JPWO2015146153A1 - Method of introducing an object into a cell - Google Patents

Abstract

Giant Unilamellar Liposomes (Giant Unilamellar Liposomes) encapsulating an object can be introduced easily even in the case of an object having a size larger than that of the prior art, and as an intracellular object introduction method that does not impair the survival of cells. ) Is electrofused with the cell. This method includes a first procedure in which an alternating electric field is applied to a mixed solution of giant unilamellar liposomes encapsulating the object and the cells, and a second pulse pulse electric field is applied to the mixed solution after the first procedure. Procedures. According to this method, an object having a diameter of 100 nm or more can be introduced into the cell.

Description

  The present invention relates to a method for introducing an object into a cell. More specifically, the present invention relates to a method of introducing a large-sized object into a cell using a giant unilamellar liposome (Giant Unilamellar Liposome).

  In recent years, microbeads and functional substances with various chemical modifications have been developed and used for research and diagnosis. If these can be introduced into cells, the environment inside living cells can be observed. Conventionally, lipofection, electroporation, particle gun, microinjection and the like have been used as methods for introducing substances into living cells.

  Giant unilamellar liposome (Giant Unilamellar Liposome or Giant Unilamellar Vesicle, hereinafter referred to as “GUV”) is an artificial membrane vesicle (liposome or vesicle) formed by a lipid bilayer closed naturally in an aqueous solution. In this case, the diameter is a micrometer order or more (see Non-Patent Documents 1 and 2). GUV is about the same size as a cell and has the advantage that it can be directly observed by various optical microscopy methods such as phase difference, fluorescence, differential interference, dark field, and the like. GUV has been used as a membrane model when studying the mechanism of morphogenesis and dynamic control of biological membranes in an in vitro system.

  Patent Document 1 and Non-Patent Document 3 describe a method of injecting a substance into a cell by preparing a liposome incorporating a connexin composed of connexin, including the substance inside the liposome, and bringing the substance into contact with the cell. Have been described. In this method, the connexon formed on the liposome membrane functions as a channel, and the substance in the liposome is injected into the cell through the channel. For this reason, the size of a substance that can be injected into cells is limited by the inner diameter (about 2 nm) of Connexon.

  Non-Patent Document 4 discloses a cryoprotective substance that is freeze-dried by electrofusion of a GUV containing a membrane-impermeable freeze-drying substance (saccharide such as trehalose) with cells for the purpose of long-term freezing / drying preservation of cells. Is introduced into cells. This document does not describe introduction of an object such as a microbead having a size larger than that of a saccharide such as trehalose into a cell, and a GUV preparation method and electrofusion conditions for that purpose.

Japanese Patent No. 5057362

Nature, 1991, 349, 475-481 Biosystems, 2003, 71, 93-100 Biomaterials, 2009, 30, 3971-3977 Cryobiology, 2006, 53 (3), 397

  In conventional methods for introducing substances into living cells such as lipofection, electroporation, and particle guns, the maximum size of substances that can be introduced is about 100 nm. In addition, in microinjection, since a substance is introduced by piercing a small injection needle into each cell, skill and time are required for the operation, and it is difficult to introduce the substance into a large number of cells.

  Therefore, the main object of the present invention is to provide a method for introducing an intracellular substance that can be easily introduced even if the object is larger in size than the conventional technique and does not impair the survival of the cell. To do.

In order to solve the above problems, the present invention provides the following intracellular object introduction method and the like.
[1] A method of introducing an object into a cell, the method comprising a step of electrofusion of a giant unilamellar liposome encapsulating the object with the cell (Giant Unilamellar Liposome).
[2] A first procedure in which an alternating electric field is applied to a mixed solution of giant unilamellar liposomes enclosing the object and the cells, and a second procedure in which a direct-current pulse electric field is applied to the mixed solution after the first procedure And the method according to [1].
[3] The first procedure is a procedure in which an alternating electric field is applied to a mixture of GUV and the cells to arrange the GUV and the cells.
The method according to [3], wherein the second procedure is a procedure in which a direct-current pulse electric field is applied to the mixed solution to at least partially fuse the GUV film and the cell film.
[4] The method according to any one of [1] to [3], wherein the GUV has a negative surface potential.
[5] The method according to [4], wherein the GUV has a surface potential of −40 mV to −20 mV.
[6] The method according to any one of [1] to [5], wherein the proportion of cholesterol in the total lipid constituting the GUV membrane is 0 to 15 mol%.
[7] In the first procedure, the AC electric field is applied at 5 to 15 V / mm for 10 to 60 seconds, and in the second procedure, the DC pulse electric field is applied at 100 to 175 V / mm and 30 to 50 microseconds. The method according to any one of [1] to [6], wherein the method is applied.
[8] The method according to any one of [1] to [7], wherein the object has a diameter of 100 nm or more.
[9] The method according to any one of [1] to [8], wherein the object is one or more selected from the group consisting of particles, nucleic acids, polymers, microorganisms, and intracellular organs.

[10] A method for producing GUV, the method comprising preparing a lipid constituting the GUV film such that a surface potential of the GUV is −40 mV to −20 mV.
[11] The production method according to [10], comprising preparing the lipid constituting the GUV membrane such that the proportion of cholesterol in the total lipid is 0 to 15 mol%.

[12] GUV having a surface potential of 40 mV to -20 mV.
[13] The GUV of [12], wherein the proportion of cholesterol in the total lipid constituting the membrane is 0 to 15 mol%.
[14] The GUV according to [12] or [13], wherein one or more objects selected from the group consisting of particles, nucleic acids, polymers, microorganisms, and organelles are encapsulated.
[15] The giant unilamellar liposome according to any one of [12] to [14], in which an object having a diameter of 100 nm or more is encapsulated.

