CN110680926B - Nano diagnosis and treatment agent and preparation method and application thereof - Google Patents
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Abstract
The invention relates to a nano diagnosis and treatment agent and a preparation method and application thereof, wherein the preparation method comprises the following steps: mixing and culturing glucose oxidase (GOx) or modified GOx with a sugar-free culture medium and a water-soluble Fenton reagent to prepare a GOx compound solution; and adding calcium salt into the GOx compound solution, and reacting to obtain the nano diagnosis and treatment agent. The preparation process of the nano diagnosis and treatment agent provided by the invention is simple and convenient to operate. In the nano diagnosis and treatment agent prepared by the invention, GOx and calcium phosphate exist in a human body and are easily absorbed and degraded by the human body, and metal ions in the Fenton reagent are all trace elements required by the human body and have good biocompatibility and biodegradability.
Description
Technical Field
The invention relates to the field of medical nano materials, in particular to a nano diagnosis and treatment agent and a preparation method and application thereof.
Background
Cancer has become one of the main diseases of human death, and how to improve the treatment effect of cancer has been a difficult problem and a hot spot in the medical field. The multifunctional nano diagnosis and treatment agent integrates different treatment modes, so that the effect of obviously improving the treatment effect of cancer is remarkably improved, and the multifunctional nano diagnosis and treatment agent becomes a popular research direction of anticancer drugs at present. However, the compositions of the currently reported multifunctional nano diagnosis and treatment agents are complex, the preparation process is complicated, the biocompatibility is poor, and the potential long-term toxicity problem exists, so that the application of the multifunctional nano diagnosis and treatment agents in clinic is severely limited. The biodegradable nano diagnosis and treatment agent can be completely degraded in organisms and participate in normal metabolism of human bodies, so that the problem of long-term toxicity of the traditional diagnosis and treatment agent is avoided, and the biodegradable nano diagnosis and treatment agent is expected to be widely applied in clinic. Therefore, the development of the biodegradable multifunctional nano diagnosis and treatment agent has good application prospect in the field of efficient treatment of tumors.
The glucose oxidase (GOx) naturally existing in organisms can catalyze and oxidize glucose in cells into gluconic acid and hydrogen peroxide (H) 2 O 2 ) Because it consumes energy and nutrients in the cell and catalyzes the production of H by oxidation of glucose 2 O 2 The free GOx has the defects of short blood circulation half-life period, high toxicity, easy inactivation and the like, so that the GOx cannot be directly injected into the body.
Accordingly, the prior art is yet to be improved and developed.
Disclosure of Invention
In view of the defects of the prior art, the invention aims to provide a nano diagnosis and treatment agent, a preparation method and application thereof, and aims to solve the problems of high blood toxicity and complex synthesis process of the conventional GOx-loaded nano diagnosis and treatment agent.
The technical scheme adopted by the invention for solving the technical problems is as follows:
a preparation method of a nano diagnosis and treatment agent comprises the following steps:
mixing and culturing GOx or modified GOx, a sugar-free culture medium and a water-soluble Fenton reagent to prepare a GOx compound solution;
and adding calcium salt into the GOx compound solution, and reacting to obtain the nano diagnosis and treatment agent.
The preparation method of the nano diagnosis and treatment agent comprises the step of preparing a water-soluble Fenton reagent, wherein the water-soluble Fenton reagent comprises one or more of manganese ions, copper ions, iron ions, cobalt ions, chromium ions, tungsten ions, aluminum ions and strontium ions.
The preparation method of the nano diagnosis and treatment agent comprises the following steps of mixing and culturing GOx or modified GOx, a sugar-free culture medium and a water-soluble Fenton reagent to prepare a GOx compound solution:
dissolving GOx and a water-soluble Fenton reagent in a sugar-free medium, and combining metal ions in the water-soluble Fenton reagent and the GOx through physical adsorption to prepare a GOx compound solution;
or dissolving the modified GOx and the water-soluble Fenton reagent in a sugar-free culture medium, and combining metal ions in the water-soluble Fenton reagent and the modified GOx through physical adsorption to prepare the GOx compound solution.
