CN115245563A - Preparation device and method of m6A demethylase FTO magnetic particles for inhibiting tumor cells - Google Patents
Preparation device and method of m6A demethylase FTO magnetic particles for inhibiting tumor cells Download PDFInfo
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Abstract
The invention discloses a device and a method for preparing m6A demethylase FTO magnetic particles for inhibiting tumor cells, wherein modified ferroferric oxide nanoparticles are obtained by using prepared equipment, so that the complicated steps in the reaction process are reduced, the control of the reaction process is facilitated, the interference of external factors in the reaction process can be avoided, the oxidation of the particles caused by the fact that external air enters the equipment is prevented, the preparation of high-quality magnetic nanoparticles which are more stable, higher in purity and uniform in particle size is facilitated, meanwhile, PEG is selected as a reaction solvent, PVP is selected as a modifier, the reaction temperature in the reaction process, the addition amount of the modifier and the grade type of the added modifier are strictly controlled, the modified magnetic ferroferric oxide nanoparticles which are moderate in size, regular in shape, good in dispersity and high in colloid stability can be prepared, a good basis is provided for the attachment of subsequent protein molecules, and the adsorbed protein molecules are not easy to fall off from the surfaces of the nanoparticles.
Description
Technical Field
The invention relates to the technical field of biosynthesis, in particular to a device and a method for preparing m6A demethylase FTO magnetic particles for inhibiting tumor cells.
Background
The targeted drug delivery system is a new hotspot of research in recent years, connects the drug with the magnetic composite microsphere through chemical action by utilizing the magnetic property and the biological compatibility of the magnetic composite microsphere, then leads the drug to reach a specific area to be released under the action of an external magnetic field, and has the advantages of low toxicity, strong drug effect and the like. Magnetic nanoparticles with an applied magnetic field and/or a magnetizable implant can deliver particles to a target area, immobilizing the particles at a local site upon drug release, and thus the drug can be released locally. Recently, targeted drug delivery using iron oxide magnetic nanoparticles has become more and more feasible. The kernel uses superparamagnetic ferroferric oxide magnetic nano particles, and has the advantages of small diameter, high sensitivity, low toxicity, stable performance and easily obtained raw materials. The iron oxide generally does not produce toxic or side effect on human bodies, the iron content of the carrier used in the whole treatment course does not exceed the total iron supplement amount of the conventional anemia patients, except that part of the carrier is utilized by the human bodies, the rest magnetic particles can be safely discharged out of the human bodies through skins, bile, kidneys and the like, and the iron oxide is an ideal targeted inner core material.
Obesity-related protein (FTO) is an N6-methyladenosine (m 6A) demethylase located on chromosome 16q12.2, and previous studies have demonstrated that FTO can affect obesity by modulating downstream m6A levels through the 3' untranslated region. With the continuous and intensive research, researchers find that the epigenetic modification of m6A methylation can regulate the generation and development of tumors by regulating the expression level of tumor genes and mRNA molecules of tumor inhibiting genes. FTO is used as an important component of m6A modification and is involved in regulating and controlling the occurrence, development and prognosis of various tumors. The current research shows that FTO can participate in regulating and controlling the occurrence and development of various tumors through different modes (influencing the growth and proliferation of tumor cells, inhibiting cell differentiation, interfering the self-renewal of tumor stem cells, influencing the tumor metastasis, the sensitivity of radiotherapy and chemotherapy and the like). Therefore, FTO is expected to become a new target point for diagnosing and treating tumors in the near future, and particularly aims at certain specific types of tumors, such as acute myelogenous leukemia, glioblastoma, breast cancer and the like, so that the FTO has important theoretical significance and application value for diagnosis and treatment of tumors.
Widder K J et al propose a magnetic reactive protein microsphere in "test method for antitumor therapy" to use denatured human serum albumin as the substrate, to encapsulate the ultramicron of anticancer drugs adriamycin and ferroferric oxide, in carrying out targeted Jitian sarcoma in vivo research shows that the magnetic reactive protein microsphere may have more significant clinical relevance in treating tumor.
