembly of metal ions or metal clusters and multidentate organic ligands. This inorganic-organic hybrid material combines the excellent properties of inorganic and organic materials, not only with high specific surface area, adjustable size and porosity, but also with high drug loading rate and easy surface modification. Therefore, it is widely used in catalysis, gas capture, sensors, drug delivery and other fields.

[0003] Zeolitic imidazole framework (ZIF) is a very important subtype of metal organic framework. The metal ions used in it are zinc ions, which will not have a great impact on the human body. At the same time, the imidazole structure used in its organic ligand has a strong acid response characteristic and can undergo a hydrophilic and hydrophobic transition in a specific weak acid environment, thereby dissociating the entire framework and releasing the drugs it carries.

[0004] ZIF-8 has high porosity, large specific surface area, high drug loading rate and pH responsiveness. It can remain stable under physiological conditions and release drugs in the weakly acidic microenvironment of the tumor. It is an ideal carrier for loading drugs and achieving sustained drug release.

[0005] ZIF-8 can load DOX (doxorubicin) in a PBS solution (phosphate buffer solution) with a pH of 6.5, and the release rate of DOX@ZIF-8 is kept within 1% at 15 days. When the pH is 6.0, 90% of DOX is slowly released in 7-9 days. Compared with unencapsulated DOX, DOX@ZIF-8 shows good biocompatibility and therapeutic effect. However, the loading rate of DOX on ZIF-8 is only 14% to 20%. The therapeutic effect of DOX@ZIF-8 on breast cancer is still not ideal. Summary of the invention

[0006] The technical problem to be solved by the present invention is that the drug loading rate of ZIF-8 for loading DOX is low, and the therapeutic effect of DOX@ZIF-8 on breast cancer is still unsatisfactory. In view of the above-mentioned deficiencies in the prior art, an anticancer drug complex and a preparation method and application thereof are provided, wherein the doxorubicin loading rate of the anticancer drug complex reaches more than 50%; the anticancer drug complex can not only release DOX to cause apoptosis of breast cancer cells, but also increase the iron ion concentration in breast cancer cells, thereby causing ferroptosis of breast cancer cells, and has a good anticancer effect; the anticancer drug complex has good in vivo safety; the raw materials of the preparation method of the anticancer drug complex are easily available, and the anticancer drug complex can be stably and reliably prepared with simple operations and processes; the anticancer drug complex can be used to prepare drugs for treating breast cancer.

[0007] In order to solve the above technical problems, the technical solution of the present invention is:

[0008] An anticancer drug complex is composed of a zeolite imidazolate skeleton structure material as a base frame and composite ferrous sulfate and anticancer active components.

[0009] As a preferred embodiment, the anticancer active component is doxorubicin.

[0010] As a preferred embodiment, the zeolite imidazolate framework material is ZIF-8.

[0011] As a preferred embodiment, the drug loading rate of doxorubicin in the anticancer drug complex reaches more than 50%.

[0012] A method for preparing an anticancer drug complex comprises:

[0013] 1) The metal salt is dissolved in the solvent to form solution A;

[0014] 2) The anticancer active components are dissolved in the solvent to form solution B;

[0015] 3) Add solution B dropwise into solution A and stir evenly to form solution C;

[0016] 4) adding the imidazole source solution to the solvent to form solution D;

[0017] 5) Solution D is added dropwise into solution C, stirred evenly, and after reaction, an anticancer drug complex is obtained.

[0018] As a preferred solution, in step 1), the metal salts are green vitriol and zinc nitrate hexahydrate, and the mass ratio of green vitriol to zinc nitrate hexahydrate is 5 to 7:100.

[0019] As a preferred embodiment, the anticancer active component in step 2) is doxorubicin hydrochloride.

[0020] As a preferred embodiment, the imidazole source in step 4) is 2-methylimidazole.

[0021] As a preferred embodiment, the solvent is methanol.

[0022] The invention discloses an application of an anticancer drug complex in preparing a drug for treating breast cancer.

[0023] The beneficial effects of the present invention are as follows: the drug loading rate of the anticancer drug complex loaded with DOX reaches more than 50%; the anticancer drug complex can release DOX to cause apoptosis of breast cancer cells, and can also increase the iron ion concentration in breast cancer cells, thereby causing ferroptosis of breast cancer cells, and has a good anticancer effect; the anticancer drug complex has good in vivo safety; the raw materials of the preparation method of the anticancer drug complex are easily available, and the anticancer drug complex can be stably and reliably prepared with simple operations and processes; the anticancer drug complex can be used to prepare drugs for treating breast cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 X-ray diffraction patterns of ZIF-8, 5FZ, 7FZ, DOX@5FZ, and DOX@7FZ;

[0025] Figure 2 It is the X-ray photoelectron spectrum of 5FZ and 7FZ;

