Radiotherapy sensitizer with multi-enzyme activity as well as preparation method and application thereof

Abstract

The invention discloses a radiotherapy sensitizer with multi-enzyme activity, a preparation method of the radiotherapy sensitizer and application of the radiotherapy sensitizer in preparation of preparations for treating tumors. The radiotherapy sensitizer comprises rhodium-copper alloy nanoparticles, trithiol-terminated polymethylacrylic acid modified on the surfaces of the rhodium-copper alloy nanoparticles, and lactate oxidase combined with the trithiol-terminated polymethylacrylic acid. The preparation method comprises the following steps: mixing rhodium-copper alloy nanoparticles of which the surfaces are combined with trithiol-terminated polymethylacrylic acid, 1-ethyl-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide ester, stirring, adding lactate oxidase, and continuously stirring, so as to obtain the radiotherapy sensitizer. The preparation method comprises the following steps: mixing rhodium-copper alloy nanoparticles of which the surfaces are combined with trithiol-terminated polymethylacrylic acid, 1-ethyl-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide ester, according to the invention, precise assembly of the nano-carrier and the enzyme active component is realized through modular design. The invention provides an efficient and safe solution for tumor diagnosis and treatment integration, and has remarkable industrial production potential.

Description

Technical Field

[0001] The present invention relates to a nano radiotherapy sensitizer, a preparation method thereof and an application thereof, belonging to the technical fields of nano material chemistry and biochemistry. Background Art

[0002] Radiotherapy is a non-invasive therapy for treating tumors clinically at present. High-energy rays (X-rays, γ-rays, α-particle beams or electron beams) are used to irradiate tumor cells to destroy the DNA of tumor cells. In addition, high-energy rays can also cause the ionization of water in tissues to generate reactive oxygen free radicals, and these reactive oxygen free radicals can react with most biomolecules (including DNA, proteins and lipids), destroy cell structures and cause cell death, thereby inhibiting the growth of tumors. According to the World Health Organization, more than 70% of tumors require radiotherapy, and 40% of tumors can be cured by radiotherapy.

[0003] Due to the non-specificity of ray irradiation, ray irradiation will also cause damage to normal tissues around tumors. Therefore, in the actual treatment process, the treatment plan often needs to consider the actual treatment effect and the side effects on the human body, which will lead to a decline in the treatment effect. In addition, solid tumors generally show a hypoxic state. Compared with normoxic cells, killing hypoxic cells requires three times higher irradiation dose, showing radiotherapy resistance. Therefore, it is necessary to develop a radiotherapy sensitizer to reduce the side effects caused by radiotherapy while improving the treatment effect.

[0004] At present, most inorganic nano materials with high atomic numbers have high X-ray absorption ability, which can effectively reduce the radiation dose and increase 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 bionic inorganic nano material with intrinsic mimetic enzyme activity. Like natural enzymes, they can carry out efficient catalytic reaction processes. Through the reasonable design of nanozymes, on the one hand, it can be used to improve the tumor microenvironment, and on the other hand, it can increase the sensitivity of tumor cells to high-energy rays. In addition, a large amount of lactic acid is produced during the development process of tumors, which is usually overproduced and accumulated in all types of tumors. Research has proved that lactic acid not only serves as a by-product of tumor development, but also promotes the proliferation and metastasis of tumors. By consuming lactic acid in tumors, the tumor microenvironment can be reconstructed, and the growth of tumor cells can be further inhibited. Remodeling the tumor microenvironment while irradiating tumor cells with high-energy rays can greatly kill tumor cells. Therefore, by multiple means of remodeling the tumor microenvironment and combining nanozymes with radiotherapy, it is expected to meet the clinical needs and provide a new strategy for the efficient treatment of cancer in the future. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a preparation method and application of a rhodium-copper alloy nanozyme with radiosensitizing effect, so as to solve the problems that existing radiosensitizers cannot improve the tumor microenvironment, tumor cells are insensitive to radiotherapy, and a large radiotherapy dose is required.

