Borated cell membrane, its preparation method and application

Abstract

The present application relates to the technical field of medicine, and more particularly to a boronized cell membrane and a preparation method and application thereof.The preparation method of the boronized cell membrane comprises the following steps: adding tumor cells into a culture medium containing a monosaccharide-modified boron drug to perform incubation, collecting cell precipitates after the incubation is completed, and then extracting cell membranes, so that the boronized cell membrane is obtained.The present application provides a boronized cell membrane and a preparation method and application thereof.By using the monosaccharide-modified BSH, the obtained boron drug is anchored on the tumor cell membrane, and then the boron drug is delivered to the tumor tissue by the homologous targeting property of the cell membrane itself, so that the boronized cell membrane has high biocompatibility and helps to better achieve BNCT treatment.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medicine, in particular to a boronated cell membrane and a preparation method and application thereof. BACKGROUND

[0002] Malignant tumor is one of the major diseases threatening human life and health. At present, the three major means of treating tumors in clinic are surgery, chemotherapy and radiotherapy. Among them, surgery is an effective means for treating primary tumors, but it is limited to cancer cells that can be reached by surgery, and cancer cells may not be completely removed. Chemotherapy is to use chemical drugs to fight cancer, and through systemic administration, the drug circulates in the body to kill rapidly dividing cells, especially cancer cells, but due to the toxicity of the drug to normal cells, the side effects are usually significant, and cancer cells can 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, and is usually used as a complementary treatment method, such as to eliminate residual cancer cells after surgery. However, traditional radiotherapy can cause damage to normal tissues near the cancer cells or within the radiation field.

[0003] Boron neutron capture therapy (BNCT) as a radiotherapy method for treating malignant tumors has received extensive attention in recent years. BNCT is a method of killing tumor cells by using a boron compound with tumor cell selectivity 10 B atom and low-energy thermal neutrons / epithermal neutrons to produce α particles and 7 Li nuclei, where the radiation range of α particles is limited to within 10 μm, which is close to the diameter of a cell, so as to produce effective tumor cell killing. The treatment logic of BNCT is different from that of traditional radiotherapy. Only cells that have absorbed a high concentration of 10 B will undergo a lethal nuclear reaction, while the neutron capture cross section of common atomic nuclei in the human body (such as 12 C and 14 N) is much smaller than that of 10 B, so normal cells, including healthy tissues similar to tumors, are less damaged.

[0004] Although BNCT has been clinically applied to the treatment of malignant brain tumors, malignant melanoma, head and neck cancer, and liver cancer, scientists have encountered some problems with boron compounds in the clinical application of BNCT. Many kinds of boron compounds have been reported for BNCT, including amino acids, nucleic acids and liposomes as boron delivery carriers, but only boron phenylalanine (BPA) and mercapto-decahydrododecaborate disodium ([B 12 H n SH] 2-Compounds 2Na and BSH are used clinically in benign neoplastic cytokines (BNCT) therapy for cancer. BPA is an essential amino acid (phenylalanine) analogue that can be absorbed by tumor cells via amino acid transporters. However, as extracellular BPA concentration decreases and intracellular BPA concentration increases, efflux mechanisms expel BPA from the cells, preventing a significant difference in accumulation between normal and tumor cells. Furthermore, BPA's low boron content (approximately 5%) limits its clinical application in BNCT. In contrast, BSH has a higher boron content, good chemical stability and biocompatibility, and enhanced permeability and retention (EPR) accumulation in tumor regions, but is less abundant in normal tissues. However, BSH exists only in the intercellular space and does not enter cells. Therefore, the therapeutic effect of BSH during BNCT remains insufficient.

[0005] To overcome this limitation, numerous studies have aimed to improve the bioavailability of BSH. For example, Futamura et al. linked 1-amino-3-fluorocyclobutane-1-carboxylic acid (ACBC), a non-natural α-amino acid, to BSH, achieving LAT-mediated specific targeting of tumor cells. Further research... 18 F-mark ACBC( 18 F-ACBC) was used to monitor the metabolism and biodistribution of boron drugs. In the F98 rat glioma model, rats treated with BNCT after ACBC-BSH delivery showed a significant increase in median survival. In 2014, Michiue et al. linked BSH to various cell-penetrating peptides (CPPs). Boron accumulation in the glioma cell line (U87-MG) was time- and concentration-dependent, and BSH could enter the cell nucleus more extensively with prolonged treatment time, enhancing the killing effect on tumor cells. The team also screened a BSH-derived compound (BSH-11R) containing eight BSH residues and a poly-11 arginine structure. When U87-MG was treated at a concentration of 10 mmol / L, the boron content in the cells increased significantly. 10 The B content reached 5623.7 ng / 10 6 Compared to BSH alone, BSH-11R has a cytotoxicity that is more than 100 times higher per cell.