[16] A method for measuring physical properties in a cell by introducing an object into the cell by the method according to [1] to [9] and detecting peristalsis of the object in the cell.
[17] A method for controlling the position of a cell by introducing a magnetic object into the cell by the method according to [1] to [9] and applying a magnetic field to the cell.
[18] Surface-enhanced Raman scattering spectroscopy including a procedure for introducing gold particles and / or silver particles into cells by the method according to [1] to [9].
[19] A method for applying a physical force to a predetermined protein in a cell or an intracellular organ constituted by the protein, wherein a first factor of a binding pair is added to the predetermined protein or the intracellular organ. A procedure for introducing a magnetic particle to which a second factor of a binding pair has been added into the cell by the method described in [1] to [9], and a binding pair formed by the first factor and the second factor. And a step of applying a magnetic field to the magnetic particles bound to the predetermined protein or the intracellular organ.
[20] The method according to [19], wherein the first factor is biotin and the second factor is streptavidin, and biotin is added to the predetermined protein or the intracellular organ by an in vivo biotinylation method.
[21] A physical force is applied to a predetermined protein in the cell or an intracellular organ composed of the protein by the method according to [19] or [20], and the gene expression level in the cell before and after the load is determined. Gene expression analysis method for detecting changes.
[22] By introducing an artificial DNA nanostructure (DNA origami) in which an enzyme involved in an enzymatic reaction and / or a functional molecule is immobilized in a cell by the method described in [1] to [9], intracellular metabolism is reduced. How to modify.
[23] A method for measuring intracellular temperature, comprising a step of introducing a temperature-sensitive polymer into cells by the method according to [1] to [9] and a step of detecting a temperature-dependent structural change of the temperature-sensitive polymer.

[24] A method for treating mitochondrial disease, comprising a step of introducing mitochondria into cells by the method according to [1] to [9].
[25] A method for producing a cell preparation for treating mitochondrial diseases, comprising a procedure for introducing normal mitochondria into cells isolated from a patient by the method according to [1] to [9].
[26] The GUV of [14], wherein the organelle is a mitochondria.
[27] A cell composition for treating mitochondrial disease, comprising the GUV according to [26] and a cell.
[28] A cell composition for treating mitochondrial disease, comprising the GUV according to [26] and a cell isolated from a patient into which normal mitochondria have been introduced.

  According to the present invention, there is provided a method for introducing an intracellular object that can be easily introduced even if the object is larger in size than the prior art and does not impair the survival of the cell.

It is a schematic diagram explaining the procedure of electrofusion of GUV and a cell in the intracellular substance introduction method according to the present invention. It is a figure which shows the base sequence of DNA origami. It is a figure which shows the base sequence of DNA origami. It is a figure which shows the base sequence of DNA origami. It is a figure which shows the base sequence of DNA origami. It is a confocal microscope picture of the cell which introduce | transduced the fluorescent bead (diameter 1 micrometer) by electrofusion with GUV. It is a confocal microscope picture of the cell electrofused with GUV encapsulating fluorescent beads of different sizes. It is the phase-contrast micrograph and the confocal micrograph of the cell which introduce | transduced the EGFP expression vector by electrofusion with GUV. It is the phase-contrast micrograph and the confocal micrograph of the cell which introduce | transduced DNA origami by electrofusion with GUV. It is the continuous photograph which image | photographed the cell which introduce | transduced the magnetic bead with the intracellular substance introduction method based on this invention, and applied the magnetic field and rotated.

  Hereinafter, preferred embodiments for carrying out the present invention will be described. In addition, embodiment described below shows an example of typical embodiment of this invention, and, thereby, the range of this invention is not interpreted narrowly.

1. Intracellular object introduction method The intracellular object introduction method according to the present invention includes a procedure for electrofusion of giant unilamellar liposomes (GUV) encapsulating an object with cells. Specifically, in the method for introducing an intracellular object according to the present invention, (i) an alternating electric field is applied to a mixed solution of the GUV in which the object is sealed and a negative surface potential is applied, and the cell, A first procedure for arranging the GUV and the cells; and (ii) after the first procedure, a DC pulse electric field is applied to the mixed solution to at least partially fuse the GUV membrane and the cell membrane. And (iii) after the second step, an alternating electric field is applied to the mixed solution to maintain a fusion state between the membrane of the giant unilamellar liposome and the membrane of the cell. Including three steps.

(1) GUV and production method thereof GUV can be produced by a known method such as static hydration method, electroformation method and interface passing method, or an improved method thereof. The static hydration method is a method for preparing GUV by adding an aqueous solution to a lipid film mainly composed of phospholipids prepared in a test tube and allowing to stand (J. Mol. Biol., 1999, 287, 293-). 300; J. Mol. Biol., 1998, 284, 1671-1681; Curr. Biol., 2009, 19, 140-145). The electro-formation method is a method for producing GUV by applying a lipid containing phospholipid as a main component to the surface of platinum or transparent conductive glass (ITO glass or the like) and applying an alternating voltage in an aqueous solution (Nature). , 2010, 464, 864-869). The interface passing method is a method for producing GUV using a lipid monolayer formed at an oil-water interface (Langmuir, 2011, 27, 11528-11535; Method Enzymol., 2009, 464, 31 -53).

  Examples of lipids used for GUV production include phospholipids, cholesterol, and synthetic lipids.

Examples of phospholipids include glyceroglycolipid and glycosphingolipid.
Examples of the glyceroglycolipid include sulfoxyribosyl glyceride, diglycosyl diglyced, digalactosyl diglyceride, galactosyl diglyceride and glycosyl diglyceride.
Examples of glycosphingolipids include galactosyl cerebroside, lactosyl cerebroside, ganglioside and the like.
Examples of phospholipids include natural or synthetic phospholipids such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidic acid, phosphatidylglycerol, phosphatidylinositol, lysophosphatidylcholine, sphingomyelin, egg yolk lecithin, soybean lecithin and hydrogenated phospholipid. Can be mentioned.

