Intracellular substance delivery and extraction technology via composite nanotubes

Abstract

The present invention delivers a substance such as a reagent into a cell. A tubular body coated with an electroconductive polymer for introducing a substance into a cell and / or recovering a substance from a cell. The use of a tubular body coated with an electroconductive polymer to introduce a substance into a cell and / or recover a substance from a cell. A device for introducing a substance into a cell and / or recovering a substance from a cell, the device being equipped with a substrate and a tubular body coated with an electroconductive polymer, and the tubular body being positioned on the substrate. A system for introducing a substance into a cell and / or recovering a substance from a cell, the system being equipped with the abovementioned device, a voltage supply unit for supplying a voltage, and, as needed, electrodes.

Description

[Technical Field]

[0001] The present invention relates to techniques for controllably transferring substances into and out of cells. [Background technology]

[0002] Conventionally, nanotube structures such as nanoneedles or nanostraws have been used to deliver reagents and other substances into cells. However, these technologies achieve substance introduction and recovery by applying a high voltage (approximately 0.1 to 10 V) to create holes in the cell membrane ("electroporation"). However, applying high voltage can cause undesirable changes in cell function, such as cell death and canceration. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Special table number 2019-528782

[0004] [Patent Document 2] Special table number 2019-517273 [Non-patent literature]

[0005] [Non-Patent Document 1] JJ Van Dersarl, AM Xu, NA Melos, Nanostraws for direct fluidic intracellular access, Nano letters 12 (8), 3881-3886, 2011.

[0006] [Non-patent document 2] A Tay, N Melosh, Nanostructured Materials for Intracellular Cargo Delivery, Accounts of chemical research 52 (9), 2462-2471, 2019.

Table 3

[0007]

Fashion 4

[0008]

Wood 5

[0009] For example, the present disclosure provides the following: (Item 1) A cylindrical body coated with a conductive polymer for introducing substances into cells and / or retrieving substances from cells. (Item 2) Use of a cylindrical body coated with a conductive polymer for introducing a substance into a cell and / or withdrawing a substance from a cell. (Item 3) 3. The cylindrical body or use according to item 1 or 2, wherein the cylindrical body is formed from a conductor. (Item 4) Item 4. The cylindrical body or use according to item 3, wherein the conductor comprises a metal. (Item 5) 5. The cylindrical body or use according to item 4, wherein the metal includes a metal that can be electrolessly plated. (Item 6) 6. The cylindrical body or use according to item 4 or 5, wherein the metal comprises at least one metal selected from the group consisting of gold, platinum, silver, and nickel, and alloys thereof. (Item 7) 5. The cylindrical body or use according to any one of items 1 to 4, wherein the conductive polymer has a property of expanding and contracting when a voltage is applied. (Item 8) 8. The cylindrical body or use according to item 7, wherein the absolute value of the voltage is 500 mV or less. (Item 9) 9. The cylindrical body or use according to any one of items 7 to 8, wherein the absolute value of the voltage is 100 mV or less. (Item 10) 10. The cylindrical body or use according to any one of items 7 to 9, wherein the absolute value of the voltage is 50 mV or less. (Item 11) 11. The cylindrical body or use according to any one of items 7 to 10, wherein the voltage is an AC voltage. (Item 12) 12. The cylindrical body or use according to any one of items 1 to 11, wherein the conductive polymer comprises at least one polymer selected from the group consisting of poly(3,4-ethylenedioxythiophene) (PEDOT), polythiophene, polyacetylene, polyaniline, polypyrrole, and combinations thereof. (Item 13) 13. The cylindrical body or use according to any one of items 1 to 12, wherein the cylindrical body has an inner diameter of 2 μm or less. (Item 14) 14. The cylindrical body or use according to any one of items 1 to 13, wherein the cylindrical body has an outer diameter of 4 μm or less. (Item 15) 15. The cylindrical body or use according to any one of items 1 to 14, wherein the cylindrical body has a length of 5 μm or more and 50 μm or less. (Item 16) 16. The cylindrical body or use according to any one of items 1 to 15, wherein the substance has an arithmetic mean diameter of 1 nm or more and 2 μm or less. (Item 17) 17. The tubular body or use according to any one of items 1 to 16, wherein the substance comprises at least one selected from the group consisting of a low molecular weight compound, a nucleic acid, a peptide, a protein, and a cellular organelle. (Item 18) A device for introducing a substance into a cell and / or recovering a substance from a cell, the device comprising a cylindrical body coated with a conductive polymer, a substrate, and an electrode, the cylindrical body being placed on the substrate. (Item 19) Item 19. The device according to item 18, wherein the device comprises a plurality of the cylindrical bodies. (Item 20) 20. The device according to item 18 or 19, wherein the device further comprises a storage section for storing the substance. (Item 21) 21. The device of any one of items 18 to 20, wherein the substrate comprises polycarbonate and / or polyethylene terephthalate. (Item 22) 22. The device according to any one of items 20 to 21, wherein the storage section is hollow and in direct contact with the substrate, and has a structure with a tube through which an electrode can be inserted into the cavity. (Item 23) 23. The device according to any one of items 18 to 22, wherein the containment section is made of at least one material selected from the group consisting of glass, ceramic, polymer, and metal. (Item 24) The device according to any one of items 18 to 23, wherein the cylindrical body further comprises the feature according to any one of items 1 to 17. (Item 25) 25. The device according to any one of items 20 to 24, wherein the storage section contains the substance. (Item 26) 26. The device according to any one of items 18 to 25, wherein the electrodes are provided in two poles for applying a voltage to the conductive polymer and the storage section. (Item 27) A device according to any one of items 18 to 26, a voltage supply unit that supplies a voltage; A system for introducing a substance into a cell and / or withdrawing a substance from a cell, comprising: (Item 28) 28. The system of item 27, further comprising a container containing the cells. (Item 29) 29. The system according to item 27 or 28, further comprising an observation unit for differential interference observation of cells in the container from below. (Item 30) 30. The system of claim 29, wherein the observation unit is realized by a differential interference prism. (Item 31) The system according to any one of items 27 to 30, further comprising a display unit for displaying an image from the observation unit. (Item 32) 1. A method for introducing a substance into a cell and / or withdrawing a substance from a cell, comprising: (a) providing a device according to any one of items 18 to 26 or a system according to any one of items 27 to 31; (b) applying a voltage to the cylinder for a desired period of time; A method comprising: (Item 33) A method for fabricating a device comprising a cylindrical body coated with a conductive polymer and a substrate, comprising: The method includes a step of fixing and covering a substrate on which a cylindrical body is placed with a film of the conductive polymer at at least one point. (Item 34) Item 34. The method according to Item 33, wherein the coating step includes a step of fixing and coating the conductive polymer film at at least two points. (Item 35) 35. The method according to claim 33 or 34, wherein the coating step is achieved by contacting the substrate with two or more working electrodes. (Item 36) 36. The method according to any one of items 33 to 35, wherein the substrate on which the cylindrical body is disposed is made by a process comprising the steps of electrolessly plating metal onto track-etched polycarbonate and etching the metal-plated surface. (Item 37) 37. The method according to any one of items 33 to 36, wherein the cylindrical body has an inner diameter of 0.01 μm to 2 μm and an outer diameter of 0.1 μm to 5 μm.

