JP2006166884A - Method for introducing substance toward cell - Google Patents

Method for introducing substance toward cell Download PDF

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JP2006166884A
JP2006166884A JP2004382462A JP2004382462A JP2006166884A JP 2006166884 A JP2006166884 A JP 2006166884A JP 2004382462 A JP2004382462 A JP 2004382462A JP 2004382462 A JP2004382462 A JP 2004382462A JP 2006166884 A JP2006166884 A JP 2006166884A
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substance
needle
cell
bond
shaped material
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JP4625925B2 (en
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Chikashi Nakamura
史 中村
Shigeo Kan
成雄 韓
Ikuo Obataya
育夫 小幡谷
Tokuyuki Nakamura
徳幸 中村
Atsushi Miyake
淳 三宅
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National Institute of Advanced Industrial Science and Technology AIST
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for introducing a substance to be introduced into a cell with a low invasiveness to the cell and in a good efficiency. <P>SOLUTION: This method for introducing the substance into the cell is provided by using a nanometer scale ultra fine needle-like material, capable of making a damage against the cell as extremely small and surely performing the introduction of a gene, protein, etc., into the cell. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

本発明は、超極細の針状材料を用いた被導入物質を細胞内に導入する方法に関する。  The present invention relates to a method for introducing a substance to be introduced into a cell using an ultrafine needle-like material.

従来、細胞内への遺伝子、タンパク質等の導入には、マイクロインジェクション法、エレクトロポレーション法、パーティクルガン法等の物理学的方法が利用されてきた。例えば、マイクロインジェクション法であれば、ガラス針を細胞に穿刺して被導入物質を注入することができる。しかしながら、例えば、動物細胞にマイクロインジェクション法を用いて導入操作を行った場合、操作後およそ９０％の確率で細胞が死に至る。すなわち３種の遺伝子を連続的に導入した場合、成功する確率はわずか０．１％である。このように、これらの既存の代表的な細胞操作技術は、細胞に多大なダメージを与えるため、被導入物質が導入されても細胞が死んでしまい、導入された細胞の生存状態及び生理状態が悪いことが問題となっていた。  Conventionally, physical methods such as microinjection, electroporation, and particle gun methods have been used to introduce genes, proteins, and the like into cells. For example, in the microinjection method, a substance to be introduced can be injected by puncturing a cell with a glass needle. However, for example, when an introduction operation is performed on an animal cell using a microinjection method, the cell dies with a probability of approximately 90% after the operation. That is, when three genes are introduced continuously, the probability of success is only 0.1%. As described above, these existing typical cell manipulation techniques cause a great deal of damage to the cells, so that even if the substance to be introduced is introduced, the cells die, and the introduced cells have a living state and physiological state. The bad thing was a problem.

また、例えば、特許文献１には、針状物に遺伝子又は遺伝子発現に関与する物質を固定化して細胞内に導入し、針状物に固定化状態で細胞内に保持して機能発現を行う方法が開示されている。しかしながら、この方法では、遺伝子又は遺伝子発現に関与する物質は針状物に固定されたままであり、針を抜去しても遺伝子等は細胞内に恒常的に保持されないため、遺伝子組換体を作製することができない。  Also, for example, in Patent Document 1, a gene or a substance involved in gene expression is immobilized on a needle-like material and introduced into the cell, and is retained in the cell in a state of being immobilized on the needle-like material to perform functional expression. A method is disclosed. However, in this method, a gene or a substance involved in gene expression remains fixed to the needle-like material, and even if the needle is removed, the gene or the like is not permanently retained in the cell, so a gene recombinant is prepared. I can't.

このような背景から、標的細胞にダメージを与えることなく、細胞内に被導入物質を残留させることが可能な導入方法が望まれている。
特開２００３−３２５１６１ From such a background, an introduction method capable of allowing a substance to be introduced to remain in a cell without damaging the target cell is desired.
JP 2003-325161 A

本発明は、被導入物質を中間的な結合力で針状材料に結合し、この針状材料を細胞内に穿刺した際に、被導入物質が残留することを特徴とする、被導入物質を細胞内に導入する方法を提供することを主な目的とする。  The present invention relates to an introduced substance, wherein the introduced substance is bonded to the needle-shaped material with an intermediate binding force, and the introduced substance remains when the needle-shaped material is punctured into a cell. The main purpose is to provide a method for introduction into cells.

本発明者らは、針状材料に中間的な結合力で被導入物質を結合させることによって、針状材料を細胞に穿刺し、抜去る際に被導入物質を針状材料から分離させて細胞内に残留させることが可能なことを見出し本発明を完成するに至った。ここで、中間的な結合力とは、被導入物質を結合させた針状材料を細胞に穿刺した際、少なくとも針状材料が細胞内に入るまでは保持され、細胞内において細胞内に存在する物質との相互作用又は、針状材料を細胞から抜去する際の抵抗によって針状材料から分離する、細胞内の環境変化、細胞内物質との反応によって分離する、外部からの間接的刺激によって分離する程度の結合力を意味する。  The present inventors bind the substance to be introduced to the needle-like material with an intermediate binding force, thereby puncturing the needle-like material into the cell and separating the substance to be introduced from the needle-like material when removing the cell. The present invention has been completed. Here, the intermediate binding force means that when the needle-like material to which the substance to be introduced is bound is punctured into the cell, it is retained at least until the needle-like material enters the cell, and exists in the cell within the cell. Separation from needle-like materials by interaction with substances or resistance when needle-like materials are removed from cells, separation by environmental changes in cells, reaction with intracellular substances, separation by external indirect stimulation It means the degree of binding.

本発明は、以下の、被導入物質を細胞内に導入する方法を提供する。
項１．針状材料に被導入物質を結合する工程、該針状材料を細胞に穿刺する工程、針状材料を細胞から抜去して該被導入物質を細胞内に残留させる工程を含む、細胞内に該被導入物質を導入する方法。
項２．２以上の被導入物質を連続して又は一定の時間間隔をおいて細胞内に導入することを特徴とする項１に記載の方法。
項３．針状材料の直径が、８００ｎｍ以下である項１又は２に記載の方法。
項４．針状材料と被導入物質間の結合が、静電気的結合、疎水性相互作用による結合、キレート結合、細胞内で切断され得る共有結合、光切断リンカーを介した結合、酵素切断リンカーを介した結合及び特異的結合からなる群より選択される少なくとも１種である、項１〜３に記載の方法。
項５．被導入物質が、遺伝子、タンパク質、糖類、多糖、脂肪酸、コレステロール、脂質、シグナル伝達物質、リガンド物質、ホルモン物質、サイトカイン、イオン、金属粒子、磁性微粒子、無機化合物、量子ドット、有機化合物及び薬剤からなる群より選択される少なくとも１種、ないしこれらの複合体である、項１〜４に記載の方法。
項６．遺伝子が、核酸、核酸様物質、ＤＮＡ、ＲＮＡ、プラスミド、ウイルス粒子、染色体である項１〜４に記載の方法。
項７．タンパク質が、アミノ酸、オリゴペプチド、ポリペプチド、マルチサブユニットタンパク質である項１〜４に記載の方法。
項８．被導入物質が、リポソーム封入体、シクロデキストリン包接体又はゲルに内包もしくは包埋される形態である請求項１〜７に記載の方法。
項９．被導入物質を、細胞内で分離可能に結合してなる針状材料。
項１０．針状材料と被導入物質間の結合が、静電気的結合、疎水性相互作用による結合、キレート結合、細胞内で切断され得る共有結合、光切断リンカーを介した結合、酵素切断リンカーを介した結合及び特異的結合からなる群より選択される少なくとも１種である、項９に記載の針状材料。
項１１．被導入物質を、必要に応じてスペーサー物質を介して細胞内で分離可能な結合により針状材料に結合させる工程を含む、被導入物質を結合させた針状材料の製造方法。
項１２．針状材料と被導入物質間の結合が、静電気的結合、疎水性相互作用による結合、キレート結合、細胞内で切断され得る共有結合、光切断リンカーを介した結合、酵素切断リンカーを介した結合及び特異的結合からなる群より選択される少なくとも１種である、項１１に記載の方法。The present invention provides the following method for introducing a substance to be introduced into a cell.
Item 1. A step of binding an introduced substance to the needle-shaped material, a step of puncturing the needle-shaped material into the cell, and a step of removing the needle-shaped material from the cell to leave the introduced substance in the cell. A method of introducing a substance to be introduced.
Item 2. The method according to Item 1, wherein the substance to be introduced of Item 2.2 or more is introduced into a cell continuously or at regular time intervals.
Item 3. Item 3. The method according to Item 1 or 2, wherein the needle-shaped material has a diameter of 800 nm or less.
Item 4. The binding between the needle-shaped material and the substance to be introduced is an electrostatic bond, a bond by hydrophobic interaction, a chelate bond, a covalent bond that can be cleaved in a cell, a bond through a photocleavable linker, a bond through an enzyme cleaved linker Item 4. The method according to Item 1 to 3, which is at least one selected from the group consisting of specific binding.
Item 5. Substances to be introduced include genes, proteins, saccharides, polysaccharides, fatty acids, cholesterol, lipids, signaling substances, ligand substances, hormone substances, cytokines, ions, metal particles, magnetic particles, inorganic compounds, quantum dots, organic compounds, and drugs Item 5. The method according to Item 1 to 4, which is at least one selected from the group consisting of these or a complex thereof.
Item 6. Item 5. The method according to Items 1 to 4, wherein the gene is a nucleic acid, a nucleic acid-like substance, DNA, RNA, a plasmid, a virus particle, or a chromosome.
Item 7. Item 5. The method according to Items 1 to 4, wherein the protein is an amino acid, an oligopeptide, a polypeptide, or a multi-subunit protein.
Item 8. The method according to any one of claims 1 to 7, wherein the substance to be introduced is in a form of being encapsulated or embedded in a liposome inclusion body, a cyclodextrin inclusion body or a gel.
Item 9. A needle-shaped material formed by binding a substance to be introduced so as to be separable within a cell.
Item 10. The binding between the needle-shaped material and the substance to be introduced is an electrostatic bond, a bond by hydrophobic interaction, a chelate bond, a covalent bond that can be cleaved in a cell, a bond through a photocleavable linker, a bond through an enzyme cleaved linker Item 10. The acicular material according to Item 9, which is at least one selected from the group consisting of specific binding.
Item 11. A method for producing an acicular material to which an introduced substance is bound, comprising a step of binding the introduced substance to the acicular material by a bond that can be separated in a cell via a spacer substance as necessary.
Item 12. The binding between the needle-shaped material and the substance to be introduced is an electrostatic bond, a bond by hydrophobic interaction, a chelate bond, a covalent bond that can be cleaved in the cell, a bond via a photocleavable linker, a bond via an enzyme cleaving linker Item 12. The method according to Item 11, wherein the method is at least one selected from the group consisting of specific binding.

