Boron-rich magnetic nanoparticles, preparation method, pharmaceutical composition and application

Abstract

The invention discloses boron-rich magnetic nanoparticles and a preparation method and application thereof.The boron element content of the boron-rich magnetic nanoparticles is as high as 9-15 wt%, the boron-rich magnetic nanoparticles are prepared through a one-step solvothermal method, an iron oleate precursor, boric acid, phenyl ether and polyethylene glycol dicarboxylic acid are mixed, and then the mixture is subjected to a three-stage temperature programming reaction at the temperature of 90-120 DEG C, 180-220 DEG C and 250-270 DEG C to obtain the boron-rich magnetic nanoparticles; the prepared boron-rich nanoparticles are uniform in particle size and good in dispersity, have both high boron loading and superparamagnetism, and can significantly improve the tumor killing efficiency of BNCT and reduce the drug toxicity; the superparamagnetism can realize active targeting delivery guided by an external magnetic field, and the magnetic resonance imaging contrast agent can be used for avoiding the risk of magnetic agglomeration at the same time; fluorescence labeling and cell membrane or specific cell membrane protein modification are easily performed on the surfaces of the particles, so that the treatment effect is enhanced, and diagnosis and treatment integration guided by multi-modal imaging is realized; the boron-rich nanoparticles are simple and convenient in preparation process and easily available in raw materials, and have wide application prospects in tumor boron neutron capture therapy drugs.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of nanomaterials and biomedicine, and in particular to a boron-rich magnetic nanoparticle, a preparation method, a pharmaceutical composition and applications, to solve the problems of existing boron neutron capture therapy drugs in terms of boron load, targeting and real-time monitoring, and to provide a new material platform for tumor treatment. BACKGROUND

[0002] Cancer is one of the major public health challenges worldwide. With the growth of the population and the extension of life expectancy, the incidence of cancer is expected to continue to rise. In this context, the development of effective cancer treatment methods is essential to alleviate the medical and socio-economic burden of the disease.

[0003] Boron neutron capture therapy (BNCT) is a new radiotherapy technology based on a binary action mechanism, with cell-scale selectivity and high linear energy transfer (LET). The basic principle is that a specific energy spectrum of neutron beam is generated by an accelerator to irradiate the tumor site, and the neutron and the pre-enriched boron (¹ 0 B) in the tumor cells undergo nuclear capture reaction, instantaneously releasing high-LET α particles and lithium (Li) particles. The range of these two secondary particles is shorter than 10 μm, which can strictly limit the energy deposition to the scale of a single tumor cell, thereby achieving precise killing of tumor cells while maximizing the protection of surrounding normal tissues. Compared with conventional X-ray, γ-ray and other external irradiation radiotherapy, BNCT has potential advantages of high biological effect, less damage to normal tissues, and fewer treatment times.

[0004] The key to achieving the efficacy of BNCT is to selectively deliver sufficient boron to tumor cells. Among the many delivery carriers, magnetic nanomaterials have attracted widespread attention in the field of biomedicine due to their unique magnetic properties, good biocompatibility, and easy surface functionalization. Such materials usually have high saturation magnetization, superparamagnetism and rapid response to external magnetic fields, and can be guided to move and accumulate in the body by an external magnetic field, and are considered as a potential targeted delivery platform. By magnetic field guidance, boron-loaded magnetic nanoparticles can be specifically aggregated in the tumor area, which is expected to significantly improve the local boron concentration in the tumor and thus improve the therapeutic effect of BNCT.

[0005] However, the current boron-loaded magnetic nanoparticles for BNCT still face several key problems: first, the boron loading is generally low, which is difficult to meet the high local boron concentration required for treatment; second, the targeting efficiency is limited, and the non-specific distribution still exists simply relying on the magnetic field guidance; third, there is a lack of real-time and non-invasive tracking means, which cannot intuitively monitor the distribution and enrichment of nanoparticles in the body before or during treatment, affecting the accurate formulation of the treatment plan. In addition, the traditional synthesis method is often difficult to balance the high magnetic responsiveness and high boron loading of the material, and the function is single, which does not have the potential of diagnosis and treatment integration.

[0006] Therefore, there is an urgent need in the prior art to develop a new boron-containing magnetic nanomaterial, which can realize high boron loading while maintaining excellent magnetic properties, and integrate real-time imaging function, thereby synergistically improving the targeting, monitorability and treatment efficiency of BNCT. SUMMARY

[0007] Therefore, the present application provides a boron-rich magnetic nanoparticle, a preparation method, a pharmaceutical composition and an application, which aims to overcome the shortcomings of the existing BNCT drug delivery system in boron loading, targeting accuracy and real-time monitoring, and provides a new material platform for accurate diagnosis and efficient treatment of tumors. The technical solution is as follows: The present application provides a boron-rich magnetic nanoparticle in a first aspect, which has a general structure (I) or a pharmaceutically acceptable salt thereof: (I) Wherein, n is any number between 0 and 4; x is any number between 0.2 and 0.5; y is any number between 0.1 and 0.3; R is selected from at least one of hydrogen, halogen, hydroxyl, amino, cyano, nitro, C1-C6 alkyl, and C1-C6 alkoxy; And the B content is 9-15wt%.

