Iron-boron nano-micelle for boron neutron capture therapy as well as preparation method and application of iron-boron nano-micelle

Abstract

The invention provides an iron-boron nano-micelle for boron neutron capture therapy as well as a preparation method and application of the iron-boron nano-micelle. And the iron-boron nano-micelle is obtained through an oxidation-reduction method. Targeted enrichment of the nano-micelles at tumors is realized by modifying penetrating peptides on the surfaces of the nano-micelles. The preparation method provided by the invention is simple and convenient in process and practical, and the obtained iron-boron nano-micelle is good in water solubility and biocompatibility. Compared with a common tumor cell targeted boron drug, the material further delivers nano-micelles to the depth of a solid tumor through penetrating peptide and enriches the nano-micelles. In addition, the nano-micelle has magnetism and can be further enriched at a tumor through magnetic targeting, boron drug enrichment at the tumor is achieved, and tumor cells are killed through BNCT and chemical kinetics combined treatment.

Description

Technical Field

[0001] The invention relates to the field of biomedical technology, in particular to an iron boron nano-micelle for boron neutron capture therapy and a preparation method and application thereof. Background Art

[0002] Boron Neutron Capture Therapy (BNCT) is an emerging technology in tumor radiotherapy. 10 After neutron irradiation, the natural non-radioactive 10 B generates alpha particles and Li particles under the action of thermal neutrons. When charged particles undergo high-line energy transfer, a radiation area of ​​about 5-9 microns will be generated, thereby killing cells. As a typical binary treatment system, BNCT requires that the tumor is enriched with 10 The B concentration cannot be lower than 20ppm. In the early BNCT clinical trials in 1950 and 1960, boric acid and some boric acid derivatives were used as boron delivery agents. However, these simple compounds are not selective for tumor cells, have high overall toxicity to the organism, and have low retention rates in tumors. These defects limit the further development of boric acid and its derivatives as boron delivery agents in BNCT. Therefore, the core challenge to improve the anti-tumor efficacy of BNCT is to achieve high selective enrichment of boron drugs in tumor cells (tumor sites). 10 At the same time, in order to reduce the toxic side effects on the surrounding normal tissues, it is required that the boron drug accumulates less in normal organs and tissues: the boron concentration ratio of tumor and normal tissue (T / N)>3, and the boron concentration ratio of tumor and blood (T / B)>3. Currently, there are only two boron drugs approved for clinical treatment of BNCT, namely (L)-4-dihydroxyborylphenylalanine (boronophenylalanine, BPA) and sodium mercaptound-ecahydrocloso-dodecaborate (sodium mercaptound-ecahydrocloso-dodecaborate, Na2B 12 H 11 SH, BSH).

[0003] Chemodynamic therapy (CDT) is a new strategy for tumor treatment that uses the highly oxidatively active and highly toxic hydroxyl radicals produced by the Fenton reaction in tumor cells. Compared with other free radical oxidative therapy methods, CDT does not need to consider the limitation of the tissue penetration depth of light waves, and this endogenously initiated treatment method has stronger tumor specificity and can avoid damage to normal tissues.

[0004] Compared with single treatment, combined therapy is the development trend of tumor treatment in the future. At present, there are also studies combining BNCT with other tumor treatment methods and have achieved better therapeutic effects than single BNCT. In 2021, Liu Zhibo's research group from the Department of Applied Chemistry, School of Chemistry and Molecular Engineering, Peking University proposed a DOX@BNNSs nanodrug delivery system for tumor treatment. Utilizing the nuclear targeting of doxorubicin (DOX) in nanomaterials, nanodrugs were successfully delivered to the cell nucleus to achieve combined treatment of chemotherapy and BNCT, achieving better therapeutic effects. Summary of the invention

[0005] The purpose of the present invention is to provide an iron boron nano-micelle for boron neutron capture therapy and a preparation method and application thereof.

[0006] In order to achieve the purpose of the present invention, in a first aspect, the present invention provides an iron boron nano-micelle for boron neutron capture therapy, which is an iron boron magnetic nano-particle with a penetrating peptide modified on the surface.

[0007] In the present invention, the penetrating peptide may be selected from iRGD, TAT, NFL-TBS.40-63 or MG2B, etc., preferably iRGD (whose amino acid sequence is CRGDKGPDC).

[0008] The particle size of the iron-boron nano-micelles used for boron neutron capture therapy is 220nm±10nm.

[0009] In a second aspect, the present invention provides a method for preparing iron boron nanomicelles for boron neutron capture therapy, wherein iron boron magnetic nanoparticles are prepared by a redox method, and then penetrating peptides are modified on the surface of the nanoparticles.

[0010] Specifically, the method comprises the following steps: Step 1: dissolve 2-20 mmol of ferrous chloride tetrahydrate in 4-40 ml of double distilled water, filter through an 800 nm filter membrane, then add 4-40 ml of PEG solution, and stir under argon atmosphere for 25-40 min to obtain solution A; The concentration of the PEG solution is 10 mg / ml, wherein the molecular weight of PEG is 200-6000, preferably 200, 4000, 6000, and more preferably 4000; Step 2: Dissolve 8-80 mmol of sodium borohydride in 16-160 ml of 0.1 M sodium hydroxide solution, and stir in a vent bottle for 25-40 min to prepare solution B; Step 3: Under ventilation conditions, solution A is slowly added to solution B, and stirred until there are no bubbles to generate a black precipitate. The precipitate is collected and washed with water, and then transferred to DMF, acetone or DMSO. After ultrasonication for 1 to 4 hours, the obtained material is transferred to the aqueous phase and freeze-dried to obtain iron boron magnetic nanoparticles, which are recorded as Fe2B@PEG. Step 4: 10 mg Fe2B@PEG, 25-50 mg DSPE-PEG-OCH3 (molecular weight of 2K-5K, preferably 2K or 5K, more preferably 2K) and 5-10 mg DSPE-PEG-iRGD (molecular weight of 2K-5K, preferably 2K or 5K, more preferably 2K) are dissolved in 2-4 ml of organic solvent and ultrasonicated for 5 min, then 10-20 ml of double distilled water is added under ultrasonic conditions, ultrasonicated for 30-120 min, and then centrifuged at 4000-14500 rpm (preferably 14500 rpm) for 30 min to collect the precipitate, washed with water twice, and freeze-dried to obtain iron boron nanomicelles for boron neutron capture therapy, recorded as Fe2B@PEG-iRGD.

