JP2010024192A - Method for producing gene transfer agent - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing a gene transfer agent by which the gene transfer agent comprising a crosslinked star-shaped branched polymer can efficiently be produced. <P>SOLUTION: The method for producing the gene transfer agent includes crosslinking a star-shaped branched polymer having an aromatic ring as a core and a plurality of cationic branched chains radially extended therefrom in a solvent containing an alcohol. Especially, the alcohol is one or more kinds selected from the group consisting of methanol, ethanol and 2-propanol. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a method for producing 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.

As a patent document 1, as a synthetic polymer vector, from a cross-linked star-shaped branched polymer in which an aromatic ring such as benzene is used as a nucleus and a star-shaped branched polymer having a branched chain in which a cationic polymer chain extends radially is crosslinked. According to this vector, DNA can be condensed at high density to form small nucleic acid complex microparticles, and genes can be efficiently introduced into cells.
WO2004 / 092388

  The gene introduction agent of Patent Document 1 has an N, N-2 substituted dithiocarbamylmethyl group at the end of a branched chain of a star-shaped branched polymer, and the radical of this N, N-2 substituted dithiocarbamylmethyl group When it is crosslinked by cleavage, but the ends of the branched chain of the star-shaped branched polymer are not crosslinked, but the ends of the branched chain and the portion other than the ends of the branched chain are crosslinked in a net shape Since it becomes a gel, the yield decreases.

  An object of this invention is to provide the manufacturing method of the gene introduction agent which can manufacture efficiently the gene introduction agent which consists of a bridge | crosslinking star-shaped branched polymer.

  The method for producing a gene transfer agent of the present invention (Claim 1) is a method of cross-linking star-shaped branched polymers in which star-shaped branched polymers having a plurality of cationic branched chains radially extended from an aromatic ring as a core are crosslinked. This is a method for producing a gene introduction agent comprising a type polymer, characterized in that the star-shaped branched polymers are crosslinked with each other in a solvent containing alcohol.

  The method for producing a gene introduction agent according to claim 2 is the method according to claim 1, wherein the star-shaped branched polymer is dissolved and / or dispersed in the solvent, and the solution and / or the dispersion is irradiated with light to thereby produce the star. The branched polymers are crosslinked with each other.

  The method for producing a gene introduction agent according to claim 3 is characterized in that, in claim 1 or 2, the alcohol comprises a lower alcohol.

  The method for producing a gene introduction agent according to claim 4 is characterized in that, in claim 3, the lower alcohol is at least one selected from the group consisting of methanol, ethanol and 2-propanol.

  The method for producing a gene introduction agent according to claim 5 is characterized in that, in any one of claims 1 to 4, the solvent consists only of alcohol.

  The method for producing a gene introduction agent according to claim 6 is characterized in that, in any one of claims 1 to 4, the solvent comprises alcohol and a solvent other than alcohol.

  The method for producing a gene introduction agent according to claim 7 is the group according to claim 6, wherein the solvent other than the alcohol is water, chloroform, toluene, benzene, xylene, ether, acetone, methyl ethyl ketone, tetrahydrofuran, methylene chloride and benzene chloride. It consists of at least 1 sort (s) selected from.

  The method for producing a gene introduction agent according to claim 8 is characterized in that, in claim 6 or 7, the alcohol concentration of the solvent is 30.0 to 99% by weight.

  The method for producing a gene introduction agent according to claim 9 is characterized in that, in any one of claims 1 to 8, the concentration of the star-shaped branched polymer in the solvent is 3 to 60% by weight. Is.

  The method for producing a gene introduction agent according to claim 10 is the method according to any one of claims 1 to 9, wherein the star-shaped branched polymer has three N, N-dialkyldithiocarbamylmethyl groups in the same molecule. A branched polymer in which a compound having the above is used as an iniferter, and a vinyl-based monomer is subjected to light irradiation living polymerization, and the branched chains of the star-shaped branched polymer are crosslinked to form a crosslinked star-shaped branched polymer. It is characterized by this.

