JP2011184391A - Nucleic acid introducing agent made of organic nanotube - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a self-assembling organic nanotube to be used as a nontoxic excellent carrier of a nucleic acid. <P>SOLUTION: The compound nanotube has an organic nanotube structure formed by mixing lipid compounds represented by chemical formula (1) or (2). In chemical formula (1) (compound 1), m indicates an integer of 12-20, and in chemical formula (2) (compound 2), m indicates an integer of 12-20 or the like, and X indicates (CH<SB>2</SB>)<SB>n</SB>, wherein n indicates an integer of 2-8 or the like. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a self-assembling organic nanotube (composite nanotube) comprising a plurality of lipid compounds suitable as a nucleic acid carrier such as a gene and a method for introducing a nucleic acid into a cell using the composite nanotube. Moreover, it is related with the novel lipid compound used as the structural component which forms the said composite nanotube, and its manufacturing method.

Introduction of exogenous genes into cells is an important technique from the viewpoints of gene therapy and recombinant protein production. However, a gene comprising a nucleic acid substance is rapidly degraded by a nuclease in a solution containing cells, and the uptake into cells is significantly reduced. Moreover, since it is a polyanionic substance derived from a phosphate group, there is little interaction with a negatively charged cell membrane, and transmembrane penetration is difficult. For this reason, the development of nucleic acid carriers for improving delivery of DNA, RNA, and oligonucleotides in vivo and in vitro has become a major issue.
Nucleic acid carriers that have been developed so far can be broadly classified into viral and non-viral systems. Among these, the virus system shows high gene expression efficiency, but there are many problems in safety such as host immune reaction and infection. This severely restricts use.
On the other hand, non-viral carriers can be produced easily and have the advantage of a very low risk of immune reaction and infection. For this reason, various artificial gene carriers have been developed. These include a cationic functional group for electrostatically interacting with a phosphate group of a gene to form an ionic complex. For example, vesicles composed of polymer phospholipid derivatives modified with polyamine (commercially available from Invitrogen as Lipofectamine 2000), cationic polymer derivatives such as poly-L-lysine, poly-L-ornithine, polyethyleneimine, chitosan, etc. Cationic dendrimers such as micelles and polyamidoamines having a controlled multi-branched structure have been reported (Patent Documents 1, 2, and 3). Although these have the advantage of being non-viral, there are some safety problems due to the recognized cytotoxicity of polyamines and the like.
The above-mentioned carrier has a spherical or particulate shape, but carbon nanotubes, silica nanotubes and the like have been reported as gene introduction agents as carriers having other unique shapes. These use single-walled and multi-walled cationic carbon nanotubes coated with a cationic functional group (Non-Patent Documents 1 to 3). In addition, a carrier in which an amino group is modified using a silane coupling agent on a silica nanotube has been reported (Non-patent Document 4).
However, in the case of these inorganic substance-based nanotubes, since they are entirely composed of covalent bonds, decomposition after introduction into cells is difficult, and there is a possibility that a safety problem may remain.

In contrast, various types of nanotube-based carriers that can be manufactured by molecular self-assembly have been developed, and these self-assembled nanotubes have the advantage of superior degradability in cells due to non-covalent bonding. Is expected. However, there are many reports that these nanotubes can transport nucleic acids into cells as conjugates with nucleic acids, but they have been introduced into cells such as gene expression and gene suppression effects in cells. There are few examples where the original functions and activities of nucleic acids have been confirmed, and there are no examples of successful expression from large nucleic acid molecules such as genes having several hundred base sequences necessary for protein expression. The effect as a nucleic acid carrier is considered to be limited.
For example, in the case of a nanotube formed by self-assembly of a cationic phenylalanylalanine derivative (Non-Patent Document 5), it has the property of changing to a nanotube and vesicle structure (spherical shape) depending on the concentration, and the actual nucleic acid after mixing with cells Since it is known that the vesicle is incorporated in a vesicle structure at the time of delivery, it is not strictly a self-assembled nanotube carrier, and the expression of the gene has not been confirmed. The nucleic acid molecules used are only single-strand short DNA.
In addition, in the case of a fiber-like structure (Non-patent Document 6) obtained by complexing lipid molecules and nucleic acids, it has been confirmed that the complex with RNA has been introduced into cells. The expression of function has not been confirmed or evaluated at all.
In an example using a nanofiber-based material consisting of self-assembly of a peptide block copolymer (Non-patent Document 7), it was reported that a conjugate with a 23-residue siRNA was introduced into a cell and functioned. However, only the low molecular weight DNA and RNA fragments in the cytoplasm have been confirmed to have functional expression, and neither large molecular weight gene expression nor nucleic acid delivery into the nucleus has been confirmed.
Based on the above, not only can nucleic acids including genes with large molecular weights be transported into cells, but they can also be delivered to the nucleus and efficiently function in the cells. There has been a strong demand for the development of self-assembled nanotubes that can be excellent nucleic acid carriers.

JP 2004-522809 A JP 2002-515418 A JP 2005-247755 A Japanese Patent No. 4174702

A. Bianco et al., Angewandte Chem., 2004, 43, 5242-5246.CNT A. Bianco et al., J. Am. Chem. Soc., 2005, 127, 4388-4396.CNT Y. Liu et al., Angewandte Chem., 2005, 44, 4782-4785.CNT Y.C.Wu, Advanced Materials, 2005, 17, 404-407. J. Li et al., Angewandte Chem., 2007, 46, 2431-2434. J. H. Fuhrhop, H. Tank, Chemistry and Physics. Of Lipids, 1987, 43, 193-213. M. Lee et al., Angewandte Chem, 2008, 47, 4525-4528. T. Shimizu, M. Masuda, H. Minamikawa, Chemical Review, 2004, 105, 1401-1443. M. Masuda, T. Shimizu, Langmuir, 2004, 20, 5969-5977. N. Kameta, M. Masuda, N. Morii, T. Shimizu, Small, 2008, 4, 561-565. N.D.Santos, C. Allen And A.M.Biochimica Biophysica Acta 1768 (2007) 1367-1377. F.K.Bedu-Addo, P.Tang, Y.Xu and L.Huang., Pharmaceutical Research 13 (1996) 710-717. Mitsutoshi Masuda, Hyakuyo Wada, Naohiro Kameda, Hiroyuki Minamikawa, Toshimi Shimizu, Proceedings of the 58th Annual Meeting of the Society of Polymer Science, Japan, 2K10, “Construction of Organic Nanotube / PEG Polymer Complex”. Y. Takeda, N. Shimada, K. Kaneko, S. Shinkai, K. Sakurai, Biomacromolecules 8 (2007) 1178-1186 D. Putnam, A.N.Zelikin, V.A.Izumrudov, R. Langer, Biomaterials 24 (2003) 4425-4433 M. Walsh, M. Tangney, M.J.O'Neill, J.O.Larkin, D.M.Soden, S.L.McKenna, R. Darcy, G.C.O'Sullivan, C.M.O'Driscoll, Molecular Pharmaceutics. G. Fotakis and J.A.Timbrell, Txocology Letters 160 (2006) 171-177

  The present invention can not only transport a nucleic acid including a gene having a large molecular weight into a cell, but also deliver the nucleic acid into the nucleus and efficiently exert the original function of the nucleic acid in the cell. It is an object of the present invention to provide a self-assembled nanotube that can be an excellent nucleic acid carrier.

The inventors have synthesized many kinds of long self-assembling lipid compounds having a hydrophilic part and a hydrophobic part (Non-patent Documents 8-10). Among these compounds, long-chain hydrocarbon chains are synthesized. It has been found that organic nanotubes composed of “wedge-shaped” lipids, which are double-headed lipids with hydrophilic parts connected at both ends and have different sizes and types of hydrophilic parts at both ends, have high dispersibility (non- Patent Document 8). Among them, a lipid having a long-chain fatty acid residue (having 6 to 20 carbon atoms) linked to the reducing end of a glucose sugar residue via an amide bond, that is, a lipid represented by the following formula (1) (Compound 1) However, it has been found and reported that organic nanotubes having an outer diameter of about 40 to 50 nm, which is stable in water and highly dispersible, can be formed (Patent Document 4, Non-Patent Document 9). The inventors focused on the high stability and dispersibility in water of organic nanotubes having biocompatibility of lipids from the beginning, and controlled release when physiologically active substances such as various medical drugs are administered to living bodies. We have been developing the use of functional carriers and so on, and examined the possibility of using them as drug carriers.
<Formula (1)> (Compound 1)
[In formula, m represents the integer of 12-20. ]

However, since these organic nanotubes are negatively charged in water, it is difficult to bind to the same negatively charged drug such as nucleic acid, and the cell surface is usually slightly negatively charged. Therefore, it is difficult to approach the cell surface by itself in the first place, and improvement is necessary to use it as a drug carrier for transporting drugs into cells.
Therefore, the present inventors have conceived of forming a composite nanotube composed of a plurality of components obtained by combining a lipid compound of the above formula (1) (compound 1) with some cationic amphiphilic lipid compound. .
As a result of intensive studies to provide a cationic amphiphilic lipid compound capable of forming a composite nanotube with the lipid compound of the above formula (1) and forming a conjugate with a nucleic acid, A novel lipid compound in which a cationic amino acid is condensed on one end of a diamine consisting of an alkylene group or an alkylene group containing an amide in the middle, and α, ω-long chain dicarboxylic acid is condensed on the other end of the diamine (the following formula ( 2) Lipid compound: Compound 2) can be synthesized, and the compound 2 alone does not have the ability to form organic nanotubes, but is mixed with the lipid compound of formula (1) (compound 1). It has been found that when self-assembled, a stable and highly dispersible nanotube structure (“composite nanotube”) is formed.
<Formula (2)> (Compound 2)
[Wherein, m represents an integer of 12 to 20, and X represents — (CH ₂ ) _n — (n is an integer of 2 to 8) or —CH ₂ —CO—NH— (CH ₂ ) ₂ —. , R represents a cationic amino acid residue. ]

Since the lipid compound (compound 2) of the above formula (2) has a cationic amino acid residue at the terminal, the resulting composite nanotube is positively charged and electrostatically associates with the phosphate group of the nucleic acid. Form a stable conjugate. In addition, this composite nanotube has the property of easily approaching the negatively charged cell membrane surface, but on the contrary, when it is taken into the positively charged cell, it quickly disperses. It also has the property of releasing nucleic acids.
The composite nanotube formed from the nanotube-forming lipid of formula (1) and the nucleic acid-binding lipid of formula (2) (compounds 1 and 2) can stably form a nucleic acid-tube conjugate organized with nucleic acid. By using the composite nanotube as a nucleic acid carrier, adhesion to the cell surface can be performed efficiently, and after the transmembrane transport of the nucleic acid, the nucleic acid is quickly released in the cell. Thus, it was confirmed that functions such as gene expression can be exhibited. Specifically, when a cell was transformed with a nucleic acid-tube conjugate in which a vector containing a reporter gene was bound to the composite nanotube, expression of the reporter gene in the cell could be confirmed.
By obtaining the above knowledge, a nucleic acid carrier capable of transporting a nucleic acid into a cell and expressing the original function of the nucleic acid, a self-assembling composite nanotube therefor, and a novel compound capable of forming the composite nanotube together with compound 1 The present invention for Compound 2 was completed.

