WO2014030601A1 - Method for producing novel nano-bubble poly-lipo-plex having anionic property - Google Patents

Method for producing novel nano-bubble poly-lipo-plex having anionic property Download PDF

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WO2014030601A1
WO2014030601A1 PCT/JP2013/072063 JP2013072063W WO2014030601A1 WO 2014030601 A1 WO2014030601 A1 WO 2014030601A1 JP 2013072063 W JP2013072063 W JP 2013072063W WO 2014030601 A1 WO2014030601 A1 WO 2014030601A1
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anionic
pdna
poly
nanobubble
complex
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PCT/JP2013/072063
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French (fr)
Japanese (ja)
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友亮 ▲黒▼▲崎▼
茂 川上
充 橋田
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国立大学法人京都大学
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    • 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
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • 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
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • 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
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A61K41/0028—Disruption, e.g. by heat or ultrasounds, sonophysical or sonochemical activation, e.g. thermosensitive or heat-sensitive liposomes, disruption of calculi with a medicinal preparation and ultrasounds
    • 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
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • 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
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
    • 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
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • 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
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • 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
    • A61K47/62—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 a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/645—Polycationic or polyanionic oligopeptides, polypeptides or polyamino acids, e.g. polylysine, polyarginine, polyglutamic acid or peptide TAT
    • A61K47/6455—Polycationic oligopeptides, polypeptides or polyamino acids, e.g. for complexing nucleic acids
    • 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
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1277—Processes for preparing; Proliposomes
    • A61K9/1278—Post-loading, e.g. by ion or pH gradient
    • 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
    • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle

Definitions

  • the present invention relates to a method for producing a novel nanobubble poly-lipoplex having an anionic property.
  • Nucleic acids such as negatively charged macromolecules such as genes and RNAs are poorly taken up into cells, and are rapidly excreted into urine when administered intravenously. It is difficult. For this reason, it is essential to develop a vector for delivering a gene into a cell. Further, since non-specific nucleic acid introduction causes unexpected side effects, development of a technique for introducing nucleic acid only into specific cells of a target organ is desired. So far, it has been reported that a positively charged complex with a positively charged polymer or a gene using a liposome as a non-viral gene vector is excellent in cellular uptake and exhibits a high gene expression effect. However, considering the clinical application of genetic drugs, the main drug is a gene and the vector is defined as an additive. In Japan, all additives used for pharmaceutical products require safety testing. On the other hand, little has been reported on the detailed safety of gene vectors developed so far. For this reason, the development of gene vectors for introducing genes completely and effectively was necessary for the realization of genetic drugs.
  • Patent Documents 1 and 2 disclose liposomes containing bubbles.
  • An object of the present invention is to further increase the efficiency of introduction of nucleic acids into cells, and if necessary, compounds such as proteins and drugs.
  • the present invention provides nanobubble poly-lipoplexes having the following anionic properties.
  • Item 1 An anionic nanobubble poly-lipoplex comprising a complex of an anionic polymer and a positively charged polymer and an anionic liposome, wherein the liposome comprises bubbles and the complex is cationic.
  • Item 2 The anionic nanobubble poly-lipoplex according to Item 1, wherein the anionic polymer is a nucleic acid.
  • Item 3 The anionic nanobubble poly-lipoplex according to Item 2, wherein the nucleic acid is DNA or RNA.
  • Item 4. The anionic property according to Item 2 or 3, wherein the nucleic acid is gene expression plasmid DNA, antisense DNA, DNA aptamer, DNA expressing siRNA or shRNA, mRNA, siRNA, miRNA, shRNA, antisense RNA, or RNA aptamer. Nano bubble poly-lipo plex.
  • the positively charged polymer is at least one selected from the group consisting of protamine, histone, Hel ⁇ 1, gelatin, polylysine, polyarginine, polyornithine, polyamidoamine dendrimer, polylysine dendrimer, diethylaminoethyl-dextran, chitosan, spermine and spermidine.
  • the anionic nanobubble poly-lipoplex according to any one of Items 1 to 4.
  • Item 6 The anionic nanobubble poly-lipoplex according to Item 5, wherein the positively charged polymer contains protamine.
  • Item 7 An anionic nanobubble poly-lipoplex comprising protamine and anionic liposomes, the liposomes containing bubbles.
  • Item 8 The anionic nanobubble poly-lipoplex according to any one of Items 1 to 7, wherein the anionic liposome comprises a protein or a drug.
  • the present invention relates to a complex that enables safe and efficient introduction of a substance such as nucleic acid and its use.
  • the complex of the present invention shows a very high gene expression effect in the liver, kidney and spleen after ultrasonic irradiation. Furthermore, targeting to specific cells is possible by introducing a ligand into the complex.
  • the complex of the present invention since the complex of the present invention is composed of components whose safety has been confirmed, it does not cause damage to cells. This is in contrast to cationic liposomes that are known to induce disorders such as cytotoxicity. Further, when no ultrasonic irradiation is performed, the complex of the present invention does not show a gene expression effect. Such a property that can be selectively introduced into cells is not found in conventional cationic liposomes.
  • An example of a method for preparing an anionic nanobubble poly-lipoplex of the present invention is schematically shown.
  • Gene transfer effect by pDNA / PEI / AL complex Gene transfer effect by pDNA / PLL / AL complex. Gene transfer effect by pDNA / PS / AL complex.
  • A AST activity in serum
  • ALT activity in serum Assessment of damage to cells (induction of cytokines).
  • TNF- ⁇ concentration in serum (b) IL-6 concentration in serum.
  • IL-6 concentration in serum Comparison of ultrasound irradiation to the abdomen or chest.
  • A Ultrasonic irradiation on the abdomen, (b) Ultrasonic irradiation on the chest. Gene transfer to cancer cells.
  • the novel anionic nanobubble poly-lipoplex of the present invention includes anionic liposomes, anionic polymers (preferably nucleic acids), positively charged polymers and bubbles (bubbles).
  • An anionic polymer (preferably a nucleic acid) and a positively charged polymer form a complex having a positive charge as a whole. This cationic complex and an anionic liposome are further complexed, and bubbles are further included in the liposome.
  • the novel nanobubble poly-lipoplex having an anionic property of the present invention can be obtained.
  • the particle size of the anionic nanobubble poly-lipoplex is about 50 to 1000 nm, about 400 to 800 nm, particularly about 500 to 600 nm. Alternatively, the thickness may be about 100 to 800 nm or about 150 to 600 nm.
  • the particle size of the anionic liposome is about 30 to 500 nm, preferably about 50 to 300 nm, particularly about 100 to 250 nm.
  • the particle size of anionic nanobubble poly-lipoplexes and anionic liposomes can be controlled using an extruder.
  • the particle size of the liposome can be measured by a dynamic light scattering method.
  • Anionic liposomes used in the present invention may be multilamellar liposomes (eg, multilamellarlamvesicles: MLV) or unilamellar liposomes (eg, small unilamellar. Vesicles: SUV, large unilamellar vesicles: LUV). .
  • Anionic liposomes are produced by sonication, reverse phase evaporation, freeze-thaw, lipid lysis, spray drying, etc., and phospholipids, glycolipids, sterols, glycols, anionic lipids, polyethylene glycol groups And lipids (eg, PEG-phospholipids).
  • the anionic liposome of the present invention may be “anionic” throughout the liposome and may contain a cationic lipid. In that case, the anionic liposome as a whole contains more anionic lipid and becomes anionic. Like that.
  • complexing includes both integration of an anionic liposome and a nucleic acid-positively charged polymer complex and integration of a nucleic acid and a positively charged polymer.
  • the anionic nanobubble poly-lipoplex can contain any physiologically active substance including drugs and proteins inside or on the surface.
  • a physiologically active substance such as a protein or drug may have a hydrophobic portion embedded in a liposome.
  • Adsorption and binding of the lipid membrane of an anionic liposome and a physiologically active substance are performed by ionic bond, hydrogen bond, hydrophobic interaction, and the like.
  • the ionic bond includes a bond by an ionic bond between an anion as a component of the liposome and a cation which is a physiologically active substance. If the surface of the anionic liposome is hydrophobic, a hydrophobic physiologically active substance (drug etc.) can be adsorbed or bound to the surface or the inside of the membrane.
  • the anionic liposome of the present invention contains an anionic phospholipid and a neutral phospholipid, and may contain a small amount of a cationic phospholipid.
  • glycolipids may be included.
  • Preferred neutral phospholipids contained in the anionic liposome of the present invention include lecithin, lysolecithin and / or hydrogenated products and hydroxide derivatives obtained from soybeans, egg yolks and the like.
  • the higher fatty acid constituting the phospholipid contained in the anionic liposome of the present invention may be either a saturated fatty acid or an unsaturated fatty acid, but is preferably a saturated fatty acid.
  • a saturated or unsaturated fatty acid derived from egg yolk, soybean, other animals or plants, or composed of a synthesized carbon chain n (n represents an integer of 3 to 30) is a constituent.
  • PC phosphatidylcholine
  • PS phosphatidylserine
  • PG phosphatidylglycerol
  • PA phosphatidyl acid
  • PE phosphatidylethanolamine
  • cardiolipin sphingosine, ceramide, sphingomyelin, ganglioside, sphingophospholipid, egg yolk
  • lecithin hydrogenated egg yolk lecithin, soybean lecithin, and hydrogenated soybean lecithin. More preferably, it is a lipid comprising a saturated fatty acid as a constituent component.
  • the phospholipid examples include dilauroyl phosphatidylcholine (DLPC), dimyristoyl phosphatidylcholine (DMPC), dipalmitoyl phosphatidylcholine (DPPC), distearoyl phosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dilinoleoylphosphatidylcholine and the like.
  • DLPC dilauroyl phosphatidylcholine
  • DMPC dimyristoyl phosphatidylcholine
  • DPPC dipalmitoyl phosphatidylcholine
  • DSPC distearoyl phosphatidylcholine
  • DOPC dioleoylphosphatidylcholine
  • Phosphatidylcholine dilauroyl phosphatidylglycerol (DLPG), dimyristoyl phosphatidylglycerol (DMPG), dipalmitoyl phosphatidylglycerol (DPPG), distearoyl phosphatidylglycerol (DSPG), dioleoylphosphatidylglycerol (DOPG), dilinoleoyl phosphatidylglycerol, etc.
  • DLPG dilauroyl phosphatidylglycerol
  • DMPG dimyristoyl phosphatidylglycerol
  • DPPG dipalmitoyl phosphatidylglycerol
  • DSPG distearoyl phosphatidylglycerol
  • DOPG dioleoylphosphatidylglycerol
  • phosphatidylglycerol dilauroylphosphatidylethanolamine (DLEA), di Phosphatidylethanolamines such as listoyl phosphatidylethanolamine (DMEA), dipalmitoylphosphatidylethanolamine (DPEA), distearoylphosphatidylethanolamine (DSEA), dioleoylphosphatidylethanolamine (DOEA), dilinoleoylphosphatidylethanolamine; Lauroylphosphatidylserine (DLPS), dimyristoylphosphatidylserine (DMPS), dipalmitoylphosphatidylserine (DPPS), distearoylphosphatidylserine (DSPS), dioleoylphosphatidylserine (DOPS), phosphatidylcholine such as dilinoleoylphosphatidylserine, etc. Can be mentioned.
  • DMEA listo
  • n2 represents an integer of 3 to 30
  • the anionic lipid membrane components that make up the lipid membrane of liposomes are saturated with carbon chain n2 (n2 is an integer from 3 to 30).
  • phosphatidic acid dicetyl phosphoric acid (DCP), dilauryl phosphoric acid, dimyristyl phosphoric acid, phosphatidyl glycerol phosphoric acid having an unsaturated fatty acid as a constituent component can be given.
  • DCP dicetyl phosphoric acid
  • dilauryl phosphoric acid dimyristyl phosphoric acid
  • phosphatidyl glycerol phosphoric acid having an unsaturated fatty acid as a constituent component can be given.
  • Examples of the cationic lipid used as needed include 3 ⁇ - [N- (N ′, N′-dimethylaminoethane) -carbamoyl] cholesterol (DC-chol), 1,2-dioleoyloxy-3 -(Trimethylammonium) propane (DOTAP), N, N-dioctadecylamide glycylspermine (DOGS), dimethyldioctadecylammonium bromide (DDAB), N- [1- (2,3-dioleyloxy) propyl]- N, N, N-trimethylammonium chloride (DOTMA), 2,3-dioleyloxy-N- [2 (spermine-carboxamido) ethyl] -N, N-dimethyl-1-propaneaminium trifluoroacetate (DOSPA) And N- [1- (2,3-dimyristyloxy) propyl] -N, N-dimethyl-N
  • glycolipids examples include glycerolipids such as digalactosyl diglyceride and galactosyl diglyceride sulfate, and sphingoglycolipids such as galactosylceramide, galactosylceramide sulfate, lactosylceramide, ganglioside G7, ganglioside G6, and ganglioside G4.
  • glycerolipids such as digalactosyl diglyceride and galactosyl diglyceride sulfate
  • sphingoglycolipids such as galactosylceramide, galactosylceramide sulfate, lactosylceramide, ganglioside G7, ganglioside G6, and ganglioside G4.
  • Anionic lipid is added so as to contain 1 to 100% by mass with respect to the total lipid amount, preferably 10 to 75% by mass with respect to the total lipid amount, more preferably 25 to 60% by mass with respect to the total lipid amount. do it.
  • physiologically active substances examples include proteins and drugs. Since the anionic liposome complex of the present invention contains a nucleic acid (DNA, RNA, etc.) as a complex with a positively charged polymer, a physiologically active substance suitable for use in combination with a nucleic acid is preferred.
  • Proteins and drugs include anticancer agents, antiallergic agents, antibacterial agents, antifungal agents, antiviral agents, immunosuppressive agents, vaccines, interferons, interleukins, growth factors, peptide hormones, enzymes, steroid hormones, antirheumatic agents , Antigens, antibodies, receptors or ligands thereof.
  • the nucleic acid may be either DNA or RNA.
  • DNA include those that express genes, such as plasmids, gene constructs containing genes linked to promoters, and artificial genes.
  • DNA include DNA that expresses RNA such as gene expression plasmid DNA, antisense DNA, DNA aptamer, siRNA and shRNA.
  • RNA include mRNA, siRNA, antisense RNA, RNA aptamer, shRNA, miRNA and the like.
  • Physiologically active substances such as nucleic acids, proteins, drugs, etc., when taken into cells or expressed in cells, damage cells such as cytotoxicity and apoptosis-inducing action, or induce cell death The thing which has is mentioned.
  • positively charged polymers examples include cationic proteins such as protamine, histone, Hel ⁇ 1, gelatin, polylysine, polyarginine, polyornithine, polyamidoamine dendrimers, dendrimers such as polylysine dendrimers, dextran such as diethylaminoethyl-dextran, and polys such as chitosan.
  • cationic polysaccharides polyamines such as spermine and spermidine. Of these, protamine, histone, spermine, and spermidine are preferred, protamine and histone are more preferred, and protamine is particularly preferred from the viewpoint that nucleic acids and the like are specifically introduced into cells at the site where ultrasonic waves are applied.
  • the complex of the nucleic acid and the positively charged polymer is such that the total positive charge of the positively charged polymer exceeds the total negative charge of the nucleic acid so that the charge of the complex becomes positive.
  • a nucleic acid and a positively charged polymer are mixed in a solution so that they can be formed.
  • a method for producing liposomes will be described in detail.
  • the above-described phospholipids containing anionic phospholipids are dissolved in an appropriate organic solvent, placed in an appropriate container, and the solvent is distilled off under reduced pressure. Then, a phospholipid film is formed on the inner surface of the container, and water, preferably a buffer solution, is added thereto and stirred to obtain an anionic liposome.
  • water preferably a buffer solution
  • the anionic nanobubble poly-lipoplex of the present invention as a whole is more preferably neutral than neutral.
  • the anionic nanobubble poly-lipoplex of the present invention can be produced by first producing a complex of anionic liposomes and cationic particles and introducing bubbles into the anionic liposomes in this complex. .
  • the generation of bubbles in the liposome can be performed according to a known method, for example, by the following method.
  • an anionic liposome is sealed in a sealed container, and a gas introduced into the liposome as a bubble is filled in the void.
  • a gas for example, fluoride gas or nitrogen gas is mentioned.
  • the pressure of the gas to be filled is about 0.1 to 1.0 MPa.
  • the fluoride gas include sulfurized hexafluoride and perfluorohydrocarbon gas.
  • ultrasonic treatment for example, ultrasonic waves of 20 to 50 kHz may be irradiated for 1 to 5 minutes.
  • the ultrasonic treatment the aqueous solution inside the liposome is replaced with fluoride gas or nitrogen gas, and gas-encapsulated liposome is obtained.
  • the anionic nanobubble poly-lipoplex of the present invention is released into liposomes by irradiating ultrasonic waves at a specific place after administration in vivo, and at the same time, nucleic acids and drugs, proteins, etc. in liposomes are stored in cells. Introduced in. Ultrasonic irradiation can be performed using an ultrasonic diagnostic apparatus or the like.
