Gold-metal oxide composite nanozyme, its preparation and application, and anti-tumor drug

Abstract

The present invention provides a gold-metal oxide composite nanozyme for anti-tumor, which is a nanozyme with gold nanomaterials as carriers and loaded with metal oxides. The gold-metal oxide composite nanozyme has photothermal and photocatalytic activities. The former can generate a thermal effect, and the latter can catalyze the production of reactive oxygen species (ROS). Under the conditions of high temperature and high reactive oxygen species in tumor cells, heat will damage the cell structure, and reactive oxygen species will oxidize biological macromolecules such as nucleic acids, proteins, and lipids. Eventually, the two work together to promote apoptosis or necrosis of tumor cells. The present invention modifies folic acid on the gold-metal oxide, which can target tumor cells. Therefore, this nano-complex has great cytotoxicity only to tumor cells and can be used to treat skin cancer, liver cancer, and lung cancer. This method of treating tumors through photothermal and photocatalytic therapy has the advantages of convenient operation, strong targeting, and good therapeutic effect, and has great application prospects in anti-tumor.

Description

Technical Field

[0001] The present invention relates to the field of anti-tumor drugs, and particularly relates to an anti-tumor drug for treating cancer. Background Art

[0002] Nanozyme is an artificial enzyme, a nanomaterial with catalytic activity. Compared with natural enzymes, it has advantages such as good stability, high catalytic activity, and low preparation cost. Since the proposal of nanozyme (L.Gao, J.Zhuang, L.Nie, et al., Nat Nanotechnol, 2(2007)577 - 583), the catalytic mechanism of nanozyme has been continuously explored (H.Wei, E.Wang, Chem Soc Rev 42(2013)6060 - 6093), and it has been widely applied in the fields of biomedicine, environmental protection, biosensors, etc. Especially in the field of tumor treatment, it has great application potential and has attracted wide attention from scientific researchers (M.Liang, X.Yan, Acc Chem Res 52(2019)2190 - 2200).

[0003] Gold nanoparticles have advantages such as good biocompatibility, high photothermal conversion efficiency, and easy modification. Compared with gold nanoparticles of other morphologies, gold nanorods have more superior physicochemical characteristics, have strong light absorption and heat dissipation effects in the near-infrared region (650 - 900nm), and have a suitable aspect ratio (A.M.Alkilany, L.B.Thompson, S.P.Boulos, et al., AdvDrug Deliver Rev 64(2012)190 - 199). Existing studies have shown that rod-shaped nanomaterials are more likely to enter cells (S.Barua, J.W.Yoo, P.Kolhar, et al., Proc Natl Acad Sci USA 110(2013)3270 - 3275). Metal oxides have activities such as peroxidase and oxidase, and they can catalyze the production of reactive oxygen species, causing damage to tumor cells. However, its low catalytic activity and poor spatiotemporality seriously affect its application in tumor treatment.

[0004] Therefore, we combined gold nanorods and metal oxides to form "thumbtack-shaped" gold-gold oxide nanoparticles, which not only have the advantages of gold nanorods, such as high photothermal conversion efficiency and easy entry into cells, but also under the photothermal effect, the enzyme activity of metal oxides has been greatly improved. Compared with gold and metal oxide nanozymes, this gold-metal oxide composite nanozyme has a better effect on killing tumor cells and has great clinical application potential. Summary of the Invention

[0005] In the combination of gold rods and metal oxides, a composite nanozyme with metal oxides loaded at one end of the gold rods is formed, and photothermal and enzymatic activities are synergistically used to improve the therapeutic effects on skin cancer, liver cancer, and lung cancer.

[0006] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0007] 1. (1) Prepare a gold seed solution by standing an aqueous solution with a final concentration of 0.01 - 0.25 mM HAuCl4, 50 - 150 mM CTAB (cetyltrimethylammonium bromide), and 0.3 - 0.6 mM NaBH4 for 2 h. Add 30 - 50 μL of the gold seed solution to 30 - 50 mL of an aqueous solution with a final concentration of 0.1 - 0.5 mM HAuCl4, 50 - 150 mM CTAB, 0.01 - 0.5 mM AgNO3, 1 - 2 mM HCl, and 0.4 - 0.8 mM L-ascorbic acid, and stand for 6 h to prepare a gold rod carrier solution; (2) Add 100 - 300 μL of 0.1 mM K2PtCl4 and 400 - 600 μL of 10 mM metal salt to 5 - 15 mL of the gold rod carrier solution, and react at 80 - 110 °C for 30 - 90 min to prepare a gold-metal oxide composite nanozyme.

[0008] 2. Further use tetramethylbenzidine (TMB), H2O2 reagents, as well as an 808 laser, a thermocouple thermometer, and an enzyme label instrument to detect temperature and oxidase (XOD) activity.

[0009] 3. Dissolve the gold-metal oxide in DPBS (Dulbecco’s Phosphate Buffered Saline), and add it to the culture media containing skin cancer cells A-375 and normal cells Hacat respectively, so that the final concentration is 0.01 - 0.25 μM, which can kill tumor cells and is non-toxic to normal cells.

[0010] 4. Dissolve the gold-metal oxide in DPBS, and add it to the culture media containing liver cancer cells Huh7 and normal cells L02 respectively, so that the final concentration is 0.01 - 0.25 μM, which can kill tumor cells and is non-toxic to normal cells.

[0011] 5. Dissolve the gold-metal oxide in DPBS, and add it to the culture media containing lung cancer cells A549 and normal cells HBE respectively, so that the final concentration is 0.01 - 0.25 μM, which can kill tumor cells and is non-toxic to normal cells.

[0012] 6. Dissolve the gold-metal oxide in DPBS and add it to the culture medium containing skin cancer cells A-375 to make its final concentration 0.01 - 0.25 μM. Use a laser with a wavelength of 650 - 900 nm, a light intensity of 1.0 - 2.5 A, and an irradiation time of 2.5 - 15 min. The effect of killing tumor cells has been greatly improved.

