WO2023195527A1 - Oligo-nucleic acid nanoparticle - Google Patents

Oligo-nucleic acid nanoparticle Download PDF

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WO2023195527A1
WO2023195527A1 PCT/JP2023/014288 JP2023014288W WO2023195527A1 WO 2023195527 A1 WO2023195527 A1 WO 2023195527A1 JP 2023014288 W JP2023014288 W JP 2023014288W WO 2023195527 A1 WO2023195527 A1 WO 2023195527A1
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oligonucleic acid
diseases
oligonucleic
core
cell internalization
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PCT/JP2023/014288
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French (fr)
Japanese (ja)
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尚樹 牧田
晃弘 上坂
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住友ファーマ株式会社
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    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
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    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
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Definitions

  • the present invention relates to oligonucleic acid nanoparticles.
  • nucleic acid medicines that can directly control the expression of various gene products expressed within cells can serve as therapeutic agents for diseases that cannot be treated with conventional medicines, and their medical applications are therefore highly anticipated.
  • nucleic acid drugs cannot spontaneously permeate cell membranes because the nucleic acid molecules themselves have large molecular weights, have many negative charges, and are highly hydrophilic.
  • As a method for transporting nucleic acid molecules into the cytoplasm, where their effects are expressed there are two methods: modifying nucleic acid molecules with hydrophobic molecules, sugars, and other cellular internalization promoters, and converting nucleic acid molecules into functional nanoparticles. A method of encapsulating it in particles is known.
  • Patent Document 1 and Non-Patent Document 1 disclose a method of preparing a nanostructure by covalently bonding a nucleic acid molecule and a cell internalization promoter to a polymer and transporting the nucleic acid molecule into the cytoplasm. has been done.
  • Patent Document 1 and Non-Patent Document 1 have room for improvement in terms of allowing the cell internalization promoter to interact efficiently with target cells.
  • the main objective of the present invention is to increase the amount of oligonucleic acid transported into the cytoplasm by allowing a cell internalization promoter to interact efficiently with target cells.
  • the present invention provides oligonucleic acid nanoparticles, which are functional nanoparticles that have a structure suitable for reducing nonspecific interactions with biological components and contain a cell internalization promoter, and a method for producing the same.
  • one embodiment of the present invention includes a core composed of a dendritic polymer, a plurality of oligonucleic acids, one or more hydrophilic polymers, and one or more cell-internal polymers arranged around the core.
  • an oligonucleic acid nanoparticle composed of a single molecule, the oligonucleic acid being bonded to the core, preferably by a covalent bond, and a hydrophilic polymer being bonded to the oligonucleic acid, preferably by a covalent bond.
  • the present invention provides oligonucleic acid nanoparticles in which the cell internalization promoting agent is preferably covalently bound to a hydrophilic polymer.
  • Another aspect of the present invention includes a core composed of a dendritic polymer, a plurality of oligonucleic acids, one or more spacers, one or more hydrophilic polymers arranged around the core, and An oligonucleic acid nanoparticle composed of a single molecule, comprising one or more cell internalization promoters, wherein the oligonucleic acid or spacer is bound to the core, preferably by a covalent bond, and the hydrophilic polymer is Oligonucleic acid nanoparticles are provided, wherein the spacer is preferably covalently bound, and the cellular internalization enhancer is preferably covalently bound to the hydrophilic polymer.
  • the reactive functional groups of the dendritic polymer forming the core are used to bind not only the oligonucleic acid or the spacer but also the capping agent.
  • the hydrophilic polymer is located outside the spatial extent (radius of gyration) of the oligonucleic acid, and the cellular internalization promoter is located on the surface of the oligonucleic acid nanoparticle. It is expected that the oligonucleic acid nanoparticles will interact more efficiently with target cells, and non-specific interactions between oligonucleic acid nanoparticles and biological components other than target cells will be reduced.
  • a core composed of a dendritic polymer, a plurality of oligonucleic acids bound to the core; one or more hydrophilic polymers bound to the plurality of oligonucleic acids;
  • An oligonucleic acid nanoparticle comprising one or more cell internalization promoters bound to the one or more hydrophilic polymers,
  • the binding between the core and the oligonucleic acid, the binding between the oligonucleic acid and the hydrophilic polymer, and the binding between the hydrophilic polymer and the cell internalization promoter are each independently performed by direct binding or via a linker.
  • oligonucleic acid nanoparticle wherein the cell internalization promoting agent is located on the surface of the oligonucleic acid nanoparticle.
  • the direct bond, or the bond between the linker and the core, the oligonucleic acid, the hydrophilic polymer, or the cell internalization promoter is not due to a covalent bond, metal coordination, or host-guest interaction.
  • the direct bond, or the bond between the linker and the core, the oligonucleic acid, the hydrophilic polymer, or the cell internalization promoter is a covalent bond or metal coordination.
  • nanoparticles [5] The oligonucleic acid nanoparticle according to any one of [1] to [4], wherein at least a portion of the reactive functional groups of the dendritic polymer are capped with a capping agent.
  • a core composed of a dendritic polymer, a plurality of oligonucleic acids bound to the core; one or more hydrophilic polymers bonded to the core via a spacer; An oligonucleic acid nanoparticle comprising one or more cell internalization promoters bound to the one or more hydrophilic polymers, The binding between the core and the oligonucleic acid, the binding between the core and the spacer, the binding between the spacer and the hydrophilic polymer, and the binding between the hydrophilic polymer and the cell internalization promoter are each independently performed.
  • the direct bond, or the bond between the linker and the core, the oligonucleic acid, the spacer, the hydrophilic polymer, or the cell internalization promoter is a covalent bond, metal coordination, or host-guest interaction.
  • the oligonucleic acid nanoparticle according to [6] which is based on [8]
  • the direct bond, or the bond between the linker and the core, the oligonucleic acid, the spacer, the hydrophilic polymer, or the cell internalization promoter is based on a covalent bond or metal coordination. 6].
  • the direct bond, or the bond between the linker and the core, the oligonucleic acid, the spacer, the hydrophilic polymer, or the cell internalization promoter is a covalent bond.
  • oligonucleic acid nanoparticle according to any one of [6] to [9], wherein at least a portion of the reactive functional groups of the dendritic polymer are capped with a capping agent.
  • capping agent is one or more molecules selected from the group consisting of hydrophilic molecules and hydrophobic molecules.
  • the capping agent is a hydrophilic molecule.
  • the capping agent is one or more types of hydrophilic molecules selected from the group consisting of electrically neutral hydrophilic molecules, polar molecules that protonate under acidic conditions, anionic molecules, and cationic molecules.
  • the capping agent is one or more types of hydrophilic molecules selected from the group consisting of electrically neutral hydrophilic molecules, polar molecules that protonate under acidic conditions, and anionic molecules. 11].
  • the oligonucleic acid nanoparticle according to [11] wherein the capping agent is a hydrophobic molecule.
  • the dendritic polymer is a poly-L-lysine dendrigraft, a polyamide amine dendrimer, or a 2,2-bis(hydroxyl-methyl)propionic acid dendrimer.
  • Oligonucleic acid nanoparticles described in [22] The oligonucleic acid nanoparticle according to any one of [1] to [21], wherein the oligonucleic acid is a gene expression control agent. [23] The oligonucleic acid nanoparticle according to [22], wherein the gene expression control agent is a molecule that suppresses mRNA expression. [24] The oligonucleic acid nanoparticle according to [22], wherein the gene expression control agent is an RNA interference-inducing nucleic acid or an antisense nucleic acid.
  • spacer is one or more spacers selected from the group consisting of polyethylene glycol, poly(2-alkyl-2-oxazoline), polypeptide, and polypeptoid.
  • the hydrophilic polymer is one or more hydrophilic polymers selected from the group consisting of polyethylene glycol, poly(2-alkyl-2-oxazoline), polypeptide, and polypeptoid, [1] to [26] ] The oligonucleic acid nanoparticle according to any one of the above.
  • the hydrophilic polymer is one or more aqueous polymers selected from the group consisting of polyethylene glycol, poly(2-methyl-2-oxazoline), EK peptide, and polysarcosine, [1] to [26] ] The oligonucleic acid nanoparticle according to any one of the above.
  • the cell internalization promoter is one or more cell internalization promoters selected from the group consisting of polypeptides, aptamers, antibodies or fragments thereof, sugars, lipids, and other low molecular weight compounds.
  • the oligonucleic acid nanoparticle according to any one of [1] to [28].
  • the cell internalization promoter is a low molecular compound with a molecular weight of 2000 or less other than a hydrophobic molecule, a polycation, a polypeptide, an aptamer, an antibody or a fragment thereof, a sugar, and a lipid, [1]
  • the oligonucleic acid nanoparticle according to any one of [28].
  • oligonucleic acid nanoparticle according to any one of [1] to [28], wherein the cell internalization promoter is a lipid.
  • [37] Containing the oligonucleic acid nanoparticle according to any one of [1] to [35] as an active ingredient, Inborn errors of metabolism, congenital endocrine diseases, single gene diseases, neurodegenerative diseases, neurological diseases, muscle diseases, meningitis, encephalitis, encephalopathy, lysosomal diseases, malignant neoplasms, fibrosis, inflammatory diseases, immunodeficiency
  • [38] comprising administering a therapeutically effective amount of the oligonucleic acid nanoparticles according to any one of [1] to [35].
  • oligonucleic acid nano-acid according to any one of [1] to [35] for use in the treatment and/or prevention of diseases selected from the group consisting of particle.
  • the oligonucleic acid nanoparticle according to any one of [1] to [35], Including a combination of one or more therapeutic agents and/or one or more preventive agents for diseases,
  • the diseases include inborn errors of metabolism, congenital endocrine diseases, monogenic diseases, neurodegenerative diseases, neurological diseases, myopathies, meningitis, encephalitis, encephalopathy, lysosomal diseases, malignant neoplasms, fibrosis, and inflammatory diseases.
  • a medicament selected from the group consisting of diseases, immunodeficiency diseases, autoimmune diseases, and infectious diseases.
  • the oligonucleic acid nanoparticle according to any one of [1] to [35], for treating a disease in combination with one or more therapeutic agents and/or one or more preventive agents for the disease.
  • the diseases include inborn errors of metabolism, congenital endocrine diseases, monogenic diseases, neurodegenerative diseases, neurological diseases, myopathies, meningitis, encephalitis, encephalopathy, lysosomal diseases, malignant neoplasms, fibrosis, and inflammatory diseases.
  • Oligonucleic acid nanoparticles selected from the group consisting of diseases, immunodeficiency diseases, autoimmune diseases, and infectious diseases.
  • (b1) A step of bonding one or more spacers to a core composed of a dendritic polymer; (b2) binding a plurality of oligonucleic acids to the core; (b3) bonding a hydrophilic polymer to the spacer; (b4) The method for producing oligonucleic acid nanoparticles according to any one of [6] to [9], comprising the step of binding a cell internalization promoter to the hydrophilic polymer.
  • (b1) A step of bonding one or more spacers to a core composed of a dendritic polymer; (b2) binding a plurality of oligonucleic acids to the core; (b3) bonding a hydrophilic polymer to the spacer; (b4) binding a cell internalization promoter to the hydrophilic polymer; (a5) The method for producing oligonucleic acid nanoparticles according to [10], comprising the step of binding a capping agent to the core.
  • the cell internalization promoting agent can efficiently interact with target cells, oligonucleic acids can be efficiently transported into cells, thereby reducing the amount of oligonucleic acids transported into the cytoplasm. can be improved.
  • non-specific interactions with other biological components other than target cells can be reduced, thereby improving pharmacokinetics (i.e., blood It is possible to extend the residence time) and improve the amount of oligonucleic acid transported into the cytoplasm.
  • oligonucleic acid nanoparticles according to one aspect of the present invention have a stable structure and can easily control their size.
  • FIG. 1 is a schematic diagram showing one embodiment of oligonucleic acid nanoparticles according to the first aspect of the present invention.
  • FIG. 2 is a schematic diagram showing one embodiment of oligonucleic acid nanoparticles according to a second aspect of the present invention.
  • 1 is a graph showing intracellular uptake of oligonucleic acid nanoparticles of Test Example 1.
  • 1 is a graph showing gene knockdown activity by oligonucleic acid nanoparticles of Test Example 1.
  • 3 is a graph showing intracellular uptake of oligonucleic acid nanoparticles of Test Example 3.
  • 3 is a graph showing gene knockdown activity by oligonucleic acid nanoparticles of Test Example 3.
  • Oligonucleic acid nanoparticles according to one aspect of the present invention have a core made of a dendritic polymer and a plurality of oligonucleic acids bound to the core. , one or more hydrophilic polymers bound to the plurality of oligonucleic acids, and one or more cell internalization promoters bound to the one or more hydrophilic polymers.
  • oligonucleic acid nanoparticles refer to nanoparticles consisting of a single molecule formed by binding an oligonucleic acid to another molecule.
  • nanoparticles mean particles of 10 to 200 nm. In this field, there is a wealth of knowledge regarding the behavior of nanoparticles in living organisms.
  • dendritic polymer refers to a polymer that is branched from the center in a dendritic manner and has regular branching.
  • Dendritic polymers may be dendrimers, dendrons or dendrigrafts.
  • Dendrimers are generally highly branched three-dimensional molecules with a dendritic structure and approximately spherical shape.
  • Dendrons have a structure in which at least one functional group in the center of the dendrimer is unbranched.
  • Dendrimers and dendrons have regular branched structures, and their repeating units are called "generations.”
  • generations molecular chains are bonded in a comb-like manner to the side chains on the main molecular chain, and molecular chains are further bonded in a comb-like manner to the side chains of the comb-shaped molecular chains, thereby spreading radially. forming a structure.
  • the comb-like repeating units are called "generations.”
  • the generation of the dendritic polymer is preferably from the 3rd generation to the 20th generation.
  • the generation is preferably from generation 5 to generation 20, more preferably from generation 5 to generation 10.
  • the generation is preferably 3rd to 6th generation, more preferably 3rd to 5th generation.
  • the generation is preferably from 4th generation to 20th generation, more preferably from 4th generation to 10th generation.
  • the dendritic polymer constitutes the core of the oligonucleic acid nanoparticle, and its average diameter (in other words, average particle diameter) is preferably 5 nm or more, more preferably 5 nm to 25 nm, and even more preferably 5 nm to 15 nm. be.
  • the average particle size of the dendritic polymer (core) means the average particle size in the particle size distribution obtained by dynamic light scattering.
  • the monomers in the above dendritic polymer are, for example, single bonds, double bonds, triple bonds, carbon-silicon bonds, amide bonds, glycosidic bonds, ester bonds, ether bonds, urethane bonds, acetal bonds, phosphate ester bonds, and thioether bonds.
  • the bond may be bonded by a bonding method such as a bond, a thioester bond, a disulfide bond, a triazole bond, a hydrazone bond, a hydrazide bond, an imine or oxime bond, a urea or thiourea bond, an amidine bond, or a sulfonamide bond.
  • a bonding method such as a bond, a thioester bond, a disulfide bond, a triazole bond, a hydrazone bond, a hydrazide bond, an imine or oxime bond, a urea or thiourea bond, an amidine bond
  • any of these bonding modes can be used, but those in which the bond is cleaved by enzymes or under specific in-vivo environments such as acidic conditions or reducing environments are Preferable from a safety standpoint.
  • Examples of preferred bonding modes include, but are not limited to, amide bonds, ester bonds, or glycosidic bonds.
  • dendritic polymers include, but are not limited to, polylysine dendrimers, polylysine dendrigrafts, PAMAM dendrimers, Bis-MPA dendrimers, or glucose dendrimers.
  • the dendritic polymer may be, for example, a poly-L-lysine dendrimer or a poly-L-lysine dendrigraft.
  • oligonucleic acids are natural or non-natural oligonucleic acids.
  • a natural oligonucleic acid is a polymer whose repeating units are nucleotides consisting of a base, sugar, and phosphoric acid.
  • the type of oligonucleic acid is not particularly limited, and the oligonucleic acid nanoparticles may contain one or more types of oligonucleic acids.
  • Examples of oligonucleic acids include single-stranded or double-stranded RNA, DNA, or a combination thereof, including oligonucleic acids in which RNA and DNA are mixed in the same strand.
  • the nucleotides contained in the oligonucleic acid may be natural nucleotides or chemically modified non-natural nucleotides, or may be added with molecules such as amino groups, thiol groups, or fluorescent compounds. It may also be a nucleotide.
  • Non-natural oligonucleic acids include peptide nucleic acids (PNA), which have a peptide structure in their main chain, and morpholino nucleic acids, which have a morpholine ring in their main chain, which control gene expression in the same way as natural oligonucleic acids. Also included are artificial molecules that have an effect.
  • oligonucleic acid examples include antisense nucleic acid, sgRNA, RNA editing nucleic acid, miRNA, siRNA, saRNA, shRNA, or dicer substrate RNA.
  • the oligonucleic acid may be, for example, a gene expression control agent.
  • a gene expression regulator is a compound that activates or suppresses the expression of a specific gene product. Gene products include, for example, mRNA or its precursor, miRNA or its precursor, ncRNA, enzyme, antibody, or other protein. Examples of gene expression control agents include molecules that positively or negatively control mRNA expression (that is, promote or suppress expression), or molecules that edit RNA or DNA.
  • Such gene expression control agents include, for example, nucleic acids that induce RNA interference (RNAi) such as miRNA and siRNA (RNAi-inducing nucleic acids), antisense nucleic acids, miRNA inhibitors, RNA activation nucleic acids, RNA editing-inducing nucleic acids, or genome editing-induced nucleic acids, but gene expression control agents are not limited to these.
  • RNAi RNA interference
  • miRNA and siRNA RNAi-inducing nucleic acids
  • antisense nucleic acids such as miRNA and siRNA (RNAi-inducing nucleic acids)
  • miRNA inhibitors such as miRNA inhibitors, RNA activation nucleic acids, RNA editing-inducing nucleic acids, or genome editing-induced nucleic acids, but gene expression control agents are not limited to these.
  • the length of the oligonucleic acid may be, for example, 4 to 200 bases (pairs), 7 to 100 bases (pairs), or 12 to 30 bases (pairs).
  • the number of oligonucleic acids in the oligonucleic acid nanoparticles is not particularly limited as long as it is plural, for example, 2 or more, 6 or more, 10 or more, 15 or more, 17 or more, 18 or more, 20 or more, 25 or more, 30 or more, 31 or more. , 35 or more, or 50 or more, and may be 400 or less, 200 or less, or 100 or less.
  • the number of oligonucleic acids is, for example, 0.5% or more, 1% or more, or 2% or more of the reactive functional groups possessed by the dendritic polymer.
  • the number of oligonucleic acids in oligonucleic acid nanoparticles can be determined, for example, by measuring the concentration of dendritic polymer and the concentration of oligonucleic acid in a solution containing oligonucleic acid nanoparticles, and from these values, calculating the ratio of oligonucleic acids to dendritic polymers. It can be determined by calculation.
  • the concentration of dendritic polymer in a solution containing oligonucleic acid nanoparticles can be measured, for example, by high performance liquid chromatography (HPLC).
  • HPLC high performance liquid chromatography
  • the concentration of oligonucleic acid can be determined, for example, from the absorption at 260 nm measured using an ultraviolet-visible spectrophotometer.
  • Oligonucleic acids can be produced by known methods. Oligonucleic acids can be produced, for example, by a phosphoramidite method or a triester method, by a solid phase synthesis method or a liquid phase synthesis method, using an automatic nucleic acid synthesizer or manually.
  • the hydrophilic polymer is a hydrophilic molecule that shields the oligonucleic acid from the outside world and exposes the cell internalization promoter to the surface of the oligonucleic acid nanoparticle.
  • a hydrophilic molecule refers to a molecule that easily forms a hydrogen bond with water and has the property of being easily soluble in water or easily miscible with water.
  • Hydrophilic molecules may be charged molecules or uncharged highly polar molecules. The charged group of a charged molecule may be a positively charged group (cation), a negatively charged group (anion), or a combination thereof.
  • Hydrophilic polymers i.e., stealth polymers
  • Hydrophilic polymers that shield oligonucleic acids from the outside world can inhibit aggregation of oligonucleic acid nanoparticles, improve solubility, avoid phagocytosis by the reticuloendothelial system, and prevent interaction with biological components. This may be advantageous in terms of avoiding specific interactions and improving pharmacokinetics (ie, prolonging blood residence time).
  • hydrophilic polymers include, but are not limited to, polyethylene glycol (PEG), poly(2-alkyl-2-oxazoline), polypeptide, polypeptoid, or polybetaine.
  • Oligonucleic acid nanoparticles may include one or more hydrophilic polymers.
  • the hydrophilic polymer is preferably one or more selected from the group consisting of PEG, poly(2-methyl-2-oxazoline) (pMeOx), polysarcosine (pSar), and EK peptide.
  • EK peptide is a peptide consisting of alternating glutamic acid and lysine.
  • One hydrophilic polymer may have multiple segments.
  • Examples of the hydrophilic polymer having multiple segments include, but are not limited to, a polymer formed by bonding EK peptide and PEG.
  • the hydrophilic polymer may have a linear or branched structure.
  • the number average molecular weight of the hydrophilic polymer may be 500 or more, 1000 or more, 2000 or more, 3400 or more, 5000 or more, 6000 or more, 8000 or more, or 10000 or more.
  • the number average molecular weight of the hydrophilic polymer may be 1000 or more, 2000 or more, 4000 or more, 7000 or more, 10000 or more, 15000 or more, or 20000 or more.
  • the hydrophilic polymer is an EK peptide
  • the hydrophilic polymer is a peptide containing 1 or more, 2 or more, 5 or more, 7 or more, 10 or more, 15 or more, or 20 or more unit structures consisting of glutamic acid and lysine. It's good.
  • the number average molecular weight is a value determined by a terminal group determination method using nuclear magnetic resonance (NMR) or size exclusion chromatography (SEC).
  • the number of hydrophilic polymers may be less than, more than, or the same as the number of oligonucleic acids or cell internalization promoters.
  • the number of hydrophilic polymers bound to one oligonucleic acid may be one or more, but is preferably one. Furthermore, there may be a hydrophilic polymer to which no cell internalization promoter is bound.
  • the number of hydrophilic polymers may be, for example, 1 or more, 2 or more, or 1% or more of the reactive functional groups possessed by the dendritic polymer, It is preferably 2% or more, more preferably 3% or more or 5% or more of the reactive functional groups possessed by the dendritic polymer. Further, the hydrophilic polymer preferably binds to 40% or more, 60% or more, 80% or more, or 85% or more of the oligonucleic acid.
  • the number of hydrophilic polymers in the oligonucleic acid nanoparticles is calculated, for example, from the ratio of the oligonucleic acid alone separated by SEC to the oligonucleic acid bound to the hydrophilic polymer by cutting the bond between the oligonucleic acid and the dendritic polymer. be able to.
  • the cell internalization promoter induces internalization of a substance bound to the cell internalization promoter into the target cell by interacting specifically or nonspecifically with the target cell. It is a molecular species.
  • the oligonucleic acid nanoparticles by binding a cell internalization promoter to a hydrophilic polymer, compared to a case where the cell internalization promoter is not included (for example, when oligonucleic acid is used alone). As a result, oligonucleic acids can be efficiently transported into target cells.
  • the cell internalization promoter is located on the surface of the oligonucleic acid nanoparticle.
  • Cell internalization promoters include substances that interact with cell surface receptors, substances that interact with membrane transporters, substances that interact with cell adhesion factors, or other substances that interact with the cell membrane surface. , but not limited to those.
  • Cell internalization promoters include, for example, substances that interact with integrin, which is a cell adhesion factor present on the surface of cell membranes, substances that interact with epithelial cell adhesion molecules, substances that interact with nucleolin, and substances that interact with vimentin, a cytoskeletal molecule.
  • Interacting substances substances that interact with prostate-specific membrane antigens, substances that interact with cell surface receptors such as epidermal growth factor receptors, somatostatin receptors, mannose receptors, asialoglycoprotein receptors, folate receptors, etc. , or a substance that interacts with a transporter such as a glucose transporter or a non-selective monoamine transporter.
  • a transporter such as a glucose transporter or a non-selective monoamine transporter.
  • cell internalization promoters include hydrophobic molecules, polycations, polypeptides, aptamers, antibodies or fragments thereof, sugars, lipids, and other low-molecular compounds; Not limited. Other low molecular weight compounds are compounds with a molecular weight of 2000 or less, other than hydrophobic molecules, polycations, polypeptides, aptamers, antibodies or fragments thereof, sugars, and lipids.
  • the oligonucleic acid nanoparticles may contain one or more cell internalization promoters.
  • the cell internalization promoter is preferably one or more selected from the group consisting of polypeptides, aptamers, sugars, and other low molecular weight compounds mentioned above.
  • the cell internalization promoting agent is more preferably a polypeptide, an aptamer, or the other low molecular weight compound mentioned above.
  • the molecular weight of the polypeptide may be, for example, 50 kDa or less, 15 kDa or less, 6 kDa or less, 2 kDa or less, or 1 kDa or less, but is not limited thereto.
  • the molecular weight of a polypeptide can be determined, for example, by mass spectrometry.
  • an antibody or a fragment thereof refers to a scaffold protein that has the function of specifically binding to a specific factor, and includes immunoglobulins such as IgA, IgD, IgE, IgG, and IgM, F(ab)'2, These include, but are not limited to, fragmented antibodies such as Fab', Fab, scFv, single domain antibodies such as shark VNAR, camel VHH, and antibody mimetics such as affibodies, affilins, monobodies, alphabodies.
  • cell internalization promoters include polypeptides represented by the following formulas (I) to (IV).
  • the polypeptide represented by formula (I) is cRGDfK (molecular weight: 603.7 Da, Pharmaceuticals, 2018, 10, 2), which is a type of cyclic peptide ligand (cRGD) containing an arginine-glycine-aspartic acid (RGD) sequence.
  • cRGDfK interacts with integrin ⁇ v ⁇ 3 .
  • cRGD other than cRGDfK can also be used as a cell internalization promoter.
  • the polypeptide shown in formula (II) is c(avb6) (molecular weight: 1046.2, ACS Omega, 2018, 3, 2428-2436), which interacts with integrin ⁇ v ⁇ 6 .
  • the polypeptide shown in formula (III) is GE11 (molecular weight: 1539.7 Da) that interacts with the epidermal growth factor receptor.
  • the polypeptide shown in formula (IV) is an octreotide derivative (OCT; molecular weight: 1577.8 Da) that interacts with the somatostatin receptor.
  • OCT octreotide derivative
  • cRGD commercially available products can be used.
  • Peptides represented by formulas (II) to (IV) are readily available by well-known synthetic methods.
  • cell internalization promoters include low molecular weight compounds represented by the following formulas (V) to (VII).
  • the low molecular compound represented by formula (V) is folic acid that interacts with folic acid receptors.
  • the low molecular compound represented by formula (VI) is DUPA that interacts with prostate-specific membrane antigen.
  • the low molecular compound represented by formula (VII) is indatraline (IND), which interacts with a non-selective monoamine transporter.
  • cell internalization promoters include sugars represented by the following formulas (VIII) to (XII).
  • the sugar represented by formula (VIII) is glucose (Glu) that interacts with glucose transporters.
  • the sugar shown in formula (IX) is mannose (Man), which interacts with the mannose receptor.
  • the sugars shown in formula (X) and formula (XI) are N-acetylgalactosamine (GalNAc) and galactose (Gal), which interact with asialoglycoprotein receptors.
  • the sugar shown in formula (XII) is N-acetylglucosamine (GlcNAc), which interacts with the cytoskeletal molecule vimentin.
  • Examples of other cell internalization promoters include aptamers having the base sequences shown in SEQ ID NOs: 1 to 6 shown in the table below.
  • DNA aptamers that interact with nucleolin AS1411 shown in SEQ ID NO: 1 (Oncotarget, 2015, 6 (26), 22270-22281), and FAN-1524dI shown in SEQ ID NO: 2 (Scientific Reports, 2016, 6, 1-12) ).
  • EpCAM Aptamer shown in SEQ ID NO: 3 Molecular Cancer Therapeutics, 2015, 14 (10), 2279-2291
  • EpCAM Aptamer shown in SEQ ID NO: 4 Theranos tics, 2015, 5( 10), 1083-1097).
  • FB4 shown in SEQ ID NO: 5 (Proc Natl Acad Sci USA., 2008, 105 (41), 15908-15913) and GS24 shown in SEQ ID NO: 6 (Mol Ther Nucleic Acids, 2014 , 3(1), e144).
  • the number of cell internalization promoters in the oligonucleic acid nanoparticles is, for example, 1 from the viewpoint of efficiently interacting the cell internalization promoters with target cells and improving the transport efficiency of the oligonucleic acid nanoparticles into cells.
  • the number may be 2 or more, 6 or more, 11 or more, 18 or more, 25 or more, 26 or more, 27 or more, or 30 or more, and may be 400 or less, 200 or less, or 100 or less.
  • the number of cell internalization promoters in the oligonucleic acid nanoparticles can be determined, for example, by measuring the concentration of the dendritic polymer and the concentration of the cell internalization promoter in the solution containing the oligonucleic acid nanoparticles, and from these values, the number of cell internalization promoters in the dendritic polymer It can be determined by calculating the ratio of the cell internalization promoter to the total amount of the cell internalization promoting agent.
  • the concentration of the dendritic polymer and the concentration of the cellular internalization promoter can be measured, for example, by HPLC or UV-visible spectrophotometry.
  • the number of cell internalization promoters bound to one hydrophilic polymer may be one or more.
  • an oligonucleic acid is bound to the dendritic polymer, and more specifically to at least a portion of the reactive functional groups (these are the terminal functional groups) of the dendritic polymer.
  • at least a portion or all of the unreacted reactive functional groups that are not bound to the oligonucleic acid may be capped with a capping agent.
  • capping a reactive functional group is to reduce the reactivity of the reactive functional group through bonding.
  • the capping agent protects the dendritic polymer from various interactions or chemical reactions by binding to the reactive functional groups of the dendritic polymer.
  • capping agents protect dendritic polymers from electrostatic interactions, decomposition reactions, condensation reactions, addition reactions, and the like.
  • the capping agent can add a function or activity that the dendritic polymer does not originally have to the dendritic polymer.
  • examples of such capping agents include, but are not limited to, molecules that improve stealth, molecules that interact with lipid bilayer membranes, and molecules that have proton buffering ability.
  • the capping agent may be, for example, one or two molecules selected from the group consisting of a) hydrophilic molecules and b) hydrophobic molecules.
  • hydrophilic molecules are a-1) electrically neutral hydrophilic molecules, a-2) polar molecules that protonate under acidic conditions, a-3) anionic molecules, or a-4) cations. It may be a sexual molecule.
  • the hydrophilic molecule may be the same type of molecule as the hydrophilic polymer, or it may be a different type of molecule from the hydrophilic polymer.
  • electrically neutral refers to a case where the number of cations and anions is equal, or the difference between the number of cations and anions is within 10% based on the number of charged groups of the larger one. Point.
  • Examples of the above a-1) electrically neutral hydrophilic molecules include hydroxyl groups, alkoxy groups, oxime groups, ester groups, amide groups, imide groups, alkoxyamide groups, carbonyl groups, sulfonyl groups, nitro groups, Hydrophilic molecules include, but are not limited to, molecules having hydrophilic groups such as pyrrolidone groups, zwitterions such as betaine, PEG, and alkoxypolyethylene glycols such as methoxypolyethylene glycol.
  • Polar molecules that are protonated under acidic conditions are molecules that have different charges depending on the acidic environment such as in endosomes and under physiological conditions such as in blood or interstitial fluid.
  • Polar molecules that protonate under acidic conditions refer to molecules having an acid dissociation constant (pK a ) of 7.4 or less, preferably from 5.0 to 7.4.
  • Examples of polar molecules that protonate under acidic conditions include tertiary amino groups, diethyltriamine (DET) groups (-NH-CH 2 -CH 2 -NH-CH 2 -CH 2 -NH 2 ), morpholino groups, Examples include molecules having polar groups such as a thiomorpholino group, an imidazolyl group, a pyridyl group, and a carboxy group, but polar molecules that are protonated under acidic conditions are not limited to these.
  • DET diethyltriamine
  • morpholino groups examples include molecules having polar groups such as a thiomorpholino group, an imidazolyl group, a pyridyl group, and a carboxy group, but polar molecules that are protonated under acidic conditions are not limited to these.
  • anionic molecule is a molecule with a negative ion valence under physiological conditions.
  • examples include molecules having functional groups such as a carboxyl group, a sulfo group, a phosphoric acid group, and a phosphoric ester group, but the anionic molecule is not limited to these.
  • the above a-4) cationic molecule is a molecule with a positive ion valence under physiological conditions.
  • Examples include molecules having functional groups such as primary amino groups, secondary amino groups, tertiary amino groups, and guanidino groups, but the cationic molecules are not limited to these.
  • hydrophobic molecule means a molecule that is difficult to form a hydrogen bond with water and has a low affinity for water.
  • Hydrophobic molecules may be nonpolar molecules or molecules with a partition coefficient of 2.0 or more.
  • Examples of hydrophobic molecules include molecules having hydrophobic groups such as aliphatic groups, trialkylamine groups, and aromatic groups, cholesterol, or steroids and their analogs, but hydrophobic molecules are not limited to these. do not have.
  • binding refers to direct or indirect, irreversible binding.
  • An irreversible bond refers to a bond in which the reaction does not proceed reversibly, that is, a bond that does not dissociate in a reverse reaction once formed, or a bond that dissociates in the reverse reaction is negligible.
  • the binding between the dendritic polymer (core) and the oligonucleic acid, the binding between the oligonucleic acid and the hydrophilic polymer, and the binding between the hydrophilic polymer and the cell internalization promoter are each independently direct binding (i.e., via a linker). bond) or via a linker.
  • the linker is not particularly limited, and may be a known linker such as PEG or an alkyl linker (eg, hexyl linker).
  • the linker may be composed of one type of linker, or may be a linker formed by bonding two or more types of linkers.
  • the linker is PEG, its number average molecular weight may be, for example, 1000 or less, 800 or less, 600 or less, or 300 or less.
  • Direct binding between a dendritic polymer (core) and an oligonucleic acid, between an oligonucleic acid and a hydrophilic polymer, or between a hydrophilic polymer and a cell internalization promoter, or between the above linker and a dendritic polymer, an oligonucleic acid , a hydrophilic polymer, or a cell internalization promoter for example, a covalent bond formed by a chemical reaction such as a nucleophilic addition reaction between functional groups, a nucleophilic substitution reaction, or an electrophilic substitution reaction, or a bond between ammonia and platinum.
  • the bond is preferably a covalent bond.
  • covalent bonds examples include single bonds, double bonds, triple bonds, amide bonds, glycosidic bonds, ester bonds, ether bonds, urethane bonds, acetal bonds, phosphate ester bonds, thioether bonds, thioester bonds, disulfide bonds, and triazole bonds.
  • Covalent bonds include bonds, hydrazone bonds, hydrazide bonds, imine or oxime bonds, urea or thiourea bonds, amidine bonds, sulfonamide bonds, or bonds formed by reverse electron request Diels-Alder reactions. Not limited.
  • An amide bond is formed between a carboxy group and an amino group.
  • Amide bonds are formed using conventional amide bond-forming reactions, e.g., between a suitably protected amino group and an activated carboxylic acid, such as an ester activated with N-hydroxysuccinimide. .
  • a disulfide bond is formed, for example, by thiol exchange between a component having a thiol group (also referred to as a mercaptan group) (-SH) and an activated thiol group of another component.
  • a component having a thiol group also referred to as a mercaptan group
  • -SH mercaptan group
  • the thioether bond (-S-) is formed using a conventional thioether bond-forming reaction, which occurs, for example, between a thiol group and a maleimide group.
  • a triazole bond is formed between an azide group and a carbon-carbon triple bond.
  • the triazole bond is formed, for example, by a so-called click reaction such as Husgen cyclization using a metal catalyst or strain-promoted alkyne-azide cycloaddition without using a metal catalyst.
  • Metal coordination is a bonding mode in which metal ions and ligands bond by forming a complex.
  • metal ions include, but are not limited to, ions of metal elements such as platinum group elements, manganese, cobalt, copper, and gadolinium.
  • ligand include, but are not limited to, ammonia, pyridine, bipyridine, ethylenediamine, ethylenediaminetetraacetic acid, acetylacetonate, and derivatives thereof.
  • Host-guest interaction is an interaction between a host molecule, which is a molecule that provides a space in which a specific molecule can be selectively recognized, and a guest molecule, which is a molecule that is accepted there.
  • Host molecules include, but are not limited to, cyclodextrin, calcerand, cavitand, crown ether, cryptand, cucurbituril, calixarene, avidin, streptavidin, and the like.
  • guest molecules include, but are not limited to, adamantane, diadamantane, cholesterol, naphthalene, biotin, and the like.
  • the single molecule constituting the oligonucleic acid nanoparticle according to this aspect may be a free form or a pharmaceutically acceptable salt.
  • the single molecules constituting the oligonucleic acid nanoparticles may be either solvates (eg, hydrates, ethanol solvates, propylene glycol solvates) or non-solvates.
  • Pharmaceutically acceptable salts may be acid addition salts or base addition salts. Acid addition salts include, for example, formate, acetate, trifluoroacetic acid (TFA) salt, propionate, succinate, lactate, malate, adipate, citrate, tartrate, methanesulfone.
  • TFA trifluoroacetic acid
  • Salts with organic acids such as acid salts, fumarates, maleates, p-toluenesulfonates, and ascorbates; salts with inorganic acids such as hydrochlorides, hydrobromides, sulfates, nitrates, phosphates, etc. salt, etc.
  • base addition salts include alkali metal salts such as sodium salts and potassium salts; alkaline earth metal salts such as calcium salts and magnesium salts; ammonium salts; trimethylamine salts; triethylamine salts; dicyclohexylamine salts, ethanolamine salts, and diethanolamine salts.
  • aliphatic amine salts such as salts, triethanolamine salts, and brocaine salts
  • aralkylamine salts such as N,N-dibenzylethylenediamine
  • heterocyclic aromatic amine salts such as pyridine salts, picoline salts, quinoline salts, and isoquinoline salts
  • Quaternary ammonium salts such as methylammonium salt, tetraethylammonium salt, benzyltrimethylammonium salt, benzyltriethylammonium salt, benzyltributylammonium salt, methyltrioctylammonium salt, tetrabutylammonium salt
  • bases such as arginine salt, lysine salt, etc. and amino acid salts.
  • FIG. 1 is a schematic diagram showing one embodiment of oligonucleic acid nanoparticles according to this aspect.
  • the oligonucleic acid nanoparticle 100 includes a core 50 (dendritic polymer) located at the center of the oligonucleic acid nanoparticle 100, a plurality of oligonucleic acids 11 arranged around the core 50, a hydrophilic polymer 12, and a cell-internal component. oxidation accelerator 13. Oligonucleic acid 11 is bound to core 50 via linker 31.
  • the hydrophilic polymer 12 binds to the oligonucleic acid 11 and shields the oligonucleic acid 11 from the outside world, while exposing the cell internalization promoter 13 bound to the hydrophilic polymer 12 on the surface of the oligonucleic acid nanoparticle 100. .
  • a capping agent 21 is also bonded to the core 50.
  • oligonucleic acid 11 and hydrophilic polymer 12 extend substantially radially from core 50, so that oligonucleic acid nanoparticles 100 take a substantially spherical shape. In FIG.
  • the oligonucleic acid 11 is bonded to the core 50 via the linker 31, but the oligonucleic acid 11 may be bonded directly to the core 50 as described above. Moreover, the oligonucleic acid 11 and the hydrophilic polymer 12, and the hydrophilic polymer 12 and the cell internalization promoter 13 may be bonded via any of the above-mentioned linkers.
  • the average particle diameter of the oligonucleic acid nanoparticles 100 is preferably 10 to 100 nm, more preferably 15 to 45 nm.
  • the average particle size of oligonucleic acid nanoparticles means the average particle size in a particle size distribution obtained by dynamic light scattering. Since the oligonucleic acid nanoparticles 100 have a dendritic polymer as the core 50, the size can be easily controlled and precisely designed.
  • the cell internalization promoter 13 In order for the oligonucleic acid nanoparticles 100 to be transported into cells, the cell internalization promoter 13 needs to interact with the cells. From the viewpoint of improving the efficiency of transporting the oligonucleic acid nanoparticles 100 into cells, it is preferable that the density of the cell internalization promoter 13 is high. According to the oligonucleic acid nanoparticles 100, the cell internalization promoter 13 is bound to the core 50 made of a highly branched dendritic polymer via the oligonucleic acid 11 and the hydrophilic polymer 12, so A high density of the internalization promoting agent 13 can be achieved, and therefore the cell internalization promoting agent 13 can efficiently interact with target cells.
  • the oligonucleic acid 11 in order to efficiently shield the oligonucleic acid 11 from the outside world through the binding of the hydrophilic polymer 12, the oligonucleic acid 11 must be attached to the core 50, which has small free mobility at the polymer end and has a three-dimensionally defined spatial configuration. Preferably, they are bonded. From this point of view as well, the oligonucleic acid nanoparticles 100 having a dendritic polymer as the core 50 are more preferable than those having a linear polymer as the core.
  • the hydrophilic polymer 12 is bonded to the oligonucleic acid 11
  • the hydrophilic polymer 12 and the cell internalization promoter 13 bonded thereto can spread the oligonucleic acid 11 spatially
  • the cell internalization promoting agent 13 can be located on the surface (ie, the outermost shell) of the oligonucleic acid nanoparticle 100.
  • the location of the cell internalization promoter 13 on the surface of the oligonucleic acid nanoparticles 100 can be determined by evaluating the binding activity using methods such as surface plasmon resonance (SPR) and enzyme-linked immunosorbent assay (ELISA). , can be confirmed.
  • SPR surface plasmon resonance
  • ELISA enzyme-linked immunosorbent assay
  • the hydrophilic polymer 12, the oligonucleic acid 11 and the cell internalization promoter 13 are the same as the hydrophilic polymer 12 and the cell internalization promoter 13. It is preferable that the oligonucleic acid 11 be bonded so that it is present at a position further away from the spatial extent of the oligonucleic acid 11.
  • the hydrophilic polymer 12 is preferably bonded to the end of the oligonucleic acid 11 that is not bonded to the core 50.
  • the cell internalization promoter 13 is preferably bound to the end of the hydrophilic polymer 12 that is not bound to the oligonucleic acid 11.
  • Oligonucleic acid nanoparticles according to one aspect of the present invention (hereinafter also referred to as oligonucleic acid nanoparticles according to a second aspect) have a core made of a dendritic polymer and a plurality of oligonucleic acids bound to the core. , one or more hydrophilic polymers bonded to the core via a spacer, and one or more cell internalization promoters bonded to the one or more hydrophilic polymers.
  • the oligonucleic acid nanoparticles according to the present aspect meet the above-mentioned first aspect in that the hydrophilic polymer to which the cell internalization promoter is bound is not bound to the oligonucleic acid but to the core via a spacer. It is different from the oligonucleic acid nanoparticles related to the side.
  • the details of the dendritic polymer, oligonucleic acid, hydrophilic polymer, and cell internalization promoter are the same as those of the oligonucleic acid nanoparticle according to the first aspect, and are as described above.
  • the spacer is a polymer for connecting the core and the hydrophilic polymer
  • the spacer is a polymer for connecting the core and the hydrophilic polymer
  • the spacer is a polymer that connects the hydrophilic polymer and the cell internalization promoter bound thereto to the extent that the spatial extent (radius of gyration) of the oligonucleic acid Place it outside.
  • the spacer is not particularly limited, and may be a polar molecule or a nonpolar molecule, and may have a positive charge or a negative charge.
  • Spacers include, but are not limited to, PEG, pMeOx, cationic or anionic polypeptides, polypeptoids, and alkyl chains.
  • the spacer is preferably a spacer having a rigid structure or a cationic spacer.
  • the spacer is preferably PEG or a cationic polypeptide.
  • the oligonucleic acid nanoparticles may contain one or more spacers as spacers.
  • the length of the spacer is appropriately adjusted depending on the length of the oligonucleic acid and the size of the hydrophilic polymer so that the cell internalization promoter can be located on the surface of the oligonucleic acid nanoparticle.
  • the length of the spacer is preferably sufficiently long compared to the length of the oligonucleic acid.
  • the number of spacers may be, for example, 1 or more, 2 or more, or 1% or more of the reactive functional groups possessed by the dendritic polymer, preferably 2% or more of the reactive functional groups possessed by the dendritic polymer, and more preferably is 3% or more or 5% or more.
  • the number of spacers in oligonucleic acid nanoparticles can be determined, for example, by binding a fluorescent dye to the terminal functional group of the spacer and dividing the concentration of the fluorescent dye in the solution containing the obtained oligonucleic acid nanoparticles by the concentration of the dendritic polymer. You can ask for it.
  • At least a portion or all of the unreacted reactive functional groups that are not bound to the oligonucleic acid or the spacer may be capped with a capping agent. Details of the capping agent are as described above.
  • the binding between the dendritic polymer (core) and the oligonucleic acid, the binding between the core and the spacer, the binding between the spacer and the hydrophilic polymer, and the binding between the hydrophilic polymer and the cell internalization promoter are each independently direct binding. (ie, binding without a linker) or binding through a linker. Details of the linker are as described above.
  • the oligonucleotide Binding to the nucleic acid, spacer, hydrophilic polymer, or cell internalization promoter may be, for example, by covalent bonding, metal coordination bonding, or host-guest interaction. Details of these bonds or interactions are as described above. From the viewpoint of achieving high structural stability and controlling the size of the oligonucleic acid nanoparticles, the bond is preferably a covalent bond.
