CN116763939A - Preparation and application of rare earth ion doped metal organic framework loaded lactate oxidase - Google Patents
Preparation and application of rare earth ion doped metal organic framework loaded lactate oxidase Download PDFInfo
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- CN116763939A CN116763939A CN202310739688.4A CN202310739688A CN116763939A CN 116763939 A CN116763939 A CN 116763939A CN 202310739688 A CN202310739688 A CN 202310739688A CN 116763939 A CN116763939 A CN 116763939A
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- 108010073450 Lactate 2-monooxygenase Proteins 0.000 title claims abstract description 69
- -1 rare earth ion Chemical class 0.000 title claims abstract description 69
- 239000012621 metal-organic framework Substances 0.000 title claims abstract description 51
- 238000002360 preparation method Methods 0.000 title claims abstract description 25
- 229910052761 rare earth metal Inorganic materials 0.000 title claims abstract description 25
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N Lactic Acid Natural products CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 claims abstract description 46
- 239000004310 lactic acid Substances 0.000 claims abstract description 33
- 235000014655 lactic acid Nutrition 0.000 claims abstract description 33
- 108090000854 Oxidoreductases Proteins 0.000 claims abstract description 22
- 102000004316 Oxidoreductases Human genes 0.000 claims abstract description 22
- 229910052751 metal Inorganic materials 0.000 claims abstract description 16
- 239000002184 metal Substances 0.000 claims abstract description 15
- 238000006243 chemical reaction Methods 0.000 claims description 34
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 28
- 229940071125 manganese acetate Drugs 0.000 claims description 21
- UOGMEBQRZBEZQT-UHFFFAOYSA-L manganese(2+);diacetate Chemical compound [Mn+2].CC([O-])=O.CC([O-])=O UOGMEBQRZBEZQT-UHFFFAOYSA-L 0.000 claims description 21
- 239000003960 organic solvent Substances 0.000 claims description 17
- 229960000583 acetic acid Drugs 0.000 claims description 14
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- 238000002156 mixing Methods 0.000 claims description 14
- 239000011572 manganese Substances 0.000 claims description 13
- 238000000034 method Methods 0.000 claims description 7
- JQBILSNVGUAPMM-UHFFFAOYSA-K terbium(3+);triacetate Chemical compound [Tb+3].CC([O-])=O.CC([O-])=O.CC([O-])=O JQBILSNVGUAPMM-UHFFFAOYSA-K 0.000 claims description 7
- 238000004519 manufacturing process Methods 0.000 claims description 6
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- VMGAPWLDMVPYIA-HIDZBRGKSA-N n'-amino-n-iminomethanimidamide Chemical compound N\N=C\N=N VMGAPWLDMVPYIA-HIDZBRGKSA-N 0.000 description 4
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- XDFNWJDGWJVGGN-UHFFFAOYSA-N 2-(2,7-dichloro-3,6-dihydroxy-9h-xanthen-9-yl)benzoic acid Chemical compound OC(=O)C1=CC=CC=C1C1C2=CC(Cl)=C(O)C=C2OC2=CC(O)=C(Cl)C=C21 XDFNWJDGWJVGGN-UHFFFAOYSA-N 0.000 description 3
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- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 3
- 230000035899 viability Effects 0.000 description 3
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- ONJSLAKTVIZUQS-UHFFFAOYSA-K manganese(3+);triacetate;dihydrate Chemical group O.O.[Mn+3].CC([O-])=O.CC([O-])=O.CC([O-])=O ONJSLAKTVIZUQS-UHFFFAOYSA-K 0.000 description 2
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- MASUWVVNWALEEM-UHFFFAOYSA-M 1-methoxy-5-methylphenazin-5-ium;methyl sulfate Chemical compound COS([O-])(=O)=O.C1=CC=C2N=C3C(OC)=CC=CC3=[N+](C)C2=C1 MASUWVVNWALEEM-UHFFFAOYSA-M 0.000 description 1
- VFNKZQNIXUFLBC-UHFFFAOYSA-N 2',7'-dichlorofluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC(Cl)=C(O)C=C1OC1=C2C=C(Cl)C(O)=C1 VFNKZQNIXUFLBC-UHFFFAOYSA-N 0.000 description 1
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
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- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/43—Enzymes; Proenzymes; Derivatives thereof
- A61K38/44—Oxidoreductases (1)
- A61K38/443—Oxidoreductases (1) acting on CH-OH groups as donors, e.g. glucose oxidase, lactate dehydrogenase (1.1)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
- A61K41/0057—Photodynamic therapy with a photosensitizer, i.e. agent able to produce reactive oxygen species upon exposure to light or radiation, e.g. UV or visible light; photocleavage of nucleic acids with an agent
- A61K41/0076—PDT with expanded (metallo)porphyrins, i.e. having more than 20 ring atoms, e.g. texaphyrins, sapphyrins, hexaphyrins, pentaphyrins, porphocyanines
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- 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
- A61K47/69—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
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- C07D487/22—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains four or more hetero rings