Examples of the phosphatidylcholine include soybean phosphatidylcholine, egg yolk phosphatidylcholine, dilauroyl phosphatidylcholine, dimyristoyl phosphatidylcholine, dioleoylphosphatidylcholine, dipalmitoylphosphatidylcholine, palmitooleoyl phosphatidylcholine and distearoylphosphatidylcholine.
Examples of the phosphatidylethanolamine include dilauroyl phosphatidylethanolamine, dimyristoyl phosphatidylethanolamine, dioleoyl phosphatidylethanolamine, dipalmitoyl phosphatidylethanolamine, palmitooleoyl phosphatidylethanolamine, and distearoyl phosphatidylethanolamine. .
Examples of the phosphatidylserine include dilauroylphosphatidylserine, dimyristoylphosphatidylserine, dioleoylphosphatidylserine, dipalmitoylphosphatidylserine, palmitooleoylphosphatidylserine, and distearoylphosphatidylserine.
Examples of the phosphatidyl glycerol include dilauroyl phosphatidyl glycerol, dimyristoyl phosphatidyl glycerol, dioleoyl phosphatidyl glycerol, dipalmitoyl phosphatidyl glycerol, palmitooleoyl phosphatidyl glycerol, and distearoyl phosphatidyl glycerol.
Examples of the phosphatidylinositol include dilauroyl phosphatidylinositol, dimyristoyl phosphatidylinositol, dioleoyl phosphatidylinositol, dipalmitoyl phosphatidylinositol, palmitooleoyl phosphatidylinositol and distearoyl phosphatidylinositol.

  Examples of synthetic lipids include alkylated ammonium salts. Examples of alkylated ammonium salts include benzyldodecyldimethylammonium bromide, benzyldimethyltetradecylammonium chloride, benzyloctadecyldimethylammonium chloride, stearyltrimethylammonium bromide, benzyldimethylhexadecylammonium chloride, hexadecyltrimethylammonium bromide, dimethyldipalmitylammonium bromide , Dioctadecyl ammonium bromide, dimethyl dioctadecyl ammonium bromide, hexadecyl pyridinium chloride monohydrate, and the like.

  Of these lipids, phosphatidylserine, phosphatidic acid, phosphatidylglycerol and phosphatidylinositol impart a negative surface potential to the GUV formed, and alkylated ammonium salts impart a positive surface potential. A GUV having a desired surface potential can be produced by adjusting the ratio of these two groups of lipids. The surface potential of GUV is preferably less than 0 mV, more preferably −20 mV or less, and further preferably −40 mV to −20 mV. Examples of the lipid composition that gives such a surface potential include dioleoylphosphatidylcholine: dioleoylphosphatidylglycerol: cholesterol = 9: 1: 1 (molar ratio). When the surface potential is too high, unintentional adhesion between the GUV and the cell occurs, and the electrofusion between the GUV and the cell becomes impossible, or the GUV and the cell cannot be separated after the electrofusion. If the surface potential is too lower than the above range, the GUV becomes unstable.

  Examples of cholesterol include cholesterol, beta-cholesterol, beta-sitosterol, and stigmasterol.

  A GUV having a desired film strength can be produced by adjusting the proportion of cholesterol in the total lipid. The greater the proportion of cholesterol in the total lipid, the greater the membrane strength. The proportion of cholesterol is, for example, 0 to 20 mol%, preferably 0 to 15 mol%, more preferably 0 to 10 mol%, still more preferably 0 to 5 mol%, and most preferably 0 mol%. That is, the GUV is most preferably composed of a lipid not containing cholesterol.

  The size of GUV is usually set to a micrometer order or more in diameter. In the present invention, the size of the GUV is preferably 10 μm or more and 50 μm or less in diameter, which is the same size as the cell in consideration of the effect of orientation with the cell due to the alternating electric field. In the intracellular object introduction method according to the present invention, an object having a diameter of 100 nm or more encapsulated in GUV in advance is introduced into the cell. Therefore, it is desirable that the size of the GUV is sufficiently larger than the object to be introduced.

(2) Object to be Encapsulated The object to be encapsulated in GUV may be one or more selected from the group consisting of particles, nucleic acids, polymers, microorganisms, and intracellular organs.

Examples of the particles include resin or metal microbeads, colloidal particles such as gold colloid and silver colloid. The microbead may be modified or contained with a compound or a polymer, and can be an affinity bead bound with an antibody, avidin / biotin, or a drug sustained-release bead. Examples of compounds or drugs include sensors, activators and inhibitors of intracellular reactions, acids and alkalis. In addition, the microbeads may have magnetism such as ferrite beads.
Examples of the nucleic acid include DNA, RNA, plasmid, and artificial DNA nanostructure (for example, DNA origami). A plasmid may incorporate a gene to be expressed in a cell and a promoter for gene expression. The artificial DNA nanostructure may be a combination of a compound or a polymer.
The polymer may be natural or artificial and includes proteins and various polymers. Examples of the protein include the above-described antibodies, enzymes involved in intracellular reactions, fluorescent proteins, and the like. The polymer may be a functional polymer whose structure changes in response to temperature and pH.
Examples of the microorganism include cyanobacterium, leiomycetes, lactic acid bacteria, and protobacteria. In addition, examples of intracellular organs include chloroplasts, mitochondria, and nuclei (nuclei of cell types different from cells into which objects are introduced). Further, the object enclosed in the GUV may be a so-called nanodevice such as a minute electric circuit, a sensor, or a mirror.

