Iron boron nanomicelles for boron neutron capture therapy and methods of making and using the same

Abstract

The application provides an iron-boron nanomicelle for boron neutron capture therapy and a preparation method and application thereof. The iron-boron nanomicelle is obtained by an oxidation-reduction method. The nanomicelle is targeted and enriched at a tumor by modifying a penetrating peptide on the surface of the nanomicelle. The preparation method provided by the application is simple in process and practical. The obtained iron-boron nanomicelle has good water solubility and good biocompatibility. Compared with a common tumor cell targeted boron drug, the material further delivers the nanomicelle to a deep part of a solid tumor through a penetrating peptide and enriches the nanomicelle. In addition, the nanomicelle has magnetism and can be further enriched at the tumor through magnetic targeting, so as to realize boron drug enrichment at the tumor, and kill tumor cells through BNCT and chemical kinetics combined therapy.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomedical technology, in particular to an iron-boron nanomicelle for boron neutron capture therapy and a preparation method and application thereof. BACKGROUND

[0002] Boron neutron capture therapy (BNCT) is a new technology in tumor radiotherapy, and its principle is that after the biological tissue containing 10 B is irradiated by neutrons, the natural non-radioactive 10 B in it produces alpha particles and Li particles under the action of thermal neutrons, and when the charged particles undergo high-state energy transfer, a 5-9 micrometer radiation area is generated, thereby killing cells. As a typical binary treatment system, BNCT requires that the 10 B concentration in the tumor site cannot be less than 20 ppm. In the 1950s and early 1960s, BNCT clinical experiments used boric acid and some boric acid derivatives as boron delivery agents, but these simple compounds had no selectivity for tumor cells, and had high overall toxicity to the organism while having low retention in the tumor site. 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 antitumor efficacy of BNCT is to achieve high selective enrichment of boron drugs in tumor cells (the concentration of 10 B in the tumor site is more than 20 ppm). At the same time, in order to reduce the toxic side effects on the surrounding normal tissues, it is required that the boron drug is less accumulated 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, only two kinds of boron drugs are approved for BNCT clinical treatment, namely (L)-4-dihydroxyboron phenylalanine (boronophenylalanine, BPA) and sodium mercaptoundecahydrocloso-dodecaborate (Na2B 12 H 11 SH, BSH).

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

[0004] Compared with single treatment, combination therapy is the development trend of future tumor treatment. At present, there are also studies on the combination of BNCT and other tumor treatment methods, and better treatment effect than single BNCT has been achieved. In 2021, Liu Zhibo's research group of the Department of Applied Chemistry, School of Chemistry and Molecular Engineering, Peking University proposed a DOX@BNNSs nano drug delivery system for tumor treatment. By using the nucleus targeting property of nanometer material doxorubicin (DOX), the nano drug delivery system is successfully delivered to the cell nucleus to realize the combination treatment of chemotherapy and BNCT, and better treatment effect is obtained. SUMMARY

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

[0006] In order to achieve the purpose of the present application, in the first aspect, the present application provides an iron-boron nanomicelle for boron neutron capture therapy, which is an iron-boron magnetic nanoparticle with penetrating peptide on the surface.

[0007] In the present application, the penetrating peptide can be selected from iRGD, TAT, NFL-TBS.40-63 or MG2B, etc., and iRGD (with an amino acid sequence of CRGDKGPDC) is preferred.

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

[0009] In the second aspect, the present application provides a preparation method of an iron-boron nanomicelle for boron neutron capture therapy, which is prepared by an oxidation-reduction method to obtain an iron-boron magnetic nanoparticle, and then a penetrating peptide is modified on the surface of the nanoparticle.

[0010] Specifically, the method comprises the following steps:

[0011] Step one: dissolve 2~20mmol of ferrous chloride tetrahydrate in 4~40ml of double distilled water, filter through an 800nm filter membrane, then add 4~40ml of PEG solution, and stir under argon atmosphere for 25~40min to obtain solution A;

[0012] The concentration of the PEG solution is 10mg / ml, and the molecular weight of PEG is 200~6000, preferably 200, 4000, 6000, and more preferably 4000;

[0013] Step two: dissolve 8~80mmol of sodium borohydride in 16~160ml of 0.1M sodium hydroxide solution, and stir in a gas bottle for 25~40min to obtain solution B;

[0014] Step three: slowly add solution A into solution B under aeration, stir until no bubble is generated, collect the black precipitate, wash with water, then transfer the precipitate into DMF or acetone or DMSO, ultrasonic for 1-4 hours, then transfer the obtained material into water phase and freeze-dry to obtain iron boron magnetic nanoparticles, denoted as Fe2B@PEG;

[0015] Step four: dissolve 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) in 2-4 ml organic solvent and ultrasonic for 5 minutes, then add 10-20 ml double distilled water under ultrasonic condition, ultrasonic for 30-120 minutes, then centrifuge at 4000-14500 rpm (preferably 14500 rpm) for 30 minutes to collect the precipitate, wash with double distilled water, then freeze-dry to obtain the iron boron nanomicelles for boron neutron capture therapy, denoted as Fe2B@PEG-iRGD.

[0016] The organic solvent can be selected from DMF, methanol or a mixture of DMF and dichloromethane at a volume ratio of 1:1, preferably DMF.

[0017] Further, in steps three-four, the ultrasonic condition is: ultrasonic power of 60 KW, 5 seconds on and 5 seconds off.

