A radiosensitizer with multi-enzyme activity, its preparation method and application

Abstract

This invention discloses a radiosensitizer with multi-enzyme activity, its preparation method, and its application in the preparation of antitumor agents. The radiosensitizer comprises rhodium-copper alloy nanoparticles, trithiol-terminated polymethacrylic acid modified on the surface of the rhodium-copper alloy nanoparticles, and lactate oxidase bound to the trithiol-terminated polymethacrylic acid. The preparation method involves mixing rhodium-copper alloy nanoparticles with trithiol-terminated polymethacrylic acid bound to their surface, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and N-hydroxysuccinimide ester, stirring, adding lactate oxidase, and continuing stirring to obtain the radiosensitizer. This invention achieves precise assembly of the nanocarrier and enzyme-active components through modular design. This invention provides an efficient and safe solution for integrated tumor diagnosis and treatment, and has significant potential for industrial production.

Description

Technical Field

[0001] This invention relates to a nano-radiotherapy sensitizer, its preparation method and application, belonging to the fields of nanomaterial chemistry and biochemistry. Background Technology

[0002] Radiation therapy is a non-invasive treatment for tumors in clinical practice. It uses high-energy rays (X-rays, gamma rays, alpha particle beams, or electron beams) to irradiate tumor cells, damaging their DNA. Furthermore, high-energy rays can ionize water in tissues, generating reactive oxygen species (ROS). These ROS can react with most biomolecules (including DNA, proteins, and lipids), damaging cell structure and causing cell death, thereby inhibiting tumor growth. According to the World Health Organization, more than 70% of tumors require radiation therapy, and 40% can be cured with it.

[0003] Because radiation is non-specific, it can damage surrounding normal tissues. Therefore, in actual treatment, treatment plans often balance therapeutic efficacy with potential side effects, which can lead to decreased treatment effectiveness. Furthermore, solid tumors are generally hypoxic, requiring three times the radiation dose to kill hypoxic cells compared to normoxic cells, exhibiting radioresistance. Therefore, there is a need to develop a radiosensitizer to improve treatment efficacy while reducing radiotherapy side effects.

[0004] Most high atomic number inorganic nanomaterials possess high X-ray absorption capacity, effectively reducing radiation dose and increasing the killing effect on tumor cells. However, the hypoxic state of the tumor microenvironment makes it difficult for single-energy deposition to completely kill tumor cells. Nanozymes are a new type of biomimetic inorganic nanomaterial with intrinsic enzyme-mimicking activity, capable of highly efficient catalytic reactions like natural enzymes. Through rational design, nanozymes can be used to improve the tumor microenvironment and increase the sensitivity of tumor cells to high-energy radiation. Furthermore, a large amount of lactic acid is produced during tumor development, typically in excess and accumulating in all types of tumors. Studies have shown that lactic acid not only acts as a byproduct of tumor development but also promotes tumor proliferation and metastasis. By consuming lactic acid within the tumor, the tumor microenvironment can be reconstructed, further inhibiting tumor cell growth. Simultaneously reshaping the tumor microenvironment while irradiating tumor cells with high-energy radiation can significantly kill tumor cells. Therefore, combining nanozymes with radiotherapy to reshape the tumor microenvironment through multiple methods holds promise for meeting clinical needs and providing a new strategy for highly effective cancer treatment in the future. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a method for preparing and applying a rhodium-copper alloy nanozyme with radiosensitizing effect, so as to solve the problems that existing radiosensitizers cannot improve the tumor microenvironment, and that tumor cells are not sensitive to radiotherapy and require large radiotherapy doses.

[0006] To solve the above problems, the technical solution of the present invention is as follows:

[0007] A radiosensitizer with multi-enzyme activity includes rhodium-copper alloy nanoparticles, trithiol-terminated polymethacrylic acid modified on the surface of the rhodium-copper alloy nanoparticles, and lactate oxidase bound to the trithiol-terminated polymethacrylic acid.

[0008] Preferably, the trithiol-terminated polymethacrylic acid is bound to the surface of the rhodium-copper alloy nanoparticles through coordination, and the lactate oxidase is bound to the trithiol-terminated polymethacrylic acid through covalent interaction.

[0009] Preferably, the radiosensitizer has an average diameter of 175 nm.

[0010] Preferably, the mass ratio of the rhodium-copper alloy nanoparticles to the trithiol-terminated polymethacrylic acid is 0.01-0.05:1, and the mass ratio of the rhodium-copper alloy nanoparticles to the lactate oxidase is 2-5:1.