  The method for producing a gene introduction agent according to claim 11 is the method according to claim 10, wherein the compound having three or more N, N-dialkyldithiocarbamylmethyl groups in the same molecule has a benzene ring as a nucleus and a branched chain in the nucleus. And three or more N, N-dialkyldithiocarbamylmethyl groups are bonded to each other.

  The method for producing a gene introduction agent according to claim 12 is the method according to claim 10 or 11, wherein the vinyl monomer is 3-N, N-dimethylaminopropylacrylamide, 2-N, N-dimethylaminoethyl methacrylate, N, N- It is at least one selected from the group consisting of dimethylacrylamide, methoxyethyl (meth) acrylate, N-isopropylacrylamide, and derivatives of N-isopropylacrylamide.

  The method for producing a gene introduction agent according to claim 13 is the method according to any one of claims 10 to 12, wherein the branched N, N-dialkyldithiocarbamylmethyl group of the branched polymer contributes as a crosslinking point. It is the characteristic characterized by this.

  The method for producing a gene introduction agent according to claim 14 is characterized in that, in any one of claims 10 to 13, the branched chain is made of a homopolymer of a vinyl monomer.

  The method for producing a gene introduction agent according to claim 15 is the method according to any one of claims 10 to 13, wherein the branched chain is a random or block copolymer of the vinyl monomer and a different monomer. It is what.

  In the method for producing a gene introduction agent of the present invention, star-shaped branched polymers having a plurality of cationic branched chains that are radially extended from an aromatic ring as a nucleus are crosslinked in a solvent containing alcohol.

  According to this method for producing a gene introduction agent, the ends of the branched chains of the star-shaped branched polymer can be reacted efficiently.

  That is, when a crosslinking reaction is performed in a solvent containing alcohol, the alcohol and the cationic moiety in the branched chain form a hydrogen bond, and the cationic moiety in the branched chain is protected by the alcohol. It is presumed that the reaction between the terminal and the cationic moiety in the branched chain is suppressed.

  When the star-shaped branched polymer is dissolved and / or dispersed in the solvent and the solution and / or dispersion is irradiated with light to crosslink the star-shaped branched polymer (Claim 2), A gene introduction agent comprising a branched polymer can be obtained with good yield.

  In the method for producing a gene introduction agent of the present invention, the alcohol is preferably composed of a lower alcohol (Claim 3), and particularly preferably at least one selected from the group consisting of methanol, ethanol and 2-propanol. (Claim 4).

  When a solvent containing the alcohol is used, the cationic part of the branched chain and the alcohol easily form a hydrogen bond.

  Further, the solvent may be composed only of alcohol (Claim 5), or may be composed of alcohol and a solvent other than alcohol (Claim 6). When the solvent comprises alcohol and a solvent other than alcohol, the solvent other than the alcohol is selected from the group consisting of water, chloroform, toluene, benzene, xylene, ether, acetone, methyl ethyl ketone, tetrahydrofuran, methylene chloride, and benzene chloride. Preferably, the solvent has an alcohol concentration of 30.0 to 99% by weight (claim 8).

  By setting the concentration within the above range, the cationic portion of the branched chain can be protected by hydrogen bonding with a small amount of alcohol.

  In the method for producing a gene introduction agent of the present invention, the concentration of the star-shaped branched polymer in the solvent is preferably 3 to 60% by weight.

  If the concentration of the star-shaped branched polymer is within the above range, the reaction between the ends of the branched chain proceeds rapidly.

  In the cross-linking reaction of a cross-linked star-shaped branched polymer, the cross-linking degree of the cross-linked star-shaped branched polymer forms an insoluble gel and the cross-linking degree of a cross-linked star-shaped branched polymer with high gene transfer activity are close. Therefore, it is difficult to control the degree of crosslinking.

