US20090130761A1 - Freeze-Dried Product for Introducing Nucleic Acid, Oligonucleic Acid or Derivative Thereof - Google Patents

Freeze-Dried Product for Introducing Nucleic Acid, Oligonucleic Acid or Derivative Thereof Download PDF

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US20090130761A1
US20090130761A1 US12/227,394 US22739407A US2009130761A1 US 20090130761 A1 US20090130761 A1 US 20090130761A1 US 22739407 A US22739407 A US 22739407A US 2009130761 A1 US2009130761 A1 US 2009130761A1
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freeze
dried product
derivative
nucleic acid
acid
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Yoshiyuki Koyama
Tomoko Ito
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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/542—Carboxylic acids, e.g. a fatty acid or an amino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/543—Lipids, e.g. triglycerides; Polyamines, e.g. spermine or spermidine
    • A—HUMAN NECESSITIES
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    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
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    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/61—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule the organic macromolecular compound being a polysaccharide or a derivative thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0091—Purification or manufacturing processes for gene therapy compositions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/141—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • 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
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • the present invention relates to a freeze-dried product of a complex containing a nucleic acid, oligonucleic acid or derivative thereof; a cationic polymer, or cationic lipid or aggregate containing the same; and, an anionic polymer, for the purpose of introducing a nucleic acid, oligonucleic acid or derivative thereof into cells, a preparation method of the same, and a preparation, reagent and kit for introducing a nucleic acid, oligonucleic acid or derivative thereof containing the same.
  • Gene therapy and antisense treatment methods are currently used practically for treating congenital genetic diseases, cancer cells or AIDS by introducing an intended gene, antisense oligonucleic acid or derivative thereof into cells and expressing that gene or function, and studies are being conducted on various types of vectors for use as carriers for introducing genes (DNA), antisense oligonucleic acids and derivatives thereof into cells.
  • cationic substances such as cationic polymers, cationic liposomes and cationic lipids for use as one such type of vector in the form of a non-viral vector that eliminates concerns over safety, has favorable efficiency, is free of immunogenicity and is easily prepared.
  • Patent Document 1 Japanese Unexamined Patent Publication No. 2005-176830
  • Patent Document 2 Japanese Unexamined Patent Publication No.
  • Non-Patent Document 1 J. Biomater. Sci. Polymer Edn., Vol. 14, No. 6, pp. 515-531 (2003)).
  • Complexes of DNA and cationic substances modified in this manner exhibit low aggregation and exhibit favorable gene expression in cells.
  • these complexes are heterogeneous suspensions, they have poor storage performance, are required to be used promptly following preparation, and end up aggregating when prepared at high concentrations, thereby having the disadvantages of difficulty in adjusting concentration and bothersome handling.
  • Non-Patent Document 2 J. Pharm. Sci., Vol. 90, pp. 1445-1455 (2001)).
  • Non-Patent Document 3 Biochim. Biophys. Acta., 2000 Sep. 29, 1468 (1-2): 127-138.
  • the amount of sugar required is 500 to 1000 times the amount of DNA in terms of the weight ratio, making this method impractical in consideration of the solution following rehydration having a much higher osmotic pressure than physiological conditions.
  • monosaccharides and disaccharides do not offer advantageous effects for gene expression.
  • the use of a neutral polysaccharide, dextran has been attempted to reduce osmotic pressure after rehydration (Non-Patent Document 4: J.
  • Non-Patent Document 4 J. Pharm. Sci., Vol. 94, pp. 1226-1236 (2005).
  • the freeze-dried product is required to be rehydrated with a small amount of water or solvent after freeze-drying to obtain the required concentration of DNA and then concentrated to a high concentration.
  • concentration of dextran following rehydration exceeds 10%, and there are limitations during the freeze-drying procedure such as on DNA concentration and cooling temperature, thereby making practical application difficult.
  • Patent Document 1 Japanese Unexamined Patent Publication No. 2005-176830
  • Patent Document 2 Japanese Unexamined Patent Publication No. 2003-231748
  • Non-Patent Document 1 J. Biomater. Sci. Polymer Edn., Vol. 14, No. 6, pp. 515-531 (2003)
  • Non-Patent Document 2 J. Pharm. Sci., Vol. 90, pp. 1445-1455 (2001)
  • Non-Patent Document 3 Biochim. Biophys. Acta., 2000 Sep. 29, 1468 (1-2): 127-138
  • Non-Patent Document 4 J. Pharm. Sci., Vol. 94, pp. 1226-1236 (2005)
  • the inventors of the present invention conducted extensive studies to overcome the aforementioned problems, and as a result they found that if a freeze-dried product of a complex containing a nucleic acid, oligonucleic acid or derivative thereof; a cationic polymer, or cationic lipid or aggregate containing the same; and, an anionic polymer is introduced into cells, the introduced gene, oligonucleic acid or derivative thereof satisfactorily expresses the function thereof, thereby leading to completion of the present invention.
