Boron-containing modified bevacizumab drug, preparation method thereof, nano-carrier and application

Abstract

The invention relates to the technical field of monoclonal antibody drugs, in particular to a boron-containing modified bevacizumab drug as well as a preparation method, a nano-carrier and application thereof. According to the technical scheme, the medicine is formed by covalent coupling of bevacizumab and a boron-containing compound through a disulfide bond. According to the invention, boron atoms are introduced into bevacizumab molecules through chemical modification, and a dual-function drug with anti-angiogenesis and boron neutron capture therapy radiosensitization functions is constructed, so that bevacizumab has anti-angiogenesis activity of anti-vascular endothelial growth factors and a boron source function of a capture therapy radiosensitizer at the same time; the design breaks through the limitation of a traditional single-function medicine, the concentration of the medicine in the brain tumor and the radiotherapy curative effect are improved through double-function cooperation, and a brand new strategy is provided for brain glioma treatment.

Description

Technical Field

[0001] The present invention relates to the technical field of monoclonal antibody drugs, and in particular to a boron-modified bevacizumab drug, a preparation method thereof, a nanocarrier and applications thereof. Background Art

[0002] As a highly malignant central nervous system tumor, glioma has an aggressive growth characteristic and the existence of blood-brain barrier, which limits the effectiveness of traditional radiotherapy and chemotherapy. Boron neutron capture therapy (BNCT) can generate high linear energy transfer α and α through the reaction of boron-10 and thermal neutrons. 7 Li particles achieve precise killing at the cellular level. However, in actual applications, the heterogeneity of tumor tissue leads to uneven distribution of boron agents in the tumor, and some tumor cells cannot obtain sufficient boron-10 concentration. At the same time, the tumor hypoxic microenvironment reduces the radiation sensitivity of neutrons, greatly limiting the therapeutic effect of BNCT.

[0003] Vascular endothelial growth factor (VEGF) antibodies specifically bind to VEGF, inhibiting tumor angiogenesis and reducing tumor blood supply, thereby inhibiting tumor growth and metastasis. This therapy has achieved some success in treating various solid tumors, but the use of VEGF antibodies alone has problems such as tumor resistance and inability to completely eliminate tumor cells.

[0004] Early technologies for introducing boron clusters into antibodies through chemical coupling were also limited by the high stability of traditional covalent bonds, which easily destroyed antibody activity; insufficient boron loading, making it difficult to reach the ≥800 μg / g threshold required for BNCT treatment in tumor tissues; and the harsh reaction conditions of the preparation process and low product purity.

[0005] Regarding the difficulty in delivering antibody drugs, conventional nanocarrier systems are easily cleared by liver phagocytes and the immune system, lack of targeting ligands leads to accumulation in normal tissues, and insufficient environmental responsiveness makes it impossible to trigger release in the tumor microenvironment, making it difficult to meet the needs of brain glioma treatment.

[0006] In summary, the present application proposes a boron-modified bevacizumab drug, a preparation method thereof, a nanocarrier, and applications thereof. Summary of the Invention

[0007] The purpose of the present invention is to address the problem in the background technology that traditional boron agents only have a single BNCT function and cannot simultaneously inhibit tumor angiogenesis, and to propose a boron-modified bevacizumab drug and its preparation method, nanocarrier and application.

[0008] In the first aspect, the present invention provides a boron-modified bevacizumab drug, which is formed by covalently coupling bevacizumab with a boron-containing compound through a disulfide bond, wherein the boron-containing compound is mercapto dodecaborane (B 12 H11 SH), boron atoms are introduced into the antibody molecule in a cage-like structure, giving the drug both anti-vascular endothelial growth factor (VEGF) activity and boron neutron capture therapy (BNCT) radiosensitization function. By modifying the cage structure of boron atoms, bevacizumab has both anti-angiogenic activity against vascular endothelial growth factor (VEGF) and the function of a boron source as a BNCT radiosensitizer, forming a synergistic treatment mechanism of "anti-angiogenesis + precision radiotherapy." This design breaks through the limitations of traditional single-function drugs and improves drug concentration and radiotherapy efficacy in brain tumors through dual-function synergy.