  • the single molecule constituting the oligonucleic acid nanoparticle according to this aspect may be a free form or a pharmaceutically acceptable salt.
  • the single molecules constituting the oligonucleic acid nanoparticles may be either solvates (eg, hydrates, ethanol solvates, propylene glycol solvates) or non-solvates. Details of the pharmaceutically acceptable salt are as described above.
  • FIG. 2 is a schematic diagram showing one embodiment of oligonucleic acid nanoparticles according to this aspect.
  • the oligonucleic acid nanoparticle 200 includes a core 50 (dendritic polymer) located at the center of the oligonucleic acid nanoparticle 200, a plurality of oligonucleic acids 11 arranged around the core 50, a spacer 14, a hydrophilic polymer 12, and cell internalization promoter 13. Oligonucleic acid 11 is bound to core 50 via linker 31.
  • the hydrophilic polymer 12 binds to the core 50 via the spacer 14 to shield the oligonucleic acid 11 from the outside world, and exposes the cell internalization promoter 13 bound to the hydrophilic polymer 12 on the surface of the oligonucleic acid nanoparticle 200. I'm letting you do it.
  • a capping agent 21 is also bonded to the core 50.
  • the oligonucleic acid nanoparticles 200 assume a substantially spherical shape due to the oligonucleic acid 11, the spacer 14, and the hydrophilic polymer 12 extending substantially radially from the core 50. Note that in FIG.
  • the oligonucleic acid 11 is bonded to the core 50 via the linker 31, but the oligonucleic acid 11 may be bonded directly to the core 50 as described above.
  • the spacer 14 and the hydrophilic polymer 12, and the hydrophilic polymer 12 and the cell internalization promoter 13 may be bonded via any of the above-mentioned linkers.
  • the average particle diameter of the oligonucleic acid nanoparticles 200 may be the same as that of the oligonucleic acid nanoparticles 100, and the details are as described above.
  • the cell internalization promoter 13 is bound to the core 50 made of a highly branched dendritic polymer via the spacer 14 and the hydrophilic polymer 12, so that cell internalization is facilitated.
  • a high density of the promoter 13 can be achieved, so that the cell internalization promoter 13 can efficiently interact with target cells.
  • the hydrophilic polymer 12 is bonded to the core 50 via the spacer 14, if the length of the spacer 14 is sufficiently long compared to the length of the oligonucleic acid 11, the hydrophilic polymer 12 is bound to the core 50 via the spacer 14.
  • the polymer 12 and the cell internalization promoter 13 bound thereto are located outside the spatial extent (radius of rotation) of the oligonucleic acid 11, and the cell internalization promoter 13 is located on the surface of the oligonucleic acid nanoparticle 200 (i.e. , outermost shell).
  • the oligonucleic acid nanoparticles 200 can more easily interact with target cells efficiently. Furthermore, by positioning the hydrophilic polymer 12 outside the spatial extent of the oligonucleic acid 11, it is expected that non-specific interactions between the oligonucleic acid nanoparticles 200 and biological components other than the target cells will be reduced. can.
  • the spacer 14 has a rigid structure or is cationic and can form a complex with the oligonucleic acid 11, the hydrophilic polymer 12 and the cell internalization promoter 13 bound thereto can more likely to be located outside of the natural expanse.
  • the present invention also provides a method for producing the oligonucleic acid nanoparticles according to the first aspect. That is, one aspect of the present invention is (a1) A step of binding a plurality of oligonucleic acids to a core composed of a dendritic polymer, (a2) a step of binding a hydrophilic polymer to the oligonucleic acid; (a3) A method for producing oligonucleic acid nanoparticles, which includes the step of binding a cell internalization promoter to a hydrophilic polymer.
  • the method for producing oligonucleic acid nanoparticles may further include the step of (a4) binding a capping agent to the core. Thereby, oligonucleic acid nanoparticles in which at least a portion of the reactive functional groups of the dendritic polymer are capped with a capping agent can be produced.
  • step (a1) may include the steps of binding a plurality of linkers to the core, and binding an oligonucleic acid to the linkers.
  • steps (a1) to (a4) can be performed in this order, it is not necessary to perform them in this order.
  • step (a4) may be performed before step (a1), simultaneously with step (a1), or between step (a1) and step (a2).
  • step (a3) may be performed before step (a2), and in step (a2), a hydrophilic polymer to which a cell internalization promoter is bound may be bound to the oligonucleic acid.
  • oligonucleic acid nanoparticles are produced by bonding a plurality of capping agents and linkers to a core composed of a dendritic polymer, bonding an oligonucleic acid to the linker, and bonding a hydrophilic polymer to the oligonucleic acid. It can be produced by a method including, in this order, a step of binding a cell internalization promoter to a hydrophilic polymer.
  • the present invention also provides a method for producing oligonucleic acid nanoparticles according to the second aspect. That is, one aspect of the present invention is (b1) bonding one or more spacers to a core composed of a dendritic polymer; (b2) a step of binding a plurality of oligonucleic acids to the core; (b3) bonding a hydrophilic polymer to the spacer; (b4) A method for producing oligonucleic acid nanoparticles, comprising the step of binding a cell internalization promoter to a hydrophilic polymer. In one embodiment, the method for producing oligonucleic acid nanoparticles may further include the step of (b5) binding a capping agent to the core. Thereby, oligonucleic acid nanoparticles in which at least a portion of the reactive functional groups of the dendritic polymer are capped with a capping agent can be produced.
  • step (b1) may include the steps of binding a plurality of linkers to the core, and binding an oligonucleic acid to the linkers.
  • steps (b1) to (b5) can be performed in this order, it is not necessary to perform them in this order.
  • step (b1) and step (b2) may be performed at the same time, or step (b5) may be performed before or at the same time as step (b1), or step (b1) and step (b2) may be performed simultaneously. It may be carried out between the steps (b2) and (b3).
  • step (b4) may be performed before step (b3), and in step (b3), the hydrophilic polymer to which the cell internalization promoter is bound may be bound to the spacer.
  • step (b3) may be performed before step (b1), and in step (b1), one or more spacers to which a hydrophilic polymer is bonded may be bonded to the core.
  • Any step of the above method for producing oligonucleic acid nanoparticles according to the first or second aspect can be performed using a known method.
  • Known methods include, for example, a method in which an amino group and a carboxy group are reacted using an activating group to form an amide bond, and a method in which thiol groups are reacted with each other using an activating group to form a disulfide bond.
  • a method of forming a bond using an inverse electron request type Diels-Alder reaction from an extremely electron-deficient heterocycle such as, and a compound having a distorted carbon multiple bond such as norbornene, trans-cyclooctene, or cyclooctyne, etc. can be mentioned.
  • the oligonucleic acid nanoparticles according to the above aspect may be produced by a known method other than the method according to the above aspect.
  • compositions include pharmaceutically acceptable excipients.
  • pharmaceutically acceptable refers to being tolerated by mammals from a pharmacological or toxicological standpoint. That is, a “pharmaceutically acceptable” substance is one that is physiologically acceptable and that does not typically produce an allergic or other harmful or toxic reaction when administered to a mammal. refers to A “pharmaceutically acceptable” substance is one that has been approved by a generally recognized regulatory agency or listed in a generally recognized pharmacopoeia for use in mammals, and more specifically humans. means a substance that is "Pharmaceutically acceptable excipient” means a pharmacologically inert material used with oligonucleic acid nanoparticles to formulate a pharmaceutical composition.
  • Additives may be liquid or solid. Excipients are selected with the intended method of administration in mind so as to obtain the desired dosage, consistency, etc. of the pharmaceutical composition. Additives are not particularly limited, but include, for example, water, physiological saline, other aqueous solvents, various carriers such as aqueous or oily bases, excipients, binders, pH adjusters, disintegrants, absorption enhancers, and lubricants. Examples include brighteners, coloring agents, flavoring agents, and fragrances. The blending ratio of additives can be appropriately set based on the range normally employed in the pharmaceutical field.
  • the pharmaceutical composition may be, for example, a sterile composition for injection.
  • Sterile compositions for injection can be prepared according to conventional pharmaceutical practice, such as dissolving or suspending the active ingredient in a solvent such as water for injection, natural vegetable oils, and the like.
  • aqueous solutions for injection include physiological saline, glucose, or isotonic solutions containing other adjuvants (eg, D-sorbitol, D-mannitol, lactose, sucrose, sodium chloride, etc.).
  • Aqueous solutions for injection may be prepared using suitable solubilizing agents, such as alcohols (e.g. ethanol), polyalcohols (e.g. propylene glycol or polyethylene glycol), nonionic surfactants (e.g.
  • Aqueous solutions for injection may also contain buffering agents (e.g., phosphate buffer or sodium acetate buffer), soothing agents (e.g., benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (e.g., human serum albumin or polyethylene glycol), preservatives (eg, benzyl alcohol, phenol, etc.), antimicrobial agents, dispersants, antioxidants, and the like, various materials known in the art.
  • the injection may be, for example, a lyophilized preparation.
  • the oligonucleic acid nanoparticles or pharmaceutical compositions according to the above aspects of the present invention can be used for the treatment and/or prevention of diseases involving specific gene products.
  • Diseases involving specific gene products include, for example, congenital metabolic disorders, congenital endocrine diseases, monogenic diseases, neurodegenerative diseases, neurological diseases, muscle diseases, meningitis, encephalitis, encephalopathy, lysosomal diseases, Diseases include, but are not limited to, malignant neoplasms, fibrosis, inflammatory diseases, immunodeficiency diseases, autoimmune diseases, or infectious diseases. Therefore, one aspect of the present invention is a therapeutic or preventive agent for the above-mentioned diseases, which contains oligonucleic acid nanoparticles as an active ingredient.
  • Another aspect of the present invention is a method for treating and/or preventing the above-mentioned diseases, which comprises administering a therapeutically effective amount of oligonucleic acid nanoparticles to a human or non-human animal.
  • the human may be a human in need of treatment, ie a patient.
  • Animals other than humans include warm-blooded mammals such as primates; birds; domestic animals or livestock such as cats, dogs, sheep, goats, cows, horses, and pigs; laboratory animals such as mice, rats, and guinea pigs; Means animals including fish; reptiles; zoo animals; and wild animals.
  • Administration methods include oral, sublingual, intravenous, intraarterial, subcutaneous, intradermal, intraperitoneal, intramuscular, intrathecal, intraventricular, intranasal, transmucosal, rectal, ophthalmic, intraocular, pulmonary, and transcutaneous.
  • Administration may be, but is not limited to, cutaneous, intraarticular, topical (dermal), intrafollicular, intravaginal, intrauterine, intratumoral, or intralymphatic administration, or combinations thereof.
  • Another aspect of the present invention is oligonucleic acid nanoparticles for use in the treatment and/or prevention of the above-mentioned diseases.
  • Another aspect of the present invention is the use of oligonucleic acid nanoparticles for producing therapeutic and/or preventive agents for the above-mentioned diseases.
  • oligonucleic acid nanoparticles or pharmaceutical compositions according to the above aspects of the present invention can also be used in combination with one or more other drugs.
  • Other drugs may be one or more therapeutic and/or prophylactic agents for diseases involving the specific gene products mentioned above.
  • examples of other drugs include pharmaceuticals that can be used for chemotherapy. That is, one aspect of the present invention is oligonucleic acid nanoparticles for treating diseases in combination with one or more therapeutic agents and/or preventive agents for the above-mentioned diseases.
  • Another aspect of the present invention is a medicament comprising a combination of an oligonucleic acid nanoparticle or a pharmaceutical composition and one or more therapeutic and/or prophylactic agents for the above-mentioned diseases.
  • the present invention is a platform technology that can efficiently transport oligonucleic acids into cells, and can be used for any disease for which oligonucleic acids can be applied as a therapeutic or preventive agent.
  • the drug is not limited to a specific drug.
  • the administration timing of the oligonucleic acid nanoparticles or pharmaceutical composition and the above-mentioned other drugs used in combination with the same is not limited, and they may be administered simultaneously to humans or non-human animals, or they may be administered at an appropriate time. Administration may be done at intervals.
  • a combination drug may be prepared by blending the above-mentioned other drugs with the pharmaceutical composition according to the above aspect.
  • the dosage and compounding amount of the above-mentioned other drugs can be appropriately determined based on the clinically used doses.
  • the blending ratio of the oligonucleic acid nanoparticles or pharmaceutical composition and the above-mentioned other drugs can be determined as appropriate depending on the administration target, administration route, target disease, symptoms, combination of other drugs, etc.
  • siRNA shown in Table 2 was prepared.
  • a thiol group was bonded to the 3' end of the sense strand RNA of siRNA via spacer 18 (hexaethylene glycol) and C6 linker (hexyl).
  • An amino group was bonded to the 5' end of the sense strand RNA of siRNA via a C6 linker.
  • siRNA and ethylenediaminetetraacetic acid trisodium salt were dissolved in 10mM phosphate buffered saline (PBS) at pH 7.4, and dithiothreitol (DTT) was added (final concentration: EDTA 0.5mM). , DTT 40mM). After heating this solution at 25° C. for 6 hours, it was purified six times by ultrafiltration (molecular weight cut off: 10 kDa) using PBS. The nucleic acid concentration of the obtained solution was determined from the measured value of absorption at 260 nm using an ultraviolet-visible spectrophotometer (manufactured by Tecan, Infinite M200 PRO).
  • the mixture was purified six times by ultrafiltration (manufactured by Merck & Co., Amicon Ultra, molecular weight cut off: 10 kDa) using PBS. Pure water was added to the collected aqueous solution to adjust the volume of the solution to 60 ⁇ L.
  • the amino group of DGL G4, Azido-PEG12-NHS ester, anionic fluorescent dye Alexa Fluor 647 NHS ester, and N-hydroxysuccinimide (NHS) of m-dPEG12-NHS ester were combined.
  • a nanoparticle compound azide-PEG12 AF6 DGL G4 was obtained.
  • the reaction solution was purified six times by ultrafiltration (Amicon Ultra, molecular weight cut off: 30 kDa) using pure water. Pure water was added to the collected aqueous solution to adjust the volume of the solution to 75 ⁇ L.
  • the azide group of azide-PEG12 AF6 DGL G4 was reacted with the DBCO group (dibenzocyclooctyne group) of NH2-siRNA-DBCO.
  • the reaction solution was purified by gel filtration using Hiprep 16/60 Sephacryl S-200 HR (manufactured by Cytiva) (eluent: PBS).
  • PBS Protein-binding protein
  • a fraction containing siRNA-bound DGL G4 was collected, and the solvent was replaced with 100 mM PBS using ultrafiltration (Amicon Ultra, molecular weight cut off: 50 kDa), and the volume was adjusted to 100 ⁇ L.
  • reaction solution was purified by gel filtration using Hiprep 16/60 Sephacryl S-200 HR (eluent: PBS).
  • fraction containing DGL G4 bound to siRNA was collected and concentrated by ultrafiltration (Amicon Ultra, molecular weight cut off: 50 kDa), and the volume of the liquid was adjusted to 100 ⁇ L.
  • the reaction solution was purified using Zeba (registered trademark) Spin Desalting Column (manufactured by Thermo Fisher Scientific, molecular weight fraction: 40 kDa). Next, purification was performed three times by ultrafiltration (Amicon Ultra, molecular weight cut off: 50 kDa) using PBS, and the volume of the solution was adjusted to 85 ⁇ L, and oligonucleic acid nanoparticles cRGD-PEG2000-siRNA-PEG12 AF6 DGL G4 were purified. A PBS solution was obtained.
  • Example 2 Production of cRGD-binding oligonucleic acid nanoparticles 2> (A) Synthesis of azide-PEG12 AF6 DGL G4 According to (B) of Example 1, azide-PEG12 AF6 DGL G4 was synthesized. However, the amount of 50 mM DMSO solution of azido-PEG12-NHS ester added was changed from 6.13 ⁇ L to 12.3 ⁇ L.
  • the azide group of azide-PEG12 AF6 DGL G4 was reacted with the DBCO group of NH2-siRNA-DBCO.
  • the reaction solution was purified by gel filtration using Hiprep 16/60 Sephacryl S-200 HR (eluent: PBS).
  • PBS Protein-binding protein
  • a fraction containing siRNA-bound DGL G4 was collected, and the solvent was replaced with 100 mM PBS using ultrafiltration (Amicon Ultra, molecular weight cut off: 50 kDa), and the volume was adjusted to 100 ⁇ L.
  • the mixture was purified six times by ultrafiltration (Amicon Ultra, molecular weight cut off: 100 kDa) using a 40 v/v% DMSO aqueous solution. A 40 v/v% DMSO aqueous solution was added to the collected solution, and the volume of the liquid was adjusted to 140 ⁇ L.
  • the recovered solution was then purified using HPLC (column: Agilent Bio SEC-5, 1000 ⁇ , 7.8 x 300 mm, eluent: PBS).
  • HPLC column: Agilent Bio SEC-5, 1000 ⁇ , 7.8 x 300 mm, eluent: PBS.
  • the recovered liquid was concentrated by ultrafiltration (Amicon Ultra, molecular weight cut off: 100 kDa), and the volume of the liquid was adjusted to 250 ⁇ L.
  • reaction solution was purified six times by ultrafiltration (Amicon Ultra, molecular weight cut off: 100 kDa) using PBS. PBS was added to the collected solution and the volume of the solution was adjusted to 80 ⁇ L.
  • the recovered solution was concentrated by ultrafiltration (Amicon Ultra, molecular weight cut off 100 kDa), and the volume of the solution was adjusted to 250 ⁇ L to obtain a PBS solution of oligonucleic acid nanoparticles cRGD-PEG2000-siRNA AF6 DGL G4. .
  • Example 4 Production of cRGD-binding oligonucleic acid nanoparticles 4>
  • A Synthesis of HOOC-PEG5000-siRNA AF6 DGL G4
  • D a PBS solution of HOOC-PEG5000-siRNA AF6 DGL G4 was obtained.
  • DBCO-PEG-SC molecular weight 5000, manufactured by Biopharma PEG Scientific
  • DBCO-PEG-SC molecular weight 2000
  • Example 3(C) instead of adding 34.0 ⁇ L of a 20 mM DMSO solution of DBCO-PEG-SC (molecular weight 2000) and 2.4 ⁇ L of PBS to 48.5 ⁇ L of the N3-siRNA AF6 DGL G4 solution in Example 3(C), Example To 189 ⁇ L of the N3-siRNA AF6 DGL G4 solution in 3(C), 132 ⁇ L of a 20 mM DMSO solution of DBCO-mPEG (molecular weight 5000, manufactured by Broadpharma) and 9.4 ⁇ L of PBS were added.
  • the mixture was purified six times by ultrafiltration (Amicon Ultra, molecular weight cut off: 100 kDa) using a 40 v/v% DMSO aqueous solution. A 40 v/v% DMSO aqueous solution was added to the collected solution, and the volume of the liquid was adjusted to 70 ⁇ L.
  • oligonucleic acid nanoparticles mPEG10000-siRNA AF5 DGL G4 were synthesized according to Comparative Example 2(D). However, instead of adding 84 ⁇ L of a 20 mM DMSO solution of DBCO-mPEG (molecular weight 5000) and 66 ⁇ L of PBS, 168 ⁇ L of a 10 mM DMSO solution of DBCO-mPEG (molecular weight 10000, manufactured by Broadpharm) and 192 ⁇ L of PBS were added.
  • the recovered liquid was concentrated by ultrafiltration (Amicon Ultra, molecular weight cut off 100 kDa), and the volume of the liquid was adjusted to 70 ⁇ L to obtain a PBS solution of the nanoparticle compound AZ4-PEG2000-siRNA AF6 DGL G4.
  • the concentrations of AZDye 405 and Alexa Fluor 647 in the resulting solution were determined from absorption measurements at 402 nm and 651 nm, respectively, using an ultraviolet-visible spectrophotometer.
  • the concentration of DGL G4 was determined by amino acid quantitative analysis using the AQC method described below. From these concentrations, the number of AZDye 405 bound to one DGL G4 was calculated to be 6. Therefore, it can be said that the number of cRGDfK modifications of the oligonucleic acid nanoparticle cRGD-PEG2000-siRNA AF6 DGL G4 of Example 3 is also 6.
  • the recovered solution was concentrated by ultrafiltration (Amicon Ultra, molecular weight cut off: 100 kDa), and the volume of the solution was adjusted to 65 ⁇ L to obtain a PBS solution of nanoparticle compound AZ4-siRNA AF6 DGL G4.
  • concentrations of AZDye 405 and Alexa Fluor 647 in the resulting solution were determined from absorption measurements at 402 nm and 651 nm, respectively, using an ultraviolet-visible spectrophotometer.
  • the concentration of DGL G4 was determined by amino acid quantitative analysis using the AQC method described below. From these concentrations, the number of AZDye 405 bound to one DGL G4 was calculated to be 33. Therefore, it can be said that the number of azide groups in N3-siRNA AF6 DGL G4 synthesized in Example 3(C) is also 33.
  • the comparative example To 25 ⁇ L of the PBS solution of mPEG5000-siRNA AF6 DGL G4 obtained in step 1, 1.9 ⁇ L of a 5 mM DMSO solution of AZDye405 DBCO and 4.3 ⁇ L of DMSO were added.
  • the concentrations of AZDye 405 and Alexa Fluor 647 in the resulting solution were determined from absorption measurements at 402 nm and 651 nm, respectively, using an ultraviolet-visible spectrophotometer.
  • the concentration of DGL G4 was determined by amino acid quantitative analysis using the AQC method described below. From these concentrations, the number of AZDye 405 bound to one DGL G4 was calculated to be 0. This number of AZDye 405 can be said to be equal to the number of unreacted azide groups in the oligonucleic acid nanoparticle mPEG5000-siRNA AF6 DGL G4 of Comparative Example 1.
  • the number of mPEG5000 modifications of the oligonucleic acid nanoparticles of Comparative Example 1 is determined from the number of AZDye 405 modifications (total number of azide groups) of AZ4-siRNA AF6 DGL G4 calculated in Reference Example 3. It can be determined by subtracting the number of AZDye 405 modifications (the number of unreacted azide groups), and this value was calculated to be 33.
  • ⁇ Reference example 5 Production of nanoparticle compounds for evaluation of oligonucleic acid nanoparticles of Comparative Examples 2 and 3> According to Reference Example 3, a PBS solution of nanoparticle compound AZ4-siRNA AF5 DGL G4 was obtained. Specifically, 1.1 ⁇ L of a 50 mM DMSO solution of AZDye405 DBCO and 3.9 ⁇ L of DMSO were added to 20 ⁇ L of the N3-siRNA AF5 DGL G4 solution shown in Comparative Example 2 (C), and reacted. As described above, the azide group of N3-siRNA AF5 DGL G4 was reacted with the DBCO group of AZDye405 DBCO.
  • the concentrations of AZDye 405 and Alexa Fluor 546 in the resulting solution were determined from absorption measurements at 402 nm and 554 nm, respectively, using an ultraviolet-visible spectrophotometer.
  • the concentration of DGL G4 was determined by amino acid quantitative analysis using the AQC method described below. From these concentrations, the number of AZDye 405 bound to one DGL G4 was calculated.
  • the number of PEG5000 determined from the number of AZDye 405 was 38.76. Therefore, it can be said that the number of azide groups in N3-siRNA AF5 DGL G4 synthesized in Comparative Example 2(C) is also 38.76.
  • the recovered solution was concentrated by ultrafiltration (Amicon Ultra, molecular weight cut off: 100 kDa), and the volume of the solution was adjusted to 70 ⁇ L to obtain a PBS solution of the nanoparticle compound AZ4-mPEG5000-siRNA AF5 DGL G4.
  • the number of AZDye 405 bound to one DGL G4 was 0.34. This number of AZDye405 can be said to be equal to the number of unreacted azide groups in the oligonucleic acid nanoparticle mPEG5000-siRNA AF5 DGL G4 of Comparative Example 2.
  • the number of mPEG5000 modifications of the oligonucleic acid nanoparticles of Comparative Example 2 is determined from the number of AZDye 405 modifications (total number of azide groups) of AZ4-siRNA AF5 DGL G4 calculated in Reference Example 5. It can be determined by subtracting the number of AZDye 405 modifications (the number of unreacted azide groups), and this value was calculated to be 38.42.
  • ⁇ Reference example 7 Production of nanoparticle compound for evaluation of oligonucleic acid nanoparticles of Comparative Example 3> According to Reference Example 6, a solution of nanoparticle compound AZ4-mPEG10000-siRNA AF5 DGL G4 was obtained. However, instead of mPEG5000-siRNA AF5 DGL G4 obtained in Comparative Example 2, mPEG10000-siRNA AF5 DGL G4 obtained in Comparative Example 3 was used. The number of AZDye 405 bound to one DGL G4 was 0.
  • the number of mPEG10000 modifications of the oligonucleic acid nanoparticle mPEG1000-siRNA AF5 DGL G4 of Comparative Example 3 is calculated from the number of AZDye 405 modifications (total number of azide groups) of AZ4-siRNA AF5 DGL G4 calculated in Reference Example 5. It can be determined by subtracting the number of AZDye 405 modifications (number of unreacted azide groups) of -siRNA AF5 DGL G4, and this value was calculated to be 37.
  • ⁇ Test example 1 Evaluation of oligonucleic acid nanoparticles> (A) Evaluation of the number of oligonucleic acids, cell internalization promoters, and fluorescent molecules The concentrations of oligonucleic acids, cell internalization promoters, and fluorescent molecules in the oligonucleic acid nanoparticle samples used in each test example below are as follows: The number of oligonucleic acids, hydrophilic polymers, cell internalization promoters, and fluorescent molecules bound to one dendritic polymer was calculated from these concentrations. Note that the number of hydrophilic polymers was considered to be the same as the number of cell internalization promoters. However, the numbers of cell internalization promoters and hydrophilic polymers in the oligonucleic acid nanoparticles used in Test Example 3 were determined as described in Reference Examples 1, 2, and 4. The calculation results are shown in each test example below.
  • concentrations of oligonucleic acids and fluorescent molecules were determined from absorption measurements at the following wavelengths using a UV-visible spectrophotometer: 260 nm for siRNA, 402 nm for AZDye405, 554 nm for Alexa Fluor 546, and 651 nm for Alexa Fluor 647.
  • the concentrations of the dendritic polymer and polypeptide-based cell internalization promoters were determined by amino acid quantitative analysis (AQC method) as follows. First, 30 ⁇ L of an aqueous solution of an oligonucleic acid nanoparticle sample and 300 ⁇ L of constant boiling point hydrochloric acid were added to a sealable glass bottle, the bottle was sealed, and the bottle was heated at 110° C. for 24 hours for hydrolysis. After hydrolysis, the solvent was removed under reduced pressure while heating the reaction solution at 45°C. AQC (manufactured by Adipogen Life Sciences) was dissolved in acetonitrile (super dehydrated) at 60°C and adjusted to 3 mg/mL.
  • AQC manufactured by Adipogen Life Sciences
  • the obtained filtrate was subjected to reverse phase HPLC (Column: AccQ-Tag Column, 60 ⁇ , 4 ⁇ m, 3.9 x 150 mm, eluent A: AccQ-Tag Eluent A/water (1/9; v/v), eluent B: water/acetonitrile (1/1; v/v)), and the concentration of DGL G4 was determined from the integral value of the peak of lysine residue, and the concentration of cRGDfK was determined from the integral value of the unique peak.
  • AccQ-Tag Column and AccQ-Tag Eluent A were purchased from Waters Corporation.
  • ⁇ Test Example 2 In vitro evaluation of cRGD ligand-bound oligonucleic acid nanoparticles> Cellular uptake of oligonucleic acid nanoparticles was evaluated as follows. First, a human glioblastoma cell line (U-87MG) was seeded in a 96-well plate and cultured at 37° C. and 5% CO 2 using a DMEM medium containing 10% FBS. The next day, the medium was replaced, and a sample was added to each well to transfect the cells, and the cells were cultured at 37° C. and 5% CO 2 . The siRNA concentration in the samples at the time of transfection was 0.01 ⁇ M, 0.1 ⁇ M or 1 ⁇ M.
  • the cells were washed with PBS, and then the fluorescence intensity was measured (excitation wavelength 640 nm, fluorescence wavelength 675 nm). Furthermore, the siRNA concentration is converted to the concentration of fluorescent molecules from the ratio of the number of siRNAs and fluorescent molecules (number of siRNAs/number of fluorescent molecules), and after approximating that the fluorescence intensity and the concentration of fluorescent molecules are directly proportional, Fluorescence intensity was calculated when the concentration of fluorescent molecules was 2 nM, 20 nM, or 200 nM. The samples used are shown in Table 4, and the results obtained are shown in FIG.
  • Gene knockdown activity by oligonucleic acid nanoparticles was evaluated as follows. First, a human glioblastoma cell line (U-87MG) was seeded in a 96-well plate and cultured at 37° C. and 5% CO 2 using a DMEM medium containing 10% FBS. The next day, the medium was replaced, and a sample was added to each well to transfect the cells, and the cells were cultured at 37° C. and 5% CO 2 . The siRNA concentration in the sample at the time of transfection was 1 ⁇ M. For the control group, PBS was added instead of the sample.
  • U-87MG human glioblastoma cell line
  • ATP5B primers the primers shown in SEQ ID NO: 9 and SEQ ID NO: 10 shown in Table 5 below were used, and as the GAPDH primers, the primers shown in SEQ ID NO: 11 and SEQ ID NO: 12 shown in Table 5 below were used.
  • PCR conditions were as follows.
  • One cycle was designed to be 1 second at 95°C and 30 seconds at 60°C, and 40 cycles were performed. Based on the results of quantitative RT-PCR, the value of "expression level of ATP5B/expression level of GAPDH (internal standard gene)" was calculated, and the calculation results for the control group and the calculation results for the sample addition group were compared. The samples used are shown in Table 4, and the results obtained are shown in FIG.
  • reaction solution is purified by gel filtration using Hiprep 16/60 Sephacryl S-200 HR (eluent: PBS), and a fraction containing siRNA-bound DGL G4 is collected.
  • PBS solution of bis(azide-PEG3) siRNA AF6 DGL G4 can be obtained by concentrating the collected solution by ultrafiltration (Amicon Ultra, molecular weight cut off 50 kDa) and adjusting the volume of the solution to 100 ⁇ L.
  • reaction solution is purified by gel filtration using Sephadex LH-20 (manufactured by Cytiva) (eluent: DMSO), and a fraction containing PEG bound to cRGDfK is collected. After removing the solvent from the collected solution by freeze-drying, a DMSO solution of cRGD-PEG2k-DBCO can be obtained by adding DMSO to adjust the concentration to 50 mM.
  • oligonucleic acid nanoparticles bis(cRGD-PEG2k) siRNA AF6 DGL G4 were purified by ultrafiltration (Amicon Ultra, molecular weight cutoff 100 kDa) three times using PBS and the volume of the solution was adjusted to 100 ⁇ L. A PBS solution can be obtained.
  • the reaction solution is purified by gel filtration using Sephadex LH-20 (eluent: DMSO), and a fraction containing CP1-bound DGL G4 is collected. After removing the solvent by freeze-drying the collected solution, a DMSO solution of azide-CP1 SPDP AF6 DGL4 can be obtained by adding DMSO to adjust the concentration to 32.2 mM.
  • the reaction solution is purified by gel filtration using Hiprep 16/60 Sephacryl S-200 HR (eluent: PBS), and a fraction containing siRNA-bound DGL G4 is collected.
  • PBS solution of azide-CP1 siRNA AF6 DGL4 can be obtained by concentrating the collected solution by ultrafiltration (Amicon Ultra, molecular weight cut off: 50 kDa) and adjusting the volume of the solution to 100 ⁇ L.
  • oligonucleic acid nanoparticles cRGD-PEG5k-CP2 siRNA AF6 DGL G4 were purified by ultrafiltration (Amicon Ultra, molecular weight cutoff 100 kDa) three times using PBS and the volume of the solution was adjusted to 100 ⁇ L. A solution can be obtained.
  • oligonucleic acid nanoparticles according to one aspect of the present invention can improve the amount of oligonucleic acids transported into the cytoplasm, they can be used as pharmaceutical compositions or medicines for treating or preventing diseases. be.

Abstract

The purpose of the present invention is to increase the amount of oligo-nucleic acid transported into a cytoplasm by bringing about an efficient interaction of a cell uptake promoter with a target cell. An oligo-nucleic acid nanoparticle according to one aspect of the present invention comprises: a core constituted by a dendritic polymer; a plurality of oligo-nucleic acids bonded to the core; one or a plurality of hydrophilic polymers bonded to the plurality of oligo-nucleic acids; and one or a plurality of cell uptake promoters bonded to the one or plurality of hydrophilic polymers. The bonds between the core and the oligo-nucleic acids, the bonds between the oligo-nucleic acids and the hydrophilic polymer(s), and the bonds between the hydrophilic polymer(s) and the cell uptake promoter(s) are each independently direct bonds or bonds facilitated by a linker. The cell uptake promoter is located on the surface of the oligo-nucleic acid nanoparticle.

Description

オリゴ核酸ナノ粒子oligonucleic acid nanoparticles
　本発明は、オリゴ核酸ナノ粒子に関する。 The present invention relates to oligonucleic acid nanoparticles.
　細胞内に発現する種々の遺伝子産物の発現を直接的に制御できる核酸医薬は、従来の医薬品では適応できない疾患に対する治療薬となり得るため、その医療応用が強く期待されている。しかし、核酸医薬は、核酸分子自体の分子量が大きく、多くの負電荷を有しており、かつ親水性が高いことから、細胞膜を自発的に透過することができない。核酸分子を、その作用が発現される場所である細胞質内に輸送させるための手法として、核酸分子に疎水性分子、糖などの細胞内在化促進剤を修飾する手法、及び核酸分子を機能性ナノ粒子に内包する手法が知られている。 Nucleic acid medicines that can directly control the expression of various gene products expressed within cells can serve as therapeutic agents for diseases that cannot be treated with conventional medicines, and their medical applications are therefore highly anticipated. However, nucleic acid drugs cannot spontaneously permeate cell membranes because the nucleic acid molecules themselves have large molecular weights, have many negative charges, and are highly hydrophilic. As a method for transporting nucleic acid molecules into the cytoplasm, where their effects are expressed, there are two methods: modifying nucleic acid molecules with hydrophobic molecules, sugars, and other cellular internalization promoters, and converting nucleic acid molecules into functional nanoparticles. A method of encapsulating it in particles is known.
　例えば、特許文献１及び非特許文献１には、ポリマーに、核酸分子と、細胞内在化促進剤とを共有結合させることでナノ構造体を調製し、核酸分子を細胞質内に輸送する方法が開示されている。 For example, Patent Document 1 and Non-Patent Document 1 disclose a method of preparing a nanostructure by covalently bonding a nucleic acid molecule and a cell internalization promoter to a polymer and transporting the nucleic acid molecule into the cytoplasm. has been done.
国際公開第２０１３／０６２９８２号International Publication No. 2013/062982
　特許文献１及び非特許文献１の技術には、細胞内在化促進剤を標的細胞と効率良く相互作用させる点において、改善の余地があった。本発明の主たる目的は、細胞内在化促進剤を標的細胞と効率良く相互作用させて、細胞質内に輸送されるオリゴ核酸の量を向上させることにある。 The techniques of Patent Document 1 and Non-Patent Document 1 have room for improvement in terms of allowing the cell internalization promoter to interact efficiently with target cells. The main objective of the present invention is to increase the amount of oligonucleic acid transported into the cytoplasm by allowing a cell internalization promoter to interact efficiently with target cells.
　本発明者らは、鋭意研究を重ねた結果、オリゴ核酸を細胞内に効率的に輸送できる技術を開発した。本発明は、生体成分との非特異的相互作用の低減に適した構造を有し、細胞内在化促進剤を含む機能性ナノ粒子である、オリゴ核酸ナノ粒子、及びその製造方法を提供する。 As a result of extensive research, the present inventors have developed a technology that can efficiently transport oligonucleotides into cells. The present invention provides oligonucleic acid nanoparticles, which are functional nanoparticles that have a structure suitable for reducing nonspecific interactions with biological components and contain a cell internalization promoter, and a method for producing the same.
　すなわち、本発明の一態様は、樹状ポリマーで構成されたコアと、コアの周りに配置された、複数個のオリゴ核酸、１又は複数個の親水性ポリマー、及び１又は複数個の細胞内在化促進剤とを含む、単分子で構成されるオリゴ核酸ナノ粒子であって、オリゴ核酸は、コアと好ましくは共有結合により結合しており、親水性ポリマーはオリゴ核酸と好ましくは共有結合により結合しており、細胞内在化促進剤は親水性ポリマーと好ましくは共有結合により結合している、オリゴ核酸ナノ粒子を提供する。本発明の別の一態様は、樹状ポリマーで構成されたコアと、コアの周りに配置された、複数個のオリゴ核酸、１又は複数個のスペーサー、１又は複数個の親水性ポリマー、及び１又は複数個の細胞内在化促進剤とを含む、単分子で構成されるオリゴ核酸ナノ粒子であって、オリゴ核酸又はスペーサーは、コアと好ましくは共有結合により結合しており、親水性ポリマーはスペーサーと好ましくは共有結合により結合しており、細胞内在化促進剤は親水性ポリマーと好ましくは共有結合により結合している、オリゴ核酸ナノ粒子を提供する。本発明の別の一態様では、コアを形成する樹状ポリマーの有する反応性官能基が、オリゴ核酸又はスペーサーに加えてキャッピング剤との結合にも用いられている。これらの態様では、親水性ポリマーが、オリゴ核酸の空間的な広がり（回転半径）の外側に位置し、また、細胞内在化促進剤がオリゴ核酸ナノ粒子の表面に位置するため、オリゴ核酸ナノ粒子が標的細胞と効率良く相互作用しやすくなるとともに、オリゴ核酸ナノ粒子と標的細胞以外の生体成分との非特異的相互作用が低減される効果が期待できる。 That is, one embodiment of the present invention includes a core composed of a dendritic polymer, a plurality of oligonucleic acids, one or more hydrophilic polymers, and one or more cell-internal polymers arranged around the core. an oligonucleic acid nanoparticle composed of a single molecule, the oligonucleic acid being bonded to the core, preferably by a covalent bond, and a hydrophilic polymer being bonded to the oligonucleic acid, preferably by a covalent bond. The present invention provides oligonucleic acid nanoparticles in which the cell internalization promoting agent is preferably covalently bound to a hydrophilic polymer. Another aspect of the present invention includes a core composed of a dendritic polymer, a plurality of oligonucleic acids, one or more spacers, one or more hydrophilic polymers arranged around the core, and An oligonucleic acid nanoparticle composed of a single molecule, comprising one or more cell internalization promoters, wherein the oligonucleic acid or spacer is bound to the core, preferably by a covalent bond, and the hydrophilic polymer is Oligonucleic acid nanoparticles are provided, wherein the spacer is preferably covalently bound, and the cellular internalization enhancer is preferably covalently bound to the hydrophilic polymer. In another embodiment of the present invention, the reactive functional groups of the dendritic polymer forming the core are used to bind not only the oligonucleic acid or the spacer but also the capping agent. In these embodiments, the hydrophilic polymer is located outside the spatial extent (radius of gyration) of the oligonucleic acid, and the cellular internalization promoter is located on the surface of the oligonucleic acid nanoparticle. It is expected that the oligonucleic acid nanoparticles will interact more efficiently with target cells, and non-specific interactions between oligonucleic acid nanoparticles and biological components other than target cells will be reduced.
