Copper / apigenin nano-drug loaded with glucose oxidase and preparation method of copper / apigenin nano-drug

Abstract

The invention relates to a copper / apigenin nano-drug (CuAp (at) GOx NSs) loaded with glucose oxidase and a preparation method of the copper / apigenin nano-drug. The nano-drug is prepared by taking CuAp NSs formed by cupric salt and apigenin as a core and loading GOx on the surface, and is prepared through coordination, pi-pi, hydrophobic interaction and electrostatic adsorption. The CuAp (at) GOx NSs can effectively disturb the glucose homeostasis of tumor cells, so that an SLC7A11 / GSH / GPX4 antioxidant axis is inhibited, double-sulfur death, copper death and ferroptosis are synchronously awakened, and the tumor treatment effect is remarkably improved. The preparation method comprises the following steps: reacting bivalent copper salt with apigenin under the conditions of specific pH, temperature, time and ultrasonic stirring to form CuAp NSs, and then stirring and loading GOx at room temperature to finally obtain a CuAp GOx NSs solution or solid powder. The nano-drug provided by the invention has the advantages of mild preparation conditions, simplicity in operation, good stability, high biological safety, remarkable treatment effect and the like, and a new strategy is provided for tumor treatment.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedical materials, and in particular relates to a copper / apigenin nanomedicine loaded with glucose oxidase and a preparation method thereof. Background Art

[0002] In today's medical field, tumors are still a major problem that seriously threatens human health and life. Although medical research and clinical practice have made significant progress in tumor treatment, the complex and powerful self-protection mechanism of tumor cells makes traditional tumor treatment methods face many challenges and it is difficult to achieve ideal treatment effects.

[0003] In order to maintain their own survival and proliferation, tumor cells have developed a sophisticated antioxidant defense system, in which the SLC7A11 / GSH / GPX4 antioxidant axis plays a key role in this process. As a cystine / glutamate antiporter, SLC7A11 is responsible for transporting extracellular cystine into cells to provide raw materials for the synthesis of glutathione (GSH); GSH, under the action of glutathione peroxidase (GPX4), can remove reactive oxygen species (ROS) in cells, thereby maintaining redox homeostasis in tumor cells. This antioxidant axis enables tumor cells to effectively resist oxidative stress damage induced by a variety of treatments, which greatly reduces the effectiveness of tumor treatment.

[0004] In recent years, researchers have gradually realized that disrupting the glucose homeostasis of tumor cells may be an effective strategy to break the antioxidant defense system of tumor cells. Glucose is the main energy source for tumor cells, and its metabolic process is closely related to the survival, proliferation and antioxidant capacity of tumor cells. By interfering with the glucose metabolism of tumor cells, it is possible to hinder the normal operation of the SLC7A11 / GSH / GPX4 antioxidant axis, thereby enhancing the effect of tumor treatment. However, how to accurately and effectively disrupt the glucose homeostasis of tumor cells while avoiding damage to normal cells is still a technical problem that needs to be solved urgently.

[0005] In the process of research and development of tumor therapeutic drugs, the emergence of nanotechnology has provided new ideas and methods for solving the above problems. Nano drugs have unique physical and chemical properties, such as high bioavailability, good targeting, controllable pharmacokinetics and relatively low toxic side effects. Nano drugs can specifically accumulate in tumor tissues through passive or active targeting, increase the concentration of drugs in the tumor site, and reduce damage to normal tissues. However, existing nano drugs still have some shortcomings in design and performance, such as low drug loading efficiency, stability and responsiveness in the tumor microenvironment need to be improved, and the killing mechanism of tumor cells is relatively simple.

[0006] Although certain progress has been made in the field of tumor treatment, existing treatment methods and drugs still cannot meet clinical needs. Therefore, it is urgent to develop a new type of nano drug that can effectively destroy the glucose homeostasis of tumor cells and has multiple tumor killing mechanisms to improve the effect of tumor treatment and bring more hope of survival to tumor patients. Based on such background, the present invention is committed to providing a copper / apigenin nano drug loaded with glucose oxidase and a preparation method thereof to solve the above technical problems. Summary of the invention

[0007] The purpose of the present invention is to provide a copper / apigenin nanodrug loaded with glucose oxidase and a preparation method thereof, which can effectively destroy the glucose homeostasis of tumor cells, hinder the operation of the SLC7A11 / GSH / GPX4 antioxidant axis, and synergistically kill tumor cells through disulfide death, copper death and iron death mechanisms. At the same time, it has the advantages of good stability, high biosafety, simple preparation method, etc., providing a new and effective means for tumor treatment.

[0008] In order to achieve the above object, the present invention provides a copper / apigenin nanodrug (CuAp@GOx NSs) loaded with glucose oxidase, which is composed of a nanodrug (CuAp NSs) formed by a divalent copper salt and apigenin and glucose oxidase (GOx) loaded on its surface. The copper / apigenin nanodrug (CuAp NSs) is formed by the coordination, π-π and hydrophobic interactions between the divalent copper salt and apigenin; and the glucose oxidase (GOx) is loaded on the surface of the CuApNSs by electrostatic interaction.

