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WO2011108624A1 - Polymère d'un composé contenant de l'azote, son procédé de fabrication et agent de transfert génique - Google Patents

Polymère d'un composé contenant de l'azote, son procédé de fabrication et agent de transfert génique Download PDF

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Publication number
WO2011108624A1
WO2011108624A1 PCT/JP2011/054855 JP2011054855W WO2011108624A1 WO 2011108624 A1 WO2011108624 A1 WO 2011108624A1 JP 2011054855 W JP2011054855 W JP 2011054855W WO 2011108624 A1 WO2011108624 A1 WO 2011108624A1
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nitrogen
polymer
containing compound
gene
containing monomer
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PCT/JP2011/054855
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English (en)
Japanese (ja)
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泰秀 中山
アレクセイ ボロフコフ
泰 根本
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独立行政法人国立循環器病研究センター
株式会社ブリヂストン
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Publication of WO2011108624A1 publication Critical patent/WO2011108624A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F2/00—Processes of polymerisation
    • C08F2/46—Polymerisation initiated by wave energy or particle radiation
    • C08F2/48—Polymerisation initiated by wave energy or particle radiation by ultraviolet or visible light
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F20/00—Homopolymers and copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride, ester, amide, imide or nitrile thereof
    • C08F20/02—Monocarboxylic acids having less than ten carbon atoms, Derivatives thereof
    • C08F20/52—Amides or imides
    • C08F20/54—Amides, e.g. N,N-dimethylacrylamide or N-isopropylacrylamide
    • C08F20/56—Acrylamide; Methacrylamide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation

Definitions

  • the present invention relates to a nitrogen-containing compound polymer and a method for producing the same.
  • the present invention also relates to a gene introduction agent comprising the nitrogen-containing compound polymer, a production method thereof, a nucleic acid complex comprising the gene introduction agent and a nucleic acid, and a gene introduction method using the nucleic acid complex.
  • gene therapy research has become increasingly important.
  • the purpose of gene therapy is to suppress the DNA expression at the target site, and to suppress or inhibit the expression by siRNA, antisense, etc., how to reach the target site, how to efficiently introduce DNA into the target site. It is important whether it is functionally expressed at the site.
  • Many vectors including retroviruses, adenoviruses, adeno-associated viruses, lentiviruses, Sendai viruses or herpes viruses, have been modified to carry therapeutic genes as vectors for the introduction of foreign DNA and RNA, Used in human clinical trials of treatment. However, the risk of infection and immune response remains.
  • Synthetic polymer vectors and cationic lipid vectors have been researched and developed as gene transfer technologies to replace viral vectors that have problems with safety, quality stability, and production costs.
  • a gene introduction agent composed of a cationic polymer having a linear structure, a branched structure, a star structure, a graft structure or the like composed of an aminoalkylene polymer has been reported (Non-patent Document 1, Patent Document 1).
  • the present applicants have a branched structure vector in which a cationic polymer chain radially extends with an aromatic ring such as benzene as a nucleus, which condenses DNA at high density and is stable in aqueous solution. It was found that complex fine particles can be formed and genes can be efficiently introduced into cells, and patent applications have been filed previously (Patent Documents 2 and 3). Furthermore, based on the knowledge that a polymer having a complex network structure is more advantageous for gene introduction, the present applicants have developed a synthetic polymer vector obtained by crosslinking the vectors having the branched structure. Invented and applied for a patent (Patent Document 4).
  • the present applicants have also introduced a gene comprising a thermosensitive cationic polymer that exhibits a cationic property at a temperature lower than a predetermined temperature (T) but is hydrophobic at a temperature higher than the predetermined temperature (T).
  • a solution containing the gene introducing agent and the nucleic acid is mixed at a temperature lower than a predetermined temperature (T), and then the solution is heated to a predetermined temperature (T) or more to combine the gene introducing agent and the nucleic acid.
  • a nucleic acid complex was obtained by bringing the nucleic acid complex and cells into contact with each other at a predetermined temperature (T) or higher in a medium containing serum (patent). Reference 5).
  • a gene transfer agent having a branched chain comprising a polymer of 2-N, N-dimethylaminoethyl methacrylamide and / or a derivative thereof having a neutral pKa value is contained in a medium containing serum.
  • a medium containing serum In US Pat. No. 5,637, they were found to be less susceptible to anionic proteins and exhibit excellent gene transfer activity, and previously filed a patent application (Patent Document 6).
  • This gene transfer agent is used to light 2-N, N-dimethylaminoethylmethacrylamide and / or a derivative thereof to an iniferter composed of a compound having three or more N, N-disubstituted dithiocarbamylmethyl groups in the same molecule. Manufactured by irradiation living polymerization.
  • Genes comprising a gene introduction agent comprising a cationic polymer described in Patent Document 1 and Non-Patent Document 1, and a vector having a branched structure in which polymer chains extend radially from a benzene ring described in Patent Documents 2 to 4
  • the introduction agent can be arranged with a higher charge density due to its structure as compared to a linear polymer composed of the same monomer unit. Therefore, a complex with a nucleic acid such as DNA or RNA can be more strongly aggregated, and more stable polyplex particles can be formed in an aqueous solution.
  • Patent Documents 1 to 4 and Non-Patent Document 1 form macroparticles by adsorbing and aggregating anionic proteins in serum in a medium containing serum due to their cationic properties. In some cases, it becomes difficult to be taken up into cells or DNA is hardly recognized by a transporter.
  • thermosensitive cationic polymer that exhibits a cationic property at a temperature lower than a predetermined temperature (T) and exhibits a hydrophobic property at a temperature higher than the predetermined temperature (T).
  • T predetermined temperature
  • T hydrophobic property
  • the gene introduction agent is cationic when the temperature is lower than a predetermined temperature (T)
  • the gene introduction agent and the nucleic acid can be mixed efficiently. Since the gene introduction agent changes to hydrophobic, that is, nonionic, the gene introduction agent and the nucleic acid are combined at a predetermined temperature (T) or higher, and then the nucleic acid complex is brought into contact with the cell. As a result, it was possible to prevent the nucleic acid complex from being complexed with the anionic substance in the serum, and as a result, the gene transfer efficiency was improved.
