JP2013237631A - Temperature-responsive molecular aggregate, composition and kit including the same, and use of the composition and kit - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a composition allowed to be used for introducing a physiologically active substance into a cell and excellent in biocompatibility and temperature responsiveness, to provide a kit containing the composition and to provide a cell introduction method utilizing the kit.SOLUTION: A molecular aggregate includes a compound (A) expressed by any one of the following formulas DL-G1 to DL-G4 and lipid (B) including a polyethylene glycol structure. DL-G1: RRNX(XHR)XHR. DL-G2: RRNX(X(XHR)XHR). DL-G3: RRNX(X(X(XHR)XHR)). DL-G4: RRNX(X(X(X(XHR)XHR))).

Description

  The present invention relates to temperature-responsive molecular assemblies, compositions and kits containing the same, and uses thereof.

  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.

  For the purpose of improving the target directionality of drugs as DDS, 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).

Kenji Kono et al., “Thermosensitive Molecular Assemblies from Poly (amidoamine) Dendron-Based Lipids”, “Angewandte Chemie International, p. 11”, 20 years. 6332-6336

  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. An 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 inventors have intensively studied to solve the above problems. 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, even when a molecular assembly is 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 invention has been completed by the inventors of the present invention based on the above findings, and has been completed as described below.

（式中、ｎは１〜１０のいずれかの整数を示し、かつＲ^５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

（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Ｒ^６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。 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,
R ³ and R ⁴ contain hydrocarbon groups containing the same or different oligoethylene glycol structures;
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):

(In the formula, n represents any 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):

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 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 a step of introducing the molecular assembly or composition according to any one of Items 1 to 7 together with the physiologically active substance into the cell.

  According to the present 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) in vivo A carrier having an improved balance of 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.