[0011] The organic solvent can be selected from DMF, methanol or a mixture of DMF and dichloromethane in a volume ratio of 1:1, preferably DMF.

[0012] Furthermore, in steps 3 and 4, the ultrasonic conditions are: ultrasonic power 60 KW, on 5s, off 5s.

[0013] In a third aspect, the present invention provides the use of the iron-boron nano-micelles or the iron-boron nano-micelles prepared according to the method in preparing boron-containing drugs for boron neutron capture therapy.

[0014] In a fourth aspect, the present invention provides the use of the iron-boron nano-micelles or the iron-boron nano-micelles prepared according to the method in the preparation of anti-tumor drugs.

[0015] By means of the above technical solution, the present invention has at least the following advantages and beneficial effects: (I) The present invention provides a method for preparing magnetic iron boron nano-micelles, wherein the iron boron nano-micelles are obtained by a redox method. The nano-micelles are modified with penetrating peptides on the surface to achieve targeted enrichment of the nano-micelles at the tumor. The preparation method provided by the present invention has simple procedures and is practical. The obtained iron boron nano-micelles have good water solubility and good biocompatibility, and the hydrated particle size is 220nm±10nm.

[0016] (II) Compared with ordinary tumor cell-targeted boron drugs, this material further delivers nano-micelles to the deep of solid tumors and enriches them through penetrating peptides. In addition, the nano-micelles have magnetic properties and can be further enriched in the tumor through magnetic targeting, thus achieving boron drug enrichment in the tumor.

[0017] (III) Enriching iron-boron nano-micelles in large quantities at the tumor site, in addition to achieving a large amount of 10 In addition to enriching the B element for BNCT treatment, a large number of iron ions are enriched in the tumor to catalyze and accelerate the Fenton-like reaction in tumor cells, produce a large amount of reactive oxygen in tumor cells, and achieve chemodynamic therapy. The tumor cells are killed through the combined treatment of BNCT and chemodynamic therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a diagram of the hydrated particle size of the Fe2B@PEG-iRGD nanomicelles prepared in Example 1 of the present invention.

[0019] Figure 2 The infrared spectra of Fe2B@PEG, Fe2B@PEG-DSEP and Fe2B@PEG-iRGD prepared in Comparative Examples 7, 8 and Example 1 of the present invention.

[0020] Figure 3 This is the full XPS test spectrum of the Fe2B nanoparticles prepared in Comparative Example 6 of the present invention.

[0021] Figure 4 The Fe2B@PEG and F prepared in Comparative Example 7 and Example 1 of the present invention e2 Full spectrum of XPS test of B@PEG-iRGD nanoparticles.

[0022] Figure 5 This is an XPS fine spectrum data analysis chart of B 1s of the Fe2B nanoparticles prepared in Comparative Example 6 of the present invention.

[0023] Figure 6 It is an XPS fine spectrum data analysis diagram of B1s of Fe2B@PEG and Fe2B@PEG-iRGD nanoparticles prepared in Comparative Example 7 and Example 1 of the present invention.

[0024] Figure 7 This is an XPS fine spectrum data analysis diagram of Fe 2p of the Fe2B nanoparticles prepared in Comparative Example 6 of the present invention.

[0025] Figure 8 It is an XPS fine spectrum data analysis diagram of Fe2p of Fe2B@PEG and Fe2B@PEG-iRGD nanoparticles prepared in Comparative Example 7 and Example 1 of the present invention.

[0026] Fig. 9 These are the X-ray diffraction patterns of Fe2B, Fe2B@PEG, and Fe2B@PEG-iRGD prepared in Comparative Examples 6, 7 and Example 1 of the present invention.

[0027] Fig.10Magnetic hysteresis loop diagrams of Fe2B, Fe2B@PEG, and Fe2B@PEG-iRGD prepared in Comparative Examples 6 and 7 and Example 1 of the present invention.

[0028] Fig.11 The graphs show the results of chemical kinetic experiments of Fe2B@PEG-iRGD prepared in Example 1 of the present invention in pH=6.5 and pH=7.4 environments, respectively.

[0029] Fig.12 This is a graph showing the results of a chemical kinetic experiment of Fe2B@PEG-iRGD prepared in Example 1 of the present invention under different hydrogen peroxide concentrations.

[0030] Fig.13 This is a graph showing the results of chemical kinetic experiments of Fe2B@PEG-iRGD prepared in Example 1 of the present invention at different concentrations.

[0031] Fig.14 The graphs show the results of chemical kinetic experiments of Fe2B@BSA-TPP prepared in Comparative Example 4 of the present invention in pH=6.5 and pH=7.4 environments, respectively.

[0032] Fig.15 This is a graph showing the results of a chemical kinetic experiment of Fe2B@BSA-TPP prepared in Comparative Example 4 of the present invention under different hydrogen peroxide concentrations.