　すなわち、本発明は、以下のとおりである。
［１］　樹状ポリマーで構成されたコアと、
　該コアに結合した複数個のオリゴ核酸と、
　該複数個のオリゴ核酸に結合した１又は複数個の親水性ポリマーと、
　該１又は複数個の親水性ポリマーに結合した１又は複数個の細胞内在化促進剤と、を含むオリゴ核酸ナノ粒子であって、
　前記コアと前記オリゴ核酸との結合、前記オリゴ核酸と前記親水性ポリマーとの結合、及び前記親水性ポリマーと前記細胞内在化促進剤との結合は、それぞれ独立に、直接結合又はリンカーを介した結合であり、
　前記細胞内在化促進剤は前記オリゴ核酸ナノ粒子の表面に位置する、オリゴ核酸ナノ粒子。
［２］　前記直接結合、又は前記リンカーと前記コア、前記オリゴ核酸、前記親水性ポリマー、若しくは前記細胞内在化促進剤との結合が、共有結合、金属配位又はホスト－ゲスト相互作用によるものである、［１］に記載のオリゴ核酸ナノ粒子。
［３］　前記直接結合、又は前記リンカーと前記コア、前記オリゴ核酸、前記親水性ポリマー、若しくは前記細胞内在化促進剤との結合が、共有結合又は金属配位によるものである、［１］に記載のオリゴ核酸ナノ粒子。
［４］　前記直接結合、又は前記リンカーと前記コア、前記オリゴ核酸、前記親水性ポリマー、若しくは前記細胞内在化促進剤との結合が、共有結合によるものである、［１］に記載のオリゴ核酸ナノ粒子。
［５］　前記樹状ポリマーの反応性官能基の少なくとも一部がキャッピング剤によりキャッピングされている、［１］～［４］のいずれか一つに記載のオリゴ核酸ナノ粒子。
［６］　樹状ポリマーで構成されたコアと、
　該コアに結合した複数個のオリゴ核酸と、
　前記コアにスペーサーを介して結合した１又は複数個の親水性ポリマーと、
　該１又は複数個の親水性ポリマーに結合した１又は複数個の細胞内在化促進剤と、を含むオリゴ核酸ナノ粒子であって、
　前記コアと前記オリゴ核酸との結合、前記コアと前記スペーサーとの結合、前記スペーサーと前記親水性ポリマーとの結合、及び前記親水性ポリマーと前記細胞内在化促進剤との結合は、それぞれ独立に、直接結合又はリンカーを介した結合であり、
　前記細胞内在化促進剤は前記オリゴ核酸ナノ粒子の表面に位置する、オリゴ核酸ナノ粒子。
［７］　前記直接結合、又は前記リンカーと前記コア、前記オリゴ核酸、前記スペーサー、前記親水性ポリマー、若しくは前記細胞内在化促進剤との結合が、共有結合、金属配位又はホスト－ゲスト相互作用によるものである、［６］に記載のオリゴ核酸ナノ粒子。
［８］　前記直接結合、又は前記リンカーと前記コア、前記オリゴ核酸、前記スペーサー、前記親水性ポリマー、若しくは前記細胞内在化促進剤との結合が、共有結合又は金属配位によるものである、［６］に記載のオリゴ核酸ナノ粒子。
［９］　前記直接結合、又は前記リンカーと前記コア、前記オリゴ核酸、前記スペーサー、前記親水性ポリマー、若しくは前記細胞内在化促進剤との結合が、共有結合によるものである、［６］に記載のオリゴ核酸ナノ粒子。
［１０］　前記樹状ポリマーの反応性官能基の少なくとも一部がキャッピング剤によりキャッピングされている、［６］～［９］のいずれか一つに記載のオリゴ核酸ナノ粒子。
［１１］　前記キャッピング剤が、親水性分子及び疎水性分子からなる群から選択される１種以上の分子である、［５］又は［１０］記載のオリゴ核酸ナノ粒子。
［１２］　前記キャッピング剤が親水性分子である、［１１］に記載のオリゴ核酸ナノ粒子。
［１３］　前記キャッピング剤が、電気的に中性な親水性分子、酸性条件下でプロトン化する極性分子、アニオン性分子、及びカチオン性分子からなる群から選択される１種類以上の親水性分子である、［１１］に記載のオリゴ核酸ナノ粒子。
［１４］　前記キャッピング剤が、電気的に中性な親水性分子、酸性条件下でプロトン化する極性分子、及びアニオン性分子からなる群から選択される１種類以上の親水性分子である、［１１］に記載のオリゴ核酸ナノ粒子。
［１５］　前記キャッピング剤が疎水性分子である、［１１］に記載のオリゴ核酸ナノ粒子。
［１６］　前記キャッピング剤が、脂肪族化合物、芳香族化合物、トリアルキルアミン、及びステロイド又はその類縁体からなる群から選択される１種類以上の疎水性分子である、［１１］に記載のオリゴ核酸ナノ粒子。
［１７］　前記キャッピング剤が脂肪族化合物である、［１１］に記載のオリゴ核酸ナノ粒子。
［１８］　前記樹状ポリマーが、デンドリグラフト又はデンドリマーである、［１］～［１７］のいずれか一つに記載のオリゴ核酸ナノ粒子。
［１９］　前記樹状ポリマーにおけるモノマー同士が、アミド結合、エステル結合、又はグリコシド結合により結合している、［１］～［１７］のいずれか一つに記載のオリゴ核酸ナノ粒子。
［２０］　前記樹状ポリマーにおけるモノマー同士がアミド結合又はエステル結合により結合している、［１］～［１７］のいずれか一つに記載のオリゴ核酸ナノ粒子。
［２１］　前記樹状ポリマーがポリ－Ｌ－リシンデンドリグラフト、ポリアミドアミンデンドリマー、又は２，２－ビス（ヒドロキシル－メチル）プロピオン酸デンドリマーである、［１］～［１７］のいずれか一つに記載のオリゴ核酸ナノ粒子。
［２２］　前記オリゴ核酸が遺伝子発現制御剤である、［１］～［２１］のいずれか一つに記載のオリゴ核酸ナノ粒子。
［２３］　前記遺伝子発現制御剤が、ｍＲＮＡの発現を抑制する分子である、［２２］に記載のオリゴ核酸ナノ粒子。
［２４］　前記遺伝子発現制御剤が、ＲＮＡ干渉誘導核酸又はアンチセンス核酸である、［２２に記載のオリゴ核酸ナノ粒子。
［２５］　前記スペーサーが、ポリエチレングリコール、ポリ（２－アルキル－２－オキサゾリン）、ポリペプチド及びポリペプトイドからなる群から選択される１種以上のスペーサーである、［６］～［１０］のいずれか一つに記載のオリゴ核酸ナノ粒子。
［２６］　前記スペーサーが、ポリエチレングリコール、又はカチオン性ポリペプチドである、［６］～［１０］のいずれか一つに記載のオリゴ核酸ナノ粒子。
［２７］　前記親水性ポリマーが、ポリエチレングリコール、ポリ（２－アルキル－２－オキサゾリン）、ポリペプチド及びポリペプトイドからなる群から選択される１種以上の親水性ポリマーである、［１］～［２６］のいずれか一つに記載のオリゴ核酸ナノ粒子。
［２８］　前記親水性ポリマーが、ポリエチレングリコール、ポリ（２－メチル－２－オキサゾリン）、ＥＫペプチド及びポリサルコシンからなる群から選択される１種以上の水性ポリマーである、［１］～［２６］のいずれか一つに記載のオリゴ核酸ナノ粒子。
［２９］　前記細胞内在化促進剤が、ポリペプチド、アプタマー、抗体若しくはそのフラグメント、糖、脂質、及びその他の低分子化合物からなる群から選択される１種以上の細胞内在化促進剤である、［１］～［２８］のいずれか一つに記載のオリゴ核酸ナノ粒子。
［３０］　前記細胞内在化促進剤が、疎水性分子、ポリカチオン、ポリペプチド、アプタマー、抗体若しくはそのフラグメント、糖、及び脂質以外の、分子量が２０００以下の低分子化合物である、［１］～［２８］のいずれか一つに記載のオリゴ核酸ナノ粒子。
［３１］　前記細胞内在化促進剤がポリペプチドである、［１］～［２８］のいずれか一つに記載のオリゴ核酸ナノ粒子。
［３２］　前記細胞内在化促進剤がアプタマーである、［１］～［２８］のいずれか一つに記載のオリゴ核酸ナノ粒子。
［３３］　前記細胞内在化促進剤が抗体若しくはそのフラグメントである、［１］～［２８］のいずれか一つに記載のオリゴ核酸ナノ粒子。
［３４］　前記細胞内在化促進剤が糖である、［１］～［２８］のいずれか一つに記載のオリゴ核酸ナノ粒子。
［３５］　前記細胞内在化促進剤が脂質である、［１］～［２８］のいずれか一つに記載のオリゴ核酸ナノ粒子。
［３６］　［１］～［３５］のいずれか一つに記載のオリゴ核酸ナノ粒子を有効成分として含有する、医薬組成物。
［３７］　［１］～［３５］のいずれか一つに記載のオリゴ核酸ナノ粒子を有効成分として含有する、
　先天性代謝異常症、先天性内分泌疾患、単一遺伝子疾患、神経変性疾患、神経疾患、筋疾患、髄膜炎、脳炎、脳症、ライソゾーム病、悪性新生物、繊維症、炎症性疾患、免疫不全疾患、自己免疫疾患、及び感染症からなる群から選択される疾患に対する治療剤又は予防剤。
［３８］　治療上の有効量の［１］～［３５］のいずれか一つに記載のオリゴ核酸ナノ粒子を投与することを含む、
　先天性代謝異常症、先天性内分泌疾患、単一遺伝子疾患、神経変性疾患、神経疾患、筋疾患、髄膜炎、脳炎、脳症、ライソゾーム病、悪性新生物、繊維症、炎症性疾患、免疫不全疾患、自己免疫疾患、及び感染症からなる群から選択される疾患を治療及び／又は予防するための方法。
［３９］　先天性代謝異常症、先天性内分泌疾患、単一遺伝子疾患、神経変性疾患、神経疾患、筋疾患、髄膜炎、脳炎、脳症、ライソゾーム病、悪性新生物、繊維症、炎症性疾患、免疫不全疾患、自己免疫疾患、及び感染症からなる群から選択される疾患に対する治療剤及び／又は予防剤を製造するための、［１］～［３５］のいずれか一つに記載のオリゴ核酸ナノ粒子の使用。
［４０］　先天性代謝異常症、先天性内分泌疾患、単一遺伝子疾患、神経変性疾患、神経疾患、筋疾患、髄膜炎、脳炎、脳症、ライソゾーム病、悪性新生物、繊維症、炎症性疾患、免疫不全疾患、自己免疫疾患、及び感染症からなる群から選択される疾患の治療及び／又は予防に使用するための、［１］～［３５］のいずれか一つに記載のオリゴ核酸ナノ粒子。
［４１］　［１］～［３５］のいずれか一つに記載のオリゴ核酸ナノ粒子と、
　疾患に対する１種以上の治療剤及び／又は１種以上の予防剤と、の組み合わせを含み、
　前記疾患は、先天性代謝異常症、先天性内分泌疾患、単一遺伝子疾患、神経変性疾患、神経疾患、筋疾患、髄膜炎、脳炎、脳症、ライソゾーム病、悪性新生物、繊維症、炎症性疾患、免疫不全疾患、自己免疫疾患、及び感染症からなる群から選択される、医薬。
［４２］　疾患に対する１種以上の治療剤及び／又は１種以上の予防剤と併用して疾患を治療するための、［１］～［３５］のいずれか一つに記載のオリゴ核酸ナノ粒子であって、
　前記疾患は、先天性代謝異常症、先天性内分泌疾患、単一遺伝子疾患、神経変性疾患、神経疾患、筋疾患、髄膜炎、脳炎、脳症、ライソゾーム病、悪性新生物、繊維症、炎症性疾患、免疫不全疾患、自己免疫疾患、及び感染症からなる群から選択される、オリゴ核酸ナノ粒子。
［４３］　（ａ１）樹状ポリマーで構成されたコアに複数個のオリゴ核酸を結合させる工程と、
　（ａ２）前記オリゴ核酸に親水性ポリマーを結合させる工程と、
　（ａ３）前記親水性ポリマーに細胞内在化促進剤を結合させる工程と、を含む、［１］～［４］のいずれか一つに記載のオリゴ核酸ナノ粒子の製造方法。
［４４］　（ｂ１）樹状ポリマーで構成されたコアに１又は複数個のスペーサーを結合させる工程と、
　（ｂ２）前記コアに複数個のオリゴ核酸を結合させる工程と、
　（ｂ３）前記スペーサーに親水性ポリマーを結合させる工程と、
　（ｂ４）前記親水性ポリマーに細胞内在化促進剤を結合させる工程と、を含む、［６］～［９］のいずれか一つに記載のオリゴ核酸ナノ粒子の製造方法。
［４５］　（ａ１）樹状ポリマーで構成されたコアに複数個のオリゴ核酸を結合させる工程と、
　（ａ２）前記オリゴ核酸に親水性ポリマーを結合させる工程と、
　（ａ３）前記親水性ポリマーに細胞内在化促進剤を結合させる工程と、
　（ａ４）前記コアにキャッピング剤を結合させる工程と、を含む、［５］に記載のオリゴ核酸ナノ粒子の製造方法。
［４６］　（ｂ１）樹状ポリマーで構成されたコアに１又は複数個のスペーサーを結合させる工程と、
　（ｂ２）前記コアに複数個のオリゴ核酸を結合させる工程と、
　（ｂ３）前記スペーサーに親水性ポリマーを結合させる工程と、
　（ｂ４）前記親水性ポリマーに細胞内在化促進剤を結合させる工程と、
　（ａ５）前記コアにキャッピング剤を結合させる工程と、を含む、［１０］に記載のオリゴ核酸ナノ粒子の製造方法。 That is, the present invention is as follows.
[1] A core composed of a dendritic polymer,
a plurality of oligonucleic acids bound to the core;
one or more hydrophilic polymers bound to the plurality of oligonucleic acids;
An oligonucleic acid nanoparticle comprising one or more cell internalization promoters bound to the one or more hydrophilic polymers,
The binding between the core and the oligonucleic acid, the binding between the oligonucleic acid and the hydrophilic polymer, and the binding between the hydrophilic polymer and the cell internalization promoter are each independently performed by direct binding or via a linker. is a combination,
An oligonucleic acid nanoparticle, wherein the cell internalization promoting agent is located on the surface of the oligonucleic acid nanoparticle.
[2] The direct bond, or the bond between the linker and the core, the oligonucleic acid, the hydrophilic polymer, or the cell internalization promoter is not due to a covalent bond, metal coordination, or host-guest interaction. The oligonucleic acid nanoparticle according to [1].
[3] In [1], the direct bond, or the bond between the linker and the core, the oligonucleic acid, the hydrophilic polymer, or the cell internalization promoter is a covalent bond or metal coordination. The oligonucleic acid nanoparticles described.
[4] The oligonucleic acid according to [1], wherein the direct bond or the bond between the linker and the core, the oligonucleic acid, the hydrophilic polymer, or the cell internalization promoter is a covalent bond. nanoparticles.
[5] The oligonucleic acid nanoparticle according to any one of [1] to [4], wherein at least a portion of the reactive functional groups of the dendritic polymer are capped with a capping agent.
[6] A core composed of a dendritic polymer,
a plurality of oligonucleic acids bound to the core;
one or more hydrophilic polymers bonded to the core via a spacer;
An oligonucleic acid nanoparticle comprising one or more cell internalization promoters bound to the one or more hydrophilic polymers,
The binding between the core and the oligonucleic acid, the binding between the core and the spacer, the binding between the spacer and the hydrophilic polymer, and the binding between the hydrophilic polymer and the cell internalization promoter are each independently performed. , a direct bond or a bond via a linker,
An oligonucleic acid nanoparticle, wherein the cell internalization promoting agent is located on the surface of the oligonucleic acid nanoparticle.
[7] The direct bond, or the bond between the linker and the core, the oligonucleic acid, the spacer, the hydrophilic polymer, or the cell internalization promoter, is a covalent bond, metal coordination, or host-guest interaction. The oligonucleic acid nanoparticle according to [6], which is based on
[8] The direct bond, or the bond between the linker and the core, the oligonucleic acid, the spacer, the hydrophilic polymer, or the cell internalization promoter, is based on a covalent bond or metal coordination. 6].
[9] According to [6], the direct bond, or the bond between the linker and the core, the oligonucleic acid, the spacer, the hydrophilic polymer, or the cell internalization promoter is a covalent bond. oligonucleic acid nanoparticles.
[10] The oligonucleic acid nanoparticle according to any one of [6] to [9], wherein at least a portion of the reactive functional groups of the dendritic polymer are capped with a capping agent.
[11] The oligonucleic acid nanoparticle according to [5] or [10], wherein the capping agent is one or more molecules selected from the group consisting of hydrophilic molecules and hydrophobic molecules.
[12] The oligonucleic acid nanoparticle according to [11], wherein the capping agent is a hydrophilic molecule.
[13] The capping agent is one or more types of hydrophilic molecules selected from the group consisting of electrically neutral hydrophilic molecules, polar molecules that protonate under acidic conditions, anionic molecules, and cationic molecules. The oligonucleic acid nanoparticle according to [11], which is
[14] The capping agent is one or more types of hydrophilic molecules selected from the group consisting of electrically neutral hydrophilic molecules, polar molecules that protonate under acidic conditions, and anionic molecules. 11].
[15] The oligonucleic acid nanoparticle according to [11], wherein the capping agent is a hydrophobic molecule.
[16] The oligo according to [11], wherein the capping agent is one or more hydrophobic molecules selected from the group consisting of aliphatic compounds, aromatic compounds, trialkylamines, and steroids or analogs thereof. Nucleic acid nanoparticles.
[17] The oligonucleic acid nanoparticle according to [11], wherein the capping agent is an aliphatic compound.
[18] The oligonucleic acid nanoparticle according to any one of [1] to [17], wherein the dendritic polymer is a dendrigraft or a dendrimer.
[19] The oligonucleic acid nanoparticle according to any one of [1] to [17], wherein the monomers in the dendritic polymer are bonded to each other through an amide bond, an ester bond, or a glycosidic bond.
[20] The oligonucleic acid nanoparticle according to any one of [1] to [17], wherein the monomers in the dendritic polymer are bonded to each other through an amide bond or an ester bond.
[21] Any one of [1] to [17], wherein the dendritic polymer is a poly-L-lysine dendrigraft, a polyamide amine dendrimer, or a 2,2-bis(hydroxyl-methyl)propionic acid dendrimer. Oligonucleic acid nanoparticles described in .
[22] The oligonucleic acid nanoparticle according to any one of [1] to [21], wherein the oligonucleic acid is a gene expression control agent.
[23] The oligonucleic acid nanoparticle according to [22], wherein the gene expression control agent is a molecule that suppresses mRNA expression.
[24] The oligonucleic acid nanoparticle according to [22], wherein the gene expression control agent is an RNA interference-inducing nucleic acid or an antisense nucleic acid.
[25] Any one of [6] to [10], wherein the spacer is one or more spacers selected from the group consisting of polyethylene glycol, poly(2-alkyl-2-oxazoline), polypeptide, and polypeptoid. The oligonucleic acid nanoparticles described in one.
[26] The oligonucleic acid nanoparticle according to any one of [6] to [10], wherein the spacer is polyethylene glycol or a cationic polypeptide.
[27] The hydrophilic polymer is one or more hydrophilic polymers selected from the group consisting of polyethylene glycol, poly(2-alkyl-2-oxazoline), polypeptide, and polypeptoid, [1] to [26] ] The oligonucleic acid nanoparticle according to any one of the above.
[28] The hydrophilic polymer is one or more aqueous polymers selected from the group consisting of polyethylene glycol, poly(2-methyl-2-oxazoline), EK peptide, and polysarcosine, [1] to [26] ] The oligonucleic acid nanoparticle according to any one of the above.
[29] The cell internalization promoter is one or more cell internalization promoters selected from the group consisting of polypeptides, aptamers, antibodies or fragments thereof, sugars, lipids, and other low molecular weight compounds. The oligonucleic acid nanoparticle according to any one of [1] to [28].
[30] The cell internalization promoter is a low molecular compound with a molecular weight of 2000 or less other than a hydrophobic molecule, a polycation, a polypeptide, an aptamer, an antibody or a fragment thereof, a sugar, and a lipid, [1] The oligonucleic acid nanoparticle according to any one of [28].
[31] The oligonucleic acid nanoparticle according to any one of [1] to [28], wherein the cell internalization promoting agent is a polypeptide.
[32] The oligonucleic acid nanoparticle according to any one of [1] to [28], wherein the cell internalization promoting agent is an aptamer.
[33] The oligonucleic acid nanoparticle according to any one of [1] to [28], wherein the cell internalization promoting agent is an antibody or a fragment thereof.
[34] The oligonucleic acid nanoparticle according to any one of [1] to [28], wherein the cell internalization promoter is a sugar.
[35] The oligonucleic acid nanoparticle according to any one of [1] to [28], wherein the cell internalization promoter is a lipid.
[36] A pharmaceutical composition containing the oligonucleic acid nanoparticle according to any one of [1] to [35] as an active ingredient.
[37] Containing the oligonucleic acid nanoparticle according to any one of [1] to [35] as an active ingredient,
Inborn errors of metabolism, congenital endocrine diseases, single gene diseases, neurodegenerative diseases, neurological diseases, muscle diseases, meningitis, encephalitis, encephalopathy, lysosomal diseases, malignant neoplasms, fibrosis, inflammatory diseases, immunodeficiency A therapeutic or prophylactic agent for a disease selected from the group consisting of a disease, an autoimmune disease, and an infectious disease.
[38] comprising administering a therapeutically effective amount of the oligonucleic acid nanoparticles according to any one of [1] to [35].
Inborn errors of metabolism, congenital endocrine diseases, single gene diseases, neurodegenerative diseases, neurological diseases, muscle diseases, meningitis, encephalitis, encephalopathy, lysosomal diseases, malignant neoplasms, fibrosis, inflammatory diseases, immunodeficiency A method for treating and/or preventing a disease selected from the group consisting of a disease, an autoimmune disease, and an infectious disease.
[39] Inborn errors of metabolism, congenital endocrine diseases, single gene diseases, neurodegenerative diseases, neurological diseases, muscle diseases, meningitis, encephalitis, encephalopathy, lysosomal diseases, malignant neoplasms, fibrosis, inflammatory diseases The oligo according to any one of [1] to [35] for producing therapeutic and/or preventive agents for diseases selected from the group consisting of , immunodeficiency diseases, autoimmune diseases, and infectious diseases. Use of nucleic acid nanoparticles.
[40] Inborn errors of metabolism, congenital endocrine diseases, single gene diseases, neurodegenerative diseases, neurological diseases, muscle diseases, meningitis, encephalitis, encephalopathy, lysosomal diseases, malignant neoplasms, fibrosis, inflammatory diseases The oligonucleic acid nano-acid according to any one of [1] to [35] for use in the treatment and/or prevention of diseases selected from the group consisting of particle.
[41] The oligonucleic acid nanoparticle according to any one of [1] to [35],
Including a combination of one or more therapeutic agents and/or one or more preventive agents for diseases,
The diseases include inborn errors of metabolism, congenital endocrine diseases, monogenic diseases, neurodegenerative diseases, neurological diseases, myopathies, meningitis, encephalitis, encephalopathy, lysosomal diseases, malignant neoplasms, fibrosis, and inflammatory diseases. A medicament selected from the group consisting of diseases, immunodeficiency diseases, autoimmune diseases, and infectious diseases.
[42] The oligonucleic acid nanoparticle according to any one of [1] to [35], for treating a disease in combination with one or more therapeutic agents and/or one or more preventive agents for the disease. And,
The diseases include inborn errors of metabolism, congenital endocrine diseases, monogenic diseases, neurodegenerative diseases, neurological diseases, myopathies, meningitis, encephalitis, encephalopathy, lysosomal diseases, malignant neoplasms, fibrosis, and inflammatory diseases. Oligonucleic acid nanoparticles selected from the group consisting of diseases, immunodeficiency diseases, autoimmune diseases, and infectious diseases.
[43] (a1) A step of binding a plurality of oligonucleic acids to a core composed of a dendritic polymer,
(a2) bonding a hydrophilic polymer to the oligonucleic acid;
(a3) The method for producing oligonucleic acid nanoparticles according to any one of [1] to [4], comprising the step of binding a cell internalization promoter to the hydrophilic polymer.
[44] (b1) A step of bonding one or more spacers to a core composed of a dendritic polymer;
(b2) binding a plurality of oligonucleic acids to the core;
(b3) bonding a hydrophilic polymer to the spacer;
(b4) The method for producing oligonucleic acid nanoparticles according to any one of [6] to [9], comprising the step of binding a cell internalization promoter to the hydrophilic polymer.
[45] (a1) A step of binding a plurality of oligonucleic acids to a core composed of a dendritic polymer,
(a2) bonding a hydrophilic polymer to the oligonucleic acid;
(a3) binding a cell internalization promoter to the hydrophilic polymer;
(a4) The method for producing oligonucleic acid nanoparticles according to [5], comprising the step of binding a capping agent to the core.
[46] (b1) A step of bonding one or more spacers to a core composed of a dendritic polymer;
(b2) binding a plurality of oligonucleic acids to the core;
(b3) bonding a hydrophilic polymer to the spacer;
(b4) binding a cell internalization promoter to the hydrophilic polymer;
(a5) The method for producing oligonucleic acid nanoparticles according to [10], comprising the step of binding a capping agent to the core.
　本発明によれば、細胞内在化促進剤が標的細胞と効率良く相互作用することができるため、オリゴ核酸を細胞内に効率的に輸送でき、よって、細胞質内に輸送されるオリゴ核酸の量を向上させることができる。 According to the present invention, since the cell internalization promoting agent can efficiently interact with target cells, oligonucleic acids can be efficiently transported into cells, thereby reducing the amount of oligonucleic acids transported into the cytoplasm. can be improved.
　また、本発明によれば、細網内皮系における生体成分等、標的細胞以外の他の生体成分との非特異的相互作用を低減する事ができるため、薬物動態を向上させ（すなわち、血中滞留時間を延長させ）るとともに、細胞質内に輸送されるオリゴ核酸の量を向上させることができる。 Furthermore, according to the present invention, non-specific interactions with other biological components other than target cells, such as biological components in the reticuloendothelial system, can be reduced, thereby improving pharmacokinetics (i.e., blood It is possible to extend the residence time) and improve the amount of oligonucleic acid transported into the cytoplasm.
　さらに、本発明によれば、従来の機能性ナノ粒子の代表である自己会合型ナノ粒子の構造的欠陥を回避することができる。例えば、リポソーム又はミセルを利用した機能性ナノ粒子は構造が不安定であり、有機溶媒、界面活性剤、希釈、せん断応力、又は生体成分との相互作用などで破壊され得る。また、これらの粒子は、５０ｎｍ未満の厳密なサイズの制御が困難であった。これに対し、本発明の一側面に係るオリゴ核酸ナノ粒子は、安定的な構造を有し、かつそのサイズを容易に制御できる。 Furthermore, according to the present invention, structural defects in self-assembling nanoparticles, which are typical of conventional functional nanoparticles, can be avoided. For example, functional nanoparticles using liposomes or micelles have unstable structures and can be destroyed by organic solvents, surfactants, dilution, shear stress, or interaction with biological components. Furthermore, it was difficult to strictly control the size of these particles to be less than 50 nm. In contrast, oligonucleic acid nanoparticles according to one aspect of the present invention have a stable structure and can easily control their size.
本発明の第一の側面に係るオリゴ核酸ナノ粒子の一態様を示す模式図である。FIG. 1 is a schematic diagram showing one embodiment of oligonucleic acid nanoparticles according to the first aspect of the present invention. 本発明の第二の側面に係るオリゴ核酸ナノ粒子の一態様を示す模式図である。FIG. 2 is a schematic diagram showing one embodiment of oligonucleic acid nanoparticles according to a second aspect of the present invention. 試験例１のオリゴ核酸ナノ粒子の細胞内取り込みを示すグラフである。1 is a graph showing intracellular uptake of oligonucleic acid nanoparticles of Test Example 1. 試験例１のオリゴ核酸ナノ粒子による遺伝子ノックダウン活性を示すグラフである。1 is a graph showing gene knockdown activity by oligonucleic acid nanoparticles of Test Example 1. 試験例３のオリゴ核酸ナノ粒子の細胞内取り込みを示すグラフである。3 is a graph showing intracellular uptake of oligonucleic acid nanoparticles of Test Example 3. 試験例３のオリゴ核酸ナノ粒子による遺伝子ノックダウン活性を示すグラフである。3 is a graph showing gene knockdown activity by oligonucleic acid nanoparticles of Test Example 3.
　以下、本発明の好適な実施形態について詳細に説明する。 Hereinafter, preferred embodiments of the present invention will be described in detail.
　本発明の一側面に係るオリゴ核酸ナノ粒子（以下、第一の側面に係るオリゴ核酸ナノ粒子ともいう。）は、樹状ポリマーで構成されたコアと、該コアに結合した複数個のオリゴ核酸と、該複数個のオリゴ核酸に結合した１又は複数個の親水性ポリマーと、該１又は複数個の親水性ポリマーに結合した１又は複数個の細胞内在化促進剤と、を含む。本明細書において、オリゴ核酸ナノ粒子とは、オリゴ核酸と他の分子とが結合することにより形成される単一の分子からなるナノ粒子を意味する。ここで、ナノ粒子とは、１０～２００ｎｍの粒子を意味する。本分野において、生体内におけるナノ粒子の挙動に関する知見は豊富である。 Oligonucleic acid nanoparticles according to one aspect of the present invention (hereinafter also referred to as oligonucleic acid nanoparticles according to the first aspect) have a core made of a dendritic polymer and a plurality of oligonucleic acids bound to the core. , one or more hydrophilic polymers bound to the plurality of oligonucleic acids, and one or more cell internalization promoters bound to the one or more hydrophilic polymers. As used herein, oligonucleic acid nanoparticles refer to nanoparticles consisting of a single molecule formed by binding an oligonucleic acid to another molecule. Here, nanoparticles mean particles of 10 to 200 nm. In this field, there is a wealth of knowledge regarding the behavior of nanoparticles in living organisms.
　本明細書において、樹状ポリマーとは、中心から樹状に分岐したポリマーであって、分岐に規則性のあるポリマーを意味する。樹状ポリマーは、デンドリマー、デンドロン又はデンドリグラフトであってよい。デンドリマーは一般に、樹状構造を有する、３次元的に高度に枝分かれした分子であり、ほぼ球状の形状を有する。デンドロンはデンドリマーの中心部の少なくとも一つの官能基が分岐していない構造を有する。デンドリマー及びデンドロンは、規則的な分岐構造を有し、その繰り返し単位を「世代」と呼ぶ。デンドリグラフトでは、主鎖となる分子鎖上の側鎖に分子鎖が櫛状に結合し、さらに櫛状の分子鎖の側鎖に分子鎖が櫛状に結合することで、放射状に広がった構造を形成している。デンドリグラフトの場合、櫛状の繰り返し単位を「世代」と呼ぶ。 As used herein, the term dendritic polymer refers to a polymer that is branched from the center in a dendritic manner and has regular branching. Dendritic polymers may be dendrimers, dendrons or dendrigrafts. Dendrimers are generally highly branched three-dimensional molecules with a dendritic structure and approximately spherical shape. Dendrons have a structure in which at least one functional group in the center of the dendrimer is unbranched. Dendrimers and dendrons have regular branched structures, and their repeating units are called "generations." In dendrigrafts, molecular chains are bonded in a comb-like manner to the side chains on the main molecular chain, and molecular chains are further bonded in a comb-like manner to the side chains of the comb-shaped molecular chains, thereby spreading radially. forming a structure. In the case of dendrigrafts, the comb-like repeating units are called "generations."
　樹状ポリマーの世代は、好ましくは第３世代～第２０世代である。例えば、エチレンジアミンコアを有するポリアミドアミン（ＰＡＭＡＭ）デンドリマーの場合、世代は、好ましくは第５世代～第２０世代であり、より好ましくは第５世代～第１０世代である。ポリリシンデンドリグラフトの場合、世代は、好ましくは第３世代～第６世代であり、より好ましくは第３世代～第５世代である。２，２－ビス（ヒドロキシル－メチル）プロピオン酸（Ｂｉｓ－ＭＰＡ）デンドリマーの場合、世代は、好ましくは第４世代～第２０世代であり、より好ましくは第４世代～第１０世代である。 The generation of the dendritic polymer is preferably from the 3rd generation to the 20th generation. For example, in the case of polyamidoamine (PAMAM) dendrimers having an ethylenediamine core, the generation is preferably from generation 5 to generation 20, more preferably from generation 5 to generation 10. In the case of polylysine dendrigrafts, the generation is preferably 3rd to 6th generation, more preferably 3rd to 5th generation. In the case of 2,2-bis(hydroxyl-methyl)propionic acid (Bis-MPA) dendrimers, the generation is preferably from 4th generation to 20th generation, more preferably from 4th generation to 10th generation.
　樹状ポリマーはオリゴ核酸ナノ粒子のコアを構成し、その平均直径（いいかえれば、平均粒子径）は、好ましくは５ｎｍ以上であり、より好ましくは５ｎｍ～２５ｎｍであり、さらに好ましくは５ｎｍ～１５ｎｍである。本明細書において、樹状ポリマー（コア）の平均粒子径は、動的光散乱によって得られる粒度分布における平均粒子径を意味する。 The dendritic polymer constitutes the core of the oligonucleic acid nanoparticle, and its average diameter (in other words, average particle diameter) is preferably 5 nm or more, more preferably 5 nm to 25 nm, and even more preferably 5 nm to 15 nm. be. In this specification, the average particle size of the dendritic polymer (core) means the average particle size in the particle size distribution obtained by dynamic light scattering.
　上記樹状ポリマーにおけるモノマー同士は、例えば、単結合、二重結合、三重結合、炭素－ケイ素結合、アミド結合、グリコシド結合、エステル結合、エーテル結合、ウレタン結合、アセタール結合、リン酸エステル結合、チオエーテル結合、チオエステル結合、ジスルフィド結合、トリアゾール結合、ヒドラゾン結合、ヒドラジド結合、イミン又はオキシム結合、尿素又はチオ尿素結合、アミジン結合、スルホンアミド結合等の結合様式により結合されていてよいが、結合様式はこれらに限られない。これらの結合様式のうちいずれの結合様式も用いることができるが、酵素により結合が切断されるか、酸性条件下、又は還元環境など特定の生体内の環境下において結合が切断されるものが、安全性の観点から好ましい。好ましい結合様式の例は、アミド結合、エステル結合、又はグリコシド結合であるが、これらに限られない。 The monomers in the above dendritic polymer are, for example, single bonds, double bonds, triple bonds, carbon-silicon bonds, amide bonds, glycosidic bonds, ester bonds, ether bonds, urethane bonds, acetal bonds, phosphate ester bonds, and thioether bonds. The bond may be bonded by a bonding method such as a bond, a thioester bond, a disulfide bond, a triazole bond, a hydrazone bond, a hydrazide bond, an imine or oxime bond, a urea or thiourea bond, an amidine bond, or a sulfonamide bond. Not limited to. Any of these bonding modes can be used, but those in which the bond is cleaved by enzymes or under specific in-vivo environments such as acidic conditions or reducing environments are Preferable from a safety standpoint. Examples of preferred bonding modes include, but are not limited to, amide bonds, ester bonds, or glycosidic bonds.
　好適な樹状ポリマーの例としては、ポリリシンデンドリマー、ポリリシンデンドリグラフト、ＰＡＭＡＭデンドリマー、Ｂｉｓ－ＭＰＡデンドリマー又はグルコースデンドリマーが挙げられるが、これらに限られない。樹状ポリマーは、例えば、ポリ－Ｌ－リシンデンドリマーであってよく、ポリ－Ｌ－リシンデンドリグラフトであってよい。 Examples of suitable dendritic polymers include, but are not limited to, polylysine dendrimers, polylysine dendrigrafts, PAMAM dendrimers, Bis-MPA dendrimers, or glucose dendrimers. The dendritic polymer may be, for example, a poly-L-lysine dendrimer or a poly-L-lysine dendrigraft.
　本明細書において、オリゴ核酸は、天然型又は非天然型のオリゴ核酸である。天然型のオリゴ核酸とは、塩基、糖、及びリン酸からなるヌクレオチドを繰り返し単位とするポリマーである。オリゴ核酸の種類は特に限定されず、オリゴ核酸ナノ粒子は１種又は２種以上のオリゴ核酸を含んでよい。オリゴ核酸としては、例えば、ＲＮＡ、ＤＮＡ、又はこれらの組み合わせからなる一本鎖又は二重鎖を挙げることができ、これには同一の鎖にＲＮＡとＤＮＡが混在したオリゴ核酸も含まれる。オリゴ核酸に含まれるヌクレオチドは、天然型のヌクレオチドであっても、化学修飾された非天然型のヌクレオチドであってもよく、また、アミノ基、チオール基、又は蛍光化合物などの分子が付加されたヌクレオチドであってもよい。非天然型のオリゴ核酸の範囲には、主鎖にペプチド構造を有するペプチド核酸（ＰＮＡ）、主鎖にモルフォリン環を有するモルフォリノ核酸など、天然型のオリゴ核酸と同様に遺伝子の発現を制御する作用を有する、人工分子も含まれる。 As used herein, oligonucleic acids are natural or non-natural oligonucleic acids. A natural oligonucleic acid is a polymer whose repeating units are nucleotides consisting of a base, sugar, and phosphoric acid. The type of oligonucleic acid is not particularly limited, and the oligonucleic acid nanoparticles may contain one or more types of oligonucleic acids. Examples of oligonucleic acids include single-stranded or double-stranded RNA, DNA, or a combination thereof, including oligonucleic acids in which RNA and DNA are mixed in the same strand. The nucleotides contained in the oligonucleic acid may be natural nucleotides or chemically modified non-natural nucleotides, or may be added with molecules such as amino groups, thiol groups, or fluorescent compounds. It may also be a nucleotide. Non-natural oligonucleic acids include peptide nucleic acids (PNA), which have a peptide structure in their main chain, and morpholino nucleic acids, which have a morpholine ring in their main chain, which control gene expression in the same way as natural oligonucleic acids. Also included are artificial molecules that have an effect.
　オリゴ核酸の機能又は作用は限定されないが、オリゴ核酸としては、例えば、アンチセンス核酸、ｓｇＲＮＡ、ＲＮＡ編集核酸、ｍｉＲＮＡ、ｓｉＲＮＡ、ｓａＲＮＡ、ｓｈＲＮＡ、又はｄｉｃｅｒ　ｓｕｂｓｔｒａｔｅ　ＲＮＡが挙げられる。 Although the function or action of the oligonucleic acid is not limited, examples of the oligonucleic acid include antisense nucleic acid, sgRNA, RNA editing nucleic acid, miRNA, siRNA, saRNA, shRNA, or dicer substrate RNA.
　オリゴ核酸は、例えば遺伝子発現制御剤であってよい。遺伝子発現制御剤とは、特定の遺伝子産物の発現を活性化又は抑制させる化合物である。遺伝子産物としては、例えば、ｍＲＮＡ若しくはその前駆体、ｍｉＲＮＡ若しくはその前駆体、ｎｃＲＮＡ、酵素、抗体、又はその他のタンパク質が挙げられる。遺伝子発現制御剤としては、例えば、ｍＲＮＡの発現を正若しくは負に制御する（すなわち、発現を促進若しくは抑制する）分子、又はＲＮＡ若しくはＤＮＡを編集する分子が挙げられる。このような遺伝子発現制御剤としては、例えば、ｍｉＲＮＡ、ｓｉＲＮＡ等のＲＮＡ干渉（ＲＮＡｉ）を誘導する核酸（ＲＮＡｉ誘導核酸）、アンチセンス核酸、ｍｉＲＮＡ阻害剤、ＲＮＡ活性化核酸、ＲＮＡ編集誘導核酸、又はゲノム編集誘導核酸が挙げられるが、遺伝子発現制御剤はこれらに限られない。 The oligonucleic acid may be, for example, a gene expression control agent. A gene expression regulator is a compound that activates or suppresses the expression of a specific gene product. Gene products include, for example, mRNA or its precursor, miRNA or its precursor, ncRNA, enzyme, antibody, or other protein. Examples of gene expression control agents include molecules that positively or negatively control mRNA expression (that is, promote or suppress expression), or molecules that edit RNA or DNA. Such gene expression control agents include, for example, nucleic acids that induce RNA interference (RNAi) such as miRNA and siRNA (RNAi-inducing nucleic acids), antisense nucleic acids, miRNA inhibitors, RNA activation nucleic acids, RNA editing-inducing nucleic acids, or genome editing-induced nucleic acids, but gene expression control agents are not limited to these.
　オリゴ核酸の長さは、例えば、４～２００塩基（対）、７～１００塩基（対）、又は１２～３０塩基（対）であってよい。 The length of the oligonucleic acid may be, for example, 4 to 200 bases (pairs), 7 to 100 bases (pairs), or 12 to 30 bases (pairs).
　オリゴ核酸ナノ粒子におけるオリゴ核酸の数は複数であれば特に限定されず、例えば、２以上、６以上、１０以上、１５以上、１７以上、１８以上、２０以上、２５以上、３０以上、３１以上、３５以上、又は５０以上であってよく、４００以下、２００以下又は１００以下であってよい。オリゴ核酸が共有結合により樹状ポリマーと結合している場合、オリゴ核酸の数は、例えば、樹状ポリマーの有する反応性官能基の０．５％以上、１％以上、又は２％以上であってよく、好ましくは樹状ポリマーの有する反応性官能基の３％以上又は５％以上である。オリゴ核酸ナノ粒子におけるオリゴ核酸の数は、例えば、オリゴ核酸ナノ粒子を含む溶液中の樹状ポリマーの濃度及びオリゴ核酸の濃度を測定し、これらの値から、樹状ポリマーに対するオリゴ核酸の割合を算出することで、求めることができる。オリゴ核酸ナノ粒子を含む溶液中の樹状ポリマーの濃度は、例えば高速液体クロマトグラフィー（ＨＰＬＣ）によって測定することができる。オリゴ核酸の濃度は、例えば、紫外可視分光光度計を用いて測定した２６０ｎｍにおける吸収から求めることができる。 The number of oligonucleic acids in the oligonucleic acid nanoparticles is not particularly limited as long as it is plural, for example, 2 or more, 6 or more, 10 or more, 15 or more, 17 or more, 18 or more, 20 or more, 25 or more, 30 or more, 31 or more. , 35 or more, or 50 or more, and may be 400 or less, 200 or less, or 100 or less. When the oligonucleic acids are bound to the dendritic polymer by covalent bonds, the number of oligonucleic acids is, for example, 0.5% or more, 1% or more, or 2% or more of the reactive functional groups possessed by the dendritic polymer. It may account for 3% or more or 5% or more of the reactive functional groups possessed by the dendritic polymer. The number of oligonucleic acids in oligonucleic acid nanoparticles can be determined, for example, by measuring the concentration of dendritic polymer and the concentration of oligonucleic acid in a solution containing oligonucleic acid nanoparticles, and from these values, calculating the ratio of oligonucleic acids to dendritic polymers. It can be determined by calculation. The concentration of dendritic polymer in a solution containing oligonucleic acid nanoparticles can be measured, for example, by high performance liquid chromatography (HPLC). The concentration of oligonucleic acid can be determined, for example, from the absorption at 260 nm measured using an ultraviolet-visible spectrophotometer.
　オリゴ核酸は、公知の方法により製造することができる。オリゴ核酸は、例えば、ホスホロアミダイト法又はトリエステル法による、固相合成法又は液相合成法により、核酸自動合成機又は手動にて製造することができる。 Oligonucleic acids can be produced by known methods. Oligonucleic acids can be produced, for example, by a phosphoramidite method or a triester method, by a solid phase synthesis method or a liquid phase synthesis method, using an automatic nucleic acid synthesizer or manually.
　本明細書において、親水性ポリマーは、オリゴ核酸を外界から遮蔽するとともに、細胞内在化促進剤をオリゴ核酸ナノ粒子の表面に露出させるための親水性分子である。親水性分子とは、水との間に水素結合を形成しやすく、水に溶解しやすいか又は水に混ざりやすい性質を有する分子を意味する。親水性分子は、荷電分子又は非荷電の高極性分子であってよい。荷電分子の荷電基は、正に荷電した基（カチオン）、負に荷電した基（アニオン）、又はこれらの組合せであってよい。オリゴ核酸を外界から遮蔽するためポリマー（すなわち、ステルスポリマー）が親水性であると、オリゴ核酸ナノ粒子の凝集の抑制、溶解性の向上、細網内皮系による貪食の回避、生体成分との非特異的相互作用の回避、及び薬物動態の向上（すなわち、血中滞留時間の延長）の点で有利となり得る。親水性ポリマーとしては、例えば、ポリエチレングリコール（ＰＥＧ）、ポリ（２－アルキル－２－オキサゾリン）、ポリペプチド、ポリペプトイド、又はポリベタインが挙げられるが、親水性ポリマーはこれらに限られない。オリゴ核酸ナノ粒子は、１種又は２種以上の親水性ポリマーを含んでよい。親水性ポリマーは、好ましくはＰＥＧ、ポリ（２－メチル－２－オキサゾリン）（ｐＭｅＯｘ）、ポリサルコシン（ｐＳａｒ）及びＥＫペプチドからなる群から選択される１種又は２種以上である。ＥＫペプチドは、グルタミン酸とリシンが交互に並んだペプチドである。 As used herein, the hydrophilic polymer is a hydrophilic molecule that shields the oligonucleic acid from the outside world and exposes the cell internalization promoter to the surface of the oligonucleic acid nanoparticle. A hydrophilic molecule refers to a molecule that easily forms a hydrogen bond with water and has the property of being easily soluble in water or easily miscible with water. Hydrophilic molecules may be charged molecules or uncharged highly polar molecules. The charged group of a charged molecule may be a positively charged group (cation), a negatively charged group (anion), or a combination thereof. Hydrophilic polymers (i.e., stealth polymers) that shield oligonucleic acids from the outside world can inhibit aggregation of oligonucleic acid nanoparticles, improve solubility, avoid phagocytosis by the reticuloendothelial system, and prevent interaction with biological components. This may be advantageous in terms of avoiding specific interactions and improving pharmacokinetics (ie, prolonging blood residence time). Examples of hydrophilic polymers include, but are not limited to, polyethylene glycol (PEG), poly(2-alkyl-2-oxazoline), polypeptide, polypeptoid, or polybetaine. Oligonucleic acid nanoparticles may include one or more hydrophilic polymers. The hydrophilic polymer is preferably one or more selected from the group consisting of PEG, poly(2-methyl-2-oxazoline) (pMeOx), polysarcosine (pSar), and EK peptide. EK peptide is a peptide consisting of alternating glutamic acid and lysine.
　１本の親水性ポリマーは複数のセグメントを有してもよい。複数のセグメントを有する親水性ポリマーとしては、例えば、ＥＫペプチドとＰＥＧとが結合してなるポリマーが挙げられるが、これに限られない。親水性ポリマーは、線状構造又は分岐構造を有してもよい。 One hydrophilic polymer may have multiple segments. Examples of the hydrophilic polymer having multiple segments include, but are not limited to, a polymer formed by bonding EK peptide and PEG. The hydrophilic polymer may have a linear or branched structure.
　一例として、親水性ポリマーがＰＥＧである場合、親水性ポリマーの数平均分子量は、５００以上、１０００以上、２０００以上、３４００以上、５０００以上、６０００以上、８０００以上、又は１００００以上であってよい。また、親水性ポリマーがｐＭｅＯｘ又はｐＳａｒである場合、親水性ポリマーの数平均分子量は、１０００以上、２０００以上、４０００以上、７０００以上、１００００以上、１５０００以上、又は２００００以上であってよい。また、親水性ポリマーがＥＫペプチドである場合、親水性ポリマーは、グルタミン酸とリシンからなる単位構造を、１以上、２以上、５以上、７以上、１０以上、１５以上、又は２０以上含むペプチドであってよい。本明細書において、数平均分子量は、核磁気共鳴（ＮＭＲ）を用いた末端基定量法、又はサイズ排除クロマトグラフィー（ＳＥＣ）により求められる値である。 As an example, when the hydrophilic polymer is PEG, the number average molecular weight of the hydrophilic polymer may be 500 or more, 1000 or more, 2000 or more, 3400 or more, 5000 or more, 6000 or more, 8000 or more, or 10000 or more. Further, when the hydrophilic polymer is pMeOx or pSar, the number average molecular weight of the hydrophilic polymer may be 1000 or more, 2000 or more, 4000 or more, 7000 or more, 10000 or more, 15000 or more, or 20000 or more. In addition, when the hydrophilic polymer is an EK peptide, the hydrophilic polymer is a peptide containing 1 or more, 2 or more, 5 or more, 7 or more, 10 or more, 15 or more, or 20 or more unit structures consisting of glutamic acid and lysine. It's good. In this specification, the number average molecular weight is a value determined by a terminal group determination method using nuclear magnetic resonance (NMR) or size exclusion chromatography (SEC).
　親水性ポリマーの数は、オリゴ核酸又は細胞内在化促進剤の数より少なくても、多くてもよく、又は同じであってよい。一つのオリゴ核酸に結合した親水性ポリマーの数は、一つであっても複数であってもよいが、好ましくは一つである。また、細胞内在化促進剤が結合していない親水性ポリマーがあってもよい。親水性ポリマーが共有結合によりオリゴ核酸と結合している場合、親水性ポリマーの数は、例えば、１以上、２以上、又は樹状ポリマーの有する反応性官能基の１％以上であってよく、樹状ポリマーの有する反応性官能基の好ましくは２％以上、より好ましくは３％以上又は５％以上である。また、親水性ポリマーは、好ましくは、オリゴ核酸の４０％以上、６０％以上、８０％以上、又は８５％以上に結合している。オリゴ核酸ナノ粒子における親水性ポリマーの数は、例えば、オリゴ核酸と樹状ポリマーとの結合を切断し、ＳＥＣによって分離されたオリゴ核酸単体と親水性ポリマーが結合したオリゴ核酸との割合から算出することができる。 The number of hydrophilic polymers may be less than, more than, or the same as the number of oligonucleic acids or cell internalization promoters. The number of hydrophilic polymers bound to one oligonucleic acid may be one or more, but is preferably one. Furthermore, there may be a hydrophilic polymer to which no cell internalization promoter is bound. When the hydrophilic polymer is bonded to the oligonucleic acid by a covalent bond, the number of hydrophilic polymers may be, for example, 1 or more, 2 or more, or 1% or more of the reactive functional groups possessed by the dendritic polymer, It is preferably 2% or more, more preferably 3% or more or 5% or more of the reactive functional groups possessed by the dendritic polymer. Further, the hydrophilic polymer preferably binds to 40% or more, 60% or more, 80% or more, or 85% or more of the oligonucleic acid. The number of hydrophilic polymers in the oligonucleic acid nanoparticles is calculated, for example, from the ratio of the oligonucleic acid alone separated by SEC to the oligonucleic acid bound to the hydrophilic polymer by cutting the bond between the oligonucleic acid and the dendritic polymer. be able to.
　本明細書において、細胞内在化促進剤は、標的となる細胞と特異的又は非特異的に相互作用することで、該細胞内在化促進剤が結合した物質の標的細胞への内在化を誘導する分子種である。本側面に係るオリゴ核酸ナノ粒子においては、親水性ポリマーに細胞内在化促進剤を結合させることにより、該細胞内在化促進剤を含まない場合（例えば、オリゴ核酸を単独で用いる場合）と比較して、オリゴ核酸を標的細胞内に効率的に輸送できる。後述するように、細胞内在化促進剤はオリゴ核酸ナノ粒子の表面に位置する。 As used herein, the cell internalization promoter induces internalization of a substance bound to the cell internalization promoter into the target cell by interacting specifically or nonspecifically with the target cell. It is a molecular species. In the oligonucleic acid nanoparticles according to this aspect, by binding a cell internalization promoter to a hydrophilic polymer, compared to a case where the cell internalization promoter is not included (for example, when oligonucleic acid is used alone). As a result, oligonucleic acids can be efficiently transported into target cells. As described below, the cell internalization promoter is located on the surface of the oligonucleic acid nanoparticle.