  • Liposomes are preferably introduced with polyethylene glycol (PEG), sugar chains, and the like to increase in vivo stealth and extend the half-life.
  • PEG polyethylene glycol
  • sugar chains, etc. phospholipids into which these are introduced may be used as a raw material for the production of anionic liposomes.
  • the anionic nanobubble poly-lipoplexes of the present invention can be recognized by specific cells by binding cell recognition ligands such as NGR and RGD, and by limiting the site to which ultrasound is applied.
  • cell recognition ligands such as NGR and RGD
  • nucleic acids, drugs that can be further combined as necessary, and cells into which proteins are introduced can be further specified, and side effects and toxicity can be reduced.
  • these peptide ligands may be used, and examples thereof include sugar chains, proteins, cytokines, chemokines, growth factors, peptide hormones, antibodies or parts thereof.
  • the zeta potential of the liposome of the present invention is about -50 to 40 mV, preferably about -50 to 30 mV, more preferably about -45 to 20 mV.
  • Nucleic acid pDNA (pCMV-Luc): plasmid DNA (used by integrating firefly luciferase cDNA excised from pGL3 control vector (Promega) with Hind III and Xba I into the polylinker site of invitrogen pcDNA3 vector did.) pDNA was amplified using Endo Free Plasmid Giga Kit (Qiagen GmbH; Hilden, Germany) and dissolved in water for injection (sterilized distilled water).
  • MRNA firefly luciferase-mRNA.
  • siRNA siRNA for firefly luciferase, the sequence of which is shown below.
  • Sense strand (5 ' ⁇ 3') CUUACGCUGAGUACUUCGAtt (SEQ ID NO: 1)
  • Antisense strand (5 ′ ⁇ 3 ′) UCGAAGUACUCAGCGUAAGtt (SEQ ID NO: 2).
  • DSPG Distearoylphosphatidylglycerol
  • DSPC Distearoylphosphatidylcholine
  • PEG-DSPE Polyethyleneglycol 2000-distearoylphosphatidylethanolamine
  • DSPA Distearoylphosphatidylic acid
  • DSPS Distearoylphosphatidylserine
  • DOTAP Dioleoyltrimethylammoniumpropane
  • Protamine (protamine sulfate) PS
  • PLL Poly-L-lysine
  • PLA Poly-L-arginine
  • PEI Polyethyleneimine
  • DPLL Poly-L-lysine dendrigraft
  • Bubble Perfluoropropane was used as a gas for bubble production.
  • ultrasound may be abbreviated as “US”.
  • Example 1 Preparation of anionic bubble liposome (1)
  • the hydrated lipid solution was sonicated with a bath sonicator for 10 minutes, and further sonicated with a probe sonicator for 3 minutes.
  • the obtained lipid solution was passed through a 0.45 ⁇ m filter to obtain an anionic liposome solution.
  • Tables 1 to 4 show pDNA dissolved in water for injection in a vial and various cationic polymers (PEI, PLA, PLL, PS). By mixing at an appropriate ratio and incubating at room temperature for 15 minutes, a polyplex of pDNA having a cationic surface on the surface and a cationic polymer was prepared.
  • the complex was described using the ratio of the phosphate group of pDNA to the amino group of the cationic polymer and the phosphate group of DSPG.
  • PS the exact amount of amino groups in the molecule is unknown, so the complex was expressed by the mass ratio of pDNA to PS and AL.
  • 1: 8: 3 of the pDNA / PEI / AL complex means that the ratio of the phosphate group of pDNA to the amino group of the cationic polymer and the phosphate group of DSPG is 1: 8: 3.
  • the particle size and ⁇ charge of composites with various compositions were measured using Zeta Sizer Nano (Malvern).
  • FIG. 2 shows the result of optical observation of the appearance of the anionic bubble liposome complex of the present invention shown in Tables 1 to 4 before and after enclosing the bubble.
  • the liposome complex before encapsulation of bubbles was almost transparent, but became cloudy due to encapsulation of bubbles.
  • the anionic nanobubble poly-lipo plexes of the present invention are stable because pDNA or a complex of pDNA and cationic polymer is not removed even by agarose gel electrophoresis. It has been shown.
  • Fig. 5 Gene transfer to the liver (Fig. 5)
  • pDNA / PS / AL + Bubble was administered into the ICR female mouse via the tail vein.
  • Ultrasonic waves were irradiated in the same manner as in FIG. 4-3.
  • the liver of the mouse was treated with collagenase to obtain hepatic parenchymal cells and non-parenchymal cells.
  • the luciferase activity per 10 6 cells was measured to confirm the gene expression cells. The results are shown in FIG.
  • anionic nanobubble poly-lipoplexes pDNA / PS / AL + Bubble
  • the anionic nanobubble poly-lipoplex (pDNA / PS / AL + Bubble) of the present invention has little gene transfer into dendritic cells and is specifically introduced into other spleen cells. It became clear.
  • NGR, RGD peptide and ARA (Scramble) peptide were prepared using solid phase synthesis method. This peptide was mixed with NHS-PEG2000-DSPE to synthesize PEG-DSPE in which the end of the PEG chain was labeled with a peptide ligand (FIG. 7).
  • the anionic nanobubble poly-lipoplex (siRNA / PS / AL + Bubble) of the present invention containing siRNA as a nucleic acid is as stable as a complex containing pDNA, and the bubble is encapsulated in an anionic liposome. It became clear.
  • the complex of the present invention was a stable complex without releasing mRNA even by agarose gel electrophoresis.
  • This hematoporphyrin-encapsulated liposome was encapsulated with perfluoropropane gas and optically observed (FIG. 11 (A)).
  • the prepared lipid thin film was hydrated with a 0.3M citric acid solution to prepare liposomes.
  • DSPG, DSPC, and PEG-DSPE were used as liposome lipids.
  • Doxorubicin was added to this liposome solution at a mass ratio of 12.5: 1, and the mixture was incubated at 60 ° C. for 30 minutes to encapsulate doxorubicin in the liposome.
  • the encapsulation rate at this time was 97.7%.
  • Perfluoropropane gas was encapsulated in the doxorubicin-encapsulated liposome and optically observed (FIG. 11 (B)).
  • Example 2 Preparation of anionic bubble liposome (2) (1) Preparation of anionic liposome (AL) In the same manner as in Example 1, an anionic liposome solution was obtained.
  • the inside of the vial was pressurized with 7.5 mL of perfluoropropane gas, and sonicated for 5 minutes with a bath sonicator, thereby constructing the anionic nanobubble poly-lipoplex of the present invention.
  • FIG. 13A shows the result of optical observation of the appearance of the anionic bubble liposome complex obtained in the above (3) before and after bubble encapsulation.
  • FIG. 13B shows a TEM observation image of the pDNA / PS / BL complex by negative staining using uranyl acetate.
  • the anionic nanobubble poly-lipo plexes of the present invention are stable because pDNA or a complex of pDNA and cationic polymer is not removed even by agarose gel electrophoresis. It has been shown.
  • the complex is a complex (polyplex; pDNA / PS, pDNA / PLL, pDNA / PLA or pDNA / PS) of the pDNA obtained above and a cationic polymer (PS, PLL, PLA or DPLL), anionic liposome complex Body (pDNA / PS / AL, pDNA / PLL / AL, pDNA / PLA / AL or pDNA / PS / AL), anionic nanobubble poly-lipoplex (pDNA / PS / BL, pDNA / PLL / BL, pDNA / PLA / BL or pDNA / PS / BL) was used.
  • BS is used interchangeably with “AL + bubble” in this specification, and represents an anionic bubble liposome encapsulating bubbles.
  • EAhy926 cells were cultured in DMEM medium supplemented with 10% FBS (fetal bovine serum), penicillin 100 IU / mL, streptomycin 100 ⁇ g / mL, L-glutamine 2 mM, non-essential amino acids 100 ⁇ M at 37 ° C. and carbon dioxide concentration 5 Maintained in% environment. After pre-incubation for 24 hours, the medium was replaced with Opti-MEM I medium containing various complexes (pDNA 10 ⁇ g). Ten minutes after the addition of various complexes, EAhy926 cells were irradiated with ultrasound (frequency, 2.0 MHz; duty, 50%; burst rate, 10 Hz; intensity, 4.0 W / cm 2 ) for 20 seconds. Ultrasonic irradiation was performed using a 6 mm diameter probe using a Sonnopore-4000 sonicator (Neppa Gene).
  • FBS fetal bovine serum
  • penicillin 100 IU / mL penicillin 100 IU
  • the medium was replaced with a culture medium, and the culture was further continued for 24 hours.
  • the cells were suspended in lysis buffer (0.05% Triton X-100, 2 mM EDTA, and 0.1 M Tris; pH 7.8), and the expression level of luciferase in the cells was evaluated. The results are shown in FIG.
  • Intracellular localization of pDNA (FIG. 16) The subcellular localization of pDNA was evaluated using fluorescein-labeled pDNA and LysoTracker Red DND-99, which is a lysosomal marker.
  • a pDNA / PS / BL complex prepared using fluorescein-labeled pDNA was added to EAhy926 cells and subjected to ultrasonic irradiation (US).
  • a group in which pDNA was introduced into cells using Lipofectamine 2000 using a conventional method and a group in which ultrasonic irradiation (US) was not performed were used as controls.
  • FIG. 16 (a) When transfection was performed with Lipofectamine® 2000, pDNA colocalized with lysosomes (FIG. 16 (a)). In addition, pDNA co-localized with lysosomes even when ultrasound was not used in the pDNA / PS / BL complex (FIG. 16 (b)), while addition of pDNA / PS / BL complex and ultrasonic irradiation (US PDNA did not localize to lysosomes in the cells treated with) (FIG. 16 (c)). This is a result suggesting that the anionic nanobubble poly-lipoplex delivers pDNA directly into the cytoplasm without going through the endocytosis pathway.
  • Erythrocytes are obtained from the mice, washed with PBS buffer solution by centrifuging at 5,000xg at 4 ° C for 5 minutes 3 times, and 2% (v / v) erythrocyte stock suspension. A suspension was prepared. Add various anionic nanobubble poly-lipoplexes (pDNA / PEI / BL, pDNA / PS / BL, pDNA / PLL / BL, pDNA / PLA / BL or pDNA / PS / BL) to the erythrosite stock suspension And left at room temperature for 15 minutes. Addition of pDNA / Lipofectamine 2000 (cationic liposome) and addition of PBS alone served as controls. A 10 ⁇ L sample was dropped onto a glass plate, and aggregation was observed under a phase contrast microscope. The results are shown in FIG.
  • Lipofectamine 2000 a commercially available gene transfer reagent, showed red blood cell hemolysis and aggregation, while polyethylenimine, which is widely used for gene transfer experiments, showed very strong aggregation, but various anionic nanobubble poly-lipo plexes showed aggregation and hemolysis. Was not recognized.
  • the obtained complex was administered to the mouse by pDNA / PS / BL in the tail vein by the same method as described above, and the abdomen was irradiated with ultrasonic waves immediately after the administration.
  • Each organ liver, kidney, spleen
  • the blood and each organ were dissolved in Soluene-350 at 60 ° C., and the resulting lysate was decolorized with isopropanol and 30% H 2 O 2 and then neutralized with 5N HCL.
  • the radiation intensity of 32 P-labeled pDNA was measured using a scintillation counter.
  • the liver of the mouse was treated with collagenase 6 hours after administration to obtain liver parenchymal cells and non-parenchymal cells.
  • the luciferase activity per 10 6 cells (Luciferase activities (pg / 106 cells)) was measured to confirm the gene-expressing cells. The results are shown in FIG.
  • luciferase mRNA expressions luciferase mRNA / GAPDH mRNA
  • GAPDH control mRNA
  • the anionic nanobubble poly-lipoplex (pDNA / PS / BL) of the present invention is often introduced into non-parenchymal cells of the liver.
  • FIG. 22 (b) it was revealed that non-parenchymal cells were introduced to the same extent into Kupffer cells and endothelial cells.
  • the particle size of pDNA / PS / BL is estimated to be about 500 nm, which was large to pass through the liver fenestra.
  • TNF- ⁇ and IL-6 serum TNF- ⁇ concentrarion, Serum IL-6 concentrarion
  • serum TNF- ⁇ concentrarion serum TNF- ⁇ concentrarion, Serum IL-6 concentrarion
  • FIG. 26 In 5 week old female Bulb / c mice, colon-26 cells were injected into the peritoneal cavity. Two weeks later, pDNA / PS / BL was administered into mice via the tail vein in the same manner as described above, and the abdomen was irradiated with ultrasonic waves immediately after administration (immediately after injection) or 5 minutes after administration (5 mim after injection). US (+)). Tumors were removed 6 hours after administration, and luciferase activity in the tumors was evaluated. The results are shown in FIG.
  • the preparation of the present invention is useful as a cirrhosis therapeutic agent, an anticancer agent, a cell selective drug / nucleic acid introduction reagent (research reagent) and the like.

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Abstract

The present invention mainly addresses the problem of providing a means for further improving the efficiency of the introduction of a nucleic acid and optionally a compound such as a protein or a medicinal agent into a cell. As the means for solving the problem, an anionic nano-bubble poly-lipo-plex is provided, which comprises a complex of an anionic polymer and a positively-charged polymer and an anionic liposome, wherein the liposome contains bubbles and the complex is cationic.

Description

アニオン性を有する新規ナノバブルポリ－リポ・プレックスの製造方法Method for producing novel nanobubble poly-lipoplex having anionic property
　本発明は、アニオン性を有する新規ナノバブルポリ－リポ・プレックスの製造方法に関する。 The present invention relates to a method for producing a novel nanobubble poly-lipoplex having an anionic property.
　負電荷を有した高分子である遺伝子、ＲＮＡなどの核酸は細胞内取り込みに乏しく、また、静脈内投与した場合には速やかに尿中へ排出されることから、それ単体での医薬品への応用は困難である。このため、遺伝子を細胞内へ送達するためのベクターの開発が必須である。また、非特異的な核酸導入は予期しない副作用の原因となることから、標的となる臓器の特定の細胞のみに核酸を導入する技術の開発が望まれている。これまでに、非ウイルス性の遺伝子ベクターとして正電荷を有した高分子やリポソームを用いた遺伝子との正電荷複合体が細胞内取り込みに優れ、高い遺伝子発現効果を示すことが報告されている。しかしながら、遺伝子医薬品の臨床応用を考えると主薬は遺伝子であり、ベクターは添加物として定義される。また、日本においては医薬品に用いる添加物は全て安全性試験が必要である。一方で、これまでに開発された遺伝子ベクターの詳細な安全性についてはほとんどが報告されていない。このため、遺伝子医薬品の実現には完全且つ効果的に遺伝子を導入するための遺伝子ベクター開発が必要であった。 Nucleic acids such as negatively charged macromolecules such as genes and RNAs are poorly taken up into cells, and are rapidly excreted into urine when administered intravenously. It is difficult. For this reason, it is essential to develop a vector for delivering a gene into a cell. Further, since non-specific nucleic acid introduction causes unexpected side effects, development of a technique for introducing nucleic acid only into specific cells of a target organ is desired. So far, it has been reported that a positively charged complex with a positively charged polymer or a gene using a liposome as a non-viral gene vector is excellent in cellular uptake and exhibits a high gene expression effect. However, considering the clinical application of genetic drugs, the main drug is a gene and the vector is defined as an additive. In Japan, all additives used for pharmaceutical products require safety testing. On the other hand, little has been reported on the detailed safety of gene vectors developed so far. For this reason, the development of gene vectors for introducing genes completely and effectively was necessary for the realization of genetic drugs.
　特許文献１，２は、バブルを含むリポソームを開示する。 Patent Documents 1 and 2 disclose liposomes containing bubbles.
特開2005-154282JP2005-154282 特開2012-36095JP2012-36095
　本発明は、細胞への核酸、必要に応じてさらにタンパク質や薬物などの化合物の導入効率をさらに高めることを目的とする。 An object of the present invention is to further increase the efficiency of introduction of nucleic acids into cells, and if necessary, compounds such as proteins and drugs.
　本発明は、以下のアニオン性を有するナノバブルポリ－リポ・プレックスを提供するものである。 The present invention provides nanobubble poly-lipoplexes having the following anionic properties.
　項１．　アニオン性高分子と正電荷高分子の複合体及びアニオン性リポソームを含み、前記リポソームは気泡を(bubble)含み、前記複合体はカチオン性であるアニオン性ナノバブルポリ－リポ・プレックス。 Item 1. An anionic nanobubble poly-lipoplex comprising a complex of an anionic polymer and a positively charged polymer and an anionic liposome, wherein the liposome comprises bubbles and the complex is cationic.
　項２．　前記アニオン性高分子は核酸である、項１に記載のアニオン性ナノバブルポリ－リポ・プレックス。 Item 2. The anionic nanobubble poly-lipoplex according to Item 1, wherein the anionic polymer is a nucleic acid.
　項３．　前記核酸は、DNAまたはRNAである、項２に記載のアニオン性ナノバブルポリ－リポ・プレックス。 Item 3. Item 3. The anionic nanobubble poly-lipoplex according to Item 2, wherein the nucleic acid is DNA or RNA.
