Synthesis method of double-targeting drug-loading superparamagnetic nanoparticles for glioma diagnosis and treatment

Abstract

The invention belongs to the technical field of preparation of diagnosis and treatment drug-loading superparamagnetic nanoparticles, and particularly relates to a synthesis method of double-targeting drug-loading superparamagnetic nanoparticles for glioma diagnosis and treatment, the synthesis method comprises the following steps: S1, preparation and characterization of RexFe3-xO4; s2, preparation and characterization of Yb < 0.015 > Fe < 2.985 > O < 4 > (at) PEI-HA are carried out; s3, preparation and characterization of MET (at) Yb < 0.015 > Fe < 2.985 > O < 4 > (at) PEI-HA are carried out; s4, the in-vitro anti-tumor effect of the MET (at) Yb < 0.015 > Fe < 2.985 > O < 4 > (at) PEI-HA is analyzed; and S5, the in-vivo MRI-T2 imaging effect of the MET (at) Yb < 0.015 > Fe < 2.985 > O < 4 > (at) PEI-HA is analyzed. According to the invention, three treatment modes of MHT-PDT-CDT can be adopted for collaborative treatment, and meanwhile, the autophagy and ferroptosis capabilities of tumor cells are enhanced through MET and magnetic heat.

Description

Technical Field

[0001] The invention belongs to the technical field of preparation of drug-loaded superparamagnetic nanoparticles for diagnosis and treatment, and specifically relates to a method for synthesizing dual-targeted drug-loaded superparamagnetic nanoparticles for diagnosis and treatment of gliomas. Background Art

[0002] Gliomas are pathological glial cells that form in the brain. They are the most common malignant tumors of the central nervous system, accounting for about 80% of primary brain malignant tumors. Among them, glioblastoma (GBM) is a glioma graded as grade IV by the WHO. The overall survival of 70%-80% of patients is only 3-6 months, the 1-year relative survival rate is 10%, and the 5-year relative survival rate is only 5%. At present, the main treatment for GBM is surgical resection plus postoperative adjuvant radiotherapy and chemotherapy. However, due to the unclear boundaries of GBM, it is often diffuse and infiltrative, making it difficult to completely remove it by surgery. When using radiotherapy and chemotherapy, the clinical treatment effect of radiotherapy and chemotherapy is poor due to the poor targeting of anti-glioma drugs, the low permeability to the blood-brain barrier (BBB), and the toxic side effects of some drugs. Therefore, the development of drugs with good targeting and the means to improve the permeability of drugs to the BBB have become a difficult problem that needs to be overcome in the treatment of gliomas.

[0003] The BBB is a glial boundary membrane surrounded by capillary endothelial cells, basement membranes, and astrocyte footplates, which restricts most drugs from entering the brain from the blood. To enhance the permeability of the BBB, current technologies include the following: (1) nanocarriers, which can enhance the passive diffusion of nanomaterials by utilizing the small size effect; (2) endothelial cell receptor ligand carriers, which mediate the endocytosis of nanomaterials by endothelial cells through the recognition of receptors and ligands; (3) focused ultrasound techniques; (4) thermal physical stimulation; (6) inducing transmembrane proteins to enhance drug transport. In addition, magnetic nanoparticles (MNPs) have shown obvious characteristics in crossing the BBB. Under the action of an external constant magnetic field, MNPs have the characteristics of aggregation and precise positioning. At the same time, in general, MNPs have the ability to absorb electromagnetic waves and convert them into heat energy under the action of an alternating magnetic field. It can use thermal physics to temporarily open the BBB, making it possible to cross the BBB. At present, the nano-delivery strategy that uses magnetic fields and magnetothermal effects to enable magnetic nano-drugs to cross the BBB and reach the tumor site has shown great application prospects in the field of glioma diagnosis and treatment. Developing MNPs of appropriate sizes and optimizing their structures to obtain performance at safe temperatures can enable drugs to cross the BBB, which will provide new materials for the treatment of gliomas.

[0004] Patent search revealed that the patent number is CN202310374772.0, a Fe 3 O4 Nano preparations and their preparation methods and applications belong to the field of medical technology. 3 O 4 As the core, the immune checkpoint inhibitor icaridostat was used as an immunomodulator, and PEG was connected to Fe by hydrophobic bonding. 3 O 4 On the surface of the nanoparticles, folic acid is linked to the outer end of PEG through an amide reaction. This design can achieve the co-delivery of magnetic materials and immunomodulators. Under the action of an external alternating magnetic field, this targeted preparation can achieve magnetic heat-immunotherapy for deep metastatic tumors.

[0005] Compared with traditional radiotherapy and chemotherapy, new catalytic therapies including photodynamic therapy (PDT), chemodynamic therapy (CDT) and magnetic hyperthermia (MHT) replace traditional drugs with safe and non-toxic catalysts, and achieve specific cancer treatment by generating therapeutic products in situ in tumor tissues, providing new ideas and new means for precise cancer treatment. However, in the actual treatment process, due to the H provided by TME, 2 O 2 The actual treatment effect of CDT is poor, and the light source currently used in PDT is generally a visible light source, which leads to its limited penetration depth of tissues and is only effective for superficial tumors. In addition, PDT relies on O 2 , which results in poor treatment effects on tumor cells in a hypoxic state. Therefore, a single treatment method is difficult to eradicate tumors, so combined treatment is used to fight difficult-to-eradicate tumors. Summary of the invention

[0006] The purpose of the present invention is to provide a method for synthesizing dual-targeted drug-loaded superparamagnetic nanoparticles for glioma diagnosis and treatment, which can adopt the three treatment modes of MHT-PDT-CDT for synergistic treatment, and at the same time enhance the autophagy and ferroptosis ability of tumor cells through MET and magnetic heat.

[0007] The technical solution adopted by the present invention is as follows:

[0008] A method for synthesizing dual-targeted drug-loaded superparamagnetic nanoparticles for glioma diagnosis and treatment, the method comprising the following steps:

[0009] S1: Perform Re x Fe 3-x O 4 Preparation and characterization of

[0010] In the S1, Fe 3 O 4The preparation of Nd / Yb / Gd / Tm is carried out by the following steps:

[0011] S11: Use an analytical balance to weigh 0.92 g of ferric chloride hexahydrate, x g of neodymium / ytterbium / gadolinium / thulium chloride hexahydrate, with a doping ratio of 5%, and calculate the X value according to the mass of different rare earths, 0.6 g of urea, 0.8 g of polyacrylamide, and 1.76 g of sodium citrate and dissolve them in 60 mL of ultrapure water;

[0012] S12: Stir the mixture at room temperature, transfer the clear solution to a reactor after it is completely dissolved, and react at 200° C. for 500 min;

[0013] S13: After the reaction is completed, centrifuge and collect the precipitate at a speed of 10000r / min for 30min, discard the supernatant and collect the precipitate;

[0014] S14: Wash the precipitate with ethanol: ultrapure water = 1:2, repeat the washing and centrifugation twice to obtain a black precipitate product, and freeze-dry it;

[0015] Fe 3 O 4 The characterization of Nd / Yb / Gd / Tm in the following steps is carried out:

[0016] S111: Characterized by X-ray diffractometer and Fourier transform infrared spectroscopy;

[0017] S112: In the X-ray diffractometer diffraction pattern, NF, YF, GF and TF are consistent with the standard Nd, Yb, Gd and Tm and ferrite standard crystal diffraction cards;

[0018] S113: In the Fourier transform infrared spectrum, NF, YF, GF and TF are at 2964, 1464, 1368 and 564 cm-1, respectively. -1 The characteristic peaks of composite ferrite appeared at , and the results proved the successful preparation of NF, YF, GF and TF.

