A 157 Gd neutron embolization microspheres and methods of making and using the same

Abstract

This invention relates to the field of neutron capture therapy drugs and medical contrast agents, specifically to a... 157 Gd neutron embolization microspheres, their preparation method, and applications. The technical solution of this invention is... 157 Gd neutron embolization microspheres, including amine oxime-based porous microspheres and microspheres loaded with amine oxime-based porous microspheres. 157 Gd. Preparation method: A nitrile-containing functional monomer is grafted and polymerized onto a porous microsphere substrate to obtain porous microspheres containing polyacrylonitrile groups; the porous microspheres containing polyacrylonitrile groups are subjected to an ammonoxime reaction to convert the nitrile groups into ammonoxime groups to obtain ammonoxime-based porous microspheres; the ammonoxime-based porous microspheres are then combined with... 157 Gd coordination chelation yields 157 Gd neutron embolization microspheres. The main application of this invention is as a drug for neutron capture therapy in local tumor embolization.

Description

Technical Field

[0001] This invention relates to the field of neutron capture therapy drugs and medical contrast agents, specifically to a... 157 Gd neutron embolization microspheres, their preparation methods, and applications. Background Technology

[0002] Radiotherapy, as an important treatment for malignant tumors, has been widely used in clinical practice. For patients with intermediate to advanced liver cancer, radiation needs to penetrate normal tissue to reach the tumor site. However, the normal liver has low tolerance to radiation (it can only withstand a radiation dose of 30 Gy; exceeding 30 Gy may cause radiation hepatitis. Therefore, the dose threshold for whole-liver external beam radiotherapy to cause a decline in liver function is 30 Gy). Even with stereotactic external beam radiotherapy (SBRT), the absorbed dose delivered to the tumor tissue is only 30-80 Gy, and the probability of killing normal liver cells is relatively high. However, the absorbed radiation dose that has a lethal effect on liver tumors needs to be at least 120 Gy. Transarterial radiotherapy embolization (TARE) can reduce the local absorbed dose to the liver tumor to 100-210 Gy, or even higher (greater than 1000 Gy), while the absorbed dose to surrounding normal tissues can be controlled below 30 Gy, ensuring the safety and effectiveness of TARE.

[0003] In summary, existing liver cancer treatment technologies (mainly external beam radiotherapy) present a contradiction between the low tolerance dose of the normal liver and the high lethal dose required by the tumor, and it is difficult to balance safety and effectiveness. Summary of the Invention

[0004] To address the problems existing in the prior art, the present invention provides a... 157 Gd neutron embolization microspheres are used to address the challenge of balancing safety and efficacy in existing radiotherapy techniques for liver cancer.

[0005] The technical solution of the present invention is as follows:

[0006] This invention provides 157 Gd neutron embolization microspheres, including amine oxime-based porous microspheres and microspheres loaded with amine oxime-based porous microspheres. 157 Gd.

[0007] The present invention provides 157 Gd neutron embolization microspheres can be used as neutron capture therapy drugs for local tumor embolization or intratumoral injection, by loading Gd neutron oxime-based porous microspheres. 157 Gd, through selective coordination and porous carrier design, achieves high tumor enrichment and good in vivo retention, generating killing particles only upon thermal neutron irradiation, thus achieving targeted attack on tumors.

[0008] 157 Gd neutron embolization microspheres achieve zero radiation pollution throughout the entire process of preparation, storage, and administration. They generate high-energy gamma rays (1330 keV) and low-energy internal conversion electrons, X-rays, and Auger electrons only during the instant of thermal neutron irradiation. The high-energy gamma rays have strong penetrating power, damaging tumor cells and inhibiting tumor growth. They can propagate several centimeters within tumor tissue, with a maximum energy of up to 7.80 MeV, thus achieving localized high-energy radiation killing of tumors. Furthermore, combined with thermal neutron irradiation, a single irradiation can achieve the highest radiation dose required for tumor treatment, while significantly reducing the additional radiation exposure dose for medical staff and patients. This solves the problem of balancing safety and effectiveness in existing liver cancer radiotherapy techniques, and is of great significance for improving cancer treatment levels and patient prognosis.

[0009] 157 Gd neutron embolization microspheres can achieve the high radiation dose required for tumor lethality in a single irradiation, while controlling the dose to surrounding normal liver tissue within the tolerance threshold to avoid radiation damage. The corresponding neutron irradiation flux is 1×10⁻⁶. 9 n / cm 2 / s.

[0010] 157 Gd has the largest pure spin magnetic moment of all elements, and Gd chelates are the most common clinical MRI contrast agents for T1 MRI. 157 Gd neutron embolization microspheres combine tumor neutron capture therapy with imaging diagnosis and treatment, and have significant clinical translational potential.

[0011] Furthermore, the surface of the geminitroxime-based porous microspheres has geminitroxime groups, and these geminitroxime groups are associated with... 157 Gd formation 157 Gd coordination complex.

[0012] Furthermore, the pores of the said geminitroxime-based porous microspheres contain geminitroxime groups, and the geminitroxime groups are related to... 157 Gd formation 157 Gd coordination complex.

[0013] Introducing a methylamine oxime group onto the surface or pores of methylamine oxime-based porous microspheres, the methylamine oxime group is associated with gadolinium-157 ( 157 Gd exhibits specific coordination chelation, forming a stable gadolinium-157 coordination complex. This gadolinium-157 coordination complex has the dual function of neutron capture therapy and magnetic resonance imaging (MRI).

[0014] Geminoxime group pair 157 The specific coordination chelation of Gd can achieve highly stable loading and significantly reduce the metal content. 157This reduces the risk of Gd escape and improves in vivo safety.

[0015] 157 Gd exists in ionic or complexable form and forms stable coordination bonds with a amine oxime group.

[0016] Furthermore, the particle diameter of the said amylopyridine porous microspheres is 20 μm-200 μm.

[0017] Furthermore, the specific gravity of the said amylopyridine porous microspheres is 1.0 g / mL to 1.5 g / mL.

[0018] Preferably, the particle diameter of the amylopyridine porous microspheres is 30 μm-100 μm.

[0019] Preferably, the particle diameter of the amylopyridine porous microspheres is 35 μm-80 μm.

[0020] Preferably, the specific gravity of the geminal oxime-based porous microspheres is 1.0 g / mL to 1.3 g / mL, close to the specific gravity of blood (1.05 g / mL), to facilitate uniform distribution and embolization in the blood.

[0021] The specific diameter, specific gravity, and pore structure of amine oxime-based porous microspheres can optimize hemodynamic distribution and in vivo retention.

[0022] The present invention also provides the above. 157 The preparation method of Gd neutron embolization microspheres includes the following steps:

[0023] S1. Grafting and polymerizing a functional monomer containing nitrile groups onto a porous substrate microsphere to obtain porous microspheres containing polyacrylonitrile groups;

[0024] S2. The porous microspheres containing polyacrylonitrile groups are subjected to an ammonium oxime reaction to convert the nitrile groups into ammonium oxime groups, thereby obtaining ammonium oxime-based porous microspheres.

[0025] S3, the aforementioned amylopyridine porous microspheres and... 157 Gd coordination chelation yields 157 Gd neutron embolization microspheres.

[0026] Based on the aforementioned technical methods, using porous microspheres as the core carrier, a series of steps including graft polymerization, ammonium oxime reaction, and coordination chelation were employed to construct... 157 Gd neutron embolization microspheres. In this preparation process, the graft polymerization of nitrile-containing functional monomers achieves the functionalization modification of the microsphere surface, while the ammonium oxime reaction is... 157 The coordination chelation of Gd provides highly active sites for the amine oxime group, while 157The coordination chelation between Gd and the amine oxime group ensures efficient gadolinium loading and enhances the structural stability of the product through strong coordination interactions. 157 The preparation method of Gd neutron embolization microspheres has the technical advantages of simple and controllable process, high loading efficiency and strong product stability. It can also meet the actual needs of industrialization and clinical application, and is suitable for large-scale production and rapid preclinical labeling. It effectively overcomes the shortcomings of existing neutron therapy embolization microspheres that are difficult to adapt to the needs of industrial-scale production and rapid preclinical labeling.

[0027] Furthermore, in S1, the porous substrate microspheres are selected from polyvinyl alcohol microspheres;

[0028] The porous microspheres of the substrate were prepared by monodisperse suspension emulsion polymerization or reverse emulsion crosslinking polymerization.

[0029] Porous microspheres containing polyacrylonitrile groups were obtained by radiation graft polymerization or chemically initiated graft polymerization.

[0030] Furthermore, the method for preparing the polyvinyl alcohol microspheres includes the following steps:

[0031] Polyvinyl alcohol was added to water, and then acid was added to obtain the aqueous phase of the monodisperse suspension emulsion polymerization reaction.

[0032] Span80 was dissolved in light liquid paraffin to obtain the oil phase of the monodisperse suspension emulsion polymerization reaction;

[0033] The oil phase was heated, and then the aqueous phase was added dropwise. The temperature was increased, and then glutaraldehyde solution was added dropwise to obtain a white emulsion.

