Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7088—Compounds having three or more nucleosides or nucleotides
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • 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
  • 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
  • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
  • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
• • 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
  • A61K47/24—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids
• • 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
  • A61K47/28—Steroids, e.g. cholesterol, bile acids or glycyrrhetinic acid
• • 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
  • A61K47/32—Macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. carbomers, poly(meth)acrylates, or polyvinyl pyrrolidone
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
  • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction

Definitions

• DDS drug delivery systems
• Lipid nanoparticles are capable of delivering nucleic acids into cells, and so they have been attracting attention as drug carriers for DDS of nucleic acid medicines.
• lipids modified with polyethylene glycol (PEG) have been used in lipid nanoparticles encapsulating nucleic acids (e.g., Patent Document 1).
• the present invention aims to provide lipid nanoparticles that can sufficiently uptake lipid nanoparticles into cells and improve the efficiency of uptake of encapsulated substances in the lipid nanoparticles into cells.
• lipid nanoparticles of the present invention are as described below in (1) to (17), etc.
• a lipid nanoparticle comprising an amphiphilic compound
• the amphipathic compound has (i) a portion (I) containing a structural unit (A) derived from a monomer (a) having two or more hydroxyl groups in the molecule and having 2 to 10 carbon atoms constituting a side chain among the carbon atoms of the structural unit, and (ii) a hydrocarbon group having 8 or more carbon atoms.
• a pharmaceutical comprising the lipid nanoparticles described in any one of (1) to (10).
• R 1 represents a hydrogen atom or a methyl group.
• FIG. 1 shows the results of cell introduction of lipid nanoparticles encapsulating mRNA.
• FIG. 1 shows the results of cell introduction of lipid nanoparticles encapsulating siRNA.
• FIG. 1 shows the results of encapsulation efficiency of siRNA into lipid nanoparticles.
• FIG. 1 shows the nucleic acid recovery rates of Examples 21 to 42.
• FIG. 13 is a graph showing the relative light emission amount and particle size of Examples 21 to 28 compared to Comparative Example 15.
• FIG. 13 is a graph showing the relative light emission intensity of Examples 21 to 28 compared to Comparative Example 15 under different pH conditions.
• FIG. 13 is a graph showing the relative light emission amount and particle size of Example 24 and Examples 29 to 36 compared to Comparative Example 15.
• FIG. 13 is a graph showing the relative light emission amount and particle size of Example 24 and Examples 37 to 42 compared to Comparative Example 15.
• FIG. 13 shows the relative luminescence intensity of Example 24 compared to Comparative Example 15 in different cell types.
• the amphipathic compound is a lipid nanoparticle having (i) a portion (I) containing a structural unit (A) derived from a monomer (a) having two or more hydroxyl groups in the molecule and having 2 to 10 carbon atoms constituting a side chain among the carbon atoms of the structural unit, and (ii) a hydrocarbon group having 8 or more carbon atoms.
• lipid nanoparticles modified with the amphiphilic compound of this embodiment have an improved efficiency of delivery into the cell compared to unmodified lipid nanoparticles (for example, comparison between Comparative Example 15 and Example 8 in FIG. 1 described later).
• the lipid nanoparticles of this embodiment are very useful as a carrier of an encapsulated substance that needs to be delivered into the cell to express a medicinal effect, such as a nucleic acid drug.
• a medicinal effect such as a nucleic acid drug.
• the lipid nanoparticles taken into the cell can easily escape from the endosome, and the lipid nanoparticles are easily transported into the cytoplasm.
• the lipid nanoparticles of this embodiment have good storage stability.
• lipid nanoparticles of this embodiment have such excellent intracellular delivery properties is unclear, but it is believed that when the amphiphilic compound and each lipid (phospholipid, sterol, and cationic lipid) of this form form lipid nanoparticles, they form a structure that is easily taken up into cells, resulting in improved efficacy of the encapsulated substance.
• X-Y means “X or more and Y or less,” and "weight” and “mass” are treated as synonyms.
• (meth)acrylate means acrylate or methacrylate
• (meth)acrylic means acrylic or methacrylic
• acrylate and methacrylate may be used alone or in combination.
• operations and measurements of physical properties are performed at room temperature (20-25°C) and relative humidity of 40-50% RH.
• the lipid nanoparticle of this embodiment contains an amphipathic compound. Furthermore, the lipid nanoparticle of this embodiment usually contains phospholipid as a component constituting the lipid nanoparticle.
• the lipid nanoparticle of this embodiment preferably contains a cationic lipid. By containing a cationic lipid, when the encapsulated substance is anionic, the cationic lipid and the encapsulated substance form a complex (aggregate), and the encapsulated substance is easily packed in the lipid nanoparticle.
• the lipid nanoparticle can be efficiently transferred from the endosome to the cytoplasm, and the function (pharmaceutical effect) of the encapsulated substance (especially nucleic acid) is easily exhibited.
• the particle diameter of the lipid nanoparticles is greater than 0 nm and less than 1000 nm.
• particle diameter means the average particle diameter measured by dynamic light scattering.
• the particle diameter can be measured by a particle diameter measuring device that utilizes dynamic light scattering.
• the particle diameter of the lipid nanoparticles is preferably 10 nm or more, and from the viewpoints of improving cellular uptake, ease of sterilization filtration, and improving storage stability, it is more preferably 10 to 300 nm, even more preferably 30 to 250 nm, even more preferably 30 to 200 nm, and particularly preferably 30 to 180 nm.
• the number average molecular weight (Mn) of the amphipathic compound is preferably 1000 or more, more preferably 2000 or more, and even more preferably 3000 or more, from the viewpoint of suppressing aggregation of lipid nanoparticles in an aqueous solvent and promoting uniform uptake into cells.
• the number average molecular weight (Mn) of the amphipathic compound is preferably 90000 or less, more preferably 30000 or less, and even more preferably 15000 or less, from the viewpoint of excretion from the body.
• the number average molecular weight (Mn) of the amphipathic compound is preferably 1000 to 90000, more preferably 2000 to 30000, and even more preferably 3000 to 15000.
• the value of the number average molecular weight (Mn) can be determined by the measurement method described in the examples described below.
• amphipathic compound according to this embodiment has a moiety (I) containing a structural unit (A) having a specific structure, and a hydrocarbon group having 8 or more carbon atoms.
• amphipathic compound is also referred to simply as an "amphipathic compound.”
• the amphiphilic compound according to the present invention has a moiety (I) including a structural unit (A) derived from a monomer (a) having two or more hydroxyl groups in the molecule and having 2 to 10 carbon atoms constituting a side chain among the carbon atoms of the structural unit.
• the moiety (I) is composed of a polymer having a structural unit (A) derived from a monomer (a) having two or more hydroxyl groups in the molecule and having 2 to 10 carbon atoms constituting a side chain among the carbon atoms of the structural unit.
• the polymer is also referred to as a "polymer constituting the moiety (I)" hereinafter.
• the "structural unit (Q) derived from a monomer (P)" (P represents any appropriate symbol, and there may be no notation of (P)) typically means a unit (Q) (Q represents any appropriate symbol, and there may be no notation of (Q)) constituting at least a part of the polymer, in which one of the bonds of the polymerizable unsaturated double bond possessed by the "monomer (P)" (or simply “monomer”) is opened by polymerization and becomes a unit.
• the "structural unit derived from monomer (P)” may be a structural unit formed by a different manufacturing method, so long as it has the same structure as the structural unit (in the specific example shown below, the structural unit represented by general formula (Q)) formed by polymerization of monomer (P) (or simply "monomer”) as described above.
• the structural unit (A) may be formed by polymerizing monomer (a) in which a hydroxyl group is protected, and then subjecting the resulting structural unit to a deprotection treatment.
• a structural unit derived from glycerin (meth)acrylate may be formed by polymerizing (2-methyl-2-ethyl-1,3-dioxolan-4-yl)methyl (meth)acrylate or glycidyl ether (meth)acrylate, followed by hydrolysis.
• Monomer (a) which has two or more hydroxyl groups in the molecule and in which the number of carbon atoms constituting the side chain among the carbon atoms of the constituent unit is 2 to 10, is preferably a vinyl monomer, and more preferably a (meth)acrylic monomer. Furthermore, monomer (a) may be a monofunctional monomer or a polyfunctional monomer, but preferably contains a monofunctional monomer, and more preferably consists of a monofunctional monomer.
• (meth)acrylates such as glycerin mono(meth)acrylate (also known as 2,3-dihydroxypropyl(meth)acrylate), 1,2-dihydroxyethyl(meth)acrylate, 2,2-dihydroxyethyl(meth)acrylate, dihydroxybutyl(meth)acrylate, trimethylolpropane mono(meth)acrylate, pentaerythritol mono(meth)acrylate, and dipentaerythritol mono(meth)acrylate are preferably used.
• glycerin monoacrylate (GLMA) and/or glycerin monomethacrylate (GLMMA) are preferred due to their industrial availability and high reactivity.
• GLMA glycerin monoacrylate
• GLMMA glycerin monomethacrylate
• the number of carbon atoms constituting the side chain among the carbon atoms of the structural unit (A) derived from monomer (a) is 2 to 10, but in this specification, the "side chain” refers to the part other than the main chain. "The number of carbon atoms constituting the side chain is 2 to 10" refers to the (total) number of carbon atoms in the entire side chain (the total (4) groups bonded to the carbon atoms of the main chain). And the "main chain” means the chain of carbon atoms bonded continuously in the polymer formed by linking structural units, which has the largest number of carbon atoms.
• the number of carbon atoms constituting the side chain among the carbon atoms of the structural unit derived from monomer (a) is 2 to 10, but this number of carbon atoms is preferably 3 to 8, and more preferably 4 to 6.
• the side chain of the structural unit derived from monomer (a) may be an unsubstituted or substituted alkyl group having 2 to 10 carbon atoms, the substituent may be a hydroxyl group, and the substituent may contain two or more hydroxyl groups.
• the structural unit (A) contains a structural unit represented by the following chemical formula (1).
• R 1 represents a hydrogen atom or a methyl group, and is preferably a methyl group
• the proportion of structural units (A) derived from monomer (a) having two or more hydroxyl groups in the molecule and having 2 to 10 carbon atoms constituting the side chain among the carbon atoms of the structural unit, in the portion (I) constituting the amphipathic compound is, for example, 1 to 100 mass%, preferably 20 to 100 mass%, more preferably 50 to 100 mass%, even more preferably 60 to 100 mass%, still more preferably 80 to 100 mass%, particularly preferably 90 to 100 mass%, and most preferably 100 mass%. If the proportion of structural units (A) is within the above range, the effect of the present invention can be achieved.
• the constituent unit (B) may be derived from any radically polymerizable monomer (hereinafter, a monomer that becomes the constituent unit (B) by copolymerization is also referred to as “monomer (b)").
• the proportion of the constituent unit (B) in the portion (I) is, for example, 99% by mass or less, preferably 80% by mass or less, more preferably 50% by mass or less, even more preferably 40% by mass or less, even more preferably 20% by mass or less, and particularly preferably 10% by mass or less.
• the portion (I) constituting the amphipathic compound does not have to contain the constituent unit (B).
• Examples of the monomer (b) include, other than the monomer (a), hydroxyl group-containing (meth)acrylates, polyoxyalkylene group-containing unsaturated monomers, alkoxyalkyl (meth)acrylates, vinyl monomers, cyclic compounds, and the like. These monomers (b) may also be used alone or in combination of two or more.
• hydroxyl group-containing (meth)acrylate examples include hydroxyalkyl (meth)acrylates in which the hydroxyalkyl group has 2 to 4 carbon atoms, such as 2-hydroxyethyl acrylate and 2-hydroxyethyl methacrylate.
• the polyoxyalkylene group-containing unsaturated monomer may, for example, be a monomer represented by the following chemical formula (2):
• polyoxyalkylene group-containing unsaturated monomers examples include unsaturated alcohol polyalkylene glycol adducts, polyalkylene glycol ester monomers, (alkoxy)polyalkylene glycol monomaleic acid esters, etc.
• Unsaturated alcohol polyalkylene glycol adducts are compounds in which a polyalkylene glycol chain is added to an alcohol having an unsaturated group.
• unsaturated alcohol polyalkylene glycol adducts include polyethylene glycol monovinyl ether, polyethylene glycol monoallyl ether, polyethylene glycol mono(2-methyl-2-propenyl) ether, polyethylene glycol mono(2-butenyl) ether, polyethylene glycol mono(3-methyl-3-butenyl) ether, polyethylene glycol mono(3-methyl-2-butenyl) ether, polyethylene glycol mono(2-methyl-3-butenyl) ether, polyethylene glycol mono(2-methyl-2-butenyl) ether, polyethylene glycol mono(1,1-dimethyl-2-propenyl) ether, polyethylene polypropylene glycol mono(3-methyl-3-butenyl) ether, and methoxypolyethylene glycol mono(3-methyl-3-butenyl)
• polyalkylene glycol ester monomer for example, an ester of an alkoxypolyalkylene glycol in which 1 to 300 moles of an oxyalkylene group having 2 to 18 carbon atoms is added to an alcohol, and (meth)acrylic acid is preferred.
• alkoxypolyalkylene glycols those containing an oxyethylene group as the main component are preferred.
• Examples of the alcohol include aliphatic alcohols having 1 to 30 carbon atoms, such as methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, 1-pentanol, 2-pentanol, 3-pentanol, 1-hexanol, 2-hexanol, 3-hexanol, octanol, 2-ethyl-1-hexanol, nonyl alcohol, lauryl alcohol, cetyl alcohol, and stearyl alcohol; alicyclic alcohols having 3 to 30 carbon atoms, such as cyclohexanol; and unsaturated alcohols having 3 to 30 carbon atoms, such as (meth)allyl alcohol, 3-buten-1-ol, and 3-methyl-3-buten-1-ol.
• aliphatic alcohols having 1 to 30 carbon atoms such as methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-but
• esterified products examples include methoxypolyethylene glycol mono(meth)acrylate, methoxy(polyethylene glycol polypropylene glycol) mono(meth)acrylate, methoxy(polyethylene glycol polybutylene glycol) mono(meth)acrylate, and methoxy(polyethylene glycol polypropylene glycol polybutylene glycol) mono(meth)acrylate.
• polyalkylene glycol ester monomers for example, (alkoxy)polyalkylene glycol mono(meth)acrylates such as methoxypolyethylene glycol monomethacrylate are preferred.
• the vinyl monomers include, for example, (meth)acrylic acid, methyl (meth)acrylate, ethyl (meth)acrylate, propyl (meth)acrylate, n-butyl (meth)acrylate, isobutyl (meth)acrylate, tert-butyl (meth)acrylate, 2-ethylhexyl (meth)acrylate, n-lauryl (meth)acrylate, n-stearyl (meth)acrylate, diaminomethyl (meth)acrylate, diaminoethyl (meth)acrylate, dimethylamino (meth)acrylate, acrylate, diethylamino (meth)acrylate, glycidyl (meth)acrylate, styrene, aziridines, 2-(meth)acryloyloxymethyl phosphorylcholine, 2-(meth)acryloyloxyethyl phosphorylcholine, tetrahydrofurfur
• Cyclic compounds include, for example, lactides such as L-lactide, lactones such as ⁇ -caprolactone, trimethyl carbonate, cyclic amino acids, and morpholine-2,5-dione.
