Iron-based metal nano diagnosis and treatment agent as well as preparation method and application thereof

Abstract

The invention discloses an iron-based metal nano diagnosis and treatment agent as well as a preparation method and application thereof. The iron-based metal nano diagnosis and treatment agent comprises an iron-based metal nano material and a diagnosis and treatment agent loaded with therapeutic nuclide 177Lu and / or a micromolecular chemotherapeutic drug F16. The iron-based metal nano diagnosis and treatment agent contains an iron element, can be used for MRI imaging and can induce tumor ferroptosis at the same time, radionuclide 177Lu can emit gamma rays for SPECT imaging and can emit beta rays for tumor internal radiotherapy, tumor mitochondria targeting small molecule medicine F16 has the fluorescence characteristic and can be used for tumor real-time fluorescence imaging, and the application range is wide. Meanwhile, a mitochondrial targeting tumor killing effect is achieved. Therefore, the iron-based metal nano diagnosis and treatment agent disclosed by the invention can realize comprehensive collaborative treatment of intratumor radiotherapy, targeted chemotherapy and ferroptosis under the guidance of MRI (Magnetic Resonance Imaging), SPECT (Single Photon Emission Computed Tomography) and multi-mode imaging of fluorescence imaging, and improves the precise diagnosis and treatment of tumors.

Description

Technical Field

[0001] The present invention relates to the technical field of biomaterials, and in particular to an iron-based metal nano-diagnostic agent and a preparation method and application thereof. Background Art

[0002] Due to the radiation resistance caused by hypoxia in the tumor microenvironment, dose-dependent toxicity of chemotherapy drugs, chemotherapy resistance, radiation damage to normal tissues and organs, and the toxic and side effects of radiotherapy and chemotherapy, the tumor treatment effect of traditional treatment options such as radiotherapy and chemotherapy is poor and the prognosis is poor. In addition, due to the high heterogeneity, low immunogenicity, high mortality rate, and low early diagnosis rate of tumors, single-mode imaging diagnosis can no longer meet clinical needs. It is necessary to develop new multimodal imaging agents and targeted tumor therapeutic drugs to improve early diagnosis and precise targeted treatment of tumors.

[0003] Therapeutic nuclides 177 Lu has a moderate half-life (T 1 / 2 =6.7d), which can release radiation through decay to damage the DNA double helix, and the gamma photons it emits can be used for single-photon computed tomography. Because tumors have the characteristics of unlimited reproduction and vigorous energy metabolism, their mitochondrial membrane potential is much higher than that of normal cells. The lipophilic cationic compound F16 can specifically target tumor cell mitochondria, accumulate in tumor cell mitochondria at a concentration of 1000 times, and induce tumor cell apoptosis by disintegrating the mitochondrial membrane potential of tumor cells and releasing cytochrome c. In addition, anti-tumor effects such as drug targeted delivery, drug controlled release, radionuclide loading internal radiotherapy, ferroptosis and copper death based on metal nanoparticles are expected to achieve multi-modal imaging-guided multi-precision comprehensive treatment of tumors. Based on the limitations and shortcomings of current chemoradiotherapy, it is necessary to construct a new type of nano-co-delivery drug system to achieve precise targeted comprehensive treatment of tumors by combining therapeutic radionuclide internal radiotherapy, targeted chemotherapy and ferroptosis, reduce related toxic side effects, improve prognosis, and enhance therapeutic efficacy.

[0004] Although the new nano-co-drug delivery system can combine therapeutic radionuclide radiotherapy, targeted chemotherapy and ferroptosis and other comprehensive treatment methods, in the preparation process of the nano-co-drug delivery system, radioactive drug labeling requires chemical group modification of the precursor compound and coupling with metal chelators to label the radioactive nuclides, which increases the initial synthesis steps and costs. Summary of the invention

[0005] The purpose of the present invention is to provide an iron-based metal nano-diagnostic agent and its preparation method and application, so as to solve the problem that the preparation of the above-mentioned diagnostic agent requires preliminary steps such as chemical group modification and coupling with metal chelators, and the preparation method is relatively complicated and the cost is high.

[0006] To achieve the above object, the first aspect of the present invention provides a method for preparing an iron-based metal nanodiagnostic agent, comprising the following steps: (1) dispersing ferric chloride and terephthalic acid in dimethylformamide, stirring evenly at room temperature, adding the mixture into a reactor for heating, and then centrifuging, washing and drying to obtain an iron-based metal nanomaterial; (2) ultrasonically dispersing the iron-based metal nanomaterial in a buffer solution to obtain a nano-dispersion liquid; (3) Adding small molecule anti-tumor drugs and therapeutic nuclides to the nano-dispersion solution in sequence, mixing thoroughly, incubating in the dark, and centrifuging to obtain an iron-based metal nano-diagnostic and therapeutic agent.

