Preparation method and application of gold-modified cerium oxide drug-loaded nano-microspheres

Abstract

The invention discloses a preparation method and application of gold-modified cerium oxide drug-loaded nanoparticles, and the preparation method comprises the following steps: dispersing soluble cerium salt in an ethylene glycol solution of a template agent, adjusting the pH value to 5-7, then carrying out a hydrothermal reaction to obtain cerium oxide nanoparticles, then dispersing the cerium oxide nanoparticles in ultrapure water, and carrying out ultrasonic treatment to obtain the gold-modified cerium oxide drug-loaded nanoparticles. And adding gold tetrachlorate into the dispersion system, adjusting the pH value to 7-11 to obtain gold-modified cerium oxide nano-microspheres, and loading chemotherapeutic drugs to obtain the gold-modified cerium oxide drug-loaded nano-microspheres. The obtained gold-modified cerium oxide drug-loaded nano-microspheres have the advantages of multiple enzyme activities, good biocompatibility, excellent antibacterial performance and excellent photo-thermal performance, can relieve an anoxic environment in tumor treatment, consume GSH, overcome tumor drug resistance and the like, and have good application prospects in the field of tumor treatment.

Description

Technical Field

[0001] The invention relates to a preparation method and application of gold-modified cerium oxide drug-loaded nano-microspheres, belonging to the technical field of biomaterial preparation. Background Art

[0002] Breast cancer is one of the most common, prevalent and deadly tumors in women. In recent years, the number of breast cancer patients worldwide has been on the rise, increasing by 2-5% each year, and the patients are getting younger, while the incidence and mortality rates have also increased. In recent decades, although the treatment of breast cancer has made great progress, the treatment of breast cancer is still similar to that of other cancers, such as surgical removal of the tumor or chemotherapy or radiotherapy. Traditional treatment methods have the problems of low efficiency and high recurrence rate. Therefore, it is necessary to develop effective new methods including photothermal therapy, nanotherapy, targeted drug delivery, etc.

[0003] Nanozymes refer to nanomaterials with activities similar to those of natural enzymes. Compared with natural enzymes, nanozymes have the characteristics of high stability, adjustable catalysis, easy scale preparation and functional modification, and therefore have attracted much attention in the biomedical field. Nanozymes mainly include peroxidase-like, catalase-like, oxidase-like and superoxide dismutase-like. Under in vitro conditions, nanozymes are stable under extreme conditions and are expected to replace natural enzymes in the fields of cell capture, biomolecule detection, and biological sewage discharge. In vivo, nanozymes can regulate the level of intracellular active ROS through multiple redox enzyme activities, which can be used to prevent and treat chronic oxidative stress diseases such as cardiovascular disease, diabetes, neurodegenerative diseases and cancer, and are currently attracting much attention in the field of nanomedicine research.

[0004] Photothermal therapy (PTT) is an emerging tumor treatment method. It uses materials with high photothermal conversion efficiency as photothermal agents (PTA). After being injected into the body, under the irradiation of near infrared light (NIR) of a specific excitation wavelength, the absorbed photon energy is converted into heat energy, causing the local tissue temperature to rise, inducing cell thermal sensitization (such as inhibition of DNA synthesis and repair, cell membrane and cytoskeleton damage, etc.), leading to cell apoptosis or necrosis, thereby achieving the purpose of killing cancer cells. Compared with traditional cancer treatment methods, PTT has a significant cancer-killing effect and strong targeting. It can overcome the serious toxic side effects of current therapies in clinical practice and maximize the treatment effect.

[0005] Localized surface plasmon resonance (LSPR) is a unique nanoscale physical phenomenon that can be described as the collective oscillation of surface free electrons in metal nanoparticles, driven by the oscillating electromagnetic field generated by the incident electromagnetic wave. LSPR is established when the frequency of the photons matches the natural frequency of the oscillating electrons. The LSPR process can decay through two pathways, including non-radiative and radiative relaxation. During the decay process, three significant effects may occur, including near-field enhancement, the generation of hot carriers (i.e., hot electrons and hot holes), and local heating (also known as the photothermal effect). The closer the wavelength of the incident light is to the LSPR peak position (i.e., the resonant wavelength), the more significant the activity enhancement effect of the simulated enzyme. Obviously, the development of efficient plasma-enhanced drug-loaded nanomaterials is one of the effective ways to treat breast cancer. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide a preparation method and application of gold-modified cerium oxide drug-loaded nano-microspheres in view of the deficiencies of the prior art. The gold-modified cerium oxide drug-loaded nano-microspheres have the characteristics of plasma-enhanced nanozyme activity, good biocompatibility and can be combined with photothermal therapy, can alleviate the hypoxic environment in tumor treatment, consume GSH, overcome tumor drug resistance, etc., and have good application prospects in the field of tumor treatment.