　細胞内在化促進剤としては、細胞表面受容体と相互作用する物質、膜輸送体と相互作用する物質、細胞接着因子と相互作用する物質、又は細胞膜表面と相互作用する他の物質が挙げられるが、それらに限られない。細胞内在化促進剤は、例えば、細胞膜表面に存在する細胞接着因子であるインテグリンと相互作用する物質、上皮細胞接着分子と相互作用する物質、ヌクレオリンと相互作用する物質、細胞骨格分子であるビメンチンと相互作用する物質、前立腺特異的膜抗原と相互作用する物質、上皮成長因子受容体、ソマトスタチン受容体、マンノース受容体、アシアロ糖タンパク受容体、葉酸受容体等の細胞表面受容体と相互作用する物質、又はグルコーストランスポーター、非選択的モノアミントランスポーター等の輸送体と相互作用する物質であってよい。 Cell internalization promoters include substances that interact with cell surface receptors, substances that interact with membrane transporters, substances that interact with cell adhesion factors, or other substances that interact with the cell membrane surface. , but not limited to those. Cell internalization promoters include, for example, substances that interact with integrin, which is a cell adhesion factor present on the surface of cell membranes, substances that interact with epithelial cell adhesion molecules, substances that interact with nucleolin, and substances that interact with vimentin, a cytoskeletal molecule. Interacting substances, substances that interact with prostate-specific membrane antigens, substances that interact with cell surface receptors such as epidermal growth factor receptors, somatostatin receptors, mannose receptors, asialoglycoprotein receptors, folate receptors, etc. , or a substance that interacts with a transporter such as a glucose transporter or a non-selective monoamine transporter.
　細胞内在化促進剤としては、例えば、疎水性分子、ポリカチオン、ポリペプチド、アプタマー、抗体若しくはそのフラグメント、糖、脂質、又はその他の低分子化合物が挙げられるが、細胞内在化促進剤はこれらに限られない。その他の低分子化合物とは、疎水性分子、ポリカチオン、ポリペプチド、アプタマー、抗体若しくはそのフラグメント、糖、及び脂質以外の、分子量が２０００以下の化合物である。オリゴ核酸ナノ粒子は、１種又は２種以上の細胞内在化促進剤を含んでよい。細胞内在化促進剤は、好ましくは、ポリペプチド、アプタマー、糖、及び上記その他の低分子化合物からなる群から選択される１種又は２種以上である。細胞内在化促進剤は、より好ましくは、ポリペプチド、アプタマー、又は上記その他の低分子化合物である。 Examples of cell internalization promoters include hydrophobic molecules, polycations, polypeptides, aptamers, antibodies or fragments thereof, sugars, lipids, and other low-molecular compounds; Not limited. Other low molecular weight compounds are compounds with a molecular weight of 2000 or less, other than hydrophobic molecules, polycations, polypeptides, aptamers, antibodies or fragments thereof, sugars, and lipids. The oligonucleic acid nanoparticles may contain one or more cell internalization promoters. The cell internalization promoter is preferably one or more selected from the group consisting of polypeptides, aptamers, sugars, and other low molecular weight compounds mentioned above. The cell internalization promoting agent is more preferably a polypeptide, an aptamer, or the other low molecular weight compound mentioned above.
　ポリペプチドの分子量は、例えば、５０ｋＤａ以下、１５ｋＤａ以下、６ｋＤａ以下、２ｋＤａ以下、又は１ｋＤａ以下であってよいが、これらに限られない。ポリペプチドの分子量は、例えば、質量分析法によって求めることができる。 The molecular weight of the polypeptide may be, for example, 50 kDa or less, 15 kDa or less, 6 kDa or less, 2 kDa or less, or 1 kDa or less, but is not limited thereto. The molecular weight of a polypeptide can be determined, for example, by mass spectrometry.
　本明細書において、抗体又はそのフラグメントは、特定の因子に特異的に結合する機能を有するスキャフォールドタンパクを指し、ＩｇＡ、ＩｇＤ、ＩｇＥ、ＩｇＧ、ＩｇＭ等の免疫グロブリン、Ｆ（ａｂ）’２、Ｆａｂ’、Ｆａｂ、ｓｃＦｖなどの断片化抗体、サメＶＮＡＲ、ラクダＶＨＨなどのシングルドメイン抗体、及びアフィボディ、アフィリン、モノボディ、アルファボディなどの抗体ミメティックを含むが、これらに限られない。 As used herein, an antibody or a fragment thereof refers to a scaffold protein that has the function of specifically binding to a specific factor, and includes immunoglobulins such as IgA, IgD, IgE, IgG, and IgM, F(ab)'2, These include, but are not limited to, fragmented antibodies such as Fab', Fab, scFv, single domain antibodies such as shark VNAR, camel VHH, and antibody mimetics such as affibodies, affilins, monobodies, alphabodies.
　細胞内在化促進剤の具体例としては、下記式（Ｉ）～（ＩＶ）に示すポリペプチドが挙げられる。式（Ｉ）に示すポリペプチドは、アルギニン－グリシン－アスパラギン酸（ＲＧＤ）配列を含む環状のペプチドリガンド（ｃＲＧＤ）の一種であるｃＲＧＤｆＫ（分子量：６０３．７Ｄａ、Ｐｈａｒｍａｃｅｕｔｉｃｓ，２０１８，１０，２）である。ｃＲＧＤｆＫは、インテグリンαＶβ３と相互作用する。ｃＲＧＤｆＫ以外のｃＲＧＤも細胞内在化促進剤として用いることが可能である。式（ＩＩ）に示すポリペプチドは、インテグリンαＶβ６と相互作用するｃ（ａｖｂ６）（分子量：１０４６．２、ＡＣＳ　Ｏｍｅｇａ，２０１８，３，２４２８－２４３６）である。式（ＩＩＩ）に示すポリペプチドは、上皮成長因子受容体と相互作用するＧＥ１１（分子量：１５３９．７Ｄａ）である。式（ＩＶ）に示すポリペプチドは、ソマトスタチン受容体と相互作用するオクトレオチド誘導体（ＯＣＴ；分子量：１５７７．８Ｄａ）である。ｃＲＧＤとしては、市販品を使用することができる。式（ＩＩ）～（ＩＶ）に示すペプチドは、周知の合成方法によって容易に入手可能である。 Specific examples of cell internalization promoters include polypeptides represented by the following formulas (I) to (IV). The polypeptide represented by formula (I) is cRGDfK (molecular weight: 603.7 Da, Pharmaceuticals, 2018, 10, 2), which is a type of cyclic peptide ligand (cRGD) containing an arginine-glycine-aspartic acid (RGD) sequence. be. cRGDfK interacts with integrin α v β 3 . cRGD other than cRGDfK can also be used as a cell internalization promoter. The polypeptide shown in formula (II) is c(avb6) (molecular weight: 1046.2, ACS Omega, 2018, 3, 2428-2436), which interacts with integrin α v β 6 . The polypeptide shown in formula (III) is GE11 (molecular weight: 1539.7 Da) that interacts with the epidermal growth factor receptor. The polypeptide shown in formula (IV) is an octreotide derivative (OCT; molecular weight: 1577.8 Da) that interacts with the somatostatin receptor. As cRGD, commercially available products can be used. Peptides represented by formulas (II) to (IV) are readily available by well-known synthetic methods.
　他の細胞内在化促進剤の具体例としては、下記式（Ｖ）～（ＶＩＩ）に示す低分子化合物が挙げられる。式（Ｖ）に示す低分子化合物は、葉酸受容体と相互作用する葉酸である。式（ＶＩ）に示す低分子化合物は、前立腺特異的膜抗原と相互作用するＤＵＰＡである。式（ＶＩＩ）に示す低分子化合物は、非選択的モノアミントランスポーターと相互作用するインダトラリン（ＩＮＤ）である。 Specific examples of other cell internalization promoters include low molecular weight compounds represented by the following formulas (V) to (VII). The low molecular compound represented by formula (V) is folic acid that interacts with folic acid receptors. The low molecular compound represented by formula (VI) is DUPA that interacts with prostate-specific membrane antigen. The low molecular compound represented by formula (VII) is indatraline (IND), which interacts with a non-selective monoamine transporter.
　他の細胞内在化促進剤の具体例としては、下記式（ＶＩＩＩ）～（ＸＩＩ）に示す糖が挙げられる。式（ＶＩＩＩ）に示す糖は、グルコーストランスポーターと相互作用するグルコース（Ｇｌｕ）である。式（ＩＸ）に示す糖は、マンノース受容体と相互作用するマンノース（Ｍａｎ）である。式（Ｘ）及び式（ＸＩ）に示す糖は、アシアロ糖タンパク受容体と相互作用するＮ－アセチルガラクトサミン（ＧａｌＮＡｃ）及びガラクトース（Ｇａｌ）である。式（ＸＩＩ）に示す糖は、細胞骨格分子であるビメンチンと相互作用するＮ－アセチルグルコサミン（ＧｌｃＮＡｃ）である。 Specific examples of other cell internalization promoters include sugars represented by the following formulas (VIII) to (XII). The sugar represented by formula (VIII) is glucose (Glu) that interacts with glucose transporters. The sugar shown in formula (IX) is mannose (Man), which interacts with the mannose receptor. The sugars shown in formula (X) and formula (XI) are N-acetylgalactosamine (GalNAc) and galactose (Gal), which interact with asialoglycoprotein receptors. The sugar shown in formula (XII) is N-acetylglucosamine (GlcNAc), which interacts with the cytoskeletal molecule vimentin.
　他の細胞内在化促進剤としては、例えば、下記表に示す配列番号１～６で示される塩基配列を有するアプタマーが挙げられる。ヌクレオリンと相互作用するＤＮＡアプタマーとして、配列番号１に示すＡＳ１４１１（Ｏｎｃｏｔａｒｇｅｔ，２０１５，６（２６），２２２７０－２２２８１）、及び配列番号２に示すＦＡＮ－１５２４ｄＩ（Ｓｃｉｅｎｔｉｆｉｃ　Ｒｅｐｏｒｔｓ，２０１６，６，１－１２）が挙げられる。上皮細胞接着分子と相互作用するアプタマーとして、配列番号３に示すＥｐＣＡＭ　Ａｐｔａｍｅｒ（Ｍｏｌｅｃｕｌａｒ　Ｃａｎｃｅｒ　Ｔｈｅｒａｐｅｕｔｉｃｓ，２０１５，１４（１０），２２７９－２２９１）及び、配列番号４に示すＥｐＣＡＭ　Ａｐｔａｍｅｒ（Ｔｈｅｒａｎｏｓｔｉｃｓ，２０１５，５（１０），１０８３－１０９７）が挙げられる。トランスフェリン受容体に相互作用するアプタマーとして、配列番号５に示すＦＢ４（Ｐｒｏｃ　Ｎａｔｌ　Ａｃａｄ　Ｓｃｉ　ＵＳＡ．，２００８，１０５（４１），１５９０８－１５９１３）、及び配列番号６に示すＧＳ２４（Ｍｏｌ　Ｔｈｅｒ　Ｎｕｃｌｅｉｃ　Ａｃｉｄｓ，２０１４，３（１），ｅ１４４）が挙げられる。 Examples of other cell internalization promoters include aptamers having the base sequences shown in SEQ ID NOs: 1 to 6 shown in the table below. As DNA aptamers that interact with nucleolin, AS1411 shown in SEQ ID NO: 1 (Oncotarget, 2015, 6 (26), 22270-22281), and FAN-1524dI shown in SEQ ID NO: 2 (Scientific Reports, 2016, 6, 1-12) ). As aptamers that interact with epithelial cell adhesion molecules, EpCAM Aptamer shown in SEQ ID NO: 3 (Molecular Cancer Therapeutics, 2015, 14 (10), 2279-2291) and EpCAM Aptamer shown in SEQ ID NO: 4 (Theranos tics, 2015, 5( 10), 1083-1097). As aptamers that interact with the transferrin receptor, FB4 shown in SEQ ID NO: 5 (Proc Natl Acad Sci USA., 2008, 105 (41), 15908-15913) and GS24 shown in SEQ ID NO: 6 (Mol Ther Nucleic Acids, 2014 , 3(1), e144).
Figure JPOXMLDOC01-appb-T000005
Figure JPOXMLDOC01-appb-T000005
　オリゴ核酸ナノ粒子における細胞内在化促進剤の数は、細胞内在化促進剤を標的細胞と効率良く相互作用させて、オリゴ核酸ナノ粒子の細胞内への輸送効率を向上させる観点から、例えば、１以上、２以上、６以上、１１以上、１８以上、２５以上、２６以上、２７以上又は３０以上であってよく、４００以下、２００以下又は１００以下であってよい。オリゴ核酸ナノ粒子における細胞内在化促進剤の数は、例えば、オリゴ核酸ナノ粒子を含む溶液中の樹状ポリマーの濃度及び細胞内在化促進剤の濃度を測定し、これらの値から、樹状ポリマーに対する、細胞内在化促進剤の割合を算出することで、求めることができる。樹状ポリマーの濃度及び細胞内在化促進剤の濃度は、例えばＨＰＬＣ又は紫外可視分光光度計によって測定することができる。一つの親水性ポリマーに結合した細胞内在化促進剤の数は、一つであっても複数であってもよい。 The number of cell internalization promoters in the oligonucleic acid nanoparticles is, for example, 1 from the viewpoint of efficiently interacting the cell internalization promoters with target cells and improving the transport efficiency of the oligonucleic acid nanoparticles into cells. The number may be 2 or more, 6 or more, 11 or more, 18 or more, 25 or more, 26 or more, 27 or more, or 30 or more, and may be 400 or less, 200 or less, or 100 or less. The number of cell internalization promoters in the oligonucleic acid nanoparticles can be determined, for example, by measuring the concentration of the dendritic polymer and the concentration of the cell internalization promoter in the solution containing the oligonucleic acid nanoparticles, and from these values, the number of cell internalization promoters in the dendritic polymer It can be determined by calculating the ratio of the cell internalization promoter to the total amount of the cell internalization promoting agent. The concentration of the dendritic polymer and the concentration of the cellular internalization promoter can be measured, for example, by HPLC or UV-visible spectrophotometry. The number of cell internalization promoters bound to one hydrophilic polymer may be one or more.
　上述のとおり、樹状ポリマー、より具体的には樹状ポリマーの反応性官能基（これらは末端官能基である）の少なくとも一部には、オリゴ核酸が結合している。一実施形態において、オリゴ核酸と結合していない未反応の反応性官能基の少なくとも一部又は全部は、キャッピング剤によりキャッピングされていてよい。反応性官能基のキャッピングとは、いいかえれば、結合により反応性官能基の反応性を低下させることである。キャッピング剤は、樹状ポリマーの反応性官能基と結合することにより、種々の相互作用又は化学反応から樹状ポリマーを保護する。例えば、キャッピング剤は、静電相互作用、分解反応、縮合反応、付加反応等から樹状ポリマーを保護する。 As mentioned above, an oligonucleic acid is bound to the dendritic polymer, and more specifically to at least a portion of the reactive functional groups (these are the terminal functional groups) of the dendritic polymer. In one embodiment, at least a portion or all of the unreacted reactive functional groups that are not bound to the oligonucleic acid may be capped with a capping agent. In other words, capping a reactive functional group is to reduce the reactivity of the reactive functional group through bonding. The capping agent protects the dendritic polymer from various interactions or chemical reactions by binding to the reactive functional groups of the dendritic polymer. For example, capping agents protect dendritic polymers from electrostatic interactions, decomposition reactions, condensation reactions, addition reactions, and the like.
　また、キャッピング剤は、樹状ポリマーと結合することにより、樹状ポリマーが本来有さない機能又は活性を、樹状ポリマーに付加し得る。このようなキャッピング剤としては、例えば、ステルス性を向上させる分子、脂質二重膜と相互作用する分子、プロトン緩衝能を有する分子などが挙げられるが、キャッピング剤はこれらに限られない。 Furthermore, by bonding with the dendritic polymer, the capping agent can add a function or activity that the dendritic polymer does not originally have to the dendritic polymer. Examples of such capping agents include, but are not limited to, molecules that improve stealth, molecules that interact with lipid bilayer membranes, and molecules that have proton buffering ability.
　キャッピング剤は、例えば、ａ）親水性分子及びｂ）疎水性分子からなる群から選択される１種又は２種の分子であってもよい。 The capping agent may be, for example, one or two molecules selected from the group consisting of a) hydrophilic molecules and b) hydrophobic molecules.
　上記ａ）親水性分子は、ａ－１）電気的に中性な親水性分子、ａ－２）酸性条件下でプロトン化する極性分子、ａ－３）アニオン性分子、又はａ－４）カチオン性分子であってよい。ａ）親水性分子は、親水性ポリマーと同じ種類の分子であってもよく、親水性ポリマーとは異なる種類の分子であってもよい。本明細書において、「電気的に中性」とは、カチオンとアニオンの数が等しいか、カチオンとアニオンの数の差が、多い方の荷電基の数を基準として１０％以内である場合を指す。 The above a) hydrophilic molecules are a-1) electrically neutral hydrophilic molecules, a-2) polar molecules that protonate under acidic conditions, a-3) anionic molecules, or a-4) cations. It may be a sexual molecule. a) The hydrophilic molecule may be the same type of molecule as the hydrophilic polymer, or it may be a different type of molecule from the hydrophilic polymer. As used herein, "electrically neutral" refers to a case where the number of cations and anions is equal, or the difference between the number of cations and anions is within 10% based on the number of charged groups of the larger one. Point.
　上記ａ－１）電気的に中性な親水性分子としては、例えば、ヒドロキシル基、アルコキシ基、オキシム基、エステル基、アミド基、イミド基、アルコキシアミド基、カルボニル基、スルホニル基、ニトロ基、ピロリドン基等の親水性基を有する分子、ベタイン等の双性イオン、ＰＥＧ、又はメトキシポリエチレングリコール等のアルコキシポリエチレングリコールが挙げられるが、親水性分子はこれらに限られない。 Examples of the above a-1) electrically neutral hydrophilic molecules include hydroxyl groups, alkoxy groups, oxime groups, ester groups, amide groups, imide groups, alkoxyamide groups, carbonyl groups, sulfonyl groups, nitro groups, Hydrophilic molecules include, but are not limited to, molecules having hydrophilic groups such as pyrrolidone groups, zwitterions such as betaine, PEG, and alkoxypolyethylene glycols such as methoxypolyethylene glycol.
　上記ａ－２）酸性条件下でプロトン化する極性分子は、エンドソーム内などの酸性環境下と、血液中、細胞間質液中などの生理的条件下とで、保有する電荷が異なる分子である。酸性条件下でプロトン化する極性分子は、７．４以下、好ましくは５．０～７．４の酸解離定数（ｐＫａ）を有する分子をいう。酸性条件下でプロトン化する極性分子としては、例えば、３級アミノ基、ジエチルトリアミン（ＤＥＴ）基（－ＮＨ－ＣＨ２－ＣＨ２－ＮＨ－ＣＨ２－ＣＨ２－ＮＨ２）、モルフォリノ基、チオモルフォリノ基、イミダゾリル基、ピリジル基、カルボキシ基等の極性基を有する分子が挙げられるが、酸性条件下でプロトン化する極性分子はこれらに限られない。 The above a-2) Polar molecules that are protonated under acidic conditions are molecules that have different charges depending on the acidic environment such as in endosomes and under physiological conditions such as in blood or interstitial fluid. . Polar molecules that protonate under acidic conditions refer to molecules having an acid dissociation constant (pK a ) of 7.4 or less, preferably from 5.0 to 7.4. Examples of polar molecules that protonate under acidic conditions include tertiary amino groups, diethyltriamine (DET) groups (-NH-CH 2 -CH 2 -NH-CH 2 -CH 2 -NH 2 ), morpholino groups, Examples include molecules having polar groups such as a thiomorpholino group, an imidazolyl group, a pyridyl group, and a carboxy group, but polar molecules that are protonated under acidic conditions are not limited to these.
　上記ａ－３）アニオン性分子は、生理的条件下においてイオンの価数が負の分子である。例えば、カルボキシ基、スルホ基、リン酸基、リン酸エステル基等の官能基を有する分子が挙げられるが、アニオン性分子はこれらに限られない。 The above a-3) anionic molecule is a molecule with a negative ion valence under physiological conditions. Examples include molecules having functional groups such as a carboxyl group, a sulfo group, a phosphoric acid group, and a phosphoric ester group, but the anionic molecule is not limited to these.
　上記ａ－４）カチオン性分子は、生理的条件下においてイオンの価数が正の分子である。例えば、１級アミノ基、２級アミノ基、３級アミノ基、グアニジノ基等の官能基を有する分子が挙げられるが、カチオン性分子はこれらに限られない。 The above a-4) cationic molecule is a molecule with a positive ion valence under physiological conditions. Examples include molecules having functional groups such as primary amino groups, secondary amino groups, tertiary amino groups, and guanidino groups, but the cationic molecules are not limited to these.
　上記ｂ）疎水性分子とは、水との間に水素結合を形成しにくく、水に対する親和性が低い分子を意味する。疎水性分子は、非極性分子又は分配係数が２．０以上の分子であってよい。疎水性分子としては、例えば、脂肪族基、トリアルキルアミン基、芳香族基等の疎水性基を有する分子、コレステロール、又はステロイド及びその類縁体が挙げられるが、疎水性分子はこれらに限られない。 The above b) hydrophobic molecule means a molecule that is difficult to form a hydrogen bond with water and has a low affinity for water. Hydrophobic molecules may be nonpolar molecules or molecules with a partition coefficient of 2.0 or more. Examples of hydrophobic molecules include molecules having hydrophobic groups such as aliphatic groups, trialkylamine groups, and aromatic groups, cholesterol, or steroids and their analogs, but hydrophobic molecules are not limited to these. do not have.
　本明細書において、「結合」は、直接的又は間接的で、不可逆的な結合を指す。不可逆的な結合とは、反応が可逆的に進行しない結合、すなわち、一旦形成された後に逆反応によって解離しない結合又は逆反応による解離が無視できる程度の結合を指す。 As used herein, "binding" refers to direct or indirect, irreversible binding. An irreversible bond refers to a bond in which the reaction does not proceed reversibly, that is, a bond that does not dissociate in a reverse reaction once formed, or a bond that dissociates in the reverse reaction is negligible.
　樹状ポリマー（コア）とオリゴ核酸との結合、オリゴ核酸と親水性ポリマーとの結合、及び親水性ポリマーと細胞内在化促進剤との結合は、それぞれ独立に、直接結合（すなわち、リンカーを介さない結合）又はリンカーを介した結合である。リンカーは特に限定されず、ＰＥＧ、アルキルリンカー（例えば、ヘキシルリンカー）等公知のリンカーであってよい。リンカーは、１種類のリンカーからなってもよく、２種以上のリンカーが結合することによって形成されたリンカーであってもよい。リンカーがＰＥＧの場合、その数平均分子量は、例えば、１０００以下、８００以下、６００以下、又は３００以下であってよい。 The binding between the dendritic polymer (core) and the oligonucleic acid, the binding between the oligonucleic acid and the hydrophilic polymer, and the binding between the hydrophilic polymer and the cell internalization promoter are each independently direct binding (i.e., via a linker). bond) or via a linker. The linker is not particularly limited, and may be a known linker such as PEG or an alkyl linker (eg, hexyl linker). The linker may be composed of one type of linker, or may be a linker formed by bonding two or more types of linkers. When the linker is PEG, its number average molecular weight may be, for example, 1000 or less, 800 or less, 600 or less, or 300 or less.
　樹状ポリマー（コア）とオリゴ核酸との間、オリゴ核酸と親水性ポリマーとの間、若しくは親水性ポリマーと細胞内在化促進剤との間の直接結合、又は上記リンカーと樹状ポリマー、オリゴ核酸、親水性ポリマー、若しくは細胞内在化促進剤との結合は、例えば、官能基間の求核的付加反応、求核置換反応、求電子置換反応等の化学反応により生じる共有結合、アンモニアと白金間の結合のような金属配位結合、又はビオチンとアビジン間の結合のようなホスト－ゲスト相互作用によるものであってよい。高い構造安定性を達成する観点、及びオリゴ核酸ナノ粒子のサイズを制御する観点から、結合は共有結合であることが好ましい。 Direct binding between a dendritic polymer (core) and an oligonucleic acid, between an oligonucleic acid and a hydrophilic polymer, or between a hydrophilic polymer and a cell internalization promoter, or between the above linker and a dendritic polymer, an oligonucleic acid , a hydrophilic polymer, or a cell internalization promoter, for example, a covalent bond formed by a chemical reaction such as a nucleophilic addition reaction between functional groups, a nucleophilic substitution reaction, or an electrophilic substitution reaction, or a bond between ammonia and platinum. may be due to metal coordination bonds, such as the binding between biotin and avidin, or host-guest interactions, such as the binding between biotin and avidin. From the viewpoint of achieving high structural stability and controlling the size of the oligonucleic acid nanoparticles, the bond is preferably a covalent bond.
　共有結合としては、例えば、単結合、二重結合、三重結合、アミド結合、グリコシド結合、エステル結合、エーテル結合、ウレタン結合、アセタール結合、リン酸エステル結合、チオエーテル結合、チオエステル結合、ジスルフィド結合、トリアゾール結合、ヒドラゾン結合、ヒドラジド結合、イミン又はオキシム結合、尿素又はチオ尿素結合、アミジン結合、スルホンアミド結合、又は逆電子要請型ディールス・アルダー反応により形成される結合が挙げられるが、共有結合はこれらに限られない。 Examples of covalent bonds include single bonds, double bonds, triple bonds, amide bonds, glycosidic bonds, ester bonds, ether bonds, urethane bonds, acetal bonds, phosphate ester bonds, thioether bonds, thioester bonds, disulfide bonds, and triazole bonds. Covalent bonds include bonds, hydrazone bonds, hydrazide bonds, imine or oxime bonds, urea or thiourea bonds, amidine bonds, sulfonamide bonds, or bonds formed by reverse electron request Diels-Alder reactions. Not limited.
　アミド結合は、カルボキシ基とアミノ基との間で形成される。アミド結合は、例えば、好適に保護されたアミノ基と、活性化カルボン酸（Ｎ－ヒドロキシスクシンイミドで活性化されたエステルなど）との間で生じる、従来のアミド結合形成反応を用いて形成される。 An amide bond is formed between a carboxy group and an amino group. Amide bonds are formed using conventional amide bond-forming reactions, e.g., between a suitably protected amino group and an activated carboxylic acid, such as an ester activated with N-hydroxysuccinimide. .
　ジスルフィド結合（－Ｓ－Ｓ－）は、例えば、チオール基（メルカプタン基ともいう）（－ＳＨ）を有する成分と、他の成分の活性化チオール基とのチオール交換によって形成される。 A disulfide bond (-SS-) is formed, for example, by thiol exchange between a component having a thiol group (also referred to as a mercaptan group) (-SH) and an activated thiol group of another component.
　チオエーテル結合（－Ｓ－）は、例えば、チオール基とマレイミド基との間で生じる、従来のチオエーテル結合形成反応を用いて形成される。 The thioether bond (-S-) is formed using a conventional thioether bond-forming reaction, which occurs, for example, between a thiol group and a maleimide group.
　トリアゾール結合は、アジド基と炭素間三重結合との間で形成される。トリアゾール結合は、例えば、金属触媒を用いるヒュスゲン環化、金属触媒を用いない歪み促進型アジド－アルキン不可環化反応（Ｓｔｒａｉｎ－ｐｒｏｍｏｔｅｄ　ａｌｋｙｎｅ－ａｚｉｄｅ　ｃｙｃｌｏａｄｄｉｔｉｏｎ）などの所謂クリック反応によって形成される。 A triazole bond is formed between an azide group and a carbon-carbon triple bond. The triazole bond is formed, for example, by a so-called click reaction such as Husgen cyclization using a metal catalyst or strain-promoted alkyne-azide cycloaddition without using a metal catalyst.
　金属配位は、金属イオンと配位子が錯体を形成することで結合する結合様式である。金属イオンとしては、白金族元素、マンガン、コバルト、銅、ガドリニウムなどの金属元素のイオンが挙げられるが、これらに限られない。また、配位子としては、アンモニア、ピリジン、ビピリジン、エチレンジアミン、エチレンジアミンテトラ酢酸、アセチルアセトナート、又はこれらの誘導体などが挙げられるが、これらに限られない。 Metal coordination is a bonding mode in which metal ions and ligands bond by forming a complex. Examples of metal ions include, but are not limited to, ions of metal elements such as platinum group elements, manganese, cobalt, copper, and gadolinium. Further, examples of the ligand include, but are not limited to, ammonia, pyridine, bipyridine, ethylenediamine, ethylenediaminetetraacetic acid, acetylacetonate, and derivatives thereof.
　ホスト－ゲスト相互作用は、特定の分子を選択的に認識できる空間を提供する分子であるホスト分子と、そこに受け入れられる分子であるゲスト分子との間の相互作用である。ホスト分子としては、シクロデキストリン、カルセランド、キャビタンド、クラウンエーテル、クリプタンド、ククルビツリル、カリックスアレン、アビジン、ストレプトアビジンなどが挙げられるが、これらに限られない。また、ゲスト分子としては、アダマンタン、ジアダマンタン、コレステロール、ナフタレン、ビオチンなどが挙げられるが、これらに限られない。 Host-guest interaction is an interaction between a host molecule, which is a molecule that provides a space in which a specific molecule can be selectively recognized, and a guest molecule, which is a molecule that is accepted there. Host molecules include, but are not limited to, cyclodextrin, calcerand, cavitand, crown ether, cryptand, cucurbituril, calixarene, avidin, streptavidin, and the like. Further, guest molecules include, but are not limited to, adamantane, diadamantane, cholesterol, naphthalene, biotin, and the like.
　本側面に係るオリゴ核酸ナノ粒子を構成する単分子は、遊離体であってもよく、薬学的に許容される塩であってもよい。オリゴ核酸ナノ粒子を構成する単分子は、溶媒和物（例、水和物、エタノール溶媒和物、プロピレングリコール溶媒和物）又は非溶媒和物のいずれであってもよい。薬学的に許容される塩は、酸付加塩であっても、塩基付加塩であってもよい。酸付加塩としては、例えば、ギ酸塩、酢酸塩、トリフルオロ酢酸（ＴＦＡ）塩、プロピオン酸塩、コハク酸塩、乳酸塩、リンゴ酸塩、アジピン酸塩、クエン酸塩、酒石酸塩、メタンスルホン酸塩、フマル酸塩、マレイン酸塩、ｐ－トルエンスルホン酸塩、アスコルビン酸塩等の有機酸との塩；塩酸塩、臭化水素塩、硫酸塩、硝酸塩、リン酸塩等の無機酸との塩等が挙げられる。塩基付加塩としては、例えば、ナトリウム塩、カリウム塩等のアルカリ金属塩；カルシウム塩、マグネシウム塩等のアルカリ土類金属塩；アンモニウム塩；トリメチルアミン塩；トリエチルアミン塩；ジシクロヘキシルアミン塩、エタノールアミン塩、ジエタノールアミン塩、トリエタノールアミン塩、ブロカイン塩等の脂肪族アミン塩；Ｎ，Ｎ－ジベンジルエチレンジアミン等のアラルキルアミン塩；ピリジン塩、ピコリン塩、キノリン塩、イソキノリン塩等の複素環芳香族アミン塩；テトラメチルアンモニウム塩、テトラエチルアモニウム塩、ベンジルトリメチルアンモニウム塩、ベンジルトリエチルアンモニウム塩、ベンジルトリブチルアンモニウム塩、メチルトリオクチルアンモニウム塩、テトラブチルアンモニウム塩等の第４級アンモニウム塩；アルギニン塩、リジン塩等の塩基性アミノ酸塩等が挙げられる。 The single molecule constituting the oligonucleic acid nanoparticle according to this aspect may be a free form or a pharmaceutically acceptable salt. The single molecules constituting the oligonucleic acid nanoparticles may be either solvates (eg, hydrates, ethanol solvates, propylene glycol solvates) or non-solvates. Pharmaceutically acceptable salts may be acid addition salts or base addition salts. Acid addition salts include, for example, formate, acetate, trifluoroacetic acid (TFA) salt, propionate, succinate, lactate, malate, adipate, citrate, tartrate, methanesulfone. Salts with organic acids such as acid salts, fumarates, maleates, p-toluenesulfonates, and ascorbates; salts with inorganic acids such as hydrochlorides, hydrobromides, sulfates, nitrates, phosphates, etc. salt, etc. Examples of base addition salts include alkali metal salts such as sodium salts and potassium salts; alkaline earth metal salts such as calcium salts and magnesium salts; ammonium salts; trimethylamine salts; triethylamine salts; dicyclohexylamine salts, ethanolamine salts, and diethanolamine salts. aliphatic amine salts such as salts, triethanolamine salts, and brocaine salts; aralkylamine salts such as N,N-dibenzylethylenediamine; heterocyclic aromatic amine salts such as pyridine salts, picoline salts, quinoline salts, and isoquinoline salts; Quaternary ammonium salts such as methylammonium salt, tetraethylammonium salt, benzyltrimethylammonium salt, benzyltriethylammonium salt, benzyltributylammonium salt, methyltrioctylammonium salt, tetrabutylammonium salt; bases such as arginine salt, lysine salt, etc. and amino acid salts.
　次に、本側面に係るオリゴ核酸ナノ粒子の構造について、図１を参照しながら説明する。図１は、本側面に係るオリゴ核酸ナノ粒子の一態様を示す模式図である。オリゴ核酸ナノ粒子１００は、オリゴ核酸ナノ粒子１００の中心部に位置するコア５０（樹状ポリマー）と、コア５０の周りに配置された複数個のオリゴ核酸１１、親水性ポリマー１２、及び細胞内在化促進剤１３とを含む。オリゴ核酸１１はリンカー３１を介してコア５０に結合している。親水性ポリマー１２はオリゴ核酸１１に結合して、オリゴ核酸１１を外界から遮蔽するとともに、親水性ポリマー１２に結合した細胞内在化促進剤１３を、オリゴ核酸ナノ粒子１００の表面に露出させている。コア５０にはキャッピング剤２１も結合している。水溶液においては、オリゴ核酸１１及び親水性ポリマー１２が、コア５０から略放射状に延びることによって、オリゴ核酸ナノ粒子１００は略球状の形状をとる。なお、図１では、オリゴ核酸１１はリンカー３１を介してコア５０に結合しているが、上述のとおり、オリゴ核酸１１はコア５０に直接結合していてもよい。また、オリゴ核酸１１と親水性ポリマー１２、及び親水性ポリマー１２と細胞内在化促進剤１３は、上述の任意のリンカーを介して結合していてもよい。 Next, the structure of the oligonucleic acid nanoparticle according to this aspect will be explained with reference to FIG. 1. FIG. 1 is a schematic diagram showing one embodiment of oligonucleic acid nanoparticles according to this aspect. The oligonucleic acid nanoparticle 100 includes a core 50 (dendritic polymer) located at the center of the oligonucleic acid nanoparticle 100, a plurality of oligonucleic acids 11 arranged around the core 50, a hydrophilic polymer 12, and a cell-internal component. oxidation accelerator 13. Oligonucleic acid 11 is bound to core 50 via linker 31. The hydrophilic polymer 12 binds to the oligonucleic acid 11 and shields the oligonucleic acid 11 from the outside world, while exposing the cell internalization promoter 13 bound to the hydrophilic polymer 12 on the surface of the oligonucleic acid nanoparticle 100. . A capping agent 21 is also bonded to the core 50. In an aqueous solution, oligonucleic acid 11 and hydrophilic polymer 12 extend substantially radially from core 50, so that oligonucleic acid nanoparticles 100 take a substantially spherical shape. In FIG. 1, the oligonucleic acid 11 is bonded to the core 50 via the linker 31, but the oligonucleic acid 11 may be bonded directly to the core 50 as described above. Moreover, the oligonucleic acid 11 and the hydrophilic polymer 12, and the hydrophilic polymer 12 and the cell internalization promoter 13 may be bonded via any of the above-mentioned linkers.
　オリゴ核酸ナノ粒子１００の平均粒子径は、好ましくは１０～１００ｎｍであり、より好ましくは１５～４５ｎｍである。本明細書において、オリゴ核酸ナノ粒子の平均粒子径は、動的光散乱によって得られる粒度分布における平均粒子径を意味する。オリゴ核酸ナノ粒子１００は、コア５０として樹状ポリマーを有するため、サイズの制御が容易であり、精密な設計が可能である。 The average particle diameter of the oligonucleic acid nanoparticles 100 is preferably 10 to 100 nm, more preferably 15 to 45 nm. In this specification, the average particle size of oligonucleic acid nanoparticles means the average particle size in a particle size distribution obtained by dynamic light scattering. Since the oligonucleic acid nanoparticles 100 have a dendritic polymer as the core 50, the size can be easily controlled and precisely designed.
　オリゴ核酸ナノ粒子１００が細胞内に輸送されるためには、細胞内在化促進剤１３が細胞と相互作用する必要がある。オリゴ核酸ナノ粒子１００の細胞内への輸送効率を向上させる観点から、細胞内在化促進剤１３の密度は高いことが好ましい。オリゴ核酸ナノ粒子１００によれば、高度に分岐した樹状ポリマーで構成されたコア５０に、オリゴ核酸１１及び親水性ポリマー１２を介して細胞内在化促進剤１３が結合しているため、細胞内在化促進剤１３の高い密度を達成することができ、よって細胞内在化促進剤１３が標的細胞と効率良く相互作用することができる。また、親水性ポリマー１２の結合によりオリゴ核酸１１を外界から効率良く遮蔽するためには、オリゴ核酸１１は、ポリマー末端の自由運動性が小さく、三次元的に明確な空間形態をとるコア５０に結合していることが好ましい。その観点からも、コア５０として樹状ポリマーを有するオリゴ核酸ナノ粒子１００は、直鎖ポリマーをコアとして有する場合と比べて好ましい。 In order for the oligonucleic acid nanoparticles 100 to be transported into cells, the cell internalization promoter 13 needs to interact with the cells. From the viewpoint of improving the efficiency of transporting the oligonucleic acid nanoparticles 100 into cells, it is preferable that the density of the cell internalization promoter 13 is high. According to the oligonucleic acid nanoparticles 100, the cell internalization promoter 13 is bound to the core 50 made of a highly branched dendritic polymer via the oligonucleic acid 11 and the hydrophilic polymer 12, so A high density of the internalization promoting agent 13 can be achieved, and therefore the cell internalization promoting agent 13 can efficiently interact with target cells. Furthermore, in order to efficiently shield the oligonucleic acid 11 from the outside world through the binding of the hydrophilic polymer 12, the oligonucleic acid 11 must be attached to the core 50, which has small free mobility at the polymer end and has a three-dimensionally defined spatial configuration. Preferably, they are bonded. From this point of view as well, the oligonucleic acid nanoparticles 100 having a dendritic polymer as the core 50 are more preferable than those having a linear polymer as the core.
　また、オリゴ核酸ナノ粒子１００においては、親水性ポリマー１２がオリゴ核酸１１に結合しているため、親水性ポリマー１２及びそれに結合した細胞内在化促進剤１３が、オリゴ核酸１１の空間的な広がり（回転半径）の外側に位置するとともに、細胞内在化促進剤１３がオリゴ核酸ナノ粒子１００の表面（すなわち、最外殻）に位置することができる。細胞内在化促進剤１３がオリゴ核酸ナノ粒子１００の表面に位置することによって、オリゴ核酸ナノ粒子１００が標的細胞と効率良く相互作用しやすくなる。また、親水性ポリマー１２が、オリゴ核酸１１の空間的な広がりの外側に位置することによって、オリゴ核酸ナノ粒子１００と標的細胞以外の生体成分との非特異的相互作用が低減される効果が期待できる。なお、細胞内在化促進剤１３がオリゴ核酸ナノ粒子１００の表面に位置することは、表面プラズモン共鳴（ＳＰＲ）法、酵素結合免疫吸着アッセイ（ＥＬＩＳＡ）法等の手法により結合活性を評価することで、確認することができる。細胞内在化促進剤１３をオリゴ核酸ナノ粒子１００の表面により露出させる観点から、親水性ポリマー１２と、オリゴ核酸１１及び細胞内在化促進剤１３とは、親水性ポリマー１２及び細胞内在化促進剤１３がオリゴ核酸１１の空間的な広がりからより離れた位置に存在するように結合していることが好ましい。例えば、図１に示すように、親水性ポリマー１２は、好ましくはオリゴ核酸１１の末端であって、コア５０と結合していない方の末端に結合している。また、親水性ポリマー１２の形状にもよるが、細胞内在化促進剤１３は、好ましくは親水性ポリマー１２の末端であって、オリゴ核酸１１と結合していない末端に結合している。 Furthermore, in the oligonucleic acid nanoparticles 100, since the hydrophilic polymer 12 is bonded to the oligonucleic acid 11, the hydrophilic polymer 12 and the cell internalization promoter 13 bonded thereto can spread the oligonucleic acid 11 spatially ( The cell internalization promoting agent 13 can be located on the surface (ie, the outermost shell) of the oligonucleic acid nanoparticle 100. By locating the cell internalization promoter 13 on the surface of the oligonucleic acid nanoparticles 100, the oligonucleic acid nanoparticles 100 can more easily interact with target cells efficiently. Furthermore, by positioning the hydrophilic polymer 12 outside the spatial extent of the oligonucleic acid 11, it is expected that non-specific interactions between the oligonucleic acid nanoparticles 100 and biological components other than the target cells will be reduced. can. The location of the cell internalization promoter 13 on the surface of the oligonucleic acid nanoparticles 100 can be determined by evaluating the binding activity using methods such as surface plasmon resonance (SPR) and enzyme-linked immunosorbent assay (ELISA). , can be confirmed. From the viewpoint of exposing the cell internalization promoter 13 to the surface of the oligonucleic acid nanoparticle 100, the hydrophilic polymer 12, the oligonucleic acid 11 and the cell internalization promoter 13 are the same as the hydrophilic polymer 12 and the cell internalization promoter 13. It is preferable that the oligonucleic acid 11 be bonded so that it is present at a position further away from the spatial extent of the oligonucleic acid 11. For example, as shown in FIG. 1, the hydrophilic polymer 12 is preferably bonded to the end of the oligonucleic acid 11 that is not bonded to the core 50. Although it depends on the shape of the hydrophilic polymer 12, the cell internalization promoter 13 is preferably bound to the end of the hydrophilic polymer 12 that is not bound to the oligonucleic acid 11.
　本発明の一側面に係るオリゴ核酸ナノ粒子（以下、第二の側面に係るオリゴ核酸ナノ粒子ともいう。）は、樹状ポリマーで構成されたコアと、該コアに結合した複数個のオリゴ核酸と、上記コアにスペーサーを介して結合した１又は複数個の親水性ポリマーと、該１又は複数個の親水性ポリマーに結合した１又は複数個の細胞内在化促進剤と、を含む。 Oligonucleic acid nanoparticles according to one aspect of the present invention (hereinafter also referred to as oligonucleic acid nanoparticles according to a second aspect) have a core made of a dendritic polymer and a plurality of oligonucleic acids bound to the core. , one or more hydrophilic polymers bonded to the core via a spacer, and one or more cell internalization promoters bonded to the one or more hydrophilic polymers.
　本側面に係るオリゴ核酸ナノ粒子は、細胞内在化促進剤が結合した親水性ポリマーが、オリゴ核酸に結合するのではなく、スペーサーを介してコアに結合しているという点で、上記第一の側面に係るオリゴ核酸ナノ粒子と異なっている。樹状ポリマー、オリゴ核酸、親水性ポリマー、及び細胞内在化促進剤の詳細は、第一の側面に係るオリゴ核酸ナノ粒子と同様であり、上述のとおりである。 The oligonucleic acid nanoparticles according to the present aspect meet the above-mentioned first aspect in that the hydrophilic polymer to which the cell internalization promoter is bound is not bound to the oligonucleic acid but to the core via a spacer. It is different from the oligonucleic acid nanoparticles related to the side. The details of the dendritic polymer, oligonucleic acid, hydrophilic polymer, and cell internalization promoter are the same as those of the oligonucleic acid nanoparticle according to the first aspect, and are as described above.
　本明細書において、スペーサーは、コアと親水性ポリマーとを連結させるためのポリマーであり、親水性ポリマー及びこれに結合した細胞内在化促進剤を、オリゴ核酸の空間的な広がり（回転半径）の外側に配置する。スペーサーは特に限定されず、極性分子であっても非極性分子であってもよく、正電荷を有していても、負電荷を有していてもよい。スペーサーとしては、例えば、ＰＥＧ、ｐＭｅＯｘ、カチオン性又はアニオン性ポリペプチド、ポリペプトイド、及びアルキル鎖が挙げられるが、スペーサーはこれらに限られない。細胞内在化促進剤をオリゴ核酸ナノ粒子の表面に露出させる観点から、スペーサーは、好ましくは、剛直な構造を有するスペーサー又はカチオン性のスペーサーである。スペーサーは、好ましくはＰＥＧ又はカチオン性ポリペプチドである。オリゴ核酸ナノ粒子は、スペーサーとして１種又は２種以上のスペーサーを含んでよい。 In the present specification, the spacer is a polymer for connecting the core and the hydrophilic polymer, and the spacer is a polymer for connecting the core and the hydrophilic polymer, and the spacer is a polymer that connects the hydrophilic polymer and the cell internalization promoter bound thereto to the extent that the spatial extent (radius of gyration) of the oligonucleic acid Place it outside. The spacer is not particularly limited, and may be a polar molecule or a nonpolar molecule, and may have a positive charge or a negative charge. Spacers include, but are not limited to, PEG, pMeOx, cationic or anionic polypeptides, polypeptoids, and alkyl chains. From the viewpoint of exposing the cell internalization promoter to the surface of the oligonucleic acid nanoparticle, the spacer is preferably a spacer having a rigid structure or a cationic spacer. The spacer is preferably PEG or a cationic polypeptide. The oligonucleic acid nanoparticles may contain one or more spacers as spacers.