The preparation method of the nano diagnosis and treatment agent comprises the following steps:
and mixing GOx, an activating agent and amino-polyethylene glycol to perform amidation reaction on the amino-polyethylene glycol and the GOx to obtain the modified GOx.
The preparation method of the nano diagnostic agent comprises the step of preparing the nano diagnostic agent, wherein the activating agent is 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide.
The preparation method of the nano diagnosis and treatment agent comprises the following steps of 1-10 mass ratio of amino-polyethylene glycol to GOx.
The preparation method of the nano diagnosis and treatment agent comprises the step of preparing a nano diagnosis and treatment agent, wherein the molecular weight of the amino-polyethylene glycol is 500-20000.
The preparation method of the nano diagnosis and treatment agent comprises the following steps of adding calcium salt into the GOx compound solution, and reacting to prepare the nano diagnosis and treatment agent:
and adding a calcium salt into the GOx compound solution, so that phosphate ions in the GOx compound solution react with calcium ions of the calcium salt to generate calcium phosphate on the surface of the GOx compound, and thus the nano diagnosis and treatment agent is prepared.
A nano diagnostic and therapeutic agent, comprising a GOx complex and calcium phosphate coated on the surface of the GOx complex, wherein the GOx complex comprises GOx and metal ions in a water-soluble fenton reagent bound to the GOx by physical adsorption; alternatively, the GOx complex comprises the modified GOx and a metal ion in a water-soluble fenton reagent bound to the modified GOx by physisorption.
The application of the nano diagnosis and treatment agent is characterized in that the nano diagnosis and treatment agent or the nano diagnosis and treatment agent prepared by the preparation method is used as a drug transport carrier.
Has the advantages that: the invention takes GOx or modified GOx as a biological template, calcium salt as a calcium source, a sugar-free culture medium as a solvent and a water-soluble Fenton reagent are added, and the nano diagnosis and treatment agent doped with the water-soluble Fenton reagent is prepared by adopting a biomineralization simulating method. The preparation process of the nano diagnosis and treatment agent provided by the invention is simple, is easy to operate, and is beneficial to industrial popularization and application. PO in the sugar-free medium 4 3- With Ca in calcium salts 2+ The calcium phosphate carrier obtained by reaction, GOx and calcium phosphate exist in a human body and are easily absorbed and degraded by the human body, and metal ions in the Fenton reagent are all metal elements required by the human body and have good biocompatibility and biodegradability.
Drawings
Fig. 1 is a flow chart of a preparation method of a nano diagnosis and treatment agent provided by an embodiment of the present invention.
FIG. 2 is a Transmission Electron Microscope (TEM) image of a sample provided in example 1 of the present invention.
FIG. 3 is a Transmission Electron Microscope (TEM) image of a sample provided in example 2 of the present invention.
Fig. 4 is a Scanning Transmission Electron Microscope (STEM) image of a sample provided in example 2 of the present invention.
FIG. 5 is a Transmission Electron Microscope (TEM) image of a sample provided in example 3 of the present invention.
FIG. 6 is a Transmission Electron Microscope (TEM) image of a sample provided in example 4 of the present invention.
Fig. 7 is a Scanning Transmission Electron Microscope (STEM) image of a sample provided in example 4 of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention clearer and clearer, the present invention is further described in detail below with reference to the accompanying drawings and examples. 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 a method for preparing a nano diagnostic agent according to a preferred embodiment of the present invention, as shown in the figure, the method includes:
s100, mixing and culturing GOx, a sugar-free culture medium and a water-soluble Fenton reagent to obtain a GOx compound solution;
s200, adding calcium salt into the GOx compound solution, and reacting to obtain the nano diagnosis and treatment agent.