　項４．　前記核酸は、遺伝子発現プラスミドDNA、アンチセンスDNA、DNAアプタマー、siRNAもしくはshRNAを発現するDNA、mRNA、siRNA、miRNA、shRNA、アンチセンスRNAまたはRNAアプタマーである、項２又は３に記載のアニオン性ナノバブルポリ－リポ・プレックス。 Item 4. Item 4. The anionic property according to Item 2 or 3, wherein the nucleic acid is gene expression plasmid DNA, antisense DNA, DNA aptamer, DNA expressing siRNA or shRNA, mRNA, siRNA, miRNA, shRNA, antisense RNA, or RNA aptamer. Nano bubble poly-lipo plex.
　項５．　前記正電荷高分子は、プロタミン、ヒストン、HelΔ１、ゼラチン、ポリリジン、ポリアルギニン、ポリオルニチン、ポリアミドアミンデンドリマー、ポリリジンデンドリマー、ジエチルアミノエチル－デキストラン、キトサン、スペルミン及びスペルミジンからなる群から選ばれる少なくとも１種である、項１～４のいずれかに記載のアニオン性ナノバブルポリ－リポ・プレックス。 Item 5. The positively charged polymer is at least one selected from the group consisting of protamine, histone, HelΔ1, gelatin, polylysine, polyarginine, polyornithine, polyamidoamine dendrimer, polylysine dendrimer, diethylaminoethyl-dextran, chitosan, spermine and spermidine. Item 5. The anionic nanobubble poly-lipoplex according to any one of Items 1 to 4.
　項６．　前記正電荷高分子はプロタミンを含む、項５に記載のアニオン性ナノバブルポリ－リポ・プレックス。 Item 6. Item 6. The anionic nanobubble poly-lipoplex according to Item 5, wherein the positively charged polymer contains protamine.
　項７．　プロタミン及びアニオン性リポソームを含み、前記リポソームは気泡を(bubble)含む、アニオン性ナノバブルポリ－リポ・プレックス。 Item 7. An anionic nanobubble poly-lipoplex comprising protamine and anionic liposomes, the liposomes containing bubbles.
　項８．　前記アニオン性リポソームが、タンパク質又は薬物を含む、項１～７のいずれかに記載のアニオン性ナノバブルポリ－リポ・プレックス。 Item 8. Item 8. The anionic nanobubble poly-lipoplex according to any one of Items 1 to 7, wherein the anionic liposome comprises a protein or a drug.
　本発明は安全且つ効率的な核酸などの物質導入を可能とする複合体及びその用途に関するものである。 The present invention relates to a complex that enables safe and efficient introduction of a substance such as nucleic acid and its use.
　本発明の複合体は超音波照射後に肝臓、腎臓、脾臓で非常に高い遺伝子発現効果を示す。さらに、複合体にリガンドを導入することで特定の細胞への標的化が可能である。 The complex of the present invention shows a very high gene expression effect in the liver, kidney and spleen after ultrasonic irradiation. Furthermore, targeting to specific cells is possible by introducing a ligand into the complex.
　細胞への導入は、超音波を与えた部位で選択的に生じるので、毒性、副作用の軽減を実現できる。 Since introduction into cells is selectively generated at the site where ultrasonic waves are applied, it is possible to reduce toxicity and side effects.
　具体的には、本発明の複合体は、安全性が確認されている成分により構成されるため、細胞への障害を引き起こすことがない。この点は、細胞毒性等の障害を誘発することが知られているカチオン性リポソームとは対照的である。また、超音波照射をしない場合は、本発明の複合体は遺伝子発現効果が見られない。このような部位選択的に細胞への導入することができる特性は、従来のカチオン性リポソームには見られない。 Specifically, since the complex of the present invention is composed of components whose safety has been confirmed, it does not cause damage to cells. This is in contrast to cationic liposomes that are known to induce disorders such as cytotoxicity. Further, when no ultrasonic irradiation is performed, the complex of the present invention does not show a gene expression effect. Such a property that can be selectively introduced into cells is not found in conventional cationic liposomes.
本発明のアニオン性ナノバブルポリ－リポ・プレックスの調製方法の一例を模式的に示す。An example of a method for preparing an anionic nanobubble poly-lipoplex of the present invention is schematically shown. 本発明のアニオン性ナノバブルポリ－リポ・プレックスのパーフルオロプロパンガスバブル封入前後の外観を観察した結果。The result of observing the appearance of the anionic nanobubble poly-lipoplex of the present invention before and after perfluoropropane gas bubble encapsulation. 本発明のアニオン性ナノバブルポリ－リポ・プレックスの安定性のアガロースゲル電気泳動による評価結果。The evaluation result by the agarose gel electrophoresis of the stability of the anionic nanobubble poly-lipoplex of the present invention. pDNA/PEI/AL複合体による遺伝子導入効果。Gene transfer effect by pDNA / PEI / AL complex. pDNA/PLL/AL複合体による遺伝子導入効果。Gene transfer effect by pDNA / PLL / AL complex. pDNA/PS/AL複合体による遺伝子導入効果。Gene transfer effect by pDNA / PS / AL complex. 肝臓への遺伝子導入後の遺伝子発現細胞。Gene expression cells after gene transfer to the liver. 脾臓への遺伝子導入後の遺伝子発現細胞。Gene expression cells after gene introduction into spleen. 標的化された細胞への遺伝子導入。Gene transfer to targeted cells. 超音波造影と崩壊の確認。Confirmation of ultrasonography and disintegration. siRNAを用いたsiRNA/PS/DSPG複合体の調製。Preparation of siRNA / PS / DSPG complex using siRNA. mRNAを用いたmRNA/PS/DSPG複合体の調製。Preparation of mRNA / PS / DSPG complex using mRNA. 薬剤封入アニオン性リポソームの調製。Preparation of drug encapsulated anionic liposomes. 本発明のアニオン性ナノバブルポリ－リポ・プレックスの調製方法の一例を模式的に示す。An example of a method for preparing an anionic nanobubble poly-lipoplex of the present invention is schematically shown. （ａ）本発明のアニオン性ナノバブルポリ－リポ・プレックスのパーフルオロプロパンガスバブル封入前後の外観を観察した結果。各パネルにおいて、右側（Bubble(+)）は、左側（Bubble(-)）を、超音波処理することで得られたアニオン性ナノバブルポリ－リポ・プレックスである。（ｂ）得られたpDNA/PS/BL複合体の、酢酸ウラニルを用いたネガティブ染色による、TEM観察像を示す。バー：200nm。(A) The result of observing the appearance of the anionic nanobubble poly-lipoplex of the present invention before and after perfluoropropane gas bubble encapsulation. In each panel, the right side (Bubble (+)) is an anionic nanobubble poly-lipoplex obtained by sonicating the left side (Bubble (-)). (B) TEM observation image of the obtained pDNA / PS / BL complex by negative staining using uranyl acetate is shown. Bar: 200nm. 本発明のアニオン性ナノバブルポリ－リポ・プレックスの安定性のアガロースゲル電気泳動による評価結果。The evaluation result by the agarose gel electrophoresis of the stability of the anionic nanobubble poly-lipoplex of the present invention. EAhy926細胞へのアニオン性ナノバブルポリ－リポ・プレックスの遺伝子導入効率。バーは、標準誤差（S.E.）を表す。**: P<0.01; *: P<0.05 （他の群全てに対して）。##: P<0.01; #: P<0.05。Gene transfer efficiency of anionic nanobubble poly-lipoplex into EAhy926 cells. Bars represent standard error (S.E.). **: P <0.01; *: P <0.05 (for all other groups). ##: P <0.01; #: P <0.05. pDNA/PS/BL複合体の細胞内局在を示す。（a）Lipofectamin 2000、(b)pDNA/PS/BL複合体；US(-)（超音波照射なし）、（c）pDNA/PS/BL複合体；US(+)群（超音波照射あり群）。The intracellular localization of pDNA / PS / BL complex is shown. (A) Lipofectamin 2000, (b) pDNA / PS / BL complex; US (-) (no ultrasound irradiation), (c) pDNA / PS / BL complex; US (+) group (with ultrasound irradiation group) ). アニオン性ナノバブルポリ－リポ・プレックスのEAhy926細胞に対する細胞毒性の評価。未処理（対照）に対する比率で示す。**: P<0.01; *: P<0.05 （コントロールに対して）。Evaluation of cytotoxicity of anionic nanobubble poly-lipoplexes against EAhy926 cells. Shown as a ratio to untreated (control). **: P <0.01; *: P <0.05 (vs. control). アニオン性ナノバブルポリ－リポ・プレックスの赤血球との凝集（位相差顕微鏡画像）。(a) Lipofectamine 2000, (b) polyethylenimine, (c) pDNA/PS/BL, (d) pDNA/PLL/BL, (e) pDNA/PLA/BL, (f) pDNA/DPLL/BL, (g) PBS。Aggregation of anionic nanobubble poly-lipoplex with erythrocytes (phase contrast microscope image). (a) Lipofectamine 2000, (b) polyethylenimine, (c) pDNA / PS / BL, (d) pDNA / PLL / BL, (e) pDNA / PLA / BL, (f) pDNA / DPLL / BL, (g) PBS. 生体へのアニオン性ナノバブルポリ－リポ・プレックスによる遺伝子導入効果。（a）肝臓、（b）腎臓、（c）脾臓。**: P<0.01; *: P<0.05 （US(-)群（超音波照射なし群）に対して）。Gene transfer effect by anionic nanobubble poly-lipo plexes to living body. (A) Liver, (b) Kidney, (c) Spleen. **: P <0.01; *: P <0.05 (for US (-) group (no ultrasound irradiation group)). アニオン性脂質の効果。(a)EAhy926細胞への遺伝子導入効率、（b）生体への遺伝子導入効率、（c）細胞毒性の評価。**: P<0.01; *: P<0.05。The effect of anionic lipids. (a) Gene transfer efficiency into EAhy926 cells, (b) Efficiency of gene transfer into living body, (c) Evaluation of cytotoxicity. **: P <0.01; *: P <0.05. pDNA/PS/AL複合体の投与後の動態。（a）血液、(b)肝臓、（c）腎臓、(d)脾臓、(e)心臓、（f）肺。Kinetics after administration of pDNA / PS / AL complex. (A) Blood, (b) Liver, (c) Kidney, (d) Spleen, (e) Heart, (f) Lung. 肝臓への遺伝子導入後の遺伝子発現細胞。（a）肝実質細胞（Parenchymal cell）と肝非実質細胞(Non parenchymal cell)、（b）クッパー細胞（Kupffer cell）及び内皮細胞（endothelial cell）。 *: P<0.05 （肝実質細胞に対して）。Gene expression cells after gene transfer to the liver. (A) Hepatocytes (Parenchymal cells) and non-hepatocytes (Non cells), (b) Kupffer cells and endothelial cells (endothelial cells). *: P <0.05 (for liver parenchymal cells). 肝臓障害の評価。（a）血清中のAST活性、（b）血清中のALT活性。Assessment of liver damage. (A) AST activity in serum, (b) ALT activity in serum. 細胞への障害（サイトカインの誘導）の評価。（a）血清中のTNF-α濃度、（b）血清中のIL-6濃度。Assessment of damage to cells (induction of cytokines). (A) TNF-α concentration in serum, (b) IL-6 concentration in serum. 腹部または胸部に対して超音波照射の比較。（a）腹部に対して超音波照射、（b）胸部に対して超音波照射。Comparison of ultrasound irradiation to the abdomen or chest. (A) Ultrasonic irradiation on the abdomen, (b) Ultrasonic irradiation on the chest. がん細胞への遺伝子導入。Gene transfer to cancer cells.
　本発明のアニオン性を有する新規ナノバブルポリ－リポ・プレックスは、アニオン性リポソーム、アニオン性高分子(好ましくは核酸)、正電荷高分子及び気泡(バブル)を含む。アニオン性高分子(好ましくは核酸)と正電荷高分子は全体として正電荷を有する複合体となり、このカチオン性の複合体とアニオン性リポソームがさらに複合体化し、さらにリポソーム内に気泡を含めることで、本発明のアニオン性を有する新規ナノバブルポリ－リポ・プレックスを得ることができる。 The novel anionic nanobubble poly-lipoplex of the present invention includes anionic liposomes, anionic polymers (preferably nucleic acids), positively charged polymers and bubbles (bubbles). An anionic polymer (preferably a nucleic acid) and a positively charged polymer form a complex having a positive charge as a whole.This cationic complex and an anionic liposome are further complexed, and bubbles are further included in the liposome. Thus, the novel nanobubble poly-lipoplex having an anionic property of the present invention can be obtained.
　アニオン性ナノバブルポリ－リポ・プレックスの粒径は、50～1000nm程度、400～800nm程度、特に500～600nm程度である。あるいは、100～800nm程度、150～600nm程度とすることもできる。 The particle size of the anionic nanobubble poly-lipoplex is about 50 to 1000 nm, about 400 to 800 nm, particularly about 500 to 600 nm. Alternatively, the thickness may be about 100 to 800 nm or about 150 to 600 nm.
　アニオン性リポソームの粒径は、30～500nm程度、好ましくは50～300nm程度、特に100～250nm程度である。アニオン性ナノバブルポリ－リポ・プレックス及びアニオン性リポソームの粒径は、エクストルーダーを使用して制御することができる。 The particle size of the anionic liposome is about 30 to 500 nm, preferably about 50 to 300 nm, particularly about 100 to 250 nm. The particle size of anionic nanobubble poly-lipoplexes and anionic liposomes can be controlled using an extruder.
　なお、リポソームの粒径は、動的光散乱法により測定をすることができる。 The particle size of the liposome can be measured by a dynamic light scattering method.
　本発明で使用されるアニオン性リポソームは、多重層リポソーム（例えば、multilamellar vesicles: MLV）、一枚膜リポソーム（例えば、small unilamellar. vesicles: SUV、large unilamellar vesicles: LUV）のいずれであってもよい。アニオン性リポソームは超音波処理法、逆相蒸発法、凍結融解法、脂質溶解法、噴霧乾燥法などにより作製され、リン脂質、糖脂質、ステロール類、グリコール類、アニオン性脂質、ポリエチレングリコール基を有する脂質（例えばＰＥＧ-リン脂質）などが含まれる。本発明のアニオン性リポソームは、リポソーム全体で「アニオン性」であればよく、カチオン性脂質を含んでいてもよいが、その場合にはアニオン性脂質をより多く含み、リポソーム全体としてアニオン性になるようにする。 Anionic liposomes used in the present invention may be multilamellar liposomes (eg, multilamellarlamvesicles: MLV) or unilamellar liposomes (eg, small unilamellar. Vesicles: SUV, large unilamellar vesicles: LUV). . Anionic liposomes are produced by sonication, reverse phase evaporation, freeze-thaw, lipid lysis, spray drying, etc., and phospholipids, glycolipids, sterols, glycols, anionic lipids, polyethylene glycol groups And lipids (eg, PEG-phospholipids). The anionic liposome of the present invention may be “anionic” throughout the liposome and may contain a cationic lipid. In that case, the anionic liposome as a whole contains more anionic lipid and becomes anionic. Like that.
　本明細書において、「複合体化」とは、アニオン性リポソームと核酸－正電荷高分子の複合体が一体化することと、核酸と正電荷高分子が一体化することの両方を包含する。 In the present specification, “complexing” includes both integration of an anionic liposome and a nucleic acid-positively charged polymer complex and integration of a nucleic acid and a positively charged polymer.
　アニオン性ナノバブルポリ－リポ・プレックスは、内部又は表面に薬物、タンパク質を含む任意の生理活性物質を包含させることができる。タンパク質、薬物などの生理活性物質は、疎水性部分がリポソームに埋め込まれていてもよい。 The anionic nanobubble poly-lipoplex can contain any physiologically active substance including drugs and proteins inside or on the surface. A physiologically active substance such as a protein or drug may have a hydrophobic portion embedded in a liposome.
　アニオン性リポソームの脂質膜と生理活性物質の吸着、結合は、イオン結合、水素結合、疎水性相互作用などにより行われる。例えば、イオン結合は、リポソームの構成要素のアニオンと、生理活性物質であるカチオンのイオン結合による結合が挙げられる。アニオン性リポソームの表面が疎水性であれば、疎水性の生理活性物質(薬物など)を表面又は膜の内部に吸着又は結合させることができる。 Adsorption and binding of the lipid membrane of an anionic liposome and a physiologically active substance are performed by ionic bond, hydrogen bond, hydrophobic interaction, and the like. For example, the ionic bond includes a bond by an ionic bond between an anion as a component of the liposome and a cation which is a physiologically active substance. If the surface of the anionic liposome is hydrophobic, a hydrophobic physiologically active substance (drug etc.) can be adsorbed or bound to the surface or the inside of the membrane.
　本発明のアニオン性リポソームには、アニオン性リン脂質と中性リン脂質が含まれ、カチオン性リン脂質が少量含まれていてもよい。また、糖脂質が含まれていてもよい。 The anionic liposome of the present invention contains an anionic phospholipid and a neutral phospholipid, and may contain a small amount of a cationic phospholipid. In addition, glycolipids may be included.