  • the present invention relates to a freeze-dried product of a complex containing a nucleic acid, oligonucleic acid or derivative thereof; a cationic polymer, or cationic lipid or aggregate containing the same; and, an anionic polymer.
  • the present invention relates to a preparation, reagent and kit containing the freeze-dried product for introducing a nucleic acid, oligonucleic acid or derivative thereof.
  • the present invention relates to a method for preparing the freeze-dried product comprising a step of forming a complex by mixing a nucleic acid, oligonucleic acid or derivative thereof; a cationic polymer, or cationic lipid or aggregate containing the same; and an anionic polymer, followed by a step of freeze-drying the complex.
  • the present invention also relates to a method for introducing a gene, oligonucleic acid or derivative thereof into cells, the method using the freeze-dried product.
  • the freeze-dried product of the present invention enables concentration to be adjusted easily, offers easy handling and has superior storage performance.
  • the freeze-dried product of the present invention contains an anionic polymer, a stable dispersion containing a complex of an extremely small size can be obtained at an arbitrary concentration even when rehydrating with a solvent to form a suspension or dilution at the time of use.
  • a nucleic acid, oligonucleic acid or derivative thereof can be efficiently introduced into cells without causing aggregation even during gene introduction, and satisfactory ability to express a function thereof is demonstrated by various types of administration methods such as local administration or intravenous administration.
  • the freeze-dried product of the present invention is a freeze-dried product of a complex containing a nucleic acid, an oligonucleic acid or a derivative thereof; a cationic polymer, or cationic lipid or aggregate containing the same; and an anionic polymer.
  • a nucleic acid, oligonucleic acid or derivative thereof in the complex forms a complex by ionic bonding with a cationic polymer, or cationic lipid or aggregate containing the same, and the cationic polymer or cationic lipid are further ionicly bonded with an anionic polymer.
  • These components form a complex that is mainly coated with anionic polymer depending on the mixing ratio, mixing sequence and the like.
  • nucleic acid or oligonucleic acid and the like introduced for the purpose of gene therapy or antisense therapy can be used for the nucleic acid, oligonucleic acid or derivative thereof able to be used in the freeze-dried product of the present invention, specific examples of which include various types of nucleic acids, oligonucleic acids and derivatives thereof, such as various DNA and RNA (single-strand or double-strand) (such as plasmid DNA, double-stranded oligo RNA, mRNA, tRNA, rRNA or cDNA), sense or antisense oligonucleotides (including recombinants) and derivatives thereof, or ribozymes or mixtures thereof.
  • DNA and RNA single-strand or double-strand
  • sense or antisense oligonucleotides including recombinants
  • derivatives thereof or ribozymes or mixtures thereof.
  • plasmid DNA can be used preferably in the case of a nucleic acid, and oligo DNA or a derivative thereof in the form of S-oligo, double-stranded RNA for RNA interference or ribozyme RNA can be used preferably in the case of an antisense nucleic acid.
  • plasmid DNA can be used particularly preferably.
  • Positively charged, naturally-occurring or synthetic polymers having a molecular weight of about 1,000 to 3,000,000 and having a plurality of, preferably five or more, functional groups capable of forming a complex with DNA in water can be used for the cationic polymer able to be used in the freeze-dried product of the present invention, and examples of such functional groups include organic amino groups such as optionally substituted amino groups, ammonium groups or salts thereof (and these groups may be mono- or poly-substituted with, for example, alkyl groups having 1 to 6 carbon atoms, phenyl groups and the like), imino groups, imidazolyl groups or guanidino groups.
  • Examples of such cationic polymers include positively charged proteins and polypeptides; positively charged dendrimers; positively charged synthetic polymers; and positively charged polysaccharide derivatives or salts thereof and combinations thereof.
  • the molecular weight of positively charged proteins or positively charged polypeptides able to be used as cationic polymers in the freeze-dried product of the present invention is preferably about 1,000 to 500,000.
  • proteins and polypeptides include proteins or polypeptides such as protamine, histone, Hel ⁇ 1 or gelatin.
  • examples also include polyamino acids containing positively charged amino acid residues.
  • polyamino acids containing positively charged amino acid residues include poly-L-lysine, polyarginine and polyornithine.
  • salts of these proteins and polypeptides include hydrochlorides, sulfates, phosphates and borates.
  • Positively charged dendrimers having functional groups like those described above able to be used as cationic polymers refer to dendrimers having an optionally substituted amino group, ammonium group or salt thereof (and these groups may also be mono- or poly-substituted with, for example, alkyl groups having 1 to 6 carbon atoms, phenyl groups and the like) on the terminal or within a branched molecular chain, and the molecular weight thereof is preferably about 1,000 to 500,000.
  • Specific examples of dendrimers include polyamide amine dendrimers and polylysine dendrimers.
