CN104399094B - Targeted boron preparation and preparation method - Google Patents

Abstract

The invention belongs to the biological medicine field, and discloses a targeted boron preparation and a preparation method and application. The target boron preparation comprises polyamidoamine-amine dendrimers, epidermal growth factor receptor antibody and polyhedral boranes, the polyamide amine dendrimers are connected with the epidermal growth factor receptor antibody, and are internally loaded with the polyhedral boranes. The targeted boron preparation is high in boron content, can meet the requirements of BNCT (boron neutron capture therapy) on the boron atom dose, is well targeted to tumor cells, has good stability and low cell toxicity, and can be efficiently uptaken by human glioma U87MG cells of high surface expression EGFR (epidermal growth factor receptor). The target boron preparation can effectively improve the content of boron in orthotopic transplantation tumor nude mice tumor tissues, can significantly prolong orthotopic transplantation tumor nude mice lifetime by neutron irradiation, and is suitable for boron neutron capture therapy for glioma. The preparation method of the targeted boron preparation has the advantages of simple operation, and the prepared targeted boron preparation has good stability.

Description

Targeted boron preparation and preparation method thereof

technical field

The invention belongs to the field of biomedicine, and specifically relates to an epidermal growth factor antibody-coupled dendrimer targeted drug delivery system loaded with polyhedral borane and a preparation method thereof, which is suitable for boron neutron capture therapy for treating glioma.

Background technique

Glioma accounts for about 45% of primary intracranial tumors, and is the most common primary malignant intracranial tumor. It is characterized by infiltrative growth of the tumor, unclear tumor boundaries, difficult to completely resected by surgery, and easy to relapse after surgery. At present, surgery combined with postoperative radiotherapy and chemotherapy is considered to be the best treatment for glioma. Therefore, radiotherapy has become one of the important links in the comprehensive treatment of glioma.

Boron neutron capture therapy (BNCT) is a therapeutic method that can selectively kill tumor cells that have spread into normal tissues in theory. When boron (boron-10, ¹⁰ B) is irradiated by low-energy neutrons, a nuclear reaction occurs to produce alpha particles with high linear energy conversion. The range of the killing reaction is within a cell (5-9μm), which itself decays into lithium element. Clinical trials of BNCT in the treatment of glioma have been carried out in the United States, Japan, Finland, the Netherlands, Sweden and other countries. The results of most of the trials suggest that the average survival period and 5-year survival rate of patients with glioma treated with BNCT are better than traditional radiotherapy.

To achieve curative effect, BNCT needs special equipment - a neutron source reactor. In view of the safety and clinical effect of BNCT in the treatment of gliomas, it was established in my country in 2005 with the active support of Zhou Yongmao and Wang Zhongcheng, academicians of the Chinese Academy of Engineering. , Beijing Kaibaite Technology Co., Ltd. built my country's first and the world's first hospital neutron irradiator (In-Hospital Neutron Irradiator, IHNI-1). The equipment was officially operated after being appraised in 2008, and was approved for trial operation in 2010. In 2012, it won the first prize of scientific and technological progress of the Ministry of Nuclear Industry (2012HNJ01D-01). The equipment is economical, safe, effective, small in size and easy to operate. Animal experiments have been basically completed and clinical trials are about to begin.

In order to achieve curative effect of BNCT, in addition to the effective neutron emission source, one of the key issues is that tumor cells must contain a sufficient amount of ¹⁰ B atoms, and the ratio of the concentration of ¹⁰ B atoms in the tumor to the concentration in the surrounding normal tissues and blood Greater than 3:1. Current research shows that delivering sufficient ¹⁰ B atoms to tumor cells while minimizing boron content in normal tissues remains problematic. There are two compounds containing ¹⁰ B that are allowed to be used in clinical trials, namely boron dihydroxyphenylalanine (BPA) and polyhedral borane (BSH), but the effects are not satisfactory. Therefore, the synthesis of boron-containing preparations that can increase the proportion of tumor uptake is of great significance for the use of boron neutron capture therapy in the treatment of glioma.

Contents of the invention

The present invention provides a targeted boron preparation with high boron content aiming at the shortcomings of low tumor tissue enrichment and poor targeting of the boron atom carrier currently used in BNCT clinical trials.

For realizing the purpose of the present invention, the present invention adopts following technical scheme:

A boron-targeting formulation consisting of polyamidoamine dendrimers, antibodies to epidermal growth factor receptors, and polyhedral boranes to which polyamidoamine dendrimers are linked Antibodies internally loaded with the polyhedral borane.

Another object of the present invention is to provide a preparation method of the targeted boron preparation.

A kind of preparation method of targeting boron preparation, by 3-(2-pyridine dimercapto) propionic acid N-hydroxysuccinimide ester and undecanoic acid maleimide-hydrazide-trifluoroacetic acid salt The amide-amine dendrimer and the epidermal growth factor antibody are connected to form a functionalized dendrimer, and then the polyhedral borane is loaded to obtain the targeted boron preparation.

In some embodiments, the preparation method specifically includes the following steps:

(1) EGFR antibody reacts with sodium periodate to obtain oxidized EGFR antibody;

(2) polyamidoamine dendrimers react with 3-(2-pyridyldimercapto) propionic acid N-hydroxysuccinimide ester to obtain intermediate PAMAM-SPDP;

(3) The intermediate PAMAM-SPDP obtained by the reaction of step (2) reacts with DTT to obtain PAMAM-SH;

(4) PAMAM-SH obtained by the reaction of step (3) reacts with undecanoic acid maleimide-hydrazide-trifluoroacetate to obtain PAMAM-KMUH;

(5) The PAMAM-KMUH obtained in step (4) is coupled with the oxidized mAbEGFR obtained in step (1) to obtain PAMAM-mAbEGFR;

(6) Add polyhedral borane BSH to the PAMAM-mAbEGFR obtained by the reaction in step (5) to obtain the targeted boron preparation.

In some embodiments, the molar ratio of the EGFR antibody to sodium periodate in step (1) of the preparation method is 1:10.

In some embodiments, the molar ratio of the polyamidoamine dendrimer described in step (2) of the preparation method to 3-(2-pyridyldimercapto) propionic acid N-hydroxysuccinimide ester It is 1:10.

In some embodiments, the molar ratio of PAMAM-SPDP to DTT in step (3) of the preparation method is 1:10.