（１）針状材料
本発明において超極細の針状材料とは、細長い形状を有するものを指し、細胞に対する侵襲性が低いものであれば特に限定されない。本発明の針状材料には、例えば、円柱形、円錐形等、細胞に穿刺することが可能なものであれば、先端が鋭利でないものも含まれる。また、支持体（例えば、原子間力顕微鏡の探針等）と本発明の針状材料が一体化されたものも含まれる。針状材料は、先端部と根本側で直径に差がない円柱形の方が細胞に対する侵襲性がより低く、導入効率を高められることから好ましい。(1) Needle-like material In the present invention, the ultrafine needle-like material refers to one having an elongated shape, and is not particularly limited as long as it has low invasiveness to cells. The needle-shaped material of the present invention includes, for example, a cylindrical shape, a conical shape, etc., as long as it can puncture cells, and the tip is not sharp. Moreover, the support body (for example, the probe of an atomic force microscope etc.) and the needle-like material of the present invention are integrated. A cylindrical material having no difference in diameter between the distal end portion and the root side is preferred because the invasiveness to cells is lower and the introduction efficiency is improved.

針状材料の材質としては、酸化ニッケル、石英、シリカ、ダイヤモンド等の無機物；金、銀、銅、白金、アルミニウム等の金属；シリコン結晶、ジルコニウム、チタン、タングステン等の金属結晶；酸化亜鉛等の金属酸化物；シリコン、窒化シリコン、ガラス、プラスチック等、細胞に毒性のない物質であればいずれも使用でき、針状の固体材料であることが好ましい。  As the material of the needle-shaped material, inorganic materials such as nickel oxide, quartz, silica and diamond; metals such as gold, silver, copper, platinum and aluminum; metal crystals such as silicon crystal, zirconium, titanium and tungsten; zinc oxide and the like Metal oxides: Any substance that is not toxic to cells, such as silicon, silicon nitride, glass, and plastic, can be used, and a needle-like solid material is preferable.

針状材料が円柱形である場合、その直径は、約８００ｎｍ以下、好ましくは約６００ｎｍ以下、より好ましくは約４００ｎｍ以下である。針状材料が円柱形でない場合は、円に換算した直径に従って製造することができる。この様な針状材料は、細胞に対して充分小さいため、穿刺時に細胞にダメージを与えることがない。また、直径４００ｎｍ以下の針状材料であれば、約１時間の穿刺に、ほぼ全ての動物細胞は耐えることが可能である。導入する細胞、細胞内に針状材料を穿刺している時間等によっては、針状材料の直径が、およそ８００ｎｍまでであれば細胞を傷つけることなく導入操作を行うことができ、１０００ｎｍ程度であっても細胞の生存に影響を与えない場合がある。針状材料は、細すぎると被導入物質を充分量結合させることが困難であるため、１００ｎｍ程度、好ましくは２００ｎｍ程度を下回らないことが望ましい。  When the acicular material is cylindrical, its diameter is about 800 nm or less, preferably about 600 nm or less, more preferably about 400 nm or less. When the acicular material is not cylindrical, it can be manufactured according to a diameter converted into a circle. Since such a needle-like material is sufficiently small with respect to the cells, the cells are not damaged at the time of puncturing. In addition, with a needle-like material having a diameter of 400 nm or less, almost all animal cells can withstand puncture for about 1 hour. Depending on the cell to be introduced and the time during which the needle-shaped material is punctured into the cell, the introduction operation can be performed without damaging the cell if the diameter of the needle-shaped material is up to about 800 nm, which is about 1000 nm. May not affect cell survival. If the needle-shaped material is too thin, it is difficult to bind a sufficient amount of the substance to be introduced. Therefore, it is desirable that the needle-shaped material should not be less than about 100 nm, preferably less than about 200 nm.

また、針状材料のアスペクト比は、２：１〜５０：１程度、好ましくは５：１〜５０：１程度、より好ましくは１０：１〜５０：１程度、さらに好ましくは２０：１〜４０：１程度である。  The aspect ratio of the acicular material is about 2: 1 to 50: 1, preferably about 5: 1 to 50: 1, more preferably about 10: 1 to 50: 1, and further preferably 20: 1 to 40. : 1.

針状材料の強度は、細胞に穿刺した際に十分維持される程度の強度を有していればよい。  The strength of the needle-shaped material only needs to be strong enough to be maintained when the cells are punctured.

上記に針状材料の材質として例示される物質を、集束イオンビーム加工装置等を用いたビーム照射によるエッチング、成型時に使用する型の窪み部分を針状の構造にする等の従来公知の方法によって加工し、本発明の針状材料とすることができる。  The substances exemplified above as the material of the needle-like material are etched by a conventionally known method such as etching by beam irradiation using a focused ion beam processing apparatus or the like, and forming a hollow portion of a mold used at the time of molding. It can process and can be set as the acicular material of this invention.

また、針状材料は、表面を化学的に修飾できるものが好ましい。化学修飾としては、例えば、アミノ基等の官能基を針状材料の表面に出すようにシラン化剤等を用いることによって修飾することで、親水化することができる。また、チオール基を導入することによって、アミノ基との架橋剤を用い、スペーサー物質を針状材料に結合することもできる。これらの化学修飾は、従来公知の方法に従って行うことができる。  The needle-shaped material is preferably one that can chemically modify the surface. As the chemical modification, for example, the modification can be performed by using a silanizing agent or the like so that a functional group such as an amino group is brought out on the surface of the needle-shaped material. Further, by introducing a thiol group, a spacer substance can be bonded to the needle-like material using a crosslinking agent with an amino group. These chemical modifications can be performed according to a conventionally known method.

（２）被導入物質の針状材料上への固定
本発明の方法では、必要に応じて針状材料の針表面上に被導入物質と結合する物質（以下、スペーサー物質と呼ぶことがある）を結合させ、さらに、これに被導入物質を結合させることによって、針状材料上に被導入物質を固定する。また、先に被導入物質とスペーサー物質を結合させておき、それを針状材料上に固定化してもよい。可能であれば、被導入物質と針状材料を直接結合してもよい。針状材料上のスペーサー物質と被導入物質の結合は、穿刺操作に伴う物理的な力によって細胞外で容易に分離しない程度の結合を意味する。(2) Immobilization of substance to be introduced on needle-shaped material In the method of the present invention, a substance that binds to the substance to be introduced on the needle surface of the needle-shaped material as needed (hereinafter sometimes referred to as spacer substance) Then, the substance to be introduced is fixed on the needle-like material by further joining the substance to be introduced to this. Alternatively, the substance to be introduced and the spacer substance may be bonded first and then immobilized on the needle-shaped material. If possible, the substance to be introduced and the acicular material may be directly bonded. The binding between the spacer substance and the substance to be introduced on the needle-like material means a binding that is not easily separated outside the cell by a physical force accompanying the puncturing operation.

