Organic framework derivative phototherapeutic agent containing ruthenium and iron metal atoms for photothermal and photodynamic therapy, its preparation method and application

Abstract

The present invention provides an organic framework derivative phototherapeutic agent containing ruthenium and iron metal atoms for photothermal and photodynamic therapy, and a preparation method and application thereof. The preparation method of the phototherapeutic agent of the present invention includes the steps of: dissolving an iron salt, a ruthenium salt, a zinc salt and 2-methylimidazole in a solvent, stirring and reacting, and then centrifuging, washing and freeze-drying to obtain a precursor; the precursor is calcined to obtain the phototherapeutic agent. The phototherapeutic agent of the present invention has good structural stability, photothermal conversion characteristics and photosensitivity; not only has the property of converting light energy into heat energy, but also has the photosensitive property of converting light energy into kinetic energy and then generating reactive oxygen species; it can be effectively used for photothermal therapy and photodynamic therapy, and thus can be used as a phototherapeutic agent for the treatment of tumor cells.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technologies, and particularly relates to a phototherapeutic agent based on an organic framework derivative containing ruthenium and iron metal atoms for photothermal and photodynamic therapy, a preparation method thereof, and an application thereof. Background Art

[0002] Metal-organic framework (MOF) materials are a class of crystalline porous materials with a periodic network structure. They have advantages such as good porosity, chemical stability, and large specific surface area, and are widely used in catalysis, energy storage, and separation. MOFs show great development potential and good development prospects in modern material research.

[0003] Ruthenium has stable properties, strong corrosion resistance, and is the cheapest metal among the platinum group metals. Ruthenium and iron elements have good therapeutic effects in the treatment of tumors. Iron is an essential trace element in the human body. It participates in the transportation of oxygen and the release of energy, and at the same time has a certain impact on the immune system. Ruthenium complexes can accurately kill tumor cells and inhibit their growth through light irradiation, and ruthenium complexes can kill tumor cells under hypoxic conditions, which is completely applicable to the hypoxic environment of tumors.

[0004] Currently, most phototherapeutic agents are either single photothermal therapeutic agents or photodynamic therapeutic agents. Single photothermal therapeutic agents have disadvantages such as poor near-infrared selectivity and limited penetration depth; while for single photodynamic therapeutic agents, there are disadvantages such as insufficient treatment range and treatment stability.

[0005] To solve the above problems, phototherapeutic agents with both photothermal therapy and photodynamic therapy functions have been reported in the prior art. For example, Chinese patent document CN109847062A discloses a quercetin metal nano-drug and a preparation method and an application thereof. The invention provides a quercetin metal nano-drug, in which quercetin and transition metal elements are coordinated through coordination bonds, and surface modification is carried out through a high molecular polymer containing a carbonyl group or a hydroxyl group to form a quercetin metal nano-drug. This nano-drug has the effects of anti-tumor in multiple modes such as photothermal therapy, photodynamic therapy, and radiotherapy. However, the photothermal therapy effect of the nano-drug prepared by this invention is poor, with poor selectivity and limited penetration depth.

[0006] Therefore, there is an urgent need to develop a phototherapeutic agent with good structural stability, photothermal conversion characteristics, and photosensitivity, so as to be effectively used for photothermal therapy and photodynamic therapy. Summary of the Invention

[0007] In view of the deficiencies of the prior art, the present invention provides a photo-therapeutic agent of an organic framework derivative containing ruthenium and iron metal atoms for photothermal and photodynamic therapy, a preparation method thereof, and an application thereof. The photo-therapeutic agent of the present invention has good structural stability, photothermal conversion characteristics and photosensitivity; it not only has the property of converting light energy into heat energy, but also has the photosensitive property of converting light energy into kinetic energy and then generating reactive oxygen species; it can be effectively used for photothermal therapy and photodynamic therapy, and thus can be used as a photo-therapeutic agent for the treatment of tumor cells.

[0008] The technical solution of the present invention is as follows:

[0009] A photo-therapeutic agent of an organic framework derivative containing ruthenium and iron metal atoms for photothermal and photodynamic therapy, the photo-therapeutic agent is an organic framework mesoporous carbon material containing ruthenium, iron and zinc metal atoms; the microscopic morphology of the photo-therapeutic agent is a monodisperse dodecahedron structure.

[0010] According to the preference of the present invention, the diameter size of the dodecahedron structure is less than 100 nm; preferably, the diameter size of the dodecahedron structure is 80-100 nm.

