Lipid particle, composition containing lipid particles, kit containing lipid particles, and active agent delivery method using lipid particles

Abstract

To provide: a lipid particle with high biodegradability, high biocompatibility, a high active agent introducibility, low cytotoxicity, and the like; a composition and a kit which use the lipid particles; and a method of delivering an active agent to a cell.SOLUTION: A lipid particle according to embodiments comprises: a compound represented by a formula (a) defined by Mal-La1-(OCH2CH2)na-O-La2Ra2; and an active agent. In the formula, Mal is a maleimide group, La1 is an unsubstituted hydrocarbon group, or a substituted hydrocarbon group, La2 is a trivalent hydrocarbon group that does not contain phosphorus, Ra is a hydrocarbon group containing at least one ester bond at an arbitrary position, and na is a number of 1 or more). In addition, a composition and a kit according to embodiments contain the lipid particles. A method of delivering an active agent to a cell according to embodiments comprises bringing the lipid particle into contact with a cell.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to lipid particles containing a biodegradable compound having a structure that is decomposed intracellularly. The present invention also relates to a composition and a kit containing the lipid particles and used for the delivery of an active agent such as a nucleic acid.

Background Art

[0002] Studies on liposomes for the treatment of various diseases are underway. Liposomes are minute capsules having a particle size on the nanometer order composed of lipids, and can encapsulate various compounds and the like inside, and are also excellent in biocompatibility and the like. Therefore, they are ideal materials for selectively delivering therapeutic agents and active agents to target sites in vivo. For such purposes, various materials have been developed for the materials constituting liposomes.

[0003] Such liposomes can be composed of a single lipid. In such a case, for example, a phospholipid having a head and a hydrophobic moiety bonded thereto is used as the lipid, and these lipids associate to form a membrane, constituting a minute capsule capable of encapsulating an active agent or the like. However, in order to impart excellent properties to liposomes, a lipid mixture is generally used to constitute them. And this lipid mixture contains a combination of lipids excellent in biodegradability, lipids that suppress aggregation of the formed liposomes, lipids having an effect of suppressing leakage of the inclusion, lipids having a membrane fusion effect, and the like.

[0004] And in order to further improve the properties of liposomes, each lipid is being studied. For example, medical liposomes specialized for gene introduction preferably satisfy high biodegradability, high biocompatibility, high active agent introducibility, and low cytotoxicity. As such liposomes, those having a surface modified with a maleimide group are known (Patent Document 1).

[0005] Although such liposomes exhibit excellent properties, it is desirable to increase the types of lipids that can be selected according to the state of the living body to which they are applied and the disease to be treated. Furthermore, lipid particles that can constitute liposomes having properties superior to those of conventional liposomes are required.

Prior Art Documents

Patent Documents

[0006]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0007] In view of the above problems, the present embodiment provides lipid particles containing a specific biodegradable compound and an active agent, a composition and a kit using the lipid particles, and a method for delivering an active agent using the lipid particles.

Means for Solving the Problems

[0008] According to the present embodiment, the following inventions are provided. [1] The following formula (a): Mal-L a1 -(OCH2CH2) na -O-L a2 R a 2(a) (In the formula, Mal is a maleimide group, L a1 is an unsubstituted hydrocarbon group or a substituted hydrocarbon group containing a linking group selected from the group consisting of -C(=O)-O-, -O-C(=O)-, -O-C(=O)-O-, -S-C(=O)-, -C(=O)-S-, -C(=O)-NH-, and -NH-C(=O)-, L a2 is a trivalent hydrocarbon group not containing phosphorus, R a are each independently C10 ~ 30 a hydrocarbon group containing at least one ester bond at any position, where na is a number of 1 or more) and a compound represented by an activator, characterized by comprising lipid particles. [2] The lipid particles according to [1], wherein na is 10 to 100. [3] The lipid particles according to [1] or [2], wherein the compound represented by formula (a) is dimyristoyl glycerol polyethylene glycol maleimide. [4] The following formula (b): Q-CHR b 2(b) (wherein, Q is a nitrogen-containing aliphatic group containing two or more tertiary nitrogens and no oxygen, R b are each independently C 12 ~C 24 aliphatic groups, and at least one R b contains a linking group selected from the group consisting of -C(=O)-O-, -O-C(=O)-, -O-C(=O)-O-, -S-C(=O)-, -C(=O)-S-, -C(=O)-NH-, and -NH-C(=O)- in its main chain or side chain) The lipid particles according to any one of [1] to [4], further comprising a compound represented by. [5] The Q is the following formula (Q’): R Q1 2N-(CR Q2 2) q1 -NR Q1 -(CR Q2 2) q2 -*2 (Q’) (wherein, R Q1 are each independently alkyl, R Q2 are each independently hydrogen or alkyl, or R Q1 and RQ2 Any two of them may form one alkylene to form a nitrogen-containing alicyclic ring, q1 is a number from 1 to 5, q2 is a number from 0 to 5, *2 represents the bonding position to -CHR b 2) The lipid particle according to [4], represented by [6] The lipid particle according to any one of [1] to [3], further comprising an additional lipid compound selected from the group consisting of a cationic lipid, a neutral lipid, a structure-forming lipid, and an aggregation-reducing lipid. [7] The lipid particle according to any one of [1] to [6], wherein the active agent is a nucleic acid selected from the group consisting of a plasmid, an oligonucleotide, a polynucleotide, siRNA, microRNA, DNA, mRNA, an aptamer, and a ribozyme. [8] The lipid particle according to [7], further comprising a compound that binds to the nucleic acid. [9] The lipid particle according to [8], wherein the compound that binds to the nucleic acid is a basic protein or a basic peptide.

[10] The lipid particle according to any one of [1] to [9], further comprising a compound that regulates the expression of the nucleic acid in cells.

[11] A composition comprising the lipid particle according to any one of [1] to

[10] and a carrier.

[12] A composition for delivering an active agent to cells, comprising the lipid particle according to any one of [1] to

[10] and a carrier.

[13] The composition according to

[12] , wherein the cell is a mammalian cell.

[14] The composition according to

[13] , wherein the mammalian cell is a hematopoietic cell.

[15] The composition according to

[14] , wherein the hematopoietic cell is selected from the group consisting of PBMC, T cell, dendritic cell, and CD34-positive cell.

[16] A method for delivering the active agent to the cell, comprising bringing the lipid particles according to any one of [1] to

[10] into contact with the cell.

[17] The method for delivering the active agent according to

[16] , wherein the cell is a cell of an animal other than a human or a cell taken out of the body.

[18] The method for delivering the active agent according to

[16] or

[17] , wherein the lipid particles further comprise a lipid forming a membrane and a lipid capable of reducing aggregation.