[0019] S2: Perform Yb 0.015 Fe 2.985 O 4 @ Preparation and characterization of PEI-HA;

[0020] In S2, Yb 0.015 Fe 2.985 O 4 @PEI-HA was prepared and characterized by electrostatic adsorption method to explore the surface active concentration. The steps are as follows:

[0021] S21: Dissolve 10 mg YF and X mg PEI in 10 mL ultrapure water respectively;

[0022] S22: Under ultrasound, the YF solution was slowly added dropwise to the PEI solution, and the mixed solution was placed in a constant temperature shaker at room temperature, 120 r, 24 h, and centrifuged at 8000 r / min for 30 min to collect the precipitate to obtain YF@PEI;

[0023] S23: 10 mg YF@PEI and X mg HA were dissolved in ultrapure water, and the YF@PEI solution was slowly added dropwise to the HA solution. The mixed solution was placed in a constant temperature shaker at room temperature, 120 r, 24 h, and centrifuged at 8000 r / min for 30 min to collect the precipitate to obtain YF@PEI-HA;

[0024] S24: The optimal surfactant ratio was selected by measuring particle size and dispersibility. 1 mg / mL YF, 0.5 mg / mL PEI and 0.1 mg / mL HA were the optimal surfactant ratios.

[0025] S3: Conduct MET@Yb 0.015 Fe 2.985 O 4 @ Preparation and characterization of PEI-HA;

[0026] In S3, MET@Yb 0.015 Fe 2.985 O 4 The preparation and characterization of @PEI-HA were carried out using the following steps:

[0027] S31: Solvent impregnation method was used to investigate the proportion of MET encapsulation;

[0028] S32: Using dialysis to explore MET@Yb 0.015 Fe 2.985 O 4 @PEI-HA released in vitro;

[0029] S33: To MET@Yb 0.015 Fe 2.985 O 4 @PEI-HA was prepared;

[0030] S34: To MET@Yb 0.015 Fe 2.985 O 4 @The basic structure of PEI-HA was characterized;

[0031] S35: To MET@Yb 0.015 Fe 2.985 O 4 @PEI-HA magnetocaloric properties were characterized;

[0032] The preparation method in S33 specifically comprises the following steps:

[0033] S331: Use an analytical balance to weigh 0.92 g of ferric chloride hexahydrate, 0.02 g of ytterbium chloride hexahydrate, 0.6 g of urea, 0.8 g of polyacrylamide, and 1.76 g of sodium citrate and dissolve them in 60 mL of ultrapure water. Stir the mixture at room temperature. After it is completely dissolved, transfer the clear solution to a reactor and react at 200°C for 8 h.

[0034] S332: After the reaction is completed, centrifuge and collect at a speed of 10000r / min for 30min, discard the supernatant and collect the precipitate, wash the precipitate with ethanol: ultrapure water = 1:2, repeat the washing and centrifugation twice to obtain YF, and freeze-dry;

[0035] S333: Disperse 20 mg of YF in 20 mL of ultrapure water and 10 mg of PEI in 20 mL of ultrapure water. Slowly add YF to PEI under ultrasound, place in a 37°C shaker for 24 h, centrifuge at 8000 r / min for 30 min, discard the supernatant, and wash the precipitate with ultrapure water three times.

[0036] S334: YF@PEI was dissolved in ultrapure water again, 2 mg HA was weighed and dispersed in 20 mL ultrapure water, HA was dissolved in ultrapure water, and the solution was weighed according to the mass ratio with YF@PEI. Under ultrasound, the YF@PEI solution was added dropwise to the HA solution, shaken on a shaker for 120 r. After 24 h, the mixture was collected by centrifugation at 8000 r / min for 30 min, the supernatant was discarded, and the mixture was washed with ultrapure water for 3 times to obtain YFH dissolved in ultrapure water;

[0037] S335: Prepare 5 mg / mL MET solution, drop the MET solution into the YFH solution, place the mixed solution in a 37°C constant temperature shaker for 24 hours, use a centrifuge at 8000 r / min for 20 minutes, wash with ultrapure water three times, collect the MYFH precipitate, and freeze-dry it for later use.

[0038] S36: To MET@Yb 0.015 Fe 2.985 O 4 @Analysis of PEI-HA imaging performance;

[0039] S37: Determine MET@Yb 0.015 Fe 2.985 O 4 @PEI-HA has multiple enzyme activities;

[0040] S38: Analysis 1 O 2 The ability to produce.

[0041] S4: MET@Yb 0.015 Fe 2.985 O 4@Analysis of the anti-tumor effect of PEI-HA in vitro;

[0042] In S4, the in vitro anti-tumor effect analysis includes MET@Yb 0.015 Fe 2.985 O 4 @PEI-HA biosafety, BBB crossing experiment, cell killing effect analysis, cell autophagy level evaluation and cell ferroptosis level detection.

[0043] S5: To MET@Yb 0.015 Fe 2.985 O 4 @PEI-HA in vivo MRI-T 2 Analyze the imaging effect;

[0044] In S5, the drug imaging effect is analyzed by using a Spectra 3.0T nuclear magnetic resonance imaging device to image the mice;

[0045] In the absence of AMF, even with the action of an external magnet, T 2 There was no enhancement signal, only the lesion signal; after applying AMF, in the case of external magnet response, T appeared at 3h. 2 The signal was enhanced, while the group without magnet also showed T at 5h. 2 The enhanced signal confirmed that AMF can effectively open the BBB briefly.

[0046] S6: To MET@Yb 0.015 Fe 2.985 O 4 @The anti-tumor effect of PEI-HA in vivo was analyzed.

[0047] In S6, analyzing the anti-tumor effect of the drug in vivo comprises the following steps:

[0048] S61: Orthotopic tumors in the brain region of C57BL / 6J magnetic mice around 7 weeks old were treated with GL261-LUC;

[0049] S62: An orthotopic glioblastoma model was successfully established. The treatment method was tail vein administration, 1064nm laser irradiation and magnetic hyperthermia therapy. During the treatment cycle, the therapeutic effect of tumor-bearing mice was monitored by intraperitoneal injection of D-luciferin potassium salt and observed in an animal living imaging device.