However, the composite nanotube composed of the nanotube-forming lipid of the formula (1) and the nucleic acid-binding lipid of the formula (2) has such excellent characteristics, but is aggregated when forming a nucleic acid tube conjugate. It also had drawbacks that were likely to occur.
Therefore, the present inventors have studied to further search for lipid compounds that are constituent components of the composite nanotube having an aggregation-inhibiting action, aiming at further improvement of the composite nanotube of the present invention. As a result, a novel lipid compound in which an alkylene group having a polyoxyethylene group consisting of a repeating sequence of 10 or more oxyethylene groups is bonded to a long-chain fatty acid residue via an amide bond (a lipid compound of the following formula (3) : Compound 3) can be synthesized, and the compound 3 alone does not have the ability to form an organic nanotube, but the nanotube-forming lipid of the formula (1) (compound 1) and the nucleic acid of the formula (2) When added as a third component in the production of composite nanotubes by mixing with binding lipid (compound 2), the composite nanotubes having higher dispersibility can be obtained while retaining the effect as the cationic composite nanotubes. Could be manufactured.
<Chemical Formula (3)> (Compound 3)
[Wherein, m represents an integer of 12 to 20, p represents an integer of 10 to 200, and Y represents a hydrogen atom or a methyl group. ]
As a result of conducting a cell introduction experiment of a reporter gene using a composite nanotube composed of the three components of compounds 1, 2 and 3 as a nucleic acid carrier, the introduction efficiency into the cell was increased, and the gene expression efficiency was also increased. Further improvements in nanotube and nucleic acid carriers could be achieved.
By obtaining the above knowledge, the present invention for a further improved composite nanotube and nucleic acid carrier was also completed.

That is, the present invention includes the following inventions.
[1] A lipid compound of the following chemical formula (1) capable of forming an organic nanotube (compound 1) or a pharmacologically acceptable salt thereof and a lipid compound of the following chemical formula (2) having a nucleic acid binding property (compound 2) Alternatively, a composite nanotube comprising an organic nanotube structure comprising a pharmacologically acceptable salt thereof;
<Chemical Formula (1)> (Compound 1)
[In formula, m is an integer of 12-20. ]
<Chemical Formula (2)> (Compound 2)
[Wherein, m represents an integer of 12 to 20, X represents an integer of (CH ₂ ) _n and n is 2 to 8, or CH ₂ —CO—NH— (CH ₂ ) ₂ , R represents arginyl] Represents a lysyl or ornithyl residue. ].
[2] The lipid compound of the chemical formula (1) and the lipid compound of the chemical formula (2) or each pharmacologically acceptable salt thereof are contained in a molar ratio of 2: 1 to 10: 1. The composite nanotube according to [1] above, wherein
[3] The above [1] or [2], further comprising a lipid compound (compound 3) having a polyoxyethylene residue and having the following chemical formula (3) or a pharmacologically acceptable salt thereof: A composite nanotube according to claim 1;
<Chemical Formula (3)> (Compound 3)
[Wherein, m represents an integer of 12 to 20, p represents an integer of 10 to 200, and Y represents a hydrogen atom or a methyl group. ].
[4] The lipid compound represented by the chemical formula (1), the lipid compound represented by the chemical formula (2), the lipid compound represented by the chemical formula (3), or a pharmacologically acceptable salt thereof in a molar conversion of 63:32. : The composite nanotube according to [3], wherein the composite nanotube is included at a mixing ratio of 5 to 87: 8: 5.
[5] A lipid compound represented by the following chemical formula (2) having a nucleic acid binding property or pharmacologically acceptable, which can be used as a component of the composite nanotube described in any one of [1] to [4] Its salt;
<Chemical Formula (2)> (Compound 2)
[Wherein, m represents an integer of 12 to 20, X represents an integer of (CH ₂ ) _n and n is 2 to 8, or CH ₂ —CO—NH— (CH ₂ ) ₂ , R represents arginyl] Represents a lysyl or ornithyl residue. ].
[6] A lipid compound of the following formula (3) having a polyoxyethylene residue that can be used as a constituent of the composite nanotube described in [3] or [4] or a pharmacologically acceptable product thereof salt;
<Chemical formula (3)>
[Wherein, m represents an integer of 12 to 20, p represents an integer of 10 to 200, and Y represents a hydrogen atom or a methyl group. ].
[7] A nucleic acid carrier comprising the composite nanotube according to any one of [1] to [4] and capable of forming a nucleic acid-tube conjugate by electrostatically binding to a nucleic acid.
[8] A nucleic acid-tube conjugate in which the composite nanotube according to any one of [1] to [4] and a nucleic acid are electrostatically bound, and the nucleic acid can be transported into a cell.
[9] A method for transforming a cell, comprising using the nucleic acid carrier according to [7] above and transporting a nucleic acid electrostatically bound to the nucleic acid carrier into the cell.
[10] A transformed cell transformed by the transformation method of [9].
[11] A composition for gene therapy comprising the nucleic acid-tube conjugate according to [8] above containing an effective amount of a gene therapy nucleic acid and a pharmacologically acceptable carrier.

The long organic nanotube structure (composite nanotube) obtained by the self-assembly of a plurality of lipid compounds provided by the present invention has low cytotoxicity and electrostatically binds to a nucleic acid to make it efficient in the cell. Since it can be delivered well and can express nucleic acid functions such as gene expression in cells, it is an excellent nucleic acid carrier for gene therapy. Moreover, since the composite nanotube of the present invention is an aggregate in a solid state, it is very stable and can be stored even in a solution dispersion state for one month or more.
In addition, the long-chain carboxylic acids and amino acids used as the raw material for the lipid compound constituting the composite nanotube are advantageous in that they are inexpensive and easy to produce.

¹ H-NMR data of compound 2 represented by general formula 2 (m = 18, X is (CH ₂ ) ₆ , R is an arginyl group) (at room temperature in deuterated DMSO). “DMSO” is dimethyl sulfoxide Abbreviation. ¹ H-NMR data of a compound represented by the general formula 2 (m = 18, X is (CH ₂ ) ₂ , R is an arginyl residue) (at room temperature in deuterated DMSO). ¹ H-NMR data (m = 18, X is CH ₂ —CO—NH— (CH ₂ ) ₂ , R is an arginyl group) ¹ H-NMR data (room temperature in deuterated DMSO) of Compound 2. ¹ H-NMR data (room temperature in deuterated DMSO) of synthesis of compound 2 (m = 18, X is (CH ₂ ) ₂ , R is lysyl residue). ¹ H-NMR data of Compound 3 (room temperature in deuterated DMSO). Observation of morphology of composite nanotubes by electron microscope. (A) Compound 1/2, Compound 2: X = (CH ₂ ) ₆ , ultrasonic untreated. (B) Compound 1/2, Compound 2: X = (CH ₂ ) ₆ , sonication. (C) Compound 1/2/3, Compound 2: X = (CH ₂ ) ₆ , ultrasonic untreated. (D) Compound 1/2/3, Compound 2: X = (CH ₂ ) ₆ , sonication. (E) Compound 1/2/3, Compound 2: X = (CH ₂ ) ₂ , ultrasonic untreated. (F) Compound 1/2/3, Compound 2: X = (CH ₂ ) ₂ , sonication. Note: Compounds 1, 2, 3 have m = 18, Compound 3 has p = 45, y = Me, Compound 2 R = arginyl group Observation of morphology of composite nanotubes by electron microscope. (A) Compound 1/2/3, Compound 2: X = CH ₂ —CO—NH— (CH ₂ ) ₂ , R = arginyl group, ultrasonic untreated. (B) After the ultrasonic treatment described above. (C) Compound 1/2, X = (CH ₂ ) ₆ , R = lysyl group (d) Compound 1/2, X = (CH ₂ ) ₂ , R = lysyl group Note) m of compounds 1, 2 and 3 = 18, p = 45 of compound 3, Y = Me Evaluation of dispersibility of composite nanotubes by introduction of oxyethylene chains. Evaluation of binding behavior of composite nanotubes with DNA by agarose electrophoresis. The upper row shows the binding of DNA to compound 1/2 composite nanotubes, and the lower row shows the binding of DNA to compound 1/2/3 composite nanotubes. Each of them was loaded with DNA at the bottom of the gel and applied with a positive charge upward to see the migration. The numerical value at the top represents the molar ratio (Arg / P) of arginine residue / DNA phosphate residue in the nanotube. (A) Compound 1/2 composite nanotube and (b) Compound 1/2/3 composite nanotube after scanning with DNA (Arg / P = 8/1) in a scanning electron microscope image (transmission) mode). Observation of a conjugate of Compound 1/2 (upper), Compound 1/2/3 composite nanotube (lower) and YOYO-1-labeled DNA with a laser confocal microscope (bright field image observed in differential interference mode) ). Arg / P = 8/1 Examination of DNA-tube conjugate degradation resistance to DNase (Compound 1/2 composite nanotube: Arg / P = 8/1, Compound 1/2/3 composite nanotube: Arg / P = 8/1, in Tris buffer, DNase 40 unit, 30 minutes every 30 seconds) Incorporation of DNA-tube conjugate (DNA fluorescently labeled with YOYO-1) into KB cells. (A) Organic nanotubes selectively stained with rhodamine B and visualized with a fluorescence microscope. (B) DNA stained with YOYO-1 and visualized in fluorescence. (C) Visualization of cells and composite nanotubes with a phase contrast optical microscope. (D) An image obtained by combining the above (a) to (c). Compound 1/2 composite nanotube and DNA mixed at Arg / P = 8/1 and then cultured for 2 hours. Concentration: 2 μg DNA / well. Excess composite nanotubes adhering to the extracellular surface were washed away twice with heparin-PBS buffer. Then, it moved to Aqua Polymount and observed with a confocal laser microscope. Semi-quantitative measurement of cellular uptake by nucleic acid-tube conjugate (Arg / P = 8/1). The nucleic acid-tube conjugate was added to cultured cells (KB cells, A549 cells) and cultured for 2 hours. Thereafter, excess composite nanotubes were washed three times with PBS (20 U / ml) buffer containing heparin. Then, after dispersing in the buffer for flow cytometry, the fluorescence from YOYO-1 was monitored using flow cytometry. Evaluation of Gene Expression in Cells Using Nucleic Acid-Tube Conjugate Nucleic acid-tube conjugate (Arg / P = 8/1) was added to cultured cells (KB cells, A549 cells) and cultured for 24 hours. This was washed twice with a heparin-containing PBS (20 U / ml) buffer, dispersed in a flow cytometry buffer, and the fluorescence of GFP emitted by the cells was monitored using flow cytometry. Comparison of GFP expression level between commercially available gene transfer agent and composite nanotube. Nucleic acid-tube conjugate (Arg / P = 8/1 or NH ₂ / P = 8/1) was added to KB cells and cultured for 24 hours, and then GFP fluorescence image (a) Lipofectamine 2000 (commercially available) If used. (B) When only the gene is added. (C) Compound 1/2 (when n = 6 in X of Compound 2), ultrasonic untreated. (D) The ultrasonic treatment of (c) above. (E) Compound 1/2/3 (in the case of n = 6 in X of Compound 2), ultrasonic untreated. (F) The ultrasonic treatment of (e) above. Note: Compounds 1, 2, and 3 are m = 18, compound 3 is p = 45, y = Me, X = (CH ₂ ) _{n of} compound 2, R = arginyl group Comparison of GFP expression level between commercially available gene transfer agent and composite nanotube. A DNA-tube conjugate (Arg / P = 8/1) was added to KB cells, cultured for 24 hours, and then GFP fluorescence image (a) Compound 1/2/3 (Compound 2 X = (CH ₂ ) ₂ , R = arginyl), unsonicated. (B) Ultrasonication of (a) above (c) Compound 1/2/3 is m = 18, Compound 2 is X═CH ₂ —CO—NH— (CH ₂ ) ₂ , and ultrasonic treatment is not performed. (D) After sonication of (c) above (e) Compound 1/2, X = (CH ₂ ) ₆ , R = lysyl group, unsonicated (d) Compound 1/2, X = (CH ₂ ) ₂ , R = lysyl group, untreated with ultrasound Note: Compounds 1, 2, 3 have m = 18, Compound 3 has p = 45, y = Me Comparison of cytotoxicity between LA2000 (commercial product) and composite nanotube. The horizontal axis shows the total number of proteins in cells without addition as 100%. Compared with LA2000, which binds to the same amount of DNA (2 micrograms / well), and composite nanotubes. In the figure, “soni” is ultrasonically treated, and “non-soni” is ultrasonically untreated.