　本発明のアニオン性リポソームに含まれる好ましい中性リン脂質として、大豆、卵黄などから得られるレシチン、リゾレシチンおよび／またはこれらの水素添加物、水酸化物の誘導体を挙げることができる。本発明のアニオン性リポソームに含まれるリン脂質を構成する高級脂肪酸は、飽和脂肪酸と不飽和脂肪酸のいずれでもよいが、飽和脂肪酸が好ましい。 Preferred neutral phospholipids contained in the anionic liposome of the present invention include lecithin, lysolecithin and / or hydrogenated products and hydroxide derivatives obtained from soybeans, egg yolks and the like. The higher fatty acid constituting the phospholipid contained in the anionic liposome of the present invention may be either a saturated fatty acid or an unsaturated fatty acid, but is preferably a saturated fatty acid.
　アニオン性リポソームに含まれるリン脂質として、卵黄、大豆またはその他の動植物に由来するか、または合成した炭素鎖n（nは3～30の整数を示す）から成る飽和または不飽和脂肪酸を構成成分に有するホスファチジルコリン（PC）、ホスファチジルセリン（PS）、ホスファチジルグリセロール（PG）、ホスファチジリックアシッド（PA）、ホスファチジルエタノールアミン（PE）、カルジオリピン、スフィンゴシン、セラミド、スフィンゴミエリン、ガングリオシド、スフィンゴリン脂質、卵黄レシチン、水素添加卵黄レシチン、大豆レシチン、水素添加大豆レシチンなどが挙げられる。より好ましくは、飽和脂肪酸を構成成分とする脂質である。 As a phospholipid contained in an anionic liposome, a saturated or unsaturated fatty acid derived from egg yolk, soybean, other animals or plants, or composed of a synthesized carbon chain n (n represents an integer of 3 to 30) is a constituent. Has phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylglycerol (PG), phosphatidyl acid (PA), phosphatidylethanolamine (PE), cardiolipin, sphingosine, ceramide, sphingomyelin, ganglioside, sphingophospholipid, egg yolk Examples include lecithin, hydrogenated egg yolk lecithin, soybean lecithin, and hydrogenated soybean lecithin. More preferably, it is a lipid comprising a saturated fatty acid as a constituent component.
　上記リン脂質の具体例としては、ジラウロイルホスファチジルコリン（DLPC）、ジミリストイルホスファチジルコリン（DMPC）、ジパルミトイルホスファチジルコリン（DPPC）、ジステアロイルホスファチジルコリン（DSPC）、ジオレオイルホスファチジルコリン（DOPC）、ジリノレオイルホスファチジルコリン等のホスファチジルコリン；ジラウロイルホスファチジルグリセロール（DLPG）、ジミリストイルホスファチジルグリセロール（DMPG）、ジパルミトイルホスファチジルグリセロール（DPPG）、ジステアロイルホスファチジルグリセロール（DSPG）、ジオレオイルホスファチジルグリセロール（DOPG）、ジリノレオイルホスファチジルグリセロール等のホスファチジルグリセロール；ジラウロイルホスファチジルエタノールアミン（DLEA）、ジミリストイルホスファチジルエタノールアミン（DMEA）、ジパルミトイルホスファチジルエタノールアミン（DPEA）、ジステアロイルホスファチジルエタノールアミン（DSEA）、ジオレオイルホスファチジルエタノールアミン（DOEA）、ジリノレオイルホスファチジルエタノールアミン等のホスファチジルエタノールアミン；ジラウロイルホスファチジルセリン（DLPS）、ジミリストイルホスファチジルセリン（DMPS）、ジパルミトイルホスファチジルセリン（DPPS）、ジステアロイルホスファチジルセリン（DSPS）、ジオレオイルホスファチジルセリン（DOPS）、ジリノレオイルホスファチジルセリン等のホスファチジルコリンなどが挙げられる。 Specific examples of the phospholipid include dilauroyl phosphatidylcholine (DLPC), dimyristoyl phosphatidylcholine (DMPC), dipalmitoyl phosphatidylcholine (DPPC), distearoyl phosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dilinoleoylphosphatidylcholine and the like. Phosphatidylcholine: dilauroyl phosphatidylglycerol (DLPG), dimyristoyl phosphatidylglycerol (DMPG), dipalmitoyl phosphatidylglycerol (DPPG), distearoyl phosphatidylglycerol (DSPG), dioleoylphosphatidylglycerol (DOPG), dilinoleoyl phosphatidylglycerol, etc. Of phosphatidylglycerol; dilauroylphosphatidylethanolamine (DLEA), di Phosphatidylethanolamines such as listoyl phosphatidylethanolamine (DMEA), dipalmitoylphosphatidylethanolamine (DPEA), distearoylphosphatidylethanolamine (DSEA), dioleoylphosphatidylethanolamine (DOEA), dilinoleoylphosphatidylethanolamine; Lauroylphosphatidylserine (DLPS), dimyristoylphosphatidylserine (DMPS), dipalmitoylphosphatidylserine (DPPS), distearoylphosphatidylserine (DSPS), dioleoylphosphatidylserine (DOPS), phosphatidylcholine such as dilinoleoylphosphatidylserine, etc. Can be mentioned.
　本発明のリポソームを構成する脂質膜のアニオン性構成成分として、炭素鎖n2（n2は3～30の整数を示す）から成る飽和または不飽和脂肪酸を構成成分に有するホスファチジルイノシトール、ホスファチジルグリセロール、ホスファチジルセリンなどが挙げられる。リポソームの脂質膜を構成するアニオン性の脂質膜成分には、ホスファチジルイノシトール、ホスファチジルグリセロールなどの負に荷電したリン脂質のほかに、炭素鎖n2（n2は3～30の整数を示す）から成る飽和または不飽和脂肪酸を構成成分に有するホスファチジン酸、ジセチルリン酸(DCP)、ジラウリルリン酸、ジミリスチルリン酸、ホスファチジルグリセロールリン酸などを挙げることができる。 As an anionic constituent of the lipid membrane constituting the liposome of the present invention, phosphatidylinositol, phosphatidylglycerol, phosphatidylserine having a saturated or unsaturated fatty acid consisting of carbon chain n2 (n2 represents an integer of 3 to 30) as a constituent Etc. In addition to negatively charged phospholipids such as phosphatidylinositol and phosphatidylglycerol, the anionic lipid membrane components that make up the lipid membrane of liposomes are saturated with carbon chain n2 (n2 is an integer from 3 to 30). Alternatively, phosphatidic acid, dicetyl phosphoric acid (DCP), dilauryl phosphoric acid, dimyristyl phosphoric acid, phosphatidyl glycerol phosphoric acid having an unsaturated fatty acid as a constituent component can be given.
　必要に応じて使用されるカチオン性脂質としては、例えば3β-［N-（N’、N’-ジメチルアミノエタン）-カルバモイル］コレステロール（DC-chol）、1、2-ジオレオイルオキシ-3-（トリメチルアンモニウム）プロパン（DOTAP）、N、N-ジオクタデシルアミドグリシルスペルミン（DOGS）、ジメチルジオクタデシルアンモニウムブロミド（DDAB）、N-［1-（2、3-ジオレイルオキシ）プロピル］-N、N、N-トリメチルアンモニウムクロリド（DOTMA）、2、3-ジオレイルオキシ-N-［2（スペルミン－カルボキサミド）エチル］-N、N-ジメチル-1-プロパンアミニウムトリフルオロアセテート（DOSPA）及びN-［1-（2、3-ジミリスチルオキシ）プロピル］-N、N-ジメチル-N-（2-ヒドロキシエチル）アンモニウムブロミド（DMRIE）、さらにジパルミトイルホスファチジン酸（DPPA）とヒドロキシエチレンジアミンとのエステル、またはジステアロイルホスファチジン酸（DSPA）とヒドロキシエチレンジアミンとのエステルなどが挙げられる。 Examples of the cationic lipid used as needed include 3β- [N- (N ′, N′-dimethylaminoethane) -carbamoyl] cholesterol (DC-chol), 1,2-dioleoyloxy-3 -(Trimethylammonium) propane (DOTAP), N, N-dioctadecylamide glycylspermine (DOGS), dimethyldioctadecylammonium bromide (DDAB), N- [1- (2,3-dioleyloxy) propyl]- N, N, N-trimethylammonium chloride (DOTMA), 2,3-dioleyloxy-N- [2 (spermine-carboxamido) ethyl] -N, N-dimethyl-1-propaneaminium trifluoroacetate (DOSPA) And N- [1- (2,3-dimyristyloxy) propyl] -N, N-dimethyl-N- (2-hydroxyethyl) ammonium bromide (DMRIE), dipalmitoylphosphatidic acid (DPPA) and Examples thereof include esters with droxyethylenediamine or esters of distearoylphosphatidic acid (DSPA) with hydroxyethylenediamine.
　糖脂質としては、ジガラクトシルジグリセリド、ガラクトシルジグリセリド硫酸エステルなどのグリセロ脂質、ガラクトシルセラミド、ガラクトシルセラミド硫酸エステル、ラクトシルセラミド、ガングリオシドG7、ガングリオシドG6、ガングリオシドG4などのスフィンゴ糖脂質などを挙げることができる。 Examples of glycolipids include glycerolipids such as digalactosyl diglyceride and galactosyl diglyceride sulfate, and sphingoglycolipids such as galactosylceramide, galactosylceramide sulfate, lactosylceramide, ganglioside G7, ganglioside G6, and ganglioside G4.
　アニオン性脂質は、全脂質量に対し1～100質量％、好ましくは全脂質量に対し10～75質量％、より好ましくは全脂質量に対し25～60質量％の割合で含有するように添加すればよい。 Anionic lipid is added so as to contain 1 to 100% by mass with respect to the total lipid amount, preferably 10 to 75% by mass with respect to the total lipid amount, more preferably 25 to 60% by mass with respect to the total lipid amount. do it.
　リポソームに包含され得る生理活性物質としては、タンパク質、薬物などが広く挙げられる。本発明のアニオン性リポソーム複合体は、核酸（ＤＮＡ、ＲＮＡなど）が正電荷高分子との複合体として含まれるので、核酸と組み合わせて使用するのに適した生理活性物質が好ましい。タンパク質、薬物としては、抗ガン剤、抗アレルギー剤、抗菌剤、抗真菌剤、抗ウイルス剤、免疫抑制剤、ワクチン、インターフェロン、インターロイキン、成長因子、ペプチドホルモン、酵素、ステロイドホルモン、抗リウマチ薬、抗原、抗体、受容体又はそのリガンドなどが挙げられる。 Examples of physiologically active substances that can be included in liposomes include proteins and drugs. Since the anionic liposome complex of the present invention contains a nucleic acid (DNA, RNA, etc.) as a complex with a positively charged polymer, a physiologically active substance suitable for use in combination with a nucleic acid is preferred. Proteins and drugs include anticancer agents, antiallergic agents, antibacterial agents, antifungal agents, antiviral agents, immunosuppressive agents, vaccines, interferons, interleukins, growth factors, peptide hormones, enzymes, steroid hormones, antirheumatic agents , Antigens, antibodies, receptors or ligands thereof.
　核酸としては、DNA、RNAのいずれでもよい。DNAは、遺伝子を発現するものが挙げられ、例えばプラスミド、或いはプロモーターに連結された遺伝子を含む遺伝子構築物、人工遺伝子などが挙げられる。DNAとしては、遺伝子発現プラスミドDNA、アンチセンスDNA、DNAアプタマー、siRNA・shRNAなどのRNAを発現するDNAが挙げられる。RNAとしては、mRNA、siRNA、アンチセンスRNA、RNAアプタマー、shRNA、miRNAなどが挙げられる。核酸、タンパク質、薬物などの生理活性物質は、細胞内に取り込まれたとき、もしくは細胞内で発現されたときに細胞毒性、アポトーシス誘導作用などの細胞に障害を与えるか、細胞死を誘導する作用を有するものが挙げられる。 The nucleic acid may be either DNA or RNA. Examples of DNA include those that express genes, such as plasmids, gene constructs containing genes linked to promoters, and artificial genes. Examples of DNA include DNA that expresses RNA such as gene expression plasmid DNA, antisense DNA, DNA aptamer, siRNA and shRNA. Examples of RNA include mRNA, siRNA, antisense RNA, RNA aptamer, shRNA, miRNA and the like. Physiologically active substances such as nucleic acids, proteins, drugs, etc., when taken into cells or expressed in cells, damage cells such as cytotoxicity and apoptosis-inducing action, or induce cell death The thing which has is mentioned.
　正電荷高分子としては、プロタミン、ヒストン、HelΔ１、ゼラチンなどのカチオン性タンパク質、ポリリジン、ポリアルギニン、ポリオルニチン、ポリアミドアミンデンドリマー、ポリリジンデンドリマーなどのデンドリマー、ジエチルアミノエチル－デキストランなどのデキストラン、キトサンなどのポリカチオン性多糖、スペルミン、スペルミジンなどのポリアミンなどが挙げられる。中でも、核酸等が超音波を当てた部位において特異的に細胞に導入されるとの観点から、プロタミン、ヒストン、スペルミン、スペルミジンが好ましく、プロタミン、ヒストンがより好ましく、プロタミンが特に好ましい。 Examples of positively charged polymers include cationic proteins such as protamine, histone, HelΔ1, gelatin, polylysine, polyarginine, polyornithine, polyamidoamine dendrimers, dendrimers such as polylysine dendrimers, dextran such as diethylaminoethyl-dextran, and polys such as chitosan. Examples include cationic polysaccharides, polyamines such as spermine and spermidine. Of these, protamine, histone, spermine, and spermidine are preferred, protamine and histone are more preferred, and protamine is particularly preferred from the viewpoint that nucleic acids and the like are specifically introduced into cells at the site where ultrasonic waves are applied.
　核酸と正電荷高分子の複合体は、複合体の電荷が正電荷になるように、正電荷高分子の総正電荷量が核酸の総負電荷量を上回り、アニオン性リポソームとの複合体が形成できるように核酸と正電荷高分子を溶液中で混合して生成させる。 The complex of the nucleic acid and the positively charged polymer is such that the total positive charge of the positively charged polymer exceeds the total negative charge of the nucleic acid so that the charge of the complex becomes positive. A nucleic acid and a positively charged polymer are mixed in a solution so that they can be formed.
　リポソームの製造法の例を具体的に説明すると、例えば前記したアニオン性リン脂質を含むリン脂質等を適当な有機溶媒に溶解し、これを適当な容器に入れて減圧下に溶媒を留去して容器内面にリン脂質膜を形成し、これに水、好ましくは緩衝液を加えて攪拌して、アニオン性リポソームを得ることができる。当該リポソームを直接、核酸と正電荷高分子の複合体と混合することにより、アニオン性リポソームとカチオン性粒子の複合粒子を得ることができる。 An example of a method for producing liposomes will be described in detail. For example, the above-described phospholipids containing anionic phospholipids are dissolved in an appropriate organic solvent, placed in an appropriate container, and the solvent is distilled off under reduced pressure. Then, a phospholipid film is formed on the inner surface of the container, and water, preferably a buffer solution, is added thereto and stirred to obtain an anionic liposome. By directly mixing the liposome with a complex of a nucleic acid and a positively charged polymer, a composite particle of an anionic liposome and a cationic particle can be obtained.
　本発明のアニオン性ナノバブルポリ－リポ・プレックスは、全体として中性より好ましくはアニオン性である。 The anionic nanobubble poly-lipoplex of the present invention as a whole is more preferably neutral than neutral.
　本発明のアニオン性ナノバブルポリ－リポ・プレックスは、アニオン性リポソームとカチオン性粒子との複合体を先に製造し、この複合体中のアニオン性リポソームにバブルを導入することにより製造することができる。 The anionic nanobubble poly-lipoplex of the present invention can be produced by first producing a complex of anionic liposomes and cationic particles and introducing bubbles into the anionic liposomes in this complex. .
　リポソーム内でのバブルの生成は公知の方法に従い実施することができ、例えば以下の方法により実施することができる。 The generation of bubbles in the liposome can be performed according to a known method, for example, by the following method.
　先ず、アニオン性リポソームを密封容器に封入し、空隙部にバブルとしてリポソームに導入されるガスを充填する。充填されるガスとしては、特に限定されないが、例えばフッ化物ガス又は窒素ガスが挙げられる。充填されるガスの圧力としては、0.1～1.0ＭＰａ程度である。フッ化物ガスとしては、硫化ヘキサフルオライド、パーフルオロ炭化水素ガスが挙げられる。 First, an anionic liposome is sealed in a sealed container, and a gas introduced into the liposome as a bubble is filled in the void. Although it does not specifically limit as gas filled, For example, fluoride gas or nitrogen gas is mentioned. The pressure of the gas to be filled is about 0.1 to 1.0 MPa. Examples of the fluoride gas include sulfurized hexafluoride and perfluorohydrocarbon gas.
