JP4991168B2 - New dendrimer - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a dendrimer useful as a transgenic carrier with high safety and capable of encapsulating DNA. <P>SOLUTION: This dendrimer is composed of a hydrophilic unit or segment consisting of a polyoxyethylene unit and a dendron having a fluorene unit. The dendron is represented by formula (1) or formula (2) wherein, X<SB>1</SB>is a nitrogen atom; X<SB>2</SB>is an imino group (-NH-); Y is a spacer containing an amide bond, etc; and R<SP>1</SP>and R<SP>2</SP>are each substituent, is a dendron of the g-generation (g=1-5), forms a micell in water and enables to encapsulate the DNA. <P>COPYRIGHT: (C)2007,JPO&amp;INPIT

Description

  The present invention relates to a dendrimer useful as a DNA encapsulating compound, in particular, a novel dendrimer in which a hydrophilic unit or segment and a dendron having a fluorene unit are bound.

  Various gene transfer carriers (synthetic gene carriers) have been studied in gene therapy. Among these, synthetic carriers have advantages such as reduced side effects and low toxicity compared to carriers using viruses. Such a synthetic carrier is often a compound having a cationic charge, such as a cationized lipid compound or a cationic polymer, because DNA is an anionic polymer having a phosphate group. Such a cationic compound interacts with DNA to form a complex. In order to more efficiently encapsulate DNA and increase gene transfer efficiency, it is necessary to increase the degree of cationization. However, the higher the degree of cationization, the higher the ability to form a complex with DNA and the higher the introduction efficiency, but the greater the toxicity to cells.

  The 53rd Polymer Symposium Proceedings Polymer Preprints, Japan, Vol. 53, No. 2 (2004), p5258 (Non-patent Document 1) reports that fluorene intercalates with DNA. However, since fluorene is highly hydrophobic, it cannot efficiently introduce genes.

In order to reduce the electrostatic interaction and reduce the toxicity to the living body, an uncharged carrier has also been proposed, but such a carrier has low DNA encapsulation efficiency and introduction efficiency.
53th Polymer Symposium Proceedings Polymer Preprints, Japan, Vol.53, No.2 (2004), p5258

  Accordingly, an object of the present invention is to provide a novel dendrimer that is highly safe and can encapsulate DNA efficiently.

  Another object of the present invention is to provide a novel dendrimer that can improve gene transfer efficiency and is useful as a gene transfer carrier.

  Still another object of the present invention is to provide a novel dendrimer that can efficiently encapsulate DNA while suppressing electrostatic interaction and is useful as a gene transfer carrier.

  As a result of intensive studies to achieve the above-mentioned problems, the present inventors have used a fluorene intercalation strength with respect to DNA (deoxyribonucleic acid) as a gene carrier, and have hydrophilic units or segments (such as polyoxyethylene segments). When a dendron (dendritic side chain) having a fluorene unit is bound to the dendrimer, the dendrimer forms micelles in water and efficiently encapsulates DNA using intercalation without using electrostatic interaction. I found what I could do and completed the present invention.

  That is, the novel dendrimer of the present invention is composed of a hydrophilic unit or segment and a dendron having a fluorene unit bonded to the unit or segment. That is, a hydrophilic unit or segment and a dendron having a fluorene unit (fluorene unit) are bonded. Since such a dendrimer includes a hydrophilic unit or segment and a highly hydrophobic fluorene unit, the encapsulation efficiency for DNA can be increased without using electrostatic interaction, and the toxicity to the living body can be increased. It can be reduced and safety is high.

  In the dendrimer, the dendron is a g-th generation (g = 1) composed of at least one repeating unit represented by the following formula (1) and a unit represented by the following formula (2) and constituting the terminal. It may be a dendron of ~ 5).

(In the formula, X ₁ is a trivalent atom or an organic group, X ₂ is an organic group, R ¹ and R ² are the same or different and each represents a hydrogen atom, a halogen atom, an alkyl group, or an alkoxy group)
X ₁ and X ₂ may be directly bonded to each other, but are usually often connected by a linking group and / or a spacer. The branching number n of the dendron from the hydrophilic unit or segment may be about 1 to 3, and the dendron may be a g-th generation (g = 1 to 4) dendron. The hydrophilic unit or segment may be composed of, for example, a polyoxyethylene unit, and the dendron is branched from the hydrophilic unit or segment at a branch number n = 1 to 3 and represented by the following formula (3). And a g-th generation (g = 1 to 3) dendron.

(In the formula, X ₁ represents a nitrogen atom, X ₂ represents an imino group (—NH—) or a substituted imino group, Y represents a spacer containing an imino group (—NH—), an amide bond, an ester bond or a urethane bond; ¹ and R ² are the same as above)
Further, the dendron may be a g-th generation (g = 1 to 3) dendron represented by the following formula (4).

Wherein X ₁ is a nitrogen atom, X ₂ is an imino group (—NH—) or a substituted imino group, R ³ and R ⁴ are each a linear or branched alkylene group, and Y ₁ is an imino group (—NH -), Represents an amide bond, an ester bond or a urethane bond, and R ¹ and R ² are the same as above)
The dendrimer has hydrophilic units or segments and highly hydrophobic fluorene units, and can form micelles in water. Therefore, micelles can be formed in water, and DNA can be efficiently taken up and encapsulated.

  In the present invention, since a hydrophilic unit or segment and a dendron (dendritic side chain) having a highly hydrophobic fluorene unit are provided, DNA can be efficiently encapsulated even if it is electrically neutral. High nature. Therefore, DNA can be efficiently encapsulated while suppressing electrostatic interaction, and it is useful as a gene transfer carrier. Further, by adjusting the balance between hydrophilicity and hydrophobicity, gene transfer efficiency can be greatly improved, and it is useful as a gene transfer carrier.