[0009] Furthermore, the divalent copper salt is selected from one or more of copper chloride, copper acetate, copper nitrate or copper sulfate.

[0010] Furthermore, the apigenin is 5,7-dihydroxy-2-(4-hydroxyphenyl)benzopyran-4-one.

[0011] Furthermore, the nanoparticle size of the CuAp@GOx NSs is 200 to 450 nanometers in the long axis and 80 to 300 nanometers in the short axis.

[0012] Another aspect of the present invention provides a method for preparing a glucose oxidase-loaded copper / apigenin nanodrug (CuAp@GOxNSs), comprising the following steps:

[0013] S1: A deionized water solution of a divalent copper salt is mixed with an organic solvent solution of apigenin, a sodium hydroxide solution is added to adjust the pH to 3.9-6.2, the mixture is reacted with ultrasonic stirring at 20-45° C. for 0.5-4 hours, and CuApNSs are obtained after centrifugal washing.

[0014] S2: Disperse CuAp NSs in deionized water, add glucose oxidase, stir at room temperature for 6 hours, and obtain CuAp@GOxNSs solution after centrifugation and washing.

[0015] Furthermore, a copper / apigenin nanodrug (CuApGOxNSs) solution loaded with glucose oxidase is prepared, and a CuApGOxNSs solid powder can be obtained by freeze-drying or drying.

[0016] Furthermore, the concentration of the deionized water solution of the divalent copper salt is 0.13-4 mmol / L, and the concentration of the organic solvent solution of apigenin is 0.05-1.54 mmol / L.

[0017] Furthermore, the organic solvent is dimethyl sulfoxide (DMSO) and / or N,N-dimethylformamide (DMF), and the proportion of the organic solvent is 5-30% of the total volume of the solution.

[0018] Furthermore, the concentration of the sodium hydroxide solution is 0.02-1 mmol / L.

[0019] Furthermore, the stirring speed of the ultrasonic stirring is 80 to 220 rpm, the ultrasonic power is 50 to 120 watts, and the centrifugal speed is 6000 to 15000 rpm.

[0020] Another aspect of the present invention is the use of a copper / apigenin nanodrug loaded with glucose oxidase in the preparation of a drug for tumor treatment, wherein the drug disrupts the glucose homeostasis of tumor cells, hinders the operation of the SLC7A11 / GSH / GPX4 antioxidant axis, and thereby induces disulfide death, copper death, and iron death.

[0021] Furthermore, the drug releases cuprous ions, apigenin and glucose oxidase in the tumor microenvironment, leading to the inhibition of cystine / cysteine ​​conversion, the reduction of GSH biosynthesis, and the accumulation of lipid peroxides, thereby activating disulfide death, copper death and iron death.

[0022] Furthermore, the CuAp@GOxNSs disintegrate in the tumor microenvironment, and the released cuprous ions react with hydrogen peroxide to generate hydroxyl radicals, further inducing ferroptosis.

[0023] Furthermore, the CuAp@GOxNSs disrupted glucose homeostasis and inhibited the operation of the SLC7A11 / GSH / GPX4 antioxidant axis by reducing intracellular glucose and GSH contents.

[0024] Furthermore, the CuAp@GOxNSs affected glucose homeostasis by downregulating GLUT1 and PKM2 protein expression, and induced copper death through downregulation of FDX1 and oligomerization of DLAT.

[0025] Furthermore, the CuAp@GOxNSs induced ferroptosis by increasing the accumulation of intracellular lipid peroxides.

[0026] Furthermore, the CuAp@GOxNSs induce disulfide death by inducing cytoskeleton contraction and disulfide stress.

[0027] Furthermore, the CuAp@GOxNSs can effectively disintegrate and release active ingredients in a simulated tumor microenvironment with pH = 5.5 and containing 2 mmol / LGSH.

[0028] Furthermore, the CuAp@GOxNSs produced hydrogen peroxide by consuming glucose, further inducing oxidative stress and death of tumor cells.

[0029] Furthermore, when the concentration of CuAp@GOxNSs was 3 μg / mL, the survival rate of SKOV3 cells was reduced to about 18%, showing a significant tumor cell killing effect.

[0030] The CuAp@GOxNSs prepared by the present invention disintegrate in the tumor microenvironment, releasing cuprous ions, apigenin and GOx. Apigenin and GOx act synergistically to disrupt glucose homeostasis, reduce the production of NADPH, hinder the SLC7A11 / GSH / GPX4 antioxidant axis, and lead to disulfide death, copper death and iron death, thereby achieving effective killing of tumor cells.

[0031] Compared with the prior art, the present invention has the following advantages:

[0032] (1) The CuAp@GOxNSs prepared by the present invention has good stability and high biosafety.

[0033] (2) The CuAp@GOx NSs prepared in the present invention significantly increased the solubility of apigenin and improved its bioavailability.

