JP2009142230A - Nucleic acid complex - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a nucleic acid complex having high gene-introducing activity. <P>SOLUTION: The nucleic acid complex is a complex of a cationic polymer and a nucleic acid, and is obtained by allowing the complex wherein the amount of positive electric charge is larger than that of negative electric charge to adsorb an anionic polymer. The cationic polymer and the anionic polymer are preferably branched polymers obtained by using a compound having three or more N,N-dialkyldithiocarbamylmethyl groups in one molecule as an iniferter, and carrying out living polymerization of cationic or anionic vinylic monomers under light radiation. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a nucleic acid complex in which a gene introduction agent and a nucleic acid are complexed.

  In recent years, gene therapy research has become more and more important as the molecular genetic factors of human diseases become clear. Gene therapy is aimed at the expression of DNA at the target site, how to make the DNA reach the target site, how to efficiently introduce the DNA into the target site and make it functionally expressed at the site It becomes important. As vectors for the introduction of foreign DNA, many viruses, including retroviruses, adenoviruses or herpes viruses, have been modified to carry therapeutic genes and are used in human clinical trials for gene therapy. However, the risk of infection and immune response remains.

Cationic star polymers are described in WO 2004/092388 and JP 2007-70579 as non-viral vectors for transporting DNA into cells.
WO2004 / 092388 JP2007-70579

  Conventionally, synthetic vectors have been researched and developed with the aim of improving gene transfer activity using parameters such as gene loading and permeability into cells. However, although the amount of gene transport into a cell of a synthetic vector is much higher than that of a virus, the actual gene expression level may be lower than that of a virus.

  An object of the present invention is to provide a nucleic acid complex having high gene transfer efficiency.

  The nucleic acid complex of the present invention (Claim 1) is a complex of a cationic polymer and a nucleic acid, and an anionic polymer with respect to a complex in which the amount of positive charges in the complex is greater than the amount of negative charges It is characterized by adsorbing.

  The nucleic acid complex according to claim 2 is characterized in that, in claim 1, the amount of positive charge in the nucleic acid complex is larger than the amount of negative charge.

  The nucleic acid complex according to claim 3 is the nucleic acid complex according to claim 1 or 2, wherein the cationic polymer includes a compound having three or more N, N-dialkyldithiocarbamylmethyl groups in the same molecule as an iniferter. It is characterized by being a branched polymer obtained by light-irradiating living polymerization of a functional monomer.

  The nucleic acid complex according to claim 4 is the compound according to any one of claims 1 to 3, wherein the anionic polymer is a compound having three or more N, N-dialkyldithiocarbamylmethyl groups in the same molecule. This is a branched polymer in which at least an anionic monomer is subjected to light irradiation living polymerization.

  The nucleic acid complex according to claim 5 is the nucleic acid complex according to claim 3 or 4, wherein the compound having three or more N, N-dialkyldithiocarbamylmethyl groups in the same molecule has a benzene ring as a nucleus and a branched chain in the nucleus. Three or more of the N, N-dialkyldithiocarbamylmethyl groups are bonded.

  The nucleic acid complex according to claim 6 is the nucleic acid complex according to any one of claims 3 to 5, wherein the cationic monomer is an acrylic monomer.

  The nucleic acid complex according to claim 7 is characterized in that, in claim 6, the acrylic monomer is 3-N, N-dimethylaminopropylacrylamide.

  The nucleic acid complex according to claim 8 is the nucleic acid complex according to any one of claims 4 to 7, wherein the anionic monomer is 4-vinylbenzoic acid.

  The nucleic acid complex according to claim 9 is the nucleic acid complex according to any one of claims 1 to 8, wherein the ratio of the positive charge amount to the negative charge amount in the nucleic acid complex is 2: 1 to 100: 1. Features.

  The reason why the nucleic acid complex of the present invention is excellent in gene transfer efficiency is considered as follows.

 (1) The cationic polymer forms an ionic complex by mixing with DNA (which is an anionic polymer derived from a phosphate residue) in an aqueous solution. When an anionic polymer is further added to the ionic complex, the anionic polymer is electrostatically adsorbed to the ionic complex to form a nucleic acid complex. This nucleic acid complex has a cationic fine particle of about 100 nm to 200 nm in the concentration range at the time of actual use (the cation / anion ratio is a positive charge rich relative to a complex of a positive charge rich cationic polymer and a nucleic acid). So as to be mixed) to be stably dispersed.