[0011] The preparation method of the above-mentioned photo-therapeutic agent of an organic framework derivative containing ruthenium and iron metal atoms for photothermal and photodynamic therapy includes the steps of: dissolving iron salt, ruthenium salt, zinc salt and 2-methylimidazole in a solvent, stirring and reacting, and then centrifuging, washing and freeze-drying to obtain a precursor; the precursor is calcined to obtain the photo-therapeutic agent.

[0012] According to the preference of the present invention, the iron salt is a trivalent iron salt; preferably ferric nitrate nonahydrate, ferric chloride or ferric sulfate.

[0013] According to the preference of the present invention, the ruthenium salt is a trivalent ruthenium salt; preferably ruthenium trichloride or ruthenium acetylacetonate.

[0014] According to the preference of the present invention, the zinc salt is a divalent zinc salt; preferably zinc nitrate hexahydrate, zinc sulfate or zinc chloride.

[0015] According to the preference of the present invention, the mass ratio of iron element in the iron salt to ruthenium element in the ruthenium salt is 2-3:1; the molar ratio of ruthenium salt to zinc salt is 1:445-460. Research shows that the mass ratio of iron to ruthenium can adjust the size and morphology of the photo-therapeutic agent. In order to make the microscopic morphology of the photo-therapeutic agent regular and the size uniform, the iron element in the iron salt and the ruthenium element in the ruthenium salt are preferably 2.73:1.

[0016] According to the preference of the present invention, the molar ratio of zinc salt to 2-methylimidazole is 1:8.

[0017] According to the preference of the present invention, the solvent is methanol; the mass ratio of 2-methylimidazole to the volume of the solvent is 0.01-0.1 g / mL.

[0018] Preferably according to the present invention, the stirring reaction temperature is room temperature and the stirring reaction time is 12 to 36 h.

[0019] Preferably according to the present invention, the calcination temperature is 800 to 1000 °C, the calcination time is 1 to 3 h, and the calcination is carried out under the protection of an inert gas. The calcination temperature affects the microstructure of the phototherapeutic agent. In order to ensure that the phototherapeutic agent is a monodisperse dodecahedron structure, the calcination temperature is preferably 900 °C.

[0020] The application of the above ruthenium- and iron-containing metal atom organic framework derivative phototherapeutic agent for photothermal and photodynamic therapy, as a phototherapeutic agent for photothermal therapy or photodynamic therapy.

[0021] The application of the above ruthenium- and iron-containing metal atom organic framework derivative phototherapeutic agent for photothermal and photodynamic therapy in the preparation of anti-tumor drugs.

[0022] The present invention also provides a phototherapeutic drug, comprising a phototherapeutic agent and a pharmaceutically acceptable carrier, and the phototherapeutic agent is the above ruthenium- and iron-containing metal atom organic framework derivative phototherapeutic agent for photothermal and photodynamic therapy.

[0023] According to the present invention, the pharmaceutically acceptable carrier is a generally pharmaceutically acceptable carrier, including but not limited to natural polymer materials (such as sugars, proteins, etc.), synthetic polymer materials (such as polylactic acid, polyethylene glycol, polyacrylate, etc.), stearic acid, phosphates, etc.

[0024] The technical features and beneficial effects of the present invention are as follows:

[0025] 1. The mass ratio of iron salt to ruthenium salt in the present invention can adjust the size of the phototherapeutic agent, and a specific mass ratio is required to obtain the phototherapeutic agent of the size of the present invention. The calcination temperature in the present invention affects the microstructure of the phototherapeutic agent, and a specific calcination temperature in the present invention is required to ensure that the phototherapeutic agent is a monodisperse dodecahedron structure. As a whole, the preparation method of the present invention can jointly realize the preparation of the phototherapeutic agent with the structure and performance of the present invention.

[0026] 2. The phototherapeutic agent prepared by the present invention is an organic framework mesoporous carbon material containing ruthenium, iron, and zinc metal atoms, and its microstructure is a monodisperse dodecahedron structure with a diameter size less than 100 nm.