[0050] S63: The groups were as follows: (1) PBS; (2) MYFH+M; (3) MYFH+AMF+M; (4) MYFH+AMF+M+1064nm; (5) MYFH+AMF+M+MHT; (6) YFH+AMF+M+MHT+1064nm; (7) MYFH+AMF+M+MHT+1064nm;

[0051] S64: The results showed that in groups (1, 2), the BBB was not opened and the treatment effect was poor. After the BBB was opened, the combined treatment effect increased and the treatment effect was obvious, inhibiting the tumor growth.

[0052] The dual-targeted drug-loaded superparamagnetic nanoparticles for the diagnosis and treatment of gliomas are prepared by the synthesis method of the dual-targeted drug-loaded magnetic nanoparticles for the diagnosis and treatment of gliomas.

[0053] The application of dual-targeted drug-loaded superparamagnetic nanoparticles for the diagnosis and treatment of gliomas enhances the ability of cell autophagy and ferroptosis by encapsulating the three treatment modes of MHT-PDT-CDT for synergistic treatment.

[0054] The technical effects achieved by the present invention are:

[0055] The synthesis method of dual-targeted drug-loaded superparamagnetic nanoparticles for glioma diagnosis and treatment of the present invention is further improved in NIR IIPDT treatment with the encapsulated MET and MYFH. In autophagy and ferroptosis, MET is regulated through the AMPK pathway, thereby further enhancing the ability of cell autophagy and ferroptosis.

[0056] The synthesis method of dual-targeted drug-loaded superparamagnetic nanoparticles for glioma diagnosis and treatment of the present invention is as follows: Most ferrites are ferrimagnetic. After ytterbium doping, MYFH has paramagnetism compared with iron oxide nanoparticles. The saturation magnetization intensity at 300K reaches 66.1emu / g, which has higher saturation magnetization intensity, MRI T 2 Imaging weighted effect and magnetocaloric properties. It can be quickly aggregated under an external static magnetic field and has favorable static magnetic field responsiveness. It is the first report that YFH achieves NIRIIPDT effect after ytterbium doping.

[0057] The synthesis method of dual-targeted drug-loaded superparamagnetic nanoparticles for glioma diagnosis and treatment of the present invention, YFH, has excellent triple enzyme-like activity (POD-like enzyme activity, CAT-like enzyme activity, and GSH-like enzyme activity). Under the acidic tumor microenvironment, through MHT therapy, it further reduces oxidative stress in tumor cells and triggers enhanced tumor autophagy. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 is the XRD pattern of TF, YF, NF and GF of the embodiments of the present invention;

[0059] Figure 2 is the FTIR spectra of GF, NF, YF, and TF of the embodiments of the present invention;

[0060] Figure 3 are the temperature rise curves and thermal imaging diagrams of the embodiments GF, NF, TF and YF of the present invention;

[0061] Figure 4 It is the embodiment of the present invention Fe 3 O 4 : Heating curves of TF and YF;

[0062] Figure 5 This is a DLS graph of the embodiment of the present invention exploring the encapsulation of PEI with different proportions;

[0063] Figure 6 It is a DLS diagram of the embodiment of the present invention to explore the packaging of different proportions of HA;

[0064] Figure 7 The zeta potential change and DLS particle size diagram of the YFH package after activation in the embodiment of the present invention;

[0065] Figure 8 It is the magnetothermal cycle curve and thermal imaging diagram of the embodiment YFH of the present invention;

[0066] Fig. 9 is the drug release performance of MYFH in the embodiment of the present invention (n=3);

[0067] Fig.10 is the SEM image of the examples YF, YFH and MYFH of the present invention (Scale bar = 1 μm);

[0068] Fig.11 are the DLS and Zeta graphs of Examples YF, YFH and MYFH of the present invention (n=3);

[0069] Fig.12 is the XRD pattern of Examples YF, YFH and MYFH of the present invention;

[0070] Fig.13 FTIR graphs of Examples YF, YFH and MYFH of the present invention;

[0071] Fig.14 is the stability of MYFH in the embodiment of the present invention (n=3);

[0072] Fig.15 It is the hysteresis loop test (illustration: magnetic adsorption) and the magnified diagram of the hysteresis loop of the embodiment of the present invention MYFH;

[0073] Fig.16The temperature rise, magnetothermal cycle curve and thermal imaging diagram of MYFH at different concentrations in the embodiment of the present invention;

[0074] Fig.17 is the T of MYFH under different iron concentrations in the embodiment of the present invention 2 Imaging and 2 / r 1 Value linear fitting curve;

[0075] Fig.18 is the ultraviolet absorption curve of TMB mixed solution at different pH values ​​and temperatures of the embodiment of the present invention;

[0076] Fig.19 1 is the ultraviolet absorption curve of the DTNB mixed solution at different pH values ​​and temperatures of the embodiment of the present invention;

[0077] Fig. 20 is the oxygen content in the solution at different pH values ​​and temperatures in the embodiments of the present invention;

[0078] Fig.21 It is the UV absorption spectrum of DPBF solution of MYFH in the embodiment of the present invention at 1064nm and different temperatures;

[0079] Fig. 22 is the cell survival rate after treatment with YFH, MET and MYFH at different concentrations in the examples of the present invention (n=3);

[0080] Fig.23 CLSM images of MYFH uptake by GL261 cells in different groups at different times in the present invention (Scale bar = 20 μm);

[0081] Fig.24 is the survival rate of GL261 under different treatment conditions in the examples of the present invention (n=3);

[0082] Fig.25 is the detection of autophagy levels of GL261 cells after treatment under different conditions in the examples of the present invention (n=3);

[0083] Fig.26 is the LPO level of GL261 cells after treatment under different conditions in the examples of the present invention (n=3);

[0084] Fig. 27 The in vivo MRI-T of MYFH under different conditions in the embodiments of the present invention 2 Weighted images and signal strength statistics;

[0085] Fig.28 is the bioluminescent signal of the brain of C57BL / 6J mice during the treatment of the embodiment of the present invention. DETAILED DESCRIPTION

[0086] In order to make the purpose and advantages of the present invention more clearly understood, the present invention is specifically described below in conjunction with embodiments. It should be understood that the following text is only used to describe one or several specific embodiments of the present invention, and does not strictly limit the scope of protection of the specific claims of the present invention.