[0007] The implementation process of the present invention is as follows: A method for preparing gold-modified cerium oxide drug-loaded nanoparticles comprises the following steps: (1) Synthesizing cerium oxide nanoparticles, specifically comprising: dissolving a template in an ethylene glycol solution to obtain an ethylene glycol solution of the template, wherein the template is selected from one or more of polyvinyl pyrrolidone, polyethylene glycol, hexadecyltrimethylammonium bromide and polyvinyl alcohol; dispersing a soluble cerium salt in the ethylene glycol solution of the template, and adjusting the pH value to 5-7 to obtain a mixed solution; transferring the mixed solution to a closed reactor, reacting it at 100-200° C., and collecting the precipitate to obtain cerium oxide nanoparticles; (2) preparing gold-modified cerium oxide nanoparticles, specifically comprising: dispersing the above-mentioned cerium oxide nanoparticles in ultrapure water to obtain a cerium oxide nanoparticle dispersion system; adding an aqueous gold tetrachloroate solution to the cerium oxide nanoparticle dispersion system, adjusting the pH value to 7-11, stirring in the dark and collecting the precipitate to obtain gold-modified cerium oxide nanoparticles; (3) Preparing gold-modified cerium oxide drug-loaded nanoparticles, specifically comprising: re-dispersing the gold-modified cerium oxide nanoparticles in ultrapure water, then adding an aqueous solution containing a chemotherapeutic drug, stirring thoroughly in the dark, and collecting the precipitate to obtain gold-modified cerium oxide drug-loaded nanoparticles.

[0008] In the above-mentioned method for preparing gold-modified cerium oxide drug-loaded nanoparticles, in step (1), the concentration of the template agent in the ethylene glycol aqueous solution is 0.01-0.2 g / mL, and the concentration of ethylene glycol in the ethylene glycol aqueous solution is 60-80%.

[0009] In the above-mentioned method for preparing gold-modified cerium oxide drug-loaded nanoparticles, in step (1), the soluble cerium salt is selected from one or more of cerium nitrate and cerium chloride.

[0010] In the above-mentioned method for preparing gold-modified cerium oxide drug-loaded nanoparticles, in step (1), the mass of the soluble cerium salt is 1 to 10 times the mass of the template.

[0011] In the above-mentioned method for preparing gold-modified cerium oxide drug-loaded nanoparticles, in step (2), the concentration of cerium oxide nanoparticles in the cerium oxide nanoparticle dispersion system is 1-200 mg / mL.

[0012] In the above-mentioned method for preparing gold-modified cerium oxide drug-loaded nanoparticles, in step (2), the mass ratio of the cerium oxide nanoparticles to gold tetrachloride is (0.1-10):(1-100).

[0013] In the above-mentioned method for preparing gold-modified cerium oxide drug-loaded nanoparticles, in step (3), the chemotherapy drug is selected from one or more of cisplatin, doxorubicin, etoposide and methotrexate.

[0014] The gold-modified cerium oxide drug-loaded nano-microspheres prepared by the above method are used in photothermal therapy for treating breast cancer.

[0015] Compared with the prior art, the present invention has the following advantages: 1. The present invention provides a gold-modified cerium oxide drug-loaded nano-microsphere with a particle size of 150-250 nm. The gold-modified cerium oxide drug-loaded nano-microsphere has the characteristics of plasma-enhanced multiple enzyme activities, good biocompatibility and can be combined with photothermal near-infrared light NIR, and can effectively achieve the treatment of breast cancer.

[0016] 2. The gold-modified cerium oxide drug-loaded nano-microspheres of the present invention can decompose H2O2 inside the cerium oxide to release oxygen, regulate the hypoxic microenvironment of the tumor site, and further inhibit the growth of tumor cells.

[0017] 3. The gold-modified cerium oxide drug-loaded nano-microspheres prepared by the present invention can effectively achieve sustained drug release and cooperate with the high-concentration GSH and high-concentration H2O2 microenvironment to quickly kill tumor cells and continuously inhibit them while avoiding damage to normal tissues.

[0018] 4. The preparation method of the present invention is simple and the raw materials are easily available. Nanospheres and chemotherapeutic drugs are used as the main raw materials. By stirring in the dark at room temperature, gold-modified cerium oxide drug-loaded nanospheres having multiple enzyme activities, good biocompatibility and the ability to be combined with photothermal therapy can be obtained. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a SEM image of cerium oxide nanoparticles prepared in Example 1 of the present invention; Figure 2 This is a SEM image of gold-modified cerium oxide nanospheres prepared in Example 1 of the present invention; Figure 3 This is a SEM image of the gold-modified cerium oxide drug-loaded nanoparticles prepared in Example 1 of the present invention; Figure 4 This is an O2 generation curve of the gold-modified cerium oxide drug-loaded nano-microspheres prepared in Example 1 of the present invention in H2O2 (pH=7.4); Figure 5 This is a diagram of the in vitro photothermal effect (NIR irradiation time-temperature curve) of gold-modified cerium oxide drug-loaded nano-microspheres prepared in Example 1 of the present invention; Figure 6 This is a graph showing the GSH oxidation-promoting ability of gold-modified cerium oxide drug-loaded nanoparticles prepared in Example 1 of the present invention; Figure 7 The gold-modified cerium oxide drug-loaded nano-microspheres prepared in Example 1 of the present invention were subjected to a tumor inhibition test (MTT) on MCF-7 cells; Figure 8 It is a graph showing the O2 production of the cerium oxide nano-microspheres and gold-modified cerium oxide nano-microspheres prepared in Example 1 of the present invention in H2O2 (pH=7.4); Fig. 9 The in vitro photothermal effect (NIR irradiation time-temperature curve) of the cerium oxide nano-microspheres and gold-modified cerium oxide nano-microspheres prepared in Example 1 of the present invention; Fig.10 This is a graph showing the GSH oxidation-promoting ability of the cerium oxide nano-microspheres and gold-modified cerium oxide nano-microspheres prepared in Example 1 of the present invention; Fig.11 The cerium oxide nanoparticles prepared in Example 1 of the present invention were subjected to a tumor inhibition test (MTT) on MCF-7 cells; Fig.12 This is a tumor inhibition experiment (MTT) of the gold-modified cerium oxide nanoparticles prepared in Example 1 of the present invention on MCF-7 cells. DETAILED DESCRIPTION