1. About Organic Nanotubes In the present invention, the term “organic nanotubes” refers to self-assembly of lipids capable of forming directional hydrogen bonds and having asymmetric carbons in addition to hydrophobic interactions derived from the hydrophobic alkylene chain. By doing so, it refers to an aggregate that forms a tube structure having an outer diameter of about 10 nm to 1000 nm and a length of 100 nm to several mm (see Non-Patent Document 8). That is, the “organic nanotube” of the present invention can be defined as “a tubular structure having a nanometer-sized width obtained by self-assembly of lipid molecules from an aqueous solution” as defined above.
Therefore, the “organic nanotube” of the present invention includes not only a case where it is composed of a single lipid component but also a case where it is composed of a plurality of lipid components. In this case, for example, “organic nanotubes obtained by self-assembly from a mixed solution of Compound 1 and Compound 2 or Compound 1, Compound 2 and Compound 3” are particularly referred to as “composite nanotubes”.
The tube structure of “organic nanotubes” is formed by weak interactions such as hydrogen bonds and van der Waals forces. Therefore, by changing the pH and temperature conditions of the environment within the range from room temperature to 100 ° C. It is possible to disassemble. Since this temperature includes a range of environmental temperatures inside and outside cells in a living body, it means that the temperature can be adjusted so as to be decomposed due to a difference in pH environment inside and outside the cells. Such degradation characteristics are one of the advantages of using lipid molecules, and in the case of nanotubes formed by covalent bonds such as carbon nanotubes and silica nanotubes, degradation in such a temperature range is not seen at all. .
In addition, what mixed the nucleic acid and the composite nanotube electrostatically by mixing the composite nanotube of this invention and a nucleic acid by a fixed ratio is called "nucleic acid-tube combination." In general, DNA is often used as the type of nucleic acid, so it may be referred to as a “DNA-tube conjugate”.

2. About lipid compounds (organic tube-forming lipids) that contribute to the formation of organic nanotubes of the present invention (2-1) Characteristics of organic tube-forming lipids: Compound of formula (1) Shape of organic tube-forming lipid molecules of the present invention As described above, one single-chain lipid having one hydrophilic portion and one hydrophobic portion, one double-chain lipid in which two hydrophobic portions are connected to one hydrophilic portion, and one in two hydrophilic portions. A double-headed lipid having a hydrophobic part is known (Non-patent Document 8). As the lipid compound used in the present invention, among the double-headed lipids, “wedge-shaped” lipids having different hydrophilic portions at both ends are effective from the viewpoint of forming stable organic nanotubes. In particular, a wedge-shaped lipid in which a glucose residue is connected to one end of an alkyl chain via an amide group and a carboxylic acid is connected to the other end as in Compound 1 provides a fine organic nanotube structure with an outer diameter of about 40 to 50 nm. Therefore, it is the best (Patent Document 4, Non-Patent Document 9). The chain length m of the alkylene group in the lipid compound represented by the following formula (1) described in Patent Document 4 was 6 to 20, but the chain length of the organic nanotube-forming lipid suitable as a component of the composite nanotube of the present invention m is in the range of m = 12 to 20, and from the viewpoint of good dispersibility during production and high stability of the obtained organic nanotube, an integer of 12 to 18 is more preferable, and it is even. preferable.
<Chemical Formula 1> (Compound 1)
(In the formula, m represents an integer of 12 to 20.)
It is effective for the present invention to form a composite nanotube by mixing a lipid compound capable of forming the organic nanotube described above and a plurality of novel compounds described below and then self-assembling. It has been reported by Shimizu et al. That organic nanotubes can be formed even if the compounds constituting them are a mixture of plural kinds (Non-Patent Document 10).

(2-2) Method for Producing Organic Tube-Forming Lipid (Compound of Formula (1)) The organic tube-forming lipid represented by Formula (1) is a known compound described in Patent Document 4, etc. Although it may be synthesized by a method, for example, it can be synthesized according to the method shown in the following reaction formula (1) (see Patent Document 4).
<Reaction Formula (1)>
[In the reaction formula, “DMT-MM” is an abbreviation of “4- (4,6-dimethoxy-1,3,5-triazin-2-yl) -4-methylmorpholinium chloride”. “DMF” is an abbreviation for “dimethylformamide”, and “Pd—C” is an abbreviation for “palladium-carbon”. The same applies below. ]

  That is, 2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl bromide (Chemical A) is treated with nanothorium azide and then recrystallized to obtain an azide. This is converted into an amine form by catalytic hydrogenation reduction, and polybenzylene carboxylic acid monobenzyl ester of (Formula B) having a benzyl group as a protecting group (wherein m = 12 to 20, preferably 12 to 18, More preferably, it is an even number of 12 to 18, and after dehydration condensation using DMT-MM or the like, it is purified by silica gel chromatography. If the benzyl ester is removed from the resulting benzyl ester intermediate by catalytic hydrogenation reduction and then deacetylated by treatment with sodium methoxide in methanol, the lipid “compound 1” of formula (1) can be obtained.

3. Regarding lipid compounds (nucleic acid-binding lipid compounds) that contribute to the formation of conjugates with nucleic acids (3-1) Features of nucleic acid-binding lipid compounds: compounds of formula (2) Cell membranes that form conjugates with nucleic acids in the present invention In order to obtain a composite nanotube for efficiently transmitting water, a cation such as a guanidyl group or an amino group capable of electrostatic mutual interaction with a phosphate group of a nucleic acid with respect to the lipid (compound 1) of the formula (1) It is necessary to complex the lipid (compound 2) of the following formula (2) having a sex group.
<Chemical Formula 2> (Compound 2)
The alkylene group (polymethylene group) chain length m of Compound 2 is preferably the same chain length as that of Compound 1 in that a composite nanotube is formed by mixing with Compound 1. Specifically, an integer of m = 12 to 20 is preferable, and further, an integer of m = 12 to 18 is more preferable from the viewpoint of high dispersibility at the time of production and high stability of the resultant composite nanotube, More preferably, it is an even number. X is an alkylene chain represented by (CH ₂ ) _n (n is an integer of 2 to 8, n is preferably an integer of 2 to 6 from the viewpoint of good dispersibility), or a hydrogen bonding amide CH ₂ —CO—NH— (CH ₂ ) ₂ having R may be a cationic amino acid residue, but is preferably an arginyl group, a lysyl group, and an ornithyl group, and among them, an arginyl group and a lysyl group are particularly preferable.

(3-2) Method for Producing Nucleic Acid-Binding Lipid Compound (Compound 2) The nucleic acid-binding lipid represented by formula (2) (compound 2) is formed into organic nanotubes as shown in the above <Reaction formula (1)> In addition to the condensation using the acid chloride of Compound B and an amine component similar to the method for synthesizing a functional lipid (described in Patent Document 4), a milder room temperature, DMT-MM capable of reacting near neutrality, An amide bond formation reaction using a condensing agent such as EDC-HCl (“EDC-HCl” is an abbreviation of “1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride”), and subsequent benzyl ester A polymethylene carboxylic acid having a carboxylic acid group at one end protected with a benzyl group can be applied to a deprotection reaction by catalytic hydrogenation in a hydrogen atmosphere at room temperature. acid A method of using a monobenzyl ester (where m = 12 to 20, preferably 12 to 18, more preferably an even number of 12 to 18) as one of the raw materials, and finally removing the benzyl group or the like of the protective group Can be synthesized.