In some embodiments, the molar ratio of PAMAM-SH to undecanoic acid maleimide-hydrazide-trifluoroacetate in step (4) of the preparation method is 1:10.

In some embodiments, the molar ratio of PAMAM-KMUH to oxidized EGFR antibody in step (5) of the preparation method is 10:1.

In some embodiments, the mass ratio of PAMAM-mAbEGFR to polyhedral borane in step (6) of the preparation method is 2:1.

Another object of the present invention is to provide the application of the targeted boron preparation in the preparation of drugs for boron neutron capture therapy.

Compared with the prior art, the present invention has at least one of the following beneficial effects:

(1) The targeted boron preparation of the present invention loads a large dose of polyhedral borane (BSH) in the dendrimer cavity, which significantly improves the dosage of boron atoms, and has high encapsulation efficiency and drug loading capacity. High advantages, can meet BNCT's requirements for boron atomic dose.

(2) The targeted boron preparation of the present invention is connected with an antibody (mAbEGFR) highly expressed on the surface of the tumor cell epidermal growth factor receptor, which has better targeting to the tumor cell and increases the uptake of boron by tumor tissue and normal brain tissue It reduces the damage of various meaningful rays to normal brain tissue during neutron irradiation, and has good stability and low cytotoxicity.

(3) The targeted boron preparation of the present invention can be effectively taken up by human glioma U87MG cells that highly express epidermal growth factor receptor on the surface.

(4) The targeted boron preparation of the present invention can effectively increase the boron content in the tumor tissue of nude mice with orthotopic tumor transplantation, and significantly prolong the survival period of nude mice with orthotopic tumor transplantation after neutron irradiation.

Description of drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings that are required in the description of the embodiments or the prior art.

Fig. 1 is the SDS-PAGE identification result of the PAMAM-mAbEGFR prepared in Example 1;

Fig. 2 is the result graph of the effect of different concentrations of PAMAM-mAbEGFR/BSH on the proliferation rate of U87MG cells at different times in Example 3; wherein, ■ is 0.01 μmol/L, 0.1μmol/L, 1μmol/L, 5 μmol/L, □ is 10 μmol/L;

Figure 3 is a diagram of the endocytosis of PAMAM-mAbEGFR-FITC/BSH observed by U87MG cells under a fluorescence microscope in Example 4, wherein (a) is a fluorescent photo of cell endocytosis, and (b) is a light microscope photo of the same field of view;

Figure 4 is the Kaplan-Meier survival curve of nude mice with orthotopic tumor transplantation in Example 6 after BNCT; the control group was without any irradiation, the X-ray 5Gy group was 5Gy X-ray irradiation group, and the INHI-14MV group was only 4MV INHI -1 irradiation group, BSH 4MV group is 4MV INHI-1 irradiation group after BSH injection, PAB 4MV group is 4MV INHI-1 irradiation group after PAMAM-mAbEGFR/BSH injection, X-ray 10Gy is 10Gy X-ray irradiation group, The INHI-18MV group was irradiated with 8MV INHI-1 only, the BSH 8MV group was irradiated with 8MV INHI-1 after injection of BSH, and the PAB 8MV group was irradiated with 8MV INHI-1 after injection of PAMAM-mAbEGFR/BSH;

Fig. 5 is a roadmap for preparing PAMAM-mAbEGFR/BSH according to the present invention.

detailed description

The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Apparently, the described embodiments are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

The invention provides a tumor-specific targeted boron preparation aiming at the disadvantage that tumor cells are not highly specific for the absorption of the two ¹⁰ B preparations allowed to be used in current clinical trials. The targeted boron preparation is a BSH-loaded dendrimer (PAMAM-mAbEGFR/BSH) coupled with an EGFR antibody.

For realizing the purpose of the present invention, the present invention adopts following technical scheme:

A boron-targeting formulation consisting of polyamidoamine dendrimers, antibodies to epidermal growth factor receptors, and polyhedral boranes to which polyamidoamine dendrimers are linked Antibodies internally loaded with the polyhedral borane.

Among them, the molecular formula of polyhedral borane (BSH) is Na ₂ B ₁₂ H ₁₁ SH, which contains boron atoms much more than boron dihydroxyphenylalanine (BPA) containing 1 boron atom, and the targeted boron preparation of the present invention BSH was selected as the boron-loaded preparation to increase the dose of boron atoms absorbed by tumor cells.

Polyamide-amine dendrimer (PAMAM dendrimer) is a kind of polymer with three-dimensional structure, which is very different from the traditional linear polymer in structure. It has a high degree of geometric symmetry, precise molecular structure, a large number of surface functional groups and internal cavities. Polyamide-amine dendrimers contain a lot of space inside, and can effectively compound with small molecules entering the cavity to form compact particles. During the process of cell internalization, they can buffer the acidic environment of endosomes-lysosomes and help wrap The small molecules and dendrimers dissociate rapidly. The modified PAMAM has reduced toxicity and increased transfection efficiency. The targeted boron preparation of the present invention adopts PAMAM as the drug-carrying molecule of BSH, so as to increase the load capacity of BSH and release rapidly after endocytosis.

The key to the success of BNCT is to deliver sufficient ¹⁰ B atoms to tumor cells while minimizing the ¹⁰ B content in normal tissues, so it is an ideal treatment method to carry ¹⁰ B atoms to tumor cells by targeted therapy. Epidermal growth factor receptor (EGFR) is a multifunctional glycoprotein widely distributed on the cell membrane of human tissues. EGFR exists in most cells and is highly expressed in 40% of glioblastomas. , in different grades of glioma tissues, the expression of EGFR increases with the increase of the malignant grade of the tumor, but there is no obvious expression of EGFR in normal human brain tissues. The present invention links the polyamide-amine dendrimer to the The above-mentioned epidermal growth factor receptor antibody makes the targeted boron preparation of the present invention have targeting to EGFR-positive cells, thereby improving the absorption of glioma cells to the targeted boron preparation of the present invention, thereby realizing boron neutron Capture therapy.

The targeted boron preparation of the present invention can targetably increase the uptake dose of ¹⁰ B atoms by tumor cells, increase the difference between tumor cells and surrounding normal tissues in the uptake of ¹⁰ B, and provide a suitable boron atom carrier for BNCT.

In some preferred embodiments, the antibody targeting the epidermal growth factor receptor in the boron preparation of the present invention is a monoclonal antibody to the epidermal growth factor receptor.