• the portion (I) is made of a polymer containing the structural unit (A) (hereinafter, also referred to simply as the polymer or the above-mentioned polymer), the polymer may have a block copolymer structure obtained by bonding the same or different types of polymers together.
• the number average molecular weight (Mn) of the polymer is preferably 1000 or more, more preferably 2000 or more, and even more preferably 3000 or more, from the viewpoint of improving the expression level of nucleic acid by uniform dispersion of particles.
• the number average molecular weight (Mn) of the polymer is preferably 90000 or less, more preferably 30000 or less, and even more preferably 15000 or less, from the viewpoint of excretion from the body.
• the number average molecular weight (Mn) of the polymer may be 1000 to 90000, 2000 to 30000, or 3000 to 15000.
• the number average molecular weight (Mn) of the amphipathic compound is measured, so that the value of Mn of the portion (I) (polymer) can be calculated by subtracting the molecular weight of the portion other than the portion (I) (polymer) from this measured value. If an accurate value can be calculated, the value of Mn may be calculated by a similar method using only the portion (I) (polymer).
• the molecular weight distribution of the polymer is preferably 1 to 5, more preferably 1 to 3, even more preferably 1 to 2, even more preferably 1 to 1.5, and even more preferably 1 to 1.3, from the viewpoint of the modifying ability of the amphiphilic compound (polymer) to the lipid nanoparticles.
• the polymer constituting portion (I) can be obtained by polymerizing a monomer composition containing monomer (a) and, if necessary, monomer (b).
• Methods for polymerizing the monomer composition include, for example, radical polymerization, atom transfer radical polymerization, reversible addition-fragmentation chain transfer (RAFT) polymerization, and other living radical polymerization methods, ionic polymerization, ring-opening polymerization, coordination polymerization, and polycondensation, but the present invention is not limited to these examples.
• a solvent When polymerizing the monomer composition, a solvent may be used.
• the solvent include aromatic solvents such as benzene, toluene, and xylene; alcohol solvents such as methanol, ethanol, isopropanol, n-butanol, and tert-butanol; halogen-containing solvents such as dichloroethane and dichloromethane; ether solvents such as diethyl ether, propylene glycol methyl ether, dipropylene glycol methyl ether, ethyl cellosolve, and butyl cellosolve; ester solvents such as ethyl acetate, butyl acetate, and cellosolve acetate; ketone solvents such as acetone, methyl ethyl ketone, methyl isobutyl ketone, and diacetone alcohol; and amide solvents such as dimethylformamide.
• aromatic solvents such as benzene, tol
• an alcohol solvent and/or water from the viewpoint of reactivity.
• These solvents may be used alone or in combination of two or more.
• the amount of the solvent may be appropriately determined taking into consideration the polymerization conditions, the composition of the monomer composition, the concentration of the resulting polymer, and the like.
• a chain transfer agent can be used to adjust the molecular weight of the polymer or to introduce a hydrocarbon group (including a hydrocarbon group having 8 or more carbon atoms, as described below).
• a polymerization initiator When polymerizing the monomer composition, a polymerization initiator can be used.
• Polymerization initiators include radical polymerization initiators and living radical polymerization initiators. These polymerization initiators may be used alone or in combination of two or more.
• the amount of polymerization initiator can be set appropriately depending on the desired physical properties of the resulting polymer.
• the polymerization conditions for polymerizing the monomer composition may be set appropriately depending on the polymerization method, and are not particularly limited.
• the polymer constituting the site (I) can be obtained by polymerizing the monomer composition as described above.
• the polymer obtained may have a functional group at its end. If the polymer constituting the site (I) has a functional group at its end, it is easy to modify a medicine or to link it to a specific hydrocarbon group described later via the functional group. However, the polymer constituting the site (I) does not have to have a functional group at its end.
• a functional group-containing compound for introducing a functional group into the polymer can be used.
• functional group-containing compounds for introducing a functional group into the terminal of the polymer include alkali metal thioacetate salts such as sodium thioacetate and potassium thioacetate, thiol-based chain transfer agents such as cysteine, cysteamine, mercaptoethanol, thioglycerol, thioglycolic acid, mercaptopropionic acid, 2-mercaptopropionic acid, 3-mercaptopropionic acid, thioacetic acid, thiomalic acid, 2-mercaptoethanesulfonic acid, and sodium and potassium salts thereof; RAFT agents such as 4-cyano-4-(phenylcarbonothioylthio)pentanoic acid; 4,4'-azobis(4-cyanopentanoic acid), 2,2'-azobis[2-methyl-
• These functional group-containing compounds may be used alone or in combination of two or more.
• the functional group-containing compounds described above include those that fall under the category of the chain transfer agent and polymerization initiator described above. However, the functional group-containing compounds that fall under the category of the chain transfer agent and polymerization initiator may be used either as a chain transfer agent or a polymerization initiator or as a functional group-containing compound, or may be used for both purposes.
• a functional group may be introduced at the end of the polymer prepared using the living polymerization initiator by reacting a functional group-containing compound with a halogen atom present at the end of the polymer.
• Examples of functional group-containing compounds that can react with such halogen atoms to introduce functional groups at the end of the polymer include amine compounds such as ethylenediamine and propyldiamine, dithiol compounds such as ethanedithiol, propanedithiol, and hexadecanedithiol, allyl mercaptan, and thiol compounds such as cysteine, cysteamine, mercaptoethanol, thioglycerol, thioglycolic acid, mercaptopropionic acid, 2-mercaptopropionic acid, 3-mercaptopropionic acid, thioacetic acid, thiomalic acid, 2-mercaptoethanesulfonic acid, and sodium and potassium salts thereof.
• amine compounds such as ethylenediamine and propyldiamine
• dithiol compounds such as ethanedithiol, propanedithiol, and hexadecanedithiol
• the amount of the functional group-containing compound for introducing a functional group to the end of the polymer is not particularly limited and may be appropriately set depending on the type of monomer (structural unit) that constitutes the polymer, polymerization conditions such as polymerization temperature, the molecular weight of the target polymer, etc.
• the amount of the chain transfer agent is preferably 0.1 to 20 parts by mass, and more preferably 0.5 to 15 parts by mass per 100 parts by mass of monomer.
• the above-mentioned hydrocarbon group is preferably a group contained in an organic compound that has the ability to form molecular aggregates through hydrophobic interactions in an aqueous solution.
• organic compounds include hydrocarbons, hydrophobic polymers, lipids, and other organic molecules.
• the organic compound is a hydrocarbon and/or a lipid, and more preferably, it is a hydrocarbon.
• the hydrocarbon may be an aliphatic hydrocarbon having 8 to 50 carbon atoms or an aromatic hydrocarbon having 8 to 50 carbon atoms. That is, in a preferred embodiment of the present invention, the number of carbon atoms in the hydrocarbon group is 8 to 50, more preferably 8 to 40, even more preferably 8 to 30, and particularly preferably 8 to 20.
• the hydrocarbon is an aliphatic hydrocarbon having 8 to 50 carbon atoms (the hydrocarbon group is an aliphatic hydrocarbon group having 8 to 50 carbon atoms).
• Examples of aliphatic hydrocarbons having 8 to 50 carbon atoms include linear alkanes such as octane, nonane, decane, undecane, dodecane, tridecane, tetradecane, pentadecane, hexadecane, heptadecane, octadecane, nonadecane, and icosane, branched alkanes thereof, and cyclic alkanes thereof, with linear alkanes being preferred.
• linear alkanes such as octane, nonane, decane, undecane, dodecane, tridecane, tetradecane, pentadecane, hexadecane, heptadecane, octadecane, nonadecane, and icosane, branched alkanes thereof, and cyclic alkanes thereof, with linear alkanes being preferred.
• aromatic hydrocarbons having 8 to 50 carbon atoms examples include 2-phenylethane, 1,3,5-trimethylbenzene, naphthalene, anthracene, fluorescein, and positional isomers of these.
• Hydrophobic polymers include polymers obtained by polymerizing vinyl monomers that can be the constituent units of the above-mentioned portion (I) and that have a hydrocarbon group with 8 or more carbon atoms in the side chain as the main component.
• Lipid refers to an organic compound that has a hydrophobic portion (e.g., a long-chain fatty acid, a hydrocarbon chain, or a sterol group), is poorly soluble in water, and is easily soluble in an organic solvent. Lipids usually have hydrophilic groups such as hydroxyl groups, carboxylic acids (carboxyl groups), and amino groups. In this case, the hydrophobic portion preferably has 8 or more carbon atoms. Therefore, the amphipathic compound of the present invention is also included in the category of lipids.
• a hydrophobic portion e.g., a long-chain fatty acid, a hydrocarbon chain, or a sterol group
• the hydrophobic portion preferably has 8 or more carbon atoms. Therefore, the amphipathic compound of the present invention is also included in the category of lipids.
• Lipids can be further broadly classified into phospholipids, glycolipids (glyceroglycolipids and sphingoglycolipids), sphingolipids, sterols, neutral lipids, saturated or unsaturated fatty acids, etc.
• Phospholipids are broadly classified into glycerophospholipids and sphingophospholipids.
• Representative glycerophospholipids include phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), and phosphatidic acid (PA).
• a representative sphingophospholipid is sphingomyelin.
• Specific examples of phospholipids include the lipids listed in (a) to (g) below.
• Phosphatidylcholines include dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), dimyristylphosphatidylcholine (DMPC), dioleoylphosphatidylcholine (DOPC), dilauroylphosphatidylcholine (DLPC), didecanoylphosphatidylcholine (DDPC), dioctanoylphosphatidylcholine, dihexanoylphosphatidylcholine (DHPC), dibutyrylphosphatidylcholine (DBPC), and diethylphosphatidylcholine (DHPC).
• DPPC dipalmitoylphosphatidylcholine
• DSPC distearoylphosphatidylcholine
• DMPC dimyristylphosphatidylcholine
• DOPC dioleoylphosphatidylcholine
• DLPC dide
• Lydoyl phosphatidylcholine dilinoleoyl phosphatidylcholine, diarachidonoyl phosphatidylcholine, diicosenoyl phosphatidylcholine (DEPC), diheptanoyl phosphatidylcholine, dicaproyl phosphatidylcholine, diheptadecanoyl phosphatidylcholine, dibehenoyl phosphatidylcholine, eleostearoyl phosphatidylcholine, hydrogenated egg phosphatidylcholine (HEPC), hydrogenated soy phosphatidylcholine (HSPC), 1-palmitoyl-2-arachidyl 1-palmitoyl-2-oleoylphosphatidylcholine, 1-palmitoyl-2-linoleoylphosphatidylcholine, 1-palmitoyl-2-myristoylphosphatidylcholine, 1-
• Phosphatidylserines include distearoylphosphatidylserine (DSPS), dimyristoylphosphatidylserine (DMPS), dilauroylphosphatidylserine (DLPS), dipalmitoylphosphatidylserine (DPPS), dioleoylphosphatidylserine (DOPS), lysophosphatidylserine, eleostearoylphosphatidylserine, and 1,2-di-(9-cis-octadecenoyl)-3-sn-phosphatidylserine.
• DSPS distearoylphosphatidylserine
• DMPS dimyristoylphosphatidylserine
• DLPS dilauroylphosphatidylserine
• DPPS dipalmitoylphosphatidylserine
• DOPS dioleoylphosphatidy
• Phosphatidylinositols include dipalmitoylphosphatidylinositol (DPPI), distearoylphosphatidylinositol (DSPI), dilauroylphosphatidylinositol (DLPI), and the like.
• DPPI dipalmitoylphosphatidylinositol
• DSPI distearoylphosphatidylinositol
• DLPI dilauroylphosphatidylinositol
• Phosphatidylglycerols include dipalmitoylphosphatidylglycerol (DPPG), distearoylphosphatidylglycerol (DSPG), dioleoylphosphatidylglycerol (DOPG), dilauroylphosphatidylglycerol (DLPG), dimyristoylphosphatidylglycerol (DMPG), lysophosphatidylglycerol, hydrogenated soybean phosphatidylglycerol (HSPG), hydrogenated egg phosphatidylglycerol (HEPG), and cardiolipin (diphosphatidylglycerol).
• DPPG dipalmitoylphosphatidylglycerol
• DSPG distearoylphosphatidylglycerol
• DOPG dioleoylphosphatidylglycerol
• DLPG dimyristoylphosphatidylglycerol
• DMPG
• Phosphatidylethanolamines include dipalmitoylphosphatidylethanolamine (DPPE), distearoylphosphatidylethanolamine (DSPE), dioleoylphosphatidylethanolamine (DOPE), dilauroylphosphatidylethanolamine (DLPE), dimyristoylphosphatidylethanolamine (DMPE), didecanoylphosphatidylethanolamine (DDPE), N-glutarylphosphatidylethanolamine (NGPE), lysophosphatidylethanolamine, N-(7-nitro-2,1,3-benzoxydiazol-4-yl)-1,2-dioleoyl-sn-phosphatidylethanolamine, eleostearoylphosphatidylethanolamine, N-succinyldioleoylphosphatidylethanolamine, and 1-hexadec
• distearoyl N-(3-maleimido-1-oxopropyl)-L- ⁇ -phosphatidylethanolamine (COATSOME (registered trademark) FE-8080MA3, manufactured by NOF Corporation), which is a distearoylphosphatidylethanolamine (DSPE) derivative having a maleimide group
• DSPE distearoylphosphatidylethanolamine
• the maleimide group can react with a thiol group that can be introduced at the end of the polymer that constitutes the site (I) to bond with the polymer that constitutes the site (I).
• phosphatidylethanolamines (and other lipid derivatives) that have functional groups such as maleimide groups and succinimide groups for linking with the site (I) together with hydrocarbon groups having various numbers of carbon atoms of 8 or more can also be used in a similar manner.
• Phosphatidic Acids include dipalmitoylphosphatidic acid (DPPA), distearoylphosphatidic acid (DSPA), dimyristoylphosphatidic acid (DMPA), and dioleylphosphatidic acid (DOPA).
• DPPA dipalmitoylphosphatidic acid
• DSPA distearoylphosphatidic acid
• DMPA dimyristoylphosphatidic acid
• DOPA dioleylphosphatidic acid
• Sphingophospholipids include sphingomyelin, dipalmitoylsphingomyelin, distearoylsphingomyelin, ceramide ciliatine, ceramide phosphorylethanolamine, and ceramide phosphorylglycerol.
• the most representative sterol is cholesterol.
