WO2013172358A1 - Functional compound, molecular assembly containing functional compound, composition containing molecular assembly, kit, and use of molecular assembly, composition or kit - Google Patents

Functional compound, molecular assembly containing functional compound, composition containing molecular assembly, kit, and use of molecular assembly, composition or kit Download PDF

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WO2013172358A1
WO2013172358A1 PCT/JP2013/063459 JP2013063459W WO2013172358A1 WO 2013172358 A1 WO2013172358 A1 WO 2013172358A1 JP 2013063459 W JP2013063459 W JP 2013063459W WO 2013172358 A1 WO2013172358 A1 WO 2013172358A1
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lipid
group
molecular assembly
xhr
dendron
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PCT/JP2013/063459
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French (fr)
Japanese (ja)
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河野　健司
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公立大学法人大阪府立大学
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C237/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups
    • C07C237/02—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton
    • C07C237/04—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C237/10—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being acyclic and saturated having the nitrogen atom of at least one of the carboxamide groups bound to an acyclic carbon atom of a hydrocarbon radical substituted by nitrogen atoms not being part of nitro or nitroso groups
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G73/00—Macromolecular compounds obtained by reactions forming a linkage containing nitrogen with or without oxygen or carbon in the main chain of the macromolecule, not provided for in groups C08G12/00 - C08G71/00
    • C08G73/02—Polyamines
    • C08G73/028—Polyamidoamines
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G83/00—Macromolecular compounds not provided for in groups C08G2/00 - C08G81/00
    • C08G83/002—Dendritic macromolecules
    • C08G83/003—Dendrimers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • the present invention relates to a functional compound, a molecular assembly containing the compound, a composition and kit containing them, and uses thereof.
  • DDS drug delivery system
  • DDS for the purpose of improving target directivity of drugs, construction of so-called temperature-responsive carriers having a function of releasing drugs in response to temperature has been attempted. Specifically, an attempt has been made to use, as a carrier, a molecular assembly that shows a vesicle shape when not heated, but changes its shape when heated. After the drug is held inside the vesicle and administered into the living body, the structure of the molecular assembly is greatly changed by selectively heating a desired site such as an affected part, for example.
  • Non-patent Document 1 Attempts have been made to develop technology for releasing drugs only in More specifically, an attempt to use a molecular assembly composed of a compound in which a lipid having a polyamide dendron structure (polyamide dendron lipid) is modified with a temperature-responsive isobutylamide (IBAM) group for the above purpose. has been reported (Non-patent Document 1).
  • HLA major histocompatibility complex
  • HLA which is a human MHC
  • a peptide capable of acting on HLA Human Leukocyte Antigen
  • an immune-related disease can be treated by controlling HLA function.
  • cancer can be selectively treated by promoting antigen presentation by HLA and activating immunity.
  • the antigen is encapsulated in a liposome that has been modified with a pH-responsive polymer and can acquire a fusion ability with a lipid membrane in accordance with a change in pH, and then introduced into a dendritic cell, whereby the antigen is It has been reported that cell-mediated immunity was induced by delivery of the protein to the cytosol (Non-patent Document 2). More specifically, the liposome is once taken up into a cell by endocytosis, fuses with the endosome in response to the pH in the endosome, and finally releases the inclusion (antigen) into the cytosol.
  • Kenji Kono et al. “Thermosensitive Molecular Assembly from Poly (amidoamine) Dendron-Based Lipids”, “Angewandte Chemie International, 50 Years” 6332-6336 Eiji Yuba et al., “PH-Sensitive fusogenic polymer-modified liposomes as a carrier of antigenic protein for activation of Biomass, 31”. 943-951
  • the first object of the present invention is to improve the balance of (1) temperature responsiveness, (2) in vivo stability and (3) biocompatibility in a temperature responsive carrier that can be used for the above purpose. .
  • the present invention selectively delivers a target substance, specifically, a physiologically active substance, to an intracellular organelle of any one of (1) early endosome, (2) late endosome, and (3) lysosome.
  • the second problem is to provide a means that can be released by the above-mentioned method, that is, to provide a carrier as the means, a method of using the carrier, and the like.
  • a molecular assembly can be formed using a compound obtained by modifying a polyamide dendron lipid with a hydrocarbon group containing an oligoethylene glycol structure, which is expected to be excellent in biocompatibility.
  • the present inventors have found that there is difficulty in in vivo stability. Therefore, the present inventors formed a molecular assembly using a lipid containing a polyethylene glycol structure in addition to the above compound. In such a molecular assembly, (1) temperature responsiveness, (2) in vivo It has been found that the balance between stability and (3) biocompatibility is improved compared to the conventional one.
  • the present inventors have intensively studied to solve the second problem.
  • the above problem can be solved by using a compound obtained by modifying a predetermined dendron lipid with a pH-responsive polymer. More specifically, it is as follows.
  • a desired substance is encapsulated in a liposome composed of the above compound and then introduced into a cell, (1) it is efficiently incorporated into the initial endosome by endocytosis.
  • the liposome has a property of releasing inclusions in response to a specific pH range (response pH range), and it is easy to adjust the response pH range.
  • the present invention has been completed by the inventors of the present invention based on the above findings, and has been completed and includes the following embodiments.
  • An embodiment of the invention (first invention) as a solution to the first problem is as follows.
  • Item 1. (A) a compound represented by any of the following formulas DL-G1 to DL-G4; and (B) a molecular assembly DL-G1: R 1 R 2 NX (XHR containing a lipid containing a polyethylene glycol structure 3 ) XHR 4 DL-G2: R 1 R 2 NX (X (XHR 3 ) XHR 4 ) 2 DL-G3: R 1 R 2 NX (X (X (XHR 3 ) XHR 4 ) 2 ) 2 DL-G4: R 1 R 2 NX (X (X (X (XHR 3 ) XHR 4 ) 2 ) 2 ) 2 (Wherein R 1 and R 2 are the same or different and represent a saturated or unsaturated long chain hydrocarbon group, and R 3 and R 4 are hydrocarbons containing the same or different oligoethylene glycol structure.
  • R 1 to R 4 may contain a cyclic structure, and one or more carbon atoms may be substituted with a hetero atom, and X represents —CH 2 CH 2 CONHCH 2 CH 2 N—. Show. ).
  • Item 2. The molecular assembly according to Item 1, wherein R 3 and R 4 are represented by the following formula (I):
  • Item 3. The molecular assembly according to Item 1 or 2, wherein the lipid (B) is represented by the following formula (II):
  • Y is a hydrocarbon group having 10 to 50 carbon atoms (one or more carbon atoms may be substituted with a heteroatom), and n represents any integer of 5 to 200, and R 6 represents a hydrocarbon group having 1 to 10 carbon atoms (one or more carbon atoms may be substituted with a hetero atom).
  • Item 4. The molecular assembly according to any one of Items 1 to 3, which is used for introducing a physiologically active substance into a cell.
  • Item 5. The molecular assembly according to any one of Items 1 to 3, which is used for treating a disease by introducing a physiologically active substance into a cell.
  • Item 6. Item 4. A composition containing the molecular assembly according to any one of Items 1 to 3.
  • Item 7. Item 7.
  • Item 8. Use of the molecular assembly, composition or kit according to any one of Items 1 to 7 in a method for introducing a physiologically active substance into a cell.
  • Item 9. Item 8.
  • An embodiment of the invention as means for solving the second problem is as follows.
  • Item 1 Compound DL-G1: R 1 R 2 NX (XHR 3 ) XHR 4 represented by any of the following formulas DL-G1 to DL-G4 DL-G2: R 1 R 2 NX (X (XHR 3 ) XHR 4 ) 2 DL-G3: R 1 R 2 NX (X (X (XHR 3 ) XHR 4 ) 2 ) 2 DL-G4: R 1 R 2 NX (X (X (XHR 3 ) XHR 4 ) 2 ) 2 ) 2 (Wherein R 1 and R 2 are the same or different and represent a saturated or unsaturated long chain hydrocarbon group, R 3 and R 4 represent the same or different pH-responsive carboxyl group-containing hydrocarbon group, R 1 to R 4 may contain a cyclic structure, and one or more carbon atoms are heteroatoms And X represents —CH 2 CH 2 CONHCH 2 CH 2
  • R 5 represents a saturated or unsaturated hydrocarbon group having 1 to 10 carbon atoms (which may contain a cyclic structure, and one or more carbon atoms may be substituted with a hetero atom).
  • the pH-responsive carboxyl group-containing hydrocarbon group is (A) contains a cyclohexyl group, a phenyl group, a pyridyl group or a naphthyl group, or (B) one or more carbon atoms having 1 or 2 carbon atoms (one or more carbon atoms may be substituted with a heteroatom). It is a chain structure that may have a branch, Item 3. The compound according to Item 1 or 2.
  • Item 5. The composition according to Item 4, comprising a phospholipid.
  • Item 6. The composition according to Item 4 or 5, which is used for introducing a physiologically active substance into cells.
  • Item 7. Item 6.
  • Item 8. Item 6.
  • Item 9. Item 6.
  • Item 10. Item 10.
  • Item 11 Use of the compound, composition or kit according to any one of Items 1 to 9 in a method for introducing a physiologically active substance into a cell.
  • Item 11. A method for introducing a physiologically active substance into a cell, comprising a step of introducing the compound, composition or kit according to any one of Items 1 to 9 into the cell together with the physiologically active substance.
  • a temperature-responsive carrier used for the purpose of improving the target directivity of a physiologically active substance that can be administered to a living body as a drug, comprising (1) temperature responsiveness, (2) A carrier having an improved balance between in vivo stability and (3) biocompatibility can be provided.
  • the carrier by using the carrier, it is possible to provide a method for delivering a physiologically active substance with improved target directivity and reduced harm that can be given to a living body.
  • the target substance can be selectively delivered to any desired intracellular organelle of (1) early endosome, (2) late endosome, and (3) lysosome.
  • a physiologically active substance that can become an antigen or generate an antigen when used as a physiologically active substance, cell immunity can be achieved through delivery of the substance to intracellular organelles by targeting the antigen-presenting cell. It has an excellent effect that can be induced.
  • Differential scanning calorimetry of MDEG-DL-G1 assembly dispersion, MDEG-DL-G2 assembly dispersion, and MDEG-DL-G1-U2 assembly dispersion (10 mM phosphoric acid 140 m NaCl aqueous solution, pH7.4) It is a measurement (DSC) chart.
  • Differential scanning calorimetry of EDEG-DL-G1 assembly dispersion, EDEG-DL-G2 assembly dispersion, and EDEG-DL-G1-U2 assembly dispersion (10 mM phosphate 140 m NaCl aqueous solution, pH7.4) It is a measurement (DSC) chart.
  • EDEG-DL-G1-2C 18 / PEG-Chol (95/5) and MDEG-DL-G1-2C 18 / PEG-Chol (98/2) aggregates both have a cloud point at about 40 ° C.
  • FIG. 5 is a graph showing the results of plotting pyranine release (%) against pH after incubation of various liposomes at 37 ° C. for 15 minutes (UMG1.25, UCG1.25, SCG1.25 and EYPC are respectively MGlu- DL-G1-2C 18 -U2 / EYPC (25/75, mol / mol), CHex-DL-G1-2C 18 -U2 / EYPC (25/75, mol / mol), CHex-DL-G1-2C 18 / EYPC (25/75, mol / mol) and liposomes with EYPC as components).
  • UG1.25, UCG1.25, SCG1.25 and EYPC are respectively MGlu- DL-G1-2C 18 -U2 / EYPC (25/75, mol / mol), CHex-DL-G1-2C 18 -U2 / EYPC (25/75, mol / mol), CHex-DL-G1-2C 18 / EYPC
  • FIG. 6 is a graph showing the results of plotting pyranine release (%) against pH after incubation of various liposomes at 37 ° C. for 15 minutes (UMG1.25, UMG1.40 and EYPC are respectively MGMG-DL-U2 / Liposomes with EYPC mole ratios of 25, 40 and 0 are shown). It is a graph showing the results of plotting the release of pyranine (%) against pH after incubating liposomes containing CHexDL-U2 / EYPC for 15 minutes at 37 ° C (UCG1.25, UCG1.10 and EYPC are , Liposomes with CHexDL-U2 / EYPC molar ratios of 25, 10 and 0, respectively).
  • CHex-DL-2C 18 -U2 / EYPC 25/75, mol / mol
  • CHex-DL-2C 18 -U2 / EYPC 25/75, mol / mol
  • CHex-DL-2C 18 / EYPC 25/75, mol / mol
  • CHex-DL-2C 18 / EYPC 25/75, mol / mol
  • CHex-DL-2C 18 / EYPC 25/75, mol / mol
  • CHex-DL-2C 18 / EYPC 25/75, mol / mol
  • EYPC The pyranine release behavior at various pH 7.4 encapsulating pyranine is shown (EYPC, UMCG1.1525, UMCG1.2020 and UMG1.40 are respectively EYPC liposome, MGlu-G1-2C 18 -U2 / CHex-G1- 2C 18 -U2 / EYPC (15/25/60, mol / mol / mol) Liposome, MGlu-G1-2C 18 -U2 / CHex-G1-2C 18 -U2 / EYPC (20/20/60, mol / mol / mol) Liposomes and CHex-G1-2C 18 -U2 / EYPC (40/60, mol / mol / mol) liposomes).
  • EYPC UMCG1.1525, UMCG1.2020 and UMG1.40 are respectively EYPC liposome, MGlu-G1-2C 18 -U2 / CHex-G1- 2C 18 -U2 / EYPC (15/25/60, mol / mol / mol) Liposome, MGlu-G1-2C 18 -U2 / CHex-G1-2C 18 -U2 / EYPC (20/20/60, mol / mol / mol) Liposomes and CHex-G1-2C 18 -U2 / EYPC (40/60, mol / mol / mol) liposomes).
  • EYPC UMCG1.1525, UMCG1.2020 and UMG1.40 are respectively EYPC liposome, MGlu-G1-2C 18 -U2 / CHex-G1- 2C 18 -U2 / EYPC (15/25/60, mol / mol / mol) Liposome, MGlu-G1-2C 18 -U2 / CHex-G1-2C 18 -U2 / EYPC (20/20/60, mol / mol / mol) Liposomes and CHex-G1-2C 18 -U2 / EYPC (40/60, mol / mol / mol) liposomes).
  • Various liposome encapsulated pyranine shows pyranine release behavior after 15 minutes incubation at various pH (EYPC, UMCG1.1525, UMCG1.2020 and UMG1.40 each EYPC liposomes, MGlu-G1-2C 18 - U2 / CHex-G1-2C 18 -U2 / EYPC (15/25/60, mol / mol / mol) Liposomes, MGlu-G1-2C 18 -U2 / CHex-G1-2C 18 -U2 / EYPC (20/20 / 60, mol / mol / mol) liposome and CHex-G1-2C 18 -U2 / EYPC (40/60, mol / mol / mol) liposome).
  • the molecular assembly of the present invention comprises: (A) a compound represented by any one of the following formulas DL-G1 to DL-G4; and (B) a molecular assembly DL-G1: R 1 R 2 NX (XHR containing a lipid containing a polyethylene glycol structure 3 ) XHR 4 DL-G2: R P R 2 NX (X (XHR 3) XHR 4) 2 DL-G3: R 1 R 2 NX (X (X (XHR 3 ) XHR 4 ) 2 ) 2 DL-G4: R 1 R 2 NX (X (X (X (XHR 3 ) XHR 4 ) 2 ) 2 ) 2 (Wherein R 1 and R 2 are the same or different and represent a saturated or unsaturated long chain hydrocarbon group, and R 3 and R 4 are hydrocarbons containing the same or different oligoethylene glycol structure. Containing groups, R 1 to R 4 may
  • X represents —CH 2 CH 2 CONHCH 2 CH 2 N—, and the terminal N usually has two hydrogen atoms. However, one hydrogen atom may be substituted with a hydrophobic amino acid or an alkyl group.
  • hydrophobic amino acids include leucine, valine, isoleucine, norleucine, phenylalanine and tyrosine.
  • alkyl group include a methyl group, an ethyl group, a normal propyl group, an isopropyl group, and a cyclopropyl group.
  • R 1 and R 2 are long chain, saturated or unsaturated hydrocarbon group.
  • the long-chain hydrocarbon group may be naturally derived, may be modified from naturally derived, or may be artificially synthesized.
  • the number of carbon atoms of the long-chain hydrocarbon group is not limited as long as the main chain is a long chain.
  • the number of carbon atoms in the main chain is preferably 8 to 30, more preferably 10 to 22, and still more preferably 12 to 20.
  • the long chain hydrocarbon group may have a branch.
  • the main chain is determined based on the IUPAC nomenclature, or when it is difficult or impossible, it is determined based on a similar method.
  • the long chain hydrocarbon group may have a cyclic structure. Although it does not specifically limit as a cyclic structure, For example, a phenyl group, a cholesteryl group, a cyclohexyl group etc. are mentioned. Particularly preferred is a cholesteryl group.
  • the long chain hydrocarbon group may be one in which at least one carbon atom is substituted with a hetero atom.
  • the hetero atom means an oxygen atom, a nitrogen atom or a sulfur atom.
  • the long chain hydrocarbon group includes an oxygen-containing hydrocarbon group, a nitrogen-containing hydrocarbon group, and a sulfur-containing hydrocarbon group.
  • the hydrocarbon group means an alkyl group, an alkenyl group or an alkynyl group.
  • oxygen-containing hydrocarbon group examples include an oxygen-containing hydrocarbon group having at least one bond selected from the group consisting of an ether bond and a carbonyl bond.
  • oxygen-containing hydrocarbon group having a carbonyl bond examples include a group having at least one structure selected from the group consisting of aldehyde, ketone, carboxylic acid, ester, amide, enone, acid chloride, or anhydride. Can be mentioned.
  • nitrogen-containing hydrocarbon group examples include a nitrogen-containing hydrocarbon group having at least one structure selected from the group consisting of nitrile, amine, amide and imide.
  • sulfur-containing hydrocarbon group examples include thiol, thioether, thioacetal, sulfide, disulfide, dithiocarboxylic acid, thioester, thioketone, thioaldehyde, thiocarbamate, thiourethane, phosphine sulfide, thiophosphate, thiophosphonate, sulfonate, sulfone and And sulfur-containing hydrocarbon groups having at least one bond selected from the group consisting of sulfonamides.
  • the long chain hydrocarbon group may further have at least one substituent in the group having the structure as exemplified above.
  • substituent those showing hydrophobicity are preferable.
  • substituent include a phenyl group, a cholesteryl group, and a pyrenyl group.
  • a cholesteryl group is particularly preferable.
  • the unsaturated long chain hydrocarbon group is preferably a hydrocarbon group having an unsaturated bond in the main chain and the main chain having a chain structure.
  • the type of unsaturated bond may be a double bond or a triple bond.
  • a double bond is preferred.
  • the number of unsaturated bonds should just be at least 1 or more, and is not limited. Moreover, you may have both a double bond and a triple bond. It preferably has one double bond.
  • the double bond may be a cis-type double bond or a trans-type double bond.
  • a cis type double bond is preferred.
  • unsaturated long-chain hydrocarbon groups include hexadecenyl, octadecenyl, octadecadienyl, octadecatrienyl, icosatrienyl, icosatetraenyl, octadecatrienyl, icosapentaenyl, or docosa A hexaenyl group is preferred.
  • unsaturated long chain hydrocarbon groups include 9-hexadecenyl group, 9-octadecenyl group (oleyl group), 12-octadecadienyl group, 6,9,12-octadecatrienyl group, 8,11,14- Eicosatrienyl group, 5,8,11,14-icosatetraenyl group, 9,12,15-octadecatrienyl group, 5,8,11,14,17-icosapentaenyl group or 4,7,13 , 16,19-docosahexaenyl group is more preferred.
  • a 9-octadecenyl group (oleyl group) is more preferable.
  • Preferred examples of the unsaturated long-chain hydrocarbon group include those in which at least one carbon atom is further substituted with a hetero atom in the group having the structure exemplified above as a preferred example.
  • Preferred examples of the unsaturated long chain hydrocarbon group are specific to oxygen-containing hydrocarbon groups, nitrogen-containing hydrocarbon groups, or sulfur-containing hydrocarbon groups on the basis of the group having the structure exemplified above as a preferred example.
  • the structure may further include a structure as exemplified above.
  • the group having the structure as exemplified above as a preferred example may further have at least one substituent.
  • substituent those showing hydrophobicity are preferable.
  • substituent include a phenyl group, a phenylene group, a cholesteryl group, and a pyrenyl group.
  • a cholesteryl group is particularly preferable.
  • hydrocarbon group containing oligoethylene glycol structure is oligoethylene glycol. It only needs to contain a structure, and may further contain other structures. In the oligoethylene glycol-containing hydrocarbon group, other structures may be interposed between the oligoethylene glycol structures, or the oligoethylene glycol structure may be interposed between the other structures. Good.
  • the other structure may be a linear structure which may have a branch.
  • a structure is not particularly limited, and examples thereof include those having a straight chain portion having 1 to 10 carbon atoms which may have a branch having 1 to 10 carbon atoms.
  • the other structure may be a ring structure.
  • the cyclic structure is not particularly limited, and examples thereof include a cyclopropyl group, an epoxy group, a cycloheptyl group, a cyclobutyl group, a phenyl group, a cholesteryl group, and a cyclohexyl group.
  • a cyclohexyl group is particularly preferable.
  • one or more carbon atoms may be substituted with a heteroatom.
  • the oligoethylene glycol structure is not particularly limited as long as the effects of the present invention can be achieved.
  • the oligoethylene glycol structure has 1 to 10 oxyethylene units continuously, in other words, without any other structure interposed. Structure is mentioned.
  • the oligoethylene glycol structure preferably has 1 to 4 oxyethylene units in succession.
  • the oligoethylene glycol structure is not particularly limited as long as the effects of the present invention can be achieved.
  • a structure having two or more divided oxyethylene units in other words, through another structure, a total of 1 Examples include structures having up to 10 oxyethylene units. In this case, those having a total of 1 to 4 oxyethylene units are preferred.
  • the branched chain of the oligoethylene glycol-containing hydrocarbon group may further have an oligoethylene glycol structure.
  • Oligoethylene glycol-containing hydrocarbon group is preferably represented by the following formula (I):
  • n represents an integer of 1 to 10 and R 5 represents a hydrocarbon group having 1 to 10 carbon atoms (one or more carbon atoms may be substituted with a hetero atom)).
  • R 5 represents a hydrocarbon group having 1 to 10 carbon atoms (one or more carbon atoms may be substituted with a hetero atom)
  • Specific examples of the oligoethylene glycol-containing hydrocarbon group include those represented by the following formula (III).
  • n an integer of 1 to 10
  • lipid (B) Containing Polyethylene Glycol Structure only needs to contain a polyethylene glycol structure, and as long as the effects of the present invention are exhibited, It is not limited.
  • the lipid (B) containing a polyethylene glycol structure is divided into a part containing a polyethylene glycol structure and a lipid part.
  • the part containing the polyethylene glycol structure only needs to contain a polyethylene glycol structure, and may further contain other hydrocarbon structures.
  • another hydrocarbon structure may be interposed between the polyethylene glycol structures, or the polyethylene glycol structure may be interposed between the other hydrocarbon structures.
  • the other hydrocarbon structure may have a branch or a straight chain structure.
  • Such a structure is not particularly limited, and examples thereof include those having a straight chain portion having 1 to 10 carbon atoms which may have a branch having 1 to 10 carbon atoms.
  • the other hydrocarbon structure may be a cyclic structure.
  • the cyclic structure is not particularly limited, and examples thereof include a cyclopropyl group, an epoxy group, a cycloheptyl group, a cyclobutyl group, a phenyl group, a cholesteryl group, and a cyclohexyl group.
  • a cyclohexyl group is particularly preferable.
  • one or more carbon atoms may be substituted with a hetero atom.
  • the polyethylene glycol structure is not particularly limited as long as the effects of the present invention can be achieved.
  • a structure having 3 to 200 oxyethylene units continuously in other words, without any other structure interposed therebetween.
  • the polyethylene glycol structure preferably has 3 to 200 oxyethylene units in succession, more preferably 10 to 100.
  • the lipid portion is not particularly limited as long as the effect of the present invention is exhibited.
  • Examples of the lipid moiety include a hydrocarbon group having 10 to 50 carbon atoms (one or more carbon atoms may be substituted with a hetero atom).
  • the hydrocarbon group may be saturated or unsaturated, and may have a cyclic structure.
  • lipid portion examples include phospholipids such as cholesterol and phosphatidylethanolamine, glycolipids, sphingolipids, long-chain fatty acids such as arachidonic acid, diacylglycerol, and the like.
  • the lipid (B) containing a polyethylene glycol structure is preferably represented by the following formula (II)
  • Y is a hydrocarbon group having 5 to 50 carbon atoms (one or more carbon atoms may be substituted with a heteroatom), and n represents any integer of 10 to 20, and R 6 represents a hydrocarbon group having 1 to 10 carbon atoms (one or more carbon atoms may be substituted with a hetero atom).
  • R 6 represents a hydrocarbon group having 1 to 10 carbon atoms (one or more carbon atoms may be substituted with a hetero atom).
  • Y is a hydrocarbon group having 5 to 50 carbon atoms (one or more carbon atoms may be substituted with a hetero atom), and n represents any integer of 10 to 20) .
  • the molecular assembly of the present invention is a molecular assembly containing a compound (A) and a lipid (B).
  • the molecular aggregate refers to an aggregate of at least the compound (A) and the lipid (B).
  • the molecular assembly of the present invention has a vesicle shape or an inverted hexagonal shape. Whether the molecular assembly is a vesicle shape or an inverted hexagonal shape can be confirmed using an atomic force microscope (AFM). If a spherical molecular assembly is observed by AFM observation, it can be evaluated that the molecular assembly has a vesicle shape. Although not particularly limited, for example, a sphere having a particle size of about 150 to 200 nm can be observed as a vesicle-shaped molecular assembly. On the other hand, if a rod-like molecular assembly is observed by AFM observation, it can be evaluated that the molecular assembly has an inverted hexagonal shape.
  • the compound (A) of the present invention exhibits temperature responsiveness due to the action of the oligoethylene glycol structure.
  • the molecular assembly of the present invention has a vesicle shape under an environment of a predetermined temperature, but changes to an inverted hexagonal shape when the temperature of the environment increases.
  • the vesicle-shaped molecular assembly of the present invention forms a lipid bilayer in an aqueous solution so that the R 1 and R 2 sides face the inside and the R 3 and R 4 sides face the outside. Since the surface of this vesicle-shaped molecular assembly is hydrophilic, it is weakly adsorbed on the cell surface when administered in vivo. A desired hydrophobic substance can be held inside the lipid bilayer membrane of the vesicle-shaped molecular assembly.
  • the reverse hexagonal-shaped molecular assembly of the present invention has a hydrophobic surface and is easily taken up into cells by endocytosis.
  • a vesicle-shaped molecular assembly is administered to a living body in a state where a desired hydrophobic substance is held inside the lipid bilayer membrane, and the environmental temperature of the desired site among the sites to which the molecular assembly is delivered is set.
  • the substance can be selectively introduced into the cell at the site by raising and changing to an inverted hexagonal shape.
  • the molecular assembly of the present invention may be in a dry state or a frozen state.
  • each component containing the compound (A) and lipid (B) is once dissolved in an organic solvent such as chloroform and then dried under reduced pressure using an evaporator or a spray dryer. It can be manufactured by spray drying.
  • the compound (A) of the present invention can be obtained, for example, by adding R 3 and R 4 to a polyamide dendron (DL) obtained as follows.
  • DL-G1 to DL-G4 indicate first to fourth generation polyamide dendrons, respectively.
  • DL-G0 is the 0th generation and does not have a dendron structure.
  • R 1 and R 2 are unsaturated hydrocarbon groups, they can be produced as follows. Oleylamine and oleyl chloride are reacted to synthesize oleyloleylamide. Next, dioleylamine is synthesized by hydride reduction, and a polyamidoamine dendron is synthesized by repeating a Michael addition reaction using methyl acrylate and an ester amide exchange reaction using ethylenediamine. This polyamidoamine dendron is represented as DL-G1-2C 18 -U2.
  • R ⁇ 1 > and R ⁇ 2 > is a saturated hydrocarbon group
  • Polyamideamine dendron is synthesized by repeating Michael addition reaction using methyl acrylate and ester amide exchange reaction using ethylenediamine using dioctadecylamine as a starting material.
  • the polyamidoamine dendron denoted as DL-G1-2C 18.
  • R 3 and R 4 can be performed as follows, for example, as shown in the following formula.
  • MDEG methoxydiethylene glycol
  • EDEG ethoxydiethylene glycol
  • composition of the present invention further contains other components in addition to the molecular assembly of the present invention.
  • an aqueous solvent (dispersion medium) is contained.
  • other components include water, sugar aqueous solutions such as glucose, lactose, and sucrose, polyhydric alcohol aqueous solutions such as glycerin and propylene glycol, phosphate buffer, and citrate buffer. Liquid, buffer solution such as phosphate buffered saline, physiological saline, medium for cell culture, and the like.
  • an aqueous solution of sugar when freeze-preserving or spray-drying preservation, an aqueous solution of sugar can be used, and when storing frozen, an aqueous solution of sugar or aqueous polyhydric alcohol can be used for effective preservation.
  • the concentration of these aqueous solvent additives is not particularly limited.
  • an aqueous sugar solution 2 to 20% (W / V) is preferable, and 5 to 10% (W / V) is more preferable. preferable.
  • a polyhydric alcohol aqueous solution 1 to 5% (W / V) is preferable, and 2 to 2.5% (W / V) is more preferable.
  • the concentration of the buffer is preferably 5 to 50 mM, more preferably 10 to 20 mM.
  • the concentration of the molecular assembly of the present invention in the aqueous solvent is not particularly limited, but is preferably 0.01 mM to 100 mM, more preferably 0.1 mM to 10 mM.
  • the form in which the molecular assembly of the present invention is dispersed in an aqueous solvent is obtained by adding the dried lipid mixture to an aqueous solvent and further emulsifying with an emulsifier such as a homogenizer, an ultrasonic emulsifier, a high-pressure jet emulsifier, or the like.
  • an emulsifier such as a homogenizer, an ultrasonic emulsifier, a high-pressure jet emulsifier, or the like.
  • an emulsifier such as a homogenizer, an ultrasonic emulsifier, a high-pressure jet emulsifier, or the like.
  • emulsifier such as a homogenizer, an ultrasonic emulsifier, a high-pressure jet emulsifier, or the like.
  • it can manufacture also by the method well-known as a method of manufacturing a liposome, for example, a reverse phase evaporation method etc., It should
  • examples of a method for further drying the molecular assembly dispersed in the aqueous solvent include ordinary freeze drying and spray drying.
  • a sugar aqueous solution preferably a sucrose aqueous solution or a lactose aqueous solution may be used.
  • the molecular assembly can be stored for a long period of time, and when an aqueous solution containing a desired substance is added to the dried molecular assembly. This is preferable because the substance is efficiently retained in the molecular assembly.
  • the molecular assemblies and compositions of the present invention are preferably each used to introduce a desired hydrophobic substance into a cell.
  • the hydrophobic desired substance is not particularly limited, and examples thereof include physiologically active substances such as low molecular weight compounds, peptides, lipids, hormones, proteins, and nucleic acid derivatives.
  • physiologically active substances such as low molecular weight compounds, peptides, lipids, hormones, proteins, and nucleic acid derivatives.
  • various anticancer agents such as doxorubicin, hormone preparations, and antigen molecules for cancer immunotherapy such as WT1 peptide and its derivatives can be used.
  • the molecular assembly and composition of the present invention may be directly introduced into the individual to be treated together with the physiologically active substance to be introduced for the purpose of treating the above-mentioned diseases.
  • the molecular assembly or composition of the present invention may be administered to an individual.
  • the means for administration to an individual may be oral administration or parenteral administration, but parenteral administration is preferred.
  • the dosage form may be a conventionally known dosage form, and examples of the dosage form for oral administration include tablets, powders, granules, syrups and the like.
  • Examples of the dosage form for parenteral administration include injections, eye drops, ointments, suppositories and the like. Among these, an injection is preferable, and an administration method is preferably intravenous injection or local injection into a target cell or organ.
  • the blending ratio of the desired hydrophobic substance and the compound (A) of the present invention is such that the compound (A) of the present invention is 1 to 1000 parts by weight, preferably 10 to 500 parts by weight per 1 part by weight of the substance. Preferably 10 to 100 parts by weight are used.
  • Second invention Compound of the Present Invention
  • the compound of the present invention is a compound DL-G1: R 1 R 2 NX (XHR 3 ) XHR 4 represented by any of the following formulas DL-G1 to DL-G4 DL-G2: R 1 R 2 NX (X (XHR 3 ) XHR 4 ) 2 DL-G3: R 1 R 2 NX (X (X (XHR 3 ) XHR 4 ) 2 ) 2 DL-G4: R 1 R 2 NX (X (X (X (XHR 3 ) XHR 4 ) 2 ) 2 ) 2 (Wherein R 1 and R 2 are the same or different and represent a saturated or unsaturated long chain hydrocarbon group, R 3 and R 4 represent the same or different pH-responsive carboxyl group-containing hydrocarbon group, R 1 to R 4 may contain a cyclic structure, and one or more carbon atoms are heteroatoms And X represents —CH 2 CH 2 CONHCH 2 CH 2 N—.
  • X represents —CH 2 CH 2 CONHCH 2 CH 2 N—, and the terminal N usually has two hydrogen atoms, but one hydrogen atom is a hydrophobic amino acid. May be substituted.
  • hydrophobic amino acids include leucine, valine, isoleucine, norleucine, phenylalanine and tyrosine.
  • R 1 and R 2 for R 1 and R 2 are long chain, saturated or unsaturated hydrocarbon group.
  • the long-chain hydrocarbon group may be naturally derived, may be modified from naturally derived, or may be artificially synthesized.
  • the number of carbon atoms of the long-chain hydrocarbon group is not limited as long as the main chain is a long chain.
  • the number of carbon atoms in the main chain is preferably 8 to 30, more preferably 10 to 22, and still more preferably 12 to 20.
  • the long chain hydrocarbon group may have a branch.
  • the main chain is determined based on the IUPAC nomenclature, or when it is difficult or impossible, it is determined based on a similar method.
  • the long chain hydrocarbon group may have a cyclic structure. Although it does not specifically limit as a cyclic structure, For example, a cyclohexyl group, a phenyl group, a pyridyl group, a naphthyl group, etc. are mentioned. A cyclohexyl group is particularly preferable.
  • the long chain hydrocarbon group may be one in which at least one carbon atom is substituted with a hetero atom.
  • the hetero atom means an oxygen atom, a nitrogen atom or a sulfur atom.
  • the long chain hydrocarbon group includes an oxygen-containing hydrocarbon group, a nitrogen-containing hydrocarbon group, and a sulfur-containing hydrocarbon group.
  • the hydrocarbon group means an alkyl group, an alkenyl group or an alkynyl group.
  • oxygen-containing hydrocarbon group examples include an oxygen-containing hydrocarbon group having at least one bond selected from the group consisting of an ether bond and a carbonyl bond.
  • oxygen-containing hydrocarbon group having a carbonyl bond examples include a group having at least one structure selected from the group consisting of aldehyde, ketone, carboxylic acid, ester, amide, enone, acid chloride, or anhydride. Can be mentioned.
  • nitrogen-containing hydrocarbon group examples include a nitrogen-containing hydrocarbon group having at least one structure selected from the group consisting of nitrile, amine, amide and imide.
  • sulfur-containing hydrocarbon group examples include thiol, thioether, thioacetal, sulfide, disulfide, dithiocarboxylic acid, thioester, thioketone, thioaldehyde, thiocarbamate, thiourethane, phosphine sulfide, thiophosphate, thiophosphonate, sulfonate, sulfone and And sulfur-containing hydrocarbon groups having at least one bond selected from the group consisting of sulfonamides.
  • the long chain hydrocarbon group may further have at least one substituent in the group having the structure as exemplified above.
  • substituent those showing hydrophobicity are preferable.
  • substituent include a phenyl group, a cholesteryl group, and a pyrenyl group.
  • a cholesteryl group is particularly preferable.
  • the unsaturated long chain hydrocarbon group is preferably a hydrocarbon group having an unsaturated bond in the main chain and the main chain having a chain structure.
  • the type of unsaturated bond may be a double bond or a triple bond.
  • a double bond is preferred.
  • the number of unsaturated bonds should just be at least 1 or more, and is not limited. Moreover, you may have both a double bond and a triple bond. It preferably has one double bond.
  • the double bond may be a cis-type double bond or a trans-type double bond.
  • a cis type double bond is preferred.
  • unsaturated long-chain hydrocarbon groups include hexadecenyl, octadecenyl, octadecadienyl, octadecatrienyl, icosatrienyl, icosatetraenyl, octadecatrienyl, icosapentaenyl, or docosa A hexaenyl group is preferred.
  • unsaturated long chain hydrocarbon groups include 9-hexadecenyl group, 9-octadecenyl group (oleyl group), 12-octadecadienyl group, 6,9,12-octadecatrienyl group, 8,11,14- Eicosatrienyl group, 5,8,11,14-icosatetraenyl group, 9,12,15-octadecatrienyl group, 5,8,11,14,17-icosapentaenyl group or 4,7,13 , 16,19-docosahexaenyl group is more preferred.
  • a 9-octadecenyl group (oleyl group) is more preferable.
  • Preferred examples of the unsaturated long-chain hydrocarbon group include those in which at least one carbon atom is further substituted with a hetero atom in the group having the structure exemplified above as a preferred example.
  • Preferred examples of the unsaturated long chain hydrocarbon group are specific to oxygen-containing hydrocarbon groups, nitrogen-containing hydrocarbon groups, or sulfur-containing hydrocarbon groups on the basis of the group having the structure exemplified above as a preferred example.
  • the structure may further include a structure as exemplified above.
  • the group having the structure as exemplified above as a preferred example may further have at least one substituent.
  • substituent those showing hydrophobicity are preferable.
  • substituent include a phenyl group, a cholesteryl group, and a pyrenyl group.
  • a cholesteryl group is particularly preferable.
  • pH-responsive carboxyl group-containing hydrocarbon group may contain a cyclic structure.
  • one or more carbon atoms may be substituted with a hetero atom.
  • the pH-responsive carboxyl group-containing hydrocarbon group is not particularly limited, but preferably has 2 to 20 carbon atoms, more preferably 2 to 10 carbon atoms, and still more preferably 2 to 6 carbon atoms.
  • the hetero atom is counted as a carbon atom.
  • the pH-responsive carboxyl group-containing hydrocarbon group is preferably represented by the following formula (I)
  • R 5 represents a saturated or unsaturated hydrocarbon group having 1 to 10, preferably 1 to 5, more preferably 1 to 3 carbon atoms (which may contain a cyclic structure and has one or more carbon atoms). Carbon atoms may be substituted with heteroatoms).
  • the pH-responsive carboxyl group-containing hydrocarbon group is more preferably (A) contains a cyclohexyl group, a phenyl group, a pyridyl group or a naphthyl group, or (B) one or more carbon atoms having 1 or 2 carbon atoms (one or more carbon atoms may be substituted with a heteroatom). It is a chain structure which may have a branch.
  • pH-responsive carboxyl group-containing hydrocarbon group corresponding to the above (A) those containing a cyclohexyl group are preferable.
  • pH-responsive carboxyl group-containing hydrocarbon group corresponding to the above (A) include those represented by the following formula (II).
  • the pH-responsive carboxyl group-containing hydrocarbon group corresponding to the above (B) more preferably has one or more branches having 1 carbon atom (the carbon atom may be substituted with a hetero atom). It may be a chain structure.
  • the pH-responsive carboxyl group-containing hydrocarbon group corresponding to the above (B) is more preferably 1 to 2 having 1 or 2 carbon atoms (one or more carbon atoms may be substituted with a hetero atom). It is a chain structure which may have the following branches.
  • the pH-responsive carboxyl group-containing hydrocarbon group corresponding to the above (B) more preferably has 1 to 2 branches having 1 carbon atom (the carbon atom may be substituted with a hetero atom). It may be a chain structure.
  • the pH-responsive carboxyl group-containing hydrocarbon group corresponding to (B) has one branch having 1 carbon atom (the carbon atom may be substituted with a heteroatom). It is a good chain structure.
  • pH-responsive carboxyl group-containing hydrocarbon group corresponding to (B) above include those represented by the following formula (III).
  • the compound of the present invention forms a vesicle in an aqueous solution by forming a lipid bilayer with the R 1 and R 2 sides facing inward and the R 3 and R 4 sides facing outward. To do.
  • This vesicle is excellent in the ability to penetrate into cells.
  • a physiologically active substance or the like can be encapsulated in the vesicle. Therefore, the compound of the present invention can be used for introducing a physiologically active substance into cells.
  • the compound of the present invention exhibits pH responsiveness due to the action of a pH responsive carboxyl group-containing hydrocarbon group.
  • PH responsiveness means that a vesicle constituted by the compound of the present invention is destabilized under an environment of a predetermined pH and releases inclusions.
  • the compound of the present invention is derived from a pH-responsive carboxyl group-containing hydrocarbon group and has a carboxyl group (A) that can exhibit a negative charge when protons dissociate.
  • the compound of this invention has the tertiary amine (B) of the polyamide dendron which can receive a protonation. Therefore, in the compound of the present invention, when the pH is continuously changed from the basic side to the acidic side, the states of the carboxyl group (A) and the tertiary amine (B) on the left change as follows. It will follow.
  • the ⁇ potential of the compound is considered to change from a negative value to a positive value while continuously changing from the basic side to the acidic side.
  • the carboxyl group is protonated, the surface of the vesicle becomes hydrophobic, and further, the internal tertiary amine is protonated, resulting in electrostatic repulsion between molecules. Thereby, it is considered that the vesicle is destabilized and the inclusion is released.
  • the compound of the present invention exhibiting desired pH responsiveness can be provided.
  • foreign substances taken into cells via phagocytosis are first retained in (1) intracellular organelles called early endosomes, and eventually, early endosomes change to (2) late endosomes. . If kept in the late endosome, the foreign substance can finally be delivered to an intracellular organelle called (3) lysosome. And (1) the early endosome, (2) the late endosome, and (3) the interior of the lysosome have different pH values of about pH 6.2, pH 6.0 to 5.0, and pH 5.0 to 4.0, respectively. ing.
  • the compound of the present invention when it is used as a vesicle, it is stabilized in a pH range higher than, for example, pH 6.2, and is designed to be destabilized only at about pH 6.2.
  • a carrier is obtained that can deliver the substance and release the encapsulated substance in the early endosome.
  • it is stabilized in a pH region higher than pH 6.0 when it is used as a vesicle, and is designed to be destabilized at pH 6.0 for the first time to deliver encapsulated substances to late endosomes, and A carrier that can release the inclusion substance in the late endosome is obtained.
  • delivery of encapsulated material to the cytosol may cause cytotoxicity.
  • delivery of the antigen to the cytosol may inhibit cellular immunity induction.
  • the binding between the MHC class II molecule and the antigen leading to humoral immunity induction is considered to occur in the late endosome to lysosome. For this reason, when the carrier is decomposed in the lysosome and the antigen is released, it may not lead to effective antigen presentation.
  • Vesicles composed of the compounds of the present invention recognize the difference in acidity in the process from early endosome to late endosome to lysosome after being taken up by cells, and efficiently release inclusions at appropriate timing it can. Such a function can be useful for various applications. For example, it can help in developing vaccines that effectively induce humoral immunity. More details are as follows. In order to effectively induce humoral immunity, (a) antigen recognition in endosomes and lysosomes, (b) effective interaction of Toll-like receptors and antigens involved in immune activation, and (c ) It is considered necessary to induce high-efficiency binding of MHC molecules that perform antigen presentation and antigens.
  • the inclusion (antigen) is completely released at an early stage in the endosome, whereby the above-mentioned mutual The action can be efficiently caused and effective immunity induction can be performed.
  • a vesicle composed of a compound of the present invention containing a pH-responsive carboxyl group-containing hydrocarbon group containing a cyclohexyl group, a phenyl group, a pyridyl group or a naphthyl group exhibits responsiveness in a higher pH range. . For example, responsiveness at pH 6.0 or higher is shown. For this reason, by using a vesicle composed of the compound of the present invention containing a pH-responsive carboxyl group-containing hydrocarbon group (A), a carrier capable of releasing inclusions in the initial endosome can be obtained.
  • vesicles composed of the compounds of the present invention are taken up by antigen-presenting cells (dendritic cells) in an extremely large amount, inclusions can be reliably released inside the endosome if they have the above characteristics. It is effective as a vaccine for inducing humoral immunity and as an immunosuppressant delivery system.
  • Vesicles composed of compounds of the present invention containing hydrocarbon groups show responsiveness in the lower pH range. For example, responsiveness at pH 5.0 or lower is shown. Therefore, by using a vesicle composed of the compound of the present invention containing a pH-responsive carboxyl group-containing hydrocarbon group (B), a carrier capable of releasing inclusions in lysosomes can be obtained.
  • a vesicle having pH responsiveness can be obtained.
  • the compound of the present invention can be obtained, for example, by adding R 3 and R 4 to polyamide dendron (DL) obtained as follows.
  • DL-G1 to DL-G4 indicate first to fourth generation polyamide dendrons, respectively.
  • DL-G0 is the 0th generation and does not have a dendron structure.
  • R 1 and R 2 are unsaturated hydrocarbon groups, they can be produced as follows. Oleylamine and oleyl chloride are reacted to synthesize oleyloleylamide. Next, dioleylamine is synthesized by hydride reduction, and a polyamidoamine dendron is synthesized by repeating a Michael addition reaction using methyl acrylate and an ester amide exchange reaction using ethylenediamine. This polyamidoamine dendron is represented as DL-G1-2C 18 -U2.
  • R ⁇ 1 > and R ⁇ 2 > is a saturated hydrocarbon group
  • Polyamideamine dendron is synthesized by repeating Michael addition reaction using methyl acrylate and ester amide exchange reaction using ethylenediamine using dioctadecylamine as a starting material.
  • the polyamidoamine dendron denoted as DL-G1-2C 18.
  • R 3 and R 4 are performed as follows, for example. By reacting polyamidoamine dendron with 3-methylglutaric anhydride, a compound having —CO—CH 2 CH (CH 3 ) CH 2 —COOH as R 3 and R 4 is obtained.
  • composition of the present invention can suitably contain a phospholipid in addition to the compound of the present invention.
  • phospholipids include phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, cardiolipin, sphingomyelin, plasmalogen, and phosphatidic acid.
  • phosphatidylethanolamine and phosphatidylcholine are preferably used alone or in combination.
  • the fatty acid residue of these phospholipids is not particularly limited, and examples thereof include saturated or unsaturated fatty acid residues having 12 to 18 carbon atoms.
  • lauroyl group myristoyl group , Palmitoyl group, stearoyl group, oleoyl group, linoleyl group and the like, and dioleoylphosphatidylethanolamine (DOPE) is particularly preferable.
  • DOPE dioleoylphosphatidylethanolamine
  • the blending amount of the phospholipid is not particularly limited, but when the total amount of the phospholipid and the compound of the present invention is 100 parts by weight, 30 to 90 parts by weight of the phospholipid, 70 to 10 parts by weight of the compound of the present invention, preferably phosphorous It is 50 to 80 parts by weight of lipid, 50 to 20 parts by weight of the compound of the present invention, more preferably 60 to 70 parts by weight of phospholipid, and 40 to 30 parts by weight of the compound of the present invention.
  • the compound of the present invention and the phospholipid may be present merely as a mixture, and the compound of the present invention and the phospholipid may be present. They may be combined to form a lipid membrane structure.
  • the existence form of the lipid membrane structure and the production method thereof are not particularly limited.
  • the existence form includes a dried lipid mixture form, a dispersed form in an aqueous solvent, and a dried form thereof. And a frozen form.
  • the dried lipid mixture can be produced, for example, by dissolving the lipid component to be used once in an organic solvent such as chloroform and then performing vacuum drying with an evaporator or spray drying with a spray dryer.
  • an organic solvent such as chloroform
  • Examples of the form in which the lipid membrane structure is dispersed in an aqueous solvent include monolayer liposomes, multilamellar liposomes, O / W emulsions, W / O / W emulsions, spherical micelles, string micelles, and irregular layered structures And so on.
  • the size of the lipid membrane structure in a dispersed state is not particularly limited.
  • the particle diameter is 50 nm to several ⁇ m
  • the particle diameter Is from 5 nm to 50 nm.
  • the thickness per layer is 5 nm to 10 nm and these form a layer.
  • the composition of the aqueous solvent is not particularly limited, but in addition to water, sugar aqueous solutions such as glucose, lactose and sucrose, polyhydric alcohol aqueous solutions such as glycerin and propylene glycol, and phosphate buffer And buffer solutions such as citrate buffer solution and phosphate buffered physiological saline solution, physiological saline, medium for cell culture, and the like.
  • sugar aqueous solutions such as glucose, lactose and sucrose
  • polyhydric alcohol aqueous solutions such as glycerin and propylene glycol
  • phosphate buffer And buffer solutions such as citrate buffer solution and phosphate buffered physiological saline solution, physiological saline, medium for cell culture, and the like.
  • the pH of the aqueous solvent From the viewpoint of chemical stability of the lipid, it is important to set the pH of the aqueous solvent from weakly acidic to neutral (pH 3.0 to 8.0) or to remove dissolved oxygen by nitrogen bubbling. . Further, when lyophilized storage or spray-dried storage is used, effective storage is possible by using a sugar aqueous solution, and when storing frozen, a sugar aqueous solution or a polyhydric alcohol aqueous solution is used.
  • the concentration of these aqueous solvent additives is not particularly limited.
  • an aqueous sugar solution 2 to 20% (W / V) is preferable, and 5 to 10% (W / V) is more preferable. preferable.
  • a polyhydric alcohol aqueous solution 1 to 5% (W / V) is preferable, and 2 to 2.5% (W / V) is more preferable.
  • the concentration of the buffer is preferably 5 to 50 mM, more preferably 10 to 20 mM.
  • the concentration of the lipid membrane structure in the aqueous solvent is not particularly limited.
  • the concentration of the total amount of phospholipid used as the lipid membrane structure is preferably 0.001 mM to 100 mM. More preferred is 01 mM to 20 mM.
  • the form in which the lipid membrane structure is dispersed in an aqueous solvent is produced by adding the dried lipid mixture to the aqueous solvent and further emulsifying with an emulsifier such as a homogenizer, an ultrasonic emulsifier, a high-pressure jet emulsifier, or the like. be able to. Moreover, it can also manufacture by the method well-known as a method of manufacturing a liposome, for example, a reverse phase evaporation method etc., It should not be specifically limited. When it is desired to control the size of the lipid membrane structure, extrusion (extrusion filtration) may be performed under high pressure using a membrane filter having a uniform pore size.
  • the compounds, compositions and kits of the present invention can be used for the uses described below.
  • the compounds, compositions and kits of the present invention are preferably used for introducing physiologically active substances into cells.
  • the cell may be a cell in the living body or a cell taken out of the living body. That is, the compound, composition and kit of the present invention are used for introducing a physiologically active substance into cells in vivo or in vitro.
  • the physiologically active substance is not particularly limited, and is, for example, a low molecular compound, a peptide or a gene.
  • a physiologically active substance encapsulated in a vesicle composed of the compound of the present invention can be contained in any of early endosomes, late endosomes or lysosomes. Can be released.
  • the peptide or gene is not particularly limited, but if it can be an antigen or can generate an antigen, it is encapsulated in a vesicle composed of the compound of the present invention and then introduced into an antigen-presenting cell. They can be released in early or late endosomes. This is preferable because it can be used for inducing antibody production by the humoral immunity response system through antigen presentation via MHC class II and thus treating the disease.
  • a gene capable of producing an antigen a gene used as a so-called DNA vaccine can be used as a so-called DNA vaccine can be used.
  • the DNA vaccine is a technique for inoculating DNA encoding antigen information as a vaccine.
  • a vector containing a DNA fragment encoding antigen information is usually used as a DNA vaccine.
  • vectors used as DNA vaccines contain unmethylated cytosine and guanine-rich sequence regions called unmethylated CpG motifs.
  • MAGE for the treatment of malignant melanoma
  • HER2 / neu for the treatment of breast cancer
  • CEA for the treatment of colorectal cancer
  • WT1 for the treatment of leukemia or various cancers. Etc. can be used.
  • Physiologically active substances that can act as immune inhibitors can be used. What can act as an immune inhibitor can be used regardless of whether the inhibitory action is selective or non-selective for a specific immune response.
  • the compound, composition and kit of the present invention of the present invention may be used for in vivo introduction into cells together with the physiologically active substance to be introduced for the purpose of treating the above diseases.
  • the compound or composition of the present invention may be administered to an individual together with a physiologically active substance.
  • the means for administration to an individual may be oral administration or parenteral administration, but parenteral administration is preferred.
  • the dosage form may be a conventionally known dosage form, and examples of the dosage form for oral administration include tablets, powders, granules, syrups and the like. Examples of the dosage form for parenteral administration include injections, eye drops, ointments, suppositories and the like. Among these, an injection is preferable, and an administration method is preferably intravenous injection or local injection into a target cell or organ.
  • the compounds, compositions and kits of the present invention may be used for in vitro introduction into cells together with the physiologically active substance to be introduced for the purpose of treating the above-mentioned diseases.
  • a physiologically active substance is released in the endosome at an early stage after the vesicle is taken into the endosome, and effective immune induction is performed.
  • antibody production by the humoral immune system is induced in the cells through antigen presentation via MHC class II.
  • the compounding ratio of the physiologically active substance and the compound of the present invention is 1 to 20 parts by weight, preferably 3 to 15 parts by weight of the compound of the present invention with respect to 1 part by weight of the physiologically active substance. More preferably, 5 to 7 parts by weight are used.
  • the compounding ratio of the physiologically active substance and the compound of the present invention is 1 to 50 parts by weight, preferably 1 to 50 parts by weight, preferably 1 part by weight of the physiologically active substance. 5 to 30 parts by weight, more preferably 10 to 15 parts by weight is used.
  • any of oligonucleotide, DNA and RNA may be used.
  • a gene for introduction in vitro such as transformation
  • a gene which acts by expression in vivo for example, gene therapy
  • genes for use in breeding industrial animals such as laboratory genes and laboratory animals and livestock are preferred.
  • genes for gene therapy include genes encoding physiologically active substances such as antisense oligonucleotides, antisense DNAs, antisense RNAs, enzymes, and cytokines.
  • FBS Fetal Bovine Serum
  • DMEM Dulbecco's modified Eagle's medium
  • N N-dimethylformamide, tetrahydrofuran, petroleum ether, sodium cyanide, lithium aluminum hydride, disodium hydrogen phosphate, potassium dihydrogen phosphate, potassium benzylpenicillin, and streptomycin sulfate were purchased from Wako Pure Chemical.
  • Pyranine was purchased from Tokyo Kasei Co., Ltd.
  • Chloroform, ethyl acetate, methanol, n-hexane, sodium sulfate, calcium chloride, magnesium chloride hexahydrate, potassium chloride, and disodium ethylenediaminetetraacetate (EDTA) were purchased from Kishida Chemical.
  • Triethylamine, methyl acrylate, ethylenediamine, diethyl ether, sodium chloride, RPMI-1640 liquid medium, and MEM non-essential amino acid solution were purchased from Nacalai Tesque. Trypsin was purchased from DIFCOLABORATORIES (USA).
  • Dioctadecylamine and calcein were purchased from Sigma.
  • Oleylamine, oleoyl chloride, cyclohexanedicarboxylic anhydride and 3-methylglutaric anhydride were purchased from ALDRICH.
  • Dichloromethane was purchased from Sigma-ALDRICH®. 2-mercaptoethanol, DPX, Hoechst, Lysotracker Green DND-26, Lysotracker Red DND-99, and Tf-Alexa555 were purchased from Invitrogen.
  • DPX 2-mercaptoethanol
  • DPX Hoechst
  • Lysotracker Green DND-26 Lysotracker Red DND-99
  • Tf-Alexa555 Tf-Alexa555
  • As a dialysis membrane Spectra / Por 6 (fractional molecular weight 2000, FE-0526-33) was purchased from Spectrum Laboratories Inc. Merck Kieselgel 60 (230-400 mesh) was used for silica gel chromatography.
  • DL-G1 and DL-G2 were synthesized using dioctadecylamine as a starting material by alternately performing a Michael addition reaction with methyl acrylate followed by an ester amide exchange reaction with ethylenediamine. This is the method reported by Tomalia et al. In the synthesis of dendrimers.
  • DL-G1-U2 reacts with oleylamine and oleyl chloride, and hydride reduction with lithium aluminum hydride (LiAlH 4 ), followed by Michael addition reaction with methyl acrylate and subsequent ester amide exchange reaction with ethylenediamine. To be synthesized.
  • the terminal amino groups of the synthesized DL-G1, DL-G2, and DL-G1-U2 were reacted with MDEG groups and EDEG groups activated with paranitrophenyl carbonate groups, and introduced at the ends to introduce MDEG-DL-G1, EDEG- DL-G1, MDEG-DL-G2, EDEG-DL-G2, MDEG-DL-G1-U2, and EDEG-DL-G1-U2 were synthesized.
  • the peak near 0.88 ppm derived from the methyl group at the end of the octadecyl group or the methyl group at the end of the oleyl chain From the integration ratio of the peak near 0.96 ppm derived from, the integration ratio of the peak near 1.11 ppm derived from the terminal ethyl group of the introduced ethoxydiethylene glycol group, and the peak near 3.41 ppm, the ethoxydiethylene glycol group introduced into the dendrimer lipid Numbers were calculated. As a result, 2.0, 4.0, and 2.4 methoxydiethylene glycol groups were introduced at the ends of EDEG-DL-G1, EDEG-DL-G2, and EDEG-DL-G1-U2, respectively.
  • the first and second generation PAMAM dendron lipids having two alkyl chains and the first generation PAMAM dendron lipids having two oleyl chains were synthesized.
  • MDEG-DL-G1 and EDEG-DL-G1 which are various OEG group-binding dendron lipids in the first and second generation, by introducing various OEG groups with temperature responsiveness to their terminal amino groups MDEG-DL-G2, EDEG-DL-G2, MDEG-DL-G1-U2, and EDEG-DL-G1-U2 were synthesized.
  • dendron lipids are amphipathic molecules, it is thought that when they are dispersed in water, they form molecular aggregates, which causes temperature-responsive groups introduced into the hydrophilic region to accumulate on the aggregate surface. . And, it can be expected that the lipid molecule aggregate exhibits temperature responsiveness due to the interaction between the accumulated temperature responsive groups. Then, next, the temperature responsiveness of the lipid molecule assembly was examined using the synthesized temperature-responsive dendron lipid dispersion.
  • lipid dispersion The solvent was removed from each lipid chloroform solution using a rotary evaporator to form a lipid thin film.
  • a 10 mM phosphate buffer solution was added thereto to adjust the pH to 3.0 and a concentration of 2 mg / ml. This was irradiated with ultrasonic waves at 45 ° C. for 10 minutes using a bath-type ultrasonic irradiation apparatus, and then allowed to stand at 45 ° C. for 20 minutes. Then, it was left still for 60 minutes or more under ice cooling.
  • the MDEG-DL-G2 Decrease in transmittance at around 68 ° C, EDEG-DL-G2 at around 32 ° C, MDEG-DL-G1-U2 at around 44 ° C, and EDEG-DL-G1-U2 at around 24 ° C. It was. These reductions in transmittance indicate that the molecular assembly has become unstable and aggregated.
  • the cloud point (Cloud Point)
  • the cloud points of -G1-U2 and EDEG-DL-G1-U2 were 34.2 ° C, 22.5 ° C, 67.6 ° C, 34.2 ° C, 45.4 ° C and 26.1 ° C, respectively. From the above, it was shown that temperature responsiveness can be imparted by integrating various OEG groups on the surface of the molecular assembly.
  • the cloud point temperature decreased as the end group became more hydrophobic, and the cloud point temperature increased as the generation number increased. This is considered to be because when the hydrophobicity is increased, aggregation due to hydrophobic interaction is likely to occur during dehydration due to temperature response. In addition, as the number of generations increases, the number of tertiary amines also increases and becomes more hydrophilic, so it is considered that the temperature response on the high temperature side was achieved. In addition, in the case of an alkyl chain and an oleyl chain, the cloud point of the molecular assembly of dendron lipids having an oleyl chain shifted to a higher temperature side. This is presumably because the lipid bilayer created by the molecular assembly is more fluid and has improved stability due to having a double bond.
  • FIG. 23 shows a part of the result at that time
  • FIG. 24 shows the result of plotting the cloudiness against pH.
