Surface functionalized iron phosphide nanoparticles and nanomedicine containing the same, as well as preparation method and application thereof

Abstract

The present invention belongs to the field of nanobiomedical materials and relates to a surface-functionalized iron phosphide nanoparticle and a nanomedicine containing the same, as well as a preparation method and application thereof. The preparation method of the surface-functionalized iron phosphide nanoparticle comprises the following steps: using iron phosphide nanoparticles and molecules containing thiol groups and other groups that can coordinate with iron to obtain surface-thiolated iron phosphide nanoparticles, and then reacting with active ingredients containing thiol groups to obtain. The nanomedicine is composed of the surface-functionalized iron phosphide nanoparticles and exosomes. The present invention modifies the surface of the iron phosphide nanoparticles and further prepares nanomedicines, which can target brain tissue and cooperate with multiple pathways to significantly improve the efficacy of tumors (especially temozolomide-resistant tumors) while having good biosafety.

Description

Technical Field

[0001] The present invention belongs to the field of nano-biomedical materials, and relates to surface-functionalized iron phosphide nanoparticles, nano-medicine containing the same, and a preparation method and application thereof. Background Art

[0002] Glioblastoma is the most common primary malignant tumor of the central nervous system (CNS) in adults worldwide, with an annual incidence of approximately 3 to 6.4 per 100,000 people. The incidence of World Health Organization (WHO) grade IV glioblastoma (GBM) is approximately 4.03 per 100,000 people, accounting for 49.1% of all primary malignant CNS tumors. Currently, the standard treatment for glioma is the Stupp regimen, which combines surgical resection, radiation therapy, and adjuvant oral chemotherapy with temozolomide (TMZ). This regimen has increased the median survival of glioblastoma patients to 16 months. While this treatment regimen can prolong survival, the overall prognosis for GBM remains poor, with most clinical data showing a five-year survival rate of less than 10%. Currently, TMZ is the only effective oral chemotherapy drug for glioma, but at least 50% of patients do not respond to TMZ, and nearly all patients experience tumor recurrence after standard treatment.

[0003] TMZ induces cell cycle arrest and apoptosis by generating diazomethane to induce methylation of specific sites of guanine and adenine in genomic DNA. Studies have shown that TMZ resistance is not caused by a single molecular event, but by multiple events. It is currently widely recognized that TMZ resistance is related to the expression levels of DNA alkylation proteins and DNA repair enzymes. 6 β-Methylguanine DNA methyltransferase (MGMT) plays a key role. Overexpressed MGMT in tumor cells can remove the methyl group at this site and repair DNA damage caused by TMZ. Studies have shown that downregulation of glutathione (GSH) in cells can inhibit the WNT / β-catenin signaling pathway, thereby inhibiting the expression of MGMT.

[0004] In addition, while TMZ produces diazomethane, another metabolite, 5-aminoimidazole-4-carboxamide (AICA), catalyzed by hypoxanthine phosphoribosyltransferase 1 (HPRT1), ultimately induces DNA repair through a series of cascade reactions. 6-Mercaptopurine (6-MP) is a potent HPRT1 inhibitor that can inhibit the DNA repair process caused by AICA.

[0005] Iron phosphide (FeP) is a transition metal phosphide with low cost and excellent comprehensive performance. It has good biosafety as a nanomedicine. Since non-metallic phosphorus and transition metal iron have multiple valence states, FeP can realize the cycle between multiple valence states.2+ Can mediate the Fenton reaction to form hydroxyl radicals, Fe 3+ It can react with reducing substances, deplete GSH in cells, downregulate the expression of glutathione peroxidase 4 (GPX4) and enhance lipid peroxidation, leading to enhanced ferroptosis.

[0006] Exosomes are a type of extracellular vesicle with a diameter ranging from 30 to 150 nm. They are secreted by virtually all cells and serve as important mediators of intercellular signaling and potential drug delivery vehicles. Microglia, the primary immune cells in the central nervous system, secrete exosomes that not only cross the blood-brain barrier but also modulate exosome content by regulating microglial polarization, potentially enabling the development of new therapeutic strategies. FeP nanoparticles coated with microglia-derived exosomes can cross the blood-brain barrier and be delivered to tumor tissue, improving therapeutic accuracy and mitigating damage to healthy tissue.

[0007] FeP nanoparticles have great potential for application in tumor treatment. However, single FeP nanoparticles are not only monofunctional and inefficient, but also unable to reach the lesions. Given these issues, it is necessary to prepare surface-functionalized FeP nanoparticles and design an optimized biomimetic nanomedicine to address these challenges in glioma treatment. Summary of the Invention

[0008] Problems to be solved by the invention

[0009] The present invention aims to provide surface-functionalized iron phosphide nanoparticles and nanomedicines containing the same, as well as a preparation method and use thereof in the treatment of tumors (especially drug-resistant tumors). After surface modification, the iron phosphide nanoparticles not only have efficient photothermal conversion capabilities, but can also significantly reduce the glutathione concentration level and MGMT protein expression level in tumor cells and inhibit HPRT1 protein activity. The nanomedicine can penetrate the blood-brain barrier and deliver the drug to tumor tissue in a targeted manner, reducing damage to healthy tissue.

[0010] Solutions for solving problems

[0011] In a first aspect, the present invention provides a method for preparing surface-functionalized iron phosphide nanoparticles, comprising the following steps:

[0012] (1) Surface-thiolated iron phosphide nanoparticles are obtained by using iron phosphide nanoparticles and molecules containing thiol groups and other groups that can coordinate with iron;

[0013] (2) reacting the surface-thiolated iron phosphide nanoparticles obtained in step (1) with an active ingredient containing a thiol group to obtain;

[0014] Wherein, the active ingredient containing a thiol group is a small molecule drug containing a thiol group or a thiolated nucleic acid; preferably, the small molecule drug containing a thiol group is 6-mercaptopurine.

[0015] Preferably, the iron phosphide nanoparticles are prepared by chemical vapor deposition or organic solution phase reaction.

[0016] Preferably, in step (1), the iron phosphide nanoparticles and molecules containing thiol groups and other groups that can coordinate with iron undergo coordination exchange to obtain the surface thiol-modified iron phosphide nanoparticles.

[0017] Preferably, the molecule containing a thiol group and other groups capable of coordinating with iron is 2,3-dimercaptosuccinic acid.

[0018] Preferably, step (1) comprises: adjusting the pH of the aqueous solution of the molecules containing thiol groups and other groups that can coordinate with iron to 9-11, dripping the solution into the dispersion of the iron phosphide nanoparticles, ultrasonicating or stirring, centrifuging, washing, and freeze-drying to obtain surface thiol-modified iron phosphide nanoparticles.

[0019] More preferably, the concentration of the aqueous solution of the molecules containing thiol groups and other groups that can coordinate with iron is 10 to 50 mg mL -1 The concentration of the dispersion of the iron phosphide nanoparticles is 1 to 10 mg mL -1 .

[0020] More preferably, the iron phosphide nanoparticles are prepared by chemical vapor deposition, and the dispersant in the dispersion of the iron phosphide nanoparticles is water; alternatively, the iron phosphide nanoparticles are prepared by organic solution phase reaction, and the dispersant in the dispersion of the iron phosphide nanoparticles is tetrahydrofuran or chloroform.

[0021] Preferably, in step (2), the small molecule drug containing a thiol group is 6-mercaptopurine, and the 6-mercaptopurine is oxidized to form a dimer, which is reacted with the surface thiol-functionalized iron phosphide nanoparticles obtained in step (1) under the protection of an inert gas, and then centrifuged, washed, and freeze-dried to obtain surface functionalized iron phosphide nanoparticles.

[0022] More preferably, the mass ratio of the 6-mercaptopurine to the surface-thiolated iron phosphide nanoparticles is (0.5-1):1, preferably 1:1.

[0023] More preferably, the reaction is carried out at room temperature.

[0024] More preferably, the reaction time is 24 to 48 hours.

[0025] In a second aspect, the present invention provides surface-functionalized iron phosphide nanoparticles, which are prepared by the preparation method described in the first aspect.

[0026] Preferably, the size of the surface-functionalized iron phosphide nanoparticles is 1 to 300 nm.

[0027] More preferably, the iron phosphide nanoparticles are prepared by chemical vapor deposition, and the size of the surface-functionalized iron phosphide nanoparticles prepared using the iron phosphide nanoparticles is 200-300 nm, preferably 244.01±15.93 nm; or the iron phosphide nanoparticles are prepared by organic solution phase reaction, and the size of the surface-functionalized iron phosphide nanoparticles prepared using the iron phosphide nanoparticles is 2-10 nm, preferably 6.46±2.82 nm.

[0028] In a third aspect, the present invention provides a nanomedicine, which is composed of the surface-functionalized iron phosphide nanoparticles described in the second aspect and microglial cell membranes or microglial cell exosomes coated with the surface-functionalized iron phosphide nanoparticles.

[0029] Preferably, the nanomedicine is a brain-targeted nanomedicine.

[0030] Preferably, the microglial cell membrane or microglial exosomes are derived from primary microglial cells or one or more of the microglial cell lines BV2, N9, HMC3, HMO6 and C8-B4.

[0031] Preferably, the size of the nanomedicine is 1 to 300 nm.

[0032] More preferably, the iron phosphide nanoparticles are prepared by chemical vapor deposition, and the size of the nanomedicine prepared using the iron phosphide nanoparticles is 200-300 nm, preferably 263.33±8.01 nm, or the iron phosphide nanoparticles are prepared by organic solution phase reaction, and the size of the nanomedicine prepared using the iron phosphide nanoparticles is 5-50 nm, preferably 20.95±1.64 nm.

