A type of HYP@GOx@Fe3O4@Lf nanoparticles and their preparation and application

Abstract

This invention discloses a HYP@GOx@Fe3O4@Lf nanoparticle and its preparation and application. The preparation method includes the following steps: (1) obtaining hollow porous Fe3O4 nanoparticles; (2) obtaining citric acid-modified Fe3O4 nanoparticles; (3) obtaining HYP@GOx@Fe3O4 nanoparticles; (4) obtaining HYP@GOx@Fe3O4@Lf nanoparticles. This invention provides the application of the HYP@GOx@Fe3O4@Lf nanoparticles in the preparation of drugs for treating glioma. The HYP@GOx@Fe3O4@Lf nanoparticles synthesized in this invention, as nanomedicines, can cut off the energy supply of glioma and inhibit tumor growth; compared with using a single treatment method to treat glioma, the effect is stronger, and it is not limited by the blood-brain barrier, which can greatly improve the treatment effect.

Description

Technical Field

[0001] This invention relates to a HYP@GOx@Fe3O4@Lf nanoparticle, its preparation method, and its application in the preparation of drugs for glioma. Background Technology

[0002] Gliomas are the most common malignant intracranial tumors, prone to recurrence and with a high mortality rate. Traditional treatments for gliomas often result in high recurrence rates and short patient survival. Therefore, researchers have explored various novel treatment modalities, such as photodynamic therapy, sonodynamic therapy, chemodynamic therapy, and photothermal therapy. While these treatments have shown some efficacy, they also come with many adverse effects, and the extension of survival time has not yet met expectations.

[0003] Photosensitizers are the most important element in photodynamic therapy (PDT) and play a decisive role in the treatment. However, the therapeutic effect of PDT is also weakened due to limitations such as the blood-brain barrier and microenvironment hypoxia.

[0004] The blood-brain barrier (BBB) ​​plays a crucial role in glioma treatment, restricting the transport of radiotherapy and chemotherapy drugs and thus reducing their effectiveness. This renders many anti-tumor drugs ineffective in glioma treatment, even leading to a situation where no effective treatments are available for gliomas.

[0005] Zhu et al. [Zhu X, H Zhou, Y Liu, et al. Transferrin / aptamer conjugated mesoporous ruthenium nanosystem for redox-controlled and targeted chemo-photodynamic therapy of glioma[J]. Acta biomaterialia, 2018, 82:143-157.] loaded transferrin (TF) and aptamer AS1411 (APT) onto the surface of mesoporous ruthenium nanoparticles (MRN), and further combined them with the antitumor drug [Ru(bpy)2(tip)]. 2+ The combination of (RBT) forms a mesoporous ruthenium nanosystem RBT@MRN-SS-TF / APT with dual targeting functions. It can effectively penetrate the BBB and target gliomas, generate ROS under laser irradiation, induce tumor cell apoptosis, and further prolong the median survival of cancer patients.

[0006] Pang et al. [Pang L,Y Zhu,J Qin,et al. Primary M1 macrophages as multifunctional carrier combined with PLGA nanoparticle delivering anticanced drug for efficient glioma therapy[J].Drug delivery,2018,25(1):1922-1931.] demonstrated the therapeutic potential of TAMs by combining M1 macrophages as multifunctional carriers with PLGA nanoparticles in the treatment of gliomas. Using T cells, TAMs, and other immune cells as targets for PSs (immune cell desertification) can overcome the effects of immune cell desertification and improve overall treatment efficiency.

[0007] Wang et al. [Wang X, Y Tian, ​​X Liao, et al. Enhancing selective photosensitizer accumulation and oxygen supply for high-efficacy photodynamic therapy toward gliomaby 5-aminolevulinic acid loaded nanoplatform[J]. J Colloid Interface Sci, 2020, 565:483-493.] constructed a Prussian blue nanoparticle (PB@PMOs) loaded with 5-ALA and coated with organosilica. This PB@PMO-5-ALA induced the accumulation of PpIX in glioma cells more effectively than free 5-ALA. Simultaneously, it exhibited good biocompatibility, decomposing hydrogen peroxide into oxygen, and the oxygen supply further promoted the effect of 5-ALA-PDT. Therefore, the photodynamic effect of PB@PMO-5-ALA was significantly improved, and its killing effect on glioma cells was enhanced. Summary of the Invention

[0008] The purpose of this invention is to provide HYP@GOx@Fe3O4@Lf nanoparticles, their preparation method, and their application in the preparation of drugs for glioma, so as to improve the therapeutic effect of drugs on glioma.