[0033] Fig.16 This is a graph showing the results of chemical kinetic experiments at different concentrations of Fe2B@BSA-TPP prepared in Comparative Example 4 of the present invention.

[0034] Fig.17 This is a stability data diagram of Fe2B@PEG-iRGD prepared in Example 1 of the present invention.

[0035] Fig.18 This is a biosafety diagram of Fe2B@PEG-iRGD prepared in Example 1 of the present invention.

[0036] Fig.19 This is a graph showing the results of an MTT cytotoxicity experiment in mouse melanoma cells (B16 cells) using Fe2B@PEG-iRGD prepared in Example 1 of the present invention.

[0037] Fig. 20 This is a graph showing the results of an uptake experiment of Fe2B@PEG-iRGD prepared in Example 1 of the present invention in mouse melanoma cells (B16 cells).

[0038] Fig.21This is a graph showing the distribution of Fe2B@PEG-iRGD prepared in Example 1 of the present invention in mice at different time points after administration as a nanomedicine.

[0039] Fig. 22 The boron content in the tumor at different time points after administration of Fe2B@PEG-iRGD prepared in Example 1 of the present invention.

[0040] Fig.23 Graph showing particle size distribution of Fe2B@PEG-iRGD prepared in Examples 1, 2 and 3 of the present invention in water.

[0041] Fig.24 This is the particle size distribution diagram of Fe2B@PEG-iRGD prepared in Examples 1, 4 and 5 of the present invention in water. DETAILED DESCRIPTION

[0042] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0043] DSPE-PEG-OCH3 and DSPE-PEG-iRGD used in the following examples were purchased from Shanghai Tuoyang Biotechnology Co., Ltd.

[0044] Example 1

[0045] This embodiment provides a method for preparing iron boron nano-micelles, comprising the following steps: Step 1: 2 mmol of ferrous chloride tetrahydrate was dissolved in 4 ml of secondary water and filtered through an 800 nm filter membrane, then 4 ml of PEG (molecular weight 4K, concentration 10 mg / ml) was added thereto, and stirred under argon atmosphere for 25 min to prepare solution A. Solution A was transferred to a 10 ml syringe for later use; Step 2: 8 mmol of sodium borohydride was dissolved in 16 ml of sodium hydroxide (0.1 M), and stirred in a 500 ml ventilation bottle for 25 min to prepare solution B; Step 3: Under ventilation conditions, slowly add solution A to solution B and stir until there are no bubbles to generate a black precipitate. Collect the precipitate and quickly wash it with water several times before transferring it to DMF (DMF can be replaced with acetone or DMSO). Insert the Fe2B@PEG magnetic particles after ultrasonic treatment for 2 hours, and transfer the material to the aqueous phase for freeze-drying and storage. Step 4: Dissolve 10 mg Fe2B@PEG, 25 mg DSPE-PEG-OCH3 (molecular weight 2K) and 5 mg DSPE-PEG-iRGD (molecular weight 2K) in 2 ml DMF and insert sonicate for 5 min. Then, add 8 ml of secondary water step by step under the condition of insert sonication, and sonicate for 30 min. Centrifuge at 14500 rpm for 30 min to collect the precipitated Fe2B@PEG-iRGD nanomicelles, wash them with secondary water 3 times, and freeze-dry for storage.

[0046] Example 2

[0047] Step 1: 2 mmol of ferrous chloride tetrahydrate was dissolved in 4 ml of secondary water and filtered through an 800 nm filter membrane, then 4 ml of PEG (molecular weight 4K, concentration 10 mg / ml) was added thereto, and stirred under argon atmosphere for 25 min to prepare solution A. Solution A was transferred to a 10 ml syringe for later use; Step 2: 8 mmol of sodium borohydride was dissolved in 16 ml of sodium hydroxide (0.1 M), and stirred in a 500 ml ventilation bottle for 25 min to prepare solution B; Step 3: Under ventilation conditions, slowly add solution A to solution B and stir until there are no bubbles to generate a black precipitate. Collect the precipitate and quickly wash it with water several times before transferring it to DMF (DMF can be replaced with acetone or DMSO). Insert the Fe2B@PEG magnetic particles after ultrasonication for 2 hours, centrifuge at 6000 rpm for 10 minutes, and take the supernatant and freeze-dry it for storage. Step 4: Dissolve 10 mg Fe2B@PEG, 25 mg DSPE-PEG-OCH3 (molecular weight 2K) and 5 mg DSPE-PEG-iRGD (molecular weight 2K) in 2 ml DMF and insert sonicate for 5 min. Then, add 8 ml of secondary water step by step under the condition of insert sonication, and sonicate for 30 min. Centrifuge at 14500 rpm for 30 min to collect the precipitated Fe2B@PEG-iRGD nanomicelles, wash them with secondary water 3 times, and freeze-dry for storage.

[0048] Example 3

[0049] Step 1: 2 mmol of ferrous chloride tetrahydrate was dissolved in 4 ml of secondary water and filtered through an 800 nm filter membrane, then 4 ml of PEG (molecular weight 4K, concentration 10 mg / ml) was added thereto, and stirred under argon atmosphere for 25 min to prepare solution A. Solution A was transferred to a 10 ml syringe for later use; Step 2: 8 mmol of sodium borohydride was dissolved in 16 ml of sodium hydroxide (0.1 M), and stirred in a 500 ml ventilation bottle for 25 min to prepare solution B; Step 3: Under ventilation conditions, slowly add solution A to solution B and stir until there are no bubbles to generate a black precipitate. Collect the precipitate and quickly wash it with water several times before transferring it to DMF (DMF can be replaced with acetone or DMSO). Insert the Fe2B@PEG magnetic particles after ultrasonication for 2 hours, centrifuge at 4000 rpm for 10 minutes, and take the supernatant for freeze-drying and storage. Step 4: Dissolve 10 mg Fe2B@PEG, 25 mg DSPE-PEG-OCH3 (molecular weight 2K) and 5 mg DSPE-PEG-iRGD (molecular weight 2K) in 2 ml DMF and insert sonicate for 5 min. Then, add 8 ml of secondary water step by step under the condition of insert sonication, and sonicate for 30 min. Centrifuge at 14500 rpm for 30 min to collect the precipitated Fe2B@PEG-iRGD nanomicelles, wash them with secondary water 3 times, and freeze-dry for storage.

