Tumor-targeting boron-containing Fe3O4-based nano-enzyme as well as preparation method and application thereof

Abstract

The invention discloses a tumor targeting boron-containing Fe3O4-based nano-enzyme and a preparation method and application thereof, and belongs to the technical field of tumor treatment.The tumor targeting boron-containing Fe3O4-based nano-enzyme takes boron-10 doped Fe3O4 nano-enzyme as a boron-containing drug group and an enzyme activity center, and the surface of the tumor targeting boron-containing Fe3O4-based nano-enzyme is modified with a targeting group; the targeting group is chitosan-folic acid-polyethylene glycol. The tumor targeting boron-containing Fe3O4-based nano-enzyme has relatively strong selective enrichment capacity on tumor cells such as melanoma, shows good biocompatibility and low toxicity, and has relatively strong apoptosis induction and killing effects on the melanoma cells in boron neutron capture therapy; meanwhile, active oxygen free radicals can be generated in boron neutron capture therapy to generate an oxidative damage effect.

Description

Technical Field

[0001] This invention relates to the field of tumor treatment technology, specifically to a tumor-targeting boron-containing Fe3O4-based nanozyme, its preparation method, and its application. Background Technology

[0002] Malignant tumors pose a significant global health threat. Traditional treatments for malignant tumors, such as surgery, radiotherapy, and chemotherapy, have limitations. Despite significant advancements in cancer treatment in modern medicine, the mortality rate from malignant tumors remains high. Therefore, precision and personalized treatment have become a major challenge for the medical community.

[0003] Boron neutron capture therapy (BNCT) is an emerging binary, cell-level targeted radiotherapy technique. Its therapeutic principle is based on the reaction of thermal or hyperthermal neutrons with the stable isotope boron-10 accumulated at the tumor site (…). 10 B) A nuclear reaction occurs, generating alpha particles with high energy transfer linear density and lithium-7 (B) 7 Li) ions are used to precisely kill cancer cells. Since the path length of alpha particles and recoil nuclei is approximately the diameter of a cell (5-9 μm), and their energy is as high as 2.4 MeV, BNCT can selectively kill tumor cells at the cellular level while causing minimal damage to surrounding normal tissues. This is achieved through the use of Li) ions. 10 By individualizing and targeting the uptake of drug B and delivering thermal neutron irradiation to the tumor site at the optimal concentration and time, BNCT can significantly improve the precision and effectiveness of treatment while minimizing side effects. Furthermore, BNCT offers advantages such as a shorter treatment cycle, high drug safety, no drug resistance issues, and lower treatment costs. Based on these advantages, BNCT demonstrates enormous development potential and application value in clinical oncology treatment, especially for tumors that are difficult to treat with conventional therapies.

[0004] However, the clinical application of BNCT still faces some technical bottlenecks, including: (1) the targeted accumulation efficiency of boron-containing drugs at the tumor site needs to be further improved; (2) there is a lack of effective real-time monitoring methods for boron distribution; and (3) the therapeutic effect still needs to be further improved.

[0005] Therefore, developing novel boron-containing drugs and their boron delivery systems to improve tumor targeting and therapeutic efficacy is a key direction for the development of BNCT technology. Summary of the Invention

[0006] The purpose of this invention is to provide a tumor-targeting boron-containing Fe3O4-based nanozyme to solve the problems mentioned in the background art.

[0007] To achieve the above objectives, the embodiments of the present invention provide the following technical solutions:

[0008] A tumor-targeting boron-containing Fe3O4-based nanozyme, which uses boron-10-doped Fe3O4 nanozyme as the boron-containing drug group and enzyme active center, and is modified with a targeting group on its surface; the targeting group is chitosan-folic acid-polyethylene glycol.

[0009] Another objective of this invention is to provide a method for preparing the above-mentioned tumor-targeting boron-containing Fe3O4-based nanozyme, which includes the following steps:

[0010] Ferric chloride, sodium acetate, polyethylene glycol, and boron-10 acid were dissolved in ethylene glycol and stirred to obtain a mixed solution. The mixed solution was then placed at 190-210℃ for reaction. After the reaction was completed, the solution was cooled and ethanol was added, resulting in the precipitation of a black precipitate. Precipitate A was collected by centrifugation, washed, and dried. The precipitate A was then separated by a magnet to obtain boron-10-doped Fe3O4 nanozyme.