[0087] like Figure 1 As shown, a method for synthesizing dual-targeted drug-loaded superparamagnetic nanoparticles for glioma diagnosis and treatment comprises the following steps:

[0088] S1: Perform Re x Fe 3-x O 4 Preparation and characterization of

[0089] In S1, Fe 3 O 4 The preparation of Nd / Yb / Gd / Tm is carried out by the following steps:

[0090] S11: Use an analytical balance to weigh 0.92 g of ferric chloride hexahydrate, x g of neodymium / ytterbium / gadolinium / thulium chloride hexahydrate, with a doping ratio of 5%, and calculate the X value according to the mass of different rare earths, 0.6 g of urea, 0.8 g of polyacrylamide, and 1.76 g of sodium citrate and dissolve them in 60 mL of ultrapure water;

[0091] S12: Stir the mixture at room temperature, transfer the clear solution to a reactor after it is completely dissolved, and react at 200° C. for 500 min;

[0092] S13: After the reaction is completed, centrifuge and collect the precipitate at a speed of 10000r / min for 30min, discard the supernatant and collect the precipitate;

[0093] S14: Wash the precipitate with ethanol: ultrapure water = 1:2, repeat the washing and centrifugation twice to obtain a black precipitate product, and freeze-dry it;

[0094] Fe 3 O 4 The characterization of Nd / Yb / Gd / Tm in the following steps is carried out:

[0095] Since we need to obtain doped ferrites with better magnetocaloric properties, we mainly studied the magnetocaloric properties of ferrites doped with four rare earth elements. 3 O 4 :Nd / Yb / Gd / Tm (hereinafter referred to as NF, YF, GF, TF);

[0096] S111: Characterized by X-ray diffractometer and Fourier transform infrared spectroscopy;

[0097] S112: Figure 1 As shown, in the X-ray diffractometer diffraction pattern, NF, YF, GF and TF are consistent with the standard Nd, Yb, Gd and Tm and ferrite standard crystal diffraction cards;

[0098] S113: Figure 3 As shown in the Fourier infrared spectrum, NF, YF, GF and TF are at 2964, 1464, 1368 and 564 cm -1 The characteristic peaks of composite ferrite appeared at , and the results proved the successful preparation of NF, YF, GF and TF.

[0099] like Figure 3 As shown in FIG. 1 , different doped rare earth ferrites were dispersed with ultrapure water, placed in an alternating magnetic field with a frequency of 498 kHz and a field strength of 1.6 mT for 10 minutes, and their temperature rise was monitored. Figure 3 It can be seen that TF and YF have relatively excellent magnetocaloric properties, and the temperature rises to above 60°C after 10 minutes. Therefore, TF and YF were selected for the next screening.

[0100] like Figure 4 As shown, for Fe 3 O 4 :Yb / Tm is characterized by different doping amounts:

[0101] The rare earth doping amount affects the dispersion and magnetocaloric properties of ferrite, so the next step of screening is to verify the resolubility and magnetocaloric properties of ferrite. With the increase of rare earth element doping amount, the temperature rise shows a trend of first increasing and then decreasing. Figure 4 From the statistics in Tables 1.1 and 1.2, it can be seen that the magnetothermal heating effect of YF under different rare earth doping ratios is better than that of TF, so Yb doping is selected. In addition, when the rare earth doping amount is greater than 2%, the obtained material will have reduced solubility and stability, so the final Yb doping amount is selected to be 1.5%.

[0102] Table 1.1 Effect of different ratios of Tm doping on magnetocaloric properties

[0103]

[0104] Table 1.2 Effect of different proportions of Yb doping on magnetocaloric effect

[0105]

[0106] S2: Perform Yb 0.015 Fe 2.985 O 4 @ Preparation and characterization of PEI-HA;

[0107] In S2, for Yb 0.015 Fe2.985 O 4 @PEI-HA was prepared and characterized by electrostatic adsorption method to explore the surface active concentration. The steps are as follows:

[0108] S21: Dissolve 10 mg YF and X mg PEI in 10 mL ultrapure water respectively;

[0109] S22: Under ultrasound, the YF solution was slowly added dropwise to the PEI solution, and the mixed solution was placed in a constant temperature shaker at room temperature, 120 r, 24 h, and centrifuged at 8000 r / min for 30 min to collect the precipitate to obtain YF@PEI;

[0110] S23: 10 mg YF@PEI and X mg HA were dissolved in ultrapure water, and the YF@PEI solution was slowly added dropwise to the HA solution. The mixed solution was placed in a constant temperature shaker at room temperature, 120 r, 24 h, and centrifuged at 8000 r / min for 30 min to collect the precipitate to obtain YF@PEI-HA (hereinafter referred to as YFH);

[0111] S24: The optimal surfactant ratio was selected by measuring particle size and dispersibility. 1 mg / mL YF, 0.5 mg / mL PEI and 0.1 mg / mL HA were the optimal surfactant ratios.

[0112] like Figure 7-Figure 8 As shown in the figure, the potential change of YFH also proves that the surfactant is successfully encapsulated and has good stability within a certain period of time. After encapsulating the surfactant, 2 mg YFH still maintains excellent magnetocaloric performance during 5 repeated magnetocaloric cycles.

[0113] S3: Conduct MET@Yb 0.015 Fe 2.985 O 4 @ Preparation and characterization of PEI-HA;

[0114] In S3, MET@Yb 0.015 Fe 2.985 O 4 The preparation and characterization of @PEI-HA were carried out using the following steps:

[0115] S31: Solvent impregnation method was used to investigate the proportion of MET encapsulation;

[0116] Drug loading is accomplished through the drug diffusion effect, whereby high concentrations of drug molecules diffuse from the solution into the mesopores and hollow cavities of the nanocarrier until equilibrium is reached.

[0117] First, 10 mg of YF was ultrasonically dispersed in 10 mL of ultrapure water to prepare a 1 mg / mL dispersion. An appropriate amount of MET was weighed and dissolved in ultrapure water. 10 mL of the nanocarrier dispersion and 10 mL of the MET solution were mixed in equal volumes. The mixed solution was placed in a 37°C constant temperature shaker for 24 h, centrifuged at 8000 r / min for 20 min, and washed with ultrapure water three times to ensure that there were no drug molecules adsorbed on the surface of the nanoparticles. All supernatants were collected, and the absorbance was measured by UV spectrophotometer to calculate the encapsulation efficiency (EE%) and drug loading (DL%). As shown in Table 3, according to the encapsulation efficiency and drug loading, we finally chose a ratio of YFH:MET = 1:5, with an encapsulation efficiency of 40% and a drug loading of 66.7%. The encapsulation efficiency was low probably because MET had good water solubility and YFH occupied part of the pores when encapsulating the surface active agent.

[0118] Table 3 Drug loading rate and encapsulation efficiency of YFH loaded with different proportions of MET

[0119]

[0120] S32: Using dialysis to explore MET@Yb 0.015 Fe 2.985 O 4 @PEI-HA released in vitro;

[0121] The dialysis method was used to explore the release of the drug in vitro. Considering the acidic environment of tumor tissue and the normal physiological environment of the human body, the drug release experiment was carried out in buffer solutions of pH = 5.5, 6.5, and 7.4. 5 mg of MYFH was weighed and completely dispersed in 2 mL of different pH solutions. The solution was transferred to a dialysis bag with a molecular weight cutoff of 1000, and the dialysis bag was immersed in 40 mL of solutions with different pH values. It was placed in a 37°C constant temperature shaker, and 2 mL of solution was taken out from PBS at a certain interval, and 2 mL of blank buffer was added to the release medium, keeping the total volume of PBS unchanged. The drug absorption value in the PBS solution taken out at different times was measured by a spectrophotometer, repeated 3 times, and the average value was taken. The drug release curve was calculated.