  In the intracellular object introduction method according to the present invention, the object to be enclosed in the GUV is not limited to those exemplified above, and may be any object as long as it can be enclosed in the GUV. According to the method for introducing an intracellular object according to the present invention, an object having a diameter of 100 nm or more can be introduced into a cell. However, the size of the object is determined by encapsulation into GUV and introduction into the cell. From the viewpoint of efficiency, the diameter is preferably about 1 μm. However, it has been confirmed that an object up to about 2 μm in diameter can be enclosed in GUV and introduced into cells (see Examples), and an object up to about 5 μm in diameter can be introduced into cells. there is a possibility.

(3) Encapsulation of an object in GUV When manufacturing GUV by using a static hydration method, an electro-formation method, an interface passing method, etc., an object is contained inside by using a solution or dispersion containing the object. Can be formed. When introducing an object having a high dielectric constant into the GUV, it is preferable to use a method other than the electroformation method, and it is particularly preferable to use an interface passing method.

  The GUV according to the present invention thus produced preferably contains one or more objects selected from the group consisting of particles, nucleic acids, macromolecules, microorganisms and organelles, and has a potential of −40 mV to −20 mV. It has a surface potential, and the proportion of cholesterol in the total lipid constituting the membrane is 0 to 15 mol%.

(4) Electrofusion Introduction of an object enclosed in GUV into cells can be performed by electrofusion of GUV and cells. In electrofusion, a first procedure for applying an alternating electric field to a mixture of GUV and cells encapsulating an object, a second procedure for applying a direct current pulse electric field to the mixture, and optionally applying an alternating electric field to the mixture again Including a third step. The electrofusion procedure is shown in FIG.

[First step]
The first procedure is a procedure for arranging GUV1 and cells 3 by applying an alternating electric field to a mixture of GUV1 and cells 3. First, GUV1 enclosing the object 2 and the cell 3 are suspended and mixed in an appropriate buffer. The buffer solution is not particularly limited, but a buffer solution having an osmotic pressure equivalent to or close to the intracellular solution is desirable. For example, a solution of 300 mM mannitol, 0.1 mM calcium chloride, and 0.1 mM magnesium chloride can be given (see FIG. (A)).

  Next, an alternating electric field is applied to the mixture of GUV 1 and cells 3 enclosing the object 2 (see FIG. (B)). This procedure can be performed by introducing a mixed solution between a pair of electrodes 4 and 4 and applying an AC electric field, and is performed using a commercially available cell electrofusion device (including electrodes and a voltage regulator). be able to. Although it does not specifically limit as an alternating voltage, For example, it is set as about 5 to 15V / mm and about 10 to 60 second. By applying an AC electric field, the GUV 1 and the cells 3 are arranged in a line between the electrodes 4, 4 and come into contact with each other to form a bead shape (pearl chain shape).

[Second step]
The second procedure is a procedure in which a DC pulse electric field is applied to the mixed solution to fuse at least a part of the GUV1 membrane and the cell 3 membrane. (See Figure (C)). In this procedure, the lipid membrane of GUV1 and the cell membrane of the cell 3 fuse with each other by applying a DC pulse voltage. Thereby, the object 2 in GUV1 is introduce | transduced in the cell 3 (refer figure (D)). Although it does not specifically limit as direct-current pulse voltage, For example, it is set as about 30-50 microseconds at about 100-175 V / mm.

[Third procedure]
The third procedure is a procedure in which an AC electric field is applied to the mixed solution to maintain a fusion state between the GUV1 membrane and the cell 3 membrane. Although this procedure is performed as necessary, it can usually be omitted and it may be preferable to omit it. Although it does not specifically limit as an alternating voltage, For example, it is about 10 seconds or less at about 5-30 V / mm. After electrofusion, the cells 3 are washed and separated from GUV1.

According to the above procedure, an object having a diameter of 100 nm or more can be simultaneously introduced into a large number of living cells (about 10 ⁵ cells / ml) without requiring a complicated operation (see Examples described later). In addition, since the substance introduced into the cell is expected to partially move inside the nucleus, endoplasmic reticulum, mitochondria, according to the intracellular substance introducing method according to the present invention, cells such as the nucleus, endoplasmic reticulum and mitochondria It is also possible to introduce objects into the internal organs.

2. Application Example (1) Measurement of Physical Properties in Cells The method for introducing intracellular objects according to the present invention can be applied to measure physical properties in cells (including in intracellular organs such as nuclei). This intracellular physical property measuring method includes a procedure of introducing an object into a cell by the intracellular object introducing method according to the present invention and detecting peristalsis of the object in the cell.

  The object to be introduced into the cell may be particles such as general-purpose resin microbeads. The movement of particles can be detected by imaging a cell under an optical microscope and analyzing the movement of the particle in the cell. Based on the detected movement of the particles, physical properties such as cytoplasm and nucleus viscoelasticity can be measured.

  In addition, the optical tweezers technique can be used to measure viscoelasticity and the like while controlling the movement of the particles because the particles can be captured or moved in the vicinity of the focal position by the focused laser beam.

  In addition, commercially available magnetic microbeads such as ferrite particles can be introduced into the cells, and the physical properties in the cells can be measured. For example, the movement (speed, acceleration, etc.) of the magnetic microbead is detected while applying a magnetic field to the cell and pulling the magnetic microbead in the cell with a magnetic force. Based on the detected velocity and acceleration, it is possible to measure the viscoelasticity of the cytoplasm and nucleus.

(2) Cell position control The intracellular substance introduction method according to the present invention can be applied to control the position of a cell. This cell position control method includes a procedure of applying a magnetic object to a cell by introducing a magnetic object into the cell by the intracellular object introduction method according to the present invention.