[0008] Preferably, R is a fluorescent dye containing an amino group.

[0009] Further, the surface of the boron-rich magnetic nanoparticle is modified with tumor homologous cell membrane or specific cell membrane protein.

[0010] The present application provides a preparation method of a boron-rich magnetic nanoparticle in a second aspect, comprising the following steps: S1. Mix iron oleate precursor, boric acid, and polyethylene glycol dicarboxylic acid in a solvent of phenyl ether; S2. Heat the mixture to a first temperature of 90-120℃ for 20-40min; S3. Further heating the mixture to a second temperature of 180-220℃ for 40-80min; S4. Further heating the mixture to a third temperature of 250-270℃ for 40-80min; S5. Washing and drying to obtain the boron-rich magnetic nanoparticles, wherein the B content is 9-15wt%.

[0011] Preferably, in step S1, the iron oleate precursor is prepared by condensing and refluxing iron chloride and sodium oleate in a mixed solvent of anhydrous ethanol, n-hexane and water at 60-80℃ for 5 hours or less, and then washing and rotary evaporation.

[0012] Preferably, further comprising the steps of: S6. Dispersing the boron-rich magnetic nanoparticles obtained in S5 in a buffer solution, adding a carbodiimide-type activator and an N-succinimidyl-type catalyst for activation reaction to obtain an activated nanoparticle dispersion; S7. Adding an amine-reactive fluorescent dye to the activated nanoparticle dispersion for coupling reaction; S8. Purifying the product after coupling reaction to obtain fluorescent boron-rich magnetic nanoparticles.

[0013] Further comprising the steps of: S9. Extracting homologous tumor cell membrane proteins; S10. Mixing the boron-rich magnetic nanoparticles obtained in S5 or the fluorescent boron-rich magnetic nanoparticles obtained in S8 with the homologous tumor cell membrane proteins by ultrasonic mixing, and then using a liposome extruder to fuse the two to obtain tumor homologous cell membrane protein-modified boron-rich magnetic nanoparticles or tumor homologous cell membrane protein-modified fluorescent boron-rich magnetic nanoparticles.

[0014] Preferably, the molar ratio of the iron oleate precursor, polyethylene glycol dicarboxylic acid and boric acid is 1:3:(20-30).

[0015] Preferably, the molar ratio of the iron chloride and sodium oleate is 1:3.

[0016] In a third aspect, the present application provides a boron-rich diagnosis and treatment integrated nanomedicine composition, comprising the boron-rich magnetic nanoparticles of any of the above embodiments, and a pharmaceutically acceptable carrier, diluent or auxiliary agent.

[0017] In a fourth aspect, the present application provides the use of the boron-rich magnetic nanoparticles of any of the above embodiments in tumor boron neutron capture therapy.

[0018] Compared with the prior art, the at least one technical scheme adopted by the embodiments of the present specification can achieve the beneficial effects at least including: First, the boron-rich magnetic nanoparticles of specific molecular structure synthesized by the solvent thermal method of the present application have a boron element content of 9-15%, which is significantly higher than that of the existing clinical drug BPA (boron element content of 4.8%). This can not only enhance the targeting and enrichment efficiency, optimize the treatment ratio, and enhance the cell killing efficiency, but also reduce the drug dosage and reduce the toxicity of non-target organs.

[0019] Second, the boron-rich magnetic nanoparticles of the present application do not exhibit hysteresis, and the remanence and coercivity are both 0. This indicates that the boron-rich magnetic nanoparticles have paramagnetism, which can not only achieve efficient and controllable external magnetic field guided targeting, but also avoid magnetic agglomeration, ensure good colloidal stability and biological safety, and also serve as an excellent magnetic resonance imaging (MRI) contrast agent to realize integrated diagnosis and treatment. At the same time, the regular displacement of the boron-rich magnetic nanoparticles over time under the control of an external magnetic field indicates that the boron-rich magnetic nanoparticles have good magnetic response and can be used for magnetic manipulation of targeted delivery.

[0020] Third, the boron-rich magnetic nanoparticles of the present application can be labeled with fluorescent dyes, and the transportation of the drug in the body and the accumulation of the drug at the tumor site can be observed, further enhancing the integration of diagnosis and treatment.

[0021] Fourth, the surface of the boron-rich magnetic nanoparticles and the fluorescent boron-rich magnetic nanoparticles of the present application can be further modified by tumor homologous cell membrane or specific cell membrane protein. The modification of the homologous cell membrane surface or the specific cell membrane protein retains the complete adhesion protein and recognition molecules, allowing the boron-rich magnetic nanoparticles and the fluorescent boron-rich magnetic nanoparticles to inherit the biological interface properties and functions of the source tumor cells, thereby enhancing precise targeting of tumor cells and improving therapeutic efficacy. In addition, it can also improve biocompatibility and reduce the amount of normal cells entering, thereby reducing the side effects of BNCT treatment.

[0022] Fifth, the boron-rich magnetic nanoparticles of the present application are synthesized by a one-step method, which is simple, convenient, fast, and efficient, and the synthesis raw materials are cheap and easy to obtain, realizing the unification of high boron content and magnetic performance. The synthesized boron-containing magnetic nanoparticles are uniform and have good dispersibility. The boron-containing magnetic nanoparticles have good stability, high boron content, and tumor tissue targeting and enrichment ability in vivo, and also have good blood safety and do not cause hemolysis. BRIEF DESCRIPTION OF DRAWINGS

[0023] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed to be used in the embodiments will be briefly introduced as follows. Obviously, the drawings in the following description only constitute some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without any creative effort on the basis of these drawings.

