Diagnosis and treatment integrated tumor efficient targeting boron carrier and preparation and application thereof

Abstract

The invention discloses a diagnosis and treatment integrated efficient tumor targeting boron carrier, a preparation method thereof and application of the boron carrier in preparation of anti-tumor drugs and magnetic resonance imaging contrast agents in the field of BNCT. The diagnosis and treatment integrated efficient tumor targeting boron carrier is obtained by condensing chelating agent molecules containing a plurality of carboxyl groups and boron phenylalanine amide and then chelating a developing agent by using the carboxyl groups of boron phenylalanine. The preparation method comprises the following steps: carrying out carboxyl protection reaction on boron phenylalanine to obtain carboxyl-protected boron phenylalanine; carrying out an amide condensation reaction on the carboxyl-protected boron phenylalanine and a chelating agent molecule containing a plurality of carboxyl groups to obtain a carboxyl-protected condensation product; removing carboxyl protection from a condensation product to obtain boron phenylalanine-chelating agent molecules; the boron phenylalanine-chelating agent molecule is chelated with a developing agent by using carboxyl of boron phenylalanine on the boron phenylalanine-chelating agent molecule, so that the diagnosis and treatment integrated efficient tumor targeting boron carrier is obtained.

Description

Technical Field

[0001] The present invention relates to the field of diagnostic and therapeutic preparations, and particularly to a tumor highly targeted boron carrier with both therapeutic molecules and imaging functions, a preparation method thereof, and an application in Boron Neutron Capture Therapy (BNCT). Background Art

[0002] As a new generation of cell-level precise binary targeted tumor radiotherapy technology, Boron Neutron Capture Therapy combines biotargeting and heavy ion effects to selectively and precisely kill tumor cells at the cell scale. BNCT involves the interaction of two elements: boron and thermal neutrons. The boron carrier preferentially accumulates in tumor cells, and then the area or tumor is irradiated with a thermal neutron beam or an epithermal neutron beam. The neutrons interact with boron-10 (which preferentially accumulates in cancer cells) to produce high-energy alpha particles and recoil 7-Li atomic nuclei, and these atomic nuclei release energy along the path of one cell diameter, thereby selectively destroying cancer cells from the inside. The therapeutic effect of BNCT depends on 10 whether B atoms can be highly selectively and highly concentrated in tumor cells. The research and clinical transformation of boron-containing targeted drugs are the key issues to be solved for the progress and development of BNCT technology, and are crucial for improving the efficacy of BNCT, expanding the indications, and increasing the survival rate of patients, etc. The therapeutic effect of BNCT depends on 10 whether B atoms can be highly selectively and highly concentrated in tumor cells. The key performance indicators of boron-containing drugs are the 10 B concentration ratio of tumor / normal tissue (T / N) and tumor / blood (T / B) is greater than 3:1, and each tumor cell contains at least 10 9 pieces 10 B atoms, so as to reduce the side effects of treatment while ensuring the killing effect of BNCT on tumor cells.

[0003] So far, two important boron compounds, boronophenylalanine (4-boron-L-phenylalanine, BPA) and mercaptoundecaborate dodecaborate-10B (BSH), have been widely used in BNCT clinical trials. In addition, clinical studies of BNCT have been widely reported and are ongoing globally. These trials cover a range of cancers, including glioblastoma, head and neck cancer, and melanoma. The active research and development of new boron compounds and the introduction of accelerators as neutron sources that can be installed in hospitals highlight the status of BNCT as a novel and effective radiotherapy modality, bringing hope for the future of cancer treatment. Among them, the structure of BPA is similar to tyrosine and participates in the synthesis of specific proteins. Therefore, during the rapid proliferation of tumors, it selectively accumulates in proteins related to tumor growth, thus selectively aggregating in tumor cells. The amino acid transporter (LAT1) causes the bidirectional transport of BPA, and finally reaches an equilibrium concentration. Therefore, inhibiting the efflux of BPA is also a research focus. In addition, because BPA is poorly soluble in water, it is often clinically administered intravenously as a complex with fructose, BPA-F.

[0004] Sialic acid is a family of negatively charged nine-carbon monosaccharides that cover glycan chains on glycoproteins and glycolipids on the cell membrane. A key feature of malignant tumors is the upregulation of sialic acid glycans on the surface of cancer cells, a process called hypersialylation. Sialoglycans on tumor cells can participate in tumor cell-extracellular matrix interactions and tumor cell-cell interactions, and can also be used as drug recognition targets for tumor cells.

[0005] In addition, for patients preparing to receive BNCT treatment, it is necessary to perform planning design and optimization from a dosimetric perspective in the treatment planning system based on medical imaging data to evaluate and ensure the effectiveness and safety of BNCT treatment for patients. The distribution data of boron drug dose in the body has a significant impact on the results of BNCT dose calculation. When the currently widely used BNCT treatment planning system designs treatment plans, almost all assume that the concentration of the targeted boron-containing drug in each tissue and organ is evenly distributed, and the ratio of boron concentration in the tumor to that in the blood is also set according to a constant value, ignoring the pharmacokinetic changes caused by the specific uptake and metabolism of different cells of different individuals to the targeted boron-containing drug, resulting in certain deviations in dose calculation. Therefore, it is necessary to visualize the drug in the body. This study combines nuclear magnetic resonance (MRI) technology with BNCT to achieve visual tracking of boron drugs in the body, that is, a new boron carrier for integrated diagnosis and treatment. Summary of the Invention

[0006] The present invention provides a tumor highly targeted boron carrier for integrated diagnosis and treatment (new integrated diagnosis and treatment molecule), its preparation method and application.