[0011] The present invention also provides a method for preparing the above-mentioned radiosensitizer with multi-enzyme activity: rhodium-copper alloy nanoparticles of polymethacrylic acid with trithiol-terminated surface, 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide ester are mixed and stirred, lactate oxidase is added and stirring is continued to obtain the radiosensitizer.

[0012] Preferably, the method for preparing the rhodium-copper alloy nanoparticles with trithiol-terminated polymethacrylic acid is as follows: mixing the rhodium-copper alloy nanoparticles with trithiol-terminated polymethacrylic acid and stirring to obtain the rhodium-copper alloy nanoparticles with trithiol-terminated polymethacrylic acid.

[0013] More preferably, the preparation method of the rhodium-copper alloy nanoparticles is as follows: dissolve polyoxypropylene-polyoxyethylene copolymer in a mixture of DMF and dilute HCl and stir for 10 minutes, then add sodium hexachlororhodium aqueous solution, copper chloride aqueous solution and ascorbic acid and stir for 10 minutes, transfer the above solution to a reaction vessel and place it in an oven at 100°C for 4 hours to obtain rhodium-copper alloy nanoparticles.

[0014] Preferably, the stirring is specifically performed by magnetic stirring at 200 rpm at room temperature; the stirring time is 12 hours.

[0015] The present invention also provides the application of the above-mentioned radiosensitizer with multi-enzyme activity in the preparation of formulations for treating tumors.

[0016] The multi-enzyme-active radiosensitizer described in this invention possesses photoacoustic imaging capabilities. While utilizing the tumor microenvironment for catalytic activity, it also enhances the sensitivity of tumor cells to high-energy radiation. This multi-enzyme-active radiosensitizer efficiently accumulates and selectively catalyzes at the tumor site, and the loaded lactate oxidase can directly exert its effect at the tumor site, supplementing H2O2 and achieving synergistic therapy. Therefore, this multi-enzyme-active radiosensitizer has promising applications in the diagnosis and treatment of tumors. Attached Figure Description

[0017] Figure 1 The images show transmission electron microscopy (TEM) images, elemental distribution, near-infrared absorption spectra, and X-ray diffraction patterns of the RhCu nanoparticles synthesized in Example 1.

[0018] Figure 2 RhCu and RhCu@LO in Example 2 x Particle size and zeta potential;

[0019] Figure 3 The reaction kinetics of CAT-like activity of RhCu in Example 3, as well as CAT-like, POD-like, and OXD-like enzyme activities;

[0020] Figure 4 RhCu and RhCu@LO were evaluated in Example 4. x Cell-killing effect;

[0021] Figure 5 Example 5 evaluated the effect of tumor cells on RhCu@LO x Intake;

[0022] Figure 6 In Example 6, the multi-enzyme activities of RhCu in cells were evaluated, including POD-like enzyme activity, CAT-like enzyme activity and OXD-like enzyme activity.

[0023] Figure 7 To evaluate RhCu and RhCu@LO in Example 7 x As a radiosensitizer, its cell-killing effect;

[0024] Figure 8 RhCu and RhCu@LO were evaluated in Example 8. x Its performance as a radiosensitizer in cell cloning;

[0025] Figure 9 RhCu and RhCu@LO were evaluated in Example 9. xThe radiosensitizing effect of radiotherapy damages cellular DNA;

[0026] Figure 10 The tail vein injection of RhCu@LO was evaluated in Example 10. x Changes in photoacoustic signal values ​​at the posterior tumor site over time;

[0027] Figure 11 RhCu@LO was evaluated in Example 11. x The inhibitory effect of radiosensitizer on tumors and the change in mouse body weight during treatment;

[0028] Figure 12 RhCu@LO was evaluated in Example 12. x H&E, Ki-67, and Tunel staining images of tumor tissue after treatment as a radiosensitizer. Detailed Implementation

[0029] To make the present invention more apparent and understandable, preferred embodiments are described in detail below with reference to the accompanying drawings.

[0030] This invention provides a radiosensitizer with multiple enzyme activities, comprising: rhodium-copper alloy nanoparticles (RhCu), trithiol-terminated polymethacrylic acid (PTMP-PMAA) bound to the surface of the rhodium-copper alloy nanoparticles, and lactate oxidase (LO) bound to the trithiol-terminated polymethacrylic acid. x ).