• other sterols include, for example, cholesterol succinate, dihydrocholesterol, lanosterol, dihydrolanosterol, desmosterol, stigmasterol, sitosterol, campesterol, brassicasterol, zymosterol, ergosterol, campesterol, fucosterol, 22-ketosterol, 20-hydroxycholesterol, 7-hydroxycholesterol, 19-hydroxycholesterol, 22-hydroxycholesterol, 25-hydroxycholesterol, 7-dehydrocholesterol, 5 ⁇ -cholest-7-en-3 ⁇ -ol, epicholesterol, and dehydrocholesterol.
• hydroergosterol cholesterol sulfate, cholesterol hemisuccinate, cholesterol phthalate, cholesterol phosphate, cholesterol valerate, cholesterol hemisuccinate, 3 ⁇ N-(N',N'-dimethylaminoethane)-carbamoyl cholesterol, cholesterol acetate, cholesteryl oleate, cholesteryl linoleate, cholesteryl myristate, cholesteryl palmitate, cholesteryl arachidate, coprostanol, cholesterol ester, cholesteryl phosphorylcholine, and 3,6,9-trioxaoctane-1-ol-cholesteryl-3e-ol.
• Neutral lipids include, for example, diglycerides (e.g., diolein, dipalmitolein) and mixed caprylin-caprin diglycerides, triacylglycerols (triolein, tripalmitolein, trimyristolein, trilaurin, tricaprin, tricaprylin, tricaproin, etc.), squalene, and tocopherol.
• Saturated and unsaturated fatty acids include saturated or unsaturated fatty acids with 5 to 30 carbon atoms.
• organic molecules that have a hydrocarbon group with 8 or more carbon atoms can also be used as a source of the hydrocarbon group with 8 or more carbon atoms in the amphiphilic compound of the present invention.
• the hydrocarbon group having 8 or more carbon atoms is preferably bonded directly or via a divalent linking group to the moiety (I) containing the structural unit (A), or is present as part of a lipid (e.g., a phospholipid) that is bonded to the moiety (I) containing the structural unit (A) directly or via a divalent linking group.
• a lipid e.g., a phospholipid
• the hydrocarbon group having 8 or more carbon atoms may have a functional group at its end, similar to the polymer constituting the portion (I).
• the hydrocarbon group having 8 or more carbon atoms has a functional group at its end, it becomes possible to modify medicines and the like via the functional group.
• the hydrocarbon group having 8 or more carbon atoms does not have to have a functional group at its end.
• the hydrocarbon group having 8 or more carbon atoms has a functional group at its end, the functional group may be present at only one end of the hydrocarbon group having 8 or more carbon atoms, or may be present at both ends.
• the functional group present at the end of the hydrocarbon group having 8 or more carbon atoms may be located on the side where the hydrocarbon group having 8 or more carbon atoms is bonded to the portion (I), or it may be located on the opposite side.
• the molecular weight of the lipid is preferably 100 to 2000, and more preferably 150 to 1000.
• the ratio of the molecular weight of the lipid to the molecular weight of the amphipathic compound is preferably 2 to 50%, more preferably 5 to 35%, and even more preferably 10 to 20%.
• the divalent linking group is a group that links the portion (I) to a hydrocarbon group having 8 or more carbon atoms, and the structure is not particularly limited, and may be included as a part of the structure of the portion (I).
• R a is a hydrogen atom or a hydrocarbon group having 1 to 30 carbon atoms.
• the R b is a hydrocarbon group having 1 to 30 carbon atoms.
• a residue obtained by removing one hydrogen atom and one hydrocarbon group having 8 or more carbon atoms from a lipid (including modified lipids) is also one of the preferred forms of the divalent linking group.
• the divalent linking group preferably has a molecular weight of 5,000 or less, more preferably 2,000 or less, and even more preferably 1,000 or less.
• examples of a method for linking a hydrocarbon group having 8 or more carbon atoms to the site (I) (the polymer constituting the site (I)) via a divalent linking group include a method in which, when a monomer composition is polymerized in the presence of a chain transfer agent and/or a polymerization initiator to obtain a polymer constituting the site (I), a chain transfer agent or polymerization initiator containing a structure in the molecule that can form a site containing a hydrocarbon group having 8 or more carbon atoms is used as the chain transfer agent and/or polymerization initiator.
• a chain transfer agent octanethiol, decanethiol, dodecanethiol, hexadecanethiol, octadecanethiol, etc.
• a chain transfer agent octanethiol, decanethiol, dodecanethiol, hexadecanethiol, octadecanethiol, etc.
• a thiol group bonded to a hydrocarbon group having 8 or more carbon atoms (preferably 8 to 50 carbon atoms, more preferably 8 to 40, even more preferably 8 to 30, and particularly preferably 8 to 20)
• a hydrocarbon group having 8 or more carbon atoms preferably 8 to 50 carbon atoms, more preferably 8 to 40, even more preferably 8 to 30, and particularly preferably 8 to 20
• the amphiphilic compound is obtained by polymerizing a monomer composition containing a vinyl monomer (preferably a meth(acrylic monomer, more preferably glycerin mono(meth)acrylate) and a chain transfer agent having a thiol group (-SH group) bonded to a hydrocarbon group having 8 or more carbon atoms.
• a vinyl monomer preferably a meth(acrylic monomer, more preferably glycerin mono(meth)acrylate
• a chain transfer agent having a thiol group (-SH group) bonded to a hydrocarbon group having 8 or more carbon atoms preferably a meth(acrylic monomer, more preferably glycerin mono(meth)acrylate
• a compound having a functional group e.g., a maleimide group, etc.
• a functional group e.g., a maleimide group, etc.
• thiol group e.g., a maleimide group, etc.
• This method is particularly useful when the hydrocarbon group having 8 or more carbon atoms is present in an amphiphilic compound as part of a lipid or other organic molecule.
• the polymer constituting the portion (I) or the polymer in the case where a hydrocarbon group having 8 or more carbon atoms is present in the compound as part of the polymer may have a crosslinked structure.
• the modification rate (% (mol)) of the lipid nanoparticle with the amphipathic compound is 0.005% or more, 0.01% or more, 0.05% or more, 0.1% or more, 0.2% or more, 0.3% or more, 0.4% or more, 0.5% or more, 0.6% or more, 0.7% or more, 0.8% or more, or 1% or more.
• the modification rate (%) of the lipid nanoparticle with the amphipathic compound is less than 40%, 30% or less, particularly 20% or less, 18% or less, 16% or less, 15% or less, 12% or less, or 10% or less.
• the modification rate (%) of the lipid nanoparticle with the amphipathic compound may be 5% or less (e.g., 0.005% or more but less than 5%, or 0.01% or more but less than 5%).
• the modification rate (% (mol)) of the lipid nanoparticles with the amphipathic compound is 0.005% or more and less than 40%, 0.01% or more and less than 40%, 0.05% or more and less than 40%, 0.1 to 30%, 0.1 to 20%, 0.2 to 18%, 0.3 to 16%, 0.4 to 15%, 0.5 to 12%, 0.6 to 10%, 0.7 to 10%, 0.8 to 10%, 1 to 10%, 0.005 to 5%, or 0.005% or more and less than 5%.
• the modification rate is equal to or more than the lower limit
• the effect of improving the introduction of lipid nanoparticles into cells by the amphipathic compound is easily obtained.
• the modification rate is equal to or more than the lower limit
• the encapsulation efficiency of the encapsulated substance e.g., nucleic acid
• the blood retention of the lipid nanoparticles is improved.
• the modification rate is equal to or lower than the above upper limit
• the particle stability of the lipid nanoparticles is improved, the effect of improving the introduction of lipid nanoparticles into cells by the amphipathic compound is easily obtained, and the function is easily exerted (for example, when the encapsulated substance is a nucleic acid, high expression efficiency is easily obtained).
• the total of each lipid contained in the lipid nanoparticle is, for example, the total of phospholipids, cationic lipids, amphipathic compounds, and sterols. Therefore, in a preferred embodiment of the present invention, the modification rate (A) (%) shown below is 0.01% or more and less than 40%.
• the modification rate (A) (% (mol%)) may be any of 0.005% or more and less than 40%, 0.01% or more and less than 40%, 0.05% or more and less than 40%, 0.1 to 30%, 0.1 to 20%, 0.2 to 18%, 0.3 to 16%, 0.4 to 15%, 0.5 to 12%, 0.6 to 10%, 0.7 to 10%, 0.8 to 10%, 1 to 10%, 0.005 to 5%, or 0.005% or more and less than 5%.
• Modification rate (A) (%) ⁇ amphiphilic compound (mol) / total of phospholipids, cationic lipids, amphiphilic compound, and sterols contained in lipid nanoparticles (mol) ⁇ x 100 (%).
• the amount of amphipathic compound and the total amount of lipid in the lipid nanoparticles are the same as the amount of each lipid added during the manufacturing process, and the amount of amphipathic compound and the total amount of lipid can be measured by extracting the lipid nanoparticles using an organic solvent (e.g., ethanol or methanol) or a surfactant (e.g., Triton (registered trademark) X100 or Tween 80) and then using HPLC.
• an organic solvent e.g., ethanol or methanol
• a surfactant e.g., Triton (registered trademark) X100 or Tween 80
• phospholipids examples include the lipids described above in the section on amphipathic compounds.
• the phospholipids used in the lipid nanoparticles may be of one type alone or two or more types in combination.
• the phospholipids used in the lipid nanoparticles include phosphatidylcholines and phosphatidylethanolamines.
• phosphatidylcholines include those listed above. Phosphatidylcholines may be used alone or in combination of two or more types.
• phospholipids with an unsaturated structure since the function of the encapsulated substance is easily exerted (for example, when the encapsulated substance is a nucleic acid, high expression efficiency is easily obtained).
• phospholipids with an unsaturated structure include dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylserine (DOPS), dioleoylphosphatidylglycerol (DOPG), dioleoylphosphatidylethanolamine (DOPE), and dioleylphosphatidic acid (DOPA).
• DOPC dioleoylphosphatidylcholine
• DOPS dioleoylphosphatidylserine
• DOPG dioleoylphosphatidylglycerol
• DOPE dioleoylphosphatidylethanolamine
• DOPA dioleylphosphatidic acid
• phospholipid used in lipid nanoparticles is a combination of phosphatidylcholines and phospholipids having an unsaturated structure, preferably a combination of distearoylphosphatidylcholine (DSPC), hydrogenated egg phosphatidylcholine (HEPC), hydrogenated soy phosphatidylcholine (HSPC) or a mixture thereof and a phospholipid having an unsaturated structure, more preferably a combination of distearoylphosphatidylcholine (DSPC) and/or hydrogenated soy phosphatidylcholine (HSPC) and dioleoylphosphatidylethanolamine (DOPE).
• DSPC distearoylphosphatidylcholine
• HEPC hydrogenated egg phosphatidylcholine
• HSPC hydrogenated soy phosphatidylcholine
• DOPE dioleoylphosphatidylethanolamine
• the ratio (mol %) of phospholipids to the total lipids present in the lipid nanoparticles is, for example, 1 to 80 mol %, 2 to 70 mol %, or 2 to 60 mol %, and in one embodiment, 3 to 30 mol %, 5 to 20 mol %, or 8 to 18 mol %. Only one type of the above phospholipids may be used, or two or more types may be used in combination. When multiple phospholipids are used, it is preferable that the phospholipids as a whole have the above ratio. Moreover, the total lipids are preferably the sum of phospholipids, cationic lipids, amphipathic compounds, and sterols.
• ⁇ Cationic lipids When the substance encapsulated in the lipid nanoparticles is a negatively charged substance (e.g., nucleic acid, etc.), combining it with a cationic lipid makes it possible to encapsulate the substance when the dispersion medium of the lipid nanoparticles is hydrophilic, and also makes it possible to reduce the particle size of the resulting lipid nanoparticles.
• a negatively charged substance e.g., nucleic acid, etc.
• Cationic lipids are broadly composed of a hydrophobic portion and a hydrophilic portion.
• the hydrophobic portion contains hydrophobic groups such as fatty acid groups and sterol groups, while the hydrophilic portion contains cationic groups such as amino groups.
• Known cationic lipid compositions include a structure in which one hydrophobic group is included per hydrophilic group (hereinafter referred to as “single-chain cationic lipid”) and a structure in which one hydrophilic group contains two hydrophobic groups (hereinafter referred to as "double-chain cationic lipid”).
• Cationic lipids include, but are not limited to, heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate (SM-102), (4-hydroxybutyl)azanediyl)di(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), (6Z,9Z,28Z,3lZ)-heptatriaconta-6,9,28,3l-tetraen-19-yl-4-(dimethylamino)butanoate, (9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyloctadeca-9,12-dienoate, di((Z)-
• the cationic lipid is preferably a tertiary amine, more preferably a compound having no unsaturated bonds in the molecule and multiple ester bonds, even more preferably heptadecane-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate (SM-102) and/or (4-hydroxybutyl)azanediyl)di(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), and even more preferably heptadecane-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate (SM-102).
• SM-102 heptadecane-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(
• the cationic lipid is preferably a cationic lipid (double-chain cationic lipid) having two or more hydrophobic parts each having 8 or more carbon atoms.
• examples of cationic lipids include lipid molecules (ionized lipids) that are charge neutral at physiological pH (around pH 7.4) and protonated (up to H + ) in the acidic region (for example, pH 6.5 or lower).
• ionized lipids are usually tertiary amines.
• the fact that the cationic lipid is an ionic lipid is a preferred embodiment in terms of the ease with which an anionic encapsulated substance (particularly nucleic acid) can be functionalized and the retention in blood. Using such compounds, uptake into cells can also be controlled.
• cationic lipids may be used alone or in combination of two or more types.
• the molar ratio of the cationic lipid to the total lipids present in the lipid nanoparticles is 0.5 to 80 mol%, 0.5 to 75 mol%, 5 to 75 mol%, 5 to 70 mol%, or 10 to 70 mol%.
• the molar ratio of the cationic lipid to the total lipids present in the lipid nanoparticles may be 10 to 80 mol%, 15 to 80 mol%, 20 to 80 mol%, 25 to 75 mol%, or 40 to 75 mol%.
• the cationic lipid is at or above the lower limit, migration from the endosome to the cytoplasm is facilitated, and the encapsulation efficiency of the anionic encapsulated substance (particularly nucleic acid) is improved and/or the function is easily expressed, and when it is at or below the upper limit, particle formation is facilitated.
• Sterols include cholesterol, cholesterol succinate, dihydrocholesterol, lanosterol, dihydrolanosterol, desmosterol, stigmasterol, sitosterol, campesterol, brassicasterol, zymosterol, ergosterol, campesterol, fucosterol, 22-ketosterol, 20-hydroxycholesterol, 7-hydroxycholesterol, 19-hydroxycholesterol, 22-hydroxycholesterol, 25-hydroxycholesterol, 7-dehydrocholesterol, 5 ⁇ -cholest-7-en-3 ⁇ -ol, epicholesterol, and dehydrocholesterol.