[19] The lipid forming the membrane is 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (PO PC), 1,2-di-O-octadecyl-3-trimethylammonium propane (DOTMA), 1,2-dioleoyl-3-dimethylammonium propane (DODAP), 1,2-dimyristoyl-3-dimethylammonium propane (14:0 DAP), 1,2-dipalmitoyl-3-dimethylammonium propane (16:0 DAP), 1,2-distearoyl-3-dimethylammonium propane (18:0 DAP), N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy) propane (DOBAQ), 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), and cholesterol, selected from the group consisting of, A method for delivering an activator according to

[18] , wherein the lipid capable of reducing the aggregation is a polyethylene glycol (PEG)-modified lipid.

[20] A method for delivering an activator according to any one of

[16] to

[19] , wherein the activator is a nucleic acid selected from the group consisting of a plasmid, an oligonucleotide, a polynucleotide, siRNA, microRNA, DNA, mRNA, an aptamer, and a ribozyme.

[21] A method for delivering an activator according to any one of

[16] to

[19] , wherein the activator comprises a combination of at least one DNA and at least one RNA.

[22] A method for delivering an activator according to

[20] or

[21] , wherein the lipid particles further comprise a compound that binds to a nucleic acid.

[23] A method for delivering an activator according to

[22] , wherein the compound that binds to the nucleic acid is a basic protein or a basic peptide.

[24] A method for delivering an activator according to

[22] , wherein the compound that binds to the nucleic acid is protamine or histone.

[25] A method for delivering an activator according to claim 22, wherein the lipid particles further comprise a compound that regulates the expression of a nucleic acid intracellularly.

[26] A method for delivering an activator according to any one of

[16] to

[25] , wherein a carrier is contacted with the cell together with the lipid particles.

[27] A method for delivering an activator according to any one of

[16] to

[26] , wherein the cell is a tumor cell.

[28] Performing a method for delivering an activator according to any one of

[16] to

[27] , Culturing the obtained cells, A method for producing cells comprising

[29] A kit comprising lipid particles according to any one of [1] to

[10] and an introducing agent for introducing the lipid particles into cells.

[30] The kit according to

[29] , further comprising a substance for improving the storage stability of the lipid particles.

Brief Description of Drawings

[0009]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

[0010] [Definitions] In the present embodiment, when a numerical range is indicated using ~, unless otherwise particularly limited, these include both endpoints and the units are common. For example, 10 to 25 mol% means 10 mol% or more and 25 mol% or less.

[0011] In this embodiment, "C" x ~C y ", and descriptions such as "C" x " mean the number of carbons in a molecule or substituent. For example, C1-C6 alkyl means an alkyl having 1 to 6 carbons. Further, in this embodiment, a halogenated alkyl means one in which one or more hydrogens in the alkyl are replaced by a halogen such as fluorine, and for example, a fluoroaryl means one in which one or more hydrogens in the aryl are replaced by fluorine.

[0012] In this embodiment, unless otherwise particularly limited, alkyl means a monovalent group obtained by removing one hydrogen from any carbon of an alkane. And the term alkyl includes linear or branched alkyl. Further, cycloalkyl means an alkyl containing a cyclic structure. Even those in which a linear or branched alkyl is substituted on the cyclic structure are also referred to as cycloalkyl.

[0013] Also, alkenyl means a monovalent group obtained by removing one hydrogen from any carbon of an alkene.

[0014] Also, an unsubstituted hydrocarbon group means a group containing carbon and hydrogen that is monovalent or divalent or higher. Also, a substituted hydrocarbon means a group containing carbon, hydrogen, and in addition, oxygen, nitrogen, or sulfur that is monovalent or divalent or higher. A substituted hydrocarbon group may contain a hetero element such as a halogen as necessary. Also, when simply referring to a hydrocarbon group, it includes both unsubstituted hydrocarbons and substituted hydrocarbons. And an aliphatic group is a hydrocarbon group that does not contain an aromatic ring and may have any structure of linear, branched, or cyclic, or a combination thereof. Also, unless otherwise particularly limited, the aliphatic group may contain an unsaturated bond. Further, unless otherwise particularly limited, the aliphatic group may contain a hetero atom such as nitrogen, oxygen, sulfur, selenium, fluorine, chlorine, or bromine. Also, the aliphatic group may be a monovalent group or a polyvalent group. Also, an aromatic hydrocarbon group contains an aromatic ring and may have an aliphatic hydrocarbon group as a substituent as necessary. Tertiary nitrogen means nitrogen to which three carbons are bonded. Therefore, tertiary nitrogen constitutes a tertiary amine structure having electron-donating properties.

[0015] [Lipid compound] The compound according to the embodiment is a compound suitable as a lipid constituting liposomes, which contains a maleimide group and a polyethylene glycol chain. This compound is represented by the following formula (a). Mal-L a1 -(OCH2CH2) na -O-L a2 R a 2(a) (In the formula, Mal is a maleimide group, L a1 is an unsubstituted hydrocarbon group or a substituted hydrocarbon group containing a linking group selected from the group consisting of -C(=O)-O-, -O-C(=O)-, -O-C(=O)-O-, -S-C(=O)-, -C(=O)-S-, -C(=O)-NH-, and -NH-C(=O)-, L a2 is a trivalent hydrocarbon group not containing phosphorus, R a are each independently a hydrocarbon group containing at least one ester bond at an arbitrary position of C 10 ~ 30 and na is a number of 1 or more). na is a number of 1 or more)

[0016] The maleimide group Mal has a structure represented by the following formula (m).

Chemical formula

[0017] L a1is an unsubstituted hydrocarbon group or a substituted hydrocarbon group containing a linking group selected from the group consisting of -C(=O)-O-, -O-C(=O)-, -O-C(=O)-O-, -S-C(=O)-, -C(=O)-S-, -C(=O)-NH-, and -NH-C(=O)-.

[0018] L a1 When L is a substituted hydrocarbon group, it is preferably bonded to the hydrocarbon chain without a linking group to the maleimide group. Further, the number of carbon atoms of the unsubstituted hydrocarbon group and the number of carbon atoms of the hydrocarbon chain contained in the substituted hydrocarbon group are preferably 1 to 5, more preferably 2 to 3. The most preferred unsubstituted hydrocarbon group L a1 is -(CH2)2-. The linking group in the substituted hydrocarbon group may contain oxygen or nitrogen. Particularly, -C(=O)-NH- is preferred. The most preferred L containing a substituted carbon group a1 is -(CH2)2-C(=O)-NH-.