[0026] Figure 3 Thermogravimetric analysis diagrams of 5FZ, 7FZ, DOX@5FZ and DOX@7FZ;

[0027] Figure 4 This is a scanning electron micrograph of 5FZ at 1 μm;

[0028] Figure 5 This is a scanning electron microscope image of 7FZ at 5 μm;

[0029] Figure 6 This is the scanning electron microscopy image of DOX@5FZ at 1 μm;

[0030] Figure 7 This is the scanning electron microscopy image of DOX@7FZ at 1 μm;

[0031] Figure 8 The cytotoxicity of ZIF-8, DOX, 5FZ, 7FZ, DOX@5FZ and DOX@7FZ to MCF-7 cells at different concentrations;

[0032] Fig. 9 The cytotoxicity of ZIF-8, DOX, 5FZ, 7FZ, DOX@5FZ and DOX@7FZ to LO2 cells at different concentrations;

[0033] Fig.10This is a graph showing the effect of ZIF-8, 5FZ and 7FZ at the same concentration on the intracellular iron concentration of MCF-7 cells;

[0034] Fig.11 Graph showing the reactive oxygen species (ROS) levels of cells treated with ZIF-8, 5FZ, 7FZ, DOX@5FZ, DOX@7FZ and DOX for 24 h at the same concentration;

[0035] Fig.12 Fluorescence imaging of the dynamic changes of reactive oxygen species (ROS) after cells were treated with DOX@5FZ and DOX@7FZ at the same concentration;

[0036] Fig.13 The relative volume of tumors in mice in the blank control group, 5FZ group, 7FZ group, ZIF-8 group, DOX@5FZ group, and DOX@7FZ group;

[0037] Fig.14 HE staining of tumor sections of mice in the blank control group, 5FZ group, 7FZ group, ZIF-8 group, DOX@5FZ group, and DOX@7FZ group

[0038] Fig.15 HE staining images of organ sections of mice in blank control group, 5FZ group, 7FZ group, ZIF-8 group, DOX@5FZ group and DOX@7FZ group. DETAILED DESCRIPTION

[0039] The structural principle and working principle of the present invention are further described in detail below in conjunction with the accompanying drawings.

[0040] Example 1

[0041] Preparation of anticancer drug complex DOX@5FZ

[0042] 1) Dissolve 0.282 g zinc nitrate hexahydrate and 0.014 g green vitriol in 18.7 mL methanol, seal a 50 mL beaker with a sealing film, and stir for 5 min to obtain solution A;

[0043] 2) Weigh 7.5 mg of doxorubicin hydrochloride and disperse it in 1.3 mL of methanol by ultrasonication to obtain solution B;

[0044] 3) Slowly drop solution B into solution A and stir for 2 minutes to obtain a mixed solution C;

[0045] 4) Dissolve 0.328 g of 2-methylimidazole in 20 mL of methanol, seal a 50 mL beaker with a sealing film, and stir evenly to obtain solution D;

[0046] 5) Liquid D is slowly dripped into liquid C, and the 50 mL beaker is sealed with a sealing film. After stirring for 2 hours, it is allowed to stand for 24 hours to obtain solid E.

[0047] 6) After washing solid E with methanol for 3 times, the solid was placed in a centrifuge and centrifuged at a speed of 12000 r / min for 10 min. The obtained solid was placed in a vacuum dryer and vacuum dried at a temperature of 60° C. for 24 hours to obtain the anticancer drug complex DOX@5FZ.

[0048] Example 2

[0049] Preparation of anticancer drug complex DOX@7FZ

[0050] 1) Dissolve 0.275 g zinc nitrate hexahydrate and 0.021 g green vitriol in 18.7 mL methanol, seal a 50 mL beaker with a sealing film, and stir for 5 min to obtain solution A;

[0051] 2) Weigh 7.5 mg of doxorubicin hydrochloride and disperse it in 1.3 mL of methanol by ultrasonication to obtain solution B;

[0052] 3) Slowly drop solution B into solution A and stir for 2 minutes to obtain a mixed solution C;

[0053] 4) Dissolve 0.328 g of 2-methylimidazole in 20 mL of methanol, seal a 50 mL beaker with sealing film, and stir evenly to obtain solution D;

[0054] 5) Liquid D is slowly dripped into liquid C, and the 50 mL beaker is sealed with a sealing film. After stirring for 2 hours, it is allowed to stand for 24 hours to obtain solid E.

[0055] 6) After washing solid E with methanol for 3 times, the solid was placed in a centrifuge and centrifuged at a speed of 12000 r / min for 10 min. The obtained solid was placed in a vacuum dryer and vacuum dried at a temperature of 60° C. for 24 hours to obtain the anticancer drug complex DOX@7FZ.