[0013] 7. Dissolve the gold-metal oxide in DPBS and add it to the culture medium containing liver cancer cells Huh7 to make its final concentration 0.01 - 0.25 μM. Use a laser with a wavelength of 650 - 900 nm, a light intensity of 1.0 - 2.5 A, and an irradiation time of 2.5 - 15 min. The effect of killing tumor cells has been greatly improved.

[0014] 8. Dissolve the gold-metal oxide in DPBS and add it to the culture medium containing lung cancer cells A549 to make its final concentration 0.01 - 0.25 μM. Use a laser with a wavelength of 650 - 900 nm, a light intensity of 1.0 - 2.5 A, and an irradiation time of 2.5 - 15 min. The effect of killing tumor cells has been greatly improved.

[0015] The gold-metal oxide composite nanozyme has photothermal and photocatalytic activities. The former can produce a thermal effect, and the latter can catalyze the production of reactive oxygen species (ROS). Under the conditions of high temperature and high reactive oxygen species, heat will damage the cell structure, and reactive oxygen species will oxidize biological macromolecules such as nucleic acids, proteins, and lipids. Eventually, the two work together to promote apoptosis or necrosis of tumor cells. In the present invention, folic acid is modified on the gold-metal oxide, which can target tumor cells. Therefore, this nano - composite has great cytotoxicity only to tumor cells and can be used to treat skin cancer, liver cancer, lung cancer, etc. This method of treating tumors by photothermal and photocatalytic therapy has the advantages of convenient operation, strong targeting, and good treatment effect, and has great application prospects in anti - tumor treatment.

[0016] The gold-metal oxide composite nanozyme described in the present invention has obvious advantages:

[0017] 1. The gold-metal oxide composite nanozyme described in the present invention has a high photothermal conversion efficiency due to the inclusion of gold nanorods.

[0018] 2. The gold-metal oxide composite nanozyme described in the present invention can greatly improve the XOD enzyme activity of metal oxides due to the inclusion of gold nanorods and metal oxides.

[0019] 3. The gold-metal oxide composite nanozyme described in the present invention is toxic only to tumor cells due to the modification of folic acid.

[0020] 4. The gold-metal oxide composite nanozyme of the present invention contains gold nanorods and metal oxides, and the synergistic effect of photothermal and enzymatic activities can greatly improve the killing effect on tumor cells. Description of the Drawings

[0021] To more clearly illustrate the specific technical solutions of the present invention, the following will provide a description of the drawings for this invention. The drawings here are only example figures, solely used to explain the invention patent and not to limit the invention patent.

[0022] Figure 1 . Electron micrograph of the gold-metal oxide composite nanozyme;

[0023] Figure 2 . Ultraviolet-visible absorption spectrum of the gold-metal oxide composite nanozyme;

[0024] Figure 3 . Photothermal conversion relationship of the gold-metal oxide composite nanozyme;

[0025] Figure 4 . XOD enzyme activity of the gold-metal oxide composite nanozyme;

[0026] Figure 5 . Folate modification of the gold-metal oxide composite nanozyme;

[0027] Figure 6 . Effects of the gold-metal oxide composite nanozyme on the survival rates of skin cancer cells and normal cells;

[0028] Figure 7 . Effects of the gold-metal oxide composite nanozyme on the survival rates of liver cancer cells and normal cells;

[0029] Figure 8 . Effects of the gold-metal oxide composite nanozyme on the survival rates of lung cancer cells and normal cells;

[0030] Figure 9 . Effects of the near-infrared enhanced gold-metal oxide composite nanozyme on the survival rate of skin cancer cells;

[0031] Figure 10 . Effects of the near-infrared enhanced gold-metal oxide composite nanozyme on the survival rate of liver cancer cells;

[0032] Figure 11 . Effects of the near-infrared enhanced gold-metal oxide composite nanozyme on the survival rate of lung cancer cells. Detailed Embodiments

[0033] In order to make the objectives, technical solutions, and advantages of this invention patent clearer and more understandable, the following further elaborates on this invention patent in conjunction with the attached drawings and examples. It should be understood that the specific embodiments described herein are merely used to explain this invention patent and are not used to limit this invention patent.

[0034] Example 1

[0035] 1. Preparation of gold-metal oxide composite nanozyme:

[0036] (1) Preparation of gold rods: A gold seed solution was prepared by allowing a 10 mL aqueous solution with a final concentration of 0.01 mM HAuCl4, 150 mM CTAB (cetyltrimethylammonium bromide), and 0.6 mM NaBH4 to stand for 2 h to obtain gold seeds. 30 μL of the gold seeds was added to a 30 mL aqueous solution with a final concentration of 0.5 mM HAuCl4, 150 mM CTAB, 0.5 mM AgNO3, 2 mM HCl, and 0.8 mM L-ascorbic acid and allowed to stand for 6 h. By observing with an electron microscope, gold rod carriers with a length of 30 - 60 nm and an aspect ratio of 2 - 3 were prepared.

[0037] (2) Take 100 μL of 0.1 mM K2PtCl4 and 400 μL of 10 mM metal salts (cerium acetate, ferrous acetate, ferric nitrate, zirconium acetate, and manganese acetate), add them to 5 mL of the gold rod carrier solution, and react at 80 °C for 30 min. By observing with an electron microscope, a composite nanozyme with a metal oxide particle size of 20 - 50 nm (cerium oxide, iron(III) oxide, iron(II,III) oxide, zirconium oxide, and manganese dioxide are all in this range) was prepared, and the metal oxide was loaded onto one end of the gold rod. After centrifugation, a gold-metal oxide composite nanozyme was finally obtained.

[0038] Take 100 μL of 0.1 mM K2PtCl4 and 400 μL of 10 mM metal salts (respectively cerium acetate, ferrous acetate, ferric nitrate, zirconium acetate, and manganese acetate), add them to 5 mL of water, and react at 80 °C for 30 min. By observing with an electron microscope, a composite nanozyme with a metal oxide particle size of 20 - 50 nm (cerium oxide, iron(III) oxide, iron(II,III) oxide, zirconium oxide, and manganese dioxide are all in this range) was prepared. After centrifugation, an aqueous solution of metal oxide particles was finally obtained.