[0020] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials and reagents used, unless otherwise specified, can all be obtained from commercial sources. Cisplatin was purchased from Shandong Qilu Pharmaceutical Co., Ltd., and doxorubicin and methotrexate were purchased from Shanghai MacLean Biochemical Technology Co., Ltd. The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. Example 1

[0021] A method for preparing gold-modified cerium oxide drug-loaded nanoparticles for treating breast cancer comprises: Step 1: Synthesize cerium oxide nano-microspheres, specifically including: dissolving 0.3g of template polyvinyl pyrrolidone in 15mL of ethylene glycol solution under magnetic stirring at room temperature to obtain an ethylene glycol solution of the template; the relative molecular mass of the polyvinyl pyrrolidone is 8000Da; the ethylene glycol solution is an aqueous solution of ethylene glycol with a volume percentage of 75%; adding 0.5g of cerium nitrate tetrahydrate to the ethylene glycol solution of the template, ultrasonicating until dissolved, adjusting the pH to 7 with a 1mM HCl solution, stirring for 30min, and obtaining a mixed system A; transferring the mixed system A to a hydrothermal autoclave, reacting at 160°C for 4h, cooling and centrifuging, and collecting the precipitate. Wash the precipitate with ultrapure water and anhydrous ethanol 3 times each, and dry it to obtain powdered cerium oxide nano-microspheres; Step 2: providing gold-modified cerium oxide nanospheres, specifically comprising: dispersing the cerium oxide nanospheres in 30 mL of ultrapure water to obtain a cerium oxide nanosphere dispersion system with a concentration of 1 mg / mL; adding 30 mL of a 1 mg / mL gold tetrachloroate aqueous solution to the cerium oxide nanosphere dispersion system under stirring, adjusting the pH to 11, and stirring for 12 hours in the dark; centrifuging the system after stirring in the dark, washing 3 times with ultrapure water, and drying to obtain gold-modified cerium oxide nanospheres.

[0022] Step 3: providing gold-modified cerium oxide drug-loaded nano-microspheres, specifically comprising: re-dispersing the gold-modified cerium oxide nano-microspheres in ultrapure water, then adding 30 mL of a 1 mg / mL cisplatin aqueous solution, stirring for 48 hours in a dark place; centrifuging the system after stirring in a dark place, washing three times with ultrapure water, and drying to obtain gold-modified cerium oxide drug-loaded nano-microspheres.

[0023] Material characterization and performance evaluation:

[0024] Figure 1 This is a SEM image of the cerium oxide nanospheres prepared in step 1. Figure 1 It can be seen that the diameter of cerium oxide nanospheres is 100~200nm and the surface is rough.

[0025] Figure 2 This is the SEM image of gold-modified cerium oxide nanoparticles in step 2. Figure 2 It can be seen that the diameter of the gold-modified cerium oxide drug-loaded nanoparticles is 150~250nm, and the surface is rougher than that of cerium oxide nanoparticles.

[0026] Figure 3 This is the SEM image of gold-modified cerium oxide drug-loaded nanoparticles in step 3. Figure 3 It can be seen that the diameter of the gold-modified cerium oxide drug-loaded nanoparticles is 150~250nm.

[0027] Figure 4 The figure is a schematic diagram of the O2 production performance of gold-modified cerium oxide drug-loaded nanoparticles in hydrogen peroxide solution. The test method includes: dispersing gold-modified cerium oxide drug-loaded nanoparticles in 5mL PBS (pH=7.4) at a concentration of 5mg / mL, adding 0.5mL 1M H2O2 solution, stirring continuously, and measuring the O2 concentration at different times. The results are as follows: Figure 4 As shown, according to Figure 4 It can be seen that O2 is continuously released within 20 minutes.

[0028] Figure 5 The results of the in vitro photothermal effect test of gold-modified cerium oxide drug-loaded nanoparticles were shown in Figure 2. The test method included dispersing the gold-modified cerium oxide drug-loaded nanoparticles in this example in 2 mL PBS at a concentration of 5 mg / mL and using NIR (808 nm, 1.0 W / cm 2 ) for 10 minutes, and record the temperature every 30 seconds. The results are as follows Figure 5 As shown. Figure 5 It can be seen that under NIR irradiation, the PBS solution dispersed with gold-modified cerium oxide drug-loaded nanomicrospheres can reach the expected photothermal temperature of 45° C. in 150 s, indicating that the gold-modified cerium oxide drug-loaded nanomicrospheres of the present invention have excellent photothermal effect.