[0010] (Item 1A) A cylindrical body coated with a conductive polymer for introducing substances into cells and / or retrieving substances from cells. (Item 2A) Use of a cylindrical body coated with a conductive polymer for introducing a substance into a cell and / or withdrawing a substance from a cell. (Item 3A) The cylindrical body or use according to item 1A or 2A, wherein the cylindrical body is formed from a conductor. (Item 4A) The cylindrical body or use according to item 3A, wherein the conductor comprises a metal. (Item 5A) The cylindrical body or use according to item 4A, wherein the metal includes a metal that can be electrolessly plated. (Item 6A) The cylindrical body or use according to item 4A or 5A, wherein the metal comprises at least one metal selected from the group consisting of gold, platinum, silver, and nickel, and alloys thereof. (Item 7A) The cylindrical body or use according to any one of items 1A to 4A, wherein the conductive polymer has a property of expanding and contracting when a voltage is applied. (Item 8A) The cylindrical body or use according to Item 7A, wherein the absolute value of the voltage is 500 mV or less. (Item 9A) The cylindrical body or use according to any one of items 7A to 8A, wherein the absolute value of the voltage is 100 mV or less. (Item 10A) The cylindrical body or use according to any one of items 7A to 9A, wherein the absolute value of the voltage is 50 mV or less. (Item 11A) The cylindrical body or use according to any one of Items 7A to 10A, wherein the voltage is an AC voltage. (Item 12A) The cylindrical body or use according to any one of items 1A to 11A, wherein the conductive polymer comprises at least one polymer selected from the group consisting of poly(3,4-ethylenedioxythiophene) (PEDOT), polythiophene, polyacetylene, polyaniline, polypyrrole, and combinations thereof. (Item 13A) The cylindrical body or use according to any one of items 1A to 12A, wherein the cylindrical body has an inner diameter of 2 μm or less. (Item 14A) The cylindrical body or use according to any one of items 1A to 13A, wherein the cylindrical body has an outer diameter of 4 μm or less. (Item 15A) The cylindrical body or use according to any one of items 1A to 14A, wherein the cylindrical body has a length of 5 μm or more and 50 μm or less. (Item 16A) The cylindrical body or use according to any one of items 1A to 15A, wherein the substance has an arithmetic mean diameter of 1 nm or more and 2 μm or less. (Item 17A) The tubular body or use according to any one of items 1A to 16A, wherein the substance includes at least one selected from the group consisting of a low molecular weight compound, a nucleic acid, a peptide, a protein, and an organelle. (Item 18A) A device for introducing a substance into a cell and / or recovering a substance from a cell, the device comprising a cylindrical body coated with a conductive polymer, a substrate, and an electrode, the cylindrical body being placed on the substrate. (Item 19A) Item 18B, the device comprising a plurality of the cylindrical bodies. (Item 20A) 19A, wherein the device further comprises a storage section for storing the substance. (Item 21A) 20A. The device of any one of items 18A to 20A, wherein the substrate comprises polycarbonate and / or polyethylene terephthalate. (Item 22A) The device according to any one of items 20A to 21A, wherein the storage section is hollow and in direct contact with the substrate, and has a structure with a tube through which an electrode can be inserted into the cavity. (Item 23A) The device of any one of items 18A to 22A, wherein the containment portion is composed of at least one material selected from the group consisting of glass, ceramic, polymer, and metal. (Item 24A) The device according to any one of items 18A to 23A, wherein the cylindrical body further comprises the feature according to any one of items 1A to 17A. (Item 25A) The device according to any one of items 20A to 24A, wherein the storage section contains the substance. (Item 26A) The device according to any one of items 18A to 25A, wherein the electrodes are provided in two poles for applying a voltage to the conductive polymer and the storage section. (Item 27A) A device according to any one of items 18A to 26A; a voltage supply unit that supplies a voltage; A system for introducing a substance into a cell and / or withdrawing a substance from a cell, comprising: (Item 28A) Item 27A, further comprising a container containing the cells. (Item 29A) The system according to Item 27A or 28A, further comprising an observation unit for differential interference observation of cells in the container from below. (Item 30A) Item 29A, the system wherein the observation unit is realized by a differential interference prism. (Item 31A) The system according to any one of Items 27A to 30A, further comprising a display unit for displaying an image from the observation unit. (Item 32A) The system according to any one of items 27A to 31A, wherein the insertion of the cylindrical body contained in the device into the cell is adjustable in increments of 0.1 μm. (Item 33A) 1. A method for introducing a substance into a cell and / or withdrawing a substance from a cell, comprising: (a) providing a device according to any one of items 18A to 26A or a system according to any one of items 27A to 32A; (b) applying a voltage to the cylinder for a desired period of time; A method comprising: (Item 34A) A method for fabricating a device comprising a cylindrical body coated with a conductive polymer and a substrate, comprising: The method includes a step of fixing and covering a substrate on which a cylindrical body is placed with a film of the conductive polymer at at least one point. (Item 35A) The method according to Item 34A, wherein the coating step includes a step of fixing and coating the conductive polymer film at at least two points. (Item 36A) The method according to item 34A or 35A, wherein the coating step is achieved by contacting the substrate with two or more working electrodes. (Item 37A) The method of any one of Items 34A to 36A, wherein the substrate on which the cylinder is disposed is made by a process including electroless plating of metal onto track-etched polycarbonate and etching the metal-plated surface. (Item 38A) The method according to any one of Items 34A to 37A, wherein the cylindrical body has an inner diameter of 0.01 μm to 2 μm and an outer diameter of 0.1 μm to 5 μm. (Item 39A) A method for activating cells, comprising the step of introducing mitochondria into the cells using the device described in any one of items 18A to 26A or the system described in any one of items 27A to 32A. (Item 40A) A method for controlling a cell function, comprising the step of introducing a gene product (protein) into the cell using the device described in any one of Items 18A to 26A or the system described in any one of Items 27A to 32A. (Item 41A) The method of item 40A, wherein the gene product comprises the gene product of Oct4. (Item 42A) A cylindrical body coated with a conductive polymer for introducing a substance into a cell and / or retrieving a substance from a cell, the conductive polymer comprising kinesin. (Item 43A) Use of a cylindrical body coated with a conductive polymer comprising kinesin for introducing a substance into a cell and / or retrieving a substance from a cell. (Item 44A) A device for introducing a substance into a cell and / or retrieving a substance from a cell, the device comprising a cylindrical body coated with a conductive polymer comprising kinesin, a substrate, and an electrode, the cylindrical body being placed on the substrate. (Item 45A) A device according to item 44A; a voltage supply unit that supplies a voltage; A system for introducing a substance into a cell and / or withdrawing a substance from a cell, comprising: (Item 46A) 1. A method for introducing a substance into a cell and / or withdrawing a substance from a cell, comprising: (a) providing a device according to item 44A or a system according to item 45A; (b) providing microtubules having a substance bound thereto; (c) applying a voltage to the cylinder for a desired period of time; A method comprising: (Item 47A) A method for producing a cylindrical body coated with a conductive polymer comprising kinesin, comprising the steps of: A method comprising the step of binding kinesin to the cylindrical body. (Item 48A) 1. A stamp system for inserting composite nanotubes into cells, comprising: a stamp having a cylindrical body coated with a conductive polymer; An electrode; a voltage supply unit that supplies a voltage; A system comprising: (Item 49A) 1. An insertion observation system for inserting a composite nanotube into a cell, comprising: A device according to any one of items 18A to 26A and 44A; a voltage supply unit that supplies a voltage; A microscope and A system comprising: (Item 50A) A composite nanotube membrane and stamp kit for delivering a substance into a cell or extracting a substance from a cell, comprising: a composite nanotube thin film having a cylindrical body coated with a conductive polymer; Stamp and A kit comprising: (Item 51A) A method for evaluating cell activity, comprising: A step of measuring the amount of a marker substance in a cell using a cylindrical body coated with a conductive polymer. A method comprising: (Item 52A) The method of item 50A, further comprising the step of introducing mitochondria into the cells. (Item 53A) A cell introduction method using microspheres, comprising: (a) providing a device according to any one of items 18A to 26A and 44A or a system according to any one of items 27A to 32A and 45A; (b) providing microspheres; (c) applying a voltage to the cylinder for a desired period of time; A method comprising: (Item 54A) Item 53A, further comprising the step of attaching a substance to the microspheres. (Item 55A) 1. A method for extracting intracellular material, comprising: (a) providing a device according to any one of items 18A to 26A and 44A or a system according to any one of items 27A to 32A and 45A; (b) applying a voltage to the cylinder for a desired period of time; A method comprising: (Item 56A) 1. A method of cellular programming, comprising: (a) providing a device according to any one of items 18A to 26A and 44A or a system according to any one of items 27A to 32A and 45A; (b) providing a gene or gene product; (c) applying a voltage to the cylinder for a desired period of time; A method comprising: (Item 57A) 1. A method for introducing a functional protein into a cell to modify the phenotype of the cell, comprising: (a) providing a device according to any one of items 18A to 26A and 44A or a system according to any one of items 27A to 32A and 45A; (b) providing a functional protein; (c) applying a voltage to the cylinder for a desired period of time; A method comprising:

[0011] According to this disclosure, while the problem of conventional methods is that holes are created in the cell membrane using high voltage, which is harmful to cells, this method has a significant advantage in that it can transport or extract substances without causing or minimizing harmful events to cells, and it can be said to be safe because it can use minute voltages. We have successfully inserted composite nanotubes into cells and controlled their transport by applying electricity. We have successfully worked on creating hollow nanotubes and developing a stamping system simultaneously.

[0012] The present disclosure is advantageous in that it is possible to control the transport of substances by turning on / off electricity to an example of a composite nanotube in which a metal nanotube is coated with a conductive polymer film, or to a composite nanotube.

[0013] In a specific embodiment, metallic nanotubes coated with a conductive polymer can be inserted into cells to deliver substances such as genes and reagents into the cells. This is useful in that substances can be introduced or withdrawn by applying a minute voltage (±0.1 V or less, a potential lower than the membrane potential). It is also useful in that the amount of substance released can be controlled by turning the applied voltage on and off.

[0014] The present disclosure is useful for providing advanced biological cells (smart cells). Such cells can be used in industry, the food industry, the environment, medicine, and more. They can be used to produce cultured foods, pharmaceuticals, and polymers, as well as cultured tissues, and are useful in the fields of regenerative medicine and cosmetics. Multiple substances can be safely introduced / extracted from many cells multiple times, enabling function control, substance production, functional analysis, and substance utilization. Biomolecular analysis in the cellular environment (in cell NMR) becomes possible. The degree of cellular aging can be improved, cancerous cells can be killed, and mitochondria can be introduced. Cell function can be controlled and directly induced into differentiated cells (direct reprogramming) becomes possible. As smart cells, they are useful in pharmaceuticals, industrial products, seasonings, perfumes, and more.

[0015] It is contemplated that one or more of the above features may be provided in combinations other than those explicitly stated. Still further embodiments and advantages of the present invention will be recognized by those skilled in the art upon reading and understanding the following detailed description, if necessary. [Effects of the Invention]

[0016] It is possible to deliver reagents and other substances into cells. Specifically, the following effects are expected:

[0017] In one embodiment, the present disclosure has made it possible to control substance transport and collection by inserting metallic nanotubes coated with a conductive polymer and applying a small voltage, and also to enable long-term insertion. With metallic nanotubes alone, it is not possible to control the amount of substance released by applying a voltage, and there are also problems with the release of intracellular substances to the outside, which can lead to cell death over time, but these problems have been improved.

[0018] Currently, technologies for controlling cell functions, including iPS cells, are required to safely deliver substances (such as genes and reagents) into cells, and are used in a wide range of applications, including cell therapy and regenerative medicine. The technology disclosed herein is expected to be put to practical use in these cosmetic and medical fields.

[0019] In one embodiment, by increasing the amount of material introduced, even more possibilities become possible. For example, iPS cells can be established by gene introduction or protein introduction to create iPS cells. Furthermore, by combining this with other technologies (isolated mitochondria, LucaScience), mitochondria can be introduced into cell sheets, making them applicable to regenerative medicine.

[0020] While conventional nanotubes create holes in cells using electroporation, which involves applying a high voltage, this method can potentially lead to cell death or cancer. The nanotubes of the present invention are coated with a polymer membrane to prevent the release of intracellular substances from the cells, allowing them to be inserted directly into the cells and processed with a minute applied voltage, thereby solving the problems associated with applying high voltages.

[0021] No other technology has been developed that controls intracellular transport of substances with minute voltages and achieves long-term insertion of composite nanotubes, as disclosed herein. Patent documents 1-2 and non-patent documents 1-3, as well as non-patent documents 4-5, all report the use of track-etched membranes to form nanotube structures and conductive polymer films using metals or metal oxides, but no attempt has been made to insert composite nanotubes into cells and control transport by applying electricity. Furthermore, conventional methods are completely different from the present invention, including the transport principle, in that they use high voltage to create holes in the cell membrane. The method disclosed herein is safer because it can utilize minute voltages.

[0022] Currently, cell function control technologies, including iPS cells, are required to safely deliver substances (such as genes and reagents) into cells, and are used in a wide range of applications, including cell therapy and regenerative medicine. Technologies for introducing or extracting substances into cells can be broadly divided into chemical and physical methods (Figure 14). Conventional methods are similar to the present invention in that they use nanoneedles, but have the disadvantage of requiring the use of electroporation in combination. In comparison, the method of the present invention has excellent features in all respects, and is expected to have a wide range of applications in the future.

[0023] If this invention can be used to insert needles into a group of cells being transported on a conveyor belt all at once and apply electricity to safely deliver substances into the cells, it will be possible to provide a new cell manufacturing tool. If it becomes possible to safely introduce any substance into cells, it could be used in regenerative medicine and treatment to create organs and tissues from individual cells, as well as in the food industry, such as cultured cell meat and plants. [Brief explanation of the drawings]

[0024] [Figure 1] Figure 1 shows the nanotube stamp intracellular insertion system incorporating DIC observation. [Figure 2] FIG. 2 is a diagram illustrating the substance introduction efficiency and cell survival rate with and without a DIC observation mechanism. [Figure 3]FIG. 3 shows the cell viability upon intracellular insertion of gold nanotubes and composite nanotubes. [Figure 4] FIG. 4 shows the control of intracellular delivery of calcein fluorescent substance using composite nanotubes. [Figure 5] Figure 5 shows the fluorescence intensity and frequency after calcein introduction. [Figure 6] Figure 6 shows the controlled intracellular delivery of GFP protein using composite nanotubes. [Figure 7] FIG. 7 shows transport of isolated mitochondria through composite nanotubes. [Figure 8] Figure 8 shows the structural analysis of the PEDOT electropolymerized composite nanotube thin film. [Figure 9] Figure 9 shows the calcein concentration measurements that permeated the gold nanotube (0 min) and the composite nanotube thin film. [Figure 10] FIG. 10 shows the amount of calcein permeated depending on whether the applied voltage is on or off. [Figure 11] Figure 11 shows the fabrication of nanostraw membranes. (a) Two steps in nanostraw fabrication: (1) electroless Au plating on a track-etched polycarbonate (TEPC) membrane and (2) etching of the top surface. (b-d) Optical images of an 8 mm diameter sample at different stages of nanostraw fabrication. The TEPC membrane contains size-controlled pores. 0.4 μm, 0.6 μm, and 1.0 μm diameter pores were used. (b) Images of the original TEPC membrane, face up and face down. (c) Au is plated on the TEPC membrane surface and the pore surface. (d) The top surface of the Au membrane was etched in two steps: (1) the Au surface was etched with aqua regia (containing nitric and hydrochloric acids), and (2) the PC surface was etched with 02RIE. (e-h) Au / TEPC nanostraws after RIE for 0 (e), 10 (f), 20 (g), and 30 (h) minutes. [Figure 12] Figure 12 shows the process flow for fabricating Au nanostraws on a polycarbonate film. [Figure 13]Figure 13 shows an overview of the intracellular substance introduction and extraction technology via composite nanotubes (conductive polymer and gold). [Figure 14] FIG. 14 shows the classification and superiority of the disclosed method compared to other methods. [Figure 15] FIG. 15 shows an exemplary stamp-shaped device (stamping system) of the present disclosure. [Figure 16] FIG. 16 is a schematic diagram of an exemplary stamp-shaped device of the present disclosure in operation. [Figure 17] FIG. 17 shows the fluorescence image and intensity profile of calcein-stained cells. [Figure 18] Figure 18 shows fluorescent images of live and dead cells in the extracted area. [Figure 19] FIG. 19 shows the experimental setup for introducing microspheres into cells by stamping. [Figure 20] FIG. 20 shows optical and fluorescent images after intracellular introduction of microspheres via composite nanotubes. [Figure 21] Figure 21 shows the intracellular transport mechanism of substances using biological molecular motors. [Figure 22] FIG. 22 shows the transport of a biomolecular motor on a gold membrane and its passage through the membrane via a nanotube. [Figure 23] FIG. 23 shows the transport of substance-labeled microtubules. [Figure 24] FIG. 24 shows the intracellular introduction of Oct4 protein. [Figure 25] FIG. 25 shows the introduction of mitochondria into cells. [Figure 26] FIG. 26 is a diagram illustrating the difference between sedimentation velocity and the velocity of a biological molecular motor. DETAILED DESCRIPTION OF THE INVENTION