That is, a specific production process of “Compound 2” is as follows.
For a polymethylene dicarboxylic acid having one end represented by the following chemical formula (4) protected with a benzyl group (corresponding to “Compound B (Chemical Formula B)” in the following Reaction Formula 2),
<Chemical formula (4)>
[Wherein m represents the same meaning as in chemical formula (2). ]

After binding one end-protected diamine compound (corresponding to “Compound C (Chemical C)” in the following reaction formula 2), the Boc group is removed, and the resulting terminal amino group is protected with a protected cationic amino group. Add a residue,
<Chemical formula (5)>
[Wherein X represents the same meaning as in chemical formula (2). Here, the Boc group is preferably used as the protecting group, but other protecting groups that can be removed under conditions other than hydrogenation, that is, 9-fluorenylmethyloxycarbonyl group, trifluoroacetyl group, 2,2,2- It may be a trichloroethoxycarbonyl group or the like. Here, the “Boc group” is an abbreviation for “tertiary butoxycarbonyl group”. ]

Alternatively, a pre-protected cationic amino residue is added to the amino group at the free end of the diamine compound represented by the chemical formula (5), and the glycine residue is introduced after removing the protecting group at the other end of the amino group. And a compound having a cationic amino acid residue represented by the following chemical formula (6) (corresponding to “compound E (compound E)” in the following reaction formula 3):
<Chemical formula (6)>
[In formula, X and R show the same meaning as Chemical formula (2). The protecting group here is a benzyloxycarbonyl group.

After obtaining a precursor having a protecting group represented by the following chemical formula (7) by any of the above steps,
<Chemical formula (7)>
[Wherein, m, X and R have the same meaning as in chemical formula (2). ].

By removing all protecting groups, the following “Compound 2” can be obtained.
<Chemical Formula (2)> (Compound 2)
[Wherein, m represents an integer of 12 to 20, X represents an integer of (CH ₂ ) _n and n is 2 to 8, or CH ₂ —CO—NH— (CH ₂ ) ₂ , R represents arginyl] Represents a lysyl or ornithyl residue. ]

Among the “compounds 2”, the case where X in the chemical formula (2) is an alkylene chain represented by (CH ₂ ) _n is represented as “compound 2a”, and X represents “CH ₂ —CO—NH— (CH ₂ )”. The case of “ ₂ ” is referred to as “compound 2b”, and typical production steps of each are shown below as “reaction formula (2)” and “reaction formula (3)”, respectively.
In the case of compound 2b, an active ester of glycine protected with a Boc group and a Z—NH—X precursor—NH ₂ previously protected with a Z group with an analog of (Chemical D) (Boc-Gly-OSu) Can be combined with a cationic amino acid residue R (protecting group-R) having a side chain and an amino group previously protected after the removal of the Z group. Since it is preferable to take the step of “formula (3)” because of high production efficiency, “reaction formula (3)” is shown as a typical example. The method for producing compound 2b of the present invention is not limited to this step.

In the case of Compound 2a: In the case of X = (CH ₂ ) _n (n is an integer of 2 to 8) <Reaction Formula (2)>
[In the reaction formula, “EDC-HCl” is an abbreviation for “1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride”, “EtOAc” is an abbreviation for “ethyl acetate”, and “Et ₃ “N” is an abbreviation for “triethylamine”. ]

That is, “Compound 2a” is a cation in which “Compound B (Chemical B)” and “Compound C (Chemical C)” are dehydrated and condensed with EDC-HCl, the Boc group is removed, and the side chain and the amino group are protected. All of the protective groups after mixing with a protective amino acid residue R ("protecting group -R", where the protecting group is a benzyloxycarbonyl group) and dehydrating condensation to obtain a precursor with a protecting group. Can be produced by deprotection by catalytic hydrogenation reduction or the like.

For compounds _{2b: X = CH 2 -CO-} NH- (CH 2) For ₂ <Reaction Formula (3)>
[In the reaction formula, “Boc-Gly-OSu” represents that the amino group of glycine is protected with the Boc group and the carboxyl group is esterified with the Su group. The Su group is an abbreviation for succinimide group. ]

That is, in order to produce “compound 2b”, first, a cationic amino acid residue in which a side chain and an amino group are protected in advance on “X precursor, ie, ethylene group” in “compound D (compound D)”. R (protecting group-R) is dehydrated and condensed with EDC-HCl to obtain an intermediate. After removing the Boc group of this intermediate, an active ester of glycine protected with the Boc group (Boc-Gly-OSu) is added to obtain “Compound E (Chemical E)”. After removing the Boc group of “Compound E” again, it was subjected to dehydration condensation with “Compound B (Chemical B)”, which is another raw material, to obtain a precursor to which a protecting group was added. To obtain “compound 2b”.

(3-3) Specific synthesis example of compound 2 (3-3-1) When X of compound 2 is (CH ₂ ) _n and R is an arginyl group (“compound 2a-1”), It can be synthesized in the process.
<Reaction Formula (4)>
[In the reaction formula, “Z group” is an abbreviation for benzyloxycarbonyl group. ]

Α, ω-alkyldiamine (for example, N- (Boc) -1,6 hexanediamine) of “Compound F (Chemical F)”, which is protected at one end with a Boc group or the like, with respect to “Compound B (Chemical B)” An intermediate represented by “Compound G (Chemical G)” is obtained after dehydration condensation.
The intermediate “compound G” is acid-treated with hydrochloric acid-ethyl acetate or the like to deprotect the Boc group to obtain an amine derivative as a hydrochloride. This was neutralized with triethylamine and subjected to dehydration condensation with tris (carbobenzoxy) -L-arginine and EDC-HCl to obtain a precursor to which a protecting group was added, and then by catalytic hydrogenation reduction, a benzyl group, Z By simultaneously deprotecting the group, “Compound 2a-1” (X═ (CH ₂ ) _n , R is an arginyl group) can be produced.

(3-3-2) When X of Compound 2 is (CH ₂ ) _n and R is a lysyl group (in the case of “Compound 2a-2”)
By using the same method as in (3-3-1) above and using di (carbobenzoxy) -L-lysine as the amino acid, X = (CH ₂ ) _n , wherein R is a lysyl group, “compound 2a— 2 "can be obtained.

(3-3-3) When X of Compound 2 is CH ₂ —CO—NH— (CH ₂ ) ₂ and R is an arginyl group (in the case of “Compound 2b-1”), it can be synthesized by the following steps.
<Reaction Formula (5)>
Specifically, N-Boc-1,2-diaminoethane and tris (carbobenzoxy) -L-arginine were dehydrated and condensed in DMF in advance to obtain “Compound H (chemical H)”, which was converted into hydrochloric acid-acetic acid. The Boc group is deprotected by acid treatment with ethyl, and the resulting hydrochloride is neutralized with triethylamine, and then reacted with N- (Boc) glycine succinimide ester, which is represented by “Compound I (Chemical I)”. An intermediate is obtained. After this was treated with hydrochloric acid-ethyl acetate to deprotect the Boc group, polymethylenecarboxylic acid monobenzyl ester (m = 12 to 20) of “compound B (chemical B)” as one of the raw materials was added. , Dehydration condensation, and purification by column chromatography, a precursor to which a protecting group is added is obtained, and by simultaneously deprotecting the benzyl group and the carbobenzoxy group by catalytic hydrogenation reduction, “compound 2b-1” (X is CH _{2 -CO-NH- (CH 2)} 2, R is obtained guanidine).

4). About lipid compounds (lipid compounds for preventing aggregation) that contribute to improving the dispersibility of composite nanotubes:
(4-1) Features of anti-aggregation lipid (compound 3) having a polyoxyethylene chain A complex nanotube comprising the organic nanotube-forming lipid (compound 1) and the nucleic acid-binding lipid (compound 2) of the present invention is a nucleic acid. When mixed to form a nucleic acid-tube conjugate by electrostatic interaction, aggregation tends to occur. This is thought to be due to charge neutralization and increase in size due to the combination of the two. In order to suppress aggregation at the time of producing this nucleic acid-tube conjugate (in order to enhance dispersibility), a lipid compound (compound 3) having a polyoxyethylene chain represented by the following formula (3) is converted to compound 1 and compound 2. Is preferably added to the step of producing a composite nanotube to form a composite nanotube containing at least three components (Non-Patent Documents 11 and 12).
<Chemical Formula 3> (Compound 3)
The alkylene group (polymethylene group) chain length m of the compound 3 is preferably the same chain length as that of the compound 1 and 2 in that a composite nanotube is formed. Specifically, an integer of m = 12 to 20 is preferable, and further, an integer of m = 12 to 18 is more preferable from the viewpoint of high dispersibility at the time of production and high stability of the resultant composite nanotube, More preferably, it is an even number.
p represents the repetition of the oxyethylene group, and is desirably an integer of 10 to 200 in order to increase the efficiency of introduction into the composite nanotube to be produced and the dispersibility of the composite nanotube. When p exceeds 200, it becomes difficult to efficiently introduce into the composite nanotubes during production due to phase separation accompanying an increase in water solubility. On the other hand, when the number is less than 10, improvement in dispersibility of the composite nanotube cannot be expected. A similar phenomenon has been reported for liposomes (Non-Patent Documents 11 and 12). That is, the introduction of oxyethylene chains suppresses the association of liposomes and improves dispersibility. Protein adsorption is reduced in liposomes with molecular weight of oxyethylene chain of 700-2000 (16-45 by repetition of oxyethylene groups) in plasma. Further, it has been reported that this effect is saturated when the molecular weight is about 5000 (about 114) and decreases when the molecular weight is 10,000 or more (227 or more).
Y in the formula represents a hydrogen atom or a methyl group.