  • this gene introduction method is required to improve the following (1) to (4).
  • T a predetermined temperature
  • the gene introduction agent changes to hydrophobicity
  • the nucleic acid complex composed of this gene introduction agent and nucleic acid also changes to hydrophobicity.
  • the gene introduction agent changes to hydrophobicity, it may be affected by components that solubilize hydrophobic substances such as cholesterol and albumin contained in serum and ionic lipids.
  • the hydrophobic gene transfer agent can be easily adsorbed to the hydrophobic petri dish made of polystyrene. There is sex.
  • Nucleic acid complexes composed of gene transfer agents that have changed to hydrophobicity tend to aggregate in an aqueous solution. This hydrophobic aggregation phenomenon is a factor that reduces the introduction efficiency in introducing a gene into cultured cells.
  • Patent Document 6 solves these problems, and a gene transfer agent having a branched chain composed of a polymer chain of 2-N, N-dimethylaminoethylmethacrylamide has excellent gene transfer activity in a medium containing serum. showed that.
  • This gene transfer agent exhibits excellent gene transfer activity and is highly useful as a gene transfer agent.
  • the number of production steps is further reduced and the production efficiency is increased. Improvements and reductions in manufacturing costs are always desired.
  • the present invention has been made in view of the above-described conventional situation, and an object thereof is to provide a nitrogen-containing compound polymer that can be synthesized in a shorter time, easily and inexpensively, and a method for producing the same. Another object of the present invention is to provide a gene introduction agent comprising this nitrogen-containing compound polymer and exhibiting high gene introduction activity in a medium containing serum, and a method for producing the gene introduction agent. Furthermore, an object of the present invention is to provide a method for introducing a gene into a medium containing the gene introduction agent and nucleic acid complex and serum.
  • the nitrogen-containing monomer represented by the following general formula (I) is cleaved by light irradiation to generate a radical. Therefore, when an attempt was made to synthesize a nitrogen-containing compound polymer made of a polymer material using this radical, it was possible to obtain a branched polymer quickly, and this branched polymer was excellent in gene transfer even in a medium containing serum. It was found to show activity.
  • the present invention has been achieved on the basis of such knowledge, and the gist thereof is as follows.
  • the nitrogen-containing compound polymer of the first aspect of the present invention is obtained by polymerizing at least a nitrogen-containing monomer represented by the following general formula (I).
  • R 1 and R 2 each independently represent an alkyl group
  • R 3 represents an alkylene group
  • R 4 represents a hydrogen atom or a methyl group.
  • the nitrogen-containing compound polymer of the second aspect is characterized in that, in the first aspect, R 3 in the general formula (I) is an alkylene group having 1 to 10 carbon atoms.
  • the nitrogen-containing compound polymer of the third aspect is characterized in that, in the first or second aspect, the nitrogen-containing compound polymer is obtained by polymerizing only the nitrogen-containing monomer.
  • the nitrogen-containing compound polymer of the fourth aspect is characterized in that, in any one of the first to third aspects, R 3 in the general formula (I) is a methylene group or an ethylene group.
  • a nitrogen-containing compound polymer according to a fifth aspect is the nitrogen-containing compound polymer according to any one of the first to fourth aspects, wherein the nitrogen-containing monomer is 2-N, N-dimethylaminomethyl (meth) acrylamide and / or 2-N, N- It is characterized by being dimethylaminoethyl (meth) acrylamide.
  • the gene introduction agent according to the sixth aspect of the present invention is composed of the nitrogen-containing compound polymer according to any one of the first to fifth aspects.
  • the gene introduction agent according to the seventh aspect is characterized in that, in the sixth aspect, the nitrogen-containing compound polymer has a molecular weight of 30,000 to 5,000,000.
  • the nucleic acid complex according to the eighth aspect of the present invention is obtained by combining the gene introduction agent according to the sixth or seventh aspect and a nucleic acid.
  • the gene introduction method according to the ninth aspect of the present invention is a method for introducing a gene into a cell by contacting the nucleic acid complex according to the eighth aspect and the cell in the presence of serum.
  • a nitrogen-containing monomer is irradiated by irradiating light to a reaction solution mainly containing a nitrogen-containing monomer represented by the following general formula (I). And a step of performing the polymerization reaction of.
  • R 1 and R 2 each independently represent an alkyl group
  • R 3 represents an alkylene group
  • R 4 represents a hydrogen atom or a methyl group.
  • the method for producing a nitrogen-containing compound polymer of the eleventh aspect is characterized in that, in the tenth aspect, the reaction solution contains 80% by weight or more of the nitrogen-containing monomer.
  • the light has a wavelength of 290 to 390 nm and an irradiation intensity of 0.01 to 50.0 mW / cm 2. It is a feature.
  • the method for producing a nitrogen-containing compound polymer of the thirteenth aspect is characterized in that, in any one of the tenth to twelfth aspects, the reaction solution is irradiated with the light for 10 seconds to 360 minutes.
  • the gene introduction agent according to the fourteenth aspect comprises a polymer produced by the method for producing a nitrogen-containing compound polymer according to any one of the tenth to thirteenth aspects.
  • the method for producing a gene introduction agent according to the fifteenth aspect of the present invention comprises polymerizing a nitrogen-containing monomer by irradiating light to a reaction solution containing a nitrogen-containing monomer represented by the following general formula (I) as a main component.
  • the method includes a step of performing a reaction.
  • R 1 and R 2 each independently represent an alkyl group
  • R 3 represents an alkylene group
  • R 4 represents a hydrogen atom or a methyl group.
  • the nitrogen-containing compound polymer of the present invention is obtained by polymerizing a specific nitrogen-containing monomer, and can be synthesized easily and inexpensively in a short time.