[0034] (3) The CuAp@GOxNSs prepared by the present invention can disintegrate in the tumor microenvironment, releasing cuprous ions, apigenin and GOx. Among them, apigenin and GOx can disrupt glucose homeostasis and reduce the production of NADPH, thereby leading to the key stage of the SLC7A11 / GSH / GPX4 antioxidant axis, namely, the blockage of cystine / cysteine ​​conversion. Therefore, a large amount of accumulated cystine can cause disulfide death; the reduction of cysteine ​​will reduce the biosynthesis of GSH, and more active cuprous ions can induce copper death; and the Fenton reaction will occur, activating ferroptosis; low levels of GSH cannot combine with GPX4 to exert an antioxidant effect, resulting in a large accumulation of lipid peroxides and enhanced ferroptosis.

[0035] (4) This experiment has the advantages of mild conditions, simple operation, good experimental repeatability, controllable reaction, stimulus responsiveness, and safety and reliability.

[0036] The preparation method of the invention does not require complicated equipment and harsh reaction conditions, thus reducing production costs and providing convenience for the industrial production and clinical promotion of nano-drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 : Transmission electron microscopy photograph of CuAp@GOxNSs prepared in Example 1, the major axis of the nanoparticles is 259 nanometers and the minor axis is 99 nanometers.

[0038] Figure 2 : Transmission electron microscopy photograph of CuAp@GOxNSs prepared in Example 2, the major axis of the nanoparticles is 356 nanometers and the minor axis is 142 nanometers.

[0039] Figure 3 : Transmission electron microscopy photograph of CuAp@GOxNSs prepared in Example 3, the major axis of the nanoparticles is 342 nanometers and the minor axis is 131 nanometers.

[0040] Figure 4 : Transmission electron microscopy photograph of CuAp@GOxNSs prepared in Example 4, the major axis of the nanoparticles is 314 nanometers and the minor axis is 126 nanometers.

[0041] Figure 5 : Transmission electron microscopy photograph of CuAp@GOxNSs prepared in Example 5, the major axis of the nanoparticles is 247 nanometers and the minor axis is 128 nanometers.

[0042] Figure 6 : Transmission electron microscopy photograph of CuAp@GOxNSs prepared in Example 6, the major axis of the nanoparticles is 218 nanometers and the minor axis is 106 nanometers.

[0043] Figure 7 : Transmission electron microscopy photograph of CuAp@GOxNSs prepared in Example 7, the major axis of the nanoparticles is 339 nanometers and the minor axis is 149 nanometers.

[0044] Figure 8 : Transmission electron microscopy photograph of CuAp@GOxNSs prepared in Example 8, the major axis of the nanoparticles is 205 nanometers and the minor axis is 81 nanometers.

[0045] Fig. 9 : Transmission electron microscopy photograph of CuAp@GOxNSs prepared in Example 9, the major axis of the nanoparticles is 290 nanometers and the minor axis is 135 nanometers.

[0046] Fig.10: Transmission electron microscopy photograph of CuAp@GOx NSs prepared in Example 10, the major axis of the nanoparticles is 262 nanometers and the minor axis is 101 nanometers.

[0047] Fig.11 : UV-visible absorption spectra of CuAp@GOxNSs, apigenin and GOx monomer measured in accordance with Example 11.

[0048] Fig.12 : Fourier transform infrared spectra of CuAp@GOxNSs, apigenin and GOx monomer measured in accordance with Example 12.

[0049] Fig.13 : Transmission electron microscopy images corresponding to Example 13. Among them, CuAp@GOx NSs were tested by transmission electron microscopy after using various interaction destroyers.

[0050] Fig.14 : Transmission electron microscopy images corresponding to Example 14. Among them, CuAp@GOx NSs were incubated in 2 mmol / L GSH solution at 37°C for 24 hours before transmission electron microscopy testing.

[0051] Fig.15 : The standard curve measured in accordance with Example 15. Wherein, the absorbance values ​​of cuprous ion solutions of different concentrations at 457 nanometers were measured using the new cuprous reagent as an indicator.

[0052] Fig.16 : The cuprous ion release histogram obtained in accordance with Example 16. In which, the new cuprous reagent is used as an indicator, and after CuAp@GOxNSs is incubated in a GSH solution at pH = 5.5 for different time periods, its absorbance at 457 nm is measured, and the cuprous ion release histogram is calculated.

[0053] Fig.17 : The UV absorption spectrum of CuAp@GOx NSs measured in Example 17 after incubation in pH=5.5 and GSH solution proves the release of apigenin.

[0054] Fig.18 : The electron paramagnetic resonance spectrum corresponding to Example 18. After incubation of CuAp@GOxNSs in a pH=5.5, GSH and hydrogen peroxide solution, the spin trapping agent 5,5-dimethyl-1-pyrroline-N-oxide was used to capture hydroxyl radicals, and electron paramagnetic resonance detection was performed.

[0055] Fig.19: The hydrogen peroxide production measured in Example 19. CuAp@GOxNSs can consume glucose to produce hydrogen peroxide. Tetramethylbenzidine is used to react with hydrogen peroxide to generate a dimeric charge transfer complex, which is then detected by ultraviolet absorption spectroscopy. The solid line in the figure is the water group, and the dotted line is the CuAp@GOxNSs+glucose group.

[0056] Fig. 20 : CCK-8 cytotoxicity test graph of CuAp@GOx NSs on human ovarian cancer cells (SKOV3) measured in Example 20.

