JP2008195687A - Nucleic acid complex - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a nucleic acid complex with gene transfer activity having a temperature-responding polymer block which is hydrophilic at temperatures lower than a prescribed temperature (T) lower than the temperature of a living body and is hydrophobic at temperatures higher than the prescribed temperature (T). <P>SOLUTION: This nucleic acid complex uses a gene transfer agent comprising a branched chain-having polymer material having a temperature-responding polymer block which is hydrophilic at temperatures lower than a prescribed temperature (T) lower than the temperature of a living body and is hydrophobic at temperatures higher than the prescribed temperature (T). The polymer material is obtained by living-polymerizing a vinylic monomer in the presence of an iniferter comprising a compound having three or more N,N-dialkyl-dithiocarbamoylmethyl molecular groups in the same molecule under irradiation of light to produce a homopolymer comprising the branched polymer and then block-polymerizing N-isopropylacrylamide to the obtained homopolymer. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a nucleic acid complex using a gene introduction agent.

Synthetic polymer vectors and cationic lipid vectors have been researched and developed as gene transfer techniques to replace virus vectors that have problems with safety, quality stability, and production costs.
Applicants have a synthetic polymer vector with a branched structure in which a cationic polymer chain radially extends with an aromatic ring such as benzene as a nucleus, condensing DNA at high density to form small nucleic acid complex microparticles. Invented that gene can be introduced into cells well (Patent Document 1 below). The mechanism by which the composite microparticles permeate the cell membrane is largely dependent on the action that the positive charge due to the cationic polymer chain electrostatically binds to the negative charge on the cell membrane surface and is taken into the cell by endocytosis. Conceivable.
In contrast, the cationic lipid vector represented by Lipofectamine 2000 has high expression by permeating the membrane so that the lipid on the surface of the liposome particles formed with DNA dissolves or fuses to the cell membrane of the cell by hydrophobic interaction. Is demonstrating.
In recent years, studies on imparting hydrophobic interaction by introducing a hydrophobic segment such as cholesterol into a cationic polymer chain have been actively conducted even in synthetic polymer vectors. However, a synthetic polymer in which a hydrophobic substance such as cholesterol is introduced into a cationic polymer chain has a micelle-like structure in which an aqueous solution contains a large number of hydrophilic cationic polymer chains on the outer peripheral side and a large amount of hydrophobic substances in the inner shell. It is thought that a micelle structure is formed. Therefore, in the complex formed with DNA, the cationic block is preferentially oriented to the aqueous solution side, and the hydrophobic interaction of the polyion complex is considered to be very inefficient. It is done.
WO2004 / 092388

  The present invention eliminates the above-mentioned conventional problems, expresses both the hydrophobic interaction with the cell membrane and the electrostatic action of the cationic block, and provides a nucleic acid complex that can permeate the cell membrane more efficiently. The purpose is to provide.

  The nucleic acid complex of the present invention is a nucleic acid complex comprising a fine particle complex of a gene introduction agent and a nucleic acid comprising a polymer material having a plurality of branched chains radially extending from an aromatic ring as a nucleus. The polymer material is at least a cationic polymer block and a temperature sensitive material that is hydrophilic at a temperature lower than a predetermined temperature (T) lower than a living body temperature and is hydrophobic at a temperature higher than the predetermined temperature (T). And a nucleic acid, a cationic polymer block, and a temperature-sensitive polymer block are present on the surface of the fine particles. The polymer referred to in the present invention includes oligomers such as monomer dimers.

  The polymer material is preferably a block copolymer of a cationic monomer and N-isopropylacrylamide or a derivative thereof, and is a block copolymer of a cationic monomer, a nonionic monomer, and N-isopropylacrylamide or a derivative thereof. It may be.

  The molecular weight of the cationic homopolymer block is preferably 2,000 to 500,000, and the molecular weight of the nonionic polymer block is preferably 2,000 to 500,000.

  The molecular weight of this block copolymer is preferably 3,000 to 600,000.

