Nano boron medicine for boron neutron capture therapy as well as preparation method and application of nano boron medicine

Abstract

The invention belongs to the technical field of tumor treatment, and particularly relates to a nano boron drug for boron neutron capture therapy and a preparation method and application thereof, the nano boron drug is PAMAM-BSH, and PAMAM is a third-generation (G3), fourth-generation (G4) or fifth-generation (G5) dendritic macromolecule. The PAMAM-BSH provided by the invention has good biocompatibility and can be well internalized into tumor cells, the cell entering mode of the PAMAM-BSH is energy-dependent, and the boron concentration in B16F10 cells can reach 5300ng / 106 cells and is 61% higher than that of BSH. The nano boron drug provided by the invention is PAMAM-BSH and has a wide application prospect in the field of boron neutron capture therapy.

Description

Technical Field

[0001] The present invention belongs to the technical field of tumor treatment, and in particular relates to a nano-boron medicine for boron neutron capture therapy, a preparation method thereof, and an application thereof. Background Art

[0002] Malignant tumors are one of the major diseases that threaten human life and health. Currently, the three main clinical treatments for tumors are surgery, chemotherapy, and radiotherapy. Surgery is an effective treatment for primary tumors, but it is limited to surgically accessible cancer cells and may not completely eliminate them. Chemotherapy uses chemical drugs to fight cancer. Systemic administration circulates the drugs throughout the body to kill rapidly dividing cells, particularly cancer cells. However, because the drugs are also toxic to normal cells, they often have significant side effects and can cause cancer cells to develop drug resistance. Traditional radiotherapy uses high-energy ionizing particles, such as X-rays, gamma rays, or electron beams, to destroy cells at the molecular level. It is often used as a supplemental treatment, such as to eliminate remaining cancer cells after surgery. However, traditional radiotherapy can damage normal tissue near cancer cells or within the radiation field. Boron neutron capture therapy (BNCT) is a dual radiotherapy modality that combines strong targeting at the cellular scale with a high linear energy transfer density. The working principle is that 10B with a high neutron capture cross section captures low-energy thermal neutrons and then undergoes a nuclear reaction to produce high-energy and short-range alpha particles and recoil 7 Li nuclei, alpha particles, and recoil 7 The range of Li nuclei is about 10μm and 5μm respectively, which is approximately the diameter of a cell. They only kill tumor cells and do not damage normal tissue cells.

[0003] In order to achieve good therapeutic effects of BNCT therapy, boron carriers need to meet the following requirements: (1) low intrinsic toxicity to increase the patient's maximum tolerated dose of boron carriers. (2) 10 The B content is greater than 20ug / g, which means that each tumor cell contains at least 10 9 indivual 10B atoms; (3) high tumor specificity, the ratio of boron concentration in tumor tissue to normal tissue (T / N) and the ratio of boron concentration in tumor to blood (T / B) are both greater than 3; to reduce the side effects of treatment; (4) can be retained in tumor cells for a long time and cleared from blood and normal tissues at a relatively fast rate. The current bottleneck problem in the development of BNCT is the development of highly effective boron-containing drugs. In the nearly 100 years of development of BNCT, boron carriers have undergone three generations of changes. The first generation is borax, boric acid and its derivatives. Due to their systemic distribution, tumor targeting is poor, so T / N and T / B are very low. The second generation is 4-dihydroxyboryl-L-phenylalanine (BPA) and sodium 11-hydrogen mercapto-12-boronide (BSH). The third generation includes boron-containing small molecules (amino acid derivatives, porphyrin derivatives, etc.), boron-containing biomacromolecules (specific ligands, monoclonal antibodies, etc.), and boron-containing nanomedicines (liposomes, boron carbide nanomaterials, boron nitride nanomaterials, etc.). BPA and BSH are currently the only BNCT drugs used in clinical practice. However, BPA and BSH still have certain disadvantages. BPA has poor water solubility; BPA can selectively accumulate in tumors through tumor-enriched amino acid transporters such as LAT1, but due to the influence of the reverse transport mechanism, BPA is easily excreted by tumor cells and has a short retention time; and BPA contains low boron. BSH has poor tumor specificity. Over the past few decades, a lot of human and financial resources have been devoted to developing new boron delivery methods. Unfortunately, apart from BSH and BPA, no boron delivery molecules have been tested in clinical trials (K.Hu et al, Drug Chemistry Reviews, 405(2020), 1-20).

