CN112717147B - Preparation method and application of Fe and Pt double-active-site single-atom diagnosis and treatment agent - Google Patents

Preparation method and application of Fe and Pt double-active-site single-atom diagnosis and treatment agent Download PDF

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CN112717147B
CN112717147B CN202011608307.1A CN202011608307A CN112717147B CN 112717147 B CN112717147 B CN 112717147B CN 202011608307 A CN202011608307 A CN 202011608307A CN 112717147 B CN112717147 B CN 112717147B
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active site
diagnosis
monoatomic
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atom
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CN112717147A (en
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胡尊富
郑秀文
戴志超
孙运强
王姗
魏秋连
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Linyi University
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    • A61K49/221—Echographic preparations; Ultrasound imaging preparations ; Optoacoustic imaging preparations characterised by the targeting agent or modifying agent linked to the acoustically-active agent
    • A—HUMAN NECESSITIES
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    • A61K33/243—Platinum; Compounds thereof
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    • A61K33/00—Medicinal preparations containing inorganic active ingredients
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    • A61K33/26—Iron; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/22—Echographic preparations; Ultrasound imaging preparations ; Optoacoustic imaging preparations
    • A61K49/222—Echographic preparations; Ultrasound imaging preparations ; Optoacoustic imaging preparations characterised by a special physical form, e.g. emulsions, liposomes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
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    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J23/00—Catalysts comprising metals or metal oxides or hydroxides, not provided for in group B01J21/00
    • B01J23/70—Catalysts comprising metals or metal oxides or hydroxides, not provided for in group B01J21/00 of the iron group metals or copper
    • B01J23/89—Catalysts comprising metals or metal oxides or hydroxides, not provided for in group B01J21/00 of the iron group metals or copper combined with noble metals
    • B01J23/8906—Iron and noble metals
    • B01J35/391—
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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    • B01J37/00—Processes, in general, for preparing catalysts; Processes, in general, for activation of catalysts
    • B01J37/0009—Use of binding agents; Moulding; Pressing; Powdering; Granulating; Addition of materials ameliorating the mechanical properties of the product catalyst
    • B—PERFORMING OPERATIONS; TRANSPORTING
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    • B01J37/08—Heat treatment
    • B01J37/082—Decomposition and pyrolysis
    • B01J37/086—Decomposition of an organometallic compound, a metal complex or a metal salt of a carboxylic acid
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    • B22—CASTING; POWDER METALLURGY
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    • B22F9/00—Making metallic powder or suspensions thereof
    • B22F9/16—Making metallic powder or suspensions thereof using chemical processes
    • B22F9/30—Making metallic powder or suspensions thereof using chemical processes with decomposition of metal compounds, e.g. by pyrolysis

Abstract

The invention belongs to the field of medical nano materials, and particularly relates to a preparation method and application of a Fe and Pt double-active-site single-atom diagnosis and treatment agent. The method is realized by the following steps: transferring the bimetallic active site monoatomic precursor into a tubular furnace, evacuating argon, calcining at high temperature, cooling to room temperature after calcining, and grinding for later use; dispersing the bimetallic active site monoatomic catalyst in DMF, performing ultrasonic dispersion, and adding DSPE-PEG-FA. Based on the excellent performances of the monatomic catalyst such as high-efficiency catalytic efficiency, 100% atom utilization rate and the like, the invention respectively realizes high-efficiency Fenton catalysis and disproportionation reaction by utilizing Fe and Pt monatomic, realizes high-efficiency tumor inhibition and tumor hypoxia alleviation, and simultaneously effectively improves the selectivity and the biological safety of tumor treatment by introducing the specific targeting reagent.

Description

Preparation method and application of Fe and Pt double-active-site single-atom diagnosis and treatment agent
Technical Field
The invention belongs to the field of medical nano materials, and particularly relates to a preparation method and application of a Fe and Pt double-active-site single-atom diagnosis and treatment agent.