¹ H NMR spectrum of DL-G-0.5-2C ₁₈ (Solvent CDCl ₃ ). ¹ H NMR spectrum of DL-G0-2C ₁₈ (Solvent CDCl ₃ ). ¹ H NMR spectrum of DL-G0.5-2C ₁₈ (Solvent CDCl ₃ ). ¹ H NMR spectrum of DL-G1-2C ₁₈ (Solvent CDCl ₃ ). ¹ H NMR spectrum of DL-G1.5-2C ₁₈ (Solvent CDCl ₃ ). ¹ H NMR spectrum of DL-G2-2C ₁₈ (Solvent CDCl ₃ ). ¹ H NMR spectrum of oleyl oleyl amide (Solvent CDCl ₃ ). ¹ H NMR spectrum of dioleylamine. (Solvent CDCl ₃ ). ¹ H NMR spectrum of DL-G-0.5-2C ₁₈ -U2 (Solvent CDCl ₃ ). ¹ H NMR spectrum of DL-G0-2C ₁₈ -U2 (Solvent CDCl ₃ ). ¹ H NMR spectrum of DL-G0.5-2C ₁₈ -U2 (Solvent CDCl ₃ ). ¹ H NMR spectrum of DL-G1-2C ₁₈ -U2 (Solvent CDCl ₃ ). ¹ H NMR spectrum of MDEG-DL-G1 (Solvent CDCl ₃ ). This is the mass spectrum of MDEG-DL-G1. MDEG-DL-G2 ¹ H NMR spectrum of (Solvent CDCl ₃ ). This is the mass spectrum of MDEG-DL-G2. ¹ H NMR spectrum of MDEG-DL-G1-U2 (Solvent CDCl ₃ ). ¹ H NMR spectrum of EDEG-DL-G1 (Solvent CDCl ₃ ). This is the mass spectrum of EDEG-DL-G1. ¹ H NMR spectrum of EDEG-DL-G2 (Solvent CDCl ₃ ). ¹ H NMR spectrum of EDEG-DL-G1-U2 (Solvent CDCl ₃ ). It is a graph which shows the temperature dependence of the transmittance | permeability of various dendron lipid aggregate dispersion liquid (10 mM phosphoric acid 140 mM NaCl aqueous solution, pH7.4). It is a graph which shows the temperature dependence of the transmittance | permeability of the EDEG-DL-G2 dendron lipid aggregate dispersion liquid (10 mM phosphoric acid 140 mM NaCl aqueous solution) in various pH. It is a graph which shows the pH dependence of the cloud point of an EDEG-DL-G2 dendron lipid assembly dispersion liquid (10 mM phosphoric acid 140 mM NaCl aqueous solution). Differential scanning calorimetry of MDEG-DL-G1 assembly dispersion, MDEG-DL-G2 assembly dispersion, and MDEG-DL-G1-U2 assembly dispersion (10 mM 140 mM NaCl aqueous solution, pH 7.4) It is a measurement (DSC) chart. Differential scanning calorimetry of EDEG-DL-G1 aggregate dispersion, EDEG-DL-G2 aggregate dispersion, and EDEG-DL-G1-U2 aggregate dispersion (10 mM 140 mM NaCl aqueous solution, pH 7.4) It is a measurement (DSC) chart. It is the photograph of 10 degreeC and 40 degreeC of EDEG-DL-G1 aggregate dispersion liquid (10 mM phosphoric acid 140 mM NaCl aqueous solution, pH7.4). It is the phase-contrast micrograph of 10 degreeC and 40 degreeC of an EDEG-DL-G1 aggregate dispersion liquid (10 mM phosphoric acid 140 mM NaCl aqueous solution, pH7.4). It is a graph which shows the temperature dependence of the transmittance | permeability of the EDEG-G1 / PEG-Chol aggregate dispersion liquid (10 mM phosphoric acid 140 mM NaCl aqueous solution, pH7.4) of various composition ratios. It is a graph which shows the influence of the PEG-Chol content rate on the cloud point of EDEG-G1 / PEG-Chol aggregate dispersion (10 mM phosphoric acid 140 mM NaCl aqueous solution, pH 7.4). It is a graph which shows the temperature dependence (10 mM phosphoric acid 140 mM NaCl aqueous solution) of the transmittance | permeability of the EDEG-G1 / PEG-Chol (5%) aggregate dispersion liquid in various pH. It is a graph which shows the pH dependence of the cloud point of EDEG-G1 / PEG-Chol (95/5) aggregate dispersion liquid and EDEG-DL-G1 aggregate dispersion liquid (10 mM phosphoric acid 140 mM NaCl aqueous solution). 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). It is an atomic force microscope image of the EDEG-DL-G1 / PEG-Chol (95/5) assembly at 10 ° C and 50 ° C. Confocal laser micrograph of HeLa cells incubated with aggregates of EDEG-G1 / PEG-Chol (95/5) labeled with rhodamine lipid (0.6 mol%) (incubation temperature is 36 ° C or 42 ° C). Confocal laser micrograph of HeLa cells incubated with aggregates of EDEG-G1 / PEG-Chol (95/5) labeled with rhodamine lipid (0.6 mol%) (incubation temperature is 36 ° C or 42 ° C. High in Figure 35) Magnification photo). 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 mM NaCl aqueous solution, pH7.4) of various composition ratios (PEG-Chol content (mol %) Is 0, 2, 5, and 10%.) It is a graph which shows the influence of the PEG-Chol content rate on the cloud point of MDEG-G1 / PEG-Chol aggregate dispersion (10 mM phosphoric acid 140 mM NaCl aqueous solution, pH 7.4). 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.).

1. Molecular assembly of the present invention The molecular assembly of the present invention comprises:
(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,
R ³ and R ⁴ contain hydrocarbon groups containing the same or different oligoethylene glycol structures;
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 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.

Description R ¹ and ^{R 2} for 1.1.2 R ¹ and ^{R 2} are long chain, saturated or unsaturated hydrocarbon group.

  The long-chain hydrocarbon group may be naturally derived, may be a naturally derived one, 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 carbon number of the main chain is preferably 8-30, more preferably 10-22, and still more preferably 12-20.

  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 heteroatom. 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.

  In the present invention, the hydrocarbon group means an alkyl group, an alkenyl group or an alkynyl group.

  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.

  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.

  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.

  The long chain hydrocarbon group may further have at least one or more substituents 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.

  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.

  Examples of unsaturated long-chain hydrocarbon groups include hexadecenyl, octadecenyl, octadecadienyl, octadecatrienyl, icosatrienyl, icosatetraenyl, octadecatrienyl, icosapentaenyl, or docosa A hexaenyl group is preferred.

  Examples of the unsaturated long chain hydrocarbon group 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 preferable.

  As the unsaturated long chain hydrocarbon group, a 9-octadecenyl group (oleyl group) is more preferable.

  Preferable examples of the unsaturated long-chain hydrocarbon group include those in which at least one carbon atom is further substituted with a heteroatom in a group having a structure as 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.

  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 Description of Hydrocarbon Group Containing Oligoethylene Glycol Structure 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.