[0009] Optionally, the coupling molar ratio of bevacizumab to mercaptododecaborane is 1:15–1:25, the boron atom enrichment in the drug molecule is ≥800 μg / g, and the monomer rate is maintained at ≥95% in pH 7.4 phosphate buffer (PBS).

[0010] In a second aspect, the present invention provides a method for preparing the boron-modified bevacizumab drug according to the first aspect, comprising the following steps:

[0011] a. Mix 1.0±0.2 mg / mL bevacizumab PBS solution (pH 7.4) with 100±1 mM mercaptododecaborane ethanol solution at a volume ratio of 10:1;

[0012] b. Using pulsed ultrasonic coupling technology: a 500W probe is inserted 1.0±0.2cm below the liquid surface, with a cycle of 2s on and 3s off for 3 times, each cycle lasting 120±5 seconds;

[0013] c. Purification was performed using a tangential flow filtration system: 100 kDa molecular weight cutoff membrane, flow rate 60 ± 5 mL / min, transmembrane pressure 0.8 ± 0.1 bar, to obtain the boron-modified bevacizumab drug.

[0014] Optionally, step b is carried out under nitrogen protection, and the temperature of the reaction system is always maintained at 3-5°C.

[0015] Optionally, the pulsed ultrasound uses a titanium alloy conical probe with a probe tip diameter of 3±0.5 mm and a cavitation effect intensity of 0.35±0.05 MPa.

[0016] Optionally, a buffer replacement step is added after purification in step c, using a PBS solution containing 5% trehalose for replacement three times at a flow rate of 5-10 times the column volume / min.

[0017] In a third aspect, the present invention provides a nanocarrier loaded with the boron-modified bevacizumab drug of the first aspect, wherein the carrier is composed of sodium alginate / polyethyleneimine (PEI) modified with phenylboronic acid, wherein:

[0018] Sodium alginate concentration 2.0 ± 0.3% w / v, PEI concentration 1.5 ± 0.2 mg / mL;

[0019] The degree of substitution of phenylboronic acid is 18-22% of the total number of PEI amino groups;

[0020] The drug-to-carrier mass ratio is 1:5, and dynamic drug loading is achieved through boronate bonds.

[0021] Optionally, the particle size of the carrier is 130-170 nm and the Zeta potential is 20-30 mV.

[0022] Optionally, a targeting peptide sequence (RGD or Angiopep-2) is covalently linked to the surface of the carrier, and the density of the targeting peptide is 8-12 molecules per nanoparticle.

[0023] In a fourth aspect, the present invention provides a use of the boron-modified bevacizumab drug described in the first aspect in the preparation of a drug for treating brain tumor BNCT.

[0024] Compared with the prior art, this application has at least one of the following beneficial technical effects:

[0025] 1. Through innovative design, the present invention enables the boron-modified bevacizumab drug to have both anti-VEGF activity and BNCT radiosensitization function. The boron atom enrichment reaches 850μg / g and the monomer rate exceeds 95%, solving the problem of insufficient boron loading in traditional BNCT drugs.

[0026] 2. By adopting pulsed ultrasonic coupling technology, combined with nitrogen protection and low-temperature control, the binding rate is improved and more antibody activity is retained, significantly optimizing coupling efficiency and drug stability compared to traditional methods.

[0027] 3. The responsive nanodelivery system achieves pH-responsive drug release through phenylboronic acid-modified carriers, and is combined with RGD targeting peptides to increase the boron concentration in tumors, break through the blood-brain barrier and enhance targeting.

[0028] 4. The drug system achieves synergistic anti-angiogenesis and radiosensitization in the treatment of brain gliomas, and has good in vivo stability and safety, providing an efficient and feasible new solution for clinical BNCT combined treatment.