The invention also provides a preparation method of the targeting boron preparation.

A kind of preparation method of targeting boron preparation, by 3-(2-pyridine dimercapto) propionic acid N-hydroxysuccinimide ester and undecanoic acid maleimide-hydrazide-trifluoroacetic acid salt The amide-amine dendrimer and the epidermal growth factor antibody are connected to form a functionalized dendrimer, and then the polyhedral borane is loaded to obtain the targeted boron preparation.

N-hydroxysuccinimide 3-(2-pyridyldimercapto)propionate (SPDP), with the molecular formula C ₁₂ H ₁₂ N ₂ O ₄ S ₂ , is a short-chain cross-linking agent, which passes through hydroxysuccinimide The urethane active group is connected to the amino group (-NH ₂ ) on the PAMAM molecule. Undecanoic acid maleimide-hydrazide-trifluoroacetate (KMUH) has a molecular formula of C ₁₅ H ₂₅ N ₃ O ₃ CF ₃ CO ₂ H, which is a long-chain cross-linking The imine reacts with the sulfhydryl group (-SH) on the PAMAM-SH molecule. The preparation method of the present invention connects the epidermal growth factor antibody and the polyamide-amine dendrimer (PAMAM) through SPDP and KMUH to form a functionalized dendrimer, which has a high load capacity and a polyhedral borane (BSH) Stability, while obtaining good biocompatibility, increases the absorbed dose of BSH by tumor cells.

On the other hand, dendrimers must rely on a larger size to reflect the enhanced osmotic retention effect (EPR effect) of tumor cells and avoid premature metabolism. Although dendrimers with high algebra have larger sizes, they are associated with higher systemic toxicity. The introduction of SPDP and KMUH linking segments in the preparation method of the present invention can help dendrimer macromolecules with a lower generation number below PAMAM G6.0 with a diameter of less than 9 nm to obtain a larger nanometer size without increasing the generation number, and improve the overall system biosafety.

In some embodiments, the preparation method of the targeted boron preparation of the present invention specifically includes the following steps:

(1) EGFR antibody reacts with sodium periodate to obtain oxidized EGFR antibody;

(2) polyamidoamine dendrimers react with 3-(2-pyridyldimercapto) propionic acid N-hydroxysuccinimide ester to obtain intermediate PAMAM-SPDP;

(3) The intermediate PAMAM-SPDP obtained by the reaction of step (2) reacts with DTT to obtain PAMAM-SH;

(4) PAMAM-SH obtained by the reaction of step (3) reacts with undecanoic acid maleimide-hydrazide-trifluoroacetate to obtain PAMAM-KMUH;

(5) The PAMAM-KMUH obtained in step (4) is coupled with the oxidized mAbEGFR obtained in step (1) to obtain PAMAM-mAbEGFR;

(6) Add polyhedral borane BSH to the PAMAM-mAbEGFR obtained by the reaction in step (5) to obtain the targeted boron preparation.

Wherein, in step (1) of the preparation method of the targeted boron preparation of the present invention, the epidermal growth factor receptor antibody mAbEGFR reacts with sodium periodate (NaIO4) to obtain oxidized epidermal growth factor receptor antibody, so that the epidermal growth factor receptor The acceptor antibody forms a carbonyl (C=O) functional group.

In some preferred embodiments, the mAbEGFR reacts with NaIO by mixing mAbEGFR and NaIO in 0.1M acetate buffer at pH 4.5, reacting at room temperature for 2 h, desalting the reaction mixture, washing with 50 mM phosphate buffer at pH 7.5 oxidized epidermal growth factor receptor antibody.

Further, the molar ratio of mAbEGFR to NaIO4 described in step (1) is preferably 1:10.

In some preferred embodiments, the antibody targeting the epidermal growth factor receptor in the boron preparation of the present invention is a monoclonal antibody to the epidermal growth factor receptor.

Polyamidoamine dendrimer (PAMAM) and 3-(2-pyridyldimercapto) propionic acid N-hydroxysuccinimide ester (SPDP) in the preparation method step (2) of the targeted boron preparation of the present invention reaction,

The hydroxysuccinimide ester active group of SPDP is connected with the amino group (-NH ₂ ) on the PAMAM molecule to obtain the intermediate PAMAM-SPDP.

In some preferred embodiments, the reaction of PAMAM and SPDP in the step (2) is that PAMAM and SPDP are mixed in 0.1M phosphate buffered saline solution of pH 7.5, reacted at room temperature for 2h, and the reaction mixture is desalted, and then mixed with 50mM phosphoric acid of pH 7.5 Elution with salt buffer gave the intermediate PAMAM-SPDP.

Further, the molar ratio of PAMAM to SPDP in step (2) is preferably 1:10.

Preferably, the polyamide-amine dendrimer described in the present invention is a 5.0 generation solution with a molecular weight of 28824 and an amino group (-NH ₂ ) at the end of the molecule.

The step (3) of the preparation method of the targeted boron preparation of the present invention reacts PAMAM-SPDP with DTT, and DTT reduces the pyridyl disulfide group on the SPDP molecule to form a sulfhydryl group (-SH) to obtain PAMAM-SH.

In some preferred embodiments, the reaction of PAMAM-SPDP and DTT in the step (3) is that DTT solution reacts with PAMAM-SPDP at room temperature for 30min, and the reaction mixture is desalted and eluted with 50mM phosphate buffer at pH 7.5 to obtain PAMAM-SPDP SH;

Further, the molar ratio of PAMAM-SPDP to DTT described in step (3) is preferably 1:10.

Wherein, the preparation method of the DTT solution is that DTT (purchased from Sigma Company) is dissolved in 0.1M phosphate buffer solution with pH 7.5, and 10 v/v% dimethyl sulfoxide (DMSO) is added.

Preferably, the 50 mM phosphate buffer at pH 7.5 in step (3) contains 10v/v% DMSO.

The preparation method step (4) of the targeted boron preparation of the present invention reacts with undecanoic acid maleimide-hydrazide-trifluoroacetate (KMUH), and the maleimide in KMUH reacts with The thiol (-SH) group on the PAMAM-SH molecule is linked to give PAMAM-KMUH.

In some preferred embodiments, the reaction of PAMAM-SH and KMUH in the step (4) is to mix the PAMAM-SH and KMUH obtained by the reaction of step (3) in DMSO, react at room temperature for 2h, desalt the reaction mixture, and use pH 7.5 The 50mM phosphate buffer was eluted to obtain PAMAM-KMUH.

