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WO2012117971A1 - Structure à membrane lipidique, procédé de production de structure à membrane lipidique et procédé pour l'encapsulation d'une molécule de substance cible à l'aide d'une seule membrane lipidique - Google Patents

Structure à membrane lipidique, procédé de production de structure à membrane lipidique et procédé pour l'encapsulation d'une molécule de substance cible à l'aide d'une seule membrane lipidique Download PDF

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WO2012117971A1
WO2012117971A1 PCT/JP2012/054602 JP2012054602W WO2012117971A1 WO 2012117971 A1 WO2012117971 A1 WO 2012117971A1 JP 2012054602 W JP2012054602 W JP 2012054602W WO 2012117971 A1 WO2012117971 A1 WO 2012117971A1
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lipid
modified
lipid membrane
liposome
cationic surfactant
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PCT/JP2012/054602
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Japanese (ja)
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原島　秀吉
勇磨 山田
亮佑 鈴木
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国立大学法人北海道大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • 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
    • 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—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers
    • A61K9/1272—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers comprising non-phosphatidyl surfactants as bilayer-forming substances, e.g. cationic lipids or non-phosphatidyl liposomes coated or grafted with polymers

Definitions

  • the present invention relates to a lipid membrane structure, a method for producing a lipid membrane structure, and a method for encapsulating one target substance in a single lipid membrane, and more specifically, a lipid obtained by modifying a cationic surfactant
  • the present invention relates to a lipid membrane structure having a single membrane or a plurality of outermost membranes, a method for producing the lipid membrane structure, and a method for encapsulating one target substance with a single lipid membrane.
  • Vectors developed so far are roughly classified into viral vectors and non-viral vectors.
  • Viral vectors are generally excellent in gene transfer ability to introduce the target gene into the nucleus of the target cell, but there are problems such as difficulty in mass production, antigenicity, and toxicity.
  • lipid membrane structures represented by liposomes have attracted attention.
  • Liposomes are lipid membrane structures based on lipid membranes consisting of artificially prepared lipid bilayers, and have the ability to encapsulate various substances and send them into target cells.
  • liposomes can improve the directivity to the target site by introducing functional molecules such as antibodies, proteins, sugar chains, etc. on the surface, liposomes can be decomposed in vivo. It has the advantage of being protected from action and metabolic action, and the advantage of preventing the substance from acting at a site other than the target (side effects). So far, for example, a liposome having a peptide containing a plurality of continuous arginine residues on its surface and having a nuclear translocation ability has been developed (Patent Document 1).
  • SPLP Stabilized plasmid-lipid-particles
  • the particle diameter is preferably about 100 nm to 150 nm (Liu D. et al., Biochim Biophys Acta., Vol. 1104, No. 1). 95-101, 1992; Akinc A. et al., Mol. Ther., 17, No. 5, 872-879, 2009).
  • the liposome described in Patent Document 1 is easily produced by a method (hydration method) in which a lipid film is prepared and then hydrated and stirred or sonicated.
  • the average particle diameter becomes excessively about 300 nm, which can be efficiently delivered to the diseased site. It is difficult.
  • Non-Patent Document 1 The SPLP described in Non-Patent Document 1 is so small that it can be efficiently delivered to a disease site, but it takes a long time for the dialysis operation and changes in slight conditions such as pH of the buffer solution. This makes it difficult to put it to practical use because the efficiency of gene encapsulation into the lipid membrane structure is significantly reduced by the above (Jeffs LB, Pharm Res., Vol. 22, No. 3, pages 362-72). 2005).
  • the present invention has been made in order to solve such problems, and can be easily produced and includes a lipid membrane structure having a minute and uniform particle diameter and a substance to be encapsulated (target substance). It is an object to provide a lipid membrane structure in which the number and the number of lipid membranes are controlled, a method for producing the same, and a method for encapsulating one target substance with one lipid membrane.
  • the present inventors have a fine and uniform particle size by performing a hydration method using a lipid film modified with a cationic surfactant, as shown in the lower part of FIG.
  • the ability to obtain liposomes in which the number of liposomes and substances to be encapsulated (target substances) and the number of lipid membranes are controlled, the ability to encapsulate one target substance with a single lipid membrane, and cationic surface activity The inventors found that the transfection activity of liposomes having a lipid membrane modified with an agent as a single membrane or as the outermost membrane of a plurality of membranes was remarkably improved, and completed the following inventions.
  • a lipid membrane structure having a lipid membrane modified with a cationic surfactant as a single membrane or as an outermost membrane of a plurality of membranes.
  • the cationic surfactant is represented by the following formula (Formula 1) [Wherein, R 1 is an alkyl group, and R 2 is a hydrophobic group.
  • R 1 is an alkyl group having 12 or more carbon atoms
  • R 2 is represented by the following formula (Formula 2): Or the following formula (Formula 3)
  • R 1 is an alkyl group having 16 carbon atoms
  • R 2 is represented by the following formula (Formula 4)
  • the lipid membrane structure according to (2) which is a group of
  • a method for producing a lipid membrane structure having a lipid membrane modified with a cationic surfactant as a single membrane or as an outermost membrane of a plurality of membranes comprising the following (i), (ii) Or (iii) the method having the step; (i) a step of preparing a lipid film in which a cationic surfactant is modified by mixing a lipid and a cationic surfactant; A step of preparing a lipid film in which the cationic surfactant is modified by adding a cationic surfactant; (iii) a single membrane or a plurality of lipid membrane structures having a single membrane or a plurality of membranes A step of modifying the outermost membrane of the membrane with a cationic surfactant.
