WO2021054407A1 - Introduction device, method for cellular introduction of macromolecule, dna nanoparticle crystal, and method for producing dna nanoparticle crystal inclusion body - Google Patents

Abstract

Provided is an introduction device that can introduce a macromolecule into a cell with high efficiency while maintaining the function of the macromolecule and having a high survival rate of the cell, and can be used to express the function of the macromolecule inside the cell. Also provided are a method for cellular introduction, a DNA nanoparticle crystal, and a method for producing a DNA nanoparticle crystal inclusion body. In regards to the introduction device, method for cellular introduction, DNA nanoparticle crystal, and method for producing a DNA nanoparticle crystal inclusion body, the introduction device perforates the surface of a cell through physical impact including crushing by air bubbles, and introduces a macromolecule into the cell. The introduction device comprises a storage unit for storing a solution that includes the macromolecule, and a liquid feeding part that feeds the solution towards the surface of the perforated cell, and introduces the macromolecule into the cell, and when the macromolecule is to be introduced into the cell, the introduction device introduces the macromolecule at a bottom section inside a container accommodating a solution containing the cells.

Description

Introduction device, polymer cell introduction method, DNA nanoparticle crystal and DNA nanoparticle crystal inclusion body production method

The present invention relates to an apparatus for introducing a polymer such as a nucleic acid or a protein into a cell, and a DNA nanoparticle crystal and a method used therein.
The present application claims priority based on US Provisional Application No. 62 / 901,803 filed in the United States on September 18, 2019, the contents of which are incorporated herein by reference.

In recent years, there has been a demand for technology for handling biopolymers such as nucleic acids and proteins, for example, particularly large polymers such as long-chain DNA and high molecular weight proteins. With the development of synthetic biology, research using long-chain DNA is accelerating worldwide, but research and development that goes into genome design guidelines for controlling cells arbitrarily is limited. At present, the functions of many genes are unknown only by sequence information, and there are repeated, homologous, and similar sequences, and it is difficult to modify them, which makes it difficult to analyze with conventional genome editing and recombination techniques. in the process of. Against this background, the establishment of low-cost genome-scale DNA synthesis technology and easy introduction technology of long-chain DNA into cells has become a rate-determining point for research and development, and is an urgent issue.

A problem when using long-chain DNA in cells is that it is difficult to handle long-chain DNA and the introduction technique is limited. For example, artificial chromosomes exceeding 100 kbp are introduced into cells mainly by using cell fusion by PEG or electricity, but physical introduction means such as electroporation or particle gun can be used as they are for long-chain DNA. It is not easy to apply for the following reasons. First, long-chain DNA has a large exclusion volume. Even supercoil plasmid DNA having a higher-order structure has a volume of several tens of nm to 200 nm, and the number of cells that can be physically introduced is limited. For example, in genome editing of untreated fertilized eggs using electroporation, there is no report that long-chain DNA or plasmid DNA having a large exclusion volume is introduced with high efficiency.
In addition, long-chain DNA is physically fragile. Long-chain DNA is also known to be cleaved by mechanical stimuli such as pipetting. When a physical phenomenon that causes a hole in a cell occurs around the long-chain DNA, the long-chain DNA is likely to be adversely affected.

Other biopolymers may have the same or similar problems as DNA when introduced into cells. For example, RNA, like DNA, has problems of exclusion volume and physical fragility, and is more chemically and physically unstable than DNA. Proteins are similarly physically vulnerable because bulk due to higher-order structures is also a matter of excluded volume, and when these higher-order structures are altered by physical impact, they easily lose their biological activity. There is a problem.

Therefore, in order to efficiently introduce a polymer into cells, a polymer having a large exclusion volume is introduced, so that a large-sized perforation can be temporarily and efficiently inserted without killing the cells. Introductory method is required. In addition, there is a need for a method for stably introducing physically fragile polymers, for example, a method for introducing these polymers without directly handling them.

The present inventors are developing an injection method using bubbles as a method for introducing molecules, particles, etc. into cells. For example, in Patent Document 1, the present inventors include a bubble ejection member and an outer outer shell portion, and the bubble ejection member is formed of a core material formed of a conductive material and an insulating material, and extends from the tip of the core material. An outer shell portion that includes the stretched portion and at least a part of which is in close contact with the core material to cover the core material, and a void formed between the stretched portion and the tip of the core material and having a bubble spout. A thick portion thicker than the other stretched portion is formed at the tip of the stretched portion, and the outer outer shell portion is formed outside the outer shell portion of the bubble ejection member and is between the outer shell portion and the outer shell portion. The gas-liquid ejection member is formed at a position separated from the outer shell portion so as to have a space in the outer shell portion, and the outer outer shell portion and the bubble ejection member are formed so as to be able to move relative to each other. , A gas-liquid ejection member using the same, a local ablation device, and a local injection device are disclosed. This technology thickens the tip of the stretched part created by cutting the insulating material so that the tip will not be damaged even if a high voltage is applied to the bubble ejection member, and the tip will be thick. , It is hard to break by piercing the object to be processed, and when the tip part is processed to be thick, the diameter of the bubble outlet can be reduced, so the object to be processed can be finely processed without damaging the tip part. By manufacturing the gas-liquid ejection member so that the bubble ejection member and the outer outer shell can move relative to each other, it becomes easy to adjust the positions of the bubble ejection member and the object to be processed at the time of local ablation or local injection. By pressing the outer outer shell against the object to be processed, it is possible to prevent the solution containing the injection substance from leaking out from the outer outer shell, and in that state, the positional relationship between the bubble ejection member and the object to be processed can be adjusted. Therefore, it is intended to provide a technique of a bubble ejection member, a gas-liquid ejection member, a local ablation device, or a local injection device, which can be used in the atmosphere and can be applied to, for example, a needleless injection device.

The present inventors are also developing a physically stable nano-sized structure. For example, in Patent Document 2, in the method for producing a superlattice structure in which nanoparticles are regularly arranged, the present inventors have a complementary binding portion that complementarily binds to at least a part of the base sequence with the nanoparticles. By mixing the above-mentioned nucleic acid in a solution, modifying the surface of the nanoparticles with the nucleic acid, heating to a predetermined temperature, and then slowly cooling the nanoparticles, the nanoparticles are complementarily bonded by the complementary binding portion of the nanoparticles. , The nanoparticles are bonded to each other to form a superlattice structure so that the volume ratio of the nanoparticles in the superlattice structure is 6% or more, and the superlattice structure is taken out from the solution. A method for producing a superlattice structure, which is characterized by drying while maintaining the symmetry of the superlattice, and a superlattice structure obtained by the method are disclosed. This technique can maintain superlattice symmetry even when the superlattice structure is taken out of solution and dried by controlling the volume fraction of nanoparticles in the superlattice structure in solution. Therefore, it is intended to obtain a structure that is physically stable even outside the solution.

Patent No. 6385453 JP-A-2018-149615

The present inventors examined the application of the above-mentioned injection method using bubbles as a method for introducing a polymer into cells. The introduction efficiency is not evaluated by the method described in Patent Document 1. Considering the introduction of DNA or protein having a large exclusion volume into cells, a large-sized perforation is required, and it cannot be said that a method for stable introduction has been sufficiently studied. In addition, a polymer that is physically more fragile than particles such as beads may not be stably introduced while maintaining a sufficient structure and function through the injection method using bubbles as in Patent Document 1.

In addition, the conventional bubble injection method mainly injects cells floating in a suspension of cells stored in a chamber or the like or cells adhering to a flat plate. In order to obtain high transfection efficiency, the cell suspension required a high cell concentration and a high concentration of the polymer to be introduced. Further, in the case of injection in cells attached to a flat plate, since the injection region is small, it is necessary to increase the number of injections. However, highly designed polymers generally have a limited amount that can be adjusted, and it is preferable that the concentration and frequency are small. Therefore, as high as possible introduction efficiency and transfection efficiency into cells are required. Be done.

Patent Document 2 discloses stable nano-sized crystals, but does not disclose or suggest application to a technique for introducing a polymer into cells, and how can it be applied? It is unknown whether it will be applied. For example, the purpose of using the nanoparticle structure of Patent Document 2 includes the development of materials such as optical materials and nanotechnology engineering, and its application to the biological field has not been shown.

In addition, when encapsulating a polymer in a structure, introducing it into a cell, and expressing its function in the cell, what kind of polymer is suitable for such an operation, and the structure. It is also unknown whether the function is expressed intracellularly from the state of being encapsulated in the body.

The present invention has been made in view of the above background, and the function of the polymer is maintained, the cell viability is high, the polymer can be introduced into the cell with high efficiency, and the polymer can be introduced into the cell. It is an object of the present invention to provide an introduction device, a cell introduction method, a method for producing a DNA nanoparticle crystal and a DNA nanoparticle crystal enclosure, which can be used for the functional expression of the above.