[0033] In a fourth aspect, the present invention provides a method for preparing the nanomedicine described in the third aspect, comprising the following steps: mixing the microglial cell membrane or microglial cell exosomes and the surface-functionalized iron phosphide nanoparticles, and extruding them through a liposome extruder to obtain the nanomedicine.

[0034] Preferably, the pore sizes of the filter membranes of the liposome extruder are 400 nm, 200 nm and 100 nm respectively.

[0035] In a fifth aspect, the present invention provides a pharmaceutical composition comprising the nanodrug described in the third aspect and at least one additional anti-tumor drug.

[0036] Preferably, the pharmaceutical composition comprises the nanodrug described in the third aspect and an additional anti-tumor drug.

[0037] More preferably, the antitumor drug is an alkylating antitumor drug, preferably temozolomide.

[0038] In a sixth aspect, the present invention provides use of the surface functionalized iron phosphide nanoparticles according to the second aspect, the nanomedicine according to the third aspect, or the pharmaceutical composition according to the fifth aspect in the preparation of a drug for treating tumors.

[0039] Preferably, the tumor is a glioma.

[0040] More preferably, the tumor is a drug-resistant glioma.

[0041] Further preferably, the tumor is a temozolomide-resistant glioma.

[0042] Effects of the Invention

[0043] By modifying the surface of iron phosphide nanoparticles or further preparing them into nanomedicines, the present invention can synergize multiple pathways to significantly improve the efficacy of tumors (especially TMZ-resistant tumors), providing a new approach for treating drug-resistant tumors, reversing tumor resistance, and reducing the development of drug resistance. Specifically, the present invention has the following beneficial effects:

[0044] (1) The present invention is the first to use iron phosphide nanoparticles as biopharmaceutical carriers for tumor treatment. After surface modification (surface thiolization and attachment of active ingredients containing thiol groups, such as 6-MP), or further preparation into nanomedicines, the nanoparticles can significantly reduce the concentration of glutathione and the expression level of MGMT protein in tumor cells, while inhibiting the protein activity of HPRT1, thereby inhibiting the DNA repair process in tumor cells.

[0045] (2) The iron phosphide nanoparticles of the present invention, after surface modification or further processed into nanomedicine, have high-efficiency photothermal conversion performance, with a power of 0.8 W cm -2 Under 808nm laser irradiation, it has a selective killing effect on drug-resistant brain glioma cells, significantly improving the survival rate of TMZ-resistant mice, and has a significant anti-tumor effect. In addition, it can induce tumor immunogenic death, stimulate a strong immune response, and resist tumor recurrence.

[0046] (3) The iron phosphide nanoparticles of the present invention, after surface modification or further processed into nanomedicines, have a strong ability to generate hydroxyl radicals, which can further promote ferroptosis of tumor cells;

[0047] (4) The iron phosphide nanoparticles of the present invention, after surface modification or further preparation into nanomedicines, have no obvious hemolytic behavior and cytotoxicity and have good biocompatibility and safety;

[0048] (5) The nanomedicine of the present invention uses microglial cell-derived exosomes for camouflage, which can mediate the nanoparticles therein to cross the blood-brain barrier and target the tumor site, providing treatment precision, thereby reducing damage to healthy tissues and reducing adverse reactions;

[0049] (6) The iron phosphide nanoparticles of the present invention, after surface modification or further processed into nanomedicines, have good dispersibility and stability in various aqueous solutions;

[0050] (7) The preparation method of the present invention is simple, low-cost, mild in conditions, has a small particle size, and can be mass-produced or industrialized. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 A is a transmission electron microscopy image of FeP nanoparticles prepared in Example 1;

[0052] Figure 1 B is the transmission electron microscopy image of FeP-MP@Exo prepared in Example 7;

[0053] Figure 1 C is a transmission electron microscopy image of FeP nanoparticles prepared in Example 2;

[0054] Figure 1 D is the transmission electron microscopy image of FeP-MP@Exo prepared in Example 8;

[0055] Figure 2 1 is the XRD pattern of the Fe2O3 nanoparticles and the FeP nanoparticles prepared in Example 1;

[0056] Figure 3 This is the XPS graph of the FeP nanoparticles prepared in Example 1;

[0057] Figure 4 is the XRD pattern of FeP nanoparticles prepared in Example 2;

[0058] Figure 5 A is the zeta potential measurement result of the FeP nanoparticles prepared in Example 1, the FeP-SH nanoparticles prepared in Example 3, the FeP-MP nanoparticles prepared in Example 5, the exosomes (Exo) in Example 7, and the FeP-MP@Exo nanomedicine prepared in Example 7;

[0059] Figure 5B is the hydrodynamic diameter measurement results of FeP nanoparticles prepared in Example 1, FeP-SH nanoparticles prepared in Example 3, FeP-MP nanoparticles prepared in Example 5, and FeP-MP@Exo nanomedicine prepared in Example 7;

[0060] Figure 6 A is the infrared spectrum measurement results of FeP nanoparticles prepared in Example 1, FeP-SH nanoparticles prepared in Example 3, DMSA, FeP-MP nanoparticles prepared in Example 5, and 6-MP;

[0061] Figure 6 B is the infrared spectrum measurement results of FeP nanoparticles prepared in Example 2, FeP-SH nanoparticles prepared in Example 4, DMSA, FeP-MP nanoparticles prepared in Example 6, and 6-MP;

[0062] Figure 7 Zeta potential measurement results of FeP nanoparticles prepared in Example 2, FeP-SH nanoparticles prepared in Example 4, FeP-MP nanoparticles prepared in Example 6, exosomes (Exo) in Example 8, FeP@Exo prepared in Comparative Example 1, and FeP-MP@Exo nanomedicine prepared in Example 8;

[0063] Figure 8 A is the FeP-MP@Exo aqueous solution prepared in Example 7 at 0.8 W cm -2 Temperature rise and fall curves under 808nm laser irradiation;

[0064] Figure 8 B is a photothermal conversion stability test graph of FeP-MP@Exo prepared in Example 7;

[0065] Figure 9 A is the absorption intensity results of four mixed solutions of TMB+H2O2, TMB+FeP, H2O2+FeP, and TMB+H2O2+FeP at 350-720nm;

[0066] Figure 9 B is the paramagnetic resonance spectrum of hydroxyl radicals generated by FeP nanoparticles prepared in Example 1;

[0067] Figure 10 The concentration change of reduced glutathione in the solution after the FeP nanoparticles prepared in Example 1 were treated;

[0068] Figure 11 A represents the change in the concentration of intracellular reduced glutathione after treatment with the FeP nanoparticles in Example 1, the FeP-MP nanoparticles in Example 5, and the FeP-MP@Exo in Example 7;

[0069] Figure 11 B shows the changes in the intracellular reduced glutathione concentration after treatment with the FeP nanoparticles in Example 2, the FeP-MP nanoparticles in Example 6, and the FeP-MP@Exo in Example 8;

[0070] Figure 12 A is the cell viability determination result of the FeP-MP@Exo nanomedicine prepared in Example 7;

[0071] Figure 12 B is the cell viability determination result of the FeP-MP@Exo nanomedicine prepared in Example 8;

[0072] Figure 13 The hemolysis rate of FeP-MP@Exo prepared in Example 7 is as follows;

[0073] Figure 14 A shows the changes in MGMT protein expression in U87 cells after treatment with various drugs;

[0074] Figure 14 B shows the changes in MGMT protein expression in T98G cells after treatment with various drugs;

[0075] Figure 15 The cell survival results at different concentrations of FeP-MP@Exo;

[0076] Figure 16 The results of U87TR cell survival in different treatment groups;

[0077] Figure 17 A is a schematic diagram of the preparation of FeP-SH-Cy3;

[0078] Figure 17 B is the fluorescence intensity result of FeP-SH nanoparticles prepared in Example 3 and FeP-SH-Cy3 prepared therefrom;

[0079] Figure 17 C is the fluorescence intensity result of FeP-SH nanoparticles prepared in Example 4 and FeP-SH-Cy3 prepared therefrom;

[0080] Figure 18 The accumulation of FeP-SH-Cy3 and FeP-SH-Cy3@Exo in U87TR cells;

[0081] Figure 19 The experimental effects of ROS generation in GL261TR cells;

[0082] Figure 20 is the result of MDA content determination;

[0083] Figure 21 The results are for GPX4 protein expression level;

[0084] Figure 22 is the expression level of PTGS2 encoding cyclooxygenase 2;

[0085] Figure 23 is the exposure effect of CRT on the cell surface in GL261TR cells;

[0086] Figure 24 A is the result of HMGB1 level determination;

[0087] Figure 24 B is the result of ATP level determination;

[0088] Figure 25 The survival rate of mice in each group was used to evaluate the therapeutic effect in vivo. DETAILED DESCRIPTION

[0089] Various exemplary embodiments, features, and aspects of the present invention will be described in detail below. The word "exemplary" is used herein to mean "serving as an example, embodiment, or illustration." Any embodiment described herein as "exemplary" is not necessarily to be construed as superior or preferred over other embodiments.

[0090] In addition, numerous specific details are provided in the following detailed description to better illustrate the present invention. Those skilled in the art will appreciate that the present invention can be practiced without certain specific details. In other instances, methods, means, equipment, and steps well known to those skilled in the art are not described in detail in order to highlight the main points of the present invention.

[0091] Unless otherwise stated, the units used in this specification are international standard units, and the numerical values ​​and numerical ranges appearing in the present invention should be understood to include the inevitable systematic errors in industrial production.

[0092] In this specification, the use of "may" includes both the meaning of performing a certain process and the meaning of not performing a certain process.