[0122] according to Fig. 9 It can be seen that the drug-loaded nanoparticles have a fast release rate in 0-24h, and the drug release is relatively slow in 24-48h. In the early stage of diffusion, there is a large concentration difference between the nano-loaded drug and the release medium, so the early release rate is fast. As the drug is released, the concentration difference between the two tends to become smaller and smaller, and the release rate slows down.

[0123] S33: To MET@Yb 0.015 Fe 2.985 O4 @PEI-HA was prepared;

[0124] The preparation method in S33 specifically comprises the following steps:

[0125] S331: Use an analytical balance to weigh 0.92 g of ferric chloride hexahydrate, 0.02 g of ytterbium chloride hexahydrate, 0.6 g of urea, 0.8 g of polyacrylamide, and 1.76 g of sodium citrate and dissolve them in 60 mL of ultrapure water. Stir the mixture at room temperature. After it is completely dissolved, transfer the clear solution to a reactor and react at 200°C for 8 h.

[0126] S332: After the reaction is completed, centrifuge and collect at a speed of 10000r / min for 30min, discard the supernatant and collect the precipitate, wash the precipitate with ethanol: ultrapure water = 1:2, repeat the washing and centrifugation twice to obtain YF, and freeze-dry;

[0127] S333: Disperse 20 mg of YF in 20 mL of ultrapure water and 10 mg of PEI in 20 mL of ultrapure water. Slowly add YF to PEI under ultrasound, place in a 37°C shaker for 24 h, centrifuge at 8000 r / min for 30 min, discard the supernatant, and wash the precipitate with ultrapure water three times.

[0128] S334: YF@PEI was dissolved in ultrapure water again, 2 mg HA was weighed and dispersed in 20 mL ultrapure water, HA was dissolved in ultrapure water, and the solution was weighed according to the mass ratio with YF@PEI. Under ultrasound, the YF@PEI solution was added dropwise to the HA solution, shaken on a shaker for 120 r. After 24 h, the mixture was collected by centrifugation at 8000 r / min for 30 min, the supernatant was discarded, and the mixture was washed with ultrapure water for 3 times to obtain YFH dissolved in ultrapure water;

[0129] S335: Prepare 5 mg / mL MET solution, drop the MET solution into the YFH solution, place the mixed solution in a 37°C constant temperature shaker for 24 hours, use a centrifuge at 8000 r / min for 20 minutes, wash with ultrapure water three times, collect the MYFH precipitate, and freeze-dry it for later use.

[0130] S34: To MET@Yb 0.015 Fe 2.985 O 4 @The basic structure of PEI-HA was characterized;

[0131] The morphology of synthesized MYFH was characterized by SEM. Fig.10 , 11, it can be seen that the prepared nano drug-carrying particles are similar to raspberry morphology and have a relatively uniform size. The particle size of the nano drug-carrying particles is 160nm. In the XRD diffraction pattern ( Fig.12 ), in Yb and Fe3 O 4 The crystal diffraction card is completely consistent with that of Fig.13 ) also showed obvious MET characteristic peaks, which indicated the successful preparation of MYFH. Moreover, MYFH had good biocompatibility and stability in ultrapure water, PBS and DMEM complete culture medium.

[0132] S35: To MET@Yb 0.015 Fe 2.985 O 4 @PEI-HA magnetocaloric properties were characterized;

[0133] like Fig.15 As shown in the figure, in order to explore the magnetic effect of MYFH, the hysteresis loop of MYFH was detected by a vibrating sample magnetometer. At room temperature, the saturation magnetization of MYFH reached 66.1emu / g, and the coercive force was 21.6Oe, which is lower than 25Oe. [1] , which shows that MYFH has a superparamagnetic effect. After placing a magnet next to the MYFH solution, it can be clearly observed that the nanoparticles are successfully adsorbed on one side of the magnet after 30 seconds, and the solution becomes clear and transparent, indicating that it has good magnetic responsiveness. We placed different concentrations of MYFH in an alternating magnetic field of 498kHz and 1.6mT and recorded once a minute to study its magnetocaloric properties. Fig.16 It can be seen that MYFH has excellent magnetocaloric performance, and the temperature and concentration are correlated, that is, as the concentration of MYFH increases, the magnetocaloric heating increases. In 5 magnetocaloric cycles, it still has excellent heating efficiency.

[0134] The above references are: [1] Miao W, Qian C, Dong X, et al. Self-cycling redox nanoplatformin

[0135] synergy with mild magnetothermal and autophagy inhibition for efficient cancer therapy[J].

[0136] Nano Today, 2022, 43: 101374.

[0137] S36: To MET@Yb 0.015 Fe 2.985 O 4 @Analysis of PEI-HA imaging performance;

[0138] To investigate whether MYFH has excellent MRI T 2Imaging performance: MYFH was prepared into dispersions of different concentrations, dispersed in 1.5% agarose gel, and nuclear magnetic resonance imaging was performed in a Siemens Magnetom Spectra 3.0T.

[0139] In MRI imaging, Fe ions are usually T 2 Weighted imaging, in Fig.17 It can be seen that MYFH has T 2 Weighted effect, it is obvious that the image becomes darker as the concentration increases, T 2 The signal is getting stronger and stronger. The imaging ability of MYFH can be further quantified by r 2 / r 1 The numerical results show that MYFH has excellent T 2 Imaging capabilities.

[0140] S37: Determine MET@Yb 0.015 Fe 2.985 O 4 @PEI-HA has multiple enzyme activities;

[0141] MET@Yb 0.015 Fe 2.985 O 4 @PEI-HAPOD-Like enzyme activity:

[0142] The POD-Like catalytic activity of MYFH was evaluated by colorimetric method, with 3,3',5,5'-tetramethylbenzidine (TMB) as the chromogenic substrate and H 2 O 2 The redox reaction of the oxidant oxidizes TMB from colorless to blue. Blue TMB has an absorption peak at 652nm, such as Fig.18 As shown in the figure, under the simulated tumor microenvironment and normal physiological conditions, pH was set at 7.4, 6.5, and 5.5, respectively. As the pH value decreased, MYFH showed stronger POD-Like enzyme activity. In order to maintain a constant temperature, a water bath was used to simulate a constant thermal environment. As the temperature increased, the POD-Like enzyme activity was further improved.

[0143] MET@Yb 0.015 Fe 2.985 O 4 @PEI-HAGSHOx enzyme activity:

[0144] The colorimetric method was used to evaluate the GSHox enzyme activity of MYFH. 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) reacted rapidly with GSH to generate a pale yellow reaction product. Yellow DTNB has an absorption peak at 412nm. Fig.19As shown in the figure, as the pH decreases, the GSHox enzyme activity produced by MYFH becomes stronger and the GSH consumption gradually increases. Under the simulated temperature, temperature can also affect the GSHox enzyme activity. The higher the temperature, the stronger the catalytic activity of the enzyme.