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- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
- C12N11/089—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
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Abstract
The invention belongs to the technical field of biological materials, and particularly relates to a preparation method and application of rare earth ion doped metal organic framework loaded lactate oxidase. The metal organic framework loaded lactic acid oxidase provided by the invention comprises a rare earth ion doped metal organic framework and milk loaded on the metal organic framework through the coordination effect of the rare earth ionsAcid oxidase; the rare earth ion doped metal organic frame is Tb 3+ Doped 5,10,15, 20-tetrakis (4-carboxyphenyl) manganese porphyrin. The metal organic frame loaded lactate oxidase provided by the invention loads lactate oxidase on the metal organic frame through the coordination of rare earth metal ions, and the lactate oxidase and the metal organic frame carrier are combined through chemical bonds, so that the acting force is strong and the stability is good, thereby ensuring that more lactate oxidase enters solid tumor cells, and effectively improving H in tumor microenvironment 2 O 2 Is effective in significantly enhancing the therapeutic effect of PDT and/or CDT.
Description
Technical Field
The invention belongs to the technical field of biological materials, and particularly relates to a preparation method and application of rare earth ion doped metal organic framework loaded lactate oxidase.
Background
Photodynamic therapy (PDT) is an emerging cancer treatment as an important treatment modality for subcutaneous tumor tissue. The main principle of photodynamic therapy is that a photosensitizer can transfer excited state energy to oxygen molecules in surrounding tissue cells after being irradiated by light with a specific wavelength, so that the photosensitizer generates Reactive Oxygen Species (ROS) with cytotoxicity, and apoptosis and necrosis of tumor cells are induced. PDT has a number of advantages over other treatments: non-invasive, low drug resistance, rapid curative effect, no accumulation of toxicity, repeated administration, low side effect, etc. Chemical Dynamic Therapy (CDT) mainly uses the slightly acidic environment of tumor cells to trigger Fenton or Fenton-like reaction to catalyze endogenous H 2 O 2 Is converted into hydroxyl free radical (OH) with cytotoxicity, thereby inducing apoptosis.
However, within solid tumor cells, there are still many factors that limit the performance of photodynamic and chemodynamic therapy. Such as: the rapid growth of tumors results in severe hypoxic conditions within solid tumors, while H in the tumor microenvironment 2 O 2 The content of (1-0 mM) is low, and the ideal PDT and CDT effects cannot be achieved; second, most conventional photosensitizers are hydrophobic, which also results in photosensitizer aggregation-induced energy dissipation and reduced efficacy.
In order to solve the above problems, it has been reported that loading biological enzymes on MOFs improves tumor chemokinetics and photodynamic therapeutic effects. However, the acting force between the enzyme and the carrier is weak, and the leakage is easy; the tumor treatment effect of PDT/CDT cannot be effectively improved.
Disclosure of Invention
The invention aims to provide a preparation method and application of rare earth ion doped metal organic frame loaded lactate oxidase, and the metal organic frame loaded lactate oxidase provided by the invention has good stability and can effectively improve H in tumor microenvironment 2 O 2 Is effective in significantly enhancing the therapeutic effect of PDT and/or CDT.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a metal organic framework loaded lactate oxidase, which comprises a rare earth ion doped metal organic framework and lactate oxidase loaded on the metal organic framework through the coordination of the rare earth ions;
the rare earth ion doped metal organic frame is Tb 3+ Doped 5,10,15, 20-tetrakis (4-carboxyphenyl) manganese porphyrin.
Preferably, the particle size of the metal organic framework loaded lactate oxidase is 190-210 nm.
Preferably, the Tb 3+ Doped 5,10,15, 20-tetrakis (4-carboxyphenyl) manganese porphyrin: the weight percentage of Tb element is 0.08-0.125 wt%; the mass percentage of Mn element is 0.15-0.2wt%;
in the metal organic framework loaded lactate oxidase: the mass percentage of the lactic acid oxidase is 10-20wt%.
The invention provides a preparation method of the metal organic framework loaded lactate oxidase, which comprises the following steps:
lactic acid oxidase solution and Tb 3+ And mixing the doped 5,10,15, 20-tetra (4-carboxyphenyl) manganese porphyrin solution for coordination reaction to obtain the metal organic framework loaded lactate oxidase.