[0018] In a third aspect, the present application provides the use of the iron boron nanomicelles or the iron boron nanomicelles prepared by the method in the preparation of boron-containing drugs for boron neutron capture therapy.

[0019] In a fourth aspect, the present application provides the use of the iron boron nanomicelles or the iron boron nanomicelles prepared by the method in the preparation of anti-tumor drugs.

[0020] By the above technical solution, the present application has at least the following advantages and beneficial effects:

[0021] (I) The present application provides a preparation method of iron boron nanomicelles with magnetism, which obtains iron boron nanomicelles by redox method. The nanomicelles are targeted to accumulate in tumors by modifying penetrating peptides on the surface of the nanomicelles. The preparation method provided by the present application is simple and practical, and the obtained iron boron nanomicelles have good water solubility and good biocompatibility, and the hydrated particle size is 220 nm±10 nm.

[0022] (II) Compared with ordinary tumor cell targeted boron drugs, the material further delivers the nanomicelles to the deep part of the solid tumor and accumulates by penetrating peptides. In addition, the nanomicelles have magnetism and can further accumulate in tumors by magnetic targeting, so as to realize the accumulation of boron drugs in tumors.

[0023] (iii) The large enrichment of iron boron nanomicelles at the tumor site, in addition to achieving a large amount of 10 B element enrichment for BNCT treatment, also catalyzes and accelerates the Fenton-like reaction in tumor cells by enriching a large amount of iron ions at the tumor site, generating a large amount of active oxygen in tumor cells, achieving chemical kinetic therapy, and achieving tumor cell killing through BNCT and chemical kinetic combined therapy. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 The hydrated particle size diagram of the Fe2B@PEG-iRGD nanomicelles prepared in Example 1 of the present application.

[0025] Figure 2 The infrared spectrograms of Fe2B@PEG, Fe2B@PEG-DSEP and Fe2B@PEG-iRGD prepared in Comparative Example 7 and 8 and Example 1 of the present application.

[0026] Figure 3 The XPS test full spectrum diagram of the Fe2B nanoparticles prepared in Comparative Example 6 of the present application.

[0027] Figure 4 The XPS test full spectrum diagram of the Fe2B@PEG and Fe2B@PEG-iRGD nanoparticles prepared in Comparative Example 7 and Example 1 of the present application. e2 B@PEG-iRGD nanoparticles prepared in Comparative Example 7 and Example 1 of the present application.

[0028] Figure 5 The XPS fine spectrum data analysis diagram of B 1s of the Fe2B nanoparticles prepared in Comparative Example 6 of the present application.

[0029] Figure 6 The XPS fine spectrum data analysis diagram of B 1s of the Fe2B@PEG and Fe2B@PEG-iRGD nanoparticles prepared in Comparative Example 7 and Example 1 of the present application.

[0030] Figure 7 The XPS fine spectrum data analysis diagram of Fe 2p of the Fe2B nanoparticles prepared in Comparative Example 6 of the present application.

[0031] Figure 8 The XPS fine spectrum data analysis diagram of Fe 2p of the Fe2B@PEG and Fe2B@PEG-iRGD nanoparticles prepared in Comparative Example 7 and Example 1 of the present application.

[0032] Figure 9 The X-ray diffraction diagram of Fe2B, Fe2B@PEG, Fe2B@PEG-iRGD prepared in Comparative Example 6, 7 and Example 1 of the present application.

[0033] Figure 10 Hysteresis loop diagram of Fe2B, Fe2B@PEG, Fe2B@PEG-iRGD prepared in Comparative Example 6, 7 and Example 1 of the present application.

[0034] Figure 11 Result diagram of chemical kinetics experiment of Fe2B@PEG-iRGD prepared in Example 1 of the present application in pH=6.5 and pH=7.4 environment respectively.

[0035] Figure 12 Result diagram of chemical kinetics experiment of Fe2B@PEG-iRGD prepared in Example 1 of the present application under different hydrogen peroxide concentration conditions.

[0036] Figure 13 Result diagram of chemical kinetics experiment of Fe2B@PEG-iRGD prepared in Example 1 of the present application under different concentrations.

[0037] Figure 14 Result diagram of chemical kinetics experiment of Fe2B@BSA-TPP prepared in Comparative Example 4 of the present application in pH=6.5 and pH=7.4 environment respectively.

[0038] Figure 15 Result diagram of chemical kinetics experiment of Fe2B@BSA-TPP prepared in Comparative Example 4 of the present application under different hydrogen peroxide concentration conditions.

[0039] Figure 16 Result diagram of chemical kinetics experiment of Fe2B@BSA-TPP prepared in Comparative Example 4 of the present application under different concentrations.

[0040] Figure 17 Stability data diagram of Fe2B@PEG-iRGD prepared in Example 1 of the present application.

[0041] Figure 18 Biological safety diagram of Fe2B@PEG-iRGD prepared in Example 1 of the present application.

[0042] Figure 19 Result diagram of MTT cytotoxicity experiment of Fe2B@PEG-iRGD prepared in Example 1 of the present application in murine melanoma cells (B16 cells).

[0043] Figure 20 Result diagram of Fe2B@PEG-iRGD prepared in Example 1 of the present application in murine melanoma cells (B16 cells) uptake experiment.

[0044] Figure 21The figure of the distribution of Fe2B@PEG-iRGD prepared in Example 1 of the present application in mice at different time points after administration as a nanomedicine.

