Cerium-based upconversion nanolzyme composite material and preparation method thereof

Abstract

The application discloses a cerium-based upconversion nanolaser composite material and a preparation method thereof, and belongs to the technical field of medical nanocarriers.The application solves the problems of limited single treatment effect and single function of the existing upconversion nanomaterials.The method comprises the following steps: firstly, reacting tetrachloroauric acid and sodium citrate to obtain Au;secondly, reacting Au and polyvinylpyrrolidone to obtain Au-PVP;thirdly, reacting Au-PVP, citric acid, a surfactant, sodium salicylate, cerium chloride, ytterbium chloride, thulium chloride and cyclenexylmethyl tetraamine to obtain Au@DMCeO2:Yb / Tm;fourthly, loading glucose oxidase;and finally, dispersing the obtained CeAG into a sodium hyaluronate solution to react, so as to obtain the cerium-based upconversion nanolaser composite material.The composite material has a special mesoporous structure, good light conversion capacity and nanolaser characteristics, low biological toxicity, can be specifically modified, and can improve the targeting effect, so as to improve the treatment effect.

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 also does not have catalytic ability and cannot complete the function of nano-enzyme.

[0004] Therefore, it is of great significance to design an up-conversion nano-composite material with the functions of carrier, catalysis and therapeutic agent to radically cure primary tumors and prevent cancer metastasis. SUMMARY

[0005] In order to solve the problems of simple function and limited single treatment effect of the existing rare earth up-conversion nano material, the application provides a cerium-based up-conversion nano-enzyme composite material and a preparation method thereof.

[0006] The technical solution adopted by the application to solve the above technical problems is as follows.

[0007] In a first aspect, the application provides a preparation method of a cerium-based up-conversion nano-enzyme composite material (CeAGH), comprising:

[0008] S1. Adding tetrachloroauric acid and sodium citrate into boiling deionized water to react, centrifuging, washing and drying the product after reaction to obtain Au;

[0009] S2. Dispersing Au in deionized water, adding polyvinylpyrrolidone, stirring and reacting at room temperature, centrifuging, washing and drying the product to obtain Au-PVP;

[0010] S3. Dispersing Au-PVP in deionized water, adding citric acid and a surfactant, ultrasonic dissolving, then adding sodium salicylate, cerium chloride, ytterbium chloride, thulium chloride and cyclen to react, centrifuging, washing and drying the brownish yellow turbid liquid obtained by reaction to obtain Au@DMCeO2:Yb / Tm, and calcining the obtained solid to obtain Au@DMCeO2:Yb / Tm;

[0011] S4. Dispersing Au@DMCeO2:Yb / Tm in deionized water, adding glucose oxidase, stirring and reacting at room temperature, centrifuging, washing and drying the product to obtain CeAG;

[0012] S5. Dispersing CeAG in sodium hyaluronate solution to react, centrifuging, washing and drying the product after reaction to obtain CeAGH.

[0013] Preferably, in S1, the ratio of tetrachloroauric acid to sodium citrate is (10-25) μmol:(5-15) mg.

[0014] Preferably, in S1, the tetrachloroauric acid is added in the form of a tetrachloroauric acid solution, and the concentration of the tetrachloroauric acid solution is 0.05-0.1 M; the sodium citrate is added in the form of a sodium citrate solution, and the concentration of the sodium citrate solution is 1-1.5 wt%;

[0015] 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.

[0016] Preferably, in S1, the reaction time is 30-40 minutes.

[0017] Preferably, in S2, the mass ratio of Au to polyvinylpyrrolidone is (2-5):(1-2).

[0018] More preferably, the ratio of deionized water to Au is 50 mL: (2-5) mg.

[0019] Preferably, in S2, the reaction time is 1-2 hours.

[0020] Preferably, in S3, the surfactant is hexadecyltrimethylammonium bromide.

[0021] 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).

[0022] Preferably, in step S3, the reaction temperature is 70°C. o C-75 o C, the reaction time is 6-7 hours.

[0023] 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.

[0024] Preferably, in S4, the mass ratio of Au@DMCeO2:Yb / Tm to glucose oxidase is (5-10):(5-10).

[0025] More preferably, the ratio of the deionized water to Au@DMCeO2:Yb / Tm is (5-10) mL:(5-10) mg.