[0007] The object of the present invention is to synthesize a new type of integrated diagnosis and treatment molecule for efficient targeting of tumors in view of the deficiencies of boron drugs in the prior art. The carboxyl groups of the chelating agent molecule containing multiple carboxyl groups are subjected to amide bonding with the amino groups of the clinically used BPA molecule, and then the carboxyl groups carried by BPA are used to replace the original carboxyl groups of the chelating agent to chelate the imaging agent. Thanks to the phenylboronic acid groups contained in BPA itself, several phenylboronic acid groups are exposed on the surface of the new type of integrated diagnosis and treatment molecule for targeting. First, compared with BPA-F, it can avoid the efflux and saturation phenomena caused by LAT1 transport; secondly, through the multivalent binding of phenylboronic acid and sialic acid, targeting is achieved.

[0008] Since the new type of integrated diagnosis and treatment tumor highly targeted boron carrier molecule of the present invention is a small molecule, similar in molecular weight to 9-glycoside sialic acid, there is a possibility of spatial binding, meeting the requirements of steric hindrance and chemical bond binding. And several phenylboronic acid groups outside the new type of integrated diagnosis and treatment molecule are evenly and spaced on the molecular surface, and may connect two adjacent sialic acid glycoside chains, or connect to different glycosides of a sialic acid chain at the same time, so as to achieve more tight and efficient sialic acid specific recognition. Compared with nanoparticles with surface sialic acid, it has more advantages in steric hindrance. The new type of integrated diagnosis and treatment molecule in the present invention achieves the purpose of integrated tumor diagnosis and treatment while realizing more efficient tumor targeting ability and tumor cell accumulation ability, and further moves towards personalized precision medicine.

[0009] The specific technical solutions are as follows:

[0010] [1] An integrated diagnosis and treatment tumor highly targeted boron carrier, obtained by condensing a chelating agent molecule containing multiple carboxyl groups with boron phenylalanine amide and then chelating an imaging agent with the carboxyl group of boron phenylalanine.

[0011] In some embodiments, the chelating agent molecule containing multiple carboxyl groups contains more than three carboxyl groups, and specifically may include at least one of DO3A (1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid sodium salt), DTPA (diethylenetriaminepentaacetic acid), BOPTA (beptumide), DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), etc.

[0012] The imaging agent has an imaging function. In some embodiments, the imaging agent may include at least one of a gadolinium (Gd)-based imaging agent, an iron-based imaging agent, a manganese-containing imaging agent, a copper-containing imaging agent, a calcium-containing imaging agent, and a magnesium-containing imaging agent. When the imaging agent includes a gadolinium-based imaging agent, the gadolinium-based imaging agent contains Gd 3+, the tumor high-efficiency targeted boron carrier for integrated diagnosis and treatment is obtained by condensing a chelating agent molecule containing multiple carboxyl groups with borophenylalanine amide and then chelating Gd in the imaging agent using the carboxyl group of borophenylalanine. 3+ Obtained.

[0013] The above-listed chelating agents and imaging agents have great structural similarity and meet the functional group and structural requirements in the present invention. Through the combination of each chelating agent and imaging agent, the object of the present invention can be achieved to varying degrees.

[0014] The present invention uses the boron drug borophenylalanine (BPA) used clinically, which can be at least one of ordinary boron-abundant BPA, boron-10 enriched BPA, etc.

[0015] [2] The preparation method of the tumor high-efficiency targeted boron carrier for integrated diagnosis and treatment according to [1] includes the steps:

[0016] (1) Carry out a carboxyl protection reaction on borophenylalanine to obtain carboxyl-protected borophenylalanine;

[0017] (2) Carry out an amide condensation reaction between the carboxyl-protected borophenylalanine and a chelating agent molecule containing multiple carboxyl groups to obtain a carboxyl-protected condensation product;

[0018] (3) Remove the carboxyl protection from the condensation product to obtain a borophenylalanine-chelating agent molecule;

[0019] (4) Chelate the imaging agent using the carboxyl group of borophenylalanine on the borophenylalanine-chelating agent molecule to obtain the tumor high-efficiency targeted boron carrier for integrated diagnosis and treatment.

[0020] In some embodiments, in step (1), the carboxyl group of borophenylalanine can be protected using isobutene, which specifically can include: adding sulfuric acid and isobutene to the dichloromethane mixture of borophenylalanine under stirring, maintaining the stirring reaction, extracting and washing the organic layer after the reaction ends, then drying and filtering, and concentrating the organic layer under reduced pressure by rotary evaporation to obtain carboxyl-protected borophenylalanine.

[0021] In some embodiments, in step (2), the molar ratio of the carboxyl-protected borophenylalanine to the chelating agent molecule containing multiple carboxyl groups can be 1-5:1, such as 2:1, 3:1, 4:1, etc.

[0022] In some embodiments, step (2) can specifically include: adding an amide reaction condensing agent and carboxyl-protected borophenylalanine to the N,N-dimethylformamide (DMF) mixture of the chelating agent molecule containing multiple carboxyl groups under stirring, maintaining the stirring reaction, and concentrating under reduced pressure after the reaction ends to obtain a carboxyl-protected condensation product.

[0023] Further, the amide reaction condensing agent may include at least one of EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide), DCC (dicyclohexylcarbodiimide), ECS (chloroacetyl chloride), BOP (benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate), TBTU (2-(1H-benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate), DIPEA (N,N-diisopropylethylamine), HBTU (O-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate), etc.

[0024] In some embodiments, step (3) may specifically include: adding trifluoroacetic acid (TFA) to the dichloromethane mixture of the carboxyl-protected condensation product under stirring, maintaining the stirring reaction, and concentrating under reduced pressure after the reaction ends to obtain boron phenylalanine-chelator molecule.

[0025] In some embodiments, step (4) may specifically include: mixing the developer with the boron phenylalanine-chelator molecule, adjusting the pH to 5.5 - 7, and stirring and reacting at 55 - 65 °C (such as 60 °C, etc.), dialyzing to remove free impurities after the reaction ends, and then freeze-drying to obtain the tumor highly efficient targeted boron carrier for the integrated diagnosis and treatment. Further, the dialysis may use a dialysis bag with a molecular weight cut-off of 200 Da.