　スペーサーの長さは、細胞内在化促進剤がオリゴ核酸ナノ粒子の表面に位置することができるように、オリゴ核酸の長さ及び親水性ポリマーの大きさに応じて適宜調整される。スペーサーの長さは、オリゴ核酸の長さと比較して十分に長いことが好ましい。 The length of the spacer is appropriately adjusted depending on the length of the oligonucleic acid and the size of the hydrophilic polymer so that the cell internalization promoter can be located on the surface of the oligonucleic acid nanoparticle. The length of the spacer is preferably sufficiently long compared to the length of the oligonucleic acid.
　スペーサーの数は、例えば、１以上、２以上、又は樹状ポリマーの有する反応性官能基の１％以上であってよく、樹状ポリマーの有する反応性官能基の好ましくは２％以上、より好ましくは３％以上又は５％以上である。オリゴ核酸ナノ粒子におけるスペーサーの数は、例えば、スペーサーの末端官能基に蛍光色素を結合させ、得られたオリゴ核酸ナノ粒子を含む溶液中の蛍光色素濃度を樹状ポリマーの濃度で除することで求めることができる。 The number of spacers may be, for example, 1 or more, 2 or more, or 1% or more of the reactive functional groups possessed by the dendritic polymer, preferably 2% or more of the reactive functional groups possessed by the dendritic polymer, and more preferably is 3% or more or 5% or more. The number of spacers in oligonucleic acid nanoparticles can be determined, for example, by binding a fluorescent dye to the terminal functional group of the spacer and dividing the concentration of the fluorescent dye in the solution containing the obtained oligonucleic acid nanoparticles by the concentration of the dendritic polymer. You can ask for it.
　一実施形態において、オリゴ核酸又はスペーサーと結合していない未反応の反応性官能基の少なくとも一部又は全部は、キャッピング剤によりキャッピングされていてよい。キャッピング剤の詳細は、上述のとおりである。 In one embodiment, at least a portion or all of the unreacted reactive functional groups that are not bound to the oligonucleic acid or the spacer may be capped with a capping agent. Details of the capping agent are as described above.
　樹状ポリマー（コア）とオリゴ核酸との結合、コアとスペーサーとの結合、スペーサーと親水性ポリマーとの結合、及び親水性ポリマーと細胞内在化促進剤との結合は、それぞれ独立に、直接結合（すなわち、リンカーを介さない結合）又はリンカーを介した結合である。リンカーの詳細は、上述のとおりである。コアとオリゴ核酸との間、コアとスペーサーとの間、スペーサーと親水性ポリマーとの間、若しくは親水性ポリマーと細胞内在化促進剤との間の直接結合、又は上記リンカーと樹状ポリマー、オリゴ核酸、スペーサー、親水性ポリマー、若しくは細胞内在化促進剤との結合は、例えば、共有結合、金属配位結合、又はホスト－ゲスト相互作用によるものであってよい。これらの結合又は相互作用の詳細は、上述のとおりである。高い構造安定性を達成する観点、及びオリゴ核酸ナノ粒子のサイズを制御する観点から、結合は共有結合であることが好ましい。 The binding between the dendritic polymer (core) and the oligonucleic acid, the binding between the core and the spacer, the binding between the spacer and the hydrophilic polymer, and the binding between the hydrophilic polymer and the cell internalization promoter are each independently direct binding. (ie, binding without a linker) or binding through a linker. Details of the linker are as described above. Direct binding between the core and the oligonucleic acid, between the core and the spacer, between the spacer and the hydrophilic polymer, or between the hydrophilic polymer and the cell internalization promoter, or between the linker and the dendritic polymer, the oligonucleotide Binding to the nucleic acid, spacer, hydrophilic polymer, or cell internalization promoter may be, for example, by covalent bonding, metal coordination bonding, or host-guest interaction. Details of these bonds or interactions are as described above. From the viewpoint of achieving high structural stability and controlling the size of the oligonucleic acid nanoparticles, the bond is preferably a covalent bond.
　本側面に係るオリゴ核酸ナノ粒子を構成する単分子は、遊離体であってもよく、薬学的に許容される塩であってもよい。オリゴ核酸ナノ粒子を構成する単分子は、溶媒和物（例、水和物、エタノール溶媒和物、プロピレングリコール溶媒和物）又は非溶媒和物のいずれであってもよい。薬学的に許容される塩の詳細は、上述のとおりである。 The single molecule constituting the oligonucleic acid nanoparticle according to this aspect may be a free form or a pharmaceutically acceptable salt. The single molecules constituting the oligonucleic acid nanoparticles may be either solvates (eg, hydrates, ethanol solvates, propylene glycol solvates) or non-solvates. Details of the pharmaceutically acceptable salt are as described above.
　次に、本側面に係るオリゴ核酸ナノ粒子の構造について、図２を参照しながら説明する。図２は、本側面に係るオリゴ核酸ナノ粒子の一態様を示す模式図である。オリゴ核酸ナノ粒子２００は、オリゴ核酸ナノ粒子２００の中心部に位置するコア５０（樹状ポリマー）と、コア５０の周りに配置された複数個のオリゴ核酸１１、スペーサー１４、親水性ポリマー１２、及び細胞内在化促進剤１３とを含む。オリゴ核酸１１はリンカー３１を介してコア５０に結合している。親水性ポリマー１２はスペーサー１４を介してコア５０に結合し、オリゴ核酸１１を外界から遮蔽するとともに、親水性ポリマー１２に結合した細胞内在化促進剤１３を、オリゴ核酸ナノ粒子２００の表面に露出させている。コア５０にはキャッピング剤２１も結合している。水溶液においては、オリゴ核酸１１、スペーサー１４、及び親水性ポリマー１２が、コア５０から略放射状に延びることによって、オリゴ核酸ナノ粒子２００は略球状の形状をとる。なお、図２では、オリゴ核酸１１はリンカー３１を介してコア５０に結合しているが、上述のとおり、オリゴ核酸１１はコア５０に直接結合していてもよい。また、スペーサー１４と親水性ポリマー１２、及び親水性ポリマー１２と細胞内在化促進剤１３は、上述の任意のリンカーを介して結合していてもよい。 Next, the structure of the oligonucleic acid nanoparticle according to this aspect will be explained with reference to FIG. 2. FIG. 2 is a schematic diagram showing one embodiment of oligonucleic acid nanoparticles according to this aspect. The oligonucleic acid nanoparticle 200 includes a core 50 (dendritic polymer) located at the center of the oligonucleic acid nanoparticle 200, a plurality of oligonucleic acids 11 arranged around the core 50, a spacer 14, a hydrophilic polymer 12, and cell internalization promoter 13. Oligonucleic acid 11 is bound to core 50 via linker 31. The hydrophilic polymer 12 binds to the core 50 via the spacer 14 to shield the oligonucleic acid 11 from the outside world, and exposes the cell internalization promoter 13 bound to the hydrophilic polymer 12 on the surface of the oligonucleic acid nanoparticle 200. I'm letting you do it. A capping agent 21 is also bonded to the core 50. In an aqueous solution, the oligonucleic acid nanoparticles 200 assume a substantially spherical shape due to the oligonucleic acid 11, the spacer 14, and the hydrophilic polymer 12 extending substantially radially from the core 50. Note that in FIG. 2, the oligonucleic acid 11 is bonded to the core 50 via the linker 31, but the oligonucleic acid 11 may be bonded directly to the core 50 as described above. Moreover, the spacer 14 and the hydrophilic polymer 12, and the hydrophilic polymer 12 and the cell internalization promoter 13 may be bonded via any of the above-mentioned linkers.
　オリゴ核酸ナノ粒子２００の平均粒子径は、オリゴ核酸ナノ粒子１００と同様であってよく、詳細は上述のとおりである。 The average particle diameter of the oligonucleic acid nanoparticles 200 may be the same as that of the oligonucleic acid nanoparticles 100, and the details are as described above.
　オリゴ核酸ナノ粒子２００によれば、高度に分岐した樹状ポリマーで構成されたコア５０に、スペーサー１４及び親水性ポリマー１２を介して細胞内在化促進剤１３が結合しているため、細胞内在化促進剤１３の高い密度を達成することができ、よって細胞内在化促進剤１３が標的細胞と効率良く相互作用することができる。 According to the oligonucleic acid nanoparticles 200, the cell internalization promoter 13 is bound to the core 50 made of a highly branched dendritic polymer via the spacer 14 and the hydrophilic polymer 12, so that cell internalization is facilitated. A high density of the promoter 13 can be achieved, so that the cell internalization promoter 13 can efficiently interact with target cells.
　また、オリゴ核酸ナノ粒子２００においては、コア５０にスペーサー１４を介して親水性ポリマー１２が結合しているため、スペーサー１４の長さがオリゴ核酸１１の長さと比較して十分に長い場合、親水性ポリマー１２及びそれに結合した細胞内在化促進剤１３が、オリゴ核酸１１の空間的な広がり（回転半径）の外側に位置するとともに、細胞内在化促進剤１３がオリゴ核酸ナノ粒子２００の表面（すなわち、最外殻）に位置することができる。細胞内在化促進剤１３がオリゴ核酸ナノ粒子２００の表面に位置することによって、オリゴ核酸ナノ粒子２００が標的細胞と効率良く相互作用しやすくなる。また、親水性ポリマー１２が、オリゴ核酸１１の空間的な広がりの外側に位置することによって、オリゴ核酸ナノ粒子２００と標的細胞以外の生体成分との非特異的相互作用が低減される効果が期待できる。スペーサー１４が、剛直な構造を有する場合、又はカチオン性であり、オリゴ核酸１１と複合体を形成し得る場合、親水性ポリマー１２及びそれに結合した細胞内在化促進剤１３が、オリゴ核酸１１の空間的な広がりの外側により位置しやすい。 In addition, in the oligonucleic acid nanoparticles 200, since the hydrophilic polymer 12 is bonded to the core 50 via the spacer 14, if the length of the spacer 14 is sufficiently long compared to the length of the oligonucleic acid 11, the hydrophilic polymer 12 is bound to the core 50 via the spacer 14. The polymer 12 and the cell internalization promoter 13 bound thereto are located outside the spatial extent (radius of rotation) of the oligonucleic acid 11, and the cell internalization promoter 13 is located on the surface of the oligonucleic acid nanoparticle 200 (i.e. , outermost shell). By locating the cell internalization promoter 13 on the surface of the oligonucleic acid nanoparticles 200, the oligonucleic acid nanoparticles 200 can more easily interact with target cells efficiently. Furthermore, by positioning the hydrophilic polymer 12 outside the spatial extent of the oligonucleic acid 11, it is expected that non-specific interactions between the oligonucleic acid nanoparticles 200 and biological components other than the target cells will be reduced. can. When the spacer 14 has a rigid structure or is cationic and can form a complex with the oligonucleic acid 11, the hydrophilic polymer 12 and the cell internalization promoter 13 bound thereto can more likely to be located outside of the natural expanse.
　本発明は、上記第一の側面に係るオリゴ核酸ナノ粒子を製造する方法も提供する。すなわち、本発明の一側面は、
　（ａ１）樹状ポリマーで構成されたコアに複数個のオリゴ核酸を結合させる工程と、
　（ａ２）オリゴ核酸に親水性ポリマーを結合させる工程と、
　（ａ３）親水性ポリマーに細胞内在化促進剤を結合させる工程と、を含む、オリゴ核酸ナノ粒子を製造する方法である。一実施形態において、オリゴ核酸ナノ粒子を製造する方法は、（ａ４）コアにキャッピング剤を結合させる工程をさらに含んでよい。これにより、樹状ポリマーの反応性官能基の少なくとも一部がキャッピング剤によりキャッピングされた、オリゴ核酸ナノ粒子を製造することができる。 The present invention also provides a method for producing the oligonucleic acid nanoparticles according to the first aspect. That is, one aspect of the present invention is
(a1) A step of binding a plurality of oligonucleic acids to a core composed of a dendritic polymer,
(a2) a step of binding a hydrophilic polymer to the oligonucleic acid;
(a3) A method for producing oligonucleic acid nanoparticles, which includes the step of binding a cell internalization promoter to a hydrophilic polymer. In one embodiment, the method for producing oligonucleic acid nanoparticles may further include the step of (a4) binding a capping agent to the core. Thereby, oligonucleic acid nanoparticles in which at least a portion of the reactive functional groups of the dendritic polymer are capped with a capping agent can be produced.
　コアとオリゴ核酸、オリゴ核酸と親水性ポリマー、及び親水性ポリマーと細胞内在化促進剤とは、それぞれ独立に、直接又はリンカーを介して結合させることができる。例えば、工程（ａ１）は、コアに複数個のリンカーを結合させる工程と、該リンカーにオリゴ核酸を結合させる工程と、を含んでよい。 The core and the oligonucleic acid, the oligonucleic acid and the hydrophilic polymer, and the hydrophilic polymer and the cell internalization promoter can each be independently bonded directly or via a linker. For example, step (a1) may include the steps of binding a plurality of linkers to the core, and binding an oligonucleic acid to the linkers.
　工程（ａ１）～（ａ４）はこの順に行うことができるが、この順に行うことは必須ではない。例えば、工程（ａ４）を、工程（ａ１）の前に、工程（ａ１）と同時に、又は工程（ａ１）と工程（ａ２）との間に行ってもよい。また、工程（ａ３）を工程（ａ２）の前に行い、工程（ａ２）において、細胞内在化促進剤が結合した親水性ポリマーをオリゴ核酸に結合させてもよい。 Although steps (a1) to (a4) can be performed in this order, it is not necessary to perform them in this order. For example, step (a4) may be performed before step (a1), simultaneously with step (a1), or between step (a1) and step (a2). Alternatively, step (a3) may be performed before step (a2), and in step (a2), a hydrophilic polymer to which a cell internalization promoter is bound may be bound to the oligonucleic acid.
　一例として、オリゴ核酸ナノ粒子は、樹状ポリマーで構成されたコアに複数個のキャッピング剤及びリンカーを結合させる工程と、該リンカーにオリゴ核酸を結合させる工程と、オリゴ核酸に親水性ポリマーを結合させる工程と、親水性ポリマーに細胞内在化促進剤を結合させる工程と、をこの順に含む方法により製造することができる。 As an example, oligonucleic acid nanoparticles are produced by bonding a plurality of capping agents and linkers to a core composed of a dendritic polymer, bonding an oligonucleic acid to the linker, and bonding a hydrophilic polymer to the oligonucleic acid. It can be produced by a method including, in this order, a step of binding a cell internalization promoter to a hydrophilic polymer.
　本発明は、上記第二の側面に係るオリゴ核酸ナノ粒子を製造する方法も提供する。すなわち、本発明の一側面は、
　（ｂ１）樹状ポリマーで構成されたコアに１又は複数個のスペーサーを結合させる工程と、
　（ｂ２）コアに複数個のオリゴ核酸を結合させる工程と、
　（ｂ３）スペーサーに親水性ポリマーを結合させる工程と、
　（ｂ４）親水性ポリマーに細胞内在化促進剤を結合させる工程と、を含む、オリゴ核酸ナノ粒子を製造する方法である。一実施形態において、オリゴ核酸ナノ粒子を製造する方法は、（ｂ５）コアにキャッピング剤を結合させる工程をさらに含んでよい。これにより、樹状ポリマーの反応性官能基の少なくとも一部がキャッピング剤によりキャッピングされた、オリゴ核酸ナノ粒子を製造することができる。 The present invention also provides a method for producing oligonucleic acid nanoparticles according to the second aspect. That is, one aspect of the present invention is
(b1) bonding one or more spacers to a core composed of a dendritic polymer;
(b2) a step of binding a plurality of oligonucleic acids to the core;
(b3) bonding a hydrophilic polymer to the spacer;
(b4) A method for producing oligonucleic acid nanoparticles, comprising the step of binding a cell internalization promoter to a hydrophilic polymer. In one embodiment, the method for producing oligonucleic acid nanoparticles may further include the step of (b5) binding a capping agent to the core. Thereby, oligonucleic acid nanoparticles in which at least a portion of the reactive functional groups of the dendritic polymer are capped with a capping agent can be produced.
　コアとオリゴ核酸、コアとスペーサー、スペーサーと親水性ポリマー、及び親水性ポリマーと細胞内在化促進剤とは、それぞれ独立に、直接又はリンカーを介して結合させることができる。例えば、工程（ｂ１）は、コアに複数個のリンカーを結合させる工程と、該リンカーにオリゴ核酸を結合させる工程と、を含んでよい。 The core and the oligonucleic acid, the core and the spacer, the spacer and the hydrophilic polymer, and the hydrophilic polymer and the cell internalization promoter can each be independently bonded directly or via a linker. For example, step (b1) may include the steps of binding a plurality of linkers to the core, and binding an oligonucleic acid to the linkers.
　工程（ｂ１）～（ｂ５）はこの順に行うことができるが、この順に行うことは必須ではない。例えば、工程（ｂ１）と工程（ｂ２）を同時に行ってもよいし、工程（ｂ５）を、工程（ｂ１）の前に、工程（ｂ１）と同時に、工程（ｂ１）と工程（ｂ２）との間に、又は工程（ｂ２）と工程（ｂ３）との間に行ってもよい。また、工程（ｂ４）を工程（ｂ３）の前に行い、工程（ｂ３）において、細胞内在化促進剤が結合した親水性ポリマーをスペーサーに結合させてもよい。また、工程（ｂ３）を工程（ｂ１）の前に行い、工程（ｂ１）において、親水性ポリマーが結合した１又は複数個のスペーサーをコアに結合させてもよい。 Although steps (b1) to (b5) can be performed in this order, it is not necessary to perform them in this order. For example, step (b1) and step (b2) may be performed at the same time, or step (b5) may be performed before or at the same time as step (b1), or step (b1) and step (b2) may be performed simultaneously. It may be carried out between the steps (b2) and (b3). Alternatively, step (b4) may be performed before step (b3), and in step (b3), the hydrophilic polymer to which the cell internalization promoter is bound may be bound to the spacer. Alternatively, step (b3) may be performed before step (b1), and in step (b1), one or more spacers to which a hydrophilic polymer is bonded may be bonded to the core.
　第一又は第二の側面に係るオリゴ核酸ナノ粒子を製造する上記方法のいずれの工程も、公知の方法を用いて行うことができる。公知の方法としては、例えば、アミノ基とカルボキシ基を、活性化基を用いて反応させてアミド結合を形成する方法、チオール基同士を、活性化基を用いて反応させてジスルフィド結合を形成する方法、チオール基とマレイミド基を反応させてチオエーテル結合を形成する方法、アジド基とアルキニル基から、触媒若しくは活性化基を用いたクリックケミストリーを活用して、トリアゾール結合を形成する方法、テトラジン、トリアジンなどの極めて電子不足な複素環と、ノルボルネン、ｔｒａｎｓ－シクロオクテン、シクロオクチンなどの歪んだ炭素多重結合を有する化合物とから、逆電子要請型ディールス・アルダー反応を活用して結合を形成する方法等が挙げられる。 Any step of the above method for producing oligonucleic acid nanoparticles according to the first or second aspect can be performed using a known method. Known methods include, for example, a method in which an amino group and a carboxy group are reacted using an activating group to form an amide bond, and a method in which thiol groups are reacted with each other using an activating group to form a disulfide bond. method, method of forming a thioether bond by reacting a thiol group and a maleimide group, method of forming a triazole bond from an azide group and an alkynyl group by utilizing click chemistry using a catalyst or an activated group, tetrazine, triazine A method of forming a bond using an inverse electron request type Diels-Alder reaction from an extremely electron-deficient heterocycle such as, and a compound having a distorted carbon multiple bond such as norbornene, trans-cyclooctene, or cyclooctyne, etc. can be mentioned.
　上記側面に係るオリゴ核酸ナノ粒子は、上記側面に係る方法以外の公知の方法で製造してもよい。 The oligonucleic acid nanoparticles according to the above aspect may be produced by a known method other than the method according to the above aspect.
　本発明の一側面は、上記側面に係るオリゴ核酸ナノ粒子を有効成分として含有する、医薬組成物である。医薬組成物には、薬学的に許容される添加剤が含まれる。本明細書において、「薬学的に許容される」とは、薬理学的又は毒性学的観点から哺乳動物に許容されることを指す。すなわち、「薬学的に許容される」物質とは、生理学的に許容可能であり、かつ哺乳動物に投与した場合、アレルギー性反応又は他の有害な若しくは毒性の反応を典型的には生じない物質を指す。「薬学的に許容される」物質は、哺乳動物、より具体的にはヒトにおける使用のために、一般的に認識される監督官庁によって認可されたか、一般的に認識される薬局方に列挙されている物質を意味する。「薬学的に許容される添加剤」は、医薬組成物を製剤化するためにオリゴ核酸ナノ粒子と一緒に使用される、薬理学的に不活性な材料を意味する。 One aspect of the present invention is a pharmaceutical composition containing the oligonucleic acid nanoparticle according to the above aspect as an active ingredient. Pharmaceutical compositions include pharmaceutically acceptable excipients. As used herein, "pharmaceutically acceptable" refers to being tolerated by mammals from a pharmacological or toxicological standpoint. That is, a "pharmaceutically acceptable" substance is one that is physiologically acceptable and that does not typically produce an allergic or other harmful or toxic reaction when administered to a mammal. refers to A “pharmaceutically acceptable” substance is one that has been approved by a generally recognized regulatory agency or listed in a generally recognized pharmacopoeia for use in mammals, and more specifically humans. means a substance that is "Pharmaceutically acceptable excipient" means a pharmacologically inert material used with oligonucleic acid nanoparticles to formulate a pharmaceutical composition.
　添加剤は、液体又は固体であってよい。添加剤は、所望の用量、軟度等の医薬組成物が得られるように、計画された投与方法を念頭において選択される。添加剤は特に限定されないが、例えば、水、生理食塩水、その他の水性溶媒、水性又は油性基材等の各種担体、賦形剤、結合剤、ｐＨ調整剤、崩壊剤、吸収促進剤、滑沢剤、着色剤、矯味剤、香料等が挙げられる。添加剤の配合割合は、医薬品分野において通常採用される範囲に基づいて、適宜設定することができる。 Additives may be liquid or solid. Excipients are selected with the intended method of administration in mind so as to obtain the desired dosage, consistency, etc. of the pharmaceutical composition. Additives are not particularly limited, but include, for example, water, physiological saline, other aqueous solvents, various carriers such as aqueous or oily bases, excipients, binders, pH adjusters, disintegrants, absorption enhancers, and lubricants. Examples include brighteners, coloring agents, flavoring agents, and fragrances. The blending ratio of additives can be appropriately set based on the range normally employed in the pharmaceutical field.
　医薬組成物は、例えば、注射のための無菌組成物であってよい。注射のための無菌組成物は、通常の製剤業務（例えば有効成分を注射用水、天然植物油等の溶媒に溶解又は懸濁させる等）に従って調製することができる。注射用の水性液としては、例えば、生理食塩水、ブドウ糖、又はその他の補助役（例えば、Ｄ－ソルビトール、Ｄ－マンニトール、ラクトース、スクロース、塩化ナトリウムなど）を含む等張液などが用いられる。注射用の水性液は、例えば、アルコール（例．エタノール）、ポリアルコール（例．プロピレングリコール又はポリエチレングリコール）、非イオン性界面活性剤（例．ポリソルベート８０ＴＭ又はＨＣＯ－５０）などの適当な溶解補助剤をさらに含んでよい。また、注射用の水性液は、緩衝剤（例えば、リン酸塩緩衝液又は酢酸ナトリウム緩衝液）、無痛化剤（例えば、塩化ベンザルコニウム、塩酸プロカインなど）、安定剤（例えば、ヒト血清アルブミン又はポリエチレングリコール）、保存剤（例えば、ベンジルアルコール、フェノールなど）、抗菌剤、分散剤、酸化防止剤など、当該技術分野においての公知の様々な材料を含んでもよい。注射剤は、例えば、凍結乾燥製剤であってよい。 The pharmaceutical composition may be, for example, a sterile composition for injection. Sterile compositions for injection can be prepared according to conventional pharmaceutical practice, such as dissolving or suspending the active ingredient in a solvent such as water for injection, natural vegetable oils, and the like. Examples of aqueous solutions for injection include physiological saline, glucose, or isotonic solutions containing other adjuvants (eg, D-sorbitol, D-mannitol, lactose, sucrose, sodium chloride, etc.). Aqueous solutions for injection may be prepared using suitable solubilizing agents, such as alcohols (e.g. ethanol), polyalcohols (e.g. propylene glycol or polyethylene glycol), nonionic surfactants (e.g. polysorbate 80TM or HCO-50), and the like. It may further contain an agent. Aqueous solutions for injection may also contain buffering agents (e.g., phosphate buffer or sodium acetate buffer), soothing agents (e.g., benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (e.g., human serum albumin or polyethylene glycol), preservatives (eg, benzyl alcohol, phenol, etc.), antimicrobial agents, dispersants, antioxidants, and the like, various materials known in the art. The injection may be, for example, a lyophilized preparation.
　本発明の上記側面に係るオリゴ核酸ナノ粒子又は医薬組成物は、特定の遺伝子産物が関与する疾患の治療及び／又は予防に利用することができる。特定の遺伝子産物が関与する疾患としては、例えば、先天性代謝異常症、先天性内分泌疾患、単一遺伝子疾患、神経変性疾患、神経疾患、筋疾患、髄膜炎、脳炎、脳症、ライソゾーム病、悪性新生物、繊維症、炎症性疾患、免疫不全疾患、自己免疫疾患、又は感染症が挙げられるが、疾患はこれらに限られない。したがって、本発明の一側面は、オリゴ核酸ナノ粒子を有効成分として含有する、上記疾患に対する治療剤又は予防剤である。 The oligonucleic acid nanoparticles or pharmaceutical compositions according to the above aspects of the present invention can be used for the treatment and/or prevention of diseases involving specific gene products. Diseases involving specific gene products include, for example, congenital metabolic disorders, congenital endocrine diseases, monogenic diseases, neurodegenerative diseases, neurological diseases, muscle diseases, meningitis, encephalitis, encephalopathy, lysosomal diseases, Diseases include, but are not limited to, malignant neoplasms, fibrosis, inflammatory diseases, immunodeficiency diseases, autoimmune diseases, or infectious diseases. Therefore, one aspect of the present invention is a therapeutic or preventive agent for the above-mentioned diseases, which contains oligonucleic acid nanoparticles as an active ingredient.
　本発明の別の一側面は、治療上の有効量のオリゴ核酸ナノ粒子を、ヒト又はヒト以外の動物に投与することを含む、上記疾患を治療及び／又は予防するための方法である。ヒトは、治療を必要とするヒト、すなわち患者であってよい。ヒト以外の動物は、霊長類などの温血哺乳動物；鳥類；ネコ、イヌ、ヒツジ、ヤギ、ウシ、ウマ、ブタなどの、家庭用の動物若しくは家畜；マウス、ラット、モルモットなどの実験動物；魚類；は虫類；動物園の動物：及び野生動物などを含む動物を意味する。投与方法は、経口、舌下、静脈内、動脈内、皮下、皮内、腹腔内、筋肉内、髄腔内、脳室内、鼻腔内、経粘膜、直腸、点眼、眼内、経肺、経皮、関節内、局所（皮膚）、毛包内、膣内、子宮内、腫瘍内、若しくはリンパ内投与、又はこれらの組合せであってよいが、これらに限られない。 Another aspect of the present invention is a method for treating and/or preventing the above-mentioned diseases, which comprises administering a therapeutically effective amount of oligonucleic acid nanoparticles to a human or non-human animal. The human may be a human in need of treatment, ie a patient. Animals other than humans include warm-blooded mammals such as primates; birds; domestic animals or livestock such as cats, dogs, sheep, goats, cows, horses, and pigs; laboratory animals such as mice, rats, and guinea pigs; Means animals including fish; reptiles; zoo animals; and wild animals. Administration methods include oral, sublingual, intravenous, intraarterial, subcutaneous, intradermal, intraperitoneal, intramuscular, intrathecal, intraventricular, intranasal, transmucosal, rectal, ophthalmic, intraocular, pulmonary, and transcutaneous. Administration may be, but is not limited to, cutaneous, intraarticular, topical (dermal), intrafollicular, intravaginal, intrauterine, intratumoral, or intralymphatic administration, or combinations thereof.
　本発明の別の一側面は、上記疾患の治療及び／又は予防に使用するためのオリゴ核酸ナノ粒子である。本発明の別の一側面は、上記疾患に対する治療剤及び／又は予防剤を製造するための、オリゴ核酸ナノ粒子の使用である。 Another aspect of the present invention is oligonucleic acid nanoparticles for use in the treatment and/or prevention of the above-mentioned diseases. Another aspect of the present invention is the use of oligonucleic acid nanoparticles for producing therapeutic and/or preventive agents for the above-mentioned diseases.
　本発明の上記側面に係るオリゴ核酸ナノ粒子又は医薬組成物は、１種以上の他の薬剤と併用することもできる。他の薬剤は、上述した特定の遺伝子産物が関与する疾患に対する１種以上の治療剤及び／又は予防剤であってよい。例えば、対象の疾患が悪性新生物の場合、他の薬剤の例としては、化学療法に使用できる医薬品が挙げられる。すなわち、本発明の一側面は、上記疾患に対する１種以上の治療剤及び／又は予防剤と併用して疾患を治療するための、オリゴ核酸ナノ粒子である。本発明の別の一側面は、オリゴ核酸ナノ粒子又は医薬組成物と、上記疾患に対する１種以上の治療剤及び／又は予防剤との組み合わせを含む、医薬である。しかしながら、本発明は、オリゴ核酸を細胞内に効率的に輸送できるプラットフォーム技術であり、オリゴ核酸を治療剤又は予防剤として適用できる疾患であれば、いかなる疾患にも使用可能であるから、他の薬剤は特定の薬剤に限られない。 The oligonucleic acid nanoparticles or pharmaceutical compositions according to the above aspects of the present invention can also be used in combination with one or more other drugs. Other drugs may be one or more therapeutic and/or prophylactic agents for diseases involving the specific gene products mentioned above. For example, if the disease of interest is malignant neoplasm, examples of other drugs include pharmaceuticals that can be used for chemotherapy. That is, one aspect of the present invention is oligonucleic acid nanoparticles for treating diseases in combination with one or more therapeutic agents and/or preventive agents for the above-mentioned diseases. Another aspect of the present invention is a medicament comprising a combination of an oligonucleic acid nanoparticle or a pharmaceutical composition and one or more therapeutic and/or prophylactic agents for the above-mentioned diseases. However, the present invention is a platform technology that can efficiently transport oligonucleic acids into cells, and can be used for any disease for which oligonucleic acids can be applied as a therapeutic or preventive agent. The drug is not limited to a specific drug.
　オリゴ核酸ナノ粒子若しくは医薬組成物及びこれと組み合わせて使用される上記他の薬剤の投与時期は限定されず、これらをヒト又はヒト以外の動物に対して、同時に投与してもよいし、適当な間隔をおいて投与してもよい。あるいは、上記側面に係る医薬組成物に上記他の薬剤を配合して、合剤を調製してもよい。上記他の薬剤の投与量及び配合量は、臨床上用いられている用量を基準として、適宜決定することができる。また、オリゴ核酸ナノ粒子若しくは医薬組成物と、上記他の薬剤との配合比は、投与対象、投与ルート、対象疾患、症状、他の薬剤の組み合わせ等により、適宜決定することができる。 The administration timing of the oligonucleic acid nanoparticles or pharmaceutical composition and the above-mentioned other drugs used in combination with the same is not limited, and they may be administered simultaneously to humans or non-human animals, or they may be administered at an appropriate time. Administration may be done at intervals. Alternatively, a combination drug may be prepared by blending the above-mentioned other drugs with the pharmaceutical composition according to the above aspect. The dosage and compounding amount of the above-mentioned other drugs can be appropriately determined based on the clinically used doses. Furthermore, the blending ratio of the oligonucleic acid nanoparticles or pharmaceutical composition and the above-mentioned other drugs can be determined as appropriate depending on the administration target, administration route, target disease, symptoms, combination of other drugs, etc.
　以下に実施例及び試験例を挙げて本発明を詳しく説明するが、本発明はこれら実施例に限定されるものではない。なお、以下の文中における「％」は、別段の定めがない限り、全て重量％を意味する。 The present invention will be explained in detail with reference to Examples and Test Examples below, but the present invention is not limited to these Examples. In addition, all "%" in the following text means weight % unless otherwise specified.
＜オリゴ核酸の合成＞
　表２に示すｓｉＲＮＡを準備した。ｓｉＲＮＡのセンス鎖ＲＮＡの３’末端には、ｓｐａｃｅｒ１８（ヘキサエチレングリコール）及びＣ６　ｌｉｎｋｅｒ（ヘキシル）を介してチオール基を結合させた。ｓｉＲＮＡのセンス鎖ＲＮＡの５’末端には、Ｃ６　ｌｉｎｋｅｒを介してアミノ基を結合させた。これらの核酸は株式会社ジーンデザインにより製造された。
Figure JPOXMLDOC01-appb-T000006
 <Synthesis of oligonucleic acid>
siRNA shown in Table 2 was prepared. A thiol group was bonded to the 3' end of the sense strand RNA of siRNA via spacer 18 (hexaethylene glycol) and C6 linker (hexyl). An amino group was bonded to the 5' end of the sense strand RNA of siRNA via a C6 linker. These nucleic acids were manufactured by Gene Design Co., Ltd.
Figure JPOXMLDOC01-appb-T000006
　ｓｉＲＮＡとエチレンジアミン四酢酸・３ナトリウム塩（ＥＤＴＡ　３Ｎａ）をｐＨ７．４の１０ｍＭリン酸緩衝生理食塩水（ＰＢＳ）に溶解し、さらにジチオトレイトール（ＤＴＴ）を添加した（終濃度：ＥＤＴＡ　０．５ｍＭ、ＤＴＴ　４０ｍＭ）。この溶液を２５℃で６時間加熱した後、ＰＢＳを用いて限外ろ過（分画分子量１０ｋＤａ）により６回精製した。得られた溶液の核酸濃度を、紫外可視分光光度計（テカン社製、Ｉｎｆｉｎｉｔｅ　Ｍ２００　ＰＲＯ）を用いて、２６０ｎｍにおける吸収の測定値から求めた。 siRNA and ethylenediaminetetraacetic acid trisodium salt (EDTA 3Na) were dissolved in 10mM phosphate buffered saline (PBS) at pH 7.4, and dithiothreitol (DTT) was added (final concentration: EDTA 0.5mM). , DTT 40mM). After heating this solution at 25° C. for 6 hours, it was purified six times by ultrafiltration (molecular weight cut off: 10 kDa) using PBS. The nucleic acid concentration of the obtained solution was determined from the measured value of absorption at 260 nm using an ultraviolet-visible spectrophotometer (manufactured by Tecan, Infinite M200 PRO).
＜実施例１．ｃＲＧＤ結合型オリゴ核酸ナノ粒子１の製造＞
（Ａ）ＮＨ２－ｓｉＲＮＡ－ＤＢＣＯの合成
　表２に示すｓｉＲＮＡの５．７４ｍＭ　ＰＢＳ溶液５８μＬに、ｐＨ７．０の１０ｍＭ　ＰＢＳを４１．９μＬ、及びｓｕｌｆｏ　ＤＢＣＯ－ＰＥＧ４－ｍａｌｅｉｍｉｄｅ（Ｂｒｏａｄｐｈａｒｍ社製）の１００ｍＭ　ＤＭＳＯ溶液を６６．６μＬ添加し、４℃で１４時間攪拌することによって、ｓｉＲＮＡのＳＨ基とｓｕｌｆｏ　ＤＢＣＯ－ＰＥＧ４－ｍａｌｅｉｍｉｄｅのマレイミド基とを反応させて、ＮＨ２－ｓｉＲＮＡ－ＤＢＣＯを得た。反応液にＰＢＳを３００μＬ添加して混合した後、ＰＢＳを用いて限外ろ過（メルク社製、Ａｍｉｃｏｎ　Ｕｌｔｒａ、分画分子量１０ｋＤａ）により６回精製した。回収した水溶液に純水を添加し、液の体積を６０μＬに調整した。 <Example 1. Production of cRGD-binding oligonucleic acid nanoparticles 1>
(A) Synthesis of NH2-siRNA-DBCO To 58 μL of a 5.74 mM PBS solution of the siRNA shown in Table 2, add 41.9 μL of 10 mM PBS at pH 7.0, and 100 mM DMSO of sulfo DBCO-PEG4-maleimide (manufactured by Broadpharm). By adding 66.6 μL of the solution and stirring at 4° C. for 14 hours, the SH group of siRNA and the maleimide group of sulfo DBCO-PEG4-maleimide were reacted to obtain NH2-siRNA-DBCO. After adding and mixing 300 μL of PBS to the reaction solution, the mixture was purified six times by ultrafiltration (manufactured by Merck & Co., Amicon Ultra, molecular weight cut off: 10 kDa) using PBS. Pure water was added to the collected aqueous solution to adjust the volume of the solution to 60 μL.
（Ｂ）ａｚｉｄｅ－ＰＥＧ１２　ＡＦ６　ＤＧＬ　Ｇ４の合成
　樹状ポリマーとしては、ＣＯＬＣＯＭ社製の、表面にアミノ基を有する第４世代ポリＬ－リシンデンドリグラフト（ＤＧＬ　Ｇ４、反応性官能基数：３６６）を使用した。ＤＧＬ　Ｇ４の５０ｍｇ／ｍＬジメチルスルホキシド（ＤＭＳＯ）溶液４μＬに、Ａｌｅｘａ　Ｆｌｕｏｒ　６４７　ＮＨＳ　ｅｓｔｅｒ（サーモフィッシャーサイエンティフィック社製）の８ｍＭ　ＤＭＳＯ溶液を１．９１μＬ、及びトリエチルアミン（ＴＥＡ）の１０ｖ／ｖ％ＤＭＳＯ溶液を２．３４μＬ添加し、室温で５時間撹拌した。次いで、この反応液にＡｚｉｄｏ－ＰＥＧ１２－ＮＨＳ　ｅｓｔｅｒ（Ｂｒｏａｄｐｈａｒｍ社製）の５０ｍＭ　ＤＭＳＯ溶液を６．１３μＬ添加し、さらに室温で１４時間撹拌した。次いで、ｍ－ｄＰＥＧ（登録商標）１２－ＮＨＳ　ｅｓｔｅｒ（Ｑｕａｎｔａ　ＢｉｏＤｅｓｉｇｎ社製）の２００ｍＭ　ＤＭＳＯ溶液を１１．２μＬ添加し、さらに室温で８時間撹拌した。以上のようにして、ＤＧＬ　Ｇ４のアミノ基と、Ａｚｉｄｏ－ＰＥＧ１２－ＮＨＳ　ｅｓｔｅｒ、アニオン性の蛍光色素であるＡｌｅｘａ　Ｆｌｕｏｒ　６４７　ＮＨＳ　ｅｓｔｅｒ、及びｍ－ｄＰＥＧ１２－ＮＨＳ　ｅｓｔｅｒのＮ－ヒドロキシこはく酸イミド（ＮＨＳ）基とを反応させることにより、ナノ粒子化合物ａｚｉｄｅ－ＰＥＧ１２　ＡＦ６　ＤＧＬ　Ｇ４を得た。反応液に純水を１０００μＬ添加して混合した後、純水を用いて限外ろ過（Ａｍｉｃｏｎ　Ｕｌｔｒａ、分画分子量３０ｋＤａ）により６回精製した。回収した水溶液に純水を添加し、液の体積を７５μＬに調整した。 (B) Synthesis of azide-PEG12 AF6 DGL G4 As the dendritic polymer, a fourth generation poly-L-lysine dendrigraft (DGL G4, number of reactive functional groups: 366) having amino groups on the surface manufactured by COLCOM was used. used. To 4 μL of a 50 mg/mL dimethyl sulfoxide (DMSO) solution of DGL G4, 1.91 μL of an 8 mM DMSO solution of Alexa Fluor 647 NHS ester (manufactured by Thermo Fisher Scientific), and a 10 v/v% DMSO solution of triethylamine (TEA). 2.34 μL of was added and stirred at room temperature for 5 hours. Next, 6.13 μL of a 50 mM DMSO solution of Azido-PEG12-NHS ester (manufactured by Broadpharm) was added to this reaction solution, and the mixture was further stirred at room temperature for 14 hours. Next, 11.2 μL of a 200 mM DMSO solution of m-dPEG (registered trademark) 12-NHS ester (manufactured by Quanta BioDesign) was added, and the mixture was further stirred at room temperature for 8 hours. As described above, the amino group of DGL G4, Azido-PEG12-NHS ester, anionic fluorescent dye Alexa Fluor 647 NHS ester, and N-hydroxysuccinimide (NHS) of m-dPEG12-NHS ester were combined. By reacting with the group, a nanoparticle compound azide-PEG12 AF6 DGL G4 was obtained. After adding 1000 μL of pure water to the reaction solution and mixing, the reaction solution was purified six times by ultrafiltration (Amicon Ultra, molecular weight cut off: 30 kDa) using pure water. Pure water was added to the collected aqueous solution to adjust the volume of the solution to 75 μL.
（Ｃ）ＮＨ２－ｓｉＲＮＡ－ＰＥＧ１２　ＡＦ６　ＤＧＬ　Ｇ４の合成
　（Ｂ）に示したａｚｉｄｅ－ＰＥＧ１２　ＡＦ６　ＤＧＬ　Ｇ４の水溶液１０μＬに対して、（Ａ）に示したＮＨ２－ｓｉＲＮＡ－ＤＢＣＯのＰＢＳ溶液１５．６μＬ、及びＤＭＳＯ　３．７７μＬを添加し、室温で攪拌した。次いで、３Ｍの塩化ナトリウム水溶液を、攪拌から３０分後に１．９９μＬ、攪拌から１時間後に２．２２μＬ、及び攪拌から２時間後に４．０９μＬ添加した後、さらに室温で１４時間攪拌した。以上のようにして、ａｚｉｄｅ－ＰＥＧ１２　ＡＦ６　ＤＧＬ　Ｇ４のアジド基とＮＨ２－ｓｉＲＮＡ－ＤＢＣＯのＤＢＣＯ基（ジベンゾシクロオクチン基）とを反応させた。反応液を、Ｈｉｐｒｅｐ　１６／６０　Ｓｅｐｈａｃｒｙｌ　Ｓ－２００　ＨＲ（Ｃｙｔｉｖａ社製）を用いて、ゲルろ過により精製した（溶離液：ＰＢＳ）。ｓｉＲＮＡが結合したＤＧＬ　Ｇ４を含むフラクションを回収し、限外ろ過（Ａｍｉｃｏｎ　Ｕｌｔｒａ、分画分子量５０ｋＤａ）を用いて溶媒を１００ｍＭ　ＰＢＳに置換した後、体積を１００μＬに調整した。 (C) Synthesis of NH2-siRNA-PEG12 AF6 DGL G4 For 10 μL of the azide-PEG12 AF6 DGL G4 aqueous solution shown in (B), 15.6 μL of the PBS solution of NH2-siRNA-DBCO shown in (A), and 3.77 μL of DMSO were added and stirred at room temperature. Next, 1.99 μL of a 3M sodium chloride aqueous solution was added 30 minutes after stirring, 2.22 μL after 1 hour from stirring, and 4.09 μL after 2 hours from stirring, and the mixture was further stirred at room temperature for 14 hours. As described above, the azide group of azide-PEG12 AF6 DGL G4 was reacted with the DBCO group (dibenzocyclooctyne group) of NH2-siRNA-DBCO. The reaction solution was purified by gel filtration using Hiprep 16/60 Sephacryl S-200 HR (manufactured by Cytiva) (eluent: PBS). A fraction containing siRNA-bound DGL G4 was collected, and the solvent was replaced with 100 mM PBS using ultrafiltration (Amicon Ultra, molecular weight cut off: 50 kDa), and the volume was adjusted to 100 μL.