The embodiment of the present application has the following embodiments.
(1) An introduction device for perforating the surface of a cell by a physical impact including crushing of bubbles to introduce a polymer into the cell, a storage portion for storing a solution containing the polymer, and the perforation. The solution is fed toward the surface of the cells, and the polymer is provided with a liquid feeding unit for introducing the polymer into the cells. When the polymer is introduced into the cells, the cells are introduced. An introduction device performed on the bottom of a container containing a solution containing a polymer.
(2) The introduction device, wherein the introduction is performed on a cell mass formed at the bottom of the container by centrifuging the solution containing the cells in the container.
(3) The introduction is the introduction device in which the solution containing the cells has a shear viscosity of 0.01 Pa · s or more at a shear rate of 1.0 / s or less.
(4) The introduction device in which the container containing the solution containing the cells has a pyramid-shaped bottom in the container.
(5) The introduction device includes a cell perforation portion that introduces the porous crystal into a bubble ejection member and applies a voltage to the bubble ejection member to generate bubbles.
(6) The introduction device in which the polymer is enclosed in a porous crystal.
(7) The introduction device, wherein the porous crystal is a DNA nanoparticle crystal composed of a nanomaterial and a DNA molecule.
(8) The introduction device for introducing the porous crystal into the cell and then slowly releasing the encapsulated polymer.
(9) The introduction device, wherein the polymer contains a nucleic acid molecule (DNA molecule, RNA molecule, artificial nucleic acid), a protein, and a polyamide having a molecular weight of 1000 or more.
(10) The introduction device, wherein the polymer is Cas9 or a long-chain DNA molecule.
(11) A method for introducing a polymer cell, comprising an encapsulation step of encapsulating a polymer in a porous crystal and a cell introduction step of introducing the porous crystal into a cell.
(12) The method for introducing a polymer cell, wherein the cell introduction step is a step of perforating the surface of cells by a physical impact including crushing of atmospheric pressure.
(13) The porous crystal is introduced into a bubble ejection member, a voltage is applied to the bubble ejection member to generate a bubble, and a physical impact including crushing of the cell is used to perforate the cell membrane and the porous structure. A method for introducing a polymer into cells, wherein the porous crystals are introduced into the cells by an electric field-induced bubble method in which the sex crystals are introduced into the cells.
(14) The method for introducing a polymer cell into a cell mass formed at the bottom of a container containing a solution containing the cell when the porous crystal is introduced into the cell.
(15) The method for introducing a polymer into cells, wherein the porous crystal is a DNA nanoparticle crystal containing a nanomaterial and a DNA molecule.
(16) A method for introducing a polymer into cells, wherein the polymer contains a nucleic acid molecule (DNA molecule, RNA molecule, artificial nucleic acid), a protein, and a polyamide having a molecular weight of 1000 or more.
(17) A method for introducing a polymer into cells, wherein the polymer is a protein.
(18) A method for introducing a polymer into cells, further comprising a sustained-release step of cutting the porous crystal and slowly releasing the polymer from the porous crystal into the cell.
(19) The sustained-release step is a method for introducing cells of the polymer, wherein the porous crystals are irradiated with light to cleave the porous crystals, or the porous crystals are cleaved by an enzyme.
(20) A DNA nanoparticle crystal modified with a molecule having a specific interaction function that interacts with a specific molecule.
(21) The DNA nanoparticle crystal, which is configured to be able to hold molecules or particles inside the DNA nanoparticle crystal by the specific interaction.
(22) The DNA nanoparticle crystal, wherein the specific interaction is an interaction between biotin and another molecule.
(23) A method for producing a DNA nanoparticle crystal inclusion body, in which an inclusion target modified with the specific molecule is encapsulated in the DNA nanoparticle crystal.
(24) A DNA nanoparticle crystal containing a nanomaterial and a DNA molecule, which comprises a structure capable of diminishing the binding force between the molecules constituting the DNA nanoparticle crystal, and the structure is such that the DNA molecule has a photoresponse. A DNA nanoparticle crystal having a sex functional group or a configuration having a nuclease recognition sequence.

The embodiment of the present application also has the following aspects.
(1B) An encapsulation step of encapsulating a polymer in a porous crystal, a cell introduction step of perforating the surface of a cell by a physical impact including crushing of pressure, and introducing the porous crystal into a cell, and the porous. A method for introducing a polymer cell, comprising a sustained release step of cutting a sex crystal and slowly releasing the polymer from the porous crystal into the cell.
(2B) In the cell introduction step, the porous crystal is introduced into a bubble ejection member, a voltage is applied to the bubble ejection member to generate a bubble, and a physical impact including crushing of the cell causes a membrane of the cell. A method for introducing a polymer cell into the cell by an electric field-induced bubble method in which the porous crystal is introduced into the cell while perforating the cell.
(3B) The method for introducing a cell, wherein the polymer contains a nucleic acid molecule (DNA molecule, RNA molecule, artificial nucleic acid), a protein, and a polyamide having a molecular weight of 450,000 or more.
(4B) The method for introducing a polymer into cells, wherein the porous crystal is a DNA nanoparticle crystal containing a nanomaterial and a DNA molecule.
(5B) The sustained-release step is a method for introducing cells of the polymer, wherein the porous crystal is irradiated with light to cleave the porous crystal, or the porous crystal is cleaved by an enzyme.
(6B) An introduction device in which a polymer is enclosed in a porous crystal, the cell membrane is perforated by crushing bubbles, and the polymer is introduced into cells.
(7B) The introduction device is the introduction device for introducing the porous crystal into a bubble ejection member and applying a voltage to the bubble ejection member to generate bubbles.
(8B) The introduction device, wherein the porous crystal is a DNA nanoparticle crystal composed of a nanomaterial and a DNA molecule.
(9B) The introduction device for introducing the porous crystal into cells and then slowly releasing the encapsulated polymer.
(10B) The introduction device, wherein the polymer contains a nucleic acid molecule (DNA molecule, RNA molecule, artificial nucleic acid), protein, and polyamide having a molecular weight of 450,000 or more.

According to one embodiment of the present invention, the function of the polymer is maintained, the survival rate of the cell is high, the polymer can be introduced into the cell with high efficiency, and the polymer is used for the expression of the function in the cell. It is possible to obtain an introduction device, a cell introduction method, and a method for producing a DNA nanoparticle crystal and a DNA nanoparticle crystal enclosure.

It is the schematic which shows the introduction apparatus of this embodiment. It is the schematic which shows the porous crystal (DNA nanoparticle crystal) of this embodiment, the unit lattice and the DNA nanoparticle crystal inclusion body. It is a micrograph which shows the cell introduction of the polymer of Test Example 1 of this Example. It is a graph which shows the transfection efficiency of the cell introduction of FIG. It is a micrograph which shows the cell introduction of the polymer of Test Example 2 of this Example. It is a graph which shows the transfection efficiency of the cell introduction of FIG. It is a graph which shows the number of transfection cells of the cell introduction of FIG. It is a micrograph which shows the cell introduction of the polymer of Test Example 3 of this Example. It is a photograph figure of the gel electrophoresis which shows the molecular weight of the cell introduction plasmid of Test Example 4 of this Example. It is a graph which shows the transfection efficiency of introduction into the poorly introduced cell of Test Example 5 of this Example. It is a micrograph which shows the cell introduction of the polymer of Test Example 6 of this Example. It is a micrograph which shows the porous crystal of Test Example 7 of this Example. It is a micrograph which shows the streptavidin modified quantum dot of the test example 7 of this example. It is a micrograph showing the encapsulation of streptavidin-modified quantum dots in the porous crystal of Test Example 7 of this example. It is a graph which shows the encapsulation of streptavidin-modified quantum dots in the porous crystal of Test Example 7 of this Example. It is a schematic diagram which shows Cas9-gRNA and DNA nanoparticle conjugate used for Test Example 8 of this Example. It is a micrograph which shows Cas9-gRNA encapsulation in the porous crystal of Test Example 8 of this Example. It is a micrograph which shows the binding property of Cas9-gRNA to the porous crystal and the gold particle of Test Example 9 of this Example. It is a micrograph which shows the cell introduction of the polymer of Test Example 10 of this Example. It is a micrograph which shows the cell introduction of the polymer of Test Example 11 of this Example. It is a figure which shows the result of the positive control of the genome editing by the cell introduction of the polymer of Test Example 12 of this Example. It is a figure which shows the result of the introduction of the RNP-encapsulated crystal of genome editing by the cell introduction of the polymer of Test Example 12 of this Example. It is a figure which shows the result of the negative control of genome editing by cell introduction of the polymer of Test Example 12 of this Example. It is a micrograph which shows the enzymatic decomposition of the DNA nanoparticle crystal of Test Example 13 of this Example. It is a micrograph which shows the photolysis of the DNA nanoparticle crystal of Test Example 14 of this Example. It is a micrograph which shows the cell introduction (Hyfrector Type B) of the polymer of Test Example 15 of this Example. It is a micrograph which shows the cell introduction (Hyfrector Type A) of the polymer of Test Example 15 of this Example. It is a micrograph which shows the cell introduction of the polymer of the reference example of this Example.

Hereinafter, the present invention will be described in detail with reference to embodiments.
(Introduction device)
The introduction device 100 of the present invention includes an injector 20, and the injector 20 includes a storage unit 21 and a liquid feeding unit 22. The liquid feeding unit 22 faces the bottom of the container 30.

The injector 20 is a member that perforates the surface of a cell by a physical impact including crushing of bubbles to introduce a polymer into the cell.
The storage unit 21 is a storage means included in the injector 20, and is a container capable of storing a liquid in the present embodiment. The reservoir 21 contains a solution containing the polymer to be introduced into cells.
The liquid feeding unit 22 is formed so that the solution can flow. In the present embodiment, the liquid feeding unit 22 is a tubular member connected to the storage unit 21.
When the amount of the solution is small, the liquid feeding unit 22 may also serve as the storage unit 21. That is, the solution may be stored in the space inside the pipe of the liquid feeding unit 22.

The injector 20 of the present embodiment includes a bubble ejection member that generates bubbles. In the present embodiment, the bubble ejection member is provided at the end of the liquid feeding unit 22. Specifically, the tubular member of the liquid feeding portion 22 is made of an insulating material, and the tubular member is provided with a core portion (none of which is shown) made of a conductive material connected to the power supply means 40 described later. The tubular member and the core form the bubble generating member. The inner diameter of the core portion is sufficiently smaller than that of the liquid feeding portion 22 so that a gap through which the solution or air bubbles can flow is sufficiently secured in the liquid feeding portion 22. That is, as will be described later, by applying a voltage to the core portion, bubbles are generated and circulate in the pipe portion, and the bubbles are ejected from the vicinity of the end portion of the liquid feeding portion 22.

The end portion of the liquid feeding portion 22 is a cell perforation portion. When the polymer is introduced into the cells, the solution containing the bubbles and the porous crystals is circulated in the liquid feeding part in a state where the cell perforation part at the end of the liquid feeding part 22 is in contact with the cells. When a bubble collides with a cell, the bubble creates a physical shock. Physical impacts include, for example, crushing of air bubbles. This physical impact perforates the surface of the cell. By sending a solution containing the porous crystals to the perforated cells, the solution is introduced into the cells.
The condition of the electric power applied to the electrode at the tip of the liquid feeding unit 22 at the time of introducing the cells varies depending on the conditions such as the structure of the injector 20 and the type of cells, but it is preferably 2 to 15 W.

The container 30 contains a solution containing cells. The solution component of the solution containing cells can be appropriately selected as long as it is a solution that does not damage cells through the introduction operation, but may be, for example, a cell culture solution.
The number of cells per volume of the solution, that is, the cell concentration is preferably ^{0.25 to 3.0 × 10 6 cells per 7 μL as a guide.} When the number is 0.75 × 10 ⁶ or more, the viscosity of the cell mass or cell suspension becomes high, and the physical impact of bubbles is easily transmitted to the cells, which is preferable. 1.0 × 10 ⁶ or more is more preferable, and 1.5 × 10 ⁶ or more is particularly preferable.