[0019] -(OCH2CH2) na -O- is a linear polyethylene glycol chain. The polyethylene glycol chain improves the hydrophilicity of the compound, and when the liposome is constituted by this compound, the cytotoxicity to cells tends to be low and the introduction rate of an active agent such as nucleic acid tends to be high.

[0020] na is a number indicating the degree of polymerization of the ethyleneoxy group, which is 1 or more, preferably 20 to 70, more preferably 30 to 60.

[0021] L a2 is a trivalent linking group that links the polyethylene glycol chain and two Rs a and. L a2 needs to contain no phosphorus. According to the study by the present inventors, it has been found that when L a2 contains phosphorus, the introduction rate of the active agent tends to be low. For this reason, L a2Preferably, it typically consists only of carbon and hydrogen, and may contain oxygen as necessary. Further, in the embodiment, the compound represented by the formula (a) preferably contains no phosphorus in its structure.

[0022] Hydrocarbon group L a2 Generally has a structure in which three hydrogens are excluded from a hydrocarbon. Optionally, at least one of the methylene -CH2- contained in the hydrocarbon may be substituted with a divalent group containing oxygen, such as a carboxylato group -C(=O)-O-, an oxy group -O-, or a carbonyl group -C(=O)-.

[0023] R a Forms the hydrophobic part of the compound represented by the formula (a), and the two Rs a May be the same or different. R a Are each independently a hydrocarbon group containing at least one ester bond at an arbitrary position of C 10 ~ 30 This ester group functions as a biodegradable group when the compound according to the embodiment is used in liposomes.

[0024] A preferred example of R a Can be represented by the following formula (ra). -L ra1 -C(=O)-O-L ra2 (ra) (In the formula, L ra1 Is alkylene, L ra2 Is a hydrocarbon group)

[0025] L ra1 And L ra2 May have a branched-chain structure or a cyclic structure, but when having a branched structure, it is preferable that the side chains are few, and most preferably it is linear. Also, L ra2 May be a saturated hydrocarbon group or an unsaturated hydrocarbon group.

[0026] Each part of the compound represented by the formula (a) used for the lipid particles according to the embodiment has the structure as described above, but the compound according to the embodiment preferably has a structure represented by the following formulas (a-01) to (a-12).

[0027] [Chemical formula] [Chemical formula]

[0028] Among the compounds represented by the formula (a), dimyristoyl glycerol polyethylene glycol maleimide represented by the formula (a-01) is most preferred.

[0029] [Additional lipid compound] The lipid particles according to the embodiment contain the compound represented by the formula (a) as a lipid compound, but preferably further contain an additional lipid compound. Such an additional lipid compound can be arbitrarily selected from those generally used for lipid particles.

[0030] For example, the lipid particles according to the embodiment preferably further contain, as an additional lipid compound, a compound represented by the following formula (b). Q-CHR b 2(b) (In the formula, Q is a nitrogen-containing aliphatic group containing two or more tertiary nitrogens and no oxygen, R b each independently represents a C 12 ~C 24 aliphatic group, and at least one R b contains a linking group selected from the group consisting of -C(=O)-O-, -O-C(=O)-, -O-C(=O)-O-, -S-C(=O)-, -C(=O)-S-, -C(=O)-NH-, and -NH-C(=O)- in its main chain or side chain)

[0031] One feature of the compound represented by formula (b) is that the head Q contains two or more tertiary nitrogens and does not contain oxygen. The head Q may further contain nitrogen constituting unsubstituted amino, quaternary ammonium, etc. as long as the effects of the embodiment are not impaired, but it is preferably free of nitrogen other than the tertiary nitrogen. Also, the head Q does not contain oxygen. Therefore, the head Q does not contain structures such as oxy, hydroxy, carboxy, alkoxy, carboxylato. Further, the head Q can contain an ionic group, but is preferably a neutral group free of polar groups.

[0032] One example of preferable Q can be represented by the following general formula (Q’). R Q1 2N-(CR Q2 2) q1 -NR Q1 -(CR Q2 2) q2 -*2 (Q’) (In the formula, R Q1 are each independently alkyl, R Q2 are each independently hydrogen or alkyl, R Q1 and R Q2 any two of may combine to form a nitrogen-containing alicyclic ring, q1 is a number from 1 to 5, preferably from 1 to 4, q2 is a number from 0 to 5, preferably from 1 to 4, *2 represents the bonding position to -CHR b 2) Here, the alkyl is preferably C1 - C3 alkyl.

[0033] Also, R Q1 and R Q2Any two of them can combine to form a nitrogen-containing alicyclic ring. The number of members of the nitrogen-containing alicyclic ring is not particularly limited, but is preferably 4 to 10, and preferably 5 to 8. Typically, the nitrogen-containing alicyclic rings formed include piperidine, piperazine, pyrrolidine, imidazolidine, hexamethyleneimine, homopiperazine, heptamethyleneimine, and the like.

[0034] Such Q has, for example, the following structure.

Chemical formula

[0035] The compound represented by formula (b) has -CHR b 2 bonded to the head. Here, R b is a hydrophobic group, and the two R b may be the same or different. Hydrophobic groups generally contain a relatively long hydrocarbon chain. And a linking group containing carboxylato or the like in a part thereof, specifically, a linking group selected from the group consisting of -C(=O)-O-, -O-C(=O)-, -O-C(=O)-O-, -S-C(=O)-, -C(=O)-S-, -C(=O)-NH-, and -NH-C(=O)-. These linking groups function as biodegradable groups when the compound according to the embodiment is used in liposomes.

[0036] An example of a preferred R b can be represented by the following formula (rb). -L rb1 -C(=O)-O-L rb2 (rb) (In the formula, L rb1 is alkylene, L rb2 is alkenyl)

[0037] L rb1 and L rb2It may have a branched-chain structure or a cyclic structure. When it has a branched structure, it is preferably low in side chains and most preferably linear.

[0038] More specifically, L rb1 and L rb are preferably represented by the following formulas (rb-1) and (rb-2), respectively. -(CH2) rb1 - (rb-1) -CH2-CH=CH-(CH2) rb2 -H (rb-2) (In the formula, rb1 is a number from 1 to 10, rb2 is a number from 1 to 10)

[0039] Here, in order for R b to exhibit sufficient hydrophobicity, rb1 is preferably a number from 4 to 8, and the longest molecular chain contained in R b is preferably 8 atoms or more.

[0040] Each part of the compound according to the embodiment has the structure as described above. However, the compound according to the embodiment preferably has a structure represented by the following formulas (b-01) to (b-21).

Chemical formula

Chemical formula

Chemical formula

[0041] Among these, (b-01) and (b-02) are particularly preferred because they can exhibit excellent properties when used in liposomes in combination with the compound of formula (a).