However, the following problems are involved in the preparation of m6A demethylase FTO magnetic particles using the above method: 1) In the preparation process of the ferroferric oxide magnetic nanoparticles, the ferroferric oxide is easily oxidized by oxygen, so the preparation process needs to be carried out in an oxygen-free environment, argon is generally introduced, but the existing reaction vessel has poor tightness, the preparation process is difficult to be ensured to be always in the oxygen-free environment, and the particle purity is reduced; 2) The prepared ferroferric oxide magnetic nanoparticles have small particle size, large specific surface area, strong aggregation tendency, poor biocompatibility and are not beneficial to coating of protein molecules, in order to solve the problem, the biocompatibility and the reaction characteristic of the magnetic nanoparticles can be improved by carrying out appropriate surface modification on the magnetic nanoparticles, so that the magnetic nanoparticles have good dispersibility, but the existing modification solvents have more types, the reaction parameter control in the preparation process by adopting different modification solvents has larger difference, and the particle size and the drug loading rate of the ferroferric oxide magnetic nanoparticles can be directly influenced by the factors such as the type of the modification solvents, the parameter control in the reaction and the like.
Disclosure of Invention
The invention mainly aims to provide a device and a method for preparing m6A demethylase FTO magnetic particles for inhibiting tumor cells, wherein modified ferroferric oxide nanoparticles are obtained by using prepared equipment, so that the complicated steps in the reaction process are reduced, the control of the reaction process is facilitated, the interference of external factors in the reaction process can be avoided, the oxidation of particles caused by the fact that external air enters the equipment is prevented, the preparation of high-quality magnetic nanoparticles which are more stable, higher in purity and uniform in particle size is facilitated, meanwhile, PEG is selected as a reaction solvent, PVP is selected as a modifier, the reaction temperature, the addition amount of the modifier and the grade type of the added modifier in the reaction process are strictly controlled, the modified magnetic ferroferric oxide nanoparticles which are good in size, regular in shape and dispersity and high in colloid stability can be prepared, a good foundation is provided for the attachment of subsequent protein molecules, the adsorbed protein molecules are not easy to fall off from the surfaces of the nanoparticles, and the problems in the background technology can be effectively solved.
In order to achieve the purpose, the invention adopts the technical scheme that:
a preparation device and a method for m6A demethylase FTO magnetic particles for inhibiting tumor cells comprise the following steps:
the method comprises the following steps: preparing modified ferroferric oxide magnetic nanoparticles;
the specific process is as follows: accurately weighing a certain amount of PEG and PVP by a balance, transferring into preparation equipment, stirring and mixing at 90 ℃ until the liquid is clear and transparent, then weighing a certain amount of ferric acetylacetonate, adding into the mixed liquid, continuously stirring for 10min at the current temperature (90 ℃), heating to the reaction temperature, continuously reacting for 1h, stopping heating, removing a heat source, cooling to 60 ℃ at room temperature, uniformly stirring in the reaction process, introducing argon to remove oxygen, adding sufficient toluene to disperse the cooled reactant, adsorbing the precipitate after the reactant is dispersed, discarding the supernatant when the black magnetic substance is basically precipitated to the bottom of a beaker, cleaning twice by using deionized water, cleaning twice by using acetone, and vacuum drying at 60 ℃ to obtain a black powder sample.
Step two: preparing magnetic responsiveness FTO magnetic nanoparticles;
the specific process is as follows: preparing demethylase FTO protein into a small-volume solution, preparing a black powder sample obtained in the step one into an aqueous suspension, fully mixing, adding sufficient toluene, homogenizing to generate protein-like spheres, adding a hydrophobic cross-linking agent into the mixed solution to harden the protein-like spheres, repeatedly washing, removing residual oil, separating the hardened protein-like spheres for 3-5 times, drying at 40 ℃ in a vacuum state, and storing at 4 ℃.
The preparation equipment include organism group, reaction flask, rabbling mechanism, sensor module, filling tube, argon gas supply mechanism, vacuum pump and controller, the reaction flask is connected with the organism group, rabbling mechanism installs in reaction flask middle part position, the filling tube passes through the solenoid valve with the reaction flask and is connected, all through the tube coupling between vacuum pump and reaction flask and the filling tube, and be equipped with the control valve on the connecting pipeline.