[0034] The white emulsion was centrifuged to obtain a precipitate, which was then washed to obtain polyvinyl alcohol microspheres.

[0035] Furthermore, when radiation graft polymerization is employed, the radiation source for the radiation graft polymerization is selected from a cobalt-60 γ source or an electron beam accelerator.

[0036] Furthermore, when chemically initiated graft polymerization is used, the chemical initiator is selected from cerium ammonium nitrate or persulfate initiators.

[0037] Furthermore, in S2, the ammonium oxime reaction is carried out in a solvent using hydroxylamine or hydroxylamine salt as a reagent, combined with a salt, and then combined with an acid solution or an alkaline solution;

[0038] S3 includes: mixing the metamine oxime-based porous microspheres with... 157 Gd solution mixed, so that 157 Gd coordinates with the aforementioned amine oxime group and is adsorbed and loaded, eluted, and dried to obtain... 157 Gd neutron embolization microspheres.

[0039] Furthermore, the hydroxylamine salt is selected from hydroxylamine hydrochloride;

[0040] Furthermore, the salt is selected from one or both of sodium carbonate and sodium bicarbonate.

[0041] Furthermore, the acid solution is selected from dilute hydrochloric acid solution.

[0042] Furthermore, the alkali is selected from dilute sodium hydroxide solution.

[0043] Adding a base can promote the reaction.

[0044] Furthermore, the solvent is selected from at least one of water, methanol, DMF (N,N-dimethylformamide) and DMSO (N,N-dimethyl sulfoxide).

[0045] Furthermore, the reaction temperature for the ammonium oximation reaction is 45°C. o C-80 o C, the time is 12-72 hours.

[0046] Furthermore, the aforementioned 157 Gd solution is 157 Gd salt solution or 157 The Gd complex solution was used for adsorption loading at a pH of 4-8, a temperature of 0-80 °C, and a time of 5 min-24 h.

[0047] Ideal loading efficiency and coordination stability can be obtained by setting the pH to 4-8, the temperature to 0-50 ℃, and the time to 5 min-24 h for adsorption loading.

[0048] Furthermore, in S1, the nitrile-containing functional monomer is selected from one or more of acrylonitrile, acrylic acid, itaconic acid, methacrylic acid and their derivatives;

[0049] Furthermore, the graft polymerization is carried out using one or more of the following: radiation graft polymerization, chemically initiated graft polymerization, monodisperse suspension emulsion polymerization, and reverse emulsion crosslinking polymerization.

[0050] Furthermore, the porous microspheres of the substrate are selected from one or more of the following: polystyrene microspheres, polymethyl methacrylate microspheres, polyethylene microspheres, polyacrylic acid microspheres, polymethyl methacrylate microspheres, polyethylene oxide microspheres, polyamide microspheres, polyvinyl alcohol microspheres, polyethylene glycol microspheres, chitosan microspheres and their derivatives, polylactic acid and its derivatives, sodium alginate and its derivatives, hyaluronic acid microspheres, gelatin microspheres, carrageenan and its derivatives, starch and its derivatives, and cellulose and its derivatives.

[0051] Preferably, in S1, the nitrile-containing functional monomer is acrylonitrile, a copolymer of acrylonitrile and acrylic acid, a copolymer of acrylonitrile and methacrylic acid, or a copolymer of acrylonitrile and itaconic acid.

[0052] The functional monomers containing nitrile groups are grafted onto the substrate porous microspheres. The grafting polymerization can be carried out by one or more of the following methods: radiation grafting polymerization, chemically initiated grafting polymerization, monodisperse suspension emulsion polymerization and reverse emulsion crosslinking polymerization. This can enable the substrate porous microspheres to form the desired pore structure and finally obtain porous microspheres containing polyacrylonitrile groups.

[0053] Furthermore, in S1, the irradiation dose of the cobalt-60 γ source is 20 kGy-500 kGy, and the irradiation dose of the electron beam accelerator is 10 kGy-1000 kGy.

[0054] Specifically, the aforementioned 157 The preparation method of Gd neutron embolization microspheres includes the following steps:

[0055] S1. Mix acrylonitrile monomer, acrylic acid monomer, dimethylformamide and porous microspheres, stir, first introduce inert gas and bubble, add initiator, second introduce inert gas, seal, first reaction, first centrifugation, remove supernatant, first ultrasonic cleaning, first drying, to obtain porous microspheres containing polyacrylonitrile groups.

[0056] S2. Dissolve ultrapure water and hydroxylamine hydrochloride, adjust pH for the first time, sonicate for the first time, adjust pH for the second time, add methanol and porous microspheres containing polyacrylonitrile groups, sonicate for the second time, carry out the first ammonium oxime reaction, centrifuge for the second time, remove the supernatant, add fresh reaction solution, carry out the second ammonium oxime reaction, centrifuge for the third time, remove the supernatant, sonicate for the third time, and dry for the second time to obtain ammonia oxime-based porous microspheres;

[0057] S3, towards 157 Gd₂O₃ was dissolved in concentrated hydrochloric acid, diluted once, and the pH was adjusted to 5-6 using sodium hydroxide. A second dilution was then performed to obtain… 157 Gd solution, mixed 157 Gd solution and geminitroxime-based porous microspheres, second reaction; fourth centrifugation, removal of supernatant; fourth ultrasonic cleaning; fifth centrifugation, removal of supernatant; third drying, to obtain 157 Gd neutron embolization microspheres.

[0058] Further, in step S1, the total volume of the acrylonitrile monomer, acrylic acid monomer, and dimethylformamide is 100-300 mL; the mass fraction of the acrylonitrile monomer is 30-40%, the mass fraction of the acrylic acid monomer is 10-20%, and the mass fraction of the dimethylformamide is 40-60%; the porous microspheres of the substrate are polyvinyl alcohol microspheres with a mass of 2-5 g; the stirring temperature is room temperature, the stirring speed is 500-600 r / min, and the stirring time is 30-120 min; the inert gas is argon, and the bubbling time is 30-60 min; the amount of the initiator is 10-25 mL, and the initiator contains nitric acid and cerium ammonium nitrate, with the mass fraction of the nitric acid and the mass fraction of the cerium ammonium nitrate being 10-15%; the second inert gas introduction time is 20-40 min; the conditions for the first reaction are stirring at 500-1000 r / min in an oil bath at 65-85℃ for 4-12 minutes. h; the first centrifugation speed is 6000-8000 r / min, the first ultrasonic cleaning solvent is dimethylformamide and deionized water, the specific operation is to first ultrasonically clean with dimethylformamide 4-5 times, and then ultrasonically clean with deionized water 4-5 times; the first drying time is 24-48 h.

[0059] Further, in step S2, the volume of ultrapure water is 60-200 mL, and the mass of hydroxylamine hydrochloride is 10-30 g; sodium carbonate is added for the first pH adjustment, and the mass of sodium carbonate is 7.4-18 g; the first ultrasonication time is 30-60 min; the solution used for the second pH adjustment is dilute hydrochloric acid or dilute sodium hydroxide solution, adjusting the pH to 7; the volume of methanol is 40-60 mL, and the mass of the porous microspheres containing polyacrylonitrile groups is 1-5 g; the second ultrasonication time is 30-60 min; the conditions for the first ammonium oxime reaction are a stirring reaction at 500-800 r / min at a temperature of 65-85 ℃ for 24-48 h; the conditions for the second ammonium oxime reaction are the same as the first ammonium oxime reaction; the speed of the third centrifugation is 6000-8000 r / min, and the solvent for the third ultrasonic cleaning is ultrapure water; the second drying time is 24 h.

[0060] Furthermore, in S3, the 157 The mass of Gd₂O₃ is 10-20 mg; the volume of concentrated hydrochloric acid is 0.2-0.4 mL; the first dilution is to dilute the solution to 5-10 mL; 157The volume of the Gd solution is 10-20 mL; the mass of the geminal oxime-based porous microspheres is 200-250 mg; the second reaction conditions are: stirring at 500 r / min for 0.5-0.8 h at 50-60 °C; the fourth centrifugation speed is 5000-6000 r / min; the solvent for the fourth ultrasonic cleaning is ultrapure water; the third drying is vacuum drying for 24 h. This invention also provides the above... 157 Gd neutron embolization microspheres or those prepared by the above methods 157 Application of Gd neutron embolization microspheres in drugs for tumor diagnosis or tumor treatment.

[0061] Furthermore, the tumor treatment is primary liver treatment or thermal neutron capture therapy (TAE-NCT) for metastatic tumors.

[0062] 157 When Gd neutron embolization microspheres are used for primary liver treatment or thermal neutron capture therapy for metastatic tumors, they can reduce additional radiation exposure to surrounding healthy tissues.

[0063] The present invention also provides the above. 157 Gd neutron embolization microspheres or those prepared by the above methods 157 Application of Gd neutron embolization microspheres in drugs for neutron capture therapy of local tumor embolization.