[0056] Comparative Example 1

[0057] Preparation of Ferrous Sulfate-Zeolite Imidazolyl Framework Composite Material 5FZ

[0058] 1) Dissolve 0.282 g zinc nitrate hexahydrate and 0.014 g green vitriol in 20 mL methanol, seal a 50 mL beaker with a sealing film, and stir for 5 min to obtain solution F;

[0059] 2) Dissolve 0.328 g of 2-methylimidazole in 20 mL of methanol, seal a 50 mL beaker with a sealing film, and stir evenly to obtain solution G;

[0060] 3) Slowly drip solution G into solution F, seal the beaker with sealing film, stir for 2 hours, and let stand for 24 hours to obtain solid H;

[0061] 4) After washing the solid H with methanol for 3 times, the solid was placed in a centrifuge and centrifuged at a speed of 12000 r / min for 10 min. The obtained solid was placed in a vacuum dryer and vacuum dried at a temperature of 60° C. for 24 hours to obtain the ferrous sulfate-zeolite imidazolate framework structure composite material 5FZ.

[0062] Comparative Example 2

[0063] Preparation of Ferrous Sulfate-Zeolite Imidazolyl Framework Composite Material 7FZ

[0064] 1) Dissolve 0.275 g zinc nitrate hexahydrate and 0.021 g green vitriol in 20 mL methanol, seal a 50 mL beaker with a sealing film, and stir for 5 min to obtain solution F;

[0065] 2) Dissolve 0.328 g of 2-methylimidazole in 20 mL of methanol, seal a 50 mL beaker with a sealing film, and stir evenly to obtain solution G;

[0066] 3) Slowly drip solution G into solution F, seal the beaker with sealing film, stir for 2 hours, and let stand for 24 hours to obtain solid H;

[0067] 4) After washing the solid H with methanol for 3 times, the solid was placed in a centrifuge and centrifuged at a speed of 12000 r / min for 10 min. The obtained solid was placed in a vacuum dryer and vacuum dried at a temperature of 60° C. for 24 hours to obtain the ferrous sulfate-zeolite imidazolate framework structure composite material 7FZ.

[0068] DOX@5FZ used in the following experimental examples was prepared from Example 1. DOX@7FZ was prepared from Example 2. 5FZ was prepared from Comparative Example 1. 7FZ was prepared from Comparative Example 2. ZIF-8 was obtained from commercial sources.

[0069] Experimental Example 1

[0070] X-ray diffraction experiments

[0071] Experimental steps: Use an X-ray diffractometer to perform X-ray diffraction measurements on ZIF-8, 5FZ, 7FZ, DOX@5FZ and DOX@7FZ respectively.

[0072] Experimental results:

[0073] Depend on Figure 1 It can be seen that the crystal structure of the zeolite imidazolate framework material ZIF-8 was not affected after being doped with 5% and 7% ferrous sulfate, and the overall material structure remained unchanged after the drug was loaded.

[0074] Experimental Example 2

[0075] X-ray photoelectron spectroscopy experiment

[0076] Experimental steps: Use an X-ray photoelectron spectrometer to measure the X-ray photoelectron spectra of 5FZ and 7FZ respectively.

[0077] Experimental results:

[0078] Depend on Figure 2 It can be seen that the X-ray photoelectron energy spectra of 5FZ and 7FZ both show iron element energy spectrum peaks, which indicates that iron is successfully doped into ZIF-8.

[0079] Experimental Example 3

[0080] Thermogravimetric experiment

[0081] Experimental steps: Weigh 10 mg 5FZ, 10 mg 7FZ, 10 mg DOX@5FZ and 10 mg negative DOX@7FZ respectively and place them in a crucible (specially for thermogravimetric analysis), and then put them into a thermogravimetric analyzer (model HCT-2, manufactured by Beijing Hengjiu Scientific Instrument Factory) for thermogravimetric experiment. During the experiment, the thermogravimetric analyzer was set according to the following conditions: nitrogen flow rate was 150 mL / min, reaction temperature was increased from 25°C to 800°C, and the heating rate was 10°C / min.

[0082] The experimental results are as follows Figure 3 shown.

[0083] Experimental Example 4

[0084] Scanning electron microscopy experiments

[0085] Experimental steps: Scanning electron microscopy was used to perform scanning electron microscopy on 5FZ, 7FZ, DOX@5FZ and DOX@7FZ respectively.

[0086] Experimental results:

[0087] Depend on Figures 4 to 7 It can be seen that 5FZ and 7FZ still have the twelve-section morphological characteristics of ZIF-8. When 5FZ and 7FZ are loaded with DOX, the surface morphology of the two materials changes to varying degrees, but the skeleton can basically be maintained.