  The predetermined temperature (T) is preferably a temperature between 25 and 35 ° C.

  The cationic homopolymer is a branched polymer in which a compound having three or more N, N-dialkyl-dithiocarbamylmethyl molecular groups in the same molecule is used as an iniferter, and a vinyl-based monomer is subjected to light irradiation living polymerization. Preferably there is.

  The compound having three or more N, N-dialkyl-dithiocarbamylmethyl groups in the same molecule has a benzene ring as a nucleus and three or more N, N-dialkyl-dithio as a branched chain in this nucleus. Those having a carbamylmethyl molecular group bonded thereto are preferred.

  The vinyl monomer is at least selected from the group consisting of 3-N, N-dimethylaminopropylacrylamide, 2-N, N-dimethylaminoethyl methacrylate, N, N-dimethylacrylamide, and methoxyethyl (meth) acrylate. One is preferred.

  The gene introduction agent used in the nucleic acid complex of the present invention has a temperature-sensitive polymer block, and is hydrophilic and water-soluble at a temperature lower than the predetermined temperature. It can be dissolved and complexed with the nucleic acid. This gene introduction agent becomes hydrophobic when the temperature is higher than a predetermined temperature.

  The gene introduction agent used in the present invention is a synthetic polymer vector having a branched structure in which a cationic polymer chain radially extends with an aromatic ring such as benzene as a nucleus. This gene introduction agent can condense DNA with high density due to its structural advantage. On the surface of the fine particles of the nucleic acid complex of the present invention in which the gene introduction agent and the nucleic acid are complexed, DNA, a cationic polymer chain block, and a temperature functional polymer chain block are randomly aligned. When this nucleic acid complex is used to introduce a gene into a cell, the temperature-functional block chain exposed on the surface of the nucleic acid complex microparticles under the culture condition undergoes a phase transition to a hydrophobic block, resulting in hydrophobicity with the cell membrane. It exhibits both the sex interaction and the electrostatic action of the cationic block, and can efficiently penetrate the cell membrane.

  The gene introduction agent used in the present invention is hydrophilic at a temperature lower than a predetermined temperature (T) lower than the living body temperature and is hydrophobic at a temperature higher than the predetermined temperature (T). It consists of a polymer material having a temperature sensitive polymer block.

  As the above polymer material, a compound having three or more N, N-dialkyl-dithiocarbamylmethyl molecular groups in the same molecule is used as an iniferter, and a vinyl monomer is irradiated with the light by living polymerization, and further a temperature sensitive polymer. A branched polymer into which a block is introduced is preferred.

  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 molecular groups in the same molecule, three or more N, N-dialkyl-dithiocarbamylmethyl molecular groups are bonded as a branched chain to the benzene ring. These are preferred, and specific examples are as follows. That is, as a 3-branched chain, 1,3,5-tri (bromomethyl) benzene and sodium N, N-dithiocarbamate (sodium N, N-dithiocarbamate) can be obtained by addition reaction in ethanol. , 3,5-tri (N, N-dithiocarbamylmethyl) benzene, and four branched chains include 1,2,4,5-tetrakis (bromomethyl) benzene and sodium N, N-dithiocarbamylate ( 1,2,4,5-tetrakis (N, N-dithiocarbamylmethyl) benzene obtained by addition reaction with sodium N, N-dithiocarbamate) in ethanol. Hexakis (N, N-dithio) obtained by addition reaction of bromomethyl) benzene and sodium N, N-dithiocarbamate in ethanol Rubamirumechiru) is a benzene.

  The above iniferter is almost insoluble in polar solvents such as alcohol, but is easily soluble in nonpolar solvents. The nonpolar solvent is preferably a hydrocarbon, an alkyl halide or an alkylene halide, and particularly preferably benzene, toluene, chloroform or methylene chloride, particularly toluene.