[0050] Example 4

[0051] This embodiment provides a method for preparing iron boron nano-micelles, comprising the following steps: Step 1: 2 mmol of ferrous chloride tetrahydrate was dissolved in 4 ml of secondary water and filtered through an 800 nm filter membrane, then 4 ml of PEG (molecular weight 4K, concentration 10 mg / ml) was added thereto, and stirred under argon atmosphere for 25 min to prepare solution A. Solution A was transferred to a 10 ml syringe for later use; Step 2: 8 mmol of sodium borohydride was dissolved in 16 ml of sodium hydroxide (0.1 M), and stirred in a 500 ml ventilation bottle for 25 min to prepare solution B; Step 3: Under ventilation conditions, slowly add solution A to solution B and stir until there are no bubbles to generate a black precipitate. Collect the precipitate and quickly wash it with water several times before transferring it to DMF (DMF can be replaced with acetone or DMSO). Insert the Fe2B@PEG magnetic particles after ultrasonication for 2 hours, centrifuge at 14500 rpm for 10 minutes, and take the supernatant and freeze-dry it for storage. Step 4: Dissolve 10mg Fe2B@PEG, 25mg DSPE-PEG-OCH3 (molecular weight 2K) and 5mg DSPE-PEG-iRGD (molecular weight 2K) in 2ml DMF and DCM mixture (volume ratio 1:1) and insert ultrasonic for 5min. Then, add 8ml of secondary water step by step under the condition of insert ultrasonic, and ultrasonic for 30min. Centrifuge at 14500rpm for 30min to collect the precipitated Fe2B@PEG-iRGD nanomicelles and wash them with secondary water for 3 times, and freeze-dry for storage.

[0052] Example 5

[0053] This embodiment provides a method for preparing iron boron nano-micelles, comprising the following steps: Step 1: 2 mmol of ferrous chloride tetrahydrate was dissolved in 4 ml of secondary water and filtered through an 800 nm filter membrane, then 4 ml of PEG (molecular weight 4K, concentration 10 mg / ml) was added thereto, and stirred under argon atmosphere for 25 min to prepare solution A. Solution A was transferred to a 10 ml syringe for later use; Step 2: 8 mmol of sodium borohydride was dissolved in 16 ml of sodium hydroxide (0.1 M), and stirred in a 500 ml ventilation bottle for 25 min to prepare solution B; Step 3: Under ventilation conditions, slowly add solution A to solution B and stir until there are no bubbles to generate a black precipitate. Collect the precipitate and quickly wash it with water several times before transferring it to DMF (DMF can be replaced with acetone or DMSO). Insert the Fe2B@PEG magnetic particles after ultrasonication for 2 hours, centrifuge at 14500 rpm for 10 minutes, and take the supernatant and freeze-dry it for storage. Step 4: Dissolve 10 mg Fe2B@PEG, 25 mg DSPE-PEG-OCH3 (molecular weight 2K) and 5 mg DSPE-PEG-iRGD (molecular weight 2K) in 2 ml methanol and insert ultrasonic for 5 min. Then, add 8 ml of secondary water step by step under the condition of insert ultrasonic, and ultrasonic for 30 min. Centrifuge at 14500 rpm for 30 min to collect the precipitated Fe2B@PEG-iRGD nanomicelles and wash them with secondary water for 3 times, and freeze-dry for storage.

[0054] The hydrated particle size of Fe2B@PEG-iRGD nanomicelles prepared in Example 1 is shown in Figure 1 .

[0055] The infrared spectra of Fe2B@PEG, Fe2B@PEG-DSEP and Fe2B@PEG-iRGD prepared in Comparative Examples 7 and 8 and Example 1 are shown in Figure 2 .

[0056] The full spectrum of XPS test of Comparative Example 6 is shown in Figure 3 .

[0057] The full XPS spectra of Fe2B@PEG and Fe2B@PEG-IRGD nanoparticles prepared in Comparative Example 7 and Example 1 are shown in Figure 4 .

[0058] The XPS fine spectrum data analysis of B 1s of Fe2B nanoparticles prepared in Comparative Example 6 is shown in Figure 5 .

[0059] The XPS fine spectrum data analysis of B 1s of Fe2B@PEG and Fe2B@PEG-iRGD nanoparticles prepared in Comparative Example 7 and Example 1 is shown in Figure 6 .

[0060] The XPS fine spectrum data analysis diagram of Fe 2p of Fe2B nanoparticles prepared in Comparative Example 6 is shown in Figure 7 .

[0061] The XPS fine spectrum data analysis of Fe 2p of Fe2B@PEG and Fe2B@PEG-iRGD nanoparticles prepared in Comparative Example 7 and Example 1 is shown in Figure 8 .

[0062] The X-ray diffraction patterns of Fe2B, Fe2B@PEG, and Fe2B@PEG-iRGD prepared in Comparative Examples 6 and 7 and Example 1 are shown in Fig. 9 .

[0063] The hysteresis loops of Fe2B, Fe2B@PEG, and Fe2B@PEG-iRGE prepared in Comparative Examples 6 and 7 and Example 1 are shown in Fig.10 .