[0024] Figure 1 Molecular structure diagram of fluorescent boron-rich magnetic nanoparticles (PFB-Cy5) of the preferred embodiment of the boron-rich magnetic nanoparticles (PFB) of the present application; Figure 2 Transmission electron microscope (TEM) diagram of the boron-rich magnetic nanoparticles (PFB) of the present application; Figure 3 Thermogravimetric diagram of the boron-rich magnetic nanoparticles (PFB) of the present application; Figure 4 Hysteresis curve diagram of the boron-rich magnetic nanoparticles (PFB) of the present application; Figure 5 Motion trajectory diagram of the boron-rich magnetic nanoparticles (PFB) of the present application; Figure 6 Fluorescence spectrum diagram of the fluorescent boron-rich magnetic nanoparticles (PFB-Cy5) of the present application; Figure 7 In-vitro fluorescence imaging diagram of the aqueous solution of the fluorescent boron-rich magnetic nanoparticles (PFB-Cy5) of the present application; Figure 8 Cytotoxicity diagram of the boron-rich magnetic nanoparticles (PFB) / tumor homologous cell membrane protein modified boron-rich magnetic nanoparticles (CM@PFB) of the present application; Figure 9 Blood compatibility diagram of the boron-rich magnetic nanoparticles (PFB) / tumor homologous cell membrane protein modified boron-rich magnetic nanoparticles (CM@PFB) of the present application; Figure 10 Fluorescence imaging diagram of the fluorescent boron-rich magnetic nanoparticles (PFB-Cy5) and the tumor homologous cell membrane protein modified fluorescent boron-rich magnetic nanoparticles (CM@PFB-Cy5) after being absorbed by tumor cells 4T1 of the present application; Figure 11 Fluorescence imaging diagram of the tumor homologous cell membrane protein modified fluorescent boron-rich magnetic nanoparticles (CM@PFB-Cy5) of the present application after being administered through the tail vein of a mouse, and the red circle represents the subcutaneous 4T1 tumor in the right armpit; Figure 12 Diagram of the change of the fluorescence intensity of the tumor homologous cell membrane protein modified fluorescent boron-rich magnetic nanoparticles (CM@PFB-Cy5) of the present application with time after being administered through the tail vein of a mouse; Figure 13The main organs and tumor ex vivo diagram of the tumor homologous cell membrane protein modified fluorescent boron-rich magnetic nanoparticles (CM@PFB-Cy5) of the application 1h after injection. DETAILED DESCRIPTION

[0025] The technical solutions in the embodiments of the application will be described clearly and completely below. Obviously, the described embodiments are only a part of the embodiments of the application, rather than all the embodiments. Based on the embodiments in the application, all other embodiments obtained by a person of ordinary skill in the art without creative work are within the protection scope of the application.

[0026] In addition, the technical solutions of various embodiments of the application can be combined with each other, but it must be based on that a person of ordinary skill in the art can realize. When the combination of technical solutions appears contradictory or cannot be realized, it should be considered that the combination of technical solutions does not exist and is not within the protection scope of the application.

[0027] The application provides a boron-rich magnetic nanoparticle (PFB) having a general structure (I): (I) wherein n is 0-4; x is 0.2-0.5; y is 0.1-0.3; R is selected from at least one of hydrogen, halogen, hydroxyl, amino, cyano, nitro, C1-C6 alkyl, and C1-C6 alkoxy; and wherein the B content is 9-15wt%.

[0028] Compared with the existing clinical drug BPA (boron element content is 4.8%), the boron content is significantly improved, and the efficacy of BNCT directly depends on the intracellular ¹ 0The number of alpha particles generated after the boron atom captures a neutron is proportional to the number of lithium ions. Higher boron loading means that more nuclear reactions can be triggered in each tumor cell under the same neutron irradiation, thereby significantly increasing the local energy deposition density and causing more complete lethal damage to cancer cells, especially to hypoxic cells that are insensitive to traditional radiotherapy. In order to achieve an effective therapeutic concentration, nanoparticles with low boron loading often need to be injected in a larger dose, which can increase the non-specific accumulation of drugs in organs such as the liver and spleen in the mononuclear phagocyte system, thereby causing potential systemic or organ-specific toxic side effects. In contrast, nanoparticles with high boron loading can achieve high boron concentration in the tumor target area with fewer particles and lower total drug dose, thereby systematically reducing non-target toxicity and improving the therapeutic safety window. Meanwhile, for the magnetic guidance delivery system of the present application, nanoparticles with high boron loading have higher therapeutic value per particle. This means that even with the same tumor enrichment percentage, the total amount of boron delivered by high-loading nanoparticles is much higher than that of low-loading nanoparticles, which can more efficiently deliver therapeutic agents to the lesion, thereby significantly increasing the boron concentration ratio between the tumor and normal tissue.