[0039] 2. Characterization of the gold-metal oxide composite nanozyme by electron microscopy and ultraviolet-visible absorption spectroscopy:

[0040] Characterize the gold nanorods and gold-metal oxide composite nanozymes obtained in Example 1. Observe a small amount of the nanomaterials under an electron microscope to check whether the size and morphology of the gold nanorods and the gold-metal oxide composite nanozyme conform to the "thumbtack shape", as Figure 1Show the electron microscopy images of gold nanorods and gold-metal oxide composite nanozymes. The results show that five kinds of gold-metal oxides are successfully synthesized. At the same time, the ultraviolet-visible absorption spectra of the gold nanorods and the gold-metal oxide composite enzymes are measured to characterize the structure of the nanomaterials, such as Figure 2 shown, gold nanorods with appropriate sizes are successfully synthesized, and metal oxides are successfully synthesized onto one end of the carrier gold nanorods.

[0041] 3. Photothermal conversion measurement of gold-metal oxide composite nanozymes:

[0042] S1: Control group: 1 mL of H2O. Experimental groups: 1 mL of an aqueous solution of 0.125 mM gold nanorods, 1 mL of an aqueous solution of 0.125 mM metal oxides (the metal oxides are cerium oxide, iron(III) oxide, iron(II,III) oxide, zirconium oxide, and manganese dioxide respectively), 1 mL of an aqueous solution of 0.125 mM gold-gold oxide (the gold-metal oxides are gold-cerium oxide, gold-iron(III) oxide, gold-iron(II,III) oxide, gold-zirconium oxide, and gold-manganese dioxide respectively).

[0043] S2: Use a laser with a wavelength of 808 nm and a power of 1.75 A to irradiate the above systems. The irradiation times are: 0 min, 5 min, 10 min, 15 min, 20 min. Use a thermocouple thermometer to detect the temperature.

[0044] S3: Record the temperature and plot the time-temperature curve.

[0045] As Figure 3 shown, with the irradiation of the 808 nm laser, the temperature of the metal oxides does not change, while the temperature of the gold nanorods and the gold nanorod-metal oxides gradually increases with the increase of the irradiation time, and the temperature rises to more than fifty degrees Celsius. The results show that this composite material has a high photothermal conversion efficiency.

[0046] 4. Oxidase activity measurement of gold-metal oxide composite nanozymes:

[0047] S1: Prepare a buffer system using 0.2 M Na2HPO4 and 0.1 M citric acid, and the pH of the buffer system is 3.0.

[0048] S2: Prepare 50 mM TMB (3,3',5,5'-tetramethylbenzidine) using DMSO (dimethyl sulfoxide), and prepare suspensions of 2.5 mM gold nanorods, metal oxides (the metal oxides are cerium oxide, iron(III) oxide, iron(II,III) oxide, zirconium oxide, and manganese dioxide respectively), and gold-metal oxide complexes (the gold-metal oxides are gold-cerium oxide, gold-iron(III) oxide, gold-iron(II,III) oxide, gold-zirconium oxide, and gold-manganese dioxide respectively) using deionized water.

[0049] S3: Prepare a 1 mL reaction system: for the experimental group, 1.25 mM TMB + 0.125 mM gold, metal oxide, or gold-metal oxide; for the control group, 1.25 mM TMB.

[0050] S4: Use a laser with a wavelength of 808 nm and a power of 1.75 A to irradiate the reaction system obtained in S3 for the following irradiation times: 0 min, 5 min, 10 min, 15 min, and 20 min.

[0051] S5: Measure the absorbance value of the reaction product solution at 652 nm and record the data.

[0052] As Figure 4 shown, the higher the absorbance value of the reaction product, the higher the enzyme activity. Under laser irradiation, the XOD activity of the gold-metal oxide complex is much higher than that of gold rods and metal oxides, and its enzyme activity continuously increases with the increase of irradiation time, indicating that the 808 nm laser can increase the XOD enzyme activity of the gold-metal oxide.

[0053] 5. Characterization of folic acid modification of gold-metal oxide composite nanozyme:

[0054] S1: Take 240 μL of 10 μg / mL FA-PEG-SH (folic acid-polyethylene glycol-thiol), add it to 1.5 mL of 2.5 mM gold-metal oxide composite nanozyme, mix well, and react at 1000 rmp and 25 °C for 2 h.

[0055] S2: Centrifuge at 10000 g for 10 min, discard the supernatant, wash once with DPBS (Dulbecco's phosphate buffered saline), and finally suspend it with sterile DPBS to form a gold-metal oxide composite nanozyme modified with folic acid.

[0056] S3: Take the above-mentioned modified gold-metal oxide, FA-PEG-SH, and gold-metal oxide without folic acid modification, and measure the ultraviolet-visible absorption spectrum.

[0057] S5: Record the data and plot the absorption curve.

[0058] As Figure 5 shown, gold-metal oxide (folic acid modified) and FA-PEG-SH have the same absorption peaks at 280 nm and 372 nm, while gold-metal oxide without folic acid modification has no absorption peaks at these wavelengths. This indicates that folic acid has been modified onto the gold-metal oxide.

[0059] 6. Evaluation of the skin cancer cell targeting ability of gold-metal oxide composite nanozyme (modified with folic acid):

[0060] S1: Prepare 2.5 mM gold-metal oxide composite nanozyme (modified with folic acid, obtained from the above preparation) under sterile conditions using DPBS for later use.

[0061] S2: Digest and count two types of cells (A-375 and Hacat) in the logarithmic growth phase, seed them in a 96-well plate and allow them to adhere and grow for 24 h.

[0062] S3: Add 10 μL of DPBS to each well in the negative control group; add 10 μL of 2.5 mM gold-metal oxide composite nanozyme (modified with folic acid) to the experimental group to make the final concentration 250 μM, and continue the treatment for 48 h. S4: Add 10 μL of CCK8 to each well, incubate for 3 h, and detect using a microplate reader.