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

[0007] A radiosensitizer with multi-enzyme activity, comprising rhodium-copper alloy nanoparticles, tris-thiol-capped poly(methacrylic acid) modified on the surface of the rhodium-copper alloy nanoparticles, and lactate oxidase combined with the tris-thiol-capped poly(methacrylic acid).

[0008] Preferably, the tris-thiol-capped poly(methacrylic acid) is combined with the surface of the rhodium-copper alloy nanoparticles through coordination, and the lactate oxidase is combined with the tris-thiol-capped poly(methacrylic acid) through covalent bonding.

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

[0010] Preferably, the mass ratio of the rhodium-copper alloy nanoparticles to the tris-thiol-capped poly(methacrylic 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 preparation method of the above radiosensitizer with multi-enzyme activity: Mix rhodium-copper alloy nanoparticles with tris-thiol-capped poly(methacrylic acid) bound to the surface, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and N-hydroxysuccinimide ester, stir, add lactate oxidase and continue to stir to obtain the radiosensitizer.

[0012] Preferably, the preparation method of the rhodium-copper alloy nanoparticles with tris-thiol-capped poly(methacrylic acid) bound to the surface is: Mix the rhodium-copper alloy nanoparticles with tris-thiol-capped poly(methacrylic acid) and stir to obtain the rhodium-copper alloy nanoparticles with tris-thiol-capped poly(methacrylic acid) bound to the surface.

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

[0014] Preferably, the stirring is specifically: Magnetic stirring at 200 rpm at room temperature; the time for continuous stirring is 12 h.

[0015] The present invention also provides the application of the above radiotherapy sensitizer with multi-enzyme activity in the preparation of a preparation for treating tumors.

[0016] The radiotherapy sensitizer with multi-enzyme activity of the present invention has the function of photoacoustic imaging, enhances the sensitivity of tumor cells to high-energy rays while catalyzing using the tumor microenvironment, efficiently accumulates and selectively catalyzes at the tumor site, and the loaded lactate oxidase can directly act at the tumor site to supplement H 2 O 2 to achieve synergistic therapy. Therefore, this radiotherapy sensitizer with multi-enzyme activity will have good application prospects in the field of tumor diagnosis and treatment. Description of the Drawings

[0017] Figure 1 are the transmission electron microscope images, elemental distributions, absorption spectra in the near-infrared region, and X-ray diffraction patterns of the RhCu nanoparticles synthesized in Example 1;

[0018] Figure 2 are the particle sizes and zeta potentials of RhCu and RhCu@LO in Example 2 x ;

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

[0020] Figure 4 are the killing effects of RhCu and RhCu@LO on cells evaluated in Example 4 x ;

[0021] Figure 5 are the uptake of tumor cells for RhCu@LO evaluated in Example 5 x ;

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

[0023] Figure 7 are the killing effects of RhCu and RhCu@LO evaluated as radiotherapy sensitizers on cells in Example 7 x ;

[0024] Figure 8 are the performances of RhCu and RhCu@LO evaluated as radiotherapy sensitizers on cell cloning in Example 8 x ;

[0025] Figure 9For Example 9, the damage to cell DNA caused by the radiosensitizing effect of RhCu and RhCu@LO was evaluated. x

[0026] Figure 10 For Example 10, the change in the photoacoustic signal value at the tumor site over time after intravenous injection of RhCu@LO was evaluated. x

[0027] Figure 11 For Example 11, the inhibitory effect of RhCu@LO on tumors and the change in the body weight of mice during the treatment process as a radiosensitizer were evaluated. x

[0028] Figure 12 For Example 12, the H&E, Ki-67, and Tunel staining images of tumor tissues after treatment with RhCu@LO as a radiosensitizer were evaluated. x Detailed implementation

[0029] To make the present invention more obvious and understandable, preferred embodiments are hereby described in detail in conjunction with the accompanying drawings as follows.

[0030] The present invention provides a radiosensitizer with multi-enzyme activity, which includes: rhodium-copper alloy nanoparticles (RhCu), trithiol-capped polymethacrylic acid (PTMP-PMAA) bound to the surface of the rhodium-copper alloy nanoparticles, and lactate oxidase (LO x ) bound to the trithiol-capped polymethacrylic acid.