  • the cloud point increased. This is because as the pH decreases, the degree of protonation of tertiary amino groups in polar groups increases and the hydrophilicity of the molecules increases, and electrostatic repulsion occurs between molecules and between molecules due to charge. Since the surface density of various OEG groups decreased, it was thought that cloud points were shown at higher temperatures. Further, when the pH became too high, the cloud point did not continue to decrease, but the cloud point was exhibited at a certain temperature. This is probably because the tertiary amino group in the polar group is no longer protonated.
  • the temperature response of the dendron lipid aggregate was evaluated by measuring the permeability of the lipid dispersion.
  • the transmittance decreased at a certain temperature or higher. This showed that the molecular assembly was aggregated.
  • the cloud point increased with a decrease in pH. This is considered to be because the protonation of the tertiary amine inside the dendron lipid was promoted and the molecular assembly became more hydrated.
  • Temperature control was performed using ETC-717. At this time, the temperature at which the transmittance dropped rapidly was taken as the cloud point. After the measurement, the pH of the solution was measured at room temperature, and the value was taken as the pH at the time of turbidity measurement.
  • FIG. 30 shows the result of plotting the cloud point against the ratio of introducing PEG lipid.
  • the cloud point increased. This is because the molecular assembly surface became more hydrophilic with the increase in PEG lipids, and the amount of PEG chains introduced on the surface increased, the surface density between EDEG groups decreased, and the interaction was reduced. This is considered to be due to the fact that the fluidity of the hydrophobic part of the molecular assembly was enhanced by the suppression and further the introduction of cholesterol.
  • FIG. 32 shows the results for EDEG-DL-G1 alone as a comparison.
  • the cloud point hardly changed. This is thought to be because the effect of increasing the hydrophilicity of the molecule due to the protonation of the tertiary amino group in the polar group accompanying the decrease in pH was slight compared to the hydration of the surface by the PEG chain. . It was also found that at any pH, the PEG lipid introduced showed a cloud point on the higher temperature side.
  • FIG. 34 shows the results of morphological observation of lipid molecular aggregates (pH 7.4). At 10 ° C. below the cloud point, a spherical molecular assembly having a particle size of about 150 to 200 nm was observed (FIG. 34 (A)). This aggregate is considered to have a vesicle structure because of its particle size.
  • the temperature responsiveness evaluation of the EDEG-DL-G1 / PEG-Chol lipid molecular assembly was performed by measuring the permeability of the lipid dispersion.
  • the transmittance decreased rapidly above a certain temperature. This showed that the molecular assembly was aggregated. That is, it was found that the introduction of PEG lipid did not impair the temperature response function.
  • the introduction rate of PEG lipid was increased, the cloud point of the molecular assembly increased. This is probably because the introduction of PEG-Chol hydrated the surface of the molecular assembly and required more energy to dehydrate and transfer.
  • the particle size was about 200 nm, and when the temperature was above the cloud point, the particle size increased. This is because when the temperature is below the cloud point, the molecular aggregate is hydrated and stable while maintaining a certain particle size, and when the cloud point is exceeded, the hydrophilicity / hydrophobicity balance becomes unstable due to dehydration due to temperature response. This is probably because the hydrophobic interaction worked to form aggregates.
  • FBS Fetal Bovine Serum
  • Dulbecco's modified Eagle's medium (DMEM) was purchased from Nissui Pharmaceutical.
  • Disodium hydrogen phosphate, potassium dihydrogen phosphate, benzylpenicillin potassium, streptomycin sulfate, acetyl CoA, ampicillin sodium, chloramphenicol, ethidium bromide were purchased from Wako Pure Chemical.
  • Calcium chloride, magnesium chloride hexahydrate, potassium chloride, trishydroxymitylaminomethane (Tris), and disodium ethylenediaminetetraacetate (EDTA) were purchased from Kishida Chemical.
  • Cell lysing agent Luc-PGC-50 was purchased from Toyo Ink.
  • Sodium chloride was purchased from Nacalai Tesque. Trypsin was purchased from DIFCO LABORATORIES (USA).
  • Rhodamine-PE (0.6 mol%) was added to a chloroform solution in which EDEG-DL-G1 and PEG lipids were mixed at a ratio of 95/5, and the solvent was removed using a rotary evaporator. Was removed to form a lipid film.
  • a 10 mM phosphate buffer solution was added thereto to adjust the pH to 3.0 and a concentration of 1.0 mM. This was irradiated with ultrasonic waves at 45 ° C. for 10 minutes using a bath-type ultrasonic irradiation apparatus, and then allowed to stand at 45 ° C. for 20 minutes. Then, it was left still for 60 minutes or more under ice cooling. Finally, the pH was adjusted to 7.4 to prepare a dispersion.
  • the vesicle surface is hydrophilic before showing a temperature response, so it only weakly adsorbs to the cell surface, but when the cloud point is exceeded, the vesicle dehydrates due to the temperature response and changes its shape. Therefore, it is thought that it was taken in in the cell. At this time, the vesicle changes its shape, interacts strongly with the cell membrane, is efficiently taken up into the cell by endocytosis, or moves through the cell membrane directly by hydrophobic interaction. It is thought that it was taken in.
  • the EDEG-DL-G1 / PEG-Chol lipid molecular assembly into which the constructed PEG lipid was introduced was labeled with a fluorescent label, and the delivery function of the vesicle in the cell was evaluated.
  • the vesicle surface is hydrophilic before showing a temperature response, so it only weakly adsorbs to the cell surface, but when the cloud point is exceeded, the vesicle dehydrates due to the temperature response and changes its shape. Therefore, it is thought that it was taken in in the cell. At this time, the vesicle changes its shape, interacts strongly with the cell membrane, is efficiently taken up into the cell by endocytosis, or moves through the cell membrane directly by hydrophobic interaction. It is thought that it was taken in.
  • Dendron lipid / PEG-Chol aggregate dispersions (10 M phosphoric acid, 140 M NaCl, pH3.0) obtained by dispersing mixed thin films of MDEG-G1 and PEG-Chol (PEG molecular weight 1000) in various ratios
  • the temperature responsiveness was evaluated by measuring the temperature dependence of the transmittance in addition to a buffer of 10 mM mM phosphoric acid, 140 mM mM NaCl, and pH 7.4. Temperature increase rate 4 °C / min.
  • the cloud point of MDEG-G1 / PEG-Chol aggregate increased with increasing PEG-Chol content. It was found that the response temperature (cloud point) of the aggregate can be adjusted by adjusting the PEG-Chol content. It was also found that a dendron lipid aggregate having a cloud point near 40 ° C. can be obtained by containing 2% of PEG-Chol.
  • HeLa cells derived from human cervical cancer
  • HeLa cells were seeded in a 12-well dish at 100,000 / well and cultured for 24 hours. Wash twice with PBS (+) and once with PBS (-), add 500 ⁇ L of DMEM (without serum), and add 500 ⁇ L of dendron lipid / PEG-Chol aggregate dispersion to a lipid concentration of 0.5 mM.
  • the cells were incubated at 37 ° C., 42 ° C. and 44 ° C. for 15 minutes and 30 minutes in a CO 2 incubator. Thereafter, the cells were washed twice with PBS (+) and once with PBS (-), and the cells were detached with trypsin.
  • the MDEG-DL-G1-2C 18 / PEG-Chol (98/2) aggregate did not change much even when the temperature and contact time were changed. This is because the dendron lipid MDEG-DL-G1-2C 18 terminal group has a lower hydrophobicity than EDEG-DL-G1-2C 18 , and so the interaction with the cell works very strongly even when morphological changes occur. It is thought that there was not.
  • temperature-responsive groups are given temperature responsiveness by introducing oligoethylene glycol chains with excellent biocompatibility as temperature-responsive groups into polar groups of dendron lipids and accumulating them on the surface of the molecular assemblies. It was confirmed that it was possible. Also, the cloud point temperature was different depending on the generation and end group. This is considered due to the balance between hydrophobicity and hydrophilicity in the molecular structure. Therefore, it is considered that it is possible to construct a molecular assembly that responds at a desired temperature by designing the balance between the hydrophobic part and the hydrophilic part in the molecular structure.
  • the temperature-responsive dendron lipid aggregate into which polyethylene glycol lipid is introduced changes its shape dramatically above the cloud point near body temperature, and has both high stability and biocompatibility due to the effect of polyethylene glycol lipid.
  • a vesicle was constructed. This PEG-Chol complex type dendron lipid vesicle can control the delivery function into cells by temperature.
  • Second invention Preparation of compounds of the invention wherein R 1 and R 2 are unsaturated hydrocarbon groups
  • FBS Fetal Bovine Serum
  • DMEM Dulbecco's modified Eagle's medium
  • N N-dimethylformamide, tetrahydrofuran, petroleum ether, sodium cyanide, lithium aluminum hydride, disodium hydrogen phosphate, potassium dihydrogen phosphate, potassium benzylpenicillin, and streptomycin sulfate were purchased from Wako Pure Chemical.
  • Pyranine was purchased from Tokyo Kasei Co., Ltd.
  • Chloroform, ethyl acetate, methanol, n-hexane, sodium sulfate, calcium chloride, magnesium chloride hexahydrate, potassium chloride, and disodium ethylenediaminetetraacetate (EDTA) were purchased from Kishida Chemical.
  • Triethylamine, methyl acrylate, ethylenediamine, diethyl ether, sodium chloride, RPMI-1640 liquid medium, and MEM non-essential amino acid solution were purchased from Nacalai Tesque. Trypsin was purchased from DIFCOLABORATORIES (USA).
  • Dioctadecylamine and calcein were purchased from Sigma.
  • Oleylamine, oleoyl chloride, cyclohexanedicarboxylic anhydride and 3-methylglutaric anhydride were purchased from ALDRICH.
  • Dichloromethane was purchased from Sigma-ALDRICH®. 2-mercaptoethanol, DPX, Hoechst, Lysotracker Green DND-26, Lysotracker Red DND-99, and Tf-Alexa555 were purchased from Invitrogen.
  • DPX 2-mercaptoethanol
  • DPX Hoechst
  • Lysotracker Green DND-26 Lysotracker Red DND-99
  • Tf-Alexa555 Tf-Alexa555
  • As a dialysis membrane Spectra / Por 6 (fractional molecular weight 2000, FE-0526-33) was purchased from Spectrum Laboratories Inc. Merck Kieselgel 60 (230-400 mesh) was used for silica gel chromatography.
  • DL-G1 by synthesizing oleyl oleoylamide with oleylamine and oleoyl chloride, then synthesizing dioleylamine by hydride reduction, repeating Michael addition reaction using methyl acrylate and ester amide exchange reaction using ethylenediamine -2C 18 -U2 was synthesized. This is the method reported by Tomalia et al. In the synthesis of dendrimers. Further, CHexDL-U2 and MGluDL-U2 were synthesized by reacting the synthesized DL-G1-2C 18 -U2 with 3-Methylglutaric Anhydride or Cyclohexanedicarboxylic Anhydride.
  • MGluAn 3-Methylglutaric Anhydride
  • DMF 3 mL
  • 430 mg (0.909 mmol) of DL-G1-2C 18 -U2 was dissolved in 4 mL of DMF and mixed, 0.370 mL of triethylamine (TEA) was added, and the mixture was stirred at 50 ° C. for 7 days under a nitrogen atmosphere.
  • TEA triethylamine
  • a dendron lipid solution in chloroform was spread on it and left to stand for 20 minutes to blow off the chloroform.
  • LB membrane measurement device In ⁇ -A mode, the surface pressure of the lipid monolayer was measured with a pressure gauge when the molecular surface area of the lipid surface on the solvent surface was reduced by laterally compressing the solvent surface with a barrier. .
  • the solvent on the trough was collected, the trough and the barrier were washed with ethanol, and the solvent was developed.
  • CHex-DL-G1-2C 18 -U2 liposome (hereinafter referred to as UCG1) is prepared by mixing a predetermined amount of EYPC (10 mg / ml) in chloroform and a predetermined amount of CHex-DL-G1-2C 18 -U2 (3.65 mg / ml). ml) of chloroform solution was mixed and the solvent was removed by a rotary evaporator to form a thin film, followed by vacuum drying for 4 hours to completely remove the solvent.
  • EYPC liposomes a predetermined amount of EYPC in chloroform was added, the solvent was removed with a rotary evaporator to form a thin film, and the solvent was completely removed by vacuum drying for 4 hours.
  • the particle size and ⁇ potential of liposomes were determined by a dynamic light scattering method.
  • the liposome solution was added to PBS (-) or 0.1 mM phosphate buffer (final volume 2.5 ml) adjusted to each pH so that the lipid concentration in the cell was 0.1 mM.
  • the mixture was allowed to stand for 15 minutes, and the particle size and ⁇ potential were measured.
  • the measurement was performed at 25 ° C., and was measured using ELS-8000F manufactured by Otsuka Electronics Co., Ltd.
  • Liposomes are dispersed in PBS (-) pH 7.4 so that the lipid concentration is 0.5 mM, and the pH is continuously changed at 37 ° C.
  • the transmittance of light at 500 nm was measured using a V-560 type ultraviolet / visible spectrophotometer manufactured by JASCO Corporation.
  • mice-derived cell culture strain DC2.4 cells Mouse-derived cell culture strain DC2.4 cells Is RPMI-1640 medium containing 10% FBS, 0.1 mg / ml benzylpenicillin potassium, 0.1 mg / ml streptomycin sulfate, 2 mM L-glutamine, 0.1 mM MEM non essential amino acid solution and 0.55 mM 2-mercaptoethanol.
  • the cells were cultured in a CO 2 incubator (MCO-96) manufactured by Sanyo Electric Co. at a CO 2 concentration of 5% at 37 ° C.
  • the cells After washing 3 times with HBSS, the cells are detached using 300 ⁇ l of trypsin aqueous solution (trypsin (DIFCO) 250 mg, disodium ethylenediaminetetraacetate (EDTA) 1 mg, PBS 100 ml) per well, and a flow cytometer It collected in the tube for use.
  • trypsin (DIFCO) 250 mg
  • EDTA disodium ethylenediaminetetraacetate
  • PBS 100 ml a flow cytometer
  • HeLa cells were seeded at 1 ⁇ 10 5 per well of a 12-well dish and cultured at 37 ° C for 48 hours in 0.5 ml of 10% FBS-containing DMEM medium. . Then, after washing twice with PBS containing 0.36 mM CaCl 2 and 0.42 mM MgCl 2 (PBS (+)), 0.5 ml of DMEM medium containing 10% FBS was added. The liposome solution was added to each well so that the lipid concentration was 0.5 mM, and incubated at 37 ° C. for 4 hours. Then, wash twice with PBS (+) and once with PBS (-) (PBS without Ca2 + and Mg2 +).
  • trypsin aqueous solution 250 mg trypsin (DIFCO) per well, disodium ethylenediaminetetraacetate (EDTA) 1 mg, PBS 100 ml
  • EDTA disodium ethylenediaminetetraacetate
  • Hela cells are seeded on a Matsunami glass bottom dish at 2 ⁇ 10 5 cells per plate, in 10 ml FBS-containing DMEM medium at 37 ° C. Incubated overnight. Thereafter, after washing twice with PBS (+) and once with PBS ( ⁇ ), 1.0 ml of 10% FBS-containing DMEM medium was added. The liposome solution was added to each well so that the lipid concentration was 0.5 mM (total volume: 2 mL), and incubated at 37 ° C. for 4 hours or 24 hours. Then, after washing twice with PBS (+) and once with PBS ( ⁇ ), OPTI-MEM was added, and intracellular dynamics were observed with a confocal laser microscope (LSM5 EXCITER (ZEISS)).
  • LSM5 EXCITER confocal laser microscope
  • Lysotracker staining of late endosomes / lysosomes Take 1 ⁇ L of Lysotracker (Invitrogen, Lysotracker Green DND-26, Lysotracker RedDND-99, DMSO solution: 1 mM) and RPMI 1640 or DMEM without FBS A staining solution was prepared by adding 99 ⁇ L of medium. Liposomes were taken up by the method described above, and then 1985 ⁇ L of RPMI 1640 or DMEM medium not containing FBS and 15 ⁇ L of staining solution were added and incubated for 5 minutes. Thereafter, after washing three times, PBS was added again, and intracellular dynamics were observed with a confocal laser microscope (LSM 5EXCITER (ZEISS)).
  • LSM 5EXCITER confocal laser microscope
  • a desired responsiveness can be obtained by constructing a vesicle by appropriately combining two or more compounds of the present invention having different pH responsiveness. It was verified as follows whether or not vesicles having the same could be obtained.
  • pH -responsive dendron lipids could be synthesized by the appearance of peaks derived from the introduced terminal groups.
  • MGluDL-U2 the peak derived from the methyl group of 3-methylglutaric acid (6H) near 1.03 ppm appeared, and in CHexDL-U2 and CHexDL-S, the peak derived from cyclohexanedicarboxylic acid (16H) at 1.2-2.0 ppm It was confirmed that synthesis was possible by the appearance of.
  • mass spectrometry was performed in the ESI-negative mode, it was confirmed that the synthesis was surely performed because it showed a value equivalent to the theoretical value.
  • FIG. 58 shows a plot of the surface area for each pH when the surface pressure is 25 mN / m.
  • CHexDL-U2 tended to have a larger area at the same surface pressure than MGluDL-U2. This is because CHexDL-U2 has a larger excluded volume of terminal groups. However, there is no clear difference between these two types. Since it is considered that there is no clear difference in G1 because only two terminal groups can be introduced, it is considered that this difference in terminal groups will occur by increasing the number of generations.
  • the area of the saturated type was larger at the same surface pressure (Fig. 59). Further, when compared at the same surface pressure, the surface area was larger in the case of acidity than neutrality. As shown in FIG. 60, this is thought to be due to the fact that the pH group decreases and the carboxy group is protonated to be distributed to the gas-liquid interface. Furthermore, when the internal tertiary amine is protonated, intermolecular electrostatic repulsion is also generated, and the surface area is thought to have increased when compared at the same surface pressure.
  • SCG1 liposomes The effect of pH on the pyranine release of UCG1 liposomes and saturated CHexDL liposomes (hereinafter SCG1 liposomes) was examined.
  • the prepared UMG1, UCG1, and SCG1 liposomes had the following lipid composition ratios.
  • release does not occur over time in a high pH environment, whereas inclusions are released from the liposomes over time in a low pH environment.
  • the inclusion release under neutrality did not occur so much, but the inclusion release occurred in any liposome under weak acidity.
  • the maximum release rate from liposomes was about 30% for UMG1, 90% or more for UCG1, and about 60% for SCG1.
  • the maximum value of the release rate of inclusions differs between MGlu -introduced and CHex -introduced, which is thought to be due to the difference in the hydrophobicity of the end groups. That is, it is considered that the CHex ⁇ ⁇ group having a higher degree of hydrophobicity interacts more strongly with the membrane, whereas the MGlu ⁇ group having a lower degree of hydrophobicity has a weak interaction with the membrane, so that the release rate remains low.
  • the reason why the release rate of inclusions is different between the saturated type and the unsaturated type even when the same CHex is introduced is that the saturated dendron has an alkyl chain (octadecyl chain) which is a hydrophobic site as an unsaturated type. It has high crystallinity compared to the oleyl chain, and it is thought that in the saturated type, dendron lipids are separated and easily assembled in the lipid membrane. It is thought that the lipid membrane became partially crystal-like, and the liposome membrane became hard and leakage of inclusions did not occur easily.
  • alkyl chain octadecyl chain
  • the release rate was maximized when 25% of the dendron lipid was modified on the liposome, and no further improvement in the release rate was observed even when the amount of the dendron lipid charged was increased.
  • the effect of the dendron lipid end group on the liposome lipid membrane is considered to be sufficiently achieved by 25% modification.
  • the amount of dendron lipid charged was 50%, UMG1 dissolved, aggregates precipitated in UCG1 and SCG1 liposomes, and liposomes were not formed. At this time, it is considered that MGluDL-U2 has reached micelle formation and dissolved, and CHexDL-U2 and CHexDL-S have self-aggregated dendron lipids.
  • the particle size was about 100 nm under neutral and weak acidity, but the particle size increased around pH5.0 and became maximum at pH4.5, and when pH was further lowered, it was again 100 nm. The particles became about the size. Also in turbidity measurement, the transmittance starts to decrease at around pH 5.0 mm, the transmittance becomes the lowest at pH 4.4 to pH 4.5 mm, and the permeability is again about the same as under neutral by lowering the pH value. Value. From this, in UMG1UM liposomes, when pH is lowered, the interaction between the hydrophobic part of its own liposome membrane and the hydrophobic part of the end group does not occur so much, and it is thought that aggregation interacting with other liposomes occurs .
  • EYPC liposome ⁇ potential did not change greatly depending on pH, and was almost neutral.
  • the effect of pH on the zeta potential of UMG1 ⁇ liposome, UCG1 ⁇ liposome and SCG1 ⁇ liposome was investigated. All liposomes had negatively similar values when weakly basic, the ⁇ potential increased with decreasing pH, and were positively similar values under acidic conditions.
  • the ⁇ potential was positive at pH 5-6, and in the UCG1 and SCG1 liposomes, it was already positive near neutrality.
  • the ⁇ potential of EYPC liposomes does not change even when the pH is changed, it is considered that the change in the ⁇ potential of the liposome occurred because the charge state of the pH responsive dendron lipid was changed. Therefore, the difference in the ⁇ potential depending on the pH of the liposome can be explained as follows from the difference in the charged state of the dendron lipid. It is considered that the charge state of the dendron lipid at each pH of the UMG1 liposome is as shown in FIG.
  • the terminal carboxy group Under neutral and weak basicity, the terminal carboxy group has a negative charge, so the ⁇ potential is considered to be negative.
  • the ⁇ potential since protonation of tertiary amines inside dendron occurs at pH 6.0 to pH 7.0%, protonation of tertiary amino groups inside dendron lipids occurs at pH 5.5 to pH 7.0%. It is considered that the ⁇ potential became neutral at around pH 5.5.
  • the terminal carboxy group is protonated, and the ⁇ potential is considered to be a positive value.
  • the charge state of the pH-responsive dendron lipid at each pH of the UCG1 and SCG1 liposomes is considered as shown in FIG.
  • the zeta potential is already positive. Since the protonation of the tertiary amino group in the dendron occurs at pH 6.0 to pH 7.0, the increase in ⁇ potential up to pH 7.0 is thought to be due to the protonation of the terminal carboxy group. . Further, it is considered that the ⁇ potential became positive due to the protonation of the tertiary amino group in the dendron due to a further decrease in pH.
  • FIG. 31 shows the results for MGluDL-U2
  • FIG. 32 shows the results for CHexDL-U2
  • FIG. 33 shows the results for CHexDL-S.
  • the ⁇ potential is negative and maximum, and the area is large.
  • the ⁇ potential increased, and the area area was minimized in the unsaturated type, and the area area was also decreased in the saturated type. This is considered to be due to the fact that the repulsion of the dendron portion of the dendron lipid became neutral due to the decrease in the ⁇ potential, so that charge repulsion disappeared and the dendrons packed more closely.
  • the pH value decreased further, the area of the area increased as the ⁇ potential increased. This is because the dendron site has been charged again. As described above, it was found that the area of the area also changed in synchronization with the change in the ⁇ potential.
  • FIGS. 73-75 show the results for UMG1.25, UCG1.25 and SCG1.25, respectively.
  • the ⁇ potential is negative and no release occurs.
  • release occurs when the ⁇ potential rises (the charge state of the dendron lipid changes), and that the inclusion release is also maximized in the pH range where the ⁇ potential is positive and maximum.
  • UMG1 has a low release rate, making it difficult to confirm the correlation, but this is true for all liposomes.
  • the carboxy group at the end of the dendron lipid is negatively charged under weak basicity, and since it does not interact with the liposome membrane, the liposome membrane is considered to be stably held.
  • the dendron under neutral conditions, from the results of monomolecular film measurement, the dendron can be packed more densely, so the inclusion is considered to be stably held.
  • the pH decreases, the hydrophobic interaction of the terminal membrane derived from 3-methylglutaric acid or 1.2-cyclohexanedicarboxylic acid increases due to the protonation of the carboxy group at the end of the dendron lipid, increasing the hydrophobic interaction with the liposome membrane.
  • the liposome membrane is destabilized by the formation of a hydrophilic site in the liposome membrane, which was hydrophobic in basic and neutralization, due to the protonation of the tertiary tertiary amino group inside the dendron lipid. It is thought that the membrane was destabilized due to the packing of lipid molecules (egg yolk phosphatidylcholine) entering the pH -responsive dendron lipid end group, resulting in destabilization of the membrane and release.
  • lipid molecules egg yolk phosphatidylcholine
  • UMG1.25 liposomes Compared to the amount of EYPC® liposomes taken into cells, the amount of UMG1.25 liposomes was slightly less, and the amount of UCG1.25 and SCG1.25 liposomes was 3-5 times higher.
  • uptake after adsorption due to hydrophobic interaction with the cell membrane may be considered. From the comparison of ⁇ potential under physiological conditions of these liposomes, this difference is considered as follows. Since the surface of the cell membrane is negatively charged, UMG1.25, which had a negative ⁇ potential, has little uptake due to electrostatic repulsion, and the hydrophobicity of the dendron end group is not so high. Uptake by hydrophobic interaction with can not be expected.
  • UMG1.25 liposomes with negative ⁇ potential had the least uptake.
  • the terminal group (MGlu group) of MGluDL-U2 incorporated in the liposome is not so large in excluded volume (steric hindrance), and the hydrophobicity is not so high. From this, it is thought that the interaction (electrostatic interaction or hydrogen bond) between the terminal carboxy group and the hydrophilic site on the outside of the liposome occurred, and it seems that it was in a state of being bound to the choline component in the liposome. It is done.
  • FIGS. Show. 77 to 80 show the results for DC 2.4 cells
  • FIGS. 81 to 85 show the results for Hella cells. 38 and 42 are 4 hours after the addition of SCG1.25, FIGS. 39 and 43 are 4 hours after the addition of UCG1.25, and FIGS. 40 and 83 are 4 after the addition of UMG1.25.
  • FIGS. 80 and 84 are the results 4 hours after the addition for EYPC
  • FIG. 85 are the results 24 hours after the addition for UMG1.25.
  • Rhodoamine- labeled liposomes and late endosome / lysosomal calcein-encapsulated UCG1.25 liposomes were incorporated, and late endosome / lysosomes were stained.
  • the results are shown in FIG.
  • the lyso tracker was red. This figure shows that the positions of late endosomes / lysosomes (intracellular acidic vesicles) and released calcein coincide.
  • CHexDL-G2-2C 18 hereinafter sometimes CHexDL-G1-2C 18 and CHexDL-G2-2C 18, respectively referred to as CHexDL-G1-S and CHexDL-G2-S.
  • CHexDL-G2-S Various evaluations were performed on CHex-DL-G2-S as follows. In the following, unless otherwise described, the same operation as described for CHex-DL-G1-S was performed.

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Abstract

The present invention addresses the problem of improving the balance among (1) temperature responsiveness, (2) in vivo stability and (3) biocompatibility in a temperature-responsive carrier that is used for the purpose of improving the target directivity of a drug. The present invention provides, as a solution for the problem, a compound that is represented by one of the following formulae DL-G1 to DL-G4. DL-G1:R1R2NX(XHR3)XHR4 DL-G2:R1R2NX(X(XHR3)XHR4)2 DL-G3:R1R2NX(X(X(XHR3)XHR4)2)2 DL-G4:R1R2NX(X(X(X(XHR3)XHR4)2)2)2

Description

機能性化合物及びその化合物を含有する分子集合体、並びにそれらを含有する組成物及びキット並びにそれらの使用Functional compounds, molecular assemblies containing the compounds, compositions and kits containing them, and uses thereof
　本発明は、機能性化合物及びその化合物を含有する分子集合体、並びにそれらを含有する組成物及びキット並びにそれらの使用に関する。 The present invention relates to a functional compound, a molecular assembly containing the compound, a composition and kit containing them, and uses thereof.
　先進医療技術の開発において、薬物を細胞内の標的部位に正確に送達するドラッグデリバリーシステム（ＤＤＳ）が求められている。ＤＤＳを利用することにより、治療効果の向上、副作用の低減又は患者の利便性向上等に資することができる。 In the development of advanced medical technology, a drug delivery system (DDS) that accurately delivers a drug to a target site in a cell is required. By using DDS, it is possible to contribute to improvement of therapeutic effect, reduction of side effects or improvement of patient convenience.
　ＤＤＳとして、薬物の標的指向性を向上することを目的として、温度に応答して薬物を放出する機能を有する、いわゆる温度応答性キャリアの構築が試みられている。具体的には、非加温時にはベシクル形状を示すが、加温により形状が変化する分子集合体をキャリアとして利用する試みがなされている。このベシクル内部に薬物を保持させた上で生体内に投与した後、例えば患部等の所望の部位を選択的に加温することにより、分子集合体の構造を大きく変化させ、これにより所望の部位においてのみ薬物を放出させる技術の開発が試みられている。より具体的には、ポリアミドデンドロン構造を有する脂質（ポリアミドデンドロン脂質）を、温度応答性を示すイソブチルアミド（ＩＢＡＭ）基で修飾した化合物で構成される分子集合体を上記目的に利用しようとする試みが報告されている（非特許文献１）。 As DDS, for the purpose of improving target directivity of drugs, construction of so-called temperature-responsive carriers having a function of releasing drugs in response to temperature has been attempted. Specifically, an attempt has been made to use, as a carrier, a molecular assembly that shows a vesicle shape when not heated, but changes its shape when heated. After the drug is held inside the vesicle and administered into the living body, the structure of the molecular assembly is greatly changed by selectively heating a desired site such as an affected part, for example. Attempts have been made to develop technology for releasing drugs only in More specifically, an attempt to use a molecular assembly composed of a compound in which a lipid having a polyamide dendron structure (polyamide dendron lipid) is modified with a temperature-responsive isobutylamide (IBAM) group for the above purpose. Has been reported (Non-patent Document 1).
　また、例えば免疫治療においては、キャリアにより抗原を免疫担当細胞へと正確に送達し、さらに主要組織適合遺伝子複合体（Ｍａｊｏｒ　Ｈｉｓｔｏｃｏｍｐａｔｉｂｉｌｉｔｙ　Ｃｏｍｐｌｅｘ；ＭＨＣ）への抗原提示を促進する必要がある。ヒトのＭＨＣであるＨＬＡは免疫誘導において中心的な役割を担っており、その機能を制御することによりアレルギー等の様々な免疫関連疾患又はがん等を治療できる。具体的には、ＨＬＡ（Human Leukocyte Antigen=ヒト白血球抗原）に対して作用しうるペプチドを用いることにより、ＨＬＡ機能を制御して免疫関連疾患を治療できる。また、ＨＬＡによる抗原提示を促進して、免疫を活性化することにより、がんを選択的に治療できる。 Also, for example, in immunotherapy, it is necessary to accurately deliver an antigen to an immunocompetent cell by a carrier, and further promote antigen presentation to a major histocompatibility complex (MHC). HLA, which is a human MHC, plays a central role in immunity induction, and can control various immune-related diseases such as allergies or cancer by controlling its function. Specifically, by using a peptide capable of acting on HLA (Human Leukocyte Antigen = human leukocyte antigen), an immune-related disease can be treated by controlling HLA function. Moreover, cancer can be selectively treated by promoting antigen presentation by HLA and activating immunity.
　これまでに、ｐＨ応答性高分子で修飾されており、ｐＨ変化に応じて脂質膜との融合能を獲得しうるリポソームに抗原を内包させ、これを樹状細胞内に導入することにより、抗原をサイトゾルへと送達することにより細胞性免疫を誘導したことが報告されている（非特許文献２）。より詳細には、上記リポソームはエンドサイトーシスにより細胞内にいったん取り込まれ、エンドソーム内におけるｐＨに応答してエンドソームと融合し、最終的に内包物（抗原）をサイトゾルへと放出する。 So far, the antigen is encapsulated in a liposome that has been modified with a pH-responsive polymer and can acquire a fusion ability with a lipid membrane in accordance with a change in pH, and then introduced into a dendritic cell, whereby the antigen is It has been reported that cell-mediated immunity was induced by delivery of the protein to the cytosol (Non-patent Document 2). More specifically, the liposome is once taken up into a cell by endocytosis, fuses with the endosome in response to the pH in the endosome, and finally releases the inclusion (antigen) into the cytosol.
　本発明者らは、薬物の標的指向性を向上することを目的として、温度応答性キャリアを利用しようとする場合、温度応答性キャリアにおいては鋭敏な温度応答性だけでなく、生体内において安定であること、及び生体適合性を有していることが求められることに着目した。本発明は、上記目的で利用されうる温度応答性キャリアにおいて、（１）温度応答性、（２）生体内安定性及び（３）生体適合性のバランスを改良することを第一の課題とする。 When the present inventors intend to use a temperature-responsive carrier for the purpose of improving the drug targeting property, the temperature-responsive carrier is not only sensitive to temperature but also stable in vivo. We focused on being there and being required to have biocompatibility. The first object of the present invention is to improve the balance of (1) temperature responsiveness, (2) in vivo stability and (3) biocompatibility in a temperature responsive carrier that can be used for the above purpose. .
　また、本発明は、目的物、具体的には生理活性物質、を（１）初期エンドソーム、（２）後期エンドソーム及び（３）リソソームのいずれかの細胞内オルガネラに選択的に送達してその内部で放出しうる手段を提供すること、すなわち具体的には当該手段としてのキャリア、及びその使用方法等を提供すること、を第二の課題とする。 In addition, the present invention selectively delivers a target substance, specifically, a physiologically active substance, to an intracellular organelle of any one of (1) early endosome, (2) late endosome, and (3) lysosome. The second problem is to provide a means that can be released by the above-mentioned method, that is, to provide a carrier as the means, a method of using the carrier, and the like.
　本発明者らは上記第一の課題を解決するため鋭意検討を行った。この検討の過程で本発明者らは、温度応答性キャリアにおいて（２）生体内安定性及び（３）生体適合性を同時に向上させようとしてもうまくいかないことを見出した。詳細には、生体適合性に優れることが期待される、オリゴエチレングリコ－ル構造を含有する炭化水素基でポリアミドデンドロン脂質を修飾することにより得られる化合物を用いて分子集合体を形成しても、生体内安定性に難があることを本発明者らは見出した。そこで、本発明者らは、上記化合物に加えてさらにポリエチレングリコール構造を含有する脂質を用いて分子集合体を形成し、かかる分子集合体においては、（１）温度応答性、（２）生体内安定性及び（３）生体適合性のバランスが従来のものに比べて改良されていることを見出した。 The present inventors have intensively studied to solve the first problem. In the course of this study, the inventors have found that it is not possible to improve (2) in vivo stability and (3) biocompatibility simultaneously in a temperature-responsive carrier. Specifically, a molecular assembly can be formed using a compound obtained by modifying a polyamide dendron lipid with a hydrocarbon group containing an oligoethylene glycol structure, which is expected to be excellent in biocompatibility. The present inventors have found that there is difficulty in in vivo stability. Therefore, the present inventors formed a molecular assembly using a lipid containing a polyethylene glycol structure in addition to the above compound. In such a molecular assembly, (1) temperature responsiveness, (2) in vivo It has been found that the balance between stability and (3) biocompatibility is improved compared to the conventional one.
　また、本発明者らは上記第二の課題を解決するため鋭意検討を行った。その結果、所定のデンドロン脂質をｐＨ応答性高分子で修飾して得られる化合物を利用することにより、上記課題を解決できることを見出した。より具体的には以下の通りである。上記化合物で構成されるリポソームに所望の物質を内包させた上でこれを細胞内に導入すると、エンドサイトーシスにより（１）初期エンドソームに効率良く取り込まれる。このリポソームは特定のｐＨ領域（応答ｐＨ領域）に応答して内包物を放出する性質を有しており、かつ応答ｐＨ領域を調整することは容易である。このリポソームを包含する（１）初期エンドソームが（２）後期エンドソームを経て（３）リソソームへと変化していく過程において、これら細胞内オルガネラ内のｐＨは連続的に変化していく。したがって、応答ｐＨ領域を適宜調整することにより、いずれかの所望の細胞内オルガネラにおいて選択的に内包物を放出させることができる。 In addition, the present inventors have intensively studied to solve the second problem. As a result, it has been found that the above problem can be solved by using a compound obtained by modifying a predetermined dendron lipid with a pH-responsive polymer. More specifically, it is as follows. When a desired substance is encapsulated in a liposome composed of the above compound and then introduced into a cell, (1) it is efficiently incorporated into the initial endosome by endocytosis. The liposome has a property of releasing inclusions in response to a specific pH range (response pH range), and it is easy to adjust the response pH range. In the process of (1) early endosomes including these liposomes changing to (2) late endosomes and (3) lysosomes, the pH in these intracellular organelles changes continuously. Therefore, by appropriately adjusting the response pH region, inclusions can be selectively released in any desired intracellular organelle.
　本発明は、本発明者らが上記知見に基づいてさらに鋭意検討を加えることにより完成されたものであり、以下の実施態様を含む。 The present invention has been completed by the inventors of the present invention based on the above findings, and has been completed and includes the following embodiments.
　第一の課題に対する解決手段としての発明（第一の発明）の実施態様は、以下の通りである。
項１．
（Ａ）下記式ＤＬ－Ｇ１～ＤＬ－Ｇ４のいずれかで表される化合物；及び
（Ｂ）ポリエチレングリコール構造を含有する脂質
を含有する、分子集合体
ＤＬ－Ｇ１：Ｒ１Ｒ２ＮＸ（ＸＨＲ３）ＸＨＲ４
ＤＬ－Ｇ２：Ｒ１Ｒ２ＮＸ（Ｘ（ＸＨＲ３）ＸＨＲ４）２
ＤＬ－Ｇ３：Ｒ１Ｒ２ＮＸ（Ｘ（Ｘ（ＸＨＲ３）ＸＨＲ４）２）２
ＤＬ－Ｇ４：Ｒ１Ｒ２ＮＸ（Ｘ（Ｘ（Ｘ（ＸＨＲ３）ＸＨＲ４）２）２）２
（式中、Ｒ１及びＲ２は、同一または異なって飽和又は不飽和の長鎖炭化水素基を示し、Ｒ３及びＲ４は、同一のまたは異なるオリゴエチレングリコ－ル構造を含有する炭化水素基を含有し、
Ｒ１～Ｒ４は、環状構造を含有していてもよく、かつ一以上の炭素原子がヘテロ原子で置換されていてもよく、かつＸは、－ＣＨ２ＣＨ２ＣＯＮＨＣＨ２ＣＨ２Ｎ－を示す。）。
項２．
前記Ｒ３及びＲ４が、以下の式（Ｉ）で表される、項１に記載の分子集合体 An embodiment of the invention (first invention) as a solution to the first problem is as follows.
Item 1.
(A) a compound represented by any of the following formulas DL-G1 to DL-G4; and (B) a molecular assembly DL-G1: R 1 R 2 NX (XHR containing a lipid containing a polyethylene glycol structure 3 ) XHR 4
DL-G2: R 1 R 2 NX (X (XHR 3 ) XHR 4 ) 2
DL-G3: R 1 R 2 NX (X (X (XHR 3 ) XHR 4 ) 2 ) 2
DL-G4: R 1 R 2 NX (X (X (X (XHR 3 ) XHR 4 ) 2 ) 2 ) 2
(Wherein R 1 and R 2 are the same or different and represent a saturated or unsaturated long chain hydrocarbon group, and R 3 and R 4 are hydrocarbons containing the same or different oligoethylene glycol structure. Containing groups,
R 1 to R 4 may contain a cyclic structure, and one or more carbon atoms may be substituted with a hetero atom, and X represents —CH 2 CH 2 CONHCH 2 CH 2 N—. Show. ).
Item 2.
Item 2. The molecular assembly according to Item 1, wherein R 3 and R 4 are represented by the following formula (I):
Figure JPOXMLDOC01-appb-C000003
Figure JPOXMLDOC01-appb-C000003
（式中、ｎは１～１０のいずれかの整数を示し、かつＲ５は炭素数１～１０の炭化水素基（一以上の炭素原子がヘテロ原子で置換されていてもよい）を示す。）。
項３．
前記脂質（Ｂ）が、以下の式（ＩＩ）で表される、項１又は２に記載の分子集合体 (Wherein n represents an integer of 1 to 10 and R 5 represents a hydrocarbon group having 1 to 10 carbon atoms (one or more carbon atoms may be substituted with a hetero atom)). ).
Item 3.
Item 3. The molecular assembly according to Item 1 or 2, wherein the lipid (B) is represented by the following formula (II):
Figure JPOXMLDOC01-appb-C000004
Figure JPOXMLDOC01-appb-C000004
（式中、Ｙは炭素数１０～５０の炭化水素基（一以上の炭素原子がヘテロ原子で置換されていてもよい）であり、かつｎは５～２００のいずれかの整数を示し、かつＲ６は炭素数１～１０の炭化水素基（一以上の炭素原子がヘテロ原子で置換されていてもよい）を示す。）。
項４．
生理活性物質を細胞内に導入するために使用される、項１～３のいずれかに記載の分子集合体。
項５．生理活性物質を細胞内に導入することにより疾患を治療するために使用される、項１～３のいずれかに記載の分子集合体。
項６．
項１～３のいずれかに記載の分子集合体を含有する組成物。
項７．
項１～６のいずれかに記載の分子集合体又は組成物を含有するキット。
項８．
項１～７のいずれかに記載の分子集合体、組成物又はキットの、生理活性物質を細胞内に導入する方法における使用。
項９．
項１～７のいずれかに記載の分子集合体又は組成物を生理活性物質とともに細胞内に導入する工程を含有する、生理活性物質を細胞内に導入する方法。 Wherein Y is a hydrocarbon group having 10 to 50 carbon atoms (one or more carbon atoms may be substituted with a heteroatom), and n represents any integer of 5 to 200, and R 6 represents a hydrocarbon group having 1 to 10 carbon atoms (one or more carbon atoms may be substituted with a hetero atom).
Item 4.
Item 4. The molecular assembly according to any one of Items 1 to 3, which is used for introducing a physiologically active substance into a cell.
Item 5. Item 4. The molecular assembly according to any one of Items 1 to 3, which is used for treating a disease by introducing a physiologically active substance into a cell.
Item 6.
Item 4. A composition containing the molecular assembly according to any one of Items 1 to 3.
Item 7.
Item 7. A kit containing the molecular assembly or composition according to any one of Items 1 to 6.
Item 8.
Item 8. Use of the molecular assembly, composition or kit according to any one of Items 1 to 7 in a method for introducing a physiologically active substance into a cell.
Item 9.
Item 8. A method for introducing a physiologically active substance into a cell, comprising the step of introducing the molecular assembly or composition according to any one of Items 1 to 7 into the cell together with the physiologically active substance.
　第二の課題に対する解決手段としての発明（第二の発明）の実施態様は、以下の通りである。
項１．
下記式ＤＬ－Ｇ１～ＤＬ－Ｇ４のいずれかで表される化合物
ＤＬ－Ｇ１：Ｒ１Ｒ２ＮＸ（ＸＨＲ３）ＸＨＲ４
ＤＬ－Ｇ２：Ｒ１Ｒ２ＮＸ（Ｘ（ＸＨＲ３）ＸＨＲ４）２
ＤＬ－Ｇ３：Ｒ１Ｒ２ＮＸ（Ｘ（Ｘ（ＸＨＲ３）ＸＨＲ４）２）２
ＤＬ－Ｇ４：Ｒ１Ｒ２ＮＸ（Ｘ（Ｘ（Ｘ（ＸＨＲ３）ＸＨＲ４）２）２）２
（式中、Ｒ１及びＲ２は、同一又は異なって飽和又は不飽和の長鎖炭化水素基を示し、
Ｒ３及びＲ４は、同一又は異なってｐＨ応答性のカルボキシル基含有炭化水素基を示し、Ｒ１～Ｒ４は、環状構造を含有していてもよく、かつ一以上の炭素原子がヘテロ原子で置換されていてもよく、かつ
Ｘは、－ＣＨ２ＣＨ２ＣＯＮＨＣＨ２ＣＨ２Ｎ－を示す。）。
項２．
前記ｐＨ応答性のカルボキシル基含有炭化水素基が、以下の式（Ｉ）で表される、項１に記載の化合物 An embodiment of the invention (second invention) as means for solving the second problem is as follows.
Item 1.
Compound DL-G1: R 1 R 2 NX (XHR 3 ) XHR 4 represented by any of the following formulas DL-G1 to DL-G4
DL-G2: R 1 R 2 NX (X (XHR 3 ) XHR 4 ) 2
DL-G3: R 1 R 2 NX (X (X (XHR 3 ) XHR 4 ) 2 ) 2
DL-G4: R 1 R 2 NX (X (X (X (XHR 3 ) XHR 4 ) 2 ) 2 ) 2
(Wherein R 1 and R 2 are the same or different and represent a saturated or unsaturated long chain hydrocarbon group,
R 3 and R 4 represent the same or different pH-responsive carboxyl group-containing hydrocarbon group, R 1 to R 4 may contain a cyclic structure, and one or more carbon atoms are heteroatoms And X represents —CH 2 CH 2 CONHCH 2 CH 2 N—. ).
Item 2.
Item 2. The compound according to Item 1, wherein the pH-responsive carboxyl group-containing hydrocarbon group is represented by the following formula (I):
Figure JPOXMLDOC01-appb-C000005
Figure JPOXMLDOC01-appb-C000005
（式中、Ｒ５は、飽和又は不飽和の炭素数１～１０の炭化水素基（環状構造を含有していてもよく、かつ一以上の炭素原子がヘテロ原子で置換されていてもよい。）を示す。）。
項３．
前記ｐＨ応答性のカルボキシル基含有炭化水素基が、
（Ａ）シクロヘキシル基、フェニル基、ピリジル基又はナフチル基を含有するか、又は
（Ｂ）炭素数１又は２（一以上の炭素原子がヘテロ原子で置換されていてもよい。）の一以上の分岐を有していてもよい鎖状構造である、
項１又は２に記載の化合物。
項４．
項１～３のいずれかに記載の化合物を含有する組成物。
項５．
リン脂質を含有する、項４に記載の組成物。
項６．
生理活性物質を細胞内に導入するために使用される、項４又は５に記載の組成物。
項７．
前記生理活性物質が、低分子化合物、ペプチド又は遺伝子である、項４又は５に記載の組成物。
項８．
抗原となりうるか又は抗原を生じうる生理活性物質を抗原提示細胞内に導入することにより疾患を治療するために使用される、項４又は５に記載の組成物。
項９．
項４又は５に記載の組成物を含有するキット。
項１０．
項１～９のいずれかに記載の化合物、組成物又はキットの、生理活性物質を細胞内に導入する方法における使用。
項１１．
項１～９のいずれかに記載の化合物、組成物又はキットを生理活性物質とともに細胞内に導入する工程を含有する、生理活性物質を細胞内に導入する方法。 (Wherein R 5 represents a saturated or unsaturated hydrocarbon group having 1 to 10 carbon atoms (which may contain a cyclic structure, and one or more carbon atoms may be substituted with a hetero atom). ).
Item 3.
The pH-responsive carboxyl group-containing hydrocarbon group is
(A) contains a cyclohexyl group, a phenyl group, a pyridyl group or a naphthyl group, or (B) one or more carbon atoms having 1 or 2 carbon atoms (one or more carbon atoms may be substituted with a heteroatom). It is a chain structure that may have a branch,
Item 3. The compound according to Item 1 or 2.
Item 4.
Item 4. A composition containing the compound according to any one of Items 1 to 3.
Item 5.
Item 5. The composition according to Item 4, comprising a phospholipid.
Item 6.
Item 6. The composition according to Item 4 or 5, which is used for introducing a physiologically active substance into cells.
Item 7.
Item 6. The composition according to Item 4 or 5, wherein the physiologically active substance is a low molecular compound, a peptide, or a gene.
Item 8.
Item 6. The composition according to Item 4 or 5, which is used for treating a disease by introducing a physiologically active substance capable of becoming an antigen or generating an antigen into an antigen-presenting cell.
Item 9.
Item 6. A kit containing the composition according to item 4 or 5.
Item 10.
Item 10. Use of the compound, composition or kit according to any one of Items 1 to 9 in a method for introducing a physiologically active substance into a cell.
Item 11.
Item 10. A method for introducing a physiologically active substance into a cell, comprising a step of introducing the compound, composition or kit according to any one of Items 1 to 9 into the cell together with the physiologically active substance.
　第一の発明によれば、薬物として生体に投与されうる生理活性物質の標的指向性を向上することを目的として利用される温度応答性キャリアであって、（１）温度応答性、（２）生体内安定性及び（３）生体適合性のバランスが改良されたキャリアを提供できる。また、本発明によれば、上記キャリアを利用することにより、標的指向性がより向上し、かつ生体に与えうる害が低減された生理活性物質の送達方法を提供できる。 According to the first invention, there is provided a temperature-responsive carrier used for the purpose of improving the target directivity of a physiologically active substance that can be administered to a living body as a drug, comprising (1) temperature responsiveness, (2) A carrier having an improved balance between in vivo stability and (3) biocompatibility can be provided. In addition, according to the present invention, by using the carrier, it is possible to provide a method for delivering a physiologically active substance with improved target directivity and reduced harm that can be given to a living body.
　また、第二の発明によれば、（１）初期エンドソーム、（２）後期エンドソーム及び（３）リソソームのいずれかの所望の細胞内オルガネラに選択的に目的物を送達できる。特に、本発明は、生理活性物質として抗原となりうるか又は抗原を生じうる生理活性物質を用いた場合には、抗原提示細胞を標的とすることにより、当該物質の細胞内オルガネラへの送達を通じて細胞免疫を誘導できるという優れた効果を有する。 In addition, according to the second invention, the target substance can be selectively delivered to any desired intracellular organelle of (1) early endosome, (2) late endosome, and (3) lysosome. In particular, in the present invention, when a physiologically active substance that can become an antigen or generate an antigen is used as a physiologically active substance, cell immunity can be achieved through delivery of the substance to intracellular organelles by targeting the antigen-presenting cell. It has an excellent effect that can be induced.
