Preparation method and application of diagnosis and treatment agent for tumor radiotherapy sensitization

Abstract

The invention provides a preparation method and application of a diagnosis and treatment agent for tumor radiotherapy sensitization, and belongs to the field of pharmacy. According to the method, a one-pot method is innovatively adopted to load D-mannose and manganese-coated gold nanoparticles (Au (at) MnO2NPs) into ZIF-8 nanoparticles, and the nano diagnosis and treatment agent (Dman / Au (at) MnO2ZIF-8) for tumor radiotherapy sensitization is prepared. The prepared composite nano diagnosis and treatment agent is enriched in tumor tissues by enhancing permeation and retention effects, releases D-mannose, gold nanoparticles and Mn < 2 + > capable of generating T1 magnetic resonance (MR) imaging signals, and realizes tumor radiotherapy sensitization under the guidance of MR imaging. Besides, D-mannose can further increase the sensitivity of tumor cells to ionizing radiation by inducing protein degradation of a tumor cell programmed death receptor-ligand 1, so that a synergistic treatment effect is achieved, and a new thought is expected to be provided for integration of radiotherapy sensitization and diagnosis and treatment of tumors.

Description

Technical Field

[0001] The invention belongs to the field of pharmacy, and specifically relates to a preparation method and application of a diagnostic and therapeutic agent for tumor radiotherapy sensitization. Background Art

[0002] In recent years, tumors have become an important factor in global population mortality and the leading cause of death among people over 70 years old in my country. Among them, breast cancer is the tumor with the highest incidence rate in women. It is also the malignant tumor with the highest diagnosis rate in Chinese women. It is currently the second leading cause of cancer death in Chinese women, seriously threatening women's life and health. In particular, triple-negative breast cancer has a poor prognosis and a short average survival period due to the lack of effective treatment methods. Therefore, research and development of diagnostic and therapeutic agents for triple-negative breast cancer is an important research direction.

[0003] As an important means to control and prevent the occurrence and development of tumors, radiotherapy plays an important role in postoperative adjuvant radiotherapy and local control of advanced tumors in triple-negative breast cancer. Radiotherapy uses high-energy radiation generated by radiotherapy equipment to destroy the DNA structure of tumor cells while producing a large amount of reactive oxygen species (ROS) in tumor cells to destroy the tumor microenvironment, thereby causing tumor tissue necrosis. However, the low radiation attenuation coefficient of tumor tissue makes it resistant to radiation. Therefore, in order to achieve effective dose accumulation in the tumor area, it is necessary to increase the irradiation dose. However, the increase in irradiation dose will cause additional radiation damage to non-target areas, resulting in inevitable side effects and a heavy treatment burden on patients. For this reason, the concept of radiotherapy sensitizers came into being, aiming to increase the dose accumulation of tumor tissue without changing the radiation dose, or to induce tumor tissue to be sensitive to radiation and reduce the effective irradiation dose.

[0004] As a monosaccharide, D-mannose plays an important role in the glycosylation process of proteins. Studies have shown that D-mannose can directly induce the degradation of programmed death receptor-ligand 1 (PD-L1) protein in breast cancer tumor cells, causing the degradation of mRNA related to DNA damage repair mediated by PD-L1 in cells, thereby blocking the repair of DNA damage caused by radiation and making tumor cells sensitive to ionizing radiation. However, the characteristic of D-mannose that it is difficult to be absorbed and utilized by living tissues under physiological conditions makes it difficult to be directly used in tumor treatment to play a therapeutic role. Summary of the invention

[0005] The purpose of the present invention is to provide a method for preparing a diagnostic and therapeutic agent for tumor radiotherapy sensitization and its application, so as to achieve the effect of tumor radiotherapy sensitization and integrated diagnosis and treatment while ensuring efficient loading and cell delivery of D-mannose.