(4-2) Method for Producing Aggregation-Preventing Lipid (Compound 3) Having Polyoxyethylene Chain The aggregation-preventing lipid (compound 3) having a polyoxyethylene chain is shown in <Reaction Formula (1)> above. Reaction conditions similar to the synthesis method of organic nanotube-forming lipids (as described in Patent Document 4), specifically, dehydration condensation reaction of carboxylic acid component and amine component using EDC-HCl at room temperature or catalytic hydrogenation By applying a deprotection reaction such as debenzylation, it can be synthesized by the following steps.
That is, for a compound of the following chemical formula (4) protected at one end with a benzyl group (corresponding to “compound B (chemical formula B)” of the following reaction formula (6)),
<Chemical formula (4)>
[Wherein m represents the same meaning as in chemical formula (3). ]

Bonding a polyethylene ethylene compound modified at one end with an aminopropyl group represented by the following chemical formula (8),
<Chemical formula (8)>
[Wherein, p and Y represent the same meaning as in chemical formula (3). ]

Next, by removing the benzyl group, a compound of the following formula (3) (compound 3) can be obtained.
<Chemical formula (3)>
[Wherein, m represents an integer of 12 to 20, p represents an integer of 10 to 200, and Y represents a hydrogen atom or a methyl group. ]

The reaction formula is as follows.
<Reaction Formula (6)>
That is, polymethylenecarboxylic acid monobenzyl ester represented by “Chemical B” (m = 12 to 20) and one-end aminopropyl modified polyethylene glycol represented by “Chemical J” (p = 45, Y = methyl group) ) To give an intermediate. Subsequently, the compound 3 can be produced by deprotecting the benzyl group by catalytic hydrogenation reduction.

5. Production of Composite Nanotubes A composite nanotube suitable for a nucleic acid carrier for nucleic acid introduction of the present invention comprises compounds 1 and 2, and preferably a plurality of lipids such as compound 1/2/3 further comprising compound 3 or It consists of a composite nanotube-like aggregate that can be produced by heating and dissolving a composition comprising a pharmacologically acceptable salt in an isopropanol / water solution or ethanol / water solution and evaporating the alcohol from the solution. The composite nanotube can form a nucleic acid-tube conjugate by electrostatic binding with the nucleic acid to be introduced into the cell, and is used as a nucleic acid carrier for introducing the nucleic acid into the cell and exhibiting its function. Therefore, the pharmacologically acceptable salt herein refers to any salt that does not hinder electrostatic binding to nucleic acids and has no harmful effects on the living body such as cytotoxicity. Examples thereof include salts, sulfates, carbonates, phosphates, citrates, and lactates.
By controlling the mixing ratio of each compound constituting the composite nanotube, the density of cationic groups on the composite tube surface can be controlled and the dispersibility can also be controlled. Can be controlled to increase nucleic acid introduction efficiency and minimize cytotoxicity.

(5-1) Method for Producing Composite Nanotubes Composed of Compound 1/2 The method for producing composite nanotubes using Compound 1/2 produced by the above-described method is as follows.
A solid mixture of Compound 1 and Compound 2 adjusted at a molar ratio of 10: 1 to 2: 1 is dissolved in ethanol / water solution, isopropanol / water solution, etc., and nitrogen gas, argon gas, dry air or the like is blown in, or rotary A composite nanotube can be produced by preferentially distilling off low boiling alcohols at room temperature or lower by vacuum concentration using a vacuum evaporator (Non-patent Document 13).
The mixing ratio can be efficiently combined within the above range, but in order to maintain a more homogeneous tube-like form, particularly when m = 18 of compounds 1 and 2, it is preferably around 5: 1.

(5-2) Method for producing composite nanotube comprising compound 1/2/3 When producing the composite nanotube comprising compound 1/2/3, compound 2 and compound 3 are mixed in a solution containing compound 1 Thus, a composite nanotube having both an oxyethylene chain and a cationic functional group on its surface can be constructed (Non-patent Document 13). The dispersibility of the composite nanotube in water is improved by the oxyethylene chain. In particular, when composite nanotubes and nucleic acids are mixed, precipitation due to aggregation of both tends to occur. However, the presence of oxyethylene chains can prevent this precipitation. Increasing the mixing ratio of Compound 3 improves the dispersibility, but no improvement in dispersibility is observed due to the addition exceeding 5 mol%. If it exceeds 5 mol%, the compound 3 partially begins to separate from the composite nanotube, and another form is observed.
When X of compound 2 is an integer of (CH ₂ ) n (n = 2 to 6), compound 1: compound 2: compound 2: compound 3 = 63: 32: 5-87: 8: 5 (molar ratio) It is desirable that Particularly in compounds 1 to 3, when m is all 18, in order to increase the amount of binding of nucleic acid and cause gene expression more efficiently, 77: 18: 5 to 81: 14: 5, Most preferred is around 79: 16: 5.
When X of Compound 2 is CH ₂ —CO—NH— (CH ₂ ) ₂ , Compound 1: Compound 2: Compound 3 = a range of 75: 20: 5 to 87: 8: 5 (molar ratio). Is desirable. If the mixing ratio is in the above range, it can be efficiently complexed. Especially in compounds 1 to 3, when m is all 18, the binding amount of nucleic acid is larger and gene expression is more efficiently caused. Is preferably 77: 18: 5-81: 14: 5, particularly around 79: 16: 5.
Shortening the length of the composite nanotube is important for improving the dispersibility of the nucleic acid-tube conjugate and efficiently permeating the cell membrane. In the case of the composite nanotube used this time, it is possible to shorten the length from a maximum of 10 μm before untreatment to about 100 nm by ultrasonic treatment of the aqueous dispersion.
In the present invention, in order to enhance the dispersibility of the composite tube, it is preferable that the length is about 100 nm to 5 μm by ultrasonic treatment, and it is more preferable that the length is about 100 nm to 2 μm in order to efficiently penetrate the cell membrane. Further, about 100 nm to 800 nm is more preferable.

6). Evaluation of composite nanotubes Concerning the homogeneity of lipid components in composite nanotubes, composite nanotubes composed of compound 1/2 and compound 1/2/3 are formed within the above-mentioned mixing ratio, and other forms are formed. It was confirmed from the transmission image in the observation with a scanning microscope. In a sample subjected to negative staining treatment using phosphotungstic acid or the like, the inner space of the tube is stained black, whereby the tube-like structure can be confirmed. (According to the method described in Non-Patent Document 9 etc.)
The effect of improving the dispersibility of composite nanotubes by introducing compound 3 having an oxyethylene chain is measured by measuring turbidity at 410 to 540 nm under the same concentration conditions as composite nanotubes composed of compounds 1/2/3 and 1/2. Confirmed by doing.

7). Use of the composite nanotube of the present invention as a nucleic acid carrier The composite nanotube of the present invention electrostatically binds to a nucleic acid, transports the nucleic acid into a cell, releases the nucleic acid in the cell, and functions as a nucleic acid. Expression). That is, it can be used as a nucleic acid carrier for introducing a nucleic acid into a cell.
Therefore, the “nucleic acid” referred to here is not limited as long as it can electrostatically bind to the cationic composite nanotube of the present invention, and includes not only DNA but also RNA, and a large-sized gene having physiological activity in vivo ( For example, it may be a gene of various hormones or cytokines to compensate for a deficiency or deficiency in a genetic disease, an antigen polypeptide gene for enhancing immune action, and the like. It may be a short nucleic acid fragment such as siRNA, miRNA or antisense oligonucleotide for acting by a sense mechanism. Typically, these are nucleic acids for gene therapy, and these nucleic acids are usually used in a state of being linked to an appropriate vector such as a plasmid vector, but in the case of gene vaccination or antisense therapy, naked nucleic acids are used. Sometimes. The nucleic acid generally refers to DNA, but RNA may also be used.
In the present invention, the “cell” typically refers to a mammalian cell including a human. Cultured cells in vitro may be used, but for gene therapy, cells in tissues and organs constituting the living body, immune cells such as blood cells and lymphocyte cells in body fluids such as blood, lung cancer cells, etc. Of cancer cells. At that time, it is possible to directly introduce it into the living body, but it is also possible to apply the gene introduction method using the nucleic acid carrier of the present invention to the cells once taken out and return them to the living body again.

8). Method for Producing Nucleic Acid-Tube Conjugate When nucleic acid and the composite nanotube of the present invention are mixed, both of them are caused by electrostatic interaction between an anionic phosphate group in the nucleic acid and a cationic amino group or guanidine group on the tube surface. Join. From the observation of the actually obtained bond by an optical microscope, the tube-like form remains as it is, and a form in which these are associated in a bundle is obtained. The formation of a polyion complex of the composite nanotube and nucleic acid can be confirmed by the disappearance of the free nucleic acid migration band in polyacrylamide electrophoresis.
Also, after mixing DNA stained with a fluorescent dye (YOYO-1 (491 nm excitation, 509 nm emission) and a composite nanotube, the laser confocal microscope matches the observation image (differential interference mode) with the fluorescence image of the DNA. It can be confirmed (according to Non-Patent Document 14).
One of the important functions of the nucleic acid carrier is to protect the nucleic acid from a degradation reaction by a degrading enzyme by complexing or encapsulation. For this evaluation, for example, in the case of DNA, the state of DNA degradation when a DNA-degrading enzyme (Dnase I) or the like is added may be evaluated by a change in absorbance at around 260 nm (according to Non-Patent Document 15). This is because when DNA has a double helix structure and bases are stacked, it has a light color effect, but when this is decomposed, the stack disappears, the light color effect disappears, and the absorbance increases as a result. It is due.

9. Introduction of nucleic acid-tube conjugates into cells, expression of genes and their confirmation Introduction into cells can be carried out using the same technique as in the case of using commercially available cell introduction agents (according to Non-patent Document 16). It was. That is, after culturing the cells in DMEM medium or the like, the DNA-tube conjugate was added, and after culturing for 1 to 2 hours, the excess conjugate was washed and removed several times with PBS buffer as necessary.
Confirmation of introduction can be confirmed by a laser confocal microscope (CLSM). That is, the composite nanotube and DNA were previously stained with fluorescent dyes rhodamine B and YOYO-1. After forming a DNA-tube conjugate using this, it is introduced into cells by the method described above. After introduction, the location of the composite nanotube, DNA, and cell is clarified by fluorescence image observation using CLSM, and it is confirmed that it is taken into the cell.
Semi-quantitative analysis of the amount introduced into the cells was performed by flow cytometry. That is, after introducing the conjugate into the cell, the excess conjugate outside the cell is washed and removed as described above, and then the fluorescence from the YOYO-1-stained DNA is measured by flow cytometry.