[0006] Since the 1990s, monosaccharide-modified drug molecules have been used in tumor treatment due to the dependence of tumor cells on enhanced glucose metabolism: a) improved drug delivery efficiency: due to the differences in the types and structures of glycoproteins and glycolipids on the surface of tumor cells from normal cells, monosaccharide-modified drugs designed for these changes can selectively target tumor cells, thereby increasing drug concentration in tumor sites and reducing drug accumulation in normal cells, thereby reducing toxic side effects; b) increased tumor cell uptake: due to the high metabolic requirements of tumor cells, including the need for monosaccharides such as glucose, which is manifested by the high expression of corresponding transporters, drugs combined with these monosaccharides can enter the cell through these transporters; c) potential for diagnostic applications: monosaccharide-modified diagnostic markers can also be used for in vivo diagnosis of tumor occurrence, development, distribution, and specific location, thereby improving diagnostic capabilities and providing a good foundation for subsequent tumor treatment.

[0007] However, the above method still has room for further improvement: the targeting of monosaccharide small molecule drugs is insufficient, and the targeting ability of boron drugs in tumor tissues needs to be further improved to improve treatment efficacy and reduce toxic side effects caused by off-target effects.

[0008] Therefore, the present application is proposed. SUMMARY

[0009] The present application aims to provide a boronized cell membrane and its preparation method and application to solve the above problems and achieve efficient delivery of boron drugs, ultimately meeting the requirements of BNCT treatment.

[0010] Based on this, the present application has the following technical solutions:

[0011] In a first aspect, the present application first provides a method for preparing a boronized cell membrane, comprising: adding tumor cells to a culture medium containing monosaccharide-modified boron drugs for incubation, collecting cell precipitates after incubation, and then extracting cell membranes to obtain the boronized cell membrane.

[0012] The present application uses the homologous targeting of tumor cell membranes to improve the targeting ability of boron drugs; specifically: by incubating monosaccharide-modified BSH with tumor cells, BSH can be anchored on the surface of tumor cell membranes through the sugar metabolism process, and then by extracting cell membranes, boronized cell membranes can be obtained, which have natural tumor homing ability, i.e. high tumor targeting ability, which can achieve the purpose of efficient delivery of boron drugs to tumor tissues.

[0013] In the present application, the culture medium can be the existing, conventional cell culture medium in the art, such as RPMI-1640, Minimum Essential Medium (MEM), DMEM-high glucose, DMEM-low glucose, DMEM / F12, McCoy's 5A, Iscove's Modified Dulbecco Medium (IMDM), M-199, Leibovitz Medium (L-15), Ham's F-10, Ham's F-12, etc.

[0014] Preferably, the glucose content in the culture medium is 0.5-5 g / L, for example, 0.5 g / L, 1 g / L, 1.5 g / L, 2 g / L, 2.5 g / L, 3 g / L, 3.5 g / L, 4 g / L, 4.5 g / L, and 5 g / L, etc.

[0015] It is found in the present application that when the glucose content in the culture medium is in the above range, the boron content on the boronated cell membrane is higher.

[0016] Preferably, the monosaccharide-modified boron drug is prepared from a monosaccharide derivative; the monosaccharide derivative includes a compound of any one of the following structural formulas:

[0017]

[0018] Preferably, the preparation method of the monosaccharide derivative includes: mixing 4-(N-maleimide methyl) cyclohexyl carboxylic acid, DIPEA, HATU, a solvent, and a monosaccharide and performing an amidation reaction; after the reaction is completed, purification treatment is performed to obtain the monosaccharide derivative; the monosaccharide includes acetylglucosamine, acetylazido-mannose, acetyl-fucose, or acetyl-galactosamine.

[0019] In the present application, the acetylglucosamine has a CAS number of 17460-45-6; the acetylazido-mannose has a CAS number of 1213701-11-1; the acetyl-galactosamine has a CAS number of 653600-56-7; and the acetyl-fucose has a CAS number of 115509-87-0.