As the monomer to be polymerized to this iniferter, vinyl monomers such as vinyl monomers, acrylic acid derivatives, styrene derivatives and the like are particularly suitable. Specifically, 3-N, N-dimethylaminopropylacrylamide, 2-N, N - dimethylaminoethyl methacrylate, N, N- dimethylacrylamide, and at least one especially 3-N is selected from the group consisting of methoxyethyl (meth) acrylate, N- dimethylaminopropyl acrylamide _{CH 2 = CHCONHC 3 H 6 N} ( CH ₃ ) ₂ is preferred.

  In order to cause the iniferter to react with the monomer, a raw material solution containing the iniferter and the monomer is prepared, and a reaction product in which the monomer is bonded to the iniferter is generated by irradiating the material solution with light.

  The concentration of this monomer in the raw material solution is preferably 0.5 M% or more, for example, 0.5 M to 2.5 M.

  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. 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.

  In addition, you may perform a 2nd light irradiation process further after this light irradiation process (1st light irradiation process). That is, the solution containing the reaction product is diluted with an alcohol, preferably an alcohol solution of the above monomer. As the alcohol, methanol or ethanol, particularly methanol is preferable. The monomer concentration in the alcohol solution is preferably about 100 mM to 5 M as the final concentration.

  It is preferable to add 5 to 500 parts by volume of the alcohol solution to 1 part by volume of the reaction product-containing liquid from the first light irradiation step.

  The diluted solution thus diluted with the alcohol solution is subjected to the second light irradiation step, and the monomer is further polymerized with respect to the reaction product. In this case, any irradiation light source may be used as long as it includes light having a wavelength of 240 to 400 nm. For example, a low-pressure mercury lamp or a high-pressure mercury lamp can be used. The light irradiation time is preferably about 10 minutes to 120 minutes.

  By this light irradiation, a branched polymer is produced in the reaction solution, and thus a cationic homopolymer comprising the branched polymer is obtained by purification as necessary.

  The molecular weight of this branched polymer depends on the number of branched chains, but is preferably 2,000 to 500,000, particularly 2,000 to 150,000, and particularly 2,000 to 100,000.

  In the present invention, it is preferable to first block-polymerize N-isopropylacrylamide or a derivative thereof to a cationic polymer made of a branched polymer to introduce a temperature-sensitive polymer block to obtain a target polymer material. However, a branched polymer having a temperature sensitive polymer block may be formed first, and then a cationic polymer block may be introduced.

  In any case, the polymer chain of N-isopropylacrylamide or a derivative thereof has a temperature dependency that becomes hydrophilic at a low temperature and hydrophobic at a high temperature. As a result, the polymer material has the temperature responsiveness.

In order to block copolymerize N-isopropylacrylamide or a derivative thereof with a cationic polymer block, the branched polymer synthesized as described above is dissolved in a solvent such as methanol, and N-isopropylacrylamide is mixed therein. What is necessary is just to superpose | polymerize by irradiating light. The concentration of the branched polymer in the solution at the start of this polymerization reaction is preferably about 0.01 to 10% by weight, and the concentration of N-isopropylacrylamide is preferably about 0.3 to 30% by weight. The light irradiation conditions are preferably a light wavelength of 250 to 400 nm, an irradiation time of 1 to 150 minutes, and an irradiation intensity of about 100 to 10,000 μW / cm ² .

  In another embodiment of the present invention, as the monomer, a cationic monomer, N-isopropylacrylamide or a derivative thereof, and a nonionic monomer are used. The order of polymerization with respect to the iniferter is arbitrary. That is, the order of arrangement of the cationic block, N-isopropylacrylamide block or derivative thereof, and nonionic polymer block constituting one branched chain is arbitrary.

  As the nonionic monomer, polyethylene glycol (meth) acrylate, polyethylene glycol alkyl ester (meth) acrylate, polyvinylpyrrolidone, or the like can be used.

  The molecular weight of the block copolymer (polymer material) is preferably 3,000 to 600,000, particularly preferably 3,000 to 150,000.

