JP2019151589A - Lipid nanoparticle - Google Patents

Abstract

To provide lipid nanoparticles useful as a carrier for drug active ingredients such as siRNA and having excellent delivery efficiency even if the particle diameter has become small.SOLUTION: Provided is a lipid nanoparticle containing a cationic lipid represented by the following general formula (I) [a is an integer from 3 to 5; b is 0 or 1; Rand Rare each independently a group having 20 or more carbon atoms represented by the following general formula (A) (q represents an integer from 1 to 9, r represents 0 or 1, s represents an integer from 1 to 3, t represents 0 or 1, u represents an integer from 1 to 8, c represents 0 or 1, v represents an integer from 4 to 12); X represents a group represented by the following general formula (B) (d is an integer of 0 to 3, Rand Rare each independently a Calkyl group or a Calkenyl group) or a 5- to 7-membered non-aromatic heterocyclic group]; and a neutral lipid with a hydrophilic group having 2 or more carbon atoms and a linear hydrophobic group.SELECTED DRAWING: None

Description

  The present invention relates to lipid nanoparticles useful as a carrier for medicinal ingredients such as siRNA (small interfering RNA).

  Nucleic acid molecules that control the expression of genes encoded by binding to target DNA or RNA in a base sequence specific manner are attracting a great deal of attention as new therapeutic modalities for intractable diseases such as cancer. In particular, siRNA (short interfering RNA) is a functional nucleic acid capable of powerfully knocking down a target gene via RNA interference, and its application to pharmaceuticals is anxious. However, since siRNA does not have tissue migration ability, development of a technique that can be efficiently delivered to target cells is essential for application to medicine. To date, a large number of lipid nanoparticles capable of realizing efficient in vivo delivery of siRNA have been reported. In particular, pH-sensitive cationic lipids that are electrically neutral at physiological pH and change cationically under a weakly acidic pH environment such as endosomes (see, for example, Patent Document 1). Has significantly contributed to improving the efficiency of siRNA delivery by lipid nanoparticles. For example, it has been reported that F7 knockdown succeeded with high efficiency by administering lipid nanoparticles containing pH-sensitive cationic lipid encapsulating siRNA targeting factor 7 (F7) in mouse liver. . (For example, refer nonpatent literature 1.).

  On the other hand, siRNA delivery to cancer tissues is more difficult than delivery to tissues such as liver. One of the major causes is interstitial components (stromal barrier) including collagen and hyaluronic acid, which are very abundant in cancer tissues. Since the penetration of the nanoparticles in the cancer tissue is physically hindered by the interstitial barrier, the lipid nanoparticles cannot move to the deep part of the cancer tissue, so that sufficient gene knockdown efficiency and drug efficacy cannot be obtained.

  Miniaturization of nanoparticles (drug carrier nanoparticles) that deliver medicinal ingredients to target cells is a very effective strategy for breaking through the stromal barrier. For example, it has been reported that by controlling the diameter of a polymer preparation-encapsulated polymer micelle to be about 30 nm, the penetration into cancer tissue is improved and the antitumor effect is improved (see Non-Patent Document 2). ). The same strategy is considered to be very effective for lipid nanoparticles that deliver siRNA to target cells (siRNA carrier lipid nanoparticles). However, it is technical to control the particle size of siRNA carrier lipid nanoparticles to be small. It is difficult. In recent years, it has been reported that lipid nanoparticles having a diameter of about 30 nm can be produced with good reproducibility by using a microchannel with a built-in micromixer that can achieve instantaneous mixing of two liquids (see, for example, Non-Patent Document 3). The technical barrier of manufacturing itself is being eliminated. However, there is a problem that siRNA delivery activity is remarkably reduced when lipid nanoparticles are miniaturized (see, for example, Non-Patent Documents 4 and 5). As a cause of the decrease in activity due to the downsizing of lipid nanoparticles, the lipid molecules on the surface of lipid nanoparticles become unstable due to sparseness (Non-patent Document 4), and accompanyingly, lipid nanoparticles are constituted. It has been clarified that lipid molecules to be detached and lipid nanoparticles disintegrate (Non-patent Document 5).

International Publication No. 2015/178343

Sato et al., Molecular Therapy, 2016, vol.24 (4), p.788-795. Cabral et al., Nature Nanotechnology, 2011, vol. 6, p.815-823. Leung et al., Journal of Physical Chemistry C Nanomater Interfaces, 2012, vol.116 (34), p.18440-18450. Sato et al., Journal of Controlled Release, 2016, vol.229, p.48-57. Chen et al., Journal of Controlled Release, 2016, vol.235, p.236-244.

  It is important that drug carrier nanoparticles that can be applied clinically can be stably delivered to target cells in order to achieve better delivery efficiency (efficiency in which drug efficacy is achieved by the delivered medicinal component). In particular, when delivering an anticancer drug, the penetration of cancer tissue into the cancer tissue is high enough to penetrate the interstitial barrier and efficiently migrate to the deep part of the cancer tissue. It is important that the stability is so high that it can be maintained until it is taken in.

  The present invention is useful as a carrier for medicinal ingredients such as siRNA, and an object thereof is to provide lipid nanoparticles having excellent delivery efficiency even when the particle diameter is reduced.

  The present inventors, as a constituent lipid, a pH-sensitive cationic lipid having a long hydrocarbon chain and a neutral lipid having a relatively bulky hydrophilic group and a long hydrocarbon chain, We found that lipid nanoparticles with a small particle size can be produced, and that siRNA carrier lipid nanoparticles containing siRNA in this lipid nanoparticle can achieve high knockdown efficiency even when the particle size is reduced, completing the present invention. I let you.

That is, the present invention provides the following lipid nanoparticles.
[1] The following general formula (I)

[In the formula (I), a represents an integer of 3 to 5; b represents 0 or 1; R ¹ and R ² each independently represents the following general formula (A):

(In formula (A), q represents an integer of 1 to 9; r represents 0 or 1; s represents an integer of 1 to 3; t represents 0 or 1; u represents an integer of 1 to 8] C represents 0 or 1; v represents an integer of 4 to 12; q + 2r + s + 2t + u + c + v is an integer of 19 or more, but when b and c are simultaneously 0, q is an integer of 3 to 5 Except that r and t are 1, s is 1, and u + v is an integer of 6 to 10)
X represents a group represented by the following general formula (B):

(In formula (B), d represents an integer of 0 to 3; R ³ and R ⁴ each independently represent a C _1-4 alkyl group or a C _2-4 alkenyl group (the C _1-4 alkyl group or C _{2 -4} alkenyl group represents one or two hydrogen atoms optionally substituted with a phenyl group, but R ³ and R ⁴ are bonded to each other to form a 5- to 7-membered non-aromatic heterocycle 1 or 2 hydrogen atoms of the ring may be substituted with a C _1-4 alkyl group or a C _2-4 alkenyl group).
Or a 5- to 7-membered non-aromatic heterocyclic group (wherein the group is bonded to (O-CO) b- by a carbon atom, and one or two hydrogen atoms of the ring are C _1-4 alkyl group or _C2-4 alkenyl group may be substituted)]
And a neutral lipid in which a linear hydrophobic group is extended from a hydrophilic group having 2 or more carbon atoms, and the total lipid amount constituting the lipid nanoparticle Lipid nanoparticles, wherein the proportion of the total amount of the pH-sensitive cationic lipid and the neutral lipid is 50 mol% or more.
[2] A linear hydrophobic group in the neutral lipid is a linear hydrocarbon group having 16 to 20 carbon atoms (between one or more carbon-carbon single bonds in the group, an ether The lipid nanoparticle according to claim 1, wherein one or more bonds selected from the group consisting of a bond, a carbonyl bond, an ester bond, and a carbonate bond may be inserted.
[3] The lipid nanoparticles according to claim 1 or 2, wherein the neutral lipid is glycerolipid or sphingolipid.
[4] The lipid nanoparticles according to any one of [1] to [3], wherein the number average particle diameter measured by a dynamic light scattering method is 50 nm or less.
[5] The lipid nanoparticle according to any one of [1] to [4], wherein the ratio of the amount of the pH sensitive cationic lipid to the total amount of lipid constituting the lipid nanoparticle is 20 mol% or more.
[6] The lipid nanoparticle according to any one of [1] to [5], which contains one or more selected from the group consisting of a low molecular compound, a low molecular nucleic acid, and a peptide.
[7] The lipid nanoparticle according to any one of [1] to [6], which contains a medicinal ingredient and is a carrier that delivers the medicinal ingredient to a target cell.

  A lipid nanoparticle according to the present invention comprises a pH-sensitive cationic lipid having a long hydrocarbon chain, and a neutral lipid having a relatively bulky hydrophilic group and a long hydrocarbon chain. Since it is a constituent lipid, it is excellent in stability of the lipid membrane on the particle surface even if the particle size is reduced. Therefore, by incorporating a medicinal component such as siRNA into the lipid nanoparticle, drug carrier nanoparticles that are sufficiently small and have an excellent ability to deliver the medicinal component can be obtained.

In Example 1, it is the figure which showed the result of having measured the firefly luciferase gene expression rate (%) of the HeLa-dluc cell which added the lipid nanoparticle from which the composition of a constituent lipid differs. In Example 1, it is the figure which showed the result of having measured the firefly luciferase gene expression rate (%) of the HeLa-dluc cell which added the lipid nanoparticle from which the composition of a constituent lipid differs. In Example 2, it is the figure which showed the result of having measured the firefly luciferase gene expression rate (%) of the HeLa-dluc cell which added the lipid nanoparticle from which the composition of a constituent lipid differs. In Example 2, it is the figure which showed the result of having measured the number average particle diameter (nm) of the lipid nanoparticle from which the composition of a constituent lipid differs. In Example 3, it is the figure which showed the result of having measured the firefly luciferase gene expression rate (%) of the HeLa-dluc cell which added the lipid nanoparticle from which the composition of a constituent lipid differs. In Example 4, it is the figure which showed the result of having measured the number average particle diameter (nm) of the lipid nanoparticle from which the composition of a constituent lipid differs. In Example 5, it is the figure which showed the time-dependent change of the number average particle diameter (nm) of the lipid nanoparticle preserve | saved under light-shielding at 4 degreeC after manufacture. In Example 5, it is the figure which showed the time-dependent change of siRNA encapsulation rate (%) of the lipid nanoparticle preserve | saved under light-shielding at 4 degreeC after manufacture.

  The lipid nanoparticles according to the present invention include a pH-sensitive cationic lipid represented by the following general formula (I) (hereinafter sometimes referred to as “the pH-sensitive cationic lipid of the present invention”), and a C 2 or more carbon atom. And a neutral lipid in which a linear hydrophobic group extends from a hydrophilic group (hereinafter, also referred to as “neutral lipid of the present invention”). The ratio of the total amount of the pH-sensitive cationic lipid of the present invention and the neutral lipid of the present invention to the total amount of lipid constituting the lipid nanoparticles according to the present invention is 50 mol% or more.