  • a method of encapsulating one target substance with a single lipid membrane comprising the following step (i) or (ii): (i) mixing a lipid and a cationic surfactant A step of preparing a lipid film modified with a cationic surfactant, and (ii) a step of preparing a lipid film modified with a cationic surfactant by adding a cationic surfactant to the lipid film. .
  • the cationic surfactant is represented by the following formula (Formula 5): [Wherein, R 1 is an alkyl group, and R 2 is a hydrophobic group. ] The method in any one of (7) to (10) which is a cationic surfactant.
  • R 1 is an alkyl group having 12 or more carbon atoms
  • R 2 is represented by the following formula (Formula 6): Or the following formula (Formula 7) The method according to (11), wherein
  • R 1 is an alkyl group having 16 carbon atoms
  • R 2 is represented by the following formula (Formula 8): The method according to (11), wherein
  • lipid membrane structure and the method for producing the same According to the lipid membrane structure and the method for producing the same according to the present invention, a fine and uniform lipid membrane structure, and a lipid membrane structure in which the number of substances to be encapsulated (target substance) and the number of lipid membranes are controlled are obtained. be able to. Moreover, according to the minute and uniform lipid membrane structure, it can be efficiently delivered to a disease site such as a tumor or liver parenchyma, and according to the lipid membrane structure in which the number of lipid membranes is controlled, the target site Can be delivered efficiently. For example, when the number of the lipid membranes of the lipid membrane structure according to the present invention is 3, the membrane or mitochondria is obtained by membrane fusion three times, and when the number of lipid membranes is one, the membrane is one time.
  • the target substance can be efficiently delivered to the cytoplasm.
  • the lipid membrane structure according to the present invention can deliver a target substance to a target site without damaging the target substance, and can exert the function of the target substance at the target site. Can be applied.
  • a lipid membrane structure having the above-described characteristics can be produced easily and inexpensively under mild preparation conditions.
  • a lipid membrane structure corresponding to the size of the target substance can be obtained, and this method is performed one or more times.
  • the method for producing a lipid membrane structure according to the present invention and the method for encapsulating one target substance with a single lipid membrane can be performed regardless of the type of lipid.
  • the lipid composition of the lipid membrane can be appropriately selected according to the above.
  • FIG. 2 is a diagram showing the structures of (TB) and n-Octyl ⁇ -D-glucopyranoside (OG).
  • TB unmodified liposomes (a), and TB modified liposomes (b, c, d, e and f) with modified concentrations of 11, 16.5, 22, 27.5 and 55 mol%
  • a diagram showing the result of measuring the particle size (A)
  • a diagram showing the result of measuring the zeta potential (B)
  • Lipofectamine PLUS (LFN PLUS; A, B), TB unmodified liposome (C, D), 11 mol% TB modified liposome (E, F), 16.5 mol% TB modified liposome (G, H) and 22 mol% TB modified liposome (I, J) shows the results of measuring luciferase activity in HeLa cells transfected with a plasmid DNA (pDNA) encoded with Enhanced Green luorescenceesProtein (EGFP) -luciferase fusion protein gene (right diagram); It is a figure (left figure) which shows the result of having calculated the cell viability.
  • pDNA plasmid DNA
  • EGFP Enhanced Green luorescenceesProtein
  • TB-modified liposomes (a), TB-unmodified liposomes (b), multilamellar liposomes (MLV; c), and unilamellar liposomes (SUV; labeled with Nitro-2-1,3-Benzoxadiazol-4-yl (NBD);
  • NBD Nitro-2-1,3-Benzoxadiazol-4-yl
  • the TB modified positively charged lipid film (a), the TB unmodified positively charged lipid film (b), the TB modified negatively charged lipid film (c) and the TB unmodified negatively charged lipid film (d) are negatively charged. It is a figure which shows the result of having measured the fluorescence intensity of NBD continuously about each sample which sealed the membrane liposome. It is a figure which shows the outline
  • the fluorescence intensity of NBD was continuously measured for the TB-modified trilamellar liposome encapsulating the PLL-stabilized bilayer liposome (a) and the BSA-stabilized bilayer liposome (b) and the negatively charged bilayer liposome, respectively. It is a figure which shows the measurement result.
  • the lipid membrane structure according to the present invention has a lipid membrane modified with a cationic surfactant as a single membrane or as an outermost membrane of a plurality of membranes.
  • the “lipid membrane” refers to a membrane having a lipid membrane structure, the main component of which is lipid.
  • the lipid membrane in the present invention may be composed of a lipid bilayer in which lipid molecules are associated with each other to form a hydrophobic portion, and the hydrophobic portion is formed inward or outward. It may consist of a single layer.
  • lipid membrane structure in the present invention a closed vesicle having a lipid membrane composed of a lipid bilayer can be mentioned, and as such a lipid membrane structure, for example, a liposome can be mentioned.
  • the number of lipid membranes included in the lipid membrane structure may be one, or two or more.
  • the lipid membrane structure according to the present invention has a plurality of membranes, at least the outermost membrane has a lipid membrane in which a cationic surfactant is modified, but not only the outermost membrane but also one other than the outermost membrane.
  • a plurality of membranes may be lipid membranes obtained by modifying a cationic surfactant.
  • the “cationic surfactant” refers to a surfactant having a cationic hydrophilic group, and the ionic value is not particularly limited, but is preferably monovalent.
  • Specific examples of the cationic surfactant include alkyltrimethylammonium salts such as trimethyltetradecylammonium chloride and cetyltrimethylammonium bromide, dialkyldimethylammonium such as dodecylethyldimethylammonium bromide and ethylhexadecyldimethylammonium bromide.
