CN110743012A - Preparation method and application of glucose oxidase modified mesoporous manganese dioxide pharmaceutical composition - Google Patents
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Abstract
The invention relates to a preparation method and application of a glucose oxidase modified mesoporous manganese dioxide medicinal composition, which effectively solve the problems that the preparation of the glucose oxidase modified mesoporous manganese dioxide medicinal composition realizes the synergistic inhibition of tumor cell growth by the acoustic kinetic treatment and the hunger therapy, and provides a new medicament for treating tumor diseases. Modifying the surface of the mesoporous manganese dioxide nanoparticles by glucose oxidase through an amido bond reaction, and then physically loading a small-molecular sound-sensitive agent in a mesoporous manganese dioxide pore channel to form a mesoporous manganese dioxide nanoparticle pharmaceutical composition with the interior loaded with the sound-sensitive agent and the exterior covalently modified by the glucose oxidase; the particle size of the mesoporous manganese dioxide nanoparticles is 100-300 nm; the mass ratio of the glucose oxidase to the manganese dioxide is 1: 5, the preparation method is stable and reliable, the cost is low, and the glucose oxidase modified mesoporous manganese dioxide pharmaceutical composition can be used as a magnetic resonance imaging contrast agent for monitoring tumors in real time, and is an innovation in tumor treatment medicines.
Description
Technical Field
The invention belongs to the field of medicines, and relates to a preparation method and application of a glucose oxidase modified mesoporous manganese dioxide medicinal composition.
Background
In recent years, sonodynamic therapy (SDT), which is a preferred treatment modality developed on the basis of photodynamic therapy (PDT), has become an emerging means for treating tumors. Compared with PDT, SDT has the characteristics of deep tissue penetration, no wound, low toxicity to normal tissues and the like. SDT refers to administering a certain dose of sound-sensitive agent to a patient, under the excitation of ultrasonic waves with certain frequency and intensity, the sound-sensitive agent transits from a ground state to an excited state, and then energy is released when the excited state returns to the ground state and is transferred to oxygen molecules to generate Reactive Oxygen Species (ROS), so that tumor cells are irreversibly damaged, and the purpose of treating tumors is achieved. It follows that SDT is oxygen dependent, however, compared to normal tissue, tumor sites present a hypoxic environment, and thus increasing oxygen content in tumor tissue is a key factor in increasing the effect of SDT.
Manganese dioxide nanoparticles are used as an in-situ oxygen generation material, and have catalase-like activity, so that hydrogen peroxide can be catalytically oxidized to generate O2And Mn2+Ions. Based on the tumor acidic microenvironment, the manganese dioxide nanoparticles are expected to pass through O2Self-supporting property and Mn2+T of1Weighted Magnetic Resonance Imaging (MRI) pairSpecific properties to amplify the effect of sonodynamic therapy. It is worth noting that the hollow manganese dioxide nanoparticles with mesoporous pore canals and high specific surface area can be used as nano-carriers to improve the loading capacity of the small-molecule sonosensitizer. However, the limited hydrogen peroxide content in tumor cells limits the oxygen production levels of manganese dioxide. Therefore, increasing the intracellular hydrogen peroxide level is an important point in solving this problem.
Glucose oxidase (GOx), a natural oxidoreductase, has been extensively studied in the biomedical field. GOx can consume oxygen, catalyzing the oxidation of glucose to hydrogen peroxide and gluconic acid. Based on the method, GOx is modified on the surface of the hollow manganese dioxide nanoparticles or circulation supplement of oxygen and hydrogen peroxide can be formed in a permitted way, and cascade catalytic treatment is successfully realized. In addition, this approach can improve the stability of GOx, preventing it from being degraded during the blood circulation. In general, unlike the oxidative phosphorylation of normal tissues, tumor cells have aerobic glycolysis as the main energy supply mode and are strongly dependent on glucose. Therefore, the mesoporous manganese dioxide pharmaceutical composition based on GOx modification tends to consume glucose, block energy supply of cells, and further 'starve' the cells to promote apoptosis.
Modifying GOx on the surface of the mesoporous manganese dioxide nanoparticles through amide bonds, and physically loading a small-molecular sound-sensitive agent in pore channels of the mesoporous manganese dioxide nanoparticles to obtain the glucose oxidase-modified mesoporous manganese dioxide pharmaceutical composition. GOx effectively oxidizes glucose to gluconic acid and hydrogen peroxide, which "starves" tumor cells that rely on glucose for energy. Meanwhile, manganese dioxide catalyzes hydrogen peroxide to generate oxygen, so that the efficiency of the acoustodynamic therapy can be greatly improved, and the manganese dioxide serving as a magnetic resonance imaging contrast agent can be used for positioning tumors and can be used for monitoring in real time in the therapy process. Therefore, the glucose oxidase modified mesoporous manganese dioxide medicinal composition has great significance and value in tumor diagnosis and treatment, but has no public report until now.
Disclosure of Invention
In view of the above situation, in order to overcome the defects of the prior art, the present invention aims to provide a preparation method and an application of a glucose oxidase modified mesoporous manganese dioxide pharmaceutical composition, which can effectively solve the problems of preparation of the glucose oxidase modified mesoporous manganese dioxide pharmaceutical composition, realization of acoustic dynamic therapy and starvation therapy for synergistically inhibiting tumor cell growth, and provision of a new drug for treating tumor diseases.