  Commercially available magnetic microbeads such as ferrite particles can be used for magnetic objects to be introduced into cells. By applying a magnetic field to a cell into which magnetic microbeads have been introduced using a neodymium magnet or the like, the cell position can be controlled by moving or rotating the cell. According to this cell position control method, it is possible to build a three-dimensional structure by stacking a plurality of types of cells.

(3) Surface-enhanced Raman scattering measurement of living cells The intracellular substance introduction method according to the present invention can be applied for observation of living cells by surface-enhanced Raman scattering spectroscopy. This surface-enhanced Raman scattering spectroscopy includes a procedure for introducing gold particles and / or silver particles into cells by the intracellular object introduction method according to the present invention.

  According to the method for introducing an intracellular substance according to the present invention, gold particles or silver particles can be introduced into the cytoplasm or nucleus of a living cell, so that enhanced Raman scattering on the particle surface can be detected and measured in living cells. it can. According to this surface-enhanced Raman scattering spectroscopy, it becomes possible to measure a single protein molecule present in the cytoplasm or nucleus of a living cell.

(4) Method of applying a physical force to an intracellular organ The method for introducing an intracellular substance according to the present invention can be applied to apply a physical force to a specific organelle (intracellular organ) in the cell. It is. That is, the method of applying a physical force to an intracellular organ according to the present invention is a method of applying a physical force to a predetermined protein in a cell or an intracellular organ constituted by the protein. A procedure for adding a first factor of a binding pair to the protein of the above or the intracellular organ (first procedure), and a procedure for electrofusion of GUV encapsulating magnetic particles added with a second factor of the binding pair to the cells (first procedure) 2 procedures), and a procedure (third procedure) for applying a magnetic field to the magnetic particles bound to the predetermined protein or the intracellular organ through a binding pair formed by the first factor and the second factor, It is characterized by including.

[First procedure]
The first procedure is a procedure for adding a first factor of a binding pair to a predetermined protein in the cell or an intracellular organ constituted by the protein.

  The first factor only needs to be capable of binding to the second factor described below, and for example, biotin that binds to avidin can be used. As a method for adding the first factor, various methods can be appropriately employed depending on the factor to be used. For example, when biotin is used, it can be performed by an in vivo biotinylation method. A biotin tag is added to cDNA encoding a predetermined protein, and a biotin tag fusion protein is expressed in cells by gene introduction, and at the same time, biotin ligase is also expressed. Thereby, biotin can be added to the fusion protein. Furthermore, a protein to which biotin is added constitutes an organelle in the cell, whereby an organelle to which biotin is added is obtained.

[Second procedure]
The second procedure is a procedure in which GUV encapsulating magnetic particles to which the second factor of the binding pair is added is electrofused with the cell, and is a procedure corresponding to the intracellular object introduction method according to the present invention.

  The first factor only needs to be capable of binding to the second factor, and for example, avidin can be used. Addition of avidin to the magnetic particles can be performed by a known method, and commercially available magnetic microbeads in which streptavidin is modified to magnetic particles such as ferrite particles may be used.

[Third procedure]
The third procedure is a procedure in which a magnetic field is applied to magnetic particles bound to a predetermined protein or intracellular organ via a binding pair formed by the first factor and the second factor.

  By applying a magnetic field to cells into which magnetic microbeads have been introduced using a neodymium magnet or the like, a physical force can be applied to the protein or organelle to which the magnetic microbeads are bound via avidin / biotin.

  This method can be applied to a gene expression analysis method. Specifically, it can be used for identification of a gene whose expression level changes in response to a mechanical stimulation load on a specific organelle. For example, various nuclear proteins such as actin and chromatin are biotinylated, and streptavidin-modified magnetic microbeads are bound to apply physical force. And the change of the gene expression level in the cell before and after loading is compared. This makes it possible to identify genes whose expression increases or decreases in response to mechanical stimulation to the actin skeleton and chromatin. The gene expression level can be measured by a known method such as RNA sequencing or microarray.

(5) Intracellular metabolism modification [artificial DNA nanostructure]
The intracellular substance introduction method according to the present invention can be applied to modify intracellular metabolism. This method for altering intracellular metabolism includes a procedure for introducing an artificial DNA nanostructure (for example, DNA origami) in which an enzyme and / or a functional molecule is immobilized in a cell by the method for introducing an intracellular substance according to the present invention.

  It has been shown that DNA origami immobilized with enzymes and / or functional molecules involved in a series of enzyme reactions can advance the reaction outside the cell (Single-step rapid assembly of DNA origami nanostructures for addressable nanoscale bioreactors. J. Am. Chem. Soc., 2013, 135: 696-702). The enzyme or the like is fixed to the DNA origami by setting coordinates on the surface of the DNA origami and arranging molecules involved in the reaction at respective coordinate positions so as to form a two-dimensional network. In Examples described later, it has been confirmed that DNA origami having 282 fluorescent molecules arranged on the surface by designating coordinates can be introduced into cells. The size of DNA origami is not limited as long as it can be encapsulated in GUV, but may be a rectangle having a side of about 100 nm, for example.

  By introducing or functioning DNA origami immobilizing enzymes and / or functional molecules involved in a series of enzyme reactions into cells, the metabolic pathways inherent in the cells can be activated or suppressed while maintaining the pathways. , The route can be modified. In addition, a new metabolic function that the cell originally does not have can be imparted to the cell. This method for modifying intracellular metabolism can be used, for example, to analyze digestion of fat globules and changes in cell cycle when the sugar metabolism system is activated.