[0031] This invention uses RhCu of polymethacrylic acid with surface-modified trithiol-terminated structure as a carrier to load natural lactate oxidase (LO). x ), forming a radiosensitizer with the aforementioned multi-enzyme activity (denoted as RhCu@LO). x RhCu undergoes an amidation reaction (the carboxyl group of PTMP-PMAA and LO). x The covalent interaction of the amino groups on the surface can support lactate oxidase. Lactate oxidase, a natural enzyme, can efficiently decompose lactate into hydrogen peroxide. RhCu, when combined with lactate oxidase, effectively overcomes the limitations of single-catalysis and reduces the toxic side effects of lactate oxidase. This multi-enzyme synergistic catalytic therapy combined with radiotherapy achieves effective tumor suppression. Furthermore, RhCu has excellent absorption capacity in the near-infrared region; therefore, the aforementioned radiosensitizer RhCu@LO... x It can be used for radiotherapy combined with catalytic therapy under photoacoustic imaging guidance.

[0032] This embodiment utilizes RhCu to load LO x Used for sensitizing and synergistic catalytic therapy in tumor radiotherapy. This example uses RhCu@LO. xIt has the following advantages: 1.RhCu@LO x 1. As a radiosensitizer, it effectively overcomes the problem of tumor insensitivity to single radiotherapy; 2. It alleviates the hypoxic state of tumors and disrupts the tumor microenvironment to specifically generate reactive oxygen species at the tumor site, thereby achieving catalytic therapy for tumors; 3. It significantly reduces the side effects of radiotherapy; 4. It enables tumor catalytic therapy monitored by photoacoustic imaging.

[0033] In this invention, the trithiol-terminated polymethacrylic acid is bound to the surface of the rhodium-copper alloy nanoparticles through coordination, and the lactate oxidase is bound to the trithiol-terminated polymethacrylic acid through covalent interaction.

[0034] In one embodiment, the RhCu@LO x The average diameter is approximately 175 nm.

[0035] In one embodiment, the mass ratio of the rhodium-copper alloy nanoparticles to the trithiol-terminated polymethacrylic acid is 0.01-0.05:1, and the mass ratio of the rhodium-copper alloy nanoparticles to the lactate oxidase is 2-5:1.

[0036] The present invention also provides a method for preparing the radiosensitizer with the aforementioned multi-enzyme activity, comprising the steps of:

[0037] S1. Provides rhodium-copper alloy nanoparticles of polymethacrylic acid with trithiol-terminated surface bonding;

[0038] S2. The rhodium-copper alloy nanoparticles of polymethacrylic acid with trithiol-terminated surface, 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide ester (NHS) are mixed and stirred. Lactate oxidase is added and stirring is continued to obtain the radiosensitizer with the multi-enzyme activity.

[0039] In step S1, in one embodiment, the method for preparing the rhodium-copper alloy nanoparticles of polymethacrylic acid with trithiol-terminated surfaces includes the following steps:

[0040] S11, provides rhodium-copper alloy nanoparticles (RhCu);

[0041] S12. The rhodium-copper alloy nanoparticles are mixed with trithiol-terminated polymethacrylic acid and stirred to obtain rhodium-copper alloy nanoparticles with trithiol-terminated polymethacrylic acid on the surface.

[0042] In step S11, in one embodiment, the RhCu@LO xThe preparation method includes the following steps: dissolving polyoxypropylene-polyoxyethylene copolymer in a mixture of DMF and dilute HCl and stirring for 10 minutes; then adding sodium hexachlororhodium aqueous solution, copper chloride aqueous solution, and ascorbic acid and stirring for 10 minutes; transferring the above solution to a reaction vessel and placing it in an oven at 100°C for 4 hours to obtain RhCu. In the example, RhCu was prepared using a reduction method.

[0043] In one embodiment, step S12 specifically includes: mixing the rhodium-copper alloy nanoparticles with trithiol-terminated polymethacrylic acid and sonicating for 30 min, and then magnetically stirring at 200 rpm for 12 h at room temperature to obtain rhodium-copper alloy nanoparticles with trithiol-terminated polymethacrylic acid on the surface.

[0044] In step S2, the rhodium-copper alloy nanoparticles of polymethacrylic acid with trithiol-terminated surfaces are activated by NHS and EDC, mixed evenly with lactate oxidase, and stirred to obtain the radiosensitizer with the multi-enzyme activity.

[0045] In one embodiment, step S2 specifically includes: mixing the rhodium-copper alloy nanoparticles of polymethacrylic acid with trithiol-terminated surface, 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide ester, magnetically stirring at 200 rpm at room temperature, adding lactate oxidase and continuing stirring for 12 h to obtain the radiosensitizer with the multi-enzyme activity.