[0042] The lipid particles according to the embodiment contain the compound represented by the formula (a) and, if necessary, an additional lipid compound represented by the formula (b), but may also contain other additional lipid compounds. Such additional lipid compounds can generally be arbitrarily selected from those commonly used for lipid particles.

[0043] For example, it is also preferable to use dimyristoyl glycerol polyethylene glycol, which is a lipid compound having a structure excluding the maleimide group, in combination with the compound of the formula (a).

[0044] In addition, it is preferable to further combine an additional lipid compound selected from the group consisting of cationic lipids, neutral lipids, structure-forming lipids, and aggregation-reducing lipids. Such lipid compounds are preferably lipids with excellent biodegradability.

[0045] Such lipid compounds act effectively, for example, in forming the membrane of liposomes. Specific examples include diacyl phosphatidylcholine, diacyl phosphatidylethanolamine, ceramide, sphingomyelin, dihydrosphingomyelin, kephalin, and cerebroside. The lipid used to form the membrane of the lipid particles in the embodiment is appropriately selected in consideration of the size of the target liposome and the stability of the liposome in the living body. Among these, diacyl phosphatidylcholine and diacyl phosphatidylethanolamine are preferable. Here, the length of the hydrocarbon chain of the acyl group contained in the lipid is preferably 10 to 20. This hydrocarbon chain may be a saturated hydrocarbon group or an unsaturated hydrocarbon group.

[0046] There are various such lipids for forming the membrane, for example, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-Dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC), 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC), 1,2-Di-O-octadecyl-3-trimethylammonium propane (DOTMA), 1,2-Dioleoyl-3-dimethylammonium propane (DODAP), 1,2-Dimyristoyl-3-dimethylammonium propane (14:0 DAP), 1,2-Dipalmitoyl-3-dimethylammonium propane (16:0 DAP), 1,2-Distearoyl-3-dimethylammonium propane (18:0 DAP), N-(4-Carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propane (DOBAQ), 1,2-Dioleoyl-3-trimethylammonium propane (DOTAP), 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-Dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-Dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), and Cholesterol etc. can be mentioned as preferred ones. Among these, DOPE, DOTAP, or cholesterol is more preferred, and the combination of DOPE and cholesterol, the combination of DOTAP and cholesterol, and the combination of DOPE, DOTAP, and cholesterol are particularly preferred. These can exert a membrane fusion effect in addition to the function of forming a membrane such as a liposome.

[0047] The lipids that can reduce aggregation and are used in the embodiments are those that exhibit functions including aggregation between particles during the preparation of lipid particles. Various such lipids are known, and any of them can be selectively used for the lipid particles according to the embodiments. Examples of such lipids include polyethylene glycol (PEG)-modified lipids, polyamide oligomers derived from omega-amino (oligoethylene glycol) alkanoic acid monomers (U.S. Patent No. 6,320,017), monosialogangliosides, and the like. More specifically, ATTA lipids such as ATTA8-DPSE listed in U.S. Patent No. 6,320,017 and polyethylene glycol lipid conjugates described in U.S. Patent Nos. 5,820,873, 5,534,499, and 5,885,613 can be used.

[0048] When lipid particles are formed, the PEG-modified lipid can form an anchoring lipid moiety on the surface of the lipid particles. Examples of such PEG-modified lipids include PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-ceramide conjugates (e.g., C14 PEG-Cer or C20 PEG-Cer described in Patent No. 3920330), PEG-modified dialkylamine, PEG-modified 1,2-diacyl-oxypropane-3-amine, PEG-modified diacylglycerol (e.g., 1,2-dimyristoyl-sn-glycerol-methoxypolyethylene glycol; PEG-DMG) and PEG-modified dialkylglycerol. Among these, PEG-modified diacylglycerol and PEG-modified dialkylglycerol are particularly preferred.

[0049] When a bulky modifying group such as PEG is bound to a lipid, the binding between the modifying group and the lipid particles affects the stability of the lipid particles or liposomes. For example, U.S. Patent No. 5,820,873 shows that characteristics such as the length of the acyl chain, the degree of saturation of the acyl chain, and the size of the steric hindrance head group in PEG-modified lipids affect the stability of lipid particles. Therefore, by adjusting these characteristics, lipid particles suitable for the purpose can be obtained. For example, by shortening the modifying group in PEG-modified lipids, the lipid particles can be lost more quickly, or by lengthening the modifying group, the residence time in plasma can be prolonged, etc. As a result, in some cases, the delivery of lipid particles to the target tissue can be improved.

[0050] Lipid particles can further contain other lipids. Such other lipids can be arbitrarily selected from those commonly used in lipid particles and used. For example, in order to adjust toxicity, relatively low-toxic lipids can be combined. Also, in order to introduce a functional group for binding a ligand to the lipid particles, lipids having a specific structure can be combined.

[0051] Furthermore, when using lipid particles as liposomes, they can also contain a sterol, such as cholesterol, as a lipid for suppressing the leakage of the inclusion. Furthermore, a targeting agent can also be coupled to the lipid particles. In such a case, any conventionally known method can be adopted as the coupling method.

[0052] [Active agent] The lipid particles according to the embodiment further contain an active agent. In the embodiment, the active agent is a substance capable of exerting a specific effect on cells, tissues, organs, or a subject. The specific effect may be any of a biological, physiological, or cosmetic effect. By using the lipid particles according to the embodiment, various active agents can be delivered to the target site in the living body. This active agent may be encapsulated inside the lipid particles, bound to the outer or inner lipid surface, or disposed inside the lipid layer.

[0053] Typical examples of the active agent are nucleic acids, such as nucleic acids selected from the group consisting of plasmids, oligonucleotides, polynucleotides, mRNA, small interfering RNA (siRNA), microRNA (miRNA), DNA, aptamers, and ribozymes. In addition, antisense oligonucleotides, antagomirs, aDNA, plasmids, ribosomal RNA (rRNA), transfer RNA (tRNA), small nuclear RNA (snRNA), etc. can also be used. Different types of DNA and RNA may be used in combination.