• the sterol examples include hydroergosterol, cholesterol sulfate, cholesterol hemisuccinate, cholesterol phthalate, cholesterol phosphate, cholesterol valerate, cholesterol hemisuccinate, 3 ⁇ N-(N',N'-dimethylaminoethane)-carbamoyl cholesterol, cholesterol acetate, cholesteryl oleate, cholesteryl linoleate, cholesteryl myristate, cholesteryl palmitate, cholesteryl arachidate, coprostanol, cholesterol ester, cholesteryl phosphorylcholine, and 3,6,9-trioxaoctane-1-ol-cholesteryl-3e-ol.
• the sterol may be used alone or in combination of two or more. Of these, the sterol is preferably cholesterol.
• the content of sterol relative to the total lipid is 5 to 80 mol%, 10 to 75 mol%, 15 to 70 mol%, or 20 to 65 mol%. Having the sterol content within the above range facilitates the formation of lipid nanoparticles and ensures an appropriate particle size.
• a molar ratio improves the efficiency of uptake into cells, and makes it easier to obtain high expression efficiency, especially when the encapsulated substance is a nucleic acid.
• the molar ratio of the cationic lipid to the total lipids present in the lipid nanoparticles is 10 to 80 mol%, 15 to 80 mol%, 20 to 80 mol%, 25 to 75 mol%, or 40 to 75 mol%.
• the efficiency of uptake into cells is improved, and high expression efficiency is easily obtained, particularly when the encapsulated substance is a nucleic acid.
• the amount of nucleic acid encapsulated in the lipid nanoparticles is, for example, 0.01 to 20% by mass, or may be 0.05 to 15% by mass, or may be 0.1 to 10% by mass.
• the encapsulated substance is a nucleic acid, since this is an effective substance. That is, one embodiment of the present invention is a lipid nanoparticle that contains the above-mentioned amphiphilic compound and encapsulates a nucleic acid.
• nucleic acids include DNA, mRNA, siRNA, microRNA, gapmers, ribozymes, aptamers, artificial nucleic acids, CpG oligodeoxynucleotides, and antisense DNA/RNA.
• the total number of amine groups (N) of the cationic lipid relative to the total number of nucleic acid phosphate groups (P) (hereinafter also simply referred to as N/P ratio) is 0.01 to 55, 0.01 to 50, 0.03 to 50, 0.05 to 25, 1 to 25, 4 to 25, or 4 to 10.
• the molar ratio of cationic lipid to nucleic acid is, for example, 2,000 to 200,000, and may be 5,000 to 100,000.
• the lipid nanoparticles may contain other components.
• other components include water, physiological saline, medicamentically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymer, sodium carboxymethylcellulose, sodium polyacrylate, sodium alginate, water-soluble dextran, sodium carboxymethyl starch, pectin, methylcellulose, ethylcellulose, xanthan gum, gum arabic, casein, gelatin, agar, diglycerin, propylene glycol, polyethylene glycol, petrolatum, paraffin, stearyl alcohol, stearic acid, human serum albumin, mannitol, sorbitol, lactose, phosphate buffered saline, biodegradable polymers, serum-free medium, surfactants, oils and fats, pH adjusters, and physiological pH buffer solutions acceptable in vivo.
• These additives may be used alone or in combination of two or more.
• the method for producing lipid nanoparticles is not particularly limited, and conventionally known methods can be used.
• the encapsulated substance is nucleic acid
• a method can be used in which a lipid solution is obtained by dissolving phospholipid, cationic lipid, sterol and amphipathic compound in an organic solvent, and the lipid solution is mixed with a nucleic acid solution in which nucleic acid is dissolved in an aqueous solvent to obtain a lipid nanoparticle dispersion.
• the concentration of the nucleic acid in the aqueous solvent is, for example, 0.1 to 10,000 ⁇ g/mL, may be 0.5 to 1,000 ⁇ g/mL, or may be 0.5 to 100 ⁇ g/mL.
• the pH of the aqueous solvent is preferably pH 3.5 to 4.5.
• the temperature of the aqueous solvent is preferably 20 to 45° C., preferably 25 to 40° C.
• the organic solvent is preferably a polar organic solvent, such as ethanol, isopropanol, butanol, chloroform, etc.
• the organic solvent may be used alone or in combination of two or more kinds. Among them, it is preferable to use ethanol from the viewpoint of particle formability.
• the lipid concentration in the organic solvent is, for example, 0.01 to 100 mg/mL, may be 0.05 to 10 mg/mL, or may be 0.05 to 1 mg/mL.
• the lipid solution and the nucleic acid solution can be mixed using a microfluidic mixing system (e.g., Asia microfluidic system (Syrris) or Nanoassemblyr (Precision Nanosystems)).
• a microfluidic mixing system e.g., Asia microfluidic system (Syrris) or Nanoassemblyr (Precision Nanosystems)
• the flow rate is, for example, 0.1 to 20 mL/min.
• the mixing temperature is, for example, 4 to 45°C, and may be 10 to 35°C.
• the hydrophilic parts of the amphipathic compound and phospholipids are placed on the surface of the particles, creating a hydrophobic environment inside the particles, allowing complexes of hydrophobic nucleic acid and cationic lipid nanoparticles to be encapsulated.
• the dispersion medium is preferably an aqueous solvent.
• the aqueous solvent is not particularly limited, and examples thereof include water (sterile water), saline, phosphate buffered saline (PBS), citrate buffer, Tris buffer, carbonate buffer, acetate buffer, etc.
• the lipid nanoparticles may also be in the form of a powder obtained by freeze-drying or the like after removing the dispersion or the dispersion medium.
• the lipid nanoparticles of the present embodiment are excellent in intracellular uptake, and therefore can easily deliver a desired component or compound into cells. Therefore, the lipid nanoparticles of the present embodiment are preferably lipid nanoparticles for delivering an encapsulated substance into cells.
• compositions for intracellular delivery that contains the above-mentioned amphipathic compound, a nucleic acid, a phospholipid, a sterol, and a cationic lipid.
• the contents of each component in the composition are the same as those described for the lipid nanoparticles.
• the lipid nanoparticles of this embodiment are excellent in intracellular uptake and can therefore be used particularly for medicinal purposes.
• another embodiment of the present invention is a medicine containing the lipid nanoparticles.
• the pharmaceutical may be composed of the lipid nanoparticles according to the present disclosure, or may be a composition further containing other components.
• the other components include water, physiological saline, pharma- ceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymers, sodium carboxymethylcellulose, sodium polyacrylate, sodium alginate, water-soluble dextran, sodium carboxymethyl starch, pectin, methylcellulose, ethylcellulose, xanthan gum, gum arabic, casein, gelatin, agar, diglycerin, propylene glycol, polyethylene glycol, petrolatum, paraffin, stearyl alcohol, stearic acid, human serum albumin, mannitol, sorbitol, lactose, phosphate-buffered saline, biodegradable polymers, serum-free media, surfactants acceptable as pharmaceutical additives, and physiological pH buffers acceptable in vivo.
• the pharmaceutical containing the lipid nanoparticles of the present disclosure is used as a liquid formulation or a gel formulation.
• the liquid formulation preferably contains water, saline, phosphate buffered saline, citrate-phosphate buffer, etc.
• the gel formulation preferably contains a neutralized anionic polymer such as polyacrylic acid, a thickener such as carboxypolymethylene and carboxymethylcellulose, pemulene, a polymeric emulsifier, polycarbophil, or the like, a lower alcohol such as ethanol or isopropanol, and water.
• a neutralized anionic polymer such as polyacrylic acid
• a thickener such as carboxypolymethylene and carboxymethylcellulose
• pemulene such as carboxypolymethylene and carboxymethylcellulose
• a polymeric emulsifier such as polycarbophil, or the like
• a lower alcohol such as ethanol or isopropanol
• lipid nanoparticles or pharmaceuticals (compositions) containing lipid nanoparticles can be used in either in vitro or in vivo tests.
• the method of administration of lipid nanoparticles and the like to a living body is preferably parenteral administration, i.e., local administration such as intra-articular administration, intravenous administration, intraperitoneal administration, subcutaneous administration, intramuscular administration, intrathecal administration, intracisternal administration, intraventricular administration, or intradermal administration. They can also be used in ex vivo treatments in which cells or tissues are removed from an organism for treatment or therapy, and then returned to the original organism. Intravenous or intraperitoneal administration of the (pharmaceutical) composition can also be performed by bolus injection.
• lipid nanoparticles or pharmaceuticals containing lipid nanoparticles disclosed herein are expected to be pharmaceuticals that have high intracellular transport and good retention in the blood.
• amphiphilic compound (polymer) An amphiphilic compound having a polymeric form was prepared by the following procedure.
• the precipitated portion of the reaction solution obtained was dissolved in 1.0 g of ethanol, and further dropped into diethyl ether for purification, to obtain polyglycerin monomethacrylate containing an alkyl group (n-octadecyl group) at the end.
• the number average molecular weight of the obtained amphiphilic compound (polymer) was 14,000. Of these, the molecular weight of the hydrocarbon group (n-octadecyl group) having 8 or more carbon atoms was 253.5.
• nucleic acid-containing test materials ⁇ Nucleic Acid>
• siRNA and mRNA were used as the nucleic acid.
• Lusiferase siRNA was synthesized by Hokkaido System Science Co., Ltd. and annealed from the sequence (sense strand: CUUACGCUGAGUACUUCGAdTdT, antisense strand: UCGAAGUACUCAGCGUAAGdTdT) described in a known document (prior document JP 2010-77091).
• Lusiferase mRNA was purchased from TriLink BioTechnologies.
• nucleic acid encapsulation efficiency The nucleic acid encapsulation efficiency and encapsulation amount in lipid particles were measured by Ribogreen Assay using Quant-iT(TM) RiboGreen(TM) (ThermoFisher SCIENTIFIC). That is, the lipid particle suspension was diluted with HEPES buffer A (10 mM HEPES, 300 mM sucrose, pH 7.4) and HEPES buffer B (HEPES buffer A + 0.1% Triton X-100), respectively.
• HEPES buffer A 10 mM HEPES, 300 mM sucrose, pH 7.4
• HEPES buffer B HEPES buffer A + 0.1% Triton X-100
• Quant-iT RiboGreen RNA reagent diluted 2000 times with TE buffer was added, and the fluorescence intensity (excitation wavelength 485 nm, fluorescence wavelength 520 nm) was measured in a 96-well plate.
• the encapsulation efficiency (%) was calculated by the following formula.
• Encapsulation efficiency (%) (B - A) ⁇ B ⁇ 100 A (fluorescence intensity without Triton X-100 added), B (fluorescence intensity with Triton X-100 added)
• the amount of encapsulation was calculated from the measured values of each sample diluted with HEPES buffer B and measured, using a calibration curve prepared from values measured by the Ribogreen Assay method using siRNA or mRNA solutions of known concentrations.
• Luciferase mRNA (TriLink BioTechnologies) was dissolved in 25 mM acetate buffer pH 4.0 to obtain a nucleic acid solution of 1.8 ⁇ g/mL.
• the obtained lipid solution and nucleic acid solution were mixed at room temperature by NanoAssemblr (registered trademark) (Precision NanoSystems) at a flow rate ratio of 1.33 mL/min:1.33 mL/min to obtain a dispersion containing lipid particles encapsulating nucleic acids.
• the obtained dispersion was concentrated by centrifugation at 3,000 ⁇ G for 10 minutes using a centrifugal filter unit (NMWL: 30 kDa, Amicon (registered trademark) Ultra, Merck Ltd.), resuspended in PBS, washed again by centrifugation at 3,000 ⁇ G for 20 minutes, and stored as a lipid particle suspension at 4°C until use.
• the particle size of the lipid particles was measured using a Zetasizer Ultra (Malvern Panalytical).
• Luciferase mRNA (TriLink BioTechnologies) was dissolved in 25 mM acetate buffer pH 4.0 to obtain a nucleic acid solution of 1.8 ⁇ g/mL.
• the obtained lipid solution and nucleic acid solution were mixed at room temperature at a flow rate ratio of 1.33 mL/min:1.33 mL/min using NanoAssemblr (registered trademark) (Precision NanoSystems) to obtain a dispersion containing lipid particles encapsulating nucleic acids.
• the obtained dispersion was concentrated, washed, and subjected to particle size measurement in the same manner as in Example 1, and then stored at 4° C. until use.
• Luciferase mRNA (TriLink BioTechnologies) was dissolved in 25 mM acetate buffer pH 4.0 to obtain a nucleic acid solution of 3.5 ⁇ g/mL.
• the obtained lipid solution and nucleic acid solution were mixed at room temperature at a flow rate ratio of 1.33 mL/min:1.33 mL/min using NanoAssemblr (registered trademark) (Precision NanoSystems) to obtain a dispersion containing lipid particles encapsulating nucleic acids.
• the obtained dispersion was concentrated, washed, and subjected to particle size measurement in the same manner as in Example 1, and then stored at 4° C. until use.
• a lipid mixture hydrogenated soybean-derived phosphatidylcholine (Avanti Polar Lipids), cholesterol (Tokyo Chemical Industry Co., Ltd.), SM-102 (Funakoshi Co., Ltd.), and the polymer of Production Example 4 were mixed in a molar ratio of 13.9:48.5:27.6:10.0, and the molar ratio of SM-102 to nucleic acid was 10,000 (N/P ratio 5.2)) was dissolved in 99.5% (v/v) ethanol (Fujifilm Wako Pure Chemical Industries, Ltd.) to obtain a lipid solution of about 0.11 mg/mL.
• Luciferase mRNA (TriLink BioTechnologies) was dissolved in 25 mM acetate buffer pH 4.0 to obtain a nucleic acid solution of 3.5 ⁇ g/mL.
• the obtained lipid solution and nucleic acid solution were mixed at room temperature at a flow rate ratio of 1.33 mL/min:1.33 mL/min using NanoAssemblr (registered trademark) (Precision NanoSystems) to obtain a dispersion containing lipid particles encapsulating nucleic acids.
• the obtained dispersion was concentrated, washed, and subjected to particle size measurement in the same manner as in Example 1, and then stored at 4° C. until use.
• a lipid mixture hydroogenated soybean-derived phosphatidylcholine (Avanti Polar Lipids), cholesterol (Tokyo Chemical Industry Co., Ltd.), SM-102 (Funakoshi Co., Ltd.), and the polymer of Production Example 4 were mixed in a molar ratio of 10.6:37.1:42.2:20.0, and the molar ratio of SM-102 to nucleic acid was 10,000 (N/P ratio 5.2)) was dissolved in 99.5% (v/v) ethanol (Fujifilm Wako Pure Chemical Industries, Ltd.) to obtain a lipid solution of about 0.23 mg/mL.
• Luciferase mRNA (TriLink BioTechnologies) was dissolved in 25 mM acetate buffer pH 4.0 to obtain a nucleic acid solution of 1.8 ⁇ g/mL.