[0009] In a first aspect, the present invention provides a method for preparing HYP@GOx@Fe3O4@Lf nanoparticles, comprising the following steps:

[0010] (1) Obtain hollow porous Fe3O4 nanoparticles;

[0011] (2) The hollow porous Fe3O4 nanoparticles obtained in step (1) were modified with citric acid to obtain citric acid-modified Fe3O4 nanoparticles.

[0012] (3) Citric acid-modified Fe3O4 nanoparticles were dissolved in ultrapure water to obtain a Fe3O4 nanoparticle solution. Hypericin (HYP) solution and glucose oxidase (GOx) aqueous solution were added, and the mixture was reacted in the dark at 2-4℃ and 100-125 rpm for 20-24 h. After the reaction was completed, the supernatant was discarded by centrifugation, and the nanoparticles were washed (preferably with methanol and ultrapure water). The nanoparticles were then freeze-dried to obtain HYP@GOx@Fe3O4 nanoparticles, which were stored in the dark at 4℃ for later use. The solvent of the hypericin solution was methanol or DMSO.

[0013] (4) HYP@GOx@Fe3O4 nanoparticles were dispersed evenly in 0.1-0.2 mol / L citric acid solution, and EDC and NHS were added for reaction activation. After magnetic separation, the supernatant was removed. The activated HYP@GOx@Fe3O4 nanoparticles were added to phosphate buffer and dispersed evenly. Then lactoferrin (Lf) was added and reacted in the dark at 20-26℃ for 12-16 h. After the reaction was completed, the supernatant was discarded by centrifugation, and the nanoparticles were washed (preferably with PBS buffer). The nanoparticles were freeze-dried to obtain HYP@GOx@Fe3O4@Lf nanoparticles and stored in the dark at 4℃ for later use.

[0014] The hollow porous Fe3O4 nanoparticles described in step (1) of this invention can be prepared according to methods reported in existing literature. Specifically, the following method is recommended: FeCl3·6H2O, C6H5Na3O7·2H2O, and urea are dissolved in ultrapure water. Polyacrylamide is added under stirring, and after mixing evenly, the mixture is placed in a reaction vessel, sealed, and reacted at 180-200℃ for 12-14 hours. After the reaction vessel has completely cooled to room temperature, the hollow porous Fe3O4 nanoparticles are obtained by centrifugation, washing (preferably with anhydrous ethanol and ultrapure water), and freeze-drying. Preferably, the molar ratio of FeCl3·6H2O, C6H5Na3O7·2H2O, and urea is 1:2:3, and the amount of polyacrylamide is 7-8 mg / mL. The hollow porous Fe3O4 nanoparticles prepared by this method have high purity, good dispersibility, and excellent biocompatibility, making them a good nanocarrier for photosensitizers and bioenzymes.

[0015] The modification of hollow porous Fe3O4 nanoparticles with citric acid in step (2) of this invention can be carried out using methods reported in existing literature. Specifically, the following method is recommended: In an inert atmosphere, the hollow porous Fe3O4 nanoparticles are dissolved in ultrapure water. The resulting solvent is heated to 80-90°C, and a citric acid solution is added. The reaction is terminated after 80-100 min. The nanoparticles are then obtained by centrifugation, washing (preferably with anhydrous ethanol and ultrapure water), and freeze-drying. Preferably, the concentration of citric acid in the reaction system is 0.1-0.2 M.

[0016] In step (3) of the present invention, the preferred mass ratio of the citric acid-modified Fe3O4 nanoparticle solution, hypericin, and glucose oxidase is 1-10:1:1, more preferably 1.5-2.5:1:1, and most preferably 2:1:1.

[0017] In step (4) of this invention, the preferred mass ratio of HYP@GOx@Fe3O4 nanoparticles to lactoferrin is 10-20:1, more preferably 10:1. The preferred molar ratio of EDC to NHS is 2:5, and the preferred molar ratio of HYP@GOx@Fe3O4 nanoparticles to EDC is 20mg:0.01-0.03mmol, more preferably 20mg:0.02mmol.

[0018] In a second aspect, the present invention provides HYP@GOx@Fe3O4@Lf nanoparticles prepared according to the preparation method described in the first aspect.

[0019] Thirdly, the present invention provides the application of the HYP@GOx@Fe3O4@Lf nanoparticles described in the second aspect in the preparation of drugs for treating glioma.