Background
In recent years, cancer has become one of three killers in human, and the incidence and mortality of cancer have risen year by year, and the quality of life of cancer patients is worried. Therefore, how to realize early detection, early diagnosis and early treatment of cancer is important. The traditional cancer treatment means (operation, radiotherapy and chemotherapy) has many defects, and the nano anti-cancer medicine is greatly promoted by researchers and is called as a feasible means for diagnosing and treating the cancer in the future. The nano diagnosis and treatment agent can realize high-efficiency cancer treatment while realizing cancer diagnosis and treatment. Through the development of decades, the nano diagnosis and treatment agent has been developed to a certain extent, but the tumor inhibition efficacy, the tumor specific targeting ability and the biocompatibility of the nano diagnosis and treatment agent are all required to be further improved. Therefore, the development of the nano diagnosis and treatment agent with high biological safety and high tumor inhibition performance has wide application prospect.
The single-atom catalyst has 100% atom utilization rate, and the catalytic efficiency is greatly improved. Therefore, based on the preparation technology of the monatomic catalyst, the tumor hypoxia environment is improved in the process of realizing the efficient chemokinetic treatment of the tumor by constructing the monatomic with the Fenton catalytic effect and the monatomic with the dismutase-like effect in a synergistic manner, so that the tumor treatment efficiency is further improved. Meanwhile, the photoacoustic imaging effect is used for realizing high-efficiency chemical kinetic treatment and hypoxic improvement under the guidance of imaging, and the method has important value.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a preparation method and application of a Fe and Pt double-active-site single-atom diagnosis and treatment agent.
The invention also provides an application of the Fe and Pt double-active-site single-atom diagnosis and treatment agent prepared by the method.
The technical scheme adopted by the invention for realizing the purpose is as follows:
the invention provides a preparation method of a Fe and Pt double-active-site single-atom diagnosis and treatment agent, which comprises the following steps:
(1) transferring the precursor of the single atom of the double metal active site into a tubular furnace, evacuating argon, calcining at high temperature, cooling to room temperature after calcining to obtain the catalyst of the single atom of the double metal active site, and fully grinding for later use;
(2) dispersing the double-metal active site monoatomic catalyst in dimethyl formamide DMF, fully performing ultrasonic dispersion, and adding a proper amount of DSPE-PEG-FA to obtain the double-metal active site monoatomic diagnosis and treatment agent.
The bimetallic active site monoatomic precursor used in the invention is prepared by the following two methods:
a, preparing hydrogen peroxide-like mimic enzyme metal salt, dismutase-like metal salt and zinc nitrate (Zn (NO) 3 ) 2 ) Mixing the dimethyl imidazole and the methanol fully, transferring the mixture to a reaction kettle for hydrothermal reaction, cooling to room temperature after the reaction is finished, and performing centrifugal washing;
or
b adding ZnO 3 Mixing dimethyl imidazole and methanol completely, transferring to a polytetrafluoroethylene reaction kettle, keeping the temperature constant for a certain time, cooling to room temperature to obtain a metal organic framework compound, centrifugally washing, suspending in a methanol solution, adding a hydrogen peroxide-like mimic enzyme metal salt and a dismutase-like metal salt, stirring completely, and centrifugally washing.
Further, the molar ratio of the hydrogen peroxide-like mimic enzyme metal salt to the dismutase-like metal salt to the zinc nitrate is 1: (0.5-2): (8-15); the molar ratio of the zinc nitrate to the dimethyl imidazole is 1: (6-10).
Further, the metal salt of the hydrogen peroxide mimetic enzyme is ferric acetylacetonate, iron phthalocyanine or ferric chloride; the dismutase-like metal salt is acetylacetone platinum, phthalocyanine platinum, chloroplatinic acid or potassium chloroplatinate.
Further, the hydrothermal reaction is carried out for 3-6h at 110-130 ℃.
Further, in the step (1), the emptying time of the tubular furnace is 30-60 min; the high-temperature calcination is carried out at the temperature of 900-1100 ℃ for 1-4 h.
The mass ratio of the bimetallic active site single-atom catalyst to the DSPE-PEG-FA is 1: (0.5 to 1.5); the molecular weight of the DSPE-PEG-FA is 500-10000.
Further, the concentration of the bimetallic active site monoatomic catalyst in DMF is 10-200 mg/mL.
The invention also provides an application of the bimetallic active site monoatomic diagnosis and treatment agent prepared by the preparation method, and the bimetallic active site monoatomic diagnosis and treatment agent is used as an anti-tumor medicament, a tumor imaging agent or a medicament carrier.
