JPH09278726A - New liposome - Google Patents

Abstract

PROBLEM TO BE SOLVED: To prepare a new compound expressed by a specified formula. capable of efficiently forming a complex in combination with an expression vector including a gene of a biological activator, capable of efficiently transfecting the gene into a target cell and useful as a component of a liposome membrane. SOLUTION: This compound is expressed by the formula [R is a 10∼22C (un)saturated aliphatic hydrocarbon; (n)>=3; Z is N(R<1> )2 or N<+> (R<1> )3 (R<1> is a 1∼6C alkyl)]. The compound is preferably obtained by e.g. reaction of dihydroxyacetone as a starting material with a fatty acid for acylation to produce diacetoxyacetone, followed by its reduction to produce 1,3-diacylated glycerol, by esterification of a free OH group with a preferred halogenated product of a fatty acid and then by introducing an ammonium or amino group into a terminal site of the esterified product of the fatty acid. It is preferable to make one, or two or more selected from a group of the resultant compounds include in a quantity of 5∼50mol% based on the total quantity of lipid to produce the objective liposome.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel cationic lipid compound as a membrane component of a liposome, and a liposome using the same.

[0002]

2. Description of the Related Art When phospholipids and glyceroglycolipids are suspended in at least 50% or more of water at a temperature above the gel-liquid crystal phase transition temperature specific to the lipids, closed vesicles composed of lipid bilayers are automatically formed. This vesicle is called a liposome.

Since liposomes have a high affinity for biological membranes, various drugs, hormones, lymphokines and other biologically active substances are contained in the internal aqueous phase and lipids of membrane components to improve their stability and activity. Attempts have been made to improve the sustainability of and the reach of target tissues. Recently, a gene encoding a bioactive substance as described above is mixed with a liposome to form a complex, or it is encapsulated and held in a liposome, and this is administered to a patient to inject the gene into the cells of the target tissue. However, gene therapy for producing bioactive substances on the spot has been studied. However, it has not been put to practical use because of problems in terms of the efficiency of complex formation with liposomes, the efficiency of gene encapsulation in liposomes, the efficiency of gene transfection into target tissue cells, and the toxicity of liposomes.

[0004]

Therefore, an object of the present invention is to efficiently form a complex with an expression vector containing a gene of a biologically active substance, and to transfect a target cell with the gene with high efficiency, and to obtain toxicity. To provide liposomes having a low viscosity. Furthermore, another object of the present invention is to provide a novel compound constituting the membrane component of the liposome as described above.

[0005]

The inventor has designed a biodegradable glyceride derivative (I) having a quaternary ammonium or tertiary amine structure as a membrane component compound capable of forming a desired liposome. This design was performed considering the association of amphipathic molecules, the structure and toxicity of natural phospholipids.

Amphiphilic molecules are said to be in various association states such as emulsions, micelles, liposomes, etc. depending on the shape of the molecule (JN Israelachvili et al., Bioche
M. Biophys. Acta, 470. 185-201 (1977)). In other words, cone-type and inverse-cone type amphiphiles with a large difference in the balance between hydrophilic and hydrophobic groups form micelles, and the cylinder type, which has a relatively good balance between hydrophilic and hydrophobic groups, is lamellar in water. Form a structure. Lipids capable of forming liposomes often belong to this cylinder type.

The biological membrane is composed of a lipid bilayer membrane,
Among them, phosphatidylcholines having long-chain fatty acids are known as cylindrical amphipathic molecules. Therefore, the structure of natural phospholipid (phosphatidylcholine) was used as a model of the compound of the present invention.

Therefore, as the basic skeleton of the compound (I), the same glycerin skeleton as the natural phospholipid is selected, and each of the three hydroxyl groups has a cation or a neutral hydrophilic group and a hydrophobic group consisting of two fatty acids. Decided to introduce. Further, in the case of a lipid having a large fatty acid moiety which is a hydrophobic group, transfer of phospholipids between liposomes can be avoided. Further, if the phase transition temperature of the lipid is unnecessarily high, it becomes difficult to prepare a liposome. Therefore, for example, oleic acid having a cis-type double bond or a trans-type double bond is added to this long-chain fatty acid. Attempts were made to use unsaturated fatty acids such as elaidic acid. For the cationic hydrophilic group portion, a quaternary ammonium cation also contained in phosphatidylcholine was used. Furthermore, the distance from the glycerin skeleton to this cation part was also considered as a model of natural lipids.