• the obtained lipid solution and nucleic acid solution were mixed at room temperature at a flow rate ratio of 1.33 mL/min:1.33 mL/min using NanoAssemblr (registered trademark) (Precision NanoSystems) to obtain a dispersion containing lipid particles encapsulating nucleic acid.
• the obtained dispersion was concentrated, washed, and subjected to particle size measurement in the same manner as in Example 1, and then stored at 4° C. until use.
• a lipid mixture hydrogenated soybean-derived phosphatidylcholine (Avanti Polar Lipids), cholesterol (Tokyo Chemical Industry Co., Ltd.), SM-102 (Funakoshi Co., Ltd.), and the polymer of Production Example 4 were mixed in a molar ratio of 11.7:40.8:46.5:1.0, and the molar ratio of SM-102 to nucleic acid was 20,000 (N/P ratio 10.4)) was dissolved in 99.5% (v/v) ethanol (Fujifilm Wako Pure Chemical Industries, Ltd.) to obtain a lipid solution of about 0.23 mg/mL.
• Luciferase mRNA (TriLink BioTechnologies) was dissolved in 25 mM acetate buffer pH 4.0 to obtain a nucleic acid solution of about 3.5 ⁇ g/mL.
• the obtained lipid solution and nucleic acid solution were mixed at room temperature at a flow rate ratio of 1.33 mL/min:1.33 mL/min using NanoAssemblr (registered trademark) (Precision NanoSystems) to obtain a dispersion containing lipid particles encapsulating nucleic acids.
• the obtained dispersion was concentrated, washed, and subjected to particle size measurement in the same manner as in Example 1, and then stored at 4° C. until use.
• a lipid mixture hydroogenated soybean-derived phosphatidylcholine (Avanti Polar Lipids), cholesterol (Tokyo Chemical Industry Co., Ltd.), SM-102 (Funakoshi Co., Ltd.), and the polymer of Production Example 4 were mixed in a molar ratio of 11.2:39.2:44.6:5.0, and the molar ratio of SM-102 to nucleic acid was 20,000 (N/P ratio 10.4)) was dissolved in 99.5% (v/v) ethanol (Fujifilm Wako Pure Chemical Industries, Ltd.) to obtain a lipid solution of about 0.23 mg/mL.
• a lipid mixture hydrogenated soybean-derived phosphatidylcholine (Avanti Polar Lipids), cholesterol (Tokyo Chemical Industry Co., Ltd.), SM-102 (Funakoshi Co., Ltd.), and the polymer of Production Example 4 were mixed in a molar ratio of 10.6:37.1:42.2:10.0, and the molar ratio of SM-102 to nucleic acid was 20,000 (N/P ratio 10.4)) was dissolved in 99.5% (v/v) ethanol (Fujifilm Wako Pure Chemical Industries, Ltd.) to obtain a lipid solution of about 0.23 mg/mL.
• Luciferase mRNA (TriLink BioTechnologies) was dissolved in 25 mM acetate buffer pH 4.0 to obtain a nucleic acid solution of 1.8 ⁇ g/mL.
• the obtained lipid solution and nucleic acid solution were mixed at room temperature at a flow rate ratio of 1.33 mL/min:1.33 mL/min using NanoAssemblr (registered trademark) (Precision NanoSystems) to obtain a dispersion containing lipid particles encapsulating nucleic acid.
• the obtained dispersion was concentrated, washed, and subjected to particle size measurement in the same manner as in Example 1, and then stored at 4° C. until use.
• a lipid mixture hydroogenated soybean-derived phosphatidylcholine (Avanti Polar Lipids), cholesterol (Tokyo Chemical Industry Co., Ltd.), SM-102 (Funakoshi Co., Ltd.), and the polymer of Production Example 4 were mixed in a molar ratio of 9.4:33.0:37.6:20.0, and the molar ratio of SM-102 to nucleic acid was 20,000 (N/P ratio 10.4)) was dissolved in 99.5% (v/v) ethanol (Fujifilm Wako Pure Chemical Industries, Ltd.) to obtain a lipid solution of about 0.23 mg/mL.
• Luciferase mRNA (TriLink BioTechnologies) was dissolved in 25 mM acetate buffer pH 4.0 to obtain a nucleic acid solution of 1.8 ⁇ g/mL.
• the obtained lipid solution and nucleic acid solution were mixed at room temperature at a flow rate ratio of 1.33 mL/min:1.33 mL/min using NanoAssemblr (registered trademark) (Precision NanoSystems) to obtain a dispersion containing lipid particles encapsulating nucleic acids.
• the obtained dispersion was concentrated, washed, and subjected to particle size measurement in the same manner as in Example 1, and then stored at 4° C. until use.
• a lipid mixture hydrogenated soybean-derived phosphatidylcholine (Avanti Polar Lipids), cholesterol (Tokyo Chemical Industry Co., Ltd.), SM-102 (Funakoshi Co., Ltd.), and the polymer of Production Example 4 were mixed in a molar ratio of 8.0:27.8:63.2:1.0, and the molar ratio of SM-102 to nucleic acid was 40,000 (N/P ratio 20.8)) was dissolved in 99.5% (v/v) ethanol (Fujifilm Wako Pure Chemical Industries, Ltd.) to obtain a lipid solution of about 0.23 mg/mL.
• a lipid mixture hydrogenated soybean-derived phosphatidylcholine (Avanti Polar Lipids), cholesterol (Tokyo Chemical Industry Co., Ltd.), SM-102 (Funakoshi Co., Ltd.), and the polymer of Production Example 4 were mixed in a molar ratio of 7.6:26.7:60.7:5.0, and the molar ratio of SM-102 to nucleic acid was 40,000 (N/P ratio 20.8)) was dissolved in 99.5% (v/v) ethanol (Fujifilm Wako Pure Chemical Industries, Ltd.) to obtain a lipid solution of about 0.23 mg/mL.
• Luciferase mRNA (TriLink BioTechnologies) was dissolved in 25 mM acetate buffer pH 4.0 to obtain a nucleic acid solution of 3.5 ⁇ g/mL.
• the obtained lipid solution and nucleic acid solution were mixed at room temperature at a flow rate ratio of 1.33 mL/min:1.33 mL/min using NanoAssemblr (registered trademark) (Precision NanoSystems) to obtain a dispersion containing lipid particles encapsulating nucleic acids.
• the obtained dispersion was concentrated, washed, and subjected to particle size measurement in the same manner as in Example 1, and then stored at 4° C. until use.
• a lipid mixture hydrogenated soybean-derived phosphatidylcholine (Avanti Polar Lipids), cholesterol (Tokyo Chemical Industry Co., Ltd.), SM-102 (Funakoshi Co., Ltd.), and the polymer of Production Example 4 were mixed in a molar ratio of 7.2:25.3:57.5:10.0, and the molar ratio of SM-102 to nucleic acid was 40,000 (N/P ratio 20.8)) was dissolved in 99.5% (v/v) ethanol (Fujifilm Wako Pure Chemical Industries, Ltd.) to obtain a lipid solution of about 0.23 mg/mL.
• Luciferase mRNA (TriLink BioTechnologies) was dissolved in 25 mM acetate buffer pH 4.0 to obtain a nucleic acid solution of 3.5 ⁇ g/mL.
• the obtained lipid solution and nucleic acid solution were mixed at room temperature at a flow rate ratio of 1.33 mL/min:1.33 mL/min using NanoAssemblr (registered trademark) (Precision NanoSystems) to obtain a dispersion containing lipid particles encapsulating nucleic acid.
• the obtained dispersion was concentrated, washed, and subjected to particle size measurement in the same manner as in Example 1, and then stored at 4° C. until use.
• Luciferase mRNA (TriLink BioTechnologies) was dissolved in 25 mM acetate buffer pH 4.0 to obtain a nucleic acid solution of 3.5 ⁇ g/mL.
• the obtained lipid solution and nucleic acid solution were mixed at room temperature at a flow rate ratio of 1.33 mL/min:1.33 mL/min using NanoAssemblr (registered trademark) (Precision NanoSystems) to obtain a dispersion containing lipid particles encapsulating nucleic acids.
• the obtained dispersion was concentrated, washed, and subjected to particle size measurement in the same manner as in Example 1, and then stored at 4° C. until use.
• a lipid mixture (hydrogenated soybean-derived phosphatidylcholine (Avanti Polar Lipids), cholesterol (Tokyo Chemical Industry Co., Ltd.), SM-102 (Funakoshi Co., Ltd.), PEG-phospholipid (NOF Corp., SUNBRIGHT (registered trademark) DSPE-020CN) in a molar ratio of 18.2:63.6:18.1:0.1, and a molar ratio of SM-102 to nucleic acid of 5000) was dissolved in 99.5% (v/v) ethanol (Fujifilm Wako Pure Chemical Industries, Ltd.) to obtain a lipid solution of about 0.11 mg/mL.
• a lipid mixture (hydrogenated soybean-derived phosphatidylcholine (Avanti Polar Lipids), cholesterol (Tokyo Chemical Industry Co., Ltd.), SM-102 (Funakoshi Co., Ltd.), PEG-phospholipid (NOF Corp., SUNBRIGHT (registered trademark) DSPE-020CN) in a molar ratio of 18.1:63.3:18.0:0.5, and a molar ratio of SM-102 to nucleic acid of 5000) was dissolved in 99.5% (v/v) ethanol (Fujifilm Wako Pure Chemical Industries, Ltd.) to obtain a lipid solution of about 0.11 mg/mL.
• a lipid mixture (hydrogenated soybean-derived phosphatidylcholine (Avanti Polar Lipids), cholesterol (Tokyo Chemical Industry Co., Ltd.), SM-102 (Funakoshi Co., Ltd.), PEG-phospholipid (NOF Corp., SUNBRIGHT (registered trademark) DSPE-020CN) in a molar ratio of 15.3:53.4:30.3:1.0, and a molar ratio of SM-102 to nucleic acid of 10,000) was dissolved in 99.5% (v/v) ethanol (Fujifilm Wako Pure Chemical Industries, Ltd.) to obtain a lipid solution of about 0.11 mg/mL.
• Luciferase mRNA (TriLink BioTechnologies) was dissolved in 25 mM acetate buffer pH 4.0 to obtain a nucleic acid solution of 1.8 ⁇ g/mL.
• the obtained lipid solution and nucleic acid solution were mixed at room temperature at a flow rate ratio of 1.33 mL/min:1.33 mL/min using NanoAssemblr (registered trademark) (Precision NanoSystems) to obtain a dispersion containing lipid particles encapsulating nucleic acids.
• the obtained dispersion was concentrated, washed, and subjected to particle size measurement in the same manner as in Example 1, and then stored at 4° C. until use.
• a lipid mixture (hydrogenated soybean-derived phosphatidylcholine (Avanti Polar Lipids), cholesterol (Tokyo Chemical Industry Co., Ltd.), SM-102 (Funakoshi Co., Ltd.), PEG-phospholipid (NOF Corp., SUNBRIGHT (registered trademark) DSPE-020CN) in a molar ratio of 14.7: 51.2: 29.1: 5.0, and a molar ratio of SM-102 to nucleic acid of 10000) was dissolved in 99.5% (v/v) ethanol (Fujifilm Wako Pure Chemical Industries, Ltd.) to obtain a lipid solution of about 0.11 mg/mL.
• a lipid mixture (hydrogenated soybean-derived phosphatidylcholine (Avanti Polar Lipids), cholesterol (Tokyo Chemical Industry Co., Ltd.), SM-102 (Funakoshi Co., Ltd.), PEG-phospholipid (NOF Corp., SUNBRIGHT (registered trademark) DSPE-020CN) in a molar ratio of 13.9: 48.5: 27.6: 10.0, and a molar ratio of SM-102 to nucleic acid of 10000) was dissolved in 99.5% (v/v) ethanol (Fujifilm Wako Pure Chemical Industries, Ltd.) to obtain a lipid solution of about 0.11 mg/mL.
• Luciferase mRNA (TriLink BioTechnologies) was dissolved in 25 mM acetate buffer pH 4.0 to obtain a nucleic acid solution of 3.5 ⁇ g/mL.
• the obtained lipid solution and nucleic acid solution were mixed at room temperature at a flow rate ratio of 1.33 mL/min:1.33 mL/min using NanoAssemblr (registered trademark) (Precision NanoSystems) to obtain a dispersion containing lipid particles encapsulating nucleic acids.
• the obtained dispersion was concentrated, washed, and subjected to particle size measurement in the same manner as in Example 1, and then stored at 4° C. until use.
• a lipid mixture (hydrogenated soybean-derived phosphatidylcholine (Avanti Polar Lipids), cholesterol (Tokyo Chemical Industry Co., Ltd.), SM-102 (Funakoshi Co., Ltd.), PEG-phospholipid (NOF Corp., SUNBRIGHT (registered trademark) DSPE-020CN) in a molar ratio of 12.4: 43.1: 24.5: 20.0, and a molar ratio of SM-102 to nucleic acid of 10000) was dissolved in 99.5% (v/v) ethanol (Fujifilm Wako Pure Chemical Industries, Ltd.) to obtain a lipid solution of about 0.23 mg/mL.
• Luciferase mRNA (TriLink BioTechnologies) was dissolved in 25 mM acetate buffer pH 4.0 to obtain a nucleic acid solution of 1.8 ⁇ g/mL.
• the obtained lipid solution and nucleic acid solution were mixed at room temperature at a flow rate ratio of 1.33 mL/min:1.33 mL/min using NanoAssemblr (registered trademark) (Precision NanoSystems) to obtain a dispersion containing lipid particles encapsulating nucleic acids.
• the obtained dispersion was concentrated, washed, and subjected to particle size measurement in the same manner as in Example 1, and then stored at 4° C. until use.
• a lipid mixture (hydrogenated soybean-derived phosphatidylcholine (Avanti Polar Lipids), cholesterol (Tokyo Chemical Industry Co., Ltd.), SM-102 (Funakoshi Co., Ltd.), PEG-phospholipid (NOF Corp., SUNBRIGHT (registered trademark) DSPE-020CN) in a molar ratio of 11.7: 40.8: 46.5: 1.0, and a molar ratio of SM-102 to nucleic acid of 20,000) was dissolved in 99.5% (v/v) ethanol (Fujifilm Wako Pure Chemical Industries, Ltd.) to obtain a lipid solution of about 0.23 mg/mL.
• a lipid mixture hydrogenated soybean-derived phosphatidylcholine (Avanti Polar Lipids), cholesterol (Tokyo Chemical Industry Co., Ltd.), SM-102 (Funakoshi Co., Ltd.), PEG-phospholipid (NOF Corp., SUNBRIGHT (registered trademark)
• Luciferase mRNA (TriLink BioTechnologies) was dissolved in 25 mM acetate buffer pH 4.0 to obtain a nucleic acid solution of about 3.5 ⁇ g/mL.