[0145] MET@Yb 0.015 Fe 2.985 O 4 @PEI-HACAT enzyme activity:

[0146] Use a dissolved oxygen meter to test the CAT enzyme performance of MYFH. Fig. 20 As shown, in H 2 O 2 (100 μM), more O was produced under neutral conditions than under acidic conditions. 2 , indicating that MYFH has CAT enzyme activity. In the temperature simulation, as the temperature increases, the amount of O 2 The more MYFH is, the more it indicates that MYFH can affect CAT enzyme activity as the temperature increases, and the enzyme activity is excellent.

[0147] S38: Analysis 1 O 2 The ability to produce.

[0148] The fluorescent molecule 1,3-diphenylisobenzofuran (DPBF) can be used with 1 O 2 The reaction generates a new compound, 1,2-dibenzoylbenzene, which shows a characteristic of decreased absorption at 410nm of UV-visible light. Fig.21 As shown in the figure, under 1064nm laser irradiation, the absorption light of DPBF decreased, indicating that MYFH has the ability to produce 1 O 2 In the simulated thermal environment in vitro, DPBF decreased and increased with increasing temperature. This is because the increase in heat will increase the activity of CAT enzyme and produce more O 2 , providing sufficient raw materials for the PDT reaction, thus producing 1 O 2 capacity enhancement.

[0149] S4: MET@Yb 0.015 Fe 2.985 O 4 @Analysis of the anti-tumor effect of PEI-HA in vitro;

[0150] In S4, in vitro antitumor effect analysis included MET@Yb 0.015 Fe 2.985 O 4 @PEI-HA biosafety, BBB crossing experiment, cell killing effect analysis, cell autophagy level evaluation and cell ferroptosis level detection.

[0151] MET@Yb 0.015 Fe 2.985 O 4 @PEI-HA Biosafety:

[0152] L929, Bend.3 and GL261 cells in the logarithmic growth phase were collected and cultured at 1×10 4 The concentration of 100 μg / well was inoculated in a 96-well plate and adhered to the wall in a 37°C incubator overnight. 20 μL of MET, YFH and MYFH culture medium of different concentrations were added to each well, and the final concentrations in the well plate were (400 μg / mL, 300 μg / mL, 200 μg / mL, 100 μg / mL, 50 μg / mL, 25 μg / mL, 12.5 μg / mL, 6.25 μg / mL, 3.125 μg / mL, 0 μg / mL). Incubate in an incubator at 37°C for 24 hours, add 20 μL of MTT solution (5 mg / mL), continue incubation for 4 hours, discard the culture medium, add 200 μL of DMSO solution to each well, shake at room temperature for 20 minutes, and measure the absorbance at 490 nm using an enzyme reader. Experiments were performed using mouse fibroblasts (L929), mouse glioma cell lines (GL261), and mouse brain endothelial cell lines (bEnd.3). like Fig. 22 As shown in the figure, when the final concentration of YFH and MYFH was below 300 μg / mL, the cell survival rate was greater than 80%, indicating that both had good biosafety. MET basically had no corresponding toxicity to cells, which may be due to the relatively poor permeability of MET, short half-life, and the need to enter the mitochondria to exert its ability to kill tumor cells. In summary, we selected a material concentration of 200 μg / mL for subsequent experiments.

[0153] Crossing BBB experiment:

[0154] Disperse 10 mg of Rhodamine B (RHB) into 25 mL of ultrapure water, and add 5 mg of MYFH into the solution. Transfer the mixed solution to a shaker and continue shaking at 120 rpm / min for 12 hours. After the mixture is centrifuged at 10,000 r / min for 20 minutes, discard the supernatant, add 20 mL of ultrapure water, repeat the washing twice, collect the precipitate, and freeze-dry for 12 hours to obtain MYFH-RHB (MYFHB) powder.

[0155] The BBB model was simulated in vitro using a cell Transwell chamber. bEnd.3 cells were cultured in the upper chamber until the transepithelial electrical resistance (TEER) of the upper chamber was ΔTEER = TEER cell group -TEERBlank ≥ 180 Ω / cm 2When GL261 cells were cultured in the lower chamber, the in vitro BBB was successfully constructed. Different stimulation conditions were applied: (1) AFM+MYFHB group; (2) AFM+MYFHB+M group. M is the magnet group, specifically a rubidium magnet with a diameter of 1 cm and a magnetic field strength of 0.04 T was used at the bottom of a 24-well plate. The final concentration of MYFHB was 200 μg / mL. The groups after treatment were placed in a confocal microscope to record fluorescence images. Fig.23 As shown in the figure, after applying AMF, red fluorescence was visible at 2h, and a large amount of fluorescence was enriched at 4h. After applying AMF and external magnets, fluorescence signals were observed at 1h, and a large amount of red fluorescence signals were visible at 2h, indicating that MYFH can effectively cross the BBB in a short time. When an exogenous magnetic field is applied, the magnetic targeting ability of MYFH further accelerates its ability to cross the BBB and improves the uptake of MYFH by GL261 cells.

[0156] Analysis of cell killing effect:

[0157] The MTT method was used to detect the cell killing effect, and the groups were set as follows: (1) PBS group; (2) single stimulation group; (3) MET group; (4) YFH group; (5) MYFH group; (6) CDT treatment group; (7) PDT treatment group; (8) CDT+PDT treatment group; (9) MHT group; (10) MHT+CDT group; (11) MHT+PDT group; (12) MHT+CDT+PDT group. The concentrations of MET, YFH, and MYFH were all 200 μg / mL; the CDT group H 2 O 2 The concentration was 50 μM; the PDT group used a 1064 nm laser with a laser power of 0.33 W / cm 2 , time 10min; the magnetic field intensity of the MHT group was 498KHz, the field strength was 1.6mT, and the time was 10min. Fig.24 As shown, the cell survival rates in the MET, YFH, and MYFH groups were all >80%, indicating that GL261 cells were relatively biosafe; the cell survival rates in the MYFH+H and MYFH+L groups were 70%, and the cell viability decreased; the cell survival rate in the MYFH+H+L group was 60%; the cell survival rate in the MYFH+A group was 43%; the cell survival rates in the MYFH+A+H and MYFH+A+L groups were 22% and 32%; and the cell survival rate in the MYFH+A+H+L group was 12%. All of them were lower than the control group, with significant differences. In summary, under the combined treatment of CDT-PDT-MHT, MYFH can have a significant tumor therapeutic effect on GL261.