[0020] More preferably, the preparation method of the monosaccharide derivative includes: mixing 4-(N-maleimide methyl) cyclohexyl carboxylic acid, DIPEA, HATU, DMF, and the monosaccharide and performing an amidation reaction at 60-80°C; after the reaction is completed, water is added to the reaction system, and the organic phase is collected after extraction with ethyl acetate, and then the crude product obtained after reduced pressure distillation is purified by a reverse phase purification instrument.

[0021] More preferably, in the purification process, phase A is acrylonitrile, phase B is 0.7-0.9% trifluoroacetic acid aqueous solution; the gradient includes: starting from 10 vol% phase B, gradually increasing to 90 vol% phase B.

[0022] More preferably, the molar ratio of 4-(N-maleimide methyl) cyclohexyl carboxylic acid and 1,3,4,6-tetra-O-acetyl-B-D-glucosamine is (1-1.2):(1-1.2).

[0023] As a preferred technical solution of the present application, the preparation method of the single derivative comprises: mixing 90-110 μmol of 4-(N-maleimide methyl) cyclohexyl carboxylic acid, 290-310 μmol of DIPEA, 110-130 μmol of HATU and DMF, stirring at 20-30°C for 20-40 min to obtain a mixed solution; then adding 90-110 μmol of the monosaccharide to the mixed solution, and stirring at 60-80°C overnight to carry out an amidation reaction; after the reaction is completed, water is added to the reaction system, and the organic phase is collected after extraction with ethyl acetate, and then the obtained crude product is subjected to reduced pressure distillation and then purified by a reverse phase purification instrument; in the purification process, phase A is acrylonitrile, phase B is 0.7-0.9% trifluoroacetic acid aqueous solution; the gradient includes: starting from 10 vol% phase B, gradually increasing to 90 vol% phase B.

[0024] As a preferred, in the monosaccharide modified boron drug, the boron drug is disodium mercaptodecahydrododecaborate.

[0025] As a preferred, the monosaccharide modified boron drug comprises any one of the following structural formulae:

[0026]

[0027]

[0028] As a preferred, the preparation method of the monosaccharide modified boron drug comprises: mixing the monosaccharide derivative, DIPEA, BSH and a solvent, and carrying out a mercapto addition reaction at 40-60°C; after the reaction is completed, the monosaccharide modified boron drug is obtained after purification treatment.

[0029] More preferably, after the reaction is completed, water is added to the reaction system, and the organic phase is collected after extraction with ethyl acetate, and then the obtained crude product is subjected to reduced pressure distillation and then purified by a reverse phase purification instrument.

[0030] More preferably, in the purification process, phase A is acrylonitrile, phase B is 0.7-0.9% trifluoroacetic acid aqueous solution; the gradient comprises: starting from 10 vol% phase B, gradually increasing to 90 vol% phase B; more preferably, the molar ratio of monosaccharide and BSH is (1-1.2):(1-1.2).

[0031] As a preferred technical solution of the present application, the preparation method of the monosaccharide-modified boron drug comprises: mixing 0.05-0.15 mmol of monosaccharide derivative, 0.25-0.35 mmol of DIPEA, 0.05-0.15 mmol of BSH, THF and DMF, and performing a thiol addition reaction at 40-60°C; after the reaction is completed, purification treatment is performed to obtain a monosaccharide-modified boron drug; preferably, after the reaction is completed, water is added to the reaction system, and after extraction with ethyl acetate, the organic phase is collected, and then the obtained crude product is distilled under reduced pressure, and then purified by a reverse phase purification instrument; in the purification process, phase A is acrylonitrile, phase B is 0.7-0.9% trifluoroacetic acid aqueous solution; the gradient comprises: starting from 10 vol% phase B, gradually increasing to 90 vol% phase B.

[0032] In a second aspect, the present application provides a boronized cell membrane prepared by the above-mentioned preparation method.

[0033] In a third aspect, the present application provides the use of the boronized cell membrane in the preparation of a BNCT drug.

[0034] In a fourth aspect, the present application provides a boron delivery preparation comprising the boronized cell membrane.

[0035] In the present application, when the boron delivery preparation is used in BNCT treatment, it is not limited to injection methods, and can include intravenous injection, intravenous infusion, intraperitoneal injection, intramuscular injection, subcutaneous injection, intradermal injection, intratumoral injection, etc.