[0029] Figure 6 The figure is a schematic diagram of the ability of gold-modified cerium oxide drug-loaded nano-microspheres to promote GSH oxidation under the presence or absence of NIR irradiation in this embodiment. The test method includes: dispersing gold-modified cerium oxide drug-loaded nano-microspheres in 3mL PBS (pH=7.4) at a concentration of 5mg / mL, adding 0.1μL of 10mM GSH, dispersing evenly, reacting for 6h, adding 100μL of 10mM DTNB, taking the supernatant after ten minutes of reaction, recording its absorbance at 412nm with an ELISA reader and calculating its oxidation degree.

[0030] Figure 7 This is a cytological experiment on gold-modified cerium oxide drug-loaded nanoparticles. The gold-modified cerium oxide drug-loaded nanoparticles are used as the research object to investigate their efficacy in inhibiting or killing cancer. The test methods include: (1) Preparation of DMEM extract of gold-modified cerium oxide drug-loaded nanoparticles ① DMEM complete cell culture medium: add 10% fetal bovine serum, 100 μg / mL streptomycin, and 100 U / mL penicillin to 500 mL of DMEM basal medium, mix well, and place in a 4°C refrigerator for later use; ② Preparation of PBS solution: Weigh 0.01 g potassium chloride, 0.40 g sodium chloride, 0.01 g dipotassium hydrogen phosphate, and 0.17 g disodium hydrogen phosphate, respectively, add 1000 ml double distilled water, dissolve by ultrasonication, adjust the pH to 7.40, sterilize at 121°C for 30 min, and store in a refrigerator at 4°C for later use; ③ Preparation of 0.25% pancreatic enzyme: weigh 100 mg of pancreatic enzyme, add 40 mL of PBS, filter with a 0.22 μm filter membrane, and store in a 4°C refrigerator for later use; ④ Preparation of DMEM extract of gold-modified cerium oxide drug-loaded nanomicrospheres: Weigh the gold-modified cerium oxide drug-loaded nanomicrospheres in Example 1 and soak them in DMEM complete cell culture medium, place them in a 37°C constant temperature box for 24 hours, and then filter them with a 0.22 μm filter membrane to obtain cerium oxide nanomicrosphere extract and gold-modified cerium oxide nanomicrosphere extract, seal them, and place them in a 4°C refrigerator for use.

[0031] (2) Cell culture MCF-7 cells were selected as model cells of cancer cells, and were cultured with cerium oxide nanosphere extracts and gold-modified cerium oxide nanosphere extracts to investigate the effects of the extracts on the growth of model cells. ①Cell passaging Transfer the MCF-7 cells into a culture dish containing DMEM culture medium and place it in a 37°C, 5% CO2 incubator for 12 hours. Discard the culture medium, add 3 mL PBS to wash the cells, add 2 mL trypsin, place it in an incubator for digestion for 1.5 minutes, take out the culture dish and add 3 mL culture medium to terminate the digestion. Use a pipette to blow the cell culture medium several times, transfer the culture medium containing MCF-7 cells into 15 mL centrifuge tubes, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, add 2 mL culture medium to each centrifuge tube, blow evenly, divide it into two parts, add it to a new culture dish containing 9 mL DMEM culture medium, shake it crosswise, and place it flat in a 37°C, 5% CO2 incubator for 24 hours. ②Cell layout Take the cells cultured in step ① out of the incubator, discard the culture medium, add 3 mL PBS to wash the cells, add 2 mL trypsin, and put them into the incubator for digestion for 1.5 min. After digestion is completed, blow the cell culture medium several times with a pipette, add 5-8 mL culture medium and blow the cells several times, then pipette 50 μL of each cell sample into the cell counting plate, count the cells, dilute the cells according to a certain ratio, and then plate the cells in a 96-well plate. The cell density is about 1×10 4 / well, and cultured in a 37°C, 5% CO2 incubator for another 24 h; ③Cell culture in the extract of grafted modified mesoporous bioactive glass The 96-well plate in which the cells were cultured in ② was taken out, the culture medium was discarded, and the cerium oxide nanosphere extract and gold-modified cerium oxide nanosphere extract prepared in this example (experimental group) and an equal volume of DMEM culture medium (control group) were added thereto, respectively, and placed in an incubator for culture, with 5 replicate wells set for each concentration. After 24 h of culture, 50 μL of MTT was added to each well, and after continuing to culture for 2 to 4 h, 150 μL of DMSO was added to each well, and the absorbance (OD) value corresponding to each well was detected at 490 nm using an ELISA instrument, and the cell survival rate was calculated based on the OD value. Example 2