[0063] Results: As Figure 6 shown, five gold-metal oxide composite nanozymes modified with folic acid (the gold-metal oxides are gold-cerium oxide, gold-iron(III) oxide, gold-iron(II,III) oxide, gold-zirconium oxide, and gold-manganese dioxide respectively) have cytotoxicity to tumor cells (A-375); while they have almost no toxicity to normal cells (Hacat). Therefore, it can be considered that these five gold-metal oxide composite nanozymes modified with folic acid have good targeting to skin cancer cells.

[0064] 7. Evaluation of the targeting of gold-metal oxide composite nanozyme (modified with folic acid) to liver cancer cells:

[0065] S1: Prepare 2.5 mM gold-metal oxide composite nanozyme (modified with folic acid) under sterile conditions using DPBS for later use.

[0066] S2: Digest and count two types of cells (Huh7 and L02) in the logarithmic growth phase, seed them in a 96-well plate and allow them to adhere and grow for 24 h.

[0067] S3: Add 10 μL of DPBS to each well in the negative control group; add 10 μL of 2.5 mM gold-metal oxide composite nanozyme (modified with folic acid) to the experimental group to make the final concentration 250 μM, and continue the treatment for 48 h. S4: Add 10 μL of CCK8 to each well, incubate for 3 h, and detect using a microplate reader.

[0068] Results: As Figure 7 shown, five gold-metal oxide composite nanozymes modified with folic acid (the gold-metal oxides are gold-cerium oxide, gold-iron(III) oxide, gold-iron(II,III) oxide, gold-zirconium oxide, and gold-manganese dioxide respectively) have cytotoxicity to tumor cells (Huh7); while they have almost no toxicity to normal cells (L02). Therefore, it can be considered that these five gold-metal oxide composite nanozymes modified with folic acid have good targeting to liver cancer cells.

[0069] 8. Targeting Evaluation of Gold-Metal Oxide Composite Nanozymes (Modified with Folic Acid) against Lung Cancer Cells:

[0070] S1: Prepare 2.5 mM gold-metal oxide composite nanozymes (modified with folic acid) using DPBS under sterile conditions for later use.

[0071] S2: Digest and count two types of cells (A549 and HBE) in the logarithmic growth phase, seed them in a 96-well plate, and allow them to adhere and grow for 24 h.

[0072] S3: Add 10 μL of DPBS to each well in the negative control group; add 10 μL of 2.5 mM gold-metal oxide composite nanozymes (modified with folic acid) to the experimental group to make the final concentration 250 μM, and continue the treatment for 48 h.

[0073] S4: Add 10 μL of CCK8 to each well, incubate for 3 h, and detect using an enzyme-linked immunosorbent assay (ELISA) reader.

[0074] Results: As Figure 8 shown, five kinds of gold-metal oxide composite nanozymes modified with folic acid (the gold-metal oxides are gold-ceria, gold-ferric oxide, gold-ferroferric oxide, gold-zirconia, and gold-manganese dioxide) have cytotoxicity to tumor cells (A549); while they have almost no toxicity to normal cells (HBE). Therefore, it can be considered that these five kinds of gold-metal oxide composite nanozymes modified with folic acid have good targeting to lung cancer cells.

[0075] 9. Cytotoxicity Detection of Gold-Metal Oxide Composite Nanozymes (Modified with Folic Acid) against Skin Cancer Cells:

[0076] S1: Prepare 2.5 mM gold-metal oxide composite nanozymes (modified with folic acid) using DPBS under sterile conditions for later use.

[0077] S2: Digest and count A-375 cells in the logarithmic growth phase, seed them in a 96-well plate, and allow them to adhere and grow for 24 h.

[0078] S3: Add 10 μL of DPBS to each well in the negative control group; add 10 μL of 2.5 mM gold-metal oxide composite nanozymes (modified with folic acid) to the experimental group to make the final concentration 250 μM, and continue the treatment for 24 h.

[0079] S4: Irradiate with an 808 nm, 1.75 A laser for 10 min and continue culturing for 24 h.

[0080] S5: Add 10 μL of CCK8 to each well, incubate for 3 h, and detect using an enzyme-linked immunosorbent assay (ELISA) reader.

[0081] Results: As Figure 9As shown, under 808 nm laser irradiation, five kinds of gold-metal oxide composite nanozymes (modified with folic acid) are highly toxic to A-375 tumor cells, and the cell survival rates are very low. Therefore, it can be considered that these five kinds of gold-metal oxide composite nanozymes (modified with folic acid) have the potential to treat skin cancer.

[0082] 10. Cytotoxicity detection of gold-metal oxide composite nanozymes (modified with folic acid) against liver cancer cells:

[0083] S1: Prepare 2.5 mM gold-metal oxide composite nanozymes (modified with folic acid) using DPBS under sterile conditions for later use.

[0084] S2: Digest and count the Huh7 cells in the logarithmic growth phase, and seed them on a 96-well plate to adhere and grow for 24 h.

[0085] S3: Add 10 μL of DPBS to each well in the negative control group; add 10 μL of 2.5 mM gold-metal oxide composite nanozymes (modified with folic acid) to the experimental group to make the final concentration 250 μM, and continue to treat for 24 h.

[0086] S4: Irradiate with 808 nm, 1.75 A laser for 10 min, and continue to culture for 24 h.

[0087] S5: Add 10 μL of CCK8 to each well, incubate for 3 h, and detect using an enzyme-labeling instrument.

[0088] Results: As Figure 10 shown, under 808 nm laser irradiation, five kinds of gold-metal oxide composite nanozymes (modified with folic acid) are highly toxic to Huh7 tumor cells, and the cell survival rates are very low. Therefore, it can be considered that these five kinds of gold-metal oxide composite nanozymes (modified with folic acid) have the potential to treat liver cancer.

[0089] 11. Cytotoxicity detection of gold-metal oxide composite nanozymes (modified with folic acid) against lung cancer cells:

[0090] S1: Prepare 2.5 mM gold-metal oxide composite nanozymes (modified with folic acid) using DPBS under sterile conditions for later use.

[0091] S2: Digest and count the A549 cells in the logarithmic growth phase, and seed them on a 96-well plate to adhere and grow for 24 h.