 (2) On the other hand, since the cell membrane of animal cells is negatively charged, the nucleic acid complex composed of cationic fine particles is electrostatically adsorbed to the cell membrane. At this time, since the particle size of the fine particles is approximately 100 nm to 200 nm, which is close to the size of the virus, the cells take this into the cells and aim for detoxification in the digestive organs. This digestive tract is called endosome and is in a strongly acidic environment.

 (3) The anionic polymer on the surface of the nucleic acid complex incorporated into the endosome changes to H-type in a strongly acidic atmosphere and becomes hydrophobic. This improves the permeability of the endosome membrane and facilitates escape from the endosome. As a result, gene transfer activity is improved.

 (4) Cell membranes and endosomal membranes of cells are mainly composed of double phospholipids, and hydrophobic substances have the property of permeating the membranes so as to dissolve the cell membranes. The nucleic acid complex in which the anionic polymer on the surface layer becomes hydrophobic as described above penetrates the cell membrane so as to fuse with the cell membrane of the cell.

  When the anionic polymer is a vinyl benzoic acid polymer, it is Na-type and hydrophilic under cell culture conditions (in an environment of physiological pH = 7.4), but becomes H-type under acidic conditions and becomes a benzene ring. The hydrophobic nature of Therefore, it is hydrophilic at the time of cell membrane permeation (permeability is sufficient even if it is conventional, and lipid fusion to the cell membrane does not cause cytotoxicity), and after being taken into endosomes in cells (strongly acidic environment) (Below) for the first time it becomes hydrophobic and efficiently transfects the endosomal membrane for lipid fusion and transports foreign genes to the nucleus.

  Embodiments of the nucleic acid complex of the present invention will be described in detail below.

The nucleic acid complex of the present invention is a complex of a cationic polymer and a nucleic acid, and an anionic polymer is adsorbed to a complex in which the positive charge amount in the complex is larger than the negative charge amount. It is characterized by this.
The cationic polymer and the anionic polymer are preferably made of a polymer material having a branched chain.

As the cationic polymer material, a branched polymer in which a compound having three or more N, N-dialkyldithiocarbamylmethyl groups in the same molecule is used as an iniferter, and at least a cationic monomer is subjected to light irradiation living polymerization, is preferable. It is.
As an anionic polymer material, a branched polymer in which a compound having three or more N, N-dialkyldithiocarbamylmethyl groups in the same molecule is used as an iniferter, and at least an anionic monomer is light-irradiated by living polymerization is preferable. It is.

  In this specification, the iniferter is a polymerization initiator that generates radicals upon irradiation with light, a function as a chain transfer agent, a function that bonds with the growth terminal to stop the growth, and a polymerization when light irradiation stops. It is a molecule that functions as a polymerization initiator / termination agent for stopping.

  As a compound having three or more N, N-dialkyl-dithiocarbamylmethyl groups in the same molecule as an iniferter, three or more N, N-dialkyl-dithiocarbamylmethyl groups are bonded to the benzene ring as a branched chain. The following are preferred, and specific examples are as follows. That is, the tri-branched compound is obtained by addition reaction of 1,3,5-tri (bromomethyl) benzene and sodium N, N-dialkyldithiocarbamate (sodium N, N-dialkyldithiocarbamate) in ethanol. 1,3,5-tri (N, N-dialkyldithiocarbamylmethyl) benzene, and four-branched compounds include 1,2,4,5-tetrakis (bromomethyl) benzene and N, N-dialkyldithiocarbamic acid 1,2,4,5-tetrakis (N, N-dialkyldithiocarbamylmethyl) benzene obtained by addition reaction of sodium (sodium N, N-dialkyldithiocarbamate) in ethanol, 6-branched compound As hexakis (bromomethyl) benzene and N, N-dialkyldithio Rubamin sodium (sodium N, N-dialkyldithiocarbamate) and the hexakis obtained by addition reaction in ethanol (N, N-dialkyldithiocarbamate carbamylmethyl) include benzene. Here, the alkyl group of the dialkyl moiety contained in the N, N-dialkyl-dithiocarbamylmethyl group is preferably an alkyl group having 2 to 18 carbon atoms such as an ethyl group, but an aromatic group such as a phenyl group. It may be a hydrocarbon group.