  • alkyldimethylbenzylammonium salt such as benzyldodecyldimethylammonium bromide, benzyltetradecyldimethylammonium bromide, benzylhexadecyldimethylammonium bromide, benzyloctadecyldimethylammonium bromide, benzalkonium chloride, tonzonium bromide, benzethonium chloride, cetyl chloride Pyridinium, decalinium chloride, second Grade amine salts, secondary amine salts, there may be mentioned cations such as from tertiary amine salts such as N-methyl-bis-hydroxyethyl amine fatty acid ester hydrochloride salt, of which the following formula (Formula 1) [Wherein, R 1 is an alkyl group, and R 2 is a hydrophobic group.
  • R 1 is an alkyl group having 12 or more carbon atoms
  • R 2 is represented by the following formula (Formula 2).
  • R 2 represents the following formula (Formula 3)
  • the surfactant be a cationic surfactant.
  • the “hydrophobic group” in the present invention includes, for example, groups represented by the above formulas (Chemical Formula 2) and (Chemical Formula 3), alkyl groups such as methyl group, ethyl group, propyl group, and isopropyl group, and alkenyl groups.
  • a hydrocarbon group such as vinyl group, aryl group such as phenyl group or naphthyl group, a heterocyclic ring, or a combination of a hydrophilic group and a hydrophobic group can be cited as a group which is hydrophobic as a whole.
  • the cationic surfactant is located in the gap between the lipid molecules constituting the lipid membrane.
  • a cationic surfactant is bound to a lipid molecule constituting the lipid membrane by a covalent bond, a hydrogen bond, an ionic bond, a hydrophobic bond, or a van der Waals bond.
  • the lipid membrane structure may be any of positive chargeability, nonchargeability, both (positive / negative) chargeability, and negative chargeability, but is preferably negatively chargeable.
  • the “negatively charged lipid membrane structure” in the present invention refers to a lipid membrane structure that is negatively charged as a whole.
  • positively charged lipids or both (positive and negatively) chargeable lipids as lipids constituting the lipid membrane. Because it contains lipids, non-charged lipids, or because the cationic surfactant is modified, even if it is locally positively charged, both (positive and negative) charged, or uncharged, If it is negatively charged as a whole, it is included in the “negatively charged lipid membrane structure”.
  • the lipid constituting the lipid membrane structure in the present invention may be any of positively charged lipids, neutral (including both positive and negative) charged lipids and uncharged lipids, and negatively charged lipids. Examples thereof include phospholipids, glycolipids, sterols, long-chain aliphatic alcohols and glycerin fatty acid esters, and one or more of these can be used.
  • phospholipid examples include phosphatidylcholine (for example, dioleoylphosphatidylcholine, dilauroylphosphatidylcholine, dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine), phosphatidylglycerol (for example, dioleoylphosphatidylglycerol, dilauroylphosphatidylglycerol, Dimyristoyl phosphatidylglycerol, dipalmitoyl phosphatidylglycerol, distearoyl phosphatidylglycerol, etc., phosphatidylethanolamine (eg dioleoylphosphatidylethanolamine, dilauroylphosphatidylethanolamine, dimyristoylphosphatidylethanolamine, dipasto) Mitoylphosphatidylethanolamine, di
  • glycolipids examples include glyceroglycolipids such as sphingomyelin, sulfoxyribosyl glyceride, diglycosyl diglyceride, digalactosyl diglyceride, galactosyl diglyceride and glycosyl diglyceride, and sphingoglycolipids such as galactosyl cerebroside, lactosyl cerebroside and ganglioside. 1 type, or 2 or more types of these can be used.
  • glyceroglycolipids such as sphingomyelin, sulfoxyribosyl glyceride, diglycosyl diglyceride, digalactosyl diglyceride, galactosyl diglyceride and glycosyl diglyceride
  • sphingoglycolipids such as galactosyl cerebroside, lactosyl cerebroside and ganglioside. 1 type, or 2
  • sterols examples include sterols derived from animals such as cholesterol, cholesterol succinic acid, lanosterol, dihydrolanosterol, desmosterol, dihydrocholesterol, sterols derived from plants such as stigmasterol, sitosterol, campesterol, and brassicasterol (tytosterol). And sterols derived from microorganisms such as ergosterol, and one or more of these can be used. In addition, these sterols can generally be used to physically or chemically stabilize the lipid bilayer or to adjust the fluidity of the membrane.
  • long-chain fatty acid or long-chain aliphatic alcohol a fatty acid having 10 to 20 carbon atoms or an alcohol thereof can be used.
  • long-chain fatty acids or long-chain aliphatic alcohols include palmitic acid, stearic acid, lauric acid, myristic acid, pentadecylic acid, arachidic acid, margaric acid, tuberculostearic acid and other saturated fatty acids, palmitoleic acid, Mention of unsaturated fatty acids such as oleic acid, arachidonic acid, vaccenic acid, linoleic acid, linolenic acid, arachidonic acid, eleostearic acid, oleyl alcohol, stearyl alcohol, lauryl alcohol, myristyl alcohol, cetyl alcohol, linolyl alcohol 1 type, or 2 or more types of these can be used.
  • glycerin fatty acid ester examples include monoacyl glycerides, diacyl glycerides, and triacyl glycerides, and one or more of these can be used.
  • Examples of the positively charged lipid include dioctadecyldimethylammonium chloride (DODAC), N- (2,3-oleyloxy) propyl-N, N, N-trimethylammonium (N-) in addition to the above-described lipids.
  • DODAC dioctadecyldimethylammonium chloride
  • N- N- (2,3-oleyloxy) propyl-N
  • N- N-trimethylammonium
  • Examples of neutral lipids including both (positive and negative) charged lipids and non-charged lipids include diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide and the like in addition to the above-described lipids. Or 2 or more types can be used.