[0025] The present invention will be described in more detail below. Throughout this specification, singular expressions should be understood to include the plural concept unless otherwise specified. Therefore, singular articles (e.g., "a," "an," "the," etc. in English) should be understood to include the plural concept unless otherwise specified. Furthermore, it should be understood that terms used in this specification are used in the sense commonly used in the art unless otherwise specified. Therefore, unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In the case of conflict, the present specification (including definitions) will prevail. (definition)

[0026] The following provides definitions of terms particularly used in this specification and / or explains basic technical content as appropriate.

[0027] As used herein, the term "cylinder" refers to a thin, rod-like body or entity with a hollow center, and is also referred to as a thin, hollow body or hollow tube. Cylinders are also sometimes referred to as tubes or straws. The "outer diameter" and "inner diameter" of a cylinder are used in the usual sense in the art, with the outer diameter referring to the diameter of the largest part of the cylinder and the inner diameter referring to the diameter of the hollow interior. If the cross section of the cylinder is circular, the diameter is in the usual sense; otherwise, the diameter refers to the arithmetic mean diameter when approximated to a circle. The inner and outer diameters can be measured by any method known in the art. Since the object is nanoscale, it can be observed and measured, for example, using a scanning electron microscope. The length of a cylinder is used in the usual sense and can be measured by any method known in the art.

[0028] As used herein, the term "cell" is defined in the broadest sense to refer to a microscopic chamber-like substructure found in all living organisms. As used herein, the term "cell" refers to any cell that is intended for the transfer of substances, and preferably includes cells that contain organelles. Examples of cells include eukaryotic cells, prokaryotic cells, bacterial cells, fungal cells, etc.

[0029] As used herein, the term "substance" is defined in the broadest sense and refers to any entity composed of molecules or atoms, including, for example, low-molecular-weight compounds (typically, but not limited to, those with a molecular weight of 500 or less), medium-molecular-weight compounds (typically, but not limited to, those with a molecular weight of 500 to 10,000), high-molecular-weight compounds (typically, but not limited to, those with a molecular weight of 10,000 or more), nucleic acids, peptides, proteins, and organelles. When discussing a substance to be introduced or recovered (or extracted) herein, the substance can be defined as one that can be taken up into the interior of a cell. As used herein, the diameter of a substance is expressed as the arithmetic mean diameter, typically the volume-average diameter. Methods for calculating such mean diameters are well known in the art.

[0030] As used herein, "introduction" of a substance into a cell is defined in the broadest sense and refers to the movement of any substance from outside the cell into the cell.

[0031] As used herein, the terms "recovery" or "removal" of a substance from a cell are used interchangeably and are defined in the broadest sense to refer to the movement of any substance from inside to outside the cell.

[0032] In this specification, the term "conductive polymer" is broadly defined as any polymer that has electrical conductivity. Here, "conductivity" refers to the property of conducting and guiding electricity, as is known in the art. The term "polymer" refers to a molecule with a large molecular weight (usually defined as a molecular weight of 10,000 or more). Conductive polymers, such as riaacetylene, are insulators and semiconductors upon synthesis. However, through a process called doping (described below), they transform from semiconductors to conductors, becoming electrically conductive despite being polymers. The main chain of conductive polymers, such as polyacetylene, generally has a conjugated structure consisting of alternating single and double bonds (σ and π bonds), and this conjugated structure is believed to determine whether or not the polymer will be conductive. To impart conductivity, a process (or reaction) called doping is required. This is called chemical doping. Conductivity is achieved by adding a small amount of a reagent (an acceptor that readily accepts electrons or a donor that readily donates electrons) to the conductive polymer. Doping can also be performed electrochemically. In this case, a conducting polymer is used as the positive or negative electrode, and a voltage is applied in an electrolyte solution containing a dissolved electrolyte. This results in acceptor doping at the positive electrode and donor doping at the negative electrode.

[0033] Examples of the conductive polymer include the following. Representative examples of conjugated polymers Aliphatic conjugated polyacetylene Aromatic conjugated system Poly(p-phenylene) Mixed conjugated poly(p-phenylene vinylene) Heterocyclic conjugated systems: polypyrrole, polythiophene, polyethylenedioxythiophene (PEDOT) Heteroatom-containing conjugated polyaniline Double-chain conjugated polyacene (virtual molecule) Two-dimensional conjugated system graphene

[0034] A preferred embodiment of the conductive polymer is PEDOT. PEDOT has recently been the subject of worldwide research and development. In some sense, PEDOT can be considered an ideal conductive polymer, notable for its excellent stability, high conductivity, hole injection properties, and doping properties, but the present disclosure is not limited thereto. PEDOT is a polymer dopant, and when dispersed in water or an organic solvent (commonly known as PEDOT / PSS (available, for example, as Aldrich product numbers 483095 and 560596)), it is believed that conductive polymer thin films of any desired thickness can be easily formed by spin coating. Furthermore, in this field, it is believed that the particle size of PEDOT can be made uniform, allowing for the formation of uniform films, which may be advantageous in this respect.

[0035] In a preferred embodiment, the conductive polymer may have the property of expanding and contracting when a voltage is applied.

[0036] As used herein, the term "expanding and contracting when a voltage is applied" refers to a conductive polymer or the like that has the property of expanding or contracting in volume when a voltage is applied, and vice versa when the voltage is stopped (those that expand in volume when a voltage is applied will contract when the voltage is stopped, and those that contract will expand when the voltage is stopped). Examples of polymers that have this property include, but are not limited to, 3,4-ethylenedioxythiophene (PEDOT), polythiophene, polyacetylene, polyaniline, polypyrrole, and combinations thereof.

[0037] As used herein, the term "conductor" is broadly defined to refer to any electrically conductive material. Typical examples include, but are not limited to, metals and semiconductors, preferably metals. Metals are preferred for the main body of the cylindrical body. Examples of semiconductors include silicon. In a preferred embodiment, the conductive polymer is used as a coating rather than constituting the main body of the cylindrical body.

[0038] The metals used herein are preferably electrolessly plateable, since it is advantageous to be able to plate the conductive polymer without electrolytic plating. Metals that can be electrolessly plated include, but are not limited to, gold, platinum, silver, and nickel, and alloys thereof.

[0039] As used herein, the term "electrode" refers to a pole for applying electricity and can be composed of any conductive material. Any shape can be used as long as a voltage can be applied to the conductive polymer and the storage section. Typically, two electrodes are provided to apply electricity, but one, three or more electrodes may be provided in the device of the present disclosure as needed.

[0040] As used herein, the term "substrate" refers to any shape and material on which the cylindrical body of the present disclosure is placed. In a preferred embodiment, the substrate can be configured into any shape after the cylindrical body of the present disclosure is placed, and is preferably made of a material and shape that allows easy access to cells. The substrate may be made of, for example, but is not limited to, polycarbonate and / or polyethylene terephthalate.

[0041] As used herein, the term "storage section" refers to a section for storing a substance, and is arranged so as to be in communication with the cylindrical body and the substrate. Preferably, the storage section is hollow, in direct contact with the substrate, and has a structure with a tube through which an electrode can be inserted into the cavity. The storage section may be made of, for example, glass, ceramic, polymer, and / or metal.

[0042] In this specification, the term "voltage supply unit" may refer to any device that can supply voltage. In addition to a dry cell battery or a storage battery, electricity may be supplied from a household outlet.

[0043] As used herein, the term "differential interference prism," also known as a Nomarski prism or a Nomarski-type Wollaston prism, refers to a prism that splits light into two polarized beams with mutually orthogonal vibration planes. A Wollaston prism is made by bonding two birefringent crystals, such as calcite, with their crystal axes offset. This prism can split light by utilizing the difference in refractive index based on the polarization of the light. A Nomarski-type prism, an improved version of a Wollaston prism, has the focal points of the two split polarized beams outside the prism. This allows the prism's imaging position to be aligned with the condenser's imaging plane, enabling dramatically flexible optical path design. The optical path configuration of a differential interference microscope is a standard bright-field microscope with the addition of two polarizing plates and two Nomarski prisms. By removing only the Nomarski prisms, the microscope can be used as a polarizing microscope. In other words, the present disclosure also provides a device that can be used as a polarizing microscope.

[0044] (Preferred embodiment) Preferred embodiments of the present invention will be described below. The embodiments provided below are provided for a better understanding of the present invention, and it is understood that the scope of the present invention should not be limited to the following description. Therefore, it is clear that those skilled in the art can make appropriate modifications within the scope of the present invention in consideration of the description in this specification. It is also understood that the following embodiments of the present invention can be used alone or in combination.

[0045] (1) A cylindrical body coated with a conductive polymer The present disclosure provides a cylindrical body for introducing a substance into a cell and / or retrieving a substance from a cell. The cylindrical body is coated with a conductive polymer. The conductive polymer-coated cylindrical body can turn on / off the flow of a substance through the cylindrical body by applying a voltage. Since the flow of a substance can be turned on / off, it is possible to prevent the outflow of undesired intracellular substances from the cell, and the cylindrical body can be inserted for a long period of time.

[0046] In one aspect, the present disclosure provides a cylindrical body coated with a conductive polymer for introducing a substance into a cell and / or retrieving a substance from a cell. In another aspect, the present disclosure provides use of a cylindrical body coated with a conductive polymer for introducing a substance into a cell and / or retrieving a substance from a cell.

[0047] In one advantageous embodiment of the present disclosure, it has been demonstrated that inserting metallic nanotubes coated with a conductive polymer into cells enables the long-term introduction and retrieval of substances by applying a small voltage (e.g., ±0.1 V or less, a potential lower than the membrane potential) (Figure 13). In an exemplary embodiment, the metallic nanotube shown here is a cylindrical object made of gold or platinum, e.g., with an outer diameter of 0.3 to 2.0 μm, an inner diameter of 0.1 to 1.9 μm, and a height (length) of 1 to 40 μm. Previously, material introduction and retrieval was achieved without inserting nanotubes into cells by applying a high voltage (approximately 0.1 to 10 V) to create holes in the cell membrane, a technique known as electroporation. However, applying a high voltage to cells can cause undesirable changes in cellular function, such as cell death and cancer. Therefore, the present disclosure develops metallic nanotubes coated with a conductive polymer, enabling the insertion of nanotubes into cells for long periods of time and controlling material transport and retrieval by applying a small voltage to the nanotubes, a feat not previously achieved with conventional techniques.

