Method for introducing target substance into cytoplasm of cell

Abstract

The present invention addresses the problem of providing a method for introducing a target substance into the cytoplasm of a cell. The problem is solved by providing a method for introducing a target substance into the cytoplasm of a cell, the method including a step for introducing a magnetic carrier into the endosomes of the cell, where the magnetic carrier is the target substance or the magnetic carrier has the target substance, and a step for irradiating the endosomes into which the magnetic carrier has been introduced with light, and the method being characterized in that the maximum diameter of the magnetic carrier that is the target substance or the entire magnetic carrier having the target substance is 1 μm or more and the wavelength of the light irradiated in the step for irradiating with light is 600 nm or less.

Description

A method for introducing a target substance into the cytoplasm of a cell.

[0001] This disclosure relates to a method for introducing a target substance into the cytoplasm of a cell.

[0002] The introduction of substances into living cells is an essential technology in research fields that deal with cells, such as modifying cell function through genome editing and cell therapy through drug introduction. In particular, chemical introduction methods using transfection reagents have been actively studied not only in analysis using cultured cells but also in applied research targeting organisms, such as drug delivery system development. This is because they can introduce substances into many cells at once with less damage compared to electroporation, which introduces substances by physically creating holes in cells, or microinjection, which introduces substances by inserting glass capillaries into individual cells. However, conventional research has mainly focused on the introduction of minute substances with a diameter of less than 200 nm, with a resolution below that of a normal optical microscope, and because the morphology of the introduced substance changes within the cell, the intracellular dynamics of minute substances remain a black box.

[0003] On the other hand, regarding the introduction of micrometer-scale macromolecules into cells, while there are numerous studies on the process of uptake into endosomes (or phagosomes), there have been no previous examples of macromolecules penetrating the cell membrane (endosomal membrane) and reaching the cytoplasm. For this reason, methods based on "cell fusion" developed in the 1970s (micronucleus cell fusion method) or its variations are still used for the delivery of macromolecules such as artificial chromosome vectors into cells (Non-Patent Literature 1). However, cell fusion methods are susceptible to the significant impact of introducing unintended substances, and suffer from limitations in applicable cell types and low introduction efficiency. Therefore, there is a strong need for the development of technologies that can efficiently introduce micrometer-scale macromolecules into cells and maintain them stably. In the future, when miniaturized and highly integrated semiconductor chips and other devices are implemented inside cells and their functions can be controlled from outside the cell, how to stably and efficiently introduce and maintain such micrometer-scale substances inside cells will be extremely important.

[0004] Non-patent document 2 discloses a method (vapor nanobubble (VNB)-mediated photoporation) in which a shock wave is generated by irradiating gold particles or the like, which are in contact with the cell membrane, with pulsed laser light, thereby rupturing the cell membrane and allowing surrounding substances to be taken into the cell. This method has been used to introduce small molecule compounds and nucleic acids into cells, but in the case of DNA introduction, for example, most of the DNA bound to the surface of the gold particles is degraded, so the percentage of cells that ultimately express the target gene is very low, at about 1-3%, and there is insufficient evidence to show that the introduced DNA is exposed in the cytoplasm.

[0005] Non-patent document 3 discloses a photochemical internalization (PCI) method using photosensitizers such as fluorescent molecules and porphyrins, which is similarly used for introducing small molecule compounds and nucleic acids into cells. Non-patent document 4 describes the introduction of microbeads by PCI, but concludes that PCI is insufficient for releasing microbeads from endosomes, that is, for introducing substances into the cytoplasm.

[0006] Patent Document 1 discloses the uptake of particles into endosomes by a transfection reagent, suggesting that the transfection reagent causes endosomes to disintegrate, exposing the uptaken particles to the cytoplasm. However, the transfection reagent method has several problems, including the fact that the probability of endosome disintegration is only about 50-60%, and if disintegration does not occur, the particles will proceed to the degradation pathway, and the timing of endosome disintegration cannot be controlled.

[0007] Thus, there was a need for methods to introduce relatively large substances, such as DNA and beads, into cells, particularly the cytoplasm. Furthermore, there was a need for methods to introduce such substances while controlling their temporal and spatial properties.

[0008] Japanese Patent Publication No. 2017-006070

[0009] Uno et al, Sci Rep12, 3009 (2022) Vermeulen LM, et al (2018) International journal of Molecular Sciences Ohtsuki, T. et al. (2015) Sci Rep, 5:18577, doi: 10.1038 / srep18577. Dec 21 Mora-Espi I. et al. Sci. Rep 2018, 8 (1), 17617.

[0010] This disclosure aims to provide a method for introducing a target substance into the cytoplasm of a cell. As one aspect, this disclosure aims to provide a method for efficiently introducing a target substance into the cytoplasm of a cell. Also, as one aspect, this disclosure aims to provide a method for introducing a target substance into the cytoplasm of a cell while controlling the timing.

[0011] The inventors have found that a target substance can be introduced into the cytoplasm of a cell by irradiating light using a magnetic carrier. That is, this disclosure provides the following aspects.

[0012] [1] A method for introducing a target substance into the cytoplasm of a cell, comprising the steps of: introducing a magnetic carrier into the endosome of the cell, wherein the magnetic carrier is the target substance or the magnetic carrier contains the target substance; and irradiating the endosome into which the magnetic carrier has been introduced with light, wherein the maximum diameter of the magnetic carrier that is the target substance or the magnetic carrier containing the target substance is 1 μm or more, and the wavelength of the light irradiated in the light irradiation step is 600 nm or less. [2] The method of [1], wherein the magnetic carrier is a magnetic bead containing iron oxide and a polymer. [3] The method of [1] or [2], wherein the step of introducing the carrier into the endosome of the cell is performed by promoting endocytosis using an endocytosis induction reagent. [4] The method of [3], wherein the endocytosis induction reagent is one or more cell membrane-binding peptides selected from the group consisting of poly-L-arginine (PLA), Pep-1 peptide, TAT, Penetratin, and poly-L-lysine (PLL). [5] In the step of irradiating with light, the light is directed onto the magnetic carrier at a distance of 0.05 μm 2 A method of [1] to [4] which involves irradiating the above range. [6] A method of [1] to [5] in which the target substance comprises one or more substances from among polypeptides, DNA, RNA, glycans, lipids, synthetic polymer compounds, or combinations thereof.