[0020] Compared with existing technologies, the beneficial effects of this invention are as follows: This invention improves the water solubility and targeting of photosensitizers by synthesizing hollow porous nanomaterials loaded with photosensitizers; simultaneously, it incorporates biological enzymes to improve the insufficient endogenous H2O2 level in tumor cells, thus accelerating the efficiency of the Fenton / Fenton-like reaction. HYP@GOx@Fe3O4@Lf nanoparticles, as nanomedicines, can cut off the energy supply to gliomas and inhibit tumor growth; compared with using a single treatment method to treat gliomas, the effect is stronger, and it is not limited by the blood-brain barrier, which can greatly improve the therapeutic effect. Attached Figure Description

[0021] Figure 1 This is a flowchart illustrating the preparation process of the HYP@GOx@Fe3O4@Lf nanoparticles described in this invention.

[0022] Figure 2Here is a SEM image of the HYP@GOx@Fe3O4@Lf nanoparticles prepared in Example 1;

[0023] Figure 3 This is a particle size distribution diagram of the HYP@GOx@Fe3O4@Lf nanoparticles prepared in Example 1;

[0024] Figure 4 These are transmission electron microscope (TEM) images of the HYP@GOx@Fe3O4@Lf nanoparticles prepared in Example 1 at scales of 100 nm, 50 nm, 20 nm, and 5 nm.

[0025] Figure 5 The image shows the XRD pattern of the HYP@GOx@Fe3O4@Lf nanoparticles prepared in Example 1, which proves that the synthesized material is Fe3O4 nanoparticles.

[0026] Figure 6 The infrared spectra of Fe3O4, Fe3O4-CA, and HYP@GOx@Fe3O4@Lf nanoparticles prepared in Example 1 are shown; the infrared spectra of Fe3O4, Fe3O4-CA, and HYP@GOx@Fe3O4@Lf are all above 590 cm⁻¹. -1 There is a characteristic Fe-O peak at 1626 cm⁻¹. -1 COO - The antisymmetric stretching vibrations demonstrate that the Fe3O4 surface was successfully modified with CA at 1635 cm⁻¹. -1 1535cm -1 The peak at this point is a characteristic peak of the amide bond, indicating that Lf has been successfully coupled with HYP@GOx@Fe3O4;

[0027] Figure 7 This is a DCFH-DA fluorescence intensity diagram after HYP@GOx@Fe3O4@Lf was incubated with glucose for 3 hours;

[0028] Figure 8 This is a DCFH-DA fluorescence intensity diagram after HYP@GOx@Fe3O4@Lf was incubated with glucose for 6 hours;

[0029] Figure 9 The concentrations of H2O2 produced after different concentrations of HYP@GOx@Fe3O4@Lf are incubated with glucose;

[0030] Figure 10 This is a graph showing the results of in vitro cell viability testing;

[0031] Figure 11 This is a graph showing the results of in vitro cytotoxicity testing;

[0032] Figure 12 It was prepared in Example 1. Infrared spectra of HYP@GOx@Fe3O4@Lf nanoparticles and nanoparticles prepared in Comparative Example 1. Detailed Implementation

[0033] The technical solution of the present invention will be further described below through specific embodiments, but the scope of protection of the present invention is not limited thereto.

[0034] Unless otherwise specified in the embodiments of this invention, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained through conventional technical means or commercially available.

[0035] Example 1

[0036] 0.5406 g FeCl3·6H2O, 1.1764 g C6H5Na3O7·2H2O, and 0.36 g urea were dissolved in 40 mL of ultrapure water. Once completely dissolved, 0.3 g polyacrylamide was added to the solution under rapid magnetic stirring. After mixing for 1 hour, the mixture was transferred to a 50 mL reaction vessel, sealed, and placed in a 200℃ electric heating drying oven for 12 hours. After the reaction vessel had completely cooled to room temperature, the black solution was centrifuged (9000 rpm, 5 min), the supernatant was discarded, and the magnetic black precipitate was washed three times each with anhydrous ethanol and ultrapure water. The precipitate was then freeze-dried in a freeze dryer to obtain a black powder, yielding hollow porous Fe3O4 nanoparticles.