[0048] In the present disclosure, the cylindrical body is typically formed from a conductor. Because the cylindrical body is intended to coat the conductive polymer, the conductor used for the body is preferably a material other than the conductive polymer, such as a metal. Metals are preferred as conductors, including materials that can be electrolessly plated, such as gold, platinum, silver, and / or nickel.

[0049] In one preferred embodiment, the conductive polymer has the property of expanding and contracting when a voltage is applied. Preferred conductive polymers include, but are not limited to, poly(3,4-ethylenedioxythiophene) (PEDOT), polythiophene, polyacetylene, polyaniline, or polypyrrole, and mixtures thereof. By being coated with a material that expands and contracts when a voltage is applied, the cylindrical body of the present disclosure can control the introduction and withdrawal of substances into and from cells, i.e., the introduction and recovery, by applying and stopping the application of voltage. This has not been possible with conventional technology.

[0050] In one preferred embodiment, the voltage is 500 mV or less, 400 mV or less, 300 mV or less, 200 mV or less, 100 mV or less, 90 mV or less, 80 mV or less, 70 mV or less, 60 mV or less, 50 mV or less, 40 mV or less, 30 mV or less, 20 mV or less, or 10 mV or less. At approximately 100 mV or less, cell viability is relatively high, and at 70 mV or less, viability approaches 100%. On the other hand, since nearly 100% efficiency has been achieved for substance transfer, it can be said that one significance of the present disclosure lies in the fact that substances can be transferred into and out of cells even at such a small voltage.

[0051] In the present disclosure, the voltage may be either direct current or alternating current. Preferably, an alternating current voltage may be used. Without wishing to be bound by theory, this is because, but not limited to, the use of alternating current increases the efficiency of material transfer and / or increases the survival rate.

[0052] In one preferred embodiment, the cylindrical body has an inner diameter of 3 μm or less, 2 μm or less, or 1 μm or less. Such inner diameter may be changed depending on the substance to be transferred. When transferring organelles such as mitochondria, the inner diameter may be 2 μm or less.

[0053] In one preferred embodiment, the cylindrical body has an outer diameter of 5 μm or less, 4 μm or less, or 3 μm or less. Such outer diameter may be changed depending on the target cell to be transferred. When transferring organelles such as mitochondria, the outer diameter may be 4 μm or less.

[0054] In one preferred embodiment, the cylindrical body has a length of 5 μm or more and 50 μm or less. For example, the length may be 6 μm or more, 7 μm or more, 8 μm or more, 9 μm or more, 10 μm or more, 15 μm or more, 20 μm or more, 25 μm or more, 30 μm or more, 35 μm or more, 40 μm or more, 45 μm or more, or 49 μm or less, 48 ​​μm or less, 47 μm or less, 46 μm or less, 45 μm or less, 40 μm or less, 45 μm or less, 40 μm or less, 35 μm or less, 30 μm or less, 25 μm or less, 20 μm or less, 15 μm or less, or 10 μm or less. The length may be any range between these values. This length may be varied depending on the target cell to be inserted or removed. Without being bound by theory, this is because it is an advantageous size when configuring a stamping system.

[0055] In one preferred embodiment, the substance has an arithmetic mean diameter (typically volume average diameter) of 1 nm or more and 2 μm or less. For example, 5 nm or more, 10 nm or more, 15 nm or more, 20 nm or more, 25 nm or more, 30 nm or more, 35 nm or more, 40 nm or more, 45 nm or more, 50 nm or more, 60 nm or more, 70 nm or more, 80 nm or more, 90 nm or more, 100 nm or more, 150 nm or more, 200 nm or more, 250 nm or more, 300 nm or more, 350 nm or more, 400 nm or more, 450 nm or more, 500 nm or more, 600 nm or more, 700 nm or more, 800 nm or more, 900 nm or more, 1000 nm or more, 1100 nm or more, 1200 nm or more, 1300 nm or more, 1400 nm or more, 1500 nm or more, 1600 nm or more, 1700 nm or more, 1800 nm or more, 1900 nm or more, 2000 nm or more, 2100 nm or more, 2200 nm or more, 2300 nm or more, 2400 nm or more, 2500 nm or more, 2600 nm or more, 2700 nm or more, 2800 nm or more, 2900 nm or more, 3000 nm or more, 3100 nm or more, 3200 nm or more, 3300 nm or more, 3400 nm or more, 3500 nm or more, 3600 nm or more, 3700 nm or more, 3800 nm or more, 3900 nm or more, The diameter may be 00 nm or more, 1800 nm or more, 1900 nm or more, or 2 μm or less, 1900 nm or less, 1800 nm or less, 1700 nm or less, 1600 nm or less, 1500 nm or less, 1400 nm or less, 1300 nm or less, 1200 nm or less, 1100 nm or less, 1000 nm or less, 900 nm or less, 800 nm or less, 700 nm or less, 600 nm or less, 500 nm or less, 400 nm or less, 300 nm or less, 200 nm or less, 100 nm or less, 90 nm or less, 80 nm or less, 70 nm or less, 60 nm or less, 50 nm or less, 40 nm or less, 30 nm or less, 20 nm or less, 10 nm or less, or 5 nm or less. Any range between these values ​​may be used. These appropriate diameters may vary depending on the target cells to be introduced or removed. The present disclosure is advantageous in that relatively large sizes corresponding to nucleic acids, peptides, proteins, and organelles can be used.

[0056] In one preferred embodiment, the substance comprises at least one selected from the group consisting of a small molecule, a nucleic acid, a peptide, a protein, and an organelle.

[0057] This disclosure makes it possible to prolong the time it takes for nanotubes to be inserted into cells, and furthermore, by applying a voltage smaller than the membrane potential, a change in the volume of the conductive polymer can be caused, allowing for arbitrary control of the transport of substances into the cells.

[0058] While not wishing to be bound by theory, it is generally believed that inserting metal nanotubes alone into cells results in the release of intracellular substances, resulting in cell death over time (Figure 3). Coating the metal nanotubes with a conductive polymer film suppresses substance release and improves cell viability. Furthermore, substance release from the composite nanotubes can be controlled by turning electricity on and off (see Example, Figure 9). Conventional gold nanotubes alone cannot control substance transport by turning electricity on and off. As illustrated in the Examples, evaluations using calcein low-molecular-weight compounds (622.55 g / mol in the Examples) and human cancer cells (HeLa) showed that no substance transport into the cells was observed when electricity was off, but calcein-stained cells were observed when a voltage of ±50 mV was applied (see Example, Figure 4). Furthermore, the cell viability after electricity application was as high as 98.78%. The present invention has demonstrated that even when the introduced substance is changed to a polymer (green fluorescent protein, 26.9 kDa), intracellular transport can be controlled electrically, resulting in high survival rates. Interestingly, as shown in the examples, applying a voltage of ±100 mV above the cell membrane potential (approximately 70 mV) reduced survival rates (to approximately 84.10%, as shown in the examples).

[0059] (2) Device The present disclosure provides a device for introducing a substance into a cell and / or retrieving a substance from a cell. The device includes a cylindrical body and a substrate, and the cylindrical body is disposed on the substrate. The cylindrical body is coated with a conductive polymer. By providing the device, it becomes easy to introduce a substance into a cell and / or retrieve a substance from a cell.

[0060] In one aspect, the present disclosure provides a device for introducing a substance into a cell and / or retrieving a substance from a cell, the device comprising a cylindrical body coated with a conductive polymer, a substrate, and an electrode, the cylindrical body being disposed on the substrate.

[0061] The cylindrical body of the present disclosure penetrates the substrate, allowing a substance to pass downward from the upper surface of the substrate through the cylindrical body, and allowing a substance to be sucked up upward through the cylindrical body. Each cylindrical body may be any one of or a combination of any of the embodiments described above in "(1) Cylindrical body coated with a conductive polymer."

[0062] In one preferred embodiment, the device comprises a plurality of the cylindrical bodies.

[0063] The cylindrical body of the present disclosure is disposed on the substrate of the present disclosure. While not wishing to be bound by theory, as an exemplary shape, the substrate is a film-like body as illustrated in Fig. 8d, and multiple cylindrical bodies are disposed on the substrate like a pin holder. It is desirable, but not limited to, that the cylindrical bodies be disposed approximately vertically to the substrate. Electrodes may be disposed on the substrate.

[0064] Therefore, an example of a device of the present disclosure is a stamp-shaped device (also referred to as a stamping system) as shown in Figure 15. In Figure 15, 1 indicates the handle portion of the stamp. 2 indicates a (substance) storage section. 3 indicates a (membrane-like) substrate. 4 indicates a substance. 5 indicates a penetrating cylindrical body. The handle portion of the stamp can be manually or mechanically grasped to insert and remove the cylindrical body into and from a cell.

[0065] 16 shows an enlarged view of the storage section 2, substrate 3, and cylindrical body 5 of FIG. 15. The substance 4 contained in the storage section can be introduced into the cell through the cylindrical body 5 that penetrates the substrate 3. The intracellular substance can be recovered from inside the cell through the cylindrical body 5. The storage section 2 can contain the substance 4 and a solvent (e.g., water) that dissolves or suspends the substance.

[0066] In one preferred embodiment, the cylindrical body and the substrate further include a storage section for storing the substance. The storage section may contain a substance to be introduced (e.g., a low-molecular-weight compound, a peptide, a protein, a nucleic acid, an intracellular organelle (mitochondria, etc.)). Other than the substance to be introduced, the storage section may be filled with substances having the same component composition as the target cells. On the other hand, when the purpose is recovery, substances having the same component composition as the target cells other than the substance to be recovered may be stored.

[0067] In one preferred embodiment, the substrate is made of, but not studded with, polycarbonate or polyethylene terephthalate.

[0068] In one preferred embodiment, the storage section has a hollow interior that is in direct contact with the substrate, and preferably has a structure with a tube that allows the electrode to be inserted into the hollow.

[0069] In a preferred embodiment, the containment section is made of, but is not limited to, glass, ceramic, polymer, metal, etc. The polymer or other material may be any material capable of retaining a solution within the containment section. Examples of polymers include, but are not limited to, ABS resin, polyethylene (including cross-linked polyethylene), ethylene vinyl acetate copolymer, acrylic resin, polyacrylic acid, polyamide (nylon, aramid, etc.), polybutylene terephthalate, polycarbonate, polyether ether ketone, polyester (polytrimethylene terephthalate, polyethylene naphthalate, polybutylene naphthalate, etc.), polyethylene, polyethylene terephthalate, polyimide, polylactic acid, polyacetal, polyphenylene ether, polypropylene, polystyrene, polyethersulfone, polytetrafluoroethylene, polyurethane, polyvinyl chloride, polyvinylidene chloride, styrene maleic anhydride, AS resin, etc. The containment section may also be made of waterproof thick paper (cellulose).

[0070] (3) System The present disclosure provides a system for applying a voltage to a device of the present disclosure to introduce a substance into a cell and / or withdraw a substance from a cell.

[0071] In one aspect, the present disclosure provides a system for introducing a substance into a cell and / or withdrawing a substance from a cell, the system comprising the device of the present disclosure and a voltage supply unit that supplies a voltage. It is understood that the device may have any of the shapes described in "(1) Cylindrical body coated with a conductive polymer" and "(2) Device" described herein.

