CN110423783B - Gene transfection/drug targeting/signal conduction method and system based on negative magnetophoresis - Google Patents

Gene transfection/drug targeting/signal conduction method and system based on negative magnetophoresis Download PDF

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CN110423783B
CN110423783B CN201910619981.0A CN201910619981A CN110423783B CN 110423783 B CN110423783 B CN 110423783B CN 201910619981 A CN201910619981 A CN 201910619981A CN 110423783 B CN110423783 B CN 110423783B
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曹全梁
陈竞舸
韩小涛
丁安梓
李亮
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Huazhong University of Science and Technology
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Abstract

The invention belongs to the technical fields of biomedicine, magnetic materials and nanometer, and particularly relates to a gene transfection/drug delivery/signal conduction method and system based on a negative magnetophoresis technology. The method is characterized in that a magnetic fluid culture solution in which cells are cultured is placed in a magnetic field environment without preparing a special magnetic carrier or performing complex magnetic modification work on the carrier, the magnetic fluid culture solution also comprises nonmagnetic target particles carrying genes, medicines or signals, and by utilizing the negative magnetophoresis characteristic, the direction of the gradient magnetic field force borne by the nonmagnetic target particles is a magnetic field gradient direction corresponding to the magnetic field intensity from large to small, so that the gene transfection/medicine targeting/signal conduction on the cells is realized, and the technical problems of complex magnetic modification operation process, high technical threshold and inconvenience for large-scale practical application existing in the traditional marked gene transfection/medicine targeting/signal conduction method are solved.

Description

Gene transfection/drug targeting/signal conduction method and system based on negative magnetophoresis
Technical Field
The invention belongs to the technical fields of biomedicine, magnetic materials and nanometer, and particularly relates to a gene transfection/drug delivery/signal conduction method and system based on a negative magnetophoresis technology.
Background
The positive magnetophoresis refers to a movement behavior that a magnetic substance in a solution (generally, a non-magnetic solution) migrates toward a direction of increasing the magnetic field strength under the action of an external non-uniform magnetic field due to the action of a gradient magnetic field.
The magnetic fluid is a novel functional material which has the liquidity of liquid and the magnetism of a solid magnetic material at the same time. It has no magnetic attraction in the absence of an external magnetic field, and exhibits magnetism in the presence of an external magnetic field.
When the non-magnetic substance is acted by an external magnetic field in the magnetic fluid, the non-magnetic or diamagnetic substance is also acted by the gradient magnetic field force because of the magnetization difference between the substance and the magnetic fluid, and the action direction of the force is opposite to the stress direction of the magnetic substance under positive magnetophoresis, so that the non-magnetic substance moves towards the direction of reducing the magnetic field intensity. This phenomenon is commonly referred to as negative magnetophoresis.
Under negative magnetophoresis, the gradient magnetic field force borne by a substance is as follows:
Figure BDA0002125182190000011
in the formula, mu0Is magnetic permeability in vacuum, VPVolume of the target particle, MPAnd MfThe magnetization of the target particles and the magnetic fluid respectively,
Figure BDA0002125182190000012
the negative sign in front of the equation for the magnetic field strength around the target particle indicates that the magnetic force is directed in the direction of decreasing magnetic field strength.
The magnitude of the gradient magnetic field force (i.e. negative magnetophoretic force) is related to the gradient magnitude of the external magnetic field, the difference of the magnetization intensity of the magnetic fluid and the target particles, and the volume of the target particles.
The key to generating negative magnetophoresis is that the magnetic permeability of the magnetofluid solution is higher than that of the target particles. The negative magnetophoresis technology has the greatest advantage that the target particles can be subjected to non-connection type motion guidance without being subjected to magnetic modification.
Gene transfection refers to the transfer or transport of a biologically functional nucleic acid into a cell and the maintenance of the biological function of the nucleic acid within the cell.
The drug targeting aims at a specific pathological change part to form relatively high concentration locally, so that the damage to normal tissues and cells is reduced.
Signal transduction refers to the process of transmitting various signals into cells through the cell membrane, gradually causing changes in the cellular material, primarily proteins.
The common technique required by gene transfection/drug targeting/signal transduction is how to directionally and efficiently deliver target particles (particles carrying genes, drugs or signals) to specific target regions.
To achieve the above objective, one effective method is to magnetically modify the microparticles and then achieve the directional movement of the microparticles under the guidance of a magnetic field, i.e., a labeled gene transfection/drug targeting/signaling method.
However, the magnetic modification operation process is complicated, the technical threshold is high, and the large-scale practical application is not facilitated. In addition, after entering the cell, the magnetic nanoparticles may need to be dissociated from the carrier in order to make the gene, signal and drug normally function, the process is complex, and the dissociated magnetic nanoparticles still bear the cell. Therefore, the non-marker gene transfection/drug targeting/signal transduction method has important scientific research value and clinical treatment significance.
Disclosure of Invention
Aiming at the defects or improvement requirements of the prior art, the invention provides a non-labeled gene transfection/drug targeting/signal conduction method and a system based on a negative magnetophoresis technology, which do not need to prepare a special magnetic carrier or carry out complicated magnetic modification work on the carrier, and realize the gene transfection/drug targeting/signal conduction on cells by utilizing the negative magnetophoresis characteristic to enable non-magnetic target particles carrying genes, drugs or signals to be subjected to gradient magnetic field force opposite to the direction of magnetic fluid under the action of a magnetic field, thereby solving the technical problems of complicated magnetic modification operation process, high technical threshold and inconvenience for large-scale practical application in the traditional labeled gene transfection/drug targeting/signal conduction method.