[0092] S3: Add 10 μL of DPBS to each well in the negative control group; add 10 μL of 2.5 mM gold-metal oxide composite nanozymes (modified with folic acid) to the experimental group to make the final concentration 250 μM, and continue to treat for 24 h.

[0093] S4: Irradiate with a 808 nm, 1.75 A laser for 10 min and continue culturing for 24 h.

[0094] S5: Add 10 μL of CCK8 to each well, incubate for 3 h, and detect using a microplate reader.

[0095] Results: As Figure 11 shown, under 808 nm laser irradiation, the five gold-metal oxide composite nanozymes (modified with folic acid) are highly toxic to AA549 tumor cells and the cell survival rates are very low. Therefore, it can be considered that these five gold-metal oxide composite nanozymes (modified with folic acid) have the potential to treat lung cancer.

[0096] Example 2

[0097] 1. Preparation of gold-metal oxide composite nanozymes:

[0098] (1) Preparation of gold rods: Let a 10 mL aqueous solution with a final concentration of 0.01 mM HAuCl4, 150 mM CTAB (cetyltrimethylammonium bromide), and 0.6 mM NaBH4 stand for 2 h to prepare a gold seed solution, and obtain gold seeds. Add 40 μL of the gold seeds to a 40 mL aqueous solution with a final concentration of 0.5 mM HAuCl4, 150 mM CTAB, 0.5 mM AgNO3, 2 mM HCl, and 0.8 mM L-ascorbic acid and let it stand for 6 h. Observe with an electron microscope to prepare a gold rod carrier with a length of 30 - 60 nm and an aspect ratio of 2 - 3.

[0099] (2) Take 200 μL of 0.1 mM K2PtCl4 and 500 μL of 10 mM metal salts (cerium acetate, ferrous acetate, ferric nitrate, zirconium acetate, and manganese acetate), add them to 10 mL of the gold rod carrier solution, react at 100 °C for 60 min, observe with an electron microscope to prepare a composite nanozyme with metal oxide particles having a particle size of 20 - 50 nm (cerium oxide, iron(III) oxide, iron(II,III) oxide, zirconium oxide, and manganese dioxide are all in this range), and load the metal oxide onto one end of the gold rod. Centrifuge to finally obtain the gold-metal oxide composite nanozyme.

[0100] Take 200 μL of 0.1 mM K2PtCl4 and 500 μL of 10 mM metal salts (cerium acetate, ferrous acetate, ferric nitrate, zirconium acetate, and manganese acetate), add them to 10 mL of water, react at 100 °C for 60 min, observe with an electron microscope to prepare a composite nanozyme with metal oxide particles having a particle size of 20 - 50 nm (cerium oxide, iron(III) oxide, iron(II,III) oxide, zirconium oxide, and manganese dioxide are all in this range). Centrifuge to finally obtain an aqueous solution of metal oxide particles.

[0101] 2. Characterize the obtained gold, metal oxides, and gold-metal oxide composite nanozymes by electron microscopy, ultraviolet-visible absorption spectroscopy, photothermal conversion measurement, oxidase activity, folic acid modification, and skin cancer, liver cancer, lung cancer targeting and near-infrared enhanced toxicity. The results show that gold nanorods with appropriate sizes are successfully synthesized, and metal oxides are successfully synthesized onto one end of the carrier gold nanorods, and they have excellent skin cancer targeting and cell killing effects.

[0102] Example 3

[0103] 1. Preparation of gold-metal oxide composite nanozymes:

[0104] (1) Preparation of gold nanorods: A gold seed solution was prepared by allowing a 10 mL aqueous solution with a final concentration of 0.01 mM HAuCl4, 150 mM CTAB (cetyltrimethylammonium bromide), and 0.6 mM NaBH4 to stand for 2 h to obtain gold seeds. 50 μL of the gold seeds was added to a 50 mL aqueous solution with a final concentration of 0.5 mM HAuCl4, 150 mM CTAB, 0.5 mM AgNO3, 2 mM HCl, and 0.8 mM L-ascorbic acid and allowed to stand for 6 h. By observing with an electron microscope, gold nanorod carriers with a length of 30 - 60 nm and an aspect ratio of 2 - 3 were prepared.

[0105] (2) Take 300 μL of 0.1 mM K2PtCl4 and 600 μL of 10 mM metal salts (cerium acetate, ferrous acetate, ferric nitrate, zirconium acetate, and manganese acetate), add them to 15 mL of the gold nanorod carrier solution, and react at 110 °C for 90 min. By observing with an electron microscope, composite nanozymes with a metal oxide particle size of 20 - 50 nm (cerium oxide, iron(III) oxide, iron(II,III) oxide, zirconium oxide, and manganese dioxide are all in this range) were prepared, and the metal oxides were loaded onto one end of the gold nanorods. After centrifugation, gold-metal oxide composite nanozymes were finally obtained.

[0106] Take 300 μL of 0.1 mM K2PtCl4 and 600 μL of 10 mM metal salts (cerium acetate, ferrous acetate, ferric nitrate, zirconium acetate, and manganese acetate), add them to 15 mL of water, and react at 110 °C for 90 min. By observing with an electron microscope, composite nanozymes with a metal oxide particle size of 20 - 50 nm (cerium oxide, iron(III) oxide, iron(II,III) oxide, zirconium oxide, and manganese dioxide are all in this range) were prepared. After centrifugation, an aqueous solution of metal oxide particles was finally obtained.

[0107] 2. Characterize the gold, metal oxides, and gold-metal oxide composite nanozymes obtained above by electron microscopy, ultraviolet-visible absorption spectroscopy, photothermal conversion measurement, oxidase activity, folic acid modification, and the targeting of skin cancer, liver cancer, and lung cancer and the enhancement of their toxicity by near-infrared light. The results show that gold rods with appropriate sizes are successfully synthesized, and metal oxides are successfully synthesized onto one end of the carrier gold rods, and they have excellent targeting and cancer cell killing effects on liver cancer.