　次いでガスを封入した密閉容器を超音波処理する。超音波処理は、例えば、２０～５０kHzの超音波を１～５分照射すればよい。当該超音波処理により、リポソーム内部の水溶液とフッ化物ガス又は窒素ガスとが置換し、ガス封入リポソームが得られる。 Next, sonicate the sealed container filled with gas. For the ultrasonic treatment, for example, ultrasonic waves of 20 to 50 kHz may be irradiated for 1 to 5 minutes. By the ultrasonic treatment, the aqueous solution inside the liposome is replaced with fluoride gas or nitrogen gas, and gas-encapsulated liposome is obtained.
　本発明のアニオン性ナノバブルポリ－リポ・プレックスは、生体内に投与後に、特定の場所で超音波を照射することで、リポソームからガスが放出され、同時に核酸とリポソーム内の薬物、タンパク質等が細胞内に導入される。超音波の照射は、超音波診断装置などを用いて行うことができる。 The anionic nanobubble poly-lipoplex of the present invention is released into liposomes by irradiating ultrasonic waves at a specific place after administration in vivo, and at the same time, nucleic acids and drugs, proteins, etc. in liposomes are stored in cells. Introduced in. Ultrasonic irradiation can be performed using an ultrasonic diagnostic apparatus or the like.
　リポソームは、ポリエチレングリコール（ＰＥＧ）、糖鎖などを導入して生体内でのステルス性を高めて半減期を長くしておくことが好ましい。ＰＥＧ、糖鎖などは、これらを導入したリン脂質をアニオン性リポソーム製造の原料として使用すればよい。 Liposomes are preferably introduced with polyethylene glycol (PEG), sugar chains, and the like to increase in vivo stealth and extend the half-life. For PEG, sugar chains, etc., phospholipids into which these are introduced may be used as a raw material for the production of anionic liposomes.
　本発明のアニオン性ナノバブルポリ－リポ・プレックスは、ＮＧＲ、ＲＧＤなどの細胞認識リガンドを結合することで、特定の細胞に認識されるようになり、これと超音波を当てる部位を限定することで、核酸、必要に応じてさらに組み合わせられる薬物、タンパク質を導入する細胞をさらに特定することができ、副作用、毒性の低減を実現できる。細胞認識リガンドとしては、これらのペプチド性リガンドであってもよく、糖鎖、タンパク質、サイトカイン、ケモカイン、増殖因子、ペプチドホルモン、抗体又はその一部などが挙げられる。 The anionic nanobubble poly-lipoplexes of the present invention can be recognized by specific cells by binding cell recognition ligands such as NGR and RGD, and by limiting the site to which ultrasound is applied. In addition, nucleic acids, drugs that can be further combined as necessary, and cells into which proteins are introduced can be further specified, and side effects and toxicity can be reduced. As the cell recognition ligand, these peptide ligands may be used, and examples thereof include sugar chains, proteins, cytokines, chemokines, growth factors, peptide hormones, antibodies or parts thereof.
　本発明のリポソームのゼータ電位は、-50～40mV程度、好ましくは-50～30mV程度、より好ましくは-45～20mV程度である。 The zeta potential of the liposome of the present invention is about -50 to 40 mV, preferably about -50 to 30 mV, more preferably about -45 to 20 mV.
　以下、本発明を実施例に基づきより詳細に説明する。 Hereinafter, the present invention will be described in more detail based on examples.
　実施例で使用した材料を以下に示す。 The materials used in the examples are shown below.
　核酸
　pDNA（pCMV-Luc）：plasmid DNA（pGL3 controlベクター(Promega)からHind IIIとXba Iを用いて切り出したホタルルシフェラーゼcDNAをinvitrogen社のpcDNA3ベクターのポリリンカー部位に組み込むことで作製したものを使用した。）
　pDNAは、Endo Free Plasmid Giga Kit（Qiagen GmbH社製；Hilden, Germany）を使用して増幅し、注射用水（滅菌した蒸留水）に溶解した。 Nucleic acid pDNA (pCMV-Luc): plasmid DNA (used by integrating firefly luciferase cDNA excised from pGL3 control vector (Promega) with Hind III and Xba I into the polylinker site of invitrogen pcDNA3 vector did.)
pDNA was amplified using Endo Free Plasmid Giga Kit (Qiagen GmbH; Hilden, Germany) and dissolved in water for injection (sterilized distilled water).
　mRNA：ホタルルシフェラーゼ mRNA。 MRNA: firefly luciferase-mRNA.
　siRNA：ホタルルシフェラーゼに対するsiRNAであり、その配列を以下に示す。
センス鎖（5'→3'）CUUACGCUGAGUACUUCGAtt(配列番号1)
アンチセンス鎖（5'→3'）UCGAAGUACUCAGCGUAAGtt(配列番号2)。 siRNA: siRNA for firefly luciferase, the sequence of which is shown below.
Sense strand (5 '→ 3') CUUACGCUGAGUACUUCGAtt (SEQ ID NO: 1)
Antisense strand (5 ′ → 3 ′) UCGAAGUACUCAGCGUAAGtt (SEQ ID NO: 2).
　リポソームを構成する脂質
Distearoylphosphatidylglycerol (DSPG)
Distearoylphosphatidylcholine (DSPC)
Polyethyleneglycol 2000-distearoylphosphatidylethanolamine (PEG-DSPE)
Distearoylphosphatidylic acid (DSPA)
Distearoylphosphatidylserine (DSPS)
Dioleoyltrimethylammoniumpropane (DOTAP)。 Lipids that make up liposomes
Distearoylphosphatidylglycerol (DSPG)
Distearoylphosphatidylcholine (DSPC)
Polyethyleneglycol 2000-distearoylphosphatidylethanolamine (PEG-DSPE)
Distearoylphosphatidylic acid (DSPA)
Distearoylphosphatidylserine (DSPS)
Dioleoyltrimethylammoniumpropane (DOTAP).
　カチオン性ポリマー
プロタミン（プロタミン硫酸塩）(PS)
ポリ-L-リジン(PLL)
ポリ-L-アルギニン(PLA)
ポリエチレンイミン(PEI)
ポリ-L-リジンデンドリグラフト(DPLL)（COLCOM社製；Montpellieer, France）。 Cationic polymer <br/> Protamine (protamine sulfate) (PS)
Poly-L-lysine (PLL)
Poly-L-arginine (PLA)
Polyethyleneimine (PEI)
Poly-L-lysine dendrigraft (DPLL) (manufactured by COLCOM; Montpellieer, France).
　バブル
バブル作製用のガスとしてパーフルオロプロパンを使用した。 Bubble Perfluoropropane was used as a gas for bubble production.
　また、超音波を「US」と略すことがある。 Also, ultrasound may be abbreviated as “US”.
　実施例１：アニオン性バブルリポソームの作製（１）
　（１）アニオン性リポソーム(AL)の作製
　アニオン性のリポソームはDSPG, DSPC, PEG-DSPEを脂質として用いて調製した。まず、各脂質をクロロホルムに溶解し、モル比がDSPG:DSPC:PEG-DSPE=7:2:1となるようにナスフラスコで混合した。ロータリーエバポレーターを用いてクロロホルムを除去し、ナスフラスコ内に脂質の薄膜を形成した。また、デシケーターでクロロホルムを完全に除去した。この脂質薄膜に注射用水を添加し、65℃で30分間インキュベートし、水和した。水和後の脂質溶液をバス型ソニケーターで10分間超音波処理し、さらに、プローブ型ソニケーターを用いて3分間超音波処理した。得られた脂質溶液を0.45μmのフィルターに通し、アニオン性リポソーム溶液とした。 Example 1: Preparation of anionic bubble liposome (1)
(1) Preparation of anionic liposome (AL) Anionic liposomes were prepared using DSPG, DSPC, and PEG-DSPE as lipids. First, each lipid was dissolved in chloroform and mixed in an eggplant flask so that the molar ratio was DSPG: DSPC: PEG-DSPE = 7: 2: 1. Chloroform was removed using a rotary evaporator, and a lipid thin film was formed in the eggplant flask. In addition, chloroform was completely removed with a desiccator. Water for injection was added to the lipid film, incubated at 65 ° C. for 30 minutes, and hydrated. The hydrated lipid solution was sonicated with a bath sonicator for 10 minutes, and further sonicated with a probe sonicator for 3 minutes. The obtained lipid solution was passed through a 0.45 μm filter to obtain an anionic liposome solution.
　（２）pDNAとカチオン性ポリマーの複合体(polyplex)の作製
　バイアル瓶内で注射用水に溶解したpDNAと様々なカチオン性の高分子(PEI, PLA, PLL, PS)を表１～４に示す適当な割合で混合し、室温で15分間インキュベートすることで、表面がカチオン性を帯びたpDNAとカチオン性ポリマーの複合体（polyplex）を調製した。 (2) Preparation of pDNA and cationic polymer complex (polyplex) Tables 1 to 4 show pDNA dissolved in water for injection in a vial and various cationic polymers (PEI, PLA, PLL, PS). By mixing at an appropriate ratio and incubating at room temperature for 15 minutes, a polyplex of pDNA having a cationic surface on the surface and a cationic polymer was prepared.
（３）アニオン性ナノバブルポリ－リポ・プレックスの作製(図１)
　上記（２）で得られたpolyplexを上記（１）で得られたアニオン性リポソームと表１～４に示す適切な割合で混合し、さらに15分間インキュベートした。次に、10倍濃度のPBS(リン酸緩衝生理食塩水)を用いて浸透圧を調整した。バイアル瓶内にパーフルオロプロパンガスを封入し、バイアル瓶を密封した。さらに、7.5mLのパーフルオロプロパンガスを用いてバイアル内を加圧し、バス型ソニケーターを用いて5分間超音波処理することで本発明のアニオン性ナノバブルポリ－リポ・プレックスを構築した (3) Preparation of anionic nanobubble poly-lipo plexes (Figure 1)
The polyplex obtained in the above (2) was mixed with the anionic liposome obtained in the above (1) at an appropriate ratio shown in Tables 1 to 4, and further incubated for 15 minutes. Next, the osmotic pressure was adjusted using 10 times concentration of PBS (phosphate buffered saline). Perfluoropropane gas was sealed in the vial and the vial was sealed. Furthermore, the inside of the vial was pressurized with 7.5 mL of perfluoropropane gas, and the anionic nanobubble poly-lipoplex of the present invention was constructed by sonicating for 5 minutes using a bath sonicator.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000004
　PEIとPLL, PLAについてはpDNAのリン酸基とカチオン性高分子のアミノ基、DSPGのリン酸基の比を用いて複合体を表記した。一方で、PSに関しては分子中のアミノ基の正確な量が不明なため、pDNAとPS, ALの質量比で複合体を表記した。すなわち、pDNA/PEI/AL複合体の1:8:3とはpDNAのリン酸基とカチオン性高分子のアミノ基、DSPGのリン酸基の比が1:8:3となるように各成分を混合した物である。様々な組成の複合体の粒子径とζ電荷をゼーターサイザーナノ（マルバーン社）を用いて測定した。ALの添加によってζ電位が添加量依存的に減少し、プラトーに達した。特記しない場合はこのプラトーに達した比率を用いて複合体を調製した。すなわち、pDNA/PEI/AL複合体は1:8:3, pDNA/PLL/AL複合体は1:4:3, pDNA/PLA/AL複合体は1:4:4, pDNA/PS/AL複合体は1:1.25:2.5の割合で調製した。 For PEI, PLL, and PLA, the complex was described using the ratio of the phosphate group of pDNA to the amino group of the cationic polymer and the phosphate group of DSPG. On the other hand, for PS, the exact amount of amino groups in the molecule is unknown, so the complex was expressed by the mass ratio of pDNA to PS and AL. In other words, 1: 8: 3 of the pDNA / PEI / AL complex means that the ratio of the phosphate group of pDNA to the amino group of the cationic polymer and the phosphate group of DSPG is 1: 8: 3. Is a mixed product. The particle size and ζ charge of composites with various compositions were measured using Zeta Sizer Nano (Malvern). With the addition of AL, the ζ potential decreased depending on the amount added and reached a plateau. Unless otherwise stated, complexes were prepared using the ratio that reached this plateau. That is, pDNA / PEI / AL complex is 1: 8: 3, pDNA / PLL / AL complex is 1: 4: 3, pDNA / PLA / AL complex is 1: 4: 4, pDNA / PS / AL complex The body was prepared at a ratio of 1: 1.25: 2.5.
　（４）複合体の外観(図２)
　表１～４に示される本発明のアニオン性バブルリポソーム複合体のバブル封入前後の外観を光学的に観察した結果を図２に示す。図２に示されるように、バブル(パーフルオロプロパンガス)の封入前のリポソーム複合体はほぼ透明であるが、バブルの封入により白濁した。 (4) Appearance of the composite (Figure 2)
FIG. 2 shows the result of optical observation of the appearance of the anionic bubble liposome complex of the present invention shown in Tables 1 to 4 before and after enclosing the bubble. As shown in FIG. 2, the liposome complex before encapsulation of bubbles (perfluoropropane gas) was almost transparent, but became cloudy due to encapsulation of bubbles.
　（５）複合体の安定性(図３)
　上記で得られた本発明のアニオン性ナノバブルポリ－リポ・プレックスの安定性をアガロースゲル電気泳動により評価した。 (5) Stability of the composite (Figure 3)
The stability of the anionic nanobubble poly-lipoplex of the present invention obtained above was evaluated by agarose gel electrophoresis.
　すなわち、1%アガロースゲルに各複合体をpDNAとして1μgアプライし、100Vで30分間電気泳動した。核酸はGelRedTM Nucleic Acid Gel Stain を用いて染色し、Image Quant LAS4000 (GEヘルスケア社)を用いて測定した。結果を図３に示す。 That is, 1 μg of each complex as pDNA was applied to a 1% agarose gel and electrophoresed at 100 V for 30 minutes. Nucleic acids were stained using GelRed ™ Nucleic Acid Gel Stain and measured using Image Quant LAS4000 (GE Healthcare). The results are shown in FIG.
　図３に示されるように、本発明のアニオン性ナノバブルポリ－リポ・プレックスは、アガロースゲル電気泳動によってもpDNA或いはpDNAとカチオン性ポリマーの複合体(polyplex)は外れることはなく、安定であることが示された。 As shown in FIG. 3, the anionic nanobubble poly-lipo plexes of the present invention are stable because pDNA or a complex of pDNA and cationic polymer is not removed even by agarose gel electrophoresis. It has been shown.
　（６）遺伝子導入の効果(図４－１～図４－３)
　ホタルルシフェラーゼを発現するpCMV-LucをpDNAとして用いた。ICR系雌性マウスに上記で得られた各pDNAとカチオン性ポリマー（PEI、PLL又はPS）の複合体(polyplex；pDNA/PEI、pDNA/PLL又はpDNA/PS)、アニオン性リポソーム複合体（pDNA/PEI/AL、pDNA/PLL/AL又はpDNA/PS/AL）、アニオン性ナノバブルポリ－リポ・プレックス（pDNA/PEI/AL＋bubble、pDNA/PLL/AL＋bubble又はpDNA/PS/AL＋bubble）をDNA量が50 μgとなるように投与した。US(+)群（超音波照射群）では、投与から5分後にSonnopore-4000 sonicator（ネッパジーン社）を用いて直径20mmのプローブを利用して体外から腹部に対して超音波(frequency, 1.045 MHz; duty, 50%; burst rate, 10 Hz; intensity, 1.0 W/cm2)を2分間照射した。複合体投与6時間後に各臓器を摘出し、臓器中のルシフェラーゼ発現量をLuc assayによって評価した。結果を図４－１～図４－３に示す。 (6) Effects of gene transfer (Figs. 4-1 to 4-3)
PCMV-Luc expressing firefly luciferase was used as pDNA. In ICR female mice, the complex of each pDNA obtained above and a cationic polymer (PEI, PLL or PS) (polyplex; pDNA / PEI, pDNA / PLL or pDNA / PS), an anionic liposome complex (pDNA / PEI / AL, pDNA / PLL / AL or pDNA / PS / AL), anionic nanobubble poly-lipoplex (pDNA / PEI / AL + bubble, pDNA / PLL / AL + bubble or pDNA / PS / AL + bubble) with a DNA amount of 50 μg It administered so that it might become. In the US (+) group (ultrasound irradiation group), 5 minutes after administration, ultrasound (frequency, 1.045 MHz) was applied to the abdomen from the outside of the body using a Sonnopore-4000 sonicator (Neppa Gene) using a 20 mm diameter probe. duty, 50%; burst rate, 10 Hz; intensity, 1.0 W / cm 2 ) for 2 minutes. Each organ was removed 6 hours after administration of the complex, and the expression level of luciferase in the organ was evaluated by Luc assay. The results are shown in FIGS. 4-1 to 4-3.
　pDNAとカチオン性ポリマーの複合体(polyplex)及びアニオン性リポソーム複合体では、超音波照射の有無（US(+)群とUS(-)群）でpDNAの導入効果に実質的な差異はないが、アニオン性ナノバブルポリ－リポ・プレックスでは超音波照射の有無（US(+)群とUS(-)群）でpDNAの導入効果に有意な差があった。特に、カチオン性ポリマーにプロタミン(PS)を用いた場合、polyplex（pDNA/PS）とアニオン性リポソーム複合体（pDNA/PS/AL）は、ほとんどpDNAの遺伝子導入効果はないが、アニオン性バブルリポソーム複合体のみ超音波照射群（US(+)群）で特異的なpDNAの遺伝子導入効果が得られることが明らかになった（図４-３)。従って、アニオン性バブルリポソーム複合体を生体内に投与し、特定の部位に超音波を照射することで、超音波の照射箇所に特異的に核酸、薬物などを導入可能である。 In pDNA and cationic polymer complex (polyplex) and anionic liposome complex, there is no substantial difference in pDNA introduction effect with or without ultrasound irradiation (US (+) group and US (-) group) In anionic nanobubble poly-lipoplexes, there was a significant difference in the pDNA introduction effect with and without ultrasonic irradiation (US (+) group and US (-) group). In particular, when protamine (PS) is used as the cationic polymer, polyplex (pDNA / PS) and anionic liposome complex (pDNA / PS / AL) have almost no pDNA gene transfer effect, but anionic bubble liposomes. It was revealed that a pDNA gene introduction effect specific to the complex-only ultrasonic irradiation group (US (+) group) was obtained (FIG. 4-3). Therefore, by administering the anionic bubble liposome complex in a living body and irradiating a specific site with ultrasonic waves, a nucleic acid, a drug, or the like can be specifically introduced into the ultrasonic irradiation site.