[0037] Hollow porous Fe3O4 nanoparticles and 50 mL of ultrapure water were mixed in a 100 mL round-bottom flask and stirred under ultrasonication for 15 minutes. The solution was then heated to 90 °C, and 4.5 mL of 2.0 M citric acid was added to the flask. The reaction was terminated after 90 minutes. The entire experiment was conducted under nitrogen atmosphere. After completely cooling to room temperature, the solution was centrifuged (9000 rpm, 5 min), the supernatant was discarded, and the precipitate was washed three times each with anhydrous ethanol and ultrapure water. The precipitate was then transferred to a freeze dryer and freeze-dried to obtain citric acid-modified Fe3O4 nanoparticles.

[0038] 20 mg of citric acid-modified Fe3O4 NPs powder was weighed and dissolved completely in 20 mL of ultrapure water by sonication. Then, 10 mg of hypericin (HYP) was weighed and dissolved in 10 mL of anhydrous methanol, and 10 mg of glucose oxidase (GOx) was dissolved in 10 mL of ultrapure water. The solutions were stirred thoroughly until completely dissolved. Then, the HYP and GOx solutions were added to the Fe3O4 NPs solution. The mixture was placed in a horizontal shaker at 4°C in the dark and subjected to rapid shaking at 125 rpm for 24 h. After the reaction was complete, the solution was centrifuged (13000 rpm, 10 min), the supernatant was discarded, and the precipitate was washed three times each with anhydrous methanol and ultrapure water. The precipitate was then transferred to a freeze dryer and freeze-dried for 48 h to obtain a black powder, yielding HYP@GOx@Fe3O4 NPs, which was stored in a 4°C refrigerator in the dark for later use.

[0039] Function: Loading photosensitizers and biological enzymes

[0040] Weigh 20 mg of HYP@GOx@Fe3O4 NPs and add 10 mL of 0.1 M citric acid solution, then sonicate until homogeneous. Add EDC and NHS to achieve final concentrations of 2 mM and 5 mM, respectively. Activate the mixture by reacting for 4 h, then remove the supernatant after magnetic separation. Add HYP@GOx@Fe3O4 NPs to 20 mL of phosphate-buffered saline (PBS, 0.1 M, pH 7.4) and sonicate until homogeneous. Add lactoferrin (Lf) (mass ratio HYP@GOx@Fe3O4:Lf = 10:1). Incubate the reaction overnight at room temperature (20-26 °C) in the dark. After the reaction is complete, centrifuge the solution (13000 rpm, 10 min), discard the supernatant, wash three times with PBS, and then freeze-dry for 48 h to obtain a black powder, yielding HYP@GOx@Fe3O4@Lf NPs. Store in a dark environment at 4 °C for later use. SEM images of HYP@GOx@Fe3O4@LfNPs are shown below. Figure 2 See particle size distribution diagram. Figure 3 Transmission electron microscope images at different scales are shown below. Figure 4 XRD pattern can be found Figure 5 Infrared image Figure 6 .

[0041] Example 2

[0042] Weigh 12.15g of DCFH-DA powder and dissolve it in 250μL of anhydrous ethanol to prepare DCFH-DA stock solution. Weigh 0.02g of NaOH granules and dissolve them in 50mL of ultrapure water. Add 1μL of DCFH-DA stock solution to 139μL of NaOH solution, let it stand in the dark for 30min, and then add 700μL of phosphate buffer (PBS, pH=7.2-7.4) to prepare DCFH-DA working solution. Prepare 125, 250, 500, and 1000μg / mL HYP@GOx@Fe3O4@Lf glucose solutions using 2mg / mL glucose solution. Add 20 μL of HYP@GOx@Fe3O4@Lf solution of different concentrations and 80 μL of DCFH-DA working solution diluted 10 times to each well of a 96-well plate. After mixing thoroughly in the dark, the samples were detected on a microplate reader at 0, 3, and 6 h. The excitation wavelength was 480 nm and the emission wavelength was 500-600 nm to obtain the DCF fluorescence emission spectrum.

[0043] The fluorescence intensity of DCFH-DA after HYP@GOx@Fe3O4@Lf incubated with glucose for 3h and 6h is as follows: Figure 7 and Figure 8 As shown, the results indicate that the ability of HYP@GOx@Fe3O4@Lf to generate ROS is concentration-dependent and time-dependent. Under the triggering of a fixed concentration of glucose solution, the longer the time, the higher the DCF fluorescence intensity of the ROS generated by HYP@GOx@Fe3O4@Lf; the higher the concentration, the higher the DCF fluorescence intensity of the ROS generated by HYP@GOx@Fe3O4@Lf.