  According to the present invention, since the cationic part of the branched chain is protected by a hydrogen bond with alcohol, the crosslinking reaction does not proceed excessively. That is, an insoluble gel is hardly formed and the yield is increased.

  In addition, since the cationic part of the branched chain is protected by a hydrogen bond with alcohol, the side chain is hardly decomposed by light irradiation. That is, the exposure of polar groups due to side chain degradation can be suppressed, and adverse effects on proteins (for example, activation of complement C3a) by polar groups can be prevented. Furthermore, the expression of cytotoxicity and ion exchange of the polymer itself can be prevented. A decrease in capacity can also be prevented.

  The gene introduction agent produced by the method for producing a gene introduction agent of the present invention is a synthetic compound comprising a crosslinked product obtained by crosslinking branched polymers in which cationic branched chains extend radially using an aromatic ring such as benzene as a nucleus. It is a molecular vector. This gene introduction agent can condense nucleic acids such as DNA with high density due to its structural advantage. Since this gene introduction agent is obtained by crosslinking a plurality of branched polymers, it can contain nucleic acids such as DNA in a wider network compared to a gene introduction agent consisting of one branched polymer, Excellent gene transfer activity is exhibited.

  The method for producing a gene introduction agent of the present invention comprises a cross-linked star-shaped branched polymer in which star-shaped branched polymers having a plurality of cationic branched chains radially extended from an aromatic ring as a core are crosslinked. This is a method for producing a gene introduction agent, in which star-shaped branched polymers are crosslinked with each other in a solvent containing alcohol.

  This star-shaped branched polymer will be described in detail later.

<Crosslinking of star-shaped branched polymer>
In the present invention, it is preferable to dissolve and / or disperse the star-shaped branched polymer in the solvent and to crosslink the star-shaped branched polymers by irradiating the solution and / or dispersion with light.

  As the alcohol used as the solvent, lower alcohols such as methanol, ethanol, 1-propanol, 2-propanol, butanol, pentanol, and hexanol are preferable, and methanol, ethanol, and 2-propanol are particularly preferable. These alcohols may be used alone or in combination of two or more.

  When such an alcohol is used, the alcohol and the cationic part of the branched chain of the star-shaped branched polymer tend to form a hydrogen bond.

  In the present invention, the solvent may be composed of only the alcohol, or may be composed of alcohol and a solvent other than alcohol. Examples of the solvent other than alcohol include water, chloroform, toluene, benzene, xylene, and diethyl ether. And ether, acetone, methyl ethyl ketone, tetrahydrofuran, methylene chloride and benzene chloride. Solvents other than this alcohol may be used alone or in combination of two or more.

  When the solvent comprises an alcohol and a solvent other than alcohol, the proportion of alcohol in the solvent is extremely small, and if the proportion of apolar solvent such as toluene or chloroform is large, the cationic portion of the branched chain is sufficiently bonded by hydrogen bonding. It is difficult to protect, and when the proportion of water is large, the radical reaction of dithiocarbamate described later does not proceed easily. Therefore, the concentration of the alcohol in the solvent is preferably about 30.0 to 99% by weight, particularly about 50.0 to 99% by weight.

  The concentration of the star-shaped branched polymer in the solvent is preferably 3 to 60% by weight, particularly 6 to 30% by weight. If the concentration of the star-shaped branched polymer is extremely high, the reaction is difficult to proceed, and if the concentration of the branched polymer is extremely low, the collision probability between the ends of the branched chain is unfavorable.

In the method for producing a gene introduction agent of the present invention, the light irradiation conditions for crosslinking the star-shaped branched polymers are as follows: light wavelength 180 to 700 nm, irradiation time 1 minute to 30,000 hours, irradiation intensity 0. A range of about 001 to 10,000 μW / cm ² is suitable.