[0029] The present invention introduces boron atoms into the bevacizumab molecule through chemical modification, constructing a dual-functional drug with both anti-angiogenesis and boron neutron capture therapy radiosensitization functions. Bevacizumab has both anti-angiogenic activity against vascular endothelial growth factor and the function of a boron source as a BNCT radiosensitizer, forming a synergistic treatment mechanism of "anti-angiogenesis + precision radiotherapy". This design breaks through the limitations of traditional single-functional drugs, and through dual-functional synergy, it improves the drug concentration and radiotherapy efficacy in brain tumors, providing a new strategy for the treatment of gliomas. DETAILED DESCRIPTION

[0030] The following describes the embodiments of the present invention through specific examples. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments. The details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that the following embodiments and features in the embodiments can be combined with each other unless they conflict.

[0031] Example 1: Preparation and characterization of boron-modified bevacizumab drugs

[0032] Drug conjugation reaction: A 1.0 mg / mL bevacizumab solution in PBS (pH 7.4) was mixed with a 100 mM thiol dodecaborane solution in ethanol at a volume ratio of 10:1. The mixture was transferred to a reaction vessel under nitrogen protection, and the system temperature was maintained at 3-5°C. Pulsed ultrasonic conjugation was performed using a 500W titanium alloy conical probe (tip diameter 3 mm). The probe was inserted 1.0 cm below the liquid surface. A cycle of 2 seconds on and 3 seconds off was repeated three times, each cycle lasting 120 seconds. The cavitation effect intensity was controlled at 0.35 MPa.

[0033] Purification and buffer exchange: The reaction solution was purified by tangential flow filtration with a 100 kDa molecular weight cutoff at a flow rate of 60 mL / min and a transmembrane pressure of 0.8 bar. The buffer was then exchanged three times with 5% trehalose in PBS at a flow rate of 8 column volumes / min and lyophilized to obtain boron-modified bevacizumab.

[0034] Performance characterization: The boron atom enrichment was determined to be 850 μg / g by inductively coupled plasma mass spectrometry (ICP-MS), and the monomer rate in pH 7.4 PBS was 96.3% by size exclusion chromatography (SEC-HPLC). Western blot confirmed that the antibody VEGF binding activity retention rate reached 89%.

[0035] Example 2: Preparation and optimization of responsive nanodelivery system

[0036] Carrier synthesis: 2.0% w / v sodium alginate solution and 1.5 mg / mL phenylboronic acid-modified PEI (phenylboronic acid substitution degree 20%) solution were mixed in a volume ratio of 1:1 and stirred at room temperature for 30 minutes to form a polymer precursor.

[0037] Drug loading: The drug solution was added dropwise to the polymer precursor at a mass ratio of boron-modified bevacizumab to carrier of 1:5 and incubated at 4°C for 2 hours to dynamically load the drug through the boronate bond.

[0038] Targeting peptide coupling: RGD peptide (final concentration 10 μM) was added to the system and covalently linked to the carrier surface under EDC / NHS catalysis. Unbound peptide segments were removed by ultrafiltration to obtain targeted nanocarriers.

[0039] Physical and chemical properties: Dynamic light scattering (DLS) determined the particle size to be 150 nm and the zeta potential to be 25 mV; transmission electron microscopy (TEM) showed the carrier to have a spherical uniform structure; flow cytometry measured the density of targeting peptide per nanoparticle to be 10 molecules.

[0040] Example 3: In vitro experimental verification of BNCT treatment of brain glioma

[0041] Cell model: U87MG human glioma cell line was cultured in DMEM medium containing 10% FBS and seeded in 96-well plates (1×10 4 cells / well).

[0042] Drug treatment: A blank control group, a free bevacizumab group (10 μg / mL), a free thiol dodecaborane group (boron content 800 μg / g), and a boron-modified bevacizumab group (boron content 850 μg / g) were set up, with 3 replicates in each group, and the cells were incubated for 24 hours.

[0043] Neutron irradiation: using thermal neutron beams (flux 1×10 9 n / cm 2 ·s) The cells were irradiated for 30 minutes and then cultured for 48 hours.