[0011] Chitosan was dissolved in acetic acid solution and heated until the solution became clear. After cooling, a dimethyl sulfoxide solution containing folic acid, polyethylene glycol carboxylic acid, N-hydroxysuccinimide, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride was added to the solution, and the reaction was carried out in the dark. After the reaction was completed, the supernatant was collected by centrifugation. The pH of the supernatant was adjusted to 8.7-9.3, and precipitate B was collected by centrifugation. Precipitate B was dissolved in methanol and subjected to H2O reaction. + The pH of the cation exchange resin was adjusted to neutral, and ether was added to precipitate the precipitate C. The precipitate C was then redissolved in dimethylformamide and dialyzed to obtain folic acid-functionalized chitosan nanoparticles.

[0012] Folic acid-functionalized chitosan nanoparticles were dissolved in acetic acid solution, heated until dissolved, and then cooled. Boron-10-doped Fe3O4 nanozymes were then added to the solution, stirred, and the pH was adjusted to 8.7-9.3. The precipitate D was collected by centrifugation. Precipitate D was then resuspended in methanol solution and subjected to H2O2... + The pH was adjusted to neutral using a cation exchange resin, and the precipitate E was collected by centrifugation. After washing and freeze-drying the precipitate E, it was separated by a magnet to obtain the tumor-targeting boron-containing Fe3O4-based nanozyme.

[0013] Preferably, the concentration of the acetic acid solution is 0.05-0.15M.

[0014] Preferably, the mass ratio of ferric chloride, sodium acetate, polyethylene glycol and boric-10 acid is (14-18):(640-680):(0.5-1.5):(2-4).

[0015] Preferably, the mass of chitosan, folic acid, polyethylene glycol carboxylic acid, N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride is (38-42):(10-12):(38-42):(10-12):(18-20).

[0016] Preferably, the mass ratio of the folic acid-functionalized chitosan nanoparticles to the boron-10-doped Fe3O4 nanozyme is (2-4):(1-3).

[0017] Another objective of this invention is to provide a tumor-targeting boron-containing Fe3O4-based nanozyme prepared by the above-described method.

[0018] Another objective of this invention is to provide the application of the above-mentioned tumor-targeting boron-containing Fe3O4-based nanozyme in the preparation of tumor BNCT drugs.

[0019] Preferably, the tumor-targeting boron-containing Fe3O4-based nanozyme is used to selectively deliver boron-10 into tumor cells, and to induce tumor cell apoptosis and slow tumor growth, while generating reactive oxygen species to cause oxidative damage.

[0020] Preferably, the tumor includes, but is not limited to, melanoma.

[0021] The tumor-targeting boron-containing Fe3O4-based nanozyme provided by this invention is prepared using surface-modified chitosan-folic acid-polyethylene glycol as the targeting group and boron-10-doped Fe3O4 nanozyme as the boron-containing drug group and enzyme active center. This tumor-targeting boron-containing Fe3O4-based nanozyme exhibits strong selective enrichment ability against melanoma and other tumor cells, while also demonstrating good biocompatibility and low toxicity. In boron neutron capture therapy, it exhibits strong apoptosis-inducing and killing effects on melanoma cells, and can also generate reactive oxygen species during boron neutron capture therapy, causing oxidative damage. Furthermore, it can significantly slow down tumor growth and prolong survival time in tumor-bearing mice, showing promising potential for application in boron neutron capture therapy. Attached Figure Description

[0022] Figure 1 This is a transmission electron microscope (TEM) schematic diagram of the tumor-targeting boron-containing Fe3O4-based nanozyme prepared according to an embodiment of the present invention.

[0023] Figure 2 This is a schematic diagram of the particle size distribution of the tumor-targeting boron-containing Fe3O4-based nanozyme prepared according to an embodiment of the present invention.