[0093] In this specification, references to "some specific / preferred embodiments," "other specific / preferred embodiments," "embodiments," etc., mean that the specific elements (e.g., features, structures, properties, and / or characteristics) described in connection with the embodiments are included in at least one embodiment described herein, and may or may not be present in other embodiments. In addition, it should be understood that the elements may be combined in various embodiments in any suitable manner.

[0094] In this specification, the numerical range expressed using "a numerical value A to a numerical value B" means a range including the endpoints A and B.

[0095] In this specification, when "normal temperature" or "room temperature" is used, the temperature may be 15°C to 30°C, for example, 20°C.

[0096] In this specification, "FeP nanoparticles" or "FeP" refers to iron phosphide nanoparticles.

[0097] In this specification, "FeP-SH nanoparticles", "FeP-SH" or "FeP-SH nanoparticles" all refer to iron phosphide nanoparticles with surface thiols.

[0098] In this specification, "FeP-MP nanoparticles", "FeP-MP" or "FeP-6-MP" refers to functionalized iron phosphide nanoparticles obtained by attaching 6-MP to surface-thiolated iron phosphide nanoparticles.

[0099] In this specification, "FeP-MP@Exo", "FeP-6-MP@Exo", "Exo@FeP-6-MP" or "FeP-6-MP-Exo" refers to the nanomedicine obtained by encapsulating the functionalized iron phosphide nanoparticles of the present invention into exosomes.

[0100] After extensive research, the inventors discovered that FeP nanoparticles have efficient photothermal conversion capabilities. The heat energy generated by external light can kill cancer cells and induce immunogenic cell death in tumors, thereby stimulating a strong immune response, attacking remaining or distant tumor cells, and resisting tumor recurrence. Therefore, FeP nanoparticles have great application potential in tumor treatment. This invention is the first to use FeP nanoparticles as a biopharmaceutical carrier for tumor treatment to address the challenge posed by temozolomide resistance in traditional tumor treatment (particularly brain glioma).

[0101] The mechanism by which the nanomedicine of the present invention reverses the drug resistance of tumors (especially gliomas) is: on the one hand, it depletes glutathione, inhibits the WNT / β-catenin signaling pathway, and reduces the expression level of MGMT protein; on the other hand, it releases 6-MP in situ, inhibits the activity of HPRT1 protein, and increases the sensitivity of gliomas to TMZ.

[0102] In addition, the present invention also builds a platform for adjusting tumor treatment plans / preparing different anti-tumor drugs. This platform is scalable and can connect FeP nanoparticles with other thiol-containing small molecule drugs or thiol-modified nucleic acids according to actual needs, thereby achieving flexible adjustment of treatment plans and efficient synergistic treatment.

[0103] <Method for preparing surface-functionalized FeP nanoparticles>

[0104] The present invention provides a method for preparing surface-functionalized iron phosphide nanoparticles, which comprises the following steps:

[0105] (1) using iron phosphide nanoparticles and molecules containing thiol groups and other groups that can coordinate with iron to obtain surface thiol-modified iron phosphide nanoparticles;

[0106] (2) reacting the surface-thiolated iron phosphide nanoparticles obtained in step (1) with an active ingredient containing a thiol group to obtain;

[0107] Wherein, the active ingredient containing thiol groups is a small molecule drug containing thiol groups or a thiol-modified nucleic acid.

[0108] In some specific embodiments, the small molecule drug containing a thiol group is 6-mercaptopurine.

[0109] In the present invention, the molecules containing sulfhydryl groups and other groups that can coordinate with iron, the active ingredients containing sulfhydryl groups, the small molecule drugs containing sulfhydryl groups or the thiolated nucleic acids all include their isomers (such as tautomers). For example, the small molecule drug containing sulfhydryl groups can be 6-mercaptopurine Tautomers

[0110] In some specific embodiments, the iron phosphide nanoparticles are prepared by chemical vapor deposition or organic solution phase reaction.

[0111] In some specific embodiments, the iron phosphide nanoparticles are prepared by chemical vapor deposition. NaH2PO2, a representative phosphorus source for preparing FeP nanoparticles by chemical vapor deposition, decomposes and releases PH3 when heated in a tube furnace. The PH3 gas can directly react with precursors such as iron-based oxides or iron-based hydroxides.

[0112] In some specific embodiments, the raw materials of the chemical vapor deposition method are iron-based oxide or iron-based hydroxide nanoparticles and sodium hypophosphite or its hydrate.

[0113] In some specific embodiments, the diameter of the iron-based oxide or iron-based hydroxide nanoparticles is 10 to 100 nm, preferably 20 to 50 nm, such as 25 nm, 30 nm, 35 nm, 40 nm, etc.

[0114] In some preferred embodiments, the iron-based oxide nanoparticles are α-Fe2O3 nanoparticles.

[0115] In some specific embodiments, the steps of the chemical vapor deposition method include: grinding and mixing iron-based oxide or iron-based hydroxide nanoparticles and sodium hypophosphite or its hydrate, heating to 350-420°C under the protection of inert gas, keeping warm for 2-4 hours, cooling, grinding, adding water, ultrasonicating, centrifuging, washing, and freeze-drying to obtain the iron phosphide nanoparticles.

[0116] In some preferred embodiments, the mass ratio of the iron-based oxide or iron-based hydroxide nanoparticles to the sodium hypophosphite or its hydrate is 1:(5-10), preferably 1:(5-7).

[0117] In some preferred embodiments, the hydrate of sodium hypophosphite is sodium hypophosphite monohydrate.

[0118] In some preferred embodiments, the heating rate is 2-3°C min -1 , preferably 3℃min -1 .

[0119] In some preferred embodiments, the temperature is raised to 380-420°C, preferably 390-410°C.

[0120] In some preferred embodiments, the temperature is maintained for 2 to 3 hours, preferably 3 hours.

[0121] In some preferred embodiments, the cooling is to room temperature.

[0122] In some preferred embodiments, the washing is washing with water, preferably washing with water 3 to 5 times.

[0123] In some preferred embodiments, the water is deionized water.

[0124] In some preferred embodiments, the lyophilization is followed by grinding. The grinding step after lyophilization is not a critical step, and its main purpose is to make the subsequent characterization and testing more uniform and dispersed, so the iron phosphide nanoparticles can be preserved after grinding.

[0125] In some specific embodiments, the iron-based oxide and iron-based hydroxide nanoparticles can be synthesized using a hydrothermal method, preferably using FeCl3·6H2O as the iron source. For example, to synthesize α-Fe2O3 nanoparticles, FeCl3·6H2O, Na2SO4, and NaH2PO4·H2O are fully dissolved in water, hydrothermally treated at 180-200°C for 12-24 hours, centrifuged, washed, and dried to obtain α-Fe2O3 nanoparticles. α-Fe2O3 nanoparticles can also be purchased commercially.

[0126] In other specific embodiments, the iron phosphide nanoparticles are prepared by an organic solution phase reaction.

[0127] In some specific embodiments, the raw materials for the organic solution phase reaction are ferric acetylacetonate and trialkylphosphine.

[0128] In some specific embodiments, the solvent for the organic solution phase reaction is oleylamine.

[0129] In some preferred embodiments, the trialkylphosphine is tri-n-octylphosphine.

[0130] In some preferred embodiments, in the organic solution phase reaction, the concentration of ferric acetylacetonate is not higher than 300mM. In the organic solution phase reaction, ferric acetylacetonate can form iron nanoparticles, and the temperature, time and material concentration of this reaction all can affect the size of the nanoparticles formed. In this reaction, the concentration of ferric acetylacetonate is limited to be not higher than 300mM. If concentration is too high, the nanoparticle size that may cause to form becomes large, and the phosphating of iron nanoparticles is difficult for carrying out.

[0131] In some specific embodiments, the step of the organic solution phase reaction includes: dispersing ferric acetylacetonate in oleylamine, maintaining the temperature at 100-150°C for 15-30 minutes (for example, 120°C for 0.5 hours) under inert gas protection, slowly adding trialkylphosphine, and heating to 330-385°C, reacting for 2-5 hours, cooling, adding an alcohol solvent, centrifuging, washing, and freeze-drying to obtain the iron phosphide nanoparticles.

[0132] In some preferred embodiments, the molar ratio of the trialkylphosphine to the ferric acetylacetonate is (3-5):1.

[0133] In some preferred embodiments, the temperature is raised to 330-350°C, preferably 340-350°C.

[0134] In some preferred embodiments, the reaction lasts 2 to 4 hours, preferably 2 to 3 hours.

[0135] In some preferred embodiments, the cooling is to room temperature.

[0136] In some preferred embodiments, the alcohol solvent is ethanol or isopropanol.

[0137] In some preferred embodiments, the amount of the alcohol solvent added is 2 to 3 times the volume of the reaction solution.

[0138] In some preferred embodiments, the washing is washing with chloroform or ethanol, preferably 3 to 5 times.

[0139] In some specific embodiments, in step (1), the iron phosphide nanoparticles and molecules containing thiol groups and other groups that can coordinate with iron can obtain the surface thiol-modified iron phosphide nanoparticles through coordination exchange.

[0140] In some preferred embodiments, the molecule containing a thiol group and other groups that can coordinate with iron is 2,3-dimercaptosuccinic acid.

[0141] In some specific embodiments, step (1) specifically comprises: adjusting the pH of the aqueous solution of the molecules containing thiol groups and other groups that can coordinate with iron to 9-11, dropping the solution into the dispersion of the iron phosphide nanoparticles, ultrasonicating or stirring, centrifuging, washing, and freeze-drying to obtain surface thiol-modified iron phosphide nanoparticles.

