JP4701414B2 - Nucleic acid molecule delivery carrier - Google Patents

Description

  The present invention relates to a nucleic acid molecule delivery carrier formed from a sugar-containing copolymer.

  Recently, gene therapy, which has been attracting attention, is mainly implemented by the “ex vivo gene transfer method” in which a foreign gene is introduced into cells taken from the patient's body in a culture system, and the transformed cells are expanded and then re-transplanted into the patient. ing. In this case, since the cells that can be isolated from the patient are limited, peripheral blood lymphocytes are often used. However, target cells vary depending on the target disease, and in particular, when targeting somatic cells or organ tissue cells, it is necessary to administer plasmid DNA encoding a foreign gene directly to the living body (In vivo gene). Introduction method).

  In vivo gene transfer methods have been studied by many researchers, and development of a nucleic acid molecule delivery carrier that can efficiently introduce a gene into a living body and ensure safety is strongly desired.

  On the other hand, adenoviruses and retroviruses are used in 85% or more of gene therapy protocols approved by the US FDA (see Non-Patent Document 1). Although the gene transfer effect by the virus is extremely high and effective, the risk of virus propagation has been pointed out in the gene therapy conducted in the United States, such as an accident that may be caused by an immune response to the virus.

  Therefore, the use of non-viral carriers such as cationic liposomes (see Non-Patent Document 2) and cationic polymers (see Non-Patent Document 3 and Non-Patent Document 4) as nucleic acid molecule delivery carriers has been studied. Specifically, conventionally, linear polycations such as diethylaminoethyldextran (DEAE-dex) and poly-L-lysine (PLL) have been studied.

  The gene expression efficiency of polycations is lower than that of liposomes, but has the advantage that it is less likely to accumulate in the liver when administered directly in vivo than liposomes, and pharmacokinetics is relatively easy to control. However, poly-L-lysine, which is one of polycations and has been studied vigorously as a carrier for nucleic acid molecule delivery, has a low efficiency of gene introduction.

In view of such problems, the present inventors have invented a technology relating to a sugar-containing copolymer having a specific structure and a nucleic acid molecule delivery carrier formed from the sugar-containing copolymer, and previously filed a patent application. Although it went (refer patent document 1), the further improvement of gene expression efficiency etc. were calculated | required.
JP 2004-26866 A Annu. Rev. Microbiol., 49, 807, 1995 Proc. Natl. Acad. Sci. USA, 84, 7413, 1987 Proc. Natl. Acad. Sci. USA, 92, 7297, 1995 Bioconjugate Chem., 6, 7, 1995

  The main object of the present invention is to provide a carrier for nucleic acid molecule delivery, which is formed from a sugar-containing copolymer and has excellent gene expression efficiency.

  As a result of intensive studies on a carrier for nucleic acid molecule delivery capable of efficiently performing gene expression, the present inventor has excellent effects when it is formed using a sugar-containing copolymer having a specific structure. It has been found that a carrier for molecular delivery can be obtained, and further studies have been made to complete the present invention.

That is, the present invention relates to the following nucleic acid molecule delivery carrier.
1. Formula (I)

(In the formula, R ₂ represents H or CH ₃ .

Y is, -C (= O) O- ( CH 2) n y -, - OC (= O) - (CH 2) n y -, - OC (= O) - (CH 2) n y -C ( = O), or -CONH- _(CH 2) n _y - shows the. n _y is an integer of 1 to 10.

Z represents —NR ₃ R ₄ (R ₃ and R ₄ are the same or different and each represents a hydrocarbon group having 1 to 10 carbon atoms), —N ⁺ R ₅ R ₆ R ₇ (R ₅ , R ₆ And R ₇ are the same or different and each represents a hydrocarbon group having 1 to 10 carbon atoms.) Or a nitrogen-containing heterocyclic group. )
A repeating unit (A) having a cationic group represented by formula (II):

(In the formula, R ₁ represents —H or —CH ₃ .
Sugar is a single NH ₂ from sugar (when sugar is bound by the amino group of an amino sugar, monosaccharide, disaccharide or polysaccharide) or OH (sugar is bound by a sugar hydroxyl group, a monosaccharide or disaccharide) Alternatively, the sugar residues excluding polysaccharides) are shown.
X is —C (═O) Z ¹ —, —C (═O) O—R _a —, —CONH—, —CONH—R _b —, —OC (═O) —R _c —C (═O ) Z ¹ -or -Ph-R _d -Z ^1- . here,
R _a is -Ph-O-, or - _(CH 2) shows a n _a -O-. n _a represents an integer from 1 to 10.
R _b represents —Ph—O— or — (CH ₂ ) n _b —O—. n _b represents an integer of 1 to 10.
The _{R c - (CH 2) n} c -, or _{- (CH 2) n c -Ph-} (CH 2) n c - shows the. n _c is 2 to 18, preferably an integer of 2 to 10.
R _d represents —CH ₂ — or —SO ₂ —.
Z ¹ represents —O— or —NH—.
Ph represents an (o-, m- or p-) phenylene group. ).
A sugar-containing copolymer having a repeating unit (B) containing a sugar represented by formula (wherein the repeating unit represented by the general formula (I) is represented by the following formula (IA)

(In the formula, n represents an integer of 1 to 10.)
When the molar ratio of the repeating units represented by the general formulas (IA) and (II) is 1: 9 to 9: 1 and the weight average molecular weight is 1 × 10 ^{4 to} 5 × 10 ⁸ except for the sugar-containing copolymer. )
A nucleic acid molecule delivery carrier comprising:
2. A saccharide-containing copolymer having a repeating unit (A) having a cationic group, a repeating unit (B) containing a sugar (Sugar), and a repeating unit (C) having a hydrophobic substituent, The repeating unit (A) having the general formula (I)

(In the formula, R ₂ represents H or CH ₃ .

Y is, -C (= O) O- ( CH 2) n y -, - OC (= O) - (CH 2) n y -, - OC (= O) - (CH 2) n y -C ( = O), or -CONH- _(CH 2) n _y - shows the. n _y represents 1 to 10.

Z represents —NR ₃ R ₄ (R ₃ and R ₄ are the same or different and each represents a hydrocarbon group having 1 to 10 carbon atoms), —N ⁺ R ₅ R ₆ R ₇ (R ₅ , R ₆ And R ₇ are the same or different and each represents a hydrocarbon group having 1 to 10 carbon atoms.) Or a nitrogen-containing heterocyclic group. )
The repeating unit (B) containing sugar (Sugar) is represented by the general formula (II):

(In the formula, R ₁ represents —H or —CH ₃ .
Sugar is a single NH ₂ from sugar (when sugar is bound by the amino group of an amino sugar, monosaccharide, disaccharide or polysaccharide) or OH (sugar is bound by a sugar hydroxyl group, a monosaccharide or disaccharide) Alternatively, the sugar residues excluding polysaccharides) are shown.
X is —C (═O) Z ¹ —, —C (═O) O—R _a —, —CONH—, —CONH—R _b —, —OC (═O) —R _c —C (═O ) Z ¹ -or -Ph-R _d -Z ^1- . here,
R _a is -Ph-O-, or - _(CH 2) shows a n _a -O-. n _a represents an integer from 1 to 10.
R _b represents —Ph—O— or — (CH ₂ ) n _b —O—. n _b represents an integer of 1 to 10.
The _{R c - (CH 2) n} c -, or _{- (CH 2) n c -Ph-} (CH 2) n c - shows the. n _c is 2 to 18, preferably an integer of 2 to 10.
R _d represents —CH ₂ — or —SO ₂ —.
Z ¹ represents —O— or —NH—.
Ph represents an (o-, m- or p-) phenylene group. ).
The carrier for nucleic acid molecule delivery represented by these.
3. The repeating unit (B) containing a sugar is represented by the following general formula (III)

In the formula, Sugar is one NH ₂ from sugar (when Sugar is monosaccharide, disaccharide or polysaccharide bonded with amino group of amino sugar) or OH (Sugar is bonded with hydroxyl group of sugar) (In the case of saccharide, disaccharide or polysaccharide), the sugar residue is excluded, m is an integer of 2 to 10. Z is O or NH.)
The carrier for nucleic acid molecule delivery according to claim 1, wherein the carrier is a repeating unit represented by the formula:
4). The repeating unit (A) having a cationic group is represented by the following general formula (IV):

(In the formula, n represents 1 to 10. R ^a and R ^b may be the same or different and represent an alkyl group having 1 to 4 carbon atoms.)
The carrier for nucleic acid molecule delivery according to any one of claims 1 to 3, wherein the carrier is a repeating unit represented by the formula:
5. The carrier for nucleic acid molecule delivery according to any one of claims 2 to 4, wherein the repeating unit (C) having a hydrophobic substituent is a repeating unit represented by the following general formula (V).
Formula (V):

(Wherein R ₈ represents —H or —CH ₃ .
W is -C (= O) O -, - OC (= O) -, - OC (= O) - and _{(CH 2) n w -C (} = O) O-, or -C (= O) NH Show. n _w is 2 to 18, preferably an integer of 2 to 10.
R ₉ represents a saturated or unsaturated aliphatic or alicyclic hydrocarbon group having 3 to 30 carbon atoms. )
6). The nucleic acid molecule delivery according to any one of claims 1 to 5, wherein the molar ratio of the repeating unit (A) to the repeating unit (B) in the sugar-containing copolymer is A: B = 1: 99 to 99: 1. Carrier.
7). The molar ratio of the repeating unit (C) to the repeating unit (A) + the repeating unit (B) in the sugar-containing copolymer is (A + B): C = 99.9: 0.1 to 0.1: 99.9. 7. The nucleic acid molecule delivery carrier according to any one of Items 2 to 6.
8). The carrier for nucleic acid molecule delivery according to any one of claims 1 to 7, wherein the sugar-containing copolymer has a weight average molecular weight of 10,000 to 1,000,000.
9. Item 9. A transfection reagent using the nucleic acid molecule delivery carrier according to any one of Items 1 to 8.
10. Item 9. A carrier for gene therapy using the carrier for nucleic acid molecule delivery according to any one of Items 1 to 8.