  In the present invention, the molecular weight of the crosslinked star-shaped branched polymer is preferably about 30,000 to 300,000, particularly about 35,000 to 150,000. If the molecular weight of the crosslinked star-shaped branched polymer is extremely large, cytotoxicity is strongly expressed, which is not preferable from the viewpoint of metabolism and excretion, and if it is extremely small, the ability to embed DNA decreases.

  In addition, in this specification, molecular weight means the number average molecular weight of polyethylene glycol conversion by GPC (gel permeation chromatography).

  The molecular weight distribution Mw / Mn of the crosslinked star-shaped branched polymer is preferably about 1.3 to 3.0, particularly preferably about 1.5 to 2.5.

  By performing the crosslinking reaction in this way, the ends of the branched chain of the star-shaped branched polymer are crosslinked to form 2 to 10, particularly 2 to 5 to form a crosslinked product. In this crosslinking reaction, it is presumed that the thio group of the branched N, N-dialkyldithiocarbamylmethyl group of the branched polymer is opened and the ends of the branched chain are crosslinked.

  According to the method for producing a gene introduction agent of the present invention, since the reaction between the cationic moiety in the branched chain and the end of the branched chain can be suppressed, the cationic moiety in the branched chain that is a by-product, A gel-like cross-linked product in which the end of the branched chain is cross-linked in a net form is hardly formed. Therefore, the yield is about 90 to 100%.

  In the present invention, since the amino group which is a cationic moiety in the branched chain is protected by alcohol, coloring due to oxidation of the amino group by light irradiation hardly occurs, and the quality of the crosslinked star-shaped branched polymer is improved. To do.

<Star-branched polymer>
As the star-shaped branched polymer, a branched polymer in which a compound having three or more N, N-dialkyldithiocarbamylmethyl groups in the same molecule is used as an iniferter, and a vinyl-based monomer is subjected to light irradiation living polymerization on this compound is used. Is preferred. When the N, N-dialkyldithiocarbamylmethyl groups are bonded to each other, the star-shaped branched polymers are cross-linked.

  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.

  Examples of the compound having three or more N, N-dialkyldithiocarbamylmethyl groups in the same molecule include those having three or more N, N-dialkyldithiocarbamylmethyl groups bonded to the benzene ring as a branched chain. Specifically, the following is exemplified. 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, particularly preferably benzene, toluene, chloroform or methylene chloride, especially 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 CH ₂ ═CHCONHC ₃ H Selected from the group consisting of ₆ N (CH ₃ ) ₂ , 2-N, N-dimethylaminoethyl methacrylate, N, N-dimethylacrylamide, methoxyethyl (meth) acrylate, N-isopropylacrylamide and N-isopropylacrylamide derivatives. At least one of these is preferred.

  In order to react the iniferter with the monomer, a raw material solution containing the iniferter and the monomer is prepared, and a light-irradiated product is produced to produce a reaction product in which the monomer is bonded to the iniferter.

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

  The concentration of the iniferter is preferably about 0.1 to 100 mM.

The wavelength of the irradiated light is preferably 300 to 400 nm. 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.

  Since the target branched polymer is produced in the reaction solution by this light irradiation, the cationic homopolymer comprising the branched polymer is obtained by purification as necessary.

  The molecular weight of this star-shaped 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, the branched chain may be the homopolymer described above, or may be a block copolymer or a random copolymer with a different polymer. For example, N, N-dimethylacrylamide 2-N, N-dimethylaminoethyl methacrylate, methoxyethyl (meth) acrylate, or the like may be introduced into the homopolymer. Alternatively, a temperature sensitive polymer block may be introduced by block copolymerization of N-isopropylacrylamide or a derivative thereof. A branched polymer having a branched chain made of N, N-dimethylacrylamide or N-isopropylacrylamide or a polymer thereof is formed first, and then a cationic polymer block is introduced at the tip side of each branched chain. You may make it do.