[0026] Preferably, in step S4, the reaction time is 12-24 hours.

[0027] Preferably, in S5, the mass ratio of CeAG to sodium hyaluronate is (20-30):(7.5-30).

[0028] Preferably, in step S5, the concentration of the sodium hyaluronate solution is 0.5-1 mg / mL.

[0029] Preferably, the reaction time in S5 is 12-24h.

[0030] In a second aspect, the present application also provides the cerium-based upconversion nanoszyme composite material prepared by the preparation method.

[0031] Preferably, the cerium-based upconversion nanoszyme composite material has a mesoporous structure of spherical nanoparticles with a particle size of 200-250nm.

[0032] Compared with the prior art, the present application has the following beneficial effects:

[0033] 1. The cerium-based upconversion nanoszyme composite material provided by the present application has light conversion capability under near-infrared light irradiation, and provides photothermal effect.

[0034] 2. The cerium-based upconversion nanoszyme composite material provided by the present application has good nanoszyme characteristics, can directly catalyze the production of reactive oxygen species (ROS) and consumption of glutathione (GSH) in tumor cells, and at the same time, the cerium-based upconversion nanoszyme composite material itself can catalyze the overexpression of hydrogen peroxide at the tumor site to produce ·OH and O2, and also can deplete the overexpression of GSH, further enhancing the effect of reactive oxygen therapy.

[0035] 3. The cerium-based upconversion nanoszyme composite material provided by the present application has a special mesoporous structure, which can effectively carry a variety of functional substances.

[0036] 4. The cerium-based upconversion nanoszyme composite material provided by the present application realizes the controllable adjustment of the tumor microenvironment response, reduces the intrinsic biological toxicity of the material. At the same time, through specific modification, the targeting effect of the nanoszyme composite is improved, thereby improving the treatment effect. In addition, the intelligence of endogenous (pH, glutathione, enzyme, etc.) stimulation and the high penetration of exogenous (near-infrared light) stimulation are also used to trigger the nanoszyme composite that targets and accumulates at the tumor site, release high heat, ROS and other substances with therapeutic function, activate the body's anti-tumor immune response to achieve the purpose of eradicating tumors and preventing metastasis and recurrence. BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1 Scanning electron microscope photos of CeAGH prepared in Example 1;

[0038] Figure 2 N2 adsorption-desorption isotherms of Au@DMCeO2:Yb / Tm, CeAG and CeAGH prepared in Example 1;

[0039] Figure 3 Pore size distribution diagrams of Au@DMCeO2:Yb / Tm, CeAG and CeAGH prepared in Example 1;

[0040] Figure 4 Figure 6 is a graph showing the change in equivalent series resistance (ESR) of a solution containing CeAGH prepared in Example 1 after treatment under different conditions;

[0041] Figure 5 Figure 7 is a graph showing the change in absorbance of a solution containing CeAGH prepared in Example 1 after treatment under different conditions;

[0042] Figure 6 Figure 8 is a graph showing the change in O2concentration in a solution containing Au@DMCeO2:Yb / Tm and CeAGH prepared in Example 1, respectively, with time after addition of H2O2to the solution;

[0043] Figure 7 Figure 9 is a graph showing the change in absorbance of reduced GSH in a solution containing CeAGH prepared in Example 1 after reaction with reduced glutathione (GSH);

[0044] Figure 8 Figure 10 is a graph showing the change in temperature of an aqueous solution containing CeAGH prepared in Example 1 with different concentrations after illumination for a certain period of time. DETAILED DESCRIPTION

[0045] In order to further understand the present application, the preferred embodiments of the present application are described below, but it should be understood that these descriptions are only for further illustrating the features and advantages of the present application, and are not limitations on the claims of the present application.