[0024] Figure 3 The infrared spectrum (IR) characterization image shows the tumor-targeting boron-containing Fe3O4-based nanozyme prepared according to an embodiment of the present invention.

[0025] Figure 4 The image shows the X-ray photoelectron spectroscopy (XPS) characterization of the tumor-targeting boron-containing Fe3O4-based nanozyme prepared in an embodiment of the present invention.

[0026] Figure 5 The figures show the cell uptake results of the tumor-targeting boron-containing Fe3O4-based nanozyme prepared in the embodiments of the present invention and the control group; in the figure, a represents the uptake by mouse fibroblasts (L929) and b represents the uptake by mouse skin melanoma cells (B16-F10).

[0027] Figure 6 The figures show the cytotoxicity test results of the tumor-targeting boron-containing Fe3O4-based nanozyme prepared in the embodiments of the present invention and the control group; in the figure, a is the L929 cytotoxicity test and b is the B16-F10 cytotoxicity test.

[0028] Figure 7 The figures show the detection results of reactive oxygen species generation in B16-F10 cells during treatment of the tumor-targeting boron-containing Fe3O4-based nanozyme prepared in the embodiments of the present invention and the control group; in the figure, a is before thermal neutron irradiation and b is after thermal neutron irradiation.

[0029] Figure 8 The figures show the treatment results of tumor-targeting boron-containing Fe3O4-based nanozymes prepared in the embodiments of the present invention and the control group on tumor-bearing mice during the treatment period; in the figure, a is the change in tumor volume of tumor-bearing mice, and b is the survival time of tumor-bearing mice. Detailed Implementation

[0030] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0031] This invention relates to a novel boron drug system based on peroxidase-like boron-containing iron tetroxide (Fe3O4)-based nanozymes. To improve tumor targeting and biocompatibility, this invention further employs folic acid-modified polyethylene glycol-based chitosan to achieve nanozyme stability and long-term in vivo circulation. Furthermore, it enhances the tumor selectivity of the boron-containing nanozyme and reduces its accumulation in normal tissue. In boron neutron capture therapy combined with catalytic therapy, the boron-containing iron tetroxide-based nanozyme catalyzes the generation of hydroxyl radicals from highly expressed hydrogen peroxide within tumors, which, combined with BNCT-induced DNA damage, can significantly enhance therapeutic efficacy for precision tumor treatment.

[0032] Specifically, in one embodiment of the present invention, a tumor-targeting boron-containing Fe3O4-based nanozyme is provided, which uses boron-10 doped Fe3O4 nanozyme as the boron-containing drug group and enzyme active center, and the surface is modified with a targeting group; the targeting group is chitosan-folic acid-polyethylene glycol.

[0033] The tumor-targeting boron-containing Fe3O4-based nanozyme provided in this invention can exert a synergistic catalytic effect of BNCT to enhance the efficacy of tumor treatment. It has high selectivity for skin melanoma cells and high safety, and can induce tumor cell apoptosis through catalytic reactions within tumor cells.

[0034] The following examples are some specific implementation cases of the present invention in practical applications, but are not limited thereto. It should be noted that the amount of each solvent involved in each step of the following examples is not limited, as long as it can fully dissolve or disperse the corresponding raw materials.

[0035] Example 1: This example provides a tumor-targeting boron-containing Fe3O4-based nanozyme, the preparation method of which specifically includes the following steps:

[0036] S1. Ferric chloride, sodium acetate, polyethylene glycol, and boron-10 acid were dissolved in an appropriate amount of ethylene glycol at a mass ratio of 16:660:1:3 and stirred vigorously for 30 min to obtain a mixed solution. The mixed solution was then reacted in a high-pressure reactor at 200°C for 2 h under nitrogen atmosphere. After the reaction was completed, the mixture was cooled to room temperature and ethanol was added, resulting in the precipitation of a black precipitate. Precipitate A was collected by centrifugation, washed three times with water and ethanol respectively, dried at 60°C for 6 h, and separated by a magnet to obtain boron-10 doped Fe3O4 nanozyme.