[0064] The present invention also provides the above. 157 Gd neutron embolization microspheres or those prepared by the above methods 157 The application of Gd neutron embolization microspheres in MRI contrast agents, the 157 Gd neutron embolization microspheres have MRI contrast enhancement capabilities and are suitable for preoperative localization, intraoperative localization, dose assessment, or in vivo distribution tracing.

[0065] Furthermore, the drug can be administered via selective transarterial delivery through a catheter, tumor injection, or intratumoral injection.

[0066] The present invention also provides the above. 157 Gd neutron embolization microspheres or those prepared by the above methods 157 Application of Gd neutron embolization microspheres in drugs for neutron capture therapy.

[0067] Furthermore, the drug can be administered via selective transarterial delivery through a catheter, tumor injection, or intratumoral injection.

[0068] The present invention also provides the above. 157 Gd neutron embolization microspheres or those prepared by the above methods 157Application of Gd neutron embolization microspheres in drugs for preoperative pulmonary embolism testing and gastrointestinal shunt assessment.

[0069] The beneficial effects of this invention are:

[0070] This invention provides 157 Gd neutron-embolized microspheres possess the following excellent properties:

[0071] (1) 157 Gd neutron embolization microspheres can be used as neutron capture therapy drugs for local tumor embolization or intratumoral injection, by loading Gd neutron oxime-based porous microspheres. 157 Gd, through selective coordination and porous carrier design, achieves high tumor enrichment and good in vivo retention, and generates killing particles only when irradiated by thermal neutrons, thus achieving targeted attack on tumors;

[0072] (2) 157 Gd neutron embolization microspheres achieve zero radiation pollution throughout the entire process of preparation, storage, and administration. They generate high-energy gamma rays (1330 keV) and low-energy internal conversion electrons, X-rays, and Auger electrons only during the instant of thermal neutron irradiation. The high-energy gamma rays have strong penetrating power, damaging tumor cells and inhibiting tumor growth. They can propagate several centimeters within tumor tissue, with a maximum energy of up to 7.80 MeV, thus achieving localized high-energy radiation killing of tumors. Furthermore, combined with thermal neutron irradiation, a single irradiation can achieve the highest radiation dose required for tumor treatment, while significantly reducing the additional radiation exposure dose for medical staff and patients. This solves the problem of balancing safety and effectiveness in existing liver cancer radiotherapy techniques, and is of great significance for improving cancer treatment levels and patient prognosis.

[0073] (3) 157 Gd neutron embolization microspheres can achieve the high radiation dose required for tumor lethality in a single irradiation, while controlling the dose to surrounding normal liver tissue within the tolerance threshold to avoid radiation damage. The corresponding neutron irradiation flux is 1×10⁻⁶. 9 n / cm 2 / s;

[0074] (4) 157 Gd has the largest pure spin magnetic moment of all elements, and Gd chelates are the most common clinical MRI contrast agents for T1 MRI. 157 Gd neutron embolization microspheres combine tumor neutron capture therapy with imaging diagnosis and treatment, and have significant clinical translational potential.

[0075] (5) Further analysis of the embodiments shows that this invention... 157 Gd neutron embolization microspheres have high labeling efficiency (labeling efficiency > 99%) and good in vitro serum stability. 157The advantages of Gd leakage rate being preferably much lower than 0.1%, good biocompatibility, neutron embolization microspheres that can achieve zero radiation pollution throughout the entire process of preparation, storage and administration, generating killing rays only at the moment of thermal neutron irradiation, monodispersity and uniform particle size, rapid labeling kinetics, long shelf life and easy engineering scale-up preparation.

[0076] The present invention provides this 157 The preparation process of Gd neutron embolization microspheres has the following advantages:

[0077] Using porous microspheres as the core carrier, a series of steps including graft polymerization, ammonium oxime reaction, and coordination chelation were employed to construct... 157 Gd neutron embolization microspheres. In this preparation process, the graft polymerization of nitrile-containing functional monomers achieves the functionalization modification of the microsphere surface, while the ammonium oxime reaction is... 157 The coordination chelation of Gd provides highly active sites for the amine oxime group, while 157 The coordination chelation between Gd and the amine oxime group ensures efficient gadolinium loading and enhances the structural stability of the product through strong coordination interactions. 157 The preparation method of Gd neutron embolization microspheres has the technical advantages of simple and controllable process, high loading efficiency and strong product stability. It can also meet the actual needs of industrialization and clinical application, and is suitable for large-scale production and rapid preclinical labeling. It effectively overcomes the shortcomings of existing neutron therapy embolization microspheres that are difficult to adapt to the needs of industrial-scale production and rapid preclinical labeling. Attached Figure Description

[0078] Figure 1 The images show scanning electron microscope (SEM) images and EDS-Mapping area scans. A represents the SEM image of PVA-Ms prepared in Example 1 and its corresponding EDS-Mapping area scan; B represents the SEM image of PAN-Ms prepared in Example 1 and its corresponding EDS-Mapping area scan; C represents the SEM image of PAN-Ms prepared in Example 1 and its corresponding EDS-Mapping area scan; and D represents the SEM image of PAN-Ms prepared in Example 1 and its corresponding EDS-Mapping area scan.

[0079] Figure 2 This is a graph showing the results of the in vitro biosafety evaluation. In this graph, A represents HepG2 cells reacting with different concentrations of [a specific substance / method / etc.]. 157 Cell viability graphs after co-incubation of Gd-PAO-Ms and GdCl3 solution at a concentration of 500 μg / mL for 24, 48, and 72 h; B represents... 157 Image of cell viability and mortality staining results for Gd-PAO-Ms;

[0080] Figure 3The graph shows the chelation stability efficiency results, where A is... 157 The serum chelation stability efficiency of Gd-PAO-Ms is shown in Figure B, which represents the serum chelation stability efficiency after neutron irradiation.

[0081] Figure 4 for 157 Figure 1 shows the results of Gd-PAO-Ms in vitro cell neutron capture therapy: where A represents... 157 Cell viability of Gd-PAO-Ms+NCT group cells after 24, 48, and 72 h of incubation; cell viability of Control group cells after 24, 48, and 72 h of incubation; cell viability of PBS+NCT group cells after 24, 48, and 72 h of incubation; the left figure of B shows the cell viability of Control group cells, PBS+NCT group cells, and... 157 Figure 1 shows the cell colony formation results of the Gd-PAO-Ms+NCT group; Figure B (right side) shows the Control group cells, PBS+NCT group cells, and... 157 Statistical chart of clone number in Gd-PAO-Ms+NCT group;

[0082] Figure 5 The image shows the MRI imaging performance results, where A represents... 157 The longitudinal relaxation rate (r1) of Gd-PAO-Ms at different concentrations was measured using a 7.0T small animal magnetic resonance scanner, where B is... 157 The transverse relaxation rate (r2) of Gd-PAO-Ms at different concentrations was measured using a 7.0T small animal magnetic resonance scanner. Detailed Implementation

[0083] The present invention will be further described in detail below through embodiments, but in no way is the invention limited.

[0084] Example 1

[0085] A type of neutron capture therapy embolization microsphere ( 157 The preparation method of Gd neutron embolization microspheres includes the following steps:

[0086] S1. Preparation of polyvinyl alcohol microspheres (PVA-Ms) with controllable and uniform particle size and good sphericity: The specific steps are as follows: Weigh 10 g of polyvinyl alcohol (PVA) and add it to 100 mL of pure water. Heat to 80°C and stir until the PVA is completely dissolved. Then add concentrated hydrochloric acid to adjust the pH of the PVA aqueous solution to 1, which will be used as the aqueous phase for the monodisperse suspension emulsion polymerization reaction. Weigh 7.2 g of Span80 and dissolve it in 200 mL of light liquid paraffin, which will be used as the oil phase for the monodisperse suspension emulsion polymerization reaction. Add the oil phase to a 500 mL glass flask and stir for 30 min at a stirring speed of 1000 r / min and a temperature of 55°C to ensure uniform mixing of the dispersant. Then, add 80 mL of the aqueous phase dropwise at a rate of 0.8 mL / min using a syringe pump. Keep the stirring speed constant and continue stirring for 30 min. Then raise the system temperature to 65°C. o C. 50% glutaraldehyde solution was added dropwise at a rate of 0.8 mL / min using a syringe pump. After stirring for 2 h, the mixture was allowed to cool naturally to obtain a white emulsion. The white emulsion was centrifuged at 6000 r / min to remove the supernatant. Acetone, anhydrous ethanol, and pure water were added sequentially for ultrasonic cleaning of the bottom microspheres. The mixture was then centrifuged and the supernatant was removed. This process of washing, centrifuging, and removing the supernatant was repeated three times for each solvent (acetone, anhydrous ethanol, and pure water). Finally, the bottom microspheres were vacuum dried for 24 h to obtain white powdery polyvinyl alcohol microspheres (PVA-Ms).