10. Confirmation of gene expression in cells Confirmation of protein expression from a reporter gene is often quantitatively evaluated by the luminescence intensity of luciferase expressed by the gene or the fluorescence intensity of GFP. In this example, it was confirmed by fluorescence of expressed GFP. That is, after culturing cells in a DMEM medium or the like as described above, a DNA-nanotube conjugate consisting of a GFP reporter gene (plasmid vector pEGFP) is added and cultured for about 24 hours to transfer the gene into the nucleus and protein Expression of (GFP) was performed. Thereafter, the fluorescence fluorescence from GFP can be evaluated semi-quantitatively by flow cytometry.
Moreover, although there was no quantitative property, it confirmed that GFP was expressed in the cell also by the laser confocal microscope (CLSM).

11. Confirmation of cytotoxicity For cytotoxicity, add a nucleic acid-tube conjugate, culture for a certain period of time, measure the number of cells or something else (intracellular protein content, mitochondrial activity, etc.), and add anything as a control experiment. This can be done by comparing with other systems or commercially available gene transfer agents. As a measurement method at that time, typically, a method of measuring the mitochondrial activity of a cell (MTT assay, Non-patent Document 17) can be used. In this example, after adding and culturing a DNA-tube conjugate to cells, the cells were destroyed, and the amount of total protein was quantified according to the above method.

12 About the composition for gene therapy of the present invention The nucleic acid carrier of the present invention comprises the above-mentioned 7. As described above, a nucleic acid-tube conjugate containing an effective amount of a nucleic acid for gene therapy can be used as a gene therapy composition together with a pharmacologically acceptable carrier. The gene therapy composition includes a gene vaccine composition.
Here, pharmaceutically acceptable carriers include diluents or excipients such as water or physiologically acceptable buffers.
The gene therapy composition of the present invention can be administered orally, such as a tablet, but can also be administered parenterally, such as intravenously or directly into the affected area, or an external preparation.
The dose for administering the gene therapy composition of the present invention to a patient can be appropriately determined according to the type of nucleic acid used, the age, weight and medical condition of the patient to be medically treated, the route of administration, etc. The effective amount of nucleic acid per administration is in the range of 1 μg to 1 g, preferably 100 μg to 10 mg.

13. Others Other terms and concepts in the present invention are based on the meanings of terms that are conventionally used in the field, and various techniques used to implement the present invention are those that clearly indicate the source. Except for, a person skilled in the art can easily and surely implement the method based on known documents and the like. For example, genetic engineering and molecular biology techniques are described in J. Sambrook, EF Fritsch & T. Maniatis, "Molecular Cloning: A Laboratory Manual (2nd edition)", Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989) DM Glover et al. Ed., "DNA Cloning", 2nd ed., Vol. 1 to 4, (The Practical Approach Series), IRL Press, Oxford University Press (1995); Ausubel, FM et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY, 1995; edited by the Japanese Biochemical Society, “Sequel Biochemistry Experiment Course 1, Genetic Research Method II”, Tokyo Chemical Doujin (1986); Laboratory Lecture 2, Nucleic Acid III (Recombinant DNA Technology) ", Tokyo Chemical Doujin (1992); R. Wu ed.," Methods in Enzymology ", Vol. 68 (Recombinant DNA), Academic Press, New York (1980); R. Wu et al. Ed., "Methods in Enzymology", Vol. 100 (Recombinant DNA, Part B) & 101 (Recombinant DNA, Part C), Academic Press, New York (1983); R. Wu et al. ed., "Methods in Enzymology", Vol. 153 (Recomb inant DNA, Part D), 154 (Recombinant DNA, Part E) & 155 (Recombinant DNA, Part F), Academic Press, New York (1987), etc. It can be carried out by substantially the same method or modification method. In addition, various proteins and peptides used in the present invention or DNAs encoding them can be obtained from existing databases (URL: http://www.ncbi.nlm.nih.gov/ etc.).
In addition, the description content in the technical literature, the patent gazette, and the patent application specification cited in this specification shall be referred to as the description content of the present invention.

EXAMPLES Next, although an Example demonstrates this invention further in detail, this invention is not limited at all by these examples.
Example 1 Synthesis of Compound 2 (m = 18, X is (CH ₂ ) ₆ —NH, R is Arginyl Group) <Reaction Scheme 7>
In dichloromethane, N-Boc-1,6 was added to polymethylenedicarboxylic acid (2 g, 4.63 mmol) protected with a benzyl group, which is a compound represented by Compound B (m = 18) such as Reaction Formula (2). -Hexanediamine (1.1 mL, 4.88 mmol) was mixed, EDC-HCl (1 g, 5.21 mmol) was added, and the mixture was stirred at room temperature for 2 hours for coupling. After distilling off the solvent, the solid was recrystallized from methanol (80 mL) containing 10% by volume of water to obtain an intermediate corresponding to compound G (n = 6) in reaction formula (4) as a powder (2 .5g, 86%).
The obtained solid was dissolved in dichloromethane, and treated with 4N hydrochloric acid-ethyl acetate solution (20 mL) to remove the Boc group, thereby removing the amine hydrochloride (1 g, 1.76 mmol). Z-Arg (Z) ₂ —OH (1.19 g, 2.0 mmol) and EDC-HCl (440 mg, 2.3 mmol) were dissolved in dichloromethane, and after cooling to 0 ° C., triethylamine (200 μL) was added. . After gradually raising the temperature and stirring at room temperature for 72 hours, the solvent was distilled off, dissolved in hot methanol, and then cooled to obtain a precursor of Compound 2 having a protecting group as a solid. This was dissolved in DMF, subjected to catalytic hydrogenation reduction using palladium on carbon (10%) for 12 hours, and then filtered through Celite to obtain Compound 2 (800 mg, 76%).
¹ H-NMR data (at room temperature in deuterated DMSO) of this compound is shown in FIG.

Example 2 Synthesis of Compound 2 (m = 18, X is (CH ₂ ) ₂ —NH, R is Arginyl Residue) <Reaction Scheme 8>
Benzyl-protected polymethylene dicarboxylic acid (Compound B, m = 18) (2.5 g, 5.8 mmol) in dichloromethane was added to N-Boc-1,2-diaminoethane (0.99 mL, 6.3 mL). Coupling) was performed by adding EDC-HCl (1.3 g, 6.7 mmol) and stirring at room temperature for 3 hours. After distilling off the solvent, the solid was recrystallized from methanol (80 mL) containing 10% by volume of water to obtain an intermediate corresponding to compound G (n = 2) in reaction formula (4) as a powder (3 0.0 g, 91%).
The obtained solid was dissolved in dichloromethane and treated with 4N hydrochloric acid-ethyl acetate solution (20 mL) to deprotect the Boc group, thereby obtaining the hydrochloride of the amine compound (0.650 g, 1.27 mmol). ), Z-Arg (Z) ₂ —OH (0.80 g, 1.38 mmol), EDC-HCl (300 mg, 1.56 mmol) were dissolved in dichloromethane, cooled to 0 ° C., and triethylamine (180 μL) was added. Added. After gradually heating up and stirring at room temperature for 72 hours, the solvent was distilled off, dissolved in hot methanol, and then cooled to obtain a precursor of Compound 2 having a protecting group as a solid (0.950 g, 75%). This was dissolved in DMF, subjected to catalytic hydrogenation reduction using palladium on carbon (10%) for 12 hours, and then filtered through Celite to obtain Compound 2 (410 mg, 83%).
FIG. 2 shows ¹ H-NMR data (at room temperature in deuterated DMSO) of this compound.

Example 3 Synthesis of Compound 2 (m = 18, X is CH ₂ —CO—NH— (CH ₂ ) ₂ , R is an Arginyl Group)
<Reaction Scheme 9>
Z-Arg (Z) ₂ —OH (2 g, 3.46 mmol) and N-Boc-1,2-diaminoethane (580 microliters, 3.54 mmol) were dissolved in dichloromethane and cooled to 0 ° C. , EDC-HCl (800 mg, 4.0 mmol) was added. After gradually returning to room temperature, the mixture was stirred for 3 hours, and the solvent was distilled off. The obtained solid was reprecipitated in methanol to obtain a compound represented by Compound H in the reaction formula (5) as a white solid (2.1 g, 84%). This was dissolved in dichloromethane / methanol, 4N hydrochloric acid-ethyl acetate solution (15 mL) was added, and the mixture was stirred at room temperature for 1 hr. The solution was concentrated under reduced pressure and then reprecipitated with ethyl ether to obtain a debocylated amine hydrochloride of compound H (1.8 g, 92%). This hydrochloride (1.2 g, 1.83 mmol) was dissolved in dichloromethane and then N-Boc-glycine-succinimide ester (0.50 g, 1.84 mmol) and triethylamine (0.26 ml, 1.9 mmol). And stirred for 2 hours. The reaction solution was concentrated under reduced pressure and purified by silica gel chromatography (eluent: chloroform / methanol = 20/1, volume ratio) to obtain the compound represented by the same compound I (1.1 g, yield 77%). This was dissolved in dichloromethane, 4N hydrochloric acid-ethyl acetate solution (15 mL) was added, and the mixture was stirred at room temperature for 1 hr. The solution was concentrated under reduced pressure and then reprecipitated with ethyl ether to obtain the de-Boc amine salt hydrochloride (0.82 g, 74%). Further, this hydrochloride (0.82 g, 1.15 mmol), Compound B (m = 18, 0.47 g, 1.1 mmol), EDC-HCl (0.24 g, 1.25 mmol) were dissolved in dichloromethane. And stirred at room temperature for 48 hours. Thereafter, the solvent was distilled off, and reprecipitation from a hot methanol solution gave a precursor of Compound 2 with a protecting group as a white precipitate. This was dissolved in DMF, Pd / C was added, catalytic hydrogen reduction was performed for 6 hours, and then Pd / C was removed using Celite. The reaction solution was concentrated under reduced pressure and reprecipitated with diethyl ether to obtain Compound 2 (0.55 g, 84%).
FIG. 3 shows ¹ H-NMR data (at room temperature in deuterated DMSO) of this compound.