[Dendrimer]
The dendrimer of the present invention is composed of a hydrophilic unit or segment and a fluorene unit (dendron (dendritic side chain) having a fluorene unit as a hydrophobic unit) bonded to the hydrophilic unit or segment. Have a branched structure. The hydrophilic unit or segment and the dendron may be directly bonded or indirectly bonded via a linking group (or spacer). Further, the hydrophilic unit or segment and the dendron may be bonded with an appropriate number of branches. The fluorene unit may be a fluorenyl group alone or may be composed of a fluorenyl group and a linking group and / or a spacer.

  The dendrimer of the present invention is composed of a hydrophilic unit or segment and a fluorene unit, and has a dendritic structure. Such a dendrimer can be represented by the following formula, for example.

_{^{A - [[S 1 - (}} R-S 2) r] p -D] n
(In the formula, A represents a residue of a hydrophilic compound, S ¹ and S ² are the same or different and represent a linking group, R represents a hydrocarbon group, and D represents a hydrophobic unit (fluorene unit such as dendron)) R represents 0 or 1, p represents 0 or 1, n represents the number of branches of a fluorene unit such as dendron)
The hydrocarbon group R is not particularly limited and may be a linear or branched C _1-10 alkylene group which may have a substituent, or a C _3-10 cycloalkylene group which may have a substituent. _{Or a} C _6-12 arylene group which may have a substituent.

In the above formula, examples of the linking group represented by S ¹ and S ² include an oxygen atom (ether bond), a sulfur atom (sulfide bond), a nitrogen atom or an imino group, and an ester bond (—COO— or —OCO—). ), An amide bond (—CONH— or —NHCO—), a urethane bond (—NHCOO— or —COONH—), a urea bond, or the like, or a combination thereof.

In the formula, the alkylene group (or alkylidene group) represented by R is a linear or branched C _1-10 alkylene or alkylidene group (preferably C ₁ ) such as methylene, ethylene, propylene, trimethylene, butylene group or the like. _-6 alkylene or alkylidene group).

  In the case where a dendrimer is obtained by a reaction between a hydrophilic compound and a compound corresponding to the fluorene unit [dendron (or dendron precursor) or the like], in the above formula, (i) p is 0, or (ii) p is 1 and r is 0. That is, in the case of the former (i) (p = 0), the reactive group of the hydrophilic compound has two or more bonds (valence) (for example, an amino group containing a nitrogen atom having a valence of 3). Etc.), this reactive group may constitute a branching site (or a binding site with a dendron) of a fluorene unit (such as a dendron).

Further, when a dendrimer is obtained by a reaction between a hydrophilic compound and a compound corresponding to the fluorene unit [dendron (or dendron precursor) and the like] and a spacer compound, in the above formula, r = p = 1, and unit-S ^1- (R—S ² ) _r — corresponds to the residue of the spacer compound. In addition, the connection of a hydrophilic compound and a fluorene unit (such as a dendron) by such a spacer compound (that is, the connection between a hydrophilic unit and a fluorene unit such as a dendron) involves a single or a plurality of known chemical reactions. Available.

  The spacer compound corresponds to the group R depending on the type of the reactive group (or functional group) of the hydrophilic compound and the reactive group of the compound corresponding to the fluorene unit [dendron (or dendron precursor, etc.)]. Various polyfunctional compounds such as alkanedicarboxylic acid, alkanediol, alkanediamine, hydroxyalkanecarboxylic acid, hydroxylalkylamine, amino acid and the like can be mentioned. The constituent components of dendrimer are described in detail below.

(Hydrophilic unit or segment)
The type of the hydrophilic unit or segment is not particularly limited, and may be, for example, an anionic group (such as a carboxyl group, a sulfonic acid group, or a salt thereof), a cationic group (such as an amino group or a salt thereof), and the like. And a nonionic group (or ether group). Such a hydrophilic unit or segment (hereinafter sometimes simply referred to as a hydrophilic unit) is composed of polyoxyalkylene units (polyoxy C _2-4 alkylene units such as polyoxyethylene units and polyoxypropylene units). It is often done. The dendrimer may have one of these hydrophilic units, or may have a plurality of the same or different hydrophilic units. The dendrimer preferably has at least a polyoxyethylene unit among the hydrophilic units.

  The dendrimer includes at least a compound having the hydrophilic unit [that is, a compound corresponding to the hydrophilic unit or segment (hydrophilic compound)] and a compound corresponding to the fluorene unit [dendron (or dendron precursor) and the like]. A reaction between the hydrophilic compound, a compound corresponding to the fluorene unit [dendron (or dendron precursor), etc.] and a spacer compound (a compound capable of forming the linking group or spacer) if necessary. May be obtained.

  The hydrophilic compound corresponding to the residue of the hydrophilic compound has, together with the hydrophilic unit, a compound corresponding to the fluorene unit [dendron (or dendron precursor) or the like] or a reactive group for the spacer compound. The type of the reactive group is not particularly limited as long as it can be bonded to a compound corresponding to the fluorene unit [dendron (or dendron precursor) or the like] or a spacer compound. For example, a halogen atom (chlorine, bromine, iodine) Atoms, etc.), hydroxyl groups, mercapto groups or their reactive derivative groups (such as lower alkoxy groups), carboxyl groups or their reactive derivative groups (such as acyl halide groups (haloformyl groups), acid anhydride groups), amino groups or imino groups Group, epoxy group (or glycidyl group) and the like. In the hydrophilic compound, the reactive group is often a hydroxyl group, an amino group or an imino group, an epoxy group (or glycidyl group) and the like.