[PH-sensitive cationic lipid]

In the general formula (I), a represents an integer of 3 to 5, but is preferably 4.
b represents 0 or 1; When b is 0, it means that the —O—CO— group does not exist and is a single bond.

In general formula (I), R ¹ and R ² each independently represent a group represented by the following general formula (A). In the general formula (A), q represents an integer of 1 to 9; r represents 0 or 1; s represents an integer of 1 to 3; t represents 0 or 1; u represents an integer of 1 to 8 C represents 0 or 1; v represents an integer of 4 to 12. However, when b and c are simultaneously 0, q is an integer of 3-5, r and t are 1, s is 1, and u + v is an integer of 6-10. except.

In the pH-sensitive cationic lipid of the present invention, R ¹ and R ² are groups having 20 or more carbon atoms. That is, in the general formula (A), q + 2r + s + 2t + u + c + v is an integer of 19 or more. The relatively long hydrocarbon chains of R ¹ and R ² contribute to the stability of the lipid nanoparticles according to the present invention. In the pH-sensitive cationic lipid of the present invention, q + 2r + s + 2t + u + c + v is preferably an integer of 19 to 33, more preferably an integer of 19 to 31, and further preferably an integer of 21 to 31. More preferably, it is an integer of 27.

As R ¹ and R ² of the pH-sensitive cationic lipid of the present invention, in general formula (A), r and t are 0, q + s + u is an integer of 13 to 23, preferably 15 to 21, and c is A group in which v is an integer of 4 to 12, q + s + u + v is an integer of 18 or more; r and t are 0, q + s + u is an integer of 13 to 23, preferably 15 to 21, and c is A group in which v is an integer of 6 to 10 and q + s + u + v is an integer of 18 or more; r is 1, t is 0, and q is an integer of 5 to 11, preferably 6 to 10 A group in which s + u is an integer of 5 to 11, preferably an integer of 6 to 10, c is 1, v is an integer of 4 to 12, and q + s + u + v is an integer of 16 or more; 1; t is 0; q is an integer from 5 to 11; preferred Is an integer of 6-10, s + u is an integer of 5-11, preferably 6-10, c is 1, v is an integer of 6-10, q + s + u + v is an integer of 16 or more Certain groups are preferred.

In the pH-sensitive cationic lipid of the present invention, R ¹ and R ² may be groups represented by the general formula (A) and may be groups different from each other, but are preferably the same group.

  In the general formula (I), X represents a group represented by the following general formula (B) or a 5- to 7-membered non-aromatic heterocyclic group. The 5- to 7-membered non-aromatic heterocyclic group represented by X is bonded to (O—CO) b— by a carbon atom.

In general formula (B), d shows the integer of 0-3. When d is 0, it means that a — (CH ₂ ) — group does not exist and is a single bond.

In general formula (B), R ³ and R ⁴ each independently represent a C _1-4 alkyl group (an alkyl group having 1 to 4 carbon atoms) or a C _2-4 alkenyl group (an alkenyl group having 1 to 4 carbon atoms). Show. In the C _1-4 alkyl group or C _2-4 alkenyl group represented by R ³ and R ⁴ , one or two hydrogen atoms may be substituted with a phenyl group.

_{Examples of the} C _1-4 alkyl group include a methyl group, an ethyl group, a propyl group, an isopropyl group, an n-butyl group, an isobutyl group, and a t-butyl group. C _2-4 alkenyl groups include vinyl, 1-propenyl, 2-propenyl, 1-methylvinyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl, and 3-butenyl. Groups.

In general formula (B), R ³ and R ⁴ may be bonded to each other to form a 5- to 7-membered non-aromatic heterocycle. Examples of the 5- to 7-membered non-aromatic heterocycle formed by combining R ³ and R ⁴ with each other include 1-pyrrolidinyl group, 1-piperidinyl group, 1-morpholinyl group, and 1-piperazinyl group. . In the 5- to 7-membered non-aromatic heterocycle formed by combining R ³ and R ⁴ with each other, one or two hydrogen atoms in the ring are _substituted with a C _1-4 alkyl group or a C _2-4 alkenyl group. May be substituted. When two hydrogen atoms in the ring are substituted with a C _1-4 alkyl group or a C _2-4 alkenyl group, they may be substituted with the same group or with different groups. Also good.

  In the general formula (I), when X is a 5- to 7-membered non-aromatic heterocyclic group, examples of the hetero atom contained in the heterocyclic group include a nitrogen atom, an oxygen atom, and a sulfur atom. . The number of heteroatoms constituting the heterocycle in the heterocyclic group may be one, or two or more heteroatoms may be the same or different. The heterocyclic ring in the heterocyclic group may be a saturated heterocyclic ring and may contain one or more double bonds, but the heterocyclic ring does not become an aromatic ring.

As the pH-sensitive cationic lipid of the present invention, in general formula (I), a is an integer of 3 to 5, b is 1, and X is a 5- to 7-membered non-aromatic heterocyclic group (however, hetero Bonded to (O-CO) b- by a carbon atom in the ring group.), Preferably a 1-pyrrolidinyl group, 1-piperidinyl group, 1-morpholinyl group, or 1-piperazinyl group (depending on the carbon atoms in the ring ( O—CO) b— and one hydrogen atom may be substituted with a C _1-4 alkyl group or a C _2-4 alkenyl group, and R ¹ and R ² are each Independently, in general formula (A), r and t are 0, q + s + u is an integer of 13 to 23, preferably 15 to 21, c is 1, and v is an integer of 4 to 12. A group in which q + s + u + v is an integer of 18 or more; in the general formula (I) a is an integer from 3 to 5, b is 1, X is in the general formula (B), d is an integer of 1 to 3, _{C 1-4} alkyl groups ^{R 3} and ^{R 4} are each independently Or a C _2-4 alkenyl group (a C _1-4 alkyl group or a C _2-4 alkenyl group represented by R ³ and R ⁴ may have one or two hydrogen atoms substituted with a phenyl group). R ¹ and R ² are each independently, in general formula (A), r and t are 0, q + s + u is an integer of 13 to 23, preferably 15 to 21, and c is 1. a lipid in which v is an integer of 4 to 12 and q + s + u + v is an integer of 18 or more; in formula (I), a is an integer of 3 to 5, b is 1, and X is 5 ~ 7-membered non-aromatic heterocyclic group (however, bonded to (O-CO) b- by a carbon atom in the heterocyclic group), Preferably, a 1-pyrrolidinyl group, 1-piperidinyl group, 1-morpholinyl group, or 1-piperazinyl group (bonded to (O-CO) b- by a carbon atom in the ring, and one hydrogen atom is C _{1 -4} alkyl group or C _2-4 alkenyl group.), R ¹ and R ² are each independently, in general formula (A), r is 1 and t is 0. Q is an integer of 5 to 11, preferably an integer of 6 to 10, s + u is an integer of 5 to 11, preferably an integer of 6 to 10, c is 1, and v is 4 to 12 A lipid in which q + s + u + v is an integer of 16 or more; in general formula (I), a is an integer of 3 to 5, b is 1, and X is in general formula (B) , d is an integer of 1 to 3, _{C 1-4} alkyl ^{R 3} and ^{R 4} are each independently Groups or _{C 2-4} alkenyl group _{(C 1-4} alkyl or _{C 2-4} alkenyl group and ^{R 3} and ^{R 4} represents are one or two hydrogen atoms may be substituted with a phenyl group) R ¹ and R ² are each independently, in general formula (A), r is 1, t is 0, and q is an integer of 5 to 11, preferably 6 to 10. S + u is an integer of 5 to 11, preferably an integer of 6 to 10, c is 1, v is an integer of 4 to 12, and q + s + u + v is a group having an integer of 16 or more; In Formula (I), a is an integer of 3 to 5, b is 0, X is General Formula (B), d is 0, and R ³ and R ⁴ are each independently C _1-4 It shows an alkyl group or a _{C 2-4} alkenyl group ^{(R 3} and ^{R 4} _{C 1-4} alkyl group or a _{C 2-} Alkenyl group is one or two hydrogen atoms may be substituted with a phenyl group), independently of R ¹ and R ² are each, among the general formula (A), with r and t is 0 A lipid in which q + s + u is an integer of 13 to 23, preferably 15 to 21, c is 1, v is an integer of 4 to 12, and q + s + u + v is an integer of 18 or more; In (I), a is an integer of 3 to 5, b is 0, X is General Formula (B), d is 0, and R ³ and R ⁴ are bonded to each other to form a 1-pyrrolidinyl group , 1-piperidinyl group, 1-morpholinyl group, and 1-piperazinyl group (even if one or two hydrogen atoms in these rings are substituted by a C _1-4 alkyl group or a C _2-4 alkenyl group) forms a good), ^{R 1} and ^{R 2} are each independently In the general formula (A), r and t are 0, q + s + u is an integer of 13 to 23, preferably 15 to 21, c is 1, v is an integer of 4 to 12, q + s + u + v Is a group wherein is an integer greater than or equal to 18. Among these lipids, a lipid in which R ¹ and R ² are the same group is more preferable as the pH-sensitive cationic lipid represented by the general formula (I), and R ¹ and R ² are the same group, A lipid in which a is 4 is particularly preferred.

  The pKa of the pH-sensitive cationic lipid represented by the general formula (I) is not particularly limited, but can be selected, for example, from about 4.0 to 9.0, preferably from about 4.5 to 8.5. It is preferred to select the type of each substituent so as to give a range of pKa.

  The pH-sensitive cationic lipid represented by the general formula (I) can be easily produced, for example, by the method specifically shown in the examples of the present specification. By referring to this production method and appropriately selecting the raw material compounds, reagents, reaction conditions, and the like, those skilled in the art can easily produce any lipid included in the range of the general formula (I).

[Neutral lipids]
The neutral lipid of the present invention is a neutral lipid in which a linear hydrophobic group extends from a hydrophilic group having 2 or more carbon atoms. The neutral lipid of the present invention is stably present in the lipid membrane by the interaction of the linear hydrophobic group with the hydrophobic site of other lipid molecules constituting the lipid membrane. The number of linear hydrophobic groups extending from the hydrophilic group may be one, or two or more. When there are two or more linear hydrophobic groups extending from the hydrophilic group, they may all be the same linear hydrophobic group or different linear hydrophobic groups.

  From the viewpoint of interaction with other lipid molecules constituting the lipid nanoparticles, the linear hydrophobic group is preferably a linear hydrocarbon group. The linear hydrocarbon group may be a saturated hydrocarbon group or an unsaturated hydrocarbon group. In the case of an unsaturated hydrocarbon group, a linear hydrocarbon group having one or two unsaturated bonds is preferable.