[0013] According to the present disclosure, a method for introducing a target substance into the cytoplasm of a cell can be provided. In particular, according to the present disclosure, even a relatively large substance such as a target substance, a particle having the same, or the whole including these, with a size of 1 μm or more can be introduced into the cytoplasm. Further, according to the present disclosure, it is possible to control when and which substance is introduced into the cytoplasm. As a result, the state of the cell before and after the foreign substance enters the cytoplasm can be analyzed with high temporal and spatial resolution, and effects such as analysis of the mechanism when the foreign substance enters the cytoplasm, analysis of the mechanism of drug uptake into the cell, or analysis of the pathogen infection pathway are expected. In addition, it becomes possible to introduce an arbitrary substance into the cytoplasm at a targeted time and at a targeted location, and for example, effects of mechanism analysis in cell biology such as the relationship between the cell cycle and the foreign substance introduction efficiency are expected. Since the present disclosure can achieve the introduction of micrometer-scale artifacts into the cytoplasm and its spatial and temporal control, in the future, it is expected to become an important basic technology for the development of communication interface technology between cells and artifacts, such as embedding and using highly integrated and tiny semiconductor chips and the like into cells.

[0014] Images taken by live imaging before light irradiation, immediately after light irradiation, and 60 seconds after light irradiation (photographs substituting for drawings). A figure (photograph substituting for drawing) showing the results of staining with Hoechst33,342 and immunostaining with an anti-emerin antibody and observing together with GFP-BAF after light irradiation. Images taken by live imaging before light irradiation, immediately after light irradiation, and 60 seconds after light irradiation (photographs substituting for drawings). A graph showing the correlation between the intensity (μW) of the irradiated light (laser) and the ratio (%) of endosomes broken for light with a wavelength of 561 nm. A graph showing the correlation between the intensity (μW) of the irradiated light (laser) and the ratio (%) of endosomes broken for light with a wavelength of 405 nm.

[0015] <1> Method for introducing a target substance into the cytoplasm of a cell The present disclosure provides a method for introducing a target substance into the cytoplasm of a cell, comprising: introducing a magnetic carrier into an endosome of the cell, wherein the magnetic carrier is the target substance or the magnetic carrier contains the target substance; and irradiating the endosome into which the magnetic carrier has been introduced with light. This method is also referred to as the method for introducing a target substance into the cytoplasm of a cell according to the present disclosure. The method for introducing a target substance into the cytoplasm of a cell according to the present disclosure may be carried out in vitro, at least in part or in whole.

[0016] In this disclosure, "introducing the target substance into the cytoplasm of a cell" may more specifically mean that the target substance is in direct contact with the cytoplasm of a cell, or in other words, that the target substance is exposed to the cytoplasm.

[0017] The procedure can be carried out using human cells, more specifically, HeLa cells or HEK293 cells. Theoretically, the cells are not particularly limited, and any cells may be used. Among any cells, it is preferable that the cells have the property of taking in foreign substances into endosomes. Theoretically, the cells may be derived from, for example, fungal cells, plant cells, animal cells, or insect cells. The cells may be derived from, for example, animal cells, especially mammalian cells. Furthermore, the cells may be derived from, for example, humans, mice, cattle, pigs, sheep, horses, dogs, cats, or chickens. The cells may be cultured cells or cells collected from living organisms. More specifically, epithelial-like adherent cells can be used as cells. Even more specifically, the cells may be, for example, the HeLa cells or HEK293 cells mentioned above. The cell culture method is not particularly limited, and the cells may be cultured using an appropriate method according to the cell type.

[0018] The target substance can be a polypeptide, such as an antibody, or DNA. Theoretically, the target substance is not particularly limited, and any substance that can be introduced into the cytoplasm of a cell may be selected. Such any substance includes the magnetic carrier itself, which will be described later. Theoretically, the target substance may be a biomolecule or a non-biomolecule, and examples include polypeptides such as antibodies, any nucleic acids including DNA such as chromosomes and RNA such as mRNA or siRNA, glycans, lipids such as phospholipids and steroid hormones, small molecule compounds including compounds that can be used in pharmaceuticals, polymers including synthetic polymer compounds, and other organic or inorganic compounds, or combinations thereof. The target substance may be relatively large, on a micrometer scale, although this is not limited to these examples. The target substance may include, for example, one or more substances from polypeptides, DNA, RNA, glycans, lipids, synthetic polymer compounds, or combinations thereof. Polypeptides, although this is not limited to these examples, specifically include antibodies, oligonucleotides, fluorescent proteins, water-soluble proteins including BAF protein and galectin-3 (Gal-3) protein, or membrane proteins. DNA may be single-stranded DNA, double-stranded DNA, a hybrid of DNA and other nucleic acids such as RNA, and may be modified in any way; there are no particular restrictions. Examples of DNA, though not limited to these, include plasmids, chromosomes, genomic DNA or a part thereof, complementary DNA (cDNA), or viral genomic DNA. Furthermore, DNA may be biotin-modified, for example. RNA may be single-stranded RNA, double-stranded RNA, a hybrid of RNA and other nucleic acids such as DNA, and may be modified in any way; there are no particular restrictions. Examples of RNA, though not limited to these, include mRNA, siRNA, microRNA (miRNA), or viral genomic RNA. Glycans may be bound to proteins or lipids, for example.Lipids are not limited to these, but examples include fatty acids, glycerolipids or sphingolipids including phospholipids, and steroids including sterols and steroid hormones. Specifically, lipids may be phospholipids, steroid hormones, or fat-soluble vitamins such as vitamins A, D, E, and K. Lipids may also be bound to proteins or sugar chains, for example. Synthetic polymer compounds mean synthesized polymer compounds, and may be any polymer, for example. Specific examples of such polymers include polyethylene glycol (PEG). Polymers may also refer to polymers contained in magnetic carriers, which will be described later. Target substances include polypeptides or DNA, for example. Target substances may also be magnetic carriers. Target substances may be the magnetic carrier itself, substances contained in the magnetic carrier, or substances such as functional groups that the magnetic carrier has on its surface or other parts.