[0072] In a preferred embodiment, the system of the present disclosure further includes a container containing cells. Any container can be used as long as it can support the cells. The container can be made of sterilized plastic or glass.

[0073] In one preferred embodiment, the device further includes an observation unit for differential interference observation of the cells in the container from below. For differential interference observation, for example, a conventional microscope (e.g., a polarizing microscope) can be equipped with a differential interference prism (e.g., a Nomarski prism or a Nomarski-type Wollaston prism, which splits light into two polarized beams with orthogonal vibration planes). This prism is made by bonding two birefringent crystals together with their crystal axes offset, and can split light based on the difference in refractive index due to the polarization of the light. Typically, the optical path configuration of a differential interference microscope is a conventional bright-field microscope with two polarizing plates and two differential interference prisms added, and such a configuration can be used (for example, an optional mirror unit called U-MDIC3 manufactured by Olympus can be used).

[0074] In one preferred embodiment, the apparatus further includes a display unit for displaying an image from the observation unit. Any display or the like capable of displaying a microscopic image can be used as the display unit.

[0075] In one preferred embodiment, the insertion of the cylindrical body included in the device into the cell can be adjusted in increments of 0.01 μm, 0.02 μm, 0.03 μm, 0.04 μm, 0.05 μm, 0.06 μm, 0.07 μm, 0.08 μm, 0.09 μm, 0.1 μm, 0.011 μm, 0.012 μm, 0.013 μm, 0.014 μm, 0.015 μm, 0.016 μm, 0.017 μm, 0.018 μm, 0.019 μm, 0.2 μm, 0.3 μm, 0.4 μm, 0.5 μm, 0.6 μm, 0.7 μm, 0.8 μm, 0.9 μm, or 1.0 μm.

[0076] (4) Introduction and collection method The present disclosure provides methods for introducing and / or withdrawing (or removing) substances into and / or from cells. This can be done using the devices or systems of the present disclosure. By applying a voltage for a desired period of time, the introduction and / or withdrawal of substances into and / or from cells can be controlled.

[0077] In one aspect, the present disclosure provides a method for introducing a substance into a cell and / or retrieving a substance from a cell, the method comprising the steps of (a) providing a device or system of the present disclosure and (b) applying a voltage to the cylindrical body for a desired period of time. While not wishing to be bound by theory, the present disclosure believes that the gate opens and the substance disperses and migrates during the period in which the voltage is applied. This allows the substance to be introduced into the cell or retrieved (removed). It is understood that the devices, systems, and cylindrical bodies used in the present disclosure can employ any embodiment or combination of those described in "(1) Cylinder coated with a conductive polymer," "(2) Device," and "(3) System."

[0078] In the present disclosure, the time for which the voltage is applied to the cylindrical body can be adjusted as appropriate depending on the degree to which the desired substance introduction and / or recovery is achieved, and may also depend on the voltage and the type of voltage (such as AC voltage or DC voltage).

[0079] In the introduction and recovery method of the present disclosure, attention may also be required to the following points. Regarding viability, attention should be paid to, but is not limited to, degassing and sterilizing the needle before insertion, the speed and depth of insertion into the cell, and suppressing vibration during the substance introduction process. Regarding recovery rate, attention should be paid to, but is not limited to, degassing and sterilizing the needle before insertion, the speed and depth of insertion into the cell, and suppressing vibration during the substance introduction process. Regarding the size of the target substance, attention should be paid to, but is not limited to, degassing and sterilizing the needle before insertion, the speed and depth of insertion into the cell, and suppressing vibration during the substance introduction process. Regarding the size of the target substance, although there are no limitations, small molecules such as fluorescent dyes are susceptible to fading and degradation due to light, so it is recommended to store them in a dark room and handle them at an appropriate temperature. Furthermore, proteins and organelles are delicate materials that are easily deactivated by vibration or during manipulation. Without wishing to be bound by theory, larger molecular size tends to slow substance diffusion, resulting in longer introduction and recovery times.

[0080] (5) Device manufacturing method The present disclosure provides a method for fabricating a device comprising a cylindrical body coated with a conductive polymer and a substrate.

[0081] In one aspect, the present disclosure provides a method for producing a device comprising a cylindrical body coated with a conductive polymer and a substrate, the method including a step of fixing and coating a film of the conductive polymer onto the substrate on which the cylindrical body is placed, preferably at at least two points.

[0082] In one preferred embodiment, the coating step can be achieved by contacting the working electrode with the substrate at two or more points. Without wishing to be bound by theory, it has been found that stable on / off control can be achieved by fixing the working electrode at two or more points, and it is understood that the present disclosure is not limited thereto.

[0083] In one preferred embodiment, the substrate on which the cylinder is disposed is made by a process comprising electrolessly plating metal onto track-etched polycarbonate and etching the metal-plated surface.

[0084] In one preferred embodiment, the metal straw coated with the conductive polymer has an inner diameter of 0.01 μm to 2 μm and an outer diameter of 0.1 μm to 5 μm.

[0085] (Method for activating cells) The present disclosure provides a method for activating cells. The method includes introducing mitochondria into cells using a device or system described herein. The device and system described herein can efficiently and safely introduce macromolecules exceeding 1 MDa, such as mitochondria, into cells. Introducing fresh mitochondria into cells is expected to have an anti-aging effect. The method, device, and system described herein are advantageous in that they are simple and unlikely to cause cell death when introducing mitochondria into cells. Fresh mitochondria can be introduced into organs for organ transplantation or cell sheets, resulting in cell activation that is particularly beneficial in fields such as regenerative medicine (cell therapy) and cosmetics (Figure 25). Meanwhile, cells that continue to proliferate, such as cancer cells, have reduced mitochondrial function, so introducing fresh mitochondria can kill cancer cells.

[0086] (Method for controlling cell function) The present disclosure provides a method for controlling cell function. The method includes the step of introducing a gene product (protein) into the cell using a device or system described in the present disclosure. By introducing the gene product into the cell, the cell function can be directly induced (direct reprogramming). It is possible to directly induce somatic cells into specific differentiated cells.

[0087] In one preferred embodiment, the gene product includes a gene product for inducing cell differentiation, such as a reprogramming factor such as Sox2, Klf4, c-Myc, Oct4, etc. Factors such as Oct4 are transcription factors, and it is known that regulating the expression level of such factors can control the induction of cell differentiation. The method, device, or system described herein can control the induction of cell differentiation.

[0088] (Intracellular transport mechanisms using biological molecular motors) The present disclosure provides an intracellular transport mechanism using a biological molecular motor. When microparticles or the like are introduced into cells by sedimentation, the number of particles is approximately 1.1 × 10 -5 The sedimentation velocity is about μm / s. In contrast, a biological molecular motor that combines kinesin and microtubules is expected to have a molecular motor speed of 0.4 to 4.0 μm (Figure 26). The biological molecular motor of the present disclosure can quickly and safely introduce substances into cells. In addition to kinesin molecules, dynein can also be used. Furthermore, the mechanism for accelerating transport can include a transport function using actin filaments and myosin.

[0089] The present disclosure provides a cylindrical body coated with a conductive polymer for introducing a substance into a cell and / or retrieving a substance from a cell, the conductive polymer comprising kinesin. This cylindrical body can be used as a biological molecular motor in combination with a microtubule.

[0090] The present disclosure provides the use of a cylindrical body coated with a conductive polymer comprising kinesin for introducing a substance into a cell and / or retrieving a substance from a cell.

[0091] The present disclosure provides a device for introducing a substance into a cell and / or retrieving a substance from a cell, the device comprising a cylindrical body coated with a conductive polymer comprising kinesin, a substrate, and an electrode, the cylindrical body being disposed on the substrate.

[0092] The present disclosure provides a system for introducing a substance into a cell and / or withdrawing a substance from a cell, comprising a device described in the present disclosure and a voltage supply unit that supplies a voltage.

[0093] The present disclosure provides a method for introducing a substance into a cell and / or withdrawing a substance from a cell, the method comprising: (a) providing a device or system as described herein; (b) providing microtubules having a substance bound thereto; (c) applying a voltage to the cylinder for a desired period of time; The present invention provides a method comprising:

[0094] Any substance can be introduced here. Substances with the following properties can be introduced and retrieved effectively. Almost all intracellular contents, excluding the nucleus, can be retrieved; however, the smaller the substance, the greater the recovery rate. Specifically, carbohydrates, lipids, proteins (amino acids), and nucleic acids can be introduced and retrieved. In addition to small molecules, it is also possible to introduce and retrieve organelles (such as vesicles, Golgi apparatus, and mitochondria) as they are.

[0095] The cylindrical bodies used are as described in this specification, with preferred examples being as follows: for the introduction and collection of small molecules, a diameter of approximately 50 to 400 nm is preferred, for medium molecules such as proteins, a diameter of approximately 400 to 1000 nm is preferred, and for larger organelles, a diameter of approximately 1000 to 1500 nm is preferred.

[0096] The present disclosure provides a method for producing a cylindrical body coated with a conductive polymer comprising kinesin, the method comprising the step of binding kinesin to the cylindrical body.

[0097] (Stamp system) The present disclosure provides a stamp system for inserting composite nanotubes into cells, comprising: a stamp having a cylindrical body coated with a conductive polymer; An electrode; a voltage supply unit that supplies a voltage; The present invention provides a system that can be mounted on a microscope, and that enables a substance to be introduced into a cell simply and safely using the microscope.

[0098] Substances that can be introduced and recovered with the stamp system include intracellular contents except for the nucleus, and it is possible to recover almost all of them. From the perspective of substance transport speed, it is thought that the smaller the substance, the more can be recovered. Mainly carbohydrates, lipids, proteins (amino acids), and nucleic acids can be introduced and recovered. In addition to small molecules, it is also possible to introduce and recover cell organelles (vesicles, Golgi apparatus, mitochondria, etc.) as they are.

[0099] Suitable examples of the voltage supply unit include a two-electrode power supply capable of applying a voltage of approximately the cell membrane potential, or a three-electrode electrochemical power supply capable of accurately controlling the potential in the solution.

[0100] The present disclosure provides an insertion and observation system for inserting a composite nanotube into a cell, comprising: a device as described herein; and a voltage supply unit that supplies a voltage; A microscope and The present invention provides a system comprising:

[0101] Substances that can be introduced and retrieved with the observation system include intracellular contents excluding the nucleus, and it is possible to retrieve almost all of these. Considering the speed of substance transport, it is thought that the smaller the substance, the more likely it is that a larger amount can be retrieved. Mainly carbohydrates, lipids, proteins (amino acids), and nucleic acids can be introduced and retrieved. In addition to small molecules, it is also possible to introduce and retrieve cellular organelles (vesicles, Golgi apparatus, mitochondria, etc.) as they are. Suitable examples of voltage supply units include a two-pole power supply capable of applying a voltage similar to the cell membrane potential, or a three-pole electrochemical power supply capable of precisely controlling the potential in the solution.

[0102] The present disclosure provides a composite nanotube film and stamp kit for delivering a substance into a cell or extracting a substance from a cell, the kit comprising: a composite nanotube thin film having a cylindrical body coated with a conductive polymer; Stamp and The present invention provides a kit comprising: a method for introducing a substance into a cell to control the cell's function or to cause the cell to produce a substance; and a method for extracting intracellular substances to enable functional analysis of the cell or utilization of the extracted substance.

[0103] In this way, using the kit disclosed herein, metallic nanotubes coated with conductive polymers (composite nanotubes) can be developed, allowing the nanotubes to be inserted into cells for extended periods of time, and by applying a minute voltage to the nanotubes, highly efficient substance transport and extraction can be achieved.Furthermore, a stamp system (microscope-mounted type) can be provided for inserting this composite nanotube into cells, making it possible, for example, to safely and efficiently provide signaling substances.