  The polymer chain of N-isopropylacrylamide or a derivative thereof has temperature dependency that becomes hydrophilic at a low temperature and hydrophobic at a high temperature. As a result, the gene introduction agent has the above-mentioned temperature responsiveness.

In order to block copolymerize N, N-dimethylacrylamide or N-isopropylacrylamide or a derivative thereof with a cationic polymer block, the star-shaped branched polymer synthesized as described above is preferably used in a solvent such as alcohol such as methanol. It may be dissolved, mixed with N, N-dimethylacrylamide or N-isopropylacrylamide or a derivative thereof, and irradiated with light for polymerization. The concentration of the branched polymer in the solution at the start of the polymerization reaction is preferably about 0.01 to 10% by weight, and the concentration of N, N-dimethylacrylamide or N-isopropylacrylamide or a derivative thereof is 0.3 to 30% by weight. The degree is preferred. 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 .

  The molecular weight of the star-shaped branched polymer is preferably 3,000 to 600,000, particularly 3,000 to 150,000.

  As described above, the gene introduction agent obtained by block copolymerization of N-isopropylacrylamide or a derivative thereof is hydrophobic at a temperature higher than about 30 ° C. and hydrophilic at a temperature lower than about 30 ° C. Therefore, the nucleic acid can be combined with the gene introduction agent by mixing the nucleic acid and the aqueous solution of the gene introduction agent at a temperature lower than 30 ° C.

  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 and becomes insoluble in water.

<Nucleic acid-containing complex>
When the gene introduction agent (vector) produced by the method for producing a gene introduction agent of the present invention surrounds a nucleic acid as a nucleic acid-containing complex, it is possible to suppress inactivation and degradation of the nucleic acid by enzymes in the living body.

  In order to combine the gene introduction agent and the nucleic acid, the nucleic acid may be added to and mixed with a solution of the gene introduction agent having a concentration of about 1 to 1000 μg / mL. 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 to the nucleic acid in a saturated state as a nucleic acid-containing complex.

  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. 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 method using a laser. If the particle size is smaller than this, the action of the enzyme may reach the nucleic acid inside the nucleic acid complex, or it may be filtered out by the kidney. Moreover, when larger than this, there exists a possibility that it may become difficult to introduce | transduce into a cell.

  The nucleic acid is used in such a form that when introduced into a cell, the function can be expressed in the cell. For example, in the case of DNA, 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.

  Such a nucleic acid complex 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.

  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.), infectious diseases (HIV, etc.), single genetic diseases (cystic fibrosis, chronic granulomas, α1-antitrypsin deficiency, Gaucher disease, etc.) .

[Synthesis of 4-branch 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.

  5.0 g of 1,2,4,5-tetrakis (bromomethylbenzene) and 34.0 g of sodium N, N-diethyldithiocarbamate were added to 100 mL of ethanol and stirred at room temperature for 4 days under light shielding. The precipitate was filtered, poured into 3 liters of methanol, stirred for 30 minutes and filtered. This operation was repeated a total of 4 times. After the precipitate was dissolved in 200 mL of toluene, 100 mL of methanol was added and heated to 50 ° C., stored in a refrigerator for 15 hours for recrystallization, and the crystals were separated by filtration and washed with a large amount of methanol. The crystals were dried at room temperature under reduced pressure to obtain white needle-like crystals of 1,2,4,5-tetrakis (N, N-diethyldithiocarbamylmethyl) benzene (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 star-shaped four-branched polymer by photopolymerization According to the following reaction formula, 1,2,4,5-tetrakis [(N, N-diethyldithiocarbamyl) ) -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-tetrakis (N, N-diethyldithiocarbamylmethyl) benzene synthesized by i) above was dissolved in 20 mL of toluene, and 3-N, N-dimethylaminopropylacrylamide was dissolved. 7.9 g of (3-N, N-DMAPAAm) was added and mixed, and the total amount was adjusted to 50 mL with toluene. After purging with high purity nitrogen gas for 5 minutes with vigorous stirring in a 3 mm thick soft glass cell, a mixed ultraviolet ray of 250 nm to 400 nm was irradiated for 25 minutes with a 300 W short arc xenon lamp (manufactured by Asahi Spectroscopic Co., Ltd., MAX-301). The irradiation intensity was adjusted to 2.5 mW / cm ² by attaching UVD-C405 (detection wavelength range of 320 nm to 470 nm) to UIT-150 manufactured by USHIO. The polymerization solution is concentrated with an evaporator, the polymer is reprecipitated with diethyl ether, purified by repeated reprecipitation three times with chloroform / diethyl ether system, ether is evaporated and dissolved in a small amount of water, 0.2 μm The solution was filtered through a filter and freeze-dried to obtain a cationic homopolymer comprising a star-shaped four-branched homopolymer pDMAPAAm (polymerization rate: 32%). The molecular weight was measured to be 41,000 (Mw / Mn = 1.3) by GPC in terms of polyethylene glycol.

