HYP (at) GOx (at) Fe3O4 (at) Lf nanoparticle and preparation and application thereof

Abstract

The invention discloses an HYP (at) GOx (at) Fe3O4 (at) Lf nano particle as well as a preparation method and application thereof. The preparation method comprises the following steps: (1) obtaining hollow porous Fe3O4 nanoparticles; (2) obtaining Fe3O4 nano particles modified by citric acid; (3) obtaining HYP (at) GOx (at) Fe3O4 nano particles; and (4) obtaining the HYP (at) GOx (at) Fe3O4 (at) Lf nanoparticles. The invention provides an application of the HYP (at) GOx (at) Fe3O4 (at) Lf nano particle in preparation of a medicine for treating brain glioma. The synthesized HYP (at) GOx (at) Fe3O4 (at) Lf nanoparticles can cut off the supply of brain glioma energy and inhibit the growth of tumors as a nano-drug; compared with a single treatment method, the traditional Chinese medicine composition has a better treatment effect on brain glioma, is not limited by the blood-brain barrier, and can greatly improve the treatment effect.

Description

Technical Field

[0001] The invention relates to HYP@GOx@Fe3O4@Lf nanoparticles and a preparation method thereof and application of the nanoparticles in preparing brain glioma drugs. Background Art

[0002] Gliomas are the most common malignant tumors in the brain, prone to recurrence and high mortality. After traditional treatment, the recurrence rate of gliomas is high and the survival of patients is short, so researchers have explored a variety of new treatment methods, such as photodynamic therapy, sonodynamic therapy, chemodynamic therapy and photothermal therapy. These treatments have certain therapeutic effects, but are also accompanied by many adverse effects, and the extension of survival has not yet achieved the expected effect.

[0003] Photosensitizer is the most important element in photodynamic therapy and plays a decisive role in the treatment. However, it is also restricted by the blood-brain barrier, microenvironment hypoxia, etc., which weakens the therapeutic effect of PDT.

[0004] The shielding effect of the blood-brain barrier (BBB) ​​also plays an important role in the treatment of gliomas. It will limit the transport of radiotherapy and chemotherapy drugs, thereby reducing the effect of radiotherapy and chemotherapy. This has led to the inability of many anti-tumor drugs to play a role in the treatment of gliomas, and even caused the treatment of gliomas to reach a point where there is no drug available.

[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 (MRNs) and also co-administered with antitumor drugs [Ru(bpy)2(tip)] 2+ (RBT) combination forms a mesoporous ruthenium nanosystem RBT@MRN-SS-TF / APT with dual targeting function, which can effectively penetrate the BBB and target gliomas, produce ROS under laser irradiation, induce tumor cell apoptosis, and further prolong the median survival time 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 anticancer drugs for efficient glioma therapy [J]. Drug delivery, 2018, 25 (1): 1922-1931.] used M1 macrophages as multifunctional carriers combined with PLGA nanoparticles for anti-glioma drug treatment, demonstrating the therapeutic potential of TAMs. Using immune cells such as T cells and TAMs as PSs targets to overcome the effects of immune cell desertification can improve the 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 an organic silica-coated Prussian blue nanoparticle (PB@PMOs) loaded with 5-ALA. This PB@PMO-5-ALA can induce the accumulation of PpIX in glioma cells better than free 5-ALA. At the same time, it has good biocompatibility and can decompose hydrogen peroxide into oxygen. The supply of oxygen further promotes the effect of 5-ALA-PDT. Therefore, the photodynamic effect of PB@PMO-5-ALA is significantly improved, and the killing effect on glioma cells is enhanced. Summary of the invention

[0008] The purpose of the present invention is to provide a HYP@GOx@Fe3O4@Lf nanoparticle and a preparation method thereof and application thereof in the preparation of a drug for treating glioma, so as to improve the therapeutic effect of the drug 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) Obtaining hollow porous Fe3O4 nanoparticles;

[0011] (2) modifying the hollow porous Fe3O4 nanoparticles obtained in step (1) with citric acid to obtain citric acid-modified Fe3O4 nanoparticles;