[0054] As the miRNA, miRNA with 17 to 25 linked nucleotide units can be used.). In one more preferred embodiment, the nucleic acid is an oligonucleotide with 15 to 50 or 20 to 30 linked nucleotide units. The siRNA can, for example, contain 16 to 30 nucleotide units and have a double-stranded region. In another embodiment, the nucleic acid is an immunostimulatory oligonucleotide, a decoy oligonucleotide, a supermir, a miRNA mimic, or a miRNA inhibitor. A supermir refers to an oligomer or polymer of RNA or deoxyribonucleic acid DNA, or both, or variants thereof, which is single-stranded, double-stranded, or partially double-stranded and has a nucleotide sequence substantially identical to that of the miRNA and is antisense to its target. miRNA mimics represent a group of molecules that can be used for the purpose of mimicking the gene silencing ability of one or more miRNAs. Therefore, the term "miRNA mimic" refers to a synthetic non-coding RNA that can enter the RNAi pathway and regulate gene expression (i.e., miRNA mimics cannot be obtained by purifying from an endogenous miRNA source).

[0055] When combining a nucleic acid with a lipid particle, the form of the nucleic acid is not particularly limited. This nucleic acid can be, for example, single-stranded DNA or RNA, double-stranded DNA or RNA, or a DNA-RNA hybrid. Examples of double-stranded RNA include siRNA. Examples of single-stranded nucleic acids include antisense oligonucleotides, ribozymes, miRNAs, and triplex-forming oligonucleotides. When the nucleic acid is mRNA, it is preferably modified to have resistance to degradation. For example, the modification is preferably a known modification that prevents RNA from being degraded by RNase or the like. Such modifications include, for example, the use / introduction of natural or unnatural nucleotides into RNA, the use / addition of unnatural sequences, or the addition of natural / unnatural CAP structures. Natural modified nucleotides are, for example, pseudouridine, 5-methylcytidine, 1-methyladenosine, etc. Unnatural nucleotides are, for example, BNA (Bridged nucleic acid), LNA (Locked nucleic acid), or PNA (Peptide nucleic acid), etc. Unnatural sequences are, for example, artificially created base sequences that do not exist in nature, such as random base sequences, or hybrid sequences of natural / unnatural amino acids and nucleic acids. Unnatural sequences are preferably added, for example, to the ends of RNA. Natural CAP structures are, for example, CAP0 (m7GpppN), CAP1 (m7GpppNm), etc. Unnatural CAP structures are, for example, ARCA (Anti-Reverse Cap Analog) or LNA-guanosine, etc. Unnatural CAP structures are preferably added to the 5' end of RNA.

[0056] When the lipid particles according to the embodiment contain nucleic acids, they can further contain a compound that binds to the nucleic acids. Examples of such compounds include basic proteins or basic peptides, preferably protamine, histone, and their salts. For example, histone and its salts have the property of binding to nucleic acids and folding nucleic acid molecules. Protamine, on the other hand, has the property of binding to nucleic acids and further wrapping the nucleic acids. Therefore, these compounds are effective for encapsulating nucleic acids in lipid particles.

[0057] In addition, the lipid particles according to the embodiment can further contain a compound that regulates the expression of nucleic acids in cells. Adjusting the expression of nucleic acids in cells is preferable because it can provide visualization and cell death effects on the cells to which the liposomes are delivered. Examples of such compounds include retinoic acid, cyclic adenosine monophosphate (cAMP), ascorbic acid, and the like.

[0058] In addition, the lipid particles according to the embodiment may contain lipoproteins, apolipoproteins, and the like.

[0059] Other therapeutic agents can also be used as the active agent. Specific examples of therapeutic agents that can be used include peptides, polypeptides, cytokines, growth factors, apoptosis factors, differentiation-inducing factors, cell surface receptors and their ligands, hormones, and the like. More specifically, the therapeutic agents include anti-inflammatory compounds, antidepressants, stimulants, analgesics, antibiotics, contraceptives, antipyretics, vasodilators, angiogenesis inhibitors, cytovascular agents, signal transduction inhibitors, cardiovascular drugs, tumor drugs, hormones, and steroids.

[0060] When combining an activator with lipid particles, it is preferable that the activator be introduced into the lipid particles at a higher introduction rate. It is also preferable that cell death due to cytotoxicity depending on the properties of the lipid be low. When introducing nucleic acid using conventionally known lipid particles, generally the introduction rate is low and the proportion of cell death due to cytotoxicity is also high. In contrast, when using the lipid particles according to this embodiment, the introduction rate of nucleic acid is high and cell death can also be reduced. Specifically, in conventional lipid particles, the introduction rate is about 10% and cell death due to electroporation is 60 - 70%, whereas when using the lipid particles according to the embodiment, they are reduced to 70% or more and 30% or less, respectively.

[0061] The lipid particles according to the embodiment can be formed into any size according to the purpose. However, when the lipid particles according to the embodiment are to be used for pharmaceutical purposes, the lipid particles are generally particles of nano - order size. Specifically, the average particle diameter of the lipid particles according to the embodiment is generally 50 nm to 300 nm, preferably 50 nm to 200 nm. The size of the lipid particles can be adjusted by any method. For example, the lipid particles can be made smaller by ultrasonic treatment. Also, size adjustment can be performed by passing through a polycarbonate membrane or a ceramic membrane and fractionating the lipid particles. In the embodiment, the average particle diameter of the lipid particles can be measured, for example, by a zeta - sizer using the dynamic light scattering method.

[0062] [Method for producing lipid particles] The lipid particles according to the embodiment can be produced by any conventionally known method. As methods for producing lipid particles or liposomes, the Bangham method, ethanol injection method, organic solvent extraction method, surfactant removal method, freeze - thaw method, etc. are known, and these can also be adopted. Also, for example, a compound represented by formula (a), further a lipid forming a membrane, and a lipid capable of reducing aggregation are introduced into an organic solvent such as alcohol, and lipid particles can be formed spontaneously by adding an aqueous buffer solution. By combining an activator in this aqueous buffer solution, it is possible to introduce the activator into the lipid particles.

[0063] [Use of Lipid Particles] The lipid particles according to the present embodiment can be used for delivering an active agent to cells. In particular, the delivery of active agents such as nucleic acids to cells is used in all fields such as genetic engineering, production of recombinant proteins, and medical techniques known as gene therapy and cell diagnosis. In one embodiment, there is provided a composition for delivering an active agent to cells, characterized by comprising the lipid particles according to the embodiment and a carrier. In another embodiment, there is provided the lipid particles according to the embodiment for delivering an active agent to cells. In another embodiment, there is provided a method for delivering an active agent to cells, comprising contacting the cells with the lipid particles according to the embodiment containing the active agent (for example, administering the lipid particles to a subject). In another embodiment, there is provided the use of the lipid particles according to the embodiment for delivering an active agent to cells. In one embodiment, the cells are tumor cells. The subject is preferably an animal in need of such treatment, more preferably a mammal, and most preferably a human. These uses will be specifically described below.

[0064] [Composition] The lipid particles according to the present embodiment can be used as a composition. For example, there is provided a composition comprising the lipid particles and a carrier of the present embodiment. Such a composition is also applicable to pharmaceutical uses.

