Nanoparticles for regulating opening and closing of ion transport membrane protein and method for controlling opening and closing of ion transport membrane protein and inhibiting cell activity using the same

Abstract

The present invention relates to a nanoparticle for regulating cell activity and a method for regulating cell activity using the same, comprising: a core; a shell composed of a plurality of magnetic particles disposed on the surface of the core; and a ligand coupled to the surface of the core and capable of specifically binding to an ion transport membrane protein of a cell surface target. The nanoparticle according to the present invention can bind to a specific ion transport membrane protein in which a labeled protein is expressed, and by rotating the nanoparticle according to a magnetic field formed outside the organism, the opening / closing or activity of the ion transport membrane protein is regulated, thereby regulating cell activity. According to the present invention, since the opening / closing, activity, or cell activity of the ion transport membrane protein can be regulated by a non-invasive method, it can be usefully employed in magnetogenetic therapy.

Description

Technology Field

[0001] The present invention relates to nanoparticles for regulating cell activity and a method for regulating cell activity using the same. More specifically, the invention relates to nanoparticles comprising a core, a shell composed of a plurality of magnetic particles disposed on the surface of the core, and a ligand capable of specifically binding to a target ion transport membrane protein on the cell surface, and a method for regulating cell activity using the same. The nanoparticles rotate according to the formation of a magnetic field, thereby regulating the opening and closing of the bound ion transport membrane protein, which enables the regulation of the activity of the target cell. Background Technology

[0002] Techniques for inhibiting neural activity have been attempted in various ways in the past. While much research has been conducted on the inhibition of neural activity through drugs, electrical stimulation, or stimulation using only magnetic fields, and these methods are currently used as treatments, these three methods have limitations in that they cannot target specific neurons.

[0003] Methods for regulating cell-specific neural activity through optogenetics or chemogenetics have also been developed in recent years. Optogenetic methods, which activate ion transport membrane proteins using light, offer the advantages of high speed and precise targeting. However, regarding the delivery of light into the body, most mammalian tissues have low light permeability, which limits the ability to deliver light to target cells through relatively invasive techniques. In contrast, magnetic fields can penetrate bone or tissue without loss of magnetic force, and can reach ion transport membrane proteins and magnetic nanoparticles present in the body even from a magnetic field generator installed at a distance (~70 cm) from the target. These characteristics suggest that magnetogenetics offers the potential to be a more non-invasive method than optogenetics for regulating proteins within the body.

[0004] Meanwhile, chemogenetic methods involve injecting chemicals into the blood vessels to activate membrane proteins expressed in target cells, thereby enabling the regulation of the nervous system; they can be considered a relatively non-invasive method. However, chemogenetic methods have limitations in that they cannot precisely regulate neural activity for a specific period of time, as the regulation of neural activity occurs over a long period (approximately 30 minutes to 2 hours) after the injection of chemicals.

[0005] Magnetogenetic methods activate ion transport membrane proteins expressed in target cells through the rotation of an ex vivo magnetic field generator and the rotation of an internal magnetic field. Since this enables the inhibition of neural activity for relatively precise durations in the second, there is a need for research to regulate cell activity using magnetogenetic methods. The problem to be solved

[0006] The present invention aims to solve the problems described above, and one of the various objectives of the present invention is to provide a non-contact / non-invasive ion transport membrane protein opening and closing control system and a method for controlling cell activity.

[0007] One of the various objectives of the present invention is to provide a cell activity regulation system and method capable of regulating cell activity while maintaining biological activity.

[0008] One of the various objectives of the present invention is to provide a composition of nanoparticles for controlling the opening and closing of ion channels and a composition for the expression of ion channels.

[0009] One of the various objectives of the present invention is to provide a method for generating ion current and regulating cell membrane potential through the control of opening and closing of ion transport membrane proteins.

[0010] One of the various objectives of the present invention is to provide a method for regulating the activity of a cell (neurocell) through the regulation of the opening and closing of an ion transport membrane protein capable of inhibiting neural activity.

[0011] The present invention also aims to provide a method for inhibiting neural activity using the above-described system and method. means of solving the problem

[0012] To achieve the above objective, the present invention provides a nanoparticle for controlling the opening and closing of an ion transport membrane protein and regulating cell activity, comprising: a core; a shell composed of a plurality of magnetic particles disposed on the surface of the core; and a ligand coupled to the surface of the core and capable of specifically binding to a target ion transport membrane protein on the cell surface.

[0013] In the present invention, “cell” refers to a cell present within a target body into which nanoparticles are injected. While the type of cell present within the target body is not limited, the cell refers to a cell that is excitable and whose function can be regulated by changes in cell membrane potential. Preferably, the cell of the present invention is a cell whose function can be regulated by the inflow or outflow of ions through the cell membrane. The cell of the present invention may include nerve cells, muscle cells (e.g., skeletal muscle cells, cardiac cells, smooth muscle cells, etc.), and endocrine cells (e.g., insulin-secreting pancreatic β cells, etc.).

[0014] The term “regulation of cell activity” in the present invention means promoting or inhibiting the operation or function of a cell, and in the present invention, preferably means inhibiting the activity of a cell.

[0015] In the present invention, “specific binding” refers to binding that is not random, and, for example, means that nanoparticles selectively bind only to a target ion transport membrane protein.

[0016] In the present invention, the core may be made of at least one inorganic material selected from the group consisting of polymers, ceramics, metal matrix composites (MMC) and ceramic matrix composites (CMC).

[0017] The magnetic particles of the present invention can generate mechanical force by a magnetic field and, in terms of structure, include but are not limited to an inverse spinel structure or an octahedral structure.

[0018] An example of the ion transport membrane protein of the present invention includes an anion transport membrane protein, and a label protein capable of binding to the ligand may be expressed on the surface (preferably, an extracellular domain) of the ion transport membrane protein.

[0019] According to one embodiment of the present invention, when a magnetic field is formed outside an object into which nanoparticles are injected, the nanoparticles rotate due to a magnetic reaction, and depending on the rotation, the ion transport membrane protein to which the nanoparticles are bound opens, closes, or is activated, thereby allowing the activity of the cell in which the ion transport membrane protein is expressed to be regulated.

[0020] The present invention also provides a cell activity control system comprising: a nanoparticle having a core-shell structure, comprising a core, a shell composed of a plurality of magnetic particles disposed on the surface of the core, and a ligand coupled to the surface of the core and capable of specifically binding to a target ion transport membrane protein on the cell surface; an ion transport membrane protein expressed on the cell membrane of an organism and to which the nanoparticle specifically binds; and a magnetic field generator having an internal space in which the organism can be disposed and forming a rotating magnetic field, wherein the ion transport membrane protein is opened / closed or its activity is regulated according to the rotation of the coupled nanoparticle when the magnetic field is generated.

[0021] In the system of the present invention, the nanoparticle rotates according to the formation of an external magnetic field, and when the nanoparticle rotates, the ion transport membrane protein opens and closes, thereby allowing the activity of the cell to be regulated.