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) Solubility of Cationic Homopolymer Consisting of Star-shaped Four-branched Polymer 0.2 g of cationic homopolymer composed of star-shaped four-branched polymer synthesized in the above ii) and 3.0 g of methanol were mixed. The mixed solution was dropped into 1000 mL of diethyl ether to evaluate the solubility of the cationic homopolymer (Example 1).

  In Example 1, precipitation of the polymer component was not confirmed only by diethyl ether becoming slightly cloudy. This is because the star-shaped four-branched polymer and methanol stubborn hydrogen bonds prevent the methanol molecules solvating the star-shaped four-branched polymer from diffusing into the diethyl ether phase. It is considered that the coalescence is stably dissolved and / or dispersed without forming secondary particles.

  A mixed liquid (Example 2) in which 0.2 g of a cationic homopolymer composed of the star-shaped four-branched polymer synthesized in ii) and 3.0 g of ethanol were mixed, and 0.2 g and 3 of the above cationic homopolymer. Although the same evaluation as Example 1 was performed also about the liquid mixture (Example 3) which mixed 0.0 g of 2-propanol, precipitation was not confirmed in all.

  As a comparative example, a mixed liquid (Comparative Example 1) in which 0.2 g of a cationic homopolymer composed of a star-shaped four-branched polymer synthesized in ii) and 50 g of chloroform were mixed, and 0.2 g of the cationic homopolymer described above. A mixed solution (Comparative Example 2) in which 50 g of toluene and 50 g of toluene were mixed was evaluated in the same manner as in the example.

  In Comparative Examples 1 and 2, a sticky bulk polymer precipitated quickly in diethyl ether. This is thought to be because chloroform and toluene are diffused rapidly into the diethyl ether phase due to weak solvation with the star-shaped four-branched polymer.

iv) Photocrosslinking of star-shaped 4-branched polymer (4-branched pDMAPAAm) 1,2,4,5-tetrakis [(N, N-diethyldithiocarbamyl) -poly- (3-N, N-dimethylamino) Propylacrylamide) -methyl] benzene was photocrosslinked.

That is, 0.2 g of the 4-branched pDMAPAAm homopolymer having a molecular weight of 41,000 synthesized in the above ii) was put into a 100 mL sterilization bottle, and 3.0 g of methanol, ethanol, 2-propanol (Examples 1 to 3) and toluene (Examples 1 to 3) It melt | dissolved in the comparative example 2), and light irradiation superposition | polymerization was performed with the same light irradiation apparatus as ii) stirring gently with a rotor. The light irradiation was performed by an epi-illumination through a 2 mm thick soft glass plate, the irradiation intensity was 1.0 mW / cm ² , and the light irradiation time was 180 minutes. After completion of the irradiation, the pastes obtained in Examples 1 to 3 were almost colorless and transparent, and no foreign matter was observed. On the other hand, in the case of Comparative Example 2 using toluene as a solvent, almost all of the polymer became an insoluble gel after 180 minutes of irradiation. The gel was yellowish brown with a strong amine odor.