[0064] The results of chemical kinetic experiments of Fe2B@PEG-iRGD prepared in Example 1 at pH = 6.5 and pH = 7.4 are shown in the figure. Fig.11 .

[0065] The results of the chemical kinetics experiment of Fe2B@PEG-iRGD prepared in Example 1 under different hydrogen peroxide concentrations are shown in FIG. Fig.12 .

[0066] The results of chemical kinetic experiments of Fe2B@PEG-iRGD prepared in Example 1 at different concentrations are shown in FIG. Fig.13 .

[0067] The results of the chemical kinetics experiments of Fe2B@BSA-TPP prepared in Comparative Example 4 at pH = 6.5 and pH = 7.4 are shown in the figure. Fig.14 .

[0068] The results of the chemical kinetics experiment of Fe2B@BSA-TPP prepared in Comparative Example 4 under different hydrogen peroxide concentrations are shown in FIG. Fig.15 .

[0069] The results of the chemical kinetics experiment of Fe2B@BSA-TPP prepared in Comparative Example 4 at different concentrations are shown in Fig.16 .

[0070] The stability data of Fe2B@PEG-iRGD prepared in Example 1 is shown in Fig.17 .

[0071] The biosafety of Fe2B@PEG-iRGD prepared in Example 1 is shown in Fig.18 .

[0072] The results of the MTT cytotoxicity test of Fe2B@PEG-iRGD prepared in Example 1 in mouse melanoma cells (B16 cells) are shown in FIG. Fig.19 .

[0073] The results of the Fe2B@PEG-iRGD uptake experiment in mouse melanoma cells (B16 cells) prepared in Example 1 are shown in FIG. Fig. 20 The distribution of Fe2B@PEG-iRGD prepared in Example 1 in mice at different time points after administration as a nanomedicine is shown in the figure below. Fig.21 .

[0074] The boron content in the tumor at different time points after administration of Fe2B@PEG-iRGD prepared in Example 1 is shown in FIG. Fig. 22 .

[0075] The particle size distribution of Fe2B@PEG-iRGD prepared in Examples 1, 2 and 3 in water is shown in Fig.23 .

[0076] The particle size distribution of Fe2B@PEG-iRGD prepared in Examples 1, 4 and 5 in water is shown in Fig.24 .

[0077] Fig.11 Chemical kinetics experiments were conducted for Fe2B@PEG-iRGD in pH=6.5 and pH=7.4 environments: The generation of hydroxyl radicals was evaluated by terephthalic acid degradation experiments. We dispersed Fe2B@PEG-iRGD in phosphate buffer solutions with pH values ​​of 6.5 and 7.4, respectively, and then added 20μL TA solution and 1% H2O2. After reacting for 1 hour, the ultraviolet absorption value was detected in the wavelength range of 350-500nm using a fluorescence spectrometer under 310nm excitation light. The difference in absorbance values ​​shows the ability to generate ·OH.

[0078] Fig.12 Chemical kinetics experiments were conducted for Fe2B@PEG-iRGD under different hydrogen peroxide concentrations: Fe2B@PEG-iRGD was dispersed in a phosphate buffer solution with a pH value of 6.5, and then 20μL of TA solution and different concentrations of H2O2 were added. After reacting for 1 hour, the ultraviolet absorption value was detected in the wavelength range of 350-500nm using a fluorescence spectrometer under 310nm excitation light. The difference in absorbance values ​​shows the ability to produce ·OH.

[0079] Fig.13 Chemical kinetics experiments were conducted for Fe2B@PEG-iRGD at different concentrations: Fe2B@PEG-iRGD at different concentrations was dispersed in a phosphate buffer solution with a pH value of 6.5, and then 20μL of TA solution and 1% H2O2 were added. After reacting for 1 hour, the ultraviolet absorption value was detected in the wavelength range of 350-500nm using a fluorescence spectrometer under 310nm excitation light. The difference in absorbance values ​​shows the ability to produce ·OH.

[0080] Fig.14 The chemical kinetics experiment of Fe2B@BSA-TPP in pH=6.5 and pH=7.4 respectively: Fe2B@BSA-TPP was dispersed in phosphate buffer solution with pH value of 6.5 and 7.4 respectively, and then 20μL TA solution and 1% H2O2 were added. After reacting for 1h, the ultraviolet absorption value was detected in the wavelength range of 350-500nm under the condition of 310nm excitation light using a fluorescence spectrometer. The difference in absorbance value shows the ability to produce ·OH.

[0081] Fig.15 Conduct chemical kinetics experiments for Fe2B@BSA-TPP under different hydrogen peroxide concentrations: disperse Fe2B@BSA-TPP in phosphate buffer solution with a pH value of 6.5, then add 20μL TA solution and different concentrations of H2O2. After reacting for 1 hour, use a fluorescence spectrometer to detect ultraviolet absorption values ​​in the wavelength range of 350-500nm under 310nm excitation light conditions. The difference in absorbance values ​​shows the ability to produce ·OH.

[0082] Fig.16 Chemical kinetics experiments were conducted for Fe2B@BSA-TPP at different concentrations: Fe2B@BSA-TPP at different concentrations was dispersed in a phosphate buffer solution with a pH value of 6.5, and then 20μL of TA solution and 1% H2O2 were added. After reacting for 1 hour, the ultraviolet absorption value was detected in the wavelength range of 350-500nm using a fluorescence spectrometer under 310nm excitation light. The difference in absorbance values ​​shows the ability to produce ·OH.