[0142] In some preferred embodiments, the concentration of the aqueous solution of the molecule containing thiol and other groups that can coordinate with iron is 10-50 mg mL -1 The concentration of the dispersion of the iron phosphide nanoparticles is 1 to 10 mg mL -1 .

[0143] In some specific embodiments, the iron phosphide nanoparticles are produced by chemical vapor deposition, and the dispersant in the dispersion of the iron phosphide nanoparticles is water. In other specific embodiments, the iron phosphide nanoparticles are produced by organic solution phase reaction, and the dispersant in the dispersion of the iron phosphide nanoparticles is tetrahydrofuran or chloroform.

[0144] In some preferred embodiments, the washing is washing with water, preferably washing with water 3 to 5 times.

[0145] In some preferred embodiments, the water is deionized water.

[0146] In some specific embodiments, in step (2), the small molecule drug containing a thiol group is 6-mercaptopurine, and the 6-mercaptopurine is oxidized to form a dimer, which is reacted with the surface thiol-functionalized iron phosphide nanoparticles obtained in step (1) under the protection of an inert gas (e.g., a disulfide bond exchange reaction), centrifuged, washed, and freeze-dried to obtain surface-functionalized iron phosphide nanoparticles.

[0147] In some preferred embodiments, the mass ratio of the 6-mercaptopurine to the surface-thiolated iron phosphide nanoparticles is (0.5-1):1, preferably 1:1.

[0148] In some preferred embodiments, the reaction is carried out at room temperature.

[0149] In some preferred embodiments, the reaction time is 24 to 48 hours, preferably 24 to 36 hours.

[0150] In some preferred embodiments, the 6-mercaptopurine is oxidized by iodine to form a dimer.

[0151] In some preferred embodiments, the washing is washing with water, preferably washing with water 3 to 5 times.

[0152] In some preferred embodiments, the water is deionized water.

[0153] <Surface Functionalized FeP Nanoparticles>

[0154] The present invention provides surface-functionalized iron phosphide nanoparticles, which are prepared by the above-mentioned <method for preparing surface-functionalized FeP nanoparticles>.

[0155] In some embodiments, the surface-functionalized iron phosphide nanoparticles have a size of 1 to 300 nm.

[0156] In some specific embodiments, the iron phosphide nanoparticles are prepared by chemical vapor deposition, and the size of the surface-functionalized iron phosphide nanoparticles prepared using the iron phosphide nanoparticles is 200-300 nm (e.g., 210 nm, 220 nm, 230 nm, 240 nm, 250 nm, 260 nm, 270 nm, 280 nm, 290 nm, etc.), preferably 244.01±15.93 nm.

[0157] In other specific embodiments, the iron phosphide nanoparticles are prepared by an organic solution phase reaction, and the size of the surface functionalized iron phosphide nanoparticles prepared using the iron phosphide nanoparticles is 2 to 10 nm (e.g., 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, etc.), preferably 6.46±2.82 nm.

[0158] <Nanomedicine>

[0159] The present invention provides a nanomedicine comprising the above-mentioned <surface-functionalized iron phosphide nanoparticles> and microglial cell membranes or microglial cell exosomes.

[0160] In some preferred embodiments, the nanomedicine consists of the above-mentioned <surface-functionalized iron phosphide nanoparticles> and microglial cell membranes or microglial cell exosomes.

[0161] In some specific embodiments, the nanomedicine is obtained by encapsulating the surface-functionalized iron phosphide nanoparticles by the microglial cell membrane or microglial cell exosomes.

[0162] In some specific embodiments, the nanodrug is a brain-targeted nanodrug.

[0163] In the present invention, the microglial cell membrane or microglial exosomes are derived from primary microglial cells or one or more of the microglial cell lines BV2, N9, HMC3, HMO6 and C8-B4.

[0164] In some embodiments, the size of the nanomedicine is 1 to 300 nm.

[0165] In some specific embodiments, the iron phosphide nanoparticles are prepared by chemical vapor deposition, and the size of the nanomedicine prepared using the iron phosphide nanoparticles is 200-300 nm (for example, 210 nm, 220 nm, 230 nm, 240 nm, 250 nm, 260 nm, 270 nm, 280 nm, 290 nm, etc.), preferably 263.33±8.01 nm.

[0166] In other specific embodiments, the iron phosphide nanoparticles are prepared by an organic solution phase reaction, and the size of the nanomedicine prepared using the iron phosphide nanoparticles is 5 to 50 nm (for example, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 12 nm, 15 nm, 20 nm, 25 nm, 30 nm, 40 nm, etc.), preferably 20.95 ± 1.64 nm.

[0167] <Method for preparing nanomedicine>

[0168] The present invention provides a method for preparing the above-mentioned nanomedicine, which comprises the following steps:

[0169] The microglial cell membrane or microglial cell exosomes and the surface functionalized iron phosphide nanoparticles are mixed and extruded through a liposome extruder to obtain the product.

[0170] In some specific embodiments, the microglial cell membrane or microglial cell exosomes and the surface-functionalized iron phosphide nanoparticles are mixed and then extruded through a liposome extruder with filter membrane pore sizes of 400 nm, 200 nm and 100 nm, respectively, to obtain the nanomedicine.

[0171] In some specific embodiments, the number of extrusions is 50 or more times, such as 50 to 100 times.

[0172] In some specific embodiments, the microglial cell membranes or microglial exosomes and the surface-functionalized iron phosphide nanoparticles are mixed in a PBS solution.

[0173] <Pharmaceutical Composition>

[0174] The present invention provides a pharmaceutical composition comprising the above-mentioned <nanomedicine> and at least one additional anti-tumor drug.

[0175] In some specific embodiments, the pharmaceutical composition comprises the above-mentioned <nanomedicine> and an additional anti-tumor drug.

[0176] In some specific embodiments, the anti-tumor drug is an alkylating anti-tumor drug, such as temozolomide.

[0177] <Medical Use>

[0178] The present invention also provides medical uses of the above-mentioned <surface-functionalized iron phosphide nanoparticles>, the above-mentioned <nanomedicine> or the above-mentioned <pharmaceutical composition>.

[0179] Specifically, the present invention provides use of the above-mentioned <surface-functionalized iron phosphide nanoparticles>, the above-mentioned <nanomedicine> or the above-mentioned <pharmaceutical composition> in the preparation of a drug for treating tumors.

[0180] In some specific embodiments, the tumor is a glioma.

[0181] In some preferred embodiments, the tumor is a drug-resistant glioma.

[0182] In some more preferred embodiments, the tumor is a temozolomide-resistant glioma.

[0183] <Abbreviation>

[0184] In this manual, the English abbreviations and Chinese full names are as follows:

[0185] English abbreviation Full Chinese name English abbreviation Full Chinese name 6-MP 6-Mercaptopurine TEER Transendothelial electrical resistance TMZ Temozolomide BBB blood-brain barrier GSH Glutathione PBS Phosphate-buffered saline Exo exosomes GBM Grade IV glioblastoma DMSA Succinic Acid TEM Transmission electron microscopy DMSO dimethyl sulfoxide XRD X-ray polycrystal diffraction TMB 3,3',5,5'-Tetramethylbenzidine XPS X-ray photoelectron spectroscopy DMPO 5,5-Dimethyl-1-pyrroline N-oxide EDS Energy Dispersive Spectrometer

[0186] Example

[0187] The embodiments of the present invention will be described in detail below with reference to the examples, but it will be understood by those skilled in the art that the following examples are merely illustrative of the present invention and should not be construed as limiting the scope of the invention. Where specific conditions are not specified in the examples, the methods were performed according to conventional conditions or the conditions recommended by the manufacturer. Where the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be obtained commercially.

[0188] The α-Fe2O3 nanoparticles used in the embodiments of the present invention are commercially available.

[0189] The present invention proves that the FeP nanoparticles in the embodiment are successfully prepared through transmission electron microscopy (TEM), energy dispersive spectrometer (EDS), X-ray polycrystal diffraction (XRD), and X-ray photoelectron spectroscopy (XPS).

[0190] The present invention demonstrated the successful modification of FeP nanoparticles and exosome encapsulation in the examples by transmission electron microscopy (TEM), zeta potential, infrared spectroscopy and fluorescence spectroscopy.

[0191] Example 1: Preparation of FeP nanoparticles by chemical vapor deposition

[0192] (1) 0.3 g of α-Fe2O3 nanoparticles with an average particle size of 30 nm were mixed with 1.8 g of NaH2PO2·H2O particles, ground into a uniform powder, and heated at 3 °C min under an argon atmosphere. -1 The mixture was heated to 400 °C at a speed of 100 °C and maintained for 3 h, and then cooled to room temperature naturally to obtain FeP product;

[0193] (2) Grind the obtained FeP uniformly, add 10 mL of deionized water, and sonicate for 2 h to obtain a uniformly distributed FeP solution;

[0194] (3) The FeP solution was centrifuged at 8000 rpm for 10 min, and the precipitate was washed three times with deionized water, freeze-dried, and then ground to obtain FeP nanoparticles.

[0195] The transmission electron microscopy images of the prepared FeP nanoparticles are shown in Figure 1 A, indicating that the size of FeP nanoparticles is uniform. The XRD patterns of FeP nanoparticles and α-Fe2O3 nanoparticles in step (1) are shown in Figure 2. Figure 2 , XPS of FeP nanoparticles Figure 3 , indicating that FeP nanoparticles have been successfully prepared.

[0196] Example 2: Preparation of FeP Nanoparticles by Organic Solution Phase Reaction

[0197] (1) Weigh 2 g of ferric acetylacetonate and dissolve it in 20 mL of oleylamine. Add 9 mL of tri-n-octylphosphine while stirring. Heat the reaction system to 120 °C under argon and maintain for 30 min.

