Cerium-based up-conversion nano-enzyme composite material and preparation method thereof

Abstract

The invention discloses a cerium-based upconversion nano-enzyme composite material and a preparation method thereof, and belongs to the technical field of medical nano-carriers. The problems that an existing up-conversion nano material is single, limited in treatment effect and single in function are solved. According to the method, Au is obtained after reaction of tetrachloroauric acid and sodium citrate, Au-PVP is obtained after reaction of Au and polyvinylpyrrolidone, Au-PVP, citric acid, a surfactant, sodium salicylate, cerium chloride, ytterbium chloride, thulium chloride and hexamethylenetetramine are subjected to reaction to obtain Au-coated DMCeO2: Yb / Tm, glucose oxidase is loaded, and the gold-coated DMCeO2: Yb / Tm / Au-coated DMCeO2: Yb / Tm / Au-coated DMCeO2: Yb / Tm / Au-coated DMCeO2: Yb / Tm / Au-coated DMCeO2 is obtained. And finally, dispersing the obtained CeAG into a sodium hyaluronate solution for reaction to obtain the cerium-based up-conversion nano-enzyme composite material. The composite material has a special mesoporous structure, is good in light conversion capability and nano-enzyme characteristic and low in biotoxicity, and can be subjected to specific modification and improve the targeting effect, so that the treatment effect is improved.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medical nanocarriers, and particularly relates to a cerium-based upconversion nanosenzyme composite material and a preparation method thereof. BACKGROUND

[0002] At present, cancer is still one of the main killers threatening human life and health, and with the rapid development of nanotechnology and nanomedicine, a large number of treatment strategies have been created for tumor treatment.

[0003] Compared with conventional treatment methods such as surgery, chemotherapy, and radiotherapy, optical therapy has become the focus of attention due to its advantages of non-invasive, precision, spatiotemporal controllability, and low toxicity to the body. Among them, rare earth upconversion luminescence nanomaterials can realize the control of light emission from near-infrared to visible to ultraviolet, and have been widely used in the field of tumor optical therapy. However, the function of rare earth upconversion nanomaterials is single, and they can only be used as light conversion reagents and cannot be used as drug carriers. Moreover, the single treatment method cannot completely achieve the ideal treatment effect. For example, in the prior art, Indian patent application IN202421051746A discloses a novel upconversion nanoparticle and its synthesis and application. The chemical formula of the novel upconversion nanoparticle is NaMF3:(L)3, wherein M is a metal selected from at least one alkaline earth metal and at least one transition metal; L is a lanthanide selected from lanthanum (La), cerium (Ce), praseodymium (Pr), neodymium (Nd), promethium (Pm), samarium (Sm), europium (Eu), gadolinium (Gd), terbium (Tb), dysprosium (Dy), holmium (Ho), erbium (Er), thulium (Tm), ytterbium (Yb), lutetium (Lu), and combinations thereof, and (L)3 is doped in NaMF3; and further comprising at least one coating agent selected from polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), poly(ethylene-vinyl acetate), poly(lactic-glycolic acid) (PLGA), poly(ethylene glycol) (PEG), poly(N-isopropylacrylamide) (PNIPAM), metal coating, gold, silver, polyacrylic acid, polyacrylamide, carboxymethyl cellulose (CMC), dextran, gelatin, chitosan, amylopectin, polyethyleneimine, fatty acid, oleic acid, linoleic acid, palmitic acid, myristic acid, citrate, alendronate, ethylenediamine, silicon dioxide, and combinations thereof. The upconversion nanoparticles are tested to have a cubic structure and no large pores on the surface, which cannot function as drug carriers. For another example, Chinese patent application CN119909041A discloses a rare earth upconversion nanoparticle and its preparation method and application. The preparation method comprises the following steps: mixing sodium trifluoroacetate, ytterbium trifluoroacetate, yttrium trifluoroacetate, and thulium trifluoroacetate, adding oleic acid and 1-octadecene, crystallizing after heating, and then dispersing in cyclohexane to obtain cubic phase nanoparticle aC solution; mixing oleic acid, 1-octadecene, and the prepared cubic phase nanoparticle aC solution, adding sodium trifluoroacetate, crystallizing after heating, and then dispersing in cyclohexane to obtain core nanoparticle βC solution; mixing oleic acid, 1-octadecene, and the prepared core nanoparticle βC solution, adding sodium trifluoroacetate and yttrium trifluoroacetate, crystallizing after heating, and then dispersing in cyclohexane to obtain the core-shell structure nanoparticle βCS solution; and the core-shell structure nanoparticle βCS is modified by BF4 ligand to prepare water-soluble nanoparticles, i.e. the rare earth upconversion nanoparticles. The upconversion nanoparticles are tested to have a hexagonal phase structure and no large pores, which cannot function as drug carriers.At the same time, it does not have catalytic ability and cannot perform the function of nanozymes.

