An iron-based metal nanodiagnostic agent and its preparation method and application

Abstract

The present invention discloses an iron-based metal nano-diagnostic agent and its preparation method and application, wherein the iron-based metal nano-diagnostic agent comprises an iron-based metal nano-material and a loaded therapeutic nuclide. 177 Lu and / or small molecule chemotherapy drug F16 diagnostic and therapeutic agents. Iron-based metal nanodiagnostic and therapeutic agents contain iron elements and can be used for MRI imaging and can induce tumor iron death and radionuclides. 177 Lu can emit gamma rays for SPECT imaging and beta rays for internal radiotherapy of tumors. The tumor mitochondria-targeted small molecule drug F16 has its own fluorescent properties and can be used for real-time fluorescence imaging of tumors. It also has a mitochondrial-targeted tumor-killing effect. Therefore, the iron-based metal nanodiagnostic and therapeutic agent of the present invention can achieve a comprehensive synergistic treatment of tumor internal radiotherapy, targeted chemotherapy, and ferroptosis under the guidance of MRI, SPECT, and fluorescence imaging multimodal imaging, improving the precision 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 nanodiagnostic agent and a preparation method and application thereof. Background Art

[0002] Traditional treatment options, such as chemotherapy and radiotherapy, are ineffective and result in poor prognosis due to factors such as 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 side effects of chemotherapy and radiotherapy. Furthermore, due to the high heterogeneity, low immunogenicity, high mortality rate, and low early diagnosis rates of tumors, single-mode imaging diagnosis is no longer sufficient for clinical needs. It is necessary to develop novel multimodal imaging agents and targeted tumor therapeutics 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), can release radiation through decay to damage the DNA double strand, and the gamma photons it emits can be used for single-photon computed tomography. Due to the characteristics of unlimited reproduction of tumors and vigorous energy metabolism, their mitochondrial membrane potential is much higher than that of normal cells. The lipophilic cationic compound F16 can specifically target the mitochondria of tumor cells, accumulate in the mitochondria of tumor cells at a concentration of 1000 times, and induce tumor cell apoptosis by disrupting the mitochondrial membrane potential of tumor cells and releasing cytochrome c. In addition, metal nanoparticles-based drug targeted delivery, drug controlled release, radionuclide-loaded internal radiotherapy, ferroptosis and copper death and other anti-tumor effects are expected to achieve multimodal imaging-guided multiple 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 treatment efficacy.

[0004] Although the new nano-co-delivery drug system can combine therapeutic radionuclide radiotherapy, targeted chemotherapy and ferroptosis and other comprehensive treatment methods, during the preparation of the nano-co-delivery drug system, radioactive drug labeling requires chemical group modification of the precursor compound and coupling with metal chelators to label the radionuclide, 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 objectives, the present invention provides a first aspect of a method for preparing an iron-based metal nanodiagnostic agent, comprising the following steps:

[0007] (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;

[0008] (2) ultrasonically dispersing the iron-based metal nanomaterial in a buffer solution to obtain a nanodispersion liquid;

[0009] (3) Add small molecule anti-tumor drugs and therapeutic nuclides to the nano-dispersion solution in sequence, mix thoroughly, incubate in the dark, and centrifuge to obtain an iron-based metal nano-diagnostic and therapeutic agent.

[0010] Preferably, the molar ratio of ferric chloride to terephthalic acid is (2-4): (1-2).

[0011] Preferably, in step (1), the heating temperature is 100-120° C. and the heating time is 20-28 h.

[0012] Preferably, in step (1), after centrifugation, the product is washed three times in a DMF solution at 70° C. 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.

[0013] Preferably, in step (2), the buffer solution is a phosphate buffer solution with a pH of 7.4, and the mass-to-volume ratio of the iron-based metal nanomaterial to the buffer solution is (8-12) mg: (8-12) mL.

[0014] Preferably, in step (3), the small molecule antitumor drug is F16, and the therapeutic nuclide is 177 Lu.

[0015] 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 nuclide is labeled per 1 mg of the iron-based metal nanomaterial.

[0016] The second aspect of the present invention provides an iron-based metal nano-diagnostic and therapeutic agent, which is prepared by the above-mentioned preparation method.