[0046] The preparation method of the cerium-based upconversion nanoszyme composite material of the present application is as follows:

[0047] S1. Adding tetrachloroauric acid and sodium citrate into boiling deionized water to react, centrifuging, washing and drying the product after reaction to obtain Au;

[0048] S2. Dispersing Au in deionized water, adding polyvinylpyrrolidone, stirring and reacting at room temperature, centrifuging, washing and drying the product to obtain Au-PVP;

[0049] S3. Dispersing Au-PVP in deionized water, adding citric acid and a surfactant, ultrasonically dissolving, then adding sodium salicylate, cerium chloride, ytterbium chloride, thulium chloride and cyclen, reacting, centrifuging, washing and drying the brownish yellow turbid liquid obtained to obtain Au@DMCeO2:Yb / Tm, and calcining the obtained solid;

[0050] S4. Dispersing Au@DMCeO2:Yb / Tm in deionized water, adding glucose oxidase, stirring and reacting at room temperature, centrifuging, washing and drying the product to obtain CeAG;

[0051] S5. The CeAG was dispersed in the sodium hyaluronate solution for reaction, and the product after reaction was centrifuged, washed and dried to obtain the CeAGH.

[0052] In S1 of the present application, the ratio of tetrachloroauric acid to sodium citrate is preferably (10-25) μmol:(5-15) mg; for example, in some embodiments, 10 μmol, 15 μmol, 20 μmol, 25 μmol of tetrachloroauric acid and 5 mg, 10 mg, 15 mg of sodium citrate are used. Preferably, tetrachloroauric acid is added in the form of a tetrachloroauric acid solution, the solvent is water, 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, 0.1 M are used in some embodiments. Sodium citrate is added in the form of a sodium citrate solution, the solvent is water, 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%, 1.5 wt% are used in some embodiments. Preferably, the volume ratio of deionized water, tetrachloroauric acid solution and sodium citrate solution is 50 mL:(200-250) μL:(0.5-1) mL, but is not limited thereto. Preferably, the reaction time is 30-40 min.

[0053] In S2 of the present application, the mass ratio of Au to polyvinylpyrrolidone is preferably (2-5):(1-2); for example, 2:1, 2:2, 3:1, 3:2, 4:1, 5:1, 5:2 are used in some embodiments. Preferably, the ratio of deionized water to Au is 50 mL:(2-5) mg, but is not limited thereto. Preferably, the reaction time is 1-2 h.

[0054] In S3 of the present application, the surfactant is preferably hexadecyl trimethyl ammonium bromide. The mass ratio of Au-PVP, citric acid, surfactant, sodium salicylate, cerium chloride, ytterbium chloride, thulium chloride and cyclen is preferably (2-5):(10-15):(150-200):(100-120):(150-200):(18-23):(1.4-1.8):(30-35); in some embodiments, the Au-PVP is 2 parts, 3 parts, 4 parts, 5 parts, the citric acid is 10 parts, 11 parts, 12 parts, 13 parts, 14 parts, 15 parts, the surfactant is 150 parts, 160 parts, 170 parts, 180 parts, 190 parts, 200 parts, the sodium salicylate is 100 parts, 105 parts, 110 parts, 115 parts, 120 parts, the cerium chloride is 150 parts, 160 parts, 170 parts, 180 parts, 190 parts, 200 parts, the ytterbium chloride is 18 parts, 19 parts, 20 parts, 21 parts, 22 parts, 23 parts, the thulium chloride is 1.4 parts, 1.5 parts, 1.6 parts, 1.7 parts, 1.8 parts, and the cyclen is 30 parts, 31 parts, 32 parts, 33 parts, 34 parts, 35 parts. The reaction temperature is preferably 70 o C-75 o C, and the reaction time is 6-7h. The calcination conditions are preferably as follows: first, argon is used for calcination for 5-6h, and then air is used for calcination for 2-3h at 600-700 o C.

[0055] In S4 of the present application, the mass ratio of Au@DMCeO2:Yb / Tm and glucose oxidase is preferably (5-10):(5-10); in some embodiments, the Au@DMCeO2:Yb / Tm is 5 parts, 6 parts, 7 parts, 8 parts, 9 parts, 10 parts, and the glucose oxidase is 5 parts, 6 parts, 7 parts, 8 parts, 9 parts, 10 parts, and the ratio of deionized water to Au@DMCeO2:Yb / Tm is preferably (5-10)mL:(5-10)mg. The reaction time is preferably 12-24h.

[0056] In S5 of the present application, the mass ratio of CeAG and sodium hyaluronate is preferably (20-30):(7.5-30); in some embodiments, the CeAG is 20 parts, 22 parts, 25 parts, 28 parts, 30 parts, and the sodium hyaluronate is 7.5 parts, 10 parts, 15 parts, 20 parts, 25 parts, 30 parts. The concentration of the sodium hyaluronate solution is preferably 0.5-1mg / mL, and the solvent is usually water. The reaction time is preferably 12-24h.