[0007] Preferably, the molar ratio of ferric chloride to terephthalic acid is (2-4): (1-2).

[0008] Preferably, in step (1), the heating temperature is 100-120° C. and the heating time is 20-28 h.

[0009] Preferably, in step (1), after centrifugation, the product is washed three times in a 70° C. DMF solution for purification, and the purified light orange product is dried in a vacuum oven at 70° C. overnight to obtain an iron-based metal nanomaterial.

[0010] Preferably, in step (2), the buffer solution is a phosphate buffer solution with a pH value of 7.4, and the mass volume ratio of the iron-based metal nanomaterial to the buffer solution is (8-12) mg: (8-12) mL.

[0011] Preferably, in step (3), the small molecule anti-tumor drug is F16 and the therapeutic nuclide is 177 Lu.

[0012] Preferably, in step (3), the mass ratio of the iron-based metal nanomaterial to the small molecule anti-tumor drug is (1-2): (1-2), and 1-2 mCi of therapeutic radionuclide is labeled for every 1 mg of the iron-based metal nanomaterial.

[0013] The second aspect of the present invention provides an iron-based metal nano-diagnostic and therapeutic agent prepared by the above-mentioned preparation method.

[0014] Preferably, the particle size of the iron-based metal nano-diagnostic agent is 100~180nm.

[0015] The third aspect of the present invention provides an application of an iron-based metal nano-diagnostic and therapeutic agent in a diagnostic and therapeutic agent for tumor diseases.

[0016] Therefore, the present invention adopts the above-mentioned iron-based metal nano-diagnostic agent and its preparation method and application, which has the following beneficial effects: (1) The iron-based metal nanomaterial prepared by the present invention is a metal organic framework with a large surface area. It can load radioactive compounds through molecular self-assembly without coupling with a metal chelator, and the preparation steps are simple.

[0017] (2) The iron-based metal nanomaterials prepared by the present invention can load tumor-killing drugs and therapeutic nuclides through molecular self-assembly, deliver chemotherapy drugs and therapeutic nuclides to tumors, and play a triple synergistic therapeutic role of ferroptosis, chemotherapy and internal radiotherapy, with good therapeutic effects.

[0018] (3) The present invention is due to radionuclides 177 Lu has a long half-life (6.7 days) and can decay to produce beta rays, causing single-strand breaks in cell DNA to kill cells. It can also emit gamma rays for nuclear medicine SPECT imaging, so it can be used to monitor the efficacy of treatment.

[0019] (4) The iron-based metal nanodiagnostic and therapeutic agent of the present invention can not only improve the precision diagnosis and treatment of tumors and meet clinical needs, but also has broad application prospects in many fields such as nanobiomedicine, multimodal imaging and imaging-guided tumor treatment.

[0020] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a scanning electron microscope image of an iron-based metal nanomaterial; Figure 2 The ultraviolet absorption spectrum of the iron-based metal nano-diagnostic agent loaded with small molecule targeted drug F16 and the labeling rate of therapeutic nuclide 177Lu; Figure 3 This is the fluorescence imaging of targeted cell mitochondria of the iron-based metal nanodiagnostic agent loaded with the small molecule targeted drug F16; Figure 4 Cytotoxicity diagram of iron-based metal nanodiagnostic agents before and after loading with therapeutic nuclides and small molecule targeted drug F16; Figure 5 This is an in situ SPECT imaging image of a tumor of an iron-based metal nanodiagnostic agent co-loaded with therapeutic nuclides and small molecule targeted drugs; Figure 6 This is a diagram showing the in vivo synergistic anti-tumor therapeutic effect of iron-based metal nanodiagnostic agents co-loaded with therapeutic nuclides and small molecule targeted drugs. DETAILED DESCRIPTION

[0022] The present invention will be further described below. It should be noted that this embodiment is based on the technical solution and provides a detailed implementation method and a specific operation process, but the present invention is not limited to this embodiment.

[0023] Example 1 The method for preparing an iron-based metal nanomaterial comprises the following steps: (1) Dissolve 670.3 mg of FeCl3·6H2O in 14 mL of DMF.

[0024] (2) Ultrasonic dispersion of 206 mg of terephthalic acid in the solution of step (1) was performed and stirred evenly.

[0025] (3) The above solution was transferred to a Teflon reactor and heated at 110°C for 24 hours.

[0026] (4) After the reaction is completed, the precipitate is removed by centrifugation, washed three times with a 70°C DMF solution, and dried in a vacuum oven at 70°C overnight to obtain an iron-based metal nanomaterial, which can be used as an iron-based metal nanodiagnostic agent containing only iron elements without loading.