Preferably, tb 3+ The preparation method of the doped 5,10,15, 20-tetra (4-carboxyphenyl) manganese porphyrin comprises the following steps:
5,10,15, 20-tetra (4-carboxyphenyl) manganese porphyrin,Tb is prepared by mixing terbium acetate, glacial acetic acid and organic solvent 3+ Doping to obtain Tb 3+ Doped 5,10,15, 20-tetrakis (4-carboxyphenyl) manganese porphyrin.
Preferably, lactate oxidase and Tb 3+ The mass ratio of the doped 5,10,15, 20-tetra (4-carboxyphenyl) manganese porphyrin is (1-2): 1.
preferably, the molar ratio of the 5,10,15, 20-tetra (4-carboxyphenyl) manganese porphyrin to the terbium acetate is 1 (1-8).
Preferably, the preparation method of the 5,10,15, 20-tetra (4-carboxyphenyl) manganese porphyrin comprises the following steps:
mixing manganese acetate, glacial acetic acid and an organic solvent to obtain a manganese acetate solution;
adding an organic solution of 5,10,15, 20-tetra (4-carboxyphenyl) porphyrin into the manganese acetate solution, and carrying out coordination reaction to obtain the 5,10,15, 20-tetra (4-carboxyphenyl) manganese porphyrin.
The invention provides a photodynamic and/or chemodynamic therapeutic reagent, which comprises the metal-organic framework loaded lactate oxidase prepared by the technical scheme or the preparation method.
The invention provides application of the metal-organic framework loaded lactate oxidase or the metal-organic framework loaded lactate oxidase prepared by the preparation method in the technical scheme in preparation of antitumor drugs.
The invention provides a metal organic framework loaded lactate oxidase, which comprises a rare earth ion doped metal organic framework and lactate oxidase loaded on the metal organic framework through the coordination of the rare earth ions; the rare earth ion doped metal organic frame is Tb 3+ Doped 5,10,15, 20-tetrakis (4-carboxyphenyl) manganese porphyrin. The metal organic frame loaded lactic acid oxidase provided by the invention loads the lactic acid oxidase on the metal organic frame through the coordination of rare earth metal ions, the lactic acid oxidase and the metal organic frame carrier are combined through chemical bonds, the acting force is strong, the stability is good, thereby ensuring that more lactic acid oxidase enters into solid tumor cells,after entering tumor cells: on the one hand, lactate Oxidase (LOX) catalyzes the production of pyruvic acid and H from lactic acid 2 O 2 Providing more H for Fenton-like reaction 2 O 2 Reaction substrate, H 2 O 2 Participate in Fenton-like reaction, thereby generating more OH with cytotoxicity and inducing apoptosis of tumor cells; generated H 2 O 2 Can also be combined with Mn 3+ The reaction generates oxygen, which upon illumination generates more cytotoxic ROS, thereby simultaneously enhancing PDT/CDT therapeutic effects. Mn on the other hand 3+ Reacts with glutathione to increase the amount of ROS and H 2 O 2 The reaction produces O 2 PDT effect can also be improved. In conclusion, the metal organic framework loaded lactic acid oxidase provided by the invention has good stability, and can effectively improve H in tumor microenvironment 2 O 2 Is effective in significantly enhancing the therapeutic effect of PDT and/or CDT.
The invention provides a preparation method of the metal organic framework loaded lactate oxidase, which comprises the following steps: lactic acid oxidase solution and Tb 3+ And mixing the doped 5,10,15, 20-tetra (4-carboxyphenyl) manganese porphyrin solution for coordination reaction to obtain the metal organic framework loaded lactate oxidase. The preparation method provided by the invention is simple and suitable for industrial application, and the lactate oxidase loaded on the metal organic frame is obtained through the coordination reaction of the lactate oxidase and rare earth metal ions in the solution.
Drawings
FIG. 1 is a TEM image of LOX-Mn-TCPP (Tb) prepared in example 1 of the present invention;
FIG. 2 is H 2 Ultraviolet absorption spectra of TCPP, mn-TCPP (Tb), LOX-Mn-TCPP (Tb);
FIG. 3 is a graph showing mass fractions of Mn and Tb in Mn-TCPP (Tb) prepared in various examples;
FIG. 4 is a graph of fluorescence emission spectra under different conditions;
FIG. 5 shows the measurement of cell activity with CCK-8 after incubation of different materials with LO2 cells (left) and MCF-7 (right).