The invention has the beneficial effects that: the preparation method takes a metal organic framework compound (ZIF-8) as a support, fixes the double-metal active site monoatomic atoms by loading high-activity metal salt into the support through coordination and space confinement, and prepares the double-metal site monoatomic catalyst under the high-temperature reduction action, so that the specific targeting ability and the biocompatibility of a monoatomic diagnosis and treatment agent can be effectively improved after a specific targeting agent DSPE-PEG-FA is coupled to the surface. Under the high-efficiency catalytic action of the monatomic catalyst, the high-concentration hydrogen peroxide in the tumor can be efficiently catalyzed to decompose and generate high-cytotoxicity hydroxyl radicals, and then the high-efficiency tumor inhibition is realized. Therefore, the method has a wider application prospect.
Drawings
Fig. 1 is a flow chart of the preparation of the bimetallic active site single atom therapeutic agent provided by the embodiment of the invention.
FIG. 2 is a Transmission Electron Microscope (TEM) picture of a metal organic framework compound (ZIF-8) prepared in example 1 of the present invention.
Fig. 3 is a Transmission Electron Microscope (TEM) image of the dual metal active site single atom therapeutic agent prepared in example 1 of the present invention.
FIG. 4 is a spherical aberration corrected transmission electron microscope (STEM) of the double metal active site single atom therapeutic agent prepared in example 1 of the present invention.
FIG. 5 shows that the double metal active site single atom diagnostic reagent prepared in example 1 of the present invention induces the survival rate of human breast cancer cells.
FIG. 6 is a graph showing the tumor-inhibiting effect of the bimetallic active site monatomic diagnostic agent prepared in example 1 on the living bodies of tumor-bearing mice.
FIG. 7 is a graph showing the change in body weight of tumor-bearing mice when the bimetallic active site monatomic diagnostic agent prepared in example 1 of the present invention is used to treat the tumor-bearing mice.
Fig. 8 is a Transmission Electron Microscope (TEM) image of the bimetallic active site single atom therapeutic agent prepared in example 2 of the present invention.
Fig. 9 is a Transmission Electron Microscope (TEM) image of the dual metal active site single atom therapeutic agent prepared in example 3 of the present invention.
Detailed Description
In order to make the objects, technical paths and advantages of the present invention more obvious and clear, the following description will be made in detail with reference to fig. 1 and specific examples to illustrate the preparation process and application of a single-atom diagnostic agent with bimetallic active sites. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Referring to fig. 1, fig. 1 is a flowchart illustrating an embodiment of a route optimization method for preparing a single-atom diagnostic agent with bimetallic active sites according to the present invention, as shown in fig. 1.
The ZIF-8 carrier prepared by the embodiment has a large number of amino groups and hydroxyl groups, and can effectively coordinate iron salts and platinum salts, and meanwhile, the ZIF-8 carrier has a large number of extremely tiny pore channels, and can effectively anchor the iron salts and the platinum salts through the adsorption confinement effect, so that the possibility of preparing the single atoms of the bimetallic active sites is provided.
The content of monatomic Fe and monatomic platinum in the double-metal active site monatomic diagnosis and treatment agent prepared by the high-temperature thermal reduction method is very low (< 2%), the dosage of the diagnosis and treatment agent is small, the image of the physiological environment of a living body is very tiny, and meanwhile, the specific targeting capability and the biocompatibility of the diagnosis and treatment agent can be effectively improved by the DSPE-PEG-FA specifically coated on the surface.
The carbonized ZIF-8 skeleton in the double-metal active site single-atom diagnosis and treatment agent prepared by the embodiment has a high-efficiency photothermal conversion effect, and can realize the photothermal treatment effect of tumors. Meanwhile, the monatomic Fe can realize the high-efficiency Fenton catalytic action in the slightly acidic environment of the tumor, and the high-efficiency chemical kinetic treatment is realized; the monatomic Pt can realize the effect of dismutase, catalyze the decomposition of high-concentration hydrogen peroxide in the tumor into oxygen, realize the hypoxic improvement of the tumor microenvironment, improve the concentration of active oxygen and induce apoptosis efficiency, and therefore, the monatomic Pt has wide application prospect in the field of tumor inhibition.
The double-metal active site monoatomic diagnosis and treatment agent prepared by the embodiment can realize the high-efficiency photoacoustic imaging and photothermal imaging effects of a tumor region based on the high-efficiency photothermal conversion effect of the carbonized ZIF-8, can realize the high-efficiency diagnosis of the tumor and the high-efficiency tumor inhibition effect under the guide of a dual-mode image, and has a great application value in the fields of tumor diagnosis, treatment and monitoring.