  The other structure may be a linear structure which may have a branch. Although it does not specifically limit as such a structure, For example, what has a C1-C10 linear part which may have a C1-C10 branch is mentioned.

  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.

  In the other structure, one or more carbon atoms may be substituted with a heteroatom.

  The oligoethylene glycol structure is not particularly limited as long as the effect of the present invention is 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. As the oligoethylene glycol structure, those having 1 to 4 oxyethylene units in succession are preferred.

  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, a total of 1 via other structures. Examples include a structure having 10 to 10 oxyethylene units. In this case, what has 1-4 oxyethylene units in total is preferable.

  The branched portion of the oligoethylene glycol-containing hydrocarbon group may further have an oligoethylene glycol structure.

  The oligoethylene glycol-containing hydrocarbon group is preferably represented by the following formula (I):

(In the formula, n represents any 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).

(In formula, n shows the integer in any one of 1-10.).

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.

  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 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. Although it does not specifically limit as such a structure, For example, what has a C1-C10 linear part which may have a C1-C10 branch is mentioned.

  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.

  In the other hydrocarbon structure, one or more carbon atoms may be substituted with a heteroatom.

  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. As the polyethylene glycol structure, those having 3 to 200 oxyethylene units in succession are preferred, and those having 10 to 100 are more preferred.

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.

  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.

  Examples of the lipid moiety 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):

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).

(In the formula, Y represents a hydrocarbon group having 5 to 50 carbon atoms (one or more carbon atoms may be substituted with a hetero atom), and n represents an integer of 10 to 20). .

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).

  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 has 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.

  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.

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.

  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.

  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.

  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 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.

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. The polyamidoamine dendron, denoted as _DL-G1-2C 18 -U2.

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.

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.

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 polyamidoamine 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.

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.

  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. .

  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. 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.

  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, and 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. 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.

  In addition, examples of the 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. 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.

  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.

  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.

  The compounding ratio of the desired hydrophobic substance and the compound (A) of the present invention is 1 to 1000 parts by weight, preferably 10 to 500 parts by weight of the compound (A) of the present invention with respect to 1 part by weight of the substance. Preferably 10 to 100 parts by weight are used.

  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.

1. Production of molecular assembly 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 Industries. 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. As a dialysis membrane, Spectra / Por 6 (fraction 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 Synthesis of Compound (A) of the Present Invention 1.2.1 Overview
Synthetic pathway of first and second generation dendron lipids (DL-G1, DL-G2) with two alkyl chains and first generation dendron lipid (DL-G1-U2) with two oleyl chains Are represented by the following chemical formulas 8 and 9, and a dendron lipid (MDEG-DL-G1, EDEG-DL-G1, MDEG-DL) in which a methoxydiethylene glycol (MDEG) group or an ethoxydiethylene glycol (EDEG) group is introduced at the amino group terminal -G2, EDEG-G2, MDEG-DL-G1-U2, and EDEG-DL-G1-U2) are shown in the following formula.

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.

  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.