[0004] With the continuous development of nanotechnology, polymer materials have also been widely used as drug carriers. Microparticle dispersion systems with a particle size of 10-1000 nm obtained by loading drugs with pharmaceutical polymer materials can be collectively referred to as nanodrug delivery systems, including nanoaggregates, polymer micelles, vesicles, etc. Drug delivery methods using polymer materials can be divided into two categories: physical encapsulation and chemical coupling. Physical encapsulation methods use non-covalent bonds between drug molecules and carriers, such as hydrogen bonds, electrostatic attraction, and hydrophobic interactions, to deliver drugs. Chemical coupling methods mainly utilize reactions between drugs and active groups of carriers to covalently bind drugs to carriers through chemical bonds such as ester bonds, ether bonds, and amide bonds. Dendrimers are a class of polymers with precise molecular structures and high geometric symmetry. They have a large number of functional groups on their surfaces, which can be structurally modified. Dendrimers are widely used in nanomedicine fields such as drug delivery, gene delivery, and biomedical imaging. Common dendrimers include polyamidoamine (PAMAM), polypropyleneimine (PPI), and polyether-co-polyester (PEPE). Currently, the biological applications of dendrimers are primarily limited by the following issues: cytotoxicity, interactions with plasma proteins and cells, toxicity to biological systems, and in vivo distribution. Numerous studies have reported on the cytotoxicity of dendrimers, demonstrating that cytotoxicity depends primarily on the nature of the surface groups and the structure of the dendrimer. Surface groups on dendrimers have a significant influence on toxicity, with amino groups generally believed to be more toxic than dendrimers modified with carboxyl, hydroxyl, or PEG (Han Yuxin, Zhejiang University, Preparation and Imaging of Novel Dendrimer Magnetic Resonance Contrast Agents, PhD thesis, 2017). Currently, no clinical success has been achieved using dendrimers to deliver boron or its analogs.

[0005] Therefore, there is a need to develop new boron compounds that can more easily enter cells, have long retention times in tumors, and selectively target and destroy tumor cells with minimal damage to normal tissues. Summary of the Invention

[0006] In order to solve the problems existing in the prior art, the present invention provides a nanoboron drug for boron neutron capture therapy, wherein the nanoboron drug is PAMAM-BSH, wherein PAMAM is generation 3 (G3), generation 4 (G4) or generation 5 (G5).

[0007] Preferably, the PAMAM in the nanoboron drug is G4.

[0008] Preferably, the molar ratio of PAMAM to BSH in the nanoboron drug is 1:(20-60), preferably, the molar ratio of PAMAM to BSH is 1:(30-50), further preferably, the molar ratio of PAMAM to BSH is 1:(35-40), and in a preferred embodiment, the molar ratio of AMAM to BSH is 1:38.

[0009] Another aspect of the present invention provides a method for preparing the nano-boron drug PAMAM-BSH, which is prepared using BSH as a boron source and 3rd generation (G3), 4th generation (G4) or 5th generation (G5) dendritic macromolecule PAMAM as a drug carrier.

[0010] Preferably, the nanoboron drug PAMAM-BSH is prepared by a Michael addition reaction between BSH and 4-(N-carboxycyclohexylmethyl)maleimide to obtain an intermediate BSM compound, which is then condensed with PAMAM to obtain the compound, wherein the PAMAM is a 3rd generation (G3), 4th generation (G4) or 5th generation (G5) dendrimer.

[0011] The Michael addition reaction is carried out under alkaline conditions, and the base forming the alkaline conditions is selected from triethylamine, pyridine, sodium carbonate, potassium carbonate, sodium hydroxide, potassium hydroxide and lithium hydroxide.

[0012] The reaction solvent of the Michael addition reaction is selected from N,N-dimethylformamide, N,N-dimethylacetamide, DMSO and the like.

[0013] The method further includes preparing BSM into an active ester, wherein the active ester is N-hydroxysuccinimidyl ester, N-hydroxyphthalimidyl ester, N-hydroxythiophthalimidyl ester, o-nitrophenyl ester, p-nitrophenyl ester, 2,4-dinitrophenyl ester, 3-sulfonyl-4-nitrophenyl ester, 3-carboxyl-4-nitrophenyl ester, pentafluorophenyl ester or sulfonyltetrafluorophenyl ester. Preferably, the active ester is N-hydroxysuccinimidyl ester.

[0014] Preferably, the step of preparing BSM into active ester is carried out in PBS buffer.

[0015] Another aspect of the present invention provides a use of the nanoboron drug in the preparation of an anti-tumor drug. Preferably, the tumor is melanoma.