[0019] A method for introducing the target substance of this disclosure into the cytoplasm of a cell includes the step of introducing a magnetic carrier into the cell's endosomes. In this step, the magnetic carrier is either the target substance or the magnetic carrier contains the target substance. In other words, a method for introducing the target substance of this disclosure into the cytoplasm of a cell may mean that it includes the step of introducing a magnetic carrier that is the target substance or a magnetic carrier containing the target substance into the cell's endosomes. When the magnetic carrier is the target substance, it is also referred to as the "magnetic carrier that is the target substance." When the magnetic carrier contains the target substance, it is also referred to as the "magnetic carrier containing the target substance."

[0020] The magnetic carrier is not particularly limited as long as it contains a magnetic material. Specifically, for example, magnetic beads may be used as the magnetic carrier. As described above, the magnetic carrier may also be the target substance. In other words, in one embodiment, the target substance introduced into the cytoplasm of a cell in the method of introducing the target substance of this disclosure may be a magnetic carrier. Examples of magnetic carriers include magnetic beads containing a magnetic material such as iron oxide and a polymer such as polystyrene or a ceramic such as silica. The magnetic carrier may preferably be a magnetic bead containing a magnetic material and a polymer. Examples of magnetic materials include iron oxide (triiron tetroxide, diiron trioxide, etc.), iron, manganese, nickel, nickel oxide, various ferrites, cobalt, cobalt iron oxide, chromium, barium ferrite, carbon steel, tungsten steel, KS steel, rare earth cobalt magnets, hematite, etc. The magnetic carrier preferably contains iron oxide, and more preferably contains triiron tetroxide. That is, the magnetic carrier may be a magnetic bead containing iron oxide and a polymer. Furthermore, the magnetic susceptibility of the magnetic carrier is not particularly limited, but for example, a volume magnetic susceptibility of 0.1 emu / cm² is acceptable. ３ Above, 0.3 emu / cm ３ Above or equal to 0.5 emu / cm² ３ The magnetic properties described above may be observed. Examples of polymers include polystyrene, polypropylene, and acrylic resin, but are not particularly limited. Furthermore, the magnetic beads may or may not contain additional molecules for purposes such as bonding with or supporting the target substance.

[0021] The magnetic carrier having the target substance is not particularly limited as long as the target substance and the magnetic carrier can move together as a single unit. For example, the target substance may be attached to the magnetic carrier by covalent bonds, antigen-antibody reactions, biotin-avidin reactions, hybridization with nucleic acids, lectin-glycan bonds, or other non-covalent bonds, or supported by voids or charges. When using a magnetic carrier having the target substance, the magnetic carrier having the target substance may be prepared in advance, or a magnetic carrier that has undergone a process to possess the target substance may be used. That is, in one embodiment, the method for introducing the target substance into the cytoplasm of a cell according to this disclosure may include a process to possess the target substance in the magnetic carrier. When using a magnetic carrier having the target substance, the magnetic carrier may be appropriately selected to possess the target substance.

[0022] A magnetic carrier having the target substance means that it has the target substance in any manner depending on the target substance, and is not particularly limited, but examples include binding between molecules bound to the magnetic carrier and the target substance by biotin-avidin reaction or antigen-antibody reaction, hybridization by complementary base pairing between nucleic acids bound to the magnetic carrier and the target substance, lectin-glycan bonding between lectins and / or glycans bound to the magnetic carrier and the target substance, covalent bonding between molecules on the surface of the magnetic carrier and the target substance, and / or support of the target substance by voids or charges of the magnetic carrier. Specifically, a magnetic carrier having the target substance means, for example, binding by interaction between avidin or streptavidin bound to the magnetic carrier and the target substance containing biotin-labeled polypeptide or DNA or biotinylated compounds, binding by interaction between an antibody bound to the magnetic carrier and the target substance containing an antigen to which the antibody specifically binds, binding by interaction between nucleic acids bound to the magnetic carrier and the target substance having a base sequence that binds complementaryly to the nucleic acid, and COOH groups or NH on the surface of the magnetic carrier. ２ The base and the NH contained in the target substance ２This includes bonding with a group or COOH group, bonding through interaction between lectins and / or sugar chains bound to the magnetic carrier and the target substance containing the sugar chain or lectin, physical support of the target substance in the voids of the magnetic carrier, or support of the target substance by ionic bonding due to the charge of the magnetic carrier.

[0023] The method for introducing the target substance into the cytoplasm of a cell according to this disclosure allows the target substance, even if the magnetic carrier or magnetic carrier containing the target substance is relatively large on a micrometer scale, to be exposed to the cytoplasm after being introduced into an endosome by the rupture of the endosome membrane. Therefore, in the method for introducing the target substance into the cytoplasm of a cell according to this disclosure, the maximum diameter of the magnetic carrier or the magnetic carrier containing the target substance may be 1 μm or more. In other words, the method for introducing the target substance into the cytoplasm of a cell according to this disclosure may be characterized by the fact that the maximum diameter of the magnetic carrier or the magnetic carrier containing the target substance is 1 μm or more.