  The temperature sensitive polymer block in the gene transfer agent made of this polymer material is hydrophobic at a temperature higher than about 30 ° C. and hydrophilic at a temperature lower than about 30 ° C.

  Therefore, at a temperature higher than about 30 ° C., the gene introduction agent combined with the nucleic acid has a hydrophobic portion made of a temperature-sensitive nucleic acid.

  The gene introduction agent (vector) generated in this way surrounds the nucleic acid as a nucleic acid-containing complex, whereby the inactivation and degradation of the nucleic acid by the enzyme in the living body can be suppressed.

  In order to combine the gene introduction agent and the nucleic acid, the nucleic acid may be added to and mixed with an aqueous solution having a concentration of about 1 to 1000 μg / mL of the gene introduction agent at a temperature lower than 30 ° C. It is preferable that an excessive amount of a gene introduction agent is added to the nucleic acid, and the cationic polymer in the gene introduction agent is complexed as a nucleic acid-containing complex in a saturated state with respect to the nucleic acid.

  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. In addition to the introduction of DNA and the like as described above, gene expression, and repressing, RNA interference that destroys intracellular mRNA can also be performed 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 complex is preferably about 50 to 400 nm. This particle size is measured by, for example, a dynamic light scattering apparatus. When the particle size is smaller than this, there is a possibility that the enzyme action may reach the nucleic acid inside the nucleic acid complex, or when it is used in vivo, it may be quickly filtered out by the kidney. Moreover, when larger than this, there exists a possibility that microparticles may aggregate and settle, or 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, it 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 complex of the present invention can be administered to a living body by any method besides being used for a culture test.

  Intravenous or intraarterial injection is particularly preferred as a method of administration to a living body, but intramuscular, adipose tissue, subcutaneous, intradermal, intralymphatic, intralymphatic, body cavity (pericardial cavity, thoracic cavity, abdominal cavity, brain) In addition to administration into the spinal cavity and the like, it is also possible to administer directly into the diseased tissue.

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

  Moreover, the medicine which contains this nucleic acid complex as an active ingredient includes those containing two or more kinds of nucleic acid-containing complexes having 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 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.), infection (HIV, etc.), single genetic disease (cystic fibrosis, chronic granulomas, α1-antitrypsin deficiency, Gaucher disease, etc.), etc. .

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

  1.0 g of 1,2,4,5-tetrakis (bromomethylbenzene) and 4.0 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, dried under reduced pressure, dissolved in 200 mL of chloroform, and extracted and separated by adding 150 mL of water to remove sodium bromide. After repeating this operation three times, the chloroform layer was dried over magnesium sulfate for 24 hours, filtered, added with n-hexane, recrystallized and purified, and white 1,2,4,5-tetrasakis ( Acicular crystals of (N, N-diethyldithiocarbamylmethyl) benzene were obtained (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. (Hereafter, it may be described as 3-N, N-DMAPAAm.) 19.0 g 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 quartz cell, ultraviolet light was irradiated with a 200 W high-pressure mercury lamp for 35 minutes. The irradiation intensity was adjusted to 1 mW / cm ² (250 nm) using an illuminometer (UVR-1, TOPCON, Tokyo, Japan). The polymerization solution is concentrated with an evaporator, purified by reprecipitation of the polymer with diethyl ether, dissolved in a small amount of water, filtered through a 0.2 μm filter, and lyophilized to obtain a 4-branched star homopolymer 1,2. , 4,5-tetrakis [(N, N-diethyldithiocarbamyl) poly (3-N, N-dimethylaminopropylacrylamide) -methyl] benzene (pDMAPAAm) was obtained (40% polymerization rate). ). The molecular weight was determined to be 50,000 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 of polymer material (4-branched pDMAPAAm-b-pNIPAM) by block copolymerization of N-isopropylacrylamide with cationic homopolymer According to the following reaction formula, tetrakis [(N, N-diethyl) Synthesis of dithiocarbamyl) -poly (N-isopropylacrylamide) -block-poly (3-N, N-dimethylaminopropylacrylamide) -methyl] benzene (hereinafter sometimes referred to as pDMAPAAm-b-pNIPAM). went.