[0057] It should be noted that the centrifugation, washing and drying in the present application are not particularly limited, and the product performance is not changed. The drying can be freeze-drying.

[0058] The application further provides the cerium-based upconversion nanzyme composite material prepared by the preparation method.

[0059] In order to make the technical personnel in the art better understand the technical solutions of the present application, the present application will be further described in detail below in combination with the drawings.

[0060] In the following examples, various processes and methods not described in detail are conventional methods known in the art. The materials, reagents, devices, instruments, equipment, etc. used in the following examples, if not specifically stated, can be obtained commercially.

[0061] Example 1

[0062] S1. Preparation of Au

[0063] In a 100 mL three-necked flask, 50 mL of deionized water was added and heated to boiling, then 200 μL of 0.05 mol / L aqueous tetrachloroauric acid solution and 0.5 mL of 1 wt% aqueous sodium citrate solution were added, and the reaction was carried out for 0.5 h. The product after reaction was centrifuged, washed and dried to obtain Au;

[0064] S2. Preparation of Au-PVP

[0065] 2 mg of Au was dispersed in 50 mL of deionized water, 1 mg of polyvinylpyrrolidone was added, and the reaction was carried out at room temperature for 1.5 h. The product was centrifuged, washed and dried to obtain Au-PVP;

[0066] S3. Preparation of Au@DMCeO2:Yb / Tm

[0067] In a 100 mL three-necked flask, 100 mL of deionized water was added, then 10 mg of citric acid and 200 mg of cetyltrimethylammonium bromide were added, and after ultrasonic dissolution, 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 cyclohexanehexamethylenetetramine were added, and then the temperature was increased to 70 o C and the reaction was carried out for 7 h. The product after reaction was centrifuged, washed and dried to obtain a brownish yellow powder;

[0068] The brownish yellow powder was calcined at 600 o C for 5 h, and then calcined in air for 2 h. The product was collected to obtain Au@DMCeO2:Yb / Tm;

[0069] S4. Preparation of CeAG

[0070] CeAG was prepared by dispersing 5 mg Au@DMCeO2:Yb / Tm in 5 mL deionized water, then adding 5 mg glucose oxidase powder, stirring at room temperature for 12 h, centrifuging, washing and freeze-drying the product to obtain CeAG;

[0071] S5. Preparation of CeAGH

[0072] CeAGH was prepared by dispersing 20 mg CeAG in 15 mL 0.5 mg / mL sodium hyaluronate solution, stirring at room temperature for 12 h, centrifuging, washing and freeze-drying the product to obtain a cerium-based upconversion nanoscale enzyme composite material, denoted as CeAGH.

[0073] The morphology and surface composition of CeAGH prepared in Example 1 were characterized.

[0074] Figure 1 The scanning electron microscope photograph of CeAGH prepared in Example 1 can prove that CeAGH is a spherical nanoparticle with a mesoporous structure, and the particle size is about 200 nm.

[0075] Figure 2 The N2 adsorption-desorption isotherm of Au@DMCeO2:Yb / Tm, CeAG and CeAGH prepared in Example 1. Figure 3 The pore size distribution of Au@DMCeO2:Yb / Tm, CeAG and CeAGH prepared in Example 1. Figure 2 and Figure 3 It can be seen that after loading glucose oxidase, the pore size and specific surface area of the material decrease significantly, and the gas adsorption amount also decreases accordingly. After modifying with hyaluronic acid, the pore size and specific surface area of the material decrease again, and the gas adsorption amount further decreases, further proving that the CeAGH material is successfully synthesized.

[0076] The performance of CeAGH prepared in Example 1 was characterized.

[0077] CeAGH prepared in Example 1 was configured into an aqueous solution with a concentration of 1 mg / mL, then an equal volume was divided into two parts, one part added with hydrogen peroxide (H2O2) with a final concentration of 1 mM as an experimental group, the other part as a blank group, and H2O2 was used as a control group. After 10 min, the ESR change curves of the experimental group, the blank group and the control group were detected, and the results are shown in Figure 4 It can be seen from Figure 4 that no hydroxyl radicals (·OH) are produced when no H2O2 is added; on the contrary, the production of ·OH is observed after the addition of H2O2, which confirms that CeAGH can catalyze H2O2 to produce highly toxic ·OH, confirming its peroxidase activity, which makes it able to kill tumor cells.