On the other hand, most of the cationic liposomes currently used have a structure that is difficult to decompose in cells. Originally, if a cationic lipid, which is not a component of a living body, is present at a high concentration for a long period of time, it is considered that it inhibits the activity of cell ion channels, receptors, enzymes and the like, resulting in toxicity. From this, the compound (I) has an ester bond which is considered to be easily decomposed in cells.

For reference, some of the known cationic lipids and typical natural lipids are shown in FIGS. 1 and 2.

From this point of view, the inventor synthesized the compound (I) represented by the following formula.

Embedded image In the formula, R has 10 to 22 carbon atoms, preferably 14 to 18,
More preferably, it is a 16-18 saturated or unsaturated aliphatic hydrocarbon group. These aliphatic hydrocarbon groups include alcoyl, oleyl, elaidyl, oleoyl and the like. Further, n in the formula is an integer of 3 or more, preferably 3 to
5, and more preferably an integer of 3-4. Z is -N (R
¹ ) ₂ or -N ⁺ (R ¹ ) ₃ where R ¹ has ¹ carbon atom
Is an alkyl group having 6 to 6 carbon atoms, preferably an alkyl group having 1 to 3 carbon atoms such as methyl, ethyl and propyl, and most preferably methyl.

The target compound (I) is obtained by reacting dihydroxyacetone as a starting material with a fatty acid for acylation to obtain diacetoxyacetone, which is reduced to 1,3-diacylated glycerol, and free hydroxyl groups are further added. It can be synthesized by esterifying with a desired fatty acid halide and introducing an ammonium group or an amino group at the terminal of the esterified fatty acid.

Compound (I) of the present invention prepared by this method
Some of them and their compound numbers are shown in FIG. Appropriately combining the novel compound synthesized in the present invention and a known fat or oil,
Liposomes are prepared by a conventional method. The conventional method is not particularly limited, and an ultrasonic method, a thin film shaking method, a reverse phase evaporation method, a surfactant removal method or the like can be used. For example, the liposome can be prepared by the following method.

A mixed lipid of the compound (I) and DOPE is dissolved in chloroform to prepare a solution thereof, and chloroform is distilled off from the solution under reduced pressure to form a lipid thin film. Water is added to this thin film, the mixture is stirred under mild heating, and further ultrasonicated to obtain a liposome solution. If necessary, this solution is passed through, for example, a sterilizing filter for sterilization and sizing to obtain the desired liposome (SUV).
Type). The preparation conditions can be appropriately selected according to a conventional method. Further, the liposome prepared in the present invention may be a unilamellar or multilamellar.

As the raw material lipid, the compound (I) of the present invention is used.
One or more of the above are used as all or part of the membrane components. Compound (I) and a known neutral phospholipid such as dioleoyl phosphatidyl ethanolamine (DOP
E), dioleoyl phosphatidyl choline (DOP
C), dilauroyl phosphatidyl choline (DLP
It can also be used as a mixture with C) and the like. Usually, the mixture is in a molar ratio of 1: 1 to 1:10, preferably 1: 2 to 1: 7, more preferably 1: 3 to 1: 5.

The compound represented by the structural formula (I) of the present invention is another known cationic lipid such as N- (α-trimethylammonioacetyl) didodecyl-D-glutamate chloride (TMAG), N-. [1- (2,3-
Oleyloxy) propyl] -N, N, N-trimethylammonium chloride (DOTMA), 2,3-dioleyloxy-N- [2- (sperminecarboxamido) ethyl] -N, N-dimethyl-1-propaneaminium tri It can also be used in combination with fluoroacetate (DOSPA) or the like.

The liposome thus obtained is suspended in water, and an expression vector containing the desired gene is added thereto and mixed, and the mixture is allowed to stand for a desired time, whereby a liposome-gene complex can be obtained.

The liposomes that have formed a complex with a gene are
It can be suspended in water or physiological saline or the like and then injected, for example, intravenously.