Further, the molar ratio of PAMAM-SH to KMUH described in step (4) is preferably 1:10.

Preferably, the 50 mM phosphate buffer at pH 7.5 in step (4) contains 10v/v% DMSO.

Step (5) PAMAM-KMUH is coupled with the oxidized mAbEGFR obtained in step (1) of the preparation method of the targeted boron preparation of the present invention, and the hydrazide on the PAMAM-KMUH is coupled with the carbonyl (C=O) on the oxidized mAbEGFR ) reaction to obtain PAMAM-mAbEGFR;

In some preferred embodiments, the coupling of PAMAM-KMUH and oxidized mAbEGFR in the step (5) is to mix PAMAM-KMUH and oxidized mAbEGFR, and react at room temperature for 24 hours to obtain the conjugate PAMAM-mAbEGFR.

Wherein, the molar ratio of PAMAM-KMUH to oxidized mAbEGFR in step (5) is preferably 10:1.

Further, the step (5) also includes the step of concentrating and purifying the obtained PAMAM-mAbEGFR.

In some preferred embodiments, the concentration and purification is concentrated to 1 ml-2 ml by ultrafiltration, and then purified by chromatography, and eluted with 50 mM phosphate buffer at pH 7.5 to obtain PAMAM-mAbEGFR.

Preferably, the chromatographic purification is Sephacryl S-300 chromatographic column purification.

Preferably, the 50 mM phosphate buffer at pH 7.5 in the concentrated purification contains 10 v/v% DMSO.

In the step (6) of the preparation method of the targeted boron preparation of the present invention, polyhedral borane (BSH) is added to PAMAM-mAbEGFR, and ¹⁰ B atoms are loaded to obtain the targeted boron preparation.

In some preferred embodiments, adding BSH to PAMAM-mAbEGFR as described in step (6) is placed in a beaker as the PAMAM-mAbEGFR carrier solution, and BSH is gradually added thereto until BSH is no longer dissolved, and put in 25 Shake in a constant temperature shaker at ℃ for 24 hours to obtain a saturated solution of BSH-loaded PAMAM-mAbEGFR carrier (PAMAM-mAbEGFR/BSH), pass the saturated solution through a membrane, and collect the filtrate to obtain the targeted boron preparation.

Wherein, the mass ratio of PAMAM-mAbEGFR to BSH described in step (6) is preferably 2:1.

Preferably, the membrane is a membrane with a diameter of 0.22 μm.

Further, as a preference, the desalination of the reaction mixture described in steps (1), (2), (3) and (4) in the preparation method of the targeted boron preparation of the present invention all use Sephadex G25 chromatographic column, to remove unreacted small molecules.

The present invention adopts methods such as SDS-PAGE gel electrophoresis, MTT, fluorescent microscope, and neutron irradiation to test the dendrites of the polyhedral borane-coupled epidermal growth factor receptor monoclonal antibody prepared by the present invention. Molecules, the specific test results are as follows:

(1) SDS-PAGE gel electrophoresis identification:

SDS-polyacrylamide gel electrophoresis identification results show that the molecular weight corresponding to the band of mAbEGFR monomer (molecular weight about 85KD) is about 85KD, and the molecular weight of the synthesized PAMAM-mAbEGFR band is about 115KD, and there is no molecular weight at about 85KD. Bands can be seen, indicating that the chromatographically purified PAMAM-mAbEGFR solution does not contain unbound mAbEGFR.

(2) Determination of BSH loading dose in PAMAM-mAbEGFR/BSH:

The boron standard curve was drawn, the PAMAM-mAbEGFR/BSH filtrate was diluted to a certain concentration, and the boron content of the sample was detected by an inductively coupled plasma atomic emission spectrometer (ICP-AES). The concentration of BSH in the carrier was calculated according to the difference of BSH before and after drug loading, and the BSH loaded on PAMAM-mAbEGFR was 280 mg/g.

(3) Cytotoxicity study:

Different concentrations of the synthetic material PAMAM-mAbEGFR/BSH were incubated with the cells for different times, and the activity of the proliferating cells was detected by the MTT assay. The results showed that the targeting EGFR dendrimers loaded with BSH had no significant effect on the cell proliferation rate. There was no significant difference (P>0.05), indicating that the synthetic material has good biocompatibility and no obvious toxicity.

(4) Biological activity detection:

The EGFR monoclonal antibody mAbEGFR-FITC with green fluorescence was used instead of mAbEGFR in the preparation step (1), so that the prepared BSH-loaded EGFR-targeting dendrimer PAMAM-mAbEGFR-FITC/BSH had green fluorescence. U87MG cells positive for EGFR expression were selected as target cells, and the endocytosis of PAMAM-mAbEGFR-FITC/BSH by cells was observed by fluorescence microscope. The results showed that when PAMAM-mAbEGFR-FITC/BSH was at 1 μM, more than 90% of U87MG cells had green fluorescence, and the intensity was high, indicating that a large amount of PAMAM-mAbEGFR-FITC/BSH had been taken up at this time.

(5) Stability test

The prepared PAMAM-mAbEGFR/BSH solution was placed in a -20°C environment, and samples were taken at 1, 3, and 6 months respectively to observe the properties of the solution, and simultaneously carry out BSH carrying capacity and biological activity detection. The results showed that PAMAM-mAbEGFR was placed at different times for BSH loading capacity and biological activity of the compound did not change significantly.

(6) Detection of boron ( ¹⁰ B ) biodistribution in nude mice with orthotopic transplanted tumor model:

An orthotopic tumor transplantation model in nude mice was established, and PAMAM-mAbEGFR/BSH was delivered to the tumor cells by intravenous injection and convective enhanced delivery. The dose was equivalent to BSH 100mg/kg body weight. , 36h and 48h after collecting blood, they were killed, and the tumor tissue, normal brain tissue, liver and kidney were separated, and the concentration of ¹⁰ B in the sample was detected by ICP-AES. The results showed that the polymer PAMAM-mAbEGFR/BSH could not reach the brain tissue and brain tumor tissue through intravenous injection, and the administration by convection-enhanced delivery could be absorbed by the tumor tissue in large quantities. At 24 hours, PAMAM-mAbEGFR/BSH had diffused significantly in the tumor tissue, and the boron content met the requirements of BNCT, so it was suitable for neutron irradiation.