[0044] Example 3

[0045] To investigate the in vitro catalytic ability of HYP@GOx@Fe3O4@Lf to produce H2O2 from glucose, the H2O2 release was determined using a hydrogen peroxide content detection kit (Solarbio hydrogen peroxide content detection kit, catalog number: BC3595). The principle is that H2O2 reacts with titanium sulfate to form a yellow titanium peroxide complex, which exhibits characteristic absorption at 415 nm. HYP@GOx@Fe3O4@Lf NPs aqueous solutions were prepared at different concentrations (400, 200, 100, 50, 25, 12.5 μg / mL). 500 μL of each solution was added to a glucose solution containing 500 μL of 2 mg / mL glucose and incubated in a 37°C oven for 30 min. Reagent 1 (acetone) from the kit was pre-cooled, while reagents 2 (dissolved in 6 mL of concentrated HCl), 3, and 4 were placed in a 37°C water bath for 10 min. The H2O2 standard from the kit was diluted to 1 μmol / mL. Mix 100 μL of each concentration sample with 900 μL of reagent I, centrifuge at 4℃ (8000g, 10 min), and collect all supernatant on ice. Add reagents to each tube in the order specified in the instructions. After mixing thoroughly, centrifuge at room temperature (4000g, 10 min) and discard the supernatant. Add 1 mL of reagent IV to each precipitate tube, mix thoroughly, and let stand at room temperature for 5 min. Measure the absorbance at 415 nm using a microplate reader. Calculate the amount of H2O2 released. The results are as follows: Figure 9 As shown: Under the triggering effect of a fixed glucose solution, the higher the concentration of HYP@GOx@Fe3O4@Lf, the more H2O2 is produced.

[0046] Example 4: In vitro cell viability detection

[0047] To investigate the effects of Light, HYP, the hollow porous Fe3O4 nanoparticles synthesized in Example 1, and HYP@GOx@Fe3O4@Lf on the activity of human astrocytes, NHA cells (Shanghai Yaji Biotechnology Co., Ltd.) were digested with 0.05% trypsin, centrifuged, and resuspended in 1 mL of Gibco DMEM medium in centrifuge tubes. 10 μL of the cell suspension was then aspirated and counted under a microscope. Cells were then centrifuged at 8 × 10⁻⁶ cells / mL. 3Cell density per well: NHA cells were seeded into 96-well plates (100 μL). After culturing in a humidified incubator for 24 h, the old medium was removed (37℃, 5% CO2), and 100 μL of HYP, Fe3O4, and HYP@GOx@Fe3O4@Lf solutions of different concentrations (400, 200, 100, 50, 25, 12.5, 6.25 μg / mL) prepared with culture medium (glucose concentration of 2 mg / mL) were added. The control group and the light-treated group were only added with 100 μL of culture medium. After incubation in a humidified incubator at 37℃ and 5% CO2 for 3 h, the old medium was removed, and 100 μL of CCK-8 reagent diluted with serum-free culture medium (preparation ratio of 1 mL culture medium + 100 μL CCK-8 reagent) was added to each well. After incubation in a humidified incubator at 37℃ and 5% CO2 for another 3 h, the absorbance at 450 nm was measured on a microplate reader to calculate cell viability. The results are as follows Figure 10 As shown.

[0048] Example 5: In vitro cytotoxicity detection

[0049] To investigate the cytotoxicity of Light, HYP, the hollow porous Fe3O4 nanoparticles synthesized in Example 1, HYP@GOx@Fe3O4, and HYP@GOx@Fe3O4@Lf, U87 cells (Hangzhou Yiheng Biotechnology Co., Ltd.) were digested with 0.05% trypsin, centrifuged, and resuspended in 1 mL of culture medium in centrifuge tubes. 10 μL of the cell suspension was then aspirated and counted under a microscope. Cells were then sputtered at 8 × 10⁻⁶ cells / mL. 3 Cell density per well: NHA cells were seeded into 96-well plates (100 μL). After 24 hours of incubation in a humidified incubator, the old medium was removed (37℃, 5% CO2). 100 μL of HYP, Fe3O4, HYP@GOx@Fe3O4, and HYP@GOx@Fe3O4@Lf solutions of different concentrations (200, 100, 50, 25, 12.5, 6.25, 3.125 μg / mL) prepared with culture medium (glucose concentration 2 mg / mL) were added. The control and light-treated groups received only 100 μL of culture medium. After 3 hours of incubation in a humidified incubator, the old medium was removed, and serum-free culture medium was added. After 2-4 hours of light exposure, the old medium was removed, and serum-free culture medium was added. 100 μL of CCK-8 reagent diluted with serum-free culture medium (preparation ratio: 1 mL culture medium + 100 μL CCK-8 reagent) was added to each well. After another 3 hours of incubation in a humidified incubator, the absorbance at 450 nm was measured using a microplate reader to calculate cell viability.