  As shown in the following results, it was revealed that the carrier for nucleic acid molecule delivery formed from the sugar-containing copolymer of the present invention exhibits high gene expression efficiency.

  The carrier for nucleic acid molecule delivery according to the present invention can appropriately introduce a nucleic acid molecule into a cell according to the cellular uptake mechanism, and is particularly suitable for nucleic acid molecule delivery by receptor-mediated endocytosis. ing.

  A complex (complex of a nucleic acid molecule and a sugar-containing copolymer) incorporated by endocytosis mediated by a receptor has a high ability to migrate into the nucleus. Delivery into the nucleus is particularly efficient.

  Moreover, the delivery carrier for nucleic acid molecules of the present invention can be suitably used particularly as a carrier for gene delivery. The gene carried by the delivery carrier for nucleic acid molecules of the present invention is gene-expressed with high efficiency after being delivered into the cell. In particular, the carrier for nucleic acid molecule delivery of the present invention, which is formed from a sugar-containing copolymer having a repeating unit having a hydrophobic substituent, has a high DNA dissociation ability and gene expression of a gene incorporated into cells. Can be performed particularly efficiently.

  The nucleic acid molecule delivery carrier of the present invention has such excellent characteristics and can be effectively used for gene transfer into various cells.

  The carrier for nucleic acid molecule delivery of the present invention comprises a repeating unit (A) having a cationic group and a sugar-containing copolymer having a repeating unit (B) containing a sugar, or repeating units (A) and (B). In addition, it is formed by a sugar-containing copolymer having a repeating unit (C) having a hydrophobic substituent.

Repeating unit (A) having a cationic group
The structure of the repeating unit having a cationic group can be appropriately set as desired, but in the present invention, a structure represented by the following general formula (I) is preferable.

(Wherein R ₂ , Y and Z are as defined above.)
In one preferred embodiment of the present invention, the repeating unit (A) having a cationic group is represented by the following general formula (IV):

(Wherein R ^a , R ^b and n are as defined above.)
It is represented by

Examples of the hydrocarbon group represented by R _{3 to} R ₇ include a linear or branched saturated hydrocarbon group or unsaturated hydrocarbon group, or a cyclic hydrocarbon group. Examples of the cyclic hydrocarbon group include an aromatic hydrocarbon group and an alicyclic hydrocarbon group. Specific examples of the hydrocarbon group include a methyl group, an ethyl group, a propyl group, and a butyl group. R3 and R4 or R5 and R6 can be combined with the nitrogen atom to which they are bonded to form a pyrrolidine ring or piperidine ring.

  Examples of the nitrogen-containing heterocyclic group include pyrrolidinyl, piperidyl, piperidino, piperazinyl, N-methylpiperazino, morpholino, pyrrolyl, imidazolyl, pyridyl, pyrimidinyl, imidazolidinyl, quinolyl, isoquinolyl and the like.

Examples of the alkyl group having 1 to 4 carbon atoms represented by R ^a and R ^b include straight chain or branched such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl An alkyl group having

  The repeating unit can be obtained by polymerizing a monomer having a cationic group.

As a monomer having a preferable cationic group, for example,
_{CH 2 = CH-C (=} O) O- (CH 2) 3 -N (CH 3) 2,
_{CH 2 = CH-C (=} O) O- (CH 2) 3 -N + (CH 3) 3,
_{CH 2 = CH-OC (=} O) - (CH 2) 3 -N (CH 3) 2,
_{CH 2 = CH-OC (=} O) - (CH 2) 3 -N + (CH 3) 3,
_{CH 2 = CH-CONH- (CH} 2) 3 -N (CH 3) 2,
_{CH 2 = CH-OC (=} O) - (CH 2) 3 -C (= O) O-N (CH 3) 2,
_{CH 2 = CH-OC (=} O) - (CH 2) 3 -C (= O) O-N (CH 2 CH 3) 2
Etc.

Repeating unit (B) containing sugar
The structure of the repeating unit containing a saccharide can be appropriately set as desired. In particular, a structure represented by the following general formula (II) is preferably used.
General formula (II):

(Wherein R ₁ , X and Sugar are as defined above).

In the general formula (II), “Sugar” refers to one NH ₂ (in the case of amino sugar) or one OH (in the case of most sugars other than amino sugar) from sugar (monosaccharide, disaccharide, polysaccharide). The sugar residue is shown by removing. When the sugar residue (Sugar) is bonded with an OH group, in the case of hexose, it binds to the anomeric carbon (position 1) or the hydroxyl group at position 2 or the primary hydroxyl group at position 6 and corresponds to these in addition to hexose. Join at the position you want. Further, if the sugar residue (Sugar) binds with NH ₂ group, for example, carbon glucosamine, galactosamine, and binding of the NH ₂ group, such as mannosamine is usually 2-position.

Sugar includes both a sugar represented by Sugar- (OH) and an amino sugar represented by Sugar- (NH ₂ ).

  The type of sugar represented by Sugar- (OH) is not particularly limited. For example, glucose, fructose, mannose, galactose, xylose, erythrose, sorbose, ribose, ribulose, xylulose and other monosaccharides, sucrose, maltose, lactose Cellobiose, agarobiose, isomaltose, xylobiose, gentiobiose, cordobiose, sofolose, taranose, trehalose and other disaccharides, maltotriose, raffinose, lacto-N-tetraose, dextrin, amylose, amylopectin, chitosan, starch, cellulose, α-cyclo Examples thereof include polysaccharides such as dextrin, β-cyclodextrin, γ-cyclodextrin, and hydrolysis products of polysaccharides. They may be natural or synthetic sugars.

Examples of the amino sugar represented by Sugar- (NH ₂ ) include monosaccharides such as glucosamine, galactosamine, mannosamine, neuraminic acid, and disaccharides or polysaccharides having these amino monosaccharides.

  The type of sugar can be appropriately set according to the type of biological sample suitable for the carrier for nucleic acid molecule delivery. For example, when a gene is introduced into a cell having a galactose receptor such as a hepatocyte, galactose is preferably used.

  The repeating unit (B) containing a sugar can be obtained by polymerizing the corresponding sugar-containing monomer.

  Preferred sugar-containing monomers include, for example, vinyladipoyl-D-Galactose, vinyladipoyl-D-Glucose, acryloylgalactose, acryloylglucose, vinylsebacilglucose, azide Poil mannose etc. are mentioned.

Repeating unit (C) having a hydrophobic substituent
The structure of the repeating unit having a hydrophobic substituent can be appropriately set as desired. Particularly, a structure represented by the following general formula (V) is preferably used.
Formula (V):

(Wherein R ₈ , R ₉ and W are as defined above).

Examples of the hydrocarbon group for R ₉ include linear or branched saturated hydrocarbon groups (propyl, n-butyl, isobutyl, t-butyl, pentyl, hexyl, octyl, decyl, dodecyl, tetradecyl, hexadecyl, octadecyl (stearyl). ) alkyl group)) or an unsaturated hydrocarbon group (allyl _C 3 _{-C 30,} such as eicosyl, butenyl, pentenyl, hexenyl, octenyl, decenyl, dodecenyl, tetradecenyl, hexadecenyl, octadecenyl, _C 3 _{-C 30,} such as Aikoseniru Alkenyl group, etc.), or a cyclic hydrocarbon group. Examples of the cyclic hydrocarbon group include aromatic hydrocarbon groups (eg, phenyl, xylyl, toluyl, naphthyl, anthranyl, phenanthryl), saturated or unsaturated alicyclic hydrocarbon groups (cyclopentyl, cyclohexyl, cyclohexenyl, cholesteryl, Group derived from plant sterols, etc.). Examples of plant sterols include stigmasterol, stigmasteranol, sitosterol (α ₁ , β, γ, etc.), campesterol, and the like. R ⁹ is preferably stearyl, palmityl, pentadecanoyl, heptadecanoyl or cholesteryl.

  The repeating unit (C) having a hydrophobic substituent can be obtained by polymerizing a monomer having a hydrophobic substituent.

  Examples of the monomer having a hydrophobic substituent include acrylic acid esters of higher fatty acids having 10 to 20 carbon atoms, vinyl esters of higher fatty acids having 10 to 20 carbon atoms, vinyl monomers having a cholesterol ring, and the like. It is done. More specifically, examples include stearyl acrylate and vinyl monomers having a cholesterol ring represented by the following general formula (VI).

  Formula (VI)

(In the formula, n1 represents an integer of 2 to 18, preferably 2 to 10.)
The cholesterol derivative can be easily synthesized by a method using an ester-type polymerizable substituent and an enzyme.

  An organic solvent such as tetrahydrofuran, toluene, pyridine, or dioxane is used as the solvent, and a lipase derived from Pseudomonas sp. (LPL-311), an immobilized enzyme of LPL-311 (LIP-301), or the like is used as the enzyme. Examples of the polymerizable substituent include divinyl dicarboxylates such as divinyl succinate, divinyl adipate, and divinyl sebacate. Similar to cholesterol, the corresponding plant sterols can be similarly synthesized using the corresponding plant sterols.