[0032] A method for preparing gold-modified cerium oxide drug-loaded nanoparticles for treating breast cancer comprises: Step 1: Provide cerium oxide nanoparticles, specifically including: dissolving 0.45g of template polyvinyl pyrrolidone in 15mL of ethylene glycol solution under magnetic stirring at room temperature to obtain an ethylene glycol solution of the template; the relative molecular mass of the polyvinyl pyrrolidone is 8000Da; the ethylene glycol solution is an aqueous solution of ethylene glycol with a volume percentage of 80%; adding 1g of cerium nitrate tetrahydrate to the ethylene glycol solution of the template, ultrasonicating until dissolved, adjusting the pH to 7 with a 1mM HCl solution, stirring for 60min, and obtaining a mixed system A; transferring the mixed system A to a hydrothermal autoclave, reacting at 160°C for 6h, cooling and centrifuging, and collecting the precipitate. Wash the precipitate with ultrapure water and anhydrous ethanol 3 times each, and dry it to obtain powdered cerium oxide nanoparticles; Step 2: providing gold-modified cerium oxide nanospheres, specifically comprising: dispersing the cerium oxide nanospheres in 30 mL of ultrapure water to obtain a cerium oxide nanosphere dispersion system with a concentration of 30 mg / mL; adding 30 mL of a 40 mg / mL gold tetrachloroate aqueous solution to the cerium oxide nanosphere dispersion system under stirring, adjusting the pH to 11, and stirring for 8 hours in the dark; centrifuging the system after stirring in the dark, washing it 3 times with ultrapure water, and drying it to obtain gold-modified cerium oxide nanospheres.

[0033] Step 3: providing gold-modified cerium oxide drug-loaded nano-microspheres, specifically comprising: re-dispersing the gold-modified cerium oxide nano-microspheres in ultrapure water, then adding 30 mL of a 2 mg / mL cisplatin aqueous solution, stirring for 24 hours in a dark place; centrifuging the system after stirring in a dark place, washing three times with ultrapure water, and drying to obtain gold-modified cerium oxide drug-loaded nano-microspheres.

[0034] The physical and chemical properties of the gold-modified cerium oxide drug-loaded nano-microspheres of this example are basically consistent with those of Example 1. Example 3

[0035] A method for preparing gold-modified cerium oxide drug-loaded nanoparticles for treating breast cancer comprises: Step 1: Provide cerium oxide nanoparticles, specifically including: dissolving 0.6g of template polyvinyl pyrrolidone in 15mL of ethylene glycol solution under magnetic stirring at room temperature to obtain an ethylene glycol solution of the template; the relative molecular mass of the polyvinyl pyrrolidone is 8000Da; the ethylene glycol solution is an aqueous solution of ethylene glycol with a volume percentage of 70%; adding 1.3g of cerium nitrate tetrahydrate to the ethylene glycol solution of the template, ultrasonicating until dissolved, adjusting the pH to 6 with a 1mM HCl solution, stirring for 100min, and obtaining a mixed system A; transferring the mixed system A to a hydrothermal autoclave, reacting at 180°C for 8h, cooling and centrifuging, and collecting the precipitate. Wash the precipitate with ultrapure water and anhydrous ethanol 3 times each, and dry it to obtain powdered cerium oxide nanoparticles; Step 2: providing gold-modified cerium oxide nanospheres, specifically comprising: dispersing the cerium oxide nanospheres in 30 mL of ultrapure water to obtain a cerium oxide nanosphere dispersion system with a concentration of 60 mg / mL; adding 30 mL of a 60 mg / mL gold tetrachloroate aqueous solution to the cerium oxide nanosphere dispersion system under stirring, adjusting the pH to 10, and stirring for 6 hours in the dark; centrifuging the system after stirring in the dark, washing 3 times with ultrapure water, and drying to obtain gold-modified cerium oxide nanospheres.

[0036] Step 3: providing gold-modified cerium oxide drug-loaded nano-microspheres, specifically comprising: re-dispersing the gold-modified cerium oxide nano-microspheres in ultrapure water, then adding 30 mL of a 4 mg / mL cisplatin aqueous solution, stirring for 12 hours in a dark place; centrifuging the system after stirring in a dark place, washing three times with ultrapure water, and drying to obtain gold-modified cerium oxide drug-loaded nano-microspheres.

[0037] The physical and chemical properties of the gold-modified cerium oxide drug-loaded nano-microspheres of this example are basically consistent with those of Example 1. Example 4

[0038] A method for preparing gold-modified cerium oxide drug-loaded nanoparticles for treating breast cancer comprises: Step 1: Provide cerium oxide nanoparticles, specifically including: dissolving 0.9g of template polyvinyl pyrrolidone in 15mL of ethylene glycol solution under magnetic stirring at room temperature to obtain an ethylene glycol solution of the template; the relative molecular mass of the polyvinyl pyrrolidone is 8000Da; the ethylene glycol solution is an aqueous solution of ethylene glycol with a volume percentage of 75%; adding 1.6g of cerium nitrate tetrahydrate to the ethylene glycol solution of the template, ultrasonicating until dissolved, adjusting the pH to 6 with a 1mM HCl solution, stirring for 400min, and obtaining a mixed system A; transferring the mixed system A to a hydrothermal autoclave, reacting at 180°C for 10h, cooling and centrifuging, and collecting the precipitate. Wash the precipitate with ultrapure water and anhydrous ethanol 3 times each, and dry it to obtain powdered cerium oxide nanoparticles; Step 2: providing gold-modified cerium oxide nanospheres, specifically comprising: dispersing the cerium oxide nanospheres in 30 mL of ultrapure water to obtain a cerium oxide nanosphere dispersion system with a concentration of 80 mg / mL; adding 30 mL of an 80 mg / mL gold tetrachloroate aqueous solution to the cerium oxide nanosphere dispersion system under stirring, adjusting the pH to 10, and stirring for 8 hours in the dark; centrifuging the system after stirring in the dark, washing it 3 times with ultrapure water, and drying it to obtain gold-modified cerium oxide nanospheres.