[0027] 3. The present invention introduces iron element to endow the obtained phototherapeutic agent with photothermal and photodynamic properties. The addition of ruthenium element can enhance its photodynamic property and enable the therapeutic agent to kill tumor cells under anaerobic conditions. The organic framework is formed by high-temperature firing to link up various elements, enabling the photothermal and photodynamic properties to play a synergistic role. Only when the components and structures of the present invention work together synergistically can the excellent effects of the present invention be achieved. The absence of any component or the change of the structure cannot achieve the effects of the present invention. The phototherapeutic agent of the present invention has good structural stability, photothermal conversion characteristics and photosensitivity; it not only has the property of converting light energy into heat energy, but also has the photosensitive property of converting light energy into kinetic energy and then generating reactive oxygen species; it can be effectively used for photothermal therapy and photodynamic therapy, and thus can be used as a phototherapeutic agent for treating tumor cells; and it can kill and inhibit the growth of tumor cells under hypoxic conditions, and is completely applicable to the hypoxic environment of tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Scanning electron microscope image of FeRu 5:1 -N-C prepared in Example 1;

[0029] Figure 2 Transmission electron microscope image of FeRu 5:1 -N-C prepared in Example 1;

[0030] Figure 3 High-resolution XPS spectrum of FeRu prepared in Example 1 5:1 -N-C; wherein, the abscissa is binding energy / ev and the ordinate is intensity / a.u.;

[0031] Figure 4 Scanning electron microscope image of FeRu prepared in Comparative Example 1 10:1 -N-C;

[0032] Figure 5 Photothermal conversion curves of FeRu -N-C aqueous dispersions with different concentrations prepared in Example 1 in Test Example 1 under laser irradiation at 808 nm and a power of 2 W·cm 5:1 for 10 minutes; wherein, the abscissa is time / min and the ordinate is temperature / °C; -2

[0033] Figure 6 Photothermal conversion curves of FeRu -N-C aqueous dispersions with different concentrations prepared in Comparative Example 1 in Test Example 1 under laser irradiation at 808 nm and a power of 2 W·cm 10:1 for 10 minutes; wherein, the abscissa is time / min and the ordinate is temperature / °C; -2

[0034] Figure 7For FeRu prepared in Example 1 of Test Example 2 5:1 -N-C (a) and the control group (b) of the ultraviolet absorption spectrogram of the detection of reactive oxygen species; wherein, the abscissa is the wavelength and the ordinate is the absorbance / a.u.;

[0035] Figure 8 For FeRu prepared in Example 1 at different concentrations in Test Example 3 5:1 -N-C (0.0, 0.5, 2.5, 5, 10, 15, 20, 40, 50, 80, 100, 200, 300 μg·mL -1 ) MTT toxicity test on Hela cells; wherein, the abscissa is the concentration / μg mL -1 , and the ordinate is the viable cell rate of cells / %. Detailed implementation mode

[0036] The present invention will be further described below in conjunction with specific embodiments, but not limited thereto.

[0037] At the same time, in the methods described in the following embodiments, unless otherwise specified, they are all conventional methods; the materials, unless otherwise specified, can all be obtained from commercial channels.

[0038] Example 1

[0039] A preparation method of an organometallic framework derivative phototherapeutic agent containing ruthenium and iron metal atoms for photothermal and photodynamic therapy, the steps are as follows:

[0040] Dissolve 3.36 g of zinc nitrate hexahydrate, 50 mg of iron nitrate nonahydrate, and 10 mg of ruthenium acetylacetonate in 160 mL of methanol to form solution A; dissolve 7.4 g of 2-methylimidazole in 160 mL of methanol to form solution B. Add solution A to solution B, continuously stir evenly at room temperature for 18 hours, and the reaction solution is centrifuged, washed with methanol, and freeze-dried to obtain the product, which is FeRu 5：1 -ZIF-8 (5:1 represents the mass ratio of iron nitrate nonahydrate and ruthenium acetylacetonate). Place the obtained FeRu 5：1 -ZIF-8 nanocrystals in a tube furnace, protect with N2, pyrolyze at 900 °C, cool down after 2 h, and the obtained phototherapeutic agent is FeRu 5：1 -N-C.

[0041] The scanning electron microscope image and transmission electron microscope image of the phototherapeutic agent FeRu 5：1 -N-C prepared in this example are respectively as Figure 1 , 2 shown. It can be seen from the figure that the microscopic morphology of the phototherapeutic agent prepared in this example is a monodisperse dodecahedron structure. The diameter size of the dodecahedron structure is 80-100 nm.

[0042] Figure 3 FeRu prepared for this example 5：1 -N-C high-resolution XPS spectrum of the phototherapeutic agent. It can be seen from the figure that the phototherapeutic agent of the present invention contains elements such as C, N, O, Zn, Ru, and Fe.