  The vinyl ester used in the present invention has a polymer chain of polyvinyl alcohol after polymerization. Polyvinyl alcohol has long been used as a biomaterial. Safety is high.

Sugar-containing copolymer In the present invention, the sugar-containing copolymer having the repeating unit (A) and the repeating unit (B) is obtained by copolymerizing a monomer having a cationic group and a sugar-containing monomer. Obtainable. In the present invention, the copolymer containing the repeating unit (A), the repeating unit (B), and the repeating unit (C) includes a monomer having a cationic group, a sugar-containing monomer, and a hydrophobic substituent. It can obtain by copolymerizing the monomer which has this.

  As a method of copolymerizing each monomer, a known method can be appropriately used. For example, it can be performed by radical polymerization using a polymerization initiator.

  As the polymerization initiator, a normal radical polymerization initiator can be used. For example, an azo initiator such as azoisobutyl nitrile (AIBN), an organic peroxide, or the like can be used.

  The molar ratio of each repeating unit in the sugar-containing copolymer can be appropriately set for the purpose of obtaining a sugar-containing copolymer or a nucleic acid molecule delivery carrier having desired properties.

  Usually, in the copolymer composed of the repeating unit (A) and the repeating unit (B), the molar ratio of (A) to (B) is about A: B = 1: 99 to 99: 1, preferably 10: It is about 90-90: 10, More preferably, it is A: B = 15-85: 50-50, More preferably, it is A: B = 20-80: 30-70.

  Moreover, when it has a repeating unit (C), the molar ratio of the repeating unit (C) with respect to the sum total of a repeating unit (A) and a repeating unit (B) is A + B: C = 99.9: 0.1-0.1. : About 99.9, preferably about 99.5: 0.5 to 0.5: 99.5. In addition, the ratio of each repeating unit (A) and (B) of the sum (A + B) of the repeating unit (A) and the repeating unit (B) is the ratio of the repeating unit (A) and the repeating unit (B). It is the same as the ratio of A and B above. More preferably, (A + B): C = 99.5 to 0.5: 90 to 10; and still more preferably (A + B): C = 99.3 to 0.7: 95 to 5.

  The molecular weight of the sugar-containing copolymer can be appropriately set as desired, but is usually about 10,000 to 1,000,000, preferably about 15,000 to 50,000, more preferably about 20,000 to 50,000 in terms of weight average molecular weight.

  Further, the sugar-containing copolymer of the present invention may contain other repeating units or structural units other than the repeating unit (A), (B) or (C) within the scope of the object of the present invention.

Nucleic acid molecule delivery carrier The nucleic acid molecule delivery carrier of the present invention is formed using the sugar-containing copolymer of the present invention.

  The sugar-containing copolymer in the nucleic acid molecule delivery carrier condenses the nucleic acid molecules by electrostatic action to form a complex with the nucleic acid molecules. This complex is taken up by the cell and the nucleic acid molecule is delivered into the cell.

  A method of forming a complex of a sugar-containing copolymer and a nucleic acid molecule can be appropriately performed by a known method. For example, a solution containing a nucleic acid molecule and a solution containing a sugar-containing copolymer are mixed. Can be adjusted.

  Examples of cells into which nucleic acids are introduced include mammalian cells including humans, animal cells such as insect cells, plant cells, bacteria such as yeast and Escherichia coli, fungi and the like, preferably animal cells, particularly mammalian cells. is there.

  Introduction into cells can be carried out by allowing the cell introduction agent of the present invention and nucleic acid molecules such as DNA and RNA to act on the cells. The cell introduction agent and nucleic acid molecules such as DNA and RNA may form a complex in advance and then act on the cell to introduce the nucleic acid.

  The ratio of the sugar-containing copolymer to the nucleic acid molecule in the complex is the ratio of the number of moles of cationic groups (C) of the sugar-containing copolymer to the number of moles of phosphate groups of DNA or RNA (A). It is usually 0.5 or more, preferably 1.0 or more, and more preferably 1.5 or more.

  It is known that cells have a phenomenon called endocytosis in which high molecular weight proteins and the like are encapsulated by part of the cell membrane. Endocytosis includes liquid phase endocytosis with no specificity for a ligand and receptor-mediated endocytosis (RME). The former is non-specific and has a low uptake rate, so it is not useful as a substance transport into the cell. The latter uses this mechanism to recognize a low concentration of ligand and efficiently take it into the cell. The applicability of delivery carriers for nucleic acid molecules is high. RME is known to be a mechanism existing in many cells such as liver, kidney, small intestine, lung, muscle, fat cell, placenta, erythrocyte, leukocyte fiber. Although each ligand has a different ligand, galactose and N-acetylgalactosamine-mediated uptake of liver parenchymal cells and mannose-mediated uptake of liver non-parenchymal cells are well known. The carrier for nucleic acid molecule delivery of the present invention is particularly suitable for nucleic acid molecule delivery by endocytosis (RME) mediated by such a receptor.

  The kind of nucleic acid molecule carried by the nucleic acid molecule delivery carrier of the present invention is not particularly limited, and examples thereof include DNA or RNA. More specifically, a gene encoding a specific protein, an antisense DNA, a plasmid, an expression construct containing the gene, iRNA and the like can be mentioned.

  The nucleic acid molecule delivery carrier of the present invention can be suitably used particularly as a gene delivery carrier. The gene carried by the delivery carrier for nucleic acid molecules of the present invention is gene-expressed with high efficiency after being delivered into the cell.

  In particular, a copolymer having a hydrophobic repeating unit (C) is particularly preferable because the nucleic acid and the introduction agent are separated in the cell after introduction into the cell, and the expression efficiency of the introduced nucleic acid is increased.

  Hereinafter, the present invention will be described more specifically with reference to Examples, Experimental Examples, and Comparative Examples, but the present invention is not limited to these Examples.

Example 1:
Poly (dimethylaminopropylacrylamide-co-6-o-vinyladipoil-D-galactose) Poly (dimethylaminopropylacrylamide-co-6-O-vinyladipoyl-D-galactose)
Or gene expression by poly (dimethylaminopropylacrylamide-co-6-O-vinyladipoyl-D-glucose) -formed carrier for nucleic acid molecule formation using Poly (dimethylaminopropylacrylamide-co-6-o-vinyladipoil-D-glucose) Example.

(1-1) Synthesis of sugar-containing copolymer
Poly (dimethylaminopropylacrylamide-co-6-o-vinyladipoil-D-galactose) (poly (dimethylaminopropylacrylamide-co-6-O-vinyladipoil-D-galactose)) was prepared as follows.

  DMAPAA (dimethylaminopropylacrylamide) and 6-O-vinyladipoyl-D-galactose (6-O-vinyladipoyl-D-galactose) are used as monomers, and 2,2-Azobis (4-methoxy-2,4-dimethylbaleronitrile) ( AMDVN) was added as an initiator in an amount of 1.0 mol%, and dimethylsulfoxide (DMSO) was used as a solvent to make the total volume 1 ml. They were charged in a sealed test tube that could be sealed, capped with a three-way cock and frozen with liquid nitrogen, and the inside of the test tube was purged with nitrogen three times. This was returned to room temperature, thawed, frozen again, and nitrogen substitution was repeated. This operation was repeated a total of three times, and then degassed using a vacuum line as it was without freezing. Then, it was immersed in a 60 degreeC water bath in a vacuum state for 4 hours, and was made to react. After 4 hours, the membrane was placed in a dialysis membrane having a molecular weight of 10,000 cut and dialyzed for 24 hours at room temperature in pure water. After 24 hours, it was placed in a glass tube and freeze-dried to obtain a polymer powder. The monomer charge ratio of DMAPAA and 6-O-vinyladipoyl-D-galactose is 20: 80, 35: 65, 50: 50, 65: 35, 80: 20 (all units are mol%). The same operation was performed.

Table 1 below shows the molecular weight, the molar ratio of the repeating units, and the yield of the obtained galactose-containing copolymer. Hereinafter, the molar ratio of the repeating unit in the polymer was calculated by an integral value obtained by ¹ H-NMR measurement. ^{For the 1} H-NMR measurement, 10 mg of the dried polymer was weighed, dissolved in 700 μl of D ₂ O, and used with a 200 MHz ¹ H-NMR apparatus. The weight average molecular weight of the polymer was determined by GPC measurement, respectively, by preparing a 5 mg / ml solution of the dried polymer, using a 1/15 M phosphate buffer (pH = 7.42) as the mobile phase, a flow rate of 0.5 ml / min, and a column temperature. 20 μl was injected at 40 ° C. and measured using pullulan as a standard sample.

  Further, in place of the galactose-containing monomer, a glucose-containing monomer (6-O-vinyladipoyl-D-gulcose (6-O-vinyladipoyl-D-glucose)) is used in the same manner as described above. A containing copolymer was obtained.

  Table 2 below shows the molecular weight of the obtained glucose-containing copolymer and the molar ratio of the repeating units.

(1-2) Formation and confirmation of complex of nucleic acid molecule and sugar-containing copolymer Plasmid pCMV-Luc (Promega) was used as a nucleic acid molecule.

  A stock solution was prepared by dissolving 2.4 mg of the sugar-containing copolymer prepared in (1-1) above in 1000 μl of D-MEM medium. By mixing 5 μl of this stock solution and 45 μl of pCMV-Luc solution (containing 100 ng of pCMV-Luc), the C / A (Cation / Anion) ratio was set to 50. Stock solutions were serially diluted and mixed with pCMV-Luc solution of the same concentration to prepare sample solutions with different C / A ratios (C / A ratio = 0.5, 1, 1.5, 2, 3, 4, 5) .