According to the technical scheme, glucose oxidase is modified on the surface of mesoporous manganese dioxide nanoparticles through an amido bond reaction, and then a micromolecular sound-sensitive agent is physically loaded in a mesoporous manganese dioxide pore passage to form a mesoporous manganese dioxide nanoparticle drug composition with the interior loaded with the sound-sensitive agent and the exterior covalently modified by the glucose oxidase; the particle size of the mesoporous manganese dioxide nanoparticles is 100-300 nm; the mass ratio of the glucose oxidase to the manganese dioxide is 1: 5, and the specific preparation method comprises the following steps:
(1) preparing mesoporous manganese dioxide nanoparticles: ultrasonically dissolving 7.5-8.0g of sodium dodecyl benzene sulfonate into 90-110mL of ultrapure water to obtain a dodecyl benzene sulfonic acid solution; dissolving 1.3-1.5g of sodium bicarbonate in 12-18mL of ultrapure water to prepare a colorless transparent solution; 1-2g of MnCl2·4H2Dissolving O in 6-12mL of ultrapure water to prepare a light pink transparent solution; stirring the dodecyl benzene sulfonic acid solution in 50 deg.C oil bath for 5-10min, adding sodium bicarbonate solution, stirring for 30-40min, and dropwise adding MnCl2·4H2Stirring the O solution for 3-4h, centrifuging at 10000-;
(2) preparing an aminated manganese dioxide compound: ultrasonically dispersing 15-25mg of mesoporous manganese dioxide powder by using 7-9mL of absolute ethanol, adding 70-90 mu L of 3-aminopropyltriethoxysilane, 0.3-0.5mL of ultrapure water and 0.3-0.5mL of concentrated ammonia water, stirring for 10h at room temperature, centrifuging to obtain a precipitate, washing the precipitate to be neutral by using the absolute ethanol, drying at 50-60 ℃, taking out the precipitate, grinding the precipitate into powder, and obtaining an aminated manganese dioxide compound;
(3) preparing the glucose oxidase modified mesoporous manganese dioxide nanoparticles: dissolving 8-12mg of glucose oxidase in 1-3mL of morpholine ethanesulfonic acid monohydrate buffer solution to prepare bright yellow transparent liquid; dissolving 7-9mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide in 0.5-1.5mL of morpholine ethanesulfonic acid monohydrate buffer solution to obtain a 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide solution; dissolving 5.5-6.5mg of N-hydroxysuccinimide in 0.5-1.5mL of morpholine ethanesulfonic acid buffer solution to obtain N-hydroxysuccinimide solution; dissolving 4.5-5.5mg of aminated manganese dioxide in 1-3mL of morpholine ethanesulfonic acid monohydrate buffer solution to obtain an aminated manganese dioxide solution; adding a 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide solution and an N-hydroxysuccinimide solution into a glucose oxidase solution, stirring for 25-30min at room temperature, adding an aminated manganese dioxide solution, stirring for reaction for 4-6h at room temperature, centrifuging to obtain a precipitate, washing the precipitate for 3-5 times with a phosphate buffer solution, drying at 30-40 ℃, taking out and grinding into powder to obtain glucose oxidase modified mesoporous manganese dioxide nanoparticles;
(4) preparing a glucose oxidase modified mesoporous manganese dioxide medicinal composition: dissolving 5-7mg of micromolecular sonosensitizer in 1-3mL of absolute ethyl alcohol; super-dispersing 5-7mg of glucose oxidase modified mesoporous manganese dioxide nanoparticles in 5-7mL of ultrapure water, mixing the two, stirring at room temperature for 24-48h, centrifuging at 10000-;
the micromolecule sonosensitizer is one of chlorin, metal phthalocyanine and hematoporphyrin monomethyl ether of porphyrin compounds.
The glucose oxidase modified mesoporous manganese dioxide pharmaceutical composition is an internal loaded sound sensitive agent, and external covalent modification is carried out on mesoporous manganese dioxide nanoparticles of glucose oxidase.
The glucose oxidase modified mesoporous manganese dioxide pharmaceutical composition is applied to the preparation of a pharmaceutical injection, an oral agent or an implanted pharmaceutical agent for treating tumors.
The glucose oxidase modified mesoporous manganese dioxide pharmaceutical composition can synergistically inhibit the growth of tumor cells in the acoustic kinetic treatment and the hunger therapy, wherein the degraded Mn2+Can be used as the contrast agent for the tumor location imaging by magnetic resonance.
The preparation method is stable and reliable, the cost is low, the prepared glucose oxidase modified mesoporous manganese dioxide pharmaceutical composition can realize the cooperative treatment of hunger therapy and acoustic dynamic therapy in the aspect of preparing anti-tumor drugs, and can be used as a magnetic resonance imaging contrast agent to monitor tumors in real time, so that the preparation method is an innovation in tumor treatment drugs, and has great economic and social benefits.
Detailed Description
The following examples and specific examples will explain the present invention in detail.
The invention may be embodied in the form of the following examples.
Example 1
The invention relates to a preparation method of a glucose oxidase modified mesoporous manganese dioxide medicinal composition, which comprises the following steps:
(1) preparing mesoporous manganese dioxide nanoparticles: ultrasonically dissolving 7.8g of sodium dodecyl benzene sulfonate in 100mL of ultrapure water to obtain a dodecyl benzene sulfonic acid solution; 1.4g of sodium bicarbonate is dissolved in 15mL of ultrapure water to prepare a colorless transparent solution; 1.5g of MnCl2·4H2Dissolving O in 9mL of ultrapure water to prepare a light pink transparent solution; stirring dodecyl benzene sulfonic acid solution in 50 deg.C oil bath for 7min, adding sodium bicarbonate solution, stirring for 35min, and dropwise adding MnCl2·4H2Stirring the O solution for 3.5h, centrifuging at 13000rpm for 10min, taking a precipitate, washing the precipitate with ultrapure water for 4 times, then washing with absolute ethyl alcohol for 4 times, drying at 55 ℃ for 24h, grinding, and then putting into a muffle furnace to calcine at 400 ℃ for 6h to obtain black solid powder, namely the mesoporous manganese dioxide nanoparticles;
(2) preparing an aminated manganese dioxide compound: ultrasonically dispersing 20mg of mesoporous manganese dioxide powder by using 8mL of absolute ethyl alcohol, adding 80 mu L of 3-aminopropyltriethoxysilane, 0.4mL of ultrapure water and 0.4mL of strong ammonia water, stirring for 10h at room temperature, centrifuging to obtain a precipitate, washing the precipitate to be neutral by using the absolute ethyl alcohol, drying at 55 ℃, taking out the precipitate, and grinding the precipitate into powder to obtain an aminated manganese dioxide compound;
(3) preparing the glucose oxidase modified mesoporous manganese dioxide nanoparticles: dissolving 10mg of glucose oxidase in 2mL of morpholine ethanesulfonic acid monohydrate buffer solution to prepare bright yellow transparent liquid; dissolving 8mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide in 1mL of morpholine ethanesulfonic acid monohydrate buffer solution to obtain a 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide solution; dissolving 6mg of N-hydroxysuccinimide in 1mL of morpholine ethanesulfonic acid monohydrate buffer solution to obtain an N-hydroxysuccinimide solution; dissolving 5mg of aminated manganese dioxide in 2mL of morpholine ethanesulfonic acid monohydrate buffer solution to obtain an aminated manganese dioxide solution; adding a 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide solution and an N-hydroxysuccinimide solution into a glucose oxidase solution, stirring for 28min at room temperature, adding an aminated manganese dioxide solution, stirring for reaction for 5h at room temperature, centrifuging to obtain a precipitate, washing the precipitate for 4 times by using a phosphate buffer solution, drying at 35 ℃, taking out and grinding into powder to obtain glucose oxidase modified mesoporous manganese dioxide nanoparticles;
(4) preparing a glucose oxidase modified mesoporous manganese dioxide medicinal composition: dissolving 6mg of micromolecular sonosensitizer hematoporphyrin monomethyl ether or chlorin in 2mL of absolute ethyl alcohol; ultra-dispersing 6mg of glucose oxidase modified mesoporous manganese dioxide nanoparticles into 6mL of ultra-pure water, mixing the two, stirring at room temperature for 36h, centrifuging at 12000rpm for 8min to obtain a precipitate, re-dissolving the precipitate by using an absolute ethanol-water mixed solution with a volume ratio of 1: 1, repeating the steps for 2 times, and freeze-drying to obtain the glucose oxidase modified mesoporous manganese dioxide pharmaceutical composition.