[0036] In a fifth aspect, the present application provides an antitumor drug comprising the boronized cell membrane.

[0037] In a sixth aspect, the present application provides the use of the boronized cell membrane in BNCT treatment.

[0038] The boronized cell membrane and the preparation method and application thereof provided by the present application can anchor the obtained boron drug on the tumor cell membrane by using the monosaccharide-modified BSH, and then deliver the boron drug to the tumor tissue through the homologous targeting of the cell membrane itself, and have high biocompatibility, which helps to better achieve BNCT treatment. BRIEF DESCRIPTION OF DRAWINGS

[0039] In order to make the technical solutions in the present application or prior art clearer, the accompanying drawings needed in the embodiments or prior art description will be briefly introduced. Obviously, the accompanying drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without any creative effort based on these drawings.

[0040] Figure 1 is a MALDI-TOF MS graph of the monosaccharide-modified boron drug (glu-BSH) described in Example 1 of the present application. 1 H-NMR graph.

[0041] Figure 2 is a MALDI-TOF MS graph of (2R, 3S, 4S, 5R, 6S)-6-(acetoxymethyl)-3-(4-((2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl)methyl)cyclohexane-1-carboxamido)tetrahydro-2H-pyran-2, 4, 5-triyl triacetate in Example 1 of the present application.

[0042] Figure 3 is a MALDI-TOF MS graph of the monosaccharide-modified boron drug (glu-BSH) described in Example 1 of the present application.

[0043] Figure 4 is the cytotoxicity result of glu-BSH in Example 1 of the present application.

[0044] Figure 5 is the boron content result of the cell membrane obtained by incubating glu-BSH under different conditions provided by the present application.

[0045] Figure 6 is the boron content result of tumor cells after uptaking the boronized cell membrane under different concentrations provided by the present application.

[0046] Figure 7 is the boron content result of tumor, blood and muscle at different time points after the boronized cell membrane is given to tumor-bearing mice provided by the present application. DETAILED DESCRIPTION

[0047] In order to make the objects, technical solutions and advantages of the present application clearer, the technical solutions in the present application will be described clearly and completely below in conjunction with the drawings in the present application. Obviously, the described embodiments are only some of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative effort should fall into the scope of protection of the present application.

[0048] The tumor cell membrane extraction kit used in the following examples is Biyun Tian P0033.

[0049] Example 1

[0050] The present embodiment provides a boronized cell membrane, and a preparation method thereof comprises the following steps:

[0051] The tumor cell line (4T1-luc) is passaged into a 10 cm culture dish at a relatively sparse density, and about 15 mL of DMEM medium containing glu-BSH at concentrations of 50, 100, 200 and 500 μg / mL is added. Incubation is carried out at 37°C and 5% CO2 for 24 hours. After the incubation is completed, the cells are trypsinized and counted, and the cell precipitate is collected. According to the kit instructions, the cell membrane is extracted, and the specific operation is as follows: the cell precipitate is gently resuspended with an appropriate amount of PBS pre-cooled in an ice bath, a small amount of cells are taken for counting, and the remaining cells are centrifuged at 600g for 5 minutes at 4°C to precipitate the cells. Discard the supernatant, then centrifuge at 600g for 1 minute at 4°C to precipitate the residual liquid on the wall of the centrifuge tube and further precipitate the cells, and try to absorb as much residual liquid as possible. Add 1 mL of membrane protein extraction reagent A added with PMSF before use to 200-500 million cells, gently and thoroughly suspend the cells, and place them in an ice bath for 10-15 minutes. Add 1 mL of membrane protein extraction reagent A added with PMSF before use to 200-500 million cells, gently and thoroughly suspend the cells, and place them in an ice bath for 10-15 minutes. If a shiny ring around the nuclei or intact cell morphology is observed, it indicates that the cells are still intact. If 70-80% of the cells do not have a shiny ring around the nuclei and intact cell morphology, it indicates that the cells have been sufficiently broken, and the next step is performed. If the degree of cell breakage is less than 70%, the number of freeze-thaw cycles can be increased until the degree of cell breakage is greater than 70%. Centrifuge at 700g for 10 minutes, and carefully collect the supernatant into a new centrifuge tube. About 30-50 microliters of supernatant can be left unabsorbed to ensure that the absorbed supernatant has high purity. Centrifuge at 14000g for 30 minutes to precipitate the cell membrane fragments.