  • the gene introduction agent comprising this nitrogen-containing compound polymer has high gene introduction efficiency in a medium containing serum.
  • the nitrogen-containing monomer represented by the general formula (I) (hereinafter, the nitrogen-containing monomer represented by the general formula (I) is referred to as “nitrogen-containing monomer (I)”) is not easily hydrolyzed. And the property that the property of the compound hardly changes due to a change in temperature. Further, when the alkylene group of R 3 in the general formula (I) is a methylene group or an ethylene group, the value of pKa is It is neutral and is not easily affected by anionic proteins in a medium containing serum. Therefore, the gene introduction agent comprising this nitrogen-containing monomer (I) can efficiently introduce genes into cells. In addition, the nitrogen-containing compound polymer obtained by polymerizing the nitrogen-containing monomer (I) does not gel even if the molecular weight is about 1,000,000, and is easily soluble in water, so it is easy to handle as a gene introduction agent.
  • R 3 in the general formula (I) is preferably an alkylene group having 1 to 10 carbon atoms (second embodiment). Since the nitrogen-containing monomer in which the number of carbon atoms of the alkylene group is within the above range easily generates radicals by light irradiation, a nitrogen-containing compound polymer can be produced in a short time using this radical.
  • the nitrogen-containing compound polymer is preferably composed of a polymer obtained by polymerizing only the nitrogen-containing monomer (third embodiment).
  • R 3 in the general formula (I) is methylene. It is preferable that it is group or ethylene group (4th aspect). When R 3 in the general formula (I) is a methylene group or an ethylene group, the gene transfer activity in serum is improved.
  • the nitrogen-containing monomer (I) is preferably 2-N, N-dimethylaminomethyl (meth) acrylamide and / or 2-N, N-dimethylaminoethyl (meth) acrylamide (fifth embodiment).
  • the gene introduction agent according to the sixth aspect of the present invention is a gene introduction agent comprising the aforementioned nitrogen-containing compound polymer.
  • the molecular weight of the gene introduction agent is preferably 30,000 to 5,000,000 (seventh embodiment). When the molecular weight is within the above range, DNA can be effectively aggregated, resulting in improved gene transfer efficiency.
  • the nucleic acid complex according to the eighth aspect of the present invention is obtained by combining the gene introduction agent and a nucleic acid.
  • the gene introduction method according to the ninth aspect of the present invention is a method for introducing a gene into a cell by bringing the nucleic acid complex into contact with the cell in the presence of serum.
  • the method for producing a nitrogen-containing compound polymer of the present invention includes a step of polymerizing the nitrogen-containing monomer (I) by irradiating the reaction liquid containing the nitrogen-containing monomer (I) as a main component with light. Therefore, a polymer having a complicated structure can be produced in a short time and at a low cost (tenth embodiment).
  • the polymerization reaction since the polymerization reaction is carried out utilizing the property that the nitrogen-containing monomer (I) generates radicals by light irradiation, it is not necessary to use the above-described iniferter or polymerization initiator. Cost can be reduced. Further, it is efficient because a nitrogen-containing compound polymer having a complicated structure can be obtained without going through a plurality of reaction steps.
  • the reaction solution in the present invention preferably contains 80% by weight or more of a nitrogen-containing monomer (11th embodiment).
  • the higher the concentration of the nitrogen-containing monomer in the reaction solution the faster the reaction proceeds.
  • the process of removing the solvent from the target polymer can be simplified, and the production cost can be reduced.
  • radical movement to the solvent molecule does not occur, or radical movement to the solvent molecule can be significantly suppressed.
  • the light irradiated in the polymerization reaction preferably has a wavelength of 290 to 390 nm and an irradiation intensity of 0.01 to 50.0 mW / cm 2 (a twelfth aspect). Such light is applied to the reaction solution. On the other hand, irradiation is preferably performed for 10 seconds to 360 minutes (13th embodiment).
  • a gene introduction agent comprising a polymer produced by such a method for producing a nitrogen-containing compound polymer exhibits excellent gene introduction activity in a medium containing serum (fourteenth aspect).
  • the method for producing a gene introduction agent of the present invention comprises a step of performing a polymerization reaction of the nitrogen-containing monomer by irradiating light to the reaction solution containing the nitrogen-containing monomer (I) as a main component. Since it is characterized by containing, the gene introduction agent which shows high gene introduction activity in the culture medium containing serum can be manufactured in a short time easily and cheaply.
  • the nitrogen-containing compound polymer of the present invention is obtained by polymerizing at least a nitrogen-containing monomer represented by the following general formula (I).
  • R 1 and R 2 each independently represent an alkyl group
  • R 3 represents an alkylene group
  • R 4 represents a hydrogen atom or a methyl group.
  • the nitrogen-containing compound polymer according to the present invention is synthesized by utilizing the property that the nitrogen-containing monomer (I) easily generates radicals when irradiated with light, it is produced in a very short time. Is possible.
  • the nitrogen-containing compound polymer of the present invention will be described in accordance with the production procedure in the case of producing the nitrogen-containing compound polymer of the present invention. It is not limited to the following method.
  • the nitrogen-containing compound of the present invention can be easily produced by irradiating the reaction liquid containing the nitrogen-containing monomer (I) as a main component with light for a short time.
  • the reason why a branched high molecular weight product can be obtained by light irradiation for a short time is presumed to be that the main reaction caused by light irradiation proceeds according to the following formulas (II) to (V). . That is, first, a radical is generated by photocleaving the amide group of the nitrogen-containing monomer irradiated with light (formula (II)), and the polymerization reaction starts when this radical reacts with the vinyl group of the nitrogen-containing monomer. (Formula (III)).
  • the unreacted amide group of the pendant side chain of the polymer produced by this polymerization reaction (the side chain constituting a part of the basic structure of the molecular chain produced by the polymerization reaction of formula (III)) is photocleaved.