[0026] [3] Use of the tumor highly efficient targeted boron carrier for the integrated diagnosis and treatment according to [1] or the tumor highly efficient targeted boron carrier for the integrated diagnosis and treatment prepared by the preparation method according to [2] in the preparation of anti-tumor drugs in the field of BNCT.

[0027] [4] Use of the tumor highly efficient targeted boron carrier for the integrated diagnosis and treatment according to [1] or the tumor highly efficient targeted boron carrier for the integrated diagnosis and treatment prepared by the preparation method according to [2] in magnetic resonance imaging contrast agents.

[0028] The novel integrated diagnosis and treatment molecule provided by the present invention can be administered by intravenous injection and is generally made into a freeze-dried powder preparation. In addition, those skilled in the art can determine the dosage with reference to the dosage of existing anti-tumor drugs and the dosage of the developer, and adjust it up and down according to individual conditions.

[0029] The novel integrated diagnosis and treatment molecule of the present invention is a molecule with good water solubility, has high tumor cell targeting ability and accumulation effect, and contains a developer for nuclear magnetic resonance imaging. It can be used as a reagent for BNCT to make anti-tumor drugs, and at the same time as a magnetic resonance imaging contrast agent to achieve the purpose of diagnosis.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] 1. Based on the fact that the boron drug molecule BPA used clinically in the BNCT field contains a phenylboronic acid group with a targeting function for sialic acid overexpressed on the surface of tumor cells, the present invention targets tumor cells through the groups of the molecule itself to obtain a new type of therapeutic molecule, improving the targeting and accumulation ability of the boron drug for tumors.

[0032] 2. According to the characteristic that the boron drug molecule BPA used clinically in the BNCT field contains a carboxyl group, the present invention replaces the carboxyl group in the original chelating agent molecule, chelates it with the imaging agent, and obtains a drug-imaging agent chelate, enabling it to have the effect of integrated diagnosis and treatment of simultaneously killing tumor cells and magnetic resonance imaging. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 1H NMR spectrum of DOTA-BPA.

[0034] Figure 2 1H NMR spectrum of BPA-F.

[0035] Figure 3 1H NMR spectrum of the chelating agent DOTA.

[0036] Figure 4 Mass spectrum of the new integrated diagnosis and treatment molecule DOTA-BPA-Gd.

[0037] Figure 5 Infrared spectra, where: Figure A is the infrared spectrum of BPA; Figure B is the infrared spectrum of DOTA; Figure C is the infrared spectrum of DOTA-BPA; Figure D is the infrared spectrum of DOTA-BPA-Gd.

[0038] Figure 6 Affinity interaction diagram of the new integrated diagnosis and treatment molecule DOTA-BPA-Gd with sialic acid. In the figure: Figure A is the emission spectrum of BPA (0.1 M) in PBS pH = 6.5 containing different concentrations of Neu5Ac (0 - 200 mM) at room temperature, with an excitation wavelength λ ex = 302 nm; Figure B is the emission spectrum of DOTA-BPA-Gd (0.1 M) under the same conditions; Figure C is the emission spectrum of BPA at pH = 7.4; Figure D is the emission spectrum of DOTA-BPA-Gd under the same conditions; Figures E and F are the relative fluorescence of BPA and DOTA-BPA-Gd as a function of Neu5Ac concentration, measured in buffer at pH = 6.5 (E) and pH = 7.4 (F), respectively.

[0039] Figure 7Molecular simulation diagrams of the binding ability of the novel theranostic molecule DOTA-BPA-Gd, as well as BPA-F and N-glcan. In the figures: Figure A shows BPA and N-glcan placed in an 8nm×8nm×8nm water box at a molecular ratio of 1:1 for kinetic molecular docking simulation; Figure B shows the binding interaction between BPA and Neu5Ac; Figure C shows DOTA-BPA-Gd and N-glcan placed in an 8nm×8nm×8nm water box at a molecular ratio of 1:1 for kinetic molecular docking simulation; Figure D shows the binding interaction between DOTA-BPA-Gd and N-glcan.

[0040] Figure 8 Molecular simulation data diagrams of the binding ability of the novel theranostic molecule DOTA-BPA-Gd, as well as BPA-F and N-glcan. In the figures: Figure A shows the RMSD results; Figure B shows the SASA; Figure C shows the number of borate ester bonds; Figure D shows the Rg.

[0041] Figure 9 MRI in vitro imaging results diagrams of the novel theranostic molecules DOTA-BPA-Gd and DOTA-Gd. In the figures: Figure A shows the corresponding in vitro imaging images of DOTA-Gd and DOTA-BPA-Gd with a concentration range of 0.02 mM to 0.3 mM; Figure B shows the longitudinal relaxation rate (1 / T1) diagrams of DOTA-Gd and DOTA-BPA-Gd.

[0042] Figure 10 Cell uptake results diagrams of the novel theranostic molecules DOTA-BPA-Gd and BPA-F. In the figures: Figure A shows the uptake of boron-10 in the 4T1, U87, and HePa1-6 cell lines; Figure B shows the uptake of Gd in the 4T1, U87, and HePa1-6 cell lines.

[0043] Figure 11 Cell colony formation diagrams before and after BNCT treatment of the novel theranostic molecules DOTA-BPA-Gd, BPA-F, and the control group. In the figures: Figure A shows the colony formation images; Figure B shows the quantification of the number of colonies consisting of at least 50 cells (n = 3, ***p < 0.001); Figure C shows the quantification of the number of colonies of 50 cells < colony number (n = 3, ns p > 0.05).

[0044] Figure 12 DNA damage results diagrams after BNCT treatment of the novel theranostic molecules DOTA-BPA-Gd, BPA-F, and the control group. In the figures: Figure A shows the fluorescence confocal images of DNA damage; Figure B shows the fluorescence semi-quantification results in Figure A.