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 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 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 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- ), δ 2.44 (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 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 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 Synthesis of Oleyloleoylamide
Oleoylchloride 7.7 mL (20 mmol) was dissolved in dichloromethane 100 mL and stirred in an ice-water bath. Thereafter, the mixture was refluxed for 71 hours under 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). 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 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 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). 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 synthesis of DL-G0-2C ₁₈ -U2
Dissolve 1.32 g (2.18 mmol) of DL-G-0.5-2C ₁₈ -U2 in 100 mL of methanol and pass it to 70 mL (1.05 mol) of distilled and purified ethylenediamine containing 32.2 mg (0.659 mmol) of sodium cyanide. It was dripped slowly using a tool pipette. 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. 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 Synthesis of DL-G0.5-2C ₁₈ -U2
DL-G0-2C ₁₈ -U2 (1.41 mg, 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 then 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 Synthesis of DL-G1-2C ₁₈ -U2
Dissolve 1.74 g (2.88 mmol) of DL -G0.5-2C ₁₈ -U2 in 50 mL of methanol and distill it into 92.8 mL (1.39 mol) of distilled and purified ethylenediamine containing 47.9 mg (0.98 mmol) of sodium cyanide. It was slowly dropped 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 Synthesis of MDEG-DL-G1
DL-G1 (100 mg, 0.12 mmol) was dissolved in dichloromethane (6 mL), and MDEG-4-nitrophenyl carbonate (131 mg, 0.48 mmol) dissolved in dichloromethane (2 mL) was gradually added. And stirred at room temperature for 6 days. 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 Synthesis of MDEG-DL-G2
DL-G2 (110 mg, 83.3 μmol) was dissolved in DMF (6 mL), and MDEG-4-nitrophenyl carbonate (190 mg, 666.4 μmol) dissolved in DMF (2 mL) was gradually added. And stirred at room temperature for 6 days. 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 Synthesis of MDEG-DL-G1-U2
DL-G1-U2 (400 mg, 0.50 mmol) was dissolved in DMF (6 mL), MDEG-4-nitrophenyl carbonate (713 mg, 2.50 mmol) dissolved in DMF (3 mL) was gradually added, and nitrogen atmosphere was added. The mixture was stirred at room temperature 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 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, 0.76 mmol) dissolved in dichloromethane (3 mL) was gradually added. And stirred at room temperature for 7 days. 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 Synthesis of EDEG-DL-G2
Dissolve DL-G2 (200 mg, 151.5 μmol) in dichloromethane (10 mL) and gradually add EDEG-4-nitrophenyl carbonate (544 mg, 1818 μmol) dissolved in dichloromethane (3 mL). And stirred at room temperature for 5 days. 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 Synthesis of EDEG-DL-G1-U2
DL-G1-U2 (400 mg, 0.38 mmol) was dissolved in DMF (4 mL), EDEG-4-nitrophenyl carbonate (673 mg, 2.25 mmol) dissolved in DMF (3 mL) was gradually added, and nitrogen was added. Stir for 7 days at room temperature under 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 Results
Characterization of temperature-responsive dendron lipids by ¹ H NMR As shown in the experimental section, dendron lipids were synthesized by reacting dioctadecylamine as an initiator with methyl acrylate and ethylenediamine alternately in methanol. 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.

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.

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.

  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.

  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. 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 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 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 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). ), And after stirring and holding at 10 ° C. for 30 minutes, measurement was performed. 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 Endothermic evaluation during temperature response by differential scanning calorimetry (DSC)
After adding 70 μl of pH 3.0 lipid dispersion prepared in 2.2. (0.2 mg / ml) to 630 μl of 10 mM phosphate buffer aqueous solution (140 mM NaCl), adjusting to pH 7.4 and holding at 10 ° C. for 30 minutes The measurement was performed. 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 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.

2.3 Results 2.3.1 Evaluation of temperature responsiveness of various dendron lipid aggregates
Each lipid dispersion (pH 7.4) for MDEG-DL-G1, EDEG-DL-G1, MDEG-DL-G2, EDEG-DL-G2, MDEG-DL-G1-U2, EDEG-DL-G1-U2 The temperature responsiveness was evaluated by the change in the transmittance when the temperature was raised. 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 -G1-U2,
The cloud points of 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.

  In addition, the pH dependence of temperature responsiveness 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.

2.3.2 Endothermic evaluation during temperature response of various dendron lipid molecular assemblies
Each lipid dispersion (pH 7.4) for MDEG-DL-G1, EDEG-DL-G1, MDEG-DL-G2, EDEG-DL-G2, MDEG-DL-G1-U2, EDEG-DL-G1-U2 The endothermic evaluation at the time of temperature response was performed by the endothermic energy when the temperature was raised. The results at that time are shown in Table 1, and the endothermic peaks in the temperature change are shown in FIGS.

  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.

  Furthermore, in MDEG-DL-G2 and EDEG-DL-G2, it was expected that an endothermic peak was observed at 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.

  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 at about 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.

2.3.3 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 further using 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.

  Next, in the microscopic observation under these circumstances, it was observed that the molecular assembly of EDEG-DL-G1 was slightly aggregated in a hydrated state at 10 ° C. When the temperature reached 40 ° C., the molecular aggregates were strongly aggregated to form huge aggregates.

  At low temperatures, these EDEG-DL-G1 molecular aggregates are in a hydrated state. However, since the hydrophilicity / hydrophobicity balance is poor, it is considered that they are slightly aggregated due to hydrophobic interaction. 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.

  As described above, the temperature responsiveness evaluation of aggregates of dendron lipid molecules into which various OEG groups were introduced and the morphology evaluation of the molecular aggregates were performed.

  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.

  In addition, 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 ° C. 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.

  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. 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 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 Preparation of dendron lipid dispersion
The solvent was removed from the 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 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).