  • examples of dendrimer salts include hydrochlorides, sulfates, phosphates and borates.
  • Positively charged synthetic polymers able to be used as cationic polymers are synthetic polymers having a plurality of, preferably five or more, functional groups capable of forming a complex with DNA in water in a molecule thereof as previously described, and preferably having a molecular weight of 1,000 to 3,000,000.
  • Specific examples of synthetic polymers include polyethyleneimines (including linear polyethyleneimines and branched polyethyleneimines), polymers or copolymers of 2-dimethylaminoethyl methacrylate, and polymers or copolymers of 2-trimethylaminoethyl methacrylate.
  • the molecular weight of one example of synthetic polymers in the form of polyethyleneimines is preferably about 1,000 to 500,000, more preferably about 5,000 to 200,000 and most preferably about 10,000 to 100,000.
  • examples of salts of polyethyleneimines include hydrochlorides, sulfates, phosphates and borates.
  • Positively charged polysaccharide derivatives able to be used as cationic polymers are polysaccharide derivatives having a plurality of, preferably five or more, functional groups capable of forming a complex with DNA in water in a molecule thereof, and having a molecular weight of preferably 1,000 to 3,000,000 and more preferably 5,000 to 500,000.
  • polysaccharides include chitosan and dextran derivatives introduced with functional groups as previously described.
  • the molecular weight of chitosan is preferably about 1,000 to 500,000, more preferably about 5,000 to 200,000 and most preferably about 10,000 to 100,000.
  • Examples of salts of chitosan include hydrochlorides and acetates.
  • the molecular weight of dextran derivatives is preferably 3,000 to 1,000,000. Specific examples of such dextran derivatives include diethylaminoethyl dextran.
  • the aforementioned cationic polymers are originally not positively charged, they can be used provided they become positively charged as a result of introducing a functional group such as an amino group, and may also be further modified with a sugar chain, oligopeptide or antibody and the like as necessary.
  • cationic lipids examples include DC-Chol (3 ⁇ -(N—(N′,N-dimethylaminoethane)carbamoyl)cholesterol), DDAB (N,N-distearyl-N,N-dimethylammonium bromide), DMRI (N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium bromide), DODAC (N,N-dioleyl-N,N-dimethylammonium chloride), DOGS (diheptadecylamidoglycylspermidine), DOSPA (N-(1-(2,3-dioleyloxy)propyl)-N-(2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoroacetate), DOTAP
  • mixtures of the aforementioned cationic lipids such as DOSPA
  • neutral substances such as DOPE (dioleylphosphatidylethanolamine) or cholesterol
  • aggregates containing cationic lipids include lipofectamine (liposome containing 3:1 w/w mixture of DOSPA and DOPE), lipofectin (liposome containing 1:1 w/w mixture of DOTMA and DOPE) and mixtures thereof.
  • polyethyleneimines; protamine; Hel ⁇ 1; dendrimers such as polyamide amine dendrimers or polylysine dendrimers; chitosan; polymers or copolymers of 2-dimethylaminoethyl methacrylate or polymers or copolymers of 2-trimethylaminoethyl methacrylate can be used preferably as cationic polymer
  • polyethyleneimines, polyamide amine dendrimers, polylysine dendrimers or chitosan can be used particularly preferably.
  • lipofectamine liposome containing a 3:1 w/w mixture of DOSPA and DOPE
  • Negatively charged, naturally-occurring or synthetic polymers containing an anionic group in a molecule thereof, having a molecular weight of about 500 to 4,000,000, and having a plurality of, preferably five or more, functional groups in a molecule thereof capable of forming a complex with a polycation in water can be used for the anionic polymer used in the freeze-dried product of the present invention, and examples of such functional groups include a carboxyl group, —OSO 3 H group, —SO 3 H group, phosphate group and salts thereof.
  • Examples of such anionic polymers include amphoteric polymers.
  • Glucosaminoglycans can be preferably used as a polysaccharide or derivative thereof having functional groups as described above able to be used as an anionic polymer in the freeze-dried product of the present invention.
  • the molecular weight of such glucosaminoglycans is preferably 1,000 to 4,000,000 and more preferably 4,000 to 3,000,000.
  • Specific examples of such glucosaminoglycans include hyaluronic acid, chondroitin, chondroitin sulfate, keratan sulfate, heparin and dermatan sulfate. Among them, hyaluronic acid can be used particularly preferably.
  • Hyaluronic acid can also be used in the form of a salt or negatively charged derivative thereof. Although the molecular weight thereof may be 5,000 or more, it is preferably 10,000 or more and more preferably 100,000 to 3,000,000.
  • Examples of salts of hyaluronic acid include sodium salts, potassium salts and ammonium salts.
  • examples of derivatives of hyaluronic acid include those obtained by introducing polyethylene glycol, peptide, sugar, protein, hydroiodic acid, antibody or portions thereof into hyaluronic acid, and amphoteric derivatives having a positively charged portion by introducing spermine, spermidine, and the like are also included.