Detailed Description
The invention provides a metal organic framework loaded lactate oxidase, which comprises a rare earth ion doped metal organic framework and lactate oxidase loaded on the metal organic framework through the coordination of the rare earth ions;
the rare earth ion doped metal organic frame is Tb 3+ Doped 5,10,15, 20-tetrakis (4-carboxyphenyl) manganese porphyrin.
In the present invention, all preparation materials/components are commercially available products well known to those skilled in the art unless specified otherwise.
The metal organic framework loaded lactic acid oxidase provided by the invention comprises Tb 3+ Doped 5,10,15, 20-tetrakis (4-carboxyphenyl) manganese porphyrin (herein designated Mn-TCPP (Tb)). The invention adopts 5,10,15, 20-tetra (4-carboxyphenyl) manganese porphyrin (Mn-TCPP) as MOFs carrier, wherein Mn 3+ Reacts with glutathione to increase the amount of ROS and H 2 O 2 The reaction produces O 2 PDT effect can also be improved. The invention passes through Tb 3+ Doped with Mn-TCPP to Tb 3+ As a connecting group, the lactic acid oxidase can be loaded on the Mn-TCPP molecule in a chemical bond mode through coordination reaction, so that the loading stability of the lactic acid oxidase on the Mn-TCPP molecule is improved, leakage of the lactic acid oxidase in the in-vivo transportation process is avoided, transportation of the lactic acid oxidase into tumor cells is ensured, and the PDT/CDT tumor treatment effect is improved.
In the present invention, the Tb 3+ Doped 5,10,15, 20-tetrakis (4-carboxyphenyl) manganese porphyrin: the weight percentage of the Tb element is preferably 0.08 to 0.125wt percent, more preferably 0.1 to 0.125wt percent; the mass percentage of Mn element is preferably 0.15 to 0.2wt%, more preferably 0.15 to 0.175wt%.
The metal organic framework loaded lactate oxidase provided by the invention comprises lactate oxidase loaded on the metal organic framework through the coordination of rare earth ions. In the present invention, the lactic acid oxidase and Tb 3+ Through chemical bond connection. In the present invention, the lactate oxidase is transported into tumor cells via Mn-TCPP (Tb) vector, on the one hand Lactate Oxidase (LOX) catalyzes the production of pyruvate and H 2 O 2 Providing more H for Fenton-like reaction 2 O 2 Reaction substrate, H 2 O 2 Participate in Fenton-like reaction, thereby generating more OH with cytotoxicity and inducing apoptosis of tumor cells; generated H 2 O 2 Can also be combined with Mn 3+ The reaction generates oxygen, which upon illumination generates more cytotoxic ROS, thereby simultaneously enhancing PDT/CDT therapeutic effects.
In the present invention, the metal-organic framework carries a lactate oxidase: the mass percentage (load) of the lactic acid oxidase is preferably 10 to 20wt%, more preferably 12 to 15wt%, and most preferably 12wt%. The loading amount of the lactate oxidase in the metal-organic framework loaded lactate oxidase cannot be too high or too low, and when the loading amount of the lactate oxidase is too high, the stability of the metal-organic framework loaded lactate oxidase is poor, and the lactate oxidase is easy to leak in the transportation process; the lactate oxidase loading is too low to effectively enhance the PDT/CDT therapeutic effect.
In the invention, the morphology of the metal organic framework loaded with the lactic acid oxidase is fusiform; the particle size is preferably 190 to 210nm.
The invention provides a preparation method of the metal organic framework loaded lactate oxidase, which comprises the following steps:
lactic acid oxidase solution and Tb 3+ And mixing the doped 5,10,15, 20-tetra (4-carboxyphenyl) manganese porphyrin solution for coordination reaction (hereinafter referred to as first coordination reaction) to obtain the metal-organic framework supported lactic acid oxidase.
In the present invention, the solvent in the lactate oxidase solution is preferably N, N' -Dimethylformamide (DMF). The mass concentration of the lactate oxidase solution is preferably 2mg/mL.
In the present invention, tb 3+ The solvent used for the doped 5,10,15, 20-tetra (4-carboxyphenyl) manganese porphyrin solution is preferably DMF. The Tb is 3+ The mass concentration of the doped 5,10,15, 20-tetra (4-carboxyphenyl) manganese porphyrin solution is preferably 1mg/mL.
In the present invention, the Tb 3+ The doping of 5,10,15,the preparation method of the 20-tetra (4-carboxyphenyl) manganese porphyrin comprises the following steps:
tb is prepared by mixing 5,10,15, 20-tetra (4-carboxyphenyl) manganese porphyrin, terbium acetate, glacial acetic acid (hereinafter referred to as first glacial acetic acid) and an organic solvent (hereinafter referred to as first organic solvent) 3+ Doping to obtain Tb 3+ Doped 5,10,15, 20-tetrakis (4-carboxyphenyl) manganese porphyrin.