[0016] The biosafety of nanomicelles PAMAM-BSH was investigated. PAMAM-BSH was modified with the fluorescent dye rhodamine B, and its cellular entry ability was investigated using flow cytometry and laser scanning confocal microscopy. Laser scanning confocal microscopy was also used to investigate the cellular entry mechanism of PAMAM-BSH. The uptake of PAMAM-BSH by tumor cells was investigated, and boron content in tumor cells was measured using inductively coupled plasma mass spectrometry (ICP-MS).

[0017] PAMAM-BSH was loaded into microneedles to create a complete microneedle array structure with uniformity and integrity. Mechanical strength testing was performed on the microneedles. After penetrating mouse skin, the microneedles dissolved in the interstitial fluid. The PAMAM-BSH-loaded microneedles were used to transdermally administer drug to tumor sites, and boron concentrations at the tumor sites were measured.

[0018] The present invention offers the advantage that the boron-containing drug BSH is one of the only currently available clinical drugs for BNCT. However, it still suffers from poor tumor specificity. Using the dendritic macromolecule PAMAM as a drug carrier, nanomicelles PAMAM-BSH are prepared. PAMAM-BSH is well internalized into tumor cells, effectively delivering boron to tumors and enriching it there, thereby enhancing the efficacy of boron neutron capture therapy.

[0019] PAMAM-BSH formed nanoparticles by self-assembly. The BSH connected to the amino end of PAMAM aggregated together due to hydrophobic interactions to form a hydrophobic core. The remaining amino groups on PAMAM acted as hydrophilic parts and were exposed to the outside. The synthesized dendritic macromolecule PAMAM-BSH has amphiphilic properties. As shown in the scanning electron microscope (SEM) image, the obtained NPs have a spherical shape with a monodisperse particle size. We used ICP-MS to detect the boron content ( Figure 14 ). The results show that in exemplary embodiments, the number of BSHs connected to PAMAM-BSH synthesized using the 3rd, 4th, and 5th generation PAMAMs are 15, 38, and 41, respectively. The number of BSHs coupled to PAMAM (G4)-BSH synthesized using the 4th generation PAMAM was significantly improved compared to PAMAM (G3)-BSH synthesized using the 3rd generation PAMAM. However, the number of BSHs coupled to PAMAM (G5)-BSH synthesized using the 5th generation PAMAM was not significantly improved compared to the 4th generation. The boron content in PAMAM (G4)-BSH synthesized using the 4th generation PAMAM is 15%. In other words, 38 of the 64 amino groups of PAMAM are connected to BSH, and the molar ratio of PAMAM to BSH is 1:38.

[0020] Dynamic light scattering (DLS) analysis revealed that the average hydrodynamic size of PAMAM(G4)-BSH was 390 nm and the zeta potential was +11.1 mV.

[0021] The biosafety of PAMAM-BSH was studied. CCK-8 cytotoxicity assay, cell live-dead staining assay, and cell apoptosis assay confirmed that PAMAM-BSH had good biocompatibility.

[0022] PAMAM-BSH was modified with the fluorescent dye Rhodamine B to investigate its cellular internalization and entry mechanism. Flow cytometry and laser scanning confocal microscopy were used to assess the internalization of PAMAM-BSH into tumor cells. The results showed that PAMAM-BSH was well internalized into tumor cells and that its entry mechanism was energy-dependent.

[0023] The uptake capacity of tumor cells to PAMAM-BSH was studied. The boron content in cells was detected by ICP-MS. The results showed that the boron concentration in B16F10 cells could reach 5300ng / 10 6 cells, 61% higher than BSH.

[0024] Soluble microneedles loaded with PAMAM-BSH, using hyaluronic acid (HA) as the matrix material, were prepared. These microneedles were transdermally administered to the tumor site. After 2.5 hours of microneedle transdermal administration, boron levels in the tumor, muscle, blood, and major organs were measured using ICP-MS. The results showed that boron concentrations in the tumor exceeded 20 ppm, while concentrations in major organs were very low. The boron concentration ratios (T / N) and (T / B) between tumor tissue and normal tissue, and between tumor tissue and blood, were both greater than 3:1. These results demonstrate that the boron nanoparticle PAMAM-BSH, prepared by coupling the dendrimer PAMAM with the boron drug BSH, can be used in boron neutron capture therapy (BNCT).

[0025] illustrate:

[0026] The numbers of surface functional groups of PAMAM (G3), PAMAM (G4), and PAMAM (G5) are different, namely 32, 64, and 128, respectively. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a composite image of BSM.