  Moreover, the hydrophilic compound may have a reactive group having a bond (valence) corresponding to the branch number n of the fluorene unit (or dendron). When the hydrophilic compound has a monovalent reactive group, the monovalent reactive group may be converted to a reactive group having a bond (valence) corresponding to the branch number n. The hydrophilic compound and the spacer compound The reactive group having a bond (valence) corresponding to the branch number n may be introduced into the hydrophilic compound.

Among the hydrophilic compounds, compounds having a hydroxyl group include alkane polyols (such as C _2-16 alkane polyols such as ethylene glycol, propylene glycol, glycerin and pentaerythritol), saccharides (such as monosaccharides and disaccharides), for example, Sugars such as sugar, lactose and glucose, sugar alcohols such as xylitol, erythritol and mannitol), (poly) oxyalkylene glycols [(poly) oxyethylene glycols such as diethylene glycol, triethylene glycol, tetraethylene glycol, (Poly) oxypropylene glycols such as dipropylene glycol and tripropylene glycol; (poly) oxyethylene- (poly) oxypropylene copolymer A compound such as a polyoxyethylene-polyoxypropylene block copolymer], a compound having an active hydrogen atom (the alkane polyols, saccharides, amino group-containing compounds, carboxyl group-containing compounds, etc.) and C _2-4 alkylene oxide. Examples include adducts (particularly, at least ethylene oxide) [for example, glycerin ethylene oxide adducts, sucrose ethylene oxide adducts, and the like].

  Examples of the amino group-containing compound include alkylamines such as methylamine and ethylamine, alkanepolyamines such as ethylenediamine, propylenediamine, tetramethylenediamine, and hexamethylenediamine, and alkylenediamines such as ethylenediamine, propylenediamine, and butylenediamine. Examples include poly (alkylenediamines) such as diethylenetriamine and triethylenetetramine; alicyclic or aromatic mono- or polyamines, etc. Examples of carboxyl group-containing compounds include monocarboxylic acids such as acetic acid, malonic acid, succinic acid, etc. Examples thereof include oxycarboxylic acids such as polycarboxylic acid and lactic acid.

Examples of hydrophilic compounds having an amino group, an imino group or an epoxy group (or glycidyl group) include (poly) oxy C _2-4 alkylene diamine (for example, (poly) oxyethylene diamine), hydroxy (poly) oxy C _{2− 4} alkylene monoamines (eg, hydroxy (poly) oxyethylene monoamine), alkoxy (poly) oxy C _2-4 alkylene monoamines (eg, alkoxy (poly) oxyethylene monoamine), (poly) oxy C _2-4 alkylene diamines glycidyl ether (e.g., (poly) oxyethylene diglycidyl ether, etc.), hydroxy (poly) oxy-C _2-4 alkylene monoglycidyl ethers (e.g., hydroxy (poly) oxyethylene mono glycidyl ether, etc.), alkoxy (poly) O Shi C _2-4 alkylene monoglycidyl ethers (e.g., alkoxy (poly) oxyethylene mono glycidyl ether, etc.), and others.

In addition, when the compound has a plurality of terminal reactive groups, some reactive groups may be a protective group or a capping group such as an alkyl group (methoxy group, ethoxy group, butoxy group, t-butyl group, etc. C _1-6 alkyl group), an alkoxy group (methoxy group, an ethoxy group, a propyl group, a butoxy group, _{t-C 1-6} alkoxy group such as butoxy), an acyl group (acetyl group, such as propionyl group _{C 1- 6} alkylcarbonyl group) or the like. For example, as a compound in which a part of a plurality of hydroxyl groups of the alkane polyols and saccharides are protected or sealed, a partial ether or a fatty acid ester, for example, an alkane polyol C _1-4 alkyl ether such as ethylene glycol monomethyl ether, Examples include polyoxyalkylene glycol mono _C1-4 alkyl ethers such as (poly) ethylene glycol monomethyl ether, sucrose fatty acid esters, and the like.

  These hydrophilic compounds can be used alone or in combination of two or more. A preferred compound corresponding to the hydrophilic unit or segment is an adduct obtained by adding at least ethylene oxide to polyols or saccharides, polyethylene glycol, or at least one terminal hydroxyl group of the adduct or a plurality of terminal hydroxyl groups of polyethylene glycol. Compounds substituted with amino groups, glycidyl ethers with epichlorohydrin added to at least one of the terminal hydroxyl groups of the adduct or polyethylene glycol, or a part of the terminal hydroxyl groups are protected or sealed Derivatives (such as monoalkyl ethers).

  The hydrophilic compound having a (poly) oxyethylene unit is often represented by the following formula, for example.

_{- (CH 2 CH 2 O)} m CH 2 CH 2 Z
(In the formula, Z represents a hydroxyl group, an amino group, an imino group, or a glycidyl group, and m represents an integer of about 1 to 500)
m is usually 2 to 400, preferably 3 to 300, more preferably about 5 to 250 (for example, 10 to 200), and may be about 10 to 100.

  The molecular weight of the compound corresponding to the hydrophilic unit or segment is not particularly limited, and can usually be selected from the range of about 50 to 25000 (for example, 75 to 20000) weight average molecular weight, and the weight average molecular weight is 100 to 10,000, preferably 200 to It may be about 8000 (for example, 200 to 7000), more preferably about 250 to 5000 (for example, 300 to 3000).