The neutral lipid of the present invention includes a linear hydrocarbon group extending from a hydrophilic group in terms of interaction with R ¹ and R ² in the pH-sensitive cationic lipid represented by the general formula (I). Is more preferably a saturated or unsaturated linear hydrocarbon group having 12 to 20 carbon atoms, more preferably a saturated or unsaturated linear hydrocarbon group having 14 to 20 carbon atoms, More preferably, it is a C16-20 saturated or unsaturated linear hydrocarbon group. The hydrocarbon group includes an ester bond (—COO—), an amide bond (—CONH—), an ether bond (—O—), a carbonyl bond (—CO—) between one or more carbon-carbon single bonds. And one or more bonds selected from the group consisting of carbonate bonds (—OCOO—) may be inserted.

  The hydrophilic group having 2 or more carbon atoms provided in the neutral lipid of the present invention is not particularly limited as long as the group as a whole has a neutral charge and high hydrophilicity. For example, a group in which a negatively charged group such as a phosphate group and a positively charged group such as an ammonium group or a quaternary ammonium group are linked by an appropriate linking group, or a polyethylene glycol group And a glycosyl group.

  The neutral lipid of the present invention is preferably glycerolipid or sphingolipid. Examples of glycerolipids include neutral glycerophospholipids such as phosphatidylethanolamine, phosphatidylcholine, cardiolipin, and plasmalogen; glycerosaccharides such as sulfoxyribosyl glyceride, diglycosyl diglyceride, digalactosyl diglyceride, galactosyl diglyceride, and glycosyl diglyceride It is done. Examples of sphingolipids include neutral sphingophospholipids such as sphingomyelin, ceramide phosphorylglycerol, and ceramide phosphorylethanolamine; and sphingosaccharides such as galactosyl cerebroside, lactosyl cerebroside, and ganglioside. The fatty acid residue (corresponding to a linear hydrophobic group extending from the hydrophilic group of the neutral lipid of the present invention) in these glycerolipids or sphingolipids is not particularly limited, but for example, saturated or having 12 to 24 carbon atoms An unsaturated fatty acid residue can be mentioned, A saturated or unsaturated fatty acid residue having 14 to 20 carbon atoms is preferred. Specific examples include acyl groups derived from fatty acids such as lauric acid, myristic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, linolenic acid, arachidic acid, arachidonic acid, behenic acid, and lignoceric acid. Can do. When these glycerolipids or sphingolipids have two or more fatty acid residues, all the fatty acid residues may be the same group or different from each other.

  The pH-sensitive cationic lipid of the present invention constituting the lipid nanoparticle according to the present invention may be only one type or two or more types. When there are two or more types of the pH sensitive cationic lipid of the present invention constituting the lipid nanoparticle according to the present invention, the amount of the pH sensitive cationic lipid of the present invention is the present among the lipid molecules constituting the lipid nanoparticle. It means the total amount of lipid molecules corresponding to the pH-sensitive cationic lipid of the invention. Similarly, the neutral lipid of the present invention constituting the lipid nanoparticle according to the present invention may be only one kind or two or more kinds. When there are two or more neutral lipids of the present invention constituting the lipid nanoparticles according to the present invention, the amount of the neutral lipid of the present invention is the neutrality of the present invention among the lipid molecules constituting the lipid nanoparticles. It means the total amount of lipid molecules corresponding to lipids.

  The greater the proportion of the pH-sensitive cationic lipid of the present invention in the lipid molecules constituting the lipid nanoparticles, the higher the efficiency of incorporation of the lipid nanoparticles into the target cells. Therefore, in the lipid nanoparticle according to the present invention, the ratio of the amount of the pH sensitive cationic lipid of the present invention to the total amount of lipid constituting the lipid nanoparticle ([the amount of the pH sensitive cationic lipid of the present invention (mol) ] / ([Amount of total lipid constituting lipid nanoparticles (mol)] × 100%) is preferably 20 mol% or more. On the other hand, if the proportion of the pH-sensitive cationic lipid in the lipid molecules constituting the lipid nanoparticles is too large, it may be difficult to sufficiently reduce the particle size. Since lipid nanoparticles having sufficient lipid nanoparticle uptake efficiency into target cells and sufficiently small particle diameters can be obtained, the present invention for the total lipid amount constituting the lipid nanoparticles in the lipid nanoparticles according to the present invention The ratio of the amount of the pH-sensitive cationic lipid of the invention is more preferably 30 mol% or more, more preferably 30 to 70 mol%, and even more preferably 30 to 50 mol%.

  As the ratio of the neutral lipid of the present invention to the lipid molecules constituting the lipid nanoparticle increases, the stability of the lipid membrane on the particle surface increases, and sufficient delivery efficiency can be achieved even when the particle diameter is reduced. Therefore, in the lipid nanoparticle according to the present invention, the ratio of the amount of the neutral lipid of the present invention to the total amount of lipid constituting the lipid nanoparticle ([the amount of the neutral lipid of the present invention (mol)] / ([ The total amount of lipids constituting the lipid nanoparticles (mol)]) × 100%) is preferably 1 mol% or more, more preferably 3 mol% or more, further preferably 25 mol% or more, more preferably 30 to 70 mol%. Even more preferred is 50 to 70 mol%.

  The lipid nanoparticles according to the present invention may be composed of only the pH-sensitive cationic lipid of the present invention and the neutral lipid of the present invention, or the particles containing other lipid molecules. Good. When the lipid nanoparticle according to the present invention contains other lipid molecules, the ratio of the total amount of the pH-sensitive cationic lipid of the present invention and the neutral lipid of the present invention to the total amount of lipid constituting the lipid nanoparticle is: It may be 50 mol% or more, preferably 60 mol% or more, more preferably 70 mol% or more, still more preferably 80 mol% or more, and even more preferably 90 mol% or more.

  Of the constituent lipids of the lipid nanoparticles according to the present invention, as the lipids other than the pH-sensitive cationic lipid of the present invention and the neutral lipid of the present invention, lipids generally used when forming liposomes are used. Can do. Examples of such lipids include positively or negatively charged phospholipids, sterols, saturated or unsaturated fatty acids, and the like. These can be used alone or in combination of two or more.

  Examples of positively or negatively charged phospholipids include phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, ceramide phosphorylglycerol phosphate, and phosphatidic acid. Examples of sterols include sterols derived from animals such as cholesterol, cholesterol succinic acid, lanosterol, dihydrolanosterol, desmosterol and dihydrocholesterol; sterols derived from plants such as stigmasterol, sitosterol, campesterol and brassicasterol (phytosterols); Examples include sterols derived from microorganisms such as timosterol and ergosterol.

  The size of the lipid nanoparticles according to the present invention is preferably such that the average particle diameter is 50 nm or less because high delivery efficiency is easily obtained even when the target cells are present in a relatively deep part in the living body. Or less, more preferably 30 nm or less, and even more preferably 10 to 30 nm. The average particle diameter of lipid nanoparticles means the number average particle diameter measured by dynamic light scattering (DLS). Measurement by the dynamic light scattering method can be performed by a conventional method using a commercially available DLS apparatus or the like.

  The polydispersity index (PDI) of the lipid nanoparticles according to the present invention is about 0.05 to 0.1, preferably about 0.06 to 0.08, and more preferably about 0.07. The zeta potential can be in the range of 5.5 mV to 6.0 mV, preferably about 5.8 mV.

  The form of the lipid nanoparticle according to the present invention is not particularly limited. Examples of the form dispersed in an aqueous solvent include monolayer liposomes, multilamellar liposomes, spherical micelles, and amorphous layered structures. The lipid nanoparticles according to the present invention are preferably monolayer liposomes and multilamellar liposomes.

The lipid nanoparticles according to the present invention are mainly composed of the pH-sensitive cationic lipid of the present invention and the neutral lipid of the present invention, so that the lipid membrane on the particle surface has high stability and the lipid molecules are detached. hard. The reason why the lipid nanoparticles according to the present invention are excellent in stability is not clear, but the following reason is presumed. The pH-sensitive cationic lipid of the present invention has two linear hydrocarbon groups (R ¹ and R ² in the general formula (I)), and the interaction between the pH-sensitive cationic lipids is as follows. This is due to the hydrophobic bond between the hydrocarbon groups. In the pH-sensitive cationic lipid of the present invention, the linear hydrocarbon group has 20 or more carbon atoms and a relatively long chain, and the hydrophobicity is improved as compared with a shorter chain hydrocarbon group. This hydrophobic improvement enhances the interaction between the lipid molecules of the pH-sensitive cationic lipids, and as a result, the detachment of the lipid molecules from the lipid nanoparticles is suppressed. On the other hand, the neutral lipid of the present invention has a bulky hydrophilic group having 2 or more carbon atoms. Therefore, the stability of the lipid membrane on the particle surface of the lipid nanoparticle is high. That is, the lipid nanoparticle according to the present invention is composed of the pH-sensitive cationic lipid of the present invention and the neutral lipid of the present invention, whereby the interaction between lipid molecules in the lipid membrane on the particle surface is enhanced, In addition, the lipid membrane can be stabilized even when the lipid molecule density is low. For this reason, the lipid nanoparticles according to the present invention can suppress detachment of lipid molecules even when the particle diameter is reduced to 50 nm or less, and can reach the target cells more efficiently when administered in vivo.

The lipid nanoparticles according to the present invention can be subjected to appropriate surface modification as required.
For example, in order to promote the intranuclear transfer of lipid nanoparticles according to the present invention, for example, the lipid nanoparticles can be surface-modified with an oligosaccharide compound having 3 or more sugars. The type of oligosaccharide compound having 3 or more sugars is not particularly limited. For example, an oligosaccharide compound to which about 3 to 10 sugar units are bound can be used, and preferably about 3 to 6 sugar units are bound. Oligosaccharide compounds can be used.