[0012] (3) Dissolving citric acid-modified Fe3O4 nanoparticles in ultrapure water to obtain a Fe3O4 nanoparticle solution, adding hypericin (HYP) solution and glucose oxidase (GOx) aqueous solution, placing in a dark environment at 2-4°C and 100-125rpm for reaction for 20-24h, after the reaction is completed, centrifuging and discarding the supernatant, washing (the washing reagent is preferably methanol and ultrapure water), freeze-drying to obtain HYP@GOx@Fe3O4 nanoparticles, and storing in a dark environment at 4°C refrigerator for standby use; the solvent of the hypericin solution is methanol or DMSO;

[0013] (4) HYP@GOx@Fe3O4 nanoparticles were evenly dispersed with 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, and the activated HYP@GOx@Fe3O4 nanoparticles were added to phosphate buffer and evenly dispersed. Then lactoferrin (Lf) was added and reacted at 20-26°C in a dark environment for 12-16 h. After the reaction was completed, the supernatant was discarded by centrifugation, washed (preferably the washing reagent was PBS buffer), and freeze-dried to obtain HYP@GOx@Fe3O4@Lf nanoparticles, which were stored in a dark environment at 4°C refrigerator for use.

[0014] The hollow porous Fe3O4 nanoparticles described in step (1) of the present invention can be prepared by referring to the methods reported in existing literature. It is specifically recommended to prepare them according to the following method: dissolve FeCl3·6H2O, C6H5Na3O7·2H2O and urea in ultrapure water, add polyacrylamide under stirring, mix well and place in a reactor, seal and react at 180-200°C for 12-14h, and after the reactor is completely cooled to room temperature, centrifuge, wash (the washing reagent is preferably anhydrous ethanol and ultrapure water), and freeze-dry to obtain hollow porous Fe3O4 nanoparticles. Preferably, the molar ratio of the FeCl3·6H2O, C6H5Na3O7·2H2O and urea is 1:2:3, and the feeding amount of the polyacrylamide is 7-8mg / mL. The hollow porous Fe3O4 nanoparticles prepared by this method have high purity, good dispersibility and excellent biocompatibility, and are good nanocarriers for photosensitizers and biological enzymes.

[0015] The method of modifying the hollow porous Fe3O4 nanoparticles with citric acid in step (2) of the present invention can be carried out by the method reported in the existing literature, and is specifically recommended to be carried out according to the following method: in an inert atmosphere, the hollow porous Fe3O4 nanoparticles are dissolved in ultrapure water, the resulting solvent is heated to 80-90°C, a citric acid solution is added, the reaction is terminated after 80-100 minutes, and then centrifuged, washed (the washing reagent is preferably anhydrous ethanol and ultrapure water), and freeze-dried to obtain citric acid-modified Fe3O4 nanoparticles. Preferably, the concentration of citric acid in the reaction system is 0.1-0.2M.

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

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

[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] In a third aspect, the present invention provides the use of the HYP@GOx@Fe3O4@Lf nanoparticles described in the second aspect in the preparation of drugs for treating brain gliomas.

[0020] Compared with the prior art, the present invention has the following beneficial effects: the present invention improves the water solubility and targeting of photosensitizers by synthesizing hollow porous nanomaterials to load photosensitizers; at the same time, it carries biological enzymes to improve the insufficient endogenous H2O2 level in tumor cells and accelerate the efficiency of Fenton / Fenton-like reactions. HYP@GOx@Fe3O4@Lf nanoparticles as nanomedicines can cut off the energy supply of brain gliomas and inhibit tumor growth; it is more effective than using a single treatment method to treat brain gliomas, and it will not be restricted by the blood-brain barrier, which can greatly improve the treatment effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 is a flow chart of the preparation of HYP@GOx@Fe3O4@Lf nanoparticles of the present invention;

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

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

[0024] Figure 4 It is the transmission electron microscopy image of HYP@GOx@Fe3O4@Lf nanoparticles prepared in Example 1 at 100nm, 50nm, 20nm, and 5nm scales;