Further, organism group includes base, casing, heating plate and electro-magnet, casing internally mounted has and is circular-arc heating plate, and the heating plate encircles and distributes in the reaction flask lower extreme off-plate side, the middle part of electro-magnet installation and casing for the absorption of reaction back magnetic product, the front end at the casing is installed to the controller.
Further, the sensor module comprises a temperature sensor, an oxygen content sensor and a pressure sensor, which are respectively used for measuring the temperature of the mixed solution, the oxygen content in the reaction bottle and the pressure value in the reaction process.
Further, the molecular weight grade of PVP added in the first step is K17 or K30.
Further, the reaction temperature in the first step is in the range of 260 ℃ to 300 ℃.
Further, the mass ratio of the PEG, the PVP and the ferric acetylacetonate added in the first step is 200:3-20:7.
further, the particle size range of the magnetic-responsive FTO magnetic nanoparticles obtained in the second step is 0.2 μm to 1.3 μm, and the average particle size is 1.0 μm.
Compared with the prior art, the invention has the following beneficial effects:
1) The modified ferroferric oxide nano particles are obtained by the arranged preparation equipment, so that the whole reaction process is carried out in one device, the complicated steps in the reaction process are reduced, the control of the reaction process is facilitated, meanwhile, the interference of external factors in the reaction process can be avoided, the oxidation of the particles caused by the fact that external air enters the equipment is prevented, and the preparation of high-quality magnetic nano particles which are more stable, higher in purity and uniform in particle size is facilitated;
2) PEG is selected as a reaction solvent, PVP is selected as a modifier, the reaction temperature, the addition amount of the modifier and the grade category of the added modifier in the reaction process are strictly controlled, modified magnetic ferroferric oxide nanoparticles with moderate size, regular shape, good dispersity and high colloid stability can be prepared, a good foundation is provided for the attachment of subsequent protein molecules, and the adsorbed protein molecules are not easy to fall off from the surfaces of the nanoparticles.
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In order to more clearly illustrate the technical solution of the present invention, the drawings needed to be used in the technical description of the present invention will be briefly introduced below, and it is apparent that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without inventive labor.
FIG. 1 is a flow chart of the production process of the present invention;
FIG. 2 is a schematic view of the overall structure of the production apparatus of the present invention.
In the figure: 1. a body group; 11. a base; 12. a housing; 13. a heating plate; 14. an electromagnet; 2. a reaction bottle; 3. a stirring mechanism; 4. a sensor module; 41. a temperature sensor; 42. an oxygen content sensor; 5. a feed tube; 6. an argon gas supply mechanism; 7. a vacuum pump; 8. a controller; 9. and (4) controlling the valve.
Detailed Description
The present invention will be further described with reference to the following detailed description, wherein the drawings are for illustrative purposes only and are not intended to be limiting, wherein certain elements may be omitted, enlarged or reduced in size, and are not intended to represent the actual dimensions of the product, so as to better illustrate the detailed description of the invention.
Example 1
As shown in fig. 1 and fig. 2, a device and a method for preparing m6A demethylase FTO magnetic particles for inhibiting tumor cells includes the following steps:
the method comprises the following steps: preparing modified ferroferric oxide magnetic nanoparticles;
the specific process comprises the following steps: accurately weighing a certain amount of PEG and PVP by a balance, transferring into preparation equipment, stirring and mixing at 90 ℃ until the liquid is clear and transparent, then weighing a certain amount of ferric acetylacetonate, adding into the mixed liquid, continuously stirring for 10min at the current temperature (90 ℃), heating to the reaction temperature, continuously reacting for 1h, stopping heating, removing a heat source, cooling to 60 ℃ at room temperature, uniformly stirring in the reaction process, introducing argon to remove oxygen, adding sufficient toluene to disperse the cooled reactant, adsorbing the precipitate after the reactant is dispersed, discarding the supernatant when the black magnetic substance is basically precipitated to the bottom of a beaker, cleaning twice by using deionized water, cleaning twice by using acetone, and vacuum drying at 60 ℃ to obtain a black powder sample.