Example 2
The invention relates to a preparation method of a glucose oxidase modified metal organic framework pharmaceutical composition, which is realized by the following steps:
(1) preparing mesoporous manganese dioxide nanoparticles: balanceTaking 7.75g of sodium dodecyl benzene sulfonate, and ultrasonically dissolving the sodium dodecyl benzene sulfonate in 100mL of ultrapure water; weighing 1.4g of sodium bicarbonate, and dissolving in 15mL of ultrapure water to prepare a colorless transparent solution; 1.5g of MnCl are weighed2·4H2Dissolving O in 9mL of ultrapure water to prepare a light pink transparent solution; placing the dodecyl benzene sulfonic acid solution in 50 deg.C oil bath, stirring for 7.5min, adding sodium bicarbonate solution, stirring for 35min, and dropwise adding prepared MnCl2·4H2Stirring the O solution for 3.5h, centrifuging at 12500rpm for 10min, taking a precipitate, washing the precipitate with ultrapure water for 4 times, then washing with absolute ethyl alcohol for 4 times, drying at 55 ℃ for 24h, grinding, and calcining in a muffle furnace at 400 ℃ for 6h to obtain black solid powder, namely the mesoporous manganese dioxide nanoparticles;
(2) preparing an aminated manganese dioxide compound: taking 20mg mesoporous manganese dioxide powder, ultrasonically dispersing with 8mL of absolute ethanol, adding 80 mu L of 3-aminopropyltriethoxysilane, 0.4mL of ultrapure water and 0.4mL of concentrated ammonia water, stirring for 10h at room temperature, centrifuging to obtain a precipitate, washing the precipitate with absolute ethanol to be neutral, drying at 55 ℃, taking out the precipitate, and grinding the precipitate into powder to obtain an aminated manganese dioxide compound;
(3) preparing the glucose oxidase modified mesoporous manganese dioxide nanoparticles: dissolving 10mg of glucose oxidase in 2mL of morpholine ethanesulfonic acid monohydrate buffer solution to prepare bright yellow transparent liquid; 8mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide was dissolved in 1mL of morpholine ethanesulfonic acid monohydrate buffer; 6.0mg of N-hydroxysuccinimide is dissolved in 1.0mL of morpholine ethanesulfonic acid monohydrate buffer solution; dissolving 5.0mg of aminated manganese dioxide in 2mL of morpholine ethanesulfonic acid monohydrate buffer solution; adding a 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide solution and an N-hydroxysuccinimide solution into a glucose oxidase solution, stirring at room temperature for 27min, adding an aminated manganese dioxide solution, and stirring at room temperature for reacting for 5 h; centrifuging to obtain precipitate, washing with phosphate buffer solution for 4 times, drying at 35 deg.C, taking out, and grinding into powder to obtain glucose oxidase modified mesoporous manganese dioxide nanoparticles;
(4) preparing a glucose oxidase modified mesoporous manganese dioxide medicinal composition: dissolving 6mg of micromolecular sonosensitizer metal phthalocyanine in 2mL of absolute ethyl alcohol; ultra-dispersing 6mg of glucose oxidase modified mesoporous manganese dioxide nanoparticles into 6mL of ultra-pure water, mixing the two, stirring at room temperature for 36h, centrifuging at 12500rpm for 7min to obtain a precipitate, re-dissolving the precipitate by using an absolute ethanol-water mixed solution with the volume ratio of 1: 1, repeating the steps for 2-3 times, and freeze-drying to obtain the glucose oxidase modified mesoporous manganese dioxide pharmaceutical composition.
Example 3
The invention relates to a preparation method of a glucose oxidase modified metal organic framework pharmaceutical composition, which is realized by the following steps:
(1) preparing mesoporous manganese dioxide nanoparticles: weighing 7.5g of sodium dodecyl benzene sulfonate, and dissolving the sodium dodecyl benzene sulfonate in 90mL of ultrapure water by ultrasonic; weighing 1.3g of sodium bicarbonate, and dissolving in 12mL of ultrapure water to prepare a colorless transparent solution; weighing 1g of MnCl2·4H2Dissolving O in 6mL of ultrapure water to prepare a light pink transparent solution; stirring the dodecyl benzene sulfonic acid solution in 50 deg.C oil bath for 5min, adding sodium bicarbonate solution, stirring for 30min, and dropwise adding MnCl2·4H2Stirring the O solution for 3 hours, centrifuging at 10000rpm for 10min, taking a precipitate, washing the precipitate with ultrapure water for 3 times, then washing with absolute ethyl alcohol for 3 times, drying at 50 ℃ for 24 hours, grinding, and calcining in a muffle furnace at 400 ℃ for 5 hours to obtain black solid powder, namely the mesoporous manganese dioxide nanoparticles;
(2) preparing an aminated manganese dioxide compound: taking 15mg of the prepared mesoporous manganese dioxide powder, ultrasonically dispersing the powder by using 7mL of absolute ethyl alcohol, adding 70 mu L of 3-aminopropyltriethoxysilane, 0.3mL of ultrapure water and 0.3mL of concentrated ammonia water into the powder, stirring the mixture at room temperature for 10 hours, centrifuging the mixture to obtain a precipitate, washing the precipitate to be neutral by using the absolute ethyl alcohol, drying the precipitate at 50 ℃, taking out the precipitate, and grinding the precipitate into powder to obtain an aminated manganese dioxide compound;
(3) preparing the glucose oxidase modified mesoporous manganese dioxide nanoparticles: dissolving 8mg of glucose oxidase in 1mL of morpholine ethanesulfonic acid monohydrate buffer solution to prepare bright yellow transparent liquid; dissolving 7mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide in 0.5mL of morpholine ethanesulfonic acid monohydrate buffer; dissolving 5.5mg of N-hydroxysuccinimide in 0.5mL of morpholine ethanesulfonic acid monohydrate buffer solution; dissolving 4.5mg of aminated manganese dioxide in 1mL of morpholine ethanesulfonic acid monohydrate buffer solution; adding a 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide solution and an N-hydroxysuccinimide solution into a glucose oxidase solution, stirring for 25min at room temperature, adding an aminated manganese dioxide solution, stirring for reaction for 4h at room temperature, centrifuging to obtain a precipitate, washing for 3 times by using a phosphate buffer solution, drying at 30 ℃, taking out and grinding into powder to obtain glucose oxidase modified mesoporous manganese dioxide nanoparticles;
(4) preparing a glucose oxidase modified mesoporous manganese dioxide medicinal composition: dissolving 5mg of micromolecular sonosensitizer hematoporphyrin monomethyl ether in 1mL of absolute ethyl alcohol; and (2) ultra-dispersing 5mg of mesoporous manganese dioxide nanoparticles modified by glucose oxidase into 5mL of ultra-pure water, mixing the two, stirring at room temperature for 24h, centrifuging at 10000rpm for 5min to obtain a precipitate, re-dissolving the precipitate by using an absolute ethanol-water mixed solution with the volume ratio of 1: 1, repeating the steps for 2-3 times, and freeze-drying to obtain the glucose oxidase-modified mesoporous manganese dioxide pharmaceutical composition.