The following can be considered as the principle that the introduction can be performed efficiently by introducing the container to the bottom of the container. At the bottom of the container, the concentration of cells in the solution containing cells is high, but the following effects can be considered due to the high concentration of cells. The present inventors have found that by increasing the concentration of cells, the viscosity of a solution containing cells increases, and the impact of bubbles in a liquid showing high viscosity increases. Specifically, the cell concentration in the cell number 0.75 × 10 6 ^/ 7μl or more, viscosity at a specific shear strength dramatically increased. In our study, the cell number 0.75 × ¹⁰ 6 / 7μl or more, below a shear rate of 1.0 / s, a shear viscosity is 0.01 Pa · s or more. Also, in the cell number 1.5 × ¹⁰ 6 / 7μl above, below a shear rate of 1.0 / s, a shear viscosity is 1.0 Pa · s or more.
It is known that the physical condition of the viscosity of a solution containing cells changes the properties of electric field-induced bubbles. From the results of Test Example 1 described later, the gene transfer efficiency increases when the cell concentration is above a certain level. Therefore, when the cell concentration is above a certain level, it is considered that the viscosity of the solution containing the cells is improved and the introduction efficiency is improved.
From these points of view, the introduction is for a solution containing cells having a shear viscosity of 0.01 Pa · s or more at a shear rate of 10.0 / s or less as measured by a viscoelasticity measuring device such as a rheometer. It is preferable to carry out. Further, it is more preferable that the solution containing cells is applied to a solution having a shear viscosity of 0.1 Pa · s or more at a shear rate of 10.0 / s or less.

It is preferable that the inner diameter of the container 30 becomes narrower toward the bottom. More preferably, the container 30 has a weight-like bottom shape.
Specific examples of the container 30 include a centrifuge tube and the like.
As the container 30, it is preferable to use a container having a volume of about 0.5 to 2 ml or the like because a solution containing cells is stored as described later. Further, it is preferable to use a container having a height to the bottom of about 0.5 to 3 cm. If the container is too small or too large, the physical impact of air bubbles will not be transmitted easily.

As the container having the above-mentioned volume and depth and a weight-shaped bottom, so-called microtubes, microcentrifuge tubes (microcentrifuge tubes), etc. are preferably used. Further, a container in which the vicinity of the bottom of a larger container (for example, a centrifuge tube having a length of 5 cm or more) is cut and the volume and depth are adjusted may be used. As for the larger container, a container having a weight shape near the bottom can be preferably used.

When the polymer is introduced into the cells, it is applied to the bottom of the container 30 containing the solution containing the cells.
At the bottom of the container 30, the number of cells per volume of the cell solution, that is, the so-called cell concentration, is high, so that the number of cells affected is large, and physical shocks caused by shock waves such as bubbles and their crushing are transmitted. It is easy to perforate the surface of cells, and it is easy to open pores in the cell membrane.
The cells may be introduced into a site having a high cell concentration due to natural sedimentation at the bottom of the container 30, or the container 30 may be centrifuged to further increase the cell concentration at the bottom, or the bottom may be introduced. It may be performed on a cell mass consisting of many cells.
Further, a high-concentration cell suspension in which the cell mass is suspended in a constant solution may be prepared. The high-concentration cell suspension makes it easy to adjust the cell concentration (number of cells) and has a higher cell survival rate than working with the cell mass as it is, so that injection can be performed with high efficiency as a result. ..

It is more preferable that the introduction is carried out on the cell mass formed at the bottom of the container by centrifuging the solution containing the cells in the container 30. By forming a cell mass and introducing the cell mass, the introduction can be almost certainly performed on the surface cells regardless of the concentration of the solution containing the cells before centrifugation.
The conditions for centrifugation vary depending on the cells used, the device for centrifugation, and the container, but as a guide, the concentration is 0 to 15,000 xg.
In the present embodiment, as described above, cells can be collected at the bottom by natural sedimentation instead of centrifugation. Therefore, there is no particular lower limit on the conditions for centrifugation, and the cells can be separated by 0 × g or more. When it is 100 × g or more, cells can be sufficiently accumulated in a short time of about 5 minutes, which will be described later, which is preferable.
If the upper limit of centrifugation conditions exceeds 15,000 xg, cells may be damaged. It is preferably 12000 × g or less, and more preferably 10000 × g or less. At 12000 xg, for example, 3T3 cells survive approximately 86%. At 10000 × g, 3T3 cells survive to the same extent (about 94%) as when they spontaneously settle without centrifugation, which is the upper limit when the maximum survival rate is taken.
From these results, the centrifugation is preferably performed under the condition of 100 to 12000 × g, and more preferably performed under the condition of 100 to 10000 × g.
By centrifuging at 10,000 × g or more, organelles (organelles) smaller in scale than cells, such as mitochondria, lysosomes, and peroxisomes, can be accumulated.
The time for centrifugation is not particularly limited, but can be appropriately selected from, for example, about 1 to 30 minutes, and in the case of 100 to 400 × g, it is preferably about 2 to 10 minutes. Under this condition, cells can be sufficiently accumulated at the bottom of the container in a shorter time than spontaneous sedimentation.

The polymer introduced into the cell in the present embodiment is a molecule having a molecular weight of 1000 or more as described later in the introduction method, and is mainly a biopolymer nucleic acid molecule, a protein, a polyamide, or a combination of a plurality of types thereof. Includes the complex. Further, as will be described later, it may be combined with another molecule such as a porous crystal.
When introducing the polymer into cells, it is also preferable that the polymer is encapsulated in a porous crystal described later, and a solution containing the porous crystal is stored in the storage portion for use.

The introduction device 100 may further include a power supply means 40. The power output means 40 may include a power supply device 41, an injector 20, and an electric wire 43 for forming a circuit with the counter electrode 42. The injector 20 is wired so that the tip of the liquid feeding portion 22 forms an electrode. Although not shown, the power supply means 40 may be provided with a resistor, a voltage amplifier circuit, a DIO (Digital Input-output) port, or the like, if necessary.
As the injector 20, a known so-called bubble injector 20, for example, the device described in Patent Document 1 may be used.

(Effect of the introduction device of this embodiment)
In the present embodiment, when the polymer is introduced into the cells, it is applied to the bottom of the container containing the solution containing the cells, so that the cell concentration at the time of introduction is high and the injection efficiency is high.
The bubble injection method has the advantage of higher cell viability than the electrical (electroporation) and chemical methods (PEG method, etc.). Conventionally, injection was performed on a solution in which cells were cultured, or injection was performed on cells attached to a flat plate such as a petri dish or a plate. With these means, only a small area can be taken as the injection area, and as a result, the introduction efficiency tends to be low. In addition, there were some aspects that were not suitable for a large number of cells and a large number of samples.
On the other hand, in the present embodiment, since injection is performed on the high-concentration cells at the bottom of the container, it is achieved by adding a target volume of medium to the cell pellet after centrifugation. At high cell concentrations, the impact caused by microbubbles and shock waves is transmitted, increasing the number of cells and providing sufficient hits to open the pores of the cell membrane.
The mechanical environment of the cell suspension is regulated by cell concentration, making it a suitable method for injecting a wide variety of cells (such as animal cells, algae or plant cells). The energy required for perforation of animal cells, algae, and plant cells varies depending on the thickness and composition of a specific membrane, but can be adjusted by electrical parameters and the distance between the electrode and the target, and the cell concentration is high. Then, the adjustment is easier. In addition, adjusting the cell concentration changes the mechanical environment (characteristics) of the cell suspension and the energy acting on specific types of cells, leading to an improvement in introduction efficiency.
In the present embodiment, the cells at the bottom of the container are centrifuged to form pellet-shaped cell clusters that adhere to the bottom of the container, so that the consumption of the plasmid to be used can be suppressed to the utmost limit. As a result, it can be widely used as a general-purpose injection method that can be applied to a wide range of cells and the like in research in the biomedical field.

(Other aspects)
The above has been described for the form in which the cells and the solution are supplied from the injector 20 to the cells, but the cell suspension contains the solution containing the porous crystals, and the injector 20 supplies the cells to the cell suspension. It may be an embodiment in which bubbles are generated and the porous crystals in the solution are introduced into cells.
In this case, the storage unit 21 may be a container in which cells described later are stored.
As an action of this aspect, the cell perforation portion at the tip of the liquid feeding portion 22 of the injector 20 is immersed in the cell suspension. Subsequently, when bubbles are generated from the injector 20 as described above and the surface of the cells is perforated by a physical impact including crushing the bubbles, the porous crystals in the cell suspension can be introduced into the cells.

(Method of introducing polymer cells)
Next, a method for introducing a polymer cell of the present embodiment will be described.
The method for introducing a polymer cell of the present embodiment includes an encapsulation step of encapsulating the polymer in a porous crystal and a cell introduction step of introducing the porous crystal into the cell.

(Encapsulation process)
(Porous crystal)
First, a porous crystal that encloses a polymer will be described. A porous crystal is a crystal having a large number of pores through which molecules can enter and exit. In the present embodiment, the porous crystal constitutes a crystal in which elements such as metals and nucleic acids are gathered and the interparticle distance is nano-sized.

In the present embodiment, as shown in FIG. 2, the porous crystal 500 is composed of unit lattices 50. In this embodiment, the porous crystal is a DNA nanoparticle crystal composed of a nanomaterial and a DNA molecule. The unit cell 50 is composed of nanoparticles 51 and an intracrystal DNA molecule 52 that modifies the nanoparticles 51.

Nanoparticle 51 is a particle having a particle size of nano-order (1 to 1000 nm, preferably 1 to 100 nm) and having a nanomaterial as a constituent material. As the nanomaterial which is a constituent material of the nanoparticles 51, any material capable of modifying DNA described later can be used on the surface thereof, and various materials such as metal, semiconductor, dielectric or magnetic material can be used, and the nanomaterial is inorganic. It may be a compound, an organic compound or an alloy. The nanoparticles 51 may contain these plurality of nanomaterials. Further, nanoparticles 51 of different materials may be mixed and used in the unit cell 50. As the nanomaterial which is a constituent material of the nanoparticles 51, Au, Pt, Pd, Li, Ag, Rh, Ru, V, Cu, Al, Co, Ni, Fe, Mg or the like should be used as long as it is a simple substance of metal. Can be done. If it is an alloy, a Ni—Mg alloy or the like can be used. Further, semiconductors such as Si, Cd, Se and CdS, and dielectrics such as SiO2 and TiO2 can also be used. The shape of the nanoparticles 51 may be arbitrary, such as a sphere, an ellipsoid, or a polyhedron.

In order to modify the nanoparticles 51 with the DNA molecule 52 in the crystal, a known method may be used as appropriate. For example, the nanomaterial constituting the nanoparticles 51 and the terminal of the DNA molecule 52 in the crystal may be chemically bonded. In this case, the end of the DNA molecule 52 in the crystal may be substituted with a substituent capable of binding to the nanomaterial, and the substituent may be bonded to the nanomaterial.
The intracrystal DNA molecule 52 is a single-stranded DNA, and one nanoparticle 51 is modified by at least two types of intracrystal DNA molecules 52. These two types of intracrystal DNA molecules 52 have regions that are complementary to each other, that is, complementary to each other (hybridization). By annealing these two types of intracrystal DNA molecules 52 to each other in a region where they can complementarily bind to each other, the metal elements are arranged at a certain distance from each other to form a unit lattice 50 having a lattice structure. The unit lattice 50 can have any three-dimensional structure such as a polyhedron due to the interaction of the DNA molecules 52 in the crystal.
For example, the unit lattice 50 may have a regular tetrahedron as a basic unit. The skeleton of the DNA regular tetrahedron has a rigid structure based on the DNA Double Crossover tile or DNA origami, so that single-stranded DNA that can bind to a part of the sequence of long-chain DNA grows from the center of the six sides. Structure can be made.
In the example shown in the figure, the lattice structure of the unit cell 50 is substantially cubic.