In some embodiments, the monatomic hydrogen peroxide mimetic enzyme includes one or more metal monatomic, such as an iron monatomic, a copper monatomic, a manganese monatomic, a cobalt monatomic, and the like, but is not limited thereto. In this embodiment, the monatomic hydrogen peroxide mimic enzyme has a very small content of iron atoms, and as an essential element of a living body, iron has little influence on the normal metabolism of the living body.
In some embodiments, the molar ratio of the hydrogen peroxide mimetic enzyme metal salt, the dismutase-like metal salt, and the zinc nitrate is 1: (0.5-2): (8-15). Within the range of the molar ratio, the obtained double-metal active site single-atom diagnosis and treatment agent has better chemical kinetics efficiency and dismutase-like catalysis efficiency, and when the molar ratio exceeds the range, single atoms are converted into micro particles or even metal particles due to excessive metal salt addition, so that the chemical kinetics efficiency and the dismutase-like catalysis efficiency are reduced.
In some embodiments, the hydrogen peroxide mimetic enzyme-like metal salt is ferric acetylacetonate, ferric phthalocyanine, ferric chloride, or the like; the dismutase-like metal salt is one or more of platinum acetylacetonate, platinum phthalocyanine, chloroplatinic acid, potassium chloroplatinate, and the like, but is not limited thereto.
In the embodiment, the emptying time of the tubular furnace is 30-60 minutes, and the emptying time is too short, so that metal oxide is generated, and the improvement of tumor inhibition efficiency is not facilitated.
In the embodiment, the preparation method of the bimetallic active site monoatomic diagnosis and treatment agent has the high-temperature calcination temperature of 900-1100 ℃, the low calcination temperature cannot obtain bimetallic active site monoatomic ions, and the too high calcination temperature can cause the reduction of monoatomic content, which is also not favorable for the exertion of tumor suppression efficiency.
In the embodiment of the invention, in the preparation method of the double-metal active site single-atom diagnosis and treatment agent, the molecular weight of DSPE-PEG-FA is 500-10000, and when the molecular weight of PEG is too high, the diagnosis and treatment agent is agglomerated, which is not beneficial to the biological environment application of the single-atom diagnosis and treatment agent.
In this embodiment, the preparation method of the double-metal active site monoatomic diagnostic agent includes that a mass ratio of a double-metal active site monoatomic catalyst to DSPE-PEG-FA is 1: (0.5-1.5), the stability, biocompatibility and specific targeting capability of the monatomic diagnosis and treatment agent in the range are better, and less targeting agents are coated on the surface beyond the range, so that the stability and specific targeting of the monatomic diagnosis and treatment agent are not facilitated.
In this example, the preparation of the bimetallic active site monoatomic precursor includes: hydrogen peroxide-like mimic enzyme metal salt, dismutase-like metal salt, zinc nitrate (ZnO) 3 ) And after fully blending the dimethyl imidazole and the methanol, transferring the mixture to a polytetrafluoroethylene reaction kettle, keeping the temperature constant for a certain time, cooling the mixture to room temperature, and centrifugally washing the mixture to obtain the double-metal active site monoatomic precursor.
In the embodiment, the temperature for the hydrothermal preparation of the bimetallic active site monoatomic precursor is 110-130 ℃. The hydrothermal reaction time is 3-6 hours. Within the range, the monoatomic precursor is a dodecahedron with uniform appearance, the hydraulic diameter is 150-220 nm, and beyond the range, the monoatomic precursor is uneven in appearance and too large in particle size, so that the monoatomic diagnostic agent is not stable and tumor cells are not endocytosed.
In this embodiment, the solid-liquid separation process may adopt one or more of centrifugal separation, filtration separation and fine separation, and the manner of solid-liquid separation is not limited to the above method. The washing process may use one or more of deionized water washing and absolute ethyl alcohol, and the washing solvent used for washing is not limited to the above examples. The drying process may be one or more of freeze drying, spin-drying, and vacuum drying, but is not limited to the above examples.