[Sub-micro gel beads]
In the method for modifying intracellular metabolism according to the present invention, sub-microgel beads can be used instead of the artificial DNA nanostructure. As a material capable of encapsulating various small molecules / polymers, microgel beads (artificial salmon roe) in which alginic acid is cross-linked and fixed with calcium are known. Microgel beads can be converted to submicrometer size by simple operations such as freeze-drying and grinding. In the gel, the environment where the diffusion coefficient of the encapsulated polymer becomes almost zero and the diffusion coefficient of the encapsulated small molecule is slightly lower than or comparable to that in the solution can be set. For this reason, by encapsulating an enzyme as a polymer and a substrate as a small molecule in a gel, an artificial chain enzyme reaction sphere encapsulating an enzyme and / or a functional molecule involved in a series of enzyme reactions can be produced. According to this artificial chain enzyme reaction sphere, an enzyme reaction pathway that forms a three-dimensional network larger than the two-dimensional network in the DNA origami can be introduced into cells.

(6) Measurement of intracellular temperature The method for introducing an intracellular substance according to the present invention can be applied to measure the temperature of a cell. This intracellular temperature measurement method includes a procedure for introducing a temperature-sensitive polymer into a cell by the intracellular substance introduction method according to the present invention, and a procedure for detecting a temperature-dependent structural change of the temperature-sensitive polymer.

  The temperature-sensitive polymer is a fluorescent polymer (for example, Diffusive Thermoprobe; Okabe, K., et. Al., Nat. Commune) that has been introduced into cells by microinjection and is used to measure the temperature inside the cells. , 2012, 28 (3), 705). Examples of such a temperature sensitive fluorescent polymer include a polymer composed of a temperature sensitive unit, a hydrophilic unit, and a fluorescent unit. In this polymer, the fluorescence of the fluorescent unit is weak due to the presence of water molecules in the structure under low temperature conditions, but the polymer becomes small and round due to the hydrophobic interaction of the temperature sensitive units under high temperature conditions. It is eliminated and it becomes a state of emitting strong fluorescence. Therefore, the temperature inside the cell can be evaluated and measured by detecting the temperature-dependent structural change of the polymer based on the change in fluorescence intensity from the temperature-sensitive fluorescent polymer introduced into the cell.

  The method for measuring intracellular temperature according to the present invention can be used for screening a drug that promotes fat burning of brown adipocytes, for example, because the amount of heat production of cells can be evaluated by measuring intracellular temperature.

(7) Treatment method for mitochondrial disease By introducing the intracellular substance according to the present invention, normal mitochondrion is introduced into the cell of a mitochondrial disease patient having a mutation in mitochondrial DNA, thereby enabling treatment of mitochondrial disease. For example, Pearson's disease, which is a type of mitochondrial disease, causes pancytopenia mainly consisting of anemia due to mitochondrial DNA abnormality. However, bone marrow stem cells isolated from patients, embryonic stem cells (ES cells), By introducing and transplanting normal mitochondria into bone marrow stem cells derived from stem cells such as competent cells (iPS cells), fundamental treatment is possible.

  The cell composition containing GUV and cells, which is generated in the process of introducing mitochondria into cells by the intracellular substance introduction method according to the present invention, can be used for the treatment of mitochondrial diseases. A cell composition for treating mitochondrial disease obtained by separating cells from a patient with mitochondrial disease and performing electrofusion with GUV encapsulating normal mitochondria may contain GUV and cells into which normal mitochondria have been introduced. .

  It is also possible to substitute mitochondrial functions by introducing protobacteria that are closely related to mitochondria into cells.

(8) Reprogramming of cells It has been reported that reprogramming can occur by incorporating lactic acid bacteria into cells (Lactic acid bacteria convert human fibroblasts to multipotent cells. PLOS ONE). By introducing bacteria such as lactic acid bacteria into cells by the intracellular substance introduction method according to the present invention, cell initialization and pluripotent stem cells can be induced.

<Example 1: Introduction of an object into a cell>
1. Preparation of GUV Water-in-oil emulsion passing method described in literature (PNAS, 2003, 100: 10718-10721; J. Physical Chemistry B Lett., 2008, 112: 14678-14681; PNAS, 2012, 109: 5942-5947) According to the above, a GUV was prepared. Dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylglycerol (DOPG) and cholesterol were dissolved in chloroform (1050 μl) at a molar ratio of 9: 1: 1 (total 105 mg). The solution was transferred to a glass tube, dried in an argon gas atmosphere and vacuum dried, and then mixed with liquid paraffin (500 μl). The mixture was sonicated at 60 ° C. for 60 minutes.

  Fluorescent beads, DNA origami or plasmid DNA was mixed with an internal solution (90 mM sucrose, 210 mM mannitol, 0.1 mM calcium chloride, 0.1 mM magnesium chloride) and this internal solution (50 μl) was added to the lipid mixture.

As the fluorescent beads, carboxylic acid-modified FluoSpheres (registered trademark, Invitrogen) having diameters of 0.2, 0.5, 1.0, and 2.0 μm were used. As the plasmid DNA, an EGFP expression vector prepared using a commercially available kit (NucleoBond Xtra Midi plus kit, Macherey-Nagel GmbH & Co.) was used. Shredded rectangular DNA origami (60 nm × 90 nm) was designed using software (caDNAno). FIG. 2 shows the sequence of DNA origami. In DNA origami, 282 FITC labels were arranged on a surface of 60 × 90 nm ² .

  The tube was agitated to produce a micrometer sized W / O emulsion. The emulsion was gently poured onto an external solution (300 mM mannitol, 0.1 mM calcium chloride, 0.1 mM magnesium chloride). After centrifugation (18,000 g) at 4 ° C. for 30 minutes, the emulsion was passed through a W / O interface saturated with lipid to form a bilayer membrane. To avoid mixing oil and water, GUV was extracted from a hole drilled with a needle in the bottom of the tube.

The average diameter of the obtained GUV was estimated to be 20 μm by image analysis under a microscope. By image analysis under a fluorescence microscope, the number of beads encapsulated in each GUV was measured to be on the order of 10 ¹ to 10 ⁴ . The concentration of plasmid and DNA origami encapsulated in each GUV was calculated to be 220 ng / μl and 3.36 nM, respectively.