(Dendron)
In a dendrimer (or a dendrimer having a micelle-forming ability), a dendron (a dendritic side chain or a hyperbranched side chain) is bound to a hydrophilic unit or segment, and the dendron is a fluorene unit that can intercalate with DNA. have.

  The dendron (dendron D in the above formula) may be composed of at least a fluorenyl group, and is usually represented by at least one repeating unit represented by the following formula (1), the following formula (2), and a terminal. It consists of the units that make up.

(In the formula, X ₁ is a trivalent atom or an organic group, X ₂ is an organic group, R ¹ and R ² are the same or different and each represents a hydrogen atom, a halogen atom, an alkyl group, or an alkoxy group)
R ¹ and R ² include a hydrogen atom, a halogen atom (fluorine, bromine, chlorine or iodine atom), an alkyl group (for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, s-butyl, t-butyl, pentyl) , Isopentyl, neopentyl, t-pentyl, hexyl group and other linear or branched C _1-6 alkyl groups), alkoxy groups (eg, methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, s-butoxy, Examples thereof include linear or branched C _1-6 alkoxy groups such as t-butoxy, pentyloxy, isopentyloxy, and hexyloxy groups. In many cases, R ¹ and R ² are usually hydrogen atoms.

Examples of the trivalent atom represented by X ₁ include a nitrogen atom, a phosphorus atom, a triol residue (such as a glycerin residue and a trimethylolpropane residue), and a tricarboxylic acid residue. X ₁ may be an atom derived from the reactive group of the hydrophilic compound or the reactive group of the spacer compound. Preferred X ₁ is a nitrogen atom.

Examples of the organic group represented by X ₂ include divalent organic groups such as divalent hydrocarbon groups such as ether groups, alkylene groups, cycloalkylene groups, and phenylene groups, imino groups, and substituted imino groups (for example, acylimino groups). Group, alkylimino group, etc.).

In dendron D, the unit X ₁ represented by the formula (1), the formula and X ₂ units represented by (2), it may be bonded directly, usually linking group (or spacer ) In many cases. That is, in the dendron D, a plurality of units (or dendron precursors) constituting the dendron may be linked by a linking group (or spacer), and the reactive group of the hydrophilic compound is a branched part of the dendron (that is, X _In the case of forming ₁ ), the reactive group of the hydrophilic compound and the dendron precursor may be linked by a linking group (or spacer).

  These ligation reactions, that is, reactions between a plurality of units (reactive groups of units) constituting a dendron, and reactions between a reactive group of a hydrophilic compound and a reactive group of a dendron precursor are various reactions. , For example, a reaction between a halogen atom and an active hydrogen atom (or a functional group having an active hydrogen atom such as a hydroxyl group, a carboxyl group, an amino group, an imino group, etc.), a hydroxyl group, a mercapto group, or a reactive derivative group thereof (Such as a lower alkoxy group) and a halogen atom, a carboxyl group or an acid anhydride group, an alkoxysilyl group or an isocyanate group, a carboxyl group or a reactive derivative group thereof (an acyl halide group, an acid anhydride group, etc.) Reaction with hydroxyl group, amino group or epoxy group, amino group or imino group and α, β-unsaturated hydrocarbon group (vinyl group) Alkenyl group such as propenyl group, isopropenyl group and butenyl group, alkynyl group such as ethynyl group and 2-propynyl group), reaction with carbonyl group, carboxyl group, epoxy group or isocyanate group, epoxy group (or glycidyl group) ) And a carboxyl group or an amino group. In addition, a Grignard reaction or a coupling reaction using a halogen atom can be used. Furthermore, carbonylation reaction with carbonyl group (ketone group) of fluorene unit uses carbonylation reagent or compound having amino group (compound having terminal amino group or imino group, hydrazine derivative, semicarbazide, etc.) You can also In the reaction with a hydroxyl group of a fluorene unit (9-hydroxyl group of fluorene), a reaction with a halogen atom (bromine, chlorine atom, etc.), a carboxyl group, an acid anhydride group or an isocyanate group can be used.

The type of the linking group is not particularly limited, and can be appropriately selected from the linking groups similar to the linking groups S ¹ and S ² exemplified in the section of the dendrimer, or combinations thereof. The spacer often includes at least these linking groups, particularly amide bond, ester bond, urethane bond, and the like, and often includes these linking groups and a hydrocarbon chain.

  The dendron is usually composed of a fluorenyl group and a linking group and / or a spacer. Examples of the dendron having a linking group and / or a spacer between the units (1) and (2) include a dendron represented by the following formula (3).

(In the formula, X ₁ represents a nitrogen atom, X ₂ represents an imino group (—NH—) or a substituted imino group, Y represents a spacer containing an imino group (—NH—), an amide bond, an ester bond or a urethane bond; ¹ and R ² are the same as above)
The spacer Y includes an aliphatic hydrocarbon chain (such as a linear or branched C _1-10 alkylene group), an alicyclic hydrocarbon chain (such as a cyclohexylene group, a cyclohexane-1,4-dimethylene group, and the like. C _3-10 cycloalkane-diyl group, etc.) and an aromatic hydrocarbon chain (C _6-12 arene-diyl group such as phenylene group, xylylenyl group, etc.). The spacer Y often includes a linking group such as an amide bond, an ester bond, or a urethane bond, and a linear or branched alkylene group, and the dendron is, for example, a dendron represented by the following formula (4). There may be.