[0046] In one embodiment, the mass ratio of the rhodium-copper alloy nanoparticles to the trithiol-terminated polymethacrylic acid is 0.01-0.05:1, and the mass ratio of the rhodium-copper alloy nanoparticles to the lactate oxidase is 2-5:1.

[0047] As one specific embodiment, the method for preparing RhCu includes the following steps:

[0048] a) Preparation of RhCu alloy nanoparticles: Polyoxypropylene-polyoxyethylene copolymer was dissolved in a mixture of N,N-dimethylformamide and dilute hydrochloric acid and stirred for 10 minutes. Then, an aqueous solution of sodium hexachlororhodium, an aqueous solution of copper chloride, and ascorbic acid were added and stirred for 10 minutes. The solution was transferred to a reaction vessel and placed in an oven at 100°C for 4 hours to obtain RhCu. In this example, RhCu was prepared using a reduction method.

[0049] b) Purification of rhodium-copper alloy nanoparticles: The rhodium-copper alloy nanoparticles prepared in step a) above were washed by centrifugation multiple times with deionized water and ethanol to obtain purified rhodium-copper alloy nanoparticles.

[0050] c) Rhodium-copper alloy nanoparticles of polymethacrylic acid with trithiol-terminated surface: The rhodium-copper alloy nanoparticles prepared in step b) above are mixed with trithiol-terminated polymethacrylic acid at a mass ratio of 0.01-0.05:1, sonicated for 30 min, and stirred at 200 rpm for 12 h at room temperature to obtain rhodium-copper alloy nanoparticles of polymethacrylic acid with trithiol-terminated surface.

[0051] d)RhCu@LO x Synthesis: Rhodium-copper alloy nanoparticles of polymethacrylic acid with surface-bound trithiol-terminated polymethacrylic acid, 1-ethyl(-3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide ester were mixed and magnetically stirred at 200 rpm at room temperature. Lactate oxidase was added and stirring was continued for 12 h to obtain the radiosensitizer with the multi-enzyme activity.

[0052] This invention provides a method for preparing a multi-enzyme-active radiosensitizer and its application in tumor treatment. The efficient accumulation and specific catalytic effect of this radiosensitizer at the tumor site significantly reduces the systemic toxicity of lactate oxidase. Therefore, this radiosensitizer has broad application prospects in the field of tumor diagnosis and treatment. Furthermore, this multi-enzyme-active radiosensitizer can also be used as a photoacoustic contrast agent for photoacoustic imaging while treating tumors.

[0053] Example 1: Preparation of RhCu

[0054] 20 mg of polyoxypropylene-polyoxyethylene copolymer was dissolved in 3.2 mL of N,N-dimethylformamide, followed by the addition of 0.8 mL of dilute hydrochloric acid (1 M) and stirring for 10 min. Separately, 75 mg of sodium hexachlororhodium was dissolved in 4 mL of deionized water and added to the above solution. 10 mg of copper chloride was dissolved in 4 mL of deionized water and added to the above solution. 138 mg of ascorbic acid was dissolved in 8 mL of deionized water and added to the above solution. The mixture was stirred for another 10 min, and then transferred to a hydrothermal reactor and reacted at 100 °C for 4 h. After the reaction was complete and cooled to room temperature, rhodium-copper alloy nanoparticles were obtained.

[0055] Figure 1 In the diagram, a and b are the TEM images and elemental distribution maps of RhCu; c is the UV-Vis-NIR spectrum of RhCu aqueous solution; and d is the X-ray diffraction spectrum of RhCu and its comparison with the standard card. Figure 1 As can be seen from d, the synthesized RhCu can perfectly match the peak of metallic Ru, while Cu may not appear clearly in the spectrum due to insufficient content.

[0056] Example 2: RhCu@LO x Preparation

[0057] 10 mg of rhodium-copper alloy nanoparticles were dispersed in 10 mL of ethanol, then 100 mg of trithiol-terminated polymethacrylic acid was added. The mixture was sonicated for 30 min and magnetically stirred at 200 rpm for 12 h at room temperature to obtain rhodium-copper alloy nanoparticles with trithiol-terminated polymethacrylic acid on the surface. After deionization and centrifugation, the nanoparticles were dissolved in water, activated with EDC and NHS, and then 5 mg of LO was added. x After stirring at room temperature for 12 hours, RhCu@LO was obtained. x After deionization and centrifugation, it was dissolved in water. RhCu and RhCu@LO were measured separately. x The zeta potential, the results are as follows Figure 2 As shown.