The double-metal active site single-atom diagnosis and treatment agent provided by the invention is low in metal single-atom (Fe and Pt) content, small in dosage of the diagnosis and treatment agent, small in biological environment image, coated with DSPE-PEG-FA on the surface of the diagnosis and treatment agent, capable of efficiently and specifically targeting tumors and small in biological environment image of a living body.
Furthermore, the bimetallic active site single-atom diagnosis and treatment agent obtained by the preparation method can be used for high-efficiency chemical kinetic treatment and photo-thermal treatment of malignant tumors and tumor microenvironment hypoxic improvement to realize high-efficiency tumor inhibition in a synergistic manner.
In some embodiments, the application of the double-metal active site single-atom diagnosis and treatment agent is further provided, and the single-atom diagnosis and treatment agent prepared by the invention is used as a carrier for carrying a photosensitizer to a tumor area. A combination therapy drug carrier can be achieved, including a combination therapy of chemokinetic therapy, photothermal therapy. In the combined treatment drug carrier, the photosensitizer is activated to generate singlet oxygen under the irradiation of laser with specific wavelength, and further the photodynamic treatment effect is realized. Under the action of the double-metal active site monoatomic diagnosis and treatment agent, high-concentration hydrogen peroxide in the tumor can be catalyzed and decomposed to generate oxygen, the oxygen content in the tumor is increased, oxygen is provided for photodynamic therapy, sufficient singlet oxygen is generated, and the efficiency of the chemokinetic therapy is improved. The high-efficiency synergistic tumor inhibition effect of the tumor can be realized by combining the high-efficiency chemokinetic therapy and the photothermal therapy effect of the double-metal-site monoatomic diagnosis and treatment agent.
The present invention will be described in detail below with reference to specific examples.
Example 1
(1) 27.32 mg of Fe (acac) 3 ，30.276 mg Pt(acac) 2 ，298 mg Zn(NO) 3 Fully performing ultrasonic treatment on 10 mL of methanol for 10 minutes to obtain a solution A;
(2) fully and ultrasonically mixing 656.8 mg of 2-methylimidazole and 10 mL of methanol for 10 minutes to obtain a solution B;
mixing the solution A and the solution B with the same volume, slowly stirring for 2 hours, transferring the mixture into a 100 mL polytetrafluoroethylene reaction kettle, reacting at 120 ℃ for 4 hours, cooling to room temperature, centrifuging and washing for three times to obtain a bimetallic active site monoatomic precursor, and freeze-drying for later use;
(3) transferring the bimetallic active site monoatomic precursor into a magnetic boat, placing the magnetic boat into a tube furnace, evacuating for 30 minutes by argon, gradually heating to 1000 ℃, calcining for 2 hours, and cooling to room temperature to obtain a bimetallic active site monoatomic catalyst;
(4) dispersing 50 mg of bimetallic active site monatomic catalyst in 20 mL of DMF solution, fully and ultrasonically mixing for 30 minutes, adding 30 mg of DSPE-PEG-FA (Mw = 2000), continuing to ultrasonically treat for 30 minutes, stirring for 6 hours, centrifuging and washing for three times to obtain the bimetallic active site monatomic diagnosis and treatment agent;
a Transmission Electron Microscope (TEM) picture of the metal organic framework compound (ZIF-8) obtained in the embodiment is shown in FIG. 2, and FIG. 2 shows that the ZIF-8 prepared in the embodiment has uniform appearance and size and the hydraulic radius of 150-250 nm.
A Transmission Electron Microscope (TEM) picture of the obtained bimetallic active site monatomic diagnosis and treatment agent in the embodiment is shown in fig. 3, in the picture, the bimetallic active site monatomic diagnosis and treatment agent is a dodecahedron with a hollow structure, the morphology is uniform, the size is uniform, the hydraulic radius is 150-250 nm, no obvious metal particles appear in a sample, and the approximate morphology is consistent with that of ZIF-8.
The spherical aberration corrected transmission electron microscope (STEM) image of the double metal active site monoatomic diagnostic reagent obtained in this example is shown in fig. 4, in which a large amount of uniformly dispersed iron monoatomic and platinum monoatomic ions are present in the sample, and no metal particles are present.
The efficiency of inducing human breast cancer cells by the diagnosis and treatment agent is examined based on the MTT method by co-incubating the obtained double-metal active site single-atom diagnosis and treatment agent with the human breast cancer cells, the survival rate of the human breast cancer cells induced by the obtained single-atom diagnosis and treatment agent is shown in fig. 5, and fig. 5 shows that the single-atom diagnosis and treatment agent obtained by the embodiment can effectively induce apoptosis of the human breast cancer cells.