[0017] Preferably, the particle size of the iron-based metal nano-diagnostic and therapeutic agent is 100~180nm.

[0018] 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.

[0019] Therefore, the present invention adopts the above-mentioned iron-based metal nanodiagnostic agent and its preparation method and application, which has the following beneficial effects:

[0020] (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 the need for coupling with metal chelating agents, and the preparation steps are simple.

[0021] (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.

[0022] (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.

[0023] (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.

[0024] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a scanning electron microscope image of an iron-based metal nanomaterial;

[0026] Figure 2 The UV absorption spectrum of the iron-based metal nano-theranostic agent loaded with the small molecule targeted drug F16 and the labeling rate of the therapeutic nuclide 177Lu;

[0027] Figure 3 This is a fluorescence imaging image of the targeted cell mitochondria of an iron-based metal nanodiagnostic agent loaded with the small molecule targeted drug F16;

[0028] Figure 4 Cytotoxicity graphs of iron-based metal nanotheranostics before and after loading with therapeutic nuclides and small molecule targeted drug F16;

[0029] Figure 5 In situ SPECT imaging of a tumor using an iron-based metal nano-theranostic agent co-loaded with therapeutic nuclides and small molecule targeted drugs;

[0030] Figure 6 This figure shows the in vivo synergistic anti-tumor therapeutic effect of iron-based metal nano-theranostics co-loaded with therapeutic nuclides and small molecule targeted drugs. DETAILED DESCRIPTION

[0031] 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 specific operation process, but the present invention is not limited to this embodiment.

[0032] Example 1

[0033] The preparation method of the iron-based metal nanomaterial comprises the following steps:

[0034] (1) Dissolve 670.3 mg of FeCl3·6H2O in 14 mL of DMF.

[0035] (2) Ultrasonic dispersion of 206 mg of terephthalic acid in the solution of step (1) was performed and stirred evenly.

[0036] (3) The above solution was transferred to a Teflon reactor and heated at 110°C for 24 hours.

[0037] (4) After the reaction is completed, the precipitate is removed by centrifugation, washed three times with a 70°C DMF solution, and dried overnight in a vacuum oven at 70°C to obtain an iron-based metal nanomaterial, which can be used as an iron-based metal nanodiagnostic agent containing only iron elements without loading.

[0038] like Figure 1 As shown, the iron-based metal nanomaterial prepared by the present invention has a regular polyhedron morphology, good dispersibility and uniform size.

[0039] Example 2

[0040] The preparation method of the iron-based metal nanomaterial loaded with the small molecule anti-tumor drug F16 comprises the following steps:

[0041] 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 at 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.

[0042] Example 3

[0043] Loaded with small molecule anti-tumor drug F16 and therapeutic nuclide 177 The preparation method of Lu iron-based metal nanomaterials comprises the following steps:

[0044] Add 2mCi of therapeutic radionuclide to 2mg of F16-loaded iron-based metal nanomaterial solution (calculated based on the content of iron-based metal nanomaterial) 177 Lu, shake and mix, incubate at room temperature, centrifuge and discard the supernatant after 2 hours, wash three times with PBS to remove free177 Lu and resuspended in 2 mL PBS solution to obtain therapeutic nuclide 177 Iron-based metal nanotheranostics co-loaded with Lu and F16.

[0045] Test Example 1

[0046] Determination of loading F16 and 177 Ultraviolet characteristic absorption peak and labeling rate of iron-based metal nanodiagnostic agents after Lu.

[0047] Load F16 and 177 The method for determining the characteristic ultraviolet absorption peak and labeling rate of the iron-based metal nanodiagnostic agent after Lu is as follows:

[0048] The standard concentration gradient of F16 solution (0, 5, 10, 15, 30, 50 μg mL) was established. -1 ), and the supernatant after loading, were detected by UV spectrophotometer, and the corresponding UV absorption peaks were obtained by scanning.

[0049] The above therapeutic nuclides 177 The iron-based metal nanotherapeutic agent co-loaded with Lu and F16 was resuspended by ultrasonic dispersion, and 5µL of the co-loaded iron-based metal nanotherapeutic agent was added to 20µL of saturated EDTA solution and mixed thoroughly. The labeling rate of the product was detected by radioactive thin layer chromatography using physiological saline as the developing agent.

[0050] 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.