[0198] (2) Continue heating to 350°C and maintain for 2 hours;

[0199] (3) After the mixed solution cools to room temperature, add 90 mL of isopropyl alcohol;

[0200] (4) Centrifuge at 9000 rpm for 20 min, wash the precipitate three times with ethanol, and freeze-dry to obtain FeP nanoparticles.

[0201] The transmission electron microscopy images of the prepared FeP nanoparticles are shown in Figure 1 C, indicating that the size of FeP nanoparticles is uniform. Figure 4 , indicating that FeP nanoparticles have been successfully prepared.

[0202] Example 3: Surface thiolation of FeP nanoparticles

[0203] (1) Weigh 0.3 g of 2,3-dimercaptosuccinic acid (DMSA) and dissolve it in 10 mL of deionized water. Adjust the pH to 10 with NaOH solution to obtain a DMSA solution.

[0204] (2) Weigh 0.2 g of the FeP nanoparticles prepared in Example 1, dissolve them in 40 mL of deionized water, and disperse them by ultrasonication to obtain a FeP solution;

[0205] (3) 10 mL of FeP solution was mixed with 2.5 mL of DMSA solution, sonicated for 1 h, and then stirred at room temperature for 2 h;

[0206] (4) The mixed solution obtained in step (3) was centrifuged at 8000 rpm for 10 min, the precipitate was washed three times with deionized water, and freeze-dried to obtain FeP-SH nanoparticles.

[0207] The average number of DMSA molecules per FeP-SH nanoparticle was calculated by the following formula:

[0208]

[0209] N DMSA =n DMSA ×N A

[0210]

[0211]

[0212] where ε represents the average number of DMSA per FeP-SH nanoparticle; N DMSA Indicates the total amount of DMSA; N FeP represents the total number of FeP-SH nanoparticles. n DMSA represents the total amount of DMSA. This value is calculated based on the amount of S, assuming no disulfide bonds are formed in FeP-SH. The amount of S is calculated based on the sulfur content (weight percentage) and the mass of the FeP-SH nanoparticles. The sulfur content in FeP-SH was calculated to be 0.484% using an elemental analyzer. total FeP represents the total volume of FeP-SH nanoparticles; m represents the total mass of FeP-SH nanoparticles; ρ represents the density of FeP-SH nanoparticles, which is approximately 6.07 g / cm 3 V0 represents the volume of a single FeP-SH nanoparticle.

[0213] The above calculations show that the average number of thiol groups in each FeP-SH nanoparticle prepared in this example is 2.8×10 5 indivual.

[0214] Example 4: Surface thiolation of FeP nanoparticles

[0215] Replace step (2) in Example 3 with the following: weigh 0.2 g of the FeP nanoparticles in Example 2 and dissolve them in 40 mL of tetrahydrofuran to obtain a FeP solution. The remaining steps remain unchanged to obtain FeP-SH nanoparticles.

[0216] Example 5: Preparation of FeP-MP nanoparticles

[0217] (1) Weigh 90 mg of iodine particles and dissolve them in 3 mL of dimethyl sulfoxide (DMSO) to obtain solution A.

[0218] (2) Weigh 100 mg of 6-MP and dissolve it in 7 mL of DMSO to obtain solution B;

[0219] (3) Solution A was slowly added dropwise to the stirred solution B, and the mixed solution was stirred at room temperature overnight;

[0220] (4) adding 100 mg of the FeP-SH nanoparticles prepared in Example 3 to the mixed solution after the reaction, and reacting at room temperature for 24 h under an argon atmosphere;

[0221] (5) The mixed solution obtained in step (4) was centrifuged at 8000 rpm for 10 min, the precipitate was washed three times with deionized water, and freeze-dried to obtain FeP-MP nanoparticles.

[0222] Example 6: Preparation of FeP-MP nanoparticles

[0223] Referring to the preparation method of Example 5, only the "FeP-SH nanoparticles" in step (4) are replaced with "FeP-SH nanoparticles in Example 4", and the other steps remain unchanged to obtain FeP-MP nanoparticles.

[0224] Example 7: Preparation of nanomedicine FeP-MP@Exo

[0225] (1) After culturing HMC3 human microglial cells in serum-free medium for 24 h, the cell supernatant was collected and centrifuged at 10,000 g for 1 h. After removing the precipitate, the supernatant was centrifuged at 100,000 g for 2 h, and the precipitate was resuspended in 200 μL PBS to obtain the exosome solution;

[0226] (2) Prepare 1mg mL -110 mL of PBS solution of FeP-MP nanoparticles (prepared in Example 5) was mixed with 50 μL of exosome solution and extruded repeatedly 50 times through a liposome extruder using membranes of 400 nm, 200 nm, and 100 nm, respectively;

[0227] (3) The mixed solution obtained in step (2) was centrifuged at 8000 rpm for 10 min, the precipitate was washed three times with deionized water, and then resuspended in PBS to obtain FeP-MP@Exo, which was stored at -20°C for future use.

[0228] The transmission electron microscopy image of the prepared FeP-MP@Exo is shown in Figure 1 B, showing that the size of FeP-MP@Exo is uniform.

[0229] The size of the nanodrug was measured to be 263.33±8.01nm.

[0230] Example 8: Preparation of nanomedicine FeP-MP@Exo

[0231] Referring to the preparation method of Example 7, only the "FeP-MP nanoparticles" in step (2) were replaced with "FeP-MP nanoparticles prepared in Example 6" to obtain FeP-MP@Exo.

[0232] The transmission electron microscopy image of the prepared FeP-MP@Exo is shown in Figure 1 D, showing that the size of FeP-MP@Exo is uniform.

[0233] It was determined that the size of the nanodrug was 20.95±1.64nm.

[0234] Comparative Example 1: Preparation of FeP@Exo

[0235] Referring to the preparation method of Example 7, only the "FeP-MP nanoparticles" in step (2) are replaced with "FeP nanoparticles prepared in Example 1" to obtain FeP@Exo (or Exo@FeP).

[0236] Experimental example

[0237] In the following experimental examples of the present invention, "BSO" refers to L-buthionine sulfoximine. BSO is a specific inhibitor of γ-glutamylcysteine ​​synthetase and effectively inhibits the biosynthesis of GSH by preventing the connection between glutamate and cysteine.

[0238] The following five types of glioma cells were used in the following experimental examples of the present invention: U87 (natural human TMZ-sensitive), U87TR (human induced TMZ-resistant), T98G (natural human TMZ-resistant), GL261 (natural mouse TMZ-sensitive), and GL261TR (mouse induced TMZ-resistant).

[0239] Experimental Example 1: Physical and Chemical Properties and Structural Characterization

[0240] The FeP nanoparticles prepared in Example 1, the FeP-SH nanoparticles prepared in Example 3, the FeP-MP nanoparticles prepared in Example 5, the exosomes (Exo) in Example 7, and the FeP-MP@Exo nanomedicine prepared in Example 7 were measured for Zeta potential and hydrodynamic diameter, and the results were shown in Table 1. Figure 5 A and Figure 5 B.

[0241] The FeP nanoparticles prepared in Example 1, the FeP-SH nanoparticles prepared in Example 3, DMSA, the FeP-MP nanoparticles prepared in Example 5, and 6-MP were measured for infrared spectra. Figure 6 A. The FeP nanoparticles prepared in Example 2, the FeP-SH nanoparticles prepared in Example 4, DMSA, the FeP-MP nanoparticles prepared in Example 6, and 6-MP were measured for infrared spectra respectively. The results are shown in FIG. Figure 6 B.

[0242] Zeta potential of FeP nanoparticles prepared in Example 2, FeP-SH nanoparticles prepared in Example 4, FeP-MP nanoparticles prepared in Example 6, exosomes (Exo) in Example 8, FeP@Exo prepared in Comparative Example 1, and FeP-MP@Exo nanomedicine prepared in Example 8 were measured respectively. The results are shown in FIG. Figure 7 .

[0243] The results showed that the above-mentioned FeP-SH nanoparticles, FeP-MP nanoparticles, FeP-MP@Exo nanodrugs, and FeP@Exo were successfully prepared, and the prepared nanodrugs had a stable PDI index and good dispersibility and stability.

[0244] Experimental Example 2: Photothermal Performance Evaluation

[0245] (1) At room temperature, 1W cm -2 The FeP-MP@Exo nanomedicine PBS solution was irradiated with an 808nm laser for a total of 600s, and the temperature change and thermal image were captured and recorded every 15s using an infrared thermal imager. As a blank control, the PBS solution was irradiated under the same conditions.

[0246] (2) When the temperature rise is basically stable, stop laser irradiation and use an infrared thermal imager to record the temperature change every 15 seconds during the cooling process until the temperature returns to room temperature;

[0247] (3) Calculate the photothermal conversion efficiency using the formula:

[0248]

[0249] Where T max is the equilibrium temperature of the sample solution, T surr Corresponding to the experimental environment temperature. I represents the power density of 808nm laser (1W cm -2 ), A λ Q is the absorbance of the sample solution at 808 nm. dis Indicates the heat loss caused by light absorption of the container itself, according to Q dis =(5.4×10 -4 )×I. The value of hS can be calculated using another formula:

[0250]

[0251] Where m is the mass of the sample solution, C water is the specific heat capacity of water, 4.2 J g -1 K -1 , τ s is the time constant of the system.

[0252] Combined with the above values, it was calculated that the photothermal conversion efficiency of the FeP-MP@Exo nanodrug prepared in Example 7 was 27.25%, and the photothermal conversion efficiency of the FeP-MP@Exo nanodrug prepared in Example 8 was 31.27%.