  In the following, the nucleic acid complex of the present invention will be described by exemplifying a case where light irradiation living polymerization is performed using an iniferter having the above-described branched chain, but the present invention is not limited to this method. It is not limited.

  The above iniferter is almost insoluble in polar solvents such as alcohol, but is easily soluble in nonpolar solvents. As this nonpolar solvent, hydrocarbons and halogenated hydrocarbons are suitable, and in particular, benzene, toluene, chloroform or methylene chloride, particularly toluene is preferred.

As the cationic monomer to be polymerized to this iniferter, vinyl monomers such as acrylic acid derivatives and styrene derivatives are suitable, and since 3-H, N-dimethylaminopropylacrylamide CH ₂ is particularly excellent in hydrolysis resistance. = CHCONHC ₃ H ₆ N (CH ₃ ) ₂ is preferred.

  As the anionic monomer, a polymerizable monomer having an anionic functional group having a pKa of 5 or less, such as a phenyl carboxyl group, and having a hydrophobic part in the H type having 3 or more carbon atoms, is preferable. As such, for example, aromatic carboxylic acid is preferable, and vinylbenzenecarboxylic acid such as 4-vinylbenzoic acid (4-carboxystyrene) or 3,4-dimethoxycarbonylstyrene is particularly preferable. Vinylbenzenecarboxylic acid may have other substituents.

  To react an iniferter with the above cationic monomer or anionic monomer, prepare a raw material solution containing the iniferter and the monomer, and irradiate it with light to generate a reaction product in which the monomer is bound to the iniferter. Let As the solvent of this solution, alkanes, alkenes, aromatics, and halogenated hydrocarbons are preferable, and specific examples include benzene, toluene, chloroform, carbon tetrachloride, and methylene chloride. Among these, toluene or chloroform is preferable. is there.

  The concentration of the cationic monomer or the anionic monomer in the raw material solution is preferably 0.5M or more, for example, 0.5 to 2.5M. The concentration of the iniferter is preferably about 0.1 to 100 mM.

The wavelength of the irradiated light is preferably 300 to 400 nm, and for example, a low-pressure mercury lamp or a high-pressure mercury lamp can be used. The irradiation time of the light depends on the irradiation intensity, about 1 to 60 minutes are preferred, about 1 to 30 minutes at a low irradiation intensity of about 1μW ^/ cm ² ~10mW ^/ cm 2 is particularly preferred.

  Since the target branched polymer is generated in the reaction solution by this light irradiation, the cationic homopolymer or the anionic homopolymer made of the branched polymer is obtained by purification as necessary.

  The molecular weights of the cationic homopolymer and the anionic homopolymer depend on the number of branched chains, but are preferably about 2,000 to 500,000, particularly about 2,000 to 150,000, especially about 2,000 to 100,000. .

  In another embodiment of the present invention, a cationic monomer or an anionic monomer and a nonionic monomer are used as monomers. In this case, the order of polymerization with respect to the iniferter is arbitrary. That is, the arrangement order of the cationic block or anionic monomer block constituting one branched chain and the nonionic polymer block is arbitrary.

  As the nonionic monomer, N, N-dimethylacrylamide, methoxyethyl (meth) acrylate, polyethylene glycol (meth) acrylate, polyethylene glycol alkyl ester (meth) acrylate, polyvinylpyrrolidone, or the like can be used. The molecular weight of the nonionic polymer block is preferably 2,000 to 500,000.

  The nucleic acid complex of the present invention is such that such a cationic polymer and nucleic acid are complexed to form a primary complex, and an anionic polymer is electrostatically adsorbed thereto.

  In order to combine the cationic polymer and the nucleic acid, the nucleic acid may be added and mixed at room temperature with respect to the dispersion of the cationic polymer having a concentration of about 1 to 1000 μg / mL. An excess amount of the cationic polymer is added to the nucleic acid to be complexed.