被導入物質を針状材料状に固定した態様のモデルを、図１に示す。図１中（ｉ）は被導入物質、（ｉｉ）はスペーサー物質、（ｉｉｉ）は結合を表す。図中、被導入物質とスペーサー物質との比は、１：１で表記しているが、組み合わせによって変動する。多数のスペーサー物質に導入物質が１分子結合する場合もあれば、１分子のスペーサー物質に、被導入物質が多数結合する場合もある。図１（ａ）は、被導入物質を直接、針状材料に固定した場合を示す。図中スペーサー物質は、１種（ｂ）２種（ｃ）の場合を示しており、その数は細胞内に導入が可能であれば特に限定されないが、好ましくは０〜５、より好ましくは０〜２である。  A model of an embodiment in which the substance to be introduced is fixed in the shape of a needle-like material is shown in FIG. In FIG. 1, (i) represents a substance to be introduced, (ii) represents a spacer substance, and (iii) represents a bond. In the figure, the ratio of the substance to be introduced and the spacer substance is shown as 1: 1, but varies depending on the combination. There are cases where one molecule of the introduced substance binds to a large number of spacer substances, and cases where a large number of substances to be introduced bind to a spacer substance of one molecule. FIG. 1A shows a case where the substance to be introduced is directly fixed to the needle-shaped material. In the figure, the spacer substance shows the case of 1 type (b) and 2 types (c), and the number thereof is not particularly limited as long as it can be introduced into cells, but it is preferably 0 to 5, more preferably 0. ~ 2.

（ｉ）被導入物質
被導入物質は、生理活性を有する物質（細胞に影響を与える可能性がある物質）であれば特に限定されず、核酸、核酸様物質、ＤＮＡ、ＲＮＡ、プラスミド、ウイルス粒子、染色体等の天然遺伝子又は非天然の遺伝子；アミノ酸、オリゴペプチド、ポリペプチド、マルチサブユニットタンパク質等の天然又は非天然タンパク質；糖類、多糖、脂肪酸、コレステロール、脂質、シグナル伝達物質、リガンド物質、ホルモン物質、サイトカイン、イオン、金属粒子、磁性微粒子、無機化合物、量子ドット、有機化合物、薬剤等の少なくとも１種ないしこれらの複合体等、本発明の針状材料にスペーサー物質を介して又は直接結合することが可能であり、細胞内に導入され得る程度に充分小さいものであれば、あらゆる物質が考えられる。また、核酸を構成する塩基又はタンパク質を構成するアミノ酸は、天然、非天然、修飾の有無を問わず、使用することができる。さらに、例えば、毒物等の細胞内に入りにくいと考えられている物質でも、導入することが可能である。(I) Substance to be introduced The substance to be introduced is not particularly limited as long as it is a physiologically active substance (a substance that may affect cells). Nucleic acid, nucleic acid-like substance, DNA, RNA, plasmid, virus particle Natural or non-natural genes such as chromosomes; natural or non-natural proteins such as amino acids, oligopeptides, polypeptides, multi-subunit proteins; sugars, polysaccharides, fatty acids, cholesterol, lipids, signaling substances, ligand substances, hormones At least one kind of substance, cytokine, ion, metal particle, magnetic fine particle, inorganic compound, quantum dot, organic compound, drug, etc. or a complex thereof, or the like is bonded to the needle-shaped material of the present invention via a spacer substance or directly. Any substance can be considered as long as it is small enough to be introduced into the cell. It is done. Moreover, the base which comprises a nucleic acid, or the amino acid which comprises a protein can be used regardless of the presence or absence of natural, non-natural, and modification. Furthermore, for example, it is possible to introduce a substance that is considered to be difficult to enter the cell, such as a poison.

また、複数種の被導入物質を１つの針状材料に結合させる、あるいは、被導入物質を混合して針状材料に結合させることによって、単一細胞に対して一度に多数の物質を導入することが可能である。  In addition, multiple substances can be introduced into a single cell at a time by binding multiple types of substances to be introduced to one needle-like material, or by mixing the substances to be introduced and binding them to the needle-like material. It is possible.

また、下記（ｉｉ）に述べるスペーサー物質である、リポソーム封入体等の内部に溶液相を内包させることができる構造物、シクロデキストリン包接体等の包接化合物、あるいは外部刺激によって容易にゾル化が可能なゲル材料等、内包型のスペーサー物質に１分子以上の被導入物質を内包させて、針状材料の表面に固定化し、導入操作を行ってもよい。例えば、リポソーム封入体等の場合は、細胞内に導入後、破壊刺激を外部より与えることによって、膜の擾乱により内容物を放出させることができる。また、シクロデキストリン包接体等の包接化合物を用いた場合は、外部刺激に応答する開閉機構を利用して内容物を放出させることができる。また、ゲルを用いた場合は外部刺激によりゾル化させ、包埋した内容物を放出させることが出来る。図１（ｄ）に示すように内包型スペーサー物質直接針状材料表面に結合するものと、図１（ｅ）に示すように、他の１種ないし複数種のスペーサー物質を介して針状材料にこれら内包型スペーサー物質が針状材料へ固定化するものがある。  In addition, the spacer substance described in (ii) below is a structure capable of encapsulating a solution phase inside a liposome inclusion body, an inclusion compound such as a cyclodextrin inclusion body, or easily solylated by external stimulation. One or more molecules to be introduced may be encapsulated in an encapsulated spacer material such as a gel material that can be immobilized and immobilized on the surface of the needle-shaped material, and the introduction operation may be performed. For example, in the case of liposome inclusion bodies or the like, the contents can be released by disturbance of the membrane by applying a disruptive stimulus from the outside after introduction into the cells. In addition, when an inclusion compound such as a cyclodextrin inclusion body is used, the contents can be released using an opening / closing mechanism that responds to external stimuli. Moreover, when a gel is used, it can be made into a sol by an external stimulus and the embedded contents can be released. As shown in FIG. 1 (d), the inclusion type spacer substance directly binds to the surface of the needle-like material, and as shown in FIG. 1 (e), the needle-like material passes through one or more kinds of spacer substances. In some cases, these inclusion type spacer substances are fixed to the needle-shaped material.

（ｉｉ）スペーサー物質
本発明において、スペーサー物質は、被導入物質又は別のスペーサー物質を特異的又は非特異的に結合する物質、あるいはリポソーム、シクロデキストリン、ゲル等の被導入物質を結合せず中に閉じこめる物質を指す。また、針状材料とスペーサー物質を結合する物質もスペーサー物質に含まれるものとする。スペーサー物質を介さずに被導入物質を直接、針状材料に固定する場合もあり、複数個を必要とする場合もある。スペーサー物質の種類は、被導入物質の種類に応じて適宜選択され得る。(Ii) Spacer substance In the present invention, the spacer substance is a substance that specifically or non-specifically binds a substance to be introduced or another spacer substance, or a substance that does not bind a substance to be introduced such as a liposome, cyclodextrin, or gel. It refers to the substance confined in In addition, a substance that binds the needle-shaped material and the spacer substance is also included in the spacer substance. In some cases, the substance to be introduced is directly fixed to the needle-like material without using a spacer substance, and a plurality of substances may be required. The type of the spacer substance can be appropriately selected according to the type of the substance to be introduced.

スペーサー物質としては、例えば、ポリアルギニン、ポリリジン等のカチオン性ペプチド；ポリエチレンイミン、ポリプロピレンイミン、ポリ（アミドアミン）、イミダゾールポリペプチド等のカチオン性ポリマー等のカチオン性被覆材料；ジンクフィンガー等の特異的に遺伝子配列を認識するタンパク質モチーフ等；ジチオスレイトール、システイン、チオール基含有ペプチド等のジスルフィド結合を形成し得るスペーサー物質；光切断リンカー、酵素切断リンカ−等があげられる。  Examples of spacer substances include cationic peptides such as polyarginine and polylysine; cationic coating materials such as cationic polymers such as polyethyleneimine, polypropyleneimine, poly (amidoamine) and imidazole polypeptide; and zinc fingers and the like. Examples include protein motifs that recognize gene sequences; spacer substances capable of forming disulfide bonds such as dithiothreitol, cysteine, and thiol group-containing peptides; photocleavable linkers, enzyme-cleaved linkers, and the like.