In order to achieve the above objects, according to one aspect of the present invention, there is provided a gene transfection/drug delivery/signal conduction method based on a negative magnetophoresis technique, comprising placing a magnetofluid culture system in which cells are cultured in a magnetic field environment, the magnetofluid culture system containing a magnetofluid culture solution, the magnetofluid culture solution exhibiting magnetism as a whole under the action of a magnetic field, and the magnetofluid culture solution being subjected to the action of a magnetic field force; the magnetic fluid culture solution has biocompatibility and can be used for culturing cells;
the magnetic fluid culture system also comprises non-magnetic target particles carrying genes, medicines or signals; the magnetic permeability of the non-magnetic target particles carrying the genes, the medicines or the signals is lower than that of the magnetic fluid culture solution;
under the action of an external magnetic field, according to the negative magnetophoresis characteristic, magnetization difference exists between the nonmagnetic target particles carrying genes, medicines or signals and the magnetofluid culture solution expressing magnetism, and the force direction of a gradient magnetic field borne by the nonmagnetic target particles is a magnetic field gradient direction corresponding to the magnetic field intensity from large to small;
by regulating and controlling the relative position between the magnetic fluid culture system and the magnetic field source, the non-magnetic target particles carrying genes, medicines or signals move towards the direction of cells under the action of gradient magnetic field force, thereby realizing gene transfection, medicine transmission or signal conduction to the cells.
Preferably, the cells are grown adherently, and the spatial position of the magnetofluid culture solution is between the cells and the magnetic field source.
Preferably, the magnetic fluid culture solution is a magnetic fluid which is biocompatible and suitable for cell survival.
Preferably, the magnetic fluid is suitable for cell survival of not less than 0.5 hour.
Preferably, magnetic nanoparticles are dispersed in the magnetic fluid culture solution, the particle size range of the magnetic nanoparticles in the magnetic fluid culture solution is 1-500nm, and the concentration of the magnetic nanoparticles in the magnetic fluid culture solution is 1-100 g/L.
Preferably, the non-magnetic target particles carrying the genes, the drugs or the signals are obtained by connecting the genes, the drugs or the signals with the non-magnetic target carrier particles through chemical bonds, hydrogen bonds, hydrophobic bonds, electrostatic interaction or van der waals forces, or are obtained by adsorbing, embedding or crosslinking the genes, the drugs or the signals with the non-magnetic target carrier particles.
According to another aspect of the present invention, there is provided a gene transfection/drug delivery/signaling system based on negative magnetophoresis technology, comprising:
a magnetic field supply module for providing a magnetic field environment;
the cell module is used for providing a magnetic fluid culture system for culturing cells, the magnetic fluid culture system contains a magnetic fluid culture solution, the magnetic fluid culture solution integrally shows magnetism under the action of a magnetic field, and the magnetic fluid culture solution is acted by the action of a magnetic field force; the magnetic fluid culture solution has biocompatibility and can be used for culturing cells; the magnetic fluid culture system also comprises non-magnetic target particles carrying genes, medicines or signals; the magnetic permeability of the non-magnetic target particles carrying the genes, the medicines or the signals is lower than that of the magnetic fluid culture solution;
under the action of an external magnetic field, according to the negative magnetophoresis characteristic, magnetization difference exists between the nonmagnetic target particles carrying genes, medicines or signals and the magnetofluid culture solution expressing magnetism, and the force direction of a gradient magnetic field borne by the nonmagnetic target particles is a magnetic field gradient direction corresponding to the magnetic field intensity from large to small; by regulating and controlling the relative position between the magnetic fluid culture system and the magnetic field source, the non-magnetic target particles carrying genes, medicines or signals move towards the direction of cells under the action of gradient magnetic field force, thereby realizing gene transfection, medicine transmission or signal conduction to the cells.
Preferably, the cells are grown adherently in a cell module, and the magnetic fluid culture solution in the cell module is between the magnetic field supply module and the cells in the cell module in a spatial position relation.
Preferably, the magnetic field supply module is a permanent magnet or an electromagnet for providing a gradient magnetic field.
Preferably, the magnetic field supply module is a magnet array composed of a plurality of columnar magnets; the cell module is a cell culture pore plate, and each independent pore of the cell culture pore plate is filled with a magnetic fluid culture system formed by mixing non-magnetic target particles carrying genes, medicines or signals and a magnetic fluid culture solution for culturing cells.
Preferably, the magnet array is arranged in a magnet orifice plate, the magnet orifice plate has the same structure as the cell culture orifice plate, a columnar magnet is fixed in each independent orifice of the magnet orifice plate to form the magnet array, and the independent orifice in which each columnar magnet is arranged in the magnet array corresponds to each independent orifice of the cell culture orifice plate in the cell module.
Preferably, the magnet array is restrained by using magnets, and the columnar magnets in the magnet array are prevented from being ejected from the orifice plate.
In general, compared with the prior art, the above technical solution contemplated by the present invention can achieve the following beneficial effects:
(1) the invention provides a gene transfection/drug delivery/signal conduction method based on negative magnetophoresis technology, which comprises the steps of mixing nonmagnetic target particles carrying genes, drugs or signals with a magnetofluid culture solution cultured with cells to form a magnetofluid culture system and placing the magnetofluid culture system in a magnetic field environment; by utilizing the characteristics of negative magnetophoresis, the magnetization difference exists between the nonmagnetic target particles carrying genes, medicines or signals and the magnetic fluid culture solution showing magnetism, the acting direction of magnetic field force applied to the nonmagnetic target particles is opposite to the force application direction of magnetic substances under positive magnetophoresis, namely the gradient magnetic field force direction applied to the nonmagnetic target particles is the magnetic field gradient direction corresponding to the magnetic field intensity from large to small, and the nonmagnetic target particles move towards the direction of cells, so that the gene transfection, the medicine transmission or the signal transmission of the cells are realized. The gene transfection/drug targeting/signal conduction method is simple to operate, and does not need to prepare a special magnetic carrier or carry out complex magnetic modification work on the carrier.