[0024] The maximum diameter of the target material magnetic carrier, or the entire magnetic carrier containing the target material, may be, for example, 1 μm or more, 1.5 μm or more, 2 μm or more, 3 μm or more, or 5 μm or more. The upper limit of the maximum diameter of the target material magnetic carrier, or the entire magnetic carrier containing the target material, is not particularly limited, but may be arbitrarily set according to the size of the cell, or may be, for example, 10 μm or less, 8 μm or less, 5 μm or less, or 3 μm or less. Furthermore, any combination of these upper and lower limits that is not contradictory may also be used. Specifically, the maximum diameter of the target material magnetic carrier, or the entire magnetic carrier containing the target material, may be, for example, 1 μm to 10 μm, 1 μm to 5 μm, or 1 μm to 3 μm.

[0025] Furthermore, the minimum diameter of the target material magnetic carrier, or the entire magnetic carrier containing the target material, is not particularly limited as long as it does not contradict the maximum diameter mentioned above. For example, it may be 0.1 μm or more, 0.2 μm or more, 0.5 μm or more, 0.8 μm or more, 1 μm or more, 1.5 μm or more, 2 μm or more, 3 μm or more, or 5 μm or more, or 10 μm or less, 8 μm or less, 5 μm or less, 3 μm or less, 2 μm or less, 1.5 μm or less, 1 μm or less, 0.8 μm or less, 0.5 μm or less, or 0.2 μm or less, and any combination of these that does not contradict each other is also acceptable. Specifically, the minimum diameter of the target material magnetic carrier, or the entire magnetic carrier containing the target material, may be, for example, 0.1 μm to 10 μm, 0.5 μm to 5 μm, or 1 μm to 3 μm.

[0026] In this disclosure, the method for introducing magnetic carriers, etc., into the endosomes of cells is not particularly limited. Methods for introducing magnetic carriers, etc., into the endosomes of cells include methods that utilize the functions of living cells, specifically, methods that promote endocytosis. Therefore, the step of introducing magnetic carriers, etc., into the endosomes of cells may be carried out by promoting cellular endocytosis. The method for promoting cellular endocytosis is not particularly limited, and any known method may be used. Cellular endocytosis may be promoted, for example, by using a reagent that induces cellular endocytosis. Such a reagent is also called an endocytosis-inducing reagent. The step of introducing magnetic carriers, etc., into the endosomes of cells may be carried out, for example, by promoting cellular endocytosis using an endocytosis-inducing reagent.

[0027] The endocytosis-inducing reagent is not particularly limited as long as it induces endocytosis when applied to cells, and any reagent may be used. Examples of endocytosis-inducing reagents include transfection reagents such as Effectene® and polyethyleneimine (PEI), and cell membrane-binding peptides such as poly-L-arginine (PLA). It has been reported that when transfection reagents such as Effectene or PEI are used as endocytosis-inducing reagents to promote endocytosis, spontaneous endosome disintegration may occur (Japanese Patent Application Publication No. 2017-006070). On the other hand, when cell membrane-binding peptides are used as endocytosis-inducing reagents to promote endocytosis, spontaneous endosome disintegration is considered unlikely to occur. Therefore, from the viewpoint of suppressing the disintegration of endosomes at undesirable timings, the endocytosis-inducing reagent may preferably be a cell membrane-binding peptide. Examples of cell membrane-binding peptides include PLA, Pep-1 peptide, TAT, Penetratein, and poly-L-lysine (PLL). Therefore, the endocytosis induction reagent may be one or more cell membrane-binding peptides selected from the group consisting of PLA, Pep-1 peptide, TAT, Penetratein, and PLL.

[0028] Furthermore, the step of introducing the substance into the endosomes of cells may or may not involve the use of additional reagents, depending on the conditions of the target substance, cells, magnetic carrier, or endocytosis induction reagent used. Examples of additional reagents include substances used to increase the efficiency of endocytosis induction. Specifically, for example, if the target substance is DNA, magnesium chloride (MgCl2) may be used simultaneously with the endocytosis induction reagent.

[0029] A method for introducing the target substance into the cytoplasm of a cell according to this disclosure further includes a step of irradiating light onto an area including at least a portion of the magnetic carrier introduced into the endosome. The light may be irradiated onto the entire magnetic carrier. A method for introducing the target substance into the cytoplasm of a cell according to this disclosure is characterized in that the wavelength of the light irradiated in the light irradiation step is 600 nm or less. By irradiating at least a portion of the magnetic carrier introduced into the endosome with light of this wavelength range, the membrane of the endosome is broken, and the magnetic carrier containing the target substance or the magnetic carrier containing the target substance is exposed to the cytoplasm. Furthermore, by irradiating light onto a magnetic carrier introduced into any endosome, the membrane of the endosome containing the magnetic carrier is broken, so the introduction of the target substance into the cytoplasm can be controlled on a per-endosome or carrier basis. Furthermore, by controlling the timing of light irradiation, the timing of the introduction of the target substance into the cytoplasm can also be controlled. Therefore, by controlling the selection of endosomes containing magnetic carriers to be irradiated with light during the light irradiation process, and / or the timing of light irradiation, the exposure of the target substance to the cytoplasm, i.e., the introduction of the target substance into the cytoplasm, can be controlled spatially and temporally.

[0030] In the step of irradiating the light, the wavelength of the irradiated light is not particularly limited as long as it is 600 nm or less. For example, it may be 600 nm or less, 580 nm or less, 565 nm or less, 561 nm or less, 550 nm or less, 530 nm or less, 510 nm or less, 500 nm or less, 490 nm or less, 488 nm or less, 480 nm or less, 450 nm or less, 430 nm or less, 420 nm or less, 410 nm or less, or 405 nm or less. The lower limit of the wavelength of the light is not particularly limited and may be appropriately set in consideration of the toxicity to cells and the damage to target substances, etc. For example, it may be 400 nm or more, 405 nm or more, 410 nm or more, 420 nm or more, 430 nm or more, 450 nm or more, 480 nm or more, 488 nm or more, 490 nm or more, 500 nm or more, 510 nm or more, 530 nm or more, 550 nm or more, or 561 nm or more. Also, any combination of these upper and lower limits that does not conflict may be used. The lower limit of the wavelength of the light may preferably be 400 nm. Specifically, the wavelength of the irradiated light in the step of irradiating the light may be, for example, 400 nm to 600 nm, 405 nm to 600 nm, 400 nm to 565 nm, 405 nm to 561 nm, 400 nm to 500 nm, 405 nm to 488 nm, 400 nm to 410 nm, or about 405 nm.