  More specific examples of oligosaccharide compounds include, for example, cellotriose (Cellotriose: β-D-glucopyranosyl- (1 → 4) -β-D-glucopyranosyl- (1 → 4) -D-glucose), chacotriose: α -L-rhamnopyranosyl- (1 → 2)-[α-L-rhamnopyranosyl- (1 → 4)]-D-glucose), gentianose (β-D-fructofuranosyl β-D-glucopyranosyl- (1 → 6) -α-D-glucopyranoside), isomaltotriose (α-D-glucopyranosyl- (1 → 6) -α-D-glucopyranosyl- (1 → 6) -D-glucose), isopanose : Α-D-glucopyranosyl- (1 → 4)-[α-D-glucopyranosyl- (1 → 6)]-D-glucose), maltotriose (α-D-glucopyranosyl- (1 → 4)- α-D-Glucopyranosyl- (1 → 4) -D-glucose), Manninotriose (α-D-galactopyranosyl- (1 → 6) -α-D- Galactopyranosyl- (1 → 6) -D-glucose), Melezitose (α-D-glucopyranosyl- (1 → 3) -β-D-fructofuranosyl = α-D-glucopyranoside), Panose ( Panose: α-D-glucopyranosyl- (1 → 6) -α-D-glucopyranosyl- (1 → 4) -D-glucose), planteose (Planteose: α-D-galactopyranosyl- (1 → 6) -β-D-fructofuranosyl = α-D-glucopyranoside), raffinose (β-D-fructofuranosyl = α-D-galactopyranosyl- (1 → 6) -α-D-glucopyranoside ), Solatriose (α-L-rhamnopyranosyl- (1 → 2)-[β-D-glucopyranosyl- (1 → 3)]-D-galactose), Umbelliferose (β-D-fructofurano) Trisaccharide compounds such as syl = α-D-galactopyranosyl- (1 → 2) -α-D-galactopyranoside); Lycotetraose (β-D-glucopyra) Syl- (1 → 2)-[β-D-xylopyranosyl- (1 → 3)]-β-D-glucopyranosyl- (1 → 4) -β-D-galactose), maltotetraose (α) -D-Glucopyranosyl- (1 → 4) -α-D-Glucopyranosyl- (1 → 4) -α-D-Glucopyranosyl- (1 → 4) -D-glucose), Stachyose: β-D-fructo 4 sugar compounds such as furanosyl = α-D-galactopyranosyl- (1 → 6) -α-D-galactopyranosyl- (1 → 6) -α-D-glucopyranoside); maltopentaose : Α-D-glucopyranosyl- (1 → 4) -α-D-glucopyranosyl- (1 → 4) -α-D-glucopyranosyl- (1 → 4) -α-D-glucopyranosyl- (1 → 4) -D -Glucose), Verbascose: β-D-fructofuranosyl = α-D-galactopyranosyl- (1 → 6) -α-D-galactopyranosyl- (1 → 6) -α Pentasaccharide compounds such as -D-galactopyranosyl- (1 → 6) -α-D-glucopyranoside); malto Hexaose (Maltohexaose: α-D-glucopyranosyl- (1 → 4) -α-D-glucopyranosyl- (1 → 4) -α-D-glucopyranosyl- (1 → 4) -α-D-glucopyranosyl- (1 → 4 ) -α-D-glucopyranosyl- (1 → 4) -D-glucose), but not limited thereto.

  Preferably, an oligosaccharide compound that is a trimer to hexamer of glucose can be used, and more preferably, an oligosaccharide compound that is a trimer or tetramer of glucose can be used. More specifically, isomaltotriose, isopanose, maltotriose, maltotetraose, maltopentaose, maltohexaose, etc. can be suitably used, and among these, malto in which glucose is α1-4 bonded. More preferred is triose, maltotetraose, maltopentaose, or maltohexaose. Particularly preferred is maltotriose or maltotetraose, and most preferred is maltotriose. The surface modification amount of the lipid nanoparticles by the oligosaccharide compound is not particularly limited, but for example, about 1 to 30 mol%, preferably about 2 to 20 mol%, more preferably about 5 to 10 mol% with respect to the total lipid amount. It is.

  The method for modifying the surface of lipid nanoparticles with an oligosaccharide compound is not particularly limited. For example, liposomes whose surface is modified with monosaccharides such as galactose and mannose are known (International Publication No. 2007/102481). Therefore, the surface modification method described in this publication can be adopted. The entire disclosures of the above publications are incorporated herein by reference. This means is a method in which a monosaccharide compound is bonded to a polyalkylene glycolated lipid to modify the surface of the lipid nanoparticles, and this means is preferable because the surface of the lipid nanoparticles can be simultaneously modified with polyalkylene glycol.

  In some cases, the surface of the lipid nanoparticles may be modified with a hydrophilic polymer such as polyalkylene glycol to improve the stability of liposomes such as blood retention. This means is described, for example, in JP-A-1-249717, JP-A-2-149512, JP-A-4-346918, and JP-A-2004-10482. As the hydrophilic polymer, polyalkylene glycol is preferable. As polyalkylene glycol, for example, polyethylene glycol, polypropylene glycol, polytetramethylene glycol, polyhexamethylene glycol and the like can be used. The molecular weight of the polyalkylene glycol is, for example, about 300 to 10,000, preferably about 500 to 10,000, and more preferably about 1,000 to 5,000.

  Surface modification of lipid nanoparticles with polyalkylene glycol can be easily performed by constructing lipid nanoparticles using, for example, polyalkylene glycol-modified lipid as a lipid membrane constituent lipid. For example, when the modification with polyethylene glycol is performed, stearyl-ized polyethylene glycol (for example, PEG45 stearate (STR-PEG45) or the like) can be used. Others, N- [carbonyl-methoxypolyethyleneglycol-2000] -1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, n- [carbonyl-methoxypolyethyleneglycol-5000] -1,2-dipalmitoyl -sn-glycero-3-phosphoethanolamine, N- [carbonyl-methoxypolyethyleneglycol-750] -1,2-distearoyl-sn-glycero-3-phosphoethanolamine, N- [carbonyl-methoxypolyethyleneglycol -2000] -1,2-distearoyl-sn-glycero-3-phosphoethanolamine, N- [carbonyl-methoxypolyethyleneglycol-5000] -1,2-distearoyl-sn-glycero-3-phosphoethanol Polyethylene glycol derivatives such as amines can also be used, but polyalkylene glycolated lipids are not limited to these.

  In addition, surface modification with the polyalkylene glycol and the oligosaccharide compound can be simultaneously achieved by bonding the oligosaccharide compound to the polyalkylene glycol. However, the method for surface modification of lipid nanoparticles with polyalkylene glycol or oligosaccharide compound is not limited to the above-mentioned method. For example, lipidated compounds such as stearyl polyalkylene glycol and oligosaccharide compounds may be used. In some cases, the surface modification can be performed by using the lipid nanoparticle as a constituent lipid.

  In the production of lipid nanoparticles according to the present invention, examples of lipid derivatives for enhancing retention in blood include glycophorin, ganglioside GM1, phosphatidylinositol, ganglioside GM3, glucuronic acid derivatives, glutamic acid derivatives, polyglycerin phospholipid derivatives, and the like. Can also be used. In addition to polyalkylene glycols, dextran, pullulan, ficoll, polyvinyl alcohol, styrene-maleic anhydride alternating copolymer, divinyl ether-maleic anhydride alternating copolymer as well as polyalkylene glycol Amylose, amylopectin, chitosan, mannan, cyclodextrin, pectin, carrageenan and the like can also be used for surface modification.

  Lipid nanoparticles according to the present invention include a sterol or a membrane stabilizer such as glycerin or a fatty acid ester thereof, an antioxidant such as tocopherol, propyl gallate, ascorbyl palmitate, or butylated hydroxytoluene, a charged substance, and a membrane. One or two or more substances selected from the group consisting of polypeptides and the like may be included. Examples of the charged substance that imparts a positive charge include saturated or unsaturated aliphatic amines such as stearylamine and oleylamine; saturated or unsaturated cationic synthetic lipids such as dioleoyltrimethylammoniumpropane; or cationic polymers. Examples of the charged substance that imparts a negative charge include dicetyl phosphate, cholesteryl hemisuccinate, phosphatidylserine, phosphatidylinositol, and phosphatidic acid. Examples of the membrane polypeptide include a membrane superficial polypeptide or an integral membrane polypeptide. The compounding amount of these substances is not particularly limited, and can be appropriately selected according to the purpose.

  In addition, the lipid nanoparticles according to the present invention can be provided with any one function or two or more functions such as a temperature change sensitivity function, a membrane permeation function, a gene expression function, and a pH sensitivity function. By appropriately adding these functions, for example, the retention of lipid nanoparticles encapsulating nucleic acids containing genes in blood is improved, and the capture rate by reticuloendothelial tissues such as the liver and spleen is reduced. Thus, after endocytosis in the target cell, lipid nanoparticles can be efficiently escaped from the endosome and transferred into the nucleus, and high gene expression activity can be achieved in the nucleus.

  In addition, the lipid nanoparticles according to the present invention can be modified with a substance such as an antibody that can specifically bind to a cell surface receptor or antigen, thereby improving the substance delivery efficiency into the nucleus of the cell. can do. For example, it is preferable to arrange a monoclonal antibody against a biological component specifically expressed in the target tissue or organ on the surface of the lipid nanoparticle. This method is described in, for example, STEALTH LIPOSOME (pages 233-244, CRC Press, Inc., edited by Danilo Lasic and Frank Martin). Lipid derivatives capable of reacting with a mercapto group in a monoclonal antibody or a fragment thereof (for example, Fab fragment, F (ab ') 2 fragment, Fab' fragment, etc.), for example, poly (ethylene glycol) -α-distearoylphosphatidylethanolamine-ω-maleimide, α- [N- (1,2-distearoyl-sn-glycero-3-phosphoryl-ethyl) carbamyl) -ω- [3- [2- (2 , 5-dihydro-2,5-dioxo-1H-pyrrol-1-yl) ethanecarboxamido] propyl} -poly (oxy-1,2-ethanediyl) and other lipid derivatives having a maleimide structure, Monoclonal antibodies can be bound to the surface of the lipid lipid nanoparticle membrane.

  The surface of the lipid nanoparticle according to the present invention may be modified with a polypeptide containing a plurality of continuous arginine residues (hereinafter referred to as “polyarginine”). The polyarginine is preferably a polypeptide containing 4 to 20 consecutive arginine residues, more preferably a polypeptide consisting only of 4 to 20 consecutive arginine residues, particularly preferably octaarginine. Can do. By modifying the surface of lipid nanoparticles such as liposomes with polyarginine such as octaarginine, the intracellular delivery efficiency of the target substance encapsulated in the liposomes can be improved (Journal of Controlled Release, 98, pp.317 -323, 2004; WO 2005/32593). Modification of the lipid nanoparticle surface with polyarginine can be easily performed by using, for example, lipid-modified polyarginine such as stearylated octaarginine as a constituent lipid of the lipid nanoparticle according to the method described in the above publication. Can do. The disclosures of the above publications and the disclosures of all documents cited in this publication are incorporated herein by reference.

  The method for producing lipid nanoparticles according to the present invention is not particularly limited, and any method available to those skilled in the art can be adopted. For example, all the lipid components are dissolved in an organic solvent such as chloroform, and after forming a lipid film by drying under reduced pressure with an evaporator or spray drying with a spray dryer, the above mixture is dried with an aqueous solvent. And further emulsifying with an emulsifier such as a homogenizer, an ultrasonic emulsifier, or a high-pressure jet emulsifier. Moreover, it can manufacture also by the method well-known as a method of manufacturing a liposome, for example, a reverse phase evaporation method etc. When it is desired to control the size of the lipid nanoparticles, the extrusion (extrusion filtration) may be performed under high pressure using a membrane filter having a uniform pore size.