[0045] Figure 13MRI imaging results of tumor-bearing mice with novel theranostic molecules DOTA-BPA-Gd and DOTA-Gd. In the figure: Panel A is the in vivo MRI image; Panel B is the quantitative result in Panel A.

[0046] Figure 14 In vivo tumor targeting of novel theranostic molecules DOTA-BPA-Gd, BPA-F, and the control group. In the figure: Panel A shows the boron-10 concentrations in tumors, blood, and muscle measured by ICP-MS (n = 3, *p < 0.05, ***p < 0.001); Panel B shows the tumor-to-normal tissue ratio (T / N) and tumor-to-blood ratio (T / B) in 4T1 tumor-bearing BALB / c mice (n = 3, **p < 0.01, ***p < 0.001).

[0047] Figure 15 Tumor inhibition rate results of novel theranostic molecules DOTA-BPA-Gd, BPA-F, and the control group before and after BNCT treatment. In the figure: Panel A is the experimental flow chart of the treatment protocol; Panel B is a photo of the thermal neutron beam and the custom-made mold, as well as the relative positions of the body and the tumor; Panel C is the average tumor volume of mice in different groups (n = 8, analyzing data at 30 days after treatment, ****p < 0.0001); Panel D is the average tumor volume of each group of mice; Panel E is the average body weight of each group of mice (n = 9); the data are SD ± means. Detailed implementation manners

[0048] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The operation methods without specific conditions noted in the following embodiments are usually carried out under conventional conditions or according to the conditions recommended by the manufacturer.

[0049] The preparation method of DOTA-Gd used in the following implementation cases is introduced as follows: DOTA (50.0 mg, 124 μmol, 1.0 eq) is dissolved in 5 mL of H2O, and GdCl3·6H2O (50.5 mg, 136 μmol, 1.1 eq.) is added to the above aqueous solution under stirring. Subsequently, the pH value is adjusted to 5.5 - 7 with KOH solution (1 M). Finally, the reaction mixture is stirred at 60 °C for 24 h and dialyzed for 4 h using a dialysis bag with a molecular weight cut-off of 200 Da to obtain DOTA-Gd.

[0050] The method for obtaining BPA-F used in the following implementation cases is introduced as follows: Add 600 mg of BPA to 10 mL of water, add NaOH to adjust the alkalinity until BPA is completely dissolved. Add 570 mg of fructose, stir for 10 min, and make up 10 mL of water. Finally, add HCl to adjust the pH to 7.4, continue to stir for 10 min, and filter through a 0.45 μm filter membrane to obtain BPA-F. Figure 2 The 1H NMR results of BPA-F are given.

[0051] Implementation case 1: Preparation and synthesis of the novel integrated diagnosis and treatment molecule DOTA-BPA-Gd.

[0052] Synthesis of BPA-t-Bu:

[0053] Suspend BPA (0.105 g, 0.5 mmol, 1.0 eq) in 10 mL of dichloromethane, add 1.2 mL of sulfuric acid under stirring conditions. Stir at 20 °C for 15 min until dissolved, then add isobutene (0.55 mmol, 1.1 eq), and continue to stir at this temperature for 6 h while monitoring the reaction progress. Alkalize the reaction mixture with 10 wt% aqueous sodium bicarbonate solution (4.17 mL). Extract the above aqueous solution with EtOAc (ethyl acetate) (3 × 27 mL), and take the organic layer. Wash the combined extracts with 10 wt% aqueous sodium bicarbonate solution (3 × 20 mL) and saturated brine (3 × 20 mL) respectively, and take the organic layer. Add an appropriate amount of Na2SO4 to dry the organic layer, and filter. Concentrate the organic layer by rotary evaporation under reduced pressure to obtain carboxyl-protected borophenylalanine BPA-t-Bu.

[0054] Synthesis of DOTA-BPA-t-Bu:

[0055] Suspend DOTA (0.1 g) in 2 mL of DMF. Add 18 eq of DIPEA (0.38 mL, 3.6 mmol) to the stirred mixture. Stir the solution for 5 - 10 min. Add 4.5 eq of HBTU (0.17 g, 0.9 mmol) to the solution. Add BPA-t-Bu (0.05 g) to the mixture. Stir the mixture at room temperature for 12 h. Concentrate the mixture under reduced pressure at 60 °C to obtain the carboxyl-protected condensation product DOTA-BPA-t-Bu.

[0056] Preparation of DOTA-BPA:

[0057] Deprotect the tert-butyl ester, DOTA-BPA-t-Bu:DCM (dichloromethane):TFA (trifluoroacetic acid) = 1 mmol:4 mL:1 mL. Slowly add 1.0 mL of TFA dropwise to the reaction solution of DOTA-BPA-t-Bu and DCM, and stir at room temperature for about 4 h until the reaction is complete; rotary evaporate to remove volatile substances, and purify by column chromatography to obtain the boron-phenylalanine-chelator molecule DOTA-BPA.

[0058] Synthesis of DOTA-BPA-Gd:

[0059] Add GdCl3·6H2O (50.5 mg, 1.1 eq) to DOTA-BPA. Adjust the pH value to 5.5 - 7 with KOH solution (1 M). Stir the reaction mixture at 60 °C for 24 h. Dialyze the above reactants in a dialysis bag with a molecular weight cut-off of 200 Da for 6 h to remove free impurities. Lyophilize the obtained substance to obtain the tumor highly targeted boron carrier DOTA-BPA-Gd for integrated diagnosis and treatment.