[0029] It should be understood that the boron-rich magnetic nanoparticles (PFB) of the present application include all stereoisomers, geometric isomers, tautomers that conform to the general structure, wherein each atom includes all isotopes. When there is one or more chiral centers in the molecule, the compound of the general structure (I) can be in the form of a pharmaceutically acceptable racemic mixture or a single stereoisomer.

[0030] The boron-rich magnetic nanoparticles of the present application also include all isotopic atoms, whether in intermediates or final compounds, and isotopic atoms include atoms with the same atomic number but different mass numbers. For example, the isotopes of hydrogen include tritium and deuterium.

[0031] Meanwhile, the boron-rich magnetic nanoparticles (PFB) of the present application can also exist in the form of a pharmaceutically acceptable salt. A pharmaceutically acceptable salt refers to the conversion of a group in the parent compound into a salt form. The form of a pharmaceutically acceptable salt can be a salt formed with an inorganic acid (such as a hydrochloride, a sulfate, a sulfonate, etc.), an ammonium salt formed with an amine (such as a triethylamine salt, a piperidine salt, or a basic drug, etc.), or a metal salt formed with an alkali metal or an alkaline earth metal (such as a sodium salt, a potassium salt, a calcium or magnesium salt, etc.).

[0032] The pharmaceutically acceptable salt of the present application can be synthesized from the parent compound, i.e. the basic group in the parent compound is reacted with 1-4 equivalents of acid in a solvent system. The pharmaceutically acceptable salt can be prepared from inorganic and organic acids, including but not limited to hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, benzenesulfonic acid, and the like.

[0033] In order to observe the drug transport in vivo and the tumor site accumulation, and to achieve the integration of diagnosis and treatment, the present application can preferably select the R group as an amino-containing fluorescent dye, including but not limited to cyanines, such as: Cy5-SE, Sulfo Cy2amine, etc.; fluoresceins, such as: 5-amino fluorescein, FAM amine, etc.; rhodamines, such as: 5(6)-TAMRA, etc.; BODIPY, such as: BODIPY FL amine, etc. The person skilled in the art can select the appropriate amino fluorescent dye according to the actual needs. Figure 1 The present application is a schematic diagram of the molecular structure of the fluorescent boron-rich magnetic nanoparticles, as shown in the figure, the fluorescent boron-rich magnetic nanoparticles (PFB) are on the main body of FeOx-B, the main body is modified with polyethylene glycol dicarboxylic acid (PEG), and the amino-containing fluorescent dye Cy5 is connected outside the PEG. However, it should be understood that the selection of the fluorescent dye in this embodiment should only be considered as a preferred example, and should not be understood as a limitation on the scope of protection of the claims.

[0034] In order to further improve the targeting of the boron-rich magnetic nanoparticles (PFB), the present application can further modify the surface of the boron-rich magnetic nanoparticles or the fluorescent boron-rich magnetic nanoparticles with tumor homologous cell membrane or specific cell membrane protein, and form the boron-rich magnetic nanoparticles modified by tumor homologous cell membrane protein (CM@PFB). The cell membrane protein is separated from the tumor cells cultured in the cell experiment, the tumor tissue separated from the model animal, or the tumor tissue separated from the clinical patient, and the homologous cell membrane surface or specific cell membrane protein modification retains the complete adhesion protein and recognition molecule, so that the boron-rich magnetic nanoparticles and the fluorescent boron-rich magnetic nanoparticles inherit the biological interface properties and functions of the source tumor cells, thereby enhancing the precise targeting of the tumor cells and improving the therapeutic effect; in addition, it can also improve the biocompatibility, reduce the immunogenicity, and reduce the intake amount of normal cells, thereby further reducing the side effects of BNCT treatment.

[0035] In order to prepare the boron-rich magnetic nanoparticles (PFB) / fluorescent boron-rich magnetic nanoparticles (PFB-Cy5) / tumor homologous cell membrane protein modified boron-rich magnetic nanoparticles (CM@PFB) / tumor homologous cell membrane protein modified fluorescent boron-rich magnetic nanoparticles (CM@PFB-Cy5) of each of the above embodiments, the present application also proposes a preparation method of boron-rich magnetic nanoparticles (PFB) / fluorescent boron-rich magnetic nanoparticles (PFB-Cy5) / tumor homologous cell membrane protein modified boron-rich magnetic nanoparticles (CM@PFB) / tumor homologous cell membrane protein modified fluorescent boron-rich magnetic nanoparticles (CM@PFB-Cy5), it should be understood that the volume and weight of the raw materials used in the specific embodiments below should only be regarded as preferred examples, and should not be understood as limiting the scope of protection of the claims.

[0036] I. Preparation of iron oleate precursor The mixture solvent composed of iron chloride and sodium oleate in a molar ratio of 1:3, anhydrous ethanol, n-hexane and water is placed in a reaction container, and the temperature is raised to 60-80℃, and the condensation reflux is carried out for 5h or less. After the reaction is completed, ultrapure water is added for washing, and rotary evaporation is carried out, so as to obtain the iron oleate precursor. Specifically, 5.4g FeCl3·6H2O, 18.25g sodium oleate, 40mL ethanol, 57mL n-hexane and 30mL water are added to a round-bottom flask, and the round-bottom flask is heated to 70℃, and the condensation reflux is carried out for 4h. After the reaction is completed, ultrapure water is added for washing, and rotary evaporation is carried out, so as to obtain the iron oleate precursor. It should be understood that the preparation process of the iron oleate precursor in this step is not a necessary step, and the commercially available iron oleate precursor can also be purchased by the person skilled in the art according to the needs.