[0087] S2. Preparation of porous microspheres containing polyacrylonitrile groups (PAN-Ms): In a 250 mL glass flask, add 100 mL of a mixed solution containing 30 wt% acrylonitrile monomer (AN), 10 wt% acrylic acid monomer (AA), and 60 wt% dimethylformamide (DMF), along with 2 g of PVA-Ms. Stir at room temperature for 30 min at 500 r / min. Then, purge with argon gas for 30 min to remove oxygen. Next, add 10 mL of an initiator containing 10 wt% nitric acid and 10 wt% cerium ammonium nitrate, and continue purging with argon gas for another 20 min to remove oxygen before sealing the system. Then, incubate the system at 65°C. o The reaction was stirred at 500 r / min in an oil bath for 4 h. After the reaction was completed, the reaction product was centrifuged at 6000 r / min to remove the supernatant. It was then ultrasonically washed 4 times with dimethylformamide (DMF), then ultrasonically washed 4 times with deionized water, and finally vacuum dried for 24 h to obtain PAN-Ms.

[0088] S3. Preparation of porous microspheres containing poly(amino oxime) groups (PAO-Ms): 60 mL of ultrapure water and 10 g of hydroxylamine hydrochloride were added to a 250 mL glass flask. After complete dissolution, 7.4 g of sodium carbonate was added to adjust the pH. The mixture was then sonicated to aid dissolution and remove bubbles for 30 min. Subsequently, the pH was adjusted to 7 using dilute hydrochloric acid or dilute sodium hydroxide solution. 40 mL of methanol and 1 g of PAN-Ms were added, and sonication was continued for another 30 min. The system temperature was then raised to 65 °C. o C. The reaction was stirred at 500 r / min for 24 h. The above operation was repeated to prepare a new reaction solution (the new reaction solution did not contain PAN-Ms because PAN-Ms was the solute. The preparation of the new reaction solution included adding 60 mL of ultrapure water and 10 g of hydroxylamine hydrochloride to a 250 mL glass flask, dissolving it completely, adding 7.4 g of sodium carbonate to adjust the pH, using ultrasound to aid dissolution and remove bubbles for 30 min; then, adjusting the pH to 7 with dilute hydrochloric acid or dilute sodium hydroxide solution, adding 40 mL of methanol to prepare a fresh reaction solution). After centrifugation to remove the supernatant, the new reaction solution was added, and the reaction was continued under the same conditions for 24 h. After the reaction was completed, the reaction product was centrifuged at 6000 r / min to settle, the supernatant was removed, the bottom layer of modified microspheres was ultrasonically cleaned with ultrapure water, and finally vacuum dried for 24 h to obtain PAO-Ms, also known as amine oxime porous microspheres.

[0089] S4 157 Gd-labeled amylopyridine porous microspheres ( 157 Preparation of Gd-PAO-Ms: Weigh 10 mg of 157 Gd₂O₃ was reacted with 0.2 mL of concentrated hydrochloric acid until completely dissolved. The solution was then diluted to 5 mL, and the pH was adjusted to 5-6 using sodium hydroxide solution. Finally, the solution was diluted to 10 mL to obtain the desired solution. 157 Gd solution; then... 157 Gd solution and 200 mg of PAO-Ms were added to a 50 mL glass flask. The system temperature was then raised to 50°C, and the mixture was stirred at 500 r / min for 0.5 h. After the reaction, the reaction product was centrifuged at 6000 r / min to remove the supernatant. The modified microspheres at the bottom layer were ultrasonically cleaned with ultrapure water, centrifuged again, and the supernatant was removed. Finally, the product was vacuum dried for 24 h to obtain... 157 Gd-PAO-Ms, also known as 157 Gd neutron embolization microspheres.

[0090] Example 2

[0091] A type of neutron capture therapy embolization microsphere ( 157The preparation method of Gd neutron embolization microspheres includes the following steps:

[0092] S1. Preparation of PVA-Ms with controllable and uniform particle size and good sphericity: The specific steps are as follows: Weigh 20 g of polyvinyl alcohol (PVA) and add it to 200 mL of pure water. Heat to 60°C and stir until the PVA is completely dissolved. Then add concentrated hydrochloric acid to adjust the pH of the PVA aqueous solution to 2-3, which will be used as the aqueous phase for the monodisperse suspension emulsion polymerization reaction. Weigh 10 g of Span80 and dissolve it in 400 mL of light liquid paraffin, which will be used as the oil phase for the monodisperse suspension emulsion polymerization reaction. Add the oil phase to a 1000 mL glass flask and stir at 1000 r / min and 65°C. o Stirring at C for 60 min to ensure uniform mixing of the dispersant; then, add 120 mL of aqueous phase dropwise at a rate of 1.0 mL / min using a syringe pump, maintaining a constant stirring speed, and continue stirring for another 60 min. Finally, raise the system temperature to 75°C. o C. 50% glutaraldehyde solution was added dropwise at a rate of 0.8 mL / min using a syringe pump. After stirring for 2 h, the mixture was allowed to cool naturally to obtain a white emulsion. The white emulsion was centrifuged at 6000 r / min to remove the supernatant. Acetone, anhydrous ethanol, and pure water were added sequentially for ultrasonic cleaning of the bottom layer of microspheres. The mixture was then centrifuged and the supernatant was removed. This process of washing, centrifuging, and removing the supernatant was repeated three times for each solvent (acetone, anhydrous ethanol, and pure water). Finally, the bottom layer of microspheres was vacuum dried for 24 h to obtain white powdery polyvinyl alcohol microspheres (PVA-Ms).

[0093] S2. Preparation of porous microspheres containing polyacrylonitrile groups (PAN-Ms): 100 mL of a mixed solution containing 40 wt% acrylonitrile monomer (AN), 20 wt% acrylic acid monomer (AA), and 40 wt% dimethylformamide (DMF), along with 3.5 g of PVA-Ms, was added to a 500 mL glass flask. The mixture was stirred at room temperature for 60 min at 500 r / min, followed by purging with argon gas for 60 min to remove oxygen. Radiation grafting polymerization was then carried out in a cobalt source (radiation dose of 200 kGy). After irradiation for 6 h, the reaction was completed. The reaction product was centrifuged at 6000 r / min to remove the supernatant. The product was then ultrasonically cleaned 5 times with DMF, followed by ultrasonic cleaning 5 times with deionized water. Finally, it was vacuum dried for 48 h to obtain PAN-Ms.

[0094] S3. Preparation of porous microspheres containing poly(amino oxime) groups (PAO-Ms): 100 mL of ultrapure water and 15 g of hydroxylamine hydrochloride were added to a 500 mL glass flask. After complete dissolution, 10.0 g of sodium carbonate was added to adjust the pH. The mixture was then sonicated to aid dissolution and remove bubbles for 60 min. Subsequently, the pH was adjusted to 7 using dilute hydrochloric acid or dilute sodium hydroxide solution. 50 mL of methanol and 2.0 g of PAN-Ms were added, and sonication was continued for another 60 min. The system temperature was then raised to 85 °C. o C. The reaction was stirred at 500 r / min for 24 h. The above operation was repeated to prepare a new reaction solution (the new reaction solution did not contain PAN-Ms because PAN-Ms was the solute. The preparation of the new reaction solution included adding 100 mL of ultrapure water and 15 g of hydroxylamine hydrochloride to a 500 mL glass flask, dissolving it completely, adding 10 g of sodium carbonate to adjust the pH, using ultrasound to aid dissolution and remove bubbles for 60 min; then, adjusting the pH to 7 with dilute hydrochloric acid or dilute sodium hydroxide solution, adding 50 mL of methanol to prepare a fresh reaction solution). After centrifugation to remove the supernatant, the new reaction solution was added, and the reaction was continued under the same conditions for 24 h. After the reaction was completed, the reaction product was centrifuged at 6000 r / min to settle, the supernatant was removed, the bottom layer of modified microspheres was ultrasonically cleaned with ultrapure water, and finally vacuum dried for 24 h to obtain PAO-Ms, also known as amine oxime porous microspheres.

[0095] S4 157 Gd-labeled amylopyridine porous microspheres ( 157 Preparation of Gd-PAO-Ms: Weigh 20 mg of... 157 Gd₂O₃ was reacted with 0.4 mL of concentrated hydrochloric acid until completely dissolved. The solution was then diluted to 10 mL, and the pH was adjusted to 5-6 using sodium hydroxide solution. The solution was then further diluted to 20 mL. 157 Gd solution and 250 mg of PAO-Ms were added to a 100 mL glass flask. The system temperature was then raised to 60°C, and the mixture was stirred at 500 r / min for 0.8 h. After the reaction, the reaction product was centrifuged at 5000 r / min to remove the supernatant. The modified microspheres at the bottom layer were ultrasonically cleaned with ultrapure water, centrifuged again, and the supernatant was removed. Finally, the product was vacuum dried for 24 h to obtain... 157 Gd-PAO-Ms, also known as 157 Gd neutron embolization microspheres.