  • Examples of the negatively charged lipid include diacylphosphatidylserine, diacylphosphatidic acid, N-succinylphosphatidylethanolamine (N-succinylPE), phosphatidylethylene glycol, cholesteryl hemisuccinate (CHEMS), etc. 1 type, or 2 or more types of these can be used.
  • the lipid membrane of the lipid membrane structure according to the present invention has positive charges such as tocopherol, propyl gallate, ascorbyl palmitate, butylated hydroxytoluene, stearylamine, oleylamine and the like.
  • Positive charges such as tocopherol, propyl gallate, ascorbyl palmitate, butylated hydroxytoluene, stearylamine, oleylamine and the like.
  • Charged substances to be added, charged substances to give negative charges such as dicetyl phosphate, membrane proteins such as membrane surface proteins and integral membrane proteins, and peptides that impart cell permeability and nuclear translocation ability to lipid membrane structures It can be combined or contained, and the amount and content of the bond can be adjusted as appropriate.
  • the lipid membrane structure having a lipid membrane modified with the cationic surfactant according to the present invention as a single membrane or as an outermost membrane of a plurality of membranes is a hydration method, an ultrasonic treatment method, an ethanol injection method, although it can be produced using a known method such as an ether injection method, a reverse phase evaporation method, a surfactant method, or a freezing / thawing method, in particular, as shown in the lower part of FIG.
  • a hydration method such as an ether injection method, a reverse phase evaporation method, a surfactant method, or a freezing / thawing method, in particular, as shown in the lower part of FIG.
  • the “lipid film” is mainly composed of lipid molecules formed so as to stick to the bottom surface or side surface of the container by dissolving the lipid in the organic solvent and then evaporating and removing the organic solvent.
  • This refers to a membrane as a constituent component.
  • the present invention provides a method for producing a lipid membrane structure.
  • the method for producing a lipid membrane structure according to the present invention is a method for producing a lipid membrane structure having a lipid membrane modified with a cationic surfactant as a single membrane or as an outermost membrane of a plurality of membranes.
  • a step of preparing a lipid film in which a cationic surfactant is modified by mixing a lipid and a cationic surfactant (Ii) adding a cationic surfactant to the lipid film to prepare a lipid film in which the cationic surfactant is modified; (Iii) modifying the cationic surfactant on the outermost membrane of one or more membranes of a lipid membrane structure having one or more membranes;
  • the process (i), (ii), or (iii) is included.
  • the description of the same or equivalent configuration as the configuration of the above-described lipid membrane structure according to the present invention is omitted.
  • a method for preparing a lipid film in which a cationic surfactant is modified by mixing a lipid and a cationic surfactant is obtained by, for example, dissolving a lipid in an organic solvent.
  • the cationic surfactant is added to the lipid solution.
  • examples of the organic solvent for dissolving the lipid and the cationic surfactant include hydrocarbons such as pentane, hexane, heptane and cyclohexane, halogenated hydrocarbons such as methylene chloride and chloroform, and benzene.
  • Aromatic hydrocarbons such as toluene, lower alcohols such as methanol and ethanol, esters such as methyl acetate and ethyl acetate, ketones such as acetone, etc. may be used alone or in combination of two or more. it can.
  • step (ii) as a method for preparing a lipid film in which a cationic surfactant is modified by adding a cationic surfactant to the lipid film, for example, a cationic surfactant solution is added to the lipid film.
  • a method of removing the solvent by evaporation after the addition can be mentioned.
  • step (iii) as a method of modifying the cationic surfactant on the outermost membrane of the single membrane or the plural membranes of the lipid membrane structure having a single membrane or a plurality of lipid membranes, for example, Examples thereof include a method of adding a cationic surfactant to a lipid membrane structure having a membrane or a plurality of membranes.
  • the method for producing a lipid membrane structure according to the present invention includes the step (i) or the step (ii), (Iv) hydrating the prepared lipid film to prepare a lipid membrane structure;
  • the step (iv) can also be included.
  • the lipid film structure is prepared by hydrating the lipid film.
  • a method of adding a liquid such as water to the lipid film for hydration, followed by stirring or ultrasonic treatment That is, the hydration method can be mentioned.
  • the present invention provides a method of encapsulating one target substance with one lipid membrane.
  • the method of encapsulating one target substance according to the present invention with one lipid membrane is as follows: (I) a step of preparing a lipid film in which a cationic surfactant is modified by mixing a lipid and a cationic surfactant; (Ii) adding a cationic surfactant to the lipid film to prepare a lipid film in which the cationic surfactant is modified; The step (i) or (ii) is included.
  • the method of encapsulating one target substance according to the present invention with one lipid membrane is as follows: (Iv) hydrating the prepared lipid film to prepare a lipid membrane structure; The step (iv) can also be included.
  • the “target substance” means a substance to be encapsulated in the lipid membrane structure or an encapsulated substance.
  • the target substance may be any substance, and examples thereof include various physiologically active substances such as drugs, nucleic acids, peptides, proteins, sugars or complexes thereof, and lipid membrane structures such as liposomes and micelles, It can select suitably according to the objectives, such as a diagnosis and treatment.
  • the target substance can be encapsulated in the aqueous phase inside the lipid membrane structure, for example, by adding it to an aqueous solvent used when the lipid membrane is hydrated in the production of the lipid membrane structure.
  • a liposome having one lipid membrane is the target substance
  • a liposome having two lipid films can be obtained.
  • liposomes having two lipid membranes are used as the target substance
  • liposomes having three lipid membranes can be obtained.
  • the lipid membrane structure of the target substance may be modified with various functional molecules. Examples of the modifying substance include peptides that improve migration to a target site and cell permeability, and lipid membrane structures. Examples include PLL, BSA, and charged substances that improve body stability.