(Wherein, X ₁ is a nitrogen atom, X ₂ is an imino group (—NH—) or a substituted imino group, R ³ and R ⁴ are hydrocarbon chains, Y ₁ is an imino group (—NH—), an amide bond, An ester bond or a urethane bond is shown, and R ¹ and R ² are the same as above)
The hydrocarbon chain may be the aliphatic hydrocarbon chain, alicyclic hydrocarbon chain, aromatic hydrocarbon chain or the like. The hydrocarbon group is usually an aliphatic hydrocarbon group, in particular a linear or branched alkylene group. Examples of the alkylene group include linear or branched C _1-10 alkylene groups such as a methylene group, an ethylene group, a propylene group, a trimethylene group, a tetramethylene group, a 2-methyltrimethylene group, and a hexamethylene group. it can. The alkylene group is usually a linear or branched C _2-6 alkylene group in many cases.

A typical dendron usually has a unit represented by the formula (1) (at least one repeating unit) and has a regular branch structure, but the branch structure is not a regular hyperbranch. A (hyperbranched) structure may be used. The dendron can constitute a g-th generation (Gn) dendron with n repeating units represented by the formula (1) and 2 ⁿ units represented by the formula (2). For example, the first generation (G1) dendron and the two repeating units represented by the formula (1) and the two units represented by the formula (2) are represented by the two formulas (1). The second generation (G2) dendron with the repeating unit and the four units represented by the formula (2), the three repeating units represented by the formula (1), and the eight represented by the formula (2) A third generation (G3) dendron can be configured with the unit. The generation g of dendron is about 1 to 5 (preferably 1 to 4, more preferably 1 to 3).

  Furthermore, when the valence (or the number of reactive groups or the number of reactive sites) of the hydrophilic compound is n, a g-th generation dendrimer having n branches (or bonds) can be obtained. The number of branches n is about 1 to 5, usually 1 to 4, preferably about 1 to 3 (for example, 1 or 2). A preferred dendrimer is a g-th generation (g = 1 to 5, preferably about 1 to 4 (for example, about 1 to 3)) dendrimer having a branch number n = 1, and a g-th generation (g = 1) To 4 (preferably about 1 to 3) dendrimers and g-th generation (g = 1 to 4, preferably about 1 to 3) dendrimers with n = 3 branches. The dendrimer is often a g-th generation (g = 1 to 3) dendrimer having n = 1 or 2 branches.

  Among the novel dendrimers of the present invention, a dendrimer having a first generation and a second generation dendron can be represented by the following formula, for example.

(In the formula, A represents a residue of a hydrophilic compound, n represents the number of branches, F represents a fluorene residue optionally having substituents R ¹ and R ² , and X ₁ , X ₂ , and Y are as defined above. the same)
In the dendrimer represented by this formula, the residue A of the hydrophilic compound is represented by the formula (CH ₂ CH ₂ O) _m CH ₂ CH ₂ Z (Z and m are the same as above) as described above. (Poly) oxyethylene chains may be used. In this case, one end of the (poly) oxyethylene chain may be a free hydroxyl group, and may be a protected or sealed end as described above, and may be at both ends of the (poly) oxyethylene chain. A dendron may be bonded to X1.

As long as it has the said structure, the dendrimer of this invention may be a molecular species of a low molecule | numerator or an oligomer area | region, and may be a molecular species of a polymer area | region. The dendrimer of the present invention has a fluorenyl group capable of intercalating with DNA. The fluorenyl group or fluorene unit is hydrophobic. Therefore, the balance between hydrophilicity and hydrophobicity (HLB) can be achieved by adjusting hydrophilicity and hydrophobicity by combining hydrophilic units or segments (hydrophilic compounds) and fluorene units (or generations of dendrons, dendrimers, etc.). ) Can be adjusted, and the encapsulation efficiency for DNA can be improved. The dendrimer HLB (hydrophile-lipophile-balance) of the present invention can be selected according to the type of encapsulated substance such as DNA, and is, for example, 5-30 (eg, 7-25), preferably 8-20 (eg, It may be about 10-18). The critical micelle concentration (CMC) of the dendrimer is about 1 × 10 ⁻⁷ to 1 × 10 ⁻⁴ mol, usually 3 × 10 ^{−7 to} 5 × 10 ⁻⁵ mol, preferably 5 × 10 ^−7. It may be about ˜7 × 10 ⁻⁵ mol.

[Preparation of dendrimer]
The dendron can be prepared by a known or conventional method, and methods described in various books and literatures can be referred to. Dendrimers can be obtained by publicly known or commonly used methods, for example, a divergent method in which monomers are sequentially bonded to a hydrophilic compound serving as a core, and branching dendrons are prepared in advance to form a hydrophilic core. It can be prepared using a convergent method for binding to a compound, a method combining these, and the like. For these reactions, known or conventional reactions such as the reaction of the above-mentioned hydrophilic compounds and dendrons can be used.

More specifically, using a divergent method, a dendrimer having a hydrophilic chain (hydrophilic segment) having a polyoxyethylene unit and a dendron represented by the formula (4) is produced. For example, (a) using a Mannich reaction, a polyoxyethylene compound having a reactive group (such as an amino group) corresponding to X ₁ and a group R ³ (wherein R ³ is the same as above) An intermediate that reacts with a corresponding compound (for example, acrylic acid or an ester thereof), branches, and has a residue R ^3a (for example, an acrylic acid residue or an ester residue thereof) of the compound corresponding to R ³ Is generated. Examples of acrylic acid or esters thereof include acrylic acid lower alkyl esters (such as acrylic acid _C1-4 alkyl esters) such as acrylic acid, acrylic acid methyl ester, and acrylic acid ethyl ester, and acrylic acid hydroxyalkyl esters (acrylic acid 2). -Hydroxyethyl, 2-hydroxypropyl acrylate, hydroxy _C2-6 alkyl ester such as 4-hydroxybutyl acrylate), glycidyl acrylate, and the like can be used.