[0022] When the ion transport membrane protein of the present invention is an ion channel, the ion channel is opened according to the rotation of the nanoparticle, and when the ion transport membrane protein is an ion pump, the ion pump is activated according to the rotation of the nanoparticle, so that the activity of the cell can be regulated.

[0023] The ion transport membrane protein of the present invention is preferably an anion transport membrane protein or a cation transport membrane protein that inhibits cell activity when opened, and according to the present invention, when rotation of the nanoparticle occurs, the anion transport membrane protein or the cation transport membrane protein opens so that anions flow into the cell, thereby inhibiting cell activity.

[0025] The present invention also provides a method for controlling the opening and closing of an ion transport membrane protein and a method for controlling the activity of a cell, comprising: (a) a step of preparing a core-shell structured nanoparticle comprising a core, a shell composed of a plurality of magnetic particles disposed on the surface of the core, and a ligand coupled to the surface of the core and capable of specifically binding to a target ion transport membrane protein on the cell surface; (b) a step of forming an ion transport membrane protein in a cell of a target having a label protein capable of binding to the ligand expressed on its surface; (c) a step of injecting the nanoparticle having the ion transport membrane protein formed thereon into the target having the ion transport membrane protein formed in step (b); and (d) a step of forming a magnetic field outside the target to induce rotation of the nanoparticle.

[0026] In the present invention, when a magnetic field is formed according to step (d), the nanoparticle rotates due to a magnetic reaction, and the ion transport membrane protein can be opened according to the rotation of the nanoparticle.

[0027] The present invention provides a method for generating ion current and regulating cell membrane potential through the regulation of the opening of the ion transport membrane protein, and a method for regulating cell activity through the same.

[0028] To achieve the above objective, the present invention comprises: 1) a step of producing an anion transport membrane protein capable of binding with the nanoparticles and capable of opening and closing using a magnetogenetic technique,

[0029] 2) a step of expressing the above ion transport membrane protein in target cells and binding it by treating with nanoparticles, and

[0030] 3) A method for inducing the inflow of negative ions into the cell or the outflow of positive ions into the cell of a target cell using a magnetogenetic technique comprising the step of activating an ion transport membrane protein by applying a rotating magnetic field to the above-mentioned nanoparticles.

[0031] The present invention may further perform the step of inhibiting the neural activity of the target neuron, wherein the target cell may be a neuron, and this includes application outside the body (in vitro) and inside the body (in vivo).

[0032] In step 1) above, a binding nucleotide sequence capable of mediating binding with a binding site of a nanoparticle is synthesized on the extracellular loop domain of an ion transport membrane protein that is exposed outside the cell.

[0033] The methods and sites of the above binding include, but are not limited to, antibody-antigen binding, nanobody-antigen binding, and ligand-receptor binding.

[0034] In step 2) above, genetic information of an ion transport membrane protein is introduced into a target cell in a form suitable for the target cell, so that the corresponding ion transport membrane protein is expressed (see Fig. 2). The method of introducing the gene in this step includes a method using a viral vector, which is a step of producing a viral vector having the corresponding ion transport membrane protein and injecting the virus having the gene of the corresponding ion transport membrane protein into a target cell or animal tissue.

[0035] After the above-mentioned gene introduction, a step of expressing the corresponding ion transport membrane protein within the target cell is performed. If a virus is selected as the method for gene introduction, it takes 2 to 3 weeks. After the expression of the anion transport membrane protein in the target cell, as a step to induce antigen-antibody binding between the ion transport membrane protein and the nanoparticle, the nanoparticle is injected into the environment where the target cell is present (the nanoparticle may be injected into the target in the form of a composition containing multiple nanoparticles). Binding between the ion transport membrane protein and the nanoparticle is possible and takes 1 hour to 1 day.

[0036] In step 3) above, a rotating magnetic field is generated in the environment where the target cell is present using a rotating magnetic field generator. The rotating magnetic field consists of a uniform magnetic field (25 mT to 50 mT) in the form of concentric circles rotating in a clockwise or counterclockwise direction. This rotating magnetic field induces the rotation of nanoparticles within the magnetic field region, thereby generating rotational force. This rotational force is transmitted to the anion transport membrane protein to which the nanoparticle is bound, resulting in the influx of anions into the target cell. The influx of anions into the target cell causes a change in membrane potential.

[0037] The nanoparticles of the present invention generate a rotational force of 10 pN nm or more when rotated, and preferably, can generate a rotational force sufficient not to destroy the cell membrane of the bound cell. For example, the rotational force of the nanoparticles in the present invention may be 10 pN·nm to 1 nN·nm.

[0038] If the inflow of negative ions or the outflow of positive ions is induced in a neuron through the above method, the activity of the target cell (e.g., neuron) can be inhibited as a result. Effects of the invention

[0039] The nanoparticles according to the present invention can bind to a specific ion transport membrane protein in which a labeled protein is expressed, and by rotating the nanoparticles according to a magnetic field formed outside the organism, the opening and closing of the ion transport membrane protein is controlled, thereby regulating cell activity. According to the present invention, since the opening and closing of the ion transport membrane protein and cell activity can be controlled in a non-invasive manner, it can be usefully employed in magnetogenetic therapy. Brief explanation of the drawing