特異的に遺伝子配列を認識するタンパク質モチーフをスペーサー物質とする際に、被導入物質（ＤＮＡ断片、プラスミド等）が結合配列を有さない場合は、従来公知の遺伝子に従って、タンパク質モチーフが認識する配列を挿入して用いればよい。  When a protein motif that specifically recognizes a gene sequence is used as a spacer substance, and the introduced substance (DNA fragment, plasmid, etc.) does not have a binding sequence, the sequence recognized by the protein motif according to a conventionally known gene Can be used.

また、特異的に被導入物質等を結合する場合は、被導入物質を認識する抗体、ペプチド抗体、単鎖抗体、ファージ抗体等ならびに、これらの抗体の派生物等の抗体様物質がスペーサー物質として利用できる。また、スペーサー物質の組み合わせとして、例えば、アビジン−ビオチン、プロテインＡ−ＩｇＧ、レセプター−リガンド等を用いることができる。  In addition, when specifically binding a substance to be introduced, antibody-like substances such as antibodies, peptide antibodies, single chain antibodies, phage antibodies, etc. that recognize the substance to be introduced, and derivatives of these antibodies are used as spacer substances. Available. Moreover, as a combination of spacer substances, for example, avidin-biotin, protein A-IgG, receptor-ligand and the like can be used.

以下に、シリコン結晶製の針状材料にスペーサー物質を結合する場合を例にとり、説明する。他の材質の針状材料を用いる場合であっても、当業者であれば、以下の例を参考に被導入物質を結合させた針状材料を調製することができる。  Hereinafter, a case where a spacer substance is bonded to a needle-shaped material made of silicon crystal will be described as an example. Even when other needle-shaped materials are used, those skilled in the art can prepare a needle-shaped material to which a substance to be introduced is bound with reference to the following example.

２％シラン化剤３−Ｍｅｒｃａｐｔｏｐｒｏｐｙｌｔｒｉｍｅｔｈｏｘｙｓｉｌａｎｅ（ＭＰＴＥＳ）、２％ Ｈ２Ｏを含むエタノール溶液を１時間接触させることによって針状材料表面にチオール基を導入し、チオール基とアミノ基の架橋剤であるＥＭＣＳ（Ｎ−（６−Ｍａｌｅｉｍｉｄｏｃａｐｒｏｙｌｏｘｙ）ｓｕｃｃｉｎｉｍｉｄｅ）を使用して、抗体、ペプチド等のアミノ基を針状材料表面のチオール基に結合させる。次に、１μＭ ＥＭＣＳを含むジメチルホルムアミドに３０分間接触させることにより、スクシンイミド基を表面に提示させる。続いて、別のスペーサー物質であるポリリジン、抗体等を含む溶液を１時間程度接触させる。2% silanating agent 3-Mercaptopropyltrimethylsilane (MPTES), 2 hours H 2 O containing ethanol solution is brought into contact with each other for 1 hour to introduce a thiol group on the surface of the needle-like material, and EMCS, which is a crosslinking agent for thiol group and amino group Using (N- (6-Maleimidocaproyloxy) succinimide), an amino group such as an antibody or a peptide is bonded to a thiol group on the surface of the needle-shaped material. Next, succinimide groups are presented on the surface by contacting with dimethylformamide containing 1 μM EMCS for 30 minutes. Subsequently, a solution containing another spacer substance, polylysine, antibody, etc. is contacted for about 1 hour.

（ｉｉｉ）結合
針状材料上におけるスペーサー物質と被導入物質間の結合の種類は、被導入物質を細胞内に残留させることができれば、任意のものを用いることができ、例えば、静電気的結合、疎水性相互作用による結合、キレート結合、細胞内で切断され得る共有結合、光切断リンカーを介した結合、酵素切断リンカーを介した結合、固有の物質の組み合わせによる特異的結合（アビジン−ビオチン、抗原−抗体、プロテインＡ−抗体、レセプター−リガンド等）、細胞内で切断されない共有結合、（疎水性又は親水性の）吸着等があげられ、被導入物質の種類によって適宜選択することができる。(Iii) Bonding Any kind of bond between the spacer substance and the substance to be introduced on the needle-like material can be used as long as the substance to be introduced can be left in the cell. For example, electrostatic binding, Binding by hydrophobic interaction, chelate bond, covalent bond that can be cleaved intracellularly, bond through photocleavable linker, bond through enzyme cleaved linker, specific bond by combination of unique substances (avidin-biotin, antigen -Antibodies, protein A-antibodies, receptor-ligands, etc.), covalent bonds that are not cleaved in cells, (hydrophobic or hydrophilic) adsorption, and the like, which can be appropriately selected depending on the type of the substance to be introduced.

また、細胞内において被導入物質の分離が可能であるならば、これらの結合の複数を任意に組み合わせて、針状材料上に被導入物質を固定してもよい。さらに、被導入物質の量、結合の種類等に応じて、被導入物質が細胞内において針状材料から分離・残留するように、適宜、結合の強弱を調節することが好ましい。例えば、抗原−抗体による特異的結合を利用する場合には、抗体の結合力を弱くしておき、抗原（被導入物質）が細胞内で分離されやすいようにしておくことが好ましい。  Further, if the introduced substance can be separated in the cell, the introduced substance may be fixed on the needle-like material by arbitrarily combining a plurality of these bonds. Furthermore, it is preferable to appropriately adjust the strength of the binding so that the introduced substance is separated / residual from the needle-like material in the cell according to the amount of the introduced substance, the type of binding, and the like. For example, when specific binding by an antigen-antibody is used, it is preferable to weaken the binding force of the antibody so that the antigen (substance to be introduced) is easily separated in the cell.

本発明において好ましい結合の種類としては、例えば、静電気的結合、キレート結合、疎水性相互作用による結合、細胞内で切断され得る共有結合、光切断リンカーを介した結合、酵素切断リンカーを介した結合等があげられる。  Preferred types of bonds in the present invention include, for example, electrostatic bonds, chelate bonds, bonds by hydrophobic interactions, covalent bonds that can be cleaved in cells, bonds via photocleavable linkers, bonds via enzyme cleaved linkers Etc.

（ａ）静電気的結合（図６参照）
静電気的結合は、弱い結合であるため、細胞内で容易に被導入物質を分離することができる。(A) Electrostatic coupling (see Fig. 6)
Since the electrostatic bond is a weak bond, the introduced substance can be easily separated in the cell.

一般的に、静電気的結合にはカチオン性ポリマーを使用する。フリーな状態のカチオン性ポリマーを、単純に細胞と混合させた場合は、細胞膜の表面（通常、負電荷を帯びている）に正電荷物質が吸着し、膜構造の擾乱によって、物質を導入することができる。この時、膜構造の擾乱は、すなわち、膜電位を維持できないことを意味し、結果として細胞死を招く。しかしながら、針状材料にカチオン性ポリマーを結合させて穿刺する場合には、針表面にカチオン性ポリマー（正電荷物質）が固定化されているため、膜との相互作用は局所的にしか起こらないものと考えられる。また、負電荷物質がさらにその表面に結合している場合には、電荷的に中和、もしくは負電荷よりになっている。従って、カチオン性ポリマーをスペーサー物質として用いても、細胞に対する悪影響は極めて小さいものと期待される。これは、疎水性相互作用による結合の場合も同様である。  Generally, a cationic polymer is used for electrostatic bonding. When a cationic polymer in a free state is simply mixed with cells, a positively charged substance is adsorbed on the surface of the cell membrane (usually negatively charged), and the substance is introduced by disrupting the membrane structure. be able to. At this time, disturbance of the membrane structure means that the membrane potential cannot be maintained, resulting in cell death. However, when puncturing with a cationic polymer bonded to the needle-shaped material, the cationic polymer (positively charged substance) is immobilized on the surface of the needle, so that the interaction with the membrane occurs only locally. It is considered a thing. In addition, when a negatively charged substance is further bonded to the surface, it is neutralized in terms of charge or has a negative charge. Therefore, even if a cationic polymer is used as a spacer substance, the adverse effect on cells is expected to be extremely small. The same applies to the case of binding by hydrophobic interaction.

（ｂ）キレート結合（図２の１．参照）
キレート結合を用いた場合、例えば、図２の１．に示されるものであれば、細胞内に存在するキレート物質で容易に置換され、被導入物質が分離する。(B) Chelate bond (see 1. in FIG. 2)
When a chelate bond is used, for example, 1. in FIG. If it is shown in (2), it is easily replaced with a chelating substance present in the cell, and the introduced substance is separated.