(B) Corresponds to the residue R ^3a (acrylic acid residue or ester residue thereof) of the compound produced by the above reaction and the formula Y ₁ -R ⁴ (wherein Y ₁ and R ⁴ are the same as above). a compound is reacted to produce a compound having a residue R ^4a corresponding to R ^4. In this reaction, various compounds can be used as the compound corresponding to the formula Y ₁ -R ⁴ depending on the type of the residue R ^3a . For example, in acrylic acid or acrylic acid lower alkyl ester, polyamines (particularly diamines such as C _2-10 alkane diamines such as ethylene diamine, propylene diamine and butylene diamine, alicyclic diamines such as cyclohexane diamine and diaminomethylcyclohexane) Amide bond formation reaction using aromatic diamine such as xylylenediamine), polyols (particularly diols such as C _2-10 alkanediols such as ethylene glycol, propylene glycol, butanediol, hexanediol, cyclohexane) Esterification reaction using diol, alicyclic diol such as cyclohexanedimethanol, aromatic diol such as xylylenediol, and the like can be used.

  In the hydroxyalkyl ester of acrylic acid, a carboxyl group-containing compound [polycarboxylic acid or a lower alkyl ester thereof (for example, an aliphatic dicarboxylic acid such as adipic acid, fumaric acid or maleic anhydride, an alicyclic dicarboxylic acid such as cyclohexanedicarboxylic acid, Phthalic acid, phthalic anhydride, isophthalic acid, aromatic dicarboxylic acid such as terephthalic acid), hydroxycarboxylic acid (for example, aliphatic hydroxycarboxylic acid such as lactic acid and glycolic acid, aromatic hydroxycarboxylic acid such as hydroxybenzoic acid, etc. ), Amino acids and the like], polyisocyanates (particularly diisocyanates such as aliphatic diisocyanates such as hexamethylene diisocyanate, isophorone diisocyanate, hydrogenated xylylene diisocyanate). Alicyclic diisocyanates such as tolylene diisocyanate, and a urethane reaction using, for example, aromatic diisocyanates such as xylylene diisocyanate) are available. In addition, in glycidyl acrylate, an addition reaction using the carboxyl group-containing compound or the like can be used.

Then, to obtain a dendrimer having a dendron represented by residues R ^4a and fluorenones corresponding to (c) R ⁴ (fluorenone, fluorenol, fluorene anions, etc.) by reacting the above formula (4) it can. In this reaction, if necessary, the residue R ^4a is converted to a reactive group (such as an amino group) that can react with a carbonyl group (ketone group), and then the 9-carbonyl group of the fluorenones is selected according to the residue R ^4a. Various reactions utilizing the reactivity of, for example, a reductive amination reaction, a reaction with a carbonylating reagent, a Grignard reaction and the like can be used.

  The dendrimer of the present invention can also form micelles in water. In water, the fluorene units at the hydrophobic sites are oriented inward and the hydrophilic units or segments are oriented outward to form micelles. The formation of micelles can be confirmed, for example, by adding a compound that does not exhibit fluorescence in a hydrophilic solvent but exhibits fluorescence in a hydrophobic solvent and measures the fluorescence.

  Furthermore, the dendrimer of the present invention can form micelles in water and encapsulate DNA. Encapsulation of DNA can be confirmed by measuring the ζ potential or Z potential, which is an indicator of the surface potential of the micelle, by increasing the micelle size by adding DNA. The encapsulated component is not limited to DNA, but may be a physiologically active component (including peptides and polypeptides). Useful encapsulating ingredients are pharmacologically active ingredients, especially DNA for gene therapy. In addition, when a disintegrant such as cyclodextrin is added, micelles can be disintegrated. Therefore, the encapsulated component can be released using this collapse phenomenon.

  Therefore, the dendrimer of the present invention is useful as a gene transfer carrier (synthetic gene carrier) in gene therapy.

  Hereinafter, the present invention will be described in more detail based on examples, but the present invention is not limited to these examples.

Dendrimers having first generation (2 terminal fluorenes) and second generation (4 terminal fluorenes) dendrons are converted to polyethylene glycol PEG chain (CH ₂ CH ₂ O) _m length m = 6,45. , 454 (molecular weight or weight average molecular weight M = 350, 2000, 20000). In addition, the reaction process formula of the dendrimer used in the Example below is shown.

Example 1
[Step 1: Tosylation reaction]
Polyethylene glycol monomethyl ether (molecular weight 350) (2) (n = 6) 10 g (0.028 mol) was dissolved in 100 ml of pyridine, 50 ml of chloroform was added, and 1.5 times equivalent of p-toluenesulfonate was added under ice cooling. .17 g (0.043 mol) was added and stirred for 4 hours. After stirring, it was confirmed by thin-layer liquid chromatography TLC that the starting material spot became thin and a new spot was developed. Ice was added, the mixture was stirred again for 30 minutes, concentrated under reduced pressure, and then dried in vacuo. After drying, gradient elution with chloroform-ethanol by silica gel chromatography (C-300) and removal of the solvent by concentration under reduced pressure gave compound (3) in a yield of 86% [12.5 g (0.024 mol)]. It was.