In the liposome of the present invention, a gene encoding a desired physiologically active substance or the like, for example, α-, β- or γ-
A complex can be formed with a desired gene such as an interferon gene, a G-CSF gene, a gene encoding an antisense of hepatitis virus.

The liposome of the present invention has an advantage that it has low toxicity when administered to a living body as compared with conventional liposomes. In addition, when used for encapsulating an expression vector containing a desired gene, this expression vector can be highly efficiently transfected into a predetermined cell.

[0021]

EXAMPLES Next, the present invention will be described in more detail with reference to Examples and Experimental Examples, but the present invention is not limited to these Examples.

Example 1 Synthesis of Compound (Ia) 1) Synthesis of 1,3-Dioleoyloxypropan-2-one (1) Reference [RK Dharma, A. David, GR Trevor and MS
According to Donald, J. lipid Res., 28, 403-413 (1987)], it was synthesized as follows.

Dihydroxyacetone (0.76 g, 8.4 mmol)
Solution of oleic acid (4.20 g, 17.64 ml) in CCl4 (16.8 ml).
mmol) and DMAP (2.05 g, 16.8 mmol) were added, and DCC (3.60
A solution of g, 17.64 mmol) in CCl ₄ (8.4 ml) was added dropwise at room temperature over 30 minutes, and the mixture was stirred at room temperature for 17 hours. After filtering the precipitate and concentrating the filtrate, the residue is dissolved in hexane and the temperature is 0 ° C.
It was cooled in ice for 2 hours. The precipitated crystals were filtered, the filtrate was concentrated, and the residue was recrystallized from MeOH to give colorless crystals 1 (4.15 g, 80%).

Mp 40-41.5 ° C. (MeOH). (Lit, 1) 43-44 ° C.).
IR ν (KBr): 2999, 2920, 2850, 2305, 2092, 1733,
1654, 1467, 1415, 1174, 886, 722 cm -1. 1 H-NMR (CD
Cl ₃ ) δ: 0.88 (6H, t, CH ₃ ), 1.27-1.31 (40H, br m,
CH ₂ ), 1.67 (4H, m, OCOCH ₂ C H ₂ ), 2.17, 2.02 (8H, m,
C H ₂ CH = CHC H ₂ ), 2.42 (4H, t, J = 7.5 Hz, OCOCH ₂ ), 4.7
5 (4H, s, CH ₂ COCH ₂ ), 5.34 (4H, m, CH ₂ C H = C H CH ₂ )

2) Synthesis of 1,3-Dioleoylglycerol (2) Reference [RK Dharma, A. David, GR Trevor and MS
According to Donald, J. lipid Res., 28, 403-413 (1987)], it was synthesized as follows.

Compound 1 (1.0 g, 1.6 mmol) in THF-water (15
: 1) (16 ml) solution at 0 ℃ NaBH ₄ (100 mg, 1.6 m
mol) was added and stirred for 20 minutes. After distilling off the solvent, the residue was diluted with ether, washed with water, the organic layer was dried over anhydrous magnesium sulfate, and then the solvent was distilled off. The residue was washed with pentane to obtain colorless oily substance 2 (0.86 g, 85%).

¹ H-NMR (CDCl ₃ ) δ: 0.88 (6H, t, CH ₃ ),
1.27 (40H, m, CH ₂ ), 1.60 (4H, m, OCOCH ₂ C H ₂ ), 2.00,
2.02 (8H, m, C H ₂ CH = CHC H ₂ ), 2.34 (4H, t, J = 7.6 Hz,
OCOCH ₂ ), 4.14-4.16 (5H, br. M, CH ₂ CHCH ₂ ), 5.34 (4
H, m, CH ₂ C H = C H CH ₂ ).

3) 1,3-Dioleoyl-2- (4-chlorobutanoyl) g
Synthesis of lycerol (3) Compound 2 (0.45 g, 0.73 mmol) in anhydrous methylene chloride (5
ml) solution, 2,6-lutidine (5 ml) and 4-chlorobutanoyl chloride (0.31 g, 2.19 mmol) were added, and the mixture was allowed to stand at room temperature.
Stirred for hours. After the solvent was distilled off, the residue was diluted with ethyl acetate and washed with water, saturated aqueous sodium hydrogen carbonate, water, 2% hydrochloric acid, water and saturated saline. After the organic layer was dried over anhydrous magnesium sulfate, the solvent was distilled off. The residue was purified by flash column chromatography (hexane: AcOEt = 6: 1) to obtain colorless oily substance 3 (0.45 g, 85%).