[0031] The present invention uses RhCu modified with trithiol-capped polymethacrylic acid on the surface as a carrier, and uses this carrier material to load natural lactate oxidase (LO x ) to form the multi-enzyme activity radiosensitizer (denoted as RhCu@LO x ). RhCu can load lactate oxidase through covalent bonding by amidation reaction (the carboxyl group of PTMP-PMAA reacts with the amino group on the surface of LO x ). Lactate oxidase, as a natural enzyme, can efficiently decompose lactate into hydrogen peroxide. After RhCu binds to lactate oxidase, the defect of single catalysis is effectively overcome, and the toxic side effects of lactate oxidase are reduced. Multi-enzyme cooperative catalytic therapy combined with radiotherapy realizes effective inhibition of tumors. In addition, RhCu has excellent absorption ability in the near-infrared region. Therefore, the radiosensitizer RhCu@LO x can be used for radiotherapy combined catalytic therapy guided by photoacoustic imaging.

[0032] In this example, RhCu was used to load LO xFor tumor radiotherapy sensitization and synergistic catalytic therapy. In this example, RhCu@LO x has the following advantages: 1. RhCu@LO x As a radiotherapy sensitizer, it effectively overcomes the problem of insensitivity of single radiotherapy to tumors; 2. It alleviates the hypoxic state of tumors, destroys the generation of reactive oxygen species in the tumor microenvironment specifically at the tumor site, and realizes the catalytic therapy of tumors; 3. Significantly reduces the side effects brought by radiotherapy; 4. Realizes the tumor catalytic therapy monitored by photoacoustic imaging.

[0033] In the present invention, the trithiol-capped polymethacrylic acid is combined with the surface of the rhodium-copper alloy nanoparticles through coordination, and the lactate oxidase is combined with the trithiol-capped polymethacrylic acid through covalent bonding.

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

[0035] In one embodiment, the mass ratio of the rhodium-copper alloy nanoparticles to the trithiol-capped 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 preparation method of the radiotherapy sensitizer with multi-enzyme activity, which includes the steps:

[0037] S1. Provide rhodium-copper alloy nanoparticles with trithiol-capped polymethacrylic acid bound to the surface;

[0038] S2. Mix the rhodium-copper alloy nanoparticles with trithiol-capped polymethacrylic acid bound to the surface, 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide ester (NHS), stir, and add lactate oxidase and continue to stir to obtain the radiotherapy sensitizer with multi-enzyme activity.

[0039] In step S1, in one embodiment, the preparation method of the rhodium-copper alloy nanoparticles with trithiol-capped polymethacrylic acid bound to the surface includes the steps:

[0040] S11. Provide rhodium-copper alloy nanoparticles (RhCu);

[0041] S12. Mix the rhodium-copper alloy nanoparticles with trithiol-capped polymethacrylic acid, stir, and obtain rhodium-copper alloy nanoparticles with trithiol-capped polymethacrylic acid bound to the surface.

[0042] In step S11, in one embodiment, the RhCu@LO xThe preparation method includes the steps of dissolving polyoxypropylene polyoxyethylene copolymer in a mixture of DMF and dilute HCl and stirring for 10 minutes, then adding an aqueous solution of sodium hexachlororhodate, an aqueous solution of copper chloride and ascorbic acid and stirring for 10 minutes, transferring the above solution to a reaction kettle and placing it in an oven at 100 °C for 4 h to obtain the RhCu. The RhCu is prepared by a reduction method in the examples.

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

[0044] In step S2, the rhodium-copper alloy nanoparticles with trithiols-terminated polymethacrylic acid bound to the surface are activated by NHS and EDC and then mixed uniformly with lactate oxidase and stirred to obtain the radiosensitizer with multi-enzyme activity.