• the obtained lipid solution and nucleic acid solution were mixed at room temperature at a flow rate ratio of 1.33 mL/min:1.33 mL/min using NanoAssemblr (registered trademark) (Precision NanoSystems) to obtain a dispersion containing lipid particles encapsulating nucleic acids.
• the obtained dispersion was concentrated, washed, and subjected to particle size measurement in the same manner as in Example 1, and then stored at 4° C. until use.
• a lipid mixture (hydrogenated soybean-derived phosphatidylcholine (Avanti Polar Lipids), cholesterol (Tokyo Chemical Industry Co., Ltd.), SM-102 (Funakoshi Co., Ltd.), PEG-phospholipid (NOF Corp., SUNBRIGHT (registered trademark) DSPE-020CN) in a molar ratio of 11.2: 39.2: 44.6: 5.0, and a molar ratio of SM-102 to nucleic acid of 20,000) was dissolved in 99.5% (v/v) ethanol (Fujifilm Wako Pure Chemical Industries, Ltd.) to obtain a lipid solution of about 0.23 mg/mL.
• a lipid mixture hydrogenated soybean-derived phosphatidylcholine (Avanti Polar Lipids), cholesterol (Tokyo Chemical Industry Co., Ltd.), SM-102 (Funakoshi Co., Ltd.), PEG-phospholipid (NOF Corp., SUNBRIGHT (registered trademark)
• Luciferase mRNA (TriLink BioTechnologies) was dissolved in 25 mM acetate buffer pH 4.0 to obtain a nucleic acid solution of about 3.5 ⁇ g/mL.
• the obtained lipid solution and nucleic acid solution were mixed at room temperature at a flow rate ratio of 1.33 mL/min:1.33 mL/min using NanoAssemblr (registered trademark) (Precision NanoSystems) to obtain a dispersion containing lipid particles encapsulating nucleic acids.
• the obtained dispersion was concentrated, washed, and subjected to particle size measurement in the same manner as in Example 1, and then stored at 4° C. until use.
• a lipid mixture (hydrogenated soybean-derived phosphatidylcholine (Avanti Polar Lipids), cholesterol (Tokyo Chemical Industry Co., Ltd.), SM-102 (Funakoshi Co., Ltd.), PEG-phospholipid (NOF Corp., SUNBRIGHT (registered trademark) DSPE-020CN) in a molar ratio of 10.6: 37.1: 42.2: 10.0, and a molar ratio of SM-102 to nucleic acid of 20,000) was dissolved in 99.5% (v/v) ethanol (Fujifilm Wako Pure Chemical Industries, Ltd.) to obtain a lipid solution of about 0.23 mg/mL.
• a lipid mixture hydrogenated soybean-derived phosphatidylcholine (Avanti Polar Lipids), cholesterol (Tokyo Chemical Industry Co., Ltd.), SM-102 (Funakoshi Co., Ltd.), PEG-phospholipid (NOF Corp., SUNBRIGHT (registered trademark)
• Luciferase mRNA (TriLink BioTechnologies) was dissolved in 25 mM acetate buffer pH 4.0 to obtain a nucleic acid solution of 1.8 ⁇ g/mL.
• the obtained lipid solution and nucleic acid solution were mixed at room temperature at a flow rate ratio of 1.33 mL/min:1.33 mL/min using NanoAssemblr (registered trademark) (Precision NanoSystems) to obtain a dispersion containing lipid particles encapsulating nucleic acids.
• the obtained dispersion was concentrated, washed, and subjected to particle size measurement in the same manner as in Example 1, and then stored at 4° C. until use.
• a lipid mixture (hydrogenated soybean-derived phosphatidylcholine (Avanti Polar Lipids), cholesterol (Tokyo Chemical Industry Co., Ltd.), SM-102 (Funakoshi Co., Ltd.), PEG-phospholipid (NOF Corp., SUNBRIGHT (registered trademark) DSPE-020CN) in a molar ratio of 9.5: 33.0: 37.5: 20.0, and a molar ratio of SM-102 to nucleic acid of 20,000) was dissolved in 99.5% (v/v) ethanol (Fujifilm Wako Pure Chemical Industries, Ltd.) to obtain a lipid solution of about 0.23 mg/mL.
• a lipid mixture hydrogenated soybean-derived phosphatidylcholine (Avanti Polar Lipids), cholesterol (Tokyo Chemical Industry Co., Ltd.), SM-102 (Funakoshi Co., Ltd.), PEG-phospholipid (NOF Corp., SUNBRIGHT (registered trademark)
• Luciferase mRNA (TriLink BioTechnologies) was dissolved in 25 mM acetate buffer pH 4.0 to obtain a nucleic acid solution of 1.8 ⁇ g/mL.
• the obtained lipid solution and nucleic acid solution were mixed at room temperature at a flow rate ratio of 1.33 mL/min:1.33 mL/min using NanoAssemblr (registered trademark) (Precision NanoSystems) to obtain a dispersion containing lipid particles encapsulating nucleic acids.
• the obtained dispersion was concentrated, washed, and subjected to particle size measurement in the same manner as in Example 1, and then stored at 4° C. until use.
• a lipid mixture (hydrogenated soybean-derived phosphatidylcholine (Avanti Polar Lipids), cholesterol (Tokyo Chemical Industry Co., Ltd.), SM-102 (Funakoshi Co., Ltd.), PEG-phospholipid (NOF Corp., SUNBRIGHT (registered trademark) DSPE-020CN) in a molar ratio of 8.0:27.8:63.2:1.0, and a molar ratio of SM-102 to nucleic acid of 40,000) was dissolved in 99.5% (v/v) ethanol (Fujifilm Wako Pure Chemical Industries, Ltd.) to obtain a lipid solution of about 0.23 mg/mL.
• Luciferase mRNA (TriLink BioTechnologies) was dissolved in 25 mM acetate buffer pH 4.0 to obtain a nucleic acid solution of 3.5 ⁇ g/mL.
• the obtained lipid solution and nucleic acid solution were mixed at room temperature at a flow rate ratio of 1.33 mL/min:1.33 mL/min using NanoAssemblr (registered trademark) (Precision NanoSystems) to obtain a dispersion containing lipid particles encapsulating nucleic acids.
• the obtained dispersion was concentrated, washed, and subjected to particle size measurement in the same manner as in Example 1, and then stored at 4° C. until use.
• a lipid mixture (hydrogenated soybean-derived phosphatidylcholine (Avanti Polar Lipids), cholesterol (Tokyo Chemical Industry Co., Ltd.), SM-102 (Funakoshi Co., Ltd.), PEG-phospholipid (NOF Corp., SUNBRIGHT (registered trademark) DSPE-020CN) in a molar ratio of 7.6:26.7:60.7:5.0, and a molar ratio of SM-102 to nucleic acid of 40,000) was dissolved in 99.5% (v/v) ethanol (Fujifilm Wako Pure Chemical Industries, Ltd.) to obtain a lipid solution of about 0.23 mg/mL.
• a lipid mixture (hydrogenated soybean-derived phosphatidylcholine (Avanti Polar Lipids), cholesterol (Tokyo Chemical Industry Co., Ltd.), SM-102 (Funakoshi Co., Ltd.), PEG-phospholipid (NOF Corp., SUNBRIGHT (registered trademark) DSPE-020CN) in a molar ratio of 7.2:25.3:57.5:10.0, and a molar ratio of SM-102 to nucleic acid of 40,000) was dissolved in 99.5% (v/v) ethanol (Fujifilm Wako Pure Chemical Industries, Ltd.) to obtain a lipid solution of about 0.23 mg/mL.
• Luciferase mRNA (TriLink BioTechnologies) was dissolved in 25 mM acetate buffer pH 4.0 to obtain a nucleic acid solution of 3.5 ⁇ g/mL.
• the obtained lipid solution and nucleic acid solution were mixed at room temperature at a flow rate ratio of 1.33 mL/min:1.33 mL/min using NanoAssemblr (registered trademark) (Precision NanoSystems) to obtain a dispersion containing lipid particles encapsulating nucleic acids.
• the obtained dispersion was concentrated, washed, and subjected to particle size measurement in the same manner as in Example 1, and then stored at 4° C. until use.
• a lipid mixture (hydrogenated soybean-derived phosphatidylcholine (Avanti Polar Lipids), cholesterol (Tokyo Chemical Industry Co., Ltd.), SM-102 (Funakoshi Co., Ltd.), PEG-phospholipid (NOF Corp., SUNBRIGHT (registered trademark) DSPE-020CN) in a molar ratio of 6.4:22.5:51.1:20.0, and a molar ratio of SM-102 to nucleic acid of 40,000) was dissolved in 99.5% (v/v) ethanol (Fujifilm Wako Pure Chemical Industries, Ltd.) to obtain a lipid solution of about 0.23 mg/mL.
• Luciferase mRNA (TriLink BioTechnologies) was dissolved in 25 mM acetate buffer pH 4.0 to obtain a nucleic acid solution of 3.5 ⁇ g/mL.
• the obtained lipid solution and nucleic acid solution were mixed at room temperature at a flow rate ratio of 1.33 mL/min:1.33 mL/min using NanoAssemblr (registered trademark) (Precision NanoSystems) to obtain a dispersion containing lipid particles encapsulating nucleic acid.
• the obtained dispersion was concentrated, washed, and subjected to particle size measurement in the same manner as in Example 1, and then stored at 4° C. until use.
• a lipid mixture (hydrogenated soybean-derived phosphatidylcholine (Avanti Polar Lipids), cholesterol (Tokyo Chemical Industry Co., Ltd.), and SM-102 (Funakoshi Co., Ltd.) were mixed in a molar ratio of 11.8:41.3:46.9, and the molar ratio of SM-102 to nucleic acid was 20,000) was dissolved in 99.5% (v/v) ethanol (Fujifilm Wako Pure Chemical Industries, Ltd.) to obtain a lipid solution of about 0.23 mg/mL.
• Luciferase mRNA (TriLink BioTechnologies) was dissolved in 25 mM acetate buffer pH 4.0 to obtain a nucleic acid solution of about 3.5 ⁇ g/mL.
• the obtained lipid solution and nucleic acid solution were mixed at room temperature with a flow rate ratio of 1.33 mL/min:1.33 mL/min using NanoAssemblr (registered trademark) (Precision NanoSystems) to obtain a dispersion containing lipid particles encapsulating nucleic acid.
• the obtained dispersion was concentrated, washed, and subjected to particle size measurement in the same manner as in Example 1, and then stored at 4°C until use.
• the particle size of the particles in the obtained dispersion was 424 nm.
• a lipid mixture hydroogenated soybean-derived phosphatidylcholine (Avanti Polar Lipids), cholesterol (Tokyo Chemical Industry Co., Ltd.), SM-102 (Funakoshi Co., Ltd.), and the polymer of Production Example 4 were mixed in a m
• Luciferase mRNA (TriLink BioTechnologies) was dissolved in 25 mM acetate buffer pH 4.0 to obtain a nucleic acid solution of 1.8 ⁇ g/mL.
• the obtained lipid solution and nucleic acid solution were mixed at room temperature at a flow rate ratio of 1.33 mL/min:1.33 mL/min using a NanoAssemblr (registered trademark) (Precision NanoSystems) to obtain a dispersion containing lipid particles encapsulating nucleic acids.
• the obtained dispersion was concentrated, washed, and subjected to particle size measurement in the same manner as in Example 1, and no particle formation was observed.
• ⁇ Cell introduction test of mRNA-encapsulated lipid particles using cultured cells Human breast cancer cells (MCF-7, DS Pharma Biomedical) were cultured using DMEM medium (Nacalai Tesque) supplemented with 10% final concentration of fetal bovine serum (FBS) (DS Pharma Biomedical). The cells were seeded on a 100 mm cell culture dish (BD Falcon) to a density of 5.0 x 10 3 cells/cm 2 and cultured under conditions of 37°C and 5% CO 2.
• DMEM medium Nacalai Tesque
• FBS fetal bovine serum
• MCF-7 cultured to a 70% confluent state on a 100 mm cell culture dish was treated with a 0.25% trypsin/50 mM EDTA solution, and the above-mentioned serum-supplemented DMEM medium was added to stop the trypsin reaction, to obtain an MCF-7 cell suspension.
• the number of cells in the MCF-7 cell suspension was measured using a 0.4 w/v% trypan blue solution (Wako Pure Chemical Industries).
• the cell suspension was seeded in a 96-well plate (Thermo Fisher Science) so that the number of cells per well was 3.2 ⁇ 10 3 , and cultured for 24 hours under conditions of 37 ° C. and 5% CO 2.
• Relative light intensity (%) (B ⁇ A) ⁇ 100 A (luminescence amount in the case of addition of non-polymer-modified lipid particles), B (luminescence amount in the case of addition of polymer-modified lipid particles) The results are shown in Figure 1.
• the modifying substance contributes to particle stabilization
• unmodified lipid nanoparticles have higher intracellular migration, and usually, as in Comparative Example 8, modification results in a lower expression efficiency of the encapsulated substance (e.g., nucleic acid).
• the expression efficiency of the encapsulated substance is higher than that of unmodified lipid nanoparticles. This is thought to be because the lipid nanoparticles of this embodiment penetrate (translocate) into cells more easily than unmodified or PEG-modified lipid nanoparticles, and because the expression efficiency of the encapsulated substance (e.g., nucleic acid) is higher after penetration into cells.
• lipid mixture hydrophilic soybean-derived phosphatidylcholine (Avanti Polar Lipids), cholesterol (Tokyo Chemical Industry Co., Ltd.), stearyltrimethylammonium chloride (Fujifilm Wako Pure Chemical Industries, Ltd.), and the polymer of Production Example 4 were mixed in a molar ratio of 11.0:38.4:45.7:5.0, and the molar ratio of stearyltrimethylammonium chloride to nucleic acid was 250 (N/P ratio 0.13)) was dissolved in 99.5% (v/v) ethanol (Fujifilm Wako Pure Chemical Industries, Ltd.) to obtain a lipid solution of about 0.17 mg/mL.
• Luciferase siRNA was dissolved in 25 mM acetate buffer pH 4.0 to obtain a nucleic acid solution of 5.7 ⁇ g/mL.
• the obtained lipid solution and nucleic acid solution were mixed at room temperature at a flow rate ratio of 1.33 mL/min:1.33 mL/min using NanoAssemblr (registered trademark) (Precision NanoSystems) to obtain a dispersion containing lipid particles encapsulating nucleic acids.
• the obtained dispersion was concentrated, washed, and subjected to particle size measurement in the same manner as in Example 1, and then stored at 4° C. until use.