[0057] Fig.21 : Changes in intracellular glucose content after SKOV3 was treated with CuAp@GOxNSs as measured in Example 21.

[0058] Fig. 22 : Changes in intracellular GSH content after SKOV3 was treated with CuAp@GOxNSs as measured in Example 22.

[0059] Fig.23 : Actin-tracker Green stained CLSM image of SKOV3 treated with CuAp@GOxNSs measured in Example 23.

[0060] Fig.24 : Western blot analysis of ferredoxin 1 (FDX1), glucose transporter 1 (GLUT1), pyruvate kinase M2 (PKM2), and dihydrolipoamide S-acetyltransferase (DLAT) in cells after CuAp@GOx NSs treatment measured in Example 24.

[0061] Fig.25 : After CuAp@GOxNSs treated SKOV3 cells, C11-BODIPY 581 / 591 Stained CLSM images. DETAILED DESCRIPTION

[0062] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0063] Embodiment 1:

[0064] Under ultrasonic stirring, 7.2 mL of dimethyl sulfoxide solvent, 4 mL of 10 mmol / L copper chloride deionized water solution and 0.8 mL of 10 mmol / L apigenin dimethyl sulfoxide solution were added to 28 mL of deionized water in sequence, followed by 40 μL of 100 mmol / L sodium hydroxide solution. Stirring was performed at 170 rpm and ultrasonicated at 120 watts. After 2 hours, centrifugation was performed at 8800 rpm for 15 minutes, the supernatant was discarded and the centrifuged product was washed with deionized water. Finally, 1 mL of deionized water was added to disperse the precipitate to obtain a CuAp NSs solution.

[0065] 15 mg of CuAp NSs was dispersed in 10 mL of deionized water, and 1.5 mg of glucose oxidase was added under stirring at room temperature. After stirring for 6 hours, the mixture was centrifuged at 13,300 rpm for 8 minutes. The supernatant was discarded and the centrifuged product was washed with deionized water. Finally, 1 mL of deionized water was added to disperse the precipitate to obtain a CuAp@GOxNSs solution, in which the major axis of the nanoparticles was 259 nm and the minor axis was 99 nm.

[0066] Embodiment 2:

[0067] Under ultrasonic stirring, 7.2 mL of dimethyl sulfoxide solvent, 4 mL of 10 mmol / L copper acetate deionized water solution and 0.8 mL of 10 mmol / L apigenin dimethyl sulfoxide solution were added to 28 mL of deionized water in sequence, followed by the addition of 40 μL of 100 mmol / L sodium hydroxide solution. The mixture was stirred at 170 rpm and ultrasonicated at 120 watts. After 2 hours, the mixture was centrifuged at 8800 rpm for 15 minutes, the supernatant was discarded, the centrifuged product was washed with deionized water, and finally 1 mL of deionized water was added to disperse the precipitate to obtain a CuAp NSs solution. 15 mg of CuAp NSs was dispersed in 10 mL of deionized water, 1.5 mg of glucose oxidase was added under stirring at room temperature, and after stirring for 6 hours, the mixture was centrifuged at 13300 rpm for 8 minutes, the supernatant was discarded, the centrifuged product was washed with deionized water, and finally 1 mL of deionized water was added to disperse the precipitate to obtain a CuAp@GOx NSs solution, in which the major axis of the nanoparticles was 356 nanometers and the minor axis was 142 nanometers.

[0068] Embodiment 3:

[0069] Under ultrasonic stirring, 7.2 mL of dimethyl sulfoxide solvent, 4 mL of 10 mmol / L copper nitrate deionized water solution and 0.8 mL of 10 mmol / L apigenin dimethyl sulfoxide solution were added to 28 mL of deionized water in sequence, followed by 40 μL of 100 mmol / L sodium hydroxide solution. The mixture was stirred at 170 rpm and ultrasonicated at 120 watts. After 2 hours, the mixture was centrifuged at 8800 rpm for 15 minutes, the supernatant was discarded, the centrifuged product was washed with deionized water, and finally 1 mL of deionized water was added to disperse the precipitate to obtain a CuAp NSs solution. 15 mg of CuAp NSs was dispersed in 10 mL of deionized water, 1.5 mg of glucose oxidase was added under stirring at room temperature, and after stirring for 6 hours, the mixture was centrifuged at 13300 rpm for 8 minutes, the supernatant was discarded, the centrifuged product was washed with deionized water, and finally 1 mL of deionized water was added to disperse the precipitate to obtain a CuAp@GOx NSs solution, in which the major axis of the nanoparticles was 342 nanometers and the minor axis was 131 nanometers.

[0070] Embodiment 4:

[0071] Under ultrasonic stirring, 7.2 mL of dimethyl sulfoxide solvent, 4 mL of 10 mmol / L copper sulfate deionized water solution and 0.8 mL of 10 mmol / L apigenin dimethyl sulfoxide solution were added to 28 mL of deionized water in sequence, followed by 40 μL of 100 mmol / L sodium hydroxide solution. The mixture was stirred at 170 rpm and ultrasonicated at 120 watts. After 2 hours, the mixture was centrifuged at 8800 rpm for 15 minutes, the supernatant was discarded, the centrifuged product was washed with deionized water, and finally 1 mL of deionized water was added to disperse the precipitate to obtain a CuAp NSs solution. 15 mg of CuAp NSs was dispersed in 10 mL of deionized water, 1.5 mg of glucose oxidase was added under stirring at room temperature, and after stirring for 6 hours, the mixture was centrifuged at 13300 rpm for 8 minutes, the supernatant was discarded, the centrifuged product was washed with deionized water, and finally 1 mL of deionized water was added to disperse the precipitate to obtain a CuAp@GOx NSs solution, in which the major axis of the nanoparticles was 314 nanometers and the minor axis was 126 nanometers.