[0083] Fig.17The stability experiment scheme of Fe2B@PEG-iRGD was as follows: the particle size and PDI changes of Fe2B@PEG-iRGD incubated in cell culture medium (DMEM containing L-glutamine, 4.5 g / L glucose and sodium pyruvate, HyCloneTM) containing 10% FBS at 37°C were evaluated for 0h, 0.5h, 1h, 2h, 4h, 6h, 8h, 10h, 12h, 24h, and 48h.

[0084] Fig.18 Hemolysis experimental protocol: 1mL of C57BL mouse whole blood was collected in a 2mL centrifuge tube containing 2.5μL of 2% sodium heparin, and the mixture was then shaken slowly. After that, the same volume of normal saline was added, followed by centrifugation (1500rpm, 10min). The supernatant was removed and the precipitate was dispersed with 10mL of normal saline. The suspension was centrifuged (1500rpm, 20min), and the same steps were repeated 3 times to obtain a red blood cell suspension. After that, 0.2mL of the red blood cell suspension was taken out and mixed with (-) 0.8mL PBS as a negative control, (+) 0.8mL deionized water as a positive control, and 0.8mL of Fe2B@PEG-iRGD aqueous solution in various concentration ranges (0 to 500μg / mL). The mixture was incubated at 37 ºC for another 1 h and then centrifuged (12000 rpm, 5 min). The supernatant was then measured for optical density (OD value) in a microplate reader (Spark™ 10M, Tecan, Zurich, Switzerland) using an excitation wavelength of 541 nm. The hemolysis rate (Hr) was calculated using the following formula: Hr (%) = (ODs-ODn / ODp-ODn) × 100%

[0085] Wherein, ODs, ODp and ODn are the OD values ​​of sample, positive control and negative control, respectively.

[0086] Fig.19 B16 cell toxicity assay protocol: B16 cells were cultured in a medium containing 10% fetal bovine serum (FBS, Gibco), 1.0 × 10 5U / L penicillin and 0.1mg / mL streptomycin (Sigma, Beijing, China) were cultured in cell culture medium, and the medium was changed every other day. The cytotoxicity of the nanoparticles was analyzed by MTT assay. B16 cells were seeded into 96-well plates at a density of 5000 cells per well. After 24h, the growth medium was replaced with fresh medium. After that, different concentrations of nanoparticles were added to the wells (six wells in parallel for each sample). After the predetermined time, the culture medium was removed. The excess material was washed with PBS, and then DMEM basal medium containing 10% MTT was added to the corresponding wells, incubated at 37 ºC and 5% CO2 for 4h, the culture was terminated, and the culture supernatant in the well was carefully aspirated after centrifugation. 100ul DMSO was added to each well and shaken for 10min to fully dissolve the crystals. The absorbance of each well was measured at a wavelength of 490nm to calculate the cell survival rate

[0087] Fig. 20 For the cell uptake experiment, B16 cells were cultured in a medium containing 10% fetal bovine serum (FBS, Gibco), 1.0 × 10 5 U / L penicillin and 0.1mg / mL streptomycin (Sigma, Beijing, China) were cultured in six-well plates. The next day, Fe2B@PEG-iRGD was incubated with cells in DMEM medium at concentrations of 500µg / mL and 250µg / mL at 37 ºC in 5% CO2 for 24 h. Subsequently, the cells were washed 3 times with PBS buffer to remove free samples. The cells were collected and counted, and then the cells were cold digested in 5ml MOS grade concentrated nitric acid overnight and then microwave digested. The boron content in the cells was tested by inductively coupled plasma mass spectrometry.

[0088] Fig.21 Ivis small animal live imaging protocol: purchased from Beijing Weitonglihua Animal Co., Ltd.

[0089] Female BALB / c mice (4-5 weeks, 13±2 g) were obtained. The mouse subcutaneous melanoma model was established as follows: the mice were weighed and anesthetized with 4% chloral hydrate (10 mL / kg) intraperitoneally. Then, 10 μg of chloral was injected subcutaneously into the right hind limb after hair removal. 7 B16 cells / each. When the tumor is palpable in the leg (about 60mm 3 ), the following experiment was conducted on mice: Fe2B@PEG-iRGD-Cy5 was injected through the tail vein of mice, and in vivo fluorescence imaging of mice was performed using the Lumina III in vivo imaging system (Ex / Em = 640 / 680nm) at 1, 2, 4, and 8 hours after injection.

[0090] Fig. 22 Experimental protocol for testing the boron content in the tumor after administration: Female BALB / c mice (4-5 weeks, 13±2 g) were obtained from Beijing Weitong Lihua Animal Co., Ltd. The mouse subcutaneous melanoma model was established as follows: the mice were weighed and anesthetized with 4% chloral hydrate (10 mL / kg) intraperitoneally, followed by a 10-min injection of chloral in the right hind limb after depilation. 7 B16 cells / each. When the tumor is palpable in the leg (about 60mm 3 ), the following experiment was conducted on mice: Fe2B@PEG-iRGD was injected into the mouse tail vein, and the mouse tumor was collected 2 and 4 hours after the injection. The tumor was cold digested in 5 ml of MOS-grade concentrated nitric acid overnight and then microwave digested. The boron content in the tumor was tested by inductively coupled plasma mass spectrometry.

[0091] Comparative Example 1 Synthesis of Fe2B magnetic particles (with low dosage of ferrous chloride)

[0092] 1.08mmol of ferrous chloride tetrahydrate was dissolved in 4ml of secondary water and oxygen was excluded; 8mmol of sodium borohydride was dissolved in 8ml of NaOH (concentration of 0.1 M) and oxygen was excluded before injection into the ferrous chloride (stirring was continued during the process).