  • Polyamino acids containing an amino acid residue having a negatively charged side chain able to be used as an anionic polymer in the freeze-dried product of the present invention are polyamino acids preferably having a molecular weight of 500 to 1,000,000 and containing an amino acid residue having as a side chain thereof a carboxyl group, —O—SO 3 H group, —SO 3 H group, phosphate group or salt thereof.
  • Specific examples of such polyamino acids include polyglutamic acid and polyaspartic acid.
  • PEG derivatives having a carboxyl side chain able to be used as an anionic polymer in the freeze-dried product of the present invention are PEG derivatives having a molecular weight of 500 or more, preferably 2,000 or more and more preferably 4,000 to 40,000 and having a plurality of, preferably five or more, carboxyl side chains per molecule of PEG.
  • PEG derivatives having carboxyl side chains can also be used as salts thereof or negatively charged derivatives thereof. Examples of these salts include sodium salts, potassium salts and ammonium salts.
  • Specific examples of such PEG derivatives include the PEG derivatives described in Non-Patent Document 1 (J. Biomater. Sci. Polymer Edn. Vol. 14, pp. 515-531 (2003)).
  • Synthetic polymers having functional groups selected from a carboxyl group, —O—SO 3 H group, —SO 3 H group, phosphate group and salts thereof able to be used as an anionic polymer in the freeze-dried product of the present invention are polymers or copolymers having a plurality of, preferably five or more, functional groups selected from a carboxyl group, —O—SO 3 H group, —SO 3 H group, phosphate group and salts thereof per molecule thereof, and preferably having a molecular weight of 500 to 4,000,000.
  • polymers or copolymers include polymers or copolymers of acrylic acid or methacrylic acid having a molecular weight of 1000 to 3,000,000, sulfuric acid esters of polyvinyl alcohol, and succinylated poly-L-lysine.
  • Polymers having functional groups selected from a carboxyl group, —O—SO 3 H group, —SO 3 H group, phosphate group and salts thereof, as well as optionally substituted amino groups, ammonium groups or salts thereof (and these groups may be mono- or poly-substituted with alkyl groups having 1 to 6 carbon atoms, phenyl groups and the like), able to be used as an anionic polymer in the freeze-dried product of the present invention are polymers having a molecular weight of 500 or more, preferably 2,000 or more and more preferably 4,000 to 40,000 and having a plurality of, preferably five or more, functional groups selected from a carboxyl group, —OSO 3 H group, —SO 3 H group, phosphate group and salts thereof per molecule thereof, as well as optionally substituted amino groups, ammonium groups or salts thereof as previously described.
  • Anionic polymers able to be used in the freeze-dried product of the present invention can be used even if they are usually not negatively charged provided they are made to be negatively charged by introducing functional groups such as a carboxyl group, and may be further modified with a sugar chain, oligopeptide or antibody and the like as necessary.
  • hyaluronic acid, PEG derivatives having carboxyl side chains, anionic polymers such as polyacrylic acid or salts thereof can be preferably used as anionic polymers, while hyaluronic acid, PEG derivatives having carboxyl side chains or salts thereof can be used particularly preferably.
  • an anionic polymer having the ability to specifically adhere to target cells for introducing a nucleic acid makes it possible to specifically introduce a nucleic acid into the target cells.
  • hyaluronic acid for the anionic polymer
  • cells having cell surface molecules such as CD44, which specifically bond with hyaluronic acid can be targeted.
  • an anionic polymer introduced with RGD peptide makes it possible to target numerous types of tumor cells, while the use of an anionic polymer introduced with a galactose side chain makes it possible to target liver cells or cells originating in the liver.
  • a cationic polymer or cationic lipid or aggregate containing the same and an anionic polymer include polyethyleneimines and hyaluronic acid; polyethyleneimines and PEG derivatives having carboxyl side chains; aggregates containing DOSPA (such as lipofectamine (liposome containing 3:1 w/w mixture of DOSPA and DOPE)) and hyaluronic acid; and aggregates containing DOSPA (such as lipofectamine) and PEG derivatives having carboxyl side chains.
  • the molar ratio (negative charge:positive charge ratio) of each charged group of a nucleic acid, oligonucleic acid or derivative thereof and a cationic polymer, or cationic lipid or aggregate containing the same used in the freeze-dried product of the present invention may be 1:0.8 to 1:100, preferably 1:1 to 1:50 and more preferably 1:1.2 to 1:30.
  • the blending ratio between a nucleic acid, oligonucleic acid or derivative thereof and a cationic polymer, or cationic lipid or aggregate containing the same refers to the molar ratio of each charged group, and more specifically indicates the molar ratio of negative charge attributable to phosphate anions of a nucleic acid, oligonucleic acid or derivative thereof to positive charge of a cationic polymer, or cationic lipid or aggregate containing the same, or functional groups able to be positively charged.