(2) By utilizing the negative magnetophoresis characteristic, the acting direction of the magnetic field force applied to the non-magnetic target particles is opposite to the force applied to the magnetic substance under the positive magnetophoresis. According to the invention, the magnetic fluid culture solution is arranged between the magnetic field source and the cell by setting the spatial position relationship between the magnetic fluid culture system and the magnetic field source, so that the magnetic fluid culture solution has a tendency of moving towards the gradient magnetic field direction, namely the magnetic field source direction, and the nonmagnetic target carrier particles carrying genes, medicines or signals move towards the opposite direction and away from the magnetic field source, namely the cell, so that the gene transfection/medicine targeting/signal conduction is more favorably realized.
(3) The method of the invention is adopted to carry out gene transfection/drug targeting/signal conduction, and no special magnetic carrier is required to be prepared or complex magnetic modification work is required to be carried out on the carrier, so that no magnetic nano particles enter cells to cause cell burden, and the toxicity is lower.
(4) The magnetic field supply module in the gene transfection/drug delivery/signal conduction system based on the negative magnetophoresis technology is a magnet array corresponding to the pore plate of the cell module, a rectangular magnet is arranged below the magnet array and used for restraining the columnar magnets in the pore plate and preventing the repulsion force between the columnar magnets from causing the magnets to be ejected out of the pore plate, and the columnar magnets in the pore plate are fixed in the pore plate and can also prevent the columnar magnets from being ejected out of the pore plate when the rectangular magnets are removed.
Drawings
FIG. 1 is a schematic view of a method of manufacturing a magnet array according to the present invention;
FIG. 2 is a schematic diagram of a conventional magnetic marker type gene transfection/drug targeting/signaling method;
FIG. 3 is a schematic diagram of the non-labeled gene transfection/drug targeting/signal transduction method based on negative magnetophoresis technology.
The same reference numbers will be used throughout the drawings to refer to the same or like elements or structures, wherein: 1 a blank orifice plate; 2, a cylindrical magnet matched with the hole diameter of the hole plate; 3 is a rectangular magnet; 4 is a magnet array; 5 is a pore plate with cells when in gene transfection/drug targeting/signal transmission; 6 is magnetic nano-particles; 7 is target particles carrying genes/signals/drugs; 8 is the target particle carrying the gene/signal/drug after magnetic modification; 9 is a traditional non-magnetic cell culture solution; 10 are cells cultured in well plates; 11 is magnetofluid cell culture solution.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.
The invention provides a gene transfection/drug delivery/signal conduction method based on negative magnetophoresis technology, which comprises the steps of placing a magnetofluid culture system cultured with cells in a magnetic field environment, wherein the magnetofluid culture system contains a magnetofluid culture solution, the magnetofluid culture solution integrally shows magnetism under the action of a magnetic field, and the magnetofluid culture solution is acted by a magnetic field force; the magnetic fluid culture solution has biocompatibility and can be used for culturing cells; the magnetic fluid culture system also comprises non-magnetic target particles carrying genes, medicines or signals; the magnetic permeability of the non-magnetic target particles carrying the genes, the medicines or the signals is lower than that of the magnetic fluid culture solution.
Under the action of an external magnetic field, according to the characteristics of negative magnetophoresis, magnetization difference exists between the nonmagnetic target particles carrying genes, medicines or signals and the magnetic fluid culture solution expressing magnetism, the acting direction of magnetic field force borne by the nonmagnetic target particles is opposite to the stressed direction of magnetic substances under positive magnetophoresis, namely the direction of gradient magnetic field force borne by the nonmagnetic target particles is the magnetic field gradient direction corresponding to the magnetic field intensity from large to small.
By regulating and controlling the relative position between the magnetic fluid culture system and the magnetic field source, the non-magnetic target particles carrying genes, medicines or signals move towards the direction of cells under the action of gradient magnetic field force, thereby realizing gene transfection, medicine transmission or signal conduction to the cells.
In order to ensure that the non-magnetic target particles are moved to a target area by negative magnetophoretic force, the magnetic permeability of the non-magnetic target particles carrying genes, medicines or signals is lower than that of the magnetic fluid culture solution.
The "/" is "or" in gene transfection/drug delivery/signaling as described in the present invention.
In some embodiments the cells are in adherent growth and the spatial location of the magnetofluid culture is between the cells and a magnetic field source.
The magnetic fluid culture solution is a novel functional material with magnetism and liquid fluidity. It does not exhibit magnetism in the absence of an external magnetic field, i.e., does not have a magnetic attraction, and exhibits magnetism as a whole in the presence of an external magnetic field, which is equivalent to a liquefied block magnet.
The magnetic fluid culture solution has biocompatibility and can be used for culturing cells. Biocompatibility here means that cells can survive in the magnetofluid culture for a certain period of time, for example, not less than 0.5 hour.
In some embodiments, the magnetic fluid culture fluid is magnetic fluid, and the magnetic fluid is biocompatible and generally suitable for cell survival for not less than 0.5 hour. The magnetic fluid can be obtained commercially or prepared by itself according to a conventional magnetic fluid preparation method.