In the invention, the preparation method of the 5,10,15, 20-tetra (4-carboxyphenyl) manganese porphyrin comprises the following steps:
mixing manganese acetate, glacial acetic acid (hereinafter referred to as second glacial acetic acid) and an organic solvent (hereinafter referred to as second organic solvent) to obtain a manganese acetate solution;
adding an organic solution of 5,10,15, 20-tetra (4-carboxyphenyl) porphyrin into the manganese acetate solution, and performing a coordination reaction (hereinafter referred to as a second coordination reaction) to obtain the 5,10,15, 20-tetra (4-carboxyphenyl) manganese porphyrin.
The manganese acetate solution is obtained by mixing manganese acetate, second glacial acetic acid and a second organic solvent. In a specific embodiment of the present invention, the manganese acetate is manganese (III) acetate dihydrate (Mn (OAc) 3 ·2H 2 O). The second organic solvent is N, N' -Dimethylformamide (DMF). In the present invention, the order in which the manganese acetate, the second glacial acetic acid and the second organic solvent are mixed is preferably: dissolving manganese acetate in part of the second organic solvent to obtain manganese acetate solution; mixing the second glacial acetic acid with the rest of the second organic solvent to obtain a mixed solution; and mixing the manganese acetate solution and the mixed solution. The ratio of the mass of the manganese acetate to the volume of part of the second organic solvent is preferably (85-90) mg/10 mL; the volume ratio of the second glacial acetic acid to the remaining second organic solvent is preferably 1:4; the ratio of the mass of the manganese acetate to the volume of the mixed solution is preferably (85-90) mg (25-27) mL.
After obtaining a manganese acetate solution, adding an organic solution of 5,10,15, 20-tetra (4-carboxyphenyl) porphyrin into the manganese acetate solution, and performing a second coordination reaction to obtain the 5,10,15, 20-tetra (4-carboxyphenyl) manganese porphyrin. In the present invention, the solvent in the organic solution of 5,10,15, 20-tetrakis (4-carboxyphenyl) porphyrin is preferably DMF; the mass concentration is preferably 1mg/mL. The mass ratio of the manganese acetate to the 5,10,15, 20-tetra (4-carboxyphenyl) porphyrin is preferably (85-90): 10-12. In the present invention, the second complexation reaction is preferably performed at room temperature, and the reaction time is preferably 12 to 24 hours. In the present invention, after the second coordination reaction is finished, the solid-liquid separation is preferably performed on the obtained second coordination reaction, and the obtained solid-phase product is sequentially subjected to DMF washing and ethanol washing to obtain the 5,10,15, 20-tetra (4-carboxyphenyl) manganese porphyrin. The solid-liquid separation is preferably centrifugation; the number of DMF washes is preferably 3 and the number of ethanol washes is preferably 2.
In the present invention, the molar ratio of the 5,10,15, 20-tetrakis (4-carboxyphenyl) manganese porphyrin to the terbium acetate is preferably 1 (1 to 8), more preferably 1:4. The volume ratio of the first glacial acetic acid to the first organic solvent is preferably 1:4. The invention has no special requirement on the dosage of the first organic solvent, and can ensure that the 5,10,15, 20-tetra (4-carboxyphenyl) manganese porphyrin and the terbium acetate are completely dissolved. In the present invention, the Tb 3+ Doping is preferably carried out at room temperature and the reaction time is preferably 1h. In the present invention, the Tb 3+ After doping, the invention preferably uses Tb obtained 3+ And (3) carrying out solid-liquid separation on the doped reaction liquid, and sequentially washing and drying the obtained solid product by DMF to obtain the Mn-TCPP (Tb). The solid-liquid separation is preferably centrifugation; the number of times of DMF washing is preferably 3, and the drying is preferably drying.
In the present invention, lactate oxidase and Tb 3+ The mass ratio of the doped 5,10,15, 20-tetra (4-carboxyphenyl) manganese porphyrin is (1-2): 1, more preferably 2:1.
In the present invention, the temperature of the first coordination reaction is preferably 37℃and the reaction time is preferably 3 hours. In the present invention, after the completion of the first coordination reaction, the present invention preferably performs solid-liquid separation of the obtained first coordination reaction liquid, and the obtained solid-phase product is LOX-Mn-TCPP (Tb).
The invention provides a photodynamic and/or chemodynamic therapeutic reagent, which comprises the metal-organic framework loaded lactate oxidase prepared by the technical scheme or the preparation method.