[0052] The DMEM low-sugar culture medium (Milen Biotech MA0577) contains glucose (1000 mg / L), L-glutamine (4 mM), and sodium pyruvate (110 mg / L).

[0053] The preparation method of glu-BSH is as follows:

[0054] The glucose derivative has the following structural formula:

[0055] The synthesis method includes:

[0056] (1) Experimental materials and equipment

[0057] The main reagents and instrument equipment used in this experiment are listed in Table 1 and Table 2 as follows:

[0058] Table 1 List of chemical reagents and solvents required for the experiment

[0059]

[0060] Table 2 List of instruments required for the experiment

[0061]

[0062] (2) Synthesis path

[0063] The synthesis path of (2R, 3S, 4S, 5R, 6S)-6-(acetoxymethyl)-3-(4-((2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) methyl) cyclohexane-1-carboxamido) tetrahydro-2H-pyran-2, 4, 5-triyltriacetate is shown below:

[0064] (3) Synthesis steps

[0065] Synthesis of (2R,3S,4S,5R,6S)-6-(acetoxymethyl)-3-(4-((2,5-dioxo-2,5-dihydro-1H- pyrrol-1-yl)methyl)cyclohexane-1-carboxamido)tetrahydro-2H-pyran-2,4,5-triyl triacetate: 4-(N-maleimido methyl) cyclohexylcarboxylic acid (23.7 mg, 100 pmol), DIPEA (21.9 mg, 300 pmol), HATU (45.6 mg, 120 pmol) and DMF (1 mL) were added to an 8 mL sample vial and the reaction was stirred at room temperature for 30 min. After adding 1,3,4,6-tetra-O-acetyl-B-D-glucosamine (34.7 mg, 100 pmol) to the above system, the reaction was stirred at 70 °C overnight. After the reaction was completed, water (2 mL) was added to the system, and the organic phase was collected after extraction with ethyl acetate (2 x 5 mL) and distilled under reduced pressure by a rotary evaporator. The crude product obtained after purification by a reverse phase purification instrument. In the purification process, phase A was acetonitrile, phase B was 0.8% trifluoroacetic acid aqueous solution; the gradient included: from 10 vol% phase B, gradually increased to 90 vol% phase B, the collected product was freeze-dried to obtain white solid (2R,3S,4S,5R,6S)-6-(acetoxymethyl)-3-(4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1- yl)methyl)cyclohexane-1-carboxamido)tetrahydro-2H-pyran-2,4,5-triyl triacetate (23 mg, 40.6 pmol), the yield was 40.6%. Ms = [M-H] = 565.

[0066] 1 H NMR (400 MHz, DMSO-d6) d, ppm: 7.85-7.84 (d, 1H), 7.901 (s, 2H), 5.72-5.70 (d, 2H), 5.18-5.14 (m, 2H), 4.90-4.88 (m, 1H), 4.20-4.16 (m, 2H), 4.00-3.92 (m, 3H), 3.23-3.22 (m, 2H), 2.02-2.01 (d, 6H), 1.98 (s, 3H), 1.89 (s, 3H), 1.62-1.57 (m, 4H), 1.52-1.51 (m, 1H), 1.24-1.20 (m, 2H), 0.92-0.84 (m, 2H).

[0067] (4) Characterization

[0068] Nuclear magnetic resonance hydrogen spectrum: 5-10 mg of sample was weighed and dissolved in deuterated dimethyl sulfoxide, and the sample was measured by using a 400Mz Bruker AVANCE nuclear magnetic resonance spectrometer. 1 H NMR nuclear magnetic hydrogen spectrum was analyzed by using MestReNova v5.3.1 software to determine the structure of the molecule. The characterization results are shown in Figure 1 .

[0069] Matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF MS): The target product was configured into a 1 mg / mL methanol solution, and the sample was tested for relative molecular mass. The characterization results are shown in Figure 2 .