[0088] Experimental Example 5

[0089] Drug loading test of DOX@5FZ and DOX@7FZ

[0090] Experimental steps:

[0091] 1) collecting the DOX@5FZ supernatant obtained after centrifugation and the DOX@5FZ washing solution obtained after washing in Example 1;

[0092] 2) collecting the DOX@7FZ supernatant obtained after centrifugation in Example 2 and the DOX@7FZ washing solution obtained after washing;

[0093] 3) Weigh 10 mg of doxorubicin hydrochloride standard and dissolve it in 100 mL of water to prepare a 100 mg / L standard solution, then dilute it with water to make doxorubicin standard solutions with concentrations of 10, 15, 20, 25, 30, and 35 mg / L, and use an ultraviolet spectrophotometer (UV-vis) to measure the absorbance of the above doxorubicin standard solutions at 530 nm, record the data, and draw a doxorubicin standard curve;

[0094] 4) Using an ultraviolet spectrophotometer (UV-vis) to measure the absorbance of the DOX@5FZ supernatant and the DOX@5FZ washing solution at 530 nm in step 1), and comparing with the doxorubicin standard curve obtained in step 3), the concentration of doxorubicin in the DOX@5FZ supernatant and the concentration of doxorubicin in the DOX@5FZ washing solution were calculated respectively;

[0095] 5) Using an ultraviolet spectrophotometer (UV-vis) to measure the absorbance of the DOX@7FZ supernatant and the DOX@7FZ washing solution at 530 nm in step 2), and comparing with the doxorubicin standard curve obtained in step 3), the concentration of doxorubicin in the DOX@7FZ supernatant and the concentration of doxorubicin in the DOX@7FZ washing solution were calculated respectively;

[0096] 6) Calculate the loading rates of doxorubicin at 5FZ and 7FZ respectively according to the loading rate formula of doxorubicin.

[0097] The loading rate formula of doxorubicin is as follows:

[0098]

[0099] Experimental results:

[0100] It was calculated that the loading rate of DOX@5FZ was 56.04% and that of DOX@7FZ was 57.21%.

[0101] Experimental Example 6

[0102] Tumor cell toxicity assay

[0103] Experimental steps:

[0104] 1) Prepare the experimental solution:

[0105] Preparation of experimental solution for DOX group: DOX was dissolved in PBS solution, and PBS solution was used to prepare DOX experimental solutions with concentrations of 15, 30, 45, and 60 μg / mL;

[0106] Preparation of experimental solution for ZIF-8 group: ZIF-8 was dissolved in PBS solution, and PBS solution was used to prepare ZIF-8 experimental solutions with concentrations of 15, 30, 45, and 60 μg / mL;

[0107] Preparation of experimental solution for 5FZ group: 5FZ was dissolved in PBS solution, and 5FZ experimental solutions with concentrations of 15, 30, 45, and 60 μg / mL were prepared with PBS solution;

[0108] Preparation of experimental solution for 7FZ group: 7FZ was dissolved in PBS solution, and 7FZ experimental solutions with concentrations of 15, 30, 45, and 60 μg / mL were prepared with PBS solution;

[0109] Preparation of experimental solution for DOX@5FZ group: DOX@5FZ was dissolved in PBS solution, and DOX@5FZ experimental solutions with concentrations of 15, 30, 45, and 60 μg / mL were prepared with PBS solution;

[0110] Preparation of experimental solution for DOX@7FX group: DOX@7FX was dissolved in PBS solution, and DOX@7FX experimental solutions with concentrations of 15, 30, 45, and 60 μg / mL were prepared using PBS solution.

[0111] 2) Take MCF-7 cells in the logarithmic growth phase and in good growth condition, digest the cells, remove the original culture medium, add 3 mL PBS solution to wash twice, add 2 mL trypsin and place in the incubator for digestion for 2 min. After digestion, add 2 mL DMEM complete culture medium (containing 10% fetal bovine serum and 1% double antibody), centrifuge at 900 r / min for 3 min, remove the supernatant, add 6 mL DMEM complete culture medium, blow evenly, count the cells, and prepare a cell suspension according to the experimental requirements (cell density 4×10 4 / mL), and 4 × 10 4Cells were plated in a 96-well plate and cultured in an incubator for 24 hours. After the cells adhered to the wall, the original culture medium was removed and 20 μL of PBS solution, DOX experimental solution, ZIF-8 experimental solution, 5FZ experimental solution, 7FZ experimental solution, DOX@5FZ experimental solution and DOX@7FX experimental solution were added respectively, and the drug concentration was converted to 7%. After the addition of drugs, the plates were placed in the incubator for another 24 hours. After the culture was completed, the drug-containing culture medium was removed, and 100 μL of DMEM complete culture medium without fetal bovine serum and double antibodies prepared with 10% MTT (5 mg / mL) was added to each well. After the plates were placed in the incubator for another 4 hours, the culture medium was removed, 100 μL of DMSO was added to each well and the plates were shaken in a shaker for 15 minutes. Finally, the absorbance of each well was measured at a wavelength of 490 nm using an ELISA reader, and the data were recorded. The corresponding cell survival rate was calculated using the cell survival rate calculation formula, and a line graph was drawn with the cell survival rate as the y-axis and the drug concentration as the x-axis.