The invention provides application of the metal-organic framework loaded lactate oxidase or the metal-organic framework loaded lactate oxidase prepared by the preparation method in the technical scheme in preparation of antitumor drugs.
In the present invention, the solid tumor preferably comprises breast cancer.
The technical solutions provided by the present invention are described in detail below in conjunction with examples for further illustrating the present invention, but they should not be construed as limiting the scope of the present invention.
The technical terms appearing in the present invention are explained: 1. metal organic framework (MetalOrganic Frameworks): MOFs for short are organic-inorganic hybrid materials with intramolecular pores formed by self-assembly of organic ligands and metal ions or clusters through coordination bonds. 2. Lactate oxidase: LOX; lactic acid: lacticacid (LA). 3.5,10,15,20-tetra (4-carboxyphenyl) manganese porphyrin (Mn-TCPP) belongs to one of the metal organic frame materials; tb (Tb) 3+ Doped 5,10,15, 20-tetrakis (4-carboxyphenyl) manganese porphyrin: LOX-Mn-TCPP (Tb). 4.2- (2-methoxy-4-nitrophenyl) -3- (4-nitrophenyl) -5- (2, 4-disulfonic acid benzene) -2H-tetrazolium monosodium salt (CCK-8, cellCountinning kit-8), reducing the methyl 1-methoxy-5-methylphenoxazine sulfate (1-methoxyPMS) by dehydrogenase in cell mitochondria into yellow Formazan product (Formazan) with high water solubility, wherein the quantity of the Formazan product is proportional to the quantity of living cells, and measuring the light absorption value of the Formazan product at the wavelength of 450nm by an enzyme-labeled instrument, so that the quantity of the living cells can be indirectly reacted. 5. Human breast cancer cells: MCF-7, human normal hepatocytes: LO2.6.2,7-dichlorofluorescein diacetate, also known as reactive oxygen species ROS fluorescent probe (DCFH-DA, 4091-99-0): DCFH-DA has no fluorescence, can freely pass through cell membrane, can be hydrolyzed by intracellular esterase to generate DCFH, and can not pass through cell membrane, so that the probe can be easily marked into cell, under the condition of ROS existence, the DCFH is oxidized to generate green fluorescent material DCF, and greenThe color fluorescence intensity is proportional to the ROS level.
Example 1
85mg of manganese (III) acetate dihydrate (Mn (OAc)) was weighed out 3 ·2H 2 O) and dispersed in a flat bottom flask containing 10 ln, n' -Dimethylformamide (DMF) with ultrasonic agitation to make it uniformly dispersed. 25mL of a glacial acetic acid (HAc)/DMF mixed solution in a volume ratio of 1:4 was added, and stirring was continued for 8 minutes after thorough mixing. To the above solution, 12mL of 5,10,15, 20-tetrakis (4-hydroxyphenyl) porphyrin (H) at a concentration of 1mg/mL was slowly added 2 TCPP) solution (DMF as solvent) and at room temperature for 24 hours, centrifuging the product and washing three times with DMF and twice with ethanol to obtain Mn-TCPP.
Mn-TCPP and C with a molar ratio of 1:4 are added under normal temperature conditions 6 H 9 O 6 Tb·xH 2 O was dissolved in 1mL of HAc/DMF solution at a volume ratio of 1:4 and reacted for 1 hour at room temperature. After the reaction is finished, centrifugally collecting a sample, washing with DMF three times, and drying to obtain Mn-TCPP (Tb) for later use.
100. Mu.L of the prepared LOX solution (with DMF as a solvent) at a concentration of 2mg/mL and 100. Mu.L of Mn-TCPP (Tb) solution (with DMF as a solvent) at a concentration of 1mg/mL were thoroughly mixed and reacted in an oven at 37℃for 3 hours. After the reaction, the sample was collected by centrifugation to obtain LOX-Mn-TCPP (Tb).
Test example 1
Characterization of LOX-Mn-TCPP (Tb)
The morphology and size of the LOX-Mn-TCPP (Tb) prepared in example 1 was characterized by Transmission Electron Microscopy (TEM). As shown in FIG. 1, the LOX-Mn-TCPP (Tb) has a size of 190-210nm and a shape of a shuttle.
Ultraviolet absorbance detection
For H 2 UV absorption detection of TCPP, mn-TCPP (Tb), LOX-Mn-TCPP (Tb), as shown in FIG. 2, with organic ligand H 2 Compared with TCPP, all ultraviolet absorption peaks of Mn-TCPP are red shifted because of the increase of conjugation area of porphyrin, and this also proves successful synthesis of Mn-TCPP. In the process of doping Tb 3+ And after loading LOX, the ultraviolet absorption peak of the material is hardly changed, which indicates the crystal structure of Mn-TCPPNo change occurs.