In this embodiment, since GOx has a certain adsorption effect on metal ions, when GOx and a water-soluble fenton reagent are dissolved in a sugar-free medium, metal ions in the water-soluble fenton reagent can be physically adsorbed and combined with GOx to prepare a GOx complex solution; adding calcium salt to the GOx compound solution, wherein calcium ions in the calcium salt are adsorbed on the surface of the GOx compound, a sugar-free medium (DMEM) in the GOx compound solution contains phosphate, and PO in the DMEM 4 3- With said Ca 2+ A calcification reaction occursAnd generating calcium phosphate on the surface of the GOx composite, and coating the calcium phosphate on the surface of the GOx composite to prepare the nano diagnosis and treatment agent.
The nano diagnostic and therapeutic agent prepared in this embodiment includes a GOx complex including GOx and metal ions in a water-soluble fenton reagent bound to the GOx by physical adsorption, and calcium phosphate coated on the surface of the GOx complex. In this example, PO in the sugar-free Medium 4 3- With Ca in calcium salts 2+ The calcium phosphate carrier is obtained through reaction, the GOx and the calcium phosphate exist in a human body and are easily absorbed and degraded by the human body, metal ions in the Fenton reagent are all metal elements required by the human body, and the nano diagnosis and treatment agent has good biocompatibility and biodegradability. Therefore, the nano diagnosis and treatment agent prepared by the embodiment can be used as a drug delivery carrier, and the nuclear magnetic resonance imaging of the tumor and the combined treatment combining the starvation treatment, the chemical kinetic treatment and the chemotherapy of the tumor can be simultaneously realized by loading the anti-tumor drug, so that the nano diagnosis and treatment agent has a good application prospect in the field of diagnosis and treatment of the tumor.
In some embodiments, the water-soluble fenton reagent includes one or more of manganese ions, copper ions, iron ions, cobalt ions, chromium ions, tungsten ions, aluminum ions, and strontium ions, but is not limited thereto. In this embodiment, the metal ions included in the water-soluble fenton reagent are all trace elements necessary for a human body, and the metal ions in the water-soluble fenton reagent physically adsorbed and combined with GOx may also partially participate in a biomimetic calcification (generation of calcium phosphate), and the metal ions may exist in the prepared nano diagnosis and treatment agent particles in an ion-doped form, so that the prepared nano diagnosis and treatment agent is easily degraded after entering the human body, has good biocompatibility and biodegradability, and has little influence on the health of the human body.
In some embodiments, the molar ratio of the water-soluble fenton's reagent to the calcium salt is from 0.001 to 1. In the range of the molar ratio, the obtained nano diagnosis and treatment agent has good effect, and if the effect exceeds the range of the molar ratio, certain side effects can be caused due to excessive human body trace elements in the Fenton reagent, for example, the trace element manganese can cause neurotoxicity and damage to a nervous system when the amount of the trace element manganese in a human body is too high; the excessive copper content will cause damage to kidney function. In the range that the mol ratio of the Fenton reagent to the calcium source is 0.001-1.
In some embodiments, the calcium salt is one or more of calcium chloride, calcium chloride hydrate, calcium nitrate hydrate, calcium acetate, and calcium acetate hydrate, but is not limited thereto.
In some embodiments, there is also provided a method for preparing a nano therapeutic agent, comprising the steps of:
s11, dissolving the modified GOx and a water-soluble Fenton reagent in a sugar-free culture medium, and combining metal ions in the water-soluble Fenton reagent and the modified GOx through physical adsorption to prepare a GOx compound solution;
s21, adding calcium salt into the GOx compound solution, and reacting to obtain the nano diagnosis and treatment agent.
The nano diagnostic and therapeutic agent prepared in this embodiment includes a GOx complex including modified GOx and metal ions in a water-soluble fenton reagent bound to the modified GOx by physisorption, and calcium phosphate coated on the surface of the GOx complex.
In this example, the preparation of the modified GOx comprises the steps of: and mixing GOx, an activating agent and amino-polyethylene glycol to perform amidation reaction on the amino-polyethylene glycol and the GOx to obtain the modified GOx. Specifically, the activator comprises 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), and the GOx is reacted with amino-polyethylene glycol (NH) under the action of the activator 2 PEG) amidation reaction, NH 2 -reacting the amino group of PEG with the carboxyl group of GOx to form an amide bond linkage, obtaining a PEG-modified GOx, i.e. a modified GOx, coupling said modified GOxThe modified GOx is used as a bionic calcification biological template, and the dispersibility of the finally prepared calcium phosphate nano diagnosis and treatment agent particles can be improved.