[0112] The cell viability calculation formula is as follows:

[0113]

[0114] Experimental results:

[0115] Depend on Figure 8 It can be seen that the drug-loaded material has a greater killing effect on tumor cells than the unloaded material, and the survival rate of tumor cells added with DOX@7FZ experimental solution at concentrations of 45g / mL and 60ug / mL is the lowest. This shows that DOX@7FZ can exert the greatest ability to kill cancer cells at concentrations of 45 and 60ug / mL.

[0116] Experimental Example 7

[0117] Normal liver cell toxicity test

[0118] Experimental steps:

[0119] 1) Prepare the experimental solution:

[0120] Preparation of experimental solution for DOX group: DOX was dissolved in PBS solution, and PBS solution was used to prepare DOX experimental solutions with concentrations of 15, 30, 45, and 60 μg / mL;

[0121] Preparation of experimental solution for ZIF-8 group: ZIF-8 was dissolved in PBS solution, and PBS solution was used to prepare ZIF-8 experimental solutions with concentrations of 15, 30, 45, and 60 μg / mL;

[0122] Preparation of experimental solution for 5FZ group: 5FZ was dissolved in PBS solution, and 5FZ experimental solutions with concentrations of 15, 30, 45, and 60 μg / mL were prepared with PBS solution;

[0123] Preparation of experimental solution for 7FZ group: 7FZ was dissolved in PBS solution, and 7FZ experimental solutions with concentrations of 15, 30, 45, and 60 μg / mL were prepared with PBS solution;

[0124] Preparation of experimental solution for DOX@5FZ group: DOX@5FZ was dissolved in PBS solution, and DOX@5FZ experimental solutions with concentrations of 15, 30, 45, and 60 μg / mL were prepared with PBS solution;

[0125] Preparation of experimental solution for DOX@7FX group: DOX@7FX was dissolved in PBS solution, and DOX@7FX experimental solutions with concentrations of 15, 30, 45, and 60 μg / mL were prepared using PBS solution.

[0126] 2) Take LO2 cells in the logarithmic growth phase and in good growth condition, digest the cells, remove the original culture medium, add 3 mL PBS solution to wash twice, add 2 mL trypsin and place in the incubator for digestion for 2 min. After digestion, add 2 mL DMEM complete culture medium (containing 10% fetal bovine serum and 1% double antibody), centrifuge at 900 r / min for 3 min, remove the supernatant, add 6 mL DMEM complete culture medium, blow evenly, count the cells, and prepare a cell suspension according to the experimental requirements (cell density 4×10 4 / mL), and 4 × 10 4 Cells were plated in a 96-well plate and cultured in an incubator for 24 hours. After the cells adhered to the wall, the original culture medium was removed and 20 μL of PBS solution, DOX experimental solution, ZIF-8 experimental solution, 5FZ experimental solution, 7FZ experimental solution, DOX@5FZ experimental solution and DOX@7FX experimental solution were added respectively, and the drug concentration was converted to 7%. After the addition of drugs, the plates were placed in the incubator for another 24 hours. After the culture was completed, the drug-containing culture medium was removed, and 100 μL of DMEM complete culture medium without fetal bovine serum and double antibodies prepared with 10% MTT (5 mg / mL) was added to each well. After the plates were placed in the incubator for another 4 hours, the culture medium was removed, 100 μL of DMSO was added to each well and the plates were shaken in a shaker for 15 minutes. Finally, the absorbance of each well was measured at a wavelength of 490 nm using an ELISA reader, and the data were recorded. The corresponding cell survival rate was calculated using the above cell survival rate calculation formula, and a line graph was drawn with the cell survival rate as the y-axis and the drug concentration as the x-axis.

[0127] Experimental results:

[0128] Depend on Fig. 9It can be seen that the toxicity of the material to liver cells after drug loading is slightly greater than that before drug loading, but it is still within the safe range. The cell activity of the DOX@7FZ group at a concentration of 45ug / mL is 86.23%. This shows that DOX@7FZ at a concentration of 45ug / mL can not only exert the maximum ability to kill cancer cells, but also have less toxicity to liver cells.