  To the light irradiators obtained in Examples 1 to 3, 30 mL of toluene was mixed and then concentrated with an evaporator, and dropped into diethyl ether to precipitate a polymer component. Then, it refine | purified by performing the reprecipitation process 3 times by chloroform / diethyl ether system. The light irradiator obtained in Comparative Example 2 was diluted with 50 mL of toluene, the insoluble gel was removed by filtration, concentrated with an evaporator, and dropped into diethyl ether to precipitate a polymer component. Then, it refine | purified by performing the reprecipitation process 3 times by chloroform / diethyl ether system.

  The molecular weights of the photocrosslinked bodies of Examples 1 to 3 were 48,000 (Mw / Mn = 1.4) and 50,000 (Mw / Mn = Mw / Mn = 1.4, respectively) in methanol, ethanol and 2-propanol by GPC in terms of polyethylene glycol. 1.5) and 53,000 (Mw / Mn = 1.5), and an increase in molecular weight was confirmed. The dispersion did not become broad, and the peak shape became a normal distribution as before photocrosslinking. This crosslinked product showed good solubility in a good solvent for the polymer before photocrosslinking such as water, alcohol, toluene and chloroform. Further, the yield of the photocrosslinked product was almost 100%, and no insoluble component was formed.

  In contrast, the molecular weight of the photocrosslinked product of Comparative Example 2 was 68,000 (Mw / Mn = 2.7), the molecular weight increased, and the peak shape was broad with a large shoulder on the high molecular weight side. became.

  The yield of the intended crosslinked star-shaped branched polymer was about 5%, and the remainder became an insoluble gel composed of the star-shaped branched polymer. This is presumed to be a network structure in which the ω end of the branched chain is bonded to the side chain of the polymer, and the crosslinking points are intricately scattered.

[Gene transfer experiment]
COS-1 derived from African green monkey kidney cells was used for the cells, and pGL3 control vector was used for the DNA. The number of COS-1 cells was adjusted to 40,000 cells / mL, seeded in a 24 Well culture dish, and gene transfer was performed after 24 hours of culture.

  The photopolymer cross-linked product of the cationic polymer synthesized in the above iv) 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 incubated with DNA in 150 μL of OPTI-MEM for 30 minutes. The mixing ratio was adjusted so that the number of positive charges was 20 times the number of negative charges, the solution was adjusted so that 0.5 μg of DNA was administered to each well, and added to the cultured cells. After culturing for 3 hours, OPTI-MEM was removed, and after washing with PBS, complete medium was added and culturing was further performed for 48 hours.

[Evaluation of gene transfer activity]
Forty-eight hours after transfection, gene transfer activity was evaluated by luciferase assay (Promega, assay kit reagent). Correction was performed at protein concentration, and protein quantification was performed with Bradford reagent from BioRad. The results are shown in FIG.

<Comparative Example 3>
A gene introduction experiment was conducted in the same manner as in Example except that the star-shaped 4-branched polymer synthesized in the above synthesis process iii) was used as a vector without being crosslinked. The results are shown in FIG.

[Discussion]
As verified in iii) above, the effect of side chain protection is expressed independently of the carbon number, but in this gene transfer activity test, the gene transfer activity is improved as the carbon number of alcohol increases. ing. This is because the N, N-diethyldithiocarbamate group that radically cleaves at the ω end of the branched chain is a hydrophobic functional group, so the solvent with a higher number of carbon atoms and higher lipophilicity is more likely to come off. It is considered that the property and the lifetime of radicals are advantageous for crosslinking. Thereby, it is thought that the density | concentration of the light irradiation body which the terminal of the branched chain bridge | crosslinked becomes high.