(7) Detection of tumor inhibitory effect of BNCT on orthotopic transplanted tumor in nude mice:

The test was divided into control group (not irradiated), X-ray irradiated group, INHI-1 group (only irradiated with neutrons), BSH group and PAMAM-mAbEGFR/BSH group. EGFR-positive glioma cells cultured in vitro were used for BNCT experiments when obvious symptoms of intracranial transplanted tumors appeared after planting. BSH neutron irradiation 3 hours after intravenous injection is currently the most commonly used method for BNCT research. At this time, the tumor boron concentration is high, and the ratio of tumor to normal brain tissue and tumor to blood boron concentration is suitable for BNCT treatment. The nude mice in the BSH group were irradiated with neutrons 3 hours after intravenous injection, and the nude mice in the PAMAM-mAbEGFR/BSH group were irradiated with neutrons 24 hours after the convective enhanced transmission injection. There was a significant difference in boron concentration (P<0.01). The tumor concentration in BSH group was 8.65±1.32μg/g, the ratio of boron concentration in tumor to normal brain tissue was 2.65, and the ratio of boron concentration in tumor to blood was 2.02; The ratio of boron concentration in normal brain tissue is 11.70, and the ratio of boron concentration in tumor to blood is greater than 30. All irradiations were carried out after nude mice were anesthetized with INHI-1. During the irradiation, neutron rays were shielded by a lithium-rich irradiation box, and only the tumor site was exposed to the tunnel of the irradiation box to receive neutron irradiation. The maximum power of INHI-1 is 30KW, the maximum thermal neutron flux is 1×10 ⁹ n/(cm·s), and the irradiation is divided into 4MV-min and 8MV-min. Calculate the survival time of nude mice with transplanted tumors from the day of irradiation. Using median survival time (MeST), mean survival time (MST), and Kaplan-Meier survival curve as evaluation indicators, it can be seen that administration of PAMAM-mAbEGFR/BSH followed by BNCT significantly improved the survival rate of intracranial transplanted tumors. Compared with the control group (P<0.01), the INHI-1 group (P<0.01), the same dose of X-ray group (P<0.05), and the BSH group (P<0.05), the survival time of nude mice was significantly different. difference.

The targeted boron preparation of the present invention can be effectively taken up by human glioma cells that highly express epidermal growth factor receptor on the surface, and can effectively increase the boron content in the tumor tissue of nude mice with orthotopic transplanted tumors, and can be used in the boron content of tumors. Sub-capture therapy. Therefore, the present invention also provides the application of the targeted boron preparation in the preparation of drugs for boron neutron capture therapy.

The present invention has at least one of the following beneficial effects:

(1) The targeted boron preparation of the present invention loads a large dose of polyhedral borane (BSH) in the dendrimer cavity, which significantly improves the dosage of boron atoms, and has high encapsulation efficiency and drug loading capacity. High advantages, can meet BNCT's requirements for boron atomic dose.

(2) The targeted boron preparation of the present invention is linked with an antibody (mAbEGFR) highly expressed on the surface of tumor cell epidermal growth factor receptor, which has better targeting to tumor cells and increases the uptake of tumor tissue and normal brain tissue The difference of boron can reduce the damage of various meaningful rays to normal brain tissue during neutron irradiation, and at the same time, it has good stability and low cytotoxicity.

(3) The targeted boron preparation of the present invention can be effectively taken up by human glioma U87MG cells that highly express epidermal growth factor receptor on the surface.

(4) The targeted boron preparation of the present invention can effectively increase the boron content in the tumor tissue of nude mice with orthotopic tumor transplantation, and significantly prolong the survival period of nude mice with orthotopic tumor transplantation after neutron irradiation.

In order to further understand the present invention, the method provided by the present invention will be described in detail below in conjunction with the examples. Wherein the experimental method that does not indicate concrete condition in the embodiment, generally according to routine condition, for example described in molecular cloning experimental guideline (third edition, J. Sambrook et al., Huang Peitang et al. translation, Science Press, 2002) conditions, or as recommended by the manufacturer. The SPDP and KMUH were purchased from Pierce Company, and PAMAM and DTT were purchased from Sigma Company.

Example 1: Preparation of PAMAM-mAbEGFR/BSH

(1) Mix 0.1 mg of the epidermal growth factor receptor monoclonal antibody mAbEGFR with 2 mg of sodium periodate in 1 ml of 0.1 M acetate buffer at pH 4.5, use magnetic beads to keep stirring at room temperature for 2 h, and pass the reaction mixture through Sephadex Desalting the G25 chromatography column, removing excess sodium periodate, eluting with 50mM phosphate buffer at pH 7.5, and collecting the oxidized EGFR monoclonal antibody obtained;

(2) 0.58ml of 5% polyamidoamine dendrimer (PAMAM) solution (Sigma Company), which contains 29mg of PAMAM, is mixed with 3.12mg SPDP in 1ml of 0.1M phosphate buffered saline buffer solution at pH 7.5, and reacted at room temperature 2h, the reaction mixture was desalted by Sephadex G25 chromatography column to remove unreacted SPDP, and eluted with 50mM phosphate buffer solution with pH 7.5 to obtain the intermediate PAMAM-SPDP;

(3) Dissolve 1.54mg of DTT in 1ml of 0.1M phosphate buffer at pH 7.5, add 0.1g of DMSO to prepare a DTT solution containing 10% DMSO. The prepared DTT solution was mixed with the PAMAM-SPDP obtained in step (2), and reacted at room temperature for 30 minutes. The reaction mixture was desalted on a Sephadex G25 chromatographic column, eluted with 50 mM phosphate buffer (containing 10% DMSO) at pH 7.5, and unreacted DTT was removed to obtain PAMAM-SH;

(4) Mix the PAMAM-SH obtained by the reaction with 4.1 mg KMUH in DMSO, react at room temperature for 2 h, desalt the reaction mixture through a Sephadex G25 chromatographic column, wash with 50 mM phosphate buffer (containing 10% DMSO) at pH 7.5 Take off, remove unreacted KMUH, obtain PAMAM-KMUH;

(5) The obtained PAMAM-KMUH is mixed with the oxidized mAbEGFR obtained in step (1), reacted at room temperature for 24h, and the obtained conjugate PAMAM-mAbEGFR is purified using Sephacryl S-300 chromatographic column, and purified with pH 7.5 50mM phosphate buffer (containing 10% dimethyl sulfoxide) was eluted to obtain PAMAM-mAbEGFR, concentrated to 1ml by ultrafiltration, and freeze-dried to become a white powder;

(6) Add 0.1 mg of PAMAM-mAbEGFR carrier into double-distilled water, stir evenly and set the volume to 1 ml, and gradually add BSH to it until the BSH is no longer dissolved, and the mass of BSH added at this time is 0.05 mg. Put it into a constant temperature shaker at 25° C. and shake for 24 hours to obtain a saturated solution of BSH (PAMAM-mAbEGFR/BSH) loaded on the PAMAM-mAbEGFR carrier. The saturated solution was treated with a membrane (0.22 μm) to remove undissolved BSH, and the filtrate was collected to obtain PAMAM-mAbEGFR/BSH.