[0037] II. Preparation of boron-rich magnetic nanoparticles (PFB) S1. The iron oleate precursor, polyethylene glycol dicarboxylic acid, boric acid, and the solvent phenyl ether are placed in a reaction container in a molar ratio of 1:3:(20-30) and mixed; specifically, 1.8g iron oleate precursor, 1.24g boric acid, 10g phenyl ether and 1.8g polyethylene glycol divinyl ether are added to a three-necked flask and mixed; S2. The mixture is heated to a first temperature, and the first temperature is 90-120℃, and the heating is continued for 20-40min; preferably, the first temperature is 110℃, and the heating is continued for 30min; S3. The mixture is further heated to a second temperature, and the second temperature is 180-220℃, and the heating is continued for 40-80min; preferably, the second temperature is 200℃, and the heating is continued for 60min; S4. The mixture is then heated to a third temperature of 250-270°C for 40-80 minutes; preferably, the third temperature is 260°C for 60 minutes; S5. After the reaction is completed, n-hexane is added for washing and centrifugation, and the obtained waxy solid is transferred to a beaker and dried at 80°C and 100 Pa. Finally, the prepared boron-rich magnetic nanoparticles have a B content of 9-15wt%.

[0038] III. Preparation of fluorescent boron-rich magnetic nanoparticles (PFB-Cy5) In order to observe the drug transport in vivo and the tumor site accumulation, and to achieve the purpose of diagnosis and treatment integration, the boron-rich magnetic nanoparticles can be further labeled with fluorescent dyes. Since polyethylene glycol dicarboxylic acid is added during the synthesis of the nanoparticles, it has carboxyl groups, which can make the nanoparticles have good water solubility and can also undergo amidation reaction with fluorescent dyes with amino groups, thereby coupling the fluorescent dyes to the boron-rich magnetic nanoparticles. Specifically, the method can further include the following steps: S6. The boron-rich magnetic nanoparticles obtained in S5 are dispersed in a buffer solution, and a carbodiimide activator and an N-succinimidyl catalyst are added for activation reaction to obtain an activated nanoparticle dispersion. The carbodiimide activator is preferably 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide (EDC), and the N-succinimidyl catalyst is preferably N-hydroxysuccinimidyl (NHS). EDC first activates the carboxyl groups of the boron-rich nanoparticles to generate an O-acylisourea intermediate, and then NHS reagent is added to form an N-hydroxysuccinimidyl (sulfonic acid) ester intermediate. Specifically, 30 mg of the boron-rich magnetic nanoparticles are dissolved in 2 mL of PBS buffer, EDC / NHS are added in a molar ratio of 1:2 for activation for 1 hour, and the activation operation is completed.

[0039] S7. Amine-reactive fluorescent dyes are added to the activated boron-rich magnetic nanoparticle dispersion for coupling reaction. The N-hydroxysuccinimidyl (sulfonic acid) ester intermediate of S6 can undergo amidation reaction with the amino groups of the fluorescent dyes to form an amide bond, thereby successfully coupling the boron-rich magnetic nanoparticles with the amino fluorescent dyes. Preferably, 0.3 mg of Cy5-SE is added after the activation reaction in step S6 is completed, and the coupling is completed by avoiding light reaction at room temperature for 24 hours.

[0040] S8. Finally, the product after coupling reaction is purified, and preferably dialyzed for 24 hours using a 3500 Da dialysis bag to obtain fluorescent boron-rich magnetic nanoparticles.

[0041] Four, preparation of tumor homologous cell membrane protein modified boron-rich magnetic nanoparticles (CM@PCB) / tumor homologous cell membrane protein modified fluorescent boron-rich magnetic nanoparticles (CM@PCB-Cy5) In order to improve the targeting of the boron-rich magnetic nanoparticles, the surface of the boron-rich magnetic nanoparticles can be further modified by tumor homologous cell membrane protein, which is isolated from the same tumor cells cultured in cell experiments, the same tumor tissues isolated from model animals, or the autologous tumor tissues isolated from clinical patients. On the one hand, the immunogenicity can be reduced, and on the other hand, the boron-rich magnetic nanoparticles can enter the cells by passing through the cell membrane of the tumor cells. The steps include the following steps: S9. Extracting the homologous tumor cell membrane, specifically, extracting the 4T1 tumor cell membrane cultured; S10. Mixing the boron-rich magnetic nanoparticles obtained in S5 or the fluorescent boron-rich magnetic nanoparticles obtained in S8 with the 4T1 tumor cell membrane at a mass ratio of 1: (1-3) by ultrasonic mixing, and then using a liposome extruder to fuse the two, to obtain tumor homologous cell membrane modified boron-rich magnetic nanoparticles or tumor homologous cell membrane modified fluorescent boron-rich magnetic nanoparticles.

[0042] It should be understood that the above tumor cell strain model is only a preferred example, and those skilled in the art can isolate the same cultured or autologous tumor cell membrane protein according to the type of tumor to be treated or experimented. The 4T1 tumor cells in the specific embodiments should not be understood as limiting the scope of protection of the claims.