[Step 2: Azido reaction]
12.5 g (0.024 mol) of compound (3) was dissolved in 200 ml of dimethylformamide DMF and stirred at room temperature. To the solution, 2.8 g (0.04 mol) of sodium azide dissolved in 100 ml of DMF was added dropwise and stirred at 80 ° C. overnight. After confirming the disappearance of the compound 3 spot by TLC, the temperature was returned to room temperature, the solution was dispersed in excess acetone, and the precipitate (crystal) was removed. The solution was concentrated under reduced pressure at 98 ° C. to obtain Compound (4) in a yield of 78% [7.02 g (0.02 mol)].

[Step 3: Amination reaction]
7.02 g (0.02 mol) of compound (4) was dissolved in 200 ml of ethanol and poured into an autoclave. After activating 200 g of palladium carbon, it was added to an autoclave, 100 ml of ethanol was added, and catalytic reduction with hydrogen was performed at 60 ° C. for 2 days. The solution was filtered, concentrated under reduced pressure, and dried under reduced pressure. After drying, gradient elution with chloroform-ethanol by silica gel chromatography (C-300) gave Compound (5) in a yield of 72% [5.03 g (0.015 mol)].

[Step 4: Addition reaction with acrylate ester]
2.5 g (7.16 × 10 ⁻³ mol) of the compound (5) was dissolved in 100 ml of dehydrated ethanol and replaced with nitrogen. 1.95 g (0.022 mol) of methyl acrylate was dissolved in 50 ml of dehydrated ethanol and slowly added dropwise with stirring under ice cooling. After the dropwise addition, the temperature was slowly returned to room temperature, 50 ml of dehydrated chloroform was added, and the mixture was stirred for 60 hours. After stirring, the mixture was concentrated under reduced pressure, and eluted with a gradient of chloroform-ethanol using silica gel chromatography (C-300) to give Compound (6) in a yield of 68% [2.54 g (4.89 × 10 ⁻³ mol). ] Was obtained.

[Step 5: Amidation with diamine]
2.54 g (4.89 × 10 ⁻³ mol) of the compound (6) was dissolved in 100 ml of dehydrated ether and purged with nitrogen. 1 ml (0.017 mol) of ethylenediamine was dissolved in 50 ml of ether and slowly added dropwise. Stir at room temperature for 72 hours. After stirring, the solvent was removed by concentration under reduced pressure, and silica gel chromatography (C-300) was used, and gradient elution was performed with chloroform-ethanol to obtain Compound (7) in a yield of 55% [1.55 g (2.7 × 10 ^-3 mol)].

[Step 6: Preparation of first generation dendrimer]
1.55 g (2.7 × 10 ⁻³ mol) of the compound (7) was dispersed in 50 ml of toluene, 1.8 g (0.01 mol) of fluorenone was added, 5 ml of pyridine was added dropwise, and the mixture was stirred for 3 days. The solvent was removed by concentration under reduced pressure, 50 ml of dehydrated DMF was added, 0.7 g (0.01 mol) of cyanoborohydride was added, and the mixture was stirred for 4 days. After stirring, the reaction solution was dispersed in 200 ml of ether and extracted with ethanol. Since three spots were confirmed by TLC, the solvent was removed by concentration under reduced pressure to obtain yellowish white crystals. The crystals were recrystallized from ethanol-water and suction filtered. The filtrate was concentrated under reduced pressure, recrystallized from hexane-toluene, and suction filtered. Using a silica gel chromatography (C-300), the crystals were gradient with chloroform-ethanol to give a white (slightly cream) crystalline compound (1-2: 350) in a yield of 73% [1.82 g (2.0 × 10 ⁻³ mol)].

  All of the compounds (4) to (7) were oily. Moreover, the production | generation of each compound was confirmed by NMR.

Example 2 (second generation dendrimer)
After repeating the reaction of Step 4 of Example 1 (addition reaction with acrylate ester) and the reaction of Step 5 (amidation reaction with diamine) from compound (7), reductive amination with fluorenone gives compound (1 -4: 350). The purification method was performed in the same manner as in the compound (1-2: 350). Formation of the compound (1-4: 350) was confirmed by NMR. The reaction process formula is shown below.

Examples 3 and 4
Compound (1-2: 2000) and compound (1) were used in the same manner as in Examples 1 and 2 except that polyethylene glycol monomethyl ether (molecular weight 2000, n = 45) was used instead of polyethylene glycol monomethyl ether (molecular weight 350). -4: 2000).

Examples 5 and 6
In place of polyethylene glycol monomethyl ether (molecular weight 350), polyethylene glycol monomethyl ether (molecular weight 20000, n = 454) was used in the same manner as in Examples 1 and 2, except that compound (1-2: 20000) and compound (1 -4: 20000).

Example 7 (Formation of micelles)
In a dendrimer having a hydrophilic part and a hydrophobic part, it is considered that a terminal fluorenyl group (or fluorenyl moiety) serves as a core, and an amphiphilic polyethylene glycol PEG chain or a micelle having a PEG moiety as a shell is formed. On the other hand, 8-anilino-1-naphthalenesulfonic acid (ANS) does not show much fluorescence in a hydrophilic solvent and has a property of showing fluorescence in a hydrophobic solvent. Therefore, it is considered that the fluorescence intensity increases when the concentration of the dendrimer is increased. Therefore, in order to confirm whether or not micelles are formed in water, the compound (1-2) is added to an aqueous solution having an ANS concentration of 2 × 10 ⁻⁶ M, 2 × 10 ⁻⁵ M, 2 × 10 ⁻⁴ M. 350) was added and the fluorescence intensity was measured.