[0044] Cytotoxicity assay: Cell viability was determined by the MTT assay. The results showed that the cell viability in the boron-modified bevacizumab + neutron irradiation group was 18.7%, significantly lower than that in the single-drug group (65.2% in the free bevacizumab group and 52.1% in the free boron agent group) and the non-irradiation group (72.3% in the boron-modified bevacizumab group), demonstrating that the drug possesses both anti-VEGF activity and BNCT radiosensitization effects.

[0045] Example 4: In vivo targeting experiment of nano-delivery system

[0046] Animal model: A nude mouse U87MG glioma orthotopic transplantation model was established and the mice were randomly divided into two groups (n=5): a boron-modified bevacizumab group and a drug-loaded nanodelivery system group.

[0047] Administration: The drug was injected into the tail vein at a dose of 5 mg / kg (calculated as bevacizumab). The animals were killed 24 hours later and the brain tumor tissues were collected.

[0048] Boron content determination: ICP-MS detection showed that the boron concentration in tumor tissue of the nanodelivery system group was 25.6μg / g, significantly higher than that of the free drug group (12.3μg / g). Immunofluorescence staining showed that the inhibition rate of VEGF expression in tumor blood vessels in the nanocarrier group reached 78%, significantly higher than that of the free drug group (55%), confirming that the nanosystem can enhance the drug's brain tumor targeting and anti-angiogenic effects.

[0049] Example 5: Comparative experiment on drug stability

[0050] Simulated physiological environment: Boron-modified bevacizumab was placed in pH 7.4 PBS, PBS containing 10% fetal bovine serum, and mouse plasma, respectively, and incubated at 37°C for 72 hours.

[0051] Structural characterization: SEC-HPLC analysis showed that the monomer rates of the drug in the three systems were maintained at 95.1%, 92.3%, and 89.7%, respectively. In contrast, the monomer rate of the traditional amide bond-coupled boron-modified antibody in mouse plasma was only 68.5%, demonstrating that disulfide bond coupling can significantly improve the drug's in vivo stability.

[0052] The above examples verify the feasibility of the preparation process of boron-modified bevacizumab drugs, the nanocarrier delivery efficiency and the BNCT treatment effect through specific parameters and experimental data. All technical parameters fall within the range defined by the claims (such as coupling molar ratio 1:15-1:25, boron atom enrichment ≥800μg / g, nanoparticle size 130-170nm, etc.), and the experimental results show that this solution effectively solves the problems of insufficient boron loading and poor targeting in the existing technology.

[0053] Comparative example design and experimental verification

[0054] 1. Comparative Example 1: Effect of coupling molar ratio on drug performance

[0055] Experimental design:

[0056] Control group A: bevacizumab and mercapto dodecaborane in a molar ratio of 1:10, and the remaining steps were the same as in Example 1;

[0057] Control group B: molar ratio 1:30, other steps are the same as Example 1;

[0058] Experimental group: molar ratio 1:20 (parameters in Example 1).

[0059] Detection indicators: boron atom enrichment, monomer rate in PBS, and VEGF binding activity.

[0060]

[0061]

[0062] From the above comparison, it can be seen that when the molar ratio is lower than 1:15, the boron loading is insufficient, and when it is higher than 1:25, the antibody aggregation increases significantly, and 1:15-1:25 is the optimal range.

[0063] 2. Comparative Example 2: Comparison between Ultrasonic Technology and Traditional Coupling Methods

[0064] Experimental design:

[0065] Control group C: conventional magnetic stirring (300 rpm, 25° C., reaction for 4 hours) was used, and the remaining steps were the same as in Example 1;

[0066] Experimental group: pulsed ultrasound coupling (parameters of Example 1).

[0067] Detection indicators: coupling efficiency (HPLC determination of binding rate), antibody structural integrity (circular dichroism spectroscopy α-helix content).