[0039] Step 3: providing gold-modified cerium oxide drug-loaded nano-microspheres, specifically comprising: re-dispersing the gold-modified cerium oxide nano-microspheres in ultrapure water, then adding 30 mL of a 6 mg / mL cisplatin aqueous solution, stirring for 72 hours in a dark place; centrifuging the system after stirring in a dark place, washing three times with ultrapure water, and drying to obtain gold-modified cerium oxide drug-loaded nano-microspheres.

[0040] The physical and chemical properties of the gold-modified cerium oxide drug-loaded nano-microspheres of this example are basically consistent with those of Example 1. Example 5

[0041] A method for preparing gold-modified cerium oxide drug-loaded nanoparticles for treating breast cancer comprises: Step 1: Provide cerium oxide nanoparticles, specifically including: dissolving 1.2g of template polyvinyl pyrrolidone in 15mL of ethylene glycol solution under magnetic stirring at room temperature to obtain an ethylene glycol solution of the template; the relative molecular mass of the polyvinyl pyrrolidone is 8000Da; the ethylene glycol solution is an aqueous solution of ethylene glycol with a volume percentage of 80%; adding 1.9g of cerium nitrate tetrahydrate to the ethylene glycol solution of the template, ultrasonicating until dissolved, adjusting the pH to 5 with a 1mM HCl solution, stirring for 600min, and obtaining a mixed system A; transferring the mixed system A to a hydrothermal autoclave, reacting at 200°C for 12h, cooling and centrifuging, and collecting the precipitate. Wash the precipitate with ultrapure water and anhydrous ethanol 3 times each, and dry it to obtain powdered cerium oxide nanoparticles; Step 2: providing gold-modified cerium oxide nanospheres, specifically comprising: dispersing the cerium oxide nanospheres in 30 mL of ultrapure water to obtain a cerium oxide nanosphere dispersion system with a concentration of 120 mg / mL; adding 30 mL of a 100 mg / mL gold tetrachloroate aqueous solution to the cerium oxide nanosphere dispersion system under stirring, adjusting the pH to 9, and stirring for 4 hours in the dark; centrifuging the system after stirring in the dark, washing 3 times with ultrapure water, and drying to obtain gold-modified cerium oxide nanospheres.

[0042] Step 3: providing gold-modified cerium oxide drug-loaded nano-microspheres, specifically comprising: re-dispersing the gold-modified cerium oxide nano-microspheres in ultrapure water, then adding 30 mL of an aqueous solution of cisplatin with a concentration of 8 mg / mL, stirring in the dark for 48 hours; centrifuging the system after stirring in the dark, washing it three times with ultrapure water, and drying it to obtain gold-modified cerium oxide drug-loaded nano-microspheres.

[0043] The physical and chemical properties of the gold-modified cerium oxide drug-loaded nano-microspheres of this example are basically consistent with those of Example 1. Example 6

[0044] A method for preparing gold-modified cerium oxide drug-loaded nanoparticles for treating breast cancer comprises: Step 1: Provide cerium oxide nanoparticles, specifically including: dissolving 1.5g of template polyvinyl pyrrolidone in 15mL of ethylene glycol solution under magnetic stirring at room temperature to obtain an ethylene glycol solution of the template; the relative molecular mass of the polyvinyl pyrrolidone is 8000Da; the ethylene glycol solution is an aqueous solution of ethylene glycol with a volume percentage of 75%; adding 2g of cerium nitrate tetrahydrate to the ethylene glycol solution of the template, ultrasonicating until dissolved, adjusting the pH to 5 with a 1mM HCl solution, stirring for 200min, and obtaining a mixed system A; transferring the mixed system A to a hydrothermal autoclave, reacting at 200°C for 12h, cooling and centrifuging, and collecting the precipitate. Wash the precipitate with ultrapure water and anhydrous ethanol 3 times each, and dry it to obtain powdered cerium oxide nanoparticles; Step 2: providing gold-modified cerium oxide nanospheres, specifically comprising: dispersing the cerium oxide nanospheres in 30 mL of ultrapure water to obtain a cerium oxide nanosphere dispersion system with a concentration of 150 mg / mL; adding 30 mL of a 200 mg / mL gold tetrachloroate aqueous solution to the cerium oxide nanosphere dispersion system under stirring, adjusting the pH to 9, and stirring for 3 hours in the dark; centrifuging the system after stirring in the dark, washing 3 times with ultrapure water, and drying to obtain gold-modified cerium oxide nanospheres.

[0045] Step 3: providing gold-modified cerium oxide drug-loaded nano-microspheres, specifically comprising: re-dispersing the gold-modified cerium oxide nano-microspheres in ultrapure water, then adding 30 mL of a 10 mg / mL cisplatin aqueous solution, stirring for 24 hours in a dark place; centrifuging the system after stirring in a dark place, washing three times with ultrapure water, and drying to obtain gold-modified cerium oxide drug-loaded nano-microspheres.