[0027] like Figure 1 As shown, the iron-based metal nanomaterial prepared by the present invention has a regular polyhedron shape, good dispersibility and uniform size.

[0028] Example 2 The preparation method of the iron-based metal nanomaterial loaded with the small molecule anti-tumor drug F16 comprises the following steps: 10 mg of iron-based metal nanomaterials and 10 mg of small molecule anti-tumor drug F16 were ultrasonically dispersed in 10 mL of PBS buffer with pH = 7.4. After incubation at 4°C for 24 hours, the supernatant was collected by centrifugation, washed three times with PBS, and freeze-dried to obtain iron-based metal nanomaterials loaded with F16, which can be used as iron-based metal nanodiagnostic and therapeutic agents containing iron elements and loaded with small molecule anti-tumor drugs.

[0029] Example 3 Loaded with small molecule anti-tumor drug F16 and therapeutic nuclide 177 The method for preparing Lu iron-based metal nanomaterials comprises the following steps: Add 2 mCi of therapeutic radionuclide to a solution of 2 mg of iron-based metal nanomaterials loaded with F16 (calculated based on the content of iron-based metal nanomaterials) 177 Lu, shake and mix, incubate at room temperature, centrifuge and discard the supernatant after 2 hours, wash three times with PBS to remove free 177 Lu, and resuspended in 2 mL PBS solution to obtain therapeutic nuclide 177 Iron-based metal nanotheranostics co-loaded with Lu and F16.

[0030] Test Example 1 Determination of loading F16 and 177 Ultraviolet characteristic absorption peak and labeling rate of iron-based metal nanodiagnostic agents after Lu.

[0031] Load F16 and 177 The method for determining the ultraviolet characteristic absorption peak and labeling rate of the iron-based metal nanodiagnostic agent after Lu is as follows: The standard concentration gradient of F16 solution (0, 5, 10, 15, 30, 50 μg mL -1 ), as well as the supernatant after loading, were detected by UV spectrophotometer, and the corresponding UV absorption peaks were obtained by scanning.

[0032] The above therapeutic nuclides 177 The iron-based metal nano-therapeutic agent co-loaded with Lu and F16 was resuspended by ultrasonic dispersion, 5 µL of the co-loaded iron-based metal nano-therapeutic agent was added to 20 µL of saturated EDTA solution and mixed thoroughly, and the labeling rate of the product was detected by radioactive thin layer chromatography using physiological saline as the developing agent.

[0033] from Figure 2 It can be seen that F16 and 177 Lu has been successfully loaded onto iron-based metal nanomaterials to prepare co-loaded iron-based metal nanodiagnostic and therapeutic agents.

[0034] Test Example 2 Cell mitochondria-targeted fluorescence imaging of iron-based metal nanotheranostics loaded with F16.

[0035] 4T1 cells in logarithmic growth phase were cultured at 5×10 5 Count and plate the cells in a 35 mm dish and culture them until the next day when the cells adhere to the wall. Incubate the cells with 50 µg / mL concentration of F16 and F16-loaded iron-based metal nanodiagnostic agents at 37°C in the dark for 1 hour. After incubation, wash with phosphate buffer three times. Finally, take pictures of the cells with a Leica inverted fluorescence microscope to collect cell bright field and fluorescence images. Figure 3 As shown, the iron-based metal nano-diagnostic and therapeutic agent loaded with F16 is consistent with F16, and can specifically target the mitochondria of breast cancer cells to achieve tumor cell killing effects.

[0036] Test Example 3 Cytotoxicity detection of iron-based metal nanodiagnostic agents before and after loading with therapeutic nuclides and F16.

[0037] Breast cancer cells in the logarithmic growth phase were incubated with different concentrations of iron-based metal nanotheranostics, iron-based metal nanotheranostics loaded with F16, and iron-based metal nanotheranostics co-loaded with therapeutic nuclides and F16, and the cell survival rate was detected by CCK-8. The experimental results are shown in Figure 4 As shown in the figure, the loading of iron-based metal nanodiagnostic agents reduced the cytotoxicity of F16, which is beneficial to reduce the toxic side effects of small molecule chemotherapeutic drugs. 177The iron-based metal nanotheranostics after Lu and F16 showed the greatest tumor cell killing effect.

[0038] Test Example 4 In situ SPECT imaging of tumors with iron-based metal nanotheranostics co-loaded with therapeutic radionuclides and F16.