Example 4
The invention relates to a preparation method of a glucose oxidase modified metal organic framework pharmaceutical composition, which is realized by the following steps:
(1) preparing mesoporous manganese dioxide nanoparticles: weighing 8.0g of sodium dodecyl benzene sulfonate, and ultrasonically dissolving the sodium dodecyl benzene sulfonate in 110mL of ultrapure water; weighing 1.5g of sodium bicarbonate, and dissolving in 18mL of ultrapure water to prepare a colorless transparent solution; weighing 2g of MnCl2·4H2Dissolving O in 12mL of ultrapure water to prepare a light pink transparent solution; stirring the dodecyl benzene sulfonic acid solution in 50 deg.C oil bath for 10min, adding sodium bicarbonate solution, stirring for 40min, and dropwise adding MnCl2·4H2Stirring the O solution for 4 hours, centrifuging at 15000rpm for 10min, taking a precipitate, washing the precipitate with ultrapure water for 5 times, then washing with absolute ethanol for 5 times, drying at 60 ℃ for 24 hours, grinding, and calcining in a muffle furnace at 400 ℃ for 7 hours to obtain black solid powder, namely the mesoporous manganese dioxide nanoparticles;
(2) preparing an aminated manganese dioxide compound: taking 25mg of the prepared mesoporous manganese dioxide powder, ultrasonically dispersing the mesoporous manganese dioxide powder by using 9mL of absolute ethanol, adding 90 mu L of 3-aminopropyltriethoxysilane, 0.5mL of ultrapure water and 0.5mL of concentrated ammonia water into the mesoporous manganese dioxide powder, stirring the mixture at room temperature for 10 hours, centrifuging the mixture to obtain a precipitate, washing the precipitate to be neutral by using the absolute ethanol, drying the precipitate at 60 ℃, taking out the precipitate, and grinding the precipitate into powder to obtain an aminated manganese dioxide compound;
(3) preparing the glucose oxidase modified mesoporous manganese dioxide nanoparticles: dissolving 12mg of glucose oxidase in 3mL of morpholine ethanesulfonic acid monohydrate buffer solution to prepare bright yellow transparent liquid; dissolving 9mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide in 1.5mL of morpholine ethanesulfonic acid monohydrate buffer; 6.5mg of N-hydroxysuccinimide is dissolved in 1.5mL of morpholine ethanesulfonic acid monohydrate buffer solution; dissolving 5.5mg of aminated manganese dioxide in 3mL of morpholine ethanesulfonic acid monohydrate buffer solution; adding a 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide solution and an N-hydroxysuccinimide solution into a glucose oxidase solution, stirring at room temperature for 30min, adding an aminated manganese dioxide solution, and stirring at room temperature for reacting for 6 h; centrifuging to obtain precipitate, washing with phosphate buffer solution for 5 times, drying at 40 deg.C, taking out, and grinding into powder to obtain glucose oxidase modified mesoporous manganese dioxide nanoparticles;
(4) preparing a glucose oxidase modified mesoporous manganese dioxide medicinal composition: dissolving 7mg of micromolecular sonosensitizer chlorin or hematoporphyrin monomethyl ether in 3mL of absolute ethyl alcohol; and (2) ultra-dispersing 7mg of mesoporous manganese dioxide nanoparticles modified by glucose oxidase into 7mL of ultra-pure water, mixing the two, stirring at room temperature for 48h, centrifuging at 15000rpm for 5-10min to obtain a precipitate, re-dissolving the precipitate by using an absolute ethanol-water mixed solution with the volume ratio of 1: 1, repeating the step for 2-3 times, and freeze-drying to obtain the glucose oxidase-modified mesoporous manganese dioxide pharmaceutical composition.
The glucose oxidase modified metal organic framework pharmaceutical composition prepared by the method of examples 1-4 can be effectively used for preparing an anti-tumor drug, wherein the anti-tumor drug is an injection, an oral agent or an implanted agent, and a magnetic resonance imaging contrast agent for tumor localization is prepared, so that the application of the glucose oxidase modified metal organic framework pharmaceutical composition in preparing the anti-tumor drug is realized, and the glucose oxidase modified metal organic framework pharmaceutical composition is used for preparing a tumor treatment drug injection, an oral agent or an implantApplication of the medicine in the acoustic dynamic therapy and the hunger therapy for synergistically inhibiting the growth of tumor cells and degraded Mn2+The magnetic resonance imaging contrast agent is applied to the tumor location imaging contrast agent.
The method is simple, easy to operate and low in cost, the prepared glucose oxidase modified metal organic framework drug composition is internally loaded with the acoustic sensitizer, mesoporous manganese dioxide nanoparticles of the glucose oxidase are covalently modified outside, and field tests show that the prepared glucose oxidase modified metal organic framework drug composition can effectively oxidize glucose into gluconic acid and hydrogen peroxide after reaching a tumor part, so that tumor cells depending on glucose energy supply are starved. Meanwhile, manganese dioxide catalyzes hydrogen peroxide to generate oxygen, so that the efficiency of the acoustodynamic therapy can be greatly improved, and the manganese dioxide serving as a magnetic resonance imaging contrast agent can be used for positioning tumors and can be used for monitoring in real time in the therapy process. The pharmaceutical composition combines the sonodynamic therapy with the hunger therapy, can make up the deficiency of the sonodynamic therapy, can synergistically enhance the treatment effect, is convenient to operate, has stable and reliable method, and has the advantages of high efficiency and controllability compared with the traditional chemotherapy. The data are as follows (example 1 is used as an example):
firstly, characterization experiment of mesoporous manganese dioxide nano composite modified by glucose oxidase loaded with hematoporphyrin monomethyl ether:
1. determination of hematoporphyrin monomethyl ether content in glucose oxidase modified mesoporous manganese dioxide drug composition
The content of hematoporphyrin monomethyl ether is measured at the wavelength of 396nm by adopting an ultraviolet spectrophotometry. The drug loading of the sample was calculated by equation (1). The drug loading rate reaches 32 percent, and the calculation formula is as follows:
2. determination of particle size and potential of hematoporphyrin monomethyl ether-loaded glucose oxidase-modified mesoporous manganese dioxide nanocomposite
A proper amount of mesoporous manganese dioxide Nano composite modified by glucose oxidase loaded with hematoporphyrin monomethyl ether is dispersed in water, and the particle size and the potential of the mesoporous manganese dioxide Nano composite are respectively 290.3 +/-5.8 nm and-18.8 +/-1.7 mV by using a Nano-ZS90 type laser Nano particle size analyzer.