[0072] Embodiment 5:

[0073] Under ultrasonic stirring, 7.2 mL of dimethyl sulfoxide solvent, 8 mL of 10 mmol / L copper chloride deionized water solution and 0.8 mL of 10 mmol / L apigenin dimethyl sulfoxide solution were added to 24 mL of deionized water in sequence, followed by 40 μL of 100 mmol / L sodium hydroxide solution. The mixture was stirred at 170 rpm and ultrasonicated at 120 watts. After 2 hours, the mixture was centrifuged at 8800 rpm for 15 minutes, the supernatant was discarded, the centrifuged product was washed with deionized water, and finally 1 mL of deionized water was added to disperse the precipitate to obtain a CuAp NSs solution. 15 mg of CuAp NSs was dispersed in 10 mL of deionized water, 1.5 mg of glucose oxidase was added under stirring at room temperature, and after stirring for 6 hours, the mixture was centrifuged at 13300 rpm for 8 minutes, the supernatant was discarded, the centrifuged product was washed with deionized water, and finally 1 mL of deionized water was added to disperse the precipitate to obtain a CuAp@GOx NSs solution, in which the major axis of the nanoparticles was 247 nanometers and the minor axis was 128 nanometers.

[0074] Embodiment 6:

[0075] Under ultrasonic stirring, 7.6 mL of dimethyl sulfoxide solvent, 4 mL of 10 mmol / L copper chloride deionized water solution and 0.4 mL of 10 mmol / L apigenin dimethyl sulfoxide solution were added to 28 mL of deionized water in sequence, followed by 40 μL of 100 mmol / L sodium hydroxide solution. The mixture was stirred at 170 rpm and ultrasonicated at 120 watts. After 2 hours, the mixture was centrifuged at 8800 rpm for 15 minutes, the supernatant was discarded, the centrifuged product was washed with deionized water, and finally 1 mL of deionized water was added to disperse the precipitate to obtain a CuAp NSs solution. 15 mg of CuAp NSs was dispersed in 10 mL of deionized water, 1.5 mg of glucose oxidase was added under stirring at room temperature, and after stirring for 6 hours, the mixture was centrifuged at 13300 rpm for 8 minutes, the supernatant was discarded, the centrifuged product was washed with deionized water, and finally 1 mL of deionized water was added to disperse the precipitate to obtain a CuAp@GOx NSs solution, in which the major axis of the nanoparticles was 218 nanometers and the minor axis was 106 nanometers.

[0076] Embodiment 7:

[0077] Under ultrasonic stirring, 7.2 mL of dimethyl sulfoxide solvent, 4 mL of 10 mmol / L copper chloride deionized water solution and 0.8 mL of 10 mmol / L apigenin N,N-dimethylformamide solution were added to 28 mL of deionized water in sequence, followed by 40 μL of 100 mmol / L sodium hydroxide solution. The mixture was stirred at 170 rpm and ultrasonicated at 120 watts. After 2 hours, the mixture was centrifuged at 8800 rpm for 15 minutes, the supernatant was discarded, the centrifuged product was washed with deionized water, and finally 1 mL of deionized water was added to disperse the precipitate to obtain a CuAp NSs solution. 15 mg of CuAp NSs was dispersed in 10 mL of deionized water, 1.5 mg of glucose oxidase was added under stirring at room temperature, and after stirring for 6 hours, the mixture was centrifuged at 13300 rpm for 8 minutes, the supernatant was discarded, the centrifuged product was washed with deionized water, and finally 1 mL of deionized water was added to disperse the precipitate to obtain a CuAp@GOx NSs solution, in which the major axis of the nanoparticles was 339 nanometers and the minor axis was 149 nanometers.

[0078] Embodiment 8:

[0079] Under ultrasonic stirring, 1.2 mL of dimethyl sulfoxide solvent, 4 mL of 10 mmol / L copper chloride deionized water solution and 0.8 mL of 10 mmol / L apigenin dimethyl sulfoxide solution were added to 34 mL of deionized water in sequence, followed by 40 μL of 100 mmol / L sodium hydroxide solution. The mixture was stirred at 170 rpm and ultrasonicated at 120 watts. After 2 hours, the mixture was centrifuged at 8800 rpm for 15 minutes, the supernatant was discarded, the centrifuged product was washed with deionized water, and finally 1 mL of deionized water was added to disperse the precipitate to obtain a CuAp NSs solution. 15 mg of CuAp NSs was dispersed in 10 mL of deionized water, 1.5 mg of glucose oxidase was added under stirring at room temperature, and after stirring for 6 hours, the mixture was centrifuged at 13300 rpm for 8 minutes, the supernatant was discarded, the centrifuged product was washed with deionized water, and finally 1 mL of deionized water was added to disperse the precipitate to obtain a CuAp@GOxNSs solution, in which the major axis of the nanoparticles was 205 nanometers and the minor axis was 81 nanometers.