In some embodiments, when the water-soluble fenton reagent contains Mn 2+ In the meantime, the GOx can be used for preparing a nano diagnosis and treatment agent with better dispersibility without PEG modification; when the water-soluble Fenton reagent contains Cu 2+ In the meantime, compared with the nano diagnosis and treatment agent prepared by the unmodified GOx, the nano diagnosis and treatment agent prepared by the PEG-modified GOx has better dispersibility. Due to different doped trace elements (such as manganese and copper), the prepared nanoparticles have different dispersion effects. The method can optionally adopt NH used in advance according to the type of metal ions contained in the water-soluble Fenton reagent 2 And (3) a method for modifying GOx by PEG, and then the modified GOx participates in the preparation of the nano diagnosis and treatment agent so as to obtain the nano particles with better dispersion effect.
In some embodiments, the mass ratio of the amino-polyethylene glycol to the GOx is 1 to 10. In this embodiment, the GOx undergoes an amidation reaction with amino-PEG, and the carboxyl site on GOx forms an amide bond with the amino group of PEG, and if the amount of amino-PEG used for modification is too large (greater than 10: 1), the carboxyl site on GOx is greatly reduced, thereby reducing the effect of GOx on metal ions (such as Ca 2+ 、Mn 2+ 、Cu 2+ ) The adsorption of (2) results in that calcium phosphate carriers cannot be generated on the surface of GOx, so that the nano diagnosis and treatment agent cannot be prepared.
In some embodiments, the amino-polyethylene glycol has a molecular weight of 500 to 20000. NH with molecular weight of 500-20000 2 And the GOx modified by PEG is used as a bionic calcification biological template, so that the size and the shape of the synthesized calcium phosphate nano carrier can be regulated and controlled.
Furthermore, the nano diagnosis and treatment agent obtained by the method is of a spherical structure, and the diameter of the nano diagnosis and treatment agent is 10-500 nanometers. Wherein, the diameter of the nano diagnosis and treatment agent is influenced by the reaction time, the reaction temperature, whether the PEG is modified or not, the molecular weight of the PEG and the concentration of the GOx. The diameter of the calcium phosphate nanoparticles of the present invention can be adjusted according to practical circumstances by adjusting the above-mentioned factors. In the specific embodiment provided by the invention, the diameter of the nano diagnosis and treatment agent is 50-300 nanometers.
In some embodiments, calcium salt is added into the GOx compound solution, the GOx compound solution is cultured for 24 hours at the temperature of 37 ℃, and the solid-liquid separation, washing and drying are carried out to obtain the nano diagnosis and treatment agent.
In this embodiment, the solid-liquid separation may be one or more of centrifugal separation, filtration separation and standing separation, and the manner of the solid-liquid separation is not limited to the above examples. The solvent used for washing may be one or more of deionized water and absolute ethyl alcohol, and the solvent used for washing is not limited to the above examples. The drying may be one or more of freeze drying and vacuum drying, and the drying manner is not limited to the above examples.
In the nano diagnosis and treatment agent provided by the invention, GOx and calcium phosphate are both originally present in a human body, and the adopted Fenton reagents are all trace elements required by the human body, so that the nano diagnosis and treatment agent has good biocompatibility and biodegradability after entering the human body, and has small influence on the internal environment.
Furthermore, the nano diagnosis and treatment agent prepared by the preparation method can be used for hunger treatment, chemodynamics treatment and chemotherapy of tumors and combined treatment of the hunger treatment, the chemodynamics treatment and the chemotherapy of the tumors.