[0108] Example 4

[0109] 1. Preparation of gold-metal oxide composite nanozymes:

[0110] (1) Preparation of gold rods: A gold seed solution was prepared by allowing a 10 mL aqueous solution with a final concentration of 0.125 mM HAuCl4, 150 mM CTAB (cetyltrimethylammonium bromide), and 0.6 mM NaBH4 to stand for 2 h to obtain gold seeds. 30 μL of the gold seeds were added to a 30 mL aqueous solution with a final concentration of 0.5 mM HAuCl4, 150 mM CTAB, 0.5 mM AgNO3, 2 mM HCl, and 0.8 mM L-ascorbic acid and allowed to stand for 6 h. By observing with an electron microscope, gold rod carriers with a length of 30 - 60 nm and an aspect ratio of 2 - 3 were prepared.

[0111] (2) Take 100 μL of 0.1 mM K2PtCl4 and 400 μL of 10 mM metal salts (cerium acetate, ferrous acetate, ferric nitrate, zirconium acetate, and manganese acetate), add them to 5 mL of the gold rod carrier solution, and react at 80 °C for 30 min. By observing with an electron microscope, composite nanozymes with a metal oxide particle size of 20 - 50 nm (cerium oxide, iron(III) oxide, iron(II,III) oxide, zirconium oxide, and manganese dioxide are all in this range) were prepared, and the metal oxides were loaded onto one end of the gold rods. Centrifugation finally yielded gold-metal oxide composite nanozymes.

[0112] Take 100 μL of 0.1 mM K2PtCl4 and 400 μL of 10 mM metal salts (cerium acetate, ferrous acetate, ferric nitrate, zirconium acetate, and manganese acetate), add them to 5 mL of water, and react at 80 °C for 30 min. By observing with an electron microscope, composite nanozymes with a metal oxide particle size of 20 - 50 nm (cerium oxide, iron(III) oxide, iron(II,III) oxide, zirconium oxide, and manganese dioxide are all in this range) were prepared. Centrifugation finally yielded an aqueous solution of metal oxide particles.

[0113] 2. Characterize the obtained gold, metal oxides, and gold-metal oxide composite nanozymes by electron microscopy, ultraviolet-visible absorption spectroscopy, photothermal conversion measurement, oxidase activity, folic acid modification, and skin cancer, liver cancer, lung cancer targeting and near-infrared enhanced toxicity. The results show that gold nanorods with appropriate sizes are successfully synthesized, and metal oxides are successfully synthesized onto one end of the carrier gold nanorods, and they have excellent skin cancer targeting and cell killing effects.

[0114] Example 5

[0115] 1. Preparation of gold-metal oxide composite nanozymes:

[0116] (1) Preparation of gold nanorods: A gold seed solution was prepared by allowing a 10 mL aqueous solution with a final concentration of 0.125 mM HAuCl4, 150 mM CTAB (cetyltrimethylammonium bromide), and 0.6 mM NaBH4 to stand for 2 h to obtain gold seeds. 40 μL of the gold seeds were added to a 40 mL aqueous solution with a final concentration of 0.5 mM HAuCl4, 150 mM CTAB, 0.5 mM AgNO3, 2 mM HCl, and 0.8 mM L-ascorbic acid and allowed to stand for 6 h. By electron microscopy observation, gold nanorod carriers with a length of 30 - 60 nm and an aspect ratio of 2 - 3 were prepared.

[0117] (2) Take 200 μL of 0.1 mM K2PtCl4 and 500 μL of 10 mM metal salts (cerium acetate, ferrous acetate, ferric nitrate, zirconium acetate, and manganese acetate), add them to 5 mL of the gold nanorod carrier solution, and react at 100 °C for 60 min. By electron microscopy observation, composite nanozymes with a metal oxide particle size of 20 - 50 nm (cerium oxide, iron(III) oxide, iron(II,III) oxide, zirconium oxide, and manganese dioxide are all in this range) were prepared, and the metal oxides were loaded onto one end of the gold nanorods. After centrifugation, gold-metal oxide composite nanozymes were finally obtained.

[0118] Take 200 μL of 0.1 mM K2PtCl4 and 500 μL of 10 mM metal salts (cerium acetate, ferrous acetate, ferric nitrate, zirconium acetate, and manganese acetate), add them to 10 mL of water, and react at 100 °C for 60 min. By electron microscopy observation, composite nanozymes with a metal oxide particle size of 20 - 50 nm (cerium oxide, iron(III) oxide, iron(II,III) oxide, zirconium oxide, and manganese dioxide are all in this range) were prepared. After centrifugation, an aqueous solution of metal oxide particles was finally obtained.

[0119] 2. Characterize the obtained gold, metal oxides, and gold-metal oxide composite nanozymes by electron microscopy, ultraviolet-visible absorption spectroscopy, photothermal conversion measurement, oxidase activity, folic acid modification, and the targeting of skin cancer, liver cancer, and lung cancer and the enhancement of their toxicity by near-infrared light. The results show that gold rods with appropriate sizes are successfully synthesized, and metal oxides are successfully synthesized onto one end of the carrier gold rods, and they have excellent skin cancer targeting and cell killing effects.

[0120] Example 6

[0121] 1. Preparation of gold-metal oxide composite nanozymes:

[0122] (1) Preparation of gold rods: A gold seed solution is prepared by allowing a 10 mL aqueous solution with a final concentration of 0.125 mM HAuCl4, 150 mM CTAB (cetyltrimethylammonium bromide), and 0.6 mM NaBH4 to stand for 2 h to obtain gold seeds. 50 μL of the gold seeds is added to a 50 mL aqueous solution with a final concentration of 0.5 mM HAuCl4, 150 mM CTAB, 0.5 mM AgNO3, 2 mM HCl, and 0.8 mM L-ascorbic acid and allowed to stand for 6 h. By observing with an electron microscope, gold rod carriers with a length of 30-60 nm and an aspect ratio of 2-3 are prepared.