[0068] Comparison Item Coupling method Binding rate (%) α-helix content (%) Control group C Magnetic stirring 58 32 Experimental group Pulsed ultrasound 92 41

[0069] Conclusion: Pulsed ultrasound improves the coupling efficiency by 34% through cavitation effect and maintains the secondary structure of antibodies more effectively.

[0070] 3. Comparative Example 3: Optimization comparison of the degree of substitution of phenylboronic acid in nanocarriers

[0071] Experimental design:

[0072] Control group D: phenylboronic acid substitution degree 10%;

[0073] Control group E: degree of substitution 30%;

[0074] Experimental group: degree of substitution 20% (parameters of Example 2).

[0075] Detection indicators: nanoparticle size, pH-responsive drug release rate (pH6.5 vs pH7.4).

[0076]

[0077] When the degree of substitution is 18-22%, the nanoparticles have both suitable particle size and tumor microenvironment responsiveness. If the substitution is too low, the drug release is insufficient, while if it is too high, the stability is reduced.

[0078] IV. Comparative Example 4: Effect of Targeting Peptide Modification on Nanocarrier Delivery Efficiency

[0079] Experimental design:

[0080] Control group F: nanocarriers were not coupled with RGD peptide;

[0081] Experimental group: coupled with RGD peptide (parameters of Example 4).

[0082] Detection indicators: in vivo tumor boron concentration (ICP-MS), tumor vascular VEGF inhibition rate.

[0083] Comparison Item Targeted peptide modification Tumor boron concentration (μg / g) VEGF inhibition rate (%) Control group F none 12.3 55 Experimental group have 25.6 78

[0084] Conclusion: Targeted peptide modification increased boron accumulation in tumors by 108% and significantly enhanced the anti-angiogenic effect.

[0085] V. Comparative Example 5: Effect of Coupling Bond Type on Drug Stability

[0086] Experimental design:

[0087] Control group G: amide bond coupling of mercapto dodecaborane and bevacizumab;

[0088] Experimental group: disulfide bond coupling (parameters of Example 1).

[0089] Detection indicators: monomer rate in mouse plasma at 37℃ (72h) and boron release rate.

[0090] Comparison Item Coupling bond type Monomer rate (%) Boron release rate (%) Control group G Amide bond 68.5 32 Experimental group disulfide bonds 89.7 58

[0091] From the above data, it can be seen that disulfide bonds are more stable in physiological environments and can trigger boron release through the reducing environment in tumors, with the release rate increased by 81% compared to amide bonds.

[0092] Data aggregation and optimization analysis

[0093] Molar ratio preference: Within the range of 1:15-1:25, the boron loading meets the standard and the antibody structure is stable. Beyond this range, "insufficient loading" or "aggregation and inactivation" occurs;

[0094] The necessity of pulsed ultrasound: Compared with traditional methods, it improves coupling efficiency and increases the retention rate of α-helical structure by 28%, confirming its protective effect on antibody activity;

[0095] The key value of the degree of substitution of phenylboronic acid: 18-22% is the balance point between pH responsiveness and nanoparticle stability. The degree of substitution affects the dynamic equilibrium of the boronate ester bond.

[0096] Targeted peptide synergistic effect: The introduction of RGD / Angiopep-2 breaks through the BBB through an active targeting mechanism, allowing the tumor boron concentration to reach the BNCT therapeutic threshold (≥20μg / g);

[0097] Advantages of disulfide bond design: It combines plasma stability with tumor microenvironment responsiveness, providing a solution for the dual needs of drugs to achieve "stability in circulation - release within tumors".

[0098] 1. Drug molecule design achieves dual therapeutic function synergy

[0099] By coupling caged thiol dodecaborane to bevacizumab via a disulfide bond, the drug possesses both anti-vascular endothelial growth factor (VEGF) activity and BNCT radiosensitization function. In vitro experiments showed that the drug inhibited the survival rate of U87MG cells under neutron irradiation by 18.7%, significantly better than single bevacizumab (65.2%) or free boron agent (52.1%), proving that the synergistic effect of anti-angiogenesis and radiosensitization can enhance the tumor killing effect. In addition, the boron atom enrichment in the drug reached 850μg / g, far exceeding the 20μg / g tumor enrichment threshold required for BNCT, and the reduction responsiveness of the disulfide bond achieved controlled release of boron in the tumor microenvironment. The monomer rate in mouse plasma remained at 89.7% after 72 hours, which was significantly improved compared to the traditional amide bond conjugate (68.5%).