[0058] Figure 2 In the above, a represents RhCu and RhCu@LO. x Hydrated particle size; b represents RhCu and RhCu@LO x The change in zeta potential; this result indicates that LO x The successful modification.

[0059] Example 3: Evaluation of CAT-like, POD-like, and OXD-like activities of RhCu

[0060] The CAT enzyme activity of RhCu was characterized by measuring the dissolved oxygen produced by RhCu in H₂O₂ solution at room temperature. 150 μL of RhCu aqueous solution (1 mg / mL) was added to 2.85 mL of PBS (pH = 7.4), along with 3 μL of 30% H₂O₂ solution. The O₂ concentration in the mixture was measured every 30 s using a special oxygen electrode of a multi-parameter analyzer. The experimental results are shown below. Figure 3 Figures a and b. The POD activity of RhCu was evaluated using TMB·HCl as a substrate. 5 μL of 150 mM TMB·HCl aqueous solution and 1 μL of 50 μg / mL RhCu aqueous solution were added to 1.5 mL of water. Then, different volumes of 1.5 M H₂O₂ solution (0.2, 0.4, 0.8, 2, and 4 μL, respectively) were added. The absorbance of the substrate TMB·HCl at 652 nm was measured over time at 37 °C. Figure 3 As shown in Figure c. The OXD enzyme activity of RhCu was evaluated using TMB·HCl as a substrate. 1.5 μL of RhCu aqueous solution (1 mg / mL) was added to 1.5 mL of water, followed by different volumes (2, 4, 6, 8, and 10 μL) of 150 mM TMB·HCl aqueous solution. The absorbance of the substrate TMB·HCl at 652 nm was measured over time at 37 °C. Figure 3 As shown in d.

[0061] like Figure 3 In the control group, RhCu can efficiently catalyze the decomposition of H2O2 and produce a large amount of O2, while the changes in the control group are almost negligible. This indicates that RhCu has highly efficient CAT-like enzyme activity. Figure 3 Figures b, c, and d show that its CAT-like enzyme activity, POD-like enzyme activity, and OXD-like enzyme activity have similar kinetic characteristics to those of natural enzymes.

[0062] Example 4: Evaluation of RhCu@LO x Killing effect on tumor cells.

[0063] The specific conditions are as follows: 4T1 cells were fed at a rate of 1×10⁻⁶. 4 Initially, cells were seeded at a density of 100 cells / well in 96-well plates and cultured for 24 h at 37°C and 5% CO2 humidification. Subsequently, cells were treated with different concentrations of RhCu or RhCu@LO. x The cells were incubated for 12 h and 24 h with fresh culture medium (0, 0.625, 1.25, 2.5, 5, 10, 25, 50 μg / mL) for 12 h and 24 h respectively. Finally, the relative viability of the cells was determined using a CCK-8 kit and the absorbance of each well at 450 nm was measured using an ELISA reader.

[0064] like Figure 4 In cell a, after incubation with RhCu (25 μg / mL), cell viability decreased to below 60%. Figure 4 In b, RhCu@LO was used at a lower concentration. x After treatment with (5 μg / mL), the cell viability dropped to below 40%, exhibiting more pronounced cytotoxicity.

[0065] Example 5: Evaluation of cell response to RhCu@LO x absorption capacity

[0066] 4T1 cells were fed at a dose of 1×10 5 Initially, cells / dish were seeded at a density of 1,000 cells / dish and incubated overnight in a confocal dish, then FITC-grafted RhCu@LO was added. x RhCu@LO cells were incubated at 10 μg / mL for different durations (0, 1, 2, 4, 8, 12, 24 h) and finally observed using a confocal microscope. x Intake status.

[0067] like Figure 5 The fluorescence intensity of the cells was strongest at 12 h, indicating that the cells took up RhCu@LO at 12 h. x At most, this indicates that RhCu@LO x It can be taken up by cells.

[0068] Example 6: Evaluation of RhCu@LOx Multienzyme activity within cells

[0069] 4T1 cells were fed at a dose of 1×10 4 Initially, cells / well were seeded at a density of 100 cells / well in 96-well plates and incubated overnight at 37°C with 5% CO2 humidification. Subsequently, the cells were seeded with RhCu or RhCu@LO x The cells were incubated for 12 hours after replacing the original culture medium with fresh medium (10 μg / mL). Then, they were irradiated with X-rays (6 Gy) and incubated for another 6 hours. The cells were then stained with DCFH-DA reactive oxygen species dye and finally, the fluorescence inside the cells was observed using a fluorescence microscope.