• Amphiphilic compound modification rate 10.0 mol%
• a lipid mixture (hydrogenated soybean-derived phosphatidylcholine (Avanti Polar Lipids), cholesterol (Tokyo Chemical Industry Co., Ltd.), stearyltrimethylammonium chloride (Fujifilm Wako Pure Chemical Industries, Ltd.), and the polymer of Production Example 4 were mixed in a molar ratio of 10.4:36.3:43.3:10.0, and the molar ratio of stearyltrimethylammonium chloride to nucleic acid was 250 (N/P ratio 0.13)) was dissolved in 99.5% (v/v) ethanol (Fujifilm Wako Pure Chemical Industries, Ltd.) to obtain a lipid solution of about 0.17 mg/mL.
• Luciferase siRNA was dissolved in 25 mM acetate buffer pH 4.0 to obtain a nucleic acid solution of 5.7 ⁇ g/mL.
• the obtained lipid solution and nucleic acid solution were mixed at room temperature at a flow rate ratio of 1.33 mL/min:1.33 mL/min using NanoAssemblr (registered trademark) (Precision NanoSystems) to obtain a dispersion containing lipid particles encapsulating nucleic acids.
• the obtained dispersion was concentrated, washed, and subjected to particle size measurement in the same manner as in Example 1, and then stored at 4° C. until use.
• Amphiphilic compound modification rate 20.0 mol%
• a lipid mixture (hydrogenated soybean-derived phosphatidylcholine (Avanti Polar Lipids), cholesterol (Tokyo Chemical Industry Co., Ltd.), stearyltrimethylammonium chloride (Fujifilm Wako Pure Chemical Industries, Ltd.), and the polymer of Production Example 4 were mixed in a molar ratio of 9.3:32.3:38.5:20.0, and the molar ratio of stearyltrimethylammonium chloride to nucleic acid was 250 (N/P ratio 0.13)) was dissolved in 99.5% ethanol (Fujifilm Wako Pure Chemical Industries, Ltd.) to obtain a lipid solution of about 0.17 mg/mL.
• Luciferase siRNA was dissolved in 25 mM acetate buffer pH 4.0 to obtain a nucleic acid solution of 5.7 ⁇ g/mL.
• the obtained lipid solution and nucleic acid solution were mixed at room temperature at a flow rate ratio of 1.33 mL/min:1.33 mL/min using NanoAssemblr (registered trademark) (Precision NanoSystems) to obtain a dispersion containing lipid particles encapsulating nucleic acids.
• the obtained dispersion was concentrated, washed, and subjected to particle size measurement in the same manner as in Example 1, and then stored at 4° C. until use.
• Amphiphilic compound modification rate 10.0 mol%
• a lipid mixture (hydrogenated soybean-derived phosphatidylcholine (Avanti Polar Lipids), cholesterol (Tokyo Chemical Industry Co., Ltd.), stearyltrimethylammonium chloride (FUJIFILM Wako Pure Chemical Industries, Ltd.), PEG-phospholipid (NOF Corp., SUNBRIGHT (registered trademark) DSPE-020CN) in a molar ratio of 10.4: 36.3: 43.3: 10.0, and a molar ratio of stearyltrimethylammonium chloride to nucleic acid of 250 (N / P ratio 0.13) was mixed) was dissolved in 99.5% ethanol (FUJIFILM Wako Pure Chemical Industries, Ltd.) to obtain a lipid solution of about 0.17 mg / mL.
• Luciferase siRNA was dissolved in 25 mM acetate buffer pH 4.0 to obtain a nucleic acid solution of 5.7 ⁇ g / mL.
• the obtained lipid solution and nucleic acid solution were mixed at room temperature at a flow rate ratio of 1.33 mL/min:1.33 mL/min using NanoAssemblr (registered trademark) (Precision NanoSystems) to obtain a dispersion containing lipid particles encapsulating nucleic acids.
• the obtained dispersion was concentrated, washed, and subjected to particle size measurement in the same manner as in Example 1, and then stored at 4° C. until use.
• ⁇ Cell introduction test of siRNA-encapsulated lipid particles using cultured cells Luciferase-expressing human breast adenocarcinoma-derived cells (MCF-7/Luc, Cosmo Bio Co., Ltd.) were cultured using DMEM medium (Nacalai Tesque) supplemented with 10% final concentration of fetal bovine serum (FBS) (DS Pharma Biomedical Co., Ltd.). The cells were seeded on a 100 mm cell culture dish (BD Falcon) to a density of 5.0 x 10 3 cells/cm 2 and cultured under conditions of 37°C and 5% CO 2.
• DMEM medium Nacalai Tesque
• FBS fetal bovine serum
• Example 18 A lipid mixture (hydrogenated soybean-derived phosphatidylcholine (Avanti Polar Lipids), cholesterol (Tokyo Chemical Industry Co., Ltd.), and the polymer of Production Example 4 mixed in a molar ratio of 21.5:75.0:3.5) was dissolved in 99.5% (v/v) ethanol (Fujifilm Wako Pure Chemical Industries, Ltd.) to obtain a lipid solution of about 0.17 mg/mL. Luciferase siRNA was dissolved in 25 mM acetate buffer pH 4.0 to obtain a nucleic acid solution of 4.0 ⁇ g/mL.
• the obtained lipid solution and nucleic acid solution were mixed at room temperature by NanoAssemblr (registered trademark) (Precision NanoSystems) at a flow rate ratio of 1.33 mL/min:1.33 mL/min to obtain a dispersion containing lipid particles encapsulating nucleic acids.
• the resulting dispersion was concentrated, washed, and subjected to particle size measurement in the same manner as in Example 1, and then stored at 4° C. until use.
• Luciferase siRNA was dissolved in 25 mM acetate buffer pH 4.0 to obtain a nucleic acid solution of 4.0 ⁇ g/mL.
• the obtained lipid solution and nucleic acid solution were mixed at room temperature at a flow rate ratio of 1.33 mL/min:1.33 mL/min using NanoAssemblr (registered trademark) (Precision NanoSystems) to obtain a dispersion containing lipid particles encapsulating nucleic acid.
• the obtained dispersion was concentrated, washed, and subjected to particle size measurement in the same manner as in Example 1, and then stored at 4° C. until use.
• Luciferase siRNA was dissolved in 25 mM acetate buffer pH 4.0 to obtain a nucleic acid solution of 4.0 ⁇ g/mL.
• the obtained lipid solution and nucleic acid solution were mixed at room temperature at a flow rate ratio of 1.33 mL/min:1.33 mL/min using NanoAssemblr (registered trademark) (Precision NanoSystems) to obtain a dispersion containing lipid particles encapsulating nucleic acids.
• the obtained dispersion was concentrated, washed, and subjected to particle size measurement in the same manner as in Example 1, and then stored at 4° C. until use.
• Luciferase mRNA (TriLink BioTechnologies) was dissolved in 25 mM acetate buffer pH 4.0 to obtain a 3.5 ⁇ g/mL nucleic acid solution.
• the obtained lipid solution and nucleic acid solution were mixed at room temperature with NanoAssemblr (registered trademark) (Precision NanoSystems) at a flow rate ratio of 1.33 mL/min:1.33 mL/min to obtain a dispersion containing lipid particles encapsulating nucleic acid.
• the obtained dispersion was concentrated, washed, and subjected to particle size measurement in the same manner as in Example 1, and then stored at 4 ° C. until use.
• Luciferase mRNA (TriLink BioTechnologies) was dissolved in 25 mM acetate buffer pH 4.0 to obtain a 3.5 ⁇ g/mL nucleic acid solution.
• the obtained lipid solution and nucleic acid solution were mixed at room temperature with NanoAssemblr (registered trademark) (Precision NanoSystems) at a flow rate ratio of 1.33 mL/min:1.33 mL/min to obtain a dispersion containing lipid particles encapsulating nucleic acid.
• the obtained dispersion was concentrated, washed, and subjected to particle size measurement in the same manner as in Example 1, and then stored at 4 ° C. until use.
• Luciferase mRNA (TriLink BioTechnologies) was dissolved in 25 mM acetate buffer pH 4.0 to obtain a 3.5 ⁇ g/mL nucleic acid solution.
• the obtained lipid solution and nucleic acid solution were mixed at room temperature with NanoAssemblr (registered trademark) (Precision NanoSystems) at a flow rate ratio of 1.33 mL/min:1.33 mL/min to obtain a dispersion containing lipid particles encapsulating nucleic acid.
• the obtained dispersion was concentrated, washed, and subjected to particle size measurement in the same manner as in Example 1, and then stored at 4 ° C. until use.
• Amphiphilic compound modification rate 1.5 mol% (phospholipid:sterol approximately 1:0.7 (molar ratio))
• Luciferase mRNA (TriLink BioTechnologies) was dissolved in 25 mM acetate buffer pH 4.0 to obtain a 3.5 ⁇ g/mL nucleic acid solution.
• the obtained lipid solution and nucleic acid solution were mixed at room temperature with NanoAssemblr (registered trademark) (Precision NanoSystems) at a flow rate ratio of 1.33 mL/min:1.33 mL/min to obtain a dispersion containing lipid particles encapsulating nucleic acid.
• the obtained dispersion was concentrated, washed, and subjected to particle size measurement in the same manner as in Example 1, and then stored at 4 ° C. until use.
• Luciferase mRNA (TriLink BioTechnologies) was dissolved in 25 mM acetate buffer pH 4.0 to obtain a 3.5 ⁇ g/mL nucleic acid solution.
• the obtained lipid solution and nucleic acid solution were mixed at room temperature with NanoAssemblr (registered trademark) (Precision NanoSystems) at a flow rate ratio of 1.33 mL/min:1.33 mL/min to obtain a dispersion containing lipid particles encapsulating nucleic acid.
• the obtained dispersion was concentrated, washed, and subjected to particle size measurement in the same manner as in Example 1, and then stored at 4 ° C. until use.
• Luciferase mRNA (TriLink BioTechnologies) was dissolved in 25 mM acetate buffer pH 4.0 to obtain a 3.5 ⁇ g/mL nucleic acid solution.
• the obtained lipid solution and nucleic acid solution were mixed at room temperature with NanoAssemblr (registered trademark) (Precision NanoSystems) at a flow rate ratio of 1.33 mL/min:1.33 mL/min to obtain a dispersion containing lipid particles encapsulating nucleic acid.
• the obtained dispersion was concentrated, washed, and subjected to particle size measurement in the same manner as in Example 1, and then stored at 4 ° C. until use.
• Luciferase mRNA (TriLink BioTechnologies) was dissolved in 25 mM acetate buffer pH 4.0 to obtain a 3.5 ⁇ g/mL nucleic acid solution.
• the obtained lipid solution and nucleic acid solution were mixed at room temperature with NanoAssemblr (registered trademark) (Precision NanoSystems) at a flow rate ratio of 1.33 mL/min:1.33 mL/min to obtain a dispersion containing lipid particles encapsulating nucleic acid.
• the obtained dispersion was concentrated, washed, and subjected to particle size measurement in the same manner as in Example 1, and then stored at 4 ° C. until use.
• Luciferase mRNA (TriLink BioTechnologies) was dissolved in 25 mM acetate buffer pH 4.0 to obtain a 3.5 ⁇ g/mL nucleic acid solution.
• the obtained lipid solution and nucleic acid solution were mixed at room temperature with NanoAssemblr (registered trademark) (Precision NanoSystems) at a flow rate ratio of 1.33 mL/min:1.33 mL/min to obtain a dispersion containing lipid particles encapsulating nucleic acid.
• the obtained dispersion was concentrated, washed, and subjected to particle size measurement in the same manner as in Example 1, and then stored at 4 ° C. until use.
• Luciferase mRNA (TriLink BioTechnologies) was dissolved in 25 mM acetate buffer pH 4.0 to obtain a 3.5 ⁇ g/mL nucleic acid solution.
• the obtained lipid solution and nucleic acid solution were mixed at room temperature with NanoAssemblr (registered trademark) (Precision NanoSystems) at a flow rate ratio of 1.33 mL/min:1.33 mL/min to obtain a dispersion containing lipid particles encapsulating nucleic acid.
• the obtained dispersion was concentrated, washed, and subjected to particle size measurement in the same manner as in Example 1, and then stored at 4 ° C. until use.
• Phospholipid:sterol about 1:0.2 (molar ratio) (modification rate of amphiphilic compound: 0.45 mol%)
• Luciferase mRNA (TriLink BioTechnologies) was dissolved in 25 mM acetate buffer pH 4.0 to obtain a 3.5 ⁇ g/mL nucleic acid solution.
• the obtained lipid solution and nucleic acid solution were mixed at room temperature with NanoAssemblr (registered trademark) (Precision NanoSystems) at a flow rate ratio of 1.33 mL/min:1.33 mL/min to obtain a dispersion containing lipid particles encapsulating nucleic acid.
• the obtained dispersion was concentrated, washed, and subjected to particle size measurement in the same manner as in Example 1, and then stored at 4 ° C. until use.
• Phospholipid:sterol about 1:0.5 (molar ratio) (modification rate of amphiphilic compound: 0.45 mol%)
• Luciferase mRNA (TriLink BioTechnologies) was dissolved in 25 mM acetate buffer pH 4.0 to obtain a 3.5 ⁇ g/mL nucleic acid solution.
• the obtained lipid solution and nucleic acid solution were mixed at room temperature with NanoAssemblr (registered trademark) (Precision NanoSystems) at a flow rate ratio of 1.33 mL/min:1.33 mL/min to obtain a dispersion containing lipid particles encapsulating nucleic acid.
• the obtained dispersion was concentrated, washed, and subjected to particle size measurement in the same manner as in Example 1, and then stored at 4 ° C. until use.
• Phospholipid:sterol about 1:1 (molar ratio) (modification rate of amphiphilic compound: 0.45 mol%)
• Luciferase mRNA (TriLink BioTechnologies) was dissolved in 25 mM acetate buffer pH 4.0 to obtain a 3.5 ⁇ g/mL nucleic acid solution.
• the obtained lipid solution and nucleic acid solution were mixed at room temperature with NanoAssemblr (registered trademark) (Precision NanoSystems) at a flow rate ratio of 1.33 mL/min:1.33 mL/min to obtain a dispersion containing lipid particles encapsulating nucleic acid.
• the obtained dispersion was concentrated, washed, and subjected to particle size measurement in the same manner as in Example 1, and then stored at 4 ° C. until use.
• Phospholipid:sterol about 1:4 (molar ratio) (modification rate of amphiphilic compound: 0.45 mol%)
• Phospholipid:sterol about 1:10 (molar ratio) (modification rate of amphiphilic compound: 0.45 mol%)
• Luciferase mRNA (TriLink BioTechnologies) was dissolved in 25 mM acetate buffer pH 4.0 to obtain a 3.5 ⁇ g/mL nucleic acid solution.
• the obtained lipid solution and nucleic acid solution were mixed at room temperature with NanoAssemblr (registered trademark) (Precision NanoSystems) at a flow rate ratio of 1.33 mL/min:1.33 mL/min to obtain a dispersion containing lipid particles encapsulating nucleic acid.
• the obtained dispersion was concentrated, washed, and subjected to particle size measurement in the same manner as in Example 1, and then stored at 4 ° C. until use.