[0065] The carrier can be arbitrarily selected from conventionally known ones and used, and examples thereof include water, saline such as physiological saline, glycine aqueous solution, buffer solution, and the like. Further, in addition to these carriers, glycoproteins such as albumin, lipoprotein, apolipoprotein, and globulin may be combined for the purpose of improving stability.

[0066] The composition according to the embodiment can be prepared by standard methods. As the carrier, physiological saline is generally used. In a composition containing saline or other salt-containing carriers, the carrier is preferably added after the formation of lipid particles. Therefore, after combining the lipid particles with an active agent such as nucleic acid, it is common to replace or dilute the composition with a pharmaceutically acceptable carrier such as physiological saline.

[0067] The composition according to the embodiment may contain adjuvants as needed. For example, in the case of pharmaceutical use, by including pharmaceutically acceptable adjuvants such as pH adjusters, buffering agents, tonicity adjusters, etc. as adjuvants, the pharmaceutical composition can be brought closer to a physiological state. Examples of adjuvants having such functions include sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, hydroxyethylpiperazineethanesulfonic acid (HEPES), etc. In addition, the composition according to the embodiment can also contain a lipid protectant for improving storage stability. Examples of such protectants include lipophilic free radical quenchers such as α-tocopherol that suppress damage by free radicals, and water-soluble chelators such as deferoxamine that suppress peroxidative damage of lipids.

[0068] In addition, the above-mentioned active agents, etc. can also be added to the composition. This active agent may be the same as or different from the active agent combined with the lipid particles. Also, a compound that binds to nucleic acid or a compound that regulates the expression of nucleic acid can be added to the composition.

[0069] The concentration of the lipid particles contained in the composition according to the embodiment is not particularly limited, and the content rate of the lipid particles contained in the composition is generally 0.01 to 30% by mass, preferably 0.05 to 10% by mass. The concentration of the lipid particles can be appropriately selected according to the purpose.

[0070] The composition according to the embodiment can be sterilized by conventional well-known methods. The sterilized composition can be packaged as a ready-to-administer formulation, or can also be dried and packaged. The dried composition can be made into a ready-to-administer preparation by combining it with a sterile aqueous solution immediately before administration.

[0071] The composition according to the embodiment can also be in the form of a kit. The kit according to the embodiment includes the lipid particles described above and an introducing agent for introducing the lipid particles into cells, and its form is arbitrary. For example, a kit in which a dispersion in which lipid particles not containing an active agent are dispersed in a carrier and the active agent are accommodated in separate containers, a kit in which the dried lipid particles, the active agent, and the carrier are accommodated in separate containers, etc. can be mentioned. Furthermore, the dried lipid particles or lipid particle dispersion and the active agent can be made into separate products so that the user can select each product according to the purpose. Reagents used in nucleic acid introduction can be combined in the kit.

[0072] [Method of using the pharmaceutical composition] When the lipid particles according to the embodiment are used for pharmaceutical purposes, the composition can be used for the treatment or diagnosis of various diseases in humans or animals. For example, by applying a therapeutic agent as an active agent to be combined with the lipid particles, the therapeutic agent can be delivered to target cells, enabling treatment.

[0073] For example, it is possible to deliver and contact various nucleic acids to cells for disease prevention or treatment. Such nucleic acids include oligonucleotides, siRNA, plasmids, antisense, or ribozymes. The lipid compounds according to these embodiments can efficiently and rapidly take in these nucleic acids. For example, although it has conventionally been difficult to introduce RNA into lipid particles in a short time and safely, by using the lipid compounds according to the embodiment, it becomes possible to easily do so.

[0074] In addition, the delivery of active agents such as nucleic acids can be carried out either in vitro or in vivo. As a method of in vivo administration, the pharmaceutical composition is preferably administered parenterally, that is, intra-articular administration, intravenous administration, intraperitoneal administration, subcutaneous administration, or intramuscular administration is employed. Intravenous or intraperitoneal administration of the pharmaceutical composition can also be carried out by bolus injection.

[0075] In addition, the pharmaceutical preparation according to the embodiment can be directly applied to the target tissue to bring the pharmaceutical composition into contact with the target tissue. In addition, administration by drip to the meninges or the like, and administration by an endoscopic instrument are also possible.

[0076] In certain embodiments, the treatment with the pharmaceutical composition is generally carried out at physiological temperature (about 37°C) for a period of 1 to 24 hours, preferably 2 to 8 hours. In in vitro applications, the cells to be targeted are not particularly limited. For example, they may be vertebrate cells, invertebrate cells, or plant cells. However, in preferred embodiments, the cells are animal cells, more preferably mammalian cells, and most preferably human cells. Also, the mammalian cells are preferably hematopoietic cells, and more preferably are selected from the group consisting of PBMC, T cells, dendritic cells, and CD34-positive cells.

Examples

[0077] [Method for preparing liposomes] Liposomes encapsulating GFP gene mRNA (GFP mRNA, TriLink) were prepared by the ethanol injection method. First, a lipid solution was prepared to have the lipid composition (unit: %nmol) shown in Table 1. The compounds used are as follows.

Chemical formula

[0078]

Table 1

[0079] [(1) Measurement of GFP mRNA Expression Level in Liposome-administered Cells] Using the lipid solutions of Comparative Examples 101 to 104 and Example 101, liposomes encapsulating GFP-mRNA were prepared. After dispensing the lipid solution into microtubes, the GFP mRNA solution was mixed. 10 mM HEPES (pH 7.3) was added to this mixture, and buffer exchange and concentration were performed using an ultrafiltration filter (Amicon Ultra) to prepare liposomes.

[0080] As cells, human peripheral blood mononuclear cells (PBMC) were used. PBMC were seeded at 2×10 5 cells in a 96-well culture plate, and then liposomes encapsulating GFP-mRNA adjusted with the above lipid composition were added. After adding the liposomes, the cells were cultured in an incubator at 37 °C and 5% CO2, and the GFP fluorescence intensity of the cells after 48 hours was measured with a fluorescence-activated cell sorter (SH800S, Sony). The results are shown in Table 2 and Figure 1. The results were expressed as the fold-change, which is the relative ratio obtained by dividing the fluorescence intensities of Example 101 and Comparative Examples 102 - 104 to be measured by the fluorescence intensity of Comparative Example 101 as a control. From Figure 1, the highest fluorescence intensity (expression of the GFP gene) was confirmed in PBMC administered with liposomes encapsulating GFP-mRNA containing formula (a-01) as the lipid compound (Example 101).