[0039] One million logarithmic growth phase 4T1 cells were injected into the fat pad of the third mammary gland of 6-week-old female BALB / c mice to construct an orthotopic model of mouse breast cancer. Ten days later, when the tumor volume reached 150 mm 3 50 μg of therapeutic nuclide and F16 co-loaded iron-based metal nano-diagnostic agent were injected into the tumor of tumor-bearing mice, and small animal SPECT / CT scanning imaging was performed using MOLECUBES, Belgium instrument at 0h, 24h, 72h, and 168h after injection. Figure 5 It can be seen that the tumor imaging effect is good.

[0040] Test Example 5 Evaluation of the synergistic therapeutic effect of iron-based metal nanotheranostics co-loaded with therapeutic nuclides and F16 on tumor-bearing mice.

[0041] The orthotopic breast cancer model mice were injected with 50μL of normal saline (control group), iron-based metal nanodiagnostic agent (200μg), F16 (200μg), 177 Lu (200µCi), iron-based metal nano-therapeutic agents loaded with F16 (200µg), and iron-based metal nano-therapeutic agents co-loaded with therapeutic nuclides and F16 (200µg). Changes in tumor volume were observed and measured every 2 days after injection, and the monitoring lasted for 14 days. After the treatment, the tumor growth curve was drawn, the tumor was peeled off and photographed. Figure 6 As shown, compared with the single treatment mode of other treatment groups, the iron-based metal nano-diagnostic agent co-loaded with therapeutic nuclides and F16 showed the best tumor inhibition effect. The iron-based metal nano-diagnostic agent prepared by the present invention can simultaneously deliver therapeutic nuclides and anti-tumor compound F16, play a synergistic role in killing tumors, and can be used to solve the problems of drug resistance or radiation resistance in some chemotherapy or radiotherapy.

[0042] Therefore, the present invention adopts the above-mentioned iron-based metal nanodiagnostic and therapeutic agent and its preparation method and application, which can not only improve the precise diagnosis and treatment of tumors to meet clinical needs, but also has broad application prospects in many fields such as nanobiomedicine, multimodal imaging and imaging-guided tumor treatment.

[0043] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution of the present invention.

Claims

1. A method for preparing an iron-based metal nanodiagnostic agent, characterized in that: The following steps are involved: (1) dispersing ferric chloride and terephthalic acid in dimethylformamide, stirring evenly at room temperature, adding the mixture into a reactor for heating, and then centrifuging, washing and drying to obtain an iron-based metal nanomaterial; (2) ultrasonically dispersing the iron-based metal nanomaterial in a buffer solution to obtain a nano-dispersion liquid; (3) Adding small molecule anti-tumor drugs and therapeutic nuclides to the nano-dispersion solution in sequence, mixing thoroughly, incubating in the dark, and centrifuging to obtain an iron-based metal nano-diagnostic and therapeutic agent.

2. The method for preparing an iron-based metal nano-diagnostic agent according to claim 1, characterized in that: In step (1), the molar ratio of ferric chloride to terephthalic acid is (2-4): (1-2).

3. The method for preparing an iron-based metal nano-diagnostic agent according to claim 1 is characterized in that: In step (1), the heating temperature is 100-120°C and the heating time is 20-28 hours.

4. The method for preparing an iron-based metal nano-diagnostic agent according to claim 1, characterized in that: In step (1), after centrifugation, the product is washed three times in a 70° C. DMF solution for purification, and the purified light orange product is dried in a vacuum oven at 70° C. overnight to obtain an iron-based metal nanomaterial.

5. The method for preparing an iron-based metal nano-diagnostic agent according to claim 1, characterized in that: In step (2), the buffer solution is a phosphate buffer solution with a pH value of 7.4, and the mass volume ratio of the iron-based metal nanomaterial to the buffer solution is (8-12) mg: (8-12) mL.

6. The method for preparing an iron-based metal nano-diagnostic agent according to claim 1, characterized in that: In step (3), the small molecule antitumor drug is F16 and the therapeutic nuclide is 177 Lu.

7. The method for preparing an iron-based metal nano-diagnostic agent according to claim 1, characterized in that: In step (3), the mass ratio of the iron-based metal nanomaterial and the small molecule anti-tumor drug is (1~2): (1~2), and 1 mCi of therapeutic radionuclide is labeled for every 1 mg of the iron-based metal nanomaterial.

8. An iron-based metal nano-diagnostic agent, characterized in that: Prepared by the preparation method according to any one of claims 1 to 7.

9. The iron-based metal nano-diagnostic agent according to claim 8, characterized in that: The particle size of iron-based metal nano-therapeutic agents is 100~180nm.

10. Use of an iron-based metal nano-diagnostic and therapeutic agent according to any one of claims 8 to 9 in a diagnostic and therapeutic agent for tumor diseases.

Images