  In the present invention, since the complex of the cationic polymer and the nucleic acid further adsorbs the anionic polymer, the ratio of the positive charge derived from the cationic polymer to the negative charge derived from the nucleic acid is preferably 2: 1 to 100: 1 and excessive positive charge. As a result, the nucleic acid complex obtained by adsorbing the anionic polymer to this complex can be positively charged as a whole.

  The adsorbed amount of the anionic polymer with respect to the complex of the cationic polymer and the nucleic acid is preferably a ratio of the positive charge derived from the cationic polymer and the negative charge derived from the nucleic acid and the anionic polymer from 2: 1 to 100. : 1 and excessive positive charge. As a result, the nucleic acid complex is positively charged as a whole, and is adsorbed to the cell membrane and transferred to the endosome. As described above, the anionic polymer in the endosome changes to the H type, and is easily escaped from the endosomal membrane mainly composed of double phospholipids.

  When the anionic polymer block is polycarboxystyrene, it is Na-type and hydrophilic under cell culture conditions (in the environment of physiological pH = 7.4), but it becomes H-type under acidic conditions and the hydrophobicity of the benzene ring. The nature of As a result, it is hydrophilic only when permeating the cell membrane (under the conditions of cell culture) (permeability is sufficient even with the conventional one as described above, and it does not cause lipid fusion to the cell membrane, so the cytotoxicity is weak), and the cell It becomes hydrophobic only after being taken into the endosome (in a strongly acidic environment), and efficiently transfects the endosomal membrane so as to fuse the lipid to carry the foreign gene to the nucleus.

  Preferred examples of the nucleic acid include herpes simplex virus thymidine kinase gene (HSV1-TK gene), p53 tumor suppressor gene, BRCA1 tumor suppressor gene and cytokine gene such as TNF-α gene, IL-2 gene, IL-4 gene, HLA- Cytokine genes such as B7 / IL-2 gene, HLA-B7 / B2M gene, IL-7 gene, GM-CSF gene, IFN-γ gene and IL-12 gene, and gp-100, MART-1 and MAGE-1 These cancer antigen peptide genes can be used for cancer treatment.

  In addition, cytokine genes such as VEGF gene, HGF gene and FGF gene, c-myc antisense, c-myb antisense, cdc2 kinase antisense, PCNA antisense, E2F decoy and p21 (sdi-1) gene are used for vascular treatment. Available. Further, not only DNA introduction and gene expression as described above, but also RNA interference that destroys intracellular mRNA can be carried out by introduction of siRNA. Such a series of genes is well known to those skilled in the art.

  The particle size of the nucleic acid-containing complex is preferably 50 to 400 nm, particularly about 100 to 200 nm. If it is smaller than this, there is a possibility that the action of the enzyme may reach the nucleic acid inside the nucleic acid-containing complex, or it may be filtered out by the kidney. Moreover, when larger than this, there exists a possibility that it may become difficult to introduce | transduce into a cell.

  The nucleic acid is used in such a form that when introduced into a cell, the function can be expressed in the cell. For example, in the case of DNA, the DNA is used as a plasmid in which the DNA is placed so that the DNA is transcribed in the introduced cell and functionally expressed through production of the polypeptide encoded thereby. Preferably, a promoter region, a start codon, DNA encoding a protein having a desired function, a stop codon, and a terminator region are sequentially arranged.

  If desired, two or more nucleic acids can be included in one plasmid.

  In the present invention, “cells” that are desirable as a target for introduction of nucleic acids are those in which functional expression of the nucleic acids is desired. Examples thereof include cardiomyocytes, smooth muscle cells, fibroblasts, skeletal muscle cells, vascular endothelial cells, bone marrow cells, bone cells, blood cell stem cells, blood cell cells and the like. In addition, monocytes, dendritic cells, macrophages, histocytes, Kupffer cells, osteoclasts, synovial A cells, microglia, Langerhans cells, epithelioid cells, multinucleated giant cells, etc., gastrointestinal epithelial cells / tubule epithelium Such as cells.

  The nucleic acid-containing complex of the present invention can be administered to a living body by any method.