[0104] Furthermore, this disclosure is expected to be used in the field of life sciences, and can be applied to technologies for measuring or controlling cellular activity; the technology disclosed herein can be used to introduce substances into cells and extract intracellular information substances.The technology disclosed herein can be applied to measurement and control technologies used in the fields of basic science or medicine, and can be applied to engineering fields that use cells, such as regenerative medicine, which creates organs from cells, or cultured food efforts, which create meat from cells.

[0105] (Cell activity evaluation method) The present disclosure provides a method for evaluating cell activity, comprising: A step of measuring the amount of a marker substance in a cell using a cylindrical body coated with a conductive polymer. The present invention provides a method for detecting a marker substance, comprising: detecting a marker substance that is a gene product (protein), but is not limited to such a marker substance. The marker substance may be a gene product (protein), or may be a substance that is known as an indicator of cellular activity.

[0106] In one preferred embodiment, the method further comprises the step of introducing mitochondria into the cells.

[0107] (Cell introduction method using microspheres) The present disclosure provides a method for cell introduction using microspheres, comprising: (a) providing a device or system as described herein; (b) providing microspheres; (c) applying a voltage to the cylinder for a desired period of time; The present invention provides a method comprising:

[0108] In one preferred embodiment, the method further comprises the step of attaching a substance to the microspheres.

[0109] (Method for extracting intracellular substances) The present disclosure provides a method for extracting intracellular material, comprising: (a) providing a device or system as described herein; (b) applying a voltage to the cylinder for a desired period of time; The present invention provides a method comprising: Examples of intracellular substances include, but are not limited to, proteins, nucleic acids, lipids, sugars, complexes thereof, organelles, and the like.

[0110] (Cellular programming) The present disclosure provides a method of cellular programming, comprising: (a) providing a device or system as described herein; (b) providing a gene or gene product; (c) applying a voltage to the cylinder for a desired period of time; The present invention provides a method comprising: Here, genes can include DNA, RNA, or derivatives thereof, and gene products include, but are not limited to, mRNA, proteins, post-translationally modified proteins, etc. Metabolites are also possible.

[0111] In this way, the present disclosure enables cells to be freely designed and created by programming them, realizing advanced biological cells (smart cells). Such cells can be used in industry, the food industry, the environment, medicine, and other fields by utilizing the cell's program. They can be used to produce cultured foods, pharmaceuticals, and polymers, and to produce cultured tissues, making them useful in the fields of regenerative medicine and cosmetics. Multiple substances can be safely introduced / extracted from many cells multiple times, enabling function control, substance production, functional analysis, and substance use. Biomolecular analysis in the cellular environment (in cell NMR) becomes possible. It is possible to improve the degree of cellular aging, kill cancerous cells, and introduce mitochondria. It is possible to control cell functions and directly induce differentiated cells (direct reprogramming). As smart cells, they are useful in pharmaceuticals, industrial products, seasonings, perfumes, and other applications.

[0112] (Method for modifying cell phenotype) The present disclosure provides a method for introducing a functional protein into a cell to modify the phenotype of the cell, comprising: (a) providing a device or system as described herein; (b) providing a functional protein; (c) applying a voltage to the cylinder for a desired period of time; The present invention provides a method comprising: Functional proteins include, but are not limited to, various enzymes, such as reprogramming factors such as Sox2, Klf4, c-Myc, and Oct4, antibodies such as anti-Bcr-Abl, and enzymes and enzyme groups involved in metabolism.

[0113] Thus, the present disclosure is useful for providing advanced biological cells (smart cells). By modifying the cellular phenotype, such cells can be used in industry, the food industry, the environment, medicine, and other fields. They can be used to produce cultured foods, pharmaceuticals, and polymers, as well as cultured tissues, making them useful in the fields of regenerative medicine and cosmetics. Multiple substances can be safely introduced / extracted from many cells multiple times, enabling functional control, substance production, functional analysis, and substance utilization. Biomolecular analysis in the cellular environment (in cell NMR) becomes possible. It is possible to improve the degree of cellular senescence, kill cancerous cells, and introduce mitochondria. It is possible to control cellular functions and directly induce differentiated cells (direct reprogramming). As smart cells, they are useful in pharmaceuticals, industrial products, seasonings, perfumes, and other applications.

[0114] (Fabrication of a substrate having a cylindrical body) As an exemplary embodiment, the fabrication of a substrate is described below. Metal (e.g., Au) is electrolessly plated onto a substrate (e.g., a track-etched polycarbonate (TEPC) template), followed by etching of the top surface (Figure 11a). A nanolayer of metal (e.g., Au) is electrolessly plated onto a porous membrane. This process is a well-established plating process consisting of four steps: (1) sensitization, (2) activation, (3) displacement plating, and (4) electroless plating (Figure 12). To activate the surface of a substrate (e.g., a TEPC membrane (e.g., pore size: 600 nm, thickness: 23 μm)), the substrate (e.g., the TEPC membrane) is immersed in, for example, a 10.55 mM SnCl2 solution at 24°C, followed by an 11.28 mM PdCl2 solution at 24°C. After washing the membrane with water, a tin-palladium metal layer is formed on the TEPC surface and used as a catalyst for electroless plating. The amount of metal catalyst on the TEPC surface is controlled by adjusting the immersion time and the number of activation cycles. The catalyst-coated substrate (e.g., TEPC film) is then immersed in a 2 g / L metal (e.g., gold) plating solution at 40 °C for 24 hours. After a metal (e.g., Au) nanolayer is coated onto this film, the color of the substrate (e.g., TEPC film) (Figure 11a) changes to a metallic (e.g., gold) color on both the top and bottom surfaces (Figure 11b). Examining the metal / substrate (e.g., Au / TEPC film) using a scanning electron microscope (SEM) confirms the formation of nanotubes with metal (e.g., Au) inside.

[0115] After forming the metal / substrate (e.g., Au / TEPC film), only the top surface of the metal (e.g., Au)-plated nanolayer is etched with aqua regia (a 1:3 molar mixture of nitric and hydrochloric acids) to expose both the substrate material (e.g., polycarbonate) and the internal metal (e.g., Au) nanotubes on the film surface (Figures 11a and 11e). After wet etching, a color change from metallic (e.g., gold) to brown can be observed on the etched surface only (Figure 11d). The outer and inner diameters of the exposed metal (e.g., Au) nanotubes can be confirmed using SEM (Figure 11e). Immersion in the metal (e.g., gold) plating solution for 24 hours can form a 100±30 nm metal (e.g., Au) layer on the substrate (e.g., TEPC). Further etching of the exposed substrate (e.g., TEPC) with O2 plasma produces hollow metal (e.g., Au) nanoneedles (nanostraws). The height (H) of the metal (e.g., Au) nanostraws can be controlled by adjusting the O plasma exposure time: for example, 0 min (Figure 11e, H: 0 μm), 10 min (Figure 11f, H: 1.3 μm), 20 min (Figure 2g, H: 2.4 μm), or 30 min (Figure 2h, H: 5.0 μm).

[0116] (Method of electrolytically polymerizing a conductive polymer monomer on a cylindrical body) The cylindrical body can be coated with a conductive polymer by electropolymerizing a conductive polymer on the substrate provided with the cylindrical body prepared as described above. The electropolymerization can be carried out under conditions normally used for conductive polymers. For example, in the case of PEDOT, a constant voltage of 1 V is applied for a certain period of time in a polymerization solvent (a mixed aqueous solution containing 50 mM EDOT (C6H6O2S) monomer and a dopant (100 mM KNO3 or 100 mM LiCiO4)). The application time depends on the area and shape of the exposed gold, but is usually between 1 and 10 minutes.

[0117] (Application example) This discovery makes it possible to safely introduce any substance into cells. This could be used in regenerative medicine and treatments to create organs and tissues from individual cells, as well as in the food sector, such as cultured cell meat and plants.

[0118] (Note) In this specification, "or" is used when "at least one or more" of the items listed in the sentence can be employed. The same applies to "alternative." In this specification, when it is stated that "within a range of two values," the range includes the two values ​​themselves.

[0119] All references cited herein, including scientific literature, patents, patent applications, and the like, are incorporated by reference in their entirety to the same extent as if each were specifically set forth.

[0120] The present disclosure has been described above by showing preferred embodiments for ease of understanding. The present disclosure will be described below based on examples. However, the above description and the following examples are provided for illustrative purposes only and are not intended to limit the present disclosure. The present invention will be further described in detail below by reference examples, examples, and test examples. However, these are provided for illustrative purposes only and do not limit the present disclosure. The compound names shown in the following reference examples and examples do not necessarily conform to IUPAC nomenclature. Abbreviations may be used to simplify the description, but these abbreviations have the same meanings as those described above. The scope of the present disclosure is not limited to the embodiments or examples specifically described herein, but is limited only by the claims. [Example]

[0121] Examples are described below. The cells used in the following examples were handled in compliance with Waseda University and other legal regulations. While the specific reagents used were the products described in the examples, equivalent products from other manufacturers (Sigma-Aldric, Wako Pure Chemical Industries, Nakarai, R&D Systems, etc.) can also be used.

[0122] (Example 1: Method for producing a cylindrical body coated with a conductive polymer) Au nanotubes were fabricated using a method similar to that previously described (Scientific Reports, 9, 6806, 2019). (Electroless Au thin film plating) TEPC membranes (it4ip SA) were treated in 1.25 M NaOH solution for 20 minutes at 40°C. After rinsing the membranes with water, they were immersed in 1.25 M SnCl2 solution for 10 minutes at 25°C and then rinsed with water. 2+ The coated TEPC film was immersed in a 1.25M PdCl2 solution for 10 minutes at 25°C to deposit Sn on the film surface. 4+ A Pd metal catalyst was formed. This metal formation cycle was repeated 1 to 4 times. The catalyst-coated TEPC membrane was immersed in an electroless gold plating solution containing 200 ml / L NC gold PDII (NC gold II; Kojima Chemical) and 20 ml / L gold(I) sodium gold(I) sulfite for 24 hours at 40°C. The Au / TEPC membrane was washed with water and then dried in a vacuum chamber.

[0123] (Au nanostraw fabrication using wet and dry etching) The Au / TEPC film was floated on aqua regia (ITO-02; Kanto Chemical Co., Inc.) for 4 minutes to etch the top surface of the Au nanolayer on the Au / TEPC film. The etched Au / TEPC film was washed with distilled water and then dried in a vacuum chamber. To fabricate Au nanostraws, the TEPC surface on the Au / TEPC film was etched using oxygen-based reactive ion etching. The height of the Au nanostraws could be controlled by varying the etching time. After etching, scanning electron microscope (SEM) images of the Au nanostraws were obtained using a HITACH SEM S-3400N and HITACH SEM software, and the outer and inner diameters of over 100 Au nanostraws were measured.

[0124] (Example 2: Coating of conductive polymer with monomer) The method for electropolymerizing the conductive polymer monomer (PEDOT) onto the Au nanotube was a known technique (ACS Macro Lett. 2012, 1, 3, 400-403). In this case, the surface area of ​​the Au nanotube was approximately 88.7 cm. 2 PEDOT was electropolymerized onto Au by applying a constant voltage of 1 V in a monomer aqueous solution (0.05 M PEDOT, 0.1 M LiClO4). Here, the polymerization time was controlled, and PEDOT films were fabricated after 3 minutes (polymerization amount: approximately 1.75 C), 5 minutes (polymerization amount: approximately 2.49 C), and 7 minutes (polymerization amount: approximately 3.05 C).