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

[0046] In one embodiment, the mass ratio of the rhodium-copper alloy nanoparticles to the trithiols-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 preparation method of the RhCu includes the steps:

[0048] a) Preparing rhodium-copper alloy nanoparticles: Dissolving polyoxypropylene polyoxyethylene copolymer in a mixture of N,N-dimethylformamide and dilute hydrochloric acid and stirring for 10 minutes, then adding an aqueous solution of sodium hexachlororhodate, an aqueous solution of copper chloride and ascorbic acid and stirring for 10 minutes, transferring the above solution to a reaction kettle and placing it in an oven at 100 °C for 4 h to obtain the RhCu. The RhCu is prepared by a reduction method in the examples.

[0049] b) Purifying the rhodium-copper alloy nanoparticles: Centrifugally washing the rhodium-copper alloy nanoparticles prepared in the above step a) with deionized water and ethanol respectively for multiple times to obtain purified rhodium-copper alloy nanoparticles;

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

[0051] d) RhCu@LO x Synthesis: Mix the rhodium-copper alloy nanoparticles surface-bound with trithiol-terminated polymethacrylic acid, 1-ethyl-(-3-dimethylaminopropyl)carbodiimide, and N-hydroxysuccinimide ester, stir magnetically at 200 rpm at room temperature, add lactate oxidase and continue stirring for 12 h to obtain the radiotherapy sensitizer with multi-enzyme activity.

[0052] The embodiments of the present invention provide a preparation method of a radiotherapy sensitizer with multi-enzyme activity and its application in tumor treatment. The efficient accumulation and specific catalytic action of the radiotherapy sensitizer at the tumor site significantly reduce the systemic toxic side effects of lactate oxidase. Therefore, the radiotherapy sensitizer has broad application prospects in the field of tumor diagnosis and treatment. In addition, while treating tumors, the radiotherapy sensitizer with multi-enzyme activity can also be used as a photoacoustic contrast agent for photoacoustic imaging.

[0053] Example 1: Preparation of RhCu

[0054] Weigh 20 mg of polyoxypropylene-polyoxyethylene copolymer and dissolve it in 3.2 mL of N,N-dimethylformamide. Subsequently, add 0.8 mL of dilute hydrochloric acid (1 M) and stir for 10 min. Separately, dissolve 75 mg of sodium hexachlororhodate in 4 mL of deionized water and add it to the above solution. Dissolve 10 mg of copper chloride in 4 mL of deionized water and add it to the above solution. Dissolve 138 mg of ascorbic acid in 8 mL of deionized water and add it to the above solution. Continue to stir the solution for 10 min, and then transfer the solution to a hydrothermal reaction kettle and react at 100 °C for 4 h. After the reaction is completed and cooled to room temperature, rhodium-copper alloy nanoparticles are obtained.

[0055] Figure 1 In, a and b are the TEM images and elemental distribution maps of RhCu; c is the ultraviolet-visible-near-infrared spectrum of the RhCu aqueous solution; d is the X-ray diffraction spectrum of RhCu and the comparison spectrum with the standard card. From Figure 1 d, it can be seen that the synthesized RhCu can perfectly match the peaks of metal Ru, while Cu may not appear clearly in the spectrum due to too little content.

[0056] Example 2: Preparation of RhCu@LO x Preparation

[0057] Disperse 10 mg of rhodium copper alloy nanoparticles in 10 mL of ethanol, then add 100 mg of trithiol-terminated polymethacrylic acid, sonicate for 30 min, and magnetically stir at 200 rpm at room temperature for 12 h to obtain rhodium copper alloy nanoparticles with trithiol-terminated polymethacrylic acid bound to the surface. After centrifugation with deionized water, dissolve them in water, activate with EDC and NHS, and then add 5 mg of LO x , stir at room temperature for 12 h to obtain RhCu@LO x , after centrifugation with deionized water, dissolve them in water. Measure the zeta potentials of RhCu and RhCu@LO x respectively. The results are as Figure 2 shown.

[0058] Figure 2 In, a is the hydrodynamic particle size of RhCu and RhCu@LO x ; b is the change in the zeta potential of RhCu and RhCu@LO x ; This result indicates the successful modification of LO x .