In the unit cell 50, the nanoparticles 51 are arranged by the complementary bonds between the intracrystal DNA molecules 52. Therefore, the distance between the nanoparticles 51 can be determined by designing the region where the intracrystal DNA molecules 52 can be complementarily bonded. Has been decided. That is, depending on this distance, the unit lattice 50 has pores 53, which are spaces in which molecules can enter and exit the lattice.
In addition to the design of the DNA molecule 52 in the crystal, the unit cell 50 can expand the unit cell 50 by adding these because the distance between the molecules increases due to the presence of water molecules, anions, and the like.
On the other hand, since the distance between the molecules is shortened by drying, addition of cations, addition of PEG, etc., the unit cell 50 can be contracted by these.

By using the in-crystal DNA molecule 52 in combination with the nanoparticles 51, the unit crystal 50 and the porous crystal 500 can be subjected to structures such as the size, crystal structure, and shape of the pores 53, which will be described later, by designing the in-crystal DNA molecule. It can be designed in various ways, and it is easy to control physical properties such as rigidity and viscoelasticity, and it is suitable for achieving the purposes of encapsulation of polymers, introduction into cells, and sustained release, which will be described later. Further, as will be described later, the DNA molecule 52 in the crystal can be modified to impart a specific interaction, and can be imparted with a function suitable for encapsulation of a specific molecule.

The size of the pores 53 is the so-called nano size, but in the present embodiment, it is mainly 1-400 nm, preferably 1-100 nm, and more preferably designed in the range of 5-50 nm. Designed at this size, it is suitable for introducing and retaining proteins, long-chain DNA, and protein-nucleic acid complexes.

Since the unit lattice 50 has a lattice structure, the porous crystal 500 in which a large number of lattice structures of the unit lattice are gathered is also called a superlattice structure. Further, since the porous crystal 500 is composed of a unit cell 50 having pores 53, it is porous having pores 53 as pores. The structure of the porous crystal 500 is also called a crystal sponge because it has pores 53 and therefore has a structure similar to a sponge having many fine voids inside. It can be said that the porous crystal 500 is self-assembled by gathering unit lattices 50 having pores 53. The shape of the porous crystal 500 can also be any three-dimensional structure such as a polyhedron due to the interaction of the DNA molecules 52 in the crystal. In the example shown in the figure, the structure of the porous crystal 500 is almost a regular dodecahedron.
The size of the porous crystal 500 in which the unit lattices 50 are gathered can be appropriately selected, but in the present embodiment, the porous crystal 500 on the order of several hundred nm to several μm may be used.
Specific examples of the superlattice structure are described in, for example, Patent Document 2.

Since the porous crystal 500 can be produced with a uniform crystal shape and a uniform particle size due to the design of the DNA molecule 52 in the crystal, the resistance at the time of cell introduction is small, and the conditions required for the operation at the time of introduction are satisfied. Easy to optimize.

The unit cell 50 is configured to be able to hold the polymer 60 via the pores 53.
The polymer 60 preferably contains a nucleic acid molecule (DNA molecule, RNA molecule, artificial nucleic acid) having a molecular weight of 1000 or more, a protein, a polyamide, or a complex obtained by combining a plurality of types thereof. Among them, the polymer 60 is also preferably RNP, that is, a complex of RNA and protein.
Nucleic acid molecules having a molecular weight of 1000 or more (so-called long-chain DNA or long-chain RNA) have a large exclusion volume, are physically fragile, and tend to lose their primary to higher-order structures. In the cell introduction method of the present embodiment, the polymer is compactly organized in the porous crystal by encapsulating the polymer in the porous crystal, and physical impact or the like is formed by encapsulating the polymer in the porous crystal. Protected from. Therefore, the cell introduction method of the present embodiment is particularly effective when introducing so-called long-chain DNA or long-chain RNA into cells.
Further, proteins and polyamides having a particularly large molecular weight have a large exclusion volume and are physically fragile. Therefore, the cell introduction method of the present embodiment is particularly effective when introducing these molecules into cells.
Further, the polymer preferably has a molecular weight of 450,000 or more. A large molecule having a molecular weight of 450,000 or more has a particularly large exclusion volume and is physically particularly fragile, and its structure or the like may change due to a physical force and lose its function. The cell introduction method of the present embodiment can be applied to such a molecule having a large molecular weight.

The polymer 60 is also preferably a molecule used for genome editing. Examples of the molecule used for genome editing include genome editing factors such as Cas family protein and TAL protein as candidates.
A more specific example of a genome editing factor is Cas9. Cas9 is a protein known as a DNA endonuclease and a DNA cleavage factor. By encapsulating Cas9 as a polymer 60 in a porous crystal and introducing it into cells, it can be widely used for editing intracellular DNA or similarly, editing DNA introduced into cells.
Cas9 of the present embodiment contains a mutant of Cas9 protein, a substitute, and a complex with other molecules (for example, deaminase, reverse transcriptase, etc.). In particular, it is preferable to use Cas9 of the present embodiment which is an RNA-bound RNP. More specifically, it is more preferable that Cas9 is a Cas9-gRNA complex. Here, gRNA (guide RNA, or sgRNA) refers to RNA used in the nucleic acid editing process. The gRNA can specify the target site on the nucleic acid of the Cas9 protein. The Cas9 protein is preferable because it can cleave the target DNA and can be used for DNA editing by designing an RNA to be a gRNA and forming a complex with the gRNA.
DNA cleavage using RNP such as Cas9-gRNA is a DNA-free (DNA-free) genome editing technology, and since it does not contain foreign DNA, it does not correspond to so-called genetic recombination, especially in the breeding of plants and livestock. It is useful.

The polymer 60 is also preferably a long-chain DNA molecule.
By introducing a long-chain DNA molecule into a cell, DNA containing a large amount of genetic information can be introduced into the cell and widely used for editing the genetic information of the cell.

When the porous crystal 500 is a DNA nanoparticle crystal composed of a nanomaterial and a DNA molecule, the DNA nanoparticle crystal is modified with a molecule having a specific interaction function that interacts with a specific molecule. It is also preferable that It is also preferable that the DNA nanoparticle crystal is configured to be able to hold molecules or particles inside the DNA nanoparticle crystal by the specific interaction.
Here, a specific molecule refers to a molecule having a certain elemental sequence, particularly an amino acid sequence or a nucleic acid sequence. A specific interaction that interacts with a particular molecule is one that interacts with this particular elemental sequence. For example, when a specific molecule is a nucleic acid having a specific nucleic acid sequence, as a molecule having a specific interaction function that interacts with the specific molecule, a nucleic acid sequence complementary to the specific nucleic acid sequence is used. There are nucleic acids and the like.
The porous crystal 500 has a site modified with a molecule having a specific interaction function that interacts with a specific molecule, so that the specific molecule is selectively and highly retained and porous. It can be held and encapsulated in the sex crystal 500.
In the present embodiment, one of the intracrystal DNA molecules 52 modifying the nanoparticles 51 has a sequence complementary to the nucleic acid having the specific nucleic acid sequence. Therefore, the nucleic acid having the specific nucleic acid sequence can be selectively held and encapsulated in the porous crystal 500 with high holding power.

(Operation of encapsulation process)
Next, the action of the porous crystal, that is, the operation of the encapsulation step will be described. As described above, the porous crystal 500 is in a state where the distance between the molecules of the unit cell 50 is large and the diameter of the pores 53 is large in an aqueous solution or the like. In this state, the porous crystal 500 is immersed in a solution containing the polymer 60 or the like, passed through the pores 53, and the polymer 60 is incorporated into the unit cell 50.

At this time, a specific interaction in the solution may be used for encapsulation. In that case, the unit cell 50 is modified with a molecule having a specific interaction function that interacts with a specific polymer 60. Therefore, the specific polymer 60 incorporated into the unit cell 50 is also retained in the unit cell 50 by the specific interaction.
The specific interaction broadly refers to an action in which a molecule having a certain structure specifically interacts (bonds, etc.) with another certain structure. Specific examples thereof include biotin-streptavidin interaction, RNA-protein interaction, metal complex-protein interaction, lectin-polysaccharide interaction, protein-protein interaction, or click chemical interaction. Among these actions, the interaction between biotin and other molecules, such as the biotin-streptavidin interaction, can be preferably used.

Further, the specific polymer 60 can be encapsulated in the porous crystal 500 by performing an operation of shrinking the porous crystal 500 by drying or the like as described above. Due to the shrinkage of the porous crystal 500, the polymer 60 is sealed in the unit cell 50 in a state where it does not easily fall off. By encapsulating the polymer 60, the unit lattice 50 and the porous crystal 500) become DNA nanoparticle crystal inclusion bodies 50A and 500A.
The methods of these encapsulation steps may be used separately or in combination.

When the porous crystal 500 is configured to hold molecules or particles inside the DNA nanoparticle crystal by the specific interaction described above, this operation modifies the DNA nanoparticle crystal with the specific molecule. This is a method for producing a DNA nanoparticle crystal encapsulation body that encapsulates the encapsulation target.

(Cell introduction process)
Next, a cell introduction step of introducing the porous crystal 500 (DNA nanoparticle crystal inclusion body 500A) into cells is performed.
The cells used in the cell introduction step of the present embodiment can be any type of cells. That is, both animal cells and plant cells can be used. In addition, both cells on biological tissues and cultured cells can be used.
The protoplast from which the cell wall of the plant cell was removed was able to introduce a molecule having a large molecular weight relatively easily using PEG. In addition, the particle gun method was also able to introduce porous crystals into plant cells. The plant cell has an advantage that the cell size is large and a large amount of porous crystals of the present embodiment can be contained.