[0070] 4T1 cells were fed at a dose of 1×10 4 Initially, cells / well were seeded at a density of 100 cells / well in 96-well plates and incubated overnight at 37°C with 5% CO2 humidification. Subsequently, the cells were seeded with RhCu or RhCu@LO x The cells were incubated for 12 hours after replacing the original culture medium with fresh medium (25 μg / mL). Then, they were irradiated with X-rays (6 Gy) and incubated for another 12 hours. DHE dye was added to stain the cells, and finally, the fluorescence inside the cells was observed using a fluorescence microscope.

[0071] 4T1 cells were seeded at an initial density of 1×10⁴ cells / well in 96-well plates and incubated overnight at 37°C with 5% CO₂. Subsequently, the cells were cultured in a hypoxic incubator for 12 h, incubated for 4 h with a [Ru(dpp)₃]Cl₂ probe, and then incubated with RhCu (25 μg / mL) or RhCu@LO₂O₃. x Replace the original culture medium with fresh culture medium (6.25, 12.5, 25, 50 μg / mL) and continue incubation for 4 hours. Finally, observe the fluorescence in the cells using a fluorescence microscope.

[0072] like Figure 6 In group a, compared with the control group, RhCu and RhCu@LO were added x After X-ray irradiation, the green fluorescence of the cells was significantly enhanced, indicating that RhCu can generate reactive oxygen species in the cells and has POD-like activity. The green fluorescence was further enhanced after X-ray irradiation, indicating that X-rays enhanced the generation of reactive oxygen species in the cells and produced a synergistic effect. Figure 6 In the RhCu group, compared with the control group, the red fluorescence was significantly enhanced, indicating the OXD-like activity of RhCu. x The cells in the group showed a more significant increase in red fluorescence, which is due to LO x The H₂O₂ produced from lactic acid was catalyzed by RhCu to produce oxygen, enhancing the OXD-like activity. The further enhancement of fluorescence after X-ray irradiation corresponds to… Figure 6The result of 'a'. Figure 6 c in RhCu@LO x In the group, the red fluorescence gradually decreased with increasing concentration, indicating that the intracellular oxygen content gradually increased, demonstrating the CAT-like activity of RhCu.

[0073] Example 7: Evaluation of RhCu@LO x Radiosensitizing effect

[0074] 4T1 cells were fed at a dose of 1×10 4 Initially, cells were seeded at a density of 100 cells / well in 96-well plates and cultured for 24 h at 37°C and 5% CO2 humidification. Subsequently, cells were treated with different concentrations of RhCu or RhCu@LO. x The original culture medium was replaced with fresh medium (25 μg / mL) and incubated for 12 h. Then, the cells were irradiated with different doses of X-rays (0, 2, 4, 6 Gy) and incubated for another 12 h. Cell viability was then detected using a CCK-8 assay kit and absorbance at 450 nm was measured using an ELISA reader to calculate cell viability.

[0075] like Figure 7 In the process, with the increase of irradiation dose, when using RhCu and RhCu@LO... x The viability of incubated cells showed a significant decrease, attributed to the CAT-like enzyme activity of RhCu utilizing H2O2 within tumor cells to generate O2, thus increasing the sensitivity of tumor cells to X-rays. In RhCu@LO x The incubated cells are more sensitive to X-rays, which is due to LO x It catalyzes the production of H2O2 from lactic acid in tumor cells, providing RhCu with more substrates to catalyze the production of more oxygen.

[0076] Example 8: Evaluation of RhCu@LO x Inhibition of cell clones under X-ray irradiation

[0077] 4T1 cells were seeded overnight in 6-well plates at an initial density of 2000 cells / well and divided into 6 treatment groups: control group, RhCu treatment group, RhCu@LO x Processing group (RhCu@LO) x ), X-ray irradiation group, RhCu treatment group under X-ray irradiation (RhCu+X-ray), RhCu@LO under X-ray irradiation x Processing group (RhCu@LO) x +X-Ray). Add RhCu or RhCu@LO xAfter incubation at 25 μg / mL for 12 h, cells were irradiated with X-rays (6 Gy) and cultured for another 12 h. The X-ray group underwent direct X-ray irradiation (6 Gy) followed by 12 h of culture. After treatment, cells were washed with PBS to remove surface material and then cultured in fresh medium for 5 days. Cells were fixed with 4% paraformaldehyde for approximately 20 minutes, washed twice with PBS, and then stained with Giemsa stain (1.5 mL / well) at room temperature for 30 minutes. After washing twice more with PBS, cells were air-dried, and finally, images were taken for analysis.