[0059] Example 3: Evaluate the CAT-like activity, POD-like activity, and OXD-like activity of RhCu

[0060] The CAT enzyme activity of RhCu was characterized by measuring the amount of dissolved oxygen generated by RhCu in H2O2 solution at room temperature. Add 150 μL of RhCu aqueous solution (1 mg / mL) to 2.85 mL of PBS (pH = 7.4), and at the same time add 3 μL of 30% H 2 O 2 solution. Use the special oxygen electrode of a multi-parameter analyzer to measure the concentration of O2 in the mixture every 30 s. The experimental results are shown in Figure 3 a and b. Use TMB·HCl as the substrate to evaluate the POD enzyme activity of RhCu. Add 5 μL of TMB·HCl aqueous solution (150 mM) and 1 μL of RhCu aqueous solution (50 μg / mL) to 1.5 mL of water, and then add different volumes of 1.5 M H 2 O 2 solution (volumes are 0.2, 0.4, 0.8, 2, 4 μL respectively). Measure the change in the absorbance of the substrate TMB·HCl at 652 nm over time at 37 °C, as shown in Figure 3 c. Use TMB·HCl as the substrate to evaluate the OXD enzyme activity of RhCu. Add 1.5 μL of RhCu aqueous solution (1 mg / mL) to 1.5 mL of water, and then add different volumes of 150 mM TMB·HCl aqueous solution (2, 4, 6, 8, 10 μL). Measure the change in the absorbance of the substrate TMB·HCl at 652 nm over time at 37 °C, as shown inFigure 3 as shown in d.

[0061] As Figure 3 in a, RhCu can efficiently catalyze the decomposition of H 2 O 2 and produce a large amount of O 2 . In contrast, the change in the control group is almost negligible, indicating that RhCu has high CAT-like enzyme activity. Figure 3 As shown in b, c, and d, its CAT-like enzyme activity, POD-like enzyme activity, and OXD-like enzyme activity have kinetic characteristics similar to those of natural enzymes.

[0062] Example 4: Evaluate the killing effect of RhCu@LO x on tumor cells.

[0063] The specific conditions are as follows: 4T1 cells were seeded in 96-well plates at an initial density of 1×10 4 cells / well and cultured for 24 h at 37 °C in a humidified atmosphere of 5% CO 2 . Subsequently, the original medium was replaced with fresh medium containing different concentrations of RhCu or RhCu@LO x (0, 0.625, 1.25, 2.5, 5, 10, 25, 50 μg / mL) and incubated for another 12 h and 24 h. Finally, the relative cell viability was determined using a CCK-8 kit, and the absorbance of each well at 450 nm was measured using a microplate reader.

[0064] As Figure 4 shown in a, after incubation with RhCu (25 μg / mL), the cell survival rate decreased to less than 60%. Figure 4 In b, after treatment with RhCu@LO x (5 μg / mL) at a lower concentration, the cell survival rate decreased to less than 40%, showing more obvious cytotoxicity.

[0065] Example 5: Evaluate the uptake ability of cells to RhCu@LO x

[0066] 4T1 cells were seeded in confocal dishes at an initial density of 1×10 5 cells / dish overnight. FITC-grafted RhCu@LO x (10 μg / mL) was added and incubated with the cells for different times (0, 1, 2, 4, 8, 12, 24 h). Finally, the uptake of RhCu@LO x was observed using a confocal microscope.

[0067] As Figure 5 shown, the fluorescence intensity of the cells was the strongest at 12 h, indicating that the cells took up the most RhCu@LO at 12 hx At most, it shows RhCu@LO x can be taken up by cells.

[0068] Example 6: Evaluation of RhCu@LO x Multi-enzyme activity in cells

[0069] 4T1 cells were seeded in 96-well plates at an initial density of 1×10 4 cells / well and incubated overnight at 37 °C in a humidified atmosphere of 5% CO 2 2. Subsequently, the original medium was replaced with fresh medium containing RhCu or RhCu@LO x (10 μg / mL) and incubated for an additional 12 h. Then, the cells were irradiated with X-rays (6 Gy) and incubated for another 6 h. DCFH-DA, a reactive oxygen species dye, was added to stain the cells, and finally, the fluorescence inside the cells was observed using a fluorescence microscope.