  • a radical is generated (formula (IV)), and this radical and an unreacted nitrogen-containing monomer further react to introduce a branched chain composed of a polymer chain into the polymer (formula (V)). Since the polymerization reaction proceeds sequentially as described above, it is considered that a polymer having a plurality of branch points and having a complicated structure can be obtained in a short time.
  • the polymer is branched by reacting the acyl radicals represented by the formula (IV) or the acyl radical and a part of the polymer chain to be crosslinked.
  • a branched polymer can be easily obtained by one polymerization reaction, it is not necessary to carry out a reaction for crosslinking the produced polymer again.
  • R 1, R 2, R 3 , R 4 have the same meaning as R 1, R 2, R 3 , R 4 in each formula (I).
  • m and n represent an integer of 2 or more, q represents an integer less than m or n.
  • the nitrogen-containing monomer is not particularly limited as long as it is represented by the general formula (I), but the nitrogen-containing monomer (I) described later is preferably used.
  • the polymerization reaction of the nitrogen-containing monomer (I) is carried out in the presence of a compound that does not participate in the polymerization reaction other than the nitrogen-containing monomer (I), for example, in the presence of a solvent.
  • the reaction may be carried out, it is preferable to carry out the polymerization in a reaction solution consisting essentially only of the nitrogen-containing monomer (I) without using a compound which does not participate in the polymerization reaction. This is because even if the compound does not directly participate in the polymerization reaction, it may absorb the generated radicals and reduce the reaction rate of the polymerization reaction.
  • Examples of the compound that does not participate in the polymerization reaction that can be used in the presence of a compound that does not participate in the polymerization reaction such as a solvent include a radical scavenger that inhibits photocleavage of the nitrogen-containing monomer (I), a polymerization reaction Any chain transfer agent that does not inhibit itself may be used, and examples thereof include nonpolar solvents such as benzene and toluene.
  • a solvent that does not affect the polymerization reaction is used as a solvent, it is possible to improve the stirring efficiency because the viscosity of the entire reaction solution is reduced, and after completion of the reaction, Collection becomes easy.
  • the amount used is preferably 20% by weight or less as the content in the reaction solution.
  • the reaction is carried out in the presence of a compound that does not participate in the polymerization reaction such as a solvent, the polymerization yield or the molecular weight of the obtained polymer may decrease, so that the production capacity of the production process or the production purpose is Accordingly, it is important to appropriately select the type and amount of the compound to be used.
  • the nitrogen-containing monomer (I) plays not only a role as a monomer but also a role as a polymerization initiator. Therefore, it is possible to synthesize a polymer having a complicated structure with many branches and leaves without separately using a polymerization initiator such as the above-described iniferter or AIBN.
  • the concentration of the nitrogen-containing monomer (I) contained in the reaction solution is preferably 80% by weight or more, particularly 90% by weight or more.
  • the gene introduction agent according to the present invention may be a branched polymer composed of one kind of nitrogen-containing monomer (I), but comprises two or more kinds of nitrogen-containing monomers (I) as described later. Further, it may be a branched polymer containing a monomer other than the nitrogen-containing monomer (I) and the nitrogen-containing monomer (I).
  • the gene introduction agent containing a monomer other than the nitrogen-containing monomer (I) can be produced by irradiating a reaction solution containing the nitrogen-containing monomer (I) and a monomer other than the nitrogen-containing monomer (I) with light. In this case as well, it is preferable to prepare the reaction solution so that the nitrogen-containing monomer (I) is 80% by weight or more.
  • the light irradiated to the reaction solution may be monochromatic light or mixed light, but the present inventors have repeatedly investigated the relationship between the wavelength of the irradiated light and the polymerization reactivity. It was found that the nitrogen-containing monomer (I) can be efficiently polymerized by irradiating with light of a specific wavelength. That is, when light having a long wavelength exceeding 390 nm is irradiated, the reaction does not proceed or the reaction rate is extremely reduced, so that the production efficiency is lowered. This is because the light having a long wavelength exceeding 390 nm does not have energy sufficient to efficiently cause radical cleavage of the side chain of the nitrogen-containing monomer (I), or an excitation band in which the energy of the light is quantized.
  • the wavelength of light irradiated to the reaction liquid is preferably about 290 to 390 nm, particularly about 300 to 320 nm, and particularly 310 nm.
  • a short arc xenon lamp, a low pressure mercury lamp, a high pressure mercury lamp, a black light, etc. are used. Can do.
  • the light irradiation intensity depends on the scale of the reaction vessel and the irradiation intensity, but is preferably about 0.01 to 50.0 mW / cm 2 , particularly preferably about 0.1 to 10.0 mW / cm 2 .
  • a high irradiation intensity is not preferable because the reaction may be difficult to control.
  • the irradiation intensity is low, the polymerization time becomes long, which is not efficient.
  • the light irradiation time depends on the scale of the reaction vessel and the irradiation intensity, it is preferably 10 seconds to 1440 minutes, particularly 1 minute to 360 minutes, particularly about 3 to 60 minutes. That is, according to the method for producing a nitrogen-containing compound polymer of the present invention, a nitrogen-containing compound polymer can be easily produced even by such short-time light irradiation.
  • This light irradiation produces the target nitrogen-containing compound polymer in the reaction solution, and the nitrogen-containing compound polymer of the present invention can be obtained by purification as necessary.
  • a coating material that covers the surface of a tablet such as a pharmaceutical product can be manufactured at low cost. That is, a tablet coating material is generally formed by using a rolling powder coating device, scattering chemical particles in an ascending current, and spraying and drying an aqueous coating material solution there. Manufacturing requires large amounts of heat to cure the coating material and fresh air to remove moisture. As the coating material, when the nitrogen-containing compound polymer of the present invention is used, it is not necessary to use water as a solvent, heat for removing water, and fresh air, so the cost can be reduced.
  • the manufacturing apparatus can be simplified.
  • the nitrogen-containing compound polymer and the method for producing the same of the present invention can be used in various fields such as the above-mentioned field of DDS (Drug Delivery System) in addition to the gene introduction agent described later.