Examples 2 to 4
The preparation process was essentially the same as in example 1, except that: in example 2, n (Mn-TCPP): n (Tb) 3+ ) =1:1, n (Mn-TCPP) n (Tb) in example 3 3+ ) =1:2, n (Mn-TCPP) n (Tb 3+ )＝1:8。
Test example 2
Inductively coupled plasma characterization
The Mn-TCPP prepared in example 1 and Mn-TCPP (Tb) prepared in examples 1 to 4 were analyzed for the content of metal elements by Inductively Coupled Plasma (ICP), as shown in FIG. 3, according to the different molar ratios of Mn-TCPP and Tb (n (Mn-TCPP): n (Tb) 3+ ) Mn-TCPP (Tb) with different doping levels were synthesized by =1:0, 1:1,1:2,1:4, 1:8), the ICP results are shown in fig. 3, with Tb 3+ The concentration is increased, the content of Tb element in Mn-TCPP (Tb) is also increased, and the content of Mn element is reduced, which indicates the successful doping of Tb element.
Test example 3
Reactive oxygen species detection
Further detection of OH production was performed by DCFH. Firstly, 0.97mg of DCFH-DA was weighed and dissolved in dimethyl sulfoxide (DMSO) to obtain a 1mM DCFH-DA solution, then 0.5mL of 1mM DCFH-DA solution was added to 2mL of 0.01M NaOH solution, and the well-mixed solution was left to react in a dark environment for 30 minutes. After the reaction was completed, 10ml of a PBS buffer solution having ph=4 was added to terminate the reaction, to obtain a DCF solution, which was stored at-20 ℃ in a dark place. Experiments were designed into the following groups: mn-TCPP (Tb), mn-TCPP (Tb) +illumination, mn-TCPP (Tb) +lactic acid, mn-TCPP (Tb) +lactic acid+illumination, LOX-Mn-TCPP (Tb) +illumination, LOX-Mn-TCPP (Tb) +lactic acid, LOX-Mn-TCPP (Tb) +lactic acid+illumination. Wherein lactic acid was formulated with NaOH solution at ph=6 and the light time was 1 minute. Finally, equal amounts of DCF solution were added to each of the above experimental groups, and the fluorescence intensity of the supernatant was measured at an excitation wavelength of 488 nm. The test results are shown in fig. 4.
Since 2',7' -dichlorofluorescein diacetate (DCFH-DA) can be oxidized by ROS to produce DC with green fluorescenceF, as shown in FIG. 4, the LOX-Mn-TCPP (Tb) +Lactic acid+laser group prepared in example 1 has the strongest fluorescence intensity because LOX catalyzes the production of H by Lactic acid 2 O 2 While more ROS are produced by manganese ions and photocatalysis.
Test example 4
Cytotoxicity detection
The cytotoxicity of different materials on human breast cancer cells (MCF-7) and human normal liver cells (LO 2) is studied through CCK-8. MCF-7 and LO2 cells were seeded into 96-well plates at a cell density of 8X10 4 Each well was incubated in a constant temperature incubator at 37℃for 12 hours. After cell attachment, the old cell culture medium is discarded. Fresh culture solutions containing different concentrations of Mn-TCPP (Tb) and LOX-Mn-TCPP (Tb) were added to the 96-well plates, respectively, and the culture was continued with the concentration gradients of Mn-TCPP (Tb) and LOX-Mn-TCPP (Tb) being 0. Mu.g/mL, 1. Mu.g/mL, 2. Mu.g/mL, 4. Mu.g/mL, 6. Mu.g/mL and 8. Mu.g/mL. For phototoxicity experiments, mn-TCPP (Tb) prepared in example 1 was incubated with LOX-Mn-TCPP (Tb) for 4 hours, followed by a further incubation of 18 hours with 650nm laser irradiation for 10 minutes. After incubation, the viability of the different groups of cells was measured by a microplate reader and the results are shown in FIG. 5, in which the viability of MCF-7 cells was about 75% and the viability of LO2 cells was more than 80% at Mn-TCPP (Tb) concentration of 8. Mu.g/mL, due to the over-expressed H in the tumor cells 2 O 2 Is catalyzed by Fenton-like materials to generate ROS. Following laser irradiation, the Mn-TCPP (Tb) +L group (where "L" stands for light) of both cells had decreased cell viability due to the co-treatment of CDT with PDT. Importantly, the LOX-Mn-TCPP (Tb) group has lower cell viability than Mn-TCPP (Tb) because LOX catalyzes the production of lactate to pyruvate versus H 2 O 2 The CDT treatment effect is improved. Likewise, the LOX-Mn-TCPP (Tb) +L (where "L" represents light) group showed lower cell viability compared to the LOX-Mn-TCPP (Tb) group, indicating better synergistic therapeutic effects of CDT/PDT.