DL-G-0.5-2C18の1H NMR スペクトル (溶媒CDCl3 )である。It is the 1 H NMR spectrum (solvent CDCl 3 ) of DL-G-0.5-2C 18 . DL-G0-2C18の1H NMR スペクトル (溶媒CDCl3 ) である。 1 H NMR spectrum of DL-G0-2C 18 (solvent CDCl 3 ). DL-G0.5-2C18の1H NMR スペクトル (溶媒CDCl3 ) である。 1 H NMR spectrum (solvent CDCl 3 ) of DL-G0.5-2C 18 DL-G1-2C18の1H NMR スペクトル (溶媒CDCl3 ) である。It is a 1 H NMR spectrum of DL-G1-2C 18 (solvent CDCl 3). DL-G1.5-2C18の1H NMR スペクトル (溶媒CDCl3 ) である。It is the 1 H NMR spectrum (solvent CDCl 3 ) of DL-G1.5-2C 18 . DL-G2-2C18の1H NMR スペクトル (溶媒CDCl3 ) である。 1 H NMR spectrum of DL-G2-2C 18 (solvent CDCl 3 ). オレイルオレイルアミドの1H NMR スペクトル (溶媒CDCl3 ) である。 1 H NMR spectrum of oleyl oleylamide (solvent CDCl 3 ). ジオレイルアミンの1H NMR スペクトル (溶媒CDCl3 ) である。 1 H NMR spectrum of dioleylamine (solvent CDCl 3 ). DL-G-0.5-2C18-U2の1H NMR スペクトル (溶媒CDCl3 ) である。It is a 1 H NMR spectrum (solvent CDCl 3 ) of DL-G-0.5-2C 18 -U2. DL-G0-2C18-U2の1H NMR スペクトル (溶媒CDCl3 ) である。It is a 1 H NMR spectrum (solvent CDCl 3 ) of DL-G0-2C 18 -U2. DL-G0.5-2C18-U2の1H NMR スペクトル (溶媒CDCl3 ) である。It is a 1 H NMR spectrum (solvent CDCl 3 ) of DL-G0.5-2C 18 -U2. DL-G1-2C18-U2の1H NMR スペクトル (溶媒CDCl3 ) である。It is a 1 H NMR spectrum (solvent CDCl 3 ) of DL-G1-2C 18 -U2. MDEG-DL-G1の1H NMR スペクトル (溶媒CDCl3 ) である。 1 H NMR spectrum (solvent CDCl 3 ) of MDEG-DL-G1. MDEG-DL-G1 のマススペクトルである。This is the mass spectrum of MDEG-DL-G1. MDEG-DL-G2 の1H NMR スペクトル (溶媒CDCl3 ) である。 1 H NMR spectrum (solvent CDCl 3 ) of MDEG-DL-G2. MDEG-DL-G2 のマススペクトルである。This is the mass spectrum of MDEG-DL-G2. MDEG-DL-G1-U2の1H NMR スペクトル (溶媒CDCl3 ) である。It is a 1 H NMR spectrum (solvent CDCl 3 ) of MDEG-DL-G1-U2. EDEG-DL-G1の1H NMR スペクトル (溶媒CDCl3 ) である。 1 H NMR spectrum (solvent CDCl 3 ) of EDEG-DL-G1. EDEG-DL-G1 のマススペクトルである。This is the mass spectrum of EDEG-DL-G1. EDEG-DL-G2の1H NMR スペクトル (溶媒CDCl3 ) である。 1 H NMR spectrum (solvent CDCl 3 ) of EDEG-DL-G2. EDEG-DL-G1-U2の1H NMR スペクトル (溶媒CDCl3 ) である。 1 H NMR spectrum (solvent CDCl 3 ) of EDEG-DL-G1-U2. 種々のデンドロン脂質集合体分散液（10 mM リン酸140 mM NaCl水溶液、pH7.4）の透過率の温度依存性を示すグラフである。It is a graph which shows the temperature dependence of the transmittance | permeability of various dendron lipid aggregate dispersion liquids (10 M phosphoric acid 140 mM NaCl aqueous solution, pH 7.4). 種々のpHにおけるEDEG-DL-G2デンドロン脂質集合体分散液（10 mM リン酸140 mM NaCl水溶液）の透過率の温度依存性を示すグラフである。It is a graph which shows the temperature dependence of the transmittance | permeability of the EDEG-DL-G2 dendron lipid aggregate dispersion liquid (10 M phosphoric acid 140 m NaCl aqueous solution) at various pHs. EDEG-DL-G2デンドロン脂質集合体分散液（10 mM リン酸140 mM NaCl水溶液）の曇点のpH依存性を示すグラフである。It is a graph which shows the pH dependence of the cloud point of EDEG-DL-G2 dendron lipid aggregate dispersion liquid (10 mM mM phosphoric acid 140 mM mM NaCl aqueous solution). MDEG-DL-G1集合体分散液 , MDEG-DL-G2集合体分散液、および MDEG-DL-G1-U2集合体分散液（10 mM リン酸140 mM NaCl水溶液、pH7.4）の示差走査熱量測定（DSC）チャートである。Differential scanning calorimetry of MDEG-DL-G1 assembly dispersion, MDEG-DL-G2 assembly dispersion, and MDEG-DL-G1-U2 assembly dispersion (10 mM phosphoric acid 140 m NaCl aqueous solution, pH7.4) It is a measurement (DSC) chart. EDEG-DL-G1集合体分散液, EDEG-DL-G2集合体分散液、および EDEG-DL-G1-U2集合体分散液（10 mM リン酸140 mM NaCl水溶液、pH7.4）の示差走査熱量測定（DSC）チャートである。Differential scanning calorimetry of EDEG-DL-G1 assembly dispersion, EDEG-DL-G2 assembly dispersion, and EDEG-DL-G1-U2 assembly dispersion (10 mM phosphate 140 m NaCl aqueous solution, pH7.4) It is a measurement (DSC) chart. EDEG-DL-G1 集合体分散液（10 mM リン酸140 mM NaCl水溶液、pH7.4）の10 ℃および40 ℃の写真である。It is a photograph at 10 ° C and 40 ° C of EDEG-DL-G1G assembly dispersion (10 mM phosphoric acid 140 mM NaCl aqueous solution, pH 7.4). EDEG-DL-G1 集合体分散液（10 mM リン酸140 mM NaCl水溶液、pH7.4）の10 ℃および40 ℃の位相差顕微鏡写真である。It is a phase-contrast micrograph at 10 ° C. and 40 ° C. of an EDEG-DL-G1 assembly dispersion (10 mM phosphoric acid 140 m NaCl aqueous solution, pH 7.4). 種々の組成比のEDEG-G1/PEG-Chol集合体分散液（10 mM リン酸140 mM NaCl水溶液、pH7.4）の透過率の温度依存性を示すグラフである。It is a graph which shows the temperature dependency of the transmittance | permeability of the EDEG-G1 / PEG-Chol aggregate dispersion liquid (10 mM mM phosphoric acid 140 mM mM NaCl aqueous solution, pH 7.4) of various composition ratios. EDEG-G1/PEG-Chol 集合体分散液（10 mM リン酸140 mM NaCl水溶液、pH7.4）の曇点に及ぼすPEG-Chol 含率の影響を示すグラフである。It is a graph which shows the influence of the content rate of PEG-Chol which has on the cloud point of EDEG-G1 / PEG-Chol * aggregate dispersion liquid (10 mM sodium phosphate 140 mM mM aqueous NaCl solution, pH 7.4). 種々のpHにおけるEDEG-G1/PEG-Chol (5%) 集合体分散液の透過率の温度依存性（10 mM リン酸140 mM NaCl水溶液）を示すグラフである。It is a graph which shows the temperature dependence (10 M phosphoric acid 140 mM NaCl aqueous solution) of the transmittance of EDEG-G1 / PEG-Chol (5%) assembly dispersion at various pHs. EDEG-G1/PEG-Chol (95/5) 集合体分散液および EDEG-DL-G1集合体分散液（10 mM リン酸140 mM NaCl水溶液）の曇点のpH依存性を示すグラフである。It is a graph which shows the pH dependence of the cloud point of EDEG-G1 / PEG-Chol (95/5) assembly dispersion liquid and EDEG-DL-G1 assembly dispersion liquid (10 mM phosphoric acid 140 m NaCl aqueous solution). EDEG-DL-G1/PEG-Chol (95/5)集合体の粒子径の温度依存性を示すグラフである（粒子径は動的光散乱によって求めた。）。3 is a graph showing the temperature dependence of the particle size of an EDEG-DL-G1 / PEG-Chol® (95/5) aggregate (the particle size was determined by dynamic light scattering). EDEG-DL-G1/PEG-Chol (95/5)集合体の10℃および50℃における原子間力顕微鏡画像である。It is an atomic force microscope image of the EDEG-DL-G1 / PEG-Chol® (95/5) assembly at 10 ° C. and 50 ° C. ローダミン脂質（0.6 mol%）でラベル化したEDEG-G1/PEG-Chol (95/5) 集合体とインキュベートしたHeLa細胞 の共焦点レーザー顕微鏡写真である（インキュベート温度は36 ℃または42 ℃）。It is a confocal laser scanning micrograph of the HeLa cell cage | basket incubated with the EDEG-G1 / PEG-Chol (95/5) assembly labeled with the rhodamine lipid (0.6 mol%) (incubation temperature is 36 ℃ or 42 ℃). ローダミン脂質（0.6 mol%）でラベル化したEDEG-G1/PEG-Chol (95/5) 集合体とインキュベートしたHeLa細胞 の共焦点レーザー顕微鏡写真（インキュベート温度は36 ℃または42 ℃。図35の高倍率写真）。Confocal laser micrographs of HeLa cells incubated with EDEG-G1 / PEG-Chol (95/5) labeled with rhodamine lipid (0.6 mol%) (incubation temperature is 36 ° C or 42 ° C. High in Fig. 35 Magnification photo). 種々の組成比のMDEG-G1/PEG-Chol集合体分散液（10 mM リン酸140 mM NaCl水溶液、pH7.4）の透過率の温度依存性を示すグラフである（PEG-Chol含率（mol%）は、0,2,5,および10%。）。It is a graph which shows the temperature dependence of the transmittance | permeability of MDEG-G1 / PEG-Chol aggregate dispersion liquid (10 mM phosphoric acid 140 m NaCl aqueous solution, pH7.4) of various composition ratios (PEG-Chol content (mol %) Is 0, 2, 5, and 10%.) MDEG-G1/PEG-Chol 集合体分散液（10 mM リン酸140 mM NaCl水溶液、pH7.4）の曇点に及ぼすPEG-Chol 含率の影響を示すグラフである。It is a graph which shows the influence of the content rate of PEG-Chol on the cloud point of MDEG-G1 / PEG-Chol® aggregate dispersion (10 mM mM phosphoric acid 140 mM mM NaCl aqueous solution, pH 7.4). EDEG-DL-G1-2C18/PEG-Chol (95/5)集合体およびMDEG-DL-G1-2C18/PEG-Chol(98/2)集合体(いずれも約40℃に曇点を示す)と細胞との相互作用に及ぼすインキュベーション温度の影響を示すグラフである（デンドロン脂質／PEG-Chol集合体はローダミン脂質(0.6 mol%)でラベル化した。インキュベーション後の細胞からのローダミン蛍光強度を示した。）。EDEG-DL-G1-2C 18 / PEG-Chol (95/5) and MDEG-DL-G1-2C 18 / PEG-Chol (98/2) aggregates (both have a cloud point at about 40 ° C. Is a graph showing the influence of the incubation temperature on the interaction between the cell and the cell (the dendron lipid / PEG-Chol aggregate is labeled with rhodamine lipid (0.6 mol%). The intensity of rhodamine fluorescence from the cells after incubation is shown in FIG. Indicated.). Oleyloleoylamideの1H-NMRスペクトラムである(400 MHz, CDCl3)。 1 H-NMR spectrum of Oleyloleoylamide (400 MHz, CDCl 3 ). Dioleylamine の1H-NMRスペクトラムである(400 MHz, CDCl3)。 1 H-NMR spectrum of dioleylamine (400 MHz, CDCl 3 ). DL-G-0.5-2C18-U2の1H-NMRスペクトラムである(400 MHz, CDCl3) 。This is a 1 H-NMR spectrum of DL-G-0.5-2C 18 -U2 (400 MHz, CDCl 3 ). DL-G0-2C18-U2の1H-NMRスペクトラムである(400 MHz, CDCl3) 。This is a 1 H-NMR spectrum of DL-G0-2C 18 -U2 (400 MHz, CDCl 3 ). DL-G0.5-2C18-U2の1H-NMRスペクトラムである(400 MHz, CDCl3) 。This is a 1 H-NMR spectrum of DL-G0.5-2C 18 -U2 (400 MHz, CDCl 3 ). DL-G1-2C18-U2の1H-NMRスペクトラムである(400 MHz, CDCl3) 。It is a 1 H-NMR spectrum of DL-G1-2C 18 -U2 (400 MHz, CDCl 3 ). CHex-DL-G1-2C18-U2の1H-NMRスペクトラムである(400 MHz, CDCl3) 。It is a 1 H-NMR spectrum of CHex-DL-G1-2C 18 -U2 (400 MHz, CDCl 3 ). CHex-DL-G1-2C18-U2のMassスペクトラムである((A) Experimental, (B) theoretical) 。Mass spectrum of CHex-DL-G1-2C 18 -U2 ((A) Experimental, (B) theoretical). MGlu-DL-G1-2C18-U2の1H-NMRスペクトラムである(400 MHz, CDCl3) 。 1 H-NMR spectrum of MGlu-DL-G1-2C 18 -U2 (400 MHz, CDCl 3 ). MGlu-DL-G1-2C18-U2のMassスペクトラムである(experimental) 。Mass spectrum of MGlu-DL-G1-2C 18 -U2 (experimental). MGlu-DL-G1-2C18-U2のMassスペクトラムである(theoretical) 。Mass spectrum of MGlu-DL-G1-2C 18 -U2 (theoretical). DL-G-0.5-2C18の1H-NMRスペクトラムである(400 MHz, CDCl3) 。 1 H-NMR spectrum of DL-G-0.5-2C 18 (400 MHz, CDCl 3 ). DL-G0-2C18の1H-NMRスペクトラムである(400 MHz, CDCl3) 。This is a 1 H-NMR spectrum of DL-G0-2C 18 (400 MHz, CDCl 3 ). DL-G0.5-2C18の1H-NMRスペクトラムである(400 MHz, CDCl3) 。It is a 1 H-NMR spectrum of DL-G0.5-2C 18 (400 MHz, CDCl 3 ). C2-DL-G1-2C18の1H-NMRスペクトラムである(400 MHz, CDCl3) 。 1 H-NMR spectrum of C2-DL-G1-2C 18 (400 MHz, CDCl 3 ). CHex-DL-G1-2C18-U2の1H-NMRスペクトラムである(400 MHz, CD3OD) 。This is a 1 H-NMR spectrum of CHex-DL-G1-2C 18 -U2 (400 MHz, CD 3 OD). CHex-DL-G1-2C18のMassスペクトラムである((A) Experimental, (B) theoretical)。Mass spectrum of CHex-DL-G1-2C 18 ((A) Experimental, (B) theoretical). (A) CHex-DL-G1-2C18, (B) CHex-DL-G1-2C18-U2及び (C) MGlu-DL-G1-2C18-U2それぞれの単分子膜 の異なるｐＨにおけるπ-A isotherms (37℃, PBS)である。(A) CHex-DL-G1-2C 18 , (B) CHex-DL-G1-2C 18 -U2 and (C) MGlu-DL-G1-2C 18 -U2 π- A isotherms (37 ℃, PBS). 表面圧が25 mN/m となるときの表面積を各pH についてプロットしたものである（三角形、四角形及びひし型は、MGlu-DL-G1-2C18-U2、CHex-DL-G1-2C18-U2 及びCHex-DL-G1-2C18をそれぞれ示す）。Surface pressure is plotted for each pH the surface area of when the 25 mN / m (triangle, square and sebum type, MGlu-DL-G1-2C 18 -U2 , CHex-DL-G1-2C 18 - shown U2 and CHex-DL-G1-2C 18, respectively). 様々なデンドロン脂質の単分子膜の模式図である。It is a schematic diagram of the monomolecular film of various dendron lipids. 様々なデンドロン脂質の単分子膜の、異なるｐＨにおける模式図である。It is the schematic diagram in different pH of the monomolecular film of various dendron lipids. 各種リポソームを37℃、15 分間インキュベーションした後のピラニンのリリース（％）をpHに対してプロットした結果を示すグラフである（UMG1.25, UCG1.25, SCG1.25 及びEYPC は、それぞれMGlu-DL-G1-2C18-U2/EYPC (25/75, mol/mol), CHex-DL-G1-2C18-U2/EYPC (25/75, mol/mol), CHex-DL-G1-2C18/EYPC (25/75, mol/mol)及びEYPCをコンポーネントとしたリポソームを示している）。FIG. 5 is a graph showing the results of plotting pyranine release (%) against pH after incubation of various liposomes at 37 ° C. for 15 minutes (UMG1.25, UCG1.25, SCG1.25 and EYPC are respectively MGlu- DL-G1-2C 18 -U2 / EYPC (25/75, mol / mol), CHex-DL-G1-2C 18 -U2 / EYPC (25/75, mol / mol), CHex-DL-G1-2C 18 / EYPC (25/75, mol / mol) and liposomes with EYPC as components). 各種リポソームを37℃、15 分間インキュベーションした後のピラニンのリリース（％）をpHに対してプロットした結果を示すグラフである（UMG1.25, UMG1.40 及びEYPC は、それぞれ MGlu-DL-U2/EYPC モル比が25, 40及び0のリポソームを示している）。FIG. 6 is a graph showing the results of plotting pyranine release (%) against pH after incubation of various liposomes at 37 ° C. for 15 minutes (UMG1.25, UMG1.40 and EYPC are respectively MGMG-DL-U2 / Liposomes with EYPC mole ratios of 25, 40 and 0 are shown). CHexDL-U2/EYPCをコンポーネントとしたリポソームを37℃、15 分間インキュベーションした後のピラニンのリリース（％）をpHに対してプロットした結果を示すグラフである（UCG1.25, UCG1.10 及びEYPC は、それぞれ CHexDL-U2/EYPC モル比が25, 10及び0のリポソームを示している）。It is a graph showing the results of plotting the release of pyranine (%) against pH after incubating liposomes containing CHexDL-U2 / EYPC for 15 minutes at 37 ° C (UCG1.25, UCG1.10 and EYPC are , Liposomes with CHexDL-U2 / EYPC molar ratios of 25, 10 and 0, respectively). CHexDL-U2/EYPCをコンポーネントとしたリポソームを37℃、15 分間インキュベーションした後のピラニンのリリース（％）をpHに対してプロットした結果を示すグラフである（SCG1.25, SCG1.40 及びEYPC は、それぞれ CHexDL-U2/EYPC モル比が25, 40及び0のリポソームを示している）。It is a graph showing the results of plotting the release of pyranine (%) against pH after incubating liposomes containing CHexDL-U2 / EYPC for 15 minutes at 37 ° C (SCG1.25, SCG1.40 and EYPC , Liposomes having a CHexDL-U2 / EYPC mole ratio of 25, 40 and 0, respectively. 各種リポソームの各ｐＨにおける粒子径を示すグラフである（三角形、四角形及びひし形は、EYPC/MGlu-DL-G1-2C18-U2をコンポーネントとしたリポソーム、EYPC/CHex-DL-G1-2C18-U2をコンポーネントとしたリポソーム及びEYPC /CHex-DL-G1-2C18をコンポーネントとしたリポソームをそれぞれ示している。）。It is a graph showing the particle diameter at each pH of the various liposomes (triangles, squares and diamonds, liposomes of EYPC / MGlu-DL-G1-2C 18 -U2 and components, EYPC / CHex-DL-G1-2C 18 - the U2 and components liposomes and EYPC / CHex-DL-G1-2C 18 and the components with liposomes are shown, respectively.). 各種リポソームを各ｐＨのリン酸緩衝液中に懸濁した際の濁度を示すグラフである（三角形、四角形及びひし形は、EYPC/MGlu-DL-G1-2C18-U2をコンポーネントとしたリポソーム、EYPC/CHex-DL-G1-2C18-U2をコンポーネントとしたリポソーム及びEYPC /CHex-DL-G1-2C18をコンポーネントとしたリポソームをそれぞれ示している。）。It is a graph showing turbidity when various liposomes are suspended in a phosphate buffer solution of each pH (triangles, squares and rhombuses are liposomes having EYPC / MGlu-DL-G1-2C 18 -U2 as components, Liposome with EYPC / CHex-DL-G1-2C 18 -U2 as a component and liposome with EYPC / CHex-DL-G1-2C 18 as a component are shown). 各種リポソームの各ｐＨにおけるζ電位を示すグラフである（三角形、四角形及びひし形は、EYPC/MGlu-DL-G1-2C18-U2をコンポーネントとしたリポソーム、EYPC/CHex-DL-G1-2C18-U2をコンポーネントとしたリポソーム及びEYPC /CHex-DL-G1-2C18をコンポーネントとしたリポソームをそれぞれ示している。）。It is a graph showing the ζ potential at each pH of the various liposomes (triangles, squares and diamonds, liposomes of EYPC / MGlu-DL-G1-2C 18 -U2 and components, EYPC / CHex-DL-G1-2C 18 - the U2 and components liposomes and EYPC / CHex-DL-G1-2C 18 and the components with liposomes are shown, respectively.). MGlu-DL-2C18-U2をコンポーネントとしたリポソームの各pHにおけるデンドロン脂質の荷電状態を示す模式図である。It is a schematic view showing a charged state of the dendron lipids MGlu-DL-2C 18 -U2 at each pH of the liposomes with the component. CHex-DL-2C18-U2をコンポーネントとしたリポソーム及びCHex-DL-2C18をコンポーネントとしたリポソームの各pHにおけるデンドロン脂質の荷電状態を示す模式図である。Is a schematic view showing a charged state of the dendron lipids CHex-DL-2C 18 -U2 liposomes and CHex-DL-2C 18 with the components of the respective pH of liposomes and the component. MGlu-DL-2C18-U2/EYPCをコンポーネントとしたリポソームのζ電位とデンドロン脂質の表面圧が25 mN/m となるときのMGlu-DL-2C18-U2の表面積の関係を示すグラフである。It is a graph showing the relationship between the ζ potential of liposomes with MGlu-DL-2C 18 -U2 / EYPC as a component and the surface area of MGlu-DL-2C 18 -U2 when the surface pressure of the dendron lipid is 25 mN / m . CHex-DL-2C18-U2/EYPCをコンポーネントとしたリポソームのζ電位とデンドロン脂質の表面圧が25 mN/m となるときのCHex-DL-2C18-U2の表面積の関係を示すグラフである。It is a graph showing the relationship between the ζ potential of liposomes with CHex-DL-2C 18 -U2 / EYPC as a component and the surface area of CHex-DL-2C 18 -U2 when the surface pressure of the dendron lipid is 25 mN / m . CHex-DL-2C18 /EYPCをコンポーネントとしたリポソームのζ電位とデンドロン脂質の表面圧が25 mN/m となるときのCHex-DL-2C18の表面積の関係を示すグラフである。It is a graph which shows the relationship between the surface area of CHex-DL-2C 18 when the ζ potential of the liposome having CHex-DL-2C 18 / EYPC as a component and the surface pressure of the dendron lipid is 25 mN / m. MGlu-DL-2C18-U2/EYPC(25/75, mol/mol)をコンポーネントとしたリポソームのζ電位とピラニン放出の関係を示すグラフである。3 is a graph showing the relationship between ζ potential and pyranine release of liposomes containing MGlu-DL-2C 18 -U2 / EYPC (25/75, mol / mol) as a component. CHex-DL-2C18-U2/EYPC (25/75, mol/mol)をコンポーネントとしたリポソームのζ電位とピラニン放出の関係を示すグラフである。 CHex-DL-2C 18 -U2 / EYPC (25/75, mol / mol) is a graph showing the relationship between the ζ potential and pyranine release of liposomes as a component. CHex-DL-2C18/EYPC (25/75, mol/mol)をコンポーネントとしたリポソームのζ電位とピラニン放出の関係を示すグラフである。 CHex-DL-2C 18 / EYPC (25/75, mol / mol) is a graph showing the relationship between the ζ potential and pyranine release of liposomes as a component. Rh-PEでラベルした種々のリポソームで処理された(A) DC2.4 細胞及び(B) HeLa細胞をフローサイトメーターにより解析した結果を示すグラフである。It is a graph which shows the result of having analyzed the (A) <> DC2.4 <> cell and (B) <> HeLa cell processed with the various liposome labeled with Rh-PE with the flow cytometer. Rh-PEでラベルし、かつカルセインを内包する、CHex-DL-2C18/EYPC (25/75, mol/mol) をコンポーネントとしたリポソームで処理されたDC2.4細胞の共焦点レーザー走査型顕微鏡(CLSM)像を示す図面に代わる写真である（Rh-PE は赤色、カルセインは緑色及び核は青色に染色されている。スケールバーは10 μmを示している。）。Confocal laser scanning microscope of DC2.4 cells treated with liposomes with CHex-DL-2C 18 / EYPC (25/75, mol / mol) as a component, labeled with Rh-PE and encapsulating calcein (CLSM) This is a photograph in place of a drawing (Rh-PE is red, calcein is green, and nucleus is blue. Scale bar indicates 10 μm). Rh-PEでラベルし、かつカルセインを内包する、CHex-DL-2C18-U2/EYPC (25/75, mol/mol) をコンポーネントとしたリポソームで処理されたDC2.4細胞の共焦点レーザー走査型顕微鏡(CLSM)像を示す図面に代わる写真である（Rh-PE は赤色、カルセインは緑色及び核は青色に染色されている。スケールバーは10 μmを示している。）。Confocal laser scanning of DC2.4 cells treated with liposomes with CHex-DL-2C 18 -U2 / EYPC (25/75, mol / mol) components, labeled with Rh-PE and encapsulating calcein It is a photograph in place of a drawing showing a scanning microscope (CLSM) image (Rh-PE is red, calcein is green, and nucleus is blue. Scale bar indicates 10 μm). Rh-PEでラベルし、かつカルセインを内包する、MGlu-DL-2C18-U2/EYPC (25/75, mol/mol) をコンポーネントとしたリポソームで処理されたDC2.4細胞の共焦点レーザー走査型顕微鏡(CLSM)像を示す図面に代わる写真である（Rh-PE は赤色、カルセインは緑色及び核は青色に染色されている。スケールバーは10 μmを示している。）。Confocal laser scanning of DC2.4 cells treated with liposomes with Rh-PE labeled and encapsulating calcein with MGlu-DL-2C 18 -U2 / EYPC (25/75, mol / mol) as a component It is a photograph in place of a drawing showing a scanning microscope (CLSM) image (Rh-PE is red, calcein is green, and nucleus is blue. Scale bar indicates 10 μm). Rh-PEでラベルし、かつカルセインを内包するEYPCをコンポーネントとしたリポソームで処理されたDC2.4細胞の共焦点レーザー走査型顕微鏡(CLSM)像を示す図面に代わる写真である（Rh-PE は赤色、カルセインは緑色及び核は青色に染色されている。スケールバーは10 μmを示している。）。This is a photograph instead of a drawing showing a confocal laser scanning microscope (CLSM) image of DC2.4 cells labeled with Rh-PE and treated with liposomes containing calcein-encapsulated EYPC as a component (Rh-PE) (Red, calcein is green and nuclei are stained blue. Scale bar indicates 10 μm.) Rh-PEでラベルし、かつカルセインを内包する、CHex-DL-2C18/EYPC (25/75, mol/mol) をコンポーネントとしたリポソームで処理されたHeLa細胞の共焦点レーザー走査型顕微鏡(CLSM)像を示す図面に代わる写真である（Rh-PE は赤色、カルセインは緑色及び核は青色に染色されている。スケールバーは10 μmを示している。）。Confocal laser scanning microscope (CLSM) of HeLa cells labeled with Rh-PE and treated with liposomes with CHex-DL-2C 18 / EYPC (25/75, mol / mol) as a component encapsulating calcein ) This is a photograph instead of a drawing showing the image (Rh-PE is red, calcein is green, and nucleus is blue. Scale bar indicates 10 μm). Rh-PEでラベルし、かつカルセインを内包する、CHex-DL-2C18-U2/EYPC (25/75, mol/mol) をコンポーネントとしたリポソームで処理されたHeLa細胞の共焦点レーザー走査型顕微鏡(CLSM)像を示す図面に代わる写真である（Rh-PE は赤色、カルセインは緑色及び核は青色に染色されている。スケールバーは10 μmを示している。）。Confocal laser scanning microscope of HeLa cells treated with liposomes containing CHex-DL-2C 18 -U2 / EYPC (25/75, mol / mol) as a component, labeled with Rh-PE and encapsulating calcein (CLSM) This is a photograph in place of a drawing (Rh-PE is red, calcein is green, and nucleus is blue. Scale bar indicates 10 μm). Rh-PEでラベルし、かつカルセインを内包する、MGlu-DL-2C18-U2/EYPC (25/75, mol/mol) をコンポーネントとしたリポソームで処理されたHeLa細胞の共焦点レーザー走査型顕微鏡(CLSM)像を示す図面に代わる写真である（Rh-PE は赤色、カルセインは緑色及び核は青色に染色されている。スケールバーは10 μmを示している。）。Confocal laser scanning microscope of HeLa cells treated with liposomes containing MGlu-DL-2C 18 -U2 / EYPC (25/75, mol / mol) as a component, labeled with Rh-PE and encapsulating calcein (CLSM) This is a photograph in place of a drawing (Rh-PE is red, calcein is green, and nucleus is blue. Scale bar indicates 10 μm). Rh-PEでラベルし、かつカルセインを内包する、EYPC をコンポーネントとしたリポソームで処理されたHeLa細胞の共焦点レーザー走査型顕微鏡(CLSM)像を示す図面に代わる写真である（Rh-PE は赤色、カルセインは緑色及び核は青色に染色されている。スケールバーは10 μmを示している。）。This is a photograph instead of a drawing showing a confocal laser scanning microscope (CLSM) image of HeLa cells labeled with Rh-PE and encapsulating calcein and containing EYPC as a component. (Rh-PE is red) Calcein is stained green and nuclei are stained blue, scale bar indicates 10 μm). Rh-PEでラベルし、かつカルセインを内包する、MGlu-DL-2C18-U2/EYPC (25/75, mol/mol) をコンポーネントとしたリポソームで処理されたHeLa細胞の共焦点レーザー走査型顕微鏡(CLSM)像を示す図面に代わる写真である（Rh-PE は赤色、カルセインは緑色及び核は青色に染色されている。スケールバーは10 μmを示している。）。Confocal laser scanning microscope of HeLa cells treated with liposomes containing MGlu-DL-2C 18 -U2 / EYPC (25/75, mol / mol) as a component, labeled with Rh-PE and encapsulating calcein (CLSM) This is a photograph in place of a drawing (Rh-PE is red, calcein is green, and nucleus is blue. Scale bar indicates 10 μm). 細胞内における、リポソームの分布及び内包物質のリリースについての模式図である。It is a schematic diagram about the distribution of a liposome and release of the inclusion substance in a cell. Rh-PE由来の蛍光及びカルセイン由来の蛍光の共局在率を計測した結果を示すグラフである。It is a graph which shows the result of having measured the co-localization rate of fluorescence derived from Rh-PE and fluorescence derived from calcein. Rh-PEでラベルし、かつカルセインを内包する、CHex-DL-2C18-U2/EYPC (25/75, mol/mol) をコンポーネントとしたリポソームで処理したHela細胞の共焦点レーザー走査型顕微鏡(CLSM)像を示す図面に代わる写真である（後期エンドソーム／リソソームは赤色、カルセインは緑色に染色されている。スケールバーは10 μmを示している。）。Confocal laser scanning microscope of Hela cells labeled with Rh-PE and treated with liposomes with CHex-DL-2C 18 -U2 / EYPC (25/75, mol / mol) as a component, encapsulating calcein ( (CLSM) A photograph in place of a drawing showing an image (late endosome / lysosome is stained red, calcein is stained green, scale bar indicates 10 μm). ピラニンを内包した種々のpH7.4におけるリポソームのピラニン放出挙動を示す（EYPC、UMCG1.1525、UMCG1.2020及びUMG1.40は、それぞれEYPCリポソーム、MGlu-G1-2C18-U2/CHex-G1-2C18-U2/EYPC (15/25/60, mol/mol/mol) リポソーム、MGlu-G1-2C18-U2/CHex-G1-2C18-U2/EYPC (20/20/60, mol/mol/mol) リポソーム及びCHex-G1-2C18-U2/EYPC (40/60, mol/mol/mol) リポソームを示す。）。The pyranine release behavior at various pH 7.4 encapsulating pyranine is shown (EYPC, UMCG1.1525, UMCG1.2020 and UMG1.40 are respectively EYPC liposome, MGlu-G1-2C 18 -U2 / CHex-G1- 2C 18 -U2 / EYPC (15/25/60, mol / mol / mol) Liposome, MGlu-G1-2C 18 -U2 / CHex-G1-2C 18 -U2 / EYPC (20/20/60, mol / mol / mol) Liposomes and CHex-G1-2C 18 -U2 / EYPC (40/60, mol / mol / mol) liposomes). ピラニンを内包した種々のpH5.0におけるリポソームのピラニン放出挙動を示す（EYPC、UMCG1.1525、UMCG1.2020及びUMG1.40は、それぞれEYPCリポソーム、MGlu-G1-2C18-U2/CHex-G1-2C18-U2/EYPC (15/25/60, mol/mol/mol) リポソーム、MGlu-G1-2C18-U2/CHex-G1-2C18-U2/EYPC (20/20/60, mol/mol/mol) リポソーム及びCHex-G1-2C18-U2/EYPC (40/60, mol/mol/mol) リポソームを示す。）。The pyranine release behavior of liposomes at various pH 5.0 containing pyranine is shown (EYPC, UMCG1.1525, UMCG1.2020 and UMG1.40 are respectively EYPC liposome, MGlu-G1-2C 18 -U2 / CHex-G1- 2C 18 -U2 / EYPC (15/25/60, mol / mol / mol) Liposome, MGlu-G1-2C 18 -U2 / CHex-G1-2C 18 -U2 / EYPC (20/20/60, mol / mol / mol) Liposomes and CHex-G1-2C 18 -U2 / EYPC (40/60, mol / mol / mol) liposomes). ピラニンを内包した種々のpH6.0におけるリポソームのピラニン放出挙動を示す（EYPC、UMCG1.1525、UMCG1.2020及びUMG1.40は、それぞれEYPCリポソーム、MGlu-G1-2C18-U2/CHex-G1-2C18-U2/EYPC (15/25/60, mol/mol/mol) リポソーム、MGlu-G1-2C18-U2/CHex-G1-2C18-U2/EYPC (20/20/60, mol/mol/mol) リポソーム及びCHex-G1-2C18-U2/EYPC (40/60, mol/mol/mol) リポソームを示す。）。The pyranine release behavior of liposomes at various pH 6.0 containing pyranine is shown (EYPC, UMCG1.1525, UMCG1.2020 and UMG1.40 are respectively EYPC liposome, MGlu-G1-2C 18 -U2 / CHex-G1- 2C 18 -U2 / EYPC (15/25/60, mol / mol / mol) Liposome, MGlu-G1-2C 18 -U2 / CHex-G1-2C 18 -U2 / EYPC (20/20/60, mol / mol / mol) Liposomes and CHex-G1-2C 18 -U2 / EYPC (40/60, mol / mol / mol) liposomes). ピラニンを内包した種々のリポソームの、種々のpHにおける15分間インキュベーション後におけるピラニン放出挙動を示す（EYPC、UMCG1.1525、UMCG1.2020及びUMG1.40は、それぞれEYPCリポソーム、MGlu-G1-2C18-U2/CHex-G1-2C18-U2/EYPC (15/25/60, mol/mol/mol) リポソーム、MGlu-G1-2C18-U2/CHex-G1-2C18-U2/EYPC (20/20/60, mol/mol/mol) リポソーム及びCHex-G1-2C18-U2/EYPC (40/60, mol/mol/mol) リポソームを示す。）。Various liposome encapsulated pyranine shows pyranine release behavior after 15 minutes incubation at various pH (EYPC, UMCG1.1525, UMCG1.2020 and UMG1.40 each EYPC liposomes, MGlu-G1-2C 18 - U2 / CHex-G1-2C 18 -U2 / EYPC (15/25/60, mol / mol / mol) Liposomes, MGlu-G1-2C 18 -U2 / CHex-G1-2C 18 -U2 / EYPC (20/20 / 60, mol / mol / mol) liposome and CHex-G1-2C 18 -U2 / EYPC (40/60, mol / mol / mol) liposome). 37℃での、15分間インキュベーション後における、修飾リポソーム（2.0×10-5 M）からのpH依存的なピラニン放出挙動を示す図面である。It is a figure which shows the pH dependent pyranine release | release behavior from a modified liposome (2.0 * 10 <-5> M) after incubation for 15 minutes at 37 degreeC. デンドロン脂質／EYPC(25/75 又は 10/90)のζ電位をpHの関数として示した図面である。It is the figure which showed the zeta potential of dendron lipid / EYPC (25/75 ^ or 10/90) as a function of pH. 単分子膜測定（圧力は25mN/m）の結果を示す図面である。It is drawing which shows the result of a monomolecular film measurement (a pressure is 25 mN / m). Rh-PEでラベルされたリポソームの細胞による取り込みを評価した結果を示す図面である。It is a figure which shows the result of having evaluated the uptake | capture by the cell of the liposome labeled with Rh-PE. 抗腫瘍効果の検討結果を示す図面である（各種リポソームを投与したマウスと、非投与マウス（ネガティブコントロール）との比較を示している）。It is drawing which shows the examination result of an anti-tumor effect (The comparison with the mouse | mouth which administered various liposome and the non-administration mouse | mouth (negative control) is shown).
Ｉ．第一の発明
１．本発明の分子集合体
　本発明の分子集合体は、
（Ａ）下記式ＤＬ－Ｇ１～ＤＬ－Ｇ４のいずれかで表される化合物；及び
（Ｂ）ポリエチレングリコール構造を含有する脂質
を含有する、分子集合体
ＤＬ－Ｇ１：Ｒ１Ｒ２ＮＸ（ＸＨＲ３）ＸＨＲ４
ＤＬ－Ｇ２：ＲPＲ２ＮＸ（Ｘ（ＸＨＲ３）ＸＨＲ４）２
ＤＬ－Ｇ３：Ｒ１Ｒ２ＮＸ（Ｘ（Ｘ（ＸＨＲ３）ＸＨＲ４）２）２
ＤＬ－Ｇ４：Ｒ１Ｒ２ＮＸ（Ｘ（Ｘ（Ｘ（ＸＨＲ３）ＸＨＲ４）２）２）２
（式中、Ｒ１及びＲ２は、同一または異なって飽和又は不飽和の長鎖炭化水素基を示し、Ｒ３及びＲ４は、同一のまたは異なるオリゴエチレングリコ－ル構造を含有する炭化水素基を含有し、
Ｒ１～Ｒ４は、環状構造を含有していてもよく、かつ一以上の炭素原子がヘテロ原子で置換されていてもよく、かつＸは、－ＣＨ２ＣＨ２ＣＯＮＨＣＨ２ＣＨ２Ｎ－を示す。）である。 I. First invention 1. Molecular assembly of the present invention The molecular assembly of the present invention comprises:
(A) a compound represented by any one of the following formulas DL-G1 to DL-G4; and (B) a molecular assembly DL-G1: R 1 R 2 NX (XHR containing a lipid containing a polyethylene glycol structure 3 ) XHR 4
DL-G2: R P R 2 NX (X (XHR 3) XHR 4) 2
DL-G3: R 1 R 2 NX (X (X (XHR 3 ) XHR 4 ) 2 ) 2
DL-G4: R 1 R 2 NX (X (X (X (XHR 3 ) XHR 4 ) 2 ) 2 ) 2
(Wherein R 1 and R 2 are the same or different and represent a saturated or unsaturated long chain hydrocarbon group, and R 3 and R 4 are hydrocarbons containing the same or different oligoethylene glycol structure. Containing groups,
R 1 to R 4 may contain a cyclic structure, and one or more carbon atoms may be substituted with a hetero atom, and X represents —CH 2 CH 2 CONHCH 2 CH 2 N—. Show. ).
　１．１　化合物（Ａ）についての説明
　１．１．１　Ｘについての説明
　Ｘは、－ＣＨ２ＣＨ２ＣＯＮＨＣＨ２ＣＨ２Ｎ－を表し、その末端のＮは、通常２個の水素原子を有するが、１個の水素原子が疎水性アミノ酸又はアルキル基で置換されていてもよい。疎水性アミノ酸としては、ロイシン、バリン、イソロイシン、ノルロイシン、フェニルアラニン及びチロシン等が挙げられる。また、アルキル基としては、メチル基、エチル基、ノルマルプロピル基、イソプロピル基及びシクロプロピル基等が挙げられる。 1.1 Description of Compound (A) 1.1.1 Description of X X represents —CH 2 CH 2 CONHCH 2 CH 2 N—, and the terminal N usually has two hydrogen atoms. However, one hydrogen atom may be substituted with a hydrophobic amino acid or an alkyl group. Examples of hydrophobic amino acids include leucine, valine, isoleucine, norleucine, phenylalanine and tyrosine. Examples of the alkyl group include a methyl group, an ethyl group, a normal propyl group, an isopropyl group, and a cyclopropyl group.
　１．１．２　Ｒ １ 及びＲ ２ についての説明
　Ｒ１及びＲ２は、飽和又は不飽和の長鎖炭化水素基である。 Description R 1 and R 2 for 1.1.2 R 1 and R 2 are long chain, saturated or unsaturated hydrocarbon group.
　長鎖炭化水素基は、天然由来であってもよく、天然由来のものを修飾したものでもよく、又は人工的に合成されたものであってもよい。 The long-chain hydrocarbon group may be naturally derived, may be modified from naturally derived, or may be artificially synthesized.
　長鎖炭化水素基の炭素数は、主鎖が長鎖であればよく限定されない。主鎖の炭素数は好ましくは８～３０であり、より好ましくは１０～２２であり、さらに好ましくは１２～２０である。 The number of carbon atoms of the long-chain hydrocarbon group is not limited as long as the main chain is a long chain. The number of carbon atoms in the main chain is preferably 8 to 30, more preferably 10 to 22, and still more preferably 12 to 20.
　長鎖炭化水素基は、分岐を有していてもよい。なお、主鎖についてはＩＵＰＡＣ命名法に基づいて、又はそれが困難若しくは不可能な場合はそれに準ずる方法に基づいて決定する。 The long chain hydrocarbon group may have a branch. The main chain is determined based on the IUPAC nomenclature, or when it is difficult or impossible, it is determined based on a similar method.
　長鎖炭化水素基は、環状構造を有していてもよい。環状構造としては、特に限定されないが、例えば、フェニル基、コレステリル基、シクロヘキシル基等が挙げられる。特に、コレステリル基が好ましい。 The long chain hydrocarbon group may have a cyclic structure. Although it does not specifically limit as a cyclic structure, For example, a phenyl group, a cholesteryl group, a cyclohexyl group etc. are mentioned. Particularly preferred is a cholesteryl group.
　長鎖炭化水素基は、少なくとも１つ以上の炭素原子がヘテロ原子で置換されているものであってもよい。本発明において、ヘテロ原子とは、酸素原子、窒素原子又は硫黄原子をいう。したがって、長鎖炭化水素基には、酸素含有炭化水素基、窒素含有炭化水素基及び硫黄含有炭化水素基が含まれる。 The long chain hydrocarbon group may be one in which at least one carbon atom is substituted with a hetero atom. In the present invention, the hetero atom means an oxygen atom, a nitrogen atom or a sulfur atom. Accordingly, the long chain hydrocarbon group includes an oxygen-containing hydrocarbon group, a nitrogen-containing hydrocarbon group, and a sulfur-containing hydrocarbon group.
　本発明において、炭化水素基とは、アルキル基、アルケニル基又はアルキニル基をいう。 In the present invention, the hydrocarbon group means an alkyl group, an alkenyl group or an alkynyl group.
　酸素含有炭化水素基としては、例えば、エーテル結合及びカルボニル結合からなる群より選択される少なくとも１種の結合を有する酸素含有炭化水素基が挙げられる。カルボニル結合を有する酸素含有炭化水素基としては、例えば、アルデヒド、ケトン、カルボン酸、エステル、アミド、エノン、酸塩化物又は無水物等からなる群より選択される少なくとも１種の構造を有する基が挙げられる。 Examples of the oxygen-containing hydrocarbon group include an oxygen-containing hydrocarbon group having at least one bond selected from the group consisting of an ether bond and a carbonyl bond. Examples of the oxygen-containing hydrocarbon group having a carbonyl bond include a group having at least one structure selected from the group consisting of aldehyde, ketone, carboxylic acid, ester, amide, enone, acid chloride, or anhydride. Can be mentioned.
　窒素含有炭化水素基としては、例えばニトリル、アミン、アミド及びイミドからなる群より選択される少なくとも１種の構造を有する窒素含有炭化水素基が挙げられる。 Examples of the nitrogen-containing hydrocarbon group include a nitrogen-containing hydrocarbon group having at least one structure selected from the group consisting of nitrile, amine, amide and imide.
　硫黄含有炭化水素基としては、例えばチオール、チオエーテル、チオアセタール、スルフィド、ジスルフィド、ジチオカルボン酸、チオエステル、チオケトン、チオアルデヒド、チオカルバメート、チオウレタン、ホスフィンスルフィド、チオホスフェート、チオホスホネート、スルホネート、スルホン及びスルホンアミドからなる群より選択される少なくとも１種の結合を有する硫黄含有炭化水素基が挙げられる。 Examples of the sulfur-containing hydrocarbon group include thiol, thioether, thioacetal, sulfide, disulfide, dithiocarboxylic acid, thioester, thioketone, thioaldehyde, thiocarbamate, thiourethane, phosphine sulfide, thiophosphate, thiophosphonate, sulfonate, sulfone and And sulfur-containing hydrocarbon groups having at least one bond selected from the group consisting of sulfonamides.
　長鎖炭化水素基は、上に例示したような構造の基においてさらに少なくとも１つ以上の置換基を有していてもよい。置換基としては、疎水性を示すものが好ましい。置換基としては、例えば、フェニル基、コレステリル基及びピレニル基等が挙げられる。特にコレステリル基が好ましい。 The long chain hydrocarbon group may further have at least one substituent in the group having the structure as exemplified above. As the substituent, those showing hydrophobicity are preferable. Examples of the substituent include a phenyl group, a cholesteryl group, and a pyrenyl group. A cholesteryl group is particularly preferable.
　不飽和長鎖炭化水素基は、不飽和結合を主鎖に有しており、かつ主鎖が鎖状構造を有する炭化水素基であれば好ましい。 The unsaturated long chain hydrocarbon group is preferably a hydrocarbon group having an unsaturated bond in the main chain and the main chain having a chain structure.
　不飽和結合の種類は、二重結合であってもよく、又は三重結合であってもよい。二重結合が好ましい。不飽和結合の数は、少なくとも１つ以上であればよく、限定されない。また、二重結合と三重結合の両方を有していてもよい。二重結合を１つ有していることが好ましい。 The type of unsaturated bond may be a double bond or a triple bond. A double bond is preferred. The number of unsaturated bonds should just be at least 1 or more, and is not limited. Moreover, you may have both a double bond and a triple bond. It preferably has one double bond.
　二重結合としては、シス型二重結合であってもよいし、トランス型二重結合であってもよい。シス型二重結合が好ましい。 The double bond may be a cis-type double bond or a trans-type double bond. A cis type double bond is preferred.
　不飽和長鎖炭化水素基としては、ヘキサデセニル基、オクタデセニル基、オクタデカジエニル基、オクタデカトリエニル基、イコサトリエニル基、イコサテトラエニル基、オクタデカトリエニル基、イコサペンタエニル基又はドコサヘキサエニル基が好ましい。 Examples of unsaturated long-chain hydrocarbon groups include hexadecenyl, octadecenyl, octadecadienyl, octadecatrienyl, icosatrienyl, icosatetraenyl, octadecatrienyl, icosapentaenyl, or docosa A hexaenyl group is preferred.
　不飽和長鎖炭化水素基としては、９－ヘキサデセニル基、９－オクタデセニル基（オレイル基）、１２－オクタデカジエニル基、６，９，１２－オクタデカトリエニル基、８，１１，１４－イコサトリエニル基、５，８，１１，１４－イコサテトラエニル基、９，１２，１５－オクタデカトリエニル基、５，８，１１，１４，１７－イコサペンタエニル基又は４，７，１３，１６，１９－ドコサヘキサエニル基がより好ましい。 Examples of unsaturated long chain hydrocarbon groups include 9-hexadecenyl group, 9-octadecenyl group (oleyl group), 12-octadecadienyl group, 6,9,12-octadecatrienyl group, 8,11,14- Eicosatrienyl group, 5,8,11,14-icosatetraenyl group, 9,12,15-octadecatrienyl group, 5,8,11,14,17-icosapentaenyl group or 4,7,13 , 16,19-docosahexaenyl group is more preferred.
　不飽和長鎖炭化水素基としては、９－オクタデセニル基（オレイル基）がさらに好ましい。 As the unsaturated long chain hydrocarbon group, a 9-octadecenyl group (oleyl group) is more preferable.
　不飽和長鎖炭化水素基の好適例としては、上に好適例として例示したような構造の基においてさらに少なくとも1つ以上の炭素原子がヘテロ原子で置換されているものも挙げられる。 Preferred examples of the unsaturated long-chain hydrocarbon group include those in which at least one carbon atom is further substituted with a hetero atom in the group having the structure exemplified above as a preferred example.
　不飽和長鎖炭化水素基の好適例としては、上に好適例として例示したような構造の基を基礎として、酸素含有炭化水素基、窒素含有炭化水素基、又は硫黄含有炭化水素基に特有な構造として上に例示したような構造をさらに有しているものであってもよい。 Preferred examples of the unsaturated long chain hydrocarbon group are specific to oxygen-containing hydrocarbon groups, nitrogen-containing hydrocarbon groups, or sulfur-containing hydrocarbon groups on the basis of the group having the structure exemplified above as a preferred example. The structure may further include a structure as exemplified above.
　不飽和長鎖炭化水素基の好適例としては、上に好適例として例示したような構造の基においてさらに少なくとも１つ以上の置換基を有していてもよい。置換基としては、疎水性を示すものが好ましい。置換基としては、例えば、フェニル基、フェニレン基、コレステリル基、及びピレニル基等が挙げられる。特にコレステリル基が好ましい。 As a preferred example of the unsaturated long chain hydrocarbon group, the group having the structure as exemplified above as a preferred example may further have at least one substituent. As the substituent, those showing hydrophobicity are preferable. Examples of the substituent include a phenyl group, a phenylene group, a cholesteryl group, and a pyrenyl group. A cholesteryl group is particularly preferable.
　１．１．３　オリゴエチレングリコ－ル構造を含有する炭化水素基についての説明
　オリゴエチレングリコ－ル構造を含有する炭化水素基（オリゴエチレングリコ－ル含有炭化水素基）は、オリゴエチレングリコ－ル構造を含有していればよく、他の構造をさらに含有していてもよい。オリゴエチレングリコ－ル含有炭化水素基は、オリゴエチレングリコ－ル構造の間に他の構造が介在していてもよいし、他の構造の間にオリゴエチレングリコ－ル構造が介在していてもよい。 1.1.3 Description of Hydrocarbon Group Containing Oligoethylene Glycol Structure The hydrocarbon group containing oligoethylene glycol structure (oligoethylene glycol-containing hydrocarbon group) is oligoethylene glycol. It only needs to contain a structure, and may further contain other structures. In the oligoethylene glycol-containing hydrocarbon group, other structures may be interposed between the oligoethylene glycol structures, or the oligoethylene glycol structure may be interposed between the other structures. Good.
　上記他の構造は、分岐を有していてもよい直鎖構造であってもよい。そのような構造として、特に限定されないが、例えば、炭素数１～１０の分岐を有していてもよい、炭素数１～１０の直鎖部分を有するものが挙げられる。 The other structure may be a linear structure which may have a branch. Such a structure is not particularly limited, and examples thereof include those having a straight chain portion having 1 to 10 carbon atoms which may have a branch having 1 to 10 carbon atoms.
　上記他の構造は、環状構造であってもよい。環状構造としては、特に限定されないが、例えば、シクロプロピル基、エポキシ基、シクロへプチル基、シクロブチル基、フェニル基、コレステリル基及びシクロヘキシル基等が挙げられる。特に、シクロヘキシル基が好ましい。 The other structure may be a ring structure. The cyclic structure is not particularly limited, and examples thereof include a cyclopropyl group, an epoxy group, a cycloheptyl group, a cyclobutyl group, a phenyl group, a cholesteryl group, and a cyclohexyl group. A cyclohexyl group is particularly preferable.
　上記他の構造は、一以上の炭素原子がヘテロ原子で置換されていてもよい。 In the other structures described above, one or more carbon atoms may be substituted with a heteroatom.
　オリゴエチレングリコ－ル構造としては、本発明の効果が奏されればよく特に限定されないが、例えば、オキシエチレンユニットを連続して、言い換えれば他の構造が介在することなく、１～１０個有する構造が挙げられる。オリゴエチレングリコ－ル構造としては、オキシエチレンユニットを連続して１～４個有するものが好ましい。 The oligoethylene glycol structure is not particularly limited as long as the effects of the present invention can be achieved. For example, the oligoethylene glycol structure has 1 to 10 oxyethylene units continuously, in other words, without any other structure interposed. Structure is mentioned. The oligoethylene glycol structure preferably has 1 to 4 oxyethylene units in succession.
　オリゴエチレングリコ－ル構造としては、本発明の効果が奏されればよく特に限定されないが、例えば、二以上の分断されたオキシエチレンユニットを有する構造、言い換えれば他の構造を介して、合計１～１０個のオキシエチレンユニットを有する構造が挙げられる。この場合、オキシエチレンユニットを合計１～４個有するものが好ましい。 The oligoethylene glycol structure is not particularly limited as long as the effects of the present invention can be achieved. For example, a structure having two or more divided oxyethylene units, in other words, through another structure, a total of 1 Examples include structures having up to 10 oxyethylene units. In this case, those having a total of 1 to 4 oxyethylene units are preferred.
　オリゴエチレングリコ－ル含有炭化水素基は、分岐部分がさらにオリゴエチレングリコ－ル構造を有していてもよい。 The branched chain of the oligoethylene glycol-containing hydrocarbon group may further have an oligoethylene glycol structure.
　オリゴエチレングリコ－ル含有炭化水素基は、好ましくは、以下の式（Ｉ）で表される　　　 Oligoethylene glycol-containing hydrocarbon group is preferably represented by the following formula (I):
Figure JPOXMLDOC01-appb-C000006
Figure JPOXMLDOC01-appb-C000006
（式中、ｎは１～１０のいずれかの整数を示し、かつＲ５は炭素数１～１０の炭化水素基（一以上の炭素原子がヘテロ原子で置換されていてもよい）を示す。）
　オリゴエチレングリコ－ル含有炭化水素基は、具体的には、下記の式（ＩＩＩ）で表されるものが挙げられる (Wherein n represents an integer of 1 to 10 and R 5 represents a hydrocarbon group having 1 to 10 carbon atoms (one or more carbon atoms may be substituted with a hetero atom)). )
Specific examples of the oligoethylene glycol-containing hydrocarbon group include those represented by the following formula (III).
Figure JPOXMLDOC01-appb-C000007
Figure JPOXMLDOC01-appb-C000007
（式中、ｎは１～１０のいずれかの整数を示す。）。 (Wherein n represents an integer of 1 to 10).
　１．２　ポリエチレングリコール構造を含有する脂質（Ｂ）についての説明
　ポリエチレングリコール構造を含有する脂質（Ｂ）は、ポリエチレングリコール構造を含有していればよく、本発明の効果が奏される限り、特に限定されない。 1.2 Description of Lipid (B) Containing Polyethylene Glycol Structure The lipid (B) containing a polyethylene glycol structure only needs to contain a polyethylene glycol structure, and as long as the effects of the present invention are exhibited, It is not limited.
　ポリエチレングリコール構造を含有する脂質（Ｂ）は、ポリエチレングリコール構造を含有する部分、及び脂質部分に分けられる。 The lipid (B) containing a polyethylene glycol structure is divided into a part containing a polyethylene glycol structure and a lipid part.
　１．２．１　ポリエチレングリコール構造を含有する部分についての説明
　ポリエチレングリコール構造を含有する部分は、ポリエチレングリコール構造を含有していればよく、他の炭化水素構造をさらに含有していてもよい。ポリエチレングリコール構造を含有する部分は、ポリエチレングリコール構造の間に他の炭化水素構造が介在していてもよいし、他の炭化水素構造の間にポリエチレングリコール構造が介在していてもよい。　上記他の炭化水素構造は、分岐を有していてもよく直鎖構造であってもよい。そのような構造として、特に限定されないが、例えば、炭素数１～１０の分岐を有していてもよい、炭素数１～１０の直鎖部分を有するものが挙げられる。 1.2.1 Description of Part Containing Polyethylene Glycol Structure The part containing the polyethylene glycol structure only needs to contain a polyethylene glycol structure, and may further contain other hydrocarbon structures. In the part containing the polyethylene glycol structure, another hydrocarbon structure may be interposed between the polyethylene glycol structures, or the polyethylene glycol structure may be interposed between the other hydrocarbon structures. The other hydrocarbon structure may have a branch or a straight chain structure. Such a structure is not particularly limited, and examples thereof include those having a straight chain portion having 1 to 10 carbon atoms which may have a branch having 1 to 10 carbon atoms.
　上記他の炭化水素構造は、環状構造であってもよい。環状構造としては、特に限定されないが、例えば、シクロプロピル基、エポキシ基、シクロへプチル基、シクロブチル基、フェニル基、コレステリル基及びシクロヘキシル基等が挙げられる。特に、シクロヘキシル基が好ましい。 The other hydrocarbon structure may be a cyclic structure. The cyclic structure is not particularly limited, and examples thereof include a cyclopropyl group, an epoxy group, a cycloheptyl group, a cyclobutyl group, a phenyl group, a cholesteryl group, and a cyclohexyl group. A cyclohexyl group is particularly preferable.
　上記他の炭化水素構造は、一以上の炭素原子がヘテロ原子で置換されていてもよい。 In the other hydrocarbon structure, one or more carbon atoms may be substituted with a hetero atom.
　ポリエチレングリコ－ル構造としては、本発明の効果が奏されればよく特に限定されないが、例えば、オキシエチレンユニットを連続して、言い換えれば他の構造が介在することなく、３～２００個有する構造が挙げられる。ポリエチレングリコ－ル構造としては、オキシエチレンユニットを連続して３～２００個有するものが好ましく、１０～１００個有するものがより好ましい。 The polyethylene glycol structure is not particularly limited as long as the effects of the present invention can be achieved. For example, a structure having 3 to 200 oxyethylene units continuously, in other words, without any other structure interposed therebetween. Is mentioned. The polyethylene glycol structure preferably has 3 to 200 oxyethylene units in succession, more preferably 10 to 100.
　１．２．２　脂質部分についての説明
　脂質部分は、本発明の効果が奏される限り、特に限定されない。 1.2.2 Description of Lipid Portion The lipid portion is not particularly limited as long as the effect of the present invention is exhibited.
　脂質部分としては、例えば、炭素数１０～５０の炭化水素基（一以上の炭素原子がヘテロ原子で置換されていてもよい）が挙げられる。上記において炭化水素基は、飽和であってもよく不飽和であってもよく、また環状構造を有していてもよい。 Examples of the lipid moiety include a hydrocarbon group having 10 to 50 carbon atoms (one or more carbon atoms may be substituted with a hetero atom). In the above, the hydrocarbon group may be saturated or unsaturated, and may have a cyclic structure.
　脂質部分としては、例えば、コレステロール、ホスファチジルエタノールアミンなどのリン脂質、糖脂質、スフィンゴ脂質、アラキドン酸などの長鎖脂肪酸、ジアシルグリセロール、等が挙げられる。 Examples of the lipid portion include phospholipids such as cholesterol and phosphatidylethanolamine, glycolipids, sphingolipids, long-chain fatty acids such as arachidonic acid, diacylglycerol, and the like.
　ポリエチレングリコール構造を含有する脂質（Ｂ）は、好ましくは、以下の式（ＩＩ）で表される The lipid (B) containing a polyethylene glycol structure is preferably represented by the following formula (II)
Figure JPOXMLDOC01-appb-C000008
Figure JPOXMLDOC01-appb-C000008
（式中、Ｙは炭素数５～５０の炭化水素基（一以上の炭素原子がヘテロ原子で置換されていてもよい）であり、かつｎは１０～２０のいずれかの整数を示し、かつＲ６は炭素数１～１０の炭化水素基（一以上の炭素原子がヘテロ原子で置換されていてもよい）を示す。
）。　ポリエチレングリコール構造を含有する脂質（Ｂ）としては、具体的には、以下の式（ＩＶ）で表されるものが挙げられる Wherein Y is a hydrocarbon group having 5 to 50 carbon atoms (one or more carbon atoms may be substituted with a heteroatom), and n represents any integer of 10 to 20, and R 6 represents a hydrocarbon group having 1 to 10 carbon atoms (one or more carbon atoms may be substituted with a hetero atom).
). Specific examples of the lipid (B) containing a polyethylene glycol structure include those represented by the following formula (IV).
Figure JPOXMLDOC01-appb-C000009
Figure JPOXMLDOC01-appb-C000009
（式中、Ｙは炭素数５～５０の炭化水素基（一以上の炭素原子がヘテロ原子で置換されていてもよい）であり、かつｎは１０～２０のいずれかの整数を示す。）。 (In the formula, Y is a hydrocarbon group having 5 to 50 carbon atoms (one or more carbon atoms may be substituted with a hetero atom), and n represents any integer of 10 to 20) .
　１．３　分子集合体についての説明
　本発明の分子集合体は、化合物（Ａ）及び脂質（Ｂ）を含有する分子集合体である。 1.3 Description of Molecular Assembly The molecular assembly of the present invention is a molecular assembly containing a compound (A) and a lipid (B).
　本発明において、分子集合体とは、少なくとも化合物（Ａ）及び脂質（Ｂ）が集合したものをいう。溶液中では、本発明の分子集合体は、ベシクル形状又は逆ヘキサゴナル形状である。分子集合体がベシクル形状であるか逆ヘキサゴナル形状であるかは、原子間力顕微鏡（Ａｔｏｍｉｃ　Ｆｏｒｃｅ　Ｍｉｃｒｏｓｃｏｐｅ；ＡＦＭ）を用いて確認できる。ＡＦＭ観察により球状の分子集合体が観察されれば、分子集合体はベシクル形状であると評価できる。特に限定されるものではないが、例えば、ベシクル形状分子集合体として、粒径が約１５０～２００ｎｍの球状が観察されうる。これに対して、ＡＦＭ観察により棒状の分子集合体が観察されれば、分子集合体は逆ヘキサゴナル形状であると評価できる。 In the present invention, the molecular aggregate refers to an aggregate of at least the compound (A) and the lipid (B). In solution, the molecular assembly of the present invention has a vesicle shape or an inverted hexagonal shape. Whether the molecular assembly is a vesicle shape or an inverted hexagonal shape can be confirmed using an atomic force microscope (AFM). If a spherical molecular assembly is observed by AFM observation, it can be evaluated that the molecular assembly has a vesicle shape. Although not particularly limited, for example, a sphere having a particle size of about 150 to 200 nm can be observed as a vesicle-shaped molecular assembly. On the other hand, if a rod-like molecular assembly is observed by AFM observation, it can be evaluated that the molecular assembly has an inverted hexagonal shape.
　また、本発明の化合物（Ａ）は、オリゴエチレングリコ－ル構造の作用により、温度応答性を示す。このため、本発明の分子集合体は、所定の温度の環境下においてベシクル形状であるが、環境の温度が上昇すると逆ヘキサゴナル形状へと変化する。 In addition, the compound (A) of the present invention exhibits temperature responsiveness due to the action of the oligoethylene glycol structure. For this reason, the molecular assembly of the present invention has a vesicle shape under an environment of a predetermined temperature, but changes to an inverted hexagonal shape when the temperature of the environment increases.
　本発明のベシクル形状分子集合体は、水溶液中で、Ｒ１及びＲ２側が内側を、そしてＲ３及びＲ４側が外側を向くようにして脂質二重膜を形成している。このベシクル形状分子集合体は、表面が親水性を示すため、生体内に投与すると、細胞表面に弱く吸着する。このベシクル形状分子集合体の脂質二重膜の内部には、所望の疎水性物質を保持させることができる。 The vesicle-shaped molecular assembly of the present invention forms a lipid bilayer in an aqueous solution so that the R 1 and R 2 sides face the inside and the R 3 and R 4 sides face the outside. Since the surface of this vesicle-shaped molecular assembly is hydrophilic, it is weakly adsorbed on the cell surface when administered in vivo. A desired hydrophobic substance can be held inside the lipid bilayer membrane of the vesicle-shaped molecular assembly.
　これに対して、本発明の逆ヘキサゴナル形状分子集合体は、表面が疎水性であり、エンドサイトーシスにより細胞内により取り込まれやすい性質を有する。 In contrast, the reverse hexagonal-shaped molecular assembly of the present invention has a hydrophobic surface and is easily taken up into cells by endocytosis.
　したがって、脂質二重膜の内部に所望の疎水性物質を保持させた状態でベシクル形状分子集合体を生体内に投与し、当該分子集合体が送達された部位のうち所望の部位の環境温度を上昇させて逆ヘキサゴナル形状に変化させることにより、当該部位において選択的に当該物質を細胞内に導入できる。本発明の分子集合体は、乾燥した状態であってもよいし、凍結した状態であってもよい。 Therefore, a vesicle-shaped molecular assembly is administered to a living body in a state where a desired hydrophobic substance is held inside the lipid bilayer membrane, and the environmental temperature of the desired site among the sites to which the molecular assembly is delivered is set. The substance can be selectively introduced into the cell at the site by raising and changing to an inverted hexagonal shape. The molecular assembly of the present invention may be in a dry state or a frozen state.
　乾燥した状態の本発明の分子集合体は、例えば、化合物（Ａ）及び脂質（Ｂ）を含有する各成分をいったんクロロホルム等の有機溶媒に溶解させ、次いでエバポレータによる減圧乾固や噴霧乾燥機による噴霧乾燥を行うことで製造できる。 In the dried molecular assembly of the present invention, for example, each component containing the compound (A) and lipid (B) is once dissolved in an organic solvent such as chloroform and then dried under reduced pressure using an evaporator or a spray dryer. It can be manufactured by spray drying.
　１．４　製法についての説明
　本発明の化合物（Ａ）は、例えば以下のようにして得られたポリアミドデンドロン（ＤＬ）に対して、Ｒ３及びＲ４を付加することにより得られる。なお、下記においてＤＬ－Ｇ１～ＤＬ－Ｇ４とあるのは、第１～４世代のポリアミドデンドロンをそれぞれ示している。なお、ＤＬ－Ｇ０は、第０世代であり、デンドロン構造を有していない。 1.4 Description of Production Method The compound (A) of the present invention can be obtained, for example, by adding R 3 and R 4 to a polyamide dendron (DL) obtained as follows. In the following, DL-G1 to DL-G4 indicate first to fourth generation polyamide dendrons, respectively. DL-G0 is the 0th generation and does not have a dendron structure.
Figure JPOXMLDOC01-appb-C000010
Figure JPOXMLDOC01-appb-C000010
　具体的には、Ｒ１及びＲ２が不飽和炭化水素基である場合、以下のようにして製造することができる。オレイルアミン及びオレイルクロリドを反応させて、オレイルオレイルアミドを合成する。次にヒドリド還元によりジオレイルアミンを合成し、アクリル酸メチルを用いたマイケル付加反応と、エチレンジアミンを用いたエステルアミド交換反応を繰り返すことによりポリアミドアミンデンドロンを合成する。このポリアミドアミンデンドロンを、ＤＬ－Ｇ１－２Ｃ１８－Ｕ２と表記する。 Specifically, when R 1 and R 2 are unsaturated hydrocarbon groups, they can be produced as follows. Oleylamine and oleyl chloride are reacted to synthesize oleyloleylamide. Next, dioleylamine is synthesized by hydride reduction, and a polyamidoamine dendron is synthesized by repeating a Michael addition reaction using methyl acrylate and an ester amide exchange reaction using ethylenediamine. This polyamidoamine dendron is represented as DL-G1-2C 18 -U2.
Figure JPOXMLDOC01-appb-C000011
Figure JPOXMLDOC01-appb-C000011
　また、Ｒ１及びＲ２が飽和炭化水素基である場合、以下のようにして製造することができる。ジアオクタデシルアミンを開始物質として、アクリル酸メチルを用いたマイケル付加反応と、エチレンジアミンを用いたエステルアミド交換反応を繰り返すことによりポリアミドアミンデンドロンを合成する。このポリアミドアミンデンドロンを、ＤＬ－Ｇ１－２Ｃ１８と表記する。 Moreover, when R < 1 > and R < 2 > is a saturated hydrocarbon group, it can manufacture as follows. Polyamideamine dendron is synthesized by repeating Michael addition reaction using methyl acrylate and ester amide exchange reaction using ethylenediamine using dioctadecylamine as a starting material. The polyamidoamine dendron, denoted as DL-G1-2C 18.
Figure JPOXMLDOC01-appb-C000012
Figure JPOXMLDOC01-appb-C000012
　本発明の化合物を製造する際に中間物質として用いられる、Ｒ３及びＲ４を付加する前の状態のポリアミドアミンデンドロンの例を以下に示す。 Examples of the polyamidoamine dendron in the state before adding R 3 and R 4 used as an intermediate substance in the production of the compound of the present invention are shown below.
Figure JPOXMLDOC01-appb-C000013
Figure JPOXMLDOC01-appb-C000013
　Ｒ３及びＲ４の付加は、例えば下記式に示すように、次のようにして行うことができる。ポリアミドアミンデンドロンの末端アミノ基に、パラニトロフェニルカーボネート基で活性化したメトキシジエチレングリコール（ＭＤＥＧ）基又はエトキシジエチレングリコール（ＥＤＥＧ）を反応させることにより、それぞれＭＤＥＧ基及びＥＤＥＧ基を導入する。 The addition of R 3 and R 4 can be performed as follows, for example, as shown in the following formula. By reacting the terminal amino group of the polyamide amine dendron with a methoxydiethylene glycol (MDEG) group or ethoxydiethylene glycol (EDEG) activated with a paranitrophenyl carbonate group, an MDEG group and an EDEG group are introduced, respectively.