[0006] The nanosystem designed by the invention uses ZIF-8 as a nanoplatform and loads Au@MnO2 nanoparticles and D-mannose to achieve integrated diagnosis and treatment of tumors. The nanosystem is enriched in tumor tissues through the tumor high permeability and high retention effect (EPR), decomposes the ZIF-8 shell in the tumor microenvironment, and releases the loaded Au@MnO2 and D-mannose. Au@MnO2 decomposes to produce gold nanoparticles and Mn under the high GSH concentration in the tumor microenvironment. 2+ , achieving D-mannose, gold nanoparticles and Mn 2+ delivery and release.

[0007] To achieve the above-mentioned object of the invention, the present invention provides a method for preparing a diagnostic and therapeutic agent for tumor radiotherapy sensitization, comprising the following steps:

[0008] (1) Add 8-15 mg of chloroauric acid to 70-100 mL of ultrapure water and stir magnetically at 40-80°C for 20-50 min to obtain solution A; add 30-45 mg of sodium citrate to 3-6 mL of deionized water, dissolve 0.1-0.4 mL of anhydrous potassium carbonate solution with a concentration of 12.5-15.5 mg / mL and 5-9 mg of tannic acid in 13-19 mL of ultrapure water in sequence, and stir in a water bath at 40-100°C for 20-60 min to obtain solution B; transfer solution B to solution A and continue stirring in a water bath at 55-75°C for 20-50 min. After the reaction is complete, centrifuge to obtain gold nanoparticles (AuNPs);

[0009] (2) Add 0.2-0.7 mL of a potassium hydroxide solution with a concentration of 0.05-0.07 mg / mL dropwise into 30-80 mL of an AuNPs aqueous solution with a concentration of 3-4 mg / mL until the pH is 8.5-10.5. Next, add 2.5-4.5 mg of potassium permanganate and 15-25 mg of potassium oxalate to the AuNPs aqueous solution in sequence. After the solution has been allowed to stand in a water bath at 0°C for 20-60 min, the solution is heated to 40-80°C and allowed to stand for 1-4 h. After the reaction is complete, centrifuge to obtain composite nanoparticles of gold and manganese dioxide (Au@MnO2);

[0010] (3) Add 2-5 mL of 6.5-7.5 mg / mL Au@MnO2 NPs solution and 2-5 mg of polyvinyl pyrrolidone (PVP) into 2-5 mL of ultrapure water. Stir the mixture at 20-40°C for 10-20 h, and then centrifuge to prepare Au@MnO2-PVP.

[0011] (4) Dissolve 4-6 g of D-mannose (Dman) in 3-6 mL of 150-250 mg / mL methylimidazole solution and stir in a 30-60 °C water bath for 20-40 min. Then, add 2.5-3.9 mL of methanol and 1.1-2.2 mL of 8.5-9.9 mg / mL Au@MnO2-PVP solution to the solution in sequence, and continue stirring at 20-40 °C for 3-10 min. Then, add 50-100 mg of zinc nitrate to the solution again, and continue stirring for 20-50 min. After the reaction is completed, the product is centrifuged, washed with methanol and naturally air-dried to obtain a nano-diagnostic agent (Dman / Au@MnO2@ZIF-8) for tumor radiosensitization.

[0012] Furthermore, in the step (1), the mass ratio of chloroauric acid to sodium citrate is 1:2 to 1:5.625.

[0013] Furthermore, the diameter of the gold nanoparticles prepared in step (1) is 8 to 12 nm, and the surface charge is -20 to -30 mV.

[0014] Furthermore, in the step (2), the mass ratio of AuNPs to potassium permanganate is 20:1 to 128:1.

[0015] Furthermore, the diameter of the Au@MnO2NPs prepared in step (2) is 10 to 18 nm, and the surface charge is -30 to -39 mV.

[0016] Furthermore, in the step (3), the mass ratio of Au@MnO2NPs to polyvinyl pyrrolidone is 2.6:1 to 18.75:1.

[0017] Furthermore, the diameter of Au@MnO2-PVP prepared in step (3) is 12 to 20 nm, and the surface charge is -20 to -29 m.

[0018] Furthermore, in the step (4), the mass ratio of Au@MnO2-PVP to D-mannose is 1:183.6 to 1:642; and the mass ratio of Au@MnO2-PVP to zinc nitrate is 1:2.3 to 1:11.