[0253] (4) Repeat steps (1) and (2) alternately for at least five times to verify the photothermal stability of the nanomedicine.

[0254] The experimental results are shown in Figure 8 , Figure 8 A is the FeP-MP@Exo aqueous solution prepared in Example 7 at 0.8 W cm -2 Temperature rise and fall curves under 808nm laser irradiation, Figure 8 B is a photothermal conversion stability test diagram. From the above results, it can be seen that the FeP-MP@Exo nanomedicine in the present invention has high photothermal conversion efficiency and good photothermal stability.

[0255] Experimental Example 3: Evaluation of the effect of generating active oxygen

[0256] The activity of FeP nanoparticles (prepared in Example 1) in catalyzing hydrogen peroxide to produce hydroxyl radicals was detected by ultraviolet spectrophotometry. TMB can be oxidized to oxTMB by hydroxyl radicals, and the oxidation product has a significant absorption peak at 652 nm. The concentration of 1 mg mL was prepared with deionized water. -1The absorption intensity of the four mixed solutions of TMB+H2O2, TMB+FeP, H2O2+FeP and TMB+H2O2+FeP at 350-720nm was detected. The results are shown in Figure 9 A, indicating that FeP nanoparticles can effectively catalyze hydrogen peroxide to produce hydroxyl radicals.

[0257] Electron paramagnetic resonance (EPR) was used to detect the ability of FeP nanoparticles (prepared in Example 1) to generate hydroxyl radicals. 10 mg of FeP nanoparticles were weighed and dissolved in 1 mL of deionized water. 10 μL of the capture agent DMPO was added to the FeP nanoparticle solution, mixed well, and then aspirated with a capillary tube and placed in a paramagnetic tube for testing. For comparison, α-Fe2O3 and Fe 2+ The signal strength generated. The experimental results are shown in Figure 9 B, the results show that the signal intensity generated by FeP nanoparticles is stronger than that of α-Fe2O3 and Fe 2 + The signal intensity generated indicates that FeP nanoparticles have a stronger ability to generate hydroxyl radicals.

[0258] The FeP nanoparticles were replaced with the FeP nanoparticles prepared in Example 2, and the experimental results were similar to the above results.

[0259] Experimental Example 4: Evaluation of the effect on GSH

[0260] In order to verify the GSH consumption ability of FeP nanoparticles, FeP nanoparticles (prepared in Example 1) with different concentrations were mixed with 200 μg mL -1 After 4 h of reaction at 37°C, the mixed solution was centrifuged and the GSH content in the supernatant was determined according to the instructions of the GSH content determination kit. Figure 10 , indicating that FeP nanoparticles can reduce the concentration of GSH in the solution, and the higher the concentration of FeP nanoparticles, the more significant the effect of reducing GSH concentration. The FeP nanoparticles were replaced with the FeP nanoparticles prepared in Example 2, and the experimental results were similar to the above results.

[0261] The concentration is 100 μg mL -1 FeP, 2 μg mL -1 6-MP, 100 μg mL -1 FeP-MP, 100 μg mL -1 FeP-MP@Exo, 2 μg mL -1 BSO, 8 μg mL -1 BSO cell-specific culture medium. U87, U87TR, T98G, GL261, and GL261TR cells were cultured at 1×105 Cells were seeded at a density of 100 μg / mL in a 6-well plate. After 24 hours of culture, the medium was replaced with complete medium containing the drug and incubated for 24 hours. The cells were collected and lysed on ice for 30 minutes using RIPA lysis buffer. After sonication for 30 seconds, the supernatant was centrifuged at 10,000 rpm for 5 minutes and the GSH content in the supernatant was measured using a GSH content assay kit. The results are shown in Figure 2. Figure 11 As shown, Figure 11 The FeP in A is the FeP nanoparticles in Example 1, the FeP-MP is the FeP-MP nanoparticles in Example 5, and the FeP-MP@Exo is the FeP-MP@Exo in Example 7; Figure 11 The FeP in B is the FeP nanoparticles in Example 2, the FeP-MP is the FeP-MP nanoparticles in Example 6, and the FeP-MP@Exo is the FeP-MP@Exo in Example 8.

[0262] Depend on Figure 11 It can be seen that FeP, FeP-MP, and FeP-MP@Exo can significantly reduce the concentration level of GSH in brain glioma cells, and after FeP nanoparticles are modified into FeP-MP or further encapsulated by exosomes, their ability to reduce the concentration level of GSH is further enhanced, even better than or reaching the effect of BSO in reducing GSH.

[0263] Experimental Example 5: Biocompatibility Evaluation

[0264] The cell validation experiments were conducted in accordance with the relevant provisions of the National Standard of the People's Republic of China GB / T16886.5 (Biological Evaluation of Medical Devices: In Vitro Cytotoxicity Test) and the international standard for biological evaluation of medical devices ISO10993-5. The in vitro biosafety of the nanomedicine was evaluated using mouse brain microvascular endothelial cells, Bend.3.

[0265] 1 mL of FeP-MP@Exo nanodrug solution was sterilized by UV for 4 h and added into Bend.3 special culture medium (containing 10% fetal bovine serum) with the concentrations of 10, 50, 100, 200, and 500 μg mL -1 Actively growing Bend.3 cells were seeded at approximately 50,000 cells / well in a 96-well plate. 100 μL of each concentration of experimental culture medium was added to each well of the experimental group. 100 μL of complete culture medium was added to each well of the control group. The blank group received only complete culture medium without cells. After incubating the cells at 37°C, 5% CO2 for 6 hours, 10 μL of CCK8 reagent was added. After incubation for 4 hours, the absorbance of each well at 450 nm was measured using a microplate reader. The absorbance was calculated according to the following formula:

[0266]

[0267] Calculate the cell survival rate. As the concentration of FeP-MP@Exo nanomedicine increases, the cell survival rate of Bend.3 cells does not change significantly. Figure 12 . Figure 12 A is the determination result of FeP-MP@Exo nanomedicine prepared in Example 7, with concentrations of 10, 50, 100, 200, and 500 μg mL -1 The cell viability rates were 86.36±1.15%, 80.93±3.07%, 84.13±3.73%, 90.19±6.30% and 80.04±22.19% respectively. Figure 12 B is the test result of FeP-MP@Exo nanomedicine prepared in Example 8, the cell survival rate is Figure 12 The above results indicate that FeP-MP@Exo nanomedicine has no obvious cytotoxicity and good biosafety.

[0268] Experimental Example 6: In vitro hemolysis rate evaluation

[0269] Fresh mouse red blood cells were taken and diluted to 2.5% with normal saline. A series of FeP-MP@Exo solutions were prepared as in Example 7, with concentrations of 50, 100, 250, 500, and 1000 μg mL -1 , added to the red blood cell solution and incubated at 37°C for 3 hours. Normal saline and deionized water served as negative and positive controls, respectively. After incubation, the solution was centrifuged at 1500 g for 5 minutes. The absorbance of the supernatant was measured at 450 nm using a UV spectrophotometer, and the hemolysis rate was calculated according to the following formula:

[0270]

[0271] The experimental results are shown in Figure 13 The hemolysis rate of FeP-MP@Exo solution at each concentration was low, even 0, and basically did not exceed 5%. When the concentration of FeP-MP@Exo solution was as high as 1000 μg mL -1 The hemolysis rate did not exceed 10%, which shows that FeP-MP@Exo has basically no hemolytic behavior and has good biosafety.

[0272] Experimental Example 7: Evaluation of the effect on MGMT protein expression

[0273] The concentration is 100 μg mL -1 FeP, 2 μg mL -1 6-MP, 100 μg mL -1 FeP-MP@Exo, 8 μg mL -1BSO cell-specific culture medium (FeP is the FeP nanoparticles in Example 1, and FeP-MP@Exo is the FeP-MP@Exo in Example 7). U87, U87TR, and T98G cells were cultured at a rate of 1×10 5 Cells were seeded at a density of 100 cells / mL in 6-well plates. After 24 hours of culture, the medium was replaced with complete medium containing the drug and incubated for a total of 24 hours. The cells were harvested and lysed on ice for 30 minutes using RIPA lysis buffer containing protease inhibitors. After sonication for 30 seconds, the supernatant was centrifuged at 10,000 rpm for 10 minutes and the supernatant was collected. Total protein concentration was determined using a BCA assay, with β-actin as an internal reference protein. MGMT protein expression in each sample was determined by Western blotting.

[0274] The experimental results are shown in Figure 14 , Figure 14 A is the result of MGMT protein expression changes in U87 cells. Figure 14 B shows the expression changes of MGMT protein in T98G cells. Figure 14 It can be seen that both FeP and FeP-MP@Exo can reduce the expression level of MGMT protein in brain glioma cells, and the effect of FeP-MP@Exo is more significant, even better than that of BSO.

[0275] Experimental Example 8: In vitro therapeutic effect evaluation

[0276] Actively growing U87, U87TR, T98G, GL261, and GL261TR cells were seeded into 96-well plates at a concentration of approximately 5 × 10 4 After culturing for 24 hours in a 37°C, 5% CO2 incubator, discard the original culture medium and prepare a series of complete culture medium with TMZ concentrations ranging from 100 to 10,000 μM. Add 100 μL of complete culture medium with each TMZ concentration to each well. After culturing for 12 hours, add 10 μL of CCK8 reagent to each well. After another 2 hours of incubation, use a microplate reader to measure the absorbance of each well at 450 nm and calculate the cell survival rate and median lethal dose (IC) of each well. 50 The result is that U87 IC 50 =983.8μM, U87TR IC 50 =4350μM, T98G IC 50 =4312μM, GL261 IC 50 =645μM, GL261TR IC 50 =2351μM.