[0031] In the step of irradiating the light, the range of light irradiation is not particularly limited as long as the target substance can be introduced into the cytoplasm of the cell. For example, the light may be irradiated so that a certain area in the plan view projected onto a plane in the direction of light irradiation is covered for the magnetic carrier. For example, in the step of irradiating the light, the light is irradiated onto an area of 0.05 μm ２ or more, 0.075 μm ２ or more, 0.10 μm ２ or more, 0.15 μm ２ or more, 0.20 μm ２ or more, 0.25 μm ２ or more, 0.30 μm ２ or more, 0.40 μm ２ or more, 0.50 μm ２ or more, 0.70 μm ２ or more, 1.00 μm ２ or more, 1.25 μm ２Above, 1.50μm ２ More than 2.00 μm ２ Above, 2.50μm ２ Above, 3.00μm ２ Above, 4.00μm ２ or more, 5.00 μm ２ Above, 6.00μm ２ Above, 7.00μm ２ Above, 8.00μm ２ Above, 9.00μm ２ Above, 10.00μm ２ Above or above, or 15.00 μm ２ The above range may be irradiated, up to 15.00 μm. ２ Below, 10.00μm ２ Below, 9.00μm ２ Below, 8.00μm ２ Below, 7.00μm ２ Below, 6.00μm ２ Below, 5.00μm ２ Below, 4.00μm ２ Below, 3.00μm ２ Below, 2.50μm ２ Below, 2.00μm ２ Below, 1.50μm ２ Below, 1.25 μm ２ Below, 1.00μm ２ Below, 0.70μm ２ Below, 0.50μm ２ Below, 0.40μm ２ Below, 0.30μm ２ Below, 0.25 μm ２ Below, 0.20μm ２ Below, 0.15 μm ２ Below, 0.10 μm ２ The following, or 0.075 μm ２ The following are also possible, and any non-contradictory combination thereof. Specifically, for example, in the step of irradiating with light, the light is directed onto 0.05 μm of the magnetic carrier. ２ ~15.00 μm ２ ,0.10~15.00μm ２ , 0.15 μm ２ ~15.00 μm ２ , 0.25 μm ２ ~15.00 μm２ , 0.25 μm ２ ~10.00 μm ２ , 1.00 μm ２ ~10.00 μm ２ , 5.00 μm ２ ~10.00 μm ２ , or 9.00 μm ２ ~10.00 μm ２ This is also acceptable. These areas may refer to the area on the magnetic carrier in a plan view projected onto a plane in the direction from which the light is irradiated. However, the upper limit of the above range is the total area of ​​the magnetic carrier. Furthermore, it is sufficient for light to be irradiated to the above-mentioned range on the magnetic carrier, and other areas may or may not be irradiated, and there are no particular restrictions on those areas.

[0032] In the process of irradiating with light, the intensity of the irradiated light is not particularly limited as long as the target substance can be introduced into the cytoplasm of the cells, but it may be appropriately determined depending on the wavelength of the irradiated light, the area on the magnetic carrier that is irradiated, the cells used, the cell culture conditions, and various other circumstances. The intensity of the light irradiated in the process of irradiating light is not limited to these, but may be, for example, 0.5 μW or more, 1 μW or more, 5 μW or more, 10 μW or more, 15 μW or more, 20 μW or more, 30 μW or more, 40 μW or more, 50 μW or more, 60 μW or more, 70 μW or more, 80 μW or more, 85 μW or more, 90 μW or more, or 95 μW or more, and may also be 100 μW or less, 95 μW or less, 90 μW or less, 85 μW or less, 80 μW or less, 70 μW or less, 60 μW or less, 50 μW or less, 40 μW or less, 30 μW or less, 20 μW or less, 10 μW or less, 5 μW or less, or 1 μW or less. The intensity of the light irradiated in the process described above is not limited to these values, but specifically, for example, it may be 0.5 μW to 100 μW, 1 μW to 100 μW, 10 μW to 100 μW, 50 μW to 100 μW, 80 μW to 100 μW, 0.5 μW to 10 μW, 10 μW to 50 μW, or 50 μW to 100 μW. The unit "W" in the numerical value of the light intensity may mean the power applied to the light irradiating device, especially the laser device.

[0033] In the process of irradiating with light, the duration of light irradiation is not particularly limited as long as the target substance can be introduced into the cytoplasm of the cells. The duration of light irradiation may be set appropriately, taking into consideration damage to cells and the target substance, the irradiation method, the irradiated area, etc. In the process of irradiating with light, the time for irradiating with light is not limited to these, but may be 0.5 milliseconds (msec) or more, 1 msec or more, 2 msec or more, 3 msec or more, 5 msec or more, 7 msec or more, 10 msec or more, 20 msec or more, 30 msec or more, 50 msec or more, 75 msec or more, 100 msec or more, or 200 msec or more, and may also be 500 msec or less, 300 msec or less, 200 msec or less, 150 msec or less, 100 msec or less, 75 msec or less, 50 msec or less, 30 msec or less, 20 msec or less, 10 msec or less, or 7 msec or less. In the process of irradiating with light, the duration of light irradiation is not limited to these, but specifically, for example, it may be 0.5 msec to 500 msec, 1 msec to 300 msec, 2 msec to 200 msec, 5 msec to 150 msec, 1 msec to 10 msec, 3 msec to 20 msec, 75 msec to 200 msec, or 100 msec to 150 msec.

[0034] The method for confirming that the target substance has been introduced into the cytoplasm of a cell is not particularly limited, and any method can be used. For example, one method for confirming that the target substance has been introduced into the cytoplasm of a cell is the method described later in "Method for confirming that the target substance has been introduced into the cytoplasm of a cell."