[0004] Therefore, designing an upconversion nanocomposite material that combines the functions of a carrier, catalyst, and therapeutic agent is of great significance for radical treatment of primary tumors and prevention of cancer metastasis. Summary of the Invention

[0005] To address the limitations of existing rare-earth upconversion nanomaterials in terms of simple functionality and limited single therapeutic effects, this invention proposes a cerium-based upconversion nanoenzyme composite material and its preparation method.

[0006] The technical solution adopted by the present invention to solve the above-mentioned technical problems is as follows.

[0007] In a first aspect, the present invention provides a method for preparing a cerium-based upconversion nanozyme composite material (CeAGH), comprising: S1. Add tetrachloroauric acid and sodium citrate to boiling deionized water and react. Centrifuge, wash and dry the product after reaction to obtain Au. S2. Au was dispersed in deionized water, polyvinylpyrrolidone was added, and the mixture was stirred at room temperature. The product was centrifuged, washed and dried to obtain Au-PVP. S3. Disperse Au-PVP in deionized water, add citric acid and surfactant, dissolve by sonication, then add sodium salicylate, cerium chloride, ytterbium chloride, thulium chloride and cyclohexamethylenetetramine to react. Centrifuge, wash and dry the brownish-yellow turbid liquid obtained from the reaction, and calcine the obtained solid to obtain Au@DMCeO2:Yb / Tm; S4. Disperse Au@DMCeO2:Yb / Tm in deionized water, add glucose oxidase, stir the reaction at room temperature, centrifuge, wash and dry the product to obtain CeAG; S5. Disperse CeAG in sodium hyaluronate solution and react. Centrifuge, wash and dry the product after reaction to obtain CeAGH.

[0008] Preferably, in S1, the ratio of tetrachloroauric acid to sodium citrate is (10-25) μmol: (5-15) mg.

[0009] Preferably, in step S1, the tetrachloroauric acid is added in the form of a tetrachloroauric acid solution with a concentration of 0.05-0.1M; the sodium citrate is added in the form of a sodium citrate solution with a concentration of 1-1.5wt%. More preferably, in S1, the volume ratio of the deionized water, tetrachloroauric acid solution and sodium citrate solution is 50 mL: (200-250) μL: (0.5-1) mL.

[0010] Preferably, in S1, the reaction time is 30-40 minutes.

[0011] Preferably, in S2, the mass ratio of Au to polyvinylpyrrolidone is (2-5):(1-2). More preferably, the ratio of deionized water to Au is 50 mL: (2-5) mg.

[0012] Preferably, in S2, the reaction time is 1-2 hours.

[0013] Preferably, in S3, the surfactant is hexadecyltrimethylammonium bromide.

[0014] Preferably, in S3, the mass ratio of Au-PVP, citric acid, surfactant, sodium salicylate, cerium chloride, ytterbium chloride, thulium chloride and cyclohexamethylenetetramine is (2-5):(10-15):(150-200):(100-120):(150-200):(18-23):(1.4-1.8):(30-35).

[0015] Preferably, in step S3, the reaction temperature is 70°C. o C-75 o C, the reaction time is 6-7 hours.

[0016] Preferably, in step S3, the calcination conditions are: 600-700 °C. o C. First, calcine with argon for 5-6 hours, then calcine with air for 2-3 hours.

[0017] Preferably, in S4, the mass ratio of Au@DMCeO2:Yb / Tm to glucose oxidase is (5-10):(5-10). More preferably, the ratio of the deionized water to Au@DMCeO2:Yb / Tm is (5-10) mL:(5-10) mg.