  • the gene introduction agent of the present invention is composed of the nitrogen-containing compound polymer, and can be produced easily and in a very short time by the above-described production method, and is similar to a dendrimer-like gene introduction agent. , And can be produced without a complicated operation of repeating the protection reaction / elimination reaction / main reaction. Moreover, since the gene introduction agent of this invention can be manufactured without using a polymerization initiator and the above-mentioned iniferter, manufacturing cost can be held down.
  • the nitrogen-containing monomer (I) has a pKa value that is neutral when R 3 in the general formula (I) is an alkylene group or an ethylene group, and is difficult to hydrolyze. It has the property that the property is difficult to change (especially, change such as desolvation and globulization by temperature change confirmed with N, N-dimethylaminoethyl methacrylate). Therefore, the gene introduction agent of the present invention comprising a nitrogen-containing compound polymer obtained by polymerizing this nitrogen-containing monomer (I) is highly gene-transferable in a medium containing serum without being affected by anionic proteins and lipids. Shows activity.
  • the nitrogen-containing monomer is not particularly limited as long as it is represented by the general formula (I), but R 3 in the general formula (I) is an alkylene having 1 to 10 carbon atoms, particularly 1 to 2 carbon atoms. If such an alkylene group is used, the pKa is almost neutral, and when used as a gene introduction agent, it can be made less susceptible to the influence of components such as anionic proteins in serum. It becomes. Moreover, it is preferable that the nitrogen atom which forms an amide bond has couple
  • R 1 and R 2 in the general formula (I) are preferably alkyl groups having about 1 to 5 carbon atoms.
  • nitrogen-containing monomer (I) include 2-N, N-dimethylaminomethyl (meth) acrylamide and / or 2-N, N-dimethylaminoethyl (meth) acrylamide.
  • (meth) acryl means “acryl and / or methacryl”.
  • the molecular weight of the gene introduction agent comprising this nitrogen-containing compound polymer is preferably about 30,000 to 5,000,000, particularly preferably about 100,000 to 1,000,000.
  • This molecular weight can be adjusted by controlling the time of light irradiation and the concentration of the nitrogen-containing monomer contained in the reaction solution. That is, by extending the irradiation time, the polymerization reaction can be advanced to obtain a branched polymer having a large molecular weight.
  • the reaction may be controlled by selecting the wavelength of the irradiated light.
  • the gene introduction agent of the present invention is easy to handle as a gene introduction agent because it does not gel even if it becomes an ultra-high molecular weight body having a molecular weight of 1 million or more and is easily soluble in water.
  • molecular weight means the number average molecular weight of polyethylene glycol conversion by GPC (gel permeation chromatography).
  • the gene introduction agent according to the present invention may be a branched polymer composed of one kind of nitrogen-containing monomer (I), but may be composed of two or more kinds of nitrogen-containing monomers (I), and further contains You may comprise so that monomers other than nitrogen monomer (I) may be included.
  • the gene introduction agent composed of a plurality of monomers can be produced by irradiating the reaction solution composed of the nitrogen-containing monomer (I) and a monomer other than the nitrogen-containing monomer (I) with light, Also in this case, it is preferable to prepare so that the nitrogen-containing monomer (I) contained in the reaction solution is 80% by weight or more.
  • vinyl monomers such as acrylic acid derivatives and styrene derivatives are suitable.
  • the gene introduction agent (vector) of the present invention includes a nucleic acid
  • the inactivation and decomposition of the nucleic acid by an in vivo enzyme can be suppressed.
  • the nitrogen-containing monomer (I) which is a preferred form of the present invention is 2-N, N-dimethylaminomethyl (meth) acrylamide and / or 2-N, N-dimethylaminoethyl.
  • a pKa value is a polymer showing a neutral region, and since it does not have a strong charge, a cationic polymer (pKa is an alkaline region) well known to those skilled in the art is a polyanion.
  • the mechanism for forming electrostatically simple ionic bonds with nucleic acids may be slightly different, but the details are currently unknown and will be clarified in future studies.
  • the nucleic acid is added to the solution of the gene introduction agent at a concentration of about 0.1 to 3,000 ⁇ g / mL, preferably by adding dropwise and mixing. . It is preferable to add an excessive amount of the gene introduction agent to the nucleic acid and to saturate the gene introduction agent with respect to the nucleic acid so that the gene introduction agent and the nucleic acid are combined.
  • the mixing ratio between the gene introduction agent and the nucleic acid is CA ratio (number of moles of cation charge (C) / number of moles of anion charge (A)) or NP ratio (number of moles of nitrogen atom (N) / number of nucleic acids). It is expressed in terms of moles of phosphate residues (P)).
  • the mixing ratio is calculated using such a ratio because the charge amount of the cation in the gene transfer agent depends on the weight and molecular structure of the gene transfer agent, and the anion charge amount in the nucleic acid depends on the weight of the nucleic acid and the base. This is because it depends on the logarithm and the nucleic acid species.
  • the NP ratio has a similar meaning to the CA ratio.
  • N-dimethylaminomethyl (meth) acrylamide and / or 2-N, N-dimethylaminoethyl (meth) acrylamide is used as the nitrogen-containing monomer (I) which is a preferred embodiment of the present invention
  • the gene introduction agent does not have a strong charge
  • a nucleic acid is added to an excessive amount of the gene introduction agent, and the gene introduction agent is supersaturated with respect to the nucleic acid to combine the gene introduction agent and the nucleic acid. To do.
  • a specific mixing ratio is recommended to have a CA ratio of about 5 to 10, and gene transfer activity is improved even with a higher CA ratio.
  • the toxicity is strongly expressed and is not preferable.
  • 2-N, N-dimethylaminomethyl (meth) acrylamide and / or 2-N, N-dimethylaminoethyl (meth) acrylamide is used as the nitrogen-containing monomer (I) which is a preferred embodiment of the present invention.
