WO2014030601A1 - Méthode de fabrication d'un nouveau lipoplexe à nanobulles constitué de polymères et de liposomes aux propriétés anioniques - Google Patents

Méthode de fabrication d'un nouveau lipoplexe à nanobulles constitué de polymères et de liposomes aux propriétés anioniques 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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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

Cette invention concerne tout particulièrement un moyen permettant d'augmenter l'efficacité d'introduction d'un acide nucléique et éventuellement d'un composé tel qu'une protéine ou un agent médicinal dans une cellule. L'invention concerne un nouveau lipoplexe à nanobulles constitué de polymères et de liposomes, ledit complexe étant formé d'un polymère anionique, d'un polymère chargé positivement et d'un liposome anionique, ledit liposome contenant des bulles et le complexe étant cationique.
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WO2020261464A1 (fr) * 2019-06-26 2020-12-30 武田薬品工業株式会社 Procédé de transfection
KR102431378B1 (ko) * 2021-03-25 2022-08-09 인천대학교 산학협력단 세포밖 소포체의 표면 단백질 및 핵산 분석방법

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JPWO2016199430A1 (ja) * 2015-06-10 2018-04-05 学校法人帝京大学 セラノスティクス用のバブル製剤（ｔｂ）及びその使用方法
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US10688199B2 (en) 2015-06-10 2020-06-23 Teikyo University Theranostic bubble preparation (TB), and method for using same
WO2020262540A1 (fr) * 2019-06-26 2020-12-30 武田薬品工業株式会社 Méthode de transfection
WO2020261464A1 (fr) * 2019-06-26 2020-12-30 武田薬品工業株式会社 Procédé de transfection
JPWO2020261464A1 (fr) * 2019-06-26 2020-12-30
JP7314270B2 (ja) 2019-06-26 2023-07-25 武田薬品工業株式会社 トランスフェクション方法
KR102431378B1 (ko) * 2021-03-25 2022-08-09 인천대학교 산학협력단 세포밖 소포체의 표면 단백질 및 핵산 분석방법

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