[0028] Figure 2 This is the synthesis diagram of PAMAM(G4)-BSH.

[0029] Figure 3 This is the 1H NMR spectrum of N-[4-(-carboxycyclohexylmethyl)]maleimide.

[0030] Figure 4 1H NMR spectrum of BSH.

[0031] Figure 5 1H NMR spectrum of BSM.

[0032] Figure 6 1H NMR comparison chart of N-[4-(-carboxycyclohexylmethyl)]maleimide and BSM.

[0033] Figure 7 This is the MS spectrum of BSM.

[0034] Figure 8 1H NMR spectrum of PAMAM (G4).

[0035] Figure 9 1H NMR comparison chart of BSH, PAMAM (G4) and PAMAM (G4)-BSH.

[0036] Figure 10 This is the SEM image of PAMAM (G4)-BSH.

[0037] Figure 11 TEM image of PAMAM(G4)-BSH.

[0038] Figure 12 This is the particle size distribution diagram of PAMAM (G4)-BSH.

[0039] Figure 13 This is the potential diagram of PAMAM(G4)-BSH.

[0040] Figure 14 is the number of BSH coupled with different generations of PAMAM.

[0041] Figure 15 This is the CCK-8 cytotoxicity test data of PAMAM-BSH synthesized from different generations of PAMAM.

[0042] Figure 16 This is a live-dead staining image of PAMAM (G4)-BSH cells.

[0043] Figure 17 This is the data of the cell apoptosis experiment of PAMAM (G4)-BSH.

[0044] Figure 18 The confocal images and flow cytometry data of the internalization of PAMAM(G4)-BSH are shown.

[0045] Figure 19The confocal images and flow cytometry data of the cell invasion of PAMAM(G4)-BSH are shown.

[0046] Figure 20 The graph shows the data of boron content in tumor cells taking up PAMANM(G4)-BSH cells.

[0047] Figure 21 The boron content in the tumor, muscle, blood, and major organs was determined by ICP-MS 2.5 hours after microneedle transdermal administration of PAMAM(G4)-BSH loaded microneedles.

[0048] Figure 22 The chemical structure is PAMAM(G4)-BSH.

[0049] Figure 23 is the chemical structure of PAMAM (G4). DETAILED DESCRIPTION

[0050] To address the poor cell entry of BSH, in a specific embodiment, the present invention couples the dendritic macromolecule PAMAM with the boron drug BSH to produce the nanoboron drug PAMAM-BSH. In this invention, N-[4-(-carboxycyclohexylmethyl)]maleimide and the boron drug BSH are coupled via a Michael addition reaction, resulting in the product named BSM. BSH is then coupled to the dendritic macromolecule PAMAM via the carboxyl group of BSM and the amino group on the dendritic macromolecule PAMAM, resulting in the product named PAMAM-BSH. PAMAM-BSH forms nanomicelles through self-assembly. The BSH attached to the amino end of PAMAM aggregates due to hydrophobic interactions, forming a hydrophobic core. The remaining amino groups on PAMAM act as hydrophilic moieties, remaining exposed externally. The prepared PAMAM-BSH exhibits amphiphilic properties. The present invention first characterizes the prepared nanomicelles PAMAM-BSH. The biosafety of PAMAM-BSH is then investigated. The cellular internalization ability of PAMAM-BSH and its uptake by tumor cells were investigated. A soluble microneedle transdermal drug delivery system loaded with PAMAM-BSH and based on hyaluronic acid was constructed. Tumor sites in tumor-bearing mice were then treated with these microneedles. The ability of this microneedle transdermal drug delivery system to achieve the boron concentration in the tumor required for BNCT (20 ppm or higher) was investigated. Furthermore, the boron concentration ratios (T / N and T / B) between tumor tissue and normal tissue, and between tumor tissue and blood, were determined to be greater than 3:1. The potential for boron drug delivery using this microneedle transdermal drug delivery system for BNCT was investigated.

[0051] (1) Preparation of nanomicelles PAMAM(G4)-BSH

[0052] 4-(N-carboxycyclohexylmethyl)maleimide (100.00 mg, 0.42 mmol) and BSH (93.00 mg, 0.21 mmol) were dissolved in N,N-dimethylformamide (10 mL), and triethylamine (21.00 mg, 0.21 mmol) was slowly added dropwise to the above system. The reaction system was stirred overnight at room temperature. Water (30 mL) was added to the reaction and extracted with EA (3x 50 mL). The obtained organic phase was washed with saturated brine (50 mL) and concentrated under reduced pressure distillation. The obtained crude product was purified by MeOH / DCM (0-20%) Prep-thin layer chromatography. The filtered filtrate was freed from the solvent under reduced pressure. After freeze-drying, the product BSM (145 mg, yield: 83%) was obtained as a yellow solid (reaction equation as shown in FIG. Figure 1 ).