In some embodiments, the invention further provides application of the nano diagnosis and treatment agent, and the nano diagnosis and treatment agent is used as a drug transportation carrier. Particularly useful as drug delivery vehicles for combination therapies including starvation, chemokinetic and chemotherapy for tumors. The nanometer diagnosis and treatment agent of the embodiment takes GOx as a biological template, takes water-soluble calcium salt as a calcium source, and adopts a biomimetic mineralization strategy to synthesize the calcium phosphate nanometer carrier wrapped with the GOx, so that the problem of high toxicity of free GOx is avoided. In the nano diagnosis and treatment agent, a calcium phosphate system is degraded in an acid response manner, and because the pH value of cells is weak acid compared with the physiological pH value of normal tissues, after entering a tumor target area, the nano diagnosis and treatment agent is degraded in an acid response manner based on the reduction of the environmental pH valueDuring the process, metal ions of GOx and Fenton reagent are released. The released GOx can decompose glucose in the organism, thereby consuming energy sources and nutrient substances in cells, further cutting off the nutrient supply of tumor cells, and achieving the effect of hunger treatment. GOx catalyzes the decomposition of glucose in vivo to gluconic acid and hydrogen peroxide (H) 2 O 2 ) Of H in the tumor environment 2 O 2 The content of the generated H is higher than that of normal tissue cells 2 O 2 Reacts with the released Fenton reagent to generate active Oxygen (OH) with high cytotoxicity, activates a cell death mode caused by oxidative damage, promotes lipid peroxidation, kills tumor cells and achieves the effect of treating cancer through chemical power.
The nanometer diagnosis and treatment agent provided by the invention can also be used for carrying PH-responsive chemotherapy drugs, because the pH value of tumor cells is weakly acidic, and the physiological pH value of normal tissues is neutral, a calcium phosphate system in the nanometer diagnosis and treatment agent provided by the invention is degraded in an acid response manner, when entering a tumor target area, calcium phosphate is degraded, and in the degradation process, the carried pH-responsive drugs can be released to act on the tumor cells in the tumor target area.
The present invention will be described in detail with reference to the following examples.
Example 1
A GOx mixed solution was prepared by completely dissolving 2 mg of GOx in 1 ml of a sugar free medium (DMEM) at room temperature, and it was incubated in an incubator at 37 ℃ for 24 hours. And then adding 10 microliters of 1 mol/liter calcium chloride solution into the GOx mixed solution, continuously incubating for 24 hours, performing centrifugal separation, washing for 2 times by using deionized water, and drying for 24 hours by using a 60-DEG C oven to obtain a powder sample.
The Transmission Electron Microscope (TEM) photograph of the powder sample prepared in this example is shown in fig. 2, and fig. 2 shows that the sample prepared in this example is a hollow nanoparticle with a diameter of 100-300 nm.
Example 2
A GOx mixed solution was prepared by completely dissolving 2 mg of GOx in 1 ml of a sugar free medium (DMEM) at room temperature. To the prepared GOx mixed solution, 10. Mu.l of a manganese chloride solution having a concentration of 0.1 mmol/l was added, and it was incubated in an incubator at 37 ℃ for 24 hours. And then adding 10 microliters of 1 mol/liter calcium chloride solution into the solution, continuously incubating at 37 ℃ for 24 hours, performing centrifugal separation, washing for 2 times by using deionized water, and drying in a 60 ℃ oven for 24 hours to obtain a powder sample.
A Transmission Electron Microscope (TEM) photograph and a Scanning Transmission Electron Microscope (STEM) photograph of the powder sample prepared in this example are shown in FIG. 3 and FIG. 4, respectively.
FIG. 3 shows that the sample prepared in this example has a spherical structure and good dispersibility, and the diameter thereof is 100-200 nm. FIG. 4 shows that the main elements of the sample prepared in this example are C, N, O, mn, ca, P.
Example 3
GOx solution was prepared by completely dissolving 2 mg of GOx in 1 ml of a sugar free medium (DMEM) at room temperature. To the prepared GOx solution, 10. Mu.l of a 0.1 mmol/l cupric chloride solution was added and incubated in a 37 ℃ incubator for 24 hours. Then 10 microliter of calcium chloride solution with the concentration of 1 mol/liter is added into the solution, after incubation for 24 hours at 37 ℃, centrifugal separation is carried out, deionized water is used for washing for 2 times, and the powder sample is obtained after drying in an oven at 60 ℃ for 24 hours.