As the animal cells, for example, mouse-derived cells, human cultured cells and the like can be used. As mouse-derived cells, 3T3 cells and the like can be used. As the rat-derived cell, bone cell UMR-106 or the like can be used. In addition, mammalian-derived cultured cells and insect-derived cultured cells can be used, and MEF, HeLa, 293-T, CHO, Jurkat, MCF-7, Vero cells, YAC-1, and the like can be considered. As the animal cells, not only the immortalized cultured cells but also cells of living tissues such as sperm, sperm cells, eggs or egg progenitor cells, primary cultured cells, tumor cells or diseased tissue-derived cells, which are difficult to transform, are used. You can also do it. Plant cells are generally known to be difficult transformative, algae cells (eg, petroleum-producing algae such as clamidmonas, euglena, or oranthiochitrium), and land plants (wheat, barley, oat wheat). , Grains such as soybeans or corn, fruit or vegetables such as tomato, eggplant, pumpkin, spinach, quinoa or hakusai, medicinal plants or industrial crops such as cotton, flax, switchgrass, sunflower, pine, paragom tree, urushi, auren or chosen carrot, Alternatively, fruit trees such as grapes, peaches, bananas, quinoa fruits, citrus fruits, and oysters) can be used. In addition, aspergillus or mushroom fungi, for which a gene transfer system does not exist in the prior art, can also be used. In addition, as the protoplast, the algae and the plant-derived mesophyll cells, hypocotyl cells, root cells, cells obtained from fungi and the like can be used.
As cells on biological tissues, epidermal cells, sheep meat cells, shoot apical meristems, callus or adventitious embryos on mature leaves, which are particularly difficult to introduce into genes, can be used, and quiescent center cells, pollen or embryo cells. Etc. can also be used.

In the cell introduction step, the means used for introducing the molecule into the cell and the conditions such as the number of cells may be appropriately selected, but in the present embodiment, the surface of the cell is perforated by a physical impact including crushing of atmospheric pressure. It is preferable to carry out the step of causing. An electric field-induced bubble method is used in which a voltage is applied to the bubble ejection member to generate bubbles, the membrane of the cells is perforated by a physical impact including crushing of the cells, and the porous crystals are introduced into the cells. Is particularly preferred.
The electric field-induced bubble method may be carried out by using the above-mentioned introduction device or other device described in, for example, Patent Document 1, but it is particularly preferable to use the above-mentioned introduction device and the device shown in FIG. .. When using cultured cells, it is particularly preferable to introduce them using the above-mentioned introduction device and the method of use which can be applied to the accumulated cells.
In the present embodiment, since the porous crystal 500 has a size on the order of several hundred nm to several μm as described above, the influence of gravity cannot be ignored, and the porous crystal 500 may sink in the solution. There is sex. In this case, for example, depending on the introduction method, it is expected that it will be difficult to introduce an appropriate number of porous crystals 500 into cells. According to the above-mentioned introduction device and the method of using the porous crystal 500, the porous crystal 500 is introduced into the cell surface by a physical impact, so that a certain number of the porous crystal 500 having a relatively large mass can be introduced into the cell. It can be done and is particularly suitable.

(Slow release process)
The method for introducing a polymer cell of the present embodiment further includes a step of introducing the porous crystal into cells and then slowly releasing the encapsulated polymer.
The step of slowly releasing the polymer enclosed in the porous crystal is not particularly limited as a means for separating the porous crystal and the polymer, but for example, a means for decomposing the structure of the porous crystal can be used. ..
By the step of slowly releasing the polymer enclosed in the porous crystal, the polymer is slowly released into the cells into which the polymer has been introduced, and the function can be exhibited. For example, when the polymer is Cas9-gRNA having a DNA editing function, the editing of intracellular DNA is started by sustained release. By this sustained release step, the timing at which the polymer exerts its function can also be controlled.

For this step, it is preferable that the porous crystal has a structure capable of reducing the bonding force between the molecules constituting the crystal. A structure in which the binding force between molecules can be attenuated is a structure in which the bonds of the molecules constituting the porous crystal can be weakened and the distance can be increased by adding an external factor. That is what the molecule does. For example, in the case of DNA nanoparticles, the structure is such that the bonds between the nanoparticles can be weakened, the distance can be increased, and the nanoparticles can be dispersed. Further, it is preferable that the external factor has a small influence on other molecules on the porous crystal in the cell.
In the present embodiment, a specific example of such a configuration is that when the porous crystal is a DNA nanoparticle, the DNA molecule has a sequence that decomposes or dissociates due to an external factor. In addition, examples of this external factor include light and enzymes.
Specifically, when the porous crystal is a DNA nanoparticle crystal including DNA molecules and nanoparticles, the sustained-release step uses means for decomposing or dissociating the DNA molecules constituting the porous crystal. be able to. At this time, the DNA molecules constituting the porous crystal may be cleaved to decompose the porous crystal. In addition, when the polymer is encapsulated in the porous crystal by the interaction between the polymer and the DNA of the porous crystal, even if the polymer is slowly released from the porous crystal by cutting the DNA molecule of the porous crystal. Good. In addition, when the polymer is encapsulated in the porous crystal by a specific interaction molecule in the DNA of the polymer and the porous crystal, a factor that cleaves the DNA molecule of the porous crystal or inhibits the specific interaction. The introduction may dissociate the polymer from the porous crystal.
As a means for decomposing or dissociating the DNA molecules constituting the porous crystal, the porous crystal is irradiated with light to cleave the porous crystal, or the porous crystal is cleaved by an enzyme. Is also preferable. For this means, the DNA nanoparticle crystal, which is a porous crystal, preferably has a photoresponsive functional group or has a nuclease recognition sequence, as described in detail below.

As a method of irradiating the porous crystal with light to decompose the porous crystal, a method of irradiating various types of light (electromagnetic waves), for example, ultraviolet rays, can be taken. When the porous crystal is composed of nanoparticles 51 and an intracrystal DNA molecule 52 and a о-nitrobenzyl group is added to a part of the intracrystal DNA, the intracrystal DNA molecule 52 can be cleaved by light. The structure of the porous crystal can be decomposed, and the polymer can be slowly released into the cell. Since the DNA molecule 52 in the crystal is cleaved by light, it is less likely to damage other molecules in the cell. As the functional group for decomposing the porous crystal by light, not only the o-nitrobenzyl group but also CNVK and the like can be used.

As a method for cleaving the porous crystal with an enzyme, an enzyme (nuclease) that cleaves DNA can be used. As the enzyme that cleaves DNA, an enzyme specific to or non-specific to specific DNA can be appropriately used. In particular, it is preferable to use a restriction enzyme specific for a specific sequence of the DNA molecule 52 in the crystal. By using a restriction enzyme specific for a specific sequence of the DNA molecule 52 in the crystal, only the specific sequence of the DNA molecule 52 in the crystal is cleaved, so that other molecules including other DNA in the cell are cleaved. Can be sustained-release without affecting. In addition, intracellular nucleases can be used as non-specific cleavage enzymes.

(Effect of the polymer cell introduction method of the present embodiment)
According to the method for introducing a polymer cell of the present embodiment, the function of the polymer is maintained, the cell viability is high, the polymer can be introduced into the cell with high efficiency, and the polymer can be introduced into the cell. It can be used for functional expression.

When introducing a polymer into cells, the present inventors introduce a polymer having a large exclusion volume into the cells, so that the cells are not irreversibly damaged as much as possible, and a large-sized perforation is performed without causing cell death in particular. We proceeded with research on a method for stably introducing polymers that can be excessively and efficiently inserted and are physically fragile, for example, a method for introducing these polymers without directly handling them. Then, as a means for introducing the polymer into cells without directly handling the polymer, it was examined to enclose the polymer in a physically stable structure. Therefore, we focused on the porous crystal of this embodiment as a target for encapsulating the polymer. By encapsulating the polymer in the porous crystal, the polymer having a large exclusion volume can be compactly enclosed in the crystal, and there is no need to perform an operation that imposes a heavy burden on the cells at the time of introduction, for example, perforation of an excessively large size. .. Since the polymer is encapsulated in the crystal, it is not necessary to directly handle the physically fragile polymer, and it is possible to perform an operation for introducing the polymer into the cell. In addition, the porous crystal can be subjected to an operation for encapsulating the polymer and an operation for sustained release. In particular, in the porous crystal which is a DNA nanoparticle crystal, the polymer can be released into the cell by means such as cutting the DNA without damaging the cell.

According to the polymer cell introduction method of the present embodiment, it is possible to control the length and arrangement of the DNA molecules forming the skeleton to create a crystal structure (superlattice structure) of nanoparticles of various sizes. The ability to arrange nanoparticles and design superlattice structures with nanoscale accuracy can greatly contribute to easy introduction technology. By encapsulating vulnerable long-chain DNA in a physically stable superlattice structure, a physical introduction method is possible, and the unit and crystal structure are controlled to control the physical rigidity. , Enables introduction to a wide range of biological objects.

According to the polymer cell introduction method of the present embodiment, the DNA sequence used in the DNA nanoparticle crystal introduced into the cell is decomposed by using a molecular biological tool including genome editing or a reversible photolinking reaction. Alternatively, by dissociating it, the long-chain DNA in the DNA nanoparticle crystal can be sustained-release and the function can be expressed. Crystals can be decomposed and cleaved in the process of isothermal or slight temperature rise, released inside the cell without destroying the long-chain DNA, and transported into the nucleus to make it functional in the cell.

Hereinafter, the present embodiment will be described in more detail based on Examples, but the present invention is not limited by these Examples, and various material changes, design changes, setting adjustments, etc. are made without departing from the gist of the present invention. Needless to say, is possible.

(Adjustment of cells)
The NIH / 3T3 cell solution cultured in OPTI-MEM medium for 48 hours was centrifuged in a container. An Eppendorf tube having a capacity of 1.5 ml, which is a microcentrifuge tube, was used as a container. The vessel, the number of cells per one container containing the cell suspension so that 0.25 ~ 3.0 × 10 ⁶ cells were centrifuged for 5 min at 1000rpm (200 × g). After aspirating and removing the OPTI-MEM medium solution, the final concentration of the pEGFP-N1 plasmid becomes 2.1 μg / ml and the total amount of the suspension becomes 7 μl with respect to the cell mass at the bottom of the container. And suspended again to give a cell suspension.

(Test Example 1)
(Evaluation of introduction efficiency: number of cells)
Using the apparatus of FIG. 1, a voltage was applied to the injector 20 to the bottom of the tube of the cell suspension to generate bubbles. The voltage was applied at 12 W and the exposure time was 600 ms. And when the number of cells were respectively 0.25,0.75,1.5,3.0 ⁽¹⁰⁶ ×, respectively), under the conditions of a cell number 1.0 × 10 ⁶ 48 hours stationary culture , FIG. 3 shows a comparative example in which lipofectamine treatment was performed without using an injector. The upper row is a fluorescence micrograph of cells into which the plasmid pEGFP-N1 has been introduced after 48 hours, and the lower row is a bright field (BF) micrograph. The transfection efficiency is shown graphically in FIG. The horizontal axis is the number of cells used ⁽¹⁰⁶ ×, respectively), the percentage of cells that vertical axis is introduced (%).
Using an injector on the bottom of the cell suspension tube increased efficiency with a large number of cells, but between 0.25 × 10 ⁶ and 0.75 × 10 ⁶ 1.5 × 10 was no significant difference in the ^six and 3.0 × 10 ⁶ between. That is, according to the introduction device and method of this example, it was shown that a certain efficiency can be obtained even if the concentration of the cells used is low.