[0025] Figure 5 is the XRD pattern of HYP@GOx@Fe3O4@Lf nanoparticles prepared in Example 1, proving 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 in Figure 1. The infrared spectra of Fe3O4, Fe3O4-CA, and HYP@GOx@Fe3O4@Lf nanoparticles are all at 590 cm -1 There is a characteristic peak of Fe-O at 1626cm -1 COO - The antisymmetric stretching vibration of Fe3O4 proves that the surface of Fe3O4 is successfully modified with CA, 1635cm -1 、1535cm -1 The peak at the center is the characteristic peak of amide bond, indicating that Lf is successfully coupled with HYP@GOx@Fe3O4;

[0027] Figure 7 is the DCFH-DA fluorescence intensity graph of HYP@GOx@Fe3O4@Lf incubated with glucose for 3 h;

[0028] Figure 8 is the DCFH-DA fluorescence intensity graph of HYP@GOx@Fe3O4@Lf incubated with glucose for 6 h;

[0029] Fig. 9 is the concentration of H2O2 produced after incubation of different concentrations of HYP@GOx@Fe3O4@Lf with glucose;

[0030] Fig.10 This is a graph showing the results of in vitro cell activity assay;

[0031] Fig.11 It is a graph of the results of in vitro cytotoxicity assay;

[0032] Fig.12 Prepared in Example 1 Infrared spectra of HYP@GOx@Fe3O4@Lf nanoparticles and nanoparticles prepared in Comparative Example 1. DETAILED DESCRIPTION

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

[0034] If no specific conditions are specified in the examples of the present invention, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be obtained by conventional technical means or purchased commercially.

[0035] Example 1

[0036] Dissolve 0.5406g FeCl3·6H2O, 1.1764g C6H5Na3O7·2H2O and 0.36g urea in 40mL ultrapure water. When dissolved completely, add 0.3g polyacrylamide to the above solution under rapid magnetic stirring. After mixing evenly for 1h, transfer to a 50mL reactor, seal and place in a 200℃ electric heating blast drying oven for 12h. After the reactor is completely cooled to room temperature, centrifuge the black solution in the reactor (9000rpm, 5min), discard the supernatant, wash the magnetic black precipitate with anhydrous ethanol and ultrapure water 3 times each, and then transfer to a freeze dryer to freeze-dry into black powder to obtain hollow porous Fe3O4 nanoparticles.

[0037] The hollow porous Fe3O4 nanoparticles and 50mL ultrapure water were mixed in a 100mL round-bottom flask and stirred for 15 minutes under ultrasound. The solution was then heated to 90°C and 2.0M citric acid (4.5mL) was added to the flask. After 90 minutes, the reaction was terminated. The entire experimental process was carried out under nitrogen. After being completely cooled to room temperature, the solution was centrifuged (9000rpm, 5min), the supernatant was discarded, and the precipitate was washed with anhydrous ethanol and ultrapure water 3 times each, and then transferred to a freeze dryer for freeze drying to obtain citric acid-modified Fe3O4 nanoparticles.

[0038] Weigh 20 mg of citric acid-modified Fe3O4 NPs powder and add 20 mL of ultrapure water to dissolve it completely by ultrasound. Then weigh 10 mg of hypericin (HYP) and add 10 ml of anhydrous methanol and 10 mg of glucose oxidase (GOx) to 10 ml of ultrapure water, stir them thoroughly to dissolve them completely, and then add HYP solution and GOx solution to the Fe3O4 NPs solution. Place it in a horizontal shaker in a 4 ° C refrigerator in a dark environment and shake it quickly at low temperature for 24 h (125 rpm). After the reaction is completed, centrifuge the solution (13000 rpm, 10 min), discard the supernatant, wash the precipitate with anhydrous methanol and ultrapure water 3 times each, and then transfer it to a freeze dryer and freeze-dry it for 48 hours to obtain a black powder, and store it in a dark environment at 4 ° C refrigerator for use.