[0037] S2. Chitosan was dissolved in an appropriate amount of acetic acid solution (acetic acid concentration 0.1M, pH=4.7), heated to 55℃, and cooled to room temperature after the solution became transparent. Then, a dimethyl sulfoxide solution containing folic acid, polyethylene glycol carboxylic acid, N-hydroxysuccinimide, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride was added to the solution. The reaction was carried out at room temperature in the dark for 24 hours. The mass ratio of chitosan, folic acid, polyethylene glycol carboxylic acid, N-hydroxysuccinimide, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride was 40:11:40:11:19. After the reaction, the supernatant was collected by centrifugation. 1M sodium hydroxide solution was added to adjust the pH of the supernatant to 9.0. Precipitate B was collected by centrifugation, dissolved in an appropriate amount of methanol, and then subjected to H2O2 reaction. +The pH of the cation exchange resin was adjusted to neutral, and ether was added to precipitate the precipitate C. After drying, the precipitate C was redissolved in an appropriate amount of dimethylformamide and placed in a dialysis bag (Mn cutoff: 3.5 kDa) for dialysis for 24 h. Then, the precipitate was dialyzed with water for 5 days, with the water changed every 12 h. Finally, the suspension in the dialysis bag was freeze-dried to obtain folic acid-functionalized chitosan nanoparticles.

[0038] S3. Dissolve the above-mentioned folic acid-functionalized chitosan nanoparticles in an appropriate amount of acetic acid solution (acetic acid concentration: 0.1M, pH=4.7), heat to 55℃ and stir to dissolve, then cool to room temperature. Next, add the above-mentioned boron-10-doped Fe3O4 nanozyme to the solution and stir at room temperature for 48 hours. The mass ratio of folic acid-functionalized chitosan nanoparticles to boron-10-doped Fe3O4 nanozyme is 3:2. Then, add 1M sodium hydroxide solution to adjust the pH to 9.0, centrifuge to collect precipitate D; suspend precipitate D in an appropriate amount of methanol solution, and then use H... + The pH was adjusted to neutral using a cation exchange resin, and the precipitate E was collected by centrifugation. The precipitate E was washed three times with water and ethanol, respectively, and then freeze-dried. After separation by magnetization, the tumor-targeting boron-containing Fe3O4-based nanozyme, denoted as B-Fe3O4-CFPEG, was obtained.

[0039] Example 2: This example provides a tumor-targeting boron-containing Fe3O4-based nanozyme, the preparation method of which specifically includes the following steps:

[0040] S1. Ferric chloride, sodium acetate, polyethylene glycol, and boron-10 acid were dissolved in an appropriate amount of ethylene glycol at a mass ratio of 14:680:0.5:2 and stirred vigorously for 30 min to obtain a mixed solution. The mixed solution was then reacted in a high-pressure reactor at 190°C for 2 h under nitrogen atmosphere. After the reaction was completed, the mixture was cooled to room temperature and ethanol was added, resulting in the precipitation of a black precipitate. Precipitate A was collected by centrifugation, washed three times with water and ethanol respectively, dried at 60°C for 6 h, and separated by a magnet to obtain boron-10 doped Fe3O4 nanozyme.

[0041] S2. Chitosan was dissolved in an appropriate amount of acetic acid solution (acetic acid concentration 0.1M, pH=4.7), heated to 55℃, and cooled to room temperature after the solution became transparent. Then, a dimethyl sulfoxide solution containing folic acid, polyethylene glycol carboxylic acid, N-hydroxysuccinimide, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride was added to the solution. The reaction was carried out at room temperature in the dark for 24 hours. The mass ratio of chitosan, folic acid, polyethylene glycol carboxylic acid, N-hydroxysuccinimide, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride was 38:12:42:12:20. After the reaction, the supernatant was collected by centrifugation. 1M sodium hydroxide solution was added to adjust the pH of the supernatant to 8.7. Precipitate B was collected by centrifugation, dissolved in an appropriate amount of methanol, and then subjected to H2O2 reaction. + The pH of the cation exchange resin was adjusted to neutral, and ether was added to precipitate the precipitate C. After drying, the precipitate C was redissolved in an appropriate amount of dimethylformamide and placed in a dialysis bag (Mn cutoff: 3.5 kDa) for dialysis for 24 h. Then, the precipitate was dialyzed with water for 5 days, with the water changed every 12 h. Finally, the suspension in the dialysis bag was freeze-dried to obtain folic acid-functionalized chitosan nanoparticles.