The nanometer material LOX-Mn-TCPP (Tb) provided by the invention is synthesized into Mn-TCPP at normal temperature, and is doped with Tb 3+ LOX is supported by the coordination of rare earth ions to obtain LOX-Mn-TCPP (Tb). After the nanomaterial LOX-Mn-TCPP (Tb) enters the body, LOX is catalyzedFormation of pyruvic acid and H by lactic acid 2 O 2 Wherein a part of H 2 O 2 Participate in Fenton-like reaction to generate OH, a part of which is combined with Mn 3+ The reaction generates oxygen and enhances PDT/CDT therapeutic effects. Mn on the other hand 3+ Reacts with glutathione to increase the amount of ROS and H 2 O 2 The reaction produces O 2 The PDT effect is improved. The LOX-Mn-TCPP (Tb) nanomaterial provided by the invention has the advantages of simple synthesis method, no dangerous operation, stable structure and capability of enhancing the synergistic treatment effect of chemical power and photodynamic power by LOX load.
Although the foregoing embodiments have been described in some, but not all embodiments of the invention, other embodiments may be obtained according to the present embodiments without departing from the scope of the invention.
Claims (10)
1. The lactate oxidase loaded on the metal organic framework is characterized by comprising a rare earth ion doped metal organic framework and lactate oxidase loaded on the metal organic framework through coordination of the rare earth ions;
the rare earth ion doped metal organic frame is Tb 3+ Doped 5,10,15, 20-tetrakis (4-carboxyphenyl) manganese porphyrin.
2. The metal-organic framework supported lactate oxidase according to claim 1, wherein the metal-organic framework supported lactate oxidase has a particle size of 190-210 nm.
3. The metal-organic framework supported lactate oxidase according to claim 1, wherein the Tb 3+ Doped 5,10,15, 20-tetrakis (4-carboxyphenyl) manganese porphyrin: the weight percentage of Tb element is 0.08-0.125 wt%; the mass percentage of Mn element is 0.15-0.2wt%;
in the metal organic framework loaded lactate oxidase: the mass percentage of the lactic acid oxidase is 10-20wt%.
4. A method for producing a metal-organic framework-supported lactate oxidase according to any one of claims 1 to 3, comprising the steps of:
lactic acid oxidase solution and Tb 3+ And mixing the doped 5,10,15, 20-tetra (4-carboxyphenyl) manganese porphyrin solution for coordination reaction to obtain the metal organic framework loaded lactate oxidase.
5. The method according to claim 4, wherein Tb 3+ The preparation method of the doped 5,10,15, 20-tetra (4-carboxyphenyl) manganese porphyrin comprises the following steps:
mixing 5,10,15, 20-tetra (4-carboxyphenyl) manganese porphyrin, terbium acetate, glacial acetic acid and organic solvent to obtain Tb 3+ Doping to obtain Tb 3+ Doped 5,10,15, 20-tetrakis (4-carboxyphenyl) manganese porphyrin.
6. The method according to claim 4, wherein the lactic acid oxidizing enzyme and Tb 3+ The mass ratio of the doped 5,10,15, 20-tetra (4-carboxyphenyl) manganese porphyrin is (1-2): 1.
7. the method according to claim 5, wherein the molar ratio of 5,10,15, 20-tetra (4-carboxyphenyl) manganese porphyrin to terbium acetate is 1 (1-8).
8. The preparation method of the 5,10,15, 20-tetra (4-carboxyphenyl) manganese porphyrin according to claim 5, comprising the following steps:
mixing manganese acetate, glacial acetic acid and an organic solvent to obtain a manganese acetate solution;
adding an organic solution of 5,10,15, 20-tetra (4-carboxyphenyl) porphyrin into the manganese acetate solution, and carrying out coordination reaction to obtain the 5,10,15, 20-tetra (4-carboxyphenyl) manganese porphyrin.
9. A photodynamic and/or chemodynamic therapeutic agent comprising a metal-organic framework-supported lactate oxidase according to any one of claims 1 to 3 or prepared by a method according to any one of claims 4 to 8.
10. Use of the metal-organic framework-supported lactate oxidase according to any one of claims 1 to 3 or the metal-organic framework-supported lactate oxidase prepared by the preparation method according to any one of claims 4 to 8 in the preparation of antitumor drugs.
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