[0051] Test Example 2

[0052] Cell mitochondria-targeted fluorescence imaging of iron-based metal nanotheranostics loaded with F16.

[0053] 4T1 cells in logarithmic growth phase were cultured at 5×10 5 Count and plate the cells in a 35mm dish and culture them until the next day when the cells adhere to the wall. Incubate the cells with 50µg / 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, and collect cell bright field and fluorescence images respectively. 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.

[0054] Test Example 3

[0055] Cytotoxicity detection of iron-based metal nanotheranostics before and after loading with therapeutic nuclides and F16.

[0056] 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. Cell viability was measured using CCK-8 after 48 hours. The experimental results are shown in Figure 2. 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 chemotherapy drugs. 177 The iron-based metal nanotheranostics after Lu and F16 showed the greatest tumor cell killing effect.

[0057] Test Example 4

[0058] In situ SPECT imaging of tumors with iron-based metal nanotheranostics co-loaded with therapeutic nuclides and F16.

[0059] One million 4T1 cells in the logarithmic growth phase were injected into the fat pad of the third mammary gland of 6-week-old female BALB / c mice to establish an orthotopic model of mouse breast cancer. After 10 days, when the tumor volume reached 150 mm 3 50µg of therapeutic nuclide and F16 co-loaded iron-based metal nano-theranostic agent were injected into the tumor of tumor-bearing mice, and small animal SPECT / CT scanning imaging was performed using MOLECUBES, Belgium instruments at 0h, 24h, 72h, and 168h after injection. Figure 5 It can be seen that the tumor imaging effect is good.

[0060] Test Example 5

[0061] Evaluation of the synergistic therapeutic effect of iron-based metal nanotheranostics co-loaded with therapeutic nuclides and F16 in tumor-bearing mice.

[0062] The orthotopic breast cancer model mice were injected with 50μL normal saline (control group), iron-based metal nanodiagnostic agent (200μg), F16 (200μg), 177 Lu (200µCi), F16-loaded iron-based metal nano-theranostics (200µg), and iron-based metal nano-theranostics co-loaded with therapeutic nuclides and F16 (200µg) were injected. Changes in tumor volume were observed and measured every 2 days after injection for 14 days. After the treatment, the tumor growth curve was drawn, the tumor was peeled off and photographed. Figure 6As shown, compared with other single-treatment models, the iron-based metal nanotheranostic agent co-loaded with therapeutic nuclides and F16 showed the best tumor suppression efficacy. The iron-based metal nanotheranostic agent prepared by the present invention can simultaneously deliver therapeutic nuclides and the anti-tumor compound F16, exerting a synergistic tumor-killing effect, and can be used to address some chemotherapy or radiotherapy problems such as drug resistance or radiation resistance.

[0063] 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.

[0064] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the same. 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 solutions of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions 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 uniformly at room temperature, adding the mixture to 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 nanodispersion liquid; The buffer solution is a phosphate buffer solution with a pH of 7.4, and the mass-to-volume ratio of the iron-based metal nanomaterial to the buffer solution is (8-12) mg: (8-12) mL; (3) Add small molecule antitumor drugs and therapeutic nuclides to the nanodispersion solution in sequence. The small molecule antitumor drug is F16 and the therapeutic nuclides are 177 The mass ratio of Lu, iron-based metal nanomaterials and small molecule anti-tumor drugs is (1~2): (1~2), and each 1 mg of iron-based metal nanomaterial is labeled with 1 mCi of therapeutic radionuclide. The mixture is fully mixed, incubated in the dark, and centrifuged to obtain an iron-based metal nanodiagnostic and therapeutic agent.

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

4. The method for preparing an iron-based metal nanodiagnostic agent according to claim 1, characterized in that: In step (1), after centrifugation, the product is washed three times in a DMF solution at 70° C. 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. An iron-based metal nanodiagnostic agent, characterized by: Prepared by the preparation method according to any one of claims 1 to 4.

6. The iron-based metal nanodiagnostic agent according to claim 5, characterized in that: The particle size of iron-based metal nano-therapeutic agents is 100~180nm.

7. Use of an iron-based metal nanodiagnostic and therapeutic agent according to any one of claims 5 to 6 in the preparation of a diagnostic and therapeutic agent for tumor diseases.

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