Figure JPOXMLDOC01-appb-C000014
Figure JPOXMLDOC01-appb-C000014
Figure JPOXMLDOC01-appb-C000015
Figure JPOXMLDOC01-appb-C000015
Figure JPOXMLDOC01-appb-C000016
Figure JPOXMLDOC01-appb-C000016
　２．本発明の組成物
　本発明の組成物は、本発明の分子集合体のほか、さらに他の成分を含有する。例えば、水系溶媒（分散媒）を含有する。特に限定されるべきものではないが、他の成分としては水のほかに、グルコース、乳糖、ショ糖などの糖水溶液、グリセリン、プロピレングリコールなどの多価アルコール水溶液、リン酸緩衝液、クエン酸緩衝液、リン酸緩衝化生理食塩液等の緩衝液、生理食塩水、細胞培養用の培地などを挙げることができる。 2. Composition of the Invention The composition of the present invention further contains other components in addition to the molecular assembly of the present invention. For example, an aqueous solvent (dispersion medium) is contained. Although not particularly limited, other components include water, sugar aqueous solutions such as glucose, lactose, and sucrose, polyhydric alcohol aqueous solutions such as glycerin and propylene glycol, phosphate buffer, and citrate buffer. Liquid, buffer solution such as phosphate buffered saline, physiological saline, medium for cell culture, and the like.
　この水系溶媒に分散した本発明の分子集合体を安定に長期間保存するには、凝集などの物理的安定性の面から、水系溶媒中の電解質を極力なくすことが重要である。また、脂質の化学的安定性の面から、水系溶媒のｐＨを弱酸性から中性付近（ｐＨ３．０から８．０）に設定したり、窒素バブリングにより溶存酸素を除去することが重要である。 In order to stably store the molecular assembly of the present invention dispersed in the aqueous solvent for a long period of time, it is important to eliminate the electrolyte in the aqueous solvent as much as possible from the viewpoint of physical stability such as aggregation. From the viewpoint of chemical stability of the lipid, it is important to set the pH of the aqueous solvent from weakly acidic to neutral (pH 3.0 to 8.0) or to remove dissolved oxygen by nitrogen bubbling. .
　さらに凍結乾燥保存や噴霧乾燥保存をする場合には、糖水溶液を、凍結保存する場合には、糖水溶液や多価アルコール水溶液をそれぞれ用いると効果的な保存が可能である。 Further, when freeze-preserving or spray-drying preservation, an aqueous solution of sugar can be used, and when storing frozen, an aqueous solution of sugar or aqueous polyhydric alcohol can be used for effective preservation.
　これらの水系溶媒の添加物の濃度は特に限定されるべきものではないが、例えば、糖水溶液においては、２から２０％（Ｗ／Ｖ）が好ましく、５から１０％（Ｗ／Ｖ）がさらに好ましい。また、多価アルコール水溶液においては、１から５％（Ｗ／Ｖ）が好ましく、２から２．５％（Ｗ／Ｖ）がさらに好ましい。緩衝液においては、緩衝剤の濃度が５から５０ｍＭが好ましく、１０から２０ｍＭがさらに好ましい。 The concentration of these aqueous solvent additives is not particularly limited. For example, in an aqueous sugar solution, 2 to 20% (W / V) is preferable, and 5 to 10% (W / V) is more preferable. preferable. Moreover, in a polyhydric alcohol aqueous solution, 1 to 5% (W / V) is preferable, and 2 to 2.5% (W / V) is more preferable. In the buffer solution, the concentration of the buffer is preferably 5 to 50 mM, more preferably 10 to 20 mM.
　水系溶媒中の本発明の分子集合体の濃度は、特に限定されるべきものではないが、０．０１ｍＭから１００ｍＭが好ましく、０．１ｍＭから１０ｍＭがさらに好ましい。 The concentration of the molecular assembly of the present invention in the aqueous solvent is not particularly limited, but is preferably 0.01 mM to 100 mM, more preferably 0.1 mM to 10 mM.
　本発明の分子集合体が水系溶媒に分散した形態は、上記の乾燥した脂質混合物を水系溶媒に添加し、さらにホモジナイザー等の乳化機、超音波乳化機、高圧噴射乳化機等により乳化することで製造できる。また、リポソームを製造する方法としてよく知られている方法、例えば逆相蒸発法などによっても製造でき、特に限定されるべきものではない。本発明の分子集合体の大きさを制御したい場合には、孔径のそろったメンブランフィルター等を用いて、高圧下でイクストルージョン（押し出し濾過）を行えばよい。 The form in which the molecular assembly of the present invention is dispersed in an aqueous solvent is obtained by adding the dried lipid mixture to an aqueous solvent and further emulsifying with an emulsifier such as a homogenizer, an ultrasonic emulsifier, a high-pressure jet emulsifier, or the like. Can be manufactured. Moreover, it can manufacture also by the method well-known as a method of manufacturing a liposome, for example, a reverse phase evaporation method etc., It should not be specifically limited. When it is desired to control the size of the molecular assembly of the present invention, extrusion (extrusion filtration) may be performed under high pressure using a membrane filter having a uniform pore size.
　また、上記の水系溶媒に分散した分子集合体をさらに乾燥させる方法としては、通常の凍結乾燥や噴霧乾燥を挙げることができる。この時の水系溶媒としては、上記したように、糖水溶液、好ましくはショ糖水溶液、乳糖水溶液を用いるとよい。ここで、水系溶媒に分散した分子集合体をいったん製造した上でさらに乾燥すると、分子集合体の長期保存が可能となるほか、この乾燥した分子集合体に所望の物質を含有する水溶液を添加すると、当該物質が効率よく分子集合体に保持されるため好ましい。 In addition, examples of a method for further drying the molecular assembly dispersed in the aqueous solvent include ordinary freeze drying and spray drying. As the aqueous solvent at this time, as described above, a sugar aqueous solution, preferably a sucrose aqueous solution or a lactose aqueous solution may be used. Here, once a molecular assembly dispersed in an aqueous solvent is manufactured and further dried, the molecular assembly can be stored for a long period of time, and when an aqueous solution containing a desired substance is added to the dried molecular assembly. This is preferable because the substance is efficiently retained in the molecular assembly.
　３．　用途についての説明
　本発明の分子集合体及び組成物は、好ましくは、疎水性の所望の物質を細胞内に導入するためにそれぞれ使用される。 3. Description of Use The molecular assemblies and compositions of the present invention are preferably each used to introduce a desired hydrophobic substance into a cell.
　疎水性の所望の物質としては、特に限定されないが、例えば低分子化合物、ペプチド、脂質、ホルモン、タンパク質、及び核酸誘導体等の生理活性物質が挙げられる。　特に限定されないが、例えば、がん治療を目的として、ドキソルビシンなどの各種抗がん剤、ホルモン製剤、及びWT1ペプチドとその誘導体などの癌免疫治療用抗原分子を用いることができる。 The hydrophobic desired substance is not particularly limited, and examples thereof include physiologically active substances such as low molecular weight compounds, peptides, lipids, hormones, proteins, and nucleic acid derivatives. Although not particularly limited, for example, for the purpose of cancer treatment, various anticancer agents such as doxorubicin, hormone preparations, and antigen molecules for cancer immunotherapy such as WT1 peptide and its derivatives can be used.
　本発明の分子集合体及び組成物は、上記疾患の治療目的で、導入しようとする生理活性物質とともに治療対象である個体にそれぞれ直接導入してもよい。この場合、本発明の分子集合体又は組成物を個体に投与すればよい。個体への投与手段としては、経口投与でも、非経口投与でもよいが、非経口投与が好ましい。剤形としては、通常知られたものでよく、経口投与の剤形としては、例えば、錠剤、散剤、顆粒剤、シロップ剤等を挙げることができる。また、非経口投与の剤形としては、例えば、注射剤、点眼剤、軟膏剤、坐剤等を挙げることができる。中でも、注射剤が好ましく、投与方法としては、静脈注射、標的とする細胞や臓器に対しての局所注射が好ましい。 The molecular assembly and composition of the present invention may be directly introduced into the individual to be treated together with the physiologically active substance to be introduced for the purpose of treating the above-mentioned diseases. In this case, the molecular assembly or composition of the present invention may be administered to an individual. The means for administration to an individual may be oral administration or parenteral administration, but parenteral administration is preferred. The dosage form may be a conventionally known dosage form, and examples of the dosage form for oral administration include tablets, powders, granules, syrups and the like. Examples of the dosage form for parenteral administration include injections, eye drops, ointments, suppositories and the like. Among these, an injection is preferable, and an administration method is preferably intravenous injection or local injection into a target cell or organ.
　疎水性の所望の物質と本発明の化合物（Ａ）の配合割合は、当該物質１重量部に対し、本発明の化合物（Ａ）を１～１０００重量部、好ましくは１０～５００重量部、より好ましくは１０～１００重量部使用する。 The blending ratio of the desired hydrophobic substance and the compound (A) of the present invention is such that the compound (A) of the present invention is 1 to 1000 parts by weight, preferably 10 to 500 parts by weight per 1 part by weight of the substance. Preferably 10 to 100 parts by weight are used.
ＩＩ．第二の発明
１．本発明の化合物
　本発明の化合物は、下記式ＤＬ－Ｇ１～ＤＬ－Ｇ４のいずれかで表される化合物
ＤＬ－Ｇ１：Ｒ１Ｒ２ＮＸ（ＸＨＲ３）ＸＨＲ４
ＤＬ－Ｇ２：Ｒ１Ｒ２ＮＸ（Ｘ（ＸＨＲ３）ＸＨＲ４）２
ＤＬ－Ｇ３：Ｒ１Ｒ２ＮＸ（Ｘ（Ｘ（ＸＨＲ３）ＸＨＲ４）２）２
ＤＬ－Ｇ４：Ｒ１Ｒ２ＮＸ（Ｘ（Ｘ（Ｘ（ＸＨＲ３）ＸＨＲ４）２）２）２
（式中、Ｒ１及びＲ２は、同一又は異なって飽和又は不飽和の長鎖炭化水素基を示し、
Ｒ３及びＲ４は、同一又は異なってｐＨ応答性のカルボキシル基含有炭化水素基を示し、Ｒ１～Ｒ４は、環状構造を含有していてもよく、かつ一以上の炭素原子がヘテロ原子で置換されていてもよく、かつＸは、－ＣＨ２ＣＨ２ＣＯＮＨＣＨ２ＣＨ２Ｎ－を示す。）である。 II. Second invention 1. Compound of the Present Invention The compound of the present invention is a compound DL-G1: R 1 R 2 NX (XHR 3 ) XHR 4 represented by any of the following formulas DL-G1 to DL-G4
DL-G2: R 1 R 2 NX (X (XHR 3 ) XHR 4 ) 2
DL-G3: R 1 R 2 NX (X (X (XHR 3 ) XHR 4 ) 2 ) 2
DL-G4: R 1 R 2 NX (X (X (X (XHR 3 ) XHR 4 ) 2 ) 2 ) 2
(Wherein R 1 and R 2 are the same or different and represent a saturated or unsaturated long chain hydrocarbon group,
R 3 and R 4 represent the same or different pH-responsive carboxyl group-containing hydrocarbon group, R 1 to R 4 may contain a cyclic structure, and one or more carbon atoms are heteroatoms And X represents —CH 2 CH 2 CONHCH 2 CH 2 N—. ).
　１．１　Ｘについての説明
Ｘは、－ＣＨ２ＣＨ２ＣＯＮＨＣＨ２ＣＨ２Ｎ－を表し、その末端のＮは、通常２個の水素原子を有するが、１個の水素原子が疎水性アミノ酸で置換されていてもよい。疎水性アミノ酸としては、ロイシン、バリン、イソロイシン、ノルロイシン、フェニルアラニン及びチロシン等が挙げられる。 1.1 Description of X X represents —CH 2 CH 2 CONHCH 2 CH 2 N—, and the terminal N usually has two hydrogen atoms, but one hydrogen atom is a hydrophobic amino acid. May be substituted. Examples of hydrophobic amino acids include leucine, valine, isoleucine, norleucine, phenylalanine and tyrosine.
　１．２　Ｒ １ 及びＲ ２ についての説明
Ｒ１及びＲ２は、飽和又は不飽和の長鎖炭化水素基である。 1.2 Description R 1 and R 2 for R 1 and R 2 are long chain, saturated or unsaturated hydrocarbon group.
　長鎖炭化水素基は、天然由来であってもよく、天然由来のものを修飾したものでもよく、又は人工的に合成されたものであってもよい。 The long-chain hydrocarbon group may be naturally derived, may be modified from naturally derived, or may be artificially synthesized.
　長鎖炭化水素基の炭素数は、主鎖が長鎖であればよく限定されない。主鎖の炭素数は好ましくは８～３０であり、より好ましくは１０～２２であり、さらに好ましくは１２～２０である。 The number of carbon atoms of the long-chain hydrocarbon group is not limited as long as the main chain is a long chain. The number of carbon atoms in the main chain is preferably 8 to 30, more preferably 10 to 22, and still more preferably 12 to 20.
　長鎖炭化水素基は、分岐を有していてもよい。なお、主鎖についてはＩＵＰＡＣ命名法に基づいて、又はそれが困難若しくは不可能な場合はそれに準ずる方法に基づいて決定する。 The long chain hydrocarbon group may have a branch. The main chain is determined based on the IUPAC nomenclature, or when it is difficult or impossible, it is determined based on a similar method.
　長鎖炭化水素基は、環状構造を有していてもよい。環状構造としては、特に限定されないが、例えば、シクロヘキシル基、フェニル基、ピリジル基及びナフチル基等が挙げられる。特に、シクロヘキシル基が好ましい。 The long chain hydrocarbon group may have a cyclic structure. Although it does not specifically limit as a cyclic structure, For example, a cyclohexyl group, a phenyl group, a pyridyl group, a naphthyl group, etc. are mentioned. A cyclohexyl group is particularly preferable.
　長鎖炭化水素基は、少なくとも１つ以上の炭素原子がヘテロ原子で置換されているものであってもよい。本発明において、ヘテロ原子とは、酸素原子、窒素原子又は硫黄原子をいう。したがって、長鎖炭化水素基には、酸素含有炭化水素基、窒素含有炭化水素基及び硫黄含有炭化水素基が含まれる。 The long chain hydrocarbon group may be one in which at least one carbon atom is substituted with a hetero atom. In the present invention, the hetero atom means an oxygen atom, a nitrogen atom or a sulfur atom. Accordingly, the long chain hydrocarbon group includes an oxygen-containing hydrocarbon group, a nitrogen-containing hydrocarbon group, and a sulfur-containing hydrocarbon group.
　本発明において、炭化水素基とは、アルキル基、アルケニル基又はアルキニル基をいう。 In the present invention, the hydrocarbon group means an alkyl group, an alkenyl group or an alkynyl group.
　酸素含有炭化水素基としては、例えば、エーテル結合及びカルボニル結合からなる群より選択される少なくとも１種の結合を有する酸素含有炭化水素基が挙げられる。カルボニル結合を有する酸素含有炭化水素基としては、例えば、アルデヒド、ケトン、カルボン酸、エステル、アミド、エノン、酸塩化物又は無水物等からなる群より選択される少なくとも１種の構造を有する基が挙げられる。 Examples of the oxygen-containing hydrocarbon group include an oxygen-containing hydrocarbon group having at least one bond selected from the group consisting of an ether bond and a carbonyl bond. Examples of the oxygen-containing hydrocarbon group having a carbonyl bond include a group having at least one structure selected from the group consisting of aldehyde, ketone, carboxylic acid, ester, amide, enone, acid chloride, or anhydride. Can be mentioned.
　窒素含有炭化水素基としては、例えばニトリル、アミン、アミド及びイミドからなる群より選択される少なくとも１種の構造を有する窒素含有炭化水素基が挙げられる。 Examples of the nitrogen-containing hydrocarbon group include a nitrogen-containing hydrocarbon group having at least one structure selected from the group consisting of nitrile, amine, amide and imide.
　硫黄含有炭化水素基としては、例えばチオール、チオエーテル、チオアセタール、スルフィド、ジスルフィド、ジチオカルボン酸、チオエステル、チオケトン、チオアルデヒド、チオカルバメート、チオウレタン、ホスフィンスルフィド、チオホスフェート、チオホスホネート、スルホネート、スルホン及びスルホンアミドからなる群より選択される少なくとも１種の結合を有する硫黄含有炭化水素基が挙げられる。 Examples of the sulfur-containing hydrocarbon group include thiol, thioether, thioacetal, sulfide, disulfide, dithiocarboxylic acid, thioester, thioketone, thioaldehyde, thiocarbamate, thiourethane, phosphine sulfide, thiophosphate, thiophosphonate, sulfonate, sulfone and And sulfur-containing hydrocarbon groups having at least one bond selected from the group consisting of sulfonamides.
　長鎖炭化水素基は、上に例示したような構造の基においてさらに少なくとも１つ以上の置換基を有していてもよい。置換基としては、疎水性を示すものが好ましい。置換基としては、例えば、フェニル基、コレステリル基及びピレニル基等が挙げられる。特にコレステリル基が好ましい。 The long chain hydrocarbon group may further have at least one substituent in the group having the structure as exemplified above. As the substituent, those showing hydrophobicity are preferable. Examples of the substituent include a phenyl group, a cholesteryl group, and a pyrenyl group. A cholesteryl group is particularly preferable.
　不飽和長鎖炭化水素基は、不飽和結合を主鎖に有しており、かつ主鎖が鎖状構造を有する炭化水素基であれば好ましい。 The unsaturated long chain hydrocarbon group is preferably a hydrocarbon group having an unsaturated bond in the main chain and the main chain having a chain structure.
　不飽和結合の種類は、二重結合であってもよく、又は三重結合であってもよい。二重結合が好ましい。不飽和結合の数は、少なくとも１つ以上であればよく、限定されない。また、二重結合と三重結合の両方を有していてもよい。二重結合を１つ有していることが好ましい。 The type of unsaturated bond may be a double bond or a triple bond. A double bond is preferred. The number of unsaturated bonds should just be at least 1 or more, and is not limited. Moreover, you may have both a double bond and a triple bond. It preferably has one double bond.
　二重結合としては、シス型二重結合であってもよいし、トランス型二重結合であってもよい。シス型二重結合が好ましい。 The double bond may be a cis-type double bond or a trans-type double bond. A cis type double bond is preferred.
　不飽和長鎖炭化水素基としては、ヘキサデセニル基、オクタデセニル基、オクタデカジエニル基、オクタデカトリエニル基、イコサトリエニル基、イコサテトラエニル基、オクタデカトリエニル基、イコサペンタエニル基又はドコサヘキサエニル基が好ましい。 Examples of unsaturated long-chain hydrocarbon groups include hexadecenyl, octadecenyl, octadecadienyl, octadecatrienyl, icosatrienyl, icosatetraenyl, octadecatrienyl, icosapentaenyl, or docosa A hexaenyl group is preferred.
　不飽和長鎖炭化水素基としては、９－ヘキサデセニル基、９－オクタデセニル基（オレイル基）、１２－オクタデカジエニル基、６，９，１２－オクタデカトリエニル基、８，１１，１４－イコサトリエニル基、５，８，１１，１４－イコサテトラエニル基、９，１２，１５－オクタデカトリエニル基、５，８，１１，１４，１７－イコサペンタエニル基又は４，７，１３，１６，１９－ドコサヘキサエニル基がより好ましい。 Examples of unsaturated long chain hydrocarbon groups include 9-hexadecenyl group, 9-octadecenyl group (oleyl group), 12-octadecadienyl group, 6,9,12-octadecatrienyl group, 8,11,14- Eicosatrienyl group, 5,8,11,14-icosatetraenyl group, 9,12,15-octadecatrienyl group, 5,8,11,14,17-icosapentaenyl group or 4,7,13 , 16,19-docosahexaenyl group is more preferred.
　不飽和長鎖炭化水素基としては、９－オクタデセニル基（オレイル基）がさらに好ましい。 As the unsaturated long chain hydrocarbon group, a 9-octadecenyl group (oleyl group) is more preferable.
　不飽和長鎖炭化水素基の好適例としては、上に好適例として例示したような構造の基においてさらに少なくとも1つ以上の炭素原子がヘテロ原子で置換されているものも挙げられる。 Preferred examples of the unsaturated long-chain hydrocarbon group include those in which at least one carbon atom is further substituted with a hetero atom in the group having the structure exemplified above as a preferred example.
　不飽和長鎖炭化水素基の好適例としては、上に好適例として例示したような構造の基を基礎として、酸素含有炭化水素基、窒素含有炭化水素基、又は硫黄含有炭化水素基に特有な構造として上に例示したような構造をさらに有しているものであってもよい。 Preferred examples of the unsaturated long chain hydrocarbon group are specific to oxygen-containing hydrocarbon groups, nitrogen-containing hydrocarbon groups, or sulfur-containing hydrocarbon groups on the basis of the group having the structure exemplified above as a preferred example. The structure may further include a structure as exemplified above.
　不飽和長鎖炭化水素基の好適例としては、上に好適例として例示したような構造の基においてさらに少なくとも１つ以上の置換基を有していてもよい。置換基としては、疎水性を示すものが好ましい。置換基としては、例えば、フェニル基、コレステリル基、及びピレニル基等が挙げられる。特にコレステリル基が好ましい。 As a preferred example of the unsaturated long chain hydrocarbon group, the group having the structure as exemplified above as a preferred example may further have at least one substituent. As the substituent, those showing hydrophobicity are preferable. Examples of the substituent include a phenyl group, a cholesteryl group, and a pyrenyl group. A cholesteryl group is particularly preferable.
　１．３　ｐＨ応答性のカルボキシル基含有炭化水素基についての説明
　ｐＨ応答性のカルボキシル基含有炭化水素基は、環状構造を含有していてもよい。 1.3 Description of pH-responsive carboxyl group-containing hydrocarbon group The pH-responsive carboxyl group-containing hydrocarbon group may contain a cyclic structure.
　ｐＨ応答性のカルボキシル基含有炭化水素基は、一以上の炭素原子がヘテロ原子で置換されていてもよい。 In the pH-responsive carboxyl group-containing hydrocarbon group, one or more carbon atoms may be substituted with a hetero atom.
　ｐＨ応答性のカルボキシル基含有炭化水素基は、特に限定されないが、好ましくは炭素数が２～２０であり、より好ましくは２～１０であり、さらに好ましくは２～６である。
なお、左記においては、炭素原子がヘテロ原子で置換されている場合、当該へテロ原子を炭素原子として算入する。 The pH-responsive carboxyl group-containing hydrocarbon group is not particularly limited, but preferably has 2 to 20 carbon atoms, more preferably 2 to 10 carbon atoms, and still more preferably 2 to 6 carbon atoms.
In the left column, when a carbon atom is substituted with a hetero atom, the hetero atom is counted as a carbon atom.
　ｐＨ応答性のカルボキシル基含有炭化水素基は、好ましくは、以下の式（Ｉ）で表される The pH-responsive carboxyl group-containing hydrocarbon group is preferably represented by the following formula (I)
Figure JPOXMLDOC01-appb-C000017
Figure JPOXMLDOC01-appb-C000017
（式中、Ｒ５は、飽和又は不飽和の炭素数１～１０、好ましくは１～５、より好ましくは１～３の炭化水素基（環状構造を含有していてもよく、かつ一以上の炭素原子がヘテロ原子で置換されていてもよい。）を示す。）。 (In the formula, R 5 represents a saturated or unsaturated hydrocarbon group having 1 to 10, preferably 1 to 5, more preferably 1 to 3 carbon atoms (which may contain a cyclic structure and has one or more carbon atoms). Carbon atoms may be substituted with heteroatoms).
　ｐＨ応答性のカルボキシル基含有炭化水素基は、より好ましくは、
（Ａ）シクロヘキシル基、フェニル基、ピリジル基又はナフチル基を含有するか、又は
（Ｂ）炭素数１又は２（一以上の炭素原子がヘテロ原子で置換されていてもよい。）の一以上の分岐を有していてもよい鎖状構造である。 The pH-responsive carboxyl group-containing hydrocarbon group is more preferably
(A) contains a cyclohexyl group, a phenyl group, a pyridyl group or a naphthyl group, or (B) one or more carbon atoms having 1 or 2 carbon atoms (one or more carbon atoms may be substituted with a heteroatom). It is a chain structure which may have a branch.
　上記（Ａ）に該当するｐＨ応答性のカルボキシル基含有炭化水素基としては、シクロヘキシル基を含有するものが好ましい。 As the pH-responsive carboxyl group-containing hydrocarbon group corresponding to the above (A), those containing a cyclohexyl group are preferable.
　上記（Ａ）に該当するｐＨ応答性のカルボキシル基含有炭化水素基は、具体的には、下記の式（II）で表されるものが挙げられる。 Specific examples of the pH-responsive carboxyl group-containing hydrocarbon group corresponding to the above (A) include those represented by the following formula (II).
Figure JPOXMLDOC01-appb-C000018
  
Figure JPOXMLDOC01-appb-C000018
  
　上記（Ｂ）に該当するｐＨ応答性のカルボキシル基含有炭化水素基は、さらに好ましくは、炭素数１（炭素原子がヘテロ原子で置換されていてもよい。）の一以上の分岐を有していてもよい鎖状構造である。 The pH-responsive carboxyl group-containing hydrocarbon group corresponding to the above (B) more preferably has one or more branches having 1 carbon atom (the carbon atom may be substituted with a hetero atom). It may be a chain structure.
　上記（Ｂ）に該当するｐＨ応答性のカルボキシル基含有炭化水素基は、さらに好ましくは、炭素数１又は２（一以上の炭素原子がヘテロ原子で置換されていてもよい。）の１～２の分岐を有していてもよい鎖状構造である。 The pH-responsive carboxyl group-containing hydrocarbon group corresponding to the above (B) is more preferably 1 to 2 having 1 or 2 carbon atoms (one or more carbon atoms may be substituted with a hetero atom). It is a chain structure which may have the following branches.
　上記（Ｂ）に該当するｐＨ応答性のカルボキシル基含有炭化水素基は、さらに好ましくは、炭素数１（炭素原子がヘテロ原子で置換されていてもよい。）の１～２の分岐を有していてもよい鎖状構造である。 The pH-responsive carboxyl group-containing hydrocarbon group corresponding to the above (B) more preferably has 1 to 2 branches having 1 carbon atom (the carbon atom may be substituted with a hetero atom). It may be a chain structure.
　上記（Ｂ）に該当するｐＨ応答性のカルボキシル基含有炭化水素基は、さらに好ましくは、炭素数１（炭素原子がヘテロ原子で置換されていてもよい。）の１の分岐を有していてもよい鎖状構造である。 More preferably, the pH-responsive carboxyl group-containing hydrocarbon group corresponding to (B) has one branch having 1 carbon atom (the carbon atom may be substituted with a heteroatom). It is a good chain structure.
　上記（Ｂ）に該当するｐＨ応答性のカルボキシル基含有炭化水素基は、具体的には、下記の式（III）で表されるものが挙げられる。 Specific examples of the pH-responsive carboxyl group-containing hydrocarbon group corresponding to (B) above include those represented by the following formula (III).
Figure JPOXMLDOC01-appb-C000019
Figure JPOXMLDOC01-appb-C000019
　１．４　効果についての説明
　本発明の化合物は、水溶液中で、Ｒ１及びＲ２側が内側を、そしてＲ３及びＲ４側が外側を向くようにして脂質二重膜を形成し、ベシクルを構成する。このベシクルは、細胞内へと侵入する能力に優れている。このベシクル内には、生理活性物質等を内包させることができる。したがって、本発明の化合物は、生理活性物質を細胞内に導入するために使用できる。 1.4 Description of Effect The compound of the present invention forms a vesicle in an aqueous solution by forming a lipid bilayer with the R 1 and R 2 sides facing inward and the R 3 and R 4 sides facing outward. To do. This vesicle is excellent in the ability to penetrate into cells. A physiologically active substance or the like can be encapsulated in the vesicle. Therefore, the compound of the present invention can be used for introducing a physiologically active substance into cells.
　また、本発明の化合物は、ｐＨ応答性のカルボキシル基含有炭化水素基の作用により、ｐＨ応答性を示す。「ｐＨ応答性」とは、本発明の化合物により構成されたベシクルが、所定のｐＨの環境下において不安定化し、内包物を放出することを意味する。例えば、本発明の化合物は、ｐＨ応答性のカルボキシル基含有炭化水素基に由来し、プロトンが解離することにより負電荷を示しうるカルボキシル基（Ａ）を有している。さらに、本発明の化合物は、プロトネーションを受けうるポリアミドデンドロンの３級アミン（Ｂ）を有している。したがって、本発明の化合物は、ｐＨを塩基性側から酸性側へと連続的に変化させていくと、左記カルボキシル基（Ａ）及び３級アミン（Ｂ）の状態がそれぞれ以下のように変化していくことになる。 In addition, the compound of the present invention exhibits pH responsiveness due to the action of a pH responsive carboxyl group-containing hydrocarbon group. “PH responsiveness” means that a vesicle constituted by the compound of the present invention is destabilized under an environment of a predetermined pH and releases inclusions. For example, the compound of the present invention is derived from a pH-responsive carboxyl group-containing hydrocarbon group and has a carboxyl group (A) that can exhibit a negative charge when protons dissociate. Furthermore, the compound of this invention has the tertiary amine (B) of the polyamide dendron which can receive a protonation. Therefore, in the compound of the present invention, when the pH is continuously changed from the basic side to the acidic side, the states of the carboxyl group (A) and the tertiary amine (B) on the left change as follows. It will follow.
Figure JPOXMLDOC01-appb-T000020
Figure JPOXMLDOC01-appb-T000020
　この場合において、化合物のζ電位は、塩基性側から酸性側へと連続的に変化させていく間に、負の値から正の値へと変化していくと考えられる。カルボキシル基がプロトン化されると、ベシクルの表面が疎水性となり、さらに内部の３級アミンがプロトン化されることにより、分子間の静電反発が生じる。これにより、ベシクルは不安定化し、内包物を放出すると考えられる。 In this case, the ζ potential of the compound is considered to change from a negative value to a positive value while continuously changing from the basic side to the acidic side. When the carboxyl group is protonated, the surface of the vesicle becomes hydrophobic, and further, the internal tertiary amine is protonated, resulting in electrostatic repulsion between molecules. Thereby, it is considered that the vesicle is destabilized and the inclusion is released.
　所望のｐＨ応答性を示すカルボキシル基含有炭化水素基を用いることによって、所望のｐＨ応答性を示す本発明の化合物を提供できる。 By using a carboxyl group-containing hydrocarbon group exhibiting desired pH responsiveness, the compound of the present invention exhibiting desired pH responsiveness can be provided.
　一般に、食作用（エンドサイトーシス）を介して細胞内に取り込まれた外来物質は、まず（１）初期エンドソームと呼ばれる細胞内オルガネラに保持され、やがて初期エンドソームは（２）後期エンドソームへと変化する。後期エンドソームに保持されたままであれば、当該外来物質は最終的に（３）リソソームと呼ばれる細胞内オルガネラまで送達されうる。そして、（１）初期エンドソーム、（２）後期エンドソーム及び（３）リソソームの内部はそれぞれｐＨ６．２程度、ｐＨ６．０～５．０及びｐＨ５．０～４．０程度のそれぞれ異なるｐＨを有している。したがって、本発明の化合物において、ベシクルとしたときに例えばｐＨ６．２よりも高ｐＨ領域では安定化しており、ｐＨ６．２程度ではじめて不安定化するように設計することによって、初期エンドソームにまで内包物質を送達し、かつ初期エンドソームにおいて内包物質をリリースしうるようなキャリアが得られる。
同様にして、ベシクルとしたときにｐＨ６．０よりも高ｐＨ領域では安定化しており、ｐＨ６．０ではじめて不安定化するように設計することによって、後期エンドソームにまで内包物質を送達し、かつ後期エンドソームにおいて内包物質をリリースしうるようなキャリアが得られる。さらに、ベシクルとしたときにｐＨ５．０よりも高ｐＨ領域では安定化しており、ｐＨ５．０ではじめて不安定化するように設計することによって、リソソームにまで内包物質を送達し、かつリソソームにおいて内包物質をリリースしうるようなキャリアが得られる。 In general, foreign substances taken into cells via phagocytosis (endocytosis) are first retained in (1) intracellular organelles called early endosomes, and eventually, early endosomes change to (2) late endosomes. . If kept in the late endosome, the foreign substance can finally be delivered to an intracellular organelle called (3) lysosome. And (1) the early endosome, (2) the late endosome, and (3) the interior of the lysosome have different pH values of about pH 6.2, pH 6.0 to 5.0, and pH 5.0 to 4.0, respectively. ing. Therefore, in the compound of the present invention, when it is used as a vesicle, it is stabilized in a pH range higher than, for example, pH 6.2, and is designed to be destabilized only at about pH 6.2. A carrier is obtained that can deliver the substance and release the encapsulated substance in the early endosome.
Similarly, it is stabilized in a pH region higher than pH 6.0 when it is used as a vesicle, and is designed to be destabilized at pH 6.0 for the first time to deliver encapsulated substances to late endosomes, and A carrier that can release the inclusion substance in the late endosome is obtained. Furthermore, when it is used as a vesicle, it is stabilized in a pH range higher than pH 5.0, and is designed to be destabilized for the first time at pH 5.0, thereby delivering the inclusion substance to the lysosome and encapsulating in the lysosome. A carrier that can release a substance is obtained.
　一般に、内包物質をサイトゾルに送達すると、細胞毒性を引き起こすおそれがある。また、内包物質として抗原を用い、免疫誘導を企図する場合には、抗原をサイトゾルに送達すると、細胞性免疫誘導を阻害するおそれもある。 In general, delivery of encapsulated material to the cytosol may cause cytotoxicity. In addition, when an antigen is used as the inclusion substance and immunity induction is intended, delivery of the antigen to the cytosol may inhibit cellular immunity induction.
　内包物質として抗原を用い、免疫誘導を企図する場合には、液性免疫誘導につながるＭＨＣクラスＩＩ分子と抗原との結合が、後期エンドソーム～リソソームにおいて起こるとされている。このため、リソソーム内でキャリアが分解し抗原が放出された場合、効果的な抗原提示につながらないおそれがある。 When an antigen is used as the inclusion substance and immunity induction is intended, the binding between the MHC class II molecule and the antigen leading to humoral immunity induction is considered to occur in the late endosome to lysosome. For this reason, when the carrier is decomposed in the lysosome and the antigen is released, it may not lead to effective antigen presentation.
　本発明の化合物により構成されたベシクルは、細胞に取り込まれた後に、初期エンドソーム～後期エンドソーム～リソソームに至る過程で、その酸性度の違いを認識して、適切なタイミングで効率よく内包物を放出できる。このような機能は種々の用途に役立ちうる。例えば、液性免疫を効果的に誘導するワクチンを開発する上で役立ちうる。より詳細には、次の通りである。液性免疫を効果的に誘導するためには、（ａ）エンドソーム内及びリソソーム内での抗原認識、（ｂ）免疫活性化にかかわるＴｏｌｌ様受容体及び抗原の効果的な相互作用、並びに（ｃ）抗原提示を行うＭＨＣ分子及び抗原の高効率での結合を誘導する必要があると考えられる。したがって、本発明の化合物により構成された、抗原を内包するベシクルがエンドサイトーシスによって細胞に取り込まれた後、エンドソーム内において早い段階で完全に内包物（抗原）を放出することで、上記の相互作用を効率よく引き起こし、効果的な免疫誘導を行うことができる。 Vesicles composed of the compounds of the present invention recognize the difference in acidity in the process from early endosome to late endosome to lysosome after being taken up by cells, and efficiently release inclusions at appropriate timing it can. Such a function can be useful for various applications. For example, it can help in developing vaccines that effectively induce humoral immunity. More details are as follows. In order to effectively induce humoral immunity, (a) antigen recognition in endosomes and lysosomes, (b) effective interaction of Toll-like receptors and antigens involved in immune activation, and (c ) It is considered necessary to induce high-efficiency binding of MHC molecules that perform antigen presentation and antigens. Therefore, after the vesicle composed of the compound of the present invention, which encapsulates the antigen, is taken up by the cells by endocytosis, the inclusion (antigen) is completely released at an early stage in the endosome, whereby the above-mentioned mutual The action can be efficiently caused and effective immunity induction can be performed.
　さらに、抗原ではなく、免疫阻害剤を本発明の化合物により構成されたベシクルに内包させることにより、生体本来の機能として生じている上記の相互作用（ａ）～（ｃ）を上記とは反対に阻害でき、ひいては免疫誘導を阻害できる。 Furthermore, the above-described interactions (a) to (c) that occur as a natural function of the living body can be reversed by encapsulating an immune inhibitor instead of an antigen in a vesicle composed of the compound of the present invention. It can inhibit and thus immune induction can be inhibited.
　（Ａ）シクロヘキシル基、フェニル基、ピリジル基又はナフチル基を含有するｐＨ応答性のカルボキシル基含有炭化水素基を含有する本発明の化合物により構成されたベシクルは、より高いｐＨ領域における応答性を示す。例えば、ｐＨ６．０以上における応答性を示す。このため、ｐＨ応答性のカルボキシル基含有炭化水素基（Ａ）を含有する本発明の化合物により構成されたベシクルを用いることによって、初期エンドソームにおいて内包物を放出しうるキャリアを得ることができる。本発明の化合物により構成されたベシクルは抗原提示細胞（樹状細胞）に著しく多量に取り込まれるため、さらに上のような特性を有していると、エンドソーム内部で確実に内包物を放出できるため、液性免疫誘導用ワクチンとして、また、免疫抑制剤デリバリーシステムとして有効である。 (A) A vesicle composed of a compound of the present invention containing a pH-responsive carboxyl group-containing hydrocarbon group containing a cyclohexyl group, a phenyl group, a pyridyl group or a naphthyl group exhibits responsiveness in a higher pH range. . For example, responsiveness at pH 6.0 or higher is shown. For this reason, by using a vesicle composed of the compound of the present invention containing a pH-responsive carboxyl group-containing hydrocarbon group (A), a carrier capable of releasing inclusions in the initial endosome can be obtained. Since vesicles composed of the compounds of the present invention are taken up by antigen-presenting cells (dendritic cells) in an extremely large amount, inclusions can be reliably released inside the endosome if they have the above characteristics. It is effective as a vaccine for inducing humoral immunity and as an immunosuppressant delivery system.
　（Ｂ）炭素数１又は２（一以上の炭素原子がヘテロ原子で置換されていてもよい。）の一以上の分岐を有していてもよい鎖状構造であるｐＨ応答性のカルボキシル基含有炭化水素基を含有する本発明の化合物により構成されたベシクルは、より低いｐＨ領域における応答性を示す。例えば、ｐＨ５．０以下における応答性を示す。このため、ｐＨ応答性のカルボキシル基含有炭化水素基（Ｂ）を含有する本発明の化合物により構成されたベシクルを用いることによって、リソソームにおいて内包物を放出しうるキャリアを得ることができる。 (B) pH-responsive carboxyl group-containing chain structure which may have one or more branches having 1 or 2 carbon atoms (one or more carbon atoms may be substituted with a hetero atom) Vesicles composed of compounds of the present invention containing hydrocarbon groups show responsiveness in the lower pH range. For example, responsiveness at pH 5.0 or lower is shown. Therefore, by using a vesicle composed of the compound of the present invention containing a pH-responsive carboxyl group-containing hydrocarbon group (B), a carrier capable of releasing inclusions in lysosomes can be obtained.
　所望のｐＨ応答性を有する本発明の化合物を用いてベシクルを構成することにより、又はｐＨ応答性の異なる２種以上の本発明の化合物を適宜組み合わせて用いてベシクルを構成することにより、所望のｐＨ応答性を有するベシクルを得ることができる。 By constructing a vesicle by using a compound of the present invention having a desired pH responsiveness, or by constructing a vesicle by appropriately combining two or more compounds of the present invention having different pH responsiveness, A vesicle having pH responsiveness can be obtained.
　１．５　製法についての説明
　本発明の化合物は、例えば以下のようにして得られたポリアミドデンドロン（ＤＬ）に対して、Ｒ３及びＲ４を付加することにより得られる。なお、下記においてＤＬ－Ｇ１～ＤＬ－Ｇ４とあるのは、第１～４世代のポリアミドデンドロンをそれぞれ示している。なお、ＤＬ－Ｇ０は、第０世代であり、デンドロン構造を有していない。 1.5 Description of Production Process The compound of the present invention can be obtained, for example, by adding R 3 and R 4 to polyamide dendron (DL) obtained as follows. In the following, DL-G1 to DL-G4 indicate first to fourth generation polyamide dendrons, respectively. DL-G0 is the 0th generation and does not have a dendron structure.
Figure JPOXMLDOC01-appb-C000021
Figure JPOXMLDOC01-appb-C000021
　具体的には、Ｒ１及びＲ２が不飽和炭化水素基である場合、以下のようにして製造することができる。オレイルアミン及びオレイルクロリドを反応させて、オレイルオレイルアミドを合成する。次にヒドリド還元によりジオレイルアミンを合成し、アクリル酸メチルを用いたマイケル付加反応と、エチレンジアミンを用いたエステルアミド交換反応を繰り返すことによりポリアミドアミンデンドロンを合成する。このポリアミドアミンデンドロンを、ＤＬ－Ｇ１－２Ｃ１８－Ｕ２と表記する。 Specifically, when R 1 and R 2 are unsaturated hydrocarbon groups, they can be produced as follows. Oleylamine and oleyl chloride are reacted to synthesize oleyloleylamide. Next, dioleylamine is synthesized by hydride reduction, and a polyamidoamine dendron is synthesized by repeating a Michael addition reaction using methyl acrylate and an ester amide exchange reaction using ethylenediamine. This polyamidoamine dendron is represented as DL-G1-2C 18 -U2.
Figure JPOXMLDOC01-appb-C000022
Figure JPOXMLDOC01-appb-C000022
　また、Ｒ１及びＲ２が飽和炭化水素基である場合、以下のようにして製造することができる。ジアオクタデシルアミンを開始物質として、アクリル酸メチルを用いたマイケル付加反応と、エチレンジアミンを用いたエステルアミド交換反応を繰り返すことによりポリアミドアミンデンドロンを合成する。このポリアミドアミンデンドロンを、ＤＬ－Ｇ１－２Ｃ１８と表記する。 Moreover, when R < 1 > and R < 2 > is a saturated hydrocarbon group, it can manufacture as follows. Polyamideamine dendron is synthesized by repeating Michael addition reaction using methyl acrylate and ester amide exchange reaction using ethylenediamine using dioctadecylamine as a starting material. The polyamidoamine dendron, denoted as DL-G1-2C 18.
Figure JPOXMLDOC01-appb-C000023
Figure JPOXMLDOC01-appb-C000023
　本発明の化合物を製造する際に中間物質として用いられる、Ｒ３及びＲ４を付加する前の状態のポリアミドアミンデンドロンの例を以下に示す。 Examples of the polyamidoamine dendron in the state before adding R 3 and R 4 used as an intermediate substance in the production of the compound of the present invention are shown below.
Figure JPOXMLDOC01-appb-C000024
Figure JPOXMLDOC01-appb-C000024
　Ｒ３及びＲ４の付加は、例えば次のようにして行う。ポリアミドアミンデンドロンに、３－メチルグルタル酸無水物を反応させることにより、Ｒ３及びＲ４として－ＣＯ－ＣＨ２ＣＨ（ＣＨ３）ＣＨ２－ＣＯＯＨを有する化合物が得られる。 The addition of R 3 and R 4 is performed as follows, for example. By reacting polyamidoamine dendron with 3-methylglutaric anhydride, a compound having —CO—CH 2 CH (CH 3 ) CH 2 —COOH as R 3 and R 4 is obtained.
　本発明の化合物として用いられる、Ｒ３及びＲ４を付加した状態のポリアミドアミンデンドロンの例を以下に示す。 Examples of the polyamidoamine dendron with R 3 and R 4 added as the compound of the present invention are shown below.
Figure JPOXMLDOC01-appb-C000025
Figure JPOXMLDOC01-appb-C000025
　２．本発明の組成物
本発明の組成物は、本発明の化合物の他に、リン脂質を好適に含むことができる。このようなリン脂質としては、ホスファチジルエタノールアミン、ホスファリジルコリン、ホスファチジルセリン、ホスファチジルイノシトール、ホスファチジルグリセロール、カルジオリピン、スフィンゴミエリン、プラスマロゲン及びホスファチジン酸等を挙げることができ、これらは１種または２種以上を組み合わせて用いることができる。このうち、ホスファチジルエタノールアミンおよびホスファリジルコリンをそれぞれ単独で、または組み合わせて用いるのが好ましい。これらのリン脂質の脂肪酸残基は、特に限定されるべきものではないが、炭素数１２から１８の飽和または不飽和の脂肪酸残基を挙げることができ、具体的には、ラウロイル基、ミリストイル基、パルミトイル基、ステアロイル基、オレオイル基、リノレイル基等を挙げることができ、ジオレオイルホスファチジルエタノールアミン（ＤＯＰＥ）が特に好ましい。 2. Composition of the present invention The composition of the present invention can suitably contain a phospholipid in addition to the compound of the present invention. Examples of such phospholipids include phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, cardiolipin, sphingomyelin, plasmalogen, and phosphatidic acid. Can be used in combination. Of these, phosphatidylethanolamine and phosphatidylcholine are preferably used alone or in combination. The fatty acid residue of these phospholipids is not particularly limited, and examples thereof include saturated or unsaturated fatty acid residues having 12 to 18 carbon atoms. Specifically, lauroyl group, myristoyl group , Palmitoyl group, stearoyl group, oleoyl group, linoleyl group and the like, and dioleoylphosphatidylethanolamine (DOPE) is particularly preferable.
　リン脂質の配合量は特に限定されないが、リン脂質と本発明の化合物の合計量を１００重量部とした場合にリン脂質３０～９０重量部、本発明の化合物７０～１０重量部、好ましくはリン脂質５０～８０重量部、本発明の化合物５０～２０重量部、より好ましくはリン脂質６０～７０重量部、本発明の化合物４０～３０重量部である。 The blending amount of the phospholipid is not particularly limited, but when the total amount of the phospholipid and the compound of the present invention is 100 parts by weight, 30 to 90 parts by weight of the phospholipid, 70 to 10 parts by weight of the compound of the present invention, preferably phosphorous It is 50 to 80 parts by weight of lipid, 50 to 20 parts by weight of the compound of the present invention, more preferably 60 to 70 parts by weight of phospholipid, and 40 to 30 parts by weight of the compound of the present invention.
　リン脂質の他に、本発明の組成物に含有され得る添加剤としては、コレステロールなどが例示される。 In addition to phospholipid, examples of additives that can be contained in the composition of the present invention include cholesterol.
　本発明の組成物の形態としては、本発明の化合物のみが存在していてもよく、本発明の化合物とリン脂質が単に混合物として存在していてもよく、本発明の化合物とリン脂質とが組み合わさって脂質膜構造体を形成していてもよい。該脂質膜構造体の存在形態およびその製造方法は特に限定されるべきものではないが、例えば、存在形態としては、乾燥した脂質混合物形態、水系溶媒に分散した形態、さらにこれを乾燥させた形態や凍結させた形態等を挙げることができる。 As the form of the composition of the present invention, only the compound of the present invention may be present, the compound of the present invention and the phospholipid may be present merely as a mixture, and the compound of the present invention and the phospholipid may be present. They may be combined to form a lipid membrane structure. The existence form of the lipid membrane structure and the production method thereof are not particularly limited. For example, the existence form includes a dried lipid mixture form, a dispersed form in an aqueous solvent, and a dried form thereof. And a frozen form.
　乾燥した脂質混合物は、例えば、使用する脂質成分をいったんクロロホルム等の有機溶媒に溶解させ、次いでエバポレータによる減圧乾固や噴霧乾燥機による噴霧乾燥を行うことで製造することができる。 The dried lipid mixture can be produced, for example, by dissolving the lipid component to be used once in an organic solvent such as chloroform and then performing vacuum drying with an evaporator or spray drying with a spray dryer.
　脂質膜構造体が水系溶媒に分散した形態としては、一枚膜リポソーム、多重層リポソーム、Ｏ／Ｗ型エマルション、Ｗ／Ｏ／Ｗ型エマルション、球状ミセル、ひも状ミセル、不定型の層状構造物などを挙げることができる。分散した状態の脂質膜構造体の大きさは、特に限定されるべきものではないが、例えば、リポソームやエマルションの場合には、粒子径が５０ｎｍから数μｍであり、球状ミセルの場合、粒子径が５ｎｍから５０ｎｍである。ひも状ミセルや不定型の層状構造物の場合は、その１層あたりの厚みが５ｎｍから１０ｎｍでこれらが層を形成していると考えればよい。 Examples of the form in which the lipid membrane structure is dispersed in an aqueous solvent include monolayer liposomes, multilamellar liposomes, O / W emulsions, W / O / W emulsions, spherical micelles, string micelles, and irregular layered structures And so on. The size of the lipid membrane structure in a dispersed state is not particularly limited. For example, in the case of liposomes and emulsions, the particle diameter is 50 nm to several μm, and in the case of spherical micelles, the particle diameter Is from 5 nm to 50 nm. In the case of a string-like micelle or an irregular layered structure, it can be considered that the thickness per layer is 5 nm to 10 nm and these form a layer.
　水系溶媒（分散媒）の組成も特に限定されるべきものではないが、水のほかに、グルコース、乳糖、ショ糖などの糖水溶液、グリセリン、プロピレングリコールなどの多価アルコール水溶液、リン酸緩衝液、クエン酸緩衝液、リン酸緩衝化生理食塩液等の緩衝液、生理食塩水、細胞培養用の培地などを挙げることができる。この水系溶媒に分散した脂質膜構造体を安定に長期間保存するには、凝集などの物理的安定性の面から、水系溶媒中の電解質を極力なくすことが重要である。また、脂質の化学的安定性の面から、水系溶媒のｐＨを弱酸性から中性付近（ｐＨ３．０から８．０）に設定したり、窒素バブリングにより溶存酸素を除去することが重要である。さらに凍結乾燥保存や噴霧乾燥保存をする場合には、糖水溶液を、凍結保存する場合には、糖水溶液や多価アルコール水溶液をそれぞれ用いると効果的な保存が可能である。 The composition of the aqueous solvent (dispersion medium) is not particularly limited, but in addition to water, sugar aqueous solutions such as glucose, lactose and sucrose, polyhydric alcohol aqueous solutions such as glycerin and propylene glycol, and phosphate buffer And buffer solutions such as citrate buffer solution and phosphate buffered physiological saline solution, physiological saline, medium for cell culture, and the like. In order to stably store the lipid membrane structure dispersed in the aqueous solvent for a long period of time, it is important to eliminate the electrolyte in the aqueous solvent as much as possible from the viewpoint of physical stability such as aggregation. From the viewpoint of chemical stability of the lipid, it is important to set the pH of the aqueous solvent from weakly acidic to neutral (pH 3.0 to 8.0) or to remove dissolved oxygen by nitrogen bubbling. . Further, when lyophilized storage or spray-dried storage is used, effective storage is possible by using a sugar aqueous solution, and when storing frozen, a sugar aqueous solution or a polyhydric alcohol aqueous solution is used.
　これらの水系溶媒の添加物の濃度は特に限定されるべきものではないが、例えば、糖水溶液においては、２から２０％（Ｗ／Ｖ）が好ましく、５から１０％（Ｗ／Ｖ）がさらに好ましい。また、多価アルコール水溶液においては、１から５％（Ｗ／Ｖ）が好ましく、２から２．５％（Ｗ／Ｖ）がさらに好ましい。緩衝液においては、緩衝剤の濃度が５から５０ｍＭが好ましく、１０から２０ｍＭがさらに好ましい。 The concentration of these aqueous solvent additives is not particularly limited. For example, in an aqueous sugar solution, 2 to 20% (W / V) is preferable, and 5 to 10% (W / V) is more preferable. preferable. Moreover, in a polyhydric alcohol aqueous solution, 1 to 5% (W / V) is preferable, and 2 to 2.5% (W / V) is more preferable. In the buffer solution, the concentration of the buffer is preferably 5 to 50 mM, more preferably 10 to 20 mM.
　水系溶媒中の脂質膜構造体の濃度は、特に限定されるべきものではないが、本発明においては脂質膜構造体として用いるリン脂質の総量の濃度は、０．００１ｍＭから１００ｍＭが好ましく、０．０１ｍＭから２０ｍＭがさらに好ましい。 The concentration of the lipid membrane structure in the aqueous solvent is not particularly limited. In the present invention, the concentration of the total amount of phospholipid used as the lipid membrane structure is preferably 0.001 mM to 100 mM. More preferred is 01 mM to 20 mM.
　脂質膜構造体が水系溶媒に分散した形態は、上記の乾燥した脂質混合物を水系溶媒に添加し、さらにホモジナイザー等の乳化機、超音波乳化機、高圧噴射乳化機等により乳化することで製造することができる。また、リポソームを製造する方法としてよく知られている方法、例えば逆相蒸発法などによっても製造することもでき、特に限定されるべきものではない。脂質膜構造体の大きさを制御したい場合には、孔径のそろったメンブランフィルター等を用いて、高圧下でイクストルージョン（押し出し濾過）を行えばよい。 The form in which the lipid membrane structure is dispersed in an aqueous solvent is produced by adding the dried lipid mixture to the aqueous solvent and further emulsifying with an emulsifier such as a homogenizer, an ultrasonic emulsifier, a high-pressure jet emulsifier, or the like. be able to. Moreover, it can also manufacture by the method well-known as a method of manufacturing a liposome, for example, a reverse phase evaporation method etc., It should not be specifically limited. When it is desired to control the size of the lipid membrane structure, extrusion (extrusion filtration) may be performed under high pressure using a membrane filter having a uniform pore size.
　また、上記の水系溶媒に分散した脂質膜構造体をさらに乾燥させる方法としては、通常の凍結乾燥や噴霧乾燥を挙げることができる。この時の水系溶媒としては、上記したように、糖水溶液、好ましくはショ糖水溶液、乳糖水溶液を用いるとよい。ここで、水系溶媒に分散した脂質膜構造体をいったん製造した上でさらに乾燥すると、脂質膜構造体の長期保存が可能となるほか、この乾燥した脂質膜構造体に生理活性物質を含有する水溶液を添加すると、効率よく脂質混合物が水和されるために生理活性物質自身も効率よく、脂質膜構造体に保持させることができるといったメリットがある。 In addition, examples of a method for further drying the lipid membrane structure dispersed in the aqueous solvent include normal freeze drying and spray drying. As the aqueous solvent at this time, as described above, a sugar aqueous solution, preferably a sucrose aqueous solution or a lactose aqueous solution may be used. Here, once a lipid membrane structure dispersed in an aqueous solvent is produced and further dried, the lipid membrane structure can be stored for a long period of time, and an aqueous solution containing a physiologically active substance in the dried lipid membrane structure. Is effective in that the lipid mixture is efficiently hydrated, so that the physiologically active substance itself can be efficiently retained in the lipid membrane structure.
　３．　用途についての説明
　本発明の化合物、組成物及びキットは、以下に説明する用途に使用できる。
本発明の化合物、組成物及びキットは、好ましくは、生理活性物質を細胞内に導入するために使用される。この場合、細胞は生体内の細胞であってもよいし、生体外に取り出された細胞であってもよい。すなわち、本発明の化合物、組成物及びキットは、生理活性物質を細胞内にin vivo又はin vitroで導入するために使用される。 3. Description of Uses The compounds, compositions and kits of the present invention can be used for the uses described below.
The compounds, compositions and kits of the present invention are preferably used for introducing physiologically active substances into cells. In this case, the cell may be a cell in the living body or a cell taken out of the living body. That is, the compound, composition and kit of the present invention are used for introducing a physiologically active substance into cells in vivo or in vitro.
　生理活性物質は、特に限定されないが、例えば低分子化合物、ペプチド又は遺伝子である。 The physiologically active substance is not particularly limited, and is, for example, a low molecular compound, a peptide or a gene.
　前述のように、ｐＨ応答性のカルボキシル基含有炭化水素基を設計することによって、本発明の化合物で構成したベシクルに内包させた生理活性物質を、初期エンドソーム、後期エンドソーム又はリソソームのいずれかの内部で放出できる。 As described above, by designing a pH-responsive carboxyl group-containing hydrocarbon group, a physiologically active substance encapsulated in a vesicle composed of the compound of the present invention can be contained in any of early endosomes, late endosomes or lysosomes. Can be released.
　ペプチド又は遺伝子としては、特に限定されないが、抗原となりうるか又は抗原を生じうるものであれば、本発明の化合物で構成したベシクルにこれらを内包させた上で抗原提示細胞内に導入することにより、これらを初期エンドソーム又は後期エンドソーム内においてリリースできる。これによりＭＨＣ　ｃｌａｓｓ　ＩＩを介した抗原提示を通じて液性免疫（ｈｕｍｏｒａｌ　ｉｍｍｕｎｅ　ｒｅｓｐｏｎｓｅ）系による抗体産生を誘導し、ひいては疾患を治療するために使用できるので好ましい。　抗原を生じうる遺伝子としては、いわゆるＤＮＡワクチンとして利用されるものを用いることができる。ＤＮＡワクチンとは、抗原情報をコードしたＤＮＡをワクチンとして接種する技術である。特に限定されないが、通常、ＤＮＡワクチンとしては、抗原情報をコードしたＤＮＡ断片を含有するベクターが用いられる。通常、ＤＮＡワクチンとして利用されるベクターには、非メチル化ＣｐＧモチーフと呼ばれる、メチル化されていないシトシン及びグアニンに富む配列領域が含まれている。 The peptide or gene is not particularly limited, but if it can be an antigen or can generate an antigen, it is encapsulated in a vesicle composed of the compound of the present invention and then introduced into an antigen-presenting cell. They can be released in early or late endosomes. This is preferable because it can be used for inducing antibody production by the humoral immunity response system through antigen presentation via MHC class II and thus treating the disease. As a gene capable of producing an antigen, a gene used as a so-called DNA vaccine can be used. The DNA vaccine is a technique for inoculating DNA encoding antigen information as a vaccine. Although not particularly limited, a vector containing a DNA fragment encoding antigen information is usually used as a DNA vaccine. Usually, vectors used as DNA vaccines contain unmethylated cytosine and guanine-rich sequence regions called unmethylated CpG motifs.
　抗原に関し、特に限定されないが、例えば、悪性黒色種（メラノーマ）治療を目的としてＭＡＧＥ、乳癌治療を目的としてＨＥＲ２／ｎｅｕ、大腸癌治療を目的としてＣＥＡ、及び白血病又は各種癌の治療を目的としてＷＴ1等を用いることができる。 There are no particular restrictions on the antigen, but for example, MAGE for the treatment of malignant melanoma, HER2 / neu for the treatment of breast cancer, CEA for the treatment of colorectal cancer, and WT1 for the treatment of leukemia or various cancers. Etc. can be used.
　生理活性物質としては、免疫阻害剤として作用しうるものを用いることができる。免疫阻害剤として作用しうるものは、その阻害作用が特定の免疫反応に対して選択的であるか非選択的であるかを問わず用いることができる。 Physiologically active substances that can act as immune inhibitors can be used. What can act as an immune inhibitor can be used regardless of whether the inhibitory action is selective or non-selective for a specific immune response.
　　　本発明の本発明の化合物、組成物及びキットを、上記疾患の治療目的で、導入しようとする生理活性物質とともにin vivoで細胞に導入するために使用してもよい。この場合、本発明の化合物又は組成物を生理活性物質とともに個体に投与してもよい。個体への投与手段としては、経口投与でも、非経口投与でもよいが、非経口投与が好ましい。剤形としては、通常知られたものでよく、経口投与の剤形としては、例えば、錠剤、散剤、顆粒剤、シロップ剤等を挙げることができる。また、非経口投与の剤形としては、例えば、注射剤、点眼剤、軟膏剤、坐剤等を挙げることができる。中でも、注射剤が好ましく、投与方法としては、静脈注射、標的とする細胞や臓器に対しての局所注射が好ましい。 The compound, composition and kit of the present invention of the present invention may be used for in vivo introduction into cells together with the physiologically active substance to be introduced for the purpose of treating the above diseases. In this case, the compound or composition of the present invention may be administered to an individual together with a physiologically active substance. The means for administration to an individual may be oral administration or parenteral administration, but parenteral administration is preferred. The dosage form may be a conventionally known dosage form, and examples of the dosage form for oral administration include tablets, powders, granules, syrups and the like. Examples of the dosage form for parenteral administration include injections, eye drops, ointments, suppositories and the like. Among these, an injection is preferable, and an administration method is preferably intravenous injection or local injection into a target cell or organ.
　本発明の本発明の化合物、組成物及びキットを、上記疾患の治療目的で、導入しようとする生理活性物質とともにin vitroで細胞に導入するために使用してもよい。この場合、本発明の化合物又は組成物を、次のような工程を含有する方法において使用してもよい。
（１）治療対象である個体から樹状細胞等の抗原提示細胞を採取する工程；
（２）工程（１）により採取された細胞に、本発明の化合物を含有するベシクルであって、生理活性物質を内包するベシクルを導入する工程；及び
（３）工程（２）により得られた細胞を治療対象である個体に戻す工程。 The compounds, compositions and kits of the present invention may be used for in vitro introduction into cells together with the physiologically active substance to be introduced for the purpose of treating the above-mentioned diseases. In this case, you may use the compound or composition of this invention in the method containing the following processes.