[0018] Preferably, in step S4, the reaction time is 12-24 hours.

[0019] Preferably, in S5, the mass ratio of CeAG to sodium hyaluronate is (20-30):(7.5-30).

[0020] Preferably, in step S5, the concentration of the sodium hyaluronate solution is 0.5-1 mg / mL.

[0021] Preferably, in step S5, the reaction time is 12-24 hours.

[0022] Secondly, the present invention also provides a cerium-based upconversion nanozyme composite material prepared by the above preparation method.

[0023] Preferably, the cerium-based upconversion nanoenzyme composite material has spherical nanoparticles with a mesoporous structure and a particle size of 200nm-250nm.

[0024] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. The cerium-based upconversion nanoenzyme composite material provided by the present invention has photoconversion capability under near-infrared light irradiation, providing photothermal effect.

[0025] 2. The cerium-based upconversion nanozyme composite material provided by the present invention has good nanozyme characteristics. It can directly catalyze the production of reactive oxygen species (ROS) from substances in tumor cells and consume glutathione (GSH). At the same time, the cerium-based upconversion nanozyme composite material itself can catalyze the production of ·OH and O2 from hydrogen peroxide overexpressed in the tumor site, and can also deplete the overexpressed GSH, further enhancing the effect of reactive oxygen species therapy.

[0026] 3. The cerium-based upconversion nanoenzyme composite material provided by the present invention has a special mesoporous structure, which can effectively support a variety of functional substances.

[0027] 4. The cerium-based upconversion nanozyme composite material provided by this invention achieves controllable regulation of the tumor microenvironment response, reducing the intrinsic biotoxicity of the material. Simultaneously, through specific modification, the targeting effect of the nanozyme complex is enhanced, thereby improving the therapeutic efficacy. Furthermore, it utilizes both the intelligence of endogenous stimulation (pH, glutathione, enzymes, etc.) and the high penetrability of exogenous stimulation (near-infrared light) to trigger the nanozyme complex to target and accumulate at the tumor site, releasing therapeutically active substances such as high-temperature radiation and ROS, activating the body's anti-tumor immune response to eradicate the tumor and prevent its metastasis and recurrence. Attached Figure Description

[0028] Figure 1 Scanning electron microscope image of CeAGH prepared in Example 1; Figure 2 The N2 adsorption-desorption isotherms of Au@DMCeO2:Yb / Tm, CeAG and CeAGH prepared in Example 1; Figure 3 Pore ​​size distribution diagrams of Au@DMCeO2:Yb / Tm, CeAG, and CeAGH prepared in Example 1; Figure 4 The graph shows the changes in equivalent series resistance (ESR) of a solution containing CeAGH prepared in Example 1 after treatment under different conditions. Figure 5 The graph shows the change in absorbance of a solution containing CeAGH prepared in Example 1 after treatment under different conditions. Figure 6The curves showing the change of O2 concentration in solutions containing Au@DMCeO2:Yb / Tm and CeAGH prepared in Example 1 over time after the addition of H2O2; Figure 7 The graph shows the change in the absorption peak of reduced GSH in the solution over time after reacting with reduced glutathione (GSH) in a solution containing CeAGH prepared in Example 1. Figure 8 Temperature changes of aqueous solutions of CeAGH prepared in Example 1 with different concentrations after illumination for a certain period of time. Detailed Implementation

[0029] To further understand the present invention, preferred embodiments of the present invention are described below. However, it should be understood that these descriptions are only for further illustrating the features and advantages of the present invention, and not for limiting the scope of the claims of the present invention.

[0030] The preparation method of the cerium-based upconversion nanoenzyme composite material of the present invention comprises the following steps: S1. Add tetrachloroauric acid and sodium citrate to boiling deionized water and react. Centrifuge, wash and dry the product after reaction to obtain Au. S2. Au was dispersed in deionized water, polyvinylpyrrolidone was added, and the mixture was stirred at room temperature. The product was centrifuged, washed and dried to obtain Au-PVP. S3. Disperse Au-PVP in deionized water, add citric acid and surfactant, dissolve by sonication, then add sodium salicylate, cerium chloride, ytterbium chloride, thulium chloride and cyclohexamethylenetetramine to react. Centrifuge, wash and dry the brownish-yellow turbid liquid obtained from the reaction, and calcine the obtained solid to obtain Au@DMCeO2:Yb / Tm; S4. Disperse Au@DMCeO2:Yb / Tm in deionized water, add glucose oxidase, stir the reaction at room temperature, centrifuge, wash and dry the product to obtain CeAG; S5. Disperse CeAG in sodium hyaluronate solution and react. Centrifuge, wash and dry the product after reaction to obtain CeAGH.