Secondly, an experiment for generating active oxygen in vitro by the mesoporous manganese dioxide nano composite modified by glucose oxidase loaded with hematoporphyrin monomethyl ether:
selecting MCF-7 human breast cancer cells in logarithmic growth phase, adjusting cell number to 3 × 105Inoculating each/mL of the cells in a six-hole culture plate, wherein each hole is 2mL, adding the medicine after the cells grow for 24 hours in an adherent manner, and sequentially adding blank groups and MnO2Group of GOx, HMME/MnO2GOx group and separate ultrasound groups, wherein MnO2Setting the final concentration to be 20 mu g/mL, culturing for 24h, discarding the old culture solution, washing each hole with fresh phosphate buffer salt solution for 2-3 times, adding 1mL of active oxygen probe culture medium containing DCFH-DA to each hole, culturing at 37 ℃ for 30min, washing with fresh phosphate buffer salt solution for 2-3 times, observing under a microscope, and performing HMME ultrasonic group and HMME/MnO2GOx ultrasound groups all detected a higher intensity green DCFH-DA fluorescence signal, consistent with literature reports that HMME as sonosensitizer produced ROS, especially singlet molecular oxygen for effective sonodynamic therapy: (1O2) Thereby achieving the effect of eliminating tumor cells.
And thirdly, a cell uptake experiment of the hematoporphyrin monomethyl ether-loaded glucose oxidase-modified mesoporous manganese dioxide nanocomposite:
selecting MCF-7 human breast cancer cells in logarithmic growth phase, adjusting cell number to 3 × 105Inoculating each/mL of the strain to a 6-well culture plate, wherein each well is 2mL, adding medicines after the strain grows for 24 hours in an adherent manner, and sequentially adding HMME group and HMME/MnO2GOx group, final concentration of drug 10 μ g/mL, incubation time 1h, 4h, after dosing at 37 deg.C, 5% CO2After culturing under the condition, discarding the drug-containing culture medium in the holes, washing each hole for 2-3 times by using 1mL of phosphate buffer saline solution, adding 500 mu L of pancreatin digestive cells without EDTA, adding 1mL of fresh culture medium to stop digestion, blowing and beating until the cells are separated from the wall, transferring the cell suspension into 10mL of centrifugal culture mediumIn a tube, centrifuging to remove supernatant, adding phosphate buffer salt solution for heavy suspension, and detecting by using a flow cytometer, finding MCF-7 human breast cancer cells for 1h to HMME, HMME/MnO2-the uptake of GOx is: 27.18 percent and 39.88 percent; the intake at 4h was: 46.93 percent and 89.12 percent. The results show that HMME/MnO2GOx nano-preparations can carry drugs more and more quickly into cells, and improve the cellular uptake efficiency.
Fourthly, cell proliferation inhibition experiment of the mesoporous manganese dioxide nano composite modified by glucose oxidase loaded with hematoporphyrin monomethyl ether:
adopting SRB method, selecting MCF-7 human breast cancer cells in logarithmic growth phase, and adjusting cell number to 5 × 104Inoculating each/mL of the cells in a 96-well culture plate, wherein each well is 100 μ L (the marginal well is filled with sterile phosphate buffered saline), adding the medicine after the cells grow for 24h in an adherent manner, and sequentially preparing a blank group, a blank + US group, HMME + US group and MnO2Group GOx, MnO2-GOx + US group, HMME/MnO2GOx group and HMME/MnO2-GOx + US group. HMME final concentration was set at 10. mu.g/mL and sonication groups (1W/cm)21min) and non-ultrasound group. Each group is provided with 6 multiple holes. Adding medicine, incubating for 24h, adding 50 μ L of 50% trichloroacetic acid (TCA) pre-cooled at 4 deg.C into each well, fixing for 10min, fixing in a refrigerator at 4 deg.C for 1h, taking out, discarding the fixing solution, washing with deionized water for 5 times, spin-drying, and naturally drying at room temperature. Drying at room temperature, adding 50 μ L of SRB dye solution into each well, standing at room temperature in dark for 15-30 min for dyeing, discarding the dye solution, washing with 1% glacial acetic acid for 5 times, drying at room temperature, dissolving the dye bound with cell protein with 150 μ L of unbuffered Tris alkali solution (10mM, pH 10.5), shaking by shaking table at 37 deg.C, 100rpm, 10min, measuring OD of each small hole at 515nm wavelength of microplate reader, calculating the tumor cell growth inhibition rate (%) (1-OD of experimental group/OD of control group) x 100%, and calculating to obtain blank group, blank + US group, HMME + US group, MnO group2Group GOx, MnO2-GOx + US group, HMME/MnO2GOx group, HMME/MnO2-inhibition of cell growth in the GOx + US groups: 2.1%, 4.7%, 3.1%, 42.5%, 19.8%, 23.4%, 26.7%, 81.6%. The results show that HMME has no obvious cytotoxicity in the absence of ultrasoundBut active oxygen generated under the ultrasonic condition can inhibit the growth of the tumor, and has obvious acoustic dynamic effect; the cytotoxicity of the preparation group is not greatly different from that of the raw medicine group in the absence of ultrasound, and HMME/MnO is adopted in the ultrasound2GOx is most effective in cell killing and is the result of the synergy of starvation therapy and sonodynamic therapy.
And fifthly, pharmacodynamic research experiment of the hematoporphyrin monomethyl ether-loaded glucose oxidase-modified mesoporous manganese dioxide nanocomposite:
purchasing Kunming mice (female, 3-4 weeks old), inoculating S-180 ascites tumor cells to the back of the right upper limb of the mice subcutaneously, measuring the tumor volume 7 days later, and taking 36 tumors with the tumor volume more than or equal to 100mm3And mice with similar tumor volumes and weights were randomized into 6 groups of 6 mice each. The specific grouping is as follows: blank group, blank + US group, HMME + US group, MnO2Group GOx, MnO2-GOx + US group, HMME/MnO2GOx group, HMME/MnO2-GOx + US group. The injection is administered by tail vein once every two days for 7 times. Ultrasonic treatment of tumor part (1W/cm) 6h after ultrasonic group administration21 min). The mice are guaranteed to normally eat every day in the whole experiment process, the mice are weighed every two days, the long diameter (A) and the short diameter (B) of sarcoma of the tumor-bearing mice are measured by using a digital display vernier caliper, and the tumor volume V is defined as A multiplied by B according to a formula2The tumor volume was calculated 2. The recorded data show the blank, blank + US, HMME + US, MnO2Group GOx, MnO2-GOx + US group, HMME/MnO2GOx group, HMME/MnO2The tumor inhibition rates of the GOx + US groups are 3.21%, 3.45%, 5.95%, 41.46%, 34.76%, 36.37%, 39.75% and 87.63%, respectively. The results show that HMME/MnO2The efficacy of the-GOx + US group is remarkable, which shows that HMME/MnO2Sonodynamic therapy of GOx under ultrasound can significantly enhance the therapeutic effect of tumors.