（Ｄ）Ｎ３－ＰＥＧ２０００－ｓｉＲＮＡ－ＰＥＧ１２　ＡＦ６　ＤＧＬ　Ｇ４の合成
　（Ｃ）に示したＮＨ２－ｓｉＲＮＡ－ＰＥＧ１２　ＡＦ６　ＤＧＬ　Ｇ４のＰＢＳ溶液５０μＬに対して、ＤＭＳＯを１９．４μＬ、及びＮ３－ＰＥＧ－ＮＨＳ（Ｂｉｏｐｈａｒｍａ　ＰＥＧ　Ｓｃｉｅｎｔｉｆｉｃ社、ＰＥＧの数平均分子量２０００）の５０ｍＭ　ＤＭＳＯ溶液を１３．９μＬ添加し、室温で１７時間攪拌することによって、ＮＨ２－ｓｉＲＮＡ－ＰＥＧ１２　ＡＦ６　ＤＧＬ　Ｇ４のアミノ基とＮ３－ＰＥＧ－ＮＨＳのＮＨＳ基とを反応させた。反応液を、Ｈｉｐｒｅｐ　１６／６０　Ｓｅｐｈａｃｒｙｌ　Ｓ－２００　ＨＲを用いて、ゲルろ過により精製した（溶離液：ＰＢＳ）。ｓｉＲＮＡが結合したＤＧＬ　Ｇ４を含むフラクションを回収し、限外ろ過（Ａｍｉｃｏｎ　Ｕｌｔｒａ、分画分子量５０ｋＤａ）により濃縮し、液の体積を１００μＬに調整した。 (D) Synthesis of N3-PEG2000-siRNA-PEG12 AF6 DGL G4 To 50 μL of the PBS solution of NH2-siRNA-PEG12 AF6 DGL G4 shown in (C), 19.4 μL of DMSO and N3-PEG-NHS ( By adding 13.9 μL of 50 mM DMSO solution of Biopharma PEG Scientific, PEG number average molecular weight 2000) and stirring at room temperature for 17 hours, the amino groups of NH2-siRNA-PEG12 AF6 DGL G4 and N3-PEG-NHS were separated. Reacted with NHS group. The reaction solution was purified by gel filtration using Hiprep 16/60 Sephacryl S-200 HR (eluent: PBS). A fraction containing DGL G4 bound to siRNA was collected and concentrated by ultrafiltration (Amicon Ultra, molecular weight cut off: 50 kDa), and the volume of the liquid was adjusted to 100 μL.
（Ｅ）ｃＲＧＤ－ＤＢＣＯの合成
　Ｃｙｃｌｏ（－ＲＧＤｆＫ）（Ｃｈｅｍｓｃｅｎｅ社製）の２００ｍＭ　ＤＭＳＯ溶液６５．４μＬに対して、ＤＢＣＯ－ＮＨＣＯ－ＰＥＧ４－ＮＨＳ（ＢｒｏａｄＰｈａｒｍ社製）の３００ｍＭ　ＤＭＳＯ溶液を４３．６μＬと、ＴＥＡ　５．５μＬを添加し、２５℃で２５時間撹拌することによって、Ｃｙｃｌｏ（－ＲＧＤｆＫ）の有するアミノ基と、ＤＢＣＯ－ＮＨＣＯ－ＰＥＧ４－ＮＨＳの有するＮＨＳ基とを反応させた。反応液を４５℃で加熱しながら溶媒を減圧除去して濃縮し、逆相ＨＰＬＣ（カラム：Ｗａｔｅｒｓ社製Ｘｂｒｉｄｇｅ　Ｐｅｐｔｉｄｅ　ＢＥＨ　Ｃ１８，３００Å，４．６×１００ｍｍ、溶離液Ａ：０．１ｖ／ｖ％トリフルオロ酢酸（ＴＦＡ）／アセトニトリル（９０／１０；ｖ／ｖ）、溶離液Ｂ：０．１ｖ／ｖ％ＴＦＡ／アセトニトリル（１０／９０；ｖ／ｖ））を用いて精製を行った。回収した溶液を４５℃で加熱しながら溶媒を減圧除去した後、ＤＭＳＯを添加して５０ｍＭの濃度に調整した。 (E) Synthesis of cRGD-DBCO To 65.4 μL of a 200 mM DMSO solution of Cyclo(-RGDfK) (manufactured by Chemscene), add 43.6 μL of a 300 mM DMSO solution of DBCO-NHCO-PEG4-NHS (manufactured by BroadPharm). , TEA (5.5 μL) was added and stirred at 25° C. for 25 hours to cause the amino group of Cyclo(-RGDfK) to react with the NHS group of DBCO-NHCO-PEG4-NHS. While heating the reaction solution at 45°C, the solvent was removed under reduced pressure and concentrated, followed by reverse phase HPLC (column: Waters Xbridge Peptide BEH C18, 300 Å, 4.6 x 100 mm, eluent A: 0.1 v/v% Purification was performed using trifluoroacetic acid (TFA)/acetonitrile (90/10; v/v), eluent B: 0.1 v/v% TFA/acetonitrile (10/90; v/v). After removing the solvent under reduced pressure while heating the recovered solution at 45° C., DMSO was added to adjust the concentration to 50 mM.
（Ｆ）ｃＲＧＤ－ＰＥＧ２０００－ｓｉＲＮＡ－ＰＥＧ１２　ＡＦ６　ＤＧＬ　Ｇ４の合成
　（Ｄ）に示したＮ３－ＰＥＧ２０００－ｓｉＲＮＡ－ＰＥＧ１２　ＡＦ６　ＤＧＬ　Ｇ４のＰＢＳ溶液１００μＬに対して、ＤＭＳＯを０．２６μＬ、及び（Ｅ）に示したｃＲＧＤ－ＤＢＣＯの４ｍＭ　ＤＭＳＯ溶液１０．９μＬを添加し、室温で１５時間攪拌することによって、Ｎ３－ＰＥＧ２０００－ｓｉＲＮＡ－ＰＥＧ１２　ＡＦ６　ＤＧＬ　Ｇ４のアジド基とｃＲＧＤ－ＤＢＣＯのＤＢＣＯ基とを反応させた。反応液を、Ｚｅｂａ（登録商標）　Ｓｐｉｎ　Ｄｅｓａｌｔｉｎｇ　Ｃｏｌｕｍｎ（サーモフィッシャーサイエンティフィック社製、分子量分画：４０ｋＤａ）を用いて精製した。次いで、ＰＢＳを用いて限外ろ過（Ａｍｉｃｏｎ　Ｕｌｔｒａ、分画分子量５０ｋＤａ）により３回精製を行い、液の体積を８５μＬに調整して、オリゴ核酸ナノ粒子ｃＲＧＤ－ＰＥＧ２０００－ｓｉＲＮＡ－ＰＥＧ１２　ＡＦ６　ＤＧＬ　Ｇ４のＰＢＳ溶液を得た。 (F) Synthesis of cRGD-PEG2000-siRNA-PEG12 AF6 DGL G4 Add 0.26 μL of DMSO to 100 μL of the PBS solution of N3-PEG2000-siRNA-PEG12 AF6 DGL G4 shown in (D), and (E). The azide group of N3-PEG2000-siRNA-PEG12 AF6 DGL G4 and the DBCO group of cRGD-DBCO were reacted by adding 10.9 μL of the indicated 4mM DMSO solution of cRGD-DBCO and stirring at room temperature for 15 hours. . The reaction solution was purified using Zeba (registered trademark) Spin Desalting Column (manufactured by Thermo Fisher Scientific, molecular weight fraction: 40 kDa). Next, purification was performed three times by ultrafiltration (Amicon Ultra, molecular weight cut off: 50 kDa) using PBS, and the volume of the solution was adjusted to 85 μL, and oligonucleic acid nanoparticles cRGD-PEG2000-siRNA-PEG12 AF6 DGL G4 were purified. A PBS solution was obtained.
＜実施例２．ｃＲＧＤ結合型オリゴ核酸ナノ粒子２の製造＞
（Ａ）ａｚｉｄｅ－ＰＥＧ１２　ＡＦ６　ＤＧＬ　Ｇ４の合成
　実施例１の（Ｂ）に準じて、ａｚｉｄｅ－ＰＥＧ１２　ＡＦ６　ＤＧＬ　Ｇ４を合成した。ただし、ａｚｉｄｏ－ＰＥＧ１２－ＮＨＳ　ｅｓｔｅｒの５０ｍＭ　ＤＭＳＯ溶液の添加量は、６．１３μＬから１２．３μＬに変更した。 <Example 2. Production of cRGD-binding oligonucleic acid nanoparticles 2>
(A) Synthesis of azide-PEG12 AF6 DGL G4 According to (B) of Example 1, azide-PEG12 AF6 DGL G4 was synthesized. However, the amount of 50 mM DMSO solution of azido-PEG12-NHS ester added was changed from 6.13 μL to 12.3 μL.
（Ｂ）ＮＨ２－ｓｉＲＮＡ－ＰＥＧ１２　ＡＦ６　ＤＧＬ　Ｇ４の合成
　（Ａ）に示したａｚｉｄｅ－ＰＥＧ１２　ＡＦ６　ＤＧＬ　Ｇ４の水溶液１０μＬに対して、実施例１の（Ａ）に示したＮＨ２－ｓｉＲＮＡ－ＤＢＣＯのＰＢＳ溶液３１．３μＬ、及びＤＭＳＯ　６．０２μＬを添加し、室温で攪拌した。次いで、３Ｍの塩化ナトリウム水溶液を、攪拌から３０分後に２．８６μＬ、攪拌から１時間後に３．５６μＬ、及び攪拌から２時間後に６．５３μＬ添加した後、さらに室温で１４時間攪拌した。以上のようにして、ａｚｉｄｅ－ＰＥＧ１２　ＡＦ６　ＤＧＬ　Ｇ４のアジド基とＮＨ２－ｓｉＲＮＡ－ＤＢＣＯのＤＢＣＯ基とを反応させた。反応液を、Ｈｉｐｒｅｐ　１６／６０　Ｓｅｐｈａｃｒｙｌ　Ｓ－２００　ＨＲを用いて、ゲルろ過により精製した（溶離液：ＰＢＳ）。ｓｉＲＮＡが結合したＤＧＬ　Ｇ４を含むフラクションを回収し、限外ろ過（Ａｍｉｃｏｎ　Ｕｌｔｒａ、分画分子量５０ｋＤａ）を用いて溶媒を１００ｍＭ　ＰＢＳに置換した後、体積を１００μＬに調整した。 (B) Synthesis of NH2-siRNA-PEG12 AF6 DGL G4 A PBS solution of NH2-siRNA-DBCO shown in (A) of Example 1 was added to 10 μL of the azide-PEG12 AF6 DGL G4 aqueous solution shown in (A). 31.3 μL and 6.02 μL of DMSO were added, and the mixture was stirred at room temperature. Next, 2.86 μL of a 3M sodium chloride aqueous solution was added 30 minutes after stirring, 3.56 μL after 1 hour from stirring, and 6.53 μL after 2 hours from stirring, and the mixture was further stirred at room temperature for 14 hours. As described above, the azide group of azide-PEG12 AF6 DGL G4 was reacted with the DBCO group of NH2-siRNA-DBCO. The reaction solution was purified by gel filtration using Hiprep 16/60 Sephacryl S-200 HR (eluent: PBS). A fraction containing siRNA-bound DGL G4 was collected, and the solvent was replaced with 100 mM PBS using ultrafiltration (Amicon Ultra, molecular weight cut off: 50 kDa), and the volume was adjusted to 100 μL.
（Ｃ）Ｎ３－ＰＥＧ２０００－ｓｉＲＮＡ－ＰＥＧ１２　ＡＦ６　ＤＧＬ　Ｇ４の合成
　（Ｂ）に示したＮＨ２－ｓｉＲＮＡ－ＰＥＧ１２　ＡＦ６　ＤＧＬ　Ｇ４を用いて、実施例１の（Ｄ）に準じて、Ｎ３－ＰＥＧ２０００－ｓｉＲＮＡ－ＰＥＧ１２　ＡＦ６　ＤＧＬ　Ｇ４を合成した。ただし、ＤＭＳＯを１９．４μＬとＮ３－ＰＥＧ－ＮＨＳの５０ｍＭ　ＤＭＳＯ溶液を１３．９μＬ添加する代わりに、ＤＭＳＯを５．５６μＬとＮ３－ＰＥＧ－ＮＨＳの５０ｍＭ　ＤＭＳＯ溶液を２７．８μＬ添加した。 (C) Synthesis of N3-PEG2000-siRNA-PEG12 AF6 DGL G4 Using NH2-siRNA-PEG12 AF6 DGL G4 shown in (B), N3-PEG2000-siRNA- PEG12 AF6 DGL G4 was synthesized. However, instead of adding 19.4 μL of DMSO and 13.9 μL of a 50 mM DMSO solution of N3-PEG-NHS, 5.56 μL of DMSO and 27.8 μL of a 50 mM DMSO solution of N3-PEG-NHS were added.
（Ｄ）ｃＲＧＤ－ＰＥＧ２０００－ｓｉＲＮＡ－ＰＥＧ１２　ＡＦ６　ＤＧＬ　Ｇ４の合成
　（Ｃ）に示したＮ３－ＰＥＧ２０００－ｓｉＲＮＡ－ＰＥＧ１２　ＡＦ６　ＤＧＬ　Ｇ４と、実施例１の（Ｅ）に示したｃＲＧＤ－ＤＢＣＯを用いて、実施例１の（Ｆ）に準じて、ｃＲＧＤ－ＰＥＧ２０００－ｓｉＲＮＡ－ＰＥＧ１２　ＡＦ６　ＤＧＬ　Ｇ４を合成した。 (D) Synthesis of cRGD-PEG2000-siRNA-PEG12 AF6 DGL G4 Using N3-PEG2000-siRNA-PEG12 AF6 DGL G4 shown in (C) and cRGD-DBCO shown in (E) of Example 1, According to (F) of Example 1, cRGD-PEG2000-siRNA-PEG12 AF6 DGL G4 was synthesized.
＜実施例３．ｃＲＧＤ結合型オリゴ核酸ナノ粒子３の製造＞
（Ａ）ＳＰＤＰ　ＡＦ６　ＤＧＬ　Ｇ４の合成
　ＤＧＬ　Ｇ４の５０ｍｇ／ｍＬ　ＤＭＳＯ溶液８μＬに、Ａｌｅｘａ　Ｆｌｕｏｒ（登録商標）　６４７　ＮＨＳ　ｅｓｔｅｒの８ｍＭ　ＤＭＳＯ溶液を３．０６μＬ、ＰＥＧ１２－ＳＰＤＰ（サーモフィッシャーサイエンティフィック社製）の６０ｍＭ　ＤＭＳＯ溶液を８．１７μＬ、及びＴＥＡの１０ｖ／ｖ％ＤＭＳＯ溶液を６．２５μＬ添加し、室温で２２時間撹拌した。次いで、この反応液にＭＳ（ＰＥＧ）１２　Ｍｅｔｈｙｌ－ＰＥＧ－ＮＨＳ－Ｅｓｔｅｒ　Ｒｅａｇｅｎｔ（サーモフィッシャーサイエンティフィック社製）の２００ｍＭ　ＤＭＳＯ溶液を２２．４μＬ添加し、さらに室温で８時間撹拌した。以上のようにして、ＤＧＬ　Ｇ４のアミノ基と、Ａｌｅｘａ　Ｆｌｕｏｒ　６４７　ＮＨＳ　ｅｓｔｅｒ、ＰＥＧ１２－ＳＰＤＰ及びＭＳ（ＰＥＧ）１２　Ｍｅｔｈｙｌ－ＰＥＧ－ＮＨＳ－Ｅｓｔｅｒ　ＲｅａｇｅｎｔのＮＨＳ基とを反応させることにより、ナノ粒子化合物ＳＰＤＰ　ＡＦ６　ＤＧＬ　Ｇ４を得た。反応液に純水を１０００μＬ添加して混合した後、４０ｖ／ｖ％ＤＭＳＯ水溶液を用いて限外ろ過（Ａｍｉｃｏｎ　Ｕｌｔｒａ、分画分子量１００ｋＤａ）により６回精製した。回収した溶液に４０ｖ／ｖ％ＤＭＳＯ水溶液を添加し、液の体積を１４０μＬに調整した。 <Example 3. Production of cRGD-binding oligonucleic acid nanoparticles 3>
(A) Synthesis of SPDP AF6 DGL G4 To 8 μL of a 50 mg/mL DMSO solution of DGL G4, add 3.06 μL of an 8 mM DMSO solution of Alexa Fluor (registered trademark) 647 NHS ester to PEG12-SPDP (manufactured by Thermo Fisher Scientific). 8.17 μL of a 60 mM DMSO solution of TEA and 6.25 μL of a 10 v/v% DMSO solution of TEA were added, and the mixture was stirred at room temperature for 22 hours. Next, 22.4 μL of a 200 mM DMSO solution of MS(PEG)12 Methyl-PEG-NHS-Ester Reagent (manufactured by Thermo Fisher Scientific) was added to this reaction solution, and the mixture was further stirred at room temperature for 8 hours. As described above, by reacting the amino group of DGL G4 with the NHS group of Alexa Fluor 647 NHS ester, PEG12-SPDP and MS(PEG)12 Methyl-PEG-NHS-Ester Reagent, the nanoparticle compound SPDP Obtained AF6 DGL G4. After adding and mixing 1000 μL of pure water to the reaction solution, the mixture was purified six times by ultrafiltration (Amicon Ultra, molecular weight cut off: 100 kDa) using a 40 v/v% DMSO aqueous solution. A 40 v/v% DMSO aqueous solution was added to the collected solution, and the volume of the liquid was adjusted to 140 μL.
（Ｂ）ＮＨ２－ｓｉＲＮＡ　ＡＦ６　ＤＧＬ　Ｇ４の合成
　（Ａ）に示したＳＰＤＰ　ＡＦ６　ＤＧＬ　Ｇ４溶液１００μＬに、表２に示すｓｉＲＮＡの６．０ｍＭ　ＰＢＳ溶液２３３μＬ、及びＤＭＳＯ１４．５μＬを添加し、室温で撹拌した。次いで、撹拌から３０分後に３Ｍの塩化ナトリウム水溶液を９．８μＬ及びＤＭＳＯを３．２μＬ、撹拌から１時間後に３Ｍの塩化ナトリウム水溶液を１２．６μＬ及びＤＭＳＯを４．２μＬ、撹拌から１．５時間後に３Ｍの塩化ナトリウム水溶液を１８．８μＬ及びＤＭＳＯを６．２μＬ添加した後、さらに室温で１６．５時間撹拌した。以上のようにして、ＳＰＤＰ　ＡＦ６　ＤＧＬ　Ｇ４の有するピリジルジスルフィド基とｓｉＲＮＡの有するＳＨ基を反応させた。反応液を、Ｈｉｐｒｅｐ　２６／６０　Ｓｅｐｈａｃｒｙｌ　Ｓ－２００　ＨＲを用いて、ゲルろ過により精製した（溶離液：ＰＢＳ）。ｓｉＲＮＡが結合したＤＧＬ　Ｇ４を含むフラクションを回収し、限外ろ過（Ａｍｉｃｏｎ　Ｕｌｔｒａ、分画分子量１００ｋＤａ）を用いて、体積を５００μＬに調整した。 (B) Synthesis of NH2-siRNA AF6 DGL G4 To 100 μL of the SPDP AF6 DGL G4 solution shown in (A), 233 μL of a 6.0 mM PBS solution of siRNA shown in Table 2 and 14.5 μL of DMSO were added, and the mixture was stirred at room temperature. . Then, 30 minutes after stirring, 9.8 μL of 3M sodium chloride aqueous solution and 3.2 μL of DMSO, and 1 hour after stirring, 12.6 μL of 3M sodium chloride aqueous solution and 4.2 μL of DMSO, 1.5 hours after stirring. After adding 18.8 μL of 3M aqueous sodium chloride solution and 6.2 μL of DMSO, the mixture was further stirred at room temperature for 16.5 hours. As described above, the pyridyl disulfide group of SPDP AF6 DGL G4 was reacted with the SH group of siRNA. The reaction solution was purified by gel filtration using Hiprep 26/60 Sephacryl S-200 HR (eluent: PBS). A fraction containing DGL G4 bound to siRNA was collected, and the volume was adjusted to 500 μL using ultrafiltration (Amicon Ultra, molecular weight cut off: 100 kDa).
（Ｃ）Ｎ３－ｓｉＲＮＡ　ＡＦ６　ＤＧＬ　Ｇ４の合成
　（Ｂ）に示したＮＨ２－ｓｉＲＮＡ　ＡＦ６　ＤＧＬ　Ｇ４溶液４９５μＬに、ａｚｉｄｏ－ＰＥＧ４－ＮＨＳ　ｅｓｔｅｒ（Ｂｒｏａｄｐｈａｒｍ社製）の２００ｍＭ　ＤＭＳＯ溶液１３９μＬ、及びＰＢＳ　５９μＬを添加し、室温で１８時間撹拌した。以上のようにして、ＮＨ２－ｓｉＲＮＡ　ＡＦ６　ＤＧＬ　Ｇ４の有するアミノ基とａｚｉｄｏ－ＰＥＧ４－ＮＨＳ　ｅｓｔｅｒの有するＮＨＳ基を反応させた。反応液を、ＮＡＰ－１０　Ｃｏｌｕｍｎ（Ｃｙｔｉｖａ社製）を用いて精製した。次いで、回収液をＨＰＬＣ（カラム：アジレント社製Ｂｉｏ　ＳＥＣ－５，１０００Å，７．８×３００ｍｍ、溶離液：ＰＢＳ）を用いて精製した。回収液を限外ろ過（Ａｍｉｃｏｎ　Ｕｌｔｒａ、分画分子量１００ｋＤａ）により濃縮し、液の体積を２５０μＬに調整した。 (C) Synthesis of N3-siRNA AF6 DGL G4 To 495 μL of the NH2-siRNA AF6 DGL G4 solution shown in (B), 139 μL of a 200 mM DMSO solution of azido-PEG4-NHS ester (manufactured by Broadpharm) and 59 μL of PBS were added. Added and stirred at room temperature for 18 hours. As described above, the amino group of NH2-siRNA AF6 DGL G4 was reacted with the NHS group of azido-PEG4-NHS ester. The reaction solution was purified using NAP-10 Column (manufactured by Cytiva). The recovered solution was then purified using HPLC (column: Agilent Bio SEC-5, 1000 Å, 7.8 x 300 mm, eluent: PBS). The recovered liquid was concentrated by ultrafiltration (Amicon Ultra, molecular weight cut off: 100 kDa), and the volume of the liquid was adjusted to 250 μL.
（Ｄ）ＨＯＯＣ－ＰＥＧ２０００－ｓｉＲＮＡ　ＡＦ６　ＤＧＬ　Ｇ４の合成
　（Ｃ）に示したＮ３－ｓｉＲＮＡ　ＡＦ６　ＤＧＬ　Ｇ４溶液４８．５μＬに、ＤＢＣＯ－ＰＥＧ－ＳＣ（分子量２０００、Ｂｉｏｐｈａｒｍａ　ＰＥＧ　Ｓｃｉｅｎｔｉｆｉｃ社製）の２０ｍＭ　ＤＭＳＯ溶液３４．０μＬ、及びＰＢＳ　２．４μＬを添加し、室温で１７時間撹拌した。以上のようにして、Ｎ３－ｓｉＲＮＡ　ＡＦ６　ＤＧＬ　Ｇ４の有するアジド基とＤＢＣＯ－ＰＥＧ－ＳＣの有するＤＢＣＯ基を反応させた。また、ＤＢＣＯ－ＰＥＧ－ＳＣの有するＮＨＳ基をＣＯＯＨ基に変換した。反応液を、ＰＢＳを用いて限外ろ過（Ａｍｉｃｏｎ　Ｕｌｔｒａ、分画分子量１００ｋＤａ）により６回精製した。回収した溶液にＰＢＳを添加し、液の体積を８０μＬに調整した。 (D) Synthesis of HOOC-PEG2000-siRNA AF6 DGL G4 Add 20 mM DM of DBCO-PEG-SC (molecular weight 2000, manufactured by Biopharma PEG Scientific) to 48.5 μL of the N3-siRNA AF6 DGL G4 solution shown in (C). SO solution 34.0 μL and 2.4 μL of PBS were added, and the mixture was stirred at room temperature for 17 hours. As described above, the azide group of N3-siRNA AF6 DGL G4 was reacted with the DBCO group of DBCO-PEG-SC. Furthermore, the NHS group of DBCO-PEG-SC was converted to a COOH group. The reaction solution was purified six times by ultrafiltration (Amicon Ultra, molecular weight cut off: 100 kDa) using PBS. PBS was added to the collected solution and the volume of the solution was adjusted to 80 μL.
（Ｅ）Ｎ３－ＰＥＧ２０００－ｓｉＲＮＡ　ＡＦ６　ＤＧＬ　Ｇ４の合成
　（Ｄ）に示したＨＯＯＣ－ＰＥＧ２０００－ｓｉＲＮＡ　ＡＦ６　ＤＧＬ　Ｇ４溶液３５μＬに、Ａｚｉｄｏ－ＰＥＧ４－ａｍｉｎｅ（東京化成工業社製）のＤＭＳＯ溶液１．５μＬ、ＮＨＳ（富士フイルム和光純薬社製）の３００ｍＭ　ＤＭＳＯ溶液３．０μＬ、及び１－エチル－３－（３－ジメチルアミノプロピル）カルボジイミド塩酸塩（ＥＤＣ－ＨＣｌ、ナカライテスク社製）の３００ｍＭ　ＤＭＳＯ溶液３．０μＬを添加し、室温で１７時間攪拌した。以上のようにして、Ａｚｉｄｏ－ＰＥＧ４－ａｍｉｎｅの有するアミノ基とＨＯＯＣ－ＰＥＧ２０００－ｓｉＲＮＡ　ＡＦ６　ＤＧＬ　Ｇ４の有するＣＯＯＨ基とを反応させた。反応液を、ＰＢＳを用いて限外ろ過（Ａｍｉｃｏｎ　Ｕｌｔｒａ、分画分子量１００ｋＤａ）により６回精製した。回収した溶液にＰＢＳを添加し、液の体積を８０μＬに調整した。 (E) Synthesis of N3-PEG2000-siRNA AF6 DGL G4 To 35 μL of the HOOC-PEG2000-siRNA AF6 DGL G4 solution shown in (D), 1.5 μL of a DMSO solution of Azido-PEG4-amine (manufactured by Tokyo Chemical Industry Co., Ltd.), 3.0 μL of a 300mM DMSO solution of NHS (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) and a 300mM DMSO solution of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC-HCl, manufactured by Nacalai Tesque) 3 .0 μL was added and stirred at room temperature for 17 hours. As described above, the amino group of Azido-PEG4-amine and the COOH group of HOOC-PEG2000-siRNA AF6 DGL G4 were reacted. The reaction solution was purified six times by ultrafiltration (Amicon Ultra, molecular weight cut off: 100 kDa) using PBS. PBS was added to the collected solution and the volume of the solution was adjusted to 80 μL.
（Ｆ）ｃＲＧＤ－ＰＥＧ２０００－ｓｉＲＮＡ　ＡＦ６　ＤＧＬ　Ｇ４の合成
　（Ｅ）に示したＮ３－ＰＥＧ２０００－ｓｉＲＮＡ　ＡＦ６　ＤＧＬ　Ｇ４溶液６０μＬに、実施例１（Ｅ）に示したｃＲＧＤ－ＤＢＣＯの５ｍＭ　ＤＭＳＯ溶液２．２μＬ及びＤＭＳＯ４．４μＬを添加し、室温で７時間撹拌した。反応液を、ＮＡＰ－５　Ｃｏｌｕｍｎ（Ｃｙｔｉｖａ社製）を用いて精製した。次いで、回収液を限外ろ過（Ａｍｉｃｏｎ　Ｕｌｔｒａ、分画分子量１００ｋＤａ）により濃縮し、液の体積を２５０μＬに調整することで、オリゴ核酸ナノ粒子ｃＲＧＤ－ＰＥＧ２０００－ｓｉＲＮＡ　ＡＦ６　ＤＧＬ　Ｇ４のＰＢＳ溶液を得た。 (F) Synthesis of cRGD-PEG2000-siRNA AF6 DGL G4 Add 2.2 μL of the 5mM DMSO solution of cRGD-DBCO shown in Example 1 (E) to 60 μL of the N3-PEG2000-siRNA AF6 DGL G4 solution shown in (E). and 4.4 μL of DMSO were added, and the mixture was stirred at room temperature for 7 hours. The reaction solution was purified using NAP-5 Column (manufactured by Cytiva). Next, the recovered solution was concentrated by ultrafiltration (Amicon Ultra, molecular weight cut off 100 kDa), and the volume of the solution was adjusted to 250 μL to obtain a PBS solution of oligonucleic acid nanoparticles cRGD-PEG2000-siRNA AF6 DGL G4. .
＜実施例４．ｃＲＧＤ結合型オリゴ核酸ナノ粒子４の製造＞
（Ａ）ＨＯＯＣ－ＰＥＧ５０００－ｓｉＲＮＡ　ＡＦ６　ＤＧＬ　Ｇ４の合成
　実施例３（Ｄ）に準じて、ＨＯＯＣ－ＰＥＧ５０００－ｓｉＲＮＡ　ＡＦ６　ＤＧＬ　Ｇ４のＰＢＳ溶液を得た。ただし、ＤＢＣＯ－ＰＥＧ－ＳＣ（分子量２０００）の代わりに、ＤＢＣＯ－ＰＥＧ－ＳＣ（分子量５０００、Ｂｉｏｐｈａｒｍａ　ＰＥＧ　Ｓｃｉｅｎｔｉｆｉｃ社製）を用いた。 <Example 4. Production of cRGD-binding oligonucleic acid nanoparticles 4>
(A) Synthesis of HOOC-PEG5000-siRNA AF6 DGL G4 According to Example 3 (D), a PBS solution of HOOC-PEG5000-siRNA AF6 DGL G4 was obtained. However, DBCO-PEG-SC (molecular weight 5000, manufactured by Biopharma PEG Scientific) was used instead of DBCO-PEG-SC (molecular weight 2000).
（Ｂ）Ｎ３－ＰＥＧ５０００－ｓｉＲＮＡ　ＡＦ６　ＤＧＬ　Ｇ４の合成
　実施例３（Ｅ）に準じて、Ｎ３－ＰＥＧ５０００－ｓｉＲＮＡ　ＡＦ６　ＤＧＬ　Ｇ４のＰＢＳ溶液を得た。ただし、実施例３（Ｄ）のＨＯＯＣ－ＰＥＧ２０００－ｓｉＲＮＡ　ＡＦ６　ＤＧＬ　Ｇ４の代わりに、（Ａ）のＨＯＯＣ－ＰＥＧ５０００－ｓｉＲＮＡ　ＡＦ６　ＤＧＬ　Ｇ４を用いた。 (B) Synthesis of N3-PEG5000-siRNA AF6 DGL G4 According to Example 3(E), a PBS solution of N3-PEG5000-siRNA AF6 DGL G4 was obtained. However, instead of HOOC-PEG2000-siRNA AF6 DGL G4 in Example 3 (D), HOOC-PEG5000-siRNA AF6 DGL G4 in (A) was used.
（Ｃ）ｃＲＧＤ－ＰＥＧ５０００－ｓｉＲＮＡ　ＡＦ６　ＤＧＬ　Ｇ４の合成
　実施例３（Ｆ）に準じて、オリゴ核酸ナノ粒子ｃＲＧＤ－ＰＥＧ５０００－ｓｉＲＮＡ　ＡＦ６　ＤＧＬ　Ｇ４のＰＢＳ溶液を得た。ただし、実施例３（Ｅ）のＮ３－ＰＥＧ２０００－ｓｉＲＮＡ　ＡＦ６　ＤＧＬ　Ｇ４の代わりに、（Ｂ）のＮ３－ＰＥＧ５０００－ｓｉＲＮＡ　ＡＦ６　ＤＧＬ　Ｇ４を用いた。 (C) Synthesis of cRGD-PEG5000-siRNA AF6 DGL G4 According to Example 3 (F), a PBS solution of oligonucleic acid nanoparticles cRGD-PEG5000-siRNA AF6 DGL G4 was obtained. However, instead of N3-PEG2000-siRNA AF6 DGL G4 in Example 3 (E), N3-PEG5000-siRNA AF6 DGL G4 in (B) was used.
＜比較例１．細胞非結合型オリゴ核酸ナノ粒子１の製造＞
　実施例３（Ｄ）に準じて、オリゴ核酸ナノ粒子ｍＰＥＧ５０００－ｓｉＲＮＡ　ＡＦ６　ＤＧＬ　Ｇ４のＰＢＳ溶液を得た。ただし、実施例３（Ｃ）のＮ３－ｓｉＲＮＡ　ＡＦ６　ＤＧＬ　Ｇ４溶液４８．５μＬに、ＤＢＣＯ－ＰＥＧ－ＳＣ（分子量２０００）の２０ｍＭ　ＤＭＳＯ溶液３４．０μＬ、及びＰＢＳ２．４μＬを添加する代わりに、実施例３（Ｃ）のＮ３－ｓｉＲＮＡ　ＡＦ６　ＤＧＬ　Ｇ４溶液１８９μＬに、ＤＢＣＯ－ｍＰＥＧ（分子量５０００、Ｂｒｏａｄｐｈａｒｍａ社製）の２０ｍＭ　ＤＭＳＯ溶液１３２μＬ、及びＰＢＳ９．４μＬを添加した。 <Comparative example 1. Production of cell-non-binding oligonucleic acid nanoparticles 1>
According to Example 3 (D), a PBS solution of oligonucleic acid nanoparticles mPEG5000-siRNA AF6 DGL G4 was obtained. However, instead of adding 34.0 μL of a 20 mM DMSO solution of DBCO-PEG-SC (molecular weight 2000) and 2.4 μL of PBS to 48.5 μL of the N3-siRNA AF6 DGL G4 solution in Example 3(C), Example To 189 μL of the N3-siRNA AF6 DGL G4 solution in 3(C), 132 μL of a 20 mM DMSO solution of DBCO-mPEG (molecular weight 5000, manufactured by Broadpharma) and 9.4 μL of PBS were added.
＜比較例２．細胞非結合型オリゴ核酸ナノ粒子２の製造＞
（Ａ）ＳＰＤＰ　ＡＦ５　ＤＧＬ　Ｇ４の合成
　ＤＧＬ　Ｇ４の５０ｍｇ／ｍＬ　ＤＭＳＯ溶液４μＬに、Ａｌｅｘａ　Ｆｌｕｏｒ（登録商標）　５４６　ＮＨＳ　ｅｓｔｅｒ（サーモフィッシャーサイエンティフィック社製）の１０ｍＭ　ＤＭＳＯ溶液を１．２３μＬ、ＰＥＧ１２－ＳＰＤＰの６０ｍＭ　ＤＭＳＯ溶液を４．０８μＬ、及びＴＥＡの１０ｖ／ｖ％ＤＭＳＯ溶液を３．１２μＬ添加し、室温で１６時間撹拌した。次いで、この反応液にＭＳ（ＰＥＧ）１２　Ｍｅｔｈｙｌ－ＰＥＧ－ＮＨＳ－Ｅｓｔｅｒ　Ｒｅａｇｅｎｔの２００ｍＭ　ＤＭＳＯ溶液を１１．２μＬ添加し、さらに室温で１０時間撹拌した。以上のようにして、ＤＧＬ　Ｇ４のアミノ基と、Ａｌｅｘａ　Ｆｌｕｏｒ　５４６　ＮＨＳ　ｅｓｔｅｒ、ＰＥＧ１２－ＳＰＤＰ及びＭＳ（ＰＥＧ）１２　Ｍｅｔｈｙｌ－ＰＥＧ－ＮＨＳ－Ｅｓｔｅｒ　ＲｅａｇｅｎｔのＮＨＳ基とを反応させることにより、ナノ粒子化合物ＳＰＤＰ　ＡＦ５　ＤＧＬ　Ｇ４を得た。反応液に純水を１０００μＬ添加して混合した後、４０ｖ／ｖ％ＤＭＳＯ水溶液を用いて限外ろ過（Ａｍｉｃｏｎ　Ｕｌｔｒａ、分画分子量１００ｋＤａ）により６回精製した。回収した溶液に４０ｖ／ｖ％ＤＭＳＯ水溶液を添加し、液の体積を７０μＬに調整した。 <Comparative example 2. Production of cell-non-binding oligonucleic acid nanoparticles 2>
(A) Synthesis of SPDP AF5 DGL G4 To 4 μL of a 50 mg/mL DMSO solution of DGL G4, add 1.23 μL of a 10 mM DMSO solution of Alexa Fluor (registered trademark) 546 NHS ester (manufactured by Thermo Fisher Scientific) and PEG12-SPDP. 4.08 μL of a 60 mM DMSO solution of TEA and 3.12 μL of a 10 v/v% DMSO solution of TEA were added, and the mixture was stirred at room temperature for 16 hours. Next, 11.2 μL of a 200 mM DMSO solution of MS(PEG)12 Methyl-PEG-NHS-Ester Reagent was added to this reaction solution, and the mixture was further stirred at room temperature for 10 hours. As described above, by reacting the amino group of DGL G4 with the NHS group of Alexa Fluor 546 NHS ester, PEG12-SPDP and MS(PEG)12 Methyl-PEG-NHS-Ester Reagent, the nanoparticle compound SPDP Obtained AF5 DGL G4. After adding and mixing 1000 μL of pure water to the reaction solution, the mixture was purified six times by ultrafiltration (Amicon Ultra, molecular weight cut off: 100 kDa) using a 40 v/v% DMSO aqueous solution. A 40 v/v% DMSO aqueous solution was added to the collected solution, and the volume of the liquid was adjusted to 70 μL.
（Ｂ）ＮＨ２－ｓｉＲＮＡ　ＡＦ５　ＤＧＬ　Ｇ４の合成
　（Ａ）に示したＳＰＤＰ　ＡＦ５　ＤＧＬ　Ｇ４溶液５０μＬに、表２に示すｓｉＲＮＡの６．０ｍＭ　ＰＢＳ溶液１１７μＬ、及びＤＭＳＯ　２９μＬを添加し、室温で撹拌した。次いで、撹拌から３０分後に３Ｍの塩化ナトリウム水溶液を９．８μＬ及びＤＭＳＯを３．２μＬ、撹拌から１時間後に３Ｍの塩化ナトリウム水溶液を１２．５μＬ及びＤＭＳＯを４．１μＬ、撹拌から１．５時間後に３Ｍの塩化ナトリウム水溶液を１８．８μＬ及びＤＭＳＯを６．３μＬ添加した後、さらに室温で１６．５時間撹拌した。以上のようにして、ＳＰＤＰ　ＡＦ５　ＤＧＬ　Ｇ４の有するピリジルジスルフィド基とｓｉＲＮＡの有するＳＨ基を反応させた。反応液を、Ｈｉｐｒｅｐ　１６／６０　Ｓｅｐｈａｃｒｙｌ　Ｓ－２００　ＨＲを用いて、ゲルろ過により精製した（溶離液：ＰＢＳ）。ｓｉＲＮＡが結合したＤＧＬ　Ｇ４を含むフラクションを回収し、限外ろ過（Ａｍｉｃｏｎ　Ｕｌｔｒａ、分画分子量１００ｋＤａ）を用いて、体積を２５０μＬに調整した。 (B) Synthesis of NH2-siRNA AF5 DGL G4 To 50 μL of the SPDP AF5 DGL G4 solution shown in (A), 117 μL of a 6.0 mM PBS solution of siRNA shown in Table 2 and 29 μL of DMSO were added and stirred at room temperature. Next, 30 minutes after stirring, add 9.8 μL of 3M sodium chloride aqueous solution and 3.2 μL of DMSO, and 1 hour after stirring, add 12.5 μL of 3M sodium chloride solution and 4.1 μL of DMSO for 1.5 hours after stirring. Afterwards, 18.8 μL of a 3M aqueous sodium chloride solution and 6.3 μL of DMSO were added, and the mixture was further stirred at room temperature for 16.5 hours. As described above, the pyridyl disulfide group of SPDP AF5 DGL G4 was reacted with the SH group of siRNA. The reaction solution was purified by gel filtration using Hiprep 16/60 Sephacryl S-200 HR (eluent: PBS). A fraction containing siRNA-bound DGL G4 was collected, and the volume was adjusted to 250 μL using ultrafiltration (Amicon Ultra, molecular weight cut off: 100 kDa).
（Ｃ）Ｎ３－ｓｉＲＮＡ　ＡＦ５　ＤＧＬ　Ｇ４の合成
　（Ｂ）に示したＮＨ２－ｓｉＲＮＡ　ＡＦ５　ＤＧＬ　Ｇ４溶液２５０μＬに、ａｚｉｄｏ－ＰＥＧ４－ＮＨＳ　ｅｓｔｅｒの２００ｍＭ　ＤＭＳＯ溶液７０μＬ、及びＰＢＳ３０μＬを添加し、室温で２０時間撹拌した。以上のようにして、ＮＨ２－ｓｉＲＮＡ　ＡＦ５　ＤＧＬ　Ｇ４の有するアミノ基とａｚｉｄｏ－ＰＥＧ４－ＮＨＳ　ｅｓｔｅｒの有するＮＨＳ基を反応させた。反応液を、ＮＡＰ－５　Ｃｏｌｕｍｎを用いて精製した。次いで、回収液を限外ろ過（Ａｍｉｃｏｎ　Ｕｌｔｒａ、分画分子量１００ｋＤａ）により濃縮し、液の体積を２５０μＬに調整した。 (C) Synthesis of N3-siRNA AF5 DGL G4 To 250 μL of the NH2-siRNA AF5 DGL G4 solution shown in (B), 70 μL of a 200 mM DMSO solution of azido-PEG4-NHS ester and 30 μL of PBS were added, and the mixture was stirred at room temperature for 20 hours. did. As described above, the amino group of NH2-siRNA AF5 DGL G4 was reacted with the NHS group of azido-PEG4-NHS ester. The reaction solution was purified using NAP-5 Column. Next, the recovered liquid was concentrated by ultrafiltration (Amicon Ultra, molecular weight cut off: 100 kDa), and the volume of the liquid was adjusted to 250 μL.
（Ｄ）ｍＰＥＧ５０００－ｓｉＲＮＡ　ＡＦ５　ＤＧＬ　Ｇ４の合成
　（Ｃ）に示したＮ３－ｓｉＲＮＡ　ＡＦ５　ＤＧＬ　Ｇ４溶液６０μＬに、ＤＢＣＯ－ｍＰＥＧ（分子量５０００、Ｂｒｏａｄｐｈａｒｍ社製）の２０ｍＭ　ＤＭＳＯ溶液８４μＬ、及びＰＢＳ６６μＬを添加し、室温で２３時間撹拌した。以上のようにして、Ｎ３－ｓｉＲＮＡ　ＡＦ５　ＤＧＬ　Ｇ４の有するアジド基とＤＢＣＯ－ｍＰＥＧの有するＤＢＣＯ基を反応させた。反応液を、ＰＢＳを用いて限外ろ過（Ａｍｉｃｏｎ　Ｕｌｔｒａ、分画分子量１００ｋＤａ）により６回精製した。回収した溶液にＰＢＳを添加し、液の体積を２００μＬに調整することで、オリゴ核酸ナノ粒子ｍＰＥＧ５０００－ｓｉＲＮＡ　ＡＦ５　ＤＧＬ　Ｇ４のＰＢＳ溶液を得た。 (D) Synthesis of mPEG5000-siRNA AF5 DGL G4 To 60 μL of the N3-siRNA AF5 DGL G4 solution shown in (C), 84 μL of a 20 mM DMSO solution of DBCO-mPEG (molecular weight 5000, manufactured by Broadpharm) and 66 μL of PBS were added. Stirred at room temperature for 23 hours. As described above, the azide group of N3-siRNA AF5 DGL G4 was reacted with the DBCO group of DBCO-mPEG. The reaction solution was purified six times by ultrafiltration (Amicon Ultra, molecular weight cut off: 100 kDa) using PBS. PBS was added to the collected solution and the volume of the solution was adjusted to 200 μL to obtain a PBS solution of oligonucleic acid nanoparticles mPEG5000-siRNA AF5 DGL G4.
＜比較例３．細胞非結合型オリゴ核酸ナノ粒子３の製造＞
　比較例２（Ｃ）に示したＮ３－ｓｉＲＮＡ　ＡＦ５　ＤＧＬ　Ｇ４溶液を用いて、比較例２（Ｄ）に準じて、オリゴ核酸ナノ粒子ｍＰＥＧ１００００－ｓｉＲＮＡ　ＡＦ５　ＤＧＬ　Ｇ４を合成した。ただし、ＤＢＣＯ－ｍＰＥＧ（分子量５０００）の２０ｍＭ　ＤＭＳＯ溶液８４μＬ及びＰＢＳ６６μＬを添加する代わりに、ＤＢＣＯ－ｍＰＥＧ（分子量１００００、Ｂｒｏａｄｐｈａｒｍ社製）の１０ｍＭ　ＤＭＳＯ溶液１６８μＬ及びＰＢＳ１９２μＬを添加した。 <Comparative example 3. Production of cell-non-binding oligonucleic acid nanoparticles 3>
Using the N3-siRNA AF5 DGL G4 solution shown in Comparative Example 2(C), oligonucleic acid nanoparticles mPEG10000-siRNA AF5 DGL G4 were synthesized according to Comparative Example 2(D). However, instead of adding 84 μL of a 20 mM DMSO solution of DBCO-mPEG (molecular weight 5000) and 66 μL of PBS, 168 μL of a 10 mM DMSO solution of DBCO-mPEG (molecular weight 10000, manufactured by Broadpharm) and 192 μL of PBS were added.