[0046] The physical and chemical properties of the gold-modified cerium oxide drug-loaded nano-microspheres of this example are basically consistent with those of Example 1. Example 7

[0047] A method for preparing gold-modified cerium oxide drug-loaded nanoparticles for treating breast cancer comprises: Step 1: Provide cerium oxide nano-microspheres, specifically including: dissolving 2.4g of template polyvinyl pyrrolidone in 15mL of ethylene glycol solution under room temperature magnetic stirring conditions to obtain an ethylene glycol solution of the template; the relative molecular mass of the polyvinyl pyrrolidone is 8000Da; the ethylene glycol solution is an aqueous solution of ethylene glycol with a volume percentage of 75%; adding 24g of cerium nitrate tetrahydrate to the ethylene glycol solution of the template, ultrasonicating until dissolved, adjusting the pH to 4 with a 1mM HCl solution, stirring for 500min, and obtaining a mixed system A; transferring the mixed system A to a hydrothermal autoclave, 120°C, reacting for 24h, cooling and centrifuging, and collecting the precipitate. The precipitate is washed with ultrapure water and anhydrous ethanol 3 times each, and dried to obtain powdered cerium oxide nano-microspheres; Step 2: providing gold-modified cerium oxide nanospheres, specifically comprising: dispersing the cerium oxide nanospheres in 30 mL of ultrapure water to obtain a cerium oxide nanosphere dispersion system with a concentration of 180 mg / mL; adding 30 mL of a 240 mg / mL gold tetrachloroate aqueous solution to the cerium oxide nanosphere dispersion system under stirring, adjusting the pH to 8, and stirring for 2 hours in the dark; centrifuging the system after stirring in the dark, washing 3 times with ultrapure water, and drying to obtain gold-modified cerium oxide nanospheres.

[0048] Step 3: providing gold-modified cerium oxide drug-loaded nano-microspheres, specifically comprising: re-dispersing the gold-modified cerium oxide nano-microspheres in ultrapure water, then adding 30 mL of a 12 mg / mL cisplatin aqueous solution, stirring for 12 hours in a dark place; centrifuging the system after stirring in a dark place, washing three times with ultrapure water, and drying to obtain gold-modified cerium oxide drug-loaded nano-microspheres.

[0049] The physical and chemical properties of the gold-modified cerium oxide drug-loaded nano-microspheres of this example are basically consistent with those of Example 1. Example 8

[0050] A method for preparing gold-modified cerium oxide drug-loaded nanoparticles for treating breast cancer comprises: Step 1: Provide cerium oxide nano-microspheres, specifically including: dissolving 3g of template polyvinyl pyrrolidone in 15mL of ethylene glycol solution under magnetic stirring at room temperature to obtain an ethylene glycol solution of the template; the relative molecular mass of the polyvinyl pyrrolidone is 8000Da; the ethylene glycol solution is an aqueous solution of ethylene glycol with a volume percentage of 75%; adding 30g of cerium nitrate tetrahydrate to the ethylene glycol solution of the template, ultrasonicating until dissolved, adjusting the pH to 4 with a 1mM HCl solution, stirring for 60min, and obtaining a mixed system A; transferring the mixed system A to a hydrothermal autoclave, reacting at 100°C for 24h, cooling and centrifuging, and collecting the precipitate. Wash the precipitate with ultrapure water and anhydrous ethanol 3 times each, and dry it to obtain powdered cerium oxide nano-microspheres; Step 2: providing gold-modified cerium oxide nanospheres, specifically comprising: dispersing the cerium oxide nanospheres in 30 mL of ultrapure water to obtain a cerium oxide nanosphere dispersion system with a concentration of 200 mg / mL; adding 30 mL of a 400 mg / mL gold tetrachloroate aqueous solution to the cerium oxide nanosphere dispersion system under stirring, adjusting the pH to 7, and stirring for 1 hour in the dark; centrifuging the system after stirring in the dark, washing 3 times with ultrapure water, and drying to obtain gold-modified cerium oxide nanospheres.

[0051] Step 3: providing gold-modified cerium oxide drug-loaded nano-microspheres, specifically comprising: re-dispersing the gold-modified cerium oxide nano-microspheres in ultrapure water, then adding 30 mL of a 14 mg / mL cisplatin aqueous solution, stirring for 24 hours in a dark place; centrifuging the system after stirring in a dark place, washing three times with ultrapure water, and drying to obtain gold-modified cerium oxide drug-loaded nano-microspheres.

[0052] The physical and chemical properties of the gold-modified cerium oxide drug-loaded nano-microspheres of this example are basically consistent with those of Example 1. Example 9

[0053] This example is a comparative example. A method for preparing gold-modified cerium oxide nanoparticles for treating breast cancer includes: Step 1: Provide cerium oxide nano-microspheres, specifically including: dissolving 6g of template polyvinyl pyrrolidone in 300mL of ethylene glycol solution under magnetic stirring at room temperature to obtain an ethylene glycol solution of the template; the relative molecular mass of the polyvinyl pyrrolidone is 8000Da; the ethylene glycol solution is an aqueous solution of ethylene glycol with a volume percentage of 75%; adding 8g of cerium nitrate tetrahydrate to the ethylene glycol solution of the template, ultrasonicating until dissolved, adjusting the pH to 7 with a 1mM HCl solution, stirring for 3h, and obtaining a mixed system A; transferring the mixed system A to a hydrothermal autoclave, reacting at 160°C for 4h, cooling and centrifuging, and collecting the precipitate. Wash the precipitate with ultrapure water and anhydrous ethanol 3 times each, and dry it to obtain powdered cerium oxide nano-microspheres; Step 2: providing gold-modified cerium oxide nanospheres, specifically comprising: dispersing the cerium oxide nanospheres in 60 mL of ultrapure water to obtain a cerium oxide nanosphere dispersion system with a concentration of 1 mg / mL; adding 30 mL of a 2 mg / mL gold tetrachloroate aqueous solution to the cerium oxide nanosphere dispersion system under stirring, adjusting the pH to 10, and stirring for 8 hours in the dark; centrifuging the system after stirring in the dark, washing 3 times with ultrapure water, and drying to obtain gold-modified cerium oxide nanospheres.