  • the lipid membrane structure according to the present invention can be used by being dispersed in an appropriate aqueous solvent such as physiological saline, phosphate buffer, citrate buffer, and acetate buffer.
  • Additives such as saccharides, polyhydric alcohols, water-soluble polymers, nonionic surfactants, antioxidants, pH adjusters, and hydration accelerators may be appropriately added to the dispersion.
  • the lipid membrane structure according to the present invention can be stored in a state in which the dispersion is dried.
  • the lipid membrane structure according to the present invention can be administered orally, and can also be administered parenterally to veins, intraperitoneally, subcutaneously, nasally and the like.
  • the method for producing a lipid membrane structure, and the method of encapsulating one target substance in one lipid membrane, a cationic surfactant to be added to the lipid film can be controlled in the range of approximately 50 nm to 200 nm by adjusting the amount of the lipid to the range of 10 to 30 mol% with respect to the total lipid. Is possible.
  • lipid membrane structure according to the present invention a method for producing the lipid membrane structure, and a method for encapsulating one target substance with a single lipid membrane will be described based on examples. Note that the technical scope of the present invention is not limited to the features shown by these examples.
  • pDNA plasmid DNA
  • EGFP Enhanced Green luminescence Protein
  • the second E. coli suspension was centrifuged at 4 ° C. and 4000 ⁇ g for 15 minutes, and then the supernatant was removed to prepare an E. coli pellet. Subsequently, DNA was extracted and purified using EndoFree Plasmid Mega Kit according to the attached specifications to prepare a crude purified pDNA aqueous solution.
  • the concentration of the purified pDNA aqueous solution was adjusted to 1.0 mg / mL by diluting with sterilized water. Thereafter, 100 ⁇ L each was dispensed into an Eppendorf tube and stored at ⁇ 20 ° C.
  • nucleic acid nanoparticles of [1-2] were subjected to a dynamic light scattering method using Zetasizer Nano ZS (Malvern Instruments). Were used to measure the particle size, and the zeta potential was measured by electrophoresis.
  • DOPE Dioleoyl glycerophosphoethanolamine
  • a DOPE solution was prepared by dissolving in an ethanol / chloroform solution mixed so as to be 15 mmol / L.
  • CHEMS cholesteryl hemisuccinate
  • CHEMS cholesteryl hemisuccinate
  • lipid film with an unmodified surfactant ( a), BB-1 modified lipid film (b and c), BB-2 modified lipid film (d and e), BB-3 modified lipid film (f and g), BB- Lipid films modified with 4 (h and i) and lipid films modified with OG (j and k) were prepared.
  • Example (1) [1-2] to (4) was repeated three times or more, and the average value and standard deviation (SD) of the results obtained for each sample were calculated.
  • SD standard deviation
  • the particle size of the liposomes is small for b, c, d, e, f, g, h and i compared to a, while j and k are a and It was almost the same in comparison.
  • b> c, d> e, f> g and h> i In comparison with liposomes having lipid membranes modified with the same type of surfactant, b> c, d> e, f> g and h> i, whereas j ⁇ k.
  • the liposome having a lipid membrane modified with a cationic surfactant has a smaller particle size than the liposome having a lipid membrane not modified with a surfactant, and a cationic interface. It was found that the particle size decreases as the modified concentration of the active agent increases. On the other hand, liposomes having lipid membranes modified with nonionic surfactants, compared with liposomes having lipid membranes not modified with surfactants, regardless of the modified concentration of nonionic surfactants, It became clear that the particle size was almost the same.
  • liposomes having a minute particle diameter can be obtained by performing a hydration method using a lipid film modified with a cationic surfactant.
  • the nucleic acid nanoparticles are positive for zeta potential, whereas a, b, c, d, e, f, g, h, i, j and k. Were both negative. From these results, the liposome having a lipid membrane modified with a cationic surfactant has a lipid membrane with no surfactant modified when the modification concentration is at least 11 mol% and 16.5 mol%. It was also revealed that negatively charged liposomes that maintain the negative chargeability derived from the constituent lipids were obtained in the same manner as liposomes having lipid membranes modified with nonionic surfactants.
  • Example 2 Examination of particle diameter of liposome having lipid membrane modified with tonzonium bromide (TB) (1) Preparation of nucleic acid nanoparticles Example 1 (1) [1-2] and [1-3] ], Nucleic acid nanoparticles were prepared, and the particle diameter and zeta potential were measured.
  • Example (2) [2-3] Preparation of lipid film About a, b, c, d, e and f of Example (2) [2-2], the method described in Example 1 (2) [2-3] A lipid film was prepared. However, instead of the BB-1 solution, the BB-2 solution, the BB-3 solution, the BB-4 solution, and the OG solution, the TB solution of Example (2) [2-1] was used. The amount of TB solution added and the modification concentration of TB were as follows.
  • Modification concentration (mol%) a: not added (control) 0 b: TB solution 5 ⁇ L 11 c: TB solution 7.5 ⁇ L 16.5 d: TB solution 10 ⁇ L 22 e; TB solution 12.5 ⁇ L 27.5 f; TB solution 25 ⁇ L 55
  • Example 1 (2) For a, b, c, d, e and f of [2-3], Example 1 According to the method described in 3), as a liposome encapsulating a nucleic acid nanoparticle, a liposome having a lipid membrane not modified with TB (TB unmodified liposome; a) and a liposome having a lipid membrane modified with TB (TB modified liposome; b, c, d, e and f) were prepared. Thereafter, the particle diameter and zeta potential were measured by the method described in Example 1 (1) [1-3].