  • the molar ratio (negative charge:negative charge ratio) of each charged group between a nucleic acid, oligonucleic acid or derivative thereof to anionic polymer used in the freeze-dried product of the present invention may be 1:0.2 to 1:1000, preferably 1:0.2 to 1:100 and more preferably 1:1 to 1:60.
  • the blending ratio between nucleic acid, oligonucleic acid or derivative thereof and anionic polymer refers to the molar ratio of each charged group, and more specifically indicates the molar ratio of negative charge attributable to phosphate anions of a nucleic acid, oligonucleic acid or derivative thereof to negative charge of the anionic polymer or functional groups able to be negatively charged.
  • the blending ratio of nucleic acid to hyaluronic acid is 1:0.2 to 1:1000, preferably 1:0.2 to 1:100 and more preferably 1:1 to 1:60.
  • the blending ratio of nucleic acid to PEG derivative having carboxyl side chains may be 1:0.2 to 1:1000, preferably 1:0.2 to 1:100 and more preferably 1:1 to 1:60.
  • the blending ratio of nucleic acid to polyethyleneimine to hyaluronic acid is 1:2 to 60:1 to 240, preferably 1:4 to 24:1 to 160, more preferably 1:7 to 20:2 to 60 and particularly preferably 1:8 to 14:2 to 32.
  • the blending ratio of nucleic acid to polyethyleneimine to PEG derivative having carboxyl side chains is 1:2 to 60:1 to 240, preferably 1:4 to 24:2 to 160, more preferably 1:7 to 20:2 to 60 and particularly preferably 1:8 to 14:4 to 32.
  • the blending ratio of nucleic acid to lipofectamine to hyaluronic acid is 1:1 to 50:0.2 to 240, preferably 1:1.2 to 48:0.2 to 160, more preferably 1:1.5 to 30:0.5 to 60, and particularly preferably 1:1.8 to 16:1 to 32.
  • the blending ratio of nucleic acid to lipofectamine to PEG derivative having carboxyl side chains is 1:1 to 50:0.1 to 160, preferably 1:1.2 to 48:0.2 to 160, more preferably 1:1.5 to 30:0.5 to 60 and particularly preferably 1:1.8 to 16:2 to 32.
  • nucleic acid, oligonucleic acid or a derivative thereof; cationic polymer or cationic lipid or aggregate containing the same; and anionic polymer contained in the freeze-dried product of the present invention are as described above, since optimum conditions fluctuate according to the number and type of cells introduced with nucleic acid and the like, the blending ratio can be suitably determined by a person with ordinary skill in the art according to the types of cells and nucleic acid used.
  • the freeze-dried product of the present invention can be prepared by a step of forming a complex by mixing the aforementioned nucleic acid, oligonucleic acid or derivative thereof, cationic polymer or cationic lipid or aggregate containing the same, and anionic polymer in the blending ratios described above, and a step of freeze-drying the complex.
  • the order of mixing is preferably [1] nucleic acid, oligonucleic acid or derivative thereof, [2] cationic polymer or cationic lipid or aggregate containing the same, and [3] anionic polymer; or [1] nucleic acid, oligonucleic acid or derivative thereof, [2] anionic polymer, and [3] cationic polymer or cationic lipid or aggregate containing the same.
  • the complex is formed as a result of the nucleic acid, oligonucleic acid or derivative thereof bonding with the cationic polymer or cationic lipid or aggregate containing the same by ionic bonding, followed by the cationic polymer or cationic lipid or aggregate containing the same bonding with the anionic polymer by ionic bonding.
  • the outer shell of such a complex may be coated mainly with the anionic polymer resulting in the formation of a mode having a negative surface potential.
  • Freeze-drying can be carried out under ordinary freeze-drying conditions such as under conditions consisting of drying under reduced pressure (preferably 5 to 100 mmHg and more preferably 10 mmHg) at an external temperature of ⁇ 78 to 60° C. and preferably ⁇ 30 to 40° C.
  • reduced pressure preferably 5 to 100 mmHg and more preferably 10 mmHg
  • the time required for drying varies according to the degree of depressurization and the amount of solvent, and is normally completed in 1 hour to 2 days.
  • the freeze-dried product of the present invention prepared in this manner can be used for various types of gene therapy, antisense therapy or introduction of a specific gene into humans and animals, and for the production of controlled, knockdown and knockout experimental animals and cells. More specifically, the freeze-dried product of the present invention can be used after converting to a rehydrate by suspending or dissolving in a solvent such as water, physiological saline, buffer, glucose solution or liquid medium prior to use. When rehydrating, the freeze-dried product is suspended or diluted using 100 to 10000 times (weight ratio) more of solvent than the nucleic acid, oligonucleic acid or derivative thereof, for example.
  • rehydrated freeze-dried product of the present invention can be used for introducing a nucleic acid and the like into cells by using any arbitrary method normally used to introduce a nucleic acid, oligonucleic acid or derivatives thereof into cells of the living body.