[0043] Five, physical and chemical performance test of boron-rich magnetic nanoparticles (PFB) / fluorescent boron-rich magnetic nanoparticles (PFB-Cy5) Figure 2 TEM imaging of the boron-rich magnetic nanoparticles (PFB) of the present application is shown. The figure shows that the boron-rich magnetic nanoparticles (PFB) synthesized by the one-step solvothermal method of the present application have a relatively uniform distribution and good dispersibility, with a particle size of about 5 nm.

[0044] Further, the B element content in the product was detected by ICP-MS, as shown in Table 1. When the feeding ratio of Fe and B was 1:20, the B element content of the boron-rich magnetic nanoparticles (PFB) of the present application reached a peak of 13.46 ± 0.70%. When the feeding ratio of Fe and B was 1:30, the B element content of the boron-rich magnetic nanoparticles (PFB) of the present application also reached 9.31 ± 0.04%. Basically, the high boron loading of 9-15wt% can be achieved. Compared with the conventional clinical drug BPA (4.8%) in the prior art, the boron content of the present application is significantly improved, which can not only strengthen the targeting and enrichment efficiency, optimize the treatment ratio, and enhance the cell killing efficiency, but also reduce the drug dosage and reduce the toxicity to non-target organs.

[0045] Table 1 The organic matter content in the boron-rich magnetic nanoparticles (PFB) was detected using thermal gravimetric analysis (TGA), as shown in Figure 3 The reaction rate is the fastest at 440℃, indicating that the organic matter PEG is rapidly decomposed at 440℃, and the content of the organic matter, especially the content of PEG, accounts for 23.79 ± 1.82%.

[0046] Figure 4 As shown in the hysteresis curve of the boron-rich magnetic nanoparticles (PFB) of the present application, the hysteresis loop of the boron-rich magnetic nanoparticles was detected by using a vibrating sample magnetometer (VSM), and it was found that the boron-rich magnetic nanoparticles of the present application did not have a hysteresis phenomenon, and the remanence and coercivity were both 0, which indicated that the boron-rich magnetic nanoparticles had paramagnetism, could realize efficient and controllable external magnetic field guided targeting, could avoid magnetic aggregation, could ensure good colloidal stability and biological safety, and could also be used as an excellent magnetic resonance imaging (MRI) contrast agent to realize diagnosis and treatment integration. Further, the boron-rich magnetic nanoparticles (PFB) of the present application were subjected to external magnetic field control, as shown in Figure 5 Under the guidance of the magnetic field, the boron-rich magnetic nanoparticles (PFB) regularly moved over time, the motion path was fitted, R2=0.999, and had a good linear relationship, which indicated that the boron-rich magnetic nanoparticles (PFB) had good magnetic response and could be used for magnetic manipulation targeted delivery.

[0047] In order to analyze the fluorescence imaging effect of the boron-rich magnetic nanoparticles coupled with fluorescent dyes, the present application performed fluorescence spectrum analysis on the fluorescent boron-rich magnetic nanoparticles (PFB-Cy5) and the blank control group of the boron-rich magnetic nanoparticles (PFB) not coupled with fluorescent dyes, and the results are shown in Figure 6 The fluorescent boron-rich magnetic nanoparticles (PFB-Cy5) showed a fluorescence intensity peak at a wavelength of 675 nm, which was exactly the same as the fluorescence peak position of the Cy5 fluorescent dye, indicating that the Cy5 fluorescent dye had been successfully coupled to the boron-rich magnetic nanoparticles and showed good fluorescence characteristics. Further, the present application coupled the boron-rich magnetic nanoparticles with different concentrations of Cy5 (0.5 mg / mL, 1 mg / mL, 2 mg / mL, 5 mg / mL, 10 mg / mL) fluorescent dyes, and detected the fluorescence intensity of the coupled fluorescent boron-rich magnetic nanoparticles (PFB-Cy5) aqueous solution, as shown in Figure 7 The fluorescence intensity increased with the increase of the concentration of the fluorescent dye, and the regression coefficient R2 was 0.992, indicating that the fluorescence intensity of the fluorescent boron-rich magnetic nanoparticles (PFB-Cy5) had good linear correlation with its concentration, which could meet the needs of quantitative analysis of optical imaging.

[0048] It can be seen from the above results that the boron-rich magnetic nanoparticles (PFB) synthesized by the one-step solvothermal method of the application have the advantages of simple and convenient synthesis method, high synthesis efficiency, cheap and easily available raw materials, good dispersibility and uniform particle size, and the unification of high boron content and magnetic performance; meanwhile, the synthesis method of the fluorescent boron-rich magnetic nanoparticles (PFB-Cy5) is simple and meets the needs of optical imaging quantitative analysis, thereby providing platform support for observing the transportation of drugs in the biological body and the accumulation of drugs in tumor sites and strengthening the integration of diagnosis and treatment.