  Complex formation between a sugar-containing copolymer mixed with various C / A ratios and a nucleic acid molecule was confirmed by using 0.8% agarose gel electrophoresis as follows. A 0.8% agarose gel was prepared using 0.5 × TBE solution containing Ethydium bromide (EtBr). C / A = 0.5, 1, 1.5, 2, 3, 4 and 5 polymer (D40glu60, D42gal58) solution 10μl, pCMV-Luc (200ng / μl) 1μl and BPB solution 2μl to 13μl Left for 30 minutes. After 30 minutes, the sample was applied to the slot, and electrophoresis was performed at 100 V for 30 minutes using 0.5 × TBE with EtBr as the running buffer.

(1-3) Gene expression experiment using a complex Gene in the case of using a pCMV-Luc-galactose-containing polymer complex and a pCMV-Luc-glucose-containing polymer complex for hepatocyte HepG2 cells and renal epithelial cells COS-1 cells The relationship between expression efficiency and C / A ratio was examined.

  Gene expression efficiency was measured as follows.

  As a carrier for nucleic acid molecule delivery, a complex of the plasmid DNA (pCMV-Luc) prepared in (1-2) above and a sugar-containing copolymer (D40glu60, D42gal58), that is, a pCMV-Luc-galactose-containing polymer ( Poly (DMAPAA-co-6-O-vinyladipoyl-D-galactose) (D42gal58) complex and pCMV-Luc-glucose-containing polymer (Poly (DMAPAA-co-6-O-vinyladipoyl-D-glucose) (D40glu60) ) The composite was used.

  As cells for delivering nucleic acid molecules, HepG2 cells, which are human liver cancer cells, and COS-1 cells, which are kidney epithelial cells of green monkeys, were used.

Add 50 μl / well of complex to COS-1 cells and HepG2 cells seeded at 1 × 10 ⁴ cells / well in a 96-well plate, and add 50 μl / well of 200 μM chloroquine solution (FBS free) for 8 hours. Incubated. After 8 hours, the plate was washed with PBS, 100 μl / well of D-MEM (10% FBS) was added, and further incubated for 40 hours. After 40 hours, wash with PBS, add 50 μl / well of cell lysate (Triton X-100, etc.), leave it at 37 ° C for 30 minutes, take it out 20 μl at a time, and add fluorescent substrate solution (ATP, D-Luciferin, etc.) In a bottle containing 100 μl, and the gene expression efficiency to the cells was measured by Luciferase activity using a mill counter.

  The results for hepatocyte HepG2 cells are shown in FIG. 1 (A), and the results for renal epithelial cells COS-1 cells are shown in FIG. 1 (B).

  As a result, it became clear that the carrier for nucleic acid molecule delivery using Poly (DMAPAA-co-6-O-vinyladipoyl-D-galactose) shows high gene expression efficiency in hepatocyte HepG2 cells with galactose receptor. (FIG. 1 (A)). The expression efficiency was high at C / A = 1.5-2. On the other hand, the results of the same experiment on COS cells did not show uptake promotion (FIG. 1 (B)). Poly (DMAPAA-co-6-O-vinyladipoyl-D-galactose) is cell-specific by receptor-mediated galactose receptors, because galactose receptors are present in HepG2 cells but galactose receptors are not present in COS cells. It was highly possible that the gene could be expressed.

  In contrast, the carrier for nucleic acid molecule delivery using Poly (DMAPAA-co-6-O-vinyladipoyl-D-glucose) did not show uptake-up action in HepG2 cells, but in COS cells In addition, an uptake promoting action was observed (FIGS. 1A and 1B).

(1-4) Toxicity evaluation with respect to cells Moreover, the toxicity to cells was also examined by performing protein quantification by the DC protein method. Protein quantification was performed based on the Lowry method.

  Toxicity comparison was performed for each polymer (Poly (DMAPAA-co-6-O-vinyladipoyl-D-galactose) (D42gal58) and (Poly (DMAPAA-co-6-O-vinyladipoyl-D-glucose)) (D40glu60)) The results are shown in Fig. 2. When cells die, the amount of protein decreases because the cells fall off the plate, but as shown in Fig. 2, the total protein amount changes in both polymers. As a result, both polymers were hardly toxic, and it was considered that the toxicity did not affect the expression efficiency.

(1-5) Competition inhibition experiment by addition of sugar Next, in order to confirm whether the gene uptake is mediated by galactose receptor, in the gene expression experiment of (1-3) above, D-galactose was added, We decided to conduct competition inhibition experiments by adding sugar. If Poly (DMAPAA-co-6-O-vinyladipoyl-D-galactose) has been introduced into HepG2 cells via the galactose receptor, Poly (( DMAPAA-co-6-O-vinyladipoyl-D-galactose) is difficult to bind to the receptor, and the addition of D-galactose to Poly (DMAPAA-co-6-O-vinyladipoyl-D-galactose) gene to HepG2 cells Expression efficiency is thought to decrease.

  In the experiment, Poly (DMAPAA-co-6-O-vinyladipoyl-D-galactose) C / A = 2 having high gene expression efficiency was used.

  The competition inhibition experiment by sugar addition was performed as follows.

Add 5 μl / well of various concentrations of D-galactose or D-glucose to COS-1 cells and HepG2 cells seeded at 1 × 10 ⁴ cells / well in a 96-well plate and adhere to them. Plasmid DNA (pCMV-Luc) And a complex of polymer was added at 50 μl / well. Thereto, 55 μl / well of 200 μM chloroquine solution (FBS free) was added and incubated for 8 hours. After 8 hours, the plate was washed with PBS, 100 μl / well of D-MEM (10% FBS) was added, and further incubated for 40 hours. Gene expression efficiency was examined by measuring Luciferase activity.

  The result of the competitive inhibition experiment by the addition of galactose in the gene expression efficiency of Poly (DMAPAA-co-6-O-vinyladipoyl-D-galactose) (D42Gal58) to HepG2 cells is shown in FIG.

  As a result, as shown in FIG. 3, the gene expression efficiency of Poly (DMAPAA-co-6-O-vinyladipoyl-D-galactose) in HepG2 cells was reduced to about 20% by adding galactose.

(1-6) Measurement of gene uptake position by RI Next, in the mechanism of gene uptake in the presence of sugar addition, clarify whether the gene is taken into the nucleus or stays in the cytoplasm Therefore, 32P-pCMV-Luc activity was measured under the competitive inhibition condition by sugar addition, and the amount of uptake into the nucleus was examined. The conditions for competitive inhibition by addition of sugar were the same as in (1-5) above.

(1) Labeling of 32P-pCMV-Luc α-dCTP-32P was labeled to pCMV-Luc by the nick translation method.

(2) Measurement of uptake by 32P-pCMV-Luc The radioactivity of 32P-pCMV-Luc introduced into cells was measured by a liquid scintillation counter, and the uptake into the nucleus and cytoplasm was measured. 32P-pCMV-Luc formed by adding various concentrations of D-galactose or D-glucose to COS-1 cells and HepG2 cells seeded and adhered to a 96-well plate at 1 × 10 ⁴ cells / well 50 μl / well of complex with polymer (D40glu60, D42gal58) was added. Thereto, 50 μl / well of 200 μM chloroquine solution (FBS free) was added and incubated for 8 hours. After 8 hours, the cells were washed with a PBS solution, a trypsin solution was added to detach the cells, and then an SDS solution was added to lyse the cells. After centrifugation, the supernatant and the precipitate were separated, placed in a liquid scintillation cocktail, and the radioactivity of 32P-pCMV-Luc introduced into the cells was measured by a liquid scintillation counter. The supernatant at this time was taken as the amount taken up into the cytoplasm, and the precipitate was taken as the amount taken up into the nucleus.

  FIG. 4 shows the results of examining the effect of galactose on gene uptake into HepG2 cells by complex using Poly (DMAPAA-co-6-O-vinyladipoyl-D-galactose) (D42Gal58).

  As shown in FIG. 4, the nuclear uptake of Poly (DMAPAA-co-6-O-vinyladipoyl-D-galactose) increased as the C / A ratio increased in HepG2 cells. Also, the amount of uptake into the cytoplasm increased as the C / A ratio increased.

  As shown in the experimental results of FIG. 1, the gene expression efficiency is highest when C / A is 1.5 to 2. On the other hand, as shown in the experimental results of FIG. 4, the amount of gene uptake into cells is increased even when C / A> 1.5. From these results, it was considered that there was no simple correlation between the expression efficiency and the uptake of the polymer-gene complex.

  Moreover, when Poly (DMAPAA-co-6-O-vinyladipoyl-D-galactose) was used by adding D-galactose, the amount of 32P-pCMV-Luc incorporated was reduced.

  In this way, the amount of gene uptake into cells due to the addition of galactose was reduced as a result of gene uptake using Poly (DMAPAA-co-6-O-vinyladipoyl-D-galactose) in HepG2 cells. It was thought to indicate that it was sex uptake.

  FIG. 5 shows the results of examining the effects of glucose and DNA uptake into HepG2 cells by a complex using Poly (DMAPAA-co-6-O-vinyladipoyl-D-glucose) (D40glu60).

  As shown in FIG. 5, Poly (DMAPAA-co-6-O-vinyladipoyl-D-glucose) increased uptake into the cell as the C / A ratio increased in HepG2 cells, but to the nucleus. Uptake was low.