  The composition of the aqueous solvent (dispersion medium) is not particularly limited, and examples thereof include a buffer solution such as a phosphate buffer solution, a citrate buffer solution, and a phosphate buffered saline solution, a physiological saline solution, a medium for cell culture, and the like. Can do. These aqueous solvents (dispersion media) can stably disperse lipid nanoparticles, but also glucose, galactose, mannose, fructose, inositol, ribose, xylose sugar monosaccharides, lactose, sucrose, cellobiose, trehalose, Disaccharides such as maltose, trisaccharides such as raffinose and merezinose, polysaccharides such as cyclodextrin, sugars (aqueous solutions) such as sugar alcohols such as erythritol, xylitol, sorbitol, mannitol, maltitol, glycerin, diglycerin, polyglycerin , Propylene glycol, polypropylene glycol, ethylene glycol, diethylene glycol, triethylene glycol, polyethylene glycol, ethylene glycol monoalkyl ether, diethylene glycol -Alkyl ether, 1,3-polyhydric alcohol (aqueous solution), such as butylene glycol and the like may be added. In order to stably store the lipid nanoparticles dispersed in the aqueous solvent for a long period of time, it is desirable to eliminate the electrolyte in the aqueous solvent as much as possible from the viewpoint of physical stability such as aggregation suppression. In terms of chemical stability of the lipid, the pH of the aqueous solvent should be set from weakly acidic to near neutral (about pH 3.0 to 8.0) and / or dissolved oxygen can be removed by nitrogen bubbling or the like. Is desirable.

  When the resulting aqueous dispersion of lipid nanoparticles is freeze-dried or spray-dried, for example, glucose, galactose, mannose, fructose, inositol, ribose, xylose sugar monosaccharide, lactose, sucrose, cellobiose, trehalose, When stability can be improved by using sugars (aqueous solutions) such as disaccharides such as maltose, trisaccharides such as raffinose and merezinose, polysaccharides such as cyclodextrin, erythritol, xylitol, sorbitol, mannitol, maltitol, etc. There is. When the aqueous dispersion is frozen, for example, the saccharides, glycerin, diglycerin, polyglycerin, propylene glycol, polypropylene glycol, ethylene glycol, diethylene glycol, triethylene glycol, polyethylene glycol, ethylene glycol monoalkyl ether If a polyhydric alcohol (aqueous solution) such as diethylene glycol monoalkyl ether or 1,3-butylene glycol is used, stability may be improved.

  Since the lipid nanoparticle according to the present invention contains the pH-sensitive cationic lipid of the present invention as a constituent lipid, the incorporation efficiency into the cell is high, and the pH-sensitive cationic lipid of the present invention is converted to the neutral lipid of the present invention. In addition, since the lipid membrane is formed together, the stability of the lipid membrane on the particle surface is high even when the particle diameter is reduced. Therefore, the lipid nanoparticles according to the present invention can efficiently deliver the components encapsulated in the lipid membrane into the target cells. Therefore, the lipid nanoparticles according to the present invention are useful as a carrier for delivering a target component to target cells.

  The lipid nanoparticle according to the present invention preferably contains a target component to be delivered into a target cell inside a particle covered with a lipid membrane. The component that the lipid nanoparticle according to the present invention encapsulates inside the particle is not particularly limited as long as it has a size capable of being encapsulated, and the lipid nanoparticle according to the present invention includes nucleic acids, saccharides, peptides, Any substance such as a low molecular compound or a metal compound can be encapsulated. The component to be encapsulated in the lipid nanoparticle according to the present invention is preferably at least one selected from the group consisting of a low molecular compound, a low molecular nucleic acid, and a peptide. Examples of the low molecular nucleic acid include siRNA, microRNA, nucleic acid aptamer, decoy nucleic acid, ribozyme, CpG oligonucleic acid and the like.

  siRNA is a small double-stranded RNA consisting of 21 to 23 base pairs, is involved in RNA interference (RNAi), and suppresses gene expression in a sequence-specific manner by destroying mRNA. Synthetic siRNA has been reported to cause RNA interference in human cells, and it can be knocked down by RNA interference using siRNA, so that it can be used in medicine and in therapeutic fields such as cancer. Expected. The type of siRNA included in the lipid nanoparticle according to the present invention is not particularly limited, and any siRNA can be used as long as it can cause RNA interference. It is a pair of double-stranded RNAs, each of which has a structure in which the 3 ′ portion of the RNA strand protrudes by 2 bases, and each strand has a structure having a phosphate group at the 5 ′ end and a hydroxyl group at the 3 ′ end. It can be used as siRNA in the present invention. Also included are siRNAs in which the hydroxyl group at the 2 'position of the ribose backbone is partially substituted with a methoxy group, a fluoro group, or a methoxyethyl group, and a phosphodiester bond is partially substituted with a phosphorothioate bond.

  The lipid nanoparticles according to the present invention are particularly useful as carriers for delivering medicinal ingredients to target cells. The medicinal component contained in the lipid nanoparticles according to the present invention is not particularly limited, and any active pharmaceutical ingredient such as an anticancer agent, an anti-inflammatory agent, an antibacterial agent, and an antiviral agent can be used. Since the lipid nanoparticles according to the present invention can efficiently reach cancer cells deep in the tumor tissue beyond the interstitial barrier, they are carriers that contain medicinal ingredients used in cancer treatment. Preferably there is. In addition, the lipid nanoparticles according to the present invention can be expected to realize high permeability not only in cancer tissues but also in other normal tissues. Specifically, excellent penetrability in subcutaneous tissue and lung tissue is expected, and it is also useful as a nucleic acid delivery preparation for subcutaneous administration or transpulmonary administration (inhalation) that is relatively less invasive.

  Patent Document 1 discloses a method for synthesizing a lipid compound containing YSK12, a method for preparing a lipid membrane structure using the lipid compound, and a THP-1 cell (human monocyte strain) for the obtained lipid membrane structure. In particular, the gene expression inhibitory effect on Jurkat cells and the gene expression inhibitory effect on Jurkat cells (human T cell line) are specifically shown. The entire disclosure of Patent Document 1 is included in the disclosure of the present specification by reference.

  EXAMPLES Next, although an Example is shown and this invention is demonstrated further in detail, this invention is not limited to a following example.

<Synthesis of pH-sensitive cationic lipid represented by general formula (I)>
The reaction formula in the case of synthesizing a lipid in which c in general formula (A) is 0 using linoleic acid (Compound A) as a starting material is shown below. After linoleic acid is reduced with lithium aluminum hydride (Compound B), it is activated by mesylating the hydroxyl group (Compound C) and brominated by the action of magnesium bromide (Compound D). Two linoleic acid-derived hydrophobic scaffolds are linked by performing Grignard reaction using δ-Valerolactone as a substrate (Compound E). When a tertiary amino group is directly bonded to a hydrocarbon chain, the primary hydroxyl group is activated by tosylation (Compound F), and the amino group is introduced by a nucleophilic substitution reaction.

On the other hand, when a tertiary amino group is bonded through an ester bond, the amino acid is linked to the primary hydroxyl group by dehydration condensation. In the synthesis of lipids in which c in general formula (A) is 1 and R ¹ and R ² contain an ester bond, hydrogen atoms bonded to both terminal carbon atoms of a linear alkane (having 6 to 10 carbon atoms) A compound (Compound G) in which is substituted with a bromine atom and a hydroxyl group one by one is used as a starting material. The hydroxyl group is protected by tert-butyldimethylsilyl etherification (Compound H), and the two molecules are linked by the Grignard reaction as described above (Compound I). As above, after introducing a tertiary amino group directly to the primary hydroxyl moiety or via an ester bond (Compound J, M), the silyl ether is deprotected (Compound K, N) and optionally dehydrated by condensation. Of straight chain fatty acids.

<Cationic lipid>
In the following examples, the following five types of cationic lipids (CL15A6, CL15H6, CL15C6, CL12H6, CL4H6), four types of known cationic lipids (YSK05 (JP 2013-245190 A), Oil trimethylammonium propane (DOTAP), dimethyl dioctadecyl ammonium bromide (DDAB), and dimethylaminoethanecarbamoyl cholesterol (DC-chol) were used.

[Synthesis Example 1] Synthesis of CL15A6 In the above synthesis reaction 1, Compound E was synthesized using linoleic acid as Compound A, and CL15A6 was synthesized from Compound E by dehydration condensation reaction with 1-methyl-4-carboxylic acid. .
Specifically, in the same manner as the synthesis of YSK12 in Patent Document 1, 4-[(9z, 12z) -octadecadienyl]-(13z, 16z) -tricosadiene-1,4-diol (Compound E) was synthesized. Subsequently, Compound E (842 mg, 1.40 mmol) was dissolved in 10 mL of 1,2-dichloroethane. To this solution, 1-methyl-4-carboxylic acid 200 mg (1.40 mmol), 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride 383 mg (2.00 mmol) and N, N-dimethyl-4- Aminopyridine 17.1 mg (0.14 mmol) was added and the resulting mixture was stirred at room temperature overnight. Next, the mixture was diluted with 50 mL of ethyl acetate and then separated and washed with 1 N aqueous sodium hydroxide solution and saturated brine. Subsequently, anhydrous sodium sulfate was added to the washed organic layer for dehydration. After filtering this, the solvent was distilled off using a rotary evaporator to obtain a crude product. The crude product was purified by silica gel chromatography {elution solvent; dichloromethane: methanol (continuous gradient)} to give (14z, 17z) -5-hydroxy-5-[(9z, 12z) -octadeca-9, 12 -Dien-1-yl] tricosa-14,17-dien-1-yl 1-methylpiperazine-4-carboxylate (CL15A6) 877 mg (1.21 mmol) was obtained as a colorless oil. The yield was 86%.

[Synthesis Example 2] Synthesis of CL4H6

(1) ((6-Bromohexyl) oxy) (tert-butyl) dimethylsilane
6-Bromohexane-1-ol 20.0 g (110.5 mmol) was dissolved in 150 mL of 1,2-dichloroethane and cooled to 4 ° C. After adding 18.0 g (120 mmol) of tert-butyldimethylchlorosilane (TBSCl), 19.5 mL (140 mmol) of triethylamine (TEA) was added dropwise and stirred overnight at room temperature. The solvent was distilled off using a rotary evaporator, 300 mL of hexane was added and suspended, and the insoluble material was removed by Celite filtration to obtain a crude product. The crude product was purified by subjecting to silica gel chromatography {elution solvent; hexane: ethyl acetate (continuous gradient)} to give 26.0 g (88.0 mmol) of ((6-bromohexyl) oxy) (tert-butyl) dimethylsilane. Was obtained as a colorless oil. The yield was 80%.

¹ H NMR; 400 MHz δ = 0.05 (s, 6H), 0.89 (s, 9H), 1.31-1.54 (m, 6H), 1.86 (m, 2H), 3.40 (t, 2H), 3.96 (t, 2H ).