[0277] The FeP-MP@Exo prepared in Example 7 was sterilized by UV for 4 h, and the concentrations were 0, 50, 75, 100, and 200 μg mL -1The experimental group culture medium was used. The vigorously growing U87, U87TR, T98G, GL261, and GL261TR cells were seeded in 96-well plates at a concentration of approximately 5×10 4 100 μL of each concentration of culture medium was added to each well of the experimental group, 100 μL of complete culture medium was added to each well of the control group, and only complete culture medium was added to the blank group. After culturing for 6 h, PBS was added to rinse gently 3 times, and 100 μL of complete culture medium was added to each well. -2 After 6 hours of culture, 10 μL of CCK8 reagent was added to each well. After 2 hours of culture, the absorbance of each well at 450 nm was measured using a microplate reader to calculate the cell survival rate. Figure 15 ,exist Figure 15 The darker the color, the lower the survival rate. Figure 15 It can be seen that with the increase of FeP-MP@Exo concentration, the survival rate of each cell gradually decreased. When the concentration of FeP-MP@Exo reached 100 μg mL -1 When treated with FeP-MP@Exo, the survival rate of each cell was less than 50%, indicating that FeP-MP@Exo has a very significant killing effect on TMZ-sensitive or resistant cells in humans and mice.

[0278] The vigorously growing U87, U87TR, T98G, GL261, and GL261TR cells were seeded in 96-well plates at a concentration of approximately 5×10 4 / well. Prepared with 1mM TMZ, 100μg mL -1 The experimental culture medium of FeP-MP@Exo was divided into control group, TMZ treatment group, TMZ+phototherapy group, FeP-MP@Exo group, FeP-MP@Exo+phototherapy group, TMZ+FeP-MP@Exo group, and TMZ+FeP-MP@Exo+phototherapy group. After culturing for 24 hours, the original culture medium was aspirated and discarded, and 100 μL of the culture medium of each experimental group was added to each well. After culturing for 4 hours, the cells were gently rinsed with PBS three times, and then 100 μL of complete culture medium was added to each well. The phototherapy group was treated with 0.8W cm -2 After 6 hours of culture, 10 μL of CCK8 reagent was added to each well. After 2 hours of culture, the absorbance of each well at 450 nm was measured using a microplate reader to calculate the cell survival rate. Figure 16 The results of other cells were similar, and the results showed that the TMZ+FeP-MP@Exo+phototherapy group achieved the best therapeutic effect, with cell survival rates below 10%.

[0279] Experimental Example 9: In vitro blood-brain barrier crossing ability assessment

[0280] 1mL 0.1mg mL -1 Add 50 μL of 1 mg mL FeP-SH -1 Cy3-maleimide (solvent is DMSO), ultrasonically mixed in pH 7.4 PBS buffer at room temperature for 2 hours, and centrifuged to obtain fluorescently labeled FeP-SH-Cy3. Figure 17 A is a schematic diagram of the preparation of FeP-SH-Cy3, Figure 17 B is the fluorescence intensity result of FeP-SH nanoparticles prepared in Example 3 and FeP-SH-Cy3 prepared therefrom, Figure 17 Figure C shows the fluorescence intensity of FeP-SH nanoparticles prepared in Example 4 and FeP-SH-Cy3 obtained from them. This result demonstrates the successful preparation of FeP-SH-Cy3 and the successful thiolation of FeP nanoparticles. The preparation method for FeP-SH-Cy3@Exo refers to the preparation method for FeP-MP@Exo.

[0281] Bend.3 cells were seeded into Transwell chambers at a concentration of approximately 1×10 5 pcs / well, used to construct an in vitro blood-brain barrier model. When TEER exceeds 200Ωcm -2 The in vitro blood-brain barrier model was successfully constructed. U87TR cells were seeded in 24-well plates, and the culture medium in the Transwell chamber was replaced with 100 μg mL -1 Fresh culture medium of fluorescently labeled FeP-SH-Cy3 and FeP-SH-Cy3@Exo was added. After culturing for 8 h, the TEER value was measured to verify the integrity of the BBB in vitro. Flow cytometry and laser confocal microscopy were used to detect the accumulation of FeP-SH-Cy3 and FeP-SH-Cy3@Exo in U87TR cells. The results are shown in Figure 2. Figure 18 As shown, the abscissa represents the Cy3 fluorescence intensity. The results show that the nanoparticles of the present invention (i.e., the nanomedicine of the present invention) can smoothly penetrate the blood-brain barrier after being coated with exosomes.

[0282] Experimental Example 10: Evaluation of the Effect of Inducing Ferroptosis

[0283] Intracellular lipid peroxide accumulation induces ferroptosis. Experimental culture media containing RSL3 (ferroptosis inducer), FeP, 6-MP, and FeP-MP@Exo were prepared. U87TR, T98G, and GL261TR cells were seeded in 6-well plates. After the cells attached, the culture medium was replaced with complete medium containing the drug. The cells were incubated for 24 hours, rinsed three times with DPBS, and then the following procedures were performed:

[0284] (1) Detection of intracellular ROS generation: Add 10 μM DCFH-DA and incubate at room temperature for 20 min, rinse three times with PBS, and collect cells for fluorescence confocal imaging or flow cytometry. Figure 19 The results showed that the nanomedicine FeP-MP@Exo of the present invention can induce a significant increase in ROS in cells (enhanced green fluorescence), which is consistent with the effect of the standard ferroptosis-inducing drug RSL3.

[0285] (2) Lipid peroxidation detection: Add 10 μM BODIPY 581 / 591C11 working solution, incubate at room temperature for 30 minutes, wash three times with PBS, and collect cells for fluorescence confocal imaging or flow cytometry detection.

[0286] (3) Malondialdehyde (MDA) content detection: Cells were collected, ultrasonically disrupted, and centrifuged at 10,000 rpm for 10 minutes, and the supernatant was collected. According to the formula in the instructions of the MDA content detection kit, the absorbance of the supernatant was measured at 450, 532, and 600 nm using an enzyme reader to calculate the MDA content. The results are shown in Figure 20 The results showed that the nanomedicine of the present invention can significantly increase the content of malondialdehyde in each cell, proving that it can significantly induce cell ferroptosis.

[0287] (4) PTGS2 and GPX4 protein expression: The cells were lysed on ice for 30 min using RIPA lysis buffer containing protease inhibitors, sonicated for 30 s, and centrifuged at 10,000 rpm for 10 min to collect the supernatant. The total protein concentration was determined using a BCA kit, and β-actin was used as an internal reference protein. The expression levels of PTGS2 and GPX4 proteins in each sample were detected by Western Blot. The results of GPX4 protein expression levels are shown in Figure 21 The results showed that in the nanomedicine treatment group of the present invention, the GPX4 protein level was significantly downregulated. At the same time, the expression level of PTGS2 encoding cyclooxygenase 2 was significantly downregulated. Figure 22 The PTGS2 protein level was significantly upregulated, indicating that the nanomedicine FeP-MP@Exo can induce the onset of cell ferroptosis.

[0288] Experimental Example 11: Evaluation of the Effect of Inducing Immunogenic Death

[0289] To evaluate the ability of FeP-MP@Exo to photothermally induce immunogenic cell death (ICD), damage-associated molecular patterns (DAMPs), namely, the surface exposure of CRT and the release of HMGB1 and ATP, were first investigated.

[0290] (1) U87TR, T98G, and GL261TR cells were seeded in glass-bottomed culture dishes and treated with the corresponding culture medium, FeP-MP@Exo, and FeP-MP@Exo combined with light, respectively. After 24 hours, they were washed three times with PBS, and then 4% paraformaldehyde was added and fixed at 4°C for 30 minutes. The cells were washed, blocked with 10% goat serum, incubated with CRT antibody at 4°C overnight, and then incubated with Alexa Fluor594-conjugated secondary antibody at 37°C for 2 hours. The cells were stained with DAPI and imaged with a fluorescence confocal microscope. The experimental results of GL261TR cells are as follows: Figure 23 The results showed that FeP-MP@Exo combined with light irradiation more significantly induced the exposure of CRT on the cell surface, proving that the nanomedicine of the present invention can induce immunogenic cell death under the condition of combined light irradiation.

[0291] (2) U87TR, T98G, and GL261TR cells were grown at 2×10 4 Cells were seeded at a density of 1000 cells / well in a 96-well plate. After 24 hours, FeP-MP@Exo and FeP-MP@Exo combined with light treatment were performed. After 24 hours, the supernatant was collected and centrifuged at 12000g for 10 minutes at 4°C. The level of HMGB1 released by cells in the supernatant was then detected using an enzyme-linked immunosorbent assay kit for HMGB1. The absorbance was measured at a wavelength of 450nm using a microplate reader. The results are shown in the figure. Figure 24 A, shows that FeP-MP@Exo combined with light treatment can significantly increase the level of HMGB1 released by cells.

[0292] (3) U87TR, T98G, and GL261TR cells were grown at 2×10 4 The cells were seeded at a density of 1000 cells / well in a black 96-well plate. After 24 hours, FeP-MP@Exo and FeP-MP@Exo combined with light treatment were performed. The supernatant was collected after 24 hours and centrifuged at 12000g for 10 minutes at 4°C. The ATP level in the supernatant was then detected using an ATP detection kit based on the luciferin-luciferase reaction, and the chemiluminescence of the sample was monitored using a microplate reader. The results are shown in Figure 24 B, indicating that FeP-MP@Exo combined with light treatment can significantly increase the level of ATP released by cells.

[0293] The above experimental results show that photothermal treatment leads to CRT surface exposure and HMGB1 and ATP release in the three cell lines, which fully proves that the photothermal treatment of the nanomedicine of the present invention can induce immunogenic cell death of tumor cells.