Step two: preparing magnetic responsiveness FTO magnetic nanoparticles;
the specific process comprises the following steps: preparing demethylase FTO protein into a small-volume solution, preparing a black powder sample obtained in the step one into an aqueous suspension, fully mixing, adding sufficient toluene, homogenizing to generate protein-like spheres, adding a hydrophobic cross-linking agent into the mixed solution to harden the protein-like spheres, repeatedly washing, removing residual oil, separating the hardened protein-like spheres for 3-5 times, drying at 40 ℃ in a vacuum state, and storing at 4 ℃.
The preparation equipment comprises a machine body group 1, a reaction bottle 2, a stirring mechanism 3, a sensor module 4, a feeding pipe 5, an argon supply mechanism 6, a vacuum pump 7 and a controller 8, wherein the reaction bottle 2 is connected with the machine body group 1, the stirring mechanism 3 is installed at the middle position of the reaction bottle 2, the feeding pipe 5 is connected with the reaction bottle 2 through an electromagnetic valve, the vacuum pump 7 is connected with the reaction bottle 2 and the feeding pipe 5 through pipelines, and a control valve 9 is arranged on the connecting pipeline.
The machine body group 1 comprises a base 11, a shell 12, heating sheets 13 and electromagnets 14, wherein the heating sheets 13 in an arc shape are arranged inside the shell 12, the heating sheets 13 are distributed on the outer side of the lower end face of the reaction flask 2 in a surrounding manner, the electromagnets 14 are arranged in the middle of the shell 12, and the controller 8 is arranged at the front end of the shell 12.
The sensor module 4 comprises a temperature sensor 41, an oxygen content sensor 42 and a pressure sensor 43, which are respectively used for measuring the temperature of the mixed solution, the oxygen content inside the reaction bottle and the pressure value in the reaction process.
By adopting the technical scheme: when preparing modified ferroferric oxide magnetic nanoparticles, after equipment is assembled, argon gas is introduced into a reaction bottle 2 at a constant speed through a pressure supply mechanism 6, the oxygen content in the reaction bottle 2 is detected in real time through an oxygen content sensor 42, when the oxygen content in the reaction bottle 2 is reduced to a lower level, the flow of the introduced argon gas can be properly reduced, the existing gas composition structure in the reaction bottle 2 is maintained, if the reactant is required to be added in the reaction process, the reactant is firstly added into a feeding pipe 5, an opening at the upper end of the feeding pipe 5 is closed, a vacuum pump 7 is started through a controller 8, a control valve 9 is adjusted, air in the feeding pipe 5 is pumped out, then an electromagnetic valve at the connection part of the feeding pipe 5 and the reaction bottle 2 is opened, the reactant enters the reaction bottle 2 through the electromagnetic valve, the flow of the introduced argon gas is adjusted according to the oxygen content value measured by the oxygen content sensor 42, the phenomenon that the oxygen is mixed into the reaction bottle 2 to cause oxidation of products in the feeding process is prevented, and the purity of reaction products can be improved.
Example 2
As shown in fig. 1, a device and a method for preparing m6A demethylase FTO magnetic particles for inhibiting tumor cells comprises the following steps:
the method comprises the following steps: preparing modified ferroferric oxide magnetic nanoparticles;
the specific process is as follows: accurately weighing a certain amount of PEG and PVP by a balance, transferring into preparation equipment, stirring and mixing at 90 ℃ until the liquid is clear and transparent, then weighing a certain amount of ferric acetylacetonate, adding into the mixed liquid, continuously stirring for 10min at the current temperature (90 ℃), heating to the reaction temperature, continuously reacting for 1h, stopping heating, removing a heat source, cooling to 60 ℃ at room temperature, uniformly stirring in the reaction process, introducing argon to remove oxygen, adding sufficient toluene to disperse the cooled reactant, adsorbing the precipitate after the reactant is dispersed, discarding the supernatant when the black magnetic substance is basically precipitated to the bottom of a beaker, cleaning twice by using deionized water, cleaning twice by using acetone, and vacuum drying at 60 ℃ to obtain a black powder sample.
Step two: preparing magnetic responsiveness FTO magnetic nanoparticles;
the specific process is as follows: preparing demethylase FTO protein into a small-volume solution, preparing a black powder sample obtained in the step one into an aqueous suspension, fully mixing, adding sufficient toluene, homogenizing to generate protein-like spheres, adding a hydrophobic cross-linking agent into the mixed solution to harden the protein-like spheres, repeatedly washing, removing residual oil, separating the hardened protein-like spheres for 3-5 times, drying at 40 ℃ in a vacuum state, and storing at 4 ℃.