  As the administration method, intravenous or intraarterial injection is particularly preferable, but intramuscular, adipose tissue, subcutaneous, intradermal, intralymphatic, intralymphatic, body cavity (pericardial cavity, thoracic cavity, abdominal cavity, cerebrospinal cavity) Etc.) In addition to administration into the bone marrow, administration into the diseased tissue is also possible.

  The pharmaceutical comprising this nucleic acid-containing complex as an active ingredient is further a pharmaceutically acceptable carrier (osmotic pressure regulator, stabilizer, preservative, solubilizer, pH adjuster, thickener, etc.) as necessary. It is possible to mix with. Any known carrier can be used.

  Moreover, the medicine containing this nucleic acid-containing complex as an active ingredient also includes those containing two or more nucleic acid-containing complexes with different types of nucleic acids. Such a medicine having a plurality of therapeutic purposes is particularly useful in the field of diversifying gene therapy.

  As for the dosage, the dosage administered to animals, particularly humans, varies depending on various factors such as the target nucleic acid, the method of administration, and the specific site to be treated. However, the dosage should be sufficient to produce a therapeutic response.

  This nucleic acid-containing complex is preferably applied to gene therapy. Applicable diseases vary depending on the type of nucleic acid included in the complex, but in addition to diseases in the cardiovascular region that cause lesions such as peripheral arterial disease, coronary artery disease, restenosis after arterial dilation, cancer (malignant Melanoma, brain tumor, metastatic malignant tumor, breast cancer, etc.), infectious diseases (HIV, etc.), single genetic diseases (cystic fibrosis, chronic granulomas, α1-antitrypsin deficiency, Gaucher disease, etc.) .

  In addition, an aqueous solution of the nucleic acid complex is attached to a substrate by coating or the like, and dried as necessary to form a coating of a polymer carrying nucleic acids.

  A gene transfer material comprising a substrate carrying a nucleic acid complex gene transfer agent is placed on a sheet-like substrate so as to surround a subcutaneous tissue, a myocardial tissue, a diseased tissue, or a diseased blood vessel, or is applied to a film of a covered stent. Therefore, it is used in such a manner that it is placed in the living body or is attached to the outer surface of the living body using an adhesive tape.

  Hereinafter, the present invention will be described more specifically with reference to examples.

Example 1
i) Synthesis of iniferter According to the following reaction formula, 1,2,4,5-tetrakis (NN-diethyldithiocarbamylmethyl) benzene was synthesized as follows.

5.0 g of 1,2,4,5-tetrakis (bromomethylbenzene) and 34.0 g of sodium N, N-diethyldithiocarbamate were added to 100 mL of ethanol and stirred at room temperature for 4 days under light shielding. The precipitate was filtered, poured into 3 liters of methanol, stirred for 30 minutes and filtered. This operation was repeated a total of 4 times. After the precipitate was dissolved in 200 mL of toluene, 100 mL of methanol was added and heated to 50 ° C., stored in a refrigerator for 15 hours for recrystallization, and the crystals were separated by filtration and washed with a large amount of methanol. The crystals were dried under reduced pressure at room temperature to obtain white 1,2,4,5-tetrasakis (N, N-diethyldithiocarbamylmethyl) benzene needle-like crystals (yield 90%). High-performance liquid chromatography confirmed that the raw material peak disappeared and the purified product was a single substance.
The measurement result of ¹ H NMR (in CDCl ₃ ) is δ1.26-1.31 ppm (t, 24H, CH ₂ CH ₃ ), δ 3.69-3.77 ppm (q, 8H, N (CH ₂ CH ₃ ) ₂ ), Δ 3.99-4.07 ppm (q, 8H, N (CH ₂ CH ₃ ) ₂ ), δ 4.57 ppm (s, 8H, Ar—CH ₂ ), δ 7.49 ppm (s, 2H, Ar—H) It became.

ii) Synthesis of cationic homopolymer comprising 4-branched star polymer by photopolymerization According to the following reaction formula, 1,2,4,5-tetrakis [(N, N-diethyldithiocarbamyl) ) A cationic homopolymer comprising poly (3-N, N-dimethylaminopropylacrylamide) -methyl] benzene (hereinafter sometimes referred to as pDMAPAAm) was synthesized.