[0078] H2O2 as blank group, pH 7.4 PBS buffer as control group 1 (pH 7.4), pH 5.5 acetic acid-sodium acetate buffer as control group 2 (pH 5.5), the CeAGH prepared in Example 1 was configured into a solution with a CeAGH concentration of 1 mg / mL using pH 7.4 PBS buffer as the solvent (CeAGH (pH 7.4)), the CeAGH prepared in Example 1 was configured into a solution with a CeAGH concentration of 1 mg / mL using pH 5.5 acetic acid-sodium acetate buffer as the solvent (CeAGH (pH 5.5)), the CeAGH prepared in Example 1 was configured into a solution with a CeAGH concentration of 1 mg / mL using pH 7.4 PBS buffer as the solvent and 1 mM H2O2 was added (H2O2+CeAGH (pH 7.4)), the CeAGH prepared in Example 1 was configured into a solution with a CeAGH concentration of 1 mg / mL using pH 5.5 acetic acid-sodium acetate buffer as the solvent and 1 mM H2O2 was added (H2O2+CeAGH (pH 5.5)), and 10 μg / mL 3,3',5,5'-tetramethylbenzidine (TMB) was added as an indicator in all solutions, and the absorbance of each solution was detected after 10 min, and the results are shown in Figure 5 It can be seen from Figure 5 that the results are consistent with the ESR results, further confirming the ·OH production ability of the material.

[0079] The Au@DMCeO2:Yb / Tm and CeAGH prepared in Example 1 were respectively configured into aqueous solutions with a concentration of 1 mg / mL, and 1 mM H2O2 was added, and H2O was used as a control group, and the change curves of the dissolved oxygen content in the solutions with time were respectively determined, and the results are shown in Figure 6 It can be seen from Figure 6 that Au@DMCeO2:Yb / Tm and CeAGH can both catalyze H2O2 to produce O2, but the modification of hyaluronic acid has a certain influence on the O2 production ability of the material.

[0080] The CeAGH prepared in Example 1 was configured into an aqueous solution with a concentration of 1 mg / mL, and 1 mM GSH was added, and the ultraviolet spectrum of the solution was detected after 0.5 h, 1 h, 2 h, 4 h, 6 h, and 8 h, and the results are shown in Figure 7 . Figure 7 It shows that CeAGH can remove GSH to a certain extent.

[0081] The CeAGH prepared in Example 1 was configured into aqueous solutions with concentrations of 0 mg / mL, 125 mg / mL, 250 mg / mL, 500 mg / mL and 1000 mg / mL respectively, and the temperature change of the solution was measured under 808 nm laser irradiation, and the results are shown in Figure 8 Figure 8 It is proved that CeAGH has good photothermal effect and the higher the concentration, the better the photothermal effect.

[0082] In summary, the cerium-based upconversion nanosenzyme composite material of the present application can not only be used as a photothermal conversion reagent to provide photothermal effect, but also be used as a photocatalytic reagent to produce a variety of active oxygen under near-infrared light irradiation. At the same time, the cerium-based upconversion nanosenzyme composite material itself can catalyze the overexpressed hydrogen peroxide at the tumor site to produce ·OH and O2, and also can deplete the overexpressed GSH, further enhancing the effect of active oxygen therapy. In addition, the special structure also endows the composite material with the function of a nanocarrier. The photothermal effect and the generated active oxygen can realize multi-modal combination therapy based on optical therapy. In addition, the nanosenzyme composite material can also be used as a photothermal and upconversion fluorescence imaging reagent to realize the positioning of the material and guide the treatment of tumors.

[0083] Obviously, the above embodiments are only preferred examples for clearly illustrating the application, and are not intended to limit the application. Based on the above description, those skilled in the art can make other different forms of changes or modifications. Here, it is not necessary and impossible to enumerate all the embodiments. The obvious changes or modifications derived therefrom are still within the protection scope of the present application.​

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 a sodium hyaluronate solution and reacted. The product was centrifuged, washed, and dried to obtain a cerium-based upconversion nanoenzyme composite material. The surfactant is hexadecyltrimethylammonium bromide.

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 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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