  • the particle size of the liposome was a> b> c> d ⁇ e ⁇ f.
  • the particle diameter of the TB modified liposome is smaller than that of the TB unmodified liposome.
  • the modification concentration of TB is at least 22 mol%
  • the liposome particle size decreases as the modification concentration increases.
  • the modification concentration is at least 22 mol%, the liposome particle size does not decrease any more. It became clear that it was almost constant.
  • the particle diameters of d, e, and f were about 130 nm, which was a value very close to about 100 nm, which is the particle diameter of nucleic acid nanoparticles.
  • the thickness of the lipid membrane is about 4 nm, and the total thickness of the inner aqueous phase is about 15 nm. Therefore, one nucleic acid nanoparticle is added to one lipid membrane.
  • d, e, and f are liposomes in which one nucleic acid nanoparticle is encapsulated with one lipid membrane. That is, it is clear that the liposome obtained by performing the hydration method using a lipid film in which TB is modified by at least about 22 mol% is a liposome in which one target substance is encapsulated by one lipid membrane. became.
  • one target substance can be encapsulated with one lipid membrane by performing a hydration method using a lipid film modified with a cationic surfactant.
  • the zeta potential was negative for nucleic acid nanoparticles, whereas a, b, c, d, e, and f were all negative. From these results, it becomes clear that TB-modified liposomes having a modified TB concentration of less than 55 mol% become negatively charged liposomes that maintain the negative chargeability derived from the constituent lipids, as in the case of TB-unmodified liposomes. It was.
  • Example (4) [4-1] to [4-3] was repeated three times or more, and the average value and standard deviation (SD) of the results obtained for each sample were calculated.
  • SD standard deviation
  • FIG. 6A shows a graph of the measurement result of the particle diameter
  • FIG. 6B shows a graph of the measurement result of the zeta potential.
  • the particle size of the empty liposome was smaller than the particle size of g in all of h, i, j, k, and l. Further, h> i> k> j> l. From these results, it was revealed that TB-modified empty liposomes have a smaller particle size than TB unmodified empty liposomes.
  • a liposome having a minute particle size can be obtained regardless of whether the target substance is encapsulated or not. It was revealed.
  • the zeta potentials of g, h, i and j were all negative, whereas k and l were positive. From these results, TB-modified empty liposomes having a modified TB concentration of at least 55 mol% or more are positively charged liposomes, whereas TB-modified empty liposomes having a modified TB concentration of less than 55 mol% are not modified with TB. It became clear that it becomes a negatively charged liposome in which the negatively charged property derived from the constituent lipid is maintained, like the empty liposome.
  • Example 3 Examination of transfection activity of TB modified liposome (1) Preparation of R8 / TB modified liposome [1-1] Preparation of TB modified liposome and TB unmodified liposome Example 2 (1), (2) [ 2-1], [2-2], [2-3] and (3), as a nucleic acid nanoparticle-encapsulated liposome, TB unmodified liposome, modified concentrations of 11 mol%, 16.5 mol% and 22 mol% TB modified liposomes (11 mol% TB modified liposome, 16.5 mol% TB modified liposome and 22 mol% TB modified liposome) were prepared.
  • R8 R8 (RRRRRRRR; SEQ ID NO: 6), a peptide known to confer a certain cell permeability to liposomes (Kentaro Kogure, Pharmaceutical Journal, Vol. 127, No. 10, 1685-1691 (2007), a peptide-modified fatty acid (STR-R8) in which stearic acid (STR) is added to the C-terminal is purchased from KUROBO and dissolved in sterile water to 2.0 mg / mL. Thus, an STR-R8 solution was prepared.
  • each liposome-containing solution 100 mL was mixed with 2.33 mL of the STR-R8 solution, and then incubated at room temperature for 30 minutes, whereby the total lipids of the liposomes were obtained.
  • R8 was modified to 5 mol%.
  • luciferase assay reagent Promega
  • the amount of luminescence was measured using a luminometer (Lumensecens-PSN; ATTO) according to the attached specifications.
  • the protein concentration of the cell lysate was measured using BCA protein assay kit (PIERCE) according to the attached specifications. Based on these results, the value of luciferase activity was determined using the following formula (1).
  • cell viability is calculated using the following formula (2), assuming that K is set to 100% for A, C, E, G and I, and L is set to 100% for B, D, F, H and J Calculated.
  • the luciferase activity when 0.04 ⁇ g of DNA was transfected was C ⁇ A ⁇ E ⁇ G ⁇ I. Further, the luciferase activity when 0.1 ⁇ g of DNA was transfected was D ⁇ F ⁇ H ⁇ B ⁇ J.
  • the TB modified liposome has an activity to introduce a gene into the nucleus of the cell (transfection activity) as compared with the TB unmodified liposome and a commercially available transfection reagent. Became clear.
  • TB modified liposomes were found to have higher transfection activity than TB unmodified liposomes.
  • the cell viability was higher than 80% for all of A, B, C, D, E, F, G, H, I, J, K, and L. From these results, it was revealed that TB modified liposomes having modified concentrations of at least 11 mol%, 16.5 mol% and 22 mol% are not cytotoxic.
  • lipid film DOPE (NBD-labeled DOPE; conjugated with fluorescent dye Nitro-2-1,3-Benzoxadiazol-4-yl (NBD); NBD-labeled DOPE solution was prepared by dissolving (Avanti Polar Lipids) in ethanol so as to be 0.1 mmol / L.
  • NBD-labeled DOPE solution was added to the mixed lipid solution prepared by the method described in Example 1 (2) [2-2], and 1 mol% of the total lipid was NBD-labeled DOPE.