[0042] S3. Dissolve the above-mentioned folic acid-functionalized chitosan nanoparticles in an appropriate amount of acetic acid solution (acetic acid concentration: 0.1M, pH=4.7), heat to 55℃ and stir to dissolve, then cool to room temperature. Next, add the above-mentioned boron-10-doped Fe3O4 nanozyme to the solution and stir at room temperature for 48 hours. The mass ratio of folic acid-functionalized chitosan nanoparticles to boron-10-doped Fe3O4 nanozyme is 2:1. Then, add 1M sodium hydroxide solution to adjust the pH to 8.7, centrifuge to collect precipitate D; suspend precipitate D in an appropriate amount of methanol solution, and then use H... + The pH was adjusted to neutral using a cation exchange resin, and the precipitate E was collected by centrifugation. The precipitate E was washed three times with water and ethanol, respectively, and then freeze-dried. After separation by magnet, the tumor-targeting boron-containing Fe3O4-based nanozyme was obtained.

[0043] Example 3: This example provides a tumor-targeting boron-containing Fe3O4-based nanozyme, the preparation method of which specifically includes the following steps:

[0044] S1. Ferric chloride, sodium acetate, polyethylene glycol, and boron-10 acid were dissolved in an appropriate amount of ethylene glycol at a mass ratio of 18:640:1.5:4 and stirred vigorously for 30 min to obtain a mixed solution. The mixed solution was then reacted in a high-pressure reactor at 210°C for 2 h under nitrogen atmosphere. After the reaction was completed, the mixture was cooled to room temperature and ethanol was added, resulting in the precipitation of a black precipitate. Precipitate A was collected by centrifugation, washed three times with water and ethanol respectively, dried at 60°C for 6 h, and separated by a magnet to obtain boron-10 doped Fe3O4 nanozyme.

[0045] S2. Chitosan was dissolved in an appropriate amount of acetic acid solution (acetic acid concentration 0.1M, pH=4.7), heated to 55℃, and cooled to room temperature after the solution became transparent. Then, a dimethyl sulfoxide solution containing folic acid, polyethylene glycol carboxylic acid, N-hydroxysuccinimide, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride was added to the solution. The reaction was carried out at room temperature in the dark for 24 hours. The mass ratio of chitosan, folic acid, polyethylene glycol carboxylic acid, N-hydroxysuccinimide, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride was 42:10:38:10:18. After the reaction, the supernatant was collected by centrifugation. 1M sodium hydroxide solution was added to adjust the pH of the supernatant to 9.3. Precipitate B was collected by centrifugation, dissolved in an appropriate amount of methanol, and then subjected to H2O2 reaction. + The pH of the cation exchange resin was adjusted to neutral, and ether was added to precipitate the precipitate C. After drying, the precipitate C was redissolved in an appropriate amount of dimethylformamide and placed in a dialysis bag (Mn cutoff: 3.5 kDa) for dialysis for 24 h. Then, the precipitate was dialyzed with water for 5 days, with the water changed every 12 h. Finally, the suspension in the dialysis bag was freeze-dried to obtain folic acid-functionalized chitosan nanoparticles.