（ｃ）細胞内で切断され得る共有結合（図２の２．参照）
ジスルフィド結合（Ｓ−Ｓ結合）等の細胞内で切断され得る共有結合によって針状材料上に被導入物質を固定化した場合、細胞内が還元状態であることから、共有結合は容易に還元され被導入物質が針状材料から分離する。(C) Covalent bond that can be cleaved intracellularly (see 2 in FIG. 2)
When a substance to be introduced is immobilized on a needle-like material by a covalent bond that can be cleaved in a cell such as a disulfide bond (SS bond), the covalent bond is easily reduced because the cell is in a reduced state. The substance to be introduced separates from the acicular material.

（ｄ）光切断リンカーを介した結合（図２の３．参照）
光切断リンカーを介した結合の場合、被導入物質を固定化した針状材料を細胞内に穿刺した後、リンカーと被導入物質間の結合を切断することが可能であって細胞に悪影響を与えない程度の光を照射することにより、リンカーと被導入物質間の結合を切断することができる。(D) Bonding via a photocleavable linker (see 3 in FIG. 2)
In the case of binding via a photocleavable linker, the needle-like material with the introduced substance immobilized thereon can be punctured into the cell, and then the bond between the linker and the introduced substance can be cleaved, adversely affecting the cell. The bond between the linker and the substance to be introduced can be cleaved by irradiating the light to a certain extent.

（ｅ）酵素切断リンカーを介した結合（図２の４．参照）
酵素切断リンカーを介した結合の場合、細胞内には、例えばエステラーゼ等の酵素が豊富に存在するため、エステラーゼが認識する配列を有する公知のリンカーを使用することで、リンカーと被導入物質間の結合は容易に切断され得る。(E) Binding via an enzyme-cleaving linker (see 4 in FIG. 2)
In the case of binding via an enzyme-cleaving linker, for example, an enzyme such as esterase is abundant in the cell. Therefore, by using a known linker having a sequence recognized by esterase, the linker and the substance to be introduced can be used. The bond can be easily cleaved.

被導入物質が本来目的とする生理活性を有している限り、被導入物質が分離される部位は特に限定されず、被導入物質にスペーサー物質が結合された状態で針状材料から分離され、細胞内に残留してもよい。  As long as the introduced substance has the originally intended physiological activity, the site where the introduced substance is separated is not particularly limited, and is separated from the needle-like material in a state where the spacer substance is bound to the introduced substance, It may remain in the cell.

針状材料に結合させる被導入物質の量は、特に限定されず、その種類及び結合力（付着力）、針状材料の材質、導入する細胞種等によって異なる。例えば、遺伝子、タンパク質等の高分子であれば１〜１０８分子程度、好ましくは１０３〜１０５分子程度である。また、低分子であれば、その分子量に応じて、より多くの分子を結合させることができる。The amount of the substance to be introduced to be bound to the needle-shaped material is not particularly limited, and varies depending on the type and binding force (adhesive force), the material of the needle-shaped material, the cell type to be introduced, and the like. For example, in the case of macromolecules such as genes and proteins, the number is about 1 to 10 8 molecules, preferably about 10 3 to 10 5 molecules. Moreover, if it is a low molecule, more molecules can be combined according to the molecular weight.

（３）細胞
本発明の針状材料を用いて被導入物質を導入する細胞としては、動物、植物を問わず真核生物由来のものがあげられ、細胞の種類は特に限定されない。また、細胞の直径は、１０〜１００μｍ程度、好ましくは１０〜５０μｍ程度、より好ましくは２０〜３０μｍ程度であることが望ましい。酵母等の細胞***が活発な細胞であれば、短時間で大量の遺伝子組換え体を作製することが可能である。(3) Cells The cells into which the substance to be introduced is introduced using the needle-shaped material of the present invention include those derived from eukaryotes regardless of animals or plants, and the type of cells is not particularly limited. The cell diameter is desirably about 10 to 100 μm, preferably about 10 to 50 μm, and more preferably about 20 to 30 μm. If cells such as yeast are active in cell division, a large amount of gene recombinants can be produced in a short time.

本発明によれば、例えば、ｉｎ ｖｉｔｒｏで維持されている組織片の単一細胞に対して導入操作を行い、被導入物質が導入された細胞が、周囲の細胞又は組織全体に対してどのような影響を及ぼすのかを観察することも可能である。  According to the present invention, for example, an introduction operation is performed on a single cell of a tissue piece that is maintained in vitro, and how a cell into which a substance to be introduced is introduced is introduced to surrounding cells or the entire tissue. It is also possible to observe whether it has a significant effect.

（４）細胞に被導入物質を導入する方法
本発明の方法は、被導入物質を結合した針状材料を細胞に穿刺し、細胞内で被導入物質が針状材料から分離し、残留させることによって導入を達成するものである。本発明において、被導入物質を針状材料から分離するとは、被導入物質が針状材料から分かれること、又は細胞内で放出されることを指し、切断、解離等の分離に至る過程は問わない。(4) Method of Introducing Substance to be Introduced into Cell In the method of the present invention, the needle-like material to which the substance to be introduced is bound is punctured into the cell, and the substance to be introduced is separated from the needle-like material and remains in the cell. To achieve the introduction. In the present invention, separating the substance to be introduced from the needle-shaped material means that the substance to be introduced is separated from the needle-shaped material or released inside the cell, and any process that leads to separation such as cutting and dissociation is not considered. .

（ｉ）標的細胞の固定
針状材料の穿刺操作を行うに当たり、標的となる細胞を動かないように固定することが好ましい。このような手段としては、例えば、付着性の細胞の場合はポリリジン、コラーゲン等で被覆した基板表面で培養又は該基板に吸着させる等の方法があげられる。(I) Fixation of target cell In performing the puncturing operation of the needle-shaped material, it is preferable to fix the target cell so as not to move. As such means, for example, in the case of adherent cells, there is a method of culturing on the surface of a substrate coated with polylysine, collagen or the like, or adsorbing to the substrate.

吸着性に乏しい細胞であれば、細胞膜結合性の高い両親媒性ポリマー等で被覆した基板に接触させることで固定化することが可能である。また、細胞の大きさによってはキャピラリによる吸引によって固定することもできる。  If the cell has poor adsorptivity, it can be immobilized by contacting it with a substrate coated with an amphiphilic polymer having high cell membrane binding properties. Further, depending on the size of the cell, it can be fixed by suction with a capillary.

さらに、針状材料の貫通できる薄さの厚みを有し、貫通用の小孔を有した薄膜材料と基板によって細胞懸濁液を挟み、細胞位置を固定することも可能である。薄膜材料には、例えば、ミリング、エッチングにより加工されたシリコン等のウエハや、薄く劈開したマイカ、伝導性薄膜Ｈｉｇｈｌｙ Ｏｒｉｅｎｔｅｄ Ｐｙｒｏｌｙｔｉｃ Ｇｒａｐｈｉｔｅ等を使用することができる。小孔は集束イオンビームや光リソグラフィによって、１〜１５μｍ程度、好ましくは１〜１０μｍ程度の大きさに調整したものが望ましい。  Furthermore, it is also possible to fix the cell position by sandwiching the cell suspension between the thin film material having a small thickness for penetrating the needle-shaped material and the substrate and the substrate. As the thin film material, for example, a wafer made of silicon or the like processed by milling or etching, a thinly cleaved mica, a conductive thin film Highly Pyrolytic Graphite, or the like can be used. The small holes are preferably adjusted to a size of about 1 to 15 μm, preferably about 1 to 10 μm by a focused ion beam or optical lithography.

また、ｉｎ ｖｉｔｒｏで維持されている組織片の単一細胞に対して導入操作を行う場合、使用する組織片として、生物体から取り出したもの（初代細胞）又は培養したものを使用することができる。初代細胞を用いる場合は、適当な基板上に組織片のスライスを乗せて導入操作を行うことができる。培養組織の場合は組織状になるように、例えば、ポリマー材料等の専用のスキャホールド（足場材料）を使用して固定することができる。  Moreover, when performing introduction | transduction operation | movement with respect to the single cell of the tissue piece currently maintained in vitro, what was taken out from the living body (primary cell) or what was cultured can be used as a tissue piece to be used. . When primary cells are used, the introduction operation can be performed by placing a slice of a tissue piece on an appropriate substrate. In the case of a cultured tissue, it can be fixed using, for example, a dedicated scaffold (scaffold material) such as a polymer material so as to form a tissue.