[0045] Figure 22 The boron content in tumors at different time points after administration of Fe2B@PEG-iRGD prepared in Example 1 of the present application.

[0046] Figure 23 The figure of the particle size distribution of Fe2B@PEG-iRGD prepared in Examples 1, 2 and 3 of the present application in water.

[0047] Figure 24 The figure of the particle size distribution of Fe2B@PEG-iRGD prepared in Examples 1, 4 and 5 of the present application in water. DETAILED DESCRIPTION

[0048] The following examples are used to illustrate the present application, but are not used to limit the scope of the present application. If not specifically indicated, the technical means used in the examples are the conventional means known to those skilled in the art, and the raw materials used are commercially available.

[0049] DSPE-PEG-OCH3 and DSPE-PEG-iRGD used in the following examples were purchased from Shanghai Taoxiang Biotechnology Co., Ltd.

[0050] Example 1

[0051] The present embodiment provides a preparation method of iron-boron nanomicelles, comprising the following steps:

[0052] Step one: 2 mmol of ferrous chloride tetrahydrate is dissolved in 4 ml of double distilled water and filtered through a 800 nm filter membrane, and then 4 ml of PEG (molecular weight of 4K, concentration of 10 mg / ml) is added thereto, and a solution A is prepared by stirring for 25 min under an argon atmosphere. The solution A is transferred to a 10 ml syringe for standby;

[0053] Step two: 8 mmol of sodium borohydride is dissolved in 16 ml of sodium hydroxide (0.1 M), and a solution B is prepared by stirring for 25 min in a 500 ml gas bottle;

[0054] Step three: under the condition of aeration, the A solution is slowly added to the B solution, and the black precipitate is collected after stirring until no gas bubbles are generated. The precipitate is quickly washed with water for several times, and then transferred to DMF (DMF can be replaced by acetone, DMSO), and Fe2B@PEG magnetic particles are obtained after inserting type ultrasonic for 2 h. The material is transferred to an aqueous phase for freeze-drying storage;

[0055] Step four: 10mg Fe2B@PEG, 25mg DSPE-PEG-OCH3 (molecular weight 2K) and 5mg DSPE-PEG-iRGD (molecular weight 2K) were dissolved in 2ml DMF and inserted with ultrasound for 5min. Then 8ml double distilled water was added step by step under the condition of inserted ultrasound and ultrasonic for 30min. The precipitate Fe2B@PEG-iRGD nanomicelles were collected by centrifugation at 14500rpm for 30min and washed with double distilled water for 3 times, and freeze-dried for storage.

[0056] Example 2

[0057] Step one: ferrous chloride tetrahydrate 2mmol was dissolved in 4ml double distilled water and filtered through an 800nm filter membrane, then 4ml PEG (molecular weight 4K, concentration 10mg / ml) was added, and the solution A was prepared by stirring for 25min under argon atmosphere.

[0058] Step two: sodium borohydride 8mmol was dissolved in 16ml sodium hydroxide (0.1M), and solution B was prepared by stirring for 25min in a 500ml gas bottle.

[0059] Step three: under the condition of aeration, A solution was slowly added to B solution and stirred until no bubbles were generated. Black precipitate was collected and washed with water several times. Then the precipitate was transferred to DMF (DMF can be replaced by acetone, DMSO), and Fe2B@PEG magnetic particles were obtained after inserted ultrasound for 2h. The supernatant was collected by centrifugation at 6000rpm for 10min and freeze-dried for storage.

[0060] Step four: 10mg Fe2B@PEG, 25mg DSPE-PEG-OCH3 (molecular weight 2K) and 5mg DSPE-PEG-iRGD (molecular weight 2K) were dissolved in 2ml DMF and inserted with ultrasound for 5min. Then 8ml double distilled water was added step by step under the condition of inserted ultrasound and ultrasonic for 30min. The precipitate Fe2B@PEG-iRGD nanomicelles were collected by centrifugation at 14500rpm for 30min and washed with double distilled water for 3 times, and freeze-dried for storage.

[0061] Example 3

[0062] Step one: ferrous chloride tetrahydrate 2mmol was dissolved in 4ml double distilled water and filtered through an 800nm filter membrane, then 4ml PEG (molecular weight 4K, concentration 10mg / ml) was added, and the solution A was prepared by stirring for 25min under argon atmosphere.

[0063] Step two: 8mmol of sodium borohydride was dissolved in 16ml of sodium hydroxide (0.1M), and a solution B was prepared by stirring in a 500ml gas bottle for 25min;

[0064] Step three: under the condition of aeration, the solution A was slowly added to the solution B and stirred until no bubbles were generated to form a black precipitate. The precipitate was collected and quickly washed with water several times. Then the precipitate was transferred to DMF (DMF can be replaced by acetone, DMSO), and Fe2B@PEG magnetic particles were obtained after inserting ultrasound for 2h. The supernatant was obtained by centrifugation at 14500rpm for 10min, and then was freeze-dried for storage;

[0065] Step four: 10mg of Fe2B@PEG, 25mg of DSPE-PEG-OCH3 (molecular weight 2K) and 5mg of DSPE-PEG-iRGD (molecular weight 2K) were dissolved in 2ml of DMF and inserted ultrasound for 5min. Then 8ml of double distilled water was added step by step under the condition of inserting ultrasound, and ultrasound was performed for 30min. The precipitate Fe2B@PEG-iRGD nanomicelles were collected by centrifugation at 14500rpm for 30min and washed with double distilled water for 3 times, and then were freeze-dried for storage.