The preparation equipment comprises a machine body group 1, a reaction bottle 2, a stirring mechanism 3, a sensor module 4, a feeding pipe 5, an argon supply mechanism 6, a vacuum pump 7 and a controller 8, wherein the reaction bottle 2 is connected with the machine body group 1, the stirring mechanism 3 is installed at the middle position of the reaction bottle 2, the feeding pipe 5 is connected with the reaction bottle 2 through an electromagnetic valve, the vacuum pump 7 is connected with the reaction bottle 2 and the feeding pipe 5 through pipelines, and a control valve 9 is arranged on the connecting pipeline.
The molecular weight grade of PVP added in the first step is K17 or K30.
The reaction temperature in step one is in the range of 260 ℃ to 300 ℃.
The mass ratio of PEG, PVP and ferric acetylacetonate added in the first step is 200:3-20:7.
the particle size range of the magnetic responsiveness FTO magnetic nanoparticles obtained in the second step is 0.2-1.3 μm, and the average particle size is 1.0 μm.
By adopting the technical scheme:
TABLE 1 influence of reaction temperature on particle size of ferroferric oxide magnetic nanoparticles
Modified ferroferric oxide magnetic nanoparticle samples are prepared at different temperatures, TEM images of the prepared samples are obtained, and results are shown in the table, when the reaction temperature is in the range of 200-260 ℃, the particle size of the prepared particles is in an increasing trend, but after the reaction temperature exceeds 260 ℃, the particle size of the prepared ions is not greatly different, and the particle size of the obtained particles is about 9nm, which is probably because the reaction temperature reaches the boiling point of a reaction solvent PEG after reaching 260 ℃, the reaction temperature does not have a positive influence on the dispersion of the prepared nanoparticles, so the temperature rise has a small influence on the results, but when the reaction temperature does not reach 260 ℃, the boiling point of the PEG solvent is not reached, the temperature rise has a large influence on the particle size, so the reaction temperature range of the mixed solution is kept between 260 ℃ and 300 ℃ in the reaction process, and the particle size of the ferroferric oxide magnetic nanoparticles is most appropriate, and the subsequent protein molecule adsorption is facilitated.
Example 3
As shown in fig. 1, a device and a method for preparing m6A demethylase FTO magnetic particles for inhibiting tumor cells comprises the following steps:
the method comprises the following steps: preparing modified ferroferric oxide magnetic nanoparticles;
the specific process is as follows: accurately weighing a certain amount of PEG and PVP by a balance, transferring into preparation equipment, stirring and mixing at 90 ℃ until the liquid is clear and transparent, then weighing a certain amount of ferric acetylacetonate, adding into the mixed liquid, continuously stirring for 10min at the current temperature (90 ℃), heating to the reaction temperature, continuously reacting for 1h, stopping heating, removing a heat source, cooling to 60 ℃ at room temperature, uniformly stirring in the reaction process, introducing argon to remove oxygen, adding sufficient toluene to disperse the cooled reactant, adsorbing the precipitate after the reactant is dispersed, discarding the supernatant when the black magnetic substance is basically precipitated to the bottom of a beaker, cleaning twice by using deionized water, cleaning twice by using acetone, and vacuum drying at 60 ℃ to obtain a black powder sample.
Step two: preparing magnetic responsiveness FTO magnetic nanoparticles;
the specific process is as follows: preparing demethylase FTO protein into a small-volume solution, preparing a black powder sample obtained in the step one into an aqueous suspension, fully mixing, adding sufficient toluene, homogenizing to generate protein-like spheres, adding a hydrophobic cross-linking agent into the mixed solution to harden the protein-like spheres, repeatedly washing, removing residual oil, separating the hardened protein-like spheres for 3-5 times, drying at 40 ℃ in a vacuum state, and storing at 4 ℃.