[0046] S3. Dissolve the above-mentioned folic acid-functionalized chitosan nanoparticles in an appropriate amount of acetic acid solution (acetic acid concentration: 0.1M, pH=4.7), heat to 55℃ and stir to dissolve, then cool to room temperature. Next, add the above-mentioned boron-10-doped Fe3O4 nanozyme to the solution and stir at room temperature for 48 hours. The mass ratio of folic acid-functionalized chitosan nanoparticles to boron-10-doped Fe3O4 nanozyme is 4:3. Then, add 1M sodium hydroxide solution to adjust the pH to 9.3, centrifuge to collect precipitate D; suspend precipitate D in an appropriate amount of methanol solution, and then use H... + The pH was adjusted to neutral using a cation exchange resin, and the precipitate E was collected by centrifugation. The precipitate E was washed three times with water and ethanol, respectively, and then freeze-dried. After separation by magnet, the tumor-targeting boron-containing Fe3O4-based nanozyme was obtained.

[0047] Performance Testing: 1. The tumor-targeting boron-containing Fe3O4-based nanozyme (B-Fe3O4-CFPEG) prepared in Example 1 was observed by transmission electron microscopy, its particle size distribution was measured, and it was characterized by infrared spectroscopy (IR) and X-ray photoelectron spectroscopy (XPS). The results are as follows: Figures 1-4 As shown. From Figure 1 It can be seen that the tumor-targeting boron-containing Fe3O4-based nanozyme prepared in the embodiments of the present invention consists of uniform spherical particles; from Figure 2 It can be seen that the average particle size of the tumor-targeting boron-containing Fe3O4-based nanozyme prepared in the embodiments of the present invention is 142 nm; from Figure 3It can be seen that the tumor-targeting boron-containing Fe3O4-based nanozyme prepared in the embodiments of the present invention has amide characteristic absorption peaks, Fe-OB and Fe-N absorption peaks; from Figure 4 It can be seen that the tumor-targeting boron-containing Fe3O4-based nanozyme prepared in the embodiments of the present invention contains the elements iron, boron, carbon, nitrogen and oxygen.

[0048] II. Detection of intracellular uptake of tumor-targeting boron-containing Fe3O4-based nanozyme (B-Fe3O4-CFPEG): Mouse fibroblasts (L929) and mouse skin melanoma cells (B16-F10) were analyzed at a ratio of 1×10⁻⁶. 5 10 cells were seeded per well in a 24-well plate. After the cells adhered, they were treated with 2, 5, or 10 μg of [a specific agent / method]. 10 Cells were treated with either the control group (boron drug, BPA) or the B-Fe3O4-CFPEG prepared in Example 1 for 24 hours (four parallel experiments were set up for each group, and a blank control group was set up). After treatment, the culture medium was discarded, and 1 mL of streptomycin solution with a concentration of 2 mg / mL was added to each well to remove the conjugates on the cell surface. Cells were collected, lysed, and intracellular contents were detected by ICP-MS. 10 B content. Intracellular uptake results are as follows: Figure 5 As shown, the results indicate that the uptake of B-Fe3O4-CFPEG in both L929 and B16-F10 cells was concentration- and time-dependent. At the same administered concentration, the uptake of B-Fe3O4-CFPEG in B16-F10 cells was significantly higher than that in L929 cells. Furthermore, the uptake capacity of B-Fe3O4-CFPEG in B16-F10 cells was significantly higher than that in the control group BPA, indicating that B-Fe3O4-CFPEG exhibits a stronger tumor uptake capacity, meeting the requirement for high tumor selectivity of boron-containing drugs in BNCT treatment.

[0049] III. Cytotoxicity assay: L929 and B16-F10 cell lines were seeded into 96-well plates (2 × 10⁶ cells per well). 4 (1 cell), after the cells adhered, different concentrations of BPA or B-Fe3O4-CFPEG (2, 5 or 10 μg) prepared in Example 1 were administered. 10 Cells were treated with a ELISA reader (B / mL, 4 replicates per group, with a blank control group) for 24 h. Then, 10 μL / well of CellCounting Kit-8 solution was added and incubated for 2.5 h. After incubation, the absorbance of each well at 450 nm was measured using a microplate reader, and cell viability was calculated. The results are shown below. Figure 6As shown in the figure. Cell viability assay results indicated that the control group BPA and B-Fe3O4-CFPEG exhibited low toxicity to L929 and B16-F10 cells within the effective concentration range, with all cell viability exceeding 75%. Low toxicity is an important condition for boron-containing drugs or delivery agents in BNCT applications; therefore, B-Fe3O4-CFPEG has the potential to become a boron-containing drug for BNCT applications.