[0158] Evaluation of cell autophagy level:

[0159] The effect of MYFH nanomaterials on the level of intracellular autophagy was evaluated using a cell autophagy (MDC) staining detection kit. After MYFH (final concentration 200 μg / mL) was added to the growing GL261 cells and incubated for 6 hours, different stimulation conditions were applied, the cells were stained according to the cell autophagy staining detection kit, and observed and photographed using a confocal microscope. The groups were as follows: (1) PBS group; (2) 1064nm+H 2 O 2 ; (3) MET group; (4) YFH group; (5) MYFH group; (6) CDT treatment group; (7) PDT treatment group; (8) CDT+PDT treatment group; (9) AMF+1064nm+H 2 O 2 ; (10) MHT group; (11) MHT+CDT group; (12) MHT+PDT group; (13) MHT+CDT+PDT group (the following groups are the same as this grouping). Fig.25 As shown in the figure, compared with the control group, the final group showed a higher level of autophagy, because under the action of AMF, autophagy is a highly conservative intracellular degradation process. High temperature promotes the generation of ROS, causing oxidative stress in cells and opening the autophagy channel. For MET, MET enters the mitochondria and inhibits the mTORC1 pathway, which increases the level of autophagy.

[0160] Cellular iron death level detection:

[0161] The lipid peroxide (MDA) kit was used to detect the malondialdehyde content in different groups to further evaluate the changes in cell LPO. The MDA content in different treatment groups was obtained by detecting the absorbance value of MDA-thiobarbituric acid conjugate at 553nm and comparing it with the MDA standard curve. Fig.26 As shown, compared with the control group, group 10 (MHT group) had a significant LPO effect. And in the final group, under the synergistic effect of CDT-PDT-MHT, magnetic heat further enhanced the ferroptosis of GL261 induced by MYFH compared with group 8 (CDT-PDT group).

[0162] S5: To MET@Yb 0.015 Fe 2.985 O 4 @PEI-HA in vivo MRI-T 2 Analyze the imaging effect;

[0163] In S5, the drug imaging effect was analyzed using a Spectra 3.0T nuclear magnetic resonance imaging device and the mice were imaged; the groups were: (1) MYFH+M group; (2) MYFH+A group; (3) MYFH+A+M group.

[0164] In the absence of AMF, even with the action of an external magnet, T 2 There was no enhancement signal, only the lesion signal; after applying AMF, in the case of external magnet response, T appeared at 3h. 2 The signal was enhanced, while the group without magnet also showed T at 5h. 2 The enhanced signal confirmed that AMF can effectively open the BBB briefly.

[0165] S6: To MET@Yb 0.015 Fe 2.985 O 4 @The anti-tumor effect of PEI-HA in vivo was analyzed.

[0166] In S6, analyzing the anti-tumor effect of the drug in vivo includes the following steps:

[0167] S61: Orthotopic tumors in the brain region of C57BL / 6J magnetic mice around 7 weeks old were treated with GL261-LUC;

[0168] S62: An orthotopic glioblastoma model was successfully established. The treatment method was tail vein administration, 1064nm laser irradiation and magnetic hyperthermia therapy. During the treatment cycle, the therapeutic effect of tumor-bearing mice was monitored by intraperitoneal injection of D-luciferin potassium salt and observed in an animal living imaging device.

[0169] S63: The groups were as follows: (1) PBS; (2) MYFH+M; (3) MYFH+AMF+M; (4) MYFH+AMF+M+1064nm; (5) MYFH+AMF+M+MHT; (6) YFH+AMF+M+MHT+1064nm; (7) MYFH+AMF+M+MHT+1064nm;

[0170] S64: The results showed that in groups (1, 2), the BBB was not opened and the treatment effect was poor. After the BBB was opened, the combined treatment effect increased and the treatment effect was obvious, inhibiting the tumor growth.

[0171] The dual-targeted drug-loaded superparamagnetic nanoparticles for glioma diagnosis and treatment are prepared by the synthesis method of the dual-targeted drug-loaded superparamagnetic nanoparticles for glioma diagnosis and treatment.

[0172] The application of dual-targeted drug-loaded superparamagnetic nanoparticles for the diagnosis and treatment of gliomas enhances the ability of cell autophagy and ferroptosis by encapsulating the three treatment modes of MHT-PDT-CDT for synergistic treatment.

[0173] The above is only a preferred embodiment of the present invention. It should be noted that, for those skilled in the art, several improvements and modifications can be made without departing from the principles of the present invention, and these improvements and modifications should also be considered as the protection scope of the present invention. The structures, devices and operating methods not specifically described and explained in the present invention shall be implemented according to the conventional means in the art unless otherwise specified and limited.

Claims

1. A method for synthesizing dual-targeted drug-loaded superparamagnetic nanoparticles for glioma diagnosis and treatment, characterized in that: The synthesis method comprises the following steps: S1: Perform Re x Fe 3-x Preparation and characterization of O4; S2: Perform Yb 0.015 Fe 2.985 Preparation and characterization of O4@PEI-HA; S3: Conduct MET@Yb 0.015 Fe 2.985 Preparation and characterization of O4@PEI-HA; S4: MET@Yb 0.015 Fe 2.985 The in vitro antitumor effect of O4@PEI-HA was analyzed; S5: To MET@Yb 0.015 Fe 2.985 The in vivo MRI-T2 imaging effect of O4@PEI-HA was analyzed; S6: To MET@Yb 0.015 Fe 2.985 The in vivo antitumor effect of O4@PEI-HA was analyzed.

2. The method for synthesizing dual-targeted drug-loaded superparamagnetic nanoparticles for glioma diagnosis and treatment according to claim 1, characterized in that: In S1, the preparation of Nd / Yb / Gd / Tm in Fe3O4 is carried out by the following steps: S11: Use an analytical balance to weigh 0.92 g of ferric chloride hexahydrate, x g of neodymium / ytterbium / gadolinium / thulium chloride hexahydrate, with a doping ratio of 5%, and calculate the X value according to the mass of different rare earths, 0.6 g of urea, 0.8 g of polyacrylamide, and 1.76 g of sodium citrate and dissolve them in 60 mL of ultrapure water; S12: Stir the mixture at room temperature, transfer the clear solution to a reactor after it is completely dissolved, and react at 200° C. for 500 min; S13: After the reaction is completed, centrifuge and collect the precipitate at a speed of 10000r / min for 30min, discard the supernatant and collect the precipitate; S14: Wash the precipitate with ethanol: ultrapure water = 1:2, repeat the washing and centrifugation twice to obtain a black precipitate product, and freeze-dry it; The following steps were used to characterize Nd / Yb / Gd / Tm in Fe3O4: S111: Characterized by X-ray diffractometer and Fourier transform infrared spectroscopy; S112: In the X-ray diffractometer diffraction pattern, NF, YF, GF and TF are consistent with the standard Nd, Yb, Gd and Tm and ferrite standard crystal diffraction cards; S113: In the Fourier transform infrared spectrum, NF, YF, GF and TF are at 2964, 1464, 1368 and 564 cm-1, respectively. -1 The characteristic peaks of composite ferrite appeared at , and the results proved the successful preparation of NF, YF, GF and TF.