[0019] Furthermore, the Au@MnO2 / Dman@ZIF-8 prepared in step (4) has ZIF-8 as the skeleton, and is loaded with Au@MnO2NPs and D-mannose, with a drug loading of D-mannose of 8.5-9.9% and a particle size of 100-150 nm.

[0020] In order to achieve the above-mentioned purpose of the invention, the present invention also provides the use of the diagnostic and therapeutic agent prepared by the above-mentioned preparation method in the integration of radiotherapy sensitization and MRI diagnosis and treatment of tumors.

[0021] The zeolitic imidazolate framework material (ZIF) in the present invention is a type of metal-organic framework (MOF). In particular, ZIF-8 is a ZIF material constructed based on zinc (Zn) and methylimidazole, and has the characteristics of acidic response degradation, good biocompatibility and small pore size; in addition, the ZIF-8 material has good plasticity, and the one-pot synthesis feature makes it easy to encapsulate drugs and surface modification, and it is also easy to incorporate other metal elements into it to form a multi-element doped ZIF-8 material.

[0022] In the present invention, gold (Au) as a high-Z element has a high radiation absorption coefficient, which can effectively increase the effective dose accumulation in the target area; and manganese dioxide (MnO2) can regulate the tumor microenvironment (TME) by consuming hydrogen peroxide (H2O2) and reducing glutathione (GSH). Therefore, the present invention uses MnO2 to consume H2O2 to produce oxygen, thereby achieving the purpose of alleviating tumor hypoxia; at the same time, the production of Mn 2+ Ions can be used for magnetic resonance T1WI imaging to achieve integrated tumor diagnosis and treatment.

[0023] Compared with the prior art, the present invention has the following advantages:

[0024] (1) The composite nano-diagnostic and therapeutic agent prepared by the present invention has good biocompatibility and biosafety. It not only has radiosensitization ability and tumor immunity effect, but also has MR imaging ability, realizing the integration of radiosensitization and MRI diagnosis and treatment of tumors.

[0025] (2) The composite nano-diagnostic and therapeutic agent prepared by the present invention achieves effective loading and intracellular delivery of D-mannose.

[0026] (3) The preparation method of the present invention is stable and reliable. The prepared composite nano-diagnostic and therapeutic agent composite nanoparticles can effectively release effective ingredients in the tumor microenvironment, realize the integrated function of radiotherapy sensitization and MRI imaging, and are expected to provide new ideas for the integration of tumor radiotherapy sensitization and MRI imaging. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of this specification or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this specification. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0028] Figure 1The transmission electron microscope image and element energy spectrum analysis EDS image of the composite nanoparticles prepared by the present invention;

[0029] Figure 2 The hydrated particle size diagrams of AuNPs, AuNPs-PVP, ZIF-8, DZ (Dman@ZIF-8), AMZ (Au@MnO2@ZIF-8) and AMDZ prepared in the present invention;

[0030] Figure 3 Schematic diagram of the change of Zeta potential during the assembly process of Au@MnO2 in the present invention;

[0031] Figure 4 This is the monosaccharide component analysis diagram of Au@MnO2 / Dman@ZIF-8 of the present invention;

[0032] Figure 5A The T1WI and pseudo-color images of Au@MnO2 / Dman@ZIF-8 of the present invention at different Mn ion concentrations;

[0033] Figure 5B The Au@MnO2 / Dman@ZIF-8 of the present invention has different Mn 2+ Relaxation rate fitting plot under concentration;

[0034] Figure 6 This is a schematic diagram of the ROS results generated by Au@MnO2 / Dman@ZIF-8 of the present invention at different concentrations;

[0035] Figure 7 This is a schematic diagram of the ROS results generated by Au@MnO2 / Dman@ZIF-8 of the present invention under different ionizing radiation doses;

[0036] Fig. 8A This is a diagram of the cell clone formation experiment of Au@MnO2 / Dman@ZIF-8 of the present invention;

[0037] Figure 8B This is the cell proliferation curve of Au@MnO2 / Dman@ZIF-8 of the present invention;