[0054] Performance Evaluation

[0055] Figure 8 This is a schematic diagram of the O2 production performance of gold-modified cerium oxide drug-loaded nanoparticles in hydrogen peroxide solution in Example 9. Figure 8 It can be seen that O2 is continuously released within 20 minutes, and there is a burst of oxygen release in the gold-modified cerium oxide nanoparticles. The surface gold modification can effectively improve the CAT enzyme activity of cerium oxide nanoparticles.

[0056] Fig. 9 The in vitro photothermal effect test results of gold-modified cerium oxide drug-loaded nanoparticles in Example 9. Fig. 9 It can be seen that pure cerium oxide nanoparticles have basically no photothermal conversion ability. Under NIR irradiation, the PBS solution dispersed with gold-modified cerium oxide drug-loaded nanoparticles can reach the expected photothermal temperature of 45°C in 150s, indicating that the gold-modified cerium oxide nanoparticles of the present invention have excellent photothermal effect.

[0057] Fig.10 This is a schematic diagram of the GSH oxidation-promoting ability of gold-modified cerium oxide drug-loaded nanoparticles with and without NIR irradiation in Example 9. The results show that cerium oxide nanoparticles have a certain ability to promote GSH oxidation, and the ability of gold-modified cerium oxide nanoparticles to promote GSH oxidation is greatly improved; NIR irradiation can further promote GSH oxidation.

[0058] The cytological experiment of gold-modified cerium oxide drug-loaded nanoparticles in this example is as follows Fig.11 and Fig.12 As shown, it is based on gold-modified cerium oxide drug-loaded nano-microspheres as the research object to investigate their efficacy in inhibiting or killing cancer, and the test method is the same as that in Example 1.

Claims

1. A method for preparing gold-modified cerium oxide drug-loaded nanoparticles, characterized in that The steps include: (1) Synthesizing cerium oxide nanoparticles, specifically comprising: dissolving a template in an ethylene glycol solution to obtain an ethylene glycol solution of the template, wherein the template is selected from one or more of polyvinyl pyrrolidone, polyethylene glycol, hexadecyltrimethylammonium bromide and polyvinyl alcohol; dispersing a soluble cerium salt in the ethylene glycol solution of the template, and adjusting the pH value to 5-7 to obtain a mixed solution; transferring the mixed solution to a closed reactor, reacting it at 100-200° C., and collecting the precipitate to obtain cerium oxide nanoparticles; (2) preparing gold-modified cerium oxide nanoparticles, specifically comprising: dispersing the above-mentioned cerium oxide nanoparticles in ultrapure water to obtain a cerium oxide nanoparticle dispersion system; adding an aqueous gold tetrachloroate solution to the cerium oxide nanoparticle dispersion system, adjusting the pH value to 7-11, stirring in the dark and collecting the precipitate to obtain gold-modified cerium oxide nanoparticles; (3) Preparing gold-modified cerium oxide drug-loaded nanoparticles, specifically comprising: re-dispersing the gold-modified cerium oxide nanoparticles in ultrapure water, then adding an aqueous solution containing a chemotherapeutic drug, stirring thoroughly in the dark, and collecting the precipitate to obtain gold-modified cerium oxide drug-loaded nanoparticles.

2. The preparation method according to claim 1, characterized in that: In step (1), the concentration of the template agent in the ethylene glycol aqueous solution is 0.01-0.2 g / mL, and the concentration of ethylene glycol in the ethylene glycol aqueous solution is 60-80%.

3. The preparation method according to claim 1, characterized in that: In step (1), the soluble cerium salt is selected from one or more of cerium nitrate and cerium chloride.

4. The preparation method according to claim 1, characterized in that: In step (1), the mass of the soluble cerium salt is 1 to 10 times the mass of the template.

5. The preparation method according to claim 1, characterized in that: In step (2), the concentration of cerium oxide nanoparticles in the cerium oxide nanoparticle dispersion system is 1-200 mg / mL.

6. The preparation method according to claim 1, characterized in that: In step (2), the mass ratio of the cerium oxide nanospheres to gold tetrachloride is (0.1-10):(1-100).

7. The preparation method according to claim 1, characterized in that: In step (3), the chemotherapy drug is selected from one or more of cisplatin, doxorubicin, etoposide and methotrexate.

8. Gold-modified cerium oxide drug-loaded nanoparticles prepared by the method according to any one of claims 1 to 7.

9. Use of the gold-modified cerium oxide drug-loaded nano-microspheres according to claim 8 in the preparation of drugs for photothermal therapy of breast cancer.

Images