[0093] Comparative Example 2 Synthesis of Fe2B Magnetic Particles (Solution with Less Sodium Borohydride Dosage)

[0094] 2mmol of ferrous chloride tetrahydrate was dissolved in 4ml of secondary water, oxygen was cut off and kept in an ice bath. 4mmol of sodium borohydride was dissolved in 8ml of NaOH (concentration of 0.1 M), oxygen was cut off and then injected into the ferrous chloride (stirring was continued during the process).

[0095] Comparative Example 3 Synthesis of Fe2B magnetic particles (addition of polyvinyl alcohol)

[0096] Step 1: 2 mmol of ferrous chloride tetrahydrate was dissolved in 4 ml of secondary water and filtered through an 800 nm filter membrane, then 1 ml of polyvinyl alcohol (PVA, 10 mg / ml) was added thereto, and stirred under argon atmosphere for 25 min to prepare solution A. Solution A was transferred to a 10 ml syringe for later use; Step 2: 8 mmol of sodium borohydride was dissolved in 16 ml of sodium hydroxide (0.1 M), and stirred in a 500 ml ventilation bottle for 25 min to prepare solution B; Step 3: Under ventilation conditions, slowly add solution A to solution B and stir until there are no bubbles to form a black precipitate. Collect the precipitate, quickly wash it with water several times, and then transfer it to DMF (DMF can be replaced with acetone or DMSO). Insert the Fe2B@PVP magnetic particles after ultrasonic treatment for 2 hours, centrifuge at 14500 rpm for 10 minutes, and take the supernatant and freeze-dry it for storage.

[0097] Comparative Example 4 Synthesis of Fe2B@BSA-TPP magnetic particles (adding bovine serum albumin)

[0098] Step 1: 2 mmol of ferrous chloride tetrahydrate was dissolved in 4 ml of secondary water and filtered through an 800 nm filter membrane, then 20 mg of BSA was added thereto, and the mixture was stirred under argon atmosphere for 25 min to prepare solution A. Solution A was transferred to a 10 ml syringe for later use; Step 2: 8 mmol of sodium borohydride was dissolved in 16 ml of sodium hydroxide (0.1 M), and stirred in a 500 ml ventilation bottle for 25 min to prepare solution B; Step 3: Under ventilation conditions, slowly add solution A to solution B and stir until there are no bubbles to generate a black precipitate. Collect the precipitate and quickly wash it with water several times, then transfer it to DMF (DMF can be replaced with acetone or DMSO). Insert the Fe2B@BSA magnetic particles after ultrasonic treatment for 2 hours, centrifuge at 14500 rpm for 10 minutes, and take the supernatant and freeze-dry it for storage. Step 4: Add 0.5 ml of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) solution (concentration 0.2 mmol / mL) to 1 ml of triphenyl phosphate (TPP) solution (concentration 0.05 mmol) and stir at 1000-1200 rpm for 10 minutes, then add 0.5 ml of N-hydroxysuccinimide (NHS) solution (concentration 0.2 mmol / mL), stir at 25-30 ° C, and react at 1000-1200 rpm for 1.5 hours. Then add Fe2B@BSA dissolved in 3 ml of secondary water, stir at 25-30 ° C for 24 hours to obtain Fe2B@BSA-TPP nanoparticles, resuspend and wash with secondary water, and freeze-dry for storage.

[0099] Comparative Example 5 Synthesis of Fe2B Nanomicelles (Adding Poloxamer 188)

[0100] Step 1: 2 mmol of ferrous chloride tetrahydrate was dissolved in 4 ml of secondary water and filtered through an 800 nm filter membrane, then 4 ml of PEG (molecular weight 4K, concentration 10 mg / ml) was added thereto, and stirred under argon atmosphere for 25 min to prepare solution A. Solution A was transferred to a 10 ml syringe for later use; Step 2: 8 mmol of sodium borohydride was dissolved in 16 ml of sodium hydroxide (0.1 M), and stirred in a 500 ml ventilation bottle for 25 min to prepare solution B; Step 3: Under ventilation conditions, slowly add solution A to solution B and stir until there are no bubbles to generate a black precipitate. Collect the precipitate and quickly wash it with water several times before transferring it to DMF (DMF can be replaced with acetone or DMSO). Insert the Fe2B@PEG magnetic particles after ultrasonication for 2 hours, centrifuge at 14500 rpm for 10 minutes, and take the supernatant and freeze-dry it for storage. Step 4: Dissolve 10 mg Fe2B@PEG in 6 ml methanol / DCM mixture, then add to 30 ml (10% w / w) poloxamer 188 aqueous solution, then sonicate for 30 min. Then add to 100 ml (0.1% w / w) poloxamer 188 aqueous solution, stir overnight, centrifuge at 14500 rpm for 30 min, collect the precipitate, wash with secondary water 3 times, and freeze-dry for storage.

[0101] Comparative Example 6 Synthesis of Fe2B

[0102] Step 1: Dissolve 2 mmol of ferrous chloride tetrahydrate in 4 ml of secondary water and filter through an 800 nm filter membrane, and stir under argon atmosphere for 25 min to prepare solution A. Transfer solution A to a 10 ml syringe for later use; Step 2: 8 mmol of sodium borohydride was dissolved in 16 ml of sodium hydroxide (0.1 M), and stirred in a 500 ml ventilation bottle for 25 min to prepare solution B; Step 3: Under ventilation conditions, slowly add solution A to solution B and stir until there are no bubbles to form a black precipitate. Collect the precipitate and quickly wash it with water several times. Transfer the precipitate to DMF (DMF can be replaced with acetone or DMSO). Insert the Fe2B magnetic particles after ultrasonic treatment for 2 hours. Transfer the material to the water phase for freeze-drying and storage.