＜参考例１．実施例３のオリゴ核酸ナノ粒子の評価のためのナノ粒子化合物の製造＞
　実施例３（Ｅ）に示したＮ３－ＰＥＧ２０００－ｓｉＲＮＡ　ＡＦ６　ＤＧＬ　Ｇ４溶液２０μＬに、ＡＺＤｙｅ４０５　ＤＢＣＯ（Ｃｌｉｃｋ　Ｃｈｅｍｉｓｔｒｙ　Ｔｏｏｌｓ社製）の５ｍＭ　ＤＭＳＯ溶液を１．５μＬ、及びＤＭＳＯを５．２μＬ添加し、室温で７時間撹拌した。以上のようにして、Ｎ３－ＰＥＧ２０００－ｓｉＲＮＡ　ＡＦ６　ＤＧＬ　Ｇ４の有するアジド基とＡＺＤｙｅ４０５　ＤＢＣＯの有するＤＢＣＯ基とを反応させた。反応液を、ＮＡＰ－５　Ｃｏｌｕｍｎを用いて精製した。次いで、回収液を限外ろ過（Ａｍｉｃｏｎ　Ｕｌｔｒａ、分画分子量１００ｋＤａ）により濃縮し、液の体積を７０μＬに調整して、ナノ粒子化合物ＡＺ４－ＰＥＧ２０００－ｓｉＲＮＡ　ＡＦ６　ＤＧＬ　Ｇ４のＰＢＳ溶液を得た。得られた溶液のＡＺＤｙｅ　４０５の濃度及びＡｌｅｘａ　Ｆｌｕｏｒ　６４７の濃度を、紫外可視分光光度計を用いて、それぞれ４０２ｎｍ及び６５１ｎｍにおける吸収の測定値から求めた。また、ＤＧＬ　Ｇ４の濃度を、後述するＡＱＣ法を用いたアミノ酸定量分析により求めた。これらの濃度から、一つのＤＧＬ　Ｇ４に結合したＡＺＤｙｅ　４０５の数は６と算出された。したがって、実施例３のオリゴ核酸ナノ粒子ｃＲＧＤ－ＰＥＧ２０００－ｓｉＲＮＡ　ＡＦ６　ＤＧＬ　Ｇ４のｃＲＧＤｆＫ修飾数も同様に６であるといえる。 <Reference example 1. Production of nanoparticle compound for evaluation of oligonucleic acid nanoparticles of Example 3>
To 20 μL of the N3-PEG2000-siRNA AF6 DGL G4 solution shown in Example 3 (E), 1.5 μL of a 5 mM DMSO solution of AZDye405 DBCO (manufactured by Click Chemistry Tools) and 5.2 μL of DMSO were added, and the mixture was incubated at room temperature. Stirred for 7 hours. As described above, the azide group of N3-PEG2000-siRNA AF6 DGL G4 was reacted with the DBCO group of AZDye405 DBCO. The reaction solution was purified using NAP-5 Column. Next, the recovered liquid was concentrated by ultrafiltration (Amicon Ultra, molecular weight cut off 100 kDa), and the volume of the liquid was adjusted to 70 μL to obtain a PBS solution of the nanoparticle compound AZ4-PEG2000-siRNA AF6 DGL G4. The concentrations of AZDye 405 and Alexa Fluor 647 in the resulting solution were determined from absorption measurements at 402 nm and 651 nm, respectively, using an ultraviolet-visible spectrophotometer. In addition, the concentration of DGL G4 was determined by amino acid quantitative analysis using the AQC method described below. From these concentrations, the number of AZDye 405 bound to one DGL G4 was calculated to be 6. Therefore, it can be said that the number of cRGDfK modifications of the oligonucleic acid nanoparticle cRGD-PEG2000-siRNA AF6 DGL G4 of Example 3 is also 6.
＜参考例２．実施例４のオリゴ核酸ナノ粒子の評価のためのナノ粒子化合物の製造＞
　参考例１に準じて、ナノ粒子化合物ＡＺ４－ＰＥＧ５０００－ｓｉＲＮＡ　ＡＦ６　ＤＧＬ　Ｇ４のＰＢＳ溶液を得た。ただし、実施例３（Ｅ）に示したＮ３－ＰＥＧ２０００－ｓｉＲＮＡ　ＡＦ６　ＤＧＬ　Ｇ４の代わりに、実施例４（Ｂ）に示したＮ３－ＰＥＧ５０００－ｓｉＲＮＡ　ＡＦ６　ＤＧＬ　Ｇ４を用いた。得られた溶液を参考例１に準じて分析し、実施例４のオリゴ核酸ナノ粒子ｃＲＧＤ－ＰＥＧ５０００－ｓｉＲＮＡ　ＡＦ６　ＤＧＬ　Ｇ４のｃＲＧＤｆＫ修飾数も同様に６と算出された。 <Reference example 2. Production of nanoparticle compound for evaluation of oligonucleic acid nanoparticles of Example 4>
According to Reference Example 1, a PBS solution of nanoparticle compound AZ4-PEG5000-siRNA AF6 DGL G4 was obtained. However, N3-PEG5000-siRNA AF6 DGL G4 shown in Example 4(B) was used instead of N3-PEG2000-siRNA AF6 DGL G4 shown in Example 3(E). The obtained solution was analyzed according to Reference Example 1, and the number of cRGDfK modifications of the oligonucleic acid nanoparticle cRGD-PEG5000-siRNA AF6 DGL G4 of Example 4 was similarly calculated to be 6.
＜参考例３．比較例１のオリゴ核酸ナノ粒子の評価のためのナノ粒子化合物の製造＞
　実施例３（Ｃ）に示したＮ３－ｓｉＲＮＡ　ＡＦ６　ＤＧＬ　Ｇ４のＰＢＳ溶液１０μＬに、ＡＺＤｙｅ４０５　ＤＢＣＯの５ｍＭ　ＤＭＳＯ溶液を２．８μＬ、ＰＢＳを１０μＬ及びＤＭＳＯを２．２μＬ添加し、室温で１７時間撹拌した。以上のようにしてＮ３－ｓｉＲＮＡ　ＡＦ６　ＤＧＬ　Ｇ４の有するアジド基とＡＺＤｙｅ４０５　ＤＢＣＯの有するＤＢＣＯ基を反応させた。反応液を、ＮＡＰ－５　Ｃｏｌｕｍｎを用いて精製した。次いで、回収液を限外ろ過（Ａｍｉｃｏｎ　Ｕｌｔｒａ、分画分子量１００ｋＤａ）により濃縮し、液の体積を６５μＬに調整して、ナノ粒子化合物ＡＺ４－ｓｉＲＮＡ　ＡＦ６　ＤＧＬ　Ｇ４のＰＢＳ溶液を得た。得られた溶液のＡＺＤｙｅ　４０５の濃度及びＡｌｅｘａ　Ｆｌｕｏｒ　６４７の濃度を、紫外可視分光光度計を用いて、それぞれ４０２ｎｍ及び６５１ｎｍにおける吸収の測定値から求めた。また、ＤＧＬ　Ｇ４の濃度を、後述するＡＱＣ法を用いたアミノ酸定量分析により求めた。これらの濃度から、一つのＤＧＬ　Ｇ４に結合したＡＺＤｙｅ　４０５の数は３３と算出された。したがって、実施例３（Ｃ）で合成したＮ３－ｓｉＲＮＡ　ＡＦ６　ＤＧＬ　Ｇ４におけるアジド基の数も同様に３３であるといえる。 <Reference example 3. Production of nanoparticle compound for evaluation of oligonucleic acid nanoparticles of Comparative Example 1>
To 10 μL of the PBS solution of N3-siRNA AF6 DGL G4 shown in Example 3 (C), 2.8 μL of 5 mM DMSO solution of AZDye405 DBCO, 10 μL of PBS, and 2.2 μL of DMSO were added, and the mixture was stirred at room temperature for 17 hours. . As described above, the azide group of N3-siRNA AF6 DGL G4 was reacted with the DBCO group of AZDye405 DBCO. The reaction solution was purified using NAP-5 Column. Next, the recovered solution was concentrated by ultrafiltration (Amicon Ultra, molecular weight cut off: 100 kDa), and the volume of the solution was adjusted to 65 μL to obtain a PBS solution of nanoparticle compound AZ4-siRNA AF6 DGL G4. The concentrations of AZDye 405 and Alexa Fluor 647 in the resulting solution were determined from absorption measurements at 402 nm and 651 nm, respectively, using an ultraviolet-visible spectrophotometer. In addition, the concentration of DGL G4 was determined by amino acid quantitative analysis using the AQC method described below. From these concentrations, the number of AZDye 405 bound to one DGL G4 was calculated to be 33. Therefore, it can be said that the number of azide groups in N3-siRNA AF6 DGL G4 synthesized in Example 3(C) is also 33.
＜参考例４．比較例１のオリゴ核酸ナノ粒子の評価のためのナノ粒子化合物の製造＞
　参考例３に準じて、ナノ粒子化合物ＡＺ４－ｍＰＥＧ５０００－ｓｉＲＮＡ　ＡＦ６　ＤＧＬ　Ｇ４のＰＢＳ溶液を得た。ただし、実施例３（Ｃ）に示したＮ３－ｓｉＲＮＡ　ＡＦ６　ＤＧＬ　Ｇ４のＰＢＳ溶液１０μＬに、ＡＺＤｙｅ４０５　ＤＢＣＯの５ｍＭ　ＤＭＳＯ溶液を２．８μＬ、ＰＢＳを１０μＬ及びＤＭＳＯを２．２μＬ添加する代わりに、比較例１で得たｍＰＥＧ５０００－ｓｉＲＮＡ　ＡＦ６　ＤＧＬ　Ｇ４のＰＢＳ溶液２５μＬに、ＡＺＤｙｅ４０５　ＤＢＣＯの５ｍＭ　ＤＭＳＯ溶液を１．９μＬ及びＤＭＳＯを４．３μＬ添加した。得られた溶液のＡＺＤｙｅ　４０５の濃度及びＡｌｅｘａ　Ｆｌｕｏｒ　６４７の濃度を、紫外可視分光光度計を用いて、それぞれ４０２ｎｍ及び６５１ｎｍにおける吸収の測定値から求めた。また、ＤＧＬ　Ｇ４の濃度を、後述するＡＱＣ法を用いたアミノ酸定量分析により求めた。これらの濃度から、一つのＤＧＬ　Ｇ４に結合したＡＺＤｙｅ　４０５の数は０と算出された。このＡＺＤｙｅ　４０５の数は、比較例１のオリゴ核酸ナノ粒子ｍＰＥＧ５０００－ｓｉＲＮＡ　ＡＦ６　ＤＧＬ　Ｇ４における未反応のアジド基の数と等しいといえる。したがって、比較例１のオリゴ核酸ナノ粒子のｍＰＥＧ５０００修飾数は、参考例３で算出したＡＺ４－ｓｉＲＮＡ　ＡＦ６　ＤＧＬ　Ｇ４のＡＺＤｙｅ　４０５修飾数（アジド基の総数）から、ＡＺ４－ｍＰＥＧ５０００－ｓｉＲＮＡ　ＡＦ６　ＤＧＬ　Ｇ４のＡＺＤｙｅ　４０５修飾数（未反応のアジド基の数）を減じることによって求めることができ、この値は３３と算出された。 <Reference example 4. Production of nanoparticle compound for evaluation of oligonucleic acid nanoparticles of Comparative Example 1>
According to Reference Example 3, a PBS solution of nanoparticle compound AZ4-mPEG5000-siRNA AF6 DGL G4 was obtained. However, instead of adding 2.8 μL of 5mM DMSO solution of AZDye405 DBCO, 10 μL of PBS, and 2.2 μL of DMSO to 10 μL of the PBS solution of N3-siRNA AF6 DGL G4 shown in Example 3(C), the comparative example To 25 μL of the PBS solution of mPEG5000-siRNA AF6 DGL G4 obtained in step 1, 1.9 μL of a 5 mM DMSO solution of AZDye405 DBCO and 4.3 μL of DMSO were added. The concentrations of AZDye 405 and Alexa Fluor 647 in the resulting solution were determined from absorption measurements at 402 nm and 651 nm, respectively, using an ultraviolet-visible spectrophotometer. In addition, the concentration of DGL G4 was determined by amino acid quantitative analysis using the AQC method described below. From these concentrations, the number of AZDye 405 bound to one DGL G4 was calculated to be 0. This number of AZDye 405 can be said to be equal to the number of unreacted azide groups in the oligonucleic acid nanoparticle mPEG5000-siRNA AF6 DGL G4 of Comparative Example 1. Therefore, the number of mPEG5000 modifications of the oligonucleic acid nanoparticles of Comparative Example 1 is determined from the number of AZDye 405 modifications (total number of azide groups) of AZ4-siRNA AF6 DGL G4 calculated in Reference Example 3. It can be determined by subtracting the number of AZDye 405 modifications (the number of unreacted azide groups), and this value was calculated to be 33.
＜参考例５．比較例２及び３のオリゴ核酸ナノ粒子の評価のためのナノ粒子化合物の製造＞
　参考例３に準じて、ナノ粒子化合物ＡＺ４－ｓｉＲＮＡ　ＡＦ５　ＤＧＬ　Ｇ４のＰＢＳ溶液を得た。具体的には、比較例２（Ｃ）に示したＮ３－ｓｉＲＮＡ　ＡＦ５　ＤＧＬ　Ｇ４溶液を２０μＬに、ＡＺＤｙｅ４０５　ＤＢＣＯの５０ｍＭ　ＤＭＳＯ溶液を１．１μＬ、及びＤＭＳＯを３．９μＬ添加して反応させた。以上のようにして、Ｎ３－ｓｉＲＮＡ　ＡＦ５　ＤＧＬ　Ｇ４の有するアジド基とＡＺＤｙｅ４０５　ＤＢＣＯの有するＤＢＣＯ基を反応させた。得られた溶液のＡＺＤｙｅ　４０５の濃度及びＡｌｅｘａ　Ｆｌｕｏｒ　５４６の濃度を、紫外可視分光光度計を用いて、それぞれ４０２ｎｍ及び５５４ｎｍにおける吸収の測定値から求めた。また、ＤＧＬ　Ｇ４の濃度を、後述するＡＱＣ法を用いたアミノ酸定量分析により求めた。これらの濃度から、一つのＤＧＬ　Ｇ４に結合したＡＺＤｙｅ　４０５の数を算出した。ＡＺＤｙｅ　４０５の数から求められるＰＥＧ５０００の数は３８．７６であった。したがって、比較例２（Ｃ）で合成したＮ３－ｓｉＲＮＡ　ＡＦ５　ＤＧＬ　Ｇ４におけるアジド基の数も３８．７６であるといえる。 <Reference example 5. Production of nanoparticle compounds for evaluation of oligonucleic acid nanoparticles of Comparative Examples 2 and 3>
According to Reference Example 3, a PBS solution of nanoparticle compound AZ4-siRNA AF5 DGL G4 was obtained. Specifically, 1.1 μL of a 50 mM DMSO solution of AZDye405 DBCO and 3.9 μL of DMSO were added to 20 μL of the N3-siRNA AF5 DGL G4 solution shown in Comparative Example 2 (C), and reacted. As described above, the azide group of N3-siRNA AF5 DGL G4 was reacted with the DBCO group of AZDye405 DBCO. The concentrations of AZDye 405 and Alexa Fluor 546 in the resulting solution were determined from absorption measurements at 402 nm and 554 nm, respectively, using an ultraviolet-visible spectrophotometer. In addition, the concentration of DGL G4 was determined by amino acid quantitative analysis using the AQC method described below. From these concentrations, the number of AZDye 405 bound to one DGL G4 was calculated. The number of PEG5000 determined from the number of AZDye 405 was 38.76. Therefore, it can be said that the number of azide groups in N3-siRNA AF5 DGL G4 synthesized in Comparative Example 2(C) is also 38.76.
＜参考例６．比較例２のオリゴ核酸ナノ粒子の評価のためのナノ粒子化合物の製造＞
　比較例２で得たｍＰＥＧ５０００－ｓｉＲＮＡ　ＡＦ５　ＤＧＬ　Ｇ４のＰＢＳ溶液２５μＬに、ＡＺＤｙｅ４０５　ＤＢＣＯの１０ｍＭ　ＤＭＳＯ溶液を２．１μＬ、及びＤＭＳＯを４．２μＬ添加し、２５℃で８時間撹拌した。以上のようにして、ｍＰＥＧ５０００－ｓｉＲＮＡ　ＡＦ５　ＤＧＬ　Ｇ４の有するアジド基とＡＺＤｙｅ４０５　ＤＢＣＯの有するＤＢＣＯ基を反応させた。反応液を、ＮＡＰ－５　Ｃｏｌｕｍｎを用いて精製した。次いで、回収液を限外ろ過（Ａｍｉｃｏｎ　Ｕｌｔｒａ、分画分子量１００ｋＤａ）により濃縮し、液の体積を７０μＬに調整して、ナノ粒子化合物ＡＺ４－ｍＰＥＧ５０００－ｓｉＲＮＡ　ＡＦ５　ＤＧＬ　Ｇ４のＰＢＳ溶液を得た。一つのＤＧＬ　Ｇ４に結合したＡＺＤｙｅ　４０５の数は０．３４であった。このＡＺＤｙｅ４０５の数は、比較例２のオリゴ核酸ナノ粒子ｍＰＥＧ５０００－ｓｉＲＮＡ　ＡＦ５　ＤＧＬ　Ｇ４における未反応のアジド基の数と等しいといえる。したがって、比較例２のオリゴ核酸ナノ粒子のｍＰＥＧ５０００修飾数は、参考例５で算出したＡＺ４－ｓｉＲＮＡ　ＡＦ５　ＤＧＬ　Ｇ４のＡＺＤｙｅ　４０５修飾数（アジド基の総数）から、ＡＺ４－ｍＰＥＧ５０００－ｓｉＲＮＡ　ＡＦ５　ＤＧＬ　Ｇ４のＡＺＤｙｅ　４０５修飾数（未反応のアジド基の数）を減じることによって求めることができ、この値は３８．４２と算出された。 <Reference example 6. Production of nanoparticle compound for evaluation of oligonucleic acid nanoparticles of Comparative Example 2>
To 25 μL of the PBS solution of mPEG5000-siRNA AF5 DGL G4 obtained in Comparative Example 2, 2.1 μL of a 10 mM DMSO solution of AZDye405 DBCO and 4.2 μL of DMSO were added, and the mixture was stirred at 25° C. for 8 hours. As described above, the azide group of mPEG5000-siRNA AF5 DGL G4 was reacted with the DBCO group of AZDye405 DBCO. The reaction solution was purified using NAP-5 Column. Next, the recovered solution was concentrated by ultrafiltration (Amicon Ultra, molecular weight cut off: 100 kDa), and the volume of the solution was adjusted to 70 μL to obtain a PBS solution of the nanoparticle compound AZ4-mPEG5000-siRNA AF5 DGL G4. The number of AZDye 405 bound to one DGL G4 was 0.34. This number of AZDye405 can be said to be equal to the number of unreacted azide groups in the oligonucleic acid nanoparticle mPEG5000-siRNA AF5 DGL G4 of Comparative Example 2. Therefore, the number of mPEG5000 modifications of the oligonucleic acid nanoparticles of Comparative Example 2 is determined from the number of AZDye 405 modifications (total number of azide groups) of AZ4-siRNA AF5 DGL G4 calculated in Reference Example 5. It can be determined by subtracting the number of AZDye 405 modifications (the number of unreacted azide groups), and this value was calculated to be 38.42.
＜参考例７．比較例３のオリゴ核酸ナノ粒子の評価のためのナノ粒子化合物の製造＞
　参考例６に準じて、ナノ粒子化合物ＡＺ４－ｍＰＥＧ１００００－ｓｉＲＮＡ　ＡＦ５　ＤＧＬ　Ｇ４の溶液を得た。ただし、比較例２で得たｍＰＥＧ５０００－ｓｉＲＮＡ　ＡＦ５　ＤＧＬ　Ｇ４の代わりに、比較例３で得たｍＰＥＧ１００００－ｓｉＲＮＡ　ＡＦ５　ＤＧＬ　Ｇ４を用いた。一つのＤＧＬ　Ｇ４に結合したＡＺＤｙｅ　４０５の数は０であった。したがって、比較例３のオリゴ核酸ナノ粒子ｍＰＥＧ１０００－ｓｉＲＮＡ　ＡＦ５　ＤＧＬ　Ｇ４のｍＰＥＧ１００００修飾数は、参考例５で算出したＡＺ４－ｓｉＲＮＡ　ＡＦ５　ＤＧＬ　Ｇ４のＡＺＤｙｅ　４０５修飾数（アジド基の総数）から、ＡＺ４－ｍＰＥＧ１００００－ｓｉＲＮＡ　ＡＦ５　ＤＧＬ　Ｇ４のＡＺＤｙｅ　４０５修飾数（未反応のアジド基の数）を減じることによって求めることができ、この値は３７と算出された。 <Reference example 7. Production of nanoparticle compound for evaluation of oligonucleic acid nanoparticles of Comparative Example 3>
According to Reference Example 6, a solution of nanoparticle compound AZ4-mPEG10000-siRNA AF5 DGL G4 was obtained. However, instead of mPEG5000-siRNA AF5 DGL G4 obtained in Comparative Example 2, mPEG10000-siRNA AF5 DGL G4 obtained in Comparative Example 3 was used. The number of AZDye 405 bound to one DGL G4 was 0. Therefore, the number of mPEG10000 modifications of the oligonucleic acid nanoparticle mPEG1000-siRNA AF5 DGL G4 of Comparative Example 3 is calculated from the number of AZDye 405 modifications (total number of azide groups) of AZ4-siRNA AF5 DGL G4 calculated in Reference Example 5. It can be determined by subtracting the number of AZDye 405 modifications (number of unreacted azide groups) of -siRNA AF5 DGL G4, and this value was calculated to be 37.
＜試験例１．オリゴ核酸ナノ粒子の評価＞
（Ａ）オリゴ核酸、細胞内在化促進剤及び蛍光分子の数の評価
　以下の各試験例で用いたオリゴ核酸ナノ粒子サンプル中のオリゴ核酸、細胞内在化促進剤及び蛍光分子の濃度を以下のとおり求め、これらの濃度から、一つの樹状ポリマーに結合したオリゴ核酸、親水性ポリマー、細胞内在化促進剤及び蛍光分子の数を算出した。なお、親水性ポリマーの数は、細胞内在化促進剤の数と同じとみなした。ただし、試験例３で用いたオリゴ核酸ナノ粒子中の細胞内在化促進剤及び親水性ポリマーの数は、参考例１、２、及び４に記載のとおり求めた。算出結果は、以下の各試験例において示す。 <Test example 1. Evaluation of oligonucleic acid nanoparticles>
(A) Evaluation of the number of oligonucleic acids, cell internalization promoters, and fluorescent molecules The concentrations of oligonucleic acids, cell internalization promoters, and fluorescent molecules in the oligonucleic acid nanoparticle samples used in each test example below are as follows: The number of oligonucleic acids, hydrophilic polymers, cell internalization promoters, and fluorescent molecules bound to one dendritic polymer was calculated from these concentrations. Note that the number of hydrophilic polymers was considered to be the same as the number of cell internalization promoters. However, the numbers of cell internalization promoters and hydrophilic polymers in the oligonucleic acid nanoparticles used in Test Example 3 were determined as described in Reference Examples 1, 2, and 4. The calculation results are shown in each test example below.
　オリゴ核酸及び蛍光分子の濃度は、紫外可視分光光度計を用いて次の波長の吸収の測定値から求めた：ｓｉＲＮＡは２６０ｎｍ、ＡＺＤｙｅ４０５は４０２ｎｍ、Ａｌｅｘａ　Ｆｌｕｏｒ　５４６は５５４ｎｍ、及びＡｌｅｘａ　Ｆｌｕｏｒ　６４７は６５１ｎｍ。 The concentrations of oligonucleic acids and fluorescent molecules were determined from absorption measurements at the following wavelengths using a UV-visible spectrophotometer: 260 nm for siRNA, 402 nm for AZDye405, 554 nm for Alexa Fluor 546, and 651 nm for Alexa Fluor 647.
　樹状ポリマーとポリペプチド系の細胞内在化促進剤の濃度は、アミノ酸定量分析（ＡＱＣ法）により、次のとおり定量した。まず、密閉できるガラスビンに、オリゴ核酸ナノ粒子サンプルの水溶液を３０μＬと、定沸点塩酸を３００μＬ添加し、密閉して、１１０℃で２４時間加熱して加水分解した。加水分解後、反応液を４５℃で加熱しながら溶媒を減圧除去した。ＡＱＣ（アディポジェン　ライフ　サイエンシーズ社製）を６０℃のアセトニトリル（超脱水）で溶解し、３ｍｇ／ｍＬに調整した。乾燥したオリゴ核酸ナノ粒子サンプルの入ったガラスビンに対して、２０ｍＭ塩酸を３０μＬ、０．２Ｍホウ酸緩衝液（ｐＨ８．８）を９０μＬ、３ｍｇ／ｍＬ　ＡＱＣ　アセトニトリル溶液を３０μＬ添加して撹拌し、６０℃で静置した。１０分間静置した後、４５℃で加熱しながら溶媒を減圧除去した。得られた固体を溶離液Ａ　１５０μＬで溶解した後、フィルターろ過（メルク社製Ｕｌｔｒａｆｒｅｅ；－ＭＣ，ＧＶ，０．２２μｍ）を行った。得られたろ液を逆相ＨＰＬＣ（カラム：ＡｃｃＱ－Ｔａｇ　Ｃｏｌｕｍｎ，６０Å，４μｍ　３．９×１５０ｍｍ、溶離液Ａ：ＡｃｃＱ－Ｔａｇ　Ｅｌｕｅｎｔ　Ａ／ｗａｔｅｒ（１／９；ｖ／ｖ）、溶離液Ｂ：水／アセトニトリル（１／１；ｖ／ｖ））により分析し、リシン残基のピークの積分値からＤＧＬ　Ｇ４の濃度を定量し、またｃＲＧＤｆＫの濃度を固有のピークの積分値から定量した。ＡｃｃＱ－Ｔａｇ　Ｃｏｌｕｍｎ及びＡｃｃＱ－Ｔａｇ　Ｅｌｕｅｎｔ　Ａはウォーターズコーポレーション社より購入した。 The concentrations of the dendritic polymer and polypeptide-based cell internalization promoters were determined by amino acid quantitative analysis (AQC method) as follows. First, 30 μL of an aqueous solution of an oligonucleic acid nanoparticle sample and 300 μL of constant boiling point hydrochloric acid were added to a sealable glass bottle, the bottle was sealed, and the bottle was heated at 110° C. for 24 hours for hydrolysis. After hydrolysis, the solvent was removed under reduced pressure while heating the reaction solution at 45°C. AQC (manufactured by Adipogen Life Sciences) was dissolved in acetonitrile (super dehydrated) at 60°C and adjusted to 3 mg/mL. To the glass bottle containing the dried oligonucleic acid nanoparticle sample, add 30 μL of 20 mM hydrochloric acid, 90 μL of 0.2 M borate buffer (pH 8.8), and 30 μL of 3 mg/mL AQC acetonitrile solution and stir for 60 minutes. It was left standing at ℃. After standing for 10 minutes, the solvent was removed under reduced pressure while heating at 45°C. After dissolving the obtained solid in 150 μL of eluent A, filter filtration (Merck Ultrafree; -MC, GV, 0.22 μm) was performed. The obtained filtrate was subjected to reverse phase HPLC (Column: AccQ-Tag Column, 60 Å, 4 μm, 3.9 x 150 mm, eluent A: AccQ-Tag Eluent A/water (1/9; v/v), eluent B: water/acetonitrile (1/1; v/v)), and the concentration of DGL G4 was determined from the integral value of the peak of lysine residue, and the concentration of cRGDfK was determined from the integral value of the unique peak. AccQ-Tag Column and AccQ-Tag Eluent A were purchased from Waters Corporation.
（Ｂ）オリゴ核酸ナノ粒子の平均粒子径及びゼータ電位の評価
　比較例２のオリゴ核酸ナノ粒子ｍＰＥＧ５０００－ｓｉＲＮＡ　ＡＦ５　ＤＧＬ　Ｇ４及び、比較例３のオリゴ核酸ナノ粒子ｍＰＥＧ１００００－ｓｉＲＮＡ　ＡＦ５　ＤＧＬ　Ｇ４の平均粒子径及びゼータ電位を、粒子径分析装置（マルバーン・パナリティカル製Ｚｅｔａｓｉｚｅｒ　Ｎａｎｏ　ＺＳ）を用いて評価した。平均粒子径は、セルとしてマルバーン・パナリティカル製ＺＥＮ００４０を使用して、多角度動的光散乱法によって測定した。ゼータ電位は、セルとしてマルバーン・パナリティカル製ゼータ電位用ディスポーザブルセルＤＴＳ１０７０を使用して、電気泳動光散乱法によって測定した。得られた結果を表３に示す。 (B) Evaluation of average particle diameter and zeta potential of oligonucleic acid nanoparticles Average particle diameter of oligonucleic acid nanoparticles mPEG5000-siRNA AF5 DGL G4 of Comparative Example 2 and oligonucleic acid nanoparticles mPEG10000-siRNA AF5 DGL G4 of Comparative Example 3 and zeta potential were evaluated using a particle size analyzer (Zetasizer Nano ZS manufactured by Malvern Panalytical). The average particle diameter was measured by a multi-angle dynamic light scattering method using ZEN0040 manufactured by Malvern Panalytical as a cell. The zeta potential was measured by electrophoretic light scattering using a zeta potential disposable cell DTS1070 manufactured by Malvern Panalytical as a cell. The results obtained are shown in Table 3.
Figure JPOXMLDOC01-appb-T000007
Figure JPOXMLDOC01-appb-T000007
＜試験例２．ｃＲＧＤリガンド結合型オリゴ核酸ナノ粒子のインビトロ評価＞
　オリゴ核酸ナノ粒子の細胞内取り込みを次のようにして評価した。まず、９６ウェルプレートにヒト神経膠芽腫細胞株（Ｕ－８７ＭＧ）を播種し、１０％ＦＢＳ含有ＤＭＥＭ培地を用いて、３７℃、５％ＣＯ２の条件で培養した。翌日、培地を交換するとともに、各ウェルにサンプルを加えて細胞をトランスフェクションし、３７℃、５％ＣＯ２の条件で培養した。トランスフェクション時点のサンプルのｓｉＲＮＡ濃度は０．０１μＭ、０．１μＭ又は１μＭであった。トランスフェクションから４８時間後、細胞をＰＢＳで洗浄した後、蛍光強度を測定した（励起波長６４０ｎｍ、蛍光波長６７５ｎｍ）。さらに、ｓｉＲＮＡと蛍光分子の数の比（ｓｉＲＮＡの数／蛍光分子の数）からｓｉＲＮＡ濃度を蛍光分子の濃度に換算し、蛍光強度と蛍光分子の濃度が正比例関係にあると近似した上で、蛍光分子の濃度が２ｎＭ、２０ｎＭ又は２００ｎＭのときの蛍光強度を算出した。使用したサンプルを表４に、得られた結果を図３に示す。 <Test Example 2. In vitro evaluation of cRGD ligand-bound oligonucleic acid nanoparticles>
Cellular uptake of oligonucleic acid nanoparticles was evaluated as follows. First, a human glioblastoma cell line (U-87MG) was seeded in a 96-well plate and cultured at 37° C. and 5% CO 2 using a DMEM medium containing 10% FBS. The next day, the medium was replaced, and a sample was added to each well to transfect the cells, and the cells were cultured at 37° C. and 5% CO 2 . The siRNA concentration in the samples at the time of transfection was 0.01 μM, 0.1 μM or 1 μM. 48 hours after transfection, the cells were washed with PBS, and then the fluorescence intensity was measured (excitation wavelength 640 nm, fluorescence wavelength 675 nm). Furthermore, the siRNA concentration is converted to the concentration of fluorescent molecules from the ratio of the number of siRNAs and fluorescent molecules (number of siRNAs/number of fluorescent molecules), and after approximating that the fluorescence intensity and the concentration of fluorescent molecules are directly proportional, Fluorescence intensity was calculated when the concentration of fluorescent molecules was 2 nM, 20 nM, or 200 nM. The samples used are shown in Table 4, and the results obtained are shown in FIG.
Figure JPOXMLDOC01-appb-T000008
Figure JPOXMLDOC01-appb-T000008
　オリゴ核酸ナノ粒子による遺伝子ノックダウン活性を次のようにして評価した。まず、９６ウェルプレートにヒト神経膠芽腫細胞株（Ｕ－８７ＭＧ）を播種し、１０％ＦＢＳ含有ＤＭＥＭ培地を用いて、３７℃、５％ＣＯ２の条件で培養した。翌日、培地を交換するとともに、各ウェルにサンプルを加えて細胞をトランスフェクションし、３７℃、５％ＣＯ２の条件で培養した。トランスフェクション時点のサンプルのｓｉＲＮＡ濃度は１μＭであった。対照群に対しては、サンプルのかわりにＰＢＳを加えた。トランスフェクションから４８時間後、ＲＮｅａｓｙ　Ｍｉｎｉ　Ｋｉｔ（キアゲン社製）を使用してｍＲＮＡを抽出し、Ｈｉｇｈ　Ｃａｐａｃｉｔｙ　ＲＮＡ－ｔｏ－ｃＤＮＡ　Ｋｉｔ（Ａｐｐｌｉｅｄ　Ｂｉｏｓｙｓｔｅｍｓ（登録商標））を使用して一定量のｍＲＮＡからｃＤＮＡを合成した。続いて、得られたｃＤＮＡをテンプレートとして、ＰｏｗｅｒＵｐ　ＳＹＢＲ　Ｇｒｅｅｎ　Ｍａｓｔｅｒ　Ｍｉｘ（Ａｐｐｌｉｅｄ　Ｂｉｏｓｙｓｔｅｍｓ）を用いて定量ＲＴ－ＰＣＲを実施した。ＡＴＰ５Ｂプライマーとしては下表５に示す配列番号９及び配列番号１０のプライマーを使用し、ＧＡＰＤＨプライマーとしては下表５に示す配列番号１１及び配列番号１２のプライマーを使用した。ＰＣＲ条件（温度及び時間）は次のとおりであった。９５℃で１秒、６０℃で３０秒を１サイクルとして設計し、４０サイクルを実施した。定量ＲＴ－ＰＣＲの結果に基づいて、「ＡＴＰ５Ｂの発現量／ＧＡＰＤＨ（内部標準遺伝子）の発現量」の値を計算し、対照群についての計算結果とサンプル添加群についての計算結果を比較した。使用したサンプルを表４に、得られた結果を図４に示す。 Gene knockdown activity by oligonucleic acid nanoparticles was evaluated as follows. First, a human glioblastoma cell line (U-87MG) was seeded in a 96-well plate and cultured at 37° C. and 5% CO 2 using a DMEM medium containing 10% FBS. The next day, the medium was replaced, and a sample was added to each well to transfect the cells, and the cells were cultured at 37° C. and 5% CO 2 . The siRNA concentration in the sample at the time of transfection was 1 μM. For the control group, PBS was added instead of the sample. 48 hours after transfection, mRNA was extracted using the RNeasy Mini Kit (manufactured by Qiagen), and cDNA was extracted from a certain amount of mRNA using the High Capacity RNA-to-cDNA Kit (Applied Biosystems®). was synthesized. Subsequently, quantitative RT-PCR was performed using the obtained cDNA as a template using PowerUp SYBR Green Master Mix (Applied Biosystems). As the ATP5B primers, the primers shown in SEQ ID NO: 9 and SEQ ID NO: 10 shown in Table 5 below were used, and as the GAPDH primers, the primers shown in SEQ ID NO: 11 and SEQ ID NO: 12 shown in Table 5 below were used. PCR conditions (temperature and time) were as follows. One cycle was designed to be 1 second at 95°C and 30 seconds at 60°C, and 40 cycles were performed. Based on the results of quantitative RT-PCR, the value of "expression level of ATP5B/expression level of GAPDH (internal standard gene)" was calculated, and the calculation results for the control group and the calculation results for the sample addition group were compared. The samples used are shown in Table 4, and the results obtained are shown in FIG.
Figure JPOXMLDOC01-appb-T000009
Figure JPOXMLDOC01-appb-T000009
＜試験例３．ｃＲＧＤリガンド結合型オリゴ核酸ナノ粒子のインビトロ評価２＞
　表６に示したサンプルを用いて、試験例２に準じてオリゴ核酸ナノ粒子の細胞内取り込みを評価した。トランスフェクション時のｓｉＲＮＡ濃度は０．０５μＭ又は０．５μＭであった。ｓｉＲＮＡと蛍光分子の数の比（ｓｉＲＮＡの数／蛍光分子の数）からｓｉＲＮＡ濃度を蛍光分子の濃度に換算し、蛍光強度と蛍光分子の濃度が正比例関係にあると近似した上で、蛍光分子の濃度が５ｎＭ又は５０ｎＭのときの蛍光強度を算出した。得られた結果を図５に示す。 <Test Example 3. In vitro evaluation of cRGD ligand-bound oligonucleic acid nanoparticles 2>
Using the samples shown in Table 6, the cellular uptake of the oligonucleic acid nanoparticles was evaluated according to Test Example 2. The siRNA concentration at the time of transfection was 0.05 μM or 0.5 μM. The siRNA concentration is converted to the concentration of fluorescent molecules from the ratio of the number of siRNAs and fluorescent molecules (number of siRNAs/number of fluorescent molecules), and after approximating that the fluorescence intensity and the concentration of fluorescent molecules are in a direct proportional relationship, The fluorescence intensity was calculated when the concentration of was 5 nM or 50 nM. The obtained results are shown in FIG.
Figure JPOXMLDOC01-appb-T000010
 　表６に示したサンプルｃＲＧＤ－ＮＰ＿３を用いて、試験例２に準じてオリゴ核酸ナノ粒子による遺伝子ノックダウン活性を評価した。トランスフェクション時のｓｉＲＮＡ濃度は０．５μＭであった。得られた結果を図６に示す。
Figure JPOXMLDOC01-appb-T000010
 Using sample cRGD-NP_3 shown in Table 6, gene knockdown activity by oligonucleic acid nanoparticles was evaluated according to Test Example 2. The siRNA concentration at the time of transfection was 0.5 μM. The obtained results are shown in FIG.
＜オリゴ核酸の合成＞
　表７に示すｓｉＲＮＡを準備する。ｓｉＲＮＡのセンス鎖ＲＮＡの３’末端には、ｓｐａｃｅｒ１８（ヘキサエチレングリコール）及びＣ６　ｌｉｎｋｅｒを介してチオール基を結合させる。 <Synthesis of oligonucleic acid>
Prepare siRNA shown in Table 7. A thiol group is attached to the 3' end of the sense strand RNA of siRNA via spacer 18 (hexaethylene glycol) and a C6 linker.
Figure JPOXMLDOC01-appb-T000011
Figure JPOXMLDOC01-appb-T000011
＜実施例５．ｃＲＧＤ結合型オリゴ核酸ナノ粒子５の製造＞
（Ａ）ｂｉｓ（ａｚｉｄｅ－ＰＥＧ３）－ＰＥＧ５ｋ－ＣＯＯＨの合成
　ＡＭＩＮＯ－ＰＥＧ４－ＢＩＳ－ＰＥＧ３－ＡＺＩＤＥ（Ｃｏｎｊｕ－Ｐｒｏｂｅ社）の１００Ｍ　ＤＭＳＯ溶液５０．０μＬに、ＮＨＳ－ＰＥＧ－ＣＯＯＨ（Ｂｉｏｐｈａｒｍａ　ＰＥＧ　Ｓｃｉｅｎｔｉｆｉｃ社製、分子量５０００）の２．５ｍＭ　ＤＭＳＯ溶液４００μＬ、及びＴＥＡの１０ｖ／ｖ％ＤＭＳＯ溶液を１０．５μＬ添加し、室温で終夜撹拌することによって、ＡＭＩＮＯ－ＰＥＧ４－ＢＩＳ－ＰＥＧ３－ＡＺＩＤＥのアミノ基とＮＨＳ－ＰＥＧ－ＣＯＯＨのＮＨＳ基とを反応させる。反応液にＰＢＳを１０００μＬ添加して混合した後、純水を用いて限外ろ過（Ａｍｉｃｏｎ　Ｕｌｔｒａ、分画分子量３ｋＤａ）により６回精製し、凍結乾燥により溶媒を除去した後、ＤＭＳＯを５００μＬ添加することにより、ｂｉｓ（ａｚｉｄｅ－ＰＥＧ３）－ＰＥＧ５ｋ－ＣＯＯＨの２．０ｍＭ　ＤＭＳＯ溶液を得ることができる。 <Example 5. Production of cRGD-binding oligonucleic acid nanoparticles 5>
(A) Synthesis of bis(azide-PEG3)-PEG5k-COOH NHS-PEG-COOH (Biopharma PEG Manufactured by Scientific , molecular weight 5000) and 10.5 μL of a 10 v/v% DMSO solution of TEA and stirred overnight at room temperature, the amino groups of AMINO-PEG4-BIS-PEG3-AZIDE and NHS were added. -PEG-COOH is reacted with the NHS group. After adding 1000 μL of PBS to the reaction solution and mixing, purify it six times by ultrafiltration (Amicon Ultra, molecular weight cutoff 3 kDa) using pure water, remove the solvent by freeze-drying, and then add 500 μL of DMSO. By this, a 2.0 mM DMSO solution of bis(azide-PEG3)-PEG5k-COOH can be obtained.
（Ｂ）ｂｉｓ（ａｚｉｄｅ－ＰＥＧ３）　ＳＰＤＰ　ＡＦ６　ＤＧＬ　Ｇ４の合成
　ＤＧＬ　Ｇ４の５０ｍｇ／ｍＬ　ＤＭＳＯ溶液３．０μＬに対して、ＰＥＧ１２－ＳＰＤＰ（サーモフィッシャーサイエンティフィック社製）の６０ｍＭ　ＤＭＳＯ溶液を２．３０μＬ、Ａｌｅｘａ　Ｆｌｕｏｒ（登録商標）６４７　ＮＨＳ　ｅｓｔｅｒの８ｍＭ　ＤＭＳＯ溶液を１．４４μＬ、及びＴＥＡの１０ｖ／ｖ％ＤＭＳＯ溶液を２．１３μＬ添加し、室温で８時間撹拌する。次いで、（Ａ）に示したｂｉｓ（ａｚｉｄｅ－ＰＥＧ３）－ＰＥＧ５ｋ－ＣＯＯＨのＤＭＳＯ溶液１７２．３μＬ、ＮＨＳの２００ｍＭ　ＤＭＳＯ溶液３．４５μＬ、及びＥＤＣ－ＨＣｌの２００ｍＭ　ＤＭＳＯ溶液３．４５μＬを添加し、室温で終夜攪拌する。次いで、ｍ－ｄＰＥＧ（登録商標）１２－ＮＨＳ　ｅｓｔｅｒの２００ｍＭ　ＤＭＳＯ溶液を８．４１μＬ添加し、さらに室温で８時間撹拌することによって、ＤＧＬ　Ｇ４のアミノ基と、ｂｉｓ（ａｚｉｄｅ－ＰＥＧ３）－ＰＥＧ５ｋ－ＣＯＯＨのＣＯＯＨ基、並びにＡｌｅｘａ　Ｆｌｕｏｒ６４７　ＮＨＳ　ｅｓｔｅｒ、ｍ－ｄＰＥＧ１２－ＮＨＳ　ｅｓｔｅｒ、及びＰＥＧ１２－ＳＰＤＰのＮＨＳ基とを反応させる。反応液にＰＢＳを１０００μＬ添加して混合した後、純水を用いて限外ろ過（Ａｍｉｃｏｎ　Ｕｌｔｒａ、分画分子量１００ｋＤａ）により６回精製し、回収した水溶液にＰＢＳを添加することで、液の体積を５０μＬに調整することにより、ｂｉｓ（ａｚｉｄｅ－ＰＥＧ３）　ＳＰＤＰ　ＡＦ６　ＤＧＬ　Ｇ４のＰＢＳ溶液を得ることができる。 (B) Synthesis of bis(azide-PEG3) SPDP AF6 DGL G4 For 3.0 μL of a 50 mg/mL DMSO solution of DGL G4, add 2.30 μL of a 60 mM DMSO solution of PEG12-SPDP (manufactured by Thermo Fisher Scientific). , 1.44 μL of an 8 mM DMSO solution of Alexa Fluor® 647 NHS ester, and 2.13 μL of a 10 v/v% DMSO solution of TEA are added, and the mixture is stirred at room temperature for 8 hours. Next, 172.3 μL of a DMSO solution of bis(azide-PEG3)-PEG5k-COOH shown in (A), 3.45 μL of a 200 mM DMSO solution of NHS, and 3.45 μL of a 200 mM DMSO solution of EDC-HCl were added, and the mixture was heated to room temperature. Stir overnight. Next, 8.41 μL of a 200 mM DMSO solution of m-dPEG (registered trademark) 12-NHS ester was added and further stirred at room temperature for 8 hours, thereby converting the amino group of DGL G4 and bis(azide-PEG3)-PEG5k- The COOH group of COOH and the NHS group of Alexa Fluor647 NHS ester, m-dPEG12-NHS ester, and PEG12-SPDP are reacted. After adding 1000 μL of PBS to the reaction solution and mixing, it was purified six times by ultrafiltration (Amicon Ultra, molecular weight cut off 100 kDa) using pure water, and the volume of the solution was reduced by adding PBS to the recovered aqueous solution. By adjusting the volume to 50 μL, a PBS solution of bis(azide-PEG3) SPDP AF6 DGL G4 can be obtained.