A Transmission Electron Microscope (TEM) photograph of the powder sample prepared in this example is shown in FIG. 5.
The Transmission Electron Microscope (TEM) photograph of FIG. 5 shows that the resulting sample has a non-uniform shape and structure, and is partially agglomerated.
Example 4
20 mg of GOx, 10 mg of EDC and 7 mg of NHS were dissolved in 10 ml of deionized water completely at room temperature and stirred for 2 hours. Then 100 mg of NH2-PEG with a molecular weight of 5000 was added and stirring was continued for 20 hours. Subsequently, the reaction solution was transferred completely into a dialysis bag having a molecular weight of 6000 to 8000, and dialyzed for 24 hours. Finally, the GOx solution was concentrated to 2 ml using an ultrafiltration tube with a molecular weight of 30000, and the concentration of the prepared GOx solution was 8 mg/ml.
GOx solution was prepared by adding 0.25 ml of the above PEG-modified GOx to 0.75 ml of a sugar free medium (DMEM) at room temperature. To the prepared GOx solution, 10. Mu.l of a copper chloride solution having a concentration of 0.1 mmol/l was added, and it was incubated in an incubator at 37 ℃ for 24 hours. And then adding 10 microliters of 1 mol/liter calcium chloride solution into the solution, continuously incubating at 37 ℃ for 24 hours, performing centrifugal separation, washing for 2 times by using deionized water, and drying in a 60 ℃ oven for 24 hours to obtain a powder sample.
A Transmission Electron Microscope (TEM) photograph and a Scanning Transmission Electron Microscope (STEM) photograph of the powder sample prepared in this example are shown in fig. 6 and 7, respectively.
The Transmission Electron Microscope (TEM) photograph of FIG. 6 shows that the sample prepared in this example has a spherical structure and good dispersibility, and has a diameter of 50-150 nm.
The Scanning Transmission Electron Microscope (STEM) photograph of FIG. 7 shows that the main elements in the sample prepared in this example are C, N, O, cu, ca, and P.
In conclusion, the invention provides a nano diagnosis and treatment agent and a preparation method and application thereof. The preparation method provided by the invention has the advantages of simple process, room-temperature preparation process, simple operation, easy acquisition of reagents participating in the reaction, no use of complex and expensive equipment, low preparation cost, accordance with the requirements of industrial production, and contribution to popularization and application of the nano diagnosis and treatment agent. According to the nano diagnosis and treatment agent obtained by the preparation method, GOx and the prepared calcium phosphate originally exist in a human body, and the doped Fenton reagent is a trace element required by the human body, so that the nano diagnosis and treatment agent has good biocompatibility and biodegradability with the human body, and causes few side effects on the human body. The nanometer diagnosis and treatment agent can be applied to combined treatment combining starvation treatment, chemodynamics treatment and chemotherapy of tumors by combining biochemical characteristics of GOx, fenton reagent and generated calcium phosphate nanometer carrier, is a biodegradable multifunctional nanometer diagnosis and treatment agent, and has good application prospect.
It will be understood that the invention is not limited to the examples described above, but that modifications and variations will occur to those skilled in the art in light of the above teachings, and that all such modifications and variations are considered to be within the scope of the invention as defined by the appended claims.