[0123] (2) Take 300 μL of 0.1 mM K2PtCl4 and 600 μL of 10 mM metal salts (cerium acetate, ferrous acetate, ferric nitrate, zirconium acetate, and manganese acetate), add them to 15 mL of the gold rod carrier solution, and react at 110 °C for 90 min. By observing with an electron microscope, composite nanozymes with a metal oxide particle size of 20-50 nm (cerium oxide, iron(III) oxide, iron(II,III) oxide, zirconium oxide, and manganese dioxide are all in this range) are prepared, and the metal oxides are loaded onto one end of the gold rods. After centrifugation, gold-metal oxide composite nanozymes are finally obtained.

[0124] Take 300 μL of 0.1 mM K2PtCl4 and 600 μL of 10 mM metal salts (cerium acetate, ferrous acetate, ferric nitrate, zirconium acetate, and manganese acetate), add them to 15 mL of water, and react at 110 °C for 90 min. By observing with an electron microscope, composite nanozymes with a metal oxide particle size of 20-50 nm (cerium oxide, iron(III) oxide, iron(II,III) oxide, zirconium oxide, and manganese dioxide are all in this range) are prepared. After centrifugation, an aqueous solution of metal oxide particles is finally obtained.

[0125] 2. Characterize the obtained gold and gold-metal oxide composite nanozymes by electron microscopy, ultraviolet-visible absorption spectroscopy, photothermal conversion measurement, oxidase activity, folic acid modification, and targeting and near-infrared enhanced toxicity to skin cancer, liver cancer, and lung cancer. The results show that gold rods with appropriate sizes are successfully synthesized, and metal oxides are successfully synthesized onto one end of the carrier gold rods, and they have excellent targeting and cancer cell killing effects on liver cancer.

[0126] Example 7

[0127] 1. Preparation of gold-metal oxide composite nanozymes:

[0128] (1) Preparation of gold rods: A gold seed solution was prepared by allowing a 10 mL aqueous solution with a final concentration of 0.25 mM HAuCl4, 150 mM CTAB (cetyltrimethylammonium bromide), and 0.6 mM NaBH4 to stand for 2 h to obtain gold seeds. 30 μL of the gold seeds was added to a 30 mL aqueous solution with a final concentration of 0.5 mM HAuCl4, 150 mM CTAB, 0.5 mM AgNO3, 2 mM HCl, and 0.8 mM L-ascorbic acid and allowed to stand for 6 h. By electron microscopy observation, gold rod carriers with a length of 30-60 nm and an aspect ratio of 2-3 were prepared.

[0129] (2) Take 100 μL of 0.1 mM K2PtCl4 and 400 μL of 10 mM metal salts (cerium acetate, ferrous acetate, ferric nitrate, zirconium acetate, and manganese acetate), add them to 5 mL of the gold rod carrier solution, and react at 80 °C for 30 min. By electron microscopy observation, composite nanozymes with a metal oxide particle size of 20-50 nm (cerium oxide, iron(III) oxide, iron(II,III) oxide, zirconium oxide, and manganese dioxide are all in this range) were prepared, and the metal oxides were loaded onto one end of the gold rods. After centrifugation, gold-metal oxide composite nanozymes were finally obtained.

[0130] Take 100 μL of 0.1 mM K2PtCl4 and 400 μL of 10 mM metal salts (cerium acetate, ferrous acetate, ferric nitrate, zirconium acetate, and manganese acetate), add them to 5 mL of water, and react at 80 °C for 30 min. By electron microscopy observation, composite nanozymes with a metal oxide particle size of 20-50 nm (cerium oxide, iron(III) oxide, iron(II,III) oxide, zirconium oxide, and manganese dioxide are all in this range) were prepared. After centrifugation, an aqueous solution of metal oxide particles was finally obtained.

[0131] 2. The electron microscopy, ultraviolet-visible absorption spectrum, photothermal conversion measurement, oxidase activity, folic acid modification, and targeting to skin cancer, liver cancer, and lung cancer, as well as the enhancement of its toxicity by near-infrared light of the obtained gold, metal oxide, and gold-metal oxide composite nanozymes were characterized. The results showed that gold nanorods with appropriate sizes were successfully synthesized, and metal oxides were successfully synthesized onto one end of the carrier gold nanorods, and they had excellent targeting to skin cancer and cell killing effects.

[0132] Example 8

[0133] 1. Preparation of gold-metal oxide composite nanozymes:

[0134] (1) Preparation of gold nanorods: A gold seed solution was prepared by allowing a 10 mL aqueous solution with a final concentration of 0.25 mM HAuCl4, 150 mM CTAB (cetyltrimethylammonium bromide), and 0.6 mM NaBH4 to stand for 2 h to obtain gold seeds. 40 μL of the gold seeds was added to a 40 mL aqueous solution with a final concentration of 0.5 mM HAuCl4, 150 mM CTAB, 0.5 mM AgNO3, 2 mM HCl, and 0.8 mM L-ascorbic acid and allowed to stand for 6 h. By electron microscopy observation, gold nanorod carriers with a length of 30 - 60 nm and an aspect ratio of 2 - 3 were prepared.

[0135] (2) 200 μL of 0.1 mM K2PtCl4 and 500 μL of 10 mM metal salts (cerium acetate, ferrous acetate, iron nitrate, zirconium acetate, and manganese acetate) were added to 10 mL of the gold nanorod carrier solution, and the reaction was carried out at 100 °C for 60 min. By electron microscopy observation, composite nanozymes with a metal oxide particle size of 20 - 50 nm (cerium oxide, iron(III) oxide, iron(II,III) oxide, zirconium oxide, and manganese dioxide were all in this range) were prepared, and the metal oxides were loaded onto one end of the gold nanorods. After centrifugation, gold-metal oxide composite nanozymes were finally obtained.

[0136] 200 μL of 0.1 mM K2PtCl4 and 500 μL of 10 mM metal salts (cerium acetate, ferrous acetate, iron nitrate, zirconium acetate, and manganese acetate) were added to 10 mL of water, and the reaction was carried out at 100 °C for 60 min. By electron microscopy observation, composite nanozymes with a metal oxide particle size of 20 - 50 nm (cerium oxide, iron(III) oxide, iron(II,III) oxide, zirconium oxide, and manganese dioxide were all in this range) were prepared. After centrifugation, an aqueous solution of metal oxide particles was finally obtained.