[0031] In S1 of this invention, the preferred ratio of tetrachloroauric acid to sodium citrate is (10-25) μmol:(5-15) mg; for example, in some embodiments, 10 μmol, 15 μmol, 20 μmol, or 25 μmol of tetrachloroauric acid are used, and 5 mg, 10 mg, or 15 mg of sodium citrate are used. Preferably, tetrachloroauric acid is added in the form of a tetrachloroauric acid solution, with water as the solvent, and the concentration of the tetrachloroauric acid solution is 0.05-0.1 M, for example, 0.05 M, 0.06 M, 0.07 M, 0.08 M, 0.09 M, or 0.1 M are used in some embodiments. Sodium citrate is added in the form of a sodium citrate solution, with water as the solvent, and the concentration of the sodium citrate solution is 1-1.5 wt%, for example, 1 wt%, 1.1 wt%, 1.2 wt%, 1.3 wt%, 1.4 wt%, or 1.5 wt% are used in some embodiments. The preferred volume ratio of deionized water, tetrachloroauric acid solution, and sodium citrate solution is 50 mL:(200-250) μL:(0.5-1) mL, but it is not limited to this. The preferred reaction time is 30-40 min.

[0032] In S2 of this invention, the preferred mass ratio of Au to polyvinylpyrrolidone is (2-5):(1-2); for example, in some embodiments, 2:1, 2:2, 3:1, 3:2, 4:1, 5:1, and 5:2 are used. The preferred ratio of deionized water to Au is 50 mL:(2-5) mg, but is not limited thereto. The preferred reaction time is 1-2 h.

[0033] In S3 of this invention, the preferred surfactant is hexadecyltrimethylammonium bromide. The preferred mass ratio of Au-PVP, citric acid, surfactant, sodium salicylate, cerium chloride, ytterbium chloride, thulium chloride, and cyclohexamethylenetetramine is (2-5):(10-15):(150-200):(100-120):(150-200):(18-23):(1.4-1.8):(30-35). In some embodiments, by mass parts, Au-PVP is used in amounts of 2, 3, 4, or 5 parts; citric acid is used in amounts of 10, 11, 12, 13, 14, or 15 parts; and the surfactant is used in amounts of... The following amounts of sodium salicylate are used: 150 parts, 160 parts, 170 parts, 180 parts, 190 parts, and 200 parts; sodium salicylate is used: 100 parts, 105 parts, 110 parts, 115 parts, and 120 parts; cerium chloride is used: 150 parts, 160 parts, 170 parts, 180 parts, 190 parts, and 200 parts; ytterbium chloride is used: 18 parts, 19 parts, 20 parts, 21 parts, 22 parts, and 23 parts; thulium chloride is used: 1.4 parts, 1.5 parts, 1.6 parts, 1.7 parts, and 1.8 parts; and cyclohexamethylenetetramine is used: 30 parts, 31 parts, 32 parts, 33 parts, 34 parts, and 35 parts. The preferred reaction temperature is 70°C. o C-75 oC, the reaction time is 6-7 hours. The preferred calcination conditions are: 600-700 °C. o C. First, calcine with argon for 5-6 hours, then calcine with air for 2-3 hours.

[0034] In S4 of this invention, the preferred mass ratio of Au@DMCeO2:Yb / Tm to glucose oxidase is (5-10):(5-10); for example, in some embodiments, the mass fractions of Au@DMCeO2:Yb / Tm are 5, 6, 7, 8, 9, or 10 parts, and the mass fractions of glucose oxidase are 5, 6, 7, 8, 9, or 10 parts. The preferred ratio of deionized water to Au@DMCeO2:Yb / Tm is (5-10) mL:(5-10) mg. The preferred reaction time is 12-24 h.