　（７）肝臓への遺伝子導入(図５)
　図５に示すプロトコールに従い、pDNA/PS/AL + BubbleをICR系雌性マウスへ尾静脈内投与した。図４－３と同様の方法で超音波を照射した。複合体の投与から6時間後にマウスの肝臓をコラゲナーゼ処理し、肝実質細胞（Parenchymal cell）と肝非実質細胞(Non parenchymal cell)を得た。106個の細胞当たりのルシフェラーゼ活性を測定し、遺伝子発現細胞を確認した。結果を図５に示す。 (7) Gene transfer to the liver (Fig. 5)
According to the protocol shown in FIG. 5, pDNA / PS / AL + Bubble was administered into the ICR female mouse via the tail vein. Ultrasonic waves were irradiated in the same manner as in FIG. 4-3. Six hours after administration of the complex, the liver of the mouse was treated with collagenase to obtain hepatic parenchymal cells and non-parenchymal cells. The luciferase activity per 10 6 cells was measured to confirm the gene expression cells. The results are shown in FIG.
　図５に示されるように、本発明のアニオン性ナノバブルポリ－リポ・プレックス（pDNA/PS/AL + Bubble）は、肝臓の非実質細胞に多く導入されることが明らかになった。 As shown in FIG. 5, it was revealed that anionic nanobubble poly-lipoplexes (pDNA / PS / AL + Bubble) of the present invention are introduced in a large amount into nonparenchymal cells of the liver.
　（８）脾臓への遺伝子導入(図６)
　図６に示すプロトコールに従い、図５と同様の方法でpDNA/PS/AL + BubbleをICR系雌性マウスへ投与し、超音波を照射した。遺伝子を導入したマウスの脾臓を分散させ、脾細胞を得た。脾細胞を磁気分離システム（Robosep）を用いてCD11c陽性細胞（樹状細胞）と陰性細胞に分離し、mRNAを抽出した。各細胞中のルシフェラーゼmRNA発現量をグリセルアルデヒド三リン酸デヒドロゲナーゼ(GAPDH)発現量で除した値を比較した。結果を図６に示す。 (8) Gene transfer into the spleen (Figure 6)
In accordance with the protocol shown in FIG. 6, pDNA / PS / AL + Bubble was administered to ICR female mice in the same manner as in FIG. The spleen of the mouse into which the gene was introduced was dispersed to obtain spleen cells. Spleen cells were separated into CD11c positive cells (dendritic cells) and negative cells using a magnetic separation system (Robosep), and mRNA was extracted. The value obtained by dividing the expression level of luciferase mRNA in each cell by the expression level of glyceraldehyde triphosphate dehydrogenase (GAPDH) was compared. The results are shown in FIG.
　図６に示されるように、本発明のアニオン性ナノバブルポリ－リポ・プレックス（pDNA/PS/AL + Bubble）は、樹状細胞への遺伝子導入は少なく、他の脾臓細胞へ特異的に導入されることが明らかになった。 As shown in FIG. 6, the anionic nanobubble poly-lipoplex (pDNA / PS / AL + Bubble) of the present invention has little gene transfer into dendritic cells and is specifically introduced into other spleen cells. It became clear.
　（９）標的化された細胞への遺伝子導入(図７)
　NGR, RGDペプチドとARA(Scramble)ペプチドを固相合成法を用いて調製した。このペプチドとNHS-PEG2000-DSPEを混合し、PEG鎖の末端をペプチドリガンドで標識したPEG-DSPEを合成した(図７)。 (9) Gene transfer to targeted cells (Figure 7)
NGR, RGD peptide and ARA (Scramble) peptide were prepared using solid phase synthesis method. This peptide was mixed with NHS-PEG2000-DSPE to synthesize PEG-DSPE in which the end of the PEG chain was labeled with a peptide ligand (FIG. 7).
　In vitro実験にはヒト血管内皮系細胞EAhy926を用いた。図７に示すプロトコールに従い、48well plateで24時間前培養した細胞に各ペプチドリガンドで標識したpDNA/PS/AL + Bubbleを10μg/wellとなるように添加した。 添加から10分後にSonnopore-4000 sonicatorを用いて超音波(frequency, 2.035 MHz; duty, 50%; burst rate, 10 Hz; intensity, 6.0 W/cm2)を30秒間照射した。照射から24時間後にルシフェラーゼアッセイを行って遺伝子発現量を測定した。結果を図７に示す。 In human experiments, human vascular endothelial cells EAhy926 were used. According to the protocol shown in FIG. 7, pDNA / PS / AL + Bubble labeled with each peptide ligand was added to cells pre-cultured in 48 well plate for 24 hours so that the concentration was 10 μg / well. Ten minutes after the addition of soot, ultrasonic waves (frequency, 2.035 MHz; duty, 50%; burst rate, 10 Hz; intensity, 6.0 W / cm2) were irradiated for 30 seconds using a Sonnopore-4000 sonicator. 24 hours after irradiation, luciferase assay was performed to measure gene expression level. The results are shown in FIG.
　図７に示すようにNGRペプチド, RGDペプチドなどを本発明のアニオン性ナノバブルポリ－リポ・プレックスに導入することにより、遺伝子導入の選択性がより向上することが明らかになった。 As shown in FIG. 7, it has been clarified that introduction of NGR peptide, RGD peptide or the like into the anionic nanobubble poly-lipoplex of the present invention further improves the gene transfer selectivity.
　（１０）超音波造影と崩壊の確認(図８)
　本発明のアニオン性ナノバブルポリ－リポ・プレックス（pDNA/PS/AL + Bubble）の超音波造影効果を解析した。VEVO2100システム（プライムテック社）を用いてチューブに注入したpDNA/PS/AL + Bubbleの超音波造影画像を得た。また、この画像撮影中にfrequency, 3 MHz; duty, 100; intensity, 2.0 W/cm2の超音波を照射し、pDNA/PS/AL + Bubbleを破壊した(図８)。図８に示されるように、本発明のアニオン性ナノバブルポリ－リポ・プレックスは、超音波照射により複合体中のバブルがはじけて複合体が破壊されることが確認された。 (10) Confirmation of ultrasound contrast and collapse (Fig. 8)
The ultrasonic contrast effect of the anionic nanobubble poly-lipoplex (pDNA / PS / AL + Bubble) of the present invention was analyzed. An ultrasonic contrast image of pDNA / PS / AL + Bubble injected into the tube was obtained using VEVO2100 system (Primetech). Further, during this image photographing, ultrasonic waves of frequency, 3 MHz; duty, 100; intensity, 2.0 W / cm 2 were irradiated to destroy the pDNA / PS / AL + Bubble (FIG. 8). As shown in FIG. 8, it was confirmed that the anionic nanobubble poly-lipo plexes of the present invention were broken by the bubbles in the complex due to ultrasonic irradiation.
　（１１）siRNAを用いたsiRNA/PS/DSPG複合体の調製(図９)
　図２～３と同様の検討を、核酸として配列番号１，２で表されるホタルルシフェラーゼに対するsiRNAで行った。表５で示すsiRNAとPS, ALの様々な質量比で調製した複合体の粒子径とζ電位を測定し、1:2:5の複合体にパーフルオロプロパンガスを封入した。また、アガロースゲル電気泳動で安定性を評価した。結果を図９に示す。 (11) Preparation of siRNA / PS / DSPG complex using siRNA (FIG. 9)
Studies similar to those in FIGS. 2 to 3 were performed using siRNA against firefly luciferase represented by SEQ ID NOs: 1 and 2 as nucleic acids. The particle diameter and ζ potential of the complexes prepared at various mass ratios of siRNA and PS and AL shown in Table 5 were measured, and perfluoropropane gas was sealed in the 1: 2: 5 complex. In addition, stability was evaluated by agarose gel electrophoresis. The results are shown in FIG.
Figure JPOXMLDOC01-appb-T000005
Figure JPOXMLDOC01-appb-T000005
　siRNAを核酸として含む本発明のアニオン性ナノバブルポリ－リポ・プレックス（siRNA/PS/AL + Bubble）は、pDNAを含む複合体と同様に安定であり、バブルがアニオン性リポソーム内に封入されていることが明らかになった。 The anionic nanobubble poly-lipoplex (siRNA / PS / AL + Bubble) of the present invention containing siRNA as a nucleic acid is as stable as a complex containing pDNA, and the bubble is encapsulated in an anionic liposome. It became clear.
　（１２）mRNAを用いたmRNA/PS/DSPG複合体の調製(図１０)
　pDNAに代えてホタルルシフェラーゼのmRNAを用いて上記と同様にして、本発明のアニオン性ナノバブルポリ－リポ・プレックス（mRNA/PS/AL + Bubble）を構築し、図３と同様な方法により安定性を評価した。結果を図１０に示す。 (12) Preparation of mRNA / PS / DSPG complex using mRNA (FIG. 10)
The anionic nanobubble poly-lipoplex (mRNA / PS / AL + Bubble) of the present invention was constructed in the same manner as described above using firefly luciferase mRNA instead of pDNA, and stabilized by the same method as in FIG. Evaluated. The results are shown in FIG.
　図１０に示されるように、本発明の複合体は、アガロースゲル電気泳動でもmRNAを放出することはなく、安定な複合体であることが明らかになった。 As shown in FIG. 10, it was revealed that the complex of the present invention was a stable complex without releasing mRNA even by agarose gel electrophoresis.
　（１３）薬剤封入アニオン性リポソームの調製(図１１)
　DSPG, DSPC, PEG-DSPE, ヘマトポルフィリンのクロロホルム溶液を各成分のモル比が7:2:1:5の割合になるように混合し、ALと同様の方法でヘマトポルフィリン内包リポソームを調製した。ヘマトポルフィリンの封入率は99.2%であった。 (13) Preparation of drug-encapsulated anionic liposome (FIG. 11)
DSPG, DSPC, PEG-DSPE and hematoporphyrin in chloroform were mixed so that each component had a molar ratio of 7: 2: 1: 5, and hematoporphyrin-encapsulated liposomes were prepared in the same manner as AL. The encapsulation rate of hematoporphyrin was 99.2%.
　このヘマトポルフィリン内包リポソームにパーフルオロプロパンガスを内封し、光学的に観察した（図１１（A））。 This hematoporphyrin-encapsulated liposome was encapsulated with perfluoropropane gas and optically observed (FIG. 11 (A)).
　作製した脂質薄膜を0.3 Mのクエン酸溶液で水和し、リポソームを作製した。リポソームの脂質としてDSPG, DSPC, PEG-DSPEを用いた。 The prepared lipid thin film was hydrated with a 0.3M citric acid solution to prepare liposomes. DSPG, DSPC, and PEG-DSPE were used as liposome lipids.
　さらに、ゲル濾過によって外水層を20 mMのHEPES含有5%グルコース溶液（pH=7.5）に置換した。このリポソーム溶液にドキソルビシンを質量比で12.5:1の割合になるように添加し、60℃で30分間インキュベートし、リポソーム内へドキソルビシンを内包した。このときの封入率は97.7%であった。
このドキソルビシン内包リポソームにパーフルオロプロパンガスを内封し、光学的に観察した（図１１（B）） 。 Furthermore, the outer aqueous layer was replaced with 20 mM HEPES-containing 5% glucose solution (pH = 7.5) by gel filtration. Doxorubicin was added to this liposome solution at a mass ratio of 12.5: 1, and the mixture was incubated at 60 ° C. for 30 minutes to encapsulate doxorubicin in the liposome. The encapsulation rate at this time was 97.7%.
Perfluoropropane gas was encapsulated in the doxorubicin-encapsulated liposome and optically observed (FIG. 11 (B)).
　アニオン性リポソームの部分に種々の薬剤が封入できることが明らかになった。 It became clear that various drugs can be encapsulated in the anionic liposome portion.
　実施例２：アニオン性バブルリポソームの作製（２）
　（１）アニオン性リポソーム(AL)の作製
　実施例１と同様にして、アニオン性リポソーム溶液を得た。 Example 2: Preparation of anionic bubble liposome (2)
(1) Preparation of anionic liposome (AL) In the same manner as in Example 1, an anionic liposome solution was obtained.
　（２）pDNAとカチオン性ポリマーの複合体(polyplex)の作製
　バイアル瓶内で注射用水に溶解したpDNAと様々なカチオン性の高分子(PS, PLL, PLA, DPLL)を適当な割合で混合し、室温で15分間インキュベートすることで、表面がカチオン性を帯びたpDNAとカチオン性ポリマーの複合体（polyplex）を調製した。 (2) Preparation of pDNA and cationic polymer complex (polyplex) In a vial, pDNA dissolved in water for injection and various cationic polymers (PS, PLL, PLA, DPLL) are mixed at an appropriate ratio. By incubating for 15 minutes at room temperature, a polyplex of pDNA and cationic polymer having a cationic surface was prepared.
　（３）アニオン性ナノバブルポリ－リポ・プレックスの作製
　上記（２）で得られたpolyplexを上記（１）で得られたアニオン性リポソーム適切な割合で混合し、さらに15分間インキュベートした。
　混合割合（重量比）は、下記の通りとした。pDNA:PLL:AL、pDNA:PLA:AL、及びpDNA:DPLL:ALのそれぞれについては1.0:1.5:17.6、並びに、pDNA:PS:ALについては1.0:1.25:2.5。
　次に、10倍濃度のPBS(リン酸緩衝生理食塩水)を用いて浸透圧を調整した。バイアル瓶内にパーフルオロプロパンガスを封入し、バイアル瓶を密封した。さらに、7.5mLのパーフルオロプロパンガスを用いてバイアル内を加圧し、バス型ソニケーターを用いて5分間超音波処理することで本発明のアニオン性ナノバブルポリ－リポ・プレックスを構築した。 (3) Preparation of anionic nanobubble poly-lipo plex The polyplex obtained in (2) above was mixed at an appropriate ratio of the anionic liposome obtained in (1) above, and further incubated for 15 minutes.
The mixing ratio (weight ratio) was as follows. 1.0: 1.5: 17.6 for each of pDNA: PLL: AL, pDNA: PLA: AL, and pDNA: DPLL: AL, and 1.0: 1.25: 2.5 for pDNA: PS: AL.
Next, the osmotic pressure was adjusted using 10 times concentration of PBS (phosphate buffered saline). Perfluoropropane gas was sealed in the vial and the vial was sealed. Further, the inside of the vial was pressurized with 7.5 mL of perfluoropropane gas, and sonicated for 5 minutes with a bath sonicator, thereby constructing the anionic nanobubble poly-lipoplex of the present invention.
　（４）アニオン性ナノバブルポリ－リポ・プレックスの理化学的性質
　得られた複合体の粒子径とζ電荷をゼーターサイザーナノ（マルバーン社）を用いて測定した。結果を表６に示す。 (4) Physicochemical properties of anionic nanobubble poly-lipo plex The particle size and ζ charge of the obtained composite were measured using Zeta Sizer Nano (Malvern). The results are shown in Table 6.
Figure JPOXMLDOC01-appb-T000006
Figure JPOXMLDOC01-appb-T000006
　（５）複合体の外観（図１３）
　上記（３）で得たアニオン性バブルリポソーム複合体のバブル封入前後の外観を光学的に観察した結果を図１３（ａ）に示す。pDNA/PS/BL複合体の、酢酸ウラニルを用いたネガティブ染色による、TEM観察像を、図１３（ｂ）に示す。 (5) Appearance of composite (FIG. 13)
FIG. 13A shows the result of optical observation of the appearance of the anionic bubble liposome complex obtained in the above (3) before and after bubble encapsulation. FIG. 13B shows a TEM observation image of the pDNA / PS / BL complex by negative staining using uranyl acetate.
　（６）複合体の安定性（図１４）
　上記で得られたアニオン性バブルリポソーム複合体の安定性を、実施例１と同様にしてアガロースゲル電気泳動により評価した。比較対照として、未処理のpDNA（Naked pDNA）も泳動した。 (6) Stability of the composite (FIG. 14)
The stability of the anionic bubble liposome complex obtained above was evaluated by agarose gel electrophoresis in the same manner as in Example 1. As a comparative control, untreated pDNA (Naked pDNA) was also run.