IR ν (KBr): 2930, 2854, 2676, 2031,
1746, 1652, 1456, 1377, 1166, 1049, 877, 788cm ^-1 .
¹ HNMR (CDCl ₃ ) δ: 0.88 (6H, t, CH ₃ ), 1.27, 1.30 (40
H, m, CH ₂ ), 1.61 (4H, m, OCOCH ₂ C H ₂ ), 2.02, 2.00 (8
H, m, C H ₂ CH = CHC H ₂ ), 2.10 (2H, m, COCH ₂ C H ₂ CH ₂ Cl),
2.32 (4H, t, J = 7.6 Hz, OCOCH ₂ ), 2.53 (2H, t, J =
7.2 Hz, COC H ₂ CH ₂ CH ₂ Cl), 3.60 (2H, t, J = 6.4 Hz, CO
CH ₂ CH ₂ C H ₂ Cl), 4.14, 4.33 (4H, A ₂ B ₂ , J = 5.9, 4.1 Hz,
C H ₂ CHC H ₂ ), 5.27 (1H, m, CH ₂ C H CH ₂ ), 5.34 (4H, m, CH
₂ C H = C H CH ₂ ). ¹³ C-NMR (CDCl ₃ ) δC: 173.23, 171.73, 1
29.99, 129.69, 69.36, 61.96, 43.79, 33.98, 31.88,
31.09, 29.74, 29.67, 29.51, 29.29, 29.13, 29.06, 2
7.50, 27.19, 27.14, 24.82, 22.66, 14.09.MS (EI) m
/ z: 724 (M +, 6.1), 602 (M ⁺ -ClC ₃ H ₆ CO ₂ , 14.2), 443,
445 (M ⁺ -C ₁₇ H ₃₃ CO ₂ , base, 40.1), 339 (C ₁₇ H ₃₃ CO ⁺ +74,
12.8), 265 (C ₁₇ H ₃₃ CO ⁺ , 58.5), 179, 181 (ClC ₃ H ₆ CO ⁺ +
74,43.1, 14.3), 105, 107 (ClC ₃ H ₆ CO ⁺ , 84.3, 31.1).
Anal. Calcd for C ₄₃ H ₇₇ ClO ₆ : C, 71.19; H, 10.70; C
l, 4.89. Found: C, 71.19; H, 10.65; Cl, 4.83.

4) 1,3-Dioleoyl-2- (4-iodobutanoyl) gly
Synthesis of cerol (4) To a solution of sodium iodide (1.29 g, 8.55 mmol) in anhydrous methyl ethyl ketone (10 ml) was added compound 3 (0.62 g, 0.86 m).
mol) was added and the mixture was stirred at room temperature for 48 hours. After the solvent was distilled off, the residue was diluted with ethyl acetate and washed with water, 10% NaHSO ₄ , water, 5% saturated sodium hydrogen carbonate solution, water and saturated saline. After the organic layer was dried over anhydrous magnesium sulfate, the solvent was distilled off. The residue was flash column chromatographed (hexane: AcOEt = 5
Purification by (0: 1) gave colorless oily substance 4 (0.55 g, 78%).