(1) a step of collecting antigen-presenting cells such as dendritic cells from an individual to be treated;
(2) a step of introducing a vesicle containing the compound of the present invention and containing a physiologically active substance into the cells collected in step (1); and (3) obtained by step (2). Returning the cells to the individual being treated.
　上記方法においては、工程（２）により得られた細胞において、ベシクルがエンドソーム内へ取り込まれた後の早い段階においてエンドソーム内で生理活性物質が放出され、効果的な免疫誘導が行われる。これにより、当該細胞においてはＭＨＣ　ｃｌａｓｓ　ＩＩを介した抗原提示を通じて液性免疫系による抗体産生が誘導されている。このような細胞を治療対象である個体に戻すことにより、疾患を治療できる。 In the above method, in the cells obtained in the step (2), a physiologically active substance is released in the endosome at an early stage after the vesicle is taken into the endosome, and effective immune induction is performed. Thus, antibody production by the humoral immune system is induced in the cells through antigen presentation via MHC class II. By returning such cells to the individual to be treated, the disease can be treated.
　本発明の化合物を単独で用いる場合、生理活性物質と本発明の化合物の配合割合は、生理活性物質１重量部に対し、本発明の化合物を１～２０重量部、好ましくは３～１５重量部、より好ましくは５～７重量部使用する。また、本発明の化合物とリン脂質の混合物を用いる場合、生理活性物質と本発明の化合物の配合割合は、生理活性物質１重量部に対し、本発明の化合物を１～５０重量部、好ましくは５～３０重量部、より好ましくは１０～１５重量部使用する。 When the compound of the present invention is used alone, the compounding ratio of the physiologically active substance and the compound of the present invention is 1 to 20 parts by weight, preferably 3 to 15 parts by weight of the compound of the present invention with respect to 1 part by weight of the physiologically active substance. More preferably, 5 to 7 parts by weight are used. When a mixture of the compound of the present invention and phospholipid is used, the compounding ratio of the physiologically active substance and the compound of the present invention is 1 to 50 parts by weight, preferably 1 to 50 parts by weight, preferably 1 part by weight of the physiologically active substance. 5 to 30 parts by weight, more preferably 10 to 15 parts by weight is used.
　生理活性物質としての遺伝子としては、オリゴヌクレオチド、ＤＮＡおよびＲＮＡのいずれでもよく、特に形質転換等のイン・ビトロにおける導入用遺伝子や、イン・ビボで発現することにより作用する遺伝子、例えば、遺伝子治療用遺伝子、実験動物や家畜等の産業用動物の品種改良に用いられる遺伝子が好ましい。遺伝子治療用遺伝子としては、アンチセンスオリゴヌクレオチド、アンチセンスＤＮＡ、アンチセンスＲＮＡ、酵素、サイトカイン等の生理活性物質をコードする遺伝子等を挙げることができる。 As a gene as a physiologically active substance, any of oligonucleotide, DNA and RNA may be used. In particular, a gene for introduction in vitro such as transformation, a gene which acts by expression in vivo, for example, gene therapy Genes for use in breeding industrial animals such as laboratory genes and laboratory animals and livestock are preferred. Examples of genes for gene therapy include genes encoding physiologically active substances such as antisense oligonucleotides, antisense DNAs, antisense RNAs, enzymes, and cytokines.
　以下に実施例により本発明をさらに詳細に説明するが、本発明は以下の例にのみ限定されるものではない。
Ｉ．第一の発明
　１．本発明の分子集合体の製造
　１．１　試薬
　Fetal Bovine Serum (FBS)はMP Biomedicals,Inc から購入した。ダルベッコ変法イーグル培地(DMEM)は日水製薬（株）から購入した。N,N-ジメチルホルムアミド、テトラヒドロフラン、石油エーテル、シアン化ナトリウム、水素化リチウムアルミニウム、リン酸水素二ナトリウム、リン酸二水素カリウム、ベンジルペニシリンカリウム、ストレプトマイシン硫酸塩は和光純薬から購入した。ピラニンは東京化成(株)から購入した。クロロホルム、酢酸エチル、メタノール、n-ヘキサン、硫酸ナトリウム、塩化カルシウム、塩化マグネシウム6 水和物、塩化カリウム、エチレンジアミン四酢酸二ナトリウム(EDTA)はキシダ化学から購入した。トリエチルアミン、アクリル酸メチル、エチレンジアミン、ジエチルエーテル、塩化ナトリウム、RPMI-1640 液体培地、MEM 非必須アミノ酸溶液はナカライテスクから購入した。トリプシンはDIFCOLABORATORIES(U.S.A)から購入した。ジオクタデシルアミン、カルセインはSigma から購入した。オレイルアミン、オレオイルクロリド、シクロヘキサンジカルボン酸無水物、3-メチルグルタル酸無水物はALDRICH から購入した。 The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to the following examples.
I. First invention 1. Production of Molecular Assemblies of the Present Invention 1.1 Reagent Fetal Bovine Serum (FBS) was purchased from MP Biomedicals, Inc. Dulbecco's modified Eagle's medium (DMEM) was purchased from Nissui Pharmaceutical. N, N-dimethylformamide, tetrahydrofuran, petroleum ether, sodium cyanide, lithium aluminum hydride, disodium hydrogen phosphate, potassium dihydrogen phosphate, potassium benzylpenicillin, and streptomycin sulfate were purchased from Wako Pure Chemical. Pyranine was purchased from Tokyo Kasei Co., Ltd. Chloroform, ethyl acetate, methanol, n-hexane, sodium sulfate, calcium chloride, magnesium chloride hexahydrate, potassium chloride, and disodium ethylenediaminetetraacetate (EDTA) were purchased from Kishida Chemical. Triethylamine, methyl acrylate, ethylenediamine, diethyl ether, sodium chloride, RPMI-1640 liquid medium, and MEM non-essential amino acid solution were purchased from Nacalai Tesque. Trypsin was purchased from DIFCOLABORATORIES (USA). Dioctadecylamine and calcein were purchased from Sigma. Oleylamine, oleoyl chloride, cyclohexanedicarboxylic anhydride and 3-methylglutaric anhydride were purchased from ALDRICH.
　ジクロロメタンはSigma-ALDRICH から購入した。2-メルカプトエタノール、DPX、Hoechst、Lysotracker Green DND-26、Lysotracker Red DND-99、Tf-Alexa555はInvitrogen から購入した。透析膜はSpectra/Por 6 (分画分子量 2000,FE-0526-33)をSpectrum Laboratories Inc.から購入した。Merck Kieselgel 60(230-400 mesh) をシリカゲルクロマトグラフィーに使用した。 Dichloromethane was purchased from Sigma-ALDRICH®. 2-mercaptoethanol, DPX, Hoechst, Lysotracker Green DND-26, Lysotracker Red DND-99, and Tf-Alexa555 were purchased from Invitrogen. As a dialysis membrane, Spectra / Por 6 (fractional molecular weight 2000, FE-0526-33) was purchased from Spectrum Laboratories Inc. Merck Kieselgel 60 (230-400 mesh) was used for silica gel chromatography.
　１．２　本発明の化合物（Ａ）の合成
　１．２．１　概要
　2本のアルキル鎖を持つ第1、第2世代のデンドロン脂質(DL-G1、DL-G2)と、2本のオレイル鎖を持つ第1世代のデンドロン脂質(DL-G1-U2)の合成経路を下記式に示し、そのアミノ基末端にメトキシジエチレングリコール(MDEG)基、又は、エトキシジエチレングリコール(EDEG)基を導入したデンドロン脂質(MDEG-DL-G1、EDEG-DL-G1、MDEG-DL-G2、EDEG-G2、MDEG-DL-G1-U2、EDEG-DL-G1-U2)の合成経路を下記式に示した。 1.2 Synthesis of Compound (A) of the Present Invention 1.2.1 Overview First and second generation dendron lipids (DL-G1, DL-G2) having two alkyl chains and two oleyl chains The synthesis route of the first generation dendron lipid (DL-G1-U2) having the following formula is shown in the following formula, and the methoxydiethylene glycol (MDEG) group or the ethoxydiethylene glycol (EDEG) group is introduced into the amino group end of the dendron lipid ( The synthesis route of MDEG-DL-G1, EDEG-DL-G1, MDEG-DL-G2, EDEG-G2, MDEG-DL-G1-U2, and EDEG-DL-G1-U2) is shown in the following formula.
　DL-G1、DL-G2は、ジオクタデシルアミンを出発物質として、アクリル酸メチルによるマイケル付加反応と、それに続くエチレンジアミンによるエステルアミド交換反応を交互に行うことにより合成した。この方法は、デンドリマーの合成において、Tomaliaらが報告した方法である。又、DL-G1-U2は、オレイルアミンとオレイルクロライドとを反応させ、リチウムアルミニウムヒドリド(LiAlH4)でヒドリド還元した後、アクリル酸メチルによるマイケル付加反応と、それに続くエチレンジアミンによるエステルアミド交換反応を交互に行うことにより合成した。 DL-G1 and DL-G2 were synthesized using dioctadecylamine as a starting material by alternately performing a Michael addition reaction with methyl acrylate followed by an ester amide exchange reaction with ethylenediamine. This is the method reported by Tomalia et al. In the synthesis of dendrimers. DL-G1-U2 reacts with oleylamine and oleyl chloride, and hydride reduction with lithium aluminum hydride (LiAlH 4 ), followed by Michael addition reaction with methyl acrylate and subsequent ester amide exchange reaction with ethylenediamine. To be synthesized.
　更に合成したDL-G1、DL-G2、DL-G1-U2の末端アミノ基にパラニトロフェニルカーボネート基で活性化したMDEG基、EDEG基を反応させ末端に導入しMDEG-DL-G1、EDEG-DL-G1、MDEG-DL-G2、EDEG-DL-G2、MDEG-DL-G1-U2、EDEG-DL-G1-U2を合成した。 Furthermore, the terminal amino groups of the synthesized DL-G1, DL-G2, and DL-G1-U2 were reacted with MDEG groups and EDEG groups activated with paranitrophenyl carbonate groups, and introduced at the ends to introduce MDEG-DL-G1, EDEG- DL-G1, MDEG-DL-G2, EDEG-DL-G2, MDEG-DL-G1-U2, and EDEG-DL-G1-U2 were synthesized.
Figure JPOXMLDOC01-appb-C000026
Figure JPOXMLDOC01-appb-C000026
Figure JPOXMLDOC01-appb-C000027
Figure JPOXMLDOC01-appb-C000027
Figure JPOXMLDOC01-appb-C000028
Figure JPOXMLDOC01-appb-C000028
Figure JPOXMLDOC01-appb-C000029
Figure JPOXMLDOC01-appb-C000029
Figure JPOXMLDOC01-appb-C000030
Figure JPOXMLDOC01-appb-C000030
　１．２．２　DL-G-0.5-2C 18 の合成
　アクリル酸メチル(35 ml, 0.39 mmol)にジオクタデシルアミン(2.00 g, 3.9 mmol)を加え、加熱溶解し、窒素雰囲気下、75℃で28時間還流した。その後、未反応のアクリル酸メチルを減圧留去し、シリカゲルクロマトグラフィー(展開溶媒：石油エーテル/ジエチルエーテル＝2/1, v/v)によって精製した。(収量2.18g、収率：90.9%)
　1H NMR (CDCl3): δ 0.88 (m, CH3(CH2)15-), δ 1.22(s, CH3(CH2)15-),δ 1.40(m, -CH2CH2N-), δ 2.38 (t, -CH2COOCH3), δ 2.44 (t, -CH2N-),δ 2.77 (t,-CH2CH2COOCH3), δ 3.67 (s, -OCH3). 1.2.2 Synthesis of DL-G-0.5-2C 18 Dioctadecylamine (2.00 g, 3.9 mmol) was added to methyl acrylate (35 ml, 0.39 mmol) and dissolved by heating. At 75 ° C. under nitrogen atmosphere. Refluxed for 28 hours. Thereafter, unreacted methyl acrylate was distilled off under reduced pressure and purified by silica gel chromatography (developing solvent: petroleum ether / diethyl ether = 2/1, v / v). (Yield 2.18 g, Yield: 90.9%)
1 H NMR (CDCl 3): δ 0.88 (m, CH 3 (CH 2) 15 -), δ 1.22 (s, CH 3 (CH 2) 15 -), δ 1.40 (m, -CH 2 CH 2 N- ), δ 2.38 (t, -CH 2 COOCH 3 ), δ 2.44 (t, -CH 2 N-), δ 2.77 (t, -CH 2 CH 2 COOCH 3 ), δ 3.67 (s, -OCH 3 ).
　１．２．３　DL-G0-2C 18 の合成
　メタノール(40 ml)にDL-G-0.5(2.18g,3.58mmol)を加え、加熱溶解した。この溶液を、シアン化ナトリウム(36.2 mg, 0.734mmol)を含むエチレンジアミン(70ml,1.05mol)に徐々に加え、窒素雰囲気下、50℃で6日間撹拌した。その後、未反応のエチレンジアミンとメタノールを減圧留去し、シリカゲルクロマトグラフィー(展開溶媒：クロロホルム/メタノール/水＝60/35/5, v/v)によって精製した。(収量：1.47g、収率：64.8%)
　1H NMR (CDCl3): δ 0.88 (m, CH3(CH2)15-), δ 1.26 (s, CH3(CH2)15-),δ 1.45(m, -CH2CH2N-), δ 2.39(t, -CH2CONH-), δ 2.46(t, -CH2N-),δ 2.69(t, -CH2CH2CONH-), δ 2.82(t, -CH2NH2), δ 3.29(m, -CH2CH2NH2), δ 8.63(m, -CONH-). 1.2.3 Synthesis of DL-G0-2C 18 DL-G-0.5 (2.18 g, 3.58 mmol) was added to methanol (40 ml) and dissolved by heating. This solution was gradually added to ethylenediamine (70 ml, 1.05 mol) containing sodium cyanide (36.2 mg, 0.734 mmol) and stirred at 50 ° C. for 6 days under a nitrogen atmosphere. Thereafter, unreacted ethylenediamine and methanol were distilled off under reduced pressure and purified by silica gel chromatography (developing solvent: chloroform / methanol / water = 60/35/5, v / v). (Yield: 1.47 g, Yield: 64.8%)
1 H NMR (CDCl 3): δ 0.88 (m, CH 3 (CH 2) 15 -), δ 1.26 (s, CH 3 (CH 2) 15 -), δ 1.45 (m, -CH 2 CH 2 N- ), δ 2.39 (t, -CH 2 CONH-), δ 2.46 (t, -CH 2 N-), δ 2.69 (t, -CH 2 CH 2 CONH-), δ 2.82 (t, -CH 2 NH 2 ), δ 3.29 (m, -CH 2 CH 2 NH 2 ), δ 8.63 (m, -CONH-).
　１．２．４　DL-G0.5-2C 18 の合成
　メタノール(20 ml)にDL-G0(1.37 g, 2.16mmol)を加え、加熱溶解した。この溶液を、アクリル酸メチル(39 ml, 0.432 mol)に徐々に加え、窒素雰囲気下、35℃で48時間撹拌した。その後、未反応のアクリル酸メチルとメタノールを減圧留去し、シリカゲルクロマトグラフィー(展開溶媒：石油エーテル/ジエチルエーテル＝2/1, v/v のちクロロホルム/メタノール＝95/5, v/v)によって精製した。(収量：1.30g、収率：74.5%)
　1H NMR (CDCl3): δ 0.88 (m, CH3(CH2)15-), δ 1.26 (s, CH3(CH2)15-), δ 1.44(m, -CH2CH2N-), δ 2.41(t, -CH2CONH-), δ 2.43(m, -CH2COOCH3), δ 2.46(m, -CH2N-), δ2.54(t, -CONHCH2CH2-), δ 2.75(t, -CH2CH2CONH-), δ 2.78(t, -CH2CH2COOCH3), δ 3.28(m, -CONHCH3-), δ 3.67 (s, -OCH3), δ 7.00(m, -CONH-). 1.2.4 Synthesis of DL-G0.5-2C 18 DL-G0 (1.37 g, 2.16 mmol) was added to methanol (20 ml) and dissolved by heating. This solution was gradually added to methyl acrylate (39 ml, 0.432 mol) and stirred at 35 ° C. for 48 hours under a nitrogen atmosphere. Then, unreacted methyl acrylate and methanol were distilled off under reduced pressure, and silica gel chromatography (developing solvent: petroleum ether / diethyl ether = 2/1, v / v followed by chloroform / methanol = 95/5, v / v) Purified. (Yield: 1.30 g, Yield: 74.5%)
1 H NMR (CDCl 3): δ 0.88 (m, CH 3 (CH 2) 15 -), δ 1.26 (s, CH 3 (CH 2) 15 -), δ 1.44 (m, -CH 2 CH 2 N- ), δ 2.41 (t, -CH 2 CONH-), δ 2.43 (m, -CH 2 COOCH 3 ), δ 2.46 (m, -CH 2 N-), δ2.54 (t, -CONHCH 2 CH 2- ), δ 2.75 (t, -CH 2 CH 2 CONH-), δ 2.78 (t, -CH 2 CH 2 COOCH 3 ), δ 3.28 (m, -CONHCH 3- ), δ 3.67 (s, -OCH 3 ) , δ 7.00 (m, -CONH-).
　１．２．５　DL-G1-2C 18 の合成
　メタノール(25 ml)にDL-G0.5(1.29 g, 3.64mmol)を加え、加熱溶解した。この溶液を、シアン化ナトリウム(18.4 mg, 0.38mmol)を含むエチレンジアミン(62.5ml,0.88mol)に徐々に加え、窒素雰囲気下、45℃で76時間撹拌した。その後、未反応のエチレンジアミンとメタノールを減圧留去し、Sephadex LH-20カラム（溶離液：クロロホルム）によって精製した。(収量：0.615g、収率：44.0%)
　1H NMR (CDCl3): δ 0.88 (m, CH3(CH2)15-), δ 1.26 (s, CH3(CH2)15-), δ1.43(m, -CH2CH2N-),δ 2.17(m, -NH2),δ 2.36(m, -CH2CONHCH2CH2NH2), δ 2.40(m, -CH2CONH-), δ 2.42(m, -CH2N-), δ2.44(t,-CONHCH2CH2-), δ 2.68(t, -CH2CH2CONH-), δ 2.74 (t, -CH2CH2CONHCH2CH2NH2), δ 2.84(t, -CH2NH2), δ 3.29 (m, -CONHCH2-), δ 3.30 (m, -CH2CH2NH2), δ 7.00(m, -CONH-). 1.2.5 DL-G1-2C 18 Synthesis in methanol (25 ml) of DL-G0.5 (1.29 g, 3.64mmol ) was added and dissolved by heating. This solution was gradually added to ethylenediamine (62.5 ml, 0.88 mol) containing sodium cyanide (18.4 mg, 0.38 mmol) and stirred at 45 ° C. for 76 hours under a nitrogen atmosphere. Thereafter, unreacted ethylenediamine and methanol were distilled off under reduced pressure, and the residue was purified by Sephadex LH-20 column (eluent: chloroform). (Yield: 0.615 g, Yield: 44.0%)
1 H NMR (CDCl 3): δ 0.88 (m, CH 3 (CH 2) 15 -), δ 1.26 (s, CH 3 (CH2) 15 -), δ1.43 (m, -CH 2 CH 2 N- ), δ 2.17 (m, -NH 2 ), δ 2.36 (m, -CH 2 CONHCH 2 CH 2 NH 2 ), δ 2.40 (m, -CH 2 CONH-), δ 2.42 (m, -CH 2 N- ), δ2.44 (t, -CONHCH 2 CH 2- ), δ 2.68 (t, -CH 2 CH 2 CONH-), δ 2.74 (t, -CH 2 CH 2 CONHCH 2 CH 2 NH 2 ), δ 2.84 (t, -CH 2 NH 2 ), δ 3.29 (m, -CONHCH 2- ), δ 3.30 (m, -CH 2 CH 2 NH 2 ), δ 7.00 (m, -CONH-).
　１．２．６　DL-G1.5-2C 18 の合成
　メタノール(23 ml)にDL-G1(0.615 g, 7.11mmol)を加え、加熱溶解した。この溶液を、アクリル酸メチル(145 ml, 1.6 mol)に徐々に加え、窒素雰囲気下、35℃で65時間撹拌した。その後、未反応のアクリル酸メチルとメタノールを減圧留去し、シリカゲルクロマトグラフィー(展開溶媒：クロロホルムのちクロロホルム/メタノール＝9/1, v/v)で精製した。(収量：0.558g、収率：65.1%)
　1H NMR (CDCl3): δ 0.88 (m, CH3(CH2)15-), δ 1.25 (s, CH3(CH2)15-), δ 1.43(m, -CH2CH2N-), δ 2.36(m, -CH2COOCH3), δ 2.43 (m, -CH2N-), δ 2.54 (m, -CONHCH2CH2-), δ 2.75 (m, -CH2CH2COOCH3), δ 3.28(m, -CONHCH2-), δ 3.67 (s, -OCH3), δ 7.00and 8.04 (m, -CONH-). 1.2.6 Synthesis of DL-G1.5-2C 18 DL-G1 (0.615 g, 7.11 mmol) was added to methanol (23 ml) and dissolved by heating. The solution was gradually added to methyl acrylate (145 ml, 1.6 mol) and stirred at 35 ° C. for 65 hours under a nitrogen atmosphere. Thereafter, unreacted methyl acrylate and methanol were distilled off under reduced pressure, and the residue was purified by silica gel chromatography (developing solvent: chloroform / chloroform / methanol = 9/1, v / v). (Yield: 0.558g, Yield: 65.1%)
1 H NMR (CDCl 3): δ 0.88 (m, CH 3 (CH 2) 15 -), δ 1.25 (s, CH 3 (CH 2) 15 -), δ 1.43 (m, -CH 2 CH 2 N- ), δ 2.36 (m, -CH 2 COOCH 3 ), δ 2.43 (m, -CH 2 N-), δ 2.54 (m, -CONHCH 2 CH 2- ), δ 2.75 (m, -CH 2 CH 2 COOCH 3 ), δ 3.28 (m, -CONHCH 2- ), δ 3.67 (s, -OCH 3 ), δ 7.00and 8.04 (m, -CONH-).
　１．２．７　DL-G2-2C 18 の合成
　メタノール(12 ml)にDL-G1.5(0.558 g, 0.461mmol)を加え、加熱溶解した。この溶液を、シアン化ナトリウム(9.3 mg, 0.19mmol)を含むエチレンジアミン(40ml, 0.6mol)に徐々に加え、窒素雰囲気下、45℃で72時間撹拌した。その後、未反応のエチレンジアミンとメタノールを減圧留去し、Sephadex LH-20カラム(溶離液：メタノール)によって精製した。
(収量：0.53g、収率87.5%)
　1H NMR (CDCl3): δ 0.88 (m, CH3(CH2)15-), δ 1.25(s, CH3(CH2)15-), δ 1.42 (m, -CH2CH2N-), δ 2.09(m,-NH2), δ 2.32 (m, -CH2CONHCH2CH2NH2), δ 2.37 (m, -CH2N-), δ 2.52(m, -CONHCH2CH2-),δ 2.73 (m, -CH2CH2CONHCH2CH2NH2), δ 2.82 (m, -CH2NH2), δ 3.26(m, -CONHCH2-), δ 3.29(m, -CH2CH2NH2), δ 7.63, 7.90and 8.47(m, -CONH-). 1.2.7 Synthesis of DL-G2-2C 18 DL-G1.5 (0.558 g, 0.461 mmol) was added to methanol (12 ml) and dissolved by heating. This solution was gradually added to ethylenediamine (40 ml, 0.6 mol) containing sodium cyanide (9.3 mg, 0.19 mmol) and stirred at 45 ° C. for 72 hours under a nitrogen atmosphere. Thereafter, unreacted ethylenediamine and methanol were distilled off under reduced pressure, and the residue was purified by Sephadex LH-20 column (eluent: methanol).
(Yield: 0.53g, Yield 87.5%)
1 H NMR (CDCl 3): δ 0.88 (m, CH 3 (CH 2) 15 -), δ 1.25 (s, CH 3 (CH 2) 15 -), δ 1.42 (m, -CH 2 CH 2 N- ), δ 2.09 (m, -NH 2 ), δ 2.32 (m, -CH 2 CONHCH 2 CH 2 NH 2 ), δ 2.37 (m, -CH 2 N-), δ 2.52 (m, -CONHCH 2 CH 2 -), δ 2.73 (m, -CH 2 CH 2 CONHCH 2 CH 2 NH 2 ), δ 2.82 (m, -CH 2 NH 2 ), δ 3.26 (m, -CONHCH 2- ), δ 3.29 (m,- CH 2 CH 2 NH 2 ), δ 7.63, 7.90and 8.47 (m, -CONH-).
　１．２．８　Oleyloleoylamideの合成
　Oleoylchlorideを7.7 mL (20 mmol)をジクロロメタン100 mLに溶解させ氷水浴で撹拌し、これにジクロロメタン50 mLにOleylamine 9.4 mL (20 mmol)とトリエチルアミン3.3 mL (0.024 mol)を溶解したものをゆっくり滴下した後、室温窒素雰囲気化で71時間還流した。その後、エバポレータにより溶媒を留去し真空乾燥した後、得られた粗生成物をオープンカラムクロマトグラフィーにより分離精製した(展開溶媒クロロホルム：酢酸エチル=2：1)。その後1H NMRにより化合物の同定を行った。（収量9.38 g、収率86.4 %）
　1H NMR (CDCl3): δ 0.88 (m, CH3(CH2)6-), δ 1.23 (s, CH3(CH2)6-,-CH2(CH2)5CH2-), δ 1.41 (m, -CH2CH2CO-),δ 1.55 (m, -CH2CH2CO-),δ 1.91 (m, -CH2CH2CH-),δ 2.05 (m, -CH2CH2NH-),δ 3.15 (m, -CH2CH2NH-),δ 5.25 (m, -CH2CHCHCH2-). 1.2.8 Synthesis of Oleyloleoylamide Oleoylchloride, 7.7 mL (20 mmol), was dissolved in 100 mL of dichloromethane and stirred in an ice-water bath. After slowly adding dropwise, was refluxed in a nitrogen atmosphere at room temperature for 71 hours. Then, after evaporating the solvent with an evaporator and vacuum drying, the obtained crude product was separated and purified by open column chromatography (developing solvent chloroform: ethyl acetate = 2: 1). Thereafter, the compound was identified by 1 H NMR. (Yield 9.38 g, Yield 86.4%)
1 H NMR (CDCl 3 ): δ 0.88 (m, CH 3 (CH 2 ) 6- ), δ 1.23 (s, CH 3 (CH 2 ) 6 -,-CH 2 (CH 2 ) 5 CH 2- ), δ 1.41 (m, -CH 2 CH 2 CO-), δ 1.55 (m, -CH 2 CH 2 CO-), δ 1.91 (m, -CH 2 CH 2 CH-), δ 2.05 (m, -CH 2 CH 2 NH -), δ 3.15 (m, -CH 2 CH 2 NH -), δ 5.25 (m, -CH 2 CHCHCH 2 -).
　１．２．９　Dioleylamineの合成
　THF 84.5 mLにゆっくりとLAH 1.489 gを加え、これにOleyloleoylamide 2.99 g(5.61 mmol)をTHF 84.5 mLで溶解したものをパスツールピペットを用いてゆっくり加えた。これを50 ℃の窒素雰囲気化で83時間反応させた。その後、LAHを濾別し、この時に酢酸エチル、クロロホルム、THFで洗浄した。その後エバポレータにより溶媒を留去し、残留物を飽和食塩水で4回洗浄した。硫酸ナトリウムを用いて乾燥した後エバポレータにより溶媒を留去、粗生成物をオープンカラムクロマトグラフィーにより分離精製した(展開溶媒クロロホルム：酢酸エチル=2：1、後クロロホルム：メタノール=9：1)。得られた生成物を1H NMRにより化合物の同定を行った。（収量1.67 g、収率56.3%）
　1H NMR (CDCl3): δ 0.88 (m, CH3(CH2)6-), δ 1.23 (s, CH3(CH2)6-,-CH2(CH2)5CH2-), δ 1.41 (m, -CH2CH2NH-), δ 1.99 (m, -CH2CH2CH-), δ 2.60 (m, -CH2CH2NH-), δ 5.35 (m, -CH2CHCHCH2-). 1.2.9 Synthesis of Dioleylamine LAH (1.489 g) was slowly added to THF (84.5 mL), and Oleyloleoylamide (2.99 g, 5.61 mmol) dissolved in THF (84.5 mL) was slowly added using a Pasteur pipette. This was reacted in a nitrogen atmosphere at 50 ° C. for 83 hours. Thereafter, LAH was filtered off and washed with ethyl acetate, chloroform and THF at this time. Thereafter, the solvent was distilled off by an evaporator, and the residue was washed 4 times with saturated saline. After drying with sodium sulfate, the solvent was distilled off by an evaporator, and the crude product was separated and purified by open column chromatography (developing solvent chloroform: ethyl acetate = 2: 1, then chloroform: methanol = 9: 1). The obtained product was identified by 1 H NMR. (Yield 1.67 g, Yield 56.3%)
1 H NMR (CDCl 3 ): δ 0.88 (m, CH 3 (CH 2 ) 6- ), δ 1.23 (s, CH 3 (CH 2 ) 6 -,-CH 2 (CH 2 ) 5 CH 2- ), δ 1.41 (m, -CH 2 CH 2 NH-), δ 1.99 (m, -CH 2 CH 2 CH-), δ 2.60 (m, -CH 2 CH 2 NH-), δ 5.35 (m, -CH 2 CHCHCH 2- ).
　１．２．１０　DL-G-0.5-2C 18 -U2の合成
　Dioleylamine 1.32 g (3.45 mmol)をアクリル酸メチル98 mL (1.23 mol)に溶解して、70 ℃の窒素雰囲気化で攪拌した。反応終了後(95 時間)、エバポレータにより未反応のアクリル酸メチルをカラムクロマトグラフィーにより分離精製した(展開溶媒クロロホルム：酢酸エチル＝2：1)。得られた生成物を1H NMR により化合物の同定を行った。（収量1.32 g、収率64.9%）
　1H NMR (CDCl3): δ 0.85 (m, CH3(CH2)6-), δ 1.3 (m, CH3(CH2)6-, -CH2(CH2)5CH2-, -CH2CH2N-), δ 1.99 (m, -CH2CH2CH-), δ 2.40 (m, -CH2CH2NH-, -CH2CH2COO), δ 2.78 (m, -CH2CH2COO), δ 5.35 (m, -CH2CHCHCH2-). 1.2.10 DL-G-0.5-2C 18 -U2 synthetic Dioleylamine 1.32 g of (3.45 mmol) was dissolved in methyl acrylate 98 mL (1.23 mol), was stirred in a nitrogen atmosphere of 70 ° C.. After completion of the reaction (95 hours), unreacted methyl acrylate was separated and purified by column chromatography using an evaporator (developing solvent chloroform: ethyl acetate = 2: 1). The obtained product was identified by 1 H NMR. (Yield 1.32 g, Yield 64.9%)
1 H NMR (CDCl 3 ): δ 0.85 (m, CH 3 (CH 2 ) 6- ), δ 1.3 (m, CH 3 (CH 2 ) 6- , -CH 2 (CH 2 ) 5 CH 2 -,- CH 2 CH 2 N-), δ 1.99 (m, -CH 2 CH 2 CH-), δ 2.40 (m, -CH 2 CH 2 NH-, -CH 2 CH 2 COO), δ 2.78 (m, -CH 2 CH 2 COO), δ 5.35 (m, -CH 2 CHCHCH 2- ).
　１．２．１１　DL-G0-2C 18 -U2の合成
　DL-G-0.5-2C18-U2の1.32 g(2.18 mmol)をメタノール100 mLに溶解し、これをシアン化ナトリウム32.2 mg(0.659 mmol)を含む蒸留精製したエチレンジアミン70 mL(1.05 mol)にパスツールピペットを用いてゆっくりと滴下した。その後、70 ℃の窒素雰囲気化で93.5時間反応させた。反応終了後、エバポレータを用いて溶媒を留去し真空乾燥した。その後、1H NMRにより化合物の同定を行った。（収量1.41 g 未精製）
　1H NMR (CDCl3): δ 0.85 (m, CH3(CH2)6-), δ 1.3 (m, CH3(CH2)6-, -CH2(CH2)5CH2-, -CH2CH2N-), δ 2.0 (m, -CH2CH2CH-), δ 2.3-2.8 (m, -CH2CH2NH-, -CH2CH2COO, -CH2CH2NH2), δ 3.25 (m, -CH2CH2NH2), δ 5.35 (m, -CH2CHCHCH2-), δ 8.55 (s, -CONHCH2-). 1.2.11 DL-G0-2C 18 -U2 synthetic DL-G-0.5-2C 18 -U2 of 1.32 g of (2.18 mmol) was dissolved in methanol 100 mL, which sodium cyanide 32.2 mg (0.659 mmol ) Was slowly added dropwise to 70 mL (1.05 mol) of distilled and purified ethylenediamine using a Pasteur pipette. Then, it was made to react for 93.5 hours by 70 degreeC nitrogen atmosphere formation. After completion of the reaction, the solvent was distilled off using an evaporator and vacuum dried. Thereafter, the compound was identified by 1 H NMR. (Yield 1.41 g unpurified)
1 H NMR (CDCl 3 ): δ 0.85 (m, CH 3 (CH 2 ) 6- ), δ 1.3 (m, CH 3 (CH 2 ) 6- , -CH 2 (CH 2 ) 5 CH 2 -,- CH 2 CH 2 N-), δ 2.0 (m, -CH 2 CH 2 CH-), δ 2.3-2.8 (m, -CH 2 CH 2 NH-, -CH 2 CH 2 COO, -CH 2 CH 2 NH 2 ), δ 3.25 (m, -CH 2 CH 2 NH 2 ), δ 5.35 (m, -CH 2 CHCHCH 2- ), δ 8.55 (s, -CONHCH 2- ).
　１．２．１２　DL-G0.5-2C 18 -U2の合成
　DL-G0-2C18-U2の1.41 mg(2.22 mmol)をメタノール141 mLに溶解した。これをアクリル酸メチル94 mL(1.03 mol)にパスツールピペットを用いてゆっくりと滴下した。その後、45 ℃の窒素雰囲気化で54時間反応させた。反応終了後、エバポレータを用いて溶媒及び未反応のアクリル酸メチルを留去し真空乾燥したのち、得られた粗生成物をオープンカラムクロマトグラフィーにより分離精製した。(展開溶媒ヘキサン：酢酸エチル=10：3、のちクロロホルム：メタノール=4：1)。その後、1H NMRにより化合物の同定を行った。（収量1.74 g、収率99.3%）
　1H NMR (CDCl3): δ 0.85 (m, CH3(CH2)6-), δ 1.3 (m, CH3(CH2)6-, -CH2(CH2)5CH2-, -CH2CH2N-), δ 2.0 (m, -CH2CH2CH-), δ 2.3-2.6 (m, -CH2CH2NH-, -CH2CH2COO, -CH2CH2NH2), δ 2.8 (m, -CH2CH2COO),δ 3.25 (m, -CH2CH2NH2),δ 3.67 (s, -OCH3),  δ 5.35 (m, -CH2CHCHCH2-), δ 8.55 (s, -CONHCH2-). 1.2.12 1.41 mg of DL-G0.5-2C 18 -U2 synthetic DL-G0-2C 18 -U2 a (2.22 mmol) was dissolved in methanol 141 mL. This was slowly added dropwise to 94 mL (1.03 mol) of methyl acrylate using a Pasteur pipette. Then, it was made to react for 54 hours by 45 degreeC nitrogen atmosphere formation. After completion of the reaction, the solvent and unreacted methyl acrylate were distilled off using an evaporator and vacuum-dried, and the resulting crude product was separated and purified by open column chromatography. (Developing solvent hexane: ethyl acetate = 10: 3, then chloroform: methanol = 4: 1). Thereafter, the compound was identified by 1 H NMR. (Yield 1.74 g, 99.3% yield)
1 H NMR (CDCl 3 ): δ 0.85 (m, CH 3 (CH 2 ) 6- ), δ 1.3 (m, CH 3 (CH 2 ) 6- , -CH 2 (CH 2 ) 5 CH 2 -,- CH 2 CH 2 N-), δ 2.0 (m, -CH 2 CH 2 CH-), δ 2.3-2.6 (m, -CH 2 CH 2 NH-, -CH 2 CH 2 COO, -CH 2 CH 2 NH 2 ), δ 2.8 (m, -CH 2 CH 2 COO), δ 3.25 (m, -CH 2 CH 2 NH 2 ), δ 3.67 (s, -OCH 3 ), δ 5.35 (m, -CH 2 CHCHCH 2 -), δ 8.55 (s, -CONHCH 2- ).
　１．２．１３　DL-G1-2C 18 -U2の合成
　DL -G0.5-2C18-U2の1.74 g(2.88 mmol)をメタノール50 mLに溶解し、これをシアン化ナトリウム47.9 mg(0.98 mmol)を含む、蒸留精製したエチレンジアミン92.8 mL(1.39 mol)にパスツールピペットを用いてゆっくり滴下した。その後、50 ℃で72時間、窒素雰囲気化で反応させた。反応終了後、ロータリーエバポレータで溶媒及び未反応のエチレンジアミンを留去し、得られた粗生成物を2日間透析することで精製し、凍結乾燥により黄色のロウ状物質を得た。（収量1.03 g、収率41.5 %）
　1H NMR (CDCl3): δ 0.88 (m, CH3(CH2)6-), δ 1.2-1.4 (m, CH3(CH2)6-, -CH2(CH2)5CH2-, -CH2CH2N-), δ 2.0 (m, -CH2CH2CH-), δ 2.3-2.7 (m, -CH2CH2NH-, -CH2CH2COO), δ 2.6-2.8 (m, -CH2CH2COO, -CH2CH2NH2), δ 3.25 (m,-CH2CH2N-), δ 5.35 (m, -CH2CHCHCH2-), δ 8.55 (s, -CONHCH2-). 1.2.13 DL-G1-2C 18 -U2 synthetic DL -G0.5-2C 18 -U2 of 1.74 g of (2.88 mmol) was dissolved in methanol 50 mL, which sodium cyanide 47.9 mg (0.98 mmol ) Was slowly added dropwise to 92.8 mL (1.39 mol) of distilled and purified ethylenediamine using a Pasteur pipette. Then, it was made to react by nitrogen atmosphere for 72 hours at 50 degreeC. After completion of the reaction, the solvent and unreacted ethylenediamine were distilled off with a rotary evaporator, and the resulting crude product was purified by dialysis for 2 days, and a yellow waxy substance was obtained by lyophilization. (Yield 1.03 g, Yield 41.5%)
1 H NMR (CDCl 3 ): δ 0.88 (m, CH 3 (CH 2 ) 6- ), δ 1.2-1.4 (m, CH 3 (CH 2 ) 6- , -CH 2 (CH 2 ) 5 CH 2- , -CH 2 CH 2 N-), δ 2.0 (m, -CH 2 CH 2 CH-), δ 2.3-2.7 (m, -CH 2 CH 2 NH-, -CH 2 CH 2 COO), δ 2.6- 2.8 (m, -CH 2 CH 2 COO, -CH 2 CH 2 NH 2 ), δ 3.25 (m, -CH 2 CH 2 N-), δ 5.35 (m, -CH 2 CHCHCH 2- ), δ 8.55 ( s, -CONHCH 2- ).
　１．２．１４　MDEG-DL-G1の合成
　DL-G1(100 mg, 0.12 mmol)をジクロロメタン(6 mL)に溶解させ、ジクロロメタン(2 mL)に溶解させたMDEG-4-nitrophenyl carbonate(131 mg, 0.48 mmol)を徐々に加え、窒素雰囲気下、室温で、6日間撹拌した。その後、ジクロロメタンを減圧留去し、シリカゲルクロマトグラフィー(展開溶媒：クロロホルム/メタノール＝9/1, 8/2, v/v)によって精製した。(収量：100 mg、収率：71.7 %)
　1H NMR (CDCl3): δ 0.89(m, CH3(CH2)15-), δ 1.27(s, CH3(CH2)15-), δ1.52(m, -CH2CH2N-),δ 2.39(m, -NCH2CH2CONH-),δ 2.51(m, CH3(CH2)16CH2N-), δ 2.72(m,-NCH2CH2CONH-, -NHCH2CH2N-),δ 3.25(t, -NHCH2CH2NH-), δ 3.32(m, -NHCH2CH2NH-, -NHCH2CH2N-), δ 3.38(s, -OCH3),δ 3.56(t, -CH2OCH3), δ 3.57(t, -CH2CH2OCH3),δ 3.65(t, -CH2OCH2CH2OCH3),δ 4.21(t, -CH2CH2OCH2CH2OCH3),δ 6.04 and 7.45(s, -NH-). 1.2.14 Synthesis of MDEG-DL-G1 MDEG-4-nitrophenyl carbonate (131 mg) in which DL-G1 (100 mg, 0.12 mmol) was dissolved in dichloromethane (6 mL) and dichloromethane (2 mL) was dissolved. , 0.48 mmol) was gradually added, and the mixture was stirred at room temperature for 6 days under a nitrogen atmosphere. Thereafter, dichloromethane was distilled off under reduced pressure, and the residue was purified by silica gel chromatography (developing solvent: chloroform / methanol = 9/1, 8/2, v / v). (Yield: 100 mg, Yield: 71.7%)
1 H NMR (CDCl 3): δ 0.89 (m, CH 3 (CH 2) 15 -), δ 1.27 (s, CH 3 (CH 2) 15 -), δ1.52 (m, -CH 2 CH 2 N -), δ 2.39 (m, -NCH 2 CH 2 CONH-), δ 2.51 (m, CH 3 (CH 2 ) 16 CH 2 N-), δ 2.72 (m, -NCH 2 CH 2 CONH-, -NHCH 2 CH 2 N-), δ 3.25 (t, -NHCH 2 CH 2 NH-), δ 3.32 (m, -NHCH 2 CH 2 NH-, -NHCH 2 CH 2 N-), δ 3.38 (s, -OCH 3 ), δ 3.56 (t, -CH 2 OCH 3 ), δ 3.57 (t, -CH 2 CH 2 OCH 3 ), δ 3.65 (t, -CH 2 OCH 2 CH 2 OCH 3 ), δ 4.21 (t, -CH 2 CH 2 OCH 2 CH 2 OCH 3 ), δ 6.04 and 7.45 (s, -NH-).
　１．２．１５　MDEG-DL-G2の合成
　DL-G2(110 mg, 83.3 μmol)をDMF(6 mL)に溶解させ、DMF (2 mL)に溶解させたMDEG-4-nitrophenyl carbonate(190 mg,666.4 μmol)を徐々に加え、窒素雰囲気下、室温で、6日間撹拌した。その後、DMFを減圧留去し、シリカゲルクロマトグラフィー(展開溶媒：クロロホルム/メタノール＝85/15, 8/2, v/v)によって精製した。(収量：140 mg、収率：88.2 %)
　1H NMR (CDCl3): δ 0.89(m, CH3(CH2)15-), δ 1.27(s, CH3(CH2) 15-), δ 1.44(m, -CH2CH2N-), δ 2.38 and 2.44(m,-NCH2CH2CO-),δ 2.53(t, CH3(CH2)16CH2N-),δ2.73(m, -NHCH2CH2N-, -NCH2CH2CO-), δ 3.29(t, -NHCH2CH2NH-), δ 3.35(t, -NHCH2CH2N-), δ 3.39(s, -OCH3), δ 3.57(t, -NHCH2CH2NH-),δ 3.65(t, -O(CH2)2OCH3),δ 3.69(t, -COOCH2CH2O-),δ 4.22(t, -COOCH2CH2O-),δ 6.12, 7.57and 8.49(m, -NH-). 1.2.15 Synthesis of MDEG-DL-G2 MDEG-4-nitrophenyl carbonate (190 mg) dissolved in DMF (2 mL) was dissolved in DMF (6 mL) DL-G2 (110 mg, 83.3 μmol). , 666.4 μmol) was gradually added, and the mixture was stirred at room temperature for 6 days under a nitrogen atmosphere. Then, DMF was distilled off under reduced pressure and purified by silica gel chromatography (developing solvent: chloroform / methanol = 85/15, 8/2, v / v). (Yield: 140 mg, Yield: 88.2%)
1 H NMR (CDCl 3): δ 0.89 (m, CH 3 (CH 2) 15 -), δ 1.27 (s, CH 3 (CH 2) 15 -), δ 1.44 (m, -CH 2 CH 2 N- ), δ 2.38 and 2.44 (m, -NCH 2 CH 2 CO-), δ 2.53 (t, CH 3 (CH 2 ) 16 CH 2 N-), δ 2.73 (m, -NHCH 2 CH 2 N-, -NCH 2 CH 2 CO-), δ 3.29 (t, -NHCH 2 CH 2 NH-), δ 3.35 (t, -NHCH 2 CH 2 N-), δ 3.39 (s, -OCH 3 ), δ 3.57 ( t, -NHCH 2 CH 2 NH-), δ 3.65 (t, -O (CH 2 ) 2 OCH 3 ), δ 3.69 (t, -COOCH 2 CH 2 O-), δ 4.22 (t, -COOCH 2 CH 2 O-), δ 6.12, 7.57and 8.49 (m, -NH-).
　１．２．１６　MDEG-DL-G1-U2の合成
　DL-G1-U2(400 mg, 0.50 mmol)をDMF(6 mL)に溶解させ、DMF (3 mL)に溶解させたMDEG-4-nitrophenyl carbonate(713mg, 2.50mmol)を徐々に加え、窒素雰囲気下、室温で、6日間撹拌した。その後、DMFを減圧留去し、シリカゲルクロマトグラフィー(展開溶媒：クロロホルム/メタノール＝9/1, v/v)によって精製した。(収量：277mg、収率：48.0%)
　1H NMR (CDCl3): δ 0.88(m, CH3(CH2)7-), δ 1.26and 1.42(t, -CHCH2(CH2)6-),δ2.00(m, -CHCH2-),δ 2.37(m, -NCH2CH2CONH-),δ 2.50(m, CH3(CH2)16CH2N-), δ 2.71(m,-NCH2CH2CONH-, -NHCH2CH2N-),δ 3.22(t, -NHCH2CH2NH-), δ 3.31(m, -NHCH2CH2NH-, -NHCH2CH2N-), δ 3.37(s, -OCH3),δ 3.54(t, -CH2OCH3), δ 3.63(t, -CH2CH2OCH3), δ 3.68(t, -CH2OCH2CH2OCH3), δ 4.20(t, -CH2CH2OCH2CH2OCH3), δ 5.34(m, -(CH)2-),δ 5.95 , 7.36and 8.51(s, -NH-). 1.2.16 Synthesis of MDEG-DL-G1-U2 DL-G1-U2 (400 mg, 0.50 mmol) was dissolved in DMF (6 mL) and MDEG-4-nitrophenyl dissolved in DMF (3 mL) Carbonate (713 mg, 2.50 mmol) was gradually added, and the mixture was stirred at room temperature under a nitrogen atmosphere for 6 days. Then, DMF was distilled off under reduced pressure and purified by silica gel chromatography (developing solvent: chloroform / methanol = 9/1, v / v). (Yield: 277 mg, Yield: 48.0%)
1 H NMR (CDCl 3): δ 0.88 (m, CH 3 (CH 2) 7 -), δ 1.26and 1.42 (t, -CHCH 2 (CH 2) 6 -), δ2.00 (m, -CHCH 2 -), δ 2.37 (m, -NCH 2 CH 2 CONH-), δ 2.50 (m, CH 3 (CH 2 ) 16 CH 2 N-), δ 2.71 (m, -NCH 2 CH 2 CONH-, -NHCH 2 CH 2 N-), δ 3.22 (t, -NHCH 2 CH 2 NH-), δ 3.31 (m, -NHCH 2 CH 2 NH-, -NHCH 2 CH 2 N-), δ 3.37 (s, -OCH 3 ), δ 3.54 (t, -CH 2 OCH 3 ), δ 3.63 (t, -CH 2 CH 2 OCH 3 ), δ 3.68 (t, -CH 2 OCH 2 CH 2 OCH 3 ), δ 4.20 (t, -CH 2 CH 2 OCH 2 CH 2 OCH 3 ), δ 5.34 (m,-(CH) 2- ), δ 5.95, 7.36and 8.51 (s, -NH-).
　１．２．１７　EDEG-DL-G1の合成
　DL-G1(110 mg, 0.13 mmol)をジクロロメタン(6 mL)に溶解させ、ジクロロメタン(3 mL)に溶解させたEDEG-4-nitrophenyl carbonate(228 mg, 0.76 mmol)を徐々に加え、窒素雰囲気下、室温で、7日間撹拌した。その後、ジクロロメタンを減圧留去し、シリカゲルクロマトグラフィー(展開溶媒：クロロホルム/メタノール＝9/1, 8/2, v/v)によって精製した。(収量：100 mg、収率：66.5 %)
　1H NMR (CDCl3): δ 0.87(m, CH3(CH2)15-),δ 1.20(t, -OCH2CH3),δ 1.25 (t, CH3(CH2)15-), δ1.48(m, -CH2CH2N-),δ 2.37(t, -NCH2CH2CONH-),δ 2.50(t, CH3(CH2)16CH2N-), δ 2.70(m,-NCH2CH2CONH-, -NHCH2CH2N-),δ 3.25(t, -NHCH2CH2NH-), δ 3.31(t, -NHCH2CH2NH-),δ 3.35(t,-NHCH2CH2N-),δ 3.53(m, -OCH2CH3),δ 3.58(t, -CH2OCH2CH3), δ 3.62(t, -CH2CH2OCH2CH3), δ 3.68(t, -CH2OCH2CH2OCH2CH3), δ 4.20(t, -CH2CH2OCH2CH2OCH2CH3), δ 6.01and 7.42(s, -NH-). 1.2.17 Synthesis of EDEG-DL-G1 DL-G1 (110 mg, 0.13 mmol) was dissolved in dichloromethane (6 mL) and EDEG-4-nitrophenyl carbonate (228 mg) dissolved in dichloromethane (3 mL). , 0.76 mmol) was gradually added, and the mixture was stirred at room temperature for 7 days under a nitrogen atmosphere. Thereafter, dichloromethane was distilled off under reduced pressure, and the residue was purified by silica gel chromatography (developing solvent: chloroform / methanol = 9/1, 8/2, v / v). (Yield: 100 mg, Yield: 66.5%)
1 H NMR (CDCl 3): δ 0.87 (m, CH 3 (CH 2) 15 -), δ 1.20 (t, -OCH 2 CH 3), δ 1.25 (t, CH 3 (CH 2) 15 -), δ1.48 (m, -CH 2 CH 2 N-), δ 2.37 (t, -NCH 2 CH 2 CONH-), δ 2.50 (t, CH 3 (CH 2 ) 16 CH 2 N-), δ 2.70 ( m, -NCH 2 CH 2 CONH-, -NHCH 2 CH 2 N-), δ 3.25 (t, -NHCH 2 CH 2 NH-), δ 3.31 (t, -NHCH 2 CH 2 NH-), δ 3.35 ( t, -NHCH 2 CH 2 N-), δ 3.53 (m, -OCH 2 CH 3 ), δ 3.58 (t, -CH 2 OCH 2 CH 3 ), δ 3.62 (t, -CH 2 CH 2 OCH 2 CH 3 ), δ 3.68 (t, -CH 2 OCH 2 CH 2 OCH 2 CH 3 ), δ 4.20 (t, -CH 2 CH 2 OCH 2 CH 2 OCH 2 CH 3 ), δ 6.01and 7.42 (s, -NH -).
　１．２．１８　EDEG-DL-G2の合成
　DL-G2(200 mg, 151.5 μmol)をジクロロメタン(10 mL)に溶解させ、ジクロロメタン(3 mL)に溶解させたEDEG-4-nitrophenyl carbonate(544 mg, 1818 μmol)を徐々に加え、窒素雰囲気下、室温で、5日間撹拌した。その後、ジクロロメタンを減圧留去し、シリカゲルクロマトグラフィー(展開溶媒：クロロホルム/メタノール＝9/1, 8/2, v/v)によって精製した。(収量：120 mg、収率：40.4 %)
　1H NMR (CDCl3): δ 0.87(m, CH3(CH2)15-),δ 1.20(t, -OCH2CH3),δ 1.25(t, CH3(CH2)15-),δ 1.44(m, -CH2CH2N-), δ 2.36(m,-NCH2CH2CO-), δ 2.52(t, CH3(CH2)16CH2N-), δ 2.71(m, -NHCH2CH2N-, -NCH2CH2CO-), δ 3.27(t, -NHCH2CH2NH-), δ 3.33(t, -NHCH2CH2N-), δ 3.51(m, -OCH2CH3), δ 3.57(t, -NHCH2CH2NH-), δ 3.62(t, -O(CH2)2OCH2CH3), δ 3.67(t, -COOCH2CH2O-),δ 4.19(t, -COOCH2CH2O-), δ 6.10 and7.50(m, -NH-). 1.2.18 Synthesis of EDEG-DL-G2 DL-G2 (200 mg, 151.5 μmol) was dissolved in dichloromethane (10 mL) and EDEG-4-nitrophenyl carbonate (544 mg dissolved in dichloromethane (3 mL)). , 1818 μmol) was gradually added, and the mixture was stirred at room temperature for 5 days under a nitrogen atmosphere. Thereafter, dichloromethane was distilled off under reduced pressure, and the residue was purified by silica gel chromatography (developing solvent: chloroform / methanol = 9/1, 8/2, v / v). (Yield: 120 mg, Yield: 40.4%)
1 H NMR (CDCl 3): δ 0.87 (m, CH 3 (CH 2) 15 -), δ 1.20 (t, -OCH 2 CH 3), δ 1.25 (t, CH 3 (CH 2) 15 -), δ 1.44 (m, -CH 2 CH 2 N-), δ 2.36 (m, -NCH 2 CH 2 CO-), δ 2.52 (t, CH 3 (CH 2 ) 16 CH 2 N-), δ 2.71 (m , -NHCH 2 CH 2 N-, -NCH 2 CH 2 CO-), δ 3.27 (t, -NHCH 2 CH 2 NH-), δ 3.33 (t, -NHCH 2 CH 2 N-), δ 3.51 (m , -OCH 2 CH 3 ), δ 3.57 (t, -NHCH 2 CH 2 NH-), δ 3.62 (t, -O (CH 2 ) 2 OCH 2 CH 3 ), δ 3.67 (t, -COOCH 2 CH 2 O-), δ 4.19 (t, -COOCH 2 CH 2 O-), δ 6.10 and 7.50 (m, -NH-).
　１．２．１９　EDEG-DL-G1-U2の合成
　DL-G1-U2(400 mg, 0.38 mmol)をDMF(4 mL)に溶解させ、DMF (3 mL)に溶解させたEDEG-4-nitrophenyl carbonate(673 mg, 2.25 mmol)を徐々に加え、窒素雰囲気下、室温で、7日間撹拌した。その後、DMFを減圧留去し、シリカゲルクロマトグラフィー(展開溶媒：クロロホルム/メタノール＝9/1, v/v)によって精製した。(収量：160 mg、収率：36.8 %)
　1H NMR (CDCl3): δ 0.88(m, CH3(CH2)7-),δ 1.20 (t, -OCH2CH3),δ 1.26and 1.42(t, -CHCH2(CH2)6-),δ2.00 (m, -CHCH2-),δ 2.37 (m, -NCH2CH2CONH-),δ 2.50 (m, CH3(CH2)16CH2N-), δ 2.69(m,-NCH2CH2CONH-, -NHCH2CH2N-),δ 3.31(t, -NHCH2CH2NH-), δ 3.35(m, -NHCH2CH2NH-, -NHCH2CH2N-), δ 3.51(m, -OCH2CH3),δ 3.58(t, -CH2OCH2CH3), δ 3.63(t, -CH2CH2OCH2CH3), δ 3.68(t, -CH2OCH2CH2OCH2CH3), δ 4.20(t, -CH2CH2OCH2CH2OCH2CH3), δ 5.34(m, -(CH)2-),δ 5.98, 7.37and 8.49(s, -NH-). 1.2.19 Synthesis of EDEG-DL-G1-U2 DL-G1-U2 (400 mg, 0.38 mmol) was dissolved in DMF (4 mL) and EDEG-4-nitrophenyl dissolved in DMF (3 mL) Carbonate (673 mg, 2.25 mmol) was gradually added, and the mixture was stirred at room temperature for 7 days under a nitrogen atmosphere. Then, DMF was distilled off under reduced pressure and purified by silica gel chromatography (developing solvent: chloroform / methanol = 9/1, v / v). (Yield: 160 mg, Yield: 36.8%)
1 H NMR (CDCl 3): δ 0.88 (m, CH 3 (CH 2) 7 -), δ 1.20 (t, -OCH 2 CH 3), δ 1.26and 1.42 (t, -CHCH 2 (CH 2) 6 -), δ2.00 (m, -CHCH 2- ), δ 2.37 (m, -NCH 2 CH 2 CONH-), δ 2.50 (m, CH 3 (CH 2 ) 16 CH 2 N-), δ 2.69 ( m, -NCH 2 CH 2 CONH-, -NHCH 2 CH 2 N-), δ 3.31 (t, -NHCH 2 CH 2 NH-), δ 3.35 (m, -NHCH 2 CH 2 NH-, -NHCH 2 CH 2 N-), δ 3.51 (m, -OCH 2 CH 3 ), δ 3.58 (t, -CH 2 OCH 2 CH 3 ), δ 3.63 (t, -CH 2 CH 2 OCH 2 CH 3 ), δ 3.68 ( t, -CH 2 OCH 2 CH 2 OCH 2 CH 3 ), δ 4.20 (t, -CH 2 CH 2 OCH 2 CH 2 OCH 2 CH 3 ), δ 5.34 (m,-(CH) 2- ), δ 5.98 , 7.37and 8.49 (s, -NH-).
　１．３　結果
　温度応答性デンドロン脂質の 1 H NMRによるキャラクタリゼーション
　デンドロン脂質は実験項に示した通り、メタノール中において、開始剤であるジオクタデシルアミンにアクリル酸メチルおよびエチレンジアミンを交互に反応させることによって合成した。同定は1H NMRによって行い、DL-G-0.5からDL-G2が合成できていることを確認した。更に、末端アミノ基を修飾したデンドロン脂質の1H NMRスペクトルでは、末端アミノ基に由来するピークが消失し、修飾したOEG基に由来するピークが出現したことにより合成できていることを確認した。このとき、MDEG-DL-Gn (n=1, 2)、MDEG-DL-G1-U2は、1H NMRスペクトルで、メトキシジエチレングリコール基の末端メチル基に由来する3.24ppm付近のピークが出現したことにより合成できていることを確認した。また、EDEG-DL-Gn (n=1, 2)、EDEG-DL-G1-U2は、1H NMRスペクトルで、エトキシジエチレングリコール基の末端エチル基に由来する1.11ppm付近と、3.41ppm付近のピークが出現したことにより合成できていることを確認した。 1.3 Results Characterization of temperature-responsive dendron lipids by 1 H NMR As shown in the experimental section, dendron lipids were obtained by reacting dioctadecylamine as an initiator with methyl acrylate and ethylenediamine alternately in methanol. Synthesized. Identification was performed by 1 H NMR, and it was confirmed that DL-G2 was synthesized from DL-G-0.5. Furthermore, in the 1 H NMR spectrum of the dendron lipid modified with the terminal amino group, it was confirmed that the peak derived from the terminal amino group disappeared, and that the peak derived from the modified OEG group appeared, so that synthesis was possible. At this time, MDEG-DL-Gn (n = 1, 2) and MDEG-DL-G1-U2 had a peak near 3.24 ppm derived from the terminal methyl group of the methoxydiethylene glycol group in the 1 H NMR spectrum. It was confirmed that the synthesis was possible. EDEG-DL-Gn (n = 1, 2) and EDEG-DL-G1-U2 are 1 H NMR spectra with peaks near 1.11 ppm derived from the terminal ethyl group of the ethoxydiethylene glycol group and around 3.41 ppm. It was confirmed that it was synthesized by the appearance of.