In some embodiments, a magnetic fluid is formulated by dispersing magnetic nanoparticles in a carrier liquid having biocompatibility, and the magnetic fluid is used as the magnetic fluid culture fluid of the present invention. The carrier liquid can here be a cell culture liquid suitable for cell culture or directly a buffer solution free of nutrients. The magnetic nanoparticles are ferroferric oxide magnetic nanoparticles, gamma-iron sesquioxide magnetic nanoparticles and the like. The cell culture solution which is biocompatible and suitable for cell culture is an aqueous solution which is suitable for cell culture and contains nutrients required by cell growth and has temperature, pH value and osmotic pressure. Such as a phosphate buffer solution. The cell culture medium can be selected from the corresponding commonly used culture medium according to the cell type, such as for culturing HEK293 cells, and DMEM culture medium can be selected.
In some embodiments, the magnetic nanoparticles have a particle size in the range of 1-500nm, and too large or too small may make it difficult to form a magnetic fluid; the concentration of the magnetic nanoparticles in the magnetic fluid culture solution is 1-100g/L, when the concentration is too low, magnetic fluid is difficult to form, and when the concentration is too high, subsequent experiments can be hindered. The magnetization intensity of the magnetic fluid culture solution prepared by the method under the condition of an external magnetic field is related to the concentration of the magnetic nanoparticles, namely the magnetization intensity of the magnetic fluid culture solution can be adjusted by adjusting the size and the concentration of the magnetic nanoparticles.
In other embodiments, the magnetic fluid is a paramagnetic salt solution formed from a paramagnetic metal and an organic chelating agent, or a paramagnetic metal and a halide, such as MnCl2And (3) solution. At a suitable concentration, the magnetic fluid can also be directly used as the magnetic fluid culture solution of the invention.
In some embodiments, the non-magnetic target particles carrying genes, drugs or signals are obtained by connecting the genes, drugs or signals with the non-magnetic target carrier particles through chemical bonds, hydrogen bonds, hydrophobic bonds, electrostatic interactions, van der waals forces, and the like, or through adsorption, embedding, and crosslinking methods.
In some embodiments, the non-magnetic target particle carrier may be a gold nanoparticle, a silicon nanoparticle, or the like. The surface area is large, the biocompatibility is good, the modification is easy, and the connection with genes, medicines or signals is easy to realize. And the magnetic permeability is low, and the purpose that the magnetic permeability of the non-magnetic target particles carrying the genes, the medicines or the signals is lower than that of the magnetic fluid culture solution can be easily achieved after the non-magnetic target particles are cracked with the genes, the medicines or the signals.
When selecting the gene to be transfected, the drug to be delivered, the signal to be transmitted and the corresponding non-magnetic target particles, the invention only needs to ensure that the finally synthesized non-magnetic target particles carrying the gene, the drug or the signal have lower magnetic permeability than the selected magnetic fluid culture solution. On the basis, the genes, the medicines or the signals can select which particles to be used as non-magnetic target particle carriers, the non-magnetic target particle carriers are connected with one another by adopting which method, and the problems of selecting which magnetic fluid culture solution and the like can be optimized according to actual conditions.
The method comprises the steps of uniformly mixing a magnetic fluid culture solution cultured with cells and nonmagnetic target particles carrying genes, medicines or signals, and placing the mixture in a magnetic field environment for a period of time to achieve gene transfection, medicine targeting or signal conduction on the cells, wherein in some embodiments, the time of placing the mixture in the magnetic field environment is 10-120 minutes, the gene transfection/medicine targeting/signal conduction efficiency is low due to too short time, and the cell survival rate is low due to too long time.
In some embodiments, the method specifically includes the following steps:
(1) preparing a magnetic fluid culture solution with biocompatibility;
(2) preparing non-magnetic target particles carrying genes, drugs or signals;
(3) adding the non-magnetic target particles carrying the genes, the medicines or the signals into the magnetic fluid culture solution, blowing, beating and uniformly mixing to obtain the magnetic fluid culture solution mixed with the non-magnetic target particles carrying the genes, the medicines or the signals
(4) Cells are cultured by magnetic fluid culture solution mixed with non-magnetic target particles carrying genes, medicines or signals, and are placed in a magnetic field environment for a period of time to realize gene transfection, medicine targeting (delivery) or signal conduction.
In some embodiments, the cells can be cultured according to the cell type by conventional methods to obtain cells with proper adherence, and then the cells can be cultured by magnetofluid culture medium mixed with nonmagnetic target particles carrying genes, drugs or signals. For example, the original culture medium of the cells is aspirated and replaced with the magnetic fluid culture medium mixed with the nonmagnetic target particles carrying genes, drugs or signals, and the cells are cultured continuously.
In some embodiments, after the gene transfection, drug targeting (delivery) or signal transduction step is completed, the magnetic fluid culture solution mixed with the non-magnetic target particles carrying the gene, drug or signal is removed, and the cells are further cultured and observed according to a conventional method, so that the effect of the gene transfection, drug targeting (delivery) or signal transduction according to the above steps can be accurately measured or observed.