Example 4 Synthesis of Compound 2 (m = 18, X is (CH ₂ ) ₆ —NH, R is Lydylyl Residue) <Reaction Scheme 10>
N-Boc-1,6-hexanediamine (1.1 mL, 4.88 mmol) was mixed with polymethylenedicarboxylic acid protected with a benzyl group (compound B) (2 g, 4.63 mmol) in dichloromethane to give EDC. Coupling was performed by adding -HCl (1 g, 5.21 mmol) and stirring at room temperature for 2 hours. After the solvent was distilled off, the solid was recrystallized from methanol (80 mL) containing 10% by volume of water to obtain an intermediate represented by compound G (n = 6) in the reaction formula (4) as a powder (2 .5g, 86%).
This was dissolved in dichloromethane and treated with 4N hydrochloric acid-ethyl acetate solution (20 mL) to obtain hydrochloride of compound 8 (n = 6) (2.1 g, 94%).
Compound 8 hydrochloride (0.5 g, 0.88 mmol), Z-Lys (Z) -OH (0.40 g, 0.97 mmol), EDC-HCl (200 mg, 1.0 mmol) dissolved in dichloromethane After cooling to 0 ° C., triethylamine (140 μL) was added. The temperature was gradually raised, and the mixture was stirred at room temperature for 72 hours. Then, the solvent was distilled off, dissolved in hot methanol, and then cooled to obtain a precursor of Compound 2 to which a protecting group was added as a solid as a white solid ( 0.68 g, 84%). This was dissolved in DMF, subjected to catalytic hydrogenation reduction using palladium on carbon (10%) for 6 hours, filtered through Celite, the solvent was distilled off, and then reprecipitated from ethanol to obtain Compound 2 (45 Milligrams, 10%).
There is no NMR data because it is difficult to dissolve in DMSO.

Example 5 Synthesis of Compound 2 (m = 18, X is (CH ₂ ) ₂ —NH, R is Lydylyl Residue) Polymethylenedicarboxylic acid protected with a benzyl group (Compound B) (2. 5 g, 5.8 mmol) is mixed with N-Boc-1,6-hexanediamine (0.99 mL, 6.3 mmol), and EDC-HCl (1.3 g, 6.7 mmol) is added at room temperature. Coupling was performed by stirring for 3 hours. After distilling off the solvent, the solid was recrystallized from methanol (80 mL) containing 10% by volume of water to obtain an intermediate represented by compound G (n = 2) in reaction formula (4) as a powder (3 0.0 g, 91%).
The obtained solid was dissolved in dichloromethane, and then treated with 4N hydrochloric acid-ethyl acetate solution (20 mL) to obtain the hydrochloride (2.38 g, 90%). Dissolve this hydrochloride (0.5 g, 0.98 mmol), Z-Lys (Z) -OH (0.42 g, 1.0 mmol), EDC-HCl (0.23 g, 1.2 mmol) in dichloromethane. After cooling to 0 ° C., triethylamine (140 μL) was added. The temperature was gradually raised, and the mixture was stirred at room temperature for 72 hours. Then, the solvent was distilled off, dissolved in hot methanol, and then cooled to obtain a precursor of Compound 2 to which a protecting group was added as a solid as a white solid ( 0.69 g, 81%). This was dissolved in DMF, subjected to catalytic hydrogenation reduction using Pd / C (10%) for 6 hours, filtered through Celite, the solvent was distilled off, and then reprecipitated from ethanol to obtain Compound 2 ( 270 mg, 66%).
FIG. 4 shows ¹ H-NMR data (at room temperature in deuterated DMSO) of this compound.

<Reaction Scheme 11>

Example 6 Synthesis of Compound 3 (m = 18, p is about 45, Y is a methyl group) <Reaction Scheme 12>
Polymethylenedicarboxylic acid protected with a benzyl group (m = 18, compound B in reaction formula (6)) (0.20 g, 0.463 mmol) and the same compound J (SUNBRIGHT MEPA-20H manufactured by NOF Corporation, molecular weight 1000, p is about 45, Y is a methyl group. 1 g, 0.458 mmol) was dissolved in dichloromethane at room temperature, and then EDC-HCl (0.106 mg, 0.55 mmol) was added. After stirring for 24 hours, the reaction solution was distilled off and isolated and purified by silica gel chromatography (eluent: chloroform) to obtain a precursor of Compound 3 protected with a benzyl group.
The precursor was subjected to catalytic hydrogen reduction with Pd / C in methanol, and the resulting solid was reprecipitated with diethyl ether to obtain Compound 3 as a white solid (0.80 g, 70%).
FIG. 5 shows ¹ H-NMR data (at room temperature in deuterated DMSO) of this compound.

(Example 7) Preparation of composite nanotube (FIGS. 6 and 7)
(7-1) In the case of a composite nanotube consisting of compound 1/2 and compound 1/2/3 (when X of compound 2 is (CH ₂ ) ₆ and R is arginyl)
Compound 2 (6 mg), compound 3 (10 mg), and if necessary, compound 3 (3.75 mg) are dissolved by heating in 10 mL of isopropyl alcohol / sterile water mixture (1/1, volume ratio). Thereafter, argon gas was blown at room temperature to remove isopropyl alcohol to obtain composite nanotubes. Sterile water was added to this dispersion to reconstitute it to 10 ml. As a result of observation with an electron microscope, composite nanotubes having an outer diameter of about 40 to 60 nm and a length of about 500 nm to several hundreds of μm were obtained (FIGS. 6a and 6c). Moreover, it changed into the composite nanotube of about 40-60 nm in outer diameter and about 500 nm-2 micrometers in length by ultrasonication (FIG. 6 b, d). All the compounds used are m = 18, compound 3: p = about 45, Y = methyl group.
These obtained composite nanotubes were stable dispersions even when stored at 4 ° C. for 1 week. The zeta potential of the obtained tubes is 45.5 and 37.7 mV (composite nanotubes of compound 1/2 and compound 1/2/3, respectively) and both are positively charged and can bind to DNA. It is.

(7-2) In the case of Compound 1/2/3 described above and X of Compound 2 is (CH ₂ ) ₂ By the method described above, the outer diameter is about 40 to 60 nm, and the length varies depending on the ultrasonic treatment. As a result, composite nanotubes of about 200 nm to 2 μm were obtained (FIGS. 6e and f).

(7-3) When X of Compound 2 is CH ₂ —CO—NH— (CH ₂ ) ₂ As described above, the outer diameter is about 40 to 60 nm and the length varies depending on the ultrasonic treatment. , Composite nanotubes of about 100 nm to 1 μm were obtained (FIGS. 7a and b).

(7-4) When X of Compound 2 is (CH ₂ ) ₆ and R is a lysyl group.
Compound 1 (10 mg) and compound 2 (5.7 mg) were dispersed in 10 mL of an isopropanol / sterile water mixed solution (1/1, volume ratio). The pH was adjusted to near neutral with 100 microliters of 0.1 N hydrochloric acid solution, and then dissolved by heating. Thereafter, argon gas was blown to remove isopropyl alcohol to obtain composite nanotubes. Composite nanotubes having an outer diameter of about 40 to 60 nm and a length of about 100 nm to 1 μm were obtained (FIG. 7 c).

(7-5) In the case where X of the compound 2 is (CH ₂ ) ₂ as described above.
Compound 1 (10 mg) and compound 2 (5.1 mg) were dispersed in 10 mL of an isopropanol / sterile water mixed solution (1/1, volume ratio). The pH was adjusted with 50 microliters of 0.1 N hydrochloric acid solution, and then dissolved by heating. Thereafter, argon gas was blown in to remove isopropyl alcohol to obtain composite nanotubes (FIG. 7d).

(Example 8) Evaluation of dispersibility of composite nanotube having oxyethylene chain (FIG. 8)
By introducing the compound 3 into the composite nanotube composed of the compound 1/2, the oxyethylene chain can be introduced to the tube surface and the dispersibility of the composite nanotube can be further improved. This effect was achieved by compound 1/2 (both m = 18, compound 2: (CH ₂ ) ₆ , R: arginyl group, mixing molar ratio 1: 2) and 1/2/3 (in addition to the above compound 3: Evaluation was made by measuring the turbidity at 400 to 500 nm under the same concentration conditions of the composite nanotube consisting of m = 18, p = 45, Y = Me, and the mixing molar ratio = 63: 32: 5) (FIG. 8). And Table 1). As a result, at any wavelength, the composite nanotube containing the compound 3 had lower turbidity, and from this, it was confirmed that the dispersibility could be improved by introducing the oxyethylene chain.

<Table 1> Dispersibility evaluation by turbidity of composite nanotube

(Example 9) Evaluation of binding property between DNA and composite nanotube (FIGS. 9, 10, and 11)
pEGFP-C1 DNA (1 μg) is compounded with compound 1/2 (both m = 18, compound 2: X = (CH ₂ ) ₆ —NH, R = arginyl, mixing molar ratio = 2: 1) or compound 1 / 2/3 (compound 3: m = 18, p = 45, Y = OH, mixing molar ratio = 63: 32: 5) in addition to the above-mentioned composite nanotube solution was converted into Arg / P (arginine residue moles / The number of moles of phosphate groups in the DNA was varied from 1 to 8 and mixed in water. The result of examining the binding property by agarose gel electrophoresis after 5 minutes is shown in FIG. In the binding between the compound nanotube 1/2 nanotube and DNA, Arg / P = 3, most of the DNA is not migrated and remains at the origin, indicating that it is completely bound at this ratio (see FIG. 9 top diagram). On the other hand, in the complex nanotube system of DNA-compound 1/2/3, the binding to DNA is shown to be almost completely bound by Arg / P = 4 (lower diagram in FIG. 9).
After binding with DNA, it was confirmed with a scanning transmission electron microscope that the shape of the tube was maintained (FIG. 10).
Furthermore, in order to evaluate the binding characteristics between DNA and composite nanotubes, observation was performed with a confocal laser microscope (CLSM) after complex formation binding between DNA stained with YOYO-1 and composite nanotubes. The YOYO-1-stained DNA was bound to a composite nanotube (Arg / P = 8) in the same manner as in the above example, and a fluorescent image and a bright field image from the DNA were observed by CLSM. As a result, it was found that fluorescence was given along the contour of the composite nanotube as shown in FIG. In the composite conjugate of compound nanotube 1/2 and compound 1/2, a bundle of thick composite nanotubes (size: about 1 micrometer) derived from the association between tubes was formed, whereas compound 1/2 In the composite complex of / 3 composite nanotube and DNA, such a wide bundle formation has not been confirmed. From this point, it can be confirmed that the dispersibility is improved by the addition of Compound 3 also in the DNA-tube conjugate.