  • any arbitrary method normally used to introduce a nucleic acid, oligonucleic acid or derivatives thereof into cells of the living body Specific one includes an ex vivo method in which target cells placed in wells after having been removed from the body are treated with the rehydrated freeze-dried product of the present invention to introduce a gene or antisense nucleic acid and the cells are returned to the body to express the intended gene; or an in vivo and in situ method for directly introducing a gene or antisense nucleic acid, and so forth.
  • freeze-dried product of the present invention can also be administered without rehydrating by means such as contacting with cells into which a nucleic acid and the like is to be introduced, subcutaneously transplanting into an animal in which a nucleic acid is to be introduced, or transplanting into, onto the surface of, or in the vicinity of a target tissue in which a nucleic acid is to be introduced.
  • the amount of the freeze-dried product of the present invention applied to cells in terms of the amount of, for example, nucleic acid, oligonucleic acid or derivative thereof in an ex vivo method or in situ method is 0.2 to 10 ⁇ g/10 4 to 10 7 cells per 1 to 2 cm diameter well, and in the case of an in vivo method, 5 to 1000 ⁇ g/cm 3 of tumor, for example, in the case of local administration into a tumor, although varying considerably according to the administration site, and for example, 0.1 ⁇ g to 100 mg/organ in the case of administration into an organ such as the urinary bladder or 0.1 ng to 10 mg/kg of body weight in the case of systemic administration.
  • Any method employed in the field of gene therapy can be used as an in vivo method for directly administering into the body, examples of which include injecting a rehydrated freeze-dried product of the present invention intravenously, subcutaneously, intramuscularly, intraperitoneally, into a tumor or the vicinity of a tumor, inhaling through the nasal cavity, oral cavity or lungs, directly injecting into the urinary bladder or rectum, directly administering into tissue at the site of a lesion or a nearby blood vessel or implanting by supporting on a porous body or non-woven fabric and the like such as a gelatinous substance or sponge.
  • freeze-dried product in an amount as previously described can be used by an ex vivo method, in situ method or in vivo method as described above.
  • the neutralizing action of the positive charge of a complex of an ordinary nucleic acid, oligonucleic acid or derivative thereof and a cationic polymer or cationic lipid or aggregate containing the same is retained even after being administered into the living body or cells.
  • interactions such as agglutination occurring between the complex and serum proteins, blood cells or the extracellular matrix and the like are inhibited.
  • nucleic acids are efficiently taken up by cells and expressed with high efficiency.
  • a hydrate of the freeze-dried product of the present invention can be used as a preparation or reagent for introducing a nucleic acid, oligonucleic acid or derivative thereof, or as a kit for introducing a nucleic acid, oligonucleic acid or derivative thereof.
  • a freeze-dried complex comprised of the three components of a gene, PEI and HA was incubated with mouse melanoma cell line derived B16 to confirm the expression of luciferase gene.
  • Non-Patent Document 6 Biomacromolecules, Vol. 7, pp. 1274-1279
  • Linear PEI Polyscience, Inc.
  • Microbial hyaluronic acid Nacalai-Tesque
  • Phosphate Buffered Salts (tablet, Roman Industries) dissolved in ion exchange distilled water was used as PBS. This applies similarly to the following examples as well.
  • B16 cells were seeded into a 24-well multiplate two days prior to gene introduction and then incubated for two nights using EMEM medium.
  • 2 ⁇ l of an aqueous solution containing 1.3 ⁇ g of luciferase plasmid was mixed with 2 ⁇ l of an aqueous solution of PEI to a +/ ⁇ ratio (charge molar ratio) of 8 on the day prior to gene introduction, and after pipetting several times, 4 ⁇ l of HA solutions of various concentrations were added and stirred well followed by freezing at ⁇ 30° C. Subsequently, freeze-drying was carried out to prepare freeze-dried products of the present invention.
  • a freeze-dried product was prepared using the same method with the exception of changing the mixing order of HA and PEI.
  • 500 ⁇ l of EMEM containing, 10% FBS, 25 U of penicillin and 25 ⁇ g of streptomycin was placed in the wells.
  • 16 ⁇ l of PBS was mixed with the freeze-dried products prepared in [2] followed by incubating for 1 hour and adding to the wells.
  • the mixtures were incubated for 4 hours at 37° C. in 5% CO 2 and 95% air.
  • the medium was replaced with fresh EMEM containing 10% FBS, 25 U of penicillin and 25 ⁇ g of streptomycin followed by incubating for 20 hours at 37° C.
  • the cell lysis solution was used directly for protein assay.
  • the protein assay was carried out using a protein assay kit (Bio-Rad).
  • Freeze-dried products were obtained by mixing with addition of PEI after first adding HA to luciferase plasmid in [2] of Example 1 followed by evaluating in the same manner as Example 1.