[0080] Embodiment 9:

[0081] Under ultrasonic stirring, 7.2 mL of dimethyl sulfoxide solvent, 4 mL of 10 mmol / L copper chloride deionized water solution and 0.8 mL of 10 mmol / L apigenin dimethyl sulfoxide solution were added to 28 mL of deionized water in sequence, followed by 8 μL of 100 mmol / L sodium hydroxide solution. The mixture was stirred at 170 rpm and ultrasonicated at 120 watts. After 2 hours, the mixture was centrifuged at 8800 rpm for 15 minutes, the supernatant was discarded, the centrifuged product was washed with deionized water, and finally 1 mL of deionized water was added to disperse the precipitate to obtain a CuAp NSs solution. 15 mg of CuAp NSs was dispersed in 10 mL of deionized water, 1.5 mg of glucose oxidase was added under stirring at room temperature, and after stirring for 6 hours, the mixture was centrifuged at 13300 rpm for 8 minutes, the supernatant was discarded, the centrifuged product was washed with deionized water, and finally 1 mL of deionized water was added to disperse the precipitate to obtain a CuAp@GOxNSs solution, in which the major axis of the nanoparticles was 290 nanometers and the minor axis was 135 nanometers.

[0082] Embodiment 10:

[0083] Under ultrasonic stirring, 7.2 mL of dimethyl sulfoxide solvent, 4 mL of 10 mmol / L copper chloride deionized water solution and 0.8 mL of 10 mmol / L apigenin dimethyl sulfoxide solution were added to 28 mL of deionized water in sequence, followed by 40 μL of 100 mmol / L sodium hydroxide solution. The mixture was stirred at 170 rpm and ultrasonicated at 120 watts. After 2 hours, the mixture was centrifuged at 8800 rpm for 15 minutes, the supernatant was discarded, the centrifuged product was washed with deionized water, and finally 1 mL of deionized water was added to disperse the precipitate to obtain a CuAp NSs solution. 15 mg of CuAp NSs was dispersed in 10 mL of deionized water, 20 mg of glucose oxidase was added under stirring at room temperature, and after stirring for 6 hours, the mixture was centrifuged at 13300 rpm for 8 minutes, the supernatant was discarded, the centrifuged product was washed with deionized water, and finally 1 mL of deionized water was added to disperse the precipitate to obtain a CuAp@GOxNSs solution, in which the major axis of the nanoparticles was 262 nanometers and the minor axis was 101 nanometers.

[0084] Embodiment 11:

[0085] The CuAp@GOxNSs prepared in Example 1 was tested using a UV-visible absorption spectrometer. The characteristic peak of apigenin exists in the spectrum of CuAp@GOxNSs, proving the existence of apigenin; a new characteristic peak appears at 430 nanometers, proving the coordination effect between copper and apigenin.

[0086] Embodiment 12:

[0087] The CuAp@GOxNSs prepared in Example 1 were tested using a BrukerVERTEX 80V Fourier transform infrared spectrometer. -1 The aromatic CH bending vibration peak at 3700-3200 cm -1 The NH stretching vibration peak at 47° becomes broadened, proving the existence of GOx.

[0088] Embodiment 13:

[0089] The CuAp@GOxNSs prepared in Example 1 were incubated in different solutions, and it was found that they were significantly disassembled in ethylenediaminetetraacetic acid, dimethyl sulfoxide, and Triton X-100 solutions. The main driving forces for the formation of CuAp@GOxNSs were coordination, π-π interaction, and hydrophobic interaction.

[0090] Embodiment 14:

[0091] The CuAp@GOxNSs prepared in Example 1 were dispersed in a 2 mmol / L GSH solution and placed in a 37°C water bath for 24 hours. The transmission images showed that the CuAp@GOxNSs disassembled under the conditions of a simulated tumor microenvironment.

[0092] Embodiment 15:

[0093] Copper ion solutions of different concentrations were prepared, and after being completely reduced with 10% hydroxylamine hydrochloride, their absorbance at 457 nanometers was detected with a new cuprous reagent, and a standard curve was drawn with the concentration as the horizontal axis and the absorbance value as the vertical axis.

[0094] Embodiment 16:

[0095] The CuAp@GOx NSs prepared in Example 1 were dispersed in a PBS solution with a pH of 5.5 and containing 2 mmol / L GSH, and placed in a deionized water bath at 37°C for 1, 3, 6, 10, 12, and 24 hours, respectively. The supernatant was collected after centrifugation, and its absorbance at 457 nm was detected using a new cuprous reagent. The absorbance was substituted into the standard curve to obtain a cuprous ion release bar graph.

[0096] Embodiment 17:

[0097] The CuAp@GOxNSs prepared in Example 1 were dispersed in a simulated tumor microenvironment (pH=5.5, 2 mmol / LGSH PBS solution), placed in a deionized water bath at 37°C for 24 hours, and the supernatant was collected after centrifugation. The release of apigenin was confirmed by UV absorption spectroscopy.