[0050] IV. Detection of Intracellular Reactive Oxygen Species (ROS): B16-F10 cells treated with BPA or B-Fe3O4-CFPEG prepared in Example 1 and then irradiated with thermal neutrons were collected. The cells were incubated with the DCFH-DA fluorescent probe in the dark for 20 minutes. Cells were then collected, and the expression of intracellular ROS was detected by flow cytometry. The results are as follows: Figure 7 As shown in the figure, B-Fe3O4-CFPEG exhibits a catalytic effect within tumor cells, significantly increasing ROS expression compared to the control group and the BPA group. During treatment, tumor cells treated with B-Fe3O4-CFPEG showed the most significant increase in ROS after BNCT, indicating its strongest ability to generate reactive oxygen species.

[0051] V. Apoptosis Assay: For apoptosis analysis, B16-F10 cells prepared in Example 1, treated with B-Fe3O4-CFPEG or BPA and then irradiated with thermal neutrons, were collected. The treated cell samples were stained with Annexin V-FITC / PI at room temperature in the dark for 15 minutes. The percentage of apoptotic cells was determined by flow cytometry, and the results are shown in Table 1.

[0052] Table 1. Results of apoptosis assay

[0053]

[0054] As shown in Table 1, the tumor-targeting boron-containing Fe3O4-based nanozyme provided in this embodiment of the invention can effectively induce apoptosis in mouse melanoma cells (B16-F10) in BNCT through a combined catalytic effect.

[0055] VI. Detection of therapeutic effect in tumor-bearing mice: B16-F10 tumor-bearing mice were intraperitoneally injected with a dose of 20 mg. 10 The test sample, at a concentration of B / kg, was irradiated with thermal neutrons onto mouse tumor sites for 2 hours. Throughout the 42-day experiment, the same vernier calipers were used daily, and the volume was measured using the formula: Volume = (Length × Width) / (Length × Width). 2The tumor volume of all mice was measured at 1 / 2. Data were recorded and tumor volume change curves and survival curves were plotted. Based on the results, the therapeutic effects of different groups (blank control group; BPA group: injected with BPA but without neutron irradiation; B-Fe3O4-CFPEG group: injected with B-Fe3O4-CFPEG prepared in Example 1 but without neutron irradiation; neutron irradiation only group; BPA + neutron irradiation group: injected with BPA and neutron irradiation; B-Fe3O4-CFPEG + neutron irradiation group: injected with B-Fe3O4-CFPEG prepared in Example 1 and neutron irradiation) were evaluated. The results are as follows: Figure 8 As shown in the figure, without thermal neutron irradiation, BPA did not affect tumor growth; however, after administration of B-Fe3O4-CFPEG, tumor growth was inhibited to some extent, indicating that B-Fe3O4-CFPEG can exert a certain anti-tumor effect in vivo. In tumor-bearing mice treated with both B-Fe3O4-CFPEG and BPA, the tumor growth rate was significantly inhibited after thermal neutron irradiation, with the B-Fe3O4-CFPEG group showing the most significant tumor growth inhibition. Furthermore, compared with other experimental groups, the average survival time of mice irradiated with thermal neutrons after B-Fe3O4-CFPEG administration was significantly prolonged. Therefore, B-Fe3O4-CFPEG, in combination with its catalytic effect, can significantly inhibit tumor growth and prolong the survival time of tumor-bearing mice in BNCT, exhibiting excellent anti-tumor efficacy.

[0056] In summary, the tumor-targeting boron-containing Fe3O4-based nanozyme provided in this embodiment of the invention has a strong selective enrichment ability for melanoma and exhibits low toxicity. In BNCT, it has a strong oxidative damage and apoptosis-inducing effect on melanoma cells. At the same time, it can combine catalytic effects in BNCT to effectively inhibit tumor growth and prolong the survival time of tumor-bearing mice, showing good application prospects.