　図１４に示されるように、本発明のアニオン性ナノバブルポリ－リポ・プレックスは、アガロースゲル電気泳動によってもpDNA或いはpDNAとカチオン性ポリマーの複合体(polyplex)は外れることはなく、安定であることが示された。 As shown in FIG. 14, the anionic nanobubble poly-lipo plexes of the present invention are stable because pDNA or a complex of pDNA and cationic polymer is not removed even by agarose gel electrophoresis. It has been shown.
　（７）in vitro遺伝子導入の効果（図１５）
　ヒト静脈内皮細胞EAhy926細胞に上記で得られた複合体を導入した。 (7) Effect of in vitro gene transfer (FIG. 15)
The complex obtained above was introduced into human venous endothelial cells EAhy926 cells.
　複合体は、上記で得られたpDNAとカチオン性ポリマー（PS、PLL、PLA又はDPLL）の複合体(polyplex；pDNA/PS、pDNA/PLL、pDNA/PLA又はpDNA/PS)、アニオン性リポソーム複合体（pDNA/PS/AL、pDNA/PLL/AL、pDNA/PLA/AL又はpDNA/PS/AL）、アニオン性ナノバブルポリ－リポ・プレックス（pDNA/PS/BL、pDNA/PLL/BL、pDNA/PLA/BL又はpDNA/PS/BL）を用いた。なお、「BS」は、本明細書において「AL+bubble」と交換可能に用いられ、バブルを封入したアニオン性バブルリポソームを表す。 The complex is a complex (polyplex; pDNA / PS, pDNA / PLL, pDNA / PLA or pDNA / PS) of the pDNA obtained above and a cationic polymer (PS, PLL, PLA or DPLL), anionic liposome complex Body (pDNA / PS / AL, pDNA / PLL / AL, pDNA / PLA / AL or pDNA / PS / AL), anionic nanobubble poly-lipoplex (pDNA / PS / BL, pDNA / PLL / BL, pDNA / PLA / BL or pDNA / PS / BL) was used. “BS” is used interchangeably with “AL + bubble” in this specification, and represents an anionic bubble liposome encapsulating bubbles.
　EAhy926細胞は、10% FBS（fetal bovine serum）、ペニシリン100 IU/mL、ストレプトマイシン100μg/mL、L-グルタミン2mM、non-essential amino acids 100μMを添加したDMEM培地中で、37℃及び二酸化炭素濃度5%の環境下で維持した。24時間のプレインキュベーション後、各種複合体（pDNA 10μg）を含むOpti-MEM I培地へと培地を交換した。各種複合体を添加から10分後に、EAhy926細胞に超音波(frequency, 2.0 MHz; duty, 50%; burst rate, 10 Hz; intensity, 4.0 W/cm2)を20秒間照射した。超音波照射は、Sonnopore-4000 sonicator（ネッパジーン社）を用いて、直径6mmのプローブを利用して行った。 EAhy926 cells were cultured in DMEM medium supplemented with 10% FBS (fetal bovine serum), penicillin 100 IU / mL, streptomycin 100 μg / mL, L-glutamine 2 mM, non-essential amino acids 100 μM at 37 ° C. and carbon dioxide concentration 5 Maintained in% environment. After pre-incubation for 24 hours, the medium was replaced with Opti-MEM I medium containing various complexes (pDNA 10 μg). Ten minutes after the addition of various complexes, EAhy926 cells were irradiated with ultrasound (frequency, 2.0 MHz; duty, 50%; burst rate, 10 Hz; intensity, 4.0 W / cm 2 ) for 20 seconds. Ultrasonic irradiation was performed using a 6 mm diameter probe using a Sonnopore-4000 sonicator (Neppa Gene).
　その後、培地を培養培地へと交換し、さらに24時間培養を継続した。培養後、細胞をlysis buffer（0.05% Triton X-100, 2 mM EDTA, and 0.1 M Tris; pH 7.8）に懸濁し、細胞中のルシフェラーゼ発現量を評価した。結果を図１５に示す。 Thereafter, the medium was replaced with a culture medium, and the culture was further continued for 24 hours. After culturing, the cells were suspended in lysis buffer (0.05% Triton X-100, 2 mM EDTA, and 0.1 M Tris; pH 7.8), and the expression level of luciferase in the cells was evaluated. The results are shown in FIG.
　pDNAとカチオン性ポリマーの複合体(polyplex)及びアニオン性リポソーム複合体では、超音波照射の有無（US(+)群とUS(-)群）でpDNAの導入効果に実質的な差異はないが、アニオン性ナノバブルポリ－リポ・プレックスでは超音波照射の有無（US(+)群とUS(-)群）でpDNAの導入効果に有意な差があった。また、用いる正電荷高分子の種類は遺伝子導入効果にほとんど影響しなかった。 In pDNA and cationic polymer complex (polyplex) and anionic liposome complex, there is no substantial difference in pDNA introduction effect with or without ultrasound irradiation (US (+) group and US (-) group) In anionic nanobubble poly-lipoplexes, there was a significant difference in the pDNA introduction effect with and without ultrasonic irradiation (US (+) group and US (-) group). Moreover, the kind of positively charged polymer used hardly affected the gene transfer effect.
　（８）pDNAの細胞内局在（図１６）
　フルオレセイン標識したpDNA及びリソソームマーカーであるLysoTracker Red DND-99を用いて、pDNAの細胞内局在を評価した。 (8) Intracellular localization of pDNA (FIG. 16)
The subcellular localization of pDNA was evaluated using fluorescein-labeled pDNA and LysoTracker Red DND-99, which is a lysosomal marker.
　上記と同様にして、フルオレセイン標識したpDNAを用いて調製したpDNA/PS/BL複合体をEAhy926細胞に添加し、超音波照射（US）をした。常法によりpDNAをLipofectamine 2000を用いてpDNAを細胞に導入した群、及び、超音波照射（US）をしない群を対照とした。処理から6時間後、各群をLysoTracker Red DND-99で処理した。結果を図１６に示す。 In the same manner as described above, a pDNA / PS / BL complex prepared using fluorescein-labeled pDNA was added to EAhy926 cells and subjected to ultrasonic irradiation (US). A group in which pDNA was introduced into cells using Lipofectamine 2000 using a conventional method and a group in which ultrasonic irradiation (US) was not performed were used as controls. Six hours after treatment, each group was treated with LysoTracker Red DND-99. The results are shown in FIG.
　Lipofectamine 2000によりトランスフェクションを行った場合、pDNAはリソソームと共局在した（図１６（a））。また、pDNA/PS/BL複合体に超音波を併用しない場合にもpDNAはリソソームと共局在した（図１６(b)）一方、pDNA/PS/BL複合体の添加及び超音波照射（US）をした細胞では、pDNAはリソソームとは局在しなかった（図１６（c））。このことは、アニオン性ナノバブルポリ－リポ・プレックスがエンドサイトーシス経路を介さず、細胞質内へ直接的にpDNAを送達していることを示唆する結果である。 When transfection was performed with Lipofectamine® 2000, pDNA colocalized with lysosomes (FIG. 16 (a)). In addition, pDNA co-localized with lysosomes even when ultrasound was not used in the pDNA / PS / BL complex (FIG. 16 (b)), while addition of pDNA / PS / BL complex and ultrasonic irradiation (US PDNA did not localize to lysosomes in the cells treated with) (FIG. 16 (c)). This is a result suggesting that the anionic nanobubble poly-lipoplex delivers pDNA directly into the cytoplasm without going through the endocytosis pathway.
　（９）細胞毒性試験（図１７）
　WST-1アッセイにより、各複合体の細胞毒性を評価した。上記（７）と同様にして、各種アニオン性ナノバブルポリ－リポ・プレックス（pDNA/PS/BS、pDNA/PLL/BS、pDNA/PLA/BS又はpDNA/PS/BS）を細胞に添加し、超音波照射（US）をした。複合体の添加及び超音波照射をしない群（Control）を、対照群とした。超音波照射（US）のみをした群も実施した。その後、24時間培養を行った後に、 Cell Proliferation Reagent WST-1（Roche Diagnostics Corporation社製）を用いて、対照に対する細胞生存率（Cell viability (% of control)）を評価した。結果を図１７に示す。 (9) Cytotoxicity test (Figure 17)
The cytotoxicity of each complex was evaluated by WST-1 assay. In the same manner as in (7) above, various anionic nanobubble poly-lipoplexes (pDNA / PS / BS, pDNA / PLL / BS, pDNA / PLA / BS, or pDNA / PS / BS) are added to the cells. Sonication (US) was applied. The group not added with the complex and subjected to ultrasonic irradiation (Control) was used as a control group. A group receiving only ultrasonic irradiation (US) was also carried out. Then, after culturing for 24 hours, the cell viability (Cell viability (% of control)) relative to the control was evaluated using Cell Proliferation Reagent WST-1 (Roche Diagnostics Corporation). The results are shown in FIG.
　図１７に示されるように、各種アニオン性ナノバブルポリ－リポ・プレックスは細胞生存率を若干低下させた。一方で、pDNA/PS/BL複合体においては有意な細胞障害性は認められなかった。 As shown in FIG. 17, various anionic nanobubble poly-lipo plexes slightly decreased cell viability. On the other hand, no significant cytotoxicity was observed in the pDNA / PS / BL complex.
　（１０）エリスロサイトの凝集（図１８）
　アニオン性ナノバブルポリ－リポ・プレックスの、エリスロサイトを凝集する活性を評価した。 (10) Aggregation of erythrosite (FIG. 18)
The activity of anionic nanobubble poly-lipoplexes to aggregate erythrocytes was evaluated.
　マウスからエリスロサイト（erythrocyte）を取得し、PBS緩衝液を用いて、4℃における5,000xgでの遠心分離5分間を３回繰り返すことで洗浄し、2%(v/v)のエリスロサイトストック懸濁液を調製した。各種アニオン性ナノバブルポリ－リポ・プレックス（pDNA/PEI/BL、pDNA/PS/BL、pDNA/PLL/BL、pDNA/PLA/BL又はpDNA/PS/BL）をエリスロサイトストック懸濁液に添加し、室温で15分間静置した。pDNA/Lipofectamine 2000（カチオン性リポソーム）の添加、及び、PBSのみの添加を対照とした。ガラスプレートに10μLの試料を滴下し、位相差顕微鏡下で凝集を観察した。結果を図１８に示す。 Erythrocytes are obtained from the mice, washed with PBS buffer solution by centrifuging at 5,000xg at 4 ° C for 5 minutes 3 times, and 2% (v / v) erythrocyte stock suspension. A suspension was prepared. Add various anionic nanobubble poly-lipoplexes (pDNA / PEI / BL, pDNA / PS / BL, pDNA / PLL / BL, pDNA / PLA / BL or pDNA / PS / BL) to the erythrosite stock suspension And left at room temperature for 15 minutes. Addition of pDNA / Lipofectamine 2000 (cationic liposome) and addition of PBS alone served as controls. A 10 μL sample was dropped onto a glass plate, and aggregation was observed under a phase contrast microscope. The results are shown in FIG.
　市販の遺伝子導入用試薬であるLipofectamine 2000では赤血球の溶血と凝集が、遺伝子導入実験に汎用されるpolyethylenimineでは非常に強い凝集が認められたものの、各種アニオン性ナノバブルポリ－リポ・プレックスでは凝集や溶血は認められなかった。 Lipofectamine 2000, a commercially available gene transfer reagent, showed red blood cell hemolysis and aggregation, while polyethylenimine, which is widely used for gene transfer experiments, showed very strong aggregation, but various anionic nanobubble poly-lipo plexes showed aggregation and hemolysis. Was not recognized.
　（１１）in vivo遺伝子導入の効果（図１９）
　ICR系雌性マウスに、上記で得られた各種アニオン性ナノバブルポリ－リポ・プレックス（pDNA/PS/BL、pDNA/PLL/BL、pDNA/PLA/BL又はpDNA/PS/BL）を、pDNA（pCMV-Luc）量が50 μgとなるように、溶液400μLを26ゲージの注射針を用いて尾静脈内投与した。US(+)群（超音波照射群）では、投与直後にSonnopore-4000 sonicator（ネッパジーン社）を用いて直径20mmのプローブを利用して体外から腹部に対して超音波(frequency, 1.0 MHz; duty, 50%; burst rate, 10 Hz; intensity, 0.5 W/cm2)を１分間照射した。複合体投与から6時間後に各臓器（肝臓、腎臓、脾臓）を摘出し、臓器中のルシフェラーゼ発現量をLuc assayによって評価した。結果を図１９に示す。
各種アニオン性ナノバブルポリ－リポ・プレックスは超音波と併用しなかった場合には遺伝子導入効果がほとんど認められなかったものの、超音波と併用する事で非常に高い遺伝子導入効果が確認された。一方で、用いる正電荷高分子の種類は遺伝子導入効果に大きな影響を及ぼさなかった。 (11) Effect of in vivo gene transfer (FIG. 19)
ICR female mice were treated with the various anionic nanobubble poly-lipoplexes (pDNA / PS / BL, pDNA / PLL / BL, pDNA / PLA / BL or pDNA / PS / BL) obtained above. -Luc) 400 μL of the solution was administered into the tail vein using a 26 gauge injection needle so that the amount was 50 μg. In the US (+) group (ultrasonic irradiation group), immediately after administration, ultrasound (frequency, 1.0 MHz; duty) was applied to the abdomen from outside the body using a Sonnopore-4000 sonicator (Neppa Gene) using a 20 mm diameter probe. 50%; burst rate, 10 Hz; intensity, 0.5 W / cm 2 ) for 1 minute. Each organ (liver, kidney, spleen) was removed 6 hours after administration of the complex, and the expression level of luciferase in the organ was evaluated by Luc assay. The results are shown in FIG.
Various anionic nanobubble poly-lipo plexes showed almost no gene transfer effect when not used in combination with ultrasound, but a very high gene transfer effect was confirmed when used in combination with ultrasound. On the other hand, the type of positively charged polymer used did not have a significant effect on the gene transfer effect.
　（１２）アニオン性脂質の効果の検証（図２０）
　脂質としてDSPG、DSPA又はDSPSを用いて、上記と同様に各種pDNA/PS/BLを構築した。 (12) Verification of the effect of anionic lipid (FIG. 20)
Various kinds of pDNA / PS / BL were constructed in the same manner as described above using DSPG, DSPA or DSPS as lipids.
　得られた複合体を上記と同様の手法により、EAhy926細胞に各複合体を導入し、細胞中のルシフェラーゼ発現量を評価した。結果を図２０（a）に示す。 Each complex was introduced into EAhy926 cells by the same method as described above, and the expression level of luciferase in the cells was evaluated. The results are shown in FIG.
　また、得られた複合体を上記と同様の手法により、マウスへpDNA/PS/BLを尾静脈内投与し、投与直後に腹部に対して超音波照射をした。複合体投与から6時間後に各臓器（肝臓、腎臓、脾臓）を摘出し、臓器中のルシフェラーゼ発現量をLuc assayによって評価した。結果を図２０（b）に示す。 Further, the obtained complex was administered to the mouse by pDNA / PS / BL in the tail vein by the same method as described above, and the abdomen was irradiated with ultrasonic waves immediately after the administration. Each organ (liver, kidney, spleen) was removed 6 hours after administration of the complex, and the expression level of luciferase in the organ was evaluated by Luc に よ っ て assay. The results are shown in FIG.
　さらに、得られた複合体について、上記と同様の手法により、WST-1アッセイによる各複合体の細胞毒性を評価した。結果を図２０（c）に示す。
DSPAを用いたpDNA/PS/BLはDSPGやDSPSを用いたpDNA/PS/BLと比較して遺伝子導入効果が大きく減少した。一方で、DSPGを用いたpDNA/PS/BLは最も高い遺伝子導入効果を示しながら、細胞障害性が認められなかった。以上より、BLを構成するアニオン性脂質としてDSPGが最適であることが示された。 Furthermore, the cytotoxicity of each complex by the WST-1 assay was evaluated for the obtained complex by the same method as described above. The results are shown in FIG.
PDNA / PS / BL using DSPA greatly reduced the gene transfer effect compared to pDNA / PS / BL using DSPG and DSPS. On the other hand, pDNA / PS / BL using DSPG showed the highest gene transfer effect, but no cytotoxicity was observed. From the above, it was shown that DSPG is optimal as an anionic lipid constituting BL.