  • Example 2 (2) [2-3] the amount of TB solution added and the modification concentration of TB were changed as follows to modify TB. Lipid films (a) and lipid films (b, c and d) not modified with TB were prepared.
  • TB modification was carried out as a nucleic acid nanoparticle enclosure liposome labeled with NBD by the method as described in Example 1 (3).
  • Liposomes (a) and TB unmodified liposomes (b) were prepared.
  • Fluorescence intensity of NBD was continuously measured for 200 seconds using a luminometer for a and b in this example (2), c in this example (3), and d in this example (4). During 10 seconds from the start of the measurement, 2.75 ⁇ L of the hydrosulfite sodium solution was added to each sample and mixed for 5 seconds. Furthermore, 12.5 ⁇ L of 10% (v / v) Triton X-100 solution was added to each sample when 105 seconds had elapsed from the start of measurement, and mixed for 5 seconds. The results are shown in Table 6 below. Moreover, the measurement result of fluorescence intensity is shown in the upper diagram of FIG.
  • hydrosulfite sodium is a quencher that reduces the chromophore of NBD by a reducing action and loses its fluorescence.
  • hydrosulfite sodium added to the liposome solution is the outermost lipid bilayer of the liposome. Only the fluorescence of NBD present in the layer in contact with the aqueous phase disappears. Therefore, it is considered that the greater the number of lipid membranes possessed by the liposome, the greater the fluorescence intensity after addition of hydrosulfite sodium.
  • TB-modified liposomes are smaller than that of SUV, which is a single-membrane liposome.
  • lipid-molecule flip-flop occurs due to the modification of TB in the lipid membrane, This is probably because the NBD existing on the side in contact with the inner aqueous phase was exposed to the liposome surface and quenched.
  • An NBD-labeled DOPE solution was added thereto to prepare an NBD negatively charged mixed lipid solution (total 152.5 nmol) in which 1 mol% of the total lipid was NBD-labeled DOPE.
  • an SUV was prepared by the method described in Example 4 (4). After gently adding a half amount (v / v) of the nucleic acid nanoparticle solution prepared by the method described in Example 1 (1) [1-1] and [1-2] to this SUV-containing solution, By vortexing for 5 seconds, negatively charged bilayer liposomes encapsulating nucleic acid nanoparticles and labeled with NBD were prepared.
  • a, b, c, and d are liposomes prepared with the intention of encapsulating liposomes labeled with NBD (labeled liposomes) with lipid membranes not labeled with NBD (unlabeled lipid membranes). Therefore, when the fluorescence of NBD is reduced by the addition of sodium hydrosulfite, the labeled liposome is not encapsulated with the unlabeled lipid membrane, or the membrane fusion between the lipid membrane of the labeled liposome and the unlabeled lipid membrane occurs. Conceivable.
  • the negatively charged bilayer liposome has a constant fluorescence intensity after the addition of sodium hydrosulfite and before the addition of Triton X-100, whereas a and b The fluorescence intensity decreased significantly shortly after the addition of sodium hydrosulfite, and almost no fluorescence was detected immediately before the addition of Triton X-100. From these results, when negatively charged bilayer liposomes are encapsulated with a positively charged lipid membrane, it becomes clear that membrane fusion occurs regardless of the presence or absence of TB modification in the positively charged lipid membrane. It was.
  • negatively charged liposomes can be encapsulated with a negatively charged lipid membrane by performing a hydration method using a negatively charged lipid film modified with a cationic surfactant. became.
  • a hydration method is performed using a lipid film in which a cationic surfactant is modified, whereby one bilayer liposome is obtained. It was revealed that a triple membrane liposome can be obtained by encapsulating with a single lipid membrane.
  • Example 6 Examination of Transfection Activity of TB Modified Trilamellar Liposomes
  • Preparation of TB Modified Trilamellar Liposomes Negatively charged 2 encapsulating nucleic acid nanoparticles by the method described in Example 5 (1) Sheet membrane liposomes were prepared. However, NBD-labeled DOPE solution was not added. Subsequently, negatively chargeable bilayer liposomes were encapsulated in a TB-modified negatively charged lipid film by the method described in Example 5 (2) [2-2], [2-3] and [2-4]. This was designated as a TB-modified trilamellar liposome. Subsequently, the TB modification trilamellar liposome was modified with R8 by the method described in Example 3 (1) [1-2].
  • ⁇ -MEM ( ⁇ ) medium and ⁇ -MEM (+) medium were stored at 4 ° C. until use.
  • the luciferase activity was 3.3 ⁇ 10 6 (3258833) for A, 1.2 ⁇ 10 5 for B, and 7.5 ⁇ 10 5 for C. From these results, it was revealed that the transfection activity of TB-modified trilamellar liposomes is lower than that of commercially available transfection reagents, but is about 6 times higher than that of tetralamellar liposomes.
  • Example 7 Examination of optimization of TB-modified trilamellar liposome (1) Preparation of PLL-stabilized bilayer liposome and BSA-stabilized bilamellar liposome Negative chargeability by the method described in Example 5 (1) A solution containing bilamellar liposomes was prepared. Two samples of 100 ⁇ L each of the solution containing the negatively charged bilamellar liposomes were prepared, and 1.0 mg / mL PLL solution 4 prepared by the method described in Example 1 (1) [1-2] was added to one of them. 0.0 ⁇ L was added and 0.5 mg / mL bovine serum albumin (BSA) solution was added to the other half.
  • BSA bovine serum albumin
  • the solution containing negatively charged bilayer liposomes (PLL-stabilized bilayer liposomes) stabilized by electrostatically binding PLL and non-specifically adsorbing BSA are incubated at room temperature for 15 minutes.