[0129] Experimental Example 8

[0130] Determination of intracellular iron concentration in MCF-7 cells

[0131] Experimental steps:

[0132] 1) Prepare the experimental solution:

[0133] Preparation of experimental solution for ZIF-8 group: ZIF-8 was dissolved in PBS solution, and PBS solution was used to prepare ZIF-8 experimental solutions with concentrations of 5, 15, and 30 μg / mL;

[0134] Preparation of experimental solution for 5FZ group: 5FZ was dissolved in PBS solution, and 5FZ experimental solutions with concentrations of 5, 15, and 30 μg / mL were prepared with PBS solution;

[0135] Preparation of experimental solution for 7FZ group: 7FZ was dissolved in PBS solution, and 7FZ experimental solutions with concentrations of 5, 15, and 30 μg / mL were prepared using PBS solution.

[0136] 2) Take MCF-7 cells in the logarithmic growth phase and in good growth condition, digest the cells, remove the original culture medium, add 3 mL PBS solution to wash twice, add 2 mL trypsin and place in the incubator for digestion for 2 min. After digestion, add 2 mL DMEM complete culture medium (containing 10% fetal bovine serum and 1% double antibody), centrifuge at 900 r / min for 3 min, remove the supernatant, add 6 mL DMEM complete culture medium, blow evenly, count the cells, and prepare a cell suspension according to the experimental requirements (cell density 5×10 5 / mL), and 5 × 10 5Cells were plated in a 24-well plate and placed in an incubator for 24 hours. After the cells adhered to the wall, the original culture medium was removed and 20 μL PBS solution, ZIF-8 experimental solution, FZ experimental solution and 7FZ experimental solution were added respectively. After the addition of drugs, the cells were placed in the incubator again for 24 hours. After the culture was completed, the culture medium was discarded, and the cells were washed twice with 2 mL of cold PBS, and the PBS was discarded. 200 ul / well lysis buffer was used to lyse the cells and placed on a shaker for 2 hours. 4.5% potassium permanganate solution was prepared and mixed with the buffer in the iron ion colorimetric detection kit in a ratio of 1:1, which is called mixed solution A. 100 ul of the lysed sample solution and the diluted standard were taken, and each was mixed with 100 ul of mixed solution A, and incubated at 60°C for 1 hour. Cool to room temperature, and centrifuge the droplets on the tube cap and the tube wall into the bottom of the tube. 30 ul of iron ion detection reagent was added, mixed, and incubated at room temperature for 30 minutes. Take 200ul into a 96-well plate and measure the absorbance at 550nm. Draw a standard curve and calculate the iron ion concentration.

[0137] Experimental results:

[0138] Depend on Fig.10 It can be seen that the intracellular iron ion concentration of the 7FZ group at a concentration of 30ug / ml was the highest and significantly different from that of the other groups, indicating that 7FZ increased the intracellular iron ion concentration of MCF-7 cells, thereby causing an imbalance in intracellular iron metabolism.

[0139] Experimental Example 9

[0140] Flow cytometry experiments

[0141] Experimental steps:

[0142] 1) Prepare the experimental solution:

[0143] Preparation of experimental solution for ZIF-8 group: ZIF-8 was dissolved in PBS solution, and PBS solution was used to prepare a ZIF-8 experimental solution with a concentration of 30 μg / mL;

[0144] Preparation of experimental solution for 5FZ group: 5FZ was dissolved in PBS solution, and PBS solution was used to prepare a 5FZ experimental solution with a concentration of 30 μg / mL;

[0145] Preparation of experimental solution for 7FZ group: 7FZ was dissolved in PBS solution, and PBS solution was used to prepare 7FZ experimental solution with a concentration of 30 μg / mL;

[0146] Preparation of experimental solution for DOX@5FZ group: DOX@5FZ was dissolved in PBS solution, and a DOX@5FZ experimental solution with a concentration of 30 μg / mL was prepared using PBS solution;

[0147] Preparation of experimental solution for DOX@7FZ group: DOX@7FZ was dissolved in PBS solution, and a DOX@7FZ experimental solution with a concentration of 30 μg / mL was prepared using PBS solution;

[0148] Preparation of experimental solution for DOX group: DOX was dissolved in PBS solution, and a DOX experimental solution with a concentration of 30 μg / mL was prepared using PBS solution.