  The photocrosslinked product of Comparative Example 2 has a lower gene transfer activity than Comparative Example 3 which is a star-shaped branched polymer. This factor is considered to be because the photocrosslinked product of Comparative Example 2 has low solubility in all solvents and hardly dissolves in physiological saline. That is, the method for preparing the gene introduction agent is a method in which the polymer is filtered with a 0.2 μm filter as a physiological saline solution having a concentration of about 1 mg / mL, and then mixed with the DNA solution. It is thought that this is because it cannot reach 1:20.

  In Comparative Example 2, when the irradiation time was 120 minutes, the yield was about 45%, and the gene transfer activity was improved as compared with Comparative Example 3. From this, it can be seen that when the branched chain is not protected, it is difficult to control the degree of crosslinking of photocrosslinking and the yield is also poor.

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

Claims (15)

A method for producing a gene transfer agent comprising a cross-linked star-shaped branched polymer in which a star-shaped branched polymer having a plurality of cationic branched chains radially extended from an aromatic ring as a nucleus is crosslinked,
A method for producing a gene introduction agent, wherein star-branched polymers are crosslinked in a solvent containing an alcohol.   2. The star-shaped branched polymer is cross-linked by dissolving and / or dispersing the star-shaped branched polymer in the solvent and irradiating the solution and / or dispersion with light. A method for producing a gene introduction agent.   The method for producing a gene introduction agent according to claim 1 or 2, wherein the alcohol comprises a lower alcohol.   4. The method for producing a gene introduction agent according to claim 3, wherein the lower alcohol is at least one selected from the group consisting of methanol, ethanol and 2-propanol.   The method for producing a gene introduction agent according to any one of claims 1 to 4, wherein the solvent comprises only an alcohol.   The method for producing a gene introduction agent according to any one of claims 1 to 4, wherein the solvent comprises alcohol and a solvent other than alcohol.   7. The solvent other than the alcohol according to claim 6, wherein the solvent is at least one selected from the group consisting of water, chloroform, toluene, benzene, xylene, ether, acetone, methyl ethyl ketone, tetrahydrofuran, methylene chloride, and benzene chloride. A method for producing a gene introduction agent.   8. The method for producing a gene introduction agent according to claim 6, wherein the alcohol concentration of the solvent is 30.0 to 99% by weight.   The method for producing a gene introduction agent according to any one of claims 1 to 8, wherein the concentration of the star-shaped branched polymer in the solvent is 3 to 60% by weight. 10. The star-shaped branched polymer according to claim 1, wherein the star-shaped branched polymer uses an iniferter as a compound having three or more N, N-dialkyldithiocarbamylmethyl groups in the same molecule. Is a branched polymer obtained by photoirradiation living polymerization,
A method for producing a gene introduction agent, wherein the branched chains of the star-shaped branched polymer are crosslinked to form a crosslinked star-shaped branched polymer.   The compound having three or more N, N-dialkyldithiocarbamylmethyl groups in the same molecule according to claim 10 has a benzene ring as a nucleus and three or more N, N-dialkyldithio groups as branched chains in the nucleus. A method for producing a gene introduction agent, wherein a carbamylmethyl group is bonded.   12. The vinyl monomer according to claim 10, wherein the vinyl monomer is 3-N, N-dimethylaminopropylacrylamide, 2-N, N-dimethylaminoethyl methacrylate, N, N-dimethylacrylamide, methoxyethyl (meth) acrylate, N- A method for producing a gene introduction agent, which is at least one selected from the group consisting of isopropylacrylamide and N-isopropylacrylamide derivatives.   The method for producing a gene introduction agent according to any one of claims 10 to 12, wherein the branched N, N-dialkyldithiocarbamylmethyl group of the branched polymer contributes as a crosslinking point.   The method for producing a gene introduction agent according to any one of claims 10 to 13, wherein the branched chain is a homopolymer of a vinyl monomer.   The method for producing a gene introduction agent according to any one of claims 10 to 13, wherein the branched chain is a random or block copolymer of the vinyl monomer and a different monomer.

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