(Test Example 2)
(Evaluation of introduction efficiency: Electric power)
Using the apparatus of FIG. 1, a voltage was applied to the injector 20 to the bottom of the tube of the cell suspension to generate bubbles. The tests were conducted with the electric power when applying the voltage as 4W, 6W, 8W, 10W, and 12W, respectively. The number of cells was carried out both at 1.5 × 10 ⁶ cells. A comparative example treated with lipofectamine and a comparative example in which the plasmid was not added to the suspension are shown in FIG. 5 (Test Example 2). The upper row is a fluorescence micrograph of cells into which the plasmid pEGFP-N1 has been introduced after 48 hours, and the lower row is a bright field (BF) micrograph. The exposure time is 600 ms.

Regarding the transfection efficiency per electric power, the ratio (%) of the introduced cells is shown in FIG. 6, and the number of cells is shown in FIG. (1) to (4) and the like represent the number of tests, and the value in the bar graph represents the average value of a plurality of measurements. The numbers on the horizontal axis (4 to 15) are the power at the time of injection, "I12" is the one to which the power of 12 W is applied without adding the plasmid as shown in FIG. 7, and "P" is the one to which the plasmid is added but the injection is performed. Those that have not been introduced, "L" refers to those that have been introduced by lipofectamine treatment. The highest transfection efficiency was shown when the power at the time of transfection was 12 W. The efficiency is high at 8 to 15W, but the efficiency at 15W is slightly lower than that at 12W.

(Test Example 3)
(Evaluation of introduction efficiency: expression)
FIG. 8 shows a micrograph of GFP and RFP staining and a bright field (BF) for the test example in which the power of Test Example 2 was set to 12 W and the test example was adjusted in the same manner as the one in which lipofectamine was introduced. The upper two rows show before the selection treatment with puromycin (exposure time 600 ms), and the lower two rows show after the selection treatment with puromycin (exposure time 2000 ms). The puromycin treatment was performed for 48 hours.
It was shown that the introduction by the introduction device of this example introduced GFP and RFP as well as puromycin and expressed normally.

(Test Example 4)
(Evaluation of plasmid damage)
For each lane 1 to 8, from a cell sample subjected to the same operation as in Test Example 1 except for the following conditions (lanes 1 to 3 were introduced into cells, and 5 to 8 were cell-free plasmids only). The plasmid was purified by centrifugation at 10 krpm, and the molecular weight of the plasmid was examined by agarose gel electrophoresis.
1: 20W, 30 times, cell number 1.5 × ¹⁰ 6, 15μg DNA
2: 12W, 30 times, cell number 1.5 × ¹⁰ 6, 15μg DNA
3: 4W, 30 times, cell number 1.5 × ¹⁰ 6, 15μg DNA
4: Marker (Gene Ladder Wide 2)
5: No introduction operation, 15 μg DNA
6: 4W, 30 times, no cells, 15 μg DNA
7: 12W, 30 times, no cells, 15 μg DNA
8: 12W, 30 times, no cells, 15 μg DNA

The results are shown in FIG. There is almost no difference in the pattern of DNA molecular weight distribution between lanes 1 to 3 where the introduction operation was performed, lane 5 where the introduction operation was not performed, and lanes 6 to 8 where the operation was performed in a cell-free container. It was.
If the plasmid is degraded through the introduction operation, a band with a low molecular weight (lower part of the figure) is strongly seen, and if a structural change occurs due to cleavage of the plasmid, a band will be generated at another location. However, no such change in band strength was observed in lanes 1 to 3 where the introduction operation was performed. From this result, it was shown that the cell introduction device and the introduction method of the present embodiment have a low risk of damaging the plasmid. Therefore, in the cell introduction device and introduction method of the present embodiment, it can be expected that macromolecules such as nucleic acids and proteins can be introduced into cells while maintaining their functions without changing their primary to higher-order structures.

(Test Example 5)
(Introduction to difficult-to-introduce cells: bone cells)
Bone cells UMR-106 were used, and the cells were introduced under the same conditions as in Test Example 1 with a power of 12 W. FIG. 10 shows a comparison of the cell culture time of 24 hours and 48 hours for the case of introduction with the above power of 12 W and the case of introduction with lipofectamine, respectively. The cell introduction method of the present embodiment succeeded in transfection of 10% or more at 24h and 20% or more at 48h, as in the case of lipofectamine, showing close values. From this result, it was clarified that the cell introduction method of the present embodiment can also be used for introduction into bone cells which are difficult-to-introduce cells.

(Test Example 6)
(Introduction to difficult-to-introduce cells: algae)
As the difficult-to-introduce cells, algae cells Chlamydomonas were used, and the cells were introduced at a power of 12 W.
Suspensions of cells were prepared by centrifugation so that the final concentration of 3 to 5 k Da fluorescein isothiocytoate (FITC) -dextran was 85 μg / μl and the total suspension volume was 7 μl. The number of cells that are in suspension in 7μl was adjusted 5.0 × 10 ⁶ cells become so.
FIG. 11 shows the results of the following three conditions.
Left figure) With dextran administration, without injection Middle figure) With dextran administration, with injection Right figure) With dextran administration, with injection, sample in which the suspension was thickened with the cell culture component TAP. It was clarified that the cell introduction method of the embodiment can also be used for introduction into algae, which is a difficult-to-introduce cell.

(Encapsulation of porous crystals and polymers)
(Test Example 7)
(Creation of biotin-modified porous crystals)
Biotin-modified DNA nanoparticle crystals were prepared as porous crystals.
A colloidal solution in which gold (Au) nanoparticles having a diameter of 10 nm are mixed and dispersed is mixed with a phosphate buffer solution containing a 33-base pair single-stranded DNA (DNA # 1) having a thiol group at the end of the DNA, and the nanoparticles are mixed. DNA-AuNP (A) modified with DNA # 1 on the surface was prepared. In the same manner, DNA-AuNP (B) modified with DNA # 2 of 33 base pairs was prepared.
DNAs # 1 and 2 have complementary bonds at the ends, but are designed so that the complementary bonds of DNA # 1 and the complementary bonds of DNA # 2 do not complement each other.
In addition, a 27-base pair single-stranded DNA (DNA # 3) that complementarily binds to the complementary binding portion of DNA # 1 and a 27-base pair single-stranded DNA (DNA #) that complementarily binds to the complementary binding portion of DNA # 2. 4) was prepared. A biotin molecule is bound to the 3'end of DNA # 3 and 4 via a C6 spacer.

Next, the two types of prepared DNA-AuNP (A) (B) and DNA # ^{3 and 4} were put into a phosphate buffer solution having a sodium ion concentration of 500 × 10 -3 mol / L and mixed.
When the DNA-nanoparticle dispersion is slowly cooled (when the temperature is gradually lowered), the complementary bonds are loosely bonded to each other and crystallize when the temperature reaches 40 to 53 ° C.
The dispersion is slowly cooled from 65 ° C. to 0.01 ° C./min to form DNA nanoparticle superlattice crystals (porous crystals) (Test Example 7) at room temperature (25 ° C.), and the porous crystals are contained in the solution. Agglomerates were formed.

(Encapsulation of streptavidin-modified quantum dots in porous crystals)
A streptavidin-modified quantum dot aqueous solution (QdotTM 585 Streptavidin Conjugate, Invitrogen, Cat #: Q10113MP) having a concentration of 1 μM was prepared. Quantum dots are composed of nanometer-scale semiconductor crystals (CdSe) coated on a semiconductor shell (ZnS). The streptavidin-modified quantum dot is a complex in which approximately 5 to 10 streptavidin are covalently bonded to the quantum dot. The diameter of the complex is 15-20 nm.
The porous crystals of Test Example 7 were mixed with the streptavidin-modified quantum dot aqueous solution.
When the porous crystals were mixed with the aqueous solution and incubated for several hours, the streptavidin-modified quantum dot aqueous solution was retained in the pores of the porous crystals. FIG. 12A shows a crystal (SYBR-Safe), FIG. 12B shows a quantum dot (Q-dot) (fluorescence), and FIG. 12C shows a confocal microscope image of superposition of a crystal and a quantum dot. FIGS. 12A to 12C were photographed using an FV3000 (manufactured by Olympus Corporation). FIG. 12D shows the fluorescence profile of the quantum dots on the line in FIG. 12C. The positions of the fluorescence indicating the streptavidin-modified quantum dot aqueous solution and the porous crystal stained with SYBR-Safe correspond to each other. In addition, fluorescence derived from quantum dots was detected not only on the surface of the crystal but also on the inner part. Therefore, it was shown that the streptavidin-modified quantum dot aqueous solution was retained in the porous crystal.
From the size of DNA # 1- # 4, the length of a piece of unit cell (distance between the centers of nanoparticles) is considered to be about 40 nm. Therefore, it is considered that the porous crystal of Test Example 7 has a size sufficient to hold a polymer having a size of several nm to several tens of nm.

(Test Example 8)
(Cas9-gRNA encapsulation in porous crystals)
For Cas9-gRNA molecule (approximately 7.5 nm in diameter), it is complementary to 6 sequences of DNA # 5 and gRNA of 33 base pairs having a sequence complementary to 4 sequences of this gRNA (approximately 5.5 nm). A 27-base pair DNA # 6 having a sequence was prepared. In addition, a 33-base pair DNA # 7 having a sequence complementary to 18 sequences of DNA # 6 was prepared.
DNA # 5 and DNA # 7 were thiol-modified at the ends and bound to Au nanoparticles (10 nm in diameter on the product label) in the same manner as in Test Example 7, and DNA-AuNP (C) and DNA-AuNP (D) were obtained, respectively. Created.
When this DNA-AuNP forms a unit cell and retains the Cas9-gRNA molecule, DNA # 5 and # 6 complementarily bind to the gRNA as shown in FIG. 13, and DNA # 6 is bound to DNA # 6. 7 is combined. Since DNA # 5 and # 7 are bound to Au nanoparticles, the distance between Au nanoparticles can be adjusted by the length of DNA # 5 to # 7 and the length of the complementary sequence, and the size of the lattice unit. Can be adjusted.

The porous crystal (Test Example 8) in which the DNA-AuNP (C) (D) formed a cubic unit cell was incubated in an aqueous solution having a Cas9-gRNA concentration of 0.1 μM for 1 hour, and Cas9 was incorporated into the crystal. -GRNA was retained. The interparticle distance of this crystal was determined by SAXS (Small Angle X-ray Scattering) measurement. The length of one side of the unit cell (distance between the centers between nanoparticles) was about 40 nm.