  • the extreme difference between the optimum CA ratio of the cationic polymer known to those skilled in the art and the optimum NP ratio of the gene introduction agent comprising the nitrogen-containing compound polymer of the present invention is sufficient for the gene introduction agent of the present invention. Because it does not have a positive charge, it does not strongly distort the negatively charged cell membrane that expresses sialic acid, etc., and may be due to the difference in the inclusion mechanism between the nucleic acid and the gene introduction agent It is speculated that there is sex.
  • the nucleic acid solution is preferably added dropwise to the solution of the gene introduction agent and mixed.
  • the gene transfer agent solution is dropped and mixed into the nucleic acid solution contrary to the present invention. Order is an important factor.
  • AFM Anatomic Force Microscope
  • a cationic polymer When a cationic polymer is added here, it is electrostatically adsorbed to DNA, ionically bound to electrostatically neutralize the anion, and aggregated so that the cyclic structure is folded to form polyplex particles.
  • the gene introduction agent of the present invention and the gene introduction agent well known to those skilled in the art may be slightly different in the mechanism of nucleic acid inclusion. I think.
  • nucleic acids include various siRNAs, antisenses, decoys, herpes simplex virus thymidine kinase gene (HSV1-TK gene), p53 tumor suppressor gene, BRCA1 tumor suppressor gene and cytokine gene as TNF- ⁇ gene, IL-2 gene Cytokine genes such as IL-4 gene, HLA-B7 / IL-2 gene, HLA-B7 / B2M gene, IL-7 gene, GM-CSF gene, IFN- ⁇ gene and IL-12 gene, and gp-100, Cancer antigen peptide genes such as MART-1 and MAGE-1 can be used for cancer treatment.
  • HSV1-TK gene herpes simplex virus thymidine kinase gene
  • BRCA1 tumor suppressor gene cytokine gene
  • IL-2 gene Cytokine genes such as IL-4 gene, HLA-B7 / IL-2 gene, HLA-B7 / B2M gene, IL-7 gene, GM
  • 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. Such a series of genes is well known to those skilled in the art.
  • the particle size of the nucleic acid complex using the gene introduction agent of the present invention is preferably about 150 to 500 nm.
  • the particle diameter is measured by, for example, a dynamic light scattering method using a laser, a transmission electron microscope, or an electron force microscope.
  • the particle diameter of the nucleic acid complex known to those skilled in the art is preferably about 50 to 150 nm, although it depends on the size of the nucleic acid used. In other words, although it is obvious that smaller particles are more easily taken up into cells, the nucleic acid complex must be reversibly dissociated in the cell or the nucleic acid must be recognized as a complex, so that the smallest size can be achieved.
  • nucleic acids are aggregated, it takes time to dissociate the complex even if it is taken into the cell. For this reason, those skilled in the art generally use a particle size of about 100 nm as a particle size of a nucleic acid complex for designing a nucleic acid complex.
  • the details of the nucleic acid complex when the gene introduction agent of the present invention is used are larger than 100 ⁇ m and 150 to It has been found that 500 nm, in particular 200 to 400 nm, in particular 250 to 350 nm, is preferred. If the particle size is larger than this, there is a risk that it will be difficult to be taken up into cells, the enzyme may affect the nucleic acid inside the nucleic acid complex, or there may be a blood vessel infarction in the case of in vivo use.
  • the nucleic acid is used in such a form that when introduced into a cell, the function can be expressed in the cell.
  • 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.
  • a promoter region, a start codon, DNA encoding a protein having a desired function, a stop codon, and a terminator region are sequentially arranged.
  • two or more nucleic acids can be included in one plasmid.
  • Gene transfer method In the gene introduction method of the present invention, a gene is introduced into a cell by bringing the nucleic acid complex into contact with the cell in the presence of serum. By contacting the nucleic acid complex and the cell in the presence of serum, the nucleic acid can be transferred to the cell without imposing a burden on the cell.
  • 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 ES cells, iPS cells, hematopoietic stem cells, nerve cells, 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.
  • 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 arterial injection is particularly preferable 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.
  • the medicine containing this nucleic acid complex as an active ingredient includes those containing two or more 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.
  • the dose administered to animals, particularly humans varies depending on various factors such as the target nucleic acid, administration method, and 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.), infectious diseases (HIV, etc.), single genetic diseases (cystic fibrosis, chronic granulomas, ⁇ 1-antitrypsin deficiency, Gaucher disease, etc.) .
  • Example 1 A polymerization reaction was performed using 2-N, N-dimethylaminoethylmethacrylamide as a monomer.
  • the irradiation intensity was adjusted to 2.5 mW / cm 2 by mounting UVD-C405 (detection wavelength range of 320 to 470 nm) on UIT-150 manufactured by USHIO.
  • the viscosity of the liquid rose dramatically within a few minutes from the start of irradiation, and it was confirmed that stirring with a stirrer was not sufficiently effective after 5 minutes. 10 minutes after the start of irradiation, the movement of the rotor completely stopped, and the liquid became a solid wax, so that the light irradiation was terminated.
  • the obtained solid wax was colorless and transparent.
  • the polymer of 2-N, N-dimethylaminoethyl methacrylamide described in Example 2 of Patent Document 6 is colored yellow-brown to orange-brown. In this example, it was obtained in that it was colorless and transparent. It can be seen that there are some differences in the polymers.
  • the inside of the quartz cell was returned to normal pressure, and 5 mL of methanol was added thereto to dissolve the polymer. The methanol solution was concentrated with an evaporator, and the polymer was reprecipitated with n-hexane.
  • the polymer was purified by repeating reprecipitation three times with a methanol / (n-hexane + diethyl ether) system, and the solvent was evaporated, followed by vacuum drying.
  • the obtained polymer was dissolved in a small amount of water, filtered through a 0.2 ⁇ m filter, and freeze-dried to obtain a 2-N, N-dimethylaminoethyl methacrylamide polymer.