That is, 45.6 mg of 1,2,4,5-tetrasakis (N, N-diethyldithiocarbamylmethyl) benzene synthesized by i) above was dissolved in 20 mL of toluene, and 3-N, N-dimethylaminopropylacrylamide was dissolved. 3.9 g of (3-N, N-DMAPAAm) was added and mixed, and the total amount was adjusted to 50 mL with toluene. After purging with high-purity nitrogen gas for 5 minutes with vigorous stirring in a 3 mm thick soft glass cell, mixed ultraviolet rays of 250 nm to 400 nm were irradiated for 30 minutes with a 300 W short arc xenon lamp (manufactured by Asahi Spectroscopic Co., Ltd., MAX-301). The irradiation intensity was adjusted to 2.5 mW / cm ² by attaching UVD-C405 (detection wavelength range of 320 nm to 470 nm) to UIT-150 manufactured by USHIO. The polymerization solution is concentrated with an evaporator, the polymer is reprecipitated with diethyl ether, purified by repeated reprecipitation three times with chloroform / diethyl ether system, ether is evaporated and dissolved in a small amount of water, 0.2 μm The solution was filtered through a filter and freeze-dried to obtain a cationic homopolymer consisting of a 4-branched star homopolymer pDMAPAAm (polymerization rate 40%). The molecular weight of this product was determined to be 32,000 (Mw / Mn = 1.3) by GPC.
The measurement result of ¹ H NMR (in D ₂ O) is as follows: δ1.5-1.8 ppm (br, 2H, —CH ₂ CH ₂ CH ₂ —), δ 2.1-2.2 ppm (br, 6H, N— _{CH 3), δ2.2-2.4ppm (br,} 2H, CH 2 -N), δ3.0-3.4ppm (br, 2H, NH-CH 2), δ7.4-7.8ppm (br, 1H, -NH-).

iii) Synthesis by photopolymerization of an anionic homopolymer comprising a 4-branched star polymer 1,2,4,5-tetrakis [(N, N-diethyldithiocarbamyl) -poly (4-carboxystyrene) -methyl Benzene (hereinafter sometimes referred to as pCS) was synthesized. That is, 45.6 mg of 1,2,4,5-tetrasakis (N, N-diethyldithiocarbamylmethyl) benzene synthesized in i) above was dissolved in 20 mL of chloroform, and 2.0 g of 4-vinylbenzoic acid was added. The total amount was adjusted to 50 mL with chloroform. Photopolymerization is performed in the same manner as in ii), and the polymer component recovered by ether reprecipitation is vacuum-dried and then dissolved in 1N sodium hydroxide solution and dialyzed with 0.1N sodium hydroxide solution for 3 days and then with water for 3 days. Thereafter, freeze drying was performed to obtain tetrakis {N, N-diethyldithiocarbamyl-poly (4-carboxylstyrene) -methyl} benzene (4-branched pCS polymer) (polymerization rate 30%). The molecular weight was measured by GPC as 15,000 (Mw / Mn = 1.3).

iv) Gene transfer experiment COS-1 derived from African green monkey kidney cells was used as cells, and a pGL3 control vector was used as DNA. The number of COS-1 cells was adjusted to 40,000 cells / mL, seeded in a 24 Well culture dish, and gene transfer was performed after 24 hours of culture.
First, a gene complex (polyplex) was formed from a cationic homopolymer composed of the 4-branched star polymer synthesized in ii) and DNA.
The amount of positive charge per unit weight of the cationic homopolymer was determined by measuring the ion exchange capacity by neutralization titration. The amount of negative charge per unit weight in DNA was calculated from the number of base pairs (5256 pairs) according to the sequence map and the average molecular weight of nucleobases (660 Da).