[0096] Example 3

[0097] A type of neutron capture therapy embolization microsphere ( 157 The preparation method of Gd neutron embolization microspheres includes the following steps:

[0098] S1. Preparation of PVA-Ms with controllable and uniform particle size and good sphericity: The specific steps are as follows: Weigh 50 g of PVA and add it to 500 mL of pure water. Heat to 80°C and stir until the PVA is completely dissolved. Then add concentrated hydrochloric acid to adjust the pH of the PVA aqueous solution to 3-4, which will be used as the aqueous phase for the monodisperse suspension emulsion polymerization reaction. Weigh 20 g of Span80 and dissolve it in 500 mL of light liquid paraffin, which will be used as the oil phase for the monodisperse suspension emulsion polymerization reaction. Add the oil phase to a 1000 mL glass flask and stir at 2000 r / min and 85°C. o Stirring at C for 80 min to ensure uniform mixing of the dispersant; then, add 200 mL of aqueous phase dropwise at a rate of 2.0 mL / min using a syringe pump, maintaining a constant stirring speed, and continue stirring for 120 min. Finally, raise the system temperature to 85°C. o C. 60% glutaraldehyde solution was added dropwise at a rate of 1.2 mL / min using a syringe pump. After stirring for 4 hours, the mixture was allowed to cool naturally to obtain a white emulsion. The white emulsion was centrifuged at 8000 r / min to remove the supernatant. Acetone, anhydrous ethanol, and pure water were added sequentially for ultrasonic cleaning of the bottom microspheres. The mixture was then centrifuged and the supernatant was removed. This process of washing, centrifuging, and removing the supernatant was repeated four times for each solvent (acetone, anhydrous ethanol, and pure water). Finally, the bottom microspheres were vacuum dried for 24 hours to obtain white powdery polyvinyl alcohol microspheres (PVA-Ms).

[0099] S2. Preparation of porous microspheres containing polyacrylonitrile groups (PAN-Ms): 300 mL of a mixed solution containing 35 wt% acrylonitrile monomer (AN), 15 wt% methacrylic acid monomer (MAA), and 50 wt% dimethylformamide (DMF), along with 5 g of PVA-Ms, was added to a 500 mL glass flask. The mixture was stirred at 600 r / min at room temperature for 120 min. Argon gas was then bubbled through the flask for 40 min to remove oxygen. Next, 25 mL of an initiator containing 5 wt% nitric acid and 10 wt% potassium persulfate was added, and argon gas was bubbled through the flask for another 40 min to remove oxygen before sealing the system. The system was then incubated at 85°C. o The reaction was stirred at 1000 r / min in an oil bath for 12 h. After the reaction was completed, the reaction product was centrifuged at 8000 r / min to remove the supernatant. It was then ultrasonically cleaned 4 times with DMF and 4 times with deionized water. Finally, it was vacuum dried for 24 h to obtain PAN-Ms.

[0100] S3. Preparation of porous microspheres containing poly(aminomethylamine) oxime groups (PAO-Ms): 200 mL of ultrapure water and 30 g of hydroxylamine hydrochloride were added to a 1000 mL glass flask. After complete dissolution, 18.0 g of sodium carbonate was added to adjust the pH. The mixture was then sonicated to aid dissolution and remove bubbles for 60 min. Subsequently, the pH was adjusted to 7 using dilute hydrochloric acid or dilute sodium hydroxide solution. 60 mL of methanol and 5.0 g of PAN-Ms were added, and sonication was continued for another 45 min. The system temperature was then raised to 75°C. o C. The reaction was stirred at 800 r / min for 48 h. The above operation was repeated to prepare a new reaction solution (the new reaction solution did not contain PAN-Ms because PAN-Ms was the solute. The preparation of the new reaction solution included adding 200 mL of ultrapure water and 30 g of hydroxylamine hydrochloride to a 1000 mL glass flask, dissolving it completely, adding 18 g of sodium carbonate to adjust the pH, using ultrasound to aid dissolution and remove bubbles for 60 min; then, adjusting the pH to 7 with dilute hydrochloric acid or dilute sodium hydroxide solution, adding 60 mL of methanol to prepare a fresh reaction solution). After centrifugation to remove the supernatant, the new reaction solution was added, and the reaction was continued under the same conditions for 48 h. After the reaction was completed, the reaction product was centrifuged at 8000 r / min to settle, the supernatant was removed, the bottom layer of modified microspheres was ultrasonically cleaned with ultrapure water, and finally vacuum dried for 24 h to obtain PAO-Ms, also known as amine oxime porous microspheres.

[0101] S4 157 Gd-labeled amylopyridine porous microspheres ( 157 Preparation of Gd-PAO-Ms: Weigh 100 mg of 157 Gd₂O₃ was reacted with 0.6 mL of concentrated hydrochloric acid until completely dissolved. The solution was then diluted to 30 mL, and the pH was adjusted to 4-5 using sodium hydroxide solution. The solution was then further diluted to 50 mL. 157 Gd solution and 500 mg of PAO-Ms were added to a 100 mL glass flask. The system temperature was then raised to 70°C, and the mixture was stirred at 800 r / min for 0.3 h. After the reaction, the reaction product was centrifuged at 6000 r / min to remove the supernatant. The modified microspheres at the bottom layer were ultrasonically cleaned with ultrapure water, centrifuged again, and the supernatant was removed. Finally, the product was vacuum dried for 24 h to obtain... 157 Gd-PAO-Ms, also known as 157 Gd neutron embolization microspheres.

[0102] Example 4

[0103] A type of neutron capture therapy embolization microsphere ( 157 The preparation method of Gd neutron embolization microspheres includes the following steps:

[0104] S1. Preparation of chitosan microspheres (CS-Ms) with controllable and uniform particle size and good sphericity. The specific steps are as follows: Weigh 20 g of chitosan (CS) and add it to 200 mL of pure water. Heat to 60°C and stir until CS is completely dissolved. Then add concentrated hydrochloric acid to adjust the pH of the CS aqueous solution to 2-3, and use it as the aqueous phase for later use. Weigh 10 g of Span80 and dissolve it in 400 mL of light liquid paraffin, and use it as the oil phase for the monodisperse suspension emulsion polymerization reaction. Add the oil phase to a 1000 mL glass flask and stir at 1000 r / min and 65°C. o Stirring at C for 60 min to ensure uniform mixing of the dispersant; then, add 120 mL of aqueous phase dropwise at a rate of 1.0 mL / min using a syringe pump, maintaining a constant stirring speed, and continue stirring for another 60 min. Finally, raise the system temperature to 75°C. o C. 50% glutaraldehyde solution was added dropwise at a rate of 0.8 mL / min using a syringe pump. After stirring for 2 hours, the mixture was allowed to cool naturally to obtain a white emulsion. The white emulsion was centrifuged at 6000 r / min to remove the supernatant. Acetone, anhydrous ethanol, and pure water were added sequentially for ultrasonic cleaning of the bottom layer of microspheres. The mixture was then centrifuged and the supernatant was removed. This process of washing, centrifuging, and removing the supernatant was repeated three times for each solvent (acetone, anhydrous ethanol, and pure water). Finally, the bottom layer of microspheres was vacuum dried for 24 hours to obtain white powdery chitosan alcohol microspheres (CS-Ms).

[0105] S2. Preparation of porous microspheres containing polyacrylonitrile groups (PAN-Ms): 100 mL of a mixed solution containing 40 wt% acrylonitrile monomer (AN), 20 wt% acrylic monomer (AA), and 40 wt% dimethylformamide (DMF) and 3.5 g CS-Ms were added to a 500 mL glass flask. The mixture was stirred at room temperature for 60 min at 500 r / min, followed by purging with argon gas for 60 min to remove oxygen. Radiation grafting polymerization was then carried out in a cobalt source (radiation dose of 200 kGy). After irradiation for 6 h, the reaction was completed. The reaction product was centrifuged at 6000 r / min to remove the supernatant. The product was then ultrasonically cleaned 5 times with DMF and 5 times with deionized water. Finally, it was vacuum dried for 48 h to obtain PAN-Ms.

[0106] S3. Preparation of porous microspheres containing poly(amino oxime) groups (PAO-Ms): 100 mL of ultrapure water and 15 g of hydroxylamine hydrochloride were added to a 500 mL glass flask. After complete dissolution, 10.0 g of sodium carbonate was added to adjust the pH. The mixture was then sonicated to aid dissolution and remove bubbles for 60 min. Subsequently, the pH was adjusted to 7 using dilute hydrochloric acid or dilute sodium hydroxide solution. 50 mL of methanol and 2.0 g of PAN-Ms were added, and sonication was continued for another 60 min. The system temperature was then raised to 85 °C. o C. The reaction was stirred at 500 r / min for 24 h. The above operation was repeated to prepare a new reaction solution (the new reaction solution did not contain PAN-Ms because PAN-Ms was the solute. The preparation of the new reaction solution included adding 100 mL of ultrapure water and 15 g of hydroxylamine hydrochloride to a 500 mL glass flask, dissolving it completely, adding 10 g of sodium carbonate to adjust the pH, using ultrasound to aid dissolution and remove bubbles for 60 min; then, adjusting the pH to 7 with dilute hydrochloric acid or dilute sodium hydroxide solution, adding 50 mL of methanol to prepare a fresh reaction solution). After centrifugation to remove the supernatant, the new reaction solution was added, and the reaction was continued under the same conditions for 24 h. After the reaction was completed, the reaction product was centrifuged at 6000 r / min to settle, the supernatant was removed, the bottom layer of modified microspheres was ultrasonically cleaned with ultrapure water, and finally vacuum dried for 24 h to obtain PAO-Ms, also known as amine oxime porous microspheres.