  • the measurement result of 1 H-NMR (in chloroform-d 1 ) is ⁇ 0.7-1.2 ppm (br, 3H, —CH 3 ), ⁇ 1.5-1.9 ppm (br, 2H, —CH 2 —CH 3 — ), ⁇ 2.0-2.2ppm (br, 6H, N-CH 3), ⁇ 2.2-2.4ppm (br, 2H, -CH 2 -N), ⁇ 2.8-3.2ppm (br, 2H, NH- CH 2 ).
  • Example 1 The polymer obtained in Example 1 was hardly soluble in benzene and toluene, and was soluble in chloroform, diethyl ether, methanol and water.
  • benzene and toluene are three-dimensionally large molecules compared to chloroform and water, and are known to be disadvantageous for diffusion into tight gaps in the polymer chain of multi-branched polymers and difficult to solvate. Yes. Judging from the results and the synthesis process, the polymer obtained in this example is considered to have a high possibility of having a complicated structure such as a net or a cage.
  • Example 2 The synthesis was performed in the same manner as in Example 1 except that 3-N, N-dimethylaminopropylacrylamide was used as a monomer. It was confirmed that the viscosity of the liquid increased several minutes after the start of light irradiation, and stirring with a stirrer was not effective after 10 minutes. The rotor was stopped 15 minutes after the start of light irradiation. The resulting polymer was purified with a chloroform / diethyl ether system.
  • the solubility of the polymer of Example 2 in the solvent was the same as that of the polymer of Example 1.
  • Comparative Example 1 As Comparative Example 1, a polymerization reaction was performed using a monomer having no alkylene group of R 3 in the general formula (I).
  • the reaction was performed in the same manner as in Example 1 except that N, N-dimethylacrylamide was used as a monomer.
  • N, N-dimethylacrylamide was irradiated with light for 48 hours, the color of the reaction solution changed to pale yellow, but the viscosity hardly changed.
  • Comparative Example 2 As Comparative Example 2, a polymerization reaction was performed using a monomer in which the amide bond is an ester bond in the general formula (I).
  • reaction was performed in the same manner as in Example except that 2-N, N-dimethylaminoethyl methacrylate was used as a monomer. Although light irradiation was performed for 48 hours like the comparative example 1, reaction did not advance and a high molecular weight body was not able to be obtained.
  • COS-1 derived from African green monkey kidney cells was used for the cells, and ProGL's pGL3 control vector was used for the DNA.
  • the number of COS-1 cells was adjusted to 60,000 cells / mL, seeded in a 24 Well culture dish, and gene transfer was performed after 24 hours of culture.
  • the polymer synthesized in Example 1 was dissolved in physiological saline to a final concentration of 78 ⁇ g / 60 ⁇ L (polymer / saline).
  • DNA was dissolved in TE buffer to a concentration of 3 ⁇ g / 90 ⁇ L (DNA / buffer).
  • the nucleic acid complex was formed by mixing while dropping the DNA solution into 60 ⁇ L of the polymer solution at room temperature.
  • nucleic acid complex 25 ⁇ L was added to 1 mL of complete medium (DMEM + 10% FCS + antibiotics) and incubated for 30 minutes. About 1 mL of the nucleic acid complex complete medium solution was added to the cultured cells washed with PBS at 37 ° C., followed by additional culture for 48 hours. After 48 hours, 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. As a reference, a commercially available gene introduction reagent (Invitrogen, Lipofectamine 2000) was used according to the protocol of the product. The gene introduction agent of Example 1 showed an activity of about 85% of a commercially available gene introduction reagent.
  • Example 3- 3 Monomer concentration 80% by weight
  • a reaction was carried out in the same manner as in Example 1 except that a spectral filter was attached to the light source and monochromatic light of 310 nm was irradiated for 9 minutes.
  • the obtained polymer was recovered, purified and freeze-dried, and the yield and number average molecular weight were measured. The results are shown in FIG.
  • Examples 3-2 and 3-3 using toluene as a solvent had a lower yield and number average molecule than Example 3-1.
  • the viscosity of the reaction solution was lowest in Example 3-3, the rotor rotated stably, and the stirring efficiency was excellent.
  • the dispersity (ratio of the weight average molecular weight to the number average molecular weight) of the polymer obtained in each example was 3.7 in Example 3-1, 3.3 in Example 3-2, and Example 3- Since 3 is 2.7 and all have a small molecular weight distribution, it can be seen that the polymers obtained in each Example are excellent in homogeneity.
  • Example 4 Examination of wavelength of irradiated light ⁇ Examples 4 to 7> A spectral filter is attached to the light irradiation device, and polymerization is performed as monochromatic light having a wavelength of 310 nm (Example 4), 330 nm (Example 5), 350 nm (Example 6), and 370 nm (Example 7).
  • the reaction was performed in the same manner as in Example 1 except that the reaction was stopped when the viscosity of the reaction solution reached 1,000,000 Pa ⁇ sec.
  • FIG. 2 shows the relationship between the time until the viscosity of the reaction solution reaches 1 million Pa ⁇ sec (polymerization completion time) and the wavelength of irradiation light.
  • FIG. 2 shows that an inflection point exists in the vicinity of a wavelength of 350 nm.
  • Example 7 polymerized by irradiating light with a wavelength of 370 nm has the largest number average molecular weight
  • the polymer of Example 4 polymerized by irradiating with light with a wavelength of 310 nm
  • the number average molecular weight of the polymer of Example 5 was decreased in the order of the polymer of Example 5, and the number average molecular weight of the polymer of Example 6 polymerized by irradiation with light having a wavelength of 350 nm was the smallest.
  • the reasons why the number average molecular weight of the polymer obtained in Example 6 (wavelength 350 nm) was the smallest were i) generation of radicals as nuclei and generation of large amounts of low molecular weight polymers in a short time.
  • the viscosity of the reaction solution increased and subsequent reactions were less likely to occur; ii) the side chain was rapidly cleaved, resulting in a more complicated branched structure;
  • the reason why the number average molecular weight of the polymer obtained in Example 7 is large is that when light with a wavelength of 370 nm is irradiated, the generation of radical nuclei is slow, and the polymerization of one molecule of polymer proceeds with priority. It is possible to do.