[0049] V. Biomedical performance test of boron-rich magnetic nanoparticles (PFB) / fluorescent boron-rich magnetic nanoparticles (PFB-Cy5) / tumor homologous cell membrane protein modified boron-rich magnetic nanoparticles (CM@PFB) / tumor homologous cell membrane protein modified fluorescent boron-rich magnetic nanoparticles (CM@PFB-Cy5) In order to further understand the biomedical characteristics of the boron-rich magnetic nanoparticles (PFB) / tumor homologous cell membrane protein modified boron-rich magnetic nanoparticles (CM@PFB) synthesized by the application, first, the cytotoxicity of boric acid (BA), boron-rich magnetic nanoparticles (PFB) and tumor homologous cell membrane protein modified boron-rich magnetic nanoparticles (CM@PFB) on triple-negative breast cancer cells at 0 μg / mL, 10 μg / mL, 20 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL and 500 μg / mL B concentrations was respectively evaluated by using the CCK-8 method, and the results are shown in Figure 8 As shown in the figure, compared with the control group BA, the PFB experimental group and the CM@PFB experimental group, especially the CM@PFB experimental group, have lower cytotoxicity on triple-negative breast cancer cells, and only have low toxicity at high concentrations (≥100 μg / mL).

[0050] In order to further verify the blood safety of the boron-rich magnetic nanoparticles (PFB) / tumor homologous cell membrane protein modified boron-rich magnetic nanoparticles (CM@PFB) synthesized by the application, the hemolysis experiment method was used to respectively evaluate the change of the absorbance at 541 nm of PFB and CM@PFB at 10 μg / mL, 20 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL and 500 μg / mL B concentrations compared with the positive control deionized water group (DI), as shown in Figure 9 As shown in the figure, the PFB and CM@PFB experimental groups at different concentrations do not cause obvious hemolysis, which indicates that the boron-rich magnetic nanoparticles / tumor homologous cell membrane protein modified boron-rich magnetic nanoparticles synthesized by the application have high blood safety and can meet the basic requirements of animal experiments.

[0051] Further, as shown in Figure 10As shown, after co-culturing fluorescent boron-rich magnetic nanoparticles (PFB-Cy5) and tumor homologous cell membrane protein modified fluorescent boron-rich magnetic nanoparticles (CM@PFB-Cy5) with tumor cell line triple-negative breast cancer cells 4T1 for 4 hours, respectively, under 640 nm wavelength of laser confocal microscope, it was found that both PFB-Cy5 and CM@PFB-Cy5 could accurately target tumor cells, but compared with PFB-Cy5, CM@PFB-Cy5 was more likely to target into tumor cells and diffusely enrich in the cytoplasm of tumor cells. Since the homologous cell membrane surface or specific cell membrane protein modification retains the complete adhesion protein and recognition molecule, the fluorescent boron-rich magnetic nanoparticles can inherit the biological interface characteristics and functions of the source tumor cells, thereby enhancing the precise targeting of tumor cells, thereby providing experimental evidence at the cellular level for the tumor BNCT treatment of tumor homologous cell membrane protein modified boron-rich magnetic nanoparticles (CM@PFB-Cy5).

[0052] In order to further verify the magnetic targeting property of the tumor homologous cell membrane protein modified fluorescent boron-rich magnetic nanoparticles (CM@PFB-Cy5) synthesized by the present application in vivo, the present application uses 4T1 triple-negative breast cancer Balb / c mouse model (allogeneic subcutaneous tumor) to explore the targeting enrichment effect of CM@PFB-Cy5 in the tumor site, as shown in Figure 11 As shown, compared with the control group (CM@PFB-Cy5) not driven by external magnetic force, the fluorescence intensity in the tumor area (red dashed box) of the experimental group (CM@PFB-Cy5+MF) driven by magnetic force is higher after 1-8h of drug administration; as shown in Figure 12 As shown, by comparing the average fluorescence intensity per unit area in the tumor area (red dashed box), it was found that the average fluorescence intensity of the experimental group (CM@PFB-Cy5+MF) driven by magnetic force was significantly higher than that of the control group (CM@PFB-Cy5) not driven by external magnetic force during 1-8h after drug administration. This also suggests that under in vivo environment, the boron-rich magnetic nanoparticles / fl uorescent boron-rich magnetic nanoparticles synthesized by the present application are more likely to enrich in the tumor site under the driving of external magnetic force, and have good in vivo magnetic targeting property.

[0053] In order to characterize the biological metabolism distribution of the tumor homologous cell membrane protein modified fluorescent boron-rich magnetic nanoparticles (CM@PFB-Cy5) synthesized by the present application in vivo, the present application injects 30mg[B] / kg CM@PFB-Cy5 into the caudal vein of the mouse, and after 1h of external magnetic force guidance, the main metabolic organs are dissected in vitro, and the fluorescence intensity is analyzed, as shown in Figure 13As shown, the strong fluorescence is mainly distributed in the liver and organs with large tumor blood flow, further illustrating that the boron-rich magnetic nanoparticles and the fluorescent boron-rich magnetic nanoparticles synthesized by the application have good tumor enrichment effect, and the distribution in other organs is relatively less, further verifying the good tumor treatment prospect and biological safety thereof.

[0054] From the above results, it can be seen that the boron-rich magnetic nanoparticles and the fluorescent boron-rich magnetic nanoparticles synthesized by the one-step solvothermal method have low toxicity, high biocompatibility, and high tumor enrichment under the external magnetic guiding effect, and have high application prospect in the BNCT tumor treatment.