2. Electrofusion of GUV and cells HeLa cells were dispersed in a fusion buffer (300 mM mannitol, 0.1 mM calcium chloride, 0.1 mM magnesium chloride). A mixed solution of GUV mannitol solution (400 μl) and cell dispersion (400 μl, 1 × 10 ⁵ cells / ml) is connected to an electrofusion chamber (NEPA) connected to a generator of a cell electrofusion device (ECFG21, NEPA GENE). GENE). The GUV number was measured as 2 × 10 ⁴ / ml by image analysis under a microscope. The chamber contains two platinum parallel electrodes (2 × 80 × 5 mm) spaced 2 mm apart.

  An AC electric field of 15 V / mm was applied to the mixed solution for 30 seconds, and cells and GUV were arranged in a line (first procedure). Next, a DC pulse of 175 V / mm was applied for 50 microseconds to fuse the cells and GUV (second procedure). Fusion of cells and GUV is caused by irreversibly destroying the insulating film in the region where the cells and GUV are in contact. Subsequently, an AC electric field of 15 V / mm was applied for 10 seconds to maintain contact between the cells and the GUV (third procedure). Thereafter, the cells were washed with PBS and transferred to a culture dish containing DMEM.

3. Introduction of objects into cells (1) Fluorescent beads FIG. 3 shows cells electrofused with GUV encapsulating fluorescent beads (diameter 1 μm). The upper left of the figure shows a stereoscopic observation image, the lower left shows a vertical sectional observation image, and the right shows a horizontal sectional observation image. It can be confirmed that fluorescent beads are introduced into the cells.

  FIG. 4 shows the result of electrofusion of GUV encapsulating fluorescent beads having diameters of 0.2, 0.5, 1.0, and 2.0 μm and cells. The figure shows a fluorescence observation image of the cytoplasm, fluorescent beads and nuclei and a composite image thereof. In addition, the results of fluorescent beads with a control (no beads) and diameters of 0.2, 0.5, 1.0, and 2.0 μm are shown from the top. It can be confirmed that fluorescent beads having diameters of 0.2, 0.5, and 1.0 μm are introduced into the cells. The fluorescent beads having a diameter of 2 μm were not introduced into the cells. As a result of measuring the introduction rate of the fluorescent beads into the cells with a flow cytometer, the fluorescent beads having diameters of 0.2, 0.5, 1.0, and 2.0 μm were 73, 50, 40, and 0.38%, respectively. there were.

(2) Plasmid FIG. 5 shows cells electrofused with GUV encapsulating an EGFP expression vector. After culturing on the 1st and 5th days after electrofusion, GFP expression in the cells was confirmed, and it was confirmed that the EGFP expression vector could be introduced into the cells. The introduction efficiency of the EGFP expression vector into the cells was about 20%.

(3) DNA origami
FIG. 6 shows a cell electrofused with GUV encapsulating DNA origami. It can be confirmed that DNA origami labeled with FITC is introduced into the cells.

  It was also found that fluorescent beads and EGFP expression vector can be simultaneously introduced into cells by using a combination of GUV encapsulating fluorescent beads (diameter 1 μm) and GUV encapsulating EGFP expression vector (not shown).

<Example 2: Magnetic manipulation of cells by introduction of magnetic beads>
Magnetic beads (FG-beads, Tamagawa Seiki) were introduced into the cells by the same method as the fluorescent beads of Example 1.

  The cells were moved by applying a magnetic field to the cells into which the magnetic beads were introduced. FIG. 7 shows a series of photographs taken while rotating a cell (one turn clockwise) by applying magnetism with a magnet. The arrows in the figure indicate the progress of time. With the introduction of magnetic beads, cells could be moved freely using a magnet.

<Example 3: Examination of surface potential of GUV>
In Example 1, “1. Preparation of GUV”, the molar ratio of dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylglycerol (DOPG) and cholesterol remains at 9: 1: 1 or 9: The GUV in which the fluorescent beads (diameter 0.2 μm) used in Example 1 were encapsulated was prepared by changing to 0: 1. Further, GUV in which fluorescent beads were similarly encapsulated was prepared using dioleoylphosphatidylcholine (DOPC): dioctadecylammonium bromide (DODAB) and cholesterol at a molar ratio of 9: 1.26: 1.

  The surface potential (zeta potential) of GUV was measured by the following procedure. GUV (fluorescent beads not encapsulated) was prepared with the above lipid composition and procedure, and the diameter was adjusted to 1 μm using an extruder (Mini-Extruder, Avanti Polar Lipids) (membrane pore size 1 μm, liquid volume 1 ml). Against the pore 21 times). 300 μl of this liposome dispersion solution was introduced into a fine particle measuring device (nano Partica SZ-100 series, manufactured by Horiba, Ltd.), and the zeta potential was measured. The measured values are shown in “Table 1”.

HeLa cells and GUV were electrofused in the same manner as in “2. Electrofusion of GUV and cells” in Example 1, and fluorescent beads were introduced into the cells. After the fusion operation, the introduction rate of fluorescent beads (diameter 0.2 μm) into cells was calculated according to the following procedure. The introduced cells were centrifuged (1000 g, 3 min), the supernatant was removed, and then resuspended and washed with a fusion buffer. The cells after resuspension were analyzed by a flow cytometer (Cell Lab Quanta SC MPL, manufactured by Beckman Coulter) to determine the substance introduction efficiency into the cells. Substances having a diameter of 7 μm or more were measured. Of the number of cells counted (1.5 × 10 ⁴ cells), the ratio of those having a fluorescence intensity equal to or higher than the negative control (detected fluorescence intensity <1 × 10 ¹ ) without fusion operation was calculated. The results are shown in “Table 2”.