[0060] Among them, the structure of DOTA-BPA was determined and analyzed by 1H NMR spectroscopy. As Figures 1 - 3 shown, it can be seen that compared with the reactants DOTA and BPA, DOTA-BPA has the characteristic peaks of the two at 7.20 ppm, 7.75 ppm and 3.25 ppm respectively. In addition, according to the 1H NMR spectroscopy analysis results, it is consistent with the hydrogen atom sites of DOTA-BPA. By mass spectrometry ( Figure 4 ), there is a product peak at 1324.05 m / z, indicating the successful synthesis of DOTA-BPA. In addition, through the verification of infrared spectroscopy ( Figure 5 ), the wavelength of 1420 - 1400 cm -1 is the strong stretching vibration peak of C-N, the wavelength of 1570 - 1515 cm -1 is the bending peak of NH and the stretching vibration peak of C-N, the wavelength around 3270 cm -1 is the strong stretching vibration peak of NH, indicating the presence of amide. There is an OH stretching vibration peak between 3400 - 2500 cm -1 , and the wavelength range of 1740 - 1650 cm -1 represents the stretching vibration peak of C=O, the position at 1300 cm -1 represents the stretching vibration peak of C-O, verifying the presence of carboxylic acid. The peak between 860 - 800 cm -1 represents the out-of-plane bending vibration peak of CH for para-disubstituted benzene ring, verifying the para-disubstitution of benzene ring. The peak at 550 cm -1 is the characteristic peak of boric acid, and the multiple absorption peaks at 1326 cm -1 prove the presence of boric acid in DOTA-BPA-Gd.

[0061] Case 2: Affinity of the novel theranostic molecule DOTA-BPA-Gd with sialic acid.

[0062] In pH = 6.5 and pH = 7.4 buffer media, 0 to 200 mM Neu5Ac and 0.1 M DOTA-BPA-Gd or BPA (added in the form of BPA-F) prepared according to Case 1 were added respectively. The excitation wavelength was 302 nm and the slit width was set to 1 nm. The emission scanning range was from 310 nm to 480 nm to explore the peak value and perform fluorescence intensity measurement. Since the kinetics of fluorescence quenching follows the Stern-Volmer equation: I0 / I = 1 + Kb×[Q], to determine the affinity of sialic acid with the preparation. Wherein, I0 represents the initial fluorescence intensity of DOTA-BPA-Gd without sugar, I is the fluorescence intensity in the presence of sialic acid (quencher), K b is the binding constant (M-1), and [Q] is the concentration of the quencher.

[0063] Among them, the binding affinity of DOTA-BPA-Gd with sialic acid was evaluated by steady-state fluorescence quenching measurement. Figure 6 The fluorescence spectra of DOTA-BPA-Gd and BPA-F when adding Neu5Ac at pH = 6.5 and pH = 7.4 are shown ([[]] Figure 6 A- Figure 6 D), indicating that due to the result of DOTA-BPA-Gd complexation, fluorescence quenching occurs due to photoinduced electron transfer. The binding constant is given by the slope in the Stern-Volmer plot ([[]] Figure 6 E- Figure 6 F). The binding constant of BPA is 0.56 at pH = 6.5 and 0.49 at pH = 7.4; the binding constant of DOTA-BPA-Gd is 5.86 at pH = 6.5 and 4.14 at pH = 7.4. The binding constant indicates that DOTA-BPA-Gd has a higher binding efficiency to Neu5Ac in the tumor microenvironment.

[0064] Case 3: Molecular simulation of the binding ability of the novel theranostic molecule DOTA-BPA-Gd.

[0065] The molecular force field used in the simulation was generated by Sobtop to obtain Amber force field parameters based on GAFF.

[0066] BPA and N-glycan, and DOTA-BPA-Gd prepared according to Case 1 and N-glycan were respectively placed in an 8 nm × 8 nm × 8 nm water box at a ratio of 1:1 by the number of molecules.

[0067] The simulations were all run using the Gromacs-2024.4 software package. Periodic boundary conditions were used in all simulations to keep the number of particles constant. After constructing the system, the system was subjected to 50,000 steps of energy minimization, and the energy-minimized system was subjected to 100 ps of NVT equilibration. The cutoff distance for non-bonded interactions was set to Long-range electrostatic forces were calculated by the particle mesh Ewald (PME) summation method. The temperature was maintained at 298 K with a coupling constant of 0.2 ps using V-rescale. The pressure was maintained at 1 bar using C-rescale, and the LINCS algorithm was used to constrain hydrogen bonds. The time step was set to 2 fs. Finally, a 100 ns simulation of the entire model system was performed.

[0068] The binding energies calculated by molecular simulation were basically consistent with the results of the above fluorescence quenching experiments. The N-glycan with Neu5Ac at the end was used to simulate sialic acid on the surface of tumor cells. By simulating the molecular interaction and kinetic binding of DOTA-BPA-Gd with N-glycan, the affinity of DOTA-BPA-Gd for sialic acid was analyzed ( Figure 7 A- Figure 7 D).

[0069] Root mean square deviation (RMSD) is often used to indicate whether the simulation has reached equilibrium ( Figure 8 A). The RMSD of the N-glycan-BPA group was between 0.27 and 6.17 nm, with an average value of 4.82 nm, and its value tended to be stable after 30 ns. The RMSD of the N-glycan-DOTA-BPA-Gd group was between 0.35 and 5.09 nm, with an average value of 4.42 nm, and its value tended to be stable after 10 ns. This indicates that DOTA-BPA-Gd binds to sialic acid faster, and the system tends to be stable in a shorter time.

[0070] The solvent accessible surface area (SASA, Figure 8 B) of the N-glycan-BPA group was between 30.24 and 60.19 nm 2 with an average value of 37.28 nm 2 ; the SASA of the N-glycan-DOTA-BPA-Gd group was between 43.43 and 90.15 nm 2 with an average value of 52.19 nm 2 . The SASA in both systems showed a trend of first decreasing and then stabilizing, indicating that both DOTA-BPA-Gd and BPA-F can stably bind to N-glycan in the solution system.