[0040] FIG. 1 is a schematic diagram of a system for regulating cell activity (regulating the opening and closing of ion transport membrane proteins) according to one embodiment of the present invention. Referring to FIG. 1, it can be seen that nanoparticles in vivo (m-torquer in FIG. 1) selectively bind to ion transport membrane proteins, and that the opening and closing of ion transport membrane proteins is regulated according to the rotation of the nanoparticles when an external magnetic field is generated. Figure 2 is a schematic diagram of a magnetic particle forming the shell of a nanoparticle according to the present invention. FIG. 3 is a schematic diagram showing the structure of a nanoparticle according to the present invention. The nanoparticle of the present invention has a core-shell structure, and magnetic particles may be sporadically arranged in the shell. The magnetic particles in the present invention may be connected to the core by 1,2,3-Triazole bonds. Figure 4 is an SEM image of nanoparticles prepared according to the present invention. The nanoparticles according to the present invention have a particle size of about 100 to 900 nm. Figure 5 shows the results of comparing the magnetic moments of the octahedral magnetic particles and nanoparticles of the present invention. Referring to Figure 5, it can be seen that the nanoparticles according to the present invention have a magnetic moment that is about 470 times higher than that of the octahedral magnetic particles. FIG. 6 is a schematic diagram showing the difference in results depending on the type of cell in which the ion transport membrane protein is formed when a magnetic field is applied to a nanoparticle bound to an ion transport membrane protein according to an embodiment of the present invention. As the nanoparticle rotates, the ion transport membrane protein opens, and if the cell in which the ion transport membrane protein is formed is a neuron, the activity of the corresponding cell can be regulated. At this time, if the ion transported by the membrane protein is a cation and the transport direction is into the cell, depolarization of the cell membrane potential occurs through the opening, and the activity of the neuron can be induced. If the ion transported by the membrane protein is a cation but the transport direction is outside the cell, hyperpolarization of the cell membrane potential occurs through the opening, and the activity of the neuron can be inhibited. On the other hand, if the ion transported by the membrane protein is an anion, hyperpolarization of the cell membrane potential occurs through the opening, and the activity of the neuron can be inhibited. Figure 7 is an image confirming the formation of a label protein according to an embodiment of the present invention. DmFLYC1, an ion channel in Figure 7, is a codon-optimized DmFLYC1-mScarletI fusion protein (blue and red in Figure 7), and a label protein, Myc tag, is generated on the extracellular domain for the binding of nanoparticles (green in Figure 7). When the corresponding DmFLYC1 is expressed in HEK cells, an antibody signal (green at the bottom of Figure 7) binding to the Myc tag is observed in cells containing RFP (red at the bottom of Figure 7), indicating that the label protein (MYC) can bind to the portion of FLYC protruding outside the cell. FIG. 8 is an image confirming whether a nanoparticle according to one embodiment of the present invention binds to an ion transport membrane protein. Referring to FIG. 8, it can be seen that the nanoparticle MNP (green in FIG. 8) is bound to an extracellular protrusion (extracellular domain in the present invention) of an ion transport membrane protein (blue in FIG. 8). Figure 9 shows a method for confirming the inflow of Cl- using the MQAE quenching technique. The top image of Figure 9 shows the Cl- binding reaction equation of MQAE, and the bottom image of Figure 9 shows that the fluorescent particle MQAE is quenched when Cl- ions are introduced according to the rotation of the nanoparticle in HEK293 cells containing FLYC1 ion channels. Figure 10 is a graph comparing the fluorescence values ​​of an ion channel without a label protein (left graph of Figure 10) and an ion channel with a label protein (right graph of Figure 10) after injecting MQAE into HEK293 cells according to an embodiment of the present invention. Referring to the left graph of Figure 10, when the label protein is not present in the ion channel, it has a constant fluorescence value regardless of the presence of nanoparticles (MNP) and a magnetic field (rotating magnetic field; RMF), whereas according to the right graph of Figure 10, in the case of an ion channel with a label protein, if nanoparticles and a magnetic field are present, the F0 / F value increases, indicating that the fluorescent particles have been quenched, which indicates that the ion channel has opened and Cl- ions have entered the cell. FIG. 11 illustrates that, according to one embodiment of the present invention, the concentration of chloride accumulated in the cell increases in mutants due to the activation of DmFLYC1 by a rotating magnetic field and nanoparticles, based on the measured fluorescence level. Nanoparticle and rotating magnetic field stimulation were applied under all conditions, and each condition represents a mutant type of the genetically introduced DmFLYC1. Figure 12 shows the results of confirming the cell potential when a magnetic field is applied for a certain period of time after treating a cell with an ion channel formed containing a labeled protein according to an embodiment of the present invention with nanoparticles. Referring to the top graph of Figure 12, it can be seen that the potential decreases when a magnetic field is applied (red arrow), indicating that Cl- has flowed into the cell due to the application of the magnetic field. Through this, the ion channel has opened, and it can be inferred that this occurred due to the rotation of the nanoparticle caused by the application of the magnetic field. On the other hand, looking at the bottom graph of Figure 12, when a magnetic field is not applied, the section where the potential regularly decreases disappears, indicating that the reason for the decrease in potential shown in the top graph of Figure 12 is due to the application of the magnetic field. FIG. 13 is a schematic diagram illustrating a method for forming an ion transport membrane protein according to an embodiment of the present invention. The ion transport membrane protein of the present invention can be expressed in a cell by introducing a modified gene, and the modified gene can be directly applied to the cell or introduced into the cell by a virus (AAV or lentivirus). At this time, the modified gene may include a gene for forming a label protein. FIG. 14 is an image confirming the formation of an ion channel (FLYC) and a label protein (MYC) according to one embodiment of the present invention. Referring to FIG. 14, it can be seen that the label protein in a cell into which a gene sequence engineered according to the present invention has been introduced is expressed at the location where the ion channel is present. FIG. 15 is a schematic diagram showing the composition of an ion transport membrane protein formed in a neuron according to one embodiment of the present invention and a nanoparticle bound to the ion transport membrane protein. The nanoparticle rotates in response to the formation of a magnetic field outside the organism and opens an ion channel (preferably an anion channel), and during the period of magnetic field formation, the cell becomes hyperpolarized and neural activity is regulated. Specific details for implementing the invention

[0041] Various embodiments are presented below to aid in understanding the invention. The following embodiments are provided merely to facilitate a better understanding of the invention and do not limit the scope of protection of the invention to the following embodiments.

[0042] According to one embodiment of the present invention, a nanoparticle for controlling the opening and closing of an ion transport membrane protein is provided, comprising: a core; a shell composed of a plurality of magnetic particles disposed on the surface of the core; and a ligand coupled to the surface of the core and capable of specifically binding to a target ion transport membrane protein on the cell surface.

[0043] In the present invention, a “nanoparticle” is a magnetic particle that rotates upon the formation of a magnetic field (preferably a rotating magnetic field). The nanoparticles of the present invention have a particle diameter of 100 to 900 nm or preferably 200 to 500 nm, but are not limited thereto. The nanoparticles of the present invention have a core-shell structure consisting of a core and a shell surrounding it, and a ligand capable of specific binding to a target ion transport membrane protein may be bound to the surface of the core.

[0044] In the present invention, “core” refers to the center of a nanoparticle, and said core may be composed of a polymer, ceramic, metal matrix composite (MMC), or ceramic matrix composite (CMC), but is not particularly limited as long as it is capable of attaching magnetic particles to its surface.Specific examples of the above polymers include polyamide, polyurethane, polyethylene, polypropylene, polyacetal, polycarbonate, polybutylene terephthalate (PBT), polyethylene terephthalate (PET), polyethylene naphthalate (PEN), polytrimethylene terephthalate (PTT), polyhexamethylene terephthalate, polystyrene, polyvinyl chloride, polytetrafluoroethene, polyester (e.g., polylactic acid), polyether, polyether sulfone, polyether ether ketone, polyacrylate, polymethacrylate, polyimide, acrylonitrile butadiene styrene (ABS), polyphenylene sulfide, vinyl polymer, polyarylene ether, polyarylene sulfide, polysulfone, polyether ketone, polyamide-imide, polyetherimide, polyether ester, polyether block, and copolymer comprising polyamide block (PEBA or polyether block amide), grafted or non-grafted thermoplastic polyolefin, functionalized or non-functionalized ethylene / vinyl monomer polymer, functionalized or non-functionalized ethylene / alkyl (meth)acrylate, functionalized or non-functionalized (meth)acrylic acid polymer, functionalized or non-functionalized ethylene / vinyl monomer / alkyl (meth)acrylate terpolymer, ethylene / vinyl monomer / carbonyl terpolymer, ethylene / alkyl (meth)acrylate / carbonyl terpolymer, methyl methacrylate-butadiene-styrene (MBS)-type core-shell polymer, polystyrene-block-polybutadiene-block-poly(methyl methacrylate) (SBM) block terpolymer, chlorinated or chlorosulfonated polyethylene, polyvinylidene fluoride (PVDF), phenolic resin, poly(ethylene / vinyl acetate), polybutadiene, One or more selected from the group consisting of polyisoprene, styrene-based block copolymer, polyacrylonitrile, silicon, silica, and combinations thereof may be used, but are not limited thereto, and preferably polystyrene.