[0125] (Example 3: Cellular insertion system of composite nanotubes (conductive polymer and gold)) Typically, cell observation is performed using bright-field or confocal laser microscopes, but our cell stamping technique was unable to observe the cells due to the blocking of light (Figure 1). Previously published results (Scientific Reports, 9, 6806, 2019) demonstrated intracellular delivery of calcein molecules using a needle measuring device capable of measuring needle height with 1 μm accuracy, achieving a delivery efficiency of 85.7% and cell viability of 94.0% (Figure 2a). However, given that the height of adherent cells is approximately 1 μm, conventional stamping methods using measuring devices pose challenges, such as damaging cells by over-insertion and making delivery difficult by under-insertion. Therefore, by incorporating differential interference contrast (DIC) observation (an optional mirror unit called the Olympus U-MDIC3) that allows needle insertion while observing cells from below, we successfully achieved a delivery efficiency of 99.9% and cell viability of 99.5% (Figure 2b).

[0126] (Example 4: Change in cell viability by intracellular insertion of gold nanotubes and composite nanotubes (conductive polymer and gold)) The use of composite nanotubes makes it possible to extend the time for insertion into cells. When gold nanotubes are inserted into cells using the intracellular insertion system described above, it was found that the cell viability decreases over time, reaching approximately 80% after 30 minutes (Figure 3). In contrast, when composite nanotubes are inserted, the decrease in cell viability is gradual, reaching 93.8% after 30 minutes. We believe this is because the conductive polymer coating prevents leakage from the cells.

[0127] (Example 5: Control of intracellular delivery of calcein molecules by applying voltage to composite nanotubes) A stamp combining composite nanotubes (polymerized for 5 minutes) and a model substance (calcein small molecule (622.55 g / mol)) was inserted into human cancer cells (HeLa). The intracellular delivery of calcein molecules was observed using a fluorescence microscope by applying an on / off voltage. When the voltage was off (Figure 4a), no calcein molecules were delivered to the cells, and the viability after 10 minutes was high at 99.02%. On the other hand, when an AC voltage (±50 mV) was applied to the composite nanotubes, calcein molecules were delivered to the cells, and stained cells were observed (Figure 4b). The delivery efficiency was nearly 100%, and the cell viability was also high at 98.78%. Furthermore, when an AC voltage of ±100 mV, which was higher than the cell membrane potential (approximately 70 mV), was applied, the delivery efficiency was nearly 100%, and the viability remained high at 84.10%, although there was a slight decrease (Figure 4c). Interestingly, when DC voltages (50 mV and -50 mV) were applied to the composite nanotubes, the viability remained at 98.7%, but the amount of calcein introduced was significantly reduced (Figure 4d, e). As shown in Figure 5, the fluorescence intensity increased with increasing AC voltage. On the other hand, when DC voltages were applied, the amount of calcein introduced into the cells did not increase significantly, even though the same voltages as AC voltages were applied. Because the application of DC voltage changed the inner diameter of the composite nanotubes, the increase in calcein introduction can be attributed to an increase in flow rate due to the change in inner diameter. On the other hand, when AC voltages were applied, the PEDOT conductive polymer repeatedly expanded and contracted in volume. This volume change within the composite nanotubes likely acted like a pump, improving the introduction of calcein molecules.

[0128] (Example 6: Control of protein delivery into cells by applying voltage to composite nanotubes) Intracellular protein introduction is expected to be a technology that can modify function without gene transfer, such as by inducing skin cells into induced pluripotent stem (iPS) cells (reprogramming) and directly inducing skin cells into pluripotent hematopoietic stem cells (direct reprogramming). However, it is extremely difficult to introduce large molecules such as proteins into cells covered with a lipid bilayer membrane. Here, we confirmed that efficient protein introduction into cells can be achieved by utilizing composite nanotubes and the present invention, which improves substance introduction through the pumping effect caused by the application of an AC voltage.

[0129] In this example, the model substance was changed from the low-molecular-weight calcein to the high-molecular-weight green fluorescent protein (GFP, molecular weight: 27 kDa), and an experiment similar to that in Example 5 was carried out. When the voltage was off (Fig. 6a), no GFP protein was introduced into the cells, but it was confirmed that GFP was introduced into the cells by applying an AC voltage (±50 mV, ±100 mV). It was also confirmed that the amount introduced increased by increasing the voltage.

[0130] Example 7: Transport of isolated mitochondria through composite nanotubes This composite nanotube is also capable of transporting even larger molecules (mitochondrial organelles). When a solution of calcein-labeled isolated mitochondria, made by staining mitochondria isolated from pig hearts with calcein dye, was applied to a substrate, it was adsorbed onto the substrate, as shown in Figure 7a, and could be confirmed by fluorescence observation. Within cells, mitochondria have complex shapes due to repeated division and fusion, but isolated mitochondria are spherical with a diameter of approximately 500-1000 nm. When this mitochondrial solution was combined with composite nanotubes to confirm whether mitochondria could be transported within the nanotubes, it was confirmed that mitochondria were transported via the composite nanotube thin film and adsorbed onto the substrate, as shown in Figure 7b.

[0131] (Example 8: Evaluation of the structure and ion permeability of composite nanotubes) (Structural analysis using a scanning electron microscope (SEM)) The top and bottom surfaces of the composite nanotube thin film prepared above were observed by SEM to confirm the polymerization of PEDOT over time (Figure 8a). SEM images of the PEDOT coating on the bottom surface (needle side) confirmed that the film thickness increased with time (Figure 8c). Furthermore, observation of the top surface (flat side without needles) revealed that the inner diameter of the PEDOT coating remained constant regardless of time (Figures 8b and c). In other words, it was revealed that the PEDOT coating on the Au nanotube thin film had an asymmetric structure on the top and bottom surfaces (Figure 8d).

[0132] (Example 9: Permeability of calcein substance to gold nanotubes and composite nanotube thin films) The amount of calcein permeating the gold nanotubes and composite nanotube thin film was quantitatively evaluated using a method similar to that previously described (Scientific Reports, 9, 6806, 2019). The flow rate J of the substance can be expressed by the following equation:

number

[0133] As shown in Figure 9, when gold nanotubes (0 min coating) were used, the flow rate was 86.9 nmol after 20 minutes, whereas the flow rate decreased for PEDOT-coated composite nanotubes. For composite nanotubes coated with PEDOT for 5 minutes or more, the flow rate of calcein was found to be close to zero. This is primarily due to the reduction in the inner diameter (r) caused by the PEDOT coating. Since SEM observations were performed in a vacuum on dry composite nanotubes, the PEDOT polymers were shrunk. Therefore, considering the PEDOT film swollen with water in the solution, the actual inner diameter of the composite nanotube would be smaller.

[0134] (Example 10: On / Off control of calcein permeation by applying voltage to composite nanotubes) It is known that PEDOT conductive polymers change volume when a voltage is applied (Journal of American Chemical Society, 132, 13174-13175, 2010). Therefore, we checked whether calcein permeability changes when a voltage is applied to the composite nanotube (Figure 10a). The results showed that the amount of permeation can be controlled by turning the voltage on and off. In contrast, Applying a similar voltage to only the gold nanotubes did not stop calcein permeation (Figure 10b), demonstrating for the first time that the volume change of the PEDOT polymer contributes to the On / Off control. Furthermore, to confirm whether the shape of the PEDOT polymer is important, a needleless Au thin film coated on both sides with PEDOT can be used.

[0135] (Example 11: Experiment of "recovery" from cells) In this example, we demonstrate whether recovery from cells is possible.

[0136] In this example, calcein-AM is introduced into cells and stained in the same manner as in Example 5. After inserting a composite nanoneedle containing the same solution as the stamp used to introduce calcein-AM into the cell, except that it does not contain calcein-AM, no recovery is observed without the application of voltage, and application of voltage to the composite nanotube is confirmed to promote the recovery of small molecules of calcein.

[0137] We will clarify the conditions under which this recovery varies depending on the magnitude and type of voltage applied.

[0138] We will also demonstrate that it is possible to recover not only small molecules but also large molecules (GFP).

[0139] (Example 12: Demonstration example of "nucleic acid" (oligo DNA) by applying voltage to composite nanotubes)

[0140] In this example, we demonstrate whether nucleic acids (oligo DNA) can be taken into and out of cells.

[0141] Commercially available fluorescent dye-labeled oligo DNA is placed in the composite nanotube device, and intracellular introduction and recovery tests are performed in the same manner as in Examples 5 and 11. After inserting the composite nanoneedle into the cell, there is no introduction or recovery without applying voltage, and it is confirmed that applying voltage to the composite nanotube promotes the introduction and recovery of nucleic acids.

[0142] We clarify the conditions under which this introduction and recovery change depending on the magnitude and type of voltage applied.

[0143] (Example 12: Demonstration of "peptide" or "protein" (GFP) by applying voltage to composite nanotubes) In this example, a "peptide" or "protein" (GFP) is used to demonstrate whether it can be imported into or out of cells.

[0144] A commercially available peptide or protein labeled with a fluorescent dye is placed in the composite nanotube device, and an intracellular introduction test and a recovery test from the cell are carried out in the same manner as in Examples 5 and 11. After inserting the composite nanoneedle into the cell, there is no introduction or recovery without applying voltage, and it is confirmed that applying voltage to the composite nanotube promotes the introduction and recovery of substances.

[0145] We clarify the conditions under which this introduction and recovery change depending on the magnitude and type of voltage applied.

[0146] (Example 13: Demonstration example of "intracellular organelles" (mitochondria) by applying voltage to composite nanotubes)

[0147] In this example, we will use "intracellular organelles" (mitochondria) to demonstrate whether they can be taken in and out of cells.

[0148] Mitochondria isolated from cells are placed in the composite nanotube device, and tests for introducing mitochondria into cells and recovering them from the cells are carried out in the same manner as in Examples 5 and 11. After inserting the composite nanoneedle into the cell, no introduction or recovery occurs without applying voltage, and it is confirmed that applying voltage to the composite nanotube promotes the introduction and recovery of mitochondria.

[0149] We clarify the conditions under which this introduction and recovery change depending on the magnitude and type of voltage applied.

[0150] (Example 14: Extraction test of intracellular substances using composite nanotubes) Here, we attempted to extract intracellular substances using composite nanotubes. First, HeLa cells were cultured for 30 minutes in a culture medium containing calcein-AM (concentration 25 μM). This allowed the cell membrane-permeable calcein AM to be taken up into the cells and react with intracellular esterases to become membrane-impermeable calcein, thereby retaining the calcein fluorescent dye within the cells (Figure 17a). Calcein is a fluorescent dye, and when irradiated with 490 nm excitation light, it emits fluorescence at 515 nm. Therefore, the intracellular calcein concentration can be evaluated by measuring the fluorescence intensity. Inserting a composite nanotube into the cells for approximately 30 seconds enabled the extraction of the calcein dye contained within the cells (Figure 17b). As can be seen in Figure 17b, the fluorescence intensity decreased only in the area where the composite nanotube was inserted. After extraction, the cells were able to take up calcein dye (Fig. 17c), and staining with PI to count dead cells (Fig. 18) revealed a viability of 99.56% (viable cells: 3600, dead cells: 16) in the extraction area (insertion area). The amount of extraction can be controlled by changing the insertion time and the inner diameter of the composite nanotube.