3. The method for synthesizing dual-targeted drug-loaded superparamagnetic nanoparticles for glioma diagnosis and treatment according to claim 2, characterized in that: In S2, Yb 0.015 Fe 2.985 Preparation and characterization of O4@PEI-HA The electrostatic adsorption method was used to explore the surface active concentration, and the steps are as follows: S21: Dissolve 10 mg YF and X mg PEI in 10 mL ultrapure water respectively; S22: Under ultrasound, the YF solution was slowly added dropwise to the PEI solution, and the mixed solution was placed in a constant temperature shaker at room temperature, 120 r, 24 h, and centrifuged at 8000 r / min for 30 min to collect the precipitate to obtain YF@PEI; S23: 10 mg YF@PEI and X mg HA were dissolved in ultrapure water, and the YF@PEI solution was slowly added dropwise to the HA solution. The mixed solution was placed in a constant temperature shaker at room temperature, 120 r, 24 h, and centrifuged at 8000 r / min for 30 min to collect the precipitate to obtain YF@PEI-HA; S24: The optimal surfactant ratio was selected by measuring particle size and dispersibility. 1 mg / mL YF, 0.5 mg / mL PEI and 0.1 mg / mL HA were the optimal surfactant ratios.

4. The method for synthesizing dual-targeted drug-loaded superparamagnetic nanoparticles for glioma diagnosis and treatment according to claim 1, characterized in that: In S3, MET@Yb 0.015 Fe 2.985 The preparation and characterization of O4@PEI-HA were carried out using the following steps: S31: Solvent impregnation method was used to investigate the proportion of MET encapsulation; S32: Using dialysis to explore MET@Yb 0.015 Fe 2.985 O4@PEI-HA was released in vitro; S33: To MET@Yb 0.015 Fe 2.985 O4@PEI-HA was prepared; S34: To MET@Yb 0.015 Fe 2.985 The basic structure of O4@PEI-HA was characterized; S35: To MET@Yb 0.015 Fe 2.985 The magnetocaloric properties of O4@PEI-HA were characterized; S36: To MET@Yb 0.015 Fe 2.985 The imaging performance of O4@PEI-HA was analyzed; S37: Determine MET@Yb 0.015 Fe 2.985 O4@PEI-HA has multiple enzyme activities; S38: Analysis 1 O2 production capacity.

5. The method for synthesizing dual-targeted drug-loaded superparamagnetic nanoparticles for glioma diagnosis and treatment according to claim 4, characterized in that: The preparation method in S33 specifically comprises the following steps: S331: Use an analytical balance to weigh 0.92 g of ferric chloride hexahydrate, 0.02 g of ytterbium chloride hexahydrate, 0.6 g of urea, 0.8 g of polyacrylamide, and 1.76 g of sodium citrate and dissolve them in 60 mL of ultrapure water. Stir the mixture at room temperature. After it is completely dissolved, transfer the clear solution to a reactor and react at 200°C for 8 h. S332: After the reaction is completed, centrifuge and collect at a speed of 10000r / min for 30min, discard the supernatant and collect the precipitate, wash the precipitate with ethanol: ultrapure water = 1:2, repeat the washing and centrifugation twice to obtain YF, and freeze-dry; S333: Disperse 20 mg of YF in 20 mL of ultrapure water and 10 mg of PEI in 20 mL of ultrapure water. Slowly add YF to PEI under ultrasound, place in a 37°C shaker for 24 h, centrifuge at 8000 r / min for 30 min, discard the supernatant, and wash the precipitate with ultrapure water three times. S334: YF@PEI was dissolved in ultrapure water again, 2 mg HA was weighed and dispersed in 20 mL ultrapure water, HA was dissolved in ultrapure water, and the solution was weighed according to the mass ratio with YF@PEI. Under ultrasound, the YF@PEI solution was added dropwise to the HA solution, shaken on a shaker for 120 r. After 24 h, the mixture was collected by centrifugation at 8000 r / min for 30 min, the supernatant was discarded, and the mixture was washed with ultrapure water for 3 times to obtain YFH dissolved in ultrapure water; S335: Prepare 5 mg / mL MET solution, drop the MET solution into the YFH solution, place the mixed solution in a 37°C constant temperature shaker for 24 hours, use a centrifuge at 8000 r / min for 20 minutes, wash with ultrapure water three times, collect the MYFH precipitate, and freeze-dry it for later use.

6. The method for synthesizing dual-targeted drug-loaded superparamagnetic nanoparticles for glioma diagnosis and treatment according to claim 1, characterized in that: In S4, the in vitro anti-tumor effect analysis includes MET@Yb 0.015 Fe 2.985 O4@PEI-HA biosafety, BBB crossing experiment, cell killing effect analysis, cell autophagy level evaluation and cell ferroptosis level detection.

7. The method for synthesizing dual-targeted drug-loaded superparamagnetic nanoparticles for glioma diagnosis and treatment according to claim 1, characterized in that: In S5, the drug imaging effect is analyzed by using a Spectra 3.0T nuclear magnetic resonance imaging device to image the mice; When AMF was not provided, even with the action of an external magnet, there was no enhanced T2 signal, only a lesion signal; after AMF was applied, with the response of an external magnet, an enhanced T2 signal appeared at 3 hours, and the group without magnet also showed an enhanced T2 signal at 5 hours, which proved that AMF can effectively open the BBB temporarily.

8. The method for synthesizing dual-targeted drug-loaded superparamagnetic nanoparticles for glioma diagnosis and treatment according to claim 1, characterized in that: In S6, analyzing the anti-tumor effect of the drug in vivo comprises the following steps: S61: Orthotopic tumors in the brain region of C57BL / 6J magnetic mice around 7 weeks old were treated with GL261-LUC; S62: An orthotopic glioblastoma model was successfully established. The treatment method was tail vein administration, 1064nm laser irradiation and magnetic hyperthermia therapy. During the treatment cycle, the therapeutic effect of tumor-bearing mice was monitored by intraperitoneal injection of D-luciferin potassium salt and observed in an animal living imaging device. S63: The groups were as follows: (1) PBS; (2) MYFH+M; (3) MYFH+AMF+M; (4) MYFH+AMF+M+1064nm; (5) MYFH+AMF+M+MHT; (6) YFH+AMF+M+MHT+1064nm; (7) MYFH+AMF+M+MHT+1064nm; S64: The results showed that in groups (1, 2), the BBB was not opened and the treatment effect was poor. After the BBB was opened, the combined treatment effect increased and the treatment effect was obvious, inhibiting the tumor growth.

9. Dual-targeted drug-loaded superparamagnetic nanoparticles for glioma diagnosis and treatment, characterized in that: The dual-targeted drug-loaded superparamagnetic nanoparticles for glioma diagnosis and treatment are prepared by the synthesis method of any one of claims 1-8.

10. Application of dual-targeted drug-loaded superparamagnetic nanoparticles for glioma diagnosis and treatment, characterized in that: By encapsulating the three treatment modes of MHT-PDT-CDT for synergistic treatment, the ability of cell autophagy and ferroptosis is enhanced.

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