[0035] In S5 of this invention, the preferred mass ratio of CeAG to sodium hyaluronate is (20-30):(7.5-30); for example, in some embodiments, the CeAG is 20 parts, 22 parts, 25 parts, 28 parts, or 30 parts by mass, and the sodium hyaluronate is 7.5 parts, 10 parts, 15 parts, 20 parts, 25 parts, or 30 parts by mass. The concentration of the sodium hyaluronate solution is preferably 0.5-1 mg / mL, and water is typically used as the solvent. The preferred reaction time is 12-24 hours.

[0036] It should be noted that there are no special limitations on centrifugation, washing, and drying in this invention, as long as the properties of the product itself are not damaged. Freeze-drying can be used for drying.

[0037] The present invention also provides a cerium-based upconversion nanoenzyme composite material prepared by the above preparation method. This cerium-based upconversion nanoenzyme composite material has spherical nanoparticles with a mesoporous structure and a particle size of 200 nm-250 nm.

[0038] To enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings.

[0039] In the following embodiments, various processes and methods not described in detail are conventional methods known in the art. Unless otherwise specified, the materials, reagents, apparatus, instruments, equipment, etc., used in the following embodiments are commercially available.

[0040] Example 1 S1. Preparation of Au Add 50 mL of deionized water to a 100 mL three-necked flask, heat to boiling, then add 200 μL of 0.05 mol / L tetrachloroauric acid aqueous solution and 0.5 mL of 1 wt% sodium citrate aqueous solution, react for 0.5 h, centrifuge, wash and dry the product after reaction to obtain Au; S2. Preparation of Au-PVP Disperse 2 mg Au in 50 mL of deionized water, add 1 mg of polyvinylpyrrolidone, stir the reaction at room temperature for 1.5 h, centrifuge, wash and dry the product to obtain Au-PVP; S3. Preparation of Au@DMCeO2:Yb / Tm Add 100 mL of deionized water to a 100 mL three-necked flask, then add 10 mg of citric acid and 200 mg of cetyltrimethylammonium bromide. After sonicating to dissolve, add 120 mg of sodium salicylate, 2 mg of Au-PVP, 150 mg of cerium chloride, 18 mg of ytterbium chloride, 1.4 mg of thulium chloride, and 30 mg of cyclohexamethylenetetramine. Then raise the temperature to 70 °C. o C. Stir the reaction for 7 hours, then centrifuge, wash and dry the product to obtain a brownish-yellow powder. The brownish-yellow powder was subjected to nitrogen atmosphere for 600 hours. o After calcining C for 5 hours, it was then calcined in air for 2 hours. The product was collected to obtain Au@DMCeO2:Yb / Tm. S4. Preparation of CeAG 5 mg Au@DMCeO2:Yb / Tm was ultrasonically dispersed in 5 mL of deionized water, and then 5 mg glucose oxidase powder was added. The mixture was stirred at room temperature for 12 h. The product was centrifuged, washed and freeze-dried to obtain CeAG. S5. Preparation of CeAGH 20 mg CeAG was dispersed in 15 mL of 0.5 mg / mL sodium hyaluronate solution. After stirring and reacting at room temperature for 12 h, the product was centrifuged, washed, and freeze-dried to obtain a cerium-based upconversion nanozyme composite material, denoted as CeAGH.

[0041] The morphology, structure and surface composition of CeAGH prepared in Example 1 were characterized.

[0042] Figure 1 The scanning electron microscope image of CeAGH prepared in Example 1 shows that CeAGH is a spherical nanoparticle with a mesoporous structure and a particle size of about 200 nm.

[0043] Figure 2 The N2 adsorption-desorption isotherms are for Au@DMCeO2:Yb / Tm, CeAG, and CeAGH prepared in Example 1. Figure 3 The image shows the pore size distribution of Au@DMCeO2:Yb / Tm, CeAG, and CeAGH prepared in Example 1. From... Figure 2 and Figure 3It can be seen that after loading glucose oxidase, the pore size and specific surface area of ​​the material decreased significantly, and the gas adsorption capacity also decreased accordingly. After modifying with hyaluronic acid, the pore size and specific surface area of ​​the material decreased again, and the gas adsorption capacity also decreased further, further proving that the CeAGH material was successfully synthesized.

[0044] The CeAGH prepared in Example 1 was characterized in terms of performance.