[0045] In the present invention, the “magnetic particles” form the shell of the nanoparticles, and a plurality of them may be arranged to surround the outside of the core. The magnetic particles of the present invention are composed of an inverse spinel structure and, preferably, may be particles with an octahedral structure. The magnetic particles of the present invention may be magnetite (Fe3O4), but are not limited thereto.

[0046] In the present invention, the average particle size of the magnetic particles may be greater than 5 nm. The average particle size of the nanoparticles is not specifically limited, but may refer to the D50 particle size measured by dynamic light scattering. The average particle size of the nanoparticles may be greater than 5 nm, greater than 6 nm, greater than 8 nm, greater than 10 nm, greater than 14 nm, greater than 18 nm, greater than 20 nm, greater than 22 nm, greater than 24 nm, or greater than 25 nm, and while the upper limit is not specifically limited, it may be, for example, 100 nm or less. The magnetic particles must possess magnetic anisotropy and, at the same time, must be attached to the aforementioned core in a sufficient amount so that the magnetic particles can generate high torque. If the magnetic particles are too small, it becomes difficult to possess magnetic anisotropy, and if the magnetic particles are too large, a sufficient amount of nanoparticles may not be attached to a single nanoparticle, or dispersibility in the solution may be significantly reduced during the manufacturing process.

[0047] The magnetic particles of the present invention can be connected to the core by chemical bonding. For example, a plurality of magnetic particles can each be connected to the core by 1,2,3-triazole bonds to form a shell.

[0048] In the present invention, the “ligand” is a binding site with a target ion transport membrane protein and can be bound to the surface of a nanoparticle, such as the surface of a core. The ligand of the present invention may consist of a specific binding protein or antibody, and may be bound to the core by covalent bonding or protein-protein interactions. For example, if the ligand of the present invention is an antibody, it may be attached to the core by covalent bonding using EDC / NHS chemistry or by interaction with an already attached Protein A (or Protein G).

[0049] In the present invention, “ion transport membrane protein” refers to a transport membrane protein and includes, but is not limited to, ion channels (proteins) and ion pumps (proteins), preferably being a mechanosensitive ion channel. The ion transport membrane proteins corresponding to the present invention may include membrane proteins that transport chloride anions (Cl-), such as MSL channels, FLYC channels, CLC channels, E-CLC channels, CLIC channels, CFTR channels, GABA receptors, glutamate receptors, etc., membrane proteins that non-specifically transport anions, such as McsS channels, MscL channels, SWELL channels, ANO channels, Maxi anion channels, etc., and other anion pumps. The ion channels corresponding to the present invention may include membrane proteins that transport potassium cations (K+), such as Kv channels, Kca channels, Kna channels, K2p channels, KscA channels, etc., and proton pump membrane proteins that transport hydrogen cations (H+), such as H-type, V-type, F-type, and P-type proton pumps, etc., and other cation transport membrane proteins, etc. Additionally, mutants of the proteins may be included. Examples of the above mutants may include a mutation of the DmFLYC1 protein, which may be a single and / or multiple mutant membrane protein mutated at the R599 (arginine 599), K606 (lysine 606), V656 (valine 656), and T659 (threonine 659) sites. According to a preferred embodiment of the present invention, a single mutant (e.g., R599K, K606R, V656L, and T659L), a double mutant (e.g., V656L / T659L), a triple mutant (e.g., R599K / V656L / T659L), or a quadruple mutant (e.g., R599K / K606R / V656L / T659L) or more mutants may be used. Hereinafter, in embodiments of the present invention, a DmFLYC1 mechanostimulating anion channel that transports chloride ions is used.According to a preferred embodiment of the present invention, the ion transport membrane protein of the present invention is an ion channel in which anions flow into the cell when it opens.

[0050] The ion transport membrane protein of the present invention is preferably a membrane protein in which a label protein is bound to an extracellular domain (a domain protruding outside the cell in an ion transport membrane protein).

[0051] When the ion transport membrane protein of the present invention is an ion channel, the ion transport membrane protein may open up depending on the rotation of the bound nanoparticle, thereby inhibiting cell activity; and when the ion transport membrane protein of the present invention is an ion pump, the ion pump may be activated (or its activity promoted) depending on the rotation of the bound nanoparticle, thereby inhibiting cell activity.

[0052] In the present invention, a label protein may be located on the surface of the ion transport membrane protein protruding outside the cell (in the present invention, the extracellular domain of the membrane protein). The “label protein (protein tag)” of the present invention is a specific protein capable of specifically binding to a ligand present in the nanoparticle, and the nanoparticle may be connected to an ion channel through the ligand and the label protein. The type of label protein of the present invention is not limited as long as it is a protein capable of interacting with the ligand, but preferably may include MYC-tag, HA tag, FLAG tag, etc. According to a preferred embodiment of the present invention, the ligand and the label protein may be connected via a streptavidin-biotin binding.

[0053] In the ion transport membrane protein of the present invention, a label protein can be expressed at a site exposed to the cell surface (i.e., a site exposed to the outside of the cell, i.e., the extracellular domain of the membrane protein) by introducing a recombinant gene sequence. According to one embodiment of the present invention, a label protein gene sequence is inserted into a position corresponding to the extracellular domain of the gene sequence of the ion transport membrane protein to be expressed, and after recombining the gene, it is introduced into a cell. In the introduced cell, an ion transport membrane protein can be formed on the cell membrane in which the label protein is bound to the extracellular domain in a manner exposed by the recombinant gene.

[0054] The vectors used for intracellular introduction of recombinant genes in the present invention may include various vectors and viral vectors, such as plasmids and recombinant viruses, namely recombinant adeno-associated virus (rAAV), recombinant retrovirus, recombinant lentivirus, recombinant adenovirus, and recombinant herpes simplex virus and other viruses known in the art.

[0055] In some embodiments, the expression of the recombinant gene of the ion transport membrane protein of the present invention is induced by constitutive promoters, namely the CMV promoter, the eF1-alpha promoter, and the LTR. In other embodiments, the promoter is an inducible or cell-specific promoter. A cell-type-specific promoter that enables the expression of the recombinant ion transport membrane protein in a specific subgroup of cells, namely neurons, may be preferred. These cells may include, but are not limited to, neurons present in the nervous system in the body. Cell-type-specific promoters are widely known in the art. Particularly preferred cell-type-specific promoters include, but are not limited to, hSyn, vGat, vGlut, and TH. The method of introducing the recombinant gene sequence into a cell may utilize conventional techniques, but it is preferably done by introducing it into the cell using an adeno-associated virus or a lentivirus. In addition, in the recombinant gene sequence of the present invention, the position of the gene sequence corresponding to the label protein can be adjusted so that the label protein can be expressed on the surface of an ion transport membrane protein protruding outside the cell.