The present invention also provides a gene transfection/drug delivery/signaling system based on negative magnetophoresis technology according to the method as described above, comprising:
a magnetic field supply module for providing a magnetic field environment;
the cell module is used for providing a magnetic fluid culture system for culturing cells, the magnetic fluid culture system contains a magnetic fluid culture solution, the magnetic fluid culture solution integrally shows magnetism under the action of a magnetic field, and the magnetic fluid culture solution is acted by the action of a magnetic field force; the magnetic fluid culture solution has biocompatibility and can be used for culturing cells; the magnetic fluid culture system also comprises non-magnetic target particles carrying genes, medicines or signals; the magnetic permeability of the non-magnetic target particles carrying the genes, the medicines or the signals is lower than that of the magnetic fluid culture solution;
under the action of an external magnetic field, according to the negative magnetophoresis characteristic, magnetization difference exists between the nonmagnetic target particles carrying genes, medicines or signals and the magnetofluid culture solution expressing magnetism, and the force direction of a gradient magnetic field borne by the nonmagnetic target particles is a magnetic field gradient direction corresponding to the magnetic field intensity from large to small; by regulating and controlling the relative position between the magnetic fluid culture system and the magnetic field source, the non-magnetic target particles carrying genes, medicines or signals move towards the direction of cells under the action of gradient magnetic field force, thereby realizing gene transfection, medicine transmission or signal conduction to the cells.
The magnetic field supply module of the present invention may be any magnetic field source, such as a permanent magnet or an electromagnet, for providing a gradient magnetic field.
In some embodiments, the cells are adherently grown in a cell module, the magnetic fluid culture fluid in the cell module being between the magnetic field supply module and the cells in the cell module in a spatial positional relationship.
In some embodiments, the magnetic field supply module is a magnet array composed of a number of cylindrical magnets; the cell module is a cell culture pore plate, and each independent pore of the cell culture pore plate is filled with mixed liquid of nonmagnetic target particles carrying genes, medicines or signals and magnetic fluid culture solution for culturing cells.
In some embodiments, the cell module is a cell culture well plate, each individual well of the cell culture well plate contains a mixed solution of nonmagnetic target particles carrying genes, drugs or signals and a magnetic fluid culture solution for culturing cells, the magnetic field supply module is disposed above the cell module, under the action of negative magnetophoretic force, the magnetic fluid has a tendency of moving away from the cells under the action of a magnetic field, and the nonmagnetic target particles carrying genes, drugs or signals move towards the cells in the cell module.
In some embodiments, the magnet array is disposed in a magnet well plate, the magnet well plate has the same structure as the cell culture well plate, a columnar magnet is fixed in each independent well of the magnet well plate to form the magnet array, and each independent well of the magnet array in which the columnar magnet is disposed corresponds to each independent well of the cell culture well plate in the cell module.
In some embodiments, the magnet array is restrained by using a large magnet to prevent the columnar magnets in the magnet array from popping out of the orifice plate.
In some embodiments, the design and preparation method of the magnet array provided in the present invention is specifically as follows:
(1) selecting a pore plate which is the same as that used for cell culture, and placing a rectangular magnet with the area not smaller than that of the pore plate below the pore plate;
(2) selecting columnar magnets with the diameters and heights close to the aperture and height of each independent hole of the orifice plate, and filling the columnar magnets into the orifice plate, wherein the N/S pole directions of the magnets in each independent hole are kept consistent in the filling process; the rectangular magnets can be used for restraining the columnar magnets to prevent the magnets from popping out of the pore plate due to repulsive force among the columnar magnets;
(3) after filling, the cylindrical magnets are fixed by glue or adhesive tape and the cover is sealed, so that the cylindrical magnets in the magnet array are prevented from popping up after the rectangular magnets are removed.
FIG. 1 is a schematic diagram of a magnet array and its fabrication, wherein 1 is a blank well plate; 2, a cylindrical magnet matched with the hole diameter of the hole plate; 3 is a rectangular magnet with large area and strong magnetism; and 4, preparing a magnet array.
The invention also provides a design and manufacturing method of the magnet array. In the conventional method for preparing the magnet array, a column magnet is not placed in each well of the well plate or the violent interaction is avoided by placing magnets with different N/S poles in different wells. The preparation method of the magnet array in the preferred embodiment of the invention can realize that the magnets with the same N/S poles are arranged in each hole of the pore plate, so that the finally obtained magnet array has better uniformity of the magnetic field environment. The key point of the method is that the magnet to be placed in the pore plate is restrained by the magnet with large area and strong magnetism in the preparation process, the magnet is prevented from popping out of the pore plate due to mutual magnetic force, and after the fixation is finished, the magnet with large area and strong magnetism is taken away to obtain the required magnet array.
Gene transfection refers to the transfer or transport of a biologically functional nucleic acid into a cell and the maintenance of the biological function of the nucleic acid within the cell. The drug targeting aims at a specific pathological change part to form relatively high concentration locally, so that the damage to normal tissues and cells is reduced. Signal transduction refers to the process of transmitting various signals into cells through the cell membrane, gradually causing changes in the cellular material, primarily proteins. The common technique required by the three is how to deliver targeted particles (particles carrying genes, drugs or signals) to specific target areas in a targeted and efficient manner. At present, an effective method is to perform magnetic modification on the microparticles and then realize the directional movement of the microparticles under the guidance of a magnetic field, i.e. a labeled gene transfection/drug targeting/signal transduction method. However, the magnetic modification operation process is complicated, the technical threshold is high, and the large-scale practical application is not facilitated.
Compared with the traditional marked gene transfection/drug targeting/signal conduction method, the invention has the advantages that on one hand, the particles carrying the genes/drugs/signals do not need to be magnetically marked, and on the other hand, the traditional non-magnetic culture solution is changed into the magnetic fluid culture solution with high biocompatibility. According to the characteristics of negative magnetophoresis, under the action of a magnetic field, the nonmagnetic particles are also subjected to gradient magnetic field force in the direction opposite to the action direction of the traditional magnetic particles in the magnetofluid solution. The method is used for gene transfection/drug delivery/signal conduction experiments, is simple to operate, has small equipment dependence, and has important scientific research value and clinical treatment significance. In addition, the invention also provides a design and a manufacturing method of the columnar magnet array magnetic field source for realizing magnetic operation.