[0071] The number of borate ester bonds between and within the N-glycan-BPA groups ( Figure 8C) Between 0 and 23, with an average of 10.35. The number of borate ester bonds between and within the N-glycan-DOTA-BPA-Gd groups is between 1 and 26, with an average of 13.51. It can be seen that more chemical bonds are formed between DOTA-BPA-Gd and sialic acid, and they are more tightly bound to each other.

[0072] The radius of gyration (Rg) of the N-glycan-BPA group, Figure 8 D) is between 2.25 and 7.12 nm, with an average of 5.77 nm; the Rg of the N-glycan-DOTA-BPA-Gd group is between 2.85 and 5.72 nm, with an average of 5.44 nm. The Rg of the N-glycan-DOTA-BPA-Gd group reaches a steady state faster and the binding is more stable.

[0073] Generally speaking, DOTA-BPA-Gd shows significant advantages in targeting sialic acid on the surface of tumor cells. The molecular simulation results show that it has good binding potential in terms of spatial structure, meeting the requirements of steric hindrance and chemical bond binding. Further structural analysis shows that the DOTA-BPA-Gd structure is distributed with four phenylboronic acid groups, which can simultaneously bind to two adjacent sialic acid glycoside chains or connect different glycoside units on the same sialic acid chain, thus enhancing the tightness and efficiency of target recognition. Compared with nanoparticles with surface sialic acid, DOTA-BPA-Gd shows greater advantages in steric hindrance, effectively avoiding intermolecular interference and improving the binding affinity with the target. Therefore, the three-dimensional spatial configuration of DOTA-BPA-Gd enhances its binding affinity with sialic acid and further improves the targeting ability. This makes it have significant potential in improving the therapeutic effect and enhancing tumor targeting specificity, providing important theoretical support and practical significance for future personalized BNCT treatment.

[0074] Implementation Case 4: MRI in vitro imaging of the novel diagnostic and therapeutic integrated molecule DOTA-BPA-Gd.

[0075] The longitudinal relaxation time (T1) of the sample was measured using a 7T magnetic resonance. The relaxation rate was measured for the DOTA-BPA-Gd and DOTA-Gd solutions (with the same Gd concentration) prepared according to Implementation Case 1 with a concentration gradient. The parameters were echo time (Echo Time) 800 ms, repetition time (Repetition time) 750,000 ms, echo spacing 8000 ms, and rare factor 2. By plotting the relationship curve of 1 / T1 versus the corresponding Gd 3+ concentration, the relaxation coefficient r1 was obtained.

[0076] Among them, Figure 9Display DOTA-BPA-Gd and DOTA-Gd solutions with concentration gradients for MRI scanning ( Figure 9 A), to establish the linear relationship between the 1 / T value and the concentration. Then use the slope of the fitted linear curve to calculate the corresponding relaxation rate (r1). The r1 of DOTA-Gd is 5.11 mM -1 ·s -1 (R 2 = 0.9805), while the r1 of DOTA-BPA-GD is 5.14 mM -1 ·s -1 (R 2 = 0.9920)( Figure 9 B). Both have similar relaxation coefficients, and DOTA-BPA-Gd with BPA on the surface shows a contrast effect comparable to that of the clinical contrast agent DOTA-Gd.

[0077] Example 5: Cellular uptake of the novel theranostic molecule DOTA-BPA-Gd.

[0078] U87 cells and 4T1 cells were seeded in 6-well culture plates at a density of 1×10 5 / mL and incubated in a 37 °C, 5% CO2 cell culture incubator for 24 h. After the cells adhered, the seeded cells were treated with 2 mL (boron-10 concentration of 200 μg / mL) of the preparation (same boron-10 dose) (3 replicates for each concentration). After co-incubation at 37 °C, 5% CO2 for 6 h, the culture medium was removed, and the cells were washed 3 times with pre-cooled PBS (10.00 mM, pH = 7.4) at 4 °C to remove impurities on the cell surface. Then the cells were digested with trypsin, and the cells were collected and counted using a hemocytometer. 1 mL of a mixed solution of 0.1 M concentrated nitric acid and 0.3 mL of hydrogen peroxide was added to each cell sample for lysis, and wet-heated to 80 °C for digestion for more than 2 h. When the solution was completely clear and transparent, it indicated complete digestion. 3 mL of deionized water was added for dilution and volume fixation, and after filtration through a 0.22 μm microporous membrane, the boron-10 content was determined by ICP-MS, and the boron-10 uptake per unit cell of DOTA-BPA-Gd, DOTA-Gd, and BPA-F was calculated.

[0079] Among them, Figure 10 A shows that the boron-10 uptake of DOTA-BPA-Gd prepared according to Example 1 in U87 cells has no significant difference from that of BPA-F, while in 4T1 cells and Hepa1-6 cells, it is about 2 times that of BPA-F. At the same time, Figure 10B showed that the uptake of DOTA-BPA-Gd was higher than that of DOTA-Gd in the uptake of Gd, with a significant difference. The experimental results suggested that since DOTA-BPA-Gd targeted the tumor sialic acid receptor through the surface-exposed phenylboronic acid group, tumors with more surface sialic acid expression had better targeting. Therefore, 4T1 cells were selected as the research model in subsequent experiments.

[0080] Example 6: Cell colony formation after BNCT treatment with the novel theranostic molecule DOTA-BPA-Gd.

[0081] After trypsinizing U87 and 4T1 cells in the logarithmic growth phase, they were resuspended in a complete medium (1640 basal medium + 10% fetal bovine serum) to form a cell suspension, and the cells were counted. 1000 cells / well were inoculated in each experimental group in a 6-well culture plate and cultured until 14 days or until the number of cells in the vast majority of single colonies was greater than 50. The medium was changed every 3 days during the process, and the cell status was observed. After colony formation, 1 mL of 4% paraformaldehyde was added to each well to fix for 30 - 60 min, and then washed once with PBS. 1 mL of crystal violet staining solution was added to each well to stain the cells for 10 - 20 min, and the cells were washed several times with PBS and air-dried for photography.