IR ν (KBr): 2926, 2854, 1745, 1656,
. 1462, 1377, 1167, 875 , 723 cm -1 1 H-NMR (CDCl 3) δ:
0.88 (6H, t, CH ₃ ), 1.27, 1.30 (40H, m, CH ₂ ), 1.62
(4H, m, OCOCH ₂ C H ₂ ), 2.00, 2.02 (8H, m, C H ₂ CH = CHC
H ₂ ), 2.13 (2H, m, COCH ₂ C H ₂ CH ₂ I), 2.32 (4H, t, J =
7.6 Hz, OCOCH ₂ ), 2.48 (2H, t, J = 7.1 Hz, COC H ₂ CH ₂ C
H ₂ I), 3.24 (2H, t, J = 6.8 Hz, COCH ₂ CH ₂ C H ₂ I), 4.16,
4.32 (4H, A ₂ B ₂ , J = 5.9,4.3 Hz, C H ₂ CHC H ₂ ), 5.27 (1
H, m, CH ₂ C H CH ₂ ), 5.34 (4H, m, CH ₂ C H = C H CH ₂ ). ¹³ C-NMR
(CDCl ₃ ) δC: 173.23, 171.41, 130.01, 129.69, 69.4
0, 69.18, 34.70, 34.02, 31.88, 30.30, 29.74, 29.6
9, 29.51, 29.31, 29.15, 29.09, 28.32,27.21, 27.15,
24.82, 22.66, 14.09, 4.94.MS (EI) m / z: 816 (M ⁺ ,
7.5), 798 (M ⁺ -H ₂ O, 1.7), 602 (M ⁺ -IC ₃ H ₆ CO ₂ , 17.5),
535 (M ⁺ -C ₁₇ H ₃₃ CO ₂ , base), 339 (C ₁₇ H ₃₃ CO ⁺ +74, 14.
7), 265 (C ₁₇ H ₃₃ CO ⁺ , 51.9), 271 (IC ₃ H ₆ CO ⁺ +74, 30.
1), 197 (IC ₃ H ₆ CO ⁺ , 47.4). Anal. Calcd for C ₄₃ H ₇₇ IO ₆
: C, 63.21; H, 9.50; I, 15.53. Found: C, 63.27;
H, 9.41; I, 15.31.

5) N- [3- [2- (1,3-Dioleoyloxy) propoxyca
rbonyl] propyl] -N, N, N-trimethylammonium iodide (I
Synthesis of a) Anhydrous trimethylamine was added to a solution of 4 (0.51 g, 0.62 mmol) in anhydrous ether (3 ml), and the mixture was stirred at 0 ° C for 48 hours. After distilling off the solvent and excess trimethylamine, the residue was subjected to silica gel column chromatography (CHCl ₃ : Me.
After purification with OH = 10: 1), white waxy substance Ia (0.38
g, 69%) was obtained.

Mp 47-50 ° C. IR ν (KBr): 2924, 2852,
1738, 1466, 1412, 1364, 1252, 1176, 1117, 1067, 10
25, 968, 938, 879, 851, 798, 725 cm -1. 1 H-NMR (CDC
l ₃ ) δ: 0.88 (6H, t, CH ₃ ), 1.27, 1.30 (40H, m, CH ₂ ),
1.61 (4H, m, OCOCH ₂ C H ₂ ), 2.00, 2.22 (8H, m, C H ₂ CH
= CHC H ₂ ), 2.12 (2H, m, COCH ₂ C H ₂ CH ₂ N), 2.33 (4H, t, J
= 7.6 Hz, OCOCH ₂ ), 2.54 (2H, t, J = 6.5 Hz, COC H ₂ CH
₂ CH ₂ N), 3.48 (9H, s, N (CH ₃ ) ₃ ), 3.75 (2H, m, COCH ₂ CH
₂ C H ₂ N), 4.13, 4.40 (4H, A ₂ B ₂ , J = 5.4, 4.3 Hz, C H ₂
CHC H ₂ ), 5.16 (1H, m, CH ₂ C H CH ₂ ), 5.34 (4H, m, CH ₂ C H
= C H CH ₂ ). ¹³ C-NMR (CDCl ₃ ) δC: 173.40, 171.23, 130.
01, 129.67, 70.39, 61.58, 53.78, 34.00, 31.88, 29.
72, 29.67, 29.49, 29.29, 29.17, 29.09, 29.06, 27.1
9, 27.14, 24.82, 22.66, 18.31, 14.11.MS (EI) m / z
: 733 (M ⁺ -CH ₃ I, base), 265 (C ₁₇ H ₃₃ CO ⁺ , 5.7), 142
(CH ₃ I, 55.8). Anal. Calcd for C ₄₆ H ₈₆ INO ₆ : C, 63.0
6; H, 9.89; N, 1.60. Found: C, 62.93; H, 9.79; N,
1.57.

Examples 2-3: Synthesis of compound (Ib) and compound (Ic) The procedure was carried out except that 5-chlorovaleryl chloride was used instead of 4-chlorobutanoyl chloride in 3) of Example 1. Example 1 was repeated to obtain the target compound (Ib). (Mp =
46.5-50 ℃)

Further, Example 1 was repeated except that elaidic acid or stearic acid was used in place of the oleic acid of Example 1, and the compound (Ic) (mp = 74-77 ° C.) or (Id) (mp = 99.5-101.5 ° C) was obtained.