[0066] Example 4

[0067] The embodiment provides a preparation method of iron boron nanomicelles, which comprises the following steps:

[0068] Step one: 2mmol of ferrous chloride tetrahydrate was dissolved in 4ml of double distilled water and filtered through a 800nm filter membrane. Then 4ml of PEG (molecular weight 4K, concentration 10mg / ml) was added thereto, and a solution A was prepared by stirring in an argon atmosphere for 25min. The solution A was transferred to a 10ml syringe for standby;

[0069] Step two: 8mmol of sodium borohydride was dissolved in 16ml of sodium hydroxide (0.1M), and a solution B was prepared by stirring in a 500ml gas bottle for 25min;

[0070] Step three: under the condition of aeration, the solution A was slowly added to the solution B and stirred until no bubbles were generated to form a black precipitate. The precipitate was collected and quickly washed with water several times. Then the precipitate was transferred to DMF (DMF can be replaced by acetone, DMSO), and Fe2B@PEG magnetic particles were obtained after inserting ultrasound for 2h. The supernatant was obtained by centrifugation at 14500rpm for 10min, and then was freeze-dried for storage;

[0071] Step four: 10mg Fe2B@PEG, 25mg DSPE-PEG-OCH3(molecular weight 2K) and 5mg DSPE-PEG-iRGD(molecular weight 2K) were dissolved in 2ml DMF and DCM mixture(volume ratio 1:1) and inserted ultrasonic for 5min. Then 8ml double distilled water was added step by step under the condition of inserted ultrasonic and ultrasonic for 30min. The precipitate Fe2B@PEG-iRGD nanomicelles were collected by centrifugation at 14500rpm for 30min and washed with double distilled water for 3 times, and then freeze-dried for storage.

[0072] Example 5

[0073] The present embodiment provides a preparation method of iron boron nanomicelles, comprising the following steps:

[0074] Step one: 2mmol ferrous chloride tetrahydrate was dissolved in 4ml double distilled water and filtered through 800nm filter membrane, then 4ml PEG(molecular weight 4K, concentration 10mg / ml) was added thereto, and the solution A was prepared by stirring for 25min under argon atmosphere.

[0075] Step two: 8mmol sodium borohydride was dissolved in 16ml sodium hydroxide(0.1M), and the solution B was prepared by stirring for 25min in a 500ml gas bottle.

[0076] Step three: under the condition of aeration, the solution A was slowly added to the solution B, and the black precipitate was collected after stirring until no gas bubble was generated. The precipitate was quickly washed with water for several times, then transferred to DMF(DMF can be replaced by acetone, DMSO), and the Fe2B@PEG magnetic particles were obtained after inserted ultrasonic for 2h. The supernatant was collected by centrifugation at 14500rpm for 10min, and then freeze-dried for storage.

[0077] Step four: 10mg Fe2B@PEG, 25mg DSPE-PEG-OCH3(molecular weight 2K) and 5mg DSPE-PEG-iRGD(molecular weight 2K) were dissolved in 2ml methanol and inserted ultrasonic for 5min. Then 8ml double distilled water was added step by step under the condition of inserted ultrasonic and ultrasonic for 30min. The precipitate Fe2B@PEG-iRGD nanomicelles were collected by centrifugation at 14500rpm for 30min and washed with double distilled water for 3 times, and then freeze-dried for storage.

[0078] The hydrated particle size diagram of the Fe2B@PEG-iRGD nanomicelles prepared in Example 1 is shown in Figure 1 .

[0079] The infrared spectrograms 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 .

[0080] The XPS test full spectrum of Comparative Example 6 is shown in Figure 3 .

[0081] The XPS test full spectrum of Fe2B@PEG and Fe2B@PEG-iRGD nanoparticles prepared in Comparative Example 7 and Example 1 is shown in Figure 4 .

[0082] The XPS fine spectrum data analysis chart of B 1s of Fe2B nanoparticles prepared in Comparative Example 6 is shown in Figure 5 .

[0083] The XPS fine spectrum data analysis chart of B 1s of Fe2B@PEG and Fe2B@PEG-iRGD nanoparticles prepared in Comparative Example 7 and Example 1 is shown in Figure 6 .

[0084] The XPS fine spectrum data analysis chart of Fe 2p of Fe2B nanoparticles prepared in Comparative Example 6 is shown in Figure 7 .

[0085] The XPS fine spectrum data analysis chart of Fe 2p of Fe2B@PEG and Fe2B@PEG-iRGD nanoparticles prepared in Comparative Example 7 and Example 1 is shown in Figure 8 .

[0086] The X-ray diffraction chart of Fe2B, Fe2B@PEG, Fe2B@PEG-iRGD prepared in Comparative Examples 6, 7 and Example 1 is shown in Figure 9 .

[0087] The magnetic hysteresis loop chart of Fe2B, Fe2B@PEG, Fe2B@PEG-iRGD prepared in Comparative Examples 6, 7 and Example 1 is shown in Figure 10 .

[0088] The result chart of chemical kinetics experiment of Fe2B@PEG-iRGD prepared in Example 1 in pH=6.5 and pH=7.4 environments respectively is shown in Figure 11 .

[0089] The result chart of chemical kinetics experiment of Fe2B@PEG-iRGD prepared in Example 1 under different hydrogen peroxide concentration conditions is shown in Figure 12 .