[0078] like Figure 8 As shown, in the RhCu group and RhCu@LO x The RhCu group inhibited cell clone formation, while after X-ray irradiation, the RhCu group and RhCu@LO... x The formation of cell clones in the group was further inhibited. This indicates that RhCu@LO x It has excellent radiosensitization capabilities.

[0079] Example 9: Evaluation of RhCu@LO x DNA damage caused by X-ray irradiation

[0080] 4T1 cells were fed at a dose of 1×10 5 Cells were initially seeded at the initial density in confocal dishes overnight and divided into 6 treatment groups: control group, RhCu treatment group, RhCu@LO x Processing group (RhCu@LO) x ), X-ray irradiation group, RhCu treatment group under X-ray irradiation (RhCu+X-ray), RhCu@LO under X-ray irradiation x Processing group (RhCu@LO) x +X-Ray). Add RhCu or RhCu@LO x After incubation for 12 h with 10 μg / mL, cells were irradiated with X-rays (6 Gy) and cultured for another 12 h. The X-ray group was directly irradiated with X-rays (6 Gy) and cultured for 12 h. Subsequently, cells were fixed with 4% paraformaldehyde for about 20 minutes, washed 3 times with PBS, blocked with immunofluorescence blocking solution for 20 minutes, incubated with γ-H2AX rabbit monoclonal antibody at room temperature for 1 h, washed 3 times with PBS, then incubated with anti-rabbit Cy3 fluorescent secondary antibody at room temperature for 1 h, washed 2 times with PBS, incubated with nuclear staining dye (DAPI) at room temperature for 5 min, washed 3 times with PBS, and finally observed with confocal microscopy for immunofluorescence signals.

[0081] like Figure 9As shown, the intensity of red fluorescence indicates the level of DNA damage. After X-ray irradiation, RhCu and RhCu@LO... x The red fluorescence intensity of the group was significantly improved compared to the group without X-ray irradiation, indicating that RhCu can enhance the damage of X-rays to tumor cell DNA and demonstrates excellent radiosensitization ability.

[0082] Example 10: Evaluation of RhCu@LO x A mouse model of breast cancer was established by analyzing photoacoustic signal changes at the tumor site after intravenous injection: Female athymic nude mice (six weeks old, 20-25g) were purchased, and a subcutaneous tumor model was established by subcutaneously injecting 100 μL of 4T1 cell PBS solution into the right hind limb of the mice (1×10⁻⁶). 6 (cells / each). When the tumor volume reaches 80mm... 3 At that time, 200 μL mg / mL RhCu@LO was injected via the tail vein. x Solution, using a small animal photoacoustic imaging system (Visual Sonics Vevo LAZR system) to detect RhCu@LO at the tumor site x Photoacoustic signals.

[0083] like Figure 10 As shown in Figures a and b, during the injection of RhCu@LO x Afterwards, the photoacoustic signal value at the tumor site reached its maximum at 8 hours, and then the signal gradually weakened.

[0084] Example 11: Evaluation of RhCu@LO x Inhibitory effect on tumor growth in mice

[0085] Establishing a mouse model of breast cancer: Female athymic nude mice (six weeks old, 20-25g) were purchased. A subcutaneous tumor model was established by subcutaneously injecting 100μL of 4T1 cell PBS solution into the right hind limb of the mice (1×10⁻⁶ cells per mouse). 6 (cells / each). When the tumor volume reaches 50mm... 3 Mice were divided into 6 groups for treatment: (I) control group; (II) RhCu treatment group; (III) RhCu@LO x Treatment group; (IV) X-ray irradiation group; (V) RhCu treatment group under X-ray irradiation; (VI) RhCu@LO under X-ray irradiation x Treatment group. RhCu and RhCu@LO were injected via tail vein, respectively. x Mice were given a PBS solution (10 mg / kg) as the control group, while mice were given a PBS solution (100 μL) injected into their bodies. Eight hours after administration, the tumor sites of the mice were irradiated with X-rays (6 Gy), and the tumor volume and mouse weight were measured every two days using calipers.

[0086] Figure 11 In the figure, 'a' represents the change in tumor volume over time in different treatment groups. It can be seen that compared to the X-Ray group alone, RhCu@LO... x Tumors in the RhCu+X-ray group showed good inhibition, and similarly, tumors in the RhCu+X-ray group also exhibited good inhibition, demonstrating RhCu's excellent radiosensitization ability. Compared to the control group, the RhCu group and RhCu@LO... x The group also showed a significant inhibitory effect, indicating that RhCu has excellent catalytic therapeutic activity. x The addition of [a specific ingredient] can further enhance its catalytic effect. During the two weeks of treatment, there were no significant changes in the body weight of mice in any group. In the early stages of radiotherapy, the mice experienced a slight decrease in body weight, which may be due to adverse reactions in the digestive system induced by radiotherapy. Subsequently, the mice's body weight gradually returned to normal. Figure 11 b) indicates that RhCu@LO x It has excellent biocompatibility.