[0038] Fig. 9 This is the magnetic resonance imaging image of Au@MnO2 / Dman@ZIF-8 cells of the present invention;

[0039] Fig.10 MRI images of Au@MnO2 / Dman@ZIF-8 of the present invention at different time points after injection into a mouse tumor model;

[0040] Fig.11 This is a diagram showing the anti-tumor effects of Au@MnO2 / Dman@ZIF-8 and other groups of materials of the present invention after injection into a mouse tumor model;

[0041] Fig.12 This is a graph showing the changes in tumor mass after injection of Au@MnO2 / Dman@ZIF-8 and other groups of materials of the present invention into a mouse tumor model. DETAILED DESCRIPTION

[0042] In order to enable those skilled in the art to better understand the technical solutions in this specification, the technical solutions in the embodiments of this specification will be clearly and completely described below in conjunction with the drawings in the embodiments of this specification. Obviously, the described embodiments are only part of the embodiments of this specification, not all of the embodiments. Based on the embodiments in this specification, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of this specification.

[0043] Example

[0044] A method for preparing a diagnostic and therapeutic agent for tumor radiotherapy sensitization comprises the following steps:

[0045] (1) Add 10 mg of chloroauric acid to 80 mL of ultrapure water and stir magnetically at 60°C for 30 min to obtain solution A; add 40 mg of sodium citrate to 4 mL of deionized water, dissolve 0.2 mL of anhydrous potassium carbonate solution with a concentration of 13.8 mg / L and 4.9 mg of tannic acid in 15.8 mL of ultrapure water in sequence, and stir in a water bath at 65°C for 30 min to obtain solution B; transfer solution B to solution A and continue stirring in a water bath at 60°C for 30 min. After the reaction is complete, centrifuge to obtain gold nanoparticles (AuNPs);

[0046] (2) Add 0.3 mL of 0.056 mg / mL potassium hydroxide solution dropwise into 50 mL of 3.6 mg / mL AuNPs aqueous solution until the pH reaches 9.2. Next, add 3.318 mg of potassium permanganate and 19.32 mg of potassium oxalate to the AuNPs aqueous solution in sequence. After the solution has been allowed to stand in a 0°C water bath for 40 min, the solution is heated to 60°C and stirred for 2 h. After the reaction is complete, centrifuge to obtain Au@MnO2NPs, a composite nanoparticle of gold and manganese dioxide;

[0047] (3) Add 3 mL of 7.14 mg / mL Au@MnO2 NPs solution and 3 mg of polyvinyl pyrrolidone (PVP) into 3 mL of ultrapure water. Stir the mixture at 25 °C for 12 h and centrifuge to prepare Au@MnO2-PVP.

[0048] (4) Dissolve 5g of D-mannose (Dman) in 4mL of 200mg / mL methylimidazole solution and stir in a 35℃ water bath for 30min. Then, add 3.08mL of methanol and 1.32mL of 9.08mg / mL Au@MnO2-PVP solution to the solution in sequence, and continue stirring at 35℃ for 10min. Then, add 80mg of zinc nitrate solution to the solution again and continue stirring for 30min. After the reaction is completed, the product is centrifuged, washed with methanol and air-dried to obtain a nano-diagnostic agent (Dman / Au@MnO2@ZIF-8) for tumor radiosensitization, referred to as AMDZ. Transmission electron microscopy and EDS energy spectrum analysis show that ( Figure 1 ), the particle size of AMDZ is about 100nm, and the gold, manganese and zinc elements are evenly distributed on the surface of the nanoparticles.