  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 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 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 Sample preparation for AFM observation
20 μl of the lipid dispersion prepared in 2.5.1 was dropped onto mica and left 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. Results 3.6.1 Effect of PEG lipids on temperature response function of molecular assembly
The responsiveness of the EDEG-DL-G1 / PEG-Chol lipid with a PEG-Chol content of 5% was evaluated by the change in permeability when the lipid dispersion (pH 7.4) was 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.

  Further, 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.

  In addition, the pH dependence of the temperature response was evaluated by measuring the change in the transmittance of the dispersion of EDEG-DL-G1 / PEG-Chol lipid with a PEG-Chol content of 5% at various pHs. 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 Particle size measurement of molecular assembly composed of EDEG-DL-G1 / PEG-Chol (5%) lipids
FIG. 33 shows the results of particle size measurement at various temperatures of an EDEG-DL-G1 / PEG-Chol (5%) lipid molecular assembly (pH 7.4) into which 5% of PEG lipid was introduced using DLS. 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 Morphological evaluation of molecular assemblies composed of EDEG-DL-G1 / PEG-Chol (5%) lipids
FIG. 34 shows the results of morphological observation of EDEG-DL-G1 / PEG-Chol (5%) lipid molecular aggregate (pH 7.4) into which 5% of PEG lipid was introduced using AFM. 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.

  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.

  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 / morphological evaluation of the molecular assembly were performed. Then, by comparing this result with the result in Section 3, the effect of PEG lipids on the EDEG-DL-G1 molecular assembly was examined.

  The temperature response of the EDEG-DL-G1 / PEG-Chol lipid molecule assembly was evaluated 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.

  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 low temperatures 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). 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 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 Preparation of fluorescently labeled dendron lipid vesicles
Rhodamine-PE was added to a chloroform solution in which EDEG-DL-G1 and PEG lipid were mixed at a ratio of 95/5 (0.6 mol%), and the solvent was removed 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 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 Evaluation of intracellular delivery function
Hela cells were seeded at 2 × 10 ⁵ per 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 Results 4.4.1 Effect of temperature control of dendron lipid vesicles on intracellular delivery
FIG. 35 shows the results of confocal laser microscope images when incubating at 36 ° C. and 42 ° C., respectively. 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. Obviously, the fluorescence was stronger when incubating above the cloud point. 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.

  As described above, fluorescent labeling was applied to the EDEG-DL-G1 / PEG-Chol lipid molecular assembly into which the constructed PEG lipid was introduced, and the delivery function of the vesicle in cells was evaluated.

  In HeLa cells, when the temperature during incubation was controlled around the cloud point, a difference in the intracellular delivery function of vesicles was clearly observed. It was found that the fluorescence intensity of the labeled rhodamine lipid was significantly different before and after the cloud point temperature, and the fluorescence was stronger when incubated above the cloud point. 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.

  Based on the above, a new temperature-responsive vesicle that can control the temperature of binding and uptake into cells has been developed.

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.

  Dendron lipid / PEG-Chol aggregate dispersion (10 mM phosphoric acid, 140 mM NaCl, pH 3.0) 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 transmittance dependence of the transmittance in a buffer of 10 mM phosphoric acid, 140 mM NaCl, 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 of around 40 ° C. can be obtained by containing 2% of PEG-Chol.

6). Effect of incubation temperature on aggregate uptake by cells
HeLa cells (derived from human cervical cancer) were seeded in 12-well dishes 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 , so the interaction with the cell works very strongly even when morphological changes occur. It is thought that there was not.

  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.

  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.

  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.

  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.

Claims (9)

(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,
R ³ and R ⁴ contain hydrocarbon groups containing the same or different oligoethylene glycol structures;
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. ). The molecular assembly according to claim 1, wherein R ³ and R ⁴ are represented by the following formula (I):

(In the formula, n represents any 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). ). The molecular assembly according to claim 1 or 2, wherein the lipid (B) is represented by the following formula (II):

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). The molecular assembly according to any one of claims 1 to 3, which is used for introducing a physiologically active substance into a cell. The molecular assembly according to any one of claims 1 to 3, which is used for treating a disease by introducing a physiologically active substance into a cell. The composition containing the molecular assembly in any one of Claims 1-3. The kit containing the molecular assembly or composition in any one of Claims 1-6. Use of the molecular assembly, composition or kit according to any one of claims 1 to 7 in a method for introducing a physiologically active substance into a cell. A method for introducing a physiologically active substance into a cell, comprising a step of introducing the molecular assembly or composition according to any one of claims 1 to 6 into the cell together with the physiologically active substance.

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