[0070] 4T1 cells were seeded in 96-well plates at an initial density of 1×10 4 cells / well and incubated overnight at 37 °C in a humidified atmosphere of 5% CO 2 2. Subsequently, the original medium was replaced with fresh medium containing RhCu or RhCu@LO x (25 μg / mL) and incubated for an additional 12 h. Then, the cells were irradiated with X-rays (6 Gy) and incubated for another 12 h. 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 in 96-well plates at an initial density of 1×104 cells / well and incubated overnight at 37 °C in a humidified atmosphere of 5% CO 2 2. Subsequently, the cells were placed in an anaerobic incubator for 12 h, and [Ru(dpp) 3 Cl 2 probe was added and incubated for 4 h. Then, the original medium was replaced with fresh medium containing RhCu (25 μg / mL) or RhCu@LO x (6.25, 12.5, 25, 50 μg / mL) and incubated for an additional 4 h. Finally, the fluorescence inside the cells was observed using a fluorescence microscope.

[0072] As Figure 6 in a, compared with the control group, after adding RhCu and RhCu@LO x , the green fluorescence of the cells was significantly enhanced, indicating that RhCu can generate reactive oxygen species in cells and has POD-like activity. After X-ray irradiation, the green fluorescence was further enhanced, indicating that X-rays enhanced the generation of reactive oxygen species in cells and produced a synergistic effect. Figure 6In b, compared with the control group, the RhCu group had a significantly enhanced red fluorescence, indicating the OXD-like activity of RhCu. In the RhCu@LO x group, the red fluorescence of the cells was even more significantly enhanced. This is because LO x catalyzes the production of H 2 O 2 by lactic acid, and the production of oxygen catalyzed by RhCu enhances the OXD-like activity. The further enhancement of fluorescence after X-ray irradiation corresponds to Figure 6 the results in a. Figure 6 In c, in the RhCu@LO x group, as the concentration increased, the red fluorescence gradually decreased, indicating that the intracellular oxygen content gradually increased, showing the CAT-like activity of RhCu.

[0073] Example 7: Evaluation of the radiotherapy sensitization effect of RhCu@LO x

[0074] 4T1 cells were seeded in 96-well plates at an initial density of 1×10 4 cells / well and cultured for 24 h at 37 °C in a humidified atmosphere of 5% CO 2 . Subsequently, the original medium was replaced with fresh medium containing different concentrations of RhCu or RhCu@LO x (25 μg / mL) and incubated for another 12 h. Then, the cells were irradiated with different doses of X-rays (0, 2, 4, 6 Gy) and incubated for 12 h. Subsequently, the cell viability was detected using a CCK-8 kit, and the absorbance of each well at 450 nm was measured with a microplate reader to calculate the cell survival rate.

[0075] As Figure 7 shown, as the irradiation dose increased, the cell viability of the cells incubated with RhCu and RhCu@LO x significantly decreased. This is attributed to the CAT-like enzyme activity of RhCu, which utilizes H 2 O 2 in tumor cells to produce O 2 , increasing the sensitivity of tumor cells to X-rays. The cells incubated with RhCu@LO x were more sensitive to X-rays because LO x catalyzes the production of H 2 O 2 from lactic acid in tumor cells, providing more substrates for RhCu to catalyze the production of more oxygen.

[0076] Example 8: Evaluation of the inhibitory ability of RhCu@LO x on cell clones under X-ray irradiation

[0077] 4T1 cells were seeded in 6-well plates at an initial density of 2000 cells / well overnight and divided into 6 groups for treatment: control group (Control), RhCu treatment group (RhCu), RhCu@LO x treatment group (RhCu@LO x ), X-ray irradiation group (X-ray), RhCu treatment group under X-ray irradiation (RhCu+X-ray), RhCu@LO x treatment group under X-ray irradiation (RhCu@LO x +X-Ray). After adding RhCu or RhCu@LO x (25 μg / mL) and incubating for 12 h, the cells were irradiated with X-rays (6 Gy) and then cultured for another 12 h. The X-ray group was directly irradiated with X-rays (6 Gy) and then cultured for 12 h. After the treatment, the cells were washed with PBS to remove the materials on the cell surface, and finally replaced with fresh medium and cultured for 5 days. The cells were fixed with 4% paraformaldehyde for about 20 minutes, washed twice with PBS, and then stained with Giemsa staining solution (1.5 mL / well) at room temperature for 30 minutes. After washing twice with PBS and air-drying, the cells were finally photographed and the images were collected for analysis.