[0090] The result chart of chemical kinetics experiment of Fe2B@PEG-iRGD prepared in Example 1 with different concentrations is shown in Figure 13 .

[0091] The results of the chemical kinetics experiment of Fe2B@BSA-TPP prepared in Comparative Example 4 in pH = 6.5 and pH = 7.4 environments are shown in the following figures Figure 14 .

[0092] The results of the chemical kinetics experiment of Fe2B@BSA-TPP prepared in Comparative Example 4 under different hydrogen peroxide concentrations are shown in the following figures Figure 15 .

[0093] The results of the chemical kinetics experiment of Fe2B@BSA-TPP prepared in Comparative Example 4 under different concentrations are shown in the following figures Figure 16 .

[0094] The stability data of Fe2B@PEG-iRGD prepared in Example 1 are shown in the following figures Figure 17 .

[0095] The biological safety of Fe2B@PEG-iRGD prepared in Example 1 is shown in the following figure Figure 18 .

[0096] The results of the MTT cytotoxicity experiment of Fe2B@PEG-iRGD prepared in Example 1 on murine melanoma cells (B16 cells) are shown in the following figures Figure 19 .

[0097] The results of the uptake experiment of Fe2B@PEG-iRGD prepared in Example 1 on murine melanoma cells (B16 cells) are shown in the following figures Figure 20 . The distribution of Fe2B@PEG-iRGD prepared in Example 1 in mice at different time points after administration as a nanodrug is shown in the following figures Figure 21 .

[0098] The boron content at the tumor site of Fe2B@PEG-iRGD prepared in Example 1 at different time points after administration is shown in the following figure Figure 22 .

[0099] The particle size distribution of Fe2B@PEG-iRGD prepared in Examples 1, 2 and 3 in water is shown in the following figures Figure 23 .

[0100] The particle size distribution of Fe2B@PEG-iRGD prepared in Examples 1, 4 and 5 in water is shown in the following figures Figure 24 .

[0101] Figure 11Chemical kinetics experiment of Fe2B@PEG-iRGD in pH=6.5 and pH=7.4 environment: To evaluate the generation of hydroxyl radicals, we dispersed Fe2B@PEG-iRGD in phosphate buffer solution with pH value of 6.5 and 7.4, respectively, and then added 20 μL TA solution and 1% H2O2. After 1 h of reaction, we used a fluorescence spectrometer to detect the ultraviolet absorption value in the wavelength range of 350-500 nm under the condition of 310 nm excitation light. By comparing the difference in absorption value, we showed the ability to generate ·OH.

[0102] Figure 12 Chemical kinetics experiment of Fe2B@PEG-iRGD under different hydrogen peroxide concentration conditions: We dispersed Fe2B@PEG-iRGD in phosphate buffer solution with pH value of 6.5, and then added 20 μL TA solution and different concentrations of H2O2. After 1 h of reaction, we used a fluorescence spectrometer to detect the ultraviolet absorption value in the wavelength range of 350-500 nm under the condition of 310 nm excitation light. By comparing the difference in absorption value, we showed the ability to generate ·OH.

[0103] Figure 13 Chemical kinetics experiment of Fe2B@PEG-iRGD with different concentrations: We dispersed Fe2B@PEG-iRGD with different concentrations in phosphate buffer solution with pH value of 6.5, and then added 20 μL TA solution and 1% H2O2. After 1 h of reaction, we used a fluorescence spectrometer to detect the ultraviolet absorption value in the wavelength range of 350-500 nm under the condition of 310 nm excitation light. By comparing the difference in absorption value, we showed the ability to generate ·OH.

[0104] Figure 14 Chemical kinetics experiment of Fe2B@BSA-TPP in pH=6.5 and pH=7.4 environment: We dispersed Fe2B@BSA-TPP in phosphate buffer solution with pH value of 6.5 and 7.4, respectively, and then added 20 μL TA solution and 1% H2O2. After 1 h of reaction, we used a fluorescence spectrometer to detect the ultraviolet absorption value in the wavelength range of 350-500 nm under the condition of 310 nm excitation light. By comparing the difference in absorption value, we showed the ability to generate ·OH.

[0105] Figure 15Chemical kinetics experiment of Fe2B@BSA-TPP under different hydrogen peroxide concentration: Fe2B@BSA-TPP was dispersed in phosphate buffer solution with pH value of 6.5, then 20 μL TA solution and different concentrations of H2O2 were added, after 1h reaction, the ultraviolet absorption value in the wavelength range of 350-500 nm was detected under the condition of 310 nm excitation light using fluorescence spectrometer. Through the difference of absorption value, the ability to produce ·OH was demonstrated.

[0106] Figure 16 Chemical kinetics experiment of Fe2B@BSA-TPP under different concentrations: Fe2B@BSA-TPP with different concentrations was dispersed in phosphate buffer solution with pH value of 6.5, then 20 μL TA solution and 1% H2O2 were added, after 1h reaction, the ultraviolet absorption value in the wavelength range of 350-500 nm was detected under the condition of 310 nm excitation light using fluorescence spectrometer. Through the difference of absorption value, the ability to produce ·OH was demonstrated.