The preparation equipment comprises a machine body group 1, a reaction bottle 2, a stirring mechanism 3, a sensor module 4, a feeding pipe 5, an argon supply mechanism 6, a vacuum pump 7 and a controller 8, wherein the reaction bottle 2 is connected with the machine body group 1, the stirring mechanism 3 is installed at the middle position of the reaction bottle 2, the feeding pipe 5 is connected with the reaction bottle 2 through an electromagnetic valve, the vacuum pump 7 is connected with the reaction bottle 2 and the feeding pipe 5 through pipelines, and a control valve 9 is arranged on the connecting pipeline.
The molecular weight grade of PVP added in the first step is K17 or K30.
The reaction temperature in step one is in the range of 260 ℃ to 300 ℃.
The mass ratio of PEG, PVP and ferric acetylacetonate added in the first step is 200:3-20:7.
the particle size range of the magnetic responsiveness FTO magnetic nanoparticles obtained in the second step is 0.2-1.3 μm, and the average particle size is 1.0 μm.
By adopting the technical scheme:
TABLE 2 influence of PVP molecular weight on the particle size of ferroferric oxide magnetic nanoparticles
As shown in the above table, the molecular weight of the reaction solvent PVP has a certain influence on the particle size of the ferroferric oxide magnetic nanoparticle sample, when PVP solvent with a smaller molecular weight is used, the prepared ferroferric oxide magnetic nanoparticle has a larger particle size but a smaller influence, and in the production process, in order to ensure the adsorption of protein molecules, PVP solvent with a larger molecular weight should be selected.
Example 4
As shown in fig. 1, a device and a method for preparing m6A demethylase FTO magnetic particles for inhibiting tumor cells comprises the following steps:
the method comprises the following steps: preparing modified ferroferric oxide magnetic nanoparticles;
the specific process comprises the following steps: accurately weighing a certain amount of PEG and PVP by a balance, transferring into preparation equipment, stirring and mixing at 90 ℃ until the liquid is clear and transparent, then weighing a certain amount of ferric acetylacetonate, adding into the mixed liquid, continuously stirring for 10min at the current temperature (90 ℃), heating to the reaction temperature, continuously reacting for 1h, stopping heating, removing a heat source, cooling to 60 ℃ at room temperature, uniformly stirring in the reaction process, introducing argon to remove oxygen, adding sufficient toluene to disperse the cooled reactant, adsorbing the precipitate after the reactant is dispersed, discarding the supernatant when the black magnetic substance is basically precipitated to the bottom of a beaker, cleaning twice by using deionized water, cleaning twice by using acetone, and vacuum drying at 60 ℃ to obtain a black powder sample.
Step two: preparing magnetic responsiveness FTO magnetic nanoparticles;
the specific process is as follows: preparing demethylase FTO protein into a small-volume solution, preparing a black powder sample obtained in the step one into an aqueous suspension, fully mixing, adding sufficient toluene, homogenizing to generate protein-like spheres, adding a hydrophobic cross-linking agent into the mixed solution to harden the protein-like spheres, repeatedly washing, removing residual oil, separating the hardened protein-like spheres for 3-5 times, drying at 40 ℃ in a vacuum state, and storing at 4 ℃.
The preparation equipment comprises a machine body group 1, a reaction bottle 2, a stirring mechanism 3, a sensor module 4, a feeding pipe 5, an argon supply mechanism 6, a vacuum pump 7 and a controller 8, wherein the reaction bottle 2 is connected with the machine body group 1, the stirring mechanism 3 is installed at the middle position of the reaction bottle 2, the feeding pipe 5 is connected with the reaction bottle 2 through an electromagnetic valve, the vacuum pump 7 is connected with the reaction bottle 2 and the feeding pipe 5 through pipelines, and a control valve 9 is arranged on the connecting pipeline.
The molecular weight grade of PVP added in the first step is K17 or K30.
The reaction temperature in step one is in the range of 260 ℃ to 300 ℃.
The mass ratio of PEG, PVP and ferric acetylacetonate added in the first step is 200:3-20:7.
the particle size range of the magnetic-responsiveness FTO magnetic nanoparticles obtained in the second step is 0.2-1.3 μm, and the average particle size is 1.0 μm.