[0100] 2. Preparation technology breaks through the bottleneck of traditional coupling efficiency and activity retention

[0101] Pulsed ultrasound coupling technology enhances the thiol-disulfide exchange reaction through the cavitation effect of 0.35 MPa, resulting in a 92% binding efficiency between bevacizumab and thiol dodecaborane, a 34% increase compared to traditional magnetic stirring (58%). Furthermore, nitrogen protection and low temperature (3-5°C) control maintain an 89% retention rate of the antibody's VEGF binding activity. Tangential flow filtration combined with a trehalose buffer exchange process not only increases purification efficiency to a monomer yield of ≥95%, but also prevents antibody aggregation through the freeze-drying protection of trehalose. Experiments have confirmed that this process can be scaled to produce high-purity drugs, with batch-to-batch boron content fluctuations of ≤5%.

[0102] 3. Responsive nanodelivery systems solve the problems of blood-brain barrier penetration and targeting

[0103] The phenylboronic acid-modified sodium alginate / PEI carrier, optimized with an 18-22% degree of phenylboronic acid substitution, maintains stability in a physiological environment of pH 7.4 and achieves a 24-hour drug release rate of 78% in the tumor microenvironment (pH 6.5), achieving precise controlled release. The optimized combination of a 150nm particle size and a 25mV zeta potential not only avoids the blood-brain barrier penetration barrier of conventional nanoparticles (>200nm), but also, through the active targeting effect of the RGD targeting peptide (10 molecules per particle), elevates the boron concentration in nude mouse glioma tissue to 25.6μg / g, a 108% increase compared to the free drug (12.3μg / g). Simultaneously, the tumor vascular endothelial growth factor (VEGF) inhibition rate reached 78%, significantly enhancing the anti-angiogenic effect.

[0104] IV. Clinical translational value highlights the advantages of therapeutic precision and safety

[0105] This drug system demonstrates multiple clinical advantages in the treatment of gliomas: First, BNCT's precise cell-level killing (α particles have a range of only 1-2 cells) complements the anti-angiogenic effects of bevacizumab in both time and space, reducing damage to normal brain tissue. Second, the nanocarrier's blood-brain barrier penetration efficiency and tumor microenvironment responsiveness reduce the risk of systemic boron exposure, and no significant liver and kidney dysfunction was observed in animal experiments. Furthermore, the drug design is compatible with existing BNCT equipment and the clinical application pathways of bevacizumab, providing a transformative technical solution for the personalized combined treatment of gliomas.

[0106] It is worth noting that boron modification not only confers drug compatibility with BNCT but also improves bevacizumab's ability to penetrate the blood-brain barrier (BBB). Combined with bevacizumab's binding properties to tumor-specific receptors, this allows for active targeted enrichment of the drug at the site of brain tumors. Animal experiments have confirmed that after nanocarrier delivery, the boron concentration in tumor tissue can reach 25.6 μg / g, a 108% increase compared to free drug, significantly overcoming the BBB barrier's limitations on drug delivery.

[0107] Furthermore, the modified bevacizumab inhibits VEGF, blocking tumor angiogenesis and reducing tumor nutrient supply. Furthermore, as a boron carrier for BNCT, it triggers a boron-10 capture reaction under neutron irradiation, releasing alpha particles and lithium-7 nuclei through high linear energy transfer, achieving precise cell-level killing. In vitro experiments have shown that this synergistic effect can reduce the survival rate of glioma cells to 18.7%, significantly improving efficacy compared to monotherapy while reducing the risk of normal tissue damage.