（ｉｉ）針状材料の穿刺／抜去
針状材料の穿刺は、細胞に位置を合わせ、針状材料を機械的に移動させることによって行う。光学顕微鏡により視認可能なサイズ、形状、材質の針状材料であれば、例えば、機械式、電気駆動式の微作動可能なマニピュレーター等によって穿刺操作を行い、顕微鏡観察によって針状材料の穿刺を確認することができる。(Ii) Needle-like material puncture / withdrawal Needle-like material is punctured by aligning the cells and mechanically moving the needle-like material. If it is a needle-shaped material of size, shape, and material that can be visually recognized by an optical microscope, for example, a puncture operation is performed with a mechanical or electrically driven micro-actuable manipulator, and the puncture of the needle-shaped material is confirmed by microscopic observation. can do.

また、細胞内への針状材料の穿刺を確認する他の方法としては、例えば、原子間力顕微鏡（ＡＦＭ）を使用した針状材料の穿刺検知方法があげられ、針状材料の細胞への接触、穿刺及び引き抜きの各状態における微小な力の変化を測定することで確認してもよい。
細胞に針状材料を穿刺する場合、穿刺の深さは、針状材料の先端から１〜５０μｍ程度、好ましくは１〜２０μｍ程度である。また、針状材料を穿刺する場所は、導入物質の種類や目的に応じて選択することが好ましく、例えば、ｍＲＮＡを導入する場合には、細胞質中に残留するように、穿刺場所を調節することが好ましい。Another method for confirming the puncture of the needle-shaped material into the cell is, for example, a method for detecting the puncture of the needle-shaped material using an atomic force microscope (AFM). You may confirm by measuring the micro force change in each state of a contact, puncture, and extraction.
When the needle-like material is punctured into the cell, the depth of puncture is about 1 to 50 μm, preferably about 1 to 20 μm from the tip of the needle-like material. In addition, it is preferable to select the location for puncturing the needle-shaped material according to the type and purpose of the introduced substance. For example, when mRNA is introduced, the puncture location should be adjusted so that it remains in the cytoplasm. Is preferred.

細胞に針状材料を穿刺する際には、３７℃、５％ＣＯ２の細胞培養環境を維持して操作することが好ましい。穿刺時間が短い場合には、室温、大気環境下で操作することが可能である。When puncturing a needle-like material into a cell, it is preferable to operate while maintaining a cell culture environment at 37 ° C. and 5% CO 2 . When the puncture time is short, it can be operated at room temperature and in an atmospheric environment.

細胞内に針状物を穿刺した後、被導入物質が針状材料から分離して細胞内に残留するように、約１秒以上、好ましくは約１０秒以上、針状物を細胞内に保持する。本発明の方法であれば、細胞に与えるダメージが極めて小さく、細胞を殺すことがないため、保持時間の上限は特に限定されないが、５分程度、好ましくは３分程度である。細胞内に針状材料を保持する時間は、被導入物質と固定化材料間の結合の種類に応じて、当業者が適宜設定することができる。  After puncturing the needle-like material into the cell, the needle-like material is held in the cell for about 1 second or more, preferably about 10 seconds or more so that the substance to be introduced is separated from the needle-like material and remains in the cell. To do. According to the method of the present invention, the damage given to the cells is extremely small and the cells are not killed. Therefore, the upper limit of the holding time is not particularly limited, but is about 5 minutes, preferably about 3 minutes. The time for holding the acicular material in the cell can be appropriately set by those skilled in the art depending on the type of bond between the substance to be introduced and the immobilization material.

針状材料を細胞内に穿刺し、一定時間おいた後、必要に応じて、針状材料上のスペーサー物質と被導入物質の結合を切断する処理を行う。具体的な切断処理は、上記（２）に記載の通りである。  The needle-shaped material is punctured into the cell, and after a certain period of time, a treatment for cutting the bond between the spacer substance on the needle-shaped material and the substance to be introduced is performed as necessary. The specific cutting process is as described in (2) above.

その後、針状物を細胞から針状材料を抜去することによって、被導入物質が細胞内に残留し、目的の導入細胞を得ることができる。本発明の方法によれば、物質の導入に使用される針状材料が極めて細く、標的細胞に与えるダメージが極めて小さいため、導入後の細胞の生存状態及び生理状態を良好に保つことができる。従って、被導入物質を複数回にわたり、連続して又は一定の時間間隔をおいて同一の細胞内に導入することも可能である。また、複数の針状材料を用いて、同時に複数の細胞に物質を導入することも可能である。  Thereafter, by removing the needle-like material from the cells, the substance to be introduced remains in the cells, and the desired introduced cells can be obtained. According to the method of the present invention, since the needle-like material used for the introduction of the substance is extremely thin and the damage given to the target cells is extremely small, the viable state and physiological state of the cells after the introduction can be kept good. Therefore, it is also possible to introduce the substance to be introduced into the same cell a plurality of times, continuously or at regular time intervals. It is also possible to introduce substances into a plurality of cells simultaneously using a plurality of needle-shaped materials.

本発明の方法によれば、標的細胞にダメージを与えることがなく、１００％の確率で確実に目的の物質を細胞内に導入することが可能である。また、本発明の方法によれば、針状材料が細胞に対して充分小さいことから、細胞を殺さず、その生理状態等に影響を与えないため、確実に被導入物質が導入された細胞を得ることができる。  According to the method of the present invention, the target substance can be reliably introduced into the cells with a probability of 100% without damaging the target cells. In addition, according to the method of the present invention, since the needle-shaped material is sufficiently small with respect to the cells, the cells are not killed and do not affect the physiological state thereof. Obtainable.

（５）導入装置
導入装置は、被導入物質を結合させた針状材料を固定する手段、該針状材料の移動手段、標的細胞を固定する手段を有する。(5) Introducing device The introducing device has means for fixing the needle-like material to which the substance to be introduced is bound, means for moving the needle-like material, and means for fixing the target cells.

さらに、本発明の導入装置は、細胞内に針状材料が穿刺されたことを検知する手段を併せ持つことが好ましく、例えば、原子間力顕微鏡等があげられる。  Furthermore, the introduction device of the present invention preferably has a means for detecting that the needle-shaped material has been punctured into the cell, and examples thereof include an atomic force microscope.

このような装置としては、例えば、特開２００３−３２５１６１に記載される細胞操作装置があげられ、該細胞走査装置に上記（１）の針状材料を適用すればよい。原子間力顕微鏡（ＡＦＭ）を用いる場合は、探針（プローブ）をエッチングし、針状材料として用いることができる。  An example of such a device is a cell manipulation device described in JP-A-2003-325161, and the needle-shaped material (1) may be applied to the cell scanning device. When an atomic force microscope (AFM) is used, the probe can be etched and used as a needle-shaped material.

本発明の方法によれば、標的細胞にダメージを与えることなく、１００％の確率で確実に目的の物質を細胞内に導入することが可能である。また、物質を導入した後の細胞の生理状態も良好である。さらに、本発明の方法によれば、組織中の単一細胞に被導入物質を導入し、周囲の細胞及びその細胞を含む組織が、どのような影響を受けるかを観察することもできる。  According to the method of the present invention, it is possible to reliably introduce a target substance into cells with a probability of 100% without damaging the target cells. Moreover, the physiological state of the cell after introducing the substance is also good. Furthermore, according to the method of the present invention, the substance to be introduced can be introduced into a single cell in the tissue, and it can be observed how the surrounding cells and the tissue containing the cells are affected.

加えて、本発明の方法によれば、複数種の被導入物質を結合した針状材料を用いることによって、複数の被導入物質を、単一の細胞に対して同時に導入することや、単一細胞に対して連続的又は一定の時間間隔で複数回にわたって導入することが可能である。  In addition, according to the method of the present invention, a plurality of substances to be introduced can be introduced simultaneously into a single cell by using a needle-shaped material in which a plurality of kinds of substances to be introduced are combined, It is possible to introduce the cells multiple times continuously or at regular time intervals.

また、幹細胞の分化における制御機構が解明されれば、本発明の方法を用いて適切なタイミングで遺伝子導入等を行い、分化を制御することによって、幹細胞を目的の細胞、組織又は器官に分化させることも可能となる。  Also, if the control mechanism in stem cell differentiation is elucidated, gene introduction or the like is performed at an appropriate timing using the method of the present invention, and differentiation is controlled to differentiate stem cells into target cells, tissues or organs. It is also possible.

本発明の方法は、細胞を殺すことなく目的の物質を確実に導入することが可能であるため、遺伝子治療、細胞治療等においても有用な手段である。  The method of the present invention is a useful means in gene therapy, cell therapy, and the like because a target substance can be reliably introduced without killing cells.

以下、参考例及び実施例をあげて本発明をさらに詳細に説明するが、本発明は、これらに限定されない。  Hereinafter, although a reference example and an example are given and the present invention is explained still in detail, the present invention is not limited to these.