(2) 11-((tert-butyldimethylsilyl) oxy) -5- (6-((tert-butyldimethylsilyl) oxy) hexyl) undecane-1,5-diol
Dissolve 1.2 g (4.06 mmol) of ((6-bromohexyl) oxy) (tert-butyl) dimethylsilane in 4 mL of diethyl ether, add 2.43 g (100 mmol) of shaved magnesium, followed by iodine 1 fragment added. The mixture was allowed to stand at room temperature for 10 minutes, stirred while heating to 40 ° C. in an oil bath, and dissolved in 21 mL of diethyl ether ((6-bromohexyl) oxy) (tert-butyl) dimethylsilane (24.8 g, 83.94 mmol). ) Was added dropwise. The mixture was reacted at 40 ° C. for 2 hours and then cooled to 4 ° C. Subsequently, 3.67 mL (39.6 mmol) of δ-valerolactone was added and allowed to react overnight at room temperature. Next, it was cooled to 4 ° C. and 5% sulfuric acid was added dropwise to dissolve the remaining magnesium. The reaction solution was diluted with diethyl ether, and then separated and washed with water and saturated brine. Subsequently, anhydrous sodium sulfate was added to the washed organic layer for dehydration. After filtering this, the solvent was distilled off using a rotary evaporator to obtain a crude product. The crude product was purified by silica gel chromatography {elution solvent; hexane: ethyl acetate (continuous gradient)} to give 11-((tert-butyldimethylsilyl) oxy) -5- (6-((tert-butyl 14.0 g (26.3 mmol) of dimethylsilyl) oxy) hexyl) undecane-1,5-diol was obtained as a colorless oil. The yield based on δ-valerolactone was 66%.

¹ H NMR; 400 MHz δ = 0.05 (s, 12H), 0.89 (s, 18H), 1.25-1.56 (m, 26H), 3.59 (t, 4H), 3.65 (t, 2H).

(3) 11-((tert-butyldimethylsilyl) oxy) -5- (6-((tert-butyldimethylsilyl) oxy) hexyl) -5-hydroxyundecyl 4-methylbenzenesulfonate
11-((tert-butyldimethylsilyl) oxy) -5- (6-((tert-butyldimethylsilyl) oxy) hexyl) undecane-1,5-diol 14.0 g (26.3 mmol) dissolved in 50 mL dichloromethane Then, 321 mg (2.63 mmol) of DMAP (N, N-dimethyl-4-aminopyridine) and 5.50 mL (39.5 mmol) of diisopropylethylamine (DIPEA) were added and cooled to 4 ° C. Subsequently, 6.02 g (31.6 mmol) of p-toluenesulfonyl chloride (pTsCl) was gradually added, followed by reaction at room temperature overnight. The solvent was distilled off using a rotary evaporator, suspended in ethyl acetate, and separated and washed with water and saturated brine. Subsequently, anhydrous sodium sulfate was added to the washed organic layer for dehydration. After filtering this, the solvent was distilled off using a rotary evaporator to obtain a crude product. The crude product was purified by silica gel chromatography {elution solvent; hexane: ethyl acetate (continuous gradient)} to give 11-((tert-butyldimethylsilyl) oxy) -5- (6-((tert-butyl 12.4 g (28.0 mmol) of dimethylsilyl) oxy) hexyl) -5-hydroxyundecyl 4-methylbenzenesulfonate was obtained as a colorless oil. The yield was 69%.

(4) 11- (4- (Diisopropylamino) butyl) -2,2,3,3,19,19,20,20-octamethyl-4,18-dioxa-3,19-disilahenicosan-11-ol
11-((tert-butyldimethylsilyl) oxy) -5- (6-((tert-butyldimethylsilyl) oxy) hexyl) -5-hydroxyundecyl 4-methylbenzenesulfonate 12.4 g (18.0 mmol) in 30 mL Of tetrahydrofuran was added and cooled to 4 ° C. Then, after adding 7.38 mL (54.0 mmol) of dipropylamine, it was made to react at room temperature for 11 days. After the solvent was distilled off using a rotary evaporator, the mixture was suspended in ethyl acetate and separated and washed with a 0.5 N aqueous sodium hydroxide solution and saturated brine. The organic layer after washing was dehydrated by adding anhydrous sodium sulfate. After filtering this, the solvent was distilled off using a rotary evaporator to obtain a crude product. The crude product was purified by subjecting to silica gel chromatography {elution solvent; dichloromethane: methanol (continuous gradient)} to give 11- (4- (diisopropylamino) bull) -2,2,3,3,19,19, 20,27-Octamethyl-4,18-dioxa-3,19-disilahenicosan-11-ol 7.27 g (11.8 mmol) was obtained as a pale yellow oil. The yield was 66%.

¹ H NMR; 400 MHz δ = 0.05 (s, 12H), 0.89 (s, 18H), 1.24-1.64 (m, 30H), 2.30-2.43 (m, 6H), 3.58 (t, 4H).

(5) 7- (4- (Diisopropylamino) butyl) tridecane-1,7,13-triol
11- (4- (Diisopropylamino) butyl) -2,2,3,3,19,19,20,20-octamethyl-4,18-dioxa-3,19-disilahenicosan-11-ol 7.27 g (11.8 mmol ) Were added with 2.23 mL (39 mmol) of acetic acid and 26 mL of 1.0 M tetrabutylammonium fluoride in tetrahydrofuran and allowed to react overnight at room temperature. The solvent was distilled off using a rotary evaporator, and then purified by subjecting it to reverse phase silica gel chromatography {elution solvent; water (0.1% trifluoroacetic acid): acetonitrile 0.1% trifluoroacetic acid) (continuous gradient)}. -(4- (Diisopropylamino) butyl) tridecane-1,7,13-triol 3.43 g (8.85 mmol) was obtained as a pale yellow oil. The yield was 75%.

(6) 7- (4- (Diisopropylamino) butyl) -7-hydroxytridecane-1,13-diyl dioleate (CL4H6)
After dissolving 388 mg (1.0 mmol) of 7- (4- (diisopropylamino) butyl) tridecane-1,7,13-triol in 5 mL of dichloromethane, followed by addition of 900 mg (3.0 mmol) of oleyl chloride And cooled to 4 ° C. TEA 697 μL (5.0 mmol) was added dropwise and reacted at room temperature for 3 hours. After the solvent was distilled off using a rotary evaporator, the residue was suspended in ethyl acetate, and insoluble matters were removed by filtration. The filtrate was separated and washed with 0.5 N aqueous sodium hydroxide solution and saturated brine. The organic layer after washing was dehydrated by adding anhydrous sodium sulfate. After filtering this, the solvent was distilled off using a rotary evaporator to obtain a crude product. The crude product was purified by silica gel chromatography {elution solvent; dichloromethane: methanol (continuous gradient)} to give 7- (4- (diisopropylamino) butyl) -7-hydroxytridecane-1,13-diyl dioleate. 570 mg (0.622 mmol) of (CL4H6) was obtained as a pale yellow oil. The yield was 62%.

¹ H NMR; 400 MHz δ = 0.88 (m, 12H), 1.18-1.71 (m, 74H), 2.01 (m, 8H), 2.24-2.30 (t, 4H), 2.32-2.42 (m, 6H), 4.04 (t, 4H), 5.32 (m, 4H).

[Synthesis Example 3] Synthesis of CL15H6

(1) 11-((tert-butyldimethylsilyl) oxy) -5- (6-((tert-butyldimethylsilyl) oxy) hexyl) -5-hydroxyundecyl 1-methylpiperidine-4-carbooxylate
11-((tert-butyldimethylsilyl) oxy) -5- (6-((tert-butyldimethylsilyl) oxy) hexyl) undecane-1,5-diol 5.33 g (10.0 mmol) dissolved in 50 mL dichloromethane Then, 122 mg (1.0 mmol) of DMAP and 2.16 g (12.0 mmol) of 1-methylpiperidine-4-carboxoxy acid hydrochloride were added. Subsequently, 2.49 g (13.0 mmol) of EDCI was gradually added, followed by reaction at room temperature overnight. After the solvent was distilled off using a rotary evaporator, the residue was suspended in ethyl acetate, and insoluble matters were removed by filtration. The filtrate was separated and washed with 0.5 N aqueous sodium hydroxide solution and saturated brine. The organic layer after washing was dehydrated by adding anhydrous sodium sulfate. After filtering this, the solvent was distilled off using a rotary evaporator to obtain a crude product. The crude product was purified by silica gel chromatography {elution solvent; dichloromethane: methanol (continuous gradient)} to give 11-((tert-butyldimethylsilyl) oxy) -5- (6-((tert-butyldimethyl). Silyl) oxy) hexyl) -5-hydroxyundecyl 1-methylpiperidine-4-carbooxylate 5.01 g (7.61 mmol) was obtained as a colorless oil. The yield was 76%.

¹ H NMR; 400 MHz δ = 0.05 (s, 12H), 0.89 (s, 18H), 1.25-2.01 (m, 26H), 2.23 (br.s, 4H), 2.78 (m, 2H), 3.58 (t , 4H), 4.08 (t, 2H).

(2) 5,11-dihydroxy 5- (6-hydroxyhexyl) undecyl 1-methylpiperidine-4-carbooxylate
11-((tert-butyldimethylsilyl) oxy) -5- (6-((tert-butyldimethylsilyl) oxy) hexyl) -5-hydroxyundecyl 1-methylpiperidine-4-carbooxylate 5.01 g (7.61 mmol) was added with 1.43 mL (25 mmol) of acetic acid and 20 mL of 1.0 M tetrabutylammonium fluoride in tetrahydrofuran and allowed to react overnight at room temperature. The solvent was distilled off using a rotary evaporator, and then purified by subjecting it to reverse phase silica gel chromatography {eluting solvent; water (0.1% trifluoroacetic acid): acetonitrile 0.1% trifluoroacetic acid) (continuous gradient)}. , 11-dihydroxy 5- (6-hydroxyhexyl) undecyl 1-methylpiperidine-4-carbooxylate 2.34 g (5.45 mmol) was obtained as a pale yellow oil. The yield was 72%.

¹ H NMR; 400 MHz δ = 1.25-1.45 (m, 20H), 1.52 (m, 4H), 1.62 (m, 2H), 1.86 (m, 2H), 2.05 (m, 2H), 2.50-2.70 (m , 6H), 3.16 (m, 2H), 3.53 (t, 4H), 4.11 (t, 2H).