  In addition, the amount of 32P-pCMV-Luc uptake when Poly (DMAPAA-co-6-O-vinyladipoyl-D-glucose) was used did not change much by adding D-glucose.

  Therefore, gene uptake using Poly (DMAPAA-co-6-O-vinyladipoyl-D-glucose) in HepG2 cells is not galactose receptor-mediated endocytosis, but nonspecific uptake of cells. It was thought that it was taken in by endocytosis.

  To clarify the difference in uptake mechanism depending on the type of sugar, Figure 6 shows Poly (DMAPAA-co-6-O-vinyladipoyl-D-galactose) (D42Gal58) and Poly (DMAPAA-co-6-) in HepG2 cells. The results when the C / A ratio is different for the amount of uptake into the cytoplasm of the gene introduced by O-vinyladipoyl-D-glucose (D40Glu60) and the ratio of the amount of uptake into the nucleus are illustrated.

  From these results, it was found that most of the genes considered to have been incorporated into the cytoplasm by D40Glu60 without involving the receptor remained in the cytoplasm regardless of the difference in the C / A ratio, and the transferability to the nucleus was low. On the other hand, it was found that most of the genes thought to be taken up by D42Gal58 via the galactose receptor are transferred from the cytoplasm to the nucleus regardless of the difference in the C / A ratio. From these results, it was considered that the complex taken up by receptor-mediated endocytosis has a high ability to migrate into the nucleus.

Example 2:
Delivery of nucleic acid molecules formed with Poly (DMAPAA-co-6-O-vinyladipoyl-D-glucose-co-stearyl) or Poly (DMAPAA-co-6-O-vinyladipoyl-D-galactose-co-stearyl) Of gene expression in a carrier.

  Although it was thought that the gene expression efficiency increases with the increase of the gene uptake into the cell, as shown in the experimental results shown in FIG. 1 and FIG. It became clear that there was no correlation between the two. This is because uptake increases as the C / A ratio increases, but DNA is less likely to dissociate from the DNA-polymer complex (polyplex), thus making transcription and translation difficult. It was considered that the expression efficiency of the protein may also decrease. Therefore, dissociation of DNA from the polyplex by anionic molecules was examined (see FIG. 7). Then, we decided to investigate the change in dissociation of DNA from the polyplex by changing the shape of the polymer by synthesizing a copolymer using a monomer having a hydrophobic substituent.

  That is, a copolymer in which a repeating unit having a stearyl group which is a hydrophobic substituent is further introduced into a polymer having a cationic repeating unit and a sugar-containing repeating unit, specifically, Poly (DMAPAA-co-6-O -vinyladipoyl-D-glucose-co-stearyl) and Poly (DMAPAA-co-6-O-vinyladipoyl-D-galactose-co-stearyl) and using these polymers The correlation between DNA dissociation ability and gene expression efficiency was compared.

(2-1) Synthesis of Poly (DMAPAA-co-6-O-vinyladipoyl-D-galactose-co-stearyl) and Poly (DMAPAA-co-6-O-vinyladipoyl-D-glucose-co-stearyl) Cationic DMAPAA as a monomer having a group, 6-O-vinyladipoyl-D-glucose or 6-O-vinyladipoyl-D-galactose as a monomer containing a sugar, and stearyl as a monomer having a hydrophobic substituent Using acrylate (stearylacrylate), a polymerization reaction was performed in the same manner as the synthesis of the copolymer in the experimental example. The total monomer concentration is 0.5 × 10 ^-3 mol, and the monomer charge ratio is DMAPAA: 6-O-vinyladipoyl-D-glucose (or 6-O-vinyladipoyl-D-galactose): stearylacrylate = 35: 65: 1 All units were mol%). The initiator 2,2′-Azobis (4-methoxy-2,4-dimethylvaleronitrile) (AMDVN) was added at 1.0 mol%, and DMSO was used as a solvent to make the total volume 1 ml. They were charged in a sealed test tube that could be sealed, capped with a three-way cock and frozen with liquid nitrogen, and the inside of the test tube was purged with nitrogen three times. This is returned to room temperature, thawed, frozen again, and the nitrogen replacement is repeated. This operation was repeated a total of three times, and then degassed as it was with a vacuum line without freezing. Then, it was immersed in a 60 degreeC water bath in a vacuum state for 4 hours, and made it react. After 4 hours, the membrane was placed in a dialysis membrane having a molecular weight of 10,000 cut and dialyzed for 24 hours at room temperature in pure water. After 24 hours, it was placed in a glass tube and freeze-dried.

  Poly (DMAPAA-co-6-O-vinyladipoyl-D-galactose-co-stearyl) is shown in Table 3 and Poly (DMAPAA-co-6-) for the molecular weight, the molar ratio of repeating units and the yield of the obtained polymer. O-vinyladipoyl-D-glucose-co-stearyl) is shown in Table 4.

(2-2) Confirmation of DNA dissociation ability
Poly (DMAPAA-o-6-O-vinyladipoyl-D-galactose-co-stearyl (D50gal49s1) and DNA complex, and Poly (DMAPAA-o-6-O-vinyladipoyl-D-glucose (D38glu61s1)) and DNA The complex was added to the complex with potassium polyvinyl sulpate solution (PVSK), and the dissociation ability of DNA from the complex was confirmed by agarose gel electrophoresis, namely Poly (DMAPAA-o-6-O-vinyladipoyl- D-galactose-co-stearyl) and DNA (pCMV-Luc) complex (C / A ratio = 0.5, 1, 1.5, 2, 3, 4, 5, 10) and Poly (DMAPAA-o-6-O -vinyladipoyl-D-galactose) and DNA (pCMV-Luc) complex (C / A ratio = 0.5, 1, 1.5, 2, 3, 4, 5, 10) each 5μl, 1μl pCMV-Luc (38 μg / μl) and equilibrated with each of 3.88 μl of PVSK and left for 30 minutes at 37 ° C. As a control, 1 μl of pCMV-Luc (200 μg / Luc) was added. 1 μl of pCMV-Luc (200 μg / μl) and 5 μl of each C / A ratio polymer solution, and 3 μl of TE. The one added with 88 μl was subjected to electrophoresis.

(2-3) Gene expression efficiency measurement using Poly (DMAPAA-co-6-O-vinyladipoyl-D-galactose-co-stearyl) (D50gal49s1)
In order to confirm the correlation between the DNA dissociation ability and the gene expression efficiency, it was decided to introduce a gene using D50gal49s1. A complex of plasmid DNA (pCMV-Luc) and polymer was formed. Add 50 μl / well of complex to COS-1 cells and HepG2 cells seeded at 1 × 10 ⁴ cells / well on a 96-well plate, and add 50 μl / well of 200 μM chloroquine solution (FBS free) for 8 hours. Incubated. After 8 hours, the plate was washed with PBS, 100 μl / well of D-MEM (10% FBS) was added, and further incubated for 40 hours. After 40 hours, wash with PBS, add 50 μl / well of cell lysate (Triton X-100, etc.), leave it at 37 ° C for 30 hours, take out 20 μl of each, and then add luminescent substrate solution (ATP, D-Luciferin, etc.) It put into the bottle containing 100 microliters, and measured the gene expression efficiency to the cell by Luciferase activity with the mill counter.

  Similarly, Poly (DMAPAA-co-6-o-vinyladipoil-D-galactose) (D42gal58), Poly (DMAPAA-co-6-o-vinyladipoil-D-glucose) (D40glu60), Poly (DMAPAA-co-6 Gene expression efficiency was measured using a copolymer of -o-vinyladipoil-D-glucose-co-stearyl) (D38glu61s1).

  Specifically, the relationship between the expression efficiency of luciferase and the C / A ratio when the gene was incorporated into Hep2 cells by the complex of the above four types of copolymers and pCMV-Luc was examined. The results are shown in FIG.

  From the results shown in FIG. 8, when Poly (DMAPAA-co-6-O-vinyladipoyl-D-galactose) (D42Gal58) is used, the transferability to the nucleus improves as the C / A ratio increases. However, it was found that the expression efficiency peaks at C / A = 1.5.

  On the other hand, when Poly (DMAPAA-co-6-O-vinyladipoyl-D-galactose-co-stearyl) was used, the gene expression level in HepG2 cells increased as the C / A ratio increased.

  From this, in the case of gene delivery using a galactose-containing copolymer in HepG2 cells, the amount of gene uptake increases with an increase in the C / A ratio, but when DNA is difficult to dissociate, C / A It was suggested that the gene expression efficiency peaked at = 1.5.

(2-4) Dissociation of DNA by addition of anion molecules Furthermore, PVS from pCMV-Luc-Gal58 (pCMV-Luc-D42Gal58) complex and pCMV-Luc-Gal49 (s) (pCMV-Luc-D50Gal49s1) complex The release of the gene in the presence of (Poly (vinylsulfate)) was examined. The gene was released in the same manner as in (2-2) Confirmation of DNA dissociation ability. The results are shown in FIG.

  When C / A = 1.5, Poly (DMAPAA-co-6-O-vinyladipoyl-D-glucose) (D42gal58) stays in the slot when no anion molecule is added, but the anion molecule is added. Then, DNA was dissociated, and the band that flowed out of the slot could be confirmed. When C / A = 2, it was possible to confirm the band of DNA that had dissociated and flowed out, but it was much thinner than the band when C / A = 1, 1.5. When the C / A ratio further increased, it was not possible to confirm the DNA band that had dissociated and flowed out of the slot. From this, it is suggested that the highest value when the expression efficiency is C / A = 1.5 may be related to the dissociation ability of this DNA. On the other hand, Poly (DMAPAA-co-6-O-vinyladipoyl-D-galactose-co-stearyl) (D50gal49s1) dissociates and flows out of the slot by adding anionic molecules even when C / A = 10. DNA was confirmed.