In the experiment, the glucose oxidase modified metal organic framework pharmaceutical compositions prepared by the methods 2, 3 and 4 are tested repeatedly according to the above items, and the same or similar results are obtained, which are not described in detail herein.
Experiments show that compared with the prior art, the invention has the following outstanding beneficial technical effects:
(1) according to the glucose oxidase modified mesoporous manganese dioxide pharmaceutical composition provided by the invention, glucose oxidase consumes glucose at a tumor part to carry out a starvation therapy; with generation of H2O2The mesoporous manganese dioxide nano-particles with catalase-like activity react to generate oxygen, so that the efficiency of the acoustic kinetic treatment is improved.
(2) The glucose oxidase modified mesoporous manganese dioxide pharmaceutical composition provided by the invention can be degraded into Mn in tumor tissues2+Ion, Mn2+Can be used as a magnetic resonance imaging contrast agent to position the tumor, can be monitored in real time during the treatment process, and provides a new direction for the diagnosis and treatment of the tumor.
(3) The glucose oxidase modified mesoporous manganese dioxide medicinal composition provided by the invention can exert an anti-tumor effect only by ultrasonic stimulation, and the safety of normal tissue parts can be ensured due to the lack of ultrasonic stimulation in normal tissues, so that the glucose oxidase modified mesoporous manganese dioxide medicinal composition has a very strong medicinal value, is an innovation on anti-tumor medicaments, and has great economic and social benefits.
Claims (8)
1. A preparation method of a glucose oxidase modified mesoporous manganese dioxide drug composition is characterized in that glucose oxidase is modified on the surface of mesoporous manganese dioxide nanoparticles through an amido bond reaction, then a small molecular sound-sensitive agent is physically loaded in a mesoporous manganese dioxide pore channel to form a mesoporous manganese dioxide nanoparticle drug composition with an internally loaded sound-sensitive agent and an externally covalently modified glucose oxidase; the particle size of the mesoporous manganese dioxide nanoparticles is 100-300 nm; the mass ratio of the glucose oxidase to the manganese dioxide is 1: 5, and the specific preparation method comprises the following steps:
(1) preparing mesoporous manganese dioxide nanoparticles: ultrasonically dissolving 7.5-8.0g of sodium dodecyl benzene sulfonate into 90-110mL of ultrapure water to obtain a dodecyl benzene sulfonic acid solution; dissolving 1.3-1.5g of sodium bicarbonate in 12-18mL of ultrapure water to prepare a colorless transparent solution; will be provided with1-2g of MnCl2·4H2Dissolving O in 6-12mL of ultrapure water to prepare a light pink transparent solution; stirring the dodecyl benzene sulfonic acid solution in 50 deg.C oil bath for 5-10min, adding sodium bicarbonate solution, stirring for 30-40min, and dropwise adding MnCl2·4H2Stirring the O solution for 3-4h, centrifuging at 10000-;
(2) preparing an aminated manganese dioxide compound: ultrasonically dispersing 15-25mg of mesoporous manganese dioxide powder by using 7-9mL of absolute ethanol, adding 70-90 mu L of 3-aminopropyltriethoxysilane, 0.3-0.5mL of ultrapure water and 0.3-0.5mL of concentrated ammonia water, stirring for 10h at room temperature, centrifuging to obtain a precipitate, washing the precipitate to be neutral by using the absolute ethanol, drying at 50-60 ℃, taking out the precipitate, grinding the precipitate into powder, and obtaining an aminated manganese dioxide compound;
(3) preparing the glucose oxidase modified mesoporous manganese dioxide nanoparticles: dissolving 8-12mg of glucose oxidase in 1-3mL of morpholine ethanesulfonic acid monohydrate buffer solution to prepare bright yellow transparent liquid; dissolving 7-9mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide in 0.5-1.5mL of morpholine ethanesulfonic acid monohydrate buffer solution to obtain a 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide solution; dissolving 5.5-6.5mg of N-hydroxysuccinimide in 0.5-1.5mL of morpholine ethanesulfonic acid buffer solution to obtain N-hydroxysuccinimide solution; dissolving 4.5-5.5mg of aminated manganese dioxide in 1-3mL of morpholine ethanesulfonic acid monohydrate buffer solution to obtain an aminated manganese dioxide solution; adding a 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide solution and an N-hydroxysuccinimide solution into a glucose oxidase solution, stirring for 25-30min at room temperature, adding an aminated manganese dioxide solution, stirring for reaction for 4-6h at room temperature, centrifuging to obtain a precipitate, washing the precipitate for 3-5 times with a phosphate buffer solution, drying at 30-40 ℃, taking out and grinding into powder to obtain glucose oxidase modified mesoporous manganese dioxide nanoparticles;
(4) preparing a glucose oxidase modified mesoporous manganese dioxide medicinal composition: dissolving 5-7mg of micromolecular sonosensitizer in 1-3mL of absolute ethyl alcohol; super-dispersing 5-7mg of glucose oxidase modified mesoporous manganese dioxide nanoparticles in 5-7mL of ultrapure water, mixing the two, stirring at room temperature for 24-48h, centrifuging at 10000-;
the micromolecule sonosensitizer is one of chlorin, metal phthalocyanine and hematoporphyrin monomethyl ether of porphyrin compounds.