[0107] S4 157 Gd-labeled amylopyridine porous microspheres ( 157 Preparation of Gd-PAO-Ms: Weigh 20 mg of... 157 Gd₂O₃ was reacted with 0.4 mL of concentrated hydrochloric acid until completely dissolved. The solution was then diluted to 10 mL, and the pH was adjusted to 5-6 using sodium hydroxide solution. The solution was then further diluted to 20 mL. 157 Gd solution and 250 mg of PAO-Ms were added to a 100 mL glass flask. The system temperature was then raised to 60 °C, and the mixture was stirred at 500 r / min for 0.8 h. After the reaction, the reaction product was centrifuged at 5000 r / min to remove the supernatant. The modified microspheres at the bottom layer were ultrasonically cleaned with ultrapure water, centrifuged again, and the supernatant was removed. Finally, the microspheres were vacuum dried for 24 h to obtain the final product. 157 Gd-PAO-Ms, also known as 157 Gd neutron embolization microspheres.

[0108] Detection and Analysis

[0109] 1. Scanning electron microscopy analysis

[0110] The specific operating steps are as follows: The substrate microspheres prepared in Example 1, namely polyvinyl alcohol microspheres (PVA-Ms), porous microspheres containing polyacrylonitrile groups (PAN-Ms), and porous microspheres with amylopectin groups (PAO-Ms), are subjected to the following steps: 157 Gd-labeled amylopyridine porous microspheres ( 157 Gd-PAO-Ms) was subjected to scanning electron microscopy and corresponding EDS-Mapping area scan analysis, and the results are as follows: Figure 1 As shown.

[0111] from Figure 1 Analysis of PVA-Ms in sample A showed that PVA-Ms had good sphericity and relatively uniform size, with an average particle size of approximately 30.5 μm. Furthermore, EDS elemental distribution indicated that PVA-Ms contained only carbon (H could not be detected by EDS). Compared to PVA-Ms, from... Figure 1 B and Figure 1 Analysis of C2 particles revealed significant morphological changes in PAN-Ms and PAO-Ms, exhibiting a hierarchical porous structure with an average particle size increasing to 45.2 μm and a pore size of approximately 474 nm. These particles maintained good sphericity and excellent monodispersity. The hierarchical porous structure of PAO-Ms was preserved, although the pore size slightly increased. Furthermore, EDS elemental distribution results for PAN-Ms and PAO-Ms confirmed the successful introduction of N and O elements. The widespread distribution of amine oxime ligands across the entire surface of PAO-Ms indirectly demonstrated the success of the chemical modification. Figure 1 Analysis of D shows that 157 The porous morphology of Gd-PAO-Ms is relatively rough, and the nanopore size increases to over 900 nm.

[0112] 2. Results of in vitro biosafety evaluation

[0113] The sample prepared in Example 1 157 Gd-PAO-Ms underwent in vitro biosafety assessment.

[0114] The product obtained in Example 1 157 Gd-PAO-Ms was dissolved in PBS (phosphate buffer) to prepare solutions containing different concentrations 157 Gd-PAO-Ms culture medium ( 157 Gd-PAO-Ms concentrations of 50 μg / mL, 100 μg / mL, 250 μg / mL, 500 μg / mL, and 1000 μg / mL were sterilized and used as treatment group one; GdCl3 was dissolved in PBS medium to prepare a 500 μg / mL GdCl3 solution, which was then sterilized and used as treatment group two; sterilized PBS medium ( 157A negative control group (Gd-PAO-Ms concentration of 0 μg / mL) was used; treatment group one and treatment group two were collectively referred to as the treatment group, and the toxicity of the treatment groups to HepG2 cells was tested. Different concentrations of Gd-PAO-Ms need to be set in 96-well plates. 157 Hep G2 cells were cultured in Gd-PAO-Ms medium for 24 hours, containing different concentrations of... 157 Hep G2 cells were cultured in Gd-PAO-Ms medium for 48 hours, containing different concentrations of... 157 Parallel wells were prepared for culturing Hep G2 cells in Gd-PAO-Ms medium for 72 h, PBS medium for 24 h, PBS medium for 48 h, PBS medium for 72 h, 500 μg / mL GdCl3 solution for 24 h, GdCl3 solution for 48 h, and GdCl3 solution for 72 h. The specific procedures are as follows:

[0115] Hep G2 cells in the logarithmic growth phase were harvested, and the cells at the bottom of the culture dish were digested with trypsin. After digestion, the cells were centrifuged and resuspended in fresh complete culture medium to prepare a cell suspension with a concentration of 5 × 10⁻⁶. 4 Hep G2 cells were seeded into 96-well plates at a density of 5000 cells per well, leaving one well empty (as a blank well). The plates were then incubated at 37°C in an air incubator containing 5% CO2 for 24 hours to allow for cell adhesion. After incubation, PBS was added to the blank well, and the complete medium in the other wells was replaced with 100 µL of PBS, 100 µL of PBS containing different concentrations of different PBS, and so on. 157 The culture medium for Gd neutron embolization microspheres was prepared with 100 µL of 500 μg / mL GdCl3 solution, and then cultured for 24 h, 48 h, and 72 h, respectively. At 24 h, 48 h, and 72 h, the 96-well plates were removed, the original culture medium was discarded, and 100 µL of culture medium containing 10 µL of CCK-8 reagent was added to each well. The plates were then incubated at 37°C. o Incubate in an incubator of C for 1-2 h, then use an ELISA reader to measure the absorbance (OD) of each group at a wavelength of 450 nm, and calculate the cell viability (%) according to formula (I).

[0116] Cell viability (%) = [(OD1-OD2) / (OD3-OD2)] × 100% (I);

[0117] OD1: Absorbance at 450 nm in the treatment group; OD2: Absorbance at 450 nm in the blank well (cell-free, containing only culture medium + CCK-8); OD3: Absorbance at 450 nm in the negative control group (HepG2 cells treated with PBS).

[0118] Cell survival rate, such as Figure 2 As shown in A.

[0119] Experiments with live and dead dyes:

[0120] Detailed operation steps:

[0121] The product obtained in Example 1 157 Gd-PAO-Ms was dissolved in PBS (phosphate buffer) to prepare solutions containing different concentrations 157 Gd-PAO-Ms culture medium, 157 The concentrations of Gd-PAO-Ms were 0 μg / mL (blank control group), 50 μg / mL (experimental group 1), 100 μg / mL (experimental group 2), 500 μg / mL (experimental group 3), and 1000 μg / mL (experimental group 4).

[0122] Prepare staining working solutions (prepare fresh each time), including Calcein-AM (2 μM) and PI (4 μM).

[0123] Hep G2 cells in the logarithmic growth phase were harvested, and the cells at the bottom of the culture dish were digested with trypsin. After digestion, the cells were centrifuged and resuspended in fresh complete culture medium to prepare a cell suspension with a concentration of 5 × 10⁻⁶. 4 Hep G2 cells were seeded into 96-well plates at a density of 5000 cells per well and then incubated at 37°C in an air incubator containing 5% CO2 for 24 hours to allow cell adhesion. After cell adhesion, the complete culture medium was discarded. Then, different concentrations of [unspecified medium] were added to [the culture medium]. 157 The culture medium containing Gd-PAO-Ms was co-incubated for 1 hour, and then the medium containing different concentrations was discarded. 157 The Gd-PAO-Ms medium was gently washed 1-2 times with PBS buffer. Then, 100 μL of staining working solutions Calcein-AM (2 μM) and PI (4 μM) were added to each well of each group (96-well plate), completely covering the cells. The plates were then incubated at 37 ℃ in the dark for 30 min. Afterward, the plates were gently washed 2-3 times with PBS buffer to completely remove free dye. The medium was then replaced with PBS. Green live cells were observed through the FITC channel and red dead cells through the RFP channel under a fluorescence microscope. The results of live and dead cell staining are shown below. Figure 2 As shown in B.

[0124] Figure 2 In B, AM represents the green live cells viewed through the FITC channel, PI represents the red dead cells viewed through the RFP channel, and Merge represents the merged image of the green live cells viewed through the FITC channel and the red dead cells viewed through the RFP channel. The results indicate... 157 Even at concentrations as high as 500 μg / mL and 1000 μg / mL, and without thermal neutron irradiation, Gd-PAO-Ms do not affect cell survival, indicating that they have good cell compatibility.