  That is, the 4-branched pDMAPAAm homopolymer synthesized in the above ii) was transferred to a 1 liter flask, re-precipitated by adding about 800 mL of diethyl ether, and diethyl ether was removed by decantation. About 50 mL of toluene was added thereto to dissolve the polymer component, and diethyl ether was added again to reprecipitate the polymer component. This operation was repeated three times. The polymer component was dissolved in about 20 mL of methanol, and 0.5 g of N-isopropylacrylamide (NIPAM) was mixed therein to adjust the total amount to 60 mL with methanol. Polymerization material pDMAPAAm-b-pNIPAM consisting of a block polymer of 4-branched pDMAPAAm and poly N-isopropylacrylamide (pNIPAM) after light irradiation polymerization under the same conditions as in ii) and purification in methanol / diethyl ether system Got. The molecular weight was determined to be 55,000 by GPC.

  As a result, the intended branched star gene introduction agent was obtained.

[Composite of gene introduction agent and nucleic acid]
As DNA, a pGL3 control vector (Promega) encoding firefly luciferase was prepared at a concentration of 0.033 μg / μL.
60 μL of the physiological saline solution of the gene introduction agent having a concentration of 1 μg / μL was tapped and mixed with 90 μL. When the particle size was measured, an ionic complex of 80 to 120 nm and an average of 110 nm was formed, and was stably dispersed even after about 48 hours at a temperature of 20 to 23 ° C. When placed in an environment of 37 ° C., the particle size was 90 to 140 nm for several hours after the temperature rise, and the average was 130 nm, but there was no significant change, but after 24 hours, the particle size increased to about 400 nm, causing aggregation of fine particles. It was confirmed that Considering that the actual gene transfer operation is performed within several hours, it is considered that there is no problem if the particle diameter of the nucleic acid complex can be maintained for several hours after the temperature is raised at 37 ° C.
Next, the particle diameter was measured by the same method as described above except that the temperature of all aqueous solutions was adjusted to 37 ° C. An ion complex of 360 to 400 nm was formed, and it was confirmed that the particle diameter increased with time and settled. This is presumed that the particle diameter was increased because micelle-like fine particles were formed so as to surround the hydrophobic block.
In addition, the dynamic light-scattering apparatus (HORIBA company, LB-500) was used for the measurement of said particle diameter.

[Gene transfer experiment]
Vascular endothelial cells were used as the cells.
The solution of the gene introduction agent was prepared in an environment of 20 to 25 ° C. The DNA is prepared as a TE buffer solution with a concentration of 0.033 μg / μL, and 90 μL of the above-described gene transfer agent physiological saline solution with a concentration of 1 μg / μL is mixed with 60 μL, and then mixed with 150 μL of OPTI-MEM at room temperature. Incubated for a minute to obtain a gene transfer agent solution. 6 × 10 ⁴ COS-1 cells were seeded in a 24-well culture plate, and transfection was performed after 24 hours of culture.
Transfection was performed by removing the medium from the cultured cells, washing with PBS twice, adding 200 μL of the gene transfer agent solution, incubating for 3 hours, washing with PBS, adding complete medium, and culturing for 48 hours.
FIG. 1 shows the results of evaluating the gene transfer activity by luciferase assay after culturing for 48 hours after the transfection. For firefly luciferase activity, Promega's assay kit was used, and correction was performed at the protein concentration. Protein quantification was performed with Bradford reagent from BioRad.