[0035] <2> Method for confirming that the target substance has been introduced into the cytoplasm of a cell The disclosure also further provides a method for confirming that the target substance has been introduced into the cytoplasm of a cell. Such a method is also referred to as a method for confirming that the target substance of the disclosure has been introduced into the cytoplasm of a cell.

[0036] One embodiment of a method for confirming that the substance of the present disclosure has been introduced into the cytoplasm of a cell includes the step of performing a method for introducing the substance of the present disclosure into the cytoplasm of a cell, wherein the cell has labeled BAF protein in its cytoplasm, and the magnetic carrier has at least double-stranded DNA (dsDNA), and the method includes the step of observing the accumulation of the fluorescently labeled BAF protein overlapping with or adjacent to the magnetic carrier after performing the step of introducing the substance of the present disclosure into the cytoplasm of a cell.

[0037] BAF proteins present in the cytoplasm are known to bind to dsDNA present in the cytoplasm. In cells containing BAF proteins in the cytoplasm, accumulation of BAF proteins overlapping with or adjacent to carriers containing dsDNA can be observed, confirming that the dsDNA has been exposed to the cytoplasm, i.e., that the carrier containing dsDNA has been introduced into the cell's cytoplasm. The labeling of BAF proteins is not particularly limited as long as the location of the BAF protein within the cell can be confirmed, but examples include labeling with fluorescent proteins. Specifically, examples of BAF proteins labeled with fluorescent proteins include fusion proteins of fluorescent proteins and BAF proteins. Examples of fluorescent proteins include green fluorescent protein (GFP) and monomeric red fluorescent protein (mRFP), but are not particularly limited.

[0038] Another embodiment of a method for confirming that the substance of the present disclosure has been introduced into the cytoplasm of a cell includes a step of performing a method for introducing the substance of the present disclosure into the cytoplasm of a cell, wherein the cell has labeled Gal-3 protein in its cytoplasm, and after performing the step of introducing the substance of the present disclosure into the cytoplasm of a cell, the method includes observing the accumulation of the labeled Gal-3 protein on the membrane surrounding the magnetic carrier.

[0039] Gal-3 protein is known to localize on ruptured cell fragments. In cells containing Gal-3 protein in the cytoplasm, when the endosomes surrounding the carrier disintegrate, Gal-3 protein localizes to endosomal membrane fragments; that is, an accumulation of Gal-3 protein is observed on the membrane surrounding the carrier. Therefore, the accumulation of Gal-3 protein on the membrane surrounding the carrier confirms that the endosomes surrounding the carrier have disintegrated and the carrier has been introduced into the cell's cytoplasm. The labeling of Gal-3 protein is not particularly limited as long as the intracellular location of Gal-3 protein can be confirmed, but examples include labeling with fluorescent proteins. Specifically, examples of Gal-3 proteins labeled with fluorescent proteins include fusion proteins of fluorescent proteins and Gal-3 protein. Examples of fluorescent proteins include green fluorescent protein (GFP) and monomeric red fluorescent protein (mRFP), but are not particularly limited.

[0040] Another embodiment of a method for confirming that the substance of the present disclosure has been introduced into the cytoplasm of a cell includes a step of performing a method for introducing the substance of the present disclosure into the cytoplasm of a cell, wherein the cell has at least a protein containing a fluorescent protein in its cytoplasm, the magnetic carrier has at least an antibody capable of binding to the fluorescent protein, and after performing the step of introducing the substance of the present disclosure into the cytoplasm of a cell, the method includes observing the accumulation of the protein containing the fluorescent protein overlapping with or adjacent to the magnetic carrier.

[0041] Examples of fluorescent proteins include green fluorescent protein (GFP) and monomeric red fluorescent protein (mRFP), but there are no particular limitations. When the carrier is exposed to the cytoplasm, that is, when the carrier is introduced into the cytoplasm of a cell, the accumulation of the fluorescent protein, overlapping with or adjacent to the carrier, is confirmed by the antigen-antibody reaction between the fluorescent protein in the cytoplasm and the antibody on the carrier.

[0042] Furthermore, the methods for confirming that the substance of interest of this disclosure has been introduced into the cytoplasm of a cell are not limited to these confirmation methods, and may include other embodiments, for example, that utilize any combination of a tag and ligand that specifically interact.

[0043] The present disclosure will be described in more detail below using examples, but the present disclosure is not limited to these examples.

[0044] (Bead uptake into endosomes using poly-L-arginine (PLA)) First, place 2.5 × 10⁶ HeLa cells into a 35mm glass-bottom dish containing 2ml of normal culture medium (37°C). 5 The cells were seeded. As a standard culture medium, a medium prepared by mixing 10% bovine serum (FBS: Thermo Fisher Scentific, #16000069) with L-glutamine and pyruvate-supplemented DMEM (Nacalai Tesque, #08458-16) was used. The following day, 10 μl of distilled water and 1.6 μl of bead suspension were placed in a 1.5 ml microtube, 1 μl of MgCl2 (10 mM) was added, and the mixture was vortexed for 5-10 seconds and allowed to stand at room temperature for 5 minutes. Then, 1 μl of 0.1 μg / μl of poly-L-arginine (PLA: Merck, #P4663-10MG) was added, the mixture was vortexed for 5-10 seconds, and allowed to stand at room temperature for 30 minutes. 90 μl of 37°C DMEM was added to the suspension, vortexed for 10 seconds, and then gently added only to the glass portion of the 35 mm glass-bottom dish from which all the culture medium had been removed beforehand. The cells were then incubated in a CO2 incubator for 1 hour. They were washed twice with 2 ml of observation medium (37°C), and then another 2 ml of observation medium (37°C) was added. The observation medium used was DMEM (Nacalai Tesque, #21063-029) supplemented with 10% FBS, 1 mM pyruvate (Nacalai Tesque, #06977-34), and 80 μg / ml kanamycin (Fujifilm Wako Pure Chemical Industries, #113-00343). After 1 hour, light irradiation was started using a confocal laser microscope, and the light irradiation experiment was completed within 2 hours. This allowed for light irradiation experiments on cells that had been washed with observation medium for 1 to 3 hours.