  • Example 3 PEG-C having a molecular weight of about 10,000 and containing about 18 carboxyl groups per molecule was used as anionic polymer after synthesizing according to the method described in Non-Patent Document 1 (J. Biomater. Sci. Polymer Edn., Vol. 14, pp. 515-531 (2003)).
  • a freeze-dried three-component complex comprised of a gene, PEI and PEG-C was incubated with mouse melanoma cell line B16 followed to confirm the expression of luciferase gene.
  • B16 cells were seeded into a 24-well multiplate two days prior to gene introduction and then incubated for two nights using EMEM medium.
  • 12.5 ⁇ l of an aqueous solution containing 1.3 ⁇ g of luciferase plasmid was mixed with 12.5 ⁇ l of an aqueous solution of PEI to a +/ ⁇ ratio (charge molar ratio) of 8 on the day prior to gene introduction, and after pipetting several times, 25 ⁇ l of PEG-C solutions of various concentrations were added and stirred well followed by freezing at ⁇ 30° C. Subsequently, freeze-drying was carried out to prepare freeze-dried products of the present invention.
  • the cell lysis solution was used directly for protein assay.
  • the protein assay was carried out using a protein assay kit (Bio-Rad).
  • lipofectamine manufactured by Invitrogen was used for the lipofectamine.
  • a freeze-dried three-component complex comprised of a gene, lipofectamine and HA was incubated with mouse melanoma cell line derived B16 to confirm the expression of luciferase gene.
  • B16 cells were seeded into a 24-well multiplate two days prior to gene introduction and then incubated for two nights using EMEM medium.
  • 12.5 ⁇ l of an aqueous solution containing 1.3 ⁇ g of luciferase plasmid was mixed with 12.5 ⁇ l of an aqueous solution of lipofectamine to a weight ratio of luciferase plasmid to lipofectamine of 8 on the day prior to gene introduction, and after pipetting several times, 25 ⁇ l of HA solutions of various concentrations were added and incubated for 30 minutes followed by freezing at ⁇ 30° C. Subsequently, freeze-drying was carried out to prepare freeze-dried products of the present invention.
  • the cell lysis solution was used directly for protein assay.
  • the protein assay was carried out using a protein assay kit (Bio-Rad).
  • freeze-dried plasmid/lipofectamine binary complex In the presence of 80% serum, although expression by the freeze-dried plasmid/lipofectamine binary complex decreased further, the freeze-dried plasmid/lipofectamine/HA tertiary complex exhibited high expression of about 57% of the non-freeze-dried original plasmid/lipofectamine binary complex.
  • Example 4 The same experiment as Example 4 was carried out using PEG-C having a charge ratio of 16 relative to the plasmid DNA instead of HA. More specifically, instead of adding 25 ⁇ l of HA solution as in Example 4, an amount of PEG-C was dissolved in water so that the charge ratio was 16 times that of the plasmid DNA as described in the following graph and added at a final volume of 25 ⁇ l.
  • Gene introduction was carried out in serum-free medium or medium containing 80% serum.
  • a liquid was prepared at a low concentration using 10 times the amount of solvent as in [2] of Example 5 followed by mixing, freeze-drying, adding 50 ⁇ l of PBS in the same manner as [4] of Example 5, and similarly evaluating the rehydrated product.
  • a TE buffer solution of luciferase-encoded plasmid (0.8 mg/ml) was diluted with 400 ⁇ l of water followed by the addition of 100 ⁇ l of aqueous hyaluronic acid solution (5.8 mg/ml) and finally the addition of 50 ⁇ l of PEI solution (1.25 mg/ml). Thirty minutes after mixing the three components, the mixture was freeze-dried at ⁇ 30° C. followed by freeze-drying to obtain a solid complex.
  • mice melanoma cell line derived B16 suspended in 100 ⁇ l of medium*1 was subcutaneously transplanted into 5-week-old, male ddY mice.
  • an incision was made in the tumor portion under anesthesia and the solid DNA/PEI/HA complex described above was implanted within the tumor followed by suturing the incision.
  • mice Two days later, the mice were sacrificed with ether, the tumors and skin were excised and then homogenized in 1 ml of cell lysis solution*2. Subsequently, centrifugation was carried out for 20 minutes at 10,000 rpm and 4° C., and a substrate (Promega, 20 ⁇ l) was added to the supernatant (5 ⁇ l) to measure the luminescence of the luciferase for 30 seconds with a luminometer.
  • a substrate Promega, 20 ⁇ l
  • Total protein was quantified by adding 20 ⁇ l of supernatant of each sample diluted 1/80 to 1 ml of protein quantification reagent (Bio-Rad) followed by measuring absorbance at a wavelength of 595 nm 20 minutes later.
  • the solid DNA complex demonstrated extremely high expression within the tumor (results are shown in the table below).