（Ｃ）ｂｉｓ（ａｚｉｄｅ－ＰＥＧ３）　ｓｉＲＮＡ　ＡＦ６　ＤＧＬ　Ｇ４の合成
　（Ｂ）に示したｂｉｓ（ａｚｉｄｅ－ＰＥＧ３）　ＳＰＤＰ　ＡＦ６　ＤＧＬ　Ｇ４のＰＢＳ溶液１０μＬに対して、ＤＭＳＯ　５．９４μＬ、表７に示したｓｉＲＮＡの６ｍＭ　ＰＢＳ溶液１０．７μＬ、及び３Ｍ　ＮａＣｌ水溶液３．０３μＬを添加し、室温で終夜攪拌することによって、ｂｉｓ（ａｚｉｄｅ－ＰＥＧ３）　ＳＰＤＰ　ＡＦ６　ＤＧＬ　Ｇ４の有するピリジルジスルフィド基とｓｉＲＮＡの有するＳＨ基を反応させる。反応液を、Ｈｉｐｒｅｐ　１６／６０　Ｓｅｐｈａｃｒｙｌ　Ｓ－２００　ＨＲ（溶離液：ＰＢＳ）を用いて、ゲルろ過により精製し、ｓｉＲＮＡが結合したＤＧＬ　Ｇ４を含むフラクションを回収する。回収した溶液を限外ろ過（Ａｍｉｃｏｎ　Ｕｌｔｒａ、分画分子量５０ｋＤａ）により濃縮し、液の体積を１００μＬに調整することによって、ｂｉｓ（ａｚｉｄｅ－ＰＥＧ３）　ｓｉＲＮＡ　ＡＦ６　ＤＧＬ　Ｇ４のＰＢＳ溶液を得ることができる。 (C) Synthesis of bis(azide-PEG3) siRNA AF6 DGL G4 To 10 μL of the PBS solution of bis(azide-PEG3) SPDP AF6 DGL G4 shown in (B), add 5.94 μL of DMSO and siRNA shown in Table 7. By adding 10.7 μL of 6 mM PBS solution and 3.03 μL of 3 M NaCl aqueous solution and stirring overnight at room temperature, the pyridyl disulfide group of bis(azide-PEG3) SPDP AF6 DGL G4 and the SH group of siRNA were reacted. let The reaction solution is purified by gel filtration using Hiprep 16/60 Sephacryl S-200 HR (eluent: PBS), and a fraction containing siRNA-bound DGL G4 is collected. A PBS solution of bis(azide-PEG3) siRNA AF6 DGL G4 can be obtained by concentrating the collected solution by ultrafiltration (Amicon Ultra, molecular weight cut off 50 kDa) and adjusting the volume of the solution to 100 μL.
（Ｄ）ｃＲＧＤ－ＰＥＧ２ｋ－ＤＢＣＯの合成
　Ｃｙｃｌｏ（－ＲＧＤｆＫ）の３００ｍＭ　ＤＭＳＯ溶液５５．０μＬに対して、ＤＢＣＯ－ＰＥＧ２ｋ－ＮＨＳ（Ｂｉｏｐｈａｒｍａ　ＰＥＧ　Ｓｃｉｅｎｔｉｆｉｃ社製）の１０ｍＭ　ＤＭＳＯ溶液を３３０．０μＬと、ＴＥＡ　４．６０μＬを添加し、室温で終夜撹拌することによって、Ｃｙｃｌｏ（－ＲＧＤｆＫ）の有するアミノ基と、ＤＢＣＯ－ＰＥＧ２ｋ－ＮＨＳの有するＮＨＳ基とを反応させる。反応液を、Ｓｅｐｈａｄｅｘ　ＬＨ－２０（Ｃｙｔｉｖａ社製）を用いて、ゲルろ過により精製し（溶離液：ＤＭＳＯ）、ｃＲＧＤｆＫが結合したＰＥＧを含むフラクションを回収する。回収した溶液を凍結乾燥により溶媒を除去した後、ＤＭＳＯを添加して５０ｍＭの濃度に調整することによって、ｃＲＧＤ－ＰＥＧ２ｋ－ＤＢＣＯのＤＭＳＯ溶液を得ることができる。 (D) CRGD -PEG2K -DBCO's synthetic CYCLO (-RGDFK) 300mm DMSO solution 55.0 μL, DBCo -PEG2K -NHS (Biopharma PEG PEG SCIENTIFIC) 10mm DMSO solution is 330. .0 μL and TEA 4 By adding .60 μL and stirring overnight at room temperature, the amino group of Cyclo(-RGDfK) and the NHS group of DBCO-PEG2k-NHS are reacted. The reaction solution is purified by gel filtration using Sephadex LH-20 (manufactured by Cytiva) (eluent: DMSO), and a fraction containing PEG bound to cRGDfK is collected. After removing the solvent from the collected solution by freeze-drying, a DMSO solution of cRGD-PEG2k-DBCO can be obtained by adding DMSO to adjust the concentration to 50 mM.
（Ｅ）ｂｉｓ（ｃＲＧＤ－ＰＥＧ２ｋ）　ｓｉＲＮＡ　ＡＦ６　ＤＧＬ　Ｇ４の合成
　（Ｃ）に示したｂｉｓ（ａｚｉｄｅ－ＰＥＧ３）　ｓｉＲＮＡ　ＡＦ６　ＤＧＬ　Ｇ４のＰＢＳ溶液４０．０μＬに対して、（Ｄ）に示したｃＲＧＤ－ＰＥＧ２ｋ－ＤＢＣＯのＤＭＳＯ溶液１．４７μＬ、及びＤＭＳＯ　２．９７μＬを添加し、室温で終夜攪拌することによって、ｂｉｓ（ａｚｉｄｅ－ＰＥＧ３）　ｓｉＲＮＡ　ＡＦ６　ＤＧＬ　Ｇ４の有するアジド基とｃＲＧＤ－ＰＥＧ２ｋ－ＤＢＣＯの有するＤＢＣＯ基を反応させる。反応液を、Ｚｅｂａ（登録商標）　Ｓｐｉｎ　Ｄｅｓａｌｔｉｎｇ　Ｃｏｌｕｍｎ（分子量分画：４０ｋＤａ）を用いて精製する。さらにＰＢＳを用いて限外ろ過（Ａｍｉｃｏｎ　Ｕｌｔｒａ、分画分子量１００ｋＤａ）により３回精製を行い、液の体積を１００μＬに調整することによって、オリゴ核酸ナノ粒子ｂｉｓ（ｃＲＧＤ－ＰＥＧ２ｋ）　ｓｉＲＮＡ　ＡＦ６　ＤＧＬ　Ｇ４のＰＢＳ溶液を得ることができる。 (E) Synthesis of bis(cRGD-PEG2k) siRNA AF6 DGL G4 The cRGD-PEG2k shown in (D) was added to 40.0 μL of the PBS solution of bis(azide-PEG3) siRNA AF6 DGL G4 shown in (C). -By adding 1.47 μL of a DMSO solution of DBCO and 2.97 μL of DMSO and stirring at room temperature overnight, the azide group of bis(azide-PEG3) siRNA AF6 DGL G4 and the DBCO group of cRGD-PEG2k-DBCO were combined. react. The reaction solution is purified using Zeba® Spin Desalting Column (molecular weight fraction: 40 kDa). Furthermore, oligonucleic acid nanoparticles bis(cRGD-PEG2k) siRNA AF6 DGL G4 were purified by ultrafiltration (Amicon Ultra, molecular weight cutoff 100 kDa) three times using PBS and the volume of the solution was adjusted to 100 μL. A PBS solution can be obtained.
＜実施例６．ｃＲＧＤ結合型オリゴ核酸ナノ粒子６の製造＞
（Ａ）ａｚｉｄｅ－ＣＰ１　ＳＰＤＰ　ＡＦ６　ＤＧＬ４の合成
　ＤＧＬ　Ｇ４の５０ｍｇ／ｍＬ　ＤＭＳＯ溶液３．０μＬに対して、ＰＥＧ１２－ＳＰＤＰの６０ｍＭ　ＤＭＳＯ溶液を２．３０μＬ、Ａｌｅｘａ　Ｆｌｕｏｒ（登録商標）６４７　ＮＨＳ　ｅｓｔｅｒの８ｍＭ　ＤＭＳＯ溶液を１．４４μＬ、及びＴＥＡの１０ｖ／ｖ％ＤＭＳＯ溶液を１．８１μＬ添加し、室温で８時間撹拌する。次いで、下式（ＸＩＩＩ）に示すペプチドＣＰ１の５ｍＭ　ＤＭＳＯ溶液４５．９μＬ、及びＨＡＴＵ（富士フイルム和光純薬株式会社製）の２００ｍＭ　ＤＭＳＯ溶液１．１５μＬを添加し、室温で終夜攪拌する。次いで、ｍ－ｄＰＥＧ（登録商標）１２－ＮＨＳ　ｅｓｔｅｒの２００ｍＭ　ＤＭＳＯ溶液を８．４１μＬ添加し、さらに室温で８時間撹拌することによって、ＤＧＬ　Ｇ４のアミノ基と、ＣＰ１のＣＯＯＨ基、並びにＡｌｅｘａ　Ｆｌｕｏｒ６４７　ＮＨＳ　ｅｓｔｅｒ、ｍ－ｄＰＥＧ１２－ＮＨＳ　ｅｓｔｅｒ、及びＰＥＧ１２－ＳＰＤＰのＮＨＳ基とを反応させる。反応液を、Ｓｅｐｈａｄｅｘ　ＬＨ－２０を用いて、ゲルろ過により精製し（溶離液：ＤＭＳＯ）、ＣＰ１が結合したＤＧＬ　Ｇ４を含むフラクションを回収する。回収した溶液を凍結乾燥することで溶媒を除去した後、ＤＭＳＯを添加して３２．２ｍＭの濃度に調整することによって、ａｚｉｄｅ－ＣＰ１　ＳＰＤＰ　ＡＦ６　ＤＧＬ４のＤＭＳＯ溶液を得ることができる。 <Example 6. Production of cRGD-binding oligonucleic acid nanoparticles 6>
(A) Synthesis of azide-CP1 SPDP AF6 DGL4 For 3.0 μL of a 50 mg/mL DMSO solution of DGL G4, 2.30 μL of a 60 mM DMSO solution of PEG12-SPDP, and 8 mM DM of Alexa Fluor® 647 NHS ester. S.O. Add 1.44 μL of the solution and 1.81 μL of a 10 v/v% DMSO solution of TEA, and stir at room temperature for 8 hours. Next, 45.9 μL of a 5 mM DMSO solution of peptide CP1 shown in the following formula (XIII) and 1.15 μL of a 200 mM DMSO solution of HATU (manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.) are added, and the mixture is stirred overnight at room temperature. Next, by adding 8.41 μL of a 200 mM DMSO solution of m-dPEG (registered trademark) 12-NHS ester and further stirring at room temperature for 8 hours, the amino group of DGL G4, the COOH group of CP1, and Alexa Fluor647 NHS ester, m-dPEG12-NHS ester, and the NHS group of PEG12-SPDP. The reaction solution is purified by gel filtration using Sephadex LH-20 (eluent: DMSO), and a fraction containing CP1-bound DGL G4 is collected. After removing the solvent by freeze-drying the collected solution, a DMSO solution of azide-CP1 SPDP AF6 DGL4 can be obtained by adding DMSO to adjust the concentration to 32.2 mM.
（Ｂ）ａｚｉｄｅ－ＣＰ１　ｓｉＲＮＡ　ＡＦ６　ＤＧＬ４の合成
　（Ａ）に示したａｚｉｄｅ－ＣＰ１　ＳＰＤＰ　ＡＦ６　ＤＧＬ４のＤＭＳＯ溶液１０．０μＬに対して、表７に示したｓｉＲＮＡの６ｍＭ　ＰＢＳ溶液１１．３μＬ、３Ｍ　ＮａＣｌ水溶液３．０９μＬ、及びＤＭＳＯ　８．９９μＬを添加し、室温で終夜攪拌することによって、ａｚｉｄｅ－ＣＰ１　ＳＰＤＰ　ＡＦ６　ＤＧＬ４の有するピリジルジスルフィド基とｓｉＲＮＡの有するＳＨ基を反応させる。反応液を、Ｈｉｐｒｅｐ　１６／６０　Ｓｅｐｈａｃｒｙｌ　Ｓ－２００　ＨＲ（溶離液：ＰＢＳ）を用いて、ゲルろ過により精製し、ｓｉＲＮＡが結合したＤＧＬ　Ｇ４を含むフラクションを回収する。回収した溶液を限外ろ過（Ａｍｉｃｏｎ　Ｕｌｔｒａ、分画分子量５０ｋＤａ）により濃縮し、液の体積を１００μＬに調整することによって、ａｚｉｄｅ－ＣＰ１　ｓｉＲＮＡ　ＡＦ６　ＤＧＬ４のＰＢＳ溶液を得ることができる。 (B) Synthesis of azide-CP1 siRNA AF6 DGL4 To 10.0 μL of the DMSO solution of azide-CP1 SPDP AF6 DGL4 shown in (A), 11.3 μL of a 6mM PBS solution of the siRNA shown in Table 7, and a 3M NaCl aqueous solution. By adding 3.09 μL and 8.99 μL of DMSO and stirring overnight at room temperature, the pyridyl disulfide group of azide-CP1 SPDP AF6 DGL4 and the SH group of siRNA are reacted. The reaction solution is purified by gel filtration using Hiprep 16/60 Sephacryl S-200 HR (eluent: PBS), and a fraction containing siRNA-bound DGL G4 is collected. A PBS solution of azide-CP1 siRNA AF6 DGL4 can be obtained by concentrating the collected solution by ultrafiltration (Amicon Ultra, molecular weight cut off: 50 kDa) and adjusting the volume of the solution to 100 μL.
（Ｃ）ａｚｉｄｅ－ＣＰ２　ｓｉＲＮＡ　ＡＦ６　ＤＧＬ４の合成
　（Ｂ）に示したａｚｉｄｅ－ＣＰ１　ｓｉＲＮＡ　ＡＦ６　ＤＧＬ４のＰＢＳ溶液５０．０μＬに対して、ＴＦＡを１２．５μＬ添加し、４℃で２時間攪拌することによって、ＣＰ１に含まれるＳｅｒ側鎖のｔｅｒｔ－ブチルエーテル基及びＬｙｓ側鎖のＢｏｃ基を脱保護することでＣＰ１を下式（ＸＩＶ）に示すＣＰ２に変換する。反応液にＰＢＳを１０００μＬ加え、限外ろ過（Ａｍｉｃｏｎ　Ｕｌｔｒａ、分画分子量１００ｋＤａ）により３回精製を行い、液の体積を１００μＬに調整することによって、ａｚｉｄｅ－ＣＰ２　ｓｉＲＮＡ　ＡＦ６　ＤＧＬ４のＰＢＳ溶液を得ることができる。 (C) Synthesis of azide-CP2 siRNA AF6 DGL4 By adding 12.5 μL of TFA to 50.0 μL of the PBS solution of azide-CP1 siRNA AF6 DGL4 shown in (B) and stirring at 4°C for 2 hours. , CP1 is converted to CP2 shown in the following formula (XIV) by deprotecting the tert-butyl ether group of the Ser side chain and the Boc group of the Lys side chain contained in CP1. Add 1000 μL of PBS to the reaction solution, perform purification three times by ultrafiltration (Amicon Ultra, molecular weight cut off 100 kDa), and adjust the volume of the solution to 100 μL to obtain a PBS solution of azide-CP2 siRNA AF6 DGL4. I can do it.
（Ｄ）ｃＲＧＤ－ＰＥＧ５ｋ－ＤＢＣＯの合成
　実施例５の（Ｄ）に準じてｃＲＧＤ－ＰＥＧ５ｋ－ＤＢＣＯを合成することができる。ただし、ＤＢＣＯ－ＰＥＧ２ｋ－ＮＨＳの１０ｍＭ　ＤＭＳＯ溶液３３０．０μＬの代わりにＤＢＣＯ－ＰＥＧ５ｋ－ＮＨＳの２ｍＭ　ＤＭＳＯ溶液１０００μＬを用い、Ｃｙｃｌｏ（－ＲＧＤｆＫ）の３００ｍＭ　ＤＭＳＯ溶液の量は５５．０μＬから３３．３μＬに変更し、ＴＥＡの量は４．６μＬから２．７９μＬに変更する。 (D) Synthesis of cRGD-PEG5k-DBCO cRGD-PEG5k-DBCO can be synthesized according to (D) of Example 5. However, instead of 330.0 μL of 10 mM DMSO solution of DBCO-PEG2k-NHS, 1000 μL of 2 mM DMSO solution of DBCO-PEG5k-NHS was used, and the amount of 300 mM DMSO solution of Cyclo(-RGDfK) was changed from 55.0 μL to 33.3 μL. The amount of TEA is changed from 4.6 μL to 2.79 μL.
（Ｅ）ｃＲＧＤ－ＰＥＧ５ｋ－ＣＰ２　ｓｉＲＮＡ　ＡＦ６　ＤＧＬ　Ｇ４の合成
　（Ｃ）に示したａｚｉｄｅ－ＣＰ２　ｓｉＲＮＡ　ＡＦ６　ＤＧＬ４のＰＢＳ溶液８０．０μＬに対して、（Ｄ）に示したｃＲＧＤ－ＰＥＧ５ｋ－ＤＢＣＯの５０ｍＭ　ＤＭＳＯ溶液３．８６μＬ、及びＤＭＳＯ　５．０１μＬを添加し、室温で終夜攪拌することによって、ａｚｉｄｅ－ＣＰ２　ｓｉＲＮＡ　ＡＦ６　ＤＧＬ４の有するアジド基とｃＲＧＤ－ＰＥＧ５ｋ－ＤＢＣＯの有するＤＢＣＯ基を反応させる。反応液を、Ｚｅｂａ（登録商標）　Ｓｐｉｎ　Ｄｅｓａｌｔｉｎｇ　Ｃｏｌｕｍｎ（分子量分画：４０ｋＤａ）を用いて精製する。さらにＰＢＳを用いて限外ろ過（Ａｍｉｃｏｎ　Ｕｌｔｒａ、分画分子量１００ｋＤａ）により３回精製を行い、液の体積を１００μＬに調整することによって、オリゴ核酸ナノ粒子ｃＲＧＤ－ＰＥＧ５ｋ－ＣＰ２　ｓｉＲＮＡ　ＡＦ６　ＤＧＬ　Ｇ４のＰＢＳ溶液を得ることができる。 (E) Synthesis of cRGD-PEG5k-CP2 siRNA AF6 DGL G4 To 80.0 μL of PBS solution of azide-CP2 siRNA AF6 DGL4 shown in (C), add 50 mM DMSO of cRGD-PEG5k-DBCO shown in (D). By adding 3.86 μL of the solution and 5.01 μL of DMSO and stirring overnight at room temperature, the azide group of azide-CP2 siRNA AF6 DGL4 and the DBCO group of cRGD-PEG5k-DBCO are reacted. The reaction solution is purified using Zeba® Spin Desalting Column (molecular weight fraction: 40 kDa). Furthermore, oligonucleic acid nanoparticles cRGD-PEG5k-CP2 siRNA AF6 DGL G4 were purified by ultrafiltration (Amicon Ultra, molecular weight cutoff 100 kDa) three times using PBS and the volume of the solution was adjusted to 100 μL. A solution can be obtained.
　本発明の一側面に係るオリゴ核酸ナノ粒子は、細胞質内に輸送されるオリゴ核酸の量を向上させることができるため、疾患を治療又は予防するための医薬組成物又は医薬としての利用が可能である。 Since the oligonucleic acid nanoparticles according to one aspect of the present invention can improve the amount of oligonucleic acids transported into the cytoplasm, they can be used as pharmaceutical compositions or medicines for treating or preventing diseases. be.
　１１・・・オリゴ核酸、１２・・・親水性ポリマー、１３・・・細胞内剤化促進剤、１４・・・スペーサー、２１・・・キャッピング剤、３１・・・リンカー、５０・・・コア、１００，２００・・・オリゴ核酸ナノ粒子。 DESCRIPTION OF SYMBOLS 11... Oligonucleic acid, 12... Hydrophilic polymer, 13... Intracellular formulation promoter, 14... Spacer, 21... Capping agent, 31... Linker, 50... Core , 100,200...oligonucleic acid nanoparticles.

Claims (46)

  1. 　樹状ポリマーで構成されたコアと、
    　該コアに結合した複数個のオリゴ核酸と、
    　該複数個のオリゴ核酸に結合した１又は複数個の親水性ポリマーと、
    　該１又は複数個の親水性ポリマーに結合した１又は複数個の細胞内在化促進剤と、を含むオリゴ核酸ナノ粒子であって、
    　前記コアと前記オリゴ核酸との結合、前記オリゴ核酸と前記親水性ポリマーとの結合、及び前記親水性ポリマーと前記細胞内在化促進剤との結合は、それぞれ独立に、直接結合又はリンカーを介した結合であり、
    　前記細胞内在化促進剤は前記オリゴ核酸ナノ粒子の表面に位置する、オリゴ核酸ナノ粒子。     A core composed of dendritic polymer,
    a plurality of oligonucleic acids bound to the core;
    one or more hydrophilic polymers bound to the plurality of oligonucleic acids;
    An oligonucleic acid nanoparticle comprising one or more cell internalization promoters bound to the one or more hydrophilic polymers,
    The binding between the core and the oligonucleic acid, the binding between the oligonucleic acid and the hydrophilic polymer, and the binding between the hydrophilic polymer and the cell internalization promoter are each independently performed by direct binding or via a linker. is a combination,
    An oligonucleic acid nanoparticle, wherein the cell internalization promoting agent is located on the surface of the oligonucleic acid nanoparticle.
  2. 　前記直接結合、又は前記リンカーと前記コア、前記オリゴ核酸、前記親水性ポリマー、若しくは前記細胞内在化促進剤との結合が、共有結合、金属配位又はホスト－ゲスト相互作用によるものである、請求項１に記載のオリゴ核酸ナノ粒子。 The direct bond, or the bond between the linker and the core, the oligonucleic acid, the hydrophilic polymer, or the cell internalization promoter is a covalent bond, metal coordination, or host-guest interaction. Item 1. Oligonucleic acid nanoparticles according to item 1.
  3. 　前記直接結合、又は前記リンカーと前記コア、前記オリゴ核酸、前記親水性ポリマー、若しくは前記細胞内在化促進剤との結合が、共有結合又は金属配位によるものである、請求項１に記載のオリゴ核酸ナノ粒子。 The oligo according to claim 1, wherein the direct bond or the bond between the linker and the core, the oligonucleic acid, the hydrophilic polymer, or the cell internalization promoter is a covalent bond or metal coordination. Nucleic acid nanoparticles.
  4. 　前記直接結合、又は前記リンカーと前記コア、前記オリゴ核酸、前記親水性ポリマー、若しくは前記細胞内在化促進剤との結合が、共有結合によるものである、請求項１に記載のオリゴ核酸ナノ粒子。 The oligonucleic acid nanoparticle according to claim 1, wherein the direct bond or the bond between the linker and the core, the oligonucleic acid, the hydrophilic polymer, or the cell internalization promoter is a covalent bond.
  5. 　前記樹状ポリマーの反応性官能基の少なくとも一部がキャッピング剤によりキャッピングされている、請求項１～４のいずれか一項に記載のオリゴ核酸ナノ粒子。 The oligonucleic acid nanoparticle according to any one of claims 1 to 4, wherein at least a portion of the reactive functional groups of the dendritic polymer are capped with a capping agent.
  6. 　樹状ポリマーで構成されたコアと、
    　該コアに結合した複数個のオリゴ核酸と、
    　前記コアにスペーサーを介して結合した１又は複数個の親水性ポリマーと、
    　該１又は複数個の親水性ポリマーに結合した１又は複数個の細胞内在化促進剤と、を含むオリゴ核酸ナノ粒子であって、
    　前記コアと前記オリゴ核酸との結合、前記コアと前記スペーサーとの結合、前記スペーサーと前記親水性ポリマーとの結合、及び前記親水性ポリマーと前記細胞内在化促進剤との結合は、それぞれ独立に、直接結合又はリンカーを介した結合であり、
    　前記細胞内在化促進剤は前記オリゴ核酸ナノ粒子の表面に位置する、オリゴ核酸ナノ粒子。     A core composed of dendritic polymer,
    a plurality of oligonucleic acids bound to the core;
    one or more hydrophilic polymers bonded to the core via a spacer;
    An oligonucleic acid nanoparticle comprising one or more cell internalization promoters bound to the one or more hydrophilic polymers,
    The binding between the core and the oligonucleic acid, the binding between the core and the spacer, the binding between the spacer and the hydrophilic polymer, and the binding between the hydrophilic polymer and the cell internalization promoter are each independently performed. , a direct bond or a bond via a linker,
    An oligonucleic acid nanoparticle, wherein the cell internalization promoting agent is located on the surface of the oligonucleic acid nanoparticle.
  7. 　前記直接結合、又は前記リンカーと前記コア、前記オリゴ核酸、前記スペーサー、前記親水性ポリマー、若しくは前記細胞内在化促進剤との結合が、共有結合、金属配位又はホスト－ゲスト相互作用によるものである、請求項６に記載のオリゴ核酸ナノ粒子。 The direct bond, or the bond between the linker and the core, the oligonucleic acid, the spacer, the hydrophilic polymer, or the cell internalization promoter is due to a covalent bond, metal coordination, or host-guest interaction. The oligonucleic acid nanoparticle according to claim 6.
  8. 　前記直接結合、又は前記リンカーと前記コア、前記オリゴ核酸、前記スペーサー、前記親水性ポリマー、若しくは前記細胞内在化促進剤との結合が、共有結合又は金属配位によるものである、請求項６に記載のオリゴ核酸ナノ粒子。 According to claim 6, the direct bond, or the bond between the linker and the core, the oligonucleic acid, the spacer, the hydrophilic polymer, or the cell internalization promoter is a covalent bond or metal coordination. The oligonucleic acid nanoparticles described.
  9. 　前記直接結合、又は前記リンカーと前記コア、前記オリゴ核酸、前記スペーサー、前記親水性ポリマー、若しくは前記細胞内在化促進剤との結合が、共有結合によるものである、請求項６に記載のオリゴ核酸ナノ粒子。 The oligonucleic acid according to claim 6, wherein the direct bond or the bond between the linker and the core, the oligonucleic acid, the spacer, the hydrophilic polymer, or the cell internalization promoter is a covalent bond. nanoparticles.
  10. 　前記樹状ポリマーの反応性官能基の少なくとも一部がキャッピング剤によりキャッピングされている、請求項６～９のいずれか一項に記載のオリゴ核酸ナノ粒子。 The oligonucleic acid nanoparticle according to any one of claims 6 to 9, wherein at least a portion of the reactive functional groups of the dendritic polymer are capped with a capping agent.
  11. 　前記キャッピング剤が、親水性分子及び疎水性分子からなる群から選択される１種以上の分子である、請求項５又は１０記載のオリゴ核酸ナノ粒子。 The oligonucleic acid nanoparticle according to claim 5 or 10, wherein the capping agent is one or more molecules selected from the group consisting of hydrophilic molecules and hydrophobic molecules.
  12. 　前記キャッピング剤が親水性分子である、請求項１１に記載のオリゴ核酸ナノ粒子。 The oligonucleic acid nanoparticle according to claim 11, wherein the capping agent is a hydrophilic molecule.
  13. 　前記キャッピング剤が、電気的に中性な親水性分子、酸性条件下でプロトン化する極性分子、アニオン性分子、及びカチオン性分子からなる群から選択される１種類以上の親水性分子である、請求項１１に記載のオリゴ核酸ナノ粒子。 The capping agent is one or more types of hydrophilic molecules selected from the group consisting of electrically neutral hydrophilic molecules, polar molecules that protonate under acidic conditions, anionic molecules, and cationic molecules. The oligonucleic acid nanoparticle according to claim 11.
  14. 　前記キャッピング剤が、電気的に中性な親水性分子、酸性条件下でプロトン化する極性分子、及びアニオン性分子からなる群から選択される１種類以上の親水性分子である、請求項１１に記載のオリゴ核酸ナノ粒子。 12. The capping agent according to claim 11, wherein the capping agent is one or more types of hydrophilic molecules selected from the group consisting of electrically neutral hydrophilic molecules, polar molecules that protonate under acidic conditions, and anionic molecules. The oligonucleic acid nanoparticles described.
  15. 　前記キャッピング剤が疎水性分子である、請求項１１に記載のオリゴ核酸ナノ粒子。 The oligonucleic acid nanoparticle according to claim 11, wherein the capping agent is a hydrophobic molecule.
  16. 　前記キャッピング剤が、脂肪族化合物、芳香族化合物、トリアルキルアミン、及びステロイド又はその類縁体からなる群から選択される１種類以上の疎水性分子である、請求項１１に記載のオリゴ核酸ナノ粒子。 The oligonucleic acid nanoparticle according to claim 11, wherein the capping agent is one or more hydrophobic molecules selected from the group consisting of aliphatic compounds, aromatic compounds, trialkylamines, and steroids or analogs thereof. .
  17. 　前記キャッピング剤が脂肪族化合物である、請求項１１に記載のオリゴ核酸ナノ粒子。 The oligonucleic acid nanoparticle according to claim 11, wherein the capping agent is an aliphatic compound.
  18. 　前記樹状ポリマーが、デンドリグラフト又はデンドリマーである、請求項１～１７のいずれか一項に記載のオリゴ核酸ナノ粒子。 The oligonucleic acid nanoparticle according to any one of claims 1 to 17, wherein the dendritic polymer is a dendrigraft or a dendrimer.
  19. 　前記樹状ポリマーにおけるモノマー同士が、アミド結合、エステル結合、又はグリコシド結合により結合している、請求項１～１７のいずれか一項に記載のオリゴ核酸ナノ粒子。 The oligonucleic acid nanoparticle according to any one of claims 1 to 17, wherein monomers in the dendritic polymer are bonded to each other through an amide bond, an ester bond, or a glycosidic bond.
  20. 　前記樹状ポリマーにおけるモノマー同士がアミド結合又はエステル結合により結合している、請求項１～１７のいずれか一項に記載のオリゴ核酸ナノ粒子。 The oligonucleic acid nanoparticle according to any one of claims 1 to 17, wherein monomers in the dendritic polymer are bonded to each other through an amide bond or an ester bond.
  21. 　前記樹状ポリマーがポリ－Ｌ－リシンデンドリグラフト、ポリアミドアミンデンドリマー、又は２，２－ビス（ヒドロキシル－メチル）プロピオン酸デンドリマーである、請求項１～１７のいずれか一項に記載のオリゴ核酸ナノ粒子。 The oligonucleic acid according to any one of claims 1 to 17, wherein the dendritic polymer is a poly-L-lysine dendrigraft, a polyamidoamine dendrimer, or a 2,2-bis(hydroxyl-methyl)propionic acid dendrimer. nanoparticles.
  22. 　前記オリゴ核酸が遺伝子発現制御剤である、請求項１～２１のいずれか一項に記載のオリゴ核酸ナノ粒子。 The oligonucleic acid nanoparticle according to any one of claims 1 to 21, wherein the oligonucleic acid is a gene expression control agent.
  23. 　前記遺伝子発現制御剤が、ｍＲＮＡの発現を抑制する分子である、請求項２２に記載のオリゴ核酸ナノ粒子。 The oligonucleic acid nanoparticle according to claim 22, wherein the gene expression control agent is a molecule that suppresses mRNA expression.
  24. 　前記遺伝子発現制御剤が、ＲＮＡ干渉誘導核酸又はアンチセンス核酸である、請求項２２に記載のオリゴ核酸ナノ粒子。 The oligonucleic acid nanoparticle according to claim 22, wherein the gene expression control agent is an RNA interference-inducing nucleic acid or an antisense nucleic acid.
  25. 　前記スペーサーが、ポリエチレングリコール、ポリ（２－アルキル－２－オキサゾリン）、ポリペプチド及びポリペプトイドからなる群から選択される１種以上のスペーサーである、請求項６～１０のいずれか一項に記載のオリゴ核酸ナノ粒子。 The spacer according to any one of claims 6 to 10, wherein the spacer is one or more spacers selected from the group consisting of polyethylene glycol, poly(2-alkyl-2-oxazoline), polypeptide, and polypeptoid. Oligonucleic acid nanoparticles.
  26. 　前記スペーサーが、ポリエチレングリコール、又はカチオン性ポリペプチドである、請求項６～１０のいずれか一項に記載のオリゴ核酸ナノ粒子。 The oligonucleic acid nanoparticle according to any one of claims 6 to 10, wherein the spacer is polyethylene glycol or a cationic polypeptide.
  27. 　前記親水性ポリマーが、ポリエチレングリコール、ポリ（２－アルキル－２－オキサゾリン）、ポリペプチド及びポリペプトイドからなる群から選択される１種以上の親水性ポリマーである、請求項１～２６のいずれか一項に記載のオリゴ核酸ナノ粒子。 Any one of claims 1 to 26, wherein the hydrophilic polymer is one or more hydrophilic polymers selected from the group consisting of polyethylene glycol, poly(2-alkyl-2-oxazoline), polypeptide, and polypeptoid. Oligonucleic acid nanoparticles as described in section.
  28. 　前記親水性ポリマーが、ポリエチレングリコール、ポリ（２－メチル－２－オキサゾリン）、ＥＫペプチド及びポリサルコシンからなる群から選択される１種以上の水性ポリマーである、請求項１～２６のいずれか一項に記載のオリゴ核酸ナノ粒子。 Any one of claims 1 to 26, wherein the hydrophilic polymer is one or more aqueous polymers selected from the group consisting of polyethylene glycol, poly(2-methyl-2-oxazoline), EK peptide, and polysarcosine. Oligonucleic acid nanoparticles as described in section.
  29. 　前記細胞内在化促進剤が、ポリペプチド、アプタマー、抗体若しくはそのフラグメント、糖、脂質、及びその他の低分子化合物からなる群から選択される１種以上の細胞内在化促進剤である、請求項１～２８のいずれか一項に記載のオリゴ核酸ナノ粒子。 Claim 1, wherein the cell internalization promoter is one or more cell internalization promoters selected from the group consisting of polypeptides, aptamers, antibodies or fragments thereof, sugars, lipids, and other low molecular weight compounds. 29. The oligonucleic acid nanoparticle according to any one of 28 to 28.
  30. 　前記細胞内在化促進剤が、疎水性分子、ポリカチオン、ポリペプチド、アプタマー、抗体若しくはそのフラグメント、糖、及び脂質以外の、分子量が２０００以下の低分子化合物である、請求項１～２８のいずれか一項に記載のオリゴ核酸ナノ粒子。 Any one of claims 1 to 28, wherein the cell internalization promoter is a low molecular compound with a molecular weight of 2000 or less other than a hydrophobic molecule, a polycation, a polypeptide, an aptamer, an antibody or a fragment thereof, a sugar, and a lipid. The oligonucleic acid nanoparticle according to item 1.
  31. 　前記細胞内在化促進剤がポリペプチドである、請求項１～２８のいずれか一項に記載のオリゴ核酸ナノ粒子。 The oligonucleic acid nanoparticle according to any one of claims 1 to 28, wherein the cell internalization promoting agent is a polypeptide.
  32. 　前記細胞内在化促進剤がアプタマーである、請求項１～２８のいずれか一項に記載のオリゴ核酸ナノ粒子。 The oligonucleic acid nanoparticle according to any one of claims 1 to 28, wherein the cell internalization promoter is an aptamer.
  33. 　前記細胞内在化促進剤が抗体若しくはそのフラグメントである、請求項１～２８のいずれか一項に記載のオリゴ核酸ナノ粒子。 The oligonucleic acid nanoparticle according to any one of claims 1 to 28, wherein the cell internalization promoting agent is an antibody or a fragment thereof.
  34. 　前記細胞内在化促進剤が糖である、請求項１～２８のいずれか一項に記載のオリゴ核酸ナノ粒子。 The oligonucleic acid nanoparticle according to any one of claims 1 to 28, wherein the cell internalization promoter is a sugar.
  35. 　前記細胞内在化促進剤が脂質である、請求項１～２８のいずれか一項に記載のオリゴ核酸ナノ粒子。 The oligonucleic acid nanoparticle according to any one of claims 1 to 28, wherein the cell internalization promoter is a lipid.
  36. 　請求項１～３５のいずれか一項に記載のオリゴ核酸ナノ粒子を有効成分として含有する、医薬組成物。 A pharmaceutical composition containing the oligonucleic acid nanoparticle according to any one of claims 1 to 35 as an active ingredient.
  37. 　請求項１～３５のいずれか一項に記載のオリゴ核酸ナノ粒子を有効成分として含有する、
    　先天性代謝異常症、先天性内分泌疾患、単一遺伝子疾患、神経変性疾患、神経疾患、筋疾患、髄膜炎、脳炎、脳症、ライソゾーム病、悪性新生物、繊維症、炎症性疾患、免疫不全疾患、自己免疫疾患、及び感染症からなる群から選択される疾患に対する治療剤又は予防剤。     Containing the oligonucleic acid nanoparticle according to any one of claims 1 to 35 as an active ingredient,
    Inborn errors of metabolism, congenital endocrine diseases, single gene diseases, neurodegenerative diseases, neurological diseases, muscle diseases, meningitis, encephalitis, encephalopathy, lysosomal diseases, malignant neoplasms, fibrosis, inflammatory diseases, immunodeficiency A therapeutic or prophylactic agent for a disease selected from the group consisting of a disease, an autoimmune disease, and an infectious disease.
  38. 　治療上の有効量の請求項１～３５のいずれか一項に記載のオリゴ核酸ナノ粒子を投与することを含む、
    　先天性代謝異常症、先天性内分泌疾患、単一遺伝子疾患、神経変性疾患、神経疾患、筋疾患、髄膜炎、脳炎、脳症、ライソゾーム病、悪性新生物、繊維症、炎症性疾患、免疫不全疾患、自己免疫疾患、及び感染症からなる群から選択される疾患を治療及び／又は予防するための方法。     administering a therapeutically effective amount of oligonucleic acid nanoparticles according to any one of claims 1 to 35.
    Inborn errors of metabolism, congenital endocrine diseases, single gene diseases, neurodegenerative diseases, neurological diseases, muscle diseases, meningitis, encephalitis, encephalopathy, lysosomal diseases, malignant neoplasms, fibrosis, inflammatory diseases, immunodeficiency A method for treating and/or preventing a disease selected from the group consisting of a disease, an autoimmune disease, and an infectious disease.
  39. 　先天性代謝異常症、先天性内分泌疾患、単一遺伝子疾患、神経変性疾患、神経疾患、筋疾患、髄膜炎、脳炎、脳症、ライソゾーム病、悪性新生物、繊維症、炎症性疾患、免疫不全疾患、自己免疫疾患、及び感染症からなる群から選択される疾患に対する治療剤及び／又は予防剤を製造するための、請求項１～３５のいずれか一項に記載のオリゴ核酸ナノ粒子の使用。 Inborn errors of metabolism, congenital endocrine diseases, single gene diseases, neurodegenerative diseases, neurological diseases, muscle diseases, meningitis, encephalitis, encephalopathy, lysosomal diseases, malignant neoplasms, fibrosis, inflammatory diseases, immunodeficiency Use of the oligonucleic acid nanoparticles according to any one of claims 1 to 35 for producing therapeutic and/or preventive agents for diseases selected from the group consisting of diseases, autoimmune diseases, and infectious diseases. .
  40. 　先天性代謝異常症、先天性内分泌疾患、単一遺伝子疾患、神経変性疾患、神経疾患、筋疾患、髄膜炎、脳炎、脳症、ライソゾーム病、悪性新生物、繊維症、炎症性疾患、免疫不全疾患、自己免疫疾患、及び感染症からなる群から選択される疾患の治療及び／又は予防に使用するための、請求項１～３５のいずれか一項に記載のオリゴ核酸ナノ粒子。 Inborn errors of metabolism, congenital endocrine diseases, single gene diseases, neurodegenerative diseases, neurological diseases, muscle diseases, meningitis, encephalitis, encephalopathy, lysosomal diseases, malignant neoplasms, fibrosis, inflammatory diseases, immunodeficiency Oligonucleic acid nanoparticles according to any one of claims 1 to 35, for use in the treatment and/or prevention of diseases selected from the group consisting of diseases, autoimmune diseases, and infectious diseases.
  41. 　請求項１～３５のいずれか一項に記載のオリゴ核酸ナノ粒子と、
    　疾患に対する１種以上の治療剤及び／又は１種以上の予防剤と、の組み合わせを含み、
    　前記疾患は、先天性代謝異常症、先天性内分泌疾患、単一遺伝子疾患、神経変性疾患、神経疾患、筋疾患、髄膜炎、脳炎、脳症、ライソゾーム病、悪性新生物、繊維症、炎症性疾患、免疫不全疾患、自己免疫疾患、及び感染症からなる群から選択される、医薬。     Oligonucleic acid nanoparticles according to any one of claims 1 to 35,
    Including a combination of one or more therapeutic agents and/or one or more preventive agents for diseases,
    The diseases include inborn errors of metabolism, congenital endocrine diseases, monogenic diseases, neurodegenerative diseases, neurological diseases, myopathies, meningitis, encephalitis, encephalopathy, lysosomal diseases, malignant neoplasms, fibrosis, and inflammatory diseases. A medicament selected from the group consisting of diseases, immunodeficiency diseases, autoimmune diseases, and infectious diseases.
  42. 　疾患に対する１種以上の治療剤及び／又は１種以上の予防剤と併用して疾患を治療するための、請求項１～３５のいずれか一項に記載のオリゴ核酸ナノ粒子であって、
    　前記疾患は、先天性代謝異常症、先天性内分泌疾患、単一遺伝子疾患、神経変性疾患、神経疾患、筋疾患、髄膜炎、脳炎、脳症、ライソゾーム病、悪性新生物、繊維症、炎症性疾患、免疫不全疾患、自己免疫疾患、及び感染症からなる群から選択される、オリゴ核酸ナノ粒子。     The oligonucleic acid nanoparticle according to any one of claims 1 to 35, for treating a disease in combination with one or more therapeutic agents and/or one or more prophylactic agents for the disease,
    The diseases include inborn errors of metabolism, congenital endocrine diseases, monogenic diseases, neurodegenerative diseases, neurological diseases, myopathies, meningitis, encephalitis, encephalopathy, lysosomal diseases, malignant neoplasms, fibrosis, and inflammatory diseases. Oligonucleic acid nanoparticles selected from the group consisting of diseases, immunodeficiency diseases, autoimmune diseases, and infectious diseases.
  43. 　（ａ１）樹状ポリマーで構成されたコアに複数個のオリゴ核酸を結合させる工程と、
    　（ａ２）前記オリゴ核酸に親水性ポリマーを結合させる工程と、
    　（ａ３）前記親水性ポリマーに細胞内在化促進剤を結合させる工程と、を含む、請求項１～４のいずれか一項に記載のオリゴ核酸ナノ粒子の製造方法。     (a1) A step of binding a plurality of oligonucleic acids to a core composed of a dendritic polymer,
    (a2) bonding a hydrophilic polymer to the oligonucleic acid;
    (a3) The method for producing oligonucleic acid nanoparticles according to any one of claims 1 to 4, comprising the step of binding a cell internalization promoter to the hydrophilic polymer.
  44. 　（ｂ１）樹状ポリマーで構成されたコアに１又は複数個のスペーサーを結合させる工程と、
    　（ｂ２）前記コアに複数個のオリゴ核酸を結合させる工程と、
    　（ｂ３）前記スペーサーに親水性ポリマーを結合させる工程と、
    　（ｂ４）前記親水性ポリマーに細胞内在化促進剤を結合させる工程と、を含む、請求項６～９のいずれか一項に記載のオリゴ核酸ナノ粒子の製造方法。     (b1) bonding one or more spacers to a core composed of a dendritic polymer;
    (b2) binding a plurality of oligonucleic acids to the core;
    (b3) bonding a hydrophilic polymer to the spacer;
    (b4) The method for producing oligonucleic acid nanoparticles according to any one of claims 6 to 9, comprising the step of binding a cell internalization promoter to the hydrophilic polymer.
  45. 　（ａ１）樹状ポリマーで構成されたコアに複数個のオリゴ核酸を結合させる工程と、
    　（ａ２）前記オリゴ核酸に親水性ポリマーを結合させる工程と、
    　（ａ３）前記親水性ポリマーに細胞内在化促進剤を結合させる工程と、
    　（ａ４）前記コアにキャッピング剤を結合させる工程と、を含む、請求項５に記載のオリゴ核酸ナノ粒子の製造方法。     (a1) A step of binding a plurality of oligonucleic acids to a core composed of a dendritic polymer,
    (a2) bonding a hydrophilic polymer to the oligonucleic acid;
    (a3) binding a cell internalization promoter to the hydrophilic polymer;
    The method for producing oligonucleic acid nanoparticles according to claim 5, comprising the step of (a4) binding a capping agent to the core.
  46. 　（ｂ１）樹状ポリマーで構成されたコアに１又は複数個のスペーサーを結合させる工程と、
    　（ｂ２）前記コアに複数個のオリゴ核酸を結合させる工程と、
    　（ｂ３）前記スペーサーに親水性ポリマーを結合させる工程と、
    　（ｂ４）前記親水性ポリマーに細胞内在化促進剤を結合させる工程と、
    　（ａ５）前記コアにキャッピング剤を結合させる工程と、を含む、請求項１０に記載のオリゴ核酸ナノ粒子の製造方法。     (b1) bonding one or more spacers to a core composed of a dendritic polymer;
    (b2) binding a plurality of oligonucleic acids to the core;
    (b3) bonding a hydrophilic polymer to the spacer;
    (b4) binding a cell internalization promoter to the hydrophilic polymer;
    The method for producing oligonucleic acid nanoparticles according to claim 10, comprising: (a5) binding a capping agent to the core.
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IN-HYUN LEE; SUKYUNG AN; MI KYUNG YU; HO-KEUN KWON; SIN-HYEOG IM; SANGYONG JON;: "Targeted chemoimmunotherapy using drug-loaded aptamer dendrimer bioconjugates", JOURNAL OF CONTROLLED RELEASE, ELSEVIER, AMSTERDAM, NL, vol. 155, no. 3, 22 May 2011 (2011-05-22), AMSTERDAM, NL , pages 435 - 441, XP028317073, ISSN: 0168-3659, DOI: 10.1016/j.jconrel.2011.05.025 *

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