[0045] (Bead uptake into endosomes using Effectene) First, place 2.5 × 10⁶ HeLa cells into a 35mm glass-bottom dish containing 2ml of normal culture medium (37°C). 5 The cells were seeded. The next day, 10 μl of Buffer EC and 1 μl of bead suspension were placed in a 1.5 ml microtube, 0.32 ml of Enhancer was added, and the mixture was vortexed for 5-10 seconds and allowed to stand at room temperature for 5 minutes. Then, 1 μl of Effectene Reagent was added, the mixture was vortexed for 5-10 seconds and allowed to stand at room temperature for 10 minutes. 90 μl of standard cell culture medium (37°C) was added to the suspension, and after vortexing for 10 seconds, it was gently added only to the glass portion of the 5 mm glass-bottom dish from which all the culture medium had been removed beforehand. The cells were then cultured in a CO2 incubator for 1 hour. The cells were washed twice with 2 ml of observation medium (37°C), and another 2 ml of observation medium (37°C) was added. One hour later, light irradiation was started with a confocal laser microscope, and the light irradiation experiment was completed within 2 hours. Thus, the light irradiation experiment was performed on cells that had been elapsed for 1-3 hours after washing with observation medium.

[0046] (Light Irradiation Experiment) A confocal laser scanning microscope LSM880 (Carl Zeiss) was used for light irradiation. The objective lens used for this microscope was a C Apo 40× / 1.2W DICIII NA1.2 (Carl Zeiss). The light irradiation experiment was performed by irradiating beads in the endosomes of cells with light of a specific wavelength using the LSM880. The pixels in this experiment were square with sides of 0.17 μm.

[0047] Example 1: Observation of Endosome Disintegration by Light Irradiation Using BAF Protein and DNA-Binding Beads Following the "Bead Incorporation into Endosomes Using Poly-L-Arginine (PLA)" procedure described above, Dynabeads M-270 Streptavidin magnetic beads (Thermo Fisher), to which approximately 8 kbp of biotin-labeled double-stranded DNA was bound, were incorporated into endosomes of HeLa cells expressing a fusion protein (GFP-BAF) of green fluorescent protein (GFP) and BAF protein in the cytoplasm. Light irradiation was performed according to the "Light Irradiation Experiment" described above, by irradiating the magnetic beads with 561 nm light over a rectangular area of ​​1 × 10 pixels. Each pixel is a square with sides of 0.17 μm. Live imaging was performed before light irradiation, immediately after light irradiation, and 60 seconds after light irradiation for observation. Furthermore, for cells fixed 30 minutes after light irradiation, emerin was visualized by staining with Hoechst33342 and immunostaining with an anti-emerin antibody.

[0048] The results of live imaging are shown in Figure 1, and the results of immunostaining with anti-emerine antibody are shown in Figure 2. Sixty seconds after light irradiation, GFP-BAF showed strong fluorescence near the beads (position of the arrows in each photograph at 60 seconds in Figure 1). This is thought to be because the DNA-binding beads were exposed to the cytoplasm due to endosomal breakdown, causing cytoplasmic GFP-BAF to aggregate around the DNA-binding beads. In addition, strong fluorescence was observed near the beads in the immunostaining results with anti-emerine antibody. Since emerine is a protein that has a binding domain to BAF, the results of immunostaining with anti-emerine antibody also suggest that the endosomal membrane was ruptured after light irradiation, and cytoplasmic GFP-BAF aggregated near the beads.

[0049] Example 2: Endosome Disintegration by Light Irradiation and Subsequent Observation of Endosome Membrane Fragments Following the "Bead Incorporation into Endosomes Using Poly-L-Arginine (PLA)" procedure described above, HeLa cells expressing a fusion protein of green fluorescent protein (GFP) and galectin 3 (Gal-3) protein (GFP-gal-3) and a fusion protein of monomeric red fluorescent protein (mRFP) and BAF protein (mRFP-BAF) in the cytoplasm were subjected to the incorporation of Dynabeads M-270 Streptavidin magnetic beads (manufactured by Thermo Fisher), to which approximately 8 kbp of biotin-labeled double-stranded DNA was bound, into endosomes. Light irradiation was performed according to the "Light Irradiation Experiment" described above, by irradiating the magnetic beads with 561 nm light over a rectangular area of ​​1 × 10 pixels. Note that each pixel is a square with sides of 0.17 μm. Live imaging was performed to capture and observe images before light irradiation, immediately after light irradiation, and 60 seconds after light irradiation.

[0050] The results of live imaging are shown in Figure 3. Similar to Example 1, mRFP-BAF aggregated on the DNA-binding beads within several tens of seconds after light irradiation, indicating endosomal disintegration (Figure 3, positions indicated by arrows in each photograph at 60 seconds). Furthermore, Gal-3 protein, which is known to localize on the disintegrated cell membrane, showed strong fluorescence near the GFP-gal-3 beads within several tens of seconds after light irradiation, suggesting that it localized on endosomal membrane fragments near the beads where endosomal disintegration occurred. These results strongly suggest that light irradiation of the magnetic beads caused endosome disintegration and fragmentation of the endosomal membrane, leading to exposure of the magnetic beads to the cytoplasm.

[0051] Example 3: Verification of Endosome Disintegration by Light Irradiation Using Various Beads Using the various beads shown in Table 1, the beads were incorporated into HeLa cell endosomes according to the "Bead Incorporation into Endosomes Using Poly-L-Arginine (PLA)" or "Bead Incorporation into Endosomes Using Effectene" procedures. Light irradiation was performed by irradiating the magnetic beads with 561 nm light, according to the "Light Irradiation Experiment" procedure.