  • FIG. 4 shows the gene transfer activity evaluated in the same manner as in Example 1 for the polymers of Examples 4-7.
  • the number average molecular weight of the obtained polymer was proportional to the gene transfer activity. From these results, it can be said that the wavelengths of irradiation light preferable in terms of gene transfer activity are 310 nm and 370 nm.
  • Example 4 [Molecular weight dependence of gene transfer activity]
  • Example 6 (wavelength 350 nm), and Example 7 (wavelength 370 nm) was treated by size exclusion chromatography and separated into three fractions, as above.
  • the gene transfer activity of each fractionated polymer was measured.
  • the column pressure was set as low as 20 kPa so that the side chain was not cleaved.
  • the fractionation conditions of Examples 4, 6 and 7 and the number average molecular weight and dispersion of the polymers obtained by fractionation are shown in Tables 1 to 3, respectively.
  • the gene transfer activity of each polymer is shown in FIGS. .
  • Example 6 wavelength 350 nm
  • Example 7 wavelength 370 nm
  • gene transfer activity increased with an increase in the number average molecular weight. This is almost the same phenomenon as known knowledge.
  • FIG. 5 in Example 4 (wavelength 310 nm), the gene transfer activity decreased as the number average molecular weight increased. From this result, in the production method of the present invention, it is considered that a polymer having a different structure can be obtained depending on the wavelength of light to be irradiated.
  • the light irradiation method can be further studied. It is considered that a gene transfer agent can be produced efficiently.
  • the product irradiated for 0.5 hour was a solid having an elastic deformability enough to be deformed by pressing with a round bar, and dissolved rapidly when immersed in water. Insoluble components did not occur. Except for the product irradiated for 0.5 hours, it was a glassy hard solid. These solids were not deformed even when pressed with a round bar, but gradually dissolved from the surface layer by adding water, and all of the glassy solid was completely dissolved by immersion in water for about 20 hours. As in the case of the product irradiated for 0.5 hour, no insoluble components were observed.
  • Example 2 Gene transfer experiment
  • the cells used were COS-1 cells or Hela cells derived from cervical cancer. The number of 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.
  • the polymer obtained in Experiment 1 was used as a gene introduction agent.
  • the number of positive charges per unit weight in the gene introduction agent was calculated from the molecular weight 156 of the monomer unit of the cationic polymer.
  • the number of negative charges per unit weight in DNA was calculated from the number of base pairs according to the sequence map and the average molecular weight 660 of nucleobases.
  • This gene introduction agent was mixed with DNA in 150 ⁇ L of TE buffer solution and incubated for 30 minutes.
  • the final concentration was adjusted, 1 mL of complete medium was added, and the cells were cultured for 48 hours. Forty-eight hours later, gene transfer activity was evaluated using a luciferase assay kit. Correction was performed with the total protein concentration, and total protein quantification was performed with Bradford reagent from Bio Rad.
  • FIG. 10 shows the result of gene introduction into COS-1 cells
  • FIG. 11 shows the result of introduction into Hela cells.

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Abstract

L'invention concerne un polymère d'un composé contenant de l'azote, qui peut être facilement synthétisé sur une courte durée, et un procédé pour le produire. L'invention concerne également un agent de transfert génique, qui comprend le polymère précédemment mentionné d'un composé contenant de l'azote et qui présente une activité élevée de transfert génique dans un milieu contenant du sérum, un procédé de production dudit agent de transfert génique, un complexe d'acide nucléique composé de l'agent de transfert génique et d'un acide nucléique, et un procédé pour transférer un gène dans un milieu contenant du sérum. L'agent de transfert génique comprend un polymère d'un composé contenant de l'azote qui est obtenu par polymérisation d'au moins un monomère contenant de l'azote représenté par la formule générale (I). Le polymère est produit par irradiation par de la lumière d'un liquide réactionnel, qui comprend le monomère contenant de l'azote représenté par la formule générale (I) comme composant principal, permettant ainsi de polymériser le monomère contenant de l'azote. Dans la formule (I), R1 et R2 représentent un groupe alkyle ; R3 représente un groupe alkylène ; et R4 représente un atome d'hydrogène ou un groupe méthyle.
PCT/JP2011/054855 2010-03-03 2011-03-03 Polymère d'un composé contenant de l'azote, son procédé de fabrication et agent de transfert génique WO2011108624A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2198732A (en) * 1986-11-22 1988-06-22 Wolfen Filmfab Veb Photopolymerisable materials
JPH01310744A (ja) * 1988-03-31 1989-12-14 Merck Patent Gmbh 分離材料
JP2009275002A (ja) * 2008-05-15 2009-11-26 National Cardiovascular Center 遺伝子導入剤、並びに核酸複合体及びその製造方法
JP2010088399A (ja) * 2008-10-10 2010-04-22 National Cardiovascular Center 遺伝子導入剤及びその製造方法
JP2011083243A (ja) * 2009-10-16 2011-04-28 Bridgestone Corp 遺伝子導入剤、遺伝子導入剤の製造方法、及び核酸複合体並びに遺伝子導入方法

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2198732A (en) * 1986-11-22 1988-06-22 Wolfen Filmfab Veb Photopolymerisable materials
JPH01310744A (ja) * 1988-03-31 1989-12-14 Merck Patent Gmbh 分離材料
JP2009275002A (ja) * 2008-05-15 2009-11-26 National Cardiovascular Center 遺伝子導入剤、並びに核酸複合体及びその製造方法
JP2010088399A (ja) * 2008-10-10 2010-04-22 National Cardiovascular Center 遺伝子導入剤及びその製造方法
JP2011083243A (ja) * 2009-10-16 2011-04-28 Bridgestone Corp 遺伝子導入剤、遺伝子導入剤の製造方法、及び核酸複合体並びに遺伝子導入方法

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