[0082] Among them, Figure 11 the cell colony formation experiment showed that the number of colonies of DOTA-BPA-Gd prepared according to Example 1 was comparable to that of BPA-F without irradiation, while after neutron irradiation, the number of cell colonies decreased significantly. More colonies with less than 50 cells were present. It can be seen that DOTA-BPA-Gd + N (DOTA-BPA-Gd plus neutron irradiation) could significantly reduce the proliferation ability of cells, which was more than 5 times that of BPA-F. In addition, for U87 cells, a cell colony study on different irradiation durations in reactor BNCT was carried out. The number of colonies of DOTA-BPA-Gd and BPA-F was comparable at different irradiation durations, and the cell killing effect increased with the increase of irradiation duration.

[0083] Example 7: DNA damage after BNCT treatment with the novel theranostic molecule DOTA-BPA-Gd.

[0084] After irradiation, the cells were at 1×10 4The cells with the density were seeded in a confocal dish and cultured for 72 h, washed with PBS, and then fixed with 4% paraformaldehyde for 10 minutes at room temperature. After fixation, the cells were washed with PBS and treated with 5% fetal bovine serum for 30 minutes, and then exposed to a PBS solution of 0.3% Triton X-100 for 10 minutes at room temperature. After removing the supernatant, rabbit anti-rat γ-H2AX primary antibody (CST, 50 μL, 1:200) was added and incubated for another 2 hours at 37 °C. Then the cells were washed with PBS and incubated with goat anti-rabbit Alexa Fluor 647 secondary antibody (CST, 50 μL, 1:1000) for 1 hour at 37 °C. Subsequently, nuclear staining was performed using Hoechst 33342. Observation was carried out under the guidance of the reagent manufacturer's manual by confocal microscopy.

[0085] Among them, Figure 12 It is shown that since DNA damage is a common result of traditional radiotherapy, on this basis, it is explored whether the ionizing radiation of BNCT will cause damage to DNA. The expression of γ-H2AX caused by DOTA-BPA-Gd prepared according to Example 1 was significantly higher than that of BPA-F and the untreated group, proving that DOTA-BPA-Gd can cause stronger DNA damage in cells and induce apoptosis.

[0086] Example 8: MRI imaging of tumor-bearing mice with the novel diagnostic and therapeutic integrated molecule DOTA-BPA-Gd.

[0087] MRI scans were performed using a 7.0T magnet equipped with a custom-made mouse coil. 4T1 tumor-bearing mice were anesthetized with isoflurane, then fixed in the coil for background scanning, and then DOTA-BPA-Gd and DOTA-Gd (0.1 mmol / kg) prepared according to Example 1 were injected via the tail vein, and then T1-weighted images of the coronal plane and axial plane were recorded. The images were analyzed using RadiAnt DICOM Viewer.

[0088] Among them, Figure 13 It shows that the MRI contrast enhancement effect of DOTA-BPA-Gd in 4T1 tumor-bearing mice is also comparable to that of DOTA-Gd, further verifying the potential of DOTA-BPA-Gd for clinical tracking imaging. The imaging effect can be achieved at 4 - 6 h, which matches the administration and distribution time of the boron drug, and can effectively judge the distribution of the boron drug in the body and formulate a personalized BNCT treatment plan.

[0089] Example 9: In vivo tumor targeting of the novel diagnostic and therapeutic integrated molecule DOTA-BPA-Gd.

[0090] Twelve 4T1 tumor-bearing Balb / C mice were randomly divided into 3 groups: Group 1 (PBS), Group 2 (BPA), and Group 3 (DOTA-BPA-Gd prepared according to Example 1), with the same boron content in each group. A solution of different substances (5 mg / kg) was injected into the tail vein of each mouse. After 6 h, blood was collected from the posterior orbital venous plexus of the mice, and then the mice were sacrificed by cervical dislocation. The tumor tissue and muscle tissue were carefully removed, the attached fur on the surface was stripped off, and they were soaked in pre-cooled PBS solution. Subsequently, the weights of the blood, tumor, and muscle tissues were accurately weighed, and the surface moisture was carefully blotted to prevent affecting the experimental results. 1 mL of 0.1 M concentrated nitric acid and 0.3 mL of hydrogen peroxide solution were added, and digestion was carried out in a water bath at 80 °C for 2 h, followed by ultrasonic digestion in a water bath at 80 °C for 1 h, and finally ultrasonic treatment in a water bath at 80 °C for 1 h until the tissue was completely digested and became transparent and clear. The digestion solution was made up to 3 mL with deionized water, and then the digestion solution was filtered through a 0.22 μm filter membrane. The boron content in the tumor tissue was detected by ICP-MS, and the tumor targeting and its in vivo distribution were obtained by calculation using a formula.

[0091] Among them, Figure 14 A shows that the boron-10 accumulation value of BPA-F in the tumor measured by ICP-MS was 11.32 μg / g, while the accumulation of DOTA-BPA-Gd in the tumor was 19.65 μg / g, with a higher tumor accumulation. At the same time, Figure 14 B shows that the T / N and T / B ratios of DOTA-BPA-Gd are better than those of BPA-F, with stronger tumor targeting specificity. Thus, it can be seen that DOTA-BPA-Gd has more advantages in tumors with high sialic acid expression, but also maintains a boron-10 accumulation effect comparable to that of the clinically used preparation BPA-F in other tumors.

[0092] Example 10: Tumor inhibition rate after BNCT treatment with the novel theranostic molecule DOTA-BPA-Gd.