[0078] As Figure 8 shown, the formation of cell clones was inhibited in the RhCu group and RhCu@LO x group. After X-ray irradiation, the formation of cell clones in the RhCu group and RhCu@LO x group was further inhibited. This indicates that RhCu@LO x has excellent radiosensitizing ability.

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

[0080] 4T1 cells were seeded in confocal dishes at an initial density of 1×10 5 cells / well overnight and divided into 6 groups for treatment: control group (Control), RhCu treatment group (RhCu), RhCu@LO x treatment group (RhCu@LO x ), X-ray irradiation group (X-ray), RhCu treatment group under X-ray irradiation (RhCu+X-ray), RhCu@LO x treatment group under X-ray irradiation (RhCu@LO x +X-Ray). After adding RhCu or RhCu@LO x(10 μg / mL) After incubation for 12 h, irradiate with X-ray (6 Gy) and continue to culture for 12 h. The X-ray group was directly irradiated with X-ray (6 Gy) and then cultured for 12 h. Subsequently, fix the cells with 4% paraformaldehyde for about 20 minutes, wash 3 times with PBS, add immunofluorescence blocking solution and block for 20 minutes, then add rabbit anti-γ-H2AX monoclonal antibody and incubate at room temperature for 1 h, wash 3 times with PBS again, then add anti-rabbit Cy3 fluorescent secondary antibody and incubate at room temperature for 1 h, wash 2 times with PBS, add nuclear staining dye (DAPI) and incubate at room temperature for 5 min, wash three times with PBS, and finally observe the immunofluorescence signal with a confocal microscope.

[0081] As Figure 9 shown, the intensity of the red fluorescence indicates the level of DNA damage. After X-ray irradiation, the red fluorescence intensity of the RhCu and RhCu@LO x groups was greatly increased compared with that of the group without X-ray irradiation. It can be seen that RhCu can enhance the damage of X-ray to the DNA of tumor cells and show excellent radiotherapy sensitization ability.

[0082] Example 10: Evaluate RhCu@LO x Construct a breast cancer model of mice by evaluating the change of photoacoustic signal at the tumor site after intravenous injection: Purchase female athymic nude mice (six weeks old, 20 - 25 g), and construct a subcutaneous tumor model by subcutaneously injecting 100 μL of 4T1 cell PBS solution into the right hind limb of the mice (1×10 6 cells / mouse). When the tumor volume reaches 80 mm 3 , inject 200 μL of 1 mg / mL RhCu@LO x solution through the tail vein, and use a small animal photoacoustic imaging system (Visual Sonics Vevo LAZRsystem) to detect the photoacoustic signal of RhCu@LO x at the tumor site.

[0083] As Figure 10 shown in a and b, after injecting RhCu@LO x , the photoacoustic signal value at the tumor site reaches the maximum at 8 h, and then the signal gradually weakens.

[0084] Example 11: Evaluate RhCu@LO x The growth inhibitory effect on mouse tumors

[0085] Construct a breast cancer model of mice: Purchase female athymic nude mice (six weeks old, 20 - 25 g), and construct a subcutaneous tumor model by subcutaneously injecting 100 μL of 4T1 cell PBS solution into the right hind limb of the mice (1×10 6 cells / mouse). When the tumor volume reaches 50 mm 3When the time came, the 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 x treatment group under X-ray irradiation. PBS solutions (10 mg / kg) of RhCu and RhCu@LO x were respectively injected into the mice through the tail vein. In the control group, PBS (100 μL) was injected into the mice. 8 h after administration, the tumor parts of the mice were irradiated under X-ray (6 Gy), and the tumor volume and the weight of the mice were measured every two days with a vernier caliper.