(Cas9-gRNA encapsulation in porous crystals)
The DNA nanoparticle crystal and Cas9 protein were labeled with different fluorescent dyes, and the encapsulation of the protein in the crystal was observed three-dimensionally. The DNA # 6 in the DNA nanoparticle crystal is TAMRA-modified and can be detected at an excitation wavelength of 555 nm and a fluorescence wavelength of 580 nm. Cas9 is modified with GFP and can be detected at an excitation wavelength of 488 nm and a fluorescence wavelength of 509 nm.

FIG. 14a shows a crystal (TAMRA), b shows Cas9 (fluorescence), and c shows a micrograph in a bright field. Further, an enlarged view of one of the porous crystals is shown in d for crystals and e for Cas9. The superposition of d and e is shown in f. From the figure of f, it was shown that the signals of the crystal and Cas9 almost overlapped, and Cas9 was enclosed in the porous crystal.

(Test Example 9)
(Examination of Cas9-gRNA binding to porous crystals)
Regarding the binding property of Cas9-gRNA to the porous crystal, especially regarding gRNA, considering that the binding property to the crystal may be different if the sequence contained in RNP is different, the AML5 sequence (SEQ ID NO: 1) and the AtPDS3 sequence Each gRNA targeting (SEQ ID NO: 2) was prepared and examined for binding.
AML5 target sequence: 5'-CGAGACAACTTGAGTATGAG-3'
AtPDS3 target sequence: 5'-GCCTGACCGCCGACCATGGC-3'

RNP: Alt-R CRISPR-Cas9 tracrRNA, ATTO 550 (IDT, Cat #: 1075928) and crRNA (AML5 or AtPDS3, IDT) are mixed with RNase-free water to a final concentration of 10 μM, and mixed for 2 minutes 95. It was heated at ° C. and gradually cooled (10 μM gRNA). 10 μM gRNA was mixed with a 25 μM Cas9-GFP protein solution (Cas9-GFP Protein, SIGMA, Cat #: CAS9GFPRO-50UG) in a SEC buffer to a final concentration of 2.5 μM (2.5 μM RNP). ).
As the DNA nanoparticle crystal, the one of Test Example 8 was used. As a comparative example, 1.0 μm gold particles (BioRad 165-2263) in a microcarrier package for PDS / Helios were used as the gold particles to be encapsulated instead of RNP.

1 μL of DNA nanoparticle crystal or gold particle and 0.5 μL of 2.5 μM RNP were mixed, and 250 mM NaCl 1x Phos buffer was added to adjust to 20 μL. After pipetting, the mixture was allowed to stand for 1 hour, centrifuged in a desktop centrifuge, and the supernatant was discarded. 250 mM NaCl 1x Phos buffer 20 μL was added again for pipetting, centrifugation was performed with a desktop centrifuge, and the supernatant was discarded. This operation was repeated once again, and finally 250 mM NaCl 1x Phos buffer 20 μL was added. 2 μL was sucked by pipetting so as to contain crystals or gold particles, and the mixture was dropped onto a slide glass for microscopic observation.

FIG. 15 shows a state in which gold particles and DNA nanoparticle crystals were mixed with a fluorescent label RNP and observed under a microscope. Bright Field is a diagram in which bright field is detected, and Cas9-GFP is a diagram in which GFP is detected by fluorescence.
No fluorescence derived from RNP was detected in the gold particles, but fluorescence was observed in the DNA nanoparticle crystals. Therefore, it was considered that the DNA nanoparticle crystal has a stronger RNP-binding force than the gold particle.

(Test Example 10)
(Examination of intracellular introduction of porous crystals)
We investigated whether porous crystals could be introduced into Arabidopsis protoplasts. As the DNA nanoparticle crystal of the fluorescent molecule TAMRA label, the one of Test Example 8 was used. As a protoplast of Arabidopsis thaliana, mesophyll cells of Arabidopsis thaliana 3-4 weeks after germination were used.
5 μL of DNA nanoparticle crystals labeled as fluorescent molecule TAMRA were mixed with 5 μL of 25 mM Tris-HCl 5 μL. A 100 μL protoplast (number of cells: 5.0 × 10 ⁴ cells) was prepared there. Microscopic observation was performed 3 hours after the introduction of the molecule using a normal PEG method.

A photomicrograph of a protoplast introduced with TAMRA fluorescent label crystals is shown in FIG. Each figure is a four-time example of the same cell under the same conditions. The arrows in the figure indicate the bright spots of TAMRA fluorescence.
Fluorescence derived from TAMRA was observed in the cells, although the number of cells was very small. In addition, it is expected that the bright spots in the cells are inside the cells, as some photographs show that the bright spots in the cells move due to cytoplasmic streaming.

(Test Example 11)
(Intracellular introduction of porous crystals using a particle gun device)
We investigated whether porous crystals could be introduced into Arabidopsis leaves using a particle gun device. As the DNA nanoparticle crystal, the one of Test Example 8 was used. Mature leaves of Arabidopsis thaliana 3-4 weeks after germination were used.
5 μL of DNA nanoparticle crystals were dried on a rupture disk attached to a particle gun device (BioRad particle gun PDS-1000 / He system) and introduced by a normal protocol (pressure 1100 ps). Microscopic observation was performed 1 hour after the introduction.

The results are shown in Fig. 17. The arrows in the figure indicate the porous crystals present in the leaves of Arabidopsis thaliana. It was revealed that DNA nanoparticle crystals are ejected by wind pressure and carried into plant cells by the particle gun method.

(Test Example 12)
(Confirmation of genome editing by intracellular introduction of macromolecules)
It was confirmed whether genome editing occurs when a DNA nanoparticle crystal to which RNP is bound is introduced into a cell.
As RNP, Alt-R CRISPR-Cas9 tracrRNA (IDT, Cat #: 10725333) and crRNA (AML5 crRNA, IDT) are mixed with RNase-free water to a final concentration of 10 μM, and heated at 95 ° C. for 2 minutes. Then, it was gradually cooled (10 μM gRNA). 10 μM gRNA was mixed with a 25 μM Cas9-GFP protein solution (Cas9-GFP Protein, SIGMA, Cat #: CAS9GFPRO-50UG) in a SEC buffer to a final concentration of 2.5 μM (2.5 μM RNP). did).
As the DNA nanoparticle crystal, the same one as in Test Example 8 was used.
Arabidopsis protoplasts used Arabidopsis mesophyll cells 3-4 weeks after germination.

5 μL of DNA nanoparticle crystal and 5 μL of 2.5 μM RNP were mixed, and 1 x Cas9 buffer was added to adjust to 20 μL. After pipetting, the mixture was allowed to stand for 1 hour, centrifuged in a desktop centrifuge, and the supernatant was discarded. 20 μL of 1x Cas9 buffer was added again for pipetting, centrifugation was performed with a desktop centrifuge, and the supernatant was discarded. This operation was repeated once again, and finally adjusted to 1x Cas9 buffer 10 μL. As a positive control, the supernatant (RNP only) of the first step in the above step was used, and as a negative control, simply 1x Cas9 buffer 10 μL was used for introduction. The prepared RNP-bound DNA nanoparticle crystal was introduced into a protoplast by the PEG method and allowed to stand for 3 days. The genome was extracted from protoplast by isopropanol extraction, and the gene was amplified using KOD FX Neo Kit (TOYOBO) and the following primers. The amplified gene product was analyzed with a Miseq sequencer (Illumina) using Miseq nano Kit v3 (500 cycles).

Primer information is as follows.
AML5_amplicon_F (5'-3') :( SEQ ID NO: 3)
TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGACGGCTGTTTCTCGCACAAACA
AML5_amplicon_R (5'-3') :( SEQ ID NO: 4)
GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGCAACTGTTCCTGCACCATTTGG

The analysis results are shown in FIGS. 18A to 18C. 7 sequences from the one in which RNP was introduced in FIG. 18A (SEQ ID NO: 5 to 11), 10 sequences from the one in which the RNP-encapsulated crystal was introduced in FIG. 18B (SEQ ID NO: 12 to 21), and negative in FIG. 18C. As a control (comparative example), 10 sequences (SEQ ID NOs: 22 to 31) are shown from those in which only colloidal crystals are introduced. In FIGS. 18A to 18C, the leftmost number corresponds to the sequence number.
In the reads analyzed by Amplicon-seq, the introduction of positive control and RNP-encapsulated nanoparticle crystals shows a mutation sequence (deletion or insertion in Arabidopsis thaliana) that appears to be derived from genome editing near the target site of genome editing. ) Was detected (indicated by the frame in the figure). On the other hand, no mutant sequence was found in the negative control. Therefore, it was suggested that genome editing can be induced by introducing RNP-bound DNA nanoparticle crystals into cells.

(Test Example 13)
(Enzymatic decomposition of DNA nanoparticle crystals)
A test was conducted in which DNA nanoparticle crystals were enzymatically decomposed.
As the TAMRA fluorescent label DNA nanoparticle crystal, the same DNA nanoparticle crystal as in Test Example 8 was used, but the following sequences 12-1 (SEQ ID NO: 32), 12-2, 12-3 were used as DNA inside. A DNA sequence of (SEQ ID NO: 33) having a double strand was used.
Sequence 12-1 5'-CATCCATCCTTATCAACT-3'
Sequence 12-2 5'-AAGGAA-3'
Array 12-3 5'-AGGTGAGTATGAGTCGTT-3'
BccI, XhoI, and DpnI (NEB) were prepared as restriction enzymes.

1 μL of DNA nanoparticle crystals, 10 x CutSmart buffer, and 1 μL of restriction enzymes (BccI, XhoI, or DpnI) were added to the PCR tube, and the mixture was adjusted to 10 μL with MilliQ water. After pipetting, the mixture was allowed to stand at 37 ° C. for 6 hours and centrifuged in a tabletop centrifuge. 1 μL was taken from the bottom of the tube, dropped on a slide glass, and observed with a fluorescence microscope.

FIG. 19 shows a photomicrograph of TAMRA fluorescently labeled DNA nanoparticle crystals treated with restriction enzymes at 37 ° C. for 6 hours. The upper row is fluorescence and the lower row is bright field. Arrows are typical DNA nanoparticle crystals. The results of A: BccI treatment, B: XhoI treatment, and C: DpnI treatment are shown.
Among the restriction enzymes tested, BccI is the only enzyme having a recognition site in the sequence (SEQ ID NOs: 5 to 7) of the DNA nanoparticle crystal. Degradation of TAMRA fluorescently labeled DNA nanoparticle crystals occurred with BccI alone. From this result, it was shown that the DNA nanoparticle crystal can be decomposed by an enzyme and the encapsulated polymer can be released slowly.