[0125] from Figure 2 Analysis shows that CCK-8 and live / dead staining experiments indicate that different concentrations of... 157 Gd-PAO-Ms had almost no effect on the cell proliferation activity of HepG2 cells. Even when the concentration was increased to 1000 μg / mL and co-incubated with the cells for 24, 48, and 72 h, the cells still maintained a viability of over 98%. Compared with the control group (containing only PBS medium), different concentrations of Gd-PAO-Ms significantly reduced the cell proliferation activity. 157 HepG2 cells cultured with Gd-PAO-Ms (50 μg / mL, 100 μg / mL, 250 μg / mL, 500 μg / mL, 1000 μg / mL) proliferated normally, indicating that... 157 Gd-PAO-Ms exhibits good biocompatibility and no cytotoxicity; compared to the severe cytotoxicity of the pure GdCl3 group, this indirectly confirms that PAO-Ms is effective against cytotoxicity. 157 The ultra-stable chelation of Gd avoids the cytotoxic side effects caused by free metal Gd ions entering normal tissues.

[0126] 3. 157 Assessment of the chelation stabilization efficiency of Gd-PAO-Ms in serum

[0127] The specific operating steps are as follows:

[0128] The product prepared in Example 1 157 The chelation stabilization efficiency of Gd-PAO-Ms in serum was analyzed, and the effects of thermal neutron irradiation treatment on [the following text is incomplete and requires further context: "to investigate the effect of Gd-PAO-Ms chelation stabilization efficiency in serum"] was also investigated. 157 The effect of Gd-PAO-Ms chelation stability, specific experimental groups and detection results are as follows:

[0129] The experiment was set up with two treatment groups: Treatment group one: 157 Gd-PAO-Ms in FBS group: 157 Gd-PAO-Ms were immersed in fetal bovine serum (FBS) solution to simulate the physiological environment of serum; Treatment group two: 157 Gd-PAO-Ms in FBS+NCT group: 157Gd-PAO-Ms were immersed in fetal bovine serum solution and then subjected to 20 minutes of thermal neutron irradiation (neutron flux: 0.5 x 10⁻⁶). 9 n / cm 2 / s), simulating the clinical application conditions of neutron capture therapy (NCT).

[0130] Treatment group one and treatment group two 157 Gd-PAO-Ms were subjected to shaking in 5% fetal bovine serum (FBS) at room temperature for 1-10 days, and the chelation stability efficiency was measured sequentially from day 1 to day 10. The results are as follows: Figure 3 As shown. Figure 3 In A, the horizontal axis represents 157 Oscillation time of Gd-PAO-Ms in FBS group, with the vertical axis representing chelation stabilization efficiency; Figure 3 In B, the horizontal axis represents 157 Oscillation time of Gd-PAO-Ms in FBS+NCT group, with the vertical axis representing chelation stabilization efficiency.

[0131] Figure 3 A showed 157 Chelation stability efficiency of Gd-PAO-Ms in fetal bovine serum; Figure 3 B showed 157 The chelation stability efficiency of Gd-PAO-Ms after fetal bovine serum and thermal neutron irradiation; together they demonstrate the... 157 Gd-PAO-Ms exhibits extremely high chelation stability.

[0132] 4. Cell-level assessment 157 Neutron capture therapy efficacy of Gd-PAO-Ms

[0133] The sample prepared in Example 1 157 Cellular-level evaluation of the efficacy of neutron capture therapy using Gd-PAO-Ms.

[0134] Specific experimental steps:

[0135] (1) Set up the Control group, PBS+NCT group and 157 In the Gd-PAO-Ms+NCT group, HepG2 cells were seeded into six-well plates (103) of each group. 6 Cells / well), and the six-well plates of each group were placed in a CO2 incubator at 37℃ for 24 h. After the HepG2 cells adhered, they were transferred to... 157 2 mL of Gd-PAO-Ms+NCT solution containing 500 μg / mL was added to the 6-well plate. 157 Gd-PAO-Ms culture medium (i.e., the culture medium in which Gd-PAO-Ms) 157The concentration of Gd-PAO-Ms was 500 μg / mL. 2 mL of PBS culture medium was directly added to each well of the PBS+NCT group and the Control group. Then, trypsin was added to each well of the 6-well plate of each group to digest HepG2 cells and centrifuge them. The cells were resuspended with 2 mL of culture medium and transferred to 5 mL EP tubes.

[0136] (2) Neutron irradiation treatment: Immediately after step (1) is completed, 157 Cells in the Gd-PAO-Ms+NCT group and the PBS+NCT group were irradiated with neutrons using an RFQ accelerator neutron capture therapy device (parameters: power 28 kW, neutron flux 1 x 10⁻⁶). 9 n / cm 2 / s, irradiation time 20 min); Control group cells did not require neutron irradiation treatment.

[0137] (3) Cell colony formation experiment: Cells irradiated by neutrons in step (2) and Control group cells were serially diluted to 200 cells / mL. Each group was seeded into a six-well plate (six in parallel), and 1 mL of cells was added to each well. The cell condition was observed daily, and the medium was replaced if the color of the medium changed. The culture was stopped when the Control group cells had formed colonies and most colonies had about 50 cells. The medium was aspirated, washed three times with PBS, fixed with 1 mL of paraformaldehyde for 30 min, aspirated, washed three times with PBS, stained with 1 mL of crystal violet for 30 min, and then the crystal violet was washed off with PBS before taking pictures. The results are as follows. Figure 4 As shown in Figure B (left), the data statistics were as follows: positive clones (i.e., each clone > 50 cells) were observed under a microscope, photographed, and the number of clones (approximately 0.3-1.0 mm in size) was counted to calculate the colony formation rate and to determine the clone size. ImageJ was used to analyze and quantify colony formation. The results are shown below. Figure 4 As shown on the right in Figure B.

[0138] (4) CCK-8 assay (detecting cell viability and calculating cell survival rate): HepG2 cells irradiated by neutrons in step (2) and HepG2 cells in the Control group were diluted to 1000 cells / 100 μL. HepG2 cells were seeded in 96-well plates at a density of 1000 cells per well, leaving one well unseeded (used as a blank well). The plates were then incubated in an air incubator at 37°C containing 5% CO2. After the HepG2 cells adhered to the plate, 100 μL of medium containing CCK-8 (medium:CCK-8=9:1) was added to each well at 24, 48, and 72 h. After incubation for 2 h, the OD value at 450 nm was measured using a microplate reader. Cell survival rate was calculated using the formula shown in equation (II). The results are as follows: Figure 4 As shown in A.

[0139] Cell viability = [(OD1-OD2) / (OD3-OD2)]×100% (II)

[0140] OD1: Treatment groups (including Control group, PBS+NCT group and) 157 OD2: Absorbance at 450 nm of the Gd-PAO-Ms+NCT group; OD3: Absorbance at 450 nm of the negative control group (HepG2 cells treated with PBS, i.e., the absorbance at 450 nm of the HepG2 cells treated with the Control group 24 hours after adhesion).

[0141] from Figure 4 Analysis of A shows that the CCK-8 experimental results... 157 Gd-PAO-Ms showed a significant killing effect on HepG2 compared to the negative control group. 157 The cell viability of the Gd-PAO-Ms+NCT group decreased to 30%, 22%, and 17% after 24, 48, and 72 h, respectively, confirming that... 157 Gd-PAO-Ms exhibits significant in vitro killing effects on tumor cells after thermal neutron irradiation.

[0142] from Figure 4 B left and Figure 4 The results of the cell colony formation assay in the right B group showed that after NCT thermal neutron irradiation, the cells in the PBS group had good cell viability and no effect on cell colony formation ability, which was the same as that of the control group, and no obvious cytotoxicity occurred. Compared with the negative control group (Control) and the pure NCT group (PBS+NCT), the cell colony formation assay showed good cell viability and no effect on cell colony formation ability. 157 Gd-PAO-Ms group ( 157 Gd-PAO-Ms+NCT significantly reduced cell colony formation ability, indirectly confirming 157 Gd-PAO-Ms has a significant inhibitory and killing effect on tumor cells.

[0143] 5. Evaluate MRI imaging performance

[0144] The longitudinal relaxation rate (r1) and transverse relaxation rate (r2) were measured on a 7.0T small animal magnetic resonance scanner to evaluate 157 MRI imaging enhancement capabilities of Gd-PAO-Ms.

[0145] Specific operating steps: Use a small animal magnetic resonance scanner to... 157 The magnetic resonance imaging performance of Gd-PAO-Ms was tested. First, the Gd-PAO-Ms prepared in Example 1 was used... 157Gd-PAO-Ms were dispersed in deionized water and formulated into different concentration gradients. 157 Gd-PAO-Ms suspension (0.1 mg / ml-40 mg / ml), then different concentration gradients of... 157 Gd-PAO-Ms suspension was placed in a pre-labeled 1 mL EP tube and centered on the scanning bed, ensuring its position was aligned with the center of the coil. T1- and T2-weighted images were then acquired under identical parameters, and the longitudinal and transverse relaxation times of each concentration sample were obtained using standard T1 and T2 sequencing sequences. The obtained relaxation times were converted into longitudinal and transverse relaxation rates (r1 and r2), and the relaxation rates were expressed as a function of... 157 The slope of the linear fit of the Gd-PAO-Ms concentration change was used as... 157 The relaxation efficiency of Gd-PAO-Ms was ultimately used to evaluate its T1 / T2 MRI enhancement imaging capability. The results are as follows: Figure 5 As shown.