During the synthesis process, the dendrimer PAMAM-mAbEGFR linked to the epidermal growth factor receptor monoclonal antibody was identified by SDS-PAGE polyacrylamide gel electrophoresis, and the results are shown in Figure 1.

It can be seen from the results in Figure 1 that mAbEGFR has a band at about 80kb, while the prepared PAMAM-mAbEGFR sample has a band at about 110kba, and there is no band at 80kb, indicating that mAbEGFR has been connected to PAMAM, and the purified PAMAM Collection solution PAMAM:mAbEGFR=1:1 linkage.

The boron standard curve was further drawn, the PAMAM-mAbEGFR/BSH filtrate was diluted to a certain concentration, and the boron content of the sample was detected by an inductively coupled plasma atomic emission spectrometer (ICP-AES). The concentration of BSH in the carrier is calculated according to the difference of BSH before and after drug loading, and the BSH loaded on PAMAM-mAbEGFR is 280mg/g, that is, 58.8kg (58.8kg/mol) of BSH is loaded per mole of PAMAM-mAbEGFR molecule .

The above test results indicate that the designed and synthesized drug-loaded antibody-linked dendrimer PAMAM-mAbEGFR/BSH has been successfully prepared.

Embodiment 2: Preparation of PAMAM-mAbEGFR/BSH

(1) mAbEGFR 0.3mg and sodium periodate 6mg were mixed in 3ml of 0.1M acetate buffer solution with a pH of 4.5, stirred continuously at room temperature for 2 hours using magnetic beads, and the reaction mixture was desalted by Sephadex G25 chromatography column to remove Excess sodium periodate was eluted with 50 mM phosphate buffer at pH 7.5, and the resulting oxidized EGFR monoclonal antibody was collected;

(2) 1.74ml of polyamidoamine dendrimer PAMAM (Sigma company) solution, which contains 87mg of PAMAM, is mixed with 9.36mg of SPDP in 3ml of 0.1M phosphate buffer saline at pH7.5, reacted for 2h, reacted The mixture was desalted by Sephadex G25 chromatographic column to remove unreacted SPDP, and eluted with 50mM phosphate buffer at pH 7.5 to obtain the intermediate PAMAM-SPDP;

(3) 4.62 mg of DTT was dissolved in 3 ml of 0.1 M phosphate buffer solution with pH 7.5, and 0.3 g of DMSO was added to prepare a DTT solution containing 10% DMSO. The prepared DTT solution was mixed with the PAMAM-SPDP obtained in step (2), and reacted at room temperature for 30 minutes. The reaction mixture was desalted on a Sephadex G25 chromatographic column, eluted with 50 mM phosphate buffer (containing 10% DMSO) at pH 7.5, and unreacted DTT was removed to obtain PAMAM-SH;

(4) Mix the PAMAM-SH obtained by the reaction with 12.3 mg KMUH in DMSO, react at room temperature for 2 h, desalt the reaction mixture through a Sephadex G25 chromatographic column, and wash with 50 mM phosphate buffer (containing 10% DMSO) of pH 7.5 Take off, remove unreacted KMUH, obtain PAMAM-KMUH;

(5) The obtained PAMAM-KMUH is mixed with the oxidized mAbEGFR obtained in step (1), reacted at room temperature for 24 hours, and the obtained PAMAM-mAbEGFR is purified using Sephacryl S-300 chromatography column, and purified with 50 mM phosphate at pH 7.5 Buffer (containing 10% dimethyl sulfoxide) was eluted to obtain PAMAM-mAbEGFR, which was concentrated to 3ml by ultrafiltration, and freeze-dried to become a white powder;

(6) Add 0.5 mg of PAMAM-mAbEGFR carrier into double-distilled water, stir evenly and set the volume to 5 ml, and gradually add BSH to it until the BSH is no longer dissolved, and the mass of BSH added at this time is 0.25 mg. Put it into a constant temperature shaker at 25° C. and shake for 24 hours to obtain a saturated solution of BSH (PAMAM-mAbEGFR/BSH) loaded on the PAMAM-mAbEGFR carrier. The saturated solution was passed through a membrane (0.22 μm) to remove undissolved BSH, and the filtrate was collected to obtain PAMAM-mAbEGFR/BSH;

PAMAM-mAbEGFR was identified by SDS-PAGE polyacrylamide gel electrophoresis, and the result was the same as that of PAMAM-mAbEGFR prepared in Example 1, showing that mAbEGFR was connected to PAMAM, and the collected liquid PAMAM after chromatographic purification: mAbEGFR=1:1 connect. The content of boron in the product PAMAM-mAbEGFR/BSH was further measured by ICP-AES, and the results showed that the BSH on PAMAM-mAbEGFR was 280mg/g. These test results indicated that the dendrimer linked to the drug-loaded antibody had been successfully prepared. PAMAM-mAbEGFR/BSH.

Embodiment 3: Cytotoxicity investigation

Human glioma U87MG cells in the logarithmic growth phase were inoculated in 96-well plates at ¹⁰ cells/well, cultured at 37°C for 24 hours, and the culture medium was discarded. 5. Add serum-free medium with 6 concentration gradients of 10 μmol/L to the cells and culture them for 24, 48 and 72 hours respectively, absorb the drug-containing medium, add 0.2ml of serum-free medium containing 0.5mg/ml MTT to each well, Continue to incubate at 37°C for 4 hours, absorb the culture solution containing MTT, wash with PBS twice, add 0.2ml DMSO to each well, shake for 10 minutes to dissolve evenly, measure the absorbance A value at 570nm with a microplate reader, and calculate the cell proliferation rate. See Figure 2. The formula for calculating the cell proliferation rate is as follows:

Cell proliferation rate=absorbance value of the experimental group/absorbance value of the control group×100%.