[0107] Figure 17 Stability experiment scheme of Fe2B@PEG-iRGD: The particle size and PDI change of Fe2B@PEG-iRGD incubated in cell culture medium containing 10% FBS (DMEM containing L-glutamine, 4.5 g / L glucose and sodium pyruvate, HyCloneTM) at 37°C for 0h, 0.5h, 1h, 2h, 4h, 6h, 8h, 10h, 12h, 24h, 48h were evaluated in turn,

[0108] Figure 18For hemolysis experimental protocol: 1 mL of C57BL mouse whole blood was collected in a 2 mL centrifuge tube containing 2.5 μL of 2% sodium heparin, then the mixture was slowly shaken. After that, the same volume of normal saline was added, then centrifuged (1500 rpm, 10 min). The supernatant was removed, and the precipitate was dispersed with 10 mL of normal saline. The suspension was centrifuged (1500 rpm, 20 min), and the same procedure was repeated 3 times to obtain a red blood cell suspension. After that, 0.2 mL of the red blood cell suspension was taken and mixed with (-) 0.8 mL of PBS as a negative control, (+) 0.8 mL of deionized water as a positive control, and 0.8 mL of Fe2B@PEG-iRGD aqueous solution at various concentration ranges (0 to 500 μg / mL). After incubation at 37 °C for 1 h, the mixture was centrifuged (12000 rpm, 5 min), and then the supernatant was measured for optical density (OD value) in a microplate reader (Spark™ 10M, Tecan, Zurich, Switzerland) with an excitation wavelength of 541 nm. The hemolysis rate (Hr) was calculated by the following formula:

[0109] Hr (%) = (ODs-ODn / ODp-ODn) x 100%

[0110] where ODs, ODp and ODn are the OD values of the sample, positive control and negative control, respectively.

[0111] Figure 19 For B16 cytotoxicity experimental protocol: B16 cells were cultured in cell culture medium containing 10% fetal bovine serum (FBS, GIBCO), 1.0 x 10 5 U / L penicillin and 0.1 mg / mL streptomycin (Sigma, Beijing, China) and the medium was changed every other day. The cytotoxicity of the nanoparticles was analyzed by the MTT assay. B16 cells were seeded into 96-well plates at a density of 5000 cells per well. After 24 h, the growth medium was replaced with fresh medium. Then, different concentrations of nanoparticles were added to the wells (six wells in parallel for each sample). After the predetermined time, the medium was removed. Excess material was washed away with PBS, and then 10% MTT-containing DMEM base medium was added to the corresponding wells, which were incubated at 37 °C and 5% CO2 for 4 h. The culture was terminated, and after centrifugation, the culture supernatant in the wells was carefully aspirated. 100 ul of DMSO was added to each well, and the mixture was shaken for 10 min to fully dissolve the crystals. The absorbance of each well was measured at a wavelength of 490 nm to calculate the cell survival rate

[0112] Figure 20Cell uptake experimental protocol: B16 cells were cultured in six-well plates in cell culture medium containing 10% fetal bovine serum (FBS, GIBCO), 1.0 x 10 5 U / L penicillin and 0.1 mg / mL streptomycin (Sigma, Beijing, China) and Fe2B@PEG-iRGD was incubated with cells in DMEM medium at a concentration of 500 pg / mL and 250 pg / mL, respectively, at 37 °C in 5% CO2 for 24 h. Subsequently, cells were washed with PBS buffer for 3 times to remove free samples. Cells were collected and counted, and then digested in 5 ml MOS grade concentrated nitric acid overnight followed by microwave digestion, and the boron content in cells was tested by inductively coupled plasma mass spectrometry.

[0113] Figure 21 Ivis small animal live imaging experimental protocol: Female BALB / c mice (4-5 weeks, 13 ± 2 g) were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd. The subcutaneous melanoma model of mice was established as follows: after weighing, the female C57BL mice were intraperitoneally injected with 4% chloral hydrate (10 mL / kg) to anesthetize the mice, and then 10

[0114] Female BALB / c mice (4-5 weeks, 13 ± 2 g) were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd. The subcutaneous melanoma model of mice was established as follows: after weighing, the female C57BL mice were intraperitoneally injected with 4% chloral hydrate (10 mL / kg) to anesthetize the mice, and then 10 7 cells per mouse were subcutaneously injected into the right hind leg of the mice after depilation. When the palpable tumor was about 60 mm 3 in diameter, the following experiment was performed on the mice: Fe2B@PEG-iRGD-Cy5 was injected into the mice through the tail vein, and the live fluorescence imaging of the mice was performed by the Lumina III in vivo imaging system (Ex / Em = 640 / 680 nm) at 1, 2, 4, and 8 h after injection.

[0115] Figure 22 Experimental protocol for testing the boron content at the tumor site after administration: Female BALB / c mice (4-5 weeks, 13 ± 2 g) were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd. The subcutaneous melanoma model of mice was established as follows: after weighing, the female C57BL mice were intraperitoneally injected with 4% chloral hydrate (10 mL / kg) to anesthetize the mice, and then 10 7 cells per mouse were subcutaneously injected into the right hind leg of the mice after depilation. When the palpable tumor was about 60 mm 3 in diameter, the following experiment was performed on the mice: Fe2B@PEG-iRGD was injected into the mice through the tail vein, and the tumor was sampled at 2 and 4 h after injection, and the tumor was digested in 5 ml MOS grade concentrated nitric acid overnight followed by microwave digestion, and the boron content at the tumor site was tested by inductively coupled plasma mass spectrometry.

[0116] Synthesis of Fe2B magnetic particles (less amount of ferrous chloride)

[0117] Ferrous chloride tetrahydrate 1.08 mmol was dissolved in 4 ml of double distilled water, and oxygen was excluded. Sodium borohydride 8 mmol was dissolved in 8 ml of NaOH (0.1 M concentration) and injected into the ferrous chloride solution under oxygen exclusion (stirring was continued during the process).