[0049] After testing the hydrated particle size of the nanomaterials using a Malvern particle size tester ( Figure 2 ), it was found that the hydrated particle sizes of AuNPs, AuNPs-PVP, ZIF-8, DZ (Dman@ZIF-8), AMZ (Au@MnO2@ZIF-8) and AMDZ nanoparticles were 15.16nm, 15.76nm, 212.1nm, 232.5nm, 252.7nm and 254.7nm, respectively, which indicates that there is no significant change in the hydrated particle size of ZIF-8 before and after drug loading. In addition, Figure 3 The difference in surface charge between Au NPs (-28.1mV), Au@MnO2 (-32.2mV), PVP (+10mV) and Au@MnO2-PVP (-23.5mV) further proves the successful assembly of Au@MnO2-PVP. The monosaccharide components in AMDZ were analyzed by HPLC, and it was found that 94.019% of the monosaccharides in AMDZ were D-mannose, with only a small amount of isomerization ( Figure 4 ). Next, the present invention used a 3.0T magnetic resonance imaging device to explore the T1 imaging capability of AMDZ, and the results were as follows Figure 5A and Figure 5B Only in the solution of GSH and pH = 5.5, AMDZ can show T1 MR signal that increases with the increase of Mn concentration, and the corresponding T1 relaxation rate is 6.66mM -1 s -1 In addition, AMDZ has been shown to produce ROS under ionizing radiation ( Figure 6 ), and the amount of ROS produced increases with the increase of X-Ray dose ( Figure 7 ).

[0050] By exploring the survival curves of tumor cells co-incubated with different nanomaterials, it was found that AMDZ had the best radiosensitization effect ( Fig. 8A and Figure 8B Using 4T1 cells as a tumor cell model, the present invention further explored the MRI imaging ability of AMDZ in cells. The results are as follows Fig. 9 After co-incubation with different concentrations of AMDZ, the T1 MR signal intensity of 4T1 cells under a 3.0T magnetic resonance imaging system increased with the increase of AMDZ concentration, indicating that AMDZ can effectively perform MR imaging on tumor cells. Subsequently, the present invention used a mouse model to verify the in vivo MR imaging ability of AMDZ, and the results are shown in Fig.10 As shown. The T1 MR signal intensity of the mouse tumor site gradually increased with time, and the best imaging effect was achieved at 2 hours. Finally, the present invention used a mouse model to evaluate the anti-tumor effect of the nanomaterial, and the results were as follows Fig.11 and 12 As shown, the average tumor weight of the model group mice without any intervention was 1.93g, while the average tumor weights of the mice in the simple Dman group, simple DZ group, simple AMZ group and simple AMDZ group were 1.76g, 1.83g, 1.70g and 1.73g, respectively, which were not statistically different from the model group. The tumor entity and weight of the mice in the radiotherapy group were smaller than those in the model group. The average tumor weights of the mice in the PBS+RI group, Dman+RI group, DZ+RI group, AMZ+RI group and AMDZ+RI group were 1.63g, 1.43g, 0.83g, 0.27g and 0.10g, respectively, and the tumor inhibition rates were 15.54%, 25.91%, 56.99%, 86.01% and 94.82%, respectively. Among them, the AMDZ+RI group had the smallest tumor volume and the highest tumor inhibition rate, indicating that under the same X-Ray irradiation, AMDZ showed the best radiosensitization effect.

[0051] For those skilled in the art, the present invention is not limited to the details of the exemplary embodiments described above. The embodiments are exemplary and non-restrictive, and the scope of protection of the present invention is defined by the appended claims rather than the above description, and therefore, any reference numerals in the claims should not be regarded as limiting the claims involved.

[0052] The above description is only an example of one or more embodiments of the present specification and is not intended to limit one or more embodiments of the present specification. For those skilled in the art, one or more embodiments of the present specification may have various changes and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present specification shall be included in the scope of the claims.