2. The preparation method of the glucose oxidase-modified mesoporous manganese dioxide pharmaceutical composition according to claim 1, comprising the following steps:
(1) preparing mesoporous manganese dioxide nanoparticles: ultrasonically dissolving 7.8g of sodium dodecyl benzene sulfonate in 100mL of ultrapure water to obtain a dodecyl benzene sulfonic acid solution; 1.4g of sodium bicarbonate is dissolved in 15mL of ultrapure water to prepare a colorless transparent solution; 1.5g of MnCl2·4H2Dissolving O in 9mL of ultrapure water to prepare a light pink transparent solution; stirring dodecyl benzene sulfonic acid solution in 50 deg.C oil bath for 7min, adding sodium bicarbonate solution, stirring for 35min, and dropwise adding MnCl2·4H2Stirring the O solution for 3.5h, centrifuging at 13000rpm for 10min, taking a precipitate, washing the precipitate with ultrapure water for 4 times, then washing with absolute ethyl alcohol for 4 times, drying at 55 ℃ for 24h, grinding, and then putting into a muffle furnace to calcine at 400 ℃ for 6h to obtain black solid powder, namely the mesoporous manganese dioxide nanoparticles;
(2) preparing an aminated manganese dioxide compound: ultrasonically dispersing 20mg of mesoporous manganese dioxide powder by using 8mL of absolute ethyl alcohol, adding 80 mu L of 3-aminopropyltriethoxysilane, 0.4mL of ultrapure water and 0.4mL of strong ammonia water, stirring for 10h at room temperature, centrifuging to obtain a precipitate, washing the precipitate to be neutral by using the absolute ethyl alcohol, drying at 55 ℃, taking out the precipitate, and grinding the precipitate into powder to obtain an aminated manganese dioxide compound;
(3) preparing the glucose oxidase modified mesoporous manganese dioxide nanoparticles: dissolving 10mg of glucose oxidase in 2mL of morpholine ethanesulfonic acid monohydrate buffer solution to prepare bright yellow transparent liquid; dissolving 8mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide in 1mL of morpholine ethanesulfonic acid monohydrate buffer solution to obtain a 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide solution; dissolving 6mg of N-hydroxysuccinimide in 1mL of morpholine ethanesulfonic acid monohydrate buffer solution to obtain an N-hydroxysuccinimide solution; dissolving 5mg of aminated manganese dioxide in 2mL of morpholine ethanesulfonic acid monohydrate buffer solution to obtain an aminated manganese dioxide solution; adding a 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide solution and an N-hydroxysuccinimide solution into a glucose oxidase solution, stirring for 28min at room temperature, adding an aminated manganese dioxide solution, stirring for reaction for 5h at room temperature, centrifuging to obtain a precipitate, washing the precipitate for 4 times by using a phosphate buffer solution, drying at 35 ℃, taking out and grinding into powder to obtain glucose oxidase modified mesoporous manganese dioxide nanoparticles;
(4) preparing a glucose oxidase modified mesoporous manganese dioxide medicinal composition: dissolving 6mg of micromolecular sonosensitizer hematoporphyrin monomethyl ether or chlorin in 2mL of absolute ethyl alcohol; ultra-dispersing 6mg of glucose oxidase modified mesoporous manganese dioxide nanoparticles into 6mL of ultra-pure water, mixing the two, stirring at room temperature for 36h, centrifuging at 12000rpm for 8min to obtain a precipitate, re-dissolving the precipitate by using an absolute ethanol-water mixed solution with a volume ratio of 1: 1, repeating the steps for 2 times, and freeze-drying to obtain the glucose oxidase modified mesoporous manganese dioxide pharmaceutical composition.
3. The method for preparing a glucose oxidase-modified metal organic framework pharmaceutical composition according to claim 1, which is achieved by the following steps:
(1) preparing mesoporous manganese dioxide nanoparticles: weighing 7.75g of sodium dodecyl benzene sulfonate, and dissolving the sodium dodecyl benzene sulfonate in 100mL of ultrapure water by ultrasonic; weighing 1.4g of sodium bicarbonate, and dissolving in 15mL of ultrapure water to prepare a colorless transparent solution; 1.5g of MnCl are weighed2·4H2Dissolving O in 9mL of ultrapure water to prepare a light pink transparent solution; stirring the dodecyl benzene sulfonic acid solution in 50 deg.C oil bath for 7.5min, adding sodium bicarbonate solution, stirring for 35min, and adding dropwiseMnCl2·4H2Stirring the O solution for 3.5h, centrifuging at 12500rpm for 10min, taking a precipitate, washing the precipitate with ultrapure water for 4 times, then washing with absolute ethyl alcohol for 4 times, drying at 55 ℃ for 24h, grinding, and calcining in a muffle furnace at 400 ℃ for 6h to obtain black solid powder, namely the mesoporous manganese dioxide nanoparticles;
(2) preparing an aminated manganese dioxide compound: taking 20mg mesoporous manganese dioxide powder, ultrasonically dispersing with 8mL of absolute ethanol, adding 80 mu L of 3-aminopropyltriethoxysilane, 0.4mL of ultrapure water and 0.4mL of concentrated ammonia water, stirring for 10h at room temperature, centrifuging to obtain a precipitate, washing the precipitate with absolute ethanol to be neutral, drying at 55 ℃, taking out the precipitate, and grinding the precipitate into powder to obtain an aminated manganese dioxide compound;
(3) preparing the glucose oxidase modified mesoporous manganese dioxide nanoparticles: dissolving 10mg of glucose oxidase in 2mL of morpholine ethanesulfonic acid monohydrate buffer solution to prepare bright yellow transparent liquid; 8mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide was dissolved in 1mL of morpholine ethanesulfonic acid monohydrate buffer; 6.0mg of N-hydroxysuccinimide is dissolved in 1.0mL of morpholine ethanesulfonic acid monohydrate buffer solution; dissolving 5.0mg of aminated manganese dioxide in 2mL of morpholine ethanesulfonic acid monohydrate buffer solution; adding a 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide solution and an N-hydroxysuccinimide solution into a glucose oxidase solution, stirring at room temperature for 27min, adding an aminated manganese dioxide solution, and stirring at room temperature for reacting for 5 h; centrifuging to obtain precipitate, washing with phosphate buffer solution for 4 times, drying at 35 deg.C, taking out, and grinding into powder to obtain glucose oxidase modified mesoporous manganese dioxide nanoparticles;
(4) preparing a glucose oxidase modified mesoporous manganese dioxide medicinal composition: dissolving 6mg of micromolecular sonosensitizer metal phthalocyanine in 2mL of absolute ethyl alcohol; ultra-dispersing 6mg of glucose oxidase modified mesoporous manganese dioxide nanoparticles into 6mL of ultra-pure water, mixing the two, stirring at room temperature for 36h, centrifuging at 12500rpm for 7min to obtain a precipitate, re-dissolving the precipitate by using an absolute ethanol-water mixed solution with the volume ratio of 1: 1, repeating the steps for 2-3 times, and freeze-drying to obtain the glucose oxidase modified mesoporous manganese dioxide pharmaceutical composition.