Claims (5)
1. A preparation method of a nano diagnosis and treatment agent is characterized by comprising the following steps:
mixing and culturing GOx or modified GOx, a sugar-free culture medium and a water-soluble Fenton reagent to prepare a GOx compound solution;
adding calcium salt into the GOx compound solution, and reacting to obtain the nano diagnosis and treatment agent;
the sugar-free culture medium is DMEM;
the water-soluble Fenton reagent comprises one or more of manganese ions, copper ions, iron ions, cobalt ions, chromium ions, tungsten ions, aluminum ions and strontium ions;
the method for preparing the GOx compound solution by mixed culture of the GOx or the modified GOx, the sugar-free culture medium and the water-soluble Fenton reagent comprises the following steps:
dissolving GOx and a water-soluble Fenton reagent in a sugar-free culture medium, and combining metal ions in the water-soluble Fenton reagent and the GOx through physical adsorption to prepare a GOx compound solution;
or dissolving the modified GOx and a water-soluble Fenton reagent in a sugar-free culture medium, and combining metal ions in the water-soluble Fenton reagent and the modified GOx through physical adsorption to prepare a GOx compound solution;
the preparation of the modified GOx comprises the following steps:
mixing GOx, an activating agent and amino-polyethylene glycol to perform amidation reaction on the amino-polyethylene glycol and the GOx to prepare modified GOx;
the step of adding calcium salt into the GOx compound solution to react to prepare the nano diagnosis and treatment agent comprises the following steps:
and adding a calcium salt into the GOx compound solution, so that phosphate ions in the GOx compound solution react with calcium ions in the calcium salt to generate calcium phosphate on the surface of the GOx compound, and thus the nano diagnosis and treatment agent is prepared.
2. The method for preparing a nano diagnostic and therapeutic agent as defined in claim 1, wherein the activating agent is 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide.
3. The method for preparing a nano diagnostic and therapeutic agent according to claim 1, wherein the mass ratio of the amino-polyethylene glycol to the GOx is 1-10.
4. The method for preparing a nano diagnostic and therapeutic agent according to claim 1, wherein the amino-polyethylene glycol has a molecular weight of 500 to 20000.
5. The nano diagnostic and therapeutic agent obtained by the preparation method according to any one of claims 1 to 4, comprising GOx complex and calcium phosphate coated on the surface of the GOx complex, wherein the GOx complex comprises GOx and metal ions in water-soluble Fenton reagent bound to the GOx by physical adsorption; alternatively, the GOx complex comprises a modified GOx and a metal ion in a water-soluble fenton's reagent bound to the modified GOx by physisorption.
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| CN113398095A (en) * | 2021-05-19 | 2021-09-17 | 深圳大学 | Calcium phosphate nano diagnosis and treatment agent and preparation method and application thereof |
| CN113499430A (en) * | 2021-06-09 | 2021-10-15 | 深圳大学 | Fenton metal ion doped metal-organic framework material solidified oxidative metabolism enzyme nano diagnosis and treatment agent, preparation method and application |
| CN114146166B (en) * | 2021-11-24 | 2024-02-02 | 北京化工大学 | Multifunctional diagnosis and treatment integrated nano composite probe and preparation method and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108210931A (en) * | 2016-12-15 | 2018-06-29 | 深圳大学 | Nanometer diagnosis and treatment agent, preparation method and application |
Family Cites Families (4)
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| US20080241256A1 (en) * | 2007-03-30 | 2008-10-02 | Liisa Kuhn | Targeted active agent delivery system based on calcium phosphate nanoparticles |
| US20110135577A1 (en) * | 2009-12-03 | 2011-06-09 | National Taiwan University | Superparamagnetic nanoparticles IN MEDICAL THERAPEUTICS and manufacturing method THEREOF |
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-
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Non-Patent Citations (3)
| Title |
|---|
| "Catalytic chemistry of glucose oxidase in cancer diagnosis and treatment";Lian-Hua Fu，et al;《Chem. Soc. Rev.》;20180828;第47卷(第17期);摘要 * |
| "In Situ Growth of a Cationic Polymer from the N‑Terminus of Glucose Oxidase To Regulate H2O2 Generation for Cancer Starvation and H2O2 Therapy";Hanjun Hao，et al;《ACS APPL. Mater.interfaces》;20190227;第11卷(第10期);摘要 * |
| "Multifunctional Shell–Core Nanoparticles for Treatment of Multidrug Resistance Hepatocellular Carcinoma";Qi Wang，et al;《Adv. Funct. Mater.》;20181231;第28卷;第1706124页 * |
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