[0137] 2. Characterize the gold, metal oxides, and gold-metal oxide composite nanozymes obtained above by electron microscopy, ultraviolet-visible absorption spectroscopy, photothermal conversion measurement, oxidase activity, folic acid modification, and the targeting properties and near-infrared enhanced toxicity to skin cancer, liver cancer, and lung cancer. The results show that gold rods with appropriate sizes are successfully synthesized, and metal oxides are successfully synthesized onto one end of the carrier gold rods, and they have excellent skin cancer targeting and cell killing effects.

[0138] Example 9

[0139] 1. Preparation of gold-metal oxide composite nanozymes:

[0140] (1) Preparation of gold rods: A gold seed solution was prepared by allowing a 10 mL aqueous solution with a final concentration of 0.25 mM HAuCl4, 150 mM CTAB (cetyltrimethylammonium bromide), and 0.6 mM NaBH4 to stand for 2 h to obtain gold seeds. 50 μL of the gold seeds were added to a 50 mL aqueous solution with a final concentration of 0.5 mM HAuCl4, 150 mM CTAB, 0.5 mM AgNO3, 2 mM HCl, and 0.8 mM L-ascorbic acid and allowed to stand for 6 h. By electron microscopy observation, gold rod carriers with a length of 30 - 60 nm and an aspect ratio of 2 - 3 were prepared.

[0141] (2) Take 300 μL of 0.1 mM K2PtCl4 and 600 μL of 10 mM metal salts (cerium acetate, ferrous acetate, ferric nitrate, zirconium acetate, and manganese acetate), add them to 15 mL of the gold rod carrier solution, and react at 110 °C for 90 min. By electron microscopy observation, composite nanozymes with a metal oxide particle size of 20 - 50 nm (cerium oxide, iron(III) oxide, iron(II,III) oxide, zirconium oxide, and manganese dioxide are all in this range) were prepared, and the metal oxides were loaded onto one end of the gold rods. After centrifugation, gold-metal oxide composite nanozymes were finally obtained.

[0142] Take 300 μL of 0.1 mM K2PtCl4 and 600 μL of 10 mM metal salts (cerium acetate, ferrous acetate, ferric nitrate, zirconium acetate, and manganese acetate), add them to 15 mL of water, and react at 110 °C for 90 min. By electron microscopy observation, composite nanozymes with a metal oxide particle size of 20 - 50 nm (cerium oxide, iron(III) oxide, iron(II,III) oxide, zirconium oxide, and manganese dioxide are all in this range) were prepared. After centrifugation, an aqueous solution of metal oxide particles was finally obtained.

[0143] 2. Characterize the obtained gold, metal oxides, and gold-metal oxide composite nanozymes by electron microscopy, ultraviolet-visible absorption spectroscopy, photothermal conversion measurement, oxidase activity, folic acid modification, and targeting to skin cancer, liver cancer, and lung cancer and enhancement of their toxicity by near-infrared light. The results show that gold nanorods with appropriate sizes are successfully synthesized, and metal oxides are successfully synthesized on one end of the carrier gold nanorods, and they have excellent targeting to liver cancer and cancer cell killing effects.

Claims

1. Use of a gold-metal oxide composite nanozyme in the preparation of one or more drugs for treating skin cancer, liver cancer, lung cancer, characterized in that: The gold-metal oxide composite nanozyme is composed of a carrier gold rod and particles of metal oxide loaded at one end of the gold rod. The length of the carrier gold rod is 30-60 nm, and the aspect ratio is 2-3. The metal oxide includes one or more of iron(III) oxide, iron(II,III) oxide, zirconium oxide, and manganese dioxide. The metal oxide is loaded onto one end of the gold rod, and the particle size of the metal oxide is 20-50 nm.

2. The use according to claim 1, characterized in that: The preparation process of the gold-metal oxide composite nanozyme is as follows: (1) Prepare a gold seed solution by allowing an aqueous solution with a final concentration of 0.01~0.25 mM HAuCl4, 50~150 mM cetyltrimethylammonium bromide CTAB, and 0.3~0.6 mM NaBH4 to stand for 2 h or more. Add 30-50 μL of the gold seed solution to 30-50 mL of an aqueous solution with a final concentration of 0.1~0.5 mM HAuCl4, 50~150 mM CTAB, 0.01~0.5 mM AgNO3, 1~2 mM HCl, and 0.4~0.8 mM L-ascorbic acid, and allow it to stand for 6 h or more to prepare a gold rod carrier solution. (2) Add 100-300 μL of 0.1 mM K2PtCl4 and 400-600 μL of 10 mM metal salt to 5-15 mL of the gold rod carrier solution, and react at 80-110 °C for 30-90 min to prepare the gold-metal oxide composite nanozyme.

3. The use according to claim 1, characterized in that: Dissolve the gold-metal oxide composite nanozyme in water or DPBS to prepare a drug with a final concentration of 0.1-2.5 mM for treating one or more of skin cancer, liver cancer, and lung cancer.

4. The application of the gold-metal oxide composite nanozyme according to claim 1, wherein: The gold-metal oxide serves as a photothermal carrier. Under the excitation of near-infrared laser with a wavelength of 650-900 nm, the gold-metal oxide composite nanozyme has a thermal effect.

5. Use of the gold-metal oxide composite nanozyme according to claim 1, characterized in that: The gold-metal oxide serves as a photothermal synergistic oxidase XOD. In an acidic environment with a pH of 3~6, under the excitation of a 650~900 nm near-infrared laser, the gold-metal oxide composite nanozyme has a photothermal synergistic XOD activity.

6. A drug for treating one or more of skin cancer, liver cancer, and lung cancer, which uses the gold-metal oxide composite nanozyme according to claim 1 as an active ingredient.

7. The drug according to claim 6, characterized in that: Dissolve the gold-metal oxide composite nanozyme in water or DPBS to prepare a drug with a final concentration of 0.1-2.5 mM.

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