From these results, if DNA dissociation ability is related to expression efficiency, Poly (DMAPAA-co-6-O-vinyladipoyl-D-galactose-co-stearyl) The expression efficiency is considered to increase with the increase.
Example 3: Synthesis of a sugar-containing copolymer
(1) Methylgalactoside-containing polymer
Poly (DMAPAA-co-6-O-vinyladipoyl-methyl-D-galactoside-co-stearylacrylate)
DMAPAA (dimethylaminopropylacrylamide) as a monomer having a cationic group, 6-O-vinyladipoyl-methyl-D-galactoside as a monomer containing a sugar, and stearylacrylate as a monomer having a hydrophobic substituent (Stearyl acrylate) was used. The concentration of the whole monomer was 0.5 × 10 ⁻³ mol, and the charging ratio was set to cationic group: sugar: hydrophobicity = 50: 50: 1 (all units are mol%). 1 mol% of initiator 2,2′-Azobis (4-methoxy-2,4-dimethylvaleronitrile) (AMDVN) was added, and the total amount was adjusted to 1 ml in a glass ampoule using DMSO as a solvent. The container was degassed and reacted at 60 ° C. for 24 hours. It was put into a dialysis membrane having a molecular weight of 10,000 cut, dialyzed at room temperature for 24 hours in pure water, and freeze-dried. The obtained powder was washed with acetone and dried under vacuum to obtain a polymer. The molecular weight, the molar ratio of repeating units and the yield of the polymer obtained are shown in the table below.

Poly (DMAPAA-co-6-O-vinyladipoyl-methyl-D-galactoside-co-vinylstearate)
DMAPAA (dimethylaminopropylacrylamide) as a monomer having a cationic group, 6-O-vinyladipoyl-methyl-D-galactoside as a monomer containing a sugar, and vinylstearate as a monomer having a hydrophobic substituent (Stearic acid vinyl ester) was used and reacted in the same manner as above. The molecular weight, the molar ratio of repeating units and the yield of the polymer obtained are shown in the table below.

Poly (DMAPAA-co-6-O-vinyladipoyl-methyl-D-galactoside-co-vinylpalmitate)
DMAPAA (dimethylaminopropylacrylamide) as a monomer having a cationic group, 6-O-vinyladipoyl-methyl-D-galactoside as a monomer containing a sugar, and vinylpalmitate as a monomer having a hydrophobic substituent (Palmitic acid vinyl ester) was used and reacted in the same manner as above. The molecular weight, the molar ratio of repeating units and the yield of the polymer obtained are shown in the table below.

Poly (DMAEA-co-6-O-vinyladipoyl-methyl-D-galactoside-co-stearyl)
DMAEA (dimethylaminoethyl acrylate) as a monomer having a cationic group, 6-O-vinyladipoyl-methyl-D-galactoside as a monomer containing a sugar, and stearylacrylate as a monomer having a hydrophobic substituent (Stearyl acrylate) was used and reacted in the same manner as above. The molecular weight, the molar ratio of repeating units and the yield of the polymer obtained are shown in the table below.

Poly (DMAEA-co-6-O-vinyladipoyl-methyl-D-galactoside-co-vinylstearate)
DMAEA (dimethylaminoethyl acrylate) as a monomer having a cationic group, 6-O-vinyladipoyl-methyl-D-galactoside as a monomer containing a sugar, and vinylstearate as a monomer having a hydrophobic substituent (Stearic acid vinyl ester) was used and reacted in the same manner as above. The molecular weight, the molar ratio of repeating units and the yield of the polymer obtained are shown in the table below.

Poly (DMAEAA-co-6-O-vinyladipoyl-methyl-D-galactoside-co-vinylpalmitate)
DMAEA (dimethylaminoethyl acrylate) as a monomer having a cationic group, 6-O-vinyladipoyl-methyl-D-galactoside as a monomer containing a sugar, and vinylpalmitate as a monomer having a hydrophobic substituent (Palmitic acid vinyl ester) was used and reacted in the same manner as above. The molecular weight, the molar ratio of repeating units and the yield of the polymer obtained are shown in the table below.

(2) Mannose-containing polymer
Poly (DMAPAA-co-6-O-vinyladipoyl-D-mannose-co-stearylacrylate)
DMAPAA (dimethylaminopropylacrylamide) as a monomer having a cationic group, 6-O-vinyladipoyl-D-mannose as a monomer containing a sugar, and stearylacrylate (stearylacrylate) as a monomer having a hydrophobic substituent Acrylate) was reacted in the same manner as above. The molecular weight, the molar ratio of repeating units and the yield of the polymer obtained are shown in the table below.

Poly (DMAPAA-co-6-O-vinyladipoyl-D-mannose-co-vinylstearate)
DMAPAA (dimethylaminopropylacrylamide) as a monomer having a cationic group, 6-O-vinyladipoyl-D-mannose as a monomer containing a sugar, and vinylstearate (stearin) as a monomer having a hydrophobic substituent Acid vinyl ester) and was reacted in the same manner as above. The molecular weight, the molar ratio of repeating units and the yield of the polymer obtained are shown in the table below.

Poly (DMAPAA-co-6-O-vinyladipoyl-D-mannose-co-vinylpalmitate)
DMAPAA (dimethylaminopropylacrylamide) as a monomer having a cationic group, 6-O-vinyladipoyl-D-mannose as a monomer containing a sugar, and vinylpalmitate (palmitin) as a monomer having a hydrophobic substituent Acid vinyl ester) and was reacted in the same manner as above. The molecular weight, the molar ratio of repeating units and the yield of the polymer obtained are shown in the table below.

Poly (DMAEMA-co-6-O-vinyladipoyl-D-mannose-co-stearylacrylate)
DMAEMA (dimethylaminoethyl methacrylate) as a monomer having a cationic group, 6-O-vinyladipoyl-D-mannose as a monomer containing a sugar, and stearylacrylate (monomer having a hydrophobic substituent) The reaction was conducted in the same manner as above. The molecular weight, the molar ratio of repeating units and the yield of the polymer obtained are shown in the table below.

Poly (DMAEMA-co-6-O-vinyladipoyl-D-mannose-co-vinylstearate)
DMAEMA (dimethylaminoethyl methacrylate) as a monomer having a cationic group, 6-O-vinyladipoyl-D-mannose as a monomer containing a sugar, and vinylstearate (as a monomer having a hydrophobic substituent) (Vinyl stearate) was reacted in the same manner as above. The molecular weight, the molar ratio of repeating units and the yield of the polymer obtained are shown in the table below.

Poly (DMAEMA-co-6-O-vinyladipoyl-D-mannose-co-vinylpalmitate)
DMAEMA (dimethylaminoethyl methacrylate) as a monomer having a cationic group, 6-O-vinyladipoyl-D-mannose as a monomer containing a sugar, and vinylpalmitate (monomer having a hydrophobic substituent) The reaction was carried out in the same manner as above. The molecular weight, the molar ratio of repeating units and the yield of the polymer obtained are shown in the table below.

(3) Maltose-containing polymer
Poly (DMAEA-co-6-O-vinyladipoyl-D-maltose-co-stearylacrylate)
DMAEA (dimethylaminoethyl acrylate) as a monomer having a cationic group, 6-O-vinyladipoyl-D-maltose as a monomer containing a sugar, and stearylacrylate (stearyl acrylate) as a monomer having a hydrophobic substituent Acrylate) was reacted in the same manner as above. The molecular weight, the molar ratio of repeating units and the yield of the polymer obtained are shown in the table below.

Poly (DMAEA-co-6-O-vinyladipoyl-D-maltose-co-vinylstearate)
DMAEA (dimethylaminoethyl acrylate) as a monomer having a cationic group, 6-O-vinyladipoyl-D-maltose as a monomer containing a sugar, and vinylstearate (stearin) as a monomer having a hydrophobic substituent Acid vinyl ester) and was reacted in the same manner as above. The molecular weight, the molar ratio of repeating units and the yield of the polymer obtained are shown in the table below.

Poly (DMAEA-co-6-O-vinyladipoyl-D-maltose-co-vinylpalmitate)
DMAEA (dimethylaminoethyl acrylate) as a monomer having a cationic group, 6-O-vinyladipoyl-D-maltose as a monomer containing a sugar, and vinylpalmitate (palmitin as a monomer having a hydrophobic substituent) Acid vinyl ester) and was reacted in the same manner as above. The molecular weight, the molar ratio of repeating units and the yield of the polymer obtained are shown in the table below.

(4) Trehalose-containing polymer
Poly (DMAEMA-co-6-O-vinyladipoyl-D-trehalose-co-stearylacrylate)
DMAEMA (dimethylaminoethyl methacrylate) as a monomer having a cationic group, 6-O-vinyladipoyl-D-trehalose as a monomer containing a saccharide, and stearylacrylate (monomer having a hydrophobic substituent) The reaction was conducted in the same manner as above. The molecular weight, the molar ratio of repeating units and the yield of the polymer obtained are shown in the table below.