<Comparative Example 1>
Following the synthesis process ii) of Example 1 above, a 4-branched pDMAPAAm homopolymer was synthesized, and diethyl ether reprecipitation was repeated 7 times in a 2 liter flask, and the total amount of the precipitate was dissolved in 20 mL of methanol. Styrene 1.0g was added here and the whole quantity was adjusted to 50 mL with methanol. Photoirradiation polymerization was carried out in the same manner as in the above synthesis process iii), and a 4-branched pDMAPAAm-b-St block polymer was synthesized. The molecular weight was determined to be 55,000 by GPC. From this, it was confirmed that a hydrophobic styrene block was introduced into the hydrophilic cationic polymer chain.
The particle size of the ion complex of the 4-branched pDMAPAAm-bSt block polymer and DNA was measured in the same manner as in Example 1. The temperature environment was 20 ° C. to 25 ° C., and the influence due to the introduction of the hydrophobic block was confirmed. It was suggested that an ion complex of 350 to 450 nm was formed and was stably dispersed for several hours, but the particle diameter increased with time and the aggregation phenomenon occurred.
A gene transfer experiment was conducted according to Example 1 except that this 4-branched pDMAPAAm-b-St block polymer was used as a vector. The result is shown in FIG.

<Comparative example 2>
A gene transfer experiment was conducted according to Example 1 except that the 4-branched pDMAPAAm homopolymer synthesized in the synthesis process ii) of Example 1 was used as the vector. The result is shown in FIG.

[Discussion]
From the above, it is considered that the fixed introduction of the hydrophobic block into the cationic homopolymer affects the particle size of the nucleic acid complex and its stability, and some improvement in gene introduction activity is confirmed, but it is not efficient. . In the present invention, the particle size of the nucleic acid complex is condensed and stabilized at a high density by phase transition to a hydrophobic block for the first time during the cell action to form a hydrophobic block when the nucleic acid complex is formed. It was confirmed that the hydrophobic interaction can be used efficiently.

It is a graph which shows the result of an Example and a comparative example.

Claims (10)

In a nucleic acid complex comprising a fine particle complex of a gene introduction agent and a nucleic acid comprising a polymer material having a plurality of branched chains radially extending from an aromatic ring as a nucleus,
The polymer material is at least a cationic polymer block and a temperature that is hydrophilic at a temperature lower than a predetermined temperature (T) lower than a living body temperature and is hydrophobic at a temperature higher than the predetermined temperature (T). And a sensitive polymer block,
A gene introduction agent, wherein a nucleic acid, a cationic polymer block, and a temperature sensitive polymer block are present on the surface of the fine particles.   2. The nucleic acid complex according to claim 1, wherein the polymer material is a block copolymer of a cationic monomer and N-isopropylacrylamide or a derivative thereof.   2. The nucleic acid complex according to claim 1, wherein the polymer material is a block copolymer of a cationic monomer, a nonionic monomer, and N-isopropylacrylamide or a derivative thereof.   4. The nucleic acid complex according to claim 3, wherein the molecular weight of the nonionic polymer block is 2,000 to 500,000.   The nucleic acid complex according to any one of claims 2 to 4, wherein the molecular weight of the cationic polymer block is 2,000 to 500,000.   The nucleic acid complex according to any one of claims 2 to 5, wherein the molecular weight of the block copolymer is 3,000 to 600,000.   The nucleic acid complex according to any one of claims 1 to 6, wherein the predetermined temperature (T) is a temperature of 25 to 35 ° C.   8. The block copolymer according to claim 2, wherein the block copolymer is an iniferter having a compound having three or more N, N-dialkyl-dithiocarbamylmethyl groups in the same molecule. A nucleic acid complex characterized by being a branched polymer obtained by light-irradiating living polymerized.   9. The compound according to claim 8, wherein the compound having three or more N, N-dialkyl-dithiocarbamylmethyl molecular groups in the same molecule has a benzene ring as a nucleus and three or more N, N- A nucleic acid complex comprising a dialkyl-dithiocarbamylmethyl molecular group bound thereto.   The group according to claim 8 or 9, wherein the vinyl monomer is 3-N, N-dimethylaminopropylacrylamide, 2-N, N-dimethylaminoethyl methacrylate, N, N-dimethylacrylamide, and methoxyethyl (meth) acrylate. A nucleic acid complex comprising at least one selected from the group consisting of:

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