[0103] Comparative Example 7 Synthesis of Fe2B@PEG

[0104] Step 1: 2 mmol of ferrous chloride tetrahydrate was dissolved in 4 ml of secondary water and filtered through an 800 nm filter membrane, then 4 ml of PEG (molecular weight 4K, concentration 10 mg / ml) was added thereto, and stirred under argon atmosphere for 25 min to prepare solution A. Solution A was transferred to a 10 ml syringe for later use; Step 2: 8 mmol of sodium borohydride was dissolved in 16 ml of sodium hydroxide (0.1 M), and stirred in a 500 ml ventilation bottle for 25 min to prepare solution B; Step 3: Under ventilation conditions, slowly add solution A to solution B and stir until there are no bubbles to generate a black precipitate. Collect the precipitate and quickly wash it with water several times before transferring it to DMF (DMF can be replaced with acetone or DMSO). Insert the Fe2B@PEG magnetic particles after ultrasonication for 2 hours, centrifuge at 14500 rpm for 10 minutes, and take the supernatant and freeze-dry it for storage. Comparative Example 8 Synthesis of Fe2B@PEG-DPSE

[0105] Step 1: 2 mmol of ferrous chloride tetrahydrate was dissolved in 4 ml of secondary water and filtered through an 800 nm filter membrane, then 4 ml of PEG (molecular weight 4K, concentration 10 mg / ml) was added thereto, and stirred under argon atmosphere for 25 min to prepare solution A. Solution A was transferred to a 10 ml syringe for later use; Step 2: 8 mmol of sodium borohydride was dissolved in 16 ml of sodium hydroxide (0.1 M), and stirred in a 500 ml ventilation bottle for 25 min to prepare solution B; Step 3: Under ventilation conditions, slowly add solution A to solution B and stir until there are no bubbles to generate a black precipitate. Collect the precipitate and quickly wash it with water several times before transferring it to DMF (DMF can be replaced with acetone or DMSO). Insert the Fe2B@PEG magnetic particles after ultrasonication for 2 hours, centrifuge at 14500 rpm for 10 minutes, and take the supernatant and freeze-dry it for storage. Step 4: Dissolve 10 mg Fe2B@PEG and 50 mg DSPE-PEG-OCH3 (molecular weight 2K) in 2 ml DMF and insert sonicate for 5 min. Then, add 8 ml of secondary water step by step under insert sonication conditions and sonicate for 30 min. Centrifuge at 14500 rpm for 30 min to collect the precipitated Fe2B@PEG-iRGD nanomicelles, wash them with secondary water for 3 times, and freeze-dry for storage.

[0106] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. An iron boron nano-micelle for boron neutron capture therapy, characterized in that: It is an iron-boron magnetic nanoparticle with a penetrating peptide modified on the surface; The penetrating peptide is selected from iRGD, TAT, NFL-TBS.40-63 or MG2B, preferably iRGD.

2. The iron boron nano micelle according to claim 1, characterized in that: The particle size is 220nm±10nm.

3. A method for preparing iron boron nano-micelles for boron neutron capture therapy, characterized in that: Iron-boron magnetic nanoparticles were prepared by a redox method, and then penetrating peptides were modified on the surface of the nanoparticles; The penetrating peptide is selected from iRGD, TAT, NFL-TBS.40-63 or MG2B, preferably iRGD.

4. The method according to claim 3, characterized in that The following steps are involved: Step 1: Dissolve 2-20 mmol of ferrous chloride tetrahydrate in 4-40 ml of double distilled water and filter through an 800 nm filter membrane, then add 4-40 ml of PEG solution, and stir under argon atmosphere for 25-40 min to prepare solution A; The concentration of the PEG solution is 10 mg / ml, wherein the molecular weight of PEG is 200-6000, preferably 200, 4000, 6000, and more preferably 4000; Step 2: Dissolve 8-80 mmol of sodium borohydride in 16-160 ml of 0.1 M sodium hydroxide solution, and stir in a venting bottle for 25-40 min to prepare solution B; Step 3: Under ventilation conditions, solution A is slowly added to solution B, and stirred until there are no bubbles to generate a black precipitate. The precipitate is collected and washed with water, and then transferred to DMF, acetone or DMSO. After ultrasonication for 1 to 4 hours, the obtained material is transferred to the aqueous phase and freeze-dried to obtain iron boron magnetic nanoparticles, which are recorded as Fe2B@PEG. Step 4: 10 mg Fe2B@PEG, 25-50 mg DSPE-PEG-OCH3 and 5-10 mg DSPE-PEG-iRGD were dissolved in 2-4 ml organic solvent and ultrasonicated for 5 min, then 10-20 ml double distilled water was added under ultrasonic conditions, ultrasonicated for 30-120 min, and then centrifuged at 4000-14500 rpm for 30 min to collect the precipitate, washed with secondary water, and freeze-dried to obtain iron boron nanomicelles for boron neutron capture therapy, recorded as Fe2B@PEG-iRGD; The organic solvent is DMF, methanol or a mixture of DMF and dichloromethane in a volume ratio of 1:1, preferably DMF.

5. The method according to claim 4, characterized in that In steps 3 and 4, the ultrasonic conditions are: ultrasonic power 60KW, on 5s, off 5s.

6. Use of the iron boron nanomicelles according to claim 1 or 2 or the iron boron nanomicelles prepared according to the method according to any one of claims 3 to 5 in the preparation of boron-containing drugs for boron neutron capture therapy.

7. Use of the iron boron nanomicelles according to claim 1 or 2 or the iron boron nanomicelles prepared according to the method according to any one of claims 3 to 5 in the preparation of antitumor drugs.

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