　MDEG-DL-Gn (n=1, 2)、MDEG-DL-G1-U2については1H NMRにおいて、オクタデシル基末端のメチル基に由来する0.88ppm付近のピーク、又はオレイル鎖末端のメチル基に由来する0.96ppm付近のピークの積分比と、導入されたメトキシジエチレングリコール基の末端メチル基に由来する3.24 ppm付近のピークの積分比から、デンドリマー脂質に導入されたメトキシジエチレングリコール基の数を算出した。その結果、MDEG-DL-G1とMDEG-DL-G2とMDEG-DL-G1-U2の末端には、それぞれ、2.0、4.0、2.0個のメトキシジエチレングリコール基が導入されたことがわかった。 For MDEG-DL-Gn (n = 1, 2) and MDEG-DL-G1-U2, in 1 H NMR, the peak near 0.88 ppm derived from the methyl group at the end of the octadecyl group, or the methyl group at the end of the oleyl chain The number of methoxydiethylene glycol groups introduced into the dendrimer lipid was calculated from the integral ratio of the peak around 0.96 ppm derived from the integral ratio of the peak around 3.24 ppm derived from the terminal methyl group of the introduced methoxydiethylene glycol group. As a result, it was found that 2.0, 4.0, and 2.0 methoxydiethylene glycol groups were introduced at the ends of MDEG-DL-G1, MDEG-DL-G2, and MDEG-DL-G1-U2, respectively.
　EDEG-DL-Gn (n=1, 2)、EDEG-DL-G1-U2については1H NMRにおいて、オクタデシル基末端のメチル基に由来する0.88 ppm付近のピーク、又はオレイル鎖末端のメチル基に由来する0.96ppm付近のピークの積分比と、導入されたエトキシジエチレングリコール基の末端エチル基に由来する1.11ppm付近と、3.41ppm付近のピークの積分比から、デンドリマー脂質に導入されたエトキシジエチレングリコール基の数を算出した。その結果、EDEG-DL-G1とEDEG-DL-G2とEDEG-DL-G1-U2の末端には、それぞれ、2.0、4.0、2.4個のメトキシジエチレングリコール基が導入されたことがわかった。 For EDEG-DL-Gn (n = 1, 2) and EDEG-DL-G1-U2, in 1 H NMR, the peak near 0.88 ppm derived from the methyl group at the end of the octadecyl group or the methyl group at the end of the oleyl chain From the integration ratio of the peak near 0.96 ppm derived from, the integration ratio of the peak near 1.11 ppm derived from the terminal ethyl group of the introduced ethoxydiethylene glycol group, and the peak near 3.41 ppm, the ethoxydiethylene glycol group introduced into the dendrimer lipid Numbers were calculated. As a result, 2.0, 4.0, and 2.4 methoxydiethylene glycol groups were introduced at the ends of EDEG-DL-G1, EDEG-DL-G2, and EDEG-DL-G1-U2, respectively.
　以上の通り、2本のアルキル鎖を持つ第1、第2世代までのPAMAMデンドロン脂質と、2本のオレイル鎖を持つ第1世代のPAMAMデンドロン脂質を合成した。それらの末端アミノ基に、温度応答性を持つ種々のOEG基を導入することによって、第1世代、第2世代で種々のOEG基結合デンドロン脂質であるMDEG-DL-G1、EDEG-DL-G1、MDEG-DL-G2、EDEG-DL-G2、MDEG-DL-G1-U2、EDEG-DL-G1-U2を合成した。 As described above, the first and second generation PAMAM dendron lipids having two alkyl chains and the first generation PAMAM dendron lipids having two oleyl chains were synthesized. MDEG-DL-G1 and EDEG-DL-G1, which are various OEG group-binding dendron lipids in the first and second generation, by introducing various OEG groups with temperature responsiveness to their terminal amino groups MDEG-DL-G2, EDEG-DL-G2, MDEG-DL-G1-U2, and EDEG-DL-G1-U2 were synthesized.
　前述したように、デンドロン脂質は両親媒性分子であるため、水中に分散することで分子集合体を形成し、それによって親水性領域へ導入した温度応答性基が集合体表面へ集積すると考えられる。そして、その集積した温度応答性基間の相互作用により、脂質分子集合体が温度応答性を発現すると期待できる。そこで、次に、合成した温度応答性デンドロン脂質の分散液を用いて、脂質分子集合体の温度応答性について検討した。 As mentioned above, since dendron lipids are amphipathic molecules, it is thought that when they are dispersed in water, they form molecular aggregates, which causes temperature-responsive groups introduced into the hydrophilic region to accumulate on the aggregate surface. . And, it can be expected that the lipid molecule aggregate exhibits temperature responsiveness due to the interaction between the accumulated temperature responsive groups. Then, next, the temperature responsiveness of the lipid molecule assembly was examined using the synthesized temperature-responsive dendron lipid dispersion.
　２．オリゴエチレングリコール鎖を導入したポリアミドアミンデンドロン脂質からなる集合体の温度応答性の評価
　合成された種々のデンドロン脂質は両親媒性分子であるため、水中に分散すると疎水性相互作用により自己組織化し、ミセルやベシクルといった分子集合体を形成すると考えられる。このとき、脂質の親水性部位末端に導入された種々のOEG基は、脂質分子集合体表面へ高密度に集積化されるため、脂質分子集合体が温度応答性を発現することが期待できる。そこでこの温度応答性デンドロン脂質を水中に分散させることで集合体を形成させ、その集合体の温度応答性及びその形態について評価を行った。 2. Evaluation of temperature responsiveness of aggregates composed of polyamidoamine dendron lipids with oligoethylene glycol chains introducedSince various synthesized dendron lipids are amphiphilic molecules, they are self-assembled by hydrophobic interaction when dispersed in water, It is thought to form molecular aggregates such as micelles and vesicles. At this time, since various OEG groups introduced at the end of the hydrophilic site of the lipid are accumulated at a high density on the surface of the lipid molecule assembly, the lipid molecule assembly can be expected to express temperature response. Thus, an aggregate was formed by dispersing the temperature-responsive dendron lipid in water, and the temperature responsiveness and form of the aggregate were evaluated.
　２．１　試薬
　リン酸水素二ナトリウム(12水和物)(Na2HPO4・12H2O)、リン酸二水素ナトリウム(2水和物) (Na2HPO4・2H2O)、はキシダ化学から購入した。水酸化ナトリウムは和光純薬工業株式会社から購入した。 2.1 Reagents Disodium hydrogen phosphate (12 hydrate) (Na 2 HPO 4 · 12H 2 O), Sodium dihydrogen phosphate (dihydrate) (Na 2 HPO 4 · 2H 2 O), Purchased from chemistry. Sodium hydroxide was purchased from Wako Pure Chemical Industries.
　２．２　脂質分散液の調製
　各々の脂質のクロロホルム溶液からロータリーエバポレーターを用いて溶媒を除去して、脂質薄膜を形成させた。これに10 mMリン酸緩衝溶液を加え、pH 3.0、濃度2 mg/mlに調整した。これにバス型超音波照射装置を用いて超音波を45℃で10分間照射した後、45℃で20分間静置した。その後、氷冷下において60分以上静置した。 2.2 Preparation of lipid dispersion The solvent was removed from each lipid chloroform solution using a rotary evaporator to form a lipid thin film. A 10 mM phosphate buffer solution was added thereto to adjust the pH to 3.0 and a concentration of 2 mg / ml. This was irradiated with ultrasonic waves at 45 ° C. for 10 minutes using a bath-type ultrasonic irradiation apparatus, and then allowed to stand at 45 ° C. for 20 minutes. Then, it was left still for 60 minutes or more under ice cooling.
　２．３　透過率測定による温度応答性の評価
　種々のpHの10 mM リン酸緩衝水溶液(140 mM NaCl)1.80mlに、上記で調製したpH 3.0の脂質分散液0.20mlを加え(0.2mg/ml)、10℃で30分間、撹拌及び保持した後、測定を行った。
測定は脂質分散液を10℃から昇温速度2℃/minで加熱し、そのときの透過率の変化を測定した。測定には日本分光製V-560型紫外・可視分光光度計を用いた(測定波長500 nm)。温度制御はETC-505Tを用いて行った。このとき透過率が下がり始める温度を曇点とした。測定後、室温にて溶液のpHを測定し、その値を濁度測定時のpHとした。 2.3 Evaluation of temperature responsiveness by permeability measurement To 1.80 ml of 10 mM phosphate buffer aqueous solution (140 mM NaCl) at various pHs, 0.20 ml of the pH 3.0 lipid dispersion prepared above was added (0.2 mg / ml). ), Stirred and held at 10 ° C. for 30 minutes, and then measured.
In the measurement, the lipid dispersion was heated from 10 ° C. at a heating rate of 2 ° C./min, and the change in transmittance at that time was measured. For measurement, a V-560 ultraviolet / visible spectrophotometer manufactured by JASCO Corporation was used (measurement wavelength: 500 nm). Temperature control was performed using ETC-505T. At this time, the temperature at which the transmittance began to drop was taken as the cloud point. After the measurement, the pH of the solution was measured at room temperature, and the value was taken as the pH at the time of turbidity measurement.
　２．４　示差走査熱量測定(Differential scanning calorimetry)(DSC)による温度応答時の吸熱評価
　10 mM リン酸緩衝水溶液(140 mM NaCl)630μlに、2.2.で調製したpH 3.0の脂質分散液70μlを加え(0.2mg/ml)、pH 7.4に調節した後、10℃で30分間保持した後、測定を行った。
測定はpH 7.4の10 mM リン酸緩衝水溶液(140 mM NaCl)と脂質分散液をそれぞれ600 μl用いて、昇温速度1℃/minで加熱し測定した。測定にはTA Instruments Japan Inc.製ナノDSCを用いた。 2.4 Endothermic evaluation during temperature response by differential scanning calorimetry (DSC ) To 70 mL of 10 mM phosphate buffer aqueous solution (140 mM NaCl), pH 3.0 lipid dispersion prepared in 2.2. (0.2 mg / ml), adjusted to pH 7.4, held at 10 ° C. for 30 minutes, and then measured.
The measurement was carried out using 600 μl each of a 10 mM phosphate buffer aqueous solution (140 mM NaCl) having a pH of 7.4 and a lipid dispersion at a heating rate of 1 ° C./min. Nano DSC manufactured by TA Instruments Japan Inc. was used for the measurement.
　２．５　位相差顕微鏡による分子集合体の形態観測
10 mM リン酸緩衝水溶液(140 mM NaCl) 0.90 mlに、2.2.で調製したpH 3.0のEDEG-DL-G1分散液0.10 mlを加え(0.2mg/ml)、pH 7.4に調節し、10℃で30分間、撹拌及び保持した後、観察を行った。また、40℃で1分以上湯浴した後、観察を行った。 2.5 Morphological observation of molecular assembly by phase contrast microscope
To 0.90 ml of 10 mM phosphate buffer aqueous solution (140 mM NaCl), add 0.10 ml of pH 3.0 EDEG-DL-G1 dispersion prepared in 2.2 (0.2 mg / ml), adjust to pH 7.4, and Observation was performed after stirring and holding for 30 minutes. In addition, observation was performed after bathing at 40 ° C. for 1 minute or longer.
　２．６　結果
　２．６．１　種々のデンドロン脂質集合体の温度応答性の評価
　MDEG-DL-G1、EDEG-DL-G1、MDEG-DL-G2、EDEG-DL-G2、MDEG-DL-G1-U2、EDEG-DL-G1-U2について、それぞれの脂質分散液(pH 7.4)を昇温させたときの透過率の変化によって温度応答性の評価を行った。そのときの結果を図22に示した。MDEG-DL-G1、EDEG-DL-G1について、低温時(10 ℃)の時点で、すでに透過率が低下していた。そして、分散液の温度を上昇させていくと、MDEG-DL-G1は32℃付近において、EDEG-DL-G1は22℃付近において、透過率の減少が見られた。また、MDEG-DL-G2、EDEG-DL-G2、MDEG-DL-G1-U2、EDEG-DL-G1-U2分散液についても、分散液の温度を上昇させていくと、MDEG-DL-G2は68 ℃付近において、EDEG-DL-G2は32 ℃付近において、MDEG-DL-G1-U2は44℃付近において、EDEG-DL-G1-U2は24 ℃付近において、透過率の減少が見られた。これらの透過率の減少は、分子集合体が不安定化し、凝集したことを示している。ここで、分散液の透過率が下がり始める温度を曇点(Cloud Point)と定義すると、MDEG-DL-G1、EDEG-DL-G1、MDEG-DL-G2、EDEG-DL-G2、MDEG-DL-G1-U2、EDEG-DL-G1-U2の曇点は、それぞれ、34.2 ℃、22.5 ℃、67.6 ℃、34.2 ℃、45.4℃、26.1 ℃となった。以上のことから、種々のOEG基を分子集合体表面へ集積化させることにより温度応答性を付与できることが示された。また、それぞれの曇天温度を比較すると、末端基がより疎水的になるほど、曇点温度は下がり、世代数が高くなると、曇点温度は上がった。これは、疎水性が高まると、温度応答による脱水での疎水性相互作用による凝集が起こりやすくなるためであると考えられる。また、世代数が高まると、第三級アミンの数も増え、より親水的になるために、高温側での温度応答となったと考えられる。加えて、アルキル鎖とオレイル鎖での時では、オレイル鎖を持つデンドロン脂質の分子集合体の方が、曇点は高温側へシフトした。これは、二重結合を持つことで、分子集合体が作り出す脂質二重層がより流動的になり、安定性を高めているためであると考えられる。 2.6 Results 2.6.1 Evaluation of temperature responsiveness of various dendron lipid assemblies MDEG-DL-G1, EDEG-DL-G1, MDEG-DL-G2, EDEG-DL-G2, MDEG-DL-G1 For -U2 and EDEG-DL-G1-U2, the temperature responsiveness was evaluated by the change in transmittance when each lipid dispersion (pH 7.4) was heated. The results at that time are shown in FIG. Regarding MDEG-DL-G1 and EDEG-DL-G1, the transmittance had already decreased at the time of low temperature (10 ° C.). As the temperature of the dispersion was increased, the transmittance decreased at around 32 ° C for MDEG-DL-G1 and around 22 ° C for EDEG-DL-G1. For MDEG-DL-G2, EDEG-DL-G2, MDEG-DL-G1-U2, and EDEG-DL-G1-U2 dispersions, the MDEG-DL-G2 Decrease in transmittance at around 68 ° C, EDEG-DL-G2 at around 32 ° C, MDEG-DL-G1-U2 at around 44 ° C, and EDEG-DL-G1-U2 at around 24 ° C. It was. These reductions in transmittance indicate that the molecular assembly has become unstable and aggregated. Here, when the temperature at which the transmittance of the dispersion begins to decrease is defined as the cloud point (Cloud Point), MDEG-DL-G1, EDEG-DL-G1, MDEG-DL-G2, EDEG-DL-G2, MDEG-DL The cloud points of -G1-U2 and EDEG-DL-G1-U2 were 34.2 ° C, 22.5 ° C, 67.6 ° C, 34.2 ° C, 45.4 ° C and 26.1 ° C, respectively. From the above, it was shown that temperature responsiveness can be imparted by integrating various OEG groups on the surface of the molecular assembly. Further, when the cloudy temperatures were compared, the cloud point temperature decreased as the end group became more hydrophobic, and the cloud point temperature increased as the generation number increased. This is considered to be because when the hydrophobicity is increased, aggregation due to hydrophobic interaction is likely to occur during dehydration due to temperature response. In addition, as the number of generations increases, the number of tertiary amines also increases and becomes more hydrophilic, so it is considered that the temperature response on the high temperature side was achieved. In addition, in the case of an alkyl chain and an oleyl chain, the cloud point of the molecular assembly of dendron lipids having an oleyl chain shifted to a higher temperature side. This is presumably because the lipid bilayer created by the molecular assembly is more fluid and has improved stability due to having a double bond.
　また、種々のpHにおいてEDEG-DL-G2の分散液の透過率の変化を測定することで、温度応答性のpH依存性についての評価を行った。そのときの結果の一部を図23に、また、pHに対して曇天をプロットした結果を図24に示した。分散液のpHの低下に伴い、曇点の上昇が見られた。これは、pHの低下に伴い、極性基における三級アミノ基のプロトン化の度合いが大きくなり、分子の親水性が高くなったため、また、電荷を帯びることで分子内や分子間で静電反発が起き、種々のOEG基の表面密度が低下したため、より高温に曇点を示したと考えられる。また、pHが高くなりすぎた時は、曇点が下がり続けるわけではなく、ある一定の温度に曇点を示すようになった。これは、極性基における三級アミノ基のプロトン化がされなくなったためだと、考えられる。 In addition, the pH dependence of the temperature response was evaluated by measuring the change in the transmittance of the dispersion of EDEG-DL-G2 at various pHs. FIG. 23 shows a part of the result at that time, and FIG. 24 shows the result of plotting the cloudiness against pH. As the pH of the dispersion decreased, the cloud point increased. This is because as the pH decreases, the degree of protonation of tertiary amino groups in polar groups increases and the hydrophilicity of the molecules increases, and electrostatic repulsion occurs between molecules and between molecules due to charge. Since the surface density of various OEG groups decreased, it was thought that cloud points were shown at higher temperatures. Further, when the pH became too high, the cloud point did not continue to decrease, but the cloud point was exhibited at a certain temperature. This is probably because the tertiary amino group in the polar group is no longer protonated.
　２．６．２　種々のデンドロン脂質分子集合体の温度応答時における吸熱評価
　MDEG-DL-G1、EDEG-DL-G1、MDEG-DL-G2、EDEG-DL-G2、MDEG-DL-G1-U2、EDEG-DL-G1-U2について、それぞれの脂質分散液(pH 7.4)を昇温させたときの吸熱エネルギーによって温度応答時の吸熱評価を行った。そのときの結果を表２に、温度変化における吸熱ピークを図２５及び２６に示した。 2.6.2 Endothermic evaluation of various dendron lipid molecular assemblies during temperature response MDEG-DL-G1, EDEG-DL-G1, MDEG-DL-G2, EDEG-DL-G2, MDEG-DL-G1-U2 , EDEG-DL-G1-U2 was subjected to endothermic evaluation during temperature response by endothermic energy when each lipid dispersion (pH 7.4) was heated. The results at that time are shown in Table 2, and the endothermic peaks in the temperature change are shown in FIGS.
　MDEG-DL-G1、EDEG-DL-G1について、約40 ℃付近に示す大きな吸熱ピークがみられた。
このピーク温度は、これらの曇点温度とは異なっていることから、温度応答時の脱水和による吸熱ではないことが分かった。しかし、組成の異なるデンドロン脂質を扱っているのに関わらず、ピーク温度がほぼ同じとなったのは、これらの分子集合体の疎水部としてアルキル鎖が集まってゲルとなしている部分が、液晶状態へと転移したことからの、吸熱だと考えられる。また、この時の吸熱ピークの温度は、EDEG-DL-G1の方が低温で、吸熱の熱量は低かったことから、分子集合体の疎水性度が高まることによって、応答温度領域もわずかに低温側にシフトし、脱水和した時の吸熱した熱量も低くなったのだと考えられる。 For MDEG-DL-G1 and EDEG-DL-G1, a large endothermic peak at around 40 ° C. was observed.
Since this peak temperature was different from these cloud point temperatures, it was found that the endotherm was not an endotherm due to dehydration during temperature response. However, regardless of handling dendron lipids with different compositions, the peak temperatures were almost the same because the portion of these molecular aggregates in which the alkyl chains gathered as the hydrophobic part of the molecular assembly is a liquid crystal. It is thought to be endothermic from the transition to a state. In addition, the temperature of the endothermic peak at this time was lower in EDEG-DL-G1 and the heat quantity of the endotherm was lower, so the hydrophobicity of the molecular assembly increased, resulting in a slightly lower response temperature range. It is thought that the amount of heat absorbed when shifted to the side and dehydrated also decreased.
　さらに、MDEG-DL-G2、EDEG-DL-G2においては、第一世代と同じように、40 ℃付近に吸熱ピークが見られると予想していたが、ピークが見られなかった。これは、世代数が高くなることにより、第三級アミンの数が増え、デンドロン脂質の分子集合体の親水性が高まり、親疎水性バランスが向上することで、アルキル鎖の疎水部がゲル状とならずに分子集合体を形成したことに起因していると考えられる。しかし、MDEG-DL-G2、EDEG-DL-G2においては、曇点付近温度でわずかな吸熱ピークが見られた。これは、デンドロン脂質の分子集合体の温度応答時の、脱水和における吸熱ピークだと考える。これより、第一世代では十分に水和していなかったが、第二世代となることで親水性が高まり、より水和したことで、わずかではあるがピークがみえるほどの脱水和が起こったためであると考えられる。 Furthermore, in MDEG-DL-G2 and EDEG-DL-G2, it was expected that an endothermic peak was observed around 40 ° C. as in the first generation, but no peak was observed. As the number of generations increases, the number of tertiary amines increases, the hydrophilicity of the dendron lipid molecular assembly increases, and the hydrophilicity / hydrophobicity balance improves, so that the hydrophobic part of the alkyl chain is gelled. It is thought that this is due to the formation of a molecular assembly. However, in MDEG-DL-G2 and EDEG-DL-G2, a slight endothermic peak was observed at a temperature near the cloud point. This is thought to be an endothermic peak in dehydration during the temperature response of dendron lipid molecular aggregates. From this, it was not fully hydrated in the first generation, but hydrophilicity increased by becoming the second generation, and dehydration to the extent that a slight peak could be seen by more hydration. It is thought that.
　また、MDEG-DL-G1-U2、EDEG-DL-G1-U2においては、大きな吸熱ピークがみられなかったが、35 ℃付近に小さな吸熱ピークが見られた。これは、アルキル鎖を持つデンドロン脂質の時には示していた約40 ℃付近のピークが、アルキル鎖からオレイル鎖に変わることによって、少し低温側にシフトし、吸熱の熱量が小さくなったと考えられる。これは、分子集合体の疎水部がアルキル鎖によるものでなく、オレイル鎖になることで流動性が高まり、ゲルと液晶状態となっているため、ゲルから液晶への転移が少なくなったためだと考えられる。 In MDEG-DL-G1-U2 and EDEG-DL-G1-U2, no large endothermic peak was observed, but a small endothermic peak was observed at around 35 ° C. This is thought to be due to the fact that the peak around 40 ° C., which was shown for dendron lipids with alkyl chains, was shifted to a lower temperature side by changing from alkyl chains to oleyl chains, and the endothermic heat was reduced. This is because the hydrophobic part of the molecular assembly is not due to an alkyl chain, but because it becomes an oleyl chain, the fluidity is increased and it is in a gel and liquid crystal state, so the transition from gel to liquid crystal is reduced. Conceivable.
Figure JPOXMLDOC01-appb-T000031
Figure JPOXMLDOC01-appb-T000031
　２．６．３　EDEG-DL-G1分子集合体の形態評価
　まず、目視によってEDEG-DL-G1分子集合体(pH 7.4)の様子を観察した結果を図27に、さらに、位相差顕微鏡を用いて形態観測を行った結果を図28に示す。曇点以下の10 ℃の時、EDEG-DL-G1の分散液は、わずかに濁っているのが観察された。また、曇点以上の40 ℃の時は、白い凝集体が観察された。 2.6.3 Morphological evaluation of EDEG-DL-G1 molecular assembly First, the results of visual observation of the state of EDEG-DL-G1 molecular assembly (pH 7.4) are shown in FIG. 27 and a phase contrast microscope. Fig. 28 shows the result of morphological observation. At 10 ° C. below the cloud point, the dispersion of EDEG-DL-G1 was observed to be slightly cloudy. When the temperature was 40 ° C. above the cloud point, white aggregates were observed.
　次に、これらの状況時においての顕微鏡観察では、10 ℃の時、EDEG-DL-G1の分子集合体が水和した状態で少し凝集しているのが観察された。また、40 ℃になると、分子集合体は強く凝集され、巨大な凝集体を形成していた。 Next, in the microscopic observation under these conditions, it was observed that the molecular aggregate of EDEG-DL-G1 was slightly agglomerated in a hydrated state at 10 ° C. When the temperature reached 40 ° C., the molecular assembly was strongly aggregated to form a huge aggregate.
　これらは、低温時においては、EDEG-DL-G1の分子集合体は水和している状態であるが、親疎水性バランスが悪いため、疎水性相互作用によって、少し凝集していると考えられる。また、加熱をして曇点温度を超えると、温度応答を示し、分子集合体が脱水和されることにより、強烈な疎水性相互作用が働き、巨大な凝集体を形成したと考えられる。 These are considered to be slightly aggregated due to hydrophobic interaction because the molecular assembly of EDEG-DL-G1 is in a hydrated state at low temperatures, but the hydrophilicity / hydrophobicity balance is poor. Further, when the temperature exceeds the cloud point by heating, a temperature response is exhibited, and the molecular aggregate is dehydrated, thereby causing a strong hydrophobic interaction and forming a huge aggregate.
　以上の通り、種々のOEG基を導入したデンドロン脂質分子の集合体の温度応答性評価と分子集合体の形態評価を行った。 As described above, the temperature responsiveness evaluation of the dendron lipid molecule assembly into which various OEG groups were introduced and the morphology evaluation of the molecular assembly were performed.
　デンドロン脂質の集合体の温度応答性評価は、脂質分散液の透過率測定によって行った。種々のOEG基末端を有するデンドロン脂質の分散液の温度を上昇させていくと、ある温度以上で透過率の減少を示した。このことから、分子集合体が凝集したことが示された。
つまり、種々のOEG基を分子集合体表面へ集積化させることにより、集合体へ温度応答性を付与できることがわかった。また、pHの低下に伴い、曇点は上昇した。これは、デンドロン脂質内部の第三級アミンのプロトン化が促進され、分子集合体がより水和したためであると考えられる。 The temperature response of the dendron lipid aggregate was evaluated by measuring the permeability of the lipid dispersion. When the temperature of the dispersion of dendron lipids having various OEG terminal ends was increased, the transmittance decreased at a certain temperature or higher. This showed that the molecular assembly was aggregated.
In other words, it was found that temperature responsiveness can be imparted to the aggregate by integrating various OEG groups on the molecular aggregate surface. Further, the cloud point increased with a decrease in pH. This is considered to be because the protonation of the tertiary amine inside the dendron lipid was promoted and the molecular assembly became more hydrated.
　また、EDEG-DL-G1分子集合体(pH 7.4)の様子を観察した結果、10 ℃の時、分子集合体が水和した状態で少し凝集しているのが観察され、40 ℃になると、分子集合体は強く凝集され、巨大な凝集体を形成していた。つまり、このEDEG-DL-G1分子集合体は、低温時において、水和している状態であるが、親疎水性バランスが悪いため、疎水性相互作用によって、少し凝集していると考えられる。また、曇点以上では、温度応答を示し、分子集合体が脱水和されることにより、強烈な疎水性相互作用が働き、巨大な凝集体を形成したと考えられる。 Also, as a result of observing the state of the EDEG-DL-G1 molecular assembly (pH 7.4), it was observed that the molecular assembly was slightly agglomerated in a hydrated state at 10 ℃, and when it reached 40 ℃, The molecular assembly was strongly aggregated to form a huge aggregate. That is, this EDEG-DL-G1 molecular assembly is in a hydrated state at a low temperature, but has a poor hydrophilicity / hydrophobicity balance. Above the cloud point, it shows a temperature response, and the molecular aggregate is dehydrated, so that it is considered that a strong hydrophobic interaction worked to form a huge aggregate.
　以上より、合成した種々のデンドロン脂質の性質が評価された。これらの末端にOEG鎖を有するデンドロン脂質からなる分子集合体は、特定の温度領域において温度応答を示すことが分かった。しかし、EDEG-DL-G1において、DDSでのキャリアへの応用を考えた時、デンドロン脂質ベシクルの、コロイドとしての安定性の不足が問題となる。そこで、このデンドロン脂質ベシクルのコロイドとしての安定性を向上させるために、PEG脂質を導入することで、ベシクルの安定性を向上させ、PEG鎖によるさらなる生体適合性の付与を目指した。次に、PEG脂質を導入することで、温度応答性と分子集合体にどのような影響を与えるかについて調べた。 From the above, the properties of various synthesized dendron lipids were evaluated. It was found that molecular assemblies composed of dendron lipids having an OEG chain at their ends showed a temperature response in a specific temperature range. However, in EDEG-DL-G1, when considering application to carriers in DDS, the lack of stability as a colloid of dendron lipid vesicles becomes a problem. Therefore, in order to improve the stability of the dendron lipid vesicle as a colloid, we aimed to improve the stability of the vesicle by introducing PEG lipid and to impart further biocompatibility with the PEG chain. Next, we investigated the effects of introducing PEG lipids on temperature responsiveness and molecular assembly.
　３．　ポリエチレングリコール脂質導入によるオリゴエチレングリコール鎖を末端に有するデンドロン脂質集合体の温度応答性機能評価
　前述の実験において温度応答性デンドロン脂質を構築はできたが、温度応答を示すよりも、低温の時から、すでにコロイドとして不安定な状態であった。この温度応答性デンドロン脂質をDDSへ応用するためには、よりコロイドとしての安定性を高めながら温度応答性を発現させることが重要となる。そこで、より生理的条件に近い環境におけるコロイドとしての安定性や、生体適合性を向上させるために、PEG脂質をベシクルに導入することを試みた。そこで、EDEG-DL-G1にPEG脂質を組み合わせた分子集合体の温度応答性や形態を調べることで、温度応答機能と形態に与えるPEG脂質の影響について検討し、評価した。 3. Evaluation of temperature-responsive functionalities of dendron lipid aggregates with oligoethylene glycol chains at the end by introduction of polyethylene glycol lipids Although the temperature-responsive dendron lipids were constructed in the previous experiment, the temperature response was lower than the temperature response. It was already unstable as a colloid. In order to apply this temperature-responsive dendron lipid to DDS, it is important to develop temperature-responsiveness while further improving the stability as a colloid. Therefore, we tried to introduce PEG lipid into vesicles in order to improve the stability as a colloid in an environment closer to physiological conditions and the biocompatibility. Therefore, we investigated and evaluated the effects of PEG lipids on temperature response function and morphology by examining the temperature response and morphology of molecular assemblies combining PEG lipids with EDEG-DL-G1.
　３．１　試薬
　リン酸水素二ナトリウム(12水和物)(Na2HPO4・12H2O)、リン酸二水素ナトリウム(2水和物)(NaH2PO4・2H2O)、塩酸、はキシダ化学から購入した。水酸化ナトリウムは和光純薬工業株式会社から購入した。CS-010(PEG脂質)は日油から購入した。 3.1 Reagents Disodium hydrogen phosphate (12 hydrate) (Na2HPO4 · 12H2O), Sodium dihydrogen phosphate (dihydrate) (NaH2PO4 · 2H2O), hydrochloric acid, were purchased from Kishida Chemical. Sodium hydroxide was purchased from Wako Pure Chemical Industries. CS-010 (PEG lipid) was purchased from NOF.
　３．２　デンドロン脂質分散液の調製
　EDEG-DL-G1とPEG脂質を種々の比で混合したクロロホルム溶液からロータリーエバポレーターを用いて溶媒を除去して、脂質薄膜を形成させた。これに10 mMリン酸緩衝溶液を加え、pH 3.0、濃度2 mg/mlに調整した。これにバス型超音波照射装置を用いて超音波を45℃で10分間照射した後、45℃で20分間静置した。その後、氷冷下において60分以上静置した。 3.2 Preparation of dendron lipid dispersion The solvent was removed from a chloroform solution in which EDEG-DL-G1 and PEG lipid were mixed at various ratios using a rotary evaporator to form a lipid thin film. A 10 mM phosphate buffer solution was added thereto to adjust the pH to 3.0 and a concentration of 2 mg / ml. This was irradiated with ultrasonic waves at 45 ° C. for 10 minutes using a bath-type ultrasonic irradiation apparatus, and then allowed to stand at 45 ° C. for 20 minutes. Then, it was left still for 60 minutes or more under ice cooling.
　３．３　透過率測定による温度応答性の評価
　種々のpHの10 mM リン酸緩衝水溶液(140 mM NaCl)1.80mlに、上記で調製したpH 3.0の脂質分散液0.20mlを加え(0.2mg/ml)、10℃で30分間保持した後、測定を行った。測定は脂質分散液を10℃から昇温速度2 ℃/minで加熱し、そのときの透過率の変化を測定した。測定には日本分光製V-630型紫外・可視分光光度計を用いた(測定波長500 nm)。 3.3 Evaluation of Temperature Response by Permeability Measurement To 1.80 ml of 10 mM phosphate buffer aqueous solution (140 mM NaCl) at various pHs, 0.20 ml of the pH 3.0 lipid dispersion prepared above was added (0.2 mg / ml). ), Held at 10 ° C. for 30 minutes, and then measured. In the measurement, the lipid dispersion was heated from 10 ° C. at a heating rate of 2 ° C./min, and the change in transmittance at that time was measured. For measurement, a V-630 ultraviolet / visible spectrophotometer manufactured by JASCO Corporation was used (measurement wavelength: 500 nm).
　温度制御はETC-717を用いて行った。このとき透過率が急激に下がる温度を曇点とした。測定後、室温にて溶液のpHを測定し、その値を濁度測定時のpHとした。 Temperature control was performed using ETC-717. At this time, the temperature at which the transmittance dropped rapidly was taken as the cloud point. After the measurement, the pH of the solution was measured at room temperature, and the value was taken as the pH at the time of turbidity measurement.
　３．４　動的光散乱測定(Dynamic light scattering)(DLS)による分子集合体の粒径測定
10 mM リン酸緩衝水溶液(140 mM NaCl) 1.80 mlに、2.2.で調製したpH 3.0の脂質分散液0.20 mlを加え(0.2mg/ml)、pH 7.4に調節し、10℃で30分間撹拌し保持した後、測定を行った。測定は脂質分散液を10℃から昇温しながら、各温度における、そのときの分子集合体の粒径を測定した。測定には大塚電子株式会社製DLS-6000型ダイナミック光散乱光度計を用いて行った。 3.4 Particle size measurement of molecular aggregates by dynamic light scattering (DLS)
Add 0.20 ml of pH 3.0 lipid dispersion prepared in 2.2. (0.2 mg / ml) to 1.80 ml of 10 mM phosphate buffer aqueous solution (140 mM NaCl), adjust to pH 7.4, and stir at 10 ° C for 30 minutes. After holding, measurements were taken. The measurement was performed by measuring the particle size of the molecular assembly at each temperature while raising the temperature of the lipid dispersion from 10 ° C. The measurement was performed using a DLS-6000 type dynamic light scattering photometer manufactured by Otsuka Electronics Co., Ltd.
　３．５　原子間力顕微鏡(Atomic force microscope)(AFM)による分子集合体の形態観測　３．５．１　形態観測に用いるサンプル作製
種々のpHの10 mM リン酸緩衝水溶液(140 mM NaCl)180 μlに、2.2で調製したpH 3.0のEDEG-DL-G1/PEG-Chol脂質分散液20 μlを加え(0.2mg/ml)、10℃で30分間撹拌し保持した後、AFM観測のマイカ作製に用いた。また、この溶液を50℃で10分間攪拌した後、AFM観測のマイカ作製に用いた。 3.5 Atomic force microscope (AFM) morphological observation of molecular aggregates 3.5.1 Sample preparation for morphological observation 10 mM phosphate buffer solution (140 mM) at various pH NaCl) 180 μl, pH 3.0 EDEG-DL-G1 / PEG-Chol lipid dispersion prepared in 2.2 (20 μl) was added (0.2 mg / ml) and stirred at 10 ° C. for 30 minutes. Used to make mica. The solution was stirred at 50 ° C. for 10 minutes and then used for making mica for AFM observation.
　３．５．２　AFM観測に用いるサンプル作製
　2.5.1.で調製した脂質分散液20μlをマイカ上に滴下、10分間、それぞれ10 ℃と50 ℃の雰囲気下で静置した。余分な分散液を濾紙で吸い取った後、形態観測に用いた。観測は、原子間力顕微鏡(SPI3800 Probe Station 及びSPA400 SOUNDPROOF HOUSING)を用いて観測した。 3.5.2 Preparation of sample used for AFM observation 20 μl of the lipid dispersion prepared in 2.5.1 was dropped onto mica and allowed to stand for 10 minutes in an atmosphere of 10 ° C. and 50 ° C., respectively. The excess dispersion was blotted with filter paper and used for morphology observation. Observation was performed using an atomic force microscope (SPI3800 Probe Station and SPA400 SOUNDPROOF HOUSING).
　３．６．結果
　３．６．１　分子集合体の温度応答機能に及ぼすPEG脂質の影響
　PEG-Chol含率が5 %のEDEG-DL-G1/PEG-Chol脂質について、この脂質分散液(pH 7.4)を昇温させたときの透過率の変化によって温度応答性の評価を行った。そのときの結果を図29に示した。分散液の温度を上昇させていくと、ある温度付近において急激な透過率の減少が見られた。これらの急激な透過率の減少は、分子集合体が不安定化し、凝集したことを示している。このことから、PEG脂質を加えることで分子集合体の温度応答機能が損なわれることはないということがわかった。しかし、38 ℃付近において少し減少していた透過率が増加しているのが見られた。これは、デンドロン脂質のアルキル鎖部位におけるゲル-液晶転移によって、凝集し始めた分子集合体が再分散化されたためによる、透過率の増加だと考えられる。 3.6. Results 3.6.1 Effect of PEG lipids on the temperature response function of molecular assemblies For EDEG-DL-G1 / PEG-Chol lipids with a PEG-Chol content of 5%, this lipid dispersion (pH 7.4) was increased. The temperature responsiveness was evaluated based on the change in transmittance when heated. The results at that time are shown in FIG. As the temperature of the dispersion was increased, a sharp decrease in transmittance was observed near a certain temperature. These sharp reductions in transmittance indicate that the molecular assembly has become unstable and aggregated. From this, it was found that the temperature response function of the molecular assembly was not impaired by adding PEG lipid. However, it was observed that the transmittance, which was slightly decreased near 38 ° C, increased. This is thought to be an increase in transmittance due to the re-dispersion of the molecular aggregates that started to aggregate due to the gel-liquid crystal transition at the alkyl chain site of the dendron lipid.
　また、PEG脂質を導入した割合に対して曇点をプロットした結果を図30に示した。PEG脂質の含有量が増加に伴い、曇点の上昇が見られた。これは、PEG脂質の増加に伴い、分子集合体表面がより親水的になったため、また、表面に導入されたPEG鎖の量が増加し、EDEG基間の表面密度が低下し、相互作用が抑制され、さらに、コレステロールが導入されることで、分子集合体の疎水部の流動性が高められたことによって安定化したためであると考えられる。 In addition, FIG. 30 shows the result of plotting the cloud point against the ratio of introducing PEG lipid. As the content of PEG lipid increased, the cloud point increased. This is because the molecular assembly surface became more hydrophilic with the increase in PEG lipids, and the amount of PEG chains introduced on the surface increased, the surface density between EDEG groups decreased, and the interaction was reduced. This is considered to be due to the fact that the fluidity of the hydrophobic part of the molecular assembly was enhanced by the suppression and further the introduction of cholesterol.
　また、種々のpHにおいてPEG-Chol含率が5 %のEDEG-DL-G1/PEG-Chol脂質の分散液の透過率の変化を測定することで、温度応答のpH依存性についての評価を行った。そのときの結果を図31に、そして、pHに対して曇点をプロットした結果を図32に示した。また、図32には比較として、EDEG-DL-G1のみの時の結果も、併せて載せた。分散液のpHの低下に伴い、曇点はほとんど変化しなかった。これは、pHの低下に伴う、極性基における三級アミノ基のプロトン化による分子の親水性の増加の影響が、PEG鎖による表面の水和と比べるとわずかなものであったためだと考えられる。また、どのpHにおいても、PEG脂質を導入した方がより高温側に曇点を示すことがわかった。 In addition, the pH dependence of temperature response was evaluated by measuring the change in permeability of EDEG-DL-G1 / PEG-Chol lipid dispersion with 5% PEG-Chol content at various pH. It was. The result at that time is shown in FIG. 31, and the result of plotting the cloud point against pH is shown in FIG. In addition, FIG. 32 also shows the results for EDEG-DL-G1 alone as a comparison. As the pH of the dispersion decreased, the cloud point hardly changed. This is thought to be because the effect of increasing the hydrophilicity of the molecule due to the protonation of the tertiary amino group in the polar group accompanying the decrease in pH was slight compared to the hydration of the surface by the PEG chain. . It was also found that at any pH, the PEG lipid introduced showed a cloud point on the higher temperature side.
　３．６．２　EDEG-DL-G1/PEG-Chol (5 %)脂質からなる分子集合体の粒径測定
　DLSを用いてPEG脂質を5％導入したEDEG-DL-G1/PEG-Chol (5 %)脂質分子集合体(pH 7.4)の各温度における粒径測定を行った結果を図33に示す。曇点以下の温度においては、約200 nm程度の粒径の分子集合体を形成しているのが分かる。しかし、曇点温度を超えると粒径は急激に増大した。そして、50 ℃では巨大な凝集体が観察され、DLSでの測定が出来なかった。これは、曇点以下の温度では、分子集合体がコロイドとして安定的であり、分子集合体表面が十分に水和しているため、一定の粒径を保っているのだと考えられる。そして、曇点温度となると、温度応答の脱水により分子集合体の親疎水バランスが崩れ、疎水性相互作用で凝集体を形成しているのだと考えられる。 3.6.2 EDEG-DL-G1 / PEG-Chol (5%) Particle size measurement of molecular assembly consisting of lipids EDEG-DL-G1 / PEG-Chol (5%) introduced with 5% PEG lipid using DLS %) The results of the particle size measurement at each temperature of the lipid molecular assembly (pH 7.4) are shown in FIG. It can be seen that a molecular assembly having a particle size of about 200 nm is formed at a temperature below the cloud point. However, the particle size increased rapidly above the cloud point temperature. Large aggregates were observed at 50 ° C., and measurement with DLS was not possible. This is presumably because at a temperature below the cloud point, the molecular assembly is stable as a colloid and the surface of the molecular assembly is sufficiently hydrated to maintain a constant particle size. When the cloud point temperature is reached, it is considered that the hydrophilic / hydrophobic balance of the molecular assembly is lost due to dehydration of temperature response, and aggregates are formed by hydrophobic interaction.
　３．６．３　EDEG-DL-G1/PEG-Chol (5 %)脂質からなる分子集合体の形態評価
　AFMを用いてPEG脂質を5％導入したEDEG-DL-G1/PEG-Chol (5 %)脂質分子集合体(pH 7.4)の形態観測を行った結果を図34に示す。曇点以下の10 ℃においては、粒径が約150～200nmの球状の分子集合体が観測された(図34(A))。この集合体は、その粒径の大きさより、ベシクル構造をとっていると考えられる。 3.6.3 EDEG-DL-G1 / PEG-Chol (5%) EDEG-DL-G1 / PEG-Chol (5%) EDEG-DL-G1 / PEG-Chol (5%) with 5% PEG lipid introduced using AFM FIG. 34 shows the results of morphological observation of lipid molecular aggregates (pH 7.4). At 10 ° C. below the cloud point, a spherical molecular assembly having a particle size of about 150 to 200 nm was observed (FIG. 34 (A)). This aggregate is considered to have a vesicle structure because of its particle size.
　次に、曇点以上の50℃においては、棒状構造の集合体が観測された(図34(B))。これは、以前の研究で分かっているIBAM-DL-G2の時と同様に、逆ヘキサゴナル構造をとっているものと考えられる。 Next, at 50 ° C. above the cloud point, aggregates of rod-like structures were observed (FIG. 34 (B)). This is considered to have an inverted hexagonal structure as in the case of IBAM-DL-G2, which was found in previous studies.
　以上の通り、PEG脂質を導入したEDEG-DL-G1/PEG-Chol脂質分子集合体の温度応答性評価と分子集合体の粒径及び形態の評価を行った。そして、この結果から、PEG脂質がEDEG-DL-G1分子集合体に与える影響について検討した。 As described above, the temperature responsiveness evaluation of the EDEG-DL-G1 / PEG-Chol lipid molecular assembly into which PEG lipid was introduced and the particle size and morphology of the molecular assembly were evaluated. Based on these results, the effect of PEG lipids on the EDEG-DL-G1 molecular assembly was examined.
　EDEG-DL-G1/PEG-Chol脂質分子集合体の温度応答性評価は、脂質分散液の透過率測定によって行った。EDEG-DL-G1/PEG-Chol脂質分散液の温度を上昇させていくと、ある温度以上で急激に透過率の減少を示した。このことから、分子集合体が凝集したことが示された。つまり、PEG脂質を導入することによって温度応答機能が損なわれることはないことがわかった。また、PEG脂質の導入率を増加させると、分子集合体の曇点は上昇した。これは、PEG-Cholが導入されたことにより、分子集合体表面がより水和され、脱水和して転移するためにより多くのエネルギーが必要になったためであると考えられる。 The temperature responsiveness evaluation of the EDEG-DL-G1 / PEG-Chol lipid molecular assembly was performed by measuring the permeability of the lipid dispersion. When the temperature of the EDEG-DL-G1 / PEG-Chol lipid dispersion was increased, the transmittance decreased rapidly above a certain temperature. This showed that the molecular assembly was aggregated. That is, it was found that the introduction of PEG lipid did not impair the temperature response function. Moreover, when the introduction rate of PEG lipid was increased, the cloud point of the molecular assembly increased. This is probably because the introduction of PEG-Chol hydrated the surface of the molecular assembly and required more energy to dehydrate and transfer.
　次に、EDEG-DL-G1/PEG-Chol脂質＝95/5の組成の分子集合体の粒径をDLS測定によって行った。曇点以下の低温では、約200 nm程度の粒径を示し、曇点以上の温度となると粒径は増大した。これは、曇点以下温度では分子集合体が水和した状態で一定の粒径を保ち安定しており、曇点を超えることで、温度応答による脱水和のために親疎水性バランスが不安定になり、疎水性相互作用が働き凝集体を形成したためだと考えられる。 Next, the particle size of the molecular assembly having the composition of EDEG-DL-G1 / PEG-Chol lipid = 95/5 was measured by DLS measurement. At a low temperature below the cloud point, the particle size was about 200 nm, and when the temperature was above the cloud point, the particle size increased. This is because when the temperature is below the cloud point, the molecular aggregate is hydrated and stable while maintaining a certain particle size, and when the cloud point is exceeded, the hydrophilicity / hydrophobicity balance becomes unstable due to dehydration due to temperature response. This is probably because the hydrophobic interaction worked to form aggregates.
　４．　ポリエチレングリコール脂質導入によるオリゴエチレングリコール鎖を末端に有するデンドロン脂質集合体の細胞内デリバリー機能評価
　前述の実験で、デンドロン脂質とPEG脂質とを複合化させることで、デンドロン脂質が形成する分子集合体のコロイドとしての安定化は図れ、一定の粒径を持ちながら、体温付近に曇点を持ち、温度応答によって形態変化を起こすベシクルが形成された。そこで、このベシクルが細胞内へと取り込まれるかのデリバリー機能実験を試みた。そこで、このデンドロン脂質ベシクルに蛍光ラベル化を施し、ヒト子宮頸ガン由来細胞(HeLa細胞)を用いて、温度制御を行うことによってベシクルのデリバリー機能を評価した。 4). Evaluation of intracellular delivery function of dendron lipid aggregates with oligoethylene glycol chains at the end by introduction of polyethylene glycol lipids In the previous experiment, dendron lipids and PEG lipids were combined to form molecular assemblies formed by dendron lipids. The colloid was stabilized, and a vesicle having a constant particle size, a cloud point near the body temperature, and a morphological change due to temperature response was formed. Therefore, we tried delivery function experiments on whether these vesicles are taken into cells. Therefore, this dendron lipid vesicle was fluorescently labeled, and the delivery function of the vesicle was evaluated by temperature control using human cervical cancer-derived cells (HeLa cells).
　４．１　試薬
　Fetal Bovine Serum (FBS)はMP Biomedicals,Incから購入した。ダルベッコ変法イーグル培地(DMEM)は日水製薬（株）から購入した。リン酸水素二ナトリウム、リン酸二水素カリウム、ベンジルペニシリンカリウム、ストレプトマイシン硫酸塩、アセチルCoA、アン
ピシリンナトリウム、クロラムフェニコール、臭化エチジウムは和光純薬から購入した。
塩化カルシウム、塩化マグネシウム6水和物、塩化カリウム、トリスヒドロキシミチルアミノメタン(Tris)、エチレンジアミン四酢酸二ナトリウム(EDTA)はキシダ化学から購入した。細胞溶解剤Luc-PGC-50は、東洋インキから購入した。塩化ナトリウムはナカライテスクから購入した。トリプシンはDIFCO LABORATORIES(U.S.A)から購入した。 4.1 Reagent Fetal Bovine Serum (FBS) was purchased from MP Biomedicals, Inc. Dulbecco's modified Eagle's medium (DMEM) was purchased from Nissui Pharmaceutical. Disodium hydrogen phosphate, potassium dihydrogen phosphate, benzylpenicillin potassium, streptomycin sulfate, acetyl CoA, ampicillin sodium, chloramphenicol, ethidium bromide were purchased from Wako Pure Chemical.
Calcium chloride, magnesium chloride hexahydrate, potassium chloride, trishydroxymitylaminomethane (Tris), and disodium ethylenediaminetetraacetate (EDTA) were purchased from Kishida Chemical. Cell lysing agent Luc-PGC-50 was purchased from Toyo Ink. Sodium chloride was purchased from Nacalai Tesque. Trypsin was purchased from DIFCO LABORATORIES (USA).
　４．２　蛍光ラベル化したデンドロン脂質ベシクルの調製
　EDEG-DL-G1とPEG脂質を95/5の割合で混合したクロロホルム溶液に、Rhodamine-PEを加え(0.6 mol%)、ロータリーエバポレーターを用いて溶媒を除去して、脂質薄膜を形成させた。これに10 mMリン酸緩衝溶液を加え、pH 3.0、濃度1.0 mMに調整した。これにバス型超音波照射装置を用いて超音波を45℃で10分間照射した後、45℃で20分間静置した。その後、氷冷下において60分以上静置した。最後に、pH 7.4に調節し、分散液を作製した。 4.2 Preparation of fluorescently labeled dendron lipid vesicles Rhodamine-PE (0.6 mol%) was added to a chloroform solution in which EDEG-DL-G1 and PEG lipids were mixed at a ratio of 95/5, and the solvent was removed using a rotary evaporator. Was removed to form a lipid film. A 10 mM phosphate buffer solution was added thereto to adjust the pH to 3.0 and a concentration of 1.0 mM. This was irradiated with ultrasonic waves at 45 ° C. for 10 minutes using a bath-type ultrasonic irradiation apparatus, and then allowed to stand at 45 ° C. for 20 minutes. Then, it was left still for 60 minutes or more under ice cooling. Finally, the pH was adjusted to 7.4 to prepare a dispersion.
　４．３　細胞内デリバリー機能評価
　Hela細胞をガラスボトムディッシュに1つ当たり2×105個になるように撒き、10% FBS含有DMEMメディウム2.0 ml中、37 ℃で一晩培養した。その後、PBS(+)で2回、PBS(-)で1回洗浄した後、DMEMメディウム(血清なし)1.0mlを加えた。そこに1穴当たり1 .0mlのRhodamine-PEでラベル化したPEG複合型デンドロン脂質ベシクルの分散液を加え、36℃と42 ℃で15分間インキュベートした。その後、PBS(+)で2回、PBS(-)で1回洗浄して細胞に吸着や取り込まれていないベシクルを除去した。その後、PBS(-)を1 ml再び加え、共焦点レーザー顕微鏡(LSM 5 EXCITER(ZEISS))により細胞内動態を観察した。 4.3 Evaluation of intracellular delivery function Hela cells were seeded at 2 × 10 5 in a glass bottom dish and cultured overnight at 37 ° C. in 2.0 ml of DMEM medium containing 10% FBS. Then, after washing twice with PBS (+) and once with PBS (−), 1.0 ml of DMEM medium (without serum) was added. A dispersion of PEG-conjugated dendron lipid vesicles labeled with 1.0 ml of Rhodamine-PE per well was added thereto and incubated at 36 ° C. and 42 ° C. for 15 minutes. Thereafter, the cells were washed twice with PBS (+) and once with PBS (−) to remove vesicles that were not adsorbed or taken up by the cells. Thereafter, 1 ml of PBS (−) was added again, and intracellular dynamics were observed with a confocal laser microscope (LSM 5 EXCITER (ZEISS)).
　４．４　結果
　４．４．１　デンドロン脂質ベシクルの温度制御による細胞内デリバリーへの影響
　36 ℃と42 ℃のそれぞれにおいて、インキュベートした時の共焦点レーザー顕微鏡画像の結果を、図35に示す。この結果より、曇点温度前後では、ラベル化したローダミン脂質の蛍光の強さがかなり異なることが分かる。明らかに、曇点以上でインキュベートした方が蛍光が強く光っていた。また、このときの細胞内におけるベシクルの分布を詳しく調べるために、拡大図を図36に示す。36 ℃においては、主に細胞の表面に蛍光が観察された。一方、42 ℃においては、細胞の表面だけでなく内部にも蛍光が観察された。これは、低温側ではベシクル表面が、温度応答を示す前で親水的なために、細胞表面に弱く吸着するだけなのに対し、曇点以上になると、ベシクルが温度応答により脱水和し、形態変化するために、細胞内に取り込まれたと考えられる。この時、ベシクルは形態変化し、細胞膜と強く相互作用し、効率良くエンドサイトーシスにより、細胞内に取り込まれるか、あるいは、疎水性相互作用によって、細胞膜を直接通過することによって、細胞内に移行し、取り込まれたものと考えられる。 4.4 Results 4.4.1 Effect of temperature control of dendron lipid vesicles on intracellular delivery Results of confocal laser microscope images when incubated at 36 ° C. and 42 ° C. are shown in FIG. From this result, it can be seen that the fluorescence intensity of the labeled rhodamine lipid is considerably different before and after the cloud point temperature. Apparently, the fluorescence was stronger when incubated at a cloud point or higher. In order to examine in detail the distribution of vesicles in cells at this time, an enlarged view is shown in FIG. At 36 ° C, fluorescence was mainly observed on the cell surface. On the other hand, at 42 ° C., fluorescence was observed not only on the cell surface but also inside. This is because, on the low temperature side, the vesicle surface is hydrophilic before showing a temperature response, so it only weakly adsorbs to the cell surface, but when the cloud point is exceeded, the vesicle dehydrates due to the temperature response and changes its shape. Therefore, it is thought that it was taken in in the cell. At this time, the vesicle changes its shape, interacts strongly with the cell membrane, is efficiently taken up into the cell by endocytosis, or moves through the cell membrane directly by hydrophobic interaction. It is thought that it was taken in.
　以上の通り、構築したPEG脂質を導入したEDEG-DL-G1/PEG-Chol脂質分子集合体に、蛍光ラベル化を施し、このベシクルの細胞内におけるデリバリー機能を評価した。 As described above, the EDEG-DL-G1 / PEG-Chol lipid molecular assembly into which the constructed PEG lipid was introduced was labeled with a fluorescent label, and the delivery function of the vesicle in the cell was evaluated.
　HeLa細胞において、インキュベート時の温度を曇点前後に制御すると、ベシクルの細胞内デリバリー機能の差が明らかに観察された。曇点温度前後では、ラベル化したローダミン脂質の蛍光の強さがかなり異なることが分かり、曇点以上でインキュベートした方が蛍光が強く光っていた。36 ℃においては、おもに、細胞の表面に蛍光が観察された。一方、42 ℃においては、細胞の表面だけでなく内部にも蛍光が観察された。これは、低温側ではベシクル表面が、温度応答を示す前で親水的なために、細胞表面に弱く吸着するだけなのに対し、曇点以上になると、ベシクルが温度応答により脱水和し、形態変化するために、細胞内に取り込まれたと考えられる。この時、ベシクルは形態変化し、細胞膜と強く相互作用し、効率良くエンドサイトーシスにより、細胞内に取り込まれるか、あるいは、疎水性相互作用によって、細胞膜を直接通過することによって、細胞内に移行し、取り込まれたものと考えられる。 In HeLa cells, when the incubation temperature was controlled around the cloud point, a difference in the intracellular delivery function of vesicles was clearly observed. Before and after the cloud point temperature, it was found that the fluorescence intensity of the labeled rhodamine lipid was considerably different, and the fluorescence was stronger when incubated at the cloud point or higher. At 36 ° C, fluorescence was mainly observed on the cell surface. On the other hand, at 42 ° C., fluorescence was observed not only on the cell surface but also inside. This is because, on the low temperature side, the vesicle surface is hydrophilic before showing a temperature response, so it only weakly adsorbs to the cell surface, but when the cloud point is exceeded, the vesicle dehydrates due to the temperature response and changes its shape. Therefore, it is thought that it was taken in in the cell. At this time, the vesicle changes its shape, interacts strongly with the cell membrane, is efficiently taken up into the cell by endocytosis, or moves through the cell membrane directly by hydrophobic interaction. It is thought that it was taken in.
　以上より、細胞への結合及び取り込みを温度制御できる、新しい温度応答性ベシクルが開発できた。 From the above, a new temperature-responsive vesicle that can control the temperature of binding and uptake into cells has been developed.
　５．　曇点に及ぼすPEG脂質含率の影響
　MDEG-G1/PEG-Chol 集合体の曇点に及ぼすPEG-Chol 含率の影響を調べた。実験法は、前述の方法に従った。詳細には次の通りである。 5. Effect of PEG lipid content on cloud point The effect of PEG-Chol content on the cloud point of MDEG-G1 / PEG-Chol aggregates was investigated. The experimental method followed the method described above. Details are as follows.
　種々の比率のMDEG-G1とPEG-Chol(PEG分子量1000)の混合薄膜を分散させて得られたデンドロン脂質／PEG-Chol集合体分散液（10 mM リン酸、140 mM NaCl、 pH3.0）を作成し、10 mM リン酸、140 mM NaCl、 pH7.4のバッファー中に加えてその透過率の温度依存性測定を行うことで、温度応答性を評価した。昇温速度 4 ℃/min 。 Dendron lipid / PEG-Chol aggregate dispersions (10 M phosphoric acid, 140 M NaCl, pH3.0) obtained by dispersing mixed thin films of MDEG-G1 and PEG-Chol (PEG molecular weight 1000) in various ratios The temperature responsiveness was evaluated by measuring the temperature dependence of the transmittance in addition to a buffer of 10 mM mM phosphoric acid, 140 mM mM NaCl, and pH 7.4. Temperature increase rate 4 ℃ / min.
　MDEG-G1/PEG-Chol 集合体の曇点はPEG-Chol含率の増大とともに上昇した。PEG-Chol含率を調節することで、集合体の応答温度（曇点）の調製が可能であることが分かった。また、PEG-Chol  を2％含有させることで、曇点を40℃付近にもつデンドロン脂質集合体が得られることが分かった。 The cloud point of MDEG-G1 / PEG-Chol aggregate increased with increasing PEG-Chol content. It was found that the response temperature (cloud point) of the aggregate can be adjusted by adjusting the PEG-Chol content. It was also found that a dendron lipid aggregate having a cloud point near 40 ° C. can be obtained by containing 2% of PEG-Chol.
　６．　細胞による集合体の取り込みに及ぼすインキュベーション温度の影響
　12穴dishにHeLa細胞(ヒト子宮頸ガン由来)を10万/wellで播種し、24時間培養した。PBS(+)で２回、PBS(-)で１回洗浄し、DMEM(血清なし)を500 μL加え、脂質濃度が0.5 mM となるようにデンドロン脂質／PEG-Chol集合体分散液を500 μL加えて、CO2 インキュベーターで、37℃,42℃,44℃で15分,30分間インキュベートした。その後、PBS(+)で２回、PBS(-)で１回洗浄し、トリプシンで細胞を剥がした後、フローサイトメーターにより細胞の蛍光強度を調べて、細胞による集合体の取り込み量を評価した。(n=2)
　細胞自体の蛍光強度を基準として相対蛍光強度によって評価した。結果を図３９に示す。インキュベーション温度を曇点以上にすると、EDEG-DL-G1-2C18/PEG-Chol (95/5)集合体では取り込み量が増大した。EDEG-DL-G1-2C18/PEG-Chol (95/5)集合体が疎水性化して、細胞に強く結合して効率よく取り込まれたものと考えられる。また、細胞とのインキュベーション時間を延ばすことでも、取り込みを大きく促進できた。一方、MDEG-DL-G1-2C18/PEG-Chol (98/2)集合体は、温度及び接触時間を変化させても、取り込みがあまり変化しなかった。これは、デンドロン脂質MDEG-DL-G1-2C18末端基の疎水性度がEDEG-DL-G1-2C18に比べて低いことから、形態変化を起こしても細胞との相互作用があまり強く働かなかったためと考えられる。 6). Effect of incubation temperature on aggregate uptake by cells HeLa cells (derived from human cervical cancer) were seeded in a 12-well dish at 100,000 / well and cultured for 24 hours. Wash twice with PBS (+) and once with PBS (-), add 500 μL of DMEM (without serum), and add 500 μL of dendron lipid / PEG-Chol aggregate dispersion to a lipid concentration of 0.5 mM. In addition, the cells were incubated at 37 ° C., 42 ° C. and 44 ° C. for 15 minutes and 30 minutes in a CO 2 incubator. Thereafter, the cells were washed twice with PBS (+) and once with PBS (-), and the cells were detached with trypsin. Then, the fluorescence intensity of the cells was examined with a flow cytometer, and the amount of aggregates taken up by the cells was evaluated. . (n = 2)
The relative fluorescence intensity was evaluated based on the fluorescence intensity of the cell itself. The results are shown in FIG. When the incubation temperature was higher than the cloud point, the amount of incorporation was increased in the EDEG-DL-G1-2C 18 / PEG-Chol (95/5) aggregate. It is considered that the EDEG-DL-G1-2C 18 / PEG-Chol (95/5) aggregate became hydrophobic and was strongly taken up by binding strongly to cells. In addition, the uptake could be greatly promoted by extending the incubation time with cells. On the other hand, the MDEG-DL-G1-2C 18 / PEG-Chol (98/2) aggregate did not change much even when the temperature and contact time were changed. This is because the dendron lipid MDEG-DL-G1-2C 18 terminal group has a lower hydrophobicity than EDEG-DL-G1-2C 18 , and so the interaction with the cell works very strongly even when morphological changes occur. It is thought that there was not.