(3) 7-hydroxy 7- (4-((1-methylpiperidine-4-carbonyl) oxy) butyl) tridecane-1,13-diyl dioleate (CL15H6)
430 mg (1.00 mmol) of 5,11-dihydroxy 5- (6-hydroxyhexyl) undecyl 1-methylpiperidine-4-carboxylate was dissolved in 10 mL of dichloromethane. Subsequently, 706 mg (2.50 mmol) of oleic acid, 24.4 mg (0.20 mmol) of DMAP and 671 mg (3.5 mmol) of EDCI were added and reacted at room temperature for 2 hours. After the solvent was distilled off using a rotary evaporator, the residue was suspended in ethyl acetate, and insoluble matters were removed by filtration. The filtrate was separated and washed with 0.5 N aqueous sodium hydroxide solution and saturated brine. The organic layer after washing was dehydrated by adding anhydrous sodium sulfate. After filtering this, the solvent was distilled off using a rotary evaporator to obtain a crude product. The crude product was purified by silica gel chromatography {elution solvent; dichloromethane: methanol (continuous gradient)} to give 7-hydroxy 7- (4-((1-methylpiperidine-4-carbonyl) oxy) butyl) tridecane. -1,13-Diyl dioleate (CL15H6) 569 mg (0.594 mmol) was obtained as a pale yellow oil. The yield was 59%.

¹ H NMR; 400 MHz) Data δ = 0.88 (t, 6H), 1.20-2.05 (m, 78H), 2.28 (m, 8H), 2.82 (m, 2H), 4.07 (m, 6H), 5.33 (m , 4H).

[Synthesis Example 4] Synthesis of CL15C6

(1) 7-hydroxy 7- (4-((1-methylpiperidine-4-carbonyl) oxy) butyl) tridecane-1,13-diyl ditetradecanoate (CL15C6)
5,11-Dihydroxy 5- (6-hydroxyhexyl) undecyl 1-methylpiperidine-4-carbooxylate 85.9 mg (0.20 mmol) was dissolved in 1.5 mL dichloromethane followed by myristoyl chloride 163 mg (0.60 mmol) And then cooled to 4 ° C. TEA 139 μL (1.00 mmol) was added dropwise and reacted at room temperature overnight. After the solvent was distilled off using a rotary evaporator, the residue was suspended in ethyl acetate, and insoluble matters were removed by filtration. The filtrate was separated and washed with 0.2 N aqueous sodium hydroxide solution and saturated brine. The organic layer after washing was dehydrated by adding anhydrous sodium sulfate. After filtering this, the solvent was distilled off using a rotary evaporator to obtain a crude product. The crude product was purified by silica gel chromatography {elution solvent; dichloromethane: methanol (continuous gradient)} to give 7-hydroxy 7- (4-((1-methylpiperidine-4-carbonyl) oxy) butyl) tridecane. 116 mg (0.136 mmol) of -1,13-diyl ditetradecanoate (CL15C6) was obtained as a pale yellow solid. The yield was 68%.

¹ H NMR; 400 MHz δ = 0.88 (t, 6H), 1.20-2.10 (m, 78H), 2.28 (m, 8H), 2.82 (m, 2H), 4.06 (m, 6H).

[Synthesis Example 5] Synthesis of CL12H6

(1) 11-((tert-butyldimethylsilyl) oxy) -5- (6-((tert-butyldimethylsilyl) oxy) hexyl) -5-hydroxyundecyl 3- (dimethylamino) propanoate
11-((tert-butyldimethylsilyl) oxy) -5- (6-((tert-butyldimethylsilyl) oxy) hexyl) undecane-1,5-diol 12.73 g (23.9 mmol) dissolved in 64 mL dichloromethane Then, 293 mg (2.4 mmol) of DMAP and 4.04 g (26.3 mmol) of 3-dimethylaminopropanoic acid hydrochloride were added. Subsequently, EDCI 5.50 g (28.7 mmol) was gradually added, and the mixture was reacted at room temperature overnight. The reaction mixture was distilled off the solvent using a rotary evaporator, suspended in ethyl acetate, and insolubles were removed by filtration. The filtrate was separated and washed with 0.5 N aqueous sodium hydroxide solution and saturated brine. The organic layer after washing was dehydrated by adding anhydrous sodium sulfate. After filtering this, the solvent was distilled off using a rotary evaporator to obtain a crude product. The crude product was purified by silica gel chromatography {elution solvent; dichloromethane: methanol (continuous gradient)} to give 11-((tert-butyldimethylsilyl) oxy) -5- (6-((tert-butyldimethyl). 6.66 g (10.54 mmol) of silyl) oxy) hexyl) -5-hydroxyundecyl 3- (dimethylamino) propanoate were obtained as a pale yellow oil. The yield was 44%.

¹ H NMR; 400 MHz δ = 0.05 (s, 12H), 0.89 (s, 18H), 1.25-2.01 (m, 26H), 2.22 (s, 6H), 2.47 (t, 2H), 2.62 (t, 2H ), 3.60 (t, 4H), 4.08 (t, 2H).

(2) 5,11-dihydroxy 5- (6-hydroxyhexyl) undecyl 3- (dimethylamino) propanoate
11-((tert-butyldimethylsilyl) oxy) -5- (6-((tert-butyldimethylsilyl) oxy) hexyl) -5-hydroxyundecyl 3- (dimethylamino) propanoate 6.66 g (10.54 mmol) Acetic acid (1.82 mL, 31.6 mmol) and 21.1 mL of 1.0 M tetrabutylammonium fluoride in tetrahydrofuran were added and allowed to react overnight at room temperature. The solvent was distilled off using a rotary evaporator to obtain a crude product. The crude product was purified by silica gel chromatography {elution solvent; dichloromethane: methanol (continuous gradient)} to give 2,11-dihydroxy 5- (6-hydroxyhexyl) undecyl 3- (dimethylamino) propanoate 2.40 g ( 5.95 mmol) was obtained as a pale yellow oil. The yield was 57%.

¹ H NMR; 400 MHz δ = 1.25-1.45 (m, 20H), 1.61 (m, 6H), 2.83 (s, 6H), 2.88 (t, 2H), 3.32 (t, 2H), 3.63 (t, 4H ), 4.11 (t, 2H).

(3) 7- (4-((3-Dimethylamino) propanoyl) oxy) butyl) -7-hydroxytridecane-1,13-diyl dioleate (CL12H6)
800 mg (2.00 mmol) of 5,11-dihydroxy 5- (6-hydroxyhexyl) undecyl 3- (dimethylamino) propanoate was dissolved in 10 mL of dichloromethane. Subsequently, 1.24 g (4.40 mmol) of oleic acid, 48.9 mg (0.40 mmol) of DMAP, and 959 mg (5.0 mmol) of EDCI were added and allowed to react overnight at room temperature. After the solvent was distilled off using a rotary evaporator, the residue was suspended in ethyl acetate, and insoluble matters were removed by filtration. The filtrate was separated and washed with 0.5 N aqueous sodium hydroxide solution and saturated brine. The organic layer was dehydrated by adding anhydrous sodium sulfate. After filtering this, the solvent was distilled off using a rotary evaporator to obtain a crude product. The crude product was purified by subjecting it to silica gel chromatography {elution solvent; dichloromethane: methanol (continuous gradient)} to give 7- (4-((3-dimethylamino) propanoyl) oxy) butyl) -7-hydroxytridecane. 874 mg (0.937 mmol) of -1,13-diyl dioleate (CL12H6) was obtained as a colorless oil. The yield was 47%.

¹ H NMR; 400 MHz δ = 0.88 (t, 6H), 1.20-1.47 (m, 58H), 1.62 (m, 12H), 2.01 (m, 8H), 2.30 (m, 10H), 2.51 (t, 2H ), 2.69 (t, 2H), 4.06 (m, 6H), 5.33 (m, 4H).

<Neutral lipid>
In the examples that follow, three neutral lipids were used: cholesterol (chol), egg yolk-derived sphingomyelin (ESM), and polyethylene glycol 2000 modified dimyristoylglycerol (PEG-DMG).

[Example 1]
In lipid nanoparticles encapsulating siRNA, the influence of the kind of cationic lipid and neutral lipid constituting the particle on the knockdown activity was examined.
Specifically, lipid nanoparticles composed of lipids having a molar ratio shown in Table 1 and encapsulating siRNA were produced using a microchannel (staggered herringbone mixer (SHM) built-in non-patent document 4). . As siRNA, siGL4 targeting firefly luciferase was used.

<Manufacture of lipid nanoparticles>
Specifically, the lipid nanoparticles were produced by first preparing an ethanol solution adjusted to a lipid concentration of 8 mM and an acetate buffer solution (25 mM, pH 4.0) adjusted to a siRNA concentration of 71.1 μg / mL, each of 0.375 mL / min. The solution was fed into the microchannel at 1.125 mL / min, and the lipid nanoparticle solution excreted from the channel was collected. The lipid nanoparticle solution was placed in a dialysis membrane (MWCO 12,000-14,000), and dialyzed for 20 hours or more at 4 ° C. using an outer aqueous phase as a 20 mM MES buffer (pH 6.0). Thereafter, the outer aqueous phase was replaced with PBS (−) and further dialyzed at 4 ° C. for 2 hours or more, and then the lipid nanoparticle solution was recovered from the dialysis membrane.

<Evaluation of physical properties of lipid nanoparticles>
The average particle diameter (number average value) of lipid nanoparticles in PBS (−) and the zeta potential in 10 mM HEPES buffer (pH 7.4) were analyzed using an analyzer “Zetasizer Nano ZS ZEN3600” (Malvern). It was measured. The measurement results are shown in Table 1.

<SiRNA encapsulation rate of lipid nanoparticles>
The siRNA encapsulation rate of lipid nanoparticles was measured using an RNA intercalator Ribogreen (manufactured by life technologies). The measurement results are shown in Table 1. In Table 1, “CL” means a cationic lipid, and the parentheses in this column indicate the used cationic lipid.

 As a result, the lipid nanoparticles containing chol all had an average particle size of 30 nm or more, and the average particle size was reduced when the amount of PEG-DMG added was increased. In lipid nanoparticles in which half or all of chol was substituted with ESM, the average particle size was reduced depending on the amount of substitution with ESM. In particular, in the lipid nanoparticles in which the entire amount of chol was replaced with ESM, the average particle diameter was as small as 30 nm or less. In addition, all lipid nanoparticles had a zeta potential near neutral, and the siRNA encapsulation rate was as high as 95% or more.

<Measurement of in vitro knockdown activity>
Knockdown of each lipid nanoparticle produced using a dual luciferase stable expression strain HeLa-dluc cells in which both firefly luciferase and Renilla luciferase were stably expressed in a human cervical cancer-derived cell line HeLa The activity was examined. HeLa-dluc cells were cultured in DMEM containing 10% fetal bovine serum.