The bimetallic active site monatomic diagnostic agent obtained in the present example was administered via the tail vein once every three days (20 mg/kg), 5 times in total, and tumor growth conditions of tumor-bearing mice were measured, and the tumor-inhibiting effect of the bimetallic active site monatomic diagnostic agent obtained in the present example on the tumor-bearing mouse living body is shown in fig. 6, and fig. 6 shows that the monatomic diagnostic agent obtained in the present example can effectively inhibit tumor growth in the tumor-bearing mice.
The change curve of the body weight of the tumor-bearing mouse in the process of treating the tumor-bearing mouse by using the obtained bi-metal active site single-atom diagnosis and treatment agent is shown in fig. 7, and fig. 7 shows that the single-atom diagnosis and treatment agent obtained in the embodiment can inhibit tumor with high efficiency and does not cause obvious physiological damage to the tumor-bearing mouse.
Example 2
(1) 81.96 mg of Fe (acac) 3 ，90.83 mg Pt(acac) 2 ，894 mg Zn(NO) 3 Fully performing ultrasonic treatment on 30 mL of methanol for 20 minutes to obtain a solution A;
(2) fully and ultrasonically mixing 1970.4 mg of 2-methylimidazole and 30 mL of methanol for 20 minutes to obtain a solution B;
(3) mixing the solution A and the solution B with the same volume, slowly stirring for 4 hours, transferring to a 200 mL polytetrafluoroethylene reaction kettle, reacting at 120 ℃ for 6 hours, cooling to room temperature, centrifuging and washing for three times to obtain a bimetallic active site monoatomic precursor, and freeze-drying for later use;
(4) transferring the bimetallic active site monoatomic precursor into a magnetic boat, placing the magnetic boat into a tube furnace, evacuating for 40 minutes by argon, gradually heating to 1000 ℃, calcining for 3 hours, and cooling to room temperature to obtain a bimetallic active site monoatomic catalyst;
(5) dispersing 200 mg of a bimetallic active site monoatomic catalyst in 40 mL of DMF (dimethyl formamide) solution, fully and ultrasonically mixing for 40 minutes, adding 120 mg of DSPE-PEG-FA (Mw = 3000), continuously performing ultrasonic treatment for 40 minutes, stirring for 6 hours, centrifuging and washing for three times to obtain the bimetallic active site monoatomic diagnosis and treatment agent.
A Transmission Electron Microscope (TEM) picture of the single-atom diagnostic agent with bimetallic active sites obtained in this embodiment is shown in fig. 8, and as shown in fig. 8, the single-atom diagnostic agent obtained in this embodiment has uniform morphology and uniform size.
Example 3
(1) 298 mg of Zn (NO) 3 10 mL of methanol was sonicated thoroughly for 20 minutes to give solution A.
(2) 656.8 mg of 2-methylimidazole and 0 mL of methanol were thoroughly mixed by sonication for 20 minutes to obtain solution B.
(3) And mixing the solution A and the solution B with the same volume, slowly stirring for 4 hours, transferring to a 50 mL polytetrafluoroethylene reaction kettle, reacting at 120 ℃ for 4 hours, cooling to room temperature, centrifuging and washing for three times to obtain the metal organic framework compound (ZIF-8), and freeze-drying for later use.
(4) 100 mg of ZIF-8 was ultrasonically dispersed in 10 mL of methanol solution, and 54.64 mg of FeCl was added 3 60.55 mg of chloroplatinic acid, ultrasonically mixing for 30 minutes, slowly stirring for 6 hours, and centrifugally washing for three times to obtain the precursor of the single atom of the bimetallic active site.
(5) And transferring the bimetallic active site monoatomic precursor into a magnetic boat, placing the magnetic boat into a tube furnace, evacuating for 40 minutes by argon, gradually heating to 1000 ℃, calcining for 3 hours, and cooling to room temperature to obtain the bimetallic active site monoatomic catalyst.
(6) Dispersing 50 mg of bimetallic active site monatomic catalyst in 5 mL of DMF solution, fully and ultrasonically mixing for 30 minutes, adding 50 mg of DSPE-PEG-FA (Mw = 3000), continuing to ultrasonically stir for 40 minutes, stirring for 6 hours, centrifuging and washing for three times to obtain the bimetallic active site monatomic diagnosis and treatment agent.