[0149] 2) Take MCF-7 cells in the logarithmic growth phase and in good growth condition, digest the cells, remove the original culture medium, add 3 mL PBS solution to wash twice, add 2 mL trypsin and place in the incubator for digestion for 2 min. After digestion, add 2 mL DMEM complete culture medium (containing 10% fetal bovine serum and 1% double antibody), centrifuge at 900 r / min for 3 min, remove the supernatant, add 6 mL DMEM complete culture medium, blow evenly, count the cells, and prepare a cell suspension according to the experimental requirements (cell density 1.5×10 6 / mL), and 1.5 × 10 6 Cells were plated in 6-well plates and placed in an incubator for 24 hours. After the cells adhered to the wall, the original culture medium was removed, and 20 μL PBS solution, ZIF-8 experimental solution, 5FZ experimental solution, 7FZ experimental solution, DOX@5FZ experimental solution, DOX@7FZ experimental solution and DOX experimental solution were added respectively. After the addition of drugs, the cells were placed in the incubator for further culturing for 24 hours. After the culture was completed, the original culture medium was removed, washed with PBS for 3 times, and then 500 μL trypsin was added and placed in an incubator for digestion for 2 minutes. After digestion, an equal amount of DMEM complete medium was added to terminate the digestion, centrifuged at 900 r / min for 3 minutes, the supernatant was removed, and then 500 μL PBS was added to blow and wash, and the centrifuged again at 900 r / min for 3 minutes. After the supernatant was removed, 500 μL PBS was added to blow and wash, and then the cells were passed through a cell sieve and collected in a flow tube. Finally, the fluorescence intensity was detected by flow cytometry.

[0150] Experimental results:

[0151] Depend on Fig.11 It can be seen that the fluorescence intensity of cell reactive oxygen species in the DOX@7FZ group was the highest, indicating that DOX@7FZ caused MCF-7 cells to produce the most reactive oxygen species.

[0152] Example 10

[0153] Cell imaging experiments

[0154] Experimental steps:

[0155] 1) Prepare the experimental solution:

[0156] Preparation of experimental solution for DOX@5FZ group: DOX@5FZ was dissolved in PBS solution, and a DOX@5FZ experimental solution with a concentration of 30 μg / mL was prepared using PBS solution;

[0157] Preparation of experimental solution for DOX@7FZ group: DOX@7FZ was dissolved in PBS solution, and a DOX@7FZ experimental solution with a concentration of 30 μg / mL was prepared using PBS solution.

[0158] 2) Take MCF-7 cells in the logarithmic growth phase and in good growth condition, digest the cells, remove the original culture medium, add 3 mL PBS solution to wash twice, add 2 mL trypsin and place in the incubator for digestion for 2 min. After digestion, add 2 mL DMEM complete culture medium (containing 10% fetal bovine serum and 1% double antibody), centrifuge at 900 r / min for 3 min, remove the supernatant, add 6 mL DMEM complete culture medium, blow evenly, count the cells, and prepare a cell suspension according to the experimental requirements (cell density 1.5×10 6 / mL), and 1.5 × 10 6 Cells were plated in 6-well plates and placed in an incubator for 24 hours. After the cells adhered to the wall, the original culture medium was removed and 20 μL of DOX@5FZ experimental solution and DOX@7FZ experimental solution were added respectively. After the addition of drugs, the cells were placed in the incubator for 6, 12, and 24 hours. After the culture was completed, the original culture medium was removed, washed with PBS 3 times, and then 500 μL of PBS containing fluorescent probes was added and placed in an incubator for 30 minutes. The cells were taken out and shaken every 5 minutes to mix well. After the end, they were washed with PBS twice and the cell fluorescence imaging was observed under a fluorescence microscope.

[0159] Experimental results:

[0160] Depend on Fig.12 It can be seen that the cell reactive oxygen species production and fluorescence intensity of the DOX@7FZ group at 6, 12, and 24 hours were more and stronger than those of the DOX@5FZ group, indicating that DOX@7FZ has a better ability to make MCF-7 cells produce reactive oxygen species.

[0161] Experimental Example 11

[0162] Modeling of MCF-7 breast cancer in nude mice

[0163] Experimental steps:

[0164] 1) Prepare the injection solution;

[0165] Preparation of 5FZ injection: 5FZ was dissolved in PBS solution, and 5FZ injection with a concentration of 5 mg / mL was prepared using PBS solution;

[0166] Preparation of 7FZ injection: 7FZ was dissolved in PBS solution, and 5 mg / mL 7FZ injection was prepared with PBS solution;

[0167] Preparation of DOX@5FZ injection: DOX@5FZ was dissolved in PBS solution, and 7DOX@5FZ injection with a concentration of 5 mg / mL was prepared using PBS solution;

[0168] Preparation of DOX@7FZ injection: DOX@7FZ was dissolved in PBS solution, and PBS solution was used to prepare DOX@7FZ injection with a concentration of 5 mg / mL;

[0169] Preparation of ZIF-8 injection: ZIF-8 was dissolved in PBS solution, and PBS solution was used to prepare ZIF-8 injection with a concentration of 5 mg / mL.