4. The method for preparing a glucose oxidase-modified metal organic framework pharmaceutical composition according to claim 1, which is achieved by the following steps:
(1) preparing mesoporous manganese dioxide nanoparticles: weighing 7.5g of sodium dodecyl benzene sulfonate, and dissolving the sodium dodecyl benzene sulfonate in 90mL of ultrapure water by ultrasonic; weighing 1.3g of sodium bicarbonate, and dissolving in 12mL of ultrapure water to prepare a colorless transparent solution; weighing 1g of MnCl2·4H2Dissolving O in 6mL of ultrapure water to prepare a light pink transparent solution; stirring the dodecyl benzene sulfonic acid solution in 50 deg.C oil bath for 5min, adding sodium bicarbonate solution, stirring for 30min, and dropwise adding MnCl2·4H2Stirring the O solution for 3 hours, centrifuging at 10000rpm for 10min, taking a precipitate, washing the precipitate with ultrapure water for 3 times, then washing with absolute ethyl alcohol for 3 times, drying at 50 ℃ for 24 hours, grinding, and calcining in a muffle furnace at 400 ℃ for 5 hours to obtain black solid powder, namely the mesoporous manganese dioxide nanoparticles;
(2) preparing an aminated manganese dioxide compound: taking 15mg of the prepared mesoporous manganese dioxide powder, ultrasonically dispersing the powder by using 7mL of absolute ethyl alcohol, adding 70 mu L of 3-aminopropyltriethoxysilane, 0.3mL of ultrapure water and 0.3mL of concentrated ammonia water into the powder, stirring the mixture at room temperature for 10 hours, centrifuging the mixture to obtain a precipitate, washing the precipitate to be neutral by using the absolute ethyl alcohol, drying the precipitate at 50 ℃, taking out the precipitate, and grinding the precipitate into powder to obtain an aminated manganese dioxide compound;
(3) preparing the glucose oxidase modified mesoporous manganese dioxide nanoparticles: dissolving 8mg of glucose oxidase in 1mL of morpholine ethanesulfonic acid monohydrate buffer solution to prepare bright yellow transparent liquid; dissolving 7mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide in 0.5mL of morpholine ethanesulfonic acid monohydrate buffer; dissolving 5.5mg of N-hydroxysuccinimide in 0.5mL of morpholine ethanesulfonic acid monohydrate buffer solution; dissolving 4.5mg of aminated manganese dioxide in 1mL of morpholine ethanesulfonic acid monohydrate buffer solution; adding a 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide solution and an N-hydroxysuccinimide solution into a glucose oxidase solution, stirring for 25min at room temperature, adding an aminated manganese dioxide solution, stirring for reaction for 4h at room temperature, centrifuging to obtain a precipitate, washing for 3 times by using a phosphate buffer solution, drying at 30 ℃, taking out and grinding into powder to obtain glucose oxidase modified mesoporous manganese dioxide nanoparticles;
(4) preparing a glucose oxidase modified mesoporous manganese dioxide medicinal composition: dissolving 5mg of micromolecular sonosensitizer hematoporphyrin monomethyl ether in 1mL of absolute ethyl alcohol; and (2) ultra-dispersing 5mg of mesoporous manganese dioxide nanoparticles modified by glucose oxidase into 5mL of ultra-pure water, mixing the two, stirring at room temperature for 24h, centrifuging at 10000rpm for 5min to obtain a precipitate, re-dissolving the precipitate by using an absolute ethanol-water mixed solution with the volume ratio of 1: 1, repeating the steps for 2-3 times, and freeze-drying to obtain the glucose oxidase-modified mesoporous manganese dioxide pharmaceutical composition.
5. The method for preparing a glucose oxidase-modified metal organic framework pharmaceutical composition according to claim 1, which is achieved by the following steps:
(1) preparing mesoporous manganese dioxide nanoparticles: weighing 8.0g of sodium dodecyl benzene sulfonate, and ultrasonically dissolving the sodium dodecyl benzene sulfonate in 110mL of ultrapure water; weighing 1.5g of sodium bicarbonate, and dissolving in 18mL of ultrapure water to prepare a colorless transparent solution; weighing 2g of MnCl2·4H2Dissolving O in 12mL of ultrapure water to prepare a light pink transparent solution; stirring the dodecyl benzene sulfonic acid solution in 50 deg.C oil bath for 10min, adding sodium bicarbonate solution, stirring for 40min, and dropwise adding MnCl2·4H2Stirring the O solution for 4 hours, centrifuging at 15000rpm for 10min, taking a precipitate, washing the precipitate with ultrapure water for 5 times, then washing with absolute ethanol for 5 times, drying at 60 ℃ for 24 hours, grinding, and calcining in a muffle furnace at 400 ℃ for 7 hours to obtain black solid powder, namely the mesoporous manganese dioxide nanoparticles;
(2) preparing an aminated manganese dioxide compound: taking 25mg of the prepared mesoporous manganese dioxide powder, ultrasonically dispersing the mesoporous manganese dioxide powder by using 9mL of absolute ethanol, adding 90 mu L of 3-aminopropyltriethoxysilane, 0.5mL of ultrapure water and 0.5mL of concentrated ammonia water into the mesoporous manganese dioxide powder, stirring the mixture at room temperature for 10 hours, centrifuging the mixture to obtain a precipitate, washing the precipitate to be neutral by using the absolute ethanol, drying the precipitate at 60 ℃, taking out the precipitate, and grinding the precipitate into powder to obtain an aminated manganese dioxide compound;
(3) preparing the glucose oxidase modified mesoporous manganese dioxide nanoparticles: dissolving 12mg of glucose oxidase in 3mL of morpholine ethanesulfonic acid monohydrate buffer solution to prepare bright yellow transparent liquid; dissolving 9mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide in 1.5mL of morpholine ethanesulfonic acid monohydrate buffer; 6.5mg of N-hydroxysuccinimide is dissolved in 1.5mL of morpholine ethanesulfonic acid monohydrate buffer solution; dissolving 5.5mg of aminated manganese dioxide in 3mL of morpholine ethanesulfonic acid monohydrate buffer solution; adding a 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide solution and an N-hydroxysuccinimide solution into a glucose oxidase solution, stirring at room temperature for 30min, adding an aminated manganese dioxide solution, and stirring at room temperature for reacting for 6 h; centrifuging to obtain precipitate, washing with phosphate buffer solution for 5 times, drying at 40 deg.C, taking out, and grinding into powder to obtain glucose oxidase modified mesoporous manganese dioxide nanoparticles;
(4) preparing a glucose oxidase modified mesoporous manganese dioxide medicinal composition: dissolving 7mg of micromolecular sonosensitizer chlorin or hematoporphyrin monomethyl ether in 3mL of absolute ethyl alcohol; and (2) ultra-dispersing 7mg of mesoporous manganese dioxide nanoparticles modified by glucose oxidase into 7mL of ultra-pure water, mixing the two, stirring at room temperature for 48h, centrifuging at 15000rpm for 5-10min to obtain a precipitate, re-dissolving the precipitate by using an absolute ethanol-water mixed solution with the volume ratio of 1: 1, repeating the step for 2-3 times, and freeze-drying to obtain the glucose oxidase-modified mesoporous manganese dioxide pharmaceutical composition.
6. Use of a glucose oxidase modified mesoporous manganese dioxide pharmaceutical composition prepared by the method of any one of claims 1 or 2-5 for the preparation of an anti-tumor medicament.
7. Use of glucose oxidase modified mesoporous manganese dioxide pharmaceutical composition prepared by the method of any one of claims 1 or 2-5 in preparation of injection, oral agent or implant for treating tumor.
8. The glucose oxidase modified mesoporous manganese dioxide pharmaceutical composition prepared by the method of any one of claims 1 or 2-5 can be used in the acoustic kinetic treatment and the hunger therapySynergistic tumor cell growth inhibiting medicine and degraded Mn2+The magnetic resonance imaging contrast agent is applied to the tumor location imaging contrast agent.
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