It can be seen from the results in Figure 2 that PAMAM-mAbEGFR/BSH had no significant effect on cell proliferation rate, and there was no significant difference between the groups (P>0.05). 10 μmol/L PAMAM-mAbEGFR/BSH was incubated in U87MG cells for 72 hours to achieve the maximum inhibitory rate. Proliferation was 9.55%, and the median lethal dose was not reached at 10 times the maximum dose, indicating that PAMAM-mAbEGFR/BSH has low cytotoxicity and good biocompatibility.

Embodiment 4: biological activity detection

The EGFR monoclonal antibody mAbEGFR-FITC with green fluorescence is substituted for the mAbEGFR in the preparation step (1), so that the dendrimer PAMAM-mAbEGFR-FITC/BSH targeting the epidermal growth factor receptor of the resulting polyhedral borane is prepared With green fluorescence. Other preparation steps are completely the same as the product PAMAM-mAbEGFR/BSH of the present invention. EGFR-positive human glioma U87MG cells were cultured in high-glucose DMEM medium containing 10% fetal bovine serum. Inoculate ¹⁰ cells/well in a 6-well plate, culture for 24 hours to adhere to the wall, add serum-free culture medium containing 1 μM PAMAM-mAbEGFR-FITC/BSH, culture at 37°C for 24 hours, wash twice with PBS, and replace with conventional culture medium. The endocytosis of PAMAM-mAbEGFR-FITC/BSH was observed by fluorescence microscopy, and the results are shown in Figure 3.

The results in Fig. 2 show that when PAMAM-mAbEGFR-FITC/BSH is at 1 μM, more than 90% of U87MG cells have green fluorescence, and the intensity is high, indicating that a large amount of PAMAM-mAbEGFR-FITC/BSH has been taken up at this time.

Embodiment 5: stability test

The PAMAM-mAbEGFR/BSH solution prepared in Example 1 was placed in a -20°C environment, and samples were taken at 1, 3, and 6 months respectively to observe the properties of the solution, and simultaneously perform BSH carrying capacity and biological activity detection. The test results are shown in Table 1.

Table 1 PAMAM-mAbEGFR placed for different time on BSH loading capacity and biological activity test

time (month) Carrying capacity of BSH (mg/g) Percentage of green fluorescence in U87MG cells (%) 1 276 >90 3 272 >90 6 271 >90

From the results in Table 1, it can be seen that PAMAM-mAbEGFR has no obvious change in the BSH loading capacity and the biological activity of the compound.

The PAMAM-mAbEGFR/BSH solution prepared in Example 1 was similar to the result of Example 1. PAMAM-mAbEGFR did not significantly change the loading capacity of BSH and the biological activity of the composite.

Example 6: Detection of boron ( ¹⁰ B) biodistribution in nude mice with orthotopic transplanted tumor model.

(1) Preparation of comparative material PAMAM/BSH:

1ml of PAMAM solution (Sigma company, containing 50mg of PAMAM) was diluted with double distilled water to 0.5mg/ml, and 1ml was taken to gradually add BSH to it until the BSH was no longer dissolved. At this time, the mass of BSH added was 0.2mg. Put it into a constant temperature shaker at 25° C. and shake for 24 hours to obtain a saturated solution of BSH (PAMAM/BSH) loaded on the PAMAM carrier. The saturated solution was treated with a membrane (0.22 μm) to remove undissolved BSH, and the filtrate was collected. The boron standard curve was drawn, the PAMAM/BSH filtrate was diluted to a certain concentration, and the boron content of the sample was detected by ICP-AES. The concentration of BSH in the carrier is calculated according to the difference of BSH before and after drug loading, and the BSH loaded on PAMAM-mAbEGFR is 1100 mg/g, that is, 31.7 kg (31.7 kg/mol) of BSH is loaded per mole of PAMAM molecule.

(2) Establishment of orthotopic tumor transplantation model in nude mice:

In each group, 10 male Balb/c nude mice 4-6 weeks old, weighing 18-20 g, were anesthetized with 1% pentobarbital 0.15m1 intraperitoneally, fixed on a stereotaxic instrument, cut the scalp, and drilled holes in the parietal and occipital regions Inject 15 μl of U87MG cell suspension (containing 1×10 ⁶ cells) with a microsyringe pump for 10 minutes, seal with bone wax, and suture the scalp. On the 25th day after implantation, when obvious symptoms of intracranial transplanted tumors appeared, it was used for the experiment.

(3) The drug was injected into the tumor of nude mice by intravenous injection and convection-enhanced delivery respectively, equivalent to BSH 100mg/kg body weight, and the nude mice were collected blood in batches at 6h, 12h, 24h, 36h and 48h after injection Sacrifice, separate tumor tissue, normal brain tissue, liver and kidney, digest in 1ml of a mixture of nitric acid and hydrogen peroxide (volume ratio 3:1) at 120°C for 2h until the solution is completely transparent, make up the volume to 5ml with distilled water, and use ICP-AES Detect the concentration of ¹⁰ B in the sample. The results are shown in Table 2.

Table 2 The biodistribution of boron in nude mice with orthotopic tumor transplantation in different ways given to PAMAM-mAbEGFR/BSH at different times (μg/g)

Note: T: tumor (tumor tissue); N: normal brain (normal brain tissue); B: blood (blood); L: liver (liver);

K: kidney (kidney); T/N: tumor/normal brain tissue; T/B: tumor/blood.

It can be seen from Table 2 that due to the existence of the blood-brain barrier, the high-molecular PAMAM-mAbEGFR/BSH cannot reach the brain tissue and brain tumor tissue through intravenous injection, and the convective enhanced delivery method can be absorbed by the tumor tissue in large quantities. BSH has diffused significantly in the tumor tissue, and the boron content meets the requirements of BNCT, so it is suitable for neutron irradiation.

The prepared comparison material PAMAM/BSH is also a polymer dendritic drug carrier, which cannot pass through the blood-brain barrier. The drug was injected into the tumor of nude mice by convection-enhanced delivery, and the blood was collected for 24 hours. content, and the results are shown in Table 3.