[0039] Function: Loading photosensitizer and biological enzyme

[0040] Weigh 20 mg of HYP@GOx@Fe3O4 NPs and add 10 mL of 0.1 M citric acid solution to ultrasonically disperse evenly. Add EDC and NHS to make the final concentrations 2 mM and 5 mM, respectively. React for 4 h for activation, and remove the supernatant after magnetic separation. Add HYP@GOx@Fe3O4 NPs to 20 mL of phosphate buffer (PBS, 0.1 M, pH 7.4) and ultrasonically disperse evenly, and add lactoferrin (Lf) (mass ratio HYP@GOx@Fe3O4: Lf = 10: 1). The reaction was carried out overnight at room temperature (20-26 ° C) in the dark. After the reaction was completed, the solution was centrifuged (13000 rpm, 10 min), the supernatant was discarded, and washed three times with PBS, and then transferred to a freeze dryer for freeze drying for 48 h to obtain black powder, and HYP@GOx@Fe3O4@LfNPs were stored in a dark environment at 4 ° C refrigerator for use. The SEM images of HYP@GOx@Fe3O4@LfNPs are shown in Figure 2 , particle size distribution diagram see Figure 3 , TEM images at different scales are shown in Figure 4 , XRD pattern see Figure 5 , infrared image see Figure 6 .

[0041] Example 2

[0042] Weigh 12.15g DCFH-DA powder and dissolve it in 250μL anhydrous ethanol to prepare DCFH-DA mother solution. Weigh 0.02g NaOH granules and dissolve it in 50mL ultrapure water. Take 1μLDCF mother solution and add it to 139μL NaOH solution. After standing in the dark for 30min, add 700μL 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 with 2mg / mL glucose solution prepared in Example 1. In each well of a 96-well plate, 20 μL of HYP@GOx@Fe3O4@Lf solution of different concentrations and 80 μL of 10-fold diluted DCFH-DA working solution were added in sequence, mixed evenly in the dark, and then detected on a microplate reader at 0, 3, and 6 h with an excitation wavelength of 480 nm and an emission wavelength of 500-600 nm to obtain the DCF fluorescence emission spectrum.

[0043] The DCFH-DA fluorescence intensity of HYP@GOx@Fe3O4@Lf incubated with glucose for 3h and 6h is shown in Figure 7 and Figure 8 As shown, the results show that the ability of HYP@GOx@Fe3O4@Lf to produce 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 ROS generated by HYP@GOx@Fe3O4@Lf; the higher the concentration, the higher the DCF fluorescence intensity of ROS generated by HYP@GOx@Fe3O4@Lf.

[0044] Example 3

[0045] In order to study the ability of HYP@GOx@Fe3O4@Lf to catalyze glucose to produce H2O2 in vitro, the amount of H2O2 released was determined using a hydrogen peroxide content detection kit (Solarbio hydrogen peroxide content detection kit catalog number: BC3595). The principle is that H2O2 and titanium sulfate can generate yellow titanium peroxide complexes with characteristic absorption at 415nm. The HYP@GOx@Fe3O4@LfNPs aqueous solution was prepared into aqueous solutions with different concentrations (400, 200, 100, 50, 25, 12.5μg / mL). 500μL of each was added to 500μL of 2mg / mL glucose solution and incubated in a 37℃ electric blast drying oven for 30min. Reagent 1 (acetone) in the kit was precooled, and reagents 2 (after adding 6mL concentrated HCl to dissolve), 3, and 4 were placed in a 37℃ water bath for 10min. The H2O2 standard in the kit was diluted to 1μmol / mL. Take 100μL of each concentration sample and mix with 900μL of reagent 1, centrifuge at 4℃ (8000g, 10min), take all the supernatant and put it on ice. Add reagents to each tube according to the order of the instructions. After mixing evenly, centrifuge at room temperature (4000g, 10min), and discard the supernatant. Add 1mL of reagent 4 to the precipitate in each tube, mix evenly, let stand at room temperature for 5min, and measure the absorbance at 415nm on the microplate reader. Calculate the amount of H2O2 released. The results are as follows: Fig. 9 As shown: Under the triggering of fixed glucose solution, the higher the concentration of HYP@GOx@Fe3O4@Lf, the more H2O2 is produced.