[0070] The monosaccharide-modified boron drug (glu-BSH) is obtained through the following reaction path:

[0071]

[0072] The synthesis steps include: (2R, 3S, 4S, 5R, 6S)-6-(acetoxymethyl)-3-(4-((2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) methyl) cyclohexane-1-carboxamido) tetrahydro-2H-pyran-2, 4, 5-triyl triacetate (56.6 mg, 0.1 mmol, DIPEA (303 mg, 0.3 mmol) was dissolved in THF (1 mL) and DMF (1 mL) and added to an 8 mL sample bottle, and finally BSH (22.2 mg, 0.1 mmol) was added. The reaction was stirred at 50°C overnight. After the reaction was completed, water (2 mL) was added to the system, extracted with ethyl acetate (2 x 5 mL), and the organic phase was collected. After distillation under reduced pressure by a rotary evaporator, the obtained crude product was purified by a reverse phase purification instrument. In the purification process, phase A was acrylonitrile, and phase B was 0.8% trifluoroacetic acid aqueous solution; the gradient included: starting from 10 vol% phase B, gradually increasing to 90 vol% phase B, and the collected product was freeze-dried to obtain a light yellow solid target product (50 mg, 67.5 μmol) after low temperature freeze-drying, with a yield of 67%. Ms = [M-H] = 739.

[0073] The characterization results are shown in Figure 3 .

[0074] Example 2

[0075] This example provides a boronized cell membrane, and the difference between its preparation method and example 1 is only that the incubation time is 48 h.

[0076] Example 3

[0077] This example provides a boronized cell membrane, the preparation method of which is only different from that of Example 1 in that the incubation time is 72 h.

[0078] Example 4

[0079] This example provides a boronized cell membrane, the preparation method of which is only different from that of Example 1 in that the DMEM low-sugar medium in Example 1 is replaced by a DMEM high-sugar medium (Meilunbio MA0212) containing glucose (4500 mg / L), glutamine (584 mg / L), sodium pyruvate (110 mg / L), and phenol red (15 mg / L).

[0080] Example 5

[0081] This example provides a boronized cell membrane, the preparation method of which is only different from that of Example 4 in that the incubation time is 48 h.

[0082] Example 6

[0083] This example provides a boronized cell membrane, the preparation method of which is only different from that of Example 4 in that the incubation time is 72 h.

[0084] Example 7

[0085] This example provides a boronized cell membrane, the preparation method of which is only different from that of Example 1 in that the DMEM medium is a DMEM high-sugar medium (Meilunbio MA0212) containing glucose (4500 mg / L), glutamine (584 mg / L), sodium pyruvate (110 mg / L), and phenol red (15 mg / L), and additionally supplemented with glucose, with a final glucose concentration of (5000 mg / L).

[0086] It is found in the present application that, within the same incubation time, the boron content on the boronized cell membrane obtained when the medium containing the monosaccharide-modified boron drug has a glucose content of 5000 mg / L is lower than that of Example 4. That is, as the glucose content in the medium increases, the boron content on the boronized cell membrane gradually decreases. The test results show that, within 0.5-5 g / L, the boron content on the boronized cell membrane is relatively high; more preferably, when the glucose content is 0.5-4.5 g / L, the boron content on the boronized cell membrane has a significant effect advantage.

[0087] In addition, when tumor cells are added to the medium containing other monosaccharide-modified boron drugs defined in the present application for incubation, the boronized cell membrane obtained can also efficiently deliver the boron drug to the tumor tissue, achieving the requirement of BNCT treatment, i.e., having an effect comparable to that of Example 1 under the same incubation conditions.

[0088] Test Example

[0089] 1. Cytotoxicity of glu-BSH

[0090] K7M2-luc cells were first seeded in 96-well plates at 5000 cells per well and allowed to adhere overnight. The glu-BSH was dissolved in PBS solution, filtered by 0.22 μm sterile filter membrane, and added to the cell supernatant at concentrations of 0, 50, 100, 200, 500, and 1000 μg / mL, and incubated for 24 h. After completion, the supernatant was discarded, and the cells were washed three times with PBS, and then CCK-8 was added to the DMEM basic medium, and incubated at 37°C for about 30 min, and a blank well (without cells) was set. After completion, the absorbance at 450 nm was detected by a microplate reader, and quantitative analysis was performed, and the results are shown in Figure 4 The data show that the cytotoxicity of glu-BSH is low.

[0091] 2. Determination of boron content on cell membrane

[0092] The boron content of the boronized cell membrane of Examples 1-6 was determined after digestion with concentrated nitric acid, and the cell number was used as a normalization standard, and the results are shown in Figure 5 The data show that the boron content on the cell membrane under low glucose medium is higher at the same incubation time.