[0098] Embodiment 18:

[0099] After a 2 mmol / L GSH solution was placed in a deionized water bath at 37°C for 60 minutes, 5 μL of the spin trapping agent 5,5-dimethyl-1-pyrroline-N-oxide was added and the electron paramagnetic resonance spectrum was immediately detected.

[0100] 5 μL of the spin trapping agent 5,5-dimethyl-1-pyrroline-N-oxide was added to 10 μL of 10 mmol / L hydrogen peroxide (final concentration was 100 μmol / L), and the electron paramagnetic resonance spectrum was immediately detected.

[0101] The CuAp@GOxNSs (final concentration of 100 μg / mL) prepared in Example 1 and 2 mg / mL of glucose were dispersed in a solution containing 2 mmol / LGSH and placed in a deionized water bath at 37°C for 2 hours. The mixture was centrifuged at 13,000 rpm, and 5 μL of spin trap 5,5-dimethyl-1-pyrroline-N-oxide and 10 μL of 10 mmol / L hydrogen peroxide (final concentration of 100 μmol / L) solution were added to the supernatant in sequence, and the electron paramagnetic resonance spectrum was immediately detected.

[0102] Fig.18 It shows that the addition of CuAp@GOxNSs causes the electron paramagnetic resonance spectrum to show a characteristic signal peak of 1:2:2:1 hydroxyl radicals, indicating that CuAp@GOx NSs can decompose cuprous ions in GSH, react with hydrogen peroxide to generate hydroxyl radicals, and react with spin traps to form spin adducts, showing a unique electron paramagnetic resonance spectrum.

[0103] Embodiment 19:

[0104] The CuAp@GOxNSs (final concentration 100 μg / mL) prepared in Example 1 and 2 mg / mL of glucose were dispersed in a deionized water solution, and a tetramethylbenzidine solution was added to perform UV absorption spectrum detection. The results showed that CuAp@GOx NSs could produce hydrogen peroxide, which indirectly proved that glucose was consumed.

[0105] Embodiment 20:

[0106] SKOV3 cells were seeded in a 96-well culture plate at an initial density of 7000 cells per well and incubated for 24 hours. The cells were then co-cultured with CuAp@GOx NSs at concentrations of 0 μg / mL, 1 μg / mL, 2 μg / mL, 3 μg / mL, 4 μg / mL, and 5 μg / mL for 24 hours. The original culture medium was then aspirated and 100 μL of the prepared CCK-8 solution was added to each well. After 1 hour, the cytotoxicity was tested using an ELISA instrument. After treatment with 3 μg / mL of CuAp@GOxNSs, the cell survival rate dropped to about 18%, proving that CuAp@GOxNSs has the potential to kill tumor cells.

[0107] Embodiment 21:

[0108] SKOV3 cells were seeded in a 6-well culture plate with an initial density of 600,000 cells per well and incubated for 24 hours. The cells were then co-cultured with CuAp@GOxNSs at concentrations of 0 μg / mL, 3 μg / mL, 6 μg / mL, 9 μg / mL, and 12 μg / mL for 24 hours and tested using a glucose detection kit. As the concentration of CuAp@GOxNSs increased, the intracellular glucose content gradually decreased, demonstrating that apigenin and GOx can disrupt glucose homeostasis.

[0109] Embodiment 22:

[0110] SKOV3 cells were seeded in a 6-well culture plate with an initial density of 300,000 cells per well and incubated for 24 hours. The cells were then co-cultured with CuAp@GOx NSs at concentrations of 0 μg / mL, 1 μg / mL, 2 μg / mL, 3 μg / mL, 4 μg / mL, and 5 μg / mL for 24 hours. The results were detected using a GSH detection kit. As the concentration of CuAp@GOx NSs increased, the intracellular GSH content gradually decreased, indicating that glucose homeostasis was unbalanced, the production of NADPH was reduced, cysteine ​​was reduced, and thus the biosynthesis of GSH was reduced.

[0111] Embodiment 23:

[0112] SKOV3 cells were seeded in confocal dishes at an initial density of 70,000 cells per dish and incubated for 24 hours. The cells were then co-cultured with CuAp@GOx NSs at concentrations of 0 μg / mL, 1 μg / mL, 2 μg / mL, and 3 μg / mL for 24 hours, stained with Actin-trackerGreen, and then imaged by CLSM. The gradual shrinkage of the cytoskeleton and cells in the green fluorescent part demonstrated that CuAp@GOxNSs caused cellular disulfide stress and disulfide death.

[0113] Embodiment 24:

[0114] The CuAp@GOxNSs prepared in Example 1 were used to detect the expression of FDX1, GLUT1, PKM2, and DLAT in cells by Western blot analysis. The downregulation of GLUT1 and PKM2 proteins proved that apigenin played a role in affecting glucose homeostasis; the downregulation of FDX1 and the oligomerization of DLAT proved the occurrence of copper death.