60 μL of this cationic homopolymer (physiological saline solution with a concentration of 0.8 μg / μL) was gently added to 90 μL of DNA (concentration of 0.33 μg / μL of Tris / HCl buffer solution pH = 7.4) and mixed at room temperature. For 30 minutes to obtain a polyplex (nucleic acid complex) solution having a total volume of about 150 μL. The cationic homopolymer and DNA mixing ratio in the polyplex obtained by this operation can be calculated such that the positive charge amount is 30 times the negative charge amount in relation to the number of charges. This nucleic acid complex solution was dispensed into 20 μL aliquots, and 200 μL of OPTI-MEM was added to each aliquot and mixed.
Subsequently, the anionic homopolymer composed of the 4-branched star polymer synthesized in the above iii) was dissolved in OPTI-MEM to adjust the concentration to 0.05 μg / μL. 10 to 150 μL of this solution was added to 220 μL of the polyplex OPTI-MEM dispersion previously prepared and incubated for 30 minutes, and further incubated for 10 minutes, and then added to the cultured cells. By this operation, the solution concentration was adjusted so that 0.5 μg of DNA was administered to each well of the cultured cells.
After 3 hours of culture, OPTI-MEM was removed, each well was washed twice with 1 mL of Dulbecco's PBS, and 1 mL of complete medium was added and cultured for 48 hours.
Forty-eight hours after transfection, gene transfer activity was evaluated by luciferase assay (Promega, assay kit reagent).
Correction was performed at protein concentration, and protein quantification was performed with Bradford reagent from BioRad. The results are shown in FIG.
As shown in FIG. 1, activity was achieved in a system to which 30 μL of an anionic homopolymer solution was added (positive charge amount: negative charge amount = 22: 1) and a system to which 50 μL was added (positive charge amount: negative charge amount = 18: 1). Improved.

By adding an anionic homopolymer later to a polyplex formed in excess of positive charge (positive charge: negative charge = 30: 1) and stably dispersed by electrostatic repulsion in a buffer solution, It is thought that ion adsorption to the surface of the polyplex occurs. Even if the anionic polymer is adsorbed in the above concentration range, the polyplex is considered to have a positive charge, and the polyplex is considered to adsorb to the cell membrane. After that, polyplexes taken up into endosomes in cells change the polyanion on the surface layer to H type in an acidic atmosphere and become hydrophobic, thereby improving the permeability of the cell membrane and helping escape from the endosomes. As a result, it is estimated that the activity was improved.
When the amount of the anionic homopolymer solution added is 100 μL or more, that is, when the amount of adsorbed anionic polymer is large, the activity decreases conversely.
(1) The amount of positive charge on the surface of the polyplex decreased and the adsorptivity to the cell membrane disappeared
(2) The amount of positive charge on the surface of the polyplex decreased, and the fine particles aggregated due to the decrease in electrostatic repulsion
(3) It is considered that the polyplex collapsed such as ion exchange with DNA.

3 is a graph showing the results of gene introduction experiment in Example 1.

Claims (9)

  A nucleic acid complex comprising a complex of a cationic polymer and a nucleic acid, wherein an anionic polymer is adsorbed to a complex in which the amount of positive charges in the complex is greater than the amount of negative charges .   The nucleic acid complex according to claim 1, wherein the amount of positive charge in the nucleic acid complex is greater than the amount of negative charge.   3. The cationic polymer according to claim 1, wherein the cationic polymer is a compound having three or more N, N-dialkyldithiocarbamylmethyl groups in the same molecule as an iniferter, and at least a cationic monomer is subjected to light irradiation living polymerization. A nucleic acid complex which is a branched polymer.   4. The anionic polymer according to claim 1, wherein the anionic polymer is a compound having three or more N, N-dialkyldithiocarbamylmethyl groups in the same molecule as an iniferter. A nucleic acid complex characterized by being a branched polymer polymerized by irradiation living polymerization.   The compound having 3 or more N, N-dialkyldithiocarbamylmethyl groups in the same molecule according to claim 3 or 4 having a benzene ring as a nucleus, and 3 or more N, N- A nucleic acid complex, wherein a dialkyldithiocarbamylmethyl group is bonded.   The nucleic acid complex according to any one of claims 3 to 5, wherein the cationic monomer is an acrylic monomer.   The nucleic acid complex according to claim 6, wherein the acrylic monomer is 3-N, N-dimethylaminopropylacrylamide.   The nucleic acid complex according to any one of claims 4 to 7, wherein the anionic monomer is 4-vinylbenzoic acid.   The nucleic acid complex according to any one of claims 1 to 8, wherein a ratio of a positive charge amount to a negative charge amount in the nucleic acid complex is 2: 1 to 100: 1.

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