　以上の通り、温度応答性基として生体適合性に優れるオリゴエチレングリコール鎖を、デンドロン脂質の極性基に導入し、その分子集合体の表面に集積させることにより、分子集合体に温度応答性を付与できることが確認された。また、世代及び末端基の違いによって、曇点の温度が異なった。このことは、分子構造内の疎水性、親水性のバランスが起因していると考えられる。よって、分子構造における疎水性部位、親水性部位のバランスの設計によって、所望の温度で応答する分子集合体の構築が可能であると考えられる。 As described above, temperature-responsive groups are given temperature responsiveness by introducing oligoethylene glycol chains with excellent biocompatibility as temperature-responsive groups into polar groups of dendron lipids and accumulating them on the surface of the molecular assemblies. It was confirmed that it was possible. Also, the cloud point temperature was different depending on the generation and end group. This is considered due to the balance between hydrophobicity and hydrophilicity in the molecular structure. Therefore, it is considered that it is possible to construct a molecular assembly that responds at a desired temperature by designing the balance between the hydrophobic part and the hydrophilic part in the molecular structure.
　また、この温度応答性デンドロン脂質の分子集合体にコロイドとしての安定性を持たせるために、ポリエチレングリコールを極性基にもつ脂質を導入したが、温度応答機能が損なわれることなく、形態変化も起こすことが確認された。更に、ポリエチレングリコール脂質の導入率を変化させることにより曇点の温度も変化することがわかった。 In addition, in order to make this temperature-responsive dendron lipid molecular assembly stable as a colloid, a lipid having polyethylene glycol as a polar group was introduced, but the temperature-responsive function was not impaired, and morphological changes occurred. It was confirmed. Furthermore, it has been found that the cloud point temperature also changes by changing the introduction rate of the polyethylene glycol lipid.
　よって、分子構造における疎水性部位、親水性部位のバランスの設計、ポリエチレングリコール脂質の導入率を変化させることによって、所望の温度で、そのベシクルのサイズや形状を変化させる分子集合体の構築が可能である。 Therefore, it is possible to construct a molecular assembly that changes the size and shape of the vesicle at the desired temperature by changing the design of the balance between the hydrophobic and hydrophilic sites in the molecular structure and the introduction rate of polyethylene glycol lipid. It is.
　そして、ポリエチレングリコール脂質を導入した温度応答性デンドロン脂質集合体は、体温付近の曇点以上でその形状が劇的に変化し、ポリエチレングリコール脂質の効果によって高い安定性と生体適合性とを併せもつベシクルが構築できた。この、PEG-Chol複合型デンドロン脂質ベシクルは、細胞内へのデリバリー機能を温度によって制御できる。 And the temperature-responsive dendron lipid aggregate into which polyethylene glycol lipid is introduced changes its shape dramatically above the cloud point near body temperature, and has both high stability and biocompatibility due to the effect of polyethylene glycol lipid. A vesicle was constructed. This PEG-Chol complex type dendron lipid vesicle can control the delivery function into cells by temperature.
　ＩＩ．第二の発明
　１．Ｒ １ 及びＲ ２ が不飽和炭化水素基である本発明の化合物の製造
　１．１　試薬
　Fetal Bovine Serum (FBS)はMP Biomedicals,Inc から購入した。ダルベッコ変法イーグル培地(DMEM)は日水製薬（株）から購入した。N,N-ジメチルホルムアミド、テトラヒドロフラン、石油エーテル、シアン化ナトリウム、水素化リチウムアルミニウム、リン酸水素二ナトリウム、リン酸二水素カリウム、ベンジルペニシリンカリウム、ストレプトマイシン硫酸塩は和光純薬から購入した。ピラニンは東京化成(株)から購入した。クロロホルム、酢酸エチル、メタノール、n-ヘキサン、硫酸ナトリウム、塩化カルシウム、塩化マグネシウム6 水和物、塩化カリウム、エチレンジアミン四酢酸二ナトリウム(EDTA)はキシダ化学から購入した。トリエチルアミン、アクリル酸メチル、エチレンジアミン、ジエチルエーテル、塩化ナトリウム、RPMI-1640 液体培地、MEM 非必須アミノ酸溶液はナカライテスクから購入した。トリプシンはDIFCOLABORATORIES(U.S.A)から購入した。ジオクタデシルアミン、カルセインはSigma から購入した。オレイルアミン、オレオイルクロリド、シクロヘキサンジカルボン酸無水物、3-メチルグルタル酸無水物はALDRICH から購入した。 II. Second invention 1. Preparation of compounds of the invention wherein R 1 and R 2 are unsaturated hydrocarbon groups 1.1 Reagent Fetal Bovine Serum (FBS) was purchased from MP Biomedicals, Inc. Dulbecco's modified Eagle's medium (DMEM) was purchased from Nissui Pharmaceutical. N, N-dimethylformamide, tetrahydrofuran, petroleum ether, sodium cyanide, lithium aluminum hydride, disodium hydrogen phosphate, potassium dihydrogen phosphate, potassium benzylpenicillin, and streptomycin sulfate were purchased from Wako Pure Chemical. Pyranine was purchased from Tokyo Kasei Co., Ltd. Chloroform, ethyl acetate, methanol, n-hexane, sodium sulfate, calcium chloride, magnesium chloride hexahydrate, potassium chloride, and disodium ethylenediaminetetraacetate (EDTA) were purchased from Kishida Chemical. Triethylamine, methyl acrylate, ethylenediamine, diethyl ether, sodium chloride, RPMI-1640 liquid medium, and MEM non-essential amino acid solution were purchased from Nacalai Tesque. Trypsin was purchased from DIFCOLABORATORIES (USA). Dioctadecylamine and calcein were purchased from Sigma. Oleylamine, oleoyl chloride, cyclohexanedicarboxylic anhydride and 3-methylglutaric anhydride were purchased from ALDRICH.
　ジクロロメタンはSigma-ALDRICH から購入した。2-メルカプトエタノール、DPX、Hoechst、Lysotracker Green DND-26、Lysotracker Red DND-99、Tf-Alexa555はInvitrogen から購入した。透析膜はSpectra/Por 6 (分画分子量 2000,FE-0526-33)をSpectrum Laboratories Inc.から購入した。Merck Kieselgel 60(230-400 mesh) をシリカゲルクロマトグラフィーに使用した。 Dichloromethane was purchased from Sigma-ALDRICH®. 2-mercaptoethanol, DPX, Hoechst, Lysotracker Green DND-26, Lysotracker Red DND-99, and Tf-Alexa555 were purchased from Invitrogen. As a dialysis membrane, Spectra / Por 6 (fractional molecular weight 2000, FE-0526-33) was purchased from Spectrum Laboratories Inc. Merck Kieselgel 60 (230-400 mesh) was used for silica gel chromatography.
　１．２　合成
　１．２．１　概要
　それぞれの第1 世代のデンドロン脂質（ CHex-DL-G1-2C18-U2( 以下CHexDL-U2)、MGlu-DL-G1-2C18-U2(以下MGluDL-U2)）の合成経路を以下に示した。 1.2 Synthesis 1.2.1 Overview The first generation dendron lipids (CHex-DL-G1-2C 18 -U2 (CHexDL-U2), MGlu-DL-G1-2C 18 -U2 (MGluDL-) The synthesis route of U2)) is shown below.
Figure JPOXMLDOC01-appb-C000032
Figure JPOXMLDOC01-appb-C000032
　オレイルアミン、オレオイルクロリドによりオレイルオレオイルアミドを合成した後、ヒドリド還元によりジオレイルアミンを合成し、アクリル酸メチルを用いたマイケル付加反応と、エチレンジアミンを用いたエステルアミド交換反応を繰り返すことによりDL-G1-2C18-U2 を合成した。この方法は、デンドリマーの合成において、Tomalia らが報告した方法である。さらに合成したDL-G1-2C18-U2 に3-Methylglutaric Anhydride あるいはCyclohexanedicarboxylic Anhydride を反応させることでCHexDL-U2 、MGluDL-U2 を合成した。 DL-G1 by synthesizing oleyl oleoylamide with oleylamine and oleoyl chloride, then synthesizing dioleylamine by hydride reduction, repeating Michael addition reaction using methyl acrylate and ester amide exchange reaction using ethylenediamine -2C 18 -U2 was synthesized. This is the method reported by Tomalia et al. In the synthesis of dendrimers. Further, CHexDL-U2 and MGluDL-U2 were synthesized by reacting the synthesized DL-G1-2C 18 -U2 with 3-Methylglutaric Anhydride or Cyclohexanedicarboxylic Anhydride.
　１．２．２　Oleyloleoylamide の合成
　Oleoyl chloride を7.7 mL (20 mmol)をジクロロメタン100 mL に溶解させ氷水浴で撹拌し、これにジクロロメタン50 mL にOleylamine 9.4 mL (20 mmol)とトリエチルアミン 3.3 mL (0.024 mol)を溶解したものをゆっくり滴下した後、室温窒素雰囲気化で71 時間還流した。その後、エバポレータにより溶媒を留去し真空乾燥した後、得られた粗生成物をオープンカラムクロマトグラフィーにより分離精製した(展開溶媒 クロロホルム：酢酸エチル=2：1)。 1.2.2 Synthesis of Oleyloleoylamide Oleoyl chloride (7.7 mL (20 mmol)) was dissolved in 100 mL of dichloromethane and stirred in an ice-water bath. ) Was slowly added dropwise and then refluxed for 71 hours in a nitrogen atmosphere at room temperature. Then, after evaporating the solvent with an evaporator and vacuum drying, the obtained crude product was separated and purified by open column chromatography (developing solvent: chloroform: ethyl acetate = 2: 1).
　その後1H NMR により化合物の同定を行った。結果を図４０に示す。（収量 9.38 g、収率 86.4 %）；1H NMR (CDCl3): δ0.88 (m, CH3(CH2)6-), δ 1.23 (s, CH3(CH2)6-,-CH2(CH2)5CH2-), δ 1.41 (m,-CH2CH2CO-),δ 1.55 (m, -CH2CH2CO-),δ 1.91 (m, -CH2CH2CH-),δ 2.05 (m, -CH2CH2NH-),δ 3.15 (m, -CH2CH2NH-),δ 5.25 (m, -CH2CHCHCH2-). Thereafter, the compound was identified by 1H NMR. The results are shown in FIG. (Yield 9.38 g, Yield 86.4%); 1H NMR (CDCl 3 ): δ0.88 (m, CH 3 (CH 2 ) 6- ), δ 1.23 (s, CH 3 (CH 2 ) 6 -,-CH 2 (CH 2 ) 5CH 2- ), δ 1.41 (m, -CH 2 CH 2 CO-), δ 1.55 (m, -CH 2 CH 2 CO-), δ 1.91 (m, -CH 2 CH 2 CH- ), δ 2.05 (m, -CH 2 CH 2 NH -), δ 3.15 (m, -CH 2 CH 2 NH -), δ 5.25 (m, -CH 2 CHCHCH 2 -).
　１．２．３　Dioleylamine の合成
　THF 84.5 mL にゆっくりとLAH 1.49 g を加え、これにOleyloleoylamide 2.99g(5.61 mmol)をTHF 84.5 mL で溶解しパスツールピペットを用いてゆっくり加えた。これを50 ℃の窒素雰囲気化で83 時間反応させた。その後、LAH を濾別し、この時に酢酸エチル、クロロホルム、THF で洗浄した。その後エバポレータにより溶媒を留去し、残留物を飽和食塩水で4 回洗浄した。硫酸ナトリウムを用いて乾燥した後エバポレータにより溶媒を留去、粗生成物をオープンカラムクロマトグラフィーにより分離精製した(展開溶媒 クロロホルム：酢酸エチル=2：1、後クロロホルム：メタノール=9：1)。 1.2.3 Synthesis of Dioleylamine 1.49 g of LAH was slowly added to 84.5 mL of THF, and 2.99 g (5.61 mmol) of Oleyloleoylamide was dissolved in 84.5 mL of THF and slowly added using a Pasteur pipette. This was reacted in a nitrogen atmosphere at 50 ° C. for 83 hours. Thereafter, LAH was filtered off and washed with ethyl acetate, chloroform and THF at this time. Thereafter, the solvent was removed by an evaporator, and the residue was washed 4 times with saturated saline. After drying with sodium sulfate, the solvent was removed by an evaporator, and the crude product was separated and purified by open column chromatography (developing solvent: chloroform: ethyl acetate = 2: 1, then chloroform: methanol = 9: 1).
　得られた生成物を1H NMR により化合物の同定を行った。結果を図４１に示す。（収量 1.67 g、収率 56.3%）；1H NMR (CDCl3): δ0.88 (m, CH3(CH2)6-), δ 1.23 (s, CH3(CH2)6-,-CH2(CH2)5CH2-), δ 1.41 (m, -CH2CH2NH-), δ 1.99 (m, -CH2CH2CH-), δ 2.60 (m, -CH2CH2NH-), δ 5.35 (m, -CH2CHCHCH2-). The obtained product was identified by 1H NMR. The results are shown in FIG. (Yield 1.67 g, Yield 56.3%); 1H NMR (CDCl 3 ): δ0.88 (m, CH 3 (CH 2 ) 6- ), δ 1.23 (s, CH 3 (CH 2 ) 6 -,-CH 2 (CH 2 ) 5CH 2- ), δ 1.41 (m, -CH 2 CH 2 NH-), δ 1.99 (m, -CH 2 CH 2 CH-), δ 2.60 (m, -CH 2 CH 2 NH- ), δ 5.35 (m, -CH 2 CHCHCH 2- ).
　１．２．４　DL-G-0.5-2C 18 -U2 の合成
　Dioleylamine 1.32 g (3.45 mmol)をアクリル酸メチル 98 mL (1.23 mol)に溶解して、70 ℃の窒素雰囲気化で攪拌した。反応終了後(95 時間)、エバポレータにより未反応のアクリル酸メチルをカラムクロマトグラフィーにより分離精製した(展開溶媒 クロロホルム：酢酸エチル＝2：1)。 1.2.4 Synthesis of DL-G-0.5-2C 18 -U2 Dioleylamine 1.32 g (3.45 mmol) was dissolved in 98 mL (1.23 mol) of methyl acrylate and stirred in a nitrogen atmosphere at 70 ° C. After completion of the reaction (95 hours), unreacted methyl acrylate was separated and purified by column chromatography using an evaporator (developing solvent: chloroform: ethyl acetate = 2: 1).
　得られた生成物を1H NMR により化合物の同定を行った。結果を図４２に示す。（収量 1.32 g、収率 64.9%）；1H NMR (CDCl3): δ0.85 (m, CH3(CH2)6-), δ 1.3 (m, CH3(CH2)6-, -CH2(CH2)5CH2-, -CH2CH2N-), δ1.99 (m, -CH2CH2CH-), δ 2.40 (m, -CH2CH2NH-, -CH2CH2COO), δ 2.78 (m, -CH2CH2COO), δ 5.35 (m, -CH2CHCHCH2-). The obtained product was identified by 1H NMR. The results are shown in FIG. (Yield 1.32 g, Yield 64.9%); 1H NMR (CDCl 3 ): δ0.85 (m, CH 3 (CH 2 ) 6- ), δ 1.3 (m, CH 3 (CH 2 ) 6- , -CH 2 (CH 2) 5CH 2 - , -CH 2 CH 2 N-), δ1.99 (m, -CH 2 CH 2 CH-), δ 2.40 (m, -CH 2 CH 2 NH-, -CH 2 CH 2 COO), δ 2.78 (m, -CH 2 CH 2 COO), δ 5.35 (m, -CH 2 CHCHCH 2- ).
　１．２．５　DL-G0-2C 18 -U2 の合成
　DL-G-0.5-2C18-U2 の1.32 g(2.18 mmol)をメタノール100 mL に溶解し、これをシアン化ナトリウム32.2 mg(0.659 mmol)を含む蒸留精製したエチレンジアミン70 mL(1.05 mol)にパスツールピペットを用いてゆっくりと滴下した。 1.2.5 DL-G0-2C 18 -U2 synthetic DL-G-0.5-2C 18 -U2 of 1.32 g of (2.18 mmol) was dissolved in methanol 100 mL, which sodium cyanide 32.2 mg (0.659 mmol ) Was slowly added dropwise to 70 mL (1.05 mol) of distilled and purified ethylenediamine using a Pasteur pipette.
　その後、70 ℃の窒素雰囲気化で93.5 時間反応させた。反応終了後、エバポレータを用いて溶媒を留去し真空乾燥した。その後、1H NMR により化合物の同定を行った。結果を図４３に示す。（収量 1.41 g 未精製）；1H NMR (CDCl3): δ 0.85 (m, CH3(CH2)6-), δ 1.3 (m, CH3(CH2)6-, -CH2(CH2)5CH2-, -CH2CH2N-), δ 2.0 (m, -CH2CH2CH-), δ 2.3-2.8 (m, -CH2CH2NH-,  -CH2CH2COO, -CH2CH2NH2), δ 3.25 (m, -CH2CH2NH2), δ 5.35 (m, -CH2CHCHCH2-), δ 8.55 (s, -CONHCH2-). Thereafter, the reaction was performed in a nitrogen atmosphere at 70 ° C. for 93.5 hours. After completion of the reaction, the solvent was distilled off using an evaporator and vacuum dried. Then, the compound was identified by 1H NMR. The results are shown in FIG. (Yield 1.41 g unpurified); 1H NMR (CDCl 3 ): δ 0.85 (m, CH 3 (CH 2 ) 6- ), δ 1.3 (m, CH 3 (CH 2 ) 6- , -CH 2 (CH 2 ) 5CH 2- , -CH 2 CH 2 N-), δ 2.0 (m, -CH 2 CH 2 CH-), δ 2.3-2.8 (m, -CH 2 CH 2 NH-, -CH 2 CH 2 COO, -CH 2 CH 2 NH 2 ), δ 3.25 (m, -CH 2 CH 2 NH 2 ), δ 5.35 (m, -CH 2 CHCHCH 2- ), δ 8.55 (s, -CONHCH 2- ).
　１．２．６　DL-G0.5-2C 18 -U2 の合成
　DL-G0-2C18-U2 の1.41 mg(2.22 mmol)をメタノール141 mL に溶解した。これをアクリル酸メチル94 mL(1.03 mol)にパスツールピペットを用いてゆっくりと滴下した。その後、45 ℃の窒素雰囲気化で54 時間反応させた。反応終了後、エバポレータを用いて溶媒及び未反応のアクリル酸メチルを留去し真空乾燥したのち、得られた粗生成物をオープンカラムクロマトグラフィーにより分離精製した。(展開溶媒 ヘキサン：酢酸エチル=10：3、のちクロロホルム：メタノール=4：1)。 1.2.6 1.41 mg of DL-G0.5-2C 18 -U2 synthetic DL-G0-2C 18 -U2 a (2.22 mmol) was dissolved in methanol 141 mL. This was slowly added dropwise to 94 mL (1.03 mol) of methyl acrylate using a Pasteur pipette. Then, it was made to react for 54 hours by 45 degreeC nitrogen atmosphere formation. After completion of the reaction, the solvent and unreacted methyl acrylate were distilled off using an evaporator and vacuum-dried, and the resulting crude product was separated and purified by open column chromatography. (Developing solvent hexane: ethyl acetate = 10: 3, then chloroform: methanol = 4: 1).
　その後、1H NMR により化合物の同定を行った。結果を図４４に示す。（収量 1.74 g、収率 99.3%）；1H NMR (CDCl3): δ 0.85 (m, CH3(CH2)6-), δ 1.3 (m, CH3(CH2)6-,-CH2(CH2)5CH2-, -CH2CH2N-), δ 2.0 (m, -CH2CH2CH-), δ 2.3-2.6 (m, -CH2CH2NH-,  -CH2CH2COO, -CH2CH2NH2), δ 2.8 (m, -CH2CH2COO),δ 3.25 (m, -CH2CH2NH2), δ 3.67 (s, -OCH3), δ 5.35 (m, -CH2CHCHCH2-), δ 8.55 (s, -CONHCH2-). Then, the compound was identified by 1H NMR. The results are shown in FIG. (Yield 1.74 g, 99.3% yield); 1H NMR (CDCl 3 ): δ 0.85 (m, CH 3 (CH 2 ) 6- ), δ 1.3 (m, CH 3 (CH 2 ) 6 -,-CH 2 (CH 2 ) 5CH 2- , -CH 2 CH 2 N-), δ 2.0 (m, -CH 2 CH 2 CH-), δ 2.3-2.6 (m, -CH 2 CH 2 NH-, -CH 2 CH 2 COO, -CH 2 CH 2 NH 2 ), δ 2.8 (m, -CH 2 CH 2 COO), δ 3.25 (m, -CH 2 CH 2 NH 2 ), δ 3.67 (s, -OCH 3 ), δ 5.35 (m, -CH 2 CHCHCH 2- ), δ 8.55 (s, -CONHCH 2- ).
　１．２．７　DL-G1-2C 18 -U2 の合成
　DL -G0.5-2C18-U2 の1.74 g(2.88 mmol)をメタノール50 mL に溶解し、これをシアン化ナトリウム47.9 mg(0.98 mmol)を含む、蒸留精製したエチレンジアミン92.8 mL(1.39 mol)にパスツールピペットを用いてゆっくり滴下した。その後、50 ℃で72 時間、窒素雰囲気化で反応させた。反応終了後、ロータリーエバポレータで溶媒及び未反応のエチレンジアミンを留去し、得られた粗生成物を2日間透析することで精製し、凍結乾燥により黄色のロウ状物質を得た。 1.2.7 DL-G1-2C 18 -U2 synthetic DL -G0.5-2C 18 -U2 of 1.74 g of (2.88 mmol) was dissolved in methanol 50 mL, which sodium cyanide 47.9 mg (0.98 mmol ) Was slowly added dropwise to 92.8 mL (1.39 mol) of distilled and purified ethylenediamine using a Pasteur pipette. Then, it was made to react by nitrogen atmosphere for 72 hours at 50 degreeC. After completion of the reaction, the solvent and unreacted ethylenediamine were distilled off with a rotary evaporator, and the resulting crude product was purified by dialysis for 2 days, and a yellow waxy substance was obtained by lyophilization.
　その後、1H NMR により化合物の同定を行った。結果を図４５に示す。（収量1.03 g、収率 41.5 %）；1H NMR (CDCl3): δ 0.88 (m, CH3(CH2)6-), δ 1.2-1.4 (m, CH3(CH2)6-, -CH2(CH2)5CH2-, -CH2CH2N-), δ 2.0 (m, -CH2CH2CH-), δ 2.3-2.7 (m,-CH2CH2NH-, -CH2CH2COO), δ 2.6-2.8 (m, -CH2CH2COO, -CH2CH2NH2), δ 3.25(m, -CH2CH2N-), δ 5.35 (m, -CH2CHCHCH2-), δ 8.55 (s, -CONHCH2-). Then, the compound was identified by 1H NMR. The results are shown in FIG. (Yield 1.03 g, Yield 41.5%); 1H NMR (CDCl 3 ): δ 0.88 (m, CH 3 (CH 2 ) 6- ), δ 1.2-1.4 (m, CH 3 (CH 2 ) 6 -,- CH 2 (CH 2 ) 5CH 2- , -CH 2 CH 2 N-), δ 2.0 (m, -CH 2 CH 2 CH-), δ 2.3-2.7 (m, -CH 2 CH 2 NH-, -CH 2 CH 2 COO), δ 2.6-2.8 (m, -CH 2 CH 2 COO, -CH 2 CH 2 NH 2 ), δ 3.25 (m, -CH 2 CH 2 N-), δ 5.35 (m, -CH 2 CHCHCH 2- ), δ 8.55 (s, -CONHCH 2- ).
　１．２．８　CHex-DL-G1-2C 18 -U2 の合成
　シクロヘキサンジカルボン酸無水物390.5 mg(2.53 mmol)をDMF 5 mL に溶解させた。その後、DL-G1-2C18-U2 の207.7 mg(0.234 mmol)をDMF 3 mL に溶解したのち混合し、トリエチルアミンを0.174 mL 加えて50 ℃で96 時間窒素雰囲気化で反応させた。反応終了後、エバポレータにより溶媒を留去した後、LH カラムにより精製を行った。 1.2.8 CHex-DL-G1-2C 18 -U2 synthetic cyclohexanedicarboxylic anhydride 390.5 mg of (2.53 mmol) was dissolved in DMF 5 mL. Thereafter, 207.7 mg (0.234 mmol) of DL-G1-2C 18 -U2 was dissolved in 3 mL of DMF and mixed. Then, 0.174 mL of triethylamine was added and reacted at 50 ° C. for 96 hours under nitrogen atmosphere. After completion of the reaction, the solvent was distilled off with an evaporator and then purified with an LH column.
　その後、質量分析及び1H NMR により化合物の同定を行った。結果を図４６及び図４７に示す。（収量 80.3 mg、収率 29.3%）；1H NMR (CDCl3): δ 0.88 (m, CH3(CH2)6-), δ1.2-1.4 (m, CH3(CH2)6-, -CH2(CH2)5CH2-, -CH2CH2N-), δ1.4-1.9(m,-COCH-(CH2)4-CHCOOH), δ 2.0 (m, -CH2CH2CH-), δ 2.3-2.7 (m, -CH2CH2N-, -CH2CH2COO), δ 2.6-2.8 (m, -CH2CH2COO, -CH2CH2NH2, -COCH-CH2), δ 3.25(m, -CH2CH2N-), δ 5.35 (m, -CH2CHCHCH2-), Calc [M]- (C67H121N7O9) m/z1167.9. Found ESI-MS [M- H]- m/z 1166.6 Thereafter, the compound was identified by mass spectrometry and 1H NMR. The results are shown in FIGS. 46 and 47. (Yield 80.3 mg, Yield 29.3%); 1H NMR (CDCl 3 ): δ 0.88 (m, CH 3 (CH 2 ) 6- ), δ 1.2-1.4 (m, CH 3 (CH 2 ) 6- , -CH 2 (CH 2) 5CH 2 -, -CH 2 CH 2 N-), δ1.4-1.9 (m, -COCH- (CH 2) 4 -CHCOOH), δ 2.0 (m, -CH 2 CH 2 CH-), δ 2.3-2.7 (m, -CH 2 CH 2 N-, -CH 2 CH 2 COO), δ 2.6-2.8 (m, -CH 2 CH 2 COO, -CH 2 CH 2 NH 2 ,- COCH-CH 2 ), δ 3.25 (m, -CH 2 CH 2 N-), δ 5.35 (m, -CH 2 CHCHCH 2- ), Calc [M]-(C 67 H 121 N 7 O 9 ) m / z1167.9. Found ESI-MS [M- H]-m / z 1166.6
　１．２．９　MGlu-DL-G1-2C 18 -U2 の合成
　3-Methylglutaric Anhydride (以下MGluAn) 618 mg(4.23 mmol)をDMF 3 mL に溶解させた。その後、DL-G1-2C18-U2 430 mg(0.909 mmol)をDMF 4 mL に溶解したのち混合し、triethylamine(TEA)を0.370 mL 加えて50 ℃で7 日間窒素雰囲気下で攪拌した。反応終了後、evaporator により溶媒を留去した後、LH-20column (溶離液:methanol) 及びsilica gel column chromatography (展開溶媒chloroform：methanol：water=60：35：5)により精製を行った。 1.2.9 MGlu-DL-G1-2C 18 Synthesis of -U2 3-Methylglutaric Anhydride (hereinafter MGluAn) 618 mg (4.23 mmol) was dissolved in DMF 3 mL. Thereafter, 430 mg (0.909 mmol) of DL-G1-2C 18 -U2 was dissolved in 4 mL of DMF and mixed, 0.370 mL of triethylamine (TEA) was added, and the mixture was stirred at 50 ° C. for 7 days under a nitrogen atmosphere. After completion of the reaction, the solvent was distilled off with an evaporator, followed by purification with LH-20 column (eluent: methanol) and silica gel column chromatography (developing solvent chloroform: methanol: water = 60: 35: 5).
　その後、質量分析及び1H NMR により化合物の同定を行った。結果を図４８～５０に示す。（収量 208 mg、収率20.5%）；1H NMR (CDCl3): δ 0.88 (m, CH3(CH2)6-), δ 1.03 (s, -CH(CH3)-), δ1.2-1.4 (m, CH3(CH2)6-, -CH2(CH2)5CH2-), δ 1.60 (s, -CH2CH2N-), δ 2.0 (m, -CH2CH2CH-), δ 2.03-2.45 (m, -CH2CH2N-, -CH2CH2CO, -CH(CH3)-), δ2.46-2.95 (m, -CH2CH2CO, -CH2CH2NH-) , δ 3.2-3.4 (m, -CH2CH2N-,-COCH2CH(CH3)CH2COOH), δ 5.35 (m, -CH2CHCHCH2-). Calc [M]-(C63H117N7O9) m/z 1115.9. Found ESI-MS [M- H]- m/z 1114.8 Thereafter, the compound was identified by mass spectrometry and 1H NMR. The results are shown in FIGS. (Yield 208 mg, Yield 20.5%); 1H NMR (CDCl 3 ): δ 0.88 (m, CH 3 (CH 2 ) 6- ), δ 1.03 (s, -CH (CH 3 )-), δ 1.2 -1.4 (m, CH 3 (CH 2 ) 6- , -CH 2 (CH 2 ) 5CH 2- ), δ 1.60 (s, -CH 2 CH 2 N-), δ 2.0 (m, -CH 2 CH 2 CH-), δ 2.03-2.45 (m, -CH 2 CH 2 N-, -CH 2 CH 2 CO, -CH (CH 3 )-), δ 2.46-2.95 (m, -CH 2 CH 2 CO, -CH 2 CH 2 NH-), δ 3.2-3.4 (m, -CH 2 CH 2 N-, -COCH 2 CH (CH 3 ) CH 2 COOH), δ 5.35 (m, -CH 2 CHCHCH 2- ). Calc [M]-(C 63 H 117 N 7 O 9 ) m / z 1115.9. Found ESI-MS [M- H]-m / z 1114.8
　２．Ｒ １ 及びＲ ２ が飽和炭化水素基である本発明の化合物の製造
　第1 世代のデンドロン脂質の合成経路を以下に示した。ジオクタデシルアミンを開始物質として、アクリル酸メチルを用いたマイケル付加反応と、エチレンジアミンを用いたエステルアミド交換反応を繰り返すことによりDL-G1-2C18 を合成した。 2. Production of the compound of the present invention in which R 1 and R 2 are saturated hydrocarbon groups The synthesis route of the first generation dendron lipids is shown below. Dioctadecyl amine as starting material, and the Michael addition reaction with methyl acrylate was synthesized DL-G1-2C 18 by repeating ester amide interchange reaction with ethylenediamine.
Figure JPOXMLDOC01-appb-C000033
Figure JPOXMLDOC01-appb-C000033
　２．１　DL-G-0.5-2C 18  の合成
　アクリル酸メチル(34.5 ml, 0.38 mmol)にジオクタデシルアミン(1.94 g, 3.72mmol)を溶かし、窒素雰囲気において80 ℃で18 時間還流した。その後、未反応のアクリル酸メチルとメタノールを減圧留去し、シリカゲルクロマトグラフィー（展開溶媒石油エーテル：ジエチルエーテル＝2：1, v/v）で精製した。（収量 2.18 g、収率 96.5%）；1H NMR (CDCl3): δ 0.88 (m, CH3(CH2) 15-), δ 1.23 (s,CH3 (CH2)15-), δ 1.41 (m, -CH2CH2N-), δ 2.38 (t, -CH2COOCH3), δ 2.44 (t,-CH2N-), δ 2.77 (t, -CH2CH2COOCH3), δ 3.67 (s, -OCH3). 2.1 Synthesis of DL-G-0.5-2C 18 Dioctadecylamine (1.94 g, 3.72 mmol) was dissolved in methyl acrylate (34.5 ml, 0.38 mmol) and refluxed at 80 ° C. for 18 hours in a nitrogen atmosphere. Thereafter, unreacted methyl acrylate and methanol were distilled off under reduced pressure, and the residue was purified by silica gel chromatography (developing solvent petroleum ether: diethyl ether = 2: 1, v / v). (Yield 2.18 g, 96.5% yield); 1H NMR (CDCl 3) : δ 0.88 (m, CH 3 (CH 2) 15 -), δ 1.23 (s, CH 3 (CH 2) 15 -), δ 1.41 (m, -CH 2 CH 2 N-), δ 2.38 (t, -CH 2 COOCH 3 ), δ 2.44 (t, -CH 2 N-), δ 2.77 (t, -CH 2 CH 2 COOCH 3 ), δ 3.67 (s, -OCH 3 ).
　２．２　DL-G0-2C 18  の合成
　DL-G-0.5(2.18 g, 3.59 mmol)をメタノール(60 ml)に溶かした。この溶液を、シアン化ナトリウム(40.3 mg, 0.82 mmol)を含むエチレンジアミン(120 ml, 1.77 mol)に徐々に加え、窒素雰囲気において50 ℃で7 日間撹拌した。その後、未反応のエチレンジアミンとメタノールを減圧留去し、シリカゲルクロマトグラフィー（展開溶媒クロロホルム：メタノール：水＝60：35：5, v/v）によって精製した。 2.2 Synthesis of DL-G0-2C 18 DL-G-0.5 (2.18 g, 3.59 mmol) was dissolved in methanol (60 ml). This solution was gradually added to ethylenediamine (120 ml, 1.77 mol) containing sodium cyanide (40.3 mg, 0.82 mmol) and stirred at 50 ° C. for 7 days in a nitrogen atmosphere. Thereafter, unreacted ethylenediamine and methanol were distilled off under reduced pressure and purified by silica gel chromatography (developing solvent chloroform: methanol: water = 60: 35: 5, v / v).
　その後、1H NMR により化合物の同定を行った。結果を図５２に示す。
（収量 1.71 g、収率 74.9%）；1H NMR (CDCl3): δ 0.88 (m, CH3(CH2)15-), δ1.26 (s, CH3(CH2)15-), δ 1.44 (m, -CH2CH2N-), δ 2.36 (t, -CH2CONH-), δ 2.42 (t,-CH2N-), δ 2.65 (t, -CH2CH2CONH-), δ 2.79 (t, -CH2NH2), δ 3.67 (m, -CH2CH2NH2). Then, the compound was identified by 1H NMR. The results are shown in FIG.
(Yield 1.71 g, 74.9% yield); 1H NMR (CDCl 3) : δ 0.88 (m, CH 3 (CH 2) 15 -), δ1.26 (s, CH 3 (CH 2) 15 -), δ 1.44 (m, -CH 2 CH 2 N-), δ 2.36 (t, -CH 2 CONH-), δ 2.42 (t, -CH 2 N-), δ 2.65 (t, -CH 2 CH 2 CONH-) , δ 2.79 (t, -CH 2 NH 2 ), δ 3.67 (m, -CH 2 CH 2 NH 2 ).
　２．３　DL-G0.5-2C 18  の合成
　DL-G0(1.71 g, 2.71 mmol)をメタノール(30 ml)に溶かした。この溶液を、アクリル酸メチル(48.5 ml, 0.53 mol)に徐々に加え、窒素雰囲気において35 ℃で50 時間撹拌した。その後、未反応のアクリル酸メチルとメタノールを減圧留去し、シリカゲルクロマトグラフィー（展開溶媒石油エーテル：ジエチルエーテル＝2：1, v/v のち クロロホルム：メタノール＝95：5, v/v）で精製した。 2.3 Synthesis of DL-G0.5-2C 18 DL-G0 (1.71 g, 2.71 mmol) was dissolved in methanol (30 ml). This solution was gradually added to methyl acrylate (48.5 ml, 0.53 mol) and stirred at 35 ° C. for 50 hours in a nitrogen atmosphere. Then, unreacted methyl acrylate and methanol were distilled off under reduced pressure, and purified by silica gel chromatography (developing solvent petroleum ether: diethyl ether = 2: 1, v / v, then chloroform: methanol = 95: 5, v / v). did.
　その後、1H NMR により化合物の同定を行った。結果を図５３に示す。（収量1.85 g、収率 85.0%）；1H NMR (CDCl3): δ 0.88 (m, CH3(CH2)15-), δ 1.26 (s, CH3(CH2)15-), δ 1.43 (m, -CH2CH2N-), δ 2.34 (t, -CH2CONH-), δ 2.42 (m,-CH2COOCH3), δ 2.44 (m, -CH2N-), δ 2.54 (t, -CONHCH2CH2-), δ 2.71 (t, -CH2CH2CONH-), δ 2.78 (t, -CH2CH2COOCH3), δ 3.29 (m, -CONHCH2-), δ 3.67(s, -OCH3). Then, the compound was identified by 1H NMR. The results are shown in FIG. (Yield 1.85 g, 85.0% yield); 1H NMR (CDCl 3) : δ 0.88 (m, CH 3 (CH 2) 15 -), δ 1.26 (s, CH 3 (CH 2) 15 -), δ 1.43 (m, -CH 2 CH 2 N-), δ 2.34 (t, -CH 2 CONH-), δ 2.42 (m, -CH 2 COOCH 3 ), δ 2.44 (m, -CH 2 N-), δ 2.54 (t, -CONHCH 2 CH 2- ), δ 2.71 (t, -CH 2 CH 2 CONH-), δ 2.78 (t, -CH 2 CH 2 COOCH 3 ), δ 3.29 (m, -CONHCH 2- ), δ 3.67 (s, -OCH 3 ).
　２．４　DL-G1-2C 18  の合成
　DL-G0.5(1.8 g, 2.3 mmol)をメタノール(61 ml)に溶かした。この溶液を、シアン化ナトリウム(27 mg, 0.55 mmol)を含むヒドラジン (80 ml, 1.2 mol)に徐々に加え、窒素雰囲気において45 ℃で64 時間撹拌した。その後、未反応のエチレンジアミンとメタノールを減圧留去し、Sephadex LH-20 カラム（溶離液 クロロホルム）によって精製した。 2.4 Synthesis DL-G0.5 (1.8 g, 2.3 mmol) of DL-G1-2C 18 was dissolved in methanol (61 ml). This solution was gradually added to hydrazine (80 ml, 1.2 mol) containing sodium cyanide (27 mg, 0.55 mmol) and stirred at 45 ° C. for 64 hours in a nitrogen atmosphere. Thereafter, unreacted ethylenediamine and methanol were distilled off under reduced pressure and purified by Sephadex LH-20 column (eluent chloroform).
　その後、1H NMR により化合物の同定を行った。結果を図５４に示す。
（収量 1.9 g、収率 97.0%）；1H NMR (CDCl3): δ0.88 (m, CH3(CH2)15-), δ 1.26 (s, CH3(CH2)15-), δ 1.42 (m, -CH2CH2N-), δ 2.32(m, -CH2CONHCH2CH2NH2), δ 2.36 (m, -CH2CONH-), δ 2.42 (m, -CH2N-), δ2.50 (t, -CONHCH2CH2-), δ 2.67 (t, -CH2CH2CONH-), δ 2.74 (t,-CH2CH2CONHCH2CH2NH2), δ 2.83 (t, -CH2NH2), δ 3.22 (m, -CONHCH2-), δ3.29 (m, -CH2CH2NH2). Then, the compound was identified by 1H NMR. The results are shown in FIG.
(Yield 1.9 g, 97.0% yield); 1H NMR (CDCl 3) : δ0.88 (m, CH 3 (CH 2) 15 -), δ 1.26 (s, CH 3 (CH 2) 15 -), δ 1.42 (m, -CH 2 CH 2 N-), δ 2.32 (m, -CH 2 CONHCH 2 CH 2 NH 2 ), δ 2.36 (m, -CH 2 CONH-), δ 2.42 (m, -CH 2 N -), δ2.50 (t, -CONHCH 2 CH 2- ), δ 2.67 (t, -CH 2 CH 2 CONH-), δ 2.74 (t, -CH 2 CH 2 CONHCH 2 CH 2 NH 2 ), δ 2.83 (t, -CH 2 NH 2 ), δ 3.22 (m, -CONHCH 2 -), δ3.29 (m, -CH 2 CH 2 NH 2).
　２．５　CHex-DL-G1-2C 18  の合成
　Cyclohexanedicarboxylic Anhydride (以下CHexAn) 265 mg(1.63 mmol)をDMF5 mL に溶解させた。その後、DL-G1-2C18 の145.3 mg(0.307 mmol)をDMF 5 mLに溶解したのち混合し、triethylamine(TEA)を0.125 mL 加えて50 ℃で25 時間窒素雰囲気化で反応させた。
反応終了後、エバポレータにより溶媒を留去した後、LH-20 column ( 溶離液:methanol) により精製し、silica gel columnchromatography (展開溶媒:chloroform/methanol=80/20) によりさらに精製を行った。その後、質量分析及び1H NMR により化合物の同定を行った。 2.5 CHex-DL-G1-2C 18 Synthesis Cyclohexanedicarboxylic Anhydride (hereinafter CHexAn) 265 mg of (1.63 mmol) was dissolved in DMF5 mL. Then, 145.3 mg (0.307 mmol) of DL-G1-2C18 was dissolved in 5 mL of DMF and mixed. Then, 0.125 mL of triethylamine (TEA) was added and reacted at 50 ° C. for 25 hours under nitrogen atmosphere.
After completion of the reaction, the solvent was distilled off with an evaporator, followed by purification with LH-20 column (eluent: methanol) and further purification with silica gel column chromatography (developing solvent: chloroform / methanol = 80/20). Thereafter, the compound was identified by mass spectrometry and 1H NMR.
　その後、質量分析及び1H NMR により化合物の同定を行った。結果を図５５及び図５６に示す。 Thereafter, the compound was identified by mass spectrometry and 1H NMR. The results are shown in FIGS. 55 and 56.
　（収量 80.3mg、収率 22.3 %）；1H NMR (CD3OD) : δ 0.88 (m, CH3(CH2)15-), δ 1.26 (s, CH3(CH2)15-), δ 1.42 (m, -CH2CH2N-),δ1.4-1.9 (m, -COCH-(CH2)4-CHCOOH) , δ2.32 (m, -CH2CONHCH2CH2NH2), δ 2.36 (m, -CH2CONH-), δ 2.42 (m, -CH2N-),δ 2.50 (t, -CONHCH2CH2-), δ 2.6-2.8 (m, -COCH-CH2), δ 2.67 (t, -CH2CH2CONH-), δ 2.74 (t, -CH2CH2CONHCH2CH2NH2), δ 2.83 (t, -CH2NH2), δ3.22 (m, -CONHCH2-), δ 3.29 (m, -CH2CH2NH2).Calc [M]- (C67H121N7O9) m/z1172.0. Found ESI-MS [M- H]- m/z 1170.8 (Yield 80.3 mg, 22.3% yield); 1H NMR (CD 3 OD ): δ 0.88 (m, CH 3 (CH 2) 15 -), δ 1.26 (s, CH 3 (CH 2) 15 -), δ 1.42 (m, -CH 2 CH 2 N-), δ1.4-1.9 (m, -COCH- (CH 2 ) 4 -CHCOOH), δ2.32 (m, -CH 2 CONHCH 2 CH 2 NH 2 ), δ 2.36 (m, -CH 2 CONH-), δ 2.42 (m, -CH 2 N-), δ 2.50 (t, -CONHCH 2 CH 2- ), δ 2.6-2.8 (m, -COCH-CH 2 ) , δ 2.67 (t, -CH 2 CH 2 CONH-), δ 2.74 (t, -CH 2 CH 2 CONHCH 2 CH 2 NH 2 ), δ 2.83 (t, -CH 2 NH 2 ), δ 3.22 (m , -CONHCH 2- ), δ 3.29 (m, -CH 2 CH 2 NH 2 ) .Calc [M]-(C 67 H 121 N 7 O 9 ) m / z1172.0. Found ESI-MS [M- H ]-m / z 1170.8
　３．作製したデンドロン脂質の性能評価
　３．１　デンドロン脂質の単分子膜の作製と測定
　LB 膜測定装置の圧力計を校正し(100mg 分銅に対し48.28mN/m)、トラフとバリアをエタノールによって洗浄した。pH を調整したPBS(リン酸水素二ナトリウム：10 mM、塩化ナトリウム：150 mM)を作製し、種々の脂質溶液をLB 膜測定装置のトラフ上に250 mL を張り、1cm2 のろ紙を測定機に設置したのち10 分間静地し圧力計を安定させた。10 分後の表面圧をP0 とした。クロロホルムのデンドロン脂質溶液をその上に展開し、20 分静置してクロロホルムを飛ばした。LB膜測定装置π-A モードで、溶媒表面をバリアで側方圧縮することで溶媒表面の脂質分子の分子表面積を減少させたときの脂質単分子膜の表面圧を圧力計により測定した測定した。再び測定する際は、トラフ上の溶媒を回収し、トラフとバリアをエタノールにより洗浄し、溶媒を展開した。 3. 3. Performance evaluation of the prepared dendron lipid 3.1 Preparation and measurement of dendron lipid monolayer The pressure gauge of the LB membrane measuring device was calibrated (48.28 mN / m for 100 mg weight), and the trough and barrier were washed with ethanol. Prepare pH-adjusted PBS (disodium hydrogen phosphate: 10 mM, sodium chloride: 150 mM), place 250 mL of various lipid solutions on the trough of the LB membrane measuring device, and use a 1 cm2 filter paper as the measuring machine. After installation, the pressure gauge was stabilized by standing still for 10 minutes. The surface pressure after 10 minutes was defined as P0. A dendron lipid solution in chloroform was spread on it and left to stand for 20 minutes to blow off the chloroform. LB membrane measurement device In π-A mode, the surface pressure of the lipid monolayer was measured with a pressure gauge when the molecular surface area of the lipid surface on the solvent surface was reduced by laterally compressing the solvent surface with a barrier. . When measuring again, the solvent on the trough was collected, the trough and the barrier were washed with ethanol, and the solvent was developed.
　３．２　リポソームの物性評価
　３．２．１　リポソームのpH 応答評価
　３．２．１．１　Pyranine-DPX 内包リポソームの調整
　MGlu-DL-G1-2C18-U2 修飾リポソーム(以下UMG1 と呼称する)は、所定量のEYPC(10mg/ml)のクロロホルム溶液と、所定量のMGlu-DL-G1-2C18-U2 (6.98mg/ml) のクロロホルム溶液を混合し、ロータリーエバポレーターで溶媒を除去し薄膜を形成させ、4 時間真空乾燥することで溶媒を完全に除去した。 3.2 Physical property evaluation of liposome 3.2.1 pH response evaluation of liposome 3.2.1.1 Preparation of liposome containing Pyranine-DPX MGlu-DL-G1-2C 18 -U2 modified liposome (hereinafter referred to as UMG1) Mix a predetermined amount of EYPC (10 mg / ml) in chloroform with a predetermined amount of MGlu-DL-G1-2C 18 -U2 (6.98 mg / ml) in chloroform, and remove the solvent with a rotary evaporator to remove the thin film. And the solvent was completely removed by vacuum drying for 4 hours.
　CHex-DL-G1-2C18-U2 リポソーム( 以下UCG1 と呼称する) は所定量のEYPC(10mg/ml) のクロロホルム溶液と、所定量のCHex-DL-G1-2C18-U2 (3.65mg/ml) のクロロホルム溶液を混合しロータリーエバポレーターで溶媒を除去し薄膜を形成させ、4 時間真空乾燥することで溶媒を完全に除去した。EYPC リポソームは、EYPC のクロロホルム溶液を所定量加え、ロータリーエバポレーターで溶媒を除去し薄膜を形成させ、4 時間真空乾燥することで溶媒を完全に除去した。 CHex-DL-G1-2C 18 -U2 liposome (hereinafter referred to as UCG1) is prepared by mixing a predetermined amount of EYPC (10 mg / ml) in chloroform and a predetermined amount of CHex-DL-G1-2C 18 -U2 (3.65 mg / ml). ml) of chloroform solution was mixed and the solvent was removed by a rotary evaporator to form a thin film, followed by vacuum drying for 4 hours to completely remove the solvent. For EYPC liposomes, a predetermined amount of EYPC in chloroform was added, the solvent was removed with a rotary evaporator to form a thin film, and the solvent was completely removed by vacuum drying for 4 hours.
　それぞれの薄膜の脂質量1.25×10-5mol に対して、組成がpyranine 35mM , DPX50mM , Na2HPO4 25mM の溶液を作製し1.0 mL 加え、バス型超音波照射装置により超音波を2 分間照射し薄膜を剥がした後に、pH を7.4 に調整した。凍結融解を5 回行い、エクストルーダーに膜孔100nm の膜を重ねリポソーム溶液を61回(30 往復)通すことによって、リポソーム粒径を100nm にそろえた。 PBS(phosphate 25 mM, saline 150 mM ; pH7.4)で平衡化したセファロース4B カラムによりリポソームに内包されていないピラニン, DPX を除くことで精製を行った。 Prepare a solution with a composition of pyranine 35 mM, DPX 50 mM, Na 2 HPO 4 25 mM for each thin film lipid amount of 1.25 x 10 -5 mol, add 1.0 mL, and irradiate ultrasonic waves with a bath-type ultrasonic irradiation device for 2 minutes. After removing the thin film, the pH was adjusted to 7.4. Freezing and thawing were performed 5 times, and the liposome particle size was adjusted to 100 nm by placing a membrane with a 100 nm membrane pore through the extruder and passing the liposome solution 61 times (30 reciprocations). Purification was performed by removing pyranin and DPX not encapsulated in liposomes using a Sepharose 4B column equilibrated with PBS (phosphate 25 mM, saline 150 mM; pH 7.4).
　３．２．１．２　脂質の定量
　リン脂質の定量は、リン脂質C テストワコー（和光純薬工業）を用いて、コリンオキシターゼ・DAOS 法によって行った。試料溶液（リポソ―ム溶液）、ブランク溶液及び、標準溶　液をそれぞれ発色溶液と混合し37℃で5 分間インキュベートした。波長600nm で試料溶液の吸光度を日本分光（株）製V-560 型紫外・可視光光度計を用いて測定し、得られた吸光度から試料溶液の濃度を決定した。 3.2.1.2 Quantification of lipids Phospholipids were quantified by the choline oxidase / DAOS method using Phospholipid C Test Wako (Wako Pure Chemical Industries). The sample solution (liposome solution), blank solution and standard solution were mixed with the coloring solution and incubated at 37 ° C. for 5 minutes. The absorbance of the sample solution was measured at a wavelength of 600 nm using a V-560 ultraviolet / visible photometer manufactured by JASCO Corporation, and the concentration of the sample solution was determined from the obtained absorbance.
　３．２．１．３　リポソームのpH 応答評価
　ピラニン内包リポソームから放出されるピラニンを416 nm の光で励起し、発せられる蛍光を512 nm で測定することにより、ポリマー修飾リポソームのpH 及び温度応答性の評価を行った。石英セル内に各pH に調製したPBS 溶液を加え、蛍光分光光度計内に設置した。表示温度が37℃になった事を確認した後、蛍光分光光度計で石英セル内の脂質濃度が0.05 mM となるように脂質溶液を各pH に調整したPBS に加えた(最終体積2.5ml)。15 分間インキュベーションした時のピラニンの放出量を調べた。最後に10％Toriton を25μl 加えてリポソームを破壊した。そのときの蛍光強度を100％とし各リポソームからの内包物の放出割合を求めた。蛍光強度の測定は分光蛍光光度計（JASCO 製 FP-6200、FP-6500）および温度コントローラ（JASCO 製 ETC-272T）を用いて37℃で行った。 3.2.1.3 Evaluation of pH response of liposomes The pH and temperature responsiveness of polymer-modified liposomes were determined by exciting pyranin released from pyranine-encapsulated liposomes with 416 nm light and measuring the emitted fluorescence at 512 nm. Was evaluated. A PBS solution prepared for each pH was added to a quartz cell and placed in a fluorescence spectrophotometer. After confirming that the indicated temperature was 37 ° C, the lipid solution was added to PBS adjusted to each pH so that the lipid concentration in the quartz cell was 0.05 mM with a fluorescence spectrophotometer (final volume 2.5 ml). . The amount of pyranine released after 15 minutes of incubation was examined. Finally, 25 μl of 10% Toriton was added to break the liposomes. The fluorescence intensity at that time was taken as 100%, and the release rate of inclusions from each liposome was determined. The fluorescence intensity was measured at 37 ° C. using a spectrofluorimeter (JASCO FP-6200, FP-6500) and a temperature controller (JASCO ETC-272T).
　３．２．２　動的光散乱を用いたリポソームの平均粒径及びζ電位のpH 依存性
　３．２．２．１　リポソームの調整
　先述のように薄膜を作製し、薄膜の脂質量1.25×10-5mol に対して、0.1 mM のリン酸バッファーを1.0 mL 加え、バス型超音波照射装置により超音波を2 分間照射し薄膜を剥がした後に、pH を7.4 に調整した。凍結融解を5 回行い、エクストルーダーに膜孔100nm の膜を重ねリポソーム溶液を61 回(30 往復)通すことによって、リポソーム粒径を100nm にそろえた。 3.2.2 Lipid average particle size and zeta potential pH dependence using dynamic light scattering 3.2.2.1 Preparation of liposomes A thin film was prepared as described above, and the lipid content of the thin film was 1.25 x 10 To -5 mol, 1.0 mL of 0.1 mM phosphate buffer was added, and ultrasonic waves were applied for 2 minutes with a bath-type ultrasonic irradiation device to remove the thin film, and then the pH was adjusted to 7.4. Freezing and thawing was performed 5 times, and a liposome with a 100 nm membrane pore was placed on the extruder and the liposome solution was passed 61 times (30 reciprocations) to adjust the liposome particle size to 100 nm.
　３．２．２．２　リポソームの平均粒径及びζ電位の測定
　リポソームの粒径及びζ電位は動的光散乱法によって求めた。リポソーム溶液を、セル内の脂質濃度が0.1mM となるように脂質溶液を各pH に調整したPBS(－)または0.1 mM のリン酸バッファーに加えた(最終体積2.5ml)。15 分静置し、粒径及びζ電位を測定した。
測定は25℃において行い、大塚電子(株)製ELS-8000F を用いて測定した。 3.2.2.2 Measurement of average particle size and ζ potential of liposomes The particle size and ζ potential of liposomes were determined by a dynamic light scattering method. The liposome solution was added to PBS (-) or 0.1 mM phosphate buffer (final volume 2.5 ml) adjusted to each pH so that the lipid concentration in the cell was 0.1 mM. The mixture was allowed to stand for 15 minutes, and the particle size and ζ potential were measured.
The measurement was performed at 25 ° C., and was measured using ELS-8000F manufactured by Otsuka Electronics Co., Ltd.
　３．２．２．３　リポソームの濁度のpH 依存性
　liposome を脂質濃度が0.5 mM となるようにpH7.4 のPBS(-)に分散させて37 ℃でpH を連続的に変化させてそれぞれのpH における500 nm の光の透過率を日本分光(株)製V-560 型 紫外・可視分光光度計を用いて測定した。 3.2.2.3 pH dependence of liposome turbidity Liposomes are dispersed in PBS (-) pH 7.4 so that the lipid concentration is 0.5 mM, and the pH is continuously changed at 37 ° C. The transmittance of light at 500 nm was measured using a V-560 type ultraviolet / visible spectrophotometer manufactured by JASCO Corporation.
　３．３　種々のデンドロン脂質を修飾したリポソームの細胞への影響
　３．３．１　培養細胞
　３．３．１．１　マウス由来の細胞培養株DC2.4 細胞
　マウス由来の細胞培養株DC2.4 細胞は10% FBS、0.1 mg/ml ベンジルペニシリンカリウム、0.1 mg/ml ストレプトマイシン硫酸塩、2 mM L-グルタミン、0.1mM MEM non essential amino acid solution および0.55 mM 2-mercaptoethanolを含むRPMI-1640 メディウムを培養液として、三洋電機製CO2 インキュベーター（MCO-96）内で、CO2 濃度5％、37℃で培養した。 3.3 Effects of various dendron lipid-modified liposomes on cells 3.3.1 Cultured cells 3.3.1.1 Mouse-derived cell culture strain DC2.4 cells Mouse-derived cell culture strain DC2.4 cells Is RPMI-1640 medium containing 10% FBS, 0.1 mg / ml benzylpenicillin potassium, 0.1 mg / ml streptomycin sulfate, 2 mM L-glutamine, 0.1 mM MEM non essential amino acid solution and 0.55 mM 2-mercaptoethanol. As an example, the cells were cultured in a CO 2 incubator (MCO-96) manufactured by Sanyo Electric Co. at a CO 2 concentration of 5% at 37 ° C.
　３．３．１．２　ヒト子宮頸がん由来HeLa 細胞
　ヒト子宮頸がん由来HeLa 細胞は、DMEM + 10%FBS + 抗生物質を培養液として、三洋電機製 CO2 インキュベーター MCO-96 内で、CO2 濃度5%、37 ℃で培養維持した。 3.3.1.2 human cervical cancer-derived HeLa Cells Human cervical carcinoma-derived HeLa cells, the DMEM + 10% FBS + antibiotics as cultures in Sanyo CO 2 incubator within MCO-96, The culture was maintained at 37 ° C. with a CO 2 concentration of 5%.
　３．４　フローサイトメトリーによるリポソーム取り込み量の定量
　３．４．１　マウス由来の細胞培養株DC2.4 細胞
　DC2.4 細胞を1 × 105/well となるように12well プレートに播き、二晩培養した。HBSS で2 回洗浄した後、10% FBS 含有RPMI メディウムを適量加えた。そこに1 穴当たりリポソーム溶液を脂質濃度が0.5mM となるように加え、4 時間取り込ませた。HBSS で3 回洗浄したのち、1 穴につき300 μl のトリプシン水溶液（トリプシン（DIFCO）250 mg、エチレンジアミン四酢酸二ナトリウム（EDTA）1 mg、PBS 100 ml）を用いて細胞を剥離し、フローサイトメーター用のチューブに回収した。回収した細胞トリプシン溶液を、フローサイトメーターBeckman Coulter.XL を用いて細胞の蛍光強度を測定し、細胞へのリポソームの取り込み量を評価した。 3.4 Quantification of liposome uptake by flow cytometry 3.4.1 Mouse-derived cell culture strain DC2.4 cells DC2.4 cells are seeded on a 12-well plate at 1 × 10 5 / well and cultured overnight. did. After washing twice with HBSS, an appropriate amount of RPMI medium containing 10% FBS was added. The liposome solution per well was added so that the lipid concentration was 0.5 mM, and allowed to be taken in for 4 hours. After washing 3 times with HBSS, the cells are detached using 300 μl of trypsin aqueous solution (trypsin (DIFCO) 250 mg, disodium ethylenediaminetetraacetate (EDTA) 1 mg, PBS 100 ml) per well, and a flow cytometer It collected in the tube for use. The collected cell trypsin solution was measured for the fluorescence intensity of the cells using a flow cytometer Beckman Coulter.XL, and the amount of liposomes taken into the cells was evaluated.