[0118] Synthesis of Fe2B magnetic particles (less amount of sodium borohydride)

[0119] Ferrous chloride tetrahydrate 2 mmol was dissolved in 4 ml of double distilled water, and oxygen was excluded and kept in an ice bath. Sodium borohydride 4 mmol was dissolved in 8 ml of NaOH (0.1 M concentration) and injected into the ferrous chloride solution under oxygen exclusion (stirring was continued during the process).

[0120] Synthesis of Fe2B magnetic particles (addition of polyvinyl alcohol)

[0121] Step one: Ferrous chloride tetrahydrate 2 mmol was dissolved in 4 ml of double distilled water and filtered through an 800 nm filter membrane, and then 1 ml of polyvinyl alcohol (PVA, 10 mg / ml) was added thereto, and a solution A was prepared by stirring for 25 min under an argon atmosphere. The solution A was transferred to a 10 ml syringe for standby;

[0122] Step two: Sodium borohydride 8 mmol was dissolved in 16 ml of sodium hydroxide (0.1 M), and a solution B was prepared by stirring for 25 min in a 500 ml gas washing bottle.

[0123] Step three: The A solution was slowly added to the B solution under aeration, and stirring was continued until no gas bubbles were generated, and a black precipitate was collected and washed with water several times, and then the precipitate was transferred to DMF (DMF can be replaced with acetone, DMSO), and Fe2B@PVP magnetic particles were obtained after ultrasonic treatment for 2 h, and the supernatant was obtained by centrifugation at 14500 rpm for 10 min, and then the supernatant was freeze-dried and stored.

[0124] Synthesis of Fe2B@BSA-TPP magnetic particles (addition of bovine serum albumin)

[0125] Step one: Ferrous chloride tetrahydrate 2 mmol was dissolved in 4 ml of double distilled water and filtered through an 800 nm filter membrane, and then 20 mg of BSA was added thereto, and a solution A was prepared by stirring for 25 min under an argon atmosphere. The solution A was transferred to a 10 ml syringe for standby;

[0126] Step two: Sodium borohydride 8 mmol was dissolved in 16 ml of sodium hydroxide (0.1 M), and a solution B was prepared by stirring for 25 min in a 500 ml gas washing bottle.

[0127] Step three: under the condition of ventilation, slowly add solution A into solution B, stir until no bubbles are generated, collect the black precipitate, quickly wash with water several times, then transfer the precipitate into DMF (DMF can be replaced by acetone, DMSO), after inserting ultrasonic for 2 h, centrifuge the Fe2B@BSA magnetic particles at 14500 rpm for 10 min, take the supernatant and freeze-dry for storage;

[0128] Step four: add 0.5 ml of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) solution (concentration of 0.2 mmol / mL) into 1 ml of triphenyl phosphate (TPP) solution (concentration of 0.05 mmol), stir at a speed of 1000-1200 rpm for 10 minutes, then add 0.5 ml of N-hydroxysuccinimide (NHS) solution (concentration of 0.2 mmol / mL), stir at a speed of 1000-1200 rpm under the condition of 25-30°C for 1.5 h. Then add Fe2B@BSA dissolved in 3 ml of double distilled water, stir under the condition of 25-30°C for 24 h after reaction to obtain Fe2B@BSA-TPP nanoparticles, resuspend and wash with double distilled water, and freeze-dry for storage.

[0129] Comparative example 5 synthesis of Fe2B nanomicelles (poloxamer 188 addition scheme)

[0130] Step one: dissolve 2 mmol of ferrous chloride tetrahydrate in 4 ml of double distilled water and filter through an 800 nm filter membrane, then add 4 ml of PEG (molecular weight of 4K, concentration of 10 mg / ml) thereto, and stir under the condition of argon gas for 25 min to prepare solution A. Transfer solution A into a 10 ml syringe for standby;

[0131] Step two: dissolve 8 mmol of sodium borohydride in 16 ml of sodium hydroxide (0.1 M), stir under the condition of ventilation for 25 min in a 500 ml ventilation bottle to prepare solution B;

[0132] Step three: under the condition of ventilation, slowly add solution A into solution B, stir until no bubbles are generated, collect the black precipitate, quickly wash with water several times, then transfer the precipitate into DMF (DMF can be replaced by acetone, DMSO), after inserting ultrasonic for 2 h, centrifuge the Fe2B@PEG magnetic particles at 14500 rpm for 10 min, take the supernatant and freeze-dry for storage;

[0133] Step four: 10 mg Fe2B@PEG was dissolved in 6 ml methanol / DCM mixture, then added to 30 ml (10% w / w) poloxamer 188 aqueous solution, followed by ultrasonic treatment for 30 min. Then added to 100 ml (0.1% w / w) poloxamer 188 aqueous solution and stirred overnight, then centrifuged at 14500 rpm for 30 min to collect the precipitate, washed with double distilled water for 3 times, and freeze-dried for storage.

[0134] Synthesis of Fe2B

[0135] Step one: ferrous chloride tetrahydrate 2 mmol was dissolved in 4 ml double distilled water and filtered through an 800 nm filter membrane, and a solution A was prepared by stirring under an argon atmosphere for 25 min.