[0055] Therefore, the application also provides a pharmaceutical composition comprising the above-mentioned boron-containing compound and a pharmaceutically acceptable carrier, diluent or auxiliary agent.

[0056] Furthermore, the application also provides the use of the above-mentioned boron-containing compound and the pharmaceutical composition comprising the same as an active ingredient in the preparation of a drug for boron neutron capture therapy of tumors. In the above use, the tumor can be derived from the central nervous system. Further, the tumor is a glioma, meningioma, neuroblastoma, germ cell tumor, pituitary tumor, brain metastasis, peripheral neuroepithelioma, primitive neuroectodermal tumor or arteriovenous malformation. For the treatment of glioma, the effect on glioblastoma, gliosarcoma, pilocytic astrocytoma, oligodendroglioma, anaplastic astrocytoma, low-grade astrocytoma or brain stem glioma is more significant. Further, in the above use, the tumor can be a malignant tumor or a metastatic tumor, and the treatment effect on melanoma, head and neck tumor, prostate cancer, liver cancer, lung cancer or breast cancer is optimal.

[0057] In the specification, the same or similar parts between the embodiments can be referred to each other, and each embodiment focuses on the difference from other embodiments. Especially, for the embodiments described later, the description is relatively simple, and the relevant parts can be referred to the part of the foregoing embodiments.

[0058] The above is merely a specific implementation of the present application, but the protection scope of the present application is not limited thereto, and any person skilled in the art can easily think of changes or replacements within the technical range disclosed by the present application, which should be covered in the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the protection scope of the claims.

Claims

1. A boron-rich magnetic nanoparticle, characterized in that, It has the general structural formula (I) or a pharmaceutically acceptable salt thereof: （I） Where n takes any value between 0 and 4; x can be any number between 0.2 and 0.5; y can take any value between 0.1 and 0.3; R is selected from at least one of hydrogen, halogen, hydroxyl, amino, cyano, nitro, C1-C6 alkyl, and C1-C6 alkoxy; Furthermore, the content of B is 9-15 wt%.

2. The boron-rich magnetic nanoparticle according to claim 1, characterized in that, R is an amino-containing fluorescent dye.

3. The boron-rich magnetic nanoparticle according to claim 1 or 2, characterized in that, The surface of the boron-rich magnetic nanoparticles is modified with tumor-derived cell membranes or specific cell membrane proteins.

4. A method for preparing boron-rich magnetic nanoparticles, characterized in that, Includes the following steps: S1. Mix ferric oleate precursor, boric acid, and polyethylene glycol dicarboxylic acid in the solvent phenyl ether; S2. Heat the mixture to a first temperature, which is 90°C to 120°C, and maintain the temperature for 20 min to 40 min. S3. Further heat the mixture to a second temperature, which is 180°C to 220°C, and maintain for 40 min to 80 min; S4. The mixture is then heated to a third temperature, which is 250°C to 270°C, and held for 40 to 80 minutes. S5. Wash and dry to obtain the boron-rich magnetic nanoparticles, wherein the B content accounts for 9~15wt%.

5. The method for preparing boron-rich magnetic nanoparticles according to claim 4, characterized in that, In step S1, the ferric oleate precursor is prepared by refluxing ferric chloride and sodium oleate in a mixed solvent composed of anhydrous ethanol, n-hexane and water at 60-80°C for no more than 5 hours, followed by washing and rotary evaporation.

6. The method for preparing boron-rich magnetic nanoparticles according to claim 4, characterized in that, It also includes the following steps: S6. The boron-rich magnetic nanoparticles obtained in S5 are dispersed in a buffer solution, and a carbodiimide activator and an N-succinimide catalyst are added to carry out an activation reaction to obtain an activated nanoparticle dispersion. S7. Add an amine-reactive fluorescent dye to the activated nanoparticle dispersion to carry out a coupling reaction; S8. The product after the coupling reaction was purified to obtain fluorescent boron-rich magnetic nanoparticles.

7. A method for preparing boron-rich magnetic nanoparticles according to claim 4 or 6, characterized in that, It also includes the following steps: S9. Extract homologous tumor cell membrane proteins; S10. The boron-rich magnetic nanoparticles obtained in S5 or the fluorescent boron-rich magnetic nanoparticles obtained in S8 are ultrasonically mixed with the homologous tumor cell membrane protein, and then fused together using a liposome extruder to obtain boron-rich magnetic nanoparticles modified with tumor homologous cell membrane protein or fluorescent boron-rich magnetic nanoparticles modified with tumor homologous cell membrane protein.

8. The method for preparing boron-rich magnetic nanoparticles according to claim 4, characterized in that, The molar ratio of the ferric oleate precursor, polyethylene glycol dicarboxylic acid, and boric acid is 1:3:(20~30).

9. The method for preparing boron-rich magnetic nanoparticles according to claim 5, characterized in that, The molar ratio of ferric chloride to sodium oleate is 1:

3.

10. A boron-rich therapeutic nanomedicine composition, characterized in that, It includes the boron-rich magnetic nanoparticles as described in any one of claims 1-3, as well as pharmaceutically acceptable carriers, diluents, or adjuvants.

11. The application of boron-rich magnetic nanoparticles as described in any one of claims 1-3 in boron neutron capture therapy for tumors.

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