[0056] In a preferred embodiment of the present invention, the amino acid of the ion transport membrane protein expressed according to the recombinant gene sequence is indicated by Sequence No. 1 of Table 1 below, and the amino acid sequence of the label protein may be indicated by Sequence No. 2.

[0057] A transport membrane protein expressed according to the amino acid sequence indicated by SEQ ID NO. 1 in Table 1 below may display a label protein (a protein consisting of the amino acid sequence indicated by SEQ ID NO. 2) in the extracellular domain.

[0058] Sequence number designation amino acid sequence Sequence No. 1 DmFLYC1-MYC sequence (amino acid sequence of DmFLYC1 anion transport membrane protein) MGSYLHEPPGDEPSMRIEQPKTADRAPEQVAIHICEPSKVVTESFPFSETAEPEAKSKNCPCPEIARIGPCPNKPPKIPINRGLSRISTNKSRPKSRFGEPSWPVESSLDLTSQSPVSPYREEAFSVENCGTAGSRRGSFARGTTSRAASSSRKDETKEGPDEKEVYQRVTAQLSARNQKRMTVKLMIEL SVFLCLLGCLVCSLTVDGFKRYTVIGLDIWKWFLLLLVIFSGMLITHWIVHVAVFFVEWKFLMRKNVLYFTHGLKTSVEVFIWITVVLATWVMLIKPDVNQPHEQKLISEEDLQTRKILEFVTWTIVTVLIGAFLWLVKTTLLKILASSFHLNRFFDRIQESVFHHSVLQTLAGRPVVELAQGISRTESQD GAGQVSFMEHTKTQNKKVVDVGKLHQMKQEKVPAWTMQLLVDVVSNSGLSTMSGMLDEDMVEGGVELDDDEITNEEQAIATAVRIFDNIVQDKVD QSYIDRVDLHRFLIWEEEVDHLFPLFEVNEKGQISLKAFAKWVVKVYNDQAALKHALNDNKTAVKQLNKLVTAILIVMMIVIWLIVTGIATTKLIV LLSSQLVVAAFIFGNTCKTIFEAIIFVFVMHPFDVGDRCVIDGNKMLVEEMNILTTVFLKWDKEKVYYPNSILCTKAIGNFFRSPDQGDVLEFSVDFTTPVLKIGDLKDRIKMYLEQNLNFWHPQHNMVVKEIENVNKIKMALFVNHTINFQDFAEKNRRRSELVLELKKIFEELDIKYNLLPQEISIRNM Sequence No. 2 Myc sequence (Myc tag sequence) EQKLISEEDL

[0060] According to the present invention, nanoparticles can rotate upon the formation of a magnetic field (preferably a rotating magnetic field). Accordingly, after injecting nanoparticles into an individual expressing a target ion transport membrane protein, if a magnetic field is formed outside the individual, the nanoparticles can rotate due to a magnetic reaction, and the ion transport membrane protein to which the nanoparticles are bound can be opened according to the rotation.

[0061] For example, when the ion channel of the present invention is DmFLYC1 and the labeled protein is MYC-tag, the ligand of the nanoparticle, namely the MYC antibody, binds to the MYC-tag. When a magnetic field is formed, the nanoparticle injected into the organism rotates, transmitting a mechanical force to the DmFLYC1 ion channel, and the DmFLYC1 ion channel opens in response to this stimulus. With the opening of the DmFLYC1 ion channel, Cl- ions flow into the cell, causing hyperpolarization of the cell membrane potential, which is consistent with the results of the graph in Fig. 12. In other words, it can be seen that cell activity can be controlled by utilizing the rotation of the nanoparticle of the present invention. If the target cell is a neuron, the activity of the neuron can be inhibited through the above process.

[0062] The present invention also provides a cell activity control system (or an ion transport membrane protein opening / closing control system) comprising: a nanoparticle having the core-shell structure; an ion transport membrane protein expressed on the cell membrane of an organism and to which the nanoparticle specifically binds; and a magnetic field generator having an internal space in which the organism can be placed and forming a rotating magnetic field, wherein the opening / closing of the ion transport membrane protein is controlled according to the rotation of the bound nanoparticle when the magnetic field is generated.

[0064] The magnetic field generating device in the present invention may be a rotating magnetic field generating device satisfying the following equations 1 and 2.

[0065] [Relation 1] |Mc| ≥ 1 mT

[0066] [Equation 2] |M75-Mc| / D75 ≤ 5.0 T / m

[0067] In the above equations 1 and 2, Mc is the magnetic field strength at the rotation axis position, D75 is the distance to the position 75% of the distance from the rotation axis to the magnetic force generating part, and M75 is the magnetic field strength at the D75 position.

[0068] The above Equation 1 relates to the strength of the magnetic field at the position of the rotation axis. The cell activity (opening / closing of ion transport membrane proteins) control system according to the present invention generates torque using a rotating magnetic field, and as the direction of the magnetic flux rotates, torque can be transmitted to the ion channel through magnetic particles bound to the ion transport membrane proteins. At this time, although the strength of the magnetic field weakens as it moves further away from the magnetic force generating unit, if the above Equation 1 is satisfied, torque of a certain level or higher can be generated even at the central part of the rotating magnetic field generating device, thereby controlling the cell activity (opening / closing of ion transport membrane proteins).

[0069] Equation 2 above represents the relationship between the magnetic field strength at the position of the rotation axis and the magnetic force strength at a position 75% of the distance from the rotation axis to the magnetic force generating part. Looking at Equation 2, a rotating magnetic field satisfying Equation 2 can satisfy a rate of change in magnetic field strength at a position 75% of the distance from the rotation axis to the magnetic force generating part, that is, a slope of magnetic field strength according to the distance from the rotation axis, of 5.0 T / m or less. This may mean that the change value of the rotating magnetic field strength formed by the rotating magnetic field generating device of the cell activity (opening / closing of ion transport membrane proteins) control system according to the present invention is not large. When Equation 2 is satisfied, a uniform rotating magnetic field can be formed in an area from the center to a predetermined distance, thereby maximizing the internal space of the rotating magnetic field generating device, and through this, cell activity (opening / closing of ion transport membrane proteins) can be controlled over a wide area.

[0070] The magnetic field generating device in the present invention may additionally satisfy the following relationship 3 or relationship 4.

[0071] [Equation 3] |M50-Mc| / D50 ≤ 1 T / m

[0072] In the above equation 3, Mc is the magnetic field strength at the axis of rotation position, D50 is the distance from the axis of rotation to the 50% position of the distance to the magnetic force generating part, and M50 is the magnetic field strength at the D50 position.

[0073] [Equation 4] |M75-M50| / (D75-D50) ≤ 10 T / m

[0074] In the above equation 4, D75 represents the distance to the 75% position from the axis of rotation to the magnetic force generating part, D50 represents the distance to the 50% position from the axis of rotation to the magnetic force generating part, M75 represents the magnetic field strength at the D75 position, and M50 represents the magnetic field strength at the D50 position.