Poly (DMAEMA -co-6-O-vinyladipoyl-D-trehalose-co-vinylstearate)
DMAEMA (dimethylaminoethyl methacrylate) as a monomer having a cationic group, 6-O-vinyladipoyl-D-trehalose as a monomer containing a sugar, and vinylstearate (as a monomer having a hydrophobic substituent) (Vinyl stearate) was reacted in the same manner as above. The molecular weight, the molar ratio of repeating units and the yield of the polymer obtained are shown in the table below.

Poly (DMAEMA -co-6-O-vinyladipoyl-D-trehalose-co-vinylpalmitate)
DMAEMA (dimethylaminoethyl methacrylate) as a monomer having a cationic group, 6-O-vinyladipoyl-D-trehalose as a monomer containing a sugar, and vinylpalmitate as a monomer having a hydrophobic substituent ( The reaction was carried out in the same manner as above. The molecular weight, the molar ratio of repeating units and the yield of the polymer obtained are shown in the table below.

(5) Glucose-containing polymer
Poly (DMAPAA-co-6-O-vinyladipoyl-D-glucose-co-vinyladipoyl-cholesterol)
DMAPAA (dimethylaminopropylacrylamide) as a monomer having a cationic group, 6-O-vinyladipoyl-D-mannose as a monomer containing a sugar, and vinyladipoyl-cholesterol as a monomer having a hydrophobic substituent (3-Vinyladipoylcholesterol) was used and reacted in the same manner as above. The molecular weight, the molar ratio of repeating units and the yield of the polymer obtained are shown in the table below.

3-vinyl adipoyl cholesterol was synthesized as follows.
(1) Examination of solvent 500 mg of enzyme (LIP-301) was added to 5 ml of tetrahydrofuran in which 485 mg of cholesterol and 0.95 ml of divinyladipic acid were dissolved, and the mixture was shaken at room temperature for 6 days. The reaction solution was examined by thin layer chromatography (hexane: ethyl acetate = 9: 1 sulfuric acid color development as a developing solvent) to confirm the presence or absence of the product. The results are shown in Table 5.

^a 0.5M concentration (++++: dissolves well,-: difficult to dissolve), ^b Check by TLC (+: product is present,-: product is not present)
(2) Examination of enzyme 5 ml each of toluene, DMSO, DMF, pyridine, dioxane and tetrahydrofuran was added to 485 mg of cholesterol and 0.95 ml of divinyladipic acid. As the enzyme, lipase derived from Candida Antarctica: Novozyme435 (Novo), Pseudomonas Lipase: LPL-311 (Toyobo), LPL-311 immobilized enzyme: LIP-301 (Toyobo), pig pancreas lipase: L-3126 (Sigma), alkaline protease derived from Bacillus subtilis: Bioprase conc. ), 500 mg each of alkaline protease derived from Streptomyces sp .: ALP-101 (Toyobo), and shaken at room temperature for 6 days. The reaction solution was examined by thin layer chromatography (hexane: ethyl acetate = 9: 1 sulfuric acid color development as a developing solvent) to confirm the presence or absence of the product. The results are shown in FIG. 10A.
(3) Examination of divinyl adipic acid concentration Cholesterol 485 mg, 3 times mole, 2 times mole, 1.5 times mole, 1.1 times mole of divinyl adipic acid added to 5 ml of tetrahydrofuran dissolved Then, 500 mg of enzyme (LIP-301) was added and shaken at room temperature for 6 days. The reaction solution was examined by thin layer chromatography (hexane: ethyl acetate = 9: 1 sulfuric acid color development as a developing solvent) to confirm the presence or absence of the product. The results are shown in FIG. 10B.
(4) Scale-up synthesis 2.3 g of lipase (LIP-301) was added to 23 ml of tetrahydrofuran in which 2.228 g of cholesterol and 4.36 ml of divinyladipic acid were dissolved, and the mixture was shaken overnight at room temperature. After the reaction, the reaction solution is filtered to remove insoluble enzyme, the solvent is concentrated, and the column of 50 g of silica gel is used to elute the target ester compound using hexane: ethyl acetate = 20: 1 as an eluent. 2.5 g of oily product was obtained.

SA: stearylacrylate, SV: vinylstearate, PV: vinylpalmitate, CholV: vinyladipoyl-cholesterol (3-vinyl adipoyl cholesterol)

Synthesis example 1:
(1) Synthesis of monoacryloyl α-cyclodextrin by immobilized lipase

10 g of α-cyclodextrin (Nacalai Tesque) was dissolved in 50 ml of DMF, 1 g of immobilized lipase (LIP Toyobo) and 8 g of molecular sieve 3A were added and stirred for 2 hours. 2 ml of vinyl acrylate was added and stirred at room temperature for 48 hours. After completion of the reaction, the mixture is filtered through a high flow supercell (manufactured by Nacalai Tesque), further filtered through a cartridge filter (MINISART SRP 15, manufactured by Sartorius), 300 ml of ethyl acetate is added, and 5 g of a white precipitate of monoacryloyl α-cyclodextrin is added. Obtained.
(2) Synthesis of Poly (DMAPAA-co-acryloyl a-cyclodextrin-D-galactoside-co-stearyl)
DMAPAA (dimethylaminopropylacrylamide), acryloyl a-cyclodextrin, stearylacrylate (stearyl acrylate) was used. The concentration of the whole monomer was 0.5 × 10 ⁻³ mol, and the charging ratio was set to cationic group: sugar: hydrophobicity = 50: 50: 1 (all units are mol%). Initiator 2,2'-Azobis (4-methoxy-2,4-dimethylvaleronitrile) (AMDVN) was added at 1 mol%, and the total amount was adjusted to 1 ml in a glass ampoule using DMF as a solvent. The container was degassed and reacted at 60 ° C. for 24 hours. It was put into a dialysis membrane having a molecular weight of 10,000 cut, dialyzed at room temperature for 24 hours in pure water, and freeze-dried. The obtained powder was washed with acetone and dried under vacuum to obtain a polymer.
Synthesis example 2:
・ Synthesis of vinyl adipoyl α-cyclodextrin by immobilized lipase

10 g of α-cyclodextrin (Nacalai Tesque) was dissolved in 50 ml of DMF, 1 g of immobilized lipase (LIP Toyobo) and 8 g of molecular sieve 3A were added and stirred for 2 hours. 2 ml of divinyl adipate was added and stirred for 48 hours at room temperature. After completion of the reaction, the mixture is filtered through a high flow supercell (manufactured by Nacalai Tesque) and further filtered through a cartridge filter (MINISART SRP 15, manufactured by Sartorius). It was.
(2) Synthesis of Poly (DMAPAA-co-vinyladipoyl a-cyclodextrin-D-galactoside-co-stearyl)
DMAPAA (dimethylaminopropylacrylamide), vinyladipoyl a-cyclodextrin, and stearylacrylate were used. The concentration of the whole monomer was 0.5 × 10 ⁻³ mol, and the charging ratio was DMAPAA: vinyladipoyl a-CD: stearylacrylate = 50: 50: 1 (all units are mol%). Initiator 2,2'-Azobis (4-methoxy-2,4-dimethylvaleronitrile) (AMDVN) was added at 1 mol%, and the total amount was adjusted to 1 ml in a glass ampoule using DMF as a solvent. The container was degassed and reacted at 60 ° C. for 24 hours. It was put into a dialysis membrane having a molecular weight of 10,000 cut, dialyzed at room temperature for 24 hours in pure water, and freeze-dried. The obtained powder was washed with acetone and dried under vacuum to obtain a polymer.
Test example 1
A RITC-labeled polymer (Poly (DMAPAA-co-6-) having a galactose content of stearyl group (S) (0%, 2%, 3%, 10%, 13%) synthesized as in Example 3 as a sugar residue. vinyladipoyl-D-galactoside-co-stearyl (S content 0%);
Poly (DMAPAA-co-6-vinyladipoyl-D-galactoside-co-stearyl) (S content is 2%))
Was used to form a complex with FITC-labeled DNA. Polyvinyl sulfate potassium salt (PVSK) was allowed to act on the complex to confirm whether DNA was released. The results are shown in FIG.

  A polymer consisting only of a cationic group and a galactose group that did not contain a stearyl group as a hydrophobic group could not be confirmed as an AFM image, and thus was considered to be linear (S content = 0). At this time, the release of DNA was not confirmed by agarose electrophoresis even when the anionic polymer PVSK was added. On the other hand, when 2 mol% or more of the stearyl group was introduced, it was confirmed from AFM that particles were formed in either the polymer alone or the DNA complex. Furthermore, it was found from the agarose electrophoresis that the higher the stearyl content, the easier it is to release.

  Polyvinyl sulfate potassium salt (PVSK) was allowed to act on the complex of FITC-labeled DNA and RITC-labeled polymer to confirm the presence or absence of DNA. The results are shown in the lower part of FIG. As shown in FIG. 11, it was confirmed that as the stearyl group (S) content increased, DNA was more easily released.

  Furthermore, it was confirmed that DNA was released in actual cells by FRET analysis of gene transfer using HepG2 cells of the complex (FIG. 12). In the polymer (Gal-DA) that does not contain a stearyl group, when it was excited at the FITC excitation wavelength (495 nm) of the cell, RITC fluorescence increased, confirming FRET, and DNA and polymer were combined in the cell. It turns out that the body remains formed. On the other hand, in the polymer containing the stearyl group, FITC fluorescence was observed at the excitation wavelength (495 nm) of FITC, and FRET did not occur. From this, it was confirmed that in these polymers, DNA was effectively released even in cells.