  Although GUV having a negative surface potential (DOPC: DOPG: cholesterol = 9: 1: 1 or 9: 0: 1) can introduce fluorescent beads, GUV having a positive surface potential (DOPC: DODAB: In the case of cholesterol = 9: 1.26: 1), GUV adhered to the cells after washing after electrofusion, and evaluation was impossible. Also, higher introduction efficiency of fluorescent beads was obtained in GUV having a lower negative surface potential.

<Example 4: Examination of cholesterol content of GUV>
In Example 1, “1. Preparation of GUV”, the molar ratio of dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylglycerol (DOPG) and cholesterol was 9: 1: 1 to 9: 1: Changed to 0, 9: 1: 0.1, 9: 1: 0.4, 9: 1: 2 or 9: 1: 5, and the fluorescent beads (diameter 0.5 μm) used in Example 1 were enclosed. GUV was prepared.

  HeLa cells and GUV were electrofused in the same manner as in “2. Electrofusion of GUV and cells” in Example 1, and fluorescent beads were introduced into the cells. In the same manner as in Example 3, the introduction rate of fluorescent beads into cells was calculated using a flow cytometer. The results are shown in “Table 3”. There was a tendency to obtain a higher introduction rate of fluorescent beads in GUV having a lower cholesterol ratio. The proportion of cholesterol is preferably 0 to 16.6 mol%, more preferably 0 to 9.1 mol%, still more preferably 0 to 3.8 mol%, even more preferably 0 to 0.9 mol%, most preferably Preferably it is 0 mol%.

<Example 5: Examination of application time of post-fusion AC electric field>
The application time of the alternating electric field (15 V / mm) in the third procedure of “2. Electrofusion of GUV and cells” in Example 1 was changed from 10 seconds to 0, 20, 60 seconds to obtain fluorescent beads (0.5 μm ) Was introduced into the cells. In the same manner as in Example 3, the introduction rate of fluorescent beads into cells was calculated using a flow cytometer. The results are shown in “Table 4”.

Claims (19)

A method of introducing an object into a cell,
By applying an alternating electric field to a mixture of giant unilamellar liposomes (Giant Unilamellar Liposomes) encapsulating the object and having a negative surface potential, and the cells, the giant unilamellar liposomes and the cells And a first step of arranging
After the first procedure, a second procedure in which a DC pulse electric field is applied to the mixed solution to fuse at least partially the membrane of the giant unilamellar liposome and the membrane of the cell;
Including methods.   The method according to claim 1, wherein the giant unilamellar liposome has a surface potential of −40 mV to −20 mV.   The method according to claim 1 or 2, wherein the proportion of cholesterol in the total lipid constituting the membrane of the giant unilamellar liposome is 0 to 15 mol%.   In the first procedure, the AC electric field is applied at 5 to 15 V / mm for 10 to 60 seconds, and in the second procedure, the DC pulse electric field is applied at 100 to 175 V / mm for 30 to 50 microseconds. Item 4. The method according to any one of Items 1 to 3.   The method according to claim 1, wherein the object has a diameter of 100 nm or more.   The method according to claim 1, wherein the object is one or more selected from the group consisting of particles, nucleic acids, macromolecules, microorganisms, and intracellular organelles. A method for producing giant unilamellar liposomes,
A production method comprising preparing the lipid constituting the membrane of the giant unilamellar liposome such that the surface potential of the giant unilamellar liposome is -40 mV to -20 mV.   The production method according to claim 7, comprising preparing the lipid constituting the membrane of the giant unilamellar liposome such that the proportion of cholesterol in the total lipid is 0 to 15 mol%.   Giant unilamellar liposomes having a surface potential of −40 mV to −20 mV.   The giant unilamellar liposome according to claim 9, wherein the proportion of cholesterol in the total lipid constituting the membrane is 0 to 15 mol%.   The giant unilamellar liposome according to claim 9 or 10, wherein one or more objects selected from the group consisting of particles, nucleic acids, polymers, microorganisms, and organelles are encapsulated.   A method for measuring physical properties in a cell by introducing an object into the cell by the method according to claim 1 and detecting peristalsis of the object in the cell.   A method for controlling the position of a cell by introducing a magnetic object into the cell by the method according to claim 1 and applying a magnetic field to the cell.   Surface-enhanced Raman scattering spectroscopy comprising a procedure for introducing gold particles and / or silver particles into cells by the method according to claim 1. A method of applying a physical force to a predetermined protein in a cell or an intracellular organ constituted by the protein,
Adding a first factor of a binding pair to the predetermined protein or the intracellular organ;
A procedure for introducing magnetic particles, to which a second factor of a binding pair has been added, into a cell by the method according to claims 1 to 6;
A step of applying a magnetic field to the magnetic particles bound to the predetermined protein or the intracellular organ through a binding pair formed by the first factor and the second factor;
Including methods.   The method according to claim 15, wherein the first factor is biotin and the second factor is streptavidin, and biotin is added to the predetermined protein or the intracellular organ by an in vivo biotinylation method.   A gene for applying a physical force to a predetermined protein in a cell or an intracellular organ composed of the protein by the method according to claim 15 or 16, and detecting a change in gene expression level in the cell before and after the load. Expression analysis method.   A method for modifying intracellular metabolism by introducing an artificial DNA nanostructure (DNA origami) in which an enzyme involved in an enzyme reaction and / or a functional molecule is immobilized in the cell by the method according to claim 1.   A method for measuring an intracellular temperature, comprising a step of introducing a temperature-sensitive polymer into a cell by the method according to claim 1 and a step of detecting a temperature-dependent structural change of the temperature-sensitive polymer.

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