A Transmission Electron Microscope (TEM) picture of the single-atom diagnostic agent with bimetallic active sites obtained in this embodiment is shown in fig. 9, and as shown in fig. 9, the single-atom diagnostic agent obtained in this embodiment has uniform morphology and uniform size.
In conclusion, the invention provides preparation and application of a double-metal active site single-atom diagnosis and treatment agent, a metal organic framework compound (ZIF-8) is used as a carrier, high-activity hydrogen peroxide mimic enzyme metal salt and dismutase-like metal salt are loaded in the carrier, the double-metal active site single-atom diagnosis and treatment agent is obtained under a high-temperature reduction method, and the biocompatibility, the stability and the tumor specific targeting capability of the single-atom diagnosis and treatment agent can be effectively improved by modifying a specific targeting agent and a stabilizing agent (DSPE-PEG-FA) on the surface. The obtained single-atom diagnosis and treatment agent can realize the synergistic tumor inhibition effect of high-efficiency chemodynamic therapy and photothermal therapy under the guidance of photoacoustic imaging and photothermal imaging, is a multifunctional nano diagnosis and treatment agent with high biocompatibility and tumor inhibition effect, and has a good application prospect.

Claims (4)

1. A preparation method of a Fe and Pt double-active-site single-atom diagnosis and treatment agent is characterized by comprising the following steps:
(1) transferring the precursor of the single atom of the double metal active site into a tubular furnace, evacuating argon, calcining at high temperature, cooling to room temperature after calcining to obtain the catalyst of the single atom of the double metal active site, and fully grinding for later use;
(2) dispersing a double-metal active site monoatomic catalyst in dimethyl formamide DMF (dimethyl formamide), fully performing ultrasonic dispersion, and adding a proper amount of DSPE-PEG-FA to obtain a double-metal active site monoatomic diagnosis and treatment agent;
in the step (1), the bimetallic active site monoatomic precursor is prepared by the following two methods:
a, preparing hydrogen peroxide mimetic enzyme metal salt, dismutase metal salt and zinc nitrate (Zn (NO) 3 ) 2 ) Dimethyl imidazole, methanolAfter fully blending, transferring the mixture to a reaction kettle for hydrothermal reaction, cooling to room temperature after the reaction is finished, and performing centrifugal washing;
or
b mixing ZnO 3 Fully blending dimethylimidazole and methanol, transferring the mixture to a polytetrafluoroethylene reaction kettle, keeping the temperature constant for a certain time, cooling to room temperature to obtain a metal organic framework compound, centrifugally washing, re-suspending the mixture in a methanol solution, adding a hydrogen peroxide-like mimic enzyme metal salt and a dismutase-like metal salt into the methanol solution, fully stirring, and centrifugally washing to obtain the metal organic framework compound;
the molar ratio of the hydrogen peroxide-like mimic enzyme metal salt to the dismutase-like metal salt to the zinc nitrate is 1: (0.5-2): (8-15);
the hydrogen peroxide mimetic enzyme metal salt is ferric acetylacetonate, iron phthalocyanine or ferric chloride; the dismutase-like metal salt is acetylacetone platinum, phthalocyanine platinum, chloroplatinic acid or potassium chloroplatinate;
the hydrothermal reaction is carried out for 3-6h at 110-130 ℃;
in the step (1), the emptying time of the tube furnace is 30-60 min; the high-temperature calcination is carried out at the temperature of 900-1100 ℃ for 1-4 h;
the mass ratio of the bimetallic active site monatomic catalyst to the DSPE-PEG-FA is 1: (0.5 to 1.5); the molecular weight of the DSPE-PEG-FA is 500-10000.
2. The method according to claim 1, wherein the molar ratio of zinc nitrate to dimethylimidazole is 1: (6-10).
3. The preparation method according to claim 1, wherein the concentration of the bimetallic active site monoatomic catalyst in DMF is 10-200 mg/mL.
4. Use of the bimetallic active site monoatomic diagnostic agent prepared by the preparation method according to any one of claims 1 to 3 in the preparation of an antitumor drug, a tumor imaging agent or a pharmaceutical vehicle.
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