[0151] Example 15: Microsphere introduction test into cells Figure 19 shows the setup for an experiment using a stamping system to introduce microspheres into cells. The stamping system incorporating composite nanotubes was introduced into cells while being observed in real time using a differential interference contrast (DIC) observation system on an inverted fluorescence microscope. The composite nanotube membrane stamp used in the experiment had a membrane diameter of 8 mm, a nanotube diameter of 1 μm, a nanotube density of 2.2E7, a nanotube length from the membrane of 2.4 μm, and a total nanotube length of 24 μm. This stamp was combined with a mixed solution (calcein: 1.6 mM, fluorescent dye-labeled microspheres: 1%, volume: 300 μl) and punctured into cells for 10 minutes to test the introduction of microspheres into cells. Calcein is a small molecule that is impermeable to the cell membrane and is easily introduced. It exhibits yellow-green fluorescence, which allows us to confirm that the nanotubes have penetrated the cells.

[0152] Figure 20 shows the results of microspheres introduced into cells by the stamping method. (a) is a HeLa cell into which microspheres were introduced, (b) is a fluorescent image of the microspheres introduced into the cell, and (c) is an image obtained by superimposing images (a) and (b). The results in Figure 20 show that the microspheres were introduced uniformly into the HeLa cells.

[0153] (Example 16: Promotion of substance transport using microtubules) Here, we demonstrated the potential of combining the mechanisms of biological molecular motors with a composite nanotube membrane to promote intracellular transport (Figure 21). Specifically, we modified a kinesin molecular motor onto a gold membrane and linked it to a microtubule. In the presence of ATP in solution, the microtubule moves along the substrate by converting the chemical energy of the ATP into mechanical energy. Generally, the speed of molecular motors is known to be 0.4–4.0 μm / s. On the membrane developed by the inventors, the speed was 0.4–2.0 μm / s (Figure 22). The speed can be adjusted by changing the substrate texture, modification method, and ATP concentration. We also confirmed that microtubules can pass through the membrane via nanotubes (Figure 6). Furthermore, when microspheres with a diameter of approximately 30 nm were chemically linked to the microtubules (biotin-avidin reaction), we successfully transported the microspheres at a sustained speed of 1.86 μm / s (Figure 23). When unmodified microspheres are suspended in a solution, they do not move in a fixed direction due to Brownian motion (random movement occurs). In addition, the rate at which they gradually sink due to Stokes sedimentation is 1.1 × 10 for polystyrene microparticles (100 nm diameter). -5 It is quite slow at μm / s.

[0154] Example 17: Intracellular introduction of Oct4 protein Oct4 is a transcription factor, and it is known that controlling its expression level can control cell differentiation induction. Here, we demonstrate the possibility of inducing cell differentiation by introducing the functional protein Oct4 into cells. Because Oct4 protein is colorless, it is difficult to assess whether it has been introduced into cells. Therefore, we modified Oct4 protein with green fluorescent protein (GFP), which emitted green fluorescence within the cells, confirming that Oct4 protein had been introduced into the cells (Figure 24).

[0155] As described above, the present invention has been illustrated using preferred embodiments thereof, but it is understood that the scope of the present invention should be interpreted solely by the claims. This application claims priority to Japanese Patent Application No. 2020-41164 (filed March 10, 2020), the contents of which are incorporated herein by reference in their entirety. It is understood that the patents, patent applications, and literature cited herein should be incorporated herein by reference in their entirety as if the contents themselves were specifically set forth herein. [Industrial Applicability]

[0156] It is possible to deliver and retrieve substances such as reagents into cells, and it can be applied to a variety of industries, including the cosmetics, beauty, health, biotechnology, pharmaceutical, and manufacturing industries such as seasonings and perfumes. [Explanation of symbols]

[0157] Stamp handle part 1 (Material) Containment Unit 2 Substrate (film) 3 substance 4 Penetrating cylindrical body 5

Claims

1. A device for introducing a substance into a cell and / or recovering a substance from a cell, the device comprising a cylindrical body coated with a conductive polymer, a substrate, and an electrode, the cylindrical body being positioned so as to penetrate the substrate.

2. The device of claim 1 , wherein the device comprises a plurality of the cylindrical bodies.

3. The device of claim 1 or 2, further comprising a storage section for storing the substance.

4. The device according to claim 3 , wherein the housing is hollow and in direct contact with the substrate, and has a structure with a tube through which an electrode can be inserted into the cavity.

5. The device according to claim 3 or 4, wherein the electrodes are provided in two poles for applying a voltage to the conductive polymer and the storage portion.

6. The device of any one of claims 3 to 5, wherein the enclosure is made of at least one material selected from the group consisting of glass, ceramic, polymer, and metal.

7. The device according to any one of claims 3 to 6, wherein the storage section contains the substance.

8. The device of any one of claims 1 to 7, wherein the substrate comprises polycarbonate and / or polyethylene terephthalate.

9. The device according to any one of claims 1 to 8, wherein the cylindrical body is formed from a conductor.

10. The device of claim 9 , wherein the conductor comprises a metal.

11. The device of claim 10 , wherein the metal comprises one that can be electrolessly plated.

12. 12. The device of claim 11, wherein the metal comprises at least one metal selected from the group consisting of gold, platinum, silver, and nickel, and alloys thereof.

13. The device according to any one of claims 1 to 12, wherein the conductive polymer has the property of expanding and contracting when a voltage is applied.

14. 14. The device of any one of claims 1 to 13, wherein the conductive polymer comprises at least one polymer selected from the group consisting of poly(3,4-ethylenedioxythiophene) (PEDOT), polythiophene, polyacetylene, polyaniline, polypyrrole, and combinations thereof.

15. The device according to any one of claims 1 to 14, wherein the cylindrical body has an inner diameter of 2 µm or less.

16. The device according to any one of claims 1 to 14, wherein the cylindrical body has an outer diameter of 4 µm or less.

17. The device according to any one of claims 1 to 16, wherein the cylindrical body has a length of 5 µm or more and 50 µm or less.

18. The device according to any one of claims 1 to 17, wherein the substance has an arithmetic mean diameter of 1 nm to 2 µm.

19. The device according to any one of claims 1 to 18, wherein the substance comprises at least one selected from the group consisting of a small molecule compound having a molecular weight of 500 or less, a nucleic acid, a peptide, a protein, and a cellular organelle.

20. A device according to any one of claims 1 to 19, A system for introducing a substance into a cell and / or withdrawing a substance from a cell, comprising: a voltage supply unit that supplies a voltage.

21. 21. The system of claim 20, further comprising a container containing the cells.

22. The system according to claim 21 , further comprising an observation unit for differential interference observation of cells in a container containing the cells from below.

23. 23. The system of claim 22, wherein the observation portion is realized by a differential interference prism.

24. 24. The system of claim 22 or 23, further comprising a display unit for displaying an image from the observation unit.

25. The system according to any one of claims 20 to 24, wherein the insertion of a cylindrical body contained in the device into the cell is adjustable in increments of 0.1 µm.

26. 1. A method for introducing a substance into a cell and / or withdrawing a substance from a cell, comprising: (a) providing a device according to any one of claims 1 to 19 or a system according to any one of claims 20 to 25; (b) applying a voltage to the cylinder for a desired time; A method comprising:

27. A method for fabricating a device comprising a cylindrical body coated with a conductive polymer and a substrate, comprising: The method includes a step of fixing and covering the conductive polymer film at at least one point on the substrate, which is disposed so that the cylindrical body passes through.

28. A method for fabricating a device comprising a cylindrical body coated with a conductive polymer and a substrate, comprising: The method includes a step of fixing and covering the substrate on which the cylindrical body is placed with a film of the conductive polymer at at least one point, wherein the substrate on which the cylindrical body is placed is fabricated by a process including a step of electrolessly plating metal onto track-etched polycarbonate and a step of etching the metal-plated surface.

29. The method according to claim 27 or 28, wherein the step of covering includes a step of fixing and covering the conductive polymer film at at least two points.

30. 30. The method of any one of claims 27 to 29, wherein the coating step is achieved by contacting the substrate with two or more working electrodes.

31. The method according to any one of claims 27 to 30, wherein the cylindrical body has an inner diameter of 0.01 μm to 2 μm and an outer diameter of 0.1 μm to 5 μm.

32. A method for activating cells, the method comprising the step of introducing mitochondria into the cells using a device according to any one of claims 1 to 19 or a system according to any one of claims 20 to 25.

33. A method for controlling a cell function, comprising the step of introducing a gene product (protein) into the cell using a device described in any one of claims 1 to 19 or a system described in any one of claims 20 to 25.

34. 34. The method of claim 33, wherein the gene product comprises a gene product of Oct4.

35. A cylindrical body coated with a conductive polymer for introducing a substance into a cell and / or retrieving a substance from a cell, the conductive polymer comprising kinesin.

36. Use of a cylindrical body coated with a conductive polymer comprising kinesin for introducing a substance into a cell and / or retrieving a substance from a cell.

37. A device for introducing a substance into a cell and / or retrieving a substance from a cell, comprising a cylindrical body coated with a conductive polymer comprising kinesin, a substrate, and an electrode, the cylindrical body being positioned so as to penetrate the substrate.

38. A device according to claim 37, A system for introducing a substance into a cell and / or withdrawing a substance from a cell, comprising: a voltage supply unit that supplies a voltage.

39. 1. A method for introducing a substance into a cell and / or withdrawing a substance from a cell, comprising: (a) providing a device according to claim 37 or a system according to claim 38; (b) providing microtubules having a substance bound thereto; (c) applying a voltage to the cylinder for a desired time; A method comprising:

40. A method for producing a cylindrical body coated with a conductive polymer comprising kinesin, comprising the steps of: A method comprising the step of binding kinesin to the cylindrical body.

41. 1. A stamp system for inserting composite nanotubes into cells, comprising: a stamp having a substrate with a cylindrical body coated with a conductive polymer passing through it; An electrode; a voltage supply unit that supplies a voltage.

42. 1. An insertion observation system for inserting a composite nanotube into a cell, comprising: A device according to any one of claims 1 to 19 and 37; a voltage supply unit that supplies a voltage; A system comprising: a microscope.

43. A kit comprising a composite nanotube thin film and a stamp for delivering a substance into a cell or extracting a substance from a cell, comprising: a composite nanotube thin film having a substrate penetrated by a cylindrical body coated with a conductive polymer; A kit comprising: a stamp;

44. A method for evaluating cell activity, comprising: A method comprising the step of measuring the amount of a marker substance in a cell using a substrate penetrated by a cylindrical body coated with a conductive polymer.

45. 45. The method of claim 44, further comprising the step of introducing mitochondria into the cells.

46. A cell introduction method using microspheres, comprising: (a) providing a device according to any one of claims 1 to 19 and 37 or a system according to any one of claims 20 to 25 and 38; (b) providing microspheres; (c) applying a voltage to the cylinder for a desired period of time.

47. 47. The method of claim 46, further comprising the step of attaching a substance to the microspheres.

48. 1. A method for extracting intracellular material, comprising: (a) providing a device according to any one of claims 1 to 19 and 37 or a system according to any one of claims 20 to 25 and 38; (b) applying a voltage to the cylinder for a desired period of time.

49. 1. A method of cellular programming, comprising: (a) providing a device according to any one of claims 1 to 19 and 37 or a system according to any one of claims 20 to 25 and 38; (b) providing a gene or gene product; (c) applying a voltage to the cylinder for a desired period of time.

50. 1. A method for introducing a functional protein into a cell to modify the phenotype of the cell, comprising: (a) providing a device according to any one of claims 1 to 19 and 37 or a system according to any one of claims 20 to 25 and 38; (b) providing a functional protein; (c) applying a voltage to the cylinder for a desired period of time.

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