[0045] The CeAGH prepared in Example 1 was prepared into an aqueous solution with a concentration of 1 mg / mL. The solution was then divided into two equal volumes. One volume was treated with hydrogen peroxide (H2O2) to a final concentration of 1 mM as the experimental group, and the other volume was treated as the blank group. H2O2 was used as the control group. After standing for 10 minutes, the ESR curves of the experimental group, blank group, and control group were detected. The results are as follows: Figure 4 As shown. From Figure 4 It can be seen that almost no hydroxyl radicals (·OH) are generated when no H2O2 is added; conversely, the generation of ·OH is observed after the addition of H2O2. This confirms that CeAGH can catalyze the production of highly toxic ·OH from H2O2, and confirms that it has peroxidase activity, a characteristic that enables it to kill tumor cells.

[0046] Using H2O2 as the blank control, PBS buffer at pH 7.4 as control group 1 (pH 7.4), and acetate-sodium acetate buffer at pH 5.5 as control group 2 (pH 5.5), CeAGH prepared in Example 1 was prepared into a CeAGH solution with a concentration of 1 mg / mL using PBS buffer at pH 7.4 (CeAGH (pH 7.4)). CeAGH prepared in Example 1 was prepared into a CeAGH solution with a concentration of 1 mg / mL using acetate-sodium acetate buffer at pH 5.5 (CeAGH (pH 5.5)). CeAGH prepared in Example 1 was prepared into a CeAGH solution with a concentration of 1 mg / mL using PBS buffer at pH 7.4, and H2O2 was added to a final concentration of 1 mM (H2O2 + CeAGH (pH 7.4)). A CeAGH solution with a concentration of 1 mg / mL was prepared using acetate-sodium acetate buffer at pH 5.5, and H₂O₂ was added to a final concentration of 1 mM (H₂O₂ + CeAGH (pH 5.5)). 3,3',5,5'-Tetramethylbenzidine (TMB) was added to all solutions as an indicator at a final concentration of 10 μg / mL. After incubation for 10 min, the changes in absorbance of each solution were measured. The results are as follows: Figure 5 As shown. From Figure 5 As can be seen, the results are consistent with the ESR results, further confirming the material's ability to generate ·OH.

[0047] Au@DMCeO2:Yb / Tm and CeAGH prepared in Example 1 were each prepared into aqueous solutions with a concentration of 1 mg / mL. H2O2 was added to each solution to a final concentration of 1 mM, with H2O used as a control group. The changes in dissolved oxygen content in the solutions over time were measured, and the results are as follows: Figure 6 As shown. From Figure 6 As can be seen, both Au@DMCeO2:Yb / Tm and CeAGH can catalyze the production of O2 from H2O2, but the modification with hyaluronic acid has a certain impact on the material's ability to produce O2.

[0048] The CeAGH prepared in Example 1 was prepared into an aqueous solution with a concentration of 1 mg / mL. GSH was added to a final concentration of 1 mM. The UV spectra of the solution were measured after 0.5 h, 1 h, 2 h, 4 h, 6 h, and 8 h, respectively. The results are as follows: Figure 7 As shown. Figure 7 This indicates that CeAGH can remove GSH to some extent.

[0049] The CeAGH prepared in Example 1 was prepared into aqueous solutions with concentrations of 0 mg / mL, 125 mg / mL, 250 mg / mL, 500 mg / mL, and 1000 mg / mL. The temperature changes of the solutions were measured under 808 nm laser illumination, and the results are as follows. Figure 8 As shown. Figure 8 This demonstrates that CeAGH has good photothermal effects, and the higher the concentration, the better the photothermal effect.

[0050] In summary, the cerium-based upconversion nanozyme composite material of this invention, under near-infrared light irradiation, can not only act as a photothermal conversion reagent, providing photothermal effects, but also as a photocatalytic reagent, generating a wide variety of reactive oxygen species (ROS). Simultaneously, the cerium-based upconversion nanozyme composite material itself can catalyze the production of ·OH and O2 from hydrogen peroxide overexpressed at tumor sites, and can also deplete overexpressed GSH, further enhancing the therapeutic effect of ROS. Furthermore, the unique structure also endows the composite material with the function of a nanocarrier. Utilizing the photothermal effect and the generated ROS, multimodal combined therapy based on phototherapy can be achieved. In addition, the nanozyme composite material can also serve as a photothermal and upconversion fluorescence imaging reagent, enabling material localization and guiding tumor treatment.