[0057] Based on the above-described preferred embodiments of the present invention, and through the foregoing description, those skilled in the art can make various changes and modifications without departing from the inventive concept. The technical scope of this invention is not limited to the contents of the specification.

Claims

1. A tumor-targeting boron-containing Fe3O4-based nanozyme, characterized in that, The tumor-targeting boron-containing Fe3O4-based nanozyme uses boron-10-doped Fe3O4 nanozyme as the boron-containing drug group and enzyme active center, and its surface is modified with a targeting group; the targeting group is chitosan-folic acid-polyethylene glycol.

2. A method for preparing a tumor-targeting boron-containing Fe3O4-based nanozyme as described in claim 1, characterized in that, Includes the following steps: Ferric chloride, sodium acetate, polyethylene glycol, and boron-10 acid were dissolved in ethylene glycol and stirred to obtain a mixed solution. The mixed solution was then placed at 190-210℃ for reaction. After the reaction was completed, the solution was cooled and ethanol was added, resulting in the precipitation of a black precipitate. Precipitate A was collected by centrifugation, washed, and dried. The precipitate A was then separated by a magnet to obtain boron-10-doped Fe3O4 nanozyme. Chitosan was dissolved in acetic acid solution and heated until the solution became clear. After cooling, a dimethyl sulfoxide solution containing folic acid, polyethylene glycol carboxylic acid, N-hydroxysuccinimide, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride was added to the solution, and the reaction was carried out in the dark. After the reaction was completed, the supernatant was collected by centrifugation. The pH of the supernatant was adjusted to 8.7-9.3, and precipitate B was collected by centrifugation. Precipitate B was dissolved in methanol and subjected to H2O reaction. + The pH of the cation exchange resin was adjusted to neutral, and ether was added to precipitate the precipitate C. The precipitate C was then redissolved in dimethylformamide and dialyzed to obtain folic acid-functionalized chitosan nanoparticles. Folic acid-functionalized chitosan nanoparticles were dissolved in acetic acid solution, heated until dissolved, cooled, and then boron-10-doped Fe3O4 nanozyme was added to the solution and stirred. The pH was adjusted to 8.7-9.3, and the precipitate D was collected by centrifugation. After suspending precipitate D in methanol solution, use H... + The pH was adjusted to neutral using a cation exchange resin, and the precipitate E was collected by centrifugation. After washing and freeze-drying the precipitate E, it was separated by a magnet to obtain the tumor-targeting boron-containing Fe3O4-based nanozyme.

3. The method for preparing tumor-targeting boron-containing Fe3O4-based nanozymes according to claim 2, characterized in that, The concentration of the acetic acid solution is 0.05-0.15M.

4. The method for preparing tumor-targeting boron-containing Fe3O4-based nanozymes according to claim 2, characterized in that, The mass ratio of the ferric chloride, sodium acetate, polyethylene glycol, and boric-10 acid is (14-18):(640-680):(0.5-1.5):(2-4).

5. The method for preparing tumor-targeting boron-containing Fe3O4-based nanozymes according to claim 2, characterized in that, The mass of chitosan, folic acid, polyethylene glycol carboxylic acid, N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride is (38-42):(10-12):(38-42):(10-12):(18-20).

6. The method for preparing tumor-targeting boron-containing Fe3O4-based nanozymes according to claim 2, characterized in that, The mass ratio of the folic acid-functionalized chitosan nanoparticles to the boron-10-doped Fe3O4 nanozyme is (2-4):(1-3).

7. A tumor-targeting boron-containing Fe3O4-based nanozyme prepared by any one of claims 2-6.

8. The use of a tumor-targeting boron-containing Fe3O4-based nanozyme as described in claim 1 or 7 in the preparation of tumor BNCT drugs.

9. The application according to claim 8, characterized in that, The tumor-targeting boron-containing Fe3O4-based nanozyme is used to selectively deliver boron-10 into tumor cells, and to induce tumor cell apoptosis and slow tumor growth, while generating reactive oxygen species to cause oxidative damage.

10. The application according to claim 8 or 9, characterized in that, The tumors include melanoma.

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