[0075] The above Equation 3 relates to the magnetic field strength at the position of the rotation axis and the magnetic field strength in the intermediate region between the position of the rotation axis and the magnetic force generating part. A rotating magnetic field satisfying the above Equation 3 can satisfy a gradient of magnetic field strength with respect to distance from the rotation axis of 1.0 T / m or less. When the above Equation 3 is satisfied, the magnetic field strength in the central region centered on the rotation axis can be nearly constant. The cell activity (opening / closing of ion transport membrane proteins) control system according to the present invention can form a rotating magnetic field satisfying the above Equation 3 and apply a uniform rotating magnetic field with very small changes in magnetic field strength to an area of ​​a certain area or larger within the internal space, thereby enabling precise control of cell activity (opening / closing of ion transport membrane proteins) over a large area.

[0076] The above Equation 4 relates to the strength of the magnetic force at a position that is 75% of the distance from the axis of rotation to the magnetic force generating part, and the strength of the magnetic force at a position that is 50% of the distance from the axis of rotation to the magnetic force generating part. While the aforementioned Equations 2 and 3 relate to the rate of change of the magnetic field strength at the central part of the rotating magnetic field generating device, the above Equation 4 represents the rate of change of the magnetic field strength at the outer part of the rotating magnetic field generating device. When Equation 3 is satisfied, the rate of change of the magnetic field strength at the outer part of the internal space of the rotating magnetic field generating device can be suppressed within a predetermined range, and cell activity (opening and closing of ion transport membrane proteins) can be controlled in a wider space.

[0078] In this specification, "rotating magnetic field" may refer to a magnetic field in which magnetic field lines directed perpendicular to a virtual axis of rotation rotate around a virtual axis of rotation, and may refer to a magnetic field in which the direction of the magnetic field appears to rotate with a predetermined angular velocity over time. The angular velocity of the rotating magnetic field may be, for example, 0.01 Hz or more and / or 10,000 Hz or less, but is not limited thereto. In addition, in this specification, a virtual plane in which the direction of the magnetic flux of the rotating magnetic field is horizontal is defined as a "reference plane," and "magnetic field strength" may refer to a value measured on the reference plane. In this specification, "distance from the axis of rotation to the magnetic force generating part" may refer to the arithmetic mean of the respective distances from the axis of rotation to a plurality of magnetic force generating parts.

[0080] The present invention also provides a method for controlling cell activity (controlling the opening and closing of ion channels), comprising: (a) manufacturing a core-shell structured nanoparticle comprising a shell composed of a plurality of magnetic particles disposed on the surface of a core, wherein a ligand capable of selectively binding to a target ion transport monolayer protein is located on the magnetic particles; (b) forming an ion transport membrane protein in a cell of an individual having a label protein capable of binding to the ligand expressed on its surface; (c) injecting the nanoparticle having the ion transport membrane protein formed thereon into the individual having the ion membrane protein formed in step (b); and (d) forming a magnetic field outside the individual to induce rotation of the nanoparticle.

[0081] In the present invention, the “object” may be a mammal and includes humans, primates, mice, rats, guinea pigs, cattle, pigs, horses, sheep, dogs, and cats. In the present invention, the “object” may be a bird, fish, amphibian, or insectoid animal and includes zebra finches, zebrafish, medaka, African clawed frogs, fruit flies, and bees.

[0082] According to the present invention, when a magnetic field is formed according to step (d), the nanoparticle rotates due to a magnetic reaction, and the ion transport membrane protein opens according to the rotation of the nanoparticle, and the opening and closing and activity of the ion transport membrane protein can be controlled depending on whether a magnetic field is formed, thereby allowing the activity of the target cell to be controlled.

[0084] The following examples are provided to aid in understanding the invention and do not limit the scope of protection of the invention.

[0086] [Example]

[0087] Example 1. Preparation of nanoparticles

[0088] Magnetic particles were prepared by the following method. Spherical polystyrene with an average particle size of 200 nm was used as the core (manufactured by Polysciences), and iron oxide (Fe3O4) with an inverse spinel structure having an octahedral shape and an average particle size of 25 nm was prepared and used as the nanoparticle. 1 mg of the above nanoparticles was dispersed in DMSO (Dimethyl Sulfoxide), and 0.1 mg of azido-dPEG12-TFP ester (manufactured by Quantabiodesign) was added to bind the nanoparticles with azide. Subsequently, 0.5 mg of the polystyrene core was dispersed in DMSO, and 1 mg of DBCO-PEG4-NHS (manufactured by Click Chemistry Tools) was added to bind DBCO to the polystyrene core. After separating each particle by centrifugation, the particles were stirred at 25°C for 8 hours to form a 1,2,3-triazole, thereby producing magnetic particles in which nanoparticles were attached to the surface of a polystyrene core. Figure 4 is an SEM image of the prepared magnetic particles. Referring to Figure 4, it can be seen that iron oxide nanoparticles are attached to the surface of the polystyrene core.

[0090] Example 2. Genetic recombination

[0091] 2-1. Recombination of label protein nucleotide sequences for ion transport membrane proteins

[0092] The gene for forming the ion transport membrane protein is produced using a gene recombination method known in the art. First, to ensure the smooth expression of the plant-derived anion channel gene DmFLYC1, codon optimization of the DmFLYC1 gene was performed to suit the mammalian species from which the target animal or cell originated.

[0093] To produce a protein capable of binding nanoparticles and anion channels, the nucleotide sequence of the codon-optimized anion channel gene DmFLYC1 was recombined. Specifically, a Myc-tag sequence was inserted as the binding site amino acid sequence at the H293 (histidine 293) position on the second extracellular domain of DmFLYC1, which is capable of binding nanoparticles.

[0094] To identify the expression and intracellular localization of anion channels, the nucleotide sequence of the RFP mScarlet was inserted into the C-terminus of DmFLYC1.

[0095] The recombinant gene of DmFLYC1 produced through the above process was cloned into a suitable vector (e.g., pcDNA, rLenti, rAAV, etc.) under the control of a promoter suitable for the target cell (e.g., CMV, hSyn, etc.).

[0096] 2-2. Generation of the mutant ion transport membrane protein DmFLYC1 construct

[0097] Mutations were induced on the codon-optimized anion channel gene DmFLYC1 protein to generate single and multiple mutations at the R599 (arginine 599), K606 (lysine 606), V656 (valine 656), and T659 (threonine 659) sites. Several variants were produced, e.g., single mutants, e.g., R599K, K606R, V656L, and T659L; double mutants, e.g., V656L / T659L; triple mutants, e.g., R599K / V656L / T659L; and quadruple mutants, e.g., R599K / K606R / V656L / T659L. The DmFLYC1 recombinant gene produced through the above process was cloned into a suitable vector (e.g., pcDNA, rLenti, rAAV, etc.) under the control of a promoter suitable for the target cell (e.g., CMV, hSyn, etc.) using a method known in the art.

[0099] Example 3.

[0100] 3-1. Expression of Ion Transport Membrane Proteins and Label Proteins

[0101] A method for expressing the gene vector containing the recombinant gene of the above-mentioned DmFLYC1 in target cells is described below.