In the traditional marker-type gene transfection/drug targeting/signal transmission method, in order to control the gene/drug/signal to transfer to the cell direction, the magnetic modification is generally carried out on the particle carrying the gene/drug/signal, then the modified particle carrying the gene/drug/signal is added into a cell pore plate for culturing the cells, and the cells are cultured for a period of time under the environment of an external gradient magnetic field. As shown in fig. 1, 5 is a well plate in which cells are grown at the time of gene transfection/drug targeting/signaling; 6 is magnetic nano-particles; 7 is target particles carrying genes/signals/drugs; 8 is the target particle carrying the gene/signal/drug after magnetic modification; 9 is a traditional non-magnetic cell culture solution; cells cultured in the well plate are 10. FIG. 1 is a left side view of a magnetic array prepared by a conventional method and a well plate with cells for gene transfection/drug targeting/signal transduction experiments, which are equivalent to a magnetic field supply module and a cell module in the system of the present invention, wherein the magnetic field supply module is located below the cell module; the upper right drawing of FIG. 2 is one of the independent wells in the cell well plate, and the lower right drawing of FIG. 2 is the column magnet in one of the independent wells in the magnetic field array. As can be seen from fig. 2, in the conventional marker-type gene transfection/drug targeting/signaling method, complicated magnetic modification operations must be performed on target particles carrying genes/drugs/signals, the process is difficult, and the magnetic nanoparticles used for performing magnetic modification enter cells along with the target particles, thereby hindering the subsequent steps of gene transfection/drug targeting/signaling and causing additional cytotoxicity.
However, unlike the conventional labeled gene transfection/drug targeting/signal conduction method, the non-labeled gene transfection/drug targeting/signal conduction method based on the negative magnetophoresis technology of the present invention does not require magnetic modification of the particles carrying the genes/drugs/signals when controlling the genes/drugs/signals to be transferred to the cell direction, and instead, the original non-magnetic culture solution is changed to a magnetofluid culture solution that exhibits magnetism when an external magnetic field is applied, and at the same time, the position of the external magnetic field source is changed so that the direction of the magnetic field gradient at the culture solution is opposite, as shown in fig. 3, in which 11 the magnetofluid cell culture solution of magnetic nanoparticles is dispersed; the other serial numbers are marked as same as fig. 2, and the left side of fig. 3 is a magnetic array prepared by the traditional method and a pore plate for culturing cells in gene transfection/drug targeting/signal conduction experiments, namely the magnetic field supply module and the cell module in the system of the invention, wherein the magnetic field supply module is positioned above the cell module; FIG. 3 is a right upper view of a cylindrical magnet in an individual hole in a magnetic field array; FIG. 3 right lower panel shows one of the individual wells in the cell well plate. As can be seen from fig. 3, in the non-labeled gene transfection/drug targeting/signal transduction method of the present invention, no additional complex magnetic modification operation is required for the target particles carrying the genes/drugs/signals, the process is simple, and no magnetic nanoparticles enter the cells along with the target particles to hinder the subsequent steps of gene transfection/drug targeting/signal transduction or cause toxicity to the cells, so the efficiency of gene transfection/drug targeting/signal transduction is higher and the safety is better.
The following are examples:
example 1
The invention provides a gene transfection method based on a negative magnetophoresis technology, which comprises the following steps:
1.1 cell culture
HEK293 cells in DMEM medium containing 10% fetal calf serum, 1% penicillin, streptomycin, and at 37 deg.C containing 5% CO2Culturing in an incubator.
1.2 preparing magnetic fluid culture solution with high biocompatibility
And adding ferroferric oxide magnetic nanoparticles with the particle size of about 15nm into DMEM culture solution without fetal calf serum, wherein the concentration is 5g/L, and uniformly stirring to obtain the magnetofluid culture solution.
1.3 preparation of citric acid-modified gold nanoparticles
With freshly prepared aqua regia (HCI-HNO)33: 1) was immersed in a 100mL flat-bottomed flask and thoroughly washed with ultrapure water. 50mL of ultrapure water and 2mL of 1% HAuCl were added in this order4Heating the aqueous solution to boiling, rapidly adding 1mL of 5% (w/v) trisodium citrate solution under vigorous stirring, keeping boiling and continuously and vigorously stirring, wherein the solution color changes from light yellow to colorless and transparent, gradually becomes black, and finally becomes bright wine red, stopping heating after reacting for 20min, continuously and vigorously stirring until the solution is cooled to room temperature, filtering with a 0.22 mu m filter membrane, collecting the filtrate, and storing at 4 ℃ for later use. Measured by U-3010 UV-visible spectrophotometer, the maximum absorption wavelength is 518nm according to the absorption spectrum of synthetic gold gel and the epsilon reported in literature(13nm，AuNPs)＝2.7ⅹ108cm-1M-1According to the Lambert-beer law, the concentration of the citric acid modified gold nanoparticles can be calculated.
1.4 preparation of Gene-carrying target particles
10mL of citric acid-modified gold nanoparticle solution (concentration of 5nmol/L) was added with 1mg of pEGFP-c1 plasmid and 2mg of polyethyleneimine (branched, 25kDa), mixed well, stirred, and allowed to stand for 6 hours.