[0115] Embodiment 25:

[0116] SKOV3 cells were seeded in confocal dishes at an initial density of 70,000 cells per dish and incubated for 24 hours. The cells were then co-cultured with CuAp@GOx NSs at concentrations of 0 μg / mL, 1 μg / mL, 2 μg / mL, and 3 μg / mL for 24 hours. 581 / 591 The staining was followed by CLSM imaging, and the gradual increase in the red fluorescence portion demonstrated that CuAp@GOxNSs caused lipid peroxide accumulation and ferroptosis.

[0117] Experimental Results

[0118] 1. Transmission Electron Microscope Analysis

[0119] The CuAp@GOx NSs prepared in Examples 1-10 were observed by transmission electron microscopy. The results showed that the nanomedicines had regular morphology, uniform size distribution, and the major and minor axis sizes were consistent with expectations, further verifying the feasibility and controllability of the preparation method of the present invention.

[0120] 2. Spectral analysis

[0121] The UV-visible absorption spectrum showed that CuAp@GOxNSs had characteristic absorption peaks of apigenin and GOx at specific wavelengths, indicating that apigenin and GOx were successfully loaded into the nanomedicine.

[0122] Fourier transform infrared spectroscopy analysis showed that characteristic peaks of copper-apigenin coordination appeared in CuAp@GOxNSs, proving the interaction between divalent copper salt and apigenin.

[0123] 3. Cell experiments

[0124] CCK-8 cytotoxicity test showed that CuAp@GOx NSs had a significant killing effect on SKOV3 cells, and the killing effect was positively correlated with the concentration and action time of the nanodrug.

[0125] The results of intracellular glucose and GSH content detection showed that the intracellular glucose and GSH contents were significantly reduced after CuAp@GOxNSs treatment, proving that nanomedicine can effectively disrupt the glucose homeostasis of tumor cells and hinder the operation of the SLC7A11 / GSH / GPX4 antioxidant axis.

[0126] CLSM images showed that cells treated with CuAp@GOxNSs showed cytoskeleton contraction and lipid peroxide accumulation, which further verified the occurrence of disulfide death and ferroptosis, indicating that the nanomedicine of the present invention effectively kills tumor cells through multiple mechanisms.

[0127] In summary, the copper / apigenin nanomedicine loaded with glucose oxidase and the preparation method thereof provided by the present invention have significant advantages and good application prospects, and provide a new strategy and method for tumor treatment.

[0128] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A copper / apigenin nanodrug loaded with glucose oxidase, characterized in that: The nano drug consists of the following two parts: Copper / apigenin nanoparticles (CuApNSs), which are formed through coordination, π-π and hydrophobic interactions between divalent copper salts and apigenin; Glucose oxidase (GOx) is loaded on the surface of the CuApNSs through electrostatic interaction.

2. The copper / apigenin nanodrug loaded with glucose oxidase according to claim 1, characterized in that: The divalent copper salt is selected from one or more of copper chloride, copper acetate, copper nitrate or copper sulfate.

3. The copper / apigenin nanodrug loaded with glucose oxidase according to claim 1, characterized in that: The apigenin is 5,7-dihydroxy-2-(4-hydroxyphenyl)benzopyran-4-one.

4. The copper / apigenin nanodrug loaded with glucose oxidase according to claim 1, characterized in that: The nanoparticle size of the copper / apigenin nanomedicine loaded with glucose oxidase is 200 to 450 nanometers in the long axis and 80 to 300 nanometers in the short axis.

5. A method for preparing the copper / apigenin nanodrug loaded with glucose oxidase according to claim 1, characterized in that: The following steps are involved: S1: Mix a deionized water solution of a divalent copper salt with an organic solvent solution of apigenin, add a sodium hydroxide solution to adjust the pH to 3.9-6.2, react under ultrasonic stirring at 20-45°C for 0.5-4 hours, and centrifuge and wash to obtain copper / apigenin nanomedicine (CuAp NSs); S2: Disperse CuAp NSs in deionized water, add glucose oxidase, stir at room temperature for 6 hours, and obtain the copper / apigenin nanodrug (CuAp@GOxNSs) solution loaded with glucose oxidase after centrifugation and washing.

6. The preparation method according to claim 5, characterized in that: The concentration of the deionized water solution of the divalent copper salt is 0.13-4 mmol / L, and the concentration of the organic solvent solution of apigenin is 0.05-1.54 mmol / L.

7. The preparation method according to claim 5, characterized in that: The organic solvent is dimethyl sulfoxide (DMSO) and / or N,N-dimethylformamide (DMF), and the proportion of the organic solvent is 5-30% of the total volume of the solution.

8. The preparation method according to claim 5, characterized in that: The concentration of the sodium hydroxide solution is 0.02-1 mmol / L.

9. The preparation method according to claim 5, characterized in that: The stirring speed of the ultrasonic stirring is 80 to 220 rpm, the ultrasonic power is 50 to 120 watts, and the centrifugal speed is 6000 to 15000 rpm.

10. Use of the copper / apigenin nanomedicine loaded with glucose oxidase according to claim 1 in the preparation of a drug for tumor treatment, characterized in that: The drug disrupts the glucose homeostasis of tumor cells and hinders the operation of the SLC7A11 / GSH / GPX4 antioxidant axis, thereby inducing disulfide death, copper death and iron death.

Images