[0052] Table 1 shows the results regarding whether or not the beads were taken up by endosomes, and whether or not endosomal breakdown occurred upon light irradiation of the beads.

[0053]

[0054] Regardless of size or the substance to which they bind, magnetic beads exhibited endosomal disruption upon light irradiation. Bead sizes ranging from at least 1 to 3 μm, and regardless of the substance to which they bound, all successfully resulted in endosomal disruption and subsequent introduction into the cytoplasm.

[0055] Example 4: Verification of the presence or absence of endosomal collapse by light irradiation at different wavelengths of light Following the "Incorporation of beads into endosomes using poly-L-arginine (PLA)" procedure described above, Dynabeads M-270 Streptavidin magnetic beads, to which approximately 8 kbp of biotin-labeled double-stranded DNA was bound, were incorporated into endosomes of HeLa cells expressing GFP-BAF in the cytoplasm. Light irradiation was performed according to the "Light Irradiation Experiment" described above, by irradiating the magnetic beads with light at 405, 488, 561, or 633 nm in a circular area of ​​20 x 20 pixels or a rectangular area of ​​1 x 10 pixels. Note that each pixel is a square with sides of 0.17 μm.

[0056] After light irradiation, GFP-BAF showed strong fluorescence near the beads, which was considered to indicate endosomal breakdown, and the presence or absence of endosomal breakdown was verified. The results are shown in Table 2.

[0057]

[0058] We confirmed that endosomal breakdown occurs when magnetic beads are irradiated with light in the 405-561 nm range.

[0059] Example 5: Verification of light irradiation intensity and the presence or absence of endosomal collapse First, we investigated irradiation with 561 nm light. Following the "Incorporation of beads into endosomes using poly-L-arginine (PLA)" procedure described above, Dynabeads M-270 Streptavidin magnetic beads, to which approximately 8 kbp of biotin-labeled double-stranded DNA was bound, were incorporated into endosomes of HeLa cells expressing GFP-BAF in the cytoplasm. Light irradiation was performed according to the "Light Irradiation Experiment" described above, with 561 nm light irradiated onto the magnetic beads in a circular area of ​​20 x 20 pixels at an intensity of 70 to 100 μW in 5 μW increments. Note that each pixel is a square with sides of 0.17 μm.

[0060] The results are shown in Figure 4. When 561 nm light was shone onto magnetic beads in a 20 x 20 pixel circular area, endosomal breakdown was observed with a certain probability at 80 μW or higher, and endosomal breakdown was reliably observed at 95 μW or higher.

[0061] Next, irradiation with 405 nm light was investigated. Following the "Bead Incorporation into Endosomes Using Poly-L-Arginine (PLA)" procedure described above, Dynabeads M-270 Streptavidin magnetic beads, to which approximately 8 kbp of biotin-labeled double-stranded DNA was bound, were incorporated into endosomes of HeLa cells expressing GFP-BAF in the cytoplasm. Light irradiation was performed according to the "Light Irradiation Experiment" described above, by irradiating the magnetic beads with 405 nm light in a 20 x 20 pixel circular area at intensities of 2, 2.5, 3, 4, 5, 10, 15, 20, 25, or 30 μW. Note that each pixel is a square with sides of 0.17 μm.

[0062] The results are shown in Figure 5. When 405 nm light was shone onto magnetic beads in a 20 x 20 pixel circular area, endosomal breakdown was observed with a certain probability at 3 μW or higher, and endosomal breakdown was reliably observed at 10 μW or higher.

[0063] This disclosure provides a method for introducing a target substance into the cytoplasm of a cell. In particular, this disclosure enables the introduction of relatively large substances into the cytoplasm, such as the target substance, particles containing it, or the entire substance including them, with a size of 1 μm or more. Furthermore, this disclosure allows for control over when and which substance is introduced into the cytoplasm. As a result, it becomes possible to analyze the state of the cell before and after the invasion of a foreign substance into the cytoplasm with high temporal and spatial resolution, which is expected to be effective in analyzing the mechanisms of foreign substance invasion into the cytoplasm, the mechanisms of drug uptake into cells, and the pathways of pathogen infection. In addition, it becomes possible to introduce any substance into the cytoplasm at a targeted time and location, which is expected to be effective in analyzing mechanisms in cell biology, such as the relationship between the cell cycle and the efficiency of foreign substance introduction. Because this disclosure enables the introduction of micrometer-scale artificial objects into the cytoplasm and its spatial and temporal control, it is expected to become an important foundational technology for the development of communication interface technologies between cells and artificial objects, such as the use of highly integrated miniature semiconductor chips embedded in cells in the future.

Claims

1. A method for introducing a target substance into the cytoplasm of a cell, comprising: a step of introducing a magnetic carrier into the endosome of the cell, wherein the magnetic carrier is the target substance or the magnetic carrier contains the target substance; and a step of irradiating the endosome into which the magnetic carrier has been introduced with light, wherein the maximum diameter of the magnetic carrier that is the target substance or the magnetic carrier containing the target substance is 1 μm or more, and the wavelength of the light irradiated in the light irradiation step is 600 nm or less.

2. The method according to claim 1, wherein the magnetic carrier is a magnetic bead containing iron oxide and a polymer.

3. The method according to claim 1, wherein the step of introducing the carrier into the endosomes of the cells is performed by promoting endocytosis using an endocytosis-inducing reagent.

4. The method according to claim 3, wherein the endocytosis-inducing reagent is one or more cell membrane-binding peptides selected from the group consisting of poly-L-arginine (PLA), Pep-1 peptide, TAT, Penetratin, and poly-L-lysine (PLL).

5. In the step of irradiating with light, the light is directed onto the magnetic carrier at a depth of 0.05 μm 2 The method according to claim 1, wherein the above range is irradiated.

6. The method according to claim 1, wherein the target substance comprises one or more substances selected from polypeptides, DNA, RNA, glycans, lipids, synthetic polymer compounds, or combinations thereof.

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