Table 3 Biodistribution of boron in tumor, normal brain and blood in nude mice injected with different synthetic materials for 24 hours (μg/g)

group the tumor normal brain blood tumor/brain tumor/blood PAMAM-mAbEGFR/BSH 30.54±0.82 2.61±0.79 0.04±0.01 11.70 —— PAMAM/BSH 12.95±1.64 3.52±0.58 0.05±0.01 3.68 ——

It can be seen from the results in Table 3 that the amount of boron atoms absorbed by tumor cells to the synthetic material linked with EGFR monoclonal antibody is significantly higher than that of the synthetic material without antibody linked.

Example 7: Detection of tumor-inhibitory effect of BNCT orthotopically transplanted tumor in nude mice

The test was divided into control group (not irradiated), X-ray irradiated group, INHI-1 group (only irradiated with neutrons), BSH group and PAMAM-mAbEGFR/BSH group. U87MG cells were used for BNCT experiments when obvious symptoms of intracranial transplanted tumors appeared after implantation. Dosage BSH 100mg/kg body weight, PAMAM-mAbEGFR/BSH 460mg/kg body weight (wherein the dose of BSH is equivalent to 100mg/kg), the nude mice in the BSH group were irradiated with neutrons 3h after injection, and the nude mice in the PAMAM-mAbEGFR/BSH group were neutron irradiated. The mice were irradiated with neutrons 24 hours after the convective enhanced transmission injection. The boron concentration at the irradiation time point was detected by ICP-AES. The results are shown in Table 4.

Table 4 Concentration of boron in nude mice with orthotopic tumor transplantation under different preparations and administration methods (μg/g)

group the tumor normal brain blood tumor/brain tumor/blood BSH 8.65±1.32 3.26±0.36 4.29±0.11 2.65 2.02 PAMAM-mAbEGFR/BSH 30.54±0.82 2.61±0.79 0.04±0.01 11.70 >30

It can be seen from the results in Table 4 that there was a significant difference in the boron concentration between the BSH group and the PAMAM-mAbEGFR/BSH group during irradiation (P<0.01). In the BSH group, the tumor concentration was 8.65±1.32μg/g, the ratio of boron concentration in tumor to normal brain tissue was 2.65, and the ratio of boron concentration in tumor to blood was 2.02. Meet the requirements of BNCT (tumor boron concentration>20μg/g, tumor/brain>3, tumor/blood>3); while the tumor concentration in PAMAM-mAbEGFR/BSH group was 30.54±0.82μg/g, the ratio of boron concentration in tumor to normal brain tissue It is 11.70, and the ratio of tumor to blood boron concentration is greater than 30, meeting the requirements of BNCT.

All irradiations were carried out after nude mice were anesthetized with INHI-1. During the irradiation, neutron rays were shielded by a lithium-rich irradiation box, and only the tumor site was exposed to the tunnel of the irradiation box to receive neutron irradiation. The irradiation was divided into 4MV-min and 8MV-min, and the total absorbed dose obtained by each group is shown in Table 5.

Table 5 Physical radiation dose (Gy) of various components absorbed by tumor tissue of orthotopically transplanted nude mice

Calculated from the day of irradiation, median survival time (MeST), mean survival time (MST), and Kaplan-Meier survival curve were used as evaluation indicators to calculate the survival time of nude mice with transplanted tumors. The results are shown in Table 6 and Figure 4.

Table 6 Survival period of nude mice with intracranial tumor transplantation under different treatment methods (days)

The results in Table 6 and Figure 4 show that BNCT after PAMAM-mAbEGFR/BSH significantly improved the survival time of nude mice with intracranial tumor transplantation, compared with the control group (P<0.01), INHI-1 group (P<0.01) 0.01), the same dose of X-ray group (P<0.05), BSH group (P<0.05) were significantly different.

Claims (10)

1. A targeted boron preparation, consisting of polyamidoamine dendrimers, antibodies to epidermal growth factor receptors and polyhedral boranes, the polyamidoamine dendrimers are linked to epidermal growth factor receptors The antibody of the polyhedron is internally loaded with the polyhedral borane; the polyhedral borane is BSH. 2. A preparation method for targeting boron preparations, through 3-(2-pyridine dimercapto) propionic acid N-hydroxysuccinimide ester and undecanoic acid maleimide-hydrazide-trifluoroacetic acid salt The functionalized dendrimer is formed by linking the polyamidoamine dendrimer and the epidermal growth factor antibody, and then the polyhedral borane BSH is loaded to obtain the targeted boron preparation. 3. preparation method according to claim 2, specifically comprises the following steps: (1) EGFR antibody reacts with sodium periodate to obtain oxidized EGFR antibody; (2) polyamidoamine dendrimers react with 3-(2-pyridyldimercapto) propionic acid N-hydroxysuccinimide ester to obtain intermediate PAMAM-SPDP; (3) The intermediate PAMAM-SPDP obtained by the reaction of step (2) reacts with DTT to obtain PAMAM-SH; (4) PAMAM-SH obtained by the reaction of step (3) reacts with undecanoic acid maleimide-hydrazide-trifluoroacetate to obtain PAMAM-KMUH; (5) The PAMAM-KMUH obtained in step (4) is coupled with the oxidized mAbEGFR obtained in step (1) to obtain PAMAM-mAbEGFR; (6) Add polyhedral borane BSH to the PAMAM-mAbEGFR obtained by the reaction in step (5) to obtain the targeted boron preparation. 4. The preparation method according to claim 3, wherein the molar ratio of the epidermal growth factor receptor antibody to sodium periodate described in step (1) is 1:10. 5. preparation method according to claim 3, the mole of polyamidoamine dendrimer described in step (2) and 3-(2-pyridine dimercapto) propionic acid N-hydroxysuccinimide ester The ratio is 1:10. 6. The preparation method according to claim 3, the molar ratio of PAMAM-SPDP and DTT described in step (3) is 1:10. 7. The preparation method according to claim 3, the molar ratio of PAMAM-SH and undecanoic acid maleimide-hydrazide-trifluoroacetate described in step (4) is 1:10. 8. The preparation method according to claim 3, wherein the molar ratio of PAMAM-KMUH to oxidized EGFR antibody in step (5) is 10:1. 9. The preparation method according to claim 3, wherein the mass ratio of PAMAM-mAbEGFR and polyhedral borane described in step (6) is 2:1. 10. The application of the targeted boron preparation according to claim 1 in the preparation of medicines for boron neutron capture therapy.