[0081]

Table 2

[0082] [(2) Measurement of nLuc mRNA Expression Level in Liposome-administered Cells] Using the lipid solutions of Example 201 and Comparative Example 202, liposomes encapsulating the mRNA of the NanoLuc gene (nLuc mRNA) were prepared by the ethanol injection method. After dispensing the lipid solution into a microtube, the nLuc mRNA solution was mixed. 10 mM HEPES (pH 7.3) was added to this mixture, and buffer exchange and concentration were performed using an ultrafiltration filter (Amicon Ultra) to prepare nLuc-mRNA-encapsulating liposomes.

[0083] As cells, human T cell leukemia cells Jurkat were used. After seeding 2×10 5 Jurkat cells in a 96-well culture plate, the nLuc-mRNA-encapsulating liposomes adjusted with the above lipid composition were added. After adding the liposomes, the cells were cultured in an incubator at 37°C under 5% CO2, and the nLuc activity of the cells after 48 hours was measured. The enzyme activity of nLuc was measured with a luminometer using the NanoGlo Luciferase Assay System (Promega). The results are shown in Table 3 and Figure 2. The results were expressed as the fold-change, which is the relative ratio obtained by dividing the fluorescence intensity of Example 201, the measurement target, by the fluorescence intensity of Comparative Example 201, the control. From Figure 2, the highest fluorescence intensity (expression of the nLuc gene) was confirmed in Jurkat administered with nLuc-mRNA-encapsulating liposomes containing formula (a-01) as the lipid compound.

[0084]

Table 3

[0085] [(3) Measurement of GFP mRNA Expression Level in Liposome-administered Cells] [Measurement of GFP mRNA expression level in liposome-administered cells] Liposomes encapsulating the lipid solutions of Examples 101 and 102 and Comparative Example 101 were prepared in the same manner as described above. Then, fluorescence intensity was measured under the same conditions as in [(1) Measurement of GFP mRNA expression level in liposome-administered cells], except that the measuring device was changed to a fluorescence-activated cell sorter (FACSverse, BD Biosciences). The results were expressed as the fold-change, which is the relative ratio obtained by dividing the fluorescence intensities of Examples 101 and 102, which were the measurement targets, by the fluorescence intensity of Comparative Example 101, which served as a control. From Table 4 and Figure 3, a positive correlation was observed between the content of the lipid compound represented by formula (a-01) in the liposomes administered to PBMC and the GFP fluorescence intensity (GFP gene expression level).

[0086]

Table 4

[0087] [(4) Measurement of GFP mRNA expression level in liposome-administered cells] [(1) Measurement of GFP mRNA expression level in liposome-administered cells] Liposomes encapsulating the lipid solutions of Comparative Examples 201 to 204 and Example 201 were prepared in the same manner as described above.

[0088] As the cells, human breast cancer cells MDA-MB231 were used. MDA-MB231 cells were seeded at 1×10 5After cell seeding, GFP-mRNA-encapsulating liposomes adjusted with the above lipid composition were added. After the addition of liposomes, the cells were cultured in an incubator at 37°C and 5% CO2, and the GFP fluorescence intensity of the cells after 24 hours was measured using a plate reader (infinite F200 PRO, Tecan). The results are shown in Table 5 and Figure 4. The results were expressed as fold-change, which is the relative ratio obtained by dividing the fluorescence intensity of Examples 201 and Comparative Examples 202 to 204 to be measured by the fluorescence intensity of Comparative Example 201 as a control. From Table 5 and Figure 4, the highest fluorescence intensity (expression of the GFP gene) was confirmed in MDA-MB231 administered with GFP-mRNA-encapsulating liposomes containing formula (a-01) as the lipid compound (Example 201).

[0089] [Table 5]

[0090] [(5) Measurement of GFP mRNA expression level in liposome-administered cells] Liposomes encapsulating the lipid solutions of Comparative Examples 201 to 204 and Example 201 were prepared in the same manner as [(1) Measurement of GFP mRNA expression level in liposome-administered cells].

[0091] As the cells, human T cell leukemia cells Jurkat were used. Jurkat was seeded at 2×10 5 After cell seeding, GFP-mRNA-encapsulating liposomes adjusted with the above lipid composition were added. After the addition of liposomes, the cells were cultured in an incubator at 37°C and 5% CO2, and the GFP fluorescence intensity of the cells after 24 hours was measured using a plate reader (infinite F200 PRO, Tecan). The results are shown in Table 6 and Figure 5. The results were expressed as fold-change, which is the relative ratio obtained by dividing the fluorescence intensity of Examples 201 and Comparative Examples 202 to 204 to be measured by the fluorescence intensity of Comparative Example 201 as a control. From Table 6 and Figure 5, the highest fluorescence intensity (expression of the GFP gene) was confirmed in Jurkat administered with GFP-mRNA-encapsulating liposomes containing formula (a-01) as the lipid compound (Example 201).

[0092] [Table 5]

[0093] [(6) Measurement of GFP mRNA Expression Level in Liposome-administered Cells] [(1) Measurement of GFP mRNA Expression Level in Liposome-administered Cells] In the same manner, liposomes encapsulating the lipid solutions of Comparative Example 101 and 301 to 303, and Example 301 were prepared.

[0094] Using the prepared liposomes, the fluorescence intensity was measured in the same manner as [(4) Measurement of GFP mRNA Expression Level in Liposome-administered Cells]. The results are shown in Table 7 and Figure 6. The results were expressed as the fold-change, which is the relative ratio obtained by dividing the fluorescence intensities of Example 301 and Comparative Examples 301 to 303, which were the measurement targets, by the fluorescence intensity of Comparative Example 101 as the control. From Table 7 and Figure 6, the highest fluorescence intensity (expression of the GFP gene) was confirmed in MDA-MB231 administered with the GFP-mRNA-encapsulating liposome (Example 301) containing the formula (a-01) as the lipid compound.

[0095] [Table 7]

[0096] [(7) Measurement of GFP mRNA Expression Level in Liposome-administered Cells] [(1) Measurement of GFP mRNA Expression Level in Liposome-administered Cells] In the same manner, liposomes encapsulating the lipid solutions of Comparative Example 101 and 301 to 303, and Example 301 were prepared.

[0097] Using the prepared liposomes, the fluorescence intensity was measured in the same manner as [(5) Measurement of the GFP mRNA expression level in liposome-administered cells]. The results are shown in Table 8 and Figure 7. The results were presented as the fold-change, which is the relative ratio obtained by dividing the fluorescence intensity of Examples 301 and Comparative Examples 301 to 303, which were the measurement targets, by the fluorescence intensity of Comparative Example 101, which served as the control. From Table 8 and Figure 7, the highest fluorescence intensity (expression of the GFP gene) was confirmed in Jurkat cells administered with GFP-mRNA-encapsulating liposomes (Example 301) containing the compound of formula (a-01) as the lipid compound.