[0086] Figure 11 In [Figure number not provided] a shows the change of tumor volume over time in different treatment groups. It can be seen that compared with the single X-Ray group, the tumors in the RhCu@LO x +X-ray group can be well inhibited. At the same time, the tumors in the RhCu+X-ray group also showed good inhibition, indicating that RhCu has excellent radiotherapy sensitization ability. Compared with the control group, the RhCu group and the RhCu@LO x group also showed significant inhibitory effects, indicating that RhCu has excellent catalytic therapy effect. The addition of LO x can further enhance its catalytic effect. During the two weeks of treatment, the body weights of the mice in each group did not show obvious changes. At the initial stage of radiotherapy, the body weights of the mice decreased slightly, which may be due to the induction of adverse reactions in the digestive system by radiotherapy. Subsequently, the body weights of the mice slowly returned to normal ( Figure 11 in [Figure number not provided] b), indicating that RhCu@LO x has excellent biosafety.

[0087] Example 12: Evaluate the apoptosis level of tumor tissues after RhCu@LO x treatment

[0088] After the treatment in Example 11, the tumor tissues of the mice were taken for paraffin sectioning, and the sections were stained with H&E, Ki-67 and Tunel respectively.

[0089] Figure 12 It is shown that the tumor tissues in the RhCu+X-ray group were the most severely damaged, showing a higher apoptosis level.

[0090] The present invention provides a multi-enzyme activity radiotherapy sensitizer and its preparation method and application. The radiotherapy sensitizer significantly improves radiotherapy sensitivity by targeting and regulating the tumor microenvironment, and simultaneously realizes the function of diagnosis and treatment integration. The preparation method includes the following steps: The obtained radiotherapy sensitizer has the following technical advantages: 1) Based on the high enrichment ability at the tumor site, it can specifically catalyze the decomposition of metabolites and significantly improve the hypoxic microenvironment of tumors; 2) By optimizing the spatial distribution and synergistic effect of the enzyme active components, the off-target toxicity of lactate oxidase (LO x ) is reduced; 3) It has the function of photoacoustic imaging contrast, and can monitor the drug distribution and treatment effect in real time and dynamically. The preparation process of the present invention adopts modular design, and the reaction conditions are mild, which is suitable for industrial scale-up production. Verified by animal experiments, the sensitizer improves the radiotherapy efficacy in the 4T1 breast cancer model and does not show significant systemic toxicity, indicating its excellent biosafety and clinical transformation potential.

Claims

1. A radiosensitizer with multiple enzyme activities, characterized in that: The invention comprises rhodium-copper alloy nanoparticles, trithiol-terminated polymethacrylic acid modified on the surface of the rhodium-copper alloy nanoparticles, and lactate oxidase combined with the trithiol-terminated polymethacrylic acid.

2. The radiosensitizer according to 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 action.

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

4. The radiosensitizer according to 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 having multiple enzyme activities according to any one of claims 1 to 4, characterized in that: The rhodium-copper alloy nanoparticles of polymethacrylic acid capped with trithiol on the surface, 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide ester are mixed and stirred, and lactate oxidase is added and stirred continuously to obtain the radiotherapy sensitizer.

6. The preparation method according to claim 5, characterized in that: The preparation method of the rhodium-copper alloy nanoparticles with trithiol-terminated polymethacrylic acid is as follows: the rhodium-copper alloy nanoparticles are mixed with trithiol-terminated polymethacrylic acid, and stirred 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: dissolving a polyoxypropylene-polyoxyethylene copolymer in a mixture of DMF and dilute HCl and stirring for 10 minutes, then adding an aqueous sodium hexachlororhodiumate solution, an aqueous copper chloride solution and ascorbic acid and stirring for 10 minutes, transferring the solution to a reaction kettle and placing it 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 is specifically: magnetic stirring at 200 rpm at room temperature; the stirring time is continued for 12 hours.

9. Use of the radiosensitizer with multiple enzyme activities according to any one of claims 1 to 4 in the preparation of a preparation for treating tumors.

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