[0053] BSM (46.00 mg, 1.758 μmol), EDC (13.00 mg, 112.60 mmol) and NHS (26.00 mg, 112.60 mmol) were dissolved in PBS (10.00 mL) pH = 7.2-7.4 buffer solution, and the system was stirred at room temperature for 8 hours, and then PAMAM (PAMAM (G3), PAMAM (G4), PAMAM (G5) were purchased from Weihai Chenyuan Molecular New Materials Co., Ltd.) (25.00 mg, 112.60 mmol) was added to the above system. The final reaction was stirred at room temperature for 16 hours. The reaction solution was added to a dialysis membrane (molecular weight cut-off Mw = 500) and the membrane was dialyzed in water every 8 hours. The product ((32 mg)) obtained after three membrane dialysis and freeze-drying was a light yellow solid (reaction equation as shown in FIG. Figure 2 ).

[0054] The synthesis of other generations of PAMAM-BSH, such as PAMAM(G4)-BSH and PAMAM(G4)-BSH, can refer to the above-mentioned synthesis method of PAMAM(G4)-BSH.

[0055] (2) Biosafety experiment of PAMAM-BSH

[0056] B16F10 cells were grown at 5×10 3 Cells were seeded in 96-well plates at a concentration of 100 μL and incubated overnight in a cell culture incubator. Various concentrations of PAMAM-BSH were added to the wells (final volume 100 μL per well). After 24 hours of incubation, cell viability was determined using CCK-8. Live / dead staining of the cells was performed using Calcein AM / PI, and images were captured using an inverted fluorescence microscope.

[0057] B16F10 cells were grown at 2×10 4Cells were seeded into 6-well plates at a concentration of 100 μL and incubated overnight in a cell culture incubator. Various concentrations of PAMAM-BSH were added to the wells (final volume 100 μL per well). After 24 hours of incubation, cells were stained using an apoptosis kit and analyzed by flow cytometry.

[0058] The results of cytotoxicity experiments showed that PAMAM (G4)-BSH synthesized by the 4th generation PAMAM had no cytotoxicity to B16-F10 cells. However, PAMAM-BSH synthesized by the 3rd generation (G3) and the 5th generation PAMAM (G5) showed certain cytotoxicity to B16-F10 cells. Therefore, we used the 4th generation PAMAM (G4) to synthesize PAMAM-BSH, which has good biosafety compared with the 3rd and 5th generation PAMAM ( Figure 15 ).

[0059] (3) PAMAM-BSH internalization and cell entry experiments

[0060] First, PAMAM-BSH was modified with the fluorescent dye Rhodamine B. B16F10 cells were plated at 5×10 cells per well. 4 Cells were seeded at a concentration of 100 cells / mL in confocal dishes and incubated overnight in a cell culture incubator. Different concentrations of rhodamine B-modified PAMAM-BSH were then added. After incubation for 2.5 hours, the cells were washed three times with PBS and fixed with paraformaldehyde. The nuclei were then stained with Hoechst 33342 and imaged using confocal microscopy.

[0061] B16F10 cells were grown at 2×10 5 Cells were seeded into 6-well plates at a concentration of 100 cells / mL and incubated overnight in a cell culture incubator. Different concentrations of Rhodamine B-modified PAMAM-BSH were then added and incubated for 2.5 hours. The cells were washed three times with PBS and the fluorescence intensity of the cells was measured using a flow cytometer.

[0062] B16F10 cells were grown at 5×10 4 Cells were seeded at a density of 100 cells / mL in confocal dishes and incubated overnight in a cell culture incubator. The cells were then treated with the endocytosis inhibitors dextran sulfate, sucrose, chlorpromazine, methyl-beta-cyclodextrin, nystatin, and amiloride at 4°C for one hour and then discarded. Following fixation with paraformaldehyde, the nuclei were stained with Hoechst 33342 and imaged using confocal microscopy.