FIG. 1 shows that luciferase produced by (A) a complex of pCMV-Luc and a sugar-containing copolymer on HepG2 cells and (B) a complex of pCMV-Luc and a sugar-containing copolymer on COS cells. It is drawing which shows the result of having investigated the relationship between expression efficiency and C / A ratio. PEI indicates polyethyleneimine. FIG. 2 is a drawing showing the results of examining the toxicity of a polymer to cells by performing protein quantification by the DC protein method. FIG. 3 is a drawing showing the results of examining the effect of galactose on gene expression efficiency in HepG2 cells. Poly-Gal is a galactose-containing copolymer, Poly-glu is a glucose-containing copolymer, and PEI is a case where polyethyleneimine is used. FIG. 4 is a drawing showing the results of examining the effect of galactose on DNA uptake into HepG2 cells by Poly (DMAPAA-co-6-O-vinyladipoyl-D-galactose). (A) shows the results of investigation into uptake into the nucleus, and (B) shows uptake studies into the cytoplasm. In FIG. 4, Sugar represents galactose. FIG. 5 is a drawing showing the results of examining the effects of glucose and DNA uptake into HepG2 cells by Poly (DMAPAA-co-6-O-vinyladipoyl-D-glucose). (C) shows the results of studies on uptake into the nucleus, and (D) shows uptake studies into the cytoplasm. In FIG. 5, Sugar indicates glucose. Fig. 6 shows the nuclear translocation of genes introduced into HepG2 cells by Poly (DMAPAA-co-6-O-vinyladipoyl-D-galactose) and Poly (DMAPAA-co-6-O-vinyladipoyl-D-glucose). It is drawing which showed the ratio. FIG. 7 is a diagram modeling the dissociation of DNA from a polyplex by anionic molecules. FIG. 8 is a graph showing the relationship between the expression efficiency of luciferase in HepG2 cells and the C / A ratio when a complex of pCMV-Luc and various sugar-containing copolymers is used. FIG. 9 is a diagram relating to gene release in the presence of PVS in a complex of a galactose-containing copolymer and pCMV-Lu. FIG. 10A shows the results of enzyme studies of cholesteryl ester synthesis. FIG. 10B shows the results of examining the concentration of DVA (divinyl adipate) for cholesteryl ester synthesis. FIG. 11 shows an AFM image of a polymer DNA complex with stearyl group (S) content and confirmation of DNA dissociation by addition of an anionic polymer (PVSK) using agarose electrophoresis. FIG. 12 shows confirmation of DNA dissociation in cells by FRET analysis. In FIG. 12, Gal-D-A (a): polymer without stearyl group Gal-D-A-S (b): polymer cell containing 2-10% stearyl group: HepG2 cells. Excitation wavelength = 495 nm, Bar = 20 μm

Claims (10)

Formula (I)
(In the formula, R ₂ represents H or CH ₃ .
Y is, -C (= O) O- ( CH 2) n y -, - OC (= O) - (CH 2) n y -, - OC (= O) - (CH 2) n y -C ( = O), or -CONH- _(CH 2) n _y - shows the. n _y is an integer of 1 to 10.
Z is —NR ₃ R ₄ (R ₃ and R ₄ are the same or different and each represents a hydrocarbon group having 1 to 10 carbon atoms), or —N ⁺ R ₅ R ₆ R ₇ (R ₅ , R ₆ and _{R 7} are each exhibit the same or different and represent a hydrocarbon group having 1 to 10 carbon atoms.). )
A repeating unit (A) having a cationic group represented by formula (II):
(In the formula, R ₁ represents —H or —CH ₃ .
Sugar is one NH ₂ from sugar (in the case of monosaccharide, disaccharide or polysaccharide where Sugar is bound by amino group of amino sugar) or OH (monosaccharide, disaccharide where sugar is bound by sugar hydroxyl group, disaccharide) (In the case of saccharides or polysaccharides), the sugar residues are shown.
X is —C (═O) Z ¹ —, —C (═O) O—R _a —, —CONH—, —CONH—R _b —, —OC (═O) —R _c —C (═O ) Z ¹ -or -Ph-R _d -Z ^1- . here,
R _a is -Ph-O-, or - _(CH 2) shows a n _a -O-. n _a represents an integer from 1 to 10.
R _b represents —Ph—O— or — (CH ₂ ) n _b —O—. n _b represents an integer of 1 to 10.
The _{R c - (CH 2) n} c -, or _{- (CH 2) n c -Ph-} (CH 2) n c - shows the. n _c is an integer of 2 to 18.
R _d represents —CH ₂ — or —SO ₂ —.
Z ¹ represents —O— or —NH—.
Ph represents an (o-, m- or p-) phenylene group. ).
A sugar-containing copolymer having a repeating unit (B) containing a sugar represented by formula (wherein the repeating unit represented by the general formula (I) is represented by the following formula (IA)
(In the formula, n represents an integer of 1 to 10.)
When the molar ratio of the repeating units represented by the general formulas (IA) and (II) is 1: 9 to 9: 1 and the weight average molecular weight is 1 × 10 ^{4 to} 5 × 10 ⁸ except for the sugar-containing copolymer. )
A nucleic acid molecule delivery carrier comprising: A saccharide-containing copolymer having a repeating unit (A) having a cationic group, a repeating unit (B) containing a sugar (Sugar), and a repeating unit (C) having a hydrophobic substituent, The repeating unit (A) having the general formula (I)
(In the formula, R ₂ represents H or CH ₃ .
Y is, -C (= O) O- ( CH 2) n y -, - OC (= O) - (CH 2) n y -, - OC (= O) - (CH 2) n y -C ( = O), or -CONH- _(CH 2) n _y - shows the. n _y is an integer of 1 to 10.
Z is —NR ₃ R ₄ (R ₃ and R ₄ are the same or different and each represents a hydrocarbon group having 1 to 10 carbon atoms), or —N ⁺ R ₅ R ₆ R ₇ (R ₅ , R ₆ and _{R 7} are each exhibit the same or different and represent a hydrocarbon group having 1 to 10 carbon atoms.). )
The repeating unit (B) containing sugar (Sugar) is represented by the general formula (II):
(In the formula, R ₁ represents —H or —CH ₃ .
Sugar is one NH ₂ from sugar (in the case of monosaccharide, disaccharide or polysaccharide where Sugar is bound by amino group of amino sugar) or OH (monosaccharide, disaccharide where sugar is bound by sugar hydroxyl group, disaccharide) (In the case of saccharides or polysaccharides), the sugar residues are shown.
X is —C (═O) Z ¹ —, —C (═O) O—R _a —, —CONH—, —CONH—R _b —, —OC (═O) —R _c —C (═O ) Z ¹ -or -Ph-R _d -Z ^1- . here,
R _a is -Ph-O-, or - _(CH 2) shows a n _a -O-. n _a represents 1 to 10.
R _b represents —Ph—O— or — (CH ₂ ) n _b —O—. n _b represents 1 to 10.
The _{R c - (CH 2) n} c -, or _{- (CH 2) n c -Ph-} (CH 2) n c - shows the. n _c is an integer of 2 to 18.
R _d represents —CH ₂ — or —SO ₂ —.
Z ¹ represents —O— or —NH—.
Ph represents an (o-, m- or p-) phenylene group. ).
The carrier for nucleic acid molecule delivery represented by these. The repeating unit (B) containing a sugar is represented by the following general formula (III)
In the formula, Sugar is one NH ₂ from sugar (when Sugar is monosaccharide, disaccharide or polysaccharide bonded with amino group of amino sugar) or OH (Sugar is bonded with hydroxyl group of sugar) (In the case of saccharide, disaccharide or polysaccharide), the sugar residue is excluded, m is an integer of 2 to 10. Z is O or NH.)
The carrier for nucleic acid molecule delivery according to claim 1, wherein the carrier is a repeating unit represented by the formula: The repeating unit (A) having a cationic group is represented by the following general formula (IV):
(In the formula, n represents an integer of 1 to 10. R ^a and R ^b may be the same or different and represent an alkyl group having 1 to 4 carbon atoms.)
The carrier for nucleic acid molecule delivery according to any one of claims 1 to 3, wherein the carrier is a repeating unit represented by the formula: The carrier for nucleic acid molecule delivery according to any one of claims 2 to 4, wherein the repeating unit (C) having a hydrophobic substituent is a repeating unit represented by the following general formula (V).
Formula (V):
(Wherein R ₈ represents —H or —CH ₃ .
W is -C (= O) O -, - OC (= O) -, - OC (= O) - and _{(CH 2) n w -C (} = O) O-, or -C (= O) NH Show. n _w is an integer of 2 to 18.
R ₉ represents a saturated or unsaturated aliphatic or alicyclic hydrocarbon group having 3 to 30 carbon atoms. ) The nucleic acid molecule delivery according to any one of claims 1 to 5, wherein the molar ratio of the repeating unit (A) to the repeating unit (B) in the sugar-containing copolymer is A: B = 1: 99 to 99: 1. Carrier. The molar ratio of the repeating unit (C) to the repeating unit (A) + the repeating unit (B) in the sugar-containing copolymer is (A + B): C = 99.9: 0.1 to 0.1: 99.9. The carrier for nucleic acid molecule delivery according to any one of claims 2 to 6. The carrier for nucleic acid molecule delivery according to any one of claims 1 to 7, wherein the sugar-containing copolymer has a weight average molecular weight of 10,000 to 1,000,000. A transfection reagent using the carrier for nucleic acid molecule delivery according to claim 1. A carrier for gene therapy using the carrier for nucleic acid molecule delivery according to any one of claims 1 to 8.