[0050] Comparative Example 1

[0051] HYP@GOx@Fe3O4 NPs were prepared according to the steps in Example 1.

[0052] Weigh 20 mg of HYP@GOx@Fe3O4 NPs and add 10 mL of PBS solution, then sonicate until homogeneous. Add EDC and NHS to achieve final concentrations of 2 mM and 5 mM, respectively. After 15 min, add lactoferrin (Lf) (mass ratio HYP@GOx@Fe3O4:Lf = 10:1). The reaction is carried out overnight in the dark at 4°C. After the reaction is complete, centrifuge the solution (13000 rpm, 10 min), discard the supernatant, wash three times with PBS, and then freeze-dry for 48 h to obtain a black powder. Store the sample in the dark at 4°C for later use.

[0053] The infrared spectra of the HYP@GOx@Fe3O4@Lf nanoparticles prepared in Example 1 and the nanoparticles prepared in Comparative Example 1 are shown in the figure. Figure 12 It can be found that in Example 1 at 1635cm -1 1535cm -1 The presence of amide bond characteristic peaks indicates that Lf is successfully coupled with HYP@GOx@Fe3O4; while Supplementary Example 1 lacks amide bond characteristic peaks.

Claims

1. A method for preparing HYP@GOx@Fe3O4@Lf nanoparticles, characterized in that: The preparation method includes the following steps: (1) Obtain hollow porous Fe3O4 nanoparticles; (2) The hollow porous Fe3O4 nanoparticles obtained in step (1) were modified with citric acid to obtain citric acid-modified Fe3O4 nanoparticles. (3) The Fe3O4 nanoparticles modified with citric acid were dissolved in ultrapure water to obtain a Fe3O4 nanoparticle solution. Hypericin solution and glucose oxidase aqueous solution were added, and the mixture was placed in the dark and reacted at 2-4℃ and 100-125 rpm for 20-24 h. After the reaction was completed, the supernatant was discarded by centrifugation, washed, and freeze-dried to obtain HYP@GOx@Fe3O4 nanoparticles. The solvent of the hypericin solution was methanol or DMSO. (4) HYP@GOx@Fe3O4 nanoparticles were evenly dispersed in 0.1-0.2 mol / L citric acid solution, and EDC and NHS were added for reaction activation. After magnetic separation, the supernatant was removed. The activated HYP@GOx@Fe3O4 nanoparticles were added to phosphate buffer and evenly dispersed. Then lactoferrin was added and the mixture was placed in the dark at 20-26℃ for 12-16 h. After the reaction was completed, the supernatant was discarded by centrifugation, washed, and freeze-dried to obtain HYP@GOx@Fe3O4@Lf nanoparticles.

2. The preparation method according to claim 1, characterized in that: In step (3), the mass ratio of the citric acid-modified Fe3O4 nanoparticles, hypericin, and glucose oxidase is 1-10:1:

1.

3. The preparation method according to claim 2, characterized in that: In step (3), the mass ratio of the citric acid-modified Fe3O4 nanoparticles, hypericin, and glucose oxidase is 1.5-2.5:1:

1.

4. The preparation method according to claim 1, characterized in that: In step (4), the mass ratio of HYP@GOx@Fe3O4 nanoparticles to lactoferrin is 10-20:

1.

5. The preparation method according to claim 4, characterized in that: In step (4), the mass ratio of HYP@GOx@Fe3O4 nanoparticles to lactoferrin is 10:

1.

6. The preparation method according to claim 1, characterized in that: In step (4), the molar ratio of EDC to NHS is 2:5, and the ratio of HYP@GOx@Fe3O4 nanoparticles to EDC is 20mg:0.01-0.03mmol.

7. HYP@GOx@Fe3O4@Lf nanoparticles prepared by the method according to any one of claims 1-6.

8. The use of the HYP@GOx@Fe3O4@Lf nanoparticles as described in claim 7 in the preparation of drugs for treating glioma.

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