1.5 Gene transfection
HEK293 cells were seeded in 96-well plates 10 per well4100 μ L of DMEM medium containing 10% fetal bovine serum, 1% penicillin and streptomycin was added thereto and cultured overnight. The gene-carrying target particle solution was added to the above prepared magnetic fluid culture medium (volume 1: 1), mixed well, and 100. mu.L of each well was added to a 96-well cell-cultured plate. Then, the home-made magnet array was placed on a cell culture plate, and after 60min, the solution was aspirated and replaced with a normal culture solution. After 48 hours, the gene transfer can be detected by an inverted fluorescence microscope, a flow cytometer or an enzyme labeling instrument and the likeThe efficiency of the dyeing.
Example 2
A drug delivery method based on a negative magnetophoresis technology comprises the following steps:
2.1 cell culture
Hela cells were cultured in DMEM containing 10% fetal calf serum, 1% penicillin, and streptomycin at 37 deg.C and 5% CO2Culturing in an incubator.
2.2 preparing a high-biocompatibility magnetofluid culture solution
Adding ferroferric oxide magnetic nanoparticles with the particle size of about 15nm into DMEM culture solution without fetal calf serum, wherein the concentration is 5g/L, and uniformly stirring.
2.3 preparation of citric acid-modified gold nanoparticles
With freshly prepared aqua regia (HCI-HNO)33: 1) was immersed in a 100mL flat-bottomed flask and thoroughly washed with ultrapure water. 50mL of ultrapure water and 2mL of 1% HAuCl were added in this order4Heating the aqueous solution to boiling, rapidly adding 1mL of 5% (w/v) trisodium citrate solution under vigorous stirring, keeping boiling and continuously and vigorously stirring, wherein the solution color changes from light yellow to colorless and transparent, gradually becomes black, and finally becomes bright wine red, stopping heating after reacting for 20min, continuously and vigorously stirring until the solution is cooled to room temperature, filtering with a 0.22 mu m filter membrane, collecting the filtrate, and storing at 4 ℃ for later use. Measured by U-3010 UV-visible spectrophotometer, the maximum absorption wavelength is 518nm according to the absorption spectrum of synthetic gold gel and the epsilon reported in literature(13nm，AuNPs)＝2.7ⅹ108cm-1M-1According to the Lambert-beer law, the concentration of the citric acid modified gold nanoparticles can be calculated.
2.4 preparation of drug-carrying target particles
Taking 10mL of citric acid modified gold nanoparticle solution (the concentration is 5nmol/L), adding 0.020mmol of sulfenyl PEGMA, and reacting in a water bath shaker at 37 ℃ and 200rpm for 12h for sulfenyl modification. After washing, the mixture was collected by centrifugation and redispersed in 25mL of deionized water. 2mg of water-soluble tetra-sulfo aluminophthalocyanine was added thereto, and the mixture was vortexed on a vortexer for 24 hours, and the precipitate was collected by centrifugation.
2.5 drug delivery
Hela cells were seeded in 96-well plates 10 per well4100 μ L of DMEM medium containing 10% fetal bovine serum, 1% penicillin and streptomycin was added thereto and cultured overnight. The drug-carrying target particle solution was added to the above prepared magnetic fluid culture medium (volume 1: 1), mixed well and added to 100. mu.L per well of a 96-well cell-grown plate. Then, the home-made magnet array was placed on a cell culture plate, and after 4 hours, the solution was aspirated and replaced with a normal culture solution. The efficiency of drug delivery can be measured after 48h by flow cytometry or the like.
Example 3
The signal transmission method based on the negative magnetophoresis technology comprises the following steps:
3.1 cell culture and Virus expansion
HEp-2 cells were cultured in DMEM medium containing 10% fetal bovine serum, 1% penicillin, and streptomycin at 37 deg.C and 5% CO2Culturing in an incubator.
3.2 preparing the culture solution of magnetic fluid with high biocompatibility
Adding ferroferric oxide magnetic nanoparticles with the particle size of about 15nm into DMEM culture solution without fetal calf serum, wherein the concentration is 5g/L, and uniformly stirring.
3.3 preparation of citric acid modified gold nanoparticles
With freshly prepared aqua regia (HCI-HNO)33: 1) was immersed in a 100mL flat-bottomed flask and thoroughly washed with ultrapure water. 50mL of ultrapure water and 2mL of 1% HAuCl were added in this order4Heating the aqueous solution to boiling, rapidly adding 1mL of 5% (w/v) trisodium citrate solution under vigorous stirring, keeping boiling and continuously and vigorously stirring, wherein the solution color changes from light yellow to colorless and transparent, gradually becomes black, and finally becomes bright wine red, stopping heating after reacting for 20min, continuously and vigorously stirring until the solution is cooled to room temperature, filtering with a 0.22 mu m filter membrane, collecting the filtrate, and storing at 4 ℃ for later use. Measured by U-3010 UV-visible spectrophotometer, the maximum absorption wavelength is 518nm according to the absorption spectrum of synthetic gold gel and the epsilon reported in literature(13nm，AuNPs)＝2.7ⅹ108cm-1M-1According to the Lambert-beer law, the concentration of the citric acid modified gold nanoparticles can be calculated.
3.4 preparation of target particles carrying Signal
10mL of citric acid-modified gold nanoparticle solution (5 nmol/L) was taken and adjusted to pH 9 with 0.1mol/L NaOH. Add 100. mu.L alkaline phosphatase ALP (50. mu.g mL)-1) Mixing, stirring at room temperature l h; then, 500. mu.L of 5% bovine serum albumin (w/v) was added to the above solution, and the mixture was stirred at room temperature for 0.5 hour, the active sites on the surface of the gold gel were blocked, centrifuged at 15000rpm for 15min to remove excess enzyme, and the precipitate was resuspended in a phosphate buffer containing 0.5% bovine serum albumin and stored at 4 ℃ for further use.