　３．４．２　ヒト子宮頸がん由来HeLa 細胞
　HeLa 細胞を12 穴ディッシュ1 穴当たり1×105 個になるように撒き、10% FBS含有DMEM メディウム0.5 ml 中、37 ℃で48 時間培養した。その後、0.36 mMCaCl2と0.42 mM MgCl2を含むPBS(PBS(+))で2 回洗浄した後、10% FBS 含有DMEM メディウム 0.5 ml を加えた。そこに1 穴当たりリポソーム溶液を脂質濃度が0.5mM となるように加え、37 ℃で4 時間インキュベートした。その後、PBS(+)で2 回、PBS(－) （Ca2+及びMg2+を含まないPBS）で1 回洗浄し、1 穴につき300 μl のトリプシン水溶液（トリプシン（DIFCO）250 mg、エチレンジアミン四酢酸二ナトリウム（EDTA）1 mg、PBS 100 ml）を用いて細胞を剥離し、フローサイトメーター用のチューブに回収した。回収した細胞トリプシン溶液を、フローサイトメーターBeckman Coulter.XL を用いて細胞の蛍光強度を測定し、細胞へのリポソームの取り込み量を評価した。 3.4.2 Human cervical cancer-derived HeLa cells HeLa cells were seeded at 1 × 10 5 per well of a 12-well dish and cultured at 37 ° C for 48 hours in 0.5 ml of 10% FBS-containing DMEM medium. . Then, after washing twice with PBS containing 0.36 mM CaCl 2 and 0.42 mM MgCl 2 (PBS (+)), 0.5 ml of DMEM medium containing 10% FBS was added. The liposome solution was added to each well so that the lipid concentration was 0.5 mM, and incubated at 37 ° C. for 4 hours. Then, wash twice with PBS (+) and once with PBS (-) (PBS without Ca2 + and Mg2 +). 300 μl of trypsin aqueous solution (250 mg trypsin (DIFCO) per well, disodium ethylenediaminetetraacetate (EDTA) 1 mg, PBS 100 ml) was used to detach cells and collected in a tube for a flow cytometer. The collected cell trypsin solution was measured for the fluorescence intensity of the cells using a flow cytometer Beckman Coulter.XL, and the amount of liposomes taken into the cells was evaluated.
　３．４．３　細胞内挙動の観察
　３．４．３．１　マウス由来の細胞培養株DC2.4 細胞
DC2.4 細胞を松浪ガラスボトムディッシュに1 つ当たり2×105 個になるように播き、二晩培養した。HBSS で2 回洗浄した後、10% FBS 含有RPMI メディウムを1 mL 加えた。そこに1 穴当たりリポソーム溶液を脂質濃度が0.5mM となるように加え(全体積2 mL)、4 時間取り込ませた。HBSS で3 回洗浄したのちOPTI-MEM を加え、共焦点レーザー顕微鏡(LSM 5 EXCITER(ZEISS))により細胞内動態を観察した。 3.4.3 Observation of intracellular behavior 3.4.3.1 Cell culture strain DC2.4 cells derived from mice
DC2.4 cells were seeded at 2 × 10 5 per Matsunami glass bottom dish and cultured overnight. After washing twice with HBSS, 1 mL of 10% FBS-containing RPMI medium was added. Thereto, the liposome solution was added per well so that the lipid concentration was 0.5 mM (total volume 2 mL) and allowed to be taken in for 4 hours. After washing three times with HBSS, OPTI-MEM was added, and intracellular dynamics were observed with a confocal laser microscope (LSM 5 EXCITER (ZEISS)).
　３．４．３．２　ヒト子宮頸がん由来HeLa 細胞
　Hela 細胞を松浪ガラスボトムディッシュに1 つ当たり2×105 個になるように撒き、10% FBS 含有DMEM メディウム2.0 ml 中、37 ℃で二晩培養した。その後、PBS(+)で2 回、PBS(－)で1 回洗浄した後、10% FBS 含有DMEMメディウム 1.0ml を加えた。そこに1 穴当たりリポソーム溶液を脂質濃度が0.5mM となるように加え(全体積2 mL)、37 ℃で4 時間又は24時間インキュベートした。その後、PBS(+)で2回、PBS(－)で1 回洗浄した後OPTI-MEM を加え、共焦点レーザー顕微鏡(LSM5 EXCITER(ZEISS))により細胞内動態を観察した。 3.4.3.2 Human cervical cancer-derived HeLa cells Hela cells are seeded on a Matsunami glass bottom dish at 2 × 10 5 cells per plate, in 10 ml FBS-containing DMEM medium at 37 ° C. Incubated overnight. Thereafter, after washing twice with PBS (+) and once with PBS (−), 1.0 ml of 10% FBS-containing DMEM medium was added. The liposome solution was added to each well so that the lipid concentration was 0.5 mM (total volume: 2 mL), and incubated at 37 ° C. for 4 hours or 24 hours. Then, after washing twice with PBS (+) and once with PBS (−), OPTI-MEM was added, and intracellular dynamics were observed with a confocal laser microscope (LSM5 EXCITER (ZEISS)).
　３．４．３．３　細胞オルガネラの染色
　３．４．３．３．１　Hoechst33342 による核の染色
　リポソームを上記の方法で取り込ませた後、10% FBS 含有RPMI 1640 またはDMEM メディウム 1998 μL と2.0 μL のHoechst33342（Invitrogen）を加え、37 ℃で15 分間培養した。その後、3 回洗浄した後PBS を再び加え、共焦点レーザー顕微鏡(LSM 5 EXCITER(ZEISS))により細胞内動態を観察した。 3.4.3.3 Cell organelle staining 3.4.3.3 Nuclear staining with Hoechst33342 After incorporation of liposomes by the above method, RPMI 1640 or DMEM medium containing 10% FBS 1998 μL and 2.0 μL Hoechst33342 (Invitrogen) was added and incubated at 37 ° C. for 15 minutes. Thereafter, after washing three times, PBS was added again, and intracellular dynamics were observed with a confocal laser microscope (LSM 5 EXCITER (ZEISS)).
　３．４．３．３．２　Lysotracker による後期エンドソーム/リソソームの染色
　Lysotracker (Invitrogen, Lysotracker Green DND-26, Lysotracker RedDND-99,DMSO 溶液 : 1mM）を1 μL 取り、FBS を含有しないRPMI 1640 またはDMEM メディウム99 μL 加えて染色液を調製した。リポソームを上記の方法で取り込ませた後、FBS を含有しないRPMI 1640 またはDMEM メディウム1985 μL と染色液15 μL 加え、5 分間培養した。その後、3 回洗浄した後PBS を再び加え、共焦点レーザー顕微鏡(LSM 5EXCITER(ZEISS))により細胞内動態を観察した。 3.4.3.3 Lysotracker staining of late endosomes / lysosomes Take 1 μL of Lysotracker (Invitrogen, Lysotracker Green DND-26, Lysotracker RedDND-99, DMSO solution: 1 mM) and RPMI 1640 or DMEM without FBS A staining solution was prepared by adding 99 μL of medium. Liposomes were taken up by the method described above, and then 1985 μL of RPMI 1640 or DMEM medium not containing FBS and 15 μL of staining solution were added and incubated for 5 minutes. Thereafter, after washing three times, PBS was added again, and intracellular dynamics were observed with a confocal laser microscope (LSM 5EXCITER (ZEISS)).
　３．４．３．３．３　蛍光ラベル化トランスフェリンによる初期エンドソーム染色
　Transferin Conjugates (Invitrogen, transferrin from human serum, Alexa Fulor555 conjugate)を所定の濃度(5 mg/mL, sodium azido 2 mM)となるようにイオン交換水で溶解し、染色液を調製した。リポソームを上記の方法で取り込ませた後、FBS を含有しないRPMI 1640 またはDMEM メディウムと染色液を加え、30 分間培養した。その後、3 回洗浄した後PBS を再び加え、共焦点レーザー顕微鏡(LSM 5 EXCITER(ZEISS))により細胞内動態を観察した。 3.4.3.3 Initial staining of endosome with fluorescently labeled transferrin Transferin Conjugates (Invitrogen, transferrin from human serum, Alexa Fulor555 conjugate) to a predetermined concentration (5 mg / mL, sodium azido 2 mM) The dyeing solution was prepared by dissolving in ion exchange water. After the liposomes were taken up by the above method, RPMI 1640 or DMEM medium not containing FBS and a staining solution were added and incubated for 30 minutes. Thereafter, after washing three times, PBS was added again, and intracellular dynamics were observed with a confocal laser microscope (LSM 5 EXCITER (ZEISS)).
　３．５　二種類のpH 応答性デンドロン脂質により構成されるリポソームの評価
　ｐＨ応答性の異なる２種以上の本発明の化合物を適宜組み合わせて用いてベシクルを構成することにより、所望のｐＨ応答性を有するベシクルを得ることができるか否かを下記の通り検証した。 3.5 Evaluation of liposomes composed of two kinds of pH-responsive dendron lipids A desired responsiveness can be obtained by constructing a vesicle by appropriately combining two or more compounds of the present invention having different pH responsiveness. It was verified as follows whether or not vesicles having the same could be obtained.
　３．５．１　ピラニン内包リポソームの作製
　EYPCのchloroform溶液(10 mg/mL)とCHex-DL-G1-2C18-U2のchloroform溶液(3.65 mg/mL)を所定量eggplant flaskに加えて混合しrotary evaporatorで溶媒を留去し、薄膜を作製した。ここにピラニン水溶液(35 mM pyranine, 50 mM DPX, 25 mM Na2HPO4)を500 μL加え、バス型超音波照射装置により薄膜を分散させた。凍結融解を5回行い、extruderを用いて粒径を100 nmに揃え、Sepharose 4B columnにより精製した。その後、テストワコーにより脂質濃度を測定した。 3.5.1 Preparation of pyranin- encapsulated liposomes Add EYPC chloroform solution (10 mg / mL) and CHex-DL-G1-2C 18 -U2 chloroform solution (3.65 mg / mL) to a predetermined amount of eggplant flask and mix. The solvent was distilled off with a rotary evaporator to produce a thin film. 500 μL of an aqueous pyranine solution (35 mM pyranine, 50 mM DPX, 25 mM Na 2 HPO 4 ) was added thereto, and the thin film was dispersed with a bath-type ultrasonic irradiation device. Freeze-thaw was performed 5 times, and the particle size was adjusted to 100 nm using an extruder, and purified using a Sepharose 4B column. Thereafter, the lipid concentration was measured by Test Wako.
　３．５．２　ピラニン放出挙動の測定
　上記で得られたliposomeを、PBS(-)に脂質濃度が50 μMとなるように蛍光セル中に加え、ピラニンの蛍光測定(37 ℃、λEX : 416 nm、λEM : 512 nm)を行い、放出率を算出した。 3.5.2 Measurement of pyranine release behavior The liposome obtained above was added to PBS (−) in a fluorescent cell so that the lipid concentration was 50 μM, and the fluorescence measurement of pyranine (37 ° C., λ EX : 416 nm, λ EM : 512 nm), and the release rate was calculated.
　４．　結果
　４．１　デンドロン部位の疎水性の異なる種々のデンドロン脂質の1H NMR によるキャラクタリゼーション
　デンドロン脂質は実験項に示した通り、オレイルアミン、オレオイルクロリドによりオレイルオレオイルアミドを合成した後、ヒドリド還元によりジオレイルアミンを合成し、メタノール中においてアクリル酸メチルおよびエチレンジアミンを繰り返し反応させることによって合成した。同定は世代数1 のデンドロン脂質までは1H NMR により、pH 応答機能を付与したデンドロン脂質については1H NMR 及び質量分析により行い、合成できていることを確認した。 4). Results 4.1 Characterization of various dendron lipids with different hydrophobicity at the dendron site by 1H NMR As shown in the experimental section, dendron lipids were synthesized by synthesizing oleyl oleoylamide with oleylamine and oleoyl chloride, followed by dihydride reduction. Oleylamine was synthesized and synthesized by repeatedly reacting methyl acrylate and ethylenediamine in methanol. Identification was performed by 1H NMR up to generation 1 dendron lipids, and by 1H NMR and mass spectrometry analysis of dendron lipids with a pH-responsive function.
　なお、pH 応答性デンドロン脂質は、それぞれ導入した末端基由来のピークの出現により合成ができたことを確認した。MGluDL-U2 では1.03 ppm 付近の3-メチルグルタル酸のメチル基由来のピーク(6H)の出現により、CHexDL-U2 及びCHexDL-S では1.2～2.0 ppm にシクロヘキサンジカルボン酸のシクロヘキサン由来のピーク(16H)の出現により合成ができたことを確認した。また、質量分析についてはESI-negative モードにより測定を行ったところ、理論値と同等の値を示したことから合成が確かにできていることを確認した。 It was confirmed that pH -responsive dendron lipids could be synthesized by the appearance of peaks derived from the introduced terminal groups. In MGluDL-U2, the peak derived from the methyl group of 3-methylglutaric acid (6H) near 1.03 ppm appeared, and in CHexDL-U2 and CHexDL-S, the peak derived from cyclohexanedicarboxylic acid (16H) at 1.2-2.0 ppm It was confirmed that synthesis was possible by the appearance of. In addition, when mass spectrometry was performed in the ESI-negative mode, it was confirmed that the synthesis was surely performed because it showed a value equivalent to the theoretical value.
　４．２　単分子膜測定
　作製したpH 応答性デンドロン脂質の単分子膜を作製し、その表面圧を測定した。
その結果を図５７に示す。 4.2 Measurement of monomolecular film A monomolecular film of the prepared pH-responsive dendron lipid was prepared, and its surface pressure was measured.
The result is shown in FIG.
　どのデンドロン脂質でも中性あるいは弱酸性で圧力の立ち上がる表面積が最少となり、酸性になるにつれて、あるいは塩基性で圧力の上がり始める表面積が大きくなっている。
これは、デンドロン脂質の電荷状態が変化するために起こったものであると考えられる。
また、CHexDL-S はCHexDL-U2 やMGluDL-U2に比べて圧力の上がり始める表面積が大きい。
次に、表面圧が25 mN/m となるときの表面積を各pH についてプロットしたものを図５８に示す。 In any dendron lipid, the surface area where the pressure rises is neutral or weakly acidic, and the surface area where the pressure starts increasing as it becomes acidic or basic is increased.
This is thought to have occurred because the charge state of the dendron lipid changed.
In addition, CHexDL-S has a larger surface area where pressure starts to increase than CHexDL-U2 and MGluDL-U2.
Next, FIG. 58 shows a plot of the surface area for each pH when the surface pressure is 25 mN / m.
　同じ不飽和型であってもMGluDL-U2 に比べてCHexDL-U2 の方が同じ表面圧ではエリア面積が大きい傾向にあった。これは末端基の排除体積がCHexDL-U2の方が大きいためである。しかしながら、明確な差はこれら二種にはない。G1には二つしか末端基を導入することができないことで明確な差が生まれていないということが考えられるため、世代数を上げることでこの末端基の差は生ずると考えられる。 [Even for the same unsaturated type, CHexDL-U2 tended to have a larger area at the same surface pressure than MGluDL-U2. This is because CHexDL-U2 has a larger excluded volume of terminal groups. However, there is no clear difference between these two types. Since it is considered that there is no clear difference in G1 because only two terminal groups can be introduced, it is considered that this difference in terminal groups will occur by increasing the number of generations.
　同じ末端基を持つCHexDL-S とCHexDL-U2 では飽和型のものの方が同じ表面圧ではエリア面積が大きかった（図５９）。また、同じ表面圧で比べた時に中性に比べて酸性の場合で表面積が大きくなっていた。これは図６０に示すようにpH が低下するとともにカルボキシ基がプロトン化されて疎水性となった末端基が気液界面に分配されることによるものだと考えられる。またさらに内部の3 級アミンがプロトン化されることで分子間の静電反発も生まれ、同じ表面圧で比較したときに表面積は大きくなったと考えられる。 In the case of CHexDL-Sex and CHexDL-U2 having the same end groups, the area of the saturated type was larger at the same surface pressure (Fig. 59). Further, when compared at the same surface pressure, the surface area was larger in the case of acidity than neutrality. As shown in FIG. 60, this is thought to be due to the fact that the pH group decreases and the carboxy group is protonated to be distributed to the gas-liquid interface. Furthermore, when the internal tertiary amine is protonated, intermolecular electrostatic repulsion is also generated, and the surface area is thought to have increased when compared at the same surface pressure.
　４．３　リポソームのpH 応答性評価
　４．３．１　ピラニン内包未修飾リポソームのpH 応答性評価
　pH 応答性デンドロン脂質を含有させずに調製した未修飾リポソーム（以下EYPC リポソーム）のpH 評価を行った。release においてpH に依存した大きな変化が見られないことから、pH 応答性は有していないことが分かった。 4.3 Evaluation of pH responsiveness of liposomes 4.3.1 Evaluation of pH responsiveness of unmodified liposomes encapsulating pyranine The pH of unmodified liposomes prepared without containing responsive dendron lipid (hereinafter referred to as EYPC liposomes) was evaluated. . Since no significant pH-dependent change was observed in release, it was found that there was no pH responsiveness.
　４．３．２　不飽和型MGluDL リポソーム、CHexDL リポソーム、及び飽和型CHexDLリポソーム(SCG1)の内包物放出挙動
　不飽和型MGluDL 含有ピラニン内包リポソーム（以下UMG1 リポソーム）、不飽和型CHexDL 含有ピラニン内包リポソーム（以下UCG1 リポソーム）及び飽和型CHexDL リポソーム(以下SCG1 リポソーム)のピラニン放出に及ぼすpHの影響について検討した。作製したUMG1、UCG1、SCG1 リポソームは脂質組成比が以下のものを用いた。
UMG1 ; EYPC/MGluDL-U2=75/25(以下UMG1.25) or 60/40(以下UMG1.40)
UCG1 ; EYPC/CHexDL-U2=90/10(以下UCG1.10) or 75/25(以下UCG1.25)
SCG1 ; EYPC/CHexDL-S=75/25(以下SCG1.25) or 60/40(以下SCG1.40)
　それぞれのリポソームにおいて、高pH 環境下では時間が経過してもリリースが起こらないのに対し、低pH 環境下では時間経過につれリポソームからの内包物の放出が見られる。 4.3.2 Inclusion Release Behavior of Unsaturated MGluDL Liposomes, CHexDL Liposomes, and Saturated CHexDL Liposomes (SCG1) The effect of pH on the pyranine release of UCG1 liposomes and saturated CHexDL liposomes (hereinafter SCG1 liposomes) was examined. The prepared UMG1, UCG1, and SCG1 liposomes had the following lipid composition ratios.
UMG1; EYPC / MGluDL-U2 = 75/25 (hereafter UMG1.25) or 60/40 (hereafter UMG1.40)
UCG1; EYPC / CHexDL-U2 = 90/10 (hereinafter UCG1.10) or 75/25 (hereinafter UCG1.25)
SCG1; EYPC / CHexDL-S = 75/25 (hereinafter SCG1.25) or 60/40 (hereinafter SCG1.40)
In each of the liposomes, release does not occur over time in a high pH environment, whereas inclusions are released from the liposomes over time in a low pH environment.
　これはリポソーム表面のpH 応答部位が持つカルボキシ基がpH 低下に伴いプロトン化することで、リポソーム表面が親水状態から疎水状態に変わったためリポソーム膜の不安定化を誘起し、内包物の放出が起こったと考えられる。 This is because the carboxy group of the pH response site on the liposome surface is protonated as the pH falls, causing the liposome surface to change from a hydrophilic state to a hydrophobic state, leading to destabilization of the liposome membrane and release of inclusions. It is thought.
　詳しく比較するために、それぞれのリポソームの15 分後のリリース（％）をpHに対してプロットし、各リポソーム間でのpH 応答性を比較した。その結果を図６１～６４に示す。 In order to compare in detail, the release (%) after 15 minutes of each liposome was plotted against pH, and the pH response between each liposome was compared. The results are shown in FIGS.
　いずれのリポソームでも中性下での内包物放出はそれほど起こっていないが、弱酸性下ではいずれのリポソームでも内包物の放出が起こった。リポソームからの放出率の最大値はUMG1 では30%程度、UCG1 では90%以上、SCG1 では60%程度であった。MGlu を導入したものとCHex を導入したものとで内包物の放出率の最大値が異なるが、これは、末端基の疎水性度が異なることによるものであると考えられる。すなわち、より疎水性度の高いCHex 基ではより強く膜と相互作用をするのに対し、疎水性度の低いMGlu 基では膜との相互作用が弱いため放出率が低いままであったと考えられる。さらに、同じCHex を導入したものでも飽和型と不飽和型とで内包物の放出率が異なった理由としては、飽和型のデンドロンは疎水性部位であるアルキル鎖(オクタデシル鎖)が不飽和型のオレイル鎖に比べて結晶性の高いものであり、飽和型ではデンドロン脂質が分離し、脂質膜中で集合しやすくなっていると考えられる。そして脂質膜が部分的に結晶様となり、リポソーム膜が堅くなって内包物の漏れが起こりにくくなったと考えられる。 In any liposome, the inclusion release under neutrality did not occur so much, but the inclusion release occurred in any liposome under weak acidity. The maximum release rate from liposomes was about 30% for UMG1, 90% or more for UCG1, and about 60% for SCG1. The maximum value of the release rate of inclusions differs between MGlu -introduced and CHex -introduced, which is thought to be due to the difference in the hydrophobicity of the end groups. That is, it is considered that the CHex 疎 水 group having a higher degree of hydrophobicity interacts more strongly with the membrane, whereas the MGlu が group having a lower degree of hydrophobicity has a weak interaction with the membrane, so that the release rate remains low. Furthermore, the reason why the release rate of inclusions is different between the saturated type and the unsaturated type even when the same CHex is introduced is that the saturated dendron has an alkyl chain (octadecyl chain) which is a hydrophobic site as an unsaturated type. It has high crystallinity compared to the oleyl chain, and it is thought that in the saturated type, dendron lipids are separated and easily assembled in the lipid membrane. It is thought that the lipid membrane became partially crystal-like, and the liposome membrane became hard and leakage of inclusions did not occur easily.
　また、脂質組成を変えた場合では、リポソームにデンドロン脂質を25%修飾したもので放出率は最大となり、それ以上デンドロン脂質の仕込み量を高くしても放出率の向上は認められなかった。デンドロン脂質末端基のリポソーム脂質膜へ及ぼす影響は25%修飾することで十分に行われることが考えられる。しかし、デンドロン脂質の仕込み量を50%にすると、UMG1 では溶解し、UCG1 及びSCG1 リポソームでは凝集体が沈殿し、リポソームは形成されなかった。この時、MGluDL-U2 ではミセル形成に至って溶解し、CHexDL-U2、CHexDL-Sではデンドロン脂質が自己凝集していると考えられる。 In addition, when the lipid composition was changed, the release rate was maximized when 25% of the dendron lipid was modified on the liposome, and no further improvement in the release rate was observed even when the amount of the dendron lipid charged was increased. The effect of the dendron lipid end group on the liposome lipid membrane is considered to be sufficiently achieved by 25% modification. However, when the amount of dendron lipid charged was 50%, UMG1 dissolved, aggregates precipitated in UCG1 and SCG1 liposomes, and liposomes were not formed. At this time, it is considered that MGluDL-U2 has reached micelle formation and dissolved, and CHexDL-U2 and CHexDL-S have self-aggregated dendron lipids.
　４．４　リポソームの粒径、ζ電位及び濁度測定に対する実験結果
　４．４．１　リポソームの粒径と濁度測定
　リポソームの粒径及び濁度を測定した結果を図６５及び図６６に示す。 4.4 Experimental Results for Particle Size , ζ Potential and Turbidity Measurement of Liposomes 4.4.1 Particle Size and Turbidity Measurement of Liposomes Results of measuring the particle size and turbidity of liposomes are shown in FIGS.
　UMG1.25 では中性下及び弱酸性下では粒径は約100 nm であったが、pH5.0 付近で粒径が大きくなってpH4.5 で最大となり、さらにpH を低下させると再び100 nm 程度の粒子となった。濁度測定においてもpH5.0 付近で透過率の低下がおこり始め、pH4.4～pH4.5 で透過率が最低となり、さらにpH を低下させることで透過率は再び中性下と同程度の値となった。このことから、UMG1 リポソームではpH を低下させると自身のリポソーム膜の疎水部分と末端基の疎水性部分の相互作用はあまり起こらず、他のリポソームと相互作用をしている凝集が起こると考えられる。そしてさらにpH を低下させると再び粒径は100 nm 程度となって透過率も上昇した。このことから、凝集した際にリポソーム間の膜融合は起こっていないと考えられる。一方、UCG1.25 及びSCG1.25 ではいずれのpH 下でも100 nm 程度であり、粒径の変化は起こらなかった。また、濁度測定においても透過率の変化は起こらなかった。このことから、CHexDL を修飾したリポソームでは他のリポソームと相互作用はせずに自身のリポソーム膜の疎水部分と末端基の疎水性部分が相互作用をしていると考えられる。 In UMG1.25, the particle size was about 100 nm under neutral and weak acidity, but the particle size increased around pH5.0 and became maximum at pH4.5, and when pH was further lowered, it was again 100 nm. The particles became about the size. Also in turbidity measurement, the transmittance starts to decrease at around pH 5.0 mm, the transmittance becomes the lowest at pH 4.4 to pH 4.5 mm, and the permeability is again about the same as under neutral by lowering the pH value. Value. From this, in UMG1UM liposomes, when pH is lowered, the interaction between the hydrophobic part of its own liposome membrane and the hydrophobic part of the end group does not occur so much, and it is thought that aggregation interacting with other liposomes occurs . When the pH value was further lowered, the particle size again became about 100 nm, and the transmittance increased. From this, it is considered that membrane fusion between liposomes does not occur when they aggregate. On the other hand, UCG 1.25 mm and SCG 1.25 mm were about 100 nm nm at any pH value, and the particle size did not change. Also, no change in transmittance occurred in the turbidity measurement. From this, it is considered that the liposome modified with CHexDL does not interact with other liposomes, but the hydrophobic part of its own liposome membrane and the hydrophobic part of the terminal group interact.
　４．４．２　リポソームのζ電位
　単分子膜測定の結果やリポソームの放出挙動など、pH 応答性デンドロン脂質あるいはpH 応答性リポソームのpH に依存した挙動の変化について詳しく検討するため、ζ電位測定を行い、その結果を図６７に示す。 4.4.2 In order to investigate in detail the changes in pH-dependent behavior of pH-responsive dendron lipids or pH-responsive liposomes, such as the results of ζ-potential monolayer measurement of liposomes and the release behavior of liposomes, The results are shown in FIG.
　EYPC リポソームのζ電位のpH に依存した大きな変化が見られず、ほぼ中性の値であった。UMG1 リポソーム、UCG1 リポソーム及びSCG1 リポソームのζ電位に及ぼすpH の影響について検討した。いずれのリポソームも弱塩基性では負の同様の値で、pHの低下に伴いζ電位は上昇し、酸性下では正の同様な値であった。しかし、UMG1 リポソームではpH5～6 でζ電位は正の値、UCG1 リポソーム及びSCG1 リポソームでは中性付近ですでに正の値となっていた。EYPCリポソームのζ電位はpH を変化させても変動しないことからリポソームのζ電位の変動はpH 応答性デンドロン脂質の荷電状態が変化したために起こったものと考えられる。よって、このようなリポソームのpH に依存したζ電位の違いをデンドロン脂質の荷電状態の違いから次のように説明することができる。UMG1リポソームの各pHにおけるデンドロン脂質の荷電状態は図６８のようになっていると考えられる。 EYPC liposome ζ potential did not change greatly depending on pH, and was almost neutral. The effect of pH on the zeta potential of UMG1 ~ liposome, UCG1 ~ liposome and SCG1 ~ liposome was investigated. All liposomes had negatively similar values when weakly basic, the ζ potential increased with decreasing pH, and were positively similar values under acidic conditions. However, in the UMG1G liposome, the ζ potential was positive at pH 5-6, and in the UCG1 and SCG1 liposomes, it was already positive near neutrality. Since the ζ potential of EYPC liposomes does not change even when the pH is changed, it is considered that the change in the ζ potential of the liposome occurred because the charge state of the pH responsive dendron lipid was changed. Therefore, the difference in the ζ potential depending on the pH of the liposome can be explained as follows from the difference in the charged state of the dendron lipid. It is considered that the charge state of the dendron lipid at each pH of the UMG1 liposome is as shown in FIG.
　中性及び弱塩基性下では末端カルボキシ基が負電荷をもつため、ζ電位が負の値となっていると考えられる。また、デンドロン内部の3 級アミンのプロトン化はpH6.0～pH7.0 で起こることから、pH5.5～pH7.0 の領域ではデンドロン脂質内部の3 級アミノ基のプロトネーションが起こっていると考えられ、pH5.5 付近でζ電位が中性の値となったと考えられる。そして、最後に末端カルボキシ基のプロトネーションが起こり、ζ電位は正の値となったと考えられる。 Under neutral and weak basicity, the terminal carboxy group has a negative charge, so the ζ potential is considered to be negative. In addition, since protonation of tertiary amines inside dendron occurs at pH 6.0 to pH 7.0%, protonation of tertiary amino groups inside dendron lipids occurs at pH 5.5 to pH 7.0%. It is considered that the ζ potential became neutral at around pH 5.5. Finally, the terminal carboxy group is protonated, and the ζ potential is considered to be a positive value.
　一方、UCG1 及びSCG1 リポソームの各pH におけるpH 応答性デンドロン脂質の電荷状態は図６９のようになっていると考えられる。中性から弱酸性下でζ電位はすでに正の値となっている。デンドロン内部の3 級アミノ基のプロトネーションはpH6.0～pH7.0 で起こることから、pH7.0までのζ電位の上昇は、末端のカルボキシ基がプロトネーションを受けたことによるものと考えられる。そしてさらにpH が低下することでデンドロン内部の3 級アミノ基がプロトネーションを受けてζ電位が正となったと考えられる。 On the other hand, the charge state of the pH-responsive dendron lipid at each pH of the UCG1 and SCG1 liposomes is considered as shown in FIG. Under neutral to weak acidity, the zeta potential is already positive. Since the protonation of the tertiary amino group in the dendron occurs at pH 6.0 to pH 7.0, the increase in ζ potential up to pH 7.0 is thought to be due to the protonation of the terminal carboxy group. . Further, it is considered that the ζ potential became positive due to the protonation of the tertiary amino group in the dendron due to a further decrease in pH.
　４．４．３　種々デンドロン脂質のエリア面積とζ電位の関係
　以下に種々デンドロン脂質をコンポーネントとしたリポソームのζ電位とデンドロン脂質の表面圧が25 mN/m となるときの表面積の関係を図７０～図７２に示す。図３１はＭＧｌｕＤＬ－Ｕ２、図３２はＣＨｅｘＤＬ－Ｕ２、図３３はＣＨｅｘＤＬ－Ｓについての結果をそれぞれ示している。 4.4.3 Relationship between area area and ζ potential of various dendron lipids The relationship between the ζ potential of liposomes containing various dendron lipids and the surface area when the surface pressure of the dendron lipids is 25 mN / m is shown in FIG. This is shown in FIG. FIG. 31 shows the results for MGluDL-U2, FIG. 32 shows the results for CHexDL-U2, and FIG. 33 shows the results for CHexDL-S.
　弱塩基性下ではζ電位が負で最大で、エリア面積も大きい。pH が低下し、中性となるとζ電位は上昇し、エリア面積は不飽和型では最小に、飽和型でもエリア面積は低下した。これはζ電位の低下によりデンドロン脂質のデンドロン部位の電荷がニュートラルになったことにより電荷反発がなくなり、デンドロン同士がより密にパッキングするようになったことによると考えられる。そしてさらにpH が低下するとζ電位の上昇とともにエリア面積も上昇した。これはデンドロン部位が再び電荷をもったためである。以上のようにζ電位の変化と同調してエリア面積も変化していることが分かった。 Under weak basicity, the ζ potential is negative and maximum, and the area is large. When the pH value decreased and became neutral, the ζ potential increased, and the area area was minimized in the unsaturated type, and the area area was also decreased in the saturated type. This is considered to be due to the fact that the repulsion of the dendron portion of the dendron lipid became neutral due to the decrease in the ζ potential, so that charge repulsion disappeared and the dendrons packed more closely. As the pH value decreased further, the area of the area increased as the ζ potential increased. This is because the dendron site has been charged again. As described above, it was found that the area of the area also changed in synchronization with the change in the ζ potential.
　４．４．４　不飽和型MGluDL リポソーム、CHexDL リポソーム、及び飽和型CHexDLリポソームのζ電位と内包物放出率の関係
　リポソームのζ電位と、ピラニンの放出との相関を考察する。図７３～７５は、それぞれ、ＵＭＧ１．２５、ＵＣＧ１．２５及びＳＣＧ１．２５についての結果を示している。
塩基性条件下ではζ電位は負の電位であり、放出は起こっていない。しかし、ζ電位が上昇する(デンドロン脂質の電荷状態が変化する)と放出が起こり、さらにζ電位が正で最大となったpH 領域で内包物の放出も最大となることが分かる。UMG1 では放出率が低いため相関を確認しにくいが、このことはすべてのリポソームについてあてはまる。 4.4.4 Relationship between ζ Potential of Unsaturated MGluDL Liposomes, CHexDL Liposomes, and Saturated CHexDL Liposomes and Inclusion Release Rate The correlation between the ζ potential of liposomes and the release of pyranine will be discussed. FIGS. 73-75 show the results for UMG1.25, UCG1.25 and SCG1.25, respectively.
Under basic conditions, the ζ potential is negative and no release occurs. However, it can be seen that release occurs when the ζ potential rises (the charge state of the dendron lipid changes), and that the inclusion release is also maximized in the pH range where the ζ potential is positive and maximum. UMG1 has a low release rate, making it difficult to confirm the correlation, but this is true for all liposomes.
　以上のことを踏まえ、弱塩基性下ではデンドロン脂質末端のカルボキシ基が負に帯電しており、リポソーム膜と相互作用しないためにリポソーム膜は安定に保持されると考えられる。また中性下では単分子膜測定の結果より、デンドロンがより密にパッキングをできるようになるために内包物は安定に保持されていると考えられる。しかし、pH の低下に伴いデンドロン脂質末端のカルボキシ基のプロトネーションにより3-メチルグルタル酸由来または1.2-シクロヘキサンジカルボン酸由来の末端基の疎水性度の増大によりリポソーム膜との疎水性相互作用の増大によりリポソーム膜を不安定化し、またデンドロン脂質内部の3 級アミノ基のプロトネーションにより、塩基性及び中性化では疎水性であったリポソーム膜に親水性部位が発生すること、さらにリポソーム膜中の脂質分子(卵黄ホスファチジルコリン)の配列中にpH 応答性デンドロン脂質の末端基が入り込むことにより、脂質分子のパッキングが低下することで膜が不安定化され、放出が起こったと考えられる。 Based on the above, the carboxy group at the end of the dendron lipid is negatively charged under weak basicity, and since it does not interact with the liposome membrane, the liposome membrane is considered to be stably held. In addition, under neutral conditions, from the results of monomolecular film measurement, the dendron can be packed more densely, so the inclusion is considered to be stably held. However, as the pH decreases, the hydrophobic interaction of the terminal membrane derived from 3-methylglutaric acid or 1.2-cyclohexanedicarboxylic acid increases due to the protonation of the carboxy group at the end of the dendron lipid, increasing the hydrophobic interaction with the liposome membrane. The liposome membrane is destabilized by the formation of a hydrophilic site in the liposome membrane, which was hydrophobic in basic and neutralization, due to the protonation of the tertiary tertiary amino group inside the dendron lipid. It is thought that the membrane was destabilized due to the packing of lipid molecules (egg yolk phosphatidylcholine) entering the pH -responsive dendron lipid end group, resulting in destabilization of the membrane and release.
　４．５　種々のデンドロン脂質を修飾したリポソームの細胞への影響
　４．５．１　フローサイトメトリによる種々のリポソームの細胞への取り込み評価
作製したリポソームがどれほど細胞内に取り込まれるかを評価するためにフローサイトメーターを用いて細胞内取り込みを評価し、その結果を図７６に示す。 4.5 Effect of various dendron lipid-modified liposomes on cells 4.5.1 Evaluation of incorporation of various liposomes into cells by flow cytometry How much of the prepared liposomes are incorporated into cells In order to evaluate, intracellular uptake was evaluated using a flow cytometer, and the results are shown in FIG.
　EYPC リポソームの細胞への取り込み量と比較して、UMG1.25 リポソームでは少し取り込み量が少なく、UCG1.25 リポソームやSCG1.25 リポソームでは3～5倍の取り込み量となっていた。UCG1.25 やSCG1.25 では細胞膜との疎水性相互作用による吸着の後の取り込みも考えられるが、これらリポソームの生理条件下でのζ電位の比較から、この差について次のように考えられる。細胞膜表面は負電荷を帯びていることから、負のζ電位であったUMG1.25 では静電反発により取り込みが少なく、また、デンドロン末端基の疎水性度もそれほど高いものではないことから、細胞膜との疎水性相互作用による取り込みも期待できない。よってUMG1.25 リポソームでは取り込み量が低かったものと考えられる。ただしDC2.4 細胞には、スカベンジャーレセプターが存在しているはずであるが、ζ電位が負であるUMG1.25 リポソームの取り込みが最も少ないものであった。このことについては次のように考えられる。リポソームに組み込まれたMGluDL-U2 の末端基(MGlu 基)はそれほど排除体積(立体障害)の大きいものではなく、疎水性度もそれほど高いものではない。このことから末端カルボキシ基とリポソームの外側の親水性部位との相互作用(静電相互作用あるいは水素結合)が発生し、リポソーム中のコリン成分と結合したような状態となったのではないかと考えられる。 Compared to the amount of EYPC® liposomes taken into cells, the amount of UMG1.25 liposomes was slightly less, and the amount of UCG1.25 and SCG1.25 liposomes was 3-5 times higher. In UCG1.25 and SCG1.25 取 り 込 み, uptake after adsorption due to hydrophobic interaction with the cell membrane may be considered. From the comparison of ζ potential under physiological conditions of these liposomes, this difference is considered as follows. Since the surface of the cell membrane is negatively charged, UMG1.25, which had a negative ζ potential, has little uptake due to electrostatic repulsion, and the hydrophobicity of the dendron end group is not so high. Uptake by hydrophobic interaction with can not be expected. Therefore, it is considered that the uptake amount of UMG1.25 liposome was low. However, although DC2.4 cells should have a scavenger receptor, UMG1.25 liposomes with negative ζ potential had the least uptake. This can be considered as follows. The terminal group (MGlu group) of MGluDL-U2 incorporated in the liposome is not so large in excluded volume (steric hindrance), and the hydrophobicity is not so high. From this, it is thought that the interaction (electrostatic interaction or hydrogen bond) between the terminal carboxy group and the hydrophilic site on the outside of the liposome occurred, and it seems that it was in a state of being bound to the choline component in the liposome. It is done.
　これにより、リガンドであるカルボキシ基が事実上露呈していないような状態となり、さらにリポソーム膜を水素結合で保護するような状態となり内包物の流出も阻害しているのではないかと考えられる。 It is considered that this leads to a state in which the ligand carboxy group is practically not exposed, and further protects the liposome membrane with hydrogen bonds, thereby inhibiting the outflow of inclusions.
　UCG1.25 とSCG1.25 の生理条件でのζ電位はそれほど大きな差はなく、ともに正の値となっていた。このことから予想される細胞への取り込みとしては、UMG1.25 やEYPC より多いがUCG1.25 とSCG1.25 の差は小さいと考えられる。しかし、実際にはSCG1.25 はUCG1.25 の5 倍程度の値となっていた。先の考察でSCG1.25 ではデンドロン脂質が部分的に集合して結晶様の部分がリポソーム膜中に存在するとした。すると、UCG1.25 に比べてSCG1.25 はデンドロン末端基の集合が起こっており疎水性も高いような状態となっていると考えられる。この部分が自身のリポソーム膜との相互作用をせずに細胞膜と相互作用をし、取り込みが上がったのではないかと考えられる。 Ζ potential under physiological conditions of UCG1.25 and SCG1.25 was not so big and both were positive values. From this, the expected uptake into cells is greater than UMG1.25 and EYPC, but the difference between UCG1.25 and SCG1.25 is considered to be small. However, SCG 1.25G was actually about 5 times UCG 1.25. In the previous discussion, it was assumed that dendron lipids partially gathered at SCG 1.25 and crystal-like parts exist in the liposome membrane. Then, compared to UCG1.25, SCG1.25 is considered to be in a state where dendron end groups are assembled and hydrophobicity is high. It is thought that this part interacted with the cell membrane without interacting with its own liposome membrane, and the uptake increased.
　４．５．２　共焦点レーザー顕微鏡による細胞内挙動の観察
　４．５．２．１　カルセインを内包したローダミンラベル化リポソームの細胞内挙動
　共焦点レーザー顕微鏡により観察を行った結果を図７７～８５に示す。図７７～８０はＤＣ２．４ｃｅｌｌｓ、図８１～８５はＨｅｌｌａ　ｃｅｌｌｓについての結果である。
また、図３８及び図４２はＳＣＧ１．２５についての添加後４時間後、図３９及び図４３はＵＣＧ１．２５についての添加後４時間後、図４０及び図８３はＵＭＧ１．２５についての添加後４時間後、図８０及び図８４はＥＹＰＣについての添加後４時間後、並びに図８５はＵＭＧ１．２５についての添加後２４時間後の結果である。 4.5.2 Observation of intracellular behavior with confocal laser microscope 4.5.2.1 Intracellular behavior of rhodamine-labeled liposomes encapsulating calcein The results of observation with a confocal laser microscope are shown in FIGS. Show. 77 to 80 show the results for DC 2.4 cells, and FIGS. 81 to 85 show the results for Hella cells.
38 and 42 are 4 hours after the addition of SCG1.25, FIGS. 39 and 43 are 4 hours after the addition of UCG1.25, and FIGS. 40 and 83 are 4 after the addition of UMG1.25. After time, FIGS. 80 and 84 are the results 4 hours after the addition for EYPC, and FIG. 85 are the results 24 hours after the addition for UMG1.25.
　DC2.4 細胞であってもHeLa 細胞であっても同様な傾向が確認された。すなわち、CHex を導入したもの(SCG1,UCG1 リポソーム)では放出されたカルセインの蛍光が確認されたが、EYPC とUMG1 ではカルセインの蛍光は確認されなかった。リポソームからのpH に依存した放出挙動と関連させても同じことがいえる。放出域が弱酸性で、放出率の高かったUCG1.25 やSCG1.25 ではカルセインの放出を示す強い蛍光が見られたが、放出域が酸性のUMG1.25 やpH 応答を示さないEYPC ではカルセインの放出を示す強い蛍光は確認できなかった。 The same tendency was confirmed for both DC2.4 and HeLa cells. That is, the fluorescence of released calcein was confirmed in the case of introducing CHex (SCG1, UCG1 liposome), but the fluorescence of calcein was not confirmed in EYPC and UMG1. The same can be said in relation to the release behavior depending on pH from liposomes. Strong emission indicating calcein release was observed in UCG 1.25 and SCG 1.25 た where the release range was weakly acidic and the release rate was high, but calcein was observed in EYPC な い where the release range did not show acidic UMG 1.25 and pH response Strong fluorescence indicating the release of was not confirmed.
　ここで、同一の細胞内であっても細胞膜近傍は赤い蛍光で核近傍では黄色い蛍光といったように、カルセインの蛍光が確認されない部分とされない部分が存在していることが分かる。このことはリポソームが酸性度の微妙な変化を感知して内包物を放出していることを示していると考えられる。また撮影した蛍光写真から、リポソームとカルセインの共局在率を計測した。結果を図８７に示す。どのリポソームでも共局在率は60 %程度であった。以上のことから、リポソームから放出されたカルセインはエンドソーム/リソソームから脱出せずに小胞内にとどまっていることが示唆される。 Here, even in the same cell, it can be seen that there are portions where calcein fluorescence is not confirmed, such as red fluorescence near the cell membrane and yellow fluorescence near the nucleus. This is considered to indicate that the liposome senses a subtle change in acidity and releases the inclusion. Moreover, the colocalization rate of a liposome and calcein was measured from the photographed fluorescence photograph. The results are shown in FIG. The colocalization rate for all liposomes was about 60%. From the above, it is suggested that calcein released from the liposome stays in the vesicle without escaping from the endosome / lysosome.
　４．５．２．２　ローダミンラベル化リポソームと後期エンドソーム/リソソーム
　カルセイン内包UCG1.25リポソームを取り込ませた場合については後期エンドソーム/リソソームの染色を行った。結果を図８８に示す。ライソトラッカーは赤色のものを使った。後期エンドソーム/リソソーム(細胞内酸性小胞)の位置と放出されたカルセインの位置が一致していることがこの図から判る。 4.5.2.2 Rhodoamine- labeled liposomes and late endosome / lysosomal calcein-encapsulated UCG1.25 liposomes were incorporated, and late endosome / lysosomes were stained. The results are shown in FIG. The lyso tracker was red. This figure shows that the positions of late endosomes / lysosomes (intracellular acidic vesicles) and released calcein coincide.
　４．６　二種類のpH 応答性デンドロン脂質により構成されるリポソームの評価
　CHex-G1-2C18-U2とMGlu-G1-2C18-U2を種々の比率で含有したデンドロン脂質/EYPC（40/60mol/mol）リポソームのピラニン放出挙動を図８９～９２に示す。CHex-G1-2C18-U2の含有率が大きくなるに従って、酸性での放出性能が上昇することがわかる。一方、MGlu-G1-2C18-U2の含有率が高まると、酸性領域での放出特性は低下するが、同時に、中性領域における内包物の保持特性が向上する。したがって、これらのデンドロン脂質の比率を調節することで、所望の酸性pH感受性放出特性を示すリポソームを作成することができる。 4.6 Two types of pH-responsive assessment of liposomes composed by dendron lipid CHex-G1-2C 18 -U2 and MGlu-G1-2C 18 dendrons -u2 were contained in various proportions lipid / EYPC (40 / 60mol 89-92 show the pyranine release behavior of liposomes. It can be seen that the acid release performance increases as the content of CHex-G1-2C 18 -U2 increases. On the other hand, when the content of MGlu-G1-2C 18 -U2 increases, the release characteristic in the acidic region decreases, but at the same time, the inclusion retention property in the neutral region improves. Therefore, by adjusting the ratio of these dendron lipids, liposomes exhibiting desired acidic pH-sensitive release characteristics can be created.
　５．　CHexDL-G2-2C 18 の評価
　以下、CHex-DL-G1-2C18及びCHexDL-G2-2C18をそれぞれCHex-DL-G1-S及びCHexDL-G2-Sと表記することがある。以下の通り、CHex-DL-G2-Sについて各種評価を行った。なお、以下において特に説明がない場合は、先述のCHex-DL-G1-Sについて説明したのと同様の操作を行った。 5. Evaluation of CHexDL-G2-2C 18 hereinafter sometimes CHexDL-G1-2C 18 and CHexDL-G2-2C 18, respectively referred to as CHexDL-G1-S and CHexDL-G2-S. Various evaluations were performed on CHex-DL-G2-S as follows. In the following, unless otherwise described, the same operation as described for CHex-DL-G1-S was performed.
　５．１　CHexDL-G2-2C 18 の合成
　CHexAn 8.13 g (46.19 mmol, DL-G2Sの末端アミンに対し30 等量) をTEA 1 mLを含む DMF 6 mL に溶解させ, DL-G2 594 mg (0.45 mmol) を溶かした DMF 4 mLをゆっくりと滴下した。この混合液を 38 h ,50 ℃ の Ar 雰囲気化で攪拌した。反応後, LH-20 カラム (溶離液,メタノール)で精製した。 5.1 CHexDL-G2-2C 18 Synthesis CHexAn 8.13 g of dissolved (46.19 mmol, 30 eq with respect to the terminal amine of the DL-G2S) in DMF 6 mL containing TEA 1 mL, DL-G2 594 mg (0.45 4 mL of DMF in which mmol) was dissolved was slowly added dropwise. The mixture was stirred in an Ar atmosphere at 50 ° C. for 38 h. After the reaction, it was purified with LH-20 column (eluent, methanol).
　５．２　リポソームの作製
　EYPCのクロロホルム溶液(10 mg/mL)とCHex-DL-G2-2C18のクロロホルム溶液(7.3 mg/mL)を10 mLナスフラスコに加えて溶媒を留去し、薄膜を作製した。ここにPBS又はピラニン水溶液(35 mMピラニン, 50 mM DPX, 25 mM Na2HPO4)を500 μL加え、バス型超音波照射装置により薄膜を分散させた。凍結融解を5回行い、エクストルーダーを用いて粒径を100 nmに揃え、Sepharose 4B カラムにより精製した。その後、テストワコーにより脂質濃度を測定した。 5.2 Preparation of liposomes EYPC in chloroform (10 mg / mL) and CHex-DL-G2-2C18 in chloroform (7.3 mg / mL) were added to a 10 mL eggplant flask and the solvent was removed to prepare a thin film. did. To this was added 500 μL of PBS or an aqueous pyranine solution (35 mM pyranine, 50 mM DPX, 25 mM Na 2 HPO 4 ), and the thin film was dispersed with a bath-type ultrasonic irradiation device. Freeze-thaw was performed 5 times, and the particle size was adjusted to 100 nm using an extruder, and purified using a Sepharose 4B column. Thereafter, the lipid concentration was measured by Test Wako.
　５．３　リポソームの内包物放出挙動
作製したリポソームを、PBS(-)に脂質濃度が20 μMとなるように蛍光セル中に加え、pH応答性を測定するために蛍光測定(37 ℃、λEX : 416 nm、λEM : 512 nm)を行った。結果を図９３に示す。 5.3 Inclusion release behavior of liposomes The prepared liposomes were added to PBS (-) in a fluorescent cell so that the lipid concentration was 20 μM, and fluorescence measurement (37 , Λ EX : 416 nm, λ EM : 512 nm). The results are shown in FIG.
　５．４　ζ電位測定
　作製したリポソームを20 μLずつ取り各pHに調整したphosphate Bufferに加え(全量：720 μL、終濃度：0.1 mM)、ζ電位を測定した。測定はZetasizer nano (Malvern製)を用いて行った。結果を図９４に示す。 5.4 Measurement of ζ Potential 20 μL of the prepared liposomes were added to phosphate buffer adjusted to each pH (total amount: 720 μL, final concentration: 0.1 mM), and ζ potential was measured. The measurement was performed using Zetasizer nano (Malvern). The results are shown in FIG.
　５．５　単分子膜測定
　USI-3-22を用いて, 単分子膜の表面圧と一分子の占める面積を測定した。250 mL のPBS s (NaCl 150 mM, Na2HPO410 mM)をトラフに用意し,デンドロン脂質を所定量(初期の表面積が6-8 nm2/moleculeとなるようにする)を静かに加え,単分子膜を作製した. 20分後測定を開始した. 結果を図９５に示す。 5.5 Monolayer measurement USI-3-22 was used to measure the surface pressure and the area occupied by one molecule. Prepare 250 mL of PBS s (NaCl 150 mM, Na 2 HPO 4 10 mM) in the trough, and gently add a predetermined amount of dendron lipid (the initial surface area should be 6-8 nm 2 / molecule). A monomolecular film was prepared. Measurement was started after 20 minutes. The results are shown in FIG.
　５．６　細胞による取り込み評価(フローサイト)
　12穴ディッシュにHeLa細胞を5万/wellで播種し、二日間培養を行った。PBS(+)で2回、PBS(-)で1回洗洗浄し、10 %牛胎児血清(FBS)を含むDMEMを1 mL加え、濃度を0.5 mMとしたliposome分散液を1 mL加えてCO2 incubatorで4時間静置した。PBS(+)で2回、PBS(-)で1回洗浄した後、フローサイトメーターにより取り込み量の評価を行った。結果を図９６に示す。 5.6 Uptake evaluation by cells (flow site)
HeLa cells were seeded in a 12-well dish at 50,000 / well and cultured for 2 days. Wash twice with PBS (+) and once with PBS (-), add 1 mL of DMEM containing 10% fetal bovine serum (FBS), add 1 mL of liposome dispersion with a concentration of 0.5 mM, and add CO. 2 incubator for 4 hours. After washing twice with PBS (+) and once with PBS (−), the amount of uptake was evaluated with a flow cytometer. The results are shown in FIG.
　５．７　抗腫瘍効果の検討
　E.G7-OVA 細胞 (1×106/mice)をマウス(C57BL/6, female, 7w, n = 4)左側背部皮下に播種した.担癌後 5日目と12日目に, 種々のリポソームを右側背部に皮下投与し、腫瘍サイズをモニターした. 結果を図９７に示す。 5.7 Examination of antitumor effect E.G7-OVA cells (1 × 10 6 / mice) were seeded subcutaneously in the left back of mice (C57BL / 6, female, 7w, n = 4). On day 12, various liposomes were subcutaneously administered to the right back and the tumor size was monitored. The results are shown in FIG.

Claims (11)

  1. 下記式ＤＬ－Ｇ１～ＤＬ－Ｇ４のいずれかで表される化合物
    ＤＬ－Ｇ１：Ｒ１Ｒ２ＮＸ（ＸＨＲ３）ＸＨＲ４
    ＤＬ－Ｇ２：Ｒ１Ｒ２ＮＸ（Ｘ（ＸＨＲ３）ＸＨＲ４）２
    ＤＬ－Ｇ３：Ｒ１Ｒ２ＮＸ（Ｘ（Ｘ（ＸＨＲ３）ＸＨＲ４）２）２
    ＤＬ－Ｇ４：Ｒ１Ｒ２ＮＸ（Ｘ（Ｘ（Ｘ（ＸＨＲ３）ＸＨＲ４）２）２）２
    （式中、Ｒ１及びＲ２は、同一または異なって飽和又は不飽和の長鎖炭化水素基を示し、Ｒ３及びＲ４は、同一のまたは異なる機能性炭化水素基を含有し、
    Ｒ１～Ｒ４は、環状構造を含有していてもよく、かつ一以上の炭素原子がヘテロ原子で置換されていてもよく、かつＸは、－ＣＨ２ＣＨ２ＣＯＮＨＣＨ２ＣＨ２Ｎ－を示す。）。     Compound DL-G1: R 1 R 2 NX (XHR 3 ) XHR 4 represented by any of the following formulas DL-G1 to DL-G4
    DL-G2: R 1 R 2 NX (X (XHR 3 ) XHR 4 ) 2
    DL-G3: R 1 R 2 NX (X (X (XHR 3 ) XHR 4 ) 2 ) 2
    DL-G4: R 1 R 2 NX (X (X (X (XHR 3 ) XHR 4 ) 2 ) 2 ) 2
    Wherein R 1 and R 2 are the same or different and represent a saturated or unsaturated long-chain hydrocarbon group, R 3 and R 4 contain the same or different functional hydrocarbon groups,
    R 1 to R 4 may contain a cyclic structure, and one or more carbon atoms may be substituted with a hetero atom, and X represents —CH 2 CH 2 CONHCH 2 CH 2 N—. Show. ).
  2. 前記機能性炭化水素基が、オリゴエチレングリコ－ル構造を含有する炭化水素基、又はｐＨ応答性のカルボキシル基含有炭化水素基である、請求項１に記載の化合物。 2. The compound according to claim 1, wherein the functional hydrocarbon group is a hydrocarbon group containing an oligoethylene glycol structure or a pH-responsive carboxyl group-containing hydrocarbon group.
  3. （Ａ）下記式ＤＬ－Ｇ１～ＤＬ－Ｇ４のいずれかで表される化合物；及び
    （Ｂ）ポリエチレングリコール構造を含有する脂質
    を含有する、分子集合体
    ＤＬ－Ｇ１：Ｒ１Ｒ２ＮＸ（ＸＨＲ３）ＸＨＲ４
    ＤＬ－Ｇ２：Ｒ１Ｒ２ＮＸ（Ｘ（ＸＨＲ３）ＸＨＲ４）２
    ＤＬ－Ｇ３：Ｒ１Ｒ２ＮＸ（Ｘ（Ｘ（ＸＨＲ３）ＸＨＲ４）２）２
    ＤＬ－Ｇ４：Ｒ１Ｒ２ＮＸ（Ｘ（Ｘ（Ｘ（ＸＨＲ３）ＸＨＲ４）２）２）２
    （式中、Ｒ１及びＲ２は、同一または異なって飽和又は不飽和の長鎖炭化水素基を示し、Ｒ３及びＲ４は、同一のまたは異なるオリゴエチレングリコ－ル構造を含有する炭化水素基を含有し、
    Ｒ１～Ｒ４は、環状構造を含有していてもよく、かつ一以上の炭素原子がヘテロ原子で置換されていてもよく、かつＸは、－ＣＨ２ＣＨ２ＣＯＮＨＣＨ２ＣＨ２Ｎ－を示す。）。     (A) a compound represented by any of the following formulas DL-G1 to DL-G4; and (B) a molecular assembly DL-G1: R 1 R 2 NX (XHR containing a lipid containing a polyethylene glycol structure 3 ) XHR 4
    DL-G2: R 1 R 2 NX (X (XHR 3 ) XHR 4 ) 2
    DL-G3: R 1 R 2 NX (X (X (XHR 3 ) XHR 4 ) 2 ) 2
    DL-G4: R 1 R 2 NX (X (X (X (XHR 3 ) XHR 4 ) 2 ) 2 ) 2
    (Wherein R 1 and R 2 are the same or different and represent a saturated or unsaturated long chain hydrocarbon group, and R 3 and R 4 are hydrocarbons containing the same or different oligoethylene glycol structure. Containing groups,
    R 1 to R 4 may contain a cyclic structure, and one or more carbon atoms may be substituted with a hetero atom, and X represents —CH 2 CH 2 CONHCH 2 CH 2 N—. Show. ).
  4. 前記Ｒ３及びＲ４が、以下の式（Ｉ）で表される、請求項３に記載の分子集合体
    Figure JPOXMLDOC01-appb-C000001
     （式中、ｎは１～１０のいずれかの整数を示し、かつＲ５は炭素数１～１０の炭化水素基（一以上の炭素原子がヘテロ原子で置換されていてもよい）を示す。）。     The molecular assembly according to claim 3, wherein R 3 and R 4 are represented by the following formula (I):
    Figure JPOXMLDOC01-appb-C000001
     (Wherein n represents an integer of 1 to 10 and R 5 represents a hydrocarbon group having 1 to 10 carbon atoms (one or more carbon atoms may be substituted with a hetero atom)). ).
  5. 前記脂質（Ｂ）が、以下の式（ＩＩ）で表される、請求項３又は４に記載の分子集合体
    Figure JPOXMLDOC01-appb-C000002
     （式中、Ｙは炭素数１０～５０の炭化水素基（一以上の炭素原子がヘテロ原子で置換されていてもよい）であり、かつｎは１０～２０のいずれかの整数を示し、かつＲ６は炭素数１～１０の炭化水素基（一以上の炭素原子がヘテロ原子で置換されていてもよい）を示す。）。     The molecular assembly according to claim 3 or 4, wherein the lipid (B) is represented by the following formula (II):
    Figure JPOXMLDOC01-appb-C000002
     (Wherein Y is a hydrocarbon group having 10 to 50 carbon atoms (one or more carbon atoms may be substituted with a hetero atom), and n represents any integer of 10 to 20, and R 6 represents a hydrocarbon group having 1 to 10 carbon atoms (one or more carbon atoms may be substituted with a hetero atom).
  6. 生理活性物質を細胞内に導入するために使用される、請求項３～５のいずれかに記載の分子集合体。 The molecular assembly according to any one of claims 3 to 5, which is used for introducing a physiologically active substance into a cell.
  7. 生理活性物質を細胞内に導入することにより疾患を治療するために使用される、請求項３～５のいずれかに記載の分子集合体。 The molecular assembly according to any one of claims 3 to 5, which is used for treating a disease by introducing a physiologically active substance into a cell.
  8. 請求項３～５のいずれかに記載の分子集合体を含有する組成物。 A composition comprising the molecular assembly according to any one of claims 3 to 5.
  9. 請求項３～５及び請求項８のいずれかに記載の分子集合体又は組成物を含有するキット。 A kit containing the molecular assembly or composition according to any one of claims 3 to 5 and claim 8.
  10. 請求項３～５並びに請求項８及び９のいずれかに記載の分子集合体、組成物又はキットの、生理活性物質を細胞内に導入する方法における使用。 Use of the molecular assembly, composition or kit according to any one of claims 3 to 5 and claims 8 and 9 in a method for introducing a physiologically active substance into a cell.
  11. 請求項３～５及び請求項８のいずれかに記載の分子集合体又は組成物を生理活性物質とともに細胞内に導入する工程を含有する、生理活性物質を細胞内に導入する方法。 A method for introducing a physiologically active substance into a cell, comprising the step of introducing the molecular assembly or composition according to any one of claims 3 to 5 and claim 8 into the cell together with the physiologically active substance.
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