[0102] First, for expression in HEK cells, for example, a DmFLYC1-mScarlet gene vector cloned into a pcDNA vector was used. The gene was designed to be expressed under the control of a CMV promoter. The above DmFLYC1 construct was delivered to HEK cells via a gene introduction method known in the art. After gene introduction, the HEK cells were cultured for 48 hours under conditions suitable for cell culture, and then gene expression was confirmed by observing the RFP signal of mScarlet fused to DmFLYC1.

[0103] Figure 7 illustrates, through fluorescence observation, that the Myc antibody selectively binds to the outer membrane of cells expressing DmFLYC1-Myc (DmFLYC1 with a Myc-tag). The fluorescence shown is indicated by DmFLYC1-Myc (red) and Myc antibody (green).

[0104] For expression in neurons, for example, a DmFLYC1-mScarlet gene vector cloned into a vector was used. The gene was designed to be expressed under the control of the hSyn promoter. Alternatively, a DmFLYC1-mScarlet gene vector cloned into an rAAV vector and an adeno-associated virus containing the gene can be used. In addition, to facilitate the smooth membrane transport of the DmFLYC1-mScarlet gene, a nucleotide of a membrane transport signaling peptide (i.e., 65 amino acids of a portion of the Kv2.1 protein) was additionally inserted into the C-terminus of DmFLYC1. The above DmFLYC1 construct was delivered to cultured neurons using a gene introduction method known in the art. After gene introduction, the cultured neurons were cultured for a sufficient period under conditions suitable for culture, and then gene expression was confirmed by observing the RFP signal of mScarlet fused to DmFLYC1. A sufficient amount of time is, for example, about 48 hours for direct DNA introduction through a pcDNA gene vector, and about 1 to 2 weeks for DNA introduction through an rAAV gene vector and an adeno-associated virus.

[0105] Figure 14 illustrates, through fluorescence observation, that DmFLYC1-Kv2.1, which additionally contains the membrane transport signal peptide Kv2.1, is smoothly transported to and present in the membrane of a neuron. The fluorescence shown is indicated by DmFLYC1-Myc-Kv2.1 (red) and Myc antibody (green).

[0106] 3-2. Binding of Ion Transport Membrane Proteins and Nanoparticles

[0107] The recombinant protein of the ion channel expressed in the target cell of the present invention can bind to a nanoparticle having a suitable ligand.

[0108] To observe whether nanoparticles using a Myc antibody as a ligand can bind to the extracellular domain (i.e., the region exposed on the cell surface) of DmFLYC1-Myc (DmFLYC1 with a Myc-tag sequence inserted) expressed in target cells, the following experiment was performed in HEK cells. For observation, a fluorescent sample was additionally attached to the nanoparticles. The introduction and expression of the DmFLYC1-Myc gene were performed using the method of Example 3.1 above. 48 hours after gene introduction, 1% BSA (Bovine Serum Albumin) was injected into the cell environment (i.e., the culture medium) to perform a blocking process for 1 hour under cell culture conditions (37°C, 5% CO2). Subsequently, nanoparticles at a concentration of 100 μg / mL were injected into the cell environment along with 1% BSA to induce binding between DmFLYC1 and the nanoparticles for 1 hour under cell culture conditions. Afterwards, nanoparticles unbound to the Myc-tag of DmFLYC1 were removed through a cell washing process.

[0109] Figure 8 illustrates, through fluorescence observation, that nanoparticles coated with a Myc antibody selectively bind to the outer membrane of cells expressing DmFLYC1-Myc (DmFLYC1 with a Myc-tag). The fluorescence shown is indicated by DmFLYC1-Myc (red) and nanoparticles (green).

[0111] Example 4. Confirmation of the control of opening and closing of ion transport membrane proteins

[0112] The recombinant protein of the ion channel expressed in the target cell of the present invention can be opened due to the rotation of nanoparticles generated by the application of a rotating magnetic field.

[0113] To observe whether it is possible to induce rotation of nanoparticles through the application of a rotating magnetic field and thereby induce the opening of ion channels bound to the nanoparticles, the following experiments were performed in HEK cells. The introduction and expression of the DmFLYC1-Myc gene and the binding of nanoparticles were performed using the methods of Examples 3.1 and 3.2 above. After the nanoparticle binding process, the experiments shown in Figures 10 and 12 were performed, respectively, to confirm that the opening and closing of ion channels could be controlled. In these experiments, the rotating magnetic field stimulation was applied in the form of a magnetic field of uniform intensity of 25 to 30 mT rotating at a speed of approximately 0.5 Hz.

[0114] In the experiments of Figures 10 and 16, a chemical MQAE chloride ion concentration sensor, which is one of the methods known in the art, was used. After the nanoparticle binding process, the cell sample was placed in a room equipped with cell culture conditions, and a rotating magnetic field stimulus was applied to the cells for 1 hour. Subsequently, intracellular fluid was extracted using a method known in the art. After injecting MQAE at a concentration of 10 μM into 200 μL of the extracted intracellular fluid, the fluorescence level was measured using a Multimode plate reader (manufactured by PerkinElmer).

[0115] Figure 10 illustrates the chloride concentration accumulated within the cell due to the activation of DmFLYC1 by a rotating magnetic field and nanoparticles, based on the measured fluorescence levels. Individual conditions represent the presence or absence of gene introduction, the presence or absence of nanoparticles, and the presence or absence of rotating magnetic field stimulation; it can be seen that when all conditions are met, there is a change in the fluorescence level (i.e., accumulation of chloride ions within the cell). The fluorescence level of MQAE decreases through binding with chloride ions.

[0116] Figure 11 illustrates that the concentration of chloride accumulated in cells increases in mutants due to the activation of DmFLYC1 by a rotating magnetic field and nanoparticles, based on the measured fluorescence levels. Nanoparticle and rotating magnetic field stimulation were applied under all conditions, and each condition represents a mutant type of the genetically introduced DmFLYC1.

[0117] In the experiment of Fig. 12, a bio-chloride ion concentration sensor mClY, which is one of the methods known in the art, was used. To simultaneously introduce and express the two genes DmFLYC1 and mClY, the process of Example 3.1 was performed using the two genes. After the nanoparticle binding process, the cell sample was placed in a room equipped with cell culture conditions, and while delivering a rotating magnetic field stimulus to the cell, the change in fluorescence brightness was simultaneously observed and measured.

[0118] Figure 12 illustrates the influx of chloride ions into the cell due to the activation of DmFLYC1 by a rotating magnetic field and nanoparticles, based on the measurement of the observed change in fluorescence brightness. The change in fluorescence level shows rapid changes in seconds depending on the magnetic field stimulation, suggesting that the activation of DmFLYC1 induces the influx of chloride ions at a rapid rate. The fluorescence brightness of mClY decreases through binding with chloride ions.

[0119] That is, according to the present embodiment, nanoparticles injected into a target according to the present invention bind to ion transport membrane proteins expressed within the target, and can control the opening and closing of ion transport membrane proteins according to rotation when a magnetic field is generated, and accordingly, cell activity is controlled.