[0136] Step two: sodium borohydride 8 mmol was dissolved in 16 ml sodium hydroxide (0.1 M), and a solution B was prepared by stirring in a 500 ml gas bottle for 25 min.

[0137] Step three: under the condition of aeration, the A solution was slowly added to the B solution and stirred until no bubbles were generated. A black precipitate was collected and quickly washed with water several times. The precipitate was then transferred to DMF (DMF can be replaced by acetone, DMSO), and Fe2B magnetic particles were obtained after 2 h of ultrasonic treatment. The material was transferred to an aqueous phase and freeze-dried for storage.

[0138] Synthesis of Fe2B@PEG

[0139] Step one: ferrous chloride tetrahydrate 2 mmol was dissolved in 4 ml double distilled water and filtered through an 800 nm filter membrane, and a solution A was prepared by stirring under an argon atmosphere for 25 min.

[0140] Step two: sodium borohydride 8 mmol was dissolved in 16 ml sodium hydroxide (0.1 M), and a solution B was prepared by stirring in a 500 ml gas bottle for 25 min.

[0141] Step three: under the condition of aeration, the A solution was slowly added to the B solution and stirred until no bubbles were generated. A black precipitate was collected and quickly washed with water several times. The precipitate was then transferred to DMF (DMF can be replaced by acetone, DMSO), and Fe2B magnetic particles were obtained after 2 h of ultrasonic treatment. The material was transferred to an aqueous phase and freeze-dried for storage.

[0142] Synthesis of Fe2B@PEG-DPSE

[0143] Step one: Dissolve 2mmol of ferrous chloride tetrahydrate in 4ml of double distilled water and filter through 800nm filter membrane, then add 4ml of PEG (4K molecular weight, 10mg / ml concentration) into the solution, and stir for 25min under argon atmosphere to obtain solution A. Transfer solution A into a 10ml syringe for later use.

[0144] Step two: Dissolve 8mmol of sodium borohydride in 16ml of sodium hydroxide (0.1M), and stir for 25min under argon atmosphere to obtain solution B.

[0145] Step three: Slowly add solution A into solution B under argon atmosphere, and stir until no gas bubble is generated. Collect the black precipitate and wash with water for several times. Then transfer the precipitate into DMF (DMF can be replaced by acetone or DMSO), and insert ultrasound for 2h to obtain Fe2B@PEG magnetic particles. Centrifuge at 14500rpm for 10min to obtain the supernatant, and freeze dry for storage.

[0146] Step four: Dissolve 10mg of Fe2B@PEG and 50mg of DSPE-PEG-OCH3 (2K molecular weight) in 2ml of DMF, and insert ultrasound for 5min. Then add double distilled water (8ml) under ultrasound condition, and insert ultrasound for 30min. Centrifuge at 14500rpm for 30min to collect the precipitate Fe2B@PEG-iRGD nanomicelles, and wash with double distilled water for 3 times. Freeze dry for storage.

[0147] Although the present application has been described in detail with general description and specific embodiments, some modifications or improvements can be made on the basis of the present application, which is obvious to those skilled in the art. Therefore, these modifications or improvements made on the basis of not deviating from the spirit of the present application, all belong to the scope of the present application claimed.

Claims

1. A method for preparing iron-boron nanomicelles for boron neutron capture therapy, characterized in that, Iron boron magnetic nanoparticles were prepared by redox method, and then penetrating peptides were modified on the surface of the nanoparticles. The penetrating peptide is iRGD; Includes the following steps: Step 1: Dissolve 2-20 mmol of ferrous chloride tetrahydrate in 4-40 ml of double-distilled water and filter it through an 800 nm filter membrane. Then add 4-40 ml of PEG solution and stir under an 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; Step 2: Dissolve 8-80 mmol of sodium borohydride in 16-160 ml of 0.1 M sodium hydroxide solution, and stir with air in a gas-breathing bottle for 25-40 min to obtain solution B; Step 3: Under aeration, slowly add solution A to solution B and stir until no bubbles are generated and a black precipitate forms. Collect the precipitate, wash it with water, and then transfer the precipitate to DMF, acetone, or DMSO. After sonication for 1-4 hours, transfer the obtained material to an aqueous phase for freeze-drying to obtain iron-boron magnetic nanoparticles, denoted as Fe2B@PEG. Step 4: Dissolve 10 mg Fe2B@PEG, 25-50 mg DSPE-PEG-OCH3 and 5-10 mg DSPE-PEG-iRGD in 2-4 ml of organic solvent and sonicate for 5 min. Then add 10-20 ml of double-distilled water under sonication and sonicate for 30-120 min. Centrifuge at 4000-14500 rpm for 30 min to collect the precipitate. Wash with secondary water and freeze dry to obtain iron-boron nanomicelles for boron neutron capture therapy, denoted as Fe2B@PEG-iRGD. The organic solvent is DMF, methanol, or a mixture of DMF and dichloromethane in a volume ratio of 1:

1.

2. The method according to claim 1, characterized in that, PEG has molecular weights of 200, 4000, and 6000.

3. The method according to claim 2, characterized in that, The molecular weight of PEG is 4000.

4. The method according to claim 1, characterized in that, The organic solvent is DMF.

5. The method according to claim 1, characterized in that, In steps three and four, the ultrasonic conditions are: ultrasonic power 60KW, on for 5 seconds, off for 5 seconds.

6. The method according to any one of claims 1-5, characterized in that, The prepared iron boron nanomicelles have a particle size of 220 nm ± 10 nm.

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