[0063] B16F10 cells were grown at 2×10 5Cells were seeded at a concentration of 100 cells / mL in a 6-well plate and incubated overnight in a cell culture incubator. The cells were then treated with the endocytosis inhibitors dextran sulfate, sucrose, chlorpromazine, methyl-beta-cyclodextrin, nystatin, and amiloride at 4°C for one hour and then discarded. The cells were then incubated with rhodamine B-modified PAMAM-BSH for 2.5 hours, washed three times with PBS, and fluorescence intensity was measured by flow cytometry.

[0064] (3) Cellular uptake experiment of PAMAM-BSH

[0065] B16F10 cells were grown at 2×10 5 Cells were seeded into 6-well plates at a concentration of 100 cells and incubated overnight in a cell culture incubator. Then, different concentrations of PAMAM-BSH were added, incubated for 2.5 hours, and washed three times with PBS. The cells were collected and counted. The cells were digested by microwave, and the boron content in the cells was detected by ICP-MS ( Figure 20 ).

[0066] The results showed that the boron content in PAMAM-BSH was 15%. In other words, 38 of the 64 amino groups of PAMAM were connected to BSH, and the molar ratio of PAMAM to BSH was 1:38. Dynamic light scattering (DLS) determined that the average hydrodynamic size of PAMAM-BSH was 390 nm ( Figure 12 ), zeta potential is +11.1mV( Figure 13 ).

[0067] (4) In vivo experiments of PAMAM-BSH

[0068] Microneedles loaded with PAMAM-BSH were transdermally administered to the tumor site at a boron concentration of 7 mg / ml. Two and a half hours after microneedle transdermal administration, the mice were sacrificed and samples from the tumor, muscle, blood, and major organs were collected for boron analysis to determine whether the boron concentration in the tumor reached 20 ppm or higher. Furthermore, the boron concentration ratios of tumor tissue to normal tissue (T / N) and tumor tissue to blood (T / B) were greater than 3:1.

Claims

1. A nanoboron drug for boron neutron capture therapy, wherein the nanoboron drug is PAMAM-BSH, wherein PAMAM is a 3rd generation (G3), 4th generation (G4) or 5th generation (G5) dendrimer.

2. The nanoboron drug according to claim 1, wherein PAMAM is G4.

3. The nanoboron drug according to claim 1, wherein the molar ratio of PAMAM to BSH in the nanoboron drug is 1:(20-60), preferably, the molar ratio of PAMAM to BSH is 1:(30-50), further preferably, the molar ratio of PAMAM to BSH is 1:(35-40), and in a preferred embodiment, the molar ratio of PAMAM to BSH is 1:

38.

4. A method for preparing the nanoboron drug according to any one of claims 1 to 3, wherein BSH is used as a boron source and a dendrimer macromolecule PAMAM is used as a drug carrier, wherein the dendrimer macromolecule is of generation 3 (G3), generation 4 (G4) or generation 5 (G5).

5. The method for preparing a nanoboron drug according to claim 4, wherein BSH and 4-(N-carboxycyclohexylmethyl)maleimide are subjected to a Michael addition reaction to obtain an intermediate BSM compound, which is then subjected to a condensation reaction with PAMAM to obtain the compound, wherein the PAMAM is a 3rd generation (G3), 4th generation (G4) or 5th generation (G5) dendrimer.

6. The method for preparing nanoboron medicine according to claim 5, wherein the Michael addition reaction is carried out under alkaline conditions, and the base forming the alkaline conditions is selected from triethylamine, pyridine, sodium carbonate, potassium carbonate, sodium hydroxide, potassium hydroxide and lithium hydroxide.

7. The method for preparing nanoboron medicine according to claim 5, wherein the reaction solvent of the Michael addition reaction is selected from N,N-dimethylformamide, N,N-dimethylacetamide, DMSO, etc.

8. The method for preparing a nano-boron drug according to claim 5, further comprising preparing BSM into an active ester, wherein the active ester is N-hydroxysuccinimide ester, N-hydroxyphthalimide ester, N-hydroxythiophthalimide ester, o-nitrophenyl ester, p-nitrophenyl ester, 2,4-dinitrophenyl ester, 3-sulfonyl-4-nitrophenyl ester, 3-carboxyl-4-nitrophenyl ester, pentafluorophenyl ester or sulfonyltetrafluorophenyl ester. Preferably, the active ester is N-hydroxysuccinimide ester. 9 . The method for preparing nanoboron medicine according to claim 8 , wherein the step of preparing BSM into active ester is carried out in PBS buffer.

10. Use of the nanoboron drug according to any one of claims 1 to 9 in the preparation of an anti-tumor drug, preferably, the tumor is melanoma.

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