[0093] 3. Incubation of tumor cells with boronized cell membrane

[0094] The tumor cell line (4T1-luc) was seeded in a 6-well plate and allowed to adhere overnight. The boronized cell membrane collected after incubation of 1,3,4,6-tetra-O-acetyl-B-D-glucosamine modified mercaptodecahydrododecaborate disodium at 100 μg / mL in DMEM low glucose medium for 72 h was added to the 4T1-luc cells at boron concentrations of 2, 5, 10, and 20 μg / mL, and incubated for 24 h. After completion, the supernatant was discarded, and the cells were washed with PBS and collected. After digestion, the boron content in the cells was determined by ICP-MS, and the results are shown in Figure 6 The results show that as the incubation concentration increases, the boron content in the cells also increases, and reaches a plateau at a concentration of 10 μg / mL.

[0095] 4. In vivo targeting verification of boronized cell membrane

[0096] A 4T1-luc subcutaneous tumor model was established, and each mouse was inoculated with 10 6 cells. The boronized cell membrane collected from about 10 7The boronated cell membranes extracted from 4T1-luc cells were injected into mice. The mice were sacrificed at 1 h, 3 h, 6 h, 12 h, 24 h, 48 h and 72 h, respectively. The tumor, blood and muscle tissues of the mice were collected and digested. The boron content was determined by ICP-MS. The results are shown in Table 1. Figure 7 The results show that the boron content in the tumor reached 35 ppm at 1 h, which exceeded the standard (20 ppm) for BNCT treatment. Meanwhile, the boron content in the blood and muscle was very low, indicating that the boronated cell membranes had high tumor targeting, thereby reducing the side effects of treatment.

[0097] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present application, and not to limit it. Although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that they can still modify the technical solutions described in the foregoing examples, or make equivalent substitutions for some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A method for preparing a boronized cell membrane, characterized in that, include: Tumor cells were added to a culture medium containing a monosaccharide-modified boron drug and incubated. After incubation, the cell pellet was collected, and the cell membrane was then extracted to obtain the boronized cell membrane. The monosaccharide-modified boron drug was prepared by means of a monosaccharide derivative; the structural formula of the monosaccharide derivative is: The structural formula of the monosaccharide-modified boron drug is as follows:

2. The method for preparing the boronized cell membrane according to claim 1, characterized in that, The glucose content in the culture medium is 0.5–5 g / L.

3. The method for preparing boronized cell membrane according to claim 1, characterized in that, The preparation method of the monosaccharide derivative includes: mixing 4-(N-maleimidemethyl)cyclohexylcarboxylic acid, DIPEA, HATU, DMF and the monosaccharide and carrying out an amidation reaction at 60-80°C; after the reaction is completed, water is added to the reaction system, and the organic phase is collected after extraction with ethyl acetate. The crude product obtained by vacuum distillation is then purified by a reverse purification instrument.

4. The method for preparing boronized cell membrane according to claim 3, characterized in that, In the purification process, phase A is acrylonitrile, and phase B is a 0.7-0.9% trifluoroacetic acid aqueous solution; the gradient includes: starting from 10 vol% phase B and gradually increasing to 90 vol% phase B.

5. The method for preparing boronized cell membrane according to claim 3, characterized in that, The molar ratio of 4-(N-maleimidemethyl)cyclohexylcarboxylic acid to monosaccharide is (1-1.2):(1-1.2).

6. The method for preparing boronized cell membrane according to claim 1, characterized in that, The monosaccharide derivative, DIPEA, BSH and solvent were mixed and subjected to a thiol addition reaction at 40-60°C; after the reaction, the mixture was purified to obtain the monosaccharide-modified boron drug.

7. The method for preparing boronized cell membrane according to claim 6, characterized in that, After the reaction was completed, water was added to the reaction system, and the organic phase was collected after extraction with ethyl acetate. The crude product obtained by vacuum distillation was then purified by a reverse purification system.

8. The method for preparing boronized cell membrane according to claim 7, characterized in that, In the purification process, phase A is acrylonitrile, and phase B is a 0.7-0.9% trifluoroacetic acid aqueous solution; the gradient includes starting from 10 vol% phase B and gradually increasing to 90 vol%.

9. A boronized cell membrane, characterized in that, It is prepared by the preparation method according to any one of claims 1 to 8.

10. The use of the boronized cell membrane according to claim 9 in the preparation of BNCT drugs.

11. A boron delivery formulation, characterized in that, Includes the boronized cell membrane as described in claim 9.

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