Claims

1. A method for preparing a diagnostic and therapeutic agent for tumor radiotherapy sensitization, characterized in that: The following steps are involved: (1) 8-15 mg of chloroauric acid was added to 70-100 mL of ultrapure water, and magnetically stirred at 40-80° C. for 20-50 min to obtain solution A; 30-45 mg of sodium citrate was added to 3-6 mL of deionized water, 0.1-0.4 mL of anhydrous potassium carbonate solution with a concentration of 12.5-15.5 mg / mL and 5-9 mg of tannic acid were dissolved in 13-19 mL of ultrapure water in sequence, and stirred in a water bath at 40-100° C. for 20-60 min to obtain solution B; solution B was transferred to solution A, and stirred in a water bath at 55-75° C. for 20-50 min. After the reaction was complete, centrifugation was performed to obtain gold nanoparticles (AuNPs); (2) 0.2-0.7 mL of a potassium hydroxide solution having a concentration of 0.05-0.07 mg / mL is added dropwise to a 30-80 mL of an AuNPs aqueous solution having a concentration of 3-4 mg / mL until the pH value reaches 8.5-10.5; then, 2.5-4.5 mg of potassium permanganate and 15-25 mg of potassium oxalate are added to the AuNPs aqueous solution in sequence; after the solution is allowed to stand in a water bath at 0°C for 20-60 min, the solution is heated to 40-80°C and allowed to stand for 1-4 h; after the reaction is complete, the solution is centrifuged to obtain composite nanoparticles of gold and manganese dioxide (Au@MnO2NPs); (3) 2-5 mL of 6.5-7.5 mg / mL Au@MnO2 NPs solution and 2-5 mg of polyvinyl pyrrolidone were added to 2-5 mL of ultrapure water, respectively. The mixed solution was stirred at 20-40°C for 10-20 h and then centrifuged to prepare Au@MnO2-PVP. (4) 4-6 g of D-mannose was dissolved in 3-6 mL of 150-250 mg / mL methylimidazole solution and stirred in a 30-60 °C water bath for 20-40 min. Then, 2.5-3.9 mL of methanol and 1.1-2.2 mL of 8.5-9.9 mg / mL Au@MnO2-PVP solution were added to the solution in sequence and stirred at 20-40 °C for 3-10 min. Then, 50-100 mg of zinc nitrate was added to the solution again and stirred for 20-50 min. After the reaction was completed, the product was centrifuged, washed with methanol and dried naturally to obtain a composite nano-diagnostic agent (Dman / Au@MnO2@ZIF-8) for tumor radiosensitization.

2. A method for preparing a diagnostic and therapeutic agent for tumor radiotherapy sensitization according to claim 1, characterized in that: In the step (1), the mass ratio of chloroauric acid to sodium citrate is 1:2 to 1:5.

625.

3. A method for preparing a diagnostic and therapeutic agent for tumor radiotherapy sensitization according to claim 1, characterized in that: The gold nanoparticles prepared in step (1) have a diameter of 8 to 12 nm and a surface charge of -20 to -30 mV.

4. A method for preparing a diagnostic and therapeutic agent for tumor radiosensitization according to claim 1, characterized in that: In the step (2), the mass ratio of AuNPs to potassium permanganate is 20:1 to 128:

1.

5. The method for preparing a diagnostic and therapeutic agent for tumor radiotherapy sensitization according to claim 1, characterized in that: The Au@MnO2NPs prepared in step (2) have a diameter of 10 to 18 nm and a surface charge of -30 to -39 mV.

6. A method for preparing a diagnostic and therapeutic agent for tumor radiotherapy sensitization according to claim 1, characterized in that: In the step (3), the mass ratio of Au@MnO2NPs to polyvinyl pyrrolidone is 2.6:1 to 18.75:

1.

7. A method for preparing a diagnostic and therapeutic agent for tumor radiosensitization according to claim 1, characterized in that: The diameter of Au@MnO2-PVP prepared in step (3) is 12 to 20 nm, and the surface charge is -20 to -29 mV.

8. A method for preparing a diagnostic and therapeutic agent for tumor radiotherapy sensitization according to claim 1, characterized in that: In the step (4), the mass ratio of Au@MnO2-PVP to D-mannose is 1:183.6 to 1:642; the mass ratio of Au@MnO2-PVP to zinc nitrate is 1:2.3 to 1:

11.

9. A method for preparing a diagnostic and therapeutic agent for tumor radiosensitization according to claim 1, characterized in that: The Au@MnO2 / Dman@ZIF-8 prepared in step (4) has ZIF-8 as the skeleton, and is loaded with Au@MnO2NPs and D-mannose. The drug loading amount of D-mannose is 8.5-9.9%, and the particle size is 100-150nm.

10. Use of the diagnostic and therapeutic agent prepared by the preparation method according to any one of claims 1 to 9 in the integration of radiosensitization and MRI diagnosis and treatment of tumors.

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