[0108] The present invention couples boron compounds through disulfide bonds, allowing the drug to maintain high stability in a physiological environment (72-hour monomer rate in plasma ≥89.7%), and can trigger boron release under the reducing conditions of the tumor microenvironment, achieving a dynamic balance of "circulatory stability-intratumor activation", taking into account both drug delivery safety and therapeutic precision.

[0109] In summary, the present invention systematically solves the problems of insufficient boron loading, limited antibody targeting and low brain delivery efficiency of existing BNCT drugs through collaborative innovation of molecular structure, preparation process and delivery system, opening up a new path for the precise treatment of malignant brain tumors.

[0110] The above specific embodiments are merely several optional embodiments of the present invention. Based on the technical solutions of the present invention and the relevant inspirations of the above embodiments, those skilled in the art may make various alternative improvements and combinations to the above specific embodiments.

Claims

1. A boron-modified bevacizumab drug, characterized in that: The drug is formed by covalently coupling bevacizumab and a boron-containing compound through a disulfide bond, wherein the boron-containing compound is mercaptododecaborane, and the boron atom is introduced into the antibody molecule in a cage-like structure, so that the drug has both anti-vascular endothelial growth factor activity and boron neutron capture therapy radiosensitization function.

2. A boron-modified bevacizumab drug according to claim 1, characterized in that: The coupling molar ratio of bevacizumab to mercapto dodecaborane is 1:15-1:25, and the boron atom enrichment in the drug molecule is ≥800 μg / g.

3. A method for preparing the boron-modified bevacizumab drug according to claim 1 or 2, characterized in that: The following steps are involved: a. Mix 1.0±0.2 mg / mL bevacizumab PBS solution with 100±1 mM mercaptododecaborane ethanol solution at a volume ratio of 10:1; b. Using pulsed ultrasonic coupling technology: a 500W probe is inserted 1.0±0.2cm below the liquid surface, with a cycle of 2s on and 3s off for 3 times, each cycle lasting 120±5 seconds; c. Purification was performed using a tangential flow filtration system: 100 kDa molecular weight cutoff membrane, flow rate 60 ± 5 mL / min, transmembrane pressure 0.8 ± 0.1 bar, to obtain the boron-modified bevacizumab drug.

4. The method for preparing a boron-modified bevacizumab drug according to claim 3, characterized in that: Step b is carried out under nitrogen protection, and the temperature of the reaction system is always maintained at 3-5°C.

5. The method for preparing a boron-modified bevacizumab drug according to claim 3, characterized in that: The pulse ultrasound uses a titanium alloy conical probe with a probe tip diameter of 3±0.5 mm and a cavitation effect intensity of 0.35±0.05 MPa.

6. The method for preparing a boron-modified bevacizumab drug according to claim 3, characterized in that: After purification in step c, a buffer replacement step was added, using a PBS solution containing 5% trehalose for 3 times at a flow rate of 5-10 times the column volume / min.

7. A nanocarrier loaded with the boron-modified bevacizumab drug according to claim 1 or 2, characterized in that: The carrier is composed of sodium alginate / polyethyleneimine modified with phenylboronic acid, wherein: Sodium alginate concentration 2.0 ± 0.3% w / v, PEI concentration 1.5 ± 0.2 mg / mL; The degree of substitution of phenylboronic acid is 18-22% of the total number of PEI amino groups; The drug-to-carrier mass ratio is 1:5, and dynamic drug loading is achieved through boronate bonds.

8. The boron-modified bevacizumab drug-loaded nanocarrier according to claim 7, characterized in that: The particle size of the carrier is 130-170 nm and the zeta potential is 20-30 mV.

9. A boron-modified bevacizumab-loaded nanocarrier according to claim 7, wherein a targeting peptide sequence is covalently linked to the surface of the carrier, and the density of the targeting peptide is 8-12 molecules per nanoparticle.

10. Use of the boron-modified bevacizumab drug according to claim 1 or 2 in the preparation of a drug for treating brain tumors (BNCT).

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