As a result, as shown in FIGS. 1 to 3 (FIG. 1: ANS concentration 2 × 10 ⁻⁶ M, FIG. 2: ANS concentration 2 × 10 ⁻⁵ M, FIG. 3: ANS concentration 2 × 10 ⁻⁴ M), The increase in the fluorescence intensity of ANS and the maximum value (peak) of the fluorescence intensity shifted to the lower wavelength side (blue shift). This shows that the environment around the ANS has changed from hydrophilic to hydrophobic. When the addition concentration of the compound (1-2: 350) is plotted on the horizontal axis and the change in the fluorescence wavelength of ANS and the change in fluorescence intensity are plotted on the vertical axis, the addition concentration of the compound (1-2: 350) is 1 × 10 ^{− A} critical micelle concentration (CMC), which is an inflection point, exists between ⁶ M and 1 × 10 ⁻⁵ M. From this critical micelle concentration (CMC), the compound (1-2: 350) can form micelles in water at a concentration of about 2 × 10 ⁻⁶ M to 2 × 10 ⁻⁵ M, particularly 5 × 10 ⁻⁶ M. all right. Further, the signal due to fluorene was confirmed in deuterated chloroform by NMR measurement of ¹³ C, but the signal due to fluorene disappeared in heavy water, so that compound (1-2: 350) was fluorene in water. It is suggested that the site | part forms the micelle used as a core.

  In addition, the result was obtained similarly about the compound (1-4: 350), the compound (1-2: 2000), and the compound (1-4: 2000).

Example 8 (Micellar Collapse)
The compound (1-2: 350) forms micelles, but the release of the encapsulated component or the sealing component is also an important factor in gene transfer. Therefore, when cyclodextrin is added by utilizing the fact that the hydrophobic fluorene moiety can be included in cyclodextrin, it is expected that the solubility in water is improved and the micelles are collapsed. It is known that one fluorene is taken into β-cyclodextrin (β-CD) and two fluorenes are taken into γ-CD. Therefore, it was examined whether β-CD was used to include the fluorene moiety to improve water solubility.

Addition of compound (1-2: 350) (1 × 10 ⁻² M) to water resulted in a milky white solution. When β-CD was added to this solution 10 times the concentration of the compound (1-2: 350), the solution became transparent with some turbidity. This phenomenon means the collapse of micelles present in the solution. From this, it can be said that DNA encapsulated in micelles using β-CD can be released.

Example 9 (Enclosure of DNA)
The concentration of the compound (1-2: 350) was 1 × 10 ⁻⁴ M, and the shape of the micelle was observed with a transmission electron microscope (abbreviation TEM) (FIG. 4). Moreover, salmon sperm (Salmon Sperm) DNA (SS-DNA) of the same concentration as the compound (1-2: 350) was added, and the change of the micelle shape was also observed by TEM (FIG. 5).

  From the results of TEM, the micelles before the addition of SS-DNA had a spherical structure with a size of about 100 nm. By adding SS-DNA, the micelle had a spherical structure, but the size was about It increased to around 400 nm. This is considered to be because DNA was taken up into the spherical micelle formed by the compound (1-2: 350).

Example 10 (Measurement of micelle size by Z potential measurement)
FIG. 6 shows the relationship between the change in micelle size and the change in the Z potential when the concentration of the compound (1-2: 350) is 1 × 10 ⁻⁵ M and SS-DNA is added in 10% M increments. The compound (1-2: 350) alone formed micelles having a size of about 150 nm, and the micelle size was increased to 400 nm by the addition of DNA. This result almost coincides with the result of Example 9. Also, the Z potential is larger on the negative side. The Z potential corresponds to the charge on the micelle surface, and since DNA is a polyanion, it is considered that the charge is increased in the negative direction by enclosing DNA inside the micelle. In FIG. 6, D / F on the horizontal axis is the concentration ratio (unit: mole) of DNA / dendrimer (fluorene dendrimer).

  From the above results, it can be seen that the dendron compound (1-2: 350) having a fluorenyl group forms micelles in water, and this micelle encapsulates DNA inside. It was also revealed that DNA can be released when the encapsulated micelles are disrupted with cyclodextrin.

FIG. 1 is a graph showing the measurement results of fluorescence intensity in Examples. FIG. 2 is a graph showing the measurement results of fluorescence intensity in the examples. FIG. 3 is a graph showing the measurement results of fluorescence intensity in the examples. FIG. 4 is an electron micrograph (TEM) showing the shape of the micelle before addition of DNA in Example 9. FIG. 5 is an electron micrograph (TEM) showing the shape of the micelle after addition of DNA in Example 9. FIG. 6 is a graph showing the relationship among micelle size, Z potential, and D / F.

Claims (5)

A new dendrimer composed of a hydrophilic unit or segment and a dendron bonded to the unit or segment and having a fluorene unit , wherein the dendron is represented by the following formula (4) and the g-th generation ( Dendrimer which is a dendron of g = 1-3) .
Wherein X ₁ is a nitrogen atom, X ₂ is an imino group (—NH—) or a substituted imino group, R ³ and R ⁴ are each a linear or branched alkylene group, and Y ₁ is an imino group (—NH -), An amide bond, an ester bond or a urethane bond, and R ¹ and R ² are the same or different and each represents a hydrogen atom, a halogen atom, an alkyl group or an alkoxy group. Dendrimers according to claim 1, wherein the number of branches n dendrons from hydrophilic unit or segment is 1-3. The hydrophilic units or segments are composed of polyoxyethylene units, dendrons dendrimers 請 Motomeko 1, wherein you branched at the branch number n = 1 to 3 from the hydrophilic units or segments.   The dendrimer according to claim 1, which can form micelles in water.   The dendrimer according to claim 1, which can form a micelle in water and can encapsulate DNA.