3.5 Signal transduction
HEp-2 cells were plated in 96-well plates 10 per well4100 μ L of DMEM medium containing 10% fetal calf serum, 1% penicillin and streptomycin was added thereto and cultured overnight. The target particle solution carrying the signal is added into the prepared magnetic fluid culture solution (volume is 1: 1), and after uniform mixing, 100 mu L of the target particle solution is added into each hole of a 96-hole plate with cells. Then, the home-made magnet array was placed on a cell culture plate, and after 4 hours, the solution was aspirated and replaced with a normal culture solution. The efficiency of signal transmission can be detected after 48h by a microplate reader or the like.
It will be understood by those skilled in the art that the foregoing is only a preferred embodiment of the present invention, and is not intended to limit the invention, and that any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the scope of the present invention.

Claims (10)

1. A gene transfection/drug delivery/signal conduction method based on negative magnetophoresis technology is characterized in that a magnetofluid culture system cultured with cells is placed in a magnetic field environment, the magnetofluid culture system contains a magnetofluid culture solution, the magnetofluid culture solution integrally shows magnetism under the action of a magnetic field, and the magnetofluid culture solution is acted by a magnetic field force; the magnetic fluid culture solution has biocompatibility and can be used for culturing cells;
the magnetic fluid culture system also comprises non-magnetic target particles carrying genes, medicines or signals; the magnetic permeability of the non-magnetic target particles carrying the genes, the medicines or the signals is lower than that of the magnetic fluid culture solution;
under the action of an external magnetic field, according to the negative magnetophoresis characteristic, magnetization difference exists between the nonmagnetic target particles carrying genes, medicines or signals and the magnetofluid culture solution, and the force direction of a gradient magnetic field borne by the nonmagnetic target particles is a magnetic field gradient direction corresponding to the magnetic field intensity from large to small;
by regulating and controlling the relative position between the magnetic fluid culture system and the magnetic field source, the non-magnetic target particles carrying genes, medicines or signals move towards the direction of the cells cultured by adherence under the action of the gradient magnetic field force, thereby realizing gene transfection, medicine transmission or signal conduction of the cells.
2. The method of claim 1, wherein the cells are in adherent growth and the magnetic fluid culture fluid is in a spatial position between the cells and a magnetic field source.
3. The method of claim 1, wherein the magnetic fluid culture fluid is a magnetic fluid that is biocompatible and suitable for cell survival.
4. The method of claim 1, wherein the gene, drug or signal bearing non-magnetic target particles are derived from gene, drug or signal binding to non-magnetic target carrier particles by chemical, hydrogen, hydrophobic, electrostatic or van der waals forces, or from gene, drug or signal binding to non-magnetic target carrier particles by adsorption, entrapment or cross-linking.
5. A gene transfection/drug delivery/signaling system based on negative magnetophoresis technology, comprising:
a magnetic field supply module for providing a magnetic field environment;
the cell module is used for providing a magnetic fluid culture system for culturing cells, the magnetic fluid culture system contains a magnetic fluid culture solution, the magnetic fluid culture solution integrally shows magnetism under the action of a magnetic field, and the magnetic fluid culture solution is acted by the action of a magnetic field force; the magnetic fluid culture solution has biocompatibility and can be used for culturing cells; the magnetic fluid culture system also comprises non-magnetic target particles carrying genes, medicines or signals; the magnetic permeability of the non-magnetic target particles carrying the genes, the medicines or the signals is lower than that of the magnetic fluid culture solution;
under the action of an external magnetic field, according to the negative magnetophoresis characteristic, magnetization difference exists between the nonmagnetic target particles carrying genes, medicines or signals and the magnetofluid culture solution expressing magnetism, and the force direction of a gradient magnetic field borne by the nonmagnetic target particles is a magnetic field gradient direction corresponding to the magnetic field intensity from large to small; by regulating and controlling the relative position between the magnetic fluid culture system and the magnetic field source, the non-magnetic target particles carrying genes, medicines or signals move towards the direction of the cells cultured by adherence under the action of the gradient magnetic field force, thereby realizing gene transfection, medicine transmission or signal conduction of the cells.
6. The system of claim 5, wherein the cells are adherently grown in a cell module, and the magnetic fluid culture fluid in the cell module is between the magnetic field supply module and the cells in the cell module in a spatial position relationship.
7. The system of claim 5, wherein the magnetic field supply module is a permanent magnet or an electromagnet to provide a gradient magnetic field.
8. The system of claim 5, wherein the magnetic field supply module is a magnet array consisting of a plurality of cylindrical magnets;
the cell module is a cell culture pore plate, and each independent pore of the cell culture pore plate is filled with a magnetic fluid culture system formed by mixing non-magnetic target particles carrying genes, medicines or signals and a magnetic fluid culture solution for culturing cells.
9. The system of claim 8, wherein the magnet array is disposed in a magnet well plate, the magnet well plate has the same structure as the cell culture well plate, a column magnet is fixed in each individual well of the magnet well plate to form the magnet array, and each individual well of the magnet array is located in an individual well corresponding to each individual well of the cell culture well plate in the cell module.
10. The system of claim 9, wherein the array of magnets is restrained by the use of magnets to prevent the columnar magnets in the array of magnets from ejecting from the orifice plate.
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