[0098]

Table 8

[0099] As described above, several embodiments have been explained. However, these embodiments are presented as examples and are not intended to limit the scope of the invention. These novel embodiments can be implemented in various other forms, and various combinations, omissions, replacements, changes, etc. can be made without departing from the gist of the invention. These embodiments and their modifications are included in the scope and gist of the invention, and are also included in the invention described in the claims and its equivalent scope.

Claims

1. The following formula (a): Mal-L ａ１ -(OCH ２ CH ２ ) ｎａ -O-L ａ２ R ａ ２ (a) (wherein Mal is a maleimide group, L ａ１ is an unsubstituted hydrocarbon group or a substituted hydrocarbon group containing a linking group selected from the group consisting of -C(=O)-O-, -O-C(=O)-, -O-C(=O)-O-, -S-C(=O)-, -C(=O)-S-, -C(=O)-NH-, and -NH-C(=O)-, L ａ２ is a trivalent hydrocarbon group that does not contain phosphorus, R ａ is, independently of each other, a hydrocarbon group containing at least one ester bond at an arbitrary position of C １０ to ３０ and containing at least one ester bond at an arbitrary position, na is a number of 1 or more) A compound represented by, An activator, A lipid particle, characterized by comprising

2. The lipid particle according to claim 1, wherein na is 10 to 100.

3. The lipid particle according to claim 1 or 2, wherein the compound represented by formula (a) is dimyristoyl glycerol polyethylene glycol maleimide.

4. The following formula (b): Q-CHR ｂ ２ (b) (wherein Q is a nitrogen-containing aliphatic group containing two or more tertiary nitrogens and no oxygen, R ｂ is each independently an aliphatic group of C １２ to C ２４ , and at least one R ｂ contains a linking group selected from the group consisting of -C(=O)-O-, -O-C(=O)-, -O-C(=O)-O-, -S-C(=O)-, -C(=O)-S-, -C(=O)-NH-, and -NH-C(=O)- in its main chain or side chain) The lipid particle according to claim 1 or 2, further comprising a compound represented by

5. The Q is the following formula (Q'): R Ｑ１ ２ N-(CR Ｑ２ ２ ) ｑ１ -NR Ｑ１ -(CR Ｑ２ ２ ) ｑ２ -*2 (Q') (wherein R Ｑ１ is, independently of one another, alkyl, R Ｑ２ is each independently hydrogen or alkyl, or R Ｑ１ and R Ｑ２ any two of which may form one alkylene to form a nitrogen-containing alicyclic ring, q1 is a number from 1 to 5, q2 is a number from 0 to 5, *2 represents the bonding position to -CHR ｂ ２ ) The lipid particle according to claim 4, represented by

6. The lipid particle according to claim 1 or 2, further comprising an additional lipid selected from the group consisting of cationic lipids, neutral lipids, structure-forming lipids, and aggregation-reducing lipids.

7. The lipid particle according to claim 1 or 2, wherein the activator is a nucleic acid selected from the group consisting of plasmid, oligonucleotide, polynucleotide, siRNA, microRNA, DNA, mRNA, aptamer, and ribozyme.

8. The lipid particle according to claim 7, further comprising a compound that binds to the nucleic acid.

9. The lipid particle according to claim 8, wherein the compound that binds to the nucleic acid is a basic protein or a basic peptide.

10. The lipid particle according to claim 1 or 2, further comprising a compound that regulates the expression of nucleic acid in cells.

11. A composition comprising the lipid particle according to claim 1 or 2 and a carrier.

12. A composition for delivering an activator to cells, comprising the lipid particle according to claim 1 or 2 and a carrier.

13. The composition according to claim 12, wherein the cell is a mammalian cell.

14. The composition according to claim 13, wherein the mammalian cell is a hematopoietic cell.

15. The composition according to claim 14, wherein the hematopoietic cell is selected from the group consisting of PBMC, T cell, dendritic cell, and CD34-positive cell.

16. A method for delivering the activator to the cell, comprising contacting the cell with the lipid particle according to claim 1 or 2.

17. The method for delivering an active agent according to claim 16, wherein the cell is a cell of an animal other than a human or a cell taken out of the body.

18. The method for delivering an active agent according to claim 16, wherein the lipid particles further comprise a lipid forming a membrane and a lipid capable of reducing aggregation.

19. The lipid forming the membrane is 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (PO PC), 1,2-di-O-octadecyl-3-trimethylammonium propane (DOTMA), 1,2-dioleoyl-3-dimethylammonium propane (DODAP), 1,2-dimyristoyl-3-dimethylammonium propane (14:0 DAP), 1,2-dipalmitoyl-3-dimethylammonium propane (16:0 DAP), 1,2-distearoyl-3-dimethylammonium propane (18:0 DAP), N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy) propane (DOBAQ), 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), and cholesterol, selected from the group consisting of, The method for delivering an active agent according to claim 18, wherein the lipid capable of reducing aggregation is a polyethylene glycol (PEG)-modified lipid.

20. The method for delivering an active agent according to claim 16, wherein the active agent is a nucleic acid selected from the group consisting of a plasmid, an oligonucleotide, a polynucleotide, siRNA, microRNA, DNA, mRNA, an aptamer, and a ribozyme.

21. The method for delivering an active agent according to claim 16, wherein the active agent comprises a combination of at least one DNA and at least one RNA.

22. The method for delivering an active agent according to claim 20, wherein the lipid particles further comprise a compound that binds to a nucleic acid. **Claim 23** The method for delivering an active agent according to claim 22, wherein the compound that binds to the nucleic acid is a basic protein or a basic peptide. **Claim 24** The method for delivering an active agent according to claim 22, wherein the compound that binds to the nucleic acid is protamine or histone. **Claim 25** The method for delivering an active agent according to claim 22, wherein the lipid particles further comprise a compound that regulates the expression of the nucleic acid in cells. **Claim 26** The method for delivering an active agent according to claim 16, wherein a carrier is brought into contact with the cell together with the lipid particles. **Claim 27** The method for delivering an active agent according to claim 16, wherein the cell is a tumor cell. **Claim 28** Performing the method for delivering an active agent according to claim 16, Culturing the obtained cells, A method for producing cells comprising. **Claim 29** A kit comprising the lipid particles according to claim 1 or 2 and an introducing agent for introducing the lipid particles into cells. **Claim 30** The kit according to claim 29, further comprising a substance for improving the storage stability of the lipid particles.

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