[0043] Example 2

[0044] A preparation method of an organometallic framework derivative phototherapeutic agent containing ruthenium and iron metal atoms for photothermal and photodynamic therapy, the steps are as follows:

[0045] Dissolve 3.36 g of zinc nitrate hexahydrate, 50 mg of iron nitrate nonahydrate, and 10 mg of ruthenium acetylacetonate in 160 mL of methanol to form solution A; dissolve 7.4 g of 2-methylimidazole in 160 mL of methanol to form solution B. Add solution A to solution B and continuously stir evenly at room temperature for 12 hours. The reaction solution is centrifuged, washed with methanol, and freeze-dried to obtain the product, which is FeRu 5：1 -ZIF-8 (5:1 represents the mass ratio of iron nitrate nonahydrate and ruthenium acetylacetonate). Place the obtained FeRu 5：1 -ZIF-8 nanocrystals in a tube furnace, under N2 protection, pyrolyze at 800 °C, cool down after 3 h, and obtain the phototherapeutic agent.

[0046] Example 3

[0047] A preparation method of an organometallic framework derivative phototherapeutic agent containing ruthenium and iron metal atoms for photothermal and photodynamic therapy, the steps are as follows:

[0048] Dissolve 3.36 g of zinc nitrate hexahydrate, 50 mg of iron nitrate nonahydrate, and 10 mg of ruthenium acetylacetonate in 160 mL of methanol to form solution A; dissolve 7.4 g of 2-methylimidazole in 160 mL of methanol to form solution B. Add solution A to solution B and continuously stir evenly at room temperature for 36 hours. The reaction solution is centrifuged, washed with methanol, and freeze-dried to obtain the product, which is FeRu 5：1 -ZIF-8 (5:1 represents the mass ratio of iron nitrate nonahydrate and ruthenium acetylacetonate). Place the obtained FeRu 5：1 -ZIF-8 nanocrystals in a tube furnace, under N2 protection, pyrolyze at 1000 °C, cool down after 1 h, and obtain the phototherapeutic agent.

[0049] Comparative Example 1

[0050] A preparation method of an organometallic framework derivative phototherapeutic agent containing ruthenium and iron metal atoms for photothermal and photodynamic therapy, the steps are as follows:

[0051] Dissolve 3.36 g of zinc nitrate hexahydrate, 100 mg of iron(III) nitrate nonahydrate, and 10 mg of ruthenium(III) acetylacetonate in 160 mL of methanol to form Solution A; dissolve 7.4 g of 2-methylimidazole in 160 mL of methanol to form Solution B. Add Solution A to Solution B and continuously stir evenly at room temperature for 18 hours. The reaction solution is centrifuged, washed with methanol, and freeze-dried to obtain the product, which is FeRu 10：1 -ZIF-8 (10:1 represents the mass ratio of iron(III) nitrate nonahydrate to ruthenium(III) acetylacetonate). Place the obtained FeRu 10：1 -ZIF-8 nanocrystals in a tubular furnace, protect with N2, pyrolyze at 900 °C, cool down after 2 h, and the obtained phototherapeutic agent is FeRu 10：1 -N-C.

[0052] Figure 4 The phototherapeutic agent FeRu 10：1 -N-C prepared in this example. As can be seen from the figure, the microscopic morphology of the phototherapeutic agent prepared in this example is different from that of the monodisperse dodecahedral structure of the FeRu 5：1 -N-C phototherapeutic agent. The dodecahedral morphology is not good and the size is not uniform, indicating that the mass ratio of iron element and ruthenium element has an important influence on the microscopic morphology of the obtained phototherapeutic agent.

[0053] Experimental Example 1

[0054] In vitro photothermal experiment:

[0055] Photothermal measurement: Prepare aqueous dispersions of FeRu -1 -N-C prepared in Example 1 or FeRu 5：1 -N-C prepared in Comparative Example 1 at concentrations of 50, 100, and 200 μg / mL respectively. Use a semiconductor laser with continuously adjustable output wavelength to irradiate at an output power of 2 W / cm 10：1 at 808 nm. Record the temperature of the solution every 1 minute and record the temperature change of the solution within 10 minutes. At the same time, use pure water as a control. -2 As

[0056] shown, the temperature change of the aqueous dispersion of FeRu Figure 5 -N-C after irradiation with a laser at 808 nm with a power of 2 W·cm 5：1 for 10 minutes shows that the phototherapeutic agent prepared in the present invention has excellent photothermal effects and can be applied to photothermal therapy to kill tumor cells; at the same time, as the sample concentration in the aqueous dispersion increases, the temperature change is greater. -2 As

[0057] shown, the temperature change of the aqueous dispersion of FeRu Figure 6 -N-C after irradiation with a laser at 808 nm with a power of 2 W·cm 10：1 for 10 minutes-2 The temperature change after 10 minutes of laser irradiation can show that the photothermal performance of its therapeutic agent is poorer than that of FeRu 5：1 -N-C phototherapeutic agent.