Compound (Ib) IR ν (KBr): 2924, 2853, 1739, 1653, 1457, 1418, 1
373, 1173, 1096, 965,913, 722 cm ^-1 , ¹ H-NMR (CDCl ₃ ).
δ: 0.88 (6H, t, -CH ₃ ), 1.27, 1.30 (40H, m, -CH
_2- ), 1.61 (4H, m, -OCOCH ₂ C H _2- ), 1.78 (2H, m, -COCH
₂ C H ₂ CH ₂ ) ₂ N), 1.90 (2H, m, -CO (CH ₂ ) ₂ C H ₂ CH ₂ N), 2.00,
2.33 (8H, m, -C H ₂ CH = CHC H _2- ), 2.33 (4H, t, J = 7.6 Hz,
-OCOCH _2- ), 2.45 (2H, t, J = 6.2 Hz, -OCOCH ₂ C H _2- ),
3.46 (9H, s, -N (CH ₃ ) ₃ ), 3.71 (2H, m, -CO (CH ₂ ) ₂ CH ₂ C H
₂ N), 4.11, 4.41 (4H, A ₂ B ₂ ,, J = 5.4, 4.6 Hz, -C H ₂ CHC H
_2- ), 5.17 (1H, m, -CH ₂ C H CH _2- ), 5.34 (4H, m, -CH ₂ C H
= C H CH _2- ), ¹³ C-NMR (CDCl ₃ ) δc: 173.39, 171.88, 130.0
1, 129.65, 69.72, 66.51, 61.64, 53.80, 34.04, 32.8
0, 31.86, 29.72, 29.67, 29.49, 29.29, 29.15, 29.0
9, 29.06, 27.19, 27.14, 24.85, 22.64, 22.18, 20.9
5, 14.09, MS (EI) m / z: 747 (M ⁺ -CH ₃ I, base), 265 (R
^1.3 CO, 5.1), 142 (CH ₃ I, 53.8), 128 (R ² CO-CH ₃ I, 64.
3), 58 (C ₃ H ₁₁ N, over). Anal Calcd for C ₄₇ H ₈₈ INO ₆ 1
/ 2 H ₂ O: C, 62.78; H, 9.89; I, 14.11; N, 1.56, Foun
d: C, 62.82; H, 9.63; I, 13.89; N, 1.59.

Compound (Ic) IR ν (KBr): 2920, 2850, 1754, 1465, 1416, 1363, 1
252, 1176, 1116, 1065, 1022, 964, 942, 798, 724 cm
^-1 , ¹ H-NMR (CDCl ₃ ) δ: 0.88 (6H, t, -CH ₃ ), 1.27,
1.30 (40H, m, -CH _2- ), 1.61 (4H, m, -OCOCH ₂ CH _2- ),
1.95, 1.97 (8H, m, -C H ₂ CH = CHC H _2- ), 2.14 (2H, m, -CO
CH ₂ C H ₂ CH ₂ N), 2.33 (4H, t, J = 7.6 Hz, -OCOCH _2- ), 2.5
4 (2H, t, J = 6.8 Hz, -COC H ₂ CH ₂ CH ₂ N-), 3.49 (9H, s,
-N (CH ₃ ) ₃ ), 3.76 (2H, m, -COCH ₂ CH ₂ C H ₂ N), 4.13, 4.39
(4H, A ₂ B ₂ ,, J = 5.1, 4.3 Hz, -CH ₂ CHC H _2- ), 5.17 (1H,
m, -CH ₂ C H CH _2- ), 5.38 (4H, m, -CH ₂ C H = C H CH _2- ), ¹³ CN
MR (CDCl ₃ ) δc: 173.30, 171.12, 130.39, 130.06, 70.
23, 65.52, 61.55, 53.75, 33.96, 32.51, 32.47, 31.8
1, 29.71, 29.56, 29.40, 29.38, 29.22, 29.09, 29.0
4, 28.99, 28.90, 24.76, 22.57, 18.30, 14,02, MS (E
I) m / z: 733 (M ⁺ -CH ₃ I, 21.6), 265 (R ^1.3 CO, 1.0), 1
42 (CH ₃ I, 75.9), 58 (C ₃ H ₁₁ N, base), Anal Calcd for
C ₄₆ H ₈₆ INO ₆ : C, 63.06; H, 9.89; I, 14.49; N, 1.60,
Found: C, 62.77; H, 9.65; I, 14.52; N, 1.63.