[0093] The mice used for BNCT were injected subcutaneously into the back of the neck with 4T1 cells (1×10 7 cells / 100 μL). When the tumor size reached approximately 50 - 100 mm 3After that, the mice can be used for subsequent experiments. Forty-eight mice were randomly divided into six groups: PBS, BPA-F, DOTA-BPA-Gd, PBS+N, BPA-F+N, DOTA-BPA-Gd+N (n = 8, where +N indicates neutron irradiation). A solution of different substances (5 mg / kg) was injected into the tail vein of each mouse. After 6 h, the 4T1 tumor-bearing mice were irradiated with 1.5 Gy neutrons, Gy-Phy (CBE = 1.0, RBEn = 1.0, RBEp = 1.0). Subsequently, the tumor volume and body weight of the mice were measured every two days and statistically analyzed. When the tumor size reached 2000 mm 3 or the loss was >20% of the total body weight, the mice were removed from the experimental group and euthanized.

[0094] Among them, Figure 15 It was shown that DOTA-BPA-Gd and BPA-F prepared according to Example 1 were intravenously injected into the tail vein at a boron-10 concentration of 5 mg / kg. Eight mice were in each group. After 6 h, BNCT irradiation was performed at a neutron irradiation dose of 1.5 Gy. The results showed that both the BPA-F+N and DOTA-BPA-Gd+N groups showed tumor suppression effects. Among them, the tumor suppression effect of DOTA-BPA-Gd+N was faster than that of BPA-F+N and could show a good therapeutic effect within one week. Specifically, on the 6th day, the tumor volume in DOTA-BPA-Gd+N had decreased to 1 / 10 of the initial tumor volume, which was about 1 / 4 of the BPA-F+N control group. At the same time, the later inhibition of the tumor was more significant. Finally, the tumor volume of DOTA-BPA-Gd was 1 / 3 of that of BPA-F and 1 / 2000 of the untreated group. There was no significant difference in the body weight of the mice among the groups.

[0095] In addition, it should be understood that after reading the above description of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Claims

1. An integrated diagnosis and treatment tumor highly efficient targeted boron carrier, characterized in that, It is obtained by condensing a chelating agent molecule containing multiple carboxyl groups with boron phenylalanine amide and then chelating a developer using the carboxyl group of boron phenylalanine.

2. The tumor high-efficiency targeted boron carrier for integrated diagnosis and treatment according to claim 1, wherein The chelating agent molecule containing multiple carboxyl groups includes at least one of DO3A, DTPA, BOPTA, and DOTA.

3. The tumor high-efficiency targeted boron carrier for integrated diagnosis and treatment according to claim 1, characterized in that The developer includes at least one of a gadolinium-based developer, an iron-based developer, a manganese-containing developer, a copper-containing developer, a calcium-containing developer, and a magnesium-containing developer.

4. The preparation method of the tumor highly efficient targeted boron carrier for integrated diagnosis and treatment according to any one of claims 1 to 3, characterized in that, It includes the steps: (1) Carry out a carboxyl protection reaction on boron phenylalanine to obtain carboxyl-protected boron phenylalanine; (2) Carry out an amide condensation reaction between the carboxyl-protected boron phenylalanine and a chelating agent molecule containing multiple carboxyl groups to obtain a carboxyl-protected condensation product; (3) Remove the carboxyl protection from the condensation product to obtain a boron phenylalanine-chelating agent molecule; (4) Chelate a developer using the carboxyl group of boron phenylalanine on the boron phenylalanine-chelating agent molecule to obtain the tumor highly targeted boron carrier for integrated diagnosis and treatment.

5. The preparation method according to claim 4, characterized in that In step (1), the carboxyl group of boron phenylalanine is protected using isobutene, which specifically includes: adding sulfuric acid and isobutene to a dichloromethane mixture of boron phenylalanine under stirring, maintaining the stirring reaction, extracting and washing the organic layer after the reaction ends, then drying and filtering, and concentrating the organic layer by rotary evaporation under reduced pressure to obtain carboxyl-protected boron phenylalanine.

6. The preparation method according to claim 4, wherein In step (2), the molar ratio of the carboxyl-protected boron phenylalanine to the chelating agent molecule containing multiple carboxyl groups is 1 to 5:1; Step (2) specifically includes: adding an amide reaction condensing agent and the carboxyl-protected boron phenylalanine to an N,N-dimethylformamide mixture of the chelating agent molecule containing multiple carboxyl groups under stirring, maintaining the stirring reaction, and concentrating under reduced pressure after the reaction ends to obtain a carboxyl-protected condensation product; The amide reaction condensing agent includes at least one of EDC, DCC, ECS, BOP, TBTU, DIPEA, and HBTU.

7. The preparation method according to claim 4, characterized in that, Step (3) specifically includes: adding trifluoroacetic acid to a dichloromethane mixture of the carboxyl-protected condensation product under stirring, maintaining the stirring reaction, and concentrating under reduced pressure after the reaction ends to obtain a boron phenylalanine-chelating agent molecule.

8. The preparation method according to claim 4, characterized in that, Step (4) specifically includes: mixing the developer with the boron phenylalanine-chelating agent molecule, adjusting the pH to 5.5 to 7, and carrying out a stirring reaction at 55 to 65 °C. After the reaction ends, dialyze to remove free impurities, and then lyophilize to obtain the tumor highly targeted boron carrier for integrated diagnosis and treatment; A dialysis bag with a molecular weight cut-off of 200 Da is used for dialysis.

9. Use of the tumor highly targeted boron carrier for integrated diagnosis and treatment according to any one of claims 1 to 3 or the tumor highly targeted boron carrier for integrated diagnosis and treatment prepared by the preparation method according to any one of claims 4 to 8 in the preparation of anti-tumor drugs in the field of BNCT.

10. Use of the tumor highly targeted boron carrier for integrated diagnosis and treatment according to any one of claims 1 to 3 or the tumor highly targeted boron carrier for integrated diagnosis and treatment prepared by the preparation method according to any one of claims 4 to 8 in a magnetic resonance imaging contrast agent.

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