[0046] Example 4: In vitro cell activity detection

[0047] To study 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 and centrifuged, and then 1 mL of Gibco DMEM medium was added to resuspend them in a centrifuge tube. 10 μL of the cell suspension was aspirated and counted under a microscope, and then 8×10 3NHA cells were seeded in 96-well plates (100 μL) at a cell density of 100 μL / well. After 24 h of incubation in a humidified incubator, the old solution was removed (37°C, 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 in culture medium (glucose concentration was 2 mg / mL) were added. Only 100 μL of culture medium was added to the control group and the light-irradiated group. After incubation in a humidified incubator at 37°C and 5% CO2 for 3 h, the old solution was removed, and 100 μL of CCK-8 reagent diluted with serum-free culture medium was added to each well (the preparation ratio was 1 mL culture medium + 100 μL CCK-8 reagent). After incubation in a humidified incubator at 37°C and 5% CO2 for another 3 h, the absorbance at 450 nm was measured on an ELISA reader to calculate the cell activity. The results are as follows Fig.10 shown.

[0048] Example 5: In vitro cytotoxicity assay

[0049] To study 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 and centrifuged, then 1 mL of culture medium was added and resuspended in a centrifuge tube, 10 μL of the cell suspension was aspirated and the cells were counted under a microscope, and then 8 × 10 3 NHA cells were seeded in 96-well plates at a cell density of 100 μL per well. After culturing in a humidified incubator for 24 h, the old solution was removed (37°C, 5% CO2), and 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 in culture medium (glucose concentration was 2 mg / mL) were added. Only 100 μL of culture medium was added to the control group and the light-irradiated group. After incubation in a humidified incubator for 3 h, the old solution was removed and serum-free culture medium was added. After irradiation for 2-4 h, the old solution was removed and serum-free culture medium was added. 100 μL of CCK-8 reagent diluted with serum-free culture medium was added to each well (the preparation ratio was 1 mL culture medium + 100 μL CCK-8 reagent). After further incubation in the humidified incubator for 3 h, the absorbance at 450 nm was measured on an enzyme reader to calculate the cell activity.

[0050] Comparative Example 1

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

[0052] Weigh 20 mg of HYP@GOx@Fe3O4 NPs and add 10 mL of PBS solution for ultrasonic dispersion. Add EDC and NHS to make the final concentrations 2 mM and 5 mM, respectively. After 15 min, add lactoferrin (Lf) (mass ratio HYP@GOx@Fe3O4: Lf = 10: 1). The reaction was carried out overnight at 4 ° C in a dark environment. After the reaction was completed, the solution was centrifuged (13000 rpm, 10 min), the supernatant was discarded, and washed three times with PBS, then transferred to a freeze dryer and freeze-dried for 48 hours to a black powder. The obtained sample was stored in a dark environment at 4 ° C refrigerator for 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 Fig.12 It can be found that Example 1 has a -1 、1535cm -1 There is an amide bond characteristic peak at , indicating that Lf is successfully coupled with HYP@GOx@Fe3O4; while there is no amide bond characteristic peak in Supplementary Example 1.

Claims

1. A method for preparing HYP@GOx@Fe3O4@Lf nanoparticles, characterized in that: The preparation method comprises the following steps: (1) Obtaining hollow porous Fe3O4 nanoparticles; (2) modifying the hollow porous Fe3O4 nanoparticles obtained in step (1) with citric acid to obtain citric acid-modified Fe3O4 nanoparticles; (3) Dissolving citric acid-modified Fe3O4 nanoparticles in ultrapure water to obtain a Fe3O4 nanoparticle solution, adding a hypericin solution and a glucose oxidase aqueous solution, and reacting the solution in a dark environment at 2-4°C and 100-125 rpm for 20-24 hours. After the reaction is completed, centrifuging and discarding the supernatant, washing, and freeze-drying to obtain HYP@GOx@Fe3O4 nanoparticles; the solvent of the hypericin solution is methanol or DMSO; (4) HYP@GOx@Fe3O4 nanoparticles were evenly dispersed with 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, and the activated HYP@GOx@Fe3O4 nanoparticles were added to phosphate buffer and evenly dispersed. Then lactoferrin was added and reacted at 20-26°C in a dark environment 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 nanoparticle solution, 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 nanoparticle solution, 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 feed mass ratio of HYP@GOx@Fe3O4 nanoparticles and 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 20 mg: 0.01-0.03 mmol.

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

8. Use of the HYP@GOx@Fe3O4@Lf nanoparticles as claimed in claim 7 in the preparation of drugs for treating brain glioma.

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