Compound (Id) IR ν (KBr): 2915, 2849, 1733, 1471, 1417, 1254, 1
175, 1115, 962, 719 cm ^-1 , ¹ H-NMR (CDCl ₃ ) δ: 0.88
(6H, t, -CH ₃ ), 1.25, (56H, m, -CH _2- ), 1.61 (4H, m,
-OCOCH ₂ C H _2- ), 2.12 (2H, m, -COCH ₂ C H ₂ CH ₂ N), 2.33
(4H, t, J = 7.7 Hz, -OCOCH _2- ), 2.54 (2H, t, J = 6.4 Hz,
-COC H ₂ CH ₂ CH ₂ N-), 3.47 (9H, s, -N (CH ₃ ) ₃ ), 3.73 (2
H, m, -COCH ₂ CH ₂ C H ₂ N), 4.12, 4.41 (4H, A ₂ B ₂ , J = 5.3,
3.9 Hz, -C H ₂ CHC H _2- ), 5.16 (1H, m, -CH ₂ C H CH _2- ), ¹³
C-NMR (CDCl ₃ ) δc: 173.40, 171.19,96.07, 70.33, 65.
54, 61.57, 53.77, 34.00, 31.88, 29.67, 29.45, 29.3
3, 29.24, 29.08, 24.84, 22.64, 18.30, 14.09, MS (E
I) m / z: 737 (M ⁺ -CH ₃ I, 4.4), 265 (R ^1.3 CO, 2.2), 14
2 (CH ₃ I, 23.06), 114 (R ² CO-CH ₃ I, 14.9), 58 (C ₃ H ₁₁ N,
base). Anal Calcd for C ₄₆ H ₉₀ INO ₆ : C, 62.78; H, 1
0.30; I, 14.41; N, 1.59. Found: C, 62.49; H, 10.03;
I, 14.48; N, 1.63.

Example 4: Preparation of SUV type liposome Mixed lipid of compound (I) and DOPE (total 1 μmo)
l) is dissolved in chloroform and the solution (0.5 ml)
Was prepared, put in a test tube, and chloroform was distilled off under reduced pressure with a rotary evaporator to prepare a lipid thin film. Water (0.5 ml) was added to this thin film, heated to 45 ° C., and vortexed for 2 minutes. Furthermore, under a nitrogen gas stream, ultrasonic treatment was performed for 5 minutes with a probe-type sonicator to obtain a liposome solution. 0.2 μm of the obtained liposome solution
The target liposomes (SUV type) were obtained by sterilizing and sizing through a sterilizing filter (1).

The obtained liposomes were used in the transfection assay of the following experimental example to test their properties.

Experimental Example 1: Transfection Assay The SUV type liposome prepared in Example 4 (20 nmo)
1) and plasmid DNA (0.5 μg) were mixed, and the mixture was allowed to stand in a clean bench for 15 minutes. This plasmid DNA
Was a control vector (product number: PGV-C) of Piccagene vector manufactured by Toyo Ink Mfg. Co., Ltd. and sold by Wako Pure Chemical Industries, Ltd.
This vector encodes the luciferase gene, and is a full-length 6046 bp plasmid containing an SV40-derived promoter and enhancer.

On the other hand, 1 × 10 ⁵ / well of CHO cells (6 wells) were washed with PBS and 1.9 ml of Opti-
MEMI was added. To this, the previously prepared mixture of liposome and plasmid was added, and the mixture was added at 37 ° C. and 5% CO ₂ for 24 hours.
Cultured for hours.

After culturing, the cells were collected, washed twice with PBS, and the activity of luciferase expressed in the cells was measured by a commercially available luciferase assay kit (Toyo Ink Co., Ltd.).
Was used for the measurement.