By adopting the technical scheme:
TABLE 3 influence of PVP addition amount in reactant on particle size of ferroferric oxide magnetic nanoparticles
As shown in the table above, PVP is used as a modifier to participate in the reaction, the addition amount of PVP in the reactant does not affect the particle size of the ferroferric oxide magnetic nanoparticles, but the addition of a sufficient amount of a PVP solvent during the reaction can affect the rest properties of the generated nanoparticles, such as the improvement of the dispersibility of the ferroferric oxide magnetic nanoparticles and the colloidal stability in a buffer solution.
The foregoing shows and describes the general principles and broad features of the present invention and advantages thereof. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (6)
1. A preparation method of m6A demethylase FTO magnetic particles for inhibiting tumor cells is characterized by comprising the following steps: the method comprises the following steps:
the method comprises the following steps: preparing modified ferroferric oxide magnetic nanoparticles; the specific process is as follows: accurately weighing a certain amount of PEG and PVP by a balance, transferring into preparation equipment, stirring and mixing at 90 ℃ until the liquid is clear and transparent, then weighing a certain amount of ferric acetylacetonate, adding into the mixed liquid, continuously stirring for 10min at the current temperature (90 ℃), heating to the reaction temperature, continuously reacting for 1h, stopping heating, removing a heat source, cooling to 60 ℃ at room temperature, uniformly stirring in the reaction process, introducing argon to remove oxygen, adding sufficient toluene to disperse the cooled reactant, adsorbing the precipitate after the reactant is dispersed, discarding supernatant liquid after the black magnetic substance is basically precipitated to the bottom of a beaker, cleaning twice by using deionized water, cleaning twice by using acetone for the precipitate, and performing vacuum drying at 60 ℃ to obtain a black powder sample;
step two: preparing magnetic responsiveness FTO magnetic nanoparticles; the specific process is as follows: preparing demethylase FTO protein into a small-volume solution, preparing a black powder sample obtained in the step one into an aqueous suspension, fully mixing, adding sufficient toluene, homogenizing to generate protein-like spheres, adding a hydrophobic cross-linking agent into the mixed solution to harden the protein-like spheres, repeatedly washing, removing residual oil, separating the hardened protein-like spheres for 3-5 times, drying at 40 ℃ in a vacuum state, and storing at 4 ℃.
2. The method for preparing m6A demethylase FTO magnetic particles for inhibiting tumor cells according to claim 1, wherein: the molecular weight grade of PVP added in the first step is K17 or K30; the reaction temperature in the first step is in the range of 260-300 ℃.
3. The method of claim 1, wherein the magnetic particles of m6A demethylase FTO for inhibiting tumor cells are prepared by: the mass ratio of PEG, PVP and ferric acetylacetonate added in the first step is 200:3-20:7.
4. the method for preparing m6A demethylase FTO magnetic particles for inhibiting tumor cells according to claim 1, wherein: the particle size range of the magnetic-responsive FTO magnetic nanoparticles obtained in the second step is 0.2-1.3 μm, and the average particle size is 1.0 μm.
5. A device for preparing m6A demethylase FTO magnetic particles for inhibiting tumor cells, characterized in that: the preparation device comprises a machine body group, a reaction bottle, a stirring mechanism, a sensor module, a feeding pipe, an argon gas supply mechanism, a vacuum pump and a controller, wherein the reaction bottle is connected with the machine body group; the sensor module comprises a temperature sensor, an oxygen content sensor and a pressure sensor which are respectively used for measuring the temperature of the mixed solution, the oxygen content in the reaction bottle and the pressure value in the reaction process.
6. The device for preparing m6A demethylase FTO magnetic particles for inhibiting tumor cells according to claim 5, wherein: organism group includes base, casing, heating plate and electro-magnet, casing internally mounted has and is circular-arc heating plate, and the heating plate encircles and distributes at the reaction flask lower extreme off-plate side, the middle part of electro-magnet installation and casing, the front end at the casing is installed to the controller.
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| CN116421705A (en) * | 2023-02-27 | 2023-07-14 | 迈迪速能医学技术（天津）有限公司 | Pharmaceutical preparation for reducing cervical cancer incidence risk |
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| CN116421705A (en) * | 2023-02-27 | 2023-07-14 | 迈迪速能医学技术（天津）有限公司 | Pharmaceutical preparation for reducing cervical cancer incidence risk |
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