[0170] 2) 30 female BALB / C nude mice were divided into 6 groups: blank control group, 5FZ group, 7FZ group, ZIF-8 group, DOX@5FZ group and DOX@7FZ group, with 5 mice in each group. The breast cancer model was established by injection of MCF-7 cell suspension. Injection site: subcutaneous tissue of the left axilla of nude mice. Cell quantity: 5*10 6 cells / mouse. After bilateral injection of MCF-7 cells, the mice were fed for 3 weeks. At 3, 4, and 5 weeks after the injection of cells, the drugs were injected through the tail vein. The blank control group was injected with PBS solution, the 5FZ group was injected with 5FZ injection, the 7FZ group was injected with 7FZ injection, the ZIF-8 group was injected with ZIF-8 injection, the DOX@5FZ group was injected with DOX@5FZ injection, and the DOX@7FZ group was injected with DOX@7FZ injection. The amount of drug injected into nude mice was 50 mg / Kg. The samples were weighed and collected 6 weeks after the injection of cells.

[0171] Experimental results:

[0172] Depend on Fig.13 It can be seen that the relative tumor volume of the DOX@7FZ group was the smallest, indicating that DOX@7FZ is most effective in treating breast cancer tumors.

[0173] Experimental Example 12

[0174] Mouse tumor and organ sampling and section HE staining

[0175] Experimental steps:

[0176] After sodium pentobarbital anesthesia, the nude mice in the blank control group, 5FZ group, 7FZ group, ZIF-8 group, DOX@5FZ group and DOX@7FZ group were sampled, and the axillary tumors and major organs (heart, liver, spleen, lung, and kidney) of the nude mice were removed. The tumor size was measured and the lymph nodes and organs of the nude mice were observed for tumor metastasis. The tumors and major organs were then fixed in 4% paraformaldehyde overnight. After fixation, they were embedded in paraffin, sectioned, dewaxed to water, and stained with hematoxylin and eosin (HE staining) in sequence.

[0177] Experimental results:

[0178] Depend on Fig.14 It can be seen that the cancer cell morphology of the 5FZ and 7FZ groups is more consistent with the cell morphology characteristics of ferroptosis. The cancer cell morphology of the DOX@5FZ and DOX@7FZ groups has both ferroptosis cell morphology characteristics and apoptosis cell morphology characteristics. The number of cells with both cell morphology characteristics in the DOX@7FZ group is greater than that in the DOX@5FZ group, and the overall tissue is more loose, indicating that DOX@7FZ is the most effective in treating breast cancer tumors. Fig.15 It can be seen that no lesions were observed in the major organs of mice in each group, indicating that 5FZ, 7FZ, ZIF-8, DOX@5FZ and DOX@7FZ all have good in vivo safety.

Claims

1. An anticancer drug complex, characterized in that: The zeolite imidazolate skeleton structure material is used as the base frame, and is composed of composite ferrous sulfate and anticancer active components; the anticancer active component is doxorubicin; the zeolite imidazolate skeleton structure material is ZIF-8; the doxorubicin loading rate of the anticancer drug complex reaches more than 50%; The method for preparing the anticancer drug complex comprises: 1) a metal salt is dissolved in a solvent to form a solution A, wherein the metal salt is green vitriol and zinc nitrate hexahydrate, and the mass ratio of the green vitriol to the zinc nitrate hexahydrate is 5:100; 2) the anticancer active component is dissolved in methanol to form solution B, wherein the anticancer active component is doxorubicin hydrochloride; 3) Add solution B dropwise into solution A and stir evenly to form solution C; 4) Dissolve an imidazole source in methanol to form solution D, wherein the imidazole source is 2-methylimidazole 5) Solution D is added dropwise into solution C, stirred evenly, and after reaction, an anticancer drug complex is obtained.

2. Use of the anticancer drug complex according to claim 1 in the preparation of a drug for treating breast cancer.

3. An anticancer drug complex, characterized in that: The zeolite imidazolate skeleton structure material is used as the base frame, and is composed of composite ferrous sulfate and anticancer active components; the anticancer active component is doxorubicin; the zeolite imidazolate skeleton structure material is ZIF-8; the doxorubicin loading rate of the anticancer drug complex reaches more than 50%; The method for preparing the anticancer drug complex comprises: 1) a metal salt is dissolved in a solvent to form a solution A, wherein the metal salt is green vitriol and zinc nitrate hexahydrate, and the mass ratio of the green vitriol to the zinc nitrate hexahydrate is 7:100; 2) the anticancer active component is dissolved in methanol to form solution B, wherein the anticancer active component is doxorubicin hydrochloride; 3) Add solution B dropwise into solution A and stir evenly to form solution C; 4) Dissolve an imidazole source in methanol to form solution D, wherein the imidazole source is 2-methylimidazole 5) Solution D is added dropwise into solution C, stirred evenly, and after reaction, an anticancer drug complex is obtained.

4. Use of the anticancer drug complex according to claim 3 in the preparation of a drug for treating breast cancer.

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