(Example 10) Protection function in DNA-tube conjugate (FIG. 12)
One of the important roles of the nucleic acid carrier is to protect DNA from degradation reactions derived from degrading enzymes or the like in the cytoplasm. Therefore, the degradation resistance to DNase I was examined. The DNA-tube conjugate used was the one prepared above. As a result, as shown in FIG. 12, until 5 minutes after addition of DNase I, the conjugate consisting of the composite nanotube of compound 1/2/3 showed a decrease in absorption due to slight degradation of DNA. After that, it was almost constant. This was almost the same as in the case of commercially available LA2000. Moreover, in the compound nanotube system of compound 1/2, aggregation derived from mixing of DNase was exhibited, and the turbidity was rather lowered, so that sufficient evaluation could not be performed. This seems to be the salting out effect of the Tris buffer used during the measurement. Since the difference between the compound nanotubes of compound 1/2 and 1/2/3 is only the presence or absence of oxyethylene chain, it can be expected that the 1/2 system that causes aggregation also has a protective function from the enzymatic degradation reaction. This result also means that the dispersibility of the DNA-tube conjugate is improved by the introduction of the oxyethylene chain, similar to the evaluation of dispersibility by turbidity in FIG.

(Example 11) Uptake of DNA-tube conjugate into cells (FIG. 13)
For cell uptake, DMEM medium (Dulbecco's Modified Eagle Medium: Dulbecco's modified Eagle medium) with 10% FBS (fetal bovine serum) and 100 μg / ml streptomycin at 37 ° C., 5% CO ₂ atmosphere The cell culture vessel prepared in the above was used. As a control experiment, commercially available LA2000 was evaluated by the same protocol.
The composite nanotubes used were compound 1/2 (both m = 18, compound 2: X = — (CH ₂ ) ₆ —NH—, R = arginyl, mixing molar ratio = 1: 2) or compound 1/2 / 3 (in addition to the above, compound 3: m = 18, p = 45, Y = OMe, mixing molar ratio = 63: 32: 5), respectively, unsonicated or after treatment.
KB cells (human pharyngeal cancer cells) were dispersed in the above 2 ml of DMEM medium (35 mm culture dish) at a concentration of 250,000 cells / well and cultured at 37 ° C. for 24 hours. Thereafter, the culture solution was removed, and a DNA-tube conjugate in which DNA was fluorescently labeled with YOYO-1 was added. As detailed conditions, 2 μg, 10 μL of YOYO-1-labeled DNA was added at a ratio of LA2000 (+ / − = 3/1), and compound ½ composite nanotubes were added in the same manner ( Arg / P = 8/1) and compound 1/2/3 composite nanotubes (Arg / P = 8/1, 5/1) were prepared. These DNA-tube conjugates were diluted to 1 ml with the above DMEM medium and added to the cells. After culturing at 37 ° C. in an atmosphere of 5% CO ₂ for 2 hours, the medium was washed twice with PBS buffer, and Aqua Poly / mount Polyscience Inc. (Warrington, Pennsylvania, USA).
As shown in FIG. 13, in the DNA-tube conjugate (Arg / P = 8) prepared from the composite nanotube of compound 1/2, either fluorescence from rhodamine B from composite nanotube or fluorescence of YOYO-1 from DNA Furthermore, since it showed high fluorescence intensity, it was found that the DNA-tube conjugate showed high uptake into cells.

(Example 12) Semi-quantitative evaluation of uptake (Figure 14)
A DNA-tube conjugate (Arg / P = 8/1) was added to cultured cells (KB cells, A549 cells) and cultured for 2 hours. Thereafter, excess nanotubes were washed three times with PBS (20 U / ml) buffer containing heparin. Then, after being dispersed in a buffer for flow cytometry, by monitoring the fluorescence of YOYO-1-modified DNA in the DNA-tube conjugate using flow cytometry, the DNA-tube conjugate incorporated into the cell A semi-quantitative evaluation of the amount was performed (FIG. 14). As a result, in particular, the DNA-tube conjugate composed of the composite nanotubes of Compound 1/2 showed highly efficient uptake into any cell. Especially in KB cells, it was higher than LA2000. In addition, in the case of the short composite nanotube subjected to the ultrasonic treatment, the uptake efficiency was higher than that of the untreated one. From these points, it was found that the efficiency of gene introduction into cells can be controlled by the presence or absence of oxytylene chains in the composite nanotube and the length of the tube.

(Example 13) Gene expression evaluation in cells (FIGS. 15, 16, and 17)
KB and A549 cells were dispersed in the above 2 ml of DMEM medium (35 mm culture dish) at a concentration of 250,000 cells / well and cultured at 37 ° C. for 24 hours. Thereafter, the culture solution was removed, and a DNA-tube conjugate was added. After culturing at 37 ° C. in a 5% CO ₂ atmosphere for 24 hours, dispersing in a flow cytometry buffer, and then monitoring with fluorescence from GFP expressed using flow cytometry, the gene expression level is semi-quantified. Evaluation was performed (FIG. 15). The composite nanotube used was a composite nanotube of compound 1/2, mixing molar ratio = 2: 1) or compound 1/2/3 (mixing molar ratio = 63: 32: 5, where X = CH ₂ —CO—NH—. In the case of (CH ₂ ) ₂ only 79: 16: 5), each of which was untreated with ultrasonic waves or was treated with DNA was used in combination.
As a result, it was found that the expression level of GFP in KB cells was almost equivalent to LA2000 when any composite nanotube was used. However, although GFP was expressed in A549 cells, it was suggested that it was slightly lower than LA2000.

  16 and 17 show the green fluorescent protein (GFP) expressed by the transgene expression of the cells obtained in the above-described genetic expression experiment, visualized and observed with a fluorescence microscope under the same observation conditions. A fluorescence image is given along the cell, which indicates that the introduced GFP gene is expressed in the cell as a protein. That is, it strongly suggests that the DNA-tube conjugate is expressed by transferring to the cytoplasm and nucleus.

Example 14 Cytotoxicity of DNA-tube conjugate (FIG. 18)
Commercially available Lipofectamine was mixed with 2 micrograms of DNA at the charge ratio indicated in the figure or at the ratio of arginine / phosphate groups. Lipofectamine was mixed at + / − = 3, and for composite nanotubes at Arg / P = 8. The mixing time was 5 to 30 minutes and then added to the cells. Twenty-four hours after addition, the medium was removed, and the cells were washed twice with 20 units / mL heparin in order to remove excess DNA-tube conjugate and lipofectamine. The cells were covered with 250 μL of Lysis buffer and incubated for 10 minutes at room temperature. The cells were crushed and the solution was transferred to a 1.5 milliliter tube. This was frozen at −80 ° C. and returned to room temperature to completely break the cells. After centrifugation at 12000 g for 5 seconds, the supernatant was isolated and compared as cell toxicity by a method of quantifying the amount of protein by the BCA method. BSA was used as a calibration curve.
The composite nanotubes used were compound 1/2 (both m = 18, R = arginyl, mixing molar ratio = 2: 1) or compound 1/2/3 (in addition to the above, compound 3: m = 18, p = 45, Y = OMe, mixing molar ratio = 63: 32: 5, but only when X = CH ₂ —CO—NH is 79: 16: 5), respectively, unsonicated or after treatment.
As a result, at the same DNA concentration, it became clear that LA2000 and composite nanotubes had almost the same protein amount and almost no toxicity.

Claims (11)

A lipid compound (compound 2) or pharmacology of the following chemical formula (2) having a nucleic acid binding property together with a lipid compound (compound 1) of the following chemical formula (1) or a pharmacologically acceptable salt thereof capable of forming an organic nanotube. A composite nanotube comprising an organically acceptable salt thereof and forming an organic nanotube structure;
<Chemical Formula (1)> (Compound 1)
[In formula, m is an integer of 12-20. ]

<Chemical Formula (2)> (Compound 2)
[Wherein, m represents an integer of 12 to 20, X represents an integer of (CH ₂ ) _n and n is 2 to 8, or CH ₂ —CO—NH— (CH ₂ ) ₂ , R represents arginyl] Represents a lysyl or ornithyl residue. ].   The lipid compound of the chemical formula (1) and the lipid compound of the chemical formula (2), or each pharmacologically acceptable salt thereof are included in a molar ratio of 2: 1 to 10: 1. The composite nanotube according to claim 1. The composite nanotube according to claim 1 or 2, further comprising a lipid compound (compound 3) having a polyoxyethylene residue and having the following chemical formula (3) or a pharmacologically acceptable salt thereof:
<Chemical Formula (3)> (Compound 3)
[Wherein, m represents an integer of 12 to 20, p represents an integer of 10 to 200, and Y represents a hydrogen atom or a methyl group. ].   The lipid compound represented by the chemical formula (1), the lipid compound represented by the chemical formula (2), the lipid compound represented by the chemical formula (3), or the pharmacologically acceptable salt thereof, in terms of mole, 63: 32: 5 The composite nanotube according to claim 3, comprising a mixing ratio of 87: 8: 5. A lipid compound of the following chemical formula (2) having a nucleic acid binding property or a pharmacologically acceptable salt thereof, which can be used as a constituent component of the composite nanotube according to any one of claims 1 to 4;
<Chemical formula (2)>
[Wherein, m represents an integer of 12 to 20, X represents an integer of (CH ₂ ) _n and n is 2 to 8, or CH ₂ —CO—NH— (CH ₂ ) ₂ , R represents arginyl] Represents a lysyl or ornithyl residue. ]. A lipid compound of the following formula (3) having a polyoxyethylene residue, or a pharmacologically acceptable salt thereof, which can be used as a component of the composite nanotube according to claim 3 or 4;
<Chemical formula (3)>
[Wherein, m represents an integer of 12 to 20, p represents an integer of 10 to 200, and Y represents a hydrogen atom or a methyl group. ].   A nucleic acid carrier comprising the composite nanotube according to any one of claims 1 to 4 and capable of forming a nucleic acid-tube conjugate by electrostatically binding to a nucleic acid.   A nucleic acid-tube conjugate in which the composite nanotube according to any one of claims 1 to 4 and a nucleic acid are electrostatically bound, and the nucleic acid can be transported into a cell.   A method for transforming a cell, comprising using the nucleic acid carrier according to claim 7 and transporting the nucleic acid electrostatically bound to the nucleic acid carrier into the cell.   A transformed cell transformed by the transformation method of claim 9.   A gene therapy composition comprising the nucleic acid-tube conjugate according to claim 8 and an effective amount of a gene therapy nucleic acid and a pharmacologically acceptable carrier.