　（１３）pDNA/PS/BL複合体の体内動態（図２１）
　32P標識したpDNAを用いて構築したpDNA/PS/BL複合体を用いる以外は上記と同様にして、マウスへpDNA/PS/BLを尾静脈内投与し、投与直後に腹部に対して超音波照射をした。投与から所定時間（0.25、0.5、1、3及び6時間）後に、ペントバルビタール麻酔下での大動脈からの採血、並びに、肝臓、腎臓、脾臓、心臓及び肺の各臓器を摘出した。摘出された臓器は生理食塩水で洗浄後、重量を計測した。血液及び各臓器をSoluene-350中で60℃において溶解し、得られたライセートをイソプロパノール及び30%H2O2で脱色処理、その後5N HCLで中和した。シンチレーションカウンターを用いて、32P標識pDNAの放射線強度を測定した。尾静脈内投与後の所定時間経過後（Time after intravenous administtration）における、血液中及び各臓器に取り込まれた32P標識したpDNAの投与した全量の32P標識したpDNAに対する割合（% of dose）を評価した。結果を図２１に示す。 (13) Pharmacokinetics of pDNA / PS / BL complex (Figure 21)
Except for using pDNA / PS / BL complex constructed using 32 P-labeled pDNA, pDNA / PS / BL was administered into mice via the tail vein in the same manner as described above. Irradiated. After a predetermined time (0.25, 0.5, 1, 3 and 6 hours) after administration, blood was collected from the aorta under pentobarbital anesthesia, and the liver, kidney, spleen, heart and lung organs were removed. The excised organ was washed with physiological saline and weighed. The blood and each organ were dissolved in Soluene-350 at 60 ° C., and the resulting lysate was decolorized with isopropanol and 30% H 2 O 2 and then neutralized with 5N HCL. The radiation intensity of 32 P-labeled pDNA was measured using a scintillation counter. The percentage of the total amount of 32 P-labeled pDNA administered into the blood and each organ after the prescribed time after intravenous administration (Time after intravenous administration) to the 32 P-labeled pDNA (% of dose) evaluated. The results are shown in FIG.
　pDNA/PS/BLは静脈内投与後に速やかに血液中から消失し、肝臓にそのほとんどが取り込まれることが明らかになった。一方で、超音波の照射の有無（US(+)又はUS(-)）によってpDNA/PS/BLの体内動態はほとんど影響を受けなかった。 It was revealed that pDNA / PS / BL disappeared rapidly from the blood after intravenous administration and most of it was taken up by the liver. On the other hand, the pharmacokinetics of pDNA / PS / BL was hardly affected by the presence or absence of ultrasonic irradiation (US (+) or US (-)).
　（１４）pDNA/PS/BL複合体の肝臓への遺伝子導入（図２２）
　上記と同様にして、マウスへpDNA/PS/BL（pDNA量50 μg）を尾静脈内投与した。投与直後に腹部又は胸部への超音波照射を上記と同様の条件で行った。 (14) Gene transfer into the liver of pDNA / PS / BL complex (FIG. 22)
In the same manner as described above, pDNA / PS / BL (pDNA amount 50 μg) was administered into mice via the tail vein. Immediately after administration, ultrasonic irradiation of the abdomen or chest was performed under the same conditions as described above.
　実施例１と同様の手法で、投与から6時間後にマウスの肝臓をコラゲナーゼ処理し、肝実質細胞（Parenchymal cell）と肝非実質細胞(Non parenchymal cell)を得た。106個の細胞当たりのルシフェラーゼ活性（Luciferase activities (pg/106 cells)）を測定し、遺伝子発現細胞を確認した。結果を図２２（a）に示す。 In the same manner as in Example 1, the liver of the mouse was treated with collagenase 6 hours after administration to obtain liver parenchymal cells and non-parenchymal cells. The luciferase activity per 10 6 cells (Luciferase activities (pg / 106 cells)) was measured to confirm the gene-expressing cells. The results are shown in FIG.
　さらに、得られた肝非実質細胞から免疫磁気 ビーズシステム（immunomagnetic cell isolation system）により、クッパー細胞（Kupffer cell）及び内皮細胞（endothelial cell）を分離した。リアルタイムRT-PCR法により、対照mRNA（GAPDH）に対する、ルシフェラーゼmRNAの相対量（Relative luciferase mRNA expressions (luciferase mRNA/GAPDH mRNA)）を測定した。結果を図２２（b）に示す。 Furthermore, Kupffer cells and endothelial cells were separated from the obtained liver non-parenchymal cells by an immunomagnetic cell bead system. The relative amount of luciferase mRNA (Relative luciferase mRNA expressions (luciferase mRNA / GAPDH mRNA)) relative to the control mRNA (GAPDH) was measured by real-time RT-PCR. The results are shown in FIG.
　図２２（a）に示されるように、本発明のアニオン性ナノバブルポリ－リポ・プレックス（pDNA/PS/BL）は、肝臓の非実質細胞に多く導入されることが明らかになった。図２２（b）に示されるように、非実質細胞のうちではクッパー細胞及び内皮細胞に同程度導入されることが明らかとなった。pDNA/PS/BLの粒子径は500nm程度と肝臓のフェネストラを通過するには大きかったものと推察される。 As shown in FIG. 22 (a), it was clarified that the anionic nanobubble poly-lipoplex (pDNA / PS / BL) of the present invention is often introduced into non-parenchymal cells of the liver. As shown in FIG. 22 (b), it was revealed that non-parenchymal cells were introduced to the same extent into Kupffer cells and endothelial cells. The particle size of pDNA / PS / BL is estimated to be about 500 nm, which was large to pass through the liver fenestra.
　（１５）pDNA/PS/BL複合体による肝臓障害の評価（図２３）
　pDNA（pCMV-Luc）量を25 μgとした以外は上記と同様にして、マウスへpDNA/PS/BLを尾静脈内投与し、投与直後に腹部に対して超音波照射をした。投与から所定時間（6、12、及び24時間）後に、ペントバルビタール麻酔下での大動脈からの採血をした。対照として、文献（Liu F, Song Y, Liu D. Hydrodynamics-based transfection in animals by systemic administration of plasmid DNA. Gene Ther. 1999;6:1258-66.）に記載の方法に準じて、hydrodynamic transfectionにより2.5mlの生理食塩水に溶解したpDNAを5秒以内に尾静脈内投与した。 (15) Evaluation of liver damage by pDNA / PS / BL complex (FIG. 23)
Mice were administered pDNA / PS / BL into the tail vein in the same manner as described above except that the amount of pDNA (pCMV-Luc) was 25 μg, and the abdomen was irradiated with ultrasonic waves immediately after administration. Blood was collected from the aorta under pentobarbital anesthesia at predetermined times (6, 12, and 24 hours) after administration. As a control, hydrodynamic transfection was performed according to the method described in the literature (Liu F, Song Y, Liu D. Hydrodynamics-based transfection in animals by systemic administration of plasmid DNA. Gene Ther. 1999; 6: 1258-66.). PDNA dissolved in 2.5 ml of physiological saline was administered into the tail vein within 5 seconds.
　得られた血清中のAST（アスパラギン酸アミノトランスフェラーゼ、Asparatate transaminase）及びALT（アラニンアミノトランスフェラーゼ、Alanine transaminase）の活性を、Transaminase CII-Test Wako Kit（和光純薬社製）を用いて評価した。結果を図２３に示す。 The activities of AST (aspartate aminotransferase, Asparatate transaminase) and ALT (alanine aminotransferase, Alanine transaminase) in the obtained serum were evaluated using Transaminase® CII-Test® Wako® Kit (manufactured by Wako Pure Chemical Industries, Ltd.). The results are shown in FIG.
　図２３に示すように、pDNA/PS/BL複合体（Bubble lipopolyplex）の投与及び超音波照射（US(+)）は、肝臓障害の指標となるAST及びALT活性の上昇をほとんど生じさせない。このことは、アニオン性ナノバブルポリ－リポ・プレックスの肝毒性は極めて低い事を示している。 As shown in FIG. 23, administration of pDNA / PS / BL complex (Bubble lipopolyplex) and ultrasonic irradiation (US (+)) hardly cause an increase in AST and ALT activities that are indicators of liver damage. This indicates that the hepatotoxicity of anionic nanobubble poly-lipoplexes is extremely low.
　（１６）pDNA/PS/BL複合体による炎症性サイトカイン分泌の評価（図２４）
　pDNA（pCMV-Luc）量を25 μgとした以外は上記と同様にして、マウスへpDNA/PS/BLを尾静脈内投与し、投与直後に腹部に対して超音波照射をした。投与から所定時間（6、12、及び24時間）後に、ペントバルビタール麻酔下での大動脈からの採血をした。対照として、文献（Ito Y, Kawakami S, Charoensit P, Higuchi Y, Hashida M. Evaluation of proinflammatory cytokine production and liver injury induced by plasmid DNA/cationic liposome complexes with various mixing ratios in mice. Eur J Pharm Biopharm. 2009;71:303-9.に記載の方法に準じてpDNA-DOTAP/コレステロール複合体（DOTAP/Cholリポプレックス、DOTAP/Chol lipoplex）を構築した、これを投与した。pDNA-DOTAP/コレステロール複合体は、pDNAとDOTAP/Cholリポソームのチャージ比（-:+）が1.0:2.3となるように、２成分を混合した。 (16) Evaluation of inflammatory cytokine secretion by pDNA / PS / BL complex (FIG. 24)
Mice were administered pDNA / PS / BL into the tail vein in the same manner as described above except that the amount of pDNA (pCMV-Luc) was 25 μg, and the abdomen was irradiated with ultrasonic waves immediately after administration. Blood was collected from the aorta under pentobarbital anesthesia at predetermined times (6, 12, and 24 hours) after administration. As a control, the literature (Ito Y, Kawakami S, Charoensit P, Higuchi Y, Hashida M. Evaluation of proinflammatory cytokine production and liver injury induced by plasmid DNA / cationic transcript complexes with various mixing ratios in mice.Eur J Pharm Biopharm. 2009; 71: 303-9. A pDNA-DOTAP / cholesterol complex (DOTAP / Chol lipoplex, DOTAP / Chol lipoplex) was constructed and administered according to the method described in 71: 303-9. The two components were mixed so that the charge ratio (-: +) of pDNA and DOTAP / Chol liposome was 1.0: 2.3.
　得られた血清中のTNF-α及びIL-6のレベル（Serum TNF-α concentrarion, Serum IL-6 concentrarion）をBD OptEIA ELISA Set（BD社製）を用いて測定した。結果を、図２４に示す。 The levels of TNF-α and IL-6 (Serum TNF-α concentrarion, Serum IL-6 concentrarion) in the obtained serum were measured using a BD OptEIA ELISA set (manufactured by BD). The results are shown in FIG.
　図２４に示すように、pDNA/PS/BL複合体（Bubble lipopolyplex）の投与及び超音波照射（US(+)）は、炎症の原因となるTNF-α及びIL-6のレベルの上昇を生じさせない。このことは、アニオン性ナノバブルポリ－リポ・プレックスは投与後に炎症の原因とならない事を示している。 As shown in FIG. 24, administration of pDNA / PS / BL complex (Bubble lipopolyplex) and ultrasonic irradiation (US (+)) resulted in increased levels of TNF-α and IL-6 that cause inflammation. I won't let you. This indicates that anionic nanobubble poly-lipoplexes do not cause inflammation after administration.
　（１７）腹部又は胸部への超音波照射の評価（図２５）
　上記と同様にして、マウスへpDNA/PS/BL（pDNA量50 μg）を尾静脈内投与した。投与直後に腹部又は胸部への超音波照射を上記と同様の条件で行った。投与から6時間後、肝臓、腎臓、脾臓、心臓及び肺を摘出し、各臓器におけるルシフェラーゼ活性を評価した。結果を図２５に示す。 (17) Evaluation of ultrasonic irradiation to the abdomen or chest (FIG. 25)
In the same manner as described above, pDNA / PS / BL (pDNA amount 50 μg) was administered into mice via the tail vein. Immediately after administration, ultrasonic irradiation of the abdomen or chest was performed under the same conditions as described above. Six hours after administration, the liver, kidney, spleen, heart and lung were removed, and luciferase activity in each organ was evaluated. The results are shown in FIG.
　図２５に示すように、腹部に超音波を照射した場合には肝臓や腎臓、脾臓に高い遺伝子導入効果を示すものの、胸部に存在する心臓や肺への遺伝子導入効果はほとんど認められない。一方で胸部への照射によって、胸部の臓器（心臓、肺）において、腹部への照射と比べて高いルシフェラーゼ活性が観察された。以上の結果は、pDNA/PS/BLが超音波照射部位選択的に遺伝子を導入する事が可能であることを示している。 As shown in FIG. 25, when the abdomen is irradiated with ultrasound, a high gene transfer effect is shown in the liver, kidney and spleen, but almost no gene transfer effect on the heart or lung existing in the chest is observed. On the other hand, by irradiating the chest, higher luciferase activity was observed in the organs of the chest (heart, lung) compared to irradiation to the abdomen. The above results indicate that pDNA / PS / BL can introduce a gene selectively in the ultrasonic irradiation site.
　（１８）がん細胞への遺伝子導入効果（図２６）
　５週齢の雌Bulb/cマウスにおいて、colon-26細胞を腹腔へと注入した。２週間後、上記と同様にしてマウスへpDNA/PS/BLを尾静脈内投与し、投与直後（immediately after injection）又は投与から５分後（5mim after injection）に腹部に対して超音波照射（US(+)）をした。投与から６時間後に腫瘍を摘出し、腫瘍におけるルシフェラーゼ活性を評価した。結果を図２６に示す。 (18) Gene transfer effect on cancer cells (FIG. 26)
In 5 week old female Bulb / c mice, colon-26 cells were injected into the peritoneal cavity. Two weeks later, pDNA / PS / BL was administered into mice via the tail vein in the same manner as described above, and the abdomen was irradiated with ultrasonic waves immediately after administration (immediately after injection) or 5 minutes after administration (5 mim after injection). US (+)). Tumors were removed 6 hours after administration, and luciferase activity in the tumors was evaluated. The results are shown in FIG.
　図２６に示すように、アニオン性ナノバブルポリ－リポ・プレックスを用いてがん細胞への遺伝子導入も行えることが明らかとなった。 As shown in FIG. 26, it was revealed that gene transfer into cancer cells can be performed using anionic nanobubble poly-lipoplexes.
　本発明の製剤は、肝硬変治療薬、抗癌剤、細胞選択的薬物・核酸導入試薬（研究用試薬）などとして有用である。 The preparation of the present invention is useful as a cirrhosis therapeutic agent, an anticancer agent, a cell selective drug / nucleic acid introduction reagent (research reagent) and the like.

Claims (7)

  1. （Ａ）アニオン性高分子と
    プロタミン、ヒストン、HelΔ１、ゼラチン、ポリリジン、ポリアルギニン、ポリオルニチン、ポリアミドアミンデンドリマー、ポリリジンデンドリマー、ジエチルアミノエチル－デキストラン、キトサン、スペルミン及びスペルミジンからなる群から選ばれる少なくとも１種の正電荷高分子との複合体及び
    （Ｂ）アニオン性リポソームを含み、
    前記リポソームは気泡を(bubble)含み、前記複合体はカチオン性であるアニオン性ナノバブルポリ－リポ・プレックス。     (A) At least one selected from the group consisting of an anionic polymer and protamine, histone, HelΔ1, gelatin, polylysine, polyarginine, polyornithine, polyamidoamine dendrimer, polylysine dendrimer, diethylaminoethyl-dextran, chitosan, spermine and spermidine A complex with a positively charged polymer and (B) an anionic liposome,
    The liposome comprises bubbles, and the complex is cationic anionic nanobubble poly-lipoplex.
  2. 前記正電荷高分子はプロタミンを含む、請求項１に記載のアニオン性ナノバブルポリ－リポ・プレックス。 The anionic nanobubble poly-lipoplex of claim 1, wherein the positively charged polymer comprises protamine.
  3. 前記アニオン性高分子は核酸である、請求項１又は２に記載のアニオン性ナノバブルポリ－リポ・プレックス。 The anionic nanobubble poly-lipoplex according to claim 1 or 2, wherein the anionic polymer is a nucleic acid.
  4. 前記核酸は、DNAまたはRNAである、請求項１～３のいずれか１項に記載のアニオン性ナノバブルポリ－リポ・プレックス。 The anionic nanobubble poly-lipoplex according to any one of claims 1 to 3, wherein the nucleic acid is DNA or RNA.
  5. 前記核酸は、遺伝子発現プラスミドDNA、アンチセンスDNA、DNAアプタマー、siRNAもしくはshRNAを発現するDNA、mRNA、siRNA、miRNA、shRNA、アンチセンスRNAまたはRNAアプタマーである、請求項３又は４に記載のアニオン性ナノバブルポリ－リポ・プレックス。 The anion according to claim 3 or 4, wherein the nucleic acid is a gene expression plasmid DNA, antisense DNA, DNA aptamer, siRNA or shRNA-expressing DNA, mRNA, siRNA, miRNA, shRNA, antisense RNA or RNA aptamer. Nanobubble poly-lipo plex.
  6. プロタミン及びアニオン性リポソームを含み、前記リポソームは気泡を(bubble)含む、アニオン性ナノバブルポリ－リポ・プレックス。 An anionic nanobubble poly-lipoplex comprising protamine and anionic liposomes, said liposomes containing bubbles.
  7. 前記アニオン性リポソームが、タンパク質又は薬物を含む、請求項１～６のいずれか１項に記載のアニオン性ナノバブルポリ－リポ・プレックス。 The anionic nanobubble poly-lipoplex according to any one of claims 1 to 6, wherein the anionic liposome comprises a protein or a drug.
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