  • 1.2 ⁇ 10 5 mouse melanoma cell line derived B16 suspended in 300 ⁇ l of medium*1 was seeded into the wells containing freeze-dried DNA complex. 1 ml of medium was added 4 hours later and was replaced with 1 ml of fresh medium 20 hours later. 24 hours later, 200 ⁇ l of cell lysis solution (Promega) were added and the cells were harvested followed by centrifuging for 1 minute at 15,000 rpm and 4° C., adding a substrate (Promega, 20 ⁇ l) to the supernatant (5 ⁇ l), and measuring the luminescence of the luciferase for 30 seconds with a luminometer.
  • a substrate Promega, 20 ⁇ l
  • Total protein was quantified by adding 20 ⁇ l of supernatant of each sample diluted 1/5 to 1 ml of protein quantification reagent (Bio-Rad) followed by measuring absorbance at a wavelength of 595 nm 20 minutes later.
  • EMEM medium containing 10% FBS, penicillin G sodium (100 units/ml) and streptomycin sulfate (0.1 mg/ml)).
  • 62.5 ⁇ l of a solution of luciferase-encoded plasmid (0.8 mg/ml) was diluted with 0, 500, 2000 or 8000 ⁇ l of water followed by the addition of 125 ⁇ l of aqueous hyaluronic acid solution (5.8 mg/ml) and finally the addition of 62.5 ⁇ l of PEI solution (1.25 mg/ml). Thirty minutes after mixing the three components, the mixtures were frozen at ⁇ 30° C. followed by freeze-drying.
  • the freeze-dried DNA complexes were resolvated with 250 ⁇ l of 5% glucose.
  • mice melanoma cell line derived B16 suspended in 100 ⁇ l of medium*1 was subcutaneously transplanted into 5-week-old, male ddY mice. When the tumors had reached 6 to 8 mm, a suspension of resolvated DNA complex was administered into a tail vein of the mice.
  • mice were exsanguinated under ether anesthesia 24 hours later followed by excision of the tumor, liver and lungs and homogenizing in 1 ml of cell lysis solution*2. Subsequently, centrifugation was carried out for 20 minutes at 10,000 rpm and 4° C., and a substrate (Promega, 20 ⁇ l) was added to the supernatant (5 ⁇ l) to measure the luminescence of the luciferase for 30 seconds with a luminometer.
  • a substrate Promega, 20 ⁇ l
  • Total protein was quantified by adding 20 ⁇ l of supernatant of each sample diluted 1/80 to 1 ml of protein quantification reagent (Bio-Rad) followed by measuring absorbance at a wavelength of 595 nm 20 minutes later.
  • 25 ⁇ l of a protamine aqueous solution (78 ⁇ g/ml) was added to 25 ⁇ l of an aqueous solution of anti-luciferase siRNA (Invitrogen, 21.28 ⁇ g/ml) followed by the addition of 50 ⁇ l of hyaluronic acid solution (53.7 ⁇ g/ml or 107.5 ⁇ g/ml). After mixing the three components, the mixtures were placed in the wells of a culture plate, frozen at ⁇ 30° C. for 30 minutes and subsequently freeze-dried.
  • 1.2 ⁇ 10 5 mouse melanoma cell line derived B16 suspended in 100 ⁇ l of medium*1 was seeded onto a culture plate followed by the addition of 1 ml of medium 4 hours later and the addition of a mixture of 25 ⁇ l of pDNA solution (50 ⁇ g/ml) and 25 ⁇ l of PEI solution (78 ⁇ g/ml). Moreover, the medium was replaced with 1 ml of fresh medium 20 hours later.
  • Total protein was quantified by adding 20 ⁇ l of supernatant of each sample diluted 1/5 to 1 ml of protein quantification reagent (Bio-Rad) followed by measuring absorbance at a wavelength of 595 nm 20 minutes later.
  • EMEM medium containing 10% FBS, penicillin G sodium (100 units/ml) and streptomycin sulfate (0.1 mg/ml)).
  • Example 2 1.5 ⁇ l of the same solution of luciferase plasmid as used in Example 1 (0.8 mg/ml) was diluted with 0, 12.5 or 200 ⁇ l of water followed by the addition of 3 ⁇ l of aqueous hyaluronic acid solution (5.8 mg/ml) and finally the addition of 1.5 ⁇ l of PEI solution (1.25 mg/ml). Thirty minutes after mixing the three components, the mixtures were frozen at ⁇ 30° C. followed by freeze-drying.
  • the freeze-dried DNA complexes were rehydrated with 6 ⁇ l of water followed by the addition of 800 ⁇ l of water 30 minutes later and measuring the size of the complexes with a zeta analyzer (Malvern Instruments).
  • the percentages of formed complex particles measuring 0 to 100 nm and the percentages of formed complex particles measuring 100 to 200 nm are shown in the graph below.
  • the values shown after the names of the components indicate the final DNA concentration at the time of complex preparation as nucleic acid base concentrations.

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