[0051] Obviously, the above embodiments are merely preferred examples for clear illustration and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for preparing cerium-based upconversion nanoenzyme composite materials, characterized in that, include: S1. Add tetrachloroauric acid and sodium citrate to boiling deionized water and react. Centrifuge, wash and dry the product after reaction to obtain Au. S2. Au was dispersed in deionized water, polyvinylpyrrolidone was added, and the mixture was stirred at room temperature. The product was centrifuged, washed and dried to obtain Au-PVP. S3. Disperse Au-PVP in deionized water, add citric acid and surfactant, dissolve by sonication, then add sodium salicylate, cerium chloride, ytterbium chloride, thulium chloride and cyclohexamethylenetetramine to react. Centrifuge, wash and dry the brownish-yellow turbid liquid obtained from the reaction, and calcine the obtained solid to obtain Au@DMCeO2:Yb / Tm; S4. Disperse Au@DMCeO2:Yb / Tm in deionized water, add glucose oxidase, stir the reaction at room temperature, centrifuge, wash and dry the product to obtain CeAG; S5. CeAG was dispersed in sodium hyaluronate solution and reacted. The product after reaction was centrifuged, washed and dried to obtain cerium-based upconversion nanoenzyme composite material.

2. The method for preparing the cerium-based upconversion nanoenzyme composite material according to claim 1, characterized in that, S1 possesses one or more of the following characteristics: The ratio of tetrachloroauric acid to sodium citrate is (10-25) μmol: (5-15) mg; The tetrachloroauric acid is added in the form of a tetrachloroauric acid solution with a concentration of 0.05-0.1M; The sodium citrate is added in the form of a sodium citrate solution with a concentration of 1-1.5 wt%. The reaction time is 30-40 minutes.

3. The method for preparing the cerium-based upconversion nanoenzyme composite material according to claim 1, characterized in that, S2 possesses one or more of the following characteristics: The mass ratio of Au to polyvinylpyrrolidone is (2-5):(1-2); The reaction time is 1-2 hours.

4. The method for preparing the cerium-based upconversion nanoenzyme composite material according to claim 3, characterized in that, The ratio of deionized water to Au is 50 mL: (2-5) mg.

5. The method for preparing the cerium-based upconversion nanoenzyme composite material according to claim 1, characterized in that, S3 possesses one or more of the following characteristics: The surfactant is hexadecyltrimethylammonium bromide; The mass ratio of Au-PVP, citric acid, surfactant, sodium salicylate, cerium chloride, ytterbium chloride, thulium chloride, and cyclohexamethylenetetramine is (2-5):(10-15):(150-200):(100-120):(150-200):(18-23):(1.4-1.8):(30-35). The reaction temperature is 70°C. o C-75 o C, the reaction time is 6-7 hours; The calcination conditions are: at 600-700°C. o C. First, calcine with argon for 5-6 hours, then calcine with air for 2-3 hours.

6. The method for preparing the cerium-based upconversion nanoenzyme composite material according to claim 1, characterized in that, S4 possesses one or more of the following characteristics: The mass ratio of Au@DMCeO2:Yb / Tm to glucose oxidase is (5-10):(5-10). The reaction time is 12-24 hours.

7. The method for preparing the cerium-based upconversion nanoenzyme composite material according to claim 6, characterized in that, The ratio of deionized water to Au@DMCeO2:Yb / Tm is (5-10) mL: (5-10) mg.

8. The method for preparing the cerium-based upconversion nanoenzyme composite material according to claim 1, characterized in that, S5 possesses one or more of the following characteristics: The mass ratio of CeAG to sodium hyaluronate is (20-30):(7.5-30). The concentration of the sodium hyaluronate solution is 0.5-1 mg / mL; The reaction time is 12-24 hours.

9. The cerium-based upconversion nanozyme composite material prepared by the preparation method according to any one of claims 1-8.

10. The cerium-based upconversion nanoenzyme composite material according to claim 9 is composed of spherical nanoparticles with a mesoporous structure and a particle size of 200 nm-250 nm.

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