Specifically, each lipid nanoparticle was mixed in a culture medium of HeLa-dluc cells seeded in a 96-well plate at 6 × 10 ³ cells / well so that the final concentration of siGL4 was 1 to 14 nM ( Rivers transfection), and cultured for 24 hours in an environment of 37 ° C. and 5 vol% CO ₂ . After culturing, the supernatant was removed, the cells were lysed with Passive Lysis Buffer, and lysate was prepared. The firefly luciferase activity of this lysate was measured using Dual-Luciferase (trademark) Reporter Assay System (Promega). The firefly luciferase specific activity (%) standardized with the measurement value of Renilla luciferase activity as a control was used as the firefly luciferase gene expression rate (%). The lower the firefly luciferase gene expression rate, the higher the knockdown activity. The measurement result of the firefly luciferase gene expression rate (%) of each HeLa-dluc cell added with lipid nanoparticles is shown in FIGS.

  As a result, in lipid nanoparticles using cationic lipid and chol at 50:50 (molar ratio), the amount of PEG-DMG blended is smaller and the average particle size is larger for any cationic lipid. The expression level (relative) of firefly luciferase was low and the knockdown activity was high (FIG. 1). When lipid nanoparticles containing CL15H6 and lipid nanoparticles containing CL15A6 were compared, CL15A6 having a relatively short hydrocarbon chain showed gene knockdown in lipid nanoparticles having an average particle diameter of 65.2 nm. No knockdown activity was observed with 25.1 nm lipid nanoparticles. In contrast, CL15H6, which has a relatively long hydrocarbon chain, showed concentration-dependent gene knockdown not only with lipid nanoparticles with an average particle diameter of 54.1 nm but also with lipid nanoparticles with 35.7 nm.

  When chol was substituted with ESM among the constituent lipids of lipid nanoparticles, higher knockdown activity was observed as the amount of substitution with ESM increased (FIG. 2). In lipid nanoparticles containing CL15A6, gene knockdown was observed by replacing the entire amount of chol with ESM. In addition, among lipid nanoparticles containing CL15H6, extremely small lipid nanoparticles having an average particle diameter of 24.5 nm showed the most excellent knockdown activity.

[Example 2]
In Example 1, lipid nanoparticles having the composition of the constituent lipids CL15H6 / ESM / PEG-DMG = 50/50/3 (molar ratio) had the highest knockdown activity. Therefore, lipid nanoparticles were prepared by fixing the content of PEG-DMG at 3 mol% and changing the molar ratio of CL15H6 to ESM between 30:70 and 70:30, and had an effect on knockdown activity. Examined.

  Specifically, lipid nanoparticles were produced with the composition shown in Table 2, and the number average particle diameter (nm), zeta potential (mV), siRNA encapsulation rate (%), and knockdown activity (firefly luciferase gene expression rate) (%)). Production of lipid nanoparticles and subsequent measurements were carried out in the same manner as in Example 1. The results are shown in Table 2 and FIG.

  As a result, the lipid nanoparticles containing 40 mol% of CL15H6 had the smallest number average particle diameter of 22 nm. In any composition, the siRNA encapsulation rate was as high as 90% or more. When these lipid nanoparticles were evaluated for gene knockdown activity in HeLa-dluc cells, lipid nanoparticles containing 30 mol% of CL15H6 showed relatively low activity, while other lipid nanoparticles were equivalent to each other. Activity (FIG. 3). From the above results, the lipid nanoparticles composed of CL15H6, ESM, and PEG-DMG have sufficiently high gene knockdown activity and the smallest particle size. Therefore, CL15H6 / ESM / PEG-DMG = The optimum composition ratio was 40/60/3 (mol%).

  Subsequently, PEG-DMG is set to 0, 1, or to lipid nanoparticles of CL15H6 / ESM = 40/60 (mol%) and lipid nanoparticles of CL15H6 / chol = 40/60 (mol%), respectively. The average particle diameter of lipid nanoparticles formed by adding 3 mol% was measured, and the influence of PEG-DMG was examined. Production of lipid nanoparticles and subsequent measurement of the average particle diameter were carried out in the same manner as in Example 1. The results are shown in FIG.

  As a result, the average particle size of the lipid nanoparticles of CL15H6 / chol significantly increased as the amount of PEG-DMG decreased. In contrast, the average particle size of the CL15H6 / ESM lipid nanoparticles showed a minimum particle size of 30 nm or less independent of PEG-DMG (FIG. 4). As a result, since CL15H6 and chol do not have bulky hydrophilic sites, the lipid nanoparticle surface cannot be stabilized, and the stabilization of the lipid nanoparticles is greatly increased to PEG-DMG containing PEG which is a hydrophilic polymer. It shows that it is a dependent system. At the same time, in the CL15H6 / ESM system, since the lipid nanoparticle surface is stabilized by the bulky hydrophilic portion (phosphatidylcholine) of ESM, it can be inferred that it showed a very small particle size independent of PEG-DMG. Since PEG-DMG is known to rapidly desorb in the presence of serum, the property of forming tiny particles independent of PEG-DMG is the property of dispersibility of lipid nanoparticles in vivo. This is considered to be a significant point from the viewpoint.

[Example 3]
It was examined whether or not the lipid nanoparticles obtained in Example 2 having high gene knockdown activity and extremely small (average particle diameter on the order of 20 nm) could be obtained with neutral lipids other than ESM.

  Dioleoylphosphatidylcholine (DOPC: unsaturated bond number 2), egg yolk-derived phosphatidylcholine (EPC: unsaturated bond number 1) and dipalmitoyl phosphatidylcholine (DPPC: unsaturated bond number 0) were used as neutral lipids. All of these are neutral glycerophospholipids having a bulky hydrophilic group and a relatively long fatty acid residue. In addition, ESM was also used as a comparison target.

  Specifically, lipid nanoparticles were produced with the composition shown in Table 3, and the number average particle diameter (nm), siRNA encapsulation rate (%), and knockdown activity (firefly luciferase gene expression rate (%)) were determined. It was. Production of lipid nanoparticles and subsequent measurements were carried out in the same manner as in Example 1. The results are shown in Table 3 and FIG. In Table 3, the parentheses in the column of “Neutral phospholipid” indicate the neutral phospholipid used.

  As a result, even when any phospholipid was used, extremely small lipid nanoparticles having an average particle diameter of 30 nm or less were obtained. The siRNA encapsulation rate also showed a high value of 90% or more (Table 3). Moreover, when gene knockdown activity in HeLa-dluc cells was evaluated, remarkable gene knockdown activity was observed in any lipid nanoparticles.

[Example 4]
The effect of cationic lipid on the particle size of lipid nanoparticles was investigated. CL15H6, CL15C6, CL12H6, CL4H6, YSK05, DOTAP, DDAB, and DC-chol were used as the cationic lipid.

  Specifically, lipid nanoparticles were produced with each cationic lipid, ESM, and PEG-DMG as 40: 60: 3 (mol%), and the average particle diameter (nm) was measured. Production of lipid nanoparticles and subsequent measurements were carried out in the same manner as in Example 1. The results are shown in FIG.

  As a result, all lipid nanoparticles produced using CL15H6, CL15C6, CL12H6, and CL4H6 were extremely small particles having an average particle diameter of 30 nm or less (FIG. 6). On the other hand, all lipid nanoparticles produced using YSK05 and other widely used cationic lipids were relatively large particles having an average particle diameter of more than 30 nm. From these results, it is possible to produce extremely small lipid nanoparticles with an average particle diameter of 20 nm for the first time by appropriately combining specific cationic lipids such as CL15H6 and specific neutral lipids such as ESM. It has been shown.

[Example 5]
The storage stability of the lipid nanoparticles produced using CL15H6 in Example 4 was examined.
Specifically, lipid nanoparticles produced with CL15H6 / ESM / PEG-DMG = 40/60/3 (mol%) were suspended in PBS (−) (pH 7), and each suspended at 4 ° C. under light shielding. Saved for hours. The average particle diameter (nm) and siRNA encapsulation rate (%) of the lipid nanoparticles after storage were measured in the same manner as in Example 1.

  The results are shown in FIGS. As a result, no change in the average particle diameter (nm) (FIG. 7) and siRNA encapsulation rate (%) (FIG. 8) was observed over 4 weeks from production, and the lipid nanoparticles had high stability. It has been shown.

Claims (7)

The following general formula (I)

[In the formula (I), a represents an integer of 3 to 5; b represents 0 or 1; R ¹ and R ² each independently represents the following general formula (A):

(In formula (A), q represents an integer of 1 to 9; r represents 0 or 1; s represents an integer of 1 to 3; t represents 0 or 1; u represents an integer of 1 to 8] C represents 0 or 1; v represents an integer of 4 to 12; q + 2r + s + 2t + u + c + v is an integer of 19 or more, but when b and c are simultaneously 0, q is an integer of 3 to 5 Except that r and t are 1, s is 1, and u + v is an integer of 6 to 10)
X represents a group represented by the following general formula (B):

(In formula (B), d represents an integer of 0 to 3; R ³ and R ⁴ each independently represent a C _1-4 alkyl group or a C _2-4 alkenyl group (the C _1-4 alkyl group or C _{2 -4} alkenyl group represents one or two hydrogen atoms optionally substituted with a phenyl group, but R ³ and R ⁴ are bonded to each other to form a 5- to 7-membered non-aromatic heterocycle 1 or 2 hydrogen atoms of the ring may be substituted with a C _1-4 alkyl group or a C _2-4 alkenyl group).
Or a 5- to 7-membered non-aromatic heterocyclic group (wherein the group is bonded to (O-CO) b- by a carbon atom, and one or two hydrogen atoms of the ring are C _1-4 alkyl group or _C2-4 alkenyl group may be substituted)]
And a neutral lipid in which a linear hydrophobic group is extended from a hydrophilic group having 2 or more carbon atoms, and the total lipid amount constituting the lipid nanoparticle Lipid nanoparticles, wherein the proportion of the total amount of the pH-sensitive cationic lipid and the neutral lipid is 50 mol% or more.   The linear hydrophobic group in the neutral lipid is a linear hydrocarbon group having 16 to 20 carbon atoms (an ether bond, a carbonyl is present between one or more carbon-carbon single bonds in the group). The lipid nanoparticle according to claim 1, wherein one or more bonds selected from the group consisting of a bond, an ester bond, and a carbonate bond may be inserted.   The lipid nanoparticles according to claim 1 or 2, wherein the neutral lipid is glycerolipid or sphingolipid.   The lipid nanoparticles according to any one of claims 1 to 3, wherein the number average particle diameter measured by a dynamic light scattering method is 50 nm or less.   The lipid nanoparticle according to any one of claims 1 to 4, wherein a ratio of the amount of the pH-sensitive cationic lipid to the total amount of lipid constituting the lipid nanoparticle is 20 mol% or more.   The lipid nanoparticle according to any one of claims 1 to 5, comprising one or more selected from the group consisting of a low molecular compound, a low molecular nucleic acid, and a peptide.   The lipid nanoparticle according to any one of claims 1 to 6, which contains a medicinal component and is a carrier that delivers the medicinal component to a target cell.

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