参考例１Reference example 1

細胞に対して低侵襲な針の直径を確認するため、ＡＦＭ探針を集束イオンビームでエッチングした針状材料を用意した。針状材料を用いた細胞穿刺操作を、図３に図示されるように行った。針状材料の直径は、それぞれ２００ｎｍ、４００ｎｍ、６００ｎｍ及び８００ｎｍであった（図４参照）。  In order to confirm the diameter of a needle that is minimally invasive to cells, a needle-like material was prepared by etching an AFM probe with a focused ion beam. A cell puncture operation using a needle-shaped material was performed as shown in FIG. The diameters of the acicular materials were 200 nm, 400 nm, 600 nm and 800 nm, respectively (see FIG. 4).

ＤＡＰＩを添加した培地中で穿刺操作を行い、針抜去後の蛍光強度経時変化を測定した。死細胞はＤＡＰＩにより染色され蛍光強度が上昇する。図５に示すように、ヒトメラニン細胞、ヒト乳ガン細胞等で１時間以上の穿刺操作を行い、ＤＡＰＩ染色による死細胞判定試験を行った。針状材料を穿刺しない細胞を、コントロール（ｃｏｎｔｒｏｌ）として用いた。  The puncture operation was performed in a medium to which DAPI was added, and the change over time in fluorescence intensity after removal of the needle was measured. Dead cells are stained with DAPI and the fluorescence intensity increases. As shown in FIG. 5, a puncture operation for 1 hour or more was performed with human melanocytes, human breast cancer cells, etc., and a dead cell determination test by DAPI staining was performed. Cells that did not puncture the needle-like material were used as controls.

直径４００ｎｍの針では１時間以上の穿刺行ってもいずれの細胞もＤＡＰＩ染色されなかった。刺激に敏感な新生児ヒトメラニン細胞では、６００ｎｍの針を５０分間穿刺することにより、蛍光強度の上昇が見られる。このような傾向は、動物細胞の種類を変更しても変わらないことから、細胞の穿刺には６００ｎｍ未満、４００ｎｍ以下程度の直径の針状材料であれば、いかなる動物細胞に対しても侵襲性は低いことが判明した。  None of the cells were stained with DAPI even when puncturing for 1 hour or longer with a needle having a diameter of 400 nm. In neonatal human melanocytes sensitive to stimulation, an increase in fluorescence intensity is observed when a 600 nm needle is punctured for 50 minutes. Such a tendency does not change even if the type of animal cell is changed. Therefore, puncture of a cell is invasive to any animal cell as long as it is a needle-like material having a diameter of less than 600 nm and less than 400 nm. Turned out to be low.

これに対して、既存の外径１μｍ程度のキャピラリを用いたマイクロインジェクションを用いて試験した結果、１秒以下の穿刺時間であっても、穿刺された細胞の４０％が染色され、死に至ることが判明した。従って、細胞に穿刺操作を行う場合には至適な材料直径が存在することが示された。  On the other hand, as a result of testing using microinjection using an existing capillary having an outer diameter of about 1 μm, 40% of the punctured cells are stained and die even in a puncture time of 1 second or less. There was found. Therefore, it was shown that an optimum material diameter exists when a puncturing operation is performed on a cell.

〔遺伝子の導入〕
図６の模式図に示すように、シリコン製ＡＦＭ探針をエッチングにより先鋭化した針状材料に対して、ＭＰＴＥＳ（３−Ｍｅｒｃａｐｔｏｐｒｏｐｙｌｔｒｉｍｅｔｈｏｘｙｓｉｌａｎｅ）により、チオール基を導入する。さらに２価性カップリング試薬であるＥＭＣＳ（Ｎ−（６−Ｍａｌｅｉｍｉｄｏｃａｐｒｏｙｌｏｘｙ）ｓｕｃｃｉｎｉｍｉｄｅ）を用いてスクシンイミド活性エステルを表面に提示させる。最後に、スペーサー物質として３０ｍｅｒのペプチド、ポリリジンを表面に接触させ、共有結合により針状材料表面にポリリジンを固定化した。固定化修飾した針に、プラスミドｐｈｒＧＦＰを吸着（静電気的に結合）させ、ＨＥＫ２９３に３分間穿刺し、抜去した。[Gene transfer]
As shown in the schematic diagram of FIG. 6, a thiol group is introduced by MPTES (3-Mercaptopropyltrimethylsilane) into a needle-shaped material obtained by sharpening a silicon AFM probe by etching. Furthermore, succinimide active ester is displayed on the surface using EMCS (N- (6-Maleimidocaproyloxy) succinimide) which is a bivalent coupling reagent. Finally, a 30-mer peptide, polylysine was brought into contact with the surface as a spacer substance, and polylysine was immobilized on the surface of the needle-shaped material by covalent bonding. Plasmid phrGFP was adsorbed (electrostatically bound) to the immobilized modified needle, punctured with HEK293 for 3 minutes, and then removed.

針状材料を抜去した後、２時間程度から穿刺操作した細胞はＧＦＰの緑色蛍光が発現し始め、図７に示すように、２４時間で細胞は明らかなＧＦＰの緑色蛍光が観察された。また、３６時間後の細胞は活発に移動している。細胞は活性が高いと移動し、死んでしまうと、動かなくなるか、基板等から剥離する。得られた結果より、短時間の穿刺操作によって細胞を傷つけることなくＤＮＡを導入することができることが示された。  After the needle-shaped material was removed, cells that had been punctured from about 2 hours began to express GFP green fluorescence, and as shown in FIG. 7, the cells observed clear GFP green fluorescence at 24 hours. In addition, the cells after 36 hours are actively moving. Cells move when they are highly active, and when they die, they either stop moving or detach from the substrate. From the obtained results, it was shown that DNA can be introduced without damaging cells by a short puncture operation.

〔リポソームによる物質導入〕
図８の模式図に示すように、シリコン製ＡＦＭ探針をエッチングにより先鋭化した針状材料に対して、ＭＰＴＥＳによりチオール基を導入する。さらにｂｉｏｔｉｎｙｌａｔｅｄ ｍａｌｅｉｍｉｄｅを用いてビオチンを表面に提示させる。ストレプトアビジンを表面に接触させ、アビジン−ビオチン結合により針状材料表面にストレプトアビジンを固定化した。続いて、蛍光物質封入ビオチン化リポソームをｅｇｇ ｙｏｌｋ ｐｈｏｓｐｈａｔｉｄｙｌｃｈｏｌｉｎｅ（ＥＰＣ）と１，２−ｄｉｏｌｅｏｙｌ−ｐｈｏｓｐｈａｔｉｄｙｌｅｔｈａｎｏｌａｍｉｎｅ−Ｎ−（ｃａｐ ｂｉｏｔｉｎｙｌ）（ｂｉｏｔｉｎ−ｃＰＥ）をモル比５０：１で作製した。ＥＰＣ、ｂｉｏｔｉｎ−ｃＰＥはクロロホルムに溶解させ、混合液を４０℃、１７０ｒｐｍで遠心エバポレーターにかけ、クロロホルムで洗浄を行った後、完全に乾燥した脂質薄膜を調製した。[Material introduction by liposome]
As shown in the schematic diagram of FIG. 8, a thiol group is introduced by MPTES into a needle-like material obtained by sharpening a silicon AFM probe by etching. Furthermore, biotin is presented on the surface using biotinylated maleimide. Streptavidin was brought into contact with the surface, and streptavidin was immobilized on the surface of the needle-shaped material by an avidin-biotin bond. Subsequently, fluorescent substance-encapsulated biotinylated liposomes were prepared in a molar ratio of 50: 1 such as egg yolk phosphophatylcholine (EPC) and 1,2-dioloyl-phosphatidylethanolamine-N- (cap biotinyl) (biotin-cPE). EPC and biotin-cPE were dissolved in chloroform, and the mixed solution was subjected to a centrifugal evaporator at 40 ° C. and 170 rpm, washed with chloroform, and then a completely dried lipid film was prepared.

脂質薄膜１００μｍｏｌに対して１００ｍＭのカルセイン溶液（ｐＨ７．４）４ｍｌを添加し、カルセイン内包多層膜リポソームを作製した。この多層膜リポソームは孔径２００ｎｍのフィルターを用いてエクストルーダーにより処理し、粒径のそろった単層膜リポソームを作製し、カルセイン封入ビオチン化リポソームとした。これに対して、アビジン修飾した針に、カルセイン封入ビオチン化リポソームを４℃、４時間で結合させた。  4 ml of 100 mM calcein solution (pH 7.4) was added to 100 μmol of the lipid thin film to prepare calcein-encapsulated multilayer membrane liposomes. This multilamellar liposome was treated with an extruder using a filter having a pore size of 200 nm to produce a monolamellar liposome having a uniform particle size, which was defined as a calcein-encapsulated biotinylated liposome. In contrast, calcein-encapsulated biotinylated liposomes were bound to avidin-modified needles at 4 ° C. for 4 hours.