The liposomes used and their concentrations are shown in Table 1 below, and the measured luciferase activities are shown in Tables 2 and 3.

FIG. 4 shows the results of measuring the luciferase activity when the amount of plasmid DNA was kept constant at 0.5 μg and the lipid concentration was changed from 1 μM to 80 μM. The activity value was shown as a relative ratio when the lipofectin activity was 1.

[0046]

[Table 1] (Liposome used for transfection and concentration used)

[0047]

[Table 2] (Transfection assay using SUV type liposome) (Compound (I): DOPE: DLPC composition change)

[0048]

[Table 3] (Transfection assay using liposome as SUV type) (Change in type of compound (I))

Experimental Example 2 Cytotoxicity of SUV Liposomes Containing Compound (Ia) In order to examine the cytotoxicity of SUV liposomes, the amount of protein in the cells was quantified 72 hours after the addition of liposomes.

4x10 passaged 18 hours before assay
⁴ / well CHO cells (12 wells) were washed with PBS and 1 ml Opti-MEMI was added. Liposomes were added at each concentration and incubated at 37 ° C., 5% CO ₂ for 72 hours. The amount of protein was quantified by the Bradford method.

The results are shown in FIG. The graph of FIG. 5 shows the ratio of the amount of protein when each liposome is added to the amount of protein when no liposome is added. The larger the number, the greater the amount of protein when liposomes are added and the lower the toxicity. .

[Brief description of drawings]

FIG. 1 shows the chemical structures of representative compounds of known cationic lipids.

FIG. 2 shows the chemical structures of typical compounds of known natural lipids.

FIG. 3 shows the chemical structure of the cationic lipid prepared by the present invention.

FIG. 4 is a graph showing a correlation between lipid amount and luciferase activity in a transfection assay using the SUV type liposome of the present invention.

FIG. 5 is a graph showing the cytotoxicity of the liposome of the present invention.

Claims (15)

[Claims] 1. The following structural formula (I): [In the formula, R represents a saturated or unsaturated aliphatic hydrocarbon group having 10 to 22 carbon atoms, n is an integer of 3 or more, and Z is -N.
(R ¹ ) ₂ or —N ⁺ (R ¹ ) ₃ is represented, and R ¹ has ¹ carbon atom.
To 6 alkyl groups]. 2. The compound according to claim 1, wherein n is an integer of 3-5. 3. n is an integer of 3 to 4, and R ¹ has 1 to ¹ carbon atoms.
The compound according to claim 1, which is an alkyl group of 3. 4. The compound according to claim 1, 2 or 3, wherein R is a saturated or unsaturated aliphatic hydrocarbon group having 14 to 18 carbon atoms. 5. The compound of claim 4, wherein R is selected from the group consisting of alcoyl, elaidyl and oleoyl. 6. The following structural formula (I): [In the formula, R represents a saturated or unsaturated aliphatic hydrocarbon group having 10 to 22 carbon atoms, n is an integer of 3 or more, and Z is -N.
(R ¹ ) ₂ or —N ⁺ (R ¹ ) ₃ (wherein R ¹ is an alkyl group having 1 to 6 carbon atoms)] and one or more compounds of And at least 1 mol% or more. 7. The liposome according to claim 6, which contains 5 to 50 mol% of one or more of the compounds with respect to the total lipid. 8. The liposome according to claim 6, comprising the compound and dioleoylphosphatidylethanolamine (DOPE). 9. The liposome according to claim 8, wherein the compounding ratio of the compound and DOPE is 1: 1 to 1:10. 10. The liposome according to claim 9, wherein the compounding ratio of the compound and DOPE is 1: 3 to 1: 5. 11. The liposome according to claim 10, wherein the compounding ratio of the compound and DOPE is 1: 4. 12. The compound represented by the structural formula (I), wherein n is an integer of 3 to 5, and
The liposome according to any one of items. 13. n is an integer of 3 to 4, and R ¹ has ¹ carbon atom.
The liposome according to claim 12, which is an alkyl group of 3 to 3. 14. The liposome according to any one of claims 6 to 13, wherein R is a saturated or unsaturated aliphatic hydrocarbon group having 14 to 18 carbon atoms. 15. The liposome according to claim 14, wherein R is selected from the group consisting of alcoyl, elaidyl and oleoyl.