  • a solution containing negatively charged bilayer liposomes (BSA-stabilized bilayer liposomes) that had been stabilized was prepared.
  • Example 5 (2) Preparation of TB-modified trilamellar liposomes
  • two samples of negatively charged lipid films modified with TB were prepared as a and b.
  • a is used in this Example (2).
  • a solution containing a PLL-stabilized bilayer liposome was added, and for b, a solution containing a BSA-stabilized bilayer liposome was added to prepare a TB-modified trilamellar liposome.
  • the outline of the preparation process of these TB modified trilamellar liposomes is shown in FIG.
  • Example 8 Examination of transfection activity of optimized TB-modified trilamellar liposomes (1) Preparation of TB-modified trilamellar liposomes By the method described in Example 7 (1), negatively charged bilayer liposomes Then, a solution containing each of the PLL-stabilized bilayer liposome and the BSA-stabilized bilayer liposome was prepared. However, NBD-labeled DOPE solution was not added.
  • Example 5 (2) [2-3] three samples of negatively charged lipid film modified with TB were prepared by the method described in Example 5 (2) [2-3], and designated as a, b and c.
  • a is a negatively charged bilayer membrane.
  • a solution containing liposomes, a solution containing PLL-stabilized bilayer liposomes for b, and a solution containing BSA-stabilized bilayer liposomes for c were added to prepare TB-modified trilamellar liposomes.
  • Example 6 (2) Examination of transfection activity Using a, b and c of this Example (1), it was described in Example 6 (3) [3-1], [3-2] and [3-3]. The method was used to transfect JAWSII cells. Thereafter, the luciferase activity was measured by the method described in Example 4 (3).
  • the luciferase activity when transfection is performed with a and b is lower than that of a commercially available transfection reagent, whereas the luciferase activity when transfection is performed with c is commercially available. Compared to the transfection reagent.
  • the transfection activity of TB-modified trilamellar liposomes encapsulating BSA-stabilized bilayer liposomes is as follows: TB-encapsulated PLL-stabilized bilayer liposomes and non-stabilized negatively charged bilayer liposomes It was found to be higher than the modified trilamellar liposome and equivalent to a commercially available transfection reagent.
  • Example 9 Photomicrograph of single-membrane liposome 10 ⁇ L of the TB modified liposome d / HEPES suspension of the present invention prepared in Example 2 (3) was rapidly frozen using liquid nitrogen and fixed. The observation was made with a transmission electron microscope. A part of the observation photograph is shown in FIG.
  • the average particle diameter of the particles observed in the microscope field was in the range of 50 to 100 nm. This value is consistent with the particle diameter measurement data measured in Example 2 (3), considering that the liquid encapsulated in the TB modified liposome is removed by rapid freezing.
  • all the observed particles had an inner black outline and an outer white outline.
  • the black outline is considered to correspond to the encapsulated core particle.
  • the white outline had a thickness of about 3.5 nm, and this value approximates the theoretical thickness of one lipid membrane of 4.0 nm.

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Abstract

L'invention a pour objet des structures à membrane lipidique qui peuvent être facilement produites, qui sont petites et qui ont un diamètre uniforme des particules, des structures à membrane lipidique pour lesquelles la quantité de substance encapsulée (substance cible) et le nombre de membranes lipidiques sont maîtrisés, un procédé de production s'y rapportant ainsi qu'un procédé pour l'encapsulation d'une molécule de substance cible dans une seule membrane lipidique. La structure à membrane lipidique de l'invention comprend une membrane lipidique modifiée par un tensioactif cationique comme seule membrane ou comme membrane la plus à l'extérieur de multiples membranes. L'utilisation de structures à membrane lipidique de l'invention ou du procédé de production s'y rapportant permet d'obtenir des structures à membrane lipidique petites et uniformes et des structures à membrane lipidique dans lesquelles la quantité de substance encapsulée (substance cible) et le nombre de membranes lipidiques sont maîtrisés.
PCT/JP2012/054602 2011-02-28 2012-02-24 Structure à membrane lipidique, procédé de production de structure à membrane lipidique et procédé pour l'encapsulation d'une molécule de substance cible à l'aide d'une seule membrane lipidique WO2012117971A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005092388A1 (fr) * 2004-03-26 2005-10-06 Terumo Kabushiki Kaisha Preparation de liposome
WO2006077857A1 (fr) * 2005-01-18 2006-07-27 National University Corporation Hokkaido University Procédé de revêtement de particules avec un film lipidique
WO2007037444A1 (fr) * 2005-09-30 2007-04-05 National University Corporation Hokkaido University Vecteur destiné à la délivrance d'une substance cible dans un noyau ou une cellule
JP2009221165A (ja) * 2008-03-17 2009-10-01 Hokkaido Univ 一遺伝子ナノ粒子のパッケージング法

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005092388A1 (fr) * 2004-03-26 2005-10-06 Terumo Kabushiki Kaisha Preparation de liposome
WO2006077857A1 (fr) * 2005-01-18 2006-07-27 National University Corporation Hokkaido University Procédé de revêtement de particules avec un film lipidique
WO2007037444A1 (fr) * 2005-09-30 2007-04-05 National University Corporation Hokkaido University Vecteur destiné à la délivrance d'une substance cible dans un noyau ou une cellule
JP2009221165A (ja) * 2008-03-17 2009-10-01 Hokkaido Univ 一遺伝子ナノ粒子のパッケージング法

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Title
KUCZERA, J. ET AL., APPLIED ORGANOMETALLIC CHEMISTRY, vol. 11, 1997, pages 591 - 600 *
SUZUKI, R. ET AL., BIOL. PHARM. BULL., vol. 31, no. 6, 2008, pages 1237 - 1243 *

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