[0058] Experimental Example 2

[0059] In vitro photodynamic experiment:

[0060] DPBF detection: The production level of reactive oxygen species in vitro of the material was detected using 1,3-diphenylisobenzofuran (DPBF) as an indicator. 100 μL of DPBF (1 mg / mL) ethanol solution was mixed with the aqueous dispersion of FeRu 5：1 -N-C prepared in Example 1. In the mixed solution, the concentration of FeRu 5：1 -N-C was 50 μg / mL -1 , and it was irradiated under an 808 nm 2 W·cm -2 laser. The UV absorption curve of DPBF was recorded with a microplate reader every 1 minute, as shown in Figure 7 (a). At the same time, a control group without adding FeRu 5：1 -N-C was set up, as shown in Figure 7 (b).

[0061] As shown in Figure 7 , for the DPBF ethanol solution and the mixed solution containing FeRu 5：1 -N-C, under 808 nm 2 W·cm -2 laser irradiation, as time increased, the UV absorption line decreased regularly, indicating that the material had a relatively strong ability to produce reactive oxygen species.

[0062] Experimental Example 3

[0063] Cell culture and toxicity test:

[0064] Hela cells were cultured in F-12k culture medium containing 10% fetal bovine serum and 1% double antibody in a 5% CO2 37 °C constant temperature and humidity incubator.

[0065] Hela cells were seeded onto a 96-well plate at a density of 5×10 3 cells / well and cultured overnight until the cells adhered to the wall. After removing the old culture medium, the cells were washed twice with sterile PBS, and serum-free culture medium was added. FeRu -1 -N-C prepared in Example 1 with concentrations of (0.0, 0.5, 2.5, 5, 10, 15, 20, 40, 50, 80, 100, 200, 300 μg·mL 5：1 ) was added and incubated for 4 h. After the incubation, the culture medium was removed, the cells were washed and fresh culture medium was added. The cells were then placed under an 808 nm 2 W·cm -2Irradiate with laser for 5 minutes, continue to incubate for 24 hours, add the MTT kit according to the operation guide, and detect the absorbance OD value at 490 nm with an enzyme-labeled instrument. As a control under dark conditions, the cells were incubated with FeRu 5：1 -N-C for the same time without laser irradiation.

[0066] Figure 8 The MTT toxicity of FeRu -1 )-N-C on Hela cells at different concentrations (0.0, 0.5, 2.5, 5, 10, 15, 20, 40, 50, 80, 100, 200, 300 μg·mL 5:1 ). As can be seen from the figure, at a concentration of 100 μg·mL -1 , the cell viability can still be maintained above 80%, indicating that the phototherapeutic agent prepared by the present invention has good cell compatibility.

[0067] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. Use of an organometallic framework derivative phototherapeutic agent containing ruthenium and iron metal atoms for photothermal and photodynamic therapy in the preparation of an anti-tumor drug, characterized in that, The photo-therapeutic agent is an organic framework mesoporous carbon material containing ruthenium, iron, and zinc metal atoms; the microscopic morphology of the photo-therapeutic agent is a monodisperse dodecahedron structure; The preparation method of the photo-therapeutic agent of the ruthenium- and iron-metal-atom-containing organic framework derivative for photothermal and photodynamic therapy includes the steps of dissolving iron salt, ruthenium salt, zinc salt, and 2-methylimidazole in a solvent, stirring and reacting, and then centrifuging, washing, and freeze-drying to obtain a precursor; The precursor is calcined to obtain the photo-therapeutic agent; The iron salt is ferric nitrate nonahydrate, ferric chloride, or ferric sulfate; the ruthenium salt is ruthenium trichloride or ruthenium acetylacetonate; the zinc salt is zinc nitrate hexahydrate, zinc sulfate, or zinc chloride; the mass ratio of iron element in the iron salt to ruthenium element in the ruthenium salt is 2.73:1; the molar ratio of ruthenium salt to zinc salt is 1:445 - 460; the molar ratio of zinc salt to 2-methylimidazole is 1:8; the solvent is methanol; the mass ratio of 2-methylimidazole to the volume of the solvent is 0.01 - 0.1 g / mL; the stirring reaction temperature is room temperature, and the stirring reaction time is 12 - 36 h; the calcination temperature is 800 - 1000 °C, the calcination time is 1 - 3 h, and the calcination is carried out under inert gas protection.

2. The application according to claim 1, wherein The diameter size of the dodecahedron structure is less than 100 nm.

3. The application according to claim 2, wherein The diameter size of the dodecahedron structure is 80 - 100 nm.

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