[0121] The present invention has been described above with reference to its preferred embodiments. Those skilled in the art will understand that the present invention may be embodied in modified forms without departing from the essential characteristics of the invention. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the invention is defined by the claims, not by the foregoing description, and all variations within the scope of the claims should be interpreted as being included in the invention.

Claims

Claim 1 A magnetic particle complex for controlling the opening and closing of ion transport membrane proteins on a cell surface in response to a rotating magnetic field stimulation, wherein the magnetic particle complex comprises (a) a non-magnetic core having no magnetic moment so as not to contribute to torque generation by a magnetic field, and a plurality of magnetic particles having a magnetic moment connected to the surface of the non-magnetic core (m-Torquer); and (b) a binder capable of binding to the ion transport membrane protein, wherein the magnetic particle complex generates torque on the ion transport membrane protein on the cell surface bound to the magnetic torque particle (m-Torquer) in response to a rotating magnetic field stimulation, controls the opening and closing of the ion transport membrane protein through the rotational control of the magnetic torque particle (m-Torquer) according to the application and control of an external rotating magnetic field stimulation, and controls cell activity by controlling the inflow or outflow of ions into or out of the cell in response to the rotating magnetic field stimulation. Claim 2 A magnetic particle complex for mediating rotational magnetic field stimulation according to claim 1, wherein the cell is at least one cell selected from the group consisting of nerve cells, muscle cells, and endocrine cells. Claim 3 A magnetic particle composite for mediating a rotating magnetic field, wherein the core is composed of at least one inorganic material selected from the group consisting of polymers, ceramics, metal matrix composites (MMCs), and ceramic matrix composites (CMCs). Claim 4 A magnetic particle complex for rotary magnetic field stimulation mediation, wherein the binder is an antibody, nanobody, or ligand that binds to an ion transport membrane protein or to a protein tag of an ion transport membrane protein. Claim 5 A magnetic particle complex for mediating a rotating magnetic field, wherein the ion transport membrane protein is a membrane protein that transports chloride anions (Cl-), a membrane protein that non-specifically transports anions, other anion pumps, a membrane protein that transports potassium cations (K+), a proton pump membrane protein that transports hydrogen cations (H+), other cation transport membrane proteins, or a mutant protein thereof. Claim 6 A magnetic particle complex for mediated rotational magnetic field stimulation according to claim 5, wherein the membrane protein transporting chloride anions (Cl-) comprises an MSL channel, FLYC channel, CLC channel, E-CLC channel, CLIC channel, CFTR channel, GABA receptor, or Glutamate receptor; the membrane protein transporting anions non-specifically comprises a McsS channel, MscL channel, SWELL channel, ANO channel, and Maxi anion channel; the membrane protein transporting potassium cations (K+) comprises a Kv channel, Kca channel, Kna channel, K2p channel, and KscA channel; and the proton pump membrane protein transporting hydrogen cations (H+) comprises H-type, V-type, F-type, and P-type proton pumps. Claim 7 A magnetic particle complex for mediated rotational magnetic field stimulation according to claim 5, wherein the ion transport membrane protein is a mutant protein of the DmFLYC1 membrane protein, and the mutant protein is a membrane protein mutated at one or more of the positions R599, K606, V656, and T659 in the amino acid sequence of DmFLYC1. Claim 8 In claim 1, the magnetic particle complex for rotary magnetic field stimulation mediation, wherein the ion transport membrane protein is an anion transport membrane protein. Claim 9 A magnetic particle complex for mediating rotary magnetic field stimulation, wherein the ion transport membrane protein is an anion transport membrane protein in which cell activity is inhibited upon opening. Claim 10 A magnetic particle complex for mediating rotational magnetic field stimulation according to claim 1, wherein the ion transport membrane protein has a label protein expressed in an extracellular domain, and the magnetic torque particle (m-Torquer) has a ligand connected to enable binding to the label protein. Claim 11 A magnetic particle complex for mediating rotational magnetic field stimulation according to claim 1, wherein when a rotational magnetic field is formed outside the organism, the magnetic torque particle (m-Torquer) rotates due to a magnetic reaction, and when the ion transport membrane protein to which the magnetic torque particle (m-Torquer) is bound is an ion channel, the ion channel opens according to the rotation of the magnetic torque particle (m-Torquer). Claim 12 A magnetic particle complex for mediating rotational magnetic field stimulation according to claim 1, wherein the magnetic torque particle (m-Torquer) rotates by magnetic reaction when a rotational magnetic field is formed outside the organism, and the ion transport membrane protein to which the magnetic torque particle (m-Torquer) is bound is an ion pump according to the rotation of the magnetic torque particle (m-Torquer), and the ion pump is activated. Claim 13 A cell activity control system comprising: a magnetic particle complex for mediating a rotating magnetic field stimulation described in any one of claims 1 to 12; an ion transport membrane protein expressed on the cell membrane of an organism and to which the magnetic particle complex for mediating a rotating magnetic field stimulation specifically binds; and a magnetic field generator having an internal space in which the organism can be placed and which forms a rotating magnetic field, wherein, when a rotating magnetic field is generated, the ion transport membrane protein is opened / closed or its activity is regulated according to the rotation of the magnetic particle complex for mediating a rotating magnetic field stimulation bound to it. Claim 14 delete Claim 15 delete Claim 16 delete Claim 17 delete Claim 18 delete Claim 19 delete Claim 20 delete Claim 21 (a) a step of preparing a magnetic particle complex for rotating magnetic field stimulation as described in any one of claims 1 to 12; (b) a step of forming an ion transport membrane protein having a label protein capable of binding to a binder of the magnetic particle complex for rotating magnetic field stimulation expressed on its surface within a cell of an organism; (c) a step of injecting the magnetic particle complex for rotating magnetic field stimulation into the organism in which the ion transport membrane protein formed in step (b) is formed; and (d) a step of forming a rotating magnetic field outside the organism to induce rotation of the magnetic particle complex for rotating magnetic field stimulation. A method for regulating cell activity. Claim 22 A method for regulating cell activity according to claim 21, wherein the magnetic particle complex for mediated rotational magnetic field stimulation rotates due to a magnetic reaction when a rotating magnetic field is formed according to step (d), and when the ion transport membrane protein is an ion channel, the ion channel opens according to the rotation of the magnetic particle complex for mediated rotational magnetic field stimulation. Claim 23 A method for regulating cell activity according to claim 21, wherein the magnetic particle complex for mediating the rotating magnetic field stimulation rotates due to a magnetic reaction when the rotating magnetic field is formed according to step (d), and if the ion transport membrane protein is an ion pump, the ion pump is activated according to the rotation of the magnetic particle complex for mediating the rotating magnetic field stimulation. Claim 24 delete Claim 25 delete Claim 26 delete Claim 27 delete Claim 28 delete Claim 29 A magnetic particle complex for mediated rotational magnetic field stimulation, wherein, in any one of claims 1 to 12, the magnetic particle complex is used to inhibit neural activity by inducing the opening of anion channels or the activation of anion pumps in nerve cells.

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