• Phospholipid:sterol about 1:20 (molar ratio) (modification rate of amphiphilic compound 0.45 mol%)
• Luciferase mRNA (TriLink BioTechnologies) was dissolved in 25 mM acetate buffer pH 4.0 to obtain a 3.5 ⁇ g/mL nucleic acid solution.
• the obtained lipid solution and nucleic acid solution were mixed at room temperature with NanoAssemblr (registered trademark) (Precision NanoSystems) at a flow rate ratio of 1.33 mL/min:1.33 mL/min to obtain a dispersion containing lipid particles encapsulating nucleic acid.
• the obtained dispersion was concentrated, washed, and subjected to particle size measurement in the same manner as in Example 1, and then stored at 4 ° C. until use.
• Phospholipid:sterol about 1:40 (molar ratio) (modification rate of amphiphilic compound 0.45 mol%)
• Luciferase mRNA (TriLink BioTechnologies) was dissolved in 25 mM acetate buffer pH 4.0 to obtain a 3.5 ⁇ g/mL nucleic acid solution.
• the obtained lipid solution and nucleic acid solution were mixed at room temperature with NanoAssemblr (registered trademark) (Precision NanoSystems) at a flow rate ratio of 1.33 mL/min:1.33 mL/min to obtain a dispersion containing lipid particles encapsulating nucleic acid.
• the obtained dispersion was concentrated, washed, and subjected to particle size measurement in the same manner as in Example 1, and then stored at 4 ° C. until use.
• Luciferase mRNA (TriLink BioTechnologies) was dissolved in 25 mM acetate buffer pH 4.0 to obtain a 3.5 ⁇ g/mL nucleic acid solution.
• the obtained lipid solution and nucleic acid solution were mixed at room temperature with NanoAssemblr (registered trademark) (Precision NanoSystems) at a flow rate ratio of 1.33 mL/min:1.33 mL/min to obtain a dispersion containing lipid particles encapsulating nucleic acid.
• the obtained dispersion was concentrated, washed, and subjected to particle size measurement in the same manner as in Example 1, and then stored at 4 ° C. until use.
• Cationic lipid molar ratio 21.0 mol% (phospholipid:sterol approximately 1:0.7 (molar ratio), amphipathic compound modification rate 0.45 mol%)
• Luciferase mRNA (TriLink BioTechnologies) was dissolved in 25 mM acetate buffer pH 4.0 to obtain a 3.5 ⁇ g/mL nucleic acid solution.
• the obtained lipid solution and nucleic acid solution were mixed at room temperature with NanoAssemblr (registered trademark) (Precision NanoSystems) at a flow rate ratio of 1.33 mL/min:1.33 mL/min to obtain a dispersion containing lipid particles encapsulating nucleic acid.
• the obtained dispersion was concentrated, washed, and subjected to particle size measurement in the same manner as in Example 1, and then stored at 4 ° C. until use.
• Cationic lipid molar ratio 35.0% (phospholipid:sterol approximately 1:0.7 (molar ratio), modification rate of amphipathic compound 0.45 mol%)
• Luciferase mRNA (TriLink BioTechnologies) was dissolved in 25 mM acetate buffer pH 4.0 to obtain a 3.5 ⁇ g/mL nucleic acid solution.
• the obtained lipid solution and nucleic acid solution were mixed at room temperature with NanoAssemblr (registered trademark) (Precision NanoSystems) at a flow rate ratio of 1.33 mL/min:1.33 mL/min to obtain a dispersion containing lipid particles encapsulating nucleic acid.
• the obtained dispersion was concentrated, washed, and subjected to particle size measurement in the same manner as in Example 1, and then stored at 4 ° C. until use.
• Luciferase mRNA (TriLink BioTechnologies) was dissolved in 25 mM acetate buffer pH 4.0 to obtain a 3.5 ⁇ g/mL nucleic acid solution.
• the obtained lipid solution and nucleic acid solution were mixed at room temperature with NanoAssemblr (registered trademark) (Precision NanoSystems) at a flow rate ratio of 1.33 mL/min:1.33 mL/min to obtain a dispersion containing lipid particles encapsulating nucleic acid.
• the obtained dispersion was concentrated, washed, and subjected to particle size measurement in the same manner as in Example 1, and then stored at 4 ° C. until use.
• Luciferase mRNA (TriLink BioTechnologies) was dissolved in 25 mM acetate buffer pH 4.0 to obtain a 3.5 ⁇ g/mL nucleic acid solution.
• the obtained lipid solution and nucleic acid solution were mixed at room temperature with NanoAssemblr (registered trademark) (Precision NanoSystems) at a flow rate ratio of 1.33 mL/min:1.33 mL/min to obtain a dispersion containing lipid particles encapsulating nucleic acid.
• the obtained dispersion was concentrated, washed, and subjected to particle size measurement in the same manner as in Example 1, and then stored at 4 ° C. until use.
• Luciferase mRNA (TriLink BioTechnologies) was dissolved in 25 mM acetate buffer pH 4.0 to obtain a 3.5 ⁇ g/mL nucleic acid solution.
• the obtained lipid solution and nucleic acid solution were mixed at room temperature with NanoAssemblr (registered trademark) (Precision NanoSystems) at a flow rate ratio of 1.33 mL/min:1.33 mL/min to obtain a dispersion containing lipid particles encapsulating nucleic acid.
• the obtained dispersion was concentrated, washed, and subjected to particle size measurement in the same manner as in Example 1, and then stored at 4 ° C. until use.
• nucleic acid recovery rate was calculated according to the following formula.
• Nucleic acid recovery rate [%] (amount of nucleic acid recovered by destroying the prepared lipid nanoparticles ( ⁇ g) / total amount of nucleic acid used in preparing the lipid nanoparticles ( ⁇ g)) ⁇ 100
• the “amount of nucleic acid that could be recovered by destroying the prepared lipid nanoparticles” was calculated from the product of the nucleic acid concentration calculated in the same manner as the encapsulation amount described in the encapsulation efficiency column above and the amount of lipid nanoparticle suspension recovered.
• the nucleic acid recovery rates of Comparative Example 15 and Examples 21 to 42 are shown in Figure 4.
• the nucleic acid recovery rates of the LNPs prepared in Examples 21 to 42 all showed higher values than Comparative Example 15. Focusing on the polymer modification rate, extremely high recovery rates were shown in Examples 23 to 28, which had high modification rates, while surprisingly, a significant improvement in recovery rate was observed compared to Comparative Example 15 even in Examples 21 and 22, which had low modification rates. Focusing on the phospholipid sterol ratio, a significant improvement in recovery rate was observed compared to Comparative Example 15 in all ranges examined in Examples 29 to 36.
• ⁇ Gene expression test using lipid nanoparticles with different polymer modification rates> Human breast cancer cells (MCF-7, DS Pharma Biomedical) were cultured using DMEM medium (Nacalai Tesque) supplemented with 10% final concentration of fetal bovine serum (FBS) (DS Pharma Biomedical). The cells were seeded on a 100 mm cell culture dish (BD Falcon) to a density of 5.0 x 10 3 cells/cm 2 and cultured under conditions of 37°C and 5% CO 2.
• DMEM medium Nacalai Tesque
• FBS fetal bovine serum
• MCF-7 cultured to a 70% confluent state on a 100 mm cell culture dish was treated with a 0.25% trypsin/50 mM EDTA solution, and the above-mentioned serum-supplemented DMEM medium was added to stop the trypsin reaction, to obtain an MCF-7 cell suspension.
• the number of cells in the MCF-7 cell suspension was measured using a 0.4 w/v% trypan blue solution (Wako Pure Chemical Industries).
• the cell suspension was seeded in a 96-well plate (Thermo Fisher Science) so that the number of cells per well was 3.2 ⁇ 10 3 , and cultured for 24 hours under conditions of 37 ° C. and 5% CO 2.
• the medium was replaced with serum-free DMEM medium from which FBS had been removed, and 50 ⁇ L of a dilution solution prepared with serum-free DMEM medium of the lipid particle suspension prepared in Examples 21 to 28 and Comparative Example 15 was added to each well so that the amount of nucleic acid encapsulated was 200 ng / mL.
• 50 ⁇ L of ONE-Glo (TM) EX Luciferase Assay System (Promega Corporation) was added per well, and the plate was stirred at 600 rpm for 3 minutes with a plate shaker, and the amount of luminescence was measured with a plate reader SH-9000 (Corona Electric Co., Ltd.).
• Relative light intensity (%) (B ⁇ A) ⁇ 100 A (luminescence intensity in the lipid particle-added area of Comparative Example 15), B (luminescence intensity in the lipid particle-added area with each polymer modification rate) The results are shown in Figure 5.
• the lipid particles of Examples 21 to 28 showed the same or increased gene expression levels.
• the modification rate was extremely low at 0.01%, but the particle size maintained a good size of 200 nm or less.
• the polymer modification rate is high, the stability of the particles decreases and it becomes difficult to maintain the particle shape, but lipid particles that maintained an appropriate particle size were confirmed even when the polymer modification rate was 30%.
• Powdered DMEM medium (D2902, Merck) was dissolved in 1 L of high-pressure steam sterilized ultrapure water, sodium bicarbonate (Wako Pure Chemical Industries) was added, the pH was adjusted to 6.7, and the mixture was sterilized by filtration to prepare low pH-DMEM medium (pH 6.7).
• MCF-7 Human breast cancer cells
• DMEM medium Nacalai Tesque
• FBS fetal bovine serum
• the cells were seeded on a 100 mm cell culture dish (BD Falcon) to a density of 5.0 x 10 3 cells/cm 2 and cultured under conditions of 37°C and 5% CO 2.
• MCF-7 cultured to a 70% confluent state on a 100 mm cell culture dish was treated with a 0.25% trypsin/50 mM EDTA solution, and the above-mentioned serum-supplemented DMEM medium was added to stop the trypsin reaction, to obtain an MCF-7 cell suspension.
• the number of cells in the MCF-7 cell suspension was measured using a 0.4 w/v% trypan blue solution (Wako Pure Chemical Industries).
• the cell suspension was seeded in a 96-well plate (Thermo Fisher Science) so that the number of cells per well was 3.2 ⁇ 10 3 , and cultured for 24 hours under conditions of 37 ° C. and 5% CO 2.
• the DMEM medium was removed from the well plate, the medium was replaced with low pH-DMEM medium, and 50 ⁇ L of a dilution solution prepared with serum-free DMEM medium of the lipid particle suspension prepared in Examples 21 to 28 and Comparative Example 8 was added to each well so that the nucleic acid encapsulation amount was 200 ng / mL.
• ⁇ Gene expression study using lipid nanoparticles with different phospholipid sterol ratios> Human breast cancer cells (MCF-7, DS Pharma Biomedical) were cultured using DMEM medium (Nacalai Tesque) supplemented with 10% final concentration of fetal bovine serum (FBS) (DS Pharma Biomedical). The cells were seeded on a 100 mm cell culture dish (BD Falcon) to a density of 5.0 x 10 3 cells/cm 2 and cultured under conditions of 37°C and 5% CO 2.
• DMEM medium Nacalai Tesque
• FBS fetal bovine serum
• MCF-7 cultured to a 70% confluent state on a 100 mm cell culture dish was treated with a 0.25% trypsin/50 mM EDTA solution, and the above-mentioned serum-supplemented DMEM medium was added to stop the trypsin reaction, to obtain an MCF-7 cell suspension.
• the number of cells in the MCF-7 cell suspension was measured using a 0.4 w/v% trypan blue solution (Wako Pure Chemical Industries).
• the cell suspension was seeded in a 96-well plate (Thermo Fisher Science) so that the number of cells per well was 3.2 ⁇ 10 3 , and cultured for 24 hours under conditions of 37 ° C. and 5% CO 2.
• the medium was replaced with serum-free DMEM medium without FBS, and 50 ⁇ L of a dilution solution prepared with serum-free DMEM medium of the lipid particle suspension prepared in Example 24, Examples 29 to 36, and Comparative Example 15 was added to each well so that the amount of nucleic acid encapsulated was 200 ng / mL.
• 50 ⁇ L of ONE-Glo (TM) EX Luciferase Assay System (Promega Corporation) was added per well, and the plate was stirred at 600 rpm for 3 minutes with a plate shaker, and the amount of luminescence was measured with a plate reader SH-9000 (Corona Electric Co., Ltd.).
• Relative light intensity (%) (B ⁇ A) ⁇ 100 A (luminescence amount in the lipid particle-added area of Comparative Example 15), B (luminescence amount in the lipid particle-added area of each phospholipid sterol ratio) The results are shown in Figure 7.
• the gene expression level was significantly increased in the lipid particles of Examples 24 and 29 to 36.
• the particle diameter of each lipid particle was in the range of 30 to 200 nm, and good particles were obtained.
• ⁇ Gene expression assay using lipid nanoparticles with different cationic ratios> Human breast cancer cells (MCF-7, DS Pharma Biomedical) were cultured using DMEM medium (Nacalai Tesque) supplemented with 10% final concentration of fetal bovine serum (FBS) (DS Pharma Biomedical). The cells were seeded on a 100 mm cell culture dish (BD Falcon) to a density of 5.0 x 10 3 cells/cm 2 and cultured under conditions of 37°C and 5% CO 2.
• MCF-7 cultured to a 70% confluent state on a 100 mm cell culture dish was treated with a 0.25% trypsin/50 mM EDTA solution, and the above-mentioned serum-supplemented DMEM medium was added to stop the trypsin reaction, to obtain an MCF-7 cell suspension.
• the number of cells in the MCF-7 cell suspension was measured using a 0.4 w/v% trypan blue solution (Wako Pure Chemical Industries).
• the cell suspension was seeded in a 96-well plate (Thermo Fisher Science) so that the number of cells per well was 3.2 ⁇ 10 3 , and cultured for 24 hours under conditions of 37 ° C. and 5% CO 2.
• the medium was replaced with serum-free DMEM medium without FBS, and 50 ⁇ L of a dilution solution prepared with serum-free DMEM medium of the lipid particle suspension prepared in Example 24, Examples 37 to 42, and Comparative Example 15 was added to each well so that the amount of nucleic acid encapsulated was 200 ng / mL.
• 50 ⁇ L of ONE-Glo (TM) EX Luciferase Assay System (Promega Corporation) was added per well, and the plate was stirred at 600 rpm for 3 minutes with a plate shaker, and the amount of luminescence was measured with a plate reader SH-9000 (Corona Electric Co., Ltd.).
• Relative light intensity (%) (B ⁇ A) ⁇ 100 A (luminescence amount in the area where lipid particles of Comparative Example 15 were added), B (luminescence amount in the area where lipid particles of each cationic ratio were added) The results are shown in Figure 8.
• the amount of gene expression was significantly increased in the lipid particles of Examples 24 and 37 to 42.
• the particle diameter of each lipid particle was in the range of 30 to 200 nm, and good particles were obtained.

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