[0294] In vitro bone marrow-derived dendritic cell (BMDC) maturation assessment: BMDCs were isolated from the tibia and femur of 8-week-old BALB / c female mice and cultured in a 10% FBS-containing medium (20 ng mL-1 GM-CSF and 10 ng mL -1 BMDCs were induced to differentiate by RMPI 1640 medium containing IL-4. U87TR cells were seeded into the upper chamber of Transwell and cultured overnight. Then, BMDCs (5×10 5 Finally, BMDCs were collected and stained with anti-CD11c, anti-CD80, and anti-CD86, and detected by flow cytometry.

[0295] Experimental Example 12: In vivo therapeutic effect evaluation

[0296] (1) Using a stereotaxic apparatus, GL261TR-Luc cells (5 μL, 1×10 8 The GL261TR-derived orthotopic TMZ-resistant GBM mouse model was established by implanting the GL261TR-derived TMZ-resistant GBM mouse model into the left striatum of C57BL / 6N mice. Within 3 to 7 days, the tumor-bearing mice were intraperitoneally injected with luciferin substrate (150 mg kg -1 ), after isoflurane anesthesia, the mice were placed in a small animal in vivo imaging system for detection.

[0297] (2) Weigh the animals once before inoculation and once a day thereafter to observe weight changes.

[0298] (3) Eight days after modeling, the mice were injected with luciferase in the abdomen, and the fluorescence intensity of the mouse brain was observed using a live imaging instrument. The mice were randomly divided into four groups, and the mice were injected with PBS, TMZ (40 mg kg -1 ), TMZ+FeP@Exo(5mg kg -1 )+NIR、TMZ+FeP-MP@Exo(5mg kg -1 )+NIR, phototherapy uses 0.8W cm -2 The mice were irradiated with an 808nm laser for 10 minutes. All treatments were repeated every three days for a total of four treatments. The mice's body weight was monitored daily, and tumor volume was monitored using an in vivo imaging system. The Kaplan-Meier method was used to determine the survival rate of each group. After treatment, brain tissue and other major organs of GBM mice were collected and stained with hematoxylin and eosin for histological analysis.

[0299] The results showed that the nanomedicine of the present invention had no damage to the main organs of mice and had good biosafety.

[0300] The survival rates of mice in each group are shown in Figure 25It can be seen that the nanomedicine obtained by coating the surface functionalized iron phosphide nanoparticles of the present invention with exosomes combined with phototherapy can significantly improve the survival rate of TMZ-resistant GBM mice, and is better than the therapeutic effect of the nanomedicine obtained by coating the surface functionalized iron phosphide nanoparticles with exosomes.

[0301] It should be noted that, although the technical solutions of the present invention are described with specific examples, those skilled in the art will appreciate that the present invention should not be limited thereto.

[0302] While various embodiments of the present invention have been described above, the above descriptions are intended to be illustrative, non-exhaustive, and not limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is selected to best explain the principles of the embodiments, their practical applications, or technological improvements in the marketplace, or to enable others skilled in the art to understand the embodiments disclosed herein.

Claims

1. A method for preparing surface functionalized iron phosphide nanoparticles, comprising the following steps: (1) Surface-thiolated iron phosphide nanoparticles are obtained by using iron phosphide nanoparticles and molecules containing thiol groups and other groups that can coordinate with iron; (2) reacting the surface-thiolated iron phosphide nanoparticles obtained in step (1) with an active ingredient containing a thiol group to obtain; Wherein, the active ingredient containing a thiol group is a small molecule drug containing a thiol group, and the small molecule drug containing a thiol group is 6-mercaptopurine; the molecule containing a thiol group and other groups that can coordinate with iron is 2,3-dimercaptosuccinic acid; Step (1) is: adjusting the pH of the aqueous solution containing the thiol group and other groups that can coordinate with iron to 9-11, dropping the solution into the dispersion of the iron phosphide nanoparticles, ultrasonicating or stirring, centrifuging, washing, and freeze-drying to obtain surface thiol-modified iron phosphide nanoparticles; In step (2), the 6-mercaptopurine is oxidized to form a dimer, which is reacted with the surface thiolated iron phosphide nanoparticles obtained in step (1) under the protection of an inert gas, and then centrifuged, washed, and freeze-dried to obtain surface functionalized iron phosphide nanoparticles.

2. The preparation method according to claim 1, characterized in that The iron phosphide nanoparticles are prepared by chemical vapor deposition or organic solution phase reaction.

3. The preparation method according to claim 1, characterized in that In step (1), the iron phosphide nanoparticles and molecules containing thiol groups and other groups that can coordinate with iron are subjected to coordination exchange to obtain the surface thiol-modified iron phosphide nanoparticles.

4. The preparation method according to any one of claims 1 to 3, wherein The concentration of the aqueous solution containing thiol and other molecules that can coordinate with iron is 10 ~ 50 mg·mL -1 The concentration of the dispersion of the iron phosphide nanoparticles is 1 to 10 mg·mL -1 and / or The iron phosphide nanoparticles are prepared by chemical vapor deposition, and the dispersant in the dispersion of the iron phosphide nanoparticles is water. Alternatively, the iron phosphide nanoparticles are prepared by organic solution phase reaction, and the dispersant in the dispersion of the iron phosphide nanoparticles is tetrahydrofuran or chloroform.

5. The preparation method according to any one of claims 1 to 3, wherein The mass ratio of the 6-mercaptopurine to the surface-thiolated iron phosphide nanoparticles is (0.5-1):1; The reaction is carried out at room temperature; and / or The reaction time is 24 to 48 hours.

6. The preparation method according to claim 5, characterized in that The mass ratio of the 6-mercaptopurine to the surface-thiolated iron phosphide nanoparticles is 1:

1.

7. A surface-functionalized iron phosphide nanoparticle, prepared by the preparation method according to any one of claims 1 to 6.

8. The surface-functionalized iron phosphide nanoparticles according to claim 7, characterized in that: The size of the surface functionalized iron phosphide nanoparticles is 1 to 300 nm.

9. The surface-functionalized iron phosphide nanoparticles according to claim 8, characterized in that: The iron phosphide nanoparticles are prepared by chemical vapor deposition, and the surface-functionalized iron phosphide nanoparticles prepared using the iron phosphide nanoparticles have a size of 200 to 300 nm. Alternatively, the iron phosphide nanoparticles are prepared by organic solution phase reaction, and the surface-functionalized iron phosphide nanoparticles prepared using the iron phosphide nanoparticles have a size of 2 to 10 nm.

10. The surface-functionalized iron phosphide nanoparticles according to claim 9, characterized in that: The iron phosphide nanoparticles are prepared by chemical vapor deposition, and the size of the surface-functionalized iron phosphide nanoparticles prepared using the iron phosphide nanoparticles is 244.01 ± 15.93 nm, or the iron phosphide nanoparticles are prepared by organic solution phase reaction, and the size of the surface-functionalized iron phosphide nanoparticles prepared using the iron phosphide nanoparticles is 6.46 ± 2.82 nm.

11. A nanomedicine comprising the surface-functionalized iron phosphide nanoparticles according to any one of claims 7 to 10 and microglial cell membranes or microglial cell exosomes coated with the surface-functionalized iron phosphide nanoparticles.

12. The nanomedicine according to claim 11, characterized in that The nanomedicine is a brain-targeted nanomedicine; the microglial cell membrane or microglial exosome is derived from primary microglial cells or one or more of the microglial cell lines BV2, N9, HMC3, HMO6 and C8-B4; and / or The size of the nanomedicine is 1 to 300 nm.

13. The nanomedicine according to claim 12, characterized in that The iron phosphide nanoparticles are prepared by chemical vapor deposition, and the size of the nanomedicine prepared using the iron phosphide nanoparticles is 200 to 300 nm. Alternatively, the iron phosphide nanoparticles are prepared by organic solution phase reaction, and the size of the nanomedicine prepared using the iron phosphide nanoparticles is 5 to 50 nm.

14. The nanomedicine according to claim 13, characterized in that The iron phosphide nanoparticles are prepared by chemical vapor deposition, and the size of the nanomedicine prepared using the iron phosphide nanoparticles is 263.33 ± 8.01 nm, or the iron phosphide nanoparticles are prepared by organic solution phase reaction, and the size of the nanomedicine prepared using the iron phosphide nanoparticles is 20.95 ± 1.64 nm.

15. The method for preparing the nanomedicine according to any one of claims 11 to 14, comprising the following steps: The microglial cell membrane or microglial cell exosomes and the surface functionalized iron phosphide nanoparticles are mixed and extruded through a liposome extruder to obtain the product.

16. A pharmaceutical composition comprising the nanodrug according to any one of claims 11 to 14 and at least one additional anti-tumor drug.

17. The pharmaceutical composition according to claim 16, characterized in that The pharmaceutical composition comprises the nanodrug according to any one of claims 11 to 14 and an additional anti-tumor drug.

18. The pharmaceutical composition according to claim 16 or 17, characterized in that The antitumor drug is an alkylating agent antitumor drug.

19. The pharmaceutical composition according to claim 18, characterized in that The antitumor drug is temozolomide.

20. Use of the surface-functionalized iron phosphide nanoparticles according to any one of claims 7 to 10, the nanomedicine according to any one of claims 11 to 14, or the pharmaceutical composition according to any one of claims 16 to 19 in the preparation of a medicament for treating tumors.

21. The use according to claim 20, characterized in that The tumor is a glioma.

22. The use according to claim 21, characterized in that The tumor is a drug-resistant glioma.

23. The use according to claim 22, characterized in that The tumor is a temozolomide-resistant glioma.

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