(Test Example 14)
(Photodegradation of DNA nanoparticle crystals)
A test was conducted in which DNA nanoparticle crystals were decomposed by light.
The same DNA nanoparticle crystal as in Test Example 8 was used, but a sequence containing a pc-linker (o-nitrobenzyl group) inside was used as the DNA. The above-mentioned pc-linker was added to the DNA corresponding to DNA # 6 of Test Example 8 and used.

Crystals containing a pc-linker were dropped on a slide glass and irradiated with ultraviolet rays under a fluorescence microscope.

FIG. 20 shows micrographs of DNA nanoparticle crystals before and after being irradiated with ultraviolet rays for 1 hour. The left row is before irradiation, and the lower row is after irradiation. Arrows indicate DNA nanoparticle crystals decomposed by light.
From this result, it was shown that the DNA nanoparticle crystal can be decomposed by light and the encapsulated polymer can be released slowly.

From Test Examples 7 to 14, the DNA nanoparticle crystals used in these tests can be bound to a genome editing factor, the DNA nanoparticle crystals can be introduced into cells, and the DNA nanoparticle crystals can be used. It was shown that the bound RNP can cause genome editing of plant cells and that DNA nanoparticle crystals can be degraded by enzymes and light.

(Test Example 15)
(Introduction of protein into plant cells by electric field-induced bubble method)
The introduction of proteins into plant cells was verified by the electric field-induced bubble method.
As the introduction device, the same device as in FIG. 1 was used, and the introduction was performed in the same manner as in Test Example 1 except that the cells were matured leaf cells. The mature leaves of Arabidopsis thaliana were used, and the female was approached at an angle to the leaves using a manipulator. The position of the scalpel tip and the sample was adjusted using a general manipulator for the cells and introduced.
As a power supply device (pulse generation source), Hypercator2000 (TypeA, TypeB) (Commed Japan Co., Ltd.) and an electric pulse output device (CFB) (Bex Co., Ltd.) were used. Hybridator 2000 is a high frequency that emits a pulse every 5 ms for both Type A and Type B, and CFB is a low frequency that emits a pulse every 0.5 ms for about 5 μs.
As a target to be introduced, RNP is a protein and has a volume almost equal to that of GFP. Therefore, in this experiment, an experiment was conducted to introduce the GFP protein. As the GFP protein used for introduction, sfGFP-NLS (270 amino acids, molecular weight 30637) in which Nuclear localization signal (NLS) was added to superfolder GFP (sfGFP) was expressed in Escherichia coli.

Protein was applied to the mature leaves (back) of Arabidopsis thaliana while changing parameters such as applied voltage (or energy), distance from the tip of the female to the sample, sample concentration, and power supply (voltage was applied). .. After introducing the protein, the leaves were washed with a buffer for 1 hour to wash off the GFP protein bound to the surface of the leaves. Then, observation was performed 1 to 24 hours later.
Installation conditions:
Target: Arabidopsis mate leaves
Energy: 3W
Distance: ~ 25 μm
Solution: 1 / 2MS + 2% Mannitol
Delivered: sfGFP-NLS protein, 750 ng / μL (6 hours after introduction, the nucleus glows)
Wash: 1mL of 1 / 2MS + 2% Mannitol for 1hour
Power supply: Hyfrecator 2000 or CFB

FIG. 21 shows a micrograph of sfGFP-NLS introduced in Hyprecator Type B, LO mode, taken 24 hours later. The upper and lower rows show the detection results of GFP fluorescence, the bright field, and their superposition, and the lower row is a larger view (detection of migration to the nucleus) than the upper row.
FIG. 22 shows a photomicrograph taken after 1 hour of sfGFP-NLS introduced in Hypercator Type A, LO mode. It shows ultraviolet (UV) and GFP fluorescence (GFP 500 ms and GFP 50 ms). Lower rows A and B show further enlarged views of the locations indicated by the upper rows of fluorescence signals A and B.
When Hyprecator A and B, which emit high-frequency pulse voltage, were used as a power source, it was observed that the GFP protein was introduced into plant cells. On the other hand, when a low-frequency CFB was used, many introduction experiments were performed, but no introduction was observed.
From the above results, it was shown that the GFP protein can be introduced into the leaves of plants by the electric field-induced bubble method, and that a high-frequency pulse voltage is required for the introduction. It was shown that the polymer cell introduction method of the present embodiment enables the introduction of macromolecules into animal cells and plant cells with less irreversible damage to cells (less toxicity and prevention of cell death). ..

(Reference example)
FIG. 23a shows a diagram in which DNA nanoparticle crystals were treated with restriction enzymes under the same conditions as in Test Example 13. In both cases, the crystals that can be confirmed in the target area in the left figure at the points indicated by the arrows disappear in the restriction enzyme treatment area in the right figure, indicating that the DNA nanoparticle crystals are decomposed by the restriction enzymes. Was done.
FIG. 23b shows a graph in which DNA nanoparticle crystals were fluorescently labeled and visualized under the same conditions as in Test Example 13. The left figure is a view in which fluorescence of GFP is detected, and the right figure is a figure in which fluorescence of GFP is detected. It is shown that crystals that can be confirmed in the bright field are fluorescently labeled, and smaller crystals indicated by arrows can also be confirmed by the fluorescence label.
FIG. 23c shows a diagram in which the fluorescently labeled DNA nanoparticle crystal of Test Example 13 was introduced into protoplasts under the same conditions as in Test Example 10. The left figure is a bright field, and the right figure is a figure in which fluorescence of GFP was detected, and it was shown that a fluorescence-labeled DNA nanoparticle crystal was introduced as shown by an arrow.

According to the embodiment of the present invention, the function of the polymer is maintained, the survival rate of the cell is high, the polymer can be introduced into the cell with high efficiency, and the polymer is used for the expression of the function in the cell. It is possible to obtain an introduction device, a cell introduction method, and a method for producing a DNA nanoparticle crystal and a DNA nanoparticle crystal enclosure.

20 Injector 21 Storage unit 22 Liquid supply unit 30 Container 40 Power supply means 41 Power supply device 42 Counter electrode 43 Wire wire 50 Unit lattice 50A DNA nanoparticles Crystal enclosure 51 Nanoparticles 52 Intracrystal DNA molecules 53 Pore 60 Polymer 100 Introduction device 500 porous crystals

Claims (24)

1. An introduction device that perforates the surface of a cell by a physical impact including crushing of bubbles and introduces a polymer into the cell.
   A storage unit for storing the solution containing the polymer and
   It has a liquid feeding unit for feeding the solution toward the surface of the perforated cell and introducing the polymer into the cell.
   When introducing the polymer into the cells, the introduction device is performed on the bottom of the container containing the solution containing the cells.
2. The introduction device according to claim 1, wherein the introduction is performed on a cell mass formed at the bottom of the container by centrifuging the solution containing the cells in the container.
3. The introduction device according to claim 1 or 2, wherein the solution containing the cells has a shear viscosity of 0.01 Pa · s or more at a shear rate of 10.0 / s or less.
4. The introduction device according to any one of claims 1 to 3, wherein the container containing the solution containing the cells has a weight-like bottom shape in the container.
5. The introduction device according to any one of claims 1 to 4, wherein the introduction device includes a cell perforation portion that applies a voltage to a bubble ejection member to generate bubbles.
6. The introduction device according to any one of claims 1 to 5, wherein the polymer is enclosed in a porous crystal.
7. The introduction device according to claim 6, wherein the porous crystal is a DNA nanoparticle crystal composed of a nanomaterial and a DNA molecule.
8. The introduction device according to claim 7, wherein after introducing the porous crystal into the cell, the encapsulated polymer is slowly released.
9. The introduction device according to any one of claims 1 to 8, wherein the polymer contains a nucleic acid molecule (DNA molecule, RNA molecule, artificial nucleic acid), protein, and polyamide having a molecular weight of 1000 or more.
10. The introduction device according to claim 9, wherein the polymer is Cas9 or a long-chain DNA molecule.
11. The encapsulation process of encapsulating the polymer in the porous crystal and
    The cell introduction step of introducing the porous crystal into the cell and
    A method for introducing macromolecular cells.
12. The method for introducing a polymer cell according to claim 11, wherein the cell introduction step is a step of perforating the surface of cells by a physical impact including crushing of atmospheric pressure.
13. The porous crystal is introduced into the bubble ejection member, and the porous crystal is introduced into the bubble ejection member.
    A voltage is applied to the bubble ejection member to generate bubbles, and the bubbles are generated.
    The twelfth aspect of claim 12, wherein the porous crystal is introduced into the cell by an electric field-induced bubble method in which the membrane of the cell is perforated by a physical impact including crushing of the cell and the porous crystal is introduced into the cell. Method of introducing high molecular weight cells.
14. The method for introducing a polymer cell according to claim 13, wherein when the porous crystal is introduced into the cell, the porous crystal is introduced into a cell mass formed at the bottom of a container containing the solution containing the cell. ..
15. The method for introducing a polymer cell according to any one of claims 11 to 14, wherein the porous crystal is a DNA nanoparticle crystal containing a nanomaterial and a DNA molecule.
16. The method for introducing a polymer into cells according to any one of claims 11 to 15, wherein the polymer contains a nucleic acid molecule (DNA molecule, RNA molecule, artificial nucleic acid), protein, and polyamide having a molecular weight of 1000 or more.
17. The method for introducing a polymer into cells according to claim 16, wherein the polymer is a protein.
18. The cell introduction of the polymer according to any one of claims 11 to 17, further comprising a sustained release step of cutting the porous crystal and slowly releasing the polymer from the porous crystal into the cell. Method.
19. The method for introducing a polymer cell according to claim 18, wherein the sustained-release step is a method for introducing a polymer cell according to claim 18, wherein the porous crystal is irradiated with light to cut the porous crystal, or the porous crystal is cut by an enzyme.
20. A DNA nanoparticle crystal containing nanomaterials and DNA molecules.
    A DNA nanoparticle crystal modified with a molecule having a specific interaction function that interacts with a specific molecule.
21. The DNA nanoparticle crystal according to claim 20, wherein the molecule or particle can be retained inside the DNA nanoparticle crystal by the specific interaction.
22. The DNA nanoparticle crystal according to claim 20 or 21, wherein the specific interaction is an interaction between biotin and another molecule.
23. A method for producing a DNA nanoparticle crystal inclusion body, wherein the inclusion target modified with the specific molecule is encapsulated in the DNA nanoparticle crystal according to any one of claims 20 to 22.
24. A DNA nanoparticle crystal containing nanomaterials and DNA molecules.
    The DNA nanoparticle has a structure capable of diminishing the binding force between the molecules constituting the DNA nanoparticle crystal, and the structure is such that the DNA molecule has a photoresponsive functional group or has a nuclease recognition sequence. Particle crystal.

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