ＤｓＲＥＤ２を発現させたＨｅＬａ細胞に対してこのリポソーム針状材料を穿刺し、レーザー共焦点顕微鏡で観察した垂直断面像が図９である。穿刺直後の断面像が図１０Ａ、穿刺状態を維持して１時間後に撮影した断面像が図１０Ｂであり、抜去した後に撮影した断面像が図１０Ｃである。穿刺後、核内にカルセインが漏出し、核内も蛍光色素が存在していることが、図１０Ｂより確認出来る。図１０Ｃではカルセインが退色したために核内の蛍光は見られない。  FIG. 9 shows a vertical cross-sectional image obtained by puncturing the liposome needle-like material into HeLa cells expressing DsRED2 and observing with a laser confocal microscope. FIG. 10A shows a cross-sectional image immediately after puncturing, FIG. 10B shows a cross-sectional image taken 1 hour after maintaining the puncture state, and FIG. 10C shows a cross-sectional image taken after removal. After puncturing, it can be confirmed from FIG. 10B that calcein leaks into the nucleus and the fluorescent dye is also present in the nucleus. In FIG. 10C, no fluorescence is observed in the nucleus because calcein has faded.

このことから、リポソーム内に物質を封入し、これを固定化した針状材料を細胞に導入することにより、目的の物質を導入できることが示された。  From this, it was shown that the target substance can be introduced by encapsulating the substance in the liposome and introducing the needle-like material on which the substance is immobilized into the cell.

被導入物質を固定化した針状材料のモデル図を示す。The model figure of the acicular material which fixed the to-be-introduced substance is shown. 被導入物質の針状材料への結合を、結合の種類ごとに具体例を図示する。Specific examples of the binding of the substance to be introduced to the needle-shaped material are shown for each type of binding. 針状材料を用いた細胞穿刺技術を図示する。1 illustrates a cell puncture technique using a needle-like material. 各直径の針状材料の電子顕微鏡写真を示す。The electron micrograph of the needle-shaped material of each diameter is shown. 各直径の針状材料の穿刺によってＤＡＰＩ染色された細胞の蛍光強度変化を示す。The change in fluorescence intensity of DAPI-stained cells by puncturing with needle-shaped material of each diameter is shown. ポリリジン修飾した針に静電吸着させたプラスミドＧＦＰ遺伝子を図示する。The plasmid GFP gene electrostatically adsorbed on a polylysine-modified needle is illustrated. プラスミドＧＦＰ遺伝子を導入したＨＥＫ２９３の遺伝子発現を示す。The gene expression of HEK293 introduced with the plasmid GFP gene is shown. カルセイン封入リポソームを固定化した針を図示する。A needle with immobilized calcein-encapsulated liposomes is illustrated. ＤｓＲＥＤ２を発現させたＨｅＬａ細胞に対して、カルセイン封入リポソーム結合針状材料を穿刺した際のレーザー共焦点顕微鏡による垂直断面写真を示す。The vertical cross-sectional photograph by a laser confocal microscope at the time of puncturing a calcein inclusion liposome binding needle-shaped material with respect to the HeLa cell which expressed DsRED2 is shown. ＤｓＲＥＤ２を発現させたＨｅＬａ細胞にカルセイン封入リポソーム結合針状材料を、（Ａ）穿刺直後、（Ｂ）穿刺状態維持１時間後、（Ｃ）抜去後のレーザー共焦点顕微鏡による水平断面写真を示す。The horizontal cross-sectional photograph by the laser confocal microscope after (A) immediately after puncture, (B) 1 hour after puncture state maintenance, (C) after extraction is shown in HeLa cells in which DsRED2 is expressed.

Claims (12)

針状材料に被導入物質を結合する工程、該針状材料を細胞に穿刺する工程、針状材料を細胞から抜去して該被導入物質を細胞内に残留させる工程を含む、細胞内に該被導入物質を導入する方法。A step of binding an introduced substance to the needle-shaped material, a step of puncturing the needle-shaped material into the cell, and a step of removing the needle-shaped material from the cell to leave the introduced substance in the cell. A method of introducing a substance to be introduced. ２以上の被導入物質を連続して又は一定の時間間隔をおいて細胞内に導入することを特徴とする請求項１に記載の方法。2. The method according to claim 1, wherein two or more substances to be introduced are introduced into the cell continuously or at regular time intervals. 針状材料の直径が、８００ｎｍ以下である請求項１又は２に記載の方法。The method according to claim 1 or 2, wherein the needle-shaped material has a diameter of 800 nm or less. 針状材料と被導入物質間の結合が、静電気的結合、疎水性相互作用による結合、キレート結合、細胞内で切断され得る共有結合、光切断リンカーを介した結合、酵素切断リンカーを介した結合及び特異的結合からなる群より選択される少なくとも１種である、請求項１〜３に記載の方法。The binding between the needle-shaped material and the substance to be introduced is an electrostatic bond, a bond by hydrophobic interaction, a chelate bond, a covalent bond that can be cleaved in a cell, a bond through a photocleavable linker, a bond through an enzyme cleaved linker And at least one selected from the group consisting of specific binding. 被導入物質が、遺伝子、タンパク質、糖類、多糖、脂肪酸、コレステロール、脂質、シグナル伝達物質、リガンド物質、ホルモン物質、サイトカイン、イオン、金属粒子、磁性微粒子、無機化合物、量子ドット、有機化合物及び薬剤からなる群より選択される少なくとも１種、ないしこれらの複合体である、請求項１〜４に記載の方法。Substances to be introduced include genes, proteins, saccharides, polysaccharides, fatty acids, cholesterol, lipids, signaling substances, ligand substances, hormone substances, cytokines, ions, metal particles, magnetic particles, inorganic compounds, quantum dots, organic compounds, and drugs The method according to claim 1, which is at least one selected from the group consisting of these or a complex thereof. 遺伝子が、核酸、核酸様物質、ＤＮＡ、ＲＮＡ、プラスミド、ウイルス粒子、染色体である請求項１〜４に記載の方法。The method according to claims 1 to 4, wherein the gene is a nucleic acid, a nucleic acid-like substance, DNA, RNA, a plasmid, a virus particle, or a chromosome. タンパク質が、アミノ酸、オリゴペプチド、ポリペプチド、マルチサブユニットタンパク質である請求項１〜４に記載の方法。The method according to claims 1 to 4, wherein the protein is an amino acid, an oligopeptide, a polypeptide, or a multi-subunit protein. 被導入物質が、リポソーム封入体、シクロデキストリン包接体又はゲルに内包もしくは包埋される形態である請求項１〜７に記載の方法。The method according to any one of claims 1 to 7, wherein the substance to be introduced is in a form of being encapsulated or embedded in a liposome inclusion body, a cyclodextrin inclusion body or a gel. 被導入物質を、細胞内で分離可能に結合してなる針状材料。A needle-shaped material formed by binding a substance to be introduced so as to be separable within a cell. 針状材料と被導入物質間の結合が、静電気的結合、疎水性相互作用による結合、キレート結合、細胞内で切断され得る共有結合、光切断リンカーを介した結合、酵素切断リンカーを介した結合及び特異的結合からなる群より選択される少なくとも１種である、請求項９に記載の針状材料。The binding between the needle-shaped material and the substance to be introduced is an electrostatic bond, a bond by hydrophobic interaction, a chelate bond, a covalent bond that can be cleaved in a cell, a bond through a photocleavable linker, a bond through an enzyme cleaved linker The acicular material according to claim 9, which is at least one selected from the group consisting of and specific binding. 被導入物質を、必要に応じてスペーサー物質を介して細胞内で分離可能な結合により針状材料に結合させる工程を含む、被導入物質を結合させた針状材料の製造方法。A method for producing an acicular material to which an introduced substance is bound, comprising a step of binding the introduced substance to the acicular material by a bond that can be separated in a cell via a spacer substance as necessary. 針状材料と被導入物質間の結合が、静電気的結合、疎水性相互作用による結合、キレート結合、細胞内で切断され得る共有結合、光切断リンカーを介した結合、酵素切断リンカーを介した結合及び特異的結合からなる群より選択される少なくとも１種である、請求項１１に記載の方法。The binding between the needle-shaped material and the substance to be introduced is an electrostatic bond, a bond by hydrophobic interaction, a chelate bond, a covalent bond that can be cleaved in a cell, a bond through a photocleavable linker, a bond through an enzyme cleaved linker And at least one selected from the group consisting of specific binding.
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