[0146] Figure 5 In A, the horizontal axis represents 157 The concentration of Gd-PAO-Ms suspension is shown on the vertical axis, which represents the longitudinal relaxation rate (r1). Figure 5 In B, the horizontal axis represents 157 The concentration of Gd-PAO-Ms suspension, with the vertical axis representing the transverse relaxation rate (r2).

[0147] from Figure 5 From A and B, we know that within the concentration range of 0.1 mg / ml-40 mg / ml, 157 The r1 value of Gd-PAO-Ms ranges from 0.381s. -1 The linear increase reached 3.645s. -1 (R) 2 =0.078), r2 value from 1.636s -1 Rising to 5.767s -1 (R=0.098). This indicates... 157 Gd-PAO-Ms ( 157 Gd neutron embolization microspheres can significantly enhance MRI imaging capabilities.

[0148] 6. Determine the specific gravity (i.e., true density) of the geminal oxime-based porous microspheres in Examples 1-4.

[0149] The true density of ammoxime-based porous microspheres was determined using a gas displacement method. The specific steps were as follows: First, the volume of the empty tube was measured; then, using helium as the medium, the free volume of the sample was measured after the gas displacement pressure reached equilibrium. The difference between the initial and final volumes was the volume of the ammoxime-based porous microspheres. Finally, based on the ratio of the mass to the volume of the ammoxime-based porous microspheres, the true density of the ammoxime-based porous microspheres was determined to be 1.0 g / mL–1.5 g / mL.

[0150] In summary, the present invention provides this... 157 Gd neutron embolization microspheres have the following advantages:

[0151] (1) A methylamine oxime group is introduced onto the surface or pores of the methylamine oxime porous microspheres. The methylamine oxime group has a positive effect on the surface of the microspheres. 157 Gd exhibits specific coordination chelation. The diameter of the amylopyridine-based porous microspheres is 20-200 μm, and their specific gravity is 1.0-1.5 g / mL, close to that of blood (1.05 g / mL), resulting in more uniform distribution in the blood. The specific diameter, specific gravity, and pore structure of the amylopyridine-based porous microspheres can optimize hemodynamic distribution and in vivo retention.

[0152] (2) The pair of amine oxime groups 157 The specific coordination chelation of Gd can achieve highly stable loading and significantly reduce the metal content. 157 This reduces the risk of Gd escape and improves in vivo safety.

[0153] (3) 157 Gd neutron embolization microspheres exhibit a very low leakage rate in in vitro fetal bovine serum and demonstrate excellent chelation stabilization efficiency.

[0154] (4) The amine oxime-based porous microspheres exhibit highly selective and stable coordination chelation ability for gadolinium-157. No external radiation pollution is generated during the entire process of preparation, storage, and administration. Under thermal neutron irradiation, they can produce a localized high-LET killing effect and also possess MRI imaging capabilities. 157 Gd neutron embolization microspheres; and also provide an industrially feasible preparation method for these microspheres and their application in transcatheter local embolization thermal neutron capture therapy;

[0155] (5) 157 Gd neutron embolization microspheres have both diagnostic and therapeutic capabilities, facilitating preoperative and intraoperative dose assessment;

[0156] (6) 157 Gd neutron embolization microspheres exhibit excellent in vivo and in vitro stability, making them particularly suitable for catheter-directed embolization and intratumoral brachytherapy, and showing promising clinical translational potential.

[0157] In short, 157Gd neutron embolization microspheres can be used as neutron capture therapy drugs for local tumor embolization. Through selective coordination and porous carrier design, they can achieve high tumor enrichment and good in vivo retention. They generate killing rays only when irradiated with thermal neutrons, thus achieving targeted attack on tumors. 157 Gd neutron embolization microspheres can achieve zero radiation pollution throughout the entire process of preparation, storage and administration, and generate high-energy ray killing effect only at the moment of thermal neutron irradiation, thereby achieving high radiation dose irradiation of the tumor. In addition, combined with the fact that a single thermal neutron irradiation can reach the highest dose required for tumor treatment, it can also significantly reduce the additional radiation exposure dose of medical staff and patients, which is of great significance for improving the level of cancer treatment and improving patient prognosis. 157 Gd neutron embolization microspheres can achieve a single irradiation dose that reaches the high dose required for tumor lethality, while the dose to the surrounding normal liver tissue is controlled within the tolerance threshold to avoid radiation damage. 157 Gd neutron embolization microspheres combine tumor neutron capture therapy with imaging diagnosis and treatment, and have significant clinical translational potential.

Claims

1. A 157 A method of preparing gadolinium neutron embolization microspheres, characterized by, It consists of the following steps: S1, grafting a functional monomer containing nitrile groups onto a porous substrate microsphere to obtain a porous microsphere containing polyacrylonitrile groups; the functional monomer containing nitrile groups is selected from acrylonitrile; the porous substrate microsphere is selected from polyvinyl alcohol microspheres; S2, performing an aminooxidation reaction on the porous microsphere containing polyacrylonitrile groups to convert the nitrile groups into amidoxime groups to obtain an amidoxime group-containing porous microsphere; the particle diameter of the amidoxime group-containing porous microsphere is 20 μm-200 μm, and the specific gravity of the amidoxime group-containing porous microsphere is 1.0 g / mL-1.5 g / mL; S3, the poromeric microspheres with 157 Gd coordination chelation, to obtain 157 Gd neutron embolism microspheres; The S3 comprises: mixing the amidoxime group porous microspheres with 157 Gd solution, so that 157 Gd coordinates with the amidoxime groups and is adsorbed and loaded, eluted, dried, to obtain 157 Gd neutron embolism microspheres; the 157 Gd solution is 157 a Gd salt solution or 157 a complex solution of Gd, the pH of the adsorbed and loaded is 4-8, the temperature is 0-80℃, and the time is 5 min-24 h; The 157 Gd neutron embolization microspheres include an amine oxime-based porous microsphere and a 157 Gd; The surface of the poromeric microspheres has amine oxime groups which are capable of binding to 157 Gd forms 157 Gd coordination complexes; 157 Gd coordination complexes have dual functions of neutron capture therapy and magnetic resonance imaging contrast.

2. The method of claim 1 157 A method for preparing Gd neutron embolizing microspheres, characterized in that the amidoxy group is in the pores of the amidoxy porous microspheres, and the amidoxy group is coordinated with 157 Gd to form 157 Gd coordination complex.

3. The preparation method according to claim 1, characterized in that, In the S1, the porous substrate microsphere is prepared by monodisperse suspension emulsion polymerization or inverse emulsion crosslinking polymerization method; and the porous microsphere containing polyacrylonitrile groups is obtained by radiation graft polymerization or chemical initiation graft polymerization.

4. The preparation method according to claim 3, characterized in that, The preparation method of the polyvinyl alcohol microspheres comprises the following steps: Polyvinyl alcohol is added to water, and then acid is added to obtain an aqueous phase for monodisperse suspension emulsion polymerization reaction; Span 80 is dissolved in light liquid paraffin to obtain an oil phase for monodisperse suspension emulsion polymerization reaction; The oil phase is heated, and then the aqueous phase is added dropwise, heated, and then a glutaraldehyde solution is added dropwise to obtain a white emulsion; The white emulsion is centrifuged to obtain a precipitate, which is washed to obtain polyvinyl alcohol microspheres; And / or, when radiation graft polymerization is used, the radiation source of the radiation graft polymerization is selected from a cobalt-60 gamma source or an electron beam accelerator; And / or, when chemical initiation graft polymerization is used, the chemical initiator is selected from cerium ammonium nitrate or a persulfate initiator.

5. The method of claim 1, wherein the step of forming the first and second layers is performed by a process selected from the group consisting of: sputtering, evaporation, and chemical vapor deposition. In the S2, the aminooxidation reaction is carried out in a solvent using hydroxylamine or a hydroxylamine salt as a reagent, with a salt, and then with an acid solution or a base solution.

6. The preparation method according to claim 5, characterized in that, The hydroxylamine salt is selected from hydroxylamine hydrochloride; And / or, the salt is selected from one or both of sodium carbonate and sodium bicarbonate; And / or, the acid solution is selected from a dilute hydrochloric acid solution; And / or, the base solution is selected from a dilute sodium hydroxide solution; And / or, the solvent is selected from at least one of water, methanol, N,N-dimethylformamide, and N,N-dimethyl sulfoxide; And / or, the reaction temperature of the aminooxidation reaction is 45℃-80℃, and the time is 12-72 h.

7. The product prepared by the process according to any one of claims 1 to 6 157 Use of Gd neutron-embolizing microspheres in the preparation of a medicament for tumor diagnosis or tumor therapy.

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