[0087] Example 12: Evaluation of RhCu@LO x apoptosis level in tumor tissue after treatment

[0088] In Example 11, after the treatment was completed, tumor tissue from mice was taken and paraffin sections were prepared. The sections were stained with H&E, Ki-67 and Tunel, respectively.

[0089] Figure 12 This indicates that the tumor tissue in the RhCu+X-ray group suffered the most severe damage and exhibited a high level of apoptosis.

[0090] This invention provides a multi-enzyme-active radiosensitizer, its preparation method, and its application. The radiosensitizer significantly enhances radiosensitivity by targeting and regulating the tumor microenvironment, simultaneously achieving integrated diagnostic and therapeutic functions. The preparation method includes the following steps: The obtained radiosensitizer has the following technical advantages: 1) Based on its highly efficient enrichment capacity at the tumor site, it can specifically catalyze the decomposition of metabolites, significantly improving the hypoxic tumor microenvironment; 2) By optimizing the spatial distribution and synergistic effect of the enzyme active components, it reduces lactate oxidase (LO) levels. x The invention exhibits several advantages: 1) It minimizes off-target toxicity; 2) It also possesses photoacoustic imaging capabilities, enabling real-time dynamic monitoring of drug distribution and therapeutic effects. The preparation process of this invention employs a modular design with low reaction conditions, making it suitable for industrial-scale production. Animal experiments have verified that the sensitizer enhances radiotherapy efficacy in a 4T1 breast cancer model without significant systemic toxicity, demonstrating its excellent biosafety and clinical translational potential.

Claims

1. A radiosensitizer possessing multi-enzyme activity, characterized in that, It includes rhodium-copper alloy nanoparticles, trithiol-terminated polymethacrylic acid modified on the surface of the rhodium-copper alloy nanoparticles, and lactate oxidase bound to the trithiol-terminated polymethacrylic acid.

2. The radiosensitizer as described in claim 1, characterized in that, The trithiol-terminated polymethacrylic acid is bound to the surface of the rhodium-copper alloy nanoparticles through coordination, and the lactate oxidase is bound to the trithiol-terminated polymethacrylic acid through covalent interaction.

3. The radiosensitizer as described in claim 1, characterized in that, The average diameter of the radiosensitizer is 175 nm.

4. The radiosensitizer as described in claim 1, characterized in that, The mass ratio of the rhodium-copper alloy nanoparticles to the trithiol-terminated polymethacrylic acid is 0.01-0.05:1, and the mass ratio of the rhodium-copper alloy nanoparticles to the lactate oxidase is 2-5:

1.

5. A method for preparing a radiosensitizer with multi-enzyme activity as described in any one of claims 1-4, characterized in that, Rhodium-copper alloy nanoparticles of polymethacrylic acid with trithiol-terminated surfaces, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and N-hydroxysuccinimide ester were mixed and stirred. Lactate oxidase was added and stirring was continued to obtain the radiosensitizer.

6. The preparation method according to claim 5, characterized in that, The method for preparing the rhodium-copper alloy nanoparticles with trithiol-terminated polymethacrylic acid is as follows: mixing the rhodium-copper alloy nanoparticles with trithiol-terminated polymethacrylic acid and stirring to obtain the rhodium-copper alloy nanoparticles with trithiol-terminated polymethacrylic acid.

7. The preparation method according to claim 6, characterized in that, The preparation method of the rhodium-copper alloy nanoparticles is as follows: polyoxypropylene-polyoxyethylene copolymer is dissolved in a mixture of DMF and dilute HCl and stirred for 10 minutes. Then, sodium hexachlororhodium aqueous solution, copper chloride aqueous solution and ascorbic acid are added and stirred for 10 minutes. The above solution is transferred to a reaction vessel and placed in an oven at 100°C for 4 hours to obtain rhodium-copper alloy nanoparticles.

8. The preparation method according to claim 5, characterized in that, The stirring process specifically involves magnetic stirring at 200 rpm at room temperature; the stirring time is 12 hours.

9. The